Development and characterization of humanized and human forms of ELR-CXC chemokine antagonist, bovine CXCL8(3-74)K11R/G31P

Glu-Leu-Arg (ELR)-CXC chemokine-mediated neutrophil migration and activation plays a key role in many inflammatory diseases. Dysregulated neutrophil activation often leads to inflammatory responses such as acute lung injury (ALI) or acute respiratory distress syndrome (ARDS). Previously, we generated a bovine drug (i.e., bovine CXCL8(3-74)K11R/G31P, bG31P) by mutating the first two amino acids at the beginning of the N-terminus of bovine CXCL8/IL-8 and later substituting Arg for Lys11 and Pro for Gly31. Bovine G31P was shown to be a highly effective ELR-CXC chemokine and neutrophil antagonist in cattle & guinea pigs, but a human equivalent thereof would be of significantly more use in human medicine. Published studies on the structure and function of human CXCL8 suggest that human CXCL8(3-72)K11R/G31P (i.e., hG31P) would not be a particularly effective chemokine antagonist. Thus, development of a humanized form of bG31P became a primary goal. I first examined the effect of wholesale ligation of the carboxy half of hCXCL8 onto the amino half of bG31P and generated a human-bovine chimeric G31P (hbG31P; i.e., bCXCL8(3-44)K11R/G31P-hCXCL8(45-72)). I also made substitutions at each remaining human-discrepant amino acid (i.e., T3K, H13Y, T15K, E35A, and S37T) within the 5’ half of the hbG31P cDNA. The results showed that hbG31P and its analogues blocked CXCL8-induced human neutrophil chemotactic responses, reactive oxygen intermediate (ROI) release, and intracellular calcium flux. Humanized bovine G31P was also shown to significantly block pulmonary neutrophilic pathology in a guinea pig model of airway endotoxemia. As bG31P, hbG31P and its further humanized forms showed essentially equivalent ELR-CXC chemokine antagonist activity, Dr. Fang Li, Ms Jennifer Town and I then generated a fully human form of bG31P, hG31P. In vitro, hG31P was shown to effectively inhibit CXCL-1-, -5-, and -8-induced neutrophil chemotactic responses, intracellular Ca2+ flux, and ROI release. Human G31P also desensitized heterologous G protein-coupled receptors (GPCR) including bacterial peptides (e.g., N-formyl-methionine-leucine-phenylalanine, fMLP), anaphylatoxin (e.g., complement 5a, C5a), lipid mediators (e.g., leukotriene B4, LTB4; platelet-activating factor, PAF) receptors. Moreover, hG31P, in a dose-dependent manner suppressed CXCL1 and CXCL8 expression by LPS-challenged airway epithelial cells and reversed the anti-apoptotic influence of ELR-CXC chemokines on neutrophils. In vivo, hG31P was significantly effective in blocking the pathology associated with airway endotoxemia, aspiration pneumonia, and intestinal ischemia and reperfusion injury, including neutrophil recruitment (70-95% reduction) into, and activation within, the airways or gut, chemokine or cytokine expression, and pulmonary vascular complications. The blockade of neutrophil recruitment by hG31P in aspiration pneumonia animals did not increase airway bacterial growth. The G31P treatment was protective in both mesenteric (i.e., local) and remote organ injury. These findings suggest that hG31P is not only a potent neutrophil antagonist, but an effective blocker of other inflammatory responses. These comprehensive anti-inflammatory effects indicate that hG31P could potentially provide a viable therapeutic approach for inflammatory diseases such as ALI /ARDS

Validation of the digital health literacy assessment among the university students in China

PurposeWith the development of the internet, digital health literacy (DHL) has become increasingly important for managing health. Consequently, various digital health literacy scales have been created for different groups. The purpose of this study was to verify the reliability and validity of the simplified Chinese version of the Digital Health Literacy Assessment (DHLA) scale among university students in China.MethodSnowball sampling was used to recruit the participants via an online platform (Wenjuan.com), and finally 304 university students were included in the survey. Demographic information and the status of DHL were collected through the online questionnaire. Cronbach’s alpha and split-half reliability were used to test the internal consistency of the scale, while the structural validity was verified by exploratory factor analysis and confirmatory factor analysis. Additionally, the convergence of the scale was tested by composite reliability (CR) and average variance extracted (AVE).ResultTwo dimensions were generated from 10 entries in the scale, named Self-rated Digital Health Literacy and Trust Degree of Online Health Information, respectively. The Cronbach’s alpha and split-half reliability of the total scale were 0.912 and 0.828, while the Cronbach’s alpha of the two dimensions were 0.913 and 0.830, respectively. The structural validity-related indexes of the scale met the standards (RMSEA = 0.079, GFI = 0.943, AGFI = 0.902, CFI = 0.971). In each dimension, the CR and AVE also reached critical values (CR > 0.7 and AVE > 0.5).ConclusionThe scale had high reliability and validity, indicating the simplified Chinese DHLA scale could be used to evaluate the DHL of university students in China

NF-κB1 Inhibits TLR-Induced IFN-β Production in Macrophages Through TPL-2-dependent ERK Activation

available in PMC 2012 February 15.Although NF-κB1 p50/p105 has critical roles in immunity, the mechanism by which NF-κB1 regulates inflammatory responses is unclear. In this study, we analyzed the gene expression profile of LPS-stimulated Nfkb1−/− macrophages that lack both p50 and p105. Deficiency of p50/p105 selectively increased the expression of IFN-responsive genes, which correlated with increased IFN-β expression and STAT1 phosphorylation. IFN Ab-blocking experiments indicated that increased STAT1 phosphorylation and expression of IFN-responsive genes observed in the absence of p50/p105 depended upon autocrine IFN-β production. Markedly higher serum levels of IFN-β were observed in Nfkb1−/− mice than in wild-type mice following LPS injection, demonstrating that Nfkb1 inhibits IFN-β production under physiological conditions. TPL-2, a mitogen-activated protein kinase kinase kinase stabilized by association with the C-terminal ankyrin repeat domain of p105, negatively regulates LPS-induced IFN-β production by macrophages via activation of ERK MAPK. Retroviral expression of TPL-2 in Nfkb1−/− macrophages, which are deficient in endogenous TPL-2, reduced LPS-induced IFN-β secretion. Expression of the C-terminal ankyrin repeat domain of p105 in Nfkb1−/− macrophages, which rescued LPS activation of ERK, also inhibited IFN-β expression. These data indicate that p50/p105 negatively regulates LPS-induced IFN signaling in macrophages by stabilizing TPL-2, thereby facilitating activation of ERK.National Institutes of Health (U.S.) (NIH AI52267)National Institutes of Health (U.S.) (NIH CA108854)National Institutes of Health (U.S.) (NIH CA67529)Medical Research Council (Great Britain

Detecting influenza and emerging avian influenza virus by influenza and pneumonia surveillance systems in a large city in China, 2005 to 2016.

BACKGROUND(#br)Detecting avian influenza virus has become an important public health strategy for controlling the emerging infectious disease.(#br)METHODS(#br)The HIS (hospital information system) modified influenza surveillance system (ISS) and a newly built pneumonia surveillance system (PSS) were used to monitor the influenza viruses in Changsha City, China. The ISS was used to monitor outpatients in two sentinel hospitals and to detect mild influenza and avian influenza cases, and PSS was used to monitor inpatients in 49 hospitals and to detect severe and death influenza cases.(#br)RESULTS(#br)From 2005 to 2016, there were 3,551,917 outpatients monitored by the ISS system, among whom 126,076 were influenza-like illness (ILI) cases, with the ILI proportion (ILI%) of 3.55%. After the HIS was used, the reported incident cases of ILI and ILI% were increased significantly. From March, 2009 to September, 2016, there were 5,491,560 inpatient cases monitored by the PSS system, among which 362,743 were pneumonia cases, with a proportion of 6.61%. Among pneumonia cases, about 10.55% (38,260/362,743) of cases were severe or death cases. The pneumonia incidence increased each year in the city. Among 15 avian influenza cases reported from January, 2005 to September, 2016, there were 26.7% (4/15) mild cases detected by the HIS-modified ISS system, while 60.0% (9/15) were severe or death cases detected by the PSS system. Two H5N1 severe cases were missed by the ISS system in January, 2009 when the PSS system was not available.(#br)CONCLUSIONS(#br)The HIS was able to improve the efficiency of the ISS for monitoring ILI and emerging avian influenza virus. However, the efficiency of the system needs to be verified in a wider area for a longer time span in China

The relationships of preventive behaviors and psychological resilience with depression, anxiety, and stress among university students during the COVID-19 pandemic: A two-wave longitudinal study in Shandong Province, China

IntroductionStudies have shown that the psychological impact of the COVID-19 pandemic may lead to long-term health problems; therefore, more attention should be paid to the mental health of university students. This study aimed to explore the longitudinal effects of preventive behaviors and psychological resilience on the mental health of Chinese college students during COVID-19.MethodsWe recruited 2,948 university students from five universities in Shandong Province. We used a generalized estimating equation (GEE) model to estimate the impact of preventive behaviors and psychological resilience on mental health.ResultsIn the follow-up survey, the prevalence of anxiety (44.8% at T1 vs 41.2% at T2) and stress (23.0% at T1 vs 19.6% at T2) decreased over time, whereas the prevalence of depression (35.2% at T1 vs 36.9% at T2) increased significantly (P < 0.001). Senior students were more likely to report depression (OR = 1.710, P < 0.001), anxiety (OR = 0.815, P = 0.019), and stress (OR = 1.385, P = 0.011). Among all majors, medical students were most likely to report depression (OR = 1.373, P = 0.021), anxiety (OR = 1.310, P = 0.040), and stress (OR = 1.775, P < 0.001). Students who wore a mask outside were less likely to report depression (OR = 0.761, P = 0.027) and anxiety (OR = 0.686, P = 0.002) compared to those who did not wear masks. Students who complied with the standard hand-washing technique were less likely to report depression (OR = 0.628, P < 0.001), anxiety (OR = 0.701, P < 0.001), and stress (OR = 0.638, P < 0.001). Students who maintained a distance of one meter in queues were less likely to report depression (OR = 0.668, P < 0.001), anxiety (OR = 0.634, P < 0.001), and stress (OR = 0.638, P < 0.001). Psychological resilience was a protective factor against depression (OR = 0.973, P < 0.001), anxiety (OR = 0.980, P < 0.001), and stress (OR = 0.976, P < 0.001).DiscussionThe prevalence of depression among university students increased at follow-up, while the prevalence of anxiety and stress decreased. Senior students and medical students are vulnerable groups. University students should continue to follow relevant preventive behaviors to protect their mental health. Improving psychological resilience may help maintain and promote university students' mental health

Nfkb1 inhibits LPS-induced IFN-β and IL-12 p40 production in macrophages by distinct mechanisms.

Nfkb1-deficient murine macrophages express higher levels of IFN-β and IL-12 p40 following LPS stimulation than control macrophages, but the molecular basis for this phenomenon has not been completely defined. Nfkb1 encodes several gene products including the NF-κB subunit p50 and its precursor p105. p50 is derived from the N-terminal of 105, and p50 homodimers can exhibit suppressive activity when overexpressed. The C-terminal region of p105 is necessary for LPS-induced ERK activation and it has been suggested that ERK activity inhibits both IFN-β and IL-12 p40 following LPS stimulation. However, the contributions of p50 and the C-terminal domain of p105 in regulating endogenous IFN-β(Ifnb) and IL-12 p40 (Il12b) gene expression in macrophages following LPS stimulation have not been directly compared.We have used recombinant retroviruses to express p105, p50, and the C-terminal domain of p105 (p105ΔN) in Nfkb1-deficient murine bone marrow-derived macrophages at near endogenous levels. We found that both p50 and p105ΔN inhibited expression of Ifnb, and that inhibition of Ifnb by p105ΔN depended on ERK activation, because a mutant of p105ΔN (p105ΔNS930A) that lacks a key serine necessary to support ERK activation failed to inhibit. In contrast, only p105ΔN but not p50 inhibited Il12b expression. Surprisingly, p105ΔNS930A retained inhibitory activity for Il12b, indicating that ERK activation was not necessary for inhibition. The differential effects of p105ΔNS930A on Ifnb and Il12b expression inversely correlated with the function of one of its binding partners, c-Rel. This raised the possibility that p105ΔNS930A influences gene expression by interfering with the function of c-Rel.These results demonstrate that Nfkb1 exhibits multiple gene-specific inhibitory functions following TLR stimulation of murine macrophages

Retrovirally-mediated expression of <i>Nfkb1</i> constructs.

<p>A) Immunoblotting of <i>Il10</i>^−/−<i>Rag2</i>^−/− (Control) or <i>Nfkb1</i>^−/−<i>Il10</i>^−/−<i>Rag2</i>^−/− (<i>Nfkb1</i>^−/−) BMDM infected with empty vector (Vector) or viruses expressing the indicated proteins. Total cell extracts from unstimulated cells were probed with the indicated antibodies (left) by western blotting. Arrows indicate size of <i>Nfkb1</i>-derived gene product (right). NS refers to a non-specific band. B) Relative expression of <i>Nfkb1</i> N- and C-terminal derived mRNA as determined by RT-PCR. N = 3 mice per group.</p

Domain structure of p105.

<p>Diagram showing p105, p50, and constructs p105ΔN and p105ΔNS930A.</p