Search PubMed⌕ Search

Biomedical subjects

R Holmes

Publications and source records attributed to R Holmes.

At least 55 records · Page 3Linked to original sources
PubMed1996

Long-term dual perfusion of isolated human placental lobules with improved oxygenation for infectious diseases research.

An improved method for long-term perfusion of the isolated human term placental lobule has been developed to investigate the maternofetal transfer of infectious agents, in particular the human immunodeficiency virus (HIV). The purpose of this paper is to describe those modifications that allow for substantially prolonged perfusions in in a biohazard environment. The method described has been adapted from previous models. The perfusion apparatus has been modified for use within a biohazard hood, and, intravenous bags contain the medium for circulation of perfusates in closed circuits. A Mera Silox-S 0.3 membrane oxygenator delivers more oxygen to the tissue, and, Electromedic Cardioplegia heat exchangers warm the perfusate prior to oxygenation. Viability criteria (glucose consumption, lactate production, de novo production of human placental lactogen (hPL), volume loss, flow, temperature, pressure, oxygen transfer, carbon dioxide production, absence of IgM transfer and light and electron microscopy) demonstrate that the placental tissue remains in a functional state throughout the perfusion. Oxygen and glucose consumption are both stable over time; lactate levels remain constant; and hPL continues to be produced. These significant modifications of the perfusion system have permitted the investigators to increase the duration of perfusion to 48 h while preserving normal metabolic function of ultrastructurally intact tissue as demonstrated by ultra structural observations. This perfusion model device provides biohazard precautions and may be applied to other studies of placental physiology.

Carbon Dioxide↗
PubMed1996

Induction of RANTES expression by astrocytes and astrocytoma cell lines.

The cellular infiltrate found during the acute phase of multiple sclerosis (MS) consists of monocytes and activated T cells, suggesting the presence of cell-specific chemotactic signals during the inflammatory response. We examined the ability of human astrocytoma cell lines, as well as primary human and rat astrocytes, to generate a specific member of the intercrine/chemokine family of cytokines, RANTES, when exposed to TNF-alpha, IL-1 beta and IFN-gamma. Astrocytoma cells as well as primary astrocytes produced RANTES upon incubation with TNF-alpha or IL-1 beta. IFN-gamma alone did not induce RANTES production by astrocytes, but it potentiated the effects of either TNF-alpha or IL-1 beta. Induction of RANTES by TNF-alpha was mediated by the p55 receptor since a specific anti-p55 antiserum mimicked the effect of TNF-alpha. These results indicate that human astrocytes are capable of generating a cell-specific chemokine that can account for the inflammatory cellular infiltrate observed during the acute phase of MS, in a process that is regulated by cytokines.

Animals↗
PubMed1996

Non-invasive prenatal diagnosis by isolation of both trophoblasts and fetal nucleated red blood cells from the peripheral blood of pregnant women.

OBJECTIVE: To isolate fetal trophoblasts and nucleated red blood cells from the peripheral blood of pregnant women. DESIGN: Trophoblasts were isolated from whole blood of women in the first trimester of pregnancy by a specific monoclonal antibody, 340. Nucleated red blood cells were isolated by separating whole blood on a triple gradient, staining with ferromagnetic particles coated with an antitransferrin monoclonal antibody and separated on a mini magnetic activated cell sorting (MACS) column. Sorted cells were sexed using a nested polymerase chain reaction for a specific sequence on the Y chromosome and the sex was confirmed by karyotyping of chorionic villus samples. PARTICIPANTS: Patients between 10 and 14 weeks of pregnancy who were undergoing elective chorionic villus sampling for the detection of fetal aneuploidies. MAIN OUTCOME MEASURE: Fetal sex determined by polymerase chain reaction on fetal cells sorted from maternal blood. RESULTS: When both trophoblasts and nucleated red blood cells were sorted, fetal sex was correctly predicted in 12/13 cases (92%), which included correct diagnosis of five of six male pregnancies. More importantly the two techniques were complementary, with only one male pregnancy being diagnosed on both trophoblasts and nucleated red blood cells, two being detected only with trophoblasts and two on nucleated red blood cells alone. No false positives (male signal from a female pregnancy) were diagnosed with either trophoblasts or nucleated red blood cells even with the highly sensitive nested polymerase chain reaction technique, which is very prone to contamination. This study also shows that it is possible to isolate both trophoblasts and nucleated red blood cells from the same sample of maternal blood. CONCLUSION: Fetal cells can be isolated from maternal blood at around 10 weeks of pregnancy.

Aneuploidy↗
PubMed1996

Cranial bone grafting: biodegradable versus titanium fixation in a rabbit model.

Rigid internal fixation with miniplates and screws has enabled reconstructive surgeons to position bony segments precisely. Unfortunately, our present systems have been associated with a potential adverse effect on ultimate craniofacial growth and development. Also, they may be palpable, necessitating secondary procedures for removal of the hardware. We have been involved in an ongoing study evaluating the efficacy of a commercially developed biodegradable system. In this study, we compared biodegradable lag screw fixation with traditional titanium screw fixation in full-thickness, membranous bone grafts harvested from the zygomatic arches to the snout in a rabbit model. The results were compared with standard hematoxylin and eosin stains and laser densometry. Although this study adds further credence to the resorption of bone grafts, those secured with the polyglycolic acid screws demonstrated significantly less resorption than those maintained in position by traditional methods.

Animals↗
PubMed1995

Signal transduction by the formyl peptide receptor. Studies using chimeric receptors and site-directed mutagenesis define a novel domain for interaction with G-proteins.

The binding of small peptide ligands to high affinity chemoattractant receptors on the surface of neutrophils and monocytes leads to activation of heterotrimeric G-proteins, stimulation of phosphatidylinositol-phospholipase C (PI-PLC), and subsequently to the inflammatory response. It was recently shown (Amatruda, T. T., Gerard, N. P., Gerard, C., and Simon, M. I. (1993) J. Biol. Chem. 268, 10139-10144) that the receptor for the chemoattractant peptide C5a specifically interacts with G alpha 16, a G-protein alpha subunit of the Gq class, to trigger ligand-dependent stimulation of PI-PLC in transfected cells. In order to further characterize this chemoattractant peptide signal transduction pathway, we transfected cDNAs encoding the formylmethionylleucylphenylalanine receptor (fMLPR) into COS cells and measured the production of inositol phosphates. Ligand-dependent activation of PI-PLC was seen in COS cells transfected with the fMLPR and G alpha 16 and stimulated with fMLP but not in cells transfected with receptor alone or with receptor plus G alpha q. Chimeric receptors in which the N-terminal extracellular domain, the second intracellular domain, or the intracellular C-terminal tail of the fMLP receptor was replaced with C5a receptor domains (Perez, H. D., Holmes, R., Vilander, L. R., Adams, R. R., Manzana, W., Jolley, D., and Andrews, W. H. (1993) J. Biol. Chem. 268, 2292-2295) were capable of ligand-dependent activation of PI-PLC when co-transfected with G alpha 16. A chimeric receptor exchanging the first intracellular domain of the fMLPR was constitutively activated, stimulating PI-PLC in the absence of ligand. Constitutive activation of PI-PLC, to a level 233% of that seen in cells transfected with wild-type fMLP receptors, was dependent on G alpha 16. Site-directed mutagenesis of the first intracellular domain of the fMLPR (amino acids 54-62) reveals this to be a domain necessary for ligand-dependent activation of G alpha 16. These results suggest that different receptors which mediate similar biochemical responses may utilize distinct mechanisms to activate G-proteins. Differences among the signaling pathways triggered by chemoattractant factor receptors suggest an opportunity for pharmacologic modifications of the inflammatory response.

Antineoplastic Agents↗
PubMed1995

Molecular epidemiology of hepatitis C virus infection amongst intravenous drug users in rural communities.

The prevalence of hepatitis C virus (HCV) infection amongst a group of intravenous drug users (IVDUs) resident in West Suffolk (East Anglia, England) was investigated and compared with the prevalence of infection with hepatitis B virus (HBV) and human immunodeficiency virus (HIV). In addition, both the level of HCV persistence, as defined by detection of viral RNA, and the HCV genotypes present in this population were determined. It was found that HCV antibodies were present in 59% of those tested; by comparison 22% had antibodies to HBV and 1% antibodies to HIV. HCV RNA was found in 44% of those with HCV antibody. HCV genotype 1 was the most prevalent within this population although both genotypes 2 and 3 were also represented.

Adolescent↗
PubMed1995

Characterization of microvillar membrane proteins of dog small intestine by two-dimensional electrophoresis.

A method for analysing microgram amounts of microvillar membranes by two-dimensional electrophoresis (protein mapping) is described, and has been used to characterize the microvillar proteins of the small intestine of German shepherd, corgi, and beagle dogs. Detergent-solubilized microvillar membranes were radiolabelled with 14C and separated by isoelectric focussing followed by SDS-PAGE. Proteins were detected fluorographically and glycoproteins by lectin-affinity staining. The microvillar hydrolases alkaline phosphatase and dipeptidyl aminopeptidase IV were identified by active-site labelling and aminopeptidase N by immunoprecipitation. Changes following pancreatic duct diversion were consistent with accumulation of pro-sucrase-isomaltase and diminished expression of the sucrase and isomaltase subunits. Cytoskeletal proteins were concentrated in the core fraction remaining after extraction of microvillar membranes with Triton X-100. There were no consistent differences between dogs of different breed, and the canine protein maps were similar to the human.

Alkaline Phosphatase↗
PubMed1995

Comparisons of risk factors for HIV-1 infection in Jefferson and Mobile County, Alabama.

A study of the Alabama state AIDS database was conducted to determine whether differences exist in demographic and risk characteristics between patients with HIV-1 in Jefferson and Mobile County. The authors found that the age distribution of patients with HIV-1, the percent of those having AIDS, and the percent of those surviving were very similar. However, significant differences existed in patient-reported risk factors in the two counties. Homosexuality was reported as the major risk factor in both counties. However, there was proportionately more homosexuality reported in Jefferson County and, conversely, more heterosexuality reported in Mobile. There also were significant differences in race and gender distributions in the two counties. This was due in part to the proportionately higher prevalence of African American females of reproductive age with HIV-1 in Mobile County. This may pose a significantly greater risk for pediatric AIDS among African American females in Mobile County.

Acquired Immunodeficiency Syndrome↗
PubMed1995

Deprivation and the 1991 census.

"This article looks at the patterns of relative deprivation in different types of area [in England and Wales], explores the connection between deprivation and other variables which might indicate disadvantage, and suggests some further lines of research which might be explored. It explains, in an inset, how to go about calculating an index of deprivation using a Census CD-ROM and a spreadsheet."

Censuses↗
PubMed1994

Human formyl peptide receptor ligand binding domain(s). Studies using an improved mutagenesis/expression vector reveal a novel mechanism for the regulation of receptor occupancy.

Recently, we reported the domain requirements for the binding of formyl peptide to its specific receptor. Based on experiments using receptor chimeras, we also postulated an importance for the amino-terminal domain of the receptor in ligand binding (Perez, H. D., Holmes, R., Vilander, L., Adams, R., Manzana, W., Jolley, D., and Andrews, W. H. (1993) J. Biol. Chem. 268, 2292-2295). We have begun to perform a detailed analysis of the regions within the formyl peptide receptor involved in ligand binding. To address the importance of the receptor amino-terminal domain, we substituted (or inserted) hydrophilic sequences within the amino-terminal domain, expressed the receptors, and determined their ability to bind ligand. A stretch of nine amino acids next to the initial methionine was identified as crucial for receptor occupancy. A peptide containing such a sequence specifically completed binding of the ligand to the receptor. Alanine screen mutagenesis of the second extracellular domain also identified amino acids involved in ligand binding as well as a disulfide bond (Cys98 to Cys176) crucial for maintaining the binding pocket. These studies provide evidence for a novel mechanism involved in regulation of receptor occupancy. Binding of the ligand induces conformational changes in the receptor that result in the apposition of the amino-terminal domain over the ligand, providing a lid to the binding pocket.

Amino Acid Sequence↗
PubMed1993

Formyl peptide receptor chimeras define domains involved in ligand binding.

We have begun to study the structural requirements for the binding of formyl peptides to their specific receptors. As an initial approach, we constructed C5a-formyl peptide receptor chimeras. Unique (and identical) restriction sites were introduced within the transmembrane domains of these receptors that allowed for the exchange of specific areas. Four types of chimeric receptors were generated. 1) The C5a receptor was progressively substituted by the formyl peptide receptor. 2) The formyl peptide receptor was progressively substituted by the C5a receptor. 3) Specific domains of the C5a receptor were substituted by the corresponding domain of the formyl peptide receptor. 4) Specific domains of the formyl peptide receptor were replaced by the same corresponding domain of the C5a receptor. Wild type and chimeric receptors were transfected into COS 7 cells and their ability to bind formyl peptide determined, taking into account efficiency of transfection and expression of chimeric protein. Based on these results, a ligand binding model is presented in which the second, third, and fourth extracellular (and/or their transmembrane) domains together with the first transmembrane domain form a ligand binding pocket for formyl peptides. It is proposed that the amino-terminal domain plays a role by presumably providing a "lid" to the pocket. The carboxyl-terminal cytoplasmic tail appears to modulate ligand binding by regulating receptor affinity.

Base Sequence↗
PubMed1993

Evaluation of liquid yeast-derived sucrase enzyme replacement in patients with sucrase-isomaltase deficiency.

BACKGROUND: No enzyme replacement therapy exists for patients with congenital sucrase-isomaltase deficiency (CSID). A by-product of the manufacture of baker's yeast is a liquid preparation containing high sucrase activity. The aim of the present study was to investigate the activity and stability of this preparation and its effect on breath hydrogen excretion and gastrointestinal symptoms after sucrose ingestion in 14 patients with CSID. METHODS: The homogeneity of yeast sucrase was studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and its activity was measured. Stability at various temperatures and pH ranges and in the presence of gastric aspirate, pepsin, and bovine serum albumin was assessed. Fourteen patients with CSID underwent double-blind placebo-controlled breath tests with yeast sucrase. They then completed an 8-week dose response study that used different enzyme concentrations while consuming a sucrose-containing diet. RESULTS: Liquid yeast sucrase is highly glycosylated, contains no lactase activity, and is stable at 4 degrees C and over a wide range of pH. Pepsin digestion of the enzyme in vitro can be blunted by bovine serum albumin and by increasing the pH. Yeast sucrase reduces breath hydrogen excretion in patients with CSID who are given a sucrose load (P < 0.001) and allows most patients to consume a sucrose-containing diet. CONCLUSIONS: Liquid yeast sucrase offers effective enzyme replacement therapy for patients with CSID.

Administration, Oral↗
PubMed1993

Immunoglobulin secretion by the normal and the infected newborn infant.

The development of quantitative single-cell immunoassays has provided a novel opportunity to demonstrate the isotype-specific immunoglobulin responses in normal and infected neonates. The reverse enzyme-linked immunospot assay was used to determine the number of immunoglobulin-secreting cells (IgSCs) in peripheral blood. Baseline numbers of IgSCs were established in 69 uninfected term and preterm infants within 5 days of birth; values above the 99th percentile were considered elevated. The IgSCs were also measured in 266 infants with proved or suspected infections or congenital anomalies. A subset of newborn infants was retested weekly. Few IgSCs (mostly IgMSCs) were detected within 5 days of birth in uninfected neonates, but by 1 month 77% had increased numbers of IgSCs, primarily IgASCs. Sixty-three (24%) of 266 study infants had increased IgSCs on initial sampling (predominantly IgMSCs); these included infants as immature as 25 to 27 weeks of gestational age; elevations in IgSCs were most frequent in infants with intrauterine infections. Increased numbers of IgSCs were uncommon in infants with early-onset sepsis in the first 5 days but were frequent by the second week, consistent with acquisition of infection near the time of delivery. We conclude that the presence of elevated numbers of IgSCs soon after birth may be a useful surrogate marker of untreated intrauterine infection. The development of predominantly IgASCs in the first month of life suggests postnatal exposure to common mucosal antigens.

Antibody-Producing Cells↗
PubMed1993

Alcohol dehydrogenases: gene multiplicity and differential functions of five classes of isozymes.

Mammalian alcohol dehydrogenases (ADHs) constitute an enzyme family of multiple forms (isozymes) which are differentially distributed throughout the body. Subunit types alpha, beta and gamma in dimeric combinations constitute the isozymes of human liver class I ADH, and are >94% homologous in structure. Human pi and chi subunits form homodimeric Class II and III ADH isozymes. pi-ADH is liver specific whereas chi-ADH is widely distributed throughout the body. A sixth human ADH subunit (designated mu or sigma), forming a new dimeric human stomach ADH, has been recently reported as Class IV ADH. Evidence for a seventh human ADH subunit has also been described, designated as Class V, the transcripts having been reported in the stomach and liver. All five classes of ADH represent isozymes which are homologous but exhibit at least 30% sequence differences in primary srtructure. Kinetic analyses of four of these classes of ADH indicated differential functions, serving either in the oxidative or reductive mode. Studies from various laboratories indicate the following respective functions: oxidation of aliphatic and aromatic alcohols-liver Class I and Class II, and stomach Class IV ADHs; reduction of peroxidic aldehydes-Classes I, II and IV; 'biogenic' alcohol oxidation-Classes I and II; and glutathione-dependent formaldehyde dehydrogenase-Class III.

Journal Article↗