Complex ABCC8 DNA variations in congenital hyperinsulinism: lessons from functional studies

OBJECTIVE: Congenital hyperinsulinism (CHI) is a cause of persistent and severe hypoglycaemia in infancy. Mutations in the genes ABCC8 and KCNJ11 encoding SUR1 and Kir6.2, respectively, are the commonest cause of CHI. We investigated whether the possession of two DNA variants leading to coding changes in a single allele of ABCC8 can affect the potential mechanism of disease pathogenesis. DESIGN AND PATIENTS: We studied two patients with complex mutations in the ABCC8 gene with CHI and used in vitro studies to explore the potential disease mechanism and the contribution of the various mutant allelles. RESULTS: The first case had diffuse disease and was homozygous for the mutations D1193V and R1436Q in SUR1. Channel complexes containing the D1193V mutant were delivered to the plasma membrane and were functional and those containing R1436Q were also present at the plasma membrane but were nonfunctional. Combining the two mutations (SUR1D1193V/R1436Q) led to intracellular retention of the channel complex. In a second family, the patient had histologically focal disease and was heterozygous for two mutations from his father (G228D and D1471N) and one from his mother (V1572I). SUR1 G228D and D1471N singly or in combination led to intracellular retention of the channel complex and loss of function. By contrast, V1572I is trafficked appropriately and is functional, consistent with a mechanism of reduction to hemizygosity of paternal ABCC8 in focal disease. V1572I is likely to be a benign DNA variant. CONCLUSION: In one patient the combination of two coding variants led to intracellular retention of channel complex. In a second patient, functional studies allowed us to unravel the DNA variants likely to be causing the abrogation of ATP-sensitive K(+) channel function.     

Evaluation of Antibody Response to Polysaccharide Vaccine and Switched Memory B Cells in Pediatric Patients with Inflammatory Bowel Disease

Background/Aims

Inflammatory bowel disease (IBD) is a chronic disease of the gastrointestinal tract, whose etiologies are still unknown. This study was performed to evaluate the humoral immune response in terms of B cell functions in selected IBD patients.

Methods

Eighteen pediatric patients with IBD, including 12 cases of ulcerative colitis (UC) and six with Crohn disease (CD), were enrolled in this study. The pneumococcal vaccine was injected in all patients, and the IgG antibody level to the polysaccharide antigen was measured before and 4 weeks after injection. The B cell switch-recombination process was evaluated.

Results

Five patients with IBD (three CD and two UC) had defects in B cell switching, which was significantly higher than in controls (p=0.05). Ten patients had a specific antibody deficiency and exhibited a higher frequency of bacterial infection than the healthy group. The mean increased level of IgG after vaccination was lower in IBD patients (82.9±32.5 µg/mL vs 219.8±59.0 µg/mL; p=0.001). Among the patients who had an insufficient response, no significant difference in the number of switched memory B-cell was observed.

Conclusions

A defect in B lymphocyte switching was observed in pediatric IBD patients, and especially in those patients with CD. Owing to an increased risk of bacterial infections in those patients with antibody production defects, pneumococcal vaccination could be recommended. However, not all patients can benefit from the vaccination, and several may require other prophylactic methods.     

Immune Responses against a New HIV-1 p24-gp41/pCAGGS-IL-12 DNA Vaccine in Balb/c Mice

Background: Development of an effective vaccine is highly needed in order to restrict the AIDS pandemic. DNA vaccines initiate both arms of immunity without the potential of causing disease. HIV-1 p24 and gp41 (gag and env) proteins play important roles in viral pathogenesis and are effective candidates for immune induction and vaccine design. Objective: In this study, new DNA vaccine candidates constructed from HIV-1 fused p24-gp41 or gp41 alone were evaluated in Balb/c mice for induction of cellular and humoral immune responses. Methods: Recombinant plasmids, pcDNA3.1/Hygro expression vector containing immunogenic sequences of fused p24-gp41 or gp41alone were produced. Dendrosome used as a system for carrying vectors in laboratory animals, and an IL-12 containing vector (pCAGGS-IL-12) was co-immunized with the p24-gp41 vector as a genetic adjuvant. Induction of effective immune responses against the designed vectors as DNA vaccine candidates in Balb/c mice was evaluated. Levels of total antibodies, IgG isotypes (IgG2a and IgG1); IFN-γ and IL-4 were measured by ELISA. MTT assay was used to evaluate lymphoproliferation. Results: The results confirmed that the immunogenic epitopes of both p24 and gp41 genes are highly effective inducers of immune responses, and administration of fused p24-gp41 alone or along with IL-12 resulted in further enhancement of immune responses. Group 4 that received fused fragments (p24-gp41) along with an IL-12 expressing vector demonstrated a significantly higher Stimulation Index (SI) and IFN-γ production (p<0.0001) with a significant increase in IgG2a/IgG1 ratio, indicating the stimulation of CMI towards Th1. Although gp41 containing vector (group 6) also showed significant increases in both proliferation and IFN-γ production, the responses were persistently lower than that of p24-gp41 containing vectors. Total antibody production was highest in group 6 as expected. Conclusion: Dendrosome proved to be an efficient carrier of recombinant plasmids constructed in this study. Further studies are necessary to evaluate these constructs as HIV vaccine candidates.     

Association of adiponectin with dietary factors and cardiovascular risk factors in type 2 diabetes mellitus patients

Background and aimAdiponectin, is an adipose tissue-specific adipokine, that circulates in human plasma at high levels, although lower levels are noted with insulin resistance and atherosclerosis. We investigated the relationship of adiponectin concentrations with dietary factors and some of the cardiovascular risk factors in patients with T2DM.Methods and resultsTotally 107 patients with T2DM were recruited from the out patients clinic of Shariati Hospital, Tehran, Iran. Patients were evaluated for laboratory and anthropometric measurements including serum adiponectin, fasting insulin, FPG, OGTT, HbA1c, HOMA-IR, hsCRP, weight, height, BMI and WHR. Nutrients intakes were obtained via 24-h recall from each patient in three successive days. Nutrients and data analysis were done using FPII and SPSS version 13 softwares. The mean of log 10-transformed serum adiponectin concentration was 0.79 ± 0.27 μg/ml. The univariate linear regression analysis could not show any significant relation between the log of serum adiponectin and dietary factors. In multivariate linear regression after multiple adjustment, the log of serum adiponectin was independently associated with WHR (P = 0.02, t = ?2.33), HDL-C (P = 0.050, t = 2.03) and markedly but not significantly with age of patients (P = 0.058, t = 1.92).ConclusionsOur findings showed that WHR, one of the important cardiovascular risk factors, can modulate independently adiponectin levels of T2DM patients in inverse manner. Also, the age of patients and HDL-C levels have marked positive effect on circulating levels of this adipocytokine. Thus, adiponectin might be a useful biomarker to prevent developing CVD in type 2 diabetes.     

Construction and Preparation of Three Recombinant Adenoviruses Expressing Truncated NS3 and Core Genes of Hepatitis C Virus for Vaccine Purposes

Background

In spite of dozens of clinical trials to establish effective therapeutic and/or preventive vaccine to resolve HCV infection, no real vaccine has been proved to date. Genetic vaccines based on replication-defective adenoviruses have proved to elicit strong and long lasting T-cell responses against a number of viral antigens and are even currently being used for vaccine trials in humans. According to the controversy in the immune modulatory effects of both core and NS3 full length genes, it seemed more practical to employ some parts of these HCV proteins for vaccine design.

Objectives

To generate recombinant Adenoviral vectors containing new overlapping-truncated region of NS3 gene or both the N- and C-terminal deleted parts of core gene, as well as a fusion fragment derived from both of them.

Materials and Methods

The corresponding transfer vectors expressing truncated fragments of core, NS3 or a fusion fragment of both genes were prepared. The integrity and sequence of the transfer vectors were confirmed, and followed by experiments involving homologous recombination between them and the adenovirus backbone plasmid in the bacterial host. Recombinant Ad-pNS3, Ad-pCore and Ad-pNS3pCore viruses were prepared by transfection of these new recombined constructs into 293 packaging cell lines. The virus titer was then calculated by an immunohistochemistry based method. The RT-PCR, Real-Time PCR and western blotting were used to evaluate gene expression by all recombinant constructs. The production of complete virion particles was evaluated by detailed electron microscopy in addition to the appearance of typical cytopathic effects (CPE) and GFP expression patterns in 293 cells. The RT-PCR and GFP detection were employed to monitor the integrity as well as infectivity potency of the viral particles in Hep-G2 cells.

Results

RT-PCR, Real-Time PCR or western blotting confirmed expression of truncated fragment of NS3, core or a fusion fragment of theirs by newly constructed Ad-pNS3, Ad-pCore, Ad- pNS3pCore particles. Electron microscopy, which revealed many adenovirus-like particles and characteristics of CPE in infected cells in addition to GFP detection, confirmed the infectivity, potency and integrity of recombinant adenoviral particles.

Conclusions

These adenoviruses expressing novel fragments of NS3 and core genes may be suitable tools to overcome shortcomings associated with full gene expression in the setting of HCV vaccine therapy.     

The effects of 5-azacytidine on the function and number of regulatory T cells and T-effectors in myelodysplastic syndrome

Expansion of regulatory T cells occurs in high-risk myelodysplastic syndrome and correlates with a poor prognosis. DNA methyltransferase inhibitors, particularly 5-azacytidine, have been shown to increase the survival of patients with high-risk myelodysplastic syndrome. It is not entirely clear whether this improvement in patients’ survival is related to the effects of DNA methyltransferase inhibitors on the immune system and/or the direct effect of these drugs on the dysplastic clone. In this study we investigated the effect of 5-azacytidine on the function and proliferation capability of regulatory T cells and T-helper cells. The number and function of CD4⁺ T-cell subsets in 68 patients with intermediate-2/high-risk myelodysplastic syndrome were serially assessed at diagnosis and following treatment. The in-vitro effects of 5-azacytidine on CD4⁺ T-cell subsets isolated from both healthy donors and patients with myelodysplastic syndrome were also investigated. The number of peripheral blood regulatory T cells was significantly higher in myelodysplastic syndrome patients than in healthy donors and responders to treatment (P=0.01). The absolute numbers of T-helper 1 and T-helper 2, but not T-helper 17, cells were significantly reduced following 12 months of treatment (P=0.03, P=0.03). The in vitro addition of 5-azacytidine to CD4⁺ T cells reduced the proliferative capacity of regulatory T cells (P=0.03). In addition, the 5-azacytidine-treated regulatory T cells had reduced suppressive function and produced larger amounts of interleukin-17. The FOXP3 expression in 5-azacyti-dine-treated T-effectors was also increased. Interestingly, these FOXP3⁺/interleukin-17⁺ cells originated mainly from effector T cells rather than regulatory T cells. Our data suggest that 5-azacytidine has profound effects on CD4⁺ T cells, which correlate with disease status after treatment. Furthermore, despite the demethylation of the FOXP3 promoter and increased FOXP3 expression following 5-azacytidine treatment, these phenotypic regulatory T cell-like cells lack the regulatory function and cytokine profile of regulatory T cells. These findings are important in correlating the clinically relevant immunomodulatory effects of 5-azacytidine.     

Cervical cancer screening in medically underserved California Latina and non-Latina women: Effect of age and regularity of Pap testing

Background: This study focuses on age, race/ethnicity and regular cervical cancer screening of medically underserved Latina and non-Latina women enrolled in California's Cancer Detection Programs: Every Woman Counts (CDP: EWC). Methods: Data from a cohort of women were evaluated for regularity of screening and ethnicity utilizing multi-category logistic regression models to investigate Pap test and biopsy results. Results: There was no statistically significant difference among medically underserved Latina or non-Latina women in Pap test result and stage of cervical cancer after controlling for age and screening regularity. Rarely/never Pap-tested women were more likely to have ‘SIL/ASC’ (odds ratio = 1.19; 95% confidence interval = 1.08, 1.31) compared to women who were screened regularly. Medically underserved 25–39-year-old women were also more likely to be identified with ‘SIL/ASC’ (odds ratio = 1.64; 95% confidence interval = 1.50, 1.79) than women 50 and over. Younger were more likely to have low-grade ‘HPV/Condylomata, Atypia/CINI/LSIL’ (odds ratio = 2.48; 95% confidence interval = 1.66, 3.72) and high-grade ‘CIN II/III/HSIL/CI/Other Cancers’ (odds ratio = 1.53; 95% confidence interval = 1.08, 2.16) than women age 40 and above, similar to rarely/never Pap-tested women. Conclusions: Women were more likely to be identified with high-grade precancerous cervical lesions and cancer process when they did not have regular screening, Ethnic differences in screening outcomes seem to be minimized by participation in a program that provides consistent screening resources to the medically underserved women who enroll. These findings support prevention strategies that expand screening to all medically underserved younger women or that provide HPV vaccination at an early age.     

Impact of ASC‐H terminology on the detection of HSILs in medically underserved California women

In this study we evaluated CDP: EWC, a large public health screening program for low‐income women to determine whether the ASC‐H term, introduced in the 2001 revision to the Bethesda System, has facilitated the detection of the most serious precancerous cervical lesions such as CIN‐II–III/CIS, including accuracy of detection and minimizing diagnostic delays. Pap test and biopsy data from the period 2003–2006 were compared with those from 1995–1999, and included analysis of a subset of rarely and never‐screened for each study period. More ASC‐H Pap tests were followed by CIN‐II+ biopsies (20%) than ASCUS Pap tests (5%). Thirteen percent of ASC‐H showed CIN‐III/CIS on follow‐up biopsy. Following the introduction of ASC‐H, negative and LSIL Pap tests followed by CIN‐III/CIS biopsies decreased from 5.6% to 0.37% and 9.2% to 4.4%, respectively. Fewer CIN‐III/CIS cases were preceded by Pap tests with negative and ASCUS results in 2003–2005 as compared with 1995–1999. The number of days from screening to diagnosis for women aged 25–39 years with ASC‐H Pap test results (58 days) was less than for ASCUS results (78 days) for the current study period. In this high‐risk multi‐ethnic population, ASC‐H more effectively communicates equivocal findings likely to represent HSIL leading to more rapid follow‐up. The incidence of CIN‐II or CIN‐III/CIS following ASC‐H Pap tests is lower than generally appreciated for this population, including for rarely or never‐screened high‐risk women. Fewer CIN‐III/CIS lesions were detected following negative and ASCUS Pap results. Compliance with follow‐up is less than ideal for this population. Diagn. Cytopathol. 2009. © 2008 Wiley‐Liss, Inc.     

Basilic vein aneurysm and palmar intramuscular hemangioma with peri and inter tendinous growth

In this report, we describe a case of concomitant basilic vein aneurysm and palmar hemangioma with peri- and inter-tendinous growth around the fourth and fifth flexor digitorum superficialis and flexor digitorum profundus tendons.It seems reasonable for physicians and radiologists to keep in mind the possibility of venous aneurysms in the presence of soft tissue hemangiomas; as they can present as palpable mass and be mistaken for other pathologies. Familiarity with clinical and imaging findings of this entity could be helpful to prevent misdiagnosis.