Scoring of tumor-infiltrating lymphocytes: From visual estimation to machine learning.

The extent of tumor-infiltrating lymphocytes (TILs), along with immunomodulatory ligands, tumor-mutational burden and other biomarkers, has been demonstrated to be a marker of response to immune-checkpoint therapy in several cancers. Pathologists have therefore started to devise standardized visual approaches to quantify TILs for therapy prediction. However, despite successful standardization efforts visual TIL estimation is slow, with limited precision and lacks the ability to evaluate more complex properties such as TIL distribution patterns. Therefore, computational image analysis approaches are needed to provide standardized and efficient TIL quantification. Here, we discuss different automated TIL scoring approaches ranging from classical image segmentation, where cell boundaries are identified and the resulting objects classified according to shape properties, to machine learning-based approaches that directly classify cells without segmentation but rely on large amounts of training data. In contrast to conventional machine learning (ML) approaches that are often criticized for their "black-box" characteristics, we also discuss explainable machine learning. Such approaches render ML results interpretable and explain the computational decision-making process through high-resolution heatmaps that highlight TILs and cancer cells and therefore allow for quantification and plausibility checks in biomedical research and diagnostics.

The role of E-cadherin down-regulation in oral cancer: CDH1 gene expression and epigenetic blockage.

BACKGROUND: The prognosis of the oral squamous cell carcinoma (OSCC) patients remains very poor, mainly due to their high propensity to invade and metastasize. E-cadherin reduced expression occurs in the primary step of oral tumour progression and gene methylation is a mode by which the expression of this protein is regulated in cancers. In this perspective, we investigated E-cadherin gene (CDH1) promoter methylation status in OSCC and its correlation with Ecadherin protein expression, clinicopathological characteristics and patient outcome. METHODS: Histologically proven OSCC and paired normal mucosa were analyzed for CDH1 promoter methylation status and E-cadherin protein expression by methylation-specific polymerase chain reaction and immunohistochemistry. Colocalization of E-cadherin with epidermal growth factor (EGF) receptor (EGFR) was evidenced by confocal microscopy and by immunoprecipitation analyses. RESULTS: This study indicated E-cadherin protein down-regulation in OSCC associated with protein delocalization from membrane to cytoplasm. Low E-cadherin expression correlated to aggressive, poorly differentiated, high grade carcinomas and low patient survival. Moreover, protein down-regulation appeared to be due to E-cadherin mRNA downregulation and CDH1 promoter hypermethylation. In an in vitro model of OSCC the treatment with EGF caused internalization and co-localization of E-cadherin with EGFR and the addition of demethylating agents increased E-cadherin expression. CONCLUSION: Low E-Cadherin expression is a negative prognostic factor of OSCC and is likely due to the hypermethylation of CDH1 promoter. The delocalization of E-cadherin from membrane to cytoplasm could be also due to the increased expression of EGFR in OSCC and the consequent increase of E-cadherin co-internalization with EGFR.

Association of intraoperative blood pressure instability with adverse outcomes after liver transplantation.

BACKGROUND: Blood pressure derangements are common in orthotopic liver transplantation (OLT), and are potentially associated with adverse outcomes if they are sustained. While this concept is often believed to be true, few have rigorously demonstrated the validity of this claim, especially in likely vulnerable OLT patients. METHODS: We retrospectively investigated 827 patients who underwent OLT to determine the magnitude of these hemodynamic associations with adverse outcomes. The median value of the mean arterial pressure (MAP) and the fractional change in the median MAP between subsequent epochs (FCM) were calculated for every 5-minute epoch intraoperatively. Epochs were classified according to prespecified ranges of MAP and fractional changes in MAP (lability) between epochs. Multivariate stepwise logistic regression was used to model associations of risk factors and epochs of intraoperative blood pressure (BP) instability with primary (30-day mortality and/or graft failure) and secondary adverse outcomes. RESULTS: Primary adverse outcomes occurred in 10.9% and 12.2% of patients for 30-day mortality and 30-day graft failure, respectively. Independent hemodynamic predictors for 30-day mortality and graft failure included sustained periods of MAP <50 mmHg and BP lability where the MAP changed >25%. All of these values were statistically significant. CONCLUSION: Although severe intraoperative hypotension and BP lability during OLT are often observed in current practice as consequences of major surgical manipulations and patient vulnerability, these are likely not benign conditions based on this retrospective analysis. Prospective trials are warranted to investigate the possibility that interventions tailored to avoidance of hypotension and BP lability may improve outcomes.

Gut--liver axis: the impact of gut microbiota on non alcoholic fatty liver disease.

AIM: To examine the impact of gut microbiota on non alcoholic fatty liver disease (NAFLD) pathogenesis. DATA SYNTHESIS: Emerging evidence suggests a strong interaction between gut microbiota and liver. Receiving approximately 70% of its blood supply from the intestine, the liver represents the first line of defence against gut-derived antigens. Intestinal bacteria play a key role in the maintenance of gut-liver axis health. Disturbances in the homeostasis between bacteria- and host-derived signals at the epithelial level lead to a break in intestinal barrier function and may foster "bacterial translocation", defined as the migration of bacteria or bacterial products from the intestinal lumen to mesenteric lymph nodes or other extraintestinal organs and sites. While the full repertoire of gut-derived microbial products that reach the liver in health and disease has yet to be explored, the levels of bacterial lipopolysaccharide, a component of the outer membrane of Gram-negative bacteria, are increased in the portal and/or systemic circulation in several types of chronic liver diseases. Derangement of the gut flora, particularly small intestinal bacterial overgrowth, occurs in a large percentage (20-75%) of patients with chronic liver disease. In addition, evidence implicating the gut-liver axis in the pathogenesis of metabolic liver disorders has accumulated over the past ten years. CONCLUSIONS: Complex metabolic diseases are the product of multiple perturbations under the influence of triggering factors such as gut microbiota and diet, thus, modulation of the gut microbiota may represent a new way to treat or prevent NAFLD.

Aurora B expression as a prognostic indicator and possible therapeutic target in oral squamous cell carcinoma.

The aim of this study is to investigate the expression of the chromosomal passenger protein Aurora B and its activated (phosphorylated) form in a large series of human oral squamous cell cancers (OSCC) and to evaluate its clinical and prognostic significance. Western blotting analysis revealed overexpression of both Aurora B and Thr-232 Phopsho-Aurora B in OSCC lines as compared to normal keratinocytes and bladder cancer cells. Furthermore, protein expression was analysed by immunohistochemistry in 101 OSCC of different site, stage and histological grade and in normal peritumoural areas. The intracellular localization of Aurora B in tumour cells was mainly nuclear, especially in proliferative areas, and significant overexpression was found in tumours in comparison to normal peritumoural areas (P=0.012). Staining results were correlated with clinicopathological parameters and long-term follow-up, and a significant association was found between protein expression and tumour stage (stage II, III and IV vs stage I, P=0.030) and size (<2cm vs >2cm, P=0.010). Cox regression analysis confirmed a poorer disease-free survival in cases with high expression of Aurora B protein. Kaplan-Meier curves showed shorter time to progression in patients with high levels of Aurora B expression (p<0.05). Moreover, the tumoral group with nuclear Aurora B immunolocalization had the worst prognosis (P=0.0364 in disease free survival). Our results suggest that assessing Aurora B expression might help in patients’ risk stratification and serve as a novel therapeutic target in advanced OSCCs.

Ectoparasitos de animais silvestres no Maranhão / Ectoparasites of wild animals in Maranhão

Objetivou-se identificar os ectoparasitos de animais silvestres recepcionados pelo Centro de Triagem de Animais Silvestres, São Luís, Maranhão. Os ectopararasitos identificados foram: piolhos Acidoproctus sp., Trinoton sp., Ciconiphilus sp, Austromenopon sp., Quadraceps sp., Saemundssonia sp. e Trichodectes canis; carrapato Amblyomma rotundatum e Rhipicephalus sanguineus; pulgas Ctenocephalides felis e larvas de Cochliomyia hominivorax. Os resultados apresentados documentam a infestação de mamíferos, répteis e aves silvestres por ectoparasitos.

The objective of this study was to identify the ectoparasites of wild animals received by the Center of Wild Animals of São Luís, Maranhão, Brazil. The ectoparasites identified were: the lice Acidoproctus sp., Trinoton sp., Ciconiphilus sp., Austromenopon sp., Quadraceps sp., Saemundssonia sp., and Trichodectes canis; the ticks Amblyomma rotundatum and Rhipicephalus sanguineus; the fleas Ctenocephalides felis, and larvae of Cochliomyia hominivorax. The data presented register the infestation of wild mammals, reptiles and birds by ectoparasites.

Detection of novel Human papilloma virus type 82 in laryngeal cancer: case report.

Human papilloma virus infection is thought to play a role in laryngeal carcinogenesis; the variable association reported in literature may be due to wide range of HPV genotypes. We report the case of a 51-year-old man affected by laryngeal squamous cell carcinoma; analysis of DNA extracted by cancer cells by an innovative molecular virology assay (INNO-LiPA HPV Genotyping Extra) showed the presence of two high-risk HPV genotypes, HPV-73 and -82. Immunohistochemical examination confirmed positivity for both capsid protein and viral oncogenic protein E7. Such association has never been reported in literature so far, and a brief discussion on the importance of assessing HPV status in laryngeal cancer is provided.

Culturable bacteria from Zn- and Cd-accumulating Salix caprea with differential effects on plant growth and heavy metal availability.

AIMS: To characterize bacteria associated with Zn/Cd-accumulating Salix caprea regarding their potential to support heavy metal phytoextraction. METHODS AND RESULTS: Three different media allowed the isolation of 44 rhizosphere strains and 44 endophytes, resistant to Zn/Cd and mostly affiliated with Proteobacteria, Actinobacteria and Bacteroidetes/Chlorobi. 1-Aminocyclopropane-1-carboxylic acid deaminase (ACCD), indole acetic acid and siderophore production were detected in 41, 23 and 50% of the rhizosphere isolates and in 9, 55 and 2% of the endophytes, respectively. Fifteen rhizosphere bacteria and five endophytes were further tested for the production of metal-mobilizing metabolites by extracting contaminated soil with filtrates from liquid cultures. Four Actinobacteria mobilized Zn and/or Cd. The other strains immobilized Cd or both metals. An ACCD- and siderophore-producing, Zn/Cd-immobilizing rhizosphere isolate (Burkholderia sp.) and a Zn/Cd-mobilizing Actinobacterium endophyte were inoculated onto S. caprea. The rhizosphere isolate reduced metal uptake in roots, whereas the endophyte enhanced metal accumulation in leaves. Plant growth was not promoted. CONCLUSIONS: Metal mobilization experiments predicted bacterial effects on S. caprea more reliably than standard tests for plant growth-promoting activities. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacteria, particularly Actinobacteria, associated with heavy metal-accumulating Salix have the potential to increase metal uptake, which can be predicted by mobilization experiments and may be applicable in phytoremediation.

Chronic hepatitis C virus in the state of Piauí, northeastern Brazil

Knowledge of genotype distribution of hepatitis C virus (HCV) has clinical importance due to genotype 1 lower response to treatment compared with genotypes 2 and 3. The goal of this survey was to describe clinical and laboratorial profiles of patients with chronic hepatitis C (CHC) in the State of Piauí, as well as to expand the overall awareness of the distribution of HCV genotyping in Northeast of Brazil. A retrospective cross-sectional study was carried out between April 1999 and August 2005. A total of 153 patients were included, 119 (77.8 percent) males and 34 (22.2 percent) females; mean age = 48.01 ± 9.11 years. We observed a homogeneous distribution between genotypes 1 (50.0 percent) and 3 (49.0 percent), while the most frequent subtype noticed was 3a (49.0 percent). The mean viral load among patients with subtype 1b (1,232,476 UI/mL) was significantly superior to the subtype 1a (391,204 UI/mL; p = 0.010) and to the subtype 3a (594,228 UI/mL; p = 0.047). The average levels of gamma-glutamiltransferase of genotype 1 (144 mg/dL) had statistical differences when compared to genotype 3 (74 mg/dL; p = 0.014). Most patients showed mild to moderate degrees of histopathological necroinflammatory activity and hepatic fibrosis (79.0 percent and 56.2 percent, respectively). We concluded that most candidates to treatment of CHC in the State of Piauí presented with clinically stable hepatic illness; the distribution of genotypes 1 and 3 was virtually homogeneous; and there was no significant demographic or clinical differences among genotypes or subtypes of HCV.

Chronic hepatitis C virus in the state of Piauí, northeastern Brazil.

Knowledge of genotype distribution of hepatitis C virus (HCV) has clinical importance due to genotype 1 lower response to treatment compared with genotypes 2 and 3. The goal of this survey was to describe clinical and laboratorial profiles of patients with chronic hepatitis C (CHC) in the State of Piauí, as well as to expand the overall awareness of the distribution of HCV genotyping in Northeast of Brazil. A retrospective cross-sectional study was carried out between April 1999 and August 2005. A total of 153 patients were included, 119 (77.8%) males and 34 (22.2%) females; mean age = 48.01 +/- 9.11 years. We observed a homogeneous distribution between genotypes 1 (50.0%) and 3 (49.0%), while the most frequent subtype noticed was 3a (49.0%). The mean viral load among patients with subtype 1b (1,232,476 UI/mL) was significantly superior to the subtype 1a (391,204 UI/mL; p = 0.010) and to the subtype 3a (594,228 UI/mL; p = 0.047). The average levels of gamma-glutamiltransferase of genotype 1 (144 mg/dL) had statistical differences when compared to genotype 3 (74 mg/dL; p = 0.014). Most patients showed mild to moderate degrees of histopathological necroinflammatory activity and hepatic fibrosis (79.0% and 56.2%, respectively). We concluded that most candidates to treatment of CHC in the State of Piauí presented with clinically stable hepatic illness; the distribution of genotypes 1 and 3 was virtually homogeneous; and there was no significant demographic or clinical differences among genotypes or subtypes of HCV.

Bombesin: a possible role in wound repair.

During tissue regeneration and wound healing of the skin, migration, proliferation and differentiation of keratinocytes are important processes. Here we assessed the effect of a neuropeptide, bombesin, on keratinocytes during regeneration from scratch wounding. Bombesin purified from amphibian skin, is homologous of mammalian gastrin-releasing peptide and is active in mammals. Its pharmacological effects mediate various physiological activities: hypertensive action, stimulating action on gastric secretion, hyperglycemic effect or increased insulin secretion. In vitro it shows a hyperproliferative effect on different experimental models and is involved in skin repair. The aim of this study was to elucidate the effect of Bombesin in an in vitro experimental model on a mechanically injured human keratinocyte monolayer. We evaluated different mediators involved in wound repair such as IL-8, TGFbeta, IL-1, COX-2, VEGF and Toll-like receptors 2 and 4 (TLR2 and TLR4). We also studied the effects of bombesin on cell proliferation and motility and its direct effect on wound repair by observing the wound closure after mechanical injury. The involvement of the bombesin receptors neuromedin receptor (NMBR) and gastrin-releasing peptide receptor (GRP-R) was also evaluated. Our data suggest that bombesin may have an important role in skin repair by regulating the expression of healing markers. It enhanced the expression of IL-8, TGFbeta, COX-2 and VEGF. It also enhanced the expression of TLR2, while TLR4 was not expressed. Bombesin also increased cell growth and migration. In addition, we showed that NMBR was more involved in our experimental model compared to GRP-R.

Anti-apoptotic seminal vesicle protein IV inhibits cell-mediated immunity.

The in vitro effect of seminal vesicle protein IV (SV-IV) on the cytotoxic activity of human natural or acquired cellular immunity has been investigated by standard immunological procedures, a (51)Cr-release cytotoxicity assay, and labeled-ligand binding experiments. The data obtained demonstrate that: (1) fluoresceinated or [(125)I]-labeled SV-IV binds specifically to the surface of human purified non-adherent mononuclear cells (NA-MNC); (2) SV-IV suppresses the cytotoxicity of natural killer (NK) cells against K562 target cells, that of IL-2-stimulated NK (LAK) cells against DAUDI target cells, and that of VEL antigen-sensitized cytotoxic T lymphocytes (CTLs) against VEL target cells; (3) treatment of K562 target cells alone with SV-IV decreases their susceptibility to NK-induced lysis. These findings indicate that the protein SV-IV has a marked in vitro inhibitory effect on NK, LAK and CTL cytotoxicity, providing a better understanding of its immune regulatory functions.

[Ectoparasites of small wild mammals from the adjacent areas of Itapecuru River and Environmental Preservation Area of Inhamum, state of Maranhão, Brazil]. / Ectoparasitos de pequenos mamíferos silvestres de áreas adjacentes ao Rio Itapecuru e Área de Preservação Ambiental do inhamum, estado do Maranhão, Brasil.

During fauna studies, thirty-six wild mammals were collected in adjacent areas of Itapecuru River and Environmental Preservation area of Inhamum, state of Maranhão, Brazil. They were sampled for ectoparasites. The following specimens of the order Rodentia and its respective ectoparasites were identified: Akodon sp. (Androlaelaps sp. and Laelaps sp.), Oecomys sp. (Androlaelaps sp. and Amblyomma cajennense), Oligoryzomys sp. (Androlaelaps sp. Laelaps sp. and Amblyomma sp.) e Oryzomys megacephalus (A. cajennense). In Calomys callosus no ectoparasite was found. It was observed infestation in the order Didelphimorphia as follows: Didelphis marsupialis (Androlaelaps sp., Laelaps sp. and larvae of Diptera Cyclorrhapha); Gracilinanus sp. (Laelaps sp. and larvae of Diptera Cyclorrhapha), Monodelphis domestica (Poplygenis (Polygenis)), Cummingsia sp., Amblyomma sp. and Androlaelaps sp.). Marmosa sp. e Thylamis sp. had no ectoparasites. From the captured hosts 56% were infested, 82% and 44% rodents and marsupials, respectively. Mites from the family Laelapidae presented the great diversity of hosts and genus.

[Efficacy of extracts of plants in engorged females of Boophilus microplus from the mesoregion West of Maranhão, Brazil.]. / Eficácia de fitoterápicos em fêmeas ingurgitadas de Boophilus microplus, provenientes da mesorregião oeste do Maranhão, Brasil.

The objective was to evaluate the efficacy of hydro-alcoholic extracts of nim and citronela at 20% and eucalipto at 10% in Boophilus microplus engorged females collected in cattle naturally infected from the mesoregion West of Maranhão. At the laboratory the females were separated, weighted and distributed in six groups of 10, in duplicate. Each group was immersed in 10mL of the solution of the extracts, for two minutes. In the nim and citronela extracts there was 32% e 17%, respectively, while larval emergence the eucalipto extracts demonstrated 96% of efficacy. In the groups treated by Cipermetrina + Clorpirifós + Citronetal and Deltametrina (positive controls) the mortality occurred after 48h of treatment, while the groups immersed in distilled water (negative control) showed 100% of eggs mass and larval emergence. According to the results, it can be concluded that the extract of eucalipto could be used as acaricide in the control of B. microplus females since it was efficient in vitro, however to nim and citronela showed not efficacy. B. microplus females were not resistant to the chemical compounds used in this experiment.

Experimental study on vasoactive intestinal peptide (VIP) and its diaminopropane bound (VIP-DAP) analog in solution.

Bioactive peptides represent an exciting area of research in the fields of biochemistry and medicine and in particular the VIP/PACAP network appears to be of interest. Vasoactive intestinal peptide (VIP) is a pleiotropic factor that exerts a physiological regulatory influence and is involved in the pathogenesis of several human disorders. In this paper we have reported structural characterization of VIP by experimental and computational methods as well as a comparative analysis of the peptide with its transglutaminase catalyzed analog VIP-Diaminopropane (VIP-DAP).

Cyclooxygenase isozymes in oral squamous cell carcinoma:a real-time RT-PCR study with clinic pathological correlations.

COX-2 expression in tumour cells has been associated with carcinogenesis in many human neoplasms, including head and neck cancer, while the COX-1 isoform of the cyclooxygenase enzyme is constitutively expressed in normal tissues. We measured COX-1 and COX-2 m-RNA expression in samples of both oral cancer and matched oral mucosa from 22 patients by RealTime RT-PCR; clinic pathological data (grading, TNM staging, inflammation, follow-up) of all patients were available for statistical evaluation. Most of the tumor samples in our study expressed at least one cyclooxygenase enzyme (COX-1 or COX-2 mRNA) more than their matched normal oral mucosa (p<0.05), with no correlation with the entity of inflammation, and a significant inverse relationship was found between COX-1 and COX-2 in each sample. Higher levels of COX-2 expression were associated with poor disease-free survival (p<0.05), but not with overall survival and higher tumor stage and grade. Our results suggest that COX-1 may play a role in oral carcinogenesis, and could be regarded as a potential therapeutic target by chemo preventive drugs; moreover, COX-2 expression might be addressed as a new prognostic tool in the clinical management of OSCC.

Prognostic value of human telomerase reverse transcriptase gene expression in oral carcinogenesis.

Human telomerase reverse transcriptase (hTERT) gene expression in resected specimens of oral squamous cell carcinoma (OSCC) and their surrounding tissue, either apparently normal or clearly histologically dysplastic, was evaluated by both real-time RT-PCR and immunohisto-chemical protein analyses. The expression level of hTERT in oral dysplasia and in OSCC was markedly higher than in normal tissues. The correlation between hTERT expression in OSCC and clinico-pathological parameters or survival of OSCC patients was statistically analyzed. Our study demonstrates that there is no significant relationship between hTERT expression and classical clinico-pathological parameters. Interestingly, survival analysis showed both overexpressing cases and lower survival rate in the early stage of OSCC (p=0.03 for immunohistochemistry; p=0.04 for RT real-time PCR). The histological location of hTERT in these tumors has been discussed in the context of the cancer stem cell theory.

Evidence for multiple rat VPAC1 receptor states with different affinities for agonists.

We compare the binding properties of [125I-VIP] and [125I]-Ro 25 1553 to VPAC1 receptors, expressed in stably transfected CHO cells. [125I]-VIP labelled two VPAC1 receptor states, while [125I]-Ro 25 1553 labelled selectively a limited number of high-affinity receptors. This high-affinity state probably corresponds to an agonist-receptor-Gs ternary complex as its properties (guanyl nucleotides, EC50 values and maximal effect) were affected by cholera toxin pre-treatment. Both high- and low-affinity receptors participated in the adenylate cyclase activation. This suggested that agonists activate not only low-affinity uncoupled receptors by facilitating the ternary complex formation, but also activated the high-affinity ternary complex by accelerating the GTP binding to emptied, receptor-bound G proteins.

Enzymatic synthesis of vasoactive intestinal peptide analogs by transglutaminase.

Vasoactive intestinal peptide is an amino acceptor and donor substrate for tissue transglutaminase (TGase) in vitro. This peptide contains a single glutamine residue, Gln16, which was identified as the amino acceptor substrate. Different gamma(glutamyl16)amine derivatives of vasoactive intestinal peptide were synthesized enzymatically in vitro. The modification is very fast when compared with that of many native substrates of TGase. The analogs 1,3-diaminopropane, putrescine, cadaverine, spermidine, spermine, glycine ethyl ester and mono-dansylcadaverine of the peptide were purified by high-performance liquid chromatography on a reverse-phase column and were analyzed by electrospray mass spectrometry. When amines were absent in the assay mixture as an external amino donor, lysine residue occurring in the peptide was an effective amino donor site for TGase. Only one of the three lysine residues of vasoactive intestinal peptide, namely Lys21, was demonstrated to be involved in both inter- and intramolecular cross-link formation.