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T lymphocytes expressing NK1.1 marker (NKT) have been suggested to play crucial roles in immune modulation. Aim.– To determine the role of NK1.1+ cells in induction and maintenance of pro-inflammatory and/or tolerizing responses. Methods.– Colitis was induced in C57/B6 donor mice by intracolonic instillation of trinitrobenzenesulfonic acid (TNBS). Donor mice received five oral doses of colonic proteins extracted from TNBS-colitis colonic wall. Depletion of NK1.1+ lymphocytes was performed before lymphocyte harvesting. Splenocytes were harvested and separated into T-cell subpopulations, and transplanted into recipient mice before intracolonic instillation of TNBS. Standard clinical, macroscopic, and microscopic scores, and intracellular staining, flow cytometry, and cytotoxicity assays were performed. Results.– The adoptive transfer of CD4+ and NK1.1+ cells harvested from tolerized mice markedly ameliorated the colitis in recipient mice. In contrast, the adoptive transfer of CD8+ and double negative lymphocytes failed to transfer the tolerance. Recipients of splenocytes from tolerized mice exhibited an increase in CD4+IL4+/CD4+IFNγ+ ratio. In contrast, recipients of splenocytes from NK1.1-depleted-tolerized mice exhibited severe colitis with a significant decrease of the CD4+IL4+/CD4+IFNγ+ ratio. However adoptive transfer of splenocytes from non-tolerized NKT-depleted mice led to an alleviation of colitis with a relative increase of the CD4+IL4+/CD4+IFNγ+ ratio. Conclusions: NK1.1+ lymphocytes play a critical role in immune regulation. They may be accountable for an alteration of the inflammatory response and the CD4+IL4+/CD4+IFNγ ratio immune-mediated colitis and in peripheral tolerance induction.

Oral tolerance is the induction of immune hyporesponsiveness to orally administered antigens. It was described in association with a decrease in interferon gamma (IFNgamma) production by activated T cells. To determine the role of IFNgamma and IL10 in immunemodulation via oral tolerization. Colitis was induced by intracolonic instillation of trinitrobenzene sulfonic acid. Treated mice received five oral doses of colitis-extracted proteins (CEPs) every other day, starting immediately after colitis induction. Control mice received similar doses of bovine serum albumin. Colitis was assessed in both groups by standard clinical, micro- and macroscopic scores. IFNgamma and IL10 expression in splenic lymphocytes from both groups was tested by RT-PCR immediately after oral feeding, 1, 4, 8, 12, and 24 h thereafter and then every 24 h for 2 weeks. Feeding of CEPs markedly ameliorated experimental colitis. These mice gained weight and showed markedly improved macro- and microscopic parameters of colitis. Tolerized mice exhibited IFNgamma expression in splenic lymphocytes starting immediately following oral CEP immunization and up to 14 d thereafter. IL10 was expressed starting 1 h after CEP feeding and during the first 48 h thereafter. In contrast, non-tolerized control mice manifested IFNgamma expression starting on day 6 and had no IL10 expression. Early induction of IFNgamma expression by oral antigen may be associated with systemic tolerance in the experimental colitis setting. In contrast, late expression of IFNgamma is associated with a pro-inflammatory response in non-tolerized controls.

Cyclin D1 plays an important role in the multi-step process of gastrointestinal tumorigenesis. We hypothesize that normal enterocytes over-expressing Cyclin D1 will demonstrate a transformed phenotype. The nontumorigenic intestinal epithelial cell line, IEC-18, was transfected with the vector pMV7-CCND1, encoding Cyclin D1. Three clones, with cyclin D1 levels similar to those seen in colon cancer cell lines, were further evaluated in comparison to the vector control cells. They proliferated faster and demonstrated anchorage-independent growth in soft agar, higher saturation density, and higher plating efficiency. When injected into nude mice, tumors were generated after 6–8 weeks. On the other hand these cells were more sensitive to induction of apoptosis. There was no change in the level of β-catenin protein. In conclusion, Cyclin D1 can act as an oncogene in vitro and in vivo, when produced in immortalized normal intestinal epithelial cells. This model may be useful for understanding the role and interrelationships of Cyclin D1 in colorectal tumorigenesis.

Recognition of thrombotic thrombocytopenic purpura (TTP)/hemolytic uremic syndrome (HUS) following bone marrow transplantation (BMT) has increased in recent years. The use of plasma exchange has greatly improved the outlook of de novo TTP. Fewer data are available on its use in post-BMT TTP but small uncontrolled series showed poor results with low response rates. We present here a case of a young patient who developed manifestations of TTP 10 months after BMT with complete recovery following treatment with plasma exchange for 1 month. This case suggests that plasma exchange could be life saving and should be tried in every patient with post-BMT TTP.

Cyclin D1 plays an important role in the multi-step process of gastrointestinal tumorigenesis. We hypothesize that normal enterocytes over-expressing cyclin D1 will demonstrate a transformed phenotype. The nontumorigenic intestinal epithelial cell line, IEC-18, was transfected with the vector pMV7-CCND1, encoding cyclin D1. Three clones, with cyclin D1 levels similar to those seen in colon cancer cell lines, were further evaluated in comparison to the vector control cells. They proliferated faster and demonstrated anchorage-independent growth in soft agar, higher saturation density, and higher plating efficiency. When injected into nude mice, tumors were generated after 6-8 weeks. On the other hand these cells were more sensitive to induction of apoptosis. There was no change in the level of beta-catenin protein. In conclusion, cyclin D1 can act as an oncogene in vitro and in vivo, when produced in immortalized normal intestinal epithelial cells. This model may be useful for understanding the role and interrelationships of cyclin D1 in colorectal tumorigenesis.

Background: Many colon cancers harbor mutations in the APC/beta-catenin pathway, leading to actwation of downstream genes with beta-catenin/T-cell factor (Tci)responsive promoters. The ultimate therapy of cancer is by targeting neoplastic cells without damaging normal cells. Aim: To selectively kill beta catenin transformed cells by up-regulation of lethal genes using a mutated signal transduction pathway. Materials & Methods: A Xbal fragment contains the TOP/FOP-cFos sequence was cut out from the TOP/FOP flash plasmids (a gift from Hans Clevers) and then cloned to the pGL3-basic vector (Promega), into the Nhel site. The resultant plasmid was designated as pGL3-TOP/FOP. Then, the Luc gene was cut out from this plasmid by Hindlll-Xbal digestion and a cloning site linker was inserted instead of the Luc gene to make the plasmid pGL3-TOP/FOP-HX. The TOP/FOP-HX was used to construct 3 separate genes: Bax, caspase 8 and PKG. The new plasmids were designated as TOP/ FOP-Bax, TOP/FOP-Cas8, and TOP/FOP-PKG. Exponentially growing SW 480 cells, with increased expression of beta~catenin, were transiently transfected with these constructs and then treated for 48 and 72hr with different dosages of a specific COX 2 inhibitor (Celecoxib, 0-50uM). Results: The growth of SW 480 cells was not hampered by the transfection with the FOP plasmids. The TOP-PKG and TOP-Bax plasmids inhibited cell growth by approximatly 20-35%, respectively, while the plasmid TOP-cas8 did not inhibit cell growth. The growth inhibition was associated with induction of apoptosis.Celecoxib inhibited the growth of SW480 ceils The addition of celecoxib to the TOP-Bax/cas8/PKG transfected cells, augmented the growth inhibition in a time and dose dependent manner. Conclusions: 1. This approach may be used to develop therapies to target tumor cells that have defects in the Wnt/beta-catenin/Tcf signal transduction pathway. 2. Selective over-expression of some pro-apoptotic genes, in beta-catenin transformed cells increase their sensitivity to celeco:db. 3. In this model Bax and PKG but not caspase 8 could serve as potential targets, to selectively induce turner cell death.