Non-linear models for black carbon exposure modelling using air pollution datasets.

Black carbon (BC) is a product of incomplete combustion, present in urban aerosols and sourcing mainly from road traffic. Epidemiological evidence reports positive associations between BC and cardiovascular and respiratory disease. Despite this, BC is currently not regulated by the EU Air Quality Directive, and as a result BC data are not available in urban areas from reference air quality monitoring networks in many countries. To fill this gap, a machine learning approach is proposed to develop a BC proxy using air pollution datasets as an input. The proposed BC proxy is based on two machine learning models, support vector regression (SVR) and random forest (RF), using observations of particle mass and number concentrations (N), gaseous pollutants and meteorological variables as the input. Experimental data were collected from a reference station in Barcelona (Spain) over a 2-year period (2018-2019). Two months of additional data were available from a second urban site in Barcelona, for model validation. BC concentrations estimated by SVR showed a high degree of correlation with the measured BC concentrations (R2 = 0.828) with a relatively low error (RMSE = 0.48 µg/m3). Model performance was dependent on seasonality and time of the day, due to the influence of new particle formation events. When validated at the second station, performance indicators decreased (R2 = 0.633; RMSE = 1.19 µg/m3) due to the lack of N data and PM2.5 and the smaller size of the dataset (2 months). New particle formation events critically impacted model performance, suggesting that its application would be optimal in environments where traffic is the main source of ultrafine particles. Due to its flexibility, it is concluded that the model can act as a BC proxy, even based on EU-regulatory air quality parameters only, to complement experimental measurements for exposure assessment in urban areas.

Reconstruction of phthalate exposure and DINCH metabolites from biomonitoring data from the EXHES cohort of Tarragona, Spain: A case study on estimated vs reconstructed DEHP using the PBPK model.

Phthalates are known endocrine disruptors (EDs) and are associated with potential diseases, such as obesity and diabetes. In 2002, the plasticizer 1,2-cyclohexane dicarboxylic acid diisononyl ester (DINCH) was introduced as an alternative to phthalates in the European market. The objective of this study was to evaluate the total exposure to phthalate and DINCH metabolites from EXHES Tarragona, Spain cohort of pregnant women. On the one hand, the analytical determination of phthalate and DINCH metabolites in urine was carried out. On the other hand, the reconstructed exposure was calculated for phthalates and DINCH using their metabolites concentration measured in the urine. Thirteen different phthalate metabolites and two metabolites of DINCH were measured and detected in almost all pregnant women's urine samples (n = 60). There were significant correlations between metabolites of the same parent compounds, and also between DEHP and MBzP metabolites, DiNP and BBZP metabolites, and DEHP and DiNP metabolites respectively. The exposure of pregnant women to phthalate and DINCH parent compounds were also back calculated using the levels of each metabolite found in pregnant women urine (reconstructed exposure). Besides, to demonstrate the utility of this approach, the physiologically based pharmacokinetic (PBPK) model was used to predict the cumulative amount of MEHP (a principal metabolite of DEHP in urine). To proceed with that, DEHP reconstructed exposure and estimated exposure from the same cohort (previously studied by the same authors) were simulated using the PBPK model. Results showed that the reconstructed-PBPK simulation was closer to the 24 h biomonitoring data than the estimated PBPK-simulation., This clearly shows that the combination of reconstructed exposure with the PBPK model is a good tool to predict chemicals exposure. However, some discrepancies between simulated and biomonitored values were found. This can be associated with other sources that contribute to the total exposure and emphasises the need to consider multi-routes exposure for the widely distributed chemicals like phthalates and DINCH.

Comparing dietary and non-dietary source contribution of BPA and DEHP to prenatal exposure: A Catalonia (Spain) case study.

Bisphenol A (BPA) and Di-(2-ethylhexyl) phthalate (DEHP) are two wide spread chemicals classified as endocrine disruptors (ED). The present study aims to estimate the non-dietary (dermal, non-dietary ingestion and inhalation) exposure to BPA and DEHP for a pregnant women cohort. In addition, to assess the prenatal exposure for the fetus, a physiologically based pharmacokinetic (PBPK) model was used. It was adapted for pregnancy in order to assess the internal dosimetry levels of EDs (BPA and DEHP) in the fetus. Estimates of exposure to BPA and DEHP from all pathways along with their relative importance were provided in order to establish which proportion of the total exposure came from diet and which came from non-dietary exposures. In this study, the different oral dosing scenarios (dietary and non-dietary) were considered keeping inhalation as a continuous exposure case. Total non-dietary mean values were 0.002 µg/kgbw/day (0.000; 0.004 µg/kgbw/day for 5th and 95th percentile, respectively) for BPA and 0.597 µg/kgbw/day (0.116 µg/kgbw/day and 1.506 µg/kgbw/day for 5th and 95th percentile, respectively) for DEHP. Indoor environments and especially dust ingestion were the main non-dietary contributors to the total exposure of BPA and DEHP with 60% and 81%. However, as expected, diet showed the higher contribution to total exposure with > 99.9% for BPA and 63% for DEHP. Although diet was considered the primary source of exposure to BPA and phthalates, it must be taken into account that with non-dietary sources the first-pass metabolism is lacking, so these may be of equal or even higher toxicological relevance than dietary sources. The present study is in the framework of "Health and environmental-wide associations based on large population surveys" (HEALS) project (FP7-603946).

Clinicobiological features and prognostic impact of diffuse large B-cell lymphoma component in the outcome of patients with previously untreated follicular lymphoma.

BACKGROUND: The co-existence at diagnosis of follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) components (FL/DLBCL) has been considered a transformed lymphoma and accordingly treated although clinicobiological information on these patients is scarce. The aim of this study was to analyze the initial features and outcome of FL/DLBCL patients in the rituximab era. PATIENTS AND METHODS: All patients consecutively diagnosed at a single institution with FL/DLBCL (n = 40), as well as those with pure FL (n = 328) or de novo DLBCL (n = 510) as controls. RESULTS: The proportion of the DLBCL component was highly variable (median 50%). In 29 FL/DLBCL cases analyzed, the cell of origin was GCB in 86%, ABC in 10% and unclassifiable in 4%. NOTCH1-2 was mutated in 10% of these cases. The proportion of DLBCL component did not impact on overall survival (OS). Regarding initial characteristics, patients with FL/DLBCL were closer to FL in terms of primary nodal origin, good performance status and advanced stage, whereas the other features were intermediate between FL and DLBCL. FL/DLBCL patients were treated as DLBCL with no further intensification. Complete response and primary refractory rates were 65% and 20%, respectively, with these figures being similar to DLBCL and worse than FL. Progression-free survival and OS were intermediate between FL and DLBCL (5-year OS: 85%, 73% and 63% for FL, FL/DLBCL and DLBCL, respectively). FL/DLBCL histology did not reach independent prognostic value for OS in the multivariate analyses. CONCLUSIONS: The outcome of FL/DLBCL patients is not worse than that of de novo DLBCL. These cases should be treated with immunochemotherapy as DLBCL, but intensification with ASCT may not be necessary. The biological insights of FL/DLBCL warrants further genetic and molecular studies.

Prenatal exposure estimation of BPA and DEHP using integrated external and internal dosimetry: A case study.

Prenatal exposure to Endocrine disruptors (EDs), such as Bisphenol A (BPA) and di (2-ethylhexyl) phthalate (DEHP), has been associated with obesity and diabetes diseases in childhood, as well as reproductive, behavioral and neurodevelopment problems. The aim of this study was to estimate the prenatal exposure to BPA and DEHP through food consumption for pregnant women living in Tarragona County (Spain). Probabilistic calculations of prenatal exposure were estimated by integrated external and internal dosimetry modelling, physiologically based pharmacokinetic (PBPK) model, using a Monte-Carlo simulation. Physical characteristic data from the cohort, along with food intake information from the questionnaires (concentrations of BPA and DEHP in different food categories and the range of the different food ratios), were used to estimate the value of the total dietary intake for the Tarragona pregnancy cohort. The major contributors to the total dietary intake of BPA were canned fruits and vegetables, followed by canned meat and meat products. In turn, milk and dairy products, followed by ready to eat food (including canned dinners), were the most important contributors to the total dietary intake of DEHP. Despite the dietary variations among the participants, the intakes of both chemicals were considerably lower than their respective current tolerable daily intake (TDI) values established by the European Food Safety Authority (EFSA). Internal dosimetry estimates suggest that the plasma concentrations of free BPA and the most important DEHP metabolite, mono (2-ethylhexyl) phthalate (MEHP), in pregnant women were characterized by transient peaks (associated with meals) and short half-lives (< 2h). In contrast, fetal exposure was characterized by a low and sustained basal BPA and MEHP concentration due to a lack of metabolic activity in the fetus. Therefore, EDs may have a greater effect on developing organs in young children or in the unborn child.

Cyclosporine A Inhibits the T-bet-Dependent Antitumor Response of CD8(+) T Cells.

Transplant recipients face an increased risk of cancer compared with the healthy population. Although several studies have examined the direct effects of immunosuppressive drugs on cancer cells, little is known about the interactions between pharmacological immunosuppression and cancer immunosurveillance. We investigated the different effects of rapamycin (Rapa) versus cyclosporine A (CsA) on tumor-reactive CD8(+) T cells. After adoptive transfer of CD8(+) T cell receptor-transgenic OTI T cells, recipient mice received either skin grafts expressing ovalbumin (OVA) or OVA-expressing B16F10 melanoma cells. Animals were treated daily with Rapa or CsA. Skin graft rejection and tumor growth as well as molecular and cellular analyses of skin- and tumor-infiltrating lymphocytes were performed. Both Rapa and CsA were equally efficient in prolonging skin graft survival when applied at clinically relevant doses. In contrast to Rapa-treated animals, CsA led to accelerated tumor growth in the presence of adoptively transferred tumor-reactive CD8(+) OTI T cells. Further analyses showed that T-bet was downregulated by CsA (but not Rapa) in CD8(+) T cells and that cancer cytotoxicity was profoundly inhibited in the absence of T-bet. CsA reduces T-bet-dependent cancer immunosurveillance by CD8(+) T cells. This may contribute to the increased cancer risk in transplant recipients receiving calcineurin inhibitors.

Occupational eye lens doses in interventional cardiology. A multicentric study.

New European regulation regarding radiological protection of workers and more specifically the new occupational dose limit for the eye lens recently reduced to 20 mSv yr(-1) may affect interventional cardiologists. This paper presents a set of measurements of occupational doses performed in five interventional cardiology centres and then compared with the new dose limit. The measurement of occupational doses was performed over the apron at chest level using electronic dosemeters recording H p(10). In one of the centres, scatter dose at goggles was also measured with optically stimulated luminescence dosemeters calibrated in terms of H p(0.07). An average H p(10) over the apron of 46 µSv/procedure was measured for cardiologists. Lower doses were noted in other professionals like second cardiologists, nurses or anaesthetists. Procedures for valvular and other structural heart diseases involved the highest occupational doses, averaging over 100 µSv/procedure. Important differences in occupational doses among centres may be indicative of different radiation protection habits. The new occupational dose limit for the eye lens is likely to be exceeded by those among the interventionalists who do not use protection tools (ceiling suspended screen and/or goggles) even with standard workloads.

Application of protective cultures against Listeria monocytogenes and Campylobacter jejuni in chicken products packaged under modified atmosphere.

To control the growth, or reduce the numbers, of food pathogens such as Listeria monocytogenes and Campylobacter jejuni in chicken products packaged under modified atmosphere (MAP), the effectiveness of protective cultures was evaluated in this study. Leuconostoc pseudomesenteroides PCK18 reduced the counts of L. monocytogenes by 1.22 log cfu/g in chicken burgers under MAP after 24 d. Furthermore, a reduction of 1.16 log cfu/g in C. jejuni together with a delay in the growth of lactic acid bacteria was obtained in chicken legs inoculated with Bifidobacterium longum ssp. longum PCB133 and packaged under MAP after 9 d. The combination and concentration of gases in the MAP doubled the products' shelf-life in comparison with air-packaged samples in both experiments. In conclusion, this study has shown the effectiveness of 2 protective culture strains against 2 foodborne pathogens, resulting in safer products with a longer shelf-life.

Impact of fertilisation practices on soil respiration, as measured by the metabolic index of short-term nitrogen input behaviour.

The main objective of this study was the evaluation of the impact of different sources of organic waste (used as an N source) on soil quality (as measured by CO(2) release) and N transformation processes (available inorganic N forms) in a short-term field study of an almond tree plantation. Three compost types were used as organic fertilisers: EC compost constituted from organic agriculture farm (vegetables and manure), SC compost formed from sewage sludge and pruning waste composted, and XC compost comprised a mixture of composted sewage sludge plus slurry and manure from an intensive pig farm. The two compost doses were compared according to N content, and a high dose (H), corresponding to 210 kg N ha(-1), and a low dose (L), equivalent to 105 kg N ha(-1), were used. In addition, an N rate corresponding to 130 kg N ha(-1), which resulted from the supplementation of NPK mineral fertiliser with compost application at a low dose (mixed fertilisation), was compared in a parallel study. Generally, almost all organically treated soils demonstrated an improvement in the levels of C, N and P, compared to controls (unfertilised soils). In addition, the nitrate content increased, predominating over ammonium content, with the highest values in the soils with the low dose application of SC. Furthermore, soil respiration improved in organically treated soils, which showed different responses according to the organic-exogenous source of the incorporated matter. In contrast, a mineral supplement promoted a decrease in biological activity and resulted in lower CO(2) production in soils with XC and mineral fertiliser. Contrary to the organically treated soil, in soils with mix fertilisation the NH(4)(+)-N was the primary available form of nitrogen. However, the application of SC plus mineral fertiliser to soil caused a positive effect on CO(2) emissions compared to the control soil. Soil respiration behaviour was closely related to the form of inorganic N available in the soils due to the fertilisation practice type (organic or mixed), where both parameters seemed to depend on the mobilisations of cations (Na(+) and Ca(2+)) to the soil solution.

Bisphenol A analogues (BPS and BPF) present a greater obesogenic capacity in 3T3-L1 cell line.

This study was aimed at comparing the toxicity effects on cell viability and the obesogenic activity of Bisphenol A (BPA) and its analogues, Bisphenol S (BPS) and Bisphenol F (BPF), by in vitro assays with a preadipocytic 3T3-L1 cell line. To compare the toxic potential and select the concentrations of each chemical not showing a decrease in cell viability, MTT assay was performed. The cell phenotype was determined in differentiated 3T3-L1 adipocytes by red oil O staining. To determine the expression levels of the different adipogenic proteins the Western Blot test was performed. The results from MTT assay showed a greater toxic effect of BPA - at equal and even lower concentrations-than its analogues. However, BPS followed by BPF showed a greater neutral lipid storage capacity than BPA, which was reflected in the increase of the protein expression of CCAAT/enhancer binding protein α (C/EBPα), peroxisome proliferator-activated receptor gamma γ (PPARγ) and acid-binding protein 4 (FABP4). In summary, these BPA analogues -especially BPS- present a greater endocrine potential activity than BPA.

Species composition and distribution of adult Anopheles (Diptera: Culicidae) in Panama.

Anopheles (Diptera: Culicidae) species composition and distribution were studied using human landing catch data over a 35-yr period in Panama. Mosquitoes were collected from 77 sites during 228 field trips carried out by members of the National Malaria Eradication Service. Fourteen Anopheles species were identified. The highest average human biting rates were recorded from Anopheles (Nyssorhynchus) albimanus (Wiedemann) (9.8 bites/person/night) and Anopheles (Anopheles) punctimacula (Dyar and Knab) (6.2 bites/person/night). These two species were also the most common, present in 99.1 and 74.9%, respectively, of the sites. Anopheles (Nyssorhynchus) aquasalis (Curry) was encountered mostly in the indigenous Kuna Yala Comarca along the eastern Atlantic coast, where malaria case history and average human biting rate (9.3 bites/person/night) suggest a local role in malaria transmission. An. albimanus, An. punctimacula, and Anopheles (Anopheles) vestitipennis (Dyar and Knab) were more abundant during the rainy season (May-December), whereas An. aquasalis was more abundant in the dry season (January-April). Other vector species collected in this study were Anopheles (Kerteszia) neivai (Howard, Dyar, and Knab) and Anopheles (Anopheles) pseudopunctipennis s.l. (Theobald). High diversity of Anopheles species and six confirmed malaria vectors in endemic areas of Panama emphasize the need for more detailed studies to better understand malaria transmission dynamics.

Integration of a Coagulation/Flocculation step in a biological sequencing batch reactor for COD and nitrogen removal of supernatant of anaerobically digested piggery wastewater.

The supernatant from mesophilic anaerobic digestion of piggery wastewater is characterised by a high amount of COD (4.1 g COD L(-1)), ammonium (2.3g NH(4)(+)-NL(-1)) and suspended solids (2.5 g SS L(-1)). This effluent can be efficiently treated by means of a Sequencing Batch Reactor (SBR) strategy for biological COD, SS and nitrogen removal including a Coagulation/Flocculation step. Total COD and SS reduction yields higher than 66% and 74%, respectively, and a total nitrogen removal (via nitrite) of more than 98% were reached when working with HRT 2.7 days, SRT 12 days, temperature 32 degrees C, three aerobic/anoxic periods, without external control of pH and under limited aeration flow. The inhibition of nitrite oxidizing biomass was achieved by the working free ammonia concentration and the restricted air supply (dissolved oxygen concentration below 1 mg O(2)L(-1)). Since a part of the total COD was colloidal and/or refractory, a Coagulation/Flocculation step was implemented inside the SBR operating strategy to meet a suitable effluent quality to be discharged. Several Jar-Tests demonstrated that the optimal concentration of FeCl(3) was 800 mg L(-1). A respirometric assay showed that this coagulant dosage did not affect the biological activity of nitrifying/denitrifying biomass.

Integrating genomic alterations in diffuse large B-cell lymphoma identifies new relevant pathways and potential therapeutic targets.

Genome studies of diffuse large B-cell lymphoma (DLBCL) have revealed a large number of somatic mutations and structural alterations. However, the clinical significance of these alterations is still not well defined. In this study, we have integrated the analysis of targeted next-generation sequencing of 106 genes and genomic copy number alterations (CNA) in 150 DLBCL. The clinically significant findings were validated in an independent cohort of 111 patients. Germinal center B-cell and activated B-cell DLBCL had a differential profile of mutations, altered pathogenic pathways and CNA. Mutations in genes of the NOTCH pathway and tumor suppressor genes (TP53/CDKN2A), but not individual genes, conferred an unfavorable prognosis, confirmed in the independent validation cohort. A gene expression profiling analysis showed that tumors with NOTCH pathway mutations had a significant modulation of downstream target genes, emphasizing the relevance of this pathway in DLBCL. An in silico drug discovery analysis recognized 69 (46%) cases carrying at least one genomic alteration considered a potential target of drug response according to early clinical trials or preclinical assays in DLBCL or other lymphomas. In conclusion, this study identifies relevant pathways and mutated genes in DLBCL and recognizes potential targets for new intervention strategies.

Sirolimus monotherapy as maintenance immunosuppression: single-center experience in 50 kidney transplant patients.

INTRODUCTION: Chronic allograft nephropathy, cardiovascular mortality, and posttransplant malignancy are complications of conventional immunosuppression after kidney transplantation. We reported the feasibility of maintenance monotherapy with sirolimus (SRL) in a pilot experience. The aim was to study safety and feasibility of SRL maintenance monotherapy in 50 kidney transplant patients. METHODS: All patients from our center with at least 6 months follow-up on SRL monotherapy were included. During the first month after start of SRL monotherapy, follow-up visits were performed weekly, then each month for the following 2 months. Each follow-up visit included a physical exam and laboratory screening. RESULTS: Mean follow-up on SRL monotherapy was 34.7 +/- 14.9 months. The time between transplantation until start of monotherapy was 7.7 +/- 3.3 years. No rejections occurred. During follow-up, two patients died of cardiovascular disease (already diagnosed before monotherapy); one, of previously diagnosed posttransplant malignancy and one, of hepatitis C-related liver failure. Glomerular filtration rate (GFR) was 53 mL/min x 1.73 m2 at start of monotherapy and 50 mL/min x 1.73 m2 after 4 years. Proteinuria was 632 +/- 562 mg/24 hours at 4 years. During the follow-up, no significant changes in the lipid profile, glycemia, or hemoglobin occurred. CONCLUSIONS: Sirolimus monotherapy is safe in a selected group of immunological low-risk patients without increasing the risk of rejection.

[Which surgical technique should we perform for benign renal disease in children?]. / Qué técnica quirúrgica hemos de utilizar en la nefrectomía por patología benigna?.

AIM: The aim of this paper is to analyze our experience in different surgical techniques to perform a nephrectomy for benign renal diseases in children. MATERIAL AND METHODS: From 1993 to 2005 we have performed 98 nephrectomies. We have three groups of patients depending on the surgical technique: open nephrectomy (ON), transperitoneal laparoscopic nephrectomy (TLN) and retroperitoneal laparoscopic nephrectomy (RLN). ON was performed in 36 patients. Mean age was 3.3 years. TLN was performed in 39 patients. Mean age was 4.7 years old. RLN was performed in 23 patients. Mean age was 3.6 years old. Criteria to nephrectomy was a renographic function under 19%. We have compared the three surgical techniques in relation with surgical time and mean hospital stay. RESULTS: Mean operative time was 126.2 minutes in ON, 132.3 minutes in TLN and 134.1 minutes in RLN. Mean stay was 5.02 days in ON, 2.35 days in TLN and 1.86 days in RLN. The median hospital stay of the ON group is significantly longer than that of NLT and NR groups (p < 0.05). However, there are no differences related to surgical time between all the groups. CONCLUSIONS: Nephrectomy may be performed for benign disease in children using less invasive surgical techniques. They are associated with minimal morbidity, minimal postoperative discomfort, improve cosmesis and a shorter hospital stay. However, we haven't found differences between TLN and RLN.

Association of Brain-Dead Donors' Terminal Inflammation With Delayed Graft Function in Kidney Transplant Recipients.

BACKGROUND: Systemic inflammation affects kidney function in a wide range of diseases. Even in kidney transplant recipients, higher levels of C-reactive protein (CRP) are invariably associated with both worse short- and long-term graft outcomes. However, little is known about systemic inflammation in kidney donors and, notably, brain death causes a strong systemic inflammatory response. OBJECTIVE: To analyze the role of systemic inflammation of brain-dead donors on short-term kidney graft outcomes (ie, delayed graft function [DGF], defined as the need of dialysis during the first week after transplantation). MATERIALS AND METHODS: Retrospective analysis of clinical and biochemical characteristics of all brain-dead kidney donors generated in the Hospital Clínic of Barcelona in the 2006 to 2015 period (n = 194). Donors who were tested for CRP in the 24 hours before BD declaration were included (n = 97, 50% of initial population). Clinical and biochemical features of their respective recipients (n = 165) were analyzed, comparing recipients who developed DGF (n = 30) with recipients who did not (n = 135). RESULTS: Donors whose recipients later developed DGF had much higher CRP values (10.58 [5.1-18.21] vs 4.81 [1.42-12.2] mg/dL, P = .025). Other characteristics associated with the development of DGF were renal biopsy score and recipient dialysis vintage (P = .025 and P = .002, respectively). In logistic regression analysis, PCR maintained significance in the non-expanded criteria donor (ECD) group (odds ratio [OR], 1.102; P = .027), but it lost significance in the ECD group (P = .67). CONCLUSIONS: Terminal donor CRP was associated with DGF in kidney transplant recipients and proved to be mostly significant in younger donors.

Inhibition of JAK3 and PKC via Immunosuppressive Drugs Tofacitinib and Sotrastaurin Inhibits Proliferation of Human B Lymphocytes In Vitro.

BACKGROUND: Antibody-mediated response in solid organ transplantation is critical for graft dysfunction and loss. The use of immunosuppressive agents partially inhibits the B-lymphocyte response leading to a risk of acute and chronic antibody-mediated rejection. This study evaluated the impact of JAK3 and PKC inhibitors tofacitinib (Tofa) and sotrastaurin (STN), respectively, on B-cell proliferation, apoptosis, and activation in vitro. METHODS: Human B cells isolated from peripheral blood of healthy volunteers were cocultured with CD40 ligand-transfected fibroblasts as feeder cells in the presence of interleukin (IL) 2, IL-10, and IL-21. The cocultures were treated with immunosuppressants Tofa, STN, and rapamycin (as a control), to analyze the proliferation and apoptosis of B cells by means of Cyquant and flow cytometry, respectively. CD27 and IgG staining were applied to evaluate whether treatments modified the activation of B cells. RESULTS: Tofa and STN were able to inhibit B-cell proliferation to the same extent as rapamycin, without inducing cell apoptosis. After 6 days in coculture with feeder cells, all B cells showed CD27 memory B-cell phenotype. None of the immunosuppressive treatments modified the proportion between class-switched and non-class-switched memory B cells observed in nontreated cultures. The high predominance of CD27+CD24+ phenotype was not modified by any immunosuppressive treatment. CONCLUSIONS: Our results show that Tofa and STN can suppress B-cell antibody responses to an extent similar to rapamycin, in vitro; therefore these compounds may be a useful therapy against antibody-mediated rejection in transplantation.

Diadenosine polyphosphates. A novel class of glucose-induced intracellular messengers in the pancreatic beta-cell.

Diadenosine polyphosphates are a group of low-weight compounds that increase after exposure to a wide variety of oxidants and have been suggested to act as "alarmones," alerting the cell to the onset of metabolic stress. We demonstrate here that glucose at concentrations that induce insulin release produce a 30- to 70-fold increase in the concentration of diadenosine triphosphate (Ap3A) and tetraphosphate (Ap4A) in beta-cells. Furthermore, Ap3A and Ap4A, at the concentrations found in glucose-stimulated cells, are effective inhibitors of the ATP-regulated K+ channels when applied to the intracellular side of excised membrane patches from cultured beta-cells. We suggest that Ap3A and Ap4A act as second messengers mediating a glucose-induced blockade of the pancreatic beta-cell ATP-regulated potassium channel.

Mechanisms of glucose hypersensitivity in beta-cells from normoglycemic, partially pancreatectomized mice.

Increased beta-cell sensitivity to glucose precedes the loss of glucose-induced insulin secretion in diabetic animals. Changes at the level of beta-cell glucose sensor have been described in these situations, but it is not clear whether they fully account for the increased insulin secretion. Using a euglycemic-normolipidemic 60% pancreatectomized (60%-Px) mouse model, we have studied the ionic mechanisms responsible for increased beta-cell glucose sensitivity. Two weeks after Px (Px14 group), Px mice maintained normoglycemia with a reduced beta-cell mass (0.88 +/- 0.18 mg) compared with control mice (1.41 +/- 0.21 mg). At this stage, the dose-response curve for glucose-induced insulin release showed a significant displacement to the left (P < 0.001). Islets from the Px14 group showed oscillatory electrical activity and cytosolic Ca2+ ([Ca2+]i) oscillations in response to glucose concentrations of 5.6 mmol/l compared with islets from the control group at 11.1 mmol/l. All the above changes were fully reversible both in vitro (after 48-h culture of islets from the Px14 group) and in vivo (after regeneration of beta-cell mass in islets studied 60 days after Px). No significant differences in the input resistance and ATP inhibition of ATP-sensitive K+ (K(ATP)) channels were found between beta-cells from the Px14 and control groups. The dose-response curve for glucose-induced MTT (C,N-diphenyl-N''-4,5-dimethyl thiazol 2 yl tetrazolium bromide) reduction showed a significant displacement to the left in islets from the Px14 group (P < 0.001). These results indicate that increased glucose sensitivity in terms of insulin secretion and Ca2+ signaling was not due to intrinsic modifications of K(ATP) channel properties, and suggest that the changes are most likely to be found in the glucose metabolism.

A novel real-time PCR assay for the specific identification and quantification of Weissella viridescens in blood sausages.

Weissella viridescens has been identified as one of the lactic acid bacteria (LAB) responsible for the spoilage of "morcilla de Burgos". In order to identify and quantify this bacterium in "morcilla de Burgos", a new specific PCR procedure has been developed. The primers and Taqman probe were designed on the basis of a sequence from the gene recN. To confirm the specificity of the primers, 77 strains from the genera Carnobacterium, Enterococcus, Lactobacillus, Leuconostoc, Pediococcus, Streptococcus, Vagococcus and Weissella were tested by conventional PCR. The specificity of the primers and the correct functioning of the probe was confirmed by performing real-time PCR (qPCR) with 21 W. viridescens strains and 27 strains from other LAB genera. The levels of detection and quantification for the qPCR procedure proposed herein were determined for a pure culture of W. viridescens CECT 283(T) and for "morcilla de Burgos" artificially inoculated with this species. The primers were specific for W. viridescens, with only one product of 91 bp being observed for this species. Similarly, the qPCR reactions were found to be specific, amplifying at a mean CT of 15.0±0.4 only for W. viridescens strains. The limit of detection (LOD) and quantification (LOQ) for this procedure was established in 0.082 pg for genomic DNA from W. viridescens. With regard to the artificially inoculated "morcilla", the limit of quantification was established in 80 CFU/reaction and the limit of detection in 8 CFU/reaction. Consequently, the qPCR developed herein can be considered to be a good, fast, simple and accurate tool for the specific detection and quantification of W. viridescens in meat samples.