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J Parasitol,
1994]
Shipment of infective-stage filarial larvae (L3s) usually has been accomplished by transporting living infected vectors or L3s cryopreserved in liquid nitrogen. Our objective was to find culture conditions for transporting L3s that would promote survival of Brugia malayi larvae without altering their capacity to infect susceptible animals. In preliminary studies we observed that Ham's nutrient mixture F-12, with antibiotics and 1% fetal calf serum, could support L3s without apparent development for at least 10 days. In order to evaluate the effect of culture temperatures on infectivity, fresh L3s were divided into groups that were either immediately injected into jirds (infectivity control) or incubated for 24, 48, or 120 hr in tightly sealed tubes maintained horizontally at either 0 C, 20 C, or 37 C, before they were injected into jirds. Necropsies were performed on the jirds 120-130 days after injection to recover and count adult worms. Levels of microfilaremia were also determined. We found that L3s held overnight at 0 C, although apparently viable, were unable to survive in jirds. However, larvae kept at 20 C and 37 C produced patent infections with adult worms in normal locations even after 120 hr of in vitro cultivation. There was no statistical difference in mean worm recovery or size of worms from jirds infected with freshly harvested L3s and jirds injected with larvae that were maintained overnight at 20 C or 37 C. When cultured L3s were shipped from Michigan to Connecticut by overnight air courier, along with infected living mosquitos, the L3s appeared to be 99% viable upon arrival. L3s shipped in F-12 produced patent infections in C.B.-17 scid/scid mice with worm recoveries comparable to those observed in mice injected with L3s freshly obtained from shipped mosquitos.
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Acta Trop,
1985]
Infective stage larvae (L3) of Loa loa and Brugia malayi upon in vitro incubation with normal human serum activated the alternative complement pathway. C3 conversion products were detected on larval cuticles by eosinophil adherence and by immunofluorescence with C3c antiserum. No evidence for cuticle binding of IgG, IgA, IgM, Clq, or C4 was found by immunofluorescence. L3-induced C3 activation was inhibited by 10 mM EDTA but unaffected by 10 mM Mg++-EGTA. Human sera deficient in C2, C4, or C6 incubated with L3 resulted in C3 activation. However, sera treated with zymosan or heated for 1 h, 56 degrees C were unreactive with L3. Immunoelectrophoresis of fresh serum exposed to L3 for 1 h at 37 degrees C showed C3 cleavage products. The results indicate that these nematode L3 activate the alternative complement cascade via cuticular surface components. Larval viability was unaffected by complement activation or by adherence of eosinophils.
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Am J Trop Med Hyg,
1985]
Vaccination of inbred jirds (Meriones unguiculatus) with 60cobalt radiation-attenuated Brugia malayi infective stage larvae (L3) protected against homologous challenge given either subcutaneously (sc) or by the intraperitoneal (ip) route. Groups of jirds vaccinated once sc with 75, 15 Krad L3 showed from 69% to 91% reduction in recovered worms after ip challenge infection compared to infection in non-vaccinated control jirds, while 75% reduction in mean worm burden was seen in jirds receiving sc challenge infection. A single sc vaccination with 75, 10 or 20 Krad L3 produced no protection (10 Krad) and 64% reduction in recovered worms (20 Krad). Therefore the 15 Krad dose appeared to be best. A marked increase in anti-B. malayi antibody in vaccinated jirds was seen (by ELISA) immediately after challenge infection and an immunofluorescence assay showed that L3 incubated in serum from vaccinated jirds were completely and uniformly covered with specific antibody. Eosinophil-rich granulomas containing dead and moribund L3 were recovered from vaccinated jirds. This model of protective immunity in a Brugia-susceptible small rodent may provide a useful system for identification of molecularly defined filarial-protective immunogens.
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Am J Trop Med Hyg,
1994]
Over the past several decades, epidemiologic data from filarial vectors typically has been obtained by mass dissection or by dissection of individual specimens. The former is quick and easy to do on large numbers of insects but provides no information on the frequency distribution of infection, presence of early developmental stages, or larval location; the latter is labor-intensive and tedious. We describe a new technique that can provide data comparable to those obtained by individual dissection, including calculation of infection and infective rates, and this technique is easy enough to accommodate large numbers of insects. Brief treatment of ethanol-fixed, intact mosquitoes in sodium hypochlorite, followed by treatments in increasing concentrations of ethanol and an organic solvent allowed microscopic visualization of filarial larvae within the abdomen, thorax, head, and proboscis of Brugia malayi-infected Aedes aegypti and Wuchereria bancrofti-infected Anopheles punctulatus. We compared the classic techniques to our technique using Ae. aegypti infected by feeding on jirds with B. malayi microfilaremias. Comparisons of the infective rate, total number of infective stage larvae (L3s) observed, and locations of L3s showed that this new technique was comparable to the established methods, while being faster and more precise in determining the location of larvae.
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Acta Trop,
1994]
We have used the severe combined immunodeficient C.B-17-scid/scid mouse to investigate the influences of maternal immune status and parasite burden on the susceptibility (or resistance) of offspring to infection with the human filarial parasite, Brugia malayi. C.B-17-scid/scid mice are permissive for infection while immunocompetent C.B-17(-)+/+ mice are uniformly resistant. Reciprocal matings of C.B-17-scid/scid and C.B-17(-)+/+ mice were performed. The C.B-17-scid/scid females were either naive or infected with Brugia malayi. The resulting immunocompetent C.B-17-scid/+ and C.B-17(-)+/scid progeny were challenged at weaning with an intraperitoneal injection of Brugia malayi third stage larvae known to produce patent infection in > 95% of C.B-17-scid/scid mice. We observed that 40.0%l (34/85) of the immunocompetent offspring of C.B-17-scid/scid females x C.B-17(-)+/+ males were permissive for the growth and development of Brugia malayi larvae to adults. No difference was observed in susceptibility to infection between the progeny of infected or uninfected C.B-17-scid/scid mothers mated with C.B-17(-)+/+ fathers, arguing against acquired immunological tolerance to the parasite in the former. In marked contrast, only 4.8% (2/42) of the heterozygous progeny of wild type C.B-17(-)+/+ females mated with C.B-17-scid/scid males were permissive. These observations document conversion of a 'resistant' phenotype to a 'susceptible' phenotype by manipulation of maternal immune status and provide clear evidence of maternal influence on offspring susceptibility to infection with Brugia malayi.
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Exp Parasitol,
1994]
Immunocompetent mice are nonpermissive for the development and maturation of the human filarial parasite, Brugia malayi. We and others have shown that the absence of T-lymphocytes, alone or in combination with B-lymphocytes, renders mice permissive to infection. In a previous study, we showed that mice lacking CD8+ T-lymphocytes are also completely nonpermissive for B. malayi, indicating that CD8+ T-lymphocytes are not an obligate requirement for resistance. In the present study, we have examined the role of CD4+ T-lymphocytes in resistance to filarial infection using two experimental systems. In the first, we used an anti-CD4 monoclonal antibody to deplete CD4+ T-cells in vivo in immunocompetent BALB/c mice. In the second system, we used mutant mice in which the gene encoding the CD4 antigen had been disrupted by homologous recombination, resulting in a lack of CD4+ T-cells. Challenge of either the anti-CD4 antibody depleted BALB/c mice or CD4 knockout mice with B. malayi infective-stage larvae demonstrated that mice lacking CD4+ T-lymphocytes were resistant to infection. These data indicate that CD4+ T-cells are not an obligate requirement for murine resistance to B. malayi.
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Nat Methods,
2004]
To take advantage of the potential quantitative benefits offered by tandem mass spectrometry, we have modified the method in which tandem mass spectrum data are acquired in ''shotgun'' proteomic analyses. The proposed method is not data dependent and is based on the sequential isolation and fragmentation of precursor windows (of 10 m/z) within the ion trap until a desired mass range has been covered. We compared the quantitative figures of merit for this method to those for existing strategies by performing an analysis of the soluble fraction of whole-cell lysates from yeast metabolically labeled in vivo with (15)N. To automate this analysis, we modified software (RelEx) previously written in the Yates lab to generate chromatograms directly from tandem mass spectra. These chromatograms showed improvements in signal-to-noise ratio of approximately three- to fivefold over corresponding chromatograms generated from mass spectrometry scans. In addition, to demonstrate the utility of the data-independent acquisition strategy coupled with chromatogram reconstruction from tandem mass spectra, we measured protein expression levels in two developmental stages of Caenorhabditis elegans.