Geovanni Dantas Cassali

Effects of inhibition of PDE4 and TNF‐α on local and remote injuries following ischaemia and reperfusion injury

The effects of phosphodiesterase (PDE)4 and TNF‐α inhibition were assessed on the local and remote injuries following intestinal ischaemia and reperfusion (I/R) injury in rats. The PDE4 inhibitor rolipram dose‐dependently (1 – 10 mg kg−1) suppressed the local (intestine) and remote (lung) increases in vascular permeability and neutrophil recruitment following mild I/R injury. SB207499 (ariflo), a structurally‐distinct PDE4 inhibitor, also suppressed the injuries following mild I/R injury. In a severe model of I/R injury, treatment with rolipram (10 mg kg−1) partially reversed the local and remote increases in vascular permeability, neutrophil recruitment, intestinal haemorrhage and intestinal LTB4 concentrations. The anti‐TNF‐α anti‐serum was more effective than rolipram at inhibiting local and remote injuries and prevented the lethality associated with severe I/R. Rolipram and anti‐TNF‐α prevented the increase in the concentrations of TNF‐α in the lung and intestine, but rolipram only partially inhibited the elevation of this cytokine in serum. Rolipram had little effect on the increases of IL‐1ß concentrations in lung and serum, whereas treatment with anti‐TNF‐α markedly increased the concentration of this cytokine. Concentrations of IL‐10 rose significantly in the lung and serum and these increases were blocked by rolipram or anti‐TNF‐α. The capacity of PDE4 inhibitors to block the recruitment of neutrophils into tissues, the production of LTB4 and of the pro‐inflammatory cytokines TNF‐α, IL‐1ß and IL‐6 appear to underlie their anti‐inflammatory effects in our model of I/R injury. Overall, PDE4 inhibition was less effective than inhibition of TNF‐α for protection against I/R injury.

Schistosoma mansoni Antigens Modulate Experimental Allergic Asthma in a Murine Model: a Major Role for CD4 CD25 Foxp3 T Cells Independent of Interleukin-10

ABSTRACT In areas where schistosomiasis is endemic, a negative correlation is observed between atopy and helminth infection, associated with a low prevalence of asthma. We investigated whether Schistosoma mansoni infection or injection of parasite eggs can modulate airway allergic inflammation in mice, examining the mechanisms of such regulation. We infected BALB/c mice with 30 S. mansoni cercariae or intraperitoneally injected 2,500 schistosome eggs, and experimental asthma was induced by ovalbumin (OVA). The number of eosinophils in bronchoalveolar lavage fluid was higher in the asthmatic group than in asthmatic mice infected with S. mansoni or treated with parasite eggs. Reduced Th2 cytokine production, characterized by lower levels of interleukin-4 (IL-4), IL-5, and immunoglobulin E, was observed in both S. mansoni-treated groups compared to the asthmatic group. There was a reduction in the number of inflammatory cells in lungs of S. mansoni-infected and egg-treated mice, demonstrating that both S. mansoni infection and the egg treatment modulated the lung inflammatory response to OVA. Only allergic animals that were treated with parasite eggs had increased numbers of CD4+ CD25+ Foxp3+ T cells and increased levels of IL-10 and decreased production of CCL2, CCL3, and CCL5 in the lungs compared to the asthmatic group. Neutralization of IL-10 receptor or depletion of CD25+ T cells in vivo confirmed the critical role of CD4+ CD25+ Foxp3+ regulatory T cells in experimental asthma modulation independent of IL-10.

Role of the Chemokine Receptor CXCR2 in Bleomycin-Induced Pulmonary Inflammation and Fibrosis

Pulmonary fibrosis is characterized by chronic inflammation and excessive collagen deposition. Neutrophils are thought to be involved in the pathogenesis of lung fibrosis. We hypothesized that CXCR2-mediated neutrophil recruitment is essential for the cascade of events leading to bleomycin-induced pulmonary fibrosis. CXCL1/KC was detected as early as 6 hours after bleomycin instillation and returned to basal levels after Day 8. Neutrophils were detected in bronchoalveolar lavage and interstitium from 12 hours and peaked at Day 8 after instillation. Treatment with the CXCR2 receptor antagonist, DF2162, reduced airway neutrophil transmigration but led to an increase of neutrophils in lung parenchyma. There was a significant reduction in IL-13, IL-10, CCL5/RANTES, and active transforming growth factor (TGF)-beta(1) levels, but not on IFN-gamma and total TGF-beta(1,) and enhanced granulocyte macrophage-colony-stimulating factor production in DF2162-treated animals. Notably, treatment with the CXCR2 antagonist led to an improvement of the lung pathology and reduced collagen deposition. Using a therapeutic schedule, DF2162 administered from Days 8 to 16 after bleomycin reduced pulmonary fibrosis and levels of active TGF-beta(1) and IL-13. DF2162 treatment reduced bleomycin-induced expression of von Willebrand Factor, a marker of angiogenesis, in the lung. In vitro, DF2162 reduced the angiogenic activity of IL-8 on human umbilical vein endothelial cells. In conclusion, we show that CXCR2 plays an important role in mediating fibrosis after bleomycin instillation. The compound blocks angiogenesis and the production of pro-angiogenic cytokines, and decreases IL-8-induced endothelial cell activation. An effect on neutrophils does not appear to account for the major effects of the blockade of CXCR2 in the system.

Cox-2 Expression in Canine Mammary Carcinomas: Correlation with Angiogenesis and Overall Survival

Mammary tumors are among the most common neoplastic processes in female dogs. Prostaglandin E2, the catalytic product of Cox-2, may promote tumor development and angiogenesis. It has been investigated in several human cancers and also correlated with the evolution of the disease. However, the clinical implications of tumor pathology require more investigation in veterinary medicine. Angiogenesis is essential for the growth and metastasis of major solid tumors and has been correlated with prognosis in human and canine breast cancer. The aim of this study was to evaluate Cox-2 expression and microvessel density in canine mammary carcinomas and to correlate them with overall survival of the animal. Cox-2 and angiogenesis were assessed by immunohistochemistry in 46 mammary carcinomas (19 ductal and 27 metaplastic) and in healthy mammary glands. To assess tumor angiogenesis, microvessel density (MVD) was determined by CD31 staining. Immunostaining revealed that 46/46 (100%) of the tumors were positive for Cox-2 and CD31, and there was no statistical difference among tumor types. Cox-2 protein expression correlated positively with CD31 staining (r = 0.3742, P = .0104) but did not correlate significantly with tumor type. Longer overall survival was observed in metaplastic carcinomas (P = .028), in tumors with low microvessel density (P = .0002) and with low Cox-2 score (P = .01). Our results demonstrate that increased microvessel density and increased Cox-2 expression were linearly related in the canine mammary tumors studied and were also related to worse prognosis and shorter overall survival. This suggests that Cox-2 inhibitors could be an alternative for the treatment and control of advanced neoplastic mammary disease in female dogs.

c-erbB-2 expression and nuclear pleomorphism in canine mammary tumors

The objective of the present investigation was to study the expression of c-erbB-2 and MIB-1 and try to associate them with morphological features of the cell such as nuclear pleomorphism, mitotic count and histological grade in a series of 70 canine mammary gland tumors, 22 of them benign and 48 malignant. Tumors were collected at the Veterinary Hospital of UFMG (Brazil) and the Veterinary Faculty of Porto University (Portugal). c-erbB-2 expression was determined according to the guidelines provided by the manufacturer of the HercepTest system and nuclear pleomorphism, mitotic count and histological grade according the Elston and Ellis grading system. The HercepTest is the FDA-approved in vitro diagnostic test marketed by Dako. It is a semi-quantitative immunohistochemical assay used to determine overexpression of HER2 protein (human epidermal growth factor receptor) in breast cancer tissue. MIB-1 expression was also evaluated in 28 malignant tumors. Seventeen (35.4%) of the malignant tumors were positive for c-erbB-2 expression, which was positively associated with nuclear pleomorphism (P < 0.0001), histological grade (P = 0.0017) and mitotic count (P < 0.05). Nuclear pleomorphism also showed a positive association with MIB-1 index (P < 0.0001). These results suggest that some of the biological and morphological characteristics of the tumor are associated in canine mammary gland tumors, as also reported for human breast cancer. It was also possible to show that the immunoexpression of c-erbB-2 can be a factor in mammary carcinogenesis. This fact opens the possibility of using anti-c-erbB-2 antibodies in the treatment of canine mammary tumors.

Platelet-Activating Factor Receptor Plays a Role in Lung Injury and Death Caused by Influenza A in Mice

Influenza A virus causes annual epidemics which affect millions of people worldwide. A recent Influenza pandemic brought new awareness over the health impact of the disease. It is thought that a severe inflammatory response against the virus contributes to disease severity and death. Therefore, modulating the effects of inflammatory mediators may represent a new therapy against Influenza infection. Platelet activating factor (PAF) receptor (PAFR) deficient mice were used to evaluate the role of the gene in a model of experimental infection with Influenza A/WSN/33 H1N1 or a reassortant Influenza A H3N1 subtype. The following parameters were evaluated: lethality, cell recruitment to the airways, lung pathology, viral titers and cytokine levels in lungs. The PAFR antagonist PCA4248 was also used after the onset of flu symptoms. Absence or antagonism of PAFR caused significant protection against flu-associated lethality and lung injury. Protection was correlated with decreased neutrophil recruitment, lung edema, vascular permeability and injury. There was no increase of viral load and greater recruitment of NK1.1+ cells. Antibody responses were similar in WT and PAFR-deficient mice and animals were protected from re-infection. Influenza infection induces the enzyme that synthesizes PAF, lyso-PAF acetyltransferase, an effect linked to activation of TLR7/8. Therefore, it is suggested that PAFR is a disease-associated gene and plays an important role in driving neutrophil influx and lung damage after infection of mice with two subtypes of Influenza A. Further studies should investigate whether targeting PAFR may be useful to reduce lung pathology associated with Influenza A virus infection in humans.

Recommended Guidelines for Submission, Trimming, Margin Evaluation, and Reporting of Tumor Biopsy Specimens in Veterinary Surgical Pathology

Neoplastic diseases are typically diagnosed by biopsy and histopathological evaluation. The pathology report is key in determining prognosis, therapeutic decisions, and overall case management and therefore requires diagnostic accuracy, completeness, and clarity. Successful management relies on collaboration between clinical veterinarians, oncologists, and pathologists. To date there has been no standardized approach or guideline for the submission, trimming, margin evaluation, or reporting of neoplastic biopsy specimens in veterinary medicine. To address this issue, a committee consisting of veterinary pathologists and oncologists was established under the auspices of the American College of Veterinary Pathologists Oncology Committee. These consensus guidelines were subsequently reviewed and endorsed by a large international group of veterinary pathologists. These recommended guidelines are not mandated but rather exist to help clinicians and veterinary pathologists optimally handle neoplastic biopsy samples. Many of these guidelines represent the collective experience of the committee members and consensus group when assessing neoplastic lesions from veterinary patients but have not met the rigors of definitive scientific study and investigation. These questions of technique, analysis, and evaluation should be put through formal scrutiny in rigorous clinical studies in the near future so that more definitive guidelines can be derived.

IMMUNOPHENOTYPIC FEATURES OF TUMOR INFILTRATING LYMPHOCYTES FROM MAMMARY CARCINOMAS IN FEMALE DOGS ASSOCIATED WITH PROGNOSTIC FACTORS AND SURVIVAL RATES

BackgroundThe immune system plays an important role in the multifactorial biologic system during the development of neoplasias. However, the involvement of the inflammatory response in the promotion/control of malignant cells is still controversial, and the cell subsets and the mechanisms involved are poorly investigated. The goal of this study was to characterize the clinical-pathological status and the immunophenotyping profile of tumor infiltrating lymphocytes and their association with the animal survival rates in canine mammary carcinomas.MethodsFifty-one animals with mammary carcinomas, classified as carcinomas in mixed tumors-MC-BMT = 31 and carcinomas-MC = 20 were submitted to systematic clinical-pathological analysis (tumor size; presence of lymph node and pulmonary metastasis; clinical stage; histological grade; inflammatory distribution and intensity as well as the lymphocytic infiltrate intensity) and survival rates. Twenty-four animals (MC-BMT = 16 and MC = 8) were elected to the immunophenotypic study performed by flow cytometry.ResultsData analysis demonstrated that clinical stage II-IV and histological grade was I more frequent in MC-BMT as compared to MC. Univariate analysis demonstrated that the intensity of inflammation (moderate/intense) and the proportion of CD4+ (≥ 66.7%) or CD8+ T-cells (<33.3%) were not associated with worse survival rate. Multivariate analysis demonstrated that only lymphocytic infiltrate intensity ≥ 600 (P = 0.02) remained as independent prognostic factor. Despite the clinical manifestation, the lymphocytes represented the predominant cell type in the tumor infiltrate. The percentage of T-cells was higher in animals with MC-BMT without metastasis, while the percentage of B-lymphocytes was greater in animals with metastasized MC-BMT (P < 0.05). The relative percentage of CD4+ T-cells was significantly greater in metastasized tumors (both MC-BMT and MC), (P < 0.05) while the proportion of CD8+ T-cells was higher in MC-BMT without metastasis. Consequently, the CD4+/CD8+ ratio was significantly increased in both groups with metastasis. Regardless of the tumor type, the animals with high proportions of CD4+ and low CD8+ T-cells had decreased survival rates.ConclusionThe intensity of lymphocytic infiltrate and probably the relative abundance of the CD4+ and CD8+ T-lymphocytes may represent important survival prognostic biomarkers for canine mammary carcinomas.

Evaluation of accuracy of fine needle aspiration cytology for diagnosis of canine mammary tumours: comparative features with human tumours

Objective:  The authors evaluated the accuracy of the fine needle aspiration cytology technique in the diagnosis of 77 canine mammary gland tumours using the same cytological and histological criteria currently applied to the diagnosis of human breast cancer.

Gonadotropin Stimulation Increases the Expression of Angiotensin-(1—7) and Mas Receptor in the Rat Ovary

We have previously shown the presence of immunoreactive angiotensin-(1—7) [Ang-(1—7)] in rat ovary homogenate and its stimulatory effect on estradiol and progesterone production in vitro. In the current study, we investigated the presence and cellular distribution of Ang-(1—7) and the Mas receptor, the expression of Mas and angiotensin-converting enzyme 2 (ACE2) messenger RNA (mRNA), and the enzymatic activity in the rat ovary following gonadotropin stimulation in vivo. Immature female Wistar rats (25 days old) were injected subcutaneously (SC) with equine chorionic gonadotropin (eCG, 20 IU in 0.2 mL) or vehicle 48 hours before euthanasia. Tissue distributions of Ang-(1—7), Mas receptor, and ACE2 were evaluated by immunohistochemistry, along with angiotensin II (Ang II) localization, while the mRNA expression levels of Mas receptor and ACE2 were evaluated by real-time polymerase chain reaction (PCR). In addition, we determined the activity of neutral endopeptidase (NEP), prolyl endopeptidase (PEP), and ACE by fluorometric assays. After eCG treatment, we found strong immunoreactivity for Ang-(1—7) and Mas primarily in the theca-interstitial cells, while Ang II appeared in the granulosa but not in the thecal layer. Equine chorionic gonadotropin treatment increased Mas and ACE2 mRNA expression compared with control animals (3.3- and 2.1-fold increase, respectively; P < .05). Angiotensin-converting enzyme and NEP activities were lower, while PEP activity was higher in the eCG-treated rats (P < .05). These data show gonadotropin-induced changes in the ovarian expression of Ang-(1—7), Mas receptor, and ACE2. These findings suggest that the renin-angiotensin system (RAS) branch formed by ACE2/Ang-(1—7)/Mas, fully expressed in the rat ovary and regulated by gonadotropic hormones, could play a role in the ovarian physiology.