Enhanced IL-12 transgene expression improves oncolytic viroimmunotherapy.

Introduction: Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive sarcomas with unacceptably low cure rates occurring often in patients with neurofibromatosis 1 defects. To investigate oncolytic Herpes Simplex Virus (oHSV) as an immunotherapeutic approach, we compared viral replication, functional activity, and immune response between unarmed and interleukin 12 (IL-12)-armed oncolytic viruses in virus-permissive (B109) and -resistant (67C-4) murine MPNSTs. Methods: This study compared two attenuated IL-12-oHSVs with γ134.5 gene deletions (Δγ134.5) and the same transgene expression cassette. The primary difference in the IL-12-oHSVs was in their ability to counter the translational arrest response in infected cells. Unlike M002 (Δγ134.5, mIL-12), C002 (Δγ134.5, mIL-12, IRS1) expresses an HCMV IRS1 gene and evades dsRNA activated translational arrest in infected cells. Results and discussion: Our results show that oHSV replication and gene expression results in vitro were not predictive of oHSV direct oncolytic activity in vivo. Tumors that supported viral replication in cell culture studies resisted viral replication by both oHSVs and restricted M002 transgene expression in vivo. Furthermore, two IL-12-oHSVs with equivalent transcriptional activity differed in IL-12 protein production in vivo, and the differences in IL-12 protein levels were reflected in immune infiltrate activity changes as well as tumor growth suppression differences between the IL-12-oHSVs. C002-treated tumors exhibited sustained IL-12 production with improved dendritic cells, monocyte-macrophage activity (MHCII, CD80/CD86 upregulation) and a polyfunctional Th1-cell response in the tumor infiltrates. Conclusion: These results suggest that transgene protein production differences between oHSVs in vivo, in addition to replication differences, can impact OV-therapeutic activity.

Targeting TMEM205 mediated drug resistance in ovarian clear cell carcinoma using oncolytic virus.

BACKGROUND: Ovarian clear cell carcinoma (OCCC) accounts for approximately 8-10% of epithelial ovarian cancers in the United States. Although it is rare, OCCC usually presents with treatment challenges and the overall prognosis is far worse than high grade serous ovarian cancer HGSOC. The objective of this study was to examine the therapeutic relevance of combining oncolytic virus with cisplatin for ovarian cancer clear cell carcinoma (OCCC). RESULTS: We identified that TMEM205, a recently discovered transmembrane protein, contributes to chemoresistance in OCCC cells via the exosomal pathway. Mechanistically, TMEM205 undergoes ligand-independent constitutive endocytosis and co-localizes with Rab11 to contribute to the late recycling endosomes in a clathrin-independent manner. Further, we observed that oncolytic virus (oHSV) pretreatment followed by treatment with cisplatin decreases TMEM205 expression and sensitizes cells to cisplatin in a synergistic manner in OCCC cells. TMEM205 interacts with glycoprotein-C of oHSV post-infection; both of these proteins undergo ubiquitination and ultimately get shuttled outside the cell via exosomes. Thus, we demonstrate the mechanotransduction pathway of TMEM205-mediated chemoresistance along with targeting this pathway using oHSV and cisplatin as a powerful therapeutic strategy for OCCC. oHSV combination with cisplatin inhibits OCCC tumor growth in vivo in immunodeficient and immunocompetent mice models. CONCLUSION: Our results suggest that the combination of oHSV and cisplatin in immunocompetent as well as immune deficient OCCC tumor bearing mice reduces overall tumor burden as well as metastatic disease thereby providing survival benefit. Additionally, the detection of TMEM205 in exosomal cargo early in OCCC development has potential to be exploited as a biomarker.

Aberrant expression of TMEM205 signaling promotes platinum resistance in ovarian cancer: An implication for the antitumor potential of DAP compound.

INTRODUCTION: TMEM205 is a novel transmembrane protein associated with platinum resistance (PR) in epithelial ovarian carcinoma (OC), however, the specific mechanisms associated with this resistance remain to be elucidated. METHODS: TMEM205 expression was evaluated in platinum-sensitive (PS) versus platinum resistant (PR) ovarian cancer cell lines and patient serum/tissues. Exosomal efflux of platinum was evaluated with inductively coupled plasma mass spectrometry (ICP-MS) after pre-treatment with small molecule inhibitors (L-2663/L-2797) of TMEM205 prior to treatment with platinum. Cytotoxicity of combination treatment was confirmed in vitro and in an in vivo model. RESULTS: TMEM205 expression was 10-20 fold higher in PR compared to PS ovarian cancer cell lines, serum samples, and tissues. Co-localization with CD1B was confirmed by in-situ proximity ligation assay suggesting that TMEM205 may mediate PR via the exosomal pathway. Exosomal secretion was significantly increased 5-10 fold in PR cell lines after treatment with carboplatin compared to PS cell lines. Pre-treatment with L-2663 prior to carboplatin resulted in significantly increased intracellular concentration of fluorescently-labeled cisplatin and decreased exosomal efflux of platinum. Decreased cell survival and tumor growth in vitro and in vivo was observed when PR cells were treated with a combination of L-2663 with carboplatin compared to carboplatin alone. CONCLUSION: TMEM205 appears to be involved in the development of PR in ovarian cancer through the exosomal efflux of platinum agents. This study provides pre-clinical evidence that TMEM205 could serve as a possible biomarker for PR as well as a therapeutic target in combination with platinum agents.

Eliciting an immune-mediated antitumor response through oncolytic herpes simplex virus-based shared antigen expression in tumors resistant to viroimmunotherapy.

BACKGROUND: Oncolytic virotherapy (OV) is an immunotherapy that incorporates viral cancer cell lysis with engagement of the recruited immune response against cancer cells. Pediatric solid tumors are challenging targets because they contain both an inert immune environment and a quiet antigenic landscape, making them more resistant to conventional OV approaches. Further complicating this, herpes simplex virus suppresses host gene expression during virotherapy infection. METHODS: We therefore developed a multimodal oncolytic herpes simplex virus (oHSV) that expresses ephrin A2 (EphA2), a shared tumor-associated antigen (TAA) expressed by many tumors to improve immune-mediated antitumor activity. We verified the virus genotypically and phenotypically and then tested it in an oHSV-resistant orthotopic model (including immunophenotypic analysis), in flank and in T cell-deficient mouse models. We then assessed the antigen-expressing virus in an unrelated peripheral tumor model that also expresses the shared tumor antigen and evaluated functional T-cell response from the treated mice. RESULTS: Virus-based EphA2 expression induces a robust acquired antitumor immune responses in both an oHSV-resistant murine brain and peripheral tumor model. Our new multimodal oncolytic virus (1) improves survival in viroimmunotherapy resistant tumors, (2) alters both the infiltrating and peripheral T-cell populations capable of suppressing tumor growth on rechallenge, and (3) produces EphA2-specific CD8 effector-like populations. CONCLUSIONS: Our results suggest that this flexible viral-based platform enables immune recognition of the shared TAA and improves the immune-therapeutic response, thus making it well suited for low-mutational load tumors.

Oncolytic HSV Therapy Modulates Vesicular Trafficking Inducing Cisplatin Sensitivity and Antitumor Immunity.

PURPOSE: Here we investigated the impact of oncolytic herpes simplex virus (HSV) treatment on cisplatin sensitivity of platinum-resistant ovarian cancer, and the impact of the combination on immunotherapy. EXPERIMENTAL DESIGN: Therapeutic efficacy of the combination was assessed in platinum-resistant human and murine ovarian cancer peritoneal metastatic mouse models (n = 9-10/group). RNA sequencing along with flow cytometry of splenocytes from treated mice was employed to examine the effect of antitumor immune response (n = 3/group). Anti-PD-1 antibody was performed to evaluate impact on checkpoint inhibition in vivo. RESULTS: Gene Ontology pathway analysis uncovered disruption of cellular extracellular vesicle (EV)-related pathways in infected cells (FDR = 2.97E-57). Mechanistically, we identified reduced expression of transporters expressed on EV implicated in cisplatin efflux. The increased cisplatin retention led to increased cisplatin-DNA adducts, which resulted in micronuclei and the subsequent activation of cGAS-STING pathway with a significant activation of innate immune cells and translated to an increase in antitumor immunity and efficacy. In mice bearing platinum-resistant ovarian cancer, we also observed a feedback induction of PD-L1 on tumor cells, which sensitized combination-treated mice to anti-PD-1 immune checkpoint therapy. CONCLUSIONS: To our knowledge, this is the first report to show HSV-induced cisplatin retention in infected cells. The consequential increased damaged DNA was then expelled from cells as micronuclei which resulted in induction of inflammatory responses and education of antitumor immunity. The combination therapy also created an environment that sensitized tumors to immune checkpoint therapy.

Hypoxia-induced exosomes contribute to a more aggressive and chemoresistant ovarian cancer phenotype: a novel mechanism linking STAT3/Rab proteins.

Hypoxia-mediated tumor progression, metastasis, and drug resistance are major clinical challenges in ovarian cancer. Exosomes released in the hypoxic tumor microenvironment may contribute to these challenges by transferring signaling proteins between cancer cells and normal cells. We observed that ovarian cancer cells exposed to hypoxia significantly increased their exosome release by upregulating Rab27a, downregulating Rab7, LAMP1/2, NEU-1, and also by promoting a more secretory lysosomal phenotype. STAT3 knockdown in ovarian cancer cells reduced exosome release by altering the Rab family proteins Rab7 and Rab27a under hypoxic conditions. We also found that exosomes from patient-derived ascites ovarian cancer cell lines cultured under hypoxic conditions carried more potent oncogenic proteins-STAT3 and FAS that are capable of significantly increasing cell migration/invasion and chemo-resistance in vitro and tumor progression/metastasis in vivo. Hypoxic ovarian cancer cells derived exosomes (HEx) are proficient in re-programming the immortalized fallopian tube secretory epithelial cells (FT) to become pro-tumorigenic in mouse fallopian tubes. In addition, cisplatin efflux via exosomes was significantly increased in ovarian cancer cells under hypoxic conditions. Co-culture of HEx with tumor cells led to significantly decreased dsDNA damage and increased cell survival in response to cisplatin treatment. Blocking exosome release by known inhibitor Amiloride or STAT3 inhibitor and treating with cisplatin resulted in a significant increase in apoptosis, decreased colony formation, and proliferation. Our results demonstrate that HEx are more potent in augmenting metastasis/chemotherapy resistance in ovarian cancer and may serve as a novel mechanism for tumor metastasis, chemo-resistance, and a point of intervention for improving clinical outcomes.

STAT3/PIAS3 Levels Serve as "Early Signature" Genes in the Development of High-Grade Serous Carcinoma from the Fallopian Tube.

The initial molecular events that lead to malignant transformation of the fimbria of the fallopian tube (FT) through high-grade serous ovarian carcinoma (HGSC) remain poorly understood. In this study, we report that increased expression of signal transducer and activator of transcription 3 (pSTAT3 Tyr705) and suppression or loss of protein inhibitor of activated STAT3 (PIAS3) in FT likely drive HGSC. We evaluated human tissues-benign normal FT, tubal-peritoneal junction (TPJ), p53 signature FT tissue, tubal intraepithelial lesion in transition (TILT), serous tubal intraepithelial carcinoma (STIC) without ovarian cancer, and HGSC for expression of STAT3/PIAS3 (compared with their known TP53 signature) and their target proliferation genes. We observed constitutive activation of STAT3 and low levels or loss of PIAS3 in the TPJ, p53 signature, TILT, and STIC through advanced stage IV (HGSC) tissues. Elevated expression of pSTAT3 Tyr705 and decreased levels of PIAS3 appeared as early as TPJ and the trend continued until very advanced stage HGSC (compared with high PIAS3 and low pSTAT3 expression in normal benign FT). Exogenous expression of STAT3 in FT cells mediated translocation of pSTAT3 and c-Myc into the nucleus. In vivo experiments demonstrated that overexpression of STAT3 in FT secretory epithelial cells promoted tumor progression and metastasis, mimicking the clinical disease observed in patients with HGSC. Thus, we conclude that the STAT3 pathway plays a role in the development and progression of HGSC from its earliest premalignant states.Significance: Concomitant gain of pSTAT3 Tyr705 and loss of PIAS3 appear critical for initiation and development of high-grade serous carcinoma. Cancer Res; 78(7); 1739-50. ©2018 AACR.

Targeting STAT3 by HO3867 induces apoptosis in ovarian clear cell carcinoma.

Advanced ovarian clear cell carcinoma (OCCC) carries a very poor prognosis in large part secondary to the extremely high rate of resistance to standard platinum and taxane chemotherapy. Signal transducer and activator of transcription 3(STAT3) expression and activation has been shown to regulate tumor progression in various human cancers, though has not been well studied in OCCC. Preliminary work in our lab has demonstrated constitutive activation of STAT3 (pSTAT3Tyr705 or pSTAT3727) in OCCC cell lines as well as human OCCC tumor tissue samples. Significantly, pSTAT3 is expressed in the absence of other forms of activated STAT (pSTAT1, 2, 6). Therefore, this work was planned to investigate the role of STAT3 and examine the efficacy of a novel anti-cancer compound -HO-3867, which is an inhibitor of STAT3, using known OCCC cell lines. Results demonstrate that treatment with HO-3867 decreased expression of pSTAT3 Tyr705 as well pSTAT3 Ser727, while total STAT3 remained constant. STAT3 overexpression increased the migration capability in OVTOKO cells in vitro and led to an increased tumor size when injected in vivo. The inhibitory effect of HO-3867 on cell proliferation and cell survival was accompanied by increased apoptosis, within 24 h post treatment. Treatment with HO-3867 resulted in a decrease in Bcl-2 and increase of cleavage of caspase 3, caspase 7, and PARP, confirming induction of apoptosis after treatment with HO-3867. In addition, HO-3867 significantly inhibited formation of human umbilical vein endothelial cells capillary-like structures and invasion at both 5 and 10 µM concentrations. STAT3 expression plays an important role in the spread of OCCC in vitro as well as in vivo. Thus, we can exploit the STAT3 pathway for targeted drug therapy. Inhibition of pSTAT3 using HO-3867in OCCC cell lines appears to be a promising therapy. This is of utmost importance given the poor response of OCCC to standard chemotherapy regimens.

High Glucose-Mediated STAT3 Activation in Endometrial Cancer Is Inhibited by Metformin: Therapeutic Implications for Endometrial Cancer.

OBJECTIVES: STAT3 is over-expressed in endometrial cancer, and diabetes is a risk factor for the development of type 1 endometrial cancer. We therefore investigated whether glucose concentrations influence STAT3 expression in type 1 endometrial cancer, and whether such STAT3 expression might be inhibited by metformin. METHODS: In Ishikawa (grade 1) endometrial cancer cells subjected to media with low, normal, or high concentrations of glucose, expression of STAT3 and its target proteins was evaluated by real-time quantitative PCR (qPCR). Ishikawa cells were treated with metformin and assessed with cell proliferation, survival, migration, and ubiquitin assays, as well as Western blot and qPCR. Expression of apoptosis proteins was evaluated with Western blot in Ishikawa cells transfected with a STAT3 overexpression plasmid and treated with metformin. A xenograft tumor model was used for studying the in vivo efficacy of metformin. RESULTS: Expression of STAT3 and its target proteins was increased in Ishikawa cells cultured in high glucose media. In vitro, metformin inhibited cell proliferation, survival and migration but induced apoptosis. Metformin reduced expression levels of pSTAT3 ser727, total STAT3, and its associated cell survival and anti-apoptotic proteins. Additionally, metformin treatment was associated with increased degradation of pSTAT3 ser727. No change in apoptotic protein expression was noticed with STAT3 overexpression in Ishikawa cells. In vivo, metformin treatment led to a decrease in tumor weight as well as reductions of STAT3, pSTAT3 ser727, its target proteins. CONCLUSIONS: These results suggest that STAT3 expression in type 1 endometrial cancer is stimulated by a high glucose environment and inhibited by metformin.

Elevated STAT3 expression in ovarian cancer ascites promotes invasion and metastasis: a potential therapeutic target.

Although activation of the STAT3 pathway has been associated with tumor progression in a wide variety of cancer types (including ovarian cancer), the precise mechanism of invasion and metastasis due to STAT3 are not fully delineated in ovarian cancer. We found that pSTAT3 Tyr705 is constitutively activated in patient ascites and ascites-derived ovarian cancer cells (ADOCCs), and the range of STAT3 expression could be very high to low. In vivo transplantation of ADOCCs with high pSTAT3 expression into the ovarian bursa of mice resulted in a large primary tumor and widespread peritoneal metastases. In contrast, ADOCCs with low STAT3 expression or ADOCCs with STAT3 expression knockdown, led to reduced tumor growth and an absence of metastases in vivo. Cytokines derived from the ADOCC culture medium activate the interleukin (IL)-6/STAT pathway in the STAT3 knockout (KO) cells, compensating for the absence of inherent STAT3 in the cells. Treatment with HO-3867 (a novel STAT3 inhibitor at 100 p.p.m. in an orthotopic murine model) significantly suppressed ovarian tumor growth, angiogenesis and metastasis by targeting STAT3 and its downstream proteins. HO-3867 was found to have cytotoxic effects in ex vivo cultures of freshly collected human ovarian cancers, including those resistant to platinum-based chemotherapy. Our results show that STAT3 is necessary for ovarian tumor progression/metastasis and highlight the potential for targeting STAT3 by HO-3867 as a therapeutic strategy for ovarian cancer.

Anticancer potential of diarylidenyl piperidone derivatives, HO-4200 and H-4318, in cisplatin resistant primary ovarian cancer.

We have previously developed a novel class of bi-functional compounds based on a diarylidenyl-piperidone (DAP) backbone conjugated to an N-hydroxypyrroline (-NOH; a nitroxide precursor) group capable of selectively inhibiting STAT3 activation, translocation, and DNA binding activity. HO-4200 and H-4318 are 2 such derivatives capable of inducing apoptosis in ovarian cancer cells through this mechanism and demonstrated efficacy in platinum resistant primary ovarian cancer cell populations and tumor tissues. The improved absorption and cellular uptake of HO-4200 by cancer cells was determined using optical and electron paramagnetic resonance spectrometry. Treatment of ovarian cancer cells with HO-4200 and H-4318 resulted in cleavage of caspase proteins 3, 7, and 9, as well as PARP and inhibition of the pro-survival protein, Bcl-xL, resulting in significantly decreased cell survival and increased apoptosis. HO-4200 and H-4318 significantly inhibit fatty acid synthase (FAS) and pSTAT3 and decreased the expression of STAT3 target proteins: Survivin, c-myc, Bcl-xl, Bcl-2, cyclin D1/D2, and VEGF were suppressed as analyzed using quantitative real time PCR. In addition, HO-4200 and H-4318 significantly inhibited migration/invasion, in primary ovarian cancer cell populations isolated from primary and recurrent ovarian cancer patients. Treatment of freshly collected human ovarian tumor sections with HO-4200 demonstrated significant suppression of pSTAT3 Tyr 705, angiogenesis (VEFG), and markers of proliferation (Ki-67) in ex vivo models. We have shown, for the first time, that the DAP compounds, HO-4200 and H-4318, inhibit cell migration/invasion and induce apoptosis by targeting FAS/STAT3 in human ovarian cancer cells, including primary ovarian cancer cell populations and tumor tissues. Therefore, our results highlight the clinical anti-cancer potential of HO-4200 and H-4318.

Doxorubicin synergizes with 34.5ENVE to enhance antitumor efficacy against metastatic ovarian cancer.

PURPOSE: Novel therapeutic regimens are needed to improve dismal outcomes associated with late-stage ovarian cancer. Oncolytic viruses are currently being tested in patients with ovarian cancer. Here, we tested the therapeutic efficacy of combining doxorubicin with 34.5ENVE, an oncolytic herpes simplex virus transcriptionally driven by a modified stem cell-specific nestin promoter, and encoding for antiangiogenic Vasculostatin-120 (VStat120) for use against progressive ovarian cancer. EXPERIMENTAL DESIGN: Antitumor efficacy of 34.5ENVE was assessed in ovarian cancer cell lines, mouse ascites-derived tumor cells, and primary patient ascites-derived tumor cells by standard MTT assay. The ability of conditioned medium derived from 34.5ENVE-infected ovarian cancer cells to inhibit endothelial cell migration was measured by a Transwell chamber assay. Scope of cytotoxic interactions between 34.5ENVE and doxorubicin were evaluated using Chou-Talalay synergy analysis. Viral replication, herpes simplex virus receptor expression, and apoptosis were evaluated. Efficacy of oncolytic viral therapy in combination with doxorubicin was evaluated in vivo in the murine xenograft model of human ovarian cancer. RESULTS: Treatment with 34.5ENVE reduced cell viability of ovarian cancer cell lines, and mouse ascites-derived and patient ascites-derived ovarian tumor cells. Conditioned media from tumor cells infected with 34.5ENVE reduced endothelial cell migration. When combined with doxorubicin, 34.5ENVE killed synergistically with a significant increase in caspase-3/7 activation, and an increase in sub-G1 population of cells. The combination of doxorubicin and 34.5ENVE significantly prolonged survival in nude mice bearing intraperitoneal ovarian cancer tumors. CONCLUSIONS: This study indicates significant antitumor efficacy of 34.5ENVE alone, and in combination with doxorubicin against disseminated peritoneal ovarian cancer.

Aberrantly activated pSTAT3-Ser727 in human endometrial cancer is suppressed by HO-3867, a novel STAT3 inhibitor.

OBJECTIVE: Constitutive activation of STAT3 is a hallmark of various human cancers, however an increased pSTAT3 expression in high grade human endometrial cancer has not been reported. In the present study, we examine the expression of STAT family of proteins in endometrial cancer cell lines and the efficacy of HO-3867, a novel STAT3 inhibitor designed in our lab. METHODS: Expression of STAT family proteins was evaluated via Western blot. The cell viability, post-treatment with HO-3867, was assessed using MTT, cell-cycle profile and Annexin assay. In vivo efficacy of HO-3867 was evaluated using xenograft mice. RESULTS: Expression of activated STATs was inconsistent among the cell lines and 18 human endometrial cancer specimens tested. While pSTAT3 Tyr705 was not expressed in any of the cell lines, pSTAT3 Ser727 was highly expressed in endometrial cancer cell lines and tumor specimens. HO-3867 decreased the expression of pSTAT3 Ser727 while total STAT3 remained constant; cell viability decreased by 50-80% and induced G2/M arrest in 55% of Ishikawa cells at the G2/M cell cycle checkpoint. There was an increase in p53, a decrease in Bcl2 and Bcl-xL, and cleavage of caspase-3, caspase-7 and PARP. HO-3867 mediated a dosage-dependent inhibition of the growth of xenografted endometrial tumors. CONCLUSIONS: HO-3867 treatment decreases the high levels of pSTAT3 Ser727 in endometrial cancer cells by inducing cell cycle arrest and apoptosis. This suggests a specific role of serine-phosphorylated STAT3, independent of tyrosine phosphorylation in the oncogenesis of endometrial cancer. HO-3867 could potentially serve as an adjunctive targeted therapy.

Osmotin-expressing transgenic tea plants have improved stress tolerance and are of higher quality.

Drought is a major stress that affects the yield and quality of tea, a widely consumed beverage crop grown in more than 20 countries of the world. Therefore, osmotin gene-expressing transgenic tea plants produced using earlier optimized conditions were evaluated for their tolerance of drought stress and their quality. Improved tolerance of polyethylene glycol-induced water stress and faster recovery from stress were evident in transgenic lines compared with the normal phenotype. Significant improvements in growth under in-vitro conditions were also observed. Besides enhanced reactive oxygen species-scavenging enzyme activity, the transgenic lines contained significantly higher levels of flavan-3-ols and caffeine, key compounds that govern quality and commercial yield of the beverage. The selected transgenic lines have the potential to meet the demands of the tea industry for stress-tolerant plants with higher yield and quality. These traits of the transgenic lines can be effectively maintained for generations because tea is commercially cultivated through vegetative propagation only.

Type of Valvular Heart Disease Requiring Surgery in the 21st Century: Mortality and Length-of-Stay Related to Surgery.

AIM: While the incidence of rheumatic heart disease has declined dramatically over the last half-century, the number of valve surgeries has not changed. This study was undertaken to define the most common type of valvular heart disease requiring surgery today, and determine in-hospital surgical mortality and length-of-stay (LOS) for isolated aortic or mitral valve surgery in a United States tertiary-care hospital. METHODS: Patients with valve surgery between January 2002 to June 2008 at The Ohio State University Medical Center were studied. Patients only with isolated aortic or mitral valve surgery were analyzed. RESULTS: From 915 patients undergoing at least aortic or mitral valve surgery, the majority had concomitant cardiac proce-dures mostly coronary artery bypass grafting (CABG); only 340 patients had isolated aortic (n=204) or mitral (n=136) valve surgery. In-hospital surgical mortality for mitral regurgitation (n=119), aortic stenosis (n=151), aortic insufficiency (n=53) and mitral stenosis (n=17) was 2.5% (replacement 3.4%; repair 1.6%), 3.9%, 5.6% and 5.8%, respectively (p=NS). Median LOS for aortic insufficiency, aortic stenosis, mitral regurgitation, and mitral stenosis was 7, 8, 9 (replacement 11.5; repair 7) and 11 days, respectively (p<0.05 for group). In-hospital surgical mortality for single valve surgery plus CABG was 10.2% (p<0.005 compared to single valve surgery). CONCLUSIONS: Aortic stenosis and mitral regurgitation are the most common valvular lesions requiring surgery today. Surgery for isolated aortic or mitral valve disease has low in-hospital mortality with modest LOS. Concomitant CABG with valve surgery increases mortality substantially. Hospital analysis is needed to monitor quality and stimulate improvement among Institutions.

Preconditioning mesenchymal stem cells with caspase inhibition and hyperoxia prior to hypoxia exposure increases cell proliferation.

Myocardial infarction is a leading cause of mortality and morbidity worldwide. Occlusion of a coronary artery produces ischemia and myocardial necrosis that leads to left ventricular (LV) remodeling, dysfunction, and heart failure. Stem cell therapy may decrease infarct size and improve LV function; the hypoxic environment, however, following a myocardial infarction may result in apoptosis, which in turn decreases survival of transplanted stem cells. Therefore, the effects of preconditioned mesenchymal stem cells (MSC) with hyperoxia (100% oxygen), Z-VAD-FMK pan-caspase inhibitor (CI), or both in a hypoxic environment in order to mimic conditions seen in cardiac tissue post-myocardial infarction were studied in vitro. MSCs preconditioned with hyperoxia or CI significantly decreased apoptosis as suggested by TUNEL assay and Annexin V analysis using fluorescence assisted cell sorting. These effects were more profound when both, hyperoxia and CI, were used. Additionally, gene and protein expression of caspases 1, 3, 6, 7, and 9 were down-regulated significantly in MSCs preconditioned with hyperoxia, CI, or both, while the survival markers Akt1, NF-κB, and Bcl-2 were significantly increased in preconditioned MSCs. These changes ultimately resulted in a significant increase in MSC proliferation in hypoxic environment as determined by BrdU assays compared to MSCs without preconditioning. These effects may prove to be of great clinical significance when transplanting stem cells into the hypoxic myocardium of post-myocardial infarction patients in order to attenuate LV remodeling and improve LV function.

Comparison of Impella and intra-aortic balloon pump in high-risk percutaneous coronary intervention: vascular complications and incidence of bleeding.

OBJECTIVE: Compare vascular complications and incidence of bleeding of Impella 2.5 and intra-aortic balloon pump (IABP) in high-risk percutaneous coronary interventions (PCI). BACKGROUND: Large arterial sheath size for device insertion is associated with vascular and/or bleeding complications; gastrointestinal bleeding may also occur with anti-coagulation use. METHODS: Patients with an acute coronary syndrome receiving Impella 2.5 or IABP during high-risk PCI were studied (13 Impella; 62 IABP). Vascular complications and incidence of bleeding were compared. RESULTS: Post-procedure hematocrit was similar between groups. Blood transfusion occurred in 38.4% and 32.2% of patients in the Impella and IABP groups, respectively (P = NS); 65.3%, 30.7% and 3.8% of bleeding were due to vascular access site/procedure related, gastrointestinal and genitourinary, respectively. There was no statistical significant difference in vascular complications between the Impella and IABP groups (15.3% and 6.4% of patients, respectively); mesenteric ischemia (n = 1) and aortic rupture (n = 1) were only in the IABP group. In-hospital and one-year mortality were not statistically significant between groups. CONCLUSION: Impella can be used as safely as IABP during high-risk PCI with similar vascular and bleeding complications. Importantly, approximately one third of bleeding was from the gastrointestinal system warranting careful prophylactic measures and monitoring.

Cloning and characterization of the TneDI restriction: modification system of Thermotoga neapolitana.

A putative Type II restriction-modification system of Thermotoga neapolitana, TneDI, was cloned into Escherichia coli XL1-Blue MRF' and characterized. Gene CTN_0339 specifies the endonuclease R.TneDI, while CTN_0340 encodes the cognate DNA methyltransferase M.TneDI. Both enzymes were purified simply by heating the cell lysates of E. coli followed by centrifugation. The enzymes were active over a broad range of temperatures, from 42°C to at least 77°C, with the highest activities observed at 77°C. R.TneDI cleaved at the center of the recognition sequence (CG↓CG) and generated blunt-end cuts. Overexpression of R.TneDI in BL21(DE3) was confirmed by both SDS-PAGE and Western blotting.

Chemical modification of L-glutamine to alpha-amino glutarimide on autoclaving facilitates Agrobacterium infection of host and non-host plants: A new use of a known compound.

BACKGROUND: Accidental autoclaving of L-glutamine was found to facilitate the Agrobacterium infection of a non host plant like tea in an earlier study. In the present communication, we elucidate the structural changes in L-glutamine due to autoclaving and also confirm the role of heat transformed L-glutamine in Agrobacterium mediated genetic transformation of host/non host plants. RESULTS: When autoclaved at 121°C and 15 psi for 20 or 40 min, L-glutamine was structurally modified into 5-oxo proline and 3-amino glutarimide (α-amino glutarimide), respectively. Of the two autoclaved products, only α-amino glutarimide facilitated Agrobacterium infection of a number of resistant to susceptible plants. However, the compound did not have any vir gene inducing property. CONCLUSIONS: We report a one pot autoclave process for the synthesis of 5-oxo proline and α-amino glutarimide from L-glutamine. Xenobiotic detoxifying property of α-amino glutarimide is also proposed.