When the adrenals alert us to a systemic fungal disease.

We present a patient referred for investigation of adrenal insufficiency, confirmed due to disseminated paracoccidioidomycosis (PCM), with abdominal and central nervous system (CNS) involvement. Establishing the pathogenesis and immunological processes involved in chronic or latent infections by PCM has been challenging. Medical doctors caring for patients with immunodeficiencies should learn about these fungal infections to properly guide travel planning and have this possibility in the diagnostic arsenal when the patient returns from endemic areas. After 13 months of treatment, the patient showed good clinical evolution, and we repeated imaging exams, showing partial improvement of the preview lesions. Diagnosis and treatment can prevent catastrophic events.

Release of 6-nitrodopamine from human popliteal artery and vein.

6-Nitrodopamine (6-ND) is a novel catecholamine that is released from human umbilical cord vessels, and it causes vascular relaxation by acting as a dopamine D2-receptor antagonist. Here it was investigated whether human peripheral vessels obtained from patients who have undergone surgery for leg amputation release 6-ND, and its action in these tissues. Popliteal artery and vein strips present basal release of 6-ND, as measured by liquid chromatography coupled to tandem mass spectrometry. The release was significantly reduced when the tissues were pre-treated with the nitric oxide synthase inhibitor L-NAME (100 µM), or when the endothelium was mechanically removed. In U-46619 (3 nM) pre-contracted rings, 6-ND induced concentration-dependent relaxations (pEC50 8.18 ± 0.05 and 8.40 ± 0.08, in artery and vein rings, respectively). The concentration-dependent relaxations induced by 6-ND were unaffected in tissues pre-treated with L-NAME, but significantly reduced in tissues where the endothelium has been mechanically removed. In U-46619 (3 nM) pre-contracted rings, the selective dopamine D2 receptor antagonist L-741,626 also caused concentration-dependent relaxations (pEC50 8.92 ± 0.22 and 8.79 ± 0.19, in artery and vein rings, respectively). The concentration-dependent relaxations induced by L-741,626 were unaffected in tissues pre-treated with L-NAME, but significantly reduced in tissues where the endothelium has been mechanically removed. This is the first demonstration that 6-nitrodopamine is released from human peripheral artery and vein rings. The results also indicate that endothelium-derived dopamine is a major contractile agent in the popliteal artery and vein, and that selective dopamine D2-receptor antagonists such as 6-ND, may have therapeutic potential in the treatment of human peripheral vascular diseases.

In vitro and in vivo acute toxicity of a novel citrate-coated magnetite nanoparticle.

Magnetic nanoparticles (MNps) have become powerful tools for multiple biomedical applications such as hyperthermia drivers, magnetic resonance imaging (MRI) vectors, as well as drug-delivery systems. However, their toxic effects on human health have not yet been fully elucidated, especially in view of their great diversity of surface modifications and functionalizations. Citrate-coating of MNps often results in increased hydrophilicity, which may positively impact their performance as drug-delivery systems. Nonetheless, the consequences on the intrinsic toxicity of such MNps are unpredictable. Herein, novel magnetite (Fe3O4) nanoparticles covered with citrate were synthesized and their potential intrinsic acute toxic effects were investigated using in vitro and in vivo models. The proposed synthetic pathway turned out to be simple, quick, inexpensive, and reproducible. Concerning toxicity risk assessment, these citrate-coated iron oxide nanoparticles (IONps) did not affect the in vitro viability of different cell lines (HaCaT and HepG2). Moreover, the in vivo acute dose assay (OECD test guideline #425) showed no alterations in clinical parameters, relevant biochemical variables, or morphological aspects of vital organs (such as brain, liver, lung and kidney). Iron concentrations were slightly increased in the liver, as shown by Graphite Furnace Atomic Absorption Spectrometry and Perls Prussian Blue Staining assays, but this finding was considered non-adverse, given the absence of accompanying functional/clinical repercussions. In conclusion, this study reports on the development of a simple, fast and reproducible method to obtain citrate-coated IONps with promising safety features, which may be used as a drug nanodelivery system in the short run. (263 words).

Endothelium modulates electrical field stimulation-induced contractions of Chelonoidis carbonaria aortic rings.

The role of endothelium in the electrical-field stimulation (EFS)-induced contractions of Chelonoidis carbonaria aorta was investigated. Contractions were evaluated in the presence and absence of L-NAME (100 µM), tetrodotoxin (1 µM), phentolamine (10 and 100 µM), phenoxybenzamine (1 and 10 µM), prazosin (100 µM), idazoxan (100 µM), atropine (10 µM), D-tubocurarine (10 µM) or indomethacin (10 µM). EFS-induced contraction was also carried out in endothelium-denuded rings. EFS-induced contraction was investigated by the sandwich assay. Concentration curves to endothelin-1 (0.1-100 nM) and U46619 (0.001-100 µM) were also constructed to calculate both Emax and EC50. EFS at 16 Hz contracted Chelonoidis aorta, which was almost abolished by the endothelium removal. The addition of L-NAME increased the EFS response (2.0 ± 0.4 and 8.3 ± 1.9 mN). In L-NAME treated aortic rings, tetrodotoxin did not change the EFS-response (5.1 ± 1.8 and 4.9 ± 1.7 mN). Indomethacin, atropine and d-tubucurarine also did not affect the EFS-response. Phentolamine at 10 µM did not change the EFS-induced contraction; however, at 100 µM, reduced it (3.9 ± 1 and 1.9 ± 0.3 mN). Prazosin and idazoxan did not change EFS-induced contractions. Phenoxybenzamine at 1 µM reduced by 76% (9.6 ± 3.4 and 2.3 ± 0.8 mN) and at 10 µM by 90% the EFS response. Immunohistochemistry identified tyrosine hydroxylase in the endothelium and brain, whereas S100 protein was found only in brain. In conclusion, endothelium modulates EFS-induced contractions in Chelonoidis aortic rings and this modulation may be due to endothelium-derived catecholamines, possibly dopamine.

ATP Synthase Subunit Beta Immunostaining is Reduced in the Sclerotic Hippocampus of Epilepsy Patients.

Epilepsy is a common disease presenting with recurrent seizures. Hippocampal sclerosis (HS) is the commonest histopathological alteration in patients with temporal lobe epilepsy (TLE) undergoing surgery. HS physiopathogenesis is debatable. We have recently studied, by using mass spectrometry-based proteomics, an experimental model of TLE induced by electrical stimulation. Specifically, protein expressions of both the beta subunit of mitochondrial ATP synthase (ATP5B) and of membrane ATPases were found to be reduced. Here, we investigated tissue distribution of ATP5B and sodium/potassium-transporting ATPase subunit alpha-3 (NKAα3), a protein associated with neuromuscular excitability disorders, in human hippocampi resected "en bloc" for HS treatment (n = 15). We used immunohistochemistry and the stained area was digitally evaluated (increase in binary contrast of microscopic fields) in the hippocampal sectors (CA1-CA4) and dentate gyrus. All HS samples were classified as Type 1, according to the International League Against Epilepsy (ILAE) 2013 Classification (predominant cell loss in CA1 and CA4). ATP5B was significantly decreased in all sectors and dentate gyrus of HS patients compared with individuals submitted to necropsy and without history of neurological alterations (n = 10). NKAα3 expression showed no difference. Moreover, we identified a negative correlation between frequency of pre-operative seizures and number of neurons in CA1. In conclusion, our data showed similarity between changes in protein expression in a model of TLE and individuals with HS. ATP5B reduction would be at least in part due to neuronal loss. Future investigations on ATP5B activity could provide insights into the process of such cell loss.

Intranodal Angiomyomatous Hamartoma in a Cynomolgus Monkey.

We describe here an angiomyomatous hamartoma in the right axillary lymph node of a three-year-old male cynomolgus monkey ( Macaca fascicularis), used as a control subject in a short-term toxicity study. This is a very rare lesion that has been reported almost exclusively in inguinal lymph nodes, and to date only in human beings. In the present case, light microscopy revealed partial replacement of the lymph node parenchyma by a disorganized, irregular vascular network, sparsely distributed smooth muscle cells, and a fibro-adipocytic stroma. This was considered to be fortuitous given the age of the animal, with no clinical or toxicological significance. To the best of our knowledge, this is the first report of an intranodal angiomyomatous hamartoma in a nonhuman animal species.

Electrical field-induced contractions on Crotalus durissus terrificus and Bothrops jararaca aortae are caused by endothelium-derived catecholamine.

Endothelium is the main source of catecholamine release in the electrical-field stimulation (EFS)-induced aortic contractions of the non- venomous snake Panterophis guttatus. However, adrenergic vasomotor control in venomous snakes such as Crotalus durissus terrificus and Bothrops jararaca has not yet been investigated. Crotalus and Bothrops aortic rings were mounted in an organ bath system. EFS-induced aortae contractions were performed in the presence and absence of guanethidine (30 µM), phentolamine (10 µM) or tetrodotoxin (1 µM). Frequency-induced contractions were also performed in aortae with endothelium removed. Immunohistochemical localization of both tyrosine hydroxylase (TH) and S-100 protein in snake aortic rings and brains, as well as in human tissue (paraganglioma tumour) were carried out. EFS (4 to 16 Hz) induced frequency-dependent aortic contractions in both Crotalus and Bothrops. The EFS-induced contractions were significantly reduced in the presence of either guanethidine or phentolamine in both snakes (p<0.05), whereas tetrodotoxin had no effect in either. Removal of the endothelium abolished the EFS-induced contractions in both snakes aortae (p<0.05). Immunohistochemistry revealed TH localization in endothelium of both snake aortae and human vessels. Nerve fibers were not observed in either snake aortae. In contrast, both TH and S100 protein were observed in snake brains and human tissue. Vascular endothelium is the main source of catecholamine release in EFS-induced contractions in Crotalus and Bothrops aortae. Human endothelial cells also expressed TH, indicating that endothelium- derived catecholamines possibly occur in mammalian vessels.

Decreased expression of stem cell markers by simvastatin in 7,12-dimethylbenz(a)anthracene (DMBA)-induced breast cancer.

Simvastatin, a competitive inhibitor of HMG-CoA reductase widely used in the treatment and prevention of hyperlipidemia-related diseases, has recently been associated to in vitro anticancer stem cell (CSC) actions. However, these effects have not been confirmed in vivo. To assess in vivo anti-CSC effects of simvastatin, female Sprague-Dawley rats with 7,12-dimethyl-benz(a)anthracene (DMBA)-induced mammary cancer and control animals were treated for 14 days with either simvastatin (20 or 40 mg/kg/day) or soybean oil (N = 60). Tumors and normal breast tissues were removed for pathologic examination and immunodetection of CSC markers. At 40 mg/kg/day, simvastatin significantly reduced tumor growth and the expression of most CSC markers. The reduction in tumor growth (80%) could not be explained solely by the decrease in CSCs, since the latter accounted for less than 10% of the neoplasia (differentiated cancer cells were also affected). Stem cells in normal, nonneoplastic breast tissues were not affected by simvastatin. Simvastatin was also associated with a significant decrease in proliferative activity but no increase in cell death. In conclusion, this is the first study to confirm simvastatin anti-CSC actions in vivo, further demonstrating that this effect is specific for neoplastic cells, but not restricted to CSCs, and most likely due to inhibition of cell proliferation.

Use of a novel polyvinyl alcohol membrane as a pericardial substitute reduces adhesion formation and inflammatory response after cardiac reoperation.

BACKGROUND: Adhesions may increase the incidence of lethal complications of cardiac reoperations, which account for up to 20% of all open-heart surgeries. Herein, we describe the use of a polyvinyl alcohol membrane (PVAM) as a pericardial alternative and describe its performance during reoperation in a relevant animal model. METHODS: The PVAM samples were reticulated by electron beam radiation and manipulated into a tube shape. After thoracotomy, the pericardium of Wistar rats was opened to expose the heart. Rats were treated by pushing the heart back into the thoracic cavity (Sham group), sprinkling the epicardium with talcum powder (Talc group), encircling the heart with PVAM (PVAM group), or sprinkling the epicardium with talcum powder before placing the PVAM to encircle the heart (PVAM + Talc group). Animals were recovered for 8 weeks and then euthanized. Macroscopic findings (ie, extent and severity of adhesions) were classified according to a 4-grade adhesion scale. The PVAM was tested for direct and indirect cytotoxicity with Vero cells. The water absorption capability and in vivo calcification after 8 weeks of subcutaneous implantation of the membrane were examined. Data were analyzed by analysis of variance and Bonferroni post hoc tests. RESULTS: The PVAM group had lower adhesion scores than the Talc and Sham groups, as well as reduced epicardium thickness and inflammatory cell results, compared with the Talc and PVAM + Talc groups. The PVAM exhibited no direct or indirect cytotoxicity, good water absorption capability (42.4% ± 0.9%), and negligible calcification after 8 weeks (4.42 × 10(-3) ± 2.56 × 10(-3) percentage of the total mass). CONCLUSIONS: The PVAM shows promising properties for its potential use as a novel pericardial substitute.

Image compression impact on quantitative angiogenesis analysis of ovarian epithelial neoplasms.

OBJECTIVE: This study aims to investigate the impact of digital image compression on manual and semiautomatic quantification of angiogenesis in ovarian epithelial neoplasms (including benign, borderline, and malignant specimens). DESIGN: We examined 405 digital images (obtained from a previously validated computer-assisted analysis system), which were equally divided into 5 groups: images captured in Tagged Image File Format (TIFF), low and high compression Joint Photographic Experts Group (JPEG) formats, and low and high compression JPEG images converted from the TIFF files. MEASUREMENTS: Microvessel density counts and CD34 endothelial areas manually and semiautomatically determined from TIFF images were compared with those from the other 4 groups. RESULTS: Mostly, the correlations between TIFF and JPEG images were very high (intraclass correlation coefficients >0.8), especially for low compression JPEG images obtained by capture, regardless of the variable considered. The only exception consisted in the use of high compression JPEG files for semiautomatic microvessel density counts, which resulted in intraclass correlation coefficients of <0.7. Nonetheless, even then, interconversion between TIFF and JPEG values could be successfully achieved using prediction models established by linear regression. CONCLUSION: Image compression does not seem to significantly compromise the accuracy of angiogenesis quantitation in the ovarian epithelial tumors, although low compression JPEG images should always be preferred over high compression ones.

Immunohistochemical analysis of vascular density and area in colorectal carcinoma using different markers and comparison with clinicopathologic prognostic factors.

Analysis of blood and lymphatic vessel in colorectal cancer is controversial in the literature, possibly due to variations in the methods of analysis. In this study, it was aimed to search for a reliable approach in the quantification of angio- and lymphangiovascular density and area as a prognostic factor and to compare such vessel counts in normal mucosa, adenomas and cancer. A retrospective study was performed on 60 sporadic colorectal cancer, 30 colorectal adenomas, and 10 colorectal non-neoplastic lesions. Archival tissues were submitted to immunohistochemical evaluation using antibodies to CD31, CD34, CD105, VEGF-A, VEGF-C, and D2-40. Microvessel density and total vascular area were determined by computer image analysis and values were compared in the three groups of lesions; the prognostic value of these parameters was evaluated in the group of colorectal cancer. Most markers showed progressive vessel counts from non-neoplastic tissue to carcinoma, both for microvessel density and total vascular area. Only microvessel density determined by CD34 in the central areas of the cancer correlated with recurrence/metastasis (p = 0.04) and survival (p = 0.02). Different methods of quantification (microvessel counting versus estimation of total vascular area), immunohistochemical markers (pan-endothelial marker versus neovessels and lymphatic markers), and areas of analysis (periphery versus inner portions of the lesion) were assessed using image analysis. The results corroborate the increase in vascularization of carcinoma and suggest that microvessel density determined by immunostaining for CD34 in the inner portion of the tumor might represent a prognostically relevant parameter in colorectal cancer.

Early changes in contractility indices and fibrosis in two minimally invasive congestive heart failure models.

BACKGROUND: Heart failure is a common and often fatal disease. Numerous animal models are used to study its aetiology, progression and treatment. This article aims to demonstrate two minimally invasive models of congestive heart failure in a rabbit model and a precise method to assess cardiac performance. METHODS: Fifty New Zealand White rabbits underwent cervicotomy incision and were then divided into three groups. Aortic regurgitation (AR group) was induced in 17 animals by catheter lesion through the right carotid artery, proximal aortic constriction (AC group) was created in 17 animals by metallic clip placement in the ascending aorta through a neck incision, while 16 animals served as controls (CO group). Eight weeks later, myocardial function and contractility indices were assessed by sonomicrometry crystals. Hearts were then collected for morphometric measurements and left ventricular tissues were subjected to immunohistochemical analysis of fibrosis, necrosis and apoptosis. Statistical analysis was by analysis of variance (ANOVA) with a Dunnett's post hoc test or by Kruskal-Wallis test with Dunn's post hoc test as appropriate, with significance at p< or =0.05. RESULTS: The model of aortic regurgitation indicated early stages of heart failure by volume overload with increased end-diastolic and end-systolic volumes, stroke volume, cardiac output and pressure-volume loop areas. The elastance was higher in the control group compared with that in the AC and AR groups (131.00+/-51.27 vs 88.77+/-40.11 vs 75.29+/-50.70; p=0.01). The preload recruitable stroke work was higher in the control group compared with that in the AC and AR groups (47.70+/-14.19 vs 33.87+/-7.46 vs 38.58+/-9.45; p=0.01). Aortic constriction produced left ventricular concentric hypertrophy. Fibrosis appeared in both heart failure models and was elevated by aortic constriction when compared with that in controls. Necrosis and apoptosis indices were very low in all the groups. Clinical signs of congestive heart failure were not present. CONCLUSIONS: The two heart failure models we describe were relatively simple to create and maintain, minimally invasive, accurate, inexpensive and, importantly, had a low mortality rate. These models rapidly induced deterioration of contractility indices and onset of fibrosis, the hallmarks of early myocardial dysfunction associated with heart failure. Sonomicrometry assessments were able to detect early contractility changes prior to clinical signs.

Correlation between thymidylate synthase protein expression and gene polymorphism with clinicopathological parameters in colorectal carcinoma.

5-Fluorouracil (5-FU) represents the basis of chemotherapy for colorectal carcinoma, inhibiting thymidylate synthase (TS), an essential enzyme for DNA replication. Previous studies have associated high TS protein expression by tumor cells with poor outcome of patients with colorectal carcinoma, but others have refuted these findings. In view of the potential role of TS as predictive parameter and the lack of consensus in the literature, the present study compared 2 methods: protein expression and gene polymorphism, correlating them with clinicopathological findings. Immunohistochemical detection of TS in tumor cells and detection of gene polymorphism in the blood were performed in 32 patients with colorectal carcinoma treated with 5-FU. No correlation was found between TS protein expression and gene polymorphism. Neither method correlated with survival, tumor staging, and tumor histological grading. This result possibly reflects a complex tumor response to 5-FU therapy, where TS is just one of the involved proteins.

Use of p63 and CD10 in the differential diagnosis of papillary neoplasms of the breast.

Papillary neoplasms of the breast represent a complex spectrum ranging from benign to malignant lesions. The myoepithelial cell (MEC) layer is generally continuous in papillomas and increasingly discontinuous to absent in atypical and malignant counterparts. Identification of MECs can be difficult on morphological grounds and currently relies on immunomarkers. We investigated the potential role of p63 and CD10 in 20 papillary lesions and compared them with 1A4 and calponin. In 18 cases, adjacent normal breast tissue was available for study. All four markers were diffusely positive in all samples of normal tissue and benign papillomas indicating similar sensitivity in the identification of MECs. Intense positivity was found in 100% of the cases with 1A4 and CD10, but in only 76% with calponin and in 60.5% with p63 (differences statistically significant, p < 0.05), suggesting that the former two render more reproducible results. The most specific markers were p63 and CD10 which showed cross-reactivity in 0% and in up to 33% of the cases respectively. 1A4 and calponin showed diffuse cross-reactivity in all cases. When assessing benign versus atypical papillomas, the best parameters were diffuse positivity using CD10 or p63, and continuous MEC layer, mainly using CD10. When comparing benign papillomas to carcinomas all parameters were equally useful with 1A4 and CD10. Regardless of the marker, intense positivity was the only parameter that could distinguish atypical papillomas from papillary carcinomas. p63 staining, which renders a nuclear and mostly discontinuous reactivity, was not as useful as the other markers when the parameter continuous MEC layer was evaluated. Although CD10 seems to combine the highest specificity and reproducibility with a good sensitivity, reproducibility of 1A4 is higher. Thus, a minimum panel to assess papillary lesions should include both markers. Although p63 is the most specific, its nuclear and discontinuous pattern may lead to erroneous diagnosis, especially in the differentiation between benign papillomas and atypical/malignant lesions.

p63 and CD10: reliable markers in discriminating benign sclerosing lesions from tubular carcinoma of the breast?

The immunohistochemical detection of myoepithelial cells in benign sclerosing lesions of the breast is useful in distinguishing them from tubular carcinoma. So far, this detection has been carried out using antibodies against cytoskeletal proteins, such as alpha-smooth muscle actin (1A4) and calponin. However, the specificity of these markers has been questioned since they may be expressed in stromal myofibroblasts and vascular smooth muscle. Recently, two novel myoepithelial markers have been described: the nuclear protein p63, a member of the p53 family, and the surface antigen CD10, also known as common acute lymphoblastic leukemia antigen (CALLA). The authors assessed the use of p63 and CD10 in the differential diagnosis between benign sclerosing lesions, such as sclerosing adenosis and radial scar, and tubular carcinoma, in comparison to the traditional myoepithelial markers 1A4 and calponin. p63, CD10, 1A4, and calponin were expressed in myoepithelial cells of all benign lesions and were consistently negative in all cases of tubular carcinoma. In contrast to cytoskeletal proteins, p63 and CD10 were mostly confined to myoepithelial cells and thus were more specific than the traditional counterparts. However, 1A4 was more intensely expressed and more reproducible than the novel markers. In conclusion, p63 and CD10 may be used as a complement to 1A4 in distinguishing benign sclerosing lesions from tubular carcinoma of the breast.

CD4+ T cells downregulate Bcl-2 in germinal centers.

Germinal centers (GCs) are the main site of T cell-dependent antibody responses. Upon antigen challenge, GCs comprise mostly B cells undergoing proliferation, somatic hypermutation and antigen-affinity selection. GC B cells down-modulate the expression of Bcl-2 protein and are highly sensitive to apoptosis to eliminate autoreactive or low-affinity cells. Bcl-2 is still expressed in a few GC cells, whose identity remains unclear. To address this issue, we examined by confocal microscopy the expression of Bcl-2 by different GC lymphocyte subsets in hyperplastic tonsils. We found that the vast majority of Bcl-2(+) GC cells are T lymphocytes. Conversely, while in the mantle zone and in the interfollicular areas T cells are almost exclusively Bcl-2(+), in the GC, most T lymphocytes are Bcl-2(-). In addition, most of the CD4(+) GC T cells are Bcl-2(-), while nearly 100% of the CD8(+) GC T cells are Bcl-2(+). The Bcl-2 downregulation by both B and CD4(+) T GC cells supports the concept that these two subsets may undergo a selection process in this microenvironment.