Biomedical subjects

K E Brown

Publications and source records attributed to K E Brown.

At least 19 recordsLinked to original sources

PubMed2007

Risk factors for hepatitis C fibrosis: a prospective study of United States veterans compared with nonveterans.

Chronic hepatitis C virus (HCV) infection causes cirrhosis in many infected patients; however, a better understanding of the risk factors for fibrosis progression in high HCV prevalence groups such as US veterans is needed. We wished to compare the demographic, clinical characteristics, and independent variables that influence fibrosis in US veterans vs nonveterans with chronic HCV. HCV-seropositive US veterans (n = 459) and nonveterans (n = 395) prospectively completed a detailed medical, social and occupational questionnaire. Clinical factors for progressive liver disease were compared between veterans and nonveterans and fibrosis stage assessed on liver biopsies (168 veterans and 208 nonveterans). Using polychotomous logistic regression, fibrosis was analysed as both a progressive and categorical outcome to determine independent risk factors for both patient groups. Although veterans were significantly older and had higher lifetime alcohol consumption than nonveterans, their median fibrosis scores did not differ from nonveterans. By univariate analysis, alanine aminotransferase, necroinflammatory activity (NIA), and cryoglobulin positivity were associated with fibrosis in veterans and nonveterans (P < 0.05, all comparisons), whereas steatosis was associated with fibrosis only in nonveterans (P < 0.0001). By multivariate analysis, NIA was an independent risk factor for fibrosis in both groups (P < 0.01). However, fibrosis in nonveterans was also independently associated with steatosis, significant alcohol consumption and age (P < 0.04, all comparisons). Independent risk factors for fibrosis vary among high HCV prevalence groups such as veterans when compared with nonveterans. Understanding specific patient cohort effects is important for determining independent risk factors for disease progression in chronic HCV infection.

Adult↗

PubMed2006

Brief report: no evidence for parvovirus B19 or hepatitis E virus as a cause of acute liver failure.

Viral hepatitis A and B are known to cause acute liver failure. While nearly 20% of acute liver failure cases are of indeterminate etiology, screening for other viruses has not been uniformly performed. We looked for evidence for parvovirus B19 and hepatitis E virus in sera from U.S. acute liver failure patients. For B19, 78 patients' sera, including 34 with indeterminate etiology, were evaluated by DNA dot-blot hybridization, reverse transcription polymerase chain reaction, and enzyme-linked immunosorbent assay for immunoglobin G and M antibodies; none showed evidence for infection.

Antibodies, Viral↗

PubMed2005

Immunodetection of aldose reductase in normal and diseased human liver.

Aldose reductase is an NADPH-dependent aldo-keto reductase best known as the rate-limiting enzyme of the polyol pathway that is implicated in the complications of diabetes. Aldose reductase appears to be involved in a variety of disease states other than diabetes, presumably due to its ability to catalyze the reduction of a broad spectrum of aldehydes, including some cytotoxic products of lipid peroxidation. Although the data regarding expression of aldose reductase in normal liver are conflicting, prior studies have suggested that the enzyme may be induced in diseased liver. The goal of these studies was to characterize expression of aldose reductase in normal and diseased human liver, using RT-PCR, Western analysis and immunohistochemistry. Aldose reductase transcripts and protein were detected at low levels in control human livers. In contrast, levels of aldose reductase mRNA and protein were increased in chronically diseased human livers. Immunohistochemistry demonstrated localization of aldose reductase in sinusoidal lining cells; dual immunofluorescence confocal microscopy with the macrophage marker, CD68, confirmed that the aldose reductase-positive sinusoidal lining cells were Kupffer cells. Abundant aldose reductase-positive, CD68-positive cells were present in the fibrous septa of cirrhotic livers, accounting for the increase in immunoreactive aldose reductase in diseased livers. Immunostaining of human lung, spleen and lymph node revealed that macrophages in those tissues also express aldose reductase. These data are the first to demonstrate that aldose reductase is expressed by human macrophages in various tissues and suggest that this enzyme may play a role in immune or inflammatory processes.

Aldehyde Reductase↗

PubMed2004

Mepivacaine local anaesthetic duration in equine palmar digital nerve blocks.

REASONS FOR PERFORMING STUDY: Perineural analgesics are used for lameness diagnosis but the duration of effect, knowledge of which would provide valuable information when performing subsequent blocks, is unknown. OBJECTIVE: To evaluate the duration of a palmar digital nerve block using force plate measurements. METHODS: Ten horses diagnosed with unilateral navicular syndrome were trotted at range of 3 +/- 0.15 m/sec over a force plate to record ground reaction forces for 5 trials of each forelimb. Data were recorded before nerve block, and then at 15 mins, 1, 2 and 24 h post nerve block. RESULTS: Before nerve block, peak vertical force (mean +/- s.e.) was significantly higher in the contralateral forelimb (CL = 5345 +/- 188 N) than in the lame forelimb (L = 4256 +/- 204 N; P<0.05). At 15 mins post nerve block there was no significant difference between the 2 forelimbs (CL = 5140 +/- 184 N; L = 5126 +/- 129 N), and this remained the case for 1 h. By 2 h, the mean score for the lame leg had decreased (L = 4642 +/- 182 N) but was still greater than preblock. By 24 h, vertical forces had returned to preblock values. CONCLUSIONS: The palmar digital nerve block was fully effective between 15 mins and 1 h. The analgesic effect began to subside between 1 and 2 h but sufficient analgesia persisted to affect gait characteristics beyond 2 h. POTENTIAL RELEVANCE: When using a palmar digital nerve block, it is important to perform lameness evaluations between 15 mins and 1 h to be sure of effective nerve blockade.

Anesthetics, Local↗

PubMed2004

Variants of B19.

The human parvovirus B19 (B19), the only known human pathogenic parvovirus, was discovered in 1975. Like other parvoviruses it is a small (22-24 nm diameter) non-enveloped icosahedral virus, with a single-stranded genome. As part of its life history it replicates to high titre in the bone marrow, and in acute infection high titres of infectious virus are present in the peripheral blood. Parvovirus B19 is therefore not an uncommon 'contaminant' of blood and blood products. In addition the lack of lipid membrane envelope renders parvoviruses insensitive to solvent detergents, and the small genome confers relative resistance to heat and gamma irradiation. Many medical reviews have been written on the molecular biology, clinical features, approaches to diagnosis and management, and these should be consulted for more information. This review will give an overview of the biology of parvovirus B19 infection, and the more recently described variants of parvovirus B19.

Animals↗

PubMed2001

Increased frequency of HLA-DR2 in patients with paroxysmal nocturnal hemoglobinuria and the PNH/aplastic anemia syndrome.

Many autoimmune diseases are associated with HLA alleles, and such a relationship also has been reported for aplastic anemia (AA). AA and paroxysmal nocturnal hemoglobinuria (PNH) are related clinically, and glycophosphoinositol (GPI)-anchored protein (AP)-deficient cells can be found in many patients with AA. The hypothesis was considered that expansion of a PNH clone may be a marker of immune-mediated disease and its association with HLA alleles was examined. The study involved patients with a primary diagnosis of AA, patients with myelodysplastic syndrome (MDS), and patients with primary PNH. Tests of proportions were used to compare allelic frequencies. For patients with a PNH clone (defined by the presence of GPI-AP-deficient granulocytes), regardless of clinical manifestations, there was a higher than normal incidence of HLA-DR2 (58% versus 28%; z = 4.05). The increased presence of HLA-DR2 was found in all frankly hemolytic PNH and in PNH associated with bone marrow failure (AA/PNH and MDS/PNH). HLA-DR2 was more frequent in AA/PNH (56%) than in AA without a PNH clone (37%; z = 3.36). Analysis of a second cohort of patients with bone marrow failure treated with immunosuppression showed that HLA-DR2 was associated with a hematologic response (50% of responders versus 34% of nonresponders; z = 2.69). Both the presence of HLA-DR2 and the PNH clone were independent predictors of response but the size of PNH clone did not correlate with improvement in blood count. The results suggest that clonal expansion of GPI-AP-deficient cells is linked to HLA and likely related to an immune mechanism.

Alleles↗

PubMed2001

Rabaptin-5 is a novel fusion partner to platelet-derived growth factor beta receptor in chronic myelomonocytic leukemia.

Chromosomal translocations involving the platelet-derived growth factor beta receptor (PDGFbetaR) gene have been reported in some patients with chronic myelomonocytic leukemia (CMML). The resultant fusion proteins have constitutive PDGFbetaR tyrosine kinase activity, but the partner genes previously reported (tel, Huntingtin interacting protein 1 [HIP-1], H4/D10S170) have poorly understood roles in the oncogenic activity of the fusion proteins. A novel PDGFbetaR fusion protein has been characterized in a patient with CMML and an acquired t(5;17)(q33;p13). Southern blot analysis on patient leukemia cells demonstrated involvement of the PDGFbetaR gene. Using 5' rapid amplification of complementary DNA ends-polymerase chain reaction (RACE-PCR) on patient RNA, rabaptin-5 was identified as a novel partner fused in-frame to the PDGFbetaR gene. The new fusion protein includes more than 85% of the native Rabaptin-5 fused to the transmembrane and intracellular tyrosine kinase domains of the PDGFbetaR. Transduction with a retroviral vector expressing rabaptin-5/PDGFbetaR transformed the hematopoietic cell line Ba/F3 to growth factor independence and caused a fatal myeloproliferative disease in mice. Rabaptin-5 is a well-studied protein shown to be an essential and rate-limiting component of early endosomal fusion through interaction with the Ras family GTPases Rab5 and Rab4. The fusion protein includes 3 of 4 coiled-coil domains (involved in homodimerization of native rabaptin-5), 2 caspase-3 cleavage sites, and a binding site for the tumor suppressor gene tuberin (tuberous sclerosis complex-2). Early endosomal transport is critical in regulation of various growth factor receptors, through ligand-induced clathrin-mediated endocytosis, and thus this new fusion protein links together 2 important pathways of growth regulation.

Adult↗

PubMed2001

Binding of adeno-associated virus type 5 to 2,3-linked sialic acid is required for gene transfer.

Recombinant adeno-associated viruses (AAV) are promising gene therapy vectors. Whereas AAV serotype 2-mediated gene transfer to muscle has partially replaced factor IX deficiency in hemophilia patients, its ability to mediate gene transfer to the lungs for cystic fibrosis is hindered by lack of apical receptors. However, AAV serotype 5 infects human airway epithelia from the lumenal surface. We found that in contrast to AAV2, the apical membrane of airway epithelia contains abundant high affinity receptors for AAV5. Binding and gene transfer with AAV5 was abolished by genetic or enzymatic removal of sialic acid from the cell surface. Furthermore, binding and gene transfer to airway epithelia was competed by lectins that specifically bind 2,3-linked sialic acid. These observations suggest that 2,3-linked sialic acid is either a receptor for AAV5 or it is a necessary component of a receptor complex. Further elucidation of the receptor for this virus should enhance understanding of parvovirus biology and expand the therapeutic targets for AAV vectors.

Animals↗

PubMed2001

Immunohistochemical detection of myeloperoxidase and its oxidation products in Kupffer cells of human liver.

Oxidative damage to tissue proteins has been implicated in the pathogenesis of liver disease, but the mechanisms that promote oxidation in vivo are unclear. Hydrogen peroxide is transformed into an array of potentially damaging reactants by the heme protein myeloperoxidase. This proinflammatory enzyme is expressed by circulating neutrophils and monocytes but is generally thought to be absent from tissue macrophages. To determine whether myeloperoxidase is present in Kupffer cells, the fixed-tissue macrophages of liver, Western blot analysis, and immunohistochemistry were performed. Two different antibodies monospecific for myeloperoxidase identified a 60-kd protein, the predicted molecular mass of myeloperoxidase, in human liver extracts. Immunostaining detected the enzyme in sinusoidal lining cells of normal and diseased human livers. Immunofluorescence confocal microscopy demonstrated co-localization of myeloperoxidase and CD68, a monocyte/macrophage marker, in sinusoidal lining cells. Numerous myeloperoxidase-expressing cells were also evident in the fibrous septa of cirrhotic livers. Immunostaining with an antibody to proteins modified by hypochlorous acid, a characteristic product of the enzyme, indicated that myeloperoxidase is enzymatically active in cases of acute liver injury and cirrhosis. These findings identify myeloperoxidase as a component of human Kupffer cells. Oxidative damage resulting from the action of myeloperoxidase may contribute to acute liver injury and hepatic fibrogenesis.

Animals↗

PubMed2001

Expression of alpha- and beta-globin genes occurs within different nuclear domains in haemopoietic cells.

The alpha- and beta-globin gene clusters have been extensively studied. Regulation of these genes ensures that proteins derived from both loci are produced in balanced amounts, and that expression is tissue-restricted and specific to developmental stages. Here we compare the subnuclear location of the endogenous alpha- and beta-globin loci in primary human cells in which the genes are either actively expressed or silent. In erythroblasts, the alpha- and beta-globin genes are localized in areas of the nucleus that are discrete from alpha-satellite-rich constitutive heterochromatin. However, in cycling lymphocytes, which do not express globin genes, the distribution of alpha- and beta-globin genes was markedly different. beta-globin loci, in common with several inactive genes studied here (human c-fms and SOX-1) and previously (mouse lambda5, CD4, CD8alpha, RAGs, TdT and Sox-1), were associated with pericentric heterochromatin in a high proportion of cycling lymphocytes. In contrast, alpha-globin genes were not associated with centromeric heterochromatin in the nucleus of normal human lymphocytes, in lymphocytes from patients with alpha-thalassaemia lacking the regulatory HS-40 element or entire upstream region of the alpha-globin locus, or in mouse erythroblasts and lymphocytes derived from human alpha-globin transgenic mice. These data show that the normal regulated expression of alpha- and beta-globin gene clusters occurs in different nuclear environments in primary haemopoietic cells.

Cell Nucleus↗

PubMed2001

Nonequivalent nuclear location of immunoglobulin alleles in B lymphocytes.

Individual B lymphocytes normally express immunoglobulin (Ig) proteins derived from single Ig heavy chain (H) and light chain (L) alleles. Allelic exclusion ensures monoallelic expression of Ig genes by each B cell to maintain single receptor specificity. Here we provide evidence that at later stages of B cell development, additional mechanisms may contribute to prioritizing expression of single IgH and IgL alleles. Fluorescent in situ hybridization analysis of primary splenic B cells isolated from normal and genetically manipulated mice showed that endogenous IgH, kappa and lambda alleles localized to different subnuclear environments after activation and had differential expression patterns. However, this differential recruitment and expression of Ig alleles was not typically seen among transformed B cell lines. These data raise the possibility that epigenetic factors help maintain the monoallelic expression of Ig.

Alleles↗

PubMed2001

Parvovirus B19: implications for transfusion medicine. Summary of a workshop.

This 1-day workshop showed that the infectivity of B19 DNA in donor blood and the neutralizing action of different antibodies present in the donated blood are not yet fully understood. It is possible that B19-induced anemia and reticulocytopenia are not being recognized in transfused recipients other than those in specific risk groups. The testing of blood components for any infectious agent is usually clinically driven, and, if B19 NAT were recommended at the present time in other than plasma products, a CMV-like model might prove appropriate; that is, virus screening would be performed on blood components destined for high-risk groups only. Currently, there is insufficient evidence to recommend universal testing, especially for single units. Workshop participants recommended that basic research continue in the scientific areas addressed. If clinical trials were to be developed, participants recommended that they include special risk groups such as seronegative pregnant women and children with malignancies who are receiving chemotherapy.

Animals↗

PubMed2001

Physical therapy outcomes for persons with bilateral vestibular loss.

OBJECTIVE: The purpose of the study was to assess the efficacy of physical therapy for patients with bilateral vestibular loss. STUDY DESIGN: Retrospective case series. METHODS: Twenty-four patients with a diagnosis of bilateral vestibular loss were identified by a retrospective chart review. Thirteen of the 24 patients met the inclusion criteria of having a moderate or greater loss of vestibular function bilaterally as rated by an otoneurologist based on the patient's vestibular function tests. These patients were treated with a custom-designed physical therapy program for a mean of 4.6 visits over an average period of 3.8 months. Patients completed the Dizziness Handicap Inventory and the Activities-specific Balance Confidence Scale at initial evaluation and discharge. Patients were asked to perform the balance and gait tasks of the Dynamic Gait Index, Sensory Organization Test of computerized dynamic posturography, and the Timed "Up and Go" test at their first and last physical therapy sessions. The number of falls in the previous 4 weeks and the use of an assistive device at initial evaluation and discharge were reported. Composite score, an overall score of clinical outcome, was calculated to determine clinically significant changes in physical performance and subjective information. RESULTS: On a population basis, statistically significant improvement was observed after physical therapy for each of the outcome measures including the composite score (P < .05). Clinically significant changes were demonstrated by 33% to 55% of the patients on the various outcome measures. No change was noted in the patients' risk of falling, their number of falls, and the use of assistive devices. CONCLUSION: Many patients with bilateral vestibular loss benefit from an individualized vestibular physical therapy exercise program based on improved physical function and reduced self-perceived levels of handicap.

Accidental Falls↗

PubMed2001

Adeno-associated virus serotype 4 (AAV4) and AAV5 both require sialic acid binding for hemagglutination and efficient transduction but differ in sialic acid linkage specificity.

Adeno-associated virus serotype 4 (AAV4) and AAV5 have different tropisms compared to AAV2 and to each other. We recently reported that alpha 2--3 sialic acid is required for AAV5 binding and transduction. In this study, we characterized AAV4 binding and transduction and found it also binds sialic acid, but the specificity is significantly different from AAV5. AAV4 can hemagglutinate red blood cells from several species, whereas AAV5 hemagglutinates only rhesus monkey red blood cells. Treatment of red blood cells with trypsin inhibited hemagglutination for both AAV4 and AAV5, suggesting that the agglutinin is a protein. Treatment of Cos and red blood cells with neuraminidases also indicated that AAV4 bound alpha 2--3 sialic acid. However, resialylation experiments with neuraminidase-treated red blood cells demonstrated that AAV4 binding required alpha 2--3 O-linked sialic acid, whereas AAV5 required N-linked sialic acid. Similarly, resialylation of sialic acid-deficient CHO cells supported this same conclusion. The difference in linkage specificity for AAV4 and AAV5 was confirmed by binding and transduction experiments with cells incubated with either N-linked or O-linked inhibitors of glycosylation. Furthermore, AAV4 transduction was only blocked with soluble alpha 2-3 sialic acid, whereas AAV5 could be blocked with either alpha 2--3 or alpha 2-6 sialic acid. These results suggest that AAV4 and AAV5 require different sialic acid-containing glycoproteins for binding and transduction of target cells and they further explain the different tropism of AAV4 and AAV5.

Animals↗

PubMed2000

Targeting of Ikaros to pericentromeric heterochromatin by direct DNA binding.

Ikaros is a sequence-specific DNA-binding protein that is essential for lymphocyte development. Little is known about the molecular function of Ikaros, although recent results have led to the hypothesis that it recruits genes destined for heritable inactivation to foci containing pericentromeric heterochromatin. To gain further insight into the functions of Ikaros, we have examined the mechanism by which it is targeted to centromeric foci. Efficient targeting of Ikaros was observed upon ectopic expression in 3T3 fibroblasts, demonstrating that lymphocyte-specific proteins and a lymphoid nuclear architecture are not required. Pericentromeric targeting did not result from an interaction with the Mi-2 remodeling factor, as only a small percentage of Mi-2 localized to centromeric foci in 3T3 cells. Rather, targeting was dependent on the amino-terminal DNA-binding zinc finger domain and carboxy-terminal dimerization domain of Ikaros. The carboxy-terminal domain was required only for homodimerization, as targeting was restored when this domain was replaced with a leucine zipper. Surprisingly, a detailed substitution mutant analysis of the amino-terminal domain revealed a close correlation between DNA-binding and pericentromeric targeting. These results show that DNA binding is essential for the pericentromeric localization of Ikaros, perhaps consistent with the presence of Ikaros binding sites within centromeric DNA repeats. Models for the function of Ikaros that are consistent with this targeting mechanism are discussed.

3T3 Cells↗

PubMed2000

Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates.

Retroviral insertion site analysis was used to track the contribution of retrovirally transduced primitive progenitors to hematopoiesis after autologous transplantation in the rhesus macaque model. CD34-enriched mobilized peripheral blood cells were transduced with retroviral marking vectors containing the neo gene and were reinfused after total body irradiation. High-level gene transfer efficiency allowed insertion site analysis of individual myeloid and erythroid colony-forming units (CFU) and of highly purified B- and T-lymphoid populations in 2 animals. At multiple time points up to 1 year after transplantation, retroviral insertion sites were identified by performing inverse polymerase chain reaction and sequencing vector-containing CFU or more than 99% pure T- and B-cell populations. Forty-eight unique insertion sequences were detected in the first animal and also in the second animal, and multiple clones contributed to hematopoiesis at 2 or more time points. Multipotential clones contributing to myeloid and lymphoid lineages were identified. These results support the concept that hematopoiesis in large animals is polyclonal and that individual multipotential stem or progenitor cells can contribute to hematopoiesis for prolonged periods. Gene transfer to long-lived, multipotent clones is shown and is encouraging for human gene therapy applications.

Animals↗

PubMed2000

Sequencing and characterization of A-2 plaque virus: A new member of the Picornaviridae family.

A-2 plaque virus (A2 virus) was originally isolated from the icteric-phase sera of US servicemen with viral hepatitis in the 1960s, but apart from a preliminary characterization little is known about the agent. We have now successfully cloned and sequenced the complete viral genome. A2 viral RNA consists of 7312 nucleotides, excluding the 62 nucleotide poly(A) tract at the 3' end, with one large open reading frame. Although clearly a member of the Picornaviridae, there is low homology to the available sequences, suggesting it is only loosely related to the classic rhino/enterovirus genus. In addition, there was no reactivity with group specific monoclonal antibody blends against polioviruses, enteroviruses 70 and 71, coxsackievirus B, and echoviruses. Two tamarins were inoculated with A2 virus to study viral pathogenesis. Both animals that received A2 virus became transiently viremic 1 week after the infection, as determined by RT-PCR, and they developed an antibody response to A2 virus. However, no physical signs or biochemical abnormalities, including elevated liver transaminases, were observed. In addition, no liver samples from patients with fulminant hepatitis (n = 7) or controls (n = 7) were positive for A2 viral RNA nor was anti-A2 neutralizing antibody detected in sera from hepatitis patients (n = 14), healthy laboratory donors (n = 14), or US blood donors (n = 33); however, most sera contained antibodies reactive with A2 virus proteins. These results suggest that A2 virus is a new member of the Picornaviridae but that its pathogenicity in nonhuman primates and association with human disease still need to be determined.

Anemia, Aplastic↗

PubMed2000

The interaction of heparin sulfate and adeno-associated virus 2.

Recently heparan sulfate was proposed as the host cell receptor for the dependovirus, adeno-associated virus type 2 (AAV2). We show that although heparan sulfate on the cell surface may contribute to the binding of AAV2 to permissive cells, the amount of heparan sulfate on the cell surface as determined by flow cytometry using four different monoclonal antibodies does not correlate with AAV2 binding to cells or recombinant AAV2 transduction efficiency. Experiments with either mutant CHO cells or cells treated with chlorate to remove sulfate groups showed that sulfation was not absolutely required for infection or binding: in the absence of cell surface sulfation, recombinant AAV2 was still able to be transduced in previously permissive cells. Heparin is commonly used as a substitute in studies of the interaction between heparan sulfate and ligand, and we demonstrate that the binding affinity of AAV2/heparin is low, with a K(d) value of approximately 2.0 nM. A study of the direct interaction between AAV2 and artificial glycosaminoglycans showed that a high degree of sulfation on heparin was critical for the ability to bind AAV2 and compete rAAV2 transduction and that both O- and N-sulfate groups are required. Overall, our data suggest that, as has been shown for other viruses, the presence of a high-affinity AAV2 receptor mediates AAV2 infection in addition to the low-affinity heparan sulfate binding.

Animals↗