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In humans, natural killer (NK) cell function is regulated by a series of receptors and coreceptors with either triggering or inhibitory activity. Here we describe a novel 60-kD glycoprotein, termed NTB-A, that is expressed by all human NK, T, and B lymphocytes. Monoclonal antibody (mAb)-mediated cross-linking of NTB-A results in the induction of NK-mediated cytotoxicity. Similar to 2B4 (CD244) functioning as a coreceptor in the NK cell activation, NTB-A also triggers cytolytic activity only in NK cells expressing high surface densities of natural cytotoxicity receptors. This suggests that also NTB-A may function as a coreceptor in the process of NK cell activation. Molecular cloning of the cDNA coding for NTB-A molecule revealed a novel member of the immunoglobulin superfamily belonging to the CD2 subfamily. NTB-A is characterized, in its extracellular portion, by a distal V-type and a proximal C2-type domain and by a cytoplasmic portion containing three tyrosine-based motifs. NTB-A undergoes tyrosine phosphorylation and associates with the Src homology 2 domain–containing protein (SH2D1A) as well as with SH2 domain–containing phosphatases (SHPs). Importantly, analysis of NK cells derived from patients with X-linked lymphoproliferative disease (XLP) showed that the lack of SH2D1A protein profoundly affects the function not only of 2B4 but also of NTB-A. Thus, in XLP-NK cells, NTB-A mediates inhibitory rather than activating signals. These inhibitory signals are induced by the interaction of NTB-A with still undefined ligands expressed on Epstein-Barr virus (EBV)-infected target cells. Moreover, mAb-mediated masking of NTB-A can partially revert this inhibitory effect while a maximal recovery of target cell lysis can be obtained when both 2B4 and NTB-A are simultaneously masked. Thus, the altered function of NTB-A appears to play an important role in the inability of XLP-NK cells to kill EBV-infected target cells.

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With the ultimate goal to generate suitable materials for the repair of osteochondral defects, in this work we aimed at developing composite osteochondral scaffolds organized in different integrated layers, with features which are biomimetic for articular cartilage and subchondral bone and can differentially support formation of such tissues.A biologically inspired mineralization process was first developed to nucleate Mg-doped hydroxyapatite crystals on type I collagen fibers during their self assembling. The resulting mineral phase was non-stoichiometric and amorphous, resembling chemico-physical features of newly deposited, natural bone matrix. A graded material was then generated, consisting of (i) a lower layer of the developed biomineralized collagen, corresponding to the subchondral bone, (ii) an upper layer of hyaluronic acid-charged collagen, mimicking the cartilaginous region, and (iii) an intermediate layer of the same nature as the biomineralized collagen, but with a lower extent of mineral, resembling the tidemark. The layers were stacked and freeze-dried, to obtain an integrated, monolithic composite. Culture of the material for 2 weeks after loading with articular chondrocytes yielded cartilaginous tissue formation selectively in the upper layer. Conversely, ectopic implantation in nude mice of the material after loading with bone marrow stromal cells resulted in bone formation which remained confined within the lower layer.In conclusion, we developed a composite material with cues which are biomimetic of an osteochondral tissue and with the capacity to differentially support cartilage and bone tissue generation. The results warrant test of the material as a substitute for the repair of osteochondral lesions in orthotopic animal models. Response to Reviewer #1:1) results and discussion should be separated We would prefer to maintain Results and Discussion combined, in order to avoid possible redundancies and improve readability. 2) figures should be detailed with arrows and information about specific orientations. Details and arrows have been included in the Figures3) collagen type 1 was used for the creation of the construct -in every layer? Usually collagen type 1 does not play a role in articular cartilage. This should be addressed in the discussion. According to the Reviewer's comment, a rationale for the use of type I collagen has been included in the text, as follows: "Type I collagen was selected for the synthesis of the composite layers, including the cartilaginous one, due to (i) its good physico-chemical stability and processability and (ii) high safety and biocompatibility profile, related to the removal of all Reply Letter to Editor and referee's comments telopeptides, which are potentially responsible for immunological reactions." (Section 2.1, Page 2, 1 st paragraph). Indeed, type II collagen is normally extracted porcine derivative, and is still not available either in bulk for processing or for clinical use. Nowadays there are only few papers on experimental studies desc...

The incorporation of magnesium ions (in the range 5-10 mol% in respect to Ca) into the hydroxyapatite structure, which is of great interest for the developing of artificial bone, was performed using magnesium chloride, calcium hydroxide and phosphoric acid, as reactants. Among the synthesized powders, the synthetic HA powder containing 5.7% Mg substituting for calcium was selected, due to its better chemico-physical features, and transformed into granules of 400-600 microm, for biocompatibility tests (genotoxicity, carcinogenicity, toxicity, in vitro cytotoxicity and in vivo skin irritation-sensitization tests). In vivo tests were carried out on New Zealand White rabbits using the granulate as filling for a femoral bone defect: osteoconductivity and resorption were found to be enhanced compared to commercial stoichiometric HA granulate, taken as control.

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Replacement of bone tissue by graft materials and products of tissue engineering having composition, structure, and biological features that mimic natural tissue is a goal to be pursued. A biomimetic synthesis was performed to prepare new bone-like composites constituted of hydroxyapatite nanocrystals and self-assembled type I collagen fibers. We used a biological inspired approach that proved that the biological systems stored and processed information at the molecular level. Two different methodologies were used: dispersion of synthetic hydroxyapatite in telopeptides free collagen molecules solution and direct nucleation of hydroxyapatite into reconstituted collagen fibers during their assembling. The different preparation techniques were experimented then the composites thoroughly characterized and compared. Composite obtained by direct nucleation showed an intimated interaction of the inorganic and proteic components, which modified the apatitic phase and made its composition, morphology and structure similar to the mineral component of natural bone.