Identification and Functional Analysis of a de novo IKZF3 Mutation in a Pediatric Patient with Combined Immunodeficiency.

AIOLOS, a vital member of the IKAROS protein family, plays a significant role in lymphocyte development and function through DNA binding and protein-protein interactions. Mutations in the IKZF3 gene, which encodes AIOLOS, lead to a rare combined immunodeficiency often linked with infections and malignancy. In this study, we evaluated a 1-year-4-month-old female patient presenting with recurrent infections, diarrhea, and failure to thrive. Laboratory investigations revealed decreased T lymphocyte and immunoglobulin levels. Through whole-exome and Sanger sequencing, we discovered a de novo mutation in IKZF3 (NM_012481; exon 5 c.571G > C, p.Gly191Arg), corresponding to the third DNA-binding zinc finger region of the encoded protein AIOLOS. Notably, the patient with the AIOLOS G191R mutation showed reduced recent thymic emigrants in naïve CD4+T cells compared to healthy counterparts of the same age, while maintaining normal levels of Th1, Th2, Th17, Treg, and Tfh cells. This mutation also resulted in decreased switched memory B cells and lower CD23 and IgM expression. In vitro studies revealed that AIOLOS G191R does not impact the expression of AIOLOS but compromises its stability, DNA binding and pericentromeric targeting. Furthermore, AIOLOS G191R demonstrated a dominant-negative effect over the wild-type protein. This case represents the first reported instance of a mutation in the third DNA-binding zinc finger region of AIOLOS highlighting its pivotal role in immune cell functionality.

Risk factor analysis and nomogram prediction model construction for NEC complicated by intestinal perforation.

OBJECTIVE: To investigate the clinical characteristics of neonatal necrotizing enterocolitis (NEC) complicated by intestinal perforation and predict the incidence of intestinal perforation in NEC. METHODS: Neonates diagnosed with NEC at the Affiliated Hospital of Zunyi Medical University from January 2012 to May 2022 were enrolled, and the clinical data were collected and analyzed retrospectively. The patients were divided into two groups based on intestinal perforation occurrence or not. Mann-Whitney U tests, t-tests, chi-square tests, and fisher's exact tests were performed between-group comparisons. Logistic and lasso regressions were applied to screen independent risk factors for concomitant bowel perforation, and R software (RMS package) was used to formulate the nomogram prediction model. In addition, the receiver operating curve (ROC) and the calibration curve were drawn to verify the predictive power, while decision curve analysis (DCA) was constructed to evaluate the clinical applicability of the nomogram model. RESULTS: One hundred eighty neonates with NEC were included, of which 48 had intestinal perforations, and 132 did not; the overall incidence of intestinal perforation was 26.67% (48/180). Bloody stool (OR = 5.60), APTT ≥ 50 s (OR = 3.22), thrombocytopenia (OR = 4.74), and hypoalbuminemia (OR = 5.56) were identified as independent risk variables for NEC intestinal perforation (P < 0.05) through multivariate logistic regression analysis. These factors were then applied to develop a nomogram prediction model (C-index = 0.838) by using the R software. The area under the curve (AUC) for the nomogram in the training and validation cohorts were 0.838 (95% Cl: 0.768, 0.908) and 0.802 (95% CI: 0.659, 0.944), respectively. The calibration curve shown that the nomogram has a good predictive ability for predicting the risk of intestinal perforation occurrence. And the decision curve and clinical impact curve analyses demonstrated good clinical utility of the nomogram model. CONCLUSION: We found that Bloody stool, APTT ≥ 50 s, Thrombocytopenia, and hypoalbuminemia could be used as independent risk factors for predicting intestinal perforation in neonates with NEC. The nomogram model based on these variables had high predictive values to identify NEC patients with intestinal perforation.

STAT3 couples with 14-3-3σ to regulate BCR signaling, B-cell differentiation, and IgE production.

BACKGROUND: STAT3 or dedicator of cytokinesis protein 8 (Dock8) loss-of-function (LOF) mutations cause hyper-IgE syndrome. The role of abnormal T-cell function has been extensively investigated; however, the contribution of B-cell-intrinsic dysfunction to elevated IgE levels is unclear. OBJECTIVE: We sought to determine the underlying molecular mechanism of how STAT3 regulates B-cell receptor (BCR) signaling, B-cell differentiation, and IgE production. METHODS: We used samples from patients with STAT3 LOF mutation and samples from the STAT3 B-cell-specific knockout (KO) mice Mb1CreStat3flox/flox mice (B-STAT3 KO) to investigate the mechanism of hyper-IgE syndrome. RESULTS: We found that the peripheral B-cell homeostasis in B-STAT3 KO mice mimicked the phenotype of patients with STAT3 LOF mutation, having decreased levels of follicular and germinal center B cells but increased levels of marginal zone and IgE+ B cells. Furthermore, B-STAT3 KO B cells had reduced BCR signaling following antigenic stimulation owing to reduced BCR clustering and decreased accumulation of Wiskott-Aldrich syndrome protein and F-actin. Excitingly, a central hub protein, 14-3-3σ, which is essential for the increase in IgE production, was enhanced in the B cells of B-STAT3 KO mice and patients with STAT3 LOF mutation. The increase of 14-3-3σ was associated with increased expression of the upstream mediator, microRNA146A. Inhibition of 14-3-3σ with R18 peptide in B-STAT3 KO mice rescued the BCR signaling, follicular, germinal center, and IgE+ B-cell differentiation to the degree seen in wild-type mice. CONCLUSIONS: Altogether, our study has established a novel regulatory pathway of STAT3-miRNA146A-14-3-3σ to regulate BCR signaling, peripheral B-cell differentiation, and IgE production.

AKT2 deficiency impairs formation of the BCR signalosome.

BACKGROUND: AKT2 is one of the key molecules that involves in the insulin-induced signaling and the development of cancer. In B cells, the function of AKT2 is unclear. METHODS: In this study, we used AKT2 knockout mice model to study the role of AKT2 in BCR signaling and B cell differentiation. RESULTS: AKT2 promotes the early activation of B cells by enhancing the BCR signaling and actin remodeling. B cells from AKT2 KO mice exhibited defective spreading and BCR clustering upon stimulation in vitro. Disruption of Btk-mediated signaling caused the impaired differentiation of germinal center B cells, and the serum levels of both sepecific IgM and IgG were decreased in the immunized AKT2 KO mice. In addition, the actin remodeling was affected due to the decreased level of the activation of WASP, the actin polymerization regulator, in AKT2 KO mice as well. As a crucial regulator of both BCR signaling and actin remodeling during early activation of B cells, the phosphorylation of CD19 was decreased in the AKT2 absent B cells, while the transcription level was normal. CONCLUSIONS: AKT2 involves in the humoral responses, and promotes the BCR signaling and actin remodeling to enhance the activation of B cells via regulating CD19 phosphorylation. Video Abstract.

[Clinical Characteristics of Children with Hemophagocytic Syndrome with Different EB Virus DNA Loads].

OBJECTIVE: To analyze the clinical characteristics of hemophagocytic syndrome (HLH) children with different EB virus (EBV) DNA loads, and to explore the relationship between differential indicators and prognosis. METHODS: Clinical data of 73 children with HLH treated in our hospital from January 2015 to April 2022 were collected. According to EBV DNA loads, the children were divided into negative group (≤5×102 copies/ml), low load group (>5×102-<5×105 copies/ml) and high load group (≥5×105copies/ml）. The clinical symptoms and laboratory indexes of the three groups were compared, and the ROC curve was used to determine the best cut-off value of the different indexes. Cox regression model was used to analyze the independent risk factors affecting the prognosis of children, and to analyze the survival of children in each group. RESULTS: The proportion of female children, the swelling rate of liver and spleen lymph nodes and the involvement rate of blood, liver, circulation and central nervous system in the high load group were higher than those in the negative group. The incidence of disseminated intravascular coagulation(DIC) and central nervous system(CNS) involvement in the high load group were higher than those in the low load group. The liver swelling rate and circulatory system involvement rate in the low load group were higher than those in the negative group(P<0.05). PLT counts in the high load group were significantly lower than those in the negative group, and the levels of GGT, TBIL, CK-MB, LDH, TG, SF, and organ involvement were significantly higher than those in the negative group. The levels of CK, LDH, SF and the number of organ involvement in the high load group were significantly higher than those in the low load group. The levels of GGT and TBIL in low load group were significantly higher than those in negative group. In terms of treatment, the proportion of blood purification therapy in the high and low load group was significantly higher than that in the negative group(P<0.01). ROC curve analysis showed that the best cut-off values of PLT, LDH, TG and SF were 49.5, 1139, 3.12 and 1812, respectively. The appellate laboratory indicators were dichotomized according to the cut-off value, and the differential clinical symptoms were included in the Cox regression model. Univariate analysis showed that LDH>1139 U/L, SF>1812 µg/L, dysfunction of central nervous system, number of organ damage, DIC and no blood purification therapy were the risk factors affecting the prognosis of children (P<0.05); Multivariate analysis shows that PLT≤49.5×109/L and dysfunction of central nervous system were risk factors affecting the prognosis of children (P<0.05). Survival analysis showed that there was no significant difference in the survival rate among the three groups. CONCLUSION: The incidence of adverse prognostic factors in children with HLH in the EBV-DNA high load group is higher, and there is no significant difference in the survival rate of the three groups after blood purification therapy. Therefore, early identification and application of blood purification therapy is of great significance for children with HLH in the high load group.

Progranulin regulates the development and function of NKT2 cells through EZH2 and PLZF.

T helper 2 (Th2) cytokine production by invariant natural killer T (iNKT) cells is involved in the development of asthma, but the regulation of Th2 cytokines in iNKT cells remains unknown. Although it is known that progranulin (PGRN) induces the production of Th2 cytokines in iNKT cells in vivo, the underlying mechanism is not clear. This study aims to investigate the role of PGRN in iNKT cells. The effects of PGRN on the differentiation of iNKT cells was detected by flow cytometry. Then stimulation of iNKT cells and airway resistance were carried out to evaluate the function of PGRN on iNKT cells. Furthermore, the mechanisms of PGRN in regulating iNKT cells was investigated by RT-PCR, WB, confocal and luciferase reporter assays. The absolute number of iNKT cells decreased in PGRN KO mice despite an increase in the percentage of iNKT cells. Furthermore, analyzing the subsets of iNKT cells, we found that NKT2 cells and their IL-4 production were reduced. Mechanistically, the decrease in NKT2 cells in the PGRN KO mice was caused by increased expression of enhancer of zeste homolog 2 (EZH2), that in turn caused increased degradation and altered nuclear localization of PLZF. Interestingly, PGRN signaling decreased expression of EZH2 and treatment of the PGRN KO mice with the EZH2 specific inhibitor GSK343 rescued the defect in NKT2 differentiation, IL-4 generation, and PLZF expression. Altogether, We have revealed a new pathway (PGRN-EZH2-PLZF), which regulates the Th2 responses of iNKT cells and provides a potentially new target for asthma treatment.

[Analysis of the difference between the appearance and the bony structure in the polysyndactyly of the fifth toe fused with the fourth toe].

OBJECTIVE: To investigate difference between the appearance and the bony structure in the polysyndactyly of the fifth toe fused with the fourth toe. METHODS: From Jan. 2009 to Jan. 2014, 54 patients (65 feet) with polysyndactyly of the fifth toe fused with the fourth toe were treated. The appearance, X-ray and intraoperative finding were recorded and compared to classify the deformity. Then the extra toe was excised and syndactyly was separated. The malalignment and brachydactyly of the sixth toes were corrected simultaneously. RESULTS: According to the bone and joint type, the fifth toes were neoplastic toes without joints in 17 feet, or had poor bony and joint alignment with the sixth toes in 48 feet. So the fifth toes were excised in all the cases. The patients were followed up for 1 month to 4 years. The oblique deformity of sixth toes were corrected completely with improved length. CONCLUSIONS: The polysyndactyly of the fifth toe fused with the fourth toe should be classified to design the excised toe (usually fifth toe) and correction procedure. The appearance and bony joint recovery are both important.