Changes in lymphokine receptor expression and fatty acid composition of phospholipids and triacylglycerols in rat adipocytes associated with lymph nodes following a transient immune challenge.

Single-photon counting fluorimetry was used to record the time course of the expression of interleukin-10 receptors labelled with fluorescent antibodies on the surface of adipocytes over 24h, following an immune challenge to the rat popliteal lymph node. Homologous perinodal and remote-from-node samples from the stimulated and unstimulated popliteal depots were compared in rats fed on plain chow and chow supplemented with 10% w/w suet, fish or vegetable oils. Receptor expression was maximal 6 h after stimulation, and returned to baseline after 24 h, and was similar in the stimulated and unstimulated depots. Fewer receptors were elicited in tissues from rats fed lipid-supplemented diets compared with the control diet, with fewest of all following the fish oil diet. These data suggest that interleukin-10 is involved in local interactions between perinodal adipocytes and lymph node lymphoid cells. Both triacylglycerols and phospholipids contained more polyunsaturates and fewer saturates in perinodal adipose tissue than in samples from sites not associated with lymphoid tissue. These data are consistent with paracrine interactions between perinodal adipocytes and activated lymphoid cells.

8-Cl-adenosine is an active metabolite of 8-Cl-cAMP responsible for its in vitro antiproliferative effects on CHO mutants hypersensitive to cytostatic drugs.

8-Cl-cAMP has been undergoing clinical trials as a potential chemotherapy agent, but there is much discussion in the literature as to whether the active agent is 8-Cl-cAMP itself, or its major metabolite, 8-Cl-adenosine. 8-Cl-cAMP is susceptible to the action of serum enzymes such as phosphodiesterases, and its metabolism when administered to cancer patients raises questions as to the mechanism of action of 8-Cl-cAMP. The stability of 8-Cl-cAMP when incubated with serum, and the effects of both 8-Cl-cAMP and 8-Cl-adenosine on the proliferation of variant lines of CHO cells hypersensitive to 8-Cl-cAMP were investigated. A solid-phase extraction (SPE) purification protocol and the HPLC method previously developed were used to determine 8-Cl-cAMP and 8-Cl-adenosine. Heat treatment of serum inactivated the enzymes in the culture medium responsible for activating 8-Cl-cAMP. Under these conditions 8-Cl-cAMP remained stable and there were no traces of its metabolite, 8-Cl-adenosine. Cell culture experiments showed that 8-Cl-cAMP only affected cell growth in medium that contained untreated serum. In contrast, 8-Cl-adenosine was shown to be growth inhibitory in medium containing either heat-treated or untreated serum. HPLC analysis of the culture medium from the cell culture experiments supported the hypothesis that 8-Cl-cAMP was only effective in inhibiting cell growth after metabolism to 8-Cl-adenosine. Thus further studies of this drug and its mechanism of action should focus on 8-Cl-adenosine.

Hydrophobic protein that copurifies with human brain acetylcholinesterase: amino acid sequence, genomic organization, and chromosomal localization.

The mechanism of attachment of acetylcholinesterase (AChE) to neuronal membranes in interneuronal synapses is poorly understood. We have isolated, sequenced, and cloned a hydrophobic protein that copurifies with AChE from human caudate nucleus and that we propose forms a part of a complex of membrane proteins attached to this enzyme. It is a short protein of 136 amino acids and has a molecular mass of 18 kDa. The sequence contains stretches of both hydrophobic and hydrophilic amino acids and two cysteine residues. Analysis of the genomic sequence reveals that the coding region is divided among five short exons. Fluorescence in situ hybridization localizes the gene to chromosome 6p21.32-p21.2. Northern blot analysis shows that this gene is widely expressed in the brain with an expression pattern that parallels that of AChE.

Production and characterization of separate monoclonal antibodies to human brain and erythrocyte acetylcholinesterases.

Four murine monoclonal antibodies (MAbs) of the IgM class were raised against human acetylcholinesterase (AChE; Ec 3.1.1.7). The MAbs BMS-3E4, BMS-7G10, and BMS-9F4 all recognized human erythrocyte AChE, while BMS-6D6 bound specifically to human soluble brain AChE, on the basis of immunobinding assays. Dose-response studies gave an ELISA ED50 titer of 4.5 x 10(-4) M for BMS-6D6, while BMS-3E4 gave the best titer at 8.8 x 10(-4) M. Sucrose density gradients demonstrated sedimentation of antigen-antibody complexes, consistent with earlier findings (i.e., BMS-6D6 bound with brain AChE while BMS-3E4 preferred erythrocyte (AChE). No cross-reactivity between the two MAbs against the two antigens was noted.

Comparison of 5-aminosalicylic acid and N-acetylaminosalicylic acid uptake by the isolated human colonic epithelial cell.

Isolated human colonic epithelial cell suspensions were incubated with either 0.1 mM 5-aminosalicylic acid (5-ASA) or 0.1 mM acetylaminosalicylic acid (Ac-ASA) for up to two hours. Intra- and extracellular 5-ASA and Ac-ASA were measured by high performance liquid chromatography. Mean 5-ASA uptake in one hour was 160.5 nmol/g dry weight, compared with an Ac-ASA uptake of only 5.75 nmol/g dry weight. No unchanged 5-ASA was detected inside the cell. Repeated washing had no effect on the intracellular Ac-ASA concentration. This discrepancy in drug uptake may explain why Ac-ASA seems to be ineffective when given to patients with ulcerative colitis.

Characterisation and quantification of mucosal cytokine that induces epithelial histocompatibility locus antigen-DR expression in inflammatory bowel disease.

Epithelial histocompatibility locus antigen (HLA) class II expression was studied to evaluate its induction by mucosal mononuclear cells in inflammatory bowel disease and to characterise the responsible cytokine. Unstimulated cells of the HT-29 epithelial cell line did not produce class II molecules. After being stimulated with the mitogenic lectin phytohaemagglutinin mucosal mononuclear cells released a cytokine that induced epithelial HLA-DR expression. The cytokine had the physicochemical and immunological characteristics of interferon-gamma, and no additional cytokines were detected.

Production of epithelial cell growth factors by lamina propria mononuclear cells.

The effects of lamina propria mononuclear cell culture supernatant on epithelial cell DNA synthesis were studied using cells isolated from patients with inflammatory bowel disease and normal controls. Supernatants from resting and phytohaemagglutinin stimulated cells were studied and supernatants that strongly promoted DNA synthesis were pooled, and growth factor activity partially characterised. The effects of recombinant interleukins-1 beta,2,3,interferon-gamma, and granulocyte macrophage colony stimulating factor were tested in the same system. Resting lamina propria mononuclear cells produce factors that increase DNA synthesis. Production of these factors is increased by phytohaemagglutinin stimulation. No significant differences were found in production of these factors between patients with inflammatory bowel disease and normal controls. The molecular weight of the active factor(s) lies in the region 31-48 kD. Chromatofocusing produced two peaks of activity, one in the region pk 5.5 and one around pk 6.4. The activity was heat and acid pH labile. Activity was not destroyed, however, by 0.05% trypsin. Recombinant granulocyte macrophage colony stimulating factor was a weak stimulus to epithelial DNA synthesis, interleukin-1 beta was weakly inhibitory but other cytokines tested did not have any effect. Granulocyte macrophage colony stimulating factor is probably important in controlling epithelial cell growth.

Serum and tissue autoantibodies to colonic epithelium in ulcerative colitis.

Sera and colonic tissue-bound immunoglobulin extracts from patients with ulcerative colitis and disease controls were examined immunohistochemically and by killer cell cytotoxicity assay for the presence of anticolonic epithelial autoantibodies. IgG yields in the tissue extracts from patients with colitis and control subjects were similar, and the extracts were uniformly autoantibody negative. Of 41 sera from patients with inflammatory bowel disease, 'classical' anticolon antibody was present in 41% and was commoner in patients with sclerosing cholangitis. Cytotoxic anticolon antibody was present in 20% overall and was strongly associated with disease activity; it did not correlate with the presence of 'classical' anticolon antibody. The heterogeneous and non-universal antiepithelial autoantibody response and the failure to detect tissue bound autoantibody in vivo argue against the hypothesis that humoral autoimmunity is of major importance in the pathogenesis of ulcerative colitis.

Anomalous molecular form of acetylcholinesterase in cerebrospinal fluid in histologically diagnosed Alzheimer's disease.

The possibility that the different molecular forms of acetylcholinesterase (AChE) in cerebrospinal fluid (CSF) which can be revealed by isoelectric focusing may reflect changes in AChE in pathologically affected neurons in Alzheimer's disease was tested in a retrospective study. CSF samples obtained at necropsy from 33 patients with clinically diagnosed dementia, 9 with possible dementia, and 19 without a diagnosis of dementia were examined by isoelectric focusing. An additional band indicating an anomalous molecular form of AChE was present in CSF from 19 of 23 patients with a histological diagnosis of Alzheimer's disease and no other central nervous system disorder but in none of the 19 non-demented patients (without a histological diagnosis of Alzheimer's disease). The band was also present in 2 of 8 patients with histologically defined Alzheimer's disease plus other neurological disorders and in 4 of 8 patients with possible dementia who did not meet histopathological criteria for Alzheimer's disease. The absence of the anomalous form of AChE from the CSF of non-demented patients and its presence in the CSF of the majority of patients with Alzheimer's disease has implications for our understanding of the biological basis of the disease and might form the basis of an antemortem diagnostic test.

Cellular hypersensitivity to a synthetic dodecapeptide derived from human adenovirus 12 which resembles a sequence of A-gliadin in patients with coeliac disease.

The human intestinal adenovirus serotype 12 (Ad12) may be implicated in the pathogenesis of coeliac disease by virtue of immunological cross reactivity between epitopes shared by its early region E1b protein and A-gliadin. In the present study a synthetic dodecapeptide from the corresponding viral epitope (Ad12E1b, residues 384-395) was tested for its effect on peripheral blood mononuclear cells from 22 treated and eight untreated patients with coeliac disease, 22 healthy subjects, 11 patients with ulcerative colitis, and 11 patients with Crohn's disease by an indirect leucocyte migration inhibition assay. In addition, the effect of both the viral and the gliadin synthetic peptides was studied by proliferation and migration assays simultaneously performed in an unselected subgroup of 12 treated coeliac patients and 12 healthy subjects of the study. Coeliac patients with untreated disease showed no response to the viral peptide compared with treated patients (p greater than 0.1). Treated coeliac patients showed a significantly different response from healthy control subjects and control patients with disease (p less than 0.001) which was dependent on the concentration of the viral peptide. In the subgroup of the treated coeliac patients (n = 12) there was a significant correlation between the responses in the migration and the proliferation assay using either the viral (p less than 0.02) or the gliadin (p less than 0.005) peptide at the highest concentration (33.3 micrograms/ml). Furthermore, the responses obtained using viral peptide correlated significantly with the responses obtained with gliadin peptide in both the migration (p less than 0.001) and the proliferation (p less than 0.001) assays. These results show that in coeliac patients there is pronounced cross reactivity at the level of T cell recognition between synthetic peptides derived from the Ad12 and A-gliadin. This antigenic cross reactivity may be involved in the pathogenesis of coeliac disease.

T lymphocyte responses to a synthetic peptide from the human intestinal adenovirus 12 in coeliac disease.

Peripheral blood mononuclear cells of treated coeliac patients are known to liberate leucocyte migration inhibition factor when challenged with a synthetic dodecapeptide of the E1b protein of adenovirus 12 sharing sequence homology with A gliadin. This study has compared the response of the unfractionated peripheral blood mononuclear cells with a highly purified T cell population. Coeliac mononuclear cells and T lymphocytes showed identical leucocyte migration inhibition activity. The migration indices for the coeliac group were significantly different than for the control group irrespective of the cell population tested. This work justifies the use of the indirect leucocyte migration inhibition assay using mononuclear cell supernatants as a test of in vitro cell-mediated immunity to clearly defined antigens in coeliac disease. It also offers further evidence for the possible implication of adenovirus 12 in the pathogenesis of coeliac disease.

Effect of human intestinal macrophages on immunoglobulin production by human intestinal mononuclear cells isolated from patients with inflammatory bowel disease.

The effect of macrophages on spontaneous immunoglobulin production by isolated human intestinal mononuclear cells (MNC) is unknown. Depletion of macrophages by adherence to fibronectin or by panning with macrophage-specific monoclonal antibody 3C10 lead to a significant reduction in IgA. IgG and IgM production by intestinal MNC from both normal (n = 10) and inflammatory bowel disease (IBD) (n = 13) mucosa. The reduction in immunoglobulin produced by macrophage-depleted intestinal MNC was greater in IBD patients than in normal controls. There was a significant correlation (r = 0.816, P less than 0.001) between the percentage of macrophages depleted by panning with 3C10 and the reduction in IgG produced by macrophage-depleted intestinal MNC. Addition of either fibronectin-adherent cells or the supernatant from these macrophage-enriched cells enhanced immunoglobulin production in a dose-dependent fashion. A greater increase in IgG production by macrophage-depleted cells was seen when cultured with supernatant from inflamed IBD mucosal cells, compared with that from normal mucosal cells. The soluble factor(s) responsible in the supernatant was acid and heat susceptible but was not affected by freezing and thawing. Addition of recombinant human interleukin-1 beta or human interferon-gamma to cell cultures did not influence immunoglobulin production. Thus, human intestinal macrophages enhance spontaneous immunoglobulin production by isolated intestinal MNC by secreting soluble factor(s) which remain to be fully characterized.

Acetylation of 5-aminosalicylic acid by isolated human colonic epithelial cells.

1. This study investigates the acetylation of 5-aminosalicylic acid by isolated human colonic epithelial cells. 2. After incubation of intact cells with 0.1 mmol/l 5-aminosalicylic acid, N-acetyl-5-aminosalicylic acid was detected in a concentration of 141 (+/- 23.8 SEM) nmol/g dry weight in the incubation medium, and 34.8 (+/- 5.5 SEM) nmol/g dry weight intracellularly. No unchanged 5-aminosalicylic acid was detected inside the cell. 3. Acetylation of 5-aminosalicylic acid by a cell homogenate was very poor, but the addition of 1 mmol/l acetyl-CoA resulted in complete conversion of 0.1 mmol/l 5-aminosalicylic acid to N-acetyl-5-aminosalicylic acid. 4. N-Acetyltransferase activity was detected in the cytosol, with a mean of 3.3 nmol min-1 mg-1 of protein. There was no N-acetyltransferase activity in the brush border. There was no difference in enzyme activity between epithelial cells derived from normal, Crohn's disease or ulcerative colitis patients. 5. Preliminary characterization of the N-acetyltransferase enzyme suggests a molecular weight of 150,000.

Immunoglobulin production by isolated intestinal mononuclear cells from patients with ulcerative colitis and Crohn's disease.

We studied immunoglobulin production by isolated intestinal mononuclear cells from 25 patients with active inflammatory bowel disease (IBD) and 17 controls undergoing surgical resections for intestinal tumour or other disorders. Normal ileal intestinal mononuclear cells spontaneously produced greater amounts of IgA and IgM than did normal colon cells. In cells from patients with IBD there was a significantly reduced IgA production, but production of IgG was enhanced in both colon and ileum. The alteration in IgA and IgG production in IBD was confirmed by comparing the immunoglobulin production by mononuclear cells from inflamed with that from non-inflamed areas of mucosa in six patients with distal ulcerative colitis. The proportion of IgA-containing cells in isolated intestinal mononuclear cells from IBD mucosa was less than normal. However, the proportion of IgG-containing cells from IBD mucosa was not increased in isolated intestinal mononuclear cells although they produced more IgG than normal mucosal cells. Our study showed an altered pattern of immunoglobulin production by intestinal mononuclear cells isolated from patients with inflammatory bowel disease.

Cellular hypersensitivity to gluten derived peptides in coeliac disease.

Wheat gluten derived antigens have been tested for their ability to inhibit the migration of leucocytes from healthy subjects and patients with coeliac disease. Three preparations of a water soluble fraction (Frazer's fraction III, FIII) of partial peptic tryptic digests of wheat gluten had different effects in a direct (one stage) assay. Subfractions B and B2 caused migration inhibition of leucocytes from patients with treated coeliac disease but not of leucocytes from healthy volunteers or patients with Crohn's disease or ulcerative colitis. This migration inhibition seems to be specific for gluten fractions because maize zein fraction B, beta-lactoglobulin and ovalbumin did not cause it. The sensitivity of coeliac leucocytes to fraction B is not related to factors present in coeliac serum as the migration of leucocytes from healthy individuals preincubated with coeliac sera was not inhibited. Puromycin diminished inhibition by fraction B, which was active at 1.2 micrograms/ml in an indirect (two stage) migration inhibition assay; this is consistent with a process involving elaboration of lymphokine(s). More highly purified fractions of B2, P1-P4 were prepared by reverse phase high performance liquid chromatography (HPLC) and showed differing potency in direct and indirect assays, with P4 being the most active fraction. Inhibition of migration by gluten derived peptides appears to result from the release of lymphokine by leucocytes specifically from coeliac patients.

Cell-mediated immunity to a synthetic gliadin peptide resembling a sequence from adenovirus 12.

Cell-mediated immunity to a synthetic peptide, which has a 12-residue sequence from A-gliadin analogous to part of an early-region protein (Elb) from adenovirus 12, was investigated in patients with coeliac disease, healthy subjects, and disease controls by means of an indirect leucocyte-migration-inhibition assay. Patients with coeliac disease being treated with a gluten-free diet showed a significantly greater response than healthy subjects (p less than 0.001) or patients with inflammatory bowel disease. This cellular immune response was dependent on antigen concentration and was not present in untreated coeliac patients.

Effects of olsalazine in the jejunum of the rat.

Olsalazine (ADS) is the azo-linked dimer of 5-aminosalicylic acid (5-ASA). It is of value for the management of patients with ulcerative colitis but may be associated with increasing diarrhoea in a few. This study examines the effect of 5-ASA and ADS on small intestinal transport systems of the rat. Krebs-Ringer-bicarbonate solution was circulated through the lumen of a jejunal segment and the appearance of fluid, glucose and lactate on the serosal surface was shown to be linear over a two hour period. Addition of 5-ASA (10 mmol/l) or ADS (5 mmol/l and 10 mmol/l) caused a significant inhibition both of fluid transport (p less than 0.001), and of the appearance of glucose (p less than 0.001) and lactate (p less than 0.001 for 5 mmol/l and 10 mmol/l ADS, p less than 0.01 for 10 mmol/l 5-ASA). The uptake of glucose by rings of rat jejunum was shown to be markedly reduced by ADS. Experiments substituting glucose with either sucrose of 2-aminoisobutyric acid showed that ADS (5 mmol/l, 10 mmol/l) also inhibited the serosal appearance of fructose and the amino acid. These results show that 5-ASA and ADS, at concentrations which could be expected in the jejunum of patients receiving therapeutic doses, are able to inhibit small intestinal transport systems. The resulting increase in load on the diseased colon could be important for the pathogenesis of diarrhoea.

Colonic bacterial proteases to IgA1 and sIgA in patients with ulcerative colitis.

The colonic faecal and mucosal associated bacterial populations of five patients with ulcerative colitis and four control patients were studied in detail to assess their ability to produce IgA1-proteases. A total of 330 bacterial strains were isolated from the patients with ulcerative colitis and IgA1-protease activity was unable to be reliably shown in any. It is therefore unlikely that such enzyme production by colonic bacteria plays a significant role in the pathogenesis of ulcerative colitis.