The ICF2 gene Zbtb24 specifically regulates the differentiation of B1 cells via promoting heme synthesis.

BACKGROUND: Loss-of-function mutations of ZBTB24 cause immunodeficiency, centromeric instability, and facial anomalies syndrome 2 (ICF2). ICF2 is a rare autosomal recessive disorder with immunological defects in serum antibodies and circulating memory B cells, resulting in recurrent and sometimes fatal respiratory and gastrointestinal infections. The genotype-phenotype correlation in patients with ICF2 indicates an essential role of ZBTB24 in the terminal differentiation of B cells. METHODS: We used the clustered regularly interspaced short palindromic repeats (CRISPER)/Cas9 technology to generate B cell specific Zbtb24-deficient mice and verified the deletion specificity and efficiency by quantitative polymerase chain reaction (Q-PCR) and western blotting analyses in fluorescence-activated cell sorting (FACS)-sorted cells. The development, phenotype of B cells and in vivo responses to T cell dependent or independent antigens post immunization were analyzed by flow cytometry and enzyme-linked immunosorbent assay (ELISA). Adoptive transfer experiment in combination with in vitro cultures of FACS-purified B cells and RNA-Seq analysis were utilized to specifically determine the impact of Zbtb24 on B cell biology as well as the underlying mechanisms. RESULTS: Zbtb24 is dispensable for B cell development and maintenance in naive mice. Surprisingly, B cell specific deletion of Zbtb24 does not evidently compromise germinal center reactions and the resulting primary and secondary antibody responses induced by T cell dependent antigens (TD-Ags), but significantly inhibits T cell independent antigen-elicited antibody productions in vivo. At the cellular level, Zbtb24-deficiency specifically impedes the plasma cell differentiation of B1 cells without impairing their survival, activation and proliferation in vitro. Mechanistically, Zbtb24-ablation attenuates heme biosynthesis partially through mTORC1 in B1 cells, and addition of exogenous hemin abrogates the differentiation defects of Zbtb24-null B1 cells. CONCLUSIONS: Zbtb24 seems to regulate antibody responses against TD-Ags B cell extrinsically, but it specifically promotes the plasma cell differentiation of B1 cells via heme synthesis in mice. Our study also suggests that defected B1 functions contribute to recurrent infections in patients with ICF2.

hFcγRIIa: a double-edged sword in osteoclastogenesis and bone balance in transgenic mice.

Rheumatoid arthritis (RA) is a chronic autoimmune disease accompanied by local and systemic bone loss. FcγRs, especially FcγRIIa (hFcγRIIa), have been implicated in the pathogenesis of RA. However, the contribution of hFcγRIIa to bone loss has not been fully elucidated. In the present study, we demonstrated the double-edged sword role of hFcγRIIa on osteoclast differentiation through investigations involving hFcγRIIa-transgenic (hFcγRIIa-Tg) mice. Our findings reveal that hFcγRIIa-Tg mice, previously shown to exhibit heightened susceptibility to collagen-induced arthritis (CIA), displayed increased osteoporosis during CIA or at advanced ages (40 weeks), accompanied by heightened in vivo osteoclast differentiation. Notably, bone marrow cells from hFcγRIIa-Tg mice exhibited enhanced efficiency in differentiating into osteoclasts and bone resorption in vitro compared to wild-type mice when stimulated with receptor activators of NF-κB ligand (RANKL). Additionally, hFcγRIIa-Tg mice exhibited augmented sensitivity to RANKL-induced bone loss in vivo, highlighting the osteoclast-promoting role of hFcγRIIa. Mechanistically, bone marrow cells from hFcγRIIa-Tg mice displayed heightened Syk self-activation, leading to mTOR-pS6 pathway activation, thereby promoting RANKL-driven osteoclast differentiation. Intriguingly, while hFcγRIIa crosslinking hindered RANKL-induced osteoclast differentiation, it activated the kinase cAbl, subsequently triggering STAT5 activation and inhibiting the expression of osteoclast-associated genes. This study provides novel insights into hFcγRIIa-mediated osteoclast biology, suggesting promising therapeutic targets for managing bone remodeling disorders.

Development and validation of a nomogram for predicting all-cause mortality in American adult hypertensive populations.

Backgrounds: Hypertension stands as the predominant global cause of mortality. A notable deficiency exists in terms of predictive models for mortality among individuals with hypertension. We aim to devise an effective nomogram model that possesses the capability to forecast all-cause mortality within hypertensive populations. Methods: The data for this study were drawn from nine successive cycles of the National Health and Nutrition Examination Survey (NHANES) spanning the years from 1999 to 2016. The dataset was partitioned into training and validation sets at a 7:3 ratio. We opted for clinical practice-relevant indicators, applied the least absolute shrinkage and selection operator (LASSO) regression to identify the most pertinent variables, and subsequently built a nomogram model. We also employed concordance index, receiver operating characteristic (ROC) curves, calibration curves and decision curve analysis (DCA) to assess the model's validity. Results: A total of 17,125 hypertensive participants were included in this study with a division into a training set (11,993 individuals) and a validation set (5,132 individuals). LASSO regression was applied for the training set to obtain nine variables including age, monocytes, neutrophils, serum albumin, serum potassium, cardiovascular disease, diabetes, serum creatinine and glycated hemoglobin (HbA1C), and constructed a nomogram prediction model. To validate this model, data from the training and validation sets were used for validation separately. The concordance index of the nomogram model was 0.800 (95% CI, 0.792-0.808, p < 0.001) based on the training set and 0.793 (95% CI, 0.781-0.805, p < 0.001) based on the validation set. The ROC curves, calibration curves, and DCA curves all showed good predictive performance. Conclusion: We have developed a nomogram that effectively forecasts the risk of all-cause mortality among American adults in hypertensive populations. Clinicians may use this nomogram to assess patient's prognosis and choose a proper intervention in a timely manner.

Effect of "Tongdu Qishen" acupuncture on expressions of ferroptosis-related factors in the hippocampus of APPswe/PS1dE9 Mice. / "通督启神"针法对APPswe/PS1dE9å°é¼ æµ·é©¬é“æ­»äº¡ç›¸å ³å› å­è¡¨è¾¾çš„å½±å“.

OBJECTIVES: To investigate the effect of "Tongdu Qishen" acupunctureï¼»dredging the Govern Vessel and normalizing mental activities, electroacupuncture (EA) of "Baihui" (GV20), "Yintang" (EX-HN3) and "Shuigou" (GV26) needlingï¼½on the learning-memory ability and the mechanism of ferroptosis in Alzheimer's disease (AD) mice. METHODS: Twenty-four male APPswe/PS1dE9 mice were randomly and equally divided into model group and EA group, and 12 normal C57BL/6 mice were used as the control group. In the EA group, EA (2 Hz/100 Hz, 20 min) was applied to GV20 and EX-HN3 in combination with manual acupuncture stimulation of GV26. The treatment was performed once a day, for a total of 28 days. The mice in the three groups were given the same fixation and grasping operation. Morris water maze swimming tests were used to assess the mice's learning-memory ability. Nissl staining and transmission electron microscopy were used to observe the morphological changes of neurons in the hippocampus. The activity of superoxide dismutase (SOD) in the hippocampus tissue was detected by superoxide anionic colorimetric assay kit (WST-1), and malondialdehyde (MDA) contents were detected by thiobarbituric acid (TBA) method. The expression levels of prostaglandin endoperoxide synthase 2 (ptgs2) and glutathione peroxidase 4 (GPX4) mRNA in the hippocampus were detected by fluorescent quantitative real-time PCR. RESULTS: Behavioral results showed that compared with the control group, the escape latencies at the 2nd, 3rd, 4th and 5th day of Morris water maze swimming test were significantly increased (P<0.05), and the swimming time in the original platform quadrant and the times of cros-sing the original platform were considerably decreased (P<0.05) in the model group. In comparison with the model group, the escape latencies at the 4th and 5th day were strikingly decreased (P<0.05), and the swimming time in the original platform quadrant and times of crossing the original platform significantly increased (P<0.05) in the EA group. Following modeling, the SOD activity and the expression of GPX4 mRNA were obviously down-regulated (P<0.05), and the content of MDA and the expression of ptgs2 mRNA significantly up-regulated (P<0.05) in the model group rele-vant to the control group. The SOD activity and the expression of GPX4 mRNA were apparently increased (P<0.05), and the content of MDA and the expression of ptgs2 mRNA remarkably down-regulated (P<0.05) in the EA group rele-vant to the model group. Histopathological and ultrastructural results showed scattered arrangement of cells, widened space among cells, reduction in the number of cells, and many shrunk of dissolved nucleoli, shrunking and incomplete mitochondria, and high membrane electron density in the hippocampus of the model group, which was relatively milder in the EA group. CONCLUSIONS: "Tongdu Qishen" acupuncture can improve the learning-memory ability of AD mice, which may be related to its functions in up-regulating SOD activity and GPX4 mRNA expression, and down-regulating MDA content and ptgs2 mRNA expression to reduce the lipid peroxidation in the process of ferroptosis.

Predictive value of serum creatinine and total bilirubin for long-term death in patients with ischemic heart disease: A cohort study.

BACKGROUND: Ischemic heart disease (IHD) has a high mortality in the population. Although serum creatinine (Cr) and serum total bilirubin (TBil) are rapid and readily available biomarkers in routine blood tests, there is a lack of literature on the prognostic value of combined Cr and TBil tests for IHD. This study aimed to evaluate a combined equation based on Cr and TBil to predict the long-term risk of death in IHD and to find indicators sensitive to the prognosis of IHD patients. METHOD: In this study, 2625 patients with IHD were included, and the combined value and combined equations of Cr and TBil were obtained by logistic regression analysis based on Cr and TBil collected at the time of admission. Patients were divided into four groups according to the quartiles of the combined value. COX proportional hazard regression model was used to analyze the risk factors for long-term death in IHD patients. Receiver operating characteristic (ROC) curves were used to evaluate the prognostic effect of Cr, TBil and combined value on long-term death events. RESULTS: Logistic regression analysis was performed for long-term death events with Cr and TBil as independent variables, and the logit regression model was Logit(P) = 0.0129×TBil+0.007×Cr-0.417. Multifactorial Cox regression analysis showed that high values of the equation were independent risk factors for long-term death events (all-cause death: HR 1.457, 95% CI 1.256-1.689, P<0.001; cardiovascular death: HR 1.452, 95% CI 1.244-1.695, P<0.001). Combined Cr and TBil value are more valuable in predicting long-term death (AUC: 0.609, 95% CI 0.587-0.630, P<0.001). CONCLUSION: Combined Cr and TBil assay is superior to single biomarkers for predicting long-term death in patients with IHD. High values of the equation are independent predictors of long-term death and can be used to identify patients at high risk for IHD.

The role and mechanisms of microvascular damage in the ischemic myocardium.

Following myocardial ischemic injury, the most effective clinical intervention is timely restoration of blood perfusion to ischemic but viable myocardium to reduce irreversible myocardial necrosis, limit infarct size, and prevent cardiac insufficiency. However, reperfusion itself may exacerbate cell death and myocardial injury, a process commonly referred to as ischemia/reperfusion (I/R) injury, which primarily involves cardiomyocytes and cardiac microvascular endothelial cells (CMECs) and is characterized by myocardial stunning, microvascular damage (MVD), reperfusion arrhythmia, and lethal reperfusion injury. MVD caused by I/R has been a neglected problem compared to myocardial injury. Clinically, the incidence of microvascular angina and/or no-reflow due to ineffective coronary perfusion accounts for 5-50% in patients after acute revascularization. MVD limiting drug diffusion into injured myocardium, is strongly associated with the development of heart failure. CMECs account for > 60% of the cardiac cellular components, and their role in myocardial I/R injury cannot be ignored. There are many studies on microvascular obstruction, but few studies on microvascular leakage, which may be mainly due to the lack of corresponding detection methods. In this review, we summarize the clinical manifestations, related mechanisms of MVD during myocardial I/R, laboratory and clinical examination means, as well as the research progress on potential therapies for MVD in recent years. Better understanding the characteristics and risk factors of MVD in patients after hemodynamic reconstruction is of great significance for managing MVD, preventing heart failure and improving patient prognosis.

Monocyte-to-High Density Lipoprotein Cholesterol Ratio Positively Predicts Coronary Artery Disease and Multi-Vessel Lesions in Acute Coronary Syndrome.

Purpose: We investigated the hypothesis that MHR (monocyte-to-high density lipoprotein cholesterol ratio) is related to the severity of coronary artery in ACS (acute coronary syndrome). Methods: In this case-control study, we recruited 15,853 participants undergoing the first time percutaneous coronary intervention (PCI) including 4093 normal controls, 10,518 chronic coronary artery disease (CAD), and 1242 ACS cases. Examination of demographic clinical data and biochemical profiles, as well as MHR values, were performed before PCI. The relationship between MHR and severity of coronary artery lesion in ACS was analyzed. We also used a flow cytometric assay to distinguish CD14+/CD16- classical monocyte subsets in peripheral blood mononucleated cells from CAD patients. Results: MHR was higher in patients with ACS compared with MHR in normal control and chronic CAD (normal control vs chronic CAD vs ACS: 0.46 ± 0.27 × 109/mmol vs 0.53 ± 0.29 × 109/mmol vs 0.73 ± 0.47 × 109/mmol, P < 0.001). MHR showed a significantly progressive increase as the angiographic severity of coronary lesions increased (single vessel lesion vs multi-vessel lesions in ACS: 0.54 ± 0.31 × 109/mmol vs 0.58 ± 0.35 × 109/mmol, P < 0.001), and classical monocyte subset to HDL-C ratio (CMHR) was increased in with CAD patients compared with control [4.69 (IQR, 1.06, 2.97) × 103/mmol vs 1.92 (IQR, 0.92, 3.04) × 103/mmol, P = 0.02]. Using a multivariate analysis, after adjusting for age, gender, body mass index (BMI), diabetes, and dyslipidemia, MHR was positively associated with multi-vessel lesions in ACS [OR (odds ratio): 1.28 (95% CI: 1.03-1.59, P = 0.029)]. Conclusion: MHR level could be a potential predictor of coronary artery lesion severity in ACS.

[Influence of electroacupuncture of "Dazhui"（GV14） and "Mingmen" （GV4） on expressions of NL1 and NF-200 in spinal cord injury rats].

OBJECTIVE: To observe the effect of electroacupuncture(EA) on activities of A2 type astrocytes(A2s)and A1 type astrocytes (A1s) , expressions of neurofilament protein 200 (NF-200, a marker of axon regeneration), nexin 1(NL1, a marker of synaptic regeneration), and regeneration of Nissl bodies in rats with spinal cord injury (SCI), so as to explore its mechanisms underlying improvement of SCI. METHODS: A total of 75 male SD rats were rando-mized into sham operation, model, antibody neutralizing (AN), EA and EA+AN groups, with 15 rats in each group. The SCI model was established by using an infinite field impactor to deliver an about 200 k dyne weight onto the exposed spinal cord after making a dorsal laminectomy at vertebral level T10. EA (2 Hz, 1 mA) was applied to"Dazhui"(GV14) and "Mingmen"(GV4) for 20 min, once daily for 28 days. After modeling, intraspinal injection of neutralizing antibodies IL-1α, TNF-α and complement 1q (C1q, 2 µL) to the injured spinal locus for inhibition of A1 type astrocytes (A1s) was conducted on the 1st, 7th , 14th and 21st day for rats of AN and EA+AN groups. BBB rating scale was used to evaluate hindlimb locomotor function on day 1, 7, 14, 21 and 28 after modeling. The activation of A2s (its specific marker S100a10), astrocyte (its specific marker glial fibrillary acidic protein, GFAP), and A1s (its specific marker C3) in the spinal cord was detected by immunofluorescence, and the protein expressions of NF-200 and NL1 in the spinal cord detected by Western blot and immunohistochemistry, separately, and the neuronal regeneration was observed after Nissl staining. RESULTS: After SCI, the BBB scores at 1 , 7, 14, 21 and 28 day, and the immunoactivity of NL1 and NF-200 were significantly decreased (P<0.01), and the fluorescence intensity of double labelled S100a10 (A2s)/GFAP and C3, and the expression of NF-200 were considerably increased in the model group (P<0.05, P<0.01). In contrast to the model group, the BBB scores at 7, 14, 21 and 28 day, and the immunoactivity of NL1 and NF-200, and the fluorescence intensity of A2s/GFAP in the AN, EA and AN+EA groups, and the expressions of NL1 in the EA and AN+EA groups, and expression of NF-200 protein in the AN+EA group were evidently increased (P<0.05, P<0.01), and the fluorescence intensity of C3 was strikingly decreased in the EA group (P<0.01). The effect of AN+EA was significantly superior to that of single AN and EA in increasing BBB scores at 14, 21 and 28 day, and in up-regulating the immunoactivity of NF-200(P<0.01, P<0.05). Nissl staining showed damaged structure of the gray matter of the spinal cord, atrophy of the Nissl body, and pyknosis of neurons, which was milder in the AN and EA groups, particularly in the AN+EA group. CONCLUSION: EA at GV14 and GV4 may promote activation of A2s and promote regeneration of axons and synapses in SCI model rats.

AT1R gene rs389566 polymorphism contributes to MACCEs in hypertension patients.

OBJECTIVE: To investigate the possible association between AT1R gene polymorphisms and major adverse cardiovascular and cerebrovascular events (MACCEs) in hypertension patients combined with or without coronary artery disease (CAD) in Xinjiang. METHODS: 374 CAD patients and 341 non-CAD individuals were enrolled as study participants and all of them have a hypertension diagnosis. AT1R gene polymorphisms were genotyped by SNPscan™ typing assays. During the follow-up in the clinic or by telephone interview, MACCEs were recorded. Kaplan-Meier curves and Cox survival analyses were used to explore the association between AT1R gene polymorphisms and the occurrence of MACCEs. RESULTS: AT1R gene rs389566 was associated with MACCEs. The TT genotype of the AT1R gene rs389566 had a significantly higher probability of MACCEs than the AA + AT genotype (75.2% vs. 24.8%, P = 0.033). Older age (OR = 1.028, 95% CI: 1.009-1.0047, P = 0.003) and TT genotype of rs389566 (OR = 1.770, 95% CI: 1.148-2.729, P = 0.01) were risk factors of MACCEs. AT1R gene rs389566 TT genotype may be a predisposing factor for the occurrence of MACCEs in hypertensive patients. CONCLUSION: We should also pay more attention to the prevent of MACCEs in hypertension patients combined with CAD. Especially those elderly hypertensive patients carrying AT1R rs389566 TT genotype requires avoidance of unhealthy lifestyle, better management of blood pressure control and reduce the occurrence of MACCEs.

A COVID-19 DNA Vaccine Candidate Elicits Broadly Neutralizing Antibodies against Multiple SARS-CoV-2 Variants including the Currently Circulating Omicron BA.5, BF.7, BQ.1 and XBB.

Waves of breakthrough infections by SARS-CoV-2 Omicron subvariants currently pose a global challenge to the control of the COVID-19 pandemic. We previously reported a pVAX1-based DNA vaccine candidate, pAD1002, that encodes a receptor-binding domain (RBD) chimera of SARS-CoV-1 and Omicron BA.1. In mouse and rabbit models, pAD1002 plasmid induced cross-neutralizing Abs against heterologous sarbecoviruses, including SARS-CoV-1 and SARS-CoV-2 wildtype, Delta and Omicron variants. However, these antisera failed to block the recent emerging Omicron subvariants BF.7 and BQ.1. To solve this problem, we replaced the BA.1 RBD-encoding DNA sequence in pAD1002 with that of BA.4/5. The resulting construct, namely pAD1016, elicited SARS-CoV-1 and SARS-CoV-2 RBD-specific IFN-γ+ cellular responses in BALB/c and C57BL/6 mice. More importantly, pAD1016 vaccination in mice, rabbits and pigs generated serum Abs capable of neutralizing pseudoviruses representing multiple SARS-CoV-2 Omicron subvariants including BA.2, BA.4/5, BF.7, BQ.1 and XBB. As a booster vaccine for inactivated SARS-CoV-2 virus preimmunization in mice, pAD1016 broadened the serum Ab neutralization spectrum to cover the Omicron BA.4/5, BF7 and BQ.1 subvariants. These preliminary data highlight the potential benefit of pAD1016 in eliciting neutralizing Abs against broad-spectrum Omicron subvariants in individuals previously vaccinated with inactivated prototype SARS-CoV-2 virus and suggests that pAD1016 is worthy of further translational study as a COVID-19 vaccine candidate.

Predictive Effect of Triglyceride-Glucose Index on Adverse Prognostic Events in Patients with Type 2 Diabetes Mellitus and Ischemic Cardiomyopathy.

Background: The triglyceride-glucose (TyG) index is regarded as an independent predictor of cardiovascular (CV) consequences and a reliable surrogate measure of insulin resistance (IR). However, the predictive significance of the TyG index in patients with type 2 diabetes mellitus (T2DM) and ischemic cardiomyopathy (ICM) remains unknown. Methods: This study included 1514 consecutive subjects with ICM and T2DM. The tertile of the TyG index values was used to categorize these patients into three groups. Major adverse cardiac and cerebral events (MACCEs) were also noted. The TyG index was calculated using the [fasting triglycerides (mg/dL) × fasting plasma glucose (mg/dL)/2] equation. Results: After adjusting for age, BMI, and other potential confounders, the scores of multivariate Cox proportional hazards regression models for chest pain [9.056 (4.370 to 18.767), p<0.001], acute myocardial infarction [4.437 (1.420 to 13.869), p=0.010], heart failure [7.334 (3.424 to 15.708), p<0.001], cardiogenic shock [3.707 (1.207 to 11.384), p=0.022], malignant arrhythmia [5.309 (2.367 to 11.908), p<0.001], cerebral infarction [3.127 (1.596 to 6.128), p<0.001], gastrointestinal bleeding [4.326 (1.612 to 11.613), p=0.004], all-cause death [4.502 (3.478 to 5.827), p<0.001] and cumulative incidence of MACCEs [4.856 (3.842 to 6.136), p<0.001] increased significantly with an increase in TyG index levels (all p<0.05). Time-dependent ROC analysis revealed that the area under the TyG index curve (AUC) reached 0.653 in the 3rd year, 0.688 in the 5th year, and 0.764 in the 10th year. The predictive efficiency of this model on MACCEs improved [net reclassification improvement (NRI): 0.361 (0.253 to 0.454); C-index: 0.678 (0.658 to 0.698); integrated discrimination improvement (IDI): 0.138 (0.098 to 0.175), all p<0.05] following the incorporation of the TyG index into the base risk model. Conclusion: TyG index could be useful in predicting MACCEs and initiating preventive measures in subjects with ICM and T2DM.

Potent immunogenicity and broad-spectrum protection potential of microneedle array patch-based COVID-19 DNA vaccine candidates encoding dimeric RBD chimera of SARS-CoV and SARS-CoV-2 variants.

Breakthrough infections by SARS-CoV-2 variants pose a global challenge to COVID-19 pandemic control, and the development of more effective vaccines of broad-spectrum protection is needed. In this study, we constructed pVAX1-based plasmids encoding receptor-binding domain (RBD) chimera of SARS-CoV-1 and SARS-CoV-2 variants, including pAD1002 (encoding RBDSARS/BA1), pAD1003 (encoding RBDSARS/Beta) and pAD131 (encoding RBDBA1/Beta). Plasmids pAD1002 and pAD131 were far more immunogenic than pAD1003 in terms of eliciting RBD-specific IgG when intramuscularly administered without electroporation. Furthermore, dissolvable microneedle array patches (MAP) greatly enhanced the immunogenicity of these DNA constructs in mice and rabbits. MAP laden with pAD1002 (MAP-1002) significantly outperformed inactivated SARS-CoV-2 virus vaccine in inducing RBD-specific IFN-γ+ effector and memory T cells, and generated T lymphocytes of different homing patterns compared to that induced by electroporated DNA in mice. In consistence with the high titer neutralization results of MAP-1002 antisera against SARS-CoV-2 pseudoviruses, MAP-1002 protected human ACE2-transgenic mice from Omicron BA.1 challenge. Collectively, MAP-based DNA constructs encoding chimeric RBDs of SARS-CoV-1 and SARS-CoV-2 variants, as represented by MAP-1002, are potential COVID-19 vaccine candidates worthy further translational study.

Development and validation of a nomogram to predict mortality risk in patients with ischemic heart disease.

Background: Ischemic Heart Disease (IHD) is the leading cause of death from cardiovascular disease. Currently, most studies have focused on factors influencing IDH or mortality risk, while few predictive models have been used for mortality risk in IHD patients. In this study, we constructed an effective nomogram prediction model to predict the risk of death in IHD patients by machine learning. Methods: We conducted a retrospective study of 1,663 patients with IHD. The data were divided into training and validation sets in a 3:1 ratio. The least absolute shrinkage and selection operator (LASSO) regression method was used to screen the variables to test the accuracy of the risk prediction model. Data from the training and validation sets were used to calculate receiver operating characteristic (ROC) curves, C-index, calibration plots, and dynamic component analysis (DCA), respectively. Results: Using LASSO regression, we selected six representative features, age, uric acid, serum total bilirubin, albumin, alkaline phosphatase, and left ventricular ejection fraction, from 31 variables to predict the risk of death at 1, 3, and 5 years in patients with IHD, and constructed the nomogram model. In the reliability of the validated model, the C-index at 1, 3, and 5 years was 0.705 (0.658-0.751), 0.705 (0.671-0.739), and 0.694 (0.656-0.733) for the training set, respectively; the C-index at 1, 3, and 5 years based on the validation set was 0.720 (0.654-0.786), 0.708 (0.650-0.765), and 0.683 (0.613-0.754), respectively. Both the calibration plot and the DCA curve are well-behaved. Conclusion: Age, uric acid, total serum bilirubin, serum albumin, alkaline phosphatase, and left ventricular ejection fraction were significantly associated with the risk of death in patients with IHD. We constructed a simple nomogram model to predict the risk of death at 1, 3, and 5 years for patients with IHD. Clinicians can use this simple model to assess the prognosis of patients at the time of admission to make better clinical decisions in tertiary prevention of the disease.

Membrane Bound CRT Fragment Accelerates Tumor Growth of Melanoma B16 Cell In Vivo through Promoting M2 Polarization via TLR4.

Calreticulin (CRT) is a major calcium-binding luminal resident protein on the endoplasmic reticulum that can also be released extracellular as well as anchored on surface of cells. Previously, we demonstrated that soluble recombinant CRT fragment 39-272 (CRT/39-272) exhibited potent immunostimulatory effects as well as immunoregulation effects on immune cells. Here, we constructed stable B16 melanoma cell lines expressing recombinant CRT/39-272 on the membrane (B16-tmCRT/39-272) to investigate the roles of cell surface CRT on tumor progression. We found that B16-tmCRT/39-272 cells subcutaneously inoculated into C57BL/6 mice exhibited stronger tumorigenicity than the B16-EGFP control cells. The tumor associated macrophages infiltrated in tumors were mainly M2 phenotype. Regulatory T cells (Tregs) were also expanded more in bearing mice. Consistent with the in vivo results, B16-tmCRT/39-272 promoted macrophage polarization toward F4/80+CD206+ M2 macrophages and promoted transforming growth factor beta (TGF-ß) secretion in vitro, which could promote naïve CD4+ T cell differentiation into Tregs. These results imply that the tmCRT/39-272 could accelerate tumor development by enhancing M2 macrophage polarization to induce TGF-ß secretion, and then promoted Treg differentiation in the tumor microenvironment. Our data may provide useful clues for better understanding of the potentiating roles of CRT in tumorigenesis.

Long-term prognostic value of macrophage migration inhibitory factor in ST-segment elevation myocardial infarction patients with metabolic syndrome after percutaneous coronary intervention.

Metabolic syndrome (MetS) is a major risk factor for cardiovascular disease and negatively affecting the prognosis of patients with ST elevation myocardial infarction (STEMI). Macrophage migration inhibitory factor (MIF) is a multipotent cytokine involved in various cardiovascular and inflammatory diseases. In this prospective study, we investigate the value of MIF in the long-term prognosis of STEMI combined with MetS after emergency PCI. Circulating MIF levels were measured at admission, and major adverse cardiovascular and cerebrovascular events (MACCE) were monitored during the follow-up period of 4.9 (3.9-5.8) years. MACCE occurred in 92 patients (22.9%), which was significantly higher in MetS (69/255, 27.1%) than in the non-MS subgroup (23/146, 15.8%, P < 0.05). Patients with MetS developed MACCE had the highest admission MIF level. Kaplan-Meier survival analysis using the cutoff value of admission MIF (143 ng/ml) showed that patients with a higher MIF level had a greater incidence of MACCE than those with lower MIF levels in both the MetS (P < 0.0001) and non-MetS groups (P = 0.016). After adjustment for clinical variables, the value of MIF ≥ 143 ng/ml still had the predictive power for the MetS group [HR 9.56, 95% CI (5.397-16.944),P < 0.001]; nevertheless, it was not the case in the non-MetS group. Our findings indicated that MetS is a critical risk factor for adverse clinical outcomes in patients with STEMI, and a high admission MIF level has predictive power for the long-term MACCE, which is superior in STEMI patients with MetS and better than other traditional predictors.

Calreticulin as an Adjuvant In Vivo to Promote Dendritic Cell Maturation and Enhance Antigen-Specific T Lymphocyte Responses against Melanoma.

An endoplasmic reticulum resident protein, calreticulin (CRT), participates in many cellular processes. CRT is a tumor-associated antigen with an important role in antitumor immunity. Previously, we reported that the recombinant CRT fragment 39-272 (CRT/39-272) exhibited superior immunobiological activity, activating macrophages to release cytokines and promoting dendritic cell (DC) maturation. However, the effect of CRT/39-272 in vivo, especially its adjuvant effect on in vivo antitumor immune responses, was not fully investigated. In this study, we constructed a fusion protein linking CRT/39-272 to an ovalbumin (OVA) peptide (residues 182-297, OVAp) and used the fusion protein (OVAp-CRT) to examine the adjuvant effect of CRT. We investigated whether CRT/39-272 could induce bone marrow-derived DC maturation and strongly promote the proliferation of OVA-specific T cells in vitro. Compared with OVAp, OVAp-CRT induced stronger antigen-specific T lymphocyte responses, including antigen-specific T cell proliferation, interferon-γ secretion, and cytotoxic T lymphocyte responses. OVAp-CRT-immunized mice generated significantly increased OVAp-specific antibody and CD4+/CD8+ memory T cells, which mediated long-term protective effects. OVAp-CRT upregulated CD40, CD80, and CD86 expressions in splenic conventional DCs. Furthermore, OVAp-CRT protected immunized mice against OVA-expressing B16 melanoma cells in vivo. Moreover, mice that were adoptively transferred with OVAp-CRT-pulsed DCs showed inhibited tumor growth and prolonged mouse survival. Our results demonstrate that CRT/39-272 can be used as a potential new adjuvant for tumor vaccines, and this finding may be useful in tumor vaccine development.

Neutrophils as regulators of macrophage-induced inflammation in a setting of allogeneic bone marrow transplantation.

Clinical data reveal that patients with allogeneic hematopoietic stem cell transplantation (HSCT) are vulnerable to infection and prone to developing severe sepsis, which greatly compromises the success of transplantation, indicating a dysregulation of inflammatory immune response in this clinical setting. Here, by using a mouse model of haploidentical bone marrow transplantation (haplo-BMT), we found that uncontrolled macrophage inflammation underlies the pathogenesis of both LPS- and E.coli-induced sepsis in recipient animals with graft-versus-host disease (GVHD). Deficient neutrophil maturation in GVHD mice post-haplo-BMT diminished modulation of macrophage-induced inflammation, which was mechanistically dependent on MMP9-mediated activation of TGF-ß1. Accordingly, adoptive transfer of mature neutrophils purified from wild-type donor mice inhibited both sterile and infectious sepsis in GVHD mice post-haplo-BMT. Together, our findings identify a novel mature neutrophil-dependent regulation of macrophage inflammatory response in a haplo-BMT setting and provide useful clues for developing clinical strategies for patients suffering from post-HSCT sepsis.

Altered Phenotype and Enhanced Antibody-Producing Ability of Peripheral B Cells in Mice with Cd19-Driven Cre Expression.

Given the importance of B lymphocytes in inflammation and immune defense against pathogens, mice transgenic for Cre under the control of Cd19 promoter (Cd19Cre/+ mice) have been widely used to specifically investigate the role of loxP-flanked genes in B cell development/function. However, impacts of expression/insertion of the Cre transgene on the phenotype and function of B cells have not been carefully studied. Here, we show that the number of marginal zone B and B1a cells was selectively reduced in Cd19Cre/+ mice, while B cell development in the bone marrow and total numbers of peripheral B cells were comparable between Cd19Cre/+ and wild type C57BL/6 mice. Notably, humoral responses to both T cell-dependent and independent antigens were significantly increased in Cd19Cre/+ mice. We speculate that these differences are mainly attributable to reduced surface CD19 levels caused by integration of the Cre-expressing cassette that inactivates one Cd19 allele. Moreover, our literature survey showed that expression of Cd19Cre/+ alone may affect the development/progression of inflammatory and anti-infectious responses. Thus, our results have important implications for the design and interpretation of results on gene functions specifically targeted in B cells in the Cd19Cre/+ mouse strain, for instance, in the context of (auto) inflammatory/infectious diseases.