Broad heparin-binding haemagglutinin-specific cytokine and chemokine response in infants following Mycobacterium bovis BCG vaccination

Heparin-binding haemagglutinin (HBHA)-specific immune responses have been linked to protection against tuberculosis (TB). We investigated the hypothesis that BCG vaccination of human infants primes an HBHA-specific response, using multiplex to measure secreted cytokines and chemokines following HBHA and Mycobacterium tuberculosis purified protein derivative (PPD) stimulation of diluted whole blood samples from BCG-vaccinated or -unvaccinated infants. Of 42 analytes measured, 24 and 32 significant, BCG-associated increases were detected in response to HBHA and PPD, respectively. Both response profiles included Th-1, Th-2, Th-17 and inflammatory cytokines and chemokines (e.g. IFN-γ, TNF-α, IL-5, IL-10, IL-13, IL-17, MIP-1α and MIP-1β). We also found that six of the seven responses most closely correlated with IFN-γ were common to both HBHA and PPD. Notably, all HBHA-specific secretion of cytokines and chemokines from infant samples was dependant on previous BCG vaccination. Also, long-term persistence of HBHA-specific responses was found in adolescents with evidence of infant BCG vaccination. This study demonstrates for the first time BCG priming of an HBHA-specific immune response in infants that is characterised by a broad cytokine and chemokine signature. It also suggests a number of BCG vaccination associated, HBHA-induced responses that should be useful for future studies of biomarkers of protection against TB.     

Recombinant BCG coexpressing Ag85B,ESAT-6 and Rv3620c elicits specific Th1 immune responses in C57BL/6 mice

Tuberculosis (TB) remains to be an enormous global health problem. The inconsistent protection efficacy of Bacille Calmette-Guérin (BCG) calls for new vaccines for TB. One choice to improve the efficacy of BCG vaccine is recombinant BCG (rBCG). Experimental evidences have revealed that Ag85B, ESAT-6 and Rv3620c are important immunodominant antigens of Mycobacterium tuberculosis. In this study, we have constructed a novel rBCG expressing fusion protein Ag85B-ESAT6-Rv3620c and evaluated the immunogenicity of this rBCG in C57BL/6 mice. Results show that there is a strong TB-specific CD4⁺ and CD8⁺ T lymphocytes proliferation in mice immunized with this rBCG vaccine. A single dose immunization of rBCG could induce a significantly strong Th1 immune response characterized by an increasing ratio of antigen-specific IgG2b/IgG1 as well as a high expression level of Th1 cytokines such as IFN-γ, TNF-α and IL-2. This conclusion was confirmed by a decreased secretion of Th2 cytokine IL-10. Moreover, this rBCG induced a strong humoral response in mice with an increasing antigen-specific IgG titer. Therefore, we concluded that this rBCG could significantly increase both Th1 type cellular immune response and antigen-specific humoral response compared with BCG. The above observations demonstrated that rBCG::Ag85B-ESAT6-Rv3620c is a potential candidate vaccine against M. tuberculosis for further study.     

Balance of Th1/Th2 Cytokines Associated with the Preventive Effect of Incomplete Freund's Adjuvant on the Development of Adjuvant Arthritis in LEW Rats

Complete Freund's adjuvant (CFA) could induce adjuvant arthritis (AA) in LEW rats and incomplete Freund's adjuvant (IFA) could induce oil induced arthritis (OIA) in DA but not in LEW rats. Lymph node cells (LNCs) from these AA and OIA rats showed increased mRNA expression of IFN-gamma, IL-2 and TNF-alpha but not IL-4. LNCs from IFA immunized LEW rats showed increased expression of IL-4, reduced expression of IFN-gamma and TNF-alpha and no IL-2, in contrast to IFA immunized DA rats. The pretreatment of IFA before CFA challenge could completely prevent AA in LEW rats and their LNCs showed increased expression of IL-4 and IFN-gamma but not IL-2 and TNF-alpha. In F1 (LEW x DA) rats, IFA could not induce OIA but the pretreatment of IFA before CFA challenge could induce very mild AA with 80% incidence, LNCs showing an elevated expression of all the above cytokines. These findings suggest that increased Th1 cytokine expression is associated with disease development and that increased IL-4 expression or the balance of Th2 over Th1 cytokine expression plays an important regulatory role in disease development.     

Serum Th1 and Th2 cytokine balance in patients of superficial transitional cell carcinoma of bladder pre- and post-intravesical combination immunotherapy

Combination therapy with intravesical bacillus Calmette-Guerin (BCG) plus interferon-α2b (IFN-α2b) for superficial transitional cell carcinoma (TCC) seems to be immune-dependent and activation of Th1 immune response is required for clinical efficacy. The present study evaluates circulating serum cytokine profiles (Th1/Th2 cytokines IFN-γ, IL-2 TNF-α, IL-4, IL-6 and IL-10) in 41 bladder cancer patients prior to transurethral resection of tumor (TURBT) (pre-therapy), and following intravesical combination immunotherapy (post-therapy) and their association with recurrence. Mean levels of IL-2 and TNF-α were significantly reduced while IL-4, IL-6 and IL-10 were significantly enhanced in pre-therapy samples as compared to controls. Mean levels of IFN-γ, IL-2 and TNF-α were significantly increased while IL-4 and IL-10 were significantly reduced in patients after instillation of combination immunotherapy. These findings suggest that bladder cancer patients develop Th2 dominant status with deficient type 1 immune response that shows tendency to reversal following therapy.     

Effect of glutamine on Th1 and Th2 cytokine responses of human peripheral blood mononuclear cells

Decreased glutamine concentrations are found in patients with catabolic stress and are related to susceptibility to infections. In this study, we evaluated the role of glutamine in Th1/Th2 cytokine responses. Peripheral blood mononuclear cells were stimulated with phytohemagglutinin (PHA), live attenuated bacillus Calmette-Guérin (BCG), or measles virus in the presence of different glutamine concentrations. We found that glutamine at an optimal concentration (0.6 mM) significantly enhanced PHA-stimulated lymphocyte proliferation as well as Th1 [interferon-gamma (IFN-gamma) and interleukin-2 (IL-2)] and Th2 cytokine (IL-4 and IL-10) production. In the absence of glutamine, BCG and measles virus elicited minimal lymphocyte proliferation, whereas BCG enhanced Th1 cytokine response and measles virus promoted Th2 cytokine response. Interestingly, addition of glutamine promoted the BCG-elicited Th1 cytokine response (IFN-gamma), but suppressed the measles-induced Th2 cytokine response (IL-10). These results suggest that appropriate glutamine levels may influence host responses to different antigens and microorganisms. Furthermore, predominately Th1, but not Th2, cytokine responses required the presence of optimal concentrations of glutamine.     

Vaccinations with T-helper type 1 directing adjuvants have different suppressive effects on the development of allergen-induced T-helper type 2 responses

BACKGROUND: Allergen-induced T-helper type 2 (Th2) responses can be inhibited with Th1 directing vaccines. However, studies comparing the efficacy of the different adjuvants have not been performed in detail. OBJECTIVE: For this reason we compare the effects of live Bacillus-Calmette-Guerin(BCG), heat-killed (hk)-BCG, CpG-ODN (oligodeoxynucleotide) or PPD on the development of allergen-induced Th2 responses in mice. METHODS: Ovalbumin (OVA)-specific allergic responses were induced in C57BL/6 mice by two intraperitoneally (i.p.) applications of OVA/alum followed by the intranasal challenge with OVA. The different Th1-inducing adjuvants were applied to the mice together with OVA/alum i.p. during the OVA-sensitization period and, subsequently, different parameters of allergic immune responses were evaluated. RESULTS: All the adjuvants were effective in inhibiting the development of allergen-induced airway eosinophilia, mucous production and, with the exception of PPD, also airway hyper-reactivity, when they were applied together with OVA/alum. However, allergen-specific IgG1 and IgE serum levels were only reduced in live BCG- and PPD-treated mice. Suppression of airway eosinophilia was not observed in IFN-gamma- or IL-12-deficient mice (hk-BCG, CpG-ODN and PPD). Interestingly, live BCG was still able to suppress allergen-induced Th2 responses in the absence of either IFN-gamma or IL-12. When mice vaccinated with the different adjuvants together with OVA/alum were subjected to a second period of OVA/alum immunization, only live and hk-BCG were able to efficiently suppress the development of airway inflammation. This effect could be adoptively transferred by splenic CD4(+) T cells. CONCLUSIONS: Taken together our data suggest that live BCG>hk-BCG>CpG-ODN >PPD are effective in suppressing allergen-induced Th2 responses. The degree of suppression and the component of the Th2 response affected (airway inflammation vs. the production of allergen-specific IgE and IgG1) were dependent upon the adjuvant used and how it was applied. Our results contribute to the design of novel vaccines protecting humans from developing allergic disorders.     

Polarization of PPD-specific T-cell response of patients with tuberculosis from Th0 to Th1 profile after successful antimycobacterial therapy or in vitro conditioning with interferon-alpha or interleukin-12

The T helper (Th) 1/Th2 balance in the T-lymphocyte response to purified protein derivative (PPD) was evaluated at the clonal level in six Italian and five Gambian patients with pulmonary tuberculosis (TB) before and after antimycobacterial therapy, as well as in five Gambian and four Italian healthy immune control subjects. In untreated patients, most PPD-specific clones derived from either peripheral blood or pleural effusions showed a Th0 cytokine profile (production of both interferon [IFN]-gamma and interleukin [IL]-4/IL-5). After 6 mo of therapy and clinical healing, most PPD-specific clones showed a polarized Th1 profile (production of IFN-gamma but not IL-4/IL-5) in both Italian and Gambian patients. The Th1 polarization was less marked in Gambian than in Italian patients and failed to occur in another group of four Italian patients who experienced treatment failure. The cytokine profile observed after successful therapy in patients with TB was similar to that found in healthy control subjects. T-cell clones of undefined specificity generated from PPD-stimulated cultures showed a similar Th0/Th2 bias in Gambian individuals and Italian patients with treatment failure. The Th0/Th2-biased responses in Gambian patients before therapy could be modulated in vitro by IFN-alpha or IL-12, which induced a Th1 polarization of both PPD-specific and bystander T cells. Our data show that active TB associates with a predominant Th0 response to mycobacterial antigens that could play a role in the pathogenesis of the disease. Adjunctive immunotherapy using Th1-polarizing cytokines could increase host defense against mycobacteria and accelerate healing.     

Bacille Calmette Guérin and interleukin-12 down-modulate interleukin-4-producing CD4+ NK1+ T lymphocytes

Early production of interleukin-12 (IL-12) by macrophages and of IL-4 from CD4⁺ NK1⁺ T cells influence development of the acquired immune response against infectious agents, namely differentiation of interferon-γ-secreting T helper 1 (Th1) cells against intracellular pathogens and of IL-4-producing Th2 cells against helminths. Evidence has been presented for transient convertibility of Th1 and Th2 cells in the presence of the polarizing cytokines IL-4 or IL-12, respectively. Moreover, it is likely that IL-4 dominates over IL-12, suggesting that Th2 cell development is preferred in the presence of both cytokines. Mycobacterium bovis Bacille Calmette Guérin (BCG) and IL-12 are potent inducers of Th1 responses. Here we show that BCG and IL-12 down-modulate IL-4-producing CD4⁺ NK1⁺ TCRα/β^intermediate liver lymphocytes. Our data provide further insights into the mechanisms by which BCG and IL-12 may promote unrestricted development of Th1 responses in vivo: BCG and IL-12 not only provide the positive stimuli for Th1 cell differentiation, but also interfere with antagonizing signals.     

Adjuvant effect of zinc oxide on Th2 but not Th1 immune responses in mice

Background and aim: We investigated the effect of zinc oxide (ZnO) on Th1 and Th2 immune responses in mice.

Material and methods: Mice were intraperitoneally administered with ovalbumin (OVA) with or without varying doses of ZnO (day 0). On day 21, anti-OVA IgG, IgG2a, IgG1, and IgE antibodies in sera, OVA-specific proliferative responses of spleen cells, and production of Th1 cytokines including IFN-γ as well as Th2 cytokines such as IL-4 and IL-5 were measured.

Results: The results showed that administration of OVA with ZnO was followed by greater increases in anti-OVA IgG and the antigen-specific splenocyte proliferation compared to that of OVA alone. The production of anti-OVA IgG1 and IgE and secretion of IL-4 and IL-5 were markedly enhanced by ZnO. The enhancing effect of ZnO on these Th2 responses was as strong as aluminium hydroxide (Alum) that was widely used as an adjuvant. In contrast, treatment with OVA plus ZnO failed to affect production of anti-OVA IgG2a as well as IFN-γ. It was also observed that ZnO had a stimulating effect on the secretion of the proinflammatory cytokine IL-17 from a new lineage of effector Th cells.

Conclusion: These results suggest that ZnO appears to have an adjuvant effect on the immune system, especially Th2 but not Th1 immune responses.     

泡球蚴原头蚴抗原和卡介苗对Th1/Th2转录因子和细胞因子的影响

目的探讨泡球蚴原头蚴抗原和卡介苗免疫小鼠对泡球蚴攻击感染的调节机制。方法用实时定量聚合酶链反应检测鼠脾组织中GATA-3及T-bet的mRNA表达水平;酶联免疫吸附法检测鼠血清中白介素4和γ干扰素的含量。结果卡介苗免疫攻击组与PBS对照组的转录因子(T-bet mRNA)和其标志性细胞因子(INF-γ)的表达量,差异有统计学意义(P<0.05)。结论实验证明卡介苗(BCG)有上调Th1型免疫反应的作用,用BCG可以干预或治疗由泡球蚴抗原诱导的晚期泡球蚴(AE)动物的免疫抑制状态。     

In vitro levels of interleukin 10 (IL-10) and IL-12 in response to a recombinant 32-kilodalton antigen of Mycobacterium bovis BCG after treatment for tuberculosis

Cell-mediated immunity plays a major role in conferring protection against tuberculosis (TB) on an individual. It is not known whether the immune status correlates with the bacterial load or whether the immunity improves after treatment. Also, it may be important to monitor treatment by being able to discriminate between active disease and successfully treated TB. The main aim of this study was to investigate the usefulness of a recombinant 32-kDa antigen (r32-kDa Ag) of Mycobacterium bovis BCG (Ag85A-BCG) as a diagnostic marker in patients being treated for TB. Specifically, the in vitro T-cell assays and the release of interleukin-12 (IL-12) (Th1-type cytokine) and IL-10 (Th2-type cytokine) in response to the r32-kDa Ag of BCG were assayed in patients with either pulmonary (sputum positive/negative, n = 74) or extrapulmonary TB (n = 49) and healthy controls. The proliferative responses of stimulated cells at 0, 2 to 4, and 6 months of treatment increased and were highly significant (P < 0.000) compared to the responses in controls. The increase in IL-12 and decrease in IL-10 release suggest that there is cytokine expression modification during different stages of TB, and treatment seems to have an influence on the levels of these cytokines, suggesting an augmentation in the protective responses. The in vitro response to the M. bovis BCG r32-kDa Ag may be useful in monitoring treatment of TB.     

Dose-dependent synergy of Th1-stimulating cytokines on bacille Calmette-Guérin-induced interferon-gamma production by human mononuclear cells

Successful bacille Calmette-Guérin (BCG) immunotherapy of bladder cancer depends on the proper induction of a T helper-type 1 (Th1) immune response. In this study we investigated the possible involvement of Th1-stimulating cytokines in BCG-induced interferon (IFN)-gamma production as well as their potential roles in enhancing BCG-induced IFN-gamma from human peripheral blood mononuclear cells (PBMCs). BCG efficiently induced IFN-gamma production by PBMCs in a dose-dependent manner. Neutralization of endogenous cytokines interleukin (IL)-2, IL-12 and IFN-alpha reduced BCG-induced IFN-gamma by 38%, 67% and 49%, respectively. Although single recombinant (r) IL-2, rIL-12 and rIFN-alpha induced no or a marginal amount of IFN-gamma, a combination of any two or three cytokines increased IFN-gamma production. When BCG (a subsaturated dose) was combined with mono, dual or triple cytokines, a synergy on IFN-gamma production was observed. Such a synergy was readily achievable even when minimal or low doses of cytokines were used. No saturation of IFN-gamma production was observed even when a subsaturated BCG dose was combined with very high doses of cytokines. A robust IFN-gamma production was also observed when a minimal BCG dose was combined with minimal doses of triple cytokines. In addition, we demonstrated that IL-2- and IFN-alpha-expressing rBCGs were superior to wild-type BCG for PBMC IFN-gamma induction and that combination of both rBCGs showed a synergy in IFN-gamma production. Taken together, these results suggest that combination of BCG with certain exogenous or endogenous (expressed by rBCGs) Th1-stimulating cytokines is a rational candidate for further study in bladder cancer treatment.     

Effect of interleukin-10 on dendritic cell maturation and function

The main function of dendritic cells (DC) is to induce the differentiation of naive T lymphocytes into helper cells producing a large array of lymphokines, including interleukin (IL)-2; interferon-γ (IFN-γ), IL-4, IL-5 and IL-10. The potent immunostimulatory properties of DC develop during a process of maturation that occurs spontaneously in vitro. Since IL-10 has been shown to inhibit Th1 responses, we determined its effect on DC maturation and accessory function. Our data show that DC that have undergone maturation in vitro in the presence of IL-10, have an impaired capacity to induce a Th1-type response in vivo, leading to the development of Th2 lymphocytes. Their inability to promote the synthesis of IFN-γ seems to correlate with a decreased production of IL-12, an heterodimeric cytokine necessary for optimal generation of Th1-type cells. These results suggest that IL-10 skews the Th1/Th2 balance to Th2 in vivo by selectively blocking IL-12 synthesis by the antigen-presenting cells that play a role of adjuvant of the primary immune response. The cytokines present in the environment at the presentation step may, therefore, determine the class of the immune response induced by DC in vivo, i.e. Th0 Th1 and/or Th2.     

The Changes in the T helper 1 (Th1) and T helper 2 (Th2) Cytokine Balance During HIV-1 Infection are Indicative of an Allergic Response to Viral Proteins that may be Reversed by Th2 Cytokine Inhibitors and Immune Response Modifiers – a Review and Hypothesis

The HIV-1 infection in humans induces an early cellular immune response to react to the viral proteins with a cytotoxic T cell (CTL) response that fails to inhibit virus replication and the spread of the virus. It became evident that the progression of the disease causes chronic changes to the immune system of which a gradual increase in IgE antibodies is one of its features. When the HIV-1 epidemic began, the relation between the gradual increase in IgE content and AIDS was not understood, but later it became a marker for disease prognosis. The advances in the knowledge on T helper 1 (Th1) and T helper 2 (Th2) cells revealed that Th1 cells produce cytokines that stimulate the proliferation of CTLs. Th2 cells produce cytokines that are responsible for the activation of the humoral immune response in healthy people. Studies on both Th1 and Th2 cytokine synthesis revealed an aberration in HIV-1 infected people. Clerici and Shearer presented a hypothesis (1993) whereby Th1 cell activity declines and Th2 activity increases (the Th1 --> Th2 switch hypothesis) in HIV-1 infected people. In fact, experiments concerning this hypothesis ultimately supported the premise that the switch involves a critical change in the cytokine balance, which leads to the contraction of AIDS. However, the research community must still discern why such a Th1 --> Th2 switch takes place in infected people and how it can be reversed. The present review points to the fact that a similar Th1 --> Th2 switch constitutes the response of allergic people to environmental allergens. HIV-1 patients and allergic people that are exposed to allergens respond with an increased synthesis of Th2 cytokines and IgE, together with a decrease in Th1 cytokines. The studies on allergen-induced Th2 cells revealed that the Th2 cytokine IL-4 induces B cells to synthesize IgE, and cytokine IL-5 is the inducer of eosinophilia, just as in HIV-1 infection. The difference between the HIV-1 infection and allergies is the ability of IL-4 to induce the synthesis in T cells of the HIV-1 coreceptor CXCR4 that selects from the replicating virus a syncytium-inducing (SI) virus, a variant virus that replicates rapidly. The present hypothesis implicates the viral proteins in the induction of Th2 cytokine synthesis. This suggests that in viral proteins, allergen-like domains may be responsible for the activation of Th2 cytokine synthesis. Based on the analogy of the responses of humans to allergens and HIV-1, the following hypotheses is suggested: (a) Removal of allergen-like domains from viral genes by genetic engineering may provide viral proteins for vaccine development. (b) Attempts to treat allergic patients with IL-4 receptor inhibitors suggests that the "Th2 --> Th1 Reversion" constitutes a possible approach to inhibiting the Th2 cytokines and inducing a revival of the anti-viral Th1 response.     

小鼠皮下感染着色芽生菌病皮损中局部细胞免疫功能研究

目的 利用小鼠皮下着色芽生菌病模型,研究在不同的病程发展阶段其T细胞免疫功能的变化.方法 足垫局部皮下注射法建立小鼠的裴氏着色霉感染的着色芽生菌病模型,通过免疫组织化学技术,检测正常小鼠足垫皮肤皮损局部细胞因子IFN-γ、IL-4、IL-10、TNF-α的表达并作为对照组,观察免疫正常组和环磷酰胺免疫抑制处理的免疫抑制组小鼠分别在感染上述真菌后7 d、30 d时,皮损局部细胞因子水平变化,并与对照组比较.结果 在着色芽生菌病小鼠模型中,第7天时,免疫正常组IL-4、TNF-α和IL-10较正常对照组均出现了显著的升高(P＜0.01),表现为Th1和Th2型细胞免疫均增强的模式,并以Th2型为主;30 d时IL-10表达显著下降(P＜0.01),与同期正常对照组比较差异无统计学意义(P=0.52),IFN-γ、TNF-α水平比7 d时显著升高(P＜0.01),表现为Th2型细胞免疫减弱Th1型占主导的模式.免疫功能受损组第7天时IL-10、IL-4的水平升高与其他两组相比差异均有统计学意义(P＜0.01),IFN-γ表达水平则明显下降(P＜0.01),TNF-α的表达与正常对照组相比差异无统计学意义(P=0.39),表现为Th2型细胞免疫增强Th1功能受抑模式;30 d时,IL-10表达水平较7 d时显著减少,但仍然高于同期的免疫正常组和正常对照组,IFN-γ、TNF-α水平显著升高(P＜0.01),但却低于免疫正常组,表现为Th2型免疫模式渐弱Th1功能逐渐增强模式.结论 在不同免疫状态下小鼠着色芽生菌病感染的过程中,随着病情好转存在由Th2型细胞免疫为主导转化为Th1型细胞免疫功能占主导的过程,免疫抑制下主要表现为Th1反应受抑制,Th1型细胞因子在控制着色芽生菌病的发展过程中具有关键性的保护作用,Th2型细胞因子则可能与感染的发展进程相关.     

Cold stress equally enhances in vivo pro-inflammatory cytokine gene expression in chicken lines divergently selected for antibody responses

The effects of cold stress, immunization and genetic selection on the expression of mRNA for cytokine genes in poultry have not been completely elucidated. Therefore, in the present experiment, using real-time quantitative RT-PCR, we evaluated the effect of cold stress and immunization with complete Freund's adjuvant (CFA) on expression of mRNA for pro-inflammatory (interleukin-1beta [IL-1beta], IL-6, IL-12beta), Th(1) (IFN-gamma and IL-2), and Th(2) (IL-4 and IL-10) cytokine genes in peripheral blood leukocytes (PBL) of chicken lines divergently selected for either high or low antibody responses. Irrespective of the duration, cold stress enhanced expression of mRNA for IL-1beta, IL-6, IL-12beta and IL-4 cytokine genes in both selection lines. These results indicate that cold stress stimulates both the innate and parts of the adaptive cellular immune system. Immunization with CFA resulted in higher expression of mRNA for pro-inflammatory cytokines and lower expression of mRNA for both Th(1) and Th(2) cytokines.     

Sequential production of Th1 and Th2 cytokines in response to live bacillus Calmette-Guérin.

Causes of individual variation in susceptibility to mycobacterial diseases are only partly understood. An efficient cell-mediated immune response is crucial for resistance. Macrophages and T cells interact to eliminate the mycobacteria, partially through the effects of secreted cytokines. A vigorous anti-bacterial inflammatory response is sometimes accompanied by severe tissue damage, while immunosuppression leads to progressive infection. Here, live, attenuated Mycobacterium bovis, bacillus Calmette-Guérin (BCG), was used as a model antigen to study cytokine production at the single-cell level in response to mycobacteria. Peripheral blood mononuclear cells from healthy individuals were challenged in vitro and the kinetics and frequencies of cytokine-producing cells were studied by immunofluorescent visualization of intracellular cytokines. Fourteen cytokines were assayed; interleukin-1 alpha (IL-1 alpha), IL-1 beta, IL-1 receptor antagonist (IL-1ra), IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-10, interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha), TNF-beta and granulocyte-macrophage colony-stimulating factor (GM-CSF). A sequential production of T helper-1 (Th1) and T helper-2 (Th2) cytokines was induced by BCG. Early, at days 1-2 after stimulation, the response was dominated by monokines and a low IFN-gamma and TNF-beta production. At days 4-5 there was a marked production of Th1 lymphokines, with approximately 6% IFN-gamma+ cells, 4% TNF-beta+ cells and 2% IL-2+ cells. Late in the reaction, at days 10-12, a Th2 response with IL-4, IL-5 and IL-10 was detected, while the synthesis of Th1 lymphokines and monokines declined. Overall, our results provide further evidence of IFN-gamma as the major cytokine induced by mycobacteria in healthy individuals, but also suggest that Th2 cytokines participate in the response.     

Characterization of human cellular immune responses to novel Mycobacterium tuberculosis antigens encoded by genomic regions absent in Mycobacterium bovis BCG

Comparative genomics has identified several regions of differences (RDs) between the infectious Mycobacterium tuberculosis and the vaccine strains of Mycobacterium bovis BCG. We aimed to evaluate the cellular immune responses induced by antigens encoded by genes predicted in 11 RDs. Synthetic peptides covering the sequences of RD1, RD4 to RD7, RD9 to RD13, and RD15 were tested for antigen-induced proliferation and secretion of Th1 cytokine, gamma interferon (IFN-gamma), by peripheral blood mononuclear cells (PBMC) obtained from culture-proven pulmonary tuberculosis (TB) patients and M. bovis BCG-vaccinated healthy subjects. Among the peptide pools, RD1 induced the best responses in both donor groups and in both assays. In addition, testing of TB patients' PBMC for secretion of proinflammatory cytokines (tumor necrosis factor alpha [TNF-alpha], interleukin 6 [IL-6], IL-8, and IL-1beta), Th1 cytokines (IFN-gamma, IL-2, and TNF-beta), and Th2 cytokines (IL-4, IL-5, and IL-10) showed differential effects of RD peptides in the secretion of IFN-gamma and IL-10, with high IFN-gamma/IL-10 ratios (32 to 5.0) in response to RD1, RD5, RD7, RD9, and RD10 and low IFN-gamma/IL-10 ratios (<1.0) in response to RD12, RD13, and RD15. Peptide-mixing experiments with PBMC from healthy subjects showed that secretion of large quantities of IL-10 in response to RD12 and RD13 correlated with inhibition of Th1 responses induced by RD1 peptides. In conclusion, our results suggest that M. tuberculosis RDs can be divided into two major groups--one group that activates PBMC to preferentially secrete IFN-gamma and another group that activates preferential secretion of IL-10--and that these two groups of RDs may have roles in protection against and pathogenesis of TB, respectively.     

Trichosanthin induced Th2 polarization status

Trichosanthin is extracted from the root tuber of Chinese medicinal herb Trichosanthes kirilowii maximowicz （Tian Hua Fen）. TCS has abortifacient, anti-tumor, anti-HIV and immunoregulatory functions. It has been proved that it could inhibit immune response and arouse a T helper 2 response in the draining lymph node. In the current study the effect of TCS on mouse splenocytes was investigated. We stimulated C57BL/6 mice with TCS both in vivo and in vitro and analyzed the change of type 1 and type 2 cytokines in mouse splenocytes. The results showed that TCS could induce the expression of IL-4, one of the major T helper 2 （Th2） cytokines, and inhibit the expression of IFN-γ, an important Thl cytokine in spleen lymphocytes both in vivo and in vitro. It is also shown the kinetics of Thl-to-Th2 transition after TCS stimulation in vivo in C57BL/6 mice. We found that type 2 cytokines, such as IL-10, TGF-β and IL-4 were increased regularly but IFN-γ, was decreased at day 3 and then increased. However the mechanism for cytokine change is not clear.