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Biomedical subjects

H Schellekens

Publications and source records attributed to H Schellekens.

At least 91 records · Page 5Linked to original sources
PubMed1985

Monoclonal antibodies to human immune interferon and their use in a sensitive solid-phase ELISA.

Two stable hybridoma cell lines secreting specific antibodies against human gamma interferon (HuIFN-gamma) were established. Both monoclonal antibodies (designated as MD-1 and MD-2) belong to the IgG1/kappa subclass and neutralize the antiviral activity of natural and recombinant DNA derived HuIFN-gamma (nHuIFN-gamma and rHuIFN-gamma respectively), although MD-1 is far more effective than MD-2. MD-1 and MD-2 recognize different epitopes and do not compete with each other in binding to HuIFN-gamma as concluded from competition assays. In a 'Western' blot, both antibodies reacted with the 20 kDa and 25 kDa polypeptides present in nHuIFN-gamma preparations. A sandwich enzyme immunoassay using microtiter plates coated with unlabeled MD-2 was developed. Biotinylated MD-1 was used as the second antibody. Bound MD-1 was detected by an avidin/alkaline phosphatase enzyme reaction. This immunoassay is highly specific and as sensitive as a bioassay. A radioimmunoassay using MD-2 coated on polystyrene balls and 125I-labeled MD-1 as the second antibody showed a sensitivity comparable to that of the enzyme immunoassay.

Antibodies, Monoclonal↗
PubMed1985

Cloning and expression of the chromosomal immune interferon gene of the rat.

The chromosomal immune interferon gene of the rat (IFN-gamma) was identified by screening a recombinant rat lambda phage library with a human IFN-gamma cDNA probe. In contrast to the genes of other rat IFNs, this rat IFN-gamma chromosomal gene contains introns and its structural organization closely resembles that of the human and murine IFN-gamma genes. The rat IFN-gamma gene encodes a signal sequence of 19 amino acids followed by the mature IFN-gamma protein of 137 amino acids. The gene was expressed under control of the simian virus 40 (SV40) early promoter in Chinese hamster ovary (CHO) cells deficient in dihydrofolate reductase (DHFR) after co-transformation with a plasmid containing the mouse DHFR gene. Initial transformants with a DHFR+ phenotype produced IFN-gamma titres ranging from 20 to 1600 units/ml. After stepwise increases in the concentration of methotrexate (MTX) in the growth medium of transformed CHO cells, MTX-resistant clones producing 80 000-100 000 units per ml were isolated. Protein analysis of supernatants of these MTX-resistant cells by polyacrylamide gel electrophoresis revealed a product with an apparent mol. wt. of 18 000 daltons which was not detectable in the growth medium of DHFR+ transformants that did not produce IFN. The product was identified as rat IFN-gamma and constituted approximately 5% of the proteins excreted from these cells.

Amino Acid Sequence↗
PubMed1985

Infectious hepatitis B virus from cloned DNA of known nucleotide sequence.

The infectivity of cloned hepatitis B viral DNA (HBV) has been tested in chimpanzees to identify a fully functional HBV genome and to assess the risk associated with its handling. Only one of two HBV DNA sequence variants tested was shown to be infectious. "Clone purified" virus of predicted nucleotide sequence was produced from the infectious HBV DNA, and the cloned viral genome was identical in structure with naturally occurring HBV. Infection could be initiated independent of whether circular monomeric or plasmid integrated dimeric forms of the viral genome were inoculated, but the infectivity of the DNA depended on liver cell transfection or intrahepatic injection. Intravenous injection of high doses of infectious HBV DNA did not induce hepatitis, suggesting that there is virtually no risk associated with routine laboratory handling of cloned HBV DNA.

Animals↗
PubMed1984

Structure and expression in Escherichia coli of a cloned rat interferon-alpha gene.

DNA synthesized by in vitro transcription on rat interferon (IFN) mRNA has been cloned and amplified as recombinant DNA. The nucleotide sequence of these rat IFN cDNA clones revealed i. the partial presence of the coding region of the gene and ii all cDNA clones were derived from the same subtype of rat IFN-alpha mRNA. Purified inserted fragments were used as a hybridisation probe against chromosomal "Southern blots" to show that at least twelve rat IFN-alpha-related sequences are present in the genome. A lambda-linked rat gene library was screened with the cDNA probes, resulting in an equivalent number of rat IFN-alpha-related hybrid phages. By use of a 3'-noncoding region as a probe, the chromosomal counterpart of the cDNA clones could be detected and the nucleotide sequence of its coding region has been determined. Expression of the coding region in E. coli yielded biologically active IFN, when tested for in vitro or in vivo antiviral activity.

Animals↗
PubMed1984

Antiviral activity of interferon in rats and the effect of immune suppression.

The antiviral effect of interferon was studied in a number of experimental virus infections in the rat. Interferon was shown to protect rats infected with pseudorabies virus, herpes simplex virus or vesicular stomatitis virus. The antiviral activity was not inhibited by immune suppression induced by azothioprine, prednisolone or cyclosporin A treatment. Cyclophosphamide completely blocked the in vivo activity of interferon in rats.

Animals↗
PubMed1984

The chimpanzee as a model to test the side effects of human interferons.

Interferon are naturally occurring proteins that are currently under evaluation as potential antiviral and antitumor agents. Currently all human interferons can in principle be produced in adequate amounts by recombinant DNA technology. Human interferons produce side effects, but because they are species-specific the toxicity cannot be tested in lower mammals. The chimpanzee is the only species in which the side effects of human interferon can be reproduced, and only in this species the toxicity of human interferons can be screened.

Animals↗
PubMed1983

Effect of treatment with interferon and cyclophosphamide on the growth of a spontaneous liposarcoma in rats.

A spontaneous non-immunogenic transplantable liposarcoma in BN rats was found to be sensitive to the antitumor effects of rat fibroblast interferon (RIF) when it was administered from the day of tumor implantation onwards. Treatment with RIF starting at 7 days after implantation was not effective. Tumor growth was markedly inhibited by cyclophosphamide (Cyclo). At the time and dose schedules used, Cyclo was more effective than RIF. When the growth of the tumor was inhibited by Cyclo, subsequent treatment with RIF did not lead to an additional retardation of tumor growth. Administration of RIF interfered with the beneficial effect of Cyclo when both agents were given concomitantly. RIF and Cyclo gave similar results to those obtained with RIF alone and these were inferior to those obtained with Cyclo alone.

Animals↗
PubMed1983

Chemical inactivation of hepatitis B virus: the effect of disinfectants on virus-associated DNA polymerase activity, morphology and infectivity.

The inactivation of hepatitis B virus (HBV) using two commercially available disinfectants was analysed. Indirect evidence of virus inactivation was obtained by examining the decrease in HBV-associated DNA polymerase and HBcAg activities after treatment with increasing concentrations of disinfectant. Inactivation was accompanied by the irreversible loss of all morphological forms typically found in hepatitis B-positive sera and in particular 42-nm HBV particles were absent. Physiochemical analysis confirmed that the exposure of HBV to either product resulted in the degradation of virus particle structure. Direct evidence of HBV inactivation was obtained by treatment of virus pellets prior to inoculation into susceptible chimpanzees. No evidence of hepatitis B was found in animals receiving treated HBV thereby confirming the suitability of certain disinfectants for the inactivation of potentially infectious material.

1-Propanol↗
PubMed1983

Rat cytomegalovirus: induction of and sensitivity to interferon.

Rat interferon (IFN) in a dose-dependent fashion inhibited the cytopathic effect of rat cytomegalovirus (RCMV) in rat cell cultures. Treatment of Lewis and Brown Norway (BN) rats with intraperitoneal injections of IFN reduced the amount of virus recovered from the spleens at 3 days post infection (p.i.) and from the salivary glands at 10 and 21 days p.i. In cell cultures, RCMV failed to induce detectable amounts of IFN. Small amounts of IFN were detectable in the serum of BN rats at 3 days p.i. In Lewis rats no IFN was found in serum at any time during the first month p.i.

Animals↗
PubMed1983

A phase I clinical tolerance study of rDNA alpha 2 human interferon in patients with non-reticuloendothelial system malignancies.

Twenty-seven patients with non-reticuloendothelial malignancies were treated with a single intramuscular injection of recombinant leukocyte alpha 2 interferon (rIFN) to assess clinical tolerance and define a maximum tolerated dose. A single patient in each of six increasing dosage groups (0.3 X 10(6) IU, 1 X 10(6) IU, 3 X 10(6) IU, 10 X 10(6) IU, 30 X 10(6) IU, 100 X 10(6) IU) received a low dose (0.01 X 10(6) IU) and served as a control for subjective and objective toxicity measurements. Severe fatigue proved dose-limiting at 100 X 10(6) IU, and all dosages above 3.0 X 10(6) IU produced one or more signs or symptoms, which typically resembled a 'flu-like' syndrome. Objective toxicity was mild to moderate (leukopenia, thrombopenia) and no toxicities were found not already known from work with interferon obtained directly from leukocytes. Evidence of an antitumor effect was apparent in 3/19 evaluable patients.

Adult↗
PubMed1983

Circulating interferon in rabbits and monkeys after administration of human gamma interferon by different routes.

Rabbits and rhesus monkeys were injected with 3 X 10(5) units of human gamma interferon (IFN) prepared in human leukocyte suspensions. Circulating IFN was detected up to 4 h after intravenous administration. Intramuscular injection maintained a relatively stable serum IFN level of about 50 units/ml for 7 to 9 h. The results in both species were similar. Little or no circulating IFN was detected after subcutaneous injection of 3 X 10(5) units, but 1.5 X 10(6) units maintained about 50 units per ml of serum for 30 h. Pharmacokinetically, human gamma IFN resembled human alpha interferons rather than human beta IFN.

Animals↗
PubMed1983

Reduced synthesis of pp60src and expression of the transformation-related phenotype in interferon-treated Rous sarcoma virus-transformed rat cells.

Treatment of Rous sarcoma virus-transformed rat cells with rat interferon-alpha (specific activity, 10(6) U/mg of protein) for 24 h caused a 50% reduction in intracellular pp60src-associated protein kinase activity. Staphylococcus aureus V8 protease digestion of pp60src, derived from 32P-labeled monolayer cultures incubated with or without interferon, revealed no differences either in the phosphopeptide pattern or in the phosphoserine-phosphotyrosine ratio. However, [3H]leucine pulse-labeling experiments showed that the synthesis of pp60src was reduced by 42 to 48%, relative to the level of bulk protein synthesis, in the interferon-treated cultures. Rat interferon-alpha also reduced the growth rate of Rous sarcoma virus-transformed rat cells in a dose-dependent manner over a 72-h period. The decrease in growth rate was accompanied by increases in the thickness and number of actin fibers per cell and by a decline in intracellular tyrosine phosphorylation by pp60src. The results suggest that interferon can inhibit the expression of the transformation-related phenotype by selectively reducing the synthesis of the Rous sarcoma virus transforming gene product. However, the interferon effects on the cytoskeletal organization and proliferation of Rous sarcoma virus-transformed cells may be due at least in part to the predominance of interferon-induced phenotypic changes over those caused by pp60src.

Actins↗
PubMed1983

Hepatitis B after infection of a chimpanzee with cloned HBV DNA.

Transfection of chimpanzee liver cells in vivo or in vitro with cloned HBV DNA induced typical hepatitis B in the inoculated animal. Intravenous inoculation of a 1000-fold greater dose of the same cloned HBV DNA did not induce infection or disease. The experiment proved that cloned HBV DNA is fully functional and that the nick gap structure, proteins covalently linked to the HBV genome, or any other HBV particle associated protein are not needed for initiation of virus replication.

Animals↗