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Gliomas are characterized by diffuse infiltration of tumor cells into surrounding brain tissue, and this highly invasive nature contributes to disease recurrence and poor patient outcomes. The molecular mechanisms underlying glioma cell invasion remain incompletely understood, limiting development of new targeted therapies. Here, we have identified phosphotyrosine adaptor protein ShcD as upregulated in malignant glioma and shown that it associates with receptor tyrosine kinase Tie2 to facilitate invasion. In human glioma cells, we find that expression of ShcD and Tie2 increases invasion, and this significant synergistic effect is disrupted with a ShcD mutant that cannot bind Tie2 or hyperphosphorylate the receptor. Expression of ShcD and/or Tie2 further increases invadopodia formation and matrix degradation in U87 glioma cells. In a coculture model, we show that U87-derived tumor spheroids expressing both ShcD and Tie2 display enhanced infiltration into cerebral organoids. Mechanistically, we identify changes in focal adhesion kinase phosphorylation in the presence of ShcD and/or Tie2 in U87 cells upon Tie2 activation. Finally, we identify a strong correlation between transcript levels of ShcD and Tie2 signaling components as well as N-cadherin in advanced gliomas and those with classical or mesenchymal subtypes, and we show that elevated expression of ShcD correlates with a significant reduction in patient survival in higher grade gliomas with mesenchymal signature. Altogether, our data highlight a novel Tie2–ShcD signaling axis in glioma cell invasion, which may be of clinical significance. Implications: ShcD cooperates with Tie2 to promote glioma cell invasion and its elevated expression correlates with poor patient outcome in advanced gliomas.

Tumor cell invasion involves targeted localization of proteins required for interactions with the extracellular matrix and for proteolysis. The localization of many proteins during these cell–extracellular matrix interactions relies on membrane trafficking mediated in part by SNAREs. The SNARE protein syntaxin4 (Stx4) is involved in the formation of invasive structures called invadopodia; however, it is unclear how Stx4 function is regulated during tumor cell invasion. Munc18c is known to regulate Stx4 activity, and here we show that Munc18c is required for Stx4-mediated invadopodium formation and cell invasion. Biochemical and microscopic analyses revealed a physical association between Munc18c and Stx4, which was enhanced during invadopodium formation, and that a reduction in Munc18c expression decreases invadopodium formation. We also found that an N-terminal Stx4-derived peptide associates with Munc18c and inhibits endogenous interactions of Stx4 with synaptosome-associated protein 23 (SNAP23) and vesicle-associated membrane protein 2 (VAMP2). Furthermore, expression of the Stx4 N-terminal peptide decreased invadopodium formation and cell invasion in vitro. Of note, cells expressing the Stx4 N-terminal peptide exhibited impaired trafficking of membrane type 1 matrix metalloproteinase (MT1-MMP) and EGF receptor (EGFR) to the cell surface during invadopodium formation. Our findings implicate Munc18c as a regulator of Stx4-mediated trafficking of MT1-MMP and EGFR, advancing our understanding of the role of SNARE function in the localization of proteins that drive tumor cell invasion.

1,25 Dihydroxyvitamin D{sub 3} (1,25D{sub 3}) primes NB4 promyelocytic leukemia cells to differentiate along the monocyte/macrophage lineage through a non-genomic mechanism. Here we show that NB4 cells express high levels of the recently identified membrane receptor for 1,25D{sub 3}, which is a distinct gene product from the classical nuclear vitamin D receptor. This 57 kDa protein, named 1,25D{sub 3}-MARRS (Membrane Activated Rapid Response to Steroids)/ERp57/PIA3 appears to associate in a complex with the transcription factor, nuclear factor kappa B (NF{kappa}B). In unstimulated cells, 1,25D{sub 3}-MARRS can be co-immunoprecipitated with antibodies directed at NF{kappa}B, and NF{kappa}B is co-precipitated when antibodies against 1,25D{sub 3}-MARRS or ERp57 are used. Confocal microscopy and subcellular fractionation studies demonstrate that both 1,25D{sub 3}-MARRS and NF{kappa}B begin translocating to the nucleus within minutes of co-stimulation with 1,25D{sub 3} and phorbol ester. The predominant nuclear localization of both proteins precedes the expression of the monocyte/macrophage phenotype and suggests that this event may be critical to the differentiation pathway. This suggests a role for 1,25D{sub 3}-MARRS in the nucleus as a regulator of gene expression. Here it may also regulate the activity of NF{kappa}B and other factors with which it may be interacting.

Detection of early carotid vascular disease in patients with systemic lupus erythematosus (SLE) is considered mandatory for evaluation of subclinical atherosclerosis (ATS), and various ultrasonographic (US) parameters have been proposed. In the present investigation, 33 SLE and 33 healthy age-matched females have been studied by colour-coded sonography of the common carotid artery, assessing intima-media thickness (IMT), vascular strain (VS), vascular distensibility (VD), vascular stiffness (VSf) and pressure-strain elastic modulus (PSEM) as possible markers of early ATS. Patients with SLE, despite equivalent exposure to “traditional” cardiovascular risk factors, presented a higher mean IMT of the common carotid artery than healthy subjects (0.7 ± 0.2 mm vs 0.5 ± 0.1 mm – P

Alexithymia is conceptualized as a disorder of emotion regulation mechanisms, which involves a dissociation of emotional and physic responses to life events and bodily sensations. Our results might suggest a possible relationship between the alexithymic construct and TNF levels in RA patients. These preliminary findings corroborate the integrated bidirectional interactions between neuropsychological mechanisms and the neuroendocrine-immune system in patients affected by autoimmune diseases and contribute to finding a common biological pathway linking alexithymia and autoimmune-inflammatory diseases.

Objective To evaluate the metabolic effect of buccal spray insulin compared with subcutaneous regular insulin in patients with type 1 diabetes. Research Design and Methods This study compared plasma glucose, insulin, and C-peptide levels in 18 patients with type 1 diabetes treated with subcutaneous regular or buccal spray insulin on 2 consecutive mornings. On day 1, patients were treated with their usual subcutaneous regular insulin regimens. On day 2, patients received buccal spray insulin. In the morning of both days 1 and 2, patients received a standard meal of 630 kJ. No intermediate or long-acting insulin was administered to patients on the morning of the test. Blood samples were collected for up to 4 hours for biokinetic analysis. In a subset of 3 patients, premeal buccal spray insulin was administered for 2 entire consecutive days. In these patients, glucose levels were monitored using the glucose sensor monitoring system. Results Overall, there were no statistically significant differences in glucose, insulin, or C-peptide levels measured after administration of subcutaneous vs buccal spray insulin. However, at 90 and 120 minutes after subcutaneous regular insulin administration, significantly higher insulin levels and more prolonged hypoglycemic effect were detected compared with buccal spray insulin administration. In the 3 patients who received 1 day of regular and 2 entire days of buccal spray insulin, no significant differences were observed in glucose levels during the 3 days of glucose sensor monitoring. Conclusions Insulin administered via the buccal spray formulation is as effective as the subcutaneous route in lowering blood glucose levels.

Cell migration occurs as a highly-regulated cycle of cell polarization, membrane extension at the leading edge, adhesion, contraction of the cell body, and release from the extracellular matrix at the trailing edge. In this study, we investigated the involvement of SNARE-mediated membrane trafficking in cell migration. Using a dominant-negative form of the enzyme N-ethylmaleimide-sensitive factor as a general inhibitor of SNARE-mediated membrane traffic and tetanus toxin as a specific inhibitor of VAMP3/cellubrevin, we conducted transwell migration assays and determined that serum-induced migration of CHO-K1 cells is dependant upon SNARE function. Both VAMP3-mediated and VAMP3-independent traffic were involved in regulating this cell migration. Inhibition of SNARE-mediated membrane traffic led to a decrease in the protrusion of lamellipodia at the leading edge of migrating cells. Additionally, the reduction in cell migration resulting from the inhibition of SNARE function was accompanied by perturbation of a Rab11-containing alpha(5)beta(1) integrin compartment and a decrease in cell surface alpha(5)beta(1) without alteration to total cellular integrin levels. Together, these observations suggest that inhibition of SNARE-mediated traffic interferes with the intracellular distribution of integrins and with the membrane remodeling that contributes to lamellipodial extension during cell migration.

Objective.To study concentrations of adipokines in patients with systemic lupus erythematosus (SLE) and the relationship among adipokines, the metabolic syndrome (MeS), and cardiovascular disease (CVD) risk factors.Methods.We enrolled 50 SLE patients and 26 controls, all women. Leptin, resistin, visfatin, and adiponectin were measured by commercial ELISA kits.Results.MeS prevalence was increased among subjects with SLE. Leptin levels were higher in patients with SLE than controls. Among SLE patients, independent determinants of leptin were insulin levels (p < 0.0001), triglycerides (p = 0.03), body mass index (p = 0.02), corticosteroid dosage (p = 0.02), and SLE Disease Activity Index (p = 0.005). Other adipokines did not differ between SLE patients and controls.Conclusion.Leptin was increased in SLE patients and could play a role in SLE-related cardiovascular diseases.

Malignant transformation of fibroblast and epithelial cells is accompanied by increased beta 1-6 N-acetylglucosaminyltransferase V (GlcNAc-TV) activity, a Golgi N-linked oligosaccharide processing enzyme. Herein, we report that expression of GlcNAc-TV in Mv1Lu cells, an immortalized lung epithelial cell line results in loss of contact-inhibition of cell growth, an effect that was blocked by swainsonine, an inhibitor of Golgi processing enzyme alpha-mannosidase II. In serum-deprived and high density monolayer cultures, the GlcNAc-TV transfectants formed foci, maintained microfilaments characteristic of proliferating cells, and also experienced accelerated cell death by apoptosis. Injection of the GlcNAc-TV transfectants into nude mice produced a 50% incidence of benign tumors, and progressively growing tumors in 2:12 mice with a latency of 6 mo, while no growth was observed in mice injected with control cells. In short term adhesion assays, the GlcNAc-TV expressing cells were less adhesive on surfaces coated with fibronectin and collagen type IV, but no changes were observed in levels of cell surface alpha 5 beta 1 or alpha v beta 3 integrins. The larger apparent molecular weights of the LAMP-2 glycoprotein and integrin glycoproteins alpha 5, alpha v and beta 1 in the transfected cells indicates that their oligosaccharide chains are substrates for GlcNAc-TV. The results suggest that beta 1-6GlcNAc branching of N-linked oligosaccharides contributes directly to relaxed growth controls and reduce substratum adhesion in premalignant epithelial cells.

Background Previous studies on pain prevalence among systemic sclerosis (SSc) patients confirms its prominent role in disease outcomes 1 . Although pain perception is receiving more importance among patient-reported outcomes in SSc research, still its interface with mood disorders and immune system need to be further elucidated. The prevalence of clinically significant symptoms of depression or anxiety in SSc varies between 36% and 65% according to the evaluation used, and are rather associated with increased disability and altered HRQoL, than with disease-specific organ manifestations. An endocrine influence on pain perception and threshold is supported by robust evidences, more specifically related to sex, adrenal and thyroid hormones. Objectives To analyze the association between pain threshold, depression and cortisol serum levels in systemic sclerosis patients, major depression disorder patients and healthy controls. Methods Thirty SSc patients, 30 major depressive disorder (MDD) patients and 30 healthy controls completed the study. All participants underwent pressure pain threshold (PPT) assessment through an algometer in three anatomical surfaces, the ungula bed (UPPT) and the metacarpophalangeal joint (MPPT) of the second finger of the right hand and the anterior portion of the quadriceps muscle (QPPT), and depression evaluation, assessed by patient health questionnaire 9 (PHQ-9). Serum cortisol was measured in SSc and MDD patients for further analysis. Patients under treatment with corticosteroids were excluded. Results PHQ-9 values showed that 15% of SSc patients had no signs of depression, while 30% had mild and another 30% moderate signs of depression, 18% moderate/severe and 9% severe signs. Among MDD patients, PHQ-9 12% had mild depression, 45% moderate, 15 moderate to severe and 28% severe depression. PHQ-9 scores were significantly higher in SSc and MDD compared to controls (p Conclusions The interface between pain, depression and cortisol could represent an additional complex aspect of care of patients with either SSc or MDD. References Schieir O, Thombs BD, Hudson M, Boivin JF, Steele R, Bernatsky S, Hanley J, Baron M; Canadian Scleroderma Research Group. Prevalence, severity, and clinical correlates of pain in patients with systemic sclerosis. Arthritis Care Res (Hoboken). 2010 Mar;62(3):409–17. Disclosure of Interest None declared

In multicellular organisms, uncontrolled movement of cells can contribute to pathological conditions, such as multiple sclerosis and cancer. In highly aggressive tumors, the expression of matrix metalloproteinases (MMPs) is linked to the capacity of tumor cells to invade surrounding tissue and current research indicates that the membrane-anchored membrane type 1-matrix metalloproteinase (MT1-MMP) has a central role in this process. Endocytosis and trafficking of MT1-MMP are essential for its proper function, and here we examine the phosphorylation, internalization, and recycling of this enzyme, and the associated biochemical signaling in HeLa and HT-1080 fibrosarcoma cells. Activation of protein kinase C with phorbol 12-myristate 13-acetate resulted in phosphorylation of endogenous MT1-MMP at Thr(567) in vivo. Mutation of Thr(567) to alanine (to mimic non-phosphorylated MT1-MMP) reduced internalization of MT1-MMP, whereas mutation of Thr(567) to glutamic acid (to mimic phosphorylation) resulted in decreased levels of MT1-MMP on the cell surface. The endosomal trafficking and recycling of MT1-MMP was found to be dependent upon Rab7 and VAMP7, and blocking the function of these proteins reduced cell migration and invasion. Intracellular trafficking of MT1-MMP was observed to be coupled to the trafficking of integrin α5 and phosphorylation of ERK that coincided with this was dependent on phosphorylation of MT1-MMP. Together, these results reveal important roles for MT1-MMP phosphorylation and trafficking in both cell signaling and cell invasion.

Neuropsychiatric involvement in patients with Systemic Lupus Erythematosus (SLE), first mentioned by Kaposi more than 100 years ago, still remains one of the main challenge facing rheumatologist and other physicians. The diagnosis of neuropsychiatric SLE (NPSLE) is complex not only because of the considerable prevalence variation (14-80%) but also because of the wide spectrum of NP manifestations. They vary from overt neurologic alterations (seizure, psychosis), to subtle abnormalities (neurocognitive dysfunctions). Different NP manifestations result from a variety of mechanisms including antibodies, vasculitis, thrombosis, hemorrhages and cytokine-mediated damages. Of note, despite the dramatic clinical manifestations, too often changes at the morphological neuroimaging techniques are minimal and non specific. There is no one diagnostic tool specific for NPSLE and diagnosis must be based on the combinated use of immunoserological tests, functional and anatomical neuroimaging and standardized specific criteria. Symptomatic, immunosuppressive and anticoagulant therapies are the main strategies available in the management of these patients. Therapy for CNS lupus should be adjusted according to the needs of the individual patients. The coming years promise to be an important time for the development of new neuroimaging techniques and for the study of disease mechanism. An early and objective identification of brain involvement will allow for appropriate treatment to avoid severe complications.

Malignant melanoma, characterized by invasive local growth and early formation of metastases, is the most aggressive type of skin cancer. Melanoma inhibitory activity (MIA), secreted by malignant melanoma cells, interacts with the cell adhesion receptors, integrins α(4)β(1) and α(5)β(1), facilitating cell detachment and promoting formation of metastases. In the present study, we demonstrate that MIA secretion is confined to the rear end of migrating cells, while in non-migrating cells MIA accumulates in the actin cortex. MIA protein takes a conventional secretory pathway including coat protein complex I (COPI)- and coat protein complex II (COPII)-dependent protein transport to the cell periphery, where its final release depends on intracellular Ca(2+) ions. Interestingly, the Ca(2+)-activated K(+)-channel, subfamily N, member 4 (KCa3.1), known to be active at the rear end of migrating cells, was found to support MIA secretion. Secretion was diminished by the specific KCa3.1 channel inhibitor TRAM-34 and by expression of dominant-negative mutants of the channel. In summary, we have elucidated the migration-associated transport of MIA protein to the cell rear and also disclosed a new mechanism by which KCa3.1 potassium channels promote cell migration.

Background To compare tumor necrosis in hepatoma induced in rats by a single percutaneous injection of ethanol (PEI) or acetic acid (PAI). Methods BW7756 hepatomas of 1 mm3 were implanted in the liver of 40 male healthy rats. After 14 days, the 36 surviving rats were treated, in a single session, by ultrasound-guided injection of 300 μl of 95% ethanol (n = 17) or 100 μl of 50% acetic acid (n = 19). They were sacrificed 14 days after treatment and explanted tumoral livers were examined. The same PAI procedure was repeated on 13 additional rats to exclude a suspected occurrence of technical failures during the experiment, due to a surprisingly high rate of deaths within 30 minutes after PAI. Results Four rats died within four days after tumor implantation; after PEI, 1/17 (6%) died, whereas after PAI 9/19 (47%) died. The remaining 26 rats, after 14 days post-percutaneous ablation, were sacrificed. Gross and microscopic examinations showed that the hepatoma's nodules treated with PEI had 45.3 ± 19.4% tumor necrosis compared to 49 ± 23.3% (P = NS) for those treated with PAI. Complete tumor necrosis was not found in any animal. Peritoneal invasion was present in 4/16 (25%) and 2/10 (20%) rats treated with PEI or PAI, respectively (P = NS). Autopsy was performed in the 5 additional rats that died within 30 minutes after PAI. Conclusion Our results show that there is no significant difference in the percentage of tumor necrosis between two local ablation methods in spite of the different dosages used. However, mortality in the PAI-treated group was greater than in PEI-treated group, presumably due to greater acetic acid systemic diffusion and its metabolic side effects. In human subjects, HCC occurs in the setting of cirrhosis, where the non-tumoral tissue is firmer than the tumor structure, with consequent reduction of drug diffusion. This could be the reason why some human studies have concluded similar or even better safety and efficacy with PAI compared to PEI.

The antiphospholipid syndrome (APS) is an autoimmune disorder, characterized by a wide spectrum of clinical manifestations. Thromboembolic events, with a greater involvement of extremities veins, are the most common features, and obstruction of abdominal vessels are sporadically reported. We present a singular case of a patient with primary APS (PAPS) that developed a spontaneous splenorenal shunt, secondary to a total portal, mesenteric and splenic vein thrombosis. Spontaneous splenorenal shunt, an uncommon circumstance reported in cirrhotic disease, to the best of our knowledge, has not been previously described in PAPS.

Type 1 diabetes is the result of the destruction of insulin-producing beta cells in the pancreatic islets. Still undefined environmental factors operating in a genetically susceptible individual with the generation of an autoimmune response towards beta cells ultimately culminate in the onset of hyperglycaemia [1–3]. In different individuals the process that leads to beta cell destruction may vary, being under the effect of different genetic, environmental, and immunological factors [4].

Objective: Aim of this study was to evaluate the prevalence of alexithymia in patients affected by SLE or RA and to investigate the correlation between alexithymia and immunoendocrine parameters (PRL, hGH, IL-6 and TNF-alfa). Methods: Twenty-five patients (12 and 13 affected by SLE and RA, respectively) were enrolled into the study. The Toronto Alexithymia Scale-20 (TAS-20) was administered. PRL, hGH, IL-6 and TNF-alfa levels were measured by commercially available ELISA kits. Results: Alexithymia prevalence (TAS-20≥51) was 54% in RA and 42% in SLE patients. hGH serum levels were 3.1±4.2 and 1.1±0.9 IU/ml in SLE and RA, respectively. PRL concentration was 18.4±6.5 ng/ml and 14.2±4.0 ng/ml in SLE and RA patients, respectively (p=0.03). In RA group, TNF-alpha was 20±36.2 whereas in SLE it was 4.9±12.8 pg/ml (p=0.03); IL-6 serum concentrations were 24.4±25.1 and 2.9±5.4 pg/ml, in RA and SLE respectively (p=0.004). The serum level of hGH showed slight increase in alexithymic group (A) compared to non alexithymic group (NA) in both SLE and RA patients. PRL serum levels in SLE-A patients was 26.7±17.3 ng/ml while in SLE-NA patients was 12.4±3.3 ng/ml (p=0.04). In RA patients increased values of IL-6 and TNF-alpha were present in the A group compared to NA group (IL-6: 35.3±28 pg/mL vs 3.5±3.9 pg/mL, p=0.01; TNF-alpha: 34.7±39 pg/mL vs 3.1±3.4 pg/mL, p=0.01). Conclusions: In this preliminary results we found an high prevalence of alexithymia and a correlation between immunoendocrine parameters and alexhytimic features in SLE and RA, suggesting that an immunomodulatory pathway could influence this cognitive style in patients with autoimmune disorders. Other studies should contribute to find a common biological pathway linking alexithymia and autoimmunity.