CpG drives human transitional B cells to terminal differentiation and production of natural antibodies

The receptor TLR9, recognizing unmethylated bacterial DNA (CpG), is expressed by B cells and plays a role in the maintenance of serological memory. Little is known about the response of B cells stimulated with CpG alone, without additional cytokines. In this study, we show for the first time the phenotypic modification, changes in gene expression, and functional events downstream to TLR9 stimulation in human B cell subsets. In addition, we demonstrate that upon CpG stimulation, IgM memory B cells differentiate into plasma cells producing IgM Abs directed against the capsular polysaccharides of Streptococcus pneumoniae. This novel finding proves that IgM memory is the B cell compartment responsible for the defense against encapsulated bacteria. We also show that cord blood transitional B cells, corresponding to new bone marrow emigrants, respond to CpG. Upon TLR9 engagement, they de novo express AID and Blimp-1, genes necessary for hypersomatic mutation, class-switch recombination, and plasma cell differentiation and produce Abs with anti-pneumococcal specificity. Transitional B cells, isolated from cord blood, have not been exposed to pneumococcus in vivo. In addition, it is known that Ag binding through the BCR causes apoptotic cell death at this stage of development. Therefore, the ability of transitional B cells to sense bacterial DNA through TLR9 represents a tool to rapidly build up the repertoire of natural Abs necessary for our first-line defense at birth.     

Isoprostanes and hepatic fibrosis

After a brief introduction to oxidative stress, the discovery of F(2)-isoprostanes as specific and reliable markers of oxidative stress is described. Isoprostanes are also agonists of important biological effects. Since a relation between oxidative stress and collagen hyperproduction has been previously suggested and since lipid peroxidation products have been proposed as possible mediators of liver fibrosis, we investigated whether collagen synthesis is induced by F(2)-isoprostanes the most proximal products of lipid peroxidation. In a rat model of carbon tetrachloride-induced hepatic fibrosis, plasma isoprostanes were markedly elevated for the entire experimental period; hepatic collagen content was also increased. When hepatic stellate cells from normal liver were cultured up to activation (expression of alpha-smooth muscle-alpha actin) and then treated with F(2)-isoprostanes in the concentration range found in the in vivo studies (10(-9)-10(-8)M), a striking increase in DNA synthesis, in cell proliferation and in collagen synthesis was observed. Moreover, F(2)-isoprostanes increased the production of transforming growth factor-beta1 by U937 cells, assumed as a model of Kupffer cells or liver macrophages. The data suggest the possibility that F(2)-isoprostanes generated by lipid peroxidation in hepatocytes mediate hepatic stellate cell proliferation and collagen hyperproduction seen in hepatic fibrosis.     

The GPI-anchored CD52 antigen of the sperm surface interacts with semenogelin and participates in clot formation and liquefaction of human semen

CD52 is a human glycosylphosphatidylinositol (GPI)-anchored antigen exclusively expressed in leukocytes and epididymal cells. It is also present in sperm, being inserted in their plasma membrane as they pass through the epididymis. In a previous paper we identified a new CD52 form without GPI anchor by fast performance liquid chromatography (FPLC) fractionation of semen components. The form has a lower negative charge than the GPI-anchored form and occurs as the only CD52 form in prostasome-free seminal plasma. It was also found associated with the ejaculated sperm, but in contrast to the GPI-anchored one, it is lost during the capacitation process. In this paper we indicate that (1) the GPI-anchored CD52 of the sperm surface serves as receptor for semenogelin I during clot formation, (2) liquefaction involves cleavage of the GPI anchor from certain CD52 molecules, releasing sperm from the clot and the soluble antigen bound to semenogelin fragments into the seminal plasma and (3) the clot is a sponge-like structure housing sperm. Soluble CD52 was immunopurified from the soluble CD52-containing FPLC fraction using CAMPATH-1G and was found to be complexed with a semenogelin-derived peptide of the carboxyl terminal portion of semenogelin I, having the sequence SQTEKLVAGKQI and starting from amino acid 376. Immunoprecipitation and immunoblot analyses using CAMPATH-1G and anti-semenogelin as immunoprecipitating antibodies and anti-gp20 and anti-semenogelin as immunoblot detectors of the corresponding antigens, confirmed that the soluble CD52 formed a complex with semenogelin. The semenogelin-CD52 soluble form was found to be a direct consequence of the liquefaction process since only the GPI-anchored CD52 was recovered in uniquefied semen after recovering sperm and seminal plasma by urea solubilization of the clot.     

Uptake and recycling of pro-BDNF for transmitter-induced secretion by cortical astrocytes

Activity-dependent secretion of brain-derived neurotrophic factor (BDNF) is thought to enhance synaptic plasticity, but the mechanisms controlling extracellular availability and clearance of secreted BDNF are poorly understood. We show that BDNF is secreted in its precursor form (pro-BDNF) and is then cleared from the extracellular space through rapid uptake by nearby astrocytes after θ-burst stimulation in layer II/III of cortical slices, a paradigm resulting in long-term potentiation of synaptic transmission. Internalization of pro-BDNF occurs via the formation of a complex with the pan-neurotrophin receptor p75 and subsequent clathrin-dependent endocytosis. Fluorescence-tagged pro-BDNF and real-time total internal reflection fluorescence microscopy in cultured astrocytes is used to monitor single endocytic vesicles in response to the neurotransmitter glutamate. We find that endocytosed pro-BDNF is routed into a fast recycling pathway for subsequent soluble NSF attachment protein receptor–dependent secretion. Thus, astrocytes contain an endocytic compartment competent for pro-BDNF recycling, suggesting a specialized form of bidirectional communication between neurons and glia.     

Predominant allylic hydroxylation at carbons 6 and 7 of 4 and 5-ene functionalized steroids by the thermophilic fungus Rhizomucor tauricus IMI23312

This paper demonstrates for the first time transformation of a series of steroids (progesterone, androst-4-en-3,17-dione, testosterone, pregnenolone and dehydroepiandrosterone) by the thermophilic fungus Rhizomucor tauricus. All transformations were found to be oxidative (monohydroxylation and dihydroxylation) with allylic hydroxylation the predominant route of attack functionalizing the steroidal skeleta. Timed experiments demonstrated that dihydroxylation of progesterone, androst-4-en-3,17-dione and pregnenolone all initiated with hydroxylation on ring-B followed by attack on ring-C. Similar patterns of steroidal transformation to those observed with R. tauricus have been observed with some species of thermophilic Bacilli and mesophilic fungi. All metabolites were isolated by column chromatography and were identified by (1)H, (13)C NMR, DEPT analysis and other spectroscopic data. The application of thermophilic fungi to steroid transformation may represent a potentially rich source for the generation of new steroidal compounds as well as for uncovering inter and intraspecies similarities and differences in steroid metabolism.     

Role of α2‐macroglobulin in regulating amyloid β‐protein neurotoxicity: protective or detrimental factor?

alpha2-Macroglobulin (alpha2M) has been identified as a carrier protein for beta-amyloid (Abeta) decreasing fibril formation and affecting the neurotoxicity of this peptide. The alpha2-macroglobulin receptor/low density lipoprotein receptor related protein (LRP) is involved in the internalization and degradation of the alpha2M/Abeta complexes and its impairment has been reported to occur in Alzheimer's disease. Previous studies have shown alpha2M to determine an enhancement or a reduction of Abeta toxicity in different culture systems. In order to clarify the role of alpha2M in Abeta neurotoxicity, we challenged human neuroblastoma cell lines with activated alpha2M in combination with Abeta. Our results show that in neuroblastoma cells expressing high levels of LRP, the administration of activated alpha2M protects the cells from Abeta neurotoxicity. Conversely, when this receptor is not present alpha2M determines an increase in Abeta toxicity as evaluated by MTT and TUNEL assays. In LRP-negative cells transfected with the full-length human LRP, the addition of activated alpha2M resulted to be protective against Abeta-induced neurotoxicity. By means of recombinant proteins we ascribed the neurotoxic activity of alpha2M to its FP3 fragment which has been previously shown to bind and neutralize transforming growth factor-beta. These studies provide evidence for both a neuroprotective and neurotoxic role of alpha2M regulated by the expression of its receptor LRP.     

Les industries magdaléniennes de l’Ardèche (France) dans le contexte du bassin méditerranéen

Ardèche, a department of Rhone Alpes region, is rich in prehistoric sites belonging to a very large chronological period dated back to 350?000 years ago. But, the prehistory of the region has been unknown for a long time, mainly, because of its distance from traditional centres of research. Jean Combier, in his abstract dated 1967, defined for the first time Upper Palaelolithic stages: only towards the acquisition of new data, we are now able to suggest a new evolution for the Magdalenian from its origins to the Alleröd climatic episod. To define Ardèche originality within the Magdalenian context, we have compared its lithic industries with those of the Adaouste Cave oriental sites, the Cornille rock shelter and of the Gazel cave in the Aude western part. Ardèche Magdalenian dwelling is peculiar compared to the South West of France. Badegoulian has been substituted by a Mediterranean Facies culture rich in bladelets, the Salpestrian. This facies limited in its geographic extention to Gard and Ardèche, evolves gradually in situ gaining Magdalenian elements (such as backed bladelets and dihedral burins) giving birth to the transitory lithic complex of Huguenots and Baume d’Oullins Cave. An established Magdalenian is certified in the Blanchisserie camp, within a cold climatic context dated back to circa 16?000 years ago. Although the lithic industry is dominated by dihedral burins and backed bladeletse it is also characterised by some archaic features (such as keel endscrapers, transverse burins and scalene bladelets). The upper Magadalenian with bone harpoons appears soon in our region, in the Colombier rock shelter, in a fairly temperate climatic context dated according to 14C back to circa 14?000 BP. We could identify six stages within the evolution of this Upper Magdalenian.which are attested in the Colombier, Ebbou and Deux Avens Caves and in the Colombier rock shelter that has been occupied during several periods. The Magdalenian gradually changed loosing his most typical elements, the bladelets and burins supremacy has been substituted by Azilian elements (such as short endscrapers and curved backed points). But even if the Azilian process happens very early (before 12?500 BP) the Magdalenian, in its fundamental features, never disappears completely and it has never been substituted by classic Azilian. After Alleröd appears a culture characterised by the recovery of Magdalenian features similar to the Epimagdalenian defined by D. Sacchi in Gazel. The described evolution can be compared, as regard to its upper stages, to that of several sites of Rhone region as well as of the North West of France, which allow to define a culturally homogeneous province having the Rhone corridor with Ardèche as its Southern border. At the end of Palaeolithic this province broke up and Ardèche opened to the South and the Mediterranean from where seems to come the retouched large blade facies and endscrapers attested by the Colombier rock shelter dating back to 12?150 BP.     

Bis-phenacetyl and phenoxyacetyl groups as substrates for penG and penV amidases

o-, m-, p-Bis-phenylacetic and -bis-phenoxyacetic acid esters with solketal are prepared and submitted to enzymatic hydrolysis with penicillin V (PVA) and G (PGA) amidases. While the para-isomers are recovered unchanged, ortho- and meta-bis-esters are completely hydrolysed. PVA shows a reversed substrate specificity, hydrolysing phenylacetates faster than its natural substrate. The use of the bis-acids as alcohol-protecting groups is proposed.