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HYBRIDOMA PRODUCTION USING IMMUNE LYMPHOCYTES AGAINST BRUGIA MALAYI ANTIGEN WITH MYELOMA CELLS  [cached]
Soeyoko Soeyoko,Noerhajati S.,Abdul Salam Sofro
Bulletin of Health Research , 2012,
Abstract: Wuchereria bancrofti, Brugia malayi, and Brugia timori are the causative agents of lymphatic hiariasis in Indonesia, but in some endemic areas, Brugia malayi is the most commonly found,Diagnosis of hiariasis is normally based on clinical and parasitological examinations, but both have limitations. Therefore now the immunological examination plays an important role in the diagnosis of filariasis. The discovery of monoclonal antibodies recently may probably give a firm scientific basis in immunology and add a new dimension to the efforts of developing a specific and sensitive immunological test for various stages of filarial infection. In this study, the production of hybridoma cells to develop monoclonal antibodies against B. malayi integrated a number of techniques: preparations of B. malayi surface antigen, immune lymphocyte cells, NSI myeloma cells and macrophage feeder layers, and a fusion of immune lymphocytes with myeloma cells. The results of this study can be concluded in three points: Protein analysis of the surface antigen was examined by Biureet and SDS-PAGE. A total of fourteen examinations were conducted by using 4000 L3 for each experiment. Three were not detected by Biureet method, but showed five protein fractions by SDS-PAGE. The protein concentration of the surface L3 was varied from 85.0/μg/ml to 769.23/μg/ml, with an average of 297.04/μg/ml. The immunoreactivifies of Balb/c mice antibodies to B. malayi L3 surface antigen were tested by ELISA and showed a gradual increase after four times immunizations at two weeks interval. The optical density (OD) after four times immunizations was varied from 0.363 to 0.878 each mouse, where as the positive control sera OD was 0.570. Hybridization using immune lymphocytes against B. malayi L3 surface antigen with myeloma cells yielded 60.41% hybrid cells and none of them produced monoclonal antibodies tested of ELISA.
Distribution of Brugia malayi larvae and DNA in vector and non-vector mosquitoes: implications for molecular diagnostics
Sara M Erickson, Kerstin Fischer, Gary J Weil, Bruce M Christensen, Peter U Fischer
Parasites & Vectors , 2009, DOI: 10.1186/1756-3305-2-56
Abstract: Parasite DNA was detected over a two week time course in 96% of pooled thoraces of vector mosquitoes. In contrast, parasite DNA was detected in only 24% of thorax pools from non-vectors; parasite DNA was detected in 56% of midgut pools and 47% of abdomen pools from non-vectors. Parasite DNA was detected in vectors in the head immediately after the blood meal and after 14 days. Parasite DNA was also detected in feces and excreta of the vector and non-vector mosquitoes which could potentially confound results obtained with field samples. However, co-housing experiments failed to demonstrate transfer of parasite DNA from infected to non-infected mosquitoes. Parasites were also visualized in mosquito tissues by immunohistololgy using an antibody to the recombinant filarial antigen Bm14. Parasite larvae were detected consistently after mf ingestion in Ae. aegypti- Liverpool. Infectious L3s were seen in the head, thorax and abdomen of vector mosquitoes 14 days after Mf ingestion. In contrast, parasites were only detected by histology shortly after the blood meal in Cx. pipiens, and these were not labeled by the antibody.This study provides new information on the distribution of filarial parasites and parasite DNA in vector and non-vector mosquitoes. This information should be useful for those involved in designing and interpreting molecular xenomonitoring studies.Human lymphatic filiarasis (LF) is caused by the mosquito-borne filarial nematodes Wuchereria bancrofti, Brugia malayi, and B. timori. These parasites are currently targeted for elimination by the Global Program for the Elimination of Lymphatic Filariasis (GPELF), and workers in this program have reported both achievements and future challenges to eliminating parasite transmission in endemic areas [1-3]. One important component of the elimination program is the ability to estimate infection prevalence and transmission rates, especially during mass drug administration (MDA), in order to accurately evaluate the pro
Regulatory T Cells in Human Lymphatic Filariasis: Stronger Functional Activity in Microfilaremics  [PDF]
Linda J. Wammes ,Firdaus Hamid,Aprilianto E. Wiria,Heri Wibowo,Erliyani Sartono,Rick M. Maizels,Hermelijn H. Smits,Taniawati Supali,Maria Yazdanbakhsh
PLOS Neglected Tropical Diseases , 2012, DOI: 10.1371/journal.pntd.0001655
Abstract: Infection with filarial parasites is associated with T cell hyporesponsiveness, which is thought to be partly mediated by their ability to induce regulatory T cells (Tregs) during human infections. This study investigates the functional capacity of Tregs from different groups of filarial patients to suppress filaria-specific immune responses during human filariasis. Microfilaremic (MF), chronic pathology (CP) and uninfected endemic normal (EN) individuals were selected in an area endemic for Brugia timori in Flores island, Indonesia. PBMC were isolated, CD4CD25hi cells were magnetically depleted and in vitro cytokine production and proliferation in response to B. malayi adult worm antigen (BmA) were determined in total and Treg-depleted PBMC. In MF subjects BmA-specific T and B lymphocyte proliferation as well as IFN-gamma, IL-13 and IL-17 responses were lower compared to EN and CP groups. Depletion of Tregs restored T cell as well as B cell proliferation in MF-positives, while proliferative responses in the other groups were not enhanced. BmA-induced IL-13 production was increased after Treg removal in MF-positives only. Thus, filaria-associated Tregs were demonstrated to be functional in suppressing proliferation and possibly Th2 cytokine responses to BmA. These suppressive effects were only observed in the MF group and not in EN or CP. These findings may be important when considering strategies for filarial treatment and the targeted prevention of filaria-induced lymphedema.
Yeast-Based High-Throughput Screens to Identify Novel Compounds Active against Brugia malayi  [PDF]
Elizabeth Bilsland?,Daniel M. Bean?,Eileen Devaney?,Stephen G. Oliver
PLOS Neglected Tropical Diseases , 2016, DOI: 10.1371/journal.pntd.0004401
Abstract: Background Lymphatic filariasis is caused by the parasitic worms Wuchereria bancrofti, Brugia malayi or B. timori, which are transmitted via the bites from infected mosquitoes. Once in the human body, the parasites develop into adult worms in the lymphatic vessels, causing severe damage and swelling of the affected tissues. According to the World Health Organization, over 1.2 billion people in 58 countries are at risk of contracting lymphatic filariasis. Very few drugs are available to treat patients infected with these parasites, and these have low efficacy against the adult stages of the worms, which can live for 7–15 years in the human body. The requirement for annual treatment increases the risk of drug-resistant worms emerging, making it imperative to develop new drugs against these devastating diseases. Methodology/Principal Findings We have developed a yeast-based, high-throughput screening system whereby essential yeast genes are replaced with their filarial or human counterparts. These strains are labeled with different fluorescent proteins to allow the simultaneous monitoring of strains with parasite or human genes in competition, and hence the identification of compounds that inhibit the parasite target without affecting its human ortholog. We constructed yeast strains expressing eight different Brugia malayi drug targets (as well as seven of their human counterparts), and performed medium-throughput drug screens for compounds that specifically inhibit the parasite enzymes. Using the Malaria Box collection (400 compounds), we identified nine filarial specific inhibitors and confirmed the antifilarial activity of five of these using in vitro assays against Brugia pahangi. Conclusions/Significance We were able to functionally complement yeast deletions with eight different Brugia malayi enzymes that represent potential drug targets. We demonstrated that our yeast-based screening platform is efficient in identifying compounds that can discriminate between human and filarial enzymes. Hence, we are confident that we can extend our efforts to the construction of strains with further filarial targets (in particular for those species that cannot be cultivated in the laboratory), and perform high-throughput drug screens to identify specific inhibitors of the parasite enzymes. By establishing synergistic collaborations with researchers working directly on different parasitic worms, we aim to aid antihelmintic drug development for both human and veterinary infections.
Differential transcript expression between the microfilariae of the filarial nematodes, Brugia malayi and B. pahangi
Michael M Kariuki, Leonard B Hearne, Brenda T Beerntsen
BMC Genomics , 2010, DOI: 10.1186/1471-2164-11-225
Abstract: Following microarray data analysis, a list of preferentially expressed genes in both microfilariae species was generated with a false discovery rate estimate of 5% and a signal intensity ratio of 2 or higher in either species. A total of 308 probes were preferentially expressed in both species with 149 probes, representing 123 genes, in B. pahangi microfilariae and 159 probes, representing 107 genes, in B. malayi microfilariae. In B. pahangi, there were 76 (62%) up-regulated transcripts that coded for known proteins that mapped into the KEGG pathway compared to 61 (57%) transcripts in B. malayi microfilariae. The remaining 47 (38%) transcripts in B. pahangi and 46 (43%) transcripts in B. malayi microfilariae were comprised almost entirely of hypothetical genes of unknown function. Twenty-seven of the transcripts in B. pahangi microfilariae coded for proteins that associate with the secretory pathway compared to thirty-nine in B. malayi microfilariae. The data obtained from real-time PCR analysis of ten genes selected from the microarray list of preferentially expressed genes showed good concordance with the microarray data, indicating that the microarray data were reproducible.In this study, we identified gene transcripts that were preferentially expressed in the microfilariae of B. pahangi and B. malayi, some of which coded for known immunomodulatory proteins. These comparative transcriptome data will be of interest to researchers keen on understanding the inherent differences, at the molecular level, between B. malayi and B. pahangi microfilariae especially because these microfilariae are capable of surviving in the same vertebrate host but elicit different immune response outcomes in the mosquito, Ar. subalbatus.Lymphatic filariasis is caused by the thread-like parasitic nematodes Wuchereria bancrofti, Brugia malayi and B. timori which are transmitted by mosquitoes. B. malayi and B. timori are the main cause of Brugian lymphatic filariasis in humans and B. pahang
Treatment Follow-up of Brugia malayi Microfilaraemic and Amicrofilaraemic Individuals with Serological Evidence of Active Infection
Rahmah, N.,Lim, B. H.,Mehdi, R.,Nazmi, M. N.
Malaysian Journal of Microbiology , 2005,
Abstract: Filariasis caused by Brugia malayi and Brugia timori affects ~13 million Asians. In order to ensure elimination of these infections in the context of the Global Programme for Elimination of Lymphatic Filariasis (GPELF), assays which are more sensitive than night blood examination must be employed. IgG4 assay using BmR1 recombinant antigen has been shown to be highly specific and sensitive for diagnosis of brugian filariasis. To provide further evidence of the diagnostic value of this assay, treatment follow-up study was performed on B. malayi microfilaraemic and amicrofilaraemic individuals who were positive by the BmR1-based IgG4-ELISA. Group 1 comprised 22 treated microfilaraemic individuals; group 2A comprised 13 treated amicrofilaraemic individuals and group 2B (control group) comprised 16 untreated amicrofilaraemic individuals. Group 1 individuals demonstrated decline in IgG4 levels with treatment and all participants were negative by the end of the 21 months study period. Group 2A also demonstrated IgG4 decline to negativity by 21 months, with re-treatment at 12 months performed on 3 individuals. In group 2B untreated individuals, at 21 months seven participants remained IgG4 positive while nine individuals were IgG4 negative, possibly through spontaneous death of adult worms. Significant difference (p=0.008) was observed when proportions between group 2A and group 2B were compared. This study showed decline of filaria-specific IgG4 post-treatment in both microfilaria positive and microfilaria negative individuals. In addition amicrofilaraemic IgG4 positive individuals were shown to be infected as evidenced by the significant difference between treated and untreated groups of individuals. Therefore, this study strengthened the reported findings that IgG4 assay based on BmR1 recombinant antigen is a good diagnostic tool for brugian filariasis.
Fitopatología en flores  [cached]
Garces de Granada Emira,Orozco de Amezquita Martha,Zapata ángela Cristina
Acta Biológica Colombiana , 1999,
Abstract: La generación de nuevos empleos y mayores divisas para el país, han sido los objetivos principales de las empresas dedicadas a la exportación de flores, actividad que ha pasado a ocupar un lugar importante en la economía colombiana; por tal razón, se hace necesario el estudio a fondo de la biología y el control de los patógenos que más afectan este sector de la agricultura ya que uno de los factores limitantes en la producción de flores de corte son las enfermedades causadas por hongos, bacterias, nernatodos y virus.
Live Brugia malayi Microfilariae Inhibit Transendothelial Migration of Neutrophils and Monocytes  [PDF]
Jan-Hendrik Schroeder,Bigboy H. Simbi,Louise Ford,Sara R. Cole,Mark J. Taylor,Charlotte Lawson,Rachel A. Lawrence
PLOS Neglected Tropical Diseases , 2012, DOI: 10.1371/journal.pntd.0001914
Abstract: Lymphatic filariasis is a major tropical disease caused by the parasite Brugia malayi. Microfilariae (Mf) circulate in the peripheral blood for 2–3 hours in synchronisation with maximal feeding of the mosquito vector. When absent from the peripheral blood, Mf sequester in the capillaries of the lungs. Mf are therefore in close contact with vascular endothelial cells (EC) and may induce EC immune function and/or wound repair mechanisms such as angiogenesis. In this study, Mf were co-cultured with human umbilical vein EC (HUVEC) or human lung microvascular EC (HLMVEC) and the transendothelial migration of leukocyte subsets was analysed. In addition, the protein and/or mRNA expression of chemokine, cytokine and angiogenic mediators in endothelial cells in the presence of live microfilariae were measured by a combination of cDNA arrays, protein arrays, ELISA and fluorescence antibody tests. Surprisingly, our findings indicate that Mf presence partially blocked transendothelial migration of monocytes and neutrophils, but not lymphocytes. However, Mf exposure did not result in altered vascular EC expression of key mediators of the tethering stage of extravasation, such as ICAM-1, VCAM-1 and various chemokines. To further analyse the immunological function of vascular EC in the presence of Mf, we measured the mRNA and/or protein expression of a number of pro-inflammatory mediators. We found that expression levels of the mediators tested were predominantly unaltered upon B. malayi Mf exposure. In addition, a comparison of angiogenic mediators induced by intact Mf and Wolbachia-depleted Mf revealed that even intact Mf induce the expression of remarkably few angiogenic mediators in vascular EC. Our study suggests that live microfilariae are remarkably inert in their induction and/or activation of vascular cells in their immediate local environment. Overall, this work presents important insights into the immunological function of the vascular endothelium during an infection with B. malayi.
PENENTUAN VEKTOR MALARIA DI FLORES  [cached]
Harijani A. Marwoto,Soeroto Atmosoedjono,Rita M. Dewi
Bulletin of Health Research , 2012,
Abstract: A field study on entomology has been conducted in 6 villages which were located in coastal and in-land areas of Sikka Regency of Central Flores since April 1990 - October 1991. The results of this study showed that the suspected malaria vectors in those areas were An. sundaicus, An. subpictus, An. barbirostris, An. aconitus and An. maculatus. Only 3 species were confirmed as vector using ELISA test, i.e. An. sundaicus, An. barbirostris and An. subpictus with sporosoite rates of 4.2%, 2.1% and 0.1% respectively. An. aconitus, a potential malaria vector in Java and in some onther places was not confirmed as vector in Flores yet. The 3 confirmed vectors were also found positive with sporozoites in West Flores and also found predominant in East Flores.
Diversity and Expression of MicroRNAs in the Filarial Parasite, Brugia malayi  [PDF]
Catherine B. Poole, Weifeng Gu, Sanjay Kumar, Jingmin Jin, Paul J. Davis, David Bauche, Larry A. McReynolds
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0096498
Abstract: Human filarial parasites infect an estimated 120 million people in 80 countries worldwide causing blindness and the gross disfigurement of limbs and genitals. An understanding of RNA-mediated regulatory pathways in these parasites may open new avenues for treatment. Toward this goal, small RNAs from Brugia malayi adult females, males and microfilariae were cloned for deep-sequencing. From ~30 million sequencing reads, 145 miRNAs were identified in the B. malayi genome. Some microRNAs were validated using the p19 RNA binding protein and qPCR. B. malayi miRNAs segregate into 99 families each defined by a unique seed sequence. Sixty-one of the miRNA families are highly conserved with homologues in arthropods, vertebrates and helminths. Of those miRNAs not highly conserved, homologues of 20 B. malayi miRNA families were found in vertebrates. Nine B. malayi miRNA families appear to be filarial-specific as orthologues were not found in other organisms. The miR-2 family is the largest in B. malayi with 11 members. Analysis of the sequences shows that six members result from a recent expansion of the family. Library comparisons found that 1/3 of the B. malayi miRNAs are differentially expressed. For example, miR-71 is 5–7X more highly expressed in microfilariae than adults. Studies suggest that in C.elegans, miR-71 may enhance longevity by targeting the DAF-2 pathway. Characterization of B. malayi miRNAs and their targets will enhance our understanding of their regulatory pathways in filariads and aid in the search for novel therapeutics.
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