Combined Use of Tocilizumab and Mesenchymal Stem Cells Attenuate the Development of an Anti-HLA-A2.1 Antibody in a Highly Sensitized Mouse Model.

Sensitization to HLA can result in allograft loss for kidney transplantation (KT) patients. Therefore, it is required to develop an appropriate desensitization (DSZ) technique to remove HLA-donor-specific anti-HLA antibody (DSA) before KT. The aim of this research was to investigate whether combined use of the IL-6 receptor-blocking antibody, tocilizumab (TCZ), and bone-marrow-derived mesenchymal stem cells (BM-MSCs) could attenuate humoral immune responses in an allo-sensitized mouse model developed using HLA.A2 transgenic mice. Wild-type C57BL/6 mice were sensitized with skin allografts from C57BL/6-Tg (HLA-A2.1)1Enge/J mice and treated with TCZ, BM-MSC, or both TCZ and BM-MSC. We compared HLA.A2-specific IgG levels and subsets of T cells and B cells using flow cytometry among groups. HLA.A2-specific IgG level was decreased in all treated groups in comparison with that in the allo-sensitized control (Allo-CONT) group. Its decrease was the most significant in the TCZ + BM-MSC group. Regarding the B cell subset, combined use of TCZ and BM-MSC increased proportions of pre-pro B cells but decreased proportions of mature B cells in BM (p < 0.05 vs. control). In the spleen, an increase in transitional memory was observed with a significant decrease in marginal, follicular, and long-lived plasma B cells (p < 0.05 vs. control) in the TCZ + BM-MSC group. In T cell subsets, Th2 and Th17 cells were significantly decreased, but Treg cells were significantly increased in the TCZ+BM-MSC group compared to those in the Allo-CONT group in the spleen. Regarding RNA levels, IL-10 and Foxp3 showed increased expression, whereas IL-23 and IFN-γ showed decreased expression in the TCZ + BM-MSC group. In conclusion, combined use of TCZ and BM-MSC can inhibit B cell maturation and up-regulate Treg cells, finally resulting in the reduction of HLA.A2-specific IgG in a highly sensitized mouse model. This study suggests that the combined use of TCZ and BM-MSC can be proposed as a novel strategy in a desensitization protocol for highly sensitized patients.

Modeling of Fabry disease nephropathy using patient derived human induced pluripotent stem cells and kidney organoid system.

OBJECTIVES: To explore the possibility of kidney organoids generated using patient derived human induced pluripotent stem cells (hiPSC) for modeling of Fabry disease nephropathy (FDN). METHODS: First, we generated hiPSC line using peripheral blood mononuclear cells (PBMCs) from two male FD-patients with different types of GLA mutation: a classic type mutation (CMC-Fb-001) and a non-classic type (CMC-Fb-003) mutation. Second, we generated kidney organoids using wild-type (WT) hiPSC (WTC-11) and mutant hiPSCs (CMC-Fb-001 and CMC-Fb-003). We then compared alpha-galactosidase A (α-GalA) activity, deposition of globotriaosylceremide (Gb-3), and zebra body formation under electromicroscopy (EM). RESULTS: Both FD patients derived hiPSCs had the same mutations as those detected in PBMCs of patients, showing typical pluripotency markers, normal karyotyping, and successful tri-lineage differentiation. Kidney organoids generated using WT-hiPSC and both FD patients derived hiPSCs expressed typical nephron markers without structural deformity. Activity of α-GalA was decreased and deposition of Gb-3 was increased in FD patients derived hiPSCs and kidney organoids in comparison with WT, with such changes being far more significant in CMC-Fb-001 than in CMC-Fb-003. In EM finding, multi-lammelated inclusion body was detected in both CMC-Fb-001 and CMC-Fb-003 kidney organoids, but not in WT. CONCLUSIONS: Kidney organoids generated using hiPSCs from male FD patients might recapitulate the disease phenotype and represent the severity of FD according to the GLA mutation type.

CRISPR-Cas9-Mediated Correction of SLC12A3 Gene Mutation Rescues the Gitelman's Disease Phenotype in a Patient-Derived Kidney Organoid System.

The aim of this study is to explore the possibility of modeling Gitelman's disease (GIT) with human-induced pluripotent stem cell (hiPSC)-derived kidney organoids and to test whether gene correction using CRISPR/Cas9 can rescue the disease phenotype of GIT. To model GIT, we used the hiPSC line CMCi002 (CMC-GIT-001), generated using PBMCs from GIT patients with SLC12A3 gene mutation. Using the CRISPR-Cas9 system, we corrected CMC-GIT-001 mutations and hence generated CMC-GIT-001corr. Both hiPSCs were differentiated into kidney organoids, and we analyzed the GIT phenotype. The number of matured kidney organoids from the CMC-GIT-001corr group was significantly higher, 3.3-fold, than that of the CMC-GIT-001 group (12.2 ± 0.7/cm2 vs. 3.7 ± 0.2/cm2, p < 0.05). In qRT-PCR, performed using harvested kidney organoids, relative sodium chloride cotransporter (NCCT) mRNA levels (normalized to each iPSC) were increased in the CMC-GIT-001corr group compared with the CMC-GIT-001 group (4.1 ± 0.8 vs. 2.5 ± 0.2, p < 0.05). Consistently, immunoblot analysis revealed increased levels of NCCT protein, in addition to other tubular proteins markers, such as LTL and ECAD, in the CMC-GIT-001corr group compared to the CMC-GIT-001 group. Furthermore, we found that increased immunoreactivity of NCCT in the CMC-GIT-001corr group was colocalized with ECAD (a distal tubule marker) using confocal microscopy. Kidney organoids from GIT patient-derived iPSC recapitulated the Gitelman's disease phenotype, and correction of SLC12A3 mutation utilizing CRISPR-Cas9 technology provided therapeutic insight.

Effect of dual inhibition of DPP4 and SGLT2 on tacrolimus-induced diabetes mellitus and nephrotoxicity in a rat model.

Sodium/glucose co-transporter-2 inhibitor (SGLT2i) or dipeptidyl peptidase IV inhibitor (DPP4i) is a newer anti-diabetic drug in type II diabetes mellitus (DM), but their use in tacrolimus (TAC)-induced DM is still undetermined. We performed this study to evaluate the effect of these two drugs in TAC-induced DM and nephrotoxicity in ex vivo and in vivo. In the experimental Sprague Dawley rat model of TAC-induced DM and nephrotoxicity, dual inhibition of DPP4 and SGLT2 significantly decreased blood glucose level, HbA1C and increased plasma insulin levels and pancreatic islet size compared with each drug. In the kidney, dual inhibition improved renal function decreased interstitial fibrosis and profibrotic cytokines compared with DPP4i and SGLT2i alone. Increased oxidative stress by TAC was remarkably decreased with DPP4i or SGLT2i in serum, pancreatic and renal tissues and this decrease was much more significant in the combination group. In in vitro study, TAC decreased the cell viability of human kidney-2(HK-2) cells and insulin-secreting beta-cell-derived line(INS-1) cells. SGLT2i protected TAC-induced cell death in HK-2 cells, but not in INS-1 cells. The addition of DPP4i to SGLT2i compensated for a lack of protective effect of SGLT2i on INS-1 cells. This finding provides the rationale for the combined treatment of SGLG2i and DPP4i in TAC-induced DM and nephrotoxicity.

Smoking and Drinking Adjusted Association between Head and Neck Cancers and Oral Health Status Related to Periodontitis: a Meta-Analysis.

BACKGROUND: Not so many reports about the association between head and neck cancer (HNC) and oral health status related to periodontitis (OHS-P) has been published in different countries with different methods. So, there is a need for an extensive meta-analysis with the total articles published until 2020. Hence, this study aimed to estimate the association between HNC and OHS-P through a meta-analysis. METHODS: Based on Preferred Reporting Items for Systematic Reviews and Meta Analyses guidelines, 22 studies were selected through PubMed and Cochrane Library databases. Meta-analysis using them was performed to evaluate the association. The risk of bias assessment using the Newcastle-Ottawa Scale (NOS) was applied to evaluate the quality of non-randomized studies. Publication bias was evaluated by funnel plot and Egger's regression test. RESULTS: Since heterogeneity was significant (I² = 88%, P < 0.001), we adopted the random effect model for 22 studies. Those with bad OHS-P, compared to those with good OHS-P, were more likely to have the risk of HNC by 2.4 times (odds ratio [OR], 2.42; 95% confidence interval [CI], 1.88-3.13) for random effect model. The association included publication bias (Egger's regression, P value < 0.001). The association among five studies (I² = 39%, P = 0.16) using alveolar bone loss (ABL) or clinical attachment level (CAL) for assessing periodontitis increased to OR of 3.85 (CI, 3.04-4.88) in the fixed effect model without publication bias (Egger's regression, P = 0.66). Moreover, the association was higher in 10 fair or good NOS studies (OR, 3.08) and in 7 Asian studies (OR, 2.68), which were from the fixed model without publication bias. CONCLUSION: Our meta-analysis showed that bad OHS-P was associated with the risk of HNC. The association was stronger in studies using ABL or CAL for assessing periodontitis.

BAFF Inhibition Effectively Suppresses the Development of Anti-HLA.A2 Antibody in the Highly Sensitized Mouse Model.

B cell activating factor (BAFF) is a cytokine that plays a role in the survival, proliferation and differentiation of B cells. We proposed to observe the effects of BAFF inhibition on the humoral immune responses of an allosensitized mouse model using HLA.A2 transgenic mice. Wild-type C57BL/6 mice were sensitized with skin allografts from C57BL/6-Tg (HLA-A2.1)1Enge/J mice and were treated with anti-BAFF monoclonal antibody (mAb) (named Sandy-2) or control IgG1 antibody. HLA.A2-specific IgG was reduced in BAFF-inhibited mice compared to the control group (Δ-13.62 vs. Δ27.07, p < 0.05). BAFF inhibition also resulted in increased pre-pro and immature B cell proportions and decreased mature B cells in the bone marrow (p < 0.05 vs. control). In the spleen, an increase in transitional B cells was observed with a significant decrease in marginal and follicular B cells (p < 0.05 vs. control). There was no significant difference in the proportions of long-lived plasma and memory B cells. Microarray analysis showed that 19 gene probes were significantly up- (>2-fold, p < 0.05) or down-regulated (≤2-fold, p < 0.05) in the BAFF-inhibited group. BAFF inhibition successfully reduced alloimmune responses through the reduction in alloantibody production and suppression of B cell differentiation and maturation. Our data suggest that BAFF suppression may serve as a useful target in desensitization therapy.

Effect of Klotho on autophagy clearance in tacrolimus-induced renal injury.

Recently, we showed that tacrolimus-induced renal injury was closely associated with impairment of autophagy clearance, and Klotho deficiency aggravated tacrolimus-induced renal injury. In this study, we evaluated the effect of Klotho treatment on autophagy clearance in tacrolimus-induced renal injury. We evaluated the effect of Klotho on tacrolimus-induced renal injury in an experimental mouse model and in vitro by treatment with tacrolimus and/or recombinant mouse Klotho. In vivo and in vitro studies showed that tacrolimus treatment impaired lysosomal acidification and decreased cathepsin B activity, expression of lysosome-associated membrane protein 2, and expression of transcription factor EB (TFEB), a master regulator for lysosomal biogenesis. These results were improved by Klotho treatment. Moreover, addition of bafilomycin A1, an inhibitor of lysosomal function, abolished the protective effect of Klotho, indicating that the protective effect of Klotho was closely associated with lysosome function. Klotho induced nuclear translocation of TFEB through inhibition of phosphorylation of glycogen synthase kinase 3ß (GSK3ß) by confirming using CHIR99021, a GSK3ß inhibitor. Collectively, our data suggest that Klotho improves autophagy clearance via activation of lysosomal function in tacrolimus-induced nephrotoxicity.-Lim, S. W., Shin, Y. J., Luo, K., Quan, Y., Ko, E. J., Chung, B. H., Yang, C. W. Effect of Klotho on autophagy clearance in tacrolimus-induced renal injury.

Host cell in vivo production of the synthetic drug anti-CD25/IL-10 using minicircle vector.

Synthetic biologic drugs are highly successful for induction therapy in transplantation, but the development of novel biologics is limited because of the high cost of synthesis and purification. In this study, we developed a novel strategy for the production of synthetic protein drugs in vivo by the host itself. We utilized minicircle (MC) technology, which can robustly express a target molecule and secrete it from cells, as an indirect method to produce a protein of interest in vivo. We designed an MC vector containing the sequences of basiliximab (anti-CD25 mAb) and IL-10. We verified the substantial production of the anti-CD25/IL-10 protein from the MC in vitro and in vivo. The therapeutic effect of MC-derived anti-CD25/IL-10 was evaluated in a skin allograft mouse model by single intravenous infusion. Mice treated with the MC encoding anti-CD25/IL-10 exhibited prolonged skin allograft survival times accompanied by improved histologic changes and immunologic regulation. These findings indicate that the anti-CD25/IL-10 protein drug obtained by MC technology is functionally active and relevant for reducing allograft rejection. This self-reproducible strategy for synthetic protein drugs using MCs is a promising tool for transplantation.-Lim, S. W., Shin, Y. J., Luo, K., Quan, Y., Ko, E. J., Chung, B. H., Yang, C. W. Host cell in vivo production of the synthetic drug anti-CD25/IL-10 using minicircle vector.

Coenzyme Q10 alleviates tacrolimus-induced mitochondrial dysfunction in kidney.

The major side effect of tacrolimus (Tac) is nephrotoxicity. We studied whether supplementation of coenzyme Q10, (CoQ10) a potent antioxidant, can reduce Tac-induced nephrotoxicity via improving mitochondrial function. In an in vitro study, CoQ10 reduced the production of Tac-induced mitochondrial reactive oxygen species and abolished the loss of mitochondrial membrane potential in proximal tubular cell line. Assessment of mitochondrial function revealed that CoQ10 decreased oxygen consumption and mitochondrial respiration rate increased by Tac, suggesting improvement of mitochondrial function to synthesize ATP with CoQ10 treatment. The effect of the CoQ10in vitro study was observed in an experimental model of chronic Tac-induced nephropathy. CoQ10 attenuated Tac-induced oxidative stress and was accompanied by function and histologic improvement. On electron microscopy, addition of CoQ10 increased not only the number but also the volume of mitochondria compared with Tac treatment only. Our data indicate that CoQ10 improves Tac-induced mitochondrial dysfunction in kidney. Supplementary CoQ10 treatment may be a promising approach to reduce Tac-induced nephrotoxicity.-Yu, J. H., Lim, S. W., Luo, K., Cui, S., Quan, Y., Shin, Y. J., Lee, K. E., Kim, H. L., Ko, E. J., Chung, B. H., Kim, J. H., Chung, S. J., Yang, C. W. Coenzyme Q10 alleviates tacrolimus-induced mitochondrial dysfunction in kidney.

Therapeutic Challenge of Minicircle Vector Encoding Klotho in Animal Model.

BACKGROUND: Klotho treatment is a promising approach against kidney injury, but its clinical application is still undetermined. We developed a novel strategy to allow self-production of Klotho protein, using minicircle (MC) technology, and evaluated its feasibility in therapeutic Klotho delivery. METHODS: We engineered MC vectors to carry cassette sequences of Klotho and verified the self-production of Klotho protein from in HEK293T cells. We evaluated the location and persistence of delivered MC in vivo, and the duration of Klotho protein production from MCs by serial measurement of Klotho protein in blood. We subsequently evaluated the therapeutic potential of Klotho-encoding MCs in experimental model of renal injury. RESULTS: We confirmed the production of Klotho from MC by its significant availability in cells transfected with the MC, as well as in its conditioned medium, compared to that in cells transfected with parent vector. MCs were delivered in vivo by hydrodynamic injection via tail vein. After a single injection of MCs, red fluorescence protein was detected until 30 days in liver, and Klotho protein was maintained until 10 days in the blood, suggesting the production of Klotho protein from MCs via protein synthesis machinery in liver. Therapeutic effect of MC was confirmed by functional and histological improvement seen in mouse model of acute ischemia-reperfusion injury and unilateral ureteral obstruction. CONCLUSION: Together, these findings implied that self-generated Klotho protein, using MC technology, is functionally active and relevant as a therapeutic approach in renal injury.

Salivary S100 proteins screen periodontitis among Korean adults.

AIM: This study aims to evaluate the association of salivary S100A8 and A9 proteins with periodontitis and its screening ability for periodontitis cross-sectionally. MATERIAL AND METHODS: We selected 326 participants from the Yangpyeong Cohort: 218 participants with periodontitis and 108 participants without periodontitis. Stage II-IV periodontitis according to the modification of new international classification of periodontitis was considered as periodontitis. S100A8 and A9 were assayed using enzyme-linked immunosorbent assay kit. Age, sex, education, smoking, drinking, exercise, and metabolic syndrome were factored as confounders. Analyses of covariance and logistic regression analysis were applied to evaluate the association of S100A8 and A9 with periodontitis. Receiver operating characteristic curve was applied for screening ability. RESULTS: Those with periodontitis compared to those without periodontitis showed higher adjusted amount of S100A8 (3694 versus 6757 ng/ml, p < 0.001), but less adjusted amount of S100A9 (1341 versus 1030 ng/ml, p = 0.015). The screening ability of S100A8 and A9 on periodontitis was c-statistics of 0.69 (p < 0.001) for both S100A8 and A9, 0.67 for S100A8 and 0.63 (p < 0.001) for S100A9. CONCLUSIONS: Overall, salivary S100A8 and S100A9 could be practical markers for periodontitis. Its screening ability for periodontitis could be beneficial in clinics and at home.

Rehabilitation of lost teeth related to maintenance of cognitive function.

OBJECTIVE: As the era of aging comes, cognitive impairment (CI) is increasing. The impact of rehabilitation of lost tooth on CI remains unclear. This study aimed to investigate whether non-rehabilitated lost teeth (NRLT) is associated with CI among Korean elders. METHODS: A total of 280 elders comprising of 140 cases and 140 age-sex-matched controls were included in this cross-sectional study. CI was assessed using the Mini-Mental Status Examination (MMSE). NRLT was evaluated using panoramic radiograph and oral examination. NRLT was categorized into low (≤4) and high (≥5). Age, sex, education, drinking, smoking, exercise, obesity, hypertension, subclinical atherosclerosis, glucose, cholesterol, depression, and denture-wearing were considered as confounders. Conditional multivariate logistic regression analysis was applied to assess the adjusted association. RESULTS: NRLT was associated with increased CI after controlling for confounders (odds ratio [OR] = 1.06, 95% confidence interval [95% CFI]: 1.00-1.13). However, lost teeth were not associated with CI. Those with high NRLT (≥5) compared to those with low NRLT (≤4) was more likely to have CI by 2.7 times (OR = 2.74, 95% CFI = 1.28-5.86). CONCLUSION: Our data showed that NRLT was independently associated with CI. Hence, rehabilitation of the lost teeth could be important for the maintenance of cognitive function.

Deep sequencing salivary proteins for periodontitis using proteomics.

OBJECTIVES: Saliva is a bodily fluid transuded from gingival crevice fluid and blood and contains many proteins. Proteins in saliva have been studied as markers for periodontal diseases. Mass spectrometric analysis is applied to investigate biomarker proteins that are related to periodontitis. MATERIAL AND METHODS: Saliva samples were collected from 207 participants including 36 pairs matched for age, sex, and smoking who joined Yangpyeong health cohort. Periodontitis was defined by 2005 5th European guideline. Shotgun proteomics was applied to detect proteins from saliva samples. Principal component analysis and Ingenuity Pathway Analysis for canonical pathway and protein pathway were applied. Protein-protein interaction was also applied. Enzyme-linked immunosorbent assay (ELISA) was used to verify the candidate protein markers among another matched participants (n = 80). RESULTS: Shotgun proteomics indicated that salivary S100A8 and S100A9 were candidate biomarkers for periodontitis. ELISA confirmed that both salivary S100A8 and S100A9 were higher in those with periodontitis compared to those without periodontitis (paired-t test, p < 0.05). CONCLUSION: Our proteomics data showed that S100A8 and S100A9 in saliva could be candidate biomarkers for periodontitis. The rapid-test-kit using salivary S100A8 and S100A9 will be a practical tool for reducing the risk of periodontitis and promotion of periodontal health. CLINICAL RELEVANCE: A rapid-test-kit using salivary biomarkers, S100A8 and S100A9, could be utilized by clinicians and individuals for screening periodontitis, which might reduce the morbidity of periodontitis and promote periodontal health.

Association of toothbrushing and proximal cleaning with periodontal health among Korean adults: Results from Korea National Health and Nutrition Examination Survey in year 2010 and 2012.

AIM: Toothbrushing (TB), dental flossing (DF) and inter-dental brushing (IDB) are regarded as fundamental self-care methods for periodontal health. Few evidences on its effectiveness on periodontal health are available. Hence, this study aimed to evaluate the association of TB, DF, IDB and interaction effect with periodontal health. MATERIALS AND METHODS: The nationally representative 4,766 Korean adults aged 19 years and older were cross-sectionally surveyed in 2010 and 2012. Periodontal health was defined as Community Periodontal Index 1-2 for gingivitis and 3-4 for periodontitis. The information about variables was from interview and blood analyses. Multivariable logistic regression analyses and the interaction effect between TB and proximal cleaning (PC: DF and/or IDB) were applied. RESULTS: Toothbrushing thrice or more per day and DF were associated with a lower prevalence of periodontitis by both 44%, while the preventive fraction of DF on gingivitis was 30%. The preventive fraction of interaction effects between TB thrice or more and PC were 78% for periodontitis and 68% for gingivitis among 40-59 year age group. CONCLUSIONS: Toothbrushing and PC are independently associated with periodontal health. Hence, periodontists should recommend TB thrice or more per day and PC such as DF and IDB to promote periodontal health.

Progressive accumulation of autofluorescent granules in macrophages in rat striatum after systemic 3-nitropropionic acid: a correlative light- and electron-microscopic study.

A variety of tissue biomolecules and intracellular structures are known to be autofluorescent. However, autofluorescent signals in brain tissues often confound analysis of the fluorescent markers used for immunohistochemistry. While investigating tissue and cellular pathologies induced by 3-nitropropionic acid, a mitochondrial toxin selective for striatal neurons, we encountered many autofluorescent signals confined to the lesion core. These structures were excited by blue (wavelength = 488 nm) and yellow-orange (555 nm), but not by red (639 nm) or violet (405 nm) lasers, indicating that this autofluorescence overlaps with the emission spectra of commonly used fluorophores. Almost all of the autofluorescence was localized in activated microglia/macrophages, while reactive astrocytes emitted no detectable autofluorescence. Amoeboid brain macrophages filled with autofluorescent granules revealed very weak expression of the microglial marker, ionized calcium-binding adaptor molecule 1 (Iba1), while activated microglia with evident processes and intense Iba1 immunoreactivity contained scant autofluorescent granules. In addition, immunolabeling with two lysosomal markers, ED1/CD68 and lysosomal-associated membrane protein 1, showed a pattern complementary with autofluorescent signals in activated microglia/macrophages, implying that the autofluorescent structures reside within cytoplasm free of intact lysosomes. A correlative light- and electron-microscopic approach finally revealed the ultrastructural identity of the fluorescent granules, most of which matched to clusters of lipofuscin-like inclusions with varying morphology. Thus, autofluorescence in the damaged brain may reflect the presence of lipofuscin-laden brain macrophages, which should be taken into account when verifying any fluorescent signals that are likely to be correlated with activated microglia/macrophages after brain insults.

Expression of SOCS2 mRNA and protein in the ischemic core and penumbra after transient focal cerebral ischemia in rats.

The suppressor of cytokine signaling 2 (SOCS2) has been reported to be involved in astroglial reactions and adult neurogenesis in the ischemic hippocampus. To elucidate whether SOCS2 is implicated in the pathophysiology of stroke, we investigate spatiotemporal regulation and identification of cell phenotypes expressing SOCS2 after transient focal cerebral ischemia. Weak hybridization signals for SOCS2 mRNA were constitutively observed in striatal neurons and upregulation of SOCS2 mRNA was induced in association with nestin-positive cells in stroke-lesioned rats. Analysis of the characteristics and phenotypes of SOCS2/nestin double-labeled cells revealed spatial differences between infarct and peri-infarct areas. SOCS2/nestin double-labeled cells in the infarct area were associated with the vasculature and were highly proliferative. In contrast, the double-labeled cells in the peri-infarct area were indeed glial fibrillary acidic protein (GFAP)-positive reactive astrocytes forming the glial scar, although nestin-negative reactive astrocytes also exhibited weak SOCS2 expression. In addition, induction of SOCS2 expression was observed in Iba1-positive cells showing a macrophage-like phenotype with amoeboid morphology; these cells were predominantly localized in the infarct area. In the peri-infarct area, only a small proportion of Iba1-positive cells with the morphology of brain macrophages expressed SOCS2 and most activated stellate microglial cells with thick and short processes exhibited weak or negligible SOCS2 expression. Thus, our results revealed the phenotypic and functional heterogeneity of SOCS2-expressing cells within infarct and peri-infarct areas, suggesting the involvement of SOCS2 in astroglial reactions and activation/recruitment of brain macrophages and its potential role in perivascular progenitors/stem cells after ischemic stroke.

Increased Expression of Slit2 and its Robo Receptors During Astroglial Scar Formation After Transient Focal Cerebral Ischemia in Rats.

Slit2, a secreted glycoprotein, has recently been implicated in the post-ischemic astroglial reaction. The objective of this study was to investigate the temporal changes and cellular localization of Slit2 and its receptors, Robo1, Robo2, and Robo4, in a rat transient focal ischemia model induced by middle cerebral artery occlusion. We used double- and triple-immunolabeling to determine the cell-specific changes in Slit2 and its receptors during a 10-week post-ischemia period. The expression profiles of Slit2 and the Robo receptors shared overlapping expression patterns in sham-operated and ischemic striatum. Constitutive expression of Slit2 and Robo receptors was observed in striatal neurons with weak intensity, whereas in rats reperfused after ischemic insults, these immunoreactivities were increased in reactive astrocytes. Astroglial induction of Slit2 and Robo in the peri-infarct region was distinct on days 7-14 after reperfusion and thereafter increased progressively throughout the 10-week experimental period. Slit2 and Robo were prominently expressed in the perinuclear cytoplasm and main processes of reactive astrocytes forming the astroglial scar. This observation was confirmed by quantification of the mean fluorescence intensity of Slit2 and Robo receptors over reactive astrocytes localized at the edge of the infarct area. However, activated microglia/macrophages in the peri-infarct area were devoid of any specific labeling for Slit2 and Robo. Thus, our data revealed a selective and sustained induction of Slit2 and Robo in astrocytes localized throughout the astroglial scar after ischemic stroke, suggesting that Slit2/Robo signaling participates in glial scar formation and brain remodeling following ischemic injury.

Induction of Krüppel-like factor 4 expression in reactive astrocytes following ischemic injury in vitro and in vivo.

Krüppel-like factor 4 (KLF4) is a transcription factor with diverse and cell type-specific functions and is associated with a variety of pathophysiological processes. Recently, it has been proposed that the regulation of KLF4 is critical to neuronal differentiation and that neural progenitors overexpressing KLF4 take on a glial identity. The present study aimed to determine whether KLF4 is involved in the astroglial reaction induced by ischemia-reperfusion injury in the brain. No specific KLF4 immunoreactivity was observed in resting astrocytes of the control hippocampus, but significant induction was detected in reactive astrocytes preferentially located in the CA1 and dentate hilar regions of the hippocampus following transient forebrain ischemia. Astroglial KLF4 expression was induced in the nuclei and cytoplasm within 3 days of ischemia and persisted for at least 4 weeks. This pattern was reproduced in an in vitro astrogliosis model of rat primary cortical astrocytes exposed to oxygen-glucose deprivation (OGD). Furthermore, immunoblot assay showed that nuclear and cytosolic extracts from cortical astrocytes subjected to OGD had significantly higher levels of KLF4 protein compared to normoxic extracts. Thus, our data demonstrate that KLF4 expression was induced in astroglia by ischemic injury both in vivo and in vitro, suggesting that KLF4 may act as a transcription factor linked to the regulation of the astroglial reaction following ischemic injury.

Prediction of diabetes mellitus after kidney transplantation using patient-specific induced pluripotent stem cells.

BACKGROUND: Multiple risk factors are involved in new-onset diabetes mellitus (DM) after organ transplantation; however, their ability to predict clinical prognosis remains unclear. Therefore, we investigated whether patient-specific induced pluripotent stem cells (iPSCs) could help predict DM development before performing kidney transplantation (KT). METHODS: We first performed whole transcriptome and functional enrichment analyses of KT patient-derived iPSCs. Our results revealed that insulin resistance, type 2 DM, and transforming growth factor beta signaling pathways are associated between the groups of DM and non-DM. We next determined whether the genetic background was associated with development of iPSCs into pancreatic progenitor (PP) cells. RESULTS: The levels of differentiation-related key markers of PP cells were significantly lower in the DM group than in the non-DM group. Moreover, the results of tacrolimus toxicity screening showed a significant decrease in the number of PP cells of the DM group compared with the non-DM group, suggesting that these cells are more susceptible to tacrolimus toxicity. CONCLUSION: Taken together, these results indicate that PP cells of the DM group showed low developmental potency accompanied by a significantly different genetic background compared with the non-DM group. Thus, genetic analysis can be used to predict the risk of DM before KT.

Single-cell RNA sequencing revealed the role of the Th17 pathway in the development of anti- human leukocyte antigen antibodies in a highly sensitized mouse model.

Background: The aim of this study is to investigate the specific pathway involved in human leukocyte antigen (HLA) sensitization using single-cell RNA-sequencing analysis and an allo-sensitized mouse model developed with an HLA.A2 transgenic mouse. Methods: For sensitization, wild-type C57BL/6 mouse received two skin grafts from C57BL/6-Tg(HLA-A2.1)1Enge/J mouse (allogeneic mouse, ALLO). For syngeneic control (SYN), skin grafts were transferred from C57BL/6 to C57BL/6. We performed single-cell RNA-sequencing analysis on splenocytes isolated from ALLO and SYN and compared the gene expression between them. Results: We generated 9,190 and 8,890 single-cell transcriptomes from ALLO and SYN, respectively. Five major cell types (B cells, T cells, natural killer cells, macrophages, and neutrophils) and their transcriptome data were annotated according to the representative differentially expressed genes of each cell cluster. The percentage of B cells was higher in ALLO than it was in SYN. Kyoto Encyclopedia of Genes and Genomes enrichment analyses indicated that the highly expressed genes in the B cells from ALLO were mainly associated with antigen processing and presentation pathways, allograft rejection, and the Th17 cell differentiation pathway. Upregulated genes in the T cells of ALLO were involved in the interleukin (IL)-17 signaling pathway. The ratio of Th17 cluster and Treg cluster was increased in the ALLO. On flow cytometry, the percentage of Th17 (IL-17+/CD4+ T) cells was higher and regulatory T cells (FOXP3+/CD4+ T) was lower in the ALLO compared to those in the SYN. Conclusion: Our results indicate that not only the B cell lineage but also the Th17 cells and their cytokine (IL-17) are involved in the sensitization to HLA.