Cholangiocytes express an isoform of soluble adenylyl cyclase that is N-linked glycosylated and secreted in extracellular vesicles.

Soluble adenylyl cyclase (sAC)-derived cAMP regulates various cellular processes; however, the regulatory landscape mediating sAC protein levels remains underexplored. We consistently observed a 85 kD (sAC85 ) or 75 kD (sAC75 ) sAC protein band under glucose-sufficient or glucose-deprived states, respectively, in H69 cholangiocytes by immunoblotting. Deglycosylation by PNGase-F demonstrated that both sAC75 and sAC85 are N-linked glycosylated proteins with the same polypeptide backbone. Deglycosylation with Endo-H further revealed that sAC75 and sAC85 carry distinct sugar chains. We observed release of N-linked glycosylated sAC (sACEV ) in extracellular vesicles under conditions that support intracellular sAC85 (glucose-sufficient) as opposed to sAC75 (glucose-deprived) conditions. Consistently, disrupting the vesicular machinery affects the maturation of intracellular sAC and inhibits the release of sACEV into extracellular vesicles. The intracellular turnover of sAC85 is extremely short (t1/2 ~30 min) and release of sACEV in the medium was detected within 3 h. Our observations support the maturation and trafficking in cholangiocytes of an N-linked glycosylated sAC isoform that is rapidly released into extracellular vesicles.

Memory CD8+ T cells upregulate glycolysis and effector functions under limiting oxygen conditions.

Memory CD8+ T cells are indispensable for maintaining long-term immunity against intracellular pathogens and tumors. Despite their presence at oxygen-deprived infected tissue sites or in tumors, the impact of local oxygen pressure on memory CD8+ T cells remains largely unclear. We sought to elucidate how oxygen pressure impacts memory CD8+ T cells arising after infection with Listeria monocytogenes-OVA. Our data revealed that reduced oxygen pressure during in vitro culture switched CD8+ T cell metabolism from oxidative phosphorylation to a glycolytic phenotype. Quantitative proteomic analysis showed that limiting oxygen conditions increased the expression of glucose transporters and components of the glycolytic pathway, while decreasing TCA cycle and mitochondrial respiratory chain proteins. The altered CD8+ T cell metabolism did not affect the expansion potential, but enhanced the granzyme B and IFN-γ production capacity. In vivo, memory CD8+ T cells cultured under low oxygen pressure provided protection against bacterial rechallenge. Taken together, our study indicates that strategies of cellular immune therapy may benefit from reducing oxygen during culture to develop memory CD8+ T cells with superior effector functions.

Donor variation in stored platelets: Higher metabolic rates of platelets are associated with mean platelet volume, activation and donor health.

BACKGROUND: Platelets (PLTs) differ in glycolytic activity, resulting in rapid acidification of 'poor' storing PLT concentrates (PCs) in plasma, or depletion of glucose when stored in PLT additive solution (PAS). We aimed to understand why PLT glycolysis rates vary between donors and how this affects storage performance. STUDY DESIGN AND METHODS: Buffy coats from donors <45, 45-70 and >70 years were selected and single-donor PCs in plasma or PAS-E were prepared. PCs were stored for 8 days at 22 ± 2°C and sampled regularly for analysis. Mitochondrial activity was analyzed with an Oroboros oxygraph. Age groups, or subgroups divided into quartiles based on glucose consumption, were analyzed with ANOVA. RESULTS: In each comparison, PCs of the different groups were not different in volume and cellular composition. PLTs with the highest glucose consumption had a higher initial mean platelet volume (MPV) and developed higher CD62P expression and Annexin A5 binding during storage. Higher glycolytic activity in these PLTs was not a compensation for lower mitochondrial ATP production, because mitochondrial ATP-linked respiration of fresh PLTs correlated positively with MPV (R2  = 0.71). Donors of high glucose-consuming PLTs had more health-related issues. Storage properties of PCs from donors over 70 were not significantly different compared to PCs from donors younger than 45 years. CONCLUSIONS: High glucose-consuming PCs developing higher activation levels, not only displayed enhanced mitochondrial activity but were also found to contain larger PLTs, as determined by MPV. Storage performance of PLTs was found to be associated with donor health, but not with donor age.

The phospholipid flippase ATP8B1 is required for lysosomal fusion in macrophages.

ATP8B1 is a phospholipid flippase and member of the type 4 subfamily of P-type ATPases (P4-ATPase) subfamily. P4-ATPases catalyze the translocation of phospholipids across biological membranes, ensuring proper membrane asymmetry, which is crucial for membrane protein targeting and activity, vesicle biogenesis, and barrier function. Here we have investigated the role of ATP8B1 in the endolysosomal pathway in macrophages. Depletion of ATP8B1 led to delayed degradation of content in the phagocytic pathway and in overacidification of the endolysosomal system. Furthermore, ATP8B1 knockdown cells exhibited large multivesicular bodies filled with intraluminal vesicles. Similar phenotypes were observed in CRISPR-generated ATP8B1 knockout cells. Importantly, induction of autophagy led to accumulation of autophagosomes in ATP8B1 knockdown cells. Collectively, our results support a novel role for ATP8B1 in lysosomal fusion in macrophages, a process crucial in the terminal phase of endolysosomal degradation.

ATP8B1 Deficiency Results in Elevated Mitochondrial Phosphatidylethanolamine Levels and Increased Mitochondrial Oxidative Phosphorylation in Human Hepatoma Cells.

ATP8B1 is a phospholipid flippase that is deficient in patients with progressive familial intrahepatic cholestasis type 1 (PFIC1). PFIC1 patients suffer from severe liver disease but also present with dyslipidemia, including low plasma cholesterol, of yet unknown etiology. Here we show that ATP8B1 knockdown in HepG2 cells leads to a strong increase in the mitochondrial oxidative phosphorylation (OXPHOS) without a change in glycolysis. The enhanced OXPHOS coincides with elevated low-density lipoprotein receptor protein and increased mitochondrial fragmentation and phosphatidylethanolamine levels. Furthermore, expression of phosphatidylethanolamine N-methyltransferase, an enzyme that catalyzes the conversion of mitochondrial-derived phosphatidylethanolamine to phosphatidylcholine, was reduced in ATP8B1 knockdown cells. We conclude that ATP8B1 deficiency results in elevated mitochondrial PE levels that stimulate mitochondrial OXPHOS. The increased OXPHOS leads to elevated LDLR levels, which provides a possible explanation for the reduced plasma cholesterol levels in PFIC1 disease.

The extracellular lactate-to-pyruvate ratio modulates the sensitivity to oxidative stress-induced apoptosis via the cytosolic NADH/NAD+ redox state.

The advantages of the Warburg effect on tumor growth and progression are well recognized. However, the relevance of the Warburg effect for the inherent resistance to apoptosis of cancer cells has received much less attention. Here, we show here that the Warburg effect modulates the extracellular lactate-to-pyruvate ratio, which profoundly regulates the sensitivity towards apoptosis induced by oxidative stress in several cell lines. To induce oxidative stress, we used the rapid apoptosis inducer Raptinal. We observed that medium conditioned by HepG2 cells has a high lactate-to-pyruvate ratio and confers resistance to Raptinal-induced apoptosis. In addition, imposing a high extracellular lactate-to-pyruvate ratio in media reduces the cytosolic NADH/NAD+ redox state and protects against Raptinal-induced apoptosis. Conversely, a low extracellular lactate-to-pyruvate ratio oxidizes the cytosolic NADH/NAD+ redox state and sensitizes HepG2 cells to oxidative stress-induced apoptosis. Mechanistically, a high extracellular lactate-to-pyruvate ratio decreases the activation of JNK and Bax under oxidative stress, thereby inhibiting the intrinsic apoptotic pathway. Our observations demonstrate that the Warburg effect of cancer cells generates an anti-apoptotic extracellular environment by elevating the extracellular lactate-to-pyruvate ratio which desensitizes cancer cells towards apoptotic insults. Consequently, our study suggests that the Warburg effect can be targeted to reverse the lactate-to-pyruvate ratios in the tumor microenvironment and thereby re-sensitize cancer cells to oxidative stress-inducing therapies.

Platelet storage properties are associated with donor age: in vitro quality of platelets from young donors and older donors with and without Type 2 diabetes.

BACKGROUND AND OBJECTIVES: Previously it has been shown that platelet (PLT) storage performance is consistent by donor. Differences involved metabolic activity, which might be caused by mitochondrial (dys)function, associated with age and age-related diseases like Type 2 diabetes (T2D). We aimed to test PLTs from young donors in comparison with PLTs from older donors with or without diagnosis for T2D. MATERIALS AND METHODS: Fifteen whole blood donors <30 year were selected, and single-donor platelet concentrates (sPC) were prepared from buffy coats (BC) and plasma. Also, 2 × 11 sPC were prepared from matched donors >45 years with and without T2D. The sPC were stored for 8 days and analysed at regular intervals for in vitro quality. RESULTS: Donors were 24 ± 3, 60 ± 7 (without T2D) and 59 ± 8 (with T2D) years old. All sPC groups had comparable volume and PLT content. On Day 8, sPC from young donors showed higher pH37°C than sPC from older donors (6.84 ± 0.15 vs. 6.40 ± 0.48, P < 0.01), due to lower lactate production. Also, CD62P expression (22.9 ± 7.4 vs. 48.8 ± 24.0%, P < 0.01) and HSR reflected better in vitro quality. PLT storage properties of sPC obtained from T2D donors (pH = 6.51 ± 0.35) were not different from sPC of matched donors (pH = 6.40 ± 0.48). No differences in mitochondrial membrane potential were detected between the groups. CONCLUSION: Platelets from young donors exhibited the best storage conditions. On average, PLTs from older donors showed poorer in vitro quality but, considering the sub-optimal storage conditions, the implications for the daily blood bank routine is probably small. No association with T2D was found.

Role of the bicarbonate-responsive soluble adenylyl cyclase in cholangiocyte apoptosis in primary biliary cholangitis; a new hypothesis.

Primary biliary cholangitis (PBC) is a chronic fibrosing cholangiopathy characterized by an autoimmune stereotype and defective biliary bicarbonate secretion due to down-regulation of anion exchanger 2 (AE2). Despite the autoimmune features, immunosuppressants are ineffective while two bile acid-based therapies (ursodeoxycholic acid and obeticholic acid) have been shown to improve biochemical and histological features of cholestasis and long-term prognosis. However, the etiology and pathogenesis of PBC is largely unknown. Recently, it has been shown that microRNA-506 (miR-506) on chromosome X is up-regulated in PBC cholangiocytes and suppresses AE2 expression, which sensitizes cholangiocytes to bile salt-induced apoptosis by activating soluble adenylyl cyclase (sAC), an evolutionarily conserved bicarbonate sensor. In this review, we discuss the experimental evidence for the emerging role of the miR-506-AE2-sAC axis in PBC pathogenesis. We further hypothesize that the initial disease trigger induces an X-linked epigenetic change, leading to a female-biased activation of the miR-506-AE2-sAC axis. This article is part of a Special Issue entitled: Cholangiocytes in Health and Diseaseedited by Jesus Banales, Marco Marzioni and Peter Jansen.

Platelet storage performance is consistent by donor: a pilot study comparing "good" and "poor" storing platelets.

BACKGROUND: In retrospective studies, it has been shown that differences in storage variables of platelet (PLT) concentrates (PCs) are partially donor dependent. It was our aim to prospectively determine the donor effect on PLT quality. STUDY DESIGN AND METHODS: Based on quality control data of outdated apheresis PCs, male donors were selected with at least one PC with a pH value of more than 7.0 ("good," n = 6) or one PC with a pH value of less than 6.7 ("poor," n = 6) on Day 8. These donors donated a PC (Trima Accel, Terumo) and completed a short questionnaire about their health and lifestyle. PCs were stored for 12 days and analyzed at regular intervals for in vitro quality. RESULTS: Donor characteristics were comparable, except that zero of six good and four of six poor donors reported high blood pressure and/or high cholesterol/fat and/or use of medicines. Lactate production in good PCs was lower than that in poor PCs (0.09 ± 0.03 mmol/day/1011 PLTs vs. 0.13 ± 0.04 mmol/day/1011 PLTs, p < 0.05) resulting in a higher pH from Day 5 onward. At the end of storage, the good PCs showed lower CD62P expression, lower phosphatidylserine exposure, and higher mitochondrial membrane potential. PLT functional properties were only slightly different. Despite having lower pH, the poor PCs also fulfilled European Guidelines during 7-day storage. CONCLUSION: Platelet storage performance is consistent when donors are dichotomized as having good or poor storing PLTs. Metabolic differences are perhaps due to different functionality of the mitochondria. More research is needed to establish the underlying causes and the implications for donors and blood products.

A novel flow cytometry-based platelet aggregation assay.

The main function of platelets is to maintain normal hemostasis. Inefficient platelet production and/or defective platelet function results in bleeding disorders resulting from a wide range of genetic traits and acquired pathologies. Several platelet function tests have been developed for use in the clinic and in experimental animal models. In particular, platelet aggregation is routinely measured in an aggregometer, which requires normal platelet counts and significant blood sample volumes. For this reason, the analysis of thrombocytopenic patients, infants, and animal models is problematic. We have developed a novel flow cytometry test of platelet aggregation, in which 10- to 25-fold lower platelet counts or sample volumes can be used, either of platelet-rich plasma or whole blood from human subjects or mice. This setup can be applied to test in small assay volumes the influence of a variety of stimuli, drugs, and plasma factors, such as antibodies, on platelet aggregation. The presented principle stands as a novel promising tool, which allows analysis of platelet aggregation in thrombocytopenic patients or infants, and facilitates studies in platelets obtained from experimental animal models without the need of special devices but a flow cytometer.

Defects in Glanzmann thrombasthenia and LAD-III (LAD-1/v) syndrome: the role of integrin ß1 and ß3 in platelet adhesion to collagen.

Patients with Glanzmann thrombasthenia or Leukocyte Adhesion Deficiency-III syndrome (LAD-III or LAD-1/variant) present with increased bleeding tendency because of the lack or dysfunction of the fibrinogen receptor GPIIb/IIIa (integrin αIIbß3), respectively. Although the bleeding disorder is more severe in LAD-III patients, classic aggregometry or perfusion of Glanzmann or LAD-III platelets over collagen-coated slides under physiologic shear rate does not discriminate between these 2 conditions. However, in a novel flow cytometry-based aggregation assay, Glanzmann platelets were still capable of forming small aggregates upon collagen stimulation, whereas LAD-III platelets were not. These aggregates required functional GPIa/IIa (integrin α2ß1) instead of integrin αIIbß3, thus explaining the clinically more severe bleeding manifestations in LAD-III patients, in which all platelet integrins are functionally defective. These findings provide genetic evidence for the differential requirements of platelet integrins in thrombus formation and demonstrate that correct integrin function assessment can be achieved with a combination of diagnostic methods.

Red blood cell storage increases hypoxia-induced nitric oxide bioavailability and methemoglobin formation in vitro and in vivo.

BACKGROUND: In this study we investigated whether storage of red blood cells (RBCs) leads to alterations in nitrite reductase activity, hence in altered hypoxia-induced nitric oxide (NO) bioavailability and methemoglobin formation. STUDY DESIGN AND METHODS: Hypoxia-induced NO bioavailability and methemoglobin formation were measured in vitro after nitrite administration to fresh (<1 week of storage) and aged (5-6 weeks of storage) human RBC units and in blood samples of hemodiluted rats subjected to hypoxic ventilation after transfusion with fresh or aged human RBCs. RESULTS: In vitro, NO and methemoglobin levels 10 minutes after nitrite administration were lower in the fresh RBC samples compared to the aged RBC samples (p = 0.026 and p = 0.022, respectively). In vivo, NO bioavailability was also significantly lower in the rats receiving fresh RBCs compared to the group receiving aged RBCs (p = 0.003). In line with NO bioavailability, methemoglobin levels were higher, albeit not significantly, in the group receiving aged RBCs compared to in the group receiving fresh RBCs (p = 0.154). The difference in methemoglobin formation after nitrite administration between fresh and aged RBCs was only present under deoxygenated conditions and not under oxygenated conditions. There were no differences in methemoglobin reductase activity between fresh and aged RBCs. CONCLUSIONS: Storage of RBCs leads to an increased rate of hypoxia-induced nitrite reduction to NO and this is associated with increased methemoglobin formation. The increased methemoglobin formation and consequent decrease in oxygen delivery capacity might contribute to the storage-related impairment of aged RBCs to oxygenate the microcirculation.

Carboxylesterase 1 mediates a distinctive metabolic profile of dendritic cells to attain an inflammatory phenotype.

The metabolic profile of dendritic cells (DCs) shapes their phenotype and functions. Carboxylestrase 1 (CES1) enzyme is highly expressed in mononuclear myeloid cells however its exact role in DCs is elusive. We used a CES1 inhibitor (WWL113) and genetic overexpression to explore the role of CES1 in DCs differentiation in inflammatory models. CES1 expression was analyzed during CD14+ monocytes differentiation to DCs (MoDCs) using quantitative PCR. CES1 Inhibitor (WWL113) was applied during MoDCs differentiation. Surface markers, secreted cytokines, lactic acid production, phagocytic and T cell polarization capacity were analyzed. Transcriptomic and metabolic profile were assessed with RNA-sequencing and mass spectrometry. Cellular respiration was assessed with seahorse respirometry. Transgenic mice were used to assess CES1 overexpression in DCs in inflammatory models. CES1 expression peaks early during MoDCs differentiation. Pharmacological inhibition of CES1 led to higher expression of CD209, CD86 and MHCII. WWL113 treated MoDCs secreted higher quantities of IL6, IL8, TNF and IL10 and demonstrated stronger phagocytic ability and higher capacity to polarize Th17 differentiation in autologous DCs-T cells co-culture model. Transcriptomic profiling revealed enrichment of multiple inflammatory and metabolic pathways. Functional metabolic analysis shows impaired maximal mitochondrial respiration capacity, increased lactate production and decreased intracellular amino acids and TCA intermediates. Transgenic human CES1 overexpression in murine DCs generated less inflammatory phenotype and increased resistance to T cell mediated colitis. In conclusion, CES1 inhibition directs DCs differentiation towards more inflammatory phenotype, that shows stronger phagocytic capacity and supports Th17 skewing. This is associated with disrupted mitochondrial respiration and amino acids depletion.

The cholesterol content of the erythrocyte membrane is an important determinant of phosphatidylserine exposure.

Maintenance of the asymmetric distribution of phospholipids across the plasma membrane is a prerequisite for the survival of erythrocytes. Various stimuli have been shown to induce scrambling of phospholipids and thereby exposure of phosphatidylserine (PS). In two types of patients, both with aberrant plasma cholesterol levels, we observed an aberrant PS exposure in erythrocytes upon stimulation. We investigated the effect of high and low levels of cholesterol on the ATP-dependent flippase, which maintains phospholipid asymmetry, and the ATP-independent scrambling activity, which breaks down phospholipid asymmetry. We analyzed erythrocytes of a patient with spur cell anemia, characterized by elevated plasma cholesterol, and the erythrocytes of Tangier disease patients with very low levels of plasma cholesterol. In normal erythrocytes, loaded with cholesterol or depleted of cholesterol in vitro, the same analyses were performed. Changes in the cholesterol/phospholipid ratio of erythrocytes had marked effects on PS exposure upon cell activation. Excess cholesterol profoundly inhibited PS exposure, whereas cholesterol depletion led to increased PS exposure. The activity of the ATP-dependent flippase was not changed, suggesting a major influence of cholesterol on the outward translocation of PS. The effects of cholesterol were not accompanied by eminent changes in cytoskeletal and membrane proteins. These findings emphasize the importance of cholesterol exchange between circulating plasma and the erythrocyte membrane as determinant for phosphatidylserine exposure in erythrocytes.

Potassium leakage primes stored erythrocytes for phosphatidylserine exposure and shedding of pro-coagulant vesicles.

During storage, erythrocytes undergo changes that alter their clearance and function after transfusion and there is increasing evidence that these changes contribute to the complications observed in transfused patients. Stored erythrocytes were incubated overnight at 37°C to mimic the temperature after transfusion. After incubation, several markers for erythrocyte damage were analysed. After overnight incubation, stored erythrocytes showed increased potassium leakage, haemolysis, PS exposure and vesicle formation, and all these effects increased with increasing storage time. Furthermore, we demonstrated that long-term stored erythrocytes develop decreased flippase activity and increased scrambling activity after overnight incubation, leading to PS exposure and the release of vesicles. Reduced intracellular potassium was identified as the cause of the decreased flippase activity. Lastly, we provide evidence that erythrocytes can return to a PS-negative state by shedding parts of their membrane as PS-containing vesicles and that these vesicles can serve as a platform for the coagulation cascade. These findings reveal that potassium leakage, a well-known phenomenon of prolonged erythrocyte storage, primes erythrocytes for PS exposure. PS exposure will lead to vesicle formation and might have an important impact on the post-transfusion function and side effects of stored erythrocytes.

Effects of a hypercaloric diet on ß-cell responsivity in lean healthy men.

OBJECTIVE: Insulin resistance and hyperinsulinaemia precede the onset of obesity-induced DM2. The early adaptation of the ß-cell during the initial phase of overfeeding and weight gain has only been partly elucidated. We studied the early changes in insulin clearance and ß-cell responsivity during a positive and negative energy balance in lean healthy men. DESIGN: We studied in nine healthy lean men [age, 37 (27-43) years; BMI, 23·6 (20·6-25·6) kg/m(2) ] insulin sensitivity, insulin clearance, insulin secretion and static and dynamic ß-cell responsivity at baseline and after the hypercaloric and subsequent hypocaloric diet. RESULTS: Participants gained 7 [5·1-7·6]% of their initial body weight on the hypercaloric diet. Compared to baseline, insulin sensitivity and insulin clearance decreased, while glucose-stimulated insulin secretion was higher. The GLP-1 response to oral glucose did not change. The dynamic ß-cell responsivity index increased but the basal and static responsivity indexes did not change. Total and static disposition indexes (DIs) in the hypercaloric state showed a trend towards a decrease. During the hypocaloric diet, insulin sensitivity, glucose-stimulated insulin secretion and insulin clearance returned to baseline. The responsivity and the DIs were not different in the hypocaloric phase compared to baseline. CONCLUSION: A positive energy balance resulting in weight gain in lean men induces hyperinsulinaemia, which is explained by a combined effect on insulin clearance and insulin secretion. Increased insulin secretion was related to insulin resistance-induced higher glucose concentrations but also to increased dynamic ß-cell responsivity. Glucose sensitivity of the ß-cell did not change. These early adaptations are completely reversible during a negative energy balance after loss of the gained weight.

Interorgan coordination of the murine adaptive response to fasting.

Starvation elicits a complex adaptive response in an organism. No information on transcriptional regulation of metabolic adaptations is available. We, therefore, studied the gene expression profiles of brain, small intestine, kidney, liver, and skeletal muscle in mice that were subjected to 0-72 h of fasting. Functional-category enrichment, text mining, and network analyses were employed to scrutinize the overall adaptation, aiming to identify responsive pathways, processes, and networks, and their regulation. The observed transcriptomics response did not follow the accepted "carbohydrate-lipid-protein" succession of expenditure of energy substrates. Instead, these processes were activated simultaneously in different organs during the entire period. The most prominent changes occurred in lipid and steroid metabolism, especially in the liver and kidney. They were accompanied by suppression of the immune response and cell turnover, particularly in the small intestine, and by increased proteolysis in the muscle. The brain was extremely well protected from the sequels of starvation. 60% of the identified overconnected transcription factors were organ-specific, 6% were common for 4 organs, with nuclear receptors as protagonists, accounting for almost 40% of all transcriptional regulators during fasting. The common transcription factors were PPARα, HNF4α, GCRα, AR (androgen receptor), SREBP1 and -2, FOXOs, EGR1, c-JUN, c-MYC, SP1, YY1, and ETS1. Our data strongly suggest that the control of metabolism in four metabolically active organs is exerted by transcription factors that are activated by nutrient signals and serves, at least partly, to prevent irreversible brain damage.

Collection and storage of red blood cells with anticoagulant and additive solution with a physiologic pH.

BACKGROUND: A donation of whole blood is most commonly collected in acidic citrate-phosphate-dextrose (CPD) variants with pH 5.2 to 6.2 as anticoagulants. Previously, we have shown that the initial pH after red blood cell (RBC) preparation can have an effect on RBCs during storage. First, we investigated the effect of the pH of the anticoagulant on RBCs. Second, we investigated the possibility of decreasing the pH of our new additive solution (AS) phosphate-adenine-glucose-guanosine-gluconate-mannitol (PAGGGM) from pH 8.2 to 7.4 in combination with an anticoagulant with a physiologic pH. STUDY DESIGN AND METHODS: Whole blood was collected in CPD (pH 5.6) or trisodiumcitrate (TNC; pH 7.4), and leukoreduced units were prepared using saline-adenine-glucose-mannitol as AS. Second, whole blood was collected in TNC (pH 7.4), and leukoreduced units were prepared using PAGGGM (pH 7.4) or PAGGGM (pH 8.2) as AS. During cold storage, several in vitro characteristics were analyzed. RESULTS: In agreement with our previous findings, the initial pH of whole blood has an effect during storage of RBCs. In the second part we show that there are no differences between PAGGGM (pH 7.4) and PAGGGM (pH 8.2) units when an anticoagulant with a physiologic pH was used. CONCLUSION: These results indicate that the pH of the anticoagulant used during whole blood collection has an effect during storage of RBCs. When an anticoagulant with a physiologic pH is used during whole blood collection, the pH of PAGGGM can be decreased to physiologic levels, while maintaining adenosine triphosphate and 2,3-diphosphoglycerate levels.

Role of mitochondrial function in insulin resistance.

The obesity pandemic increases the prevalence of type 2 diabetes (DM2).DM2 develops when pancreatic ß-cells fail and cannot compensate for the decrease in insulin sensitivity. How excessive caloric intake and weight gain cause insulin resistance has not completely been elucidated.Skeletal muscle is responsible for a major part of insulin stimulated whole-body glucose disposal and, hence, plays an important role in the pathogenesis of insulin resistance.It has been hypothesized that skeletal muscle mitochondrial dysfunction is involved in the accumulation of intramyocellular lipid metabolites leading to lipotoxicity and insulin resistance. However, findings on skeletal muscle mitochondrial function in relation to insulin resistance in human subjects are inconclusive. Differences in mitochondrial activity can be the result of several factors, including a reduced mitochondrial density, differences in insulin stimulated mitochondrial respiration, lower energy demand or reduced skeletal muscle perfusion, besides an intrinsic mitochondrial defect. The inconclusive results may be explained by the use of different techniques and study populations. Also, mitochondrial capacity is in far excess to meet energy requirements and therefore it may be questioned whether a reduced mitochondrial capacity limits mitochondrial fatty acid oxidation. Whether reduced mitochondrial function is causally related to insulin resistance or rather a consequence of the sedentary lifestyle remains to be elucidated.

LAD-1/variant syndrome is caused by mutations in FERMT3.

Leukocyte adhesion deficiency-1/variant (LAD1v) syndrome presents early in life and manifests by infections without pus formation in the presence of a leukocytosis combined with a Glanzmann-type bleeding disorder, resulting from a hematopoietic defect in integrin activation. In 7 consanguineous families, we previously established that this defect was not the result of defective Rap1 activation, as proposed by other investigators. In search of the genetic defect, we carried out homozygosity mapping in 3 of these patients, and a 13-Mb region on chromosome 11 was identified. All 7 LAD1v families share the same haplotype, in which 3 disease-associated sequence variants were identified: a putative splice site mutation in CALDAGGEF1 (encoding an exchange factor for Rap1), an intronic 1.8-kb deletion in NRXN2, and a premature stop codon (p.Arg509X) in FERMT3. Two other LAD1v patients were found to carry different stop codons in FERMT3 (p.Arg573X and p.Trp229X) and lacked the CALDAGGEF1 and NRXN2 mutations, providing convincing evidence that FERMT3 is the gene responsible for LAD1v. FERMT3 encodes kindlin-3 in hematopoietic cells, a protein present together with integrins in focal adhesions. Kindlin-3 protein expression was undetectable in the leukocytes and platelets of all patients tested. These results indicate that the LAD1v syndrome is caused by truncating mutations in FERMT3.