Optimisation of herpes simplex virus-based vectors for delivery to human peripheral blood mononuclear cells

Peripheral blood mononuclear cells (PBMCs) represent a significant target for gene delivery both for therapeutic and experimental purposes. Thus far however, it has proved difficult to develop vectors capable of high efficient gene delivery to unstimulated PBMCs. We have tested a range of different vectors derived from herpes simplex virus (HSV) which differ in their degree of disablement in terms of their gene delivery efficiency to unstimulated human PBMCs and ability to deliver a reporter gene. None of the viruses had any significant toxic effect in PBMCs. However, optimal gene delivery to unstimulated PBMCs was obtained with a semidisabled virus lacking functional genes encoding ICP34.5 and Vmw65 which was more efficient than either nondisabled or more extremely disabled viruses. Expression of green fluorescent protein (GFP) with this virus was observed in up to 50% of PBMCs 1 day after infection, and reporter gene expression was detectable by Western blotting and immunofluorescence at undiminished levels at the longest time points tested, up to 5 days after infection. This optimised HSV vector may thus represent an effective tool for gene delivery to unstimulated PBMCs in culture.     

Age at menopause and prevalence of its different types in contemporary Greek women

OBJECTIVE: This study attempted to estimate the mean age at menopause and also characterize and quantify its types and frequencies in Greek women who have recently become postmenopausal. DESIGN: A retrospective analysis of hospital records was used to record the frequency of natural, spontaneous or induced premature, and delayed natural menopause; the mean age at menopause of each group; and their possible interrelationships with major events of reproductive history in 1,747 women visiting an endocrine clinic 1 to 5 years after menopause. A group of 438 postmenopausal women drawn from 4,000 cases from the nongynecological clinics of this hospital comprised the control group. RESULTS: Natural menopause was found in 1,333 (76.3%) women with a median age of 51 (95% CI, 50.8-51.2) and mean +/- SD of 48.7 +/- 3.8 years. Medically induced menopause after the age of 40 was recorded in 204 women (11.7%), spontaneous premature in 127 (7.1%), medically induced premature in 60 (3.7%), and delayed menopause in 26 (1.5%), with mean ages of 43.6 +/- 1.7, 35.1 +/- 3.3, 37.2 +/- 1.6, and 59.6 +/- 2.1 years, respectively. No significant differences in mean age at menopause or its types were noted between the two groups. Furthermore, no significant correlations among the parameters examined could be demonstrated except between smoking and age at menopause in the spontaneous premature menopause subgroup (P < 0.05). Finally, ovarian autoantibodies were found in 6.7% of the tested women with premature menopause. CONCLUSIONS: Natural menopause occurred at a mean age of 48.7 years in Greek women, who displayed an unexpectedly high prevalence of spontaneous or induced premature cessation of menstruation.     

Detection and calibration of microdeletions and microduplications by array-based comparative genomic hybridization and its applicability to clinical genetic testing.

PURPOSE: Genome-wide telomere screening by fluorescence in situ hybridization (FISH) has revealed that approximately 6% of unexplained mental retardation is due to submicroscopic telomere imbalances. However, the use of FISH for telomere screening is labor intensive and time consuming, given that 41 telomeres are interrogated. We have evaluated the use of array-based Comparative Genomic Hybridization (aCGH) as a more efficient tool for identifying telomere rearrangements. METHODS: In this study, 102 individuals with unexplained mental retardation, with either normal or abnormal FISH results, were selected for a blinded retrospective study using aCGH. Results between the two methodologies were compared to ascertain the ability of aCGH to be used in a clinical diagnostics setting. RESULTS: We detected 100% of all imbalances previously identified by FISH (n = 17) and identified two additional abnormalities, a 10q telomere duplication and an interstitial duplication of 22q11. Interphase FISH analysis verified all abnormal array results. We also demonstrated that aCGH can accurately calibrate the size of telomere imbalances by using an array with "molecular rulers" for the telomeric regions of 1p, 16p, 17p, and 22q. CONCLUSION: This study demonstrates that aCGH is an equivalent methodology to telomere FISH for detecting submicroscopic deletions. In addition, small duplications that are not easily visible by FISH can be accurately detected using aCGH. Because aCGH allows simultaneous interrogation of hundreds to thousands of DNA probes and is more amenable to automation, it offers an efficient and high-throughput alternative for detecting and calibrating unbalanced rearrangements, both of the telomere region, as well as other genomic locations.     

Comparison of chromogenic in situ hybridisation with fluorescence in situ hybridisation and immunohistochemistry for the assessment of her-2/neu oncogene in archival material of breast carcinoma

The successful treatment of breast cancer is dependent upon a number of complex factors. Her-2/neu gene amplification is known to be one of the most common genetic alterations associated with breast cancer and its accurate determination has become necessary for the selection of patients for trastuzumab therapy.The aim of this study was to prove the consistency of chromogenic in situ hybridisation (CISH) technique after analyzing the overexpression of the Her-2/neu proto-oncogene in 100 invasive breast carcinomas and by comparing CISH results with immunohistochemistry (IHC) and fluorescence in situ hybridisation (FISH). Moreover, it was done to evaluate the possible correlation of estrogen (ERs) and progesterone receptors (PRs), the proliferation marker Ki67 and the tumour suppressor gene p53 with HER-2/neu status of these breast carcinomas.Of the 100 breast carcinomas that were analysed, 22 cases showed HER-2/neu amplification, 66 cases showed no amplification, whereas 12 cases were non-interpretable in both assays (FISH and CISH). Consequently, the overall concordance between FISH and CISH was 100%. Additionally, it was observed that when HER-2/neu gene was overexpressed, there was an association with negative PRs and ERs status, negative p53 protein expression and high Ki67 labelling index.It is concluded that patients with tumours scoring 2+ with the CBE356 antibody (borderline immunohistochemistry-tested cases) would also benefit from CISH as it is shown to be highly accurate, practical and can be easily integrated into routine testing in any histopathology laboratory. Finally, CISH represents an important addition to the HER2 testing algorithm.     

A case of digenic maturity onset diabetes of the young with heterozygous variants in both HNF1Α and HNF1Β genes

BackgroundMaturity onset diabetes of the young (MODY) is the most commonly reported form of monogenic diabetes in the pediatric population. Only a few cases of digenic MODY have been reported up to now.Case reportA female patient was diagnosed with diabetes at the age of 7 years and was treated with insulin. A strong family history of diabetes was present in the maternal side of the family. The patient also presented hypomagnesemia, glomerulocystic kidney disease and a bicornuate uterus. Genetic testing of the patient revealed that she was a double heterozygous carrier of HNF1A gene variant c.685C > T; (p.Arg229Ter) and a whole gene deletion of the HNF1B gene. Her mother was a carrier of the same HNF1A variant.ConclusionDigenic inheritance of MODY pathogenic variants is probably more common than currently reported in literature. The use of Next Generation Sequencing panels in testing strategies for MODY could unmask such cases that would otherwise remain undiagnosed.     

Endovascular Treatment of a Bleeding Secondary Aorto-Enteric Fistula. A Case Report with 1-Year Follow-up

We report a patient with life-threatening gastrointestinal bleeding caused by a secondary aorto-enteric fistula. Because the patient had several comorbid conditions, we succesfully stopped the bleeding by endovascular placement of a bifurcated aortic stent-graft. The patient developed periaortic infection 4 months later, but he was managed with antibiotics. The patient is well 1 year after the procedure.     

Effect of Cyclosporine Therapy With Low Doses of Corticosteroids on Idiopathic Nephrotic Syndrome

Cyclosporine (CyA) has an immunosuppressive effect that might suggest a therapeutic role in idiopathic glomerular conditions. We focused on the optimization of CyA treatment control in patients with idiopathic nephrotic syndrome by using trough‐level CyA measurements (C0) and the 2‐h postdose levels (C2). Twenty‐two patients (14 male, 8 female) with idiopathic nephrotic syndrome and the mean age of 51 ± 18 months (mean [M] ± standard deviation [SD]) were enrolled in our study during a period of 10 months (range: 3–18 months). All of the patients received CyA (2–3 mg/kg) in combination with methylprednisolone. In the present study protocol CyA concentrations (C0, C2), renal function, lipid profile, and degree of proteinuria were determined. The mean proteinuria of our patients before treatment was 11 972 ± 7953 mg/24 H (±SD) and the mean creatinine level (Cr) was 0.99 ± 0.37 mg/dL (±SD). Proteinuria decreased significantly already from the first month of therapy with CyA to 3578 ± 2470 mg/24 H (M± SD), and during the whole study period this reduction was significant (0.56 ± 0.37 gr/24 H (M ± SD), P < 0.05). At the same time renal function preserved, 1.09 ± 0.48 mg/dL (M ± SD). The blood levels of C0 were 135.10 ± 97.36 ng/mL (M ± SD) and the blood levels of C2 were 725 ± 256 ng/mL (M ± SD) at the first month of therapy. At the same time renal function preserved, 1.09 ± 0.48 mg/dL (M ± SD). Total cholesterol levels reduced significantly during study period (276.89 ± 45.57 to 200.67 ± 40.27 mg/dL [M ± SD]). The mean number of antihypertensive medication remained the same. The whole therapeutic protocol did not provoke any kind of side effects and CyA was quite tolerated by our patients. Treatment of idiopathic nephrotic syndrome with low doses of CyA with methylprednisolone leads to remission of proteinuria without deterioration of renal function. Blood levels of C0 for monitoring and treatment of nephrotic syndrome agrees with recent literature, while our study focus on establishing the proper levels of C2 for the treatment of nephrotic syndrome. The efficacy of CyA is combined with safety and tolerance.     

Concurrent validity and reliability of cephalometric analysis using smartphone apps and computer software

Objectives:To assess the diagnostic accuracy of two smartphone cephalometric analysis apps compared with Viewbox software.Materials and Methods:Pretreatment digital lateral cephalograms of 50 consecutive orthodontic patients (20 males, 30 females; mean age, 19.1 years; SD, 11.7) were traced twice using two apps (ie, CephNinja and OneCeph), with Viewbox used as the gold standard computer software program. Seven angular and two linear measurements, originally derived from Steiner cephalometric analysis, were performed.Results:Regarding validity, intraclass correlation coefficients (ICCs) ranged from .903–.983 and .786–.978 for OneCeph vs Viewbox and CephNinja vs Viewbox, respectively. The ICC values for intratool reliability ranged from .647–.993. None of the CephNinja measurements was below the recommended cutoff values of ICCs for reliability.Conclusions:OneCeph has a high validity compared with Viewbox, while CephNinja is the best alternative to Viewbox regarding reliability. Smartphone apps may have a great potential in supplementing traditional cephalometric analysis.     

Gender differences in anxiety and depressive symptoms in patients with primary hyperaldosteronism: A cross-sectional study

Objective. The renin-angiotensin-aldosterone-system (RAAS) has gained increasing attention in the investigation of the pathogenesis of depression. Primary hyperaldosteronism (PA) is associated with a marked aldosterone excess. Prior studies on PA describe an increased prevalence of anxiety and sub-threshold depressive symptoms in these patients. Methods. In a cross-sectional exploratory study we investigated 132 patients with PA. Twenty-seven patients were studied before initiation of specific treatment (U = untreated), 56 were studied 5.4 years after initiation of mineralocorticoid antagonist treatment (MRA) and 49 patients were studied 4.3 years after unilateral adrenalectomy (ADX). GAD-7 and PHQD self-rating questionnaires were used to assess symptoms for anxiety and depression. Results. No significant difference was found between the three investigated groups. A higher prevalence for depression and anxiety compared to the normal population was found. Women of all groups had higher mean values compared to men, for depression in untreated patients this difference was found to be significant. Correlations between the psychopathology and hormones were only found for renin. Plasma renin concentration correlated significantly with anxious symptoms of untreated females. Conclusions. This study supports the RAAS to be involved in the pathogenesis of depression as patients with PA seem to be more depressive and anxious compared to the normal population. Gender differences in the regulation of the RAAS seem to be apparent, as females were more affected by the dysregulation than males.