The effect of calcium ion concentration on osteoblast viability, proliferation and differentiation in monolayer and 3D culture

Our research group aims to develop an osteochondral composite using type II collagen gel with hydroxyapatite (HAp) deposited on one side. Soaking gels in Ca2+ and phosphate solution is indispensable to HAp deposition, so relationships between cell behavior and Ca2+ concentration were examined in two- and three-dimensional cultures. The present results indicate that 2-4 mM Ca2+ is suitable for proliferation and survival of osteoblasts, whereas slightly higher concentrations (6-8 mM) favor osteoblast differentiation and matrix mineralization in both 2- and 3-dimensional cultures. Higher concentrations (>10 mM) are cytotoxic. Purely from the perspective of calcium deposition, higher concentrations lead to increased accumulation of Ca2+. Culturing cells in phosphate-containing gel in media with Ca2+ also leads to time-dependent formation of HAp in the gel. Considering the viability of embedded cells, culturing scaffolds in media with Ca2+ concentrations around 5mM is useful for both HAp deposition and osteoblast behavior.     

Polymer conformation and NMR chemical shifts, 9. Poly(α-methylene-γ-butyrolactone)

The conformational energies of poly(α-methylene-γ-butyrolactone) are calculated and compared with those of poly(methyl methacrylate). In spite of the structural resemblance of these two polymers, the patterns of the energy contour maps are clearly distinguishable from each other; the energy barriers between rotational isomeric states are appreciably higher in the former than in the latter polymer. The calculation indicates large non-bonded interactions between the protons in one lactone ring and those in the adjacent lactone rings. The broad NMR spectrum of poly(α-methylene-γ-butyrolactone) apparently reflects its rigid conformational structure. ¹H and ¹³C NMR chemical shifts are calculated by theoretical shielding calculations based on conformational analysis. Much lower magnetic field resonances of the O? CH₂ and α-CH₂ carbons in poly(α-methylene-γ-butyrolactone) as compared with those of the O? CH₃ and α-CH₃ carbons in poly(methyl methacrylate) are well reproduced by the calculation. The shift to lower magnetic field is mainly attributed to paramagnetic shielding derived from the interaction between O? CH₂ carbon and α-CH₂ carbon. Tacticity- and conformation-dependent ¹H and ¹³C NMR chemical shifts of poly(α-methylene-γ-butyrolactone) are well interpreted on the basis of the conformational analysis.     

Microdissection is essential for gene expression profiling of clinically resected cancer tissues.

The gene expression array method enables us to achieve expression profiling with thousands of genes. Clinically resected bulk cancer tissues, however, contain not only cancer cells but also stromal cells, which may affect gene expression profiling and hamper accurate analysis of the cancer cells per se. Therefore, a procedure for dissecting specific cells, such as laser capture microdissection, is neededfor the clinical application of a gene expression array. There has been no study actually comparing 2 gene expression profiles, one obtained using RNA extractedfrom cancer cells by laser capture microdissection and one obtained using RNA extractedfrom bulk cancer tissues. Wefirst demonstrated the difference in expression patterns between them, without any amplification procedures. In addition, differential expression analysis between tumor and nontumor tissue yielded quite different patterns between the 2 methods. We conclude that microdissection is essential for gene expression profiling of clinical specimens.     

2B4 co-stimulation: NK cells and their control of adaptive immune responses

NK cells have primarily been defined by their ability to kill infected cells, tumor cells and some normal cells expressing low levels of MHC class I molecules. NK cells have also been shown to affect adaptive immune responses by their production of both pro- and anti-inflammatory cytokines. Recently it has been shown that adaptive immune responses can be enhanced or maintained also through direct lymphocyte-lymphocyte interactions. One of these interactions was identified to occur between 2B4 and CD48, where 2B4 acted as a co-stimulatory ligand for both NK cells and T cells. In the current article, we discuss the role of 2B4 in the development of adaptive immune responses and the role of NK-T cell interactions in these responses.     

Unique properties of fetal lymphoid progenitors identified according to RAG1 gene expression

RAG1/GFP knockin mice were exploited to isolate and characterize fetal lymphoid progenitors. CD11b and IL-7Ralpha are expressed in a developmental stage-dependent fashion, revealing how substantial numbers of early lymphoid progenitors were discarded or neglected in previous studies. The myeloerythroid potential of fetal progenitors in clonal assays declined in synchrony with activation of the RAG1 locus but was not completely extinguished. Lymphoid differentiation corresponded to patterns of gene expression previously found for adult marrow, but no fraction of fetal liver was enriched with respect to B + T progenitors. Also, unlike adults, fetal lymphoid progenitors transiently expressed endothelial cell markers. These findings help to reconcile discrepancies in previous reports and suggest that the fetal immune system arises via unique mechanisms.     

Diagnostic utility of galectin-3 and CD26/DPPIV as preoperative diagnostic markers for thyroid nodules

The aim of this study was to search for diagnostic markers that could correctly identify thyroid nodular lesions requiring urgent surgical treatment. We investigated whether galectin-3 and dipeptidyl peptidase IV (CD26/DPPIV) could be potential markers for improving the diagnostic accuracy of conventional cytology. Seventy-nine patients with histologically proven thyroid diseases were analyzed. The immunocytochemical staining results showed galectin-3 expression in neoplastic cells of all 37 papillary carcinomas, five of six follicular carcinomas, all three anaplastic carcinomas, one of three medullary carcinomas, and two of 14 follicular adenomas. All 16 adenomatous goiters were negative for galectin-3 immunostaining. On the other hand, all 37 papillary carcinomas, all six follicular carcinomas, and one of three anaplastic carcinomas revealed CD26/DPPIV expression, whereas all three medullary carcinomas were negative. Among benign thyroid lesions, four of 14 follicular adenomas and two of 16 adenomatous goiters exhibited varying degrees of immunoreactivity for CD26/DPPIV. RT-PCR analysis demonstrated overexpression of galectin-3 and CD26/DPPIV mRNAs in all six papillary and all three follicular carcinomas analyzed, whereas the mRNA expressions of these molecules were barely or not detectable in benign thyroid lesions and normal thyroid tissues, except for one case of follicular adenoma. In conclusion, we demonstrate that galectin-3 and CD26/DPPIV were consistently coexpressed at protein and mRNA levels in differentiated thyroid carcinomas. We propose that combined immunostaining for galectin-3 and CD26/DPPIV in the preoperative evaluation of thyroid nodules may play a role in accurate cytodiagnosis.     

Synthesis of 3-amino-ribofuranans having 1,5-α and -β structures by selective ring-opening polymerization of a 1,4-anhydro-3-azido-3-deoxy-α-D-ribopyranose derivative

Ring-opening polymerization of a new anhydro ribose-type monomer, 1,4-anhydro-3-azido-3-deoxy-2-O-tert-butyldimethylsilyl-α-D -ribopyranose (A3ASR), was investigated. The monomer was synthesized from 1,4-anhyro-α-D -xylopyranose by three steps comprising Walden inversion at the C3 position into ribose configuration. Ring-opening polymerization of A3ASR by Lewis acid catalysts such as boron trifluoride etherate and stannic chloride gave a stereoregular 3-azido-3-deoxy-2-O-tert-butyldimethylsilyl-(1→5)-α-D -ribofuranan having specific rotations of +246 ～ +271 deg · dm^?1 · g^?1 · cm³ and number-average molecular weights of 18,7 × 10³ ～ 25,1 × 10³. When the polymerization was carried out by antimony pentachloride at 0°C, the resulting polymer exhibited a negative specific rotation of ?6 deg · dm^?1 · g^?1 · cm³ and the C1 absorption in the ¹³C NMR spectrum shifted downfield to 107,5 ppm, suggesting that the polymer might consist of 1,5-β furanosidic unit. The reduction of the azido group of the 1,5-α and 1,5-β furanosidic polymers into amino group and subsequent desilylation gave 3-amino-3-deoxy-(1→5)-α- and -β-D -ribofuranans, respectively. In addition, copolymerization of A3ASR with 1,4-anhydro-2,3-di-O-tert-butyldimethylsilyl-α-D -ribopyranose (ADSR) in various feeds was performed by boron trifluoride etherate as catalyst to give copolymers with different monomeric components. The structural analysis of the homopolymers and copolymers was examined by means of ¹H and ¹³C NMR spectroscopies, IR spectroscopy, and optical rotation.     

Polymer conformation and NMR chemical shifts, 6. Alternating copolymer of α-methylene-γ-butyrolactone with styrene

In view of the structural resemblance of α-methylene-γ-butyrolactone to methyl methacrylate, conformation and NMR chemical shifts of poly(α-methylene-γ-butyrolactone-alt-styrene). are calculated and compared with those of poly(methyl methacrylate-alt-styrene). The conformational energy surfaces for the dyad sequence models of the both copolymers are analogous to each other, as is expected from the similarity in the constituting monomeric units. Agreement of calculation with observation is satisfactory with respect to the cotacticity-dependent splittings in the ¹³C NMR spectra and is improved by the fixation of a substituent in poly(α-methylene-γ-butyrolactone-alt-styrene). Calculation of ¹H chemical shifts gives an alignment of NMR signals which is in accordance with the observation.     

Frequent occurrence of in vivo clonal expansion of CD4− CD8− T cells bearing T cell receptor αβ chains in adult humans

We have previously reported 2 cases of healthy men showing in vivo monoclonal expansion of mature CD4^? CD8^? αβ T cells. In the present study, an additional 3 adults were found to exhibit such an expansion, among a total 464 adult donors studied. These 5 individuals were otherwise physiologically normal, with no history of severe illness and autoimmune disease at the time of examination. To investigate the mechanisms of the clonal expansion, further characterization of the clonal cells was attempted. No apparent preference for usage of the Tcell receptor β chain variable region was observed in the clonal T cells. These clonal T cells showed lectin-dependent or redirected antibody-dependent cell-mediated cytotoxicities, whereas they could not lyse autologous lymphoblastoid cell lines. Failure of Fas antigen expression was not observed for any of these clones. These results suggest that clonal expansion of CD4^? CD8^? αβ T cells frequently occurs in the periphery without any T cell abnormalities.