T-cell response in human leishmaniasis

In the present communication we provide evidence for the existence of a Th1/Th2 dichotomy in the T-cell response to Leishmania antigens in human leishmaniasis. Our data suggest that the pattern of IL-4 and IFN-gamma response is polarised in these patients. Lymphocytes from individuals recovered from cutaneous leishmaniasis (CL) responded by IFN-gamma production following stimulation with Leishmania antigens whereas cells from patients recovered from visceral leishmaniasis (VL) showed a mixed pattern of IFN-gamma and IL-4 responses. The cells producing these cytokines were predominantly CD4+. Furthermore, IL-10 plays an important role in the development of post kala azar dermal leishmaniasis (PKDL) from VL. The balance between the parasitic-specific T-cell response plays an important regulatory role in determining the outcome of Leishmania infections in humans.     

Flow cytometric determination of cellular sources and frequencies of key cytokine-producing lymphocytes directed against recombinant LACK and soluble Leishmania antigen in human cutaneous leishmaniasis

Leishmaniasis, caused by infection with the protozoan parasite Leishmania, affects millions of individuals worldwide, causing serious morbidity and mortality. This study directly determined the frequency of cells producing key immunoregulatory cytokines in response to the recombinant antigen Leishmania homolog of receptors for activated kinase C (LACK) and soluble leishmania antigen (SLA), and it determined relative contributions of these antigens to the overall cytokine profile in individuals infected for the first time with Leishmania braziliensis. All individuals presented with the cutaneous clinical form of leishmaniasis and were analyzed for proliferative responses to LACK antigen and SLA, frequency of lymphocyte subpopulations (analyzed ex vivo), and antigen-induced (LACK and SLA) cytokine production at the single-cell level (determined by flow cytometry). The following were determined. (i) The Th1-type response previously seen in patients with cutaneous leishmaniasis is due to gamma interferon (IFN-gamma) production by several different sources, listed in order of contribution: CD4(+) T lymphocytes, CD4(-), CD8(-) lymphocytes, and CD8(+) T lymphocytes. (ii) SLA induced a higher frequency of lymphocytes producing IFN-gamma and tumor necrosis factor alpha (TNF-alpha) than did LACK. (iii) LACK induced an activation of monocyte populations as reflected by an increased percentage of CD14-positive cells. (iv) Neither SLA nor LACK induced detectable frequencies of cells producing interleukin-4 (IL-4) or IL-5. These data demonstrated a multifaceted immune response to SLA in human leishmaniasis involving Th1 CD4(+) T lymphocytes (IFN-gamma(+) and IL-10(-)/IL-4(-)), Tc1 CD8(+) T cells (IFN-gamma(+), and IL-10(-)/IL-4(-)), and a high frequency of TNF-alpha-producing lymphocytes. Moreover, it was determined that the recombinant antigen LACK acts as a weak inducer of Th1-type lymphocyte responses compared to SLA.     

Interferon-γ- and Tumour Necrosis Factor-α-Producing Cells in Humans who are Immune to Cutaneous Leishmaniasis

Individuals infected with Leishmania major usually acquire immunity to cutaneous leishmaniasis. In this study we have investigated peripheral blood mononuclear cells (PBMC) stimulated by Leishmania antigens in two groups of Sudanese individuals, one with a history of cutaneous leishmaniasis and one living in an area without the disease. The production of interferon (IFN)-gamma, tumour necrosis factor (TNF)-alpha and interleukin (IL)-10 was investigated in culture supernatants, and the cellular sources of IFN-gamma and TNF-alpha were identified. Cells from individuals with a history of cutaneous leishmaniasis produced significantly higher levels of IFN-gamma and TNF-alpha than cells from individuals without a history of the disease. Similar levels of IL-10 were found in the two groups. Flow cytometric analysis revealed high numbers of CD3+ cells producing IFN-gamma and TNF-alpha, and only a few CD3+ cells containing IL-10, in the PBMC cultures from the individuals with a history of cutaneous leishmaniasis. Interferon-gamma and TNF-alpha were predominantly produced by CD4+ T cells rather than CD8+ T cells. The results suggest that cellular immunity against cutaneous leishmaniasis is mediated predominantly through antigen-specific CD4+ T cells in individuals with a history of cutaneous leishmaniasis.     

Nickel-induced IL-10 down-regulates Th1- but not Th2-type cytokine responses to the contact allergen nickel

Whereas the involvement of Th1- and Th2-type cytokines in contact allergy to nickel (Ni) is well documented, the role of the regulatory cytokine IL-10 is less clear. We therefore investigated the impact of IL-10 on Ni-induced Th1- (IFN-gamma) and Th2-type (IL-4 and IL-13) cytokine responses in human peripheral blood mononuclear cells (PBMC). PBMC from 15 blood donors with reactivity to Ni (Ni-PBMC) and 8 control donors devoid of reactivity (control PBMC) were stimulated with Ni and the frequency of cytokine-producing cells and the levels of secreted cytokines were analysed by ELISpot (IL-4, IL-13 and IFN-gamma) and ELISA (IL-10, IL-13 and IFN-gamma), respectively. The Ni-induced response was further assessed in the presence of recombinant IL-10 (rIL-10) or neutralizing antibody to IL-10 and the phenotype of the Ni-specific cytokine-producing cells regulated by IL-10 was determined by cell depletion experiments. Ni induced IL-10 production in Ni-PBMC (mean, (range); 33.1 pg/ml (0-93.4 pg/ml)) but not control PBMC (2.2 pg/ml (0-14.9 pg/ml)) (P = 0.002). Ni also induced significant production of IL-4, IL-13 and IFN-gamma that correlated with the IL-10 response. Addition of rIL-10 down-regulated the Ni-induced production of all cytokines but with a more pronounced effect on IFN-gamma. However, neutralization of Ni-induced IL-10 enhanced the levels of IFN-gamma induced by Ni (P = 0.004) but did not affect the number of IFN-gamma-producing cells or the production of other cytokines. Cell depletion experiments suggested that the Ni-specific IFN-gamma (and Th2-type cytokine) producing cells were CD4(+) T cells. The impact of IL-10 on Ni-induced IFN-gamma responses by CD4(+) T cells suggests that an important role of IL-10 in vivo is to counteract the allergic reactions mediated by Th1-type cytokines.     

Leishmania donovani-reactive Th1- and Th2-like T-cell clones from individuals who have recovered from visceral leishmaniasis.

Infections in humans by Leishmania donovani parasites can result in a fatal disease, visceral leishmaniasis (VL), or in a self-limiting asymptomatic infection. In murine models of the infection employing Leishmania major, the course of the disease can be directed into a VL-like syndrome by interleukin-4 (IL-4)-producing Th2 cells, or cure may result by Th1 cells secreting gamma interferon (IFN-gamma). The present study examined the potential of human T cells to generate Th1 or Th2 responses to L. donovani. The profiles of IFN-gamma, IL-4, and lymphotoxin secretion after antigen stimulation were analyzed in a panel of L. donovani-reactive CD4+ human T-cell clones generated from individuals who had recovered from VL after antimonial treatment. Two of the T-cell clones produced large amounts of IL-4 without production of IFN-gamma, seven clones produced both IFN-gamma and IL-4, and eight produced only IFN-gamma. This is the first report of a Th1- and Th2-type response in human leishmaniasis. These results suggest that in analogy with murine models, there is a dichotomy in the human T-cell response to L. donovani infections. Preferential activation of IL-4-producing Th2-like cells may be involved in the exacerbation of human VL, whereas activation of IFN-gamma-producing Th1 cells may protect the host from severe disease. Identification of leishmanial antigens activating one or the other type of T cells will be important in the development of vaccines against leishmaniasis.     

Control of Leishmania infantum Infection Is Associated with CD8+ and Gamma Interferon- and Interleukin-5-Producing CD4+ Antigen-Specific T Cells

Visceral leishmaniasis is a severe and lethal disease caused by the protozoan parasites of the genus Leishmania. In areas where leishmaniasis is endemic, most infected individuals control the infection and remain asymptomatic; chemotherapy of visceral leishmaniasis restores some immunity which protects against relapses. In the present study, Leishmania-specific T-cell clones were established from six asymptomatic and five cured patients. Cytokines production by these clones was analyzed. A large fraction of the parasite-specific T-cell clones from asymptomatic patients were CD8(+) and produced high amounts of gamma interferon (IFN-gamma). Most CD4(+) T-cell clones from two asymptomatic subjects exhibited an unusual phenotype: production of high levels of IFN-gamma low levels of interleukin-4, (IL-4), but high levels of IL-5. In contrast, only few parasite-specific CD8(+) T-cell clones were obtained from cured patients after chemotherapy; moreover, CD4(+) T-cell clones from these patients exhibited an heterogeneous profile of cytokines from Th1-like to Th2-like phenotypes. These results point to CD8(+) T cells and to IL-5- and IFN-gamma-producing CD4(+) T cells as possible contributors to human resistance to Leishmania infection. They should stimulate new immunological approaches in the control of this disease.     

LACK-specific CD4(+) T cells that induce gamma interferon production in patients with localized cutaneous leishmaniasis during an early stage of infection

The profile of cytokines induced by soluble leishmania antigen (SLA) and the Leishmania homologue of the mammalian receptor for activated C kinase (LACK), a candidate vaccine against leishmaniasis, and the cellular source of the cytokines produced in response to these antigens were analyzed in patients infected with Leishmania guyanensis. Gamma interferon (IFN-gamma) and interleukin-10 (IL-10) were produced in response to LACK. Although LACK-specific CD4(+) cells producing IFN-gamma were isolated only during the early phase of infection (less than 30 days following the onset of infection), cells producing IL-10 in response to LACK were detected in all patients. CD4(+) T cells producing IFN-gamma and IL-13 were produced in response to SLA in all patients. SLA- and LACK-specific T cells are effector memory cells, as they are CD45RA(-) CCR7(-) CD4(+) T cells. CD4(+) T cells producing IFN-gamma are CD62L(-), and CD4(+) T cells producing IL-10 are CD62L(+), indicating that these cells have different tissue-homing capacities. These findings show that SLA and LACK induce both type 1 (IFN-gamma) and type 2 (IL-10 or IL-13) cell responses.     

Induction and abrogation of LACK reactive cells in the evolution of human leishmaniasis.

Peripheral blood mononuclear cells (PBMC) from cutaneous leishmaniasis patients with ongoing Leishmania aethiopica infection and individuals cured/under treatment from L. infantum or L. donovani infection were stimulated in vitro with LACK, the Leishmania homologue of receptors for activated C kinase. The LACK protein is conserved in related leishmanial species and is expressed both in the promastigote and amastigote stages of Leishmania. Our results show that LACK induced marked NK and some CD8+ cell proliferation in PBMC from cutaneous leishmaniasis patients with active disease. These responses were coupled with high levels of IFN-gamma and IL-10 production. At the concentration tested, the proliferative responses to freeze-thawed Leishmania antigen (Ft-Leish) were higher, while the levels of IFN-gamma were consistently lower than that of LACK. Although cells from individuals cured of leishmaniasis could respond to whole Leishmania lysate by proliferation and IFN-gamma production, there was no evident response to LACK. Ethiopian controls tested at the same time also showed LACK induced proliferation with IFN-gamma and IL-10 responses. Thus LACK reactivity in terms of proliferation and cytokine induction were present in cells from some healthy donors and most of the patients with active lesions, while this response was absent in individuals cured of L. infantum or L. donovani leishmaniasis. Since cure from leishmaniasis often results in life-long protection, and active but not cured patients showed in vitro responses to LACK stimulation, questions arose as to how this highly immunodominant molecule functions during human leishmanisasis. Some possible mechanisms are discussed.     

Cellular profile of cytokine production in a patient with visceral leishmaniasis: gammadelta+ T cells express both type 1 cytokines and interleukin-10

The cytokine profile of CD4+, CD8+ T cells, gammadelta+ T cells and natural killer (NK) cells (CD94+CD3-) was studied in a patient with visceral leishmaniasis (VL). The otherwise healthy, human immunodeficiency virus-negative patient acquired the disease in Tuscany, Italy. Diagnosis was made by demonstration of high concentrations of antibodies against Leishmania antigens in serum. Flow cytometry for the detection of intracellular interferon-gamma (IFN-gamma), interleukin (IL)-2, IL-4, IL-6, IL-10, IL-13 and tumour necrosis factor (TNF)-alpha expression in peripheral blood mononuclear cells stimulated with phorbol 12-myristate 13-acetate and ionomycin was performed, followed by treatment with liposomal amphotericin B. CD4+ cells were identified as major cytokine-expressing cells, capable of producing both type 1 and type 2 cytokines. A high frequency of IL-4- and IL-13-expressing CD8+ cells was noted. NK cells and gammadelta+ T cells, thought to be involved in innate host defences against Leishmania, expressed IFN-gamma and TNF-alpha. Ten per cent of gammadelta+ T cells expressed IL-10, predominantly together with IFN-gamma, suggesting additional immune-regulatory roles for this T-cell subset in VL.     

IL-15 in human visceral leishmaniasis caused by Leishmania infantum

Interleukin (IL)-15 is a recently discovered cytokine with the ability to stimulate the proliferation activity of Th1 and/or Th2 lymphocytes. Here, we investigated the involvement of IL-15 in the immune response to Leishmania infantum infection by studying patients with visceral leishmaniasis (VL). We found that IL-15 is produced by leishmanial antigen (LAg)-stimulated peripheral blood mononuclear cells (PBMC) from active VL patients at a significantly higher level than those produced by cells from healed VL subjects or healthy controls. A significant increase in IL-15 serum blood levels was also observed in acute VL patients compared with healed ones. Furthermore, recombinant IL-15 had an appreciable effect in vitro in reducing IL-4 and increasing the production of IL-12 in response to LAg, but it was ineffective in altering the production of interferon-gamma (IFN-gamma). The production of endogenous IL-15 in acute VL patients appeared to be insufficient to activate both IFN-gamma and IL-12, as attested by the absence of modification of these two cytokines by neutralization experiments in the presence of anti-IL-15 monoclonal antibodies (MoAB). On the contrary, the neutralization of IL-15 increased IL-4 production. Together, these results indicate that endogenous IL-15 plays a role in the suppression of Th2-type cytokines, even though it does not enhance the production of Th1 cytokines in acute VL patients. Since IL-15, in the presence of anti-IL-4 MoAb, caused a further increase in IL-12 production and led to a significant production of IFN-gamma, one of its indirect effects on Th1 cell activation could be due to the latter's effect on Th2 cytokines such as IL-4. Therefore, our observations indicate that there is a potential for IL-15 to augment the T-cell response to human intracellular pathogens.     

CD4+ CCR5+ and CD4+ CCR3+ lymphocyte subset and monocyte apoptosis in patients with acute visceral leishmaniasis

The potential involvement of apoptosis in the pathogenesis of visceral leishmaniasis (VL) was examined by studying spontaneous and Leishmania antigen (LAg)-induced apoptosis using cryopreserved peripheral blood mononuclear cells (PBMC) of Sicilian patients with VL. Results indicate that monocytes and T lymphocytes from acute VL patients show a significantly higher level of apoptosis compared with that observed in healed subjects. The percentage of apoptotic cells was higher in monocytes than in T lymphocytes. T cells involved in programmed cell death (PCD) were mainly of the CD4(+) phenotype. In particular, the T helper 1-type (Th1) subset, as evaluated by chemokine receptor-5 (CCR5) expression, is involved in this process. Cell death in Th1-type uses a CD95-mediated mechanism. Furthermore, Th1-type CCR5(+) cells are prone to cell suicide in an autocrine or paracrine way, as attested by enhanced expression of CD95L in acute VL patients. The reduction in Th1-type cells by apoptosis was confirmed by the decrease in interferon-gamma secretion. In conclusion, apoptosis of monocytes, CD4(+) and CD4(+) CCR5(+) T cells could be involved in the failure of cell mediated immunity that is responsible for severe immune-depression in VL.     

Cytokine profile of bronchoalveolar lavage-derived CD4(+), CD8(+), and gammadelta T cells in people with asthma after segmental allergen challenge

T cell-derived cytokines play an important role in the pathogenesis of allergic asthma, but little is known about the cytokine profile of their different subsets. The aim of the present study was to investigate the cytokine production potential of CD4(+), CD8(+), or gammadelta(+) T cells derived from the bronchoalveolar space of mild atopic asthmatic subjects (n = 11) and nonatopic control subjects (n = 9) before and 24 h after segmental allergen challenge. The cytokine production was determined using the technique of intracellular cytokine detection by flow cytometry. Comparing asthmatic with control subjects we found no difference in the percentage of CD4(+), CD8(+), or gammadelta T cells in the bronchoalveolar lavage fluid before and after allergen challenge. Before allergen challenge the proportion of cells producing the cytokines interferon (IFN)-gamma, interleukin (IL)-2, IL-4, IL-5, and IL-13 was not different in CD4(+) and CD8(+) cells. The major difference between the groups was an increased percentage of positive-staining cells for the T helper-(Th)2-cytokines IL-5 and IL-13 in the gammadelta T-cell subset. After allergen challenge, all T-cell subsets revealed a decreased proportion of cells producing the Th1-type cytokines IFN-gamma and IL-2. The percentage of IL-4- and IL-5-positive cells did not change in all subsets, and there was a decreased proportion of IL-13- positive cells in the CD4(+) subset. These findings indicate an increased Th2-cytokine profile in gammadelta T cells. After allergen challenge, the dysbalance between Th1 and Th2 cytokines was further accentuated by a reduction in Th1 cytokine-producing T cells.     

Regulatory effects of Th1-type (IFN-γ, IL-12) and Th2-type cytokines (IL-10, IL-13) on parasite-specific cellular responsiveness in Onchocerca volvulus-infected humans and exposed endemic controls

The present study investigated in vitro the regulatory effects of T helper 1 (Th1)-type (interferon-gamma, IFN-gamma; interleukin-12, IL-12) and Th2-type cytokines (IL-10, IL-13) on Onchocerca volvulus-specific cellular reactivity in onchocerciasis patients, and in exposed endemic control individuals presenting no clinical and parasitological signs of disease. In both patients and controls, addition of IL-10 dose-dependently depressed O. volvulus antigen (OvAg)-specific cellular proliferation, and peripheral blood mononuclear cells (PBMC) from patients who were more sensitive to the suppressive effect of IL-10 than those from endemic controls. However, neutralization of IL-10 by specific antibody did not reverse cellular hyporesponsiveness. In contrast to the inhibitory effects of IL-10, exogenous IL-12 and IL-13 augmented PBMC proliferative responses to OvAg both in patients and controls (P<0. 01) and neutralizing of IL-12 or IL-13 significantly decreased OvAg-specific proliferation in both groups. Exogenous IFN-gamma did not activate OvAg-specific proliferative responses in patients, but anti-IFN-gamma antibodies abolished cellular reactivity to OvAg. Antibody to IL-10 increased (P<0.05) OvAg-specific production of IL-5, IL-12 and IFN-gamma, and inversely, anti-IFN-gamma enhanced IL-10 (in patients only) and IL-5 and IL-13 in both patients and controls. Neutralization of IL-12 activated OvAg-specific production of IL-10, IL-2 and IFN-gamma. In conclusion, despite of an overproduction of IL-10, which suppressed cellular reactivity in patients and control individuals, OvAg-specific cellular responses were activated in vitro by exogenous supplementation with IL-12 and IL-13, and cytokine neutralization experiments confirmed that distinct type 1 and type 2 T helper cytokines cross-regulate expression and magnitude of O. volvulus-specific cellular responsiveness in humans.     

The development of post-kala-azar dermal leishmaniasis (PKDL) is associated with acquisition of Leishmania reactivity by peripheral blood mononuclear cells (PBMC)

PKDL develops in about 50% of Sudanese patients treated for visceral leishmaniasis (kala-azar). Patients with kala-azar were entered into this study and followed for a period of up to 2 years. During follow up 12 patients developed PKDL and eight did not. Proliferative responses and cytokine production to Leishmania donovani and control antigens were measured in vitro using PBMC isolated at the time of diagnosis of kala-azar, after treatment of visceral leishmaniasis, during follow up, and at the time of diagnosis of PKDL. Proliferative responses and interferon-gamma (IFN-gamma) production were low at diagnosis and increased after treatment of kala-azar in both patients who developed (group 1) and those who did not develop PKDL later (group 2). In group 1, development of PKDL was always associated by an increased PBMC response to Leishmania antigen in proliferation and IFN-gamma production assays. There were no differences in Leishmania antigen-induced production of IL-4, IL-5 and IL-10 between or within the two groups. We have previously shown that Leishmania parasites spread to the skin during visceral leishmaniasis and proposed that PKDL was the result of an immunological attack on parasites, which have survived in the skin despite the drug treatment. The finding that PKDL develops after treatment of kala-azar as Leishmania-reactive T cells start to circulate in peripheral blood in sufficient numbers to be detected in in vitro assays supports this hypothesis.     

Cloning, expression and antigenicity of the L. donovani reductase

The protozoan parasite Leishmania undergoes a morphological and biochemical transformation from the promastigote to the amastigote form during its life cycle, which is reflected in the expression of stage-specific proteins. One of these proteins shows homology to a superfamily of reductase proteins. We have cloned the reductase gene from L donovani and have shown that it differs in only one nucleotide from the L. major homologue, resulting in one amino acid change. A cytosine (C) to guanine (G) transposition in the coding sequence leads to a nonconserved substitution of asparagine (N) for lysine (K). Only 2 of 22 plasma samples from patients with visceral leishmaniasis were found to have detectable anti-reductase antibodies and peripheral blood mononuclear cells (PBMC) from one of three individuals previously infected with visceral leishmaniasis proliferated in the presence of recombinant reductase protein. Interestingly, 6 of 10 PBMC isolated from Danish controls proliferated in the presence of the reductase protein. Intracellular IFNgamma was found in a significant percentage of cells in all the tested PBMC cultures from Danes, whereas IL4 was only found in a small proportion of cells, or not at all. The results indicate the presence of cross-reacting CD45R0 memory T-cells in individuals not exposed to Leishmania. Several previous studies have shown that T-cells from nonexposed individuals often respond to crude Leishmania antigen preparations. The present study suggests that this reactivity is partly caused by T-cells recognising L. donovani reductase.     

Antigen specific correlations of cellular immune responses in human leishmaniasis suggests mechanisms for immunoregulation

Regulation of the immune response directed against Leishmania is critical for the establishment of effective control of the disease. It is likely that some types of immune responses directed against Leishmania can lead to more severe clinical forms of leishmaniasis causing a poor control of the pathogen and/or pathology, while others lead to resolution of the infection with little pathology as in cutaneous leishmaniasis. To gain a better understanding of the possible role that subpopulations of T cells, and their associated cytokines have on disease progression and/or protective immune responses to L. braziliensis infection, a detailed study of the frequency of activated and memory T cells, as well as antigen specific, cytokine producing T cells was carried out. Following the determination of cytokine producing mononuclear cell populations in response to total Leishmania antigen (SLA), and to the recombinant antigen LACK, correlation analysis were performed between specific cytokine producing populations to identify models for cellular mechanisms of immunoregulation in human cutaneous leishmaniasis. These studies have shown: (1) a positive correlation between ex vivo CD45RO frequencies and antigen specific cytokine (IFN-gamma or IL-10) producing cells; (2) a negative correlation between ex vivo CD69 expression and the frequency of IFN-gamma producing cells; (3) a positive correlation amongst SLA specific, IFN-gamma or TNF-alpha and IL-10 producing lymphocytes with one another; and (4) a higher frequency of IL-10 producing, parasite specific (anti-SLA or anti-LACK), lymphocytes are correlated with a lower frequency of TNF-alpha producing monocytes, demonstrating an antigen specific delivery of IL-10 inducing negative regulation of monocyte activity.     

Cytokine profiles of patients infected with Mycobacterium ulcerans and unaffected household contacts

Mycobacterium ulcerans, the cause of Buruli ulcer, is an environmental mycobacterium with a distinct geographic distribution. The reasons why only some individuals who are exposed to M. ulcerans develop ulcers are not known but are likely to reflect individual differences in the immune response to infections with this bacterium. In this study, we investigated cytokine profiles of peripheral blood mononuclear cells (PBMC) from 23 Buruli ulcer patients and 25 household contacts in a region of Australia where Buruli ulcer is endemic. The results showed that following stimulation with M. ulcerans or Mycobacterium bovis BCG, PBMC from Buruli ulcer patients mounted a Th2-type response, which was manifested by the production of mRNA for interleukin 4 (IL-4), IL-5, IL-6, and IL-10, whereas unaffected contacts responded mainly with the Th1 cytokines gamma interferon (IFN-gamma) and IL-12. For example, mRNA for IL-4 was detected in 18 of 23 patients but in only 3 of 25 control subjects (P < 0.0001). By contrast, PBMC from 21 of 25 unaffected individuals produced IFN-gamma compared with 3 of 23 patients (P < 0.0001). IFN-gamma release following stimulation with mycobacteria was markedly reduced in affected subjects. Frequencies of antibodies to M. ulcerans in serum samples from affected and unaffected subjects were similar, indicating that many of the control subjects had been exposed to this bacterium. Together, these findings suggest that a Th1-type immune response to M. ulcerans may prevent the development of Buruli ulcer in people exposed to M. ulcerans, but a Th-2 response does not.     

Evaluation of Th1/Th2 ratio and cytokine production profile during acute exacerbation and convalescence in asthmatic children.

BACKGROUND: Th2-type cytokines, such as IL-4 and IL-5, are generally believed to play an important role in the pathogenesis of allergic asthma. In contrast, Th1-type cytokine, especially interferon (IFN)-gamma, is thought to have a downregulatory effect on Th2 immune response cells. Thus, the imbalance of Th1 and Th2 cells may be a key factor in relation to disease severity. OBJECTIVE: The aim of this study is to examine the changes in the Th1/Th2 ratios and cytokine production profiles of asthmatic children at acute attacks and convalescent stages. METHODS: Twelve asthmatic patients were included in this study. The percentages of IFN-gamma- or IL-4-producing cells were determined with a flow-cytometric method of intracellular protein detection. Fresh whole blood obtained from normal controls and patients at two stages was stimulated with brefeldin A, phorbol myristate acetate, and ionomycin for 4 hours. Cells were fixed and stained intracellularly with fluorescein isothiocyanate- or phycoerythrin-conjugated antibody specific to each cytokine in combination with anti-CD4. ELISA assays were applied to measure cytokine concentrations of supernatant from peripheral blood mononuclear cells (PBMC) activated with phorbol myristate acetate and ionomycin for 24 hours. RESULTS: Among CD4+ cells, the percentage of IL-4+ cells decreased significantly from 8.18 +/- 4.77% at acute attacks to 4.18 +/- 1.26% (P < .020) at convalescence. The percentage of IFN-gamma+ cells also decreased from 13.49 +/- 4.21% to 9.03 +/- 5.42% (P < .052). The Th1/Th2 ratios of patients at the two stages were similar, and both were lower than the normal controls. Significantly higher IL-5 and lower IFN-gamma production were detected from activated PBMC of asthmatic patients than normal controls. CONCLUSIONS: The decrease of IFN-gamma+ and IL-4+ cells detected at the single-cell level may explain the potential mechanism of convalescence from acute asthma attacks. High Th1/Th2 ratio and low IL-5 production from the PBMC of normal controls support the idea of a biased Th2 immune response in asthmatic patients.