Study of the cross-talk between Fasciola hepatica juveniles and the intestinal epithelial cells of the host by transcriptomics in an in vitro model.

Fasciolosis is a globally widespread trematodiasis with a major economic and veterinary impact. Therefore, this disease is responsible for millions of dollars in losses to the livestock industry, and also constitutes an emerging human health problem in endemic areas. The ubiquitous nature of Fasciola hepatica, the main causative agent, is one of the key factors for the success of fasciolosis. Accordingly, this parasite is able to subsist in a wide variety of ecosystems and hosts, thanks to the development of a plethora of strategies for adaption and immune evasion. Fasciolosis comprises a growing concern due to its high prevalence rates, together with the emergence of strains of the parasite resistant to the treatment of choice (triclabendazole). These facts highlight the importance of developing novel control measures which allow for an effective protection against the disease before F. hepatica settles in a niche inaccessible to the immune system. However, knowledge about the initial phases of the infection, including the migration mechanisms of the parasite and the early innate host response, is still scarce. Recently, our group developed an in vitro host-parasite interaction model that allowed the early events to be unveiled after the first contact between the both actors. This occurs shortly upon ingestion of F. hepatica metacercariae and the emergence of the newly excysted juveniles (FhNEJ) in the host duodenum. Here, we present a transcriptomic analysis of such model using an approach based on RNA sequencing (RNA-Seq), which reveals changes in gene expression related to proteolysis and uptake of metabolites in FhNEJ. Additionally, contact with the parasite triggered changes in host intestinal cells related to pseudogenes expression and host defence mechanisms, including immune response, among others. In sum, these results provide a better understanding of the early stages of fasciolosis at molecular level, and a pool of targets that could be used in future therapeutic strategies against the disease.

CYSTEINE-RICH RECEPTOR-LIKE PROTEIN KINASES: their evolution, structure, and roles in stress response and development.

Plant-specific receptor-like protein kinases (RLKs) are central components for sensing the extracellular microenvironment. CYSTEINE-RICH RLKs (CRKs) are members of one of the biggest RLK subgroups. Their physiological and molecular roles have only begun to be elucidated, but recent studies highlight the diverse types of proteins interacting with CRKs, as well as the localization of CRKs and their lateral organization within the plasma membrane. Originally the DOMAIN OF UNKNOWN FUNCTION 26 (DUF26)-containing extracellular region of the CRKs was proposed to act as a redox sensor, but the potential activating post-translational modification or ligands perceived remain elusive. Here, we summarize recent progress in the analysis of CRK evolution, molecular function, and role in plant development, abiotic stress responses, plant immunity, and symbiosis. The currently available information on CRKs and related proteins suggests that the CRKs are central regulators of plant signaling pathways. However, more research using classical methods and interdisciplinary approaches in various plant model species, as well as structural analyses, will not only enhance our understanding of the molecular function of CRKs, but also elucidate the contribution of other cellular components in CRK-mediated signaling pathways.

Germline CDH1 G212E Missense Variant: Combining Clinical, In Vitro and In Vivo Strategies to Unravel Disease Burden.

E-cadherin, encoded by CDH1, is an essential molecule for epithelial homeostasis, whose loss or aberrant expression results in disturbed cell-cell adhesion, increased cell invasion and metastasis. Carriers of CDH1 germline mutations have a high risk of developing diffuse gastric cancer and lobular breast cancer, associated with the cancer syndrome Hereditary Diffuse Gastric Cancer (HDGC). The ubiquitous availability of cancer panels has led to the identification of an increasing amount of "incidental" CDH1 genetic variants that pose a serious clinical challenge. This has sparked intensive research aiming at an accurate classification of the variants and consequent validation of their clinical relevance. The present study addressed the significance of a novel CDH1 variant, G212E, identified in an unusually large pedigree displaying strong aggregation of diffuse gastric cancer. We undertook a comprehensive pipeline encompassing family data, in silico predictions, in vitro assays and in vivo strategies, which validated the deleterious phenotype induced by this genetic alteration. In particular, we demonstrated that the G212E variant affects the stability and localization, as well as the adhesive and anti-invasive functions of E-cadherin, triggering epithelial disruption and disorganization. Our findings illustrate the clinical implication of a complementary approach for effective variant categorization and patient management.

High-fat diet decreases activity of the oxidative phosphorylation complexes and causes nonalcoholic steatohepatitis in mice.

Nonalcoholic fatty liver disease (NAFLD) is the most frequent histological finding in individuals with abnormal liver-function tests in the Western countries. In previous studies, we have shown that oxidative phosphorylation (OXPHOS) is decreased in individuals with NAFLD, but the cause of this mitochondrial dysfunction remains uncertain. The aims of this study were to determine whether feeding mice a high-fat diet (HFD) induces any change in the activity of OXPHOS, and to investigate the mechanisms involved in the pathogenesis of this defect. To that end, 30 mice were distributed between five groups: control mice fed a standard diet, and mice on a HFD and treated with saline solution, melatonin (an antioxidant), MnTBAP (a superoxide dismutase analog) or uric acid (a scavenger of peroxynitrite) for 28 weeks intraperitoneously. In the liver of these mice, we studied histology, activity and assembly of OXPHOS complexes, levels of subunits of these complexes, gene expression of these subunits, oxidative and nitrosative stress, and oxidative DNA damage. In HFD-fed mice, we found nonalcoholic steatohepatitis, increased gene expression of TNFα, IFNγ, MCP-1, caspase-3, TGFß1 and collagen α1(I), and increased levels of 3-tyrosine nitrated proteins. The activity and assembly of all OXPHOS complexes was decreased to about 50-60%. The amount of all studied OXPHOS subunits was markedly decreased, particularly the mitochondrial-DNA-encoded subunits. Gene expression of mitochondrial-DNA-encoded subunits was decreased to about 60% of control. There was oxidative damage to mitochondrial DNA but not to genomic DNA. Treatment of HFD-fed mice with melatonin, MnTBAP or uric acid prevented all changes observed in untreated HFD-fed mice. We conclude that a HFD decreased OXPHOS enzymatic activity owing to a decreased amount of fully assembled complexes caused by a reduced synthesis of their subunits. Antioxidants and antiperoxynitrites prevented all of these changes, suggesting that nitro-oxidative stress played a key role in the pathogenesis of these alterations. Treatment with these agents might prevent the development of NAFLD in humans.

Early-onset colorectal cancer is an easy and effective tool to identify retrospectively Lynch syndrome.

BACKGROUND AND OBJECTIVES: Early age of onset is a marker of a possible hereditary component in colorectal cancer (CRC). We evaluated whether early age of onset is a good marker to identify Lynch syndrome, especially retrospectively, and if there is any other feature that could improve this identification. METHODS: We selected patients with CRC aged 45 years or younger from the pathological reports of three different institutions and different periods of time. Clinical information, family history, and tumor samples were obtained. Cases were classified according to mismatch repair (MMR) proficiency. RESULTS: Of 133 tumors, 22 showed microsatellite instability (MSI). In 15 MSI cases, a germline mutation in 1 of the MMR genes was identified, 7 of which were not identified before. The positive predictive value (PPV) of right colon CRC for a positive genetic MMR test is 30.6%, whereas "signet ring" cells and fulfillment Amsterdam II criteria have PPVs of 42.9% and 47.8%, respectively. Combining right-sided CRC with mucin production, with fulfilling Amsterdam II criteria, or with "signet ring" cells, PPVs are 54.5, 64.3, and 100%. The probability of the absence of a mutation when CRC is located in the left colon is 94.7%, whereas absence of aggregation for Lynch-related neoplasm has a 100% probability. CONCLUSIONS: Early age of onset is an effective method to identify retrospectively Lynch syndrome. Taking into account the location and histology features of the tumor, and the familial history of the cases, we notably increase the a priori probability of detecting a germline MMR mutation.

Switching to sirolimus monotherapy for de novo tumors after liver transplantation. A preliminary experience.

BACKGROUND/AIMS: Alcoholic cirrhosis, smoking, and use of calcineurin inhibitors (CNI) are associated with the development of de novo tumors in liver transplant (LT) recipients. Sirolimus is an immunosuppressor with antitumoral properties. METHODOLOGY: Between April 1986 and April 2007, we performed 1231 liver transplants in 1084 recipients. A total of 128 de novo tumors were observed in 116 recipients from a sample of 850 adult recipients who survived more than 2 months. This study comprises 16 LT recipients (13 male and 3 female; mean age, 45.1 +/- 11.1 years) who were switched to sirolimus monotherapy who developed de novo tumors and were switched from CNI or mycophenolate mofetil to sirolimus monotherapy. RESULTS: De novo tumors location: 2 lymphomas, 9 upper aerodigestive, 1 skin, 1 parotid, 1 lung, 1 breast, and 1 rectum. Time from LT to sirolimus monotherapy was 86 months; time taking to switching from CNI to sirolimus monotherapy was 48 days, and mean follow-up of patients on sirolimus monotherapy was 15.7 months. Thirteen patients underwent tumor resection, 5 received chemotherapy, and 5 received radiotherapy. Five patients died during the follow-up, and patient survival after diagnosis was 42.8 months. Mean dose of sirolimus was 2.7 mg/day and the mean trough level was 8.9 ng/mL. Total cholesterol and triglycerides values increased after switching. Mean serum creatinine, glucose, AST and ALT values, and haematological parameters were similar before and after switching. No patients developed acute rejection, and adverse effects were observed in 8 patients. CONCLUSIONS: Sirolimus monotherapy can be used safely to improve survival in LT recipients with de novo tumors.

Approach to early-onset colorectal cancer: clinicopathological, familial, molecular and immunohistochemical characteristics.

AIM: To characterize clinicopathological and familial features of early-onset colorectal cancer (CRC) and compare features of tumors with and without microsatellite instability (MSI). METHODS: Forty-five patients with CRC aged 45 or younger were included in the study. Clinical information, a three-generation family history, and tumor samples were obtained. MSI status was analyzed and mismatch repair genes were examined in the MSI families. Tumors were included in a tissue microarray and an immunohistochemical study was carried out with a panel of selected antibodies. RESULTS: Early onset CRC is characterized by advanced stage at diagnosis, right colon location, low-grade of differentiation, mucin production, and presence of polyps. Hereditary forms represent at least 21% of cases. Eighty-one percent of patients who died during follow-up showed a lack of expression of cyclin E, which could be a marker of poor prognosis. beta-catenin expression was normal in a high percentage of tumors. CONCLUSION: Early-onset CRC has an important familial component, with a high proportion of tumors showing microsatellite stable. Cyclin E might be a poor prognosis factor.

Prognostic factors in cancer of unknown primary site.

AIMS AND BACKGROUND: Patients with cancer of an unknown primary site (CUP) usually have a poor outcome. The identification of prognostic factors that affect survival can help clinicians find a better approach to such cases in terms of diagnostic and therapeutic management. METHODS: We conducted a retrospective study including the cases of CUP recorded at the University Hospital 12 de Octubre Tumor Registry between 1999 and 2003. RESULTS: CUP was diagnosed in 265 patients during the analyzed period. One hundred and seventy-one were men (64.5%) and the mean age of the patients was 66.9 years (range 32-98 years). The median survival was 2.5 months, and the survival rate was 35.1% 6 months from diagnosis (95% CI: 28.9-41.3) and 24.5% 1 year from diagnosis (95% CI: 18.7-30.3). Univariate analysis revealed as significant predictive variables of a better outcome age under 70 years; involvement of a single organ; normal serum levels of alkaline phosphatase and albumin; normal erythrocyte sedimentation rate; normal levels of the serum tumor markers CEA, CA 19.9 and CA 15.3; squamous carcinoma histology; clinical presentation as lymph node enlargement; and the administration of treatment. Multivariate analysis showed that albumin and alkaline phosphatase levels, squamous carcinoma histology, age and treatment were the most important prognostic factors. Other variables analyzed (liver, bone or lung involvement, lactate dehydrogenase levels, gender) did not affect survival. CONCLUSIONS: CUP has a poor prognosis. Some prognostic factors that affect survival in these patients, however, may be identified.

Incidence, risk factors, and outcome of chronic rejection during antiviral therapy for posttransplant recurrent hepatitis C.

Antiviral therapy for recurrent hepatitis C in liver transplantation has been associated with the development of chronic rejection. The aim of this study was to assess the incidence, evolution, and risk factors associated with the development of chronic rejection during posttransplant hepatitis C virus antiviral therapy. Seventy-nine patients with posttransplant recurrent hepatitis C who were treated with pegylated interferon and ribavirin were prospectively followed. Liver biopsy was performed before antiviral therapy was initiated and when liver tests worsened during therapy. Pretransplant and posttransplant factors were analyzed as potential risk factors for the development of chronic rejection. Seven of 79 patients (9%) developed chronic rejection during antiviral therapy. The mean time from the start of treatment to the development of chronic rejection was 5.8 months (3-12 months). An analysis of factors associated with the development of chronic rejection showed that the use of cyclosporine as immunosuppression therapy (6 of 19 patients who received cyclosporine developed chronic rejection in comparison with only 1 of 57 patients who received tacrolimus; P = 0.0013), achievement of sustained virological response (P = 0.043), and ribavirin discontinuation (P = 0.027) were associated with the development of chronic rejection. In conclusion, the development of chronic rejection during posttransplant pegylated interferon and ribavirin therapy is a severe complication. The use of cyclosporine, ribavirin discontinuation, and viral infection elimination seem to be associated with the development of this complication. Liver Transpl 15:948-955, 2009. (c) 2009 AASLD.

[Bronchogenic carcinoma 2000-2001: characteristics and overall survival]. / Carcinoma broncogénico 2000-2001: características y supervivencia global.

OBJECTIVE: To describe the clinical characteristics and survival of patients diagnosed with bronchogenic carcinoma during the years 2000 and 2001 in a tertiary level hospital. PATIENTS AND METHODS: Data were collected from our hospital's tumor registry and validated with independent sources. Of all the patients diagnosed with or treated for bronchogenic carcinoma in our hospital, only those from our health care area were selected. RESULTS: During the 2-year study period, 482 patients were diagnosed. Of those, 91% were men. The mean (SD) age was 66.6 (9.65) years. Large cell carcinomas accounted for 29.4% of cases. Of all the cases of bronchogenic carcinoma, 41.3% were diagnosed in stage IV. Thirty percent of non-small cell carcinomas were classified as stage I, compared to 6% of small cell carcinomas (P< .001). The most frequent treatment was chemotherapy (42.1%) and 20% of patients underwent surgery. The overall 5-year survival rate was 13% (95% confidence interval [CI], 10%-16%), while survival was significantly lower in patients aged 68 years or older (95% CI, 3%-15%; P< .001) and in patients with small cell carcinoma (0%, P< .01). CONCLUSIONS: Our recent experience (2000-2001) confirmed the advanced age of patients with bronchogenic carcinoma, the frequency of diagnosis in advanced stages of the disease (41% in stage IV), and the low overall 5-year survival rate (13%).

Relationship between biomarker expression and allelic alteration in esophageal carcinoma.

BACKGROUND AND AIM: Expression of biomarkers and probable allelic alterations were studied in esophagus tissue samples from patients with esophageal carcinoma. METHODS: A total of 116 esophagus tissue samples were obtained from 25 patients with esophagus cancer. Histological studies revealed 23 samples were adenocarcinoma and 14 samples were epidermoid carcinoma while 79 samples were non-tumor. Expression of biomarkers was determined by enzyme immunoassay, and allelic alterations on chromosome 17p were performed by polymerase chain reaction (PCR) using primers D17S513 and D17S514. RESULTS: The adenocarcinoma group exhibited an increase of matrix metalloproteinase (MMP)-1 (P < 0.0001) and sialyl Le (a) (P < 0.001) mean levels when compared with the non-tumor group. Adenocarcinoma samples from patients with more than three positive lymph nodes had lower levels of tissue-inhibitor metalloproteinase (TIMP)-1 than those with negative nodes (P < 0.0005). Positive allelic alteration was associated with high levels of MMP-1 expression (P = 0.003). Epidermoid carcinoma samples showed higher expression of MMP-1 (P < 0.0001) and TIMP-1 (P < 0.02) than non-tumor samples. Both epidermal growth factor receptor and sialyl Le (a) levels were overexpressed in tumors of patients with more than three positive lymph nodes (P < 0.005). Carcinoembryonic antigen levels were higher in tumors associated with allelic wild type group (P = 0.0001) and patients with negative lymph nodes (P < 0.05). Furthermore, variability in expression of biomarkers was observed according to sample location, and allelic alterations were also found both in tumor and in some non-tumor samples. CONCLUSION: The data suggest that overexpression of tissue biomarkers associated with allelic alterations may have potential prognostic implications with different behavior in esophagus cancer.

Clinical and histological efficacy of pegylated interferon and ribavirin therapy of recurrent hepatitis C after liver transplantation.

Treatment of recurrent hepatitis C in liver transplant is controversial. The aim of our study was to evaluate the clinical and histological efficacy of pegylated interferon alpha 2b (PEG-IFN) and ribavirin therapy of recurrent hepatitis C after liver transplantation (LT). We prospectively included 47 liver transplant patients with: 1) a positive test for hepatitis C virus (HCV)-ribonucleic acid (RNA) in serum; 2) alanine aminotransferase (ALT) >45 UI/mL; and 3) a liver biopsy showing chronic hepatitis without rejection in the previous 2 months. Patients received PEG-IFN (1.5 microg/kg/week) and ribavirin (800-1,000 mg/day) for 12 months. Follow-up was based on biochemical (ALT), virological (RNA-HCV), and histological (liver biopsy) examinations. Follow-up lasted a minimum of 6 months after the end of antiviral therapy. Sustained virological response (SVR) was achieved in 23% of the patients. A total of 33 (70%) patients had normalized ALT levels at the end of therapy. Inflammatory portal and lobular score declined significantly in patients with SVR (P < 0.05) but not in nonresponder patients. Fibrosis did not change significantly in either group. SVR was significantly associated with low gamma-glutamyltransferase GGT (P = 0.04) and HCV-RNA levels (P = 0.03), a virological response at 12 weeks (P = 0.002) and patient's compliance (P = 0.04). Ten (21%) patients were withdrawn prematurely due to adverse effects. In conclusion, Therapy with PEG-IFN and ribavirin achieved SVR and a significant histological improvement in 23% of liver transplant recipients with chronic hepatitis C. Toxicity is an important drawback of this therapy.

Uric acid and anti-TNF antibody improve mitochondrial dysfunction in ob/ob mice.

The mechanisms responsible for low mitochondrial respiratory chain (MRC) activity in the liver of patients with nonalcoholic steatohepatitis are unknown. In this study, we examined the cause of this dysfunction in ob/ob mice. Forty-six mice were distributed in six groups: group I: C57BL/6J mice; group II: C57BL/6J Lep(-/-) mice (ob/ob); group III, ob/ob mice treated with manganese [III] tetrakis (5,10,15,20 benzoic acid) porphyrin (MnTBAP); group IV, ob/ob mice treated with IgG1 immunoglobulin; group V, ob/ob mice treated with anti-TNF antibody; group VI: ob/ob mice treated with uric acid. In liver tissue, we measured MRC activity, fatty acid beta-oxidation, tumor necrosis factor (TNF), inducible nitric oxide synthase (iNOS), 3-tyrosine-nitrated proteins, 3-tyrosine-nitrated mitochondrial proteins, including cytochrome c and ND4 subunit of complex I. MRC activity was decreased in ob/ob mice. TNF levels, iNOS protein expression, and tyrosine nitrated proteins were markedly increased in the liver of ob/ob mice. In these animals, mitochondrial proteins were markedly tyrosine nitrated, particularly the ND4 subunit of complex I and cytochrome c. Treatment of these animals with uric acid, a peroxynitrite scavenger, anti-TNF antibody, or MnTBAP decreased tyrosine nitrated proteins, improved the activity of MRC complexes, and led to a marked regression of hepatic steatosis and inflammation. In conclusion, MRC dysfunction and liver lesions found in ob/ob mice are likely to reflect the tyrosine nitration of mitochondrial proteins by peroxynitrite or a peroxynitrite-derivate radical. Increased hepatic TNF and iNOS expression might enhance peroxynitrite formation and inhibition of MRC complexes.

Factors associated with the presence of nonalcoholic steatohepatitis in patients with chronic hepatitis C.

OBJECTIVES: The aim of this study was to identify factors associated with the presence of nonalcoholic steatohepatitis (NASH) in patients with chronic hepatitis C (CHC). METHODS: We studied 98 patients with CHC [47 with NASH (group HCV/NASH), 51 without NASH (group HCV)] and 85 with NASH not infected with hepatitis C virus (HCV) (group NASH). We determined factors associated with the presence of NASH in patients with hepatitis C. RESULTS: Group HCV/NASH patients resembled those with NASH. Body mass index (BMI) was higher in group HCV/NASH than in group HCV, but was similar to group NASH. Most HCV/NASH patients had risk factors for NASH. In patients infected with HCV, NASH and NASH-related lesions were independently associated with BMI, while steatosis score was associated with HCV genotype 3 and BMI. Fibrosis stage was independently associated with steatosis, necroinflammatory activity index, and NASH lesions. CONCLUSION: While HCV genotype 3 infection and BMI are associated with the presence of steatosis in CHC, BMI is the only factor independently associated with the presence of NASH in these patients. We suggest that overweight-related factors might induce NASH in CHC patients.

Defective hepatic mitochondrial respiratory chain in patients with nonalcoholic steatohepatitis.

Mitochondrial dysfunction might play a central role in the pathogenesis of nonalcoholic steatohepatitis (NASH). The aims of this study were to evaluate whether free fatty acid (FFA) transport into the mitochondria or the activity of mitochondria respiratory chain (MRC) complexes are impaired in NASH. In patients with NASH and control subjects, we measured free carnitine, short-chain acylcarnitine (SCAC) and long-chain acylcarnitine (LCAC) esters, carnitine palmitoyltransferase (CPT) activity, and MRC enzyme activity in liver tissue as well as serum concentration of tumor necrosis factor alpha (TNF-alpha), homeostatic metabolic assessment of insulin resistance (HOMA(IR)), and body mass index (BMI). In patients with NASH, the LCAC/free carnitine ratio was significantly increased and the SCAC/free carnitine ratio was decreased. In patients with NASH, the activity of the MRC complexes was decreased to 63% +/- 20% (complex I), 58.5% +/- 16.7% (complex II), 70.6% +/- 10.3% (complex III), 62.5% +/- 13% (complex IV), and 42.4% +/- 9.1% (adenosine triphosphate synthase) of the corresponding control values. Activity of these complexes correlated significantly with serum TNF-alpha and HOMA(IR). Serum TNF-alpha (36.3 +/- 23.1 pg/mL), HOMA(IR) (4.5 +/- 2.38), and BMI (29.9 +/- 3.5 kg/m(2)) values were significantly increased in patients with NASH. In conclusion, activities of MRC complexes were decreased in liver tissue of patients with NASH. This dysfunction correlated with serum TNF-alpha, insulin resistance, and BMI values.

Colorectal cancer relapse: allelic alterations associated with tumour marker overexpression.

The objective of the present study was to assess the prognostic value of allelic alterations in comparison with clinical prognostic factors (age and gender, clinical stage, lymph node involvement, tissue tumour marker expression) and clinical outcomes (disease relapse and overall survival time) in colorectal cancer patients. Polymerase chain reaction was performed on the DNA of 72 colorectal samples (from 36 colorectal cancer patients) using primers D17S513 and D17S514. Carbohydrate antigen 19-9 (CA 19-9) marker was determined in tumour sections by enzyme immunoassay. Tumours were considered to exhibit allelic alterations if the microsatellite region adjacent to the p53 locus in chromosome 17 either gained or lost repeated sequences. Allelic alterations were detected in 44% of tumour samples. Patients with more than 3 involved lymph nodes had more frequent allelic alterations (p < 0.002). The allelic alteration status was compared with tumour CA 19-9 expression, which showed statistically significantly higher values within the allelic alterations group (p < 0.005). Multivariate analyses confirmed that tumours with allelic alterations had a higher probability of disease relapse (odds ratio 7.3, p = 0.01). This is the first report showing an association between allelic alteration and overexpression of a tissue tumour marker protein and established risk factors. These results could be considered useful additional prognostic information for colorectal cancer.

An unusual expression of hyperplastic gastropathy (Menetrier type) in twins.

Menetrier's disease is an uncommon condition of unknown aetiology. We describe two cases of male identical twins with haematemesis aged 29 and 35 years that exhibited a similar and particular form of this hyperplastic gastropathy. Their stomachs showed confluent polypoid mucosal projections affecting mainly the gastric fundus and the antrum. To the best of our knowledge, only four previous cases have been reported in a familial setting, and this is the first documented example of an occurrence in twins. These two cases suggest the possibility of a genetic predisposition for this condition.