Reconstitution of immune responses to tuberculosis in patients with HIV infection who receive antiretroviral therapy

STUDY OBJECTIVE:s: To assess the restoration of immune responses to tuberculosis, as manifested by secretion of T-helper type 1 cytokines (interferon [IFN]-gamma, interleukin [IL]-12, and IL-2) and T-helper type 2 cytokines (IL-10), in HIV-positive patients who receive antiretroviral therapy (ART). DESIGN: Prospective cohort study. SETTING: University hospital. PATIENTS: Ten HIV-positive patients, all na?ve to ART and all about to start ART for clinical indications, and 11 healthy, HIV-negative control subjects. INTERVENTIONS: Assessment of T-cell proliferation and cytokine production after administration of ART to patients with HIV infection. MEASUREMENTS AND RESULTS: All patients had a negative tuberculin skin test result at baseline and were anergic. Highly active ART reduced the viral load to very low levels in all patients within a short time after starting therapy. Blood samples were drawn every 2 months after starting therapy, and continued for 1 year while the patients continued to receive ART. There were trends toward increased proliferation of peripheral blood mononuclear cells (PBMCs) in response to Mycobacterium tuberculosis-specific stimuli, but these were delayed until several months of ART had elapsed. Similar trends were noted in relation to the secretion of IFN-gamma. Neither PBMC proliferation nor IFN-gamma secretion reached levels seen in healthy control subjects. No consistent trends in IL-2, IL-10, or IL-12 production were noted. CONCLUSION: ART restores immune responses to M tuberculosis, although this restoration is delayed and does not reach levels seen in healthy, HIV-negative control subjects. These results may explain in part the phenomenon of paradoxic reactions to antituberculosis therapy in patients with HIV infection. A larger study in which patients are followed up for a longer period of time will allow the magnitude and timing of this reconstitution to be more precisely defined.     

HIV-1-related pleural tuberculosis: elevated production of IFN-gamma, but failure of immunity to Mycobacterium tuberculosis

BACKGROUND: Pleural tuberculosis can resolve spontaneously, suggesting that the inflammatory process may represent a protective immune response. However, pleural tuberculosis is strongly associated with HIV infection. It has been suggested that cell-mediated immune responses may be reduced, and direct bacterial invasion may have a role in pathogenesis, in HIV-positive cases. To test this hypothesis, we compared production of the pro-inflammatory cytokines, interferon (IFN)-gamma and tumour necrosis factor(TNF)-alpha, production of the immunosuppressive cytokine, interleukin (IL)-10, and mycobacterial culture positivity, in HIV-negative and HIV-positive patients with pleural tuberculosis. METHODS: Cytokine levels were measured in serum and pleural fluid, and in supernatants of blood and pleural fluid stimulated in vitro using mycobacterial antigens. Intracellular IFN-gamma and TNF-alpha production was measured after stimulation with phorbol myristate acetate and ionomycin in vitro. RESULTS: IFN-gamma was strikingly elevated in serum and pleural fluid in HIV-positive, compared to HIV-negative subjects (P < or = 0.02). TNF-alpha was elevated, but this was not statistically significant. IL-10 levels were higher in serum (P < 0.001), but similar in pleural fluid. IFN-gamma responses to soluble mycobacterial antigen in vitro were reduced in peripheral blood (P = 0.006), but not pleural fluid, of HIV-positive subjects. Intracellular cytokine staining suggested that CD8+ T cells were a major source of IFN-gamma in HIV-positive subjects. The proportion of subjects with a positive culture for Mycobacterium tuberculosis from pleural fluid was higher in the HIV-positive group. CONCLUSIONS: HIV-positive patients with pleural tuberculosis show elevated production of IFN-gamma, for which CD8+ T cells may be a major source. Mycobacterium tuberculosis can proliferate despite high levels of pro-inflammatory cytokines.     

Explosion of tuberculin-specific Th1-responses induces immune restoration syndrome in tuberculosis and HIV co-infected patients

OBJECTIVES: To test the hypothesis that an acute exacerbation of mycobacteria-specific Th1 response after HIV infection control by HAART causes immune restoration syndrome (IRS) in HIV-tuberculosis (TB) coinfected patients. DESIGN: Prospective, multicenter study of 19 consecutive untreated HIV-TB coinfected patients included when initiating antimycobacterial therapy and sequentially evaluated during HAART and at time of IRS. IRS was defined according to classical clinical diagnostic criteria. Patients were declared IRS- if no IRS occurred within 3 months after HAART initiation. METHODS: Mycobacteria-specific [purified protein derivative (PPD), ESAT-6, 85B] Th1 cells producing interferon (IFN)-gamma quantified by ELISpot, in vitro production of 25 cytokines/chemokines in antigen-stimulated peripheral blood mononuclear cell (PBMC) supernatants quantified by chemiluminescence. RESULTS: Seven patients (37%) experienced IRS (IRS+). Mycobacteria-specific (PPD) Th1 IFN-gamma-producing cells increased sharply during IRS (median, 2970 spot forming cells/10 PBMC), but not the cytomegalovirus-specific responses tested as control. Only three IRS+ patients had low ESAT-6- but no 85B-specific responses. IRS- patients did not develop acute PPD-specific responses except in one case. In addition, at time of IRS a peak of PPD-specific Th1 cytokines/chemokines [interleukin (IL)-2, IL-12, IFN-gamma, IP10 and monokine-induced by IFN-gamma] without Th2 cytokines, and a peak of non-specific inflammatory cytokines/chemokines (TNF-alpha, IL-6, IL-1beta, IL-10, RANTES and MCP-1) occurred. These findings were independent from CD4 cell count, viral loads or time of HAART initiation. CONCLUSION: An acute exacerbation of Th1 responses against mycobacterial antigens appears to cause IRS in patients co-infected with HIV and TB. This key event provides new evidence valuable for the diagnosis and treatment of IRS.     

Antigen-specific cytokine response to hepatitis C virus core epitopes in HIV/hepatitis C virus-coinfected patients.

OBJECTIVE: Epidemiological data indicate that hepatitis C virus (HCV) infection runs a more rapid and severe course of disease in HIV-coinfected patients, probably because of an altered immune response. DESIGN: We investigated whether HCV-specific cytokine responses are affected by HIV coinfection. METHODS: Using triple colour flow cytometry on peripheral blood lymphocytes after stimulation with the four major immunodominant HCV core T cell epitopes, CT1-CT4, we determined intracytoplasmic production of IFN-gamma, IL-2, IL-4, IL-10 and CD30 expression, a putative surrogate marker of type 2 cells. Fifteen patients with asymptomatic HIV/HCV coinfection (group A), 15 patients with chronic HCV infection (group B) and 10 HIV-infected patients without hepatitis C (group C) were included in the study. RESULTS: In group A, HCV antigens induced significantly higher IL-2 and IFN-gamma production than groups B and C (P < 0.05). Groups A and B showed a similar induction of CD30, which was significantly higher than in group C (P < 0.001). Remarkably, in group A HCV antigens induced IL-4 production in addition to IL-10 and IFN-gamma in the CD30 subset, whereas in groups B and C no IL-4 induction was observed in this T cell subset (P < 0.002). CONCLUSION: Our data suggest that asymptomatic HIV coinfection importantly alters the HCV-specific cytokine response towards a greater production of proinflammatory type 1 cytokines. Moreover, the antiviral activity of type 1 cytokines may be modified by an increased production of type 2 cytokines in the CD30 subset. The altered cytokine pattern may contribute to the adverse natural course of hepatitis C in HIV coinfection.     

Interferon-gamma responses to candidate leprosy skin-test reagents detect exposure to leprosy in an endemic population

New tools for the detection of leprosy exposure in a community will be necessary for the eradication of leprosy. Candidate leprosy skin-test antigens derived from the fractionation of the leprosy bacillus into cytoplasmic and cell-wall proteins free of immuno-inhibitory mycobacterial lipoglycans and carbohydrates were used in an overnight blood test to determine whether exposure to leprosy can be detected by the production of the cytokine interferon gamma (IFN-gamma). Strong IFN-gamma responses were detected in leprosy contacts to both skin-test antigens compared with control subjects from the same endemic communities. There was little response in patients with tuberculosis. Responses were greatest in contacts with recent leprosy exposure. The implications of these findings for the application of these reagents in a field trial as skin tests to detect exposure to leprosy are discussed in light of the strong association between overnight IFN-gamma to PPD and the tuberculin skin-test responses previously reported.     

Analysis of the effect of IL-12 therapy on immunoregulatory T-cell subsets in patients with chronic hepatitis C infection

BACKGROUND/AIMS: Aim of this study was to investigate the influence of IL-12 therapy on the production of immunoregulatory type 1/type 2-cytokines by peripheral blood mononuclear cells from patients with chronic hepatitis C. METHODOLOGY: Peripheral blood mononuclear cells were isolated from 12 patients with chronic HCV infection before and after eight weeks of IL-12 application (0.03-0.5 micrograms/kg body weight). Peripheral blood mononuclear cells from 40 healthy blood donors served as a control group. The peripheral blood mononuclear cells were incubated for seven days with antigens stimulating specifically type 1 (tuberculin purified protein derivative) or type 2 T-cells (tetanus-toxoid). Furthermore, BCG (bacille Calmette-Guérin) was added to the cultures known to activate macrophages/antigen-presenting cells. Supernatants of peripheral blood mononuclear cells were analyzed for tuberculin purified protein derivative-induced production of the type 1 cytokine IFN-gamma, tetanus-toxoid-induced production of the type 2 cytokine IL-5 and the BCG-induced production of TNF-alpha by a double sandwich ELISA. RESULTS: IL-12 therapy hardly influenced the regulatory type 1/type 2 T-cell reactivity. In contrast, BCG induced secretion of TNF-alpha was significantly higher after eight weeks of IL-12 therapy (4348 +/- 3083 pg/mL) than before treatment (1559 +/- 988 pgmL, p < 0.01). Clinically, serum alanine transaminase levels significantly decreased during IL-12 treatment but HCV-RNA persisted in all patients. CONCLUSIONS: IL-12 therapy in patients with HCV does not alter the production of regulatory cytokines produced by type 1/type 2 TH cells. The significantly enhanced production of BCG-induced TNF-alpha may, however, indicate an activation of antigen presenting cells or natural killer cells.     

Production of interleukin-18 in human tuberculosis

To investigate the role of interleukin (IL)-18 in human tuberculosis, IL-18 production was evaluated in blood and at the site of disease in patients with tuberculosis. Mycobacterium tuberculosis-stimulated peripheral blood mononuclear cells (PBMC) from tuberculosis patients secreted less IL-18 and interferon-gamma (IFN-gamma) than did PBMC from healthy persons reactive to tuberculin. M. tuberculosis-induced IFN-gamma production was inhibited by anti-IL-18 and enhanced by recombinant IL-18. Alveolar macrophages secreted IL-18 in response to M. tuberculosis, and IL-18 and IFN-gamma concentrations were higher in pleural fluid of patients with tuberculosis than in pleural fluid of patients with nontuberculous diseases. These findings demonstrate that IL-18 production by PBMC correlates with IFN-gamma production and effective immunity to tuberculosis, suggesting that IL-18 contributes to a protective type 1 cytokine response in persons with mycobacterial infection.     

The level of PPD-specific IFN-gamma-producing CD4+ T cells in the blood predicts the in vivo response to PPD

There are no reliable means for detecting subclinical mycobacterial infections. The recent sequencing of several mycobacterial genomes has now afforded new opportunities for the development of pathogen-specific diagnostic tests, critical in the context of leprosy and tuberculosis control. In the present study, we applied a multi-parametric flow cytometric analysis that allowed the investigation of T-cell functions in order to define immunological markers that measure previous exposure to mycobacteria. We compared the in vivo response to PPD, the gold standard skin test reagent for measuring previous exposure to Mycobacterium tuberculosis, with in vitro parameters of leukocyte activation in five PPD positive and five PPD negative healthy volunteers. PPD-stimulated peripheral leukocytes expressing CD4, CD69, cutaneous lymphocyte-associated antigen (CLA) and intracellular IFN-gamma were enumerated in whole blood and compared with the size of in vivo PPD-induced induration and IFN-gamma production levels as measured by ELISA in supernatants of PPD-stimulated peripheral blood mononuclear cells. The reactivity to the tuberculin skin test (TST) was associated with markedly increased frequencies of PPD-responsive activated (CD69+) and IFN-gamma-producing CD4+T cells. Detection of PPD-specific IFN-gamma producing leukocytes was restricted to CD4+T cells and a subset of these cells was shown to express the skin homing molecule CLA. Multiple linear regression modeling of responses to PPD showed the highest association between skin test indurations and frequencies of PPD-responsive IFN-gamma-producing CD4+CD69+ T cells. Our data show that the in vitro enumeration of antigen-specific IFN-gamma-producing CD4+ T cells can provide an alternative to the in vivo tuberculin test for the detection of latent Mycobacterium tuberculosis infection. Moreover, the measurement of these immunological parameters can be useful for the screening of new specific antigens defined by the genome sequence allowing selection of the best candidates for new diagnostics (including new skin tests), and vaccines for leprosy and tuberculosis.     

T cell responses in coinfection with Onchocerca volvulus and the human immunodeficiency virus Type 1

Onchocerca volvulus and the human immunodeficiency virus (HIV) are two immunocompromising infectious agents of major public health concern in Uganda. To examine the effect of coinfection with O. volvulus and HIV on cellular immune responses, lymphocyte proliferative responses and cytokine production of peripheral blood mononuclear cells (PBMC) from persons infected with O. volvulus with and without HIV type 1 infection were compared. Proliferation of PBMC to PHA and tuberculin (PPD) in coinfection was less ( P  = 0.08, P  < 0.01) than in O. volvulus infection . O. volvulus extract stimulated lymphocyte proliferation in microfilaria-negative and HIV-negative O. volvulus infection while only an inconspicuous response was observed in microfilaria-negative coinfection. After stimulation of PBMC with PPD, the production of interferon-γ (IFN-γ), interleukin (IL)-4 and IL-5—demonstrated in O. volvulus infection—were reduced in coinfection with HIV ( P  < 0.01). While both groups failed to produce IFN-γ in response to O. volvulus extract, only O. volvulus infected persons generated pronounced IL-5 and low IL-4 levels (0.01 > P  = 0.02). The cellular immune responses in coinfection suggested an HIV-related lack of specific reactivity to O. volvulus antigen and impairment of IL-4 and IL-5 production in addition to the lack of IFN-γ response on antigenic stimulation .     

IL-2 and IFN-gamma, but not IL-4 secretion by peripheral blood mononuclear cells (PBMC) are related to CD4+ T cells and clinical status in Brazilian HIV-1-infected subjects.

It has been reported that production of IL-2 and IFN-gamma, known as T-helper type 1 cytokines, by peripheral mononuclear cells (PBMC) decreases with progression of HIV infection. In contrast, IL-4 and IL-10 production, Th2 cytokine profile, increases with HIV disease progression. PBMC were evaluated from 55 HIV-infected subjects from Divis?o de Imunologia, Hospital das Clínicas, Faculdade de Medicina da Universidade de S?o Paulo, to "in vitro" cytokines production after 24 hours of stimulation with PHA. Low levels of IL-4 production in both HIV-infected patients and normal subjects, were detected. The patients with CD4+ T cell counts < 200 showed a significant decrease of IL-2 and IFN-gamma production compared to controls. Patients with higher counts of CD4+ T cells (either between 200-500 or > 500 cells/mm3) also showed decreased production of IL-2 that was not statistically significant. There was a correlation between IL-2 and IFN-gamma release with CD4+ T cells counts. HIV-1-infected individuals with CD4+ T cells > 500 cells/mm3 showed increased levels of IL-2 and IFN-gamma, than individuals with CD4+ T cells < 500 cells/mm3. In conclusion, we observed a decline of IL-2 and IFN-gamma production at advanced HIV disease. IL-4 production was not affected during HIV infection. Taken together, these findings suggest that the cytokine profile might be influenced by the HIV infection rather than the cause of disease progression.     

HIV-1 alters T helper cytokines, interleukin-12 and interleukin-18 responses to the protozoan parasite Leishmania donovani

OBJECTIVE: To investigate the in vitro and in vivo effect of HIV-1 on lymphoproliferative and T helper (Th) cytokine responses in leishmaniasis. METHODS: Th1 [interleukin (IL)-2 and interferon (IFN)-gamma] and Th2 (IL-4 and IL-10) as well as IFN-gamma-inducing cytokines (IL-12 and IL-18) were measured in antigen and mitogen-stimulated culture supernatants of peripheral blood mononuclear cells (PBMC) of healthy donors, HIV-infected and visceral leishmaniasis (VL) patients with or without HIV co-infection. RESULTS: Proliferative responses to phytohaemagglutinin (PHA) were significantly lower in PBMC from VL and asymptomatic HIV-infected persons compared with responses in healthy individuals. VL-HIV co-infected patients showed the lowest responses. Although there was no significant difference in the Leishmania-induced proliferative responses among the healthy group and those infected with HIV only, VL patients (with or without HIV) exhibited very low proliferation. When cultured with PHA or Leishmania, PBMC from healthy donors produced high levels of a Th1 cytokine (IFN-gamma) and low levels of Th2 cytokines (IL-4 and IL-10). In addition, co-culturing PBMC from healthy donors with a killed HIV preparation abrogated the production of IFN-gamma induced by Leishmania and augmented IL-4 and IL-10 production. Cells from HIV-infected patients produced low levels of IFN-gamma, but high levels of IL-10. The addition of anti-IL-10 did not increase Leishmania-induced proliferative responses or IFN-gamma production. Both IL-12 and/or IL-18 responses were lower in VL patients, HIV-infected, or VL-HIV co-infected patients as compared with those of healthy donors. CONCLUSION: The data suggest that the inhibitory effect of HIV and VL on proliferation and IFN-gamma production is not due to IL-10 alone, but that the defect induced by HIV and VL probably operates at the level of regulation of IFN-gamma-inducing factors, such as IL-12 and IL-18.     

Decreased production of interleukin-12 and interferon-gamma is associated with renal involvement in systemic lupus erythematosus

OBJECTIVE: To analyze the type 1/type 2 cytokine balance in patients with systemic lupus erythematosus (SLE) according to the presence of renal disorder and activity status. METHODS: We measured the serum levels of type 1 (IFN-gamma, IL-12) and type 2 cytokines (IL-4, IL-10) as well as spontaneous and stimulated cytokine production from peripheral blood mononuclear cells (PBMC) in 40 patients with SLE. RESULTS: Patients with lupus nephritis (LN) showed significantly lower levels of serum IL-12 and IFN-gamma than those without LN. Production of IL-12 and IFN-gamma by stimulated PBMC were also decreased in patients with LN. The circulating IL-12 correlated positively with IFN-gamma, but inversely with IL-10. The SLEDAI scores correlated well with the ratio of IL-4/IFN-gamma levels. CONCLUSION: The reduced production of IL-12 and IFN-gamma and the resultant shift towards the type 2 cytokine phenotype may be associated with LN.     

Immunity to placental malaria. III. Impairment of interleukin(IL)-12, not IL-18, and interferon-inducible protein-10 responses in the placental intervillous blood of human immunodeficiency virus/malaria-coinfected women.

Pregnant women are highly susceptible to malaria, and human immunodeficiency virus (HIV) infection increases this susceptibility. In our previous studies, placental malaria (PM), HIV infection, and HIV/PM coinfection were all associated with decreased interferon (IFN)-gamma production by maternal placental (intervillous) blood mononuclear cells (IVBMC). This study investigated whether in vitro production of the IFN-gamma regulatory cytokines interleukin (IL)-12 and IL-18 and the chemokine IFN-inducible protein (IP)-10 by IVBMC is altered in women who have been exposed to malaria and are infected with HIV. IL-12 production from IVBMC was significantly lower in HIV-positive women, regardless of PM status, in contrast to HIV-negative, PM-negative women. IL-18 and IP-10 production by IVBMC was reduced in HIV-positive, PM-negative women but elevated in HIV-positive, PM-positive women. These results reveal a substantial impairment of IL-12 production by IVBMC in HIV-positive women, implicating this cytokine as a potentially critical regulator of malaria antigen-specific IFN-gamma responses in HIV-infected and HIV/PM-coinfected women.     

Mycobacterial T cell responses in HIV-infected patients with advanced immunosuppression

Antimycobacterial T cell reactivity at different stages of HIV infection was investigated. Subjects were screened with purified protein derivative (PPD), early secreted antigenic target (ESAT)-6, and culture filtrate protein (CFP)-10 antigens for interferon (IFN)-gamma-producing effector T cell responses by direct ex vivo enzyme-linked immunospot (ELISpot) assay. The proportion of responders to PPD tuberculin decreased with a reduction in CD4 T cell count, whereas the proportion of responder to ESAT-6 and CFP-10 did not. The main sources of IFN-gamma secretion were CD4 cells, and the relative responses to ESAT-6 and CFP-10 significantly increased in HIV-infected patients with decreasing CD4 cell count. This may reflect early signs of reactivation, reinfection, or a restricted, inefficient immune response to Mycobacterium tuberculosis.     

Antigen-specific and persistent tuberculin anergy in a cohort of pulmonary tuberculosis patients from rural Cambodia

Purified protein derivative (PPD) skin testing is used to identify persons infected with Mycobacterium tuberculosis (Mtb) and to assess cell-mediated immune responses to Mtb. However, lack of skin induration to intradermal injection of PPD or PPD anergy is observed in a subset of patients with active tuberculosis (TB). To investigate the sensitivity and persistence of PPD reactivity and its in vitro correlates during active TB disease and after successful chemotherapy, we evaluated the distribution of skin size induration after intradermal injection of PPD among 364 pulmonary TB patients in Cambodia. A subset of 25 pulmonary TB patients who had a positive skin reaction to mumps and/or candida antigens showed persistent anergy to PPD after successful completion of TB therapy. Strikingly, in vitro stimulation of T cells from persistently anergic TB patients with mumps but not PPD resulted in T cell proliferation, and lower levels of IL-2 and IFN-gamma and higher levels of IL-10 were detected in PPD-stimulated cellular cultures from PPD-anergic as compared with PPD-reactive pulmonary TB patients. These results show that anergy to PPD is antigen-specific and persistent in a subset of immunocompetent pulmonary TB patients and is characterized by antigen-specific impaired T cell proliferative responses and a distinct pattern of cytokine production including reduced levels of IL-2.     

Detection of intracellular antigen-specific cytokines in human T cell populations

Determination of antigen-specific cytokine responses of T lymphocytes after vaccination is made difficult by the low frequency of responder cells. In order to detect these responses, the profile of intracellular cytokines was analyzed using flow cytometry after antigenic expansion. Peripheral blood mononuclear cells were stimulated with antigens for 5 days, further expanded with interleukin (IL)-2, and then restimulated on day 10. Cytokine production was detected by intracellular staining with monoclonal antibodies after saponin-based permeabilization. Influenza expansion resulted in specific interferon-gamma (IFN-gamma) production of 6%-20%, with less IL-4 production (0%-2%). Tetanus toxoid resulted in even greater production. IL-4 and IFN-gamma were produced mainly by memory cells of the CD45RO+ phenotype. IFN-gamma production was contributed by both CD4 and CD8 populations. These methods were then applied to a clinical trial of a candidate human immunodeficiency virus type 1 vaccine. Antigen-specific increases in IFN-gamma were measured, which corresponded to antibody production, lymphoproliferation, and skin testing.     

Cytokine production in children with tuberculous infection and disease.

To determine if the manifestations of initial infection with Mycobacterium tuberculosis reflect changes in the balance of T cell cytokines, we evaluated cytokine production by M. tuberculosis-stimulated peripheral blood mononuclear cells (PBMCs) from 24 children with tuberculosis and 22 children who were healthy tuberculin reactors. PBMCs from patients with tuberculosis had lower production and mRNA expression of interferon gamma (IFN-gamma) than did PBMCs from healthy tuberculin reactors. IFN-gamma production was most severely depressed in patients with moderately advanced and far-advanced pulmonary disease and in malnourished patients. Production of IL-12, IL-4, and IL-10 was similar in tuberculosis patients and healthy tuberculin reactors. These results indicate that, during the initial immune response to M. tuberculosis, development of tuberculosis is associated with diminished IFN-gamma production, which is not due to reduced production of IL-12 or enhanced production of IL-4 or IL-10.     

Interleukin-10-secreting CD4 cells from aged patients with AIDS decrease in-vitro HIV replication and tumour necrosis factor alpha production

OBJECTIVE: To evaluate the impact of age on the proliferative response, cytokine profile and viral kinetics in AIDS patients treated successfully with antiretroviral drugs. METHODS: Peripheral blood mononuclear cells (PBMC), CD4 cell-depleted PBMC or CD4 T cells from young adult and aged HIV-1-infected patients were activated in vitro with anti-CD3 with or without interleukin (IL)-2. Lymphoproliferation and cytokines were measured after 3 days and in-vitro HIV-1 replication after 7 days. RESULTS: Both lymphoproliferation and cytokine [IL-1beta, tumour necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma)] secretion were higher in younger than in older AIDS patients. In cultures of cells derived from aged patients and activated by anti-CD3, IFN-gamma production was severely damage and IL-10 production was much higher. Although IL-2 addition to activated PBMC elevated IFN-gamma secretion, IL-10 production remained elevated in the aged group. The depletion of CD4 T lymphocytes from these cultures dramatically reduced released IL-10 in the older group but did not alter significantly IFN-gamma production. Interestingly, higher IL-10 levels produced by CD4 T cells were related to lower in-vitro HIV-1 replication, and the blockade of this cytokine by anti-IL-10 monoclonal antibody enhanced virus replication. This effect may be correlated with elevated TNF-alpha secretion. Finally, impaired IFN-gamma secretion detected in activated CD4 T cells obtained from aged patients was not directly correlated with high IL-10 production. CONCLUSIONS: Elevated IL-10 production by aged AIDS patients contributed considerably to control of HIV replication and to inhibition of TNF-alpha secretion but not to the reduced IFN-gamma production.     

Ex vivo evaluation of PPD-specific IFN-gamma or IL-5 secreting cells in the peripheral blood and lungs of patients with tuberculosis.

OBJECTIVES: To evaluate ex vivo purified protein derivative (PPD) specific Th1- and Th2-type functional responses in human tuberculosis (TB). DESIGN: IFN-gamma and IL-5 secreting cells were measured by a computer-assisted ELISPOT assay in the peripheral blood of patients with pulmonary TB, in patients with other respiratory diseases (control patients) and in tuberculin skin test negative or positive healthy controls. Moreover, the number of IFN-gamma or IL-5 spots was assessed in the bronchoalveolar lavage (BAL) cells of five patients with advanced TB and lung inflammation. RESULTS: The frequency of PPD-specific IFN-gamma secreting cells in TB patients was higher than in control patients and healthy subjects. Although the number of PPD-specific IL-5 spots was low, a trend towards a higher frequency was observed in the peripheral blood of TB patients. Patients with advanced TB and lung inflammation had an increased number of both PPD-specific IFN-gamma and IL-5 spots in BAL as compared to that in peripheral blood, but the IFN-gamma/IL-5 ratio was about two-fold lower. CONCLUSIONS: In human TB, the host response in the periphery is driven by a specific Th1-type cytokine response, whereas in the lungs of patients with advanced disease and lung inflammation, polarisation towards a Th2-like bias is observed.