Analysis of CTNS gene transcripts in nephropathic cystinosis

Nephropathic cystinosis (NC) is an autosomal recessive disorder caused by mutations of the CTNS gene that encodes for a cystine transmembrane transporter. Several mutations have been described in the coding and promoter regions of the CTNS gene in affected individuals. We selected three patients with NC from two unrelated families, in whom sequence analysis of the CTNS gene detected only one or no mutations. Total RNA was isolated from peripheral blood mononuclear cells or fibroblasts and CTNS transcripts were analyzed. We observed a skipping of exon 5 (85 bp) in two siblings and an intron 9 retention of 75 bp associated with partial replication of exon 9 in the third patient. Genomic DNA analysis of intron regions surrounding exon 5 showed a point mutation in the hypothetical lariat branch site of intron 4 at position –24 (c.141–24 T > C) in the first two patients and a duplication of 266 bp including a part of exon and intron 9 in the third patient. Analysis of CTNS gene transcripts allowed identification of mutations in patients in whom CTNS mutations could not be detected by traditional DNA sequencing. These results support the hypothesis that cystinosis is a monogenic disorder

Microcirculatory effects of the transfusion of leukodepleted or non-leukodepleted red blood cells in patients with sepsis: a pilot study

Introduction: Microvascular alterations impair tissue oxygenation during sepsis. A red blood cell (RBC) transfusion increases oxygen (O2)-delivery but rarely improves tissue O2 uptake in septic patients. Possible causes include RBC alterations due to prolonged storage or residual leukocyte-derived inflammatory mediators. The aim of this study was to compare the effects of two types of transfused-RBCs on microcirculation in septic patients. Methods: In a prospective randomized trial, 20 septic patients were divided into two separate groups and received either non-leukodepleted (n = 10) or leukodepleted (n = 10) RBC transfusions. Microvascular density and perfusion were assessed with sidestream dark-field (SDF) imaging sublingually, before and 1 hour after transfusions. Thenar tissue O2-saturation (StO2) and tissue haemoglobin index (THI) were determined with near-infrared spectroscopy (NIRS), and a vascular occlusion test was performed. The microcirculatory perfused boundary region was assessed in SDF images as an index of glycocalyx damage and glycocalyx compounds (syndecan-1, hyaluronan, heparan sulfate) were measured in the serum. Results: No differences were observed in microvascular parameters at baseline and after transfusion between the groups, except for the proportion of perfused vessels (PPV) and blood flow velocity, which were higher after transfusion in the leukodepleted group. Microvascular flow index in small vessels (MFI) and blood flow velocity exhibited different responses to transfusion between the two groups (P = 0.03 and P = 0.04, respectively), with a positive effect of leukodepleted RBCs. When looking at within-group changes, microcirculatory improvement was only observed in patients that received leukodepleted RBC transfusion as suggested by the increase in De Backer score (P = 0.02), perfused vessel density (P = 0.04), PPV (P = 0.01) and MFI (P = 0.04). Blood flow velocity decreased in the non-leukodepleted group (P = 0.03). THI and StO2-upslope increased in both groups. StO2 and StO2-downslope increased in patients who received non-leukodepleted RBC transfusions. Syndecan-1 increased after the transfusion of non-leukodepleted RBCs (P = 0.03). Conclusions: This study does not show a clear superiority of leukodepleted over non-leukodepleted RBC transfusions on microvascular perfusion in septic patients, although it suggests a more favourable effect of leukodepleted RBCs on microcirculatory convective flow. Further studies are needed to confirm these findings. © 2014 Donati et al.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Targeted therapies for advanced and metastatic adenocarcinoma of the gastroesophageal junction: is there something new?

Despite improvements in systemic chemotherapy (CT), the prognosis of metastatic adenocarcinoma of the gastroesophageal junction remains poor. Over the years, new targeting agents have become available and were tested, with or without CT, in first or subsequent lines of therapy. The epidermal growth factor receptor family was targeted with monoclonal antibodies (MoAbs) (trastuzumab, cetuximab, panitumumab) and tyrosin kinase inhibitors (TKIs) (lapatinib, erlotinib, gefitinib). Only trastuzumab, in combination with cisplatin and fluoropyrimidines, significantly improved overall survival (OS) in first-line therapy (13.8 vs. 11.1 months). Angiogenesis also was targeted with MoAbs (bevacizumab and ramucirumab); ramucirumab, a vascular endothelial growth factor-receptor 2 antagonist, enhanced OS in two phase III studies in the first (9.6 vs. 7.4 months) and subsequent lines of treatment (5.2 vs. 3.8 months), while the bevacizumab study was negative. TKIs (sunitinib, sorafenib, regorafenib, apatinib) were tested in this setting in phase II studies in the second/third line, only showing modest antitumor activity. The hepatocyte growth factor receptor (MET) was targeted in untreated patients in a phase III trial with MoAb rilotumumab, with or without CT, but the study was stopped because of mortality excess in the rilotumumab arm. Mammalian target of rapamycin (MTOR) pathway inhibition with everolimus was tested in pretreated patients in a placebo-controlled phase III trial who failed to improve OS (5.4 vs. 4.3 months). In conclusion, considering the modest survival gain obtained overall, the high cost of these therapies and the quality of life issue must be primarily considered in treating these patients

Impact of the number of comorbidities on cardiac sympathetic derangement in patients with reduced ejection fraction heart failure

Introduction Heart failure (HF) is frequently associated with comorbidities. 123I-metaiodobenzylguanidine (123I-mIBG) imaging constitutes an effective tool to measure cardiac adrenergic innervation and to improve prognostic stratification in HF patients, including the risk of major arrhythmic events. Although comorbidities have been individually associated with reduced cardiac adrenergic innervation, thus suggesting increased arrhythmic risk, very comorbid HF patients seem to be less likely to experience fatal arrhythmias. We evaluated the impact of the number of comorbidities on cardiac adrenergic innervation, assessed through 123I-mIBG imaging, in patients with systolic HF. Methods Patients with systolic HF underwent clinical examination, transthoracic echocardiography and cardiac 123I-mIBG scintigraphy. The presence of 7 comorbidities/conditions (smoking, chronic obstructive pulmonary disease, diabetes mellitus, peripheral artery disease, atrial fibrillation, chronic ischemic heart disease and chronic kidney disease) was documented in the overall study population. Results The study population consisted of 269 HF patients with a mean age of 66±11 years, a left ventricular ejection fraction (LVEF) of 31±7%, and 153 (57%) patients presented ≥3 comorbidities. Highly comorbid patients presented a reduced late heart to mediastinum (H/M) ratio, while no significant differences emerged in terms of early H/M ratio and washout rate. Multiple regression analysis revealed that the number of comorbidities was not associated with mIBG parameters of cardiac denervation, which were correlated with age, body mass index and LVEF. Conclusion In systolic HF patients, the number of comorbidities is not associated with alterations in cardiac adrenergic innervation. These results are consistent with the observation that very comorbid HF patients suffer lower risk of sudden cardiac death

Effects of Germline VHL Deficiency on Growth, Metabolism, and Mitochondria.

Mutations in VHL, which encodes von Hippel-Lindau tumor suppressor (VHL), are associated with divergent diseases. We describe a patient with marked erythrocytosis and prominent mitochondrial alterations associated with a severe germline VHL deficiency due to homozygosity for a novel synonymous mutation (c.222C→A, p.V74V). The condition is characterized by early systemic onset and differs from Chuvash polycythemia (c.598C→T) in that it is associated with a strongly reduced growth rate, persistent hypoglycemia, and limited exercise capacity. We report changes in gene expression that reprogram carbohydrate and lipid metabolism, impair muscle mitochondrial respiratory function, and uncouple oxygen consumption from ATP production. Moreover, we identified unusual intermitochondrial connecting ducts. Our findings add unexpected information on the importance of the VHL-hypoxia-inducible factor (HIF) axis to human phenotypes. (Funded by Associazione Italiana Ricerca sul Cancro and others.)

A Recent Experience in an Egyptian Medical Center: Strategies for the Clinical and Genetic Diagnoses of Nephropathic Cystinosis

Aims: To screen cases of infantile cystinosis among different forms of proximal renal tubular acidosis (RTA). Study Design: Cross sectional. Place and Duration of Study: From a total of 25 families of RTA followed up in Nephrology unit of Mansoura University Children's Hospital (MUCH), Egypt, two unrelated families were diagnosed as infantile nephropathic cystinosis using clinical suspicion plus mutation analysis of CTNS gene in the period between January 2008 and November 2012. Methodology: Two families with multiple cases of infantile nephropathic cystinosis have been diagnosed. In absence of high-performance liquid chromatography and tandem mass spectrometry used for measuring intraleucocyte cystine, diagnostic tools for cystinosis used in the current work were clinical and laboratory evidences of PRTA, slit lamp detection of corneal cystine crystals and finally identification of CTNS gene mutations. All patients were subjected to routine echocardiography because of accidental discovery of heart malformation in one case. Rare mutant variant of the first family was subjected to RNA analysis which unfortunately failed, alternatively an in silico study was used to predict splice site. Results: All patients with cystinosis manifested a severe clinical course. Proband of family 1 showed two known mutations; deletion in the exon 3 (c.18_21 del GACT) and substitution in acceptor splice site of intron 11 (c.971 -12G>A). In silico study predicted an anticipated splice site that modified the open reading frame in carboxy-terminal region. Probands of family 2 were affected by ventricular and atrial septal defects in younger, and mild mitral and aortic incompetence in older patient; their DNA analysis revealed a novel nonsense mutation (c.734 G>A) which caused a premature stop codon in position 245 of protein. Conclusion: Nephropathic cystinosis has been diagnosed with ease in Egyptian population without need of sophisticated investigations. A novel mutation had been added to the list of CTNS gene variants

Immunization with Usutu virus and with a chimeric West Nile virus (WNV) harboring Usutu-E protein protects immunocompetent adult mice against lethal challenges with different WNV lineage 1 and 2 strains

West Nile virus (WNV) and Usutu virus (USUV), two antigenically related flaviviruses co-circulating in Europe, can cause severe neurological disease in animals and humans. The immune response against USUV and WNV and their immunopathogenesis are still poorly investigated. Here we present results upon sequential infections of adult immunocompetent CD-1 and BALB/c mice primed with two different doses (high dose, HD or low dose, LD) of an USUV isolate and challenged with HD or LD of three different WNV isolates. CD-1 and BALB/c LD USUVprimed mice, regardless of the dose, are largely protected from lethal WNV challenges despite showing no detectable neutralizing antibodies. Furthermore, mice immunized with a chimeric virus harboring the E protein of USUV within the WNV backbone (WNVE-USUV) are protected against a lethal challenge with WNV. We believe these findings could contribute to understanding the dynamics of the interaction during sequential infection of these two flaviviruses