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Biomedical subjects

K E Brown

Publications and source records attributed to K E Brown.

At least 37 records · Page 2Linked to original sources
PubMed2000

Rhesus and pig-tailed macaque parvoviruses: identification of two new members of the erythrovirus genus in monkeys.

We have previously reported the identification of a novel simian parvovirus in cynomolgus monkeys, which causes severe anemia in immunosuppressed cynomolgus monkeys and is currently being studied as an animal model for human B19 infection. We now report two similar outbreaks of anemia in rhesus and pig-tailed macaques associated with two distinct but similar simian parvoviruses (pig-tailed macaque and rhesus parvovirus). Both viruses have been cloned and over 5000 nucleotides sequenced from each virus. The viruses show marked similarities to other members of the Erythrovirus genus in the Parvoviridae family.

Amino Acid Sequence↗
PubMed2000

Increased expression of tissue inhibitor of metalloproteinases type 1 (TIMP-1) in a more tumourigenic colon cancer cell line.

Genetic changes occurring in the late stages of colonic tumour progression have received much less attention than those occurring in the early stages. As described in the accompanying paper, SW480 and SW620 cell lines provide a useful model for studying the advanced stages of progression for colon cancer. Comparison of the two cell lines by differential display reveals that SW620 cells express lower levels of the CC3 tumour suppressor gene and also lower levels of the tissue inhibitor of metalloproteinases-3 (TIMP-3) gene. Northern blot analysis for TIMP-3 confirms this finding and shows a similar difference in the expression of TIMP-2, which seems logical since TIMPs inhibit enzymes that play a role in tumour invasion. For this reason, it was surprising to find that TIMP-1 messenger RNA expression is markedly increased in SW620 cells. Consistent with this finding, western blot analysis shows a ten-fold increase in TIMP-1 protein secretion by SW620 cells. It is noteworthy that high TIMP-1 expression is associated with poor prognosis in colorectal cancer. This association between TIMP-1 expression and tumour progression may be related to additional growth factor-like effects described for TIMP-1 in some systems.

Blotting, Northern↗
PubMed2000

Prevalence of the newly described human circovirus, TTV, in United States blood donors.

BACKGROUND: A novel nonenveloped single-stranded circular DNA virus (TTV) was recently identified. The prevalence of TTV in blood donors in the United States is, however, still unclear. STUDY DESIGN AND METHODS: Viral DNA was detected in US blood donors from five cities by using two sets of TTV primers: NG059/NG061/NG063 primers, which amplified the conserved region of strains 1 and 2, and T801/T935 primers, which amplified the 5' end region of the TTV sequence. A TTV antibody assay system was based on the detection of the truncated open reading frame (ORF)-1 (amino acids 1-411) from type 1b. The truncated ORF-1 was expressed as a fusion protein in Escherichia coli, and the fusion protein was used as the antigen in the antibody assay system. RESULTS: Viremia was detected in 21 (8. 4%) of 250 donors by use of NG059/NG061/NG063 primers and 104 (41. 6%) of 250 by use of T801/T935 primers. There was little correlation among the assays, which suggests the preferential detection of different strains with the different primers. TTV antibody was detected in 38 of 100 donors: 32 (84%) of 38 with concurrent TTV viremia and 6 (16%) of 38 without TTV viremia. TTV viremia and/or TTV antibody-positive samples were detected in 52 (52%) of 100 of US blood donors. CONCLUSION: Evidence of infection or exposure to TTV appears to be common among blood donors in United States.

Antibodies, Viral↗
PubMed2000

Haematological consequences of parvovirus B19 infection.

Parvovirus B19, a member of the Erythrovirus genus, is the only member of the Parvoviridae family known to be pathogenic in humans. Erythroviruses are so named because of their tropism and selective replication in erythroid progenitor cells. Haematological consequences of B19 infection arise due to a direct cytotoxic effect on erythroid progenitors in bone marrow with interruption of erythrocyte production. In addition, the physiology of host haematopoiesis and competence of the immune response each determines clinical manifestations of B19 infection: in individuals with underlying haemolytic disorders, B19 infection causes transient aplastic crisis; in immunocompromised patients, persistent B19 infection may develop that manifests as pure red cell aplasia and chronic anaemia; B19 infection in utero may result in fetal death, hydrops fetalis, or congenital anaemia. Diagnosis is based on examination of bone marrow and B19 virological studies. Treatment of persistent infection with immunoglobulin leads to a prompt resolution of the anaemia.

Erythropoiesis↗
PubMed2000

GB virus C/Hepatitis G virus infection is frequent in American children and young adults.

The novel flavivirus GB virus C/hepatitis G virus (GBV-C/HGV) has been detected in approximately 2% of blood donors in the United States, and neutralizing antibody to the envelope protein (E2), a marker of previous infection with GBV-C/HGV, is present in approximately 9% of donors. The rate of GBV-C/HGV infection among American children is unknown. To determine whether viral infection might occur during childhood, 160 serum specimens (obtained from blood bank samples) from children and young adults with no history of transfusion were tested. Viral RNA and antibody to E2 were detected in 6.3% and 9.4% of subjects, respectively. Evidence of previous or current infection (viremia and/or antibody to E2) was detected in 13.8% of subjects, indicating that GBV-C/HGV infection appears to be common among American children and young adults, even in the absence of blood transfusion.

Adolescent↗
PubMed2000

Primary T lymphocytes as targets for gene therapy.

Peripheral blood T lymphocytes have been considered an attractive target for gene therapy applications. They can be easily harvested and readily expanded ex vivo. The transduction efficiency of primary human lymphocytes with standard retroviral vectors approaches 50% or more using optimized methods of gene transfer. Other methods of gene transfer, including adenoviral, adeno-associated viral, and lentiviral vectors, or nonviral techniques, have also been used for gene transfer into primary lymphocytes. Despite encouraging results in vitro, human clinical trials using retroviral vectors to transduce primary lymphocytes have been hindered by low expression levels of transgenes and immune responses against transgene products. Strategies to overcome these problems need to be developed.

Animals↗
PubMed2000

Physical therapy for migraine-related vestibulopathy and vestibular dysfunction with history of migraine.

OBJECTIVES/HYPOTHESIS: To assess the efficacy of physical therapy for patients with a diagnosis of migraine-related vestibulopathy (MRV) or vestibular dysfunction with a history of migraine headache. STUDY DESIGN: Retrospective case series METHODS: Thirty-nine patients were identified through a retrospective chart review, 14 with a diagnosis of MRV and 25 with migraine headache. The patients were treated with a custom-designed physical therapy exercise program for a mean of 4.9 visits over a mean duration of 4 months. Patients completed the Dizziness Handicap Inventory (DHI), the Activities-Specific Balance Confidence Scale (ABC), and the Dynamic Gait Index (DGI), reported the number of falls they had experienced in the past 4 weeks, and rated the severity of their dizziness on an analogue scale of 0 to 100 at initial evaluation and at discharge. RESULTS: Significant differences were seen before and after therapy in each of the outcome measures used. The average decrease in DHI score was 12 points (P < .01). ABC scores increased an average of 14 points (P < .01). Subjects increased their DGI scores an average of 4 points (P < .01). The number of patients reporting more than one fall decreased by 78% at discharge. (P < .05). Baseline symptoms of dizziness decreased an average of 11 points (P < .05). CONCLUSIONS: Patients with MRV and migraine headache demonstrated improvement in physical performance measures and self-perceived abilities after vestibular physical therapy.

Adult↗
PubMed2000

GB virus C/hepatitis G virus replicates in human haematopoietic cells and vascular endothelial cells.

A novel flavivirus, GB virus C (GBV-C)/hepatitis G virus (HGV), has been detected in chronic liver disease patients. It is known that the viral RNA can be detected in approximately 5% of American blood donors. However, the implications for liver disease and the sites of virus replication remain unknown. Possible sites of virus replication were studied by using cell lines and/or primary cells derived from human lymphoid cells, myeloid cells, hepatocytes and endothelial cells. RNA was detected by virus strand-specific RT-PCR and GBV-C/HGV antigen was detected with a rabbit polyclonal anti-E2 (envelope 2) antibody by Western blot analysis. Negative-strand RNA, representative of replicating virus, was detected in lymphoid and megakaryocytoid cell lines and primary vascular endothelial cells. In addition, an increase in virus titre over time was demonstrated and viral antigen was detected, and virus could be passaged to infect fresh cells. However, viral RNA or antigen could not be detected in any of the hepatocyte lines tested. These results indicate that the replication site of GBV-C/HGV is not primarily in hepatocytes and that detection of replicating virus in hepatic tissue may reflect virus replication in haematopoietic cells and/or vascular endothelial cells present in the liver.

Animals↗
PubMed1999

Integrin alphaVbeta5 is not involved in adeno-associated virus type 2 (AAV2) infection.

alphaVbeta5 integrin was recently proposed as a coreceptor for adeno-associated virus type 2 (AAV2) infection (Summerford et al., 1999, Nat. Med. 5, 78-82), based mainly on the direct binding of AAV2 to denatured beta5 by virus overlay assay. In studies using purified natural or recombinant human integrin alphaVbeta5 we were unable to demonstrate AAV2 binding, either by virus overlay or by liquid binding assay. Furthermore, neither purified integrin alphaVbeta5, nor RGD peptides, nor functional blocking monoclonal antibody blocked rAAV2 transduction. These data strongly suggest that integrin alphaVbeta5 is not involved in AAV2 infection.

Animals↗
PubMed1999

In vivo marking of rhesus monkey lymphocytes by adeno-associated viral vectors: direct comparison with retroviral vectors.

We have compared adeno-associated virus (AAV)-based and retrovirus-based vectors for their ability to transduce primary T lymphocytes in vitro and then tracked the persistence of these genetically marked lymphocytes in vivo, using the rhesus monkey model. To avoid the complication of immune rejection of lymphocytes transduced with xenogeneic genes in tracking studies primarily designed to investigate transduction efficiency and in vivo kinetics, the vectors were designed without expressed genes. All vectors contained identically mutated beta-galactosidase gene (beta-gal) and neomycin resistance gene (neo) DNA sequences separated by different length polylinkers, allowing simple differentiation by polymerase chain reaction (PCR). Each of 2 aliquots of peripheral blood lymphocytes from 4 rhesus monkeys were transduced with either AAV or retroviral vectors. The in vitro transduction efficiency (mean vector copy number/cell) after the ex vivo culture was estimated by PCR at 0.015 to 3.0 for AAV, varying depending on the multiplicity of infection (MOI) used for transduction, and 0.13 to 0.19 for the retroviral transductions. Seven days after transduction, Southern blot analysis of AAV-transduced lymphocytes showed double-stranded and head-to-tail concatemer forms but failed to show integration of the AAV vector. AAV and retroviral aliquots were reinfused concurrently into each animal. Although the retrovirally marked lymphocytes could be detected for much longer after infusion, AAV transduction resulted in higher short-term in vivo marking efficiency compared with retroviral vectors, suggesting possible clinical applications of AAV vectors in lymphocyte gene therapy when long-term vector persistence is not required or desired.

Animals↗
PubMed1999

A 110-kDa nuclear shuttle protein, nucleolin, specifically binds to adeno-associated virus type 2 (AAV-2) capsid.

A 110-kDa protein was copurified with adeno-associated virus type 2 (AAV-2) virions after CsCl density gradient isopycnic centrifugation. Amino acid sequence of peptides derived from this protein after tryptic digestion, monoclonal antibody production, and Western blot analysis showed that the copurified protein was the major nucleolar phosphoprotein, human nucleolin. Virus overlay assays demonstrated that AAV-2 capsid specifically bound to the human nucleolin, and immunoprecipitation studies confirmed the in vitro binding of nucleolin and intact AAV-2 capsids but not denatured viral proteins. Double-immunofluorescence staining of infected cells showed that AAV capsid and nucleolin were colocalized in both cytoplasm and nucleus. In addition, when cytoplasmic and nuclear fractions were extracted from AAV-infected KB cells at different time points postinfection, immunoprecipitation data and Western blotting showed that AAV capsid formation and nucleolin interact specifically and share their subcellular localization in infected cells. With the known functions of nucleolin in the synthesis of rRNA and ribosome assembly, binding to single-stranded DNA, and acting as a shuttle between cytoplasm and nucleolus, our data showing that AAV-2 capsid binds specifically to nucleolin both in vitro and in vivo suggest a key role of nucleolin in AAV-2 replication, particularly in capsid assembly.

Animals↗
PubMed1999

Dynamic repositioning of genes in the nucleus of lymphocytes preparing for cell division.

We show that several transcriptionally inactive genes localize to centromeric heterochromatin in the nucleus of cycling but not quiescent (noncycling) primary B lymphocytes. In quiescent cells, centromeric repositioning of inactive loci was induced after mitogenic stimulation. A dynamic repositioning of selected genes was also observed in developing T cells. Rag and TdT loci were shown to relocate to centromeric domains following heritable gene silencing in primary CD4+8+ thymocytes, but not in a phenotypically similar cell line in which silencing occurred but was not heritable. Collectively, these data indicate that the spatial organization of genes in cycling and noncycling lymphocytes is different and that locus repositioning may be a feature of heritable gene silencing.

Alleles↗
PubMed1999

Quantitative analysis of neutralizing immune responses to human parvovirus B19 using a novel reverse transcriptase-polymerase chain reaction-based assay.

Infection with human parvovirus B19 causes fifth disease, acute and chronic red cell aplasia, fetal hydrops, arthropathy, and other disorders. Antiviral antibodies limit B19 infection in vivo; however, the identification of serologic markers of protection has been hampered by the lack of a quantitative assay for parvovirus neutralization. A novel in vitro test for parvovirus neutralization has been developed using reverse transcriptase-polymerase chain reaction to detect viral transcripts in a B19-permissive cell line. Parvovirus neutralizing activity was measured in sera from naturally infected individuals, and common features of sera with high neutralizing capacity were identified as protection correlates. Sera that suppressed B19 replication in vitro demonstrated IgG reactivity with capsid proteins VP1 and VP2, but no linear relationship between antibody titer and neutralizing capacity was observed. Sera from experimental animals and human volunteers immunized with a virus-like particle vaccine candidate exhibited B19 neutralizing titers equal to or greater than those observed in natural infections.

Animals↗
PubMed1999

Molecular diagnosis of viral disease in hematology patients.

Viruses, through their direct and indirect effects on hematopoietic cells, are not only etiological agents of hematologic disease, but they both complicate transfusion therapy and are major causes of morbidity and mortality in immunosuppressed patients. The development of molecular techniques, especially DNA hybridization and amplification, has allowed new viruses to be discovered, has aided in the early diagnosis of infection before the development of an antibody response, and has revolutionized the detection of viruses in blood transfusions.

Cytogenetic Analysis↗