Miri Fujita

Apoptotic process of cerebellar degeneration in experimental methylmercury intoxication of rats

Abstract This report deals with the mechanism involved in the cerebellar degeneration following experimental methylmercury poisoning of male Wistar rats. The cerebellar granule cells of animals that exhibited typical hind leg paresis were shrunken and displayed marked nuclear pyknosis. At the ultrastructural level, the nuclei of these cells were condensed and fragmented, features which are characteristic of apoptosis. In situ staining for DNA strand breaks revealed that the pyknotic nuclei were positively labeled. DNA fragmentation was confirmed by agarose gel electrophoresis; a ladder pattern of multiples of approximately 200-base pair fragments, typical of apoptosis, was observed with the cerebellar DNA of the methylmercury-treated animals. These observations suggest that the degeneration of cerebellar granule cells by alkyl mercury compounds involves an apoptotic process.

Sequential Observation of Liver Cell Regeneration after Massive Hepatic Necrosis in Auxiliary Partial Orthotopic Liver Transplantation

The morphogenesis of hepatocytes after massive hepatic necrosis to recovery through liver cell regeneration has not been fully understood. Sequential biopsies were performed on the native liver of a 22-year-old man who underwent auxiliary partial orthotopic liver transplantation 1 month after fulminant hepatitis. Auxiliary partial orthotopic liver transplantation was successful, and the biopsy samples permitted us to examine the regenerating process of hepatocytes after massive necrosis. At the time of auxiliary partial orthotopic liver transplantation (postoperative day 0), 95% of hepatocytes were lost and a few ductules were found in the portal areas. The ductules stained with cytokeratin 19. At postoperative day 7, the ductules began to increase in size and number and became dilated over a period of 1 month, when individual hepatocytes with clear cytoplasm appeared from the ductules. As the differentiation of hepatocytes increased, the expression of cytokeratin 19 was found to decrease. From 2 to 3 months, all of the ductules were transformed into hepatocytes, and they began to form round cell clusters. From 3 to 6 months, the round cell clusters became organized into trabecula with fibrosis. From 6 to 12 months, a lobular architecture was established, and by 14 months, the necrotic liver was fully recovered to normal. This study by examination of sequential biopsies demonstrates the progression of the regenerating process from total hepatic necrosis to complete recovery.

Primary extranodal marginal zone B-cell lymphoma of the mucosa-associated lymphoid tissue type in the CNS.

Extranodal marginal zone B‐cell lymphoma of the mucosa‐associated lymphoid tissue (MALT) type has been reported in various internal organs. Here a case is reported of MALT lymphoma developing in the cerebellopontine (CP) angle in a patient with Sjögren syndrome, and the concept of MALT lymphoma of the CNS is introduced. Pathologically, the tumor showed inflammatory features of reactive lymphocytic infiltration with follicle formation, but there were slightly atypical lymphocytes and plasmacytes with B‐cell markers. These cells invaded reactive follicles, showing follicular colonization, and showed aberrant expression indicating their neoplastic nature. A review of the literature revealed eight cases of MALT lymphoma originating from the dura mater and one from the CP angle. The average age of patients was 50 years (range 28–66 years), and all patients were female. The tumors were slow to develop and the patients were cured after surgical removal and/or additional therapies. It is proposed that MALT lymphoma should be considered as a differential diagnosis of inflammatory pseudotumor of the CNS.

Expression of vascular endothelial growth factor in human myocardial infarction.

SummaryTo define mechanisms that may influence collateral circulation and angiogenesis, we investigated vascular endothelial growth factor (VEGF) mRNA expression in human hearts. In non-ischemic human hearts, VEGF mRNA was not detected in vessels, but was found in cardiomyocytes. In hearts with myocardial infarction, the intensity of the VEGF signal was much higher in smooth muscle cells of arterioles adjacent to necrosis and in infiltrating macrophages than in myocytes around the site of the necrosis. This study suggests that levels of VEGF expression are high in smooth muscle cells and macrophages around infarcted areas after myocardial infarction and that VEGF may play a role in promoting collateral circulation and angiogenesis in human ischemic hearts.

An unusual case of primary effusion lymphoma in a HIV-negative patient not pathogenetically associated with HHV8.

Abstract: The development of an unusual case of primary pleural effusion in a 90‐year‐old human immunodeficiency virus (HIV)‐negative Japanese woman with no identifiable tumor mass has been described. Pleural effusion specimens contained large diffuse lymphoma cells, with the phenotype and genotype of a B‐cell lineage (positive for CD20, CD79a and clonal rearrangement of Ig heavy chain) and the c‐myc gene rearrangement, but were negative for T‐cell markers (CD45RO and CD3). The patient was negative for human herpes virus 8 (HHV8), Epstein–Barr virus (EBV) and hepatitis C virus (HCV), as well as human T‐cell lymphotropic virus type‐1 (HTLV‐1). The patient died of respiratory failure 5 months after the diagnosis of primary effusion lymphoma (PEL), and an autopsy was performed. Autopsy findings revealed no evidence of tumor mass or bone marrow involvement of lymphoma cells. This case has been considered as a PEL in a HIV‐, HHV8‐, EBV‐ and HCV‐negative patient. Although cytomorphology of lymphoma cells was classified as large‐cell lymphoma in this case, it is interesting that the present case may represent an unusual subset of Burkitt‐like disease because of clear B‐cell phenotype and c‐myc gene rearrangement.

Prolongation of canine liver allograft survival by a novel immunosuppressant, FTY720. Effect of monotherapy and combined treatment with conventional drugs

BACKGROUND The immunosuppressive effect and other properties of a novel immunosuppressant, FTY720, have been studied mostly in the experimental transplantation of various extrahepatic organs. In this experiment, we evaluated the antirejection potency and adverse effects of this agent on liver grafts using a canine liver transplantation model. METHODS Forty-eight orthotopic liver transplantations were performed by the standard technique under a veno-venous bypass. Liver recipients were divided into two studies: a single-dose study with FTY720 at various doses and a combined dose study with conventional immunosuppressants (cyclosporine or tacrolimus) alone and combined with FTY720. Survival, biochemical and hematological tests, blood levels of immunosuppressants, and postmortem histology were determined. RESULTS The median survival of untreated control animals was 9 days, whereas treatment with FTY720 at a dose of 0.1 mg/kg/day prolonged graft survival to 49.5 days. FTY720 at 1 mg/kg/day showed a slight but insignificant prolongation to 16 days, but when the dose was increased to 5 mg/kg/day, the graft was rejected at 10 days. The combination of FTY720, 0.1 mg/kg/day, with a subtherapeutic dose of cyclosporine, 5 mg/kg/ day, prolonged median animal survival from 40 days with cyclosporine alone to 74 days. A combination of FTY720 (0.1 mg/kg/day) with tacrolimus (0.5 mg/kg/ day) compromised animal survival, reducing survival from 83.5 days with tacrolimus alone to 30.5 days due to infectious complication and emaciation by overimmunosuppression. No evident drug-induced side effects were observed. CONCLUSIONS FTY720 has a potent immunosuppressive effect when used alone at 0.1 mg/kg/day in canine liver transplantation. FTY720 is a promising candidate for future clinical application in orthotopic liver transplantation.

Immunohistochemical detection of hepatocellular carcinoma in the setting of ongoing necrosis after radiofrequency ablation.

After radiofrequency ablation (RFA), hepatocellular carcinoma undergoes complete necrosis and an ongoing necrosis that is irreversible and characterized histologically by disrupted cell outlines, homogenous cytoplasmic eosinophilia, and preserved nuclear staining, with the cells appearing quite distinct from viable cancer cells. Antibody to detect single-stranded DNA (ssDNA) specifically labeled nuclei in the setting of ongoing necrosis, but not viable tumor cells, whereas human mitochondrial antibody labeled the cytoplasm of viable cells but not cells of ongoing necrosis. The results demonstrate that RFA causes denaturation of both DNA and proteins and that the immunohistochemistry of ssDNA and mitochondrial protein is useful in detection of ongoing necrosis after RFA and provides pathological information on the validity of this procedure.

Long-term acceptance of rat cardiac allografts on the basis of adenovirus mediated CD40Ig plus CTLA4Ig gene therapies

Background. We have previously demonstrated that blockade of either CD80/86-CD28 or CD40-CD154 costimulatory pathways by using adenovirus vector coding CTLA4Ig (AdCTLA4Ig) or CD40Ig (AdCD40Ig) genes induced donor-specific tolerance in rat liver transplantation. In this study, we asked whether these gene–therapy-based costimulation blockade would induce tolerance in cardiac transplantation. Methods. Heterotopic heart transplantation was performed in a full major histocompatibility complex (MHC) barrier combination of ACI (RT1avl) to Lewis (LEW, RT1l) rats. Vector (1x109 plaque forming unit [PFU]), AdLacZ, AdCTLA4Ig, or AdCD40Ig, was administered intravenously to recipient animals immediately after grafting, and graft survival, serum CTLA4Ig/CD40Ig levels, and graft histology were assessed. Tolerance was determined by secondary skin-graft challenging. Results. Allografts of both untreated and AdLacZ controls were promptly rejected within 7 days, whereas a single treatment with AdCTLA4Ig or AdCD40Ig significantly prolonged median graft survival to 55.5 and 28.5 days, respectively. In contrast, the combined AdCTLA4Ig and AdCD40Ig gene therapy maintained high CTLA4Ig and CD40Ig levels through the posttransplant period and allowed long-term cardiac allograft survival for more than 270 days. However, both donor and third-party skin grafts were rejected in the animals who harbored cardiac grafts over 150 days. Also, typical features of chronic rejection were evident in the long-term surviving grafts. Conclusion. Simultaneous blockade of CD28 and CD154 pathways by AdCTLA4Ig plus AdCD40Ig induces a strong immunosuppression that allows long-term acceptance of full MHC mismatched cardiac graft in rats. This strategy, however, was not enough to induce tolerance to skin grafts and to avoid chronic rejection, as shown in the liver-transplantation model.

Induction of donor-specific tolerance by adenovirus-mediated CD40Ig gene therapy in rat liver transplantation

Background. Blockade of CD40-CD40 ligand (CD154) costimulatory pathway with anti-CD154 antibody (Ab) prolongs allograft survival in experimental organ transplantations; however, repeated agent administration is needed to provide an adequate immunosuppression. Seeking for simple and effective approach to interfere this signaling, we applied adenovirus-mediated gene therapy by encoding CD40Ig gene (AdCD40Ig). Methods. Liver graft from ACI (RT1av1) rat was transplanted orthotopically into LEW (RT1l) rat, and AdCD40Ig was given to animals via the penile vein immediately after grafting (n=6). Results. A single treatment with AdCD40Ig at 1×109 plaque forming units induced specific expression of CD40Ig gene on allograft liver, produced substantial amount of the protein in the sera, and allowed indefinite graft survival. Whereas, LEW recipients given no treatment or control adenovirus vector (AdLacZ) promptly rejected ACI liver. In addition, AdCD40Ig-treated, long-term survivors accepted skin graft from the donor strain but not the third party graft. Histopathology revealed that liver structure of the long-term surviving animals was completely preserved in normal with no infiltration of mononuclear cells. Conclusion. Blockade of CD40-CD154 pathway by CD40Ig gene therapy is a potent alloantigen-specific immunosuppressive strategy to induce permanent acceptance of liver allograft and would be a new therapeutic candidate in a clinical liver transplantation.

Acute and chronic effects of verapamil in patients with paroxysmal supraventricular tachycardia.

Efficacy of acute intravenous verapamil, 10 mg, and chronic oral verapamil, 320 mg, daily were studied electrophysiologically in 15 patients with paroxysmal supraventricular tachycardia (PSVT). Plasma verapamil concentrations were measured concurrently. Both intravenous and oral verapamil significantly increased the AV node conduction time, the cycle length producing a Wenckebach period, and the refractory period of the AV node. These changes were reflected in changes in plasma verapamil concentration. The echo zone and the supraventricular tachycardia (SVT) zone markedly narrowed after administration of both intravenous and chronic oral verapamil. Verapamils efficacy was found to be related to the type of SVT. For instance, verapamil was more effective in SVT due to AV nodal re-entry than in SVT due to concealed accessory pathway. Fourteen patients were followed from 3 to 31 months and all except one were well controlled. In conclusion, verapamil was effective in prophylaxis of paroxysmal SVT.