Characterization of pediatric microtia cartilage: a reservoir of chondrocytes for auricular reconstruction using tissue engineering strategies.

The external ear is composed of elastic cartilage. Microtia is a congenital malformation of the external ear that involves a small reduction in size or a complete absence. The aim of tissue engineering is to regenerate tissues and organs clinically implantable based on the utilization of cells and biomaterials. Remnants from microtia represent a source of cells for auricular reconstruction using tissue engineering. To examine the macromolecular architecture of microtia cartilage and behavior of chondrocytes, in order to enrich the knowledge of this type of cartilage as a cell reservoir. Auricular cartilage remnants were obtained from pediatric patients with microtia undergoing reconstructive procedures. Extracellular matrix composition was characterized using immunofluorescence and histological staining methods. Chondrocytes were isolated and expanded in vitro using a mechanical-enzymatic protocol. Chondrocyte phenotype was analyzed using qualitative PCR. Microtia cartilage preserves structural organization similar to healthy elastic cartilage. Extracellular matrix is composed of typical cartilage proteins such as type II collagen, elastin and proteoglycans. Chondrocytes displayed morphological features similar to chondrocytes derived from healthy cartilage, expressing SOX9, COL2 and ELN, thus preserving chondral phenotype. Cell viability was 94.6 % during in vitro expansion. Elastic cartilage from microtia has similar characteristics, both architectural and biochemical to healthy cartilage. We confirmed the suitability of microtia remnant as a reservoir of chondrocytes with potential to be expanded in vitro, maintaining phenotypical features and viability. Microtia remnants are an accessible source of autologous cells for auricular reconstruction using tissue engineering strategies.

Treatment outcome with mTOR inhibitors for metastatic renal cell carcinoma with nonclear and sarcomatoid histologies.

BACKGROUND: The clinical trials that reported benefit of the rapalogs temsirolimus and everolimus in advanced renal cell carcinoma (RCC) were primarily conducted in patients with clear-cell histology (ccRCC). We assessed outcome with these mammalian target of rapamicin (mTOR) inhibitors in two subsets of kidney cancer: sarcomatoid variant ccRCC and nonclear-cell RCC. PATIENTS AND METHODS: Baseline clinical features, information on prior treatment, and histologic subtypes were collected for patients previously treated with rapalogs for metastatic RCC of either nonclear phenotype or ccRCC with sarcomatoid features. Outcome was assessed centrally by a dedicated research radiologist for determination of tumor response, progression-free survival (PFS), and overall survival (OS). RESULTS: Eighty-five patients received temsirolimus (n = 59) or everolimus (n = 26). Nonclear-cell phenotypes included papillary (n = 14), chromophobe (n = 9), collecting duct (n = 4), translocation-associated (n = 3), and unclassified (n = 32) RCC. Twenty-three patients had clear-cell histology with sarcomatoid features. The response rate in assessable patients (n = 82) was 7% (all partial responses); 49% of patients achieved stable disease, and 44% had progressive disease as their best response. Tumor shrinkage was observed in 26 patients (32%). Median PFS and OS were 2.9 and 8.7 months, respectively. Nine patients (11%) were treated for ≥1 year, including cases of papillary (n = 3), chromophobe (n = 2), unclassified (n = 3) RCC, and ccRCC with sarcomatoid features (n = 1). No tumor shrinkages were observed for patients with collecting duct or translocation-associated RCC. CONCLUSIONS: A subset of patients with nonclear-cell and sarcomatoid variant ccRCC subtypes benefit from mTOR inhibitors, but most have poor outcome. Histologic subtype does not appear to be helpful in selecting patients for rapalog therapy. Future efforts should include the identification of predictive tissue biomarkers.

Gitelman's variant of Bartter's syndrome, inherited hypokalaemic alkalosis, is caused by mutations in the thiazide-sensitive Na-Cl cotransporter.

Maintenance of fluid and electrolyte homeostasis is critical for normal neuromuscular function. Bartter's syndrome is an autosomal recessive disease characterized by diverse abnormalities in electrolyte homeostasis including hypokalaemic metabolic alkalosis; Gitelman's syndrome represents the predominant subset of Bartter's patients having hypomagnesemia and hypocalciuria. We now demonstrate complete linkage of Gitelman's syndrome to the locus encoding the renal thiazide-sensitive Na-Cl cotransporter, and identify a wide variety of non-conservative mutations, consistent with loss of function alleles, in affected subjects. These findings demonstrate the molecular basis of Gitelman's syndrome. We speculate that these mutant alleles lead to reduced sodium chloride reabsorption in the more common heterozygotes, potentially protecting against development of hypertension.

Results of the oral egg-challenge test performed on two different groups of children. One group with a history, suggestive of allergic reaction with egg intake and the other group sensitised to hen's egg without previous egg intake.

INTRODUCTION: Egg allergy is an adverse immune-system reaction of an IgE-mediated type, which can happen in children after egg intake and several times after their first egg intake. OBJECTIVES: Compare the results of the oral egg-challenge test in two groups of egg-sensitised children, with and without prior intake. PATIENTS AND METHODS: Retrospective study of two egg-sensitised groups (72 subjects). Group 1: 22 children without prior egg-intake. Group 2: 50 children with a clinical history of adverse reactions after egg intake. Skin prick tests, egg-white specific IgE (sIgE) and yolk specific IgE, were performed on all children. The oral egg-challenge tests were performed after a period of egg-avoidance diet and when egg-white specific IgE levels were lower than 1.5K U/L. RESULTS: 31.8% of the children in Group 1 did not tolerate egg-intake whereas 38% of the children in Group 2 did not tolerate egg-intake. Egg-avoidance periods lasted 19.5 and 18 months, respectively. Egg-white specific IgE levels went down in both groups after an egg-avoidance diet. No statistically significant differences were found between the groups and the positivity of oral egg-challenge test. CONCLUSIONS: No statistically significant differences were found in the behaviour of the two groups studied. Given the high risk of adverse reactions, it was recommended that any egg-introduction tests were to be performed in a hospital environment on the children who were sensitised to hen's egg (including children without prior egg intake).

[Nelson syndrome: a rare cause of generalized hyperpigmentation of the skin]. / Síndrome de Nelson: una causa infrecuente de hiperpigmentación cutánea generalizada.

Nelson syndrome is a rare cause of generalized mucocutaneous hyperpigmentation. Its clinical manifestations are due to excessive secretion of adrenocorticotropic hormone from a pituitary adenoma, which develops after bilateral therapeutic adrenalectomy. As this operation has fallen into disuse, Nelson syndrome is now extremely rare and difficult to recognize. We present a very severe case of generalized hyperpigmentation due to Nelson syndrome in a 37-year-old woman.

Quantitative study of Leydig cell populations in mice exposed to low doses of cadmium.

Leydig cell morphological changes were evaluated using morphometric and stereological methods in male mice exposed to low doses of cadmium. A possible reversibility of the changes after cadmium withdrawal was also considered. Nuclear morphological parameters and stereological densities of the Leydig cell population were lower in the cadmium-exposed groups than in the control. The withdrawal of cadmium did not lead to any significant recovery of the morphological parameters. Nevertheless, numerical density increased significantly in the withdrawn groups, suggesting that the hyperplasia of interstitial cells could try to relieve morphological damage after cadmium withdrawal.

Spontaneous trichoepithelioma in a laboratory mouse: gross, microscopic and immunohistochemical findings.

A spontaneous trichoepithelioma occurred in a Swiss OF1 outbred, four-month-old, intact, nulliparous female mouse from a breeding colony. At necropsy, the tumour was a single, well-delineated mass measuring 4.2 cm in major diameter, located in the thoracic region and had an intact haired surface. The regional lymph nodes were not enlarged and no other abnormalities were found. Microscopically, it was composed of a random admixture of budding epithelial islands and cystic structures variable in size. The epithelial islands were composed of basaloid cells. The cystic structures were lined by squamous epithelium with or without a granular cell layer and contained lamellar or amorphous keratin, as well as wide areas of matrical keratinization (ghost cells) with or without a peripheral layer of basaloid cells and calcified contents. Mitotic activity of basaloid cells was moderate to high, but nuclear or mitotic atypia were not observed. High and low molecular weight cytokeratins, profilaggrin and involucrin expression were observed in the tumour. The immunohistochemical profile of this rare type of tumour of the skin of mice, which includes a first-time description of involucrin expression, confirms the histological evidence of differentiation towards more than one segment of follicular epithelium.

Smart Release Nano-formulation of Cytochrome C and Hyaluronic Acid Induces Apoptosis in Cancer Cells.

Herein we tested a nanosized cancer-cell targeted delivery system based on cytochrome c (Cyt c) and hyaluronic acid. Cyt c was chosen since it is a per se non-toxic protein but causes apoptosis when delivered to the cytoplasm of target cells. Hyaluronic acid was employed to create the nanosized delivery system with passive targeting capability in order to exploit the enhanced permeation and retention (EPR) effect and active targeting capability of hyaluronic acid. In addition, our goal was to incorporate a smart release strategy to only promote protein release upon reaching its target. Nanoparticles were formed by a simple yet precise nanoprecipitation process based on desolvation. They were physically characterized to select precipitation conditions leading to adequate size, shape, protein bioactivity, and protein loading to produce a feasible targeted cancer treatment. We synthesized nanoparticles of around 500 nm diameter with a 60% protein loading and more than 80% of protein bioactivity. In vitro, cumulative release of 92% of Cyt c was observed after 8 h under conditions mimicking the reductive intracellular environment, while under non-denaturing conditions only 20% was released. The nanoparticles displayed a selective cytotoxic effect on cancer cells. After 6 h of incubation with the nanoparticles, hyaluronic acid receptor over expressing A549 human lung adenocarcinoma cells showed a viability of ca. 20% at 0.16 mg/ml of Cyt c concentration. Only a negligible effect was observed on viability of COS-7 African green monkey kidney fibroblast, a normal cell line notoverexpressing the hyaluronic acid receptor. Confocal microscopy confirmed that the drug delivery system indeed delivered Cyt c to the cytoplasm of the target cells. We conclude that we were able to create a smart stimuli-responsive targeted drug delivery system with significant potential in cancer therapy.

Cytotoxic chemotherapy in the treatment of advanced renal cell carcinoma in the era of targeted therapy.

BACKGROUND: Renal cell carcinoma (RCC) is a heterogeneous disease with regards to histology, progression, and response to treatment. Cytotoxic chemotherapy has been extensively studied in metastatic RCC (mRCC). Responses in most studies are modest and the mechanisms of resistance remain poorly understood. Targeted therapies have significantly improved outcomes in mRCC; however, most patients eventually relapse and die of their disease. Early clinical data suggest that combinations of chemotherapy and targeted agents are clinically active and are well tolerated. METHODS: We reviewed the available literature for published clinical trials incorporating traditional chemotherapeutic agents in the treatment of mRCC. These papers were identified through a Medline search and were included if they employed at least one chemotherapeutic agent in the treatment of mRCC. The literature was also reviewed for information regarding mechanisms of chemotherapy resistance. RESULTS: The data regarding the use of cytotoxic chemotherapy in mRCC consist of small, non-randomized phase I and II studies. The major response proportions with single agent chemotherapies are low but combination regimens either with other cytotoxic agents, cytokines, or targeted agents have demonstrated moderate activity. Disparate trial designs and lack of head to head clinical trials make it difficult to compare the efficacy of chemotherapy with that of immunotherapy or targeted agents. Chemotherapy is particularly useful in patients with collecting duct histology and predominantly sarcomatoid differentiation. Chemotherapy resistance may be mediated by overexpression of p-glycoprotein efflux pumps and the dysregulation of the microtubule-hypoxia inducible factor signaling axis. CONCLUSIONS: The role of cytotoxic chemotherapy in the treatment for clear cell RCC remains poorly defined. Cytotoxic chemotherapy is considered a standard of care in patients with mRCC with predominantly sarcomatoid differentiation and collecting duct RCC variants (Motzer et al., 2014). Early trials combining chemotherapy with targeted therapies are generally well tolerated and show clinical activity. A better understanding of the biology of aggressive subsets of RCC and mechanisms of resistance will help elucidate the role of cytotoxic agents in the current treatment paradigm of RCC.

Sunitinib objective response in metastatic renal cell carcinoma: analysis of 1059 patients treated on clinical trials.

BACKGROUND: Retrospective analyses were performed in patients with metastatic renal cell carcinoma (mRCC) to characterise the objective response (OR) rate to sunitinib and differentiate pretreatment features and outcomes of patients with early (response by ≤ 12 weeks) versus late response, and responders versus non-responders. METHODS: Data were pooled from 1059 patients in six trials. Median progression-free survival (PFS) and overall survival (OS) were estimated by Brookmeyer and Crowley method and compared between groups by log-rank test. Baseline characteristics were compared by Fisher-exact, t-, or Wilcoxon rank-sum tests. Associations between characteristics and survival were investigated by Cox proportional regression analysis. RESULTS: 398 patients (38%) had confirmed OR (12 complete responses); 26%, 61%, 79% and 86% responded by 6, 12, 18 and 24 weeks, respectively. Median (range) time to tumour response (TTR) was 10.6 (2.7-94.4) weeks and was similar in treatment-naïve and cytokine-refractory patients. Median response duration in early and late responders was 52.0 and 55.0 weeks, respectively. Median PFS in early versus late responders was 13.8 versus 20.2 months (P=0.001); however, median OS did not significantly differ (37.8 versus 40.8 months; P=0.144). Early responders had more lung metastases (P<0.01), but baseline characteristics were otherwise mostly similar. Median PFS (16.3 versus 5.3 months) and OS (40.1 versus 14.5 months) were longer in responders versus non-responders (both P<0.001); responders had more favourable prognostic factors. CONCLUSIONS: OR occurred in 38% of sunitinib-treated mRCC patients. Sixty-one percent of responses occurred by 12 weeks of therapy, and responders had favourable pretreatment features and significantly longer survival.

Neutrophil chemokines secreted by tumor cells mount a lung antimetastatic response during renal cell carcinoma progression.

The mechanism by which renal cell carcinoma (RCC) colonizes the lung microenvironment during metastasis remains largely unknown. To investigate this process, we grafted human RCC cells with varying lung metastatic potential in mice. Gene expression profiling of the mouse lung stromal compartment revealed a signature enriched for neutrophil-specific functions that was induced preferentially by poorly metastatic cells. Analysis of the gene expression signatures of tumor cell lines showed an inverse correlation between metastatic activity and the levels of a number of chemokines, including CXCL5 and IL8. Enforced depletion of CXCL5 and IL8 in these cell lines enabled us to establish a functional link between lung neutrophil infiltration, secretion of chemokines by cancer cells and metastatic activity. We further show that human neutrophils display a higher cytotoxic activity against poorly metastatic cells compared with highly metastatic cells. Together, these results support a model in which neutrophils recruited to the lung by tumor-secreted chemokines build an antimetastatic barrier with loss of neutrophil chemokines in tumor cells acting as a critical rate-limiting step during lung metastatic seeding.

Pelvic floor electrostimulation in women with urinary incontinence and/or overactive bladder syndrome: a systematic review.

CONTEXT: Electrostimulation (ES) is one of the techniques employed in conservative treatment of urinary incontinence (UI) and/or overactive bladder syndrome (OAB). Nevertheless, there is controversy in the scientific literature regarding its effectiveness as monotherapy. OBJECTIVE: To evaluate the scientific evidence on ES of the pelvic floor in women with UI and with/without OAB. EVIDENCE ACQUISITION: A systematic review of clinical trials was carried out in the following databases: PubMed, Cochrane, PEDro, Elsevier (Doyma) and EnFisPo (1980-2011). Quality of study registries was evaluated and information was obtained from those that presented the inclusion criteria established in the review. EVIDENCE SYNTHESIS: The 27 clinical trials were included in the review: 13 randomized controlled trials, 11 randomized non-controlled trials and 3 non-randomized trials. CONCLUSION: Most of the clinical trials conclude that ES is effective in the treatment of UI and OAB in women. However, better methodological quality studies are needed to obtain a higher level of scientific evidence and to know the optimal current modality, type and parameters for each type of UI and OAB.

Endogenous lipid pneumonia in rats after subacute exposure to CdCl2.

The objective of this report was to study lung cellular lesions in Wistar rats after subacute oral exposition to CdCl(2). The experimental groups were exposed to CdCl(2), through their drinking water in a concentration of 1 g/L, continuously for a period of 9 days. Histologically, all the exposed animals showed the incidence of interstitial pneumonia; hyperplasia of type II pneumocytes and Clara cells; the presence of foamy macrophages; and lesions linked to the existence of endogenous lipid pneumonia. Endogenous lipid pneumonia after CdCl(2) exposure has not been previously described; and in its pathogenesis, hyperplasia of type II pneumocytes and Clara cells activation could play an important role.

Echium vulgare and Senecio vulgaris poisoning in fighting bulls.

An unusual case of poisoning by simultaneous ingestion of Echium vulgare L. and Senecio vulgaris L. in a herd of Spanish fighting bulls is described. Ten animals died from a herd of 700 in an area located in Sierra Norte, Seville (Constantina) in Spain. The interest of this case lies both in the breed affected (this is the first report on fighting bulls) and the lack of information about bovine poisoning by these plants in Spain. Animal samples were obtained from October to March. All the dead animals were 1 year old and had grazed at the farm. The diagnosis was made by determining the plant species and studying its distribution in the pastureland, and also by performing blood analysis of the sick animals in addition to an anatomopathological study of the carcasses. Tuberculosis, brucellosis, salmonellosis, IBR/BVD and also the presence of aflatoxins in the forage were all ruled out.

Enhanced sensitivity to the photodynamic effect of 3,4-benzpyrene in ethylenediaminetetraacetate-treated Escherichia coli.

Ethylenediaminetetraacetate treatment of Escherichia coli B enhanced cell sensitivity to the lethal effect of the photodynamic action of 3,4-benzpyrene. The alteration of the shape of the survival curve suggests that such photodynamic action may be modified by membrane barriers to the drug.

Transformation as a tool for studying the epidemiology of tet determinants in Streptococcus pneumoniae.

Transformation of pneumococcus was used to detect homology among tetracycline resistance determinants of clinical isolates of Streptococcus pneumoniae. A strain of pneumococcus containing a mutated tet determinant (tet-3), of class M, integrated into the chromosome was used as a recipient in transformation experiments, where donor DNA was from the tetracycline resistant isolates. 34/34 strains appeared to have tet determinants homologous to tet-3 (i.e. tet M). Still using transformation it was possible to determine that the tet-3 transforming activity of DNA from Tn916 and S. pneumoniae BM6001 was contained in a 5 kb HincII fragment. For this purpose a transformation technique where donor DNA was directly taken from low melting point agarose gels was standardized and used.

Photodynamic effect of 3,4-benzpyrene on thermo-sensitive mutants of Escherichia coli B.

Results obtained with thermo-sensitive mutants of Escherichia coli B have shown that the presence of a protein with impaired structure may be responsible for enhanced sensitivity to the lethal photodynamic effect of 3,4-benzpyrene. Thus, the evidence has been obtained of the in vivo action of excited 3,4-benzpyrene on proteins.

[Atopic dermatitis. Allergological characteristics and association with respiratory disease]. / Dermatitis atópica: características alergológicas y asociación a patología respiratoria.

BACKGROUND: Atopic dermatitis (AD) is a chronic inflammatory skin disease that frequently precedes the development of asthma or other respiratory allergies. The aim of this study was to review allergen sensitization, type of feeding in infancy, and development of asthma or rhinitis in a group of patients with AD. METHODOLOGY: One hundred children with AD were selected. All patients underwent skin prick and patch tests to foodstuffs and inhalant allergens, total and specific IgE determination, and oral challenge tests. RESULTS: The study included 57 boys and 43 girls. The mean age at consultation was 3.77 +/- 2.81 years and mean age at onset of of AD was 1.09 +/- 1.69 years. Twenty-eight percent of the children were exclusively sensitized to food allergens, 20% to inhalant allergens and 22% to both food and inhalant allergens. Mean serum IgE levels were higher in children sensitized to Dermatophagoides pteronyssinus (DPT) (346.86 +/- 430.43 U/ml) than in non-sensitized children (78.24 +/- 132.93 U/m) (p < 0.001). Total IgE levels were also higher in patients with respiratory symptoms (283.77 +/- 336.53 U/ml) than in children without respiratory disease (124.62 +/- 285.21 U/ml) (p = 0.021). Thirty-five percent of the children developed some kind of respiratory allergic disease (asthma and/or rhinitis) in a mean interval of 2.55 years after the onset of dermatitis. Of the children sensitized to inhalant allergens (DPT), 55.26 % developed respiratory symptoms compared with 22.58 % of the non-sensitized children (p < 0.001). The odds ratio of developing respiratory allergy if the patient showed sensitization to DPT was 4.235 (95 % CI 1.768-0.147, p = 0.002). CONCLUSIONS: Children with AD that develops in the first year of life, associated with high IgE levels and early sensitization, independently of the kind of feeding, develop respiratory allergic disease more frequently than children without these factors.

Properties of an E. coli O26 mutant with modified cell wall and improved efficiency as recipient in conjugation for an FII plasmid.

Among populations of an E. coli O26 strain two types of mutants have been found to be present which act as better recipients in conjugation for an FII plasmid. Some properties of one of these mutants, O26SMB9, are here described. They indicate that a defect in the cell-wall structure has occurred which corresponds to a smooth----semirough transition.