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.     

Quantitative assay of the carbohydrate in urokinase-type plasminogen activator by lectin-enzyme immunoassay.

We developed an enzyme immunoassay (EIA) for the measurement of Asn302-linked carbohydrate in urokinase-type plasminogen activator (u-PA) using peroxidase (HRP)-labelled lectins. u-PA antigen in the sample was immunologically bound to microtitre plate wells by anti-u-PA IgG and the binding of HRP-labelled lectins [Con A (Concanavalin A), WGA (wheat germ agglutinin), PNA (peanut agglutinin), CSA (Scotch broom), GS-I (Groffonia simplicifollia) and SBA (soybean agglutinin)] to the carbohydrate of u-PA was measured. The lectin-EIA was dose-dependent in the range 6-6000 IU/ml of u-PA using Con A and WGA. The assay did not detect the carbohydrate of bovine albumin, ovalbumin, human albumin, plasminogen, tau-globulin and fibrinogen. The binding of HRP-labelled Con A and WGA to the carbohydrate of u-PA was specifically inhibited by alpha-methylmannose and N-acetylglucosamine respectively. Endo F treatment of the carbohydrate of u-PA decreased the binding of Con A and WGA. The carbohydrate of u-PA obtained from chest fluid, ascites and U937 cell culture medium reacted with Con A and WGA by this assay forming a band in the 55 kDa region. These results suggest that the lectin-EIA method is suitable for the assay of the carbohydrate in the B-chain of u-PA.     

Identification of the major lectin-binding surface proteins of human neutrophils and alveolar macrophages

Concanavalin A (Con A) and wheat germ agglutinin (WGA) are frequently used as stimuli of neutrophils and macrophages. While the effects of these lectins on cell function are presumably mediated by interaction with cell-surface molecules, the target structures on the cell surface involved are not well defined. We have used the techniques of lactoperoxidase catalyzed cell-surface iodination, lectin affinity chromatography, monoclonal antibody immunoprecipitation, and NaDodSO4- polyacrylamide gel electrophoresis to study the surface proteins of human neutrophils and alveolar macrophages that react with six lectins including Con A and WGA. We found that several major surface-labeled proteins of neutrophils bound Con A. Four of these proteins were identified by immunoprecipitation as members of the LFA-1/HMac- 1/gp150,95 adhesion glycoprotein family. Con A also bound CR1 and a 135- kd surface-labeled protein recognized by CD15 monoclonal antibodies. WGA also bound many of these proteins, but had a much lower avidity for CR1. All three of the major surface-labeled proteins of human alveolar macrophages bound to Con A, including the 183-kd mannose receptor and the 30-kd smoking-associated protein. WGA also bound the 183-kd macrophage protein, but not the 30-kd protein. These results should aid the understanding of studies using these lectins as stimuli.     

Differential recognition of microfilarial antigens by sera from immigrants into an area endemic for brugian filariasis

Studies in animal models indicate that antibodies to surface antigens of microfilariae participate in the control of parasitaemia resulting from infections with lymphatic filarial nematodes. In an attempt to identify parasite antigens that elicit such 'protective' host responses, we compared the antigen recognition patterns of persons who remained amicrofilaraemic after 3-6 years of exposure to Brugia malayi with those of individuals who developed patent filariasis during the same period. IgG antibodies in sera from immigrants identified between 0 and 25 microfilarial antigens on Western blots. The highest degree of reactivity was observed with antigens in the 65-75 kD and 20-30 kD ranges, and with a group of high mol. wt antigens (greater than 180 kD). Sera from amicrofilaraemic donors preferentially reacted with 70/75 kD microfilarial antigens. A proportion of such sera inhibited the binding of monoclonal antibody MF1 to its target epitope; eight of nine inhibitory sera were from patients with active infections, evidenced by the presence of microfilariae or filarial antigens in the donors' blood, but who were amicrofilaraemic. These results indicate that some amicrofilaraemic residents of areas where brugian filariasis is endemic develop immune reactions to a microfilarial stage-specific antigen that was previously identified as a potentially 'protective' parasite antigen in animal models of lymphatic filariasis.     

Surface properties of developing stages of Trichuris muris

Surface properties of developing stages of Trichuris muris were investigated by analysis of binding affinities for specific anti-parasite antibodies present in a range of infection sera; in vitro eosinophil adherence studies; and binding of the fluorescent-labelled lectins, Con A, WGA, PNA and RCA. In general, larvae of any one particular stage did not bind anti-parasite antibodies present in serum collected at an earlier stage of the infection, thus indicating a considerable degree of antigenic stage-specificity. Forty day and older parasites displayed similar binding properties and it is suggested that no major changes in surface antigenicity occur after the final moult at day 25-30 after infection. Surface properties were also examined by means of complement and antibody-mediated eosinophil adherence assays. Attachment of eosinophils was maximal with day 5 larvae, although even at this stage not all of the parasite surface was covered with attached cells. Despite adherence, eosinophils were unable to effect parasite killing, even after 48 h of incubation. These studies showed that eosinophil adherence-promoting antibodies were present in immune sera; that the larval parasite surface was able to activate complement by the alternate pathway with subsequent generation of C3b molecules, and that the parasite was able to withstand eosinophil adherence and secretion by an as yet unidentified evasive stratagem. Studies with fluorescent-labelled lectins showed that all larval stages (days 5-25 after infection) were positive for Con A binding. Early larval stages (days 5-10 after infection) also bound PNA and WGA. Interestingly, recently moulted individuals rarely exhibited fluorescence, but cast cuticles fluoresced brightly.     

Anti-filarial IgG4 in men and women living in Brugia malayi-endemic areas

To assess whether antifilarial IgG4 can be used to study various epidemiological facets of filarial infections, we studied this isotype in 238 individuals resident in areas endemic for brugian filariasis, focusing on the differences between men and women. In the study area, the prevalence of microfilariae was 6.7% and the prevalence of antifilarial IgG4 was 49.2%. All microfilariae carriers were positive for antifilarial IgG4, whereas a proportion of the endemic normals (94/208) and clephantiasis patients (7/14) had IgG4 antibodies to filarial antigens. Data were analysed as a function of gender in distinct clinical groups and stratified for age. The prevalence of microfilariae was higher in males in all age groups, as reflected in significantly higher antifilarial IgG4 antibody levels compared to females. The prevalence of IgG4 increased to reach a plateau at the age of 30 years in both males and females. These results indicate that antifilarial IgG4 antibodies can reflect the differences in the extent of infection in males and females as measured by microfilarial counts, and that this parameter can be used for epidemiological assessments of filarial infection.     

Localization and immunogenicity of tubulin in the filarial nematodes Brugia malayi and B. pahangi

Tubulin was identified in the filarial nematodes Brugia malayi and B. pahangi by several approaches. Initially, a monoclonal antibody (6D8) was selected for its unusual binding to B. malayi microfilariae in indirect immunofluorescence assays: 6D8 showed granular, heterogeneously dispersed fluorescence on fixed parasites but did not bind to unfixed microfilariae. The microfilarial sheath did not bind 6D8, although it did bind fluoresceinated wheatgerm agglutinin. By Western blotting against microfilarial sonicate, 6D8 reacted with a 50,000-55,000 mol. wt protein, and also bound to purified chicken brain beta-tubulin. Additionally, this monoclonal antibody reacted with a recombinant fusion protein expressed by a clone (Bpa-7) originally isolated from an adult B. pahangi cDNA expression library by its reaction with chronic human filariasis serum. This clone encodes a small 40 amino acid C-terminal segment corresponding to residues 409-449 of beta-tubulin, and shows complete amino acid sequence homology with vertebrate beta-tubulin from 409 to 430 but 55% divergence (six amino acid substitutions, four insertions and one deletion) from human and chicken beta-tubulin over positions 431-449 at the C terminus. Antibody to both parasite and vertebrate (chicken) tubulin was found in filarial infection sera, with higher levels of autoreactive antibody apparent in amicrofilaraemic individuals. Immunogold electron microscopy was then used to localize beta-tubulin in B. malayi microfilariae and adult worms. Tubulin was shown not to be exposed on the microfilarial sheath or in the cuticle of either stage, but was found to be abundant in the somatic tissues. In microfilariae, 6D8 bound myofibril structures under the hypodermal layer, and also bound within cell nuclei. In the adult stage, tubulin was associated with muscle blocks, as well as the intestinal brush border and the embryonic uterine microfilariae.     

Auto-anti-anti-DNA antibodies from SLE patients and normals

Cross reacting auto-anti-idiotypic antibodies against anti-ssDNA antibodies were investigated in patients with systemic lupus erythematosus (SLE) and normals. Sera or immunoglobulins from SLE and normals depleted of anti-ssDNA activity and DNA antigen inhibited the reaction between 125I-F(ab')2 anti-ssDNA and ssDNA. In addition, binding to F(ab')2 portions of chromatographically purified portions of anti-ssDNA coated on polystyrene wells could be measured both in depleted SLE and normal sera. Depleted sera from SLE had both greater inhibitory activity and more antibody binding capacity than depleted sera from normals. IgG from SLE sera bound to both F(ab')2 anti-ssDNA and SLE F(ab')2 non-anti-ssDNA. However, the binding of IgG to F(ab')2 anti-ssDNA was significantly inhibited by ssDNA. These results indicate that cross reacting auto-anti-anti-ssDNA as well as other antibodies to F(ab')2 portions of homologous IgG are found in higher concentration in SLE than in normals.     

Antigenic determinants in idiopathic thrombocytopenic purpura

Serum platelet bindable immunoglobulin (SPBIg) was determined in a group of 23 idiopathic thrombocytopenic purpura (ITP) patients and compared to 20 normal, healthy controls. The mean SPBIg of the ITP group was 16.1 (+/- 17.9 SD) fg/platelet, while the normals were substantially lower, 4.0 (+/- 1.2) fg/platelet. Sera from patients of both groups were then incubated with platelet fractions immobilized on nitrocellulose membrane strips (Western Blotting) to detect platelet antigen specificity using a peroxidase labelled indicator antibody. The normal patient sera did not react with platelet fractions on the nitrocellulose strips. However, 21 of 23 ITP sera bound to one or more platelet fractions with large variations in the number and molecular weights of the platelet fractions identified by ITP antibody. These observations suggest the presence of multiple antigenic binding sites for platelet specific immunoglobulin in ITP sera. This variation may reflect heterogeneous antibodies binding to diverse antigens or homogeneous antibodies to a limited number of antigenic determinants shared by several discrete platelet molecules.     

A lectin histochemical study of intrahepatic ducts in patients with hepatolithiasis

A panel of 12 biotinylated lectins was used to investigate the diversity of glycoconjugate on the epithelium of stone-containing intrahepatic bile ducts and compared to controls. Among the 12 lectins, only WGA (wheat germ agglutinin) and Con A (concanavalin agglutinin) stained the epithelium of stone-containing intrahepatic ducts. Con A, a glucose/mannose-specific lectin, bound weakly on the epithelium of the stone-bearing intrahepatic duct in 10 of the 25 specimens, but none of the controls. All stone-containing intrahepatic bile ducts were stained heavily and homogenously by WGA, theN-acetylglucosamine-specific lectin. The high columnar epithelia of both intramural and extramural glands were stained in the supranuclear region, while the serous acini of extramural glands were stained in whole cytoplasm. The epithelium of intrahepatic ducts from the controls was stained weakly by WGA only. The WGA receptors were not abolished by pretreatment of neuraminidase. This led us to conclude that the stone-containing intrahepatic ducts were rich inN-acetylglucosamine and the heavy and homogenous staining with WGA will be indicative of hypersecretion of mucus from stone-bearing intrahepatic bile ducts.     

Characterization of immunosuppressive proteins of Brugia malayi microfilariae

Inhibition of Concanavalin A-induced lymphocyte proliferation was used to monitor the partial purification and characterization of suppressor molecules from microfilariae of Brugia malayi. Suppressor activity was present in high molecular weight fractions of microfilarial extracts (Mr greater than 50 kd on SDS-PAGE) and was protease-sensitive but resisted treatment with sodium periodate, indicating that it is associated with parasite proteins. Suppressor activity was released by microfilariae cultured in vitro and could be detected in peritoneal exudates of intraperitoneally-infected jirds and in lymph and sera from athymic C3H/HeN mice with patent B. malayi infections. These findings indicate that immune unresponsiveness during patent filarial infections may result from the in vivo release by microfilariae of high molecular weight proteins that suppress host immune responses.     

Induction of tumor associated macrophage-mediated lysis of autologous tumor cells by lectins

Lectin dependent cell mediated cytotoxicity (LDCC) to autologous tumor cells (ATC) by the tumor associated macrophages (TAM) was studied by 4 h 51Cr release assays at early and late stages of growth of a murine transplantable ascites tumor, S-180, in presence of different concentrations of wheat germ agglutinin (WGA) and Concanavalin A (Con A). Induction of LDCC by the unstimulated resident macrophages (RM) to S-180 tumor cells was also assayed. Both WGA and Con A induced significant tumoricidal activity in the TAM at different states of activation and in RM in a dose-dependent manner, the activated TAM showing expression of cytotoxicity with the lowest doses of the lectins used. Addition of N-acetylglucosamine (NAcGle) or D-mannose (D-man) in the assay completely inhibited LDCC induced by WGA and Con A, respectively. Effector-target contact alone not sufficient for inducing lysis in LDCC was evident from the observations that low doses of the lectins augmented target binding by the inactivated TAM and RM with no subsequent cytolysis.     

Studies on human filariasis in Malaysia: immunodiagnosis using indirect immunofluorescence.

The indirect immunofluorescence test using sonicated microfilariae of Brugia malayi has been evaluated on 173 sera from patients and persons exposed to Wuchereria bancrofti and B. malayi in endemic areas of Peninsular Malaysia. In the microfilaria-negative group, without signs and symptoms of filariasis 55/62 sera (89%) had titers of 1:16 and less. In the microfilaremic groups and in the amicrofilaremic cases with clinical filariasis, all the sera tested were positive, with the antibody titers ranging generally from 1:16 - 1:256. Cross-reaction tests were done on 16 samples of onchocerciasis sera from West Africa using sonicated antigen as well as antigen-coated CNB1-activated sepharose. Antibody titers were detected in all the sera. The usefulness of the sonicated microfilarial antigen in serodiagnosis of filariasis is discussed.     

Cellular immune competence in bancroftian filariasis

Lymphocyte proliferative responses to homologous and heterologous filarial antigens, mitogens and purified protein derivatives (PPD) were analysed in a group of 37 subjects from an area endemic for bancroftian filarial infection. The majority of the subjects without any clinical or parasitological evidence of filariasis (endemic normals) reacted with homologous microfilarial antigens only. Non-treated patients with patent microfilaraemia, did not respond to homologous of heterologous microfilarial antigens. In contrast, diethylcarbamazine (DEC)-treated microfilaraemic patients, reacted with homologous filarial antigens. Patients with elephantiasis reacted to microfilarial and adult worm antigens. Response to PPD was marginally depressed in patent microfilaraemic patients and a rise was observed in elephantiasis cases. Endemic normals exhibited normal response to PPD. Responses to mitogens were depressed throughout the course of the infection.     

High prevalence of Brugia timori infection in the highland of Alor Island,Indonesia

To identify areas endemic for Brugia timori infection, a field survey was carried out in 2001 on Alor, East Nusa Tenggara Timor, Indonesia. Elephantiasis was reported on this island by villagers as a major health problem. Bancroftian filariasis was detected in four villages in the coastal area, whereas B. timori was identified in four rice-farming villages. No mixed infections with both species were found. In the highland village Mainang (elevation = 880 m), 586 individuals were examined for B. timori infection and 157 (27%) microfilaria carriers were detected. The prevalence of microfilaremic individuals standardized by sex and age was 25%. The geometric mean microfilarial density of microfilaremic individuals was 138 microfilariae/ml. Among teenagers and adults, males tended to have a higher microfilarial prevalence than females. Microfilaria prevalence increased with age and a maximum was observed in the fifth decade of life. In infected individuals, the microfilarial density increased rapidly and high levels were observed in those individuals 11-20 years old. The highest microfilaria density was found in a 27-year-old woman (6,028 microfilariae/ml). Brugia timori on Alor was nocturnally periodic, but in patients with high parasite loads, a small number of microfilariae was also detected in the day blood. The disease rate was high and many persons reported a history of acute filarial attacks. Seventy-seven (13%) individuals showed lymphedema of the leg that occasionally presented severe elephantiasis. No hydrocele or genital lymphedema were observed. This study showed that B. timori infection is not restricted to the lowland and indicated that it might have a wider distribution in the lesser Sunda archipelago than previously assumed.     

Evaluation of Og4C3 antigen ELISA as a tool for detection of bancroftian filariasis under lymphatic filariasis elimination programme

Diagnosis of Lymphatic Filariasis by microscopic examination of thick blood films (TBF) collected between 8.30 pm to 12 midnight, though highly specific is operationally problematic. We evaluated the TropBio Og4C3 serum ELISA as a tool for detection of W. bancrofti microfilaria carriers using Dried Blood Spots (DBS). The study was carried out in two parts (i) to test the sensitivity and specificity of the ELISA test for detection of circulating filarial antigen (CFA) in microfilaria (Mf) carriers vis-à-vis the conventional thick blood film (TBF) microscopy and its persistence in different categories of individuals during the course of disease viz., Endemic normals (n=51), microfilaria (Mf) carriers (n=27), acute cases (n=27), chronic cases (n=50) and a control group of non-endemic normals (n=48) using sera samples and ii) to study the utility of finger prick Dried Blood Spots (DBS) collected on filter paper for detection of Mf carriers and its comparison with another antigen detection assay, the Immunochromatographic test (ICT).Considering the non-endemic normals and microfilaria carriers, the ELISA test was found to have 100% sensitivity and 94.12% specificity for detection of Mf carriers in sera samples. The CFA was absent in majority of the subjects tested under other categories with a positivity of 7.8% among endemic normals, 11.12% among acute cases, 7.84% among chronic cases and 6.25% among nonendemic normals. Comparison of finger prick DBS and sera samples by ELISA vis-à-vis the ICT, carried out on Mf carriers (n=91) and endemic normals (n=97), showed a positivity of 88 (96.7%) in DBS as against 86 (94.5%) in sera samples and 88 (96.7%) by ICT, amongst Mf carriers, with a statistically significant correlation in antigen units between sera and DBS samples (r = 0.959, p = 0.000) amongst the microfilaria carriers. Out of 97 endemic normals, 19 (19.6%) sera and 17 (17.5%) DBS samples tested positive by ELISA while as 12(12.4%) tested positive by ICT, again with a statistically significant correlation between the antigen units in sera and DBS samples (r = 0.942, p = 0.000). DBS prepared from 25 microl of blood were found to be as sensitive as 50 microl for antigen detection. Antigen positivity detected from DBS collected during day and night from known microfilaria carriers (n=27) showed a statistically insignificant difference (p = 0.125) and a significant correlation in antigen units (r = 0.820 and p = 0.013).In view of the comparable results of ELISA, ICT and TBF microscopy, it is concluded that the TropBio Og4C3 ELISA using finger prick DBS can be used as an alternate to TBF microscopy for detection of bancroftian Filariasis under the LFE programme.     

Lectin-binding histochemistry of normal and osteoarthritic cartilage tissue

Fluorescein isothiocyanate (FITC) labelled lectins were applied to study the topographical localisation and distribution of cellular as well as extracellular glycoconjugates in histological sections of normal and degenerative cartilage tissue. Using a biochemically induced model of osteoarthrosis (OA) in the knee joints of rats, the binding pattern of several lectins (wheat germ agglutinin (WGA), concanavalin A (Con A), Ulex europeus agglutinin I (UEA I), soybean agglutinin (SBA), and peanut agglutinin (PNA] in degenerative cartilage was compared with results obtained from normal healthy tissue. In response to osteoarthritic metabolic lesions the binding pattern of WGA revealed an increased cellular staining throughout the whole depth of the tissue which represents the compensatory activity of chondrocytes. The increased anabolic cell activity is maintained by enzymatic degradation of pericellular glycosaminoglycans. Osteoarthritic cartilage exposed positive fluorescence with labelled Con A demonstrating alterations in the composition of matrix glycoconjugates. The incubation of normal healthy cartilage tissue with UEA I, SBA, and PNA resulted in a completely negative staining pattern. In contrast, we could show that the affinity of UEA I to the extracellular matrix was high in degenerative cartilage indicating degradation of matrix macromolecules. The obtained data revealed a significant difference of the lectin-binding pattern between osteoarthritic and normal cartilage tissue. Therefore it can be concluded that lectins are useful histochemical tools to characterize alterations in the integrity of joint tissue by their specific binding patterns.     

Brugia malayi in seven villages in South Kalimantan, Indonesia

Seven villages in South Kalimantan were visited in 1971 and night peripheral blood smears from 2,764 people examined for microfilariae. Brugia malayi was found endemic in all villages with microfilarial rates of 12--46% (average 25%) and the median microfilarial density (MfD50) of 6 to 15 microfilariae per 20 microliter of blood. The microfilariae showed a typical subperiodic pattern. The disease was more common in males than females and the prevalence increased with age. Clinical manifestations of filariasis were found in 20% of 1,099 persons examined. Mansonia species are considered important vectors and cats important reservoir hosts. In addition to B. malayi, Dirofilaria repens and an unknown microfilaria were found in cats in the area and strains of the B. malayi and D. repens have been established in the laboratory.     

Lectin binding to human colonocytes is predictive of colonic neoplasia

OBJECTIVE: The aim of this study was to determine whether lectin binding to exfoliated human colonocytes could be used as a noninvasive test for colorectal polyps or cancer. METHODS: Colonocytes were harvested from 31 patients (10 controls, 10 with adenomatous polyps, and 11 with cancer), incubated with a panel of fluorescent-labeled lectins, and assayed by flow cytometry. RESULTS: The lectins jacalin (JAC) and wheat germ agglutinin (WGA) were useful in predicting the presence of a colorectal neoplasm (p = 0.0018 for JAC and p = 0.0099 for WGA). For JAC, sensitivity reached 81% with a specificity of 80%, and for WGA the sensitivity and specificity were both 75%. CONCLUSIONS: Lectin binding to human colonocytes can predict the presence of malignant and premalignant lesions of the colon, and has potential as a noninvasive screening tool for colorectal neoplasms.     

Immunoglobulin interactions with surfaces of sheathed and unsheathed microfilariae

Summary The sheath surface of Brugia pahangi microfilariae (mf) and the cuticle surface of Dirofilaria immitis mf were compared with regard to the ultrastructural arrangement of neutral and charged polysaccharides and binding of immunoglobulins from dog sera. Brugia pahangi : mf demonstrated large amounts of neutral sugar and negatively charged sugars just above the sheath surface, projecting some distance from the surface, in addition to a dense layer of sulphated material on the sheath surface. Microfilariae from high microfilaremic dogs showed no innately bound IgG or IgM when examined fresh from serum nor did they bind IgG or IgM from normal (NDS) or infected (IDS) dog sera after 48 h maintenance in RPMI1640. Many of these mf did bind IgG and IgM from hyperimmune dog serum (ImDS) and these immunoglobulins were found binding at a distance from the sheath similar to that of the sugars. Dirofilaria immitis mf demonstrated much less neutral sugar at the cuticle surface, no negatively charged sugars and a diffuse, rather than dense, distribution of sulfated material extending from the cuticle. The majority of these mf showed innately bound IgG and IgM. After 48 h maintenance in RPMI 1640 D. immitis mf bound and shed IgG and IgM from NDS, high microfilaremia D. immitis IDS and D. immitis mf ImDS at distinctly different rates.