In the past year, we have completed a number of sudies s in lymphatic filariasis, pediatric tuberculosis and the influence of filarial infection on the prevalence of Type 1 and Type 2 diabetes mellitus. In addition, we have begun two new studies to examine the role of treatment induced latency in pulmonary tuberculosis and the identification of biomarkers/morbidity markers in lymphatic filarial pathology including both bancroftian and brugian filariasis. 1) Lymphatic filariasis pathogenesis To elucidate the role of TLRs in the development of lymphatic pathology, we examined the cytokine responses to different Toll-ligands in filarial pathology CP infected patients INF and endemic normal EN individuals. The TLR2 ligands - Pam and HKLM induced significantly elevated production of Th1 and other pro inflammatory cytokines, while TLR9 ligand-ODN induced significantly increased production of Th1, Th17 and other pro-inflammatory cytokines in CP patients in comparison to both INF and EN. TLR adaptor expression was not significantly different among the groups. However, both TLR2 and TLR9 ligands induced significantly higher levels of phosphorylation in ERK1/2 and P38 and enhanced NFkB activation in CP when compared to INF and EN individuals. In addition, inhibition of ERK and p38 phosphorylation as well as NFkB activation by pharmacological inhibitors in CP individuals significantly decreased the production of pro-inflammatory cytokines. Our data therefore, strongly suggest an important role for TLR2 and TLR9 mediated pro-inflammatory cytokine induction and activation of MAPK/ERK and NFkB pathway in the development of pathology in human lymphatic filariasis. 2) Mycobacterium tuberculosis (Mtb) and filarial coinfection Coinfection with Mtb and lymphatic filariasis is highly prevalent, and the presence of filarial infections may regulate the immune response needed to control Mtb infection. Since patent lymphatic filariasis is associated with the downregulated expression and function of Toll-like receptors (TLR) and since control of Mtb is dependent on the initiation of immune responses by TLR, we sought to determine the impact of co-existing filarial infections on TLR expression and function in latent tuberculosis (TB). By analyzing the baseline and mycobacterial Ag stimulated expression of TLR1, 2, 4 and 9 (in individuals with latent TB with or without filarial infection) by real-time RT-PCR, we were able to demonstrate that filarial infection, coincident with Mtb, significantly diminishes both baseline and Mtb antigen specific TLR2 and TLR9 expression. In addition, pro-inflammatory cytokine responses, including IL-1β, TNF-α, IL-6, IL-12p70 and IFN-g, to TLR2 and 9 ligands are significantly diminished in filarial-TB coinfected individuals compared to individuals with latent TB alone. Pro-inflammatory cytokine production in response to TLR4 ligand stimulation (used as a control TLR stimulus) is not altered in co-infected individuals. Definitive treatment of lymphatic filariasis significantly restores the pro-inflammatory cytokine responses in individuals with latent TB at one year post antifilarial therapy. Thus, coincident filarial infection exerted a profound inhibitory effect on protective mycobacteria specific TLR mediated immune responses in latent tuberculosis and suggests a novel mechanism by which concomitant filarial (and other systemic helminth) infections could predispose to the development of active tuberculosis in humans. 3)Complement activation and lymphatic filariasis The presence of circulating immune complexes (CIC) is a characteristic feature of human lymphatic filariasis. However, the role of CIC in modulating granulocyte function and complement functional activity in filarial infection is unknown. The levels of CIC in association with complement activation in clinically asymptomatic, filarial-infected patients (INF);filarial-infected patients with overt lymphatic pathology (CP) and uninfected control individuals (EN) were examined. Significantly increased levels of CIC and enhanced functional efficiency of the classical and Mannose-binding lectin (MBL) pathway of the complement system was observed in INF compared to CP and EN. PEG-precipitated CIC from INF and CP patients induced significantly increased granulocyte activation compared to those from EN, determined by the increased production of neutrophil granular proteins. Similarly, CIC from the INF and CP patients induced significantly higher production of pro-inflammatory mediators - GM-CSF, IL-17, IL-1β, TNF-αand PDGF and significantly lower levels of IL-8, CXCL1 and IL-4 from normal granulocytes compared to those from EN. Thus, CIC mediated enhanced granulocyte activation and modulation of the both the classical and the MBL pathway of complement function are important features of filarial infection and disease caused by those parasites known to cause human lymphatic filarial infections. 4) Pediatric tuberculosis Th1 responses are known to play an important role in immunity to tuberculosis (TB) in children, though little else is known about other factors that can also be important. In addition, children are more prone to develop extra-pulmonary manifestations of TB compared to adults. To identify the immune responses important in control of infection as well as in extra-pulmonary dissemination, we examined mycobacteria specific immune responses of children with pulmonary TB (PTB) (n=13) and extra-pulmonary TB (ETB) (n=17) and compared them to those of healthy children (HC) (n=18). Although there were no differences between those with PTB and ETB (active TB), children with active TB, when compared to HC, showed markedly diminished production of Type 1 (IFNγand TNFα), Type 2 (IL-4 and IL-13) and Type 17 (IL-17A, IL-21 and IL-23) - associated cytokines at homeostasis (baseline) as well as in response to TB antigens. CD4+ T cells were primarily responsible for the differential cytokine production between infected and uninfected children. This diminished cytokine production was associated with a significant increase in Type 1 interferon (IFNβ) production but not the production of IL-10 or TGFβ. Among children with ETB, those with neurological forms exhibited even more significantly diminished purified protein derivative (PPD) and culture filtrate protein (CFP) driven IFNγand IL-17 production compared to those with other forms of ETB. BCG vaccination status did not affect the production of these cytokines in children with active TB. Thus, pediatric TB is characterized by diminished Th1, Th2 and Th17 response with the most profound diminution favoring the development of neurological TB. Hence, Th1, Th2, Th17 and Type 1 interferon responses might play a crucial role in protection against pediatric tuberculosis. 5) Lymphatic filariasis and diabetes mellitus Epidemiological and animal studies have shown an inverse correlation between the incidence of helminth infections including lymphatic filariasis (LF) and the incidence of atopy and autoimmunity. However, the interrelationship between LF and Type-1 and Type-2 diabetes (T1DM and T2DM, respectively) in humans is not known and hence, two cross sectional studies to assess the baseline prevalence and the correlates of sero-positivity of LF among diabetic subjects were undertaken in Chennai, India. In the first study, there was a significantly lower prevalence (p=0.026) of LF among T1DM subjects (0%;n=200) compared to non-diabetic subjects (2.6%;n=500) providing validation for animal data showing the protective effect of filarial infection on T1DM. More importantly, in the second study, there was a significantly lower prevalence of LF among T2DM subjects (both newly diagnosed 5.7%;n=158 and those under treatment.
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