Abnormalities of follicular helper T-cell number and function in Wiskott-Aldrich syndrome.

Wiskott-Aldrich syndrome protein (WASp) is a hematopoietic-specific regulator of actin nucleation. Wiskott-Aldrich syndrome (WAS) patients show immunodeficiencies, most of which have been attributed to defective T-cell functions. T follicular helper (Tfh) cells are the major CD4(+) T-cell subset with specialized B-cell helper capabilities. Aberrant Tfh cells activities are involved in immunopathologies such as autoimmunity, immunodeficiencies, and lymphomas. We found that in WAS patients, the number of circulating Tfh cells was significantly reduced due to reduced proliferation and increased apoptosis, and Tfh cells were Th2 and Th17 polarized. The expression of inducible costimulator (ICOS) in circulating Tfh cells was higher in WAS patients than in controls. BCL6 expression was decreased in total CD4(+) T and Tfh cells of WAS patients. Mirroring the results in patients, the frequency of Tfh cells in WAS knockout (KO) mice was decreased, as was the frequency of BCL6(+) Tfh cells, but the frequency of ICOS(+) Tfh cells was increased. Using WAS chimera mice, we found that the number of ICOS(+) Tfh cells was decreased in WAS chimera mice, indicating that the increase in ICOS(+) Tfh cells in WAS KO mice was cell extrinsic. The data from in vivo CD4(+) naive T-cell adoptive transfer mice as well as in vitro coculture of naive B and Tfh cells showed that the defective function of WASp-deficient Tfh cells was T-cell intrinsic. Consistent findings in both WAS patients and WAS KO mice suggested an essential role for WASp in the development and memory response of Tfh cells and that WASp deficiency causes a deficient differentiation defect in Tfh cells by downregulating the transcription level of BCL6.

Ablation of C/EBP homologous protein increases the acute phase mortality and doesn't attenuate cardiac remodeling in mice with myocardial infarction.

Endoplasmic reticulum stress is a proapoptotic and profibrotic stimulus. Ablation of C/EBP homologous protein (CHOP) is reported to reverse cardiac dysfunction by attenuating cardiac endoplasmic reticulum stress in mice with pressure overload or ischemia/reperfusion, but it is unclear whether loss of CHOP also inhibits cardiac remodeling induced by permanent-infarction. In mice with permanent ligation of left coronary artery, we found that ablation of CHOP increased the acute phase mortality. For the mice survived to 4 weeks, left ventricular anterior (LV) wall thickness was larger in CHOP knockout mice than in the wildtype littermates, while no difference was noted on posterior wall thickness, LV dimensions, LV fractional shortening and ejection fraction. Similarly, invasive assessment of LV hemodynamics, morphological analysis of heart and lung weight indexes, myocardial fibrosis and TUNEL-assessed apoptosis showed no significant differences between CHOP knockout mice and their wildtype ones, while in mice with ischemia for 45 min and reperfusion for 1 week, myocardial fibrosis and apoptosis in the infarct area were significantly attenuated in CHOP knockout mice. These findings indicate that ablation of CHOP doesn't ameliorate cardiac remodeling induced by permanent-myocardial infarction, which implicates that early reperfusion is a prerequisite for ischemic myocardium to benefit from CHOP inhibition.

Overexpression of ankyrin repeat domain 1 enhances cardiomyocyte apoptosis by promoting p53 activation and mitochondrial dysfunction in rodents.

The Ankrd1 (ankyrin repeat domain 1) gene is known to be up-regulated in heart failure and acts as a co-activator of p53, modulating its transcriptional activity, but it remains inconclusive whether this gene promotes or inhibits cell apoptosis. In the present study, we attempted to investigate the role of Ankrd1 on AngII (angiotensin II)- or pressure-overload-induced cardiomyocyte apoptosis. In the failing hearts of mice with pressure overload, the protein expression of Ankrd1-encoded CARP (cardiac ankyrin repeat protein) was significantly increased. In NRCs (neonatal rat cardiomyocytes), AngII increased the expression of Ankrd1 and CARP. In the presence of AngII in NRCs, infection with a recombinant adenovirus containing rat Ankrd1 cDNA (Ad-Ankrd1) enhanced the mitochondrial translocation of Bax and phosphorylated p53, increased mitochondrial permeability and cardiomyocyte apoptosis, and reduced cell viability, whereas these effects were antagonized by silencing of Ankrd1. Intra-myocardial injection of Ad-Ankrd1 in mice with TAC (transverse aortic constriction) markedly exacerbated cardiac dysfunction with an increase in the lung weight/body weight ratio and a decrease in left ventricular fractional shortening. Cardiomyocyte apoptosis and the expression of phosphorylated p53 were also significantly increased in Ad-Ankrd1-infected TAC mice, whereas knockdown of Ankrd1 significantly inhibited the apoptotic signal pathway as well as cardiomyocyte apoptosis in pressure-overload mice. These findings indicate that overexpression of Ankrd1 exacerbates pathological cardiac dysfunction through enhancement of cardiomyocyte apoptosis mediated by the up-regulation of p53.

Effects of Transcranial Magnetic Stimulation Combined with Computer-Aided Cognitive Training on Cognitive Function of Children with Cerebral Palsy and Dysgnosia.

Objective: This study is aimed at researching transcranial magnetic stimulation (TMS) effects combined with computer-aided cognitive training (CACT) on cognitive function of children suffering from cerebral palsy and dysgnosia. Methods: From December 2019 to October 2021, 86 children with cerebral palsy and dysgnosia who were treated at our hospital were recruited and assigned into observation and control groups (n = 43, each) using the random number table technique. The observation group received TMS combined with CACT (TMS+CACT), whereas the control group received only TMS. Chinese Wechsler Young Children Scale of Intelligence (C-WYCSI) and Chinese-Wechsler Intelligence Scale for Children (C-WISC) were used to evaluate the intelligence level of the two groups; Gross Motor Function Measure-88 (GMFM-88) of Fudan Chinese version was employed for evaluating the gross motor function of the two groups; a comparison was drawn among the two groups for the cerebral hemodynamic parameters before and after the treatment. Results: For young children, the verbal intelligence quotient (VIQ) scores at 6 and 12 weeks of treatment in the observation group were increased when compared to those in the control group (48.91 ± 3.70 vs. 47.32 ± 3.33, 54.25 ± 4.46 vs. 49.48 ± 3.36), and the observation group's performance intelligence quotient (PIQ) score at 12 weeks of treatment was higher as to that of the control group (65.38 ± 4.23 vs. 62.81 ± 4.74, all P < 0.05). For older age children, the observation group's VIQ and PIQ scores were greater than the control group's at 6 and 12 weeks of treatment, with statistical significance (63.80 ± 3.76 vs. 59.50 ± 5.32, 74.64 ± 12.04 vs. 65.08 ± 6.30; 63.91 ± 5.96 vs. 58.42 ± 3.70, 72.73 ± 5.06 vs. 66.42 ± 5.93; all P < 0.05). The GMFM-88 scale scores in both groups were increased after 6 and 12 weeks of treatment. After treatment for 12 weeks, the observation group's A-E scores were greater than those of the control group (all P < 0.05). The peak systolic velocity (V s), end-diastolic velocity (V d), and mean velocity (V m) at the anterior cerebral artery (ACA), middle cerebral artery (MCA), and posterior cerebral artery (PCA) in the observation group were dramatically increased than those in the control group (all P < 0.05) after 12 weeks of treatment. Conclusion: TMS+CACT can effectively improve the intelligence level, cognitive ability, gross motor function, and cerebral blood flow of children suffering from cerebral palsy and intellectual disability.

Growth differentiation factor 11 attenuates cardiac ischemia reperfusion injury via enhancing mitochondrial biogenesis and telomerase activity.

It has been reported that growth differentiation factor 11 (GDF11) protects against myocardial ischemia/reperfusion (IR) injury, but the underlying mechanisms have not been fully clarified. Considering that GDF11 plays a role in the aging/rejuvenation process and that aging is associated with telomere shortening and cardiac dysfunction, we hypothesized that GDF11 might protect against IR injury by activating telomerase. Human plasma GDF11 levels were significantly lower in acute coronary syndrome patients than in chronic coronary syndrome patients. IR mice with myocardial overexpression GDF11 (oe-GDF11) exhibited a significantly smaller myocardial infarct size, less cardiac remodeling and dysfunction, fewer apoptotic cardiomyocytes, higher telomerase activity, longer telomeres, and higher ATP generation than IR mice treated with an adenovirus carrying a negative control plasmid. Furthermore, mitochondrial biogenesis-related proteins and some antiapoptotic proteins were significantly upregulated by oe-GDF11. These cardioprotective effects of oe-GDF11 were significantly antagonized by BIBR1532, a specific telomerase inhibitor. Similar effects of oe-GDF11 on apoptosis and mitochondrial energy biogenesis were observed in cultured neonatal rat cardiomyocytes, whereas GDF11 silencing elicited the opposite effects to oe-GDF11 in mice. We concluded that telomerase activation by GDF11 contributes to the alleviation of myocardial IR injury through enhancing mitochondrial biogenesis and suppressing cardiomyocyte apoptosis.

Four novel compound heterozygous mutations in C5orf42 gene in patients with pure and mild Joubert syndrome.

Joubert syndrome (JS) is a rare clinically and genetically heterogeneous disease. Using whole or targeted exome sequencing, we identified four novel compound heterozygous mutations in chromosome 5 open reading frame 42 gene (C5orf42), including c.2876C>T (missense mutation) and c.3921+1G>A (splicing mutation), c.2292 -2delA (splicing mutation) and c.4067C>T (missense mutation), c.6997_6998insT (frameshift mutation) and c.8710C>T (nonsense mutation), c.3981G>C (nonsense mutation) and c.230 _233del (frameshift mutation), in four Chinese JS families. They were all inherited from their heterozygosis parents in the autosomal recessive inheritance mode. Pure JS clinical manifestations and mild neuroimaging findings were found in these patients. These verified the previous findings that C5orf42 mutations generally resulted in a purely neurological Joubert phenotype, and neuroimaging findings were mild in JS with C5orf42 mutations. Our report analyzed these C5orf42 mutations-associated phenotypes and neuroimaging findings in JS and updated the genetic variation spectrum of JS caused by C5orf42.These will help clinicians and geneticists reach a more accurate diagnosis for JS.

Inhibition of microRNA-497 ameliorates anoxia/reoxygenation injury in cardiomyocytes by suppressing cell apoptosis and enhancing autophagy.

MiR-497 is predicted to target anti-apoptosis gene Bcl2 and autophagy gene microtubule-associated protein 1 light chain 3 B (LC3B), but the functional consequence of miR-497 in response to anoxia/reoxygenation (AR) or ischemia/reperfusion (IR) remains unknown. This study was designed to investigate the influences of miR-497 on myocardial AR or IR injury. We noted that miR-497 was enriched in cardiac tissues, while its expression was dynamically changed in murine hearts subjected to myocardial infarction and in neonatal rat cardiomyocytes (NRCs) subjected to AR. Forced expression of miR-497 (miR-497 mimic) induced apoptosis in NRCs as determined by Hoechst staining and TUNEL assay. In response to AR, silencing of miR-497 using a miR-497 sponge significantly reduced cell apoptosis and enhanced autophagic flux. Furthermore, the infarct size induced by IR in adenovirus (Ad)-miR-497 sponge infected mice was significantly smaller than in mice receiving Ad-vector or vehicle treatment, while Ad-miR-497 increased infarct size. The expression of Bcl-2 and LC3B-II in NRCs or in murine heart was significantly decreased by miR-497 mimic and enhanced by miR-497 sponge. These findings demonstrate that inhibition of miR-497 holds promise for limiting myocardial IR injury.

[Clinical characteristics and treatment responses of X-linked thrombocytopenia].

OBJECTIVE: To analyze the clinical and molecular characteristics of patients with X-linked thrombocytopenia (XLT) and their responsiveness to treatment with various doses of corticosteroids or intravenous immunoglobulin (IVIG) separately. METHOD: Data from 15 XLT patients who were hospitalized in Children's Hospital Affiliated to Chongqing Medical University from March 2010 to July 2014 were analyzed retrospectively, including clinical manifestations, scores, peripheral blood, immunological functions, responses to IVIG and steroid treatment with various doses and duration. RESULT: All 15 XLT patients met the inclusion criteria and showed microthrombocytopenia with or without mild-to-moderate eczema or minor infections. Platelet counts ranged from (8-80) × 109/L. The platelet volume value ranged between 5.6 and 10.9 fl (normal range: 9.4-12.5 fl). Raised serum IgG was found in 5 cases, while low serum IgG was found in 2 cases. WAS gene analysis revealed missense mutations in 14 patients, including 4 hotspots (V75M, R86C, R86H, R86L) and 1 novel mutation (Y107C). Flow cytometer analysis of 13 patients showed various amounts of WAS protein (WASP) expression, 2 patients had normal amounts of WASP expression, 5 had reduced amounts, and 6 had absent WASP expression. Their responses to individual steroid and IVIG treatment with various doses and duration were also reviewed. Fourteen patients who were misdiagnosed as immune thrombocytopenic purpura at first received 28 courses of steroids and (or) 47 courses of IVIG treatment. The post-treatment platelet counts of 1 000-2 000 mg/(kg × d) IVIG(25 courses) at 2-7 d and 8-14 d time points were (60 ± 10) × 109/L and (41 ± 7) × 109/L, which indicate a significantly better responsiveness than those by [(31 ± 7) × 109/L, (21 ± 2) × 109/L] of 400-500 mg/(kg·d) IVIG(22 courses) (Z = -4.419, -1.592;P = 0.002,0.011). However, there were no significant differences between the responsiveness of 3 doses [1-2 mg/(kg·d)(8 courses), 3-6 mg/(kg·d) (11 courses) and 20-30 mg/(kg × d)(9 courses)] of steroids (F = 0.387,0.252;P = 0.980,0.761) at 2-7 d and 8-14 d time points. The platelet counts gradually decreased to the primary level at 15-30 d after any doses of steroids and (or) IVIG treatment. The effective rate of 1 000-2 000 mg/(kg × d) IVIG treatment was 18/25, which was significantly higher than that (2/22) of 400-500 mg/(kg × d) (χ² = 9.836, P = 0.008). The effective rate of 20-30 mg/(kg × d) steroids treatment (7/9) was relatively higher than 1-2 mg/(kg × d) (4/8) and 3-6 mg/(kg × d) (6/11) with no significant difference (χ9 = 3.235, P = 0.581). After the treatment with steroids and /or IVIG 14 cases with hemorrhage were all improved. CONCLUSION: The clinical characteristics of X-linked thrombocytopenia were microthrombocytopenia with or without mild-to-moderate eczema or minor infections. WAS gene and WASP analysis were diagnostic methods. There were no significant differences between the responsiveness of 3 doses of steroids; 1 000-2 000 mg/(kg·d) IVIG had a significantly better responsiveness. However, IVIG and steroids with any dose and duration may only transiently increase peripheral platelet level of XLT patients.

[Construction of a recombinant adenovirus co-expressing bone morphogenic proteins 9 and 6 and its effect on osteogenesis in C3H10 cells].

OBJECTIVE: To construct a recombinant adenovirus co-expressing bone morphogenic protein (BMP) 9 and BMP6 and observe its effect on the osteogenesis in C3H10 cells. METHOD: The full-length sequences of BMP9 and BMP6 were amplified from AdEasy vector by PCR and cloned into the shuttle plasmid pASG2 vector to construct the co-expression shuttle plasmid pASG2-BMP9, 6 followed by homologous recombination with plasmid pAdeasy-1 in BJ5183. After confirmation by restriction endonuclease digestion, the recombinant vector was transfected into HEK293 cells, and high-titer recombinant adenovirus (Ad-BMP9, 6) was collected after amplification. Ad-BMP9, 6 was then transduced into C3H10 cells in vitro, and the mRNA expression of BMP9 and BMP6 was detected by RT-PCR. The osteogenic capability of the transfected cells was observed by alkaline phosphatase staining and calcium-alizarin red staining. RESULTS: AdBMP9,6 was constructed successfully and effectively infected in C3H10 cells, in which high expressions of BMP6 and BMP9 were detected. C3H10 cells infected with Ad-BMP9,6 showed stronger alkaline phosphatase and calcium-alizarin red staining than the cells transfected by either BMP9 or BMP6 alone. CONCLUSION: The recombinant adenovirus co-expressing BMP9 and BMP6 we constructed shows a more potent effect than the adenoviruses expressing either BMP9 or BMP6 alone in inducing the osteogenic differentiation of C3H10 cells into osteoblasts.