Chronic hyperammonemia induces peripheral inflammation that leads to cognitive impairment in rats: Reversed by anti-TNF-α treatment.

BACKGROUND & AIMS: Chronic hyperammonemia induces neuroinflammation which mediates cognitive impairment. How hyperammonemia induces neuroinflammation remains unclear. We aimed to assess whether: chronic hyperammonemia induces peripheral inflammation, and whether this then contributes to neuroinflammation, altered neurotransmission and impaired spatial learning - before assessing whether this neuroinflammation and impairment is reversible following hyperammonemia elimination or treatment of peripheral inflammation with anti-TNF-α. METHODS: Chronic hyperammonemia was induced by feeding rats an ammonia-containing diet. Peripheral inflammation was analyzed by measuring PGE2, TNF-α, IL-6 and IL-10. We tested whether chronic anti-TNF-α treatment improves peripheral inflammation, neuroinflammation, membrane expression of glutamate receptors in the hippocampus and spatial learning. RESULTS: Hyperammonemic rats show a rapid and reversible induction of peripheral inflammation, with increased pro-inflammatory PGE2, TNF-α and IL-6, followed at around 10 days by reduced anti-inflammatory IL-10. Peripheral anti-TNF-α treatment prevents peripheral inflammation induction and the increase in IL-1b and TNF-α and microglia activation in hippocampus of the rats, which remain hyperammonemic. This is associated with prevention of the altered membrane expression of glutamate receptors and of the impairment of spatial memory assessed in the radial and Morris water mazes. CONCLUSIONS: This report unveils a new mechanism by which chronic hyperammonemia induces neurological alterations: induction of peripheral inflammation. This suggests that reducing peripheral inflammation by safe procedures would improve cognitive function in patients with minimal hepatic encephalopathy. LAY SUMMARY: This article unveils a new mechanism by which chronic hyperammonemia induces cognitive impairment in rats: chronic hyperammonemia per se induces peripheral inflammation, which mediates many of its effects on the brain, including induction of neuroinflammation, which alters neurotransmission, leading to cognitive impairment. It is also shown that reducing peripheral inflammation by treating rats with anti-TNF-α, which does not cross the blood-brain barrier, prevents hyperammonemia-induced neuroinflammation, alterations in neurotransmission and cognitive impairment.

Sustained hyperammonemia induces TNF-a IN Purkinje neurons by activating the TNFR1-NF-κB pathway.

BACKGROUND: Patients with liver cirrhosis may develop hepatic encephalopathy. Rats with chronic hyperammonemia exhibit neurological alterations mediated by peripheral inflammation and neuroinflammation. Motor incoordination is due to increased TNF-a levels and activation of its receptor TNFR1 in the cerebellum. The aims were to assess (a) whether peripheral inflammation is responsible for TNF-a induction in hyperammonemic rats, (b) the cell type(s) in which TNF-a is increased, (c) whether this increase is associated with increased nuclear NF-κB and TNFR1 activation, (d) the time course of TNF-a induction, and (e) if TNF-a is induced in the Purkinje neurons of patients who die with liver cirrhosis. METHODS: We analyzed the level of TNF-a mRNA and NF-κB in microglia, astrocytes, and Purkinje neurons in the cerebellum after 1, 2, and 4 weeks of hyperammonemia. We assessed whether preventing peripheral inflammation by administering an anti-TNF-a antibody prevents TNF-a induction. We tested whether TNF-a induction is reversed by R7050, which inhibits the TNFR1-NF-κB pathway, in ex vivo cerebellar slices. RESULTS: Hyperammonemia induced microglial and astrocyte activation at 1 week. This was followed by TNF-a induction in both glial cell types at 2 weeks and in Purkinje neurons at 4 weeks. The level of TNF-a mRNA increased in parallel with the TNF-a protein level, indicating that TNF-a was synthesized in Purkinje cells. This increase was associated with increased NF-κB nuclear translocation. The nuclear translocation of NF-κB and the increase in TNF-a were reversed by R7050, indicating that they were mediated by the activation of TNFR1. Preventing peripheral inflammation with an anti-TNF-a antibody prevents TNF-a induction. CONCLUSION: Sustained (4 weeks) but not short-term hyperammonemia induces TNF-a in Purkinje neurons in rats. This is mediated by peripheral inflammation. TNF-a is also increased in the Purkinje neurons of patients who die with liver cirrhosis. The results suggest that hyperammonemia induces TNF-a in glial cells and that TNF-a released by glial cells activates TNFR1 in Purkinje neurons, leading to NF-κB nuclear translocation and the induction of TNF-a expression, which may contribute to the neurological alterations observed in hyperammonemia and hepatic encephalopathy.

Characterization of triple-negative breast cancer preclinical models provides functional evidence of metastatic progression.

Triple-negative breast cancer (TNBC), an aggressive, metastatic and recurrent breast cancer (BC) subtype, currently suffers from a lack of adequately described spontaneously metastatic preclinical models that faithfully reproduce the clinical scenario. We describe two preclinical spontaneously metastatic TNBC orthotopic murine models for the development of advanced therapeutics: an immunodeficient human MDA-MB-231-Luc model and an immunocompetent mouse 4T1 model. Furthermore, we provide a broad range of multifactorial analysis for both models that could provide relevant information for the development of new therapies and diagnostic tools. Our comparisons uncovered differential growth rates, stromal arrangements and metabolic profiles in primary tumors, and the presence of cancer-associated adipocyte infiltration in the MDA-MB-231-Luc model. Histopathological studies highlighted the more rapid metastatic spread to the lungs in the 4T1 model following a lymphatic route, while we observed both homogeneous (MDA-MB-231-Luc) and heterogeneous (4T1) metastatic spread to axillary lymph nodes. We encountered unique metabolomic signatures in each model, including crucial amino acids and cell membrane components. Hematological analysis demonstrated severe leukemoid and lymphoid reactions in the 4T1 model with the partial reestablishment of immune responses in the immunocompromised MDA-MB-231-Luc model. Additionally, we discovered ß-immunoglobulinemia and increased basal levels of G-CSF correlating with a metastatic switch, with G-CSF also promoting extramedullary hematopoiesis (both models) and causing hepatosplenomegaly (4T1 model). Overall, we believe that the characterization of these preclinical models will foster the development of advanced therapeutic strategies for TNBC treatment, especially for the treatment of patients presenting both, primary tumors and metastatic spread.

An immunohistochemical score to predict the outcome for oral squamous cell carcinoma.

BACKGROUND: Oral cancer is a major public health problem worldwide, with a poor survival. Our aim was to evaluate several protein markers in oral squamous cell carcinomas (OSCC) and analyse their prognostic value on patient's survival. METHODS: We analysed the expression of EGFR, p53, p27, p16, cyclin D1, cyclin A2, COX-2, Ki-67, Bcl-2, VEGFR-1 and VEGFR-2, by immunohistochemistry on 67 primary OSCC. Cancer-specific survival (CSS) analysis was evaluated by the Cox regression model. RESULTS: Markers showed variable expression between 27.9% and 95.2%. In univariate analysis for CSS, we found that four of the tested markers, namely high expression of p53 (P = .001), EGFR (P = .003), cyclin A2 (P = .005) and low expression of p16 (P = .019), along with clinical stage (P < .001), tumour size (P < .001), presence of nodal metastasis (P < .001) and perineural permeation (P = .039) were related to decreased survival. On the basis of these results, we constructed an immunohistochemical score hinging on the possibility that any tumour could express none of these four markers (score 0), one or two markers (score 1) and three or more markers (score 2). In multivariable analysis, this immunohistochemical score revealed an independent prognostic value on cancer-specific survival (P = .001; HR: 3.7: 95%CI 1.7-7.9). Moreover, we confirmed that in early-stage tumours (stage I or II) this score maintained its independent prognostic value (P = .025; HR: 7.9, 95%CI 1.3-49.1) on CSS. CONCLUSION: The expression of the markers p53, p16, EGFR and cyclin A in OSCC, combined to give an immunohistochemical score, may identify high-risk subgroups for decreased survival and to further guide therapeutic decisions.

Combined plasma rich in growth factors and adipose-derived mesenchymal stem cells promotes the cutaneous wound healing in rabbits.

BACKGROUND: The use of Plasma Rich in Growth Factors (PRGF) and Adipose Derived Mesenchymal Stem Cells (ASCs) are today extensively studied in the field of regenerative medicine. In recent years, human and veterinary medicine prefer to avoid using traumatic techniques and choose low or non-invasive procedures. The objective of this study was to evaluate the efficacy of PRGF, ASCs and the combination of both in wound healing of full-thickness skin defects in rabbits. With this purpose, a total of 144 rabbits were used for this study. The animals were divided in three study groups of 48 rabbits each depending on the administered treatment: PRGF, ASCs, and PGRF+ASCs. Two wounds of 8 mm of diameter and separated from each other by 20 mm were created on the back of each rabbit: the first was treated with saline solution, and the second with the treatment assigned for each group. Macroscopic and microscopic evolution of wounds was assessed at 1, 2, 3, 5, 7 and 10 days post-surgery. With this aim, 8 animals from each treatment group and at each study time were euthanized to collect wounds for histopathological study. RESULTS: Wounds treated with PRGF, ASCs and PRGF+ASCs showed significant higher wound healing and epithelialization rates, more natural aesthetic appearance, significant lower inflammatory response, significant higher collagen deposition and angiogenesis compared with control wounds. The combined treatment PRGF+ASCs showed a significant faster cutaneous wound healing process. CONCLUSIONS: The combined treatment PRGF+ASCs showed the best results, suggesting this is the best choice to enhance wound healing and improve aesthetic results in acute wounds.

Distinct Osteomimetic Response of Androgen-Dependent and Independent Human Prostate Cancer Cells to Mechanical Action of Fluid Flow: Prometastatic Implications.

BACKGROUND AND METHODS: Prostate cancer frequently expresses an osteomimetic phenotype, but it is unclear how it is regulated and what biological and clinical implications it confers. Because mechanical forces physiologically regulate bone-remodeling activity in osteocytes, we hypothesized that mechanical action of fluid flow (MAFF) at the cancer microenvironment may similarly foster prostate cancer cell osteomimicry. RESULTS: We showed that in vitro MAFF on androgen-dependent (LNCap) and androgen-independent (PC3) prostate cancer cells remarkably increased OPG, VEGF, RunX2, PTH1R, and PTHrP gene expression in both cell lines irrespective of their androgen dependency. MAFF also altered the cytokine secretion pattern of prostate cancer cells, including Ang2, SCF, and TNFα increase with TRAIL decrease in the supernatant of both cell lines; preferential increase of Leptin and PDGF-BB in LnCap and of VEGF, IL-8, and G-CSF in PC3; and exclusive increase of FGFß, MIF, and PECAM-1 with HGF decrease in LnCap, and of TGBß1, HGF, M-CSF, CXCL1, and CCL7 with NGF decrease in PC3. Murine MLO-Y4 osteocyte-conditioned medium (CM) abrogated M-CSF, G-CSG, IL-8, TNFα, and FGFß secretion-stimulating activity of mechanical stimulation on PC3 cells, and did the opposite effect on LnCap cells. However, MAFF fostered osteomimetic gene expression response of PC3 cells, but not of LnCap cells, to mechanically stimulated osteocyte-CM. Moreover, it abrogated TNFα and IL-8 secretion inhibitory effect of osteocyte-CM on mechanically stimulated PC3 cells and G-CSF, TNFα, and FGFß-stimulating effect on mechanically stimulated LnCap cells. CONCLUSIONS: MAFF activated osteoblast-like phenotype of prostate cancer cells and altered their responses to osteocyte soluble factors. It also induced osteocyte production of osteomimetic gene expression- and cytokine secretion-stimulating factors for prostate cancer cells, particularly, when they were mechanically stimulated. Importantly, MAFF induced a prometastatic response in androgen-independent prostate cancer cells, suggesting the interest of mechanical stimulation-dependent transcription and secretion patterns as diagnostic biomarkers, and as therapeutic targets for the screening of bone-metastasizing phenotype inhibitors upregulated during prostate cancer cell response to MAFF at the cancer microenvironment. Prostate 77:321-333, 2017. © 2016 Wiley Periodicals, Inc.

TLR4 response mediates ethanol-induced neurodevelopment alterations in a model of fetal alcohol spectrum disorders.

BACKGROUND: Inflammation during brain development participates in the pathogenesis of early brain injury and cognitive dysfunctions. Prenatal ethanol exposure affects the developing brain and causes neural impairment, cognitive and behavioral effects, collectively known as fetal alcohol spectrum disorders (FASD). Our previous studies demonstrate that ethanol activates the innate immune response and TLR4 receptor and causes neuroinflammation, brain damage, and cognitive defects in the developmental brain stage of adolescents. We hypothesize that by activating the TLR4 response, maternal alcohol consumption during pregnancy triggers the release of cytokines and chemokines in both the maternal sera and brains of fetuses/offspring, which impairs brain ontogeny and causes cognitive dysfunction. METHODS: WT and TLR4-KO female mice treated with or without 10% ethanol in the drinking water during gestation and lactation were used. Cytokine/chemokine levels were determined by ELISA in the amniotic fluid, maternal serum, and cerebral cortex, as well as in the offspring cerebral cortex. Microglial and neuronal markers (evaluated by western blotting), myelin proteins (immunohistochemical and western blotting) and synaptic parameters (western blotting and electron microscopy) were assessed in the cortices of the WT and TLR4-KO pups on PND 0, 20, and 66. Behavioral tests (elevated plus maze and passive avoidance) were performed in the WT and TLR4-KO mice on PND 66 exposed or not to ethanol. RESULTS: We show that alcohol intake during gestation and lactation increases the levels of several cytokines/chemokines (IL-1ß, IL-17, MIP-1α, and fractalkine) in the maternal sera, amniotic fluid, and brains of fetuses and offspring. The upregulation of cytokines/chemokines is associated with an increase in activated microglia markers (CD11b and MHC-II), and with a reduction in some synaptic (synaptotagmin, synapsin IIa) and myelin (MBP, PLP) proteins in the brains of offspring on days 0, 20, and 66 (long-term effects). These changes are associated with long-term behavioral impairments, in the 66-day-old alcohol-exposed pups. TLR4-deficient mice are protected against ethanol-induced cytokine/chemokine production in alcohol-treated dams and offspring, along with synaptic and myelin alterations, and the log-term behavioral dysfunction induced by ethanol in offspring. CONCLUSIONS: These results suggest that the immune system activation, through the TLR4 response, might play an important role in the neurodevelopmental defects in FASD.

Neurophysiological monitoring during acute and progressive experimentally induced compression injury of the spinal cord in pigs.

PURPOSE: To evaluate the degree of acute or progressive lateral compression needed to cause neurologic injury to the spinal cord assessed by electrophysiological monitoring. METHODS: In five domestic pigs, the spinal cord was exposed and compressed between T8-T9 roots using a precise compression device. Two sticks placed on both sides of the spinal cord were sequentially brought together (0.5 mm every 2 min), causing progressive spinal cord compression. Acute compression was reproduced by a 2.5-mm displacement of the sticks. Cord-to-cord evoked potentials were obtained with two epidural catheters. RESULTS: Increasing latency and decreasing amplitude of the evoked potentials were observed after a mean progressive displacement of the sticks of 3.2 ± 0.9 mm, disappearing after a mean displacement of 4.6 ± 1.2 mm. The potential returned after compression removal (16.8 ± 3.2 min). The potentials disappeared immediately after an acute compression of 2.5 ± 0.3 mm, without any sign of recovering after 30 min. CONCLUSIONS: The experimental model replicates the mechanism of a spinal cord injury caused by medially displaced screws into the spinal canal. The spinal cord had more ability for adaptation to progressive and slow compression than to acute mechanisms.

Local and systemic diffusion of antineoplastic drugs following vertebroplasty using acrylic cement mixed with cisplatin or methotrexate: experimental study in pigs.

PURPOSE: To determine the efficacy of cisplatin- or methotrexate-containing acrylic cement for local and systemic antineoplastic drug diffusion. Among the uses of acrylic cement or Polymethylmethacrylate (PMMA), there is the possibility to employ it as vehicle for drug diffusion. This capability is of interest in the treatment of pathological fractures: The curative effects of the cement (cytotoxicity of the monomer and increased temperature) are added to the antineoplastic effect of the drugs. METHODS: In the experimental study, two groups of ten pigs underwent vertebroplasty using cement mixed with 500 mg of powder cisplatin or 1000 mg of powder methotrexate. Vertebroplasty was performed in two non-consecutive lumbar vertebrae with bipedicular cement injection. Transpedicular bone biopsy was performed weekly to measure levels of antineoplastic agent in bone tissue and blood plasma. Cisplatin was studied by atomic absorption spectrometry and methotrexate by fluorescence polarization immunoassay. Renal and hepatic function and blood analysis were performed weekly. RESULTS: Cisplatin and methotrexate levels were found in bone tissue at more than 5 weeks following surgery. The cisplatin peak occurred at week 3 (mean 1269 µg/g bone) and the methotrexate peak at week 1 (mean 862.76 µg/g bone). Plasma drug levels were found 72 h after surgery, with a peak at 24 h for cisplatin (mean 0.23 µmol/L) and at 30 min for methotrexate (mean 0.92 µmol/L). None of the animals died during the study. Animals with intracanal cement leaks showed no neurological involvement. Renal, hepatic and hemogram studies remained within normal limits. CONCLUSIONS: There is local diffusion of antineoplastic agents from the cement to bone and plasma. We found methotrexate and cisplatin levels in bone at up to 5 weeks, comparable to previous in vitro reports. At the doses administered, there were no cases of myelosuppression, hepatotoxicity, or nephrotoxicity.

Infliximab reduces peripheral inflammation, neuroinflammation, and extracellular GABA in the cerebellum and improves learning and motor coordination in rats with hepatic encephalopathy.

BACKGROUND: Peripheral inflammation contributes to the neurological alterations in hepatic encephalopathy (HE). Neuroinflammation and altered GABAergic neurotransmission mediate cognitive and motor alterations in rats with HE. It remains unclear (a) if neuroinflammation and neurological impairment in HE are a consequence of peripheral inflammation and (b) how neuroinflammation impairs GABAergic neurotransmission. The aims were to assess in rats with HE whether reducing peripheral inflammation with anti-TNF-α (1) prevents cognitive impairment and motor in-coordination, (2) normalizes neuroinflammation and extracellular GABA in the cerebellum and also (3) advances the understanding of mechanisms linking neuroinflammation and increased extracellular GABA. METHODS: Rats with HE due to portacaval shunt (PCS) were treated with infliximab. Astrocytes and microglia activation and TNF-α and IL-1ß were analyzed by immunohistochemistry. Membrane expression of the GABA transporters GAT-3 and GAT-1 was analyzed by cross-linking with BS3. Extracellular GABA was analyzed by microdialysis. Motor coordination was tested using the beam walking and learning ability using the Y maze task. RESULTS: PCS rats show peripheral inflammation, activated astrocytes, and microglia and increased levels of TNF-α and IL-1ß. Membrane expression of GAT-3 and extracellular GABA are increased, leading to impaired motor coordination and learning ability. Infliximab reduces peripheral inflammation, microglia, and astrocyte activation and neuroinflammation and normalizes GABAergic neurotransmission, motor coordination, and learning ability. CONCLUSIONS: Neuroinflammation is associated with altered GABAergic neurotransmission and increased GAT-3 membrane expression and extracellular GABA (a); peripheral inflammation is a main contributor to the impairment of motor coordination and of the ability to learn the Y maze task in PCS rats (b); and reducing peripheral inflammation using safe procedures could be a new therapeutic approach to improve cognitive and motor function in patients with HE

Acute cellular rejection versus recurrent hepatitis C after liver transplantation: Clinical and pathological features driving a rational diagnostic approach.

AIM: The aim of our study was develop and validate an algorithm system based on morphological features for finding the differences between recurrent hepatitis C virus (HCV) and acute cellular rejection (ACR) in liver biopsies of HCV-transplanted patients. METHODS: Two hundred and eighty-eight liver biopsies were analyzed from 121 patients transplanted for HCV. A diagnostic consensus was reached between clinicians and pathologists in 214 biopsies for the diagnosis of recurrent HCV or ACR. A random sample of 114 liver biopsies (derivation cohort) was taken to generate the diagnostic tree and was subsequently evaluated using the validation cohort in 100 liver biopsies by recursive partitioning analysis of morphological variables and time since transplantation. RESULTS: The presence of endotheliitis together with a time of less than 6 weeks since LT definitely excluded recurrent HCV. After obtaining the regression tree, diagnostic accuracy was 96% and 93% in the derivation and validation cohort, respectively. Both cases surpassed the pathologist's original diagnosis, which had a diagnostic accuracy of 91% (P < 0.05, for both comparisons). CONCLUSION: A recursive partitioning analysis of the morphological features in liver biopsies from HCV-transplanted patients may be useful for easily distinguishing between recurrent HCV and ACR.

A molecular risk score based on 4 functional pathways for advanced classical Hodgkin lymphoma.

Despite improvement in the treatment of advanced classical Hodgkin lymphoma, approximately 30% of patients relapse or die as result of the disease. Current predictive systems, determined by clinical and analytical parameters, fail to identify these high-risk patients accurately. We took a multistep approach to design a quantitative reverse-transcription polymerase chain reaction assay to be applied to routine formalin-fixed paraffin-embedded samples, integrating genes expressed by the tumor cells and their microenvironment. The significance of 30 genes chosen on the basis of previously published data was evaluated in 282 samples (divided into estimation and validation sets) to build a molecular risk score to predict failure. Adequate reverse-transcription polymerase chain reaction profiles were obtained from 262 of 282 cases (92.9%). Best predictor genes were integrated into an 11-gene model, including 4 functional pathways (cell cycle, apoptosis, macrophage activation, and interferon regulatory factor 4) able to identify low- and high-risk patients with different rates of 5-year failure-free survival: 74% versus 44.1% in the estimation set (P < .001) and 67.5% versus 45.0% in the validation set (P = .022). This model can be combined with stage IV into a final predictive model able to identify a group of patients with very bad outcome (5-year failure-free survival probability, 25.2%).

Usefulness of material recovered from distal embolic protection devices after carotid angioplasty for proteomic studies.

PURPOSE: To demonstrate the usefulness of biologic material obtained from distal embolic protection devices (DEPDs) used in carotid angioplasty for the study of atherosclerosis protein markers and to establish the effect of systemic inflammation on the protein expression of carotid atheromatous plaques. MATERIALS AND METHODS: Two-dimensional gel electrophoresis and mass spectrometry were used to study proteins obtained from debris captured in DEPDs from patients who underwent carotid angioplasty. In addition, protein expression obtained from angioplasty samples in patients with different types of systemic inflammation (measured by serum levels of high-sensitivity C-reactive protein [CRP] with a cutoff value of 3 mg/L) was compared. Finally, immunohistochemistry of atherosclerotic plaques obtained by endarterectomy was used to validate the results obtained using DEPDs. RESULTS: Proteomic studies were successfully performed using debris from DEPDs. Protein expression differences were found in debris from patients with high systemic inflammation compared with debris from patients with low systemic inflammation. Annexin A5 (ANXA5), haptoglobin precursor, purine nucleoside phosphorylase, transgelin-2 (TAGLN2), and bisphosphoglycerate mutase were upregulated in debris from patients with high systemic inflammation, and proteasome subunit 8 beta type and glutathione-S-transferase kappa 1 (GSTK1) levels were higher in debris from patients with low levels of systemic inflammation. CONCLUSIONS: Atherosclerotic plaque debris captured in DEPDs is a suitable and valid source of material for proteomic studies of atherosclerosis. Protein expression in DEPD debris is affected by systemic inflammation.

Combined cytoplasmic and membranous EGFR and p53 overexpression is a poor prognostic marker in early stage oral squamous cell carcinoma.

OBJECTIVE: Our aim was to evaluate the expression of several molecules that regulate growth, the cell cycle and signalling pathways including EGFR, p53, p16 and p27 in oral squamous cell carcinomas (OSCC). We examined their utility as prognostic markers by relating to clinicopathological characteristics and the clinical outcome. PATIENTS AND METHODS: Using tissue microarray technology, we analysed 67 primary OSCC and examined immunohistochemical expression of EGFR, p53, p16 and p27. Multivariate analysis was conducted to examine their role in survival. RESULTS: Many of the markers were highly expressed in these cancers. Membranous EGFR expression in 95.2%, both membrane and cytoplasm expression in 35%, p53 expression in 61.6%, p27 expression in 89.5% and p16 expression in 27.9% of cases. In the multivariate analysis, independent prognostic influence of a lower overall survival was determined only for advanced tumour stage (P < 0.001), p53 overexpression (P = 0.004), EGFR cytoplasm and membrane co-expression location (P = 0.002) and p16 reduced expression (P = 0.002). When considering a subgroup of early stage tumours, p53 overexpression (P = 0.028) and combined membranous and cytoplasm EGFR co-expression (P = 0.039) were indicators of a lower overall survival. For disease-free survival, in addition to these three factors, the histological grade (P = 0.011) showed independent prognostic values. CONCLUSION: The independent value of EGFR subcellular location (cytoplasm and membrane) and p53 overexpression in overall survival even in early stages of OSCC suggests that these markers may serve as reliable biological markers to identify high-risk subgroups and to guide therapy.

BRCA1 mutations do not increase prostate cancer risk: results from a meta-analysis including new data.

BACKGROUND: Although in recent years deleterious BRCA1 mutations have been extensively studied as a prostate cancer risk factor, results are inconclusive. To assess the contribution of the BRCA1 Galician founder mutation c.211A>G in prostate cancer morbidity we conducted a case-control study. Moreover, to better elucidate whether deleterious BRCA1 mutations are involved in the development of prostate cancer, we performed a systematic review and a meta-analysis of BRCA1 studies on prostate cancer. METHODS: A total of 905 unselected men diagnosed with adenocarcinoma of the prostate and a control group of 936 unrelated men without history of prostate cancer were evaluated for c.211A>G. Adjusted by age Odds ratios (OR) and 95% confidence intervals (CIs) were estimated using logistic regression. To construct the meta-analysis, genotype-based epidemiological studies reporting BRCA1 founder mutations on prostate cancer were identified by comprehensive and systematic bibliographic search. After extraction of relevant data, main and subgroup analysis by mutation were performed to assess the effect of BRCA1 on prostate cancer risk. RESULTS: Four c.211A>G heterozygous individuals, one patient and three controls, were detected (OR = 0.27; 95% CI: 0.01-2.36; P = 0.28). Meta-analysis results from the integration of our data and other seven studies with BRCA1 genotyping data (5,705 prostate cancer cases and 13,218 controls) did not detect an association with prostate cancer risk (OR = 1.36; 95% CI: 0.87-2.14; P = 0.18). CONCLUSIONS: Our conclusive trial demonstrates the lack of association between Galician splicing mutation c.211A>G in the BRCA1 gene and prostate cancer risk. Moreover, the result of the meta-analysis also discards the involvement of BRCA1 mutations in the development of prostate cancer.

Quantitative endoscopic ultrasound elastography: an accurate method for the differentiation of solid pancreatic masses.

BACKGROUND & AIMS: Qualitative endoscopic ultrasound (EUS) elastography is an accurate but subjective tool for the differential diagnosis of solid pancreatic masses. Second-generation EUS elastography allows quantitative analysis of tissue stiffness. We evaluated the accuracy of quantitative, second-generation EUS elastography in the differential diagnosis of solid pancreatic masses. METHODS: The study included 86 consecutive patients who underwent EUS for the evaluation of solid pancreatic masses. EUS elastography was performed with the linear Pentax EUS and the Hitachi EUB900. Representative areas from the mass (A) and soft reference areas (B) were analyzed. The result of the elastographic evaluation was defined by the quotient B/A (strain ratio). Final diagnosis was based on histology of surgical specimens and cytology of EUS-fine-needle aspiration samples. The diagnostic accuracy of EUS elastography in detecting malignancy was calculated using receiver operating curve analysis. RESULTS: The mean size of the pancreatic masses was 31.4 ± 12.3 mm. The final diagnoses were pancreatic adenocarcinoma (n = 49), inflammatory mass (n = 27), malignant neuroendocrine tumor (n = 6), metastatic oat-cell lung cancer (n = 2), pancreatic lymphoma (n = 1), and pancreatic solid pseudopapillary tumor (n = 1). The strain ratio was significantly higher among patients with pancreatic malignant tumors compared with those with inflammatory masses. The sensitivity and specificity of strain ratio for detecting pancreatic malignancies were 100% and 92.9%, respectively (area under the receiver operating curve, 0.983). CONCLUSIONS: Quantitative, second-generation EUS elastography is useful for differential diagnosis of solid pancreatic masses. It allows for a quantitative and objective evaluation of tissue stiffness, which indicates the malignant or benign nature of the pancreatic lesion.

Bacteroides uniformis CECT 7771 Modulates the Brain Reward Response to Reduce Binge Eating and Anxiety-Like Behavior in Rat.

Food addiction (FA) is characterized by behavioral and neurochemical changes linked to loss of food intake control. Gut microbiota may influence appetite and food intake via endocrine and neural routes. The gut microbiota is known to impact homeostatic energy mechanisms, but its role in regulating the reward system is less certain. We show that the administration of Bacteroides uniformis CECT 7771 (B. uniformis) in a rat FA model impacts on the brain reward response, ameliorating binge eating and decreasing anxiety-like behavior. These effects are mediated, at least in part, by changes in the levels of dopamine, serotonin, and noradrenaline in the nucleus accumbens and in the expression of dopamine D1 and D2 receptors in the prefrontal cortex and intestine. B. uniformis reverses the fasting-induced microbiota changes and increases the abundance of species linked to healthy metabolotypes. Our data indicate that microbiota-based interventions might help to control compulsive overeating by modulating the reward response.

EGFR and Ki-67 expression in oral squamous cell carcinoma using tissue microarray technology.

OBJECTIVE: Our aim was to validate the use of tissue microarrays (TMA) in oral squamous cell carcinomas (OSCC) to analyse epidermal growth factor receptor (EGFR) and Ki-67 expression. We also analysed the relationship that the expression of these markers may have with clinical, pathological and survival variables. PATIENTS AND METHODS: The study sample comprised 39 unselected patients diagnosed and treated for OSCC. We analysed Ki-67 and EGFR expression by immunohistochemistry on formalin-fixed, paraffin-embedded surgical specimens. Whole sections (WS) were compared with double 1.5 mm core-tissue microarrays. RESULTS: High EGFR expression was observed both on TMA (in 98% of the cases) and WS (in 100% of the cases) with substantial agreement kappa value (0.720). EGFR expression was not significantly associated with clinical, pathological and survival variables on TMA and WS. Ki-67 analysis showed a Spearman correlation of 0.741 with a Ki-67 mean labelling index of 45% in TMA and 56.8% in WS. We found a significant relationship between gender and Ki-67 labelling index on WS (P = 0.022) and TMA (P = 0.002). Clinical stage was the only parameter in multivariate analysis that had a significant predictive value. CONCLUSION: We demonstrate that dual 1.5 mm core TMA is a valid, rapid, economical and tissue-saving way to study OSCC biopsies and that it presents strong correlation with the WS. EGFR overexpression in OSCC suggests that these tumours may be a candidate for therapy investigation directed to EGFR.

Fluorescent in situ hybridization heating pretreatment: the key is temperature control.

Fluorescent in situ hybridization (FISH) is a very useful tool for diagnostic and prognostic purposes in pathology. However, many laboratories still experience troubles when applying FISH to paraffin material. To overcome these difficulties, different pretreatments which include enzymatic digestion have been described. Usually, previous to digestion, a heating step is performed. The aim of this study was to compare the efficiency of the heating step with different buffers and different heating methods. We conclude that the main factor in the heating pretreatment is the temperature control, irrespective of the buffer used. Best results are obtained with any buffer by heating the slides to 99°C for 15 min followed by 10 min at room temperature.

EUS elastography for the characterization of solid pancreatic masses.

BACKGROUND: Differential diagnosis of solid pancreatic masses remains a challenge. EUS elastography, by analyzing tissue stiffness of the mass, may be of help in this setting. OBJECTIVE: To evaluate the different elastographic patterns of solid pancreatic masses and the diagnostic accuracy of EUS elastography for malignancy. DESIGN: Prospective, consecutive, descriptive study with a second blind evaluation of elastographic patterns for concordance analysis and use of a well-defined reference method for calculation of diagnostic accuracy. PATIENTS: This study involved 130 consecutive patients with solid pancreatic masses and 20 controls with normal pancreases. INTERVENTION: EUS elastography was performed by using a linear Pentax echoendoscope and Hitachi EUB-8500 US. MAIN OUTCOME MEASUREMENTS: Elastographic patterns of solid pancreatic masses and accuracy of the technique for malignancy. RESULTS: Mean (SD) size of the masses was 30.9 (12.5) mm. The final diagnosis was malignant tumor in 78 patients, inflammatory mass in 42 patients, and neuroendocrine tumor in 10 patients. Four elastographic patterns were described, with a high concordance among 2 blinded investigators. A green-predominant pattern, either homogeneous or heterogeneous, excluded malignancy with a high accuracy. On the contrary, a blue-predominant pattern, either homogeneous or heterogeneous, supported the diagnosis of malignant tumor. Sensitivity, specificity, positive and negative predictive values, and overall accuracy of EUS elastography for diagnosis of malignancy were 100%, 85.5%, 90.7%, 100%, and 94.0%, respectively. LIMITATION: Single-center study. CONCLUSION: EUS elastography is a useful tool for differential diagnosis of solid pancreatic masses. It provides specific patterns supporting the benign or malignant nature of the disease.