Loss of NLRP6 expression increases the severity of intestinal injury after syngeneic hematopoietic stem cell transplantation.

NLRP6 plays a crucial role in maintaining intestinal homeostasis by regulating the interaction between the intestinal mucosa and the microbiota. However, the impact of NLRP6 deficiency on intestinal damage following hematopoietic stem cell transplantation (HSCT) remains poorly understood. In this study, we established a syngeneic HSCT mouse model using C57BL/6 mice as donors and NLRP6-/- or C57BL/6 mice as recipients. Our findings revealed that NLRP6 deficiency had minimal influence on peripheral blood cell counts and splenic immune cell proportions in transplanted mice. However, it exacerbated pathological changes in the small intestine on day 14 post-transplantation, accompanied by increased proportions of macrophages, dendritic cells, and neutrophils. Furthermore, the NLRP6 deficiency resulted in elevated expression of MPO and CD11b, while reducing the levels mature caspase-1 and mature IL-1ß in the intestine. Moreover, the NLRP6 deficiency disturbed the expression of apoptosis-related molecules and decreased the tight junction protein occludin. Notably, recipient mice with NLRP6 deficiency exhibited lower mRNA expression levels of antimicrobial genes, such as Reg3γ and Pla2g2a. The short-term increase in inflammatory cell infiltration caused by NLRP6 deficiency was associated with intestinal damage, increased apoptosis, reduced expression of antimicrobial peptides, and impaired intestinal repair. Taken together, our findings demonstrate that the loss of NLRP6 exacerbates post-transplantation intestinal damage in recipient mice.

Association between the infections of Trichomonas vaginalis and uterine cervical human papillomavirus: a meta-analysis.

Trichomonas vaginalis (TV) may have an impact on other reproductive tract infections. Studies on the connection between the infection of TV and human papillomavirus (HPV) have been inconsistent. We performed a systematic review of the relevant articles through keywords that satisfy the criteria and filtered the articles according to the inclusion and exclusion criteria. A total of 16 eligible studies were screened for the meta-analysis, involving a total of 150,605 women. RevMan 5.4 software was used for meta-analysis of the selected literatures. The results showed that the papers included in this study had good homogeneity and no significant publication bias was found in the current analysis. The pooled estimates using a fixed-effects model showed that TV was more prevalent in HPV-infected women than in non-infected women [odds ratio (OR): 1.51, 95% confidence interval (CI): 1.29-1.75]; In turn, HPV was more widespread in TV-infected women than in uninfected women (OR: 3.62, 95% CI: 2.71-4.85). Moreover, the interaction between TV and HPV infection was insensitive to the deletion of some studies and correlation coefficients, consequently, the results were robust and reliable. These results suggested that TV is positively associated with HPV infection, and HPV is also a risk factor for TV infection.

Preparation of chitosan grafted caffeic acid coating and its effect on pompano (Trachinotus ovatus) preservation.

BACKGROUND: The present study aimed to investigate the preservative effect of chitosan-caffeic acid grafts coating (CS-g-CA) on the quality and microbial characteristics of pompano (Trachinotus ovatus) during iced storage. CS-g-CA was prepared by a 1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydro/N-hydroxysuccinimide coupling reaction. The grafting of CS-g-CA was confirmed by UV-visible and Fourier-transform infrared spectra. Samples were treated with distilled water (control), chitosan (CS), caffeic acid (CA) and CS-g-CA for 10 min, respectively. Microbiological [total viable count (TVC), H2 S-producing bacteria count, Pseudomonas bacteria count], physicochemical indicators [water holding capacity (WHC), cooking loss, pH, total volatile basic nitrogen (TVB-N), thiobarbituric acid (TBA), texture profile analysis, free amino acids] and sensory evaluation were investigated during ice storage at 4 °C for up to 27 days. RESULTS: The results showed that the antioxidant and antibacterial activities of CS could be improved by grafting CA onto CS. CS-g-CA coating could greatly slow down the speed of water loss and maintain WHC. Furthermore, CS-g-CA coating showed superior antibacterial activities by inhibiting the growth of TVC, delayed the decline of flavor amino acids and reduced sensory change. In addition, CS-g-CA coating reduced lipid oxidation and protein degradation as indicated by the decrease in TBA and TVB-N, possibly as a result of the addition of CA into CS membrane significantly improving the antioxidant activity of CS. CONCLUSION: Compared with the control group, CS-g-CA coating had the optimal effect and could enhance the shelf-life of Trachinotus ovatus for at least another 9 days. © 2021 Society of Chemical Industry.

High expression of miR-363 predicts poor prognosis and guides treatment selection in acute myeloid leukemia.

BACKGROUND: Acute myeloid leukemia (AML) is a highly heterogeneous malignancy with various outcomes, and therefore needs better risk stratification tools to help select optimal therapeutic options. METHODS: In this study, we identify miRNAs that could predict clinical outcome in a heterogeneous AML population using TCGA dataset. RESULTS: We found that MiR-363 is a novel prognostic factor in AML patients undergoing chemotherapy. In multivariable analyses, high miR-363 remained predictive for shorter OS (HR = 2.349, P = 0.012) and EFS (HR = 2.082, P = 0.001) independent of other well-known prognostic factors. More importantly, allogeneic hematopoietic stem cell transplantation (allo-HSCT) overcame the adverse outcomes related to high miR-363 expression. In gene expression profiling, high miR-363 expression was positively correlated with the amounts of leukemogenic transcription factors, including Myb, RUNX3, GATA3, IKZF3, ETS1 and MLLT3. Notably, we found that the in silico predicted target genes (EZH2, KLF6 and PTEN) of miR-363 were downregulated in association with high miR-363 expression. CONCLUSIONS: In summary, miR-363 expression may help identify patients in need of strategies to select the optimal therapy between chemotherapeutic and allo-HCST regimens. AML patients with high miR-363 expression may be highly recommended for early allo-HSCT regimen.

Loss of NLRP3 Function Alleviates Murine Hepatic Graft-versus-Host Disease.

NLRP3 is associated with multiple risks in graft-versus-host disease, though unifying principles for these findings remain largely unknown. To explore the effects and mechanisms of the absence of NLRP3 function on hepatic graft-versus-host-disease, we established an allogeneic hematopoietic cell transplantation mice model by infusing bone marrow mononuclear cells and spleno-T cells of the BALB/c mouse into either NLRP3 knockout (NLRP3-/- ) or wild-type C57BL/6 mice. Elevated inflammatory cell infiltration, liver fibrosis, and secretions of alanine aminotransferase (ALT) and aspartate transaminase (AST), together with weight loss, were observed in C57BL/6 recipients after transplantation. However, moderate injury pathology was detected in the liver of NLRP3-/- recipients at day 14, which gradually improved over time. Likewise, proinflammatory cytokine IL-1ß, a downstream effecter of NLRP3 inflammasome activation, showed significantly lower expression (P < .05) in the liver of NLRP3-/- recipients relative to C57BL/6 recipients at day 7 and day 21. Moreover, compared with C57BL/6 recipients, the expression of both TNF-α and IL-1ß were decreased 3-fold and 4.7-fold, respectively, at day 21 in NLRP3-/- recipients. Interestingly, NLRP1a was expressed at a significantly reduced level in the liver of NLRP3-/- recipients (P < .001). Furthermore, systemic inflammation was analyzed by measuring the concentration of IL-1ß and adenosine triphosphate (ATP) in serum. The concentration of IL-1ß achieved a maximum at day 14, then decreased at day 21 and day 28 in NLRP3-/- recipients. In contrast, the concentration of IL-1ß in C57BL/6 recipients gradually increased from day 7 to day 28. ATP levels reduced from day 7 to day 28 in NLRP3-/- recipients, but were extremely high in C57BL/6 recipients from day 14 to day 28 (P < .01). The decreased levels of P2X7R were connected to less ATP in NLRP3-/- recipients at day 21 and day 28. In conclusion, NLRP3 knockout in recipients could significantly relieve liver injury after transplantation and block the NLRP3 inflammasome pathway, thus providing a promising strategy for the treatment of graft-versus-host disease prophylaxis.

MiR-425 expression profiling in acute myeloid leukemia might guide the treatment choice between allogeneic transplantation and chemotherapy.

BACKGROUND: Acute myeloid leukemia (AML) is a highly heterogeneous disease. MicroRNAs function as important biomarkers in the clinical prognosis of AML. METHODS: This study identified miR-425 as a prognostic factor in AML by screening the TCGA dataset. A total of 162 patients with AML were enrolled for the study and divided into chemotherapy and allogeneic hematopoietic stem cell transplantation (allo-HSCT) groups. RESULTS: In the chemotherapy group, patients with high miR-425 expression had significantly longer overall survival (OS) and event-free survival (EFS) compared with patients with low miR-425 expression. In multivariate analyses, high miR-425 expression remained independently predictive of a better OS (HR = 0.502, P = 0.005) and EFS (HR = 0.432, P = 0.001) compared with patients with low miR-425 expression. Then, all patients were divided into two groups based on the median expression levels of miR-425. Notably, the patients undergoing allo-HSCT had significantly better OS (HR = 0.302, P < 0.0001) and EFS (HR = 0.379, P < 0.0001) compared with patients treated with chemotherapy in the low-miR-425-expression group. Mechanistically, high miR-425 expression levels were associated with a profile significantly involved in regulating cellular metabolism. Among these genes, MAP3K5, SMAD2, and SMAD5 were predicted targets of miR-425. CONCLUSIONS: The expression of miR-425 may be useful in identifying patients in need of strategies to select the optimal therapy between chemotherapy and allo-HSCT treatment regimens. Patients with low miR-425 expression may consider early allo-HSCT.

NLRP3 regulates platelet integrin αIIbß3 outside-in signaling, hemostasis and arterial thrombosis.

In addition to their hemostatic function, platelets play an important role in regulating the inflammatory response. The platelet NLRP3 inflammasome not only promotes interleukin-1ß secretion, but was also found to be upregulated during platelet activation and thrombus formation in vitro However, the role of NLRP3 in platelet function and thrombus formation in vivo remains unclear. In this study, we aimed to investigate the role of NLRP3 in platelet integrin αIIbß3 signaling transduction. Using NLRP3-/- mice, we showed that NLRP3-deficient platelets do not have significant differences in expression of the platelet-specific adhesive receptors αIIbß3 integrin, GPIba or GPVI; however, NLRP3-/- platelets transfused into wild-type mice resulted in prolonged tail-bleeding time and delayed arterial thrombus formation, as well as exhibiting impaired spreading on immobilized fibrinogen and defective clot retraction, concomitant with decreased phosphorylation of c-Src, Syk and PLCγ2 in response to thrombin stimulation. Interestingly, addition of exogenous recombinant interleukin-1ß reversed the defect in NLRP3-/- platelet spreading and clot retraction, and restored thrombin-induced phosphorylation of c-Src/Syk/PLCγ2, whereas an anti-interleukin-1ß antibody blocked spreading and clot retraction mediated by wild-type platelets. Using the direct NLRP3 inhibitor, CY-09, we demonstrated significantly reduced human platelet aggregation in response to threshold concentrations of collagen and ADP, as well as impaired clot retraction in CY-09-treated human platelets, supporting a role for NLRP3 also in regulating human platelet αIIbß3 outside-in signaling. This study identifies a novel role for NLRP3 and interleukin-1ß in platelet function, and provides a new potential link between thrombosis and inflammation, suggesting that therapies targeting NLRP3 or interleukin-1ß might be beneficial for treating inflammation-associated thrombosis.

The impact of P2X7 receptor antagonist, brilliant blue G on graft-versus-host disease in mice after allogeneic hematopoietic stem cell transplantation.

The purpose of this study was to investigate the role of P2X7 on liver inflammation in mice after HSCT. Hematopoietic stem cells obtained from C57BL/6 mice were administrated into BALB/c mice to establish GVHD model. On day 7, 14, 21 and 28 after HSCT, mice received P2X7R antagonist brilliant blue G (BBG) or not were sacrificed for analysis of weight loss, liver inflammation, cytokine secretion, P2X7, NLRP3 expression as well as caspase-1 activation. Liver inflammation with neutrophils and macrophases infiltration as well as weight loss increase was present after HSCT, but improved after administration with high dose of BBG compared with lower dose. High dose of P2X7R inhibitor administration after HSCT previously reduced levels of IL-1ß, IL-18, caspase-1, NLRP3 as well as P2X7, and the level of alanine transaminase (ALT) and the ratio of aspartate amino transferase (AST)/ALT compared with that receiving low dose of BBG. Meanwhile, P2X7R blockage also reduced infiltration of macrophages and neutrophils and levels of CXCL8 and CCL2 in peripheral blood as well as improved liver function. In conclusion, blockage of P2X7R by BBG exerts a protective effect on GVHD post HSCT and improves liver function suggesting that this receptor could be considered as an attractive target for treatment of GVHD.

Cloning and high level expression of the biologically active extracellular domain of Macaca mulatta CD40 in Pichia pastoris.

The CD40-mediated immune response contributes to a wide variety of chronic inflammatory diseases. CD40 antagonists have potential as novel therapies for immune disorders. However, the CD40 pathway has not been well characterized in the rhesus monkey Macaca mulatta, which is a valuable animal model for human immune disease. An 834 bp transcript was cloned from peripheral blood mononuclear cells (PBMCs) of rhesus monkey using specific primers designed according to the predicted sequence of M. mulatta CD40 (mmCD40) in GenBank. Sequence analysis demonstrated that mmCD40 is highly homologous to human CD40 (hCD40), with an amino acid sequence identity of 94%. Genes encoding the extracellular domain of mmCD40 and the Fc fragment of the hIgG1 were inserted into a pPIC9K plasmid to produce mmCD40Ig by Pichia pastoris. Approximately 15-20 mg of the mmCD40Ig protein with ∼90% purity could be recovered from 1 L of culture. The purified mmCD40Ig protein can form dimers and can specifically bind CD40L-positive cells. Additionally, the mmCD40Ig protein can bind hCD40L protein in phosphate buffered saline and form a stable combination in a size-exclusion chromatography assay using a Superdex 200 column. Moreover, mmCD40Ig is as efficient as M. mulatta CTLA4Ig (mmCTLA4Ig) to suppress Con A-stimulated lymphocyte proliferation. Additionally, mmCD40Ig only showed mild immunosuppressive activity in a one-way mixed lymphocyte reaction (MLR) system. These results suggest that mmCD40Ig secreted by P. pastoris was productive and functional, and it could be used as a tool for pathogenesis and therapies for chronic inflammatory diseases in a M. mulatta model.

[Recombinant expression and characterization of CD2-binding domain of Macaca mulatta lymphocyte function-associated antigen 3 in Pichia pastoris].

Human lymphocyte function-associated antigen 3 (hLFA3) has been identified as an important T cell accessory molecule. Rhesus monkeys (Macaca mulatta) have been widely used as animal models for human immune disorders. Due to the species-specificity of immune system, it is necessary to study M. mulatta LFA3 (mmLFA3). In this study, the gene encoding mmLFA3 CD2-binding domain (mmLFA3Sh) was amplified by polymerase chain reaction (PCR) and genetically fused to human IgG1 Fc fragment in pPIC9K to construct the expression plasmid pPIC9K-mmLFA3Sh-Ig. Approximately 3-4 mg mmLFA3Sh-Ig protein was recovered from 1 L of inductive media, and mmLFA3Sh-Ig produced by the P. pastoris can bind to the CD2 positive cells, and suppress the monkey and human lymphocytes proliferation induced by Con A and alloantigen in a dose-dependent manner. These results suggested that mmLFA3Sh-Ig might be used as a novel tool for pathogenesis and experimental immunotherapy of Rhesus monkey immune disorders.

Nanoindentation Test of Ion-Irradiated Materials: Issues, Modeling and Challenges.

Exposure of metals to neutron irradiation results in an increase in the yield strength and a significant loss of ductility. Irradiation hardening is also closely related to the fracture toughness temperature shift or the ductile-to-brittle transition temperature (DBTT) shift in alloys with a body-centered cubic (bcc) crystal structure. Ion irradiation is an indispensable tool in the study of the radiation effects of materials for nuclear energy systems. Due to the shallow damage depth in ion-irradiated materials, the nanoindentation test is the most commonly used method for characterizing the changes in mechanical properties after ion irradiation. Issues that affect the analysis of irradiation hardening may arise due to changes in the surface morphology and mechanical properties, as well as the inherent complexities in nanoscale indentation. These issues, including changes in surface roughness, carbon contamination, the pile-up effect, and the indentation size effect, with corresponding measures, were reviewed. Modeling using the crystal plasticity finite element method of the nanoindentation of ion-irradiated materials was also reviewed. The challenges in extending the nanoindentation test to high temperatures and to multiscale simulation were addressed.

Cavity Swelling of 15-15Ti Steel at High Doses by Ion Irradiation.

The titanium-stabilized austenitic stainless steel Fe-15Cr-15Ni, which shows enhanced resistance to irradiation swelling compared with more traditional 316Ti, has been selected as a core material for fast reactors. Data on the evolution of irradiation swelling in 15-15Ti steels at very high doses, which cannot be easily achieved by neutron irradiation, are still lacking. In this paper, the swelling behavior of the titanium-modified austenitic stainless steel 15-15Ti was investigated by pre-implantation of He at room temperature followed by Ni-ion irradiation at 580 °C to peak doses of 120, 240 and 400 dpa. Relatively small cavities were observed in the zone of helium implantation, while large cavities appeared in the region near the damage peak. A correction formula for the dpa curve was proposed and applied to samples with large swelling. It was found that the steady-state swelling rate of 15-15Ti remains at ~1%/dpa even at high doses. By comparing the swelling data of the helium-implanted and helium-free regions at same doses, 70 dpa and 122 dpa, the suppression of swelling by excessive helium can be deduced at such doses.

Production and characterization of a CD25-specific scFv-Fc antibody secreted from Pichia pastoris.

Antibodies against CD25 would be novel tools for the diagnosis and treatment of adult T cell leukemia lymphoma (ATLL) and many other immune disorders. In our previous work, we successfully produced the single-chain fragment of a variable antibody against CD25, the Dmab(scFv) antibody, using Pichia pastoris. Here, we describe a novel form of an antibody against CD25, the Dmab(scFv)-Fc antibody, also produced by P. pastoris. To construct the Dmab(scFv)-Fc antibody, the Dmab(scFv) antibody was genetically fused to the Fc fragment of a human IgG1 antibody. A fusion gene encoding Dmab(scFv)-Fc antibody was cloned into the pPIC9K plasmid and expressed at high levels, 60-70 mg/l, by P. pastoris under optimized conditions. The Dmab(scFv)-Fc antibody was similar to the Dmab(scFv) antibody in its binding specificity but different in its molecular form and Fc-mediated effector functions. The Dmab(scFv)-Fc antibody and the Dmab(scFv) antibody both bound to CD25-positive MJ cells but not to CD25-negative K562 cells. The Dmab(scFv)-Fc antibody existed as a dimer whereas the Dmab(scFv) antibody was a monomer because it lacks the Fc fragment. The Dmab(scFv)-Fc antibody enhanced the antibody-dependent cellular cytotoxicity of CD25-positive cancer cells, whereas the Dmab(scFv) antibody was inactive in the antibody-dependent cellular cytotoxicity assays. In addition, compared to the Dmab(scFv) antibody, the Dmab(scFv)-Fc antibody showed stronger immunosuppressive activity in the Con A-stimulated lymphocyte proliferation system and in the mixed lymphocyte reaction system. These results demonstrate that the Dmab(scFv)-Fc antibody produced in P. pastoris is functional, and therefore it might be developed as a novel diagnostic and therapeutic tool for ATLL and other immune disorders.

Synergistic Effect of Combined Treatment with Allicin and Antioxidant of Bamboo Leaves and Preservation of Bullfrogs (Lithobates catesbeiana) during Refrigeration Storage.

The effects of allicin and antioxidant of bamboo leaves (AOB) on the quality of bullfrogs (Lithobates catesbeiana) during refrigerated storage (4 °C) were investigated. The quality changes in samples treated with deionized water (CK), allicin solution (All), antioxidant of bamboo leaves (AOB), and allicin solution combined with AOB solution (AA) in microbiological, physicochemical, and sensory evaluation were analyzed, respectively. The results demonstrated that combination treatment inhibited the increase in total viable counts, delayed the decrease in amino acid content, and retarded the sensory deterioration. Preservative treatment has an inhibitory effect on the early storage of PBC, which can reduce PBC by about 1.0 log CFU/g. The reduction in thiobarbituric acid (TBA) content and total volatile basic nitrogen (TVB-N) content indicated that combination treatment could better restrain the lipid oxidation and degradation of protein than the CK group and single-treatment group. In addition, the TVB-N content in the AA group still did not exceed the threshold on the 14th day. As a consequence, combination treatment prolonged the shelf life of bullfrogs for another six days. Therefore, allicin and AOB with excellent antioxidant and antimicrobial activity could be an effective approach to delay the biochemical reaction of refrigerated bullfrogs. This study has provided a potential approach for increasing the shelf life of bullfrogs and preserving their quality during refrigerated storage.

Assessment of intestinal status in MPLW515L mutant myeloproliferative neoplasms mice model.

The MPLW515L mutation is a prevalent genetic mutation in patients with myeloproliferative neoplasms (MPN), and utilizing this mutation in mice model can provide important insights into the disease. However, the relationship between intestinal homeostasis and MPN mice model remains elusive. In this study, we utilized a retroviral vector to transfect hematopoietic stem cells with the MPLW515L mutation, creating mutated MPN mice model to investigate their intestinal status. Our results revealed that the MPLW515L in MPN mice model aggravated inflammation in the intestines, decreased the levels of tight junction proteins and receptors for bacteria metabolites. Additionally, there was increased activation of the caspase1/IL-1ß signaling pathway and a significant reduction in phos-p38 levels in the intestinal tissue in MPN mice. The MPLW515L mutation also led to up-expression of anti-microbial genes in the intestinal tract. Though the mutation had no impact on the alpha diversity and dominant bacterial taxa, it did influence the rare bacterial taxa/sub-communities and consequently impacted intestinal homeostasis. Our findings demonstrate the significance of MPLW515L mice model for studying MPN disease and highlight the mutation's influence on intestinal homeostasis, including inflammation, activation of the IL-1ß signaling pathway, and the composition of gut microbial communities.

The formation of percolative composites with a high dielectric constant and high conductivity.

The dielectric constant and electrical conductivity of a composite of two insulators, poly(1,1-difluoroethylene) (yellow) and K(2)CO(3) (white), increased dramatically near the percolation threshold f(c) (f=concentration of K(2)CO(3)). This intriguing phenomenon can be interpreted in terms of interface percolation caused by the formation of chemically activated interfaces.

Effects of ultrasound-assisted chitosan grafted caffeic acid coating on the quality and microbial composition of pompano during ice storage.

The effects of ultrasound-assisted chitosan grafted caffeic acid coating on the quality and microbial composition of fresh pompano (Trachinotus ovatus) fillets during ice storage for 24 days were evaluated. Samples were treated by distilled water (CK), ultrasound (US), chitosan grafted caffeic acid coating (G), and chitosan grafted caffeic acid coating with ultrasound-assisted (USG). Results showed that samples treated with USG could inhibit the formation of corrupt substances such as TVB-N, TBA, biogenic amines (BAs), hypoxanthine (Hx), and hypoxanthine riboside (HxR) when compared to the CK group.The results of high-throughput sequencing technology observed that the major bacteria genus of fresh samples was Acinetobacter.The diversity of bacterial communities at the initial stage was more diverse than that at the end of stage. With the extension of storage time, the USG treatment could maintain the microbial diversity. The dominant microbiota was Shewanella and Brochothrix in the CK group after 24 days of storage. In addition, Brochothrix in treated groups was effectively decreased. The microbial communities of samples in all treatments were changed during storage. At the end of storage, there was a significant difference in bacterial composition between the CK and treated samples, indicating that the treatment can effectively inhibit the growth of microorganisms, especially spoilage microorganisms, and reduce the quality deterioration caused by bacteria.

Production and characterization of LEA29Y, a variant of cytotoxic T-lymphocyte antigen 4-immunoglobulin, in Pichia pastoris.

Blocking the CD28/B7 costimulatory pathway is a promising strategy in the treatment of graft rejection, graft-versus-host disease and autoimmune diseases. LEA29Y, a high-affinity variant of cytotoxic T-lymphocyte antigen 4-immunoglobulin (CTLA4Ig), is a more potent inhibitor of the interaction between CD28 and B7 than is CTLA4Ig. In a previous study, LEA29Y was produced in a mammalian cell system, which is time-consuming and expensive. To obtain LEA29Y more efficiently and cost effectively, we attempted to produce LEA29Y using a Pichia pastoris expression system. The gene encoding LEA29Y, with an additional 6-His tag at the N-terminus, was cloned into the yeast vector pPIC9K and expressed in the P. pastoris strain GS115. Under the optimized induction conditions for protein expression (inoculum density, OD(600) = 80; methanol concentration added daily, 1.0-3.0%; induction time point, 72-96 h; culture medium pH = 6.0), the yield of purified LEA29Y was approximately 30 mg l(-1) by one-step Ni-agarose affinity chromatography. PNGase F treatment showed the purified LEA29Y to be post-translational modified by N-linked glycosylation. In biological function assays, LEA29Y expressed in P. pastoris demonstrated specific binding to B7-1/B7-2-positive Raji cells and also suppressed lymphocyte proliferation in a dose-dependent manner. These results suggest that LEA29Y produced in P. pastoris is biologically active and will be useful for experimental therapy on immunotherapy for transplant rejection and autoimmune diseases.

Combination of Mesenchymal Stem Cell and Endothelial Progenitor Cell Infusion Accelerates Injured Intestinal Repair by Regulating Gut Microbiota after Hematopoietic Cell Transplantation.

Mesenchymal stem cells (MSC) have been widely applied for repairing intestinal barrier function and restoring immune homeostasis for pretransplantation conditioning, yet the repair process is often impaired or delayed owing to a lack of vascularity. How combined therapy with MSC and endothelial progenitor cells (EPC) for the intestinal microenvironment and repair remain unclear. In this study, BALB/c mice received syngeneic bone marrow transplantation with or without MSC or EPC infusion. The findings show that the MSC+EPC mice had greater blood capillary distribution and higher expression of tight junction protein (occludin) in the small intestinal tract. Meanwhile, the MSC+EPC cotreatment increased IL-17A levels and decreased IFN-γ levels at the early stage after transplantation. Furthermore, the MSC+EPC treatment motivated p38 mitogen-activated protein kinase (MAPK) and enhanced heat shock protein 27 (HSP27) activation, which subsequently promoted intestinal epithelial cell proliferation and down-regulated apoptosis-related molecule caspase 3 expression. Finally, the high-throughput sequencing of gut microbiota (16S) showed that the MSC+EPC treatment can inhibit the Enterococcus population (<0.5%) and stabilize the Akkermansia population (~15%), with the Akkermansia population showing significant positive correlations with p38 MAPK/phos-p38, HSP27/phos-HSP27, IL-17A, and occludin. Taken together, our results show that MSC+EPC combined therapy is beneficial for the repair of injured intestine and drives gut microbial community stability by regulating the intestinal microenvironment.

The effects of myeloablative or non-myeloablative total body irradiations on intestinal tract in mice.

Acute radiation injury caused by high-dose radiation exposure severely impedes the application of radiotherapy in cancer management. To deeply understand the side effects of radiation on intestinal tract, an irradiation murine model was applied and evaluated. C57BL/6 mice were given 4 Gy non-myeloablative irradiation, 8 Gy myeloablative irradiation and non-irradiation (control), respectively. Results demonstrated that the 8 Gy myeloablative irradiations significantly damaged the gut barrier along with decreasing MECA32 and ZO-1. However, a slight increase in MECA32 and ZO-1 was detected in the 4 Gy non-myeloablative irradiations treatment from day 5 to day 10. Further, the irradiations affected the expression of P38 and JNK mitogen-activated protein kinase (MAPK) but not ERK1/2 MAPK signal pathway. Moreover, irradiation had adverse effects on hematopoietic system, altered the numbers and percentages of intestinal inflammatory cells. The IL-17/AhR had big increase in the gut of 4 Gy irradiation mice at day 10 compared with other groups. Both 8 Gy myeloablative and 4 Gy non-myeloablative irradiation disturbed the levels of short-chain fatty acids (SCFAs) in intestine. Meanwhile, high dosage of irradiation decreased the intestinal bacterial diversity and altered the community composition. Importantly, the fatty acids generating bacteria Bacteroidaceae and Ruminococcaceae played key roles in community distribution and SCFAs metabolism after irradiation. Collectively, the irradiation induced gut barrier damage with dosages dependent that led to the decreased p38 MAPK and increased JNK MAPK, unbalanced the mononuclear cells (MNCs) of gut, disturbed intestinal bacterial community and SCFAs level.