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Editorial BoardViorel Alexandrescu (Cantacuzino N.M.M.I.R.D., Bucharest, Romania)Gabriela Anton (Şt. S. Nicolau Virology Institute, Bucharest, Romania) Fernando Baquero (Ramón y Cajal Health Research Institute, Ramón y Cajal University Hospital, Madrid, Spain) Daniela Bădescu (Cantacuzino N.M.M.I.R.D., Bucharest, Romania) Coralia Bleotu (University of Bucharest, Faculty of Biology, Bucharest, Romania) Carmen Chifiriuc (University of Bucharest, ICUB,Bucharest, Romania) Oana Ciofu (University of Copenhagen, Institute of Immunology and Microbiology, Faculty of Health and Medical Sciences, Copenhagen, Denmark) Irina Codiţă (Cantacuzino N.M.M.I.R.D., Bucharest, Romania) Daniel I. Cohen (Tel Aviv University, School of Public Health, Sackler Faculty of Medicine, Tel Aviv, Israel)Stefan N. Constantinescu (Université Catholique de Louvain, Belgium)Victor Cristea (Iuliu Haţieganu U.M.Ph., Cluj-Napoca, Romania) Jessica Cusato (University of Torino, School of Medicine, Torino, Italy) Luminiţa Smaranda Iancu (Gr. T. Popa U.M.Ph., Iaşi, Romania) David Y. Graham (Baylor College of Medicine, Houston, Texas, USA) Kianoush Khosravi-Darani (National Nutrition and Food Technology Research Institute, Tehran, Iran)Veronica Lazăr (University of Bucharest, Faculty of Biology, Bucharest, Romania) Andreea-Roxana Lupu (Cantacuzino N.M.M.I.R.D., Bucharest, Romania) Emilia Lupulescu (Cantacuzino N.M.M.I.R.D., Bucharest, Romania) Gina Manda (Victor Babeş N.I.R.D., Bucharest, Romania) Grigore Mihăescu (University of Bucharest, Faculty of Biology, Bucharest, Romania) Roxana Moldovan (Victor Babeş U.M.Ph, Timişoara, Romania) Thierry Naas (Université Paris-Sud, APHP, Institut Pasteur, Paris, France) Monica Neagu (Victor Babeş N.I.R.D., Bucharest, Romania) Marian Neguț (Academy of Medical Sciences, Bucharest, Romania)Adrian Onu (Cantacuzino N.M.M.I.R.D., Bucharest, Romania) Marina Pană (Cantacuzino N.M.M.I.R.D., Bucharest, Romania) Giovanni di Perri (University of Torino, School of Medicine, Torino, Italy) Graţiela Grădișteanu Pîrcălăbioru (University of Bucharest, ICUB, Bucharest, Romania) Aurora Sălăgeanu (Cantacuzino N.M.M.I.R.D., Bucharest, Romania)Edit Székely (U.M.Ph Târgu-Mureş, Romania)Codruţa-Romaniţa Usein (Cantacuzino N.M.M.I.R.D., Bucharest, Romania) Cem Uzun (Trakya University, Faculty of Medicine, Edirne, Turkey)Weiwen Zhang (Tianjin University, Tianjin, P.R. China)Imola Zigoneanu (University of North Carolina at Chapel Hill, USA)

ROMANIAN ARCHIVES OF MICROBIOLOGY AND IMMUNOLOGY

145 - Updating the roUtine speciation of romanian CampylobaCter strains Mădălina Militaru, Marilena Sorokin, Simona Adriana Ciontea, Daniela Cristea, Codruța-Romanița Usein, Ileana Stoica

149 - pro-and antitUmor roLe of the interLeUKins 1 to 41 Ovidiu Farc, Victor Cristea

163 - circULating tUmor ceLLs: fascinating insights in the cLinicaL reLevance Iulia I. Niță, Valentin Vasile, Laura Zaharia, Vlad Tofan, Cătălin Țucureanu, Ramona Caraghergheopol, Aurora Sălăgeanu, Iuliana Caraş

168 - cytotoxicity evaLUation of fe-based stabiLized sUspensions for biomedicaL appLications Dora Domnica Baciu, Iulia Ioana Lungu, Andrei-Mihai Dumitraşcu, Gabriel Prodan, Aurora Sălăgeanu and Florin Dumitrache

177 - a probLem-soLving integrative approach of esCheriChia Coli o157:h7 serotype diagnosis Codruța-Romanița Usein, Mădălina Militaru, Mihaela Oprea, Sorin Dinu, Daniela Cristea, Adriana Simona Ciontea

182 - virULence and resistance profiLes of shigella strains isoLated in romania from 2016 to 2018 Daniela Cristea, Adriana Simona Ciontea, Melania Mihaela Andrei, Andrei Popa, Mădălina Zamfir, Lavinia Zota, Maria Nica, Codruța-Romanița Usein

189 - eosinophiLia and parasitic diseases Dan Steriu

201 - gUideLines for aUthors

CONTENTS

VOLUME 78 Issue 3 JULY - SEPTEMBER 2019

145

Mãdãlina Militaru1, Marilena Sorokin2, Simona Adriana Ciontea1, Daniela Cristea1, Codruþa-Romaniþa Usein1,3*, Ileana Stoica4

1Cantacuzino National Medico-Military Institute for Research and Development, Bucharest, Romania2Prahova Public Health Authority

3Carol Davila University of Medicine and Pharmacy, Bucharest, Romania4University of Bucharest, Faculty of Biology, Bucharest, Romania

ROMANIAN ARCHIVES OF MICROBIOLOGY AND IMMUNOLOGY, Vol. 78, Issue 3, pp. 145-148, July - September, 2019

UPDATING THE ROUTINE SPECIATION OF ROMANIAN CAMPyloBACteR STRAINS

* Corresponding author:Codruþa-Romaniþa Usein, e-mail: [email protected]

AbStRACtIntroduction. diarrhea caused by Campylobacter spp., especially Campylobacter jejuni and Campylobacter

coli, is one of the most frequently reported gastrointestinal infection raising public health concern in many european countries.

Objectives. in order to improve the speciation protocol of Campylobacter strains for optimal disease surveillance, a PCR approach was introduced for species specific detection of C. jejuni and C. coli at reference laboratory level.

Methods. a collection of 70 Campylobacter strains previously identified at species level based on the hippurate hydrolysis biochemical test was used to test a three-gene multiplex-pcr-based method. previously published primers were used to target the hippuricase gene (hipo) characteristic of C. jejuni, aspartokinase gene (asp) characteristic of C. coli, and a universal 16s rdna gene sequence serving as an internal positive control.

Results. the phenotypic and genotypic methods were found to be 90% in concordance as six strains initially identified as C. coli by the biochemical tests proved to be C. jejuni in the pcr tests.

Conclusion. the established multiplex pcr approach can be used for routine diagnosis as well as epidemiological purposes.

Keywords: Campylobacter jeuni, Campylobacter coli, PCR identification

REZUMAtIntroducere. Diareea produsă de speciile Campylobacter jejuni si Campylobacter coli este printre cele mai

frecvente infecţii gastrointestinale, care ridică probleme de sănătate publică în multe ţări europene.Obiective. Pentru a îmbunătăţi identificarea speciilor de Campylobacter, necesară pentru supravegherea

optimă a infecţiei, la nivelul laboratorului de referinţă a fost introdus un protocol de analiză bazat pe PCR.Metode. O colecţie formată din 70 tulpini de Campylobacter, iniţial identificate la nivel de specie cu ajutorul

testului de hidroliză a hipuratului, a fost utilizată pentru a pune la punct un protocol bazat pe reacţia PCR multiplex. S-au folosit primeri preluaţi din literatură, pentru amplificarea genei pentru hipuricază (hipo), caracteristică speciei C. jejuni, genei pentru aspartokinază (asp), specifică pentru C. coli şi genei pentru ARNr 16s, care a servit drept control intern.

Rezultate. Concordanţa dintre metoda fenotipică şi genotipică a fost de 90%, şase dintre tulpinile identificate biochimic drept C.coli dovedindu-se a fi, la testarea prin PCR, C. jejuni.

Concluzie. Protocolul bazat pe PCR multiplex poate fi aplicat atât în scop diagnostic, cât şi epidemiologic.

Cuvinte-cheie: Campylobacter jejuni, Campylobacter coli, identificare prin PCR

INtRODUCtION

the increasing body of literature dedicated to campylobacteriosis, the collective name of the infectious diseases caused by members of the bacterial genus Campylobacter, reflects

the path of these pathogens from obscurity to notoriety in the field of public health [1]. typically, the infections present as diarrhea sometimes bloody with cramps, fever and pain that cannot be distinguished from those

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caused by other enteric pathogens. Usually, the symptoms resolve without antimicrobial therapy but occasionally they can persist and result in hospitalization. Although rare, local or systemic complications can occur in some patients. the guillain-barre´ syndrome, an acute demyelinating disease of the peripheral nervous system, is the most severe clinical entity linked to a preceding Campylobacter infection [2].

Campylobacter jejuni and Campylobacter coli are the most frequently isolated campylobac-ter species from human disease [3]. Of them, C. jejuni makes the greatest contribution, but it seems that the impact of other, less prevalent species has largely been ignored. the under-standing of the epidemiology of Campylobacter infections relies on the systematic study of the different species and subtypes resulted from the routine characterization of clinical strains. The isolation and identification of Campylobac-ter species are not easy and always successful procedures. both culture-dependent and cul-ture-independent present flaws, so there is no single gold standard method for Campylobacter identification [4]. As in many clinical laborato-ries molecular typing tools are widely applied in the identification of bacterial strains, this approach is also used for the differentiation of Campylobacter at species and strain level.

This report describes the first results ob-tained by using a pcr-based protocol adopted in the laboratory in order to complement the time consuming and sometimes difficult to in-terpret biochemical test based on hippurate hy-drolysis traditionally used for discriminating between C. coli and C. jejuni.

MAtERIALS AND MEtHODS

Strains and growth conditionsthis study focused on 70 Campylobacter

strains, isolated from stool specimens. five Campylobacter strains of know species, namely three C. jejuni and two C. coli were added as controls. all strains were grown on 7% (v/v) sheep blood columbia agar (prepared in house), under microaerobic conditions generated by campygen gas generating system (oxoid) with campygen™ sachet (5%

o2, 10% co2, and 85% n2), for 48 h at 42ºc. the template dna for pcr was prepared from each culture using pure Link genomic dna kit (invitrogen), following the manufacturer’s instruction.

Biochemical tests glucose fermentation, catalase, and nitrate

reduction were tested by routine laboratory procedures [5]. Oxidase test and hippurate hydrolysis test were performed according to the manufacturers’ instructions. antimicrobial susceptibility testing was performed using a disk diffusion test for nalidixic acid and cephalotin. No zone of growth inhibition was defined as “resistant” while any zone of inhibition was defined as “susceptible”.

Multiplex PCRbacterial dna template was prepared

using pure Link genomic dna kit (invitrogen) according to the manufacturers’ instructions. previously published primers targeting hippu-ricase (hipo) and putative aspartokinase (asp) genes were used for the molecular identifica-tion of the species Campylobacter jejuni and coli, combined with universal primers used to am-plify a fragment of the 16s rdna gene, serving as PCR internal positive control [6]. The assay was performed with a total volume of 25 µl containing 5 µl of template dna and the fol-lowing: 1x PCR buffer with 1.5 mM MgCl2, 200 mm (each) datp, dctp, dgtp, and dttp, 0.4 µm of asp-primers, 0.2 µm hipo-primers, 0.05 µm 16s rdna primers, and 1.25 U of taq dna polymerase (promega).

RESULtS AND DISCUSSION

all Campylobacter strains recovered from fecal cultures were first subjected to the phenotypic analysis which indicated 53 of them as reactive in the hippurate hydrolysis test and 17 hippuricase-negative.

for the routine diagnosis purpose, this biochemical test is of paramount importance for the differentiation of C. jejuni and C. coli, the former displaying hipurricase activity by contrast with the latter. However, reports have already showed that it may not be entirely

MILItARU et al.

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Updating the routine speciation of Romanian Campylobacter strains

reliable due to the problems caused by either the strains with an atypical phenotype, such as hippuricase-negative variants of C. jejuni as well as the sensitivity limitations leading to inability to detect low-level producers of hippuricase or the influence of a low inoculum size [7, 8]. therefore, as for other microorganisms, the use of pcr-based genotypic methods helps to overcome the problematic phenotypic identification. To date, the literature indicates that different molecular strategies and genetic targets relying on the pcr technique have been applied for the identification of Campylobacter to species level [9-13].

We decided to implement a multiplex pcr protocol which allowed the co-identification of C. jejuni and C. coli based on the detection of the hipo gene, characteristic of C. jejuni, and asp gene specific for C. coli.

the assay has been designed to also amplify the 16s rdna gene in order to provide a more intense validation with respect to sensitivity [6]. To optimize the PCR protocol, we initially used five well-characterized Campylobacter strains received in the laboratory with the occasion of an external Quality assessment (eQa).

all the strains originated from the eQa panel gave the same and expected results by

both phenotypic and genotypic methods. afterwards, when the Campylobacter clinical collection was tested, a 90% concordance was obtained between the methods used as six strains lacking the hippuricase activity and initially identify to C. coli species proved to be hipo-positive and asp-negative in pcr (fig. 1).

The PCR assay identified more C. jejuni strains than the classical biochemical test by detecting those that most probably did not ex-press hippurate hydrolysis activity in vitro.

overall, the percentage of C. jejuni mem-bers among the human strains of campylobac-ter sampled in this study was 84% and even though the aim was not to provide epidemio-logical data, we could not help noticing that the prevalence of C. jejuni was greater compared to the one reported in a previous romanian study [14].

This finding deserves further investigation to ascertain the trend of C. jejuni infections in locals.

the established multiplex pcr approach can be used for routine diagnosis as well as epidemiological purposes.

Conflict of interests: No conflict of interest to declare.

Fig. 1. Multiplex PCR on DNA extracted from Romanian strains of Campylobacter: lanes 1-3 and 5, 6, 8 -11, Campylobacter jejuni strains; lanes 4 and 12, Campylobacter coli strains;

lane 13, DNA negative control (H2O); lane 7, 14, 100 bp DNA marker.

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REFERENCES1. Butzler JP. Campylobacter, from obscurity

to celebrity. clin microbiol infect. 2004 oct;10(10):868-76. review. pubmed pmid: 15373879.

2. Nachamkin I. Chronic effects of Campylobacter infection. microbes infect. 2002 apr;4(4):399-403.

3. costa d, iraola g. pathogenomics of emerging campylobacter species. clin microbiol rev. 2019 Jul 3;32(4); pii:e00072-18; doi:10.1128/cmr.00072-18.

4. Magana M, Chatzipanagiotou S, Burriel ar, ioannidis a. inquiring into the gaps of campylobacter surveillance methods. vet Sci. 2017 July 19;4(3). pii:E36. doi:10.3390/vetsci4030036.

5. Murray PR, Baron EJ, Pfaller MA, Tenover fc and yolken rh (eds.) manual of clinical microbiology. 1995; asm press, Washington dc.

6. persson s, olsen Ke. multiplex pcr for identification of Campylobacter coli and Campylobacter jejuni from pure cultures and directly on stool samples. J Med Microbiol. 2005 nov;54(pt 11):1043-7).

7. Totten PA, Patton CM, Tenover FC, Barret TJ, Stamm WE, Steigerwalt AG, Lin JY, Holmes KK and Brenner DJ. Prevalence and characterization of hippurate-negative Campylobacter jejuni in King County, Washington. J Clin Microbiol. 1987;25:1747–1752.

8. On SLW, and Holmes B.E ffect of inoculum size on the phenotypic characterization of Campylobacter species. J Clin Microbiol. 1991;29:923–926.

9. Linton D, Lawson AJ, Owen RJ, Stanley J. PCR detection, identification to species level, and fingerprinting of Campylobacter jejuni and campylobacter coli direct from diarrheic samples. J Clin Microbiol. 1997 Oct;35(10):2568-72.

10. giesendorf ba, Quint Wg. detection and identification of Campylobacter spp using the polymerase chain reaction. cell mol biol (Noisy-le-grand). 1995 Jul;41(5):625-38.

11. Kulkarni SP, Lever S, Logan JM, Lawson AJ, Stanley J, Shafi MS. Detection of Campylobacter species: a comparison of culture and polymerase chain reaction based methods. J Clin Pathol. 2002 oct;55(10):749-53.

12. Platts-Mills JA, Liu J, Gratz J, Mduma E, Amour c, swai n, taniuchi m, begum s, peñataro yori p, tilley dh, Lee g, shen z, Whary mt, fox JG, McGrath M, Kosek M, Haque R, Houpt er. detection of campylobacter in stool and determination of significance by culture, enzyme immunoassay, and PCR in developing countries. J Clin Microbiol. 2014 Apr;52(4):1074-80. doi: 10.1128/JCM.02935-13

13. Persson S, Petersen HM, Jespersgaard C, Olsen Ke. real-time taqman polymerase chain re-action-based genus-identification and pyrose-quencing-based species identification of Cam-pylobacter jejuni, C. coli, C. lari, C. upsaliensis, and c. fetus directly on stool samples. diagn microbiol infect dis. 2012 sep;74(1):6-10.doi: 10.1016/j.diagmicrobio.2012.05.029

14. sorokin m, Usein cr, irimia m, damian m. a laboratory-based survey of campylobacter infections in prahova county. roum arch Microbiol Immunol. 2007 Jul-Dec;66(3-4):85-9.

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PRO-AND ANTITUMOR ROLE OF THE INTERLEUKINS 1 TO 41

Ovidiu Farc*, Victor CristeaIuliu Hațieganu University of Medicine and Pharmacy, Cluj-Napoca, Immunology Department, Cluj, Romania

* Corresponding author: Ovidiu Farc, Iuliu Haþieganu University of Medicine and Pharmacy, Cluj-Napoca, Immunology Department, Cluj, Romania, e-mail: [email protected], tel. +40 741 165 562

AbStRACtUntil recently, cancer was considered mainly a disease of genes, in which mutations accumulate over a

long period of time, both suppressors and oncogenes, ending with malignant transformation of the cell.this is undoubtedly true, but data accumulated in the recent years showed that the process is far more

complex than that, involving not only the malignant cells, but also a lot of non-malignant cells, fibrils and blood vessels, forming together what it was called the tumor microenvironment.

these cells, far from being only sustaining and nutrition elements, as they were thought to be, are in fact active participants to the malignant growth, being connected to the tumor cells through both direct connections (cell adhesion) and paracrine signaling through a complex network of cytokines, growth and angiogenic factors, chemokines and extracellular vesicles.

The purpose of the present study is to review the current knowledge concerning the roles of the different cytokines that are involved in the dynamic relation between the tumor and its microenvironment, with either pro-or antitumoral action.

Keywords: interleukin, cancer, protumoral, antitumoral

REZUMAtPână de curând, cancerul era considerat în principal o boală a genelor, în care mutațiile se acumulează pe

o perioadă lungă de timp, atât supresoare cât și oncogene, ducând la transformarea malignă a celulei.Acest lucru este fără îndoială adevărat, dar datele acumulate în ultimii ani au arătat că procesul este mult

mai complex, implicând nu numai celulele maligne, ci și numeroase celule non-maligne, fibre conjunctive sau vase sanguine, alcătuind ceea ce a fost numit micromediul tumoral.

Aceste celule, departe de a fi doar elemente de susținere și nutriție, așa cum se credea, sunt, de fapt, participanți activi la creșterea malignă, fiind conectate la celulele tumorale, atât prin conexiuni directe (adeziune celulară), cât și prin semnalizare paracrină printr-o rețea complexă de citokine, factori de creștere și angiogeni, chemokine și vezicule extracelulare.

Scopul prezentei lucrări este de a trece în revistă cunoștințele actuale cu privire la rolurile diferitelor citokine care sunt implicate în relația dinamică dintre tumoră și micromediul acesteia, având fie o acțiune pro-, fie una antitumorală.

Cuvinte-cheie: interleukină, cancer, protumoral, antitumoral

INtRODUCtION

interleukins are small proteins which func-tion as signals between the cells involved in the immune response, being also linked to other physiologic processes like embryonic develop-ment, skin biology or nervous system function-ing. in cancer they are secreted by the immune cells within the tumor and also by the tumor cells, and they influence not only the immune

infiltrates in the tumor, but also the tumor growth, angiogenesis and invasiveness. they have the potential of being useful biomarkers and powerful therapeutical means and are ex-tensively investigated as such.

the purpose of the present study is to re-view the 41 known interleukins, their mecha-nisms and interventions and their applications in the diagnostic and therapy of cancer.


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Proinflammatory cytokines and cancerInterleukin 1 (iL1) is produced by stromal

and tumor cells, it signals through the iraK system of kinases and NfkB pathway [1] and it has the effect of increasing proliferation, angio-genesis and invasiveness of the tumor cells [2]. It also supports inflammation, which is most-ly pro-tumoral [3]. It is up-regulated in many cancers like breast, lung, colon, head and neck cancer and melanoma [4] and associated with tumor stage and aggressivity.

there is also a positive role, since iL 1 supports antigen-specific antitumor defense. in some models, iL1 was necessary for the th1-dependent rejection of the tumor [5].

it is considered mostly protumoral, but inhibition through the natural antagonist IL1Ra had not the expected results [6] .

Interleukin 6. secreted by stromal and also by tumor cells, it signals through iL 6r-gp 130 and STAT 3 [1,7]. It has a direct role in sti-mulating the growth of the tumor cells through the activation of oncogenic pathways, and an indirect one, in stimulating inflammation [8, 9]. activation of ras in some tumors was iL6-de-pendent. it also can up-regulate iL17 and iL4, and inhibit apoptosis [9]. It increases with the tumor stage [10]. Like IL1, it is seen mainly as a target for therapy [6].

iL6 is also necessary for the immune response through the T and B lymphocytes [11]. it restores the sensitivity of the endothelium which is lost through the action of vegf and it favors the treatment with adoptive cells. for these reasons, in therapy, it should be given in brief cures, for obtaining this good effect without the favourable effect on the tumor growth [11].

TNFα. It is secreted by macrophages at the beginning of the inflammatory process. It signals through TNFR 1 and 2, NfkB, JNK and MAPK pathways [1,7].

in cancer, it is secreted by many tumor cells and acts in an autocrine way to increase NfkB signaling, which is pro-tumoral. It favors angiogenesis and is associated with progression in prostate, esophageal, cervical and breast cancer (reviewed in 12). it has also an anti-tumoral effect, through the stimulation

of m1 macrophages, dcs (dendritic cells) and Lts (lymphocytes) and promoting apoptosis of the tumor cells through the death domain of TNFR 1 [12].

in therapy, to overcome pro-tumor pro-perties while preserving the anti-tumor ones, modified molecules, simultaneous inhibition of NfkB and local administration have been proposed, with good effect [13].

Interleukin 18, member of the iL1 family, signals through the iraK system of kinases and NfkB, and it has antitumor effects through the stimulation of Th1 differentiation and of ifnϒ (interferon ϒ) secretion, especially in the presence of IL 12 [14]. In other tumors it has a pro-tumoral effect similar to IL1 [8, 14, 15]. in certain tumors (esophageal, breast, stomach, lung, hepatic, renal, ovarian), iL 18 was up-regulated in relation with stage and worse prognosis (14 and references therein).

in therapy with anti pdL1, gm-csf, in vaccines and lymphocytes with car, adding IL 18 was beneficial [16].

Interleukin 20. secreted by the monocytes and other cells (keratinocytes, activated en-dothelia), it targets keratinocytes through JAK and STAT3, regulating these cells in inflamma-tion. it is also a hematopoietic cytokine for pro-genitor cells. Hypoxia is a stimulus [7, 17].

In tumors, it has the effect of increasing os-teolysis and angiogenesis. there are receptors on some tumors, and their activation leads to increases in iL1, iL6, tgf and mmps (matrix metalloproteinases) [8, 18].

in breast tumors, it led to angiogenesis, increase of tumor-associated macrophages and increased proliferation; in bladder tumors, increases in iL20 and iL20-r led to the acceleration of the tumor growth [18]. In lung cancer models, it increased, together with hgf (hepatocyte growth factor), the tumor growth; in hepatocellular cancer the inhibition of IL20 led to decreased tumor growth [19]; skin, esophageal, bone and pleural cancer are also locations where iL 20 has a protumoral activity [18].

iL 20 is seen mainly as a potential thera-peutical target.

A pro-tumoral and proinflammatory role has also been found for Interleukin 32. in can-

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cer, overexpression of iL 32 is due to hypoxia and it contributes to an inflammatory environ-ment, stimulating also proliferation, angioge-nesis and invasion through NfkB, VEGF, MMPs 2 and 9 [19].

In cervical cancer inflammation is detri-mental and iL 32 is increased with progression of tumors; same is true in hepatocellular, pan-creatic, oesophageal, gastric, breast cancer and cutaneous T-cell lymphoma [20]. In colorectal cancer, it has an anti-tumor effect through in-creasing cd8+ Lts in stroma and by diminish-ing the stemness of cancer stem cells [20].

So the anti- or pro-tumor effect of IL 32 depends on the tissue and tumor, being mostly protumoral.

Interleukin 36 has three isoforms and is secreted in the early phase of the inflammatory response, as an alarmin. it belongs to iL1 family of interleukins; its targets are Lts (with increased iL2 secretion and stimulation of th1 defense), fibroblasts and epithelia. It has a natural antagonist, IL36Ra [7, 21].

in cancer, few things are known; iL36 alpha is low in poorly differentiated cells; increased iL 36 leads to increased cd 8+Lts (not cd4+ Lts), low iL 36 being a marker of worse prognosis [21]. It suppresses ovary tumor cells growth in vitro and in vivo; in colorectal and hepatocellular cancer there is the same effect, through immune mechanism. no protumoral effect has been found for IL 36 so far.

iL 36 is overexpressed in lung, neck, eso-phageal and colorectal cancers, correlated with stage; tumor cells express iL36 which functions as an antitumor vaccine [21].

Cytokines stimulatory for the lymphocytesInterleukin 2. it is secreted by the tumor-

infiltrating lymphocytes and it has a stimulating action on the antigen-specific LTs, effect which is partly reduced by the activation of Lt regs (T regulatory lymphocytes) [1, 7]. It is used as therapy in renal carcinoma and melanoma, alone or in combination with other cytokines or vaccines [6].

Interleukin 15 is produced mainly by monocytes and dendritic cells. it signals through iL2 r and it has the same downstream signaling like iL 2.

in cancer, it can stimulate the growth of the cells of hematological malignancies, but it does not appear to have any effect on the solid tumors. instead, it is thought to strengthen the immunosurveillance and the immune antitumor defense [6]. Unlike IL2, it has no stimulatory effect on Tregs, nor does it stimulate aicd-(activation-induced cell death) -of Lts, as iL2 does, things that can be useful in immunotherapy [1, 6].

therapeutical uses are the same as for iL2- stimulation of the antitumor Lth1 response, alone or in combination with vaccines [22].

Interleukin 12. it is produced by macro-phages, and also by neutrophils, dcs, Lts and glial cells; together with iL 27, it stimulates the Th1 profile of LTs. It is important in the re-sponse to infectious pathogens and also in the antitumor response [1, 7].

in tumors, it is associated with good prognosis; it diminishes angiogenesis and stroma through fasl, it stimulates th1 Lts and inhibits m2 macrophages and mdscs (myeloid-derived suppressor cells). it stimu-lates the production of ifnϒ [23].

it is between the most potent antitumor cy-tokines. It is believed to be a possible adjuvant for tumor vaccines or other therapies [6, 23].

Interleukin 27. secreted by dcs, macro-phages and epithelial cells, it targets nK and LTh1 cells, to whose differentiation it contri-butes. it also inhibits th17 cells through stat 1 [7]. In cancer, it inhibits the tumor growth through infiltration with myeloid cells ( from which m1 macrophages, cd 11+ cells). it also inhibited pancreatic, lung, cervical and pros-tatic cancer cells, through the inhibition of iL 8 and angiogenesis and also through the up-reg-ulation of fasl and inhibition of bcl, support-ing apoptosis [24].

Interleukin 9. Formerly classified as a th2 cytokine, it is now considered separately as being produced by a special type of Lth, th9 cells. it stimulates the cd8+,th2 and th17 and inhibits Th 1 differentiation. A subset of Th9mem lymphocytes have been described [1, 7, 25].

in cancer, in some tumors like melanomas it inhibits the tumor growth; in others like

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acute leukemia, it is protumoral. intratumoral injection of IL9 slows the tumor growth, not through direct action on the tumor cells, but through the stimulation of th9 cells which are thought to be tumoricidal [26]. In tumor models, th9 recruites dcs in the tumor, which in turn stimulate cd8+ ctLs with tumoricidal effect. This effect depends on IFNϓ [27].

Interleukin 21. it is a th2 and th9 cytokine and its targets are cd4, cd8 dcs, nKt cells, macrophages and keratinocytes. it stimulates antigene-specific cells, DCs and LTh 17 cells [7].

in tumor models, it led to the regression of melanoma, renal and other tumors. iL21 in-jected in the tumor increased inborn antitumor immunity (dc, nK cells), through ifnϒ. its activity is also mediated through cd8+ t cells and B cells [28]. IL21 increased antibody-medi-ated tumor rejection.

in colorectal cancer, it is increased in the tumor tissue, due to the secretion from stromal nK and Lt; in this location it is believed to be pro-tumorigenic through the up-regulation of IL6, 22, 17 [29].

in therapy, it is used with sorafenib in a phase i/ii trial. there is also a hybrid molecule with anti her2 antibody. vaccines with cells expressing IL21 proved to be effective in reducing tumor [30].

in conclusion, many tumors (except maybe colorectal cancer), would benefit from therapy with iL 21.

Interferons (α, β and ϒ, interleukins 28 and 29) are important anti-tumor elements, with direct tumoricidal effect on the tumor cell, inducing apoptosis, increasing the expression of MCH (major histocompatibility complex), stimulating lymphocytes and m1 macrophages, at the same time inhibiting the Th2 profile [7, 31]. Interferons alone or in combination with kinases inhibitors or egfr inhibitors, are of value in cancer treatment [31].

Th-2 cytokines Interleukin 4. produced mainly by th2

lymphocytes and basophils, iL 4 stimulates the differentiation of the Th2 lymphocytes, of LBs and m2 macrophages. it signals through iL4r

and STAT 6, through AKT, MAPK and NfkB [1, 7].

iL4 increases the resistance to apoptosis, activates pro-tumor macrophages, promotes mitogenesis in some animal models [8, 32]. IL 4 r is found in a lot of tumors. iL 4 contributes also to the tumor development through the stimulation of the Th2 profile, which is not fa-vorable to the antitumor defense, and through inhibiting the Th1 profile [32].

in cancer, iL 4 is overexpressed in a lot of epithelial tumors -prostate, bladder, colon, ovary, pancreas, breast, lung, being related to stage and grade [32].

Interleukin 13 is secreted by stromal th2 lymphocytes and tumor cells, it signals through IL4R and IL13R [6], and it increases the proliferation, invasiveness, emt (epitheli-al-to mesenchimal transition) of the colon, pan-creatic, ovarian tumors and malignant glioma, (where the receptor IL13R is present) [32]; it also contributes to the immune depression of the tumor microenvironment [8, 32].

Antagonizing IL4 and 13 is likely to have a good effect on the tumor inhibition [33].

Interleukin 25 (iL 17 e). it is a product of Lth2, mast cells, eosinophils and epithelial cells.it activates iL17 br, stimulates th2 response, IgEs, NfkB, IL8 and eosinophils and inhibits Th1 and Th9 responses on models [7].

in cancer, it is a potent antitumor agent in melanoma, breast, lung, colon and pancreatic cancer; it inhibits cancer cells in culture; it sti-mulates death receptors; it is tumoricidal on cells with IL17BR [34]. Blocking IL 25 in colorec-tal cancer led to increased tumor growth.

Interleukin 5 is a hematopoietic cytokine produced by the th2 lymphocytes and by basophils. it stimulates eosinophil activation and proliferation and also b cell growth and antibody production [1].

in cancer, iL 5 increased development of metastasis. Eosinophilic deficiency abolished this effect. IL 5 had no effect on the primary tumor. suggested mechanism is secretion of CCL 22 by eosinophils, which attracts Tregs in the tumor microenvironment [35]. The same was true in esophageal carcinoma. it has also

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been shown to facilitate the invasion of bladder tumor cells [36].

in other tumor models, the eosinophils had a tumoricidal efect. this is true especially for early stages [8].

In conclusion, the pro- or antitumor effect of iL5 depends on the tissue and tumor type.

Interleukin 19. expressed by monocytes and also by keratinocytes and some Lb cells, it signals through iLr20 and stat3, and it targets mainly keratinocytes, and also Lth2 cells [7].

in tumors, it is produced by monocytes and tumor cells at inflammatory stimuli and it has an autocrine action of promoting mitogenesis, being associated with worse prognosis. it stimulates Lth2 cells, iL 9,5,10,13 and inhibits LTh; it increases IL1, 6, TGF and MMP 2 [37].

it is up-regulated in breast, tongue, skin, cervical, esophageal, ovarian cancer [37].

given these facts, iL 19 is seen as a potential therapeutic target.

Interleukin 24, secreted by lymphocytes (mainly th2), monocytes and melanocytes [7], acts by inhibiting the cell cycle and by stimulating the autophagy and apoptosis of the tumor cells. this action is thought to be independent of its cytokine receptor, which is IL 20R [38]. In therapy, it is in phase I-II trials for cancer treatment [39].

Interleukin 31. it is also a th2 cytokine; DCs and mast cells also produce it [7]. There is an increase of the levels of iLs 31 and 33 in uterine cancer with stage and metastasis [40]; it provokes the pruritus of lymphomas and mastocytosis. there are also studies that reveal an anti-tumor role in cancer, through the inhibition of angiogenesis and invasion [41]. Overall, it is seen as contributing to tumor development.

Interleukin 33. It is a Th2 polarizing cytokine, targeting also iLc2 (innate lymphoid cells type 2 ) and mast cells [7]. In cancer, giving iL 33 to mice increased the tumor growth and the stemness of the tumor cells, by activating stem genes-nanog, oct ¾ and notch. in glioma, it increases migration; in gastric cancer, it stimulates mapK, iL 6 and mmp3 [42]. In some experiments, IL 33 inhibited the

growth of colic tumors, inhibition of st2 (iL 33 receptor) and iL 33 leading to accelerated tumor growth [43].

Th-17 cytokinesInterleukin 23 is a Th 17 polarizing

cytokine [7].in cancer, it promotes tumor growth

and inflammation in the microenvironment, through iL 17, 22, mmp 9; in hepatocellular cancer, it favors proliferation and migration [44]; in colorectal cancer, together with IL 17, it produces inflammation and is associated with bad prognosis (reviewed in 45). it is seen as a possible target.

Interleukin 17 is produced by Lth 17; iL 17 family has 6 members (iL17 a-f); its role is to stimulate the immune defense of the natural barriers, by increasing NFkB stimulation [1]. in cancer, the origin of iL 17 are stromal cells. iL 17 a is pro-angiogenic through vegf and PGE2 [46]; injection of IL 17 increased the vas-cularization and proliferation of colon tumors; it also stimulates IL 6 which is protumoral [47]. some studies showed a protective role of iL 17 in colon cancer [47].

iL 17 and 23 and their receptors are up-reg-ulated in a lot of epithelial tumors: colon, ova-ry, lung, breast, stomach, skin, liver, head and neck cancer [45, 46].

Interleukin 22 is a product mainly of th22 cells. other sources are iLcs, th1 and th17 lymphocytes. the targets are non-hematopoie-tic cells. it contributes to the defense and tissue regeneration after inflammations [7].

in cancer, it promotes the tumor growth on models; growth of breast cancer cells in culture is stimulated [48]. IL 22 and Th 22 cells in pancreatic cancer are associated with stage and progression of tumor [49]. It protects co-lon cancer cells and supports stemness through stat 3 and akt.

Interleukin 26. it is produced by the Lth 17 cells and its targets are epithelia, which it activates and regulates during inflammation; it activates mapK and aKt pathways, stats 1, 3 and contributes to antibacterial and antiviral defense [7, 50].


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in colorectal cancer it stimulates icam-1, iLs 8 and 10 ; in gastric cancer it up-regulates stat 3, bcl, myc and is pro-tumoral; most of the infiltrating lymphocytes are Th17 and express IL 26 [51].

in conclusion, iL 17, together with its cytokine network, contributes through multiple mechanisms to cancer development.

Anti-inflammatory cytokinesInterleukin 10, produced by Ltregs, m2

macrophages, some tumor cells, mdscs and Th2 lymphocytes [1, 7], stimulates the prolife-ration of some cells with receptor, being also immunosuppressive [52]. In increased doses, it stimulates CD8+LTs; it also limits the inflam-mation which is mostly protumoral [53].

Interleukin 30. sources are myeloid and tumor cells; it is a subunit of iL 27 and it is anti-inflammatory [6]; in cancer, its expression increases with stage; tumor growth is stimulated and stromal immune cells are regulated. it stimulates breast cancer growth, through the mechanisms above [54]. It is regarded as a possible therapeutical target.

Interleukin 35, produced by tregs and stimulating this subset [7], is antitumoral by a direct mechanism, stimulating apoptosis and arresting the cell cycle [55], and also by the inhibition of inflammation. It also has a protumoral action, being immunosuppressive through the mentioned effect of Treg stimula-tion, and associated with aggressiveness and recurrence [56].

TGFβ (transforming growth factor beta), an important factor of immune suppression, is produced by many tumor cells and also by tregs and mdscs; it stimulates Ltregs, and, if iL 6 and iL 23 are present, it favors the th17 differentiation [1, 7]. In the initial phases of the tumor development, it is antitumoral by stimu-lating apoptosis (smad pathway), while in the more advanced stages it is a promoter of the tumor development, invasion and EMT [57]. together with iL33 and iL15, it stimulates Lt cd8+mem, important antitumoral subset.

Interleukin 37, secreted by monocytes, DCs and epithelial cells, [7] inhibits direct-ly the tumor growth and angiogenesis, part-

ly through the inhibition of STAT3 [58], and also through a stimulatory action on the nK lymphocytes. iL 37 is a promising therapeutic mean and a potential biomarker in cancer.

IL1 Ra – natural inhibitor of iL1, limits its protumoral action, therefore being considered for the therapy of some tumor types [6].

ChemokinesInterleukin 8 is secreted by many cells in

the inflammatory process and it targets neutro-phils, LTs, monocytes and endothelial cells [1, 7]. Secreted by many cell lines and for which there are receptors on many malignant cells, it acts as an angiogene on the microenvironment and as a stimulator of the proliferation on the tumor cells [74]. It also leads to the infiltration of tumors with different myeloid cells (neutro-phils and others) and, by consequence, to angi-ogenesis [59].

Interleukin 16, overexpressed in some tumors, attracts cells with CD4+, but also favors invasion in some tumors like multiple myeloma [60].

Hematopoietic cytokinesInterleukin 3 is synthesized by

lymphocytes and it supports hematopoiesis (multilineage CSF) [7]. It is angiogenic in some tumors [61]; it contributes to the bone marrow regeneration after radiotherapy. it contributes to the growth of some tumor cells with receptor, especially hematogene (but also solid tumors). it has been proposed as a recovery agent after bone marrow depression with chemoterapy. it is also considered for gene therapy of cancer [62].

Interleukin 7. produced by macrophages, dcs and keratinocytes, it is a lymphopoietic cytokine which signals through JAK 3-STAT 1 and PI3K and increases the Th1 response [1, 6]. in tumor models, it inhibits the tumor growth, stimulating the antitumor response. Injection with IL7 produced complete remission [63]. It is believed to produce immune regeneration in cancer, where there is immune depression.

Interleukin 11. produced by the stromal cells and stimulating myeloid hematopoiesis, especially in situations of infection, this cy-

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tokine, belonging to the iL 6 superfamily, sig-nals through IL6 R-gp 130 and through JAK-STAT 3 [1, 7]. In tumors, it is produced by the stromal and tumor cells and it has a favourable action on the tumor growth, both on the tumor cells and on the microenvironment. it has been associated with unfavourable prognosis in breast cancer. it is up-regulated in many can-cers [64].

Other interleukins- Interleukin 34 increases the growth of

the tumor cells with csfr (colony-stimulating factor receptor), by increasing proliferation, survival and invasiveness, through the stimu-lation of ERK [65].

- Interleukin 14 is a B cell growth factor [1].

- Interleukin 38 signals through iL 36 r and inhibits the production of iL17 and 22 and also inhibits iL 36 on the receptor, acting as an anti-inflammatory cytokine [7].

- Interleukin 39 is produced by macrophages and barrier tissues and it is a th2 cytokine.

- Interleukin 40 is expressed by b cells and stimulates antibody production, especially at the mucosal barriers.

- Interleukin 41 is secreted by macrophages and is proinflammatory.

except for implications in the pathogenesis of some hematological malignancies of iL 14, and the association of iL 38 with some solid tumors [66], there is no known involvement of interleukins14, 38, 39, 40 and 41 in cancer.


Fig. 1 - Cytokines with dominant pro- or antitumor affect

Abbreviations: IL-interleukin; TGF-Transforming growth factor; TNF-Tumor necrosis factor; IFN-interferon

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Mechanisms of interleukins in cancer the action on the tumor cell implies the

existence of the receptor. downstream the receptor, there are pathways that interfere with the oncogene modified pathway, establishing a complicated circuitry whose effect is to modulate the growth, the invasiveness and the angiogenesis of the tumor cells, either positively or negatively (fig. 2).

Cytokine networks in the tumor microenvironment

Between the tumor and the cells that infil-trate it, complicated networks are established, having as result the modulation of the im-mune response and also of the tumor cells and of the cells associated with them (fig. 3). there are differences between tumors, there existing certain profiles with the predominance of one or others of these networks [64].

Implications of the study of interleukins in diagnostic and immunotherapy

interleukins can serve, alone or in combi-nations, as biomarkers for the diagnostic of the presence, extension and response of tumors to therapy. most of the interleukins have been proposed or tested as biomarkers in different cancers (reviewed in 68, 69, 70)

in the immunotherapy of cancer, the considered strategies (reviewed in 6, 71) are:

- the use of interleukins with antitumor effect: IL2, interferon (currently in use), ILs 7, 9, 12, 15, 18, 21, 24, 25, 27, 35, 36, 37 (in different stages of clinical evaluation).

- the inhibition of protumoral interleukins; iLs 1, 6, 8, 10, 11, 13, 17, 19, 20, 22, 23, 26, 30, 33, TGFβ are seen as possible targets.

- combined therapy: iLs and vaccines, iLs and kinases inhibitors, iLs + radiotherapy + pdL 1 inhibitors, adoptive cell transfer.

Conclusions and perspectives. inter-leukins are powerful means to influence the growth of the malignant tumors and the anti-tumor capabilities of the immune system. Un-fortunately, there is no interleukin with only protumor or only antitumor effect. They all have heterogenous action, as the present and other studies have shown.

the study of the relations within the tu-mor shows that cytokines act in complex net-works, which should be known and taken into consideration when conceiving therapeutic strategies (fig. 3). indeed, it has been shown that Lts with car (chimeric antigen receptors) act efficiently only if IL 4 is also inhibited [72] and pdL-1 inhibitors act optimally in combina-tion with TGFβ inhibition [73].

a reasonable approach would be to con-sider, between the many possible targets, the key cytokines of these networks, like iL12 for the antitumoral network, iL4 for the th2 net-work and TGFβ for the inhibitory network and also for the th17 network; targeting the down-stream signaling (STAT 3, NfkB) is also taken into account.

another reasonable strategy is to inhi-bit what is increased; there is a trend towards personalization of immunotherapy, by using means like serum cytokine profiles, which can be useful to evaluate which molecule is in-creased and to direct therapy [74].

Last but not least, the interleukin therapy must be seen in the larger context of other im-munotherapies-adoptive cells, vaccines, antitu-mor antibodies. interleukins like iL 2, ifns or iL 12 can shift the balance in the tumor micro-environment, strengthening the antitumor de-fense, which is the reason why they are used or clinically tested for monotherapy; however, they are also useful to augment the effect of strong therapeutical approaches like adoptive cell therapy, immune checkpoint inhibitors or kinases inhibitors.

a lot of progress has been made, but there is still much to do in this area, concerning the development of new strategies of antitumor therapy, efforts which are based on the grow-ing knowledge of the genomic and immuno-logic processes in tumors, including interleu-kin biology.

Abbreviations: NfkB - Nuclear-factor-kb; iL-interleukin; mf - macrophage; dc - dendritic cell; Lt, Lb - t, b lymphocyte; Lth 1, 2, 9, 17, 22 - Lymphocyte polarised th...; ag-antigen; ab-antibody; ifn-interferon; fb-fibroblast; EC - endothelial cell; NK - natural killer lymphocyte; iLc 1, 2, 3 - innate lymphoid

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cell 1, 2, 3; tme - tumor microenvironment. erK-extracell-regulated kinase; egfr - epidermal growth factor receptor; stat - signal transducer and activator of transcription; mdsc - myeloid-derived suppressor cell; ido-indoleamine 2,3-dioxygenase; pdL - programmed death ligand; car - chimeric antigen receptor; csf - colony-stimulating factor; mmp - matrix metalloproteinase; emt – epithelial-to-mesenchimal transition; mch - major histocompatibility complex; CD8 - Cluster of differentiation 8.

Conflicts of interests. the autors declare that they have no conflict of interests.

Funding: this study was not funded.Author contributions: ovidiu farc -

conceptualization, writing; Victor Cristea - supervision

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CIRCULATING TUMOR CELLS: FASCINATING INSIGHTS IN THE CLINICAL RELEVANCE

Iulia I. Niþã*, Valentin Vasile, Laura Zaharia, Vlad tofan, Cãtãlin Þucureanu, Ramona Caraghergheopol, Aurora Sãlãgeanu, Iuliana Caraş

Cantacuzino National Medico-Military Institute for Research and Development, Bucharest, Romania

* Corresponding author: Iulia I. Niþã, Phone: +40 762 870 154, E-mail address: [email protected]

AbStRACtduring tumor progression, circulating tumor cells (ctcs) shed from epithelial tumor and circulate through

bloodstream to form metastasis at distant sites. ctcs are heterogeneous rare cells that hold the promise of a valuable prognosis and prediction biomarker. the detection and analysis of ctcs through enumeration provide significant information on monitoring in real time the therapy efficiency and selection of personalized therapy. a continuous improvement of the detection techniques is still required. therefore, this review focuses on significance of CTCs clinical approaches in the personalized treatment.

Keywords: circulating tumor cells, enumeration, personalized treatment

REZUMAtIn timpul progresiei tumorale, celulele tumorale circulante (CTC) se desprind din tumora epitelială și

circulă în fluxul sangvin pentru a forma metastaze la distanță. CTC-urile sunt celule rare heterogene, care dețin rolul promițător de biomarkeri prognostici și predictivi. Detecția și analiza CTC-urilor prin numărare oferă informații importante în timp real despre eficiența terapiei și ghidarea terapeutică. O continuă îmbunătățire a tehnicilor de detecție este încă necesară. Prin urmare, acest articol se focalizează pe importanța CTC-urilor în abordările clinice din cadrul tratamentului personalizat.

Cuvinte-cheie: celule tumorale circulante, numărare, tratament personalizat

INtRODUCtION

circulating tumor cells (ctcs) in the peripheral blood were observed by ashworth, T.R (1869) for the first time in a metastatic patient as particular cells that look similar to tumors. in addition, Paget claimed that tumor cells “seeds” can form secondary metastasis on predisposed certain “soil”, predominantly liver and bone [1]. However, the nature of CTCs and their role in the mechanism of metastasis process were unknown.

over one hundred and forty years of cancer research, the involvement of ctcs in the metastatic cascade [2] as well the characteristics of ctcs that illustrate partially the tumor heterogeneity were revealed [3]. overall, ctcs exhibit strong interest not only

in the knowledge of mechanisms underlying metastasis process, but also in the development of non-invasive clinical approaches for monitoring tumor evolution.

the conventional tissue biopsy is a robust approach used in diagnosis of cancer stage and disease assessment, but it is unfeasible to pro-vide information on spatial and temporal pat-tern of tumor progression. in addition, the im-aging provides limiting information on tumor while radiological techniques show high health risks. moreover, serological tumor markers frequently used in clinical monitoring of tu-mor progression during treatment exhibit lack of sensitivity and specificity [4]. An alternative to conventional clinical approaches is liquid biopsy through analysis of ctcs in blood sam-


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ples providing direct real-time insights on tu-mor evolution with minimal invasiveness, that could be repeated any time, with very limited interventions and without exposing the patient to health risks [5]. Therefore, CTCs hold the promise of a valuable biomarker in prognosis and prediction of treatment efficiency, early re-lapse and therapy selection.

ctcs were exploited in the clinical prac-tice following the development techniques with appropriate performance characteristics (e.g. sensibility, specificity, reproducibility). cellsearch is the only fda approved detection system used for clinical detection of ctcs val-idated for metastatic breast, colon and prostate cancer [7]. However, low concentration and heterogeneity hamper the detection of ctcs [6]. A continuing improvement of techniques performances is required.

in the present review, we describe the characteristics and detection techniques relevant to clinical approaches of ctcs, with a focus on the clinical utilities of ctcs as prognostic and predictive biomarkers in personalized medicine.

Physical and molecular properties of CTCs

ctcs occur at low concentration of 1ctc per 106 - 108 blood cells [8, 9]. Detection of CTCs for various metastatic cancer types, including ovarian, lung, pancreatic revealed wide rang-es of ctcs frequencies from 0 to thousands of cells [10]. In contrast, the presence of CTCs in healthy or nonmalignant patients does not exceed 1 ctc in a very limited number of pa-tients. on the other hand, the 0 ctcs in met-astatic carcinoma patients could be addressed to infrequency or limitation of separation/de-tection techniques. therefore, improvement of techniques sensitivity is further required.

in addition, ctcs exhibit a strong dynamic in the morphological and phenotypical innate heterogeneity during the disease progression [11]. CTCs diameters range from 9 to 30 µm as intact single cells, clusters, apoptotic and cells fragments with round or odd shapes [12, 13]. In general, patients exhibit single CTCs, but the simultaneous presence of single ctcs

and clusters are detectable in patients with advanced stages. however, the presence of ctcs clusters in circulatory system is associat-ed with malignancy [14, 15]. There are studies suggesting that CTCs use platelets, fibroblats or leukocytes to aggregate in clusters creating protection against immune surveillance [16].

in addition, apoptotic ctcs phenotype (cK+/m30+) is present in a significant percent-age in early or metastatic stage of breast cancer [17]. The changes in apoptotic CTCs levels be-fore and after treatment in patients with small cells lung cancer was related independently to efficiency of treatment [18]. Moreover, nu-merous immunocytochemical studies on ctcs from various carcinomas confirmed the pres-ence of large ctcs with typical high nucleus to cytoplasmic ratio [19, 20]. Morphological fea-tures of tumor cells (nucleus size and shape) are fundamentally correlated with distant metas-tasis in pathology [21]. Further classification of CTCs based on their nucleus size demonstrat-ed that the number of the smallest size nucle-us ctcs was successfully correlated with vis-ceral metastatic prostate cancer [22]. However, the presence of very small size nucleus CTCs should be also addressed to wider number of cancers. In respect to molecular profile, CTCs co-overexpress epithelial molecule of adhesion (epcam) and cytoskeletal maker cytokeratine (cK). however, the present markers show a low specificity being expressed also in normal epithelial cells. nevertheless, epcam and cK are routinely used in the clinical pathology in combination with inspection of morphological structure of the cells. during metastasis pro-cess, ctcs show a transition in their pheno-type. throughout the epithelial-mesenchymal transition mechanism, the mesenchymal mark-ers (e.g. vimentin) are upregulated while the epithelial markers are suppressed. interesting-ly, experimental studies revealed a partial tran-sition between epithelial and mesenchymal phenotype that came out to three phenotypes: epithelial, epithelial-mesenchymal and totally mesenchymal phenotypes [23]. Surprisingly, these phenotypes of ctcs demonstrate clinical relevance. for example, analysis of phenotypic populations of ctcs (cK+/vim- vs cK+/vim+)

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during post 1-cycle pazopanib treatment in small cells lung cancer patients is considered relevant for monitoring the treatment efficacy [24].

Detection of CTCsthe detection of low abundance ctcs

requires a-priori enrichment or separation of ctcs from the peripheral blood cells.

the gold standard for detection and enu-meration of ctcs, cellsearch, is based on ep-cam immunomagnetic enrichment method. the enriched epcam positive cells are con-firmed as being epithelial tumor cells whether cells express cK8, 18/19 while lack cd45, the leukocyte common antigen marker. several studies showed that cellsearch detected suc-cessfully the ctcs counts associated with pre-diction and prognosis of the patient outcome during therapy in metastatic breast, colon and prostate cancer [25, 27]. However, CTCs change their phenotype lacking epcam dur-ing EMT process. Hence, a specific antibody or label free method should be employed in sep-aration of ctcs. multiple technologies based on physical and molecular properties of ctcs were developed. for example, fluxion tech-nique uses cocktail of antibodies against epi-thelial and mesenchymal phenotypes demon-strating an improved number of CTCs [28]. In addition, methods based on physical proper-ties, especially size based filtration exhibit high sensibility in clinical practice. for instance, separation of ctcs based on iset technology (ISET: Isolation by SizE of Tumor cells) demon-strated detection of 1 ctc in the peripheral blood [29]. Comparison of CTCs enumeration through iset vs cellsearch technologies in patients with non-small lung cancer showed a higher number of negative epcam ctcs separated by ISET [30]. Therefore, CellSearch system could underestimate the number of CTCs. Currently, microfluidic based platforms with increased sensibility have been presented [31]. Particularly, a new CellMax biomimetic platform (cmx) was developed to investigate ctcs in early stage. the new platform was tested only in a single center that further needs to be validated by independent groups.

CTCs Clinical Relevancethe presence of ctcs in the peripheral

blood is associated with poor patient outcome.in addition, ctcs counts are considered

clinical prognostic biomarkers in the stratifi-cation of patients in groups that follow treat-ment response and predict the patient survival outcome before and after treatment [32]. For example, the study of preoperative non-small lung cancer patients with resectable tumors showed a shorter overall survival and poor prognosis for patients that exhibit higher ctcs number over the cut-off point (threshold). The presence of CTCs significantly correlated with poor prognosis is confirmed in other cancers, such as breast cancer [33] and pancreatic can-cer [34].

in addition, ctcs enumeration is com-monly used in clinical practice to monitor the therapy outcome and to select the appropriate therapy suitable to each patient.

for example, the dynamics of ctcs num-bers for metastatic breast, colon and pros-tate cancer predicts the overall survival and progression free survival related to real time treatment response. the patients with a con-version from CTCs counts ≤5 cut off point to CTCs counts ≥5 above cut off point or patients with persistent ctcs before and after therapy showed a worse survival. during the therapy course, CTCs counts ≤5 indicate treatment ef-ficiency through a significant improvement of patient outcome [35]. Moreover, changes in ctcs counts in response to chemotherapy were monitored in metastatic breast cancer [36]. Therefore, CTCs number is the strongest predictor of patient outcome during treatment, a useful tool to monitor in real time the treat-ment efficiency.

the populations of patients that show an increase or constant number of ctcs can be re-sistant to therapy [37]. Moreover, it has been recently shown that ctcs enumeration plays a significant role in therapy selection [38].

although the prognostic and predictive roles of ctcs are proven in metastatic patients, the detection of ctcs in non-metastatic pa-tients is quite challenging. in the early stages, the number of ctcs is extremely low < 5ctcs

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in 10mL blood [39]. Nevertheless, Bidard et al. [40] succeeded to detect 1CTC in 7.5mL blood, but it is still unclear whether 1ctc is clinically relevant.

CONCLUSIONS

circulating tumor cells represent multi-functional biomarkers, valuable in monitoring the progression of tumor in real time related to patient outcome and treatment efficacy in a non-invasive way and in prediction of patient response to therapy.

CTCs based identification and enumera-tion associated with progression free survival and overall survival profiles of patients offer the possibility to change the therapy in short period of time instead of waiting for long time conventional clinical approaches to check the progression. moreover, analysis of ctcs can predict a targeted therapy with a best response for each individual patient.

further studies should focus on basic inves-tigations of ctcs nature, necessary to improve of ctcs detection technologies and thereby, to complement current diagnostic tests.

Acknowledgementsthis work was supported by a grant

from the romanian ministry of research and Innovation (Project number PN-III-P1-1.2-pccdi-2017-0214/3pccdi).

Disclaimers: the views expressed in the submitted article are our own and not an official position of the institution or funder.

Conflict of interests: We have no conflict of interests to disclose.

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CYTOTOxICITY EVALUATION OF FE-BASED STABILIzED SUSPENSIONS FOR BIOMEDICAL APPLICATIONS

Dora Domnica baciu1, Iulia Ioana Lungu2, Andrei-Mihai Dumitraşcu1, Gabriel Prodan3, Aurora Sãlãgeanu1* and Florin Dumitrache2

1Cantacuzino National Medico-Military Institute for Research and Development, 103 Spl. Independenþei, Bucharest, Romania

2National Institute of laser, Plasma and Radiation Physics (NIlPRP), Bucharest-Mãgurele, Romania3ovidius University of Constanþa, 124 Mamaia Avenue, Constanþa, Romania, 9005127

* Corresponding author: Aurora Sãlãgeanu, Cantacuzino National Medico-Military Institute for Research and Development, 103 Spl. Independenþei, Bucharest, Romania

AbStRACtnanoparticles involved in this research are designed for use in medical purposes, as they present the

capability of being targeted in a magnetic field towards tumor area where they could be heat activated to destroy cancer cells. also, some antitumoral agents, such as doxorubicin, could be bound on their surface and targeted to reach tumor site. for this mechanism to be applicable in vivo, the iron oxide nanoparticles (ionps) have to present very low cytotoxicity/immunotoxicity. Two IONPs were synthesized by laser pyrolysis and characterized by different analytical methods. The present study aimed at two complementary directions: first, to evaluate the cytotoxicity of IONPs and second, to evaluate if the treatment with these agents could induce an inflammatory response in macrophages. The results showed that IONPs considered in this study have no significant effects on cell viability at concentrations as high as 50 µg/mL. It has been also proved that IONPs neither induce a direct inflammatory effect on macrophages nor influence LPS-induced activation.

Keywords: iron oxide nanoparticles, cytotoxicity, inflammatory cytokine

REZUMAt

Nanoparticulele magnetice pe bază de oxizi de fier (IONP) prezintă proprietăți unice care le fac deosebit de atrăgătoare pentru aplicații biomedicale. Pentru a fi folosite în domeniul biomedical, ele trebuie să prezinte o citotoxicitate/imunotoxicitate extrem de redusă. În acest studiu, au fost investigate două tipuri de nanoparticule, obținute prin piroliză laser, care diferă prin raportul precursorilor Fe/O. Folosind linia de macrofage murine RAW 264.7, s-a arătat că viabilitatea și proliferarea celulară nu sunt afectate de expunerea la aceste nanoparticule în concentrații de până la 50 µg/mL. Nanoparticulele ca atare nu induc sinteza de mediatori inflamatori (NO și TNFα) și nici nu amplifică secreția acestor mediatori în urma stimularii concomitente cu LPS. Rezultatele obținute au demonstrat că cele două tipuri de IONP sintetizate prin tehnica pirolizei laser sunt candidați promițători pentru aplicațiile biomedicale viitoare, deoarece sunt biocompatibile și nu induc producția de mediatori inflamatori în macrofage. Sunt necesare studii suplimentare pentru a elucida modul în care aceste IONP și derivații potențiali cu diferite grupuri funcționale ar putea interfera în alte mecanisme imunitare, precum și pentru a clarifica mecanismul de interacție cu LPS.

Cuvinte-cheie: nanoparticule de oxid de fier, citotoxicitate, citokină inflamatorie


INtRODUCtION

due to the unique properties of fe-based magnetic nanoparticles they have been in-tensely researched in several fields, including for biomedical-related applications. one of the

most attractive properties of iron oxide nano-particles (IONPs) is they can be easily attracted and manipulated by using external magnet-ic field; in addition, their superparamagnetic properties enable them to work as magnetic

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switches [1]. Drug delivery applications, in particular, have also received considerable attention due to the need of specifically locat-ed delivery of various pharmaceutically active components [2]. In most of the early studies, ionps were considered to be relatively safe and biocompatible especially at lower levels as these particles could be cleared from the body [3]. To the contrary, exposure to high doses of ionps may generate cellular stress and even damage. Moreover, the criteria to define toxic-ity in the case of ionps has to be reevaluated to include a broader range of effects such as inflammation, DNA damage, oxidative stress, mitochondrial membrane dysfunction, chang-es in gene expression [4, 5]. It had been sug-gested that after entering the cell by several possible mechanisms, including both passive diffusion and endocytosis (clathrin and/or re-ceptor mediated), ionps might be degraded into ferrous ions (fe+2) by lysosomal enzymes. these fe+2 ions could react with hydrogen per-oxide and oxygen to generate highly reactive oxygen species (ros) which may indirectly cause dna damage, intervene in cell signal-ing pathways which can lead to activation of inflammatory cells or alter the expression of various genes [4, 6].

the aim of this study was to evaluate bio-compatibility of two preparations of fe-based nanoparticles (namely sfnew10 and sfnew11) synthesized by laser pyrolysis. To this aim, the effect of these IONPs on cell proliferation and secretion of inflammation mediators were eval-uated after exposure of murine macrophage cell line, raW 264.7. this cell line is often used for initial screening of different products for bi-oactivity and to predict their potential effect in vivo. Due to their nature as first barrier against potentially harmful agents to the body, these cells are commonly used to evaluate the in-flammatory response [7].

1. MAtERIALS AND MEtHODS

1.1. Particle synthesis and characterization The nanosized particles were synthesized

using the laser pyrolysis technique which

was described in detail in a previous paper [8]. The method is based on the resonance between the emission line of a co2 laser and the infrared absorption band of at least one gas-phase component, in this case ethylene. iron pentacarbonyl vapors were used as precursor, a mixture between o2 and ar as oxidizer and ethylene had a double role as sensitizer and as carrier gas for Fe(CO)5 vapors. All the gas flows named reactive mixture come through the inner tube of the central nozzle (see Fig. 1). An argon flow is used to ensure the confinement of the reactive mixture and the flow value is adjusted in order to achieve comparable gas speed from both admission tubes. Also two lateral Ar flows were used to continuously flush the IR transparent windows. the two samples used in this study, sfnew10 and sfnew11, were prepared with highly different ratio between Fe/O precursors: r=0.08 and r=0.23, respectively. the fe(co)5 vapors were entrained by the ethylene flow in the first case and by ethylene and argon (from oxidized mixture) flows in the second one. The interaction between the laser beam and the gas flow takes place in the cross-shaped reactor, where the decomposition of the gas precursors generate the formation of nanosized iron based particles that are further entrained towards the collection chamber. this chamber is equipped with a microporous filter which facilitates the collection of the ….. as synthesized nanopowders (fig. 1).

The resulted nanopowders were analyzed by different analytical methods: the phase composition and crystallinity were assessed by X-ray diffraction (XRD) using a PANalytical X`Pert MPD theta-theta X-Ray diffraction apparatus using a Cu K α source (0.15418 nm); the morphology and structure were observed by transmission electron microscopy (tem) and selected area electron diffraction (SAED) analysis, using a philips cm 120st (120kv) transmission electron microscope; and the elemental analysis was performed by energy-dispersive X-ray spectroscopy (EDS) attached to a scanning electron microscope type philips xL30 cp, with an acceleration voltage of 15kv.

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1.2. Biological tests1.2.1. Sample preparationbefore use, ionps need a good dispersion

and stabilization. To this purpose, 50 mg of each were suspended in 9 mL sterile distilled water and ultrasonicated for 10 minutes. to improve dispersion sterile foetal bovine serum (FBS, Gibco) was added to a final concentration of 10% (vol/vol) [9]. Aliquotes of 1.5 mL from this suspension were transferred in a 12-well cell culture plate and the plate was left in the laminar flow cabinet with the lid open on UV irradiation o/n.

1.2.2. Cell cultureraW 264.7 cells (ecacc) were grown

in Dulbecco′s Modified Eagle′s Medium (DMEM, Lonza) supplemented with 10% FBS and 1% penicillin/streptomycin (Lonza) (complete culture medium) on 75 cm2 flasks in a humidified atmosphere at 370c and 5% co2.

1.2.3. Cell stimulationfor the cytotoxicity assay, cells were

seeded in 96-well plates at a starting density of 5×104 cells/well and grown overnight to allow adherence (100 µl/well) in complete culture medium. after 24 h the medium was discarded and the cells were washed with dmem. the cells were then exposed to samples in binary serial dilutions (starting with 500 µg/mL) in

fresh complete culture medium for 24h. cells grown in complete culture medium alone served as control. cell viability was determined after 24 h exposure.

similar conditions of cell culture were used to determine the immune mediators secreted by raW 264.7 cells after treatment with ionps. cells were exposed to serial dilutions of ionps in the presence or absence of lipopolysaccharide (1 µg/mL) (Lps from e. coli o111:b4, invivogen). cell culture supernatants were collected after 3h for TNF-α measurement and after 24h for nitric oxide (no).

1.2.4. MTT viability assaycell viability was determined as previously

described [10]. Briefly, after 24h exposure to ionps, culture medium was replaced by fresh culture medium containing 500 µg/ mL of methylthiazolyldiphenyl-tetrazolium bromide (mtt, sigma-aldrich).

the test is based on the ability of nadh mitochondrial dehydrogenases in living cells to reduce soluble tetrazolium salts from MTT (yellow) and to form insoluble crystals of formazan (purple). Cells were incubated for additional 3h, then 100 µL of lysis solution (50% dmf, 4.8% sds, 8% acetic acid, 3 mm HCl) were added to dissolve the formazan crystals formed by reducing mtt.

the plate was incubated at room tempe-rature in the dark until the cells were lysed and the purple crystals dissolved (about 48 h). the amount of water-insoluble blue formasan dye formed from mtt was proportional to the number of live cells and was determined using a 96-well plate reader (tecan) at 550 nm wavelength. the experiment was performed in triplicate and repeated three times.

1.2.5. Nitric oxide determinationthe amount of no was measured by the

accumulation of nitrite in the culture superna-tants, collected after 24 h of stimulation, using a colorimetric reaction with the griess reagent (0.1% (w/v) n-(1-naphthyl) ethylenediamine dihydrochloride and 1% (w/v) sulfanilamide containing 5% (w/v) h3po4). 80 µL of cell

Fig. 1. Experimental setup of the laser pyrolysis technique

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culture supernatants were mixed with equal volume of the griess reagent and the plate in-cubated for 10 min in the dark. the standard curve was created by using known concen-trations of sodium nitrite, and the absorbance was measured within 30 min at 540 nm using a microplate reader spectrophotometer (ther-mo Scientific, USA). The experiment was per-formed in triplicate and repeated three times.

1.2.6. TNF-α determinationTNF-α levels were measured in cell super-

natants collected after 3 h of stimulation using an eLisa kit (duoset, r&d systems inc., Usa) according to the manufacturer’s instructions. Data were normalized to cell number deter-mined by mtt assay using unstimulated cells as reference. the experiment was performed in triplicate and repeated three times.

2. RESULtS AND DISCUSSION

2.1. Nanoparticle characterizationXRD patterns of the nanopowder samples

(sfnew10 and sfnew11) are presented in Fig. 2 γ-Fe2o3- fe3o4 were detected in both samples as the main crystalline phase. the mean crystalline dimension calculated with sherrer equation using fWhm (full width half maximum) from the isolated (511) peak increase from 11.4 to 17.4 nm with r(fe/o) increasing. in addition, at high r ratio, the most relevant peaks of α-Fe and Fe3c (located only by grey arrows) were also highlighted (see fig. 2). it

is considered difficult, especially at nanoscale level, to distinguish fe3o4 and γ-Fe2o3 using xrd because they have almost the same spinel structure and lattice parameters. However, taking into account the 2θ position around 35.50 for (311) identified plane, it might be assumed that these two analyzed samples are a mixture of both crystalline phases. in the case of the sample with a higher r value, sfnew11, γ-Fe2o3 seems to be the dominant phase.

eds spectra of the nanopowder samples are presented in Fig. 3. The synthesized nano-particles were pressed using an al foil on si wafers which explains the presence of si K and Al K peaks. The EDS analyses identified the presence of the following elements:

sfnew10 (lower fe/o ratio): c 5.0 at.%, o 52.5 at.% and fe 42.5 at.%

sfnew11 (higher fe/o ratio): c 3.2 at.%, o 61.5 at.% and fe 35.3 at.%

as can be noticed, the fe/o ratio in the powder composition apparently evolves against that of the precursor composition. the main reason for this contradiction derives from the temperature difference in the reactive zone (8500c for sfnew10 and 7100c for sfnew11). at higher temperatures, over 8000c ethylene reacts with oxygen generating volatile species (co2 and h2O vapors) and consumes the major part of molecular oxygen and hides the oxidation process of freshly formed iron clusters, while at lower temperatures only the iron cluster oxidation process is activated.

Fig. 2. XRD patterns of the as synthesized powders (sfnew10 with r=0.08 - grey line; sfnew1with r=0.23 – black line)

Fig. 3. EDS spectra of the synthesized powders (sfnew10 – grey; sfnew11 – black)

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Fig. 4. TEM micrograph of SFnew10 and corresponding SAED image

Fig. 5. Cell viability of RAW 264.7 cells after 24 h exposure to different concentration of SFnew10 and SFnew 11 ionps, as determined by mtt asasy. each bar represents mean value of three replicates ± sd (standard deviation). results are representative of three independent experiments performed in triplicate in same conditions and expressed as mean ± sd (error bars) of three replicates. * p<0.05 as compared to control (unstimulated cells).

presented in fig. 4 for on sample (sfnew10) but they were similar for sfnew11. it was observed that the powder contained spherical shaped nanoparticles with a mean diameter of 5.8 nm. the nanoparticles seem to have a tendency of agglomeration in small ramified chain-like aggregates. SAED pattern highlights the spinel structure of fe3o4 and γ-Fe2o3 with homogeneous nanocrystals. the saed analysis is in agreement with the xrd analysis patterns.

2.2. Biotesting results2.2.1. Effect of IONPs on cell viability

using MTT assayin order to examine the cytotoxicity of

synthesized IONPs, RAW 264.7 cells were incubated with the samples at concentrations ranging from 500 µg/mL to 6.25 µg/mL and cell viability was measured after 24h using MTT assay. As shown in Fig. 5, a significantly

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statistic decrease in cell viability was only seen for high concentration (more than 200 µg/mL) while slight variations in cell viability was noticed for lower concentrations for both samples. it seems that sfnew11 sample was more cytotoxic than sfnew10, especially for higher range of concentration. one possible explanation might come from crystallographic data which showed that although both samples are a mixture of γ-Fe2o3- fe3o4, in sfnew11, γ-Fe2o3 seems to be the dominant phase. it has been shown that γ-Fe2o3 could induce DNA damage in murine neural stem cells [11]. this hypothesis needs however to be tested in future experiments.

2.2.2. Effect of IONPs on NO and TNF-α secretion

nitric oxide (no) is an important physiological messenger in many biological systems, including immunological, neuronal and cardiovascular tissues and plays a major role in the pathogenesis of acute and chronic inflammation [12]. The effect of IONPs on NO

secretion by macrophages was investigated by incubating raW 264.7 cells with ionps, in the presence or absence of powerful tLr4 ligand Lps (1 µg/mL). stimulation with sfnew10 or sfnew11 alone did not induce no secretion in raW 264.7 cells (data not shown). as shown in fig. 6, co-stimulation with sfnew10 or sfnew11 and Lps resulted in no production at about the same level as stimulation with Lps alone, although a slight reduction could be noticed at higher concentration.

Tumor necrosis factor (TNF-α) represents a cytokine involved in systemic inflammation and it is one of the cytokines that make up the acute phase reaction.

Having a major role in regulating immune system cells, TNF-α is considered as an indi-cator of the pro-inflammatory capacity of a substance. similar to no production, stimula-tion of raW 264.7 cells with ionps alone in concentration range from 50 to 12.5 µg/mL did not result in increased TNF-α secretion while co-stimulation with Lps induced comparable levels of TNF-α (Fig. 7).

Fig. 6. Nitric oxide secretion by RAW 264.7 cells after 24 h concomitant exposure to different concentration of sfnew10/sfnew11 in the presence of Lps. each bar represents mean value of three replicates ± sd (standard deviation). results are representative of three independent experiments performed in triplicate in same conditions and expressed as mean ± sd (error bars) of three replicates.

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Fig. 7. TNF-α secretion by RAW 264.7 cells after 3h exposure to different concentration of SFnew10/sfnew11 in the absence/presence of Lps. each bar represents mean value of three replicates ± sd (standard deviation). results are representative of three independent experiments performed in triplicate in same conditions and expressed as mean ± sd (error bars) of three replicates.

Conflicting results may be explained by different cellular type, experimental settings but also by different particle size and/or surface charge.

It has been suggested that the internaliza-tion route could be relevant for the effect of na-noparticles on cytokine release by monocytes/macrophages upon stimulation [15].

In this manuscript, the effect of two iron-based nanoparticles on viability and secretory function of raW 264.7 macrophage

3. CONCLUSIONS

interaction of nanoparticles with immune cells are particularly relevant for future biomedical applications and still limited data are available [13]. Many nanoparticles could induce secretion of proinflammatory mediators upon uptake by professional phagocytes [4, 14]. in other studies, modulation of Lps induced inflammatory responses or direct in vivo antiinflammatory effect were demonstrated [15, 16].

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cell line was investigated. Moreover, to better understand ionps interaction with immune cells, co-stimulation experiments in which cells were exposed to ionps in the presence of a powerful inflammatory stimulus, i.e. LPS, were performed.

our results demonstrated that two ionps synthesized by laser pyrolysis technique are promising candidates for future biomedical applications as they are biocompatible and do not induce production of inflammatory mediators in macrophages.

further studies are necessary to elucidate how these ionps and potential derivatives with different functional groups could interfere in other immune mechanisms.

Acknowledgementsthis study was supported by the romanian

Ministry of Research (project PN-III-P1-1.2-pccdi-2017-0728).


REFERENCES

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2. Arias L, Pessan J, Vieira A, Lima T, Delbem a, monteiro d. iron oxide nanoparticles for biomedical applications: a perspective on synthesis, drugs, antimicrobial activity, and toxicity. antibiotics. 2018;7:46. doi:10.3390/antibiotics7020046

3. Kim JS, Yoon T-J, Yu KN, Kim BG, Park SJ, Kim hW, et al. toxicity and tissue distribution of magnetic nanoparticles in mice. toxicol sci. 2006;89:338–347. doi:10.1093/toxsci/kfj027

4. shah a, dobrovolskaia ma. immunological effects of iron oxide nanoparticles and iron-based complex drug formulations: therapeutic benefits, toxicity, mechanistic insights, and translational considerations. nanomedicine nanotechnol biol med. 2018;14:977–990. doi:10.1016/j.nano.2018.01.014

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8. Morjan I, Alexandrescu R, Soare I, Dumitrache f, sandu i, voicu i, et al. nanoscale powders of different iron oxide phases prepared by continuous laser irradiation of iron pentacarbonyl-containing gas precursors. mater sci eng c. 2003;23:211–216. doi:10.1016/s0928-4931(02)00269-2

9. Ji Z, Jin X, George S, Xia T, Meng H, Wang X, et al. Dispersion and Stability Optimization of tio2 nanoparticles in cell culture media. environ sci technol. 2010;44:7309–7314. doi:10.1021/es100417s

10. chiulan i, mihaela panaitescu d, nicoleta frone A, Teodorescu M, Andi Nicolae C, Căşărică A, et al. biocompatible polyhydroxyalkanoates/bacterial cellulose composites: preparation, characterization, and in vitro evaluation. J biomed mater res a. 2016;104:2576–2584. doi:10.1002/jbm.a.35800

11. Pongrac IM, Dobrivojević M, Ahmed LB, Babič M, Šlouf M, Horák D, et al. Improved biocompatibility and efficient labeling of neural stem cells with poly(L-lysine)-coated maghemite nanoparticles. Beilstein J Nanotechnol. 2016;7: 926–936. doi:10.3762/bjnano.7.84

12. bredt ds, snyder sh. nitric oxide: a physiologic messenger molecule. annu rev biochem. 1994;63: 175–195. doi:10.1146/annurev.bi.63.070194.001135

13. patil rm, thorat nd, shete pb, bedge pa, Gavde S, Joshi MG, et al. Comprehensive cytotoxicity studies of superparamagnetic iron oxide nanoparticles. biochem biophys rep. 2018;13:63–72. doi:10.1016/j.bbrep.2017.12.002

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15. Grosse S, Stenvik J, Nilsen AM. Iron oxide nanoparticles modulate lipopolysaccharide-induced inflammatory responses in primary human monocytes. Int J Nanomedicine. 2016; Volume 11:4625–4642. doi:10.2147/IJN.S113425

16. Calero M, Gutiérrez L, Salas G, Luengo Y, Lázaro A, Acedo P, et al. Efficient and safe internalization of magnetic iron oxide nanoparticles: two fundamental requirements for biomedical applications. nanomedicine nanotechnol biol med. 2014;10:733–743. doi:10.1016/j.nano.2013.11.010

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177ROMANIAN ARCHIVES OF MICROBIOLOGY AND IMMUNOLOGY, Vol. 78, Issue 3, pp. 177-181, July - September, 2019

A PROBLEM-SOLVING INTEGRATIVE APPROACH OF eSCHeRICHIA ColI O157:H7 SEROTYPE DIAGNOSIS

Codruþa-Romaniþa Usein1,2*, Mãdãlina Militaru1, Mihaela Oprea1, Sorin Dinu1, Daniela Cristea1, Adriana Simona Ciontea1

1Cantacuzino National Medico-Military Institute for Research and Development, Bucharest, Romania2Carol Davila University of Medicine and Pharmacy, Bucharest, Romania

* Corresponding author: Codruþa-Romaniþa Usein, Cantacuzino National Medico-Military Institute for Research and Development, Bucharest, Romania; e-mail: [email protected]

AbStRACtthe diagnosis of escherichia coli o157:h7 infection needs to be considered for all the patients who pres-

ent with diarrhea and hemolytic uremic syndrome. in romania, the routine method for detecting the e. coli o157:h7 members in stool cultures is still based on the use of slide agglutination tests to screen sorbitol non- fermenting colonies recovered from sorbitol-macconkey medium.

this report focused on a sorbitol-negative e. coli strain presumptively assigned to the o157 serogroup during the routine clinical laboratory diagnosis that was referred to the reference laboratory for confirmatory testing and further genetic characterization. The PCR-based serotyping and virulence gene detection com-plemented by DNA sequencing showed that the strain did not belong to the O157 serogroup but qualified as enteropathogenic e. coli.

the screening of e. coli o157:h7 strains can result in a false-positive diagnosis and lead to inappropriate public health measures. Therefore, although the schemes of microbial detection and characterization may use various combinations of testing methods, depending on the laboratory level, the aim of investigations is to specifically identify the true pathogenic strains.

Keywords: escherichia coli pathotype, VTEC/STEC, molecular identification

REZUMAtInfecţia cu escherichia coli O157:H7 trebuie suspicionată în cazul tuturor pacienţilor cu diaree şi sindrom

hemolitico-uremic. În Romania, metoda utilizată în mod curent pentru detectarea în probele de materii fecale a membrilor serotipului O157:H7 se bazează pe teste de aglutinare pe lamă, aplicate coloniilor sorbitol-nega-tive, izolate pe mediul MacConkey cu sorbitol.

Acesta este un raport despre o tulpină de e. coli sorbitol negativă, identificată prin algoritmul curent de diagnostic al unui laborator clinic ca fiind din serogrupul O157, care a fost trimisă laboratorului de referinţă pentru confirmare şi caracterizare suplimentară. Serotipizarea bazată pe PCR şi analiza genelor de virulenţă, completate de secvenţierea AND, au arătat că tulpina nu aparţine serogrupului O157, dar este enteropatogenă.

Modul de identificare a tulpinilor de e. coli O157:H7 poate genera rezultate false, care conduc la luarea unor măsuri de sănătate publică nepotrivite. Ca urmare, chiar dacă schemele de detecţie şi caracterizare mi-crobiană pot include o combinaţie variată de teste, în functie de nivelul de expertiză al laboratorului, scopul final al investigaţiilor trebuie să fie identificarea adevăratelor tulpini patogene.

Cuvinte-cheie: prototip de escherichia coli, VTEC/STEC, identificare moleculară

MAtERIALS AND MEtHODS

E. coli strain and DNA preparationa sorbitol-negative e. coli isolate recovered

from the culture of a stool specimen collected from a child hospitalized with diarrhea was

referred to the reference laboratory for further investigation. the strain was tested in the clinical laboratory where it had been isolated by slide agglutination with o157 antiserum and gave a positive result.

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O157 and H7 serologyserotyping of both o (lipopolysaccharide)

and H (flagellar) antigens was performed with commercially available antisera (ssi diagnos-tica, hillerød, denmark). the o antigen was determined using antisera for slide aggluti-nation produced against the epec and vtec serogroups (statens serum institute). the h7 was the only flagellar antigen sought. All the antisera were used according to the manufac-turer’s instructions. the strain was initially checked for autoagglutination with saline.

PCR-based assays for genes encoding determinants for serotype and virulence factors

a conventional touchdown pcr with previously designed primers for the wzx sequences unique to serogroup o157 was used [6]. The DNA template was made by suspending a loop full of culture in 200 µl of water and heated for 15 min in boiling water. the 50 µl pcr mix contained 3 µl of crude DNA template, 1x Taq Polymerase buffer (promega) with 3 mm mgcl2, 0.3 mm of each deoxynucleoside triphosphates, 15 pmols of each primer, and 2U of gotaq g2 flexi dna polymerase (promega). the pcr conditions were 95°c for 15 min; 10 cycles of 95°c for 30 s, 68–59°c (decrease 1°c /cycle) for 20 s, 72°c for 52 s, followed by 35 cycles of 95°c for 30 s, 59°C for 20 s, 72°C for 52 s and 72°C for a final 1 min extension.

a multiplex pcr with previously pub-lished primers was additionally performed for defining the O157:H7 serotype. The main target genes were rfbEo157 and fliCh7 and the 16s rrna encoding gene as internal positive control [7]. The PCR volume adjusted to 25 µl contained: 1x reaction buffer, 1.5 mM MgCl2, 0.2 mm deoxynucleoside triphosphates, 1.5 U taq dna polymerase (promega) plus primers (0.1 µm of the e. coli 16s rrna (e16s) primers, 0.4 µM fliC primers, and 1 µM rfbE primers) and 3 µl of bacterial lysate. the pcr program consisted in an initial denaturation step at 95°c for 3 min followed by 30 cycles of amplification with denaturation at 95°c for 30 s, annealing at 58°c for 30 s, and extension at 72°c for 30 s, ending with a final extension at 72°C for 7 min.

commercial primers (ssi diagnostica, hillerød, denmark) designed to detect viru-lence encoding genes associated with the diar-rheagenic e. coli strains were used. Specifically, the following genes were targeted in a multi-plex pcr: eae (intimin), vtx1 (verotoxin 1), vtx2 (verotoxin 2), elt (heat-labile enterotoxin), est (heat-stable enterotoxin), and ipah (invasive plasmid antigen). the pcr was performed in a total reaction volume of 20 µl containing 1 x multiplex pcr master mix (Qiagen), 4 µl prim-er mix, and 4 µl of bacterial lysate. Amplifica-tion conditions comprised 95°c for 15 min, fol-lowed by 35 cycles of 94°c for 50 s, 57°c for 420 s, and 72°C for 50 s, and finally 72°C for 3 min.

pcr for the detection of the gene coding for enterohemolysin was performed using previously published primers [8].

pcr was performed by addition of 5 ml of this bacterial cell suspension to a reaction mixture containing 30 pmol of each primer per ml, 5 ml of 10-fold-concentrated polymerase synthesis buffer, 200 mM (each) deoxynucleo-side triphosphate, and 2.0 U of taq polymerase (amersham Laboratories, buckinghamshire, United Kingdom). The samples were filled up with water to a final volume of 50 ml. After an initial denaturation of 5 min at 94°c, the sam-ples were subjected to 30 cycles of amplifica-tion, each of which consisted of 30 s at 94°c, 90 s at 57°c, and 90 s at 72°c. f

Amplicons were analyzed by electropho-resis on agarose 2% w/v gels using standard conditions, followed by staining with ethidium bromide.

DNA sequencing the amplicon resulted from the pcr of the

wzx gene, previously purified with Nucleospin gel and pcr clean-up (mackerey nagel), was sequenced using the pcr primers and bigdye terminator v3.1 cycle sequencing kit (applied biosystems). cycle sequencing reaction prod-ucts were further purified using DyeEx 2.0 Spin kit (Qiagen) and analyzed on a SeqStudio Genetic Analyzer instrument (Applied Biosys-tems), according to the manufacturer’s instruc-tions. raw sequences were visually inspected and edited using bioedit free software and the

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A problem-solving integrative approach of Escherichia coli O157:H7 serotype diagnosis

consensus sequence was identified using the basic Local alignment search tool (bLast) program (https://blast.ncbi.nlm.nih.gov/).

RESULtS AND DISCUSSION

serotyping based on the combination of the principal cell surface components, the O-antigens and flagellar H-antigens, is still used for diagnosis and surveillance purposes and for distinguishing strains during outbreaks. the method is a time-consuming and demanding task with several limitations among which cross reactivity of the antisera or non-typeability of many strains. in romania, by virtue of the fact that o157:h7 clone has been considered to cause the vast majority of severe infections, o157 serotyping remains the most commonly employed method to screen for vtec in the clinical laboratories. mostly, owing to the cost of testing, the screening, performed only if a specific request is received and restricted to o157 antigen, is not confirmed by titration. Moreover, nucleic acid amplification methods that can rapidly determine the presence of genes encoding determinants for major virulence factors such as verocytotoxin genes are not routinely used.

this report chose to describe the practices used by the reference laboratory to confirm the identity of a presumptive vtec e. coli strain and the challenges posed to the identification achieved by both phenotypic and genotypic methods. The definitive characterization of the strain relied on traditional and molecular serotyping coupled with an assay for specific virulence genes enabling the determination of o- and h-group, pathotype, and the strain’s pathogenic potential.

the pcr-based virulence genotyping targeted specific virulence factors of the major diarrheagenic e. coli groups [1], including the eae, vtx1 and vtx2 genes. the intimin encoding gene is part of a pathogenicity island designated locus of enterocyte effacement (LEE), integrated in the chromosome of different clonal lineages represented mostly by enteropathogenic e. coli (epec) but also by some vtec strains. intimin is required for intimate bacterial adhesion to epithelial cells inducing a characteristic

histopathological lesion defined as “attaching and effacing” (A/E) [9]. The phage-encoded genes vtx1 and vtx2 are found specifically in vtec conferring the ability to produce verocytotoxins (also known as shiga toxins), the major virulence factors responsible for the damages of several organs as kidney, brain, liver, and pancreas [10]. The investigated strain displayed a genotype that qualified it as epec based on the presence of eae gene and lack of vtx genes. moreover, the absence of the plasmid-encoded enterohemolysin, a putative virulence factor associated with vtec, confirmed the identification [11]. The strain failed to agglutinate with all the commercial antisera used against the most common epec and vtec serogroups, including o157 serogroup. the molecular serotyping, performed as indicated by two previously published protocols, confirmed that the strain did not belong to the o157:h7 serotype. yet, the pcr-based protocol targeting only the o157 antigen generated an amplicon with a higher-than-expected molecular weight (fig. 1) which further sequenced proved to be a 161-bp dna fragment with 100% similarity with the adhE gene encoding a multifunctional protein of e. coli, amplified due to mis-priming [12]. consequently, the studied strain did not belong to o157:h7 serotype and, moreover, lacked the vtx genes characteristic of vtec. nevertheless, it qualified as an EPEC that could have caused diarrhea especially since the infected patient was a three-year old child [1]. Regarding the strain’s sorbitol negative phenotype, although usually considered as highly sensitive and predictive for o157:h7 clone, it was also observed in a small percentage of e. coli strains belonging to other serotypes and pathotypes such as EPEC [13]. This EPEC strain was such an example. therefore, the e. coli o157 could be falsely identified by the screening procedure based on conventional serotyping and the inability to ferment sorbitol. The confirmatory tests rejected the suspicion and prevented inappropriate and unnecessary public health measures. at the same time, the inconclusive result obtained in one of the molecular serotyping assays underlined that none of

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the aforementioned diagnosis approaches is without drawbacks.

CONCLUSIONS

essentially, there is no absolute character-istic of pathogenic e. coli, and therefore testing algorithms need sufficient inclusivity to cap-ture emerging strains. While the schemes of microbial detection and characterization may use various combinations of testing methods, depending on the laboratory level, the ulti-mate goals for public health investigations are aimed at specifically identifying the true path-ogenic strains.


REFERENCES1. Croxen MA, Law RJ, Scholz R, Keeney KM,

Wlodarska m, finlay bb. recent advances in understanding enteric pathogenic escherichia coli. clin microbiol rev. 2013 oct;26(4):822-80. doi: 10.1128/cmr.00022-13

2. Bryan A, Youngster I, McAdam AJ. Shiga Toxin producing escherichia coli. clin Lab med. 2015 Jun;35(2):247-72. doi: 10.1016/j.cll.2015.02.004.

3. tarr pi. shiga toxin-associated hemolytic uremic syndrome and thrombotic thrombocytopenic purpura: distinct mechanisms of pathogenesis. Kidney int suppl. 2009 feb;(112):s29-32. doi: 10.1038/ki.2008.615.

4. Scheutz F. Taxonomy Meets Public Health: The case of shiga toxin-producing escherichia coli. Microbiol Spectr. 2014 Jun;2(3). doi:10.1128/microbiolspec.ehec-0019-2013.

5. Karch H, Tarr PI, Bielaszewska M. enterohaemorrhagic escherichia coli in human medicine. Int J Med Microbiol. 2005 Oct;295(6-7):405-18.

6. Monday SR, Beisaw A, Feng PC. Identification of shiga toxigenic escherichia coli seropathotypes a and b by multiplex pcr. mol cell probes. 2007 aug;21(4):308-11.

7. Wang g, clark cg, rodgers fg. detection in escherichia coli of the genes encoding the major virulence factors, the genes defining the o157:h7 serotype, and components of the type 2 Shiga toxin family by multiplex PCR. J Clin microbiol. 2002 oct;40(10):3613-9.

8. schmidt h, beutin L, Karch h. molecular analysis of the plasmid-encoded hemolysin of escherichia coli o157:h7 strain edL 933. infect immun. 1995 mar;63(3):1055-61.

9. Franzin FM, Sircili MP. Locus of enterocyte effacement: a pathogenicity island involved in the virulence of enteropathogenic and enterohemorragic Escherichia coli subjected to a complex network of gene regulation. biomed res int. 2015;2015:534738. doi: 10.1155/2015/534738.

10. gyles cL. shiga toxin-producing escherichia coli: an overview. J Anim Sci.2007 Mar;85(13 suppl):e45-62.

11. schwidder m, heinisch L, schmidt h. genetics, toxicity, and distribution of enterohemorrhagic escherichia coli hemolysin. toxins (basel). 2019 aug 29;11(9).pii: e502. doi: 10.3390/toxins11090502.

Fig. 1. Results of the PCR for wzxO157 gene (amplicon size 133 bp). Lane 1: pcr product generated by the presumptive o157 e. coli strain, lane 2: positive control (o157:h7 strain), lane 3: negative control (h2o), lane 4: 100-bp dna ladder

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13. Ojeda A, Prado V, Martinez J, Arellano C, Borczyk A, Johnson W, et al. Sorbitol-negative phenotype among enterohemorrhagic Escherichia coli strains of different serotypes and from different sources. J Clin Microbiol. 1995 aug;33(8):2199-2201.

12. Membrillo-Hernandez J, Echave P, Cabiscol E, Tamarit J, Ros J, Lin EC. Evolution of the adhe gene product of escherichia coli from a functional reductase to a dehydrogenase. genetic and biochemical studies of the mutant proteins. J Biol Chem. 2000 Oct 27;275(43):33869-33875.

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VIRULENCE AND RESISTANCE PROFILES OF SHIgellA STRAINS ISOLATED IN ROMANIA FROM 2016 TO 2018

Daniela Cristea1*, Adriana Simona Ciontea1, Melania Mihaela Andrei1, Andrei Popa1, Mãdãlina Zamfir1, Lavinia Zota3, Maria Nica4, Codruþa Romaniþa Usein1,2

1Cantacuzino National Medico-Military Institute for Research and Development, Bucharest, Romania2Carol Davila University of Medicine and Pharmacy, Bucharest, Romania

3National Institute of Public Health, National Centre for Prevention and Control of Communicable Diseases, Bucharest, Romania4Dr. Victor Babeş Clinical Hospital of Infectious and Tropical Diseases, Bucharest, Romania

* Corresponding author: Daniela Cristea, Enteric Bacterial Infections; Cantacuzino National Medico-Military Institute for Research and Development, Splaiul Independenþei 103, sector 5, 050096, Bucharest, Romania, Tel: +40724034886, E-mail: [email protected]

AbStRACtThis study aimed to describe the virulence gene content and antibiotic resistance profiles as well as the genetic

relatedness of shigella strains originated from romanian patients, collected between 2016-2018 through the national diarrheal disease surveillance program.

a total of 60 Shigella sonnei strains and 26 S. flexneri strains were tested against a set of 16 antimicrobials, screened by pcr for several genes involved in the disease pathogenesis (ipah, ipaBCD, ial, sen, set1A, set1B, sat and pic genes), and typed by pulsed-field gel electrophoresis (PFGE) to make inferences about their genetic relatedness.

overall, the resistance most commonly acquired by the autochthonous Shigella strains investigated was against sulfonamide compounds (58/86 strains), trimethoprim (46/86 strains) or combination of trimethoprim with sulfamethoxazole (44/86 strains), and ampicillin (41/86 strains). Strains with resistance to extended spectrum cephalosporins (12/86 strains) were detected, all extended spectrum beta-lactamase producers, as well as strains that displayed resistance to nalidixic acid (3/86 strains) but not to ciprofloxacin.

the virulence markers targeted ranged in prevalence from 6% (set1A, set1B, pic) to 100% (ipah). the S. flexneri strains displayed a richer virulence gene content than s. sonnei strains. the most complex virulence genotype, represented by the association of ipah, ipaBCD, ial, sen, sat, set1A, set1B, and pic genes was found in S. flexneri serotype 2a strains.

The PFGE-based molecular typing allowed the detection of several clusters of strains displaying band-based profiles with at least 85% similarity. Eight such clusters were identified among the s. sonnei strains and four clusters among the S. flexneri strains.

continued laboratory-based surveillance is essential to generate data that help to draw the picture of the public health problem represented by shigellosis in romania.

Keywords: Shigella, virulence genes, antibiotic resistance, pfge

REZUMAtAcest studiu a urmărit să descrie conţinutul de gene de virulență şi profilurile de rezistența la antibiotice, precum

şi gradul de înrudire genetică al unor izolate de Shigella provenite de la pacienți români, colectate în perioada 2016-2018, prin programul naţional de supraveghere a bolii diareice.

Un total de 60 de tulpini de Shigella sonnei și 26 de Shigella flexneri au fost testate faţă de 16 substanţe antimicrobiene, analizate prin tehnica PCR pentru gene implicate în patogeneză (genele ipah, ipaBCD, ial, sen, set1A, set1B, sat and pic) şi tipizate prin electroforeză în câmp pulsator, pentru a se evalua gradul lor de înrudire genetică.

Cel mai comun profil de rezistenţă la antibiotice căpătat de tulpinile autohtone de Shigella a fost faţă de compuşi sulfonamidici (58/86 tulpini), trimethoprim (46/86 tulpini), trimethoprim asociat cu sulfamethoxazol (44/86 tulpini) şi ampicilină (41/86 tulpini). Au fost detectate tulpini cu rezistenţă la beta-lactamine cu spectru extins (12/86 tulpini), toate producatoare de beta-lactamaze cu spectru extins, precum şi tulpini rezistente la acid nalidixic (3/86 tulpini), dar nu şi la ciprofloxacin.

Prevalenţa markerilor de virulenţă a variat între 6% (set1A, set1B, pic) şi 100% (ipaH). Tulpinile de S. flexneri au prezentat un conţinut de gene de virulenţă mai bogat decat cele de s. sonnei. Cel mai complex genotip de virulenţă, reprezentat de asocierea genelor ipaH, ipaBCD, ial, sen, sat, set1A, set1B şi pic, a fost găsit în tulpinile de S. flexneri serotip 2.

Tipizarea moleculară bazată pe electroforeza în câmp pulsator a permis detectare mai multe clustere de tulpini care prezentau profiluri de benzi cu similaritate de cel puţin 85%. Opt astfel de clustere s-au detectat printre tulpinile de s. sonnei şi patru printre cele de S. flexneri.

Supravegherea continuă bazată pe laborator este esenţială pentru a genera datele care să ajute la întelegerea problemei de sănătate publică reprezentată de shigelozele din România.

Cuvinte-cheie: Shigella, gene de virulenţă, rezistenţă la antibiotic, PFGE


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INtRODUCtION

Shigella species is a highly virulent enteric pathogen, reported to be an important cause of diarrhea in developing countries and highly contagious due to its low infective dose [1]. shigellosis, the infection caused by Shigella spp., can progress to severe disease, depending on the virulence potential of the strain and the nutritional status of the individual [2].

shigellosis is caused by penetration of invasive Shigella into the intestinal mucosa of the colon, leading to degeneration of the epithelium and a strong inflammatory response. several virulence factors, which are the expression of arrays of virulence genes located in the chromosome or on a large virulence plasmid (pinv) are described as important for pathogenesis, being associated with colonization, invasion/penetration of the epithelium, intercellular spread and toxin-mediated disease. among them are the following: the invasion-associated locus (ial), the invasion plasmid antigen h (ipah), the invasion plasmid antigens b (ipab), c (ipaC) and d (ipad), shigella enterotoxin 1 or shet-1 (set1A and set1B genes), shigella enterotoxin 2 or shet-2 (sen gene), and the secreted auto-transporter (sat gene) [3].

chromosomal genes, set1A and set1B are encoding the factors associated with the watery phase of diarrhea. shet-1 and shet-2, in addition to their enterotoxic activity, play an important role in the transport of electrolytes and water in the intestine.

shigellosis is an invasive illness of the human colon that leads to varied clinical symptoms ranging from mild watery diarrhea to severe colitis. antimicrobial therapy is required in the cases of severe dysentery to reduce the duration of clinical illness, minimize the complications, and prevent the dissemination of infectious cases, conferring significant public health benefits [4].

Fluoroquinolones, β-lactams and cotrimoxazole were considered to be the first choice for treating shigellosis. however, in the last years, the use of these drugs has been compromised by the emergence of resistant strains. Shigella has developed mechanisms of

resistance based on different mobile genetic elements (plasmids, integrons and transposons) which play a central part in the clinical dissemination of antibiotic resistance genes and have allowed for the rapid development of multi-antibiotic resistance [5].

this study aimed to provide meaningful information about the antibiotic resistance phenotypes, virulence profiles, and the genetic relatedness of the latest Shigella strains reported through the romanian national surveillance program of diarrhea disease.

MAtERIAL AND MEtHODS

Bacterial isolatesbetween 2016 and 2018 years, a total of 86

Shigella strains were confirmed by the Reference Laboratory for bacterial enteric infections in Cantacuzino National Medico-Military institute for research and development. they were serotyped as group b/S. flexneri (26 strains) and group d/s. sonnei (60 strains), respectively. the strains assigned to s. flexneri were distributed in the following serotypes: 1b (8 strains), 2a (5 strains), 3a (1 strain), 4a (1 strain), 5 (2 strains), 6 (9 strains). all these strains originated from patients with acute diarrheal disease residing in various romanian counties. The laboratory confirmation was performed using conventional biochemical tests and commercially available antisera (denka seiken, tokyo). all the strains were stored at -20°c in Luria bertani broth containing 15% glycerol.

Antimicrobial susceptibility testingall the Shigella strains were screened by

the Kirby Bauer disk diffusion method for antibiotic susceptibility and the breakpoint values for qualitative interpretation were based on the recommendations of the european Committee on Antimicrobial Susceptibility testing (eUcast) (http://www.eucast.org/ast_of_bacteria/guidance_documents). the following antimicrobial agents were tested: ampicillin (amp, 10 µg), cefotaxime (ctx, 5µg), ceftazidime (caz,10 µg), cefoxitin (fox, 30 µg), meropenem (mem, 10 µg), imipenem (imp, 10 µg), chloramphenicol (c, 30 µg), streptomycin (s, 10 µg), gentamicin (cn,

Virulence and resistance profiles of Shigella strains isolated in Romania from 2016 to 2018

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30µg), kanamycin (K, 30 µg), sulphonamide compounds (s3, 300 µg), tetracycline (te, 30 µg), trimethoprim (W, 5 µg), trimethoprim-sulfamethoxazole (sxt, 1.25/23.75 µg), nalidixic acid (na, 30 µg) and ciprofloxacin (cip, 5 µg). escherichia coli atcc 25922 was included as a reference strain for antimicrobial susceptibility testing quality control.

the antibiotic disks were obtained from oxoid (basingstoke, United Kingdom). in this study, multidrug resistance (mdr) was defined as resistance to three or more drugs from unrelated antibiotic classes. detection of extended-spectrum beta-lactamase (esbL) production in the isolates with resistance to third-generation cephalosporins was performed using a cefotaxime/ceftazidime/amoxicillin/clavulanate synergy test previously described [6].

PCR assays for virulence markersall dna templates used for pcrs were

prepared by boiled cell lysis. the 4-5 colonies were suspended in 200 µl of sterile molecular-grade water and boiled for 15 minutes. the supernatant containing the dna, obtained after centrifugation at 5 minutes at 14,000 rpm, was stored at −20°C till further use for PCR testing.

Each sample was submitted to PCR amplification of the following virulence markers: ipah, ipaBCD, ial, set1A, set1B, sen, sat and pic, using previously described primers and protocols [7].

Pulsed-field gel electrophoresis (PFGE)Shigella strains were analyzed by pulsed-

field gel electrophoresis (PFGE) following the PulseNet standardized protocol for Shigella (https://www.cdc.gov/pulsenet/pdf/ecoli-shigella-salmonella-pfge-protocol-508c) and genomic dna was digested with the restriction endonuclease Xbai (roche). genomic dna was separated in 1% SeaKem Gold agarose (Lonza) with a chef mapper system (biorad, hercules, Usa) using a run time of 19 hours. salmonella enterica serovar braenderup h9812 strain was used as size marker. The electrophoretic profiles were analyzed using BioNumerics

software version 6.6 (applied maths, sint-Martens-Latem, Belgium). Banding patterns were compared based on the dice similarity coefficient and a dendrogram was constructed by Upgma (unweighted pair -group method with arithmetic averages) algorithm with a setting of 1.0% for optimization and position tolerance of 1.0% for band comparison. clusters designations were assigned at the ≥85% profile similarity level (approximately 3-band difference).

RESULtS

Prevalence of virulence genes in Shigella strains

overall, among the 86 Shigella strains studied, between two and eight virulence gene markers were found per strain (table 1). While all the strains harbored ipah gene only 6% of them carried set1A, set1B or pic genes. s. sonnei was represented by strains with less virulence gene markers than S. flexneri. among the s. flexneri serotypes, serotype 2a was represented by strains exhibiting the highest number of virulence genes (virulence profiles comprising 8 virulence markers) while serotype 6 displayed the lowest number. the virulence gene distribution is shown in table 2.

Antibiotic resistance phenotypes overall, ten strains were susceptible to

all the antibiotics tested and 76 strains were resistant at least to one of them. the twenty-one antibiotypes defined by resistance against one to seven antimicrobial agents did not comprise resistance to meropenem, ciprofloxacin and gentamycin (table 3). twenty-four strains, mostly assigned to S. flexneri species, were mdr strains. the most commonly acquired resistance was against sulphonamide compounds (58 strains), trimethoprim alone (46 strains) or in combination with sulfamethoxazole (44 strains), and against ampicillin (41 strains), and 18 strains (21%) were tetracycline resistant. only 3 strains (3%) displayed resistance to nalidixic acid but not to ciprofloxacin. Twelve strains (14%) were resistant to cefotaxime and/or ceftazidime and yielded a positive result in the esbL phenotypic test.

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according to the antibiotic resistance profiles, a total of 21 antibiotypes were distinguished among the studied isolates, defined by resistance against one to seven antimicrobial agents (table 3).

PFGE typing in relationship with the antibiotic resistance profiles of Shigella strains

pfge was performed in order to explore the potential dissemination of clonal groups of resistant Shigella across romania. by applying the 85% similarity criterion, the genetic relatedness of the strains allowed a cluster

distribution. eight clusters were revealed among the 60 s. sonnei strains while among the 26 S. flexneri group four clusters were detected: one cluster for serotype 1b, one cluster among the serotype 6, and two clusters among serotype 2a. there was a clear correlation between the resistance profiles and the PFGE patterns. Specifically, 5 Shigella strains with ampr, Wr, s3r, sxtr resistance profile were included in the same cluster and so were also 12 other strains that had Wr, s3r, sxtr resistance profile, which suggested their genetic relatedness.


species and serotype Virulence patterns number of strainsS. flexneri

1b

ipah, ipaBCD, ial, sat, sen 4ipah, ial, sat, sen 1ipah, sen 1ipah, sat 2

2a ipah, ipaBCD, ial, sat, pic, sen, set1A, set1B 53a ipah, ipaBCD, ial, sat, sen 14a ipah, ipaBCD, ial, sat, sen 15 ipah, ipaBCD, ial, sat, sen 15 ipah, sen 16 ipah, ial, sen 9

s. sonnei ipah 27ipah, ial 4ipah, ial, sen 23ipah, ipaBCD, ial, sen 6

Table 1. The virulence genotypes displayed by the 86 Shigella isolates

species and serotypes

no. of strains

number (%) of strains carryingipah ipaBCD ial sen sat set1A set1B pic

S. flexneri 26 26 (100) 12(46)

22(85)

24(92)

15(58)

5(19)

5(19)

5(19)

1b 8 8 4 5 6 7 0 0 0

2a 5 5 5 5 5 5 5 5 5

3a 1 1 1 1 1 1 0 0 0

4a 1 1 1 1 1 1 0 0 0

5 2 2 1 1 2 1 0 0 0

6 9 9 0 9 9 0 0 0 0

s. sonnei 60 60(100)

6(10)

33(55)

29(48) 0 0 0 0

Table 2. The distribution of the virulence markers among the 86 Shigella strains studied

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DISCUSSION

Shigella infections are a serious health problem in the world, being responsible for 125 million diarrheal episodes each year with about 160000 deaths, a third of them among the pediatric population [2, 8]. Antibiotic therapy is recommended to diminish potential com-plications hence the risk of mortality, improve the clinical status and decrease transmission by eliminating Shigella from the gut [9].

Shigella strains harbor many virulence factors, including factors that are associated with invasion of the colonic epithelium and toxins [10]. The genetic determinants associated with invasion assist Shigella in penetration of epithelial cells (i.e. ial), are the effectors of bacterial entry into the host cell (i.e. ipa BCD) or facilitate cell-to-cell spread (i.e. ipah). in our study, the ipah gene was detected in all the isolates, while the cluster ipaBCD had a much lower prevalence and was more commonly found in S. flexneri than in s. sonnei strains. a possible explanation can be that ipah gene is a much more stable marker with

multiple copies located on both plasmids and chromosome whereas the locus which includes the ipaBCD genes can be lost by spontaneous deletions [11, 12]. It is worth noting that among the autochthonous strains ipaBCD cluster was more frequently present in S. flexneri strains than in s. sonnei.

regarding the overall prevalence of the other virulence determinants targeted, ial and sen genes which displayed similar rates, were the second most prevalent virulence markers detected in the autochthonous strains after ipah. however, when considering how many strains within each species harbored these genes, a significantly higher proportion of s. flexneri strains than s. sonnei ones possessed them (92% vs. 48%). a report resulted from an eight-year Chinese study showed a major dif-ference between s. sonnei and S. flexneri only in terms of sen gene prevalence [13] and similar findings were also reported in an Indian study [14].

across the shigella strains investigated in this study, the genes coding for the shet1 enterotoxin (set1A and set1B) and pic (pic) were

Resistance profile No. of isolatessusceptible 10

ampr 2s3r 9

Wr, nar 1ampr, ctxr 6ampr, sr, s3r 3Wr, s3r, sxtr 21ampr, cr, ter 1

ampr, ctxr, cazr 1ampr, cr, ter, sr 5

ampr, Wr, s3r, sxtr 12ampr, Wr, cr, ter, sr 1

ampr, Wr, s3r, ctxr, sxtr 1Wr, ter, sr, s3r, sxtr 2

ampr, cr, ter, sr, ctxr, cazr 1ampr, Wr, s3r, ctxr, cazr, sxtr 1

ampr, Wr, ter, sr, s3r, sxtr 1Wr, ter, sr, s3r, sxtr, nar 2ampr, Wr, cr, sr, s3r, sxtr 1

ampr, cr, ter, sr, s3r, ctxr, cazr 2ampr, Wr, cr, ter, sr, s3r, sxtr 3

Table 3. Antibiotic resistance profiles detected among the 86 Shigella spp. strains

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found exclusively in S. flexneri 2a strains, a finding which contributed to the overall higher virulence potential assigned to this species. the studies of noriega et al. [15] and Vargas et al. [16] also showed that set1A and set1B were present exclusively in S. flexneri 2a. also, a malaysian study confirmed that the set of set1A and set1B genes is highly conserved in this serotype [17]. the sat gene which codes for serine protease autotransporters of the enterobacteriaceae family sat was another marker present in S. flexneri (with the exception of serotype 6 members) but absent from s. sonnei.

besides the description of the virulence genotypes, the study aimed to evaluate how many of the shigellosis cases diagnosed during 2016-2018 could have been treated efficient-ly based on the antimicrobial susceptibility expressed by the Shigella isolates that caused them. it is well-known that antimicrobial ther-apy is beneficial but microbial resistance to many of the widely used antimicrobials is lead-ing to failures in an increasing number of cases [17]. Based on the susceptibility results, useful antibiotics such as ampicillin and the combi-nation trimethoprim-sulfamethoxazole are no longer a choice for treating the autochthonous patients with shigellosis. moreover, 28% of the investigated strains proved to be mdr strains.

among them, there were strains with re-sistance to third-generation cephalosporins. Specifically, 14% of Shigella strains investigated had acquired such antibiotic resistance due to the capacity to produce extended-spectrum be-ta-lactamases. based on previous reports, sim-ilar strains also circulate in other countries [19-21]. This is concerning because the third-gener-ation cephalosporins are considered as the use-ful alternative for infections caused by Shigella strains with resistance to fluroquinolones.

fluoroquinolones, currently recommend-ed by the World Health Organization as first-line treatment for adults, proved to be still very efficient against the Shigella strains recovered from the romanian patients, as none of them displayed resistance to ciprofloxacin. Moreo-ver, based on the results of this study, even the nalidixic acid could be still used efficiently for treating the autochthonous patients as only 3%

of the strains displayed a resistance phenotype. by contrast, in other parts of the world, this

drug is no longer used due to the high rates of resistance found, this being the reason for the switch to fluoroquinolones as an alternative [17].

in conclusion, this study provided evi-dence about the circulation of resistant and virulent strains of Shigella and underlined that continued laboratory-based surveillance is es-sential to generate data that help to draw the picture of the public health problem represent-ed by shigellosis in romania.


REFERENCES1. zaidi mb, estrada-garcía t. shigella: a highly

virulent and elusive pathogen. curr trop med rep 2014. 1:81-87.

2. baker s, the hc. recent insights into Shigella: a major contributor to the global diarrhoeal disease burden. curr opin infect dis. 2018 oct;31(5):449-454.doi: 10.1097/Qco.0000000000000475.

3. Mattock E, Blocker AJ. How Do the Virulence factors of shigella Work together to cause disease? front cell infect microbiol. 2017 mar 24;7:64. doi:10.3389/fcimb.2017.00064.

4. Williams PCM, Berkley JA. Guidelines for the treatment of dysentery (shigellosis): a systematic review of the evidence. paediatr int child health. 2018; 38(suppl 1): s50–s65.

5. Ranjbar R, Farahani A. Shigella: Antibiotic-resistance mechanisms and new horizons for treatment. infect drug resist. 2019; 12: 3137–3167.

6. Jarlier V, Nicolas MH, Fournier G, Philippon a. extended broad-spectrum beta-lactamases conferring transferable resistance to newer beta-lactam agents in enterobacteriaceae: hospital prevalence and susceptibility patterns. rev infect dis 1988, 10:867-78.

7. cristea d, oprea m, ciontea as, antohe f, Usein CR. Prevalenţa markerilor de virulenţă şi a plasmidelor de tip pHS-2 in izolatele de Shigella sonnei şi Shigella flexneri provenite din cazurile de shigelloză din Romania. Revista Română de Medicină de Laborator Vol. 24, Nr. 1, martie, 2016: 103-110.

8. Black RE, Cousens S, Johnson HL, Lawn JE, Rudan I, Bassani DG, et al. Child Health


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epidemiology reference group of Who and UNICEF. Lancet. 2010 Jun 5;375(9730):1969-87.

9. bennish mL. potentially lethal complications of shigellosis. rev infect dis 1991;13 suppl 4:s319.

10. schroeder gn, hilbi h. molecular pathogenesis of Shigella spp: controlling host cell signaling, invasion, and death by type iii secretion. clin microbiol rev 2008. 21:134-56.

11. sasakawa c, Kamata K, sakai t, murayama sy, makino s, yoshikawa m. molecular alteration of the 140-megadalton plasmid associated with loss of virulence and congo red binding activity in Shigella flexneri. Infect. Immun. 1986 feb;51(2):470-521.

12. Venkatesan MM, Buysse JM, Kopecko DJ. Use of Shigella flexneri ipaC and ipaH gene sequences for the general identification of Shigella spp. and enteroinvasive Escherichia coli. J Clin microbiol. 1989 dec;27(12):2687-91.

13. Qu M, Zhang X, Liu G, Huang Y, Jia L, Liang W, et al. an eight-year study of shigella species in Beijing, China: serodiversity, virulence genes and antimicrobial resistance. J Infect Dev Ctries. 2014 Jul 14;8(7):904-8.

14. Roy S, Thanasekaran K, Dutta Roy AR, Sehgal sc. distribution of shigella enterotoxin genes and secreted autotransporter toxin gene among diverse species and serotypes of shigella isolated from andaman islands, india. tropical medicine and international health 2006;vol. 11, no 11:1694–1698.

15. noriega fr, Liao fm, formal sb, fasano a,

Levine mm. prevalence of shigella enterotoxin 1 among shigella clinical isolates of diverse serotypes. J Infect Dis. 1995;172:1408–1410.

16. Lin thong K, Ling Ling hoe s, puthucheary sd, md yasin r. detection of virulence genes in malaysian shigella species by multiplex pcr assay. bmc infect dis. 2005 feb;5:8.

17. niyogi, sK. increasing antimicrobial resistance: an emerging problem in the treatment of shigellosis. clin microbiol infect. 2007;13:1141-1143.

18. gendrel d, cohen r. bacterial diarrheas and antibiotics: european recommendations. arch pediatr 2008;15 suppl 2:s93–6.

19. aminshahidi m, arastehfar a, pouladfar g, arman e, fani f. diarrheagenic escherichia coli and shigella with high rate of extended-spectrum beta-Lactamase production: two predominant etiological agents of acute Diarrhea in Shiraz, Iran. Microb Drug Resist. 2017 dec;23(8):1037-1044. doi: 10.1089/mdr.2017.0204.

20. Radice M, Gonzalez C, Power P, Vidal MC, gutkind g. third generation cephalosporin resistance in shigella sonnei, argentina. emerg infect dis. 2001;7:442-443.

21. rahman m, shoma s, rashid h, siddique aK, nair gb, sack da. extended spectrum beta-lactamase-mediated third generation cephalosporin resistance in shigella isolates in Bangladesh. J Antimicrob Chemother. 2004;54:846-847.

189ROMANIAN ARCHIVES OF MICROBIOLOGY AND IMMUNOLOGY, Vol. 78, Issue 3, pp. 189-197, July - September, 2019

EOSINOPHILIA AND PARASITIC DISEASES

Dan Steriu*Carol Davila University of Medicine and Pharmacy, Bucharest, Romania

* Corresponding author: Dan Steriu, Carol Davila University of Medicine and Pharmacy Bucharest, Romania, e-mail: [email protected]

AbStRACtModification of eosinophil number may be due to more than one cause. One of these is a possible parasitic

infection. not every parasitic infection can cause change in peripheral blood eosinophil number. Usually, there is a growing eosinophilia in tissue helminth infections. In parasitic infections produced by protozoa or intraluminal worms eosinophilia is not significantly modified or altered. In the following are summarized the main nonparasitic diseases that can cause alteration of eosinophilia and the evolution of eosinophilia in the most common parasitic infections is analyzed. To determine the significance of eosinophilia within the broader clinical and laboratory diagnosis any investigation should start with fixing presence or lack of possible parasitic infections.

Keywords: eosinophilia, parasitic infections, nonparasitic diseases

REZUMAtModificarea numărului de eozinofile poate fi datorată mai multor cauze. Una dintre acestea este o

eventuală infecție parazitară. Nu orice infecție parazitară determină modificarea numărului eozinofilelor în sângele periferic. De regulă, are loc o creștere importantă a eozinofiliei în parazitozele tisulare produse de helminți. Infecțiile determinate de protozoare parazite sau de helminți cavitari nu modifică sau modifică nesemnificativ eozinofilia.

În cele ce urmează, sunt prezentate pe scurt principalele afecțiuni nonparazitare ce pot determina modificarea eozinofiliei și, în continuare, este analizată evoluția eozinofiliei în cele mai răspândite infecții parazitare. Pentru a stabili semnificația unei eozinofilii în cadrul mai larg al unui diagnostic clinic și de laborator, se consideră că orice investigație trebuie să înceapă cu stabilirea prezenței sau absenței unei eventuale infecții parazitare.

Cuvinte-cheie: eozinofilie, infecții parazitare, afecțiuni nonparazitare

INtRODUCtION

The notion of “eosinophilia” means an in-crease in the relative or absolute number of cir-culating eosinophils in the blood, or existing in the bone marrow or other tissues [1, 2].

in adults, normal values are usually within the range of 1-5%, or about 300-400 elements/mm3 of peripheral blood.

although physiological variations have been described depending on age, the stages of the menstrual cycle, or even the different moments of the day, it is believed that eosino-philia can be most commonly associated with parasitic or allergic diseases.

there are also non-parasitic eosinophils, such as:

Hypereosinophilic syndrome (> 1500/mm3), lasting more than 6 months, with or without splenomegaly that can affect: • cardiac (Loeffler endomyocardial fibrosis

with adiastolia, myocarditis with eosino-philia,

• neurological infiltrates (diffuse encepha-lopathy, peripheral neuropathy, diseases of the optic nerve), respiratory (chronic cough),

• polymorphic mucous membranes (urticaria, erythematous lesions),

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• abdominal (diarrhea, frequent charcot-Leyden crystals in the stool),

• kidneys (kidney failure).

Systemic diseases with eosinophilia, such as:• carrington chronic eosinophilic pneumo-

nia (more common in women), with dysp-nea, general aneurysm, radiographic evi-dence of dense peripheral infiltration with progressive extension,

• shulman eosinophilia, myalgia, arthralgia, edema at the distal extremities,

• churg-strauss syndrome – granulomatosis and angiitis (diffuse vasculitis, with asth-ma, heart failure, neuritis and paralysis).

Eosinophilic leukemiaAllergic diseases:

• urticaria, bronchial asthma, allergic rhini-tis, iatrogenic hypereosinophilia in relation to drug allergies (β-lactam, iodine-pro-duced, radiotherapy).

• dermatological disorders: eczema, her-petiform dermatitis and lichens, as well as: hodgkin’s disease, duhring-brocq disease, chronic myeloid leukemia, addi-son’s disease, liver cancer, some digestive cancers, dermatomyositis, rheumatoid ar-thritis, hemorrhagic rectocolitis, crohn’s disease, leprosy, tuberculosis.given this complex of possible etiologies,

it is considered that a diagnosis algorithm of eosinophilia begins with the exclusion of a pos-sible parasitic infection.

Features and functions of mature eosinophil

the mature eosinophil is a mobile cell ca-pable of phagocytosis. it comes from an auton-omous medullary line and has a bilobed nucle-us and intracytoplasmic granules.

after leaving the bone marrow, it enters into the circulatory system and then through the diapedesis into the tissues.

it is considered that in the peripheral blood there are normally less than 400 eosinophils/mm3. in the case of a number greater than 400-500 eosinophils/mm3, it is considered to be hypereosinophilia.

if the number of eosinophils is between:• 500/mm3 and 1500/mm3 = moderate hyper-

eosinophilia• 1500/mm3 and 3000/mm3 = forte hypereo-

sinophilia• > 3000/mm3 = massive hypereosinophilia

There are two different populations of eo-sinophils: • a population of eosinophils called “nor-

modense”, granular, present in normal subjects or during eosinophils in helmin-thiasis.

• An eosinophil population called “hypo-dense”, cytotoxic potential, responsible for tissue lesions and encountered in hypere-osinophilic syndrome, collagenase, hemo-pathies, asthma.three essential functions of eosinophils are

currently accepted: cell with cytotoxic function, the participant cell in the inflammatory process and the immunoregulatory cell.

these functions are ensured by the content of specific granules, that appear in the promye-locyte stage. the granules contain a crystalline central core and a clear peripheral matrix. at the crystalline core level, the so-called “Major Basic Protein” (MBP) involved in cytotoxic ef-fects of eosinophil, and activating function on neutrophils, mast cells and basophils was iden-tified. In the matrix other major proteins have been identified, such as: a neurotoxin (EDN/epx), a peroxidase (epo) and a cationic pro-tein (ecp) also involved in cytotoxic processes (including parasites), but also in activities rib-onuclease catalysts. Dosage of such major pro-teins from different biological fluids (serum, alveolar lavage) brings important information in establishing certain diagnoses.

the disintegration of eosinophils makes the crystalline cores associated with the forma-tion of the charcot-Leyden crystals. the occur-rence of charcot-Leyden crystals in biological products has therefore the significance of an appreciable local eosinophilia.

The role of eosinophils in the inflammato-ry process is determined by the release of:• lipid mediators: produced leukotrienes

(Lt) of arachidonic acid metabolism, pros-

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Eosinophilia and parasitic diseases

taglandin, thromboxane b2, platelet activa-tion factors (pafs)

• some chemokines: rantes (products of both t lymphocytes and chemotactic plate-lets for monocytes and eosinophils), mip-1α (macrophage inflammatory peptides), interleukins (iL-8)

• some cytokines: (IL-1, IL-6, TNF-α)

the immunoregulatory function of the eosinophil is expressed by the production of complex functions cytokines: • immunoregulatory factors ( iL-2, iL-4, iL-

10, Th-2, IFN γ),• growth and activation factors: granu-

lo-monocyte colony growth factor (gm-CSF), IL-3, IL-5, IL-16, RANTES, MIP-1α, a specific eosinophil chemoattractant (Eo-taxin)

• Factors involved in tissue and fibrous re-modeling processes: TNFα, Transforming Growth Factor (TGFα, TGFβ), IL-1, IL-6, iL-8).

there are normally less than 400 eosin-ophils/mm3 in the peripheral blood. the vast majority of eosinophils are found at the tissue level where they reach by the diapedesis. they concentrate on gastrointestinal tissues, res-piratory and genito-urinary apparatus, that is tissues that can be the input gates of potential antigens. a ratio of 1/300 between blood and eosinophilic tissues is estimated.

hypereosinophilia is relatively common in current pathology. its origin is, however, often difficult to establish. It is appreciated that be-sides a helminthic parasitic infection or a pos-sible myiasis (protozoa does not produce sig-nificant growth), hypereosinophilia can occur in the following situations: allergic diseases, dermatoses, gastrointestinal diseases, tumor processes, immune deficiencies, some systemic diseases, hereditary eosinophils, cyclic eosino-phils, syndromes due to special environmental conditions.

in the context of this complex of possible etiologies, it is considered and, in fact, it is al-ready in use that a diagnostic algorithm of hy-pereosinophilia should begin with the exclu-sion of an even tiny parasitic infection [3].

Characteristics of eosinophilia in parasitosis [4, 5, 6]

eosinophilia is a dynamic phenomenon that varies depending on:

• The time elapsed in the evolution of the infectionthe evolution of eosinophilia according

to the time factor is expressed by the so-called “curve of Lavier”. The Lavier curve records the eosinophilia values at different stages of the in-fection. its appearance depends on the parasitic load, the host reactivity, the stages of the para-sites involved and their localization. The curve has the appearance of an asymmetric “bell” with a roughly upward initial portion (corre-sponding to the initial phase of the infection), a maximum plate (state phase) and a slow down line (corresponding to the healing phase).

• Influence of the species and of the biological cycle peculiarities.depending on these factors, eosinophilia

may develop differentlyWhen parasitosis evolves linearly,

in one way (ascariasis, ancylostomiasis), eosinophilia follows the Lavier curve. When the infection develops in acute attack, with allergic phenomena (filariasis), each attack corresponds to an increase in eosinophilia. When autoinfection occurs (strongyloidiasis), eosinophilia is increased in frequent and persistent repetitions, sometimes for decades. parasites, whose biological cycle involves a tissue phase, cause important eosinophils during the course of this phase. at the end of the tissue phase and the onset of the cavity, the eosinophilia returns to moderate values (ascariasis, filariasis, schistosomiasis, fasciolosis, etc.) Some parasites from animals cause important and long-lasting eosinophils (mvLs, mcLs). in the same way, a dead tissue parasite causes a more severe reaction (including eosinophilia) than a viable one (cysticercosis, dracunculiasis).

intact or non-viable hydatid cyst does not produce an important eosinophilia, but a fis-sured hydatid cyst causes a significant increase in eosinophilia throughout the drainage of the content.

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• Localization of the parasiteThe localization of the parasite also

greatly influences how eosinophilia evolves. Thus, cavitary parasites without significant tissue phases cause moderate eosinophils and with rapid decreases (oxyurosis, trichuriasis, taeniasis). however, if the infection is strong or the progression of the parasite is rapid, significant temporal increases in eosinophils may occur. cause: massive release, in a short time, of metabolites that, being absorbable, are found to be powerful allergens.

• Parasitic load:in a certain infection, eosinophilia reaches

the maximum values for a certain number of parasites. Affected as the characteristic value, the increase in eosinophilia is no longer pro-portional to the number of parasites.

in relation: parasitic load - the value of eo-sinophilia, the metabolism factor may also be involved; a parasitic species with intense me-tabolism can cause significant eosinophilia.

• Influence of the host’s biological characteristics

there are internal and external factors that can influence eosinophilia. Among the internal factors, the following can be mentioned: adre-nal cortical hyperactivity stimulates eosinophil production, the estrogen secretion, the black race produces fewer eosinophils than the white race.

The external factors that can influence eo-sinophilia are: bacterial infections and corti-coid treatments transiently decrease eosinophil levels. first infection can produce an important eosinophilia. after healing, a possible reinfec-tion causes a moderate increase in eosinophil-ia. After effective treatment, eosinophilia de-creases at the beginning faster, then slower and returns to normal in a few weeks.

the following are the most widespread parasitic infections and their relationship with eosinophilia.

Ascariasisit is the infection produced by a geohel-

minth; Ascaris lumbricoides. the infection is widespread in all geographical areas, affecting

a high percentage of the population, especially in less-favored areas. the infective stage is the embryoning egg on the ground. the infection is caused by digestive tract by faulty food hy-giene. The final location is the small intestine, but there is an initial peripheral phase through the liver and lung. the diagnosis can be sup-ported by other elements (apart from eosino-philia): Loeffler syndrome (in the peripheral phase), mild digestive manifestations, skin allergic phenomena, respiratory, eventual di-gestive complications (intestinal occlusions, wrong migration of the adult parasite) [4, 6].

Eosinophilia: average value is 25%; respect the “curve of Lavier”.

maximum eosinophilia time: approximate-ly 20 days from infection under the conditions of the liver and lung circulation of the larvae. after the parasite is located in the intestine, the eosinophilia decreases to moderate values and then normalizes [5].

Recommended investigations: initial radi-ological examination (migratory infiltrative im-ages), sputum examination (the parasite larvae and/or charcot-Leyden crystals can be high-lighted). Later, repeated coproparasitological tests for egg evidence (the test is positive after at least 60 days from infection). often there is no correlation between peak eosinophilia and positive coproparasitological examinations. for this reason, parasitological examinations may be negative although the parasite exists, but in the hepatic and pulmonary migration phase. if suspicion persists, it is recommended to repeat the examinations after a few weeks.

Hookworm infectionthe infection is produced by two geohel-

minths with tropical and subtropical spread or possibly in temperate climate in mine biotope: Ancylostoma duodenale and Necator americanus. the infection is transcutaneous by active pen-etration of strongylid larvae (infective stage). these can be found on soil contaminated with faeces from infected persons. The final locali-zation of the parasite is the small intestine after initial hepatic and pulmonary migration phase.

the most common symptoms are dermati-tis, Loeffler syndrome under the conditions of

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perienteric migration larvae through the liver and lung, intestinal irritation, digestive disor-ders with melenic stools, severe anemia [3, 6].

Eosinophilia: the mean eosinophilia is 35-40%. this value is recorded in acute infections. at this stage, the Lavier curve is respected. maximum value, 60-70% is recorded approximately three months after the infection. it is worth noting that eosinophilia remains at high levels both in the perienteric and intestinal migration phase where the parasite, although it is located cavitary, produces tissue lesions. in chronic infections, eosinophilia no longer respects the Lavier curve and usually decreases [4].

Recommended investigations: initial ra-diological examination (migratory infiltrative images), possibly sputum examination (par-asite larvae can be highlighted). the certain-ty diagnosis consists of viewing the parasite eggs by repeated coproparasitological exami-nations. tests are positive after 30-40 days of infection.

Bothriocephalosisthe infection is due to a cestode; Diphyl-

lobothrium latum. man is infected by the con-sumption of non-heat-activated fish meat, which contains the infective stage; “plerocer-coid larvae”. The infection is particularly com-mon in riparian human communities that reg-ularly consume fish.

the parasite located in the small intestine can produce a mild nonspecific symptomatol-ogy, in more serious cases with avitaminosis b and/or anemia [4, 6].

Eosinophilia: being a cavitary parasite, there is often no increase in the number of eo-sinophils. if it does occur, it is irregular and around the 40th day of infection reaches maxi-mum values [5].

The diagnosis is made by coproparasito-logical examinations, indicating the eggs about three weeks after the infection.

Cysticercosisit is Cysticercus celullosae larvae infection of

taenia solium. it is a cosmopolitan infection in countries where pigs are grown for consump-

tion. the infection is by digestive tract and is due to defective hygiene, the infective stage being the eggs of taenia solium. self-infection is also possible if the person is parasitized by the adult stage located in the small intestine. the larval stage, the cysticercus is usually located subcutaneously, muscularly, and nervously. the most severe symptoms occur in nerve lo-cations. [4, 6].

Peripheral eosinophilia may reach 20-30%, but does not always occur. it is mainly deter-mined by the inactivated cysticercus, the viable being weakly reactive. in nerve locations, eo-sinophilia appears elevated in the CSF [5].

Diagnosis is usually imaging in corrobora-tion with serology.

Fasciolosiscosmopolitan infection produced by a

trematode helminth; Fasciola hepatica. the in-fection is particularly prevalent in herbivorous animals, but accidentally can occur in humans as it feeds on spontaneous vegetation, usually riparian, contaminated with the larvae of the parasite; the infective stage. the infection de-velops in two stages, one caused by larval mi-gration through the peritoneum and the other by the adult worms located in intrahepatic bile ducts.

The most common symptoms are: Loeffler’s syndrome, abdominal pain some-ti mes simulating an acute abdomen, acute angiocholitis, pain in the liver lobe, icteric phenomena. [4, 6]

Peripheral eosinophilia can be 40-60% and is reached in the larvae migration phase through the peritoneum, in the first 5-6 weeks after infection. the maximum value can reach 80%. evolution of peripheral eosinophilia re-spects the Lavier curve. a particular feature is that eosinophilia, although decreasing, is maintained at high levels and after the parasite becomes cavitary in intrahepatic bile ducts. [5]

The diagnosis of certainty is achieved by repeated coproparasitological examinations with the emphasis of the characteristic opercu-lum eggs. tests are positive over two months after infection. the appearance of the eggs in the faeces does not coincide with the installa-


194

tion of the peripheral eosinophilia, the latter preceding the positivity of the examinations. so, although eosinophilia and some epide-miological data might suggest fasciolosis, co-proparasitological examinations may be nega-tive for a period of time.

Lymphatic filariosisthese are tropical haemolymphatic

infections produced by nematodes with vec-to rial transmission: Wuchereria bancrofti, W. bancrofti var. Pacifica and Brugia malayi. the infective stage is “microfilaria” transmitted by the stings of several genera and species of tropical mosquitoes.

The most common symptoms are varied and target lymphatic and blood vessels. the lymphatic vessels and superficial lymph nodes are affected first and then the deep ones. The gradual narrowing of lumen of large lymph vessels leads to their closure, chronic disease and the appearance of “elephantiasis”. De-pending on the genera and species of filarial, elephantiasis affects the legs, genitalia, upper limbs, breasts. frequently, lymphatic vessels fistulate with lymph drainage in cavity organs. a special form of pathology is the so-called “Tropical Lung Eosinophilia Syndrome” [4, 6].

Peripheral eosinophilia varies between 30-50%, but can also reach 80% in the case of tropical Lung eosinophilia syndrome, all amid a pronounced leukocytosis and increased ige. in reactive processes at the level of lym-phatic vessels and adjacent tissues, eosinophils prevail [5].

Diagnosis of certainty is made by high-lighting microfilaries on Giemsa stained pe-ripheral blood stain thin and thick smear. the frequency of larvae is taken into account. serol-ogy can also be done.

Cutaneous dermal filariosisthere are tropical infections with vecto-

rial transmission. etiologic agents are oncho-cerca volvulus (transmitted by simulium dip-ter stings), loa loa (transmitted by the Crysops dipter) Dracunculus medinensis transmitted via drinking water contaminated with microfilaria infected small crustaceans.

Clinical manifestations include: derma-titis (prurigo, depigmentation, lichenal tear, lymphatic sorts), edema (calabar), allergic phenomena, subcutaneous nodules, ocular le-sions (keratite, iridocyclite, retinitis) [4, 6]

Peripheral eosinophilia can reach values of 30-60%, especially for people coming for a short time from areas without filariosis in en-demic areas. it remains at high levels all the time in infections with onchocerca and loa.

in dracunculosis, the values are lower, ap-proximately 10% and kept at this value only the first year of infection [5].

The diagnosis of certainty is made by highlighting microfilariae on Giemsa stained peripheral blood stain thin and thick smear, the eLisa serological tests, the skin biopsy, possibly the Mazzotti test.

Hydatidosishydatidosis is the larval stage infection of

Echinococcus granulosus, a small parasite in the dog’s intestine. the infection is cosmopolitan, but it is more common in breeding colleges with animals accompanied by dogs. acciden-tally, man infects with parasite eggs eliminated by faeces of the infected dog. the infection is by the digestive tract, and the larval stage; hy-datid cyst is usually located in the liver, lung, brain, and less commonly in the other organs.

Clinical manifestations depend on the af-fected organ, the hydatid cyst exerting a com-pression that causes alteration of the functions. in case of cyst cracking, allergic manifestations appear to anaphylactic shock and in the longer term secondary sowing can occur [4, 6].

Peripheral eosinophilia does not occur or if it occurs has low values in the situation where the hydatid cyst is intact. in the case of cracked or broken cysts, the eosinophilia val-ues may be very high [5].

Diagnosis is usually imaging in corrobora-tion with serology.

Enterobiosisit is an infection commonly seen in chil-

dren especially in the community and is pro-duced by a small nematode called enterobius vermicularis. Localization of the parasite is the large intestine, the area of the cecum and the

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vermicular appendix. the female deposits eggs mainly in the anal mucous membranes causing intense pruritus. originally, non-embryonic eggs quickly form the embryo in the presence of oxygen becoming infectious. autoinfections are common.

Clinical manifestations are mild and largely due to pruritus. they consist of insom-nia, agitation, nervousness, lack of concentra-tion. Rare complications like vulvovaginitis [4, 6].

Peripheral eosinophilia is mild, the para-site being cavitary. in the case of strong infec-tions and when some parasites penetrate into the mucosal thickness, eosinophilia can reach 15% [5].

Diagnosis is done through a coproparasi-tological examination and especially by “anal print” or NIH, with emphasis on characteristic eggs.

Schistosomiasistropical parasitosis is produced by sever-

al species of the genus schistosoma: s. mansoni, s. japonicum, s. intercalatum (produce intestinal schistosomiasis) and schistosoma haematobium (uro-vesicular schistosomiasis). the biological cycle involves embryogenesis in water in the presence of intermediate hosts. the infection is caused by active skin patching by the infected stage “furco cercariae”. People who wash or work in contaminated water are affected.

Clinical manifestations are severe and in-clude: dysentery, hemorrhage, anemia, fibro-sis, necrosis and hepatic failure, hepatospleno-megaly, cachexia, esophageal varices, hema-turia, kidney failure, granulomas and polyps in the bladder and intestinal mucosa, severe cough and respiratory cough [4, 6].

Peripheral eosinophilia is on average 30%, higher in pulmonary affections; These values are reached around the 60th day from infection in urinary forms, but earlier in digestive tract [5].

Diagnosis of certainty is made by pointing out the eggs in the faeces, usually after 35 days of infection or 60 to 70 days in the urine. recto-

scopy, cystoscopy, biopsy of rectal or bladder mucosa, serology.

StrongyloidiasisWidespread cosmopolitan infection. Like

all geohelminth and strongyloidiasis infec-tions, it is common where there is no running water, sewerage, in conjunction with a poor health education that leads to massive contam-ination of soil with faeces.

the parasite, Strongyloides stercoralis, is lo-cated in the small intestine.

Clinical manifestations are most often di-gestive and depend on the intensity of the in-fection. the parasite is not hematophagous, so there is no anemia. at the skin level, pruritic, urticaria eruptions susceptible to over-infec-tions and irregular tracts produced by trans-cutaneous infective strongyloides larvae may occur. transient pulmonary symptoms of the Loeffler syndrome can occur due to perienteric migration. Frequent autoinfection occurs [4, 6].

Peripheral eosinophilia. since all stages of the parasite are tissue and due to frequent au-toinfection, we expect a high peripheral eosino-philia. on average, it can often exceed 25%, but there are many cases where it can reach 90%. But it is fluctuating due to repeated autoinfec-tions. maximum values are recorded approx-imately 40 days after infection, namely when rhabditoid larvae appear in the faeces [5].

Diagnosis of certainty is obtained by rec-ognizing rhabditoid larvae in the faeces by di-rect exams or coprocultures on charcoal.

Teniasisthere are infections with adult stage of

taenia solium and T. saginata. infections spread in all geographical areas to pork or beef con-sumers in the absence of rigorous veterinary control. the infection is by larval stage; Cyst-icercus cellulosae/bovis of not adequately heat treated pork/beef. Localization is the small in-testine, and digestive symptoms are mild, un-specific or absent [4, 6].

Peripheral eosinophilia is low, on average 7-10%, not respecting the Lavier curve. max-imum eosinophilia can reach 15-20% and is


196

recorded around when proglots or eggs (diag-nostic elements) appear in the faeces, i.e. 8-10 weeks after the infection [5].

TrichinosisIn the temperate zone the infection is usu-

ally produced by the adult and larval stage of the trichinella spiralis nematode. the infective stage is the parasite larvae in the pork. in oth-er geographical areas, the disease is produced by other parasite species located in other hosts, but these cases are less prevalent.

Symptomatology is varied as forms of manifestation and intensity and is dependent on the extent of infection. there may be: fever, allergic skin and respiratory phenomena, pru-ritus, facial edema, glottis, headache, myalgia, heart attacks [4, 6].

Peripheral eosinophilia appears quite late after the onset of symptoms, has an average of 15-20%, but in severe infections it can reach 90%. follows the Lavier curve. maximum eo-sinophilia is recorded in weeks 2 to 4 after in-fection under the conditions of larvae localiza-tion, so much later to serological positivity and to the larva circulation phase which is clinically most dangerous [5].

Diagnosis can be supported by serology and epidemiological investigation, the disease being an outbreak.

Trichuriasisthe infection is caused by the trichuris

trichiura nematode. Like ascaridiosis, tricho-cephalosis is a cosmopolitan infection, wide-spread in the absence of current water, sewer-age, soil faecal contamination, and poor food hygiene. it is very widespread in tropical areas.

the digestive symptomatology is unspecif-ic. in mild infections it may be missing. some-times, a mild anemia can occur in massive in-fections [4, 6].

Peripheral eosinophilia is moderate, usu-ally in strong infections and reaches peak val-ues approximately ten days after infection [5].

Diagnosis is confirmed by repeated co-proparasitological examinations with the

emphasis on characteristic eggs. exams are positive, however, after about one month of infection, and later than the time of recording of increased eosinophilia. so although eosino-philia could signal a parasitic infection, the co-proparasitological exam might be negative for a while.

Toxocariasis (M. V. L. S.)toxocariasis or migrans visceral Larva

syndrome is an accidental digestive infection with embryonated eggs on the soil belonging to Toxocara canis nematodes, of dogs and cats. Larvae begin their perietheric cycle, but go into the so-called “parasitic deadlock.” The body’s reaction kills the larvae and in time they are re-moved.

Clinical manifestations are the ones asso-ciated with Loeffler Syndrome. The liver may be enlarged and allergic manifestations may occur. cases of ocular toxocariasis, usually uni-lateral, are reported less frequently [4, 6].

Peripheral eosinophilia has high values, but usually fluctuating without respecting Lavier’s curve.

the maximum values may exceed 60-70% and persist after the treatment is established, as the destroyed larvae are resorbed, the tissue antigens persisting for a long time. it is worth mentioning that an o cular toxocariasis as the only localization of the infection does not signif-icantly change the number of eosinophils. eo-sinophilia in toxocariasis is also accompanied by igm, igg and ige hypergammaglobuline-mia [5].

Diagnosis of an active toxocariasis is given by a positive serology for igm and igg asso-ciated with high eosinophilia. typically, toxo-cariasis releases the investigations in its active phase, the presence of the parasites being de-tected by long-standing igg antibodies.

Migrans Cutanata Larva Syndromeit is a cutaneous and temporary parasitic

infection caused by worms of some species of ancylostoma parasite in animals. accidentally, the infective strongyloides larvae transcutane-ously enter humans.

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Clinical manifestations. at the place of penetration, larvae cause the formation of a pruritic, indurated and red papule. from this point appears a red, serpentine line a few cen-timeters. this line corresponds to a tunnel in the thickness of the skin by the moving larvae. the area is pruritus, especially at night. the infection can affect any part of the body that comes into contact with the contaminated soil and can last for a few weeks, sometimes even a year [4, 6].

Peripheral and local eosinophilia are significantly increased [5].

Conflict of Interests: No conflict of interests to declare.

REFERENCES1. Eosinophil - Definition of eosinophil in English

by oxford dictionaries - english. retrieved 2018.

2. What is an Eosinophil? Definition & Function cced. www.cincinnatichildrens.org retrieved 2018.

3. Dan Steriu. Eozinofilia in infecțiile parazitare, RoEqualm, Simpozionul cu tema “Asigurarea calității rezultatelor examinărilor în laboratorul medical” 27-28 Octombrie 2018 (In Romanian).

4. Lab.diag. infec. dis. princip. and pract. balows, Hausler, Lennette (1988).

5. Dan Steriu. Infectii parazitare, (2003), Edit. brilliant.

6. topley & Wilson’s microbiology and microbial infections (1998) v 5 ninth ed. arnold.


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Journals:Hallal AH, Amortegui JD, Jeroukhimov IM,

Casillas J, Schulman CI, Manning RJ, et al. Magnetic resonance cholangiopancreatography accurately detects common bile duct stones in resolving gallstone pancreatitis. J Am Coll Surg. 2005;200(6):869-75.

Books:carlson bm. human embryology and develop-

mental biology. 4th ed. st. Louis: mosby; 2009. 541 p.Book chapters:blaxter ps, farnsworth tp. social health and class

inequalities. In: Carter C, Peel JR, editors. Equalities and inequalities in health. 2nd ed. London: academic press; 1976. p. 165-78.

Web pages:Diabetes Australia. Diabetes globally [Internet].

Canberra ACT: Diabetes Australia; 2012 [updated

Guidelines for authors

203

Guidelines for authors

2012 June 15; cited 2012 Nov 5]. Available from: http://www.diabetesaustralia.com.au/en/ Understanding-diabetes/diabetesglobally/

10. Tables with suitable captions at the top and numbered with arabic numerals should be collected at the end of the text on separate sheets (one page per table). each column should be given a short or an abbreviated heading. footnotes to tables should be marked with a) b) c) etc and *, **, *** should be reserved for p values. each table must be understood independently of the text. all tables must be cited in the text.

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