Claudius Conrad

Regeneration and orthotopic transplantation of a bioartificial lung

About 2,000 patients now await a donor lung in the United States. Worldwide, 50 million individuals are living with end-stage lung disease. Creation of a bioartificial lung requires engineering of viable lung architecture enabling ventilation, perfusion and gas exchange. We decellularized lungs by detergent perfusion and yielded scaffolds with acellular vasculature, airways and alveoli. To regenerate gas exchange tissue, we seeded scaffolds with epithelial and endothelial cells. To establish function, we perfused and ventilated cell-seeded constructs in a bioreactor simulating the physiologic environment of developing lung. By day 5, constructs could be perfused with blood and ventilated using physiologic pressures, and they generated gas exchange comparable to that of isolated native lungs. To show in vivo function, we transplanted regenerated lungs into orthotopic position. After transplantation, constructs were perfused by the recipients circulation and ventilated by means of the recipients airway and respiratory muscles, and they provided gas exchange in vivo for up to 6 h after extubation.

Mst1 and Mst2 Maintain Hepatocyte Quiescence and Suppress Hepatocellular Carcinoma Development through Inactivation of the Yap1 Oncogene

Hippo-Lats-Yorkie signaling regulates tissue overgrowth and tumorigenesis in Drosophila. We show that the Mst1 and Mst2 protein kinases, the mammalian Hippo orthologs, are cleaved and constitutively activated in the mouse liver. Combined Mst1/2 deficiency in the liver results in loss of inhibitory Ser127 phosphorylation of the Yorkie ortholog, Yap1, massive overgrowth, and hepatocellular carcinoma (HCC). Reexpression of Mst1 in HCC-derived cell lines promotes Yap1 Ser127 phosphorylation and inactivation and abrogates their tumorigenicity. Notably, Mst1/2 inactivates Yap1 in liver through an intermediary kinase distinct from Lats1/2. Approximately 30% of human HCCs show low Yap1(Ser127) phosphorylation and a majority exhibit loss of cleaved, activated Mst1. Mst1/2 inhibition of Yap1 is an important pathway for tumor suppression in liver relevant to human HCC.

STAT3 Plays a Critical Role in KRAS-Induced Pancreatic Tumorigenesis

The STAT3 transcription factor is an important regulator of stem cell self-renewal, cancer cell survival, and inflammation. In the pancreas, STAT3 is dispensable for normal development, whereas the majority of pancreatic ductal adenocarcinomas (PDAC) show constitutive activation of STAT3, suggesting its potential as a therapeutic target in this cancer. Here, we sought to define the mechanisms of STAT3 activation and its functional importance in PDAC pathogenesis. Large-scale screening of cancer cell lines with a JAK2 inhibitor that blocks STAT3 function revealed a more than 30-fold range in sensitivity in PDAC, and showed a close correlation of sensitivity with levels of tyrosine-phosphorylated STAT3 and of the gp130 receptor, an upstream signaling component. Correspondingly, upregulation of the IL6/LIF-gp130 pathway accounted for the strong STAT3 activation in PDAC subsets. To define functions of STAT3 in vivo, we developed mouse models that test the impact of conditional inactivation of STAT3 in KRAS-driven PDAC. We showed that STAT3 is required for the development of the earliest premalignant pancreatic lesions, acinar-to-ductal metaplasia (ADM) and pancreatic intraepithelial neoplasia (PanIN). Moreover, acute STAT3 inactivation blocked PDAC initiation in a second in vivo model. Our results show that STAT3 has critical roles throughout the course of PDAC pathogenesis, supporting the development of therapeutic approaches targeting this pathway. Moreover, our work suggests that gp130 and phospho-STAT3 expression may be effective biomarkers for predicting response to JAK2 inhibitors.

Hepatic Portal Venous Gas The ABCs of Management

OBJECTIVE To review the use of computed tomography (CT) and radiography in managing hepatic portal venous gas (HPVG) at a university-affiliated tertiary care center and in the literature. Hepatic portal venous gas is frequently associated with acute mesenteric ischemia, accounting for most of the HPVG-associated mortality. While early studies were necessarily dependent on plain abdominal radiography, modern high-resolution CT has revealed a host of benign conditions in which HPVG has been reported that do not require emergent surgery. DATA SOURCES Patient records from our institution over the last 10 years and relevant studies from BioMed Central, CENTRAL, PubMed, and PubMed Central. In addition, references cited in selected works were also used as source data. STUDY SELECTION Patient records were selected if the CT or radiograph findings matched the term hepatic portal venous gas. Studies were selected based on the search terms hepatic portal venous gas or portal venous gas. DATA EXTRACTION Quantitative and qualitative data were quoted directly from cited work. DATA SYNTHESIS Early studies of HPVG were based on plain abdominal radiography and a literature survey in 1978 found an associated mortality rate of 75%, primarily due to ischemic bowel disease. Modern abdominal CT has resulted in the detection of HPVG in more benign conditions, and a second literature survey in 2001 found a total mortality of only 39%. While the pathophysiology of HPVG is, as yet, unclear, changing abdominal imaging technology has altered the significance of this radiologic finding. Hepatic portal venous gas therefore predicts high risk of mortality (>50%) if detected by plain radiography or by CT in a patient with additional evidence of necrotic bowel. If detected by CT in patients after surgical or endoscopic manipulation, the clinician is advised that there is no evidence of increased risk. If HPVG is detected by CT in patients with active peptic ulcer disease, intestinal obstruction and/or dilatation, or mucosal diseases such as Crohn disease or ulcerative colitis, caution is warranted, as risk of death may approach 20% to 30%. CONCLUSION The finding of HPVG alone cannot be an indication for emergency exploration, and we have developed an evidence-based algorithm to guide the clinician in management of patients with HPVG.

Targeting tumor stroma using engineered mesenchymal stem cells reduces the growth of pancreatic carcinoma

Objective:To analyze the efficacy of engineered mesenchymal stem cell based therapy directed towards pancreatic tumor stroma. Summary Background Data:Mesenchymal stem cells (MSC) are actively recruited to tumor stroma where they enhance tumor growth and metastases. Upregulation of chemotactic cytokine (CCL5) by MSCs within the tumor stroma has been shown to play a central role in this process. Murine MSCs were engineered to express reporter genes or therapeutic genes under control of the CCL5 promoter and adoptively transferred into mice with growing pancreatic tumors. The effect on tumor growth and metastases was then evaluated. Methods:MSCs isolated from bone marrow of C57/Bl6 p53−/− mice were stably transfected with red fluorescent protein (RFP), enhanced green fluorescent protein (eGFP), or herpes simplex virus (HSV) thymidine kinase (Tk) gene driven by the RANTES promoter. MSCs were intravenously applied once per week over 3 weeks to mice carrying an orthotopic, syngeneic pancreatic Panc02 tumor. Results:eGFP and RFP signals driven by the CCL5 promoter were detected by fluorescence in treated pancreatic tumor samples. The HSV-Tk therapy group treated intraperitoneal with the prodrug ganciclovir 5 to 7 days after stem cell application lead to a 50% reduction of primary pancreatic tumor growth (P < 0.0003, student t test) and reduced liver metastases (0% vs. 60%). Conclusion:The active homing of MSCs into primary pancreatic tumor stroma and activation of the CCL5 promoter was verified using eGFP- and RFP-reporter genes. In the presence of ganciclovir, HSV-Tk transfected MSCs led to a significant reduction of primary pancreatic tumor growth and incidence of metastases.

Multipotent Mesenchymal Stem Cells Acquire a Lymphendothelial Phenotype and Enhance Lymphatic Regeneration In Vivo

Background— The importance and therapeutic value of stem cells in lymphangiogenesis are poorly understood. We evaluated the potential of human and murine mesenchymal stem cells (MSCs) to acquire a lymphatic phenotype in vitro and to enhance lymphatic regeneration in vivo. Methods and Results— We assessed the lymphendothelial differentiation of human and murine MSCs after induction with supernatant derived from human dermal microvascular endothelial cells, isolated lymphatic endothelial cells, and purified vascular endothelial growth factor (VEGF)-C in vitro. We used human or murine progenitor MSC lines and then characterized the lymphatic phenotype by morphology, migratory capacity, and the expression of lymphatic markers such as Prox-1, podoplanin, Lyve-1, VEGF receptor-2, and VEGF receptor-3. Using a murine lymphatic edema model, we assessed the potential of these cells to form a functional lymphatic vasculature in vivo after injection of syngeneic MSCs. Incubation with supernatant from lymphatic endothelial cells induced an endothelium-like morphology and the expression of lymphendothelial markers in both human and murine MSCs in vitro. MSCs showed migratory activity along a VEGF-C gradient, which was enhanced by VEGF-C conditioning. In vivo, the local application of MSCs resulted in a significant decrease in edema formation (−20.1%; P<0.01 versus untreated tails) after 3 weekly cell injections and restored the drainage of intradermally injected methylene blue after 7 weekly injections. Conclusions— MSCs were capable of expressing a lymphatic phenotype when exposed to lymph-inductive media and purified VEGF-C. Migratory activity toward VEGF-C in vitro suggests homing capability in vivo. Restoration of lymphatic drainage after injection of MSCs in a lymphedema model indicates that MSCs play a role in lymphatic regeneration. The potential clinical application of MSC in wound healing and reduction of lymphatic edema warrants further research.

Selective targeting of genetically engineered mesenchymal stem cells to tumor stroma microenvironments using tissue-specific suicide gene expression suppresses growth of hepatocellular carcinoma

Background:The use of engineered mesenchymal stem cells (MSCs) as therapeutic vehicles for the treatment of experimental pancreatic and breast cancer has been previously demonstrated. The potential application of MSCs for the treatment of hepatocellular carcinoma (HCC) has been controversial. The general approach uses engineered MSCs to target different aspects of tumor biology, including angiogenesis or the fibroblast-like stromal compartment, through the use of tissue-specific expression of therapeutic transgenes. The aim of the present study was (1) to evaluate the effect of exogenously added MSCs on the growth of HCC and (2) the establishment of an MSC-based suicide gene therapy for experimental HCC. Methods:Mesenchymal stem cells were isolated from bone marrow of C57/Bl6 p53−/− mice. The cells were injected into mice with HCC xenografts and the effect on tumor proliferation and angiogenesis was evaluated. The cells were then stably transfected with red fluorescent protein (RFP) or Herpes simplex virus thymidine kinase (HSV-Tk) gene under control of the Tie2 promoter/enhancer or the CCL5 promoter. Mesenchymal stem cells were injected intravenously into mice with orthotopically growing xenografts of HCC and treated with ganciclovir (GCV). Results:Ex vivo examination of hepatic tumors revealed tumor-specific recruitment, enhanced tumor growth, and increased microvessel density after nontherapeutic MSC injections. After their homing to the hepatic xenografts, engineered MSCs demonstrated activation of the Tie2 or CCL5 promoter as shown by RFP expression. Application of CCL5/HSV-TK transfected MSCs in combination with GCV significantly reduced tumor growth by 56.4% as compared with the control group and by 71.6% as compared with nontherapeutic MSC injections. CCL5/HSV-TK+ transfected MSCs proved more potent in tumor inhibition as compared with Tie2/HSV-TK+ MSCs. Conclusion:Exogenously added MSCs are recruited to growing HCC xenografts with concomitant activation of the CCL5 or Tie2 promoters within the MSCs. Stem cell–mediated introduction of suicide genes into the tumor followed by prodrug administration was effective for treatment of experimental HCC and thus may help fill the existing gap in bridging therapies for patients suffering from advanced HCCs.

Overture for growth hormone: Requiem for interleukin-6?

Background: Music has been used for therapeutic purposes since the beginning of cultural history. However, despite numerous descriptions of beneficial effects, the precise mechanisms by which music may improve human well‐being remain unclear. Methods: We conducted a randomized study in ten critically ill patients to identify mechanisms of music‐induced relaxation using a special selection of slow movements of Mozarts piano sonatas. These sonatas were analyzed for compositional elements of relaxation. We measured circulatory variables, brain electrical activity, serum levels of stress hormones and cytokines, requirements for sedative drugs, and level of sedation before and at the end of a 1‐hr therapeutic session. Results: Compared with controls, we found that music application significantly reduced the amount of sedative drugs needed to achieve a comparable degree of sedation. Simultaneously, among those receiving the music intervention, plasma concentrations of growth hormone increased, whereas those of interleukin‐6 and epinephrine decreased. The reduction in systemic stress hormone levels was associated with a significantly lower blood pressure and heart rate. Conclusion: Based on the effects of slow movements of Mozarts piano sonatas, we propose a neurohumoral pathway by which music might exert its sedative action. This model includes an interaction of the hypothalamic–pituitary axis with the adrenal medulla via mediators of the unspecific immune system.

Meta‐analysis of KRAS mutations and survival after resection of colorectal liver metastases

In patients with advanced colorectal cancer, KRAS mutation status predicts response to treatment with monoclonal antibody targeting the epithelial growth factor receptor (EGFR). Recent reports have provided evidence that KRAS mutation status has prognostic value in patients with resectable colorectal liver metastases (CLM) irrespective of treatment with chemotherapy or anti‐EGFR therapy. A meta‐analysis was undertaken to clarify the impact of KRAS mutation on outcomes in patients with resectable CLM.

Return to intended oncologic treatment (RIOT): A novel metric for evaluating the quality of oncosurgical therapy for malignancy

After cancer surgery, complications, and disability prevent some patients from receiving subsequent treatments. Given that an inability to complete all intended cancer therapies might negate the oncologic benefits of surgical therapy, strategies to improve return to intended oncologic treatment (RIOT), including minimally invasive surgery (MIS), are being investigated.