Search

Satellitosis or in-transit metastasis (S-ITM) has clinical outcomes comparable to node-positivity in cutaneous squamous cell carcinoma (cSCC). There is a need to stratify the risk groups. To determine which prognostic factors of S-ITM confer an increased risk of relapse and cSCC-specific–death. A retrospective, multicenter cohort study. Patients with cSCC developing S-ITM were included. Multivariate competing risk analysis evaluated which factors were associated with relapse and specific death. Of a total of 111 patients with cSCC and S-ITM, 86 patients were included for analysis. An S-ITM size of ≥20 mm, >5 S-ITM lesions, and a primary tumor deep invasion was associated with an increased cumulative incidence of relapse (subhazard ratio [SHR]: 2.89 [95% CI, 1.44-5.83; P =.003], 2.32 [95% CI, 1.13-4.77; P =.021], and 2.863 [95% CI, 1.25-6.55; P =.013]), respectively. Several >5 S-ITM lesions were also associated with an increased probability of specific death (SHR: 3.48 [95% CI, 1.18-10.2; P =.023]). Retrospective study and heterogeneity of treatments. The size and the number of S-ITM lesions confer an increased risk of relapse and the number of S-ITM an increased risk of specific-death in patients with cSCC presenting with S-ITM. These results provide new prognostic information and can be considered in the staging guidelines. [ABSTRACT FROM AUTHOR]

• Surgery is a safe treatment for GCMN. • Expanded skin flaps are the most optimal surgical option in most cases. • Surgeons, patients, and caregivers agree that surgical treatment should begin in the first months of life. • Surgical treatment has a low impact on QoL. Purpose: The aim of this study was to evaluate the outcomes, complications and psychosocial impact of surgical treatment of giant congenital melanocytic nevus (GCMN). Methods: Patients with surgically treated GCMN who attended our clinic between May 2014 and May 2018 were included. Patient demographics and data on the characteristics of the nevus, surgical treatment, and the psychosocial impact (including C-DLQI/DLQI questionnaires) were collected. Results: One hundred thirty-six patients were included (median age 9 years). Mean age at first surgery was 34 (+/- 61.45) months; 5.53 (+/- 3.69) surgical interventions were necessary to completely excise the nevus. The expanded skin flap was the preferred surgical technique in most locations. Complications were common but not severe. Of the patients studied, 70.4% reported that the surgery had a minor impact on their quality of life (QoL). Patients and caregivers stated that surgical treatment should begin as soon as possible, even in cases where early treatment did not have an impact on their QoL nor on their satisfaction with the surgery (p < 0.05). The lower the patient age at first surgery, the higher the surgeon's satisfaction (p < 0.01). Conclusions : Surgical treatment is a safe option for management of GCMN, and has a low impact on QoL. Patients, caregivers, and surgeons agree that the treatment should begin as soon as possible. This is the largest single-center study evaluating surgical treatment in GCMN patients and its psychosocial impact, and the first to take into account the patient, caregivers and dermatologists opinion of surgical results. [ABSTRACT FROM AUTHOR]

Background: Dermatofibrosarcoma protuberans (DFSP) is a low-grade malignant skin tumor that may also infiltrate dermis and subcutaneous tissue. Although the mainstay of treatment has been wide local excision, during the last decade three-dimensional (3D) histology surgery has proven very effective for the treatment of this tumor. Objective: We sought to evaluate the effectiveness of 3D histology surgery for the treatment of DFSP. Methods: We retrospectively reviewed charts of patients with DFSP treated in our unit with 3D histology surgery between April 2000 and May 2011. Age at onset, gender, duration of tumor, previous treatment, lesion site, number of surgical stages, postsurgical defect size, closure technique, and follow-up were registered. Results: A total of 29 patients were included. Mean patient age was 40.7 years. Fifteen lesions were located on the trunk, 11 on the extremities, and 3 in the head and neck region. Twelve patients had primary tumors and 17 were referred to us after incomplete excision. The average number of 3D histology surgical stages required for tumor clearance was 1.4. Mean postsurgical defect size was 26 cm2. All lateral and deep borders excised were tumor-free. Mean follow-up period was 68 months (range 12-142 months) with a 0% recurrence rate. Limitations: This was a retrospective review. Conclusion: Three-dimensional histology surgery with paraffin sections is effective for the treatment of DFSP and feasible in an outpatient setting. The low recurrence rates obtained confirm the oncologic efficacy of the procedure. Furthermore, designing closure on the basis of focally affected margins improves functional and aesthetic outcomes without compromising oncological effectiveness. [ABSTRACT FROM AUTHOR]

At least nine types of vascular malformations with specific clinical and radiologic characteristics must be distinguished in the lower limbs: Klippel–Trénaunay syndrome, port-wine stain with or without hypertrophy, cutis marmorata telangiectatica congenita, macrocephaly–capillary malformation, Parkes Weber syndrome, Stewart–Bluefarb syndrome, venous malformation, glomuvenous malformation, and lymphatic malformation. Extensive vascular malformations are often more complex than they appear and require a multidisciplinary therapeutic approach. Vascular malformations may be associated with underlying disease or systemic anomalies. Part II of this two-part series on the diagnosis and management of extensive vascular malformations of the lower limb highlights the systemic repercusions (bone, articular, visceral, and hematologic involvement), diagnosis, and treatment of these lesions. [Copyright &y& Elsevier]

There is significant confusion in the literature when describing vascular anomalies, and vascular malformations are often misnamed or incorrectly classified. Part I of this two-part series on the diagnosis and management of extensive vascular malformations of the lower limbs will discuss the dermatologist’s role in the diagnosis of these lesions. At least nine types of vascular malformations with specific clinical and radiologic characteristics must be distinguished in the lower limbs: Klippel–Trénaunay syndrome, port-wine stain with or without hypertrophy, cutis marmorata telangiectatica congenita, macrocephaly–capillary malformation, Parkes Weber syndrome, Stewart–Bluefarb syndrome, venous malformation, glomuvenous malformation, and lymphatic malformation. This article highlights the differences in clinical appearance and discusses the differential diagnosis of extensive vascular malformations in an attempt to ensure earlier diagnosis and better outcomes for these patients. [Copyright &y& Elsevier]

Background: Klippel-Trénaunay syndrome (KTS) is a capillary-lymphatic-venous malformation associated with soft tissue and skeletal hypertrophy of one or more limbs. Deep venous system (DVS) anomalies are reported to be present in 8% to 18% of patients with KTS; approximately 25% of patients with KTS have hand or foot malformations. Objective: We sought to assess whether the presence of hand or foot malformations in KTS is a predictor of DVS anomalies. Methods: Retrospective data were collected from 51 consecutive patients with KTS seen in a university hospital between January 2000 and February 2008. Patients with possible Proteus syndrome were not included. The presence and patency of the DVS was studied using conventional venography, multidetector computed tomography, or fast 3-dimensional magnetic resonance imaging venography. Results: Seventeen hand or foot malformations were present in 9 patients, consisting of: toe macrodactyly in 5 patients (two bilateral and one with plantar expansion); toe microdactyly in one patient; finger macrodactyly in one patient; finger macrodactyly and ectrodactyly in one patient; syndactyly in 4 patients; and clinodactyly with camptodactyly of the hand of one patient with lower limb KTS. Eleven patients had DVS anomalies (one with aplasia of entire DVS; one with duplication of the superficial femoral vein; 7 with hypoplasia of femoral vein; and 7 with aplasia of the popliteal vein). All patients with hand or foot malformations also had DVS anomalies (P < .001). Limitations: Small sample size was a limitation. Conclusion: The presence of hand or foot malformations in KTS may predict the presence of DVS anomalies. [ABSTRACT FROM AUTHOR]

Abstract: Endogenously expressed human canonical transient receptor potential 1 (hTRPC1) and human canonical transient receptor potential 6 (hTRPC6) have been shown to play a role in store-operated Ca2+ entry (SOCE) in human platelets, where two mechanisms for SOCE, regulated by the dense tubular system (DTS) or the acidic granules, have been identified. In cells preincubated for 1 min with 100 µM flufenamic acid we show that hTRPC6 is involved in SOCE activated by both mechanisms, as demonstrated by selective depletion of the DTS or the acidic stores, using thapsigargin (TG) (10 nM) or 2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ) (20 µM), respectively, although it is more relevant after acidic store depletion. Co-immunoprecipitation experiments indicated that depletion of both stores separately results in time-dependent interaction between hTRPC1 and hTRPC6, and also between both hTRPCs and the type II IP3 receptor (IP3RII). The latter was greater after treatment with TG. TBHQ-induced coupling between hTRPC1 and 6 was transient and decreased after 30s of treatment, while that induced by TG increased for at least 3 min. TBHQ induced association between SERCA3, located in the acidic stores, hTRPC1, hTRPC6 and Orai1. TBHQ also evoked coupling between SERCA3 and IP3RII, presumably located in the DTS, thus suggesting interplay between both Ca2+ stores. Similarly, TG induces the interaction of SERCA2b with hTRPC1 and 6 and the IP3RII. The interactions between hTRPC1, hTRPC6, IP3RII and SERCA3 were impaired by disruption of the microtubules, supporting a role for microtubules in Ca2+ homeostasis. In conclusion, the present data demonstrate for the first time that hTRPC1, hTRPC6, IP3RII and SERCA3 are parts of a macromolecular protein complex activated by depletion of the intracellular Ca2+ stores in human platelets. [Copyright &y& Elsevier]

Abstract: Two agonist-releasable Ca2+stores have been identified in human platelets differentiated by the distinct sensitivity of their SERCA isoforms to thapsigargin (TG) and 2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ). Here we have examined whether the SERCA isotypes might be involved in store-operated Ca2+entry (SOCE) activated by the physiological agonist thrombin in human platelets. Ca2+-influx evoked by thrombin (0.01 U/mL) reached a maximum after 3 min, which was consistent with the decrease in the Ca2+content in the stores; afterwards, the extent of SOCE decreased with no correlation with the accumulation of Ca2+in the stores. Inhibition of SERCA2b, by 10 nM TG, and SERCA3, with 20 μM TBHQ, individually or simultaneously, accelerated Ca2+ store discharge and subsequently enhanced the extent of SOCE stimulated by thrombin. In addition, TG and TBHQ modified the time course of thrombin-evoked SOCE from a transient to a sustained increase in Ca2+ influx, which reveals a negative role for SERCAs in the regulation of SOCE. This effect was consistent under conditions that inhibit Ca2+ extrusion by PMCA or the Na+/Ca2+ exchanger. Coimmunoprecipitation experiments revealed that thrombin stimulates direct interaction between SERCA2b and 3 with the hTRPC1 channel, an effect that was found to be independent of SERCA activity. In summary, our results suggest that SERCA2b and 3 modulate thrombin-stimulated SOCE probably by direct interaction with the hTRPC1 channel in human platelets. [Copyright &y& Elsevier]

Abstract: Phosphatidylinositol 4,5-bisphosphate (PIP2) is a versatile regulator of TRP channels. We report that inclusion of a PIP2 analogue, PIP2 1,2-dioctanoyl, does not induce non-capacitative Ca2+ entry per se but enhanced Ca2+ entry stimulated either by thrombin or by selective depletion of the Ca2+ stores in platelets, the dense tubular system, using 10 nM TG, and the acidic stores, using 20 μM 2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ). Reduction of PIP2 levels by blocking PIP2 resynthesis with Li+ or introducing a monoclonal anti-PIP2 antibody, or sequestering PIP2 using poly-lysine, attenuated Ca2+ entry induced by thrombin, TG and TBHQ, and reduced thrombin-evoked, but not TG- or TBHQ-induced, Ca2+ release from the stores. Incubation with the anti-hTRPC1 antibody did not alter the stimulation of Ca2+ entry by PIP2, whilst introduction of anti-hTRPC6 antibody directed towards the C-terminus of hTRPC6 reduced Ca2+ and Mn2+ entry induced by thrombin, TG or TBHQ, and abolished the stimulation of Ca2+ entry by PIP2. The anti-hTRPC6 antibody, but not the anti-hTRPC1 antibody or PIP2, reduced non-capacitative Ca2+ entry by the DAG analogue 1-oleoyl-2-acetyl-sn-glycerol. In summary, hTRPC6 plays a role both in store-operated and in non-capacitative Ca2+ entry. PIP2 enhances store-operated Ca2+ entry in human platelets, most probably by stimulation of hTRPC6 channels. [Copyright &y& Elsevier]

Abstract: Two mechanisms for store-operated Ca2+ entry (SOCE) regulated by two independent Ca2+ stores, the dense tubular system (DTS) and the acidic stores, have been described in platelets. We have previously suggested that coupling between the type II IP3 receptor (IP3RII) and hTRPC1, involving reorganization of the actin microfilaments, play an important role in SOCE. However, the involvement of the tubulin microtubules, located beneath the plasma membrane, remains unclear. Here we show that the microtubule disrupting agent colchicine reduced Ca2+ entry stimulated by low concentrations (0.1 U/mL) of thrombin, which activates SOCE mostly by depleting acidic Ca2+-store. Consistently, colchicine reduced SOCE activated by 2,5 di-(tertbutyl)-1,4-hydroquinone (TBHQ), which selectively depletes the acidic Ca2+ stores. In contrast, colchicine enhanced SOCE mediated by depletion of the DTS, induced by high concentrations of thapsigargin (TG), which depletes both the acidic Ca2+ stores and the DTS, the major releasable Ca2+ store in platelets. These findings were confirmed by using Sr2+ as a surrogate for Ca2+ entry. Colchicine attenuated the coupling between IP3RII and hTRPC1 stimulated by thrombin while it enhanced that evoked by TG. Paclitaxel, which induces microtubular stabilization and polymerization, exerted the opposite effects on thrombin- and TG-evoked SOCE and coupling between IP3RII and hTRPC1 compared with colchicine. Neither colchicine nor paclitaxel altered the ability of platelets to extrude Ca2+. These findings suggest that tubulin microtubules play a dual role in SOCE, acting as a barrier that prevents constitutive SOCE regulated by DTS, but also supporting SOCE mediated by the acidic Ca2+ stores. [Copyright &y& Elsevier]

Melanoma lesions can be frozen in vivo, resulting in necrotic death of malignant cells and in tumor antigen release suitable for cross-presentation by professional antigen-presenting cells. Imiquimod is a small molecule with adjuvant pro-inflammatory effects that can be topically delivered as a cream. Local cryosurgery of B16/ovalbumin (OVA)-derived subcutaneous tumor nodules leads to curative destruction of the lesions. If imiquimod is repeatedly applied on the cryo-treated lesion, a conspicuous, leukocyte-rich inflammatory infiltrate appears during the days following treatment. Mice treated by cryosurgery plus imiquimod rejected rechallenges of B16/OVA in 90% of the cases, whereas cryosurgery alone failed to prevent tumor grafting in 70% of the cases. The combination treatment of B16/OVA tumors was also able to protect 60% of the mice against outgrowth of a lethal dose of non-transfected B16 tumor cells. Addition of imiquimod to cryosurgery results in increases of the cellular immune response against tumor antigens as measured by in vitro IFN-γ production and T-cell proliferation in response to OVA. The potent memory response is not only directed against the OVA epitope, but also toward a broader range of B16 antigens. Our data indicate that these combined treatments turn the treated tumor lesion into an autologous tumor vaccine, which is even able to cause vitiligo in several cases. These preclinical data and the simplicity of the procedures warrant the design of a pilot clinical trial.Journal of Investigative Dermatology (2007) 127, 1673–1680; doi:10.1038/sj.jid.5700777; published online 22 March 2007 [ABSTRACT FROM AUTHOR]

Background: Klippel–Trénaunay syndrome (KTS) is a well-known eponym for a capillary-lymphatic-venous malformation which is associated with soft tissue and skeletal hypertrophy, usually of one or more limbs. Plain films, sonograms, conventional venograms, and arteriograms have been employed for the evaluation of the disease. Objective: To demonstrate the usefulness of multidetector computed tomography (MDCT) and fast 3-dimensional magnetic resonance imaging (3D-MR) venography for the assessment and therapeutic planning of patients with KTS. Methods: A prospective study in 16 consecutive patients with KTS using MDCT and 3D-MR venography, performed between January 2004 and January 2006 in a university hospital in Pamplona, Spain. Results: In nearly all patients, persistent embryologic veins were observed, and in one subject aplasia/atresia of the whole deep venous system of the affected extremity was seen. In four individuals hypoplasia of the femoral vein was observed; one subject had duplication of the femoral vein, and in three patients aplasia/atresia of this vein was found. Only half of the patients had normal popliteal veins. In one patient, aneurysmal dilatation of the popliteal vein was detected, and in six subjects, aplasia of this vein was observed. The presence of geographic stains was suggestive of hypoplasia and/or aplasia of femoral and popliteal veins. Limitations: The small size of the group of patients with KTS, which is related to low incidence of the disease. Conclusions: MDCT and 3D-MR venography are extremely helpful for the global evaluation of patients with KTS. Information regarding soft tissue and bony anatomy as well as information about superficial and deep venous systems may be obtained with a single exam. [Copyright &y& Elsevier]

Abstract: Recent studies propose the existence of two distinct Ca2+ compartments in human platelets based on the expression of different SERCA isoforms with distinct sensitivity to thapsigargin and 2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ). Using fura-2-loaded human platelets we have found that depletion of the TBHQ sensitive store reduces thrombin—but not ADP—or vasopressin (AVP)-induced Ca2+ release. Redistribution of cytosolic Ca2+ after thrombin stimulation resulted in overloading of the TBHQ-sensitive store. This phenomenon was not observed with ADP or AVP. We found that NAADP decreases the Ca2+ concentration into the stores in permeabilized platelets, which is prevented by depletion of the TBHQ-sensitive store. Nimodipine, an inhibitor of the NAADP receptor, reduced thrombin-induced Ca2+ release from the TBHQ-sensitive stores, without having any effect on the responses elicited by ADP or AVP. Finally, the phospholipase C inhibitor, U-73122, abolished ADP- and AVP-induced Ca2+ release, suggesting that their responses are entirely dependent on IP3 generation. In contrast, treatment with both U-73122 and nimodipine was required to abolish thrombin-induced Ca2+ release. We suggest that thrombin evokes Ca2+ release from TBHQ-sensitive and insensitive stores, which requires both NAADP and IP3, respectively, while ADP and AVP exert an IP3-dependent release of Ca2+ from the TBHQ-insensitive compartment in human platelets. [Copyright &y& Elsevier]

Abstract: Store-mediated Ca2+ entry (SMCE), which is rapidly activated by depletion of the intracellular Ca2+ stores, is a major mechanism for Ca2+ influx. Several studies have involved tyrosine kinases in the activation of SMCE, such as pp60src, although at present those involved in the early activation steps are unknown. Here we report the involvement of Bruton''s tyrosine kinase (Btk) in the early stages of SMCE in human platelets. Cell treatment with thrombin or thapsigargin (TG) plus ionomycin (Iono) results in rapid activation of Btk, which was independent of rise in intracellular Ca2+ concentration ([Ca2+]i) but dependent on H2O2 generation. Platelet treatment with Btk inhibitors, LFM-A13 or terreic acid, significantly reduced TG+Iono- and thrombin-evoked SMCE. Btk was rapidly activated by addition of low concentrations of H2O2, whose effect on Ca2+ entry was prevented by Btk inhibitors. Our results indicate that pp60src and Btk co-immunoprecipitate after platelet stimulation with TG+Iono, thrombin or H2O2. In addition, we have found that LFM-A13 impaired actin filament reorganization after store depletion and agonist-induced activation of pp60src, while the inhibitor of pp60src, a protein that requires actin reorganization for its activation, did not modify Btk activation, suggesting that Btk is upstream of pp60src. We propose a role for Btk in the early steps of activation of SMCE in human platelets. [Copyright &y& Elsevier]

Redox regulation is important for the modulation of cytosolic Ca2+ concentration. Hence, we have investigated the effect of H2O2 on store-mediated Ca2+ entry (SMCE). In fura-2-loaded human platelets treatment with H2O2 resulted in a concentration-dependent increase in Ca2+ release from intracellular stores, while the effect on Ca2+ entry was biphasic. In addition, 1 mM H2O2 reduced SMCE induced by agonists. The inhibitory effect of 1 mM H2O2 was prevented by inhibition of actin polymerization with cytochalasin D. Consistent with this, we found that 10 μM H2O2 and store depletion by treatment with thapsigargin plus ionomycin induced a similar temporal sequence of actin reorganization, while exposure to 1 mM H2O2 shifted the dynamics between polymerization and depolymerization in favor of the former. One millimolar H2O2-induced polymerization was reduced by treatment with methyl 2,5-dihydroxycinnamate and farnesylthioacetic acid, inhibitors of tyrosine kinases and Ras superfamily proteins, respectively. Finally, exposure to 1 mM H2O2 significantly increased store depletion-induced p60src activation. We conclude that H2O2 exerted a biphasic effect on SMCE. The inhibitory role of high H2O2 concentrations is mediated by an abnormal actin reorganization pattern involving both Ras- and tyrosine kinases-dependent pathways. [Copyright &y& Elsevier]

Using Fura-2-loaded human platelets we studied the nature of the mechanisms involved in Ca2+ signalling mediated by H2O2. In a Ca2+-free medium, H2O2 (10 μM–100 mM) induced a concentration-dependent increase in [Ca2+]i. Depletion of either agonist-sensitive or mitochondrial Ca2+ pools reduced this effect while depletion of both stores abolished it. Xestospongin C, an inositol 1,3,5-trisphosphate (IP3) receptor inhibitor, reduced Ca2+ release evoked by 1 mM H2O2 by 45%, indicating that H2O2-induced Ca2+ release involves interaction with IP3 receptors. Blockade of the IP3 turnover by lithium or treatment with U-73122 did not modify H2O2-induced Ca2+ release from the agonist-sensitive pool, suggesting the involvement of a mechanism independent of IP3 generation. H2O2 inhibited Ca2+ reuptake into the agonist-sensitive stores mediated by the sarcoendoplasmic reticulum Ca2+ ATPase (SERCA). Thimerosal (5 μM), a sulphydryl reagent, induced Ca2+ release from the agonist-sensitive stores. This event was impaired by treatment with 2 mM DTT, which also inhibited H2O2-induced Ca2+ release from the agonist-sensitive pool but not from mitochondria. H2O2 reduced the ability of the plasma membrane Ca2+ ATPase (PMCA) to extrude Ca2+ by 75%, an effect that was unaffected by DTT. Consistent with this, thimerosal did not modify the PMCA activity. Finally, exposure to H2O2 triggered platelet aggregation, which was slower than that observed after agonist stimulation. We conclude that H2O2 induced Ca2+ release from agonist-sensitive stores by oxidation of sulphydryl groups in SERCA and the IP3 receptors independently of IP3 generation. In addition, H2O2 induced Ca2+ release from mitochondria and inhibited the PMCA activity by different mechanisms in human platelets. [Copyright &y& Elsevier]

Reactive oxygen species, such as H[sub 2]O[sub 2], have been recognized as intracellular messengers involved in several cell functions. Here we report the activation of the tyrosine kinase pp60[sup 8rc] by H[sub 2]O[sub 2], a mechanism required for the activation of store-mediated Ca[sup 2+] entry (SMCE) in human platelets. Treatment of platelets with H[sub 2]O[sub 2] resuited in a time- and concentration-dependent activation of pp60[sup src]. Incubation with GF 109203X, a protein kinase C (PKC) inhibitor, prevented H[sub 2]O[sub 2]-induced pp60[sub src] activation. In contrast, dimethyl-BAPTA loading did not affect this response, suggesting that activation of pp60[sup src] by H[sub 2]O[sub 2] is independent of increases in [Ca[sup 2+]][sub i]. Cytochalasin D, an inhibitor of actin polymerization, significantly reduced H[sub 2]O[sub 2]-induced pp60[sup src] activation. We found that platelet stimulation with thapsigargin (TG) plus ionomycin (Iono) or thrombin induced rapid H[sub 2]O[sub 2] production, a mechanism independent of elevations in [Ca[sup 2+]][sub i]. Treatment of platelets with catalase attenuated TG plus Iono- and thrombin-induced activation of pp60[sup src]. In addition, catalase as well as the pp60[sup src] inhibitor, PP1, reduced both the activation of SMCE and the coupling between the hTrp1 and the IP[sub 3]R type II without having any effect on the maintenance of SMCE. Consistent with the role of PKC in the activation of pp60[sup src] by H[sub 2]O[sub 2], the PKC inhibitors GF 109202X and Ro-31-8220 were found to reduced SMCE in platelets. This study suggests that platelet activation with TG plus Iono or thrombin is associated with H[sub 2]O[sub 2] production, which acts as a second messenger by stimulating pp60[sup src] by a PKC-dependent pathway and is involved in the activation of SMCE in these cells. [ABSTRACT FROM AUTHOR]

Presents a letter to the editor about the role of the drug imatinib mesylate in cutaneous melanoma.

Background Sturge-Weber syndrome (SWS) is characterized by port-wine stains (PWS) affecting the face, eyes, and central nervous system. Pulsed dye laser (PDL) is the standard treatment for PWS. Unfortunately, recurrence is frequent because of reformation and reperfusion of blood vessels. Objective We sought to assess the clinical efficacy of topical rapamycin combined with PDL in PWS of patients with SWS. Methods We conducted a phase II, randomized, double-blind, intraindividual placebo-controlled, clinical trial. We recruited 23 patients with SWS and facial PWS (12 women; median age 33 years, age range 17-65 years) from the University Clinic of Navarra, Spain. Four interventions were evaluated: placebo, PDL + placebo, rapamycin, and PDL + rapamycin. Clinical and histologic responses were evaluated using a chromatographic computerized system, spectrometry, and histologic analyses at 6, 12, and 18 weeks after the intervention. Results PDL + rapamycin yielded the lowest digital photographic image score and the lowest percentage of vessels in histologic analysis, and showed a statistically significant improvement compared with the other interventions. The treatment was generally well tolerated. Limitations PDL was only applied to the lateral parts of the PWS area. Conclusion Topical rapamycin associated with PDL seems to be an effective treatment for PWS in patients with SWS. [ABSTRACT FROM AUTHOR]

Abstract: Store-operated Ca2+ entry (SOCE) is a major mechanism for Ca2+ influx in platelets and other cells activated by a reduction in Ca2+ concentration in the intracellular stores. SOCE has been reported to be regulated by extracellular Ca2+, although the underlying mechanism remains unclear. Here we have examined the involvement of plasma membrane-located STIM1 (PM-STIM1) in the regulation of SOCE by extracellular Ca2+. Treatment of platelets with the SERCA inhibitor thapsigargin (TG) induced Mn2+ entry, which was inhibited by extracellular Ca2+ in a concentration-dependent manner. Incubation of platelets with a specific antibody, which recognizes the extracellular amino acid sequence 25–139 of PM-STIM1 that contains the Ca2+-binding domain, prevented the inactivation of Ca2+ entry induced by extracellular Ca2+. TG induced translocation of STIM1 to the plasma membrane (PM), an event that was found to be Ca2+-dependent. In addition, TG stimulated association of PM-STIM1 with Orai1, an event that was not prevented by stabilization of the membrane cytoskeleton using jasplakinolide. These findings suggest that PM-STIM1 is important for the inactivation of SOCE by extracellular Ca2+, an event that is likely to be mediated by interaction with Orai1. [Copyright &y& Elsevier]

Non-capacitative calcium entry (NCCE) contributes to cell activation in response to the occupation of G protein-coupled membrane receptors. Thrombin administration to platelets evokes the synthesis of diacylglycerol downstream of PAR receptor activation. Diacylglycerol evokes NCCE through activating TRPC3 and TRPC6 in human platelets. Although it is known that immunophilins interact with TRPCs, the role of immunophilins in the regulation of NCCE remains unknown. Platelet incubation with FK506, an immunophilin antagonist, reduced OAG-evoked NCCE in a concentration-dependent manner, an effect that was independent on the inactivation of calcineurin (CaN). FK506 was unable to reduce NCCE evoked by OAG in platelets from TRPC6 −/− mice. In HEK-293 cells overexpressing TRPC6, currents through TRPC6 were altered in the presence of FK506. We have found interaction between FKBP38 and other FKBPs, like FKBP25, FKBP12, and FKBP52 that were not affected by FK506, as well as with calmodulin (CaM). FK506 modified the pattern of association between FKBP25 and TRPCs as well as impaired OAG-evoked TRPC3 and TRPC6 coupling in both human and mouse platelets. By performing biotinylation experiments we have elucidated that FKBP25 and FKBP38 might be found at different cellular location, the plasma membrane and the already described intracellular locations. Finally, FKBP25 and FKBP38 silencing significantly inhibits OAG-evoked NCCE in MEG-01 and HEK293 cells, while overexpression of FKBP38 does not modify NCCE in HEK293 cells. All together, these findings provide strong evidence for a role of immunophilins, including FKBP25 and FKBP38, in NCCE mediated by TRPC6. [ABSTRACT FROM AUTHOR]

Abstract: Cytosolic-free Ca2+ plays a crucial role in blood platelet function and is essential for thrombosis and hemostasis. Therefore, cytosolic-free Ca2+ concentration is tightly regulated in this cell. TRPC6 is expressed in platelets, and an important role for this Ca2+ channel in Ca2+ homeostasis has been reported in other cell types. The aim of this work is to study the function of TRPC6 in platelet Ca2+ homeostasis. The absence of TRPC6 resulted in an 18.73% decreased basal [Ca2+]c in resting platelets as compared to control cells. Further analysis confirmed a similar Ca2+ accumulation in wild-type and TRPC6-deficient mice; however, passive Ca2+ leak rates from agonist-sensitive intracellular stores were significantly decreased in TRPC6-deficient platelets. Biotinylation studies indicated the presence of an intracellular TRPC6 population, and subcellular fractionation indicated their presence on endoplasmic reticulum membranes. Moreover, the presence of intracellular calcium release in platelets stimulated with 1-oleoyl-2-acetyl-sn-glycerol further suggested a functional TRPC6 population located on the intracellular membranes surrounding calcium stores. However, coimmunoprecipitation assay confirmed the absence of STIM1–TRPC6 interactions in resting conditions. This findings together with the absence of extracellular Mn2+ entry in resting wild-type platelets indicate that the plasma membrane TRPC6 fraction does not play a significant role in the maintenance of basal [Ca2+]c in mouse platelets. Our results suggest an active participation of the intracellular TRPC6 fraction as a regulator of basal [Ca2+]c, controlling the passive Ca2+ leak rate from agonist-sensitive intracellular Ca2+ stores in resting platelets. [Copyright &y& Elsevier]

Abstract: Immunophilins are FK506-binding proteins that have been involved in the regulation of calcium homeostasis, either by modulating Ca2+ channels located in the plasma membrane or in the rough endoplasmic reticulum (RE). We have investigated whether immunophilins would participate in the regulation of stored-operated Ca2+ entry (SOCE) in human platelets and MEG 01. Both cell types were loaded with fura-2 for determining cytosolic calcium concentration changes ([Ca2+]c), or stimulated and fixed to evaluate the protein interaction profile by performing immunoprecipitation and western blotting. We have found that incubation of platelets with FK506 increases Ca2+ mobilization. Thapsigargin (TG)-evoked, Thr-evoked SOCE and TG-evoked Mn2+ entry resulted in significant reduction by treatment of platelets with immunophilin antagonists. We confirmed by immunoprecipitation that immunophilins interact with transient receptor potential channel 1 (TRPC1) and Orai1 in human platelets. FK506 and rapamycin reduced the association between TRPC1 and Orai1 with FK506 binding protein (52) (FKBP52) in human platelets, and between TRPC1 and the type II IP3R, which association is known to be crucial for the maintenance of SOCE in human platelets. FKBP52 role in SOCE activation was confirmed by silencing FKBP52 using SiRNA FKBP52 in MEG 01 as demonstrated by single cell configuration imaging technique. TRPC1 silencing and depletion of cell of TRPC1 and FKBP52 simultaneously, impair activation of SOCE evoked by TG in MEG 01. Finally, in MEG 01 incubated with FK506 we observed a reduction in TRPC1/FKBP52 coupling, and similarly, FKBP52 silencing reduced the association between IP3R type II and TRPC1 during SOCE. All together, these results demonstrate that immunophilins participate in the regulation of SOCE in human platelets. [Copyright &y& Elsevier]

Abstract: Stromal interaction molecule 1 (STIM1) is a key element of the store-operated Ca2+ entry mechanism (SOCE). Recently, regulation of STIM1 by glycosylation and phosphorylation on serine/threonine or proline residues has been described; however other modes of phosphorylation that are important for activating SOCE in platelets, such as tyrosine phosphorylation, have been poorly investigated. Here we investigate the latency of STIM1 phosphorylation on tyrosine residues during the first steps of SOCE activation. Human platelets were stimulated and fixed at desired times using rapid kinetic assays instruments, and immunoprecipitation and western blotting techniques were then used to investigate the pattern of STIM1 tyrosine phosphorylation during the first steps of SOCE activation. We have found that maximal STIM1 tyrosine phosphorylation occurred 2.5s after stimulation of human platelets with thapsigargin (Tg). STIM1 localized in the plasma membrane were also phosphorylated in platelets stimulated with Tg. By using chemical inhibitors that target different members of the Src family of tyrosine kinases (SKFs), two independent signaling pathways involved in STIM1 tyrosine phosphorylation during the first steps of SOCE activation were identified. We finally conclude that STIM1 tyrosine phosphorylation is a key event for the association of STIM1 with plasma membrane Ca2+ channels such as Orai1, hence it is required for conducting SOCE activation. [Copyright &y& Elsevier]

A major pathway for stimulated Ca2+ entry in nonexcitable cells is activated following depletion of intracellular Ca2+ stores. Secretion-like coupling between elements in the plasma membrane (PM) and Ca2+ stores has been proposed as the most likely mechanism to activate this store-mediated Ca2+ entry (SMCE) in several cell types. Here we identify two mechanisms for SMCE in human platelets activated by depletion of two independent Ca2+ pools, which are differentially modulated by the actin cytoskeleton. Ca2+ entry induced by depletion of a 2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ)sensitive pool is increased by disassembly of the actin cytoskeleton and that induced by a TBHQ-insensitive pool is reduced. Stabilization of the actin cytoskeleton prevented Ca2+ entry by both mechanisms. We propose that the membrane-associated actin network prevents constitutive Ca2+ entry via both pathways. Reorganization of the actin cytoskeleton permits the activation of Ca2+ entry via both mechanisms, but only SMCE activated by the TBHQ-insensitive pool requires new actin polymerization, which may support membrane trafficking toward the PM. [ABSTRACT FROM AUTHOR]

Objective: We evaluated the long-term effects of a fat-enriched diet (virgin olive oil) on calcium mobilization and amylase secretion induced by cholecystokinin-octapeptide (CCK-8) in rat pancreatic acinar cells. Olive oil is a major component of the Mediterranean diet, and its role in human health is actively being debated.Methods: Weaning male Wistar rats (21 d old) were assigned to one of two experimental groups and fed for 8 wk with a commercial chow (control group) or an experimental diet (olive group) containing 100 g/kg of virgin olive oil as dietary fat. Intracellular free calcium [Ca2+]i levels were determined by loading the pancreatic cells with the fluorescent ratio-metric calcium indicator Fura-2 on an inverted fluorescent microscope. For measurement of amylase secretion, cells were incubated with the appropriate secretagogue for 30 min, and amylase activities in the supernatant were determined by the Phadebas blue starch method. Analysis of variance was used to test differences between groups.Results: Compared with the control group, the CCK-8–induced increase in [Ca2+]i occurred in cells from rats in the olive group (P < 0.05). This stimulatory effect of dietary virgin olive oil was observed in calcium oscillations and large [Ca2+]i transients induced by low (20 pM/L) and high (10 nM/L) concentrations of CCK-8, respectively. In addition to the effects of dietary virgin olive oil on calcium mobilization, it increased (P < 0.05) amylase secretion in response to CCK-8. Olive oil treatment did not significantly alter resting [Ca2+]i or amylase release values compared with the control group. Similar results were obtained when pancreatic acinar cells were stimulated with a high concentration of acetylcholine (10 μM/L).Conclusion: The present results demonstrate that a diet supplemented with virgin olive oil can modify pancreatic cell function as assessed by [Ca2+]i mobilization and amylase release evoked by secretagogues in rat pancreatic acinar cells. A role for fatty acids in calcium signaling is suggested. [Copyright &y& Elsevier]