Accumulation of duct cells with activated YAP parallels fibrosis progression in non-alcoholic fatty liver disease.

BACKGROUND & AIMS: Mechanisms that regulate regeneration of injured livers are complex. YAP, a stem cell associated factor, controls liver growth in healthy adult mice. Increasing nuclear localization of YAP triggers accumulation of reactive-appearing ductular cells (YAP+RDC) with liver progenitor capabilities. The significance of YAP activation, and mechanisms involved, are unknown in diseased livers. We evaluated the hypothesis that YAP is more activated in injured livers that are scarring than in those that are regenerating effectively. METHODS: Immunohistochemistry and qRT-PCR analysis were used to localize and quantify changes in YAP and RDC in 52 patients with non-alcoholic fatty liver disease (NAFLD) and two mouse models of diet-induced non-alcoholic steatohepatitis (NASH). Results were correlated with liver disease severity, metabolic risk factors, and factors proven to control NAFLD progression. RESULTS: YAP increased in NAFLD where it mainly localized in nuclei of RDC that expressed progenitor markers. Accumulation of YAP+RDC paralleled the severity of hepatocyte injury and accumulation of Sonic hedgehog, but not steatosis or metabolic risk factors. YAP+RDC expressed osteopontin, a Shh-regulated fibrogenic factor. Myofibroblast accumulation, fibrosis, and numbers of YAP+RDC strongly correlated. In murine NASH models, atrophic fibrotic livers contained significantly more YAP+RDC than livers with less severe NASH. CONCLUSION: YAP+RDC promote scarring, rather than effective regeneration, during NASH.

Identification of a minority population of LMO2+ breast cancer cells that integrate into the vasculature and initiate metastasis.

Metastasis is responsible for most breast cancer-related deaths; however, identifying the cellular determinants of metastasis has remained challenging. Here, we identified a minority population of immature THY1+/VEGFA+ tumor epithelial cells in human breast tumor biopsies that display angiogenic features and are marked by the expression of the oncogene, LMO2. Higher abundance of LMO2+ basal cells correlated with tumor endothelial content and predicted poor distant recurrence-free survival in patients. Using MMTV-PyMT/Lmo2CreERT2 mice, we demonstrated that Lmo2 lineage-traced cells integrate into the vasculature and have a higher propensity to metastasize. LMO2 knockdown in human breast tumors reduced lung metastasis by impairing intravasation, leading to a reduced frequency of circulating tumor cells. Mechanistically, we find that LMO2 binds to STAT3 and is required for STAT3 activation by tumor necrosis factor-α and interleukin-6. Collectively, our study identifies a population of metastasis-initiating cells with angiogenic features and establishes the LMO2-STAT3 signaling axis as a therapeutic target in breast cancer metastasis.

Arsenic Trioxide Triggers Apoptosis of Metastatic Oral Squamous Cells Carcinoma with Concomitant Downregulation of GLI1 in Hedgehog Signaling.

Given the lack of advances in Oral Squamous Cell Carcinoma (OSCC) therapy in recent years, pharmacological strategies to block OSCC-related signaling pathways have gained prominence. The present study aimed to evaluate the therapeutic potential of Arsenic Trioxide (ATO) concerning its antitumoral effects and the inhibition of the Hedgehog (HH) pathway in OSCC. Initially, ATO cytotoxicity was assessed in a panel of cell lines. Cell viability, cell cycle, death patterns, and cell morphology were analyzed, as well as the effect of ATO on the expression of HH pathway components. After the cytotoxic assay, HSC3 cells were chosen for all in vitro assays. ATO increased apoptotic cell death and nuclear fragmentation in the sub-G1 cell cycle phase and promoted changes in cell morphology. In addition, the reduced expression of GLI1 indicated that ATO inhibits HH activity. The present study provides evidence of ATO as an effective cytotoxic drug for oral cancer treatment in vitro.

Quality of Life Assessment Among Patients Living With Hepatosplenic Schistosomiasis and Schistosomal Myeloradiculopathy.

Schistosomiasis is a major public health problem in tropical areas of the world. Health-related quality of life (HRQOL) measurement is being widely used to evaluate the impact of a disease or treatment in several aspects of daily life. However, few studies evaluated the impact of severe forms of schistosomiasis on HRQOL of affected individuals and compared them to healthy controls with a similar socio-demographic background. Our aims were to evaluate the HRQOL in patients with hepatosplenic schistosomiasis (HS) and schistosomal myeloradiculopathy (SMR) and healthy volunteers (HV) and determine if clinical complications of the disease are associated with HRQOL scores. We interviewed and evaluated the HRQOL in 49 patients with HS, 22 patients with SMR, and 26 HV from an outpatient clinic of the Federal University of Minas Gerais University Hospital using the WHOQOL-BREF questionnaire. SMR and HS patients had a significantly lower overall quality of life score when comparing with the HV control group (p = 0.003 and p = 0.005, respectively). Multivariate ordinal regression model adjusted for sex, age, and educational level indicated that HS and SMR patients have three and five times more chances of having a lower quality of life than healthy volunteers (Odds Ratio 3.13 and 5.04, respectively). There was no association between complications of HS disease and quality of life scores. In contrast, worse quality of life was observed in SMR patients that presented back or leg pain, leg paresthesia, and bladder dysfunction. In conclusion, HS and SMR significantly impact the overall quality of life of the affected individuals, reinforcing the importance of efforts to control and eradicate this debilitating disease and suggesting that multidisciplinary clinical management of schistosomiasis patients would be more appropriate and could potentially improve patient's quality of life.

EDTA-dependent pseudothrombocytopenia in patients with hepatosplenic schistosomiasis mansoni: a clinical management alert.

BACKGROUND: Hepatosplenic schistosomiasis mansoni (HS) is associated with thrombocytopenia. Accurate platelet counts are required for identification and management of HS patients. EDTA-dependent pseudothrombocytopenia (EDTA-PTCP) is an in vitro phenomenon of anticoagulant-activated platelet agglutination resulting in low platelet counts by automated methods. The prevalence of EDTA-PCTP in schistosomiasis is unknown and only one case has been described. Our aims were to determine the prevalence of EDTA-PTCP in HS and evaluate alternative methods to overcome this analytical error. METHODS: Blood samples from 56 HS patients and 56 healthy volunteers were collected, and platelet counts were obtained using standard microscopy and automated (electric impedance) methods. Automated platelet counts and the presence of platelet clumps in blood smears were evaluated in samples collected in EDTA or sodium citrate tubes 20 and 180 min after blood collection. RESULTS: EDTA-PTCP was more frequent in HS patients than healthy volunteers (8.92% vs 0.00%, p<0.0285). Platelet clumps and PTCP were also observed in samples collected in sodium citrate tubes, refuting its use as an alternative method. CONCLUSIONS: Automated platelet counts in blood samples from HS patients should be performed right after blood collection in EDTA tubes and verified by manual counts in blood smears.

Hedgehog pathway activation in oral squamous cell carcinoma: cancer-associated fibroblasts exhibit nuclear GLI-1 localization.

The purpose of this study was to evaluate the expression of Hedgehog (HH) signaling molecules (SHH and GLI-1) by cancer-associated fibroblasts (CAF) in oral squamous cell carcinoma (OSCC). Immunohistochemistry was used to detect molecular HH signaling and CAF-related protein expression, including α-SMA and S100A4, in 70 samples of human OSCC. The colocalization of α-SMA and S100A4 with SHH was also evaluated by double-staining. In vitro study was performed using primary normal oral fibroblast (NOF) and CAF through immunofluorescence and Western Blot for CAF-proteins, SHH, and GLI-1. Forty-five cases (64.28%) were positive for α-SMA exclusively in tumor stroma, and S100A4 was identified in the cytoplasm of CAFs in 94.28% (n = 66) of the cases. With respect to stromal cells, 64 (91.43%) OSCC cases were positive for SHH, and 31 were positive for GLI-1 (44.29%); positive correlations were found between SHH and α-SMA (p < 0.0001, φ = 0.51), as well as between SHH and S100A4 (p = 0.087, φ = 0.94). Protein expression of SHH and GLI-1 was observed in primary CAFs and NOFs. Although SHH was found to be localized in the cellular cytoplasm of both cell types, GLI-1 was present only in the nuclei of CAF. Our results indicate that CAFs are not only potential sources of HH ligands in tumor stroma, but may also respond to HH signaling through nuclear GLI-1 activation. We further observed that elevated SHH expression by OSCC cells was associated with higher CAF density, reinforcing the chemoattractant role played by these molecules.

Inhibition of CAL27 Oral Squamous Carcinoma Cell by Targeting Hedgehog Pathway With Vismodegib or Itraconazole.

Oral Squamous Cell Carcinoma (OSCC) presents an important challenge for the health systems worldwide. Thus, unraveling the biological mechanisms involved in OSCC pathogenesis is essential to the discovery of new drugs with anticancer potential. The Hedgehog (HH) pathway has shown promising results as a therapeutic target both in vitro and in vivo. This study aimed to investigate the effects of vismodegib and itraconazole on the expression of Hedgehog (HH) genes (PTCH1, SMO, and GLI1), cell cycle and cell death in OSCC cells. Alamar Blue assay was used to assess the cytotoxicity of vismodegib and itraconazole in a panel of oral cancer cell lines, including CAL27. The expression of HH signaling components after treatment with vismodegib and itraconazole, at concentrations of 25 or 50 µg/ml was evaluated by qPCR. Cell cycle and apoptosis were evaluated by flow cytometry after 72 h treatment with 50 µg/ml of vismodegib or itraconazole. HH signaling was activated in OSCC cell lines CAL27, SCC4, SCC9, and HSC3. Vismodegib and itraconazole significantly reduced CAL27 cell viability after 48 h of treatment. Gene expression of PTCH1, SMO, and GLI1 decreased in response to 24 h of treatment with vismodegib or itraconazole. Furthermore, CAL27 cells exhibited alterations in morphology, cell size, and cellular granularity. An increase in the DNA fragmentation was observed after treatment and both inhibitors induced apoptosis after 72 h. In conclusion, SMO inhibitors vismodegib and itraconazole demonstrably reduced the expression of HH genes in CAL27 OSCC cell line. In addition, treatment with vismodegib and itraconazole reduced cellular viability and altered the morphology of CAL27 cells, and also induced apoptosis.

Immunohistochemical Evaluation of the Hedgehog Route as a Potential Prognostic Factor in Hepatocellular Carcinoma / Avaliação imuno-histoquímica da rota Hedgehog como potencial fator prognóstico no carcinoma hepatocelular

ABSTRACT Introduction Hepatocellular carcinoma (HCC) is the most common primary malignant neoplasm in the liver. HCC develops gradually from multiple stages that control proliferation and apoptosis. In hepatocarcinogenesis, multiple signaling pathways were already described, such as the Hedgehog pathway (Hh). However, few studies have investigated the expression of Hh proteins as a potential prognostic factor in human HCC. This study aimed to investigate the expression of the Shh protein in HCC and to correlate with clinical and morphological prognostic characteristics of the tumor. Methods Immunohistochemical expression of Shh protein in tumor and cirrhotic parenchyma was performed in 36 HCC samples from patients who underwent liver transplantation at Clinical Hospital - UFMG. Correlation between the Shh tumor expression and etiology, number of nodules, size of the nodules, levels of alpha-fetus-protein (AFP), MELD score, tumor differentiation, and vascular invasion were performed. Results In our study, Shh protein labeling gradually increased from the normal to the cirrhotic and neoplastic parenchyma. Degree of tumor differentiation and vascular invasion were correlated with high Shh protein expression (p = 0.014 and p = 0.003, respectively). The other variables did not show a statistically significant correlation with Shh labeling. Conclusion Hedgehog pathway has importance in hepatocarcinogenesis. The immunohistochemical study of the Hh signaling pathway may have a promising role as a prognostic factor for HCC, especially due to the positive correlation between the Shh expression and the degree of tumor differentiation and invasion vascular.

RESUMO Introdução O carcinoma hepatocelular (CHC) é a neoplasia maligna primária mais comum no fígado. O CHC se desenvolve gradualmente a partir de múltiplos estágios que controlam a proliferação e a apoptose. Na hepatocarcinogênese, múltiplas vias de sinalização já foram descritas, como a via Hedgehog (Hh). No entanto, poucos estudos investigaram a expressão de proteínas Hh como um potencial fator prognóstico no CHC humano. Este estudo teve como objetivo investigar a expressão da proteína Shh no CHC e correlacionar com características prognósticas clínicas e morfológicas do tumor. Métodos A expressão imuno-histoquímica da proteína Shh em tumor e parênquima cirrótico foi realizada em 36 amostras de CHC de pacientes submetidos a transplante hepático no Hospital das Clínicas - UFMG. Correlação entre a expressão e etiologia do tumor Shh, número de nódulos, tamanho dos nódulos, níveis de proteína alfa-feto (AFP), pontuação MELD, diferenciação tumoral e invasão vascular foram realizadas. Resultados Em nosso estudo, a marcação da proteína Shh aumentou gradualmente do parênquima normal para o cirrótico e neoplásico. Grau de diferenciação tumoral e invasão vascular foram correlacionados com alta expressão da proteína Shh (p = 0,014 ep = 0,003, respectivamente). As demais variáveis não apresentaram correlação estatisticamente significativa com a marcação de Shh. Conclusão A via Hedgehog tem importância na hepatocarcinogênese. O estudo imuno-histoquímico da via de sinalização Hh pode ter um papel promissor como fator prognóstico para CHC, principalmente devido à correlação positiva entre a expressão de Shh e o grau de diferenciação tumoral e invasão vascular.

Sensitivity and specificity of the circulating cathodic antigen rapid urine test in the diagnosis of Schistosomiasis mansoni infection and evaluation of morbidity in a low- endemic area in Brazil.

INTRODUCTION:: The Kato-Katz technique is the standard diagnostic test for Schistosoma mansoni infection in rural areas. However, the utility of this method is severely limited by the day-to-day variability in host egg excretion in the stool. In high-transmission areas, the point-of-care circulating cathodic antigen (POC-CCA) urine assay has proven to be a reliable test. However, investigations of the reliability of the POC-CCA assay in low-transmission regions are under way. This study aimed to evaluate the sensitivity and specificity of the POC-CCA assay and the morbidity of schistosomiasis in a low-endemic area in Brazil. METHODS:: Pains City is a low-transmission zone for schistosomiasis. A total of 300 subjects aged 7-76 years were randomly selected for the POC-CCA cassette test. For S. mansoni diagnosis, three stool samples on six slides were compared with one urine sample for each subject. The sensitivity and specificity in the absence of a gold standard were calculated using latent class analysis. Clinical examinations and abdominal ultrasounds were performed in 181 volunteers to evaluate morbidity associated with schistosomiasis. RESULTS:: The sensitivity and specificity of the Kato-Katz technique were 25.6% and 94.6%, respectively. By contrast, the sensitivity and specificity of the POC-CCA assay were 68.1% and 72.8%, respectively. Hepatosplenic schistosomiasis was diagnosed in two patients (1.1%). CONCLUSIONS:: Overall, the POC-CCA urine assay proved to be a useful test for diagnosing S. mansoni in a low-endemic area in Brazil. Severe clinical forms of schistosomiasis can be present even in such low-endemic areas.

Bone marrow-derived monocyte infusion improves hepatic fibrosis by decreasing osteopontin, TGF-ß1, IL-13 and oxidative stress.

AIM: To evaluate the therapeutic effects of bone marrow-derived CD11b+CD14+ monocytes in a murine model of chronic liver damage. METHODS: Chronic liver damage was induced in C57BL/6 mice by administration of carbon tetrachloride and ethanol for 6 mo. Bone marrow-derived monocytes isolated by immunomagnetic separation were used for therapy. The cell transplantation effects were evaluated by morphometry, biochemical assessment, immunohistochemistry and enzyme-linked immunosorbent assay. RESULTS: CD11b+CD14+ monocyte therapy significantly reduced liver fibrosis and increased hepatic glutathione levels. Levels of pro-inflammatory cytokines, including tumor necrosis factor-α, interleukin (IL)-6 and IL-1ß, in addition to pro-fibrotic factors, such as IL-13, transforming growth factor-ß1 and tissue inhibitor of metalloproteinase-1 also decreased, while IL-10 and matrix metalloproteinase-9 increased in the monocyte-treated group. CD11b+CD14+ monocyte transplantation caused significant changes in the hepatic expression of α-smooth muscle actin and osteopontin. CONCLUSION: Monocyte therapy is capable of bringing about improvement of liver fibrosis by reducing oxidative stress and inflammation, as well as increasing anti-fibrogenic factors.

Serum osteopontin is a biomarker of severe fibrosis and portal hypertension in human and murine schistosomiasis mansoni.

Schistosomiasis is a major cause of fibrosis and portal hypertension. The reason 4-10% of infected subjects develops hepatosplenic schistosomiasis remains unclear. Chronically infected male CBA/J mice reproduce the dichotomic forms of human schistosomiasis. Most mice (80%) develop moderate splenomegaly syndrome (similar to hepatointestinal disease in humans) and 20% present severe hypersplenomegaly syndrome (analogous to human hepatosplenic disease). We demonstrated that the profibrogenic molecule osteopontin discriminates between mice with severe and mild disease and could be a novel morbidity biomarker in murine and human schistosomiasis. Failure to downregulate osteopontin during the chronic phase may explain why hepatosplenic subjects develop severe fibrosis.

Osteopontin Is Upregulated in Human and Murine Acute Schistosomiasis Mansoni.

BACKGROUND: Symptomatic acute schistosomiasis mansoni is a systemic hypersensitivity reaction against the migrating schistosomula and mature eggs after a primary infection. The mechanisms involved in the pathogenesis of acute schistosomiasis are not fully elucidated. Osteopontin has been implicated in granulomatous reactions and in acute hepatic injury. Our aims were to evaluate if osteopontin plays a role in acute Schistosoma mansoni infection in both human and experimentally infected mice and if circulating OPN levels could be a novel biomarker of this infection. METHODOLOGY/PRINCIPAL FINDINGS: Serum/plasma osteopontin levels were measured by ELISA in patients with acute (n = 28), hepatointestinal (n = 26), hepatosplenic (n = 39) schistosomiasis and in uninfected controls (n = 21). Liver osteopontin was assessed by immunohistochemistry in needle biopsies of 5 patients. Sera and hepatic osteopontin were quantified in the murine model of schistosomiasis mansoni during acute (7 and 8 weeks post infection, n = 10) and chronic (30 weeks post infection, n = 8) phase. Circulating osteopontin levels are increased in patients with acute schistosomiasis (p = 0.0001). The highest levels of OPN were observed during the peak of clinical symptoms (7-11 weeks post infection), returning to baseline level once the granulomas were modulated (>12 weeks post infection). The plasma levels in acute schistosomiasis were even higher than in hepatosplenic patients. The murine model mirrored the human disease. Macrophages were the major source of OPN in human and murine acute schistosomiasis, while the ductular reaction maintains OPN production in hepatosplenic disease. Soluble egg antigens from S. mansoni induced OPN expression in primary human kupffer cells. CONCLUSIONS/SIGNIFICANCE: S. mansoni egg antigens induce the production of OPN by macrophages in the necrotic-exudative granulomas characteristic of acute schistosomiasis mansoni. Circulating OPN levels are upregulated in human and murine acute schistosomiasis and could be a non-invasive biomarker of this form of disease.

Brain schistosomiasis in mice experimentally infected with Schistosoma mansoni.

INTRODUCTION: Human neuroschistosomiasis has been reported in the literature, but the possibility of modeling neuroschistosomiasis in mice is controversial. METHODS: In two research laboratories in Brazil that maintain the Schistosoma mansoni life cycle in rodents, two mice developed signs of brain disease (hemiplegia and spinning), and both were autopsied. RESULTS: S. mansoni eggs, both with and without granuloma formation, were observed in the brain and meninges of both mice by optical microscopy. CONCLUSIONS: This is the first description of eggs in the brains of symptomatic mice that were experimentally infected with S. mansoni. An investigation of experimental neuroschistosomiasis is now feasible.

Sensitivity and specificity of the circulating cathodic antigen rapid urine test in the diagnosis of Schistosomiasis mansoni infection and evaluation of morbidity in a low- endemic area in Brazil

Abstract INTRODUCTION: The Kato-Katz technique is the standard diagnostic test for Schistosoma mansoni infection in rural areas. However, the utility of this method is severely limited by the day-to-day variability in host egg excretion in the stool. In high-transmission areas, the point-of-care circulating cathodic antigen (POC-CCA) urine assay has proven to be a reliable test. However, investigations of the reliability of the POC-CCA assay in low-transmission regions are under way. This study aimed to evaluate the sensitivity and specificity of the POC-CCA assay and the morbidity of schistosomiasis in a low-endemic area in Brazil. METHODS: Pains City is a low-transmission zone for schistosomiasis. A total of 300 subjects aged 7-76 years were randomly selected for the POC-CCA cassette test. For S. mansoni diagnosis, three stool samples on six slides were compared with one urine sample for each subject. The sensitivity and specificity in the absence of a gold standard were calculated using latent class analysis. Clinical examinations and abdominal ultrasounds were performed in 181 volunteers to evaluate morbidity associated with schistosomiasis. RESULTS: The sensitivity and specificity of the Kato-Katz technique were 25.6% and 94.6%, respectively. By contrast, the sensitivity and specificity of the POC-CCA assay were 68.1% and 72.8%, respectively. Hepatosplenic schistosomiasis was diagnosed in two patients (1.1%). CONCLUSIONS: Overall, the POC-CCA urine assay proved to be a useful test for diagnosing S. mansoni in a low-endemic area in Brazil. Severe clinical forms of schistosomiasis can be present even in such low-endemic areas.

Brain schistosomiasis in mice experimentally infected with Schistosoma mansoni

Introduction Human neuroschistosomiasis has been reported in the literature, but the possibility of modeling neuroschistosomiasis in mice is controversial. Methods In two research laboratories in Brazil that maintain the Schistosoma mansoni life cycle in rodents, two mice developed signs of brain disease (hemiplegia and spinning), and both were autopsied. Results S. mansoni eggs, both with and without granuloma formation, were observed in the brain and meninges of both mice by optical microscopy. Conclusions This is the first description of eggs in the brains of symptomatic mice that were experimentally infected with S. mansoni. An investigation of experimental neuroschistosomiasis is now feasible. .