Genetic or nutritional disorders in homocysteine (Hcy) metabolism elevate Hcy-thiolactone and cause heart and brain diseases. Hcy-thiolactone has been implicated in these diseases because it has the ability to modify protein lysine residues and generate toxic N-Hcy-proteins with auto-immunogenic, pro-thrombotic, and amyloidogenic properties. Bleomycin hydrolase (Blmh) has the ability to hydrolyze L-Hcy-thiolactone (but not D-Hcy-thiolactone) to Hcy in vitro, but whether this reflects a physiological function has been unknown. Here, we show that Blmh (-/-) mice excreted in urine 1.8-fold more Hcy-thiolactone than wild-type Blmh (+/+) animals (P = 0.02). Hcy-thiolactone was elevated 2.3-fold in brains (P = 0.004) and 2.0-fold in kidneys (P = 0.047) of Blmh (-/-) mice relative to Blmh (+/+) animals. Plasma N-Hcy-protein was elevated in Blmh (-/-) mice fed a normal (2.3-fold, P < 0.001) or hyperhomocysteinemic diet (1.5-fold, P < 0.001), compared with Blmh (+/+) animals. More intraperitoneally injected L-Hcy-thiolactone was recovered in plasma in Blmh (-/-) mice than in wild-type Blmh (+/+) animals (83.1 vs. 39.3 μM, P < 0.0001). In Blmh (+/+) mice injected intraperitoneally with D-Hcy-thiolactone, D,L-Hcy-thiolactone, or L-Hcy-thiolactone, 88, 47, or 6.3%, respectively, of the injected dose was recovered in plasma. The incidence of seizures induced by L-Hcy-thiolactone injections (3,700 nmol/g body weight) was higher in Blmh (-/-) than in Blmh (+/+) mice (93.8 vs. 29.5%, P < 0.001). Using the Blmh null mice, we provide the first direct evidence that a specific Hcy metabolite, Hcy-thiolactone, rather than Hcy itself, is neurotoxic in vivo. Taken together, our findings indicate that Blmh protects mice against L-Hcy-thiolactone toxicity by metabolizing it to Hcy and suggest a mechanism by which Blmh might protect against neurodegeneration associated with hyperhomocysteinemia and Alzheimer's disease.
Elevated plasma homocysteine (Hcy) is a risk factor for Alzheimer's disease (AD). Bleomycin hydrolase (BLH), a thiol-dependent enzyme that has Hcy-thiolactonase (HTase) and aminopeptidease (APase) activities, has also been implicated in Alzheimer's disease (AD). In order to examine its role in AD, BLH activities were measured in postmortem brain tissue from twelve AD patients and twelve control patients who died from non-neurological causes. We found that HTase and APase activities in human brain extracts were strongly correlated and sensitive to the thiol reagent iodoacetamide, indicating that they are associated with BLH. Both activities were significantly decreased in brain tissue extracts from AD patients relative to controls (7.6 +/- 4.2 vs. 13.5 +/- 5.5 units, p= 0.003 for HTase, and 3.82 +/- 1.27 vs. 5.33 +/- 1.68 units, p=0.010 for APase). HTase and APase activities were positively correlated with N-linked protein Hcy, but not with tHcy, in AD and control brains. Levels of brain total Hcy and N-linked protein Hcy did not differ between AD cases and controls. These results suggest that diminished functional BLH activity could contribute to the pathology of AD.
Growing evidence indicates that elevated total homocysteine (tHcy) levels can elicit autoimmune response in vivo. Antibodies against Nepsilon-Hcy-proteins have been shown to be associated with stroke and premature myocardial infarction. The aim of the current study was to investigate the effect of treatment with folic acid on anti-Nepsilon-Hcy-albumin and -hemoglobin antibodies. We recruited 20 apparently healthy men and 12 male patients with documented coronary artery disease (CAD). All participants had plasma fasting tHcy levels >15 microM. At baseline, and after three and six months of treatment with folic acid 1 mg daily, we determined tHcy, serum folate and vitamin B12 levels, along with serum anti-Nepsilon-Hcy-albumin and -hemoglobin IgG antibodies using the home-made immunoenzymatic assays. Both groups did not differ with regard to age, tHcy, folate, lipid profile, and CRP. The only significant difference between healthy subjects and CAD patients was levels of antibodies against Nepsilon-Hcy-albumin. As expected, folic acid administration led to significant decreases in tHcy and increases in folate levels in both groups. Levels of both anti-Nepsilon-Hcy-albumin and -hemoglobin antibodies fell markedly following a three-month folic acid administration in healthy subjects, but not in CAD patients, without any changes at six months in either group. Folic acid administration resulted in a loss of significant correlations between tHcy and antibodies both following three and six months of the therapy in healthy subjects, in contrast to CAD patients. Carriers of the methylenetetrahydrofolate reductase (MTHFR) 677T allele with CAD had significantly higher levels of anti-Nepsilon-Hcy-albumin before and during folic acid administration as compared to healthy subjects. In conclusion, our findings suggest that Hcy-related autoimmune response is resistant to folic acid administration in CAD patients, while in healthy subjects reduced tHcy levels are associated with suppressed production of antibodies against Nepsilon-Hcyproteins. These observations might explain at least in part the failure of vitamin therapy to reduce the risk of cardiovascular events as recently reported.
The induction of antitumor immune responses in tumor-bearing hosts depends on efficient uptake and processing of native or modified tumors/self-antigens by dendritic cells (DCs) to activate immune effector cells, as well as the extent of the immunosuppressive network in the tumor microenvironment (TME). Because the C-X-C motif chemokine receptor 4 (CXCR4) for the C-X-C motif chemokine 12 (CXCL12) is involved in signaling interactions between tumor cells and their TME, we used oncolytic virotherapy with a CXCR4 antagonist to investigate whether targeting of the CXCL12/CXCR4 signaling axis in murine neuroblastoma cells (NXS2)-bearing syngeneic mice affects the efficacy of bone marrow (BM)-derived DCs loaded with autologous tumor cells treated with doxorubicin for induction of immunogenic cell death. Here, we show that CXCR4 antagonist expression from an oncolytic vaccinia virus delivered intravenously to mice with neuroblastoma tumors augmented efficacy of the DC vaccines compared to treatments mediated by a soluble CXCR4 antagonist or oncolysis alone. This study is the first demonstration that modulating the tumor microenvironment by an armed oncolytic virus could have a significant impact on the efficacy of DC vaccines, leading to the generation of effective protection against neuroblastoma challenge.
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