Integrated analysis reveals crosstalk between pyroptosis and immune regulation in renal fibrosis.

Purpose: The pathogenesis of renal fibrosis (RF) involves intricate interactions between profibrotic processes and immune responses. This study aimed to explore the potential involvement of the pyroptosis signaling pathway in immune microenvironment regulation within the context of RF. Through comprehensive bioinformatics analysis and experimental validation, we investigated the influence of pyroptosis on the immune landscape in RF. Methods: We obtained RNA-seq datasets from Gene Expression Omnibus (GEO) databases and identified Pyroptosis-Associated Regulators (PARs) through literature reviews. Systematic evaluation of alterations in 27 PARs was performed in RF and normal kidney samples, followed by relevant functional analyses. Unsupervised cluster analysis revealed distinct pyroptosis modification patterns. Using single-sample gene set enrichment analysis (ssGSEA), we examined the correlation between pyroptosis and immune infiltration. Hub regulators were identified via weighted gene coexpression network analysis (WGCNA) and further validated in a single-cell RNA-seq dataset. We also established a unilateral ureteral obstruction-induced RF mouse model to verify the expression of key regulators at the mRNA and protein levels. Results: Our comprehensive analysis revealed altered expression of 19 PARs in RF samples compared to normal samples. Five hub regulators, namely PYCARD, CASP1, AIM2, NOD2, and CASP9, exhibited potential as biomarkers for RF. Based on these regulators, a classifier capable of distinguishing normal samples from RF samples was developed. Furthermore, we identified correlations between immune features and PARs expression, with PYCARD positively associated with regulatory T cells abundance in fibrotic tissues. Unsupervised clustering of RF samples yielded two distinct subtypes (Subtype A and Subtype B), with Subtype B characterized by active immune responses against RF. Subsequent WGCNA analysis identified PYCARD, CASP1, and NOD2 as hub PARs in the pyroptosis modification patterns. Single-cell level validation confirmed PYCARD expression in myofibroblasts, implicating its significance in the stress response of myofibroblasts to injury. In vivo experimental validation further demonstrated elevated PYCARD expression in RF, accompanied by infiltration of Foxp3+ regulatory T cells. Conclusions: Our findings suggest that pyroptosis plays a pivotal role in orchestrating the immune microenvironment of RF. This study provides valuable insights into the pathogenesis of RF and highlights potential targets for future therapeutic interventions.

The novel HLA-A*24:02:160 allele, identified by Sanger dideoxy nucleotide sequencing in a Chinese individual.

HLA-A*24:02:160 differs from HLA-A*24:02:01:01 by one nucleotide in exon 3.

Phenotypes of CIK cells prepared by ATG-F culture system and its killing effect against K562 cells / 中国肿瘤生物治疗杂志

@# Objective: To investigate the function of CIK (cytokine induced killer) cells cultured using ATG-F (anti-human T lymphocyte rabbit immunoglobulin-Fresenius) and IFN- γ, IL-2 system and its feasibility in clinical practice. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors and were used to culture CIK cells by different activating antibodies; the total cell count was calculated on Day 7 and 14. The CIK cell composition, cell surface activation and proportion of inhibitory receptor molecular in ATG-F group, CD3 group and TG (Thymoglobulin) group were analyzed by Flow cytometry, and the cytotoxicity of CIK cells against K562 cells were also determined by flow cytometry at day 14 in ATG-F high-dose group, CD3 group and TG group. Results: CIK cells were successfully cultured by ATG-F, IFN-γ, IL-2 system. The proliferation rate of ATGF high-dose group was significantly higher than that in TG group (27.25±1.25 vs 16.60±1.72, P <0.01), but the proportion of CD3+ CD56+ cells showed no statistical difference compare with the CD3 group ( P >0.05). The percentage of CD3-CD56+ NK cells in ATG-F high-dose group was significantly higher than that in TG group and CD3 group [(11.19±2.60）% vs（5.66±1.00）%,（1.42± 0.51）% ， P <0.01], while the proportion of CD4+T cells was significantly lower than that in CD3, TG group [(4.35±1.47）% vs （26.88±5.01）%,（14.52±6.22）%, P <0.01]; the proportion of CD56+CD94+, CD56+CD158a+, CD56+CD158b cells was significantly higher than those in CD3 group (all P <0.01). The ATG-F high does group showed significantly higher cytotoxicity against K562 cells than that of CD3 group at the target/effect ratio of 1∶10. Conclusion: CIK cells cultured by ATG-F culture system has higher NK cell proportion than other ordinary culture system, and its activated receptor has more stronger cytotoxicity against K562 cells.

Enhancement of the cytotoxic activity of cytokine-induced killer cells transfected with IL3PE38KDEL gene against acute myeloid leukemia cells.

Cytokine-induced killer (CIK) cells, one of the feasible and effective methods of adoptive immunotherapy, have shown anti-leukemia activity in vivo and in vitro. But the strategy exhibits limited cytotoxic activity in clinical studies. In this study, CIK cells were transfected with an interleukin-3/Pseudomonas exotoxin gene (IL3PE38KDEL). RT-PCR and ELISA were used to verify the expression of IL3PE38KDEL in the transfected CIK cells. These cells released 1,186.7 ± 149.6 pg IL3PE38KDEL/10(4) cells over 48 h into the medium and the culture supernatant selectively killed IL3 receptor(IL3R)-positive HL60 cells, but not IL3R-negative K562 cells. Moreover, IL3PE38KDEL transfection did not influence phenotypes and cytokine production of CIK cells. Co-cultured with leukemia cells, IL3PE38KDEL transfected CIK cells showed enhanced cytotoxicity against IL3R-positive HL60 cells at all effector-to-target (E:T) ratios, but exerted a basal anti-leukemia activity against IL3R-negative K562 cells. Our findings demonstrate that IL3PE38KDEL gene transfection may be a novel strategy for improving anti-leukemia activity of CIK cells.

[Construstion of IL3-PE38KDEL fusion gene eukaryolic expression vector and its expression in pichia].

AIM: to construct a eukaryolic expression plasmid of Ppic9k-IL3-Linker-PE38KDE. METHODS: the IL3 and PE38KDEL gene were amplified by polymerase chain reaction (PCR) and cloned into the eukaryolic expression plasmid Ppic9k-Linker constructed after being sequenced.The recombinant vector confirmed by restriction endonucleases digestion, coenobium PCR and DNA sequence analysis showed that the prokaryotic expression vector Ppic9k-IL3-Linker-PE38KDEL was constructed successfully. CONCLUSION: the fusion gene IL3-PE38KDEL is successfully constructed, which laid a solid foundation for the further research.