Pregnancy outcomes following in vitro fertilization frozen embryo transfer (IVF-FET) with or without preimplantation genetic testing for aneuploidy (PGT-A) in women with recurrent pregnancy loss (RPL): a SART-CORS study.

STUDY QUESTION: Can preimplantation genetic testing for aneuploidy (PGT-A) improve the live birth rate in patients with recurrent pregnancy loss (RPL)? SUMMARY ANSWER: PGT-A use was associated with improved live birth rates in couples with recurrent pregnancy loss undergoing frozen embryo transfer (IVF-FET). WHAT IS KNOWN ALREADY: Euploid embryo transfer is thought to optimize outcomes in some couples with infertility. There is insufficient evidence, however, supporting this approach to management of recurrent pregnancy loss. STUDY DESIGN, SIZE, DURATION: This study included data collected by the Society of Assisted Reproductive Technologies Clinical Outcomes Reporting System (SART-CORS) for IVF-FET cycles between years 2010 through 2016. A total of 12 631 FET cycles in 10 060 couples were included in this analysis designed to assess the utility of PGT-A in couples with RPL undergoing FET, including 4287 cycles in couples with tubal disease who formed a control group. PARTICIPANTS/MATERIALS, SETTING, METHODS: The experimental group included couples with RPL (strictly defined as a history of 3 or more pregnancy losses) undergoing FET with or without PGT-A. The primary outcome was live birth rate. Secondary outcomes included rates of clinical pregnancy, spontaneous abortion, and biochemical pregnancy loss. Differences were analyzed using generalized estimating equations logistic regression models to account for multiple cycles per patient. Covariates included in the model were age, gravidity, geographic region, race/ethnicity, smoking history, and indication for assisted reproductive technologies. Analyses were stratified for age groups as defined by SART: <35 years, 35-37 years, 38-40 years, 41-42 years, and >42 years. MAIN RESULTS AND THE ROLE OF CHANCE: In women with a diagnosis of RPL, the adjusted odds ratio (OR) comparing IVF-FET with PGT-A versus without PGT-A for live birth outcome was 1.31 (95% CI: 1.12, 1.52) for age <35 years, 1.45 (95% CI: 1.21, 1.75) for ages 35-37 years, 1.89 (95% CI: 1.56, 2.29) for ages 38-40, 2.62 (95% CI: 1.94-3.53) for ages 41-42, and 3.80 (95% CI: 2.52, 5.72) for ages >42 years. For clinical pregnancy, the OR was 1.26 (95% CI: 1.08, 1.48) for age <35 years, 1.37 (95% CI: 1.14, 1.64) for ages 35-37 years, 1.68 (95% CI: 1.40, 2.03) for ages 38-40 years, 2.19 (95% CI: 1.65, 2.90) for ages 41-42, and 2.31 (95% CI: 1.60, 3.32) for ages >42 years. Finally, for spontaneous abortion, the OR was 0.95 (95% CI: 0.74, 1.21) for age <35 years, 0.85 (95% CI: 0.65, 1.11) for ages 35-37 years, 0.81 (95% CI: 0.60, 1.08) for ages 38-40, 0.86 (95% CI: 0.58, 1.27) for ages 41-42, and 0.58 (95% CI: 0.32, 1.07) for ages >42 years. LIMITATIONS, REASONS FOR CAUTION: The retrospective collection of data including only women with recurrent pregnancy loss undergoing FET presents a limitation of this study, and results may not be generalizable to all couples with recurrent pregnancy loss. Also, data regarding evaluation and treatment for RPL for the included women is unavailable. WIDER IMPLICATIONS OF THE FINDINGS: This is the largest study to date assessing the utility of PGT-A in women with RPL. PGT-A was associated with improvement in live birth and clinical pregnancy in women with RPL, with the largest difference noted in the group of women with age greater than 42 years. Couples with RPL warrant counseling on all management options to reduce subsequent miscarriage, which may include IVF with PGT-A for euploid embryo selection. STUDY FUNDING/COMPETING INTEREST(S): There are no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Osteoblastic protein tyrosine phosphatases inhibition and connexin 43 phosphorylation by alendronate.

Bisphosphonates (BPs), potent inhibitors of bone resorption which inhibit osteoclasts, have also been shown to act on osteocytes and osteoblasts preventing apoptosis via connexin (Cx) 43 hemichannels and activating the extracellular signal regulated kinases ERKs. We previously demonstrated the presence of a saturable, specific and high affinity binding site for alendronate (ALN) in osteoblastic cells which express Cx43. However, cells lacking Cx43 also bound BPs. Herein we show that bound [(3)H]-alendronate is displaced by phosphatase substrates. Moreover, similar to Na3VO4, ALN inhibited the activity of transmembrane and cytoplasmic PTPs, pointing out the catalytic domain of phosphatases as a putative BP target. In addition, anti-phospho-tyrosine immunoblot analysis revealed that ALN stimulates tyrosine phosphorylation of several proteins of whole cell lysates, among which the major targets of the BP could be immunochemically identified as Cx43. Additionally, the transmembrane receptor-like PTPs, RPTPµ and RPTPα, as well as the cytoplasmic PTP1B, are highly expressed in ROS 17/2.8 cells. Furthermore, we evidenced that Cx43 interacts with RPTPµ in ROS 17/2.8 and ALN decreases their association. These results support the hypothesis that BPs bind and inhibit PTPs associated to Cx43 or not, which would lead to the activation of signaling pathways in osteoblasts.

Cobalt chloride induces metaphase when topically applied to larvae and pupae of the stingless bee Melipona scutellaris (Hymenoptera, Apidae, Meliponini).

In order to optimize preparations of bee metaphases, we tested cobalt chloride, which has been used as a metaphase inducer in other organisms, such as hamsters and fish. Four microliters of 65 mM cobalt chloride aqueous solution was topically applied to larval and pupal stages of the stingless bee Melipona scutellaris. The cerebral ganglion was removed after treatment and prepared for cytogenetic analysis. Identically manipulated untreated individuals were used as controls. The number of metaphases was increased 3-fold in treated individuals compared to controls. The micronucleus test showed no mutagenic effects of cobalt chloride on M. scutellaris cells. We concluded that cobalt chloride is a metaphase-inducing agent in M. scutellaris, thus being useful for cytogenetic analyses.

Role of connexin 43 in the mechanism of action of alendronate: dissociation of anti-apoptotic and proliferative signaling pathways.

Bisphosphonates (BPs) inhibit osteocyte and osteoblast apoptosis via opening of connexin (Cx) 43 hemichannels and activating the extracellular signal regulated kinases ERKs. Previously, we hypothesized that intracellular survival signaling is initiated by interaction of BPs with Cx43. However, using whole cell binding assays with [(3)H]-alendronate, herein we demonstrated the presence of saturable, specific and high affinity binding sites in the Cx43-expressing ROS 17/2.8 osteoblastic cells, authentic osteoblasts and MLO-Y4 cells expressing Cx43 or not, as well as in HeLa cells lacking Cx43 expression and ROS 17/2.8 cells pretreated with agents that disassemble Cx channels. In addition, both BPs and the PTP inhibitor Na(3)VO(4) increased proliferation of cells expressing Cx43 or not. Furthermore, although BPs are internalized and inhibit intracellular enzymes in osteoclasts, whether the drugs penetrate non-resorptive bone cells is not known. To clarify this, we evaluated the osteoblastic uptake of AF-ALN, a fluorescently labeled analog of alendronate. AF-ALN was rapidly internalized in cells expressing Cx43 or not indicating that this process is not mediated via Cx43 hemichannels. Altogether, these findings suggest that although required for triggering intracellular survival signaling by BPs, Cx43 is dispensable for cellular BP binding, its uptake, as well as the proliferative effects of these agents.

First report of fatal autochthonous angiostrongylosis in a dog in Greece.

Canine angiostrongylosis caused by Angiostrongylus vasorum is a life-threatening disease which is emerging in regions of Europe. Thus, there is the merit for a continuous epidemiological surveillance in dog populations. This is the first description of a clinical autochthonous case of canine angiostrongylosis in Greece. A 7-month-old, male, mixed-breed dog was presented with progressively worsening anorexia, respiratory distress, coughing, bleeding diathesis and succumbed four days post admission. Gross post mortem examination revealed numerous nodular fistulated lesions in the lungs, and pulmonary cytology and histopathology showed a verminous pyogranulomatous pneumonia. The definitive diagnosis was based on the morphological identification of first stage larvae (L1) retrieved in the feces by the Baermann method, the detection of the circulating antigen by an in-clinic test and the molecular identification of L1. This report underlines the epidemiological and clinical implications, as well as the infection risks when the index of clinical suspicion is low and the disease is unexpected in a country.

Real-world efficacy and safety of lenvatinib: data from a compassionate use in the treatment of radioactive iodine-refractory differentiated thyroid cancer patients in Italy.

BACKGROUND: Lenvatinib is a multi-kinase inhibitor approved for patients with radioactive iodine (RAI)-resistant differentiated thyroid cancer (DTC). Before the drug approval from the Italian National Regulatory Agency, a compassionate use programme has been run in Italy. This retrospective study aimed to analyse data from the first series of patients treated with lenvatinib in Italy. METHODS: The primary aim was to assess the response rate (RR) and progression-free survival (PFS). Secondary end-points include overall survival (OS) and toxicity data. RESULTS: From November 2014 to September 2016, 94 patients were treated in 16 Italian sites. Seventeen percent of patients had one or more comorbidities, hypertension being the most common (60%). Ninety-eight percent of patients were treated by surgery, followed by RAI in 98% of cases. Sixty-four percent of patients received a previous systemic treatment. Lenvatinib was started at 24 mg in 64 subjects. Partial response and stable disease were observed in 36% and in 41% of subjects, respectively; progression was recorded in 14% of patients. Drug-related side-effects were common; the most common were fatigue (13.6%) and hypertension (11.6%). Overall, median PFS and OS were 10.8 months (95% confidence interval [CI], 7.7-12.6) and 23.8 months (95% CI, 19.7-25.0) respectively. CONCLUSION: Lenvatinib is active and safe in unselected, RAI-refractory, progressive DTC patients in real-life setting. RR and PFS seem to be less favourable than those observed in the SELECT trial, likely due to a negative selection that included heavily pretreated patients or with poor performance status.

Immunogenicity of advanced glycation end products in diabetic patients and in nephropathic non-diabetic patients on hemodialysis or after renal transplantation.

Advanced glycation end products (AGE) increase as a consequence of diabetic hyperglycemia and, in nephropathic patients, following renal function loss. Protein-bound AGE behave as immunogens, inducing formation of specific antibodies (Ab-AGE). In this work AGE immunogenicity was studied in 42 diabetic patients, 26 nephropathic patients on hemodialysis and 26 patients with end-stage renal disease who underwent kidney transplantation and in 20 normal subjects. Non-oxidation-derived AGE (nox-AGE), oxidation-derived AGE (ox-AGE) and Ab-AGE were measured by competitive or direct enzyme-linked immunosorbent assay (ELISA) and circulating immune complexes (CIC) by C1q ELISA. Nox- AGE increased significantly in all patient groups (p < or = 0.05 to < or = 0.0001) except in patients on hemodialysis for less than 6 yr. Ox-AGE were only significantly increased in patients transplanted more than 3 yr previously (p < 0.05). Ab-AGE were significantly lower than controls in both diabetic groups and in patients on hemodialysis for more than 6 yr (p < 0.005 to < 0.0001) and not unlike controls in the other groups. These results demonstrate that hemodialysis or renal transplantation can, initially, reduce either nox- or ox-AGE levels, which however go back to being high in time. Renal transplantation fails to normalize nox-AGE. More importantly, plasma Ab-AGE levels are reduced or unchanged in all patient groups in comparison with controls, despite higher circulating AGE levels. This suggests the importance of tissue-bound AGE as Ab-AGE targets. Additional interventions are needed to control AGE levels in treated nephropathic patients. The search and quantification of specific Ab-AGE would give more meaningful results if performed over specific tissue specimens.

Comparative study of two drying techniques used in radioactive source preparation: freeze-drying and evaporation using hot dry nitrogen jets.

Quantitative solid sources are used widely in the field of radionuclide metrology. With the aim to improve the detection efficiency for electrons and x-rays, a comparative study between two source drying techniques has been undertaken at LNE-Laboratoire National Henri Becquerel (LNE-LNHB, France). In this paper, freeze-drying using commercial equipment is compared with a system of drying using hot jets of nitrogen developed at Institute for Reference Materials and Measurements (IRMM, Belgium). In order to characterize the influence of self-absorption, the detection efficiencies for (51)Cr sources have been measured by coincidence counting and photon spectrometry.

Management of fluid balance in continuous renal replacement therapy: technical evaluation in the pediatric setting.

Fluid overload control and fluid balance management represent very important factors in critically ill children requiring renal replacement therapy. A relatively high fluid volume administration in children and neonates is often necessary to deliver adequate amounts of blood derivatives, vasopressors, antibiotics, and parenteral nutrition. Fluid balance errors during pediatric continuous renal replacement therapy (CRRT) might significantly impact therapy delivery and have been described as potentially lethal. The aim of this study was to evaluate the accuracy of delivered vs. prescribed net ultrafiltration (UF) during CRRT applied to 2 neonates and 2 small children, either as dialytic treatment alone or during extracorporeal membrane oxygenation (ECMO). In accordance with an Acute Dialysis Quality Initiative workgroup statement, net UF was defined as the ""overall amount of fluid extracted from the patient in a given time"". Mean prescribed net UF was 18.5 ml/h (SD=6.7) during neonatal treatments and 70.3 ml/h (SD=22.5) during CRRT in small children. Daily net UF ranged from 200 mL to about 600 mL in the 2 neonates and from 1,200 to 1800 mL in the 2 children. The percentage error of delivered net UF ranged from -1.6% to 5.8% of the prescribed level. The mean error of the ECMO/CRRT patients was 3.024 ml/h vs. 0.45 m/h for the CRRT patients (p<0.001). The same difference was not evident when the 2 neonates were compared with the 2 small children (without considering the presence of ECMO). CRRT and net UF delivery appeared to be accurate, safe, and effective in this small cohort of high-risk pediatric patients.

Evolutionary cytogenetics of the Hoplias lacerdae, Miranda Ribeiro, 1908 group. A particular pathway concerning the other Erythrinidae fish.

The taxonomy/systematics of the Erythrinidae fish is still imprecise, with several doubts on their relationships. Karyotypes and chromosomal characteristics of some species of the Hoplias lacerdae group (Erythrinidae), from different Brazilian hydrographic basins and pisciculture stations, were analyzed in the present study, using conventional Giemsa staining, C-banding, silver staining, Mithramycin and Distamycin/DAPI fluorochromes, and fluorescent in situ hybridization (FISH). A diploid chromosome number of 2n = 50 and karyotypes composed of meta- and submetacentric chromosomes without sex-related differences were found. Only one active NOR (Nucleolar Organizer Region) site was found, which was identified by silver staining (Ag-NOR) and FISH, located on the chromosome pair 11, although additional 45S rDNA sites were also mapped on other chromosome pairs only by FISH. The Ag-NOR of the chromosome pair 11 was found to be GC-rich, appearing positive after Mithramycin staining. Mithramycin-positive/DAPI-negative sites were also observed in the centromeric/pericentomeric regions of the chromosome pairs 4, 6, 15, and 19, which have also affinity to silver nitrate. However, these four sites were not detected by FISH with the rDNA probe, indicating to be only argentophilic GC-rich heterochromatic regions. Chromosome data show that the karyotype evolution in Hoplias lacerdae group is relatively conserved and follows a particular pathway concerning the other Erythrinidae fishes, such as Hoplias malabaricus, Hoplerythrinus unitaeniatus, and Erythrinus erythrinus, in which polytypic karyotypes are found. Thus, the H. lacerdae group shows chromosome features that are not closely related to those of the congeneric H. malabaricus group. These finds, together with genetic and morphologic data, are important tools to be considered in a major revision of the Erythrinidae family, as well as for conservation programs.

Long-term efficacy and safety of fourth-line multikinase inhibitor treatment with lenvatinib in a young papillary thyroid carcinoma patient.

Lenvatinib, a multikinase inhibitor, is approved for the treatment of patients with radioiodine-refractory metastatic thyroid cancer on the basis of a Phase III, prospective, double-blind, randomized, placebo-controlled trial that showed longer progression-free survival in the drug-treated arm. Here, we report the case of a young papillary thyroid cancer patient, pretreated with three other kinase inhibitors, who achieved a long-term clinical benefit from lenvatinib in the fourth-line setting.

The immune response of women with prolonged exposure to electromagnetic fields produced by radiotelevision broadcasting stations.

Twelve women, five of them housewives, exposed in their residences to electromagnetic fields (EMFs)emitted by radio-television broadcasting stations for a mean period of 13 years, were investigated. The EMFs in the balconies of the homes were (mean + S.D.) 4.3 + 1.4 V/m in the year 2000 and 3.7 + 1.3 V/m in 2005, while the exposure in the nearby area was <2.0 V/m. The EMF exposed women showed in 2000 reduced blood NK lymphocytes as well as PHA stimulated PBMC proliferation and IL-2 and IFN-gamma release. In the year 2005, the EMF exposed women and 48 control women with similar ages(mean 43 years), smoking habits, atopy and social level were investigated. State (temporary) and trait(tendency of the personality) anxiety were determined by STAI I and II, respectively. Blood cytotoxic activity and lymphocyte subsets were also determined. The ratio STAI I/STAI II of the EMF exposed group was lower than that of the control group. The blood cytotoxic activity of the exposed women was lower (p<0.01), percent of B CD45+-CD19+ lymphocytes higher and percent of CD45+-CD3+-CD8+ cells lower (p<0.05). Moreover, cytotoxic activity/CD45+-CD16+-56+ NK lymphocytes of the controls was negatively correlated with STAI I and STAI II (p<0.001). In conclusion, this study demonstrates reduced blood cytotoxic activity and increased trait anxiety in relation to state anxiety in EMF exposed women. An effect of EMFs on immune functions, in part mediated by nervous mechanisms, may be hypothesized. However, the influence of lifestyle may not be excluded.

Quality assessment and cataloguing of telemedicine applications.

We created a Web catalogue of approved telemedicine systems that authoritative Italian research bodies had made available for more general use. The evaluation process was divided into two stages: (1) classification of the telemedicine systems and rough preliminary evaluation; (2) assessment of the telemedicine products and services. The scoring method was applied to four well-known telemedicine systems that had been tested in health-care settings: an echocardiology teleconsulting and analysis system; a ward nursing management system; a virtual cooperative system for the management of oncology patients and a telepathology system based on remotely controlled microscopy. After technical revision during the standardization/qualification process, the applications were transferred successfully to eight new health-care facilities. The methodology achieved the main goal of providing effective tools, such as a set of quality control procedures for telemedicine and telehealth projects and a Web catalogue of telemedicine applications with a standardized level of quality, available to all interested parties.

Chromosomal analysis of Astyanax fasciatus (Pisces, Characidae) from the Araguari river, Uberlândia, MG, Brazil.

The genus Astyanax is one of the most numerous of the family Characidae, comprising a large number of similar-shaped species, but displaying innumerable karyotypic variations in its chromosome number and/or structure. The literature describes A. fasciatus populations with diploid chromosome numbers varying from 2n = 45 to 2n = 48. In this study, A. fasciatus specimens captured in the Araguari River (Alto Paraná basin) were cytogenetically characterized, revealing a diploid chromosome number of 2n = 46. The nucleolar organizing regions (NORs), detected with silver nitrate staining, showed a multiple system with two pairs of marked chromosomes. These findings are congruent with those of other studies involving populations of the same species.

Opposite effect of tumor necrosis factor alpha on granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor-dependent growth of normal and leukemic hemopoietic progenitors.

The effect of recombinant human tumor necrosis factor alpha (TNF-alpha) on normal and chronic myeloid leukemia granulocyte-macrophage progenitors (CFU-GM) growing in semisolid agar cultures in the presence of recombinant granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor was studied. Granulocyte-macrophage colony-stimulating factor-dependent growth of normal and chronic myeloid leukemia bone marrow CFU-GM was greatly enhanced by TNF-alpha at doses of 0.1 to 100 units/ml. Growth enhancement included neutrophil, eosinophil, and monocyte-macrophage colonies and clusters at 7 and 14 days of culture. Since similar results were achieved with highly enriched progenitor cell populations, devoid of accessory cells, an indirect effect on CFU-GM growth through the release by accessory cells of other cytokines upon TNF-alpha stimulation was thus ruled out. By contrast, the same doses of TNF-alpha inhibited the growth of normal CFU-GM in granulocyte colony-stimulating factor-dependent cultures. Taken together, our findings indicate that the final effect of TNF-alpha on normal bone marrow granulocyte-macrophage progenitor growth is dependent on the specific growth factor interacting with it, and that both normal and chronic myeloid leukemia CFU-GM are equally responsive to the combined effects of TNF-alpha and a given colony-stimulating factor.

A bcl-2/IgH antisense transcript deregulates bcl-2 gene expression in human follicular lymphoma t(14;18) cell lines.

The 14;18 chromosome translocation, characteristic of most human follicular B-cell lymphomas, juxtaposes the bcl-2 gene with the IgH locus, creating a bcl-2/IgH hybrid gene. By mechanisms that are still under investigation, this event increases the cellular levels of the bcl-2 mRNA and thereby induces an overproduction of the antiapoptotic BCL-2 protein which is likely responsible for neoplastic transformation. In an effort to identify potential upregulators of bcl-2 activity in t(14;18) cells, we found, by strand-specific RT-PCR, a bcl-2 antisense transcript that is present in the t(14;18) DOHH2 and SU-DHL-4 but not in the t(14;18)-negative Raji and Jurkat lymphoid cell lines, and thus appears to be dependent on the bcl-2/IgH fusion. This antisense transcript is a hybrid bc1-2/IgH RNA, that originates in the IgH locus, encompasses the t(14;18) fusion site and spans at least the complete 3' UTR region of the bcl-2 mRNA. To achieve some insight into its biological function, we treated the t(14;18) DOHH2 cell line with oligonucleotides (ODNs) by specifically targeting the bc1-2/IgH antisense strand. These ODNs lowered bcl-2 gene expression, inhibited neoplastic cell growth by inducing apoptosis. We would like to propose the hypothesis that the bc1-2/IgH antisense transcript may contribute, by an unknown mechanism, to upregulation of bcl-2 gene expression in t(14;18) cells. The possibility has been considered that the hybrid antisense transcript mask AU-rich motifs present in the 3' UTR of the bcl-2 mRNA characterized in other genes as mRNA destabilizing elements.

1,25(OH)2-vitamin D-3 stimulates phospholipase A2 activity via a guanine nucleotide-binding protein in chick myoblasts.

The steroid hormone 1,25(OH)2-vitamin D-3 [1,25(OH)2D3] stimulated phospholipase A2 (PLA2) activity in embryonic chick myoblasts releasing [3H]arachidonic acid from the sn-2 position of phospholipids. GTP-binding protein mediation of 1,25(OH)2D3-dependent PLA2 activity was investigated in cells prelabeled with [3H]arachidonic acid. AIF4-, a G-protein activator, mimicked 1,25(OH)2D3-stimulated arachidonic acid release from myoblasts in a dose-dependent manner. Consistent with the involvement of a G-protein in the activation of PLA2 by the hormone, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), a stable GTP analogue which activates G-protein mediated signals, strongly enhanced arachidonic acid release in myoblasts. Guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), which competitively inhibits G-protein activation by GTP and its analogues, abolished 1,25(OH)2D3-dependent arachidonic acid release. Bordetella pertussis toxin pretreatment significantly suppressed the hormone action whereas cholera toxin had minor effects on 1,25(OH)2D3 action. Hormone-induced activation of PLA2 was mimicked by the Ca2+ ionophore A23187 and blocked by nifedipine, but was unaffected by neomycin, a phospholipase C inhibitor, ruling out the contribution of phosphoinositide metabolism to arachidonic acid release. These results suggest that 1,25(OH)2D3-stimulation of PLA2 activity in embryonic chick myoblasts is mediated by a pertussis toxin-sensitive GTP-binding protein coupled to influx of extracellular calcium.

Parathyroid hormone activation of map kinase in rat duodenal cells is mediated by 3',5'-cyclic AMP and Ca(2+).

In a previous study, we demonstrated that parathyroid hormone (PTH) stimulates in rat duodenal cells (enterocytes) the phosphorylation and activity of extracellular signal-regulated mitogen-activated protein kinase (MAPK) isoforms ERK1 and ERK2. As PTH activates adenylyl cyclase (AC) and phospholipase C and increases intracellular Ca(2+) in these cells, in the present study we evaluated the involvement of cAMP, Ca(2+) and protein kinase C (PKC) on PTH-induced MAPK activation. We found that MAPK phosphorylation by the hormone did not depend on PKC activation. PTH response could, however, be mimicked by addition of forskolin (5-15 microM), an AC activator, or Sp-cAMP (50-100 microM), a cAMP agonist, and suppressed to a great extent by the AC inhibitor, compound Sq-22536 (0.2-0.4 mM) and the cAMP antagonist Rp-cAMP (0.2 mM). Removal of external Ca(2+) (EGTA 0.5 mM), chelation of intracellular Ca(2+) with BAPTA (5 microM), or blockade of L-type Ca(2+)-channels with verapamil (10 microM) significantly decreased PTH-activation of MAPK. Furthermore, a similar degree of phosphorylation of MAPK was elicited by the Ca(2+) mobilizing agent thapsigargin, the Ca(2+) ionophore A23187, ionomycin and membrane depolarization with high K(+). Inclusion of the calmodulin inhibitor fluphenazine (50 microM) did not prevent hormone effects on MAPK. Taken together, these results indicate that cAMP and Ca(2+) play a role upstream in the signaling mechanism leading to MAPK activation by PTH in rat enterocytes. As Ca(2+) and cAMP antagonists did not block totally PTH-induced MAPK phosphorylation, it is possible that linking of the hormone signal to the MAPK pathway may additionally involve Src, which has been previously shown to be rapidly activated by PTH. Of physiological significance, in agreement with the mitogenic role of the MAPK cascade, PTH increased enterocyte DNA synthesis, and this effect was blocked by the specific inhibitor of MAPK kinase (MEK) PD098059, indicating that hormone modulation of MAPK through these messenger systems stimulates duodenal cell proliferation.

Diffusive and convective transport through hollow fiber membranes for liver cell culture.

For an efficient membrane bioreactor design, transport phenomena determining the overall mass flux of metabolites, catabolites, cell regulatory factors, and immune-related soluble factors, need to be clarified both experimentally and theoretically. In this work, experiments and calculations aimed at discerning the simultaneous influence of both diffusive and convective mechanisms to the transport of metabolites. In particular, the transmembrane mass flux of glucose, bovine serum albumin (BSA), APO-transferrin, immunoglobulin G, and ammonia was experimentally measured, under pressure and concentration gradients, through high-flux microporous hydrophilic poly-ether-sulphone (PES-HFMs) and poly-sulphone hollow fiber membranes (PS-HFMs). These data were analyzed by means of a model based on the mechanism of capillary pore diffusion, assuming that solute spherical molecules pass through an array of solvent-filled cylindrical pores with a diffusive permeation corrected for friction and steric hindrances. Additionally, resistances to the mass transfer were taken into account. Convective permeation data were discussed in terms of morphological properties of the polymeric membranes, molecular Stokes radius, and solute-membrane interactions according to information given by contact angle measurements. The observed steady-state hydraulic permeance of PS-HFMs was 0.972 L/m2hmbar, about 15.6-fold lower than that measured for PES-HFMs (15.2 L/m2h); in general, PS-HFMs provided a significant hindrance to the transport of target species. Diffusion coefficients of metabolites were found to be similar to the corresponding values in water through PES-HFMs, but significantly reduced through PS-HFMs (D(Glucose)(Membrane)=2.8x10(-6)+/-0.6x10(-6)cm2/s, D(BSA)(Membrane)=6.4 x 10(-7)+/-1 x 10(-7)cm(/s, D(Apotransferrin)(Membrane)=2.3 x 10(-7)+/-0.25 x 10(-7)cm2/s).