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Biomedical subjects

D R Brown

Publications and source records attributed to D R Brown.

At least 163 records · Page 9Linked to original sources
PubMed1998

Expression of the human papillomavirus type 11 E5A protein from the E1E4,E5 transcript.

The abundant human papillomavirus type 11 (HPV 11) E1E4,E5 transcript potentially encodes the E1E4,E5a and E5b proteins. It is not known if either of the E5 proteins are expressed from this transcript. For HPV 16, E5 is a single open reading frame (ORF), and the E5 protein is expressed from an unspliced E2,E5 transcript but not from the spliced E1E4,E5 transcript. This study was undertaken to determine if the HPV 11 E5a protein is expressed from the E1E4,E5 transcript. To detect E5a expression in eukaryotic cells, the green fluorescent protein (GFP) gene was fused to the 3' end of the E5a gene in the pEGFP-N1 vector. Several recombinant plasmid constructs were made to determine if E5a translation is influenced by upstream sequences present in the E1E4,E5 transcript. COS-7 cells were transfected with each construct, and flow cytometry was performed after 24 h of growth. The amount of E5a-GFP expressed from each construct was determined by the mean fluorescence of 2,000 transfected cells. Although the E5a-GFP fusion was expressed by all but one construct, the quantity of expressed E5a-GFP varied considerably. The most abundant expression was detected in cells transfected with the E1E4,E5a construct that lacked the 5' noncoding sequence between nucleotides (nts) 714 and 831 that is present in the authentic transcript. Other constructs expressed E5a-GFP in variable amounts, suggesting that sequences between nt 714 and the start of the E5a ORF affect expression of the E5a protein. An E2,E5a construct was made to compare the HPV 11 E5a expression to that of HPV 16. In contrast to HPV 16, no E5a-GFP was expressed from the HPV 11 E2,E5a construct. E1wedgeE4 protein was detected by immunofluorescence in COS-7 cells transfected with a construct that expressed E1E4 as a T7-epitope-tagged protein, and E5a as a GFP fusion. We conclude that the abundant HPV 11 E1E4,E5 transcript is a functional message that can support both E1E4 and E5a expression in eukaryotic cells.

Animals↗
PubMed1998

High-grade dysplasia in genital warts from two patients infected with the human immunodeficiency virus.

Cancer-associated human papillomavirus (HPV) types are detected in genital warts removed from immunosuppressed individuals more commonly than from those occurring in otherwise healthy individuals. The prognosis of genital warts containing cancer-associated HPV types is not known. Because it is assumed that genital warts are benign lesions, they are usually treated by destructive therapies without prior knowledge of histopathology. The aim of the present study was to determine whether genital warts from individuals with or without human immunodeficiency virus (HIV) contain high-risk HPV types or areas of dysplasia. The study design was a nonrandomized analysis of genital warts removed by excision biopsy from 15 HIV-infected patients and 15 HIV-negative patients. The tissue was analyzed for HPV DNA by hybrid capture, and microscopic sections of each biopsy were examined for areas of dysplasia. Genital warts from HIV-infected patients contained cancer-associated ("high risk") HPV types in 9 of 15 cases, including 1 that contained only a high-risk type. High-grade dysplastic abnormalities were present in 2 of the 15 lesions from this group, both of which contained high-risk HPV types. Four genital warts removed from HIV-negative patients contained high-risk HPV types, but none contained dysplastic abnormalities. It is concluded that genital warts from HIV-infected patients often contain high-risk HPV types. Such lesions may exhibit dysplastic changes. The frequency of dysplastic changes in genital warts from HIV-infected patients is not known. Biopsy of genital warts may be indicated prior to additional therapy in HIV-infected patients, and surgical removal should be considered as a preferred treatment option in these patients.

Anus Diseases↗
PubMed1998

Transient alteration in intestinal permeability to technetium Tc99m diethylenetriaminopentaacetate during the prodromal stages of alimentary laminitis in ponies.

OBJECTIVES: To determine whether mucosal permeability is altered during the prodromal stages of alimentary laminitis. ANIMALS: 15 healthy adult ponies. PROCEDURES: intestinal permeability was evaluated for control ponies (n = 5) and for ponies 4 to 12 (n = 5) and 20 to 28 (n = 5) hours after administration of carbohydrate overload. Mucosal permeability was determined by measuring the percentage of orally administered technetium Tc99m diethylenetriaminopentaacetate (99mTc-DTPA) excreted in urine during an 8-hour period, then measuring blood radioactivity at hourly intervals. Plasma endotoxin-like activity was measured by use of a chromogenic Limulus amebocyte assay. RESULTS: Urinary excretion of 99mTc-DTPA was 2.45% of administered dose for control ponies, and was 16.67% of administered dose 4 to 12 hours and 3.57% of administered dose 20 to 28 hours after administration of carbohydrate. CONCLUSIONS: A marked but transient increase in intestinal permeability was observed early in the prodromal stages of alimentary laminitis. CLINICAL RELEVANCE: Absorption of substances from the intestine may be an initiating event in alimentary laminitis.

Animals↗
PubMed1997

Vaccination with DNA encoding the immunodominant LACK parasite antigen confers protective immunity to mice infected with Leishmania major.

To determine whether DNA immunization could elicit protective immunity to Leishmania major in susceptible BALB/c mice, cDNA for the cloned Leishmania antigen LACK was inserted into a euykaryotic expression vector downstream to the cytomegalovirus promoter. Susceptible BALB/c mice were then vaccinated subcutaneously with LACK DNA and challenged with L. major promastigotes. We compared the protective efficacy of LACK DNA vaccination with that of recombinant LACK protein in the presence or absence of recombinant interleukin (rIL)-12 protein. Protection induced by LACK DNA was similar to that achieved by LACK protein and rIL-12, but superior to LACK protein without rIL-12. The immunity conferred by LACK DNA was durable insofar as mice challenged 5 wk after vaccination were still protected, and the infection was controlled for at least 20 wk after challenge. In addition, the ability of mice to control infection at sites distant to the site of vaccination suggests that systemic protection was achieved by LACK DNA vaccination. The control of disease progression and parasitic burden in mice vaccinated with LACK DNA was associated with enhancement of antigen-specific interferon-gamma (IFN-gamma) production. Moreover, both the enhancement of IFN-gamma production and the protective immune response induced by LACK DNA vaccination was IL-12 dependent. Unexpectedly, depletion of CD8(+) T cells at the time of vaccination or infection also abolished the protective response induced by LACK DNA vaccination, suggesting a role for CD8(+) T cells in DNA vaccine induced protection to L. major. Thus, DNA immunization may offer an attractive alternative vaccination strategy against intracellular pathogens, as compared with conventional vaccination with antigens combined with adjuvants.

Animals↗
PubMed1997

Functional characterization of the C---C chemokine-like molecules encoded by molluscum contagiosum virus types 1 and 2.

Many viruses have evolved mechanisms for evading the host immune system by synthesizing proteins that interfere with the normal immune response. The poxviruses are among the most accomplished at deceiving their hosts' immune systems. The nucleotide sequence of the genome of the human cutaneous poxvirus, molluscum contagiosum virus (MCV) type 1, was recently reported to contain a region that resembles a human chemokine. We have cloned and expressed the chemokine-like genes from MCV type 1 and the closely related MCV type 2 to determine a potential role for these proteins in the viral life cycle. In monocyte chemotaxis assays, the viral proteins have no chemotactic activity but both viral proteins block the chemotactic response to the human chemokine, macrophage inflammatory protein (MIP)-1alpha. Like MIP-1alpha, both viral proteins also inhibit the growth of human hematopoietic progenitor cells, but the viral proteins are more potent in this activity than the human chemokine. These viral chemokines antagonize the chemotactic activity of human chemokines and have an inhibitory effect on human hematopoietic progenitor cells. We hypothesize that the inhibition of chemotaxis is an immune evasion function of these proteins during molluscum contagiosum virus infection. The significance of hematopoietic progenitor cell inhibition in viral pathogenesis is uncertain.

Amino Acid Sequence↗
PubMed1997

A role for CD4 in peripheral T cell differentiation.

Naive CD4+ T helper cells (Th) differentiate into one of two well-defined cell types during immune responses. Mature Th1 and Th2 cells regulate the type of response as a consequence of the unique cytokines that they secrete. CD4 serves a prominent role in potentiating antigen recognition by helper T cells. We have examined the role of CD4 in peripheral T cell differentiation by studying helper T cells from mice with a congenital defect in CD4 expression. After protein immunization or infection with Leishmania major, CD4-deficient mice were incapable of mounting antigen-specific Th2 responses, but retained their Th1 potency. CD4-deficient, T cell receptor transgenic T cells were also incapable of Th2 differentiation after in vitro activation. Expression of a wild-type CD4 transgene corrected the Th2 defect of CD4-deficient mice in all immune responses tested. To investigate the role of the cytoplasmic domain, mice reconstituted with a truncated CD4 molecule were also studied. Expression of the tailless CD4 transgene could not rescue the Th2 defect of CD4-deficient mice immunized with protein or CD4-deficient transgenic T cells activated in vitro, raising the possibility that the cytoplasmic domain of CD4 may influence Th2 generation. Expression of the tailless transgene was, however, capable of restoring Th2 development in CD4-deficient mice infected with L. major or CD4-deficient transgenic T cells activated in the presence of recombinant IL-4, demonstrating that the cytoplasmic domain is not absolutely required for Th2 development. Together, these results demonstrate a previously undescribed role of the CD4 molecule. The requirement for CD4 in Th2 maturation reflects the importance of molecules other than cytokines in the control of helper T cell differentiation.

Animals↗
PubMed1997

Sympathetic nervous activity and arterial pressure responses during rest and acute behavioral stress in SHR versus WKY rats.

The object of this experiment is to compare changes in renal sympathetic nerve activity (SNA), mean arterial blood pressure (MAP) and heart rate (HR) during rest and behavioral stress in 12-14 week old spontaneously hypertensive rats (SHR; N = 12) and normotensive Wistar-Kyoto (WKY; N = 12) controls. Animals were behaviorally trained by following a 15 s auditory conditional stimulus (CS+) with a 1/2 s tail shock. Resting MAP was higher (p < 0.001) in SHR (154 +/- 3 mmHg, mean +/- SEM) compared to WKY (116 +/- 3 mmHg); conversely, there was no difference in the average resting HR. The pattern of the SNA and MAP changes during the CS+ was similar across groups, but the amplitude was larger in the SHR. The CS+ stress stimulus evoked an initial transient MAP increase averaging 14 +/- 2 mmHg in the SHR compared to 4 +/- 1 mmHg in the WKY. This pressor response was preceded by a sudden burst of SNA averaging 177 +/- 22% over baseline in SHR versus 105 +/- 13% for the WKY. HR decreased in SHR only during the second component of the CS+ trial despite the large increase in SNA. We conclude that (1) SHR have higher reactivity than WKY to stress in SNA and MAP; (2) both SHR and WKY have greater SNA and MAP responses to CS+ than CS-(i.e., the discriminative paradigm was effective); (3) control of sympathetic and parasympathetic nervous activity during sustained stress differs remarkably in hypertensive and normotensive subjects; and (4) SHR blood pressure effector mechanisms may have a higher responsiveness to sympathetic nervous activity as compared to WKY.

Animals↗
PubMed1997

T helper subset differentiation in the absence of invariant chain.

The outcome of murine infection with Leishmania major is regulated by major histocompatibility complex class II-restricted T helper cells. Invariant chain-deficient (Ii -/-) mice have impaired ability to present major histocompatibility complex class II-restricted antigens, and reduced numbers of CD4+ T cells. Despite these deficits, C57BL/6 Ii -/- mice controlled L. major infection comparably to wild-type mice. As assessed by mRNA analysis and in vitro antigen restimulation for IFN-gamma, Ii -/- mice had normal induction of Th1 subset differentiation even though antigen-dependent proliferation of their lymph node cells was substantially compromised. In addition, BALB/c Ii -/- mice exhibited a progressive course of infection and Th2 effector cell development that were comparable to that seen in wild-type BALB/c mice. We wished to determine whether this unexpected efficiency of T helper subset induction despite inefficient T cell stimulation could be modeled in vitro. In the presence of rIL-12 or rIL-4 naive parasite-specific transgenic T cells could mature into IFN-gamma-or IL-4-secreting T helper cells, respectively, even when antigen presentation was suboptimal or antigen dose was submitogenic. These experiments demonstrate that activation of T helper cells to a threshold required for IL-2 production or proliferation is not required to achieve induction of disease-regulating T helper cell effector functions, and that pathogen-associated secondary activation signals may facilitate the full differentiation of T helper subsets during limiting presentation of antigenic peptides.

Animals↗
PubMed1997

Human papillomavirus type 11 neutralization in the athymic mouse xenograft system: correlation with virus-like particle IgG concentration.

Neutralization of virus is likely to be necessary for development of an effective prophylactic vaccine against genital human papillomavirus (HPV) infection. Two New Zealand white rabbits were immunized with purified HPV type 11 (HPV 11) virions in Freund's adjuvant. An enzyme linked immunoassay (ELISA) was used to determine the quantity of IgG which recognized the HPV 11 major capsid protein (L1 protein) virus-like particles (VLPs) in the two anti-HPV 11 sera (serum A and serum B). The concentration of HPV 11 L1 VLP-specific IgG in the A and B sera were determined to be 37 and 90 micrograms per ml, respectively. The A and B sera were used in neutralization experiments in the athymic mouse xenograft system with known quantities of purified HPV 11 virions. The concentration of HPV 11 L1 VLP-specific IgG required to neutralize HPV 11 was determined for each antiserum. This concentration of IgG was approximately 700 to 900 ng per ml. This study demonstrates a positive correlation between the level of HPV 11 L1 VLP-specific IgG in animals immunized with HPV 11 virions and neutralization of HPV 11 in the athymic mouse model. Further studies are needed 1) to determine if sera or genital secretions from other species are neutralizing in the athymic mouse xenograft system, and 2) to determine if the VLP ELISA can be used as a reliable substitute for more cumbersome neutralization assays.

Animals↗
PubMed1997

Increase of intracellular free Ca2+ in microglia activated by prion protein fragment.

A synthetic peptide consisting of amino acid residues 106 to 126 of the human prion protein (PrPc) that forms fibrils in vitro is toxic to cultured neurons. We have previously shown that the neurotoxic effect of this peptide is related to microglia activation (Brown et al., 1996a). For closer insight into this process of activation, we investigated the effect of the peptide on the intracellular free Ca2+ concentration ([Ca2+]i) in cultured microglia using Fura-2. Cultured microglia from wild-type as well as from PrPc gene-ablated mice (Prn-p0/0) responded to exposure to PrP106-126 with an increase in intracellular free calcium within 30 min. We observed two types of responses. Both in wild-type and Prn-p0/0 mice about half of the tested cells presented a small and often transient calcium increase after peptide application which was found to be independent of the extracellular calcium concentration. However, a further 33% of wild-type cells showed a strong and often permanent calcium increase depending on the extracellular calcium concentration, which was only rarely observed in Prn-p0/0 cells. To determine whether the response depended on the activation state of the microglia, we also examined LPS-treated activated microglia. The character of the calcium response remained unchanged, but significantly fewer cells responded. Our findings demonstrate the earliest reaction of microglia to a PrP fragment known to date.

Amino Acid Sequence↗
PubMed1997

Prion protein-deficient cells show altered response to oxidative stress due to decreased SOD-1 activity.

The cellular function of the prion protein (PrPc), a cell surface glycoprotein expressed in neurones and astrocytes, has not been elucidated. Cell culture experiments reveal that cerebellar cells lacking PrPc are more sensitive to oxidative stress and undergo cell death more readily than wild-type cells. This effect is reversible by treatment with vitamin E. In vivo studies show that the activity of Cu/Zn superoxide dismutase is reduced in Prnp gene-ablated (Prnp0/0) mice. Constitutively high Mn superoxide dismutase activity in these animals may compensate for this loss of responsiveness to oxidative stress. These findings suggest that PrPc may influence the activity of Cu/Zn superoxide dismutase and may be important for cellular resistance to oxidative stress.

Amino Acid Sequence↗
PubMed1997

Cell death in prion disease.

Prion diseases are neurodegenerative transmissible diseases. The infectious agent, termed prion, is thought to consist of an altered host-encoded protein. The pathogenesis of these diseases which typically in a very short time lead to rampant nerve cell death and astrocytic gliosis is poorly understood. Investigations using the in situ endlabeling technique and electron microscopy in a scrapie model in the mouse (79A strain) show that nerve cell death is due to apoptosis. A cell culture model using a synthetic peptide of the prion protein (PrP106-126) shows that this peptide is toxic only to normal neurons whereas nerve cells derived from PrP knock-out (PrP0/0) mice are unaffected by this neurotoxic effect. In addition, microglia play a crucial part in this process by secreting reactive oxygen species. Experiments in animals will have to show whether these cell culture findings adequately reflect the in vivo pathogenesis.

Amino Acid Sequence↗
PubMed1997

Linkage assignment of eleven genes to the porcine genome.

We report comparative linkage mapping of eleven genes in the swine genome by RFLP analysis. These genes include: Acid phosphatase type 5 (ACP5), Cholecystokinin Type B Receptor (CCKBR), Antibiotic Peptide (FALL39), Insulin-like Growth Factor 1 Receptor (IGF1R), Integrin Alpha M (ITGAM), Integrin Beta 2 (ITGbeta2), Opioid Receptor Mu-1 (OPRM1), Pro-hormone Converter (PC1/3), Retinol Binding Protein 3 (RBP3), Ribosomal DNA (RNR1), and Zona Pellucida Glycoprotein 1 (ZP1). The CCKBR and ITGbeta2 loci define the ends of the linkage groups on Chromosomes (Chro) (SSC) 9p and 13qter, respectively.

Animals↗
PubMed1997

A novel Mycoplasma sp. associated with proliferative tracheitis and pneumonia in a Burmese python (Python molurus bivittatus).

Proliferative lymphocytic tracheitis and pneumonia were observed histologically in the respiratory tract of a captive Burmese python (Python molurus bivittatus). A mycoplasma species was isolated from the respiratory tissue. Polymerase chain reaction analysis of the 16S rRNA gene sequence of the isolate showed 0.90 similarity to Mycoplasma agassizii, an organism previously shown to cause respiratory disease in reptiles. Based on these findings, a novel Mycoplasma species was suspected to be the causative agent of respiratory disease in this snake.

Animals↗
PubMed1997

Regulation of ion transport in the porcine intestinal tract by enteric neurotransmitters and hormones.

In the present paper, the mechanisms underlying the neural and hormonal regulation of mucosal ion transport in the pig intestinal tract are reviewed. The active transport of NaCl by isolated sheets of porcine intestinal mucosa is modulated by cholinergic and non-cholinergic neurons of undetermined neurochemical identity that lie in the submucosa. The application of electrical field stimulation to mucosa-submucosa preparations from porcine jejunum, ileum, or colon produces rapid elevations in short-circuit current which are inhibited by tetrodotoxin or omega-conotoxin GVIA, blockers of neuronal Na+ and Ca2+ channels, respectively. In porcine ileum, these elevations in current are mimicked in large part by cholinergic agonists and have been attributed to anion secretion. The majority of classical neurotransmitters and gut peptides that have been examined to date increase active transepithelial anion secretion through interactions with G protein-coupled receptors associated with submucosal neurons or situated on the basolateral membranes of epithelial cells. A small number of neuropeptides interact with neuronal receptors to augment NaCl absorption or decrease anion secretion. Noradrenergic control of intestinal transport differs in the porcine small and large intestines, and displays considerable inter-species variability in its cellular underpinnings. Transport regulation by bombesin-like peptides may be mediated by receptors distributed in both the apical and basolateral membrane domains of epithelial cells in porcine colon. The transport process affected by these peptides may be linked to epithelial growth and differentiation. The pig intestinal tract appears to be a useful biological model for resolving the cellular mechanisms by which gut neurotransmitters and hormones act in regulating transepithelial ion fluxes. Its general relevance to human intestinal function is discussed.

Animals↗
PubMed1997

Effects of oxidative stress on prion protein expression in PC12 cells.

PC12 cells are known to express the prion protein, a normal cell surface glycoprotein. This protein is upregulated in PC12 cells differentiated with nerve growth factor. A neurotoxic prion protein peptide, PrP106-126, is not toxic to PC12 cells alone. PrP106-126 is toxic to PC12 cells co-cultured with microglia and more so to NGF-differentiated PC12 cells. PC12 cells selected for resistance to either copper toxicity or oxidative stress have higher levels of PrP(C) expression. Both PC12 variants are more sensitive to the toxicity of PrP106-126. This suggests that PC12 sensitivity to PrP106-126 toxicity is related to prion protein expression and not to a state of high differentiation induced by NGF. Variants of PC12 cells that are more resistant to copper toxicity have higher levels of anti-oxidant enzymes, superoxide dismutase and glutathione peroxidase. Our results suggest that cells expressing higher levels of PrP(C) have higher resistance to oxidative stress or copper toxicity but are more sensitive to PrP106-126 toxicity. Prion protein expression may be involved in both the metabolism of copper and resistance to oxidative stress. Increased cellular resistance to copper toxicity may be partly related to increased activity of anti-oxidant enzymes.

Animals↗
PubMed1997

Psychosocial and spiritual care in hospice: differences between nursing, social work, and clergy.

A chart review of the most recent home visits from nursing, social work, and clergy to 37 home hospice patients indicated that all but two psychosocial and spiritual issues examined were addressed by hospice staff on home visits. Spirituality and, secondly, death anxiety were the most frequently discussed variables. Clergy addressed more spiritual issues than the other two professions, and social work addressed more psychosocial issues than the other two professions. These findings underscore the important roles that clergy and social work play on the hospice team, and imply that hospice should make a renewed commitment to psychosocial and spiritual care.

Clergy↗