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Background: In the last decades the replacement of fetal bovine serum (FBS) with human Platelet Lysate (hPL) for ATMPs expansion has been for a long time investigated to overcome FBS-related issues. Despite several studies confirming hPL safety and efficacy in Mesenchymal Stromal Cell (MSC) expansion, there are still gaps in the knowledge of hPL as a supplement, like the composition and release criteria. As growth factors are released after thrombocytes lysis during hPL production, starting platelet concentration may affect hPL quality. This study aimed to investigate hPL starting platelet concentration effects on bone marrow-derived MSC (BM-MSC) ex-vivo expansion. Methods: MSC were isolated from the bone marrow (BM) of 7 donors and cultured from passage 1 to 5 in 4 different conditions: DMEM 10% FBS and DMEM 5% hPL varying starting platelet concentration. Particularly hPL was produced by in-hospital Transfusion Service, in three different starting platelet concentrations (sPLTC): high (4x109 PLTS/ml), medium (2x109 PLTS/ml) and low (1x109 PLTS/ml). The study focused on the analysis of parameters that are mostly affected by hPL such as cell proliferation, immunophenotype, telomeric length, differentiation and senescence. Results: Evaluation of proliferation indexes (PDT and PD) underlined dose-dependent effects of sPLTC, also confirmed by flow cytometry cell cycle analysis. Immunophenotype seems not to be affected by sPLTC. Differences were instead detected by adhesion molecules markers CD10, CD106, CD166, and CD146 expression, as their expression showed a dose-dependent downregulation, based on the sPLTC. Differentiation potential seems to be unaffected by different sPLTC as all cell batches differentiated into osteoblasts, adipocytes and chondrocytes. On the contrary senescence and relative telomeric length RTL (detected by SA-β-GAL activity and PNA-FITC flow cytometry) are strongly affected by sPLTC, in a dose-dependent manner. Particularly high sPLTC results in cell senescence associated with decreased RTL. Conclusions: our data showed that sPLTC affects some BM MSCs properties, underlying its importance during hPL preparation. According to this study, we suggest a medium sPLTC for hPL preparation, as the best compromise between the increase in proliferation index and effects on senescence.

Background Manufacturing of human Mesenchymal Stromal Cells as advanced therapy medicinal product (ATMP) for clinical use involves an ex vivo expansion, which leads to a risk of contamination by microbiological agents. Even if manufacturing under Good Manufacturing Practice (GMP) license minimizes this risk, contamination of cell cultures by mycoplasmas still represents a widespread problem. Furthermore, the absence of mycoplasma contamination represents one of ATMPs release criteria. Since July 2007, European Pharmacopoeia (EuPh) offers the possibility to replace official mycoplasma detection methods with Nucleic Acid Amplification techniques, after suitable validation. As an Italian authorized Cell Factory, we developed an in-house GMP-compliant validation of real-time PCR method for mycoplasma detection in human Mesenchymal Stromal Cells, according to EuPh sec. 2.6.7 and International Conference on Harmonization Q2. Materials and methods The study was performed in compliance with GMP international requirements with MycoSEQ™ Mycoplasma Detection Assay (Thermofisher) on QuantStudio5 real-Time PCR (Applied Biosystems). Assay validation was developed to evaluate sensitivity, interferences matrix-related, specificity and robustness. Results MycoSEQ™ Mycoplasma Detection Assay has been successfully validated on human Mesenchymal Stromal Cells as results comply with validation protocol acceptance criteria. Conclusions MycoSEQ™ Mycoplasma Detection Assay is a fast, sensitive and specific PCR-based Nucleic Acid Test assay that can be used as an alternative to official mycoplasma test methods for lot release of human Mesenchymal Stromal Cells as advanced therapy medicinal product (ATMP). Moreover, our study underlines the presence of interference on real-time PCR reaction due to matrix composition, pointing out a practical approach for method validation (i.e interference removal).

Background: Hematopoietic progenitor cells (HPCs) cryopreservation have applications, especially in the autologous setting, allowing therapeutic use several years after collection. Cryopreservation...

Background PBSC collection using a blood cell separator in very low weight patients can be frequently complicated by severe adverse effects and technical difficulties. Material and methods From March 2013 to January 2017, 14 PBSC collections were performed in 12 children weighing less than 10 kg, affected by different solid tumours. PBSC collection was performed with a "homemade" aseptically assembled circuit. The circuit is composed by a 150 mL collection bag connected with a 4 stopcock ramp, perfused with ACD. This circuit allows collection of a specific total blood amount from CVC, depending on CD34+ /kg target. Results Mean CD34+ cell performance per collection was 9.3 × 106 /kg. Tolerance to the procedure was very good as none of the patients experienced complications, with the exception of a patient who showed mild cyanosis and pallor after collection. Moreover, no bleeding or thrombotic complications have been observed. To date, 16 PBSC reinfusions have been performed in 7 children with a mean CD34+ cells viability of 98.1% ± 2.7 and mean WBC viability of 57% ± 10. Cell recovery after thawing was 87% ± 10.8. A rapid graft intake for both neutrophils and platelets, between day 7 and 20 after reinfusion was observed. Discussion The procedure of total blood collection without the use of a cell separator is feasible and allows a good PBSC collection without significant side effects in very low-weight children. Moreover, this method could represent a valid and safe alternative to leukapheresis in patients where classic procedure could be difficult to apply.

What is the central question of this study? It is a challenge to discover effective therapies for fibrosis. Increasing evidence supports the antifibrotic potential of platelet-rich plasma (PRP) as a source of bioactive molecules, such as vascular endothelial growth factor (VEGF)-A. However, the effects and mechanisms of action of PRP need to be clarified. What is the main finding and its importance? This report clarifies the mechanisms mediating the antifibrotic action of PRP, strengthening the role of VEGF-A/VEGF receptor, and identifies gap junction currents and connexin 43 as novel targets of this pathway in the fibroblast-to-myofibroblast transition induced by the transforming growth factor-β1.Despite increasing experimental evidence, the antifibrotic potential of platelet-rich plasma (PRP) remains controversial, and its mechanisms of action are not fully clarified. This short report extends our previous research on the capability of PRP to prevent the in vitro differentiation of fibroblasts toward myofibroblasts, the key effectors of fibrosis, induced by the profibrotic agent transforming growth factor-β1 (TGF-β1). In particular, we focused on the involvement of signalling mediated by vascular endothelial growth factor (VEGF)-A/VEGF receptor (VEGFR) in the PRP-induced fibroblast response, highlighting gap junction features. Electrophysiological and morphological analyses revealed that PRP hindered morphofunctional differentiation of both murine NIH/3T3 and human primary adult skin fibroblasts toward myofibroblasts as judged by the analysis of membrane phenomena, α-smooth muscle actin and vinculin expression and cell morphology. Neutralization of VEGF-A by blocking antibodies or pharmacological inhibition of VEGFR by KRN633 in TGF-β1-treated fibroblasts prevented the PRP-promoted effects, such as the reduction of voltage-dependent transjunctional currents in cell pairs and a decreased expression of connexin 43, the typical connexin isoform forming voltage-dependent connexons. The role of VEGF-A in inhibiting these events was confirmed by treating TGF-β1-stimulated fibroblasts with soluble VEGF-A. The results obtained when cells were differentiated using KRN633 alone suggest an antagonistic cross-talk between TGF-β1 and VEGFR. In conclusion, this study identifies, for the first time, gap junction currents as crucial targets in the VEGF-A/VEGFR-mediated antifibrotic pathway and provides new insights into mechanisms behind the action of PRP in preventing differentiation of fibroblasts to myofibroblasts.

The persistence of activated myofibroblasts is a hallmark of fibrosis of many organs. Thus, the modulation of the generation/functionality of these cells may represent a strategical anti-fibrotic therapeutic option. Bone marrow-derived mesenchymal stromal cell (MSC)-based therapy has shown promising clues, but some criticisms still limit the clinical use of these cells, including the need to avoid xenogeneic compound contamination for ex vivo cell amplification and the identification of appropriate growth factors acting as a pre-conditioning agent and/or cell delivery vehicle during transplantation, thus enabling the improvement of cell survival in the host tissue microenvironment. Many studies have demonstrated the ability of platelet-rich plasma (PRP), a source of many biologically active molecules, to positively influence MSC proliferation, survival, and functionality, as well as its anti-fibrotic potential. Here we investigated the effects of PRP, murine and human bone marrow-derived MSCs, and of the combined treatment PRP/MSCs on in vitro differentiation of murine NIH/3T3 and human HDFα fibroblasts to myofibroblasts induced by transforming growth factor (TGF)-β1, a well-known pro-fibrotic agent. The myofibroblastic phenotype was evaluated morphologically (cell shape and actin cytoskeleton assembly) and immunocytochemically (vinculin-rich focal adhesion clustering, α-smooth muscle actin and type-1 collagen expression). We found that PRP and MSCs, both as single treatments and in combination, were able to prevent the TGF-β1-induced fibroblast-myofibroblast transition. Unexpectedly, the combination PRP/MSCs had no synergistic effects. In conclusion, within the limitations related to an in vitro experimentation, our study may contribute to providing an experimental background for supporting the anti-fibrotic potential of the combination PRP/MSCs which, once translated “from bench to bedside,” could potentially offer advantages over the single treatments.

Background & Aim Background In the last decades the replacement of fetal bovine serum (FBS)with human Platelet Lysate (hPL) for ATMPs clinical expansion has been for a long time investigated to overcome FBS related issues.Despite several studies confirm hPL safety and efficiency in Mesenchymal Stromal Cell(MSC)expansion, there are still some gaps in the knowledge of hPL as cell culture supplement, starting from composition and quality release criteria. Aim of the study As growth factors are released after thrombocytes lysis during hPL production, starting platelet concentration may affect hPL quality.The aim of this study was to investigate hPL starting platelet concentration effects on bone marrow derived MSC(BM-MSC). Methods, Results & Conclusion Material and methods MSC were isolated from the bone marrow of 7 donors and cultured from p1 to p7 in 4 different conditions: DMEM 10% FBS and DMEM 5% hPL varying starting platelet concentration.Particularly hPL was produced according to standardized protocol by in-hospital Transfusion Service, in three different starting platelet concentrations (sPLTC): high sPLTC(4 × 109 PLTS/ml), medium(2 × 109 PLTS/ml) and low(1 × 109 PLTS/ml).The study focused on analysis of parameters that are mostly affected by hPL such as proliferation, immunophenotype, telomeric length, differentiation and cell senescence. Results and Conclusions Evaluation of proliferation indexes(PDT and PD) underlined dose dependent effects of sPLTC (figure 1), also confirmed by flow cytometry cell cycle analysis of BrdU incorporation. Immunophenotype seems not to be affected by sPLTC, as classical MSC markers meet ISCT criteria (table 1). Differences were instead detected by analyzing mean fluorescence intensity (MFI) in a dose dependent manner (figure 2), both for intrinsic and extrinsic parameters. Differentiation potential seems to be unaffected by different sPLTC as all cells batches differentiated into osteoblasts, adipocytes and chondrocytes. On the contrary senescence and relative telomeric length RTL (detected by SA-β-GAL activity and PNA-FITC flow cytometry) are strongly affected by sPLTC, in a dose dependent manner. Particularly high sPLTC results in increased senescent cells associated with decreased RTL (figure 3). In conclusion our data showed that sPLTC affects some BM MSCs properties, underlying its importance during hPL preparation. According to this study we suggest a medium sPLTC for hPL preparation, as best compromise between increase in proliferation index and effects on senescence.

Background Extracellular vesicles (EVs) are secreted membranous particles intensively studied for their potential cargo of diagnostic markers. Efficient and cost-effective isolation methods need to be established for the reproducible and high-throughput study of EVs in the clinical practice. Methods We designed the nickel-based isolation (NBI) to rapidly isolate EVs and combined it with newly-designed amplified luminescent proximity homogeneous assay or digital PCR to detect biomarkers of clinical utility. Findings From plasma of 46 healthy donors, we systematically recovered small EV (~250 nm of mean diameter; ~3 × 1010/ml) and large EV (~560 nm of mean diameter; ~5 × 108/ml) lineages ranging from 50 to 700 nm, which displayed hematopoietic/endothelial cell markers that were also used in spike-in experiments using EVs from tumor cell lines. In retrospective studies, we detected picomolar concentrations of prostate-specific membrane antigen (PSMA) in fractions of EVs isolated from the plasma of prostate cancer patients, discriminating them from control subjects. Directly from oil-encapsulated EVs for digital PCR, we identified somatic BRAF and KRAS mutations circulating in the plasma of metastatic colorectal cancer (CRC) patients, matching 100% of concordance with tissue diagnostics. Importantly, with higher sensitivity and specificity compared with immuno-isolated EVs, we revealed additional somatic alterations in 7% of wild-type CRC cases that were subsequently validated by further inspections in the matched tissue biopsies. Interpretation We propose NBI-combined approaches as simple, fast, and robust strategies to probe the tumor heterogeneity and contribute to the development of EV-based liquid biopsy studies. Fund Associazione Italiana per la Ricerca sul Cancro (AIRC), Fondazione Cassa di Risparmio Trento e Rovereto (CARITRO), and the Italian Ministero Istruzione, Universita e Ricerca (Miur).

The GvHD is a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT). Extracorporeal photopheresis (ECP) represents an alternative therapeutic strategy to immunosuppressive therapy. Although ECP is used since 1990s, the mechanism of action has not yet been completely clarified. We analyzed cells collected from 20 ECP procedures of 4 patients affected by chronic GvHD and, for comparison, Peripheral Blood Mononuclear Cells (PBMCs) of 10 healthy donors undergoing from same type of photochemiotherapy, evaluating by flow cytometry, the effects before and after photoactivation with 8-MOP. The analysis showed a significant increase in cell death after ECP in particular in CD4 T lymphocytes as described in literature correlated with haematocrit value. Most interesting data emerge from the analysis of cytotoxic activity of NK cells, using flow cytometry analysis of surface expression of CD107a in the presence of target cells (K562). In all analyzed samples it was possible to document a statistically significant reduction of the cytotoxic activity of NK cells after photoactivation. The decrease of the cytotoxic activity was related to hematocrit value of leukoapheresis: in fact, lower HCT values were associated with a more marked reduction of cytotoxic activity. The study confirms literature data about the increase of cellular mortality induce by ECP. Furthermore, for the first time it is demonstrated that the ECP exerts a marked and significant inhibitory effect on the cytotoxic activity of NK cells. Our study suggests that lower values of hematocrit are associated with better treatment outcome.

Background The use of platelet lysate (PL) for the ex-vivo expansion of mesenchymal stromal/stem cells (MSCs) was initially proposed by Doucet et al. in 2005, as an alternative to animal serum. Moreover, regulatory authorities discourage the use of fetal bovine serum (FBS) or other animal derivatives, to avoid risk of zoonoses and xenogeneic immune reactions. Even if many studies investigated PL composition, there still are some open issues related to its use in ex-vivo MSC expansion, especially according to good manufacturing practice (GMP) grade protocols. Methods As an authorized cell factory, we report our experience using standardized PL produced by Azienda Ospedaliero Universitaria Meyer Transfusion Service for MSC expansion according to a GMP grade clinical protocol. As suggested by other authors, we performed an in-vitro test on MSCs versus MSCs cultured with FBS that still represents the best way to test PL batches. We compared 12 MSC batches cultured with DMEM 5% PL with similar batches cultured with DMEM 10% FBS, focusing on the MSC proliferation rate, MSC surface marker expression, MSC immunomodulatory and differentiation potential, and finally MSC relative telomere length. Results Results confirmed the literature data as PL increases cell proliferation without affecting the MSC immunophenotype, immunomodulatory potential, differentiation potential and relative telomere length. Conclusions PL can be considered a safe alternative to FBS for ex-vivo expansion of MSC according to a GMP grade protocol. Our experience confirms the literature data: a large number of MSCs for clinical applications can be obtained by expansion with PL, without affecting the MSC main features. Our experience underlines the benefits of a close collaboration between the PL producers (transfusion service) and the end users (cell factory) in a synergy of skills and experiences that can lead to standardized PL production. Electronic supplementary material The online version of this article (10.1186/s13287-018-0863-8) contains supplementary material, which is available to authorized users.

Exercise is a cornerstone of diabetes therapy in type 1 diabetes mellitus (DMT1) patients. The type of exercise is important in determining the propensity to hypoglycemia. We assessed, by continuous glucose monitoring (CGM), the glucose profiles during and in the following 20h after a session of two different types of exercise.Eight male volunteers with well-controlled DMT1 were studied. They underwent 30min of both intermittent high-intensity exercise (IHE) and moderate-intensity exercise (MOD) in random order. Expired air was recorded during exercise, while metabolic and hormonal determinations were performed before and for 120 min after exercises. The CGM system and activity monitor were applied for the subsequent 20h.Blood glucose level declined during both type of exercise. At 150 min following the start of exercise, plasma glucose content was slightly higher after IHE. No changes were observed in plasma insulin concentration. A significant increase of norepinephrine concentration was noticed during IHE. Between midnight and 6:00 a.m. the glucose levels were significantly lower after IHE than those observed after MOD (area under the curve, 23.3 ± 3 vs. 16 ± 3 mg/dL/420 min [P = 0.04]; mean glycemia at 3 a.m., 225 ± 31 vs. 147 ± 17 mg/dL [P0.05]). The number of hypoglycemic episodes after IHE was higher than that observed after MOD (seven vs. two [P0.05]).We demonstrate that (1) CGM is a useful approach in DMT1 patients who undergo an exercise program and (2) IHE is associated with delayed nocturnal hypoglycemia.

Statement of problem: The respiratory nasal effects of physical exercise have been extensively investigated; on the other hand there are no data regarding olfactory threshold modification after aerobic physical exercise. Methods: The present prospective study investigated the modifications in nasal respiratory flows and olfactory thresholds after controlled aerobic physical exercise in a cohort of 15 adult, healthy volunteers. The Peak Nasal Inspiratory Flow (PNIF), and the Sniffin’ Sticks olfactory threshold test were used for our determinations. Main results: The mean PNIF after physical exercise was significantly higher than the mean PNIF value found before physical exercise. Statistical analysis ruled out any significant difference between mean olfactory thresholds pre vs post physical exercise. Principal conclusions: These outcomes confirmed PNIF sensitivity and reliability also in determining the changes in nasal patency occurring after physical exercise. The active vasoconstriction of nasal mucosa associated with the reduction of blood flow to the olfactory epithelium due to physical exercise may be compensated for by the increase of olfactory molecules that reach the olfactory mucosa because of nasal mucosal shrinkage: this mechanism could explain the stability of mean olfactory threshold after physical exercise.

Background Leukapheresis for autologous stem cell transplantation represents an efficient technique for the reconstitution of haematopoietic system in patients subjected to a high-dose chemotherapy for the treatment of haematological malignancies. The current regulations emphasise first steps of leukapheresis procedure but do not recommend methods for thawing, only suggesting that it must be performed as soon as possible in a 37 °C thermostatic bath. Aim of the Study We compared the classic method of thawing with an innovative and fully traceable method that uses WSCFD ® Stem Cell Fast Thawer KW. Materials and Methods The first part of the study was focused on the thermodynamic process of the two methods, thawing 6 "simulated" leukapheresis (buffy coats of healthy donors cryopreserved with saline solution, 5% HSA and 5% DMSO) and analysing the thawing curve obtained, by using an inside probe. In the second part, we focused on the recovery of viable CD34+ cells and leukocytes, thawing 20 real leukapheresis from paediatric patients. In this phase we also analyse final core bag temperature, time of procedure, cellular recovery with ISHAGE single platform flow cytometry assay and clonogenic potential performing a CFU assay. Results We found no significant differences between the two methods, both for thermodynamic aspect and cellular recovery. Thawing curves were similar and the paired Student's-t test used for statistical analysis showed a CD34+ cells recovery of 92.2% ± 11.4 using WSCFD ® versus 90% ± 11.1 of thermostatic bath. Data were similar even for leukocytes recovery (80.8% ± 9.5 with WSCFD ® and 79.2% ± 14.4 with thermostatic bath). All thawed products never exceeded the core temperature of 30 °C and no differences were found about the post-thaw clonogenic potential (614 × 10 4 ± 98.3 total CFU using WSCFD ® versus 592 × 10 4 ± 78.5 using thermostatic bath). The only difference observed was about the thawing time: WSCFD method requires a slightly longer time but, on the other hand, it correlates with reduced mean increase in temperature per minute, as a result of a more linear thawing curve. Conclusions WSCFD ® can replace the 37 °C thermostatic bath thawing procedure for leukapheresis, providing more security and fully traceable process data.

PURPOSE: The American Diabetes Association states that physical activity can be performed by individuals with Type 1 diabetes. Nevertheless, extreme altitude mountaineering represents a demanding challenge. We present the metabolic and cardiovascular parameters found in individuals with Type 1 diabetes during the ascent to Cho Oyu located at a height of 8201 m. METHODS: Six individuals with Type 1 diabetes and 10 matched controls participated in the expedition. Both groups were evaluated before and after 4 h of trekking for vital indices, blood gases, acute mountain sickness, and metabolic control at 0, 3700, and 5800 m. RESULTS: No difference between the groups was observed in acute mountain sickness scores. There was a progressive elevation in basal heart rates in both groups at increasing altitude while no changes were observed in mean blood pressures. After the 3 h of trekking, a significant increase in heart rate was observed in the controls at 0 m whereas a significant decrease in blood pressure was observed at higher altitude only in controls. HbA1c levels were worse after the expedition in both groups. A progressive increase in insulin requirement was observed in subjects with Type 1 diabetes (38 +/- 6 U x d(-1) at 0 m to 51 +/- 6 at 4200 m, P < 0.001). At an altitude of 5800 m, there was a significant increase in blood lactate concentration, independently of the activity level in the two groups. CONCLUSIONS: At extreme altitude, highly motivated trekkers with Type 1 diabetes but free from long-term complications present metabolic and cardiovascular parameters comparable with those of control subjects despite a worsening in metabolic control. This type of physical activity must be accompanied by careful glucose monitoring.

At present, no information is available about the possibility that acute loss of metabolic control might be able to modify fatty acid composition in IDDM patients. Therefore, the aim of this study was to determine whether a short-term period of ketosis has any effect on the composition of fatty acid of plasma phospholipids in a group of insulin-dependent diabetes mellitus (IDDM) patients. Eleven IDDM patients and nine healthy volunteers were studied. Patients were studied over a 2-day period; each 2-day period consisted of initial baseline measurements followed by a day of hyperglycemia and mild ketosis, which was promptly alleviated by insulin infusion. No significant difference in baseline percent fatty acid composition was observed between normal controls and IDDM patients. In IDDM patients, ketosis induced a significant decrease in percent arachidonic acid (20:4 n-6) content, with a significant parallel decline in n-6 total polyunsaturated fatty acids. A significant inverse correlation between the prevailing plasma glucose and the relative content of arachidonic acid in plasma phospholipids was observed (r = -0.35; P = 0.0488). A highly significant inverse correlation was observed between the change from baseline condition to ketosis of the ratio C20:4/C20:3, the product/precursor ratio for the reaction catalyzed by delta5-desaturase, and the values of hemoglobin A1c,(r = -0.855; P = 0.0015). In conclusion, short term diabetic ketosis is associated with a significant decrease in n-6 polyunsaturated fatty acid content in plasma phospholipids, especially arachidonic acid. This decrease in arachidonic acid appears to be related to the degree of metabolic derangement, whereas the influence of short-term diabetic ketosis on the ratio of C20:4 to C20:3 seems to be due to the degree of long-term metabolic control.

The effect of ethanol (ETOH) on muscle metabolism was assessed in both normal (NC) and noninsulin-dependent (NIDDM) subjects in the basal state and during isoglycemic hyperinsulinemia (450 pmol/L) clamp studies carried out either with systemic (NC, n = 5; NIDDM, n = 5) or intrabrachially (NC, n = 5; NIDDM, n = 5)ETOH infusion. On a repeat study, each subject underwent the same experimental procedures, except that saline was infused instead of ETOH. Systemic ETOH significantly decreased whole body glucose disposal in both NC and NIDDM patients. In NC, ETOH infusion decreased basal forearm glucose uptake (FGU) from 1.22 +/- 0.20 to 0.32 +/- 0.04 mumol/min.100 mL tissue (P0.01), whereas in NIDDM, this decrement was not significant (from 0.95 +/- 0.31 to 0.66 +/- 0.23). With saline infusion, hyperinsulinemia significantly stimulated FGU to 4.09 +/- 0.46 mumol/min.100 mL tissue in NC and to 2.50 +/- 0.76 in NIDDM. During ETOH, FGU was depressed by 81% in NC (delta = 3.32 mumol/min.100 mL tissue) and by 48% (P0.05) in NIDDM (delta = 1.21 mumol/min.100 mL tissue). Local ETOH infusion did not affect FGU in either NC (1.18 +/- 0.23 vs. 1.1 +/= 0.11 mumol/min.100 mL tissue in the baseline condition and 4.12 +/- 0.65 vs. 3.97 +/- 0.35 in insulin-stimulated conditions) or NIDDM (1.05 +/- 0.29 vs. 1.1 +/- 0.19 mumol/min.100 mL tissue in baseline condition and 2.72 +/- 0.82 vs. 2.83 +/- 0.51 in insulin-stimulated conditions) subjects. With systemic ETOH, but not local infusion, there was a reduction in baseline plasma free fatty acid level and an increase in blood lactate concentration during isoglycemic hyperinsulinemia. In summary, systemic ETOH infusion impairs both whole body and forearm glucose uptake in NC and NIDDM subjects; this effect was more apparent in NC than in NIDDM at both the whole body and forearm level. On the contrary, intrabrachial ETOH infusion did not affect forearm glucose balance in either group. These results suggest that the reduction in muscle glucose disposal associated with increased systemic ETOH concentrations is not caused by a direct ETOH effect on muscle glucose metabolism.

The American Diabetes Association states that all levels of physical activity can be performed by people with type 1 diabetes who do not have complications and are in good blood glucose control (1). Extreme altitude mountaineering, defined as climbing at altitudes in excess of 5,000 m, creates physiological demands, especially in type 1 diabetic subjects, who might experience impaired pulmonary function (2). We present the metabolic control and symptoms of mountain sickness during the 2002 Alpinisti in Alta Quota (ADIQ) Expedition to Cho Oyu, which is the sixth …

It has been suggested that the hemodynamic derangements present in diabetic ketoacidosis are the results not only of profound volume depletion but also of the effects of increased production of vasodilating prostaglandins (PGs), principally PGI2, released by adipose tissue. In animal and in vitro models, prostaglandin synthesis is increased during insulin deficiency. We assessed the effects of short-term ketosis on the metabolic and hemodynamic variables of 10 IDDM patients free from long-term complications and of 9 normal control subjects after a 7-day randomized double-blind indomethacin (INDO) (50 mg q.i.d.) or placebo treatment period. Calf blood flow (CBF), postocclusive reactive hyperemia (PORH), and recovery half-time (an index of overall perfusion) after PORH were measured by plethysmography. Left ventricular and myocardial functions were also studied in each different condition during placebo and INDO treatment in IDDM patients. During placebo treatment, the increase in CBF during ketosis was higher (1.75 ± 0.29 ml · min−1 · 100 ml muscle−1) than during INDO (0.85 ± 0.17 ml · min−1 · 100 ml muscle−1; P = 0.007). PORH was similar in baseline conditions, during ketosis, and in recovery in both the placebo and INDO arms. Recovery half-time significantly increased during placebo (10 ± 2; 200%; P < 0.01) but not during INDO (1 ± 1; 106%; NS) treatment. In normal control subjects, insulin deficiency did not induce any significant effect on hemodynamic variables. In IDDM patients, during placebo treatment, ketosis increased both the cardiac index (from 3.4 ± 0.7 to 4.1 ± 0.8 1 · min−1 · m−2; P < 0.01) and the stroke index (from 42 ± 8 to 49 ± 7 ml/m2; P < 0.01) without changes in left ventricular ejection fraction but with a significant increase in both left and right ventricular end-diastolic volumes. Metabolic recovery induced a normalization of these parameters. INDO treatment significantly blunted these alterations. In summary, we showed that during acute insulin deficiency, INDO-sensitive mechanisms mediate vascular disturbances. Moreover, INDO treatment was capable of completely preventing the cardiac venous return and the left ventricular alterations. INDO does not interfere with the overall ketogenetic process or with insulin-induced metabolic recovery.

Endothelium-dependent and -independent vascular responses were assessed in 10 NIDDM patients and 6 normal subjects with no evidence of atherosclerotic disease. Changes in forearm blood flow and arteriovenous (AV) serum nitrite/nitrate (NO2-/NO3-) concentrations were measured in response to intra-arterial infusion of acetylcholine (ACh) (7.5, 15, 30 microg/min, endothelium-dependent response) and sodium nitroprusside (SNP) (0.3, 3, 10 microg/min, endothelium-independent response). Insulin sensitivity (determined by minimal model intravenous glucose tolerance test) was lower in NIDDM patients (0.82 +/- 0.20 vs. 2.97 +/- 0.29 10(4) min x microU(-1) x ml(-1); P0.01). Baseline forearm blood flow (4.8 +/- 0.3 vs. 4.4 +/- 0.3 ml x 100 ml(-1) tissue x min(-1); NS), mean blood pressure (100 +/- 4 vs. 92 +/- 4 mmHg; NS), and vascular resistance (21 +/- 1 vs. 21 +/- 1 units; NS), as well as their increments during ACh and SNP, infusion were similar in both groups. No difference existed in baseline NO2-/NO3- concentrations (4.09 +/- 0.33 [NIDDM patients] vs. 5.00 +/- 0.48 micromol/l [control subjects]; NS), their forearm net balance (0.31 +/- 0.08 [NIDDM patients] vs. 0.26 +/- 0.08 micromol/l x 100 ml(-1) tissue x min(-1); NS), and baseline forearm glucose uptake. During ACh infusion, both NO2- and NO3- concentrations and net balance significantly increased in both groups, whereas glucose uptake increased only in control subjects. When data from NIDDM and control groups were pooled together, a correlation was found between the forearm AV NO2- and NO3- differences and blood flow (r = 0.494, P = 0.024). On the contrary, no correlation was evident between NO2- and NO3- concentrations or net balance and insulin sensitivity. In summary, 1) no difference existed in basal and ACh-stimulated NO generation and endothelium-dependent relaxation between uncomplicated NIDDM patients and control subjects; 2) in both NIDDM and control groups, forearm NO2- and NO3- net balance following ACh stimulation was related to changes in the forearm blood flow; and 3) ACh-induced increase in forearm blood flow was associated with an increase in glucose uptake only in control subjects but not in NIDDM patients. In conclusion, our results argue against a role of impaired NO generation and blood flow regulation in determining the insulin resistance of uncomplicated NIDDM patients; rather, it supports an independent insulin regulation of hemodynamic and metabolic effects.