Raquel Reyes

Carcinoma‐associated fibroblasts derive from mesothelial cells via mesothelial‐to‐mesenchymal transition in peritoneal metastasis

Peritoneal dissemination is a frequent metastatic route for cancers of the ovary and gastrointestinal tract. Tumour cells metastasize by attaching to and invading through the mesothelial cell (MC) monolayer that lines the peritoneal cavity. Metastases are influenced by carcinoma‐associated fibroblasts (CAFs), a cell population that derives from different sources. Hence, we investigated whether MCs, through mesothelial–mesenchymal transition (MMT), were a source of CAFs during peritoneal carcinomatosis and whether MMT affected the adhesion and invasion of tumour cells. Biopsies from patients with peritoneal dissemination revealed the presence of myofibroblasts expressing mesothelial markers in the proximity of carcinoma implants. Prominent new vessel formation was observed in the peritoneal areas harbouring tumour cells when compared with tumour‐free regions. The use of a mouse model of peritoneal dissemination confirmed the myofibroblast conversion of MCs and the increase in angiogenesis at places of tumour implants. Treatment of omentum MCs with conditioned media from carcinoma cell cultures resulted in phenotype changes reminiscent of MMT. Adhesion experiments demonstrated that MMT enhanced the binding of cancer cells to MCs in a β1‐integrin‐dependent manner. Scanning electron microscopy imaging showed that the enhanced adhesion was mostly due to increased cell–cell interaction and not to a mere matrix exposure. Invasion assays suggested a reciprocal stimulation of the invasive capacity of tumour cells and MCs. Our results demonstrate that CAFs can derive from mesothelial cells during peritoneal metastasis. We suggest that MMT renders the peritoneum more receptive for tumour cell attachment/invasion and contributes to secondary tumour growth by promoting its vascularization. Copyright

Depletion of T-cell intracellular antigen proteins promotes cell proliferation

BackgroundT-cell intracellular antigen-1 (TIA-1) and TIA-1 related/like protein (TIAR/TIAL1), two DNA/RNA binding proteins broadly expressed in eukaryotic cells, participate in the regulation of gene expression through RNA metabolism. Despite the biological relevance of these regulators, there are no genome-wide studies assessing global transcriptomic and phenotypic impacts after changes in the expression and/or function of these proteins.ResultsUsing high-throughput gene expression profiling, we found that the TIA-1/TIAR-depleted cell phenotype is linked to a transcriptome involved in the control of inflammation, cell-cell signaling, immune-suppression, angiogenesis, metabolism and cell proliferation. Induced genes included pro-inflammatory cytokines, inflammatory chemokines, growth-stimulating factors and pro-angiogenic inducers. Repressed genes involved the RAS oncogene family member RAB40B, regulators of cytoskeleton organization and biogenesis and a mitochondrial modulator. Consistent with these observations, depletion of TIA proteins in HeLa cells results in increased cell proliferation, altered cell-cycle and anchorage-independent growth. Mechanistically, the changes associated with the steady-state target mRNA levels regulated by TIA proteins are consistent with overlapping effects on gene basal transcription rate and mRNA turnover.ConclusionsCollectively, our findings suggest a role for TIA proteins as cellular sensors that modulate gene expression control at the transcriptional and post-transcriptional levels, coupling cell proliferation responses and metabolic homeostasis to cell survival and growth.

ALCAM/CD166 adhesive function is regulated by the tetraspanin CD9

ALCAM/CD166 is a member of the immunoglobulin superfamily of cell adhesion molecules (Ig-CAMs) which mediates intercellular adhesion through either homophilic (ALCAM–ALCAM) or heterophilic (ALCAM–CD6) interactions. ALCAM-mediated adhesion is crucial in different physiological and pathological phenomena, with particular relevance in leukocyte extravasation, stabilization of the immunological synapse, T cell activation and proliferation and tumor growth and metastasis. Although the functional implications of ALCAM in these processes is well established, the mechanisms regulating its adhesive capacity remain obscure. Using confocal microscopy colocalization, and biochemical and functional analyses, we found that ALCAM directly associates with the tetraspanin CD9 on the leukocyte surface in protein complexes that also include the metalloproteinase ADAM17/TACE. The functional relevance of these interactions is evidenced by the CD9-induced upregulation of both homophilic and heterophilic ALCAM interactions, as reflected by increased ALCAM-mediated cell adhesion and T cell migration, activation and proliferation. The enhancement of ALCAM function induced by CD9 is mediated by a dual mechanism involving (1) augmented clustering of ALCAM molecules, and (2) upregulation of ALCAM surface expression due to inhibition of ADAM17 sheddase activity.

Knockdown of T-cell intracellular antigens triggers cell proliferation, invasion and tumour growth.

TIA (T-cell intracellular antigen) proteins function as DNA/RNA trans-acting regulators to expand transcriptome and proteome diversity in mammals. In the present paper we report that the stable silencing of TIA1 and/or TIAR/TIAL1 (TIA1-related/like protein 1) expression in HeLa cells enhances cell proliferation, anchorage-dependent and -independent growth and invasion. HeLa cells lacking TIA1 and/or TIAR generate larger and faster-growing epithelial tumours with high rates of proliferation and angiogenesis in nude mice xenografts. Protein array analysis of a collection of human tumours shows that TIA1 and TIAR protein expression is down-regulated in a subset of epithelial tumours relative to normal tissues. Our results suggest a link between the epigenetic control exerted by TIA proteins and the transcriptional and post-transcriptional regulation of a subset of specific genes involved in tumour progression. Taken together, these results are consistent with a role for TIA proteins as growth/tumour-suppressor genes.

Impact of Preemptive Fibrinogen Concentrate on Transfusion Requirements in Liver Transplantation: A Multicenter, Randomized, Double-Blind, Placebo-Controlled Trial

We hypothesized that preemptive fibrinogen administration to obtain an initial plasma level of 2.9 g/L would reduce transfusion requirements in liver transplantation. A randomized, multicenter, hemoglobin‐stratified, double‐blind, fibrinogen‐versus‐saline–controlled trial was conducted. The primary end point was the percentage of patients requiring red blood cells. We evaluated 51 patients allocated to fibrinogen and 48 allocated to saline; the primary end point was assessed using data for 92 patients because the electronic record forms were offline for three patients in the fibrinogen group and four in the saline group. We injected a median of 3.54 g fibrinogen preemptively in the fibrinogen group. Nine patients in the saline group (20.9%) required fibrinogen at graft reperfusion (compared with one patient [2.1%] in the fibrinogen group; p = 0.005). Blood was transfused to 52.9% (95% confidence interval [CI] 42.5–63.3%) in the fibrinogen group and 42.74% (95% CI 28.3–57.2%) in the saline group (p = 0.217). Relative risk for blood transfusion was 0.80 (95% CI 0.57–1.13). Thrombotic events occurred in one patient (2.1%) and five patients (11.4%) in the fibrinogen and saline groups, respectively. Seven patients (14.6%) in the fibrinogen group and nine (20.3%) in the saline group required reoperation. Preemptive administration of fibrinogen concentrate did not influence transfusion requirements.

Aporte de calcio, magnesio y sodio a través del agua embotellada y de las aguas de consumo público: implicaciones para la salud

Fundamento y objetivo: El aporte de calcio (Ca2+) en la dieta se obtiene, en su mayor parte, mediante el consumo de productos lacteos. Sin embargo, existen otras fuentes, como el agua, que pueden contribuir en su ingesta. Ademas, el agua contiene otros minerales, como el magnesio (Mg2+) y el sodio (Na+), con efectos potenciales para la salud. Asi, el aumento de Na+ en la ingesta contribuye al desarrollo de hipertension, mientras que el aumento del Mg2+ se ha relacionado con una disminucion de muerte subita. El creciente consumo de agua embotellada en la poblacion espanola indica la necesidad de conocer sus posibles efectos en la salud, ya que puede haber una gran variabilidad en las concentraciones de los minerales dependiendo del tipo de agua consumida. El objetivo de este trabajo fue revisar las concentraciones de Ca2+, Mg2+ y Na+ en las aguas de consumo publico y en las envasadas comercializadas y compararlas con los objetivos nutricionales de estos minerales. Metodologia: Se revisan los datos analiticos relativos al Ca2+, Mg2+ y Na+ de las aguas de consumo publico de 492 poblaciones espanolas, de 122 aguas envasadas inscritas en el Registro Sanitario de Alimentos de la Direccion General de Salud Publica y de 60 aguas envasadas europeas. Se comparan los resultados con los objetivos nutricionales de estos minerales. Resultados: Hay una gran variabilidad en las concentraciones de minerales de las diferentes aguas envasadas y en las aguas de consumo publico. Asi, en las aguas envasadas inscritas en nuestro pais, la concentracion de Ca2+ oscila entre 0,5 y 672 mg/l, el 16% tenia una concentracion de Ca2+ > 100 mg/l y dos tenian una concentracion > 300 mg/l. Algunas aguas europeas tenian concentraciones muy altas de Ca2+ (459-575 mg/l); las concentraciones de Na+ oscilan entre 0,1 y 2.000 mg/l y las de Mg2+, entre 0,1 y 128 mg/l. En las aguas de consumo publico, las concentraciones de Ca2+ oscilan entre 0 y 337 mg/l; las de Na+, entre 1 y 332 mg/l, y las de Mg2+, entre 0,3 y 315 mg/l. El 33,4% de las aguas de consumo publico tenian una concentracion de Ca2+ > 100 mg/l, y en 4 fue > 200 mg/l. Conclusiones: El agua, tanto envasada como de consumo publico, presenta una gran variabilidad en las concentraciones de Ca2+, Mg2+ y Na+. En ocasiones, el agua incluso puede suministrar los objetivos nutricionales minimos de Ca2+ y Mg2+ y exceder los de Na+. Estos datos, dadas sus repercusiones en la salud, deberian tenerse en cuenta a la hora de seleccionar el agua para el consumo.

RIAM (Rap1-interacting adaptor molecule) regulates complement-dependent phagocytosis

Phagocytosis mediated by the complement receptor CR3 (also known as integrin αMß2 or Mac-1) is regulated by the recruitment of talin to the cytoplasmic tail of the ß2 integrin subunit. Talin recruitment to this integrin is dependent on Rap1 activation. However, the mechanism by which Rap1 regulates this event and CR3-dependent phagocytosis remains largely unknown. In the present work, we examined the role of the Rap1 effector RIAM, a talin-binding protein, in the regulation of complement-mediated phagocytosis. Using the human myeloid cell lines HL-60 and THP-1, we determined that knockdown of RIAM impaired αMß2 integrin affinity changes induced by stimuli fMLP and LPS. Phagocytosis of complement-opsonized RBC particles, but not of IgG-opsonized RBC particles, was impaired in RIAM knockdown cells. Rap1 activation via EPAC induced by 8-pCPT-2′-O-Me-cAMP resulted in an increase of complement-mediated phagocytosis that was abrogated by knockdown of RIAM in HL-60 and THP-1 cell lines and in macrophages derived from primary monocytes. Furthermore, recruitment of talin to ß2 integrin during complement-mediated phagocytosis was reduced in RIAM knockdown cells. These results indicate that RIAM is a critical component of the phagocytosis machinery downstream of Rap1 and mediates its function by recruiting talin to the phagocytic complement receptors.

Prolonged bisphosphonate release after treatment in women with osteoporosis. Relationship with bone turnover

Bisphosphonates (BP), especially alendronate and risedronate, are the drugs most commonly used for osteoporosis treatment, being incorporated into the skeleton where they inhibit bone resorption and are thereafter slowly released during bone turnover. However, there are few data on the release of BP in patients who have received treatment with these drugs for osteoporosis. This information is essential for evaluating the possibility of BP cyclic therapy in these patients and for controlling their long-term presence in bone tissue. This study evaluated the urinary excretion of alendronate and risedronate in patients treated with these drugs for osteoporosis and analysed its relationship with bone turnover, time of previous drug exposure and time of treatment discontinuation. We included 43 women (aged 65±9.4 years) previously treated with alendronate (36) or risedronate (7) during a mean of 51±3 and 53±3 months, respectively, who had not been treated with other antiosteoporotic treatment and with a median time of discontinuation of 13.5 and 14 months, respectively. Both BP were detected in 24-hour urine by HPLC. In addition, bone formation (PINP) and resorption (NTx) markers were analysed. Both BP were also determined in a control group of women during treatment. Alendronate was detected in 41% of women previously treated with this drug whereas no patient previously treated with risedronate showed detectable urinary values. All control patients showed detectable values of both BP. In patients with detectable alendronate levels, the time of drug cessation was shorter than in patients with undetectable values (12 [6-19] versus 31 [7-72] months, p<0.001). Alendronate was not detected in any patient 19 months after treatment cessation. Alendronate levels were inversely related to time of treatment discontinuation (r=-0.403, p=0.01) and the latter was directly related to NTx (r=0.394, p=0.02). No relationship was observed with age, length of drug exposure, renal function or weight. In conclusion, contrary to risedronate, which was not detected in patients after cessation of treatment, alendronate was frequently detected in women previously treated with this agent up to 19 months after discontinuation of therapy. The relationship between alendronate levels and both bone resorption and time of treatment cessation further indicates a residual effect of this drug in bone, despite treatment discontinuation.

Different states of integrin LFA-1 aggregation are controlled through its association with tetraspanin CD9

The tetraspanin CD9 has been shown to interact with different members of the β1 and β3 subfamilies of integrins, regulating through these interactions cell adhesion, migration and signaling. Based on confocal microscopy co-localization and on co-immunoprecipitation results, we report here that CD9 associates with the β2 integrin LFA-1 in different types of leukocytes including T, B and monocytic cells. This association is resistant to stringent solubilization conditions which, together with data from chemical crosslinking, in situ Proximity Ligation Assays and pull-down experiments, suggest a primary/direct type of interaction mediated by the Large Extracellular Loop of the tetraspanin. CD9 exerts inhibitory effects on the adhesive function of LFA-1 and on LFA-1-dependent leukocyte cytotoxic activity. The mechanism responsible for this negative regulation exerted by CD9 on LFA-1 adhesion does not involve changes in the affinity state of this integrin but seems to be related to alterations in its state of aggregation.

Effects of intravenous pamidronate on renal function, bone mineral metabolism and bone mass in patients with severe osteoporosis.

Introduction:To analyze the effects of intravenous pamidronate (APD) on bone remodelling, bone mineral density (BMD), fractures and bone mineral metabolism parameters, and the rate of adverse events, with special attention to renal function, in patients with osteoporosis with intolerance and/or any contraindication to oral bisphosphonates. Methods:We analyzed prospectively 17 osteoporotic patients (age, 66.8 ± 9.4 years): 65% women, 82% with prevalent vertebral fractures. All patients received APD therapy (30 mg intravenously every 3 months) and were followed up for 1 year. We analyzed serum amino-terminal propeptide of type I procollagen and urinary N-terminal cross-linked telopeptide of type I collagen (as markers of bone turnover), serum calcium, phosphate, parathormone, 25OH-vitamin D, creatinine, and the creatinine clearance: at baseline, 1 week after starting APD treatment, and thereafter for every 3 months (before infusion) during 1 year. We also analyzed lumbar and femoral BMD at baseline and after 1 year, the incidence of new fractures, and the treatment-related adverse events. Results:One week after starting APD treatment, a significant decrease of N-terminal cross-linked telopeptide of type I collagen (32%) (P < 0.05) and an increase of parathormone values (72%) (P < 0.01) were observed, without significant differences found thereafter. No significant differences were observed in BMD evolution and in the other parameters analyzed throughout the study, nor in impairment of renal function. Sixty-four percent of patients suffered new skeletal fractures, 41% of patients showed flu-like syndrome after APD infusion, and 1 patient was withdrawn from treatment because of adverse events. Conclusion:Patients with severe osteoporosis receiving APD infusions had a high rate of fractures without significant changes in bone mass or in bone markers; nevertheless, such a therapeutic regimen showed a good renal safety profile, suggesting that APD at this dosage is safe but ineffective for treating severe osteoporosis.