Rui M. Gil da Costa

Limited Role of Secreted Aspartyl Proteinases Sap1 to Sap6 in Candida albicans Virulence and Host Immune Response in Murine Hematogenously Disseminated Candidiasis

ABSTRACT Candida albicans secreted aspartyl proteinases (Saps) are considered virulence-associated factors. Several members of the Sap family were claimed to play a significant role in the progression of candidiasis established by the hematogenous route. This assumption was based on the observed attenuated virulence of sap-null mutant strains. However, the exclusive contribution of SAP genes to their attenuated phenotype was not unequivocally confirmed, as the Ura status of these mutant strains could also have contributed to the attenuation. In this study, we have reassessed the importance of SAP1 to SAP6 in a murine model of hematogenously disseminated candidiasis using sap-null mutant strains not affected in their URA3 gene expression and compared their virulence phenotypes with those of Ura-blaster sap mutants. The median survival time of BALB/c mice intravenously infected with a mutant strain lacking SAP1 to SAP3 was equivalent to that of mice infected with wild-type strain SC5314, while those infected with mutant strains lacking SAP5 showed slightly extended survival times. Nevertheless, no differences could be observed between the wild type and a Δsap456 mutant in their abilities to invade mouse kidneys. Likewise, a deficiency in SAP4 to SAP6 had no noticeable impact on the immune response elicited in the spleens and kidneys of C. albicans-infected mice. These results contrast with the behavior of equivalent Ura-blaster mutants, which presented a significant reduction in virulence. Our results suggest that Sap1 to Sap6 do not play a significant role in C. albicans virulence in a murine model of hematogenously disseminated candidiasis and that, in this model, Sap1 to Sap3 are not necessary for successful C. albicans infection.

Estimation of rat mammary tumor volume using caliper and ultrasonography measurements

Mammary tumors similar to those observed in women can be induced in rats by intraperitoneal administration of N-methyl-N-nitrosourea. Determining tumor volume is a useful and quantitative way to monitor tumor progression. In this study, the authors measured dimensions of rat mammary tumors using a caliper and using real-time compound B-mode ultrasonography. They then used different formulas to calculate tumor volume from these tumor measurements and compared the calculated tumor volumes with the real tumor volume to identify the formulas that gave the most accurate volume calculations. They found that caliper and ultrasonography measurements were significantly correlated but that tumor volumes calculated using different formulas varied substantially. Mammary tumors seemed to take on an oblate spheroid geometry. The most accurate volume calculations were obtained using the formula V = (W2 × L)/2 for caliper measurements and the formula V = (4/3) × π × (L/2) × (L/2) × (D/2) for ultrasonography measurements, where V is tumor volume, W is tumor width, L is tumor length and D is tumor depth.

Bacterial Cellulose: Long-Term Biocompatibility Studies

Abstract The bacterial cellulose (BC) secreted by Gluconacetobacter xylinus is a network of pure cellulose nanofibres which has high crystallinity, wettability and mechanical strength. These characteristics make BC an excellent material for tissue-engineering constructs, noteworthy for artificial vascular grafts. In this work, the in vivo biocompatibility of BC membranes produced by two G. xylinus strains was analyzed through histological analysis of long-term subcutaneous implants in the mice. The BC implants caused a mild and benign inflammatory reaction that decreased along time and did not elicit a foreign body reaction. A tendency to calcify over time, which may be related to the porosity of the BC implants, was observed, especially among the less porous BC-1 implants. In addition, the potential toxicity of BC nanofibres — obtained by chemical-mechanical treatment of BC membranes — subcutaneously implanted in mice was analysed through bone marrow flow cytometry and histological analyses. At 2 and 4 months post-implantation, the nanofibres implants were found to accumulate intracellularly, in subcutaneous foamy macrophages aggregates. Moreover, no differences were observed between the controls and implanted animals in thymocyte populations and in B lymphocyte precursors and myeloid cells in the bone marrow.

Sesquiterpene lactones: Adverse health effects and toxicity mechanisms

Abstract Sesquiterpene lactones (STLs) present a wide range of biological activities, mostly based on their alkylating capabilities, which underlie their therapeutic potential. These compounds are the active constituents of a variety of plants, frequently used as herbal remedies. STLs such as artemisinin and its derivatives are in use as first-line antimalarials while others, such as parthenolide, have recently reached cancer clinical trials. However, the toxicological profile of these compounds must be thoroughly characterized, since the same properties that make STL useful medicines can also cause severe toxicity. STL-containing plants have long been known to induce a contact dermatitis in exposed farm workers, and also to cause several toxic syndromes in farm animals. More recently, concerns are been raised regarding the genotoxic potential of these compounds and the embryotoxicity of artemisinins. A growing number of STLs are being reported to be mutagenic in different in vitro and in vivo assays. As yet no systematic studies have been published, but the genotoxicity of STLs seems to depend not so much on direct DNA alkylation as on oxidative DNA damage and other partially elucidated mechanisms. As the medicinal use of these compounds increases, further studies of their toxic potential are needed, especially those focusing on the structural determinants of genotoxicity and embryotoxicity.

CD117 immunoexpression in canine mast cell tumours: correlations with pathological variables and proliferation markers

BackgroundCutaneous mast cell tumours are one of the most common neoplasms in dogs and show a highly variable biologic behaviour. Several prognosis tools have been proposed for canine mast cell tumours, including histological grading and cell proliferation markers. CD117 is a receptor tyrosine kinase thought to play a key role in human and canine mast cell neoplasms. Normal (membrane-associated) and aberrant (cytoplasmic, focal or diffuse) CD117 immunoexpression patterns have been identified in canine mast cell tumours. Cytoplasmic CD117 expression has been found to correlate with higher histological grade and with a worsened post-surgical prognosis. This study addresses the role of CD117 in canine mast cell tumours by studying the correlations between CD117 immunoexpression patterns, two proliferation markers (Ki67 and AgNORs) histological grade, and several other pathological variables.ResultsHighly significant (p < 0,001) correlations were found between CD117 immunostaining patterns and histological grade, cell proliferation markers (Ki67, AgNORs) and tumoral necrosis. Highly significant (p < 0,001) correlations were also established between the two cellular proliferation markers and histological grade, tumour necrosis and epidermal ulceration. A significant correlation (p = 0.035) was observed between CD117 expression patterns and epidermal ulceration. No differences were observed between focal and diffuse cytoplasmic CD117 staining patterns concerning any of the variables studied.ConclusionThese findings highlight the key role of CD117 in the biopathology of canine MCTs and confirm the relationship between aberrant CD117 expression and increased cell proliferation and higher histological grade. Further studies are needed to unravel the cellular mechanisms underlying focal and diffuse cytoplasmic CD117 staining patterns, and their respective biopathologic relevance.

N-diethylnitrosamine mouse hepatotoxicity: time-related effects on histology and oxidative stress.

Animal models, namely mice, have been used to study chemically induced carcinogenesis due to their similarity to the histological and genetic features of human patients. Hepatocellular carcinoma (HCC) is a common malignancy with poor clinical outcome. The high incidence of HCC might be related to exposure to known risk factors, including carcinogenic compounds, such as N-nitrosamines, which cause DNA damage. N-nitrosamines affect cell mitochondrial metabolism, disturbing the balance between reactive oxygen species (ROS) and antioxidants, causing oxidative stress and DNA damage, potentially leading to carcinogenesis. This work addresses the progressive histological changes in the liver of N-diethylnitrosamine (DEN)-exposed mice and its correlation with oxidative stress. Male ICR mice were randomly divided into five DEN-exposed and five matched control groups. DEN was IP administered, once a week, for eight consecutive weeks. Samples were taken 18 h after the last DEN injection (8 weeks post-exposure). The following sampling occurred at weeks 15th, 22nd, 29th and 36th after the first DEN injection. DEN resulted in early toxic lesions and, from week 29 onwards, in progressive proliferative lesions. Between 15 and 29 weeks, DEN-exposed animals showed significant changes in hepatic antioxidant (glutathione, glutathione reductase, and catalase) status (p<0.05) compared with controls. These results point to an association between increased DEN-induced oxidative stress and the early histopathological alterations, suggesting that DEN disrupted the antioxidant defense mechanism, thereby triggering liver carcinogenesis.

Abnormal Protein Glycosylation and Activated PI3K/Akt/mTOR Pathway: Role in Bladder Cancer Prognosis and Targeted Therapeutics.

Muscle invasive bladder cancer (MIBC, stage ≥T2) is generally associated with poor prognosis, constituting the second most common cause of death among genitourinary tumours. Due to high molecular heterogeneity significant variations in the natural history and disease outcome have been observed. This has also delayed the introduction of personalized therapeutics, making advanced stage bladder cancer almost an orphan disease in terms of treatment. Altered protein glycosylation translated by the expression of the sialyl-Tn antigen (STn) and its precursor Tn as well as the activation of the PI3K/Akt/mTOR pathway are cancer-associated events that may hold potential for patient stratification and guided therapy. Therefore, a retrospective design, 96 bladder tumours of different stages (Ta, T1-T4) was screened for STn and phosphorylated forms of Akt (pAkt), mTOR (pmTOR), S6 (pS6) and PTEN, related with the activation of the PI3K/Akt/mTOR pathway. In our series the expression of Tn was residual and was not linked to stage or outcome, while STn was statically higher in MIBC when compared to non-muscle invasive tumours (p = 0.001) and associated decreased cancer-specific survival (log rank p = 0.024). Conversely, PI3K/Akt/mTOR pathway intermediates showed an equal distribution between non-muscle invasive bladder cancer (NMIBC) and MIBC and did not associate with cancer-specif survival (CSS) in any of these groups. However, the overexpression of pAKT, pmTOR and/or pS6 allowed discriminating STn-positive advanced stage bladder tumours facing worst CSS (p = 0.027). Furthermore, multivariate Cox regression analysis revealed that overexpression of PI3K/Akt/mTOR pathway proteins in STn+ MIBC was independently associated with approximately 6-fold risk of death by cancer (p = 0.039). Mice bearing advanced stage chemically-induced bladder tumours mimicking the histological and molecular nature of human tumours were then administrated with mTOR-pathway inhibitor sirolimus (rapamycin). This decreased the number of invasive lesions and, concomitantly, the expression of STn and also pS6, the downstream effector of the PI3K/Akt/mTOR pathway. In conclusion, STn was found to be marker of poor prognosis in bladder cancer and, in combination with PI3K/Akt/mTOR pathway evaluation, holds potential to improve the stratification of stage disease. Animal experiments suggest that mTOR pathway inhibition could be a potential therapeutic approach for this specific subtype of MIBC.

In vivo biocompatibility and biodegradability of dextrin-based hydrogels

The in vivo biocompatibility of dextrin hydrogels obtained by polymerization of dextrin-hydroxyethylmethacrylate (dextrin-HEMA) and dextrin-vinyl acrylate (dextrin-VA) are reported in this work. The histological analysis of subcutaneous implants of these hydrogels, featuring inflammatory and reabsorption events, were carried out over a 16-week period in mice. The dextrin-HEMA hydrogel was quickly and completely degraded and reabsorbed, whereas the dextrin-VA degradation occurred slowly and a thin fibrous capsule surrounded the nondegradable hydrogel. The dextrin-HEMA was degraded after 16 weeks with only mild inflammation and a few detectable foamy macrophages around the implant. These events were followed by complete resorption and no sign of capsule formation or fibrosis associated to the implants. The results indicate that the dextrin hydrogels are biocompatible because no toxicity on the tissues surrounding the implants was found. It may be speculated that a controlled degradation rate of the hydrogels may be obtained by grafting dextrin to HEMA and VA in different proportions.

A Role for MicroRNA-155 Expression in Microenvironment Associated to HPV-Induced Carcinogenesis in K14-HPV16 Transgenic Mice

Human Papillomavirus cause a number of diseases most notably cervical cancer. K14-HPV16 transgenic mice expressing the HPV16 early genes in squamous epithelial cells provide a suitable experimental model for studying these diseases. MicroRNAs are small non-coding RNAs that play an important role in regulating gene expression and have been suggested to play an important role in cancer development. The role of miR-155 in cancer remains controversial and there is limited evidence linking this miRNA to HPV- associated diseases. We hypothesized that miR-155 expression modulates each tissue’s susceptibility to develop HPV-associated carcinogenesis. In this study, we analyzed miR-155 expression in ear and chest skin samples from 22-26 weeks old, female K14-HPV16 transgenic (HPV16+/-) and wild-type (HPV-/-) mice. Among wild-type mice the expression of miR-155 was lower in ear skin compared with chest skin (p = 0.028). In transgenic animals, in situ carcinoma was present in all ear samples whereas chest tissues only showed epidermal hyperplasia. Furthermore, in hyperplastic chest skin samples, miR-155 expression was lower than in normal chest skin (p = 0,026). These results suggest that miR-155 expression may modulate the microenvironmental susceptibility to cancer development and that high miR155 levels may be protective against the carcinogenesis induced by HPV16.

Bovine papillomavirus: opening new trends for comparative pathology

For many years, research on bovine papillomavirus (BPV) has contributed to the understanding of papillomavirus-induced pathology in humans and animals. The present review shows how recent studies on BPV keep providing evidence concerning key points in viral infection, such as the expression of viral proteins in lymphocytes and the occurrence of productive infections of the placenta. Studies on BPV-induced tumours also provide important information concerning the mechanisms of oncogenesis and immune evasion, as in the cases of connexin 43 down-regulation with loss of intercellular gap junctions and Toll-like receptor 4 (TLR4) down-regulation in equine sarcoids. The biological functions of viral proteins are also being further clarified, as in the case of E2, which was recently shown to load BPV genomes into host chromosomes during the S phase, a process mediated by the ChlR1 protein. In the near future, the ongoing efforts to characterize and classify additional emerging BPV types are likely to broaden even further the possibilities for research.