Drug Integrating Amphiphilic Nano-Assemblies: 2. Spatiotemporal Distribution within Inflammation Sites.

The need for chronic systemic immunosuppression, which is associated with unavoidable side-effects, greatly limits the applicability of allogeneic cell transplantation for regenerative medicine applications including pancreatic islet cell transplantation to restore insulin production in type 1 diabetes (T1D). Cell transplantation in confined sites enables the localized delivery of anti-inflammatory and immunomodulatory drugs to prevent graft loss by innate and adaptive immunity, providing an opportunity to achieve local effects while minimizing unwanted systemic side effects. Nanoparticles can provide the means to achieve the needed localized and sustained drug delivery either by graft targeting or co-implantation. Here, we evaluated the potential of our versatile platform of drug-integrating amphiphilic nanomaterial assemblies (DIANAs) for targeted drug delivery to an inflamed site model relevant for islet transplantation. We tested either passive targeting of intravenous administered spherical nanomicelles (nMIC; 20-25 nm diameter) or co-implantation of elongated nanofibrils (nFIB; 5 nm diameter and >1 µm length). To assess the ability of nMIC and nFIB to target an inflamed graft site, we used a lipophilic fluorescent cargo (DiD and DiR) and evaluated the in vivo biodistribution and cellular uptake in the graft site and other organs, including draining and non-draining lymph nodes, after systemic administration (nMIC) and/or graft co-transplantation (nFIB) in mice. Localized inflammation was generated either by using an LPS injection or by using biomaterial-coated islet-like bead implantation in the subcutaneous site. A cell transplant inflammation model was used as well to test nMIC- and nFIB-targeted biodistribution. We found that nMIC can reach the inflamed site after systemic administration, while nFIB remains localized for several days after co-implantation. We confirmed that DIANAs are taken up by different immune cell populations responsible for graft inflammation. Therefore, DIANA is a useful approach for targeted and/or localized delivery of immunomodulatory drugs to decrease innate and adaptive immune responses that cause graft loss after transplantation of therapeutic cells.

Parallel Evaluation of Polyethylene Glycol Conformal Coating and Alginate Microencapsulation as Immunoisolation Strategies for Pancreatic Islet Transplantation.

Pancreatic islet transplantation improves metabolic control and prevents complications in patients with brittle type 1 diabetes (T1D). However, chronic immunosuppression is required to prevent allograft rejection and recurrence of autoimmunity. Islet encapsulation may eliminate the need for immunosuppression. Here, we analyzed in parallel two microencapsulation platforms that provided long-term diabetes reversal in preclinical T1D models, alginate single and double capsules versus polyethylene glycol conformal coating, to identify benefits and weaknesses that could inform the design of future clinical trials with microencapsulated islets. We performed in vitro and in vivo functionality assays with human islets and analyzed the explanted grafts by immunofluorescence. We quantified the size of islets and capsules, measured capsule permeability, and used these data for in silico simulations of islet functionality in COMSOL Multiphysics. We demonstrated that insulin response to glucose stimulation is dependent on capsule size, and the presence of permselective materials augments delays in insulin secretion. Non-coated and conformally coated islets could be transplanted into the fat pad of diabetic mice, resulting in comparable functionality and metabolic control. Mac-2+ cells were found in conformally coated grafts, indicating possible host reactivity. Due to their larger volume, alginate capsules were transplanted in the peritoneal cavity. Despite achieving diabetes reversal, changes in islet composition were found in retrieved capsules, and recipient mice experienced hypoglycemia indicative of hyperinsulinemia induced by glucose retention in large capsules as the in silico model predicted. We concluded that minimal capsule size is critical for physiological insulin secretion, and anti-inflammatory modulation may be beneficial for small conformal capsules.

Performance of islets of Langerhans conformally coated via an emulsion cross-linking method in diabetic rodents and nonhuman primates.

Polyethylene glycol (PEG)-based conformal coating (CC) encapsulation of transplanted islets is a promising ß cell replacement therapy for the treatment of type 1 diabetes without chronic immunosuppression because it minimizes capsule thickness, graft volume, and insulin secretion delay. However, we show here that our original CC method, the direct method, requiring exposure of islets to low pH levels and inclusion of viscosity enhancers during coating, severely affected the viability, scalability, and biocompatibility of CC islets in nonhuman primate preclinical models of type 1 diabetes. We therefore developed and validated in vitro and in vivo, in several small- and large-animal models of type 1 diabetes, an augmented CC method-emulsion method-that achieves hydrogel CCs around islets at physiological pH for improved cytocompatibility, with PEG hydrogels for increased biocompatibility and with fivefold increase in encapsulation throughput for enhanced scalability.

A novel Xp22.11 deletion causing a syndrome of craniosynostosis and periventricular nodular heterotopia.

We report on a follow-up evaluation of a male with a phenotype including craniosynostosis, periventricular nodular heterotopia, and neurodevelopmental delay. He was initially assigned a clinical diagnosis of Fontaine-Farriaux syndrome (FFS) as an infant although now, with improved delineation of this entity, it is evident that this diagnosis is not applicable to this individual. Array comparative genomic hybridization has demonstrated a 300 kb interstitial deletion on Xp22.11 affecting all or part of three annotated genes, ZFX, PDK3, and PCYT1B in this subject. The deletion was inherited from the phenotypically normal mother who also exhibited markedly skewed X-inactivation. These findings implicate hemizygosity for one or all three of these genes as the cause of this phenotype.

Growth hormone secretory pattern in non-obese children and adolescents with Prader-Willi syndrome.

The aetiology of impaired growth hormone (GH) secretion in Prader-Willi syndrome (PWS) remains controversial due to the common occurrence of obesity. To further clarify whether suboptimal GH secretion in PWS is an artefact of excess weight, we evaluated both GH immunological activity and GH bioactivity after arginine administration in 23 non-obese PWS patients [seven females, aged 6.9 +/- 0.9 years, body mass index (BMI) SDS 0.63 +/- 0.26], in comparison with a control group of 32 healthy subjects, matched for age, gender and BMI (10 females, aged 7.9 +/- 0.3 years, BMI SDS 0.21 +/- 0.20). Serum GH concentration was measured with a time-resolved immunofluorometric assay (IFMA), while GH bioactivity was evaluated by the Nb2 cell bioassay. Serum IGF-I concentrations were measured by double-antibody RIA. GH mean peak after pharmacological stimulation was significantly lower in PWS individuals compared with controls when measured either by IFMA (6.05 +/- 1.23 microg/L vs. 23.7 +/- 1.06 microg/L, p < 0.0001) or by Nb2 (6.87 +/- 0.55 microg/L vs. 12.88 +/- 0.19 microg/L, p < 0.0001). Analysis of integrated GH secretion (AUC) confirmed that the PWS group differed significantly from the control subjects (387.9 +/- 76.1 microg/L/h vs. 1498.1 +/- 56.2 microg/L/h, p < 0.0001); the same result was obtained when the GH rise after arginine administration was expressed as nAUC (278.2 +/- 53.3 microg/L/h vs. 1443.6 +/- 52.5 microg/L/h, p < 0.0001). PWS patients had an IGF-I SDS significantly lower than those found in control subjects (p < 0.0001). Subnormal IGF-I values were present in 19 PWS individuals (82.6%) and two healthy controls (6.2%). These findings are in agreement with the hypothesis that a complex derangement of hypothalamus-pituitary axis occurs in PWS.

Drug-Integrating Amphiphilic Nanomaterial Assemblies: 1. Spatiotemporal control of cyclosporine delivery and activity using nanomicelles and nanofibrils.

Immunomodulatory therapies are limited by unavoidable side effects as well as poor solubility, stability, and pharmacokinetic properties. Nanomaterial-based drug delivery may overcome these limitations by increasing drug solubility, site-targeting, and duration of action. Here, we prepared innovative drug-integrating amphiphilic nanomaterial assemblies (DIANA) with tunable hydrophobicity, size, and morphology, and we evaluated their ability to deliver cyclosporine A (CsA) for immunomodulatory applications. We synthesized amphiphilic block copolymers made of poly(ethylene glycol)-poly(propylene sulfide) (PEG-PPS) and poly(ethylene glycol)-oligo(ethylene sulfide) (PEG-OES) that can self-assemble into solid core nanomicelles (nMIC, with ≈20 nm diameter) and nanofibrils (nFIB, with ≈5 nm diameter and > 500 nm length), respectively. nMIC and nFIB displayed good CsA encapsulation efficiency (up to 4.5 and 2 mg/mL, respectively in aqueous solution), superior to many other solubilization methods, and provided sustained release (>14 and > 7 days for the nMIC and nFIB) without compromising CsA's pharmacological activity. Treatment of insulin-secreting cells with unloaded DIANAs did not impair cell viability and functionality. Both CsA-loaded DIANAs inhibited the proliferation and activation of insulin-reactive cytotoxic T cells in vitro. Subcutaneous injections of CsA-loaded DIANAs in mice provided CsA sustained release, decreasing alloantigen-induced immune responses in the draining lymph node at lower doses and reduced administration frequency than unformulated CsA. While nMIC solubilized higher amounts and provided more sustained release of CsA in vitro, nFIB enhanced cellular uptake and promoted local retention due to slower trafficking in vivo. DIANAs provide a versatile platform for a local immune suppression regimen that can be applied to allogeneic cell transplantation.

Conformal Coating of Stem Cell-Derived Islets for ß Cell Replacement in Type 1 Diabetes.

The scarcity of donors and need for immunosuppression limit pancreatic islet transplantation to a few patients with labile type 1 diabetes. Transplantation of encapsulated stem cell-derived islets (SC islets) might extend the applicability of islet transplantation to a larger cohort of patients. Transplantation of conformal-coated islets into a confined well-vascularized site allows long-term diabetes reversal in fully MHC-mismatched diabetic mice without immunosuppression. Here, we demonstrated that human SC islets reaggregated from cryopreserved cells display glucose-stimulated insulin secretion in vitro. Importantly, we showed that conformally coated SC islets displayed comparable in vitro function with unencapsulated SC islets, with conformal coating permitting physiological insulin secretion. Transplantation of SC islets into the gonadal fat pad of diabetic NOD-scid mice revealed that both unencapsulated and conformal-coated SC islets could reverse diabetes and maintain human-level euglycemia for more than 80 days. Overall, these results provide support for further evaluation of safety and efficacy of conformal-coated SC islets in larger species.

Clinical and genetic characterization of Chanarin-Dorfman syndrome.

We describe the clinical features, muscle pathology features, and molecular studies of seven patients with Chanarin-Dorfman syndrome (CDS) or neutral lipid storage disease and ichthyosis (NLSDI), a multisystem triglyceride storage disease with massive accumulation of lipid droplets in muscle fibers. All patients presented with congenital ichthyosiform erythroderma, cytoplasmic lipid droplets in blood cells, mild to severe hepatomegaly, and increased serum CK levels and liver enzymes. Three patients showed muscle symptoms and three had steathorrea. Molecular analysis identified five mutations, three of which are novel. These findings expand the clinical and mutational spectrum and underline the genetic heterogeneity of this disease.

The Italian National Survey for Prader-Willi syndrome: an epidemiologic study.

Twenty-five medical centers and the Prader-Willi Syndrome (PWS) Association collaborated on a study which attempted to identify all people with genetically confirmed diagnosis of PWS living in Italy. Investigators of the participating centers contacted PWS subjects and/or their family, filled in a specially developed form with the required data and forwarded this information by email. The study identified 425 subjects (209 males and 216 females, between the ages of 0.4-46.7). Two hundred thirty-eight patients had del15, 104 had UPD15, 4 demonstrated a translocation affecting chromosome 15 and 79 showed a positive methylation test. There were fewer subjects found over the age of 35, probably due to the low rate of identification of older PWS patients as well as the high mortality rate. There were a greater number of male children and adolescents with PWS whilst, amongst adults, there were more females. As expected, the majority of subjects with PWS were obese, especially in adult life. Nevertheless, it is noteworthy that 26% of patients aged between 6 and 17 were normal weight. A total of 212 subjects had received GH treatment, of which 141 were still receiving therapy, while the remaining 71 had stopped. In children and adolescents (233 cases), 89 subjects had never undergone GH therapy. Eighteen PWS patients had died in the past 20 years. Obesity-related cardiovascular and respiratory diseases were the cause of death, both during childhood and after 18 years of age. Three children died suddenly whilst undergoing GH therapy. Respiratory infection and cardiac illness were the causes of death in two cases. There was no definitive cause of death found in the third case. Overall, there was no increase in number of deaths during GH treatment, suggesting that GH administration in patients with PWS, as a group, does not increase the risk of death.

Unusual white matter involvement in EAST syndrome associated with novel KCNJ10 mutations.

BACKGROUND: Epilepsy, ataxia, sensorineural deafness, and tubulopathy (EAST syndrome) is a rare channelopathy due to KCNJ10 mutations. So far, only mild cerebellar hypoplasia and/or dentate nuclei abnormalities have been reported as major neuroimaging findings in these patients. METHODS: We analyzed the clinical and brain MRI features of two unrelated patients (aged 27 and 23 years) with EAST syndrome carrying novel homozygous frameshift mutations (p.Asn232Glnfs*14and p.Gly275Valfs*7) in KCNJ10, detected by whole exome sequencing. RESULTS: Brain MRI examinations at 8 years in Patient 1 and at 13 years in Patient 2 revealed a peculiar brain and spinal cord involvement characterized by restricted diffusion of globi pallidi, thalami, brainstem, dentate nuclei, and cervical spinal cord in keeping with intramyelinic edema. The follow-up studies, performed, respectively, after 19 and 10 years, showed mild cerebellar atrophy and slight progression of the brain and spinal cord T2 signal abnormalities with increase of the restricted diffusion in the affected regions. CONCLUSION: The present cases harboring novel homozygous frameshift mutations in KCNJ10 expand the spectrum of brain abnormalities in EAST syndrome, including mild cerebellar atrophy and intramyelinic edema, resulting from abnormal function of the Kir4.1 inwardly rectifying potassium channel at the astrocyte endfeet, with disruption of water-ion homeostasis.

Wolfram syndrome (diabetes insipidus, diabetes, optic atrophy, and deafness): clinical and genetic study.

OBJECTIVE: Wolfram syndrome is an autosomal recessive neurodegenerative disorder characterized by diabetes insipidus, diabetes (nonautoimmune), optic atrophy, and deafness (a set of conditions referred to as DIDMOAD). The WFS1 gene is located on the short arm of chromosome 4. Wolfram syndrome prevalence is 1 in 770,000 live births, with a 1 in 354 carrier frequency. RESEARCH DESIGN AND METHODS: We evaluated six Italian children from five unrelated families. Genetic analysis for Wolfram syndrome was performed by PCR amplification and direct sequencing. RESULTS: Mutation screening revealed five distinct variants, one novel mutation (c.1346C>T; p.T449I) and four previously described, all located in exon 8. CONCLUSIONS: Phenotype-genotype correlation is difficult, and the same mutation gives very different phenotypes. Severely inactivating mutations result in a more severe phenotype than mildly inactivating ones. Clinical follow-up showed the progressive syndrome's seriousness.