Development of antigen induced colitis in SCID mice reconstituted with spleen derived memory type CD4(+) CD45RB(+) T cells.

BACKGROUND AND AIMS: Enteric bacterial and/or food antigens may be crucial in the development of colitis but little is known of the exact mechanism of antigen specific reactions in this condition. The aim of this study was to determine whether systemically primed antigen specific CD4(+) T cells containing both CD45RB(high) and CD45RB(low) populations participate as a pathogenic subset that in turn leads to inflammatory reactions selectively in the large intestine. METHODS: SCID mice were reconstituted with splenic CD4(+) CD45RB(+) T cells or CD4(+) CD45RB(low) T cells isolated from donor mice systemically primed with ovalbumin (OVA) plus CFA. The reconstituted mice were then fed OVA for several weeks. RESULTS: Reconstitution of SCID mice with OVA primed splenic CD4(+) T cells, containing populations of CD45RB(high) and CD45RB(low), resulted in the development of colitis by 4-5 weeks following repeated administration of oral OVA. Histopathological study revealed thickened wall, inflammatory cell infiltration, crypt elongation, and loss of goblet cells in the large intestine. The CD4(+) CD45RB(low) population of cells extracted from the affected large intestine secreted high levels of interferon gamma (IFN-gamma) and tumour necrosis factor alpha (TNF-alpha) at the protein and mRNA levels. Administration of neutralising antibodies to TNF-alpha, but not to IFN-gamma, prevented the development of colitis. Furthermore, adoptive transfer with OVA primed splenic CD4(+) CD45RB(low) T cells evoked severe colitis. CONCLUSIONS: These results demonstrate that systemically primed activated/memory CD4(+) CD45RB(low) T cells can mediate the development of specific antigen induced colitis in SCID mice, and also that TNF-alpha is critical in the induction of this type of colitis. Our results contrast with those from studies in some colitis models in which CD45RB(low) T cells appeared to prevent colitis through secretion of immunosuppressive cytokines.

Fifty-seven-kDa protein in royal jelly enhances proliferation of primary cultured rat hepatocytes and increases albumin production in the absence of serum.

We have previously shown that 57-kDa protein in royal jelly (RJ) was specifically degraded in proportion to both storage temperature and storage period, and we suggested that it could be useful as a marker of freshness of RJ (Kamakura, M., Fukuda, T., Fukushima, M. and Yonekura, M. (2001) Biosci. Biotechnol. Biochem. 65, 277-284.). Here, we investigated the physiological effects of 57-kDa protein on primary cultured rat hepatocytes in the absence of serum. The 57-kDa protein and RJ significantly stimulated hepatocyte DNA synthesis, whereas bovine serum albumin and RJ stored at 40 degrees C for 7 days, which lacks 57-kDa protein, did not. The mitogenic activity of 57-kDa protein was lost after treatment with trypsin. These results indicate that 57-kDa protein acts as a mitogen. The stimulatory effect of 57-kDa protein was dose-dependent and was more potent at lower than at higher cell densities. The 57-kDa protein also prolonged the cell proliferation of primary cultured rat hepatocytes, with an enhancement of albumin production compared to untreated cells. Therefore, 57-kDa protein is likely to promote liver regeneration and may have a cytoprotective action on hepatocytes.

Natriuretic peptides and nitric oxide induce endothelial apoptosis via a cGMP-dependent mechanism.

Apoptosis is a mode of cell death in which the cell participates in its own demise. We studied whether endothelium-derived relaxing factor, nitric oxide (NO), and natriuretic peptides affect apoptosis of rat vascular endothelial cells via a cGMP-dependent pathway and whether such effects are antagonized by an endothelium-derived vasoconstrictor, endothelin-1 (ET-1). Three natriuretic peptides (atrial natriuretic peptide, brain natriuretic peptide, and C-type natriuretic peptide) induced endothelial apoptosis as demonstrated by nucleosomal laddering on agarose gel electrophoresis and by the terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling method. This dose-dependent relation was assessed by quantifying the fragmented and intact DNA contents by the diphenylamine method. The atrial natriuretic peptide-induced endothelial apoptosis was completely blocked by a guanylate cyclase-coupled receptor antagonist (HS-142-1) and an inhibitor of cGMP-dependent protein kinase (KT5823). An NO donor, NOR3 ((+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexeneamide; FK409) also induced endothelial apoptosis; the effect of this compound was abrogated by KT5823 and an inhibitor of soluble guanylate cyclase, ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one). A cGMP derivative, 8-bromo-cGMP, but not the cAMP derivative 8-bromo-cAMP, caused endothelial apoptosis; the effect of ODQ was also abrogated by KT5823. Endothelial apoptosis induced by ANP, NOR3, and 8-bromo-cGMP was similarly antagonized by ET-1. ANP, NOR3, and 8-bromo-cGMP caused marked accumulations of the tumor suppressor gene product p53 but not of bcl-2, as determined by Western blot analysis. These results demonstrate for the first time that endothelium-derived NO and natriuretic peptides are proapoptotic factors for endothelial cells, whereas the endothelium-derived vasoconstrictor ET-1 is an antiapoptotic factor, suggesting that the countervailing balance between these vasodilators and vasoconstrictors, in addition to regulation of vascular tonus, may contribute to endothelial cell integrity.

Stimulative effect of non-parenchymal liver cells on ability of tyrosine aminotransferase induction in hepatocytes.

Hepatocytes and non-parenchymal liver cells were isolated from adult rat liver and co-cultured for 48 hours as a monolayer on polystyrene culture dishes. The ability of tyrosine aminotransferase (TAT) induction in hepatocytes was examined in the presence of dexamethasone and dibutyryl cAMP. Non-parenchymal cells greatly enhance the ability of TAT induction of hepatocytes. A soluble factor with molecular weight of more than 10,000 is responsible for this enhancement, because conditioned medium prepared from non-parenchymal cells is also stimulatory. Non-parenchymal cells restored the ability in hepatocytes damaged with the addition of D-galactosamine. Conditioned medium prepared from non-parenchymal cells treated with D-galactosamine had higher activity of enhancement than the medium from normal cells. The soluble factor might be released in response to some signal of injury. Hepatocytes and non-parenchymal cells were immobilized within Ca-alginate, and although immobilized hepatocytes rapidly lost the ability to induce TAT, hepatocytes co-immobilized with non-parenchymal cells maintained the ability during 4 days of culture. These results indicated that non-parenchymal liver cells, as well as hepatocytes, could be used to construct a bioartificial liver support system.