Some inborn errors of metabolism due to deficiencies of soluble lysosomal enzymes cause global neurodegenerative disease. Representative examples include the infantile and late infantile forms of the ceroid lipofuscinoses (CLN1 or CLN2 deficiency, respectively) and mucopolysaccharidoses type VII (MPS VII), a deficiency of ␤-glucuronidase. Treatment of the central nervous system component of these disorders will require widespread protein or enzyme replacement, either through dissemination of the protein or through dissemination of a gene encoding it. We hypothesize that transduction of brain microcapillary endothelium (BME) with recombinant viral vectors, with secretion of enzyme product basolaterally, could allow for widespread enzyme dissemination. To achieve this, viruses should be modified to target the BME. This requires (i) identification of a BME-resident target receptor, (ii) identification of motifs targeted to that molecule, (iii) the construction of modified viruses to allow for binding to the target receptor, and (iv) demonstrated transduction of receptor-expressing cells. In proof of principal experiments, we chose the human transferrin receptor (hTfR), a molecule found at high density on human BME. A nonamer phage display library was panned for motifs which could bind hTfR. Forty-three clones were sequenced, most of which contained an AKxxK/R, KxKxPK/R, or KxK motif. Ten peptides representative of the three motifs were cloned into the HI loop of adenovirus type 5 fiber. All motifs tested retained their ability to trimerize and bind transferrin receptor, and seven allowed for recombinant adenovirus production. Importantly, the fiber-modified viruses facilitated increased gene transfer (2-to 34-fold) to hTfR expressing cell lines and human brain microcapillary endothelia expressing high levels of endogenous receptor. Our data indicate that adenoviruses can be modified in the HI loop for expanded tropism to the hTfR.Many inherited metabolic disorders lead to central nervous system (CNS) deficits, either alone or in combination with systemic involvement (24). One approach to metabolic correction is by cellular transduction with virus vectors encoding a functional cDNA. For correction of the CNS component, therapies will likely require direct application to brain parenchyma, since closure of the blood-brain barrier (BBB) shortly after birth would restrict entry of the gene product or gene transfer vectors into the brain. In metabolic disorders due to deficiencies in soluble lysosomal proteins, genetic correction of all affected cells will not be required; secretion of overexpressed protein provides a pool of available enzyme for distribution to surrounding cells (cross-correction). Examples of such disorders include the ceroid lipofuscinoses I and II and mucopolysaccharidoses type VII (MPS VII). But even with cross-correction, spread of enzyme is limited. Thus, an important remaining problem for clinical application is how to affect global correction in these disorders.Earlier studies using MPS VII mous...

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Sex differences in the effect of diet-induced obesity (DIO) have been reported in juvenile mice. However, thorough side by side comparisons of the effects of DIO in males and females at different ages of onset have yet to be examined. We hypothesized that aged females would lose their protection, relative to males, from the effects of DIO. We examined the effect of DIO on body weight and glucose tolerance in juvenile, young adult, and middle-aged male and female mice. Our data show DIO in juvenile mice causes a greater increase in body weight and greater impairment in glucose tolerance in males than females. However, if the diet is initiated in young adult mice, these sex differences are absent. Further, if the diet is initiated in middle-aged mice, the sex difference is reversed, and females gain more weight and have greater impairment in glucose tolerance than males. Our data show that sex differences in the effect of DIO vary by age of onset; thus highlighting the importance of both age and sex as biological variables in DIO research.

1158 sentinel lymph nodes (SLNs), excised from patients with primary cutaneous melanoma, were assessed pathologically using histology with immunohistochemistry (IHC) on all nodes, and RT-PCR for Mart-1 and tyrosinase on 55 nodes. RT-PCR was compared with the histology and IHC assessed on the same nodes. The evaluation of progressively more detailed protocols for histology and IHC modulated by the RT-PCR results led to a procedure that consistently detects metastases in 34% of patients submitted to SLN biopsy for cutaneous melanomas with a vertical growth phase and a mean thickness of 2.02 mm (range 0.25, with regression, to 19 mm). As this technique is virtually free of false positives and produces only a marginally lower detection rate than RT-PCR, which was subject to false positives of 7% in our study, it is suggested that this extended protocol should be the basis on which further evaluation of the place of RT-PCR in SLN assessment takes place. The evolved protocol described here has been adopted by the EORTC as the standard procedure for pathological handling of sentinel lymph nodes for melanoma when SLN status is a criterion in their clinical trials or studies.

Type 1 Diabetes (T1D) is a chronic pro-inflammatory autoimmune disease consisting of islet-infiltrating leukocytes involved in pancreatic β-cell lysis. One promising treatment for T1D is islet transplantation; however, clinical application is constrained due to limited islet availability, adverse effects of immunosuppressants, and declining graft survival. Islet encapsulation may provide an immunoprotective barrier to preserve islet function and prevent immune-mediated rejection after transplantation. We previously demonstrated that a novel cytoprotective nanothin multilayer coating for islet encapsulation consisting of tannic acid (TA), an immunomodulatory antioxidant, and poly(N-vinylpyrrolidone) (PVPON), was efficacious in dampening in vitro immune responses involved in transplant rejection and preserving in vitro islet function. However, the ability of (PVPON/TA) to maintain islet function in vivo and reverse diabetes has not been tested. Recent evidence has demonstrated that modulation of redox status can affect pro-inflammatory immune responses. Therefore, we hypothesized that transplanted (PVPON/TA)-encapsulated islets can restore euglycemia to diabetic mice and provide an immunoprotective barrier. Our results demonstrate that (PVPON/TA) nanothin coatings can significantly decrease in vitro chemokine synthesis and diabetogenic T cell migration. Importantly, (PVPON/TA)-encapsulated islets restored euglycemia after transplantation into diabetic mice. Our results demonstrate that (PVPON/TA)-encapsulated islets may suppress immune responses and enhance islet allograft acceptance in patients with T1D.

Lower estimated insulin sensitivity was associated with risk for hyperfiltration over time, whereas increased albumin excretion was associated with hyperglycemia in youth-onset T2DM.

Adult hippocampal neurogenesis (AHN) is suppressed by high-fat (HF) diet and metabolic disease, including obesity and type 2 diabetes. Deficits in AHN may contribute to cognitive decline and increased risk of dementia and mood disorders, which have higher prevalence in women. However, sex differences in the effects of HF diet/metabolic disease on AHN have yet to be thoroughly investigated. Herein, male and female C57BL/6J mice were fed an HF or control (CON) diet from ϳ2 to 6 months of age. After 3 months on the diet, mice were injected with 5-ethynyl-2=-deoxyuridine (EdU) then killed 4 weeks later. Cell proliferation, differentiation/maturation, and survival of new neurons in the dentate gyrus were assessed with immunofluorescence for EdU, Ki67, doublecortin (DCX), and NeuN. CON females had more proliferating cells (Ki67 ϩ) and neuroblasts/immature neurons (DCX ϩ) compared with CON males; however, HF diet reduced these cells in females to the levels of males. Diet did not affect neurogenesis in males. Further, the numbers of proliferating cells and immature neurons were inversely correlated with both weight gain and glucose intolerance in females only. These effects were robust in the dorsal hippocampus, which supports cognitive processes. Assessment of microglia in the dentate gyrus using immunofluorescence for Iba1 and CD68 uncovered sex-specific effects of diet, which may contribute to observed differences in neurogenesis. These findings demonstrate sex-specific effects of HF diet/metabolic disease on AHN, and highlight the potential for targeting neurogenic deficits to treat cognitive decline and reduce the risk of dementia associated with these conditions, particularly in females.

Optical linewidth broadening through both white noise (WNS) and pseudo-random binary sequence (PBRS) phase modulation are effective techniques for suppressing stimulated Brillouin scattering (SBS) in high- power fiber amplifiers. However, detailed studies comparing both coherent beam combining and SBS suppression of these phase modulation schemes have not been reported. In this study, a passive fiber cutback experiment is performed comparing the SBS threshold enhancement factor of a PRBS and WNS broadened seed as a function of linewidth and fiber length. Particularly, assuming an optimal PRBS pattern is chosen, pseudo-random modulation provides superior SBS suppression than WNS for a given fiber length and signal linewidth. Furthermore, two WNS and PRBS modulated 150 W fiber lasers are coherently combined to measure and compare the combining efficiency, beam quality, and coherence as a function of optical path length difference. Notably, the discrete spectral density of PRBS modulation provides a re-coherence effect where the lasers periodically come back into phase. Overall, this may reduce path length matching complexity in coherently combined fiber laser systems.