Therapeutic administration of bone marrow-derived mesenchymal stromal cells reduces airway inflammation without up-regulating Tregs in experimental asthma.

BACKGROUND: Prophylactic administration of mesenchymal stromal cells (MSCs) derived from adipose (AD-MSC) and bone marrow tissue (BM-MSC) in ovalbumin-induced asthma hinders inflammation in a Treg-dependent manner. It is uncertain whether MSCs act through Tregs when inflammation is already established in asthma induced by a clinically relevant allergen. OBJECTIVE: Evaluate the effect of therapeutic administration of MSCs on inflammation and Treg cells in house dust mite (HDM)-induced asthma. METHODS: BM-MSCs and AD-MSCs were administered intratracheally to C57BL/6 mice 1 day after the last HDM challenge. Lung function, remodelling and parenchymal inflammation were assayed 3 or 7 days after MSCs treatment, through invasive plethysmography and histology, respectively. Bronchoalveolar lavage fluid (BALF) and mediastinal lymph nodes (mLNs) were assessed regarding the inflammatory profile by flow cytometry, ELISA and qRT-PCR. MSCs were studied regarding their potential to induce Treg cells from primed and unprimed lymphocytes in vitro. RESULTS: BM-MSCs, but not AD-MSCs, reduced lung influx of eosinophils and B cells and increased IL-10 levels in HDM-challenged mice. Neither BM-MSCs nor AD-MSCs reduced lung parenchymal inflammation, airway hyperresponsiveness or mucus hypersecretion. BM-MSCs and AD-MSCs did not up-regulate Treg cell counts within the airways and mLNs, but BM-MSCs decreased the pro-inflammatory profile of alveolar macrophages. Co-culture of BM-MSCs and AD-MSCs with allergen-stimulated lymphocytes reduced Treg cell counts in a cell-to-cell contact-independent manner, although co-culture of both MSCs with unprimed lymphocytes up-regulated Treg cell counts. CONCLUSIONS: MSCs therapeutically administered exert anti-inflammatory effects in the airway of HDM-challenged mice, but do not ameliorate lung function or remodelling. Although MSC pre-treatment can increase Treg cell numbers, it is highly unlikely that the MSCs will induce Treg cell expansion when lymphocytes are allergenically primed in an established lung inflammation.

Candidate predisposing germline copy number variants in early onset colorectal cancer patients.

PURPOSE: A great proportion of the heritability of colorectal cancer (CRC) still remains unexplained, and rare variants, as well as copy number changes, have been proposed as potential candidates to explain the so-called 'missing heritability'. We aimed to identify rare high-to-moderately penetrant copy number variants (CNVs) in patients suspected of having hereditary CRC due to an early onset. METHODS/PATIENTS: We have selected for genome-wide copy number analysis, 27 MMR-proficient early onset CRC patients (<50 years) without identifiable germline mutations in Mendelian genes related to this phenotype. Rare CNVs were selected by removing all CNVs detected at MAF >1% in the in-house control CNV database (n = 629 healthy controls). Copy number assignment was checked by duplex real-time quantitative PCR or multiplex ligation probe amplification. Somatic mutation analysis in candidate genes included: loss of heterozygosity studies, point mutation screening, and methylation status of the promoter. RESULTS: We have identified two rare germline deletions involving the AK3 and SLIT2 genes in two patients. The search for a second somatic mutational event in the corresponding CRC tumors showed loss of heterozygosity in AK3, and promoter hypermethylation in SLIT2. Both genes have been previously related to colorectal carcinogenesis. CONCLUSIONS: These findings suggest that AK3 and SLIT2 may be potential candidates involved in genetic susceptibility to CRC.

Tuberculosis in Anti-Tumour Necrosis Factor-treated Inflammatory Bowel Disease Patients After the Implementation of Preventive Measures: Compliance With Recommendations and Safety of Retreatment.

BACKGROUND AND AIMS: Despite having adopted preventive measures, tuberculosis (TB) may still occur in patients with inflammatory bowel disease (IBD) treated with anti-tumour necrosis factor (anti-TNF). Data on the causes and characteristics of TB cases in this scenario are lacking. Our aim was to describe the characteristics of TB in anti-TNF-treated IBD patients after the publication of the Spanish TB prevention guidelines in IBD patients and to evaluate the safety of restarting anti-TNF after a TB diagnosis. METHODS: In this multicentre, retrospective, descriptive study, TB cases from Spanish hospitals were collected. Continuous variables were reported as mean and standard deviation or median and interquartile range. Categorical variables were described as absolute and relative frequencies and their confidence intervals when necessary. RESULTS: We collected 50 TB cases in anti-TNF-treated IBD patients, 60% male, median age 37.3 years (interquartile range [IQR] 30.4-47). Median latency between anti-TNF initiation and first TB symptoms was 155.5 days (IQR 88-301); 34% of TB cases were disseminated and 26% extrapulmonary. In 30 patients (60%), TB cases developed despite compliance with recommended preventive measures; *not performing 2-step TST (tuberculin skin test) was the main failure in compliance with recommendations. In 17 patients (34%) anti-TNF was restarted after a median of 13 months (IQR 7.1-17.3) and there were no cases of TB reactivation. CONCLUSIONS: Tuberculosis could still occur in anti-TNF-treated IBD patients despite compliance with recommended preventive measures. A significant number of cases developed when these recommendations were not followed. Restarting anti-TNF treatment in these patients seems to be safe.

Regulation of cell death receptor S-nitrosylation and apoptotic signaling by Sorafenib in hepatoblastoma cells.

Nitric oxide (NO) plays a relevant role during cell death regulation in tumor cells. The overexpression of nitric oxide synthase type III (NOS-3) induces oxidative and nitrosative stress, p53 and cell death receptor expression and apoptosis in hepatoblastoma cells. S-nitrosylation of cell death receptor modulates apoptosis. Sorafenib is the unique recommended molecular-targeted drug for the treatment of patients with advanced hepatocellular carcinoma. The present study was addressed to elucidate the potential role of NO during Sorafenib-induced cell death in HepG2 cells. We determined the intra- and extracellular NO concentration, cell death receptor expression and their S-nitrosylation modifications, and apoptotic signaling in Sorafenib-treated HepG2 cells. The effect of NO donors on above parameters has also been determined. Sorafenib induced apoptosis in HepG2 cells. However, low concentration of the drug (10nM) increased cell death receptor expression, as well as caspase-8 and -9 activation, but without activation of downstream apoptotic markers. In contrast, Sorafenib (10 µM) reduced upstream apoptotic parameters but increased caspase-3 activation and DNA fragmentation in HepG2 cells. The shift of cell death signaling pathway was associated with a reduction of S-nitrosylation of cell death receptors in Sorafenib-treated cells. The administration of NO donors increased S-nitrosylation of cell death receptors and overall induction of cell death markers in control and Sorafenib-treated cells. In conclusion, Sorafenib induced alteration of cell death receptor S-nitrosylation status which may have a relevant repercussion on cell death signaling in hepatoblastoma cells.

Treatment Steps, Surgery, and Hospitalization Rates During the First Year of Follow-up in Patients with Inflammatory Bowel Diseases from the 2011 ECCO-Epicom Inception Cohort.

BACKGROUND AND AIMS: The ECCO-EpiCom study investigates the differences in the incidence and therapeutic management of inflammatory bowel diseases [IBD] between Eastern and Western Europe. The aim of this study was to analyse the differences in the disease phenotype, medical therapy, surgery, and hospitalization rates in the ECCO-EpiCom 2011 inception cohort during the first year after diagnosis. METHODS: Nine Western, five Eastern European centres and one Australian centre with 258 Crohn's disease [CD], 380 ulcerative colitis [UC] and 71 IBD unclassified [IBDU] patients [female/male: 326/383; mean age at diagnosis: 40.9 years, SD: 17.3 years] participated. Patients' data were registered and entered in the web-based ECCO-EpiCom database [www.epicom-ecco.eu]. RESULTS: In CD, 36 [19%] Western Europe/Australian and 6 [9%] Eastern European patients received biological therapy [p = 0.04], but the immunosuppressive [IS] use was equal and high in these regions [Eastern Europe vs Western Europe/Australia: 53% vs 45%; p = 0.27]. Surgery was performed in 17 [24%] CD patients in Eastern Europe and 13 [7%] in Western Europe/Australia [p < 0.001, pLogRank = 0.001]. Of CD patients from Eastern Europe, 24 [34%] were hospitalized, and 39 [21%] from Western Europe/Australia, [p = 0.02, pLogRank = 0.01]. In UC, exposure to biologicals and colectomy rates were low and hospitalization rates did not differ between these regions during the 1-year follow-up period [16% vs 16%; p = 0.93]. CONCLUSIONS: During the first year after diagnosis, surgery and hospitalization rates were significantly higher in CD patients in Eastern Europe compared with Western Europe/Australia, whereas significantly more CD patients were treated with biologicals in the Western Europe/Australian centres.

A genome-wide association study on copy-number variation identifies a 11q11 loss as a candidate susceptibility variant for colorectal cancer.

Colorectal cancer (CRC) is a complex disease, and therefore its development is determined by the combination of both environmental factors and genetic variants. Although genome-wide association studies (GWAS) of SNP variation have conveniently identified 20 genetic variants so far, a significant proportion of the observed heritability is yet to be explained. Common copy-number variants (CNVs) are one of the most important genomic sources of variability, and hence a potential source to explain part of this missing genetic fraction. Therefore, we have performed a GWAS on CNVs to explore the relationship between common structural variation and CRC development. Phase 1 of the GWAS consisted of 881 cases and 667 controls from a Spanish cohort. Copy-number status was validated by quantitative PCR for each of those common CNVs potentially associated with CRC in phase I. Subsequently, SNPs were chosen as proxies for the validated CNVs for phase II replication (1,342 Spanish cases and 1,874 Spanish controls). Four common CNVs were found to be associated with CRC and were further replicated in Phase II. Finally, we found that SNP rs1944682, tagging a 11q11 CNV, was nominally associated with CRC susceptibility (p value = 0.039; OR = 1.122). This locus has been previously related to extreme obesity phenotypes, which could suggest a relationship between body weight and CRC susceptibility.

The C-terminal fragment of parathyroid hormone-related peptide promotes bone formation in diabetic mice with low-turnover osteopaenia.

BACKGROUND AND PURPOSE: Current data suggest that parathyroid hormone (PTH)-related peptide (PTHrP) domains other than the N-terminal PTH-like domain contribute to its role as an endogenous bone anabolic factor. PTHrP-107-139 inhibits bone resorption, a fact which has precluded an unequivocal demonstration of its possible anabolic action in vivo. We thus sought to characterize the osteogenic effects of this peptide using a mouse model of diabetic low-turnover osteopaenia. EXPERIMENTAL APPROACH: PTHrP-107-139 was administered to streptozotocin-induced diabetic mice, with or without bone marrow ablation, for 13 days. Osteopaenia was confirmed by dual-energy X-ray absorptiometry and microcomputed tomography analysis. Histological analysis was performed on paraffin-embedded bone tissue sections by haematoxylin/eosin and Masson's staining, and tartrate-resistent acid phosphatase immunohistochemistry. Mouse bone marrow stromal cells and osteoblastic MC3T3-E1 cells were cultured in normal and/or high glucose (HG) medium. Osteogenic and adipogenic markers were assessed by real-time PCR, and PTHrP and the PTH(1) receptor protein expression by Western blot analysis. KEY RESULTS: PTHrP-107-139 reversed the alterations in bone structure and osteoblast function, and also promoted bone healing after marrow ablation without affecting the number of osteoclast-like cells in diabetic mice. This peptide also reversed the high-glucose-induced changes in osteogenic differentiation in both bone marrow stromal cells and the more differentiated MC3T3-E1 cells. CONCLUSIONS AND IMPLICATIONS: These findings demonstrate that PTHrP-107-139 promotes bone formation in diabetic mice. This mouse model and in vitro cell cultures allowed us to identify various anabolic effects of this peptide in this scenario.

Alterations of the Wnt/beta-catenin pathway and its target genes for the N- and C-terminal domains of parathyroid hormone-related protein in bone from diabetic mice.

Type 1 diabetes mellitus (T1D) is associated with bone loss. Given that the Wnt/beta-catenin pathway is a major regulator of bone accrual, we assessed this pathway in mice with streptozotozin-induced T1D. In diabetic mouse long bones, we found alterations favouring the suppression of this pathway by using PCR arrays and beta-catenin immunostaining. Downregulation of sclerostin, an inhibitor of this pathway, also occurred, and related to increased osteocyte apoptosis. Our data show that both N- and C-terminal parathyroid hormone-related peptide fragments might exert osteogenic effects in this setting by targeting several genes of this pathway and increasing beta-catenin in osteoblastic cells.

Amnésia retrógrada funcional grave: relato de caso / Severe functional retrograde amnesia: case report

CONTEXTO: Uma síndrome amnésica clássica caracteriza-se por evidente prejuízo da memória anterógrada, variável e temporária amnésia retrógrada, sendo as formas não-declarativas da memória poupadas. Entretanto, publicações recentes relataram casos de prejuízo desproporcional da memória retrógrada em relação à anterógrada. OBJETIVOS: Relatar o caso de um paciente de 26 anos de idade com um quadro grave de amnésia retrógrada, aparentemente sem fatores desencadeantes. MÉTODOS: Entrevista psiquiátrica e avaliação neuropsicológica. RESULTADOS: A perda de memória do paciente se estendia por toda sua vida, mas ele era capaz de adquirir e reter novas informações. Ele também apresentava prejuízos na produção e na compreensão de palavras, assim como no reconhecimento e no uso de objetos. CONCLUSÃO: A formulação diagnóstica final do caso é difícil, apontando possivelmente o contínuo existente entre a amnésia retrógrada psicogênica e a orgânica.

BACKGROUND: A classic amnestic syndrome is characterized by a significant impairment of the anterograde memory, a variable and transitory retrograde amnesia with preserved non-declarative memory. However, case reports of patients with disproportionate compromise of the retrograde memory have been described in the recent literature. OBJECTIVES: To report a 26-year-old patient with a severe global retrograde amnesia with no evident triggering factor. METHODS: Psychiatric interview and neuropsychological evaluation. RESULTS: His memory loss compromised all domains of his life, although he could acquire and retain new information. He also exhibited prominent deficits in production and comprehension of common words as well as in recognition and use of objects. DISCUSSION: The final diagnostic formulation of the present case is difficult possibly indicating a continuum between psychogenic and organic retrograde amnesia.

Diabetes mellitus: new therapeutic approaches to treat an old disease

Diabetes mellitus is a widespread disease whose frequency increases constantly and is expected to reach alarming levels by the year 2025. Introduction of insulin therapy represented a major breakthrough; however, a very strict regimen is required to maintain blood glucose levels within the normal range and to prevent or postpone chronic complications associated with this disease. Frequent hyper- and hypoglycemia seriously affect the quality of life of these patients. Reversion of this situation can only be achieved through whole organ (pancreas) transplant or pancreatic islet transplant, the former being a high-risk surgical procedure, while the latter is a much simpler and may be accomplished in only 20-40 min. The advantages and perspectives of islet cell transplantation will be discussed, in the light of tissue engineering and gene therapy. Ongoing research carried out in our laboratory, aimed at developing clinical cell and molecular therapy protocols for diabetes will also be focused

[Circumscribed acral hypokeratosis]. / Zirkumskripte akrale Hypokeratose.

Circumscribed acral hypokeratosis is a rare chronic disorder of cornification that occurs predominantly in women. Lesions are solitary and do not respond to any local conservative treatment. They have to be differentiated by biopsy from other non-healing lesions and tumors in acral skin. Clinically lesions appear as sharply circumscribed reddish macules. The histologic hallmark is a circumscribed loss of the entire stratum corneum, which can be best demonstrated at the border of the lesion as a contrast to the perilesional broad stratum corneum typical for acral sites.

DAPI binding to the DNA minor groove: a continuum solvent analysis.

A continuum solvent model based on the generalized Born (GB) or finite-difference Poisson-Boltzmann (FDPB) approaches has been employed to compare the binding of 4'-6-diamidine-2-phenyl indole (DAPI) to the minor groove of various DNA sequences. Qualitative agreement between the results of GB and FDPB approaches as well as between calculated and experimentally observed trends regarding the sequence specificity of DAPI binding to B-DNA was obtained. Calculated binding energies were decomposed into various contributions to solvation and DNA-ligand interaction. DNA conformational adaptation was found to make a favorable contribution to the calculated total interaction energy but did not change the DAPI binding affinity ranking of different DNA sequences. The calculations indicate that closed complex formation is mainly driven by nonpolar contributions and was found to be disfavored electrostatically due to a desolvation penalty that outbalances the attractive Coulomb interaction. The calculated penalty was larger for DAPI binding to GC-rich sequences compared with AT-rich target sequences and generally larger for the FDPB vs the GB continuum model. A radial interaction profile for DAPI at different distances from the DNA minor groove revealed an electrostatic energy minimum a few Angstroms farther away from the closed binding geometry. The calculated electrostatic interaction up to this distance is attractive and it may stabilize a nonspecific binding arrangement.

Low frequency of mutations in the core promoter and precore regions of hepatitis B virus in anti-HBe positive Brazilian carriers.

BACKGROUND: Mutations in the core promoter and precore regions of the hepatitis B virus (HBV) genome, notably the double substitution (AGG to TGA) at nt positions 1762-1764 in the core promoter, and the precore stop codon mutation G to A at nt 1896, can often explain the anti-HBe phenotype in chronic carriers. However, the A1896 mutation is restricted to HBV isolates that have T at nt 1858. The double substitution at positions 1762-1764 has been described to occur preferentially in patients infected with strains showing C instead of T at nt 1858. RESULTS: HBV DNAs from 29 anti-HBe Brazilian samples were characterized by nucleotide sequencing of PCR products from precore region. Among them, 18 isolates presented C at nt 1858 (mostly genotype A strains). The 11 remaining isolates (genotypes D and F) had T1858. The stop codon mutation at nt 1896 was found in seven isolates (24% of the total and 63% of the isolates that had T1858). The frequency of the double substitution at positions 1762-1764 was surprisingly low (20%) among C1858 isolates. An association between A1896 and TGA 1762-1764 mutations was observed among genotype D isolates: these showed either none of the two mutations or both. Furthermore, strains mutated at positions 1896 and/or 1762-1764 also presented an elevated number of other, less common substitutions in the core promoter and precore regions. CONCLUSIONS: The data reported here are not in accordance with some reports from other parts of the world. In half of the isolates, none of the mutations previously described could explain the anti-HBe phenotype.

[Trans-sphenoidal meningoencephaloventriculocele in an asymptomatic adult: case report]. / Meningoencefaloventriculocele transesfenoidal assintomática em adulto: relato de um caso.

We report a casual finding of a sphenoethmoidal meningoencephaloventriculocele in an adult patient that the tomography finding was misinterpreted as an expansive neoplastic lesion with malignant features. We alert for the rare site of localization of this malformative pathology.

Recognition of melanoma cell antigens with antibodies present in sera from patients with vitiligo.

BACKGROUND: Patients with vitiligo show specific losses of integumentary melanocytes, probably due to autoimmunity against melanocytes. We attempted to determine the presence of antibodies against pigment cell antigens in the sera of vitiligo patients. METHODS: Detergent-solubilized human melanoma cells were submitted to electrophoretic separation and immunoblotted against serum samples obtained from 19 patients with vitiligo and from 20 age- and sex-matched healthy individuals. RESULTS: Eighty-nine per cent of patients with vitiligo had antibodies to one or more pigment cell antigens. Similar antibodies were detected in 20% of healthy individuals. Antigens of 165, 90, and 68 kDa were recognized by the antibodies present in sera from 11%, 26%, and 37% of vitiligo patients, respectively, and in none of the normal sera. All patients with familial vitiligo also had antibodies to these three proteins. CONCLUSIONS: Proteins of 165, 90, and 68 kDa are specifically recognized by antibodies present in the sera of vitiligo patients and in all patients with genetic vitiligo. Whether or not these proteins might be implicated in the destruction of melanocytes by the immune system in vitiligo remains to be evaluated.