Title

TGF-β plasmatic levels in chromoblastomycosis patients receiving itraconazole

treatment

Authors.

Jorge Pereira da Silva1,2,5, Moisés Batista da Silva1, Ubirajara Imbiriba Salgado1,3, José

Antonio Picanço Diniz4, Sonia Rozental6 and Claudio Guedes Salgado1,5.

Affiliation:

1-Laboratório de Dermato-Imunologia Universidade do Estado do Pará (UEPA),

Universidade Federal do Pará (UFPA) and Unidade de Referência em Dermatologia

Sanitária do Estado do Pará “Dr. Marcello Candia” (MC); 2-Departamento de Farmácia,

UFPA; 3- Serviço de Dermatologia, UEPA; 4-Laboratório de Microscopia Eletrônica do

Instituto Evandro Chagas; 5- Departamento de Patologia, UFPA; 6-Laboratório de Biologia

Celular de Fungos/Universidade Federal do Rio de Janeiro (UFRJ);

Correspondence:

Dr. Claudio Guedes Salgado, MD, PhD. Laboratório de Dermato-Imunologia

UEPA/UFPA/MC. Av. João Paulo II, 113, Bairro Dom Aristides, 67200-000. Marituba,

Pará, Brasil. Tel/Fax: 55-91-3256-9097. E-mail: csalgado@ufpa.br

1

Summary Background. Chromoblastomycosis is a granulomatous mycosis of the skin and

subcutaneous tissues, normally described in rural workers from tropical and subtropical

regions. The disease evolves to a chronic state with the lesions increasing slowly and

progressively. The histological feature is characterized by a suppurative granulomatous

dermatitis, combined with variable dermal fibrosis in skin. The host defense mechanisms,

granuloma morphogenesis, and cytokines engaged in fibrosis formation, pathogenesis of

the inflammation and tissue repair in chromoblastomycosis are poorly understood.

Aim. The purpose of the present study was to quantify Transforming Growth Factor-β

(TGF-β) serum levels of chromoblastomycosis patients before, during and after

itraconazole (ITZ) treatment.

Methods. Blood serum of 12 chromoblastomycosis patients was subjected to TGF-β

titration with ELISA at 0, 3, 6 and 12 months of 200mg per day of ITZ therapy, and

correlated with the clinical aspects of the lesions in each period analyzed. Serum of 12

healthy individuals were used for control.

Results. Chromoblastomycosis patients present high serum levels of TGF-β (7.016 ± 1988

pg/ml), decreasing after 03 months (4.625 ± 645 pg/ml; p < 0.005) of ITZ treatment, which

correlates with a rapid clinical improvement, specially associated to the dryness and

thinning of the lesions. However, after 6 (6.566 ± 777 pg/ml; p < 0.001) and 12 months

(6.908 ± 776; p < 0.001) of treatment, TGF-β levels increase, returning to almost the same

levels observed before treatment, which is related to a slow clinical improvement, fungal

persistence on the lesion, and fibrotic scars.

2

Conclusion. TGF-β serum levels are high in chromoblastomycosis patients. Fungal

destruction by ITZ correlates with TGF-β downregulation, but tissue remodeling probably

raises its levels again, interfering with cellular immune responses, facilitating fungal

survival in the tissue, and inducing fibrosis for tissue scar formation, which could be related

to the slow clinical improvement observed after the third month of treatment.

3

Introduction

Chromoblatomycosis is a chronic, suppurative, granulomatous mycosis of the skin and

subcutaneous tissues, which usually shows no major tendency to disseminate to deeper

organs [1], and usually presents with verrucose lesions, which can be localized or

cutaneously disseminated [2]. It occurs worldwide, but it is more frequently observed in

tropical and sub-tropical countries [3]. The infection frequently occurs after a traumatic skin

puncture, with percutaneous implantation of hyphae or conidia [4-6].

Inside the host, infectious propagules, conidia or fragments of hyphae adhere to

subcutaneous tissues and differentiate into sclerotic cells, a specific intermediate stage of

several species of dematiaceous fungi, which have characteristic septations on more than

one plane [7]. Common species isolated from chromoblastomycosis lesions include

Fonsecaea pedrosoi, Cladophialophora carrionii, Phialophora verrucosa, Rhinocladiella

aquaspersa, Wangiella dermatitidis, Exophiala jeanselmei and Exophiala spinifera [8]. The

diagnosis can be performed by detection of sclerotic cells in KOH preparations or with

hematoxylin and eosin staining [9]. Histopathology is characterized by a fibrotic reaction,

usually extended from the superficial to the lower dermis, obliterating skin adnexa, and, in

a few cases, predominating over the active granulomatous inflammatory reaction [10].

Treatment of chromoblastomycosis is difficult; and response to oral antimycotic drugs is

limited, but can be managed by different methods. Itraconazole (ITZ) or terbinafine has

been used as single-agent therapy [11] and, for resistant cases, in combination with

flucytosine [12] or with cryotherapy, shaving and oral amphotericin B [13]. ITZ is a

member of the azole antifungal agents, which contains three nitrogen molecules in the azole

ring. It is fungistatic in vitro, and blocks the cytochrome p450-dependent enzyme

4

lanosterase demethylase (14-α-sterol demethylase), which is essential for the synthesis of

ergosterol, a critical component of fungal membranes [14].

Mechanisms of immunity in chromoblastomycosis, including humoral and cell mediated

responses, are not well understood. Some studies have demonstrated the importance of cell-

mediated immune response in the fungus-host interaction, showing a predominant cellular

response and observing that fungal persistence in situ is the principal factor responsible for

the morbidity of this subcutaneous mycosis [15].

Besides its inhibitory effects on macrophages, eosinophils, and T and B lymphocyte

populations [16], Transforming Growth Factor-β (TGF-β) is an important regulator of cell

proliferation, differentiation and formulation of the extracellular matrix [17]. It plays a role

in regulating the extracellular matrix by decreasing degradation of matrix proteins through

a reduction in protease synthesis and an increase in the synthesis of protease inhibitors [18],

participating in granuloma formation and fibrosis [19], which has been correlated with the

presence of TGF-β [20].

In many diseases, excessive expression of TGF-β contributes to a pathophysiological

fibrosis [21]. The tissue-repair process involves two distinct stages: a regenerative phase, in

which injured cells are replaced by cells of the same type, leaving no lasting evidence of

damage; and a phase known as fibroplasia or fibrotic scarring, in which connective tissue

replaces normal parenchymal tissue. Macrophages and fibroblasts operate as key effector

cells in the pathogenesis of fibrosis. However, fibrosis is not always linked with robust

inflammation, indicating that the mechanisms regulating fibrogenesis and inflammation are

distinct [22]. The participation of TGF -β and other cytokines in the fibrotic pathogenesis

or tissue-repair process in chromoblastomycosis is not still completely defined. Some

5

research has demonstrated that the chronic granulomatous inflammatory reaction occurs by

extensive and progressive dermal fibrosis, and tissue-repair is linked to the presence of

TGF-β in situ, as well as that the irreversible fibrotic process can be associated with

important tissue levels of TGF-β and at the presence of lysyl oxidase and transglutaminase

enzymes, involved with mature collagen cross-linking [23;24].

In the present study, our objective was to quantify TGF-β by ELISA in the blood serum of

patients with chromoblastomycosis, and correlate plasmatic levels of this cytokine with

clinical improvement after different duration periods of ITZ therapy.

6

Patients and Methods

The study was conducted with 11 male and 01 female chromoblastomycosis patients, with a

mean age of 46.5 years (SD: 9.5 years old), attending the Dr Marcello Candia Reference

Unit in Sanitary Dermatology of The State of Pará (UREMC), in Marituba, Pará, Brazil,

from March, 2005 to April, 2006. Eleven male and one female healthy subjects, with a

mean age of 32.5 years old (SD: 7.5 years old), served as controls. The evolution period of

the disease varied from a minimum of 02 years to a maximum of 30 years (mean: 15.9; SD:

7.8 years).

From 21 patients of chromoblastomycosis, 12 who were attending UREMC regularly, once

a month, in the last 12 months were chosen to participate in the study. Twelve healthy

subjects who came for consultation at UREMC, but had no disease, were recruited to

participate in the study. All patients were treated with 200mg/day of itraconazole, and

clinical assessment and TGF-β levels in the serum were monitored for a period of 12

months. Blood serum was obtained from 5ml of venous blood collected from each patient

with chromoblastomycosis and from each control with 2 Vacutainer tubes (Becton-

Dickinson, USA) with anticoagulant. Informed consent was obtained for venupuncture and

the study was approved by the Health Sciences Center Ethical Committee of Pará Federal

University. Serum was divided into aliquots and stored at –20oC until use. TGF-β present in

the serum of patients and controls was quantified by ELISA and the results are reported as

pg/ml based on the standard curve established for each assay, according to the

manufacturer’s instructions. Each sample was tested in triplicate.

7

Statistical analysis

The ELISA results for different duration periods of treatment (0, 3, 6 and 12 months) were

compared to each other and to controls, and the paired Student’s t-test was used to evaluate

the results, where a p value less than 0.005 was considered statistically significant.

8

Results

To confirm chromoblastomycosis, direct microscopy examination of skin scrapings

clarified by 20% KOH was performed, presenting the characteristic pathognomonic

sclerotic cells (Figure 1A). All specimens were cultured on Mycosel-agar (Becton-

Dickinson, USA), where colonies of black or dark green fungus, with a velvet surface grew

after 3 to 4 weeks of culture (Figure 1B). After microculture, dematiaceous hyphae, with

cylindrical, intercalary or terminal conidiophores, giving rise to small conidia characteristic

of F. pedrosoi were observed (Figure 1C).

All patients were clinically examined, presenting well-delimitated, wet or dry, nodular or

verrucose lesions, sometimes confluent, forming large plaques in the skin of one of the

limbs (Figure 1D, G), varying from 3 to 20 cm in diameter. The evolution period for the

disease varied from 2 to 30 years (mean: 15.9; SD: 7.8 years).

Treatment was done with ITZ at 200mg/day for at least 12 months. Hemogram, glycemia,

hepatic and renal enzyme examinations were performed at 0, 6 and 12 months, and no

significant alterations were found. After 3 months, an important improvement in skin

lesions, with dryness, thinning and softening of nodular or verrucose lesions was observed

(Figure 1E, H). Nonetheless, after the first three months lesions improved, but the progress

was very slow, resulting in the presence of active lesions at the end of twelve months of

ITZ treatment (Figure 1F, I)

TGF-β was present in the blood serum of chromoblastomycosis patients (7.016 ± 1.988

pg/ml) when compared to healthy controls (3.791 ± 209 pg/ml; p < 0.005). The serum levels

decreased to nearly the same levels of control subjects in the first 3 months of ITZ

treatment (4.625 ± 645 pg/ml; p < 0.005). However, after 6 months of treatment, TGF-β

serum levels returned to the almost the same titration found before treatment (6.566 ± 777

9

pg/ml; p < 0.001), which was maintained until 12 months of treatment (6.908 ± 776 pg/ml;

p < 0.001) (Figure 2). The presence of circulating TGF-β in healthy controls can be

explained by the release of this cytokine from platelets activated by EDTA, after

venupuncture [25].

10

Discussion

TGF-β upregulation has been implicated in hypertrophic scar formation, but it is unclear if

it is the cause or an effect of this process [26]. In chromoblastomycosis, one paper has

demonstrated the profile of the extracellular matrix with irreversible fibrosis in the lesion,

associated to a dense inflammatory infiltrate, sometimes organized in granulomas, and the

presence in situ of TGF-β [15], but the presence or absence, and the correlation between

plasmatic levels of TGF-β and chromoblastomycosis evolution is unknown.

The interactions between different cellular and extracellular host components and F.

pedrosoi sclerotic cells present in tissue are complex and might be important in the control

of dissemination of chromoblastomycosis. Our results suggest an active role for TGF-β in

the pattern of tissue response, interfering with the type and size of the lesion. At first, TGF-

β levels are high, indicating a possible role for the fungus in stimulating the production of

this cytokine by inflammatory cells, and also that TGF-β might be important in the chronic

course of chromoblastomycosis. Different infection processes such as leishmaniasis [27]

and Chagas disease [28] have detectable TGF-β in tissue, which indicates that this cytokine

is important in the maintenance of the disease, and corroborates our findings.

Thus, with ITZ therapy and destruction of the fungus, there is a TGF-β downregulation,

correlated with an important improvement in the clinical picture, followed by a new

increment in TGF-β blood serum levels, which can also be correlated to the slow clinical

improvement we observed. It is known that ribavirin treatment can reduce TGF-β plasmatic

levels, also reducing hepatic fibrosis in chronic hepatitis C patients [29] – similarly to what

we observed with ITZ and chromoblastomycosis lesions in the skin – and that progressive

bacterial regrowth occurs after the use of antimicrobial agents against bacteria of the

11

Mycobacterium avium complex associated with TGF-β persistence in tissue [30], which

could also be occurring in our study, as demonstrated by the high TGF-β levels after the

third month of treatment, associated to a slow clinical improvement, and the persistence of

sclerotic cells in the lesional skin.

Fungal persistence and the capacity of fungal multiplication as an exuberant inflammatory

response are related to the absence of development of a true granulomatous reaction,

probably as a consequence of factors that inhibited the Th1 response, with a predominance

of the Th2 response [31]. TGF-β secretion can also be stimulated by pathogens during

entry, replication, and persistence in the host [32]. These data can be related to the

important fibrosis observed in the clinic recovery of chromoblastomycosis patients.

Further studies are necessary to elucidate which inflammatory cells are responsible for

TGF-β production in chromoblastomycosis, and how the presence of the fungus stimulates

its secretion, since it is known that anti-TGF-β antibodies can enhance the healing process

in other infections [33], and could also be important for improving the treatment of

chromoblastomycosis patients.

12

Acknowledgments

This work was supported by Fundo de Ciência e Tecnologia do Estado do Pará (FUNTEC),

by Programa de Apoio à Pesquisa da Universidade do Estado do Pará, by Secretaria

Executiva de Saúde Pública (SESPA), by Secretaria de Ciência e Tecnologia, Ministério da

Saúde do Brasil and by Programa Nacional de Cooperação Acadêmica

(PROCAD/CAPES).

13

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Figure Legends

Figure 1. Macroscopic and microscopic characteristics of Fonsecaea pedrosoi and

clinical aspects of chromoblastomycosis. Direct microscopic examination after 20% KOH

clarification, showing sclerotic cells (A). Mycosel-agar culture of sclerotic cells results in

growth of black colonies, with a velvet surface (B). Microculture of these colonies shows

dematiaceous hyphae, with classic cladosporium frutification, characteristic of F. pedrosoi

(C). Clinical examination reveals classic verrucose, wet (D) or dry, hypertrofic (G), well-

delimitated lesions. After 3 months of itraconazol (ITZ) treatment, thinning (E, H) and

dryness (E) are observed. After 12 months of ITZ treatment, a verrucose lesion still can be

observed in one case (F), while an atrophic, acromic, cicatricial plaque with a very tiny,

small (1.0cm), verrucose area in the center of the lesion is seen in another patient (I). In

both cases, sclerotic cells were found. Bars: A: 15µm; B: 2.5cm; C: 20µm; D-I: 2.0cm.

Figure 2. Sequential plasma TGF-β assessment in itraconazole treated

chromoblastomycosis patients. Before treatment (month 0), plasma TGF-β levels were

significantly higher than in healthy subjects (control). After 3 months, mean levels of TGF-

β decreased to almost the same amounts as those of healthy subjects, rising again in the

next 6 and 12 months of treatment, to almost the same titration observed before treatment.

P-values are in comparison with different periods of treatment and healthy subjects

(control), according to the lines drawn.

19