Regulation of plasma membrane Ca(2+)-ATPase activity by acetylated tubulin: influence of the lipid environment.

We demonstrated previously that acetylated tubulin inhibits plasma membrane Ca(2+)-ATPase (PMCA) activity in plasma membrane vesicles (PMVs) of rat brain through a reversible interaction. Dissociation of the PMCA/tubulin complex leads to restoration of ATPase activity. We now report that, when the enzyme is reconstituted in phosphatidylcholine vesicles containing acidic or neutral lipids, tubulin not only loses its inhibitory effect but is also capable of activating PMCA. This alteration of the PMCA-inhibitory effect of tubulin was dependent on concentrations of both lipids and tubulin. Tubulin (300µg/ml) in combination with acidic lipids at concentrations >10%, increased PMCA activity up to 27-fold. The neutral lipid diacylglycerol (DAG), in combination with 50µg/ml tubulin, increased PMCA activity >12-fold, whereas tubulin alone at high concentration (≥300µg/ml) produced only 80% increase. When DAG was generated in situ by phospholipase C incubation of PMVs pre-treated with exogenous tubulin, the inhibitory effect of tubulin on PMCA activity (ATP hydrolysis, and Ca(2+) transport within vesicles) was reversed. These findings indicate that PMCA is activated independently of surrounding lipid composition at low tubulin concentrations (<50µg/ml), whereas PMCA is activated mainly by reconstitution in acidic lipids at high tubulin concentrations. Regulation of PMCA activity by tubulin is thus dependent on both membrane lipid composition and tubulin concentration.

HPA-5 typing discrepancy reveals an Ile503Leu substitution in platelet GPIa (α2 integrin).

BACKGROUND AND OBJECTIVES: In fetal/neonatal thrombocytopenia, maternal alloimmunization is diagnosed by the identification of the maternal alloantibody and the offending paternal antigen inherited by the foetus/neonate. Today, for practical reasons, most laboratories perform platelet genotyping instead of phenotyping. Here, we report the case of a human platelet antigen (HPA)-5 genotype/phenotype discrepancy observed in a mother who delivered a mildly thrombocytopenic newborn. MATERIALS AND METHODS: Platelet antibody detection and platelet phenotyping were performed using the MAIPA assay; platelet genotypes were determined using BeadChip technology (BioArray), PCR-SSP, PCR-RFLP and sequencing. RESULTS: Serological investigations revealed the presence of maternal anti-GPIIbIIIa autoantibodies. No alloantibodies were detected. No feto-maternal platelet incompatibility was observed for HPA-1 to -21. The mother and newborn were genotyped as HPA-5aa using BeadChips, but as HPA-5a (weak b) with PCR-SSP and HPA-5ab with PCR-RFLP. Mother's platelets were phenotyped as HPA-5b(+). GPIa exon 13 sequencing confirmed the HPA-5ab genotype of the mother and newborn, and revealed an NM_002203.3:c.1594A>C mutation near the HPA-5 polymorphism (5' side), leading to an I503L amino acid change. CONCLUSION: Feto-maternal alloimmunization was ruled out: the neonatal thrombocytopenia probably resulted from maternal anti-GPIIbIIIa autoantibodies. This case highlights that platelet typing should be performed using two different methods to avoid false diagnosis.

A novel engineered dermis for in vitro photodamage research.

The realization of biologically relevant human tissue equivalents as an in vitro model to investigate human diseases, as well as to test the efficacy or toxicity of novel compounds, is emerging as a new challenge in tissue engineering. Currently, the in vitro three-dimensional (3D) dermis model mainly involves the use of cells embedded in exogenous non-human matrices. However, such models feature biological and functional disparities with native dermis, therefore limiting their relevance to the in vivo situation. The purpose of this study was to provide a reliable endogenous human dermal equivalent (HDE) able to recapitulate the extracellular matrix (ECM) remodelling of the native dermis occurring after external damage. To this end, UVA irradiation was used to induce photodamage to both the HDE and to a fibroblast-populated collagen matrix. The photodamage was investigated at the cellular and ECM level and the results showed that, although a cellular response was detected in both systems, no ECM reorganization characteristic of the in vivo photo-aged dermis could be detected in the fibroblast-populated collagen matrix. In contrast in the HDE, the neosynthesized ECM recapitulated the characteristic ageing behaviour of the dermis found in vivo, in terms of collagen and hyaluronic acid synthesis as well as collagen organization remodelling. This study therefore demonstrates the role of the endogenous ECM in recapitulating in vitro the functionality of the human dermis and the proposed HDE as a novel tool for photoprotection trials. Copyright © 2016 John Wiley & Sons, Ltd.

Cab4b, the first human platelet antigen carried by glycoprotein IX discovered in a context of severe neonatal thrombocytopenia.

Essentials Life-threatening maternofetal thrombocytopenias mostly depend on αIIb ß3 antigens. We performed serological, genomic and in vitro studies of two life-threatening thrombocytopenias. Identification of a c.368C>T variation leading to Pro123Leu substitution in GPIX. A rare GPIX variant reported in a genomic database define a new alloantigen. SUMMARY: Background After three miscarriages, a 39-year-old woman gave birth, with a 1-year interval, to two severely thrombocytopenic neonates (4 ×109 L-1 and 33 ×109 L-1 ) with intracranial hemorrhages. Transfusion of platelet concentrates corrected the thrombocytopenia. The outcome was favorable for the first child, but the second one died 10 days after cesarean delivery (31 weeks of gestation + 6 days). Methods Serologic studies were performed with mAb-specific immobilization of platelet antigens and flow cytometry techniques. Human platelet alloantigen (HPA) genotyping was performed with the BioArray HPA BeadChip and PCR-sequence-specific primer techniques. Genomic DNA was studied by direct sequencing of PCR products. The mutant glycoprotein (GP) was expressed in transiently transfected HEK293 cells. Results In MAIPA assay, the maternal serum faintly reacted with GPIbIX from paternal and child 1 platelets, but not with maternal or panel platelets. No maternofetal incompatibility was found in the 22 known HPA systems, tested except for HPA-1b in child 2. A new alloantigen carried by GPIbIX was suspected. Genomic sequencing revealed a paternal GPIX variation (NM_000174.4:c.368C>T). The father and children were heterozygous and incompatible with the mother, who was NM_000174.4:c.368C homozygous. The maternal serum reacted with the GPIX NP_000165.1:p.Leu123 form coexpressed with GPIb in transfected HEK293 cells. The NM_000174.4:c.368T allele (rs202229101) has a minor allele frequency of 0.0002, and was not detected in 120 French subjects (families with fetal and neonatal alloimmune thrombocytopenia [FNAIT]), suggesting that it is rarely implicated in alloimmunization. Conclusion The NP_000165.1:p.Leu123 allele named Cab4b is the first platelet alloantigen described on GPIX. In the absence of other known maternofetal incompatibility, the child 1 case suggests that anti-Cab4b alloantibodies can induce severe thrombocytopenias.

Biophysical properties of dermal building-blocks affects extra cellular matrix assembly in 3D endogenous macrotissue.

The fabrication of functional tissue units is one of the major challenges in tissue engineering due to their in vitro use in tissue-on-chip systems, as well as in modular tissue engineering for the construction of macrotissue analogs. In this work, we aim to engineer dermal tissue micromodules obtained by culturing human dermal fibroblasts into porous gelatine microscaffold. We proved that such stromal cells coupled with gelatine microscaffolds are able to synthesize and to assemble an endogenous extracellular matrix (ECM) resulting in tissue micromodules, which evolve their biophysical features over the time. In particular, we found a time-dependent variation of oxygen consumption kinetic parameters, of newly formed ECM stiffness and of micromodules self-aggregation properties. As consequence when used as building blocks to fabricate larger tissues, the initial tissue micromodules state strongly affects the ECM organization and maturation in the final macrotissue. Such results highlight the role of the micromodules properties in controlling the formation of three-dimensional macrotissue in vitro, defining an innovative design criterion for selecting tissue-building blocks for modular tissue engineering.

One versus four heat treatments in combination with radiotherapy in metastatic mammary carcinoma.

To optimize the total and the weekly number of heat treatments to be combined with a conventional radiotherapy course, a study was designed on a 75-year-old woman with 40 cutaneous nodules of metastatic mammary carcinoma. All nodules were individually irradiated by means of orthovoltage radiation to doses of 36 to 44 Gy, given in 20 equal fractions in 4 weeks. The nodules were randomly assigned to receive radiotherapy alone or radiotherapy combined with one or four heat treatments. Eight lesions were left untreated as a control arm of the systemic therapy (endocrine manipulation). Hyperthermia at a minimum temperature of 43 degrees C was applied for 45 min once per week for four times or only once, during a course of radiotherapy. Percent mean diameter of the treated lesions continuously decreased, reaching a minimum of 25-30% of the initial value after 4 months from the beginning of treatment with no difference between the three arms. After this period, tumors treated with radiotherapy alone or radiotherapy plus one heat treatment started to regrow, whereas those treated with radiotherapy plus four heat treatments continued to decrease slowly. The actuarial analysis of freedom from local progression showed a trend of improvement of response duration with four hyperthermic treatments with respect to radiotherapy alone or combined with one hyperthermic treatment.

[Water absorption and resistance to wear of 2 newly invented composite resins]. / Assorbimento d'acqua e resistenza all'usura di due resine composite di nuova concezione.

Two recently built composites, one for posterior teeth, and the other for general employ, were evaluated in relation to their water absorption and resistance to wear by means of the "scratch test". Critical loads and attrition of the two materials were also evaluated, with statistical analysis. The results supported the good qualities of the two composites, which are to be considered very advanced in the field of esthetic reconstruction materials.

A randomized double-blind placebo-controlled clinical trial on efficacy and safety of association of simethicone and Bacillus coagulans (Colinox®) in patients with irritable bowel syndrome.

INTRODUCTION: Irritable bowel syndrome (IBS) is a chronic gastrointestinal (GI) disorder that affects 15-20% of the Western population. BACKGROUND: There are currently few therapeutic options available for the treatment of IBS. The aim of this study is to evaluate the efficacy and the safety of a medical device containing a combination of Simethicone and Bacillus coagulans in the treatment of IBS. PATIENTS AND METHODS: This is a monocentric double-blind, placebo-controlled parallel group clinical trial. Adult subjects suffering from IBS as defined by Rome III criteria were enrolled. Bloating, discomfort, abdominal pain were assessed as primary end point. Subjects received the active treatment or placebo 3 time a day after each meal for 4 weeks of study period. Subjects were submitted to visit at Day 0 (T1), at Days 14 (T2) and 29 (T3). RESULTS: Fifty-two patients were included into the study. Intragroup analysis showed a significant reduction of the bloating, discomfort and pain in Colinox® group (CG) compared to placebo group (PG). Between group analysis confirmed, at T1-T3, significant differences between CG and PG in bloating and discomfort. DISCUSSION: Simethicone is an inert antifoaming able to reduce bloating, abdominal discomfort. Literature offers increasing evidence linking alterations in the gastrointestinal microbiota and IBS and it is well known that probiotics are important to restore the native gut microbiota. The Colinox medical device is specifically targeted against most intrusive symptom of IBS (bloating) and it is also able to counteract the most accredited ethiopathogenetic factor in IBS (alterations of intestinal microbiota). CONCLUSIONS: This is the first randomized double-blind placebo-controlled clinical trial demonstrating the efficacy and safety of a combination of simethicone and Bacillus coagulans in treatment of IBS.

Mastectomía profiláctica contralateral, con técnica ahorradora de piel y del complejo aréola-pezón, como tratamiento oncológico y estético / Contralateral prophylactic mastectomy with a skin-sparing, nipple-sparing technique as an oncological and esthetic procedure

La mastectomía profiláctica contralateral (MPC) en mujeres con diagnóstico de cáncer de mama es un procedimiento quirúrgico que permite obtener un mejor resultado estético y un pronóstico oncológico más favorable, al prevenir el desarrollo contralateral del cáncer. Presentamos nuestra experiencia en 38 casos de mujeres con diagnóstico de cáncer de mama que fueron operadas de mastectomía y reconstrucción inmediata en 2 tiempos mediante expansor tisular/implante. Estas pacientes optaron por una MPC en el segundo tiempo del proceso reconstructivo por motivos psicológicos u oncológicos. Realizamos una técnica ahorradora de piel y del complejo aréola-pezón, que ofrece un resultado natural de la mama una vez reconstruida y, por tanto, un alto nivel de satisfacción de la paciente

Contralateral prophylactic mastectomy (CPM) in women with a diagnosis of breast cancer improves the esthetic outcome and oncological prognosis because it reduces the risk of developing contralateral breast cancer. We report our experience of 38 women with a diagnosis of breast cancer who underwent mastectomy and immediate two-stage tissue expander / implant reconstruction. These patients opted for a CPM in the second stage of their reconstructive process due to psychological or oncological reasons. We performed a skin-sparing, nipple-sparing technique that offers a natural result and consequently a high level of patient satisfaction

Reducción funcional y estética de los labios menores: labioplastia con técnica de resección con colgajos especulares en «S» / Functional and esthetic reduction of the labia minora: labioplasty with specular S-shaped resection

Las mujeres que presentan los labios menores hipertróficos o prominentes a menudo presentan trastornos funcionales y psicosociales. Se consideran labios menores hipertróficos aquellos que, en posición y condiciones normales, sobresalen notoriamente de los labios mayores, ocasionando irritación local, dificultades higiénicas, incomodidad mientras se camina o realizan algunas prácticas deportivas e interfiriendo en el desarrollo normal de las relaciones sexuales. Hay diversas técnicas quirúrgicas de labioplastia. Una de las más habituales es la amputación de aquellos segmentos de los labios menores que sobresalen de los labios mayores. Para evitar las complicaciones derivadas de esa técnica, nosotros hemos optado por realizar la labioplastia efectuando una resección con colgajos especulares en forma de «S», con una sutura interdigitada. De esta manera el resultado final es más natural, con bordes suaves y redondeados, lo que reduce en gran medida las complicaciones relacionadas con la contracción cicatricial. A continuación, presentamos nuestra experiencia tras haber aplicado con éxito esta técnica en 7 casos

Women with hypertrophic or enlarged labia minora usually have functional and psychosocial problems. Labial hypertrophy is defined as labia minora that extend beyond the labia majora when the woman is in a normal position. Many affected women have local irritation, interference with sexual intercourse, difficulty with hygiene, and discomfort during walking, sitting, biking or horse-riding. Several surgical techniques can be used to perform labioplasty. One of most common procedures is a simple and straight amputation of the protuberant segments and over sewing of the edge. To prevent the complications associated with this technique, we perform a specular S-shaped resection with interdigitated suturing of the protuberant labium. This technique results in a natural, softer, and more rounded labial edge, with fewer complications. We present our experience in 7 cases with successful outcomes

Anti-endomysial antibody of IgG1 isotype detection strongly increases the prevalence of coeliac disease in patients affected by type I diabetes mellitus.

A strong association between type 1 insulin-dependent diabetes mellitus (IDDM1) and coeliac disease (CD) is well documented, but it is known that prevalence values are underestimated. Serum anti-endomysial antibodies (EMA), considered diagnostic for CD because of their high sensitivity and specificity, belong to the IgA class, but the existence of EMA of IgG1 isotype in the presence or absence of IgA deficiency was reported. In order to re-evaluate the occurrence of CD in IDDM1 patients we performed a screening in IDDM1 patients using EMA of both isotypes. Ninety-four adults affected by IDDM1 (unaffected by CD before enrolling) were enrolled and 83 blood donors as controls. All subjects were on a gluten-containing diet. Histology and biopsy culture were performed. EMA IgA and IgG1 in sera and culture supernatants were detected. Serum EMA were positive in 13 of 94 IDDM1 patients (13.8%). Six of 13 presented IgA-EMA, seven of 13 presented IgG1-EMA. No EMA were found in the control population. Total intestinal atrophy was found in all six patients with serum IgA-EMA and in five of seven with serum IgG1-EMA. Diagnosis of CD was confirmed by histology and organ culture in all 13 patients with serum EMA. The prevalence of CD in the patients affected by IDDM1 was 6.4% for IgA-EMA-positive and 7.4% for IgG1-EMA-positive patients. We confirmed the prevalence of CD in the IDDM1 population obtained with IgA-EMA screening only (6.4%). This prevalence value increases dramatically to 13.8% when IgG1-EMA are also used in the screening. We conclude that IgG1-EMA should also be sought whenever an IDDM1 patient undergoes screening for CD.

Brain plasma membrane Na+,K+-ATPase is inhibited by acetylated tubulin.

Membranes from brain tissue contain tubulin that can be isolated as a hydrophobic compound by partitioning into Triton X-114. The hydrophobic behavior of this tubulin is due to the formation of a complex with the alpha-subunit of Na+,K+-ATPase. In the present work we show that the interaction of tubulin with Na+K+-ATPase inhibits the enzyme activity. We found that the magnitude of the inhibition is correlated with: (1) concentration of the acetylated tubulin isoform present in the tubulin preparation used, and (2) amount of acetylated tubulin isoform isolated as a hydrophobic compound. In addition, some compounds involved in the catalytic action of Na+K+-ATPase were assayed to determine their effects on the inhibitory capability of tubulin on this enzyme. The inhibitory effect of tubulin was only slightly decreased by ATP at relatively low nucleotide concentration (0.06 mM). NaCl (1-160 mM) and KCl (0.2-10 mM) showed no effect whereas inorganic phosphate abolished the inhibitory effect of tubulin in a concentration-dependent manner.

Carnitine resembles choline in the induction of cholinesterase, acid phosphatase, and phospholipase C and in its action as an osmoprotectant in Pseudomonas aeruginosa.

The present study demonstrates that under conditions of iso or hyperosmolarity, P. aeruginosa utilized carnitine as the carbon, nitrogen or carbon and nitrogen sources. As occurred in the case of choline, the bacteria synthesized cholinesterase (ChE), acid phosphatase (Ac.Pase) and phospholipase C (PLC) under any of these conditions and in the presence of high or low Pi concentrations. Carnitine acted as an osmoprotectant when the cells were grown in the presence of preferred carbon and nitrogen sources and high NaCl concentrations. Under these conditions the three enzyme activities were not produced. The osmotically stressed bacteria grown under any of the above conditions accumulated betaine. Its presence indicated that carnitine may be metabolized by P. aeruginosa to produce betaine which could account for the induction of the three enzyme activities or its action as an osmoprotectant. The phosphatidylcholine encountered in the host cell membranes allows the bacteria to obtain free choline by the coordinated action of PLC and Ac.Pase. Since the consequence of this action may be cell disruption, the increase of free carnitine in the natural environment of the bacteria is also possible. These two compounds, choline and carnitine, acting in conjunction or separately, may increase the production of PLC and Ac.Pase activities by P. aeruginosa and thus enhance the degradative effect upon the host cells.