Summary:Purpose: To evaluate their susceptibility to audiogenic seizures, five groups of knockout mice with various forms of fragile X genetic involvement [hemizygous males (n = 46), and homozygous (n = 38) and heterozygous females (n = 45), and their normal male (n = 45) and female (n = 52) littermates] were studied.Methods: All mouse groups were tested at ages 17, 22, 35, and 45 days. Audiogenic seizure susceptibility was scored, and the analysis of variance was used for the evaluation of the effects of age and genetic condition on seizure severity score (SSS).Results: All groups of knockout fragile X mice exhibited SSSs significantly higher than those observed in their wild-type littermates; among knockout mice, hemizygous males and homozygous females showed the highest SSSs. Hemizygous males showed higher SSSs with increasing age, from 17 to 45 days; homozygous females showed a peak at age 22 days, followed by a decrease; finally, heterozygous females had their highest SSSs at age 17 days.Conclusions: This study demonstrates that an increased susceptibility to audiogenic seizures is present in fragile X knockout mice at all the ages tested. These results support the validity of this animal model also for epilepsy and seizures in the human fragile X syndrome.

RESEARCH DESIGN -A multicenter study was performed in 70 diabetic patients. A microdialysis fiber was inserted subcutaneously into the periumbelical region and perfused with a buffer solution. Glucose concentrations in the dialysate were then measured every 3 min by the glucose sensor over a 24-h period, during which nine venous blood samples were also collected throughout the day.RESULTS -Both the insertion of the fiber and the wearing of the device were well tolerated by the patients. Subcutaneous glucose levels were well correlated with venous glucose measurements (r ϭ 0.9, P Ͻ 0.001) over a wide range (40 -400 mg/dl) for up to 24 h, with a single-point calibration. An analysis of 381 data pairs showed a linear relationship between the GlucoDay and serial venous blood glucose levels, and 97% of the data fell in the A and B regions of the error grid analysis. Percentage bias between the GlucoDay and the blood venous levels was Ϫ2.0% in the hypoglycemic range (Ͻ70 mg/dl), 6.9% in the euglycemic range (70 -180 mg/dl), and 11.2% in the hyperglycemic range (Ͼ180 mg/dl).CONCLUSIONS -The GlucoDay system demonstrated high reliability and reported values that closely agreed with venous blood glucose measurements. The system was well tolerated and thus constitutes a relatively easy method to monitor glucose excursions in diabetic patients. Diabetes Care 25:347-352, 2002T he benefits of strict metabolic control on microvascular complications of both type 1 and type 2 diabetes have been well established (1,2). In the last decade, self-monitoring of blood glucose levels has been the only available measure for diabetic patients to achieve a good metabolic control; however, glucose fluctuations during the day are often missed with this technique (3), and only continuous glucose measurements over prolonged periods can ensure optimal blood glucose management. Recently, minimally invasive techniques have been proposed for continuous monitoring of subcutaneous glucose in both normal and diabetic patients. Microdialysis of subcutaneous adipose tissue has been shown to identify glucose variations in vivo that closely mimic blood glucose patterns observed in patients on intensified insulin therapy. A monitoring system that provides automatic and frequent determinations would therefore identify the glycemic excursions and typical glucose trends in diabetic patients in a manner that is not possible even with frequent blood glucose meter readings. The ability to detect such fluctuations during the day, and particularly during the night, would enable appropriate changes in diabetes management in order to achieve the goal of optimal metabolic control in diabetic patients.We documented the efficacy of a new glucose sensor and its accuracy in monitoring glucose levels in type 1 and type 2 diabetic patients recruited in a multicenter study. The GlucoDay is composed of a subcutaneous microdialysis probe connected to a portable unit. The system takes a glucose measurement every second and stores an average value every 3 min, for a total of ...

T ime-related decline of human islet allograft (TX) function in generally immunosuppressed type 1 diabetic patients (1-5) has led us, after years of preclinical study (6), to initiate a phase 1 pilot clinical trial of microencapsulated TX into 10 nonimmunosuppressed patients with type 1 diabetes, under permission and surveillance by the Italian Ministry of Health (file no. 19382, PRE 805, 5 September 2003). RESEARCH DESIGN AND METHODS Human islet procurementHuman islets were isolated from singledonor pancreases according to the Edmonton protocol (1). Only preparations complying with standard quality control criteria (1) were considered for TX. The islets were cultured for 24 h in HAM F12 (Celbio, Milano, Italy), supplemented with antibiotics and 1.25% human albumin (Kedrion Spa, Milano, Italy) at 37°C in 95% air/CO 2 . MicroencapsulationThe islets were washed and thoroughly mixed with 1.6% endotoxin-and pyrogenfree sodium alginate (Stern Italia, Milan, Italy) that had been highly purified according to U.S. Pharmacopeia. Upon extrusion through a microdroplet generator (droplets generated by combination of air shears and mechanical pressure by a peristaltic pump), microdroplets containing the islets, upon collection in a CaCl 2 bath, turned into gel microbeads. The beads, containing 1-2 islets and measuring an average 500 m in diameter, were sequentially double-coated with 0.12% and 0.06% poly-L-ornithine (Sigma) and finally with 0.04% sodium alginate (Stern) (7). Patient recruitmentTen patients with long-standing type 1 diabetes on intensive insulin therapy regimens (lispro prebreakfast, lunch, and supper and glargine presupper) and undetectable serum C-peptide responses (sCPRs) were tested for complete blood chemistry, parameters of diabetes control (GHb, daily blood glucose profiles, and insulin requirement), anti-GAD 65 antibodies, and islet cell antibodies. Chest Xray and abdominal echography were also performed. Pre-and post-TX patient assessmentThe patients were maintained on euglycemia overnight before TX by supplementing exogenous insulin as required. After TX, the patients were monitored hourly for blood glucose (range: 80 -150 mg/dl) and insulin requirement. sCPR samples, assayed by radioimmunoassay (sCPR sensitivity: 0.06 ng/ml [intra-assay CV 3.7-4.5%, interassay CV 4.4 -5%]) (TechnoGenetics, Milan, Italy) in our laboratory, were drawn twice daily for the first 7 days and weekly thereafter, both in basal and 90 min after meals. A 75-g oral glucose tolerance test (OGTT) was scheduled for patient 1 at 60 days post-TX. At 7 days post-TX, the patients were discharged and instructed to continue blood glucose home selfmonitoring, with adjustments of insulin requirement. At 30 days post-TX, control abdominal echography was scheduled. Encapsulated human islets for TXA total of 400,000 and 600,000 microencapsulated islets (islet equivalents normalized to 150 ) were grafted in patient 1 and patient 2, respectively. The final TX volume included 50 ml of microcapsules diluted in 100 ml of saline. Empty microcaps...

Most genes associated with neurodevelopmental disorders (NDDs) were identified with an excess of de novo mutations (DNMs) but the significance in case–control mutation burden analysis is unestablished. Here, we sequence 63 genes in 16,294 NDD cases and an additional 62 genes in 6,211 NDD cases. By combining these with published data, we assess a total of 125 genes in over 16,000 NDD cases and compare the mutation burden to nonpsychiatric controls from ExAC. We identify 48 genes (25 newly reported) showing significant burden of ultra-rare (MAF < 0.01%) gene-disruptive mutations (FDR 5%), six of which reach family-wise error rate (FWER) significance (p < 1.25E−06). Among these 125 targeted genes, we also reevaluate DNM excess in 17,426 NDD trios with 6,499 new autism trios. We identify 90 genes enriched for DNMs (FDR 5%; e.g., GABRG2 and UIMC1); of which, 61 reach FWER significance (p < 3.64E−07; e.g., CASZ1). In addition to doubling the number of patients for many NDD risk genes, we present phenotype–genotype correlations for seven risk genes (CTCF, HNRNPU, KCNQ3, ZBTB18, TCF12, SPEN, and LEO1) based on this large-scale targeted sequencing effort.

Cell-based therapy holds great promise for tendon disorders, a widespread debilitating musculoskeletal condition. Even if the cell line remains to be defined, preliminary evidences have proven that amniotic-derived cells possess in vitro and in vivo a great tenogenic potential. This study investigated the efficacy of transplanted human amniotic epithelial cells (hAECs) by testing their early regenerative properties and mechanisms involved on a validated ovine Achilles tendon partial defect performed on 29 animals. The injured tendons treated with hAECs recovered rapidly, in 28 days, structural and biomechanical properties undertaking a programmed tissue regeneration, differently from the spontaneous healing tissues. hAECs remained viable within the host tendons establishing with the endogenous progenitor cells an active dialogue. Through the secretion of modulatory factors, hAECs inhibited the inflammatory cells infiltration, activated the M2 macrophage subpopulation early recruitment, and accelerated blood vessel as well as extracellular matrix remodelling. In parallel, some in situ differentiated hAECs displayed a tenocytelike phenotype. Both paracrine and direct hAECs stimulatory effects were confirmed analysing their genome profile before and after transplantation. The 49 human up-regulated transcripts recorded in transplanted hAECs belonged to tendon lineage differentiation (epithelial-mesenchymal transition, connective specific matrix components, and skeleton or muscle system development-related transcripts), as well as the in situ activation of paracrine signalling involved in inflammatory and immunomodulatory response. Altogether, these evidences support the hypothesis that hAECs are a practicable and efficient strategy for the acute treatment of tendinopathy, reinforcing the idea of a concrete use of amniotic epithelial cells towards the clinical practice.