Association of human papillomavirus (HPV), p16, p53 and p63 expression with non-bilharzia-associated squamous cell carcinoma of the bladder and algorithm construction for histopathological grading prediction.

OBJECTIVE: To investigate the expression of human papillomavirus (HPV), p16, p53, and p63 in non-schistosomiasis-related squamous cell carcinoma of the bladder and to develop an accurate and automated tool to predict histological classification based on clinicopathological features. METHODS: Twenty-eight patients with primary bladder pure squamous cell carcinoma who underwent cystectomy or transurethral resection of bladder tumor (TURBT) for bladder cancer between January 2011 and July 2017 were evaluated. Clinical data and follow-up information were obtained from medical records. Formalin-fixed, paraffin-embedded surgical specimens were used for immunohistochemical staining for p16, p53, and p63. Human papillomavirus detection was evaluated by PCR. Statistical analysis was performed, and statistical significance was set at p<0.05. Finally, decision trees were built to classify patients' prognostic features. Leave-one-out cross-validation was used to test the generalizability of the model. RESULTS: Neither direct HPV detection nor its indirect marker (p16 protein) was identified in most cases. The absence of p16 was correlated with less aggressive histological grading (p=0.040). The positive p16 staining detection found only in pT1 and pT2 cases in our sample suggests a possible role for this tumor suppressor protein in the initial stages of bladder squamous cell carcinoma. The decision trees constructed described the relationship between clinical features, such as hematuria/dysuria, the level of tumor invasion, HPV status, lymphovascular invasion, gender, age, compromised lymph nodes, and tumor degree differentiation, with high classification accuracy. CONCLUSION: The algorithm classifier approach established decision pathways for semi-automatic tumor histological classification, laying the foundation for tailored semi-automated decision support systems for pathologists.

Whole bone subcutaneous transplantation as a strategy to study precisely the bone marrow niche.

Hematopoietic stem cells are maintained in a specialized microenvironment, known as the 'niche', within the bone marrow. Understanding the contribution of cellular and molecular components within the bone marrow niche for the maintenance of hematopoietic stem cells is crucial for the success of therapeutic applications. So far, the roles of crucial mechanisms within the bone marrow niche have been explored in transgenic animals in which genetic modifications are ubiquitously introduced in the whole body. The lack of precise tools to explore genetic alterations exclusively within the bone marrow prevents our determination of whether the observed outcomes result from confounding effects from other organs. Here, we developed a new method - 'whole bone subcutaneous transplantation'- to study the bone marrow niche in transgenic animals precisely. Using immunolabeling of CD45.1 (donor) vs. CD45.2 (recipient) hematopoeitic stem cells, we demonstrated that hematopoeitic stem cells from the host animals colonize the subcutaneously transplanted femurs after transplantation, while the hematopoietic stem cells from the donor disappear. Strikinlgy, the bone marrow niche of these subcutaneously transplanted femurs remain from the donor mice, enabling us to study specifically cells of the bone marrow niche using this model. We also showed that genetic ablation of peri-arteriolar cells specifically in donor femurs reduced the numbers of hematopoietic stem cells in these bones. This supports the use of this strategy as a model, in combination with genetic tools, to evaluate how bone marrow niche specific modifications may impact non-modified hematopoietic stem cells. Thus, this approach can be utilized for genetic manipulation in vivo of specific cell types only within the bone marrow. The combination of whole bone subcutaneous transplantation with rodent transgenic models will facilitate a more precise, complex and comprehensive understanding of existing problems in the study of the hematopoietic stem cell bone marrow niche.

Association of human papillomavirus (HPV), p16, p53 and p63 expression with non-bilharzia-associated squamous cell carcinoma of the bladder and algorithm construction for histopathological grading prediction

ABSTRACT Objective To investigate the expression of human papillomavirus (HPV), p16, p53, and p63 in non-schistosomiasis-related squamous cell carcinoma of the bladder and to develop an accurate and automated tool to predict histological classification based on clinicopathological features. Methods Twenty-eight patients with primary bladder pure squamous cell carcinoma who underwent cystectomy or transurethral resection of bladder tumor (TURBT) for bladder cancer between January 2011 and July 2017 were evaluated. Clinical data and follow-up information were obtained from medical records. Formalin-fixed, paraffin-embedded surgical specimens were used for immunohistochemical staining for p16, p53, and p63. Human papillomavirus detection was evaluated by PCR. Statistical analysis was performed, and statistical significance was set at p<0.05. Finally, decision trees were built to classify patients' prognostic features. Leave-one-out cross-validation was used to test the generalizability of the model. Results Neither direct HPV detection nor its indirect marker (p16 protein) was identified in most cases. The absence of p16 was correlated with less aggressive histological grading (p=0.040). The positive p16 staining detection found only in pT1 and pT2 cases in our sample suggests a possible role for this tumor suppressor protein in the initial stages of bladder squamous cell carcinoma. The decision trees constructed described the relationship between clinical features, such as hematuria/dysuria, the level of tumor invasion, HPV status, lymphovascular invasion, gender, age, compromised lymph nodes, and tumor degree differentiation, with high classification accuracy. Conclusion The algorithm classifier approach established decision pathways for semi-automatic tumor histological classification, laying the foundation for tailored semi-automated decision support systems for pathologists.

The distribution and chemical coding of enteroendocrine cells in Trypanosoma cruzi-infected individuals with chagasic megacolon.

Chagas disease is caused by the parasite, Trypanosoma cruzi that causes chronic cardiac and digestive dysfunction. Megacolon, an irreversible dilation of the left colon, is the main feature of the gastrointestinal form of Chagas disease. Patients have severe constipation, a consequence of enteric neuron degeneration associated with chronic inflammation. Dysmotility, infection, neuronal loss and a chronic exacerbated inflammation, all observed in Chagas disease, can affect enteroendocrine cells (EEC) expression, which in turn, could influence the inflammatory process. In this study, we investigated the distribution and chemical coding of EEC in the dilated and non-dilated portion of T. cruzi-induced megacolon and in non-infected individuals (control colon). Using immunohistochemistry, EECs were identified by applying antibodies to chromogranin A (CgA), glucagon-like peptide 1 (GLP-1), 5-hydroxytryptamine (5-HT), peptide YY (PYY) and somatostatin (SST). Greater numbers of EEC expressing GLP-1 and SST occurred in the dilated portion compared to the non-dilated portion of the same patients with Chagas disease and in control colon, but numbers of 5-HT and PYY EEC were not significantly different. However, it was noticeable that EEC in which 5-HT and PYY were co-expressed were common in control colon, but were rare in the non-dilated and absent in the dilated portion of chagasic megacolon. An increase in the number of CgA immunoreactive EEC in chagasic patients reflected the increases in EEC numbers summarised above. Our data suggests that the denervation and associated chronic inflammation are accompanied by changes in the number and coding of EEC that could contribute to disorders of motility and defence in the chagasic megacolon.

Cervical cancer patients that respond to chemoradiation therapy display an intense tumor infiltrating immune profile before treatment.

Cervical cancer (CC) is a major cause of death and suffering to women globally with 570,000 new cases in 2017. It disproportionately affects those living in resource-constrained countries such as Brazil, with 90% of the deaths from CC happening in low and middle-income countries. Early detection is still the best strategy for improving response to therapy and survival and cases detected in advanced stages show variable response rates to the standard chemoradiation therapy protocols. Both the genetic landscape and the immune status of patients can dramatically affect cancer progression and response to therapy, as well as disease recurrence. Here we performed a comprehensive sequencing analysis using the cancer gene panel - Ion AmpliSeq™ Cancer hotspot Panel V2 CHPv2, as well as determined the immune infiltrate composition of a group of locally advanced CC patients with the goal of identifying genetic and immune characteristics associated with a clinical response to therapy. The expression levels of CD68+ tumor-associated macrophages (TAMs) and CD8+ tumor-infiltrating lymphocytes (TILs), as well as the immune checkpoint molecules PD-1, PD-L1 and PD-L2 in stroma and in tumor regions were analyzed by immunohistochemistry (IHC). The HPV infection status with high-risk strains was also determined. Twenty-one samples from patients with squamous cell carcinoma segregated into responder (11) and non-responder (10) groups according to standard chemoradiation therapy response were studied. Our findings indicate that responder patients showed an increase of an inflammatory tumor microenvironment as indicated by higher numbers of CD8+ and PD-L2+ TILs, as well as higher expression of PD-L1 immunoreactive area, as compared to the non-responder group. Additionally, our results demonstrate a correlation between the number of gene mutations and PD-L2+ TILs in the responder group. The genes PIK3CA and KDR/VEGFR were the most mutated genes, corroborating past findings. Together, these findings indicate an inflammatory tumor microenvironment present in patients that will respond to future chemoradiation treatment as compared to those that will not. This points to possible future predictors of response to therapy in CC patients.

Evaluation of the immunoreactivity of nerve growth factor and tropomyosin receptor kinase A in the esophagus of noninfected and infected individuals with Trypanosoma cruzi.

Megaesophagus is one of the major manifestations of the chronic phase of Chagas disease. Its primary symptom is generally dysphagia due to disturbance in the lower esophageal sphincter. Microscopically, the affected organ presents denervation, which has been considered as consequence of an inflammatory process that begins at the acute phase and persists in the chronic phase. Inflammatory infiltrates are composed of lymphocytes, macrophages, natural killer cells, mast cells, and eosinophils. In this study, we evaluated the immunoreactivity of nerve growth factor (NGF), and of its receptor tropomyosin receptor kinase A (TrkA), molecules that are well known for having a relevant role in neuroimmune communication in the gastrointestinal tract. Esophageal samples obtained via autopsy or surgery procedures from six noninfected individuals, six infected individuals without megaesophagus, and six infected individuals with megaesophagus were analyzed. Infected individuals without megaesophagus presented increased numbers of NGF immunoreactive (IR) mast cells and increased areas of TrkA-IR epithelial cells and inner muscle cells. Infected individuals with megaesophagus showed increased numbers of NGF-IR eosinophils and mast cells, TrkA-IR eosinophils and mast cells, increased area of NGF-IR epithelial cells, and increased areas of TrkA-IR epithelials cells and inner muscle cells. The data presented here point to the participation of NGF and its TrkA receptor in the pathology of chagasic megaesophagus.

Mast cell-nerve interaction in the colon of Trypanosoma cruzi-infected individuals with chagasic megacolon.

Chagas disease is an infection caused by the parasite Trypanosoma cruzi that affects millions of people worldwide and is endemic in Latin America. Megacolon is the most frequent complication of the digestive chronic form and happens due to lesions of the enteric nervous system. The neuronal lesions seem to initiate in the acute phase and persist during the chronic phase, albeit the mechanisms involved in this process are still debated. Among the cells of the immune system possibly involved in this pathological process is the mast cell (MC) due to its well-known role in the bi-directional communication between the immune and nervous systems. Using ultrastructural analysis, we found an increased number of degranulated MCs in close proximity to nerve fibers in infected patients when compared with uninfected controls. We also immunostained MCs for the two pro-inflammatory molecules tryptase and chymase, the first being also important in neuronal death. The number of MCs immunostained for tryptase or chymase was increased in patients with megacolon, whereas increased tryptase staining was additionally observed in patients without megacolon. Moreover, we detected the expression of the tryptase receptor PAR2 in neurons of the enteric nervous system, which correlated to the tryptase staining results. Altogether, the data presented herein point to the participation of MCs on the denervation process that occurs in the development of T. cruzi-induced megacolon.

Mast cells in the colon of Trypanosoma cruzi-infected patients: are they involved in the recruitment, survival and/or activation of eosinophils?

Megacolon is frequently observed in patients who develop the digestive form of Chagas disease. It is characterized by dilation of the rectum-sigmoid portion and thickening of the colon wall. Microscopically, the affected organ presents denervation, which has been considered as consequence of an inflammatory process that begins at the acute phase and persists in the chronic phase of infection. Inflammatory infiltrates are composed of lymphocytes, macrophages, natural killer cells, mast cells, and eosinophils. In this study, we hypothesized that mast cells producing tryptase could influence the migration and the activation of eosinophils at the site, thereby contributing to the immunopathology of the chronic phase. We seek evidence of interactions between mast cells and eosinophils through (1) evaluation of eosinophils, regarding the expression of PAR2, a tryptase receptor; (2) correlation analysis between densities of mast cells and eosinophils; and (3) ultrastructural studies. The electron microscopy studies revealed signs of activation of mast cells and eosinophils, as well as physical interaction between these cells. Immunohistochemistry and correlation analyses point to the participation of tryptase immunoreactive mast cells in the migration and/or survival of eosinophils at the affected organ.

Neuroimmunopathology of Trypanosoma cruzi-induced megaoesophagus: Is there a role for mast cell proteases?

Tryptase and chymase are mast cell (MC)-specific proteases, which influence in the activation of inflammatory cells. In this study, we quantified tryptase- or chymase-expressing MCs in the oesophaguses of Chagas patients, and searched for a correlation between those data with area of nerve fibres that expressed either PGP9.5 (pan-marker) or vasoactive intestinal polypeptide (VIP), which is a neuromediator that has anti-inflammatory activity. Samples from the oesophaguses of 14 individuals Trypanosoma cruzi-infected and from six uninfected individuals were analysed by immunohistochemistry. It was demonstrated that the number of tryptase-IR MCs in infected individuals increased when compared with controls, regardless of whether the individuals had megaoesophagus, whereas the number of chymase-IR MCs increased only in infected individuals without megaoesophagus. Negative correlations were observed between tryptase-IR MCs and the density of nerve fibres that expressed VIP or PGP 9.5-IR. The participation of chymase and tryptase in this type of immunopathology is discussed.

An imbalance between substance P and vasoactive intestinal polypeptide might contribute to the immunopathology of megaesophagus after Trypanosoma cruzi infection.

Megaesophagus is one of the major causes of morbidity in chronic Chagas disease, and extensive denervation, associated with an inflammatory process, is recognized as the key factor for alterations in motility and disease development. Here, we analyzed esophagus samples from necropsied, infected individuals--6 cases with megaesophagus and 6 cases without megaesophagus--for the relative areas of expression of 2 neuromediators, substance P and vasoactive intestinal peptide, which are known to activate or inhibit, respectively, local immune cells. Samples from 6 noninfected individuals were used as controls. Esophageal sections were immunohistochemically stained for protein gene product 9.5, vasoactive intestinal peptide, and substance P, and the relative areas of expression of the latter 2 were calculated. Morphometric analyses revealed increased substance P and decreased vasoactive intestinal peptide relative areas in esophageal sections from patients with megaesophagus. Furthermore, in the group of patients without megaesophagus, the loss of vasoactive intestinal peptide positively correlated with the denervation process. We suggest that an imbalance between vasoactive intestinal peptide and substance P production results in the reestablishment and maintenance of the inflammatory process, leading to denervation and, consequently, promoting the development of megaesophagus.

Efeito da laserterapia sobre modelo experimental de inflamação granulomatosa / Effect of the laser therapy on experimental model of granulomatous inflammation

Na inflamação antigênica induzida por corpo estranho, observa-se a formação de células gigantes, sendo que os mecanismos envolvidos neste processo não se encontram completamente compreendidos. Na literatura, alguns trabalhos apontam para o efeito terapêutico do laser na resposta inflamatória e na cicatrização de feridas, mas, esta ação também não foi explicada de forma completa. A ação do laser As-Ga na inflamação induzida pela introdução de lamínulas de vidro no dorso de camundongos foi avaliada. Para os dois grupos tratados e não-tratados, utilizou-se trinta e seis camundongos adultos e a laserterapia foi aplicada 24h, 48h, 72h, 5, 7, 14 e 21 dias, após a indução da inflamação. O sacrifício ocorreu 7, 14 e 21 dias após cirurgia. O grupo tratado apresentou grau mais avançado no fechamento da cirurgia em relação ao grupo não-tratado. O número de células gigantes nos animais tratados com laser foi significativamente maior, assim como os valores do material fibroso, principalmente no 14 e 21 dias. Na indução experimental de inflamação granulomatosa em camundongos, a laserterapia tem ação inflamatória nas primeiras semanas e a partir dos vinte e um dias a resposta caracteriza-se como antiinflamatória.

In the antigenic induced inflammation by foreign body, the giant cells formation is observed, and the mechanisms involved in this process are not totally understood. In literature, we find some works that therapeutic effect of laser showed good inflammatory answer and wound healing, but this action was also not well explained. The effect of the laser As-Ga in the induced inflammation was evaluated by inserting the glass coverslip on the back of the mice. For the two treated groups and non-treated, it was used thirty six male adult mice and the laser therapy was applied 24:00, 48:00, 72:00h and 5, 7, 14 and 21 days, after inflammation induction. The sacrifice was performed 7, 14 and 21 days after surgery. The treated group presented more advanced stage of closing the surgery in relation to the non-treated group. The number of giant cells in the treated mice with laser was significantly higher, as well as values of the fibrous material, in the 14 and 21 days. On experimental induction of granulomatous inflammation in mice, the laser therapy has inflammatory action in the first weeks and after twenty-one days has anti-inflammatory action.