PubMed Health⌕ Search

Biomedical subjects

J Wilhelm

Publications and source records attributed to J Wilhelm.

At least 55 records · Page 3Linked to original sources
PubMed1994

Immunogenicity of recombinant influenza virus haemagglutinin carrying peptides from the envelope protein of human immunodeficiency virus type 1.

Haemagglutinin (HA), the major surface glycoprotein of influenza virus, is a potent immunogen against which viral neutralizing antibodies are directed. Studies of the three-dimensional structure of HA have identified major antigenic sites on the molecule. We have exploited HA as a carrier for small antigenic regions (epitopes) of the HIV-1 envelope (env) glycoprotein. Using recombinant DNA techniques, the epitopes were inserted in-frame into a known antigenic site of HA to produce HA-epitope chimeras. Guinea-pigs and mice immunized with these chimeras in combination with adjuvant generated significant immune responses against the carrier HA and also produced epitope-specific antibodies that recognized the native whole HIV-1 env. One of the chimeras which contained a V3-loop sequence of HIV-1 env elicited neutralizing antibodies against the homologous strain of HIV-1. The antibodies against HA and the inserted epitopes remained at high levels for up to 72 weeks. Remarkably, these responses were generated with low doses of immunogens containing only nanogram quantities of the inserted epitopes. These results suggest the utility of HA as a carrier to allow selective antibody induction against foreign epitopes, and offer a new approach for vaccine development as well as for the production of monospecific antibodies useful in diagnostics and research.

AIDS Vaccines↗
PubMed1993

Immunogenicity of recombinant human adenovirus-human immunodeficiency virus vaccines in chimpanzees.

Recombinant human adenovirus (Ad) type 4-, 5-, and 7-vectored vaccines expressing either the HIV env or gag-protease genes were tested for immunogenicity in three chimpanzees. The first phase of the vaccination protocol consisted of a primary and two booster immunizations with Ad-HIVs by the oral route of administration, followed by a single booster immunization with Gag and/or Env subunit vaccines. The second phase of the vaccination protocol consisted of intranasal administration of Ad-HIVs previously administered by the oral route. Following the first phase adenovirus was shed into stools for only 1-7 days and modest type-specific anti-adenovirus neutralizing antibody titers were induced. Strong anti-Env binding antibody responses were detected in all three animals following the second oral booster immunization. One chimpanzee responded with a low-titered type-specific neutralizing antibody response to HIV. Cell-mediated immune responses to Env were not detected after the primary vaccination, but were detected following all booster immunizations. Administration of the Gag subunit vaccine boosted both humoral and cell-mediated immune responses to Gag antigens. In contrast, the Env subunit vaccine boosted cellular but not humoral immune responses. In the second phase of the vaccination protocol, both virus shedding and anti-adenovirus responses were enhanced. All three chimpanzees responded to the intranasal administration of Ad7-HIVs with boosted anti-HIV serum responses, including low-titered type-specific neutralizing antibodies, elicited anti-HIV antibodies at secretory sites, and stimulated cell-mediated immune responses to both Gag and Env antigens.

AIDS Vaccines↗
PubMed1993

[Extraction of a ruptured PTCA balloon catheter].

While attempting angioplasty of a very tight and long circumflex lesion with a metal-shafted monorail catheter, the flexible part of the balloon catheter shaft broke off within the guiding catheter. The lost piece of catheter was then retrieved from the coronary artery by using an on-the-wire balloon catheter that was inflated inside the distal end of the guiding catheter.

Angioplasty, Balloon, Coronary↗
PubMed1992

Adenovirus-human immunodeficiency virus (HIV) envelope recombinant vaccines elicit high-titered HIV-neutralizing antibodies in the dog model.

Recombinant human adenoviruses (Ads) (types 4, 5, and 7) expressing the HIV-1 envelope membrane glycoprotein (gp160) were tested for immunogenicity in the dog. Administration of recombinant Ad7-env by intratracheal inoculation resulted in a low serum antibody response to gp160, which developed over several weeks. A strong neutralizing antibody response to the Ad7 vector developed within 1 week of infection. A subsequent booster inoculation 12 weeks later with the heterotypic Ad4-env recombinant virus resulted in significantly enhanced humoral responses directed at the envelope antigen, as measured by both ELISA and Western blot analysis as well as high-titer type-specific neutralizing antibodies, with some animals achieving neutralization titers approaching 1000. Recombinant HIV envelope glycoprotein derived from Ad-HIV-infected cell cultures was used as a subunit booster injection for dogs that had previously received sequential immunizations with heterotypic recombinant Ads. Significant immune responses against the envelope developed as measured by ELISA, Western blot analysis, and neutralization assays. These data indicate that live recombinant Ad-HIV vaccines are capable of inducing high-titer type-specific neutralizing antibodies to gp160 in vivo. Recombinant HIV envelope glycoprotein subunit vaccines, prepared from Ad-env-infected cells, are capable of boosting these responses.

AIDS Vaccines↗
PubMed1992

Chromatography and spectrofluorometry of brain fluorophores in neuronal ceroid lipofuscinosis (NCL).

The aim of the present work was to develop a chromatographic system for the separation of individual fluorophores extracted from neuronal ceroid lipofuscinosis (NCL) brain and isolated storage bodies. Extracts from gray matter were best resolved on silica-gel HPTLC plates using a mixture of chloroform/methanol/water (55:45:10 by vol.). Two other chromatographic systems were tested which gave poorer separation. Corrected fluorescence spectra were obtained on the original extract and fluorescence intensity, especially at longer wavelengths was increased in both samples. Yellow and blue fluorophores were detected on HPTLC plates using a primary violet and secondary yellow filter with cut-off levels of 400 and 520 nm, respectively. Plates were photographed at 20 min, 2 h and 1 week after chromatography. With this filter system, up to 12 yellow bands of differing intensity were observed at 20 min but with time, some of these changed to blue as a result of autoxidation. NCL tissues emit yellow fluorescence when viewed under light microscopy, however extracted material did not demonstrate a distinct peak in this region of the spectrum which should be around 575 nm. HPTLC confirmed this observation and time studies revealed that autoxidation changes occur and must be carefully controlled to reduce artifacts. The discrepancy between extracted and non-extracted observations may be the result of superposition of multiple fluorophores with differing maxima and/or a self-absorption phenomenon. The combination of chromatographic separation and spectral analysis as described in this study, may be a valuable technique to further clarify the characteristics of compound fluorescent lipopigments. It is suggested that NCL fluorophores of human brain differ in their properties from other models.

Brain Chemistry↗
PubMed1991

Ultrastructural characterization of human immunodeficiency virus type 1 Gag-containing particles assembled in a recombinant adenovirus vector system.

The human immunodeficiency virus type 1 (HIV-1) Gag protein was expressed in A549 cells infected with recombinant adenovirus types 4 and 7, each carrying the HIV-1 gag and pro genes. The Gag protein was assembled into enveloped virus-like particles that budded from plasma and vacuolar membranes. The particles, isolated by precipitation and isopycnic density centrifugation, contained both processed and unprocessed Gag-associated proteins.

Adenoviruses, Human↗
PubMed1991

Induction of sustained patency after clot-selective coronary thrombolysis with Hybrid-B, a genetically engineered plasminogen activator with a prolonged biological half-life.

BACKGROUND: Despite the utility of tissue-type plasminogen activator (t-PA) in eliciting coronary thrombolysis clinically, early reocclusion remains a problem, occurring despite anticoagulation in 5-30% of patients with initially successful recanalization. This study evaluated the utility of Hybrid-B, a molecular variant of t-PA with a prolonged half-life in the circulation, in eliciting coronary thrombolysis and maintaining patency in the presence of a continuing thrombogenic stimulus. METHODS AND RESULTS: In intact, anesthetized dogs, either 18 mg Hybrid-B over 30 minutes (n = 15) or 50 mg t-PA (Activase) over 60 minutes (n = 8) was administered starting 60 minutes after left anterior descending coronary artery occlusion was induced with a thrombogenic copper coil. Time to lysis averaged 54 +/- 26 (means +/- SD) minutes and 64 +/- 34 minutes with Hybrid-B and t-PA, respectively (p = NS). When Hybrid-B was administered as a bolus (20 mg over 1 minute) to induce a high initial concentration in blood, time to lysis was shortened markedly and averaged 15 +/- 5 minutes. Dogs given Hybrid-B by either infusion or bolus exhibited prolonged time to reocclusion (337 +/- 192 minutes compared with 192 +/- 125 minutes in dogs given t-PA, p less than 0.03), reflecting maintenance of a subthrombolytic but persistently active concentration of activator in blood. Despite the persistence of Hybrid-B in blood, concentrations of fibrinogen and alpha 2-antiplasmin were not depleted markedly and remained at 77 +/- 25 and 56 +/- 24%, respectively, of control values. CONCLUSIONS: Thus, Hybrid-B, a novel variant of t-PA with unique pharmacokinetic properties, elicits prompt, sustained, and clot-selective coronary thrombolysis.

Animals↗
PubMed1990

Deglycosylation increases the fibrinolytic activity of a deletion mutant of tissue-type plasminogen activator.

delta 2-89 t-PA is a deletion mutant lacking the finger (F) and epidermal growth factor (EGF) domains; thus, the fibrin interaction of this molecule must be mediated solely by the kringle region. In the present study, the influence of the oligosaccharide side-chains on the activity of delta 2-89 t-PA has been investigated. delta 2-89 t-PA was secreted in two forms, designated I and II, which presumably differ by the lack of one asparagine-linked oligosaccharide in the kringle 2 domain of form II. Forms I and II of delta 2-89 t-PA were purified; form II displayed higher fibrinolytic activity than form I. When form I was partially deglycosylated or treated to remove sialic acid, fibrinolytic activity was increased. Production of delta 2-89 t-PA in the presence of tunicamycin led to secretion of a glycan-free activator with higher activity. These findings suggest that certain oligosaccharide side-chains, particularly those containing sialic acid, can interfere with the interaction between the kringle region of t-PA and fibrin.

Amides↗
PubMed1990

Biological properties of hybrid plasminogen activators.

A number of hybrid plasminogen activator genes were constructed from the t-PA and u-PA cDNAs and expressed using a bovine papilloma virus vector and mouse C-127 cells. Hybrid A was constructed by replacing the finger (F) and EGF domains of t-PA with the EGF and Ku domains of u-PA, while hybrids B and C had an extra Ku inserted before or after the double kringle (K1-K2) region of t-PA respectively. While all the hybrids showed comparable enzymatic activities towards a small substrate (S-2288), they had different activities in binding to fibrin clots as well in the fibrin-dependent plasminogen activation, the order of activities being: t-PA greater than or equal to hybrid B greater than hybrid C greater than hybrid A. Carbohydrate analysis showed that while hybrid C, like rt-PA, had at least one high-mannose type sugar chain (probably at residue 117 in K1), the other hybrids had only complex-type carbohydrates suggesting that domain interaction in t-PA might influence glycan processing. Pharmacokinetic studies in dog showed that hybrid B had a significantly longer plasma half-life than rt-PA. Thrombolytic efficacies of hybrid B and rt-PA were compared in dog model using an artificially induced coronary thrombus. Complete thrombolysis was achieved with 18 mg and 50 mg dosages for hybrid B and rt-PA respectively. These data show the superior pharmacokinetic and thrombolytic properties of hybrid B compared to rt-PA.

Animals↗
PubMed1990

Alterations in the domain structure of tissue-type plasminogen activator change the nature of asparagine glycosylation.

The formation of N-linked oligosaccharides of eukaryotic glycoproteins starts with the attachment of a common precursor at the recognition site Asn-X-Ser/Thr. Subsequent processing, by yet unknown controlling factors, leads to the formation of three different glycans: the high mannose type, the complex type and the hybrid type. In order to gain insight into the processing mechanisms, we studied the glycan pattern of a panel of related molecules constructed by insertion, duplication or deletion of the domains encoded by the cDNA of a fibrinolytic glycoprotein, tissue-type plasminogen activator (t-PA). These variant molecules are identical in regard to the glycosylation sites originally situated in particular domains, but differ with respect to the sequential alignment of the domains. The variant and native t-PA genes were transfected into mouse C127 cells and their carbohydrate structures analyzed by the susceptibility to specific endoglycosidases and by reaction with sugar-specific lectins. We found that with one exception, all mutant activators lack the high mannose glycan found at asn 117 of native t-PA. The exception was a molecule that retains the original domain arrangement up to and through the glycosylation site at asn 117. These results demonstrate for the first time that structural alterations in the primary sequence distal to the actual glycosylation site can result in altered processing of N-linked oligosacharides.

Amino Acid Sequence↗
PubMed1990

Epilepsy and pregnancy--a review of 98 pregnancies.

This study presents the clinical and management problems in 81 epileptic patients attending the Queen Victoria Hospital for 98 pregnancies between 1985 and 1988. Epileptic women were found to have a 26.1% risk of seizure deterioration during pregnancy and the single most important factor for predicting seizure prognosis was the severity of epilepsy prepregnancy. There was a significant increase in the complications of vaginal bleeding, preeclampsia, and preterm delivery, compared with a control group of women. Infants of epileptic mothers had an increased incidence of low birth-weight and lower Apgar scores at 5 minutes. The congenital abnormality rate was 14.3% (9 major and 5 minor abnormalities). These results confirm that pregnant epileptic women are a high risk group.

Adult↗