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Background Resuscitative endovascular balloon occlusion of the aorta (REBOA) is a novel method of controlling subdiaphragmatic hemorrhage while improving hemodynamic stability. This procedure achieves many of the goals of resuscitative thoracotomy (RT), but is less invasive. Here, we present the initial experience with REBOA at a level 1 academic trauma center. Study Design We performed a retrospective review. Orientation of surgeons and residents to REBOA was accomplished by an educational program including a hands-on simulation session (1.5 hours). Surgeons were not required to attend an external training course. Operating room personnel were oriented with a slide presentation. Initially, a 12-Fr introducer and aortic occlusion balloon were used. Subsequently, a 7-Fr device was used. All REBOAs were performed in a dedicated hybrid operating room. Resuscitative thoracotomy was performed in the trauma bays and operating room. Results During a 21-month period (June 2015 to March 2017), 16 patients (Injury Severity Score [ISS] 38.6 ± 22.3, Glasgow Coma Scale [GCS] 8.9 ± 5.9, lactate 4.91 ± 3.26 mmol/L) had REBOA placed. All patients were hemodynamically unstable (systolic blood pressure 96.5 ± 9.3 mmHg) due to hemorrhage. Preoperative hemoglobin ranged from 5 to 14.4 mg/dL. Etiology of hemorrhage was blunt trauma (n = 11), penetrating injury (n = 2), and nontraumatic mechanisms (n = 3). After REBOA, hemodynamic status improved in 10 of 16 patients. Fourteen patients survived the initial operative intervention and 6 survived 30 days; REBOA was successfully performed in all patients. One survivor developed a common femoral pseudoanuerysm. Survival for RT (ISS 31.3 ± 11.25) during same period was 0%. Conclusions Resuscitative endovascular balloon occlusion of the aorta is an effective method of improving hemodynamic status in patients with sub-diaphragmatic hemorrhage. Extensive training is not required to implement a REBOA program, and REBOA is a useful technique for trauma surgeons.

Surgical insult and trauma have been shown to cause dysregulation of the immune and inflammatory responses. Interaction of damage-associated molecular patterns (DAMPs) with toll-like receptors (TLRs) initiates innate immune response and systemic inflammatory responses. Given that surgical patients produce high levels of circulating damage-associated molecular patterns, we hypothesized that plasma-activated TLR activity would be correlated to injury status and could be used to predict pathological conditions involving tissue injury.An observational study was performed using samples from a single-institution prospective tissue and data repository from a Level-1 trauma center. In vitro TLR 2, 3, 4, and 9 activation was determined in a TLR reporter assay after isolation of plasma from peripheral blood. We determined correlations between plasma-activated TLR activity and clinical course measures of severity.Eighteen patients were enrolled (median Injury Severity Score 15 [interquartile range 10, 23.5]). Trauma resulted in significant elevation in circulation high mobility group box 1 as well as increase of plasma-activated TLR activation (2.8-5.4-fold) compared to healthy controls. There was no correlation between circulating high mobility group box 1 and trauma morbidity; however, the plasma-activated TLR activity was correlated with acute physiology and chronic health evaluation II scores (R square = 0.24-0.38, P 0.05). Patients who received blood products demonstrated significant increases in the levels of plasma-activated TLRs 2, 3, 4, and 9 and had a trend toward developing systemic inflammatory response syndrome.Further studies examining TLR modulation and signaling in surgical patients may assist in predictive risk modeling and reduction in morbidity and mortality.

Creatine (Cr), phosphocreatine (PCr), and creatine kinases (CK) comprise an energy shuttle linking ATP production in mitochondria with cellular consumption sites. Myocytes cannot synthesize Cr: these cells depend on uptake across the cell membrane by a specialized creatine transporter (CrT) to maintain intracellular Cr levels. Hypoxia interferes with energy metabolism, including the activity of the creatine energy shuttle, and therefore affects intracellular ATP and PCr levels. Here, we report that exposing cultured cardiomyocytes to low oxygen levels rapidly diminishes Cr transport by decreasing V max and K m . Pharmacological activation of AMP‐activated kinase (AMPK) abrogated the reduction in Cr transport caused by hypoxia. Cr supplementation increases ATP and PCr content in cardiomyocytes subjected to hypoxia, while also significantly augmenting the cellular adaptive response to hypoxia mediated by HIF‐1 activation. Our results indicate that: (1) hypoxia reduces Cr transport in cardiomyocytes in culture, (2) the cytoprotective effects of Cr supplementation are related to enhanced adaptive physiological responses to hypoxia mediated by HIF‐1, and (3) Cr supplementation increases the cellular ATP and PCr content in RNCMs exposed to hypoxia.

Heart failure is a common complication of doxorubicin (DOX) therapy. Previous studies have shown that DOX adversely impacts cardiac energy metabolism, and the ensuing energy deficiencies antedate clinical manifestations of cardiac toxicity. Brief exposure of cultured cardiomyocytes to DOX significantly decreases creatine transport, which is the cell's sole source of creatine. We present the results of a study performed to determine if physiological creatine supplementation (5 mmol/L) could protect cardiomyocytes in culture from cellular injury resulting from exposure to therapeutic levels of DOX. Creatine supplementation significantly decreased cytotoxicity, apoptosis, and reactive oxygen species production caused by DOX. The protective effect was specific to creatine and depended on its transport into the cell.

Despite the rising incidence of hepatocellular carcinoma (HCC), challenges and controversy persist in optimizing treatment. As recent randomized trials suggest that ablation can have oncologic equivalence compared with resection for early HCC, the relative morbidity of the 2 approaches is a central issue in treatment decisions. Although excellent contemporary perioperative outcomes have been reported by a few hepatobiliary units, it is not clear that they can be replicated in broader practice. Our objective was to help inform this treatment dilemma by defining perioperative outcomes in a broader set of patients as represented in NSQIP-participating institutions.Mortality and morbidity data were extracted from the 2005-2010 NSQIP Participant Use Data Files based on Current Procedural Terminology (hepatectomy and ablation) and ICD-9 (HCC). Perioperative outcomes were reviewed, and factors associated with morbidity and mortality were identified with multivariable logistic regression.Eight hundred and thirty-seven (52%) underwent minor hepatectomy, 444 (28%) underwent major hepatectomy, and 323 (20%) underwent surgical ablation. Mortality rates were 3.4% for minor hepatectomy, 3.7% for ablation, and 8.3% for major hepatectomy (p0.01). Major complication rates were 21.3% for minor hepatectomy, 9.3% for ablation, and 35.1% for major hepatectomy (p0.01). When controlling for confounders, ablation was associated with decreased mortality (adjusted odds ratio = 0.20; 95% CI, 0.04-0.97; p = 0.046) and major complications (adjusted odds ratio = 0.34; 95% CI, 0.22-0.52; p0.001).Exceedingly high complication rates after major hepatectomy for HCC exist in the broader NSQIP treatment environment. These data strongly support the use of parenchymal-sparing minor resections or ablation over major hepatectomy for early HCC when feasible.

Doxorubicin is commonly used to treat leukemia, lymphomas, and solid tumors, such as soft tissue sarcomas or breast cancer. A major side effect of doxorubicin therapy is dose-dependent cardiotoxicity. Doxorubicin's effects on cardiac energy metabolism are emerging as key elements mediating its toxicity. We evaluated the effect of doxorubicin on [14C]creatine uptake in rat neonatal cardiac myocytes and HL-1 murine cardiac cells expressing the human creatine transporter protein. A significant and irreversible decrease in creatine transport was detected after an incubation with 50–100 nmol/l doxorubicin. These concentrations are well below peak plasma levels (5 μmol/l) and within the ranges (25–250 nmol/l) for steady-state plasma concentrations reported after the administration of 15–90 mg/m2 doxorubicin for chemotherapy. The decrease in creatine transport was not solely because of increased cell death due to doxorubicin's cytotoxic effects. Kinetic analysis showed that doxorubicin decreased Vmax, Km, and creatine transporter protein content. Cell surface biotinylation experiments confirmed that the amount of creatine transporter protein present at the cell surface was reduced. Cardiomyocytes rely on uptake by a dedicated creatine transporter to meet their intracellular creatine needs. Our findings show that the cardiomyocellular transport capacity for creatine is substantially decreased by doxorubicin administration and suggest that this effect may be an important early event in the pathogenesis of doxorubicin-mediated cardiotoxicity.

Profound alterations in myocellular creatine and phosphocreatine levels are observed during human heart failure. To maintain its intracellular creatine stores, cardiomyocytes depend upon a cell membrane creatine transporter whose regulation is not clearly understood. Creatine transport capacity in the intact heart is modulated by substrate availability, and it is reduced in the failing myocardium, likely adding to the energy imbalance that characterizes heart failure. AMPK, a key regulator of cellular energy homeostasis, acts by switching off energy-consuming pathways in favor of processes that generate energy. Our objective was to determine the effects of substrate availability and AMPK activation on creatine transport in cardiomyocytes. We studied creatine transport in rat neonatal cardiomyocytes and HL-1 cardiac cells expressing the human creatine transporter cultured in the presence of varying creatine concentrations and the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-d-ribonucleoside (AICAR). Transport was enhanced in cardiomyocytes following incubation in creatine-depleted medium or AICAR. The changes in transport were due to alterations in Vmax that correlated with changes in total and cell surface creatine transporter protein content. Our results suggest a positive role for AMPK in creatine transport modulation for cardiomyocytes in culture.

Background: Creatine, Phosphocreatine, and creatine kinases, constitute an energy shuttle that links ATP production in mitochondria with cellular consumption sites. Myocytes and neurons cannot synthesize creatine and depend on uptake across the cell membrane by a specialized transporter to maintain intracellular creatine levels. Although recent studies have improved our understanding of creatine transport in cardiomyocytes, the structural elements underlying the creatine transporter protein regulation and the relevant intracellular signaling processes are unknown. Methods: The effects of pharmacological activation of kinases or phosphatases on creatine transport in cardiomyocytes in culture were evaluated. Putative phosphorylation sites in the creatine transporter protein were identified by bioinformatics analyses, and ablated using site-directed mutagenesis. Mutant transporter function and their responses to pharmacological PKC activation or changes in creatine availability in the extracellular environment, were evaluated. Results: PKC activation decreases creatine transport in cardiomyocytes in culture. Elimination of high probability potential phosphorylation sites did not abrogate responses to PKC activation or substrate availability. Conclusion: Modulation of creatine transport in cardiomyocytes is a complex process where phosphorylation at predicted sites in the creatine transporter protein does not significantly alter activity. Instead, non-classical structural elements in the creatine transporter and/or interactions with regulatory subunits may modulate its activity.

Aim: Peripheral blood-derived endothelial cells (pBD-ECs) are an attractive tool for cell therapies and tissue engineering, but have been limited by their low isolation yield. We increase pBD-EC yield via administration of the chemokine receptor type 4 antagonist AMD3100, as well as via a diluted whole blood incubation (DWBI). Materials & Methods: Porcine pBD-ECs were isolated using AMD3100 and DWBI and tested for EC markers, acetylated LDL uptake, growth kinetics, metabolic activity, flow-mediated nitric oxide production and seeded onto titanium tubes implanted into vessels of pigs. Results: DWBI increased the yield of porcine pBD-ECs 6.6-fold, and AMD3100 increased the yield 4.5-fold. AMD3100-mobilized ECs were phenotypically indistinguishable from nonmobilized ECs. In porcine implants, the cells expressed endothelial nitric oxide synthase, reduced thrombin-antithrombin complex systemically and prevented thrombosis. Conclusion: Administration of AMD3100 and the DWBI method both increase pBD-EC yield.

The purpose of this study was to determine the roles of alginate composition and gelling cations on bead swelling, which affects its durability.Using a 2-channel droplet generator, microspheres were generated with 1.5% solutions of low viscosity high-mannuronic acid (LVM), medium viscosity high-mannuronic acid (MVM), low viscosity high-guluronic acid (LVG) and medium viscosity high-guluronic acid (MVG) alginate. They were gelled by cross-linking with 1.1% solution of either BaCl2 or CaCl2. The diameters of the micro-beads were measured and recorded on day 0. The micro-beads were subsequently washed and incubated in saline at 37 degrees C for 2 weeks with size assessment every 2 days. The data were normalized by calculation of the percentage change from control (day 0) for all groups of micro-beads.Diameters of all beads were between 550 and 700 microm on day 0. Viscosity had no effect on swelling of Ba++- and Ca++-alginate micro-beads. Ca++-alginate micro-beads were more prone to swelling than the corresponding Ba++-alginate beads. High G-Ba++ beads had only a modest increase in size over time, in contrast to the high M-Ba++.Alginate composition and the gelling cation have significant effects on bead swelling.

Alginate is commonly used to microencapsulate islets in experiments with islet allografts and xenografts for the treatment of Type I diabetes. The purpose of the present study is to determine the effects of alginate composition and purity on the morphology and size of microspheres.Microcapsules produced with the impure alginate types, medium-viscosity high-guluronic acid (IMVG), low-viscosity high-G (ILVG), low-viscosity high-mannuronic acid (ILVM) and medium-viscosity high-M (IMVM) were compared with one another and others generated with a highly purified LVM (HPLVM) alginate. Droplets of 1.5% alginate from an air-syringe pump were gelled in 1.1% CaCl2 solution. While leaving the alginate pressure and needle recess constant, the air-jacket pressure was varied between 9.5-10.5 PPSI to enhance stable microcapsule generation and different batches of microbeads were made from each alginate type.The sizes of the high-guluronic acid alginate microbeads were consistently bigger than those of the corresponding high-mannuronic acid alginate beads at all air-jacket settings. At the optimal air-jacket pressure of 9.0 PPSI, the mean+SD diameter of the IMVG microbeads was 780 + 20 microm, while that of IMVM was 607 + 44 microm (p0.0001, n=30). Similarly, the mean ILVG microbead diameter was 816+28 microm compared to 656+26 microm for ILVM capsules (p0.0001, n=30). Less polymorphism was found with the HPLVM microspheres than with the ILVM microbeads.Highly purified high-mannuronic acid alginate will provide smaller, spherical microcapsules suitable for islet cell transplantation.

Antioxidant depletion is believed to be a mechanism involved in the pathophysiology of several upper gastrointestinal disorders, and H, K-ATPase inhibitors can alter free radical production by neutrophils. We hypothesized that the H, K-ATPase inhibitor esomeprazole magnesium would decrease gut free radical production with a concomitant increase in gut total antioxidant capacity. A/J mice (n = 10/group) received either vehicle (control) or one of three concentrations of esomeprazole magnesium in vehicle by once-daily gavage for 10 days. Using tissue extracts from stomach and colon, total antioxidant capacity, lipid peroxide levels, and constitutive Cu/Zn-superoxide dismutase were measured using validated assays. There was a dose-related increase in total antioxidant capacity (analysis of variance, P < 0.001) in stomach, but there was no change in the colon. In the assessment of free radical production, there was a trend toward decreased lipid peroxide levels in stomach from mice receiving esomeprazole. In stomach, Cu/Zn-superoxide dismutase activity was increased (ANOVA: p=.03) in mice receiving esomeprazole. In conclusion, gastric total antioxidant capacity and Cu/Zn-superoxide dismutase activity are increased by esomeprazole, and these changes may result in part from decreased free radical production. The present results support the notion that the pharmacological effects of this agent on upper intestinal tissue are more complex than previously thought, and appear to involve both enzymatic and nonenzymatic tissue antioxidants.

Chelated hollow microcapsules are unstable under in vitro conditions because of their hygroscopic nature. Nongel inducing cations, such as Na+, stabilize the outer membrane of the alginate-polylysine-alginate microcapsules leading to more stable beads. We made different batches of empty capsules with a mean +/- SEM diameter of 607+/-11 microns, and found that within 1 week of incubating these capsules in normal saline at 37 degrees C, they increased to 718+/-10 microns (p < 0.05, n = 5). In initial experiments, we made different batches of capsules and divided them into two groups. One group was left untreated (control) whereas the other was treated with 6 mM Na2SO4 for 30 min, before incubation in saline at 37 degrees C. Control capsules increased in weight and size, before beginning to melt in less than 1 week. In contrast, treated capsules rapidly lost weight and remained intact during 1 month of follow-up. In perifusion experiments, we found no deleterious effect of sodium sulfate treatment on the function of islets enclosed in the capsules.

Objective We sought to evaluate the effect of tumor size, location, and clinical nodal status on outcomes after thoracoscopic lobectomy for lung cancer. Methods All patients who underwent attempted thoracoscopic lobectomy for lung cancer between June 1999 and October 2010 at a single institution were reviewed. A model for morbidity including published risk factors as well as tumor size, location, and clinical N status was developed by multivariable logistic regression. Results During the study period, 916 thoracoscopic lobectomies met study criteria: 329 for peripheral, clinical N0 tumors ≤3 cm and 504 for tumors that were central, clinical node positive, or >3 cm. Tumor location could not be documented for 83 patients. Conversions to thoracotomy occurred in 36 patients (4%); patients with clinically node-positive disease had higher conversion rates (11 conversions in 153 clinical N1 to N3 patients [7.2%] vs 25 in 763 clinical N0 patients [3.3%, P = . 03]. Overall operative mortality was 1.6% (14 patients) and morbidity was 32% (296 patients). Although patients with larger tumors ( P = . 006) and central tumors ( P = . 01) had increased complications by univariate analysis, tumor size >3 cm ( P = . 17) and central location ( P = . 5) did not predict significantly overall morbidity in multivariate analysis. Clinical node status did not predict increased complications by univariate or multivariate analysis. Significant predictors of morbidity in multivariable analysis were increasing age, decreasing forced expiratory volume in 1 second, prior chemotherapy, and congestive heart failure. Conclusions Thoracoscopic lobectomy for lung cancers that are central, clinically node positive, or >3 cm does not confer increased morbidity compared with peripheral, clinical N0 cancers that are

ObjectivesAdvantages of thoracoscopic lobectomy include less postoperative pain, shorter hospitalization, and improved delivery of adjuvant chemotherapy. The incidence of postoperative complications has not been thoroughly assessed. This study analyzes morbidity after lobectomy to compare the thoracoscopic approach and thoracotomy.MethodsBy using a prospective database, the outcomes of patients who underwent lobectomy from 1999–2009 were analyzed with respect to postoperative complications. Propensity-matched groups were analyzed based on preoperative variables and stage.ResultsOf the 1079 patients in the study, 697 underwent thoracoscopic lobectomy, and 382 underwent lobectomy by means of thoracotomy. In the overall analysis thoracoscopic lobectomy was associated with a lower incidence of atrial fibrillation (P = .01), atelectasis (P = .0001), prolonged air leak (P = .0004), transfusion (P = .0001), pneumonia (P = .001), sepsis (P = .008), renal failure (P = .003), and death (P = .003). In the propensity-matched analysis based on preoperative variables, when comparing 284 patients in each group, 196 (69%) patients who underwent thoracoscopic lobectomy had no complications versus 144 (51%) patients who underwent thoracotomy (P = .0001). In addition, thoracoscopic lobectomy was associated with a lower incidence of atrial fibrillation (13% vs 21%, P = .01), less atelectasis (5% vs 12%, P = .006), fewer prolonged air leaks (13% vs 19%, P = .05), fewer transfusions (4% vs 13%, P = .002), less pneumonia (5% vs 10%, P = .05), less renal failure (1.4% vs 5%, P = .02), shorter chest tube duration (median of 3 vs 4 days, P < .0001), and shorter length of hospital stay (median of 4 vs 5 days, P < .0001).ConclusionsThoracoscopic lobectomy is associated with a lower incidence of major complications, including atrial fibrillation, compared with lobectomy by means of thoracotomy. The underlying factors responsible for this advantage should be analyzed to improve the safety and outcomes of other thoracic procedures.

The purpose of this study was to determine the roles of alginate composition and gelling cations on bead swelling, which affects its durability.Using a 2-channel droplet generator, microspheres were generated with 1.5% solutions of low viscosity high-mannuronic acid (LVM), medium viscosity high-mannuronic acid (MVM), low viscosity high-guluronic acid (LVG) and medium viscosity high-guluronic acid (MVG) alginate. They were gelled by cross-linking with 1.1% solution of either BaCl2 or CaCl2. The diameters of the microbeads were measured and recorded on day 0. The microbeads were subsequently washed and incubated in saline at 37 degrees C for 2 weeks with size assessment every 2 days. The data were normalized by calculation of the percentage change from control (day 0) for all groups of microbeads.Diameters of all beads were between 550-700 microns on day 0. Viscosity had no effect on swelling of Ba++- and Ca++-alginate microbeads. Ca++-alginate microbeads were more prone to swelling than the corresponding Ba++-alginate beads. High G-Ba++ beads had only a modest increase in size over time, in contrast to the high M-Ba++.Alginate composition and the gelling cation have significant effects on bead swelling.

Poly-L-Lysine (PLL) is the most widely used biomaterial for providing perm-selectivity in alginate microcapsules for islet transplantation. We had previously reported that Poly-L-Ornithine (PLO) is less immunogenic than PLL, and in the present study, we have compared the physical characteristics of PLO- and PLL-coated hollow alginate microcapsules. Microspheres made with 1.5% alginate were divided into 2 groups that were first coated with either 0.1% PLO or PLL, followed by a second coating with 0.25% alginate. After liquefaction of the inner alginate core with sodium citrate, the microcapsules were washed with saline and used for experiments. Pore size exclusion studies were performed with FITC-labeled lectins incubated with encapsulated pig islets followed by examination for fluorescence activity. Mechanical strength was assessed by an osmotic pressure test and by 36 h of mechanical agitation of microcapsules with inert soda lime beads. The pore size exclusion limit of microcapsules after 20 min of coating was significantly smaller with PLO. While the mean +/- SEM diameter of PLL-coated microcapsules increased from 718+/-17 to 821 +/- 17 microm (p < 0.05) during 14 days incubation at 37 degrees C, the PLO group did not change in size. Also, PLL group had a higher percentage of broken capsules (52.7 +/- 4.9%) compared to 3.1 +/- 2.05% for PLO capsules (p < 0.0001,n = 6). We conclude that PLO-coated alginate microcapsules are mechanically stronger and provide better perm-selectivity than PLL-coated microcapsules.

It has been proposed that tissue damage induced by nonsteroidal anti-inflammatory drugs is related to increased tissue free radical production with antioxidant depletion. We have shown that esomeprazole increases gastric total antioxidant capacity in mice and, therefore, hypothesized that the protective effect of esomeprazole during treatment with a nonsteroidal anti-inflammatory drug is related to increased gastric antioxidant capacity and decreased tissue free radical production. A/J mice received one of four treatments by daily gavage: saline in vehicle (control), indomethacin, esomeprazole, or indomethacin and esomeprazole. After 10 days, all mice were sacrificed and validated assays were used to measure gastric total antioxidant capacity, lipid peroxide levels, and myeloperoxidase activity. Indomethacin-treated mice developed weight loss and melena. No mice receiving indomethacin and esomeprazole died, but the death rate while receiving indomethacin was 38% (chi2, P = 0.05). Gastric lipid peroxide levels increased in mice receiving indomethacin treatment compared to treatment with esomeprazole and indomethacin (P = 0.03). There was a strong trend (P = 0.08) toward increased gastric total antioxidant capacity in mice receiving esomeprazole and indomethacin compared to mice receiving indomethacin. Gastric myeloperoxidase activities were not different among the four groups. Esomeprazole significantly improved survival in mice that received indomethacin, reduced free radical production, as estimated by lipid peroxide levels, and appeared to increase gastric total antioxidant capacity. The mechanisms for the beneficial effects of esomeprazole in the treatment of gastropathy are more complex than previously thought.

The present study was performed on the basis of the hypothesis that the low molecular weight (MW) compounds, DMSO and glycerol, permeate the cell and interact hydrophobically with intracellular proteins, thereby perturbing the cytoskeletal architecture of frozen cells and diminishing islet cell integrity and function. Isolated rat islets were cultured overnight (18-24 h) at 37 degrees C in RPMI medium supplemented with 10% fetal calf serum and 1% mixture of penicillin/streptomycin. Using a programmable temperature controller, samples of precounted islets were then frozen under liquid nitrogen, in the presence of either 2 M DMSO (MW = 0.078 kDa), 3 M glycerol (MW = 0.092 kDa), 5% polyethylene glycol (PEG, MW = 20 kDa), or 10% polyvinylpyrrolidone (PVP, MW = 40 kDa), and stored at -80 degrees C for 1 week. Following thawing and overnight (18-24 h) culture, intact islet recovery was determined by islet counting after dithizone staining. Islet function was assessed by determination of glucose-stimulated insulin secretion in perifusion experiments with Krebs-Ringer bicarbonate buffer, pH 7.4, containing either basal (3.3 mM) or high (16.7 mM) glucose concentrations. The assessment of islet recovery and function of all cryopreserved samples was performed only after thawing and overnight culture (18-24 h) of islets. The mean +/- SEM percent intact islet recovery was higher with PVP compared with DMSO (82 +/- 4.6 vs. 62.7 +/- 3.1%, respectively, p < 0.005, n = 9). Furthermore, the glucose stimulation index of insulin secretion by islets taken from samples frozen with PEG and PVP, after thawing and overnight culture, was comparable to that of freshly isolated islets, in contrast to DMSO and glycerol. There was no significant difference in intact islet recovery and function between samples frozen with PVP and those frozen with PEG. Samples frozen with DMSO and glycerol had similar results in islet recovery and function. These data show that PVP is a new and potent cryoprotectant for islet cell freezing.

Background Alginate microcapsule swelling, which occurs as a result of increased hydrophilicity owing to the Ca++ that remains after rapid chelation of the inner alginate core, is a problem in encapsulation. We have previously shown that exchange of the residual divalent Ca++ with the monovalent Na+ through the use of 6 mmol/L Na2SO4 decreases swelling in chelated alginate-polylysine-alginate microcapsules, and this process enhances their durability. The purpose of the present study was to examine the morphology of Na2SO4-treated microcapsules in long-term incubation with the use of serum-supplemented culture medium. Methods Spherical beads of purified alginate (3%) that were gelled with 1.1% CaCl2 were first coated with polylysine, and then with 0.24% alginate. After rapid chelation of the inner alginate core with 55 mmol/L sodium citrate, the capsules were either incubated for 30 minutes in 6 mmol/L Na2SO4 or left untreated (control). Each group of capsules was then placed in a flask containing Ham's culture medium supplemented with 20% porcine serum and incubated at 37°C. Results The diameters of Na2SO4-treated capsules only increased modestly from a mean±SD of 635±22.08 to 684.53±17.86 μm ( P Conclusion We have developed a new method of making more durable and stable microcapsules that can be used for islet cell xenotransplantation.

Phosphocreatine (PCr) and Creatine Cr play key roles in energy metabolism by replenishing ATP levels via creatine kinases and ADP. The sole source of Cr to skeletal and cardiac myocytes is transport by the Cr transporter (CrT), a membrane protein belonging to the neurotransmitter transporter family (SLC6) that also includes the serotonin, dopamine and norepinephrine transporters. Previous work suggests that Cr transport is modulated by AMPK and PKC. For example, Cr transport in HL-1 cardiomyocytes expressing the human CrT is decreased when the cells are treated with β-PMA, or incubated in culture media enriched for Cr. Cr transport increases if these cells are incubated in culture media depleted of Cr or supplemented with AICAR, an AMPK activator. To identify phosphorylation sites that regulate CrT function, we created single and multiple site mutants lacking putative intracellular phosphorylation sites that scored highly (greater than 0.7) when the transporter protein sequence was evaluated by the NetPhos 2.0 algorithm (CBS, Technical University of Denmark). These sites tended to cluster at the beginning of the N-terminus and the end of the C-terminus of the protein. Ablation of these potential phosphorylation sites had no effect on transport capacity, or the ability to respond to β-PMA, AICAR or substrate availability. We also generated mutants with incremental deletions of the N-or C-termini of the CrT protein. Deletions that remove the entire N or C-terminus resulted in the expression of an inactive transporter protein, while smaller truncations yielded functional Cr transporters that still responded to PKC and AMPK activation and Cr availability like the phosphorylation mutants. Therefore, either low-probability phosphorylation site(s) modulate CrT activity and/or a yet to be identified signalling molecule or interacting protein(s) are responsible for modulation of Cr transport by AMPK, PKC and substrate availability.