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Biomedical subjects

C S Crumpacker

Publications and source records attributed to C S Crumpacker.

At least 55 records · Page 3Linked to original sources
PubMed1991

Detection of human immunodeficiency virus type 1 clinical isolates with reduced sensitivity to zidovudine and dideoxyinosine by RNA.RNA hybridization.

A quantitative rapid assay to detect resistant clinical human immunodeficiency virus type 1 (HIV-1) strains remains an important medical goal. A system incorporating a quantitative RNA.RNA hybridization assay that measures the amount of intracellular HIV-1-specific RNA has been employed to detect the level of inhibition by nucleoside analogues in sensitive and resistant HIV-1 strains. The RNA.RNA hybridization assay readily distinguished previously published zidovudine (ZDV; 3'-azido-3'-deoxythymidine)-resistant isolates from ZDV-sensitive isolates of HIV-1. The 50% inhibitory concentration (IC50) of ZDV for HTLV-IIIB and sensitive clinical HIV-1 isolates is between 0.01 and 0.04 microM. HIV-1 strains from three patients on long-term ZDV therapy displayed a greater than 20-fold increase in the ZDV IC50 compared to sensitive strains. The drug sensitivity system was confirmed by showing that mutations in the HIV reverse transcriptase gene from a ZDV-resistant isolate resulted in four amino acid changes (Leu-125----Trp, Ile-142----Val, Thr-215----Tyr, and Pro-294----Thr) including one change (Thr-215----Tyr) that has been previously reported to be associated with resistance. One clinical HIV strain with high-level ZDV resistance displayed a 5-fold increase in 2',3'-dideoxyinosine IC50 compared to that of HTLV-IIIB. A drug sensitivity assay employing RNA.RNA hybridization may be useful for extensive screening of HIV isolates from patients enrolled in clinical trials and permit the correlation of in vitro resistance with clinical outcome.

Cloning, Molecular↗
PubMed1991

Analysis of the thymidine kinase gene from clinically isolated acyclovir-resistant herpes simplex viruses.

The isolation and description of acyclovir-resistant (ACVR) herpes simplex-2 viruses from patients with AIDS has recently been reported. These ACVR viruses were all markedly decreased in their thymidine kinase (TK) activity, and 6 of 10 of these TK viruses were able to establish latency. In addition, one of these isolates, ACVR-86012 was neuropathogenic in a murine encephalitis model. In this paper, the characteristics of these isolates with respect to TK polypeptide synthesis are examined. All but one isolate synthesized a detectable TK protein by immunoprecipitation, and 9/10 of the TK proteins had an altered electrophoretic mobility as compared to wild-type. The TK polypeptide from the neuropathogenic isolate ACVR-86012 was full-length and the gene was sequenced. An amino acid change from a glutamine to a proline at amino acid residue 105 was detected compared to the wild-type HSV-333 strain. These results indicate that an amino acid change in the NH2 portion of the TK protein is associated with a full-length peptide with decreased enzyme activity but the virus retains neuropathic virulence.

Acquired Immunodeficiency Syndrome↗
PubMed1991

In vitro suppression of normal human bone marrow progenitor cells by human immunodeficiency virus.

Incubation of normal human nonadherent and T-cell-depleted bone marrow cells with HIVIIIB at multiplicities of infection (MOI) ranging from 0.0001:1 to 1:1 reverse transcriptase (RT) units resulted in the dose-dependent suppression of the in vitro growth of erythroid burst-forming unit (BFU-E), granulocyte-macrophage (CFU-GM), and T-lymphocyte (CFU-TL) colonies of progenitor cells. Maximum inhibition of colony formation was observed at a 1:1 ratio of virus to bone marrow cells. At this MOI, BFU-E and CFU-GM colonies were inhibited by 60 to 80%, while CFU-TL colonies were totally suppressed. Inhibition of colony formation was also observed at an MOI of 0.1:1 but not with further log dilutions of the virus. Incubation of the virus with antibody to gp160 resulted in the complete reversal of stem cell suppression and the normalization of colony growth in vitro. For BFU-E and CFU-GM colonies, this reversal was observed with dilutions of antibody up to 1:100 and was no longer observed at titers greater than 1:500. The CFU-TL colony number normalized at titers between 1:10 and 1:50. Human immunodeficiency virus (HIV) also suppressed by 50% the growth of colonies derived from CD34+ stem cell fractions. Infection of CD34+ cells and T-cell-depleted, nonadherent cell fractions was demonstrated by detection with HIV-specific DNA probe following amplification by polymerase chain reaction. The results suggest that HIV can directly infect human bone marrow progenitor cells and affect their ability to proliferate and give rise to colonies in vitro. The results indicate a direct role for the virus in bone marrow suppression and a possible mechanism for the cytopenias observed in patients with AIDS.

Bone Marrow↗
PubMed1990

Localization of the herpes simplex virus type 1 65-kilodalton DNA-binding protein and DNA polymerase in the presence and absence of viral DNA synthesis.

Using indirect immunofluorescence, well-characterized monoclonal and polyclonal antibodies, and temperature-sensitive (ts) mutants of herpes simplex virus type 1, we demonstrated that the 65-kilodalton DNA-binding protein (65KDBP), the major DNA-binding protein (infected cell polypeptide 8 [ICP8]), and the viral DNA polymerase (Pol) colocalize to replication compartments in the nuclei of infected cells under conditions which permit viral DNA synthesis. When viral DNA synthesis was blocked by incubation of the wild-type virus with phosphonoacetic acid, the 65KDBP, Pol, and ICP8 failed to localize to replication compartments. Instead, ICP8 accumulated nearly exclusively to prereplication sites, while the 65KDBP was only diffusely localized within the nuclei. Although some of the Pol accumulated in prereplication sites occupied by ICP8 in the presence of phosphonoacetic acid, a significant amount of Pol also was distributed throughout the nuclei. Examination by double-labeling immunofluorescence of DNA- ts mutant virus-infected cells revealed that the 65KDBP also did not colocalize with ICP8 to prereplication sites at temperatures nonpermissive for virus replication. These results are in disagreement with the hypothesis that ICP8 is the major organizational protein responsible for attracting other replication protein to prereplication sites in preparation for viral DNA synthesis (A. de Bruyn Kops and D. M. Knipe, Cell 55:857-868, 1988), and they suggest that other viral proteins, perhaps in addition to ICP8, or replication fork progression per se are required to organize the 65KDBP.

Animals↗
PubMed1990

Site-specific mutagenesis of a highly conserved region of the herpes simplex virus type 1 DNA polymerase gene.

Seven point mutations were introduced into region I of the herpes simplex virus type 1 DNA polymerase, which is most highly conserved among DNA polymerases and has no drug sensitivity markers mapped to it. The functional consequences of these mutations were studied in an in vitro transcription-translation system in which T7 transcripts of cloned polymerase genes were used to generate enzymatically active polypeptides in reticulocyte lysate. Analysis of labeled polypeptides on sodium dodecyl sulfate-polyacrylamide gel electrophoresis failed to show any alterations of stability caused by these mutations. The mutations G885R, D886N, T887K, D888A, and G896V lacked polymerase activity and failed to be stimulated by cotranslation of the herpes simplex virus 65-kilodalton DNA-binding protein, whereas R881G and S889A retained both polymerase activity and the capacity to be stimulated by the 65-kilodalton DNA-binding protein.

Amino Acid Sequence↗
PubMed1989

Characteristics of lymphocytes cultured from murine viral myocarditis specimens: a preliminary and technical report.

Long-term culture of lymphocytes from myocardial biopsy specimens has been reported. To test the usefulness of this technique in experimental models of murine myocarditis, the culture and characterization of lymphocytes from mice infected with encephalomyocarditis virus or coxsackievirus B3 were studied. Lymphocytes were successfully cultured from three hearts of encephalomyocarditis virus-infected mice in interleukin 2-containing culture. After approximately 10 days, lymphocytes migrated out of myocardium in the chronic stage of myocarditis and gradually increased in numbers. More than half of the exuding cells were Thy 1.2-positive. None of the myocardial samples from coxsackievirus B3-infected mice grew out lymphocytes. Pathologic examination of all specimens showed myocardial necrosis with lymphoid infiltration. Although further studies are needed, this preliminary study suggests that the technique of lymphocyte culture may promote new insights into the pathogenesis of experimental myocarditis.

Animals↗
PubMed1989

Effect of acyclovir on radiation- and chemotherapy-induced mouth lesions.

Several chemotherapeutic regimens and radiation therapy, if delivered to the oral mucosa, are associated with a high frequency of mouth lesions. The cause of this side effect is not known for certain, but in past studies it has sometimes been associated with the ability to culture herpes simplex virus type 1 from the mouth. In a double-blind prospective trial, patients with head and neck tumors treated with chemotherapy or radiation therapy were treated with either acyclovir or placebo. Although the frequency of culture-positive herpes simplex virus was low in the untreated group, it was significantly lower, zero, in the acyclovir-treated group. However, there were no differences in the frequency or type of mouth lesions experienced by patients receiving either radiation or chemotherapy who were taking acyclovir or placebo. These results suggest that herpes simplex virus is not a frequent cause or complication of oral lesions afflicting this patient population.

Acyclovir↗
PubMed1989

The essential 65-kilodalton DNA-binding protein of herpes simplex virus stimulates the virus-encoded DNA polymerase.

The 65-kilodalton DNA-binding protein (65KDBP) of herpes simplex virus type 1 (HSV-1), the product of the UL42 gene, is required for DNA replication both in vitro and in vivo, yet its actual function is unknown. By two independent methods, it was shown that the 65KDBP stimulates the activity of the HSV-1-encoded DNA polymerase (Pol). When Pol, purified from HSV-1-infected cells, was separated from the 65KDBP, much of its activity was lost. However, addition of the 65KDBP, purified from infected cells, stimulated the activity of Pol 4- to 10-fold. The ability of a monoclonal antibody to the 65KDBP to remove the Pol-stimulating activity from preparations of the 65KDBP confirmed that the activity was not due to a trace contaminant. Furthermore, the 65KDBP did not stimulate the activity of other DNA polymerases derived from T4, T7, or Escherichia coli. The 65KDBP gene transcribed in vitro from cloned DNA and translated in vitro in rabbit reticulocyte lysates also was capable of stimulating the product of the pol gene when the RNAs were cotranslated. The product of a mutant 65KDBP gene missing the carboxy-terminal 28 amino acids exhibited wild-type levels of Pol stimulation, while the products of two large deletion mutants of the gene could not stimulate Pol activity. These experiments suggest that the 65KDBP may be an accessory protein for the HSV-1 Pol.

Animals↗
PubMed1988

Myocarditis presenting as acute myocardial infarction.

Ten patients with acute myocarditis, who were initially seen with clinical signs of acute myocardial infarction, will be discussed. All had symptoms and seven had laboratory evidence of an acute viral infection. Acute cardiac findings consisted of chest pain in nine patients, compatible ECGs and elevated creatine kinase levels in 10, positive MB fractions in eight, and regional wall motion abnormalities in eight. Acutely, the left ventricular ejection fraction was less than 55% in six patients; ventricular ectopy occurred in five patients, bundle branch block in four, transient junctional escape rhythm in three, and congestive heart failure in three. Among the nine patients followed-up for 1 to 14 months there was one death, five patients had normal results of exercise tests, and three had normal coronary angiograms. Wall motion abnormalities persisted in four patients; ejection fraction improved in five and was less than 55% in three. These findings suggest that focal myocardial damage may occur during acute viral myocarditis and mimic acute myocardial infarction resulting from atherosclerotic coronary artery disease.

Adult↗
PubMed1988

Ribavirin treatment of murine coxsackievirus B3 myocarditis with analyses of lymphocyte subsets.

The efficacy of the synthetic nucleoside ribavirin in the therapy of experimental myocarditis caused by coxsackievirus B3 and its effects on peripheral and splenic lymphocyte subsets were investigated. Two week old male C3H/He mice were inoculated with coxsackievirus B3. Ribavirin was administered subcutaneously on days 0 to 15 (experiment I) and on days 4 to 15 (experiment II) in a dose of 200 (experiment I: Group 2, n = 10; experiment II: Group 5, n = 10) or 400 mg/kg per day (experiment I: Group 3, n = 10; experiment II: Group 6, n = 10). Control mice (experiment I: Group 1, n = 15; experiment II: Group 4, n = 14) received an injection of saline solution. In experiment I, mice treated with ribavirin survived significantly longer than did control mice (p = 0.005, Group 1 versus Group 2; p = 0.001, Group 1 versus Group 3). In experiment II, the survival rate on day 15 in ribavirin-treated mice was high compared with that in control mice (Group 4 = 14.3%, Group 5 = 20%, Group 6 = 50%). Myocardial viral titers on days 5 to 8 were significantly lower in ribavirin-treated mice of both experiments than in control mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Significance of resistance of herpes simplex virus to acyclovir.

The genome of herpes simplex virus codes for several enzymes, including viral thymidine kinase and viral deoxyribonucleic acid (DNA) polymerase. When viral resistance develops, it does so by changes in these two enzymes. Three possible mechanisms of viral resistance to acyclovir include (1) selection of viral mutants that make little or no thymidine kinase and do not phosphorylate acyclovir adequately, (2) selection of mutants that can phosphorylate thymidine but cannot phosphorylate acyclovir (i.e., these viruses have thymidine kinases with altered substrate specificity), and (3) selection of viruses that have altered DNA polymerases that replicate viral DNA in the presence of acyclovir triphosphate. Thymidine kinase-deficient virus has been isolated from clinical isolates frequently, but few strains appear to be virulent for animals or humans and only a few seem to have caused clinical disease. Viruses with altered substrate specificity have been reported but viruses with an altered DNA polymerase have not occurred in clinical practice. Antiviral drugs should be used only when necessary to minimize the appearance of resistant strains of virus.

Acyclovir↗
PubMed1988

Prevention of murine coxsackie B3 viral myocarditis and associated lymphoid organ atrophy with recombinant human leucocyte interferon alpha A/D.

The effects of recombinant human leucocyte interferon alpha A/D on experimental myocarditis due to coxsackie virus B3 were investigated. Specific plaque reduction assays showed that 50% of in vitro plaque formation in VERO (kidney of African green monkey) cells was inhibited by interferon alpha A/D 48 U.ml-1 when administered 24 h before infection with coxsackie virus B3. Three week old male C3H/He mice were inoculated intraperitoneally with 3 x 10(2) plaque-forming units (pfu) of coxsackie virus B3. Controls (group 1) were injected with saline. Interferon alpha A/D 10(4) (group 2) or 10(3) (group 3) U.g-1.day-1, was administered subcutaneously daily, starting 1 d before the infection. Interferon alpha A/D 10(4) U.g-1.day-1 (group 4) was also given, starting the same day. Each group consisted of ten mice. Animals were sacrificed on d 5 for evaluation. Myocardial virus titres were significantly lower in groups 2, 3, and 4 (p less than 0.05) compared with controls. Histological examination showed extensive myocardial necrosis and cellular infiltration in all untreated mice but less severe necrosis and cellular infiltration in treated groups. Thymus and spleen weights in treated groups were greater than in the untreated group, and cellular depletion was less. Thus interferon alpha A/D effectively inhibited myocardial replication of coxsackie virus B3, reduced the myocardial inflammatory response, and prevented the disease associated lymphoid organ atrophy in this animal model.

Animals↗