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Biomedical subjects

J A Higgins

Publications and source records attributed to J A Higgins.

At least 73 records · Page 4Linked to original sources
PubMed1994

Murine typhus: updated roles of multiple urban components and a second typhuslike rickettsia.

Studies using serologic and polymerase chain reaction-(PCR) facilitated analysis of field samples from southern Texas indicate the presence of Rickettsia typhi and ELB agent infected cat fleas, Ctenocephalides felis (Bouché), and the first observation of ELB infected vertebrates (opossums). The ELB agent is a recently described typhus-like rickettsia that is not distinguished from R. typhi or R. prowazekii by currently available serologic reagents. Restriction digests of PCR products from 399 fleas revealed an ELB agent infection rate of 3.8% and a R. typhi infection rate of 0.8%. Three of nine tested opossums (Didelphis virginiana) were shown to harbor ELB agent infections. No R. typhi infected rats, Rattus norvegicus, or rat-fleas, Xenopsylla cheopis Rothschild, were detected among surveyed samples. The persistence of this murine typhus disease focus appears to be better accounted for by the presence of infected cat fleas, opossums, and other non-rat hosts found in close association with human populations. Involvement of the ELB agent in the biology of murine typhus is suggested by its prevalence among suspected vectors and reservoir hosts.

Animals↗
PubMed1994

Molecular identification of rickettsia-like microorganisms associated with colonized cat fleas (Ctenocephalides felis).

Cat fleas (Ctenocephalides felis) from eight commercial flea colonies from various regions of the USA were examined by selective PCR amplification, and subsequent restriction digest analysis and Southern hybridization of PCR products, for the presence of a rickettsia-like organism (ELB agent). These flea colonies were either started with fleas from one supplier (EL Labs), in which ELB agent was first identified, or were started with fleas from stray cats and dogs and later came into contact with ELB-infected fleas. Infection rates in the colonies ranged from 43% to 93%. The successful propagation of ELB agent in these colonies may be due to efficient trans-stadial and transovarial transmission. While ELB agent has recently been identified in blood from human murine typhus cases, attempts to infect mammalian cells and SCID mice with flea isolates were unsuccessful.

Animals↗
PubMed1994

Apolipoprotein[a] is not associated with apolipoprotein B in human liver.

The aim of this research was to determine whether apolipoprotein[a] (apo[a]) is linked to apolipoprotein B (apoB) in human liver. Four ELISAs were developed: 1) a competition assay that measures apoB; 2) a competition assay that measures apo[a]; 3) a capture assay based on capture of apo[a] by a polyclonal antibody and detection of co-immobilized apoB using a monoclonal antibody; and 4) a capture assay based on capture of apo[a] using a polyclonal antibody and detection of immobilized apo[a] using a monoclonal antibody. Assays 2 and 4, therefore, measure apo[a] either free or in complex with other proteins, while assay 3 measures apo[a] associated with apoB. The levels of apo[a] ranged from 25 to 440 micrograms/g liver in nine individual liver samples. There was no significant difference between apo[a] levels in individual human liver samples measured using ELISA 1 or 3; however, it was not possible to detect apo[a]/apoB using assay 3. ApoB was present in human liver homogenates at levels ranging from 90 to 700 micrograms/g measured using assay 1. These results suggest, therefore, that apo[a] is not coupled to apoB in the liver and may be secreted in the free form to bind with low density lipoprotein (LDL) in the extracellular fluid or plasma.

Apolipoproteins A↗
PubMed1993

Transit and sorting of apolipoprotein B within the endoplasmic reticulum and Golgi compartments of isolated hepatocytes from normal and orotic acid-fed rats.

Apolipoprotein B (apoB) secretion by isolated rat hepatocytes was dependent on addition of oleate to the incubation medium and inhibited in hepatocytes isolated from livers of orotic acid-fed rats (OA hepatocytes). To investigate the intracellular transit of newly synthesized apoB under different conditions, normal hepatocytes (with or without oleate) or OA hepatocytes (with oleate) were incubated with [35S]methionine, and subcellular fractions (rough endoplasmic reticulum, smooth endoplasmic reticulum, cis-Golgi, and trans-Golgi and membrane and lumenal contents from these) were isolated at intervals. The specific activities and pool sizes of apoB100 and apoB48 were determined. The observations indicate that there are several points at which intracellular transit of apoB is regulated. Newly synthesized apoB is either translocated to the lumen of the rough endoplasmic reticulum or remains membrane bound and is degraded. The lumenal apoB is either retained and degraded or transferred to the Golgi lumen and secreted. In OA hepatocytes degradation of the membrane-bound form of apoB is inhibited, and the protein accumulates in the trans-Golgi membranes. Although apoB is translocated to the lumen of the rough endoplasmic reticulum in OA hepatocytes, it is not packaged with lipid and is transferred to the Golgi lumen only slowly.

Animals↗
PubMed1993

Clonal analysis of the atopic immune response to the group 2 allergen of Dermatophagoides spp.: identification of HLA-DR and -DQ restricted T cell epitopes.

The group 2 allergens of Dermatophagoides spp. (house dust mite, HDM) are a major immunological target for IgE antibodies in the allergic immune response of HDM atopic individuals. In this report the heterogeneity of the T cell repertoire reactive with the group 2 allergen of Dermatophagoides pteronyssinus (Der p 2) of a HDM allergic individual was investigated using overlapping synthetic peptides. By clonal analysis four distinct T cell epitopes were identified, located within residues 16-31, 22-40, 82-100, and 111-129. The importance of these epitopes was confirmed by investigation of the peripheral T cell repertoire, with the polyclonal T cell response to Der p 2 failing to show marked variations in epitope specificity over time. Serological inhibition studies and the use of Epstein-Barr virus transformed B cell lines characterized for their expression of HLA-D region gene products demonstrated that recognition of peptides 16-31 and 111-129 was restricted by HLA-DQ (DQB1*0301), whereas peptide 82-100 is recognized in association with HLA-DR (DRB1*1101). Peptide 22-40 was presented by both HLA-DRB1*1101 and -DQB1*0301 class II molecules. The potential application of these findings lies in the design of peptide-based immunotherapeutics for the management of HDM allergic disease.

Amino Acid Sequence↗
PubMed1993

Immunological effects of intermediate purity clotting factor concentrates: failure to affect lymphocyte activation in vivo.

Concern has been expressed that intermediate purity clotting factor concentrates may cause immunological abnormalities in haemophilic patients, distinct from those related to HIV infection. Early reports of lymphocyte dysfunction in anti-HIV seronegative haemophiliacs pointed to activation of their lymphocytes; a potential cause of CD4+ ve lymphocyte decline in anti-HIV seropositive patients. Recent reports have suggested that the use of high purity FVIII concentrates might retard the rate of decline in CD4+ ve lymphocytes in haemophiliacs infected with the HIV virus. Expression of markers of acute and chronic activation of T and B lymphocytes was measured in heavily treated anti-HIV seronegative haemophiliacs using two-colour flow cytometry. No T or B lymphocyte stimulation was observed. Cellular markers of activation were absent and CD4+ ve lymphocyte counts and serum IgG levels were normal. Anti-HIV seropositive haemophiliacs showed T and B cell activation consistent with HIV infection. The extent of lymphocyte activation in individual patients was unrelated to the type, amount or frequency of FVIII received. These findings do not support the hypothesis that lymphocytes of haemophiliacs are affected directly by the regular administration of intermediate purity concentrates so as to accelerate the progression of HIV disease.

Adolescent↗
PubMed1993

Topography of apolipoprotein B in subcellular fractions of rabbit liver probed with a panel of monoclonal antibodies.

We have used a panel of anti-rabbit apolipoprotein B monoclonal antibodies in a competitive ELISA to probe the availability of apoB in rough endoplasmic reticulum, smooth endoplasmic reticulum, cis-enriched Golgi, and trans-enriched Golgi fractions from rabbit liver. The ability of each subcellular fraction to inhibit binding of monoclonal antibody to immobilized low density lipoprotein (LDL)-apoB was determined and compared with the expected inhibition based on the apoB content of the fraction. The vesicles remained closed during ELISA, demonstrated by monitoring loss of radiolabeled secretory proteins from the lumen and by measuring leakage of albumin from the vesicles. In control experiments, vesicles were permeabilized using 0.4% taurocholate. All epitopes of apoB were fully expressed in closed trans-Golgi vesicles, indicating that the membrane-bound apoB is at the cytosolic side of this fraction. In the smooth endoplasmic reticulum the epitopes were expressed between 55 and 70%, suggesting that the two pools of apoB may exist in these membranes. These results suggest that newly synthesized apoB has two possible fates. It may be incorporated into the cytosolic side of the endoplasmic reticulum from where it moves to the cytosolic side of the Golgi membrane, or newly synthesized apoB may be translocated to the lumenal surface of the endoplasmic reticulum membrane followed by assembly with lipids for secretion.

Animals↗
PubMed1992

Determination of the intracellular distribution and pool sizes of apolipoprotein B in rabbit liver.

We have investigated the intracellular distribution of apolipoprotein B (apo B) in rabbit liver by immunoblotting, radioimmunoassay (r.i.a.) and enzyme-linked immunoassay (e.l.i.s.a.). Apo B100 was detected in total microsomes, rough microsomes, smooth microsomes, trans-enriched Golgi and cis-enriched Golgi and membrane and cisternal-content subfractions prepared from these fractions. There was also evidence of degradation of apo B100 in the Golgi membrane fractions. The amount of apo B in the subcellular fractions detected by competitive r.i.a. or e.l.i.s.a. ranged from 1.5 micrograms/mg of protein in the rough endoplasmic reticulum to 13 micrograms/mg of protein in the trans-Golgi fraction. Using internal standards (NADPH-cytochrome c reductase for the endoplasmic reticulum and galactosyltransferase for the Golgi membranes) it was calculated that all the apo B of liver is recovered within the secretory compartment, with 63% of the total apo B in the endoplasmic reticulum and the remainder in the Golgi. When the subcellular fractions were separated into membranes and cisternal contents, 60%, 50%, 60% and 30% of the total apo B was recovered in the membrane of the rough microsomes, smooth microsomes, cis-Golgi and trans-Golgi respectively. Using competitive e.l.i.s.a. we found that the membrane-bound form of the apo B was exposed at the cytosolic surface of the intact subcellular fractions. These observations are consistent with a model for assembly of very-low-density lipoproteins (VLDL) in which newly synthesized apo B is incorporated into a membrane-bound pool and a lumenal pool. The membrane-bound pool not used for VLDL assembly may be degraded, possibly in the Golgi region.

Animals↗
PubMed1992

Quantification of apolipoprotein B-48 and B-100 in rat liver endoplasmic reticulum and Golgi fractions.

We have developed a method for measurement of apolipoprotein (apo) B-48 and apo B-100 in blood and subcellular fractions of rat liver based on SDS/PAGE followed by quantitative immunoblotting using 125I-Protein A. Standard curves were prepared in each assay using apo B prepared from total rat lipoproteins by extraction with tetramethylurea. Subcellular fractions (rough and smooth endoplasmic reticulum and Golgi fractions) were prepared from rat liver and separated into membrane and cisternal-content fractions. For quantification, membrane fractions were solubilized in Triton X-100, and the apo B was immunoprecipitated before separation by SDS/PAGE and immunoblotting. Content fractions were concentrated by ultrafiltration and separated by SDS/PAGE without immunoprecipitation. Quantification of apo B in subcellular fractions and detection of apo B by immunoblotting yielded consistent results. In all fractions apo B-48 was the major form, accounting for approximately three-quarters of the total apo B. By using marker enzymes as internal standards, it was calculated that all of the apo B was recovered in the endoplasmic reticulum and Golgi fractions, with approximately 80% of each form of apo B in the endoplasmic reticulum. More than 90% of the apo B of the rough- and smooth-endoplasmic-reticulum fractions was membrane-bound, whereas approx. 33 and 15% of the apo B of the cis-enriched Golgi fractions and trans-enriched Golgi fractions respectively were membrane-bound.

Animals↗
PubMed1992

Membrane-bound apolipoprotein B is exposed at the cytosolic surface of liver microsomes.

We have used a competitive enzyme-linked immunoassay with a panel of monoclonal antibodies to probe the topography of the membrane-bound form of apolipoprotein B (apo B) in rabbit microsomes. All epitopes investigated were found to be expressed at the cytosolic side of the microsomal membrane under conditions in which the vesicles remained sealed. These results indicate that the membrane-associated form of apolipoprotein B is either at the cytosolic side of the endoplasmic reticulum membrane or integrated into the membrane. From this site apo B may be translocated to the lumen for assembly into VLDL or may be degraded.

Animals↗
PubMed1992

Peptide-induced nonresponsiveness of HLA-DP restricted human T cells reactive with Dermatophagoides spp. (house dust mite).

The activation of CD4+ T lymphocytes, which play a central role in allergic inflammation, depends on the recognition of allergen-derived peptides in association with major histocompatibility complex class II gene products. In this report we demonstrate, at a clonal level, that a component of the T-cell repertoire reactive with Dermatophagoides spp. (house dust mite) in atopic individuals, is restricted by HLA-DP class II molecules. This supports the recent results emerging from genetic epidemiologic studies that indicate positive associations between the HLA-DP phenotype and immune responsiveness to a variety of common allergens. Our findings also reveal that the T cells restricted by HLA-DP recognize a species-specific epitope located in the group I allergen of Dermatophagoides pteronyssinus (residues 101-119). Furthermore, we report that the pretreatment of the T cells restricted by HLA-DP with the Der p I peptide renders them nonresponsive to an immunogenic challenge with house dust mite allergen, and the loss of antigen-dependent proliferation is associated with downregulation of membrane expression of the T-cell antigen receptor. The ability to functionally inactivate T cells restricted by HLA-DP, as well as those that recognize allergen in association with HLA-DR class II molecules, suggests that desensitization with allergen-derived peptides may have therapeutic potential in the management of allergic diseases irrespective of their HLA class II association.

Animals↗