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Epsilon toxin (ETX) from Clostridium perfringens is a pore-forming toxin (PFT) that crosses the blood-brain barrier and binds to myelin structures. In in vitro assays, ETX causes oligodendrocyte impairment, subsequently leading to demyelination. In fact, ETX has been associated with triggering multiple sclerosis. Myelin and lymphocyte protein (MAL) is widely considered to be the receptor for ETX as its presence is crucial for the effects of ETX on the plasma membrane of host cells that involve pore formation, resulting in cell death. To overcome the pores formed by PFTs, some host cells produce extracellular vesicles (EVs) to reduce the amount of pores inserted into the plasma membrane. The formation of EVs has not been studied for ETX in host cells. Here, we generated a highly sensitive clone from HeLa cells overexpressing the MAL-GFP protein in the plasma membrane. We observed that ETX induces the formation of EVs. Moreover, the MAL protein and ETX oligomers are found in these EVs, which are a very useful tool to decipher and study the mode of action of ETX and characterize the mechanisms involved in the binding of ETX to its receptor.

The epsilon toxin (Etx) from Clostridium perfringens has been identified as a potential trigger of multiple sclerosis, functioning as a pore-forming toxin that selectively targets cells expressing the plasma membrane (PM) myelin and lymphocyte protein (MAL). Previously, we observed that Etx induces the release of intracellular ATP in sensitive cell lines. Here, we aimed to re-examine the mechanism of action of the toxin and investigate the connection between pore formation and ATP release. We examined the impact of Etx on Xenopus laevis oocytes expressing human MAL. Extracellular ATP was assessed using the luciferin-luciferase reaction. Activation of calcium-activated chloride channels (CaCCs) and a decrease in the PM surface were recorded using the two-electrode voltage-clamp technique. To evaluate intracellular Ca 2+ levels and scramblase activity, fluorescent dyes were employed. Extracellular vesicles were imaged using light and electron microscopy, while toxin oligomers were identified through western blots. Etx triggered intracellular Ca 2+ mobilization in the Xenopus oocytes expressing hMAL, leading to the activation of CaCCs, ATP release, and a reduction in PM capacitance. The toxin induced the activation of scramblase and, thus, translocated phospholipids from the inner to the outer leaflet of the PM, exposing phosphatidylserine outside in Xenopus oocytes and in an Etx-sensitive cell line. Moreover, Etx caused the formation of extracellular vesicles, not derived from apoptotic bodies, through PM fission. These vesicles carried toxin heptamers and doughnut-like structures in the nanometer size range. In conclusion, ATP release was not directly attributed to the formation of pores in the PM, but to scramblase activity and the formation of extracellular vesicles., (© 2024 The Author(s). Pharmacology Research & Perspectives published by British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics and John Wiley & Sons Ltd.)

The objective has been to develop and validate a questionnaire to know patient experience in relation to nursing care during their hospital stay in the Spanish healthcare setting. To know patient experience will improve the quality of care of the healthcare system; therefore, we must count on validated tools so it can be evaluated in an accurate way., Method: a questionnaire containing 29 items alongside socio-demographic questions was developed. It was distributed to 158 patients admitted to a tertiary hospital. The psychometric properties were assessed through principal components analysis and confirmatory factor analysis to evaluate construct validity, employing Cronbach's alpha to test reliability., Results: The final tool contains 17 items grouped into 5 dimensions: interrelations, nursing care, information during hospital stay, information about patient's rights, and discharge information. Two additional questions related to pain were added. The questionnaire showed adequate validity and reliability., Conclusions: we describe a new tool validated and adapted to the Spanish healthcare setting with adequate validity and reliability to assess patient experience with nursing professionals during hospital stay. This tool will serve to identify areas for improvement in hospital nursing care and as an instrument in the management and supervision of nursing teams.

Introduction: The aim of this study is to analyze the expression of the main oxidant scavenger superoxide dismutase (EC-SOD), its main binding protein Fibulin-5 and several oxidative and nitrosative-derived products in the lung of COPD patients and controls., Materials and Methods: Lung tissue samples from 19 COPD patients and 20 control subjects were analyzed. The architecture of elastic fibres was assessed by light and electron microscope histochemical techniques, and levels of EC-SOD and fibulin-5 were analyzed by immunohistochemistry and RT-PCR. The impact of oxidative stress on the extracellular matrix was estimated by immunolocalization of 4-hydroxynonenal (4-HNE), malondialdehyde (MDA) and 3-nitrotyrosine (3-NYT) adducts., Results: Alveolar walls of COPD patients exhibited abnormal accumulations of collapsing elastic fibres, showing a pierced pattern in the amorphous component. The semiquantitative analysis revealed that COPD patients have a significantly reduced expression of both EC-SOD and fibulin-5 (0.59±0.64 and 0.62±0.61, respectively) in alveolar, bronchiolar and arteriolar walls compared to control subjects (1.39±0.63 and 1.55±0.52, respectively, p<0.05). No significant changes in mRNA levels of these proteins were observed between groups. Among the oxidation markers, malondialdehyde was the best in distinguishing COPD patients., Conclusions: COPD patients show a reduced expression of EC-SOD and fibulin-5 in the lung interstitium. Paralleling the reduction of EC-SOD levels, the decrease of fibulin-5 expression in COPD lungs supports the hypothesis of an impaired pulmonary antioxidant response in COPD patients., (Copyright © 2022 SEPAR. Published by Elsevier España, S.L.U. All rights reserved.)

This prospective and comparative study aimed to compare the use of a conjunctival autograft (CAG), plasma rich in growth factors fibrin membrane (mPRGF) or amniotic membrane transplantation (AMT) in primary pterygium surgery. Patients were assigned for surgery with CAG (group A), mPRGF (group B), or AMT (group C). Pterygium recurrence, Best Corrected Visual Acuity (BCVA), graft size (measured with anterior segment optical coherence tomography (AS-OCT)), and ocular surface symptoms (visual analogue scale (VAS) and ocular surface disease index (OSDI)) were evaluated. Thirteen eyes in group A, 26 in group B, and 10 in group C were evaluated. No changes in BCVA ( p > 0.05) were found. Recurrence cases for groups A, B, and C were none, two, and two, respectively, and three cases of pyogenic granulomas in group A. The horizontal/vertical graft size was lower in group B vs group A ( p < 0.05) from months 1 to 12. The improvement in VAS frequency for groups A, B, and C was: 35.5%, 86.2%, and 39.1%, respectively. The OSDI scale reduction for groups A, B, and C was: 12.7%, 39.0%, and 84.1%. The use of the three surgical techniques as a graft for primary pterygium surgery was safe and effective, showing similar results. The mPRGF graft represents an autologous novel approach for pterygium surgery.

Ectonucleoside triphosphate diphosphohydrolase-2 (NTPDase2/CD39L1) has been described in human non-pathological endometrium in both epithelial and stromal components without changes along the cycle. It was identified as a stromal marker of basalis. In the present study, we aimed to evaluate NTPDase2 distribution, using immunolabeling and in situ enzyme activity approaches, in endometrial carcinoma (EC) at different tumor grades. NTPDase2 was present in tumor epithelial EC cells, as in the non-pathological endometria, but the expression underwent changes in subcellular distribution and also tended to decrease with the tumor grade. In stroma, NTPDase2 was identified exclusively at the tumor-myometrial junction but this expression was lost in tumors of invasive phenotype. We have also identified in EC samples the presence of the perivascular population of endometrial mesenchymal stem cells (eMSCs) positive for sushi domain containing 2 (SUSD2) and for NTPDase2, already described in non-tumoral endometrium. Our results point to NTPDase2 as a histopathological marker of tumor invasion in EC, with diagnostic relevance especially in cases of EC coexisting with other endometrial disorders, such as adenomyosis, which occasionally hampers the assessment of tumor invasion parameters.

Endometriosis is an estrogen-dependent gynecological disease, with an associated chronic inflammatory component, characterized by the presence of endometrial tissue outside the uterine cavity. Its predominant symptom is pain, a condition notably altering the quality of life of women with the disease. This review is intended to exhaustively gather current knowledge on purinergic signaling in endometriosis-associated pain. Altered extracellular ATP hydrolysis, due to changes in ectonucleotidase activity, has been reported in endometriosis; the resulting accumulation of ATP in the endometriotic microenvironment points to sustained activation of nucleotide receptors (P2 receptors) capable of generating a persistent pain message. P2X3 receptor, expressed in sensory neurons, mediates nociceptive, neuropathic, and inflammatory pain, and is enrolled in endometriosis-related pain. Pharmacological inhibition of P2X3 receptor is under evaluation as a pain relief treatment for women with endometriosis. The role of other ATP receptors is also discussed here, e.g., P2X4 and P2X7 receptors, which are involved in inflammatory cell-nerve and microglia-nerve crosstalk, and therefore in inflammatory and neuropathic pain. Adenosine receptors (P1 receptors), by contrast, mainly play antinociceptive and anti-inflammatory roles. Purinome-targeted drugs, including nucleotide receptors and metabolizing enzymes, are potential non-hormonal therapeutic tools for the pharmacological management of endometriosis-related pain.

The pore-forming protein epsilon toxin (Etx) from Clostridium perfringens produces acute perivascular edema affecting several organs, especially the brain and lungs. Despite the toxin evident effect on microvasculature and endothelial cells, the underlying molecular and cellular mechanisms remain obscure. Moreover, no Etx-sensitive endothelial cell model has been identified to date. Here, we characterize the mouse lung endothelial cell line 1G11 as an Etx-sensitive cell line and compare it with the well-characterized Etx-sensitive Madin-Darby canine kidney epithelial cell line. Several experimental approaches, including morphological and cytotoxic assays, clearly demonstrate that the 1G11 cell line is highly sensitive to Etx and show the specific binding, oligomerization, and pore-forming activity of the toxin in these cells. Recently, the myelin and lymphocyte (MAL) protein has been postulated as a putative receptor for Etx. Here, we show the presence of Mal mRNA in the 1G11 cell line and the presence of the MAL protein in the endothelium of some mouse lung vessels, supporting the hypothesis that this protein is a key element in the Etx intoxication pathway. The existence of an Etx-sensitive cell line of endothelial origin would help shed light on the cellular and molecular mechanisms underlying Etx-induced edema and its consequences.

Endometriosis is a prevalent disease defined by the presence of endometrial tissue outside the uterus. Adenosine triphosphate (ATP), as a proinflammatory molecule, promotes and helps maintain the inflammatory state of endometriosis. Moreover, ATP has a direct influence on the two main symptoms of endometriosis: infertility and pain. Purinergic signaling, the group of biological responses to extracellular nucleotides such as ATP and nucleosides such as adenosine, is involved in the biology of reproduction and is impaired in pathologies with an inflammatory component such as endometriosis. We have previously demonstrated that ectonucleotidases, the enzymes regulating extracellular ATP levels, are active in non-pathological endometria, with hormone-dependent changes in expression throughout the cycle. In the present study we have focused on the expression of ectonucleotidases by means of immunohistochemistry and in situ activity in eutopic and ectopic endometrial tissue of women with endometriosis, and we compared the results with endometria of women without the disease. We have demonstrated that the axis CD39-CD73 is altered in endometriosis, with loss of CD39 and CD73 expression in deep infiltrating endometriosis, the most severe, and most recurring, endometriosis subtype. Our results indicate that this altered expression of ectonucleotidases in endometriosis boosts ATP accumulation in the tissue microenvironment. An important finding is the identification of the nucleotide pyrophophatase/phosphodiesterase 3 (NPP3) as a new histopathological marker of the disease since we have demonstrated its expression in the stroma only in endometriosis, in both eutopic and ectopic tissue. Therefore, targeting the proteins directly involved in ATP breakdown could be an appropriate approach to consider in the treatment of endometriosis.

Intra-articular (IA) injection of mesenchymal stem cells (MSCs) promotes articular cartilage repair. However, cell fate and action after transplantation remain unclear. This study aimed at evaluating the biodistribution and efficacy of MSCs after IA injection. We used an immunocompetent, dual transgenic rat model, which is based on donor rats ubiquitously expressing heat stable human placental alkaline phosphatase (ALPP), and recipient rats expressing a heat sensitive ALPP form. A focal cartilage defect was created in the patellofemoral groove of recipient rats. Bone marrow-derived MSCs isolated from donor rats were injected into the synovial cavity of recipients, and cell tracking was performed in distant organs and knees over 6 months post-injection. A few donor MSCs were observed in the lung of one of the recipients, 1 day post-injection. We failed to detect donor MSCs in any of the studied tissues at all later time points. IA-injected MSCs remained in the synovial cavity, engrafted within the cartilage lesion, and were detectable up to 1 month post-injection. Although the number of MSCs decreased over time, MSCs injection promoted cartilage regeneration as evidenced by histology and immunofluorescent collagen staining. Our study supports the safety and efficacy of using MSCs for cartilage repair via IA delivery.

COPD patients are prone to acute infectious exacerbations that impair their quality of life and hamper prognosis. The purpose of the present study was to investigate the in situ IFN-β response in the lungs of stable COPD and non-COPD patients. Lung samples from 70 subjects (9 control never smokers, 19 control smokers without COPD, 21 patients with moderate COPD and 21 patients with very severe COPD) were studied for the expression of IFN-β, its main transcription factor, IRF-7, and two products of its autocrine function, namely RIG-I and MDA-5, by immunohistochemical techniques and quantitative real-time PCR. IFN-β, IRF-7, RIG-I and MDA-5 were widely detected in most lung cell types. In epithelial tissues and alveolar macrophages, IFN-β and IRF-7 labeling scores were decreased up to 65% and 74%, respectively, for COPD patients, paralleling an analogous reduction (43% and 65%, respectively) in the amount of their lung mRNA. Moreover, this decreased production of IFN-β in COPD patients correlated with a similar decrease in the expression of RIG-I and MDA-5, two essential members of the innate immune system. Our study indicates that most lung cells from stable COPD patients show a constitutive decreased expression of IFN-β, IRF-7, RIG-I and MDA-5, suggesting that this deficiency is the main cause of their acute viral exacerbations., Competing Interests: The authors have declared that no competing interests exist.

The human endometrium undergoes repetitive regeneration cycles in order to recover the functional layer, shed during menses. The basal layer, which remains in charge of endometrial regeneration in every cycle, contains adult stem or progenitor cells of epithelial and mesenchymal lineage. Some pathologies such as adenomyosis, in which endometrial tissue develops within the myometrium, originate from this layer. It is well known that the balance between adenosine triphosphate (ATP) and adenosine plays a crucial role in stem/progenitor cell physiology, influencing proliferation, differentiation, and migration. The extracellular levels of nucleotides and nucleosides are regulated by the ectonucleotidases, such as the nucleoside triphosphate diphosphohydrolase 2 (NTPDase2). NTPDase2 is a membrane-expressed enzyme found in cells of mesenchymal origin such as perivascular cells of different tissues and the stem cells of adult neurogenic regions. The aim of this study was to characterize the expression of NTPDase2 in human nonpathological cyclic and postmenopausic endometria and in adenomyosis. We examined proliferative, secretory, and atrophic endometria from women without endometrial pathology and also adenomyotic lesions. Importantly, we identified NTPDase2 as the first marker of basal endometrium since other stromal cell markers such as CD10 label the entire stroma. As expected, NTPDase2 was also found in adenomyotic stroma, thus becoming a convenient tracer of these lesions. We did not record any changes in the expression levels or the localization of NTPDase2 along the cycle, thus suggesting that the enzyme is not influenced by the female sex hormones like other previously studied ectoenzymes. Remarkably, NTPDase2 was expressed by the Sushi Domain containing 2 (SUSD2) + endometrial mesenchymal stem cells (eMSCs) found perivascularly, rendering it useful as a cell marker to improve the isolation of eMSCs needed for regenerative medicine therapies.

It is currently controversially discussed whether mesenchymal stem cells (MSC) facilitate cartilage regeneration in vivo by a progenitor- or a nonprogenitor-mediated mechanism. Here, we describe a potentially novel unbiased in vivo cell tracking system based on transgenic donor and corresponding immunocompetent marker-tolerant recipient mouse and rat lines in inbred genetic backgrounds. Tolerance of recipients was achieved by transgenic expression of an immunologically neutral but physicochemically distinguishable variant of the marker human placental alkaline phosphatase (ALPP). In this dual transgenic system, donor lines ubiquitously express WT, heat-resistant ALPP protein, whereas recipient lines express a heat-labile ALPP mutant (ALPPE451G) resulting from a single amino acid substitution. Tolerance of recipient lines to ALPP-expressing cells and tissues was verified by skin transplantation. Using this model, we show that intraarticularly injected MSC contribute to regeneration of articular cartilage in full-thickness cartilage defects mainly via a nonprogenitor-mediated mechanism.

The aim of this study was to evaluate the effects of the remnants of two suture materials on osseointegration of titanium implants in a rabbit tibial model. Calibrated defects were prepared in the tibia of five Chinchilla rabbits. Filaments of nonresorbable (NR) nylon or resorbable (R) chitosan were placed at the bone to implant interface, whereas control sites had no suture material. After a healing period of 4 weeks, a pull-out test procedure was performed followed by enzymatic analyses of the wound fluid and relative quantification of mRNA levels for bone-related and cytokine markers from the peri-implant bone. A trend toward a reduced pull-out force was observed in the NR group (NR: 23.0 ± 12.8 N; R: 33.9 ± 11.3 N; control: 33.6 ± 24.0 N). Similarly, the bone resorption marker vacuolar type H+-ATPase was increased in the NR group compared with that in the control group (P = 0.041). The R group showed trends for lower alkaline phosphatase activity and osteocalcin expression and higher total protein content and RNA compared with the control group. In this submerged healing model, peri-implant bone healing was marginally affected by the two suture materials tested. However, there was a tendency toward better osseointegration and lower expression of bone resorption markers in the R group compared with the control group.

In this work, we report that Entpd1(-/-) mice, deficient for the ectonucleotidase nucleoside triphosphate diphosphohydrolase-1 (NTPDase1), produce smaller litters (27% reduction) compared with wild-type C57BL6 animals. This deficit is linked to reduced in vivo oocyte fertilization by Entpd1(-/-) males (61 ± 11% versus 88 ± 7% for Entpd1(+/+)). Normal epididymal sperm count, spermatozoa morphology, capacitation, and motility and reduced ejaculated sperm number (2.4 ± 0.5 versus 3.7 ± 0.4 million for Entpd1(+/+)) pointed to vas deferens dysfunction. NTPDase1 was localized by immunofluorescence in the tunica muscularis of the vas deferens. Its absence resulted in a major ATP hydrolysis deficiency, as observed in situ by histochemistry and in primary smooth muscle cell cultures. In vitro, Entpd1(-/-) vas deferens displayed an exacerbated contraction to ATP, a diminished response to its non-hydrolysable analog αβMeATP, and a reduced contraction to electrical field stimulation, suggesting altered P2X1 receptor function with a propensity to desensitize. This functional alteration was accompanied by a 3-fold decrease in P2X1 protein expression in Entpd1(-/-) vas deferens with no variation in mRNA levels. Accordingly, exogenous nucleotidase activity was required to fully preserve P2X1 receptor activation by ATP in vitro. Our study demonstrates that NTPDase1 is required to maintain normal P2X1 receptor functionality in the vas deferens and that its absence leads to impaired peristalsis, reduced spermatozoa concentration in the semen, and, eventually, reduced fertility. This suggests that alteration of NTPDase1 activity affects ejaculation efficacy and male fertility. This work may contribute to unveil a cause of infertility and open new therapeutic potentials., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Epsilon toxin (Etx) from Clostridium perfringens is a pore-forming protein with a lethal effect on livestock, producing severe enterotoxemia characterized by general edema and neurological alterations. Site-specific mutations of the toxin are valuable tools to study the cellular and molecular mechanism of the toxin activity. In particular, mutants with paired cysteine substitutions that affect the membrane insertion domain behaved as dominant-negative inhibitors of toxin activity in MDCK cells. We produced similar mutants, together with a well-known non-toxic mutant (Etx-H106P), as green fluorescent protein (GFP) fusion proteins to perform in vivo studies in an acutely intoxicated mouse model. The mutant (GFP-Etx-I51C/A114C) had a lethal effect with generalized edema, and accumulated in the brain parenchyma due to its ability to cross the blood-brain barrier (BBB). In the renal system, this mutant had a cytotoxic effect on distal tubule epithelial cells. The other mutants studied (GFP-Etx-V56C/F118C and GFP-Etx-H106P) did not have a lethal effect or cross the BBB, and failed to induce a cytotoxic effect on renal epithelial cells. These data suggest a direct correlation between the lethal effect of the toxin, with its cytotoxic effect on the kidney distal tubule cells, and the ability to cross the BBB.

Endometriosis, defined as the growth of endometrial tissue outside the uterus, is a common gynecologic condition affecting millions of women worldwide. It is an inflammatory, estrogen-dependent complex disorder, with broad symptomatic variability, pelvic pain, and infertility being the main characteristics. Ovarian endometriomas are frequently developed in women with endometriosis. Late diagnosis is one of the main problems of endometriosis; thus, it is important to identify biomarkers for early diagnosis. The aim of the present work is to evaluate the ecto-nucleotidases activities in the contents of endometriomas. These enzymes, through the regulation of extracellular ATP and adenosine levels, are key enzymes in inflammatory processes, and their expression has been previously characterized in human endometrium. To achieve our objective, the echo-guided aspirated fluids of endometriomas were analyzed by evaluating the ecto-nucleotidases activities and compared with simple cysts. Our results show that enzyme activities are quantifiable in the ovarian cysts aspirates and that endometriomas show significantly higher ecto-nucleotidases activities than simple cysts (5.5-fold increase for ATPase and 20-fold for ADPase), thus being possible candidates for new endometriosis biomarkers. Moreover, we demonstrate the presence of ecto-nucleotidases bearing exosomes in these fluids. These results add up to the knowledge of the physiopathologic mechanisms underlying endometriosis and, open up a promising new field of study.

One of the strategies used by tumors to evade immunosurveillance is the accumulation of extracellular adenosine, which has immunosupressive and tumor promoting effects. The study of the mechanisms leading to adenosine formation at the tumor interstitium are therefore of great interest in oncology. The dominant pathway generating extracellular adenosine in tumors is the dephosphorylation of ATP by ecto-nucleotidases. Two of these enzymes acting sequentially, CD39 and CD73, efficiently hydrolyze extracellular ATP to adenosine. They have been found to play a crucial role in a variety of tumors, but there were no data concerning endometrial cancer, the most frequent of the invasive tumors of the female genital tract. The aim of the present work is to study the expression of CD39 and CD73 in human endometrial cancer. We have analyzed protein and gene expression, as well as enzyme activity, in type I endometrioid adenocarcinomas and type II serous adenocarcinomas and their nonpathological endometrial counterparts. High levels of both enzymes were found in tumor samples, with significantly increased expression of CD39 in type II serous tumors, which also coincided with the higher tumor grade. Our results reinforce the involvement of the adenosinergic system in cancer, emphasizing the relevance of ecto-nucleotidases as emerging therapeutic targets in oncology.

Schizophrenia (SZ) is a major chronic neuropsychiatric disorder characterized by a hyperdopaminergic state. The hypoadenosinergic hypothesis proposes that reduced extracellular adenosine levels contribute to dopamine D2 receptor hyperactivity. ATP, through the action of ecto-nucleotidases, constitutes a main source of extracellular adenosine. In the present study, we examined the activity of ecto-nucleotidases (NTPDases, ecto-5'-nucleotidase, and alkaline phosphatase) in the postmortem putamen of SZ patients (n = 13) compared with aged-matched controls (n = 10). We firstly demonstrated, by means of artificial postmortem delay experiments, that ecto-nucleotidase activity in human brains was stable up to 24 h, indicating the reliability of this tissue for these enzyme determinations. Remarkably, NTPDase-attributable activity (both ATPase and ADPase) was found to be reduced in SZ patients, while ecto-5'-nucleotidase and alkaline phosphatase activity remained unchanged. In the present study, we also describe the localization of these ecto-enzymes in human putamen control samples, showing differential expression in blood vessels, neurons, and glial cells. In conclusion, reduced striatal NTPDase activity may contribute to the pathophysiology of SZ, and it represents a potential mechanism of adenosine signalling impairment in this illness.

Extracellular ATP and its hydrolysis product, adenosine, acting through specific receptors collectively named purinergic receptors, regulate female fertility by influencing the endometrial fluid microenvironment. There are four major groups of ecto-nucleotidases that control the levels of extracellular ATP and adenosine and thus their availability at purinergic receptors: ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases), ecto-nucleotide pyrophosphatase/phospho-diesterases (E-NPPs), ecto-5'-nucleotidase (5'NT), and alkaline phosphatases (APs). The aim of the present work is to characterize the expression and distribution of ecto-nucleotidases in human endometrium along the menstrual cycle and after menopause, to evaluate their potential utility as fertility markers. We examined proliferative, secretory and atrophic endometria from women without endometrial pathology undergoing hysterectomy. We show that the ecto-nucleotidases are mainly present at endometrial epithelia, both luminal and glandular, and that their expression fluctuates along the cycle and also changes after menopause. An important result was identifying NPP3 as a new biological marker of tubal metaplasia. Our results emphasize the relevance of the study of purinergic signaling in human fertility.

Background and Purpose: Ectonucleotidases control extracellular nucleotide levels and consequently, their (patho)physiological responses. Among these enzymes, nucleoside triphosphate diphosphohydrolase-1 (NTPDase1), -2, -3 and -8 are the major ectonucleotidases responsible for nucleotide hydrolysis at the cell surface under physiological conditions, and NTPDase1 is predominantly located at the surface of vascular endothelial cells and leukocytes. Efficacious inhibitors of NTPDase1 are required to modulate responses induced by nucleotides in a number of pathological situations such as thrombosis, inflammation and cancer., Experimental Approach: Here, we present the synthesis and enzymatic characterization of five 8-BuS-adenine nucleotide derivatives as potent and selective inhibitors of NTPDase1., Key Results: The compounds 8-BuS-AMP, 8-BuS-ADP and 8-BuS-ATP inhibit recombinant human and mouse NTPDase1 by mixed type inhibition, predominantly competitive with Ki values <1 μM. In contrast to 8-BuS-ATP which could be hydrolyzed by other NTPDases, the other BuS derivatives were resistant to hydrolysis by either NTPDase1, -2, -3 or -8. 8-BuS-AMP and 8-BuS-ADP were the most potent and selective inhibitors of NTPDase1 expressed in human umbilical vein endothelial cells as well as in situ in human and mouse tissues. As expected, as a result of their inhibition of recombinant human NTPDase1, 8-BuS-AMP and 8-BuS-ADP impaired the ability of this enzyme to block platelet aggregation. Importantly, neither of these two inhibitors triggered platelet aggregation nor prevented ADP-induced platelet aggregation, in support of their inactivity towards P2Y1 and P2Y12 receptors., Conclusions and Implications: The 8-BuS-AMP and 8-BuS-ADP have therefore potential to serve as drugs for the treatment of pathologies regulated by NTPDase1., (© 2013 The Authors. British Journal of Pharmacology © 2013 The British Pharmacological Society.)

Several probes have been developed to identify steroidogenic activity in the brain of vertebrates. However, the presence of the cytochrome P450 17α-hydroxylase/C(17,20)-lyase (P450C(17)), an enzyme that converts pregnenolone and progesterone into dehydroepiandrosterone and androstenedione, in specific areas of the cerebellum such as the deep cerebellar nuclei, remains virtually unexplored. Using Western blot analysis and immunohistochemistry, we found molecular expression of P450C(17) in the lateral, interposed and medial deep cerebellar nuclei. Moreover, double immunofluorescence procedures enabled localization of P450C(17) mainly in neurons, axons and glutamatergic synapses. Taken together, these data demonstrate the occurrence of P450C(17) in the deep cerebellar nuclei, and enable the chemical characterization of the cells that express the cytochrome., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Extracellular nucleotides and adenosine are biologically active molecules that bind members of the P2 and P1 receptor families, respectively. In the digestive system, these receptors modulate various functions, including salivary, gastric, and intestinal epithelial secretion and enteric neurotransmission. The availability of P1 and P2 ligands is modulated by ectonucleotidases, enzymes that hydrolyze extracellular nucleotides into nucleosides. Nucleoside triphosphate diphosphohydrolases (NTPDases) and ecto-5'-nucleotidase are the dominant ectonucleotidases at physiological pH. While there is some information about the localization of ecto-5'-nucleotidase and NTPDase1 and -2, the distribution of NTPDase3 in the digestive system is unknown. We examined the localization of these ectonucleotidases, with a focus on NTPDase3, in the gastrointestinal tract and salivary glands. NTPDase1, -2, and -3 are responsible for ecto-ATPase activity in these tissues. Semiquantitative RT-PCR, immunohistochemistry, and in situ enzyme activity revealed the presence of NTPDase3 in some epithelial cells in serous acini of salivary glands and mucous acini and duct cells of sublingual salivary glands, in cells from the stratified esophageal and forestomach epithelia, and in some enteroendocrine cells of the gastric antrum. Interestingly, NTPDase2 and ecto-5'-nucleotidase are coexpressed with NTPDase3 in salivary gland cells and stratified epithelia. In the colon, neurons express NTPDase3 and glial cells express NTPDase2. Ca(2+) imaging experiments demonstrate that NTPDases regulate P2 receptor ligand availability in the enteric nervous system. In summary, the specific localization of NTPDase3 in the digestive system suggests functional roles of the enzyme, in association with NTPDase2 and ecto-5'-nucleotidase, in epithelial functions such as secretion and in enteric neurotransmission.

The chemokine interleukin 8 (IL-8) is a major chemoattractant for human neutrophils. Here, we demonstrate novel evidence that IL-8-induced neutrophil chemotaxis requires a concurrent activation of P2 receptors, most likely the P2Y(2) which is dominantly expressed in these cells. Indeed, the migration of human neutrophils towards IL-8 was significantly inhibited by the P2Y receptor antagonists, suramin and reactive blue 2 (RB-2) and potentiated by a P2Y(2) ligand, ATP, but insensitive to specific antagonists of P2Y(1), P2Y(6) and P2Y(11) receptors. Adenosine had no effect on neutrophil migration towards IL-8 which contrasted with the stimulatory effect of this molecule on neutrophil chemotaxis caused by formyl-Met-Leu-Phe (fMLP or fMLF). Taken together, these data suggest that extracellular ATP is necessary for IL-8 to exert its chemotactic effect on neutrophils.

Voltage-dependent K(+) (Kv) currents in macrophages are mainly mediated by Kv1.3, but biophysical properties indicate that the channel composition could be different from that of T-lymphocytes. K(+) currents in mouse bone marrow-derived and Raw-264.7 macrophages are sensitive to Kv1.3 blockers, but unlike T-cells, macrophages express Kv1.5. Because Shaker subunits (Kv1) may form heterotetrameric complexes, we investigated whether Kv1.5 has a function in Kv currents in macrophages. Kv1.3 and Kv1.5 co-localize at the membrane, and half-activation voltages and pharmacology indicate that K(+) currents may be accounted for by various Kv complexes in macrophages. Co-expression of Kv1.3 and Kv1.5 in human embryonic kidney 293 cells showed that the presence of Kv1.5 leads to a positive shift in K(+) current half-activation voltages and that, like Kv1.3, Kv1.3/Kv1.5 heteromers are sensitive to r-margatoxin. In addition, both proteins co-immunoprecipitate and co-localize. Fluorescence resonance energy transfer studies further demonstrated that Kv1.5 and Kv1.3 form heterotetramers. Electrophysiological and pharmacological studies of different ratios of Kv1.3 and Kv1.5 co-expressed in Xenopus oocytes suggest that various hybrids might be responsible for K(+) currents in macrophages. Tumor necrosis factor-alpha-induced activation of macrophages increased Kv1.3 with no changes in Kv.1.5, which is consistent with a hyperpolarized shift in half-activation voltage and a lower IC(50) for margatoxin. Taken together, our results demonstrate that Kv1.5 co-associates with Kv1.3, generating functional heterotetramers in macrophages. Changes in the oligomeric composition of functional Kv channels would give rise to different biophysical and pharmacological properties, which could determine specific cellular responses.

1. Various types of anticholinesterasic agents have been used to improve the daily activities of Alzheimer's disease patients. It was recently demonstrated that Galantamine, described as a molecule with anticholinesterasic properties, is also an allosteric enhancer of human alpha4beta2 neuronal nicotinic receptor activity. We explored its effect on the human alpha7 neuronal nicotinic acetylcholine receptor (nAChR) expressed in Xenopus oocytes. 2. Galantamine, at a concentration of 0.1 microM, increased the amplitude of acetylcholine (ACh)-induced ion currents in the human alpha7 nAChR expressed in Xenopus oocytes, but caused inhibition at higher concentrations. The maximum effect of galantamine, an increase of 22% in the amplitude of ACh-induced currents, was observed at a concentration of 250 microM Ach. 3. The same enhancing effect was obtained in oocytes transplanted with Torpedo nicotinic acetylcholine receptor (AChR) isolated from the electric organ, but in this case the optimal concentration of galantamine was 1 microM. In this case, the maximum effect of galantamine, an increase of 35% in the amplitude of ACh-induced currents, occurred at a concentration of 50 microM ACh. 4. Galantamine affects not only the activity of post-synaptic receptors but also the activity of nerve terminals. At a concentration of 1 microM, quantal spontaneous events, recorded in a cholinergic synapse, increased their amplitude, an effect which was independent of the anticholinesterasic activity associated with this compound. The anticholinesterasic effect was recorded in preparations treated with a galantamine concentration of 10 microM. 5. In conclusion, our results show that galantamine enhances human alpha7 neuronal nicotinic ACh receptor activity. It also enhances muscular AChRs and the size of spontaneous cholinergic synaptic events. However, only a very narrow range of galantamine concentrations can be used for enhancing effects.

Epsilon toxin (epsilon-toxin), produced by Clostridium perfringens types B and D, causes fatal enterotoxemia, also known as pulpy kidney disease, in livestock. Recombinant epsilon-toxin-green fluorescence protein (epsilon-toxin-GFP) and epsilon-prototoxin-GFP were successfully expressed in Escherichia coli. MTT assays on MDCK cells confirmed that recombinant epsilon-toxin-GFP retained the cytotoxicity of the native toxin. Direct fluorescence analysis of MDCK cells revealed a homogeneous peripheral pattern that was temperature sensitive and susceptible to detergent. epsilon-Toxin-GFP and epsilon-prototoxin-GFP bound to endothelia in various organs of injected mice, especially the brain. However, fluorescence mainly accumulated in kidneys. Mice injected with epsilon-toxin-GFP showed severe kidney alterations, including hemorrhagic medullae and selective degeneration of distal tubules. Moreover, experiments on kidney cryoslices demonstrated specific binding to distal tubule cells of a range of species. We demonstrate with new recombinant fluorescence tools that epsilon-toxin binds in vivo to endothelial cells and renal tubules, where it has a strong cytotoxic effect. Our binding experiments indicate that an epsilon-toxin receptor is expressed on renal distal tubules of mammalian species, including human.

ATP mediates intercellular communication. Mechanical stress and changes in cell volume induce ATP release from various cell types, both secretory and non-secretory. In the present study, we stressed Xenopus oocytes with a hypertonic solution enriched in mannitol (300 mM). We measured simultaneously ATP release and ionic currents from a single oocyte. A decrease in cell volume, the activation of an inward current and ATP release were coincident. We found two components of ATP release: the first was associated with granule or vesicle exocytosis, because it was inhibited by tetanus neurotoxin, and the second was related to the inward current. A single exponential described the correlation between ATP release and the hypertonic-activated current. Gadolinium ions, which block mechanically activated ionic channels, inhibited the ATP release and the inward current but did not affect the decrease in volume. Oocytes expressing CFTR (cystic fibrosis transmembrane regulator) released ATP under hypertonic shock, but ATP release was significantly inhibited in the first component: that related to granule exocytosis. Since the ATP measured is the balance between ATP release and ATP degradation by ecto-enzymes, we measured the nucleoside triphosphate diphosphohydrolase (NTPDase) activity of the oocyte surface during osmotic stress, as the calcium-dependent hydrolysis of ATP, which was inhibited by more than 50 % in hypertonic conditions. The best-characterized membrane protein showing NTPDase activity is CD39. Oocytes injected with an antisense oligonucleotide complementary to CD39 mRNA released less ATP and showed a lower amplitude in the inward current than those oocytes injected with water.

The presence of ATP within cells is well established. However, ATP also operates as an intercellular signal via specific purinoceptors. Furthermore, nonsecretory cells can release ATP under certain experimental conditions. To measure ATP release and membrane currents from a single cell simultaneously, we used Xenopus oocytes. We simultaneously recorded membrane currents and luminescence. Here, we show that ATP release can be triggered in Xenopus oocytes by hyperpolarizing pulses. ATP release (3.2 +/- 0.3 pmol/oocyte) generated a slow inward current (2.3 +/- 0.1 microA). During hyperpolarizing pulses, the permeability for ATP(4-) was more than 4000 times higher than that for Cl(-). The sensitivity to GdCl(3) (0. 2 mm) of hyperpolarization-induced ionic current, ATP release and E-ATPase activity suggests their dependence on stretch-activated ion channels. The pharmacological profile of the current inhibition coincides with the inhibition of ecto-ATPase activity. This enzyme is highly conserved among species, and in humans, it has been cloned and characterized as CD39. The translation, in Xenopus oocytes, of human CD39 mRNA encoding enhances the ATP-supported current, indicating that CD39 is directly or indirectly responsible for the electrodiffusion of ATP.

Alpha(1,3)- and alpha(1,4)-fucosylated oligosaccharides such as sialyl-Lewis(x) (sialyl-Le(x)) and sialyl-Lewis(a) (sialyl-Le(a)) have been reported to participate in tumor cell adhesion to activated endothelium. We examined by cytofluorometry the expression of Le(x), sialyl-Le(x), sialyl-Le(x) dimeric, Le(a), and sialyl-Le(a) on the surface of two human lung adenocarcinoma cell lines with different lung colonization potential. High expression levels of all of these antigens were detected in the metastatic HAL-8Luc cells, whereas the closely related nonmetastatic HAL-24Luc cells only expressed the sialyl-Le(a) and sialyl-Le(x) dimeric antigens, both at lower level than in HAL-8Luc cells. Five alpha(1,3)-fucosyltransferases (alpha(1,3)-Fuc-T) controlling the synthesis of these molecules have been identified to date (Fuc-TIII-Fuc-IVII). The expression of these five genes was also higher in the metastatic cells than in the nonmetastatic counterparts as was shown by Northern blot analysis. In vitro adhesion assays showed that only the metastatic cell line adheres significantly to E-selectin-expressing human endothelial cells. Moreover, monoclonal antibody (mAb) blockade of E-selectin completely abolished tumor cell binding. Adhesion inhibition experiments using mAbs against sialylated fucosylated oligosaccharides expressed on tumor cells indicated that these antigens are involved in the binding. Anti-sialyl-Lex(x) mAb (CSLEX-1) inhibited adhesion by 85%; it had the most pronounced inhibitory effect. These findings suggest that the overexpression of alpha(1,3)-Fuc-T genes in the metastatic HAL-8Luc cells, compared with HAL-24Luc cells, results in an enhanced surface display of fucosylated oligosaccharides, which contributes to the adhesive capacity of these cells to the activated endothelium and correlates with their lung colonization potential.