Skeletal muscle overexpression of sAnk1.5 in transgenic mice does not predispose to type 2 diabetes.

Genome-wide association studies (GWAS) and cis-expression quantitative trait locus (cis-eQTL) analyses indicated an association of the rs508419 single nucleotide polymorphism (SNP) with type 2 diabetes (T2D). rs508419 is localized in the muscle-specific internal promoter (P2) of the ANK1 gene, which drives the expression of the sAnk1.5 isoform. Functional studies showed that the rs508419 C/C variant results in increased transcriptional activity of the P2 promoter, leading to higher levels of sAnk1.5 mRNA and protein in skeletal muscle biopsies of individuals carrying the C/C genotype. To investigate whether sAnk1.5 overexpression in skeletal muscle might predispose to T2D development, we generated transgenic mice (TgsAnk1.5/+) in which the sAnk1.5 coding sequence was selectively overexpressed in skeletal muscle tissue. TgsAnk1.5/+ mice expressed up to 50% as much sAnk1.5 protein as wild-type (WT) muscles, mirroring the difference reported between individuals with the C/C or T/T genotype at rs508419. However, fasting glucose levels, glucose tolerance, insulin levels and insulin response in TgsAnk1.5/+ mice did not differ from those of age-matched WT mice monitored over a 12-month period. Even when fed a high-fat diet, TgsAnk1.5/+ mice only presented increased caloric intake, but glucose disposal, insulin tolerance and weight gain were comparable to those of WT mice fed a similar diet. Altogether, these data indicate that sAnk1.5 overexpression in skeletal muscle does not predispose mice to T2D susceptibility.

A (single case) rehabilitation program based on cueing for freezing of speech.

BACKGROUND: Freezing of speech (FoS) and other repetitive speech behaviours can frequently occur in parkinsonian syndromes, worsening the efficacy of language functioning, hampering social interactions, and thus reducing quality of life. Pharmacological treatment are ineffective and other interventions have not specifically developed so far. AIM: The aim of this paper was to test the efficacy on a pilot rehabilitation program for freezing of speech based on cueing. SETTING: Outpatient clinic. POPULATION: Single-case. METHODS: We developed a pilot rehabilitation program based on visual, auditory and sensory cueing. The patient underwent the protocol for a 6-month period (1-hour sessions, 3 times weekly). Perceptual analyses of his speech were performed before and after the rehabilitation program by to different blinded reviewers. RESULTS: There has been a reduction of FoS and other iterative speech episodes. Intelligibility also improved according to both the patient and his relatives. CONCLUSION: Our preliminary results show that visual, auditory and sensory cueing can be effective to prevent and overcome FoS episodes. Possible underlying mechanisms of the improvement are discussed. CLINICAL REHABILITATION IMPACT: Rehabilitation programs based on cueing should be considered and offered in Parkinsonian patients exhibiting FoS or other iterative speech phenomena.

Desmoplastic melanoma: a 12-year experience with sentinel lymph node biopsy.

AIMS: Given the paucity of data regarding nodal involvement in desmoplastic melanoma (DM), we decided to review the incidence of nodal metastasis in our patients with DM to better define guidelines regarding the performance of sentinel lymph node biopsy (SLNB) in this specific melanoma subtype. METHODS: Using a prospectively maintained database, we reviewed all patients who underwent treatment for melanoma at the Yale Melanoma Unit in a twelve-year period (1998-2010), during which 3531 cases were treated. We identified 24 patients (0.7%) diagnosed with DM. These patients' records were studied for clinical and histologic parameters and clinical outcomes. RESULTS: Twenty-two patients from the DM group had SLNB, of which four (18%) were diagnosed with micro-metastasis. These four patients were all treated with completion lymphadenectomy and none had additional positive nodes in the remainder of the nodes. Patients were followed after surgery for a median of 25 months (range 2-60 months). Two patients (9%) developed local recurrence, two (9%) in-transit recurrence, and six (27%) showed distant metastases (three patients were pure DM and three patients showed mixed morphology). Patients with mixed DM had a higher rate of nodal metastasis (25%) vs those with pure DM (14%). CONCLUSIONS: Other authors have reported that patients diagnosed with pure DM were less likely to have a positive SLN (0-2%) than those patients with the mixed DM subtype (12-16%). Our findings of higher incidence rates of regional lymph node metastases in both the pure and mixed DM subtypes (14% and 25%) compel us to continue to still recommend that SLNB be considered in patients with both subcategories, pure and mixed DM. LEVEL OF EVIDENCE: Level IV.

Dendritic cells overexpressing Fas-ligand induce pulmonary vasculitis in mice.

Dendritic cells (DC) genetically engineered to express Fas (CD95) ligand (FasL-DC) have been proposed as immunotherapeutic tools to induce tolerance to allografts. However, we and others recently showed that FasL-DC elicit a vigorous inflammatory response involving granulocytes and can promote Th1-type CD4+ and cytotoxic CD8+ T lymphocytes. This prompted us to evaluate the pathology induced by intravenous injection of FasL-DC in mice. We observed that FasL-DC obtained after retroviral gene transfer of bone marrow precursors derived from Fas-deficient C57Bl/6 mice induce massive pulmonary inflammation and pleuritis one day after a single intravenous injection in C57Bl/6 mice. Two months later, all mice presented granulomatous vasculitis of small to medium sized vessels, alveolar haemorrhage and pleuritis. In these lesions, apoptotic bodies were found in large number. Anti-neutrophilic cytoplasmic and anti-myeloperoxidase autoantibodies were not detected. This study documents that intravenous injection of FasL-DC causes severe lung granulomatous vasculitis. This new animal model for vasculitis is inducible, highly reproducible and shares many features with human Wegener granulomatosis. This model may be an appropriate tool to further investigate the pathogenesis of vasculitis and test new therapeutic strategies. Moreover, our findings highlight the potential severe complications of FasL-DC-based immunotherapy.

Unexpected effects of viral interleukin-10-secreting dendritic cells in vivo: preferential inhibition of TH2 responses.

Viral interleukin (IL)-10 (vIL-10) has been widely described as an immunoregulatory cytokine that does not possess the T-cell costimulatory activities of cellular IL-10; it was therefore believed to be a more potent tolerogenic mediator. The immunosuppressive properties of this cytokine are partly attributed to its capacity to render dendritic cells (DCs) unable to undergo full maturation and to activate T cells. We reported here that myeloid DCs retrovirally transduced with vIL-10 had an impaired production of IL-12 and a decreased expression of MHC class II molecules but had minor defects in costimulatory molecule expression and no alteration on CCR5 and CCR7 expression. In mixed leukocyte reaction, vIL-10-transduced C57BL/6 bm12 (MHC class II mismatch) DCs had a reduced capacity to stimulate C57BL/6 wild-type CD4+ T-cell proliferation. We show that bm12 vIL-10-transduced DC administration in CD8-/- C57BL/6 mice promoted IFN-gamma production, down-regulated TH2-type cytokine production, and did not induce skin graft tolerance. These findings suggest that vIL-10-transduced DC may surprisingly facilitate Th1-type inflammatory responses in vivo.

Recent advances in DNA sequencing by capillary and microdevice electrophoresis.

A number of significant improvements in the electrophoretic performance and design of DNA sequencing devices have culminated in the introduction of truly industrial grade production scale instruments. These instruments have been the workhorses behind the massive increase in genomic sequencing data available in public and private databases. We highlight the recent progress in aspects of capillary electrophoresis (CE) that has enabled these achievements. In addition, we summarize recent developments in the use of microfabricated devices for DNA sequencing that promise to bring the next leap in productivity.

Optimization of high-performance DNA sequencing on short microfabricated electrophoretic devices.

We have examined the parametric performance of short microfabricated electrophoresis devices that operate with a replaceable linear poly(acrylamide) (LPA) solution for the application of DNA sequencing. A systematic study is presented of the dependence of selectivity, separation efficiency, and resolution of sequencing fragments on buffer composition, LPA concentration, LPA composition, microdevice temperature, electric field, and device length. A specific optimization is made for DNA sequencing on 11.5-cm devices. Using a separation matrix composed of 3.0% (w/w) 10 MDa plus 1.0% (w/w) 50 kDa LPA, elevated microdevice temperature (50 degrees C), and 200 V/cm, high-speed DNA sequencing of 580 bases on standard M13mp18 was obtained in only 18 min with a base-calling accuracy of 98.5%. Read lengths of 640 bases at 98.5% accuracy were achieved in approximately 30 min by reducing the electric field strength to 125 V/cm. We believe that this constitutes matrix-limited performance for microdevices of this length using LPA sieving matrix and this buffer chemistry. In addition, it was confirmed, that shorter devices are rather impractical for production sequencing applications when LPA is used as sieving matrix.

Eight hundred-base sequencing in a microfabricated electrophoretic device.

The human genome will be sequenced using capillary array electrophoresis technology. Although currently achieving only 550 base reads per run, capillary arrays have increased the efficiency and lowered the cost of sequencing by eliminating gel plate preparation, reducing sample volumes, and offering automation and speed. However, much higher throughput and greater cost reductions are needed. The next major advancement in sequencing technology is expected from the development of arrays of microfabricated channels in a plate or "chip" format. For de novo sequencing, the practical utility of the microdevice approach has been limited by device length to a read of 500-600 bases per run. We demonstrate a significant milestone for a microfabricated device by obtaining an average read length of 800 bases in 80 min (98% accuracy) for either M13 standards or DNA sequencing samples from the Whitehead Institute Center for Genomic Research (WICGR) production line. This result is achieved in 40-cm-long channels using a new class of large-scale microfabricated devices. Both microfabrication of extended structures and achievement of long reads are essential steps toward a 384-lane very-large-scale microfluidic (VLSMF) system for ultrahigh-throughput DNA sequencing analysis, currently under construction in our laboratory.

Capillary electrophoresis-based immunoassays.

This review covers the progress and developments in the field of capillary electrophoresis immunoassay (CEIA) over the past three years. Because many excellent descriptions of the principles of these methods are available (e.g., in the reviews listed in this article), no elementary introduction is given to the field of immunoassays (IAs) or CEIAs. This report focuses exclusively on experimental results, dividing the CEIA papers into the categories of direct, indirect, and microchip electrophoretic immunoassays. In the last section, a brief summary of the current status of the CEIA field is presented.

Efficacy of hepatitis A vaccine in prevention of secondary hepatitis A infection: a randomised trial.

BACKGROUND: Hepatitis A vaccination stops outbreaks of hepatitis A infection, but its efficacy against infection after exposure has not been proven. We investigated the use of hepatitis A vaccine to prevent secondary infections with hepatitis A virus (HAV). METHODS: We did a randomised controlled trial of hepatitis A vaccine in household contacts of people with sporadic HAV infection (index cases). Households (index cases and contacts) were randomly assigned to the vaccine group or unvaccinated group, according to the study week in which they were enrolled. All household contacts in the vaccine group received vaccination at the time of entry to the study. FINDINGS: During 45 days of follow-up, secondary infection had occurred in ten (13.3%) of 75 households (two families had two cases each) in the untreated group and in two (2.8%) of 71 households in the vaccine group. The protective efficacy of the vaccine was 79% (95% CI 7-95). The number of secondary infections among household contacts was 12 (5.8%) of 207 in the unvaccinated group and two (1.0%) of 197 in the vaccinated group. Therefore, 18 individuals needed to be vaccinated to prevent one secondary infection. INTERPRETATION: Hepatitis A vaccine is effective in the prevention of secondary infection of HAV and should be recommended for household contacts of primary cases of HAV infection.

Correlation between virus genotype and chronicity rate in acute hepatitis C.

BACKGROUND/AIMS: Forty-two patients with the diagnosis of acute hepatitis C virus hepatitis were studied to investigate the relationship between hepatitis C virus genotype and progression to chronic infection. METHODS: The patients were followed for more than 1 year (mean age 29 years, male/female ratio 2.5). Intravenous drug use was documented in 15 cases, blood transfusion in four, surgical intervention, dental therapy or other parenteral exposure in 15, and unknown factors in the remaining eight. The evolution to chronicity was diagnosed on the basis of a persistent increase in transaminase levels, the presence of HCV-RNA and the histological pattern of chronic hepatitis. RESULTS: The majority of cases presented hepatitis C virus infection of subtype 1a (38.1%) or 1b (33.9%). Six cases showed the presence of genotype 3a (14.3%). Subtype 2c was observed in three out of four cases infected with genotype 2. No significant association was demonstrated with documented risk factors. The overall chronicity rate was 59.5%. This value increased to 92% in individuals infected with genotype 1b. By multivariate analysis the age-adjusted odds ratio for infection with genotype 1b as compared with all other genotypes was 14.4 (95% confidence interval; 1.52-137). Moreover, significant differences (p= 0.0002) were present in this group for histological activity index (8.7 as compared with 5-7). CONCLUSIONS: The results of this prospective study are consistent with an independent association between hepatitis C virus genotype 1b and a poor prognosis.

[Clinical-echocardiography of 210 HIV-Ab positive patients: retrospective study]. / Indagine clinica-ecocardiografica su 210 pazienti HIV Ab positivi: studio retrospettivo.

Through the retrospective study of 210 HIV Ab+ patients in different disease's stages, recovered in the "D. Cotugno-Naples" hospital during the period February 1989-February 1991, the authors have valued the prevalence of cardiological alterations underlined by ecocardiograph. Pericardial pouring has been observed in four patients out of thirty, belonged to stages II, III and ARC. Ventricles' movement alterations have been observed in twenty patients out of thirty (66%), belonged prevalently to the fourth group. Kaposi's sarcoma has been observed in two patients out of thirty (6.6%), belonged to the fourth group. In the patients showing alterations, the authors have also noticed a correlation among the observed cardiological alterations and the immunital outline and the frequent homosexuality's presence. The follow up between the alterations' observation and the possible patient's death is on an average 4.3 months (range 1-12). They suggest that a wider and more precocious ecocardiographical research of HIV Ab+ patients is the only way, at the moment, to underline and follow the cardiac alterations' evolution, also in relation to a precocious antiretroviral therapy.

Pulsatile glucagon has greater hyperglycaemic, lipolytic and ketogenic effects than continuous hormone delivery in man: effect of age.

The present study aimed at investigating the hyperglycaemic, lipolytic and ketogenic effects of small doses of glucagon delivered continuously or in a pulsatile manner. The study was performed in eight healthy young volunteers (24.2 +/- 1.2 years) and in eight healthy aged subjects (69.4 +/- 2.0 years). In all the subjects, endogenous pancreatic hormone secretion was inhibited by somatostatin and only glucagon was replaced. Consequently, the effects of pulsatile and continuous glucagon delivery were studied in conditions of progressive somatostatin-induced insulin deficiency. In both the young and the aged subjects, pulsatile glucagon delivery resulted in increases in plasma glucose, non-esterified fatty acid, glycerol and beta-hydroxybutyrate levels greater than those observed when the same amount of glucagon was delivered in a continuous manner. The net increases in plasma glucose, glycerol and non-esterified fatty acid levels were similar between the young and the aged subjects when glucagon was infused continuously; in contrast, the rise in plasma beta-hydroxybutyrate in the aged was only about half that observed in the young subjects. Surprisingly, when glucagon was infused in a pulsatile manner, the rises in plasma glycerol, non-esterified fatty acid and beta-hydroxybutyrate levels were all significantly smaller in the aged subjects, while no significant differences were observed in the blood glucose responses. We conclude that, in the presence of somatostatin-induced insulin deficiency, pulsatile glucagon exerts greater effects on blood glucose, plasma non-esterified fatty acid, glycerol and beta-hydroxybutyrate levels than its continuous delivery. In the elderly, the lipolytic and ketogenic, but not the hyperglycaemic, responses to pulsatile glucagon are significantly reduced.

[Maternal-fetal transmission of hepatitis B virus. Follow-up study of 21 infants born to HBSAG-positive mothers, and 6 born to HBSAG-negative mothers with anti-HBV antibodies]. / La trasmissione materno-fetale del virus della epatite B (HBV). Osservazione nel tempo di 21 nati da madri HBsAg positive e di 6 nati da madri HBsAg negative con anticorpi anti-HBV.

Vertical transmission of hepatitis B virus (HBV) was studied in 21 mother-infant pairs. A transient HBs-antigenemia was observed in 60% of the 10 children born to HBsAg healthy carrier mothers and 91% of the 11 babies born to mothers with HBsAg-positive CAH. Among the 16 children showing HBs-antigenemia, 3 developed acute viral hepatitis two of whom showed progression to chronicity.