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Biomedical subjects

S L Vonderfecht

Publications and source records attributed to S L Vonderfecht.

At least 19 recordsLinked to original sources
PubMed1996

Release of soluble intercellular adhesion molecule 1 into bile and serum in murine endotoxin shock.

Neutrophil-induced liver injury during endotoxemia is dependent on the adhesion molecules Mac-1 (CD11b/CD18) on neutrophils and its counterreceptor on endothelial cells and hepatocytes, intercellular adhesion molecule 1 (ICAM-1). To investigate a potential release of a soluble form of ICAM-1 (sICAM-1), animals received 100 micrograms/kg Salmonella abortus equi endotoxin alone or in combination with 700 mg/kg galactosamine. In endotoxin-sensitive mice (C3Heb/FeJ), injection of endotoxin did not cause liver injury but induced a time-dependent increase of sICAM-1 in serum (300%) and in bile (615%) without affecting bile flow. In galactosamine/endotoxin-treated animals, which developed liver injury, the increase in both compartments was only 97% and 104%, respectively. In either case, the increase in sICAM-1 concentrations paralleled the enhanced ICAM-1 expression in the liver. The endotoxin-resistant strain (C3H/HeJ) did not show elevated sICAM-1 levels in serum or bile after endotoxin administration. In contrast, the intravenous injection of murine tumor necrosis factor alpha (TNF-alpha), interleukin-1 alpha (IL-1 alpha) or IL-1 beta (13-23 micrograms/kg) into endotoxin-resistant mice induced a 225% to 364% increase in serum sICAM-1 and a 370% elevation of the biliary efflux of sICAM-1, again independent of changes in bile flow. These data indicate that cytokines are major inducers of sICAM-1 formation during endotoxemia in vivo. The described experimental model can be used to investigate the role of sICAM-1 in the pathophysiology of inflammatory liver disease.

Analysis of Variance↗
PubMed1996

Phenotypic analysis of airway eosinophils and lymphocytes in a Th-2-driven murine model of pulmonary inflammation.

In order to investigate whether the pulmonary response to helminth antigens mimics that seen in allergic inflammation of the airways, we have examined the phenotypic characteristics of lymphocytes and eosinophils recruited to the airways following Nippostrongylus brasiliensis (N.b.) infection. Specifically, the cellular response was divided into an early and a late phase. During the early response there was a small but significant increase in neutrophil numbers recovered by bronchoalveolar lavage (BAL). Phenotypic analysis of BAL leukocytes revealed an early rise in the percentage of BAL lymphocytes expressing the naive T cell markers CD45RB and L-selectin, and the activation marker IL-2R. In addition, during the early response, there was an increased percentage of lymphocytes expressing the gamma delta TCR, but not the alpha beta TCR. In contrast, the late response was marked by a much larger accumulation, in the lungs and BAL, of memory CD4+ T lymphocytes and an influx of small, hypodense eosinophils which produced LTB4 and LTC4 on stimulation with calcium ionophore. At this time there was a substantial increase in the number of T lymphocytes and eosinophils expressing ICAM-1 and the integrins VLA-4 and LFA-1, implicating these adhesion molecules in inflammatory cell recruitment to the airways. We conclude that the pattern and phenotypic characteristics of the cellular recruitment seen following N.b. infection resemble those seen in early- and late-phase allergic inflammation of the airways in asthma, and therefore N.b. may be used to model these aspects of the disease.

Animals↗
PubMed1996

Role of very late activation antigen-4 in the antigen-induced accumulation of eosinophils and lymphocytes in the lungs and airway lumen of sensitized brown Norway rats.

We used flow cytometry and treatment in vivo with a monoclonal antibody (mAb), TA-2, to the alpha 4 integrin to investigate the role of alpha 4 beta 1, CD49d/CD29 (VLA-4) in antigen-induced lung inflammation in Brown Norway (BN) rats. Ovalbumin (OVA) inhalation induced an accumulation of eosinophils and lymphocytes in the lungs and bronchoalveolar lavage (BAL) fluid of sensitized BN rats at 24 h after challenge. Phenotypic analyses demonstrated that the percentages of T cells expressing detectable alpha 4 and CD25 in the bronchial lumen after antigen challenge were dramatically increased compared with blood and lymph node T cells. The mean channel fluorescence values of alpha 4 expression were also increased on BAL T cells compared with blood or lymph node T cells. Treatment of OVA-sensitized rats in vivo with total cumulative doses of 0.75 to 6 mg/kg TA-2 mAb intraperitoneally produced dose-related increases in circulating TA-2 and a peripheral blood lymphocytosis, basophilia, and eosinophilia. Flow cytometric analysis of the peripheral blood T cells after in vivo TA-2 mAb administration showed decreases in detectable alpha 4 when these cells were examined ex vivo. Treatment with TA-2, but not an isotype-matched control mouse immunoglobulin G1 mAb, markedly inhibited the OVA-induced recruitment of lymphocytes and eosinophils into the airway lumen. Very few CD3+CD49d+ cells migrated into BAL fluid following anti-alpha 4 mAb treatment in vivo. Treatment with TA-2 also significantly attenuated OVA-induced inflammatory histopathology. We conclude that VLA-4 is a critically important adhesion molecule involved in antigen-specific lung inflammation in sensitized BN rats.

Animals↗
PubMed1994

Borna disease virus p24 and p38/40 synthesized in a baculovirus expression system: virus protein interactions in insect and mammalian cells.

To facilitate studies of the individual viral proteins, two Borna disease virus proteins, p24 and p38/40, were synthesized in vitro by means of a baculovirus expression system and examined for antigenic identity to viral proteins from BDV-infected cells. Recombinant proteins p24 and p38/40 were nearly identical in size to the viral proteins from BDV-infected cells. Immunoblot and immunocytochemistry analysis of BDV proteins from infected tissue culture cells and rat brain showed binding of antisera directed against the recombinant proteins. Specific recognition of the recombinant proteins by Borna disease virus-specific convalescent antisera and monoclonal antibodies further demonstrated that the antigenic characters of the p24 and p38/40 had been conserved. Polyclonal antibody directed against either of the recombinant proteins recognized only the protein used as immunogen, without cross reactivity with the other recombinant protein, indicating no common epitopes. Moreover, these data confirmed the proposed gene coding assignments of ORF I and II of BDV p38/40 and p24, respectively. Both of the recombinant proteins were secreted into the media of insect cells in tissue culture, but secretion of recombinant p24 was evident only as a dimeric form and not with the monomeric form. Immunoprecipitation studies performed with monoclonal antibodies and BDV proteins from infected rat brain suggested that a heterodimer forms via binding of p40 to the p24.

Animals↗
PubMed1994

Group B rotavirus VP2: sequence analysis, expression, and gene coding assignment.

The second largest genomic segment of the IDIR strain (infectious diarrhea of infant rats) of group B rotavirus (GBR) was completely sequenced, cloned, and expressed in insect cells, and its gene coding assignment was determined. The sequence of IDIR virus gene 2 (IDIRg2) contained 2847 bp with a single, long open reading frame that encoded a deduced polypeptide of 934 amino acids (M(r) 106 kDa, pI 5.325). BestFit homology indicated that the predicted amino acid sequence of IDIRg2 shared 46.5% similar and 19.8% identical sequences with VP2 of the SA11 strain of group A rotavirus. The polypeptide product encoded by this gene was synthesized in insect cells by means of a baculovirus expression vector and employed to elucidate the corresponding gene to protein coding assignment. Recombinant IDIRg2 product maintained virion antigenic epitopes as evidenced by reactivity with convalescent antisera from infant rat pups infected with the IDIR agent. Reactivity with antisera from heterologous human and porcine GBR strains was also observed. Antibody directed against recombinant IDIRg2 product specifically reacted with VP2 of IDIR virus and a human strain of GBR (ADRV) obtained from fecal specimens. These experiments identified the IDIRg2 product as the VP2 core protein of the IDIR virus strain of GBR.

Amino Acid Sequence↗
PubMed1994

The 21-aminosteroid tirilazad mesylate protects against endotoxin shock and acute liver failure in rats.

The protective effect of the 21-aminosteroid tirilazad mesylate (U-74006F) was investigated in an experimental model of endotoxin shock and acute liver failure. In male Fischer rats subjected to 20 min of hepatic no-flow ischemia followed by reperfusion and injection of 0.5 mg/kg of Salmonella enteritidis endotoxin, severe hepatic injury developed, as indicated by a histological evaluation and liver enzyme release. Treatment with U-74006F (two bolus doses of 3 mg/kg each; the first dose was injected i.v. 30 min before ischemia and the second dose, at the time of reflow) reduced the hepatic injury by 60% at 4 hr of reperfusion, improved the survival rate from 18% to 55% and decreased the degree of hepatic injury at 48 hr of reperfusion. U-74006F treatment did not affect the extent of complement activation during reperfusion, the Kupffer cell-induced oxidant stress, or tumor necrosis factor-alpha formation in this model. U-74006F did not significantly reduce superoxide formation of Kupffer cells and neutrophils in vitro or in vivo. The substantial neutrophil infiltration in the liver during the pathogenesis was not affected at 4 hr of reperfusion but was attenuated by 70% at 48 hr. It was therefore concluded that, in the sequence of pathophysiological events, U74006F acted at a site distal to inflammatory cell activation and the generation of cytotoxic mediators. The protection against the initial endotoxin-enhanced reperfusion injury in the liver strongly inhibited the progression of the inflammatory response and subsequent liver failure.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Priming of phagocytes for reactive oxygen production during hepatic ischemia-reperfusion potentiates the susceptibility for endotoxin-induced liver injury.

Plasma levels of glutathione disulfide (GSSG) as an indicator of a vascular oxidant stress, tumor necrosis factor-alpha (TNF-alpha) formation, and liver injury (alanine aminotransferase activity, histology) were monitored in male Fischer rats after 30 min of hepatic ischemia followed by up to 4 hr of reperfusion. The injection of 1 mg/kg Salmonella enteritidis endotoxin at 30 min of reflow potentiated the postischemic oxidant stress and liver injury. TNF-alpha levels increased from 10 +/- 7 pg/ml (baseline) to 3,553 +/- 738 pg/ml after ischemia-reperfusion followed by endotoxin, or to 3,670 +/- 508 pg/ml after endotoxin alone. Depletion of serum complement before ischemia attenuated the endotoxin-mediated increase of reactive oxygen formation by 70% but did not affect TNF-alpha levels. Complement activation with cobra venom factor (CVF) during reperfusion had an effect similar to that of endotoxin on the oxidant stress and liver injury. CVF did not increase TNF-alpha formation during reperfusion. Kupffer cells and neutrophils isolated from the postischemic liver 2.5 hr after endotoxin injection generated 600% and 400% more superoxide, respectively, than cells isolated from control livers. The results demonstrate a substantial priming of hepatic phagocytes for reactive oxygen production but not TNF-alpha formation, even after short periods of hepatic ischemia, and the vulnerability of the postischemic liver to severe endotoxin-induced injury. Activated complement seems to be mainly responsible for the effects. These results may explain the high risk for hepatic failure after extensive liver resection and hypovolemic shock.

Adenosine Triphosphate↗
PubMed1993

Baculovirus expression of gene 6 of the IDIR strain of group B rotavirus (GBR): coding assignment of the major inner capsid protein.

The polypeptide product of gene 6 of the IDIR strain of group B rotavirus was synthesized by means of a baculovirus expression system in order to confirm the coding assignment of the gene and to develop reagents broadly reactive with heterologous strains of Group B rotaviruses (GBR). Earlier experiments indicated that IDIR virus gene 6 encoded the group-specific, major inner capsid protein, but direct confirmation of this coding assignment was not previously reported. The expression of IDIR virus gene 6 from baculovirus recombinants resulted in production of a protein with an apparent molecular weight equivalent to that deduced from the gene sequence (44 kDa). In addition, larger recombinant proteins were also observed, and these appeared to be consistent in size with oligomers of the primary VP6 product. The expressed protein reacted with antibody directed against the IDIR agent and other strains of GBR, but no reaction was observed with antibody directed against group A rotavirus. Serologic reactivity was also observed between the gene 6 product and a monoclonal antibody directed against the GBR group-specific antigen. Antibody directed against the recombinant gene 6 product specifically reacted with the IDIR virus major inner capsid protein in an immunoblot format. These experiments conclusively demonstrated that IDIR virus gene 6 encoded the major inner capsid protein and confirmed the presence of group B-specific antigenic epitopes on the protein.

Animals↗
PubMed1993

Purification of the IDIR strain of group B rotavirus and identification of viral structural proteins.

A purification scheme was developed that allowed for the partial purification of complete, double-shelled particles of the infectious diarrhea of infant rats (IDIR) virus, a group B rotavirus (GBR). Structural proteins from the isolated, complete viral particles were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and evaluated by silver staining and by immunoblotting using antisera obtained from rats that had recovered from IDIR virus infection. Analysis of the stained gels and immunoblots demonstrated the presence of IDIR virus structural proteins having estimated molecular weights of 130, 100, 88, 80, 61, 44, 32, and 25 kDa. Treatment of complete viral particles with EDTA removed the outer capsid layer producing single-shelled particles that lacked the 80, 61, 32, and 25 kDa proteins seen in the double-shelled particles. The 44-kDa protein was most abundant and was recognized by a mouse monoclonal antibody and hyperimmune guinea pig serum prepared against IDIR virus and by hyperimmune guinea pig serum prepared against adult diarrhea rotavirus (ADRV), a human strain of GBR. Convalescent serum obtained from a piglet inoculated with a porcine GBR reacted with the 61 kDa, outer capsid protein of IDIR virus. This information on the structural nature of IDIR virus should be helpful in establishing the genetic and structural relationships among the GBR and to further exploit the potential of IDIR virus as a tool for understanding GBR infections in human and animal populations.

Animals↗
PubMed1992

Immunocytochemical localization of platelets in baboon hepatic sinusoids using monoclonal mouse anti-human platelet glycoprotein IIIa following induction of thrombocytopenia.

A commercially available mouse monoclonal antibody to human platelet glycoprotein IIIa was used to demonstrate sequestration of platelets in hepatic biopsies obtained from baboons following intravenous infusion of echistatin, a novel fibrinogen receptor antagonist derived from the venom of the snake Echis carinatus. Biopsies of liver and spleen were taken prior to administration of echistatin. The hepatic biopsies were either snap-frozen in Freon-22/liquid nitrogen or fixed in 10% neutral buffered formalin. Biopsies of spleen were snap-frozen. During infusion of echistatin (2.3 micrograms/kg/min), circulating platelet counts decreased from 331,000/mm3 to 167,000/mm3. Selective sequestration within the liver was confirmed using whole body gamma camera imaging to demonstrate 111Indium-oxine labeled platelet accumulation within the liver during the thrombocytopenic episode. Hepatic biopsies were again taken and either snap-frozen in Freon-22/liquid nitrogen or fixed in 10% neutral buffered formalin. Biopsies of spleen and inguinal lymph node were also snap-frozen. Platelet rich plasma smears, included as positive controls, dewaxed paraffin sections, and cryosections of liver, spleen, and lymph node were stained with monoclonal mouse anti-human platelet glycoprotein IIIa using an avidin biotinylated peroxidase complex (ABC) technique. Prior to infusion of echistatin, platelet staining within the liver was minimal. After echistatin infusion, hepatic cryosections showed prominent platelet staining within hepatic sinusoids. No localization was shown in lymph node, however, the spleen showed prominent platelet staining both before and after echistatin infusion. Platelet rich plasma smears were intensely positive. No prominent platelet staining was observed in formalin-fixed, paraffin-embedded material. Thus, this immunocytochemical technique may help localize platelets in cryosections of tissues from baboons and other primate species.

Animals↗
PubMed1992

In vitro transcription and translation of group B rotavirus strain IDIR gene 8 and immunoprecipitation by human sera.

Group B rotaviruses (GBRs) are associated with episodes of acute diarrhea in humans and a variety of animal species. To date, these agents have not been well adapted to growth in tissue culture, and evaluation of human sera for antibodies directed against GBRs has been hindered by the inability to obtain standardized and highly purified preparations of GBR antigens. In order to evaluate the reactivities of antisera with a highly specific antigen, we prepared a full-length cDNA clone of gene 8 of the IDIR strain of GBR. This clone was transcribed with T7 RNA polymerase, and the resulting RNA was translated in vitro with rabbit erythrocyte lysates. The polypeptide expressed from IDIR gene 8 was specifically precipitated by antibody directed against IDIR but not by antibody directed against ADRV (adult diarrhea rotavirus) or bovine strains of GBR. Subsequent immunoprecipitation reactions confirmed the presence of anti-IDIR antibodies among the U.S. population. Of 129 human serum specimens, 3 specifically immunoprecipitated the IDIR gene 8 polypeptide.

Animals↗
PubMed1992

Human milk mucin inhibits rotavirus replication and prevents experimental gastroenteritis.

Acute gastrointestinal infections due to rotaviruses and other enteric pathogens are major causes of morbidity and mortality in infants and young children throughout the world. Breast-feeding can reduce the rate of serious gastroenteritis in infants; however, the degrees of protection offered against rotavirus infection vary in different populations. The mechanisms associated with milk-mediated protection against viral gastroenteritis have not been fully elucidated. We have isolated a macromolecular component of human milk that inhibits the replication of rotaviruses in tissue culture and prevents the development of gastroenteritis in an animal model system. Purification of the component indicates that the antiviral activity is associated with an acidic fraction (pI = 4.0-4.6), which is free of detectable immunoglobulins. Furthermore, high levels of antiviral activity are associated with an affinity-purified complex of human milk mucin. Deglycosylation of the mucin complex results in the loss of antiviral activity. Further purification indicated that rotavirus specifically binds to the milk mucin complex as well as to the 46-kD glycoprotein component of the complex. Binding to the 46-kD component was substantially reduced after chemical hydrolysis of sialic acid. We have documented that human milk mucin can bind to rotavirus and inhibit viral replication in vitro and in vivo. Variations in milk mucin glycoproteins may be associated with different levels of protection against infection with gastrointestinal pathogens.

Adult↗
PubMed1992

Diarrhea caused by a slow-growing Enterococcus-like agent in neonatal rats.

An enteropathogenic Enterococcus-like agent was isolated from a spontaneous outbreak of diarrhea that occurred in a colony containing neonatal rats. Diarrhea was experimentally reproduced in virus-antibody-free neonatal rats inoculated with this purified "enterococcus." Gram-positive cocci were adhered to the small intestinal villi of affected animals from which the organism was reisolated. The isolate's classification in the genus Enterococcus was confirmed by genetic probe; however, because of its unique fermentation pattern, it could not be definitively speciated. Indirect immunofluorescence assays indicate that this strain of enterococcus and Enterococcus hirae, another strain pathogenic for neonatal rats, differ antigenically. Enterococci should be considered as potential etiologic agents in outbreaks of diarrhea involving neonatal rats and future efforts directed to increasing our understanding of the pathophysiology of this disease.

Animals↗
PubMed1990

Growth of group A rotaviruses in a human liver cell line.

Recent observations in children with rotavirus gastroenteritis and in infant mice given rotavirus vaccine by oral administration suggest that this well-known gastrointestinal pathogen may infect the liver. To examine this possibility, the susceptibility of Hep G2 cells to infection with a variety of rotavirus strains was tested. These cells were used because they are considered to be well differentiated and exhibit many liver-specific functions. The Hep G2 cells supported the growth of the simian strain rhesus rotavirus (MMU 18006), a strain currently being used in vaccine trails, but did not support the growth of any human strain (D, DS1, Price or ST3). The rhesus rotavirus infection was cytopathic and resulted in release of lactate dehydrogenase. Rhesus rotavirus growth in Hep G2 cells displayed trypsin-enhanced infectivity and was inhibited by pretreatment of cells with Arthrobacter ureafaciens neuraminidase but not with neuraminidase from Clostridium perfringens. Hep G2 cells were also permissive for another simian strain (SA11), a bovine strain (UK) and single gene substitution reassortants containing VP7 (the major outer capsid neutralization protein) from a human rotavirus strain and the remaining 10 genes from either rhesus rotavirus or UK. In general, UK and its reassortants produced lower levels of antigen than did rhesus rotavirus and its reassortants. Hep G2 cells and other hepatic cell lines may prove to be useful tools to explore the hepatotropic potential of wild-type rotaviruses and candidate vaccine strains.

Antigens, Viral↗
PubMed1989

Monoclonal antibody assay for detection of double-stranded RNA and application for detection of group A and non-group A rotaviruses.

Fastidious viruses are generally detected in human body fluids by means of immunoassay or nucleic acid hybridization systems. These approaches can be difficult to apply to the detection of viruses which display variations in antigenic or genetic composition. Rotaviruses are examples of viruses which can display such variations. Recently identified antigenic variants, designated as non-group A rotaviruses, cannot be detected by immunoassays or nucleic acid hybridization assays which utilize reagents directed at group A rotavirus strains. The incomplete understanding of the extent of antigenic and genetic variation has inhibited the development of assay systems for all of the non-group A rotaviruses and has limited the study of their role in human disease. While rotaviruses display genetic variation, they all contain a genome which consists of double-stranded RNA. We utilized a monoclonal antibody to devise a sensitive assay for the measurement of double-stranded RNA and applied it to the detection of a wide range of rotaviruses. We found that the assay could detect double-stranded RNA from as few as 10 PFU of standard strains of group A rotaviruses. The assay system was also capable of detecting double-stranded RNA from several strains of group B rotaviruses isolated from calves, rats, and pigs at levels below those at which viral RNA could be visualized by means of polyacrylamide gel electrophoresis. When applied to the detection of double-stranded RNA in serial stools shed by rotavirus-infected children, the assay system was capable of detecting double-stranded RNA in samples in which antigen could not be detected by immunoassay. The specific nature of the double-stranded RNA detected by this assay system could be determined by the elution of the nucleic acids from the monoclonal antibody and the reaction of the RNA with specific nucleotide probes. The measurement of double-stranded RNA offers a potential method for the sensitive detection of a wide range of rotaviruses and other members of the family Reoviridae.

Animals↗
PubMed1989

Detection of group B rotavirus in fecal specimens by dot hybridization with a cloned cDNA probe.

Cloned cDNA copies were synthesized from the genomic RNA of the IDIR strain of group B rotavirus (GBR) isolated in Baltimore, Md. These clones were screened for hybridization with heterologous GBR to identify cDNA for use in dot hybridization experiments. In multiple screening experiments, cDNA clones derived from gene segment 3 provided the most intense hybridization signals. 32P-labeled probes were produced from one of the gene 3 clones, and these were employed in dot hybridization assays. Purified preparations of bovine GBR were detected in concentrations of greater than or equal to 0.5 ng, and GBR was detected in fecal specimens obtained from five of six infected calves. Four of six human fecal specimens containing the Baltimore strain of GBR were also positive in the hybridization assay, while GBR was identified in only one of the six specimens by means of immunoelectron microscopy. A fecal specimen obtained from a patient infected with the adult diarrhea rotavirus strain of GBR was also positive in the dot hybridization assay. Fecal specimens from uninfected humans, calves, and rats, as well as specimens containing group A rotaviruses, did not hybridize with the cloned cDNA probe.

Animals↗
PubMed1989

Pathology of infectious diarrhea of infant rats (IDIR) induced by an antigenically distinct rotavirus.

Suckling rats were inoculated with a group B rotavirus to determine the progression of the morphologic changes induced in the intestine by this virus. Several changes were observed by light microscopy 1 day after viral inoculation: shortening of small intestinal villi, villous epithelial necrosis, and villous epithelial syncytia. The lesions were most often present in the distal small intestine, although other small intestinal segments were affected to a lesser degree. By day 3 post-inoculation, epithelial necrosis, and syncytia were no longer present; however, the villous epithelium was disorganized and irregularly vacuolated, and intestinal crypt epithelium was hyperplastic. Alterations in villous height to crypt depth ratios were present in portions of the small intestine for the remainder of the 12-day study period. Epithelial syncytia appeared to form by the breakdown of the lateral interdigitating membranes of the absorptive villous epithelium. Viral particles, abundant in the syncytia, appeared to form from amorphous or reticular arrays of viral precursor material. Group B rotaviral antigens, as detected by indirect immunofluorescence, were present in large amounts in the small intestinal villous epithelium only on the first day after viral inoculation. These studies show that two important diagnostic features of group B rotaviral infections of rats, epithelial syncytia and viral antigen as determined by immunofluorescence, are present only on the first day of disease. These findings should be taken into consideration when attempting to diagnose disease induced by this agent.

Animals↗