Bcl-2 expression is moderately correlated with long-term variability of CD4 T-cell increase under...

Correspondence

AIDS 2003, 17:133–143

Decreased expression of activation markers on CD4 T lymphocytes of HIV-infected long-term non-progressors

Recent cross-sectional studies have reported strongCD4 T helper type 1 cell responses to viruses in HIV-infected long-term non progressors (LTNP) [1,2]. Ithas been suggested that the loss of CD4 Th1 cellfunction in HIV-infected individuals is related to aprocess of the activation-induced death of CD4 Th1cells [3]. In a cohort of LTNP, we have focused on thecharacterization of activated CD4 and CD8 T-cellsubsets that have been reported to have independentprognostic value for progression to AIDS [4,5]. In a 6-year prospective study, we compared the baseline levelsof T-cell subsets in LTNP and controls when enrolledinto the study. We studied six HIV LTNP (mean ageat diagnosis of HIV infection 24 years; standard error ofmean, SE 1.5) with the following criteria: asympto-matic status, at least 8 years of documented HIVinfection, CD4 T-cell number over 500 cells 3 106/lfrom the detection of HIV infection (baseline levelswhen enrolled into the study and latest CD4 T-cellcount values at the end of follow-up: 1152, SE 160 and1079, SE 78 cells 3 106/l, respectively, P ¼ 0.29) andan absence of previous antiretroviral therapy. Thedisease control group consisted of 10 asymptomaticHIV patients (Centers of Disease Control and Preven-tion category A1, mean age 23 years, SE 1.2) with aprogressive course to more advanced stages of asympto-matic HIV infection, measured by a significant declinein CD4 T cells (baseline and latest CD4 T-cell countvalues: 646, SE 61 and 461, SE 28 cells 3 106/l,respectively, P ¼ 0.027). The mean time of HIVinfection at the time of the baseline immunologicalstudy was 5.3 years in asymptomatic progressors. LTNPand A1 progressors had a history of injecting drug use.Twenty-seven HIV-uninfected injecting drug users and39 HIV-negative age-matched healthy volunteers wereincluded as HIV-negative controls [6]. Baseline levelsof HIV RNA were 791, SE 712 copies/ml and 1293,SE 556 copies/ml in LTNP and progressors, respec-tively (P . 0.05). None of the A1 progressors werereceiving antiretroviral therapy at study entry, butseven started treatment during follow-up. ActivatedCD4 and CD8 T-cell subsets were quantitated usingthree-colour flow cytometry (FACScan, Becton andDickinson, San Jose, CA, USA). We enumerated T cellsubsets using fluorescein isothiocyanate/phycoerythrin/peridin chlorophyll protein combinations of CD38/HLA-DR/CD4 or CD8; CD38/CD45RO/CD4 orCD8; HLA-DR/CD45RO/CD4 or CD8; CD3 fluor-escein isothiocyanate/CD4 peridin chlorophyll protein

or CD8; CD45/CD14 and isotype controls. Theplasma HIV-RNA level was measured using reversetranscriptase polymerase chain reaction (UltrasensitiveAmplicor HIV Test, Roche Molecular Systems,Branchburg, NJ, USA). Comparisons were made usingthe Mann–Whitney test.

Table 1 shows the percentages of activated CD4 andCD8 T-cell subsets in LTNP, asymptomatic progressorsand uninfected controls at the baseline study. In com-parison with the A1 group of patients, LTNP showeddecreased percentages of the activated T CD4 sub-sets CD4+CD45RO+CD38+, CD4+CD45RO+DR+and CD4+CD38+DR+. The CD8 cell activationantigen profile of LTNP showed a decrease in theCD8+CD45RO+CD38+ and CD8+CD38+ subsetsand an increase in the CD8+CD38-DR+ subset com-pared with A1 progressors. In comparison with unin-fected controls, LTNP demonstrated elevated levelsof CD8+CD45RO+CD38+, CD8+CD45RO+DR+,CD8+CD38+ and CD8+CD38-DR+ T cells. Withinthe subsets of CD4 T cells there were no significantdifferences in the proportions of activated T lympho-cytes between the group of LTNP and the HIV-negative controls.

We have previously described our findings that in-creased levels of CD4+CD38+DR+ T cells is anindependent predictor of progression to AIDS [4].Decreased levels of CD4+CD38+DR+ and CD8+CD38+ T cells, used as cellular prognostic factors,were observed in LTNP compared with asymptomaticprogressors. A diminished CD4 T-cell activation mightbe associated with the reduced rates of spontaneousapoptosis that have been described in lymphocytes fromLTNP [7]. LTNP had higher levels of activated CD8T-cell subsets than HIV-negative controls. A numberof studies have documented increased CD8 cytotoxicT lymphocyte activity in asymptomatic HIV-seroposi-tive individuals and in particular in LTNP, whichcould be part of the host antiviral immune response instable non-progressive HIV infection [8]. Anti-HIV-directed cytotoxic T lymphocytes have been foundamong CD8 T cells that express CD38 and HLA-DR[9]. This represents a paradox as CD8 T-cell activation,measured as elevated levels of CD8+CD38+ T cells,predicts more rapid disease progression [5]. In compari-son to asymptomatic progressors and HIV-negativecontrols, LTNP had higher levels of CD8+CD38-

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ISSN 0269-9370 & 2003 Lippincott Williams & Wilkins 133

DR+ T cells, a subset described as a marker of subse-quent stable HIV disease [10]. Although in a smallnumber of patients, these results suggest that immuneactivation changes (in particular, decreased levels ofactivated CD4 T cells) might contribute to the estab-lishment of the stable clinical and immunologicalcourse of the disease in LTNP.

Javier Carbone, Juana Gil, Jose Miguel Benito andEduardo Fernandez-Cruz, Department of Immunology,University Hospital Gregorio Maranon, Madrid, Spain

Sponsorship: This study was supported by grants from theComunidad Autonoma de Madrid and the Fondo deInvestigacion Sanitaria to E.F-C.

Received: 28 June 2002; accepted: 6 August 2002.

DOI: 10.1097/01.aids.0000042932.55529.59

References

1. Alatrakchi N, Di Martino V, Thibault V, Autran B. Strong CD4Th1 responses to HIV and hepatitis C virus in HIV-infected long-term non-progressors co-infected with hepatitis C virus. AIDS2002; 16:713–717.

2. Valdez H, Carlson NL, Post AB, Asaad R, Heeger PS, LedermanMM, et al. HIV long-term non-progressors maintain brisk CD8T cell responses to other viral antigens. AIDS 2002; 16:1113–1118.

3. Estaquier J, Idziorek T, Zou W, Emilie D, Farber CM, Bourez JM,et al. T helper type 1/T helper type 2 cytokines and T cell death:preventive effect of interleukin 12 on activation-induced andCD95 (FAS/APO-1)-mediated apoptosis of CD4+ T cells fromhuman immunodeficiency virus-infected persons. J Exp Med1995; 182:1759–1767.

4. Carbone J, Gil J, Benito JM, Navarro J, Munoz-F A, Bartolome J,et al. Increased levels of activated subsets of CD4+ T cells addto the prognostic value of low CD4+ T cell counts in a cohort ofHIV-infected drug users. AIDS 2000; 14:2823–2829.

5. Liu Z, Cumberland WG, Hultin LE, Kaplan AH, Detels R, GiorgiJV. Lymphocyte activation in HIV-1 disease reflects an aspect ofpathogenesis distinct from viral burden and immunodeficiency.J Acquir Immune Defic Syndr Hum Retrovirol 1998; 18:332–340.

6. Benito JM, Zabay JM, Gil J, Bermejo M, Escudero A, Sanchez E,et al. Quantitative alterations of the functionally distinct subsetsof CD4 and CD8 T lymphocytes in asymptomatic HIV infection:changes in the expression of CD45RO, CD45RA, CD11b, CD38,HLA-DR, and CD25 antigens. J Acquir Immune Defic Syndr HumRetrovirol 1997; 14:128–135.

7. Liegler TJ, Yonemoto W, Elbeik T, Vittinghoff E, Buchbinder SP,Greene WC. Diminished spontaneous apoptosis in lymphocytesfrom human immunodeficiency virus-infected long-term nonpro-gressors. J Infect Dis 1998; 178:669–679.

8. Harrer T, Harrer E, Kalams SA, Elbeik T, Staprans SI, FeinbergMB, et al. Strong cytotoxic T cell and weak neutralizingantibody responses in a subset of persons with stable nonpro-gressing HIV type 1 infection. AIDS Res Hum Retroviruses1996; 12:585–592.

9. Ho HN, Hultin LE, Mitsuyasu RT, Matud SL, Hausner MA,Bockstoce D, et al. Circulating HIV-specific CD8+ cytotoxic Tcells express CD38 and HLA-DR antigens. J Immunol 1993;150:3070–3079.

10. Giorgi J, Ho HN, Hirji K, Chou CH, Hultin LE, O’Rourke SO,et al. CD8+ lymphocyte activation at human immunodeficiencyvirus type 1 seroconversion: development of HLA-DR+CD38-CD8+ cells is associated with subsequent stable CD4+ celllevels. J Infect Dis 1994; 170:775–781.

Cytokines and AIDS dementia complex

In their article entitled ‘Granulocyte–macrophage col-ony-stimulating factor enhances viral load in humanbrain tissue: amelioration with stavudine’ published inFebruary 2002, Kandanearatchi et al. [1] recently re-ported on the role of granulocyte–macrophage colony-stimulating factor in inducing both HIV replication and

dementia expression. As reported by these authors, in1992 our previous study [2] suggested an importantpathogenetic role for the above growth factor and IL-6 indetermining the development of HIV-related dementia.

Our research showed an increase of these T helper


Table 1. Percentages of CD4 and CD8 T-cell subsets in HIV-positive long-term non-progressors, HIV-positive asymptomatic progressors and uninfected controls.

Lymphocyte subsetsa LTNP (n ¼ 6)b

CDC A1progressors(n ¼ 10)b

HIV-negativeIDU controls(n ¼ 27)b

HIV-negativehealthy controls

(n ¼ 39)b

CD4+CD45RO+DR+ 8 � 1c 13 � 1d,e 6 � 1 6 � 3CD4+CD45RO+CD38+ 17 � 3c 30 � 2d,e 13 � 1 17 � 2CD4+CD38+DR+ 5 � 1c 10 � 1d,e 2 � 0.2 4 � 1CD8+CD45RO+DR+ 31 � 3d,e 36 � 5d,e 10 � 1 10 � 1CD8+CD45RO+CD38+ 16 � 3c,d,e 31 � 4d,e 5 � 3 7 � 1CD8+CD38+ 44 � 5c,d,e 62 � 3d,e 28 � 3 33 � 3CD8+CD38+DR+ 30 � 5d,e 35 � 5d,e 7 � 2 6 � 1CD8+CD38�DR+ 20 � 2c,d,e 10 � 1d,e 10 � 1 10 � 1

CDC, Centers for Disease Control and Prevention; IDU, injection drug users; LTNP, long-term non-progressors.aLymphocyte subsets expressed as percentage of total CD4 or CD8 T cells.bMean values � SE.For comparisons with progressors cP , 0.05; with HIV-negative IDU dP, 0.05; with HIV-negative healthycontrols eP , 0.05.

AIDS 2003, Vol 17 No 1134

(Th) type 1-like cytokines only in the cerebrospinalfluid (CSF), suggesting an intrathecal synthesis frommacrophages. By contrast, most AIDS dementia com-plex (ADC) patients did not have detectable IL-6 andgranulocyte–macrophage colony-stimulating factor ser-um levels. More recently, the role of HIV amplificationin the brain has also been demonstrated for othersubstances with a Th1-like activity such as monocytechemoattractant protein type 1 [3], therefore a possiblemodel of ADC pathogenesis can be determined by theinteraction between cytokines and the cerebral HIVload. A balance between cytokines with different in-vivo activity and the microenvironment in which theyare produced might be important in regulating theprocesses of cerebral inflammation. Recent advancessupport the hypothesis that interactions between neuro-resident cells such as macrophages/microglia and astro-cytes may play a critical role in determining theproduction of products with both harmful and bene-fical effects in the brain [4].

On this basis, our recent study [5] indicated that theproduction of different cytokines characterized theexpression of dementia. In that study we studied 24patients (10 women and 14 men) with a mean age of33 years (range 23–51), a mean duration of disease of 7years, affected by clinically defined ADC according tothe criteria of Price and Brew [6], including eitherbrain computed tomography scan or magnetic reso-nance imaging. Twelve patients (five women and sevenmen) were classified at the time by the CSF as at the0.5 stage (mild). The other twelve ADC patients wereat the 2 stage (severe). The median CD4 cell counts ofpatients in the mild stage was 293 � 143 3 106,whereas patients in the severe stage had CD4 cellcounts of 94 � 88 3 106. The CD8 cell count was594 � 311 3 106 and 604 � 369 3 106 in mild andsevere ADC, respectively. At the time of the study, allADC patients were treated with highly active antire-troviral therapy consisting of a combination of zidovu-dine, lamivudine and one protease inhibitor such asindinavir or ritonavir. The control group was charac-terized by 18 HIV-negative individuals (seven womenand 11 men) sex and age matched, with one of thefollowing other neurological diseases: degenerative diskproblem, ataxia or headache. In the CSF of all patientsand controls we evaluated: cell counts, glucose, protein,albumin, IgG index and microbiological analyses. Wealso evaluated the HIV-RNA load by Roche AmplicorMonitor (Roche, Milan, Italy) according to a standardprocedure with a detection limit of 500 copies/ml.Serum and CSF IFN-Æ and transforming growth factorbeta (TGF-�) levels were detected by two enzyme-linked immunosorbent assay kits (Valter Occhiena,Turin, Italy and Bender Medisystem, Vienna, Austria),respectively. The limit of sensitivity was 1 pg/ml. Theresults showed that CSF HIV RNA was higher in theadvanced ADC (log 5.3 copies/ml) than in the mild

ADC (log 3.8 copies/ml), with a statistically significantdifference (P , 0.001). In particular, IFN-Æ was de-tected in the CSF of ADC patients, both in the mildand severe stage, but not in controls, with a significantdifference between the mild and severe ADC (seeTable 1). Moreover, the patients with severe ADCshowed undetectable levels of TGF-�, whereas themild stage was characterized by higher TGF-� levels. Asignificant inverse correlation was also observed be-tween these two cytokines in the CSF (r ¼ �0.921;P , 0.001). On the basis of these data, IFN-Æ seems tobe related to a higher progression of ADC, andaccording to a previous report is correlated with thegreatest neurocognitive dysfunction [7].

In our study, TGF-� seemed to downregulate HIVproduction in the brain, and we suggest that it couldinduce an inhibitory effect on other pro-inflammatorycytokines such as IFN-Æ, regulating the brain inflam-matory processes and influencing the polarization of aTh2-like immune response [8]. In this regard, criticalissues in the pathogenesis of ADC could be: first, arelationship between dementia and a complex inter-action of cytokines with a differential modulation onHIV replication; second, the role of the environmenton individual immune responses during the course ofHIV disease, and the role of new potent antiretroviraldrugs in regulating the cerebral viral load. It is possiblethat a regulation of the viral load in the cerebralnervous system by new antiretroviral drugs can deter-mine different immune mechanisms associated with adynamic process in which ADC might have severalphenotype expressions.

In conclusion, such a scenario suggests a rationale forthe possible direct use of cytokines with Th2 activity asa therapeutic tool to block the cascade of events leadingto inflammation and macrophage activation.

Oreste Perrellaa, Alessandro Perrellab, Marco Perrellac ,Costanza Sbregliaa and Gugliemo Borgiab, aVIIthDepartment of Infectious Diseases and Immunology,Hospital D. Cotugno, Naples, Italy; and Departments of


Table 1. Cerebrospinal fluid and serum levels of transforminggrowth bactor beta 1 and IFN-Æ in patients with mild to severeAIDS dementia complex and controls.

TGF-� IFN-Æ

CSF Serum CSF Serum

Mild ADC 66 � 50� 14 � 3 30 � 46 1 � 1.2Severe ADC UD 15 � 2 176 � 71 1.5 � 1.6Controls UD 16 � 3 UD 1.1 � 1.3

�P, 0.001 with respect to patients with severe AIDS dementiacomplex (ADC).UD, Undetectable.The values are expressed as (mean � SD).

Correspondence 135

bInfectious Diseases and cClinical and ExperimentalMedicine, Federico II Medical School University, Naples,Italy.

Received: 18 April 2002; revised: 25 July 2002;accepted: 12 August 2002.

DOI: 10.1097/01.aids.0000042960.95433.4c

References

1. Kandanearatchi A, Zuckerman M, Smith M, Vyakarnam A, EverallIP. Granulocyte-macrophage colony-stimulating factor enhancesviral load in human brain tissue:amelioration with stavudine.AIDS 2002; 16:413–420.

2. Perrella O, Guerriero M, Izzo E, Soscia M, Carrieri PB. Inter-leukin-6 and granulocyte macrophage-CSF in the cerebrospinalfluid from HIV-infected subjects with involvement of the cere-

bral nervous system. Arq Neuropsiquiat 1992; 50:180–182.3. Cinque P, Vago L, Mangozzi M, Torri V, Ceresa D, Vicenzi E,

et al. Elevated cerebrospinal fluid levels of monocyte chemotac-tic protein-1 correlates with HIV-1 encephalitis and local viralreplication. AIDS 1999; 12:1327–1332.

4. Merrill JE. Pro-inflammatory and anti-inflammatory cytokines inmultiple sclerosis and central nervous system acquired immuno-deficiency syndrome [Review]. J Immunother 1992; 12:167–173.

5. Perrella O, Carrieri PB, Perrella A, Sbreglia C, Gorga F, Guarnac-cia D, Tarantino G. Transforming growth factor-beta 1 andinterpheron-alpha in the AIDS dementia complex (ADC): possi-ble relationship with cerebral viral load? Eur Cytokine Netw2001; 12:51–55.

6. Price RW, Brew BJ. The AIDS dementia complex [Review].J Infect Dis 1988; 158:1079–1084.

7. Rho MB, Wesselingh S, Glass JD, McArthur JC, Choi S, Griffin J,Tyor WR. A potential role for interferon-alpha in the pathogen-esis of HIV associated dementia. Brain Behav Immun 1995;9:366–372.

8. Fargeas C, Wu CY, Nakajima T, Cox D, Nutmann T, DelespesseG. Differential effect of transforming growth factor-� on thesynthesis of Th1 and Th2-like lymphokines by human T lympho-cytes. Eur J Immunol 1992; 22:2173–2176.

Lopinavir/ritonavir absorption in a gastrectomized patient

Combination antiretroviral therapy is the standard ofcare for patients with HIV infection [1,2]. Thesemultiple drug regimens are complex, have major side-effects, and their effectiveness is limited by thedevelopment of viral resistance either as a result ofnon-adherence to the drugs or suboptimal drug con-centrations in plasma [3]. As antiretroviral drugs areapproved for clinical use following an acceleratedprocess, information regarding the factors that have thepotential for influencing pharmacokinetic profiles arerather limited.

We report here the case of a 38-year-old female HIV-positive patient who was affected by high-grade non-Hodgkin’s lymphoma (Burkitt-like, according to theWorking Formulation Classification system), whichwas first detected in the stomach. The patient under-went total gastrectomy followed by an oesophago-jejunostomy with a Roux-en-y reconstruction. Fourmonths after surgical operation, the patient was ad-mitted to our wards for a systemic cytomegalovirusinfection, and started ganciclovir (200 mg) intravenousinfusion treatment. At the time of the clinical onset ofcytomegalovirus infection, her CD4 cell count andplasma HIV-RNA level were 37 cells/mm3 and283 000 copies/ml, respectively. The patient was naiveto antiretroviral agents; a potent drug combination wasinitiated to contain HIV replication and to restore theimmunological function. The drugs chosen were dida-nosine buffered tablets (as the absence of gastric aciditywould avoid drug degradation, administered 2 h apartfrom associated drugs [4]), lamivudine oral liquidformulation and lopinavir/ritonavir oral liquid formula-tion, which was subsequently changed to the capsulatedformulation (400/100 mg twice a day) at the patient’srequest.

As the absorption of orally delivered drugs depends onseveral factors such as gastrointestinal motility and pH[5], and based on the fact that our patient had under-gone gastrectomy in the recent past, therapeutic drugmonitoring was undertaken to see whether the drugabsorption was altered.

As lopinavir/ritonavir is not available in parenteralformulations, its absolute bioavailability in humans hasnot been fully established [6]. Ritonavir markedlyincreases lopinavir exposure by inhibiting cytochromeP450 isoenzyme 3A4 [7], and therefore adequate drugabsorption is essential for the therapeutic activity to beexerted with the expected level of potency. Chemicalfactors can affect drug absorption by influencing thestate of the drug in the intestine; indeed, the absorptionof ritonavir is likely to be decreased in the absence ofgastric acidity. Both lopinavir and ritonavir absorptionis limited by their intrinsic solubility, and a change inthe pH at which the substance is 50% ionized resultingfrom a change in intestinal pH (with the associatedchanges in the rate of ionization) may lead to alteredabsorption.

Blood samples were obtained at the end of the disonginterval and trough plasma concentrations (Ctrough) oflopinavir and ritonavir were measured during the firstweek of therapy and 17 days after the initiation of theantiretroviral treatment by high performance liquidchromatography–mass spectometry [8]. The plasmaHIV-RNA level was measured on the same days usingthe Roche Amplicor Ultrasensitive Assay (Roche,Basel, Switzerland). Lopinavir Ctrough ranged from 2864to 6753 ng/ml (median 4411, n ¼ 8) and steady-stateplasma Ctrough was 4864 ng/ml. All values are compar-able to previously effective lopinavir plasma concentra-


AIDS 2003, Vol 17 No 1136

tions measured in antiretroviral-naive patients [9]. Theritonavir plasma Ctrough ranged from 199 to 413 ng/mlduring the first week of therapy and was 410 ng/ml onday 17.

The day before the initiation of anti-HIV drugs, theplasma HIV-RNA level was 430 000 copies/ml; thishad decreased by more than 2.5 logs after 17 days oftherapy (to 840 copies/ml) and the CD4 cell count hadincreased from 37 to 105 cells/mm3 after the firstmonth of therapy, both indicators showing evidence ofa significant therapeutic effect.

This information lends support to the importance ofantiretroviral drug measurement in plasma, especially incomplex conditions that have not been specificallystudied. The information gathered in the study of ourpatient might be of interest not only for patients havingthe same associated disease but also for the entirecontext of reduced gastric acidity, which includes avariety of underlying conditions affecting HIV-infectedpatients.

Acknowledgements

The authors would like to thank Richard Bertz, PhD,from Abbott Laboratories for his kind and usefulsuggestions.

Marta Boffitoa,b, Anna Lucchinia, Agostino Maielloa,Ivano Dal Contea, Patrick G. Hoggardb, David J. Backb

and Giovanni Di Perri a, aDepartment of InfectiousDiseases, University of Torino, Turin, Italy; andbPharmacology Department, University of Liverpool,Liverpool, UK.

Received: 17 July 2002; accepted: 7 August 2002.

DOI: 10.1097/01.aids.0000042961.95433.05

References

1. Panel on Clinical Practices for the Treatment of HIV Infection.Guidelines for the use of antiretroviral agents in HIV-infectedadults and adolescents. Ann Intern Med 1998; 128:1079–1100.

2. Centers for Disease Control and Prevention. 1993 Revisedclassification system for HIV infection and expanded surveil-lance case definition for AIDS among adolescents and adults.MMWR 1992; 41:1–19.

3. Back DJ, Khoo SH, Gibbons SE, Merry C. The role of therapeuticdrug monitoring in treatment of HIV infection. Br J ClinPharmacol 2001; 51:301–308.

4. Bristol-Meyers-Squibb. Videx (didanosine) package insert. Prince-ton, NJ; 1999.

5. Schanker LS, Shore PA, Brodie BB, Hogben CAM. Absorption ofdrugs from the stomach. J Pharmacol 1957; 120:528–539.

6. Abbott Laboratories. Kaletra (lopinavir/ritonavir) package insert.North Chicago, IL; 2000.

7. Sham HL, Kempf DJ, Molla A, Marsh KC, Kumar GN, Chen CM,et al. ABT-378, a highly potent inhibitor of the human immuno-deficiency virus protease. Antimicrob Agents Chemother 1998;42:3218–3224.

8. Reynolds HE, Tjia JF, Gibbons SE, Khoo SH, Back DJ. Simultane-ous determination of four HIV protease inhibitors by hplc-MS/MS for use in a therapeutic drug monitoring service. Br J ClinPharmacol 2001; 52:481P.

9. Murphy RL, Bruna S, Hicks C, Eron JJ, Gulick R, King M, et al.ABT-378/ritonavir plus stavudine and lamivudine for the treat-ment of antiretroviral-naive adults with HIV-1 infection: 48-week results. AIDS 2001, 15:F1–F9.

Rituximab and chemotherapy is highly effective in patients with CD20-positive non-Hodgkin’s lymphomaand HIV infection

HIV increases the risk of aggressive B-cell lymphoma(non-Hodgkin’s lymphoma; NHL), which often pre-sents at an advanced stage and frequently involvesextranodal sites. The combination of cyclophospha-mide, doxorubicin, vincristine, and prednisone(CHOP) is considered to be the standard treatment forpatients with diffuse large B-cell NHL, both in thegeneral population and in the HIV setting.

Recently, Coiffier et al. [1] presented the results of aphase III randomized study comparing CHOP versusCHOP plus rituximab, a chimeric monoclonal anti-body against the CD20 B-cell antigen, in elderlypatients (more than 60 years of age) with diffuse largeB-cell NHL. The results of the study showed that theaddition of rituximab to the CHOP regimen increasedthe complete response rate (76 versus 63%, P ¼ 0.005)and improved the event-free survival (EFS) at 2 years(57 versus 38%, P , 0.001) and the overall survival at 2years (70 versus 57%, P ¼ 0.007), without a significantincrease in toxic effects [1].

Infusional cyclophosphamide, doxorubicin and etopo-side (CDE) is one of the most effective chemother-apeutic regimens for HIV-associated NHL, with acomplete response rate of 46% and a median overallsurvival of 8.2 months [2]. We carried out the firstprospective study testing the combination of rituximabplus chemotherapy infusional CDE in patients withCD20-positive high-grade NHL and HIV infection. InJune 1998, we started a phase II study using cyclophos-phamide 187.5 mg/m2 per day, doxorubicin 12.5 mg/m2 per day and etoposide 60 mg/m2 per day admin-istered by continuous intravenous infusion for 4 daysevery 4 weeks for up to six cycles, and rituximab375 mg/m2 given intravenously on day 0 before eachcycle. Highly active antiretroviral therapy was givenconcomitantly with chemotherapy, irrespective of theCD4 cell count and HIV viral load, and was selectedaccording to the patient’s previous antiretroviral expo-sure. All patients received granulocyte colony-stimulat-ing factor support (5 �g/kg from day 6 to day 11) andprophylactic treatment with trimethoprim–sulfameth-


Correspondence 137

oxazole double-strength by mouth once a day andfluconazole 100 mg by mouth every day.

From June 1998 to October 2001, 41 patients wereenrolled and 38 patients are evaluable for response andtoxicity. Thirty-five patients (85%) were men and theirmedian age was 38 years (range 29–65). The majorityof our patients (13/41, 32%) were male homosexuals,12 out of 41 (12%) were intravenous drug users, and12 out of 41 (12%) reported heterosexual relationshipsas the only risk factor for HIV infection; in four out of41 patients (10%) the risk factor was not determined.The median CD4 cell count was 120 cells/mm3 (range3–578) and the median performance status (EasternCooperative Oncology Group) was 1 (range 0–2).Diffuse large B-cell NHL was diagnosed in 24 out of41 patients (59%), Burkitt’s lymphoma in 15 out of 41(36%) and anaplastic large-cell Ki-1+ NHL in two outof 41 (5%). According to the Ann Arbor stagingsystem, disease staging was as follows: stage I in 12 outof 41 patients (29%), stage II in four out of 41 (10%),stage III in seven out of 41 (17%) and stage IV in 18out of 41 (44%). Twenty out of 41 patients (49%) hadB symptoms. As for the distribution of the prognosticfactors according to the age-adjusted internationalprognostic index (IPI), this turned out to be as follows:IPI ¼ 0 in seven out of 41 patients (17%), IPI ¼ 1 in16 out of 41 (39%), IPI ¼ 2 in 12 out of 41 (29%),IPI ¼ 3 in six out of 41 (15%).

Twenty-nine out of 38 evaluable patients (76%)achieved a complete response, two out of 38 (5%) hada partial remission and seven patients progressed. Witha median follow-up of 12 months, only three patientsout of 29 complete responders (10%) have relapsed and32 out of 41 patients are alive. The major cause ofdeath was NHL (8/9 patients). Grade 3–4 neutropenia,anemia and thrombocytopenia were observed in 80, 37and 29% of patients, respectively. Twenty-nine percent of patients developed bacterial infections duringneutropenia, and two patients (5%) developed opportu-nistic infections (Pneumocystis carinii pneumonia andcytomegalovirus infection). Twelve per cent of patientshad mucositis. No toxic deaths were observed. Theactuarial overall survival and EFS at 2 years were 70and 86%, respectively.

The combination of rituximab plus CDE seems to be

very active, with a complete response rate (76%) muchhigher than that reported so far in HIV-NHL (45–65%), also with a significant increase in overall survival(70% at 2 years in our hands versus a median 7–18months in previous studies) [2–4]. Interestingly, com-paring our data and those of Coiffier et al. [1], thecomplete response rates, the 2-year overall survival andEFS are superimposable, showing that the outcome ofHIV-NHL in the era of highly active antiretroviraltherapy seems at least comparable with that of elderlypatients with high-grade NHL.

In conclusion, on the basis of our results and consider-ing the impossibility of conducting randomized studiesin this setting, we think that the association ofrituximab plus chemotherapy should be strongly re-commended as a front line treatment also for patientswith CD20-positive high-grade NHL and HIV infec-tion.

Michele Spinaa, Joseph A. Sparanob, Ulrich Jaegerc,Giuseppe Rossid and Umberto Tirelli a, aDivision ofMedical Oncology A, National Cancer Institute, Aviano,Italy; bMontefiore Medical Center, New York, USA;cUniversity of Vienna, Vienna, Austria; and d III Medi-cine, Hematology Department, Spedali Civili, Brescia,Italy.

Received: 24 July 2002; accepted: 7 August 2002.

DOI: 10.1097/01.aids.0000042962.95433.de

References

1. Coiffier B, Lepage E, Briere J, Herbrecht R, Tilly H, Bouabdallah J,et al. CHOP chemotherapy plus Rituximab compared withCHOP alone in elderly patients with diffuse large-B-cell Lymph-oma. N Engl J Med 2002; 346:235–242.

2. Sparano JA, Wiernik PH, Hu X, Sarta C, Schwartz EL, Soeiro R,et al. Pilot trial of infusional cyclophosphamide, doxorubicin,and etoposide plus didanosine and filgrastim in patients withhuman immunodeficiency virus-associated non-Hodgkin’slymphoma. J Clin Oncol 1996; 14:3026–3035.

3. Kaplan LD, Straus DJ, Testa MA, Von Roenn J, Dezube BJ, CooleyTP, et al. Low-dose compared with standard-dose m-BACODchemotherapy for non-Hodgkin’s lymphoma associated withhuman immunodeficiency virus infection. National Institute ofAllergy and Infectious Diseases AIDS Clinical Trials Group.N Engl J Med 1997; 336:1641–1648.

4. Tirelli U. Dose adjusted treatment in AIDS-related lymphoma(NHL and HD) [Abstract]. J Acquir Immune Defic Syndr 2000;23:S17.

Initation of highly active antiretroviral therapy leads to an HIV-specific immune response in a seronegativeinfant

This is a report of an HIV-1-infected child with absentHIV-specific antibodies, in whom the appearance ofHIV-specific antibodies followed after the initiation ofhighly active antiretroviral therapy (HAART).

An 11-month-old girl with a history of oral candidiasis,anaemia and failure to thrive was hospitalized fordyspnoea, tachypnoea and diarrhoea. Pneumonia wasdiagnosed by chest X-ray. Blood cultures were positive


AIDS 2003, Vol 17 No 1138

for Salmonella typhimurium. HIV-1 infection was con-sidered, and enzyme immunoassay and Western blotwere performed; HIV-1-specific antibodies were notdetected. Shortly afterwards the child was diagnosedwith hypertrophic cardiomyopathy and referred to atertiary centre. The diagnosis of HIV/AIDS was againconsidered, and HIV-1-specific polymerase chain reac-tion and p24-antigen tests were performed, which werepositive. The child had been breastfed for 6 weeks.Other possible routes of transmission besides verticaltransmission could not be identified. Genotyping re-vealed an A/B mosaic virus, which normally yields adetectable antibody response. Before the start ofHAART, CD4 T and B-cell numbers were 0.19 and0.021 3 10 E9/l, respectively, with more than 90% ofthe B cells expressing the CD5 phenotype. Immuno-globulin levels were 9.9 g/l, and adequate levels ofspecific antibodies against poliomyelitis and pertussiswere found after vaccination, indicating that the im-mune system was capable of mounting a specificantibody response. Dissociation of antigen-antibodycomplexes did not result in the detection of HIV-1-specific antibodies in enzyme immunoassay andWestern blot. However, p24 levels increased in thedissociated samples. The patient’s clinical situationgradually improved and she was discharged from thehospital after 1.5 months. B-cell counts normalizedafter 3 months of therapy (150% median normal value)and T-cell counts normalized between 6 and 9 months(80 and 110% of median normal values, respectively).T-cell function was studied during treatment in wholeblood lymfocyte culture. The proliferative response toCD3 monoclonal antibody plus CD28 monoclonalantibody was measured in vitro by means of theincorporation of 3H-thymidine [1]. Steadily an increasein T-cell reactivity was seen, from less than 50 atbaseline to 12 100 at week 48 and 33 000 counts/minat week 96 (95% confidence limit in normal adults. 17 000). HIV-1-specific antibodies could be detectedfor the first time after 6 months of therapy. TheWestern blot showed antibodies specific for gp24 andgp160. For more detailed virological and immunologi-cal parameters during the 2 year follow-up see Table 1[2].

This patient suffered from a rapidly progressive HIV-1infection, with a striking absence of HIV-1-specificantibodies. The possible hypotheses for this findingwere evaluated:

The capture of antibodies within immune complexeswas not responsible for the absence of HIV-specificantibodies. However, after dissociation p24 levels in-creased. This may have been caused by the dissociationof low avidity HIV-specific antibodies.

HIV-1-specific in-utero induced immunotolerance, asdescribed by Siegrist et al. [3] is a possibility, but one


Table1.Virologicalandimmunologicalparametersduring2-yearfollow-up.

Monthsafterstarttherap

yDay

0Wee

k1

Wee

k2

Month

1Month

2Month

3Month

5Month

6Month

9Month

12

Month

24

HIV-1-spec

ifican

tibodies

Neg

ative

Neg

ative

Neg

ative

––

–Neg

ative

Positive

Positive

––

Viral

load

(copies/ml)

252000

300000

317000

115000

22700

8190

–3230

1530

783

50

IgG

(g/l)

9.9

––

–12.3

12.9

–10.7

8.3

7.7

13.1

CD4ce

lls(3

109/l)b

0.19

19

0.20

0.44

0.49

0.44

–1.76

2.45

3.19

2.94

CD4ce

ll%

med

iana

8(#)

8(#)

9(#)

19(#)

21(#)

19(#)

–80(N

)111(N

)145(N

)226(:)

CD8ce

lls(3

109/l)b

1.42

1.02

1.00

2.23

2.50

1.63

–3.69

3.30

3.87

3.15

CD8ce

ll%

med

iana

29(N

)93(N

)91(N

)202(N

)227(N

)148(N

)307(:)

275(:)

323(:)

394(:)

CD19+ce

lltotal(3

109/l)b

–0.021

0.027

0.037

0.109

2.108

–2.204

2.974

3.090

2.500

CD19ce

ll%

med

iana

–2(#)

2(#)

3(#)

8(#)

150(N

)–

170(N

)229(:)

238(:)

313(:)

%CD5+CD19+ce

lls

–96

91

63

98

99

–96

96

90

55

aPe

rcen

tage

ofmed

ianag

e-spec

ificreference

values

(#below

norm

alrange

,:ab

ove

norm

alrange

,N

within

norm

alrange

).Norm

alvalues

arederived

from

Coman

s-Bitteretal.[2].

bAbsolute

counts.

Correspondence 139

would not expect an HIV-1-specific immune responseafter initiating HAART. Tolerance for HIV developedin utero would have resulted in the absence of HIV-specific antibodies for many years.

The presence of B-cell dysfunction has previously beenobserved in HIV-1-infected patients [4]. Despite thelow number of B cells, normal concentrations ofimmunoglobulins were present and the immune systemwas capable of mounting a specific immune responseafter vaccination. This implied a specific immunotoler-ance to HIV. Three months after the start of therapy,B-cell numbers reached normal levels, but still noHIV-1-specific antibodies could be detected. Thesewere not detected until 6 months after the start oftherapy when the CD4 T-cell numbers normalized.Therefore, the absence of HIV-1-specific antibodiesmay have been caused by a selective defect in T–B cellinteraction. On the initiation of therapy 96% of the Bcells expressed CD5 (normally 50–75%). This subpo-pulation dominates in cord blood and can be found inelevated levels in patients with autoimmune disease.They are thought to produce low-affinity polyreactiveantibodies [5]. These may have caused the increase inp24 levels after the dissociation of immune complexes.During follow-up the percentage of B cells that ex-pressed CD5 remained high, and only decreased tonormal levels 2 years after the start of therapy. Wespeculate that as a consequence of the undetectableviral load, the CD4 T-cell function fully restored,resulting in further maturation of the B-cell compart-ment.

In conclusion, p24 antigen and HIV-1-specific poly-merase chain reaction tests should always be performed

in seronegative children who are clinically suspected ofAIDS. The initial absence of HIV-1-specific antibodiesin this child may have been caused by a selective defectin T–B cell interaction.

Pieter L.A. Fraaij a, Annemarie M.C. Van Rossuma, KatjaC. Wolthersb, Ann C.T.M. Vossenb, Yvonne M.Jeuckend , Jac H.S.A.M. Kuijpersc , Ellen G. VanLochemc, Marijke T.L. Roose, Ronald De Groota andNico G. Hartwiga, Departments of aPediatrics, bVirologyand cImmunology, Erasmus MC Rotterdam, the Nether-lands; dDepartment of Pediatrics, Leiden Medical Center,the Netherlands; and eSanquin Research at CLB, Amster-dam, the Netherlands.

Received: 9 May 2002; revised: 17 July 2002; accepted:7 August 2002.

DOI: 10.1097/01.aids.0000042963.95433.97

References

1. Bloemena E, Roos MT, Van Heijst JL, Vossen JM, Schellekens PT.Whole-blood lymphocyte cultures. J Immunol Methods 1989;122:161–167.

2. Comans-Bitter WM, de Groot R, van den Beemd R, Neijens HJ,Hop WC, Groeneveld K, et al. Immunophenotyping of bloodlymphocytes in childhood. Reference values for lymphocytesubpopulations. J Pediatr 1997; 130:388–393.

3. Siegrist CA, Wyler CA, Gerritsen EJ, Perrin L, Speiser D, Suter S.Specific tolerance to HIV-1 antigens in an infant with rapidprogression to AIDS. AIDS 1993; 7:1683–1684.

4. Ammann AJ, Schiffman G, Abrams D, Volberding P, Ziegler J,Conant M. B-cell immunodeficiency in acquired immune defi-ciency syndrome. JAMA 1984; 251:1447–1449.

5. Erkeller-Yuksel FM, Deneys V, Yuksel B, Hannet I, Hulstaert F,Hamilton C, et al. Age-related changes in human blood lympho-cyte subpopulations. J Pediatr 1992; 120:216–222.

Discontinuation of secondary prophylaxis and the risk of Pneumocystis carinii pneumonia

We read with great interest the correspondence ofDegen and colleagues [1] describing a case of Pneumocys-tis carinii pneumonia (PCP) in a patient receiving highlyactive antiretroviral therapy (HAART) after discontinu-ing prophylaxis. Our data confirm this observation, andhave demonstrated the development of a number ofPCP cases in HIV-positive individuals, with and with-out prophylaxis, enrolled in a nutritional chemopreven-tion trial during the HAART era (1998–2000).

Over the course of the study, 12 cases of PCP,confirmed by hospital discharge records, direct fluor-escent identification, clinical course and treatmentresponse, were observed in the study cohort (n ¼ 250).Most of the PCP infections occurred in men (80%,P ¼ 0.003); 78% of the PCP cases reported having hada previous history of tuberculosis.

As shown in Table 1, several of the participants who

developed PCP were on HAART (n ¼ 7), had CD4cell counts greater than 350 cells/mm3 and undetect-able viral loads; prophylaxis had been discontinued inthis group. These patients exhibited enhanced immunefunction (increased CD4 T-lymphocyte counts frombelow 200 cells/mm3 to above that level), and hadachieved a low viral burden. As HAART had beenadministered for approximately 9 months, the develop-ment of PCP is not likely to be attributable to animmune rebound.

These findings are of concern, especially in the light ofthe recent Centers for Disease Control and Prevention(CDC) report [2], indicating that the incidence ofAIDS-defining opportunistic infections in the UnitedStates is no longer declining. Opportunistic infectionsof the respiratory tract, in fact, continue to have asignificant impact upon morbidity and mortality inHIV/AIDS, even in the era of HAART [3], under-


AIDS 2003, Vol 17 No 1140

scoring the need to advance our understanding of themechanisms involved in the pathogenesis of infectiousdiseases.

We strongly concur with the proposal of Degen et al.[1] that patients be monitored closely for PCP whensecondary prophylaxis is discontinued, and suggest thatthe general recommendation for discontinuing prophy-laxis in those patients receiving HAART who have aCD4 cell count greater than 200 cells/mm3 may needto be re-evaluated. We are currently investigating the

role of additional risk factors associated with thepresence of respiratory infections in HIV-infected pa-tients receiving HAART.

Maria Jose Miguez-Burbano, Harry Archer, MariaRodriguez and Gail Shor-Posner Department of Psy-chiatry and Behavioural Sciences, Division of DiseasePrevention, University of Miami School of Medicine,Miami, FL 33136, USA.

Sponsorship: This work was supported by NIH/NIDAgrant no. 1R01DA11378 (G.S.P.) and NIH/FOGARTYD43TWOO17.

Received: 2 August 2002; accepted: 12 August 2002.

DOI: 10.1097/01.aids.0000042964.95433.59

References

1. Degen O, ven Lunzen J, Horstkotte MA, Sobottka I, Stellbrink HJ.Pneumocystis carinii pneumonia after the discontinuation ofsecondary prophylaxis. AIDS 2002; 16:1433–1434.

2. McNaghten AD, Hanson L, Nakashima AK, Swerdlow DL.Incidence of AIDS-defining opportunistic infections in the USmay be leveling from 1998–1999. In: 39th Annual Meeting ofthe Infectious Diseases Society of America. San Francisco, CA,October 2001 [Abstract 751].

3. Masur H. Update: incidence, diagnosis, and clinical manifesta-tions of HIV-related opportunistic infections. In: 39th AnnualMeeting of the Infectious Diseases Society of America. SanFrancisco, CA, 25 October 2001. www.medscape.com/viewarticle/413061

Bcl-2 expression is moderately correlated with long-term variability of CD4 T-cell increase under successfulhighly active antiretroviral therapy

The objective of highly active antiretroviral therapy(HAART) is to induce a complete suppression of theplasma viral load (pVL), to achieve a CD4 T-cellincrease, and to protect against opportunistic infections.However, the cause–effect relationship between thesuppression of HIV replication and immune restorationis not completely clear. Indeed, in up to 30% ofpatients, CD4 T cell counts remain low or do notincrease further, despite complete pVL suppression,providing a margin of intervention for immunomodu-lating approaches.

We and others [1,2] have demonstrated that lympho-cyte spontaneous apoptosis (LSA) provides a predictivefactor for the immunological and clinical evolution ofHIV infection, partly independent of the CD4 T-cellcount and pVL. Among the cascade of factors thatregulate LSA, Bcl-2 plays a key role in exertingantiapoptotic activity [3]. We have also demonstratedthat HAART induces Bcl-2 hyperexpression in CD4 Tcells (particularly in the naive subset) of patients withcomplete pVL suppression, in contrast to those who

have residual HIV replication [4]. Therefore, it hasbeen suggested that Bcl-2 upregulation and, as conse-quence, a decrease in LSA might contribute to theCD4 T-cell increase induced by HAART [4,5]. Thishypothesis is supported by the findings recently re-ported by David et al. [6]. In a cross-sectional study,two groups of patients with prolonged pVL suppression(. 9 months) were evaluated, and it was shown thatthose who had previously failed to achieve a CD4 T-cell increase had lower levels of Bcl-2 expression andmore LSA at the time of analysis than those in whom agood immune recovery had occurred. It could there-fore be argued that levels of Bcl-2 expression mightpredict the long-term CD4 T-cell recovery in patientsachieving sustained pVL suppression.

Here we present the results of a prospective studyaimed at evaluating this hypothesis.

Twenty-one patients with pVL less than 50 copies/mlfor 3 months or longer were selected for this study [15men; mean age 44 years (SD 8.2); nine at first HAART


Table 1. Characteristics of patients who developed Pneumocystiscarinii pneumonia.

HAARTNot-antiretroviral

treatedVariable (n ¼ 7) (n ¼ 5) P value

Age (years)Range 32–49 32–45Mean 41 � 6 40 � 5SexFemale 25% (3) 0% (0)Male 33% (4) 42% (5) 0.003CD4 cell count (cells/mm3)Range 198–692 74–271Mean 384 � 167 165.8 � 74 0.001Viral load (RNA copies/mm3)Range 200–67 225 14 414–161062Mean 24 754 � 12 045 73 945 � 62 907 0.014

HAART, Highly active antiretroviral therapy

Correspondence 141

experience; duration of previous pVL suppression 37.8weeks (SD 29.8); four were on non-nucleoside reversetranscriptase inhibitor-containing regimens, whereas 17were on protease inhibitor-containing regimens atbaseline]. Bcl-2 expression in the CD4 T-cells wasmeasured at baseline and calculated as the molecularequivalent of soluble fluorescein (MESF) [4]. Patientswere followed with routine laboratory testing andclinical evaluation every 3 months until the most recentpVL, still undetectable for a mean of 99.7 weeks (SD52). A summary measure of CD4 T-cell variation wascalculated as the area under the curve of the percentagechange (˜%AUC) [7] during the period of observation,divided into semesters, and correlated with MESFexpression of Bcl-2 at baseline. Correlations wereanalysed using Pearson’s coefficient. All tests were two-sided. Analyses were performed using Statistica forWindows software (StatSoft Inc., Tulsa, OK, USA).

The mean Bcl-2 expression in CD4 T cells was 81 239MESF (SD 28 040). During the period of observation,the mean ˜%AUC of CD4 T cells was +26.74% (SD25.93) per semester. As shown in Fig. 1, a trend for acorrelation was found between Bcl-2 expression inCD4 T cells and their increase during the follow-up,although it did not reach statistically conventionalsignificance (r ¼ 0.40, P ¼ 0.07). The time of observa-tion, the duration of pVL suppression preceding base-line, the line of therapy, and the type of regimen atbaseline did not significantly influence the correlationbetween Bcl-2 expression and ˜%AUC of the CD4 Tcells (data not shown).

In a subgroup of 12 patients, Bcl-2 expression wasmeasured both in memory (CD4 CD45R0+) and naive

(CD4 CD45RA+) T cells, as previously described [4].No statistically significant correlations were found be-tween ˜%AUC of CD4 T cells and either Bcl-2expression in the memory T cells (r ¼ 0.24, P ¼ 0.45)or in the naive T cells (r ¼ 0.29, P ¼ 0.36).

In conclusion, the prospective study presented heresuggests that Bcl-2 expression in CD4 T cells may be apredictor of CD4 cell increase in patients with sus-tained pVL suppression induced by HAART. How-ever, the correlation reported here is only moderate.Although the prediction of a CD4 T-cell increase bymeans of Bcl-2 expression would be an attractive toolto individualize the use of immunomodulating agentsin patients who really deserve them, Bcl-2 did notexplain completely the viro-immunological discordanttrends in this study, therefore other factors should beinvestigated.

The correlation between Bcl-2 expression and CD4 T-cell increase was not attributable to a particular sub-population (either memory or naive), on the basis ofour data. It would be of interest to see whether IL-2 orother immunomodulating agents could induce Bcl-2expression particularly in naive T cells, thus promotinga more pronounced immune-recovery with respect toHAART alone.

Maria Cristina Uccelli a,b, Carlo Torti a, Eugenia Quiros-Roldana, Carmine Tinelli c , Andrea Patronia, FrancescoCastelli a, Giampiero Carosia and Paolo Airob, aInstituteof Infectious and Tropical Diseases and bDepartment ofClinical Immunology, University of Brescia, Brescia, Italy;and cBiostatistics Unit, IRCCS Policlinico S. Matteo,Pavia, Italy.

Sponsorship: This study was supported by a grant fromthe University of Brescia ‘Progetto Giovani Ricercatori2002’ to Dr Carlo Torti.

Received: 14 June 2002; accepted: 9 August 2002.

DOI: 10.1097/01.aids.0000042965.95433.10

References

1. Prati E, Gorla R, Malacarne F, Airo P, Brugnoni D, Gargiulo F,et al. Study of spontaneous apoptosis in HIV+ patients: correla-tion with clinical progression and failure of T cell homeostasis.AIDS Res Hum Retroviruses 1997; 13:1501–1508.

2. Wasmuth JC, Klein KH, Hackbarth F, Rockstroh JK, Sauerbruch T,Spengler V. Prediction of imminent complications in HIV-1-infected patients by markers of lymphocytes apoptosis. J AcquirImmune Defic Syndr 2000; 23:44–51.

3. Yang E, Korsmeyer SJ. Molecular thanatopsis: a discourse onBCL2 family and cell death. Blood 1996; 88:386–401.

4. Airo P, Torti C, Uccelli MC, Malacarne F, Palvarini L, Carosi G,Castelli F. Naive CD4+ lymphocytes express high level of Bcl-2after highly active antiretroviral therapy for HIV infection. AIDSRes Hum Retroviruses 2000; 17:1805–1807.

5. Roger PM, Breittmayer JP, Arlotto C, Pugliese P, Pradier C,Bernard-Pomier G, et al. Highly active anti-retroviral therapy


80

70

60

50

40

30

20

10

0

�10

�20

�3020000 40000 60000 80000 100000 120000 140000 160000 180000

Bcl-2 expression in CD4� Tcells (MESF)

% C

hang

e A

UC

CD

4� T

-cel

l cou

nts

Regression95% confidence interval

Fig. 1. Correlation between Bcl-2 expression and percent-age change in the area under the curve of the CD4 T cells.AUC, Area under the curve; MESF, molecular equivalent ofsoluble fluorescein.

AIDS 2003, Vol 17 No 1142

(HAART) is associated with a lower level of CD4+ T cellapoptosis in HIV-infected patients. Clin Exp Immunol 1999;118:412–416.

6. David D, Keller H, Naıt-Ighil L, Treilhou M-P, Joussemet M,Dupont B, et al. Involvement of Bcl-2 and IL-2R in HIV-positive

patients whose CD4 cell counts fail to increase rapidly withhighly active antiretroviral therapy. AIDS 2002; 16:1093–1101.

7. Senn S, Stevens L, Chaturvedi N. Repeated measures in clinicaltrials: simple strategies for analysis using summary measures.Stat Med 2000; 19:861–877.


Correspondence 143

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