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Biomedical subjects

G A Neil

Publications and source records attributed to G A Neil.

At least 19 recordsLinked to original sources
PubMed1995

Nerve growth factor synthesis by intestinal epithelial cells.

Nerve growth factor (NGF) exists in the gut of adult rats. The cells responsible for NGF synthesis in the gut remain unknown. IEC-6 and Caco-2 cells, established cell culture models of intestinal epithelial cells, were studied to determine whether intestinal epithelial cells, were studied to determine whether they synthesize and release NGF. Conditioned media from both IEC-6 and Caco-2 cells stimulated neurite outgrowth in both rat pheochromocytoma (PC-12) cells and sensory neurons derived from embryonic chick dorsal root ganglia (DRG). The addition of anti-NGF antibody blocked neurite outgrowth in PC-12 cells and partially blocked outgrowth in DRG cells. An NGF-enzyme-linked immunosorbant assay readily detected immunoreactive NGF in conditioned media from both cell lines, whereas cellular extracts from IEC-6, Caco-2, and isolated rat intestinal epithelial cells had low levels of immunoreactivity. Caco-2 monolayers primarily secreted NGF from the basolateral compartment, and interleukin-1 enhanced its secretion. IEC-6, Caco-2, and isolated rat intestinal epithelial cells expressed NGF mRNA as determined by reverse transcription polymerase chain reaction. These observations suggest that intestinal epithelial cells are capable of NGF synthesis.

Animals↗
PubMed1995

Generation of a human monoclonal antibody to hepatitis C virus, JRA1 by activation of peripheral blood lymphocytes and hypo-osmolar electrofusion.

We have generated a human monoclonal antibody with binding specificity for hepatitis C virus (HCV)-specific peptides using peripheral blood lymphocytes isolated from a HCV antibody positive patient. The B-lymphocytes were stimulated with lipopolysaccharide (LPS) for 72 hours prior to the fusion. A recently described high efficiency hypo-osmolar electrofusion technique was employed, allowing generation of a large number of human hybridomas. The hybridomas were screened for human immunoglobulin and HCV-specific peptide binding by EIA. A single HCV-positive clone, JRA1, was detected and sub-cloned. Isotype analysis showed it to secrete an IgM lambda monoclonal antibody. The antibody was positive on both first and second generation HCV antibody analysis. This study confirms that viable pathogen-specific B-cells may be recovered from the peripheral blood. Although such cells are likely to be relatively uncommon in the circulating B-cell pool, they may be successfully immortalized by high efficiency electrofusion techniques. This technique might be valuable for the generation of human monoclonal antibodies with specificity for other human pathogens.

Adult↗
PubMed1995

Variability with omeprazole-amoxicillin combinations for treatment of Helicobacter pylori infection.

OBJECTIVE: Although omeprazole co-therapy enhances the effectiveness of some antimicrobials for the treatment of Helicobacter pylori infection, results have not been uniform. A meta-analysis suggested that 20 mg of omeprazole b.i.d. and 2 g or more of amoxicillin would yield a > 80% success rate (Gastroenterology 1994; 106: 142A). Our objective in this study was to test that hypothesis. METHODS: Volunteers with H. pylori infection were studied. Anti-H. pylori therapy was administered with meals for 14 days (omeprazole 20 mg b.i.d. plus amoxicillin 1 g t.i.d., or omeprazole 20 mg b.i.d. plus amoxicillin 0.5 g t.i.d.). Endoscopy was performed 4-6 wk after antimicrobial therapy ended, and the presence or absence of H. pylori was determined with biopsy specimens by Genta stain. RESULTS: Fifty-nine volunteers completed the study; 30 were studied twice. The overall success for initial treatment with either combination of amoxicillin and omeprazole was 18 of 59 [30.5%; 95% confidence interval (CI) = 19-44%]. The success rate with 500 mg amoxicillin t.i.d. was 7 of 29 (24%; 95% CI = 10-43%). With 1 g t.i.d. amoxicillin, the cure rate was higher (36.6%) (11 of 30; 95% CI = 20-56%), or intention-to-treat result was 11 of 31 (35.4%), which includes the early dropout. Compliance was > 95% for both therapies. Side effects were experienced by eight patients, two receiving 1.5 g amoxicillin and six receiving 3 g amoxicillin (p > 0.2). German trials suggest that better results might be achieved when amoxicillin is given as suspension while fasting. Thirty treatment failures were re-treated with 1 g amoxicillin suspension t.i.d., given fasting, and omeprazole 20 mg b.i.d. The cure rate was 16.6% (95% CI = 6-35%). CONCLUSION: Amoxicillin/omeprazole combinations for treatment of H. pylori infection do not yield consistent results. The reason is unknown, but the reported high rate of success with 40 mg of omeprazole and 750 mg t.i.d. suggests that almost complete inhibition of acid secretion is necessary to obtain consistent results with this combination.

Adult↗
PubMed1994

Analysis of T-lymphocyte subpopulations in inflammatory bowel diseases by three-color flow cytometry.

In order to better define changes in the relative proportion of peripheral blood T-lymphocyte subpopulations in patients with inflammatory diseases of the bowel, we performed simultaneous three-color fluorescence-activated cytometric (FACS) analysis using fluorophore-conjugated monoclonal antibodies with specificity for CD4, CD8, Leu 8, and CD45RA on 22 normal control subjects, 28 patients with Crohn's disease (CD), 15 patients with ulcerative colitis (UC), and 11 patients with intestinal inflammation secondary to etiologies other than inflammatory bowel disease (NIBD). This staining combination allowed enumeration of distinct T-cell subpopulations as follows: virgin CD4+, recall antigen helper T cells, nonspecific B-cell helper T cell, virgin CD8+, cytotoxic effector and suppressor effector and recall antigen cytotoxic T cells based on a synthesis of published functional analyses. No differences in the proportion of CD4+ or CD8+ cells or in the CD4+/CD8+ ratios were evident when UC and NIBD patients were compared to normal subjects. A significant reduction in the proportion of CD4+ cells and an increase in CD8+ cells was observed, however, in the CD group. When two-color analysis was performed, several significant differences in the proportions of circulating lymphocytes were seen. Specifically, these included significant increases in the number of CD4+, Leu 8- (P < 0.01) cells in all disease groups and an increase in CD4+, CD45RA+ cells in the NIBD group. Conversely, significant decreases in the proportions of CD8+, Leu 8+ (P < 0.01) cells were evident in the Crohn's disease group.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1994

Induction of hepatic pathology in SCID-Hu mice engrafted with peripheral blood lymphocytes of patients with Schistosomiasis japonica.

SCID mice were engrafted with peripheral blood lymphocytes (PBL) derived from persons currently or previously infected with Schistosoma japonicum. After immunization with soluble worm antigenic preparation, the SCID-Hu mice were analyzed for a human immune response. ELISA revealed a low titer of human antibody recognizing soluble egg antigens in 2 of 10 mice. One mouse had detectable levels of interleukin (IL)-2 and gamma-interferon, TH1 phenotype cytokines. All mice had elevated levels of IL-4, a TH2 phenotype cytokine. The human cytokine profile of the mice paralleled the patient's serum profile at clinical examination. In addition, all mice had substantial hepatic pathology, including inflammatory cell infiltrates and macrovesicular fat deposition. The data indicate that activation of PBL from patients with a history of schistosomiasis japonica infection can result in focal hepatic pathology, which may be driven by specific cytokines.

Animals↗
PubMed1994

Identification and characterization of the rat intestinal epithelial cell (IEC-18) interleukin-1 receptor.

Interleukin-1 (IL-1), an important mediator of inflammation, may act in chronic inflammatory disorders of the intestine such as idiopathic inflammatory bowel diseases. Although the IL-1 receptor (IL-1R) has been studied extensively in many cell lines, including T cells, B cells, and fibroblasts, it has not been demonstrated on intestinal epithelial cells. IL-1 affects intestinal epithelial cell proliferation in vitro, suggesting the presence of IL-1Rs on intestinal epithelium. This paper demonstrates and further characterizes an IL-1R in a rat intestinal epithelial cell. Cells from rat intestinal epithelial cell line IEC-18 were grown to confluence in six-well plates, and association studies with 125I-labeled IL-1 beta to determine specific binding and equilibrium conditions were performed. A competitive-inhibition curve verified the IL-1 concentration required to saturate the IL-1R, and a Scatchard plot revealed a dissociation constant (Kd) of 1.2 x 10(-10) M, with 1,000 receptors per epithelial cell. Binding studies using increasing concentrations of 125I IL-1 beta alone confirmed the receptor density. Cross-linking 125I-IL-1 beta to the IEC-18 IL-1R demonstrated an IL-1R of approximately 80.5 kDa. cDNA transcribed from IEC-18 mRNA was used as a template for amplification of a segment of the IL-1R using complementary oligonucleotides. The resulting sequence of the IL-1R demonstrated a high degree of homology with both the human and mouse type I IL-1R.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Caco-2 cells express type I interleukin-1 receptors: ligand binding enhances proliferation.

We examined the effect of interleukin-1 (IL-1) on the rate of proliferation of the human colon carcinoma Caco-2 and characterized the human intestinal epithelial cell IL-1 receptor (IL-1R). IL-1 dose dependently increased tritiated thymidine uptake in confluent Caco-2 monolayers fed complete growth medium. An anti-IL-1 beta completely blocked the increase in tritiated thymidine uptake, whereas an IL-1 receptor antagonist human recombinant blocked it partially. In long-term culture, IL-1 increased DNA content over control, an effect similar to that of epidermal growth factor (EGF). Unlike EGF, IL-1 did not enhance tritiated thymidine uptake in Caco-2 monolayers grown in serum-free medium, implying that IL-1 needs a cofactor(s) to elicit its proliferative effect. Cross-linking 125I-IL-1 beta to Caco-2 membranes revealed a binding protein of approximately 80 kDa with binding saturated at approximately 2.5 x 10(9) M-1 consistent with that for the type I IL-1R. cDNA transcribed from Caco-2 mRNA and amplified by polymerase chain reaction, using complementary oligonucleotides, resulted in a reaction product matching the sequence of the type I IL-1R. Our results demonstrate that IL-1 enhances proliferation of Caco-2 cells. This effect requires the presence of an unidentified cofactor(s). Also, Caco-2 cells express the type I IL-1R.

Affinity Labels↗
PubMed1993

Electrofusion.

Explore the source record for details and available documents.

Animals↗
PubMed1993

Antineutrophil cytoplasmic antibody in inflammatory bowel and hepatobiliary diseases. High prevalence in ulcerative colitis, primary sclerosing cholangitis, and autoimmune hepatitis.

The prevalence of antineutrophil cytoplasmic antibodies was evaluated in patients with ulcerative colitis, primary sclerosing cholangitis, and various other gastrointestinal and hepatobiliary diseases to define the sensitivity and specificity of the test. The presence of antineutrophil cytoplasmic antibodies was detected in alcohol-fixed cytospin preparations of peripheral blood neutrophils with an indirect immunofluorescence technique. A perinuclear staining pattern was considered positive. Thirty-six of 50 patients (72%) with ulcerative colitis and/or primary sclerosing cholangitis had positive results. Twenty-two of 210 patients (10%) in the control group had positive findings, including a significant proportion of patients with autoimmune hepatitis (50%) and non-A, non-B and non-C hepatitis (27%). This test for antineutrophil cytoplasmic antibodies has a sensitivity of 72% and specificity of 90% for either ulcerative colitis or primary sclerosing cholangitis. It may be useful in the differential diagnosis of Crohn's disease and ulcerative colitis and in the early diagnosis of ulcerative colitis. It also may be employed to distinguish primary biliary cirrhosis from primary sclerosing cholangitis.

Adolescent↗
PubMed1992

Omeprazole heals mucosal ulcers associated with endoscopic injection sclerotherapy.

Endoscopic injection sclerotherapy (EIS) is a standard and definitive therapy for bleeding esophageal varices. While the overall complication rate of the procedure is low, a substantial minority of patients treated by EIS develop refractory mucosal ulceration and/or esophageal strictures. However, despite the prophylactic use of H2 blockers and sucralfate in our EIS protocol, we observed a number of patients who developed nonhealing esophageal and/or gastroduodenal ulceration. We conducted an open trial in which we enrolled nine patients who had refractory ulcer disease. Patients that enrolled in the trial exhibited complete healing of their mucosal ulcers. These results suggest that acid is an important contributory factor in the pathogenesis and perpetuation of EIS-associated mucosal ulceration. Patients with alcohol-associated liver disease may be at increased risk for the development of EIS-associated complications.

Adult↗
PubMed1992

CD5+ B cells are decreased in peripheral blood of patients with Crohn's disease.

B cells bearing the CD5 surface marker comprise a substantial minority of the circulating lymphocyte population in healthy individuals. These recently described cells have been implicated in T-independent humoral responses, immunoregulation, and autoimmunity. We undertook to enumerate circulating CD5+ B cells by three-color fluorescence activated flow cytometry in 28 patients with Crohn's disease (CD). None of the CD patients were using immunosuppressive medication. The CD patients were subdivided into "inactive" and "active" groups based upon their Crohn's disease activity index (CDAI). Thirty-two normal subjects served as a control population. The percentage of CD19+ B cells was significantly reduced in both active and inactive CD patients as compared with normal controls (P less than or equal to 0.01). CD5+ B cells were likewise found to be significantly decreased in both inactive and active CD patients (P less than or equal to 0.01) as compared with normal controls. The proportion of CD5+ B cells was significantly lower in the peripheral blood of active as compared with inactive CD patients (P less than or equal to 0.05). The finding that CD5+ B cells are reduced in CD may provide an important clue to immunological dysfunction in inflammatory bowel disease and merits further study.

Adult↗
PubMed1992

The effect of lowering faecal pH on the rate of proliferation of the normal colonic mucosa.

Epidemiological and animal studies suggest that faecal pH may be a risk factor for colorectal cancer with low faecal pH associated with a lower incidence of the disease. The aim of this study was to determine whether faecal pH (or dietary fibre) affects the short-term risk factors for colon cancer. Sixty-nine normal volunteers were randomized into three equal groups (A-C). They provided food records, faecal specimens and submitted to rectal biopsy for thymidine labelling studies before and after a 2-week intervention. Group A received a placebo of fruit juice. Group B, approximately 3.0 g d-1 sodium sulphate in juice. Group C, 30 g d-1 supplementary dietary fibre as wheat bran in bread. Age, sex, weight, height and intake of macronutrients and minerals were similar in the groups prior to intervention. Faecal pH was similar for the three groups before and was reduced in Group B after intervention (P = 0.001) with a relative reduction of 0.5 pH units. The labelling index for the three groups was similar prior to intervention; after, it was lowest in Group B with a relative reduction of 0.5% points, although this difference was not statistically significant. The results thus do not support the hypothesis that an acidification of faecal pH leads to a reduction in risk markers for colon cancer.

Adult↗
PubMed1992

Immunization of SCID-Hu mice and generation of anti-hepatitis B surface antigen-specific hybridomas by electrofusion.

Human hybridomas with specificity for recombinant hepatitis B surface antigen (HBSAg) were produced by adoptive transfer of human peripheral blood mononuclear cells prepared from HBSAg-immune donors to CB.17 mice bearing the severe combined immunodeficiency (SCID) phenotype. A total of ten SCID-Hu mice were immunized with recombinant HBSAg. Eight SCID-Hu mice found to have human HBSAg antibody in their serum were sacrificed, and single-cell suspensions were made from their spleens. The SCID-Hu spleen cells were electrofused to the mouse-human fusion partner H7. HBSAg-specific hybridomas were recovered from all fusions. This method may provide the means for the production of other human hybridomas and may, in some cases, circumvent the need for in vitro immunization or Epstein-Barr virus transformation of human B lymphocytes.

Adult↗
PubMed1992

Microscale production of hybridomas by hypo-osmolar electrofusion.

We have developed and tested a novel electrofusion chamber, the adjustable plate microchamber, that permits the successful electrofusion and production of hybridomas in a hanging droplet from as few as 1,000 B lymphocytes. Cell suspension volumes of 10 microliters may be used without excessive difficulty in aseptically recovering fused cells. With a modification of the hypo-osmolar electrofusion protocol with this microchamber, fusion efficiencies of the order of 10(-3) may be attained. These efficiencies are comparable to those attained with standard hardware and much higher numbers of input B lymphocytes. This technology should permit high efficiency hybridoma production using a variety of hitherto inaccessible B lymphocyte populations such as B cells isolated from human organ biopsies.

Animals↗
PubMed1991

Substance P but not vasoactive intestinal peptide modulates immunoglobulin secretion in murine schistosomiasis.

Neuropeptides including SP and VIP modulate Ig secretion by in vitro stimulated lymphocyte cultures. It is not known whether these neuropeptides effect the B cell directly, or if they significantly alter humoral immune responses to pathogens. We have previously shown that granulomas derived from schistosome-infected mice contain immunoglobulin secreting B cells (ISC) as well as eosinophils that secrete substance P (SP) and vasoactive intestinal peptide (VIP). It therefore seemed plausible that B cells derived from infected animals might respond to these neuropeptides, and that such responses might effect immunoregulatory signals. In this study, we addressed these issues in the murine Schistosoma mansoni model, at the level of immunoglobulin secretion in single B cells. Spontaneous ISC were observed in both splenic and granuloma cell preparations. The addition of SP resulted in a dose-dependent reduction in the number and size of plaques (a 50% reduction was observed at 10(-9) M). This effect was blocked with SP antagonists. Similar results were observed in T cell-depleted cell cultures. VIP had no effect on ISC number or plaque size. We conclude that SP, but not VIP, decreases spontaneous ISC number and Ig secretion in short-term cultures of spleen and granuloma cells. SP appears to exert its effects at the level of single B cells through a receptor-mediated mechanism and may thus play an immunoregulatory role in schistosomiasis.

Animals↗
PubMed1990

Electrofused mammalian cells analyzed by free-flow electrophoresis.

Somatic cell fusion is a powerful and widely used technique. In recent years, electrofusion has become increasingly popular because it is a gentle process that can be optically controlled and carefully monitored using appropriate fusion chambers and because it permits the efficient fusion of smaller cell numbers. However, damage of the cell membrane and cell lysis occurs during application of the electrical field and is accompanied by changes in surface charge which can be detected by free-flow electrophoresis. In this study, we evaluated free-flow electrophoresis to detect changes in cell viability after application of electric-field conditions employed in mammalian cell electrofusion and to separate dead cells and cell debris from intact unfused or fused cells.

Animals↗
PubMed1989

Efficient generation of stable antibody forming hybridoma cells by electrofusion.

A variety of modified electrofusion protocols designed to improve the efficiency of hybridoma production have recently appeared in the literature. We undertook to maximize the number of antibody secreting murine hybridomas by optimizing the temperature and fusion strength parameters of the conventional electrofusion technique. Anti-DNP secreting hybridomas were generated by fusing SP2/0 to immunized mouse splenic lymphocytes using an unmodified electrofusion protocol consisting of washing in a weakly conducting sorbitol fusion medium supplemented with bovine serum albumin, calcium and magnesium ions. This was followed by dielectrophoretic alignment and application of 3 short duration, high intensity field pulses in helical chambers. Optimal efficiencies of hybridomas were generated by the application of 2000 V/cm pulses at 25 degrees C (2.45 hybridomas x 10(-4) splenocytes) and as many as 63% of resulting hybridomas secreted anti-DNP monoclonal antibodies, the majority of which were IgG's. These data show that modification of the electrofusion protocol by pretreatment of fusion partners with proteolytic enzymes or the use of antigen bridging is not required for the successful and efficient production of specific monoclonal antibodies by electrofusion.

Animals↗