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.

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.

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.

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.

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.

Electroestimulación del suelo pélvico en mujeres con incontinencia urinaria y/o síndrome de vejiga hiperactiva: una revisión sistemática / Pelvic Floor Electrostimulation in Women With Urinary Incontinence and/or Overactive Bladder Syndrome: A Systematic Review

Contexto: La electroestimulación (EE) es una de las técnicas empleadas en el tratamiento conservador de la incontinencia urinaria (IU) y/o síndrome de vejiga hiperactiva (SVH). Sin embargo, existe controversia en la literatura científica acerca de su eficacia como monoterapia. Objetivo: Evaluar la evidencia científica sobre la EE del suelo pélvico en mujeres con IU y/o SVH. Adquisición de evidencia: Se realizó una revisión sistemática de ensayos clínicos en las bases de datos PubMed, Cochrane, PEDro, Elsevier (Doyma) y EnFisPo (1980-2011). Se evaluó la calidad de los estudios y se extrajo la información de los que reunieron los criterios de inclusión establecidos. Síntesis de evidencia: Un total de 27 ensayos clínicos han sido incluidos en la revisión: 13 ensayos controlados aleatorizados, 11 ensayos aleatorizados no controlados y 3 ensayos no aleatorizados. Conclusión: La mayor parte de los ensayos clínicos concluyen que la EE es eficaz en el tratamiento de la IU y el SVH en mujeres. Sin embargo, son necesarios más estudios de buena calidad metodológica para obtener un mayor nivel de evidencia científica y conocer cuál es la modalidad, tipo y parámetros de corriente óptimas para cada tipo de IU y el SVH (AU)

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 (AU)

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.

Modificación de biomarcadores histopatológicos e inmunohistoquímicos en las fibras musculares de cerdos tratados con clenbuterol / Modification of his topathological and immunohistochemical biomarkers in the muscle fibers of swine after clenbuterol exposure

El clenbuterol frecuentemente se ha utilizado de forma ilícita en el engorde del ganado, suponiendo ello un riesgo para la salud pública. La actual normativa prohíbe por tanto su uso en la cría del ganado. En este estudio se utilizaron 14 cerdos distribuidos al azar en dos grupos de estudio (n=7), uno control, y otro expuesto a 1 mg/kg de clenbuterol durante tres meses. Se utilizó el músculo longissimus lumbaris para la determinación de la concentración de clenbuterol, así como para la evaluación de los parámetros inmunohistoquímicos y el análisis estructural y ultraestructural de las fibras. El estudio de los distintos biomarcadores histopatológicos e inmunohistoquímicos de las fibras musculares, tras la exposición de los animales al clenbuterol, indicó la presencia de hipertrofia muscular, miofibrolisis y degeneración zenkeriana, observada tanto por microscopía óptica como electrónica. Mediante la técnica miosina ATP-asa se identificó un tipo de fibra denominada alterada en el grupo tratado que no apareció en el grupo control. Por tanto, se comprueba que el músculo longissimus lumbaris resulta una buena matriz para la investigación de la exposición a largo plazo a este compuesto en cerdos (AU)

Clenbuterol has been frequently used illicitly, causing a risk to public health; the current regulation prohibits its use to put on weight the animals. In our study we have used 14 swines radomly distributed into two groups (n=7): one control group, and another group exposed to 1 mg/kg of clenbuterol during three months. The muscle selected for the research was longissimus lumbaris and it was used for the determination of the concentration of clenbuterol, as well as for the evaluation of the immunohistochemical parameters, and the structural and ultrastructural analysis of the fibers. The study of the different histopathological and immunohistochemical biomarkers of the muscular fibers, after clenbuterol exposure, indicated the presence of muscular hypertrophy, myolysis, and zenkerian degeneration, observed at optic and electronic microscopy. By the use of the miosin ATP-ase technique, it was identified a fiber type denominated altered in the treated group that did not appeared in the control group. Therefore, it was verified that the muscle longissimus lumbaris is a good matrix for the investigation of the long-term exposure to this clenbuterol in swines (AU)

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.

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.

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.

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.

Genetic organization of the catabolic plasmid pJP4 from Ralstonia eutropha JMP134 (pJP4) reveals mechanisms of adaptation to chloroaromatic pollutants and evolution of specialized chloroaromatic degradation pathways.

Ralstonia eutropha JMP134 (pJP4) is a useful model for the study of bacterial degradation of substituted aromatic pollutants. Several key degrading capabilities, encoded by tfd genes, are located in the 88 kb, self-transmissible, IncP-1 beta plasmid pJP4. The complete sequence of the 87,688 nucleotides of pJP4, encoding 83 open reading frames (ORFs), is reported. Most of the coding sequence corresponds to a well-conserved IncP-1 beta backbone and the previously reported tfd genes. In addition, we found hypothetical proteins putatively involved in the transport of aromatic compounds and short-chain fatty acid oxidation. ORFs related to mobile elements, including the Tn501-encoded mercury resistance determinants, an IS1071-based composite transposon and a cryptic class II transposon, are also present in pJP4. These mobile elements are inefficient in transposition and are located in two regions of pJP4 that are rich in remnants of lateral gene transfer events. pJP4 plasmid was able to capture chromosomal genes and form hybrid plasmids with the IncP-1 alpha plasmid RP4. These observations are integrated into a model for the evolution of pJP4, which reveals mechanisms of bacterial adaptation to degrade pollutants.

[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.

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.

Prevalence of Streptococcus mutans and dental decay in schoolchildren living in Genoa (Italy).

In this study population (105 schoolchildren aged 5.5-11.5 yrs), dental decay was detected in 75.2% and S. mutans in 55.2% of the subjects. The presence of S. mutans was assessed - using the selective GSTB medium - in unstimulated saliva and in pooled occlusal and pooled buccal plaques from the four most posterior teeth. All three samples showed association between S. mutans presence and caries prevalence. Of the two types of plaque, the occlusal not only had a higher frequency of isolation but also a significantly higher proportion of S. mutans. The presence of S. mutans was significantly associated with both caries prevalence and extent of caries experience. Both S. mutans prevalence and S. mutans proportion in plaque increased with the number of decayed teeth present among those sampled. Sucrose consumption between meals appeared to be more correlated with the degree of caries experience rather than with caries or S. mutans prevalence. A second clinical examination was scheduled six months after the first for S. mutans-positive children who either were free of active carious lesions, or were caries-active but without signs of dental decay in the sampled teeth. Caries-active subjects proved to be more prone to new carious lesions than caries-free subjects, who tended to remain caries-free even when they had a high proportion of S. mutans in plaque, thus indicating the basic importance of the host factor in the caries process.