Haemolysin Sph2 of Leptospira interrogans induces cell apoptosis via intracellular reactive oxygen species elevation and mitochondrial membrane injury.

Leptospira interrogans causes widespread leptospirosis in humans and animals, with major symptoms of jaundice and haemorrhage. Sph2, a member of the sphingomyelinase haemolysins, is an important virulence factor for leptospire. In this study, the function and mechanism of Sph2 in the pathogenesis of leptospirosis were investigated to further understand the pathogenesis of leptospire. Real-time PCR analysis of expression levels during cell invasion showed that sph2 gene expression was transiently induced in human umbilical vein endothelial cells (HUVECs), human embryo liver cells (L02), and human epithelial lung cells (L132), with expression levels reaching a peak after 45 min of infection. Further functional analysis of recombinant Sph2 (rSph2) by LDH assays and confocal microscopy showed that rSph2 can be internalised by cells both by causing cell membrane damage and by a damage-independent clathrin-mediated endocytosis pathway. Subsequently, rSph2 is able to translocate to mitochondria, which led to an increase in the levels of reactive oxygen species (ROS) and a decrease of the mitochondrial membrane potential (ΔΨm ). Further flowcytometry analyses after rSph2 exposure showed that 28.7%, 31%, and 27.3% of the HUVEC, L02, and L132 cells, respectively, became apoptotic. Because apoptosis could be decreased with the ROS inhibitor N-acetyl cysteine, these experiments suggested that rSph2 triggers apoptosis through mitochondrial membrane damage and ROS elevation. The ability of leptospiral haemolysin rSph2 to cause apoptosis likely contributes to the pathogenesis of leptospirosis.

In vitro research of combination therapy for multidrug-resistant Klebsiella pneumoniae bloodstream infections.

OBJECTIVE: Multidrug-resistant Klebsiella pneumoniae (MDR KP) bloodstream infections are a serious problem. The objective of this study was to investigate the effects of appropriate combination therapies on MDR KP bloodstream infections. METHODS: MDR KP strains isolated from clinical samples were assessed for antibiotic susceptibility using the broth microdilution method. Twenty consecutive MDR KP clinical isolates from patients with bloodstream infections were examined in this study. The experiments were conducted at the Bacterial Laboratory of Tongde Hospital from March to August 2021. Antibiotic combination tests were performed using the minimum inhibitory concentration (MIC) test, and the sum of the fractional inhibitory concentration was used to assess synergy. RESULTS: Following treatment with a combination of two antibiotic agents, the MIC50 and MIC90 values decreased compared with that before treatment. MIC50 decreased by at least 50%, with one value reduced to 6.25% of the pretreatment value. None of the antibiotic combinations were antagonistic. Combination of polymyxin B with rifampicin or tigecycline had a synergistic effect on 70% and 65% of the strains, respectively. CONCLUSIONS: In vitro combination therapies with two active drug agents (polymyxin B plus rifampicin or tigecycline) had a better effect on MDR KP infections compared with that in other regimens.

The High Expression of PTPRH Is Associated with Poor Prognosis of Human Lung Adenocarcinoma.

OBJECTIVE: The aim of the study is to explore the prognosis value of PTPRH in patients with lung adenocarcinoma (LUAD). METHODS: Oncomine, UALCAN, and GEPIA databases were employed to examine the differential expression of PTPRH between LUAD and adjacent tissues. 100 pairs of LUAD and adjacent tissue samples were involved in this study. qRT-PCR and immunohistochemical staining were performed. Meanwhile, we analyzed The Cancer Genome Atlas (TCGA) data to investigate the correlation between PTPRH gene expression and clinicopathological characteristics. Kaplan-Meier analysis and univariate and multivariate Cox analyses were performed to estimate the relationship between PTPRH expression and LUAD prognosis. The evaluation performance was verified by drawing a ROC curve. In addition, through GSEA, the changes of PTPRH expression were analyzed by GSEA to screen out primarily affected signaling pathway. RESULTS: Oncomine, UALCAN, and GEPIA databases showed that the mRNA expression of PTPRH in LUAD tissues was significantly higher than that in adjacent tissues. qRT-PCR and immunohistochemical staining indicated the mRNA and protein levels of PTPRH in LUAD tissues were markedly upregulated. TCGA data showed that the expression of PTPRH was significantly correlated with T stage and disease stage. Kaplan-Meier analysis showed that the patients with high PTPRH expression had a poor prognosis. Univariate and multivariate Cox analyses exhibited that PTPRH expression could act as an independent prognostic factor for LUAD. The ROC curve showed that PTPRH combined with various clinicopathological features could effectively predict the prognosis of LUAD. Finally, GSEA indicated that changes in PTPRH expression level may affect p53, VEGF, Notch, and mTOR cancer-related signaling pathways. CONCLUSION: Our results demonstrated that PTPRH was highly expressed in LUAD and may be closely correlated with the poor prognosis of LUAD patients.

Safflower injection inhibits pulmonary arterial remodeling in a monocrotaline-induced pulmonary arterial hypertension rat model.

Pulmonary arterial hypertension (PAH) is a group of diseases with an increase of pulmonary artery pressure (PAP) and pulmonary vascular resistance. Here, the effects of safflower injection, a preparation of Chinese herbs, was investigated in a monocrotaline (MCT)-induced PAH rat model. PAP, carotid artery pressure (CAP), and the right ventricular hypertrophy index (RVHI) increased in the PAH group, while safflower injection was able to inhibit this increase to similar levels as observed in the normal group. The arteriole wall of the lungs and cardiac muscle were thickened and edema was observed in the PAH group, while these pathologies were improved in the herb-treated group in a dose-dependent manner. MCT treatment induced proliferation of pulmonary artery smooth muscle cells (PASMCs), which was inhibited by safflower injection in a dose-dependent manner. Our experimental results demonstrated that safflower injection can regulate pulmonary arterial remodeling through affecting the expression of connective tissue growth factor, transforming growth factor-ß, integrin, collagen or fibronectin, which subsequently affected the thicknesses of the arteriole walls of the lungs and cardiac muscle, and thereby benefits the control of PAH. This means safflower injection improved the abnormalities in PAP, CAP and RVHI, and pulmonary arterial remodeling through regulation of remodeling factors.

Predictive value of inflammation-based Glasgow prognostic score, platelet-lymphocyte ratio, and global registry of acute coronary events score for major cardiovascular and cerebrovascular events during hospitalization in patients with acute myocardial infarction.

PURPOSE: The goal of this study was to evaluate the predictive ability of the inflammation-based Glasgow Prognostic Score (GPS), platelet-lymphocyte ratio (PLR), Global Registry of Acute Coronary Events (GRACE) score and combined diagnostic models for the occurrence of major adverse cardiovascular and cerebrovascular events (MACEs) in patients with acute myocardial infarction (AMI). METHODS: In this retrospective cohort study, eligible patients were required to meet the third global definition of myocardial infarction. The primary outcome of this study was the occurrence of MACEs during hospitalization. Receiver operating characteristic (ROC) curve analysis was performed to assess the predictive ability of the GPS, PLR, GRACE scores, and joint diagnostic models for primary outcomes; univariate and multivariate logistic regression analyses were performed. FINDINGS: A total of 175 patients were enrolled. The results of the univariate ROC curve analysis for the incidence of MACEs during hospitalization showed that the area under the curve (AUC) was 0.780 (95% confidence interval (CI) 0.696-0.864) for the GPS, 0.766 (95% CI 0.682-0.850) for the redefined GPS (RGPS), 0.561 (95% CI 0.458-0.664) for the PLR score (PLRS), and 0.793 (95% CI 0.706-0.880) for GRACE. Multivariate ROC curve analysis showed that the AUC value was 0.809 (95% CI 0.726-0.893) for the GPS combined with GRACE, 0.783 (95% CI 0.701-0.864) for the GPS combined with the PLRS, 0.794 (95% CI 0.707-0.880) for GRACE combined with the PLRS, and 0.841 (95% CI 0.761-0.921) for the GPS combined with GRACE and the PLRS. The combined diagnostic model including the GPS plus GRACE and the PLRS had a higher AUC value than the GPS, RGPS and GRACE models (P = 0.014, P = 0.004, and P = 0.038, respectively). The multivariate logistic regression model showed that the odds ratio for hospitalized MACEs was 5.573 (95% CI 1.588-19.554) for GPS scores of 2 versus 0, and the GRACE score was also an independent risk factor for MACEs, with an odds ratio of 1.023 (95% CI 1.009-1.036). IMPLICATIONS: The diagnostic model combining the GPS plus GRACE and the PLRS has better predictive ability for the occurrence of MACEs during hospitalization than each single score. Thus, the use of a combined GPS plus GRACE and PLRS model will be of clinical benefit in a broad group of individuals with AMI.

Surveillance of Multidrug-Resistant Bacterial Infections in Non-Adult Patients - Zhejiang Province, China, 2014-2019.

INTRODUCTION: Antimicrobial resistance has become a major public health threat globally. The prevalence of multidrug-resistant (MDR) bacterial infections increased substantially among inpatients under 18 years of age in recent years. In Zhejiang Province, China, the trends of drug-resistance in non-adult patients from 2014 to 2019 were monitored, aiming to determine the variation patterns and epidemiological features of MDR strains. METHODS: Patient data were collected from the Annual Review of Hospital Infection Resistance Survey in Zhejiang Province, 2014-2019. Statistical analysis was performed to analyze the pattern of distribution of five key bacterial pathogens in different age groups, ward settings, and bloodstream infections. RESULTS: From 2014 to 2019, a total of 30,163 multidrug-resistant strains were identified among 212,252 clinical isolates. The prevalence of extended spectrum ß-lactamase-producing Enterobacteriaceae (ESBL-E), carbapenem-resistant Enterobacteriaceae (CRE), carbapenem-resistant Acinetobacter baumannii, carbapenem-resistant Pseudomonas aeruginosa (CRPA), and methicillin-resistant Staphylococcus aureus (MRSA) were 40.6%, 2.3%, 14.7%, 9.0%, and 27.4%, respectively. The prevalence of these key pathogens was lower than that reported in the national surveillance system (China Antimicrobial Resistance Surveillance System and Infectious Diseases Surveillance of Pediatrics). The prevalence of ESBL-E and CRE decreased since 2015 but that of CRPA and MRSA increased from 2014 to 2018. CONCLUSIONS: Despite an overall decrease in the prevalence of drug-resistant bacteria in 2019, the rising prevalence of MRSA and CRPA still warrant much attention. Multidrug-resistant bacteria prevention and control strategies should be adjusted in a timely manner based on the surveillance results.

Epidemiology and risk factors of methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci infections in Zhejiang China from 2015 to 2017.

Background: Gram-positive bacteria are dangerous and challenging agents of infection due to their increasing resistance to antibiotics. We aim to analyse the epidemiology and risk factors of methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) in Zhejiang China. Methods: Gram-positive bacteria (including S. aureus, Enterococcus faecalis and Enterococcus faecium) were collected from eighty-six hospitals of eleven cities in Zhejiang China from 2015 to 2017. The detection rates of MRSA and VRE infection were calculated for the non-duplicated isolate according to year, region, hospital level, patient age, specimen type and patient category. Meanwhile, the detected resistances of MRSA, E. faecalis and E. faecium to different antibiotics from 2015 to 2017 were compared. The risk factors and the differences in MRSA and VRE detection rates were compared using odds ratio (OR) with 95% confidence interval (95% CI) and Chi-square test respectively. Results: From 2015 to 2017, the detection rates of MRSA and VRE decreased gradually. The cities with the highest MRSA and VRE detection rates tended to be adjacent; for example, the neighbouring cities Hangzhou and Quzhou had simultaneously high rates of MRSA and VRE infection. Patients from IIIA hospital who were older than 75 years and in the intensive care unit (ICU) were most at risk. No vancomycin-resistant isolate was found in MRSA. Resistance of E. faecalis and E. faecium to vancomycin and linezolid decreased slightly and then maintained a low level. Conclusions: The detection rates of MRSA and VRE stayed at moderate and low levels during the three year period of this study, while local dissemination was found in MRSA and VRE isolates. Sustained surveillance is necessary to prevent the spread or clonal dissemination of drug-resistant strains in Zhejiang China.

Leptospiral hemolysins induce proinflammatory cytokines through Toll-like receptor 2-and 4-mediated JNK and NF-κB signaling pathways.

BACKGROUND: Infection with pathogenic Leptospira species causes serious systemic inflammation in patients. Although a few leptospiral proinflammatory molecules have been identified, Leptospira likely encodes other unidentified strong inflammation stimulators. The pathogenic L. interrogans genome encodes numerous putative hemolysin genes. Since hemolysins from other bacteria can cause inflammatory reactions, we hypothesized that leptospiral hemolysins may function as proinflammatory stimulators that contribute to the strong inflammation associated with Leptospira infection. METHODOLOGY/PRINCIPAL FINDINGS: We first used cytokine protein microarrays for systematic analysis of serum cytokine profiles in leptospirosis patients and leptospire-infected mice. We found that IL-1ß, IL-6 and TNF-α were the main proinflammatory cytokines in the sera of both the patients and the mice. We then analyzed eight putative hemolysins in L. interrogans strain Lai. The results showed that five of them, Sph1, Sph2, Sph3, HlpA and TlyA were secreted and had hemolytic activity. More importantly, these five hemolysins induced the strong production of IL-1ß, IL-6 and TNF-α in human and mouse macrophages (although a bit lower in the latter). Furthermore, blockade of TLR2 or TLR4 with either antibodies or inhibitors of the NF-κB or JNK signaling pathways significantly reduced the production of hemolysin-induced IL-1ß, IL-6 and TNF-α. Macrophages isolated from TLR2-, TLR4-or double TLR2-and 4-deficient mice also confirmed that the leptospiral hemolysins that induce proinflammatory cytokines are both TLR2-and TLR4-dependent. CONCLUSIONS/SIGNIFICANCE: Our findings demonstrate that L. interrogans secretes many hemolysins that function as powerful inducers of proinflammatory cytokines through both TLR2-and TLR4-dependent JNK and NF-κB pathways.