Antitumor effect of locally produced CD95 ligand.

Activation of the cell-surface antigen CD95 induces apoptosis of CD95-bearing tumor cells. In this study, we investigated the antitumor effect of locally produced CD95 ligand (CD95L) on CD95-negative tumor cells in vivo. Introduction of CD95L cDNA into murine tumor cells did not affect growth in vitro but caused rejection in vivo. Neutrophils were primarily responsible for this rejection. A CD8 T cell-mediated protective immunity against subsequent challenge with parental tumor cells was also elicited. These results provide evidence for the potential utility of CD95L in tumor eradication and also reveal a proinflammatory function of CD95L.

Influence of SiO2 matrix on electronic and optical properties of Si nanocrystals.

Based on ab initio density functional theory we present electronic properties and the optical response for Si nanocrystals embedded in amorphous SiO(2) networks. Quasi-spherical dots with diameters from 0.8 to 1.6 nm are investigated. The results for Si nanocrystals embedded in SiO(2) are compared with corresponding results for hydrogenated Si nanocrystals of the same size. The calculations show the influence of the interface between nanocrystal and matrix on the electronic properties. The results are compared with recent experimental data and discussed in detail. As striking features, strong reductions of the gaps and their diameter variation are predicted due to the oxide presence. Electronic confinement mainly influences the absorption edge while at higher photon energies only broad peaks at almost fixed positions occur.

Environmental risk factors associated with West Nile virus clinical disease in Florida horses.

The objective of this study was to examine the extrinsic risk factors of West Nile virus (WNV) clinical disease in Florida horses as established from confirmed and negative horses tested within the state from 2001 to 2003. An Arboviral Case Information Form (ACF) was submitted by a referring veterinarian at the time of testing to the Florida Department of Agriculture and Consumer Services on every horse suspected of a viral encephalitis in Florida. A follow-up survey that focused on arbovirus prevention and farm ecology was created and mailed to the owner of each tested horse. Data from the follow-up survey indicated peak WNV prevalence in the late summer months in Florida. Quarter horses were the most commonly affected breed. The WNV vaccine was highly protective and natural water on the property also had a protective association. Factors that increased the risk of WNV to horses were the use of fans and a stable construction of solid wood or cement. Some risk indicators were dead birds on the property and other ill animals on the property. Data from this retrospective study have helped identify factors associated with WNV transmission in equines in Florida. Horses that have not been vaccinated and show clinical signs of arboviral infection from June to November should be tested for WNV. Horses that have been vaccinated and show clinical signs should be tested when the vaccination was administered within 1 month or greater than 6 months prior to the onset of clinical symptoms associated with WN infection.

A retrospective analysis of renal carcinoma in the horse.

BACKGROUND: Renal carcinoma is a rare tumor of horses. HYPOTHESIS: Presenting complaints and clinical signs of this disease are vague and early diagnosis increases survival time. ANIMALS: Data were collected from the medical records of 4 horses presented to Washington State University as well as the 23 previously published case reports of horses with renal carcinoma. METHODS: Retrospective study. RESULTS: Renal carcinoma affects horses of all ages with most cases observed in geldings and Thoroughbreds. The most common presenting complaints are nonspecific and usually do not occur until late in the course of the disease. Routine laboratory results generally are unremarkable with no evidence of renal dysfunction. Urine and peritoneal fluid analyses are consistently abnormal, but the changes usually are nonspecific. Rectal palpation often allows detection of an abnormal kidney or a mass in the area of the kidney. Renal ultrasound examination is the most rewarding imaging procedure, and when combined with renal biopsy, antemortem diagnosis can be achieved. Renal carcinoma is both locally invasive and metastatic, necessitating careful staging for metastasis using thoracic radiography and abdominal ultrasound examination. If the tumor is localized to 1 kidney, nephrectomy is the treatment of choice. No chemotherapy or radiation treatment for renal carcinoma has been reported in the horse. Median survival for this series of cases was 11 days (0 days-1 year). CONCLUSIONS AND CLINICAL IMPORTANCE: Prognosis is poor to grave.

Safety of an attenuated West Nile virus vaccine, live Flavivirus chimera in horses.

REASON FOR PERFORMING STUDY: West Nile virus (WNV) infection is endemic and able to cause disease in naive hosts. It is necessary therefore to evaluate the safety of new vaccines. OBJECTIVES: To establish: 1) the safety of a modified live Flavivirus/West Nile virus (WN-FV) chimera by administration of an overdose and testing for shed of vaccine virus and spread to uninoculated sentinel horses; 2) that this vaccine did not become pathogenic once passaged in horses; and 3) vaccine safety under field conditions. METHODS: There were 3 protocols: 1) In the overdose/shed and spread study, horses were vaccinated with a 100x immunogenicity overdose of WN-FV chimera vaccine and housed with sentinel horses. 2) A reversion to virulence study, where horses were vaccinated with a 20x immunogenicity overdose of WN-FV chimera vaccine. Horses in both studies were evaluated for abnormal health conditions and samples obtained to detect virus, seroconversion and dissemination into tissues. 3) In a field safety test 919 healthy horses of various ages, breeds and sex were used. RESULTS: Vaccination did not result in site or systemic reactions in either experimental or field-injected horses. There was no shed of vaccine virus, no detection of vaccine virus into tissue and no reversion to virulence with passage. CONCLUSIONS: WN-FV chimera vaccine is safe to use in horses with no evidence of ill effects from very high doses of vaccine. There was no evidence of reversion to virulence. In addition, administration of this vaccine to several hundred horses that may have been previously exposed to WNV or WNV vaccine resulted in no untoward reactions. POTENTIAL RELEVANCE: These studies establish that this live attenuated Flavivirus chimera is safe to use for immunoprophylaxis against WNV disease in horses.

Efficacy, duration, and onset of immunogenicity of a West Nile virus vaccine, live Flavivirus chimera, in horses with a clinical disease challenge model.

REASON FOR PERFORMING STUDY: West Nile virus (WNF) is a Flavivirus responsible for a life-threatening neurological disease in man and horses. Development of improved vaccines against Flavivirus infections is therefore important. OBJECTIVES: To establish that a single immunogenicity dose of live Flavivirus chimera (WN-FV) vaccine protects horses from the disease and it induces a protective immune response, and to determine the duration of the protective immunity. METHODS: Clinical signs were compared between vaccinated (VACC) and control (CTRL) horses after an intrathecal WNV challenge given at 10 or 28 days, or 12 months post vaccination. RESULTS: Challenge of horses in the immunogenicity study at Day 28 post vaccination resulted in severe clinical signs of WNV infection in 10/10 control (CTRL) compared to 1/20 vaccinated (VACC) horses (P<0.01). None of the VACC horses developed viraemia and minimal histopathology was noted. Duration of immunity (DPI) was established at 12 months post vaccination. Eight of 10 CTRL exhibited severe clinical signs of infection compared to 1 of 9 VACC horses (P<0.05). There was a significant reduction in the occurrence of viraemia and histopathology lesion in VACC horses relative to CTRL horses. Horses challenged at Day 10 post vaccination experienced moderate or severe clinical signs of WNV infection in 3/3 CTRL compared to 5/6 VACC horses (P<0.05). CONCLUSIONS: This novel WN-FV chimera vaccine generates a protective immune response to WNV infection in horses that is demonstrated 10 days after a single vaccination and lasts for up to one year. POTENTIAL RELEVANCE: This is the first USDA licensed equine WNV vaccine to utilise a severe challenge model that produces the same WNV disease observed under field conditions to obtain a label claim for prevention of viraemia and aid in the prevention of WNV disease and encephalitis with a duration of immunity of 12 months.

Requirement of ceramide for adhesion of Helicobacter pylori to glycosphingolipids.

Direct adhesion of Helicobacter pylori to immobilized glycosphingolipids (GSLs) was compared to that of their corresponding oligosaccharide-conjugated neoglycoconjugates in order to clarify the roles of the carbohydrate and lipid portions of GSLs in H. pylori adhesion. These bacteria were found to adhere to sulfatide, GM3, GalCer and LacCer, but not to ceramide, sphingomyelin, or polyacrylamides conjugated with beta-galactose, lactose, 3'-sialyllactose or 3'-sulfo-beta-galactose. Furthermore, neoglycolipids or bovine serum albumin derivatives with corresponding oligosaccharides were unable to serve as the ligands. H. pylori adhesion to GalCer with alpha-hydroxyl fatty acid was much stronger than GalCer with the non-hydroxyl fatty acid. These results suggest that H. pylori recognize the conformation of GSL with alpha-hydroxyl fatty acid on solid phase.

Lack of cognate help by CD4+ T cells and anergy of CD8+ T cells are the principal mechanisms for anti-leukocyte function-associated antigen-1/intercellular adhesion molecule-1-induced cardiac allograft tolerance.

Combined treatment with anti-leukocyte function-associated antigen-1 (LFA-1) and anti-intercellular adhesion molecule-1 (ICAM-1) monoclonal antibodies leads to allograft tolerance in murine cardiac transplantation. In the present study, we analyzed the mechanisms for this tolerance induction. In the tolerant mice, proliferative response of splenic T cells against donor-type cardiac myocytes and of CD8+ T cells against donor-type alloantigens was impaired as compared with responses in naive or rejected mice, but was completely restored with exogenous interleukin 2. This suggests that class I-restricted CD8+ T cells of tolerant mice were rendered anergic against donor-type alloantigens in the periphery. In contrast, proliferative response of CD4+ T cells against donor-type alloantigens in vitro was comparable between tolerant and naive mice. When heart and skin grafts from the same donor (BALB/c [H2d]) were simultaneously transplanted to C3H mice (H2k), both were rejected within 29 days, even though the mice were similarly treated with anti-LFA-1 and anti-ICAM-1 monoclonal antibodies. In contrast, when heart graft from BALB/c and skin graft from third-party donor (C57BL/6 [H2b]) were simultaneously transplanted to C3H mice under the same condition, the heart graft was accepted indefinitely and the skin graft was rejected. These findings suggest that the peripheral tolerance against cardiac allografts could be induced by selective inactivation of alloreactive CD8+ T cells resulting from the lack of cognate help by CD4+ T cells.

Transplantation of CD95 ligand-expressing grafts: influence of transplantation site and difficulty in protecting allo- and xenografts.

BACKGROUND: CD95 and its ligand (CD95L) have been implicated in the regulation of immune responses. Recently, it was reported that CD95L expression prevented rejection of allogeneic grafts transplanted under the kidney capsule. In contrast, we reported that enforced CD95L expression in subcutaneously grafted cells induced acute rejection even in the syngeneic or immunodeficient hosts. In this study, we investigated whether the CD95L-expressing cells could be protected from rejection when transplanted under the kidney capsule. METHODS: CD95-negative cells (baby hamster kidney and L5178Y lymphoma cells) were transfected with CD95L cDNA to express functional CD95L. The cells were transplanted into skin or renal subcapsular space of immunocompetent or T cell-deficient nu/nu mice. RESULTS: The parental cells grew well in nu/nu or syngeneic mice but were rejected in allogeneic or xenogeneic immunocompetent mice. The CD95L transfectants were rejected when transplanted subcutaneously in all types of mice studied. However, when transplanted under the kidney capsule, they survived in nu/nu or syngeneic mice but were rejected in allogeneic or xenogeneic immunocompetent mice. CONCLUSIONS: These results imply that CD95L expression may not be sufficient to protect the grafts from rejection, and the survival of CD95L-bearing grafts is substantially influenced by the site of transplantation.

Pharmacologic graft protection without donor pretreatment in liver transplantation from non-heart-beating donors.

BACKGROUND: Non-heart-beating donors (NHBDs) are considered potential sources of transplant organs in an effort to alleviate the problem of donor shortage in clinical liver transplantation. We investigated the possibility of pharmacologic protection of hepatic allograft function from NHBDs without donor pretreatment. METHODS: Orthotopic liver transplantation was performed using pigs. In donors, cardiac arrest was induced by stopping the respirator. Forty-five minutes after cessation of the respirator, the liver was flushed with cold lactated Ringer's solution including heparin and with the University of Wisconsin (UW) solution, and then preserved for 8 hr at 4 degrees C in the UW solution. The pigs were divided into two groups: a control group and a treated group. In the treated group, an endothelin antagonist TAK-044 was added to the UW solutions (10 mg/L), and TAK-044 (10 mg/kg body weight) and a platelet activating factor antagonist E5880 (0.3 mg/kg body weight) were also administered to the recipients. RESULTS: TAK-044 and E5880 treatment significantly increased the 7-day survival rate of the recipients (100% vs. 17%, P<0.05). In the treated group, portal venous pressure immediately after reperfusion of the graft was significantly lower than in the control group, and postoperative increase in serum concentrations of glutamic oxaloacetic transaminase and total bilirubin was attenuated. Moreover, the energy charge and adenosine triphosphate concentration of the liver were rapidly restored after reperfusion. CONCLUSIONS: Pharmacologic modulation with TAK-044 and E5880 avoiding donor pretreatment can improve the viability of hepatic allografts procured from NHBDs.

Soluble forms of CD95 and CD95 ligand after living related liver transplantation.

BACKGROUND: Soluble forms of CD95 and CD95 ligand (sCD95 and sCD95L, respectively) can increase in the serum of patients with some inflammatory disease. In this study, we investigated the serum levels of sCD95 and sCD95L in liver transplantation recipients. METHODS: Serum levels of sCD95 and sCD95L in living related liver transplant recipients were analyzed by ELISA and their relation to the clinical findings estimated. RESULTS: Serum samples from the recipients did not show detectable levels of sCD95L but showed significantly increased levels of sCD95. The increase of sCD95 was positively associated with that of total-bilirubin and incidence of rejection, infection, and graft ischemia. CONCLUSIONS: The present results indicate an existence of sCD95 in the recipients of living related liver transplants. The increased serum levels of sCD95 may modify the immunological situation of the recipients after transplantation or represent the ongoing graft damage.

Endothelin antagonist treatment for successful liver transplantation from non-heart-beating donors.

BACKGROUND: With the shortage of cadaveric donors, non-heart-beating donors (NHBDs) are a potential source of liver allografts. However, warm ischemic injury in NHBDs seriously affects the viability of graft liver. Endothelin (ET)-1 has been reported to be involved in the hepatic microcirculatory disturbances after ischemia-reperfusion. METHODS: In a porcine orthotopic liver transplantation model, changes in the serum and liver tissue ET-1 concentration were measured and the effects of an ET receptor antagonist, TAK-044, were evaluated. After cardiac arrest of the donors, liver allografts were subjected to 90 min of warm ischemia, flushed, and preserved for 4 hr at 4 degrees C. The pigs were divided into two groups: a control group (no drug treatment) and a drug-treated group, in which donors and recipients were treated with TAK-044 (10 mg/kg body, drip intravenous injection). Both groups had six donor/recipient pairs. RESULTS: -The ET-1 concentration in the hepatic venous blood increased after reperfusion of the graft in the control group recipients. ET-1 in the graft liver significantly increased during the cold preservation period. TAK-044 treatment significantly increased recipient 7-day survival rate. After reperfusion of the graft, the concentrations of serum liver enzymes and arterial lactate in the drug-treated group were significantly lower than in the control group. The postoperative increase in portal venous pressure was significantly reduced in the drug-treated group. Measurements of liver enzymes in the washed-out preservation fluid at the time of graft rinsing indicated that TAK-044 treatment of the donors significantly suppressed liver enzyme release during ischemia. CONCLUSIONS: These findings indicate TAK-044 treatment has protective effects on postoperative function of hepatic allografts procured from NHBDs.

Transition and improvement in surgical treatment for rectal cancer during the last 21 years in our department.

The subject of this study was to examine the net effect of numerous changes in basic strategies, personnel and devices, upon the clinical courses and outcomes of rectal cancer patients. A total of 151 rectal cancer patients who underwent low anterior resection were divided into 4 groups (period 1 to 4) based upon the time period of the operation. They were compared among groups based upon the following parameters: blood loss, operation time, incidence of leakage and urinary dysfunction, incidence of ileus, duration of naso-gastric tube insertion, timing of initial oral feeding and survival. The blood loss during the operations, urinary dysfunction and duration of naso-gastric tube insertion tended to decrease in every period. Timing of initial oral feeding became faster. The operation times, incidence of leakage and ileus were nearly the same in each period. The 5-year survival rates on Dukes' C cases were 100% in period 4, 82.4% in period 3 and 50% in period 2. Survival rates became better. Our net outcome for rectal cancer treatment was satisfactory, because the survival rates became better under function preserving strategies.

Improved survival and ammonia metabolism by intraperitoneal transplantation of microencapsulated hepatocytes in totally hepatectomized rats.

BACKGROUND: We evaluated the effects of intraperitoneal transplantation of microencapsulated hepatocytes in a 3-stage total hepatectomy rat model. METHODS: A new model of total hepatectomy was created as follows. First, the infrahepatic inferior vena cava was ligated just above the right renal vein. Seven days later, the portal vein was ligated and a portacaval shunt was established using a Teflon catheter over a venipuncture needle. Another 7 days later, total hepatectomy was completed by ligating and dividing the suprahepatic inferior vena cava, the hepatic artery, and the bile duct. Next, 4 x 10(7) hepatocytes (4% of the normal liver hepatocyte mass) isolated from male Wistar rats were microencapsulated within a collagen matrix enveloped by a 3-layer membrane of sodium alginate-poly-L-lysine-sodium alginate copolymer. Capsules containing hepatocytes (diameter, 500-800 microm) and empty capsules (control) were transplanted intraperitoneally 4 days before the total hepatectomy. Survival time and selected blood chemistry concentrations after the total hepatectomy were measured. The capsules were also examined histologically with hematoxylin and eosin staining and modified Gmelin's stain for bile pigments. RESULTS: The survival time was greater in the rats given the microencapsulated hepatocytes than in the control rats (17.3 +/- 3 vs 3.7 +/- 0.1 hours; P <.01). The blood ammonia concentrations increased soon after total hepatectomy but remained significantly lower in the rats with microencapsulated hepatocytes (P <.05). The microcapsules contained numerous viable hepatocytes with abundant bile pigments and no lymphocytic infiltration. CONCLUSIONS: Microencapsulated hepatocytes with an ultrathin polymer layer that protects them from inflammatory and lymphocytic reactions may facilitate their ability to function. In this study, 4 x 10(7) hepatocytes significantly prolonged the survival of rats that underwent hepatectomy and supported ammonia metabolism. Further development of this technique may permit its use in patients with hepatic failure.

Clonogenic colony-forming ability of flow cytometrically isolated hepatic progenitor cells in the murine fetal liver.

Stem cells are defined as cells having multilineage differentiation potential and self-renewal capability. Hepatic stem cells have aroused considerable interest not only because of their developmental importance but also for their therapeutic potential. However, their presence in the liver has not yet been demonstrated. With the use of a fluorescence-activated cell sorter (FACS) and monoclonal antibodies, we attempted to ascertain whether hepatic stem cells are present in the murine fetal liver. For this purpose, we optimized a cell isolation technique for FACS sorting of fetal liver cells. When isolated CD45 TER119 cells (the non-blood cell fraction in the fetal liver) were tested for their clonogenic colony-forming ability, mechanical dissociation (pipetting) was the most suitable cell isolation technique for FACS sorting. We confirmed that these colonies contained not only cells expressing hepatocyte markers but also cells expressing cholangiocyte markers. To identify hepatic stem cells, studies must focus on CD45TER119- cells in the murine fetal liver.

Immunoregulation via adhesion molecules in allogenic and xenogenic hepatocyte transplantation to Nagase's analbuminemic rats.

We investigated the effects of monoclonal antibodies (mAbs) against lymphocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) on intrasplenic allogenic and xenogenic hepatocyte transplantation (HCTx) to analbuminemic rats. Ten to 12-wk-old male Nagase's analbuminemic rats (RT1l) were used as recipients, Wistar/Shi rats (RT1k) were used as donors for allografts and BALB/C mice were used as donors for xenografts. The experimental groups were as follows: group 1, allo-HCTx (n = 7); group 2, allo-HCTx + antirat ICAM-1/antirat LFA-1 mAbs (1.0 mg/kg/day, for 7 days, respectively) (n = 6); group 3, xeno-HCTx (n = 5); group 4, xeno-HCTx + mAbs (antimouse LFA-1/antirat ICAM-1) (n = 5). group 5, xeno-HCTx + mAbs (antirat LFA-1/antimouse ICAM-1) (n = 5). Serum rat albumin levels were measured in groups 1 and 2, and serum mouse albumin levels were measured in groups 3, 4, and 5, as indicators of the function of grafted hepatocytes. In allotransplantation groups, the serum rat albumin levels in the mAbs-treated group were significantly higher than those in the control group for 6 wk after transplantation. In xenotransplantation groups, no increase in the serum mouse albumin levels was detected in any group

Thyrotropin-releasing hormone-induced changes in electroencephalographic and vagal activity in the cross-circulated brain of the rat.

The effects of centrally administered thyrotropin-releasing hormone (TRH) on vagal efferent and cortical electroencephalographic (EEG) activity were assessed in rat brains which were functionally isolated from the body trunk and maintained via cross-circulation. Upon withdrawal of inputs from the trunk, cervical vagal activity was markedly attenuated. The decreased activity was partially restored by TRH with a latent period of a few minutes. On the other hand, TRH immediately but transiently augmented the fast waves of the EEG. The change in brain activity produced by TRH appears to simulate the state which makes possible the generation of vagal efferent activity even though inputs from the body trunk are withdrawn.

Idiopathic calcifying pancreatitis in a Japanese pediatric patient.

We recently experienced a rare case of chronic pancreatitis in a 13-year-old Japanese boy. Recently, in hereditary pancreatitis patients, some mutations have been identified in the trypsinogen gene. The purpose of this study was to investigate whether the same mutations could also be found in this patient. Polymerase chain reaction (PCR)-amplified products of his cationic and anionic trypsinogen genes were examined by direct sequence analysis. The gene analysis failed to show any mutation in any exons and their flanking intronic sequences of his trypsinogen genes. These findings indicate that the chronic calcifying pancreatitis in the present patient is "idiopathic", and thus a rare case of juvenile pancreatitis.

Treatment of advanced colorectal adenocarcinoma with weekly high-dose l-leucovorin and 5-fluorouracil.

We report the results of 5-fluorouracil (5-FU) combined with high-dose l-folinic acid (leucovorin) therapy for patients with advanced colorectal carcinoma. In each treatment course, the patients weekly received both 5-FU (600 mg/m2 by intravenous 15 min infusion) and l-folinic acid (250 mg/m2 by intravenous infusion over a period of 2 h). A total of six treatments were administered with a 14-day interval to the next course of six treatments. Forty-eight patients were evaluated for toxicity and 32 for response. The combined complete and partial response rate was 25% in 32 patients. Toxicity was within acceptable limits without grade 4 toxicity. Although the response rate was slightly lower than those reported in phase II trials in Japan, the result was satisfactory. This therapy can be the standard chemotherapy for colorectal cancer patients, even in Japan.

An endothelin receptor antagonist ameliorates injuries of sinusoid lining cells in porcine liver transplantation.

BACKGROUND: TAK-044 is an endothelin receptor antagonist. Whether the agent has protective effects on liver graft injuries from non-heart-beating donors is unknown. METHODS: In donor pigs, cardiac arrest was induced by stopping the respirator. Forty-five minutes after cessation of the respirator, the liver was flushed with University of Wisconsin (UW) solution, preserved for 8 hours at 4 degrees C, and transplanted orthotopically. The pigs were divided into two groups: a control group and a drug-treated group in which TAK-044 was given in the UW solutions (10 mg/L) and was administered to recipients (10 mg/kg body weight). RESULTS: TAK-044 treatment significantly increased recipient survival rate. After reperfusion of the graft, portal venous pressure and 15-minute retention rate of indocyanine green were significantly reduced in the drug-treated group. Electron microscopic findings indicated that TAK-044 attenuated endothelial cell injuries. CONCLUSION: TAK-044 treatment improves the viability of livers harvested from non-heart-beating donors. The main effect of the agent is protection of endothelial cells from ischemia/reperfusion injuries.