Formation of the junctions between lymph follicles in the Peyer's patches even before postweaning activation.

Peyer's patches (PPs), which contain an abundance of B and T cells, play a key role in inducing pivotal immune responses in the intestinal tract. PPs are defined as aggregated lymph follicles, which consist of multiple lymph follicles (LFs) that may interact with each other in a synergistic manner. LFs are thought to be spherical in shape; however, the characteristics of their structure are not fully understood. To elucidate changes in the structure of PPs as individuals grow, we generated serial 2D sections from entire PPs harvested from mice at 2, 4, and 10 weeks of age and performed a 3D analysis using a software, Amira. Although the number of LFs in PPs was not changed throughout the experiment, the volume and surface area of LFs increased significantly, indicating that LFs in PPs develop continuously by recruiting immune cells, even after weaning. In response to the dramatic changes in the intestinal environment after weaning, the development of germinal centers (GCs) in LFs was observed at 4 and 10 weeks (but not 2 weeks) of age. In addition, GCs gradually began to form away from the center of LFs and close to the muscle layer where export lymphatic vessels develop. Importantly, each LF was joined to the adjacent LF; this feature was observed even in preweaning nonactivated PPs. These results suggest that PPs may have a unique organization and structure that enhance immune functions, allowing cells in LFs to have free access to adjacent LFs and egress smoothly from PPs to the periphery upon stimulation after weaning.

Highly purified hypochlorous acid water facilitates glucose metabolism and memory formation in type 2 diabetic mice associated with altered-gut microbiota.

Hypochlorous acid (HOCl) is an endogenous oxidant and chlorinating agent in mammals that is effective against a broad range of microorganisms. However, the effects of exogenous HOCl on biological processes have not been reported. In this study, the effects of highly purified slightly acidic hypochlorous acid water (HP-HAW) were investigated. After the safety of oral administration of HP-HAW was confirmed, the effects of HP-HAW on glucose homeostasis were assessed in mice. HP-HAW treatment significantly improved blood glucose levels in hyperglycemic condition. Based on the 16S rRNA sequencing, HP-HAW treatment significantly increased the diversity and changed the composition of gut microbiota by decreasing the abundance of genus Romboutsia in mice fed normal chow. In obese mice, HP-HAW administration tended to improve glucose tolerance. HP-HAW also attenuated memory impairments and changes N-methyl-d-aspartate (NMDA) receptor mRNA expression in obese mice. HP-HAW treatment suppressed Il-6 mRNA expression in the hippocampus in type 2 diabetic mice. Overall, these results support HP-HAW as a potential therapeutic agent to improve or prevent glucose tolerance and memory decline via gut microbiota alteration.

Effect of age on orthodontic tooth movement in mice.

Background/purpose: The number of middle-aged and elderly orthodontic patients is increasing due to changes in age composition. It is important to investigate the detailed mechanisms of bone remodeling in orthodontic tooth movement (OTM) in the elderly. However, there are few reports on the mechanism of tooth movement in the elderly. The purpose of the present study was to analyze OTM and osteoclastogenesis in aged mice and to elucidate the mechanism. Materials and methods: It has been reported that tumor necrosis factor (TNF)-α plays an important role in osteoclast formation and OTM. First, 8-week-old and 78-week-old male C57BL/6J mice were subcutaneously injected with TNF-α into the calvaiae, and micro-CT, tartrate-resistant acid phosphatase (TRAP) staining, and real-time PCR were performed to evaluate osteoclast formation and bone resorption. Furthermore, osteoclastogenesis by TNF-α and receptor activator of nuclear factor-kappa B ligand (RANKL) using bone marrow cells was evaluated in vitro. Finally, a nickel-titanium closed-coil spring was attached, mesial movement of the maxillary left first molar was performed, and tooth movement distance and osteoclast formation were evaluated. Results: Compared to 8-week-old mice, 78-week-old mice had decreased TNF-α-induced bone resorption, osteoclastogenesis, and TRAP and cathepsin K expression in the calvariae. In vitro osteoclast formation also decreased in 78-week-old mice. Furthermore, tooth movement distance and osteoclastogenesis were reduced. Conclusion: OTM decreased in aged mice, which was shown to be caused by a decrease in osteoclastogenesis. Therefore, it was suggested that it is necessary to keep in mind that tooth movement may be suppressed when treating elderly patients.

Phospholipase D4 as a signature of toll-like receptor 7 or 9 signaling is expressed on blastic T-bet + B cells in systemic lupus erythematosus.

BACKGROUND: In systemic lupus erythematosus (SLE), autoreactive B cells are thought to develop by-passing immune checkpoints and contribute to its pathogenesis. Toll-like receptor (TLR) 7 and 9 signaling have been implicated in their development and differentiation. Although some B cell subpopulations such as T-bet + double negative 2 (DN2) cells have been identified as autoreactive in the past few years, because the upregulated surface markers of those cells are not exclusive to them, it is still challenging to specifically target autoreactive B cells in SLE patients. METHODS: Our preliminary expression analysis revealed that phospholipase D4 (PLD4) is exclusively expressed in plasmacytoid dendritic cells (pDCs) and B cells in peripheral blood mononuclear cells (PBMCs) samples. Monoclonal antibodies against human PLD4 were generated, and flow cytometry analyses were conducted for PBMCs from 23 healthy donors (HDs) and 40 patients with SLE. In vitro cell culture was also performed to study the conditions that induce PLD4 in B cells from HDs. Finally, recombinant antibodies were synthesized from subpopulations of PLD4 + B cells from a patient with SLE, and their antinuclear activity was measured through enzyme-linked immunosorbent assay. RESULTS: pDCs from both groups showed comparable frequency of surface PLD4 expression. PLD4 + B cells accounted for only a few percent of HD B cells, whereas they were significantly expanded in patients with SLE (2.1% ± 0.4% vs. 10.8% ± 1.2%, P < 0.005). A subpopulation within PLD4 + B cells whose cell size was comparable to CD38 + CD43 + plasmablasts was defined as "PLD4 + blasts," and their frequencies were significantly correlated with those of plasmablasts (P < 0.005). PLD4 + blasts phenotypically overlapped with double negative 2 (DN2) cells, and, in line with this, their frequencies were significantly correlated with several clinical markers of SLE. In vitro assay using healthy PBMCs demonstrated that TLR7 or TLR9 stimulation was sufficient to induce PLD4 on the surface of the B cells. Finally, two out of three recombinant antibodies synthesized from PLD4 + blasts showed antinuclear activity. CONCLUSION: PLD4 + B cells, especially "blastic" ones, are likely autoreactive B cells undergoing TLR stimulation. Therefore, PLD4 is a promising target marker in SLE treatment.

Platelet αIIbß3 integrin binds to SARS-CoV-2 spike protein of alpha strain but not wild type and omicron strains.

Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 causes a pandemic infectious disease, Coronavirus disease 2019 (COVID-19). It causes respiratory infection. Then, it progresses into a systemic infection by involving other organs. This progression mechanism remains to be elucidated, although thrombus formation plays an important role in its progression. Platelets is involved in the thrombus formation by aggregating each other through association of activated αIIbß3 integrin with the Arg-Gly-Asp (RGD) motif-containing its ligands such as fibrinogen and von Willebrand factor. SARS-CoV-2 enters host cells through association of the spike protein (S-protein) with its receptor, angiotensin-converting enzyme 2 (ACE-2), on the host cells. While presence of ACE2 in platelets is suspicious, S-protein harbors the RGD sequences within its receptor binding domain. Therefore, it could be possible SARS-CoV-2 enter platelets through association of S-protein with αIIbß3. In this study, we found that receptor binding domain of S-protein of WT SARS-CoV-2 strain barely bound to isolated healthy human platelets. In contrast, highly toxic alpha-strain-based N501Y substitution strongly bound to platelets in a RGD dependent manner, although binding of S protein did not induce platelet aggregation or activation. This binding may serve for transferring the infection to systemic organs.

Toward the development of a vibrant, super-aged society: The future of medicine and society in Japan.

BACKGROUND: As Japan's population continues to age, it is estimated that the number of people aged ≥75 years will exceed 20 million by 2025. Furthermore, over the past 10 years, we have not reduced the difference between life expectancy and healthy life expectancy. Therefore, the extension of healthy life expectancy and the development of a healthy society are the most urgent issues. In terms of medical care, the changing times have inevitably led to changes in disease structures and medical demands; therefore, the medical delivery system has had to be changed to meet these demands. As dementia rapidly increases, it is important to address "frailty," a condition in which people become more vulnerable to environmental factors as they age, and there is a need to provide services to older people, particularly the old-old, that emphasize quality of life in addition to medical care. To realize a super-aged society that will remain vigorous and vibrant for many years, we need to rethink the future of Japanese medicine and healthcare, and the state of society. CURRENT SITUATION AND PROBLEMS: Disparity between healthy life expectancy and average life expectancy in the realization of a healthy society It is a challenge to build a society with a long and healthy life expectancy through comprehensive prevention and management of lifestyle-related diseases, as well as the elucidation of the factors that explain sex differences in healthy life expectancy, based on the recognition that lifestyle-related diseases in midlife are risk factors for frailty and dementia in old age. Challenges in medical care for building a super-aged and healthy society The challenges include promoting clinical guidelines suitable for older people, including lifestyle-related disease management, promoting comprehensive research on aging (basic research, clinical research and community collaboration research), and embodying a paradigm shift from "cure-seeking medical care" to "cure- and support-seeking medical care." Furthermore, the key to the future of integrated community care is the development of a comprehensive medical care system for older people in each region and the development of the next generation of medical personnel. Dissemination of frailty prevention measures in a super-aged society The concept of frailty encompasses the meaning of multifacetedness and reversibility; therefore, a comprehensive approach is required, including the renewal of conventional prevention activities in each region, such as the nutritional status of older people, physical activity including exercise, and various opportunities for social participation and participation conditions. Challenges of an unstable diet and undernutrition in older people According to the National Health and Nutrition Examination Survey of Japan, energy and protein intakes are low in Japanese people aged ≥75 years; particularly in people aged ≥80 years, low and insufficient intake of nutrients are prominent. Undernutrition in older people is increasing and is more pronounced in women. There are multiple factors behind this, including social factors, such as living alone, eating alone, poverty and other social factors, as well as problems with access to food security. Pharmacotherapy for older people: measures against polypharmacy In addition to the problems of adverse drug events, drug interactions, duplication of effects and the presence of drugs that "require particularly careful administration," it is also necessary to take measures against polypharmacy in older people, as well as medical economic issues, such as high drug costs and large amounts of remaining drugs. Barriers to this measure include multiple medical institution visits for each disease, lack of coordination between professions, and lack of understanding by patients and families. Role of local communities in a healthy society The decline in the working-age population is also a major challenge; however, we need to make a shift to use this declining birthrate and aging population as an opportunity rather than a crisis. As we look ahead to the coming of the 100-year age of life, we rethink the creation of a comprehensive society and community, and aim to create an age-free society where everyone can play an active role and live in peace, regardless of age. CONTENTS OF THE PROPOSAL: In this report, we have put together a vision for the future of an aging Japanese society from a broader perspective of how the environment and local communities should be, rather than simply from the perspective of individual health. We aim to convey this proposal to the Ministry of Health, Labor and Welfare, the Ministry of Education, Culture, Sports, Science and Technology, the Cabinet Office, and various professional organizations. The paradigm shift from "cure-seeking medical care" to "cure- and support-seeking medical care" should be promoted for the development of a healthy society While further promoting pre-emptive medical care in the medical care for older people, the development of multidisciplinary medical guidelines appropriate for older people should be promoted at the same time. In addition, we should promote basic aging research, clinical research (including the long-term care field) and transitional research that cover regional areas. Furthermore, while promoting the paradigm shift from "cure-seeking medical care" to "cure- and support-seeking medical care," the development of various comprehensive medical treatment systems for older people and the strengthening of integrated community care systems should be promoted. Development of the next generation of medical personnel to comprehensively deal with geriatric care, including training geriatric specialists, should be promoted As the number of older people with multimorbidities and frailty rapidly increases in the future, we should promote the development of the next generation of medical personnel who can comprehensively handle medical care for older people, including training leading geriatricians in cooperation with multiple professions in the integrated community care system to provide sufficient medical care. Countermeasures for frailty in older people should be promoted from medical and community planning perspectives To address frailty, which requires comprehensive evaluation and intervention, the three pillars of frailty prevention (nutrition, exercise and social participation) should be incorporated and addressed as part of community development within each municipality, taking into account local characteristics. In particular, it is necessary to revise the way of thinking about nutrition management in older people and the guidelines of the societies in the field. In addition, it is important to strengthen industry-academia-government-private partnerships in each region, taking into account not only medical issues, but also social factors, and encourage the development of momentum in the entire region regarding measures against undernutrition in older people. Polypharmacy measures should be promoted in pharmacotherapy for older people It is necessary to promote cooperation between physicians and pharmacists, establish other multiprofessional cooperation systems, and develop medical and long-term care insurance systems to support this. It is also essential to change the public's mindset, and awareness-raising activities at all levels are required, including the enhancement of educational materials for medical caregivers and the general public. In addition, the economic impact of healthcare using big data should be timely clarified. Innovation in medical and urban planning perspectives should be promoted In the future, it will be necessary to modify and update multidisciplinary approaches such as social participation (e.g. participation in a salon) with a view to innovation in both medical care and community development, especially on the idea of a symbiotic community. In addition, industry-academia-government-private partnership is necessary, including all aforementioned, such as places where people can play an active role in the rest of their lives (such as employment), promotion of human connections, promotion of technology to support older people and support for daily life. Geriatr Gerontol Int 2021; 21: 601-613.

Importance of lymph node immune responses in MSI-H/dMMR colorectal cancer.

Patients with colorectal cancers (CRCs) generally exhibit improved survival through intensive lymph node (LN) dissection. However, recent progress in cancer immunotherapy revisits the potential importance of regional LNs, where T cells are primed to attack tumor cells. To elucidate the role of regional LN, we investigated the immunological status of nonmetastatic regional LN lymphocytes (LNLs) in comparison with those of the tumor microenvironment (tumor-infiltrating lymphocytes; TILs) using flow cytometry and next-generation sequencing. LNLs comprised an intermediate level of the effector T cell population between peripheral blood lymphocytes (PBLs) and TILs. Significant overlap of the T cell receptor (TCR) repertoire was observed in microsatellite instability-high/mismatch repair-deficient (MSI-H/dMMR) CRCs with high tumor mutation burden (TMB), although limited TCRs were shared between nonmetastatic LNs and primary tumors in microsatellite stable/MMR proficient (MSS/pMMR) CRC patients with low TMB. In line with the overlap of the TCR repertoire, an excessive LN dissection did not provide a positive impact on long-term prognosis in our MSI-H/dMMR CRC cohort (n = 130). We propose that regional LNs play an important role in antitumor immunity, particularly in MSI-H/dMMR CRCs with high TMB, requiring care to be taken regarding excessive nonmetastatic LN dissection in MSI-H/dMMR CRC patients.

Palladium-Induced Temporal Internalization of MHC Class I Contributes to T Cell-Mediated Antigenicity.

Palladium (Pd) is a widely used metal and extremely important biomaterial for the reconstruction of occlusions during dental restorations. However, metallic biomaterials can cause serious allergic reactions, such as Pd-related oral mucositis seen in dentistry. Metal allergy is categorized as a type IV allergy and we demonstrated that CD8 T cells play an important role in Pd allergy previously. As TCR of CD8 T cells recognizes MHC class I/peptide complex, the antigen specificity to this complex seems to be generated during Pd allergy. However, it remains unknown if Pd affects the MHC class I/peptide complex. In this study, we investigated the behavior of the MHC class I/peptide complex in response to Pd treatment. We found that PdCl2 treatment altered peptide presentation on MHC class I and that co-culture with Pd-treated DC2.4 cells induced activation of Pd-responsive TCR-expressing T cell line. Furthermore, PdCl2 treatment induced temporal MHC class I internalization and inhibition of membrane movement suppressed Pd-induced T cell-mediated antigenicity. These data suggest that Pd-induced MHC class I internalization is critical for generation of antigenicity through a mechanism including differential peptide loading on MHC class I, which results in Pd allergy.

Formation of carbon-added anatase-rich TiO2 layers on titanium and their antibacterial properties in visible light.

OBJECTIVE: To avoid risk of infections associated with dental implants, thermal oxidation processes for practical dental Ti alloys were studied for both high bonding strength and antibacterial properties in visible light. METHODS: Two-step thermal oxidation, comprising carburization (first step of treatment: in Ar-1%CO gas) and subsequent oxidation (second step of treatment: in air), was conducted on commercially pure (CP) Ti, Ti-6Al-4V (Ti64), and Ti-6Al-7Nb (Ti67) alloys to form TiO2 layers. Their bonding strengths and antibacterial properties against Escherichia coli (E. coli) in visible light (λ ≥ 400 nm) were evaluated. RESULTS: TiO2 layers formed on each metal were composed of anatase and/or rutile. Anatase fraction and carbon concentration in the layers decreased with increasing temperature in the second step of treatment. Antibacterial properties of the TiO2 layers were dependent on the temperature in the second step of treatment. An approximate antibacterial activity value of 2 (killing ∼99% bacteria) was obtained when the temperatures in the second step of treatment were 673 and 773 K for CP Ti, 773 K for Ti64, and 773 and 873 K for Ti67. It was found that the TiO2 layer must contain carbon and be anatase-rich to exhibit excellent antibacterial properties. Bonding strength between the substrate and TiO2 layers formed at 773 K in the second step of treatment exceeded 80 MPa and was independent of substrate type. SIGNIFICANCE: TiO2 layers, possessing both high bonding strength and excellent antibacterial properties, were successfully formed on practical dental Ti alloys via two-step thermal oxidation.

Point mutation bias in SARS-CoV-2 variants results in increased ability to stimulate inflammatory responses.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection induces severe pneumonia and is the cause of a worldwide pandemic. Coronaviruses, including SARS-CoV-2, have RNA proofreading enzymes in their genomes, resulting in fewer gene mutations than other RNA viruses. Nevertheless, variants of SARS-CoV-2 exist and may induce different symptoms; however, the factors and the impacts of these mutations are not well understood. We found that there is a bias to the mutations occurring in SARS-CoV-2 variants, with disproportionate mutation to uracil (U). These point mutations to U are mainly derived from cytosine (C), which is consistent with the substrate specificity of host RNA editing enzymes, APOBECs. We also found the point mutations which are consistent with other RNA editing enzymes, ADARs. For the C-to-U mutations, the context of the upstream uracil and downstream guanine from mutated position was found to be most prevalent. Further, the degree of increase of U in SARS-CoV-2 variants correlates with enhanced production of cytokines, such as TNF-α and IL-6, in cell lines when compared with stimulation by the ssRNA sequence of the isolated virus in Wuhan. Therefore, RNA editing is a factor for mutation bias in SARS-CoV-2 variants, which affects host inflammatory cytokines production.

Organogenesis of Ileal Peyer's Patches Is Initiated Prenatally and Accelerated Postnatally With Comprehensive Proliferation of B Cells in Pigs.

Morphogenesis and differentiation of organs is required for subsequent functional maturation. The morphological features of Peyer's patches vary among species. In pigs, they develop extensively in the ileum as ileal Peyer's patches (IPPs). However, the role of IPPs in the porcine immune system remains to be elucidated because of a lack of complete understanding of IPP organogenesis. Results of the present study revealed that development of porcine IPPs is initiated prenatally between embryonic days 76 and 91. The process of IPP organogenesis is concomitant with increased transcriptional patterns of CXCL13 and CCL19. IPPs undergo further development postnatally by forming central, marginal, and subepithelial zones. Importantly, a large number of proliferating B cells and apoptotic cells are found in porcine IPPs postnatally, but not prenatally. The expression level of IgM in proliferating B cells depends on the zone in which distinct B cells are separately localized after birth. Specifically, IgM+ cells are predominantly found in the central zone, whereas IgM-/low cells are abundant in the marginal zone. Importantly, the cellular feature of IPPs differs from that of mesenteric lymph nodes (MLNs) where such distinct zones are not formed both prenatally and postnatally. Our findings suggest that IPPs (not MLNs) in postnatal pigs are involved in complementing functions of the primary lymphoid tissue that promotes the differentiation and maturation of B cells.

COX-2 induces T cell accumulation and IFN-γ production during the development of chromium allergy.

Chromium (Cr) is commonly added into various metal alloys to improve some mechanical properties such as corrosion resistance, strength, and workability. However, Cr is also known to be a metal allergen for some individuals. Metal allergy is a T cell-mediated disease with symptoms of inflammation and swelling that involve inflammatory cytokines and prostaglandins. Hence, suppressing these inflammation paths by using COX-2 inhibitor might be useful in treating Cr allergy. In this study, mice were used with Cr-induced allergy challenge model. The mice were injected with celecoxib once per day for 7 days one hour after the challenge. Footpad samples were stained with haematoxylin and eosin (H&E), and lymphocytes were isolated from popliteal lymph nodes for the flow cytometric analysis. The results show that both prostaglandin E2 (PGE2), a known mediator of inflammation, and cyclooxygenases (COX)-2 have important roles in the development of Cr allergy. Further, COX-2 inhibitor, celecoxib, was effective in relieving swelling and inflammation in Cr-allergic mice concordant with suppression of IFN-γ production by CD8+ T cells and T cell accumulation in the lymph nodes. Therefore, the inhibition of COX-2 may be a therapeutic target for Cr allergy, and additional molecules in the PGE2 signalling pathway may also be an effective therapeutic target for the treatment of metal allergy.

Visible-light-responsive antibacterial activity of Au-incorporated TiO2 layers formed on Ti-(0-10)at%Au alloys by air oxidation.

Rutile TiO2 layers were formed on substrates of Ti-(0-10)at%Au alloys by a simple process of air oxidation, and their antibacterial activities were evaluated under visible-light irradiation (λ ≥ 400 nm). Au was introduced into the TiO2 layers on Ti-(1-10)at%Au alloys and existed as both metallic Au nanoparticles and dissolved Au3+ ions. The TiO2 layers that formed on Ti-5at%Au and Ti-10at%Au alloys exhibited visible-light photocatalytic activity, that is, degradation of stearic acid and antibacterial activity against Escherichia coli. These visible-light activities were attributed to the surface plasmon resonance of metallic Au nanoparticles and the decrease in bandgap energy caused by dissolved Au3+ ions. The formation of hydroxyl radicals observed under visible-light irradiation is attributable to antibacterial activity. From a cost perspective, a Ti-5at%Au alloy is more suitable as a substrate for the formation of a TiO2 layer with antibacterial properties than a Ti-10at%Au alloy. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 991-1000, 2019.

Zinc ions have a potential to attenuate both Ni ion uptake and Ni ion-induced inflammation.

Nickel ions (Ni2+) are eluted from various metallic materials, such as medical devices implanted in human tissues. Previous studies have shown that Ni2+ enters inflammatory cells inducing inflammation. However, the regulation of Ni2+ uptake in cells has not yet been reported in detail. In the present study, we investigated the effects of various divalent cations on Ni2+ uptake and Ni2+-induced interleukin (IL)-8 production in the human monocytic cell line, THP-1. We demonstrated that ZnCl2, MnCl2, and CoCl2 inhibited the Ni2+ uptake, while CuCl2, FeCl2, MgCl2, and divalent metal transporter (DMT)-1 inhibitor, Chlorazol Black, did not. Furthermore, ZnCl2 inhibited Ni2+-induced IL-8 production, correlating with the inhibition of Ni2+ uptake. These results suggested that Ni2+ uptake occurred through Zn2+, Mn2+, and Co2+-sensitive transporters and that the inhibition of Ni2+ uptake resulted in the inhibition of IL-8 production. Furthermore, using an Ni wire-implanted mouse model, we found that Ni wire-induced expression of mouse macrophage inflammatory protein-2 (MIP-2) and cyclooxygenase-2 (COX-2) mRNA in the skin tissue surrounding the wire were enhanced by low Zn conditions. These results suggested that the physiological concentration of Zn2+ modulates Ni2+ uptake by inflammatory cells, and a Zn deficient state might increase sensitivity to Ni.

Nickel ions bind to HSP90ß and enhance HIF-1α-mediated IL-8 expression.

Nickel ions (Ni2+) eluted from biomedical devices cause inflammation and Ni allergy. Although Ni2+ and Co2+ elicit common effects, Ni2+ induces a generally stronger inflammatory reaction. However, the molecular mechanism by which Ni2+ and Co2+ induce such different responses remains to be elucidated. In the present study, we compared the effects of Ni2+ and Co2+ on the expression of interleukin (IL)-8 in human monocyte THP-1 cells. We report that NiCl2 but not CoCl2 induced the expression of IL-8; in contrast, CoCl2 elicited a higher expression of hypoxia-inducible factor-1α (HIF-1α). The NiCl2-induced expression of IL-8 in late phase was blocked by a HIF-1α inhibitor, PX-478, indicating that NiCl2 targets additional factors responsible for activating HIF-1α. To identify such targets, proteins that bound preferentially to Ni-NTA beads were analyzed by LC/MS/MS. The analysis yielded heat shock protein 90ß (HSP90ß) as a possible candidate. Furthermore, Ni2+ reduced the interaction of HSP90ß with HIF-1α, and instead promoted the interaction between HIF-1α and HIF-1ß, as well as the nuclear localization of HIF-1α. Using various deletion variants, we showed that Ni2+ could bind to the linker domain on HSP90ß. These results suggest that HSP90ß plays important roles in Ni2+-induced production of IL-8 and could be a potential target for the regulation of Ni2+-induced inflammation.

TRAV7-2*02 Expressing CD8⁺ T Cells Are Responsible for Palladium Allergy.

While metallic biomaterials have led to an improvement in the quality of life, metal allergies, especially to palladium (Pd), has caused a recent increase in allergic patients. Metal allergy is known to be a T cell-mediated delayed-type hypersensitivity (DTH); however, the pathogenic T cell subsets and the specific T cell receptor (TCR) have not been identified. Therefore, we attempted to identify the pathogenic T cells responsible for Pd allergy. We found that activating CD8⁺ T cells significantly increased and that the TRAV (TCRα variable) 7-2*02 chain skewed in Pd allergic mice. Furthermore, adoptive transfer experiments revealed that in vitro-cultured Pd-stimulated antigen presenting cells (APCs) function as memory APCs with recipient mice developing Pd allergy and that the frequency of TRAV7-2*02 increases the same as conventional Pd allergic mice. In contrast, neither proliferation of CD8⁺ T cells nor increasing of TRAV7-2*02 was observed in major histocompatibility complex I (MHC I)-deficient Pd-APCs transferred to mice. Taken together, we revealed that TRAV7-2*02-expressing CD8⁺ T cells are the pathogenic T cells for the development of Pd allergy. We also identified the CDR3 consensus motif of pathogenic TCRs as CAAXSGSWQLIF in TRAV7-2*02/TRAJ (TCRα junction)22*01 positive cells. These results suggest that the specific TCRs represent novel targets for the development of diagnostics and treatments for metal allergy.

In vitro evaluation of Ag-containing calcium phosphates: Effectiveness of Ag-incorporated ß-tricalcium phosphate.

Development of bioceramics with antibacterial activity and without cytotoxicity would be beneficial for preventing infection associated with implants. This study aimed to capitalize on the antibacterial properties of silver (Ag) incorporated in or coexisting in metallic form with calcium phosphates (CaPs). The in vitro dissolution behavior, antibacterial activity, and cytotoxicity of Ag-containing CaPs with different phase fractions of hydroxyapatite (HA) and ß-tricalcium phosphate (ß-TCP) were evaluated. The antibacterial activity of Ag-containing CaPs depended on the main phase of CaP, the chemical state of Ag, and the amount of incorporated Ag. Superior antibacterial activity was obtained from sustained release of Ag ions through continuous dissolution of Ag-incorporated ß-TCP compared to that obtained for HA coexisting with metallic Ag particles. Ag-containing CaPs did not exhibit any toxic effect on V79 fibroblasts. Thus, these results demonstrated the effectiveness of Ag-incorporated ß-TCP in preventing infection, with respect to long-term applications.

The potential role of Osteopontin in the maintenance of commensal bacteria homeostasis in the intestine.

Osteopontin (Opn), a multifunctional extracellular matrix protein, is implicated in the pathogenesis of various inflammatory disorders. Under physiologic conditions, its expression is restricted to certain tissues including bone and kidney tubule. However, cellular activation during disease development induces Opn expression in various immune cells. In this study, using Opn-EGFP knock-in (KI) mice we found that CD8α+ T cells in the intestinal tissues, including Peyer's patch, lamina propria and epithelium, express Opn under steady state conditions. Therefore, we examined the role of Opn-expressing CD8α+ T cells in intestinal homeostasis. Interestingly, Opn knockout (KO) mice had altered fecal microflora concordant with a reduction of TCRγδ+ intraepithelial lymphocytes (IELs). Consistent with this result, both treatment with anti-Opn blocking antibody and deficiency of Opn resulted in decreased survival of TCRγδ+ and TCRαß+ IELs. This data suggests that a possibility that Opn may function as a survival factor for IELs in the intestinal tissue. Collectively, these data suggest the possibility that Opn might regulate the homeostasis of intestinal microflora through maintenance of TCRγδ+ IELs, possibly by support of IEL survival.

IFN-γ is required for cytotoxic T cell-dependent cancer genome immunoediting.

Genetic evolution that occurs during cancer progression enables tumour heterogeneity, thereby fostering tumour adaptation, therapeutic resistance and metastatic potential. Immune responses are known to select (immunoedit) tumour cells displaying immunoevasive properties. Here we address the role of IFN-γ in mediating the immunoediting process. We observe that, in several mouse tumour models such as HA-expressing 4T1 mammary carcinoma cells, OVA-expressing EG7 lymphoma cells and CMS5 MCA-induced fibrosarcoma cells naturally expressing mutated extracellular signal-regulated kinase (ERK) antigen, the action of antigen-specific cytotoxic T cell (CTL) in vivo results in the emergence of resistant cancer cell clones only in the presence of IFN-γ within the tumour microenvironment. Moreover, we show that exposure of tumours to IFN-γ-producing antigen-specific CTLs in vivo results in copy-number alterations (CNAs) associated with DNA damage response and modulation of DNA editing/repair gene expression. These results suggest that enhanced genetic instability might be one of the mechanisms by which CTLs and IFN-γ immunoedits tumours, altering their immune resistance as a result of genetic evolution.

The antihistamine olopatadine regulates T cell activation in palladium allergy.

Because of its corrosion resistance palladium (Pd) has been widely used in many consumer products ranging from fashion accessories to dental materials. Recently, however, an increase in Pd allergy cases has been reported. Metal allergy is categorized as a Type IV allergy, which is characterized as a delayed-type hypersensitivity reaction in which T cells are known to play an important role; however, the precise mechanism of their action remains unclear. Here we defined the relationship between histamine and the Pd allergic reaction specifically with respect to T cell responses. To verify the effects of histamine on T cells, we examined whether there is a change in IFN-γ production following stimulation of histamine or the antihistamine, olopatadine hydrochloride (OLP), in vitro. In addition, we assessed whether OLP administration affected the degree of footpad swelling or IFN-γ production during the Pd allergy response in mice. We found that histamine stimulation increased IFN-γ production in T cells, specifically enhancing IFN-γ production in CD8(+) T cells compared with CD4(+) T cells. Interestingly, OLP suppressed the production of IFN-γ in CD8(+) T cells, and this compound inhibited footpad swelling and IFN-γ production in mice with Pd allergy. These results suggest that histamine promotes the Type IV allergic reaction and thus, the histamine 1 receptor (H1R) might be useful therapeutic target for treatment of metal allergy.