Sphingomyelin in Human Breast Milk might be Essential for the Hippocampus Maturation.

BACKGROUND: It has been established that sphingomyelin present human breast milk is useful for the brain maturation and cognitive development. At 10 days of breastfeeding the sphingomyelin content is double that present in cow's milk and its content is independent of the maternal diet. The aim of the study was to analyze the content of sphingomyelin in breast milk at 3 months of breastfeeding and to consider the effect of this molecule on synaptic function and nerve conduction through the probable expansion of myelinated axons. METHODS: Therefore, to begin to define and assess this, we performed sphingolipidomic analysis in human breast milk. Then, we cultured embryonic hippocampal cells (HN9.10) in the presence of sphingomyelin at a concentration from 0.6% to 31% of human milk, estimated by considering its bioavailability and its passage into the interstitial fluid. To highlight the effect of sphingomyelin in the cells, cell viability and morphology were evaluated. Analyses of neutral sphingomyelinase gene and protein expression was performed. The entry of sphingomyelin into the cell was studied in immunofluorescence; the expression of heavy neurofilament (NF200) was tested with immunocytochemical technique. RESULTS: We demonstrated that sphingomyelin is able to enter cell nucleus and overexpress the sphingomyelin phosphodiesterase 4 (SMPD4) gene encoding for neutral sphingomyelinase (nSMase), an enzyme useful for its own metabolism. Later, cells displayed changes of the soma and the appearance of neurites supported by NF200 overexpression. CONCLUSIONS: We speculated that the sphingomyelin present in human breast milk is useful in part to regulate nuclear activity and in part to form myelin sheet to facilitate nerve cell maturation. As brain development occurs at 0-3 years, these data open a new avenue of potential intervention to integrate the infant formulas with SM to obtain a product similar to the maternal milk.

Mouse Thyroid Gland Changes in Aging: Implication of Galectin-3 and Sphingomyelinase.

Prevalence of thyroid dysfunction and its impact on cognition in older people has been demonstrated, but many points remain unclarified. In order to study the effect of aging on the thyroid gland, we compared the thyroid gland of very old mice with that of younger ones. We have first investigated the changes of thyroid microstructure and the possibility that molecules involved in thyroid function might be associated with structural changes. Results from this study indicate changes in the height of the thyrocytes and in the amplitude of interfollicular spaces, anomalous expression/localization of thyrotropin, thyrotropin receptor, and thyroglobulin aging. Thyrotropin and thyrotropin receptor are upregulated and are distributed inside the colloid while thyroglobulin fills the interfollicular spaces. In an approach aimed at defining the behavior of molecules that change in different physiopathological conditions of thyroid, such as galectin-3 and sphingomyelinase, we then wondered what was their behavior in the thyroid gland in aging. Importantly, in comparison with the thyroid of young animals, we have found a higher expression of galectin-3 and a delocalization of neutral sphingomyelinase in the thyroid of old animals. A possible relationship between galectin-3, neutral sphingomyelinase, and aging has been discussed.

Limb rescue: a new autologous-peripheral blood mononuclear cells technology in critical limb ischemia and chronic ulcers.

INTRODUCTION: Therapeutic angiogenesis by autologous-peripheral blood mononuclear cells (A-PBMNC) implantation has been shown to be a safe and effective treatment for critical limb ischemia (CLI). We herein report our investigation of the long-term efficacy of implantation of A-PBMNC produced by selective filtration to treat patients with CLI, for which surgical bypass and/or percutaneous transluminal angioplasty are not possible. MATERIALS AND METHODS: This is a prospective, and not a randomized, study based on a treated group who did not respond to conventional therapy (n=43) when implanted with A-PBMNC cells versus a historically matched control group. Patients of both groups were suffering from CLI Fontaine scale IV with chronic ulcers and various accompanying conditions (diabetes, heart disease, kidney failure, etc.). Treated patients were implanted with 12 mL of A-PBMNC, 0.2-0.3 mL for each bolus, collected by selective filtration from 120 mL of peripheral blood in the ischemic area of the limbs. Patients were not mobilized by granulocyte colony-stimulating factor, and the A-PBMNC treatment was repeated for a maximum of three times. RESULTS: The A-PBMNC-treated group showed a statistically significant improvement of limb rescue of 95.3% versus 52.2% of the control group (p<0.001), and the result had been maintained for 2 years. The A-PBMNC group also showed reduction in pain at rest, increased maximum walking distance, and healing of the wound, which led to an overall improvement in the quality of life. Post-treatment radiological studies showed an improvement of vascularization with the formation of new collateral and by histological findings. Within 2 years of follow-up, none of the patients whom we treated showed any major or systemic adverse effects. CONCLUSION: The local injection of A-PBMNC showed striking early and long-term effects together with a favorable safety profile, significantly decreasing the risk of amputation. Our results are comparable with published data obtained by injection of bone marrow mononuclear cells, but with a lot less invasive approach. Moreover the intraoperative selective filtration system we used is fast, safe, not operator dependent, and easy to use in a sterile operating theatre. This system aims to produce fresh A-PBMNC as a valuable treatment option, particularly for those difficult patients who cannot undergo revascularization.

Defective lymphocyte caspase-3 expression in type 1 diabetes mellitus.

OBJECTIVE: Activation-induced cell death (AICD) is a major mechanism in the regulation of peripheral tolerance and its impairment can determine the development of autoimmunity. In the present study, in order to evaluate the role of caspase-3 in type 1 diabetes mellitus (T1DM) AICD, caspase-3 expression was analyzed in peripheral blood lymphocytes from 37 new onset T1DM patients and from 36 normal control subjects (NC) in resting conditions and after anti-Fas-triggered apoptosis. METHODS: Caspase-3 expression was determined by semiquantitative RT-PCR and Western blot. Apoptosis was induced in activated lymphocytes by anti-Fas monoclonal antibody and quantified by flow cytometry and morphological analysis. RESULTS: Caspase-3 mRNA expression was reduced in resting lymphocytes in 18/37 T1DM patients and in 1/36 NC (P < 0.01). Patients studied for both Fas-mediated AICD and caspase-3 mRNA expression revealed that a reduced caspase-3 mRNA expression in resting lymphocytes occurred in all patients showing resistance to Fas-mediated apoptosis (T1DM vs NC, P < 0.02) with the exception of 3 patients who exhibited normal caspase-3 expression levels. Caspase-3 protein analysis confirmed mRNA data and showed an impaired expression of caspase-3 active form in T1DM subjects compared with NC. CONCLUSIONS: Our data show that defective expression and function of caspase-3 in peripheral lymphocytes of T1DM patients may contribute to the development of AICD resistance in type 1 diabetes.

Prosaposin: a new player in cell death prevention of U937 monocytic cells.

We report that prosaposin binds to U937 and is active as a protective factor on tumor necrosis factor alpha (TNFalpha)-induced cell death. The prosaposin-derived saposin C binds to U937 cells in a concentration-dependent manner, suggesting that prosaposin behaves similarly. Prosaposin binding induces U937 cell death prevention, reducing both necrosis and apoptosis. This effect was inhibited by mitogen-activated protein ERK kinase (MEK) and sphingosine kinase (SK) inhibitors, indicating that prosaposin prevents cell apoptosis by activation of extracellular signal-regulated kinases (ERKs) and sphingosine kinase. Prosaposin led to rapid ERK phosphorylation in U937 cells as detected by anti-phospho-p44/42 mitogen-activated protein (MAP) kinase and anti-phosphotyrosine reactivity on ERK immunoprecipitates. It was partially prevented by apo B-100 and pertussis toxin (PT), suggesting that both lipoprotein receptor-related protein (LRP) receptor and Go-coupled receptor may play a role in the prosaposin-triggered pathway. Moreover, sphingosine kinase activity was increased by prosaposin treatment as demonstrated by the enhanced intracellular formation of sphingosine-1-phosphate (S-1-P). The observation that the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin prevented the prosaposin effect on cell apoptosis suggests that sphingosine kinase exerts its anti-apoptotic activity by the PI3K-Akt pathway. Thus, cell apoptosis prevention by prosaposin occurs through ERK phosphorylation and sphingosine kinase. The biological effect triggered by prosaposin might be extended to primary cells because it triggers Erk phosphorylation in peripheral blood mononuclear cells (PBMCs). This is the first evidence of a biological effect consequent to a signal transduction pathway triggered by prosaposin in cells of non-neurological origin.

Hippocampal prosaposin changes during stress: a glucocorticoid-independent event.

Several studies indicate that stress can produce remarkable effects on neurotrophic factors. In this regard, hippocampus is the most interesting structure of the brain because of its broad involvement in behavioral and neuroendocrine phenomena. In the present study, we investigated the effect of stress on hippocampal prosaposin, which is known to act as a neurotrophic and neuroprotective factor. Rats subjected to restraint stress (120 min) had a significant and transient reduction of hippocampal, but not hypothalamic, prosaposin full-length protein. Indeed, when this stressful stimulus was applied daily for 3 days, no differences were detected in comparison with naive rats. To investigate the role of glucocorticoids in the stress-induced decrease in hippocampal prosaposin, adrenalectomized and corticosterone-treated rats were studied. The results indicate that adrenalectomized rats behave as intact animals. This finding indicates that the absence of endogenous corticosterone does not prevent a decrease in hippocampal prosaposin. When an increase of corticosterone was achieved through exogenous administration, hippocampal prosaposin concentrations were unchanged in comparison with vehicle-injected (sesame oil) rats. These results led to the conclusion that stress, not via an increase of glucocorticoid hormone, transiently reduces hippocampal prosaposin levels. This phenomenon is followed by rapid recovery of the neurotrophin level, even when the stress stimulus persists.

Association of cellular prion protein with gangliosides in plasma membrane microdomains of neural and lymphocytic cells.

In this report we demonstrated that cellular prion protein is strictly associated with gangliosides in microdomains of neural and lymphocytic cells. We preliminarily investigated the protein distribution on the plasma membrane of human neuroblastoma cells, revealing the presence of large clusters. In order to evaluate its possible role in tyrosine signaling pathway triggered by GEM, we analyzed PrPc presence in microdomains and its association with gangliosides, using cholera toxin as a marker of GEM in neuroblastoma cells and anti-GM3 MoAb for identification of GEM in lymphoblastoid cells. In neuroblastoma cells scanning confocal microscopical analysis revealed a consistent colocalization between PrPc and GM1 despite an uneven distribution of both on the cell surface, indicating the existence of PrPc-enriched microdomains. In lymphoblastoid T cells PrPc molecules were mainly, but not exclusively, colocalized with GM3. In addition, PrPc was present in the Triton-insoluble fractions, corresponding to GEM of cell plasma membrane. Additional evidence for a specific PrPc-GM3 interaction in these cells was derived from the results of TLC analysis, showing that prion protein was associated with GM3 in PrPc immunoprecipitates. The physical association of PrPc with ganglioside GM3 within microdomains of lymphocytic cells strongly suggests a role for PrPc-GM3 complex as a structural component of the multimolecular signaling complex involved in T cell activation and other dynamic lymphocytic plasma membrane functions.