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G J Weil

Publications and source records attributed to G J Weil.

At least 55 records · Page 3Linked to original sources
PubMed1992

Brugia malayi: antibody responses to larval antigens in infected and immunized jirds.

Vaccination with irradiated third stage Brugia malayi larvae (L3) has been reported to induce partial protective immunity to L3 challenge in jirds. The purpose of this study was to identify antigens that may be targets of protective immunity in this model. Jirds were immunized by s.c. injection of irradiated L3 and challenged either s.c. or i.p. Necropsy was performed 11 wk after challenge. Partial protection was achieved in s.c. challenged animals; worm recovery was only 41% of that observed in unvaccinated controls, and worms recovered from immunized animals were stunted. Worm recoveries in immunized animals that were challenged i.p. did not differ from those of unimmunized controls. Group differences in parasite antigen levels in sera collected 2-11 wk after larval challenge were consistent with parasitological findings obtained at necropsy. Antibody studies compared prechallenge sera from immunized animals to sera from infected (unimmunized) controls. Antibody responses to L3 surface antigens (assessed by IFA) were much stronger after immunization than after infection. Immunoblot studies showed preferential recognition of several L3 antigens (97, 54, 48, and 40 kDa) by antibodies in sera from immunized animals. Additional studies are needed to determine whether immunization with such preferentially recognized antigens can induce protection to larval challenge comparable to or better than that observed with live vaccines.

Animals↗
PubMed1992

Use of a recombinant antigen (OC 3.6 cDNA) for the serological diagnosis of onchocerciasis in exposed Nigerian children.

We evaluated a recombinant antigen (OC 3.6 cDNA) expressed in pMAL TM vector for the diagnosis of onchocerciasis in children living in an endemic focus of forest-type onchocerciasis in Nigeria. Using the Western blot with the maltose-binding fusion protein as antigen, 91 per cent of mf-positive children and 24 per cent of endemic normal children were positive. Furthermore, age was not a limitation to the assay. The practical limitations of the assay for field diagnosis of onchocerciasis is discussed.

Animals↗
PubMed1992

Field evaluation of IgG4 serology for the diagnosis of onchocerciasis in children.

IgG4 serology was evaluated for early diagnosis of onchocerciasis in children living within an endemic focus of forest-type onchocerciasis in Nigeria. IgG4 serology proved to be more sensitive than conventional skin snip examination being able to detect 42% of infected children as opposed to 24% by snip examination. Furthermore, age was not a limitation to the technique within the 8-12 y spectrum. We conclude that IgG4 serology in children is a reliable indicator of community microfilarial rate and ongoing transmission of onchocerciasis.

Age Factors↗
PubMed1991

Identification of paramyosin as a potential protective antigen against Brugia malayi infection in jirds.

Vaccination of jirds with irradiated infective larvae of Brugia malayi has been reported to provide partial immunity to larval challenge. In the present study, we found that sera from vaccinated animals recognized larval antigens with apparent molecular weights of 97, 55-60, and 10 kDa that were not recognized by sera from infected animals. A B. malayi cDNA expression library in lambda gt11 was screened to identify clones that were preferentially recognized by sera from immunized animals. One of these clones (BM-5) was chosen for further study. BM-5 contains a 2.1 kb DNA insert and produces a fusion protein with a molecular weight of 185 kDa. Antibody, affinity-purified with the BM-5 fusion protein, binds to a 97 kDa native B. malayi antigen. Immunological studies and partial DNA sequence data confirm that BM-5 encodes paramyosin. Recombinant B. malayi paramyosin is strongly recognized by antibodies in sera from jirds that have been immunized either by injection with irradiated larvae or by chemotherapy-abbreviated infection. Most sera from infected jirds do not contain antibody to paramyosin. Additional studies are needed to determine whether paramyosin is actually protective in this filariasis model.

Amino Acid Sequence↗
PubMed1991

Changes in circulating parasite antigen levels after treatment of bancroftian filariasis with diethylcarbamazine and ivermectin.

This study assessed changes in circulating parasite antigen levels after diethylcarbamazine (DEC) and ivermectin treatment of bancroftian filariasis to determine effects of these drugs on adult Wuchereria bancrofti in vivo. Thirty adult Haitians with microfilaremia were treated with 1 mg of ivermectin to reduce counts of microfilariae. Later, subjects were treated with either one or two 200 micrograms/kg doses of ivermectin or with 12 daily 6 mg/kg doses of DEC. Macrofilaricidal activity of these drugs was indirectly monitored by measuring circulating W. bancrofti antigen by EIA. Antigen levels fell by 75% after DEC and by 34% after ivermectin. These results suggest that low-dose ivermectin treatment followed by a standard course of DEC is a more effective macrofilaricidal regimen for W. bancrofti than either of the multidose ivermectin regimens used in this study.

Animals↗
PubMed1991

Molecular cloning and characterization of recombinant parasite antigens for immunodiagnosis of onchocerciasis.

Immunological cross-reactivity among nematodes has hampered the development of specific serodiagnostic assays for onchocerciasis. In the present study, an Onchocerca volvulus adult worm complementary DNA expression library was differentially screened with human sera from patients infected with O. volvulus and with an omnibus anti-nematode serum pool comprised of sera from patients infected with Brugia malayi, Loa loa, Wuchereria bancrofti, Mansonella perstans, Strongyloides stercoralis, Ancylostoma duodenale, Ascaris lumbricoides, and Dracunculus medinensis. Seven Onchocerca-specific clones were identified and screened with individual onchocerciasis patient sera. Additional studies were performed to characterize the most immunoreactive clones, OC 3.6 and OC 9.3. OC 3.6 produced a 152-kD beta-galactosidase fusion protein that was recognized in dot-immunoblots by 54 of 55 sera from onchocerciasis patients (98%). The OC 3.6 DNA insert is 996 bp long with an open reading frame of 627 bp and a 369-bp untranslated 3' end. OC 3.6 is closely related to a previously reported clone (OV 33-3), but it differs from that clone at both the 5' and 3' ends. OC 9.3 contained a novel 565-bp insert and produced a 138-kD fusion protein that was recognized by 46 of 55 sera from onchocerciasis patients (83%). Additional studies are in progress to develop and evaluate immunodiagnostic tests for onchocerciasis based on measurement of antibodies to these promising recombinant antigens.

Amino Acid Sequence↗
PubMed1991

Evaluation of a monoclonal-antibody based antigen assay for diagnosis of Wuchereria bancrofti infection in Egypt.

Conventional methods for diagnosis of Wuchereria bancrofti infection are insensitive and often impractical because of the need for night blood collections. A sensitive and specific antigen detection assay has been developed for W. bancrofti, which is based on a monoclonal antibody (AD12) that binds to a repeated epitope on a 200 kDa adult worm excretion product present in sera from infected humans. The only formal evaluation of this assay to date was performed with sera from India. In the present study, we have evaluated the performance of the AD12 antigen assay in two laboratories with sera collected in endemic and non-endemic areas in Egypt. Antigen was detected in 57 of 59 (97%) sera from microfilaremic subjects, and in 22 of 139 asymptomatic and amicrofilaremic subjects who reside in a highly endemic area. Antigen titers were significantly correlated with microfilaria counts (r = 0.41, P less than 0.01). Filarial antigen was not detected in most sera from amicrofilaremic subjects with clinical filariasis. Comparative antigen test results obtained from laboratories in Cairo and St. Louis agreed in 170 of 173 sera tested. Filarial antigen was not detected in sera from Egyptians with no history of residence in filaria-endemic areas. Specifically, nonendemic sera from patients with other parasitic infections (schistosomiasis, fascioliasis, ascariasis, etc.) were uniformly negative in the assay. We conclude that the AD12 filarial antigen assay is sensitive and specific for W. bancrofti infection in Egypt.

Animals↗
PubMed1991

Effect of CGP 20376 on Brugia malayi and parasite antigenemia in jirds.

This study was designed to investigate the activity of CGP 20376, a benzothiazole derivative, against Brugia malayi in jirds and to illustrate the utility of parasite antigen detection as a means of monitoring drug efficacy in filariasis. Drug treatment was 100% effective in jirds treated 3 or 24 days after infection. Microfilaria and adult worm counts were reduced (relative to counts in sham-treated control animals) by 96% and 95%, respectively, in animals treated 153 days after infection. Four of 6 animals in this treatment group cleared their microfilaremias and were free of adult worms 5 mo after treatment. Thus, CGP 20376 was effective against all life cycle stages of B. malayi in jirds. Parasite antigen levels in jird sera were consistent with parasitological results in all treatment groups, but antigen clearance was incomplete in some cases after apparently successful treatment of mature and immature infections.

Animals↗
PubMed1990

Simultaneous flow cytometric measurements of thrombin-induced cytosolic pH and Ca2+ fluxes in human platelets.

Human platelets exhibit an extremely rapid increase in cytoplasmic Ca2+ concentrations ((Ca2+]in) and a dose-dependent cytoplasmic pH change ((pH]in) upon thrombin stimulation. A cytoplasmic alkalinization, maximal by 60 s, is preceded by a very rapid acidification, which is masked by the alkalinization when saturating thrombin doses are used. Using the pH probe 2',7'-bis-(carboxyethyl)-5(6)-carboxyfluorescein we report here the kinetics of simultaneous cytoplasmic pH and Ca2+ changes in thrombin-stimulated platelets, measured in single cells by flow cytometry. This permits analysis of the responding subpopulation. Maximal thrombin stimulation (greater than or equal to 4.5 nM) induces a dose-dependent increase in pHin from approximately 7.0 to 7.30 and a maximal [Ca2+]in transient of up to 800 nM. The Ca2+ transient coincides temporally with the rapid initial acidification, while the alkalinization is maximal considerably later. The Ca2+ transients occur maximally in each responding cell, but occur only in a subpopulation of the platelets at subsaturating (less than 4.5 nM) thrombin doses; in contrast, the dose-dependent cytoplasmic acidification appears to occur uniformly in all platelets. The rapid increase in [Ca2+]in is not dependent on the alkalinization, and the former occurs maximally in amiloride treated, Na+/H+ exchange inhibited human platelets. These results indicate that the acidification and the rise in [Ca2+]in may be interrelated, whereas the cytoplasmic alkalinization (maximal considerably later than either the acidification or the [Ca2+]in rise) may be independent of these earlier, temporally correlated increases in H+ and Ca2+ concentrations.

Blood Platelets↗
PubMed1990

Measurement of superoxide release in the phagovacuoles of immune complex-stimulated human neutrophils.

Immune complex stimulation of human neutrophils elicits, among other events, the formation of phagocytic vacuoles into which the products of the stimulus activated oxidative burst and degranulation are released. In order to monitor burst activity in the phagocytic vacuole, we have developed a fluorochrome-coupled derivative of this neutrophil agonist. The fluorochrome 2',7'-dichlorodihydrofluorescein (DCFH) (the nonfluorescent, reduced form of 2',7'-dichlorofluorescein (DCF] has been covalently linked to bovine serum albumin (BSA), which can be used to form an immune complex with anti-BSA immunoglobulin. The resultant complex is an effective agonist for stimulating all immune complex-mediated neutrophil responses, as compared to nonderivatized controls. Upon exposure to hydrogen peroxide the stimulus-linked probe is converted to its oxidized, fully fluorescent form, the fluorescence of which is linearly related to the extent of probe oxidation. Using flow cytometry, we have demonstrated that the probe-stimulus complex is capable of monitoring the kinetics of the production of activated oxygen species by the membrane bound NADPH-oxidase enzyme, presumably within the phagocytic vacuoles of immune complex-activated neutrophils. We have found that the immune complex-mediated activation of the oxidative burst within the phagocytic compartment is preceded by a lag of approximately 30 s followed by a large sustained release of superoxide dependent hydrogen peroxide. Neutrophils from patients with chronic granulomatous disease, however, demonstrated no sustained increase in probe fluorescence, a finding consistent with the lack of oxidative burst activity in these cells. The DCFH-immune complex conjugate therefore provides an effective probe for monitoring the kinetics of the localized release of oxidative products within the forming phagocytic vacuoles of activated neutrophils, and may be used to further examine both the activation and activity of human neutrophils in response to 'physiologic' host defense agonists such as immune complexes.

Antigen-Antibody Complex↗
PubMed1990

IgG4 subclass antibody serology for onchocerciasis.

Antibodies to phosphorylcholine and carbohydrate determinants responsible for much of the cross-reactivity among nematodes are subclass restricted in humans and absent in the IgG4 subclass. Total IgG and IgG4 antibody responses to Onchocerca volvulus were examined by enzyme immunoassay and by immunoblot. Significant background IgG reactivity was detected in both assays in US control sera and sera from patients with intestinal nematode infections, but background reactivity was negligible in the IgG4 assays. IgG4 antibodies were detected by enzyme immunoassay in 17 of 18 Nigerian onchocerciasis serum samples and in 2 of 9 endemic control serum samples. Cross-reactive IgG4 antibodies were present in serum pools from patients infected with other filariae. IgG4 antibodies recognized a restricted subset of O. volvulus antigens in immunoblots relative to IgG. These results confirm the previously reported enhanced specificity of IgG4 antibody assays for filariasis and extend the observation to include onchocerciasis. Whereas IgG4 subclass antibody serology is more specific than measurement of total IgG antibodies for onchocerciasis, cross-reactivity among filariae limits the utility of this approach.

Animals↗
PubMed1990

Circulating immune complex-associated parasite antigens in human onchocerciasis.

This study identified and characterized parasite antigens in sera from humans infected with Onchocerca volvulus. Immune complexes were precipitated from human sera with polyethylene glycol and analyzed by immunoblot with rabbit antibodies to O. volvulus. A 23-kDa parasite antigen was detected in sera from 17 of 23 Nigerian onchocerciasis patients and 5 of 10 endemic controls. Other parasite antigens with apparent molecular masses of 62, 66, and 70 kDa were less consistently observed. These antigens were not present in Nigerian or US nonendemic control sera, in sera from patients with various other parasitic infections, or in sera from US patients with autoimmune diseases. Biochemical studies indicated that these antigens are nonglycosylated acidic proteins that do not contain phosphorylcholine. These antigens may be useful as targets for improved diagnostic tests for onchocerciasis based on parasite antigen detection.

Adult↗
PubMed1990

Use of parasite antigen detection to monitor macrofilaricidal therapy in Brugia malayi-infected jirds.

Improved methods are needed to evaluate new treatments for filarial infections. We have recently developed a monoclonal antibody-based enzyme immunoassay to detect circulating parasite antigen in sera from Brugia malayi-infected jirds. In the present study, parasite antigen levels were compared to parasitological parameters after treatment of B. malayi-infected jirds with CGP 20376 that has been reported to be active against both microfilariae and adult worms of this parasite. Microfilariae were cleared promptly and permanently after CGP 20376 treatment, and no adult worm was recovered in jirds at necropsy 20 wk after treatment. In contrast, untreated animals had sustained microfilaremia throughout the course of the study, and adult worms were recovered in all control animals (mean worm recovery; 24.3 +/- 7.8 SE). Parasite antigen was present in sera from all infected animals before treatment. Parasite antigen titers in sera were unchanged 5 wk after treatment but fell to undetectable levels in 4 of 6 animals by 20 wk after treatment. Low-level antigenemia was detected in 2 of 6 animals at 20 wk, perhaps suggesting incomplete killing of parasites or incomplete clearance of antigen. Parasite antigen levels were stable throughout the study in control animals. These preliminary results suggest that parasite antigen detection is useful as a means of noninvasively monitoring the efficacy of anti-filarial drug therapy.

Animals↗
PubMed1990

Circulating parasite antigen in Brugia pahangi-infected jirds.

The Mongolian jird is used widely in filariasis research for studies of protective immunity, pathogenesis, and therapy. The purpose of this study was to evaluate parasite antigen detection as a means of noninvasively monitoring Brugia pahangi infection in jirds. A parasite antigen with Mr of 105-110 kDa was identified in sera from i.p.- and s.c.-infected jirds by immunoblot with a monoclonal antibody to phosphorylcholine. The same antibody was used in a direct sandwich enzyme immunoassay to measure antigen in jird sera. Parasite antigen was detectable as early as 2 wk after i.p. or s.c. injection of L3. Antigen titers increased between 2 and 12 wk and stabilized between 12 and 36 wk after infection in s.c.-infected animals. A different pattern was seen in i.p.-infected jirds with antigen titers peaking at 16 wk and falling significantly between 16 and 32 wk after infection. Parasite antigen titers correlated significantly with adult worm infection intensities in jirds with mature i.p. and s.c. infections. Antigenemia was also detectable in sera from jirds after i.p. implantation of adult parasites of either sex. However, antigen was not detected in sera from infant offspring of antigenemic infected mothers. We conclude that parasite antigen detection allows B. pahangi development and survival as well as infection intensity to be monitored in living animals with unprecedented sensitivity and accuracy. This technique should facilitate drug and vaccine studies in this important experimental filariasis model.

Animals↗
PubMed1989

Cytoplasmic Ca2+ is necessary for thrombin-induced platelet activation.

alpha-Thrombin induces a dose-dependent rapid transient increase in platelet cytosolic Ca2+ levels, coming solely from intracellular stores, since EGTA has no effect. In contrast, the post-stimulation equilibrium [Ca2+]in depends upon an influx from the extracellular milieu, and is lower in the presence of EGTA. We measured the Ca2+ transient (with Indo-1, 1-[2-amino-5-(6-carboxyindol-2-yl)-phenoxy]-2-(2'-amino-5'-methylp henoxy)- ethane-N,N,N',N'-tetraacetic acid), cytosolic alkalinization (with BCECF, 2',7-bis-(2-carboxyethyl)-5(and 6)-carboxyfluorescein), membrane depolarization (with diS-C3-(5), 3,3'-dipropylthiodi-carbocyanide iodide), and degranulation (by beta-glucuronidase release) induced in washed human platelets by 9 nM thrombin in the absence or presence of extracellular or intracellular Ca2+ chelating agents (EGTA and BAPTA, 5,5'-dimethyl-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, respectively). Platelets loaded simultaneously with 2 microM Indo-1 and 15 microM BAPTA (each as the acetoxymethyl ester) before addition of thrombin exhibited no cytoplasmic Ca2+ transient or alkalinization, no depolarization or degranulation. Replenishment of such cells with extracellular CaCl2 restored resting [Ca2+]in. Upon stimulation with 9 nM thrombin these replenished platelets exhibited no Ca2+ transient, and a slow gradual increase in [Ca2+]in from extracellular stores, a slow alkalinization and depolarization, and partial degranulation, all abolished by extracellular EGTA. Thus thrombin-induced platelet activation exhibits a biphasic Ca2+ requirement: the initial transient increase in [Ca2+]in comes from intracellular stores only, while the later steps of depolarization, alkalinization, and degranulation can proceed, albeit more slowly, if only extracellular Ca2+ is available.

Blood Platelets↗
PubMed1989

Use of Iodogen and sulfosuccinimidobiotin to identify and isolate cuticular proteins of the filarial parasite Brugia malayi.

The cuticle of filarial nematodes is a dynamic structure which may be an important target for protective host immune responses. Prior studies have employed radioiodination of intact parasites to demonstrate that the collagenous cuticle of filariids contains relatively few exposed proteins, some of which are stage and/or species-specific. In the present study, we have used sulfo-NHS-biotin to label and affinity purify cuticular components of living adult Brugia malayi. Results obtained by this method were compared with the widely used Iodogen method of surface radioiodination by SDS-PAGE analysis of detergent-solubilized worms and by ultrastructural analysis. Both labeling methods produced very similar electrophoretic patterns with major doublets at 70 and 100 kDa, a major band at 25 kDa, and minor bands between 60-200 kDa. Ultrastructural analysis showed that both methods labeled components throughout all levels of the parasite cuticle; underlying somatic tissues were not labeled. The biotinylated components were isolated from the total parasite extract by affinity chromatography on an avidin matrix. Further characterization of these surface-associated proteins may lead to improved methods for the control of filariasis.

Animals↗
PubMed1989

Detection of Wuchereria bancrofti antigen in serum and finger prick blood samples by enzyme immunoassay: field evaluation.

We have recently reported that a monoclonal antibody-based enzyme immunoassay for detection of filarial antigen in human serum is sensitive and specific for active infection with Wuchereria bancrofti. The present studies were undertaken to assess the feasibility of testing whole blood collected by finger prick in this assay. A preliminary study was performed to compare antigen test results obtained with whole blood, blood dried on filter paper, and serum. Results obtained with anticoagulated whole blood specimens agrees with serum results in 94 of 97 cases. All whole blood and serum specimens from 28 people with positive microfilaria smears were positive in the test. Filter paper blood specimens were less satisfactory because of decreased sensitivity and specificity. A population-based survey of 1009 persons was conducted in a village near Calicut, India, to evaluate the use of finger prick blood specimens for filarial antigen detection in field studies. Thirty-eight of 39 microfilaria carriers had positive antigen tests. In addition, 10.7% of amicrofilaremic endemic controls had positive tests. Additional studies are needed to test the hypothesis that filarial antigenemia in endemic controls indicates the presence of subclinical infection.

Adolescent↗