"There is more than what meets the eye" - Extensive spinal tuberculosis presenting as ocular tuberculosis.

Ocular tuberculosis (OTB) is a rare form of extrapulmonary Tuberculosis (EPTB) and a rare presenting feature of tuberculosis (TB) in children. We report such a case in a 3-year-old boy who presented with a painless swelling over left upper eyelid. Mycobacterium tuberculosis bacilli were isolated from the swelling by a Fine Needle Aspiration Cytology (FNAC) which confirmed the diagnosis. Investigating him for the extent of disease, we found him to have intracranial extension to involve the ethmoid sinus on contrast enhance Computed Tomography and Pott's disease causing a compression fracture of L3 with bilateral paravertebral collection, epidural extension and a left psoas abscess on Magnetic Resonance Imaging. After starting antitubercular therapy, the child is doing well and on regular follow up. We are presenting this case to highlight the fact that extensive spinal tuberculosis can present without any neurological deficit and may even present only as a benign looking orbital swelling.

Increased Fas ligand expression of peripheral B-1 cells correlated with CD4+ T-cell apoptosis in filarial-infected patients.

Cellular hyporesponsiveness observed during helminth infections is attributed to factors such as antigen-presenting cells (APC) dysfunction, increased interleukin-10(IL-10), regulatory T cells and induction of CD4+ T (Th)-cell apoptosis. Increased Fas ligand (FasL) expression on the surface of B-1 cells and induction of apoptosis of Th cells by FasL-expressing B-1 cells due to helminth infection were demonstrated in murine model of helminth infection where as profile of FasL expression, Th-cell apoptosis and correlation between these two populations of cells in clinical filariasis remain unknown. In this study, we have scored the profile of apoptotic Th-cell population and FasL-expressing B-1 cells in different clinical categories of filariasis. The peripheral apoptotic T-helper cells were significantly increased in filarial patients compared to endemic controls. Expression of FasL on the surface of peripheral B-1 cells increased in filarial patients and positively correlated with peripheral apoptotic T-helper cells indicating FasL-expressing B-1 cells may be one of the important mediators of Th-cell apoptosis and immune anergy during filarial pathology.

Increased IgG antibody responses to excretory/secretory antigens in neonates born from mothers infected with filarial nematodes.

The present investigation aimed to evaluate the extent to which maternal filarial infection influences IgG subclass immune responses in the cord blood of neonates. Prevalence of antigenaemia was detected using an Og4C3 assay. Filaria-specific IgG subclasses against excretory/secretory antigens were measured by ELISA. Transplacental transfer of circulating filarial antigen (CFA) was observed from 34.8% of CFA-positive mothers to their respective cord bloods. Filaria-specific IgG1, IgG2 and IgG4 responses were significantly higher among cord bloods of infected mothers compared to cord bloods of uninfected mothers. In contrast, the IgG3 response was significantly higher among cord bloods of uninfected mothers. The study shows that transplacental transfer of filarial antigens and filaria-specific IgG4 occurs more in mothers having high worm burdens, and transfer of filaria-specific IgG3 occurs more in the cord blood of uninfected mothers. The findings of the study provide evidence for the development of prenatal sensitization to filarial antigens in utero, and high filaria-specific IgG4 in cord blood may serve as a marker for in-utero sensitization.

In utero sensitization modulates IgG isotype, IFN-γ and IL-10 responses of neonates in bancroftian filariasis.

In utero exposure has been considered as a risk factor for filarial infection. To evaluate the influence of maternal infection on filarial-specific IgG subclass response in neonates and their correlation with plasma levels IL-10 and interferon-γ, 145 pairs of mothers and their respective cord bloods were examined. Transplacental transfer of circulating filarial antigen (CFA) was observed in 34·8% cord bloods from CFA positive mothers. Filarial-specific IgG1, IgG2 and IgG4 responses of cord bloods were found to be positively correlated with CFA of mothers. In contrast, IgG3 responses negatively correlated with CFA of mothers. The % of similarity of recognition pattern in the cord blood with maternal blood was high for IgG3 response than IgG4 in all three groups. An increased levels of IL-10 and decreased levels of interferon gamma (IFN-γ) were observed in cord blood of infected mothers. Interferon gamma was positively correlated with IgG3 and negatively correlated with IgG4 level. On the other hand, IL-10 was positively correlated with IgG4 and CFA, indicating that cytokines may play a role in modulating the immune responses in cord bloods of sensitized foetus. The findings of the study reveal that in utero tolerance or sensitization may influence the filarial-specific immunity to infection in neonates.

Bancroftian filariasis: circulating B-1 cells decreased in microfilaria carriers and correlate with immunoglobulin M levels.

B-1 cells play an important role in the outcome of infection in schistosomiasis, pneumonia and experimental filariasis. However, no information exists regarding status of B-1 cells in clinical manifestations of human filariasis. We investigated the levels of B-1 cells from the total B cells by flow cytometry. Significantly low levels of B-1 cells and IgM antibodies were detected against a wide variety of autoantigens in microfilariae carriers as compared to endemic controls and patients with chronic pathology. A positive correlation was found between IgM antibodies to actin and ss-DNA. Absorption of plasma with soluble actin, myosin and lipopolysaccharides (LPS) resulted in significant removal of antifilarial antibodies. Affinity-purified anti-ss-DNA antibodies were found to be reactive to filarial antigens and various autoantigens. Further, a positive correlation was found between polyreactive antibodies and B-1 cells in filarial-infected human subjects. After antifilarial treatment, levels of IgM antibodies to ss-DNA, actin, LPS and filarial antigen increased significantly indicating a role of polyreactive naturally occurring antibodies in filarial infection. Our findings add to the existing evidence that the B-cell defect in BALB.Xid mice account for susceptibility to murine filarial infection and indicate an important role for these antibodies in providing host protection against filarial infection.

Maternal filarial infection: association of anti-sheath antibody responses with plasma levels of IFN-γ and IL-10.

Maternal filarial infection influences the risk of acquiring infection and development of immunity in children. Here we have analysed the blood samples of 60 mothers (24 infected and 36 uninfected) and their corresponding cord bloods to assess the impact of maternal infection on the anti-sheath antibodies and cytokine production in neonates born from them. About 69·4% of non-infected mothers and their cord bloods showed the presence of anti-sheath antibodies, while only 16·6% of the cord bloods from infected mothers were positive for it. The IL-10 level was significantly high in cord bloods of infected mothers compared with non-infected mothers. At the same time the IL-10 level was also observed to be remarkably high in cord bloods of both infected and non-infected mothers negative for anti-sheath antibody. In contrast, IFN-γ levels were significantly high in cord bloods of non-infected mothers compared with infected mothers and the increment was prominent in cord bloods of both infected and non-infected mothers positive for anti-sheath antibody. The study reveals that the presence or absence of anti-sheath antibodies in association with cytokines skews the filarial specific immunity to either Th1 or Th2 responses in neonates. This may affect the natural history of filarial infection in early childhood.

Bancroftian filariasis: a 13-year follow-up study of asymptomatic microfilariae carriers and endemic normals in Orissa, India.

The natural history of human filarial infections leading to development of disease has been a subject of intense debate. The models proposed so far have largely been based on cross-sectional data on microfilariae (Mf) and disease prevalence in filariasis endemic areas. In an attempt to study the parasitological and clinical consequences of filarial infection in Beldal (Orissa, India), an area endemic for Bancroftian filariasis, cohorts of 59 asymptomatic Mf carriers (AS) and 187 asymptomatic and amicrofilaraemic subjects or 'endemic normals' ('EN'), were followed-up and a fraction (73% and 46% respectively) re-examined after 13 years to monitor (a) Mf prevalence, (b) Mf density, (c) circulating filarial antigen (CFA) and (d) chronic disease manifestations. The Mf prevalence and density were also monitored in Mf carriers after 1 and 4 years. Both Mf prevalence and density decreased progressively in the cohort of Mf carriers over a period of 13 years in Beldal. Only 37% of them continued to be microfilaraemic and the Mf density in these subjects was only 10% of the original level. However, loss of circulating Mf in this cohort did not result in loss of CFA and 95% remained CFA positive regardless of Mf status. About 23% of males in the 'EN' cohort developed hydrocoele while only 5.7% of male Mf carriers, who were not treated with DEC, had developed hydrocoele after 13 years. A cohort of Mf carriers in another area, Jatni, was also examined after 10 years to study the parasitological and clinical outcome. In this area, about 59% of the Mf carriers continued to be microfilaraemic after 10 years. These results reveal that in Mf carriers adult filarial worms persist for several years and that loss of circulating Mf with or without chemotherapy with DEC (single 12-day course) does not influence adult worm survival. The findings have been discussed in the context of 'static' and 'dynamic' models describing the relationship between infection and disease in human filariasis.

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.

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.

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.

Setaria digitata infections in cattle: parasite load, microfilaraemia status and relationship to immune response.

A total of 110 cattle were examined in an area endemic for Bancroftian filariasis for the prevalence of infection of the bovine filarial parasite Setaria digitata. About 12.5% of cattle were found to harbour both adult worms in the peritoneum and microfilariae (mf) in circulation; 70% of the cattle were amicrofilaraemic but with an adult worm infection. A third group of cattle (16.5%) was free of detectable mf and adult worms. The presence of adult worms and/or mf did not influence the antibody levels to any of the four antigen preparations of S. digitata. However, there was a significant inverse relationship between the presence of antibodies to microfilarial sheaths and the absence of circulating mf as shown by the immunoperoxidase assay. Cattle immunoglobulin containing high titres of anti-sheath antibodies cleared circulating microfilariae very effectively in Mastomys coucha thus demonstrating the protective nature of anti-sheath antibodies in eliminating circulating microfilariae in vivo.

Naturally occurring alpha-galactosyl antibodies in human sera display polyreactivity.

Anti-gal is a dominant autoantibody constituting nearly 1% of total circulating IgG in humans and old world primates. Raised levels of anti-gal have been demonstrated in parasitic diseases such as malaria, leishmaniasis and Chagas disease and in a variety of autoimmune diseases. It has also been implicated as a primary cause of rejection of xenogeneic cells and organs transplanted in old world primates since Gal-alpha 1,3 Gal is thought to be the major antigenic epitope to which xenoreactive natural antibodies bind. Since polyreactive antibodies have also been widely implicated in xenotransplantation and anti-gal is yet to be demonstrated to be polyreactive, we have attempted to study this property of anti-gal antibodies. Anti-gal levels were assayed in 72 human sera and compared with DNA-binding antibodies. A significant positive correlation was found between anti-gal and DNA-binding antibodies. Absorption of sera with fresh rabbit erythrocytes (which express abundant alpha-galactose on their surface) resulted in significant removal of both anti-gal and DNA-binding antibodies. Affinity purified anti-gal were found to be reactive to DNA, actin, myosin and tubulin indicating the polyreactive nature of naturally occurring anti-gal antibodies in human sera. The observed polyreactivity was not an exclusive feature of sera collected from tropical countries-anti-gal affinity purified from sera of North Americans were also found to react with DNA. The demonstration of polyreactivity of anti-gal indicates a much wider biological role for this autoantibody in humans and old world primates.

A quantitative cell-ELISA for alpha-galactose specific antibodies in human malaria.

Naturally occurring antibodies to alpha-linked galactose (anti- gal) has been reported to be present in large quantities in normal human sera and they seem to play an important role in a variety of infectious as well as autoimmune diseases. A cell-ELISA using glutaraldehyde fixed normal rabbit erythrocytes was developed for quantification of anti-gal in human sera. This assay was compared with three other(commonly used) immunoassays viz. a) agglutination b) enhanced agglutination and c) lipid ELISA-assays for detection of anti-gal in human sera. The cell-ELISA was found to be the most sensitive assay followed by lipid-ELISA, enhanced agglutination and agglutination assay in decreasing order. Anti-gal affinity purified through a column of melibiose-agarose was tested by cell-ELISA. Monolayers of RRBC pre-treated with alpha-galactosidase was not reactive while in monolayers treated with beta-galactosidase, the anti-gal reactivity was comparable to those in untreated RRBC monolayer, thus indicating the high specificity of cell-ELISA for detection of antibodies to alpha-linked galactose.

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.

Murine malaria: anti-erythrocytic antibodies recognize N-acetyl neuraminic acid residues.

A cell-ELISA was developed using monolayers of glutaraldehyde-fixed normal as well as Plasmodium berghei-infected mouse erythrocytes for quantification and characterization of anti-erythrocytic autoantibodies in murine malaria. Testing normal (NMS) and peak parasitaemic sera (PPS) on erythrocyte monolayers treated with trypsin, sodium meta periodate, neuraminidase or heat, and competitive inhibition of antibodies with soluble sialic acid, revealed that some anti-erythrocytic antibodies (which increase during the parasitaemic phase of infection) recognize N-acetyl neuraminic acid (NANA) residues on host erythrocytes. High levels of antibodies to NANA covalently conjugated to bovine serum albumin (BSA) were detectable in PPS. Such antibodies could be significantly absorbed out by preincubation of PPS with mouse erythrocytes (MRBC). Antibodies in PPS, when affinity-purified on a column of Fetuin-Agarose, were found to be reactive to normal as well as parasitized erythrocyte monolayers. Immunoglobulin isotyping and IgG subgroup typing revealed that most of the anti-erythrocytic autoantibodies in NMS were IgM and IgA, while in PPS there was an appreciable increase in IgG2a and IgG3. Affinity-purified anti-NANA antibodies reacted with DNA when tested in an ELISA. There was a significant positive correlation between anti-erythrocytic antibody and DNA-binding levels in NMS as well as PPS. The DNA-binding antibodies in PPS could be effectively absorbed out by preincubation of sera with fresh MRBC. Affinity determination of anti-erythrocytic antibodies eluted from MRBC revealed binding characteristics in the following order: MRBC > single-stranded DNA > double-stranded DNA.

Antibodies to microfilarial sheath in bancroftian filariasis--prevalence and characterization.

Antibodies directed towards the sheaths of microfilariae have been implicated in the elimination of circulating microfilariae, both in experimental and human filariasis. In the present study antisheath antibodies have been detected in human sera by indirect immunoperoxidase assay (IPA) using fixed Wuchereria bancrofti microfilariae as antigen. One hundred and eighteen sera collected from an area endemic for Bancroftian filariasis were tested. While 80% of sera from microfilariae carriers had no demonstrable antisheath antibodies, more than 80% of amicrofilaraemic samples (chronic filariasis cases and endemic normals) had antisheath antibodies. The antibody activity was found in IgG, IgM and also IgE isotypes. IgG subclass typing with monospecific antisera revealed significantly higher antisheath activity in IgG2 in comparison with other IgG subclasses. The determinants on sheathed microfilariae reacting with antisheath antibodies were found to be thermostable (100 degrees C for 30 minutes), resistant to protease treatment and significantly sensitive to sodium periodate treatment, indicating the possible role of carbohydrate moieties in eliciting protective antisheath antibodies in Bancroftian filariasis.

Antibodies in human filariasis sera react with diethylcarbamazine.

We demonstrate by an ELISA the presence of antibodies in human filarial sera that react with diethylcarbamazine (DEC); they appear to be primarily filarial antibodies cross-reacting with DEC skeleton, since affinity-purified DEC antibodies strongly react with Wuchereria bancrofti microfilariae. These observations indicate a possible antigenic mimicry between the drug and some parasite component.