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Biomedical subjects

J A Higgins

Publications and source records attributed to J A Higgins.

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

Acquisition of the cat scratch disease agent Bartonella henselae by cat fleas (Siphonaptera:Pulicidae).

We assayed the ability of cat fleas to become infected with Bartonella henselae, using an artificial feeding device. Fleas fed a concentration of 1 x 10(5) cfu/ml in blood were examined using immunofluorescent antibody assay and polymerase chain reaction. Bacteria were present in the gut at 3 h, and persisted up to 9 d after infection. Qualitatively, the density of B. henselae was greater in the flea gut at 9 d, indicating that replication was occurring in the gut. B. henselae also was detected in the feces of infected fleas 9 d after infection, and produced viable colonies upon inoculation onto heart infusion agar/rabbit blood plates. Our results indicate that fleas can maintain infection with B. henselae, and may play a role in the transmission of this bacterium from infected cats to humans.

Animals↗
PubMed1996

Amylopectin starch induces nonreversible insulin resistance in rats.

Starches that are high in amylopectin are digested and absorbed more quickly than starches with a high amylose content and produce insulin resistance in rats during long-term feeding. The aim of this study was to determine whether amylopectin-induced insulin resistance could be prevented or reversed by a period of high amylose feeding. We employed a randomized design in which two groups of rats were fed either the high amylose and then the high amylopectin diet for two consecutive 8-wk periods or vice versa (high amylopectin and then high amylose). Four other groups were fed either a high amylose or a high amylopectin diet for 8 or 16 wk. All rats were fed two 10-g meals per day (300 kJ/d), and insulin sensitivity was assessed by intravenous glucose tolerance test (IVGTT) after 8 or 16 wk of feeding. We found no difference in glucose tolerance between any group at any time point. Insulin responses, however, were 50% higher (P < 0.01) after 16 wk of high amylopectin feeding [area under the plasma insulin curve (AUC) = 18.1 +/- 1.4 nmol.L-1 x 15 min] compared with high amylose feeding (AUC = 13.0 +/- 1.2 nmol.L-1 x 15 min). The two groups which received both diets developed a similar degree of insulin resistance, equivalent to that after 16 wk of high amylopectin feeding. The findings suggest that amylopectin-induced insulin resistance cannot be reversed or prevented by either a subsequent or previous period of amylose feeding. Taken together, the data suggest that the nature of starch in the Western diet influences the development of noninsulin-dependent diabetes mellitus in humans.

Amylopectin↗
PubMed1996

Development of insulin resistance in the rat is dependent on the rate of glucose absorption from the diet.

The effect of long-term consumption of diets of different carbohydrate composition was investigated be feeding rats for up to 52 wk on diets in which the carbohydrate was either glucose, amylose or amylopectin. A glucose-based diet was included to examine the relationship between the rate of carbohydrate absorption from the diet and the development of insulin resistance. Insulin sensitivity was assessed by subjecting animals to an intravenous glucose tolerance test (IVGTT). Amylopectin-fed animals became progressively insulin resistant from 12 to 26 wk of feeding. The area under the plasma insulin curves in response to a glucose load (IVGTT) for these animals rose progressively from 15.1 +/- 2.5 nmol/L.30 min at 8 wk to 45.8 +/- 3.5 nmol/L.30 min (P < 0.001) at 26 wk of feeding. Amylose-fed animals did not exhibit insulin resistance until 26 wk of feeding when insulin secretion in response to a glucose load was 28.3 +/- 0.9 vs. 14.6 +/- 3.2 nmol/L.30 min at 16 wk of feeding (P < 0.005). Glucose-fed animals displayed insulin resistance after only 8 wk of feeding. At this time, the area under their plasma insulin curves was almost double that for amylose- or amylopectin-fed animals (P < 0.001). We conclude that long-term consumption of a diet in which available carbohydrate is rapidly absorbed causes insulin resistance in rats. The more rapidly glucose is absorbed from the diet, the faster the insulin resistance develops.

Absorption↗
PubMed1996

Rickettsia felis: a new species of pathogenic rickettsia isolated from cat fleas.

A flea-borne rickettsia, previously referred to as ELB, has been implicated as a cause of human illness. Using sequence data obtained from a fragment of the citrate synthase gene, we compared ELB, Rickettsia australis, R. rickettsii, and R. akari with the louse-borne R. prowazekii. We tallied 24 base pair differences between ELB and R. prowazekii and 25 between R. rickettsii and R. prowazekii; there were 30 base pair differences between R. australis and R. prowazekii and 29 between R. akari and R. prowazekii. We observed 32 differences between Rickettsia typhi and ELB. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analyses of ELB, with typing sera against R. typhi indicate that ELB surface antigens are more closely related to the flea-borne R. typhi than to the mite-borne R. akari. On the basis of the results of citrate synthase gene sequence comparisons, as well as previous comparisons with 16S rRNA and 17-kDa-protein gene segments, we found that ELB is sufficiently genetically distinct from other rickettsiae to be designated a new species, Rickettsia felis.

Animals↗
PubMed1996

Investigation of the association of intracellular apolipoprotein (a) with apolipoprotein B in human liver.

Plasma lipoprotein-a(Lp-a) consist of LDL-like particles in which apolipoprotein-a (apo-a) is linked by a disulphide bond to apolipoprotein B (apo-B). There is strong evidence that apo-a is synthesized in the liver. However, little is known of the intracellular transit of apo-a and the site and mechanism of its linkage to apo-B. In this investigation we have addressed the primary question--does apo-a become linked covalently to apo-B intracellularly in human liver? For this study, we have developed competition and capture ELISA to measure apo-a either free or in complex with apo-B and applied these to human liver samples. The levels of free apo-a ranged from 25 to 440 micrograms/g liver in nine individual liver samples; those of apo B ranged from 90-700 micrograms/g liver. However, it was not possible to detect an apo-a/apo-B complex suggesting that apo-a is secreted in the free form and binds with apo-B/LDL in the extracellular fluid or plasma.

Apolipoproteins↗
PubMed1995

Intracellular events in the assembly of very-low-density-lipoprotein lipids with apolipoprotein B in isolated rabbit hepatocytes.

Isolated rabbit hepatocytes incorporated [35S]methionine into cellular and secreted apolipoprotein B (apo-B), and [3H]glycerol into cellular and secreted triacylglycerol and phospholipids. Newly synthesized apo-B was incorporated into rough endoplasmic reticulum (RER), smooth endoplasmic reticulum (SER), cis-Golgi and trans-Golgi membranes and was preferentially transferred into the lumen of the RER with specific radioactivities ten times those in the membrane. Radiolabelled apo-B did not equilibrate with pre-existing unlabelled apo-B, and pools of different specific radioactivities were established in different subcellular fractions. Only a small fraction of the newly synthesized apo-B was transferred to the Golgi lumen. In pulse-chase experiments, most of the newly synthesized apo-B in the RER membrane and the RER lumen was degraded. [3H]Glycerol was incorporated into triacylglycerol and phospholipids in the lumen of the RER, SER, cis-Golgi and trans-Golgi. However, in contrast with apo-B, all of the radiolabelled lipids in the lumen of the RER, SER and cis-Golgi were transferred to the trans-Golgi lumen or secreted. Analysis of the lipid composition of the lumenal content fractions suggests that, although very-low-density-lipoprotein (VLDL) lipids are present in the endoplasmic reticulum lumen, a large fraction of these is not associated with apo-B. Collectively these observations suggest that assembly of apo-B into complete VLDL is not cotranslational, that most lipids become associated with apo-B late in the endoplasmic reticulum compartment and that the lipids are further modified in the Golgi lumen.

Animals↗
PubMed1995

Differential induction of the NF-AT complex during restimulation and the induction of T-cell anergy.

Stimulation of human CD4+ T-cell clones through the T-cell receptor (TcR) by high doses of specific peptide results in the induction of a long-lived state of nonresponsiveness that has been called anergy. During the induction of anergy, T cells are phenotypically similar to cells responding to an immunogenic stimulus. The amount of TcR at the cell surface is downmodulated, whereas the CD2 and CD25 receptors are increased. When restimulated, however, anergic T cells fail to up-regulate transcription of the IL-2 gene and in consequence do not produce IL-2. In this study, we have compared the ability of various transcription factors to bind to their appropriate site on DNA. Factors were isolated from the nuclei of T cells that were in the induction phase of anergy or were undergoing activation. The pattern of binding activity in restimulated T cells is consistent with the pattern that has previously been shown to regulate T-cell-specific expression of the IL-2 and the beta chain of the TcR genes. The measured binding to a TCF-1 site is the same in the nuclei of resting, activated, and anergized cells. The inducible factors NK-kappa B, beta E2, CD28RC, and AP-1 are not expressed in resting cells and are twofold lower in anergized as compared with activated cells. In contrast, anergic T cells express approximately eightfold lower amounts of NF-AT, a member of the class of inducible factors that regulates IL-2 gene transcription.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1995

Use of polymerase chain reaction to detect bacteria in arthropods: a review.

Detection of bacteria in arthropod vectors traditionally has been pursued using serological and cell culture methodologies. The advent of the polymerase chain reaction (PCR) has made possible accurate, timely, and reproducible identification of bacteria in these vectors, particularly those microbes that are difficult to culture in vitro. We have reviewed the literature for PCR primers used to amplify gene segments of pathogenic bacteria from insect, tick, and mite vectors and provide the sequences of these primers, as well as notes on preparation of arthropod samples and direct sequencing of PCR products.

Animals↗
PubMed1995

Clonal analysis of CD4 mediated accessory function on the effector activity of human CD4+ T cell subsets.

BACKGROUND: It has been reported for the peripheral T cell repertoire that CD4 molecules may enhance adhesion between T cells and antigen presenting cells and, through their physical association with T cell antigen receptors, contribute to signal transduction. OBJECTIVE: The aims of this study were to determine if the modulation of CD4 molecules had differential effects on T cell recognition, antigen induced cytokine (IL-4 and IFN gamma), release and the induction of specific anergy for human TH-0, Th-1 and TH-2 cells. METHODS: A panel of anti-CD4 antibodies was examined for its ability to modulate T cell proliferation, cytokine production and tolerance induction in house dust mite (TH-0 and TH-2) and influenza haemagglutinin (TH-1) specific human CD4+ T cell clones all restricted by DRB1*1101 and isolated from dust mite allergic individuals. RESULTS: We observed that anti-CD4 antibodies may inhibit or enhance antigen mediated T cell proliferation, which may reflect the differential requirements of T cells for selective functions of CD4. Furthermore, IFN gamma and IL-4 production was differentially modulated depending on the specificity of the anti-CD4 antibody and the clone of T cells. However, pretreatment of T cells with anti-CD4 antibody alone neither induced nor enhanced the susceptibility of T cells to peptide mediated anergy. CONCLUSION: Antigen recognition by different subsets of human CD4+ T cells has differential requirements on CD4, whereas the induction of specific anergy appeared to be independent of the functions of CD4 molecules. Antigen induced IFN gamma production was more susceptible than IL-4 to the inhibitory effects of anti-CD4 antibodies. Furthermore, it appeared that certain anti-CD4 antibodies can dissociate antigen induced IFN gamma and IL-4 production, and may downregulate IFN gamma synthesis without inhibiting antigen dependent proliferation.

Antibodies↗
PubMed1995

Differential dependence of TH-0, TH-1 and TH-2 CD4+ T cells on co-stimulatory activity provided by the accessory molecule LFA-1.

BACKGROUND: The adhesion molecule LFA-1 contributes to the activation response of peripheral blood human CD4+ T cells. Less is known of its contribution to stimulation of long-term CD4+ T cell lines and clones or of its potential to co-stimulate CD4+ T cells of different functional phenotype. OBJECTIVE: This study was therefore performed to investigate co-stimulatory properties of the LFA-1 (CD11a/CD18) complex in the activation of human CD4+ T cell lines and clones of TH-0, TH-1 and TH-2 subsets. METHODS: Co-stimulatory activity was measured by cross-linking antibodies to CD11a or CD18 with anti-CD3 antibodies to plastic and then measuring the proliferative response of CD4+ T cells to these antibodies. RESULTS: A house duct mite allergen-specific CD4+ T cell line (TH-2) demonstrated much greater dependence on both CD11a and CD18 than a mycobacterial antigen-specific CD4+ T cell line (TH-1). Co-stimulatory activity through LFA-1 was also provided to a house dust mite-specific CD4+ T cell clone (DE-9; TH-2) but not to an influenza haemagglutinin-specific CD4+ T cell clone (HA1-7; TH-0). In contrast, soluble antibodies to CD18 inhibited proliferative responses of both DE-9 and HA1.7 to an immunogenic challenge of antigen and to stimulation by anti-CD3 antibodies. However, the allergen-specific T cells were more susceptible to inhibition. Signal transduction was also observed from the T-cell receptor to LFA-1. Ligation of the T-cell receptor modulated the phenotypic expression of LFA-1 and ICAM-1 on both HA1.7 and DE-9. Phenotypic modulation was observed as a result of both activation and the induction of non-responsiveness. CONCLUSION: These experiments indicate that CD4+ T cells of TH-2 functional phenotype may have a greater requirement for the co-stimulatory activity of LFA-1 than CD4+ T cells of TH-0 or TH-1 phenotypes.

Antibodies, Monoclonal↗
PubMed1995

In vitro and in vivo antibiotic susceptibilities of ELB rickettsiae.

The activities of doxycycline, rifampin, chloramphenicol, and erythromycin against ELB rickettsiae (Rickettsia azadi) were determined by dye uptake and plaque assays. Plaque formation in Vero cells was inhibited by 0.12 microgram of doxycycline per ml. The data presented demonstrate the susceptibility of ELB rickettsiae to commonly used antibiotics for the treatment of rickettsial diseases.

Animals↗
PubMed1995

Isolation, cultivation, and partial characterization of the ELB agent associated with cat fleas.

ELB rickettsiae from cat flea homogenates were recovered in tissue culture cells following sequential passage through laboratory rats and the yolk sacs of embryonated chicken eggs. Seven days after inoculation of ELB from the infected yolk sacs, Vero cells and L929 cells were observed to contain intracellular bacteria as demonstrated by Diff Quik and indirect immunofluorescence assay staining. The rickettsial and ELB identity of the cultured agent was confirmed by PCR detection of the 16S rRNA and citrate synthase genes and PCR-restriction fragment length polymorphism analysis of the 17-kDa conserved rickettsial antigen gene. The ELB rickettsiae induced plaques in Vero cells on day 11 postinfection. Rat anti-ELB serum reacted at 1:4,096 to cultured ELB and had lower reactivity to Rickettsia typhi Wilmington (1:1,024), Rickettsia akari Kaplan (1:512), and Rickettsia australis JC (1:64). Spotted fever group polyclonal sera also exhibited lower reactivity to ELB than to the homologous antigen. Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles of the ELB isolate and two R. typhi strains were identical.

Animals↗
PubMed1995

Polyclonal and clonal analysis of human CD4+ T-lymphocyte responses to nut extracts.

The induction of IgE antibodies to aeroallergens depends upon antigen-specific CD4+ helper T cells of an 'interleukin-4 (IL-4)-dominant' phenotype. Nuts also drive IgE-mediated hypersensitivity and are the most dangerous of the orally encountered allergens. We have studied the polyclonal T-cell responses of atopic and non-atopic individuals to extracts of peanut, brazilnut and hazelnut. Strong proliferative responses were observed in all patients but specific IgE was only present in the nut-allergic patients suggesting a similar pathogenic mechanism to aeroallergen-mediated hypersensitivity. To investigate this hypothesis a panel of peanut-reactive T-cell clones was raised from a peanut- and brazilnut-allergic individual without hazelnut allergy. The antigen specificity, major histocompatibility complex (MHC) class II restriction and cytokine profiles of the T-cell clones were determined. With the exception of one T-cell clone, which proliferated in response to both peanut and hazelnut extract, the peanut T-cell clones were not cross-reactive with hazelnut or brazilnut. The T-cell clones recognized antigen in association with HLA-DR and HLA-DP but not HLA-DQ class II molecules. The peanut-specific clones produced high levels of IL-4 and low levels of interferon-gamma (IFN-gamma), exhibiting the 'TH2-like' profile which dominates the aeroallergen response. In contrast, the T-cell clone that was cross-reactive on both peanut and hazelnut allergen had a Th0-like phenotype, consistent with the lack of specific serum IgE to hazelnut. These results support the importance of functionally distinct T-cell populations that recognize oral allergens. The relative production of IL-4 and IFN-gamma of the cloned T cells in the peanut-allergic patients plays a role in determining whether or not IgE antibody responses are induced with the associated potential to develop anaphylactic reactions.

Adult↗
PubMed1995

The effects of changes at peptide residues contacting MHC class II T-cell receptor on antigen recognition and human Th0 cell effector function.

Cytokines can influence the selection of functional subsets (Th1 or Th2) of CD4+ T cells. However, quantitative changes in affinity of peptide/major histocompatibility complex (MHC) class II/T-cell receptor (TCR) interactions may alter antigen density and modulate T-cell effector function. The possibility exists to use peptide analogues to induce a partial signal to dissociate production of interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) by T-helper type-0 (Th0) cells and, consequently, to regulate T-cell function. Based on binding assays and resolution of the crystalline structure of an influenza virus haemagglutinin peptide (HA 306-318) bound to the human MHC class II molecule DRB1*0101, we synthesized HA peptide analogues with amino acid substitutions predicted to modify either MHC class II/peptide density or TCR/peptide interactions. When we examined their antigenicity using cloned human Th0 cells, the analogues, in general, elicited a gradation in potency reflected by a reduction in both proliferation and cytokine production (IL-2, IL-4 and IFN-gamma). Although the analogue HA-R309 diminished IL-2 production, none of the analogues tested could selectively induce only IL-4 or IFN-gamma. Since, in general, the effector functions of the Th0 cells examined here were resistant to selective manipulation by the peptide analogues, this suggests that for some clones of chronically activated T cells modulation of selected functions may be difficult to achieve.

Amino Acid Sequence↗
PubMed1995

The effect of a single amino acid substitution within the V3 loop of HIV-1 gp120 on HLA-DR1-restricted CD4 T-cell recognition.

Viral variation has been proposed to play a role in the pathogenesis of human immunodeficiency virus type-1 (HIV-1) infection, and is an important consideration in vaccine design. During the course of an infection, isolates with sequence changes in CD8 T-cell and B-cell epitopes arise. To determine whether sequence variation within the V3 loop of HIV-1 gp120 affects HLA-DR beta 1*0101-restricted CD4 T-cell recognition, we have generated CD4 T-cell clones (TLC) specific to gp120 V3 loop peptides. Four HLA-DR beta 1*0101-restricted groups of TLC were defined by distinct patterns of responses to a panel of peptides, consistent with a highly diverse T-cell repertoire recognizing the 30 amino acid stretch (296-326) of the gp120 V3 loop. Nevertheless, a single residue change at position 311 was found to abolish the recognition of two of the four groups of TLC. This was not due to an effect of the residue at 311 on binding to major histocompatibility complex (MHC), because: (1) irrespective of the residue at 311, peptides competed well with the influenza haemagglutinin peptide 307-319 for binding to cell-bound DR1; and (2) R311-specific TLC were also HLA DR beta 1*0101 restricted. Instead, the substitution of arginine for serine at position 311 blocked the interaction of the peptide with the T-cell receptor. Thus, despite the diversity of the T-cell response to the V3 loop of HIV-1, a single amino acid change can have a considerable influence on the responding T-cell population. As residue 311 is one of the most variable of the V3 loop residues, these results suggest that CD4 recognition can also exert pressure on viral variation consistent with a role for these cells in antiviral immunity.

Amino Acid Sequence↗
PubMed1994

Immunosuppressant deoxyspergualin inhibits antigen processing in monocytes.

Deoxyspergualin (DSG) is a novel immunosuppressive agent recently shown to bind to the constitutive heat shock protein 70, which is involved in binding and intracellular transport of antigenic peptides. In this study, we show that DSG inhibits the proliferation of PBMCs to the Ags tetanus toxoid and diphtheria toxoid, but not to the mitogens PHA and PMA/ionomycin, nor to the superantigens toxic shock syndrome toxin-1 and staphylococcal enterotoxin A. DSG's effect was specific for monocytes as preincubation of T cells with DSG did not inhibit their proliferation to monocytes pulsed with tetanus toxoid Ag for 16 h, whereas the presence of DSG during Ag pulsing of the monocytes inhibited their ability to stimulate T cell proliferation. DSG did not down-regulate the expression of MHC class II molecules by monocytes, and the inhibitory effect of DSG on T cell proliferation was not reversed by the addition of IL-2, nor by the addition of the costimulatory signals IL-1, IL-6, and anti-CD28. Studies with two human T cell clones, HA1.7 and PF5, specific, respectively, to peptides spanning amino acids 307-319 and 256-270 of influenza hemagglutinin, showed that DSG inhibited the proliferation of the clones to the native hemagglutinin molecule but minimally affected their proliferation to the peptides. These data suggest that DSG interferes with Ag processing and/or presentation.

Adult↗
PubMed1994

Overlapping T-cell epitopes in the group I allergen of Dermatophagoides species restricted by HLA-DP and HLA-DR class II molecules.

The induction of IgE antibodies reactive with the group I allergen of Dermatophagoides species (house dust mite [HDM]), which comprise a major component of the allergic immune response in HDM-atopic individuals, is dependent on the functional activity of specific CD4+ T cells. In this report we demonstrate that for a particular HDM-atopic individual the T-cell response to the group I allergen of Dermatophagoides pteronyssinus (Der p I) is limited to a single region (residues 101-143) of the protein. By mapping the fine antigen specificity with T-cell clones, we observed that the sequence 101-131 of Der p I contains a cluster of at least three overlapping T-cell epitopes. Analysis of the HLA class II restriction specificity of the T-cell clones revealed that the T-cell epitope, residues 110-131, was restricted by HLA-DRB1*0101. In contrast, peptide Der p I, 110-119 was recognized in association with HLA-DPB1*0402. However, the ability of cloned T cells to proliferate to the peptide Der p I, 107-119 presented by HLA-DPB1*0401, HLA-DPB1*0402, and HLA-DPB1*0501 expressing accessory cells illustrates the heterogeneity of the restriction specificity of this region of Der p I. The application of this information in the design of peptide-based immunotherapy in the management of allergic responses to HDM is discussed.

Allergens↗