No abstract

The neuronal cytoskeleton is one of the most profoundly altered organelles in late life neuro-degenerative disorders that are characterized by progressive impairments in cognitive abilities. The elucidation of the protein building blocks of these organelles as well as advances in understanding how these proteins become altered in Alzheimer's disease (AD) and other less common dementing illnesses, i.e., diffuse Lewy body disease (DLBD) or the Lewy body variant of AD (LBVAD), will provide insights into the molecular basis of these disorders. Within, we review evidence that normal adult human tau is abnormally phosphorylated and converted into the subunits of AD paired helical filaments (PHFs), and that Lewy bodies (LBs) represent accumulation of altered neurofilament (NF) triplet subunits. Although the precise biological consequences of PHF and LB formation in neurons is unknown, growing evidence suggests that the formation of PHFs and LBs from normal neuronal cytoskeletal proteins could have deleterious effects on neuronal function and survival. Finally, insights into the composition of PHFs and LBs could lead to the development of novel strategies for the timely and accurate diagnosis of AD, DLBD and the LBVAD.

Tacrolimus (FK506) has recently been approved for immunosuppression in organ transplantation, although its use is accompanied by a wide spectrum of neurotoxic side effects. We describe the clinical, radiological, and pathological features of 3 cases of tacrolimus-related leukoencephalopathy. The syndrome of immunosuppression-related leukoencephalopathy is proposed as an uncommon neurological syndrome occurring in patients with organ transplants involving demyelination, in particular in the parieto-occipital region and centrum semiovale. Although the syndrome is not associated with a particular (absolute) serum level of tacrolimus, it resolves spontaneously upon decreasing the dose. The tacrolimus-related syndrome has a similar radiographic and pathologic appearance as the analogous syndrome that occurs in patients taking cyclosporine.

Aberrandy phosphorylated tau proteins (i.e., A68 or PHF-tau) and (-amyloid MATERIALS AND METHODS Isolation and Characterization of A68, Dephosphorylated A68 (DEP-A68), and Normal Tau. A68 was purified from the brains of four AD patients and one Down syndrome patient with . To render A68 water soluble for injection, A68 was further purified as follows. After sucrose gradient centrifugation (14,15), the 1.25-2.0 M and 2.25-2.5 M sucrose fractions were extracted in 2 M guanidine isothiocyanate at 37°C for 60 min, and the guanidine-insoluble material was removed by further centrifugation for 30 min at 100,000 x g. The supernatant was exhaustively dialyzed against distilled water, and the water-insoluble material was removed by centrifugation once again. The resulting supernatant was lyophilized and used for injection into rats as well as for the generation of DEP-A68. This guanidine-extracted, water-soluble supernatant was shown by Western blots (see below) to contain purified A68. Although the A68 preparation contained PHFs (14) prior to guanidine extraction, the guanidine-extracted, water-soluble A68 did not reassemble into PHFs or straight filaments in vitro (data not shown), as monitored by negative staining and electron microscopy (14). DEP-A68 was generated from A68 by enzymatic dephosphorylation after overnight incubation in type III-N Escherichia coli alkaline phosphatase (20 units/ml) at 37°C as described (14). Normal adult human tau was prepared exactly as described (14,15). Aliquots of the A68, DEP-A68, and normal adult human tau preparations that were used for injection were analyzed by gel electrophoresis and by Western blots with epitope-specific antibodies to A68 and tau according to described methods (2,3,14,15). The anti-tau and anti-A68 antibodies used in this study included Alz5O to residues 2-10; T60 to residues 119-150; T14 to residues 141-178; T46 to residues 404-441; Taul, which recognizes tau and DEP-A68, but not A68, and binds to a nonphosphorylated epitope within residues 189-207; T3P, which recognizes A68, but not tau or DEP-A68, and binds to an epitope within residues 389-402 that contains a phosphate at Ser-396; and PHF1, which is similar to the T3P antiserum (for further information on these antibodies, see refs. 2, 3, and 14-21 and citations therein; the numbering system for the amino acids in tau referred to here is based on the largest tau isoform, as described in ref. 22

Current therapies available to treat and heal ocular surface injuries and periocular burns are frequently inadequate, costly, and labor intensive. To address these limitations, we have employed a flexible, semitransparent ocular wound chamber (OWC) to provide protection as well as a watertight seal to allow for the constant delivery of therapeutics to the ocular surface and surrounding periocular tissue. This study demonstrates the safety and utilization of the OWC on uninjured eyes and in our exposure keratopathy model. For initial safety studies (N = 3 per group), the eyelids remained intact and the eye uninjured. A blepharotomy (N = 6 per group) was performed to remove the upper and lower eyelids surrounding the left (OS) eye to create our exposure keratopathy model. Right (OD) eyes served as uninjured controls in all studies. Following OWC placement, 0.5 mL HPMC gel or balanced saline solution (BSS) was injected into the chamber. Animals were monitored daily and fully assessed via white light, fluorescein, and OCT imaging at least through 72 hours post OWC placement. In studies that included a blepharotomy, skin samples were analyzed by multiplex cytokine analysis. Results of safety experiments revealed no significant differences between treatment groups in corneal thickness, fluorescein staining, OCT imaging, or histological eye or skin sections when compared to control eyes. In our exposure keratopathy model, OWC treated eyes showed significantly less fluorescein uptake and also were found to have significantly lower levels of cytokines IL-13 and IL-5 in skin samples. These results demonstrate for the first time that treatment using the OWC device is not only safe, but significantly protects against blepharotomy-induced exposure keratopathy. As a whole, this study advances our overall efforts to develop a feasible solution to treat ocular surface injuries, infections, and periocular burns.