Psychological distress reduces the effcacy of chemotherapy in breast cancer patients. The mechanism may be related to the altered neuronal or hormonal secretions during stress. Here, we reported that adrenaline, a hormone mediating the biological activities of stress, upregulates mdr1 gene expression in MCF-7 breast cancer cells via a2-adrenerdric receptors in a dose-dependent manner. Mdr1 upregulation can be specifically inhibited by pretreatment with mdr1-siRNA. Consequently, adrenergic stimulation enhances the pump function of P-glycoprotein and confers resistance of MCF-7 cells to paclitaxel. In vivo, restraint stress increases mdr1 gene expression in the MCF-7 cancers that are inoculated subcutaneously into the SCID mice and provokes resistance to doxorubicin in the implanted tumors. The effect can be blocked by injection of yohimbine, an a2-adrenergic inhibitor, but not by metyrapone, a corticosterone synthesis blocker. Therefore, we conclude that breast cancers may develop resistance against chemotherapeutic drugs under psychological distress by over-expressing mdr1 via adrenergic stimulation.

Background. Donor hepatocyte apoptosis that is induced by host cytotoxic T lymphocytes (CTLs) limits the application of hepatocyte transplantation. Hepatocytes from Bcl-2 transgenic mice can resist the lethal effect of anti-Fas antibody. However, the anti-apoptotic effect of Bcl-2 expression on allogeneic transplanted hepatocytes remains illusive. This study tested the feasibility of Bcl-2 gene transfer as an approach to inhibit CTL-mediated apoptosis in allogeneic transplanted hepatocytes. Methods. An adenovirus vector that encoded human Bcl-2 gene (AdCMVhBcl-2) was used to transfect cultured rat hepatocytes, which were then transplanted into allogeneic spleens. DNA fragmentation and caspase-3 activation were examined by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling assay and immunohistochemistry for active caspase-3, respectively. Cocultivation of hepatocytes and allogeneic CD8+ T lymphocytes was performed, and cytotoxicity on hepatocytes was examined by alanine transaminase release. Results. Bcl-2 gene transfer inhibited apoptosis and increased liver-associated enzyme activities in allogeneic transplanted hepatocytes, which were associated with inhibition of caspase-3 activation. Alanine transaminase release in hBcl-2 modified hepatocytes was lower compared with controls, which could not be further decreased by inhibition of Fas ligand and granzyme B. Conclusions. Adenovirus-mediated Bcl-2 gene transfer blocks CTL-mediated apoptosis in allogeneic hepatocytes by inhibition of caspase-3 activation. Bcl-2 gene transfer could be used to promote survival of transplanted hepatocytes.

ITHASBEEN well established that the spleen is an important immune organ, serving as a large phagocytic filter and a major site of antibody production. The IgM-specific antibody, generated primarily in the spleen, 1 promotes opsonized phagocytosis by activating various complement fragments, and thus contributes to the early immune response to pathogens. In addition, the spleen produces tuftsin, which is a tetrapeptide regulating the activity and migration responses of phagocytic cells. 2 Splenectomy impairs normal immune function by reducing phagocytic activity, lowering serum IgM and tuftsin levels, and diminishing suppressor T-cell numbers. 3 Hence, patients with splenectomy have been shown to bear a higher risk of overwhelming postsplenectomy infection. 4,5 This provokes interest in developing alternative operative methods for splenic preservation. Autotransplantation of splenic fragments after splenectomy has been advocated as a method of retaining normal splenic immune function in the treatment of massive splenic trauma or splenomegaly caused by hematologic disorders.6-9 However, it remains equivocal whether splenic autotransplantation may help maintain splenic immune function in patients with portal hypertension and cirrhosis. Therefore, we conducted a prospective randomized study to evaluate the influence of autologous splenic transplantation in patients with portal hypertension and cirrhosis on splenic immune function, as assessed by levels of serum IgM and tuftsin.

Background: It has been demonstrated that colorectal carcinomas rarely metastasize to diseased livers. However, this phenomenon has not been thoroughly evaluated in patients with various forms of chronic hepatitis B virus (HBV) infection. Therefore, the present study examined the relationship between the incidence of hepatic metastasis of colorectal carcinomas and chronic HBV infection, with emphasis on the influence of HBV viral replication and chronic liver damage. Methods: We analyzed the clinicopathological data of 512 patients undergoing surgical treatment of colorectal carcinomas at our department from 1992 to 1998. Among these cases, 74 had chronic HBV infection, including 28 cases with HBV replication and 21 with chronic liver damage. Results: The incidence of liver metastasis in the HBV infection group (13.5%) was significantly lower than that of the noninfection group (27.1%, P, 0.05). In addition, patients with HBV infection survived longer than those without infection (P 5 0.018). Furthermore, liver metastatic rate in patients with HBV replication (3.6%) was lower than those without virus replication (19.6%, P, 0.05). In contrast, there was no significant difference in liver metastasis between HBV infected patients with or without chronic liver damage (P .0.05). Conclusions: Chronic HBV infection with viral replication reduces hepatic metastasis of colorectal cancer, and thus prolongs the survival of patients.

HEPATOCYTE transplantation (HTx) has been proposed as a potential therapeutic modality for various liver disorders by providing metabolic support during acute and chronic liver failure and replacing specific liver functions in inherited metabolic liver diseases. However, after allogenic HTx into the spleens, the transplanted cells are rejected within a few days without immunosuppression.1 It has been documented that activation of cytotoxic T lymphocytes (CTL), engaging the Fas-mediated or perforin/granzyme cytolytic pathway, are responsible for the apoptosis of transplanted hepatocytes.2 On activation, CTL release perforin and granzyme B by exocytosis, and perforin facilitates the entry of granzyme B that induce apoptosis in target cells.3 Granzyme B inhibitor I antagonizes the activity of granzyme B, and may inhibit apoptotic cell death mediated by this pathway.4 Upregulation of granzyme B has been associated with the development of acute rejection in liver transplantation. The present study was designed to investigate the protective effects of granzyme B inhibitor I on allogeneic hepatocytes transplanted into rat spleens or cocultured with CTL.

Summary Expression of membrane-bound Fas ligand (mFasL) on colon cancer cells serves as a potential mechanism to inhibit host immune function by inducing apoptosis of host lymphocytes. Membrane-bound FasL can be cleaved and released as a soluble mediator (sFasL), which may spread the apoptosis induction effect. Our study examined whether colon adenocarcinoma cells release sFasL, and induce apoptosis of host lymphocytes without direct cell–cell contact. In 12 consecutive patients with colon adenocarcinoma mFasL was identified in the tumours, sFasL was measured in the sera and apoptosis identified in tumour-infiltrating and peripheral blood lymphocytes. To analyse the function of sFasL, colon cancer cells were primarily cultured; sFasL was isolated from supernatants, measured, incubated with Fas-bearing Jurkat cells, and the resulting apoptosis was analysed. Serum levels of sFasL were significantly elevated in all colon cancer patients with mFasL expression in tumour tissues (n=8). In these patients, the number of apoptotic lymphocytes was significantly increased within tumour and peripheral blood. Furthermore, sFasL was present in the corresponding supernatants and induced apoptosis of Jurkat cells in a dosedependent manner. These findings suggest that mFasL-positive colon cancer cells release sFasL, and thus may induce apoptosis of host lymphocytes as a potential mechanism for immune evasion.

Small interfering RNAs (siRNAs) can induce potent gene silencing by degradation of cognate mRNA. However, in dividing cells, the silencing lasts only 3 to 7 days, presumably because of siRNA dilution with cell division. Here, we investigated if sustained siRNA-mediated silencing of human immunodeficiency virus type 1 (HIV-1) is possible in terminally differentiated macrophages, which constitute an important reservoir of HIV in vivo. CCR5, the major HIV-1 coreceptor in macrophages, and the viral structural gene for p24 were targeted either singly or in combination. When transfected 2 days prior to infection, both CCR5 and p24 siRNAs effectively reduced HIV-1 infection for the entire 15-day period of observation, and combined targeting of both genes abolished infection. To investigate whether exogenously introduced siRNA is maintained stably in macrophages, we tested the kinetics of siRNA-mediated viral inhibition by initiating infections at various times (2 to 15 days) after transfection with CCR5 and p24 siRNAs. HIV suppression mediated by viral p24 siRNA progressively decreased and was lost by day 7 posttransfection. In contrast, viral inhibition by cellular CCR5 knockdown was sustained even when transfection preceded infection by 15 days, suggesting that the continued presence of target RNA may be needed for persistence of siRNA. The longer sustenance of CCR5 relative to p24 siRNA in uninfected macrophages was also confirmed by detection of internalized siRNA by modified Northern blot analysis. We also tested the potential of p24 siRNA to stably silence HIV in the setting of an established infection where the viral target gene is actively transcribed. Under these circumstances, long-term suppression of HIV replication could be achieved with p24 siRNA. Thus, siRNAs can induce potent and long-lasting HIV inhibition in nondividing cells such as macrophages.

Background. Initial insults to kidney allografts, character-ized by infiltration of mononuclear inflammatory cells, contribute to chronic allograft nephropathy. Chemokines such as RANTES (regulated upon activation, normal T cell expressed) are thought to be responsible for the recruitment and activation of infiltrating cells. The present study investigated whether early application of Met-RANTES, a chemokine receptor an-tagonist that blocks the effects of RANTES, can protect renal allografts from long-term deterioration. Methods. Fisher (F344) rat kidneys were orthotopically transplanted into Lewis recipients and treated with cyclopo-rine A (1.5mg/kg/day) for the first 10 days following trans-plantation, together with either Met-RANTES at 40g/day, 200g/day or vehicle for the first 7 days. Animals were har vested at 2 and 28 weeks after transplantation for histologic, immunohistologic and molecular analysis. Results. Met-RANTES treatment reduced the infiltration of belymphocytes and macrophages in allografts at 2 weeks after transplantation, accompanied by decreased mRNA expression of interleukin (IL)-2, IL-1, tumor necrosis factor-(TNF-) and RANTES. At post-transplantation week 28, Met-RANTES treatment at high and low doses reduced urinary protein excre-tion and significantly ameliorated glomerulosclerosis, intersti-tial fibrosis, tubular atrophy, intimal proliferation of graft arter-ies and mononuclear cell infiltration. However, creatinine clearance was not influenced by Met-RANTES. Furthermore, Met-RANTES suppressed the mRNA expression of trans-forming growth factor-(TGF-) and platelet-derived growth factor-B (PDGF-B). Conclusions. Blockade of chemokine receptors by Metby RANTES diminishes early infiltration and activation of mono nuclear cells in the grafts, and thus reduces the pace of chronic allograft nephropathy.

Background. A major obstacle in allogenic hepatocyte transplantation is increased apoptosis of grafted cells due to CTL-based cytotoxicity. However, whether blockade of Fas-and granzyme-mediated pathways of CTL-based cytotoxicity may provide immune protection to transplanted hepatocytes is poorly de

macrophages regulate fibrogenesis by providing cytokines and growth factors that modulate the proliferation and collagen synthesis of fibroblasts. However, macrophages can be activated in a classical pathway induced by LPS or IFN-g and an alternative pathway induced by IL-4 or glucocorticoid. Differently activated macrophages display distinct biological features. To clarify the difference between these two subsets of macrophages in the regulatory mechanisms controlling fibrogenesis, human peripheral blood monocytes were used as the source of macrophages and cocultivation of differently activated macrophages and a fibroblast cell line, WI-38, was performed. Alternatively activated macrophages increased the proliferation index and collagen synthesis of cocultivated WI-38 cells in comparison to untreated monocytes, while classically activated macrophages markedly reduced collagen production of cocultivated WI-38 cells. Additionally, mRNA expression and protein production of TGF-b1, PDGF-AA, and PDGF-BB were elevated in alternatively activated macrophages in parallel to their profibrogenic effects. In contrast, expression and production of TNF-a, as well as MMP-7, were enhanced in classically activated macrophages. These findings suggested that alternatively activated macrophages enhance fibrogenesis of fibroblasts by providing profibrogenic factors, while classically activated macrophages inhibit fibrogenesis of fibroblasts by releasing antifibrogenic or fibrolytic factors.