Treatment sequence in castration-resistant prostate cancer: A retrospective study in the new anti-androgen era.

In recent years, abiraterone acetate (AA) and enzalutamide (EZL) have become available for the treatment of cancer. Prior clinical trials have demonstrated the benefits of these agents in males with castration-resistant prostate cancer (CRPC). The optimal sequencing of available therapies in the context of efficacy and known cross-resistance remains uncertain. Based on the mechanisms of action and accessible clinical data, AA and EZL may be indicated for the early stages of prostate cancer. Until clinical trials are conducted to determine the best treatment sequence, individualized therapy is required for each patient based on the clinicopathological characteristics. In the present study, 46 sequential patients (median age: 77, range 59-89; median serum PSA level: 56 ng/ml, range 1.5-3,211) with CRPC treated with EZL (160 mg/day) were retrospectively analyzed between June 2014 and July 2015 at the following institutions: Yamagata Prefectural Central Hospital (Yamagata, Japan); Yamagata Tokushukai Hospital (Yamagata, Japan); Ishinomaki Red Cross Hospital (Ishinomaki, Japan); Kan-etsu Hospital (Tsurugashima, Japan); Niigata Cancer Center Hospital (Niigata, Japan); Sakado Central Hospital (Sakado, Japan). A total of 18 patients were pre-treated with Docetaxel (DOC) and 28 patients were DOC-naïve. Once EZL therapy was initiated, increases in prostate specific antigen (PSA) levels were observed in 3/18 patients (17%) pre-treated with DOC and in 6/20 (30%) who were DOC-naïve. In total, 8/28 DOC-naïve patients were treated with AA without EZL. An increase in the PSA level was observed in only 1/8 (12%) cases following AA treatment in the DOC-naïve group. It was demonstrated that AA had a better efficacy in DOC-naïve patients. The efficacy of EZL was limited in AA-pre-treated patients following DOC administration.

Porcine TCR CD3zeta-chain and eta-chain.

Complete porcine CD3zeta-chain cDNA sequence was obtained for the first time, and its genomic nucleotide sequence was investigated from exon 2 down to CD3eta-chain exon 8. The sequence of porcine CD3zeta-chain showed homologous amino acid sequence with human and murine counterparts, in contrast to CD3eta-chain exon 8 with diversity among animals previously investigated. CD3eta-chain peptide is an alternative splice form of CD3zeta-chain exon 7 splicing to CD3eta-chain exon 8 instead of CD3zeta-chain exon 8. The genomic sequences revealed that the splice acceptor sequences of CD3eta-chain exon 8 of all animals investigated to be completely uniform. Further, CD3eta-chain exon 8 amino acid sequences retained the unique characters of having high proline (Pro) and positively charged amino acid content except for rats and mice. Although the biological role of CD3eta-chain remains to be enigmatic, these evidences suggests the evolutional pressure to maintain its sequence.

Transplantation of human hepatocytes cultured with deleted variant of hepatocyte growth factor prolongs the survival of mice with acute liver failure.

BACKGROUND: Considering the scarcity of donor livers, it is extremely important to establish a functional culture method for isolated hepatocytes. As a tool for maintaining hepatocyte functions in vitro, dHGF, a variant of HGF (hepatocyte growth factor) with a deletion of five amino acids, attracted our attention because it is less cytotoxic compared with HGF. METHODS: We evaluated growth, albumin production, metabolizing abilities of ammonia, lidocaine, and diazepam of human hepatocytes in the presence of dHGF (10-1000 ng/ml). The gene expression of liver markers was comparatively analyzed. The effect of intrasplenic transplantation of dHGF-treated human hepatocytes into severe combined immunodeficient (SCID) mice was evaluated in an acute liver failure (ALF) model induced by D-galactosamine (D-gal). RESULTS: When 100 ng/ml of dHGF was utilized, metabolism rates of ammonia, lidocaine, and diazepam and albumin production per unit cell significantly increased. The gene expression analysis demonstrated the enhanced expression of albumin, HNF-4alpha, and C/EBPalpha in the hepatocytes treated with 100 ng/ml of dHGF. Transplantation of such hepatocytes prolonged the survival of the SCID mice with ALF induced by D-gal. CONCLUSIONS: The present work clearly demonstrates the usefulness of dHGF (100 ng/ml) for maintaining the differentiated functions of human hepatocytes in tissue culture.

Transplantation of nonvascularized kidney tissue fragments into the rat liver with the aim of preserving renal function.

For research in regenerative medicine, not only the study of cellular pluripotency but also knowledge of the reorganization of tissue structure is crucial. However, the latter will probably be more difficult to acquire. When small fragments of kidney (approx. 1 x 1 mm) were implanted in the liver of syngeneic LEW rats, the tissue survived at least 2 weeks with retention of normal structure including glomeruli and tubules. In contrast, no kidney structure survived when transplanted to subcutaneous sites, omentum, or spleen. Molecules involved in renal tubular function, such as megalin and glut2 transporter protein, were detectable in the implanted tissue by immunohistochemistry, suggesting that the cells were biologically active. Survival of cortex, medulla, and calyx tissues was then compared. All three components were still detectable 8 weeks after transplantation but cortex and medulla were replaced by granuloma at 6 months. Only calyx tissue survived for up to 12 months after transplantation. There was no marked difference in tissue survival, either when the recipient liver was partially resected or when infantile donor kidney was implanted instead of adult kidney. The present method opens new avenues in the development of regenerative medicine (i.e., tissue transplantation) as an intermediate modus between organ transplantation and cell transplantation.

Development of novel monoclonal antibody 4G8 against swine leukocyte antigen class I alpha chain.

A mouse monoclonal antibody (MAb) was generated against swine leukocyte antigen (SLA) class I alpha chain. A newly developed series of MAb clones that react with pan leukocytes were selected and tested by immuno-histochemistry using SLA class I alpha chain expressing Cos-7 cells. Among them, MAb 4G8 was characterized by the following features: (1) 4G8 reacted with Cos-7 cells transfected with SLA class I alpha chain from the d haplotype, (2) 4G8 recognized epitopes that were different from those of commercially available anti-SLA class I MAbs 74-11-10 and PT85A, and (3) 4G8 could be used to immunostain frozen sections of thymus, spleen, lymph node, kidney, and liver tissues with good results.

Standardization of ischemic hepatic failure in pigs as a model for bioartificial liver assessment.

PURPOSE: A standard protocol of ischemic liver failure in pigs was examined to establish a system for assessing the efficacy of a bioartificial liver, based on clinical practice. METHODS: The portal blood flow was extracorporeally bypassed into the cervical jugular vein, using a centrifugal blood pump. The portal vein and hepatic artery were then ligated. RESULTS: The maintenance protocol was established as follows: (1) the concentration of the inhaled anesthetic was decreased by 0.2% when the systolic blood pressure was <100 mmHg; (2) the volume of an infusion containing 5% glucose was increased to 10 ml/kg per hour when central venous pressure was <5 mmHg; (3) 20 ml of 50% glucose was injected intravenously when the blood glucose was <50 mg/dl; (4) 2000 units of heparin was injected intravenously when the activated clotting time was <150 s; (5) sodium bicarbonate was given when the blood pH was <7.3; (6) tidal volume was increased by 1 ml/kg when the pCO(2) was >80 mmHg; (7) oxygen was increased by 25% when the pO(2) was <100 mmHg. No vasopressors were used in the experiment. CONCLUSION: Our protocol reduced the operating time and minimized the risk of data deviation that can arise from variations in operating techniques and individual animal conditions. This experimental model is also easy to use as a bridge to transplantation.

Catalytic RAG1 mutants obstruct V(D)J recombination in vitro and in vivo.

To generate severe combined immunodeficient (SCID) livestocks for xenotransplantation, we have attempted to generate a SCID phenotype without gene knockout. Based on the reported mouse RAG1 mutants, we constructed the corresponding rabbit RAG1 mutants by mutagenesis of three residues within the catalytic domain: D602A, D710A, and E964A. As expected, these mutants each exhibited no catalytic activity on artificial substrates and inhibited recombination by the wild type RAG1. Moreover, replacement of the N-terminus of RAG1 with enhanced green fluorescent protein (EGFP) greatly increased protein stability, and the triple mutant RAG1 showed a twofold increase in its ability to inhibit wild type activity in vitro. We generated mice transgenic for the latter mutant to assess its effect on V(D)J recombination in vivo. Serum IgM levels in four out of seven transgenic mice were reduced to approximately 30-50% of control levels in four out of seven transgenic mice. Our results suggest that immunodeficient animals for regenerative medicine could be generated without gene knockout.

Amelioration of experimental autoimmune encephalomyelitis in Lewis rats by FTY720 treatment.

Experimental autoimmune encephalomyelitis (EAE) is a T-cell-dependent autoimmune disease that reproduces the inflammatory demyelinating pathology of multiple sclerosis (MS). We investigated the efficacy and mechanism of immunosuppression against EAE by administering 2-amino-[2-(4-octylphenyl) ethyl]-1,3-propanediol hydrochloride (FTY720) in Lewis rats immunized with myelin basic protein together with complete Freund's adjuvant. FTY720 treatment almost completely protected the rats against disease. The protection by FTY720 was associated with a dramatic reduction in the number of lymphocytes staining for T-cell receptors in the spinal cord as examined by immunohistochemistry. The mRNA expression of Th1 cytokines interleukin (IL)-2, IL-6, and interferon-gamma in the spinal cord was also reduced dramatically as assessed by reverse-transcription polymerase chain reaction. Furthermore, lymphocytes isolated from the spleen of FTY720-treated rats were transferred into naive recipient rats against EAE manifestation by reducing both disease incidence and clinical score. These results suggested that the protective anti-inflammatory effect of treatment with FTY720 was, to a large extent, due to the inhibition of encephalitogenic T-cell responses and/or their migration into the central nervous system and may be a potential candidate for use in treating patients with MS.

Transplantation of amniotic epithelial cells into fetal rat liver by in utero manipulation.

It has been hoped that amniotic epithelial cells would be a gene carrier to neural and hepatic tissue, because of 1) the presence of neural and hepatic stem-like cells, 2) the ability to cryopreserve them, 3) long-term survival in the transplanted site, and 4) few ethical problems concerning procurement. But transplantation of a sufficient number of cells to adult tissue needs large-scale cell supply and may lead to vascular embolism. We attempted transplantation of amniotic epithelial cells into fetal liver, because 1) the fetal liver is at the proliferative stage, 2) the number of cells required is small, and 3) the fetal stage is advantageous for the induction of immunological tolerance. Amniotic epithelial cells from day 18.5-20.5 fetuses were transfected with adenoviral AdlacZ and harvested to inject into fetal rat liver of the syngeneic strain (day 18.5-20.5). The efficacy of cell transplantation into the liver increased in the order: intraplacental < intraumbilical vein < intrahepatic route. LacZ-transfected amniotic cells (1-8 x 10(5) cells), hepatocytes (5 x 10(5) cells), or AdlacZ vector solution (1.7 x 10(7) pfu) were injected through the uterine membrane into the liver. Transplanted cells formed a cellular mass and survived for up to 14 days after birth, whereas lacZ-transfected cells were rapidly decreased after the injection of AdlacZ vector or rat hepatocytes as a gene carrier so that the use of amniotic epithelial cells as a gene carrier will result in long-term expression of exogenous genes in the liver.

Electrical connections between pulmonary veins: implication for ostial ablation of pulmonary veins in patients with paroxysmal atrial fibrillation.

BACKGROUND: Electrical disconnection of the myocardial extensions into arrhythmogenic pulmonary veins (PVs) is recognized as a curative technique for paroxysmal atrial fibrillation (AF). However, the presence of electrical connections between the PVs, which may make achievement of PV disconnection difficult, has not been systematically evaluated. METHODS AND RESULTS: Forty-nine consecutive patients with drug-resistant AF underwent ostial radiofrequency (RF) catheter ablation of arrhythmogenic PVs with foci triggering AF. Pacing from inside the targeted PV was performed after each RF delivery to identify the left atrial exit site of the residual venoatrial conduction. Successful PV disconnection was defined as achieving elimination of the PV potentials during sinus rhythm or left atrial pacing, and the loss of left atrial conduction during intra-PV pacing. A total of 112 arrhythmogenic PVs were identified. PV disconnection was achieved with 10+/-6.1 minutes of RF delivery to the ostia of 101 targeted PVs. In 7 left superior (LS) PVs from 7 patients (14%), the earliest atrial activity was recorded from the left inferior (LI) PV ostium during intra-LSPV pacing after 11+/-4.7 minutes of RF delivery to the LSPV ostium. Disconnection of these LSPVs was achieved by LIPV disconnection. In the remaining 4 PVs from 4 patients, PV disconnection could not be achieved. CONCLUSIONS: Fourteen percent of the patients had electrical connections between contiguous PVs. In these patients, ostial ablation of an untargeted PV was required for successful targeted PV disconnection.

Prolonged survival in rat liver transplantation with mouse monoclonal antibody against an inducible costimulator (ICOS).

BACKGROUND: An inducible costimulator (ICOS), a recently identified costimulatory receptor with a close structural homology to CD28 and CTLA4, is expressed on activated T cells. Interaction with its ligand on antigen-presenting cells stimulates T-cell proliferation to produce a different spectrum of cytokine. The inhibition of ICOS-mediated signal transduction by an anti-ICOS antibody is considered to be capable of protecting against graft rejection in organ transplantation. METHODS: An anti-rat ICOS antibody was intravenously administered into recipients of dark Agouti-to-Lewis liver transplantations. The recipient lymphocytes from mesenteric lymph nodes were harvested on day 7 after transplantation for fluorescence-activated cell sorting analysis, and tissue specimens from the grafts were removed for histologic evaluation. Antigen-specific T-cell proliferation responses were assessed in vitro with anti-ICOS antibody. RESULTS: Monotherapy with the antibody significantly prolonged the graft survival time by inhibiting T-cell activation and its proliferation response. The graft-infiltrating cells, both CD4 and CD8 T cells, were not completely reduced even when rats were administered the antibody, whereas the expression of ICOS almost completely disappeared in these cells. CONCLUSIONS: T-cell activation through the ICOS costimulatory pathway plays an important role in graft rejection, and manipulating its pathway is an effective method for modulating transplantation immunity.