Profound reduction of mature B cell numbers, reactivities and serum lg levels in mice which simultaneously carry the XID and CD40 deficiency gense

It has been known for some time that single mutant nude or CD40T mice have apparently normal numbers of cells in the precursor compartments of bone marrow and the mature B cell compartments of the periphery. X-linked immunodeficiency (XID) mice are deficient only in some of the slgM+slgD+ B cells. We have investigated further the contributions of the xid mutation, of the T cell deficiency of nude and of the inability of CD40T B cells to cooperate with T cells in the generation of the precursor and the mature B cell compartments in mice. Double mutant XIDInu and XIDlCD4OT mice have precursor B cell compartments that are no more deficient than the single mutant XID mice. However, the peripheral B cell compartments of both XIDInu and XIDlCD40T are even more deficient than those of single mutant XID mice. While 10% of the peripheral B cells of wild-type or CD40T, one-third of XID and half of XIDInu mice turn over rapidly, as many as threequarters of those in XIDlCD40T are short-lived. Total numbers of slgM+slgD+ B cells in the spleen are at best 1615% of normal mice at 6-8 weeks of age in XID, XIDInu and XIDICD40T mice. They remain that low at 3 months of age in XIDICD40T mice, while in XID mice these peripheral B cells slowly build up in numbers with age. As expected, double mutant XIDlCD40T mice do not respond to the T-dependent antigen keyhole limpet hemocyanin. Only the responses to the T-independent type I antigen, TNP-lipopolysaccharide (LPS), appear to be normal. In vitro, their splenic B cells respond poorly to LPS or to IgM-specific antibody in either the absence or presence of cytokines. Most notably, serum IgM, lgG2b and lgG3 levels are severely depressed, while IgG1, lgG2a and IgA levels are <I0 pglml. The results suggest a model of mature B cell development in which the peripheral, mature B cell compartments are generated in two parallel, not tandemly organized pathways. They could be selected and/or stimulated at the transition from immature to mature B cells: in btk controlled or in CD40 controlled way

Physico-Chemical, In Vitro, and In Vivo Evaluation of a 3D Unidirectional Porous Hydroxyapatite Scaffold for Bone Regeneration

The unidirectional porous hydroxyapatite HAp (UDPHAp) is a scaffold with continuous communicated pore structure in the axial direction. We evaluated and compared the ability of the UDPHAp as a three-dimensional (3D) bone tissue engineering scaffold to the interconnected calcium porous HAp ceramic (IP-CHA). To achieve this, we evaluated in vitro the compressive strength, controlled rhBMP-2 release behavior, adherent cell morphology, cell adhesion manner, and cell attachment of UDPHAp. As a further in vivo experiment, UDPHAp and IP-CHA with rhBMP-2 were transplanted into mouse calvarial defects to evaluate their bone-forming ability. The Results demonstrated that the maximum compressive strengths of the UDPHAp was 7.89 +/- 1.23 MPa and higher than that of IP-CHA (1.92 +/- 0.53 MPa) (p = 0.0039). However, the breaking energies were similar (8.99 +/- 2.72 vs. 13.95 +/- 5.69 mJ, p = 0.055). The UDPHAp released rhBMP-2 more gradually in vivo. Cells on the UDPHAp adhered tightly to the surface, which had grown deeply into the scaffolds. A significant increase in cell number on the UDPHAp was observed compared to the IP-CHA on day 8 (102,479 +/- 34,391 vs. 32,372 +/- 29,061 estimated cells per scaffold, p = 0.0495). In a mouse calvarial defect model, the percentages of new bone area (mature bone + trabecular bone) in the 2x field were 2.514% +/- 1.224% for the IP-CHA group and 7.045% +/- 2.055% for the UDPHAp group, and the percentage was significantly higher in the UDPHAp group (p = 0.0209). While maintaining the same strength as the IP-CHA, the UDPHAp with 84% porosity showed a high cell number, high cell invasiveness, and excellent bone formation. We believe the UDPHAp is an excellent material that can be applied to bone regenerative medicine.ArticleMATERIALS. 10(1):33 (2017)journal articl

In Vitro and In Vivo Evaluation of Starfish Bone-Derived -Tricalcium Phosphate as a Bone Substitute Material

We evaluated starfish-derived -tricalcium phosphate (Sf-TCP) obtained by phosphatization of starfish-bone-derived porous calcium carbonate as a potential bone substitute material. The Sf-TCP had a communicating pore structure with a pore size of approximately 10 m. Although the porosity of Sf-TCP was similar to that of Cerasorb M (CM)a commercially available -TCP bone fillerthe specific surface area was roughly three times larger than that of CM. Observation by scanning electron microscopy showed that pores communicated to the inside of the Sf-TCP. Cell growth tests showed that Sf-TCP improved cell proliferation compared with CM. Cells grown on Sf-TCP showed stretched filopodia and adhered; cells migrated both to the surface and into pores. In vivo, vigorous tissue invasion into pores was observed in Sf-TCP, and more fibrous tissue was observed for Sf-TCP than CM. Moreover, capillary formation into pores was observed for Sf-TCP. Thus, Sf-TCP showed excellent biocompatibility in vitro and more vigorous bone formation in vivo, indicating the possible applications of this material as a bone substitute. In addition, our findings suggested that mimicking the microstructure derived from whole organisms may facilitate the development of superior artificial bone.ArticleMATERIALS. 12(11):1881 (2019)journal articl

Applications of Carbon Nanotubes in Bone Regenerative Medicine

Scaffolds are essential for bone regeneration due to their ability to maintain a sustained release of growth factors and to provide a place where cells that form new bone can enter and proliferate. In recent years, scaffolds made of various materials have been developed and evaluated. Functionally effective scaffolds require excellent cell affinity, chemical properties, mechanical properties, and safety. Carbon nanotubes (CNTs) are fibrous nanoparticles with a nano-size diameter and have excellent strength and chemical stability. In the industrial field, they are used as fillers to improve the performance of materials. Because of their excellent physicochemical properties, CNTs are studied for their promising clinical applications as biomaterials. In this review article, we focused on the results of our research on CNT scaffolds for bone regeneration, introduced the promising properties of scaffolds for bone regeneration, and described the potential of CNT scaffolds.ArticleNANOMATERIALS. 10(4):659 (2020)journal articl

Expression of Interleukin-10 in Intestinal Lymphocytes Detected by an Interleukin-10 Reporter Knockin tiger Mouse

SummaryTo identify interleukin-10 (IL-10)-producing cells in vivo, we generated a knockin mouse where an internal ribosome entry site (IRES) green fluorescence protein (GFP) element was inserted immediately before the polyadenylation site of the IL-10 gene. GFP fluorescence in cells from these mice was found to correlate positively with IL-10 protein expression. With this model, we found that after multiple T cell receptor (TCR) stimulations, strong expression of IL-10 was produced specifically by intraepithelial lymphocytes (IEL) in the small intestine and colonic lamina propria lymphocytes (cLPL). We found that anti-CD3 treatment induces T regulatory cell 1 (Tr1)-like cells in small intestinal IEL (sIEL) and led to the accumulation of naturally occurring regulatory T (nTreg) cells in colonic LPL (cLPL). These findings highlight the intestine as a unique site for induction of IL-10-producing T cells, which play a critical role in the regulation of inflammation in the gut

Memory/effector (CD45RBlo) CD4 T cells are controlled directly by IL-10 and cause IL-22–dependent intestinal pathology

Interleukin-10 acts directly on CD45RBlo but not CD45RBhi cells to control colitis upon transfer into Rag1-deficient recipients

Anti-PD-1 antibody decreases tumour-infiltrating regulatory T cells

Background There are many types of therapies for cancer. In these days, immunotherapies, especially immune checkpoint inhibitors, are focused on. Though many types of immune checkpoint inhibitors are there, the difference of effect and its mechanism are unclear. Some reports suggest the response rate of anti-PD-1 antibody is superior to that of anti-PD-L1 antibody and could potentially produce different mechanisms of action. On the other hand, Treg also express PD-1; however, their relationship remains unclear. Methods In this study, we used osteosarcoma cell lines in vitro and osteosarcoma mouse model in vivo. In vitro, we analyzed the effect of IFN gamma for expression of PD-L1 on the surface of cell lines by flowcytometry. In vivo, murine osteosarcoma cell line LM8 was subcutaneously transplanted into the dorsum of mice. Mouse anti-PD-1 antibody was intraperitoneally administered. we analysed the effect for survival of anti-PD-1 antibody and proportion of T cells in the tumour by flowcytometry. Results We discovered that IFN gamma increased PD-L1 expression on the surface of osteosarcoma cell lines. In assessing the relationship between anti-PD-1 antibody and Treg, we discovered the administration of anti-PD-1 antibody suppresses increases in tumour volume and prolongs overall survival time. In the tumour microenvironment, we found that the administration of anti-PD-1 antibody decreased Treg within the tumour and increased tumour-infiltrating lymphocytes. Conclusions Here we clarify for the first time an additional mechanism of anti-tumour effect-as exerted by anti-PD-1 antibody decreasing Treg- we anticipate that our findings will lead to the development of new methods for cancer treatment.ArticleBMC CANCER. 20(1):25 (2020)journal articl

Organ accumulation and carcinogenicity of highly dispersed multi-walled carbon nanotubes administered intravenously in transgenic rasH2 mice

Purpose: Multiwalled carbon nanotubes (MWCNTs) have been known to enter the circulatory system via the lungs from inhalation exposure; however, its carcinogenicity and subsequent accumulation in other organs have not been adequately reported in the literature. Moreover, the safety of MWCNTs as a biomaterial has remained a matter of debate, particularly when the material enters the circulatory system. To address these problems, we used carcinogenic rasH2 transgenic mice to intravenously administer highly dispersed MWCNTs and to evaluate their carcinogenicity and accumulation in the organs. Methods: Two types of MWCNTs (thin-and thick-MWCNTs) were intravenously administered at a high dose (approximately 0.7 mg per kg body weight) and low dose (approximately 0.07 mg per kg body weight). Results: MWCNTs showed pancreatic accumulation in 3.2% of mice administered with MWCNTs, but there was no accumulation in other organs. In addition, there was no significant difference in the incidence of tumor among the four MWCNTs-administered groups compared to the vehicle group without MWCNTs administration. Blood tests revealed elevated levels in mean red blood cell volume and mean red blood cell hemoglobin level for the MWCNTs-administered group, in addition to an increase in eotaxin. Conclusion: The present study demonstrated that the use of current technology to sufficiently disperse MWCNTs resulted in minimal organ accumulation with no evidence of carcinogenicity.ArticleINTERNATIONAL JOURNAL OF NANOMEDICINE. 14:6465-6480 (2019)journal articl

Antitumor Effect of Sclerostin against Osteosarcoma

Various risk factors and causative genes of osteosarcoma have been reported in the literature; however, its etiology remains largely unknown. Bone formation is a shared phenomenon in all types of osteosarcomas, and sclerostin is an extracellular soluble factor secreted by osteocytes that prevents bone formation by inhibiting the Wnt signaling pathway. We aimed to investigate the antitumor effect of sclerostin against osteosarcoma. Osteosarcoma model mice were prepared by transplantation into the dorsal region of C3H/He and BALB/c-nu/nu mice using osteosarcoma cell lines LM8 (murine) and 143B (human), respectively. Cell proliferations were evaluated by using alamarBlue and scratch assays. The migratory ability of the cells was evaluated using a migration assay. Sclerostin was injected intraperitoneally for 7 days to examine the suppression of tumor size and extension of survival. The administration of sclerostin to osteosarcoma cells significantly inhibited the growth and migratory ability of osteosarcoma cells. Kaplan–Meier curves and survival data demonstrated that sclerostin significantly inhibited tumor growth and improved survival. Sclerostin suppressed the proliferative capacity and migratory ability of osteosarcoma cells. Osteosarcoma model mice inhibited tumor growth and prolonged survival periods by the administration of sclerostin. The effect of existing anticancer drugs such as doxorubicin should be investigated for future clinical applications.ArticleCancers 13(23) : 6015(2021)journal articl