Melanoma cells with acquired resistance to vemurafenib have decreased autophagic flux and display enhanced ability to transfer resistance.

Over the last years, the incidence of melanoma, the deadliest form of skin cancer, has risen significantly. Nearly half of the melanoma patients exhibit the BRAFV600E mutation. Although the use of BRAF and MEK inhibitors (BRAFi and MEKi) showed an impressive success rate in melanoma patients, durability of response remains an issue because tumor quickly becomes resistant. Here, we generated and characterized Lu1205 and A375 melanoma cells resistant to vemurafenib (BRAFi). Resistant cells (Lu1205R and A375R) exhibit higher IC50 (5-6 fold increase) and phospho-ERK levels and 2-3 times reduced apoptosis than their sensitive parents (Lu1205S and A375S). Moreover, resistant cells are 2-3 times bigger, display a more elongated morphology and have a modulation of migration capacity. Interestingly, pharmacological inhibition of sphingosine kinases, that prevents sphingosine-1-phosphate production, reduces migration of Lu1205R cells by 50 %. In addition, although Lu1205R cells showed increased basal levels of the autophagy markers LC3II and p62, they have decreased autophagosome degradation and autophagy flux. Remarkably, expression of Rab27A and Rab27B, which are involved in the release of extracellular vesicles are dramatically augmented in resistant cells (i.e. 5-7 fold increase). Indeed, conditioned media obtained from Lu1205R cells increased the resistance to vemurafenib of sensitive cells. Hence, these results support that resistance to vemurafenib modulates migration and the autophagic flux and may be transferred to nearby sensitive melanoma cells by factors that are released to the extracellular milieu by resistant cells.

Myelin-associated glycoprotein activation triggers glutamate uptake by oligodendrocytes in vitro and contributes to ameliorate glutamate-mediated toxicity in vivo.

BACKGROUND: Myelin-associated glycoprotein (MAG) is a key molecule involved in the nurturing effect of myelin on ensheathed axons. MAG also inhibits axon outgrowth after injury. In preclinical stroke models, administration of a function-blocking anti-MAG monoclonal antibody (mAb) aimed to improve axon regeneration demonstrated reduced lesion volumes and a rapid clinical improvement, suggesting a mechanism of immediate neuroprotection rather than enhanced axon regeneration. In addition, it has been reported that antibody-mediated crosslinking of MAG can protect oligodendrocytes (OLs) against glutamate (Glu) overload by unknown mechanisms. PURPOSE: To unravel the molecular mechanisms underlying the protective effect of anti-MAG therapy with a focus on neuroprotection against Glu toxicity. RESULTS: MAG activation (via antibody crosslinking) triggered the clearance of extracellular Glu by its uptake into OLs via high affinity excitatory amino acid transporters. This resulted not only in protection of OLs but also nearby neurons. MAG activation led to a PKC-dependent activation of factor Nrf2 (nuclear-erythroid related factor-2) leading to antioxidant responses including increased mRNA expression of metabolic enzymes from the glutathione biosynthetic pathway and the regulatory chain of cystine/Glu antiporter system xc- increasing reduced glutathione (GSH), the main antioxidant in cells. The efficacy of early anti-MAG mAb administration was demonstrated in a preclinical model of excitotoxicity induced by intrastriatal Glu administration and extended to a model of Experimental Autoimmune Encephalitis showing axonal damage secondary to demyelination. CONCLUSIONS: MAG activation triggers Glu uptake into OLs under conditions of Glu overload and induces a robust protective antioxidant response.

Diazepam Impairs Innate and Adaptive Immune Responses and Ameliorates Experimental Autoimmune Encephalomyelitis.

Currently there is increasing attention on the modulatory effects of benzodiazepines on the immune system. Here, we evaluate how Diazepam (DZ) affects both innate and adaptive immunity. We observed that treatment with DZ and Lipopolysaccharide (LPS) on macrophages or dendritic cells (DCs) induced a defective secretion of IL-12, TNF-α, IL-6 and a lesser expression of classical activation markers as NO production and CD40 in comparison with LPS condition. More importantly, mice pre-treated with DZ and then challenged to LPS induced-septic shock showed reduced death. The DZ treatment shifted the LPS-induced pro-inflammatory cytokine production of peritoneal cells (PCs) to an anti-inflammatory profile commanded by IL-10. In agreement with this, DZ treatment prevented LPS-induced DC ability to initiate allogeneic Th1 and Th17 responses in vitro when compared with LPS-matured DC. Since these inflammatory responses are the key in the development of the experimental autoimmune encephalomyelitis (EAE), we treated EAE mice preventively with DZ. Mice that received DZ showed amelioration of clinical signs and immunological parameters of the disease. Additionally, DZ reduced the release of IFN-γ and IL-17 by splenocytes from untreated sick mice in vitro. For this reason, we decided to treat diseased mice therapeutically with DZ when they reached the clinical score of 1. Most importantly, this treatment ameliorated clinical signs, reduced the MOG-specific inflammatory cytokine production and prevented axonal damage. Altogether, these results indicate that DZ is a potent immunomodulator capable of controlling undesired innate and adaptive immune responses, both at the beginning of these responses and also once they have started.

A Potent N-(piperidin-4-yl)-1H-pyrrole-2-carboxamide Inhibitor of Adenylyl Cyclase of G. lamblia: Biological Evaluation and Molecular Modelling Studies.

In this work, we report a derivative of N-(piperidin-4-yl)-1H-pyrrole-2-carboxamide as a new inhibitor for adenylyl cyclase of Giardia lamblia which was obtained from a study using structural data of the nucleotidyl cyclase 1 (gNC1) of this parasite. For such a study, we developed a model for this specific enzyme by using homology techniques, which is the first model reported for gNC1 of G. lamblia. Our studies show that the new inhibitor has a competitive mechanism of action against this enzyme. 2-Hydroxyestradiol was used as the reference compound for comparative studies. Results in this work are important from two points of view. on the one hand, an experimentally corroborated model for gNC1 of G. lamblia obtained by molecular modelling is presented; on the other hand, the new inhibitor obtained is an undoubtedly excellent starting structure for the development of new metabolic inhibitors for G. lamblia.

Annual program review process: an enhanced process with outcomes.

The Accreditation Council for Graduate Medical Education's required Annual Program Review of Educational Effectiveness (APREE) has helped us improve our program and change its culture to one of continuous quality improvement. This report outlines our systematic process and describes specific outcomes it has produced over a 10-year period. We identified ways to enhance our APREE after reading articles that described various ways to conduct the process found in a PubMed and OvidSP search and relevant policies from our local Graduate Medical Education Office. After discussing options, we incorporated new ideas into our APREE and tasked our Program Evaluation Committee to track outcomes from objectives developed by faculty and residents during each APREE. Objectives from faculty and residents in 10 years of our APREE led to major improvements (e.g., increased board pass rate) in our program. In addition, the enhanced APREE process gradually changed our residency's culture to one that embraces continuous quality improvement. A systematic APREE process can engage residents and faculty in improving specific components of a residency. Besides providing outcomes for Web Ads and Self-Study items, the APREE models quality improvement techniques to residents, involves a wide array of stakeholders, and helps program stakeholders embrace continuous quality improvement.

PD-L2 negatively regulates Th1-mediated immunopathology during Fasciola hepatica infection.

Macrophage plasticity is critical for controlling inflammation including those produced by helminth infections, where alternatively activated macrophages (AAM) are accumulated in tissues. AAM expressing the co-inhibitory molecule programmed death ligand 2 (PD-L2), which is capable of binding programmed death 1 (PD-1) expressed on activated T cells, have been demonstrated in different parasitic infections. However, the role of PD-L2 during F. hepatica infection has not yet been explored. We observed that F. hepatica infection or a F. hepatica total extract (TE) injection increased the expression of PD-L2 on peritoneal macrophages. In addition, the absence of PD-L2 expression correlated with an increase in susceptibility to F. hepatica infection, as evidenced by the shorter survival and increased liver damage observed in PD-L2 deficient (KO) mice. We assessed the contribution of the PD-L2 pathway to Th2 polarization during this infection, and found that the absence of PD-L2 caused a diminished Th2 type cytokine production by TE stimulated splenocytes from PD-L2 KO infected compared with WT mice. Besides, splenocytes and intrahepatic leukocytes from infected PD-L2 KO mice showed higher levels of IFN-γ than those from WT mice. Arginase expression and activity and IL-10 production were reduced in macrophages from PD-L2 KO mice compared to those from WT mice, revealing a strong correlation between PD-L2 expression and AAM polarization. Taken together, our data indicate that PD-L2 expression in macrophages is critical for AAM induction and the maintenance of an optimal balance between the Th1- and Th2-type immune responses to assure host survival during F. hepatica infection.

Fasciola hepatica Kunitz type molecule decreases dendritic cell activation and their ability to induce inflammatory responses.

The complete repertoire of proteins with immunomodulatory activity in Fasciola hepatica (Fh) has not yet been fully described. Here, we demonstrated that Fh total extract (TE) reduced LPS-induced DC maturation, and the DC ability to induce allogeneic responses. After TE fractionating, a fraction lower than 10 kDa (F<10 kDa) was able to maintain the TE properties to modulate the DC pro- and anti-inflammatory cytokine production induced by LPS. In addition, TE or F<10 kDa treatment decreased the ability of immature DC to stimulate the allogeneic responses and induced a novo allogeneic CD4+CD25+Foxp3+ T cells. In contrast, treatment of DC with T/L or F<10 kDa plus LPS (F<10/L) induced a regulatory IL-27 dependent mechanism that diminished the proliferative and Th1 and Th17 allogeneic responses. Finally, we showed that a Kunitz type molecule (Fh-KTM), present in F<10 kDa, was responsible for suppressing pro-inflammatory cytokine production in LPS-activated DC, by printing tolerogenic features on DC that impaired their ability to induce inflammatory responses. These results suggest a modulatory role for this protein, which may be involved in the immune evasion mechanisms of the parasite.

In vivo expression of recombinant pregnancy-specific glycoprotein 1a inhibits the symptoms of collagen-induced arthritis.

PROBLEM: The contribution of Pregnancy-specific glycoproteins (PSG), the major variant of PSG released into the circulation during pregnancy, to the pregnancy-dependent improvement of rheumatoid arthritis (RA) has still not been elucidated. METHOD OF STUDY: Collagen-induced arthritis (CIA) was used to test the hypothesis that PSG1a when released into circulation has a modulatory role on the Th1-pathogenic response, thus improving the CIA symptoms. In vivo expression of PSG1a was induced by injection of the vaccinia (Vac)-based expression vector harboring the complete open-reading frame of PSG1a cDNA. RESULTS: In vivo PSG1a expression during the induction of CIA ameliorated the clinical symptoms, thereby reducing the arthritis score and incidence. Significantly lower levels of IL-17, IL-6, and IFN-γ, but higher levels of TGF-ß and IL-10 were secreted by collagen type II-stimulated spleen mononuclear cells from Vac-PSG1a-treated mice compared with control mice. Moreover, Vac-PSG1a treatment promoted the increase in splenic CD4+CD25+Foxp3+ Treg cells. CONCLUSION: Pre-clinical Vac-PSG1a treatment suppressed the Th1- and Th17-type-specific responses, leading to an increase in splenic Treg cells as well as IL-10- and TGF-ß-secreting cells, with the CIA symptoms being ameliorated.

Human survivin and Trypanosoma cruzi calreticulin act in synergy against a murine melanoma in vivo.

Immune-based anti-tumor or anti-angiogenic therapies hold considerable promise for the treatment of cancer. The first approach seeks to activate tumor antigen-specific T lymphocytes while, the second, delays tumor growth by interfering with blood supply. Tumor Associated Antigens are often employed to target tumors with therapeutic drugs, but some are also essential for tumor viability. Survivin (Surv) is a member of the inhibitor of apoptosis protein family that is considered a Tumor Associated Antigen important for cancer cell viability and proliferation. On the other hand, Trypanosoma cruzi (the agent of Chagas' disease) calreticulin (TcCRT) displays remarkable anti-angiogenic properties. Because these molecules are associated with different tumor targets, we reasoned that immunization with a Surv-encoding plasmid (pSurv) and concomitant TcCRT administration should generate a stronger anti-tumor response than application of either treatment separately. To evaluate this possibility, C57BL/6 mice were immunized with pSurv and challenged with an isogenic melanoma cell line that had been pre-incubated with recombinant TcCRT (rTcCRT). Following tumor cell inoculation, mice were injected with additional doses of rTcCRT. For the combined regimen we observed in mice that: i). Tumor growth was impaired, ii). Humoral anti-rTcCRT immunity was induced and, iii). In vitro rTcCRT bound to melanocytes, thereby promoting the incorporation of human C1q and subsequent macrophage phagocytosis of tumor cells. These observations are interpreted to reflect the consequence of the following sequence of events: rTcCRT anti-angiogenic activity leads to stress in tumor cells. Murine CRT is then translocated to the external membrane where, together with rTcCRT, complement C1 is captured, thus promoting tumor phagocytosis. Presentation of the Tumor Associated Antigen Surv induces the adaptive anti-tumor immunity and, independently, mediates anti-endothelial cell immunity leading to an important delay in tumor growth.

The immunological response and post-treatment survival of DC-vaccinated melanoma patients are associated with increased Th1/Th17 and reduced Th3 cytokine responses.

INTRODUCTION: Immunization with autologous dendritic cells (DCs) loaded with a heat shock-conditioned allogeneic melanoma cell lysate caused lysate-specific delayed type hypersensitivity (DTH) reactions in a number of patients. These responses correlated with a threefold prolonged long-term survival of DTH(+) with respect to DTH(-) unresponsive patients. Herein, we investigated whether the immunological reactions associated with prolonged survival were related to dissimilar cellular and cytokine responses in blood. MATERIALS AND METHODS: Healthy donors and melanoma patient's lymphocytes obtained from blood before and after vaccinations and from DTH biopsies were analyzed for T cell population distribution and cytokine release. RESULTS/DISCUSSION: Peripheral blood lymphocytes from melanoma patients have an increased proportion of Th3 (CD4(+) TGF-ß(+)) regulatory T lymphocytes compared with healthy donors. Notably, DTH(+) patients showed a threefold reduction of Th3 cells compared with DTH(-) patients after DCs vaccine treatment. Furthermore, DCs vaccination resulted in a threefold augment of the proportion of IFN-γ releasing Th1 cells and in a twofold increase of the IL-17-producing Th17 population in DTH(+) with respect to DTH(-) patients. Increased Th1 and Th17 cell populations in both blood and DTH-derived tissues suggest that these profiles may be related to a more effective anti-melanoma response. CONCLUSIONS: Our results indicate that increased proinflammatory cytokine profiles are related to detectable immunological responses in vivo (DTH) and to prolonged patient survival. Our study contributes to the understanding of immunological responses produced by DCs vaccines and to the identification of follow-up markers for patient outcome that may allow a closer individual monitoring of patients.

Helminth antigens enable CpG-activated dendritic cells to inhibit the symptoms of collagen-induced arthritis through Foxp3+ regulatory T cells.

Dendritic cells (DC) have the potential to control the outcome of autoimmunity by modulating the immune response. In this study, we tested the ability of Fasciola hepatica total extract (TE) to induce tolerogenic properties in CpG-ODN (CpG) maturated DC, to then evaluate the therapeutic potential of these cells to diminish the inflammatory response in collagen induced arthritis (CIA). DBA/1J mice were injected with TE plus CpG treated DC (T/C-DC) pulsed with bovine collagen II (CII) between two immunizations with CII and clinical scores CIA were determined. The levels of CII-specific IgG2 and IgG1 in sera, the histological analyses in the joints, the cytokine profile in the draining lymph node (DLN) cells and in the joints, and the number, and functionality of CD4+CD25+Foxp3+ T cells (Treg) were evaluated. Vaccination of mice with CII pulsed T/C-DC diminished the severity and incidence of CIA symptoms and the production of the inflammatory cytokine, while induced the production of anti-inflammatory cytokines. The therapeutic effect was mediated by Treg cells, since the adoptive transfer of CD4+CD25+ T cells, inhibited the inflammatory symptoms in CIA. The in vitro blockage of TGF-ß in cultures of DLN cells plus CII pulsed T/C-DC inhibited the expansion of Treg cells. Vaccination with CII pulsed T/C-DC seems to be a very efficient approach to diminish exacerbated immune response in CIA, by inducing the development of Treg cells, and it is therefore an interesting candidate for a cell-based therapy for rheumatoid arthritis (RA).

Toll-like receptor 4 gene polymorphism influences dendritic cell in vitro function and clinical outcomes in vaccinated melanoma patients.

Toll-like receptor 4 (TLR4) is expressed on dendritic cells (DCs), sensing environmental danger molecules that induce their activation and maturation. Recently, we reported a method for the production of therapeutic DCs against melanoma, called tumor antigen-presenting cells (TAPCells), using a heat-shocked allogeneic melanoma cell lysate (TRIMEL) as an activation factor and antigen provider. Since TRIMEL contains endogenous TLR4 ligands, we evaluated the role of TLR4 in TAPCells differentiation by antibody neutralization and the association of a Tlr4 polymorphism (896A/G) (Asp299Gly), determined by PCR-RFLP, with the in vitro activation capacity and the clinical outcome of TAPCells-vaccinated patients. Antibody blocking of monocyte TLR4 inhibited surface expression, determined by flow cytometry, of the major histocompatibility complex class I, CCR7, CD80, CD83 and CD86 on TAPCells, reduced interleukin (IL)-6 and tumor necrosis factor -α gene expression evaluated by qRT-PCR, and also inhibited the TAPCells-mediated interferon-γ (IFN-γ) secretion of melanoma-specific CD8(+) T cells determined by ELISpot (p < 0.01). Moreover, CD8(+) T-cell activation capacity was significantly reduced in TAPCells bearing the TLR4 Asp299Gly receptor (p < 0.05). Finally, TAPCells-vaccinated stage-IV melanoma patients bearing the Tlr4 896G allele showed a shortened post-therapy median survival rate compared with those carrying the Tlr4 896A allele (p < 0.05; log-rank test). Our results indicate that TLR4 is a key receptor for the tumor lysate-mediated in vitro generation of clinically efficient antigen-presenting cells. Further analysis of patients included in different vaccine protocols is necessary for definitively establishing a role for TLR4 polymorphism in clinical responses.

Adoptive transfer of dendritic cells pulsed with Fasciola hepatica antigens and lipopolysaccharides confers protection against fasciolosis in mice.

Dendritic cells (DCs) can function as adjuvants able to mediate protection against different pathogens. Given that successful vaccination against Fasciola hepatica is mostly related to the induction of Th1 responses, we studied the potential of DCs loaded with F. hepatica antigens and lipopolysaccharide (LPS) (which promote DCs maturation) as a vaccine against fasciolosis in BALB/c mice. However, only a semimature phenotype was achieved when DCs were simultaneously cultured with an F. hepatica total extract (TE) and LPS. The activation status of TE-loaded DCs was enhanced when these cells were treated with TE 90 minutes before being stimulated with LPS (TE90 DCs). More importantly, a single vaccination of mice with TE90 DCs stimulated a systemic Th1 response and conferred protection against hepatic damage induced by F. hepatica infection. Thus, TE90 DCs may prove to be a useful new tool for vaccination against F. hepatica.

Excretory-secretory products (ESP) from Fasciola hepatica induce tolerogenic properties in myeloid dendritic cells.

Fasciola hepatica is a helminth trematode that migrates through the host tissues until reaching bile ducts where it becomes an adult. During its migration the parasite releases different excretory-secretory products (ESP), which are in contact with the immune system. In this study, we focused on the effect of ESP on the maturation and function of murine bone marrow derived-dendritic cells (DC). We found that the treatment of DC with ESP failed to induce a classical maturation of these cells, since ESP alone did not activate DC to produce any cytokines, although they impaired the ability of DC to be activated by TLR ligands and also their capacity to stimulate an allospecific response. In addition, using an in vitro ovalbumin peptide-restricted priming assay, ESP-treated DC exhibited a capacity to drive Th2 and regulatory T cell (Treg) polarization of CD4(+) cells from DO11.10 transgenic mice. This was characterized by increased IL-4, IL-5, IL-10 and TGF-beta production and the expansion of CD4(+)CD25(+)Foxp3(+) cells. Our results support the hypothesis that ESP from F. hepatica modulate the maturation and function of DC as part of a generalized immunosuppressive mechanism that involves a bias towards a Th2 response and Treg development.

Sarcoma vesical primario durante el embarazo, el primer caso publicado / Primary bladder sarcoma in pregnancy: the first case report

Los sarcomas vesicales primarios son tumores extremadamente raros, constituyendo menos del 1 por ciento de todos los tumores primarios de vejiga, con casos reportados esporádicamente. El embarazo trae consigo un problema complejo a cualquier patología urológica, especialmente oncológicas. Presentamos el primer caso publicado de sarcoma de vejiga en una mujer embarazada chilena de 28 años de edad, que dio a luz un recién nacido sano, además de una revisión de la literatura al respecto.

Primary bladder sarcomas are extremely rare tumors, representing less than 1 percent of the primary tumorsof bladder, being reported sporadically. Urologic problems become more complex to treat during pregnancy, especially oncologics diseases. We present the first known case of bladder sarcoma diagnosed in a pregnant 28 years old Chilean. Areview of literature was performed.

Efectos nocivos del uso de enalapril sobre la gestación experimental y sus implicancias sobre el uso clínico / Noxious effect of enalapril in experimental pregnancy and clinical implications

Se investigó el efecto del enalapril en la gestación en ratas. A un grupo de 11 ratas se les administró 15 mg/kg/día desde el día primeroal noveno de gestación (E10-20); 12 ratas sirvieron de control (C); 15 animales fueron sacrificados al día 20 y 19 continuaron hasta el parto. Las placentas fueron 15% menores en el grupo E10-20 (p<0.05) y tenían una estructura cordonal hipocelular simple. En el grupo E1-9 el tipo estructural predominante fue una combinación de estructuras simple y compleja. En el día 20 de gestación los fetos de los grupos tratados eran de menor tamaño que los controles (-5% en E1-9 y -16% en E10-20, p<0.05). Las diferencias desaparecieron a los 13 días postparto. En dos fetos tratados se observó calcificación incompleta del cráneo. Estimamos que estos resultados, analizados en el contexto de los daños observados en el embarazo humano, agregan información que fundamenta la abstención del uso de inhibidores de enzima convertidora en el embarazo

Carcinoides del tracto gastrointestinal: estudio anátomo-clínico / Gastrointestinal tract carcinoids: anatomo-clinical study

En el archivo de Anatomía Patológica de nuestro hospital se registraron 43 carcinoides (C) entre 1968-1988. Localización: 38 en el tracto gastrointestinal -TG- (88%) y 5 en el árbol bronquial (12%). En los C del TGI, 32 correspondieron a biopsia y 6 a necropsias, con predominio femenino (2:1) y edad X de 52 años; distribuyéndose con mayor frecuencia en apéndice (34%), íleon (32%), estómago (8%) y duodeno (8%). El 45% fue asintomático y el 55% sintomático (obstrucción, hemorragia digestiva, masa palpable y compromiso del estado general). El síndrome del carcinoide se observó en 4 casos (10%). El 45% de los tumores, especialmente los de íleon, estaban diseminados al momento del diagnóstico. Los C del TGI deben enfrentarse como cáncer, tanto en la exploración quirúrgica, como en la amplitud de la resección