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Biomedical subjects

E Furrie

Publications and source records attributed to E Furrie.

15 recordsLinked to original sources
PubMed2006

A molecular revolution in the study of intestinal microflora.

Bacterial colonisers of the colon comprise several hundred bacterial species that live in a complex ecosystem. Study of this complex ecosystem has been carried out, until recently, by traditional culture techniques with biochemical methods to identify organisms. The development of molecular techniques to investigate ecological microbial communities has provided the microbiologist with a vast array of new techniques to investigate human intestinal microflora. Metagenomics, the science of biological diversity, combines the use of molecular biology and genetics to identify and characterise genetic material from complex microbial environments. The combination of metagenomics and subsequent quantitation of each identified species using molecular techniques allows the relatively rapid analysis of whole bacterial populations in human health and disease.

Bacteria↗
PubMed2005

Mucosal bacteria in ulcerative colitis.

Ulcerative colitis (UC) is an acute and chronic inflammatory bowel disease of unknown aetiology, although bacterial species belonging to the normal colonic microbiota are known to be involved in its initiation and maintenance. Several organisms have been linked to the disease; however, mucosa-associated bacteria are more likely to be involved than their luminal counterparts, due to their close proximity to the host epithelium. Comparative bacteriological analyses were done on rectal biopsies to investigate differences in mucosal bacteria in patients with UC and healthy controls. Complex bacterial communities were found in both groups, with significant reductions in bifidobacterial numbers in UC, which suggested that they might have a protective role in the disease. Accordingly, a therapy for treating UC was designed, with the aim of modifying the mucosal microbiota to increase bifidobacterial colonisation and reduce inflammation. Ranges of mucosal and faecal bifidobacteria were tested for their substrate preferences and their abilities to survive under a variety of environmental conditions. A synbiotic comprising a probiotic (Bifidobacterium longum) isolated from healthy rectal mucosa combined with a prebiotic (oligofructose-enriched inulin - Synergy 1) was developed. The treatment was used in a randomised controlled trial involving eighteen patients with active UC, for a period of 1 month. Rectal biopsies were collected at the beginning and end of the study. Bacteriological analysis and transcription levels of epithelium-related immune markers were assessed. Results demonstrated that short-term synbiotic treatment resulted in increased bifidobacterial colonisation of the rectal mucosa and induced significant reductions in the expression of molecules that control inflammation in active UC.

Adult↗
PubMed2005

Synbiotic therapy (Bifidobacterium longum/Synergy 1) initiates resolution of inflammation in patients with active ulcerative colitis: a randomised controlled pilot trial.

BACKGROUND AND AIMS: Ulcerative colitis (UC) is an acute and chronic inflammatory disease of the large bowel with unknown aetiology. The immune response against normal commensal microorganisms is believed to drive inflammatory processes associated with UC. Therefore, modulation of bacterial communities on the gut mucosa, through the use of probiotics and prebiotics, may be used to modify the disease state. METHODS: A synbiotic was developed for use in UC patients combining a probiotic, Bifidobacterium longum, isolated from healthy rectal epithelium, and a prebiotic (Synergy 1), a preferential inulin-oligofructose growth substrate for the probiotic strain. Treatment was employed in a double blinded randomised controlled trial using 18 patients with active UC for a period of one month. Clinical status was scored and rectal biopsies were collected before and after treatment, and transcription levels of epithelium related immune markers were measured. RESULTS: Sigmoidoscopy scores (scale 0-6) were reduced in the test group (start 4.5 (1.4), end 3.1 (2.5)) compared with placebo (start 2.6 (2.1), end 3.2 (2.2)) (p=0.06). mRNA levels for human beta defensins 2, 3, and 4, which are strongly upregulated in active UC, were significantly reduced in the test group after treatment (p=0.016, 0.038, and 0.008, respectively). Tumour necrosis factor alpha and interleukin 1alpha, which are inflammatory cytokines that drive inflammation and induce defensin expression, were also significantly reduced after treatment (p=0.018 and 0.023, respectively). Biopsies in the test group had reduced inflammation and regeneration of epithelial tissue. CONCLUSIONS: Short term synbiotic treatment of active UC resulted in improvement of the full clinical appearance of chronic inflammation in patients receiving this therapy.

Adult↗
PubMed2004

Bacterial milieu and mucosal bacteria in ulcerative colitis.

The aetiology of ulcerative colitis (UC) is unknown, but there is evidence that bacteria are needed for initiation and maintenance of the disease. A number of organisms have been associated with UC, but evidence for a specific transmissible agent is weak. Despite this, there is a good case for mucosal bacterial involvement, either through pathogens colonizing the epithelial surface, by non-pathogenic commensal species occupying adhesion sites on the mucosa and preventing invasion by harmful bacteria, or by inappropriate host immune responses to members of the normal microflora. Since mucosal bacteria exist in close juxtaposition to host tissues, it might be expected that they interact to a greater extent with the immune and neuroendocrine systems than their luminal counterparts. For this reason, comparative bacteriological analyses were done on rectal biopsies from patients with active colitis, and individuals who had no inflammatory bowel disease. Complex bacterial communities colonized the rectal mucosa in all subjects and great interindividual variabilities in mucosal bacterial populations were observed in both groups. These organisms often occurred in microcolonies, which may have implications for UC, since it would result in higher localized concentrations of bacterial antigens, or toxins, than would be the case if the organisms were diffusely spread across the mucosa.

Bacterial Adhesion↗
PubMed2004

Identification and quantitation of mucosal and faecal desulfovibrios using real time polymerase chain reaction.

BACKGROUND: Desulfovibrios produce sulphide, which is toxic to colonic epithelial cells. These bacteria have previously been linked to ulcerative colitis. Traditional methods of culturing these organisms are slow, and often unreliable, while molecular approaches are either non-quantitative or lack sensitivity. AIMS: To develop a sensitive method for quantitating desulfovibrios in stools and biopsy tissue, and to investigate the effects of age and disease on these bacteria. METHODS: Rectal biopsies were taken from 10 colitis patients and 10 healthy controls. Stool samples were obtained from 10 healthy infants (mean age 1.01 (0.18) years), 10 healthy young adults (26.7 (1.2) years), and 10 healthy elderly people (71.7 (1.2) years). Primers were designed and developed for analysing Desulfovibrio populations in the bowel using real time polymerase chain reaction (PCR). RESULTS: The PCR primers were highly specific for desulfovibrios. Large numbers (approximately 10(6)-10(7)/g) occurred in biopsies in colitis patients and healthy subjects, and no disease related differences were observed. Measurements of mucosal desulfovibrios over 12 months showed marked changes in some patients. Infants (10(6)-10(7)/g) and elderly people (10(7)-10(8)/g) had significantly higher numbers of desulfovibrios in stools compared with young adults (10(5)/g). CONCLUSIONS: Real time PCR analysis of desulfovibrios was an efficient and accurate method for studying these potentially harmful microorganisms. Desulfovibrios were ubiquitous in the bowel, irrespective of age. As rectal mucosae were heavily colonised in health and disease, if these bacteria play a role in colitis, some host defect, possibly in sulphide detoxication pathways or in bacterial antigen handling, is required for manifestations of pathogenicity.

Adult↗
PubMed2004

Systemic antibodies towards mucosal bacteria in ulcerative colitis and Crohn's disease differentially activate the innate immune response.

BACKGROUND AND AIMS: The mucosa in ulcerative colitis (UC) is replete with antibody producing plasma B cells and polymorphonuclear leucocytes (PMN). This combination of effector cells requires a crosslinking antigen to evoke an antibody driven PMN inflammatory response via their Fc receptors. The stimulus for activation is thought to be commensal bacteria colonising the gut mucosa. The aim of this investigation was to compare the principal culturable bacterial populations on the rectal mucosa of UC patients, and to determine whether specific antibodies towards these bacteria can activate infiltrating PMN through opsonisation. This would provide an explanation for this chronic inflammatory condition. METHODS: Bacteria colonising rectal tissue were characterised using chemotaxonomic techniques. Systemic antibody responses were measured against total antigens and surface antigens of these organisms in UC and Crohn's disease (CD) patients, together with healthy controls. Antibody enhancement of the respiratory burst in PMN was also investigated, against a range of mucosal isolates. RESULTS: Distinct differences were observed in some bacterial populations in UC biopsies, which were generally reflected in antibody responses towards these organisms. UC patients had higher IgG responses to surface antigens, primarily IgG1, whereas the response in CD was mainly IgG2. Antibodies from UC patients greatly enhanced the respiratory burst in PMN, in response to individual bacterial species. CONCLUSIONS: Changes in mucosal bacteria, and a switch from internal to surface antigen/antibody reactivity of a predominantly IgG1 type, leads to greater opsonisation of the respiratory burst in PMN, providing a mechanism for maintaining the inflammatory state in UC.

Aged↗
PubMed2000

Antigen triggering selectively increases TCRBV gene transcription.

When the TCR binds Ag it is phosphorylated, internalized, and degraded. We wished to examine whether this process was accompanied by a specific concomitant increase in TCR mRNA levels. To do this, PBMC and a T cell clone were cultured with a series of superantigens and an alloantigen. Only T cells specifically responding to an antigenic stimulus had increased levels of TCR beta-chain variable (TCRBV)-specific mRNA. This response was apparent after 48 h, peaked around 72 h, and was still elevated after 7 days. Increased gene transcription appeared to be driven solely by Ag as specific Ag depletion prevented culture supernatants transferring this effect. The level of TCRBV mRNA elevation was not influenced by the stimulating Ag, but appeared dependent on the gene encoding the stimulated TCR. Reporter gene assays, using cloned TCRBV gene promoters, confirmed both that TCR gene transcription rises after stimulation and that basal and stimulated levels of TCR transcription vary between different TCRBV genes. These data conclusively demonstrate that there is no direct relationship between TCRBV mRNA and T cell number, and that future repertoire studies must take both factors into account.

Adult↗
PubMed1999

Oral vaccination with immune stimulating complexes.

There is a need for non-living adjuvant vectors which will induce a full range of local and systemic immune responses to orally administered purified antigens. Here we describe our experience with lipophilic immune stimulating complexes (ISCOMS) containing the saponin adjuvant Quil A. When given orally, ISCOMS containing the model protein antigen ovalbumin (OVA) induce a wide range of systemic immune responses, including Th1 and Th2 CD4 dependent activity, class I MHC restricted cytotoxic T-cell responses and local production of secretory IgA antibodies. More recent results indicate that ISCOMS may act partly by enhancing the uptake of protein from the gut. In addition, intraperitoneal injection of ISCOMS recruits and activates many components of the innate immune system. including neutrophils, macrophages, and dendritic cells. In parallel, there is increased production of nitric oxide (NO), reactive oxygen intermediates (ROI), interleukins (IL) 1, 6, 12, and gamma interferon (gammaIFN). Of these factors, only IL12 is essential for the immunogenicity of ISCOMS in vivo, as mucosal and systemic responses to ISCOMS are reduced in IL12KO mice, but not in IL4KO, IL6KO, inducible NO synthase (iNOS) KO, or gammaIFN receptor KO mice. We propose that ISCOMS act by targetting antigen and adjuvant to macrophages and/or dendritic cells. This pathway may be amenable to exploitation for vaccine development, especially if combined with another vector with a different mucosal adjuvant profile, such as cholera toxin.

Adjuvants, Immunologic↗
PubMed1996

The effect of minocycline in rat models of inflammatory arthritis: correlation of arthritis suppression with enhanced T cell calcium flux.

Adjuvant and collagen arthritis in the rat are widely accepted T-cell-dependent counterparts of rheumatoid arthritis and were used to examine the antiinflammatory properties of minocycline. Administration of oral minocycline, a semisynthetic tetracycline, significantly decreased (P < 0.01) the incidence of arthritis in both models. In vivo exposure to minocycline also significantly increased the percentage of splenocytes exhibiting a rise in free intracellular calcium concentration ([Ca2+]i) following concanavalin A stimulation (P < 0.05 in adjuvant and P < 0.01 in collagen). This enhancement was mitogen dose-dependent and supported exclusively by extracellular Ca2+. Resting [Ca2+]i levels were unaffected by minocycline and predominantly the CD4+ subset was involved. No changes were observed in weight, IgG antibodies to collagen, synoviocyte release of collagenase and prostaglandin E2, acute inflammation in an air-pouch system, or cell surface expression of activation markers (interleukin-2 and transferrin receptors) by splenocytes or lymph node cells. As a controlled [Ca2+]i rise is a critical event in normal T cell activation, minocycline's antiarthritic profile in vivo may relate to perturbed Ca2+ influx during T cell activation, an alteration that could promote the development of clinical tolerance to otherwise arthritogenic stimuli.

Animals↗
PubMed1995

Influence of MHC class I molecules on T-cell proliferation induced by CD3 or Thy-1 stimulation.

We have reported that class I- [and lymphocyte function-associated antigen-1 (LFA-1-)] specific monoclonal antibodies (mAb) inhibit anti-CD3-mediated activation of naive T cells. The present study investigated the mechanism of this inhibition. CD28-specific mAb augmented stimulation induced by soluble CD3 mAb, but this costimulation was also inhibited by anti-class I or anti-LFA-1 mAb. However, stimulation of T cells was not inhibited when activated B cells were present. Neither B7-1- nor B7-2-specific blocking mAb or soluble CTLA-4, CD40 or gp39 restored the inhibition. Thus, other molecules expressed on activated B cells are implicated for T-cell activation, which could compensate blockade of class I or LFA-1 molecules. Inhibition induced by class I-specific mAb could potentially be mediated through extracellular, transmembrane or cytoplasmic domains of the target molecules. These possibilities were evaluated by the use of mice transgenic for the Qa-2 molecule, selected for expression of Qa-2 at levels equivalent to classical class I molecules. Qa-2 is inserted in the membrane through phosphatidylinositol linkages. Antibodies directed to Qa-2 inhibited CD3-induced stimulation, demonstrating that cytoplasmic and transmembrane protein sequences of class I molecules are not necessary for the inhibitory effect. Inhibition thus presumably depends on extracellular domains. Finally, T cells from beta 2-microglobulin knock-out mice responded to CD3-specific mAb as well as their class I-positive littermates. Nevertheless, stimulation of T cells from these mice with mitogenic anti-Thy-1 mAb was markedly reduced. Signalling by Thy-1 and the CD3 complex may normally occur through pathways in which class I molecules are implicated.

Animals↗
PubMed1995

Partial characterization of a circulating tolerogenic moiety which, after a feed of ovalbumin, suppresses delayed-type hypersensitivity in recipient mice.

Serum collected 60 min after feeding ovalbumin (OVA) to BALB/c mice transfers specific tolerance to naive recipients via an unknown mechanism. We have now identified a large fragment of the OVA molecule as the putative active moiety. Specific absorption of immunoreactive OVA from tolerogenic serum by immunoaffinity chromatography removed the tolerogenic activity; following elution of the bound OVA and subsequent injection into naive recipients it was possible to demonstrate tolerogenic activity equivalent to that seen with unmanipulated serum. Passage of OVA tolerogenic serum through a bovine serum albumin (BSA)-specific affinity column had no effect on in vivo OVA tolerogenic activity. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting using anti-OVA antibodies demonstrated the presence of two bands with apparent molecular weights (MW) of 21,000 and 24,000 in the OVA-related material absorbed from the tolerogenic serum. The 24,000 MW moiety was also visualized by silver staining. These moieties are putative candidate tolerogens as they were absent from normal BALB/c mouse serum analysed 5 min after feeding OVA and from severe combined immunodeficiency (SCID) mouse serum--without tolerising activity--analysed 60 min after a similar feed.

Animals↗
PubMed1994

Failure of SCID mice to generate an oral tolerogen after a feed of ovalbumin: a role for a functioning gut-associated lymphoid system.

The role of the mucosal immune system in the generation of circulating tolerogenic ovalbumin (OVA) moieties has been investigated after a single feed of the protein. Serum collected from SCID mice 1 hr after a 25-mg feed of OVA was unable to transfer tolerance of delayed-type hypersensitivity (DTH) into naive BALB/c recipients. This is in contrast to serum collected from BALB/c mice which was able to transfer DTH tolerance to naive BALB/c recipients. The levels of circulating OVA detected in the serum of SCID mice 60 min after feeding OVA were approximately half those detected in the serum of BALB/c mice at the same time-point. However even dose adjustment of SCID mouse serum to a level of immunoreactive OVA equivalent to that found in BALB/c serum was unable to induce DTH tolerance in BALB/c recipients. This failure of SCID serum to transfer tolerance was shown to be unrelated to the germ-free conditions under which SCID mice are kept. Serum from OVA-fed germ-free BALB/c mice transferred DTH tolerance at equivalent levels to serum from conventionally reared BALB/c mice. When the intestinal morphology and intraepithelial lymphocyte (IEL) numbers in the duodenum of SCID mice were compared to conventionally reared and germ-free BALB/c controls, SCID mice were characterized by a lower number of IEL with a different morphology from the majority of IEL found in BALB/c mice.

Administration, Oral↗