Human beta-defensin-3 induction in H. pylori-infected gastric mucosal tissues.

AIM: To examine human beta-defensin-3 (hBD-3) expression in inflamed gastric mucosal tissues or MKN45 gastric cancer cells with or without H. pylori infection for better understanding the innate immune response to H. pylori. METHODS: We used reverse transcription-polymerase chain reactions and immunohistochemistry to examine hBD-3 expression in inflamed gastric mucosal tissues or MKN45 gastric cancer cells with or without H. pylori. Effects of hBD-3 against H. pylori were also evaluated. RESULTS: The mean mRNA expression of hBD-3 in H. pylori-positive specimens was significantly higher than that in H pylori-negative specimens (P = 0.0002, Mann-Whitney). In addition, unlike uninfected samples, 8 of 15 (53.33%) infected mucosal samples expressed hBD-3 protein. H. pylori dose-dependently induced mRNA expression of hBD-3 in MKN45 cells, an effect inhibited by adding anti-toll-like receptor (TLR)-4 antibody. HBD-3 protein completely inhibited H. pylori growth. CONCLUSION: Our results suggest that like hBD-2, hBD-3 may be involved in the pathophysiology of H. pylori-induced gastritis.

Identification of transformation-related antigen by monoclonal antibody on Swiss 3T3 cells induced by transfection with murine cultured colon 36 tumor DNA.

The cell surface antigen associated with the transformed state of cells that could grow in an anchorage-independent manner was analyzed by use of techniques of DNA transfection and hybridomas secreting the monoclonal antibody (MoAb). Spleen cells of C57BL/6 mice immunized with a highly tumorigenic, chemically induced murine cultured colon 36 tumor (C-C36) of BALB/c origin were hybridized with NS-1, a hypoxanthine phosphoribosyltransferase-deficient myeloma line of BALB/c mice. Screening of hybridomas revealed an antibody that reacted with C-C36 and transformed Swiss 3T3 cells growing in soft agar after transfection of 3T3 cells with C-C36 DNA. The hybridomas that did not react with nontransformed 3T3 and the less tumorigenic BALB/c hemangioendothelioma line D10 were then selected. An MoAb was designated "#71295." This MoAb immunoprecipitated the antigen that consisted of 65,000- and 14,000-molecular-weight components with soluble C-C36 membrane antigens. It also reacted with 2 other chemically induced syngeneic colon tumor lines, cultured colon 26 tumor line and cultured colon 51 tumor line, and with fibrosarcoma Meth A. However, #71295 was not found in NS-1, D14, and BALB/c normal thymus, liver, colon, and kidney tissues. In addition, this MoAb could not inhibit the anchorage-independent growth of C-C36 and transformed 3T3 cells. These results suggest that although the molecule defined by #71295 might not be associated with the anchorage independence of cell growth, it could be a newly expressed determinant on the cell surface that is related to the events of cell transformation.

Characterization of tumor rejection antigen molecules of chemically induced murine colon tumor C-C26.

The molecular nature of a tumor-specific transplantation antigen (TSTA) of a chemically induced BALB/c mouse colon tumor C-C26 was investigated. The antigen was noncytolytically extracted by 2.5% n-butanol treatment of the cells. Crude butanol extract from C-C26, but not from colon tumor C-C51 and fibrosarcoma Meth-A of BALB/c mice could provide protection against the challenged C-C26 tumor in the transplantation experiment. Crude butanol extract from another syngeneic colon tumor C-C36 also induced a cross-protection against the challenged C-C26 tumor. C-C26 crude butanol extract was characterized by biochemical procedures including the Sephadex G200 column, lens culinaris affinity column, and anion-exchange Mono Q fast protein liquid chromatography column, and by the enzyme digestion study of the antigens and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The data indicated that C-C26 TSTA was eluted into fractions containing molecules of approximately Mr 200,000 on Sephadex G200 column chromatography. This antigen was also found in unbound fractions on a lens culinaris affinity column. The antigen was further separated into the fraction that was eluted with 0.4 M NaCl in an ionic strength on Mono Q fast protein liquid chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this fraction showed the molecule with a molecular weight of 30,000. The enzyme digestion study indicated that the immunogenicity of the antigen was inactivated by papain but probably not by neuraminidase treatment. These data suggest that the immunogenic moiety of C-C26 TSTA molecules is located in the peptide portions rather than in sialic acid residues or carbohydrate portions. Furthermore, there are several similarities of the molecular characteristics between C-C26 TSTA and previously reported C-C36 TSTA, such as the amenability to n-butanol extraction. Lens culinaris lectin inaffinity, and ionic strength.

Identification of the transformation-associated cell surface antigen expressed on the rat fetus-derived fibroblast.

A WKA rat fetus-derived fibroblast cell line WFB showed strict nontransformant phenotypes in vitro such as anchorage dependency of cell growth in soft agar, contact inhibition, and serum dependency on the monolayer cell culture. Transfection of 6.6-kilobase EJras oncogene into WFB resulted in the acquisition of tumorigenicity in vitro and in vivo. The cell surface antigen that is moderately or highly expressed on these WFB transformants, designated as W14 and W31, was analyzed using monoclonal antibody 109 that was produced after the immunization of BALB/c mice with W31. Moab 109 recognized a glycoprotein with a molecular weight of 36,000 composed of a single polypeptide chain with 5.4 isoelectric point value. This antigen was highly expressed on WFB EJras and polyoma middle T-DNA transformants, but was undetectable or at the best only faintly recognized on WFB parental cells, transfectants of WFB with c-myc, and normal thymus, liver and kidney of WKA adult rats. It was also clearly expressed on the EJras transformants of Fisher rat fetus-derived 3Y1 fibroblast, but very faintly on parental 3Y1. Furthermore, this antigen was detected on some rat T-lymphoma and gliosarcoma lines. However, it was undetectable on EJras transformants on NRK-49F rat kidney cells and NIH3T3 and BALB3T3 mouse cells. In addition, this antigen appeared on the cell surface of concanavalin A-activated WKA rat lymphocytes and WKA rat on the 16th day of embryo but not on the 8th. These results suggested that the cell surface antigen detected by Moab 109 was clearly unrelated to the ras oncogene product p21 that was highly expressed on EJras-transformants of WFB or 3Y1 cells. Furthermore, it was shown that W14 and W31 cells but not parental WFB cells were susceptible to rat splenic NK cells that were induced by poly(I-C) treatment. Pretreatment of these W14 or W31 cells with Moab 109 could block the NK cell activity against W14 and W31. These data suggest that this antigen may act as one of the NK target structures, and plays an important role as a tumor antigen on the host tumor surveillance, since the antigen was expressed (a) on the cell surface after the cell transformation or enhanced DNA synthesis of some particular cells, and (b) in the W31 tumor developing progressively in the syngeneic rats.

Characterization of n-butyl alcohol solubilized, breast tumor specific antigens recognized by a human autologous cytotoxic T-cell clone.

We demonstrated previously the establishment of a human cytotoxic T-cell clone, TcHMC-1, under culturing with recombinant interleukin that showed the specific cytotoxicity against an autologous breast tumor cell line, HMC-1-8. In the present study, the autologous tumor specific antigens that could be involved in this cytotoxicity were extracted by using n-butyl alcohol and were analyzed for their biochemical profiles. The cytotoxicity of TcHMC-1 against HMC-1-8 was inhibited by adding OKT3 and OKT8 monoclonal antibodies into the cultures, or by pre-sensitizing HMC-1-8 target cells by anti-major histocompatibility complex class I monoclonal antibodies. This suggests that T-cell antigen receptor molecule complexes Ti/T3 on TcHMC-1 and corresponding specific tumor antigens on HMC-1-8 are involved in the cytotoxicity under the restriction of major histocompatibility complex class I products. Precultures of TcHMC-1 with crude n-butyl alcohol extracts from HMC-1-8 cells enhanced the cytotoxic potentials of this clone as seen as mixed lymphocyte tumor cell cultures. This enhancement was dependent on dosage of crude n-butyl alcohol extracts and these TcHMC-1 cells were still cytotoxic specifically for HMC-1-8 targets, but not for other allogenic tumor lines including K562. However, HMC-1-8 crude n-butyl alcohol extracts could not enhance DNA synthesis of TcHMC-1 as assessed by incorporation of [3H]thymidine in the cells. Biochemical purification studies demonstrated that the HMC-1-8 tumor specific antigens were eluted into fractions containing molecules with molecular weights of approximately 200,000 on Sephadex G-200 column chromatography. The antigens were further separated into the fraction that was eluted with 0.4-0.5 M NaCl in an ionic strength on Mono Q fast protein liquid chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of this fraction demonstrated three molecules with molecular weights of 26,000, 30,000, and 32,000 under reduced molecular conditions. The data suggest that these molecules could be tumor specific antigens that are involved in the cytotoxicity of cytotoxic T-cells against a human autologous tumor.

Heat- or stress-inducible transformation-associated cell surface antigen on the activated H-ras oncogene-transfected rat fibroblast.

A WKA rat fetus fibroblast (WFB) was transfected by several oncogenes including EJras (activated H-ras), polyoma middle T (PyMT), v-src, c-myc, and adenovirus type 12 E1A-E1B. We analyzed the expression of the transformation-associated cell surface antigens on WFB by developing monoclonal antibodies. One of the WFB transformation-associated cell surface antigens, recognized by monoclonal antibody 067, was constitutively expressed only on two (W31 and W70) of ten WFB-EJras-transformed clones. This antigen could not be detected on parental WFB cells as well as ten WFB-PyMT transformant clones. Furthermore, it was not expressed on several clones of partially transformed WFB-v-src and WFB-adeno E1 transfectants or nontransformed WFB-c-myc transfectants. Monoclonal antibody 067 could form an immunoprecipitate with an approximate molecular weight of 67,000 which was composed of a single polypeptide chain. It was also shown that the expression of this antigen could be enhanced by cyclic AMP or cholera toxin treatment of W31; treated cells also showed a phenotypic reversion to the nonmalignant growth characteristics of the parental WFB. Moreover, the expression of this antigen could be induced on the WFB-EJras transformants such as W14, which do not constitutively express this antigen, by treatment of these agents. Furthermore, the expression of antigen was enhanced by heat and superoxide treatment on W31. These data suggest that the monoclonal antibody 067-defined molecule is a novel transformation-associated cell surface antigen that could be induced by heat shock or other physiological stress.

Natural killer target molecules associated with the transformation of the oncogene-transfected fibroblast.

Cell surface antigens, the expression of which is highly enhanced along with the transformation of cells, were analyzed. W14 and W31, EJ-ras oncogene-induced transformants of a WKA rat fetus-derived fibroblast WFB, strongly expressed several transformation-associated antigens as defined by monoclonal antibodies 109, 061, and 081. These monoclonal antibodies recognized Mr 86,000, 62,000, and 101,000 molecules, each composed of a single polypeptide chain. The expression of these transformation-associated antigens was negligible on parental WFB cells. Transforming growth factor-beta could enhance the expression of all of these transformation-associated antigens, but platelet-derived growth factor could only enhance the Mr 86,000 kd molecule expression. In the cytotoxicity assays, poly-I:C-induced rat splenic NK cells were cytotoxic to W14 and W31, but not to WFB. The data also showed that the cytotoxicity by these NK cells against NK-sensitive YAC-1 cells was absorbed with the addition of W14, W31, platelet-derived growth factor, or transforming growth factor-beta-stimulated WFB cells. This indicates that NK cells may recognize common target antigens that are expressed among these target cells. It was also indicated that Mr 86,000 and 62,000 molecules were strongly involved in this cytotoxicity, possibly as the target antigens, since F(ab')2 fragments of monoclonal antibodies 109 and 061 strongly inhibited the cytotoxicity. The addition of monoclonal antibody 109, but not 061, inhibited the cytotoxicity even at 60 min after mixing with the effector and target cells, suggesting that the Mr 86,000 molecule may participate in the lethal hit phase of cytotoxicity by NK cells. These data may indicate that some, but not all, transformation-associated antigens are virtually important in the antitumor surveillance mechanisms by the host effector cells, such as NK cells.

Treatment with beta-SQAG9 prevents rat hepatic ischemia-reperfusion injury.

BACKGROUND: Ischemia-reperfusion (I/R) injury occurs in various situations, including transplantation, trauma, and shock. We previously reported that the synthetic beta-SQDG (18:0), which was derived from sulfoquinovosyl diacylglycerol of the sea urchin, possessed immunosuppressive effects, such as inhibition of T-cell responses in human allogenic human mixed lymphocyte reactions (MLR) and skin allograft survival in rats. beta-SQAG9 was synthesized from beta-SQDG (18:0) to improve structural stability in aqueous solution with the same biological activities to bind to CD62L (L-selectin) and CD62P (P-selectin) in vitro. We hypothesized that beta-SQAG9 might attenuate leukocyte rolling on the endothelium and neutrophil infiltration in which L-selectin and P-selectin are key molecules. We investigated the protective effect of beta-SQAG9 against hepatic I/R injury. METHODS: Male Lewis rats were divided into 6 groups: sham, control, and treatment. Rats in the control, and the treatment groups were subjected to hepatic ischemia for 30 minutes. They were injected with PBS or beta-SQAG9 at doses of 5, 10, 25, and 50 mg/kg into the penile vein immediately before reperfusion. To assess the damage to the hepatic parenchyma, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) were measured and histological evaluation was performed at 6 hours after reperfusion. RESULTS: In the group treated with beta-SQAG9 at a dose of 10 mg/kg, AST, ALT, and LDH were significantly reduced, and the amount of neutrophil infiltration also was significantly reduced. CONCLUSIONS: Our data suggest that SQAG-9 (10 mg/kg) reduces the warm hepatic I/R injury.

An immuno-polymerase chain reaction assay for human interleukin-18.

Conventional enzyme-linked immunosorbent assays (ELISA) are sufficient to measure normal and elevated serum interleukin (IL)-18 concentrations, but have limited sensitivity when measuring low concentrations of IL-18 such as in patients with the acquired immunodeficiency syndrome. We have developed a highly sensitive method for detecting human (h) IL-18 using an immuno-polymerase chain reaction (PCR). A mouse monoclonal anti-hIL-18 antibody and rabbit polyclonal anti-hIL-18 antibody was used for an indirect sandwich ELISA with a detection limit of 40 ng/l and a very low background. For immuno-PCR, biotinylated DNA was produced from the plasmid Bluescript by PCR amplification with biotinylated M13-20 primer and nonbiotinylated M13 reverse primer. Immuno-PCR for hIL-18 was performed for 40 cycles using 1 ng/l of biotinylated DNA. This immuno-PCR has a detection limit of 2.5 pg/l, 1.6x10(4) times lower than that of the ELISA. In addition, our system avoids sampling error caused by heat transfer from the ELISA plate to the PCR tube because all procedures from immobilization of the antibody to PCR amplification can be performed in the same tube. This immuno-PCR for hIL-18 is the most sensitive method for detecting hIL-18 reported to date.

Prolonged survival of rat skin allograft by treatment with FK506 ointment.

BACKGROUND: We studied the effect of FK506 ointment on rat skin allograft survival. METHODS: Lewis rat (RT1(1)) skin allografts were histologically evaluated on day 7 after transplantation to recipient ACI (RT1a) rats. We set histological gradings for rejection as follows: grade 1, intraepidermal blister formation; grade 2, incomplete epidermal separation from the dermis; and grade 3, complete epidermal separation from the epidermis. According to these histological criteria, the immunosuppressive effect of FK506 ointment was assessed. RESULTS: Our data indicated that all recipient rats without FK506 ointment showed grade 3 rejection on day 7 after transplantation. In contrast, the allografts treated with FK506 ointment showed grade 1 rejection. Furthermore the blood concentration of FK506 was below 0.5 ng/ml, minimizing the systemically adverse effects of FK506. CONCLUSIONS: Thus, the present study suggests that the topical administration of FK506 on the skin allografts may be useful and effective in the suppression of skin allograft rejection.

Early detection of graft function using hepatic venous oxygen saturation in pig liver transplantation.

In this study, we measured hepatic venous oxygen saturation (Shvo2) in pig liver transplantations in order to evaluate its usefulness as a predictor of early postoperative graft function. Shov2 of the grafts with good function was over 60% after reperfusion, and the mean Shov2 at end of the operations was 69.8+/-6.9%. Shov2 of the grafts with poor function never increased over 60%, and for most of the operation until its end, Shov2 was under 50%. At the end of the operations, the mean Shov2 was 39.7+/-5.5%. Shov2 levels of the grafts with good function were significantly higher than those of the grafts with poor function (P=0.0016). Corresponding with these Shov2 data, glutamic-oxaloacetic transaminase and lactate dehydrogenase levels of grafts with poor function were significantly higher than those of the grafts with good function. Shov2 represents a summation of the hemoglobin oxygen saturation at the venous end of the sinusoids of the liver and indicates adequate hepatic blood flow if the hepatic oxygen is constant. A decrease of Shov2 in poor graft function might indicate a disturbance of microcirculation in the sinusoids. Through the use of Shov2, we are able to recognize conditions of microcirculatory disturbance more quickly than with any other system. In conclusion, Shov2 is a useful indicator for an early and reliable prediction of outcome in liver transplantation.

CTLA4IgG treatment induces long-term acceptance of rat small bowel allografts.

BACKGROUND: CTLA4 immunoglobulin (Ig)G that binds to B7 effectively inhibits the signaling of CD28/CTLA4-B7 pathway and induces antigen specific T cell unresponsiveness in vitro and in vivo. Using CTLA4IgG, we examined induction of long-term graft survival and the mechanism of maintenance of tolerance in rat allogeneic small bowel transplantation. METHODS: Small bowels of Brown-Norway rats (RT1n) were heterotopically transplanted into Lewis rats (RT1l). Recipients were treated with an i.p. injection of either CTLA4IgG or control IgG for 7 days. RESULTS: Long-term survival was observed in rats treated with CTLA4IgG, whereas control rats died within 16 days after transplantation. To examine whether a tolerant state was established in long-term survival rats, secondary transplantation was performed using small bowels of Brown-Norway rats or ACI (RT1b) rats. It was demonstrated that small bowels of Brown-Norway rats were accepted; however, those of ACI rats were rejected within 10 days. Serum concentrations of interleukin (IL)-4 were maintained at >50 microg/ml for 7 days after transplantation in rats treated with CTLA4IgG but <15 microg/ml in control rats. IL-2 concentration was reduced to half in CTLA4IgG-treated rats compared with that in control recipients. Serum IFN-gamma in CTLA4IgG-treated recipients increased after transplantation and was not distinguishable from that of control recipients during the first 7 days after transplantation. Conclusion. We demonstrated that CTLA4IgG treatment alone for 7 days induced a long-term donor specific tolerance in rat allogeneic small bowel transplantation. The induction of long-term acceptance of small bowel allografts by CTLA4IgG is not caused by simply the shift of anti-alloimmune responses from Thl to Th2 cytokine production.

Cadaveric small bowel and small bowel-liver transplantation in humans.

Five patients had complete cadaveric small bowel transplants under FK506 immunosuppression, one as an isolated graft and the other 4 in continuity with a liver. Three were children and two were adults. The five patients are living 2-13 months posttransplantation with complete alimentation by the intestine. The typical postoperative course was stormy, with sluggish resumption of gastrointestinal function. The patient with small intestinal transplantation alone had the most difficult course of the five, including two severe rejections, bacterial and fungal translocation with bacteremia, renal failure with the rejections, and permanent consignment to renal dialysis. The first four patients (studies on the fifth were incomplete) had replacement of the lymphoreticular cells in the graft lamina propria by their own lymphoreticular cells. Although the surgical and after-care of these patients was difficult, the eventual uniform success suggests that intestinal transplantation has moved toward becoming a practical clinical service.

The adverse impact on liver transplantation of using positive cytotoxic crossmatch donors.

Because of the liver graft's ability to resist cytotoxic antibody-mediated rejection, it has become dogma that the conventional transplant crossmatch used to avoid hyperacute rejection of other organs is irrelevant to the liver. We examined this hypothesis in a consecutive series of adult primary liver recipients treated with FK506 and low-dose steroids. Twenty-five of 231 (10.8%) patients received a liver from a cytotoxic-positive crossmatch donor (more than 50% of donor T lymphocytes were killed by dithiothreitol-pretreated recipient serum). The outcome was compared with that of 50 negative crossmatch patients who had their transplantations just before and after the crossmatch positive cases. The one-year graft and patient survivals were 56% and 68%, for positive and 82% and 86% for negative crossmatch patients (P = 0.004, P = 0.03, respectively). The difference between patient and first graft survival was accounted for by retransplantation, which was 4 times more frequent in the positive-crossmatch cases. Histologically, failed allografts obtained at the time of retransplantation revealed a spectrum of pathologic findings related to vascular injury. This study showed a higher difficulty of intraoperative blood product management, a degraded prognosis, and a poorer average quality of ultimate graft function when liver transplantation was performed against positive cytotoxic crossmatches. In such patients for whom crossmatch-negative donors may never be found because of the broad extent and intensity of sensitization, special therapeutic strategies perioperatively must be evolved if results are to improve.

Regulation of HLA class II antigen expression on cultured corneal epithelium by interferon-gamma.

The effect of recombinant human interferon gamma (IFN-gamma) on the induction of HLA class II (HLA-DR, -DP, -DQ) antigen expression on human corneal epithelial (HCE) cells was examined in different stages of culture. Primary cultures were established with limbal explants without endothelium. HCE cells in Stage 1 and Stage 2, with cells negative and positive for the 64K corneal keratin (the marker for advanced corneal epithelial differentiation), respectively, were prepared. HCE cells in both stages were treated with IFN-gamma at a concentration of 0 to 1000 U/ml for two to six days and were stained by the avidin-biotin peroxidase complex method. Class II antigens were not detected on HCE cells in either stage without IFN-gamma treatment. IFN-gamma induced three class II antigens on HCE cells in both stages in a dose- and time-dependent manner but at different levels for each antigen (DR greater than DP greater than DQ). In addition, DQ expression was related to cell differentiation, with DQ extremely rare at Stage 1 and more frequent at Stage 2 (5% vs. 20%). These findings indicate that the induction of class II antigens on HCE cells may be regulated by IFN-gamma independently for each of the antigens and that DQ induction may depend upon the differentiation of HCE cells in culture.

Human leukocyte antigen-class II-positive human corneal epithelial cells activate allogeneic T cells.

PURPOSE: To achieve a better understanding of the mechanism of corneal immune diseases, including corneal allograft rejection, the authors examined the potential of human corneal epithelial (HCE) cells to activate allogeneic T lymphocytes. METHODS: The mixed lymphocyte-HCE cell reaction (MLCER) was performed as follows: HCE cells from primary cultures, with or without treatment with interferon-gamma (IFN-gamma), were treated with mitomycin C and then mixed with peripheral blood lymphocytes (PBL) from normal volunteers. Triplicate cultures were incubated for 7 days. Interleukin-1-alpha (IL-1-alpha) was added to some cultures to examine its effect on MLCER: The lymphocyte responses were measured by 3H-thymidine uptake for the last 18 hours of incubation in MLCER: RESULTS: IFN-gamma-treated, HLA-class-II-bearing HCE cells stimulated allogenic lymphocytes, whereas IFN-gamma nontreated, class-II-negative HCE cells did not. The stimulation by IFN-gamma-treated HCE cells was blocked by anti-HLA class II monoclonal antibody. In addition, exogenous IL-1-alpha reduced the lymphocyte response in MLCER: This effort was inhibited by indomethacin. CONCLUSIONS: This study demonstrates that HLA-class-II-bearing HCE cells can activate allogeneic PBL by a major histocompatibility complex class II-dependent mechanism. In addition, HCE cells may regulate immune reactions, probably through prostaglandin production caused by IL-1.

Long survival in rats after multivisceral versus isolated small-bowel allotransplantation under FK 506.

Abdominal multivisceral allotransplantation (MVTX) from Brown Norway donor rats to Lewis recipient rats was performed under a 14-day course of low (0.32 mg/kg) or high-dose (0.64 mg/kg) intramuscular FK 506 to which weekly further injections were added in some of the high-dose animals. With all three regimens, long survival was frequently achieved with good intestinal adsorption and weight gain, but histopathologic evidence of intestinal rejection existed in the most lightly treated animals. The liver, stomach, and pancreas had only minor abnormalities. Rejection of isolated intestinal grafts was more difficult to control based on histopathologic criteria, and satisfactory results were obtained only with the most aggressive treatment protocol, suggesting that the liver in the MVTX had provided an advantage to the companion organs of the graft, of which the intestine was most vulnerable. Histopathologically, the lymphoid elements of the intestine, including the Peyer's patches, appeared to be the most immunogenic component of the intestine. Epithelium near lymphoid areas was secondarily involved with villous atrophy, cryptitis, and abscess formation. Beginning within 12 days in successful MVTX experiments, the lymphoreticular components of the graft intestine, including the Peyer's patches, lamina propria, and mesenteric nodes, were shown with anti-Ia monoclonal antibodies to be repopulated with recipient cells. This finding in grafts that appeared to be permanently accepted was surprising and contrary to expectations from the literature on intestinal allotransplantation.

Heat-shock protein-73 protects against small intestinal warm ischemia-reperfusion injury in the rat.

BACKGROUND: The protective effects of heat-shock protein (hsp) in rat small intestinal warm ischemia-reperfusion (I/R) injury are poorly understood. METHODS: Hsp-73 expression was induced in rat small intestine with use of sodium arsenite injected (6 mg/kg) through a catheter cannulated into the left common carotid artery 24 hours before ischemia (group 1). In the control group an equal volume of phosphate-buffered saline solution was injected (group 2). To block the induction of hsp-73 expression, sodium arsenate and quercetin (5 mg/kg) were injected (group 3). RESULTS: The mean peak plasma levels of tumor necrosis factor-alpha and cytokine-induced neutrophil chemoattractant after reperfusion were lower in group 1 than in group 2. The tissue myeloperoxidase activity after reperfusion was lower in group 1 than in group 2. The mean peak plasma level of interleukin-10 after reperfusion was higher in group 1 than in group 2. The induction of hsp-73 expression reduced the synthesis of nitric oxide and the magnitude of the small intestinal warm I/R injury. The results in group 3 were similar to those in group 2. CONCLUSION: Hsp-73 protects against small intestinal warm I/R injury by inhibiting the synthesis of inflammatory cytokines and the activation of neutrophils and by accelerating the synthesis of anti-inflammatory cytokines.

Human beta-defensin-2 induction in Helicobacter pylori-infected gastric mucosal tissues: antimicrobial effect of overexpression.

The objective of this study was to understand more of the innate immune response to Helicobacter pylori by determining the expression of human beta-defensin-2 (hBD-2) in various gastric mucosal tissues and MKN45 gastric cancer cells with or without H. pylori. Semi-quantitative TaqMan RT-PCR and immunohistochemistry were carried out. The antimicrobial effects of a transfected hBD-2 gene against H. pylori were also evaluated. The results showed that hBD-2 was expressed in inflamed gastric mucosal tissues with H. pylori infection, but not in the absence of H. pylori infection. Expression was also detected in gastric cancers in patients with H. pylori infection. Expression was induced in the MKN45 gastric cancer cell line by H. pylori in a manner dependent on the abundance of bacteria. hBD-2-transfected 3T3J2-1 cells secreted hBD-2 protein into the culture medium and this protein inhibited growth of H. pylori completely. The results suggest that hBD-2 may be involved in the pathophysiology of H. pylori-induced gastritis.

A new peritoneal dissemination model established from the human pancreatic cancer cell line.

We established a new cell line, HPC-3P4a, with high peritoneal disseminated potential in nude mice. HPC-3P4a was derived from a human pancreatic carcinoma cell line (HPC-3) that had low capacity for peritoneal dissemination. HPC-3P4a developed peritoneal dissemination in 10 of 11 (90.9%) cases, whereas parental HPC-3 developed peritoneal dissemination in one of six (16.7%) cases. The metastatic foci in the peritoneum showed essentially the same histologic appearance of parental involvement. The tumorigenicity, motility, and adhesive activity of HPC-3P4a to the extracellular matrix were stronger than were those of the HPC-3. In FACS analysis, HPC-3P4a significantly increased the expression of alpha6 and alpha(v)beta5 integrins, while it decreased alpha2 integrin, hCD44H, and hCD44v 10, as compared with HPC-3. The VEGF production of HPC-3P4a was significantly lower than that of HPC-3. Analysis of gene macroarrays showed a variety of cytokines, interleukin, and other immunomodulatory, and their receptors were up-regulated and down-regulated on an mRNA level in HPC-3P4a cells, compared with HPC-3 cells. Intrasplenic injection of HPC-3P4a produced no liver metastasis. We named our original highly liver metastatic cell line HPC-3H4 (previously reported). This HPC-3H4 cell was established by repeated intrasplenic injection from parental cell HPC-3; thus, it developed high liver metastasis. Moreover, HPC-3H4 developed peritoneal dissemination by intra-abdominal injection. In contrast, HPC-3P4a did not develop liver metastasis by intrasplenic injection. These findings are very interesting and might suggest that the process of hematogenous metastasis differed from that of peritoneal dissemination. Thus, this cell line may be useful for investigating the mechanism of peritoneal dissemination in human pancreatic cancer.