Lysosomal membrane damage in soluble Abeta-mediated cell death in Alzheimer's disease.

Our previous studies suggest that a failure to degrade aggregated Abeta1-42 in late endosomes or secondary lysosomes is a mechanism that contributes to intracellular accumulation in Alzheimer's disease. In this study, we demonstrate that cultured primary neurons are able to internalize soluble Abeta1-42 from the culture medium and accumulate inside the endosomal/lysosomal system. The intracellular Abeta1-42 is resistant to protease degradation and stable for at least 48 h within the cultured neurons. Incubation of cultured neurons with a cytotoxic concentration of soluble Abeta1-42 invokes the rapid free radical generation within lysosomes and disruption of lysosomal membrane proton gradient which precedes cell death. The loss of lysosomal membrane impermeability is only specific to the Abeta1-42 isoform since incubation of cells with high concentrations of Abeta1-40 has no effect on lysosomal hydrolase release. To further support the role of lysosomal membrane damage in Abeta-mediated cell death, we demonstrate that photodisruption of acridine orange (AO)-loaded lysosomes with intense blue light induces a relatively rapid synchronous lysosomal membrane damage and neuronal death similar to that observed as a result of Abeta exposure. AO leaks quickly from late endosomes and lysosomes and partially shifts the fluorescence from an orange fluorescence to a diffuse, green cytoplasmic fluorescence. Such AO relocalization is due to an initial disruption of the lysosomal proton gradient, followed by the release of lysosomal hydrolases into the cytoplasmic compartment. Treatment of cells with either the antioxidant n-propyl gallate or lysosomotropic amine (methylamine) partially blocks the release of lysosomal contents suggesting that this AO relocalization is due to lysosomal membrane oxidation. Based on these findings, we propose that the cell death mediated by the soluble Abeta may be fundamentally different from the cell loss observed following extracellular Abeta deposition.

Opposite effects of lithium on proximal and distal caspases of immature and mature primary neurons correlate with earlier paradoxical actions on viability.

To provide an explanation for earlier paradoxical findings of lithium on survival of mature and immature neurons, this study monitors changes in cytosolic caspases in rat cerebellar granule cells (CGC) grown 2-7 days in vitro (DIV), or in murine E-17 cortical neurons. Data show Li+ protects mature 7-DIV CGC parallel to a decrease in proximal and distal caspases but increases levels for immature 2-DIV-CGC or E-17 cortical neurons. Caspases mirror viability based on morphological analyses (dye uptake, phase-contrast, DNA fragmentation), and suggest protection occurs by suppressing activation of a cascade resulting in distal effectors that destroy proteins essential for neuronal survival. Protection was dose-dependent with EC50 3.0 mM and extended to 64 h in K+-serum deprived apoptotic media. Neuronal extracts contain a spectrum of proximal (-2, -8, -9) and distal (-3, -6) caspases sensitive to Li+ on assay with preferred peptide substrates and by immunoblotting. The lack of direct effect on activated cytosols indicates Li+ acts upstream only on intact cells, at sites for recruitment of pivotal procaspases. Alterations of procaspase-9 p46 and membrane-bound cytochrome c (Apaf-1) point to interaction with an intrinsic Mt-mediated pathway as one of the targets. The opposite effects on caspases and viability of immature or embryological neurons point to existence of alternative pathways that alter during neurite outgrowth suggesting the use of Li+ as a probe to unravel events relevant to neurogenesis.

Adjuvant immunotherapy for patients with melanoma: are patients with melanoma of the head and neck candidates for this therapy?

BACKGROUND: Although a wealth of information is available on adjuvant immunotherapy for melanoma, little is known about adjuvant immunotherapy for head and neck melanoma. Interestingly, a few immunotherapy clinical trials report the observation of clinical responses in a subset of patients with head and neck melanoma. METHOD: An up-to-date literature search was performed to identify the current information on adjuvant immunotherapy for patients with melanoma, including head and neck melanoma. Moreover, a retrospective analysis of a subset of primary head and neck melanoma was performed using data from a phase III, randomized, double-blind, multi-institutinal, vaccinia melanoma oncolysate adjuvant immunotherapy trial that was performed in our laboratory for patients with stage III (AJCC) melanoma. RESULTS: In a passive immunotherapy trial with an antibody to melanoma ganglioside antigen GM2, a complete regression was observed in one patient with lesions of the right cheek. In three active specific immunotherapy trials, including our phase III trial, a subset of patients with head and neck primary melanoma showed a longer disease-free and overall survival with immunotherapy. Moreover, these clinical responses were correlated to the induction of immune response, delayed-type hypersensitivity response and melanoma-specific antibody response. CONCLUSIONS: The above results therefore suggest that patients with head and neck melanoma clinically respond to immunotherapy. However, these results need to be confirmed in a prospectively randomized trial for patients with head and neck melanoma.

Increased survival of patients treated with a vaccinia melanoma oncolysate vaccine: second interim analysis of data from a phase III, multi-institutional trial.

OBJECTIVE: The efficacy of vaccinia melanoma oncolysate (VMO) vaccine to increase overall survival and disease-free survival of patients with surgically resected International Union Against Cancer (UICC) stage II melanoma was studied in a phase III, randomized, multi-institutional trial. SUMMARY BACKGROUND DATA: Phase I and II trials with VMO showed minimal toxicity and clinical efficacy in patients with melanoma. In a recently completed phase III VMO trial, the first interim analysis performed in April 1994 showed an increasing trend in the survival of patients treated with VMO. The second interim analysis was performed in April 1995. METHODS: Patients with surgically resected stage II (UICC) melanoma were treated with VMO (N = 104) or placebo vaccinia vaccine virus (V) (N = 113) once a week for 13 weeks and then once every 2 weeks for a total of 12 months. Patients' clinical data were collected as of May 1995 and analyzed for survival. RESULTS: In this second interim analysis, the mean follow-up time is 42.28 months. No survival difference was observed between VMO and V treatments. However, in a retrospective subset analysis, a subset of males between the ages of 44 and 57 years and having one to five positive nodes (at 2-, 3-, and 5-year intervals, 13.6%, 15.9%, and 20.3% difference insurvival in favor of VMO [N = 20] when compared to V [N = 18] [p = 0.037]) and another subset of patients with clinical stage I (at 3- and 5-year intervals, 30% and 7% difference in survival in favor of VMO [N = 20] when compared to V [N = 23], [p = 0.05]) showed significant survival advantage with VMO. CONCLUSIONS: Although VMO vaccine therapy in surgical adjuvant setting did not produce a significant survival benefit to all patients with melanoma, patients from the above two subsets had significant survival benefit.

Favorable clinical responses in subsets of patients from a randomized, multi-institutional melanoma vaccine trial.

BACKGROUND: A phase III, randomized, double-blind, multi-institutional trial was performed evaluating active specific immunotherapy using vaccinia melanoma oncolysate (VMO) in the surgical adjuvant setting in patients with stage II melanoma (UICC staging). The first interim analysis showed no significant difference in disease-free and overall survival. The data were further analyzed to identify subsets of patients with improved outcome when treated with VMO. METHODS: Patients received either VMO or placebo of live vaccinia vaccine virus (V), once a week for 13 weeks and then once every 2 weeks for an additional 39 weeks or until recurrence. Having stratified patients according to sex, age, number of positive nodes, tumor thickness, and clinical stage, data were analyzed for disease-free survival and overall survival. RESULTS: Male patients showed a 17% difference in overall survival at 4 years when treated with VMO (p = 0.19). A subset of male patients < 57 years of age with one to five positive nodes showed a 30% difference at 4 years with VMO (p = 0.06). Patients with clinical stage I but pathological stage II disease (both male and female), who had undergone prophylactic node dissection, showed a 23% difference in survival at 3 years with VMO (p = 0.11). CONCLUSIONS: This subset analysis shows encouraging survival benefit in certain subsets of patients and an increasing trend in overall survival. Further follow-up of this phase III trial from a second interim analysis will be forthcoming.