Disruption of hepatocyte growth factor/c-Met signaling enhances pancreatic beta-cell death and accelerates the onset of diabetes

OBJECTIVE

To determine the role of hepatocyte growth factor (HGF)/c-Met on β-cell survival in diabetogenic conditions in vivo and in response to cytokines in vitro.

RESEARCH DESIGN AND METHODS

We generated pancreas-specific c-Met-null (PancMet KO) mice and characterized their response to diabetes induced by multiple low-dose streptozotocin (MLDS) administration. We also analyzed the effect of HGF/c-Met signaling in vitro on cytokine-induced β-cell death in mouse and human islets, specifically examining the role of nuclear factor (NF)-κB.

RESULTS

Islets exposed in vitro to cytokines or from MLDS-treated mice displayed significantly increased HGF and c-Met levels, suggesting a potential role for HGF/c-Met in β-cell survival against diabetogenic agents. Adult PancMet KO mice displayed normal glucose and β-cell homeostasis, indicating that pancreatic c-Met loss is not detrimental for β-cell growth and function under basal conditions. However, PancMet KO mice were more susceptible to MLDS-induced diabetes. They displayed higher blood glucose levels, marked hypoinsulinemia, and reduced β-cell mass compared with wild-type littermates. PancMet KO mice showed enhanced intraislet infiltration, islet nitric oxide (NO) and chemokine production, and β-cell apoptosis. c-Met-null β-cells were more sensitive to cytokine-induced cell death in vitro, an effect mediated by NF-κB activation and NO production. Conversely, HGF treatment decreased p65/NF-κB activation and fully protected mouse and, more important, human β-cells against cytokines.

CONCLUSIONS

These results show that HGF/c-Met is critical for β-cell survival by attenuating NF-κB signaling and suggest that activation of the HGF/c-Met signaling pathway represents a novel strategy for enhancing β-cell protection.Type I diabetes is an autoimmune disease that results from cellular cytotoxicity leading to selective and progressive destruction of insulin-secreting cells (1–3). Many growth factors known to control cell growth and survival in physiologic and pathologic conditions are expressed in the pancreas and could potentially participate in an autocrine/paracrine fashion in the final fate of β-cells in an autoimmune environment. Overexpression of IGF-1, transforming growth factor-β, or granulocyte macrophage-colony stimulating factor ameliorates islet infiltration and β-cell death in mouse models of increased islet inflammation and diabetes (4–6). However, the role of endogenous pancreatic growth factors in type I diabetes has not been examined. Because growth factors can locally affect β-cell survival, neogenesis, and regeneration, and modulate chemokine production and immune responses, alterations in the level/activation of growth factor signaling pathways might contribute to the delay/acceleration of the onset of diabetes.Hepatocyte growth factor (HGF)/c-Met signaling pathway participates in the control of multiple biological functions, including development, proliferation, survival, regeneration, and branching morphogenesis (7). HGF binds with high affinity to, and induces the dimerization of, c-Met, its transmembrane tyrosine kinase receptor (8). Deletion of exon 16 of the c-Met gene, which encodes Lys¹¹⁰⁸ (ATP binding site), essential for the kinase activity of this receptor, in knockout mice results in embryonic lethality (9). These mice display a phenotype identical to HGF knockout mice (10). Both HGF and c-Met are expressed in the pancreas; HGF localizes to endothelial, islet, and mesenchymal cells, and c-Met is expressed in islet, ductal, and pancreatic progenitor cells (11–14). Conditional ablation of the c-Met gene in mouse β-cells using RIP-Cre and lox-c-Met mice leads to deficient insulin secretion without alteration of β-cell mass (12,13). On the other hand, HGF overexpression in the β-cell of transgenic mice increases β-cell replication, mass, and function (15,16). Furthermore, HGF improves islet graft survival in animal models of diabetes (17–19). HGF positively influences autoimmune responses, reducing the severity of autoimmune myocarditis and arthritis (20,21). HGF also downregulates airway and kidney inflammation, and inflammatory bowel disease (22–24). Whether HGF plays a role in autoimmune diabetes is unknown.To address the function of c-Met in the development, growth, and maintenance of β-cells under physiologic conditions, as well as its role in β-cell survival and response to injury in vivo, we generated pancreas-specific c-Met-null (PancMet KO) mice. We report that although c-Met is dispensable for normal β-cell growth and function under basal conditions, it is critically important for β-cell survival in diabetogenic conditions. β-Cell survival is dramatically worsened in the absence of HGF/c-Met signaling, resulting in accelerated diabetes onset. These observations also apply to human β-cells, underscoring a therapeutic opportunity for the HGF/c-Met signaling pathway in human diabetes.     

Effect of various trophic factors on bacterial translocation in experimental short bowel syndrome

Massive bowel resection triggers an adaptive process in the remaining intestine in spite of which, bacterial translocation (BT) is frequent under these conditions. Several trophic factors, including growth hormone (GH), epidermal growth factor (EGF) and insuline (INS) are involved in the process of adaptation in short bowel syndrome (SBS). However, the effect of GH, EGF or INS on BT has not been investigated experimentally. The aim of the study was to test the hypothesis that GH, EGF or INS administration prevents BT in rats with SBS receiving only parenteral nutrition (PN). Thirty-seven adult Wistar rats underwent central venous cannulation and were randomly assigned to one of two groups receiving for ten days four treatment regimes: PN group (N = 10) fasting, all-in-one PN solution (300 mL/kg/24 h, 280 kcal/kg/24 h), 80% gut resection including ileo-cecal valve. GH group (N = 9) fasting, same PN regime and resection plus GH (1 mg/kg/d, s.c.). EGF group (N = 9): same PN regime and resection plus EGF (150 microgr/24 h, e.v.) INS group(N = 9): same PN regime and resection plus INS (1 U.I./100 g/24 h s.c.) At the end of the experiment the rats were exanguinated and mesenteric lymph nodes and samples of systemic and portal blood were obtained and cultured. Several samples of full-thickness jejunal wall were taken for measuring cell proliferation index (PCNA) and mucosal thickness. Jejunal mucosal thickness increased by 30%, 28% and 29% and PCNA index by 21%, 20% and 25% in GH, EGF and INS, treated rats respectively in comparison with those treated with PN alone. However, contrary to our expectations, BT expressed by positive culture of intestinal germs in systemic blood was demonstrated respectively in 44%, 40% and 28% of GH, EGF and INS animals, respectively, and in 0% of PN-only rats. Although exogenous GH, EGF or INS improves gut mucosal structure in rats with SBS treated with PN, it seems to increase rather than decrease mucosal permeability to intestinal germs in them.     

NMP 22, BTA stat test and cytology in the diagnosis of bladder cancer: a comparative study

OBJECTIVE: To evaluate the usefulness of the NMP 22 and BTA stat test in the diagnosis and follow-up of bladder cancer and to compare these tests to cytology and cystoscopy, routine diagnostic methods. METHODS: 150 patients followed up for bladder cancer or symptoms suggestive of bladder cancer underwent cystoscopy after cytology, NMP 22 and BTA stat test using a recently voided urine sample. In suspect cases, TUR and histopathological analysis were performed. RESULTS: Bladder cancer was proven in 76 patients and excluded in 74. For NMP 22 we have used the cutoff value recommended by the manufacturer (10 U/ml) and that obtained by our receiver-operating characteristic curve (6 U/ml). Sensitivity was 84.21% for NMP 22 at the cutoff value of 6 U/ml and 76.32% with 10 U/ml; 72.37% for BTA stat test; 69.74% for cytology, and 100% for cystoscopy. Specificity was 86.49% for NMP 22 at a cutoff value of 6 U/ml and 90.54% at 10 U/ml; 89.19% for the BTA stat test; 93.24% for cytology and 89.19% for cystoscopy. NMP 22 sensitivity for grades 1, 2, and 3 was 68.75, 75.86 and 100%, respectively, at a cutoff value of 6 U/ml, and 50, 68.97 and 96.77%, respectively, at a cutoff level of 10 U/ml; for BTA stat the sensitivity was 56.25% in G1, 62.07% in G2 and 90.32% in G3, and for cytology the sensitivity was 43.75, 62.07 and 90.32%, respectively. The sensitivity of NMP 22 was 68.75% in stage Ta, 84.78% in T1 and 100% in T2-T4 at a cutoff level of 6 U/ml and 50, 80.43 and 92.86%, respectively, at a cutoff level of 10 U/ml; BTA stat sensitivity was 50% in Ta, 73.91% in T1 and 92.86% in T2-T4; and in cytology the results were 37.50, 73.91 and 85.71%, respectively. Using the McNemar test, there was only a significant difference between the sensitivity of NMP 22 at a cutoff level of 6 U/ml and cytology in the overall sample. CONCLUSIONS: The high sensitivity of the NMP 22 and BTA stat test in combination with the data obtained from the parameters used for the evaluation of the test demonstrate their usefulness in the diagnosis and follow-up of bladder cancer. NMP 22 at a cutoff value of 6 U/ml is significantly more sensitive than cytology and consequently a thoroughly valid diagnostic tool in the diagnosis of bladder cancer which may substitute voided urine cytology.     

Myocardial and Coronary Effects of Propofol in Rabbits with Compensated Cardiac Hypertrophy

Background: Myocardial effects of propofol have been previously investigated but most studies have been performed in healthy hearts. This study compared the cardiac effects of propofol on isolated normal and hypertrophic rabbits hearts.

Methods: The effects of propofol (10-1,000 [mu]m) on myocardial contractility, relaxation, coronary flow and oxygen consumption were investigated in hearts from rabbits with pressure overload-induced left ventricular hypertrophy (LVH group, n = 20) after aortic abdominal banding and from sham-operated control rabbits (SHAM group, n = 10), using an isolated and erythrocyte-perfused heart model. In addition, to assess the myocardial and coronary effects of propofol in more severe LVH, hearts with a degree of hypertrophy greater than 140% were selected (severe LVH group, n = 7).

Results: The cardiac hypertrophy model induced significant left ventricular hypertrophy (136 +/- 21%, P < 0.05). The pressure-volume relation showed normal systolic function but an altered diastolic compliance in hypertrophic hearts. Propofol only decreased myocardial contractility and relaxation at supratherapeutic concentrations (>= 300 [mu]m) in SHAM and LVH groups. The decrease in myocardial performances was not significantly different in SHAM and LVH groups. Propofol induced a significant increase in coronary blood flow which was not significantly different between groups. In severe LVH group, the degree of hypertrophy reached to 157 +/- 23%. Similarly, the effects of concentrations of propofol were not significantly different from the SHAM group.     

Activation of Coagulation and Fibrinolysis during Coronary Surgery: On-pump versus Off-pump Techniques

Background : The authors studied the changes in selected hemostatic variables in patients undergoing coronary surgery with on-pump coronary artery bypass grafting (CABG) or off-pump coronary artery bypass surgery (OPCAB) techniques.

Methods : Platelet counts and plasma concentrations of antithrombin, fibrinogen, D dimer, [alpha]2 antiplasmin, and plasminogen were measured preoperatively, 5 min after administration of heparin, 10 min after arrival in the intensive care unit, and 24 h after surgery in patients scheduled to undergo OPCAB (n = 15) or CABG (n = 15). To correct for dilution, hemostatic variables and platelet counts were adjusted for the changes in immunoglobulin G plasma concentrations and hematocrit, respectively.

Results : Adjusting for dilution, antithrombin and fibrinogen concentrations decreased to a similar extent in patients undergoing OPCAB or CABG (pooled means and 95% confidence limits of the mean: 95.5% of baseline, 93-98%, P = 0.002, and 91.7% of baseline, 88-95%, P = 0.0001), respectively, whereas [alpha]2-antiplasmin concentrations were unchanged. Only CABG was associated with a reduction in platelet counts (76% of baseline, 66-85%, P = 0.0001), plasminogen concentrations (96% of baseline, 91-99%, P = 0.011), and increased D-dimer formation (476%, 309-741%, P = 0.004). Twenty-four hours after surgery, platelet counts were still lower in patients undergoing CABG (P = 0.049), but all the investigated variables adjusted for dilution were similar in the two groups.     

Mathematical Modeling of Carbon Monoxide Exposures from Anesthetic Breakdown: Effect of Subject Size, Hematocrit, Fraction of Inspired Oxygen, and Quantity of Carbon Monoxide

Background: Carbon monoxide (CO) is produced by reaction of isoflurane, enflurane, and desflurane in desiccated carbon dioxide absorbents. The inspiratory CO concentration depends on the dryness and identity of the absorbent and anesthetic. The adaptation of existing mathematical models to a rebreathing circuit allows identification of patient factors that predispose to more severe exposures, as identified by carboxyhemoglobin concentration.

Methods: From our companion study, the authors used quantitative in vitro CO production data for 60 min at 7.5% desflurane or 1.5% isoflurane at 1 l/min fresh gas flow. The carboxyhemoglobin concentration was calculated by iteratively solving the Coburn Forster Kane equation modified for a rebreathing system that incorporates the removal of CO by patient absorption. Demonstrating good fit of predicted carboxyhemoglobin concentrations to published data from animal and human exposures validated the model. Carboxyhemoglobin concentrations were predicted for exposures of various severity, patients of different sizes, hematocrit, and fraction of inspired oxygen.

Results: The calculated carboxyhemoglobin concentrations closely predicted the experimental results of other investigators, thereby validating the model. These equations indicate the severity of CO poisoning is inversely related to the hemoglobin quantity of a subject. Fraction of inspired oxygen had the greatest effect in patients of small size with low hematocrit values, where equilibrium and not the rate of uptake determined carboxyhemoglobin concentrations.     

Complex nature of the human antisperm antibody response in SCID mice

Human peripheral blood mononuclear (PBMs) cells were introduced into the peritoneal cavity of severely‐combined immunodeficient (SCID) mice in concentrations of 2.5–4.0 × 10⁷ cells per mouse. Whole mononuclear cell suspensions were used either unstimulated or following primary in vitro culture with human spermatozoa. In some experiments, immunodepletion of CD8⁺ cells was carried out prior to grafting. Lymphocytes were obtained from nonsensitized (to antigen) human subjects or from individuals who were primed in vivo (vasectomized individuals in case of sperm antigens). An enzyme‐linked immunosorbent assay was employed to assess total human immunoglobulin (G or M) levels as well as the specificity of the antibodies generated. We have been successful by generating primary and secondary immune responses with ‘naïve’ human lymphocytes, challenged with chlamydia or ovalbumin but without adjuvant or CD8⁺ immunodepletion; however, we were unable to induce specific antibodies to spermatozoa under this regime in SCID male mice. We then employed female SCID mice, treated with sperm antigen extracts (glycosylated or deglycosylated) encapsulated in liposomes and human lymphocytes obtained from ‘naïve’ or pre‐sensitized in vivo subjects. It was found that the most pronounced humoral response to sperm antigens was obtained with deglycosylated antigens and PBMs from vasectomized (in vivo pre‐primed to spermatozoa) individuals. A presented SCID mice model can be helpful at understanding of antisperm antibody development and the molecular nature of generated antibodies to modified sperm antigenic entities.     

Open bicondylar Hoffa fracture associated with extensor mechanism injury

Two cases of open bicondylar Hoffa fracture of the knee associated with extensor mechanism injury are described in two active young patients with multiple fractures. The level of the fracture was determined by the proximal insertion of the posterior cruciate ligament and anterior cruciate ligament in the medial and lateral condyle. The level of the extensor mechanism injury was determined by the degree of flexion of the knee at the moment of impact. No ligament or meniscal tears were found. Open reduction and internal fixation with four lag screws and bone-to-tendon repair of the patellar and quadriceps tendon gave excellent results after more than 2 years of follow-up. The mechanism of injury and the therapeutic implications are discussed, and the literature is reviewed.     

Human plasma as a dermal scaffold for the generation of a completely autologous bioengineered skin

BACKGROUND: Keratinocyte cultures have been used for the treatment of severe burn patients. Here, we describe a new cultured bioengineered skin based on (1) keratinocytes and fibroblasts obtained from a single skin biopsy and (2) a dermal matrix based on human plasma. A high expansion capacity achieved by keratinocytes grown on this plasma-based matrix is reported. In addition, the results of successful preclinical and clinical tests are presented. METHODS: Keratinocytes and fibroblasts were obtained by a double enzymatic digestion (trypsin and collagenase, respectively). In this setting, human fibroblasts are embedded in a clotted plasma-based matrix that serves as a three-dimensional scaffold. Human keratinocytes are seeded on the plasma-based scaffold to form the epidermal component of the skin construct. Regeneration performance of the plasma-based bioengineered skin was tested on immunodeficient mice as a preclinical approach. Finally, this skin equivalent was grafted on two severely burned patients. RESULTS: Keratinocytes seeded on the plasma-based scaffold grew to confluence, allowing a 1,000-fold cultured-area expansion after 24 to 26 days of culture. Experimental transplantation of human keratinocytes expanded on the engineered plasma scaffold yielded optimum epidermal architecture and phenotype, including the expression of structural intracellular proteins and basement-membrane components. In addition, we report here the successful engraftment and stable skin regeneration in two severely burned patients at 1 and 2 years follow-up. CONCLUSIONS: Our data demonstrate that this new dermal equivalent allows for (1) generation of large bioengineered skin surfaces, (2) restoration of both the epidermal and dermal skin compartments, and (3) functional epidermal stem-cell preservation.