Mechanism of immune tolerance induced by donor derived immature dendritic cells in rat high-risk corneal transplantation

·AIM: To study the role of immature dendritic cells (imDCs) on immune tolerance in rat penetrating keratoplasty (PKP) in high -risk eyes and to investigate the mechanism of immune hyporesponsiveness induced by donor-derived imDCs. ·METHODS: Seventy-five SD rats (recipient) and 39 Wistar rats (donor) were randomly divided into 3 groups: control, imDC and mature dendritic cell (mDC) group respectively. Using a model of orthotopic corneal transplantation in which allografts were placed in neovascularized high -risk eyes of recipient rat. Corneal neovascularization was induced by alkaline burn in the central cornea of recipient rat. Recipients in imDC group or mDC group were injected donor bone marrow-derived imDCs or mDCs of 1 ×10 6 respectively 1 week before corneal transplantation tail vein. Control rat received the same volume of PBS. In each group, 16 recipients were kept for determination of survival time and other 9 recipients were executed on day 3, 7 and 14 after transplantation. Cornea was harvested for hematoxylin eosin staining and acute rejection evaluation, Western blot was used to detect the expression level of Foxp3. ·RESULTS: The mean survival time of imDC group was significantly longer than that of control and mDC groups (all 【0.05). The expression level of Foxp3 on CD4 + CD25 + T cells of imDC group (2.24 ±0.18) was significantly higher than that in the control (1.68 ±0.09) and mDC groups (1.46±0.13) (all 【0.05).·CONCLUSION: Donor -derived imDC is an effective treatment in inducing immune hyporesponsiveness in rat PKP. The mechanism of immune tolerance induced by imDC might be inhibit T lymphocytes responsiveness by regulatory T cells. ·

Mechanism of Immune Hyporesponsiveness Induced by Recipient-derived Immature Dendritic Cells in Liver Transplantation Rat

Objective To investigate the mechanism of immune hyporesponsiveness induced by donor-antigen- unloaded recipient-derived immature dendritic cell （imDC） of liver grafts in rats. Methods Forty Sprague-Dawley rats （donor） and forty male Wistar rats （recipient） were randomly divided into 4 groups： control, cyclosporine A （CsA）, mature DC （mDC）, and imDC groups respectively, with 10 donor rats and 10 recipient rats in each group. Recipient rats in CsA group were treated with 10 mg-kg-~＇d-I CsA starting day 2 after the transplantation. Recipients in the mDC or imDC groups were given Wistar rat derived mDCs （1 × 10^6/rat） or imDCs （1 × 10^6/rat） via dorsal vein of the penis respectively 1 day before the transplantation. In each group, 5 recipients were kept for determination of survival time and the other 5 rats were executed at day 10 after transplantation. Blood samples were collected for the measurement of serum alanine aminotransferase （ALT）, total bilirubin （TBIL）, interleukin 2 （IL-2）, interferon gamma （IFN-γ）, IL-4, and IL-10 levels. Liver tissue was harvested for HE staining and acute rejection evaluation. Expression levels of Fas-L/Fas in the grafts were detected by immunohistochemical staining; andWestern blot was used to detect the expression level of Scurfm. Results The survival time of CsA and imDC groups was significantly longer than that of control and mDC groups （all P〈0.05）. The levels of serum ALT and TBIL in the control group （2072.20±217.93 IU/L and 147.42±22.02pmol/L） and mDC group （2117.00±285.13 IU/L and 141.58±20.82 pmol/L） were significantly higher than those in the CsA group （59.68±13.48 IU/L and 15.40±2.13 pmol/L） or imDC group （50.80±9.63 IU/L and 14.44±3.49 pmol/L） （all P〈0.05）. In the CsA and imDC groups, the levels of IL-2 （22.52±3.75 pg/mL and 22.12±3.90 pg/mL） and IFN-γ （309.20±25.19 pg/mL and 321.00±21.64 pg/mL） were significantly lower, but the levels of IL-4 （297.60±25.07 pg/mL and 277.00±22.47 pg/mL） and IL-10 （1226.00±140.49 pg/mL and 1423.00±106.39 pg/mL） were higher than those of the control （IL-2：147.78±12.80 pg/mL, IFN-γ： 1758.60±106.22 pg/mL, IL-4：17.40±4.77pg/mL, IL-10： 81.00＋ 9.47 pg/mL） and mDC groups （IL-2：142.34±9.29 pg/mL, IFN-7：1835.00±82.63 pg/mL, IL-4： 15.60＋ 3.96 μg/mL, IL-10： 68.80± 11.23 pg/mL） （all P〈0.01）. The expression level of Scurfin protein on CD4＋CD25 ＋ T cells of the imDC group （1.34±0.29） was significantly higher than that in the control （0.72±0.13）, CsA （0.37±0.11）, and mDC groups （0.78±0.17） （all P〈0.05）.

Sinomenine promotes differentiation of induced pluripotent stem cells into immature dendritic cells with high induction of immune tolerance

BACKGROUND Immature dendritic cells(imDCs)play an important role in the induction of donor-specific transplant immunotolerance.However,these cells have limitations,such as rapid maturation and a short lifespan in vivo.In previous studies,induced pluripotent stem cells(iPSCs)differentiated into imDCs,and sinomenine(SN)was used to inhibit the maturation of imDCs.AIM To study the capacity of SN to maintain iPSC-derived imDCs(SN-iPSCs-imDCs)in an immature state and the mechanism by which SN-iPSCs-imDCs induce immunotolerance.METHODS In this study,mouse iPSCs were induced to differentiate into imDCs in culture medium without or with SN(iPSCs-imDCs and SN-iPSCs-imDCs).The imDCrelated surface markers,endocytotic capacity of fluorescein isothiocyanate Dextran and apoptosis were analyzed by flow cytometry.The effects of iPSCs-imDCs and SNiPSCs-imDCs on T-cell stimulatory function,and regulatory T(Treg)cell proliferative function in vitro were analyzed by mixed lymphocyte reaction.Cytokine expression was detected by ELISA.The apoptosis-related proteins of iPSCs-DCs and SN-iPSCs-DCs were analyzed by western blotting.The induced immunotolerance of SN-iPSCs-DCs was evaluated by treating recipient Balb/c skin graft mice.Statistical evaluation of graft survival was performed using Kaplan–Meier curves.RESULTS Both iPSCs-imDCs and SN-iPSCs-imDCs were successfully obtained,and their biological characteristics and ability to induce immunotolerance were compared.SN-iPSCs-imDCs exhibited higher CD11c levels and lower CD80 and CD86 levels compared with iPSCs-imDCs.Reduced major histocompatibility complex II expression,worse T-cell stimulatory function,higher Treg cell proliferative function and stronger endocytotic capacity were observed with SN-iPSCs-imDCs(P<0.05).The levels of interleukin(IL)-2,IL-12,interferon-γin SN-iPSCs-imDCs were lower than those in iPSCs-imDCs,whereas IL-10 and transforming growth factor-βlevels were higher(P<0.05).The apoptosis rate of these cells was significantly higher(P<0.05),and the expression levels of cleaved caspase3,Bax and cleaved poly(ADP-ribose)polymerase were higher after treatment with lipopolysaccharides,but Bcl-2 was reduced.In Balb/c mice recipients immunized with iPSCsimDCs or SN-iPSCs-imDCs 7 d before skin grafting,the SN-iPSCs-imDCs group showed lower ability to inhibit donor-specific CD4+T-cell proliferation(P<0.05)and a higher capacity to induce CD4+CD25+FoxP3+Treg cell proliferation in the spleen(P<0.05).The survival span of C57bl/6 skin grafts was significantly prolonged in immunized Balb/c recipients with a donor-specific pattern.CONCLUSION This study demonstrated that SN-iPSCs-imDCs have potential applications in vitro and in vivo for induction of immunotolerance following organ transplantation.

Mechanism of Immune Hyporesponsiveness Induced by Recipientderived Immature Dendritic Cells in Rat Liver Transplantation

Objective： The use of donor-derived immature dendritic cells （imDC） has become a promising approach to induce immune tolerance or immune hyporesponsiveness. However, donor-derived imDC needs to be harvested for a few days and transfused into the recipient in 5-10 days before transplantation, which is practically impossible in a clinical setting where donor organs are mainly harvested from cadavers. Moreover, donor-derived imDC might be cleared by allogeneic reaction offsetting induced immune tolerance or immune hyporesponsiveness. In our study, we further explored the underlying mechanism of immune hyporesponsiveness induced by donor-antigen-unloaded recipient-derived imDC by transfusing these imDC into rats in 1 day before liver transplantation. This paper is to study the mechanism of immune hyporesponsiveness induced by donor-antigen-unloaded recipient-derived imDC and its protection of liver grafts in rats. Methods： 40 SD rats （donor） and 40 male Wistar rats （recipient） were randomly divided into 4 groups： control, cyclosporine A （CsA）, mature DC （mDC）, and imDC; with 10 SD rats and 10 Wistar rats for each group. Animal models of acute graft rejection were established with these rats. Corresponding treatments were given before or after transplantation. In the control group, Wistar rats received no treatment other than liver transplantation. In the CsA group, Wistar rats underwent liver transplantation plus CsA treatment （10 mg/kg·d） in the starting day 2 after transplantation. For the mDC group, recipient-derived mDC （1 × 10^6/rat） were infused intravenously via the dorsal vein of the penis to recipient rats. For the imDC group, imDC （1× 10^6/rat） were injected into recipient rats via the dorsal vein of the penis. In each group, 5 recipients were executed at 10 days after transplantation; the remaining five recipients were kept for the observation of survival time. Blood samples were collected for the measurement of ALT and TBIL; IL-2, IFN-γ, IL-4 and IL-10 and levels were measured with double-antibody sandwich ELISA. Liver tissue was harvested for HE staining and the observation of histological features. Acute rejection was evaluated with Banff classification. Expression levels of Fas-L/Fas in the grafts were detected by iminunohistochemieal staining; and western blot was used to detect the expression level of Scurfin. Results： The median survival times （MST） of the liver allografls in the CsA and imDC group were significantly longer than those in the control or mDC group （P〈0.05）. The serum levels of ALT and TBIL in the control and mDC groups were significantly higher than those of the CsA or imDC group （P〈0.05）. Compared with the CsA anti imDC group, the levels of IL-2 and IFN-γ were higher but the levels of IL-4 and IL-10 were lower than those of the control and mDC groups （P〈0.01）. Slight or no rejection reaction was found in the CsA anti imDC groups （P〈0.05）. The expression level of Scurfin protein in CD4^＋ CD25^＋ T cells of the imDC group was significantly higher than that of three other groups （P〈 0.05）. Conclusion： Donor-antigen-unloaded recipient- derived imDC is an effective treatment in inducing immune hyporesponsiveness by blocking indirect recognition in rat liver transplantation model. Survival span was significantly prolonged by its protective effect. The mechanism of immune hyporesponsiveness induced hy imDC transfusion may involve the preprocesses of T cell apoptosis induction, immune tolerance or hyporesponsiveness in T cells, induction of the shift in TH1/TH2 balance, selection activation of Th2 subset, or induction of regulatory T cell.

Inhibition of VEGF-A expression in hypoxia-exposed fetal retinal microvascular endothelial cells by exosomes derived from human umbilical cord mesenchymal stem cells

Objective:This study aimed to investigate the potential of human umbilical cord mesenchymal stem cell(hucMSC)-derived exosomes(hucMSC-Exos)in inhibiting hypoxia-induced cell hyper proliferation and overexpression of vascular endothelial growth factor A(VEGF-A)in immature human fetal retinal microvascular endothelial cells(hfRMECs).Methods:Exosomes were isolated from hucMSCs using cryogenic ultracentrifugation and characterized through various techniques,including transmission electron microscopy,nanoparticle tracking analysis,bicinchoninic acid assays,and western blotting.The hfRMECs were identified using von Willebrand factor(vWF)co-staining and divided into four groups:a control group cultured under normoxic condition,a hypoxic model group,a hypoxic group treated with low-concentration hucMSC-Exos(75μg/mL)and a hypoxic group treated with high-concentration hucMSC-Exos(100μg/mL).Cell viability and proliferation were assessed using Cell Counting Kit-8(CCK-8)assay and EdU(5-ethynyl-2′-deoxyuridine)assay respectively.Expression levels of VEGF-A were evaluated using RT-PCR,western blotting and immunofluorescence.Results:Hypoxia significantly increased hfRMECs’viability and proliferation by upregulating VEGF-A levels.The administration of hucMSC-Exos effectively reversed this response,with the high-concentration group exhibiting greater efficacy compared to the lowconcentration group.Conclusion:In conclusion,hucMSC-Exos can dose-dependently inhibit hypoxia-induced hyperproliferation and VEGF-A overexpression in immature fetal retinal microvascular endothelial cells.

Adult neurogenesis in the primate hippocampus

Adult hippocampal neurogenesis(AHN) is crucial for learning,memory,and emotion.Deficits of AHN may lead to reduced cognitive abilities and neurodegenerative disorders,such as Alzheimer’s disease.Extensive studies on rodent AHN have clarified the developmental and maturation processes of adult neural stem/progenitor cells.However,to what extent these findings apply to primates remains controversial.Recent advances in next-generation sequencing technologies have enabled in-depth investigation of the transcriptome of AHN-related populations at single-cell resolution.Here,we summarize studies of AHN in primates.Results suggest that neurogenesis is largely shared across species,but substantial differences also exist.Marker gene expression patterns in primates differ from those of rodents.Compared with rodents,the primate hippocampus has a higher proportion of immature dentate granule cells and a longer maturation period of newly generated granule cells.Future research on species divergence may deepen our understanding of the mechanisms underlying adult neurogenesis in primates.

Multiple Myeloma: Modification and Adaptation of Plasma Cell Morphology Algorithm for the Prognosis of Congolese Patient

Objectives: To determine the multiple myeloma (MM) prognostic formulas applicable in Congolese with many aberrant and/or immature plasma cells according to GOASGUEN’s plasma cell morphology algorithm (PMA). Methods: The prospective, observational and descriptive study was carried out at the Ngaliema Clinic in Kinshasa from July 2016 to December 2019. The variables studied were the plasma cell types and the prognosis groups according to GOASGUEN and ZANDECKI. The nucleolus, the chromatin and the N/C ratio of 2 for each plasma cell encountered in multiple myeloma are examined in order by asking three successive questions as follows: 1) Is the nucleolus present? If yes, we denote 1;if not, note 0;2) Is the chromatin thin? If yes, we score 1;if not we score 0;3) The N/C ratio is greater than 6, if so, 1 is noted;if not, we note 0. We obtain a number with 3 successive digits preceded by the letter P for each plasma cell. Results: The overall rate of immature plasma cells and aberrant plasma cells in MM among Congolese was high at 26.6%. Their integration in the prognostic formulas showed for group A" = 22 patients, group B" = 5 patients and group C" = 32 patients. Examination of plasma cell types in the deceased showed that 14/17 had a P001 plasma cell count ≥ 15%. Conclusion: The high rate of aberrant and/or immature plasma cells, the P001 plasma cell type at a rate ≥ 15%, found mostly in group C" with poor prognosis, are responsible for the aggressive nature of MM in Congolese Blacks.

The Application of Somaclonal Variation in Early Maturity,High Yield and High Quality Improvement in Wheat

Yield characters, maturity and grain protein content of somaclones derived both from immature embryo of cultivar 77(2)-Spring and single-cell culture of cultivar NE7742 in vitro were studied and the wide variation was found. Somaclones with maturity 8 days earlier than or the same as CK NE 7742 (high yield, early maturity and high quality), combining with high quality (grain protein content 15.5% - 18%) and high yield (the same as 7724 or higher) have been found and selected and now multiplied for 8 generations. The results of cultivar comparison trial in 1995 showed that several somaclones (the yields were significantly higher than CK DN120) could be used directly in wheat production. The studies confirmed that somaclonal variation is one of the effective ways for early maturity, high-yielding and high-quality improvement in wheat.

The role of myeloid cells in prevention and control of group A streptococcal infections

Group A Streptococcus(GAS;Streptococcus pyogenes)is one of the most versatile bacteria among human pathogens.Non-invasive GAS infections can cause common diseases,such as pharyngitis and impetigo.Severe invasive GAS infections can lead to rapid progressive and life-threatening manifestations,including necrotizing fasciitis and streptococcal toxic shock syndrome with high mortality rates ranging from 30%to 70%.Therefore,GAS is also known as"killer microbes"or"flesh-eating bacteria".During severe invasive GAS infections,anti-bacterial immunity is impeded by attenuation of the cellular components of innate immune responses.However,this loss of protection is compensated for by interferon-γ-producing immature myeloid cells,which are recruited upon severe invasive GAS infections in mouse models.In this review,we discuss and summarize the current knowledge on the role of interferon-γ-producing myeloid cells and other myeloid cells in the prevention and control of severe invasive GAS infections.