Perfringolysin O as a useful tool to study human sperm physiology.

OBJECTIVE: To evaluate perfringolysin O, a cholesterol-dependent pore-forming cytolysin, as a tool to study several aspects of human sperm physiology. DESIGN: Prospective study. SETTING: Basic research laboratory. PATIENT(S): Human semen samples with normal parameters obtained from healthy donors. INTERVENTION(S): Interaction of recombinant perfringolysin O with human spermatozoa. MAIN OUTCOME MEASURE(S): Assessment of perfringolysin O binding to spermatozoa, tests for acrosome and plasma membrane integrity, and acrosomal exocytosis assays. RESULT(S): Perfringolysin O associated with human spermatozoa at 4°C. The binding was sensitive to changes in cholesterol concentrations and distribution occurring in the plasma membrane of these cells during capacitation. When perfringolysin O-treated sperm were incubated at 37°C, the plasma membrane became permeable, whereas the acrosome membrane remained intact. Permeabilized spermatozoa were able to respond to exocytic stimuli. The process was inhibited by proteins that interfere with membrane fusion, indicating that large molecules, including antibodies, were able to permeate into the spermatozoa. CONCLUSION(S): PFO is a useful probe to assess changes in the amount and distribution of the active sterol fraction present in the sperm plasma membrane. The toxin can be used for the efficient and selective permeabilization of this membrane, rendering a flexible experimental model suitable for studying molecular processes occurring in the sperm cytoplasm.

Inhibition of MAPK/ERK, PKC and CaMKII signaling blocks cytolysin-induced human glioma cell death.

BACKGROUND: Cytolysins are pore-forming toxins that show anticancer activity by a mechanism hitherto poorly investigated. MATERIALS AND METHODS: To investigate how cytolysins are cytotoxic to resistant cancer cells, proliferation and cell death were evaluated on U87 glioblastoma cells treated with toxin Bc2 or equinatoxin-II (EqTx-II). RESULTS: Toxins Bc2 and EqTx-II decreased cell viability and increased lactate dehydrogenase (LDH) release in a concentration-dependent manner. Swollen, dead or dying cells were negative for TUNEL staining. The pre-treatment with inhibitors of mitogen-activated/extracellular regulated kinase (MEK1), protein kinase C (PKC) or Ca(2+)/calmodulin-dependent kinase II (CaMKII) blocked the toxic effects of toxin Bc2 and EqTx-II, suggesting that calcium entry, activation of MEK1, PKC and CaMKII pathways are involved in the cytotoxicity induced by these cytolysins. CONCLUSION: Cytolysins were shown to be toxic to glioblastoma cells by activating several intracellular signaling pathways and resulting in necrosis-like cell death.

Isolation and characterization of CD8+ regulatory T cells in multiple sclerosis.

To investigate CD8+ regulatory T cell influence on multiple sclerosis development, peripheral blood and cerebrospinal fluid (CSF) CD8+ T cell clones (TCCs) recognizing MBP(83-102) and MOG(63-87)-specific CD4+ T cells were isolated from 20 patients during acute exacerbations, 15 in remission and 15 controls. Blood and CSF CD8+ regulatory TCC cloning frequency decreased more during exacerbations than remissions or controls. Target cell pre-activation significantly enhanced CD8+ T granule-mediated cell killing of CD4+ targets, and was restricted by HLA-E. During exacerbations, killer-inhibitory receptor CD94/NKG2A expression was significantly higher in CD8+ TCCs, limiting their cytotoxic activity. Moreover, IL-15 and IFN-gamma significantly increased CD94 and NKG2A expression. These data provide evidence that CD94/NKG2A receptors play an important role in regulating T cell activity during the course of MS.