Identification of tumor-agnostic biomarkers for predicting prostate cancer progression and biochemical recurrence.

The diverse clinical outcomes of prostate cancer have led to the development of gene signature assays predicting disease progression. Improved prostate cancer progression biomarkers are needed as current RNA biomarker tests have varying success for intermediate prostate cancer. Interest grows in universal gene signatures for invasive carcinoma progression. Early breast and prostate cancers share characteristics, including hormone dependence and BRCA1/2 mutations. Given the similarities in the pathobiology of breast and prostate cancer, we utilized the NanoString BC360 panel, comprising the validated PAM50 classifier and pathway-specific signatures associated with general tumor progression as well as breast cancer-specific classifiers. This retrospective cohort of primary prostate cancers (n=53) was stratified according to biochemical recurrence (BCR) status and the CAPRA-S to identify genes related to high-risk disease. Two public cohort (TCGA-PRAD and GSE54460) were used to validate the results. Expression profiling of our cohort uncovered associations between PIP and INHBA with BCR and high CAPRA-S score, as well as associations between VCAN, SFRP2, and THBS4 and BCR. Despite low levels of the ESR1 gene compared to AR, we found strong expression of the ER signaling signature, suggesting that BCR may be driven by ER-mediated pathways. Kaplan-Meier and univariate Cox proportional hazards regression analysis indicated the expression of ESR1, PGR, VCAN, and SFRP2 could predict the occurrence of relapse events. This is in keeping with the pathways represented by these genes which contribute to angiogenesis and the epithelial-mesenchymal transition. It is likely that VCAN works by activating the stroma and remodeling the tumor microenvironment. Additionally, SFRP2 overexpression has been associated with increased tumor size and reduced survival rates in breast cancer and among prostate cancer patients who experienced BCR. ESR1 influences disease progression by activating stroma, stimulating stem/progenitor prostate cancer, and inducing TGF-ß. Estrogen signaling may therefore serve as a surrogate to AR signaling during progression and in hormone-refractory disease, particularly in prostate cancer patients with stromal-rich tumors. Collectively, the use of agnostic biomarkers developed for breast cancer stratification has facilitated a precise clinical classification of patients undergoing radical prostatectomy and highlighted the therapeutic potential of targeting estrogen signaling in prostate cancer.

Identification of HDAC4 as a potential therapeutic target and prognostic biomarker for ZFTA-fused ependymomas.

Members of the HDAC family are predictive biomarkers and regulate the tumorigenesis in several cancers. However, the role of these genes in the biology of intracranial ependymomas (EPNs) remains unexplored. Here, an analysis of eighteen HDACs genes in an EPN transcriptomic dataset, revealed significantly higher levels of HDAC4 in supratentorial ZFTA fusion (ST-ZFTA) compared with ST-YAP1 fusion and posterior fossa EPNs, while HDAC7 and SIRT2 were downregulated in ST-ZFTA. HDAC4 was also overexpressed in ST-ZFTA as measured by single-cell RNA-Seq, quantitative real time-polymerase chain reaction, and immunohistochemistry. Survival analyses showed a significantly worse outcome for EPNs with higher HDAC4 and SIRT1 mRNA levels. Ontology enrichment analysis showed an HDAC4-high signature consistent with viral processes while collagen-containing extracellular matrix and cell-cell junction were enriched in those with an HDAC4-low signature. Immune gene analysis demonstrated a correlation between HDAC4 expression and low levels of NK resting cells. Several small molecules compounds targeting HDAC4 and ABCG2, were predicted by in silico analysis to be effective against HDAC4-high ZFTA. Our results provide novel insights into the biology of the HDAC family in intracranial ependymomas and reveal HDAC4 as a prognostic marker and potential therapeutic target in ST-ZFTA.

mTOR pathway somatic variants and the molecular pathogenesis of hemimegalencephaly.

OBJECTIVES: Recently, defects in the protein kinase mTOR (mammalian target of rapamycin) and its associated pathway have been correlated with hemimegalencephaly (HME). mTOR acts as a central regulator of important physiological cellular functions such as growth and proliferation, metabolism, autophagy, death, and survival. This study was aimed at identifying specific variants in mTOR signaling pathway genes in patients diagnosed with HME. METHODS: Using amplicon and whole exome sequencing (WES) of resected brain and paired blood samples from five HME patients, we were able to identify pathogenic mosaic variants in the mTOR pathway genes MTOR, PIK3CA, and DEPDC5. RESULTS: These results strengthen the hypothesis that somatic variants in PI3K-Akt-mTOR pathway genes contribute to HME. We also describe one patient presenting with a pathogenic variant on DEPDC5 gene, which reinforces the role of DEPDC5 on cortical structural changes due to mTORC1 hyperactivation. These findings also provide insights into when in brain development these variants occurred. An early developmental variant is expected to affect a larger number of cells and to result in a larger malformation, whereas the same variant occurring later in development would cause a minor malformation. SIGNIFICANCE: In the future, numerous somatic variants in known or new genes will undoubtedly be revealed in resected brain samples, making it possible to draw correlations between genotypes and phenotypes and allow for a genetic clinical diagnosis that may help to predict a given patient's outcome.

Phosphorylation of S6 Protein as a Potential Biomarker in Surgically Treated Refractory Epilepsy.

The tuberous sclerosis complex (TSC), focal cortical dysplasia IIB (FCD IIB), and hemimegalencephaly (HME) exhibit similar molecular features that are dependent on the hyperactivation of the mTOR pathway. They are all associated with refractory epilepsy and the need for surgical resection with varying outcomes. The phosphorylated protein S6 (pS6) is a downstream target of mTOR, whose increased expression might indicate mTOR hyperactivation, but which is also present when there is no alteration in the pathway (such as in FCD type I). We have performed immunohistochemical marking and quantification of pS6 in resected brain specimens of 26 patients clinically and histologically diagnosed with TSC, FCD IIB, or HME and compared this data to a control group of 25 patients, to measure the extent of pS6 positivity and its correlation with clinical aspects. Our results suggest that pS6 may serve as a reliable biomarker in epilepsy and that a greater percentage of pS6 marking can relate to more severe forms of mTOR-dependent brain anomalies.

PTEN-deficient prostate cancer is associated with an immunosuppressive tumor microenvironment mediated by increased expression of IDO1 and infiltrating FoxP3+ T regulatory cells.

BACKGROUND: Accumulating evidence shows that tumor cell-specific genomic changes can influence the cross talk between cancer cells and the surrounding tumor microenvironment (TME). Loss of the PTEN tumor suppressor gene is observed in 20% to 30% of prostate cancers (PCa) when first detected and the rate increases with PCa progression and advanced disease. Recent findings implicate a role for PTEN in cellular type I interferon response and immunosuppression in PCa. However, the way that PTEN inactivation alters antitumor immune response in PCa is poorly understood. MATERIALS AND METHODS: To investigate the changes associated with PTEN loss and an immunosuppressive TME in PCa, we used CIBERSORT to estimate the relative abundance of 22 immune-cell types from 741 primary and 96 metastatic tumors. Our in silico findings were then validated by immunohistochemical analysis of immune cells and IDO1 and PDL1 checkpoint proteins in a cohort of 94 radical prostatectomy specimens. RESULTS: FoxP3+ T regulatory cells (Tregs) were significantly increased in PTEN-deficient PCa in all three public domain cohorts. Loss of PTEN in bone metastases was associated with lower CD8+ T-cell abundance, but in liver metastasis, FoxP3+ Tregs were present at higher levels. PTEN-deficient lymph node metastasis had a distinct profile, with high levels of CD8+ T cells. Moreover, we found that metastatic PCa presents higher abundance of FoxP3+ Treg when compared to primary lesions. Since PTEN-deficient tumors are likely to be immunosuppressed as a consequence of increased FoxP3+ Tregs, we then evaluated the localization and expression of IDO1, PDL1 immune checkpoints, and the corresponding density of FoxP3+ Treg and CD8+ T cells using our validation cohort (n = 94). We found that IDO1 protein expression and FoxP3+ Treg density were higher in neoplastic glands compared with benign adjacent tissue. Moreover, higher densities of FoxP3+ Treg cells in both stromal (P = 0.04) and tumor (P = 0.006) compartments were observed in PTEN-deficient tumors compared to tumors that retained PTEN activity. Similarly, IDO1 protein expression was significantly increased in the tumor glands of PTEN-deficient PCa (P < 0.0001). Spearman correlation analysis showed that IDO1 expression was significantly associated with FoxP3+ Treg and CD8+ T-cell density (P < 0.01). CONCLUSIONS: Our findings imply that PTEN deficiency is linked to an immunosuppressive state in PCa with distinct changes in the frequency of immune cell types in tumors from different metastatic sites. Our data suggest that determining PTEN status may also help guide the selection of patients for future immunotherapy trials in localized and metastatic PCa.

Skin cancer treatment effectiveness is improved by iontophoresis of EGFR-targeted liposomes containing 5-FU compared with subcutaneous injection.

Squamous cell carcinoma (SCC) is a malignant tumor in which epidermal growth factor receptor (EGFR) overexpression is associated with poor prognosis and malignancy. For SCC treatment, cetuximab, an anti-EGFR antibody, is administered in combination with a chemotherapeutic drug for improved efficacy. In this work, an EGFR-targeted immunoliposome loaded with 5-fluorouracil (5- FU) was developed to allow co-administration of the antibody and the chemotherapeutic agent and selective delivery to SCC cells. Topically applied iontophoresis and subcutaneous injections of the 5-FU-loaded immunoliposomes were employed in an SCC xenograft animal model to evaluate the influence of the administration route on therapeutic efficacy. In vitro, cellular uptake of cetuximab-immunoliposomes by EGFR-positive SCC cells was 3.5-fold greater than the uptake of control liposomes. Skin penetration studies showed that iontophoresis of immunoliposomes doubled the 5-FU penetration into the viable epidermis compared with the same treatment with control liposomes. In vivo, subcutaneous injection of immunoliposomes reduced tumor volume by >60% compared with the negative control and approximately 50% compared with the 5-FU solution and control liposome treatments. Interestingly, topical administration via iontophoresis improved tumor reduction by almost 2-fold compared with subcutaneous administration of 5-FU solution and control liposomes but was equally effective for the immunoliposome treatment. However, histological analysis showed that iontophoresis of immunoliposomes was more effective than subcutaneous injection in reducing cell proliferation, resulting in cells with less aggressive characteristics. In conclusion, topical administration of immunoliposomes containing 5-FU using iontophoresis is a promising strategy for SCC treatment.

Anti-HER2 immunoliposomes for co-delivery of paclitaxel and rapamycin for breast cancer therapy.

Breast cancer is the second leading cause of cancer deaths among women. Paclitaxel (PTX) is used for its treatment, however non-selectivity, rapid systemic clearance and hypersensitivity to the commercially available formulation are major drawbacks. Rapamycin (RAP), an mTOR inhibitor, acts synergistically with PTX, and thus could be used in combination with it. Drug loading into nanocarriers, particularly liposomes, has proven to enhance efficacy and reduce side-effects of chemotherapeutic drugs. Within this context, the functionalization of liposomes with antibodies for overexpressed receptors on tumor surface is a potential strategy to increase specificity and reduce side-effects. Specifically, active targeting of HER2(+) breast cancer cells can be achieved by immunoliposomes consisting of liposomes coated with an anti-HER2 monoclonal antibody, Trastuzumab. Herein, we have synthesized PTX/RAP co-loaded immunoliposomes coated with Trastuzumab, performed physicochemical characterization, and evaluated the formulations for cytotoxicity and uptake in 4T1 (triple negative) and SKBR3 (HER2 positive) cell lines. Furthermore, we aimed to compare the immunoliposomes with liposomes and solution of PTX/RAP in vivo, employing human xenograft HER2-overexpressing tumors in mouse model. The co-loaded immunoliposomes had a mean particle size of 140.3nm, a zeta potential of -9.85mV and drug encapsulation efficiency of 55.87 and 69.51, respectively for PTX and RAP. The functionalization efficiency of Trastuzumab was higher than 70% and the antibody retained HER2 binding activity. Cell studies showed increased cytotoxicity of PTX/RAP for the immunoliposome, compared to the control liposomes in SKBR3 cells, which could be attributed to enhanced uptake mediated through HER2 binding. Furthermore, immunoliposomes were better able to control tumor growth in vivo, with tumor volume averages corresponding to 25.27, 44.38 and 47.78% of tumor volumes of untreated control, PTX/RAP solution and control liposomes, respectively. Taken together, our results support the clinical development of immunoliposomes for targeted delivery of PTX and RAP to HER2-positive breast cancer.

Lymph node or perineural invasion is associated with low miR-15a, miR-34c and miR-199b levels in head and neck squamous cell carcinoma.

BACKGROUND: MicroRNAs (miRNAs or miRs) are post-transcriptional regulators of eukaryotic cells and knowledge of differences in miR levels may provide new approaches to diagnosis and therapy. METHODS: The present study measured the levels of nine miRs in head and neck squamous cell carcinomas (HNSCC) and determined whether clinical pathological features are associated with differences in miR levels. SET (I2PP2A) and PTEN protein levels were also measured, since their levels can be regulated by miR-199b and miR-21, respectively. Nine miRs (miR-15a, miR-21, miR-29b, miR-34c, miR-100, miR-125b, miR-137, miR-133b and miR-199b) were measured by real time qRT-PCR in HNSCC samples from 32 patients and eight resection margins. SET (I2PP2A) and PTEN protein levels were estimated by immunohistochemistry in paired HNSCC tissues and their matched resection margins. RESULTS: In HNSCC, the presence of lymph node invasion was associated with low miR-15a, miR-34c and miR-199b levels, whereas the presence of perineural invasion was associated with low miR-199b levels. In addition, miR-21 levels were high whereas miR-100 and miR-125b levels were low in HNSCC compared to the resection margins. When HNSCC line HN12, with or without knockdown of SET, were transfected with miR-34c inhibitor or miR-34c mimic, the miR-34c inhibitor increased cell invasion capacity while miR-34c mimic decreased the cell invasion. CONCLUSIONS: We showed that the levels of specific miRs in tumor tissue can provide insight into the maintenance and progression of HNSCC. GENERAL SIGNIFICANCE: MiRNAs are up- or down-regulated during cancer development and progression; they can be prognosis markers and therapeutic targets in HNSCC.

HOX genes: potential candidates for the progression of laryngeal squamous cell carcinoma.

Laryngeal squamous cell carcinoma (LSCC) is a very aggressive cancer, considered to be a subtype of the head and neck squamous cell carcinoma (HNSCC). Despite significant advances in the understanding and treatment of cancer, prognosis of patients with LSCC has not improved recently. In the present study, we sought to understand better the genetic mechanisms underlying LSCC development. Thirty-two tumor samples were collected from patients undergoing surgical resection of LSCC. The samples were submitted to whole-genome cDNA microarray analysis aiming to identify genetic targets in LSCC. We also employed bioinformatic approaches to expand our findings using the TCGA database and further performed functional assays, using human HNSCC cell lines, to evaluate viability, cell proliferation, and cell migration after silencing of selected genes. Eight members of the homeobox gene family (HOX) were identified to be overexpressed in LSCC samples when compared to normal larynx tissue. Quantitative RT-PCR analysis validated the overexpression of HOX gene family members in LSCC. Receiver operating characteristic (ROC) statistical method curve showed that the expression level of seven members of HOX gene family can distinguish tumor from nontumor tissue. Correlation analysis of clinical and gene expression data revealed that HOXC8 and HOXD11 genes were associated with the differentiation degree of tumors and regional lymph node metastases, respectively. Additionally, siRNA assays confirmed that HOXC8, HOXD10, and HOXD11 genes might be critical for cell colony proliferation and cell migration. According to our findings, several members of the HOX genes were overexpressed in LSCC samples and seem to be required in biological processes involved in tumor development. This suggests that HOX genes might play a critical role in the physiopathology of LSCC tumors.

Sarcoma radioinduzido pós-tratamento de glioblastoma: relato de caso / Radiation induced sarcoma after treatment of glioblastoma: case report / Sarcoma inducido por la radiación después del tratamiento de glioblastoma: reporte de un caso

Introdução: O glioblastoma multiforme é a neoplasia de sistema nervoso central mais letal, com sobrevida média em torno de 13 meses e a de pior prognóstico dentre todos os gliomas. A abordagem terapêutica do glioblastoma consiste em neurocirurgia com ressecção máxima possível do volume tumoral, seguida de radioterapia e quimioterapia. A radioterapia reduz o risco de recidiva tumoral por meio de lesão direta e indireta ao ácido desoxirribonucleico tumoral. Os efeitos em longo prazo da radioterapia incluem necrose tecidual, vasculopatia e neoplasia induzida pela radiação. Os tumores malignos intracranianos secundários mais reportados incluem meningiomas, gliomas e sarcomas. O período de latência entre a radioterapia de crânio e o surgimento de lesões radioinduzida varia na literatura entre seis meses a 47 anos, com média de 18,7 anos. Relato de caso: O presente relato descreve o surgimento de sarcoma fusocelular de alto grau radioinduzido após dez meses em paciente que recebeu tratamento de glioblastoma no Hospital das Clínicas de Ribeirão Preto da Universidade de São Paulo. Conclusão: A raridade dessa associação se deve provavelmente à baixa sobrevida dos pacientes com glioblastoma, limitando assim o tempo para desenvolvimento de neoplasias secundárias.

Introduction: Glioblastoma multiforme and neoplasia is the deadliest cancer of the central nervous system, with survival rates averaging around 13 months and it shows the worst prognosis among all gliomas. The therapeutic approach of glioblastoma is based on a full neurosurgical resection of the tumor volume followed by radiotherapy and chemotherapy. Radiotherapy reduces the risk of recurrence by direct and indirect deoxyribonucleic acid damage of the tumor. The long-term effects of radiotherapy include tissue necrosis, vascular injury and neoplasia induced by radiation. The majority of secondary intracranial tumors and most commonly reported include meningiomas, gliomas and sarcomas. The latency period between the cranial radiotherapy and the onset of radiation-induced injuries in the literature varies between six months to 47 years, with an average of 18.7 years. Case report: This report describes the onset of spindle cell sarcoma of highly radiation-induced after ten months in a patient who received treatment of Glioblastoma at the General Hospital of the University of Sao Paulo Medical School, Ribeirao Preto. Conclusion: The rarity of this association is probably due to poor survival of patients with Glioblastoma, thus limiting the time for development of secondary malignancies.

Introducción: El glioblastoma multiforme es el cáncer más mortal del sistema nervioso central, con una supervivencia media acerca de 13 meses, y tiene el peor pronóstico entre todos los gliomas. El enfoque terapéutico del glioblastoma es la resección neurocirugica completo del volumen del posible tumor, seguido por radioterapia y quimioterapia. La radioterapia reduce el riesgo de reincidencia debido a daño directo e indirecto para el ácido desoxirribonucleico del tumor. Los efectos a largo plazo de la terapia de radiación incluyen necrosis de los tejidos, lesión vascular y el cáncer inducido por radiación. Los tumores intracraneales malignos secundarios más comunes incluyen meningiomas, gliomas y sarcomas. El período de latencia entre la radioterapia craneal y la aparición de las lesiones inducidas por radio en la literatura varía entre seis meses a 47 años, con una media 18,7 años. Caso clínico: Este caso describe la aparición de sarcoma de células fusiformes de grado alto inducida por radiación después de diez meses en un paciente que recibió tratamiento de glioblastoma en el Hospital das Clínicas de Ribeirão Preto, de la Universidad de Sao Paulo. Conclusión: Esta asociación poco común es probablemente debida a la mala supervivencia de los pacientes con glioblastoma, lo que limita el tiempo para el desarrollo de neoplasias malignas secundarias.

Halofuginone inhibits phosphorylation of SMAD-2 reducing angiogenesis and leukemia burden in an acute promyelocytic leukemia mouse model.

BACKGROUND: Halofuginone (HF) is a low-molecular-weight alkaloid that has been demonstrated to interfere with Metalloproteinase-2 (MMP-2) and Tumor Growth Factor-ß (TGF-ß) function and, to present antiangiogenic, antiproliferative and proapoptotic properties in several solid tumor models. Based on the fact that high levels of Vascular Endothelial Growth Factor (VEGF) and increased angiogenesis have been described in acute myeloid leukemia and associated with disease progression, we studied the in vivo effects of HF using an Acute Promyelocytic Leukemia (APL) mouse model. METHODS: NOD/SCID mice were transplanted with leukemic cells from hCG-PML/RARA transgenic mice (TM) and treated with HF 150 µg/kg/day for 21 days. The leukemic infiltration and the percentage of VEGF+ cells were evaluated by morphology and flow cytometry. The effect of HF on the gene expression of several pro- and antiangiogenic factors, phosphorylation of SMAD2 and VEGF secretion was assessed in vitro using NB4 and HUVEC cells. RESULTS: HF treatment resulted in hematological remission with decreased accumulation of immature cell and lower amounts of VEGF in BM of leukemic mice. In vitro, HF modulated gene expression of several pro- and antiangiogenic factors, reduced VEGF secretion and phosphorylation of SMAD2, blocking TGF-ß-signaling. CONCLUSION: Taken together, our results demonstrate that HF inhibits SMAD2 signaling and reduces leukemia growth and angiogenesis.

Fas, FasL, and cleaved caspases 8 and 3 in glioblastomas: a tissue microarray-based study.

This investigation analyzed the immunoexpression of FasL, Fas, cleaved caspase-8, and cleaved caspase-3 in glioblastomas. Formalin-fixed and paraffin-embedded glioblastoma tissues and control brain tissues from 97 patients were analyzed by tissue microarrays and immunohistochemistry. Patients with glioblastomas that were negative or weakly stained (<50% of cells positive) for cleaved caspase-8 had worse cancer-specific overall survival (median=8.5 months) than did patients with tumors that highly expressed cleaved caspase-8 (median=11.7 months; P=0.0325), independent of clinical variables. There was no association of other markers with survival, treatment, sex, age, tumor size, and primary site. Among the tumors, there were reasonable to good positive correlations between the expression of FasL and Fas (r=0.47) and between Fas and cleaved caspase-8 (r=0.41), and there were poor positive correlations between Fas and cleaved caspase-3 (r=0.26), FasL and cleaved caspase-8 (r=0.22), and cleaved caspase-8 and -3 (r=0.31). Our results suggest that Fas-Fas-ligand signal transduction could be inhibited, especially at the stage of caspase-8 activation, thereby establishing a major mechanism for evasion of apoptosis by these tumors. The absence or low expression of cleaved caspase-8 in the tumors was a negative prognostic indicator for patient survival.

SAGE analysis highlights the putative role of underexpression of ribosomal proteins in GH-secreting pituitary adenomas.

BACKGROUND: Although the molecular pathogenesis of pituitary adenomas has been assessed by several different techniques, it still remains partially unclear. Ribosomal proteins (RPs) have been recently related to human tumorigenesis, but they have not yet been evaluated in pituitary tumorigenesis. OBJECTIVE: The aim of this study was to introduce serial analysis of gene expression (SAGE), a high-throughput method, in pituitary research in order to compare differential gene expression. METHODS: Two SAGE cDNA libraries were constructed, one using a pool of mRNA obtained from five GH-secreting pituitary tumors and another from three normal pituitaries. Genes differentially expressed between the libraries were further validated by real-time PCR in 22 GH-secreting pituitary tumors and in 15 normal pituitaries. RESULTS: Computer-generated genomic analysis tools identified 13,722 and 14,993 exclusive genes in normal and adenoma libraries respectively. Both shared 6497 genes, 2188 were underexpressed and 4309 overexpressed in tumoral library. In adenoma library, 33 genes encoding RPs were underexpressed. Among these, RPSA, RPS3, RPS14, and RPS29 were validated by real-time PCR. CONCLUSION: We report the first SAGE library from normal pituitary tissue and GH-secreting pituitary tumor, which provide quantitative assessment of cellular transcriptome. We also validated some downregulated genes encoding RPs. Altogether, the present data suggest that the underexpression of the studied RP genes possibly collaborates directly or indirectly with other genes to modify cell cycle arrest, DNA repair, and apoptosis, leading to an environment that might have a putative role in the tumorigenesis, introducing new perspectives for further studies on molecular genesis of somatotrophinomas.

Amygdala gene expression of NMDA and GABA(A) receptors in patients with mesial temporal lobe epilepsy.

Temporal lobe epilepsy (TLE) is the most common form of partial epilepsy and affects 40% of the patients. Seizures arising from the mesial temporal lobe structures (i.e., amygdala and hippocampus) are common, whereas neocortical seizures are rare. In recent years, many studies aimed to identify the pattern of gene expression of neurotransmitters involved in molecular mechanisms of epilepsy. We used real-time PCR to quantify the expression of GABA(A) (subunits α1, ß1, ß2) and NMDA (subunits NR1, NR2A, and NR2B) receptor genes in amygdalae of 27 patients with TLE and 14 amygdalae from autopsy controls. The NR1 subunit was increased in patients with epilepsy when compared with controls. No differences were found in expression of NMDA subunits NR2A and NR2B or in α1, ß1, and ß2 subunits of GABA(A) receptors. Our results suggest that the NR1 subunit of NMDA receptors is involved in the amygdala hyperexcitability in some of the patients with TLE.

An experimental model of meningoencephalomyelitis by Rocio flavivirus in BALB/c mice: inflammatory response, cytokine production, and histopathology.

Rocio virus (ROCV) is a flavivirus, probably transmitted by Culex mosquitoes and maintained in nature as a zoonosis of wild birds. Rocio virus caused a human epidemic of severe encephalitis that lasted from 1973 to 1980 in the Ribeira valley, in the southeastern coast of Brazil. After this outbreak, serologic evidence of ROCV circulation has been reported and public health authorities are concerned about a return of ROCV outbreaks in Brazil. We show here a study on the pathogenesis and the physiopathology of ROCV disease in the central nervous system of a Balb/C young adult mice experimental model. The animals were intraperitoneally infected by ROCV and followed from 0 to 9 days after infection, when all of them died. Nervous tissue samples were collected from infected animals for immunohistochemistry and molecular biology analysis. We observed the virus in the central nervous system, the inflammatory changes induced by Th1 and Th2 cytokines, and the final irreversible damage of nervous tissues by neuronal degeneration and apoptosis. These findings can help to better understand the pathogenesis and physiopathology of the human meningoencephalomyelitis by ROCV and other flaviviruses.

High expression of cancer testis antigens MAGE-A, MAGE-C1/CT7, MAGE-C2/CT10, NY-ESO-1, and gage in advanced squamous cell carcinoma of the larynx.

BACKGROUND: Despite diagnostic and therapeutic advances in head and neck cancer, the 5-year survival of patients with laryngeal cancer has not improved in the last 30 years. Several recent studies indicate that specific targets for immunotherapeutic approaches can be useful in the control of cancer. There is considerable interest in the expression of cancer testis antigens in human cancers since they may serve as the basis for an immunologic approach to therapy. METHODS: We evaluated by immunohistochemical analysis the expression of cancer testis antigens MAGE-A4 (57B), MAGE-C1 (CT7-33), MAGE-A1 (MA454), MAGE-A3 (M3H67), MAGE-C2 (CT10.5), NY-ESO-1 (E978), and GAGE (GAGE) in squamous cell carcinoma (SCC) of the larynx. RESULTS: A total of 63 cases (57 men and 6 women) of laryngeal SCC were available for this study. The findings were correlated with the clinical course and laboratory data. Expression of at least 1 cancer testis antigen was detected in 42 of 63 of the laryngeal SCCs (67%). In 34 of 42 of the positive cases (81%) there was simultaneous expression of ≥2 cancer testis antigens. There was significant correlation between antigen expression and advanced tumor stage (stage III/IV) in cases with reactivity to only 1 antibody (p = .01) as well as in the cases with reactivity to ≥2 primary antibodies (≥2 mAbs, p = .04). There was no association between survival and expression of any of the analyzed antigens. CONCLUSIONS: We find a high incidence of cancer testis antigen expression in SCCs of the larynx, which was correlated with advanced clinical stage. Our data indicate that cancer testis antigens could be valuable vaccine targets in laryngeal tumors, especially in those with a worse prognosis.

Central mucoepidermoid carcinoma: report of 2 cases.

Central mucoepidermoid carcinoma is a rare mandibular neoplasm. The objective of this paper was to report two cases.

Flow cytometry characterization of leukemic phase of nasal NK/T-cell lymphoma in tumor biopsies and peripheral blood.

We report the findings of the immunophenotypic profile of three cases of nasal T/NK cell lymphoma in leukemic phase. Flow cytometry analysis was carried out using cell suspensions of tumor nasal biopsies and peripheral blood. Tumor samples were composed by a mixture of a predominant subset of medium-size true NK cytCD3epsilon-, sCD3epsilon-, CD56+ cells mixed with a minor subset of medium-size T/NK sCD3epsilon+, CD56+ cells. Both subsets were also detected in peripheral blood. In addition, an infiltration of small-size sCD3epsilon+, CD56- normal T lymphocytes was also present.

Hantavirus infection induces a typical myocarditis that may be responsible for myocardial depression and shock in hantavirus pulmonary syndrome.

Despite clinical evidence of myocardial dysfunction, there is no pathological evidence of myocardial injury in hantavirus pulmonary syndrome (HPS). The dominant opinion is that the primary cardiac lesion is functional rather than structural. The present study describes hantaviral antigen and particles in the cardiac endothelium and interstitial macrophages in association with a typical myocarditis in HPS. Human hearts from 14 individuals who died of HPS were compared with hearts from 14 individuals who died of acute necrotizing pancreatitis associated with acute lung injury and 4 individuals who died accidental deaths without thoracic injury (as controls); all cases were selected from autopsies. Transmural blocks of myocardial tissue were excised from the middle portion of the left-ventricular free wall and fixed in formalin. Small samples of myocardial tissue from 4 HPS cases and 4 non-HPS controls were fixed in glutaraldehyde for electron microscopic study. Histomorphometric, immunohistochemical, and ultrastructural methods were employed to detect the presence of hantavirus in the myocardium and to evaluate interstitial edema and the minor diameter of myocytes, to characterize the immunophenotype, and to estimate the number of inflammatory cells and in situ cytokine-producing cells and the T helper cell subset 1 and 2 immune responses (tumor necrosis factor [TNF]-alpha, interferon-gamma, interleukin [IL]-10, and IL-4). Cardiac remodeling; hantaviral antigen and particles in the endothelium and macrophages; scattered foci of myofiber necrosis; greater interstitial cellular infiltration, mainly composed of macrophages and memory T lymphocytes and a significant number of T helper and B lymphocytes; and TNF-alpha protein expression in macrophage-type cells and cardiomyocytes were observed to a greater extent in HPS myocardium than in normal and acute pancreatitis control myocardium. These findings give support to the opinion that structural changes could be responsible for myocardial depression and shock in HPS, and it should be properly named as "hantavirus cardiopulmonary syndrome" (HCPS).

Myocardial structural changes in long-term human severe sepsis/septic shock may be responsible for cardiac dysfunction.

The present investigation sought to determine the cellular mechanisms directly dependent on long-term severe sepsis/septic shock that could lead to myocardial structural changes in humans. Human hearts from eight cases of long-term severe sepsis/septic shock arising from infection, as defined by the ACCP/SCCM Consensus Conference; eight cases of acute necrotizing pancreatitis and acute lung injury, a noninfectious pathologic cause of systemic inflammatory response; and three cases of accidental death without thoracic injury selected from autopsies were studied. Transmural blocks of myocardial tissue were excised from the middle portion of the left ventricular free wall and were fixed in formalin or were frozen. Histochemical and immunohistochemical methods were used to evaluate the cross-striations of the myocardial cells, the number and size of interstitial macrophages, the intracardiomyocyte accumulation of lipid, the actin/myosin contractile apparatus, and the expression of iNOS, nitrotyrosine, and TNF-alpha in the myocardia of septic and control hearts. Greater interstitial cellular infiltration composed of larger and elongated macrophages and TNF-alpha protein expression in myofibers, interstitial macrophage cell types, and smooth muscle cells and endothelial cell in the vessels; intracardiomyocyte lipid accumulation; scattered foci of actin/myosin contractile apparatus disruption; and increased expression for iNOS and nitrotyrosine in myocytes and interstitial macrophage cell types could be observed in long-term human septic myocardium as compared with normal and acute pancreatitis control myocardia. These findings give support to an opinion that structural changes could be responsible for long-term sepsis-induced myocardial dysfunction. The higher number of macrophages, most of them with morphological features of "activation," and TNF-alpha protein expression could favor the reduction of cardiac function in septic hearts. The intramyocyte lipid accumulation in these hearts very likely reflects myocardium ventricular contractile dysfunction. In addition, the increased expression of iNOS and the evidence for the significant presence of peroxynitrite in cardiomyocytes and interstitial macrophage cell types suggest that oxidative damage may play a role in actin/myosin disruption in the hearts of septic patients.