Intraoperative hypertensive crisis in a dog with functional paraganglioma of the gall bladder.

A 15-year-old spayed female mongrel presented with anorexia and an abdominal mass. The mass originated from the gall bladder and was surgically resected along with divisionectomy of the central hepatic division. Paroxysmal hypertension and tachycardia were noted during manipulation of the mass. Following resection, arterial blood pressure decreased significantly. Histopathological analysis confirmed a diagnosis of neuroendocrine neoplasm. Immunohistochemical staining for synaptophysin and chromogranin A yielded diffuse and strong positive results, while gastrin was positive in only 10% of the cells. The preoperative elevated concentrations of catecholamine in the urinalysis showed a marked decrease after surgery. Based on these findings, the tumour was diagnosed as a functional paraganglioma of the gall bladder. The patient has undergone regular thoracic radiographs and ultrasound examinations and, until 431 days after surgery, has shown no signs of metastases or recurrences. Based on our literature search, we report the first case of functional paraganglioma of the gall bladder in a dog.

Clinical relationship between histopathological necrotic/partial necrotic findings and disease condition of gallbladder mucoceles in dogs.

Gallbladder mucocele (GM) is a common extrahepatic biliary disease recognized in dogs and is defined as the expansion and extension of the gallbladder by an accumulation of semi-solid bile or bile acid. Histopathological diagnosis of necrotizing cholecystitis and transmural coagulative necrosis of the gallbladder wall shows poor prognosis. Conversely, histopathological diagnosis with partial necrotic findings is often achieved. We hypothesized that histopathological partial necrosis of the gallbladder wall is the primary lesion of necrotic cholecystitis or transmural ischemic necrosis. Therefore, we investigated the relationship between histopathological necrosis/ partial necrosis findings and their clinical conditions. We retrospectively analyzed 55 dogs diagnosed with GM that had undergone cholecystectomy at the Yamaguchi University Animal Medical Center. The group with histopathological necrosis/partial necrosis of the gallbladder wall showed elevated levels of preoperative white blood cells, alanine transaminase, alkaline phosphatase, γ-glutamyltransferase, total bilirubin, and C-reactive protein compared to the non-necrotic group. Partial necrosis of the gallbladder wall may affect the progression of the disease and hematological abnormalities. Additionally, all death cases until 2 weeks were included in the histopathological necrosis/partial necrosis group. In this study, we found that poor prognosis factors were associated with partial necrosis of the gallbladder wall. Furthermore, these cases of partial necrosis showed elevated levels of blood test parameters. These results suggest that necrosis of the gallbladder wall is associated with poor prognosis and poor pathophysiological conditions.

Transcatheter arterial embolisation in four dogs with hepatocellular carcinoma.

Four dogs with hepatocellular carcinoma were treated by transcatheter arterial embolisation. In all dogs, the tumour-supplying arteries were selectively embolised with gelatine sponge particles. Post-embolisation tumour volumes decreased relative to pre-embolisation volumes in all dogs. No adverse reactions were observed in three dogs after treatment but one dog showed pancreatitis. These results suggest that transcatheter arterial embolisation is a feasible treatment for dogs with hepatocellular carcinoma.

Oncolytic reovirus therapy: Pilot study in dogs with spontaneously occurring tumours.

Oncolytic virotherapy is a novel treatment involving replication-competent virus in the elimination of cancer. We have previously reported the oncolytic effects of reovirus in various canine cancer cell lines. This study aims to establish the safety profile of reovirus in dogs with spontaneously occurring tumours and to determine a recommended dosing regimen. Nineteen dogs with various tumours, mostly of advanced stages, were treated with reovirus, ranging from 1.0 × 108 to 5.0 × 109 TCID50 given as intratumour injection (IT) or intravenous infusion (IV) daily for up to 5 consecutive days in 1 or multiple treatment cycles. Adverse events (AEs) were graded according to the Veterinary Cooperative Oncology Group- Common Terminology Criteria for Adverse Events (VCOG-CTCAE) v1.1 guidelines. Viral shedding, neutralizing anti-reovirus antibody (NARA) production and immunohistochemical (IHC) detection of reovirus protein in the tumours were also assessed. AE was not observed in most dogs and events were limited to Grade I or II fever, vomiting, diarrhoea and inflammation of the injected tumour. No infectious virus was shed and all dogs had elevated NARA levels post-treatment. Although IHC results were only available in 6 dogs, 4 were detected positive for reovirus protein. In conclusion, reovirus is well-tolerated and can be given safely to tumour-bearing dogs according to the dosing regimen used in this study without significant concerns of viral shedding. Reovirus is also potentially effective in various types of canine tumours.

Therapeutic vaccination based on side population cells transduced by the granulocyte-macrophage colony-stimulating factor gene elicits potent antitumor immunity.

Among cancer immunotherapies, granulocyte-macrophage colony-stimulating factor (GM-CSF) gene-transduced tumor cell vaccine (GVAX) therapies appear promising and have been shown to be safe and effective in multiple clinical trials. However, the antitumor efficacies of GVAX therapy alone are in some cases limited. Here we showed that GVAX therapy targeting cancer stem cells (CSCs) substantially suppressed tumor development in syngeneic immunocompetent mice recapitulating normal immune systems. CSCs were isolated as side population (SP) cells from 4T1 murine breast carcinoma cell line and transduced with GM-CSF gene delivered by non-transmissible Sendai virus (4T1-SP/GM). Impaired tumorigenicity of subcutaneously injected 4T1-SP/GM depended on CD8+ T cells in concert with CD4+ T cells and natural killer cells. Mice therapeutically vaccinated with irradiated 4T1-SP/GM cells had markedly suppressed tumor development of subcutaneously transplanted 4T1-SP cells compared with those treated with irradiated cells of non-transduced 4T1-SP cells or non-SP (4T1-NSP/GM) cells. Tumor suppression was accompanied by the robust accumulation of mature dendritic cells at vaccination sites and T-helper type 1-skewed systemic cellular immunity. Our results suggested that CSC cell-based GVAX immunotherapy might be clinically useful for inducing potent tumor-specific antitumor immunity.

Multimodal immunogenic cancer cell death as a consequence of anticancer cytotoxic treatments.

Apoptotic cell death generally characterized by a morphologically homogenous entity has been considered to be essentially non-immunogenic. However, apoptotic cancer cell death, also known as type 1 programmed cell death (PCD), was recently found to be immunogenic after treatment with several chemotherapeutic agents and oncolytic viruses through the emission of various danger-associated molecular patterns (DAMPs). Extensive studies have revealed that two different types of immunogenic cell death (ICD) inducers, recently classified by their distinct actions in endoplasmic reticulum (ER) stress, can reinitiate immune responses suppressed by the tumor microenvironment. Indeed, recent clinical studies have shown that several immunotherapeutic modalities including therapeutic cancer vaccines and oncolytic viruses, but not conventional chemotherapies, culminate in beneficial outcomes, probably because of their different mechanisms of ICD induction. Furthermore, interests in PCD of cancer cells have shifted from its classical form to novel forms involving autophagic cell death (ACD), programmed necrotic cell death (necroptosis), and pyroptosis, some of which entail immunogenicity after anticancer treatments. In this review, we provide a brief outline of the well-characterized DAMPs such as calreticulin (CRT) exposure, high-mobility group protein B1 (HMGB1), and adenosine triphosphate (ATP) release, which are induced by the morphologically distinct types of cell death. In the latter part, our review focuses on how emerging oncolytic viruses induce different forms of cell death and the combinations of oncolytic virotherapies with further immunomodulation by cyclophosphamide and other immunotherapeutic modalities foster dendritic cell (DC)-mediated induction of antitumor immunity. Accordingly, it is increasingly important to fully understand how and which ICD inducers cause multimodal ICD, which should aid the design of reasonably multifaceted anticancer modalities to maximize ICD-triggered antitumor immunity and eliminate residual or metastasized tumors while sparing autoimmune diseases.

[Bentall operation in left sinus of valsalva aneurysm with anomalous origin of left coronary artery and severe aortic regurgitation].

We report a successful Bentall operation in a 74-year-old man with an unruptured left sinus of Valsalva aneurysm and aortic regurgitation. The coronary angiogram showed an absent left main trunk (split origination of left coronary arteries from the same left sinus of Valsalva). We implanted a stented bovine pericardial valve and a Valsalva tube graft as a composite graft. To reconstruct the coronary arteries, we made one left coronary button including both ostia of the left anterior descending coronary artery and circumflex artery, and reimplanted the coronary button with interposition of a short small synthetic graft. Excessive tension of the coronary ostia can be avoided and a new left main trunk can also be created in this manner. Postoperative course was uneventful.

[Experience of chest wall reconstruction with newly developed material].

After the chest wall resection, its reconstruction is often needed. A 45-year-old male lung adenocarcinoma patient with chest wall invasion underwent upper lobectomy of the right lung with partial resection of 4-6th ribs. The size of the removed chest wall was 11 x 6.5 cm. We reconstructed the chest wall with Bard Composix E/X Mesh. This prosthesis is consisted of a polypropylene mesh and an expanded polytetrafluoroethylene sheet This material is seems to be useful in the reconstruction of chest wall in both preventing pulmonary adhesion and enabling good wound healing.

Nonsteroidal anti-inflammatory drugs induce hypermotilinemia and disturbance of interdigestive migrating contractions in instrumented dogs.

Nonsteroidal anti-inflammatory drugs (NSAIDs) induce gastric ulcers due to inhibition of prostaglandin production. Prostaglandins have an influence on physiological gastrointestinal motility, but the relationships between NSAID-induced gastric ulcer, gastrointestinal motility and motilin are unknown. Fifteen dogs were allocated randomly to three groups in which either gelatin, meloxicam or indomethacin was administered. Fecal occult blood and gastrointestinal motility were monitored continuously for 6 days. In addition, analyses of the plasma motilin concentration, gastrointestinal endoscopy and gastric emptying, and detection of motilin cells were performed. Gastrointestinal motility was disturbed in the indomethacin group, presenting as disappearance of interdigestive migrating contractions (IMCs) 3 days before gastric ulcers were detected. Delayed gastric emptying and hypermotilinemia were observed significantly more often in the indomethacin group compared with the other groups. Motilin cell-crypt/villi ratio in the indomethacin group significantly decreased in the duodenum and jejunum, compared with the other groups. No significant changes in any tests were observed in the meloxicam group, when compared with the gelatin group. These findings suggest that the disturbance of IMCs caused by hypermotilinemia, with changes in motilin cell distribution, and delayed gastric emptying induced by indomethacin may contribute to the development of gastric ulcers.

Rb plays a role in survival of Abl-dependent human tumor cells as a downstream effector of Abl tyrosine kinase.

The retinoblastoma (Rb) gene product is a tumor suppressor that is mutated or inactivated in many types of human cancers. Although Rb is known to be an upstream negative regulator of Abl protein tyrosine kinase, we propose here that Rb also functions as a downstream effector of Abl that plays a positive role in survival of Abl-dependent human tumor cells, including Bcr/Abl-positive chronic myelogenous leukemia (CML). We show that Rb is constitutively phosphorylated at tyrosine in Abl-dependent tumor cells, and that Abl phosphorylates Rb specifically at Y805 within the C-terminal domain of the molecule. We also show that ectopic expression of Rb induces apoptosis in Abl-dependent tumor cells by inhibiting the Abl tyrosine kinase activity, and that Rb-induced apoptosis is compromised by Abl-catalysed phosphorylation of Rb at Y805. Furthermore, the silencing of endogenous Rb by RNA interference induced apoptosis in Abl-dependent tumor cells. Thus, our findings suggest that Abl-catalysed tyrosine phosphorylation of Rb is necessary for survival of Abl-dependent human tumor cells, and raises the possibility that this phosphorylated Rb can be a molecular target for cancer therapy aimed at inducing apoptosis of Abl-dependent tumor cells, such as Bcr/Abl-positive CML.

Antiangiogenic activity of BAI1 in vivo: implications for gene therapy of human glioblastomas.

Glioblastomas are the most common primary brain tumors in adults. These tumors exhibit a high degree of vascularization, and malignant progression from astrocytoma to glioblastoma is often accompanied by increased angiogenesis and the upregulation of vascular endothelial growth factor and its receptors. In this study, we investigated the in vivo antiangiogenic and antitumor effects of brain-specific angiogenesis inhibitor 1 (BAI1) using human glioblastoma cell lines. Glioblastoma cells were transduced with an adenoviral vector encoding BAI1 (AdBAI1), and Northern and Western blot analyses, respectively, demonstrated BAI1 mRNA and protein expression in the transduced tumor cells. Using an in vivo neovascularization assay, we found that angiogenesis surrounding AdBAI1-transduced glioblastoma cells transplanted into transparent skinfold chambers of SCID mice was significantly impaired compared to control treated cells. Additionally, in vivo inoculation with AdBAI1 of established subcutaneous or intracerebral transplanted tumors significantly impaired tumor growth and promoted increased mouse survival. Morphologically, the tumors exhibited signs of impaired angiogenesis, such as extensive necrosis and reduced intratumoral vascular density. Taken together, these data strongly indicate that BAI1 may be an excellent gene therapy candidate for the treatment of brain tumors, especially human glioblastomas.

Long-term follow-up of pulmonary function in bronchial asthma patients treated with pranlukast.

Clinical studies have shown that pranlukast, a selective cysteinyl leukotriene antagonist, is effective for bronchial asthma. In the present paper, we retrospectively analyzed long-term asthma control by pranlukast treatment in patients treated with inhaled corticosteroids. We analyzed medical records and asthma diaries of 21 patients (9 males, 12 females) (52.1 +/- 3.5 years of age) with bronchial asthma who experienced increase of more than 10 L/min in peak expiratory flow in the first 4 weeks of treatment with pranlukast (450 mg/day) and were subsequently treated with pranlukast for more than 1 year. They all received inhaled corticosteroids (400-1600 microg/day of beclomethasone dipropionate or equivalent). We examined clinical control in terms of time course of self-monitored peak expiratory flow. During the analyzed period, the dose of inhaled corticosteroids was tapered in 4 patients, constant in 15 patients and increased in 2 patients. In 19 patients treated with unchanged or tapered dose of inhaled corticosteroids, improvement in the increase of mean PEF at 4-week treatment was maintained for 1 year. No difference in the add-on effect of pranlukast was observed in patients treated with less than 800 microg and more than or equal to 800 microg of inhaled corticosteroids. Four patients underwent reduction of inhaled corticosteroids in the analyzed period and PEF was well-maintained and even increased by pranlukast treatment. In 11 patients in whom data for 3 years were available, the improvement in PEF persisted for 3 years. Although the present investigation is a retrospective analysis, these data may suggest that pranlukast has no tachyphylaxis and its effect continues for more than 1 year.

Herpes simplex virus infection in adult patients after unrelated cord blood transplantation: a single-institute experience in Japan.

Herpes simplex virus (HSV) infection in adult patients who underwent cord blood transplantation (CBT) from unrelated donors was studied. None of nine HSV-seronegative patients developed HSV disease after CBT. Of 28 HSV-seropositive patients, seven (25%) developed HSV disease at a median of 92 days after CBT (range, 52-239 days). The cumulative incidence of HSV disease in HSV-seropositive patients was 27% at 12 months after CBT. The manifestations of HSV disease included gingivostomatitis (three patients), herpes labialis (two patients), localized herpes facialis of the nose (one patient), and disseminated eczema herpeticum (one patient). HSV disease recurred in two patients as gingivostomatitis and disseminated eczema herpeticum. All the patients responded to antiviral therapy. The presence of grade II-IV acute graft-versus-host disease (GVHD) was significantly associated with a higher rate of HSV disease after CBT (51 vs 8%, P=0.015). These results suggest that the recovery of HSV-specific immune responses is delayed in patients who develop grade II-IV acute GVHD after CBT.

Effect of cyclophosphamide on serum cyclosporine levels at the conditioning of hematopoietic stem cell transplantation.

We retrospectively analyzed the factors that affect serum cyclosporine (CsA) concentrations up to day 14 after allogeneic hematopoietic stem cell transplantation (HSCT). In all, 103 transplant recipients who received MTX and CsA for acute GVHD prophylaxis were analyzed. No significant relationships between serum CsA concentrations and gender, age, serum creatinine levels, AST/ALT levels, or antibiotic/fluconazole administration were found by comparing median CsA concentrations or by using longitudinal or regression multivariate analyses. However, the mean of the median serum CsA concentration in patients (n=54) receiving the regimen containing cyclophosphamide (CY) (149.7 ng/ml; 95% confidence interval (CI): 132.1-167.4) was significantly (P<0.0001) lower than that in patients (n=49) receiving the non-CY regimen (217.3 ng/ml; 95% CI: 198.9-235.6). Longitudinal analysis and regression multivariate analysis showed that only administration of CY had a significant effect on the serum CsA concentration. Our results suggest that administration of CY during conditioning can reduce the effects on serum CsA concentrations during the 2 weeks following HSCT. The mechanism of this effect is not clear, but it may be due to the autoinduction of CY.

Effective transduction and stable transgene expression in human blood cells by a third-generation lentiviral vector.

Difficulty in gene transduction of human blood cells, including hematopoietic stem cells, has hampered the development of gene therapy applications for hematological disorders, encouraging the development and use of new gene delivery systems. In this study, we used a third-generation self-inactivating (SIN) lentiviral vector system based on human immunodeficiency virus type 1 (HIV-1) to improve transduction efficiency and prevent vector-related toxicity. The transduction efficiency of the HIV-1-based vector was compared directly with the Moloney murine leukemia virus (MLV) SIN vector in human leukemia cell lines. Initial transduction efficiencies were almost 100% for the HIV and less than 50% for the MLV vectors. Similar results were observed in 11 types of primary cells obtained from leukemia or myeloma patients. Transgene expression persisted for 8 weeks in cells transduced with the HIV vector, but declined with the MLV vector. In addition, resting peripheral blood lymphocytes and CD34(+) hematopoietic cells were transduced successfully with the HIV vector, but not with the MLV vector. Finally, we confirmed vector gene integration in almost all colony-forming cells transduced with the HIV vector, but not with the MLV vector. In conclusion, this lentiviral vector is an excellent gene transduction system for human blood cells because of its high gene transduction and host chromosome integration efficiency.

Acute disseminated encephalomyelitis (ADEM) after allogeneic bone marrow transplantation for acute myeloid leukemia.

Acute disseminated encephalomyelitis (ADEM) is a rare inflammatory demyelinating disease of the central nervous system. We describe here a patient who developed ADEM after allogeneic bone marrow transplantation (BMT). A 48-year-old woman with acute myeloid leukemia (M2) underwent allogeneic BMT from her HLA-identical sister. Cyclosporin for prophylaxis of acute graft-versus-host disease (GVHD) was discontinued from day 15 because of its toxicity. She was relatively well after the resolution of cytomegalovirus reactivation and chronic GVHD. Nine months after BMT, she suddenly developed diplopia, dysarthria, and gait disturbance. Computed tomography of the brain at that time revealed no abnormal findings. Leukemia recurrence was not revealed. The neurological symptoms were very mild without further deterioration. Her clinical course was carefully watched without therapy. Two weeks after onset, fluid attenuated inversion recovery magnetic resonance imaging (MRI) revealed multifocal abnormal high-signal intensity mainly in the white matter of the cerebrum as well as in the cerebellum and brainstem. Cerebrospinal fluid examination showed no abnormal findings. No laboratory findings suggested the presence of infectious agents. The typical MRI findings and an acute monophasic clinical course of this patient led to a diagnosis of ADEM. Twelve weeks after onset, the symptoms had almost resolved. Follow-up MRI showed a substantial improvement of the previous lesions without any new lesions. The symptoms had completely resolved 5 months after onset. This is a rare case of ADEM developing after allogeneic BMT.

Differential graft-versus-leukaemia effect by CD28 and CD40 co-stimulatory blockade after graft-versus-host disease prophylaxis.

Co-stimulatory blockade may be a promising strategy for tolerance induction in transplantation. In allogeneic bone marrow transplantation (BMT) for leukaemia treatment, however, preservation of the graft-versus-leukaemia (GVL) effect is another critical requirement for clinical application. In this study, we have compared the effect on GVL of using CD28 and CD40 co-stimulatory blockades as graft-versus-host disease (GVHD) prophylaxis in a murine allogeneic BMT model with simultaneous transfer of BCL1 leukaemia. Despite the relative improvement of GVHD as assessed by survival and body weight in both treatment regimes, treatment with anti-CD154 moAb clearly diminished the GVL effect, whereas treatment with anti-CD80 and CD86 MoAbs maintained this effect. Although T cell-mediated effector function at 14 days post-BMT assessed by IFNgamma expression and cytotoxicity against host alloantigen was comparable between both co-stimulatory blockades, IL-12 mRNA expression was preferentially reduced by CD40 blockade. Our results suggest the differential involvement of the CD28 and CD40 co-stimulatory pathways in the development of GVHD and GVL effects. CD28 blockade may be a favourable strategy for tolerance induction in leukaemia patients undergoing BMT.

Overexpression of the p53-inducible brain-specific angiogenesis inhibitor 1 suppresses efficiently tumour angiogenesis.

The brain-specific angiogenesis inhibitor 1 gene has been isolated in an attempt to find fragments with p53 "functional" binding sites. As reported herein and by others, brain-specific angiogenesis inhibitor 1 expression is present in some normal tissues, but is reduced or lost in tumour tissues. Such data and its particular structure prompted the hypothesis that brain-specific angiogenesis inhibitor 1 may act as a mediator in the local angiogenesis balance. We herein demonstrate that brain-specific angiogenesis inhibitor 1 over-expression suppresses tumour angiogenesis, delaying significantly the human tumour growth in immunodeficient mice. The inhibitory effect of brain-specific angiogenesis inhibitor 1 was documented using our intravital microscopy system, strongly implicating brain-specific angiogenesis inhibitor 1 as a mediator in the control of tumour angiogenesis. In contrast, in vitro tumour cell proliferation was not inhibited by brain-specific angiogenesis inhibitor 1 transfection, whereas some level of cytotoxicity was assessed for endothelial cells. Immunohistochemical analysis of tumour samples confirmed a reduction in the microvessel density index in brain-specific angiogenesis inhibitor 1-overexpressing tumours. At messenger level, moderate changes could be detected, involving the down-regulation of vascular endothelial growth factor and collagenase-1 expression. Furthermore, brain-specific angiogenesis inhibitor 1 expression that was lost in a selection of human cancer cell lines could be restored by wild-type p53 adenoviral transfection. Brain-specific angiogenesis inhibitor 1 should be considered for gene therapy and development of efficient drugs based on endogenous antiangiogenic molecules.

Transient hematopoietic stem cell rescue using umbilical cord blood for a lethally irradiated nuclear accident victim.

We performed stem cell rescue and allogeneic skin transplantation on a lethally neutron-irradiated nuclear accident victim. HLA-DRB1 mismatched unrelated umbilical cord blood cells (2.08 x 10(7)/kg recipient body weight) were transplanted to an 8-10 Gy equivalent neutron-irradiated patient because of a lack of a suitable bone marrow or peripheral blood donor. Pre-transplant conditioning consisted of anti-thymocyte gamma-globulin alone, and GVHD prophylaxis was a combination of cyclosporine (CYA) and methylprednisolone (mPSL). Granulocyte colony-stimulating factor (G-CSF), erythropoietin (EPO), and thrombopoietin (TPO) were concurrently administered after transplantation. The absolute neutrophil count reached 0.5 x 10(9)/l on day 15, the reticulocyte count rose above 1% on day 23, and the platelet count was over 50 x 10(9)/l on day 27, respectively. Cytogenetic studies of blood and marrow showed donor/recipient mixed chimerism. Rapid autologous hematopoietic recovery was recognized after withdrawal of CYA and mPSL. Repeated pathological examinations of the skin revealed no evidence of acute GVHD. Eighty-two days after the irradiation, skin transplantation was performed to treat radiation burns. Almost 90% of the transplanted skin engrafted. Immunological examination after autologous hematopoietic recovery revealed an almost normal T cell count. However, immune functions were severely impaired. The patient died from infectious complication 210 days after the accident.