Analysis of IL-4 promoter and VNTR polymorphisms in Thai patients with pulmonary tuberculosis.

Tuberculosis (TB) is a leading cause of morbidity and mortality in Thailand. Cytokines play important roles in defense against Mycobacterium tuberculosis infection. Interleukin (IL)-4 is one of the anti-inflammatory cytokines and has been found to be elevated in TB patients. The common polymorphisms in IL-4 gene, including IL-4-590C/T, IL-4-33C/T, and IL-4-variable number of tandem repeats (VNTR) intron 3 have been reported to be associated with risk for some diseases. The purpose of this study was to investigate possible associations between the above mentioned three common functional polymorphisms in the IL-4 gene in patients with pulmonary tuberculosis (PTB) in a Thai population. Forty three patients with PTB and 90 healthy control subjects were studied. The three common polymorphisms of the IL-4 gene were determined using polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (PCR-RFLP). The allele and genotype frequencies of IL-4 -590 C/T, -33 C/T, VNTR intron 3 polymorphisms did not show significant differences between PTB patients and healthy controls (genotype: p=0.88, p=0.92, p=0.40; allele: p=0.38, p=0.44, p=0.53, respectively). However, the allele distribution of the IL-4 -590 C, -33 C, and VNTR R3 was higher among PTB patients (25.58%, 25.58%, 25.58%, respectively) than among control subjects (20%, 20.48%, 19.44%, respectively). This may suggest that IL-4-590C/T, -33C/T and VNTR intron 3 might play a role in susceptibility to PTB. A larger cohort may possibly help conclude our findings.

Analysis of IL-4 promoter and VNTR polymorphisms in Thai patients with pulmonary tuberculosis

@#Tuberculosis (TB) is a leading cause of morbidity and mortality in Thailand. Cytokines play important roles in defense against Mycobacterium tuberculosis infection. Interleukin (IL)-4 is one of the anti-inflammatory cytokines and has been found to be elevated in TB patients. The common polymorphisms in IL-4 gene, including IL-4-590C/T, IL-4-33C/T, and IL-4-variable number of tandem repeats (VNTR) intron 3 have been reported to be associated with risk for some diseases. The purpose of this study was to investigate possible associations between the above mentioned three common functional polymorphisms in the IL-4 gene in patients with pulmonary tuberculosis (PTB) in a Thai population. Forty three patients with PTB and 90 healthy control subjects were studied. The three common polymorphisms of the IL-4 gene were determined using polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (PCR-RFLP). The allele and genotype frequencies of IL-4 -590 C/T, -33 C/T, VNTR intron 3 polymorphisms did not show significant differences between PTB patients and healthy controls (genotype: p=0.88, p=0.92, p=0.40; allele: p=0.38, p=0.44, p=0.53, respectively). However, the allele distribution of the IL-4 -590 C, -33 C, and VNTR R3 was higher among PTB patients (25.58%, 25.58%, 25.58%, respectively) than among control subjects (20%, 20.48%, 19.44%, respectively). This may suggest that IL-4-590C/T, -33C/T and VNTR intron 3 might play a role in susceptibility to PTB. A larger cohort may possibly help conclude our findings.

Differential regulation of IgG subclasses and IgE antimalarial antibody responses in complicated and uncomplicated Plasmodium falciparum malaria.

The aim of this study was to assess the immunoglobulin (Ig)-subclass distribution of antimalarial antibody responses in 110 and 169 Thai patients with complicated and uncomplicated Plasmodium falciparum malaria, respectively. Antimalarial plasma IgG subclasses and IgE antibody levels against a crude malaria blood stages, and antigen preparation were determined using enzyme-linked immunosorbent assay (ELISA). On admission, the levels of anti-P. falciparum IgG1, IgG2 and IgG3 were significantly lower in patients with complicated malaria than uncomplicated malaria (IgG1, P < 0.0001; IgG2, P < 0.0001; IgG3, P < 0.0001). The levels of antimalarial IgE were slightly lower, but not statistically significant (P = 0.389) in the complicated malaria. After adjusting all antibody levels and age, anti-P. falciparum IgG3 levels remained significantly associated with complicated malaria. None of the other antibody concentrations showed statistically significant associations with complicated malaria. The anti-P. falciparum IgG3 levels were related to the IgG1 as well as IgG2 levels. A correlation between anti-P. falciparum IgG2 and IgE was observed in the complicated malaria group, and this may indicate their roles in the severity of disease. Our data suggest that anti-P. falciparum IgG3 is associated with a reduced risk of complicated malaria and that antimalarial Ig-subclasses are differently regulated in patients with complicated and uncomplicated malaria.

Analysis of T-cell responses in malaria-exposed and non-exposed donors using Plasmodium falciparum asexual blood stages enriched by a simple centrifugation method.

Several studies have reported on similar in vitro cellular responses to different malaria-antigen preparations in both malaria-primed and un-primed donors. Whether intact live parasites can exert a distinct type of response in either of the two groups is not well known. In this study, we developed a simple three-step centrifugation method for simultaneous enrichment of early and late blood stages from Plasmodium falciparum cultures. Such enriched P. falciparum fractions and other antigen preparations were used to stimulate lymphocytes from malaria-exposed and non-exposed individuals to examine the proliferative activity and expansion of CD3+, gammadelta+, CD4+, and CD8+ T cells. While lymphocytes from malaria non-exposed donors proliferated relatively higher than those from malaria-exposed donors in response to most antigens tested, the enriched fractions of live parasites exerted higher proliferative responses on cells from the latter donors. This suggests the existence of memory cells in the malaria-exposed donors, but not in the non-exposed ones. Flow cytometric analysis revealed a higher percentage expansion of CD4+ T cells in the responding cells of the exposed donors than the non-exposed ones. Taken together, this study reports on a simple method that simultaneously enriches for intact live early and late blood stages of P. falciparum parasites. Moreover, the study revealed higher expansion CD4+ T cells in the exposed individuals than the non-exposed in response to live malaria parasites and not to other parasite-antigen preparations.

Natural human IgG subclass antibodies to Plasmodium falciparum blood stage antigens and their relation to malaria resistance in an endemic area of Thailand.

The immunoglobulin G (IgG) subclass antibodies to Plasmodium falciparum blood stage antigens in the sera of 181 individuals living in malaria endemic area in Kanchanaburi Province, western Thailand, were determined by enzyme-linked immunosorbent assay (ELISA). In this study, IgG3 and IgG1 were shown to be the predominant subclasses. Generally, IgG2 were coexpressed with IgG1 and IgG3 while IgG4 was found to coexpress with other three IgG subclasses. The levels of specific IgG1, IgG2, and IgG3 increased significantly with age (r = 0.295, p = 0.000; r = 0.416, p = 0.000; r = 0.320, p = 0.000, respectively). The data indicate that the higher antibody production required continuous stimulation under natural condition. Furthermore, the levels of specific IgG1, IgG2 and IgG3 increased in immune individuals without clinical malaria, reported in adolescents and adults, were associated with malaria resistance. Similar results were found in children with different patterns of IgG subclasses in which the specific IgG2 and IgG3, but not IgG1 was related to resistance.

Human gamma delta T cells that inhibit the in vitro growth of the asexual blood stages of the Plasmodium falciparum parasite express cytolytic and proinflammatory molecules.

The functional properties, regarding parasite growth inhibition in vitro, the cytotoxic potential and cytokine profiles of human gammadelta+ and alphabeta+ T cells, T-cell lines and clones stimulated with Plasmodium falciparum-antigen-or T-cell mitogen in vitro were investigated. Using reverse transcriptase-polymerase chain reaction (RT-PCR) and specific primers, mRNA for the cytolytic molecules perforin, granzyme A and B, Fas and Fas ligand (FasL) were detected in both the gammadelta- and the alphabetaT cells. Despite this fact, only gammadeltaT cells inhibited, both Vdelta1+ and Vdelta2+, the in vitro growth of the asexual blood stages in a dose dependent manner. The inhibition required cell-to-cell contact and was not observed until the second parasite replication implied that the likely gammadeltaT-cell target was the extracellular merozoite or schizont. The failure of alphabetaT cells to inhibit the growth of the parasite suggests requirement of additional cytolytic molecules/signals or different receptor specificities exhibited by the gammadeltaT cells. Both the gammadelta- and alphabetaT cells expressed mRNA for a large number of cytokines. Interferon (IFN)-gamma, interleukin (IL) IL-5, IL-6, IL-8, tumour necrosis factor alpha (TNFalpha), tumour necrosis factor beta (TNF-beta)/lymphotoxin (LT) and T-cell growth factor beta-1 (TGF-beta1) were observed in all activated clones tested. No IL-3 was detected, while IL-1beta, IL-2, IL-4, IL-10 and GM-CSF were variably expressed. In conclusion, our data show that gammadeltaT cells in malaria nonimmune individuals inhibit the asexual blood stages of P. falciparum malaria, while similarly activated alphabetaT cells do not. Thus, it is likely that the gammadeltaT cells could play a mandatory role in the elimination of parasites and/or the regulation of the early immune response to malaria infection.

Low CD8+ T lymphocyte response to P. falciparum circumsporozoite protein in naturally-exposed malaria endemic populations in Thailand.

Cytotoxic T lymphocytes (CTLs) specific for epitope(s) within the circumsporozoite (CS) protein of malaria sporozoite have been shown to play an important role in protective immunity against malaria. Human CTLs against the potential epitope at the carboxy terminal region of CS protein of Plasmodium falciparum 7G8 strain (Pf7G8CS 368-390) were determined in thirty-six falciparum malaria patients and ten healthy controls. Four of 36 individuals and none of the healthy controls developed Pf7G8CS 368-390 specific CTL activity. The CTL activity was antigen specific and CD8+ T cell dependent. Although low CTL response has been determined, the study suggested that there was a correlation between initial parasitemia and the specific Pf7G8CS 368-390 CTL activity. A correlation between such CTL activity and anti-R32tet32 antibody levels among individuals with previous malaria experience was found, which was in contrast to those among individuals with recent malaria infection. All these 4 CTL positive individuals had at least two episodes of clinical malaria experience while all 25 individuals who were exposed to malaria for the first time did not have such a specific CTL response. These results showed that individuals with a history of natural endemic exposure to P. falciparum sporozoite developed low specific CTL responses to Pf7G8CS 368-390, so that previous but recent sporozoite exposure might be a prerequisite for generation of such CS protein specific CTL response.