We established cytotoxic T-lymphocyte (CTL) lines against colonic epithelial cell line from LPLs of patients with ulcerative colitis by continuous stimulation with human lymphocyte antigen A (HLA-A) ...matched colonic epithelial cell lines and clones from LPLs using polyclonal stimulation with phytohemagglutinin. The established CTL lines and clones showed cytotoxicity against HLA-A-matched colonic epithelial cell line but not against HLA-mismatched colonic epithelial cell lines, and HLA-A-matched lung and esophagus cell lines. The CTL response was HLA class I-restricted and mediated by CD8-positive T-lymphocytes. Moreover, the CTL line showed cytotoxicity against autologous B-LCLs pulsed with peptides extracted from HLA-A-matched colonic epithelial cell line but not against other organ-derived peptides pulsed and unpulsed autologous B-LCLs. CTL lines and clones established from LPLs of patients with ulcerative colitis showed colon-specific and HLA class I-restricted killing of human colonic epithelial cell line, suggesting that these CTLs may play a role in colonic epithelial cell damage in some patients with ulcerative colitis.
In a yeast two-hybrid screening test for tobacco proteins that interact with TMV replicase using the helicase (H) domain as bait, a cDNA clone was selected that encodes a polyamine biosynthetic ...enzyme, arginine decarboxylase (ADC). In yeast cells, the C-terminal internal region of ADC interacted with the H domain. This observation was confirmed in vitro by far-Western blotting. Inhibition of the binding between the H domain and the IRnHEL (I region and N-terminus of helicase domain) region by ADC using a yeast three-hybrid assay suggested possible interference of the heterodimerization of 126 K and 183 K by ADC.
We studied the molecular genetic basis of a C1 inhibitor deficiency in a patient with type I hereditary angioneurotic edema using both the polymerase chain reaction and nucleotide sequencing. A ...single nucleotide change (T→A) at the GT 5′ donor splice recognition motif in the seventh intron of the C1 inhibitor gene was detected. A restriction site analysis of the C1 inhibitor gene in the patient’s family indicated that this mutation is correlated with a decreased level of C1 inhibitor activity. A northern blot analysis demonstrated C1 inhibitor mRNA to have a normal size, but its contents were reduced by about 50% compared with a normal subject. As the donor splice site is essential for an excising of the intron during mRNA processing, aberrant mRNA splicing may cause a rapid degradation of the transcript, thus resulting in the onset of hereditary angioneurotic edema.
We analyzed the regulation of immunoglobulin (Ig) production in short-term cultures of human (rib) bone marrow cells. In contrast to blood or tonsil cell cultures, large quantities of IgG and IgA, ...but not IgM, were secreted by unstimulated marrow cells. The addition of pokeweed mitogen or phytohemagglutinin resulted in the suppression of this Ig secretion. Both mitogens induced the production of high levels of interleukin 2 (IL 2) in marrow cultures, and the addition of IL 2 alone mimicked the suppressive effect of mitogens. Incubation of marrow cells with Epstein Barr virus resulted in enhanced Ig secretion, primarily of the IgM isotype. The addition of mitogen or IL 2 suppressed Ig production in these cultures as well. The mitogen-induced suppression of Ig secretion in stimulated or unstimulated marrow cultures was inhibited by the monoclonal anti-TAC (IL 2 receptor) antibody. Cell separation experiments indicated that the induction of suppressor activity in marrow cultures involved two distinct populations of marrow-resident T lineage cells. The first population responds to activation by mitogens with the production of IL 2. This population has a surface phenotype appropriate for helper T cells. The second T cell population expresses T8 and TAC determinants. These cells acquire suppressor cell activity after exposure to IL 2. The expression of suppressor function does not require additional (e.g., mitogenic) activation signals. The IL 2-dependent marrow suppressor T cells represent a newly recognized T lymphocyte subset. The regulatory pathway delineated may be important for the regulation of antibody formation in bone marrow, the major site of Ig production in man.
We established CTL lines and clones against colonic epithelial cells from PBLs of patients with ulcerative colitis by continuous stimulation with HLA-A locus-matched colonic epithelial cell lines. We ...developed a nonradioactive europium release cytotoxicity assay to detect CTLs. PBLs from 3 of 12 patients but not from any of 14 normal controls who shared at least one haplotype of HLA-A locus with two colonic epithelial cell lines, CW2 and ACM, showed increased cytotoxicity against these lines. Three CTL lines established from the PBLs of patients showed increased cytotoxicity against HLA-A locus-matched CW2 or ACM but not against matched lung or esophagus cell lines. The phenotypes of CTL lines were alpha beta-TCR+ CD3+ CD8+ CD16-. The CTL line MS showed increased cytotoxicity against freshly isolated colonic epithelial cells but not against cells with a different HLA-A locus. Two CTL clones were generated from MS and clone 3-2, expressing CD3+ CD8+ CD4- CD56-, showed high MHC class I-restricted cytotoxicity against the colonic epithelial cells. These results indicated that CTLs against colonic epithelial cells may contribute to epithelial cell damage in ulcerative colitis.
It is conceivable that brush border enzyme activities of the intestinal mucosa will change when bacterial toxins are exposed to the intestinal microvillous membranes. The effect of cholera toxin on ...the activity of intestinal alkaline phosphatase in rats was therefore determined in the intestinal mucosa by the histochemical method as well as in intestinal lymph by using lymph fistulated-rats. Activity of intestinal alkaline phosphatase in the intestinal mucosa and lymphatics changed biphasically after the oral administration of cholera toxin to rats. For the first three hours after the administration of cholera toxin it was depressed; it then increased and at eight hours reached a maximum. These changes in the activity of intestinal alkaline phosphatase were prevented by the administration of chlorpromazine, a known inhibitor of adenylate cyclase activity.
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