Construction of a recombinant plasmid harbouring the glyceraldenyde-3-phosphate dehydrogenase gene of periodic Brugia malayi and observation on DNA immunity.

Purpose: Controlling and eliminating lymphatic filariasis will require further research of preventative measures and implementation. Parasite is dependent on glycolysis for ATP production. The glycolytic enzyme glyceraldenyde-3-phosphate dehydrogenase (GAPDH) plays an important role in glycolysis and therefore is either a potential target for anti-parasite drug development or a vaccine candidate. Therefore, we tried to investigate the DNA vaccine-elicited immune responses in BALB/c mice. Materials and Methods: We cloned a gene encoding the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from periodic Brugia malayi into vector pcDNA3.1. Mice were injected at a dosage of 100 μg recombinant plasmid DNA with CpG intramuscular injection and immunized three times at 2-week intervals. pcDNA3.1 and normal saline were used as control. The tissue of muscles at the 4 weeks after the third injection was collected and target genes were detected using RT-PCR. The humoral responses elicited in mice by inoculation with the recombinant plasmid pcDNA3.1-BmGAPDH were detected using a standard ELISA. Two weeks after the third immunization, stimulation index (SI) was measured using the MTT method and the level of secreted IL-4 and INF-g were detected using ELISA. Results: Specific gene fragment coding GAPDH was amplified and the recombinant plasmid pcDNA3.1-BmGAPDH was constructed. Post-challenge sera from the mice immunized with the DNA vaccine had specific antibody titres of 1:1600 to 1:6400, and the highest titre was observed in the mice that were inoculated by pcDNA3.1-BmGAPDH/CpG at 6 weeks. At 4 weeks after immunization, the spleens of the mice were obviously enlarged. The proliferation of spleen T lymphocytes seen on the MTT assay was higher in the pcDNA3.1-BmGAPDH group than in the control group (P value <0.05). The levels of IL-4 and INF-g in serums from the immunized mice were significantly higher than that of the control (P value <0.05). Conclusions: We conclude that the recombinant eukaryotic plasmid pcDNA3.1-BmGAPDH could elicit humoral and cellular immune responses in mice.

T lymphocyte activation and expression of costimulatory molecules in filarial elephantiasis

This study was carried in 48 patients with positive blood films for W. bancrofti microfilaria and 12 cross matched healthy control persons. All the studied cases were submitted to flow cytometric analysis of peripheral blood mononuclear cells using monoclonal antibodies against CD3, CD4, CD8, CD28, HLA-DR. In this study, there was a significant decrease in CD3 and CD4 T lymphocytes but the changes in CD8 T cells and CD28 expression on CD8 T lymphocytes was insignificant while the activation marker HLA-DR expression on CD4 T lymphocytes was increased

Perfil protéico e reconhecimento antigênico de extratos de larvas infectantes (L3) de Wuchereria bancrofti / Proteic profile and antigenic recognition of extracts from Wuchereria bancrofti L3 infective larvae

A caracterização protéica dos extratos de larvas infectantes (L3) de Wuchereria bancrofti foi realizada por eletroforese em gel de poliacrilamida, em presença de dodecil sulfato de sódio (SDS-PAGE) e o reconhecimento antigênico das proteínas por Western-blot. O maior número de bandas protéicas reconhecidas foi evidenciado nos extratos AgSE (105, 100, 76, 55, 49, 39 e 32 kDa) e AgS (100, 76, 55, e 49 kDa) na presença de soros de indivíduos endêmicos normais. As bandas de 49 e 55 kDa foram reconhecidas pelos soros dos microfilarêmicos, endêmicos normais (residentes de área endêmica livres de infecção filarial) e portadores da forma crônica da doença. As larvas infectantes foram obtidas pela dissecção de mosquitos Culex quinquefasciatus infectados com sangue microfilarêmico de voluntários portadores de microfilaremia, residentes do Município de Olinda-PE. Nos 792 indivíduos investigados pela técnica da gota espessa mensurada (60æl de sangue) 87 foram positivos (11 por cento). A diferenca da positividade entre homens e mulheres não foi significativa e a faixa etária de 11 a 19 anos foi a de maior prevalência.

A four year follow up study of filaria specific IgE response in individuals with hydrocele.

BACKGROUND & OBJECTIVES: Hydrocele is the most common clinical manifestation in males infected with Wuchereria bancrofti. IgE response to a filarial allergen Sd30 was evaluated in hydrocele patients living in a W. bancrofti endemic region of Orissa, India. METHODS: IgE levels to Sd30, an antigenic fraction isolated from Setaria digitata, were determined by ELISA in sera from patients of filariasis (n = 93). IgG and IgM levels were also determined. RESULTS: It was observed that microfilaraemic patients with hydrocele (n = 16) exhibited considerably reduced (P < 0.01) IgE levels in comparison to hydrocele patients without microfilariae (n = 27) and to other filarial groups (elephantiasis, asymptomatic microfilaraemics). The reduced IgE response in microfilariae positive patients with hydrocele persisted even after a gap of four years in the majority of patients. INTERPRETATION & CONCLUSIONS: The results show low IgE levels in microfilaraemic patients with hydrocele in comparison to other groups in filaria endemic regions. The exact mechanism of this reduction in IgE is not known but these patients make a distinct group in filaria endemic regions and should be considered separately for immunological evaluation.

Antibodies to somatic L3 antigen not protective against Brugia malayi infection.

Western blot analysis of infective larvae (L3) antigen of Brugia malayi were performed on 200 sera from six groups of individuals: 36 samples from B. malayi microfilaremic individuals; 10 samples from individuals with elephantiasis; 50 and 20 samples from amicrofilaremic individuals in a B. malayi endemic area with no anti-filarial IgG4 antibodies (towards microfilaria and adult worm antigens) and samples with high titres of the anti-filarial IgG4 antibodies respectively; 50 samples from non-endemic normals and 34 samples from geohelminth-infected individuals. After protein transfer, PVDF membrane strips were successively incubated with blocking solution, human sera, monoclonal anti-human IgG4 antibody-HRP and developed with luminol chemiluminescence substrate. 28/36 (78%), 1/10 (10%) and 16/20(80%) of sera from individuals with microfilariae, elephantiasis and amicrofilaremic individuals with high titers of anti-filarial IgG4 antibodies respectively recognized L3 antigenic epitopes; the dominant and consistent antigenic bands were of approximately MW 43 kDa, 14 kDa, 15 kDa and 59 kDa. The rest of the sera were unreactive. This study showed that microfilaremics may or may not mount a notable antibody response to somatic L3 antigens, thus lending evidence that antibody response to this antigen is not protective against establishment of Brugia malayi infection.

Role of calcium influx & energy metabolism in histamine release by filarial antigen in Mastomys natalensis experimentally infected with Brugia malayi.

The present work was undertaken to study the biochemical pathways involved in terms of role of calcium influx and status of energy metabolism in the activation of mast cells obtained from Mastomys natalensis infected with Brugia malayi when challenged in vitro with a potentially allergenic antigen (60 kDa) of Brugia malayi. It was observed that histamine release from sensitized lung and peritoneal mast cells was associated with intracellular influx of radioactive Ca2+, thus establishing the role of calcium in histamine release. The process of activation of mast cells by 60 kDA antigen was an energy requiring process as it utilized the energy phosphates in the form of ATP and the cells followed the aerobic respiratory pathway for the generation of energy molecules.

Advances in immunology and immunopathology of lymphatic filariasis.

The lymphatic filarial parasites which affect about 90 million people worldwide have similar host-parasite relationships in man. They are all able to survive, reproduce and cause chronic infections if they can successfully evade the protective responses of the host. Studies to investigate the wide spectrum of clinical manifestations of the infection even among those living in similar endemic areas and with presumed equal exposure to infective larvae, have been hampered by the lack of animal models showing similar host-parasite responses. The recent use of the nude mouse infected with Brugia spp, and the leaf-monkey (Presbytis spp) infected with B. malayi or Wuchereria spp for the study of immune responses and the associated pathology of these infections, has elucidated some of the host protective immune responses as well as the associated immunopathological reactions. The successfully entrenched parasite elicits minimal reactions and pathology, but with the onset of effective host responses, whether assisted by chemotherapy, development of protective immunity or both, severe inflammatory responses may occur. The role of such immune mediated response in determining subsequent pathology will probably be dependent on the frequency and duration of these episodes, but these have yet to be defined. Prenatal and perinatal sensitization by filarial antigens are postulated to result in tolerance and/or modification of immune responses to subsequent infections. A role for genetic predisposition to certain clinical outcomes, for example, the development of elephantiasis, has been postulated but needs further study. Advances have also been achieved in defining those parasite antigens/products involved in eliciting or suppressing protective and other immune responses.(ABSTRACT TRUNCATED AT 250 WORDS)

Suppression of Con A induced lymphocyte transformation by sera from filarial patients with clinical manifestations.

Reduced lymphocyte transformation to Wuchereria bancrofti microfilariae excretory-secretory antigen and Con A were observed in clinical filarial patients. Pre-incubation of normal human peripheral blood mononuclear cells with sera from filarial patients with clinical manifestations such as hydrocele and elephantiasis suppressed Con A induced responses. Effect of fractionated clinical filarial serum on Con A induced lymphocyte transformation showed that the inhibitory activity was associated with high molecular weight serum fraction.

Circulating immune complexes and complement in bancroftian filariasis.

Sixty filarial cases, 30 endemic normal individuals and 10 non endemic subjects were investigated for the presence of Circulating Immune Complexes (CICs) and Complement Component C3. Using Polyethylene Glycol precipitation and Polyethylene Glycol precipitation-Complement Consumption methods, it was observed that CICs were raised significantly in chronic lymphatic filariasis and Tropical Pulmonary Eosinophilia (TPE) groups. The results observed by both the techniques for detection of CICs were comparable. Low levels of C3 were detected in chronic lymphatic filariasis cases by single radial immunodiffusion method, suggesting the utilization of complement by immune complexes.

Excretory/secretory antigens from a bovine filarial parasite cross react with human antifilarial antibodies.

Certain excretory/secretory proteins released by adult females of the bovine filarial parasite, Setaria digitata, along with the release of microfilariae when chromatographically analysed has three major protein fractions of molecular weights 70 kD (ESF1), 16.5 kD (ESF2) and 11 kD (ESF3). Of these ESF2 and ESF3 cross reacted with antibodies from Wuchereria bancrofti infected humans. ESF2 was more specific and accurate in detecting human filarial infection. Similar proteins secreted by human filarial parasites could be targets for combating the disease by cure or control.

Differential recognition of Brugia malayi antigens by bancroftian filariasis sera.

Individuals residing in an area endemic to Wuchereria bancrofti infection were broadly categorised as endemic normals (EN), microfilaraemics (mf + ve) and elephantoids i.e., chronic lymphatic filariasis (EL). The immune status of these three groups was examined in terms of (i) specific antibody levels; (ii) ability to induce antibody dependent cellular cytotoxicity (ADCC) to microfilariae; and (iii) ability to recognise different microfilarial antigens by immunoblotting. All three groups of endemic residents were indistinguishable in their antibody levels as measured by ELISA with B. malayi microfilarial antigen. Many endemic normal sera and most elephantoid sera exerted strong cytotoxicity against W. bancrofti microfilariae whereas none of the mf + ve sera had any such activity. Immunoblotting studies revealed that a protein with mol. wt of 79 KDa was the only one among the proteins of B. malayi microfilarial extracts that was consistently recognised by sera from all endemic residents. Endemic normal sera and elephantoid sera, which exerted maximum cytotoxicity, together specifically recognised three proteins with molecular weights 25, 58 and 68 KDa and these three proteins could be among the candidate antigens that induce resistance to filarial infection.

Manifestaçoës clínicas da filariose linfática bancroftiana / Clinical manifestations of lymphatic bancroftian filariasis

Os autores inicialmente detêm-se, de forma sucinta, nos aspectos básicos da filariose bancroftiana, envolvendo principalmente a biologia do parasita e os aspectos imunológicos e patogênicos. Em seguida, na base de suas experiências pessoais e dos dados da literatura, sugerem uma classificaçäo geral das formas clínicas da infecçäo, cujo amplo espectro distribuem em seis grupos: 1§) indivíduos endêmicos "normais" (negativos endêmicos); 2§) indivíduos microfilarêmicos assintomáticos; 3§) pacientes com manifestaçöes agudas (ex. linfangite, orquiepididimite); 4§) pacientes com manifestaçöes crônicas (ex. edema, elefantíase, hidrocele, quilúria); 5§) pacientes com eosinofilia pulmonar tropical (EPT); 6§) pacientes com formas frustas ou controversas. Em apítulos sucessivos, os autores procuram descrever, de forma sumária, em cada grupo de pacientes, os aspectos pertinentes a clínica e ao diagnóstico, assinalando em cada caso as prováveis respostas imunes do hospedeiro. Finalizando, os autores tecem algumas consideraçöes sobre o tratamento da infecçäo

Relationship of C3 & C4 levels with filarial immune complexes in bancroftian filariasis.

C3 and C4 levels were determined by radial immuno-diffusion technique and filarial circulating immune-complexes by anti-C3 ELISA in microfilaraemic individuals and patients with clinical filariasis. Decreased levels of C3 and C4 were observed in both groups of filarial patients. Low levels of complement components were associated with low levels of circulating immune complexes.

Fractionation and characterization of urinary filarial antigen in bancroftian filariasis.

Urine samples from microfilaraemic patients were concentrated and fractionated by gel chromatography on Ultrogel AcA 44. Four protein fractions labelled as UFA C1, UFA C2, UFA C3 and UFA C4 were tested for filarial antigenicity by sandwich ELISA. UFA C1 and UFA C2 showed antigenic activity. On further analysis by SDS-PAGE, UFA C1 and UFA C2 showed antigenic components with MW ranging from 10.4 K to 123 K. UFA C1-1 and UFA C2-2 showed high antigen titre in ELISA. Urinary albumin was observed as a major component in UFA C2. Absorption of albumin from UFA C2 enhanced its antigenic activity considerably. As little as 0.01 pg antigenic protein per test was found to be sufficient for the detection of filarial antibody in ELISA. Biochemical characterization indicated a glycoprotein nature of UFA C2.

Development of Brugia malayi in Mongolian gerbils previously exposed to Wuchereria bancrofti.

Twelve Mongolian gerbils, Meriones unguiculatus, were infected with 100 third-stage larvae of Wuchereria bancrofti. One month later these animals, along with 4 control animals, were given 100 third-stage larvae of Brugia malayi. Eleven of the 12 experimental animals and the 4 controls survived, and 8 of the experimental animals and all of the controls demonstrated microfilaremia after 3 months. The animals were killed at 6-months post-infection and examined for parasites. One W. bancrofti larva was found in one of the experimental animals, and 15% of the B. malayi given were recovered as adults from the testes, viscera, and carcass. Thirty-eight percent of the worms given to the controls were recovered from the testes, viscera, and pelt. The worms from the experimental animals also appeared to be smaller. This study suggests that gerbils are able to develop partial resistance to Brugia malayi following a previous infection with Wuchereria bancrofti.