Prakash K. Sahoo

Macrofilaricidal Activity and Amelioration of Lymphatic Pathology in Bancroftian Filariasis after 3 Weeks of Doxycycline Followed by Single-Dose Diethylcarbamazine

In a placebo controlled trial, the effects of 21- and 10-day doxycycline treatments (200 mg/day) followed by single dose diethylcarbamazine (administered 4 months post treatment) on depletion of Wolbachia endobacteria from Wuchereria bancrofti, filaricidal activity, and amerlioration of scrotal lymph vessel dilation were studied in 57 men from Orissa, India. The 21-day doxycycline course reduced Wolbachia in W. bancrofti by 94% before diethylcarbamazine administration. After 12 months, all patients with this treatment were amicrofilaremic and different from the 10-day doxycycline (42.9%) and placebo (37.5%) groups, and significantly fewer were positive for scrotal worm nests (6.7%) compared with 10-day doxycycline (60%) and placebo (66.7%). Average scrotal lymph vessel diameters were reduced from 0.7 cm pre-treatment to 0.02 cm in patients after 21 days of treatment, while no significant changes were seen in the other groups. This latter feature confirms the beneficial effects of doxycycline on lymphatic dilation and thus adds to the existing evidence that doxycycline, in addition to being macrofilaricidal, may be used to prevent or reverse lymphatic pathology.

Protective immunity in human lymphatic filariasis: problems and prospects

Abstract. Human filariasis caused by lymphatic dwelling nematodes, affecting 120 million persons worldwide, is a major public health problem. Efforts towards development of vaccines for such large tissue-dwelling nematodes depends significantly on identification and demonstration of protective immunity in the exposed population. Immunological studies conducted in human filariasis so far are essentially attempts to establish a correlation of the immune response phenotypes with presence or absence of filarial infections/disease in the host, and the cause-effect relationship between the observed immune responses in the host and protective immunity continues to be conjectural. This short review attempts to clarify the functional definition of protective immunity, problems associated with identification of putatively immune subjects in endemic areas, role of antibodies reactive to surface of microfilariae and larvae stages of filarial parasites and importance of undertaking immunological investigations on a longitudinal basis in different cohorts of subjects presenting with one or the features of infection and/or disease for more accurate delineation of protective immunity in human filariasis.

Protective immunity in human Bancroftian filariasis: inverse relationship between antibodies to microfilarial sheath and circulating filarial antigens

The existence and the nature of protective immunity in human filariasis continues to be a subject of intense debate. While there is no broad consensus on functional immunity against larval and adult stage parasites, anti‐microfilarial immunity has been demonstrated to be mediated by antibodies to the microfilarial sheath. In the present study, circulating filarial antigens (CFA), a marker of active filarial infection in human Bancroftian filariasis, was found to be inversely associated with antibodies to microfilarial sheath in a cohort of 411 subjects representing all categories of filariasis across the clinical spectrum of the disease. Approximately 80% of humans of all age groups (5–65 years) were found to have either CFA or anti‐sheath antibodies. The inverse relationship observed between these two parameters was found to be independent of the clinical manifestation; both symptomatic and asymptomatic cases were found to display similar inverse association between CFA and anti‐sheath antibodies. The prevalence of anti‐sheath antibodies in the paediatric group was found to be very high as compared to adults; 78% of children below the age of 10 years tested positive for anti‐sheath antibodies although the mf rate and CFA rate were only 4.5% and 22.7%, respectively, in this age group, indicating that developing larvae or juvenile adult stage parasites could have been the source of antigenic stimulus for induction of antibodies to the microfilarial sheath.

A role for tumour necrosis factor-alpha in acute lymphatic filariasis.

A spectrum of clinical manifestations is a feature of human lymphatic filariasis. The acute disease is characterized by periodic and self limiting episodes of adenolymphangitis, fever and associated constitutional symptoms, while the chronic disease includes long lasting manifestations such as lymphoedema and/or hydrocoele. The microfilariae carriers are generally free of clinical symptoms. In the present study circulating Tumour Necrosis Factor (TNF‐α) was measured in human bancroftian filariasis with different clinical manifestations. Significantly elevated levels were observed only in patients with acute disease and not in microfilariae carriers or in patients with chronic manifestations. A detailed analysis of the acute cases indicated an absence of correlation between TNF‐α levels and duration of the episodes. However, a significant positive correlation was observed between the severity of the disease and the TNF‐α levels. About 85% of the acute cases with severe manifestations showed raised levels of TNF‐α while only 6.5% of mild cases showed such levels. Manifestation of fever was also significantly associated with higher levels of TNF‐α—while 80% of acute cases with fever had significant levels only 24% of acute cases without fever had high levels of TNF‐α. Based on these observations we propose a mediatory role for TNF‐α in acute filariasis and the possible use of TNF‐α inhibitors for clinical management of the disease.

Human bancroftian filariasis – a role for antibodies to parasite carbohydrates

Studies on immune responses to parasites have been undertaken in filariasis with a view to understand protective immunity, pathogenesis of the disease process and mechanisms of immune deviation. However none of the investigations conducted so far on antibody responses have addressed the issue of immunogenicity of filarial carbohydrate antigens in human lymphatic filariasis. In this communication we report details on relative protein and carbohydrate contents of various developmental stages of filarial parasites and antibody responses to filarial proteins (Fil.Pro) and carbohydrates (Fil.Cho) in different clinical spectrum of human bancroftian filariasis. As expected, antibodies of IgM and IgG2 subclass recognized primarily Fil.Cho while IgG4 filarial antibodies recognized exclusively Fil.Pro. Reactivity of IgG3 to Fil.Cho was similar to that of IgG2 while IgG1 more readily recognized Fil.Pro than Fil.Cho. The IgG2 and IgG3 antibodies to Fil.Cho were found to be significantly more in patients with chronic filarial disease and in endemic normals when compared with microfilariae (mf) carriers while IgG4 antibodies to Fil.Pro were significantly more in mf carriers. The dichotomy in reactivity of filarial IgG2, IgG3 and IgG4 was dependent on active filarial infection as indicated by presence of circulating filarial antigen (CFA). Individuals with CFA were found to possess significantly more IgG4 to Fil.Pro than those without CFA while IgG2 and IgG3 levels to Fil.Cho was significantly more in CFA negative subjects when compared to those with CFA. Although IgG1 reacted more readily with Fil.Pro, unlike IgG4, their levels were significantly more in CFA negative subjects when compared to those with active filarial infection. Absorption of sera with phosphorylcholine (PC) resulted in no significant loss of reactivity to Fil.Cho indicating that most of the anticarbohydrate antibodies were recognizing non‐PC determinants in human filariasis. Elevated levels of IgG2 and IgG3 antibodies to Fil.Cho in individuals free of filarial infection indicate a possible role for carbohydrate antigens in induction of protective immunity in human filariasis.

Association of TNF level with production of circulating cellular microparticles during clinical manifestation of human cerebral malaria

Microparticles (MPs) resulting from vesiculation of different cell types in Plasmodium falciparum infection correlate with the level of proinflammatory cytokine TNF that may thereby determine the disease severity. Using TruCount tube based flow cytometric method for the exact quantification of MP and enzyme linked immunosorbent assay for the measurement of TNF, we conducted a hospital based case control study on P. falciparum malaria patients to scrutinize and infer the link between the two. In 52 cerebral malaria (CM), 21 multi-organ-dysfunction (MOD), 12 non cerebral severe malaria (NCSM) and 43 uncomplicated malaria patients, the MP level was found to be significantly elevated in febrile malaria patients compared to healthy controls and a striking decrease in MP level was observed with the clearance of the P. falciparum infection in the patients upon follow-up. The lowering of the parasite density with the level of plasma TNF and the positive correlation of the cytokine with the cell derived MPs and negative correlation with the respective cell count in human malaria patients suggests that TNF may be a key stimulant to the cells resulting in the release of MPs in malaria infection.

Protective Immunity in Human Filariasis: A Role for Parasite-Specific IgA Responses

BACKGROUND Filaria-specific antibodies of immunoglobulin (Ig) G, IgE, and IgM isotypes have been correlated with acquired immunity in the literature, but the status of filaria-specific IgA and its role in human filariasis has not been addressed. The present study attempts to fill this lacuna. METHODS Both total and filaria-specific IgA to different developmental stages of filarial parasites were quantified by solid-phase immunoassays in 412 clinically and parasitologically defined cases occurring in an area endemic for human bancroftian filariasis in Orissa, India. RESULTS Compared with other clinical categories, microfilariae carriers were deficient in total as well as filaria-specific IgA. More crucially, significantly high levels were observed in putatively immune control subjects from areas of endemicity. These associations were also related to sex; female subjects in each category displayed higher levels of filaria-specific IgA than did male subjects. CONCLUSION The study demonstrates, for the first time, a positive correlation between protective immunity and increased levels of filaria-specific IgA in human bancroftian filariasis. Furthermore, filaria-specific IgA appears to be an immunological window for the sex-related differences in susceptibility to infection observed in human filariasis.

Human Lymphatic Filariasis: Genetic Polymorphism of Endothelin-1 and Tumor Necrosis Factor Receptor II Correlates With Development of Chronic Disease

BACKGROUND Hydrocele and elephantiasis are 2 clinically very diverse and often mutually exclusive chronic manifestations of human bancroftian filariasis. Plasma levels of endothelin-1 (ET-1), a major angiogenic factor, and tumor necrosis factor receptors (TNFRs) that regulate host inflammation have been associated with development of chronic filariasis, although their genetic basis are not known. METHODS We studied polymorphisms of ET-1 (Ala288Ser) and TNFR-II (Met196Arg) genes by means of the polymerase chain reaction confronting 2 pairs primers method and restriction fragment length polymorphism, respectively. Plasma ET-1 level was measured by enzyme-linked immunosorbent assay. RESULTS Met196Arg genotype frequency of TNFR-II polymorphism was significantly greater in hydrocele patients, compared with elephantiasis patients (OR, 4.34 [95% CI, 2.04-9.20]). Conversely, a significantly high prevalence of the Ala288Ser mutation of ET-1 was observed in elephantiasis patients, compared with hydrocele cases (OR, 2.15 [95% CI, 1.13-4.10]). Decreased plasma ET-1 levels associated significantly with Ala288Ser mutation in the study population. A combined analysis indicated a 23-fold higher risk for developing elephantiasis in individuals with TNFR-II (Met196Met) and ET-1 mutants (Ala288Ser + Ser288Ser). CONCLUSIONS ET-1 (Ala288Ser) and TNFR-II (Met196Arg) polymorphisms are associated with development of one or the other form of chronic disease in bancroftian filariasis.

Bancroftian filariasis-differential reactivity of anti-sheath antibodies in microfilariae carriers

Anti‐sheath antibodies have been detected using an immunofluorescent assay (IFAT) in the sera of microfilariae carriers (AS cases) residing in areas endemic for Bancroftian filariasis. Microfilariae (mf) of Wuchereria bancrofti purified from five different mf carriers were used separately as antigen to identify anti‐sheath antibodies. The reactivity of sera from AS cases to mf sheath was found to be variable to the five different mf preparations. While as high as 25% of the sera reacted with mf purified from one individual, none of them reacted with mf purified from two other individuals. Such a differential reactivity to the sheath was found to be a feature of antibodies in AS cases only. Sera of seven amicrofilaraemic patients with elephantiasis reacted uniformly with all five mf preparations. The possible existence of polymorphic antigen (s) on the sheath of W. bancrofti mf has been proposed.

Human Bancroftian filariasis: loss of patent microfilaraemia is not associated with production of antibodies to microfilarial sheath

Antisheath antibodies have been incriminated in elimination of circulating microfilariae in human filariasis since a very significant inverse association has been consistently demonstrated between the two parameters. An attempt was made in the present study to seek empirical proof for the above proposal. Two cohorts of 43 and 73 microfilariae (mf) carriers were examined after 13 and 10 years, respectively, for mf as well as antisheath antibodies. The first cohort was also examined for the presence of circulating filarial antigen (CFA). Of the 43 mf carriers examined after 13 years, 62·8% were free of circulating mf although only 3·7% of them had demonstrable antisheath antibodies. Approximately 93% of this cohort (with or without current microfilaraemia) tested positive for CFA after 13 years indicating continued presence of adult filarial worms in the host after loss of mf in circulation. When the second cohort of 73 mf carriers were examined after 10 years, 30 were found to be amicrofilaraemic and only 6·66% of them had demonstrable antisheath antibodies. We conclude that, in human Bancroftian filariasis, elimination of circulating microfilariae may not be mediated by antibodies to microfilarial sheath.