Trans-splicing repair of CD40 ligand deficiency results in naturally regulated correction of a mouse model of hyper-IgM X-linked immunodeficiency.

X-linked immunodeficiency with hyper-IgM (HIGM1), characterized by failure of immunoglobulin isotype switching, is caused by mutations of the CD40 ligand (CD40L), which is normally expressed on activated CD4(+) T cells. As constitutive expression of CD40L induces lymphomas, we corrected the mutation while preserving the natural regulation of CD40L using pre-mRNA trans-splicing. Bone marrow from mice lacking CD40L was modified with a lentivirus trans-splicer encoding the normal CD40L exons 2-5 and was administered to syngenic CD40L-knockout mice. Recipient mice had corrected CD40L mRNA, antigen-specific IgG1 responses to keyhole limpet hemocyanin immunization, regulated CD4(+) T-cell CD40L expression after CD3 stimulation in primary and secondary transplanted mice, attenuation of Pneumocystis carinii pneumonia, and no evidence of lymphoproliferative disease over 1 year. Thus, HIGM1 can be corrected by CD40L trans-splicing, leading to functional correction of the genetic defect without the adverse consequences of unregulated expression of the CD40L gene.

OX40 ligand expressed by DCs costimulates NKT and CD4+ Th cell antitumor immunity in mice.

The exceptional immunostimulatory capacity of DCs makes them potential targets for investigation of cancer immunotherapeutics. We show here in mice that TNF-alpha-stimulated DC maturation was accompanied by increased expression of OX40 ligand (OX40L), the lack of which resulted in an inability of mature DCs to generate cellular antitumor immunity. Furthermore, intratumoral administration of DCs modified to express OX40L suppressed tumor growth through the generation of tumor-specific cytolytic T cell responses, which were mediated by CD4+ T cells and NKT cells. In the tumors treated with OX40L-expressing DCs, the NKT cell population significantly increased and exhibited a substantial level of IFN-gamma production essential for antitumor immunity. Additional studies evaluating NKT cell activation status, in terms of IFN-gamma production and CD69 expression, indicated that NKT cell activation by DCs presenting alpha-galactosylceramide in the context of CD1d was potentiated by OX40 expression on NKT cells. These results show a critical role for OX40L on DCs, via binding to OX40 on NKT cells and CD4+ T cells, in the induction of antitumor immunity in tumor-bearing mice.

Successful treatment of a case with rapidly progressive Bronchiolitis obliterans organizing pneumonia (BOOP) using cyclosporin A and corticosteroid.

A 60-year-old woman was suffering from acute onset and progressive respiratory distress. Her radiographic findings showed bilateral volume loss in her lower lobes and consolidation predominantly distributed in peribronchovascular areas. The biopsied specimens performed by video-assisted thoracoscopic surgery revealed prominent fibromyxoid connective tissue within the terminal respiratory bronchioles and the alveolar spaces along the airways without marked interstitial fibrosis. No relevant cause was determined, and she was diagnosed as having idiopathic BOOP. Although her clinical course was fulminant with a poor reaction to steroid therapy, simultaneous administration of cyclosporin A and corticosteroid elicited a rapid improvement. This case report presents the effectiveness of cyclosporin A in the treatment of progressive BOOP.

[An autopsy case of pulmonary and central nervous system metastatic osteosarcoma treated with thirty-six courses of chemotherapy over four years].

A 32-year-old man presented with cough, dyspnea and orthopnea ten years after amputation of the right humerus because of osteosarcoma. Chest radiographs and chest computed tomographs showed left pleural effusion, pericardial effusion and a giant intrathoracic mass, which was histologically diagnosed as a recurrence of the osteosarcoma. After 4 courses of chemotherapy combined with CDDP, the mass in the left upper lobe of the lung decreased in size, and it was then resected. Three months later, new metastatic lesions were detected in the thoracic area. Therefore, 29 additional courses of chemotherapy were administered (36 courses in total over 4 years; including regimens combined with CDDP, carboplatin, high-dose methotrexate, ifosfamide, dacarbazine, vindesine, etoposide, vincristine, taxotere and gemcitabine). In spite of the several courses of chemotherapy, brain and spinal cord metastases appeared, and the patient eventually died of cerebral hemorrhage. During the four years after the first recurrence he had good quality of life as a result of the chemotherapy.

Hepatocyte growth factor gene transfer to alveolar septa for effective suppression of lung fibrosis.

We examined therapeutic gene transfer of human hepatocyte growth factor (hHGF) to alveolar septa in mouse bleomycin-induced lung fibrosis using macroaggregated albumin-polyethylenimine complex (MAA-PEI). Intravenous administration of MAA-PEI along with 1 microg pCAG.hHGF to C57BL/6 mice increased the uptake of plasmids into alveolar capillary endothelial cells and epithelial cells, prolonged hHGF expression in the lung, and induced a level of hHGF expression equal to that seen with 10 microg of hHGF-expression plasmids alone. The exogenous source of hHGF gene expression increased the endogenous mouse HGF in the lungs and significantly decreased TNF-alpha, IL-6, and collagen synthesis after bleomycin injury. Because GFP-labeled bone marrow-derived stem cells after bleomycin injury were reduced in number by HGF, the primary mechanism of HGF is likely to be the prevention of apoptosis, as has been suggested by in vitro experiments. This novel HGF gene transfer method to alveolar septa with nonstimulatory MAA-PEI conjugates may have promising clinical applications.

Efficacy of induction chemoradiotherapy in thymic cancer: report of a successful case and review of the literature.

Thymic cancer is a rare tumor, the optimal treatment of which remains controversial. The efficacy of induction therapy in thymic cancer is unclear. A 51-year-old man was diagnosed as having a poorly differentiated carcinoma of the thymus with lymph node metastasis (stage IVb according to the Masaoka staging system), through an echo-guided biopsy. The patient was administrered cisplatin (CDDP) combined with paclitaxel once per week for 4 weeks, under concurrent local radiation. Once a partial response was achieved, the residual tumor was completely resected, and bilateral mediastinal lymph nodes were dissected. Histopathological examination of the resected specimen showed no evidence of viable malignant cells. At present, 15 months after surgery, the patient is doing well and shows no signs of recurrence. This case demonstrates that induction chemoradiotherapy with CDDP and paclitaxel may be well tolerated and useful for patients with advanced thymic cancer.

A newly identified AMSH-family protein is specifically expressed in haploid stages of testicular germ cells.

Associated Molecule with SH3 domain of STAM (AMSH) plays a critical role in the cytokine-mediated intracellular signal transduction downstream of the Jak2/Jak3-STAM complex. We newly identified a family molecule of AMSH, AMSH-FP (AMSH-Family Protein) in the mouse brain. AMSH-FP encodes the intracellular protein and has a highly conserved JAB1 Subdomain Homologous (JSH) region, suggesting that AMSH-FP may act as adaptor of gene transcription and/or regulation system. AMSH-FP has two splicing forms, one is expressed in various tissues, whereas the other one is restricted to expression in testis. We named the abundant type AMSH-FPalpha and the testis type AMSH-FPbeta. AMSH-FPbeta is a variant lacking N-terminal 166 amino acid residues of AMSH-FPalpha. Analysis of the 5(')-untranslated regions in AMSH-FPalpha and AMSH-FPbeta mRNAs and exon-intron structure of AMSH-FP gene suggests that testis-specific transcripts are generated due to alternative promoter usage and/or alternative splicing. Importantly, AMSH-FPbeta mRNA was not detected in juvenile and infertile mouse testis but was restrictively expressed in the haploid stage of testicular germ cells in the normal mature testis. We suggested that AMSH-FPbeta had a functional role in the spermiogenesis.

Targeting angiogenesis and HGF function using an adenoviral vector expressing the HGF antagonist NK4 for cancer therapy.

Hepatocyte growth factor (HGF) affects tumor growth/invasion and tumor neovascularization. A proposed HGF antagonist, NK4 (an amino-terminal kringle-domain peptide of HGF), inhibits tumor growth/invasion through the competition of HGF binding to its receptor, c-Met, and acts as an angiogenesis inhibitor. To investigate the in vivo effect of NK4 gene transfer, we constructed an adenovirus vector expressing human NK4 (AdCMV.NK4). Human lung cancer cell lines (A549 and H358) infected in vitro with AdCMV.NK4 yielded NK4 protein without a change in the cell growth rate. In contrast, direct injection of AdCMV.NK4 (1 x 10(9) pfu, twice) into established subcutaneous tumors in BALB/c nu/nu mice resulted in suppression of the tumors by 64% for A549 or by 91% for H358 compared with controls (P<0.02 or P<0.01, respectively). Counting of the tumor vessels revealed suppressed vascularity by 57% in H358 tumors when using AdCMV.NK4 (P<0.0001). Furthermore, systemic NK4 delivery by intraperitoneal injection of AdCMV.NK4 effectively suppressed both angiogenesis in the Matrigel assay (86% reduction, P<0.032), subcutaneous tumor growth in vivo (by 65% for H358, P<0.001), and hematogenous lung metastases without obvious side effects. These results indicate that NK4 elicits tumor-growth suppression in vivo through its anti-angiogenic activity and anti-HGF activity and that NK4 gene transfer can be an effective tool in the treatment of cancer.

Decreased microsomal triglyceride transfer protein activity contributes to initiation of alcoholic liver steatosis in rats.

BACKGROUND/AIMS: To elucidate the role of microsomal triglyceride transfer protein (MTP) in the pathogenesis of alcoholic fatty liver, the effects of ethanol on MTP activity and gene expression were investigated. METHODS AND RESULTS: Male Sprague-Dawley rats fed an ethanol-containing liquid diet for 37 days, respectively, showed 2.9- and 4.9-fold increases in hepatic cholesterol and triglyceride content in comparison with rats fed an isocaloric ethanol-free diet (P<0.01). Furthermore, a significant decrease in MTP activity and mRNA expression (by 27 and 58%, respectively) was observed after ethanol administration. Intravenous injection of human recombinant hepatocyte growth factor (hrHGF) on each of the last 7 days markedly suppressed ethanol-induced lipid accumulation in the liver. This inhibition of fatty change by hrHGF was accompanied by recovery of MTP activity and gene expression. No inhibitory effect of hrHGF on ethanol-induced acyl-CoA synthetase activation was observed. Experiments using human hepatoma-derived HepG2 cells indicated a direct positive effect of hrHGF on MTP gene expression as well as apolipoprotein B secretion. CONCLUSIONS: These results suggest that reduced MTP activity is crucial to development of alcoholic fatty liver, while promotion of MTP activity by HGF might serve as a therapeutic measure against alcoholic liver steatosis.

A new predictor of improvement in regional left ventricular function assessed by ST/HR slope derived from ST elevation on exercise testing in patients with healed myocardial infarction.

This study evaluates the clinical usefulness of ST/HR slope derived from ST elevation on exercise to predict the improvement in regional wall motion following coronary revascularization in patients with healed myocardial infarction. We studied 58 patients with a diseased, infarct-related and single-vessel coronary artery. The decline calculated from the final 12 data points relating ST-segment elevation to heart rate during exercise were derived (ST/HR slope). Hypokinesis in the infarcted region was assessed by the centerline method and expressed in terms of standard deviations (SD/chord). The increase more than 30% of the SD/chord, which was defined as the improvement in regional wall motion, was seen in 23 of the 30 patients with a ST/HR slope of > or = 5.0 (microV/bpm), and in 4 of the 28 patients with a ST/HR slope <5.0 (microV/bpm) (P <.0001). Thus, a ST/HR slope derived from ST elevation on exercise identifies subgroups of patients who show a good recovery of regional wall motion.