Nanoliposomal minocycline for ocular drug delivery.

Nanoliposomal technology is a promising drug delivery system that could be employed to improve the pharmacokinetic properties of clearance and distribution in ocular drug delivery to the retina. We developed a nanoscale version of an anionic, cholesterol-fusing liposome that can encapsulate therapeutic levels of minocycline capable of drug delivery. We demonstrate that size extrusion followed by size-exclusion chromatography can form a stable 80-nm liposome that encapsulates minocycline at a concentration of 450 ± 30 µM, which is 2% to 3% of loading material. More importantly, these nontoxic nanoliposomes can then deliver 40% of encapsulated minocycline to the retina after a subconjunctival injection in the STZ model of diabetes. Efficacy of therapeutic drug delivery was assessed via transcriptomic and proteomic biomarker panels. For both the free minocycline and encapsulated minocycline treatments, proinflammatory markers of diabetes were downregulated at both the messenger RNA and protein levels, validating the utility of biomarker panels for the assessment of ocular drug delivery vehicles. FROM THE CLINICAL EDITOR: Authors developed a nano-liposome that can encapsulate minocycline for optimized intraocular drug delivery. These nontoxic nanoliposomes delivered 40% of encapsulated minocycline to the retina after a subconjunctival injection in a diabetes model.

Exogenous ceramide-1-phosphate reduces lipopolysaccharide (LPS)-mediated cytokine expression.

Toll-like receptor 4 (TLR4) is a component of the innate immune system that recognizes a diverse group of molecular structures, such as lipopolysaccharide (LPS) from Gram-negative bacteria. TLR4 signaling ultimately leads to activation of the transcription factor, nuclear factor κB (NF-κB), and the production of cytokines. Ceramide is a bioactive sphingolipid that has been suggested to regulate TLR4-induced NF-κB signaling, although reports on the role of ceramide in TLR4 activation conflict. We investigated the possibility that ceramide metabolites, such as ceramide-1-phosphate (C-1-P), may explain these discrepancies. We now report that exogenous C-1-P, but not ceramide, reduces NF-κB-mediated gene transcription in HEK 293 cells stably transfected with human TLR4, CD14, and MD-2. We demonstrate that inhibition of NF-κB by exogenous C-1-P is dose-dependent and specific to TLR4 in a reporter assay. We further demonstrate a requirement for both the phosphate moiety and the sphingoid backbone to inhibit LPS-activated NF-κB transcription. Specifically, C-1-P prevents the degradation of IκB, the phosphorylation of the p65 subunit of NF-κB, and LPS-stimulated MAPK activation. The functional consequence of C-1-P inhibition of NF-κB is a reduction in LPS-mediated cytokine release from HEK 293 TLR4-expressing cells and human peripheral blood mononuclear cells. Taken together, these data demonstrate that C-1-P may function as an anti-inflammatory lipid mediator of immune response.

Circulating sphingolipid biomarkers in models of type 1 diabetes.

Alterations in lipid metabolism may contribute to diabetic complications. Sphingolipids are essential components of cell membranes and have essential roles in homeostasis and in the initiation and progression of disease. However, the role of sphingolipids in type 1 diabetes remains largely unexplored. Therefore, we sought to quantify sphingolipid metabolites by LC-MS/MS from two animal models of type 1 diabetes (streptozotocin-induced diabetic rats and Ins2(Akita) diabetic mice) to identify putative therapeutic targets and biomarkers. The results reveal that sphingosine-1-phosphate (So1P) is elevated in both diabetic models in comparison to respective control animals. In addition, diabetic animals demonstrated reductions in plasma levels of omega-9 24:1 (nervonic acid)-containing ceramide, sphingomyelin, and cerebrosides. Reduction of 24:1-esterfied sphingolipids was also observed in liver and heart. Nutritional stress via a high-fat diet also reduced 24:1 content in the plasma and liver of mice, exacerbating the decrease in some cases where diabetes was also present. Subcutaneous insulin corrected both circulating So1P and 24:1 levels in the murine diabetic model. Thus, changes in circulating sphingolipids, as evidenced by an increase in bioactive So1P and a reduction in cardio- and neuro-protective omega-9 esterified sphingolipids, may serve as biomarkers for type 1 diabetes and represent novel therapeutic targets.

Autoreactive human T-cell receptor initiates insulitis and impaired glucose tolerance in HLA DR4 transgenic mice.

A human T-cell receptor (TcR) derived from an autoreactive T-cell specific for GAD65, from a subject at high risk for autoimmune diabetes, was introduced into HLA-DR4 transgenic mice. The source of TcR was a CD4(+) T(H)1(+) T-cell clone which responded to an immunodominant epitope of the human islet protein GAD65, an epitope shared with both GAD65 and GAD67 in the mouse. The resulting HLA-DR4/GAD-TcR transgenic mice on a Rag2(o/o)/I-Ab(o/o)/B6 background exhibited a CD4(+) infiltrate into pancreatic islets that correlated with a loss of insulin in infiltrated islets. These mice also exhibited a subclinical impaired tolerance to exogenously fed glucose as assayed by an intraperitoneal glucose tolerance test. T cells containing the GAD65/67 (555-567) responsive TcR undergo strong negative selection as evidenced by a 10-fold lower thymocyte cellularity compared to non-TcR transgenic mice, and clonotype peripheral T cells represented approximately 1% of CD4(+) T cells in Rag2 sufficient mice. Upon in vitro stimulation, GAD65/67 555-567 responsive T cells secrete interferon-gamma, minimal interleukin (IL)-2 and tumor necrosis factor-alpha, and no IL-4, IL-5, IL-10, or IL-17, consistent with a T(H)1 profile. These data demonstrate that CD4(+) T cells specific for a naturally processed epitope within GAD can specifically home to pancreatic islets and lead to impaired islet beta-cell function in diabetes-associated HLA-DR4 transgenic mice on the relatively non-autoimmune C57BL/6 background. The relatively slow progression and patchy insulitis are reminiscent of the chronic pre-clinical phase similar to a majority of human at-risk subjects, and models these indolent features of human T1D.

Age-dependent loss of tolerance to an immunodominant epitope of glutamic acid decarboxylase in diabetic-prone RIP-B7/DR4 mice.

We have identified for the first time an age-dependent spontaneous loss of tolerance to two self-antigenic epitopes derived from putative diabetes-associated antigens glutamic acid decarboxylase (GAD65) and glial fibrillary acidic protein (GFAP) in RIP-B7/DRB1*0404 HLA transgenic mice. Diabetic and older non-diabetic mice exhibited a proliferative response to an immunodominant epitope from GAD65 (555-567) and also from GFAP (240-252) but not from an immunogenic epitope from diabetes-associated islet-specific glucose-6-phosphatase catalytic subunit-related protein. The response to both of these self-antigens is not observed in young mice but is observed in older non-diabetic mice and is accompanied by histological evidence of insulitis in the absence of overt diabetes. Islet infiltrates in older non-diabetic mice and diabetic mice contain CD4(+)/FoxP3(+) cells and suggest the presence of a regulatory mechanism prior and during diabetic disease. Diabetes penetrance in RIP-B7/DR0404 mice is 23% with a mean onset age of 40 weeks and is similar to that reported for RIP-B7/DR0401 mice. A gender preference is observed in that 38% of female mice become diabetic compared to 8% of male mice.