Multinucleated cells formed on calcified dentine from mouse bone marrow cells treated with 1 alpha,25-dihydroxyvitamin D3 have ruffled borders and resorb dentine

Osteoclast-like multinucleated cells were formed from mouse bone marrow mononuclear cells, and their morphology on coverslips and on calcified dentine slices was compared by means of transmission electron microscopy. Addition of 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3] to bone marrow cells cultured on coverslips greatly stimulated the formation of multinucleated cells within 8 days. These multinucleated cells had the cytological features of osteoclasts (abundant pleomorphic mitochondria, a large number of vacuoles and lysosomes, many stacks of Golgi membranes, and an extensive canalicular system), but they developed neither ruffled borders nor clear zones. The multinucleated cells appeared to result from direct fusion of mononuclear progenitor cells, whose structural features were similar to those of multinucleated cells. Like isolated osteoclasts, both multinucleated cells and their precursors exhibited an intense reaction for tartrate-resistant acid phosphatase (TRACP) in the cisterns of endoplasmic reticulum and lysosomes. Multinucleated cells formed from alveolar macrophages in the presence of 1 alpha,25(OH)2D3 were totally negative for TRACP reaction. When marrow cells were cultured on dentine slices in the presence of 1 alpha,25(OH)2D3, some of the multinucleated cells were located in the shallow resorption lacunae of dentine surfaces, and they developed the characteristic ruffled borders and clear zones. The narrow extracellular spaces of the ruffled borders, the adjacent pale endocytotic vacuoles, and the dark lysosomes located in the perinuclear cytoplasm of the multinucleated cells contained numerous apatite crystals delete in resorption lacunae. These results indicate that 1) the multinucleated cells formed on coverslips from mouse marrow cells treated with 1 alpha,25(OH)2D3 exhibit non-functional basic features of osteoclast morphology, and 2) differentiation of the multinucleated cells into functional osteoclasts requires some components of calcified dentine.     

α-L-fucosidase in cultured bone marrow fibroblasts from fucosidosis patients

Bone marrow fibroblasts were cultured from two patients with fucosidosis type 2, six control subjects, and three patients with other lysosomal disorders. Optimal conditions for measuring α-L-fucosidase activity in lysates of these cells with the fluorogenic substrate 4-methylumbelliferyl-α-L-fucoside were established. The pH profile of normal bone marrow fibroblasts showed three peaks and a shoulder of enzymatic activity, with maximum activity at pH 4.75. In cells derived from fucosidosis patients two peaks of apparent α-L-fucosidase activity were obtained; the pH optimum was 4.5. α-L-Fucosidase activity (mean ± SD) in the fucosidosis and control bone marrow fibroblasts was 2.5 and 312.4 ± 10.9 nmoles 4-methylumbelliferone per milligram protein per hour, respectively. A reduction in the apparent specific enzymatic activity in the fucosidosis cells was observed by using increasing concentrations of cellular protein in the assay system. Mixing experiments between normal and fucosidosis cells gave the expected activities. These findings indicate that cultured bone marrow fibroblasts can be used for the diagnosis and study of fucosidosis.     

Age-dependent stimulation or inhibition of calcium release from bone cultures by 1 alpha, 25-dihydroxyvitamin D3

The effect of 1 alpha, 25-dihydroxyvitamin D3 (1 alpha, 25-(OH)2D3) on calcium release from neonatal and young adult mouse calvaria in tissue culture was studied. At concentrations from 2.6 X 10(-6) M to 2.6 X 10(-9) M, 1 alpha 25-(OH)2D3 enhanced calcium release from 5-day-old calvaria. At the same concentrations, 1 alpha, 25-(OH)2D3 either inhibited or failed to enhance calcium release from 75-day-old calvaria. Parathyroid hormone-enhanced calcium release from 75-day-old calvaria was inhibited considerably by 1 alpha, 25-(OH)2D3 at 2.6 X 10(-6) M and 2.6 X 10(-7) M. These findings suggest that cells involved in the bone resorption process may respond differently to the same stimulus at different stages in the development of the organism. This direct inhibitory effect of 1 alpha, 25-(OH)2D3 on bone resorption in cultured calvaria of older animals may explain, in part, its beneficial effect in the treatment of osteoporosis.     

Phenotypic and functional markers for 1α,25‐dihydroxyvitamin D3‐modified regulatory dendritic cells

The clinical use of dendritic cells (DCs) to induce antigen‐specific immune tolerance has been hampered by the lack of a widely acknowledged method for generating human regulatory DCs but even more so by the non‐existence of reliable markers. Thus, we set out to find reliable markers that can be measured with simple methods to identify regulatory DCs that are applicable for future clinical studies. Human DCs were generated from peripheral blood monocytes in the presence of 1α,25‐dihydroxyvitamin D₃ (VD3), which gave rise to a phenotype that resembles immature DCs, with the exception of high CD14 and reduced CD1a on the cell surface. These VD3‐treated DCs exert a long‐lasting inefficient T cell stimulation and induce T cell hyporesponsiveness with regulatory potential. Importantly, such VD3‐treated DCs were readily distinguishable from untreated DCs by low levels of interleukin‐23 secretion and low expression of miR‐155 upon exposure to maturation stimuli. Furthermore, VD3‐treated DCs showed over‐expression of miR‐378. All these features can be used as robust markers for quality control of VD3‐treated regulatory DCs in future clinical studies.     

Skewed differentiation of bone marrow CD34+ cells of tumor bearers from dendritic toward monocytic cells,and the redirection of differentiation toward dendritic cells by 1alpha,25-dihydroxyvitamin D3

Tumor presence is detrimental to the development of antigen-presenting dendritic cells. Since dendritic cells can arise from CD34+ precursor cells, the present study assessed the capacity of bone marrow CD34+ cells from tumor bearers to develop into dendritic cells when cultured in the absence of either tumor cells or their products. Culturing bone marrow CD34+ cells from mice bearing Lewis lung carcinomas yielded a lower number of dendritic cells than arose from CD34+ cells of normal mice. This reduced yield of dendritic cells was associated with a shift to development of monocytic cells and a reduced antigen presenting capability by the cultures. When the CD34+ cell cultures from tumor bearers were supplemented with the differentiation-inducing hormone 1alpha,25-dihydroxyvitamin D3, there was the restoration of dendritic cell development and antigen presenting ability. These results show that CD34+ cells from tumor bearers remain defective in their development into dendritic cells even when cultured outside the tumor environment, but development of dendritic cells can be restored with 1alpha,25-dihydroxyvitamin D3.     

1alpha,25-dihydroxyvitamin D3 or analogue treated dendritic cells modulate human autoreactive T cells via the selective induction of apoptosis

Epidemiological evidence indicates that the vitamin D status after birth modulates the risk for development of type 1 diabetes mellitus (T1DM). We previously demonstrated that the biologically active form of vitamin D, 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3), as well as its analogue TX527 permanently alter the morphology and T cell stimulatory function of human dendritic cells (DC). Here, we studied the mechanism of T cell modulation by 1,25(OH)2D3 or analogue treated DC. By using CFSE-labelled autoreactive T cells, we observed that T cell proliferation is hampered upon coculture with modulated DCs, i.e. T cells underwent fewer cycles of cell divisions when compared to T cells stimulated by nontreated DCs. Moreover, 1,25(OH)2D3 or analogue modulated DCs induced significantly higher numbers of early apoptotic (annexin V+/PI-) and/or late apoptotic (annexin V+/PI+) T cells. Apoptosis was selectively induced in T cells activated by modulated DC, since other T cells present in the same cultures, either resting or activated by control untreated DC, were unaffected. Thus, in vitro preconditioning of DC with 1,25(OH)2D3 or analogue yields regulatory DC that may interfere with ongoing autoimmunity in vivo without affecting T cells with other specificities.     

Insoluble polyelectrolyte complex formed from chitosan and α-keratose: conformational change of α-keratose

The conformational change of α-keratose in the polyelectrolyte complex (PEC) formation with chitosan was investigated by means of differential scanning calorimetry and infrared spectroscopy. The change of the secondary structure of α-keratose in the reaction mixture was estimated quantitatively with circular dichroic spectra by the curve fitting method. The α-helix structure of 30% and above of α-keratose was found to be destructed and transformed into random structures at a mixing ratio of maximum PEC formation.     

Influence of magnesium on the in vitro synthesis of 24,25-dihydroxyvitamin D3 and 1 alpha, 25-dihydroxyvitamin D3.

The role of magnesium in the regulation of 25(OH)D3-24R-hydroxylase and 25(OH)D3-1 alpha-hydroxylase in vitro remains to be clarified. Using a radiochemical assay, the effect of substrate concentration on both enzymes was measured in rat kidney homogenates as well as the alterations in their kinetic behaviour resulting from changes in the magnesium concentration in the incubation medium. The results showed that magnesium could be a modulator of 24- and 1 alpha-hydroxylases. Variations in magnesium caused alterations in the kinetic behaviour of both enzymes, which altered the synthesis rates of 24,25(OH)2D3 and 1,25(OH)2D3. Moreover, the substrate concentration was clearly involved in the response of both enzymes to the modulation by magnesium.     

CD45RA expression on γδ and αβ T cells emigrating from the fetal and postnatal thymus

We have compared the expression of CD45RA on αβ and γδ T cells emigrating from the fetal and postnatal thymus. The fetal and postnatal thymus export both CD45RA⁺ and CD45RA^- T cells. The number of γδ⁺CD45RA⁺ T cells was remarkably constant regardless of stage of ontogeny or T cell maturity. Around 5--8% of γδ thymic emigrants, thymocytes and peripheral blood lymphocytes expressed CD45RA in both fetal and postnatal animals. In contrast to γδ T cells, up to one quarter of both fetal and postnatal αβ emigrants expressed CD45RA. Post-thymic maturation of CD45RA expression on αβ emigrants, which occurred both before and after birth, appeared to be antigen independent.     

1, 25二羟基胆钙化醇诱导大鼠骨髓单核细胞向破骨细胞转化的机制

目的:探讨1,25二羟基胆钙化醇(1,25(OH)2D3)诱导大鼠骨髓单核细胞向破骨细胞转化时明胶酶表达及其参与骨陷窝形成机制。方法:分离乳鼠骨髓内细胞,诱导生成破骨样细胞。姬姆莎、抗酒石酸酸性磷酸酶(TRAP)染色鉴定。扫描电镜观察诱导出的细胞贴附于骨片上形成的骨陷窝,明胶酶谱检测细胞培养液中明胶酶表达水平。结果:单核细胞经1,25(OH)2D3诱导,第9日生成大量的破骨样细胞。姬姆莎染色显示出多核(≥3个),TRAP染色阳性,扫描电镜观察破骨细胞在骨片上培养时产生骨陷窝。1,25(OH)2D3组基质金属蛋白酶2(MMP-2)表达水平增加显著。结论:1,25(OH)2D3诱导单核细胞产生大量破骨细胞。参与骨陷窝形成的明胶酶是MMP-2。这可能是破骨细胞通过某些机制,促进其他非破骨细胞分泌有活性的MMP-2参与噬骨。     

1α,25‐dihydroxyvitamin D3 acts via transforming growth factor‐β to up‐regulate expression of immunosuppressive CD73 on human CD4+ Foxp3– T cells

Vitamin D deficiency is associated with increased incidence and severity of various immune‐mediated diseases. Active vitamin D (1α,25‐dihydroxyvitamin D3; 1,25(OH)₂D3) up‐regulates CD4⁺ T‐cell expression of the purine ectonucleotidase CD39, a molecule that is associated with the generation of anti‐inflammatory adenosine. Here we aimed to investigate the direct impact of 1,25(OH)₂D3 on expression of the downstream ecto‐5′‐nucleotidase CD73 by human CD4 T cells, and components of the transforming growth factor‐β (TGF‐β) pathway, which have been implicated in the modulation of CD73 by murine T cells. At 10^?8 to 10^?7 m , 1,25(OH)₂D3 significantly increased expression of CD73 on peripheral human CD4⁺ T cells. Although 1,25(OH)₂D3 did not affect the mRNA expression of latent TGF‐β₁, 1,25(OH)₂D3 did up‐regulate expression of TGF‐β‐associated molecules [latency‐associated peptide (LAP), glycophorin A repetitions predominant (GARP), GP96, neuropilin‐1, thrombospondin‐1 and α_v integrin] which is likely to have contributed to the observed enhancement in TGF‐β bioactivity. CD73 was highly co‐expressed with LAP and GARP following 1,25(OH)₂D3 treatment, but unexpectedly, each of these cell surface molecules was expressed primarily on CD4⁺ Foxp3^– T cells, rather than CD4⁺ Foxp3⁺ T cells. Notably, neutralization of TGF‐β significantly impaired 1,25(OH)₂D3‐mediated induction of CD73. Collectively, we show that 1,25(OH)₂D3 enhances expression of CD73 on CD4⁺ Foxp3^– T cells in a process that is at least partially TGF‐β‐dependent. These data reveal an additional contributing mechanism by which vitamin D may be protective in immune‐mediated disease.     

Immunomodulatory and Immunosuppressive Roles of 1α,25(OH)2D3 in Autoimmune Diseases

Autoimmune diseases are pathological conditions characterized by abnormal responses, accompanied by autoantibodies to self‐molecules. The role of vitamin D in autoimmune diseases has increased significantly in the recent past from its functions in calcium and phosphate homoeostasis, and it is now involved in the regulations and proliferations of Th1 and Th17 lymphocyte. 1α,25(OH)2D3 is very important in ameliorations of inflammatory disorders arising from autoimmune diseases, but the mechanism by which this is performed is still a bone of contentions. This review aimed to highlight the existing facts about the roles of Vitamin D in the treatment and management of autoimmune diseases. An extensive online literature search was conducted using PubMed, MEDLINE and Scopus. Accumulated bodies of research evidence are available which demonstrates that Vitamin D has a very important part to play in the regulation of immune responses in autoimmune diseases. Some of the authors suggested that Vitamin D3 carry‐out its immunosuppressive and immune modulatory action, through its actions on antigen‐presenting cells and activated T and B cells with the help of Vitamin D receptors present on the each of these cells. Vitamin D supplementation assists in autoimmune disorders by making qualitative and quantitative changes in the immune system (downregulation of Th1 and upregulations of Th2 cells). This resulted in the body to be more tolerant of self and less likely to mount autoimmune responses.     

Developmental expression of the αIELβ7 integrin on T cell receptor γδ and T cell receptor αβ T cells

A novel monoclonal antibody, 2E7, was shown by immunoprecipitation to be reactive with the α_IELβ7 integrin and was employed to analyze the expression of this integrin in lymphocyte subsets and during T cell ontogeny. In adult lymph nodes, α_IEL was expressed at low levels by 40–70% of CD8⁺ T cells and < 5% of CD4⁺ T cells. However, virtually all intestinal intraepithelial lymphocytes and ?20% of lamina propria CD4⁺ T cells were 2E7⁺, indicating a preferential expression of this integrin on mucosal T cells. Examination of α_IEL integrin expression during thymus ontogeny revealed that ?3–5% of fetal or adult thymocytes were 2E7⁺. Interestingly, early in fetal thymus ontogeny, ?40% of 2E7⁺ cells expressed T cell receptor (TcR)-γδ and this subset persisted through birth. A developmental switch occurred such that 2E7⁺ TcR^? CD4^?8⁺ cells detected on fetal day 19 were followed by 2E7⁺ TcR-αβ CD4^?8⁺ cells in the neonatal thymus. The latter population persisted throughout thymus ontogeny into adulthood. Interestingly, a subset of TcR-γδ Vγ3⁺ day 16 fetal thymocyte dendritic epidermal cell (DEC) precursors were 2E7⁺, but all mature DEC expressed high levels of α_IEL integrin, suggesting that the α_IEL integrin was acquired late in DEC maturation. This possibility was strenghthened by immunohistochemical localization of the majority of 2E7⁺ γδ and αβ T cells to the medullary regions of the thymus. Overall, the results demonstrate a developmentally ordered expression pattern of the α_IELβ₇ integrin that suggests a common function for this integrin during TcR-γδ and -αβ CD4^?8⁺ T cell thymocyte development or perhaps in effector functions for these subsets.     

Inhibitory effect of CGRP on osteoclast formation by mouse bone marrow cells treated with isoproterenol

The present study was designed to elucidate the mode of action of isoproterenol (Isp; adrenergic beta-agonist) and to characterize the effect of the calcitonin gene-related peptide (CGRP; sensory neuropeptide) on osteoclast formation induced by Isp in a mouse bone marrow culture system. Treatment of mouse bone marrow cells with Isp generated tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells (MNCs) capable of excavating resorptive pits on dentine slices, and caused an increase in receptor activator of NF-kappaB ligand (RANKL) and a decrease in osteoprotegerin (OPG) production by the marrow cells. The osteoclast formation was significantly inhibited by OPG, suggesting the involvement of the RANKL-RANK system. CGRP inhibited the osteoclast formation caused by Isp or soluble RANKL (s-RANKL) but had no influence on RANKL or OPG production by the bone marrow cells treated with Isp, suggesting that CGRP inhibited the osteoclast formation by interfering with the action of RANKL produced by the Isp-treated bone marrow cells without affecting RANKL or OPG production. This in vitro data suggest the physiological interaction of sympathetic and sensory nerves in osteoclastogenesis in vivo.     

T cells recognize a peptide derived from α-gliadin presented by the celiac disease-associated HLA-DQ (α10501, β10201) heterodimer

CD is unique among the HLA-associated diseases since (a) the disease-promoting agent (gliadin) is known and (b) the disease is precipitated mainly in individuals carrying a particular cis- or trans-encoded HLA-DQ heterodimer; i.e., DQ(α1^*0501, β1^*0201). Further, a preponderance of gliadin-specific T cells derived from the small intestinal mucosa of CD patients are restricted by this DQ heterodimer. T-cell recognition of gliadin peptides presented by the DQ(α1^*, β1^*0201) heterdimer may thus be of importance in CD. Here we report that a T-cell clone from a patient with CD recognizes a synthetic α-gliadin peptide, when presented by the cis- or trans-encoded CD-associated DQ(α1^*0501, β1^*0201) heterdimer. The minimal peptide recognized by the T-cell clone corresponds to residues 31–47 of α-gliadin, which is included in the part of α-gliadin previously shown to have disease-promoting activity. When testing analogue peptides derived from other α-gliadin sequences, one peptide differing by one amino acid was recognized by the T-cell clone, whereas the other peptide differing by two amino acids was not recognized. Our findings demonstrate that the CD-associated DQ(α1^*0501, β1^*0201) heterodimer may serve as an antigen-presenting molecule to T cells for certain gliadin peptides.     

A TCR α chain transgene induces maturation of CD4− CD8− α β+ T cells from γ δ T cell precursors

The proportion of CD4⁻ CD8⁻ double-negative (DN) α β T cells is increased both in the thymus and in peripheral lymphoid organs of TCR α chain-transgenic mice. In this report we have characterized this T cell population to elucidate its relationship to α β and γ δ T cells. We show that the transgenic DN cells are phenotypically similar to γ δ T cells but distinct from DN NK T cells. The precursors of DN cells have neither rearranged endogenous TCRα genes nor been negatively selected by the Mls^a antigen, suggesting that they originate from a differentiation stage before the onset of TCR α chain rearrangements and CD4/CD8 gene expression. Neither in-frame VδDδJδ nor VγJγ rearrangements are over-represented in this population. However, since peripheral γ δ T cells with functional TCRβ gene rearrangements have been depleted in the transgenics, we propose that the transgenic DN population, at least partially, originates from the precursors of those cells. The present data lend support to the view that maturation signals to γ δ lineage-committed precursors can be delivered via TCR α β heterodimers.