Efficacy and safety of multikinase inhibitors for patients with refractory thyroid cancer: Systematic review and network meta-analysis.

CONTEXT: Multikinase inhibitors (MKIs) improve the treatment of refractory thyroid cancer, included radioactive iodine-refractory differentiated thyroid cancer (RAIR-DTC) and advanced medullary thyroid carcinoma (aMTC). OBJECTIVE: This study aims to compare the efficacy of MKIs in improving survival outcomes and safety. DATA SOURCES: Comprehensive database searches of MEDLINE via PubMed, EMBASE and Cochrane performed from inception to December 2023. STUDY SELECTION: Three independent authors selected these studies. Randomised-controlled trials that compared the use of a MKI to other MKIs or placebo were included. DATA EXTRACTION AND SYNTHESIS: This review followed PRISMA guidelines. Risk of bias was analyzed using the Cochrane RoB 2 tool. Bayesian network meta-analysis was performed. Treatments were grouped into common nodes based on the type of MKI. MAIN OUTCOMES AND MEASURES: Primary outcomes were progression-free survival (PFS) and overall survival (OS). Secondary outcomes included objective response rate, disease control rate, clinical benefit rate, and adverse events. RESULTS: Cabozantinib 60 mg/d (CAB60) was associated with the highest prolonged PFS in RAIR-DTC patients, followed by lentivatinib 18 or 24 mg/d (LEN18 or LEN24), and apatinib. PFS was also improved in in aMTC patients received CAB 140 mg/d (CAB140), CAB60, or anlotinib. A significantly greater improvement on the performance of OS was seen in CAB60, LEN24, anlotinib, and sorafenib in RAIR-DTC patients, but which in aMTC patients were lack of statistical differences. Compared with the low-dose of MKIs, high-dose of MKIs such as CAB, LEN, and vandetanib increased the incidence of adverse events. CONCLUSION: CAB60, LEN, and apatinib are promising topical MKIs with statistically significant primary outcomes in RAIR-DTC patients, while CAB and anlotinib are effective in prolonging PFS in aMTC patients.

68Ga-PSMA-11 PET and mpMRI in the diagnosis of initial lymph node staging of prostate cancer: a head-to-head comparative meta-analysis.

Purpose: This meta-analysis evaluates the comparative diagnostic efficacy of 68Ga-prostate-specific membrane antigen-11 PET (68Ga-PSMA-11 PET) and multiparametric MRI (mpMRI) for the initial lymph node staging of prostate cancer. Methods: We searched PubMed and Embase databases through October 2023 for studies that provide a head-to-head comparison of 68Ga-PSMA-11 PET and mpMRI, using pelvic lymph node dissection as the gold standard. We assessed sensitivity and specificity using the DerSimonian and Laird method, with variance stabilization via the Freeman-Tukey double inverse sine transformation. The quality of included studies was evaluated using the Quality Assessment of Diagnostic Performance Studies (QUADAS-2) tool. Results: The meta-analysis incorporated 13 articles, involving a total of 1,527 patients. 68Ga-PSMA-11 PET demonstrated an overall sensitivity of 0.73 (95% CI: 0.51-0.91) and a specificity of 0.94 (95% CI: 0.88-0.99). In comparison, mpMRI showed a sensitivity of 0.49 (95% CI: 0.30-0.68) and a specificity of 0.94 (95% CI: 0.88-0.99). Although 68Ga-PSMA-11 PET appeared to be more sensitive than mpMRI, the differences in sensitivity (p = 0.11) and specificity (p = 0.47) were not statistically significant. Conclusion: Our findings indicated that 68Ga-PSMA-11 PET and mpMRI exhibit similar sensitivity and specificity in the diagnosis of initial lymph node staging of prostate cancer. However, given that most included studies were retrospective, further prospective studies with larger sample sizes are essential to validate these results. Systematic Review Registration: PROSPERO code is CRD42023495266.

The synergy of 177Lu-FAPI-46 with tyrosine kinase inhibitor in a sarcoma patient-derived xenograft mouse model.

BACKGROUND: Given the heterogeneity and high mortality associated with metastatic soft tissue sarcoma, this study aims to evaluate the therapeutic efficacy of combining 177Lu-FAPI-46 with Pazopanib against this malignancy. METHODS: Patient-derived xenograft (PDX)-bearing mice were randomly divided into three groups: the control group, the 177Lu-FAPI-46 monotherapy group, and the 177Lu-FAPI-46 combined with Pazopanib therapy group. Therapeutic efficacy was regularly monitored. RESULTS: The microPET imaging showed a 0.84-fold decrease in the T/M ratio of 68Ga-FAPI-46 on day 7/8 post combination therapy, while the control group exhibited a 1.23-fold increase. Combination therapy significantly inhibited tumor proliferation, as evidenced by reduced Ki-67 and increased caspase 3 expressions. Notably, there was no significant body weight loss observed in any group. CONCLUSION: This study successfully demonstrated the reduction in FAP expression and suppression of tumor volume in sarcoma PDX following the combination therapy of 177Lu-FAPI-46 with Pazopanib.

A Comparative Study of Human Pluripotent Stem Cell-Derived Macrophages in Modeling Viral Infections.

Macrophages play multiple roles in innate immunity including phagocytosing pathogens, modulating the inflammatory response, presenting antigens, and recruiting other immune cells. Tissue-resident macrophages (TRMs) adapt to the local microenvironment and can exhibit different immune responses upon encountering distinct pathogens. In this study, we generated induced macrophages (iMACs) derived from human pluripotent stem cells (hPSCs) to investigate the interactions between the macrophages and various human pathogens, including the hepatitis C virus (HCV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and Streptococcus pneumoniae. iMACs can engulf all three pathogens. A comparison of the RNA-seq data of the iMACs encountering these pathogens revealed that the pathogens activated distinct gene networks related to viral response and inflammation in iMACs. Interestingly, in the presence of both HCV and host cells, iMACs upregulated different sets of genes involved in immune cell migration and chemotaxis. Finally, we constructed an image-based high-content analysis system consisting of iMACs, recombinant GFP-HCV, and hepatic cells to evaluate the effect of a chemical inhibitor on HCV infection. In summary, we developed a human cell-based in vitro model to study the macrophage response to human viral and bacterial infections; the results of the transcriptome analysis indicated that the iMACs were a useful resource for modeling pathogen-macrophage-tissue microenvironment interactions.

Ion channel TRPV2 is critical in enhancing B cell activation and function.

The function of transient receptor potential vanilloid (TRPV) cation channels governing B cell activation remains to be explored. We present evidence that TRPV2 is highly expressed in B cells and plays a crucial role in the formation of the B cell immunological synapse and B cell activation. Physiologically, TRPV2 expression level is positively correlated to influenza-specific antibody production and is low in newborns and seniors. Pathologically, a positive correlation is established between TRPV2 expression and the clinical manifestations of systemic lupus erythematosus (SLE) in adult and child SLE patients. Correspondingly, mice with deficient TRPV2 in B cells display impaired antibody responses following immunization. Mechanistically, the pore and N-terminal domains of TRPV2 are crucial for gating cation permeation and executing mechanosensation in B cells upon antigen stimulation. These processes synergistically contribute to membrane potential depolarization and cytoskeleton remodeling within the B cell immunological synapse, fostering efficient B cell activation. Thus, TRPV2 is critical in augmenting B cell activation and function.

In vivo functional immunoprotection correlates for vaccines against invasive bacteria.

Vaccination has significantly reduced the incidence of invasive infections caused by several bacterial pathogens, including Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria meningitidis. However, no vaccines are available for many other invasive pathogens. A major hurdle in vaccine development is the lack of functional markers to quantify vaccine immunity in eliminating pathogens during the process of infection. Based on our recent discovery of the liver as the major organ of vaccine-induced clearance of blood-borne virulent bacteria, we here describe a new vaccine evaluation system that quantitatively characterizes the key features of effective vaccines in shuffling virulent bacteria from the blood circulation to the liver resident macrophage Kupffer cells (KCs) and sinusoidal endothelial cells (LSECs) in mouse septic infection model. This system consists of three related correlates or assays: pathogen clearance from the bloodstream, pathogen trapping in the liver, and pathogen capture by KCs/LSECs. These readouts were consistently associated with the serotype-specific immunoprotection levels of the 13-valent pneumococcal polysaccharide conjugate vaccine (PCV13) against lethal infection of S. pneumoniae, a major invasive Gram-positive pathogen of community-acquired infections in humans. Furthermore, the reliability and sensitivity of these correlates in reflecting vaccine efficacy were verified with whole cell vaccines of Klebsiella pneumoniae and Escherichia coli, two major Gram-negative pathogens in hospital-acquired invasive infections. This system may be used as effective readouts to evaluate the immunoprotective potential of vaccine candidates in the preclinical phase by filling the current technical gap in vaccine evaluation between the conventional in vitro approaches (e.g. antibody production and pathogen neutralization/opsonophagocytosis) and survival of immunized animals.

Surgery for fibrosing mediastinitis with severe pulmonary hypertension due to pulmonary venous stenosis.

A 21-year-old woman with severe pulmonary hypertension and circulatory collapse was referred to our hospital for possible lung transplantation with extracorporeal membrane oxygenation support. Computed tomography revealed severe stenosis of all 4 pulmonary veins, and fibrosing mediastinitis was suspected. Surgical reconstruction of the pulmonary veins was performed, and extracorporeal membrane oxygenation support was weaned off. After surgery, pulmonary vascular resistance normalized. This successful case demonstrates that surgical pulmonary venous reconstruction is an important treatment for fibrosing mediastinitis induced by pulmonary venous stenosis and pulmonary hypertension.

Discussing the Overview of Typical Traditional Chinese Medicines in the Prevention and Treatment of Alzheimer's Disease Based on Pathogenesis of Amyloid β Protein / 医药导报

Alzheimer's disease(AD)is a disease with clinical manifestations of learning and memory impairment,cognitive dysfunction,and language dysfunction,the pathogenesis of AD is complex,of which Aβ theory covers various mechamisms such as oxidative stress,inflammation,apoptosis and other mechanisms.Based on the Aβ mechanism and related signaling pathways,this study discusses the overview of typical Chinese medicines and their active ingredients in the prevention and treatment of AD.The aim is to provide insights and references for the development of traditional Chinese medicines for the prevention and treatment of AD.

Multi-parametric MRI combined with 68Ga-PSMA PET/CT for the diagnosis of clinically significant prostate cancer / 中华核医学与分子影像杂志

Objective:To explore whether multi-parametric MRI (mpMRI) combined with 68Ga-prostate specific membrane antigen (PSMA) PET/CT can improve the detection efficiency of clinically significant prostate cancer (csPCa). Methods:Clinical and imaging data of 152 patients (age (68.5±8.5) years) who underwent mpMRI and 68Ga-PSMA PET/CT examination for suspected prostate cancer in the First Affiliated Hospital of the Air Force Medical University from January 2021 to November 2022 were retrospectively analyzed, with the histopathological results from transrectal ultrasound guided biopsy as reference. Lesions with Gleason scores (GS) ≥3+ 4 from the biopsy were diagnosed with csPCa, and lesions with negative biopsy or GS 6 were diagnosed with non-csPCa. MpMRI was evaluated independently by two radiologists according to the Prostate Imaging Reporting and Data System (PI-RADS) version 2.1. The radioactive uptake of 68Ga-PSMA PET/CT in prostate lesions was evaluated by SUV max. The independent-sample t test, Mann-Whitney U test and χ2 test were used to compare differences between the two groups, and then multivariate logistic regression analysis was performed. ROC curves analysis was used to analyze the diagnostic efficacies of individual and combined factors and Delong test was used. Results:There were 85 csPCa and 67 non-csPCa confirmed. Prostate specific antigen (PSA), PI-RADS score and SUV max were significantly different between the csPCa group and the non-csPCa group ( χ2=68.06, U values: -7.66, -8.98, all P<0.001). Multivariate logistic regression analysis indicated that PI-RADS score (odds ratio ( OR)=3.424, 95% CI: 1.651-7.100) and SUV max ( OR=1.931, 95% CI: 1.403-2.658) were independent predictors of csPCa (both P<0.001). ROC curves analysis revealed that the cut-off value for diagnosing csPCa was 4 for PI-RADS score and 5.6 for SUV max. The accuracy of mpMRI and PET/CT alone in csPCa diagnosis was 80%(122/152) (AUC of 0.789(95% CI: 0.711-0.866) with the sensitivity and specificity of 91%(77/85) and 67%(45/67)), and 87%(132/152) (AUC of 0.876(95% CI: 0.817-0.936) with the sensitivity and specificity of 81%(69/85) and 94%(63/67)), respectively. Several joint models incorporating 68Ga-PSMA PET/CT with mpMRI data were investigated, the model of PI-RADS 5 or PI-RADS 3-4 and SUV max>5.6 showed better performance than mpMRI and PET/CT alone and other joint models ( z values: 2.01-3.64, all P<0.05), with the accuracy of 91%(138/152) (AUC of 0.910(95% CI: 0.857-0.962) with the sensitivity and specificity of 89%(76/85) and 93%(62/67)). Conclusion:MpMRI combined with 68Ga-PSMA PET/CT can significantly improve the detection efficiency of csPCa, with the principal effect being improved in risk stratification of PI-RADS 3-4 lesions in mpMRI.

XEN drainage tube implantation combined with mitomycin C for open angle glaucoma / 国际眼科杂志(Guoji Yanke Zazhi)

AIM: To investigate the clinical efficacy and safety of XEN drainage tube implantation combined with mitomycin C(MMC)for open angle glaucoma(OAG).METHODS:A total of 37 OAG patients(37 eyes)were retrospectively included, grouped by anti-glaucoma surgical treatment as the first choice or not, with 17 patients(17 eyes)in the group with primary surgical treatment, and 20 patients(20 eyes)in the group with the numerous surgeries. The intraocular pressure(IOP), kinds of IOP-lowering drugs, and complications were collected and analyzed in 1 a follow-up postoperatively.RESULTS:Upon the one-year follow-up, IOP had decreased from 27.56±9.94, 28.43±14.18 mmHg to 15.16±3.65, 17.18±5.83 mmHg in both groups, respectively, representing a reduction of 55.01% and 60.43%, respectively(t=4.863, P&#x003C;0.001; t=3.255, P=0.004). The IOP at various follow up points were lower than preoperative points in both groups(Ftime=6.876, Ptime&#x003C;0.001; Fintergroup=0.242, Pintergroup=0.626; Ftime×intergroup=0.959, Ptime×intergroup=0.458). The complete success rate was 47% and 45%, the qualified success rate was 76% and 75%(Z=-0.115, P=0.909), respectively, and there was no significant difference in the cumulative survival rate between two groups(χ2=0.042, P=0.838; χ2=0.004, P=0.949). At the last follow up, IOP-lowering drugs were reduced from 3(2, 3)to 1(0, 2)in both groups(Z=-3.289, -3.796, all P&#x003C;0.001), and no significant difference between groups(Z=-0.581, P=0.561). Hypotony is the most common short-term complications, anterior chamber haemorrhage followed, while, filtering bleb encapsulation is the most frequent long-term complication, no serious complications occurred, but with XEN drainage tube exposure in 1 eye and drop in 1 eye.CONCLUSION:Initial XEN drainage tube implantation combined with MMC and numerous glaucoma surgeries are both safe and effective treatment for OAG patients, while the incidence of filtering bleb encapsulation is high in those with numerous glaucoma surgeries.

Diagnosis and treatment of prostate mucosa adenocarcinoma under multidisciplinary diagnosis and treatment mode: 2 cases report and literature review / 现代泌尿外科杂志

【Objective】 To explore the clinicopathological characteristics and comprehensive treatment strategies of prostate mucosa adenocarcinoma under multidisciplinary diagnosis and treatment (MDT) mode. 【Methods】 Data of two patients with typical prostate mucosa adenocarcinoma treated in our hospital during Sep.2020 and Apr.2023 were retrospectively analyzed. 【Results】 In case 1, the clinical manifestation was macroscopic hematuria; multiparametric magnetic resonance imaging (mpMRI) indicated solid prostatic nodules, clinical stage T4N1Mx; initial prostate specific antigen (PSA) was 1.2 ng/mL; 6868Ga-prostate specific membrane antigen PET/CT (68Ga-PSMA PET/CT) suggested abnormal uptake of nuclear lesions in the prostate (SUV4.2-5.3); biopsy results indicated invasive mucinous adenocarcinoma.After prostate and pelvic field radiotherapy + androgen deprivation therapy (ADT) + antihypertensive treatment, lesions were significantly reduced, and hematuria symptoms were relieved.In case 2, the clinical manifestation was dysuria; initial PSA was 91.78 ng/mL; mpMRI suggested invasion of prostate mass into the bladder and clinical stage of T4N1M1b; 68Ga-PSMA PET/CT indicated prostate and pelvic lymph nodes, and multiple bone lesions showed increased nuclide uptake; biopsy results indicated prostate adenocarcinoma with mucinous adenocarcinoma.Initial endocrine treatment was performed.After 3 months, PSA was reduced to 0.083 ng/mL, and imaging showed the tumor was significantly reduced.Robotic-assisted laparoscopic tumor prostatectomy with extended pelvic lymph node dissection was performed, and endocrine adjuvant therapy was continued after surgery. 【Conclusion】 Prostate mucosa adenocarcinoma has different clinicopathological characteristics and prognosis from conventional acinar adenocarcinoma, and the whole-process management under MDT mode is of great value in the diagnosis and treatment of this disease.

Growth Differentiation Factor 5 and Metabolic Diseases / 生物化学与生物物理进展

Growth/differentiation factor-5 (GDF-5) belongs to transforming growth factor-β (TGF-β) family, which is expressed in bone, cartilage, heart, brain, kidney, skeletal muscle and tendon, liver, fat and other organs and tissues as well. GDF-5 binds to receptor BMPR-I/BMPR-II and activates different signaling pathways such as smad1/5/8, PI3K/Akt, p38-MAPK. For a long time, numerous studies have shown that GDF-5 plays an important role in protecting joints. However, researchers have found GDF-5 also plays significant biological functions in other organs. For example, GDF-5 improves cardiac function by reducing oxidative stress and fibrosis in infarcted hearts. GDF-5 can also reduce oxidative stress in the brain and increase the number of neurons in effort to delay the progression of Alzheimer’s disease and Parkinson’s disease. It is a situation, research on GDF-5, at present, mainly focuses on the growth and repair of bone, cartilage and tendons, while there are few reports on its biological effects in other organs. Therefore, this article reviews and summarizes the research progress on GDF-5 and metabolic diseases in recent years in order to provide new insights and theoretical basis for the role of GDF-5 in improving metabolic diseases.

Liver macrophages and sinusoidal endothelial cells execute vaccine-elicited capture of invasive bacteria.

Vaccination has substantially reduced the morbidity and mortality of bacterial diseases, but mechanisms of vaccine-elicited pathogen clearance remain largely undefined. We report that vaccine-elicited immunity against invasive bacteria mainly operates in the liver. In contrast to the current paradigm that migrating phagocytes execute vaccine-elicited immunity against blood-borne pathogens, we found that invasive bacteria are captured and killed in the liver of vaccinated host via various immune mechanisms that depend on the protective potency of the vaccine. Vaccines with relatively lower degrees of protection only activated liver-resident macrophage Kupffer cells (KCs) by inducing pathogen-binding immunoglobulin M (IgM) or low amounts of IgG. IgG-coated pathogens were directly captured by KCs via multiple IgG receptors FcγRs, whereas IgM-opsonized bacteria were indirectly bound to KCs via complement receptors of immunoglobulin superfamily (CRIg) and complement receptor 3 (CR3) after complement C3 activation at the bacterial surface. Conversely, the more potent vaccines engaged both KCs and liver sinusoidal endothelial cells by inducing higher titers of functional IgG antibodies. Endothelial cells (ECs) captured densely IgG-opsonized pathogens by the low-affinity IgG receptor FcγRIIB in a "zipper-like" manner and achieved bacterial killing predominantly in the extracellular milieu via an undefined mechanism. KC- and endothelial cell-based capture of antibody-opsonized bacteria also occurred in FcγR-humanized mice. These vaccine protection mechanisms in the liver not only provide a comprehensive explanation for vaccine-/antibody-boosted immunity against invasive bacteria but also may serve as in vivo functional readouts of vaccine efficacy.

An anoikis-related gene signature predicts prognosis and reveals immune infiltration in hepatocellular carcinoma.

Background: Hepatocellular carcinoma (HCC) is a global health burden with poor prognosis. Anoikis, a novel programmed cell death, has a close interaction with metastasis and progression of cancer. In this study, we aimed to construct a novel bioinformatics model for evaluating the prognosis of HCC based on anoikis-related gene signatures as well as exploring the potential mechanisms. Materials and methods: We downloaded the RNA expression profiles and clinical data of liver hepatocellular carcinoma from TCGA database, ICGC database and GEO database. DEG analysis was performed using TCGA and verified in the GEO database. The anoikis-related risk score was developed via univariate Cox regression, LASSO Cox regression and multivariate Cox regression, which was then used to categorize patients into high- and low-risk groups. Then GO and KEGG enrichment analyses were performed to investigate the function between the two groups. CIBERSORT was used for determining the fractions of 22 immune cell types, while the ssGSEA analyses was used to estimate the differential immune cell infiltrations and related pathways. The "pRRophetic" R package was applied to predict the sensitivity of administering chemotherapeutic and targeted drugs. Results: A total of 49 anoikis-related DEGs in HCC were detected and 3 genes (EZH2, KIF18A and NQO1) were selected out to build a prognostic model. Furthermore, GO and KEGG functional enrichment analyses indicated that the difference in overall survival between risk groups was closely related to cell cycle pathway. Notably, further analyses found the frequency of tumor mutations, immune infiltration level and expression of immune checkpoints were significantly different between the two risk groups, and the results of the immunotherapy cohort showed that patients in the high-risk group have a better immune response. Additionally, the high-risk group was found to have higher sensitivity to 5-fluorouracil, doxorubicin and gemcitabine. Conclusion: The novel signature of 3 anoikis-related genes (EZH2, KIF18A and NQO1) can predict the prognosis of patients with HCC, and provide a revealing insight into personalized treatments in HCC.

Amino Acid Starvation-Induced Glutamine Accumulation Enhances Pneumococcal Survival.

Bacteria are known to cope with amino acid starvation by the stringent response signaling system, which is mediated by the accumulation of the (p)ppGpp alarmones when uncharged tRNAs stall at the ribosomal A site. While a number of metabolic processes have been shown to be regulatory targets of the stringent response in many bacteria, the global impact of amino acid starvation on bacterial metabolism remains obscure. This work reports the metabolomic profiling of the human pathogen Streptococcus pneumoniae under methionine starvation. Methionine limitation led to the massive overhaul of the pneumococcal metabolome. In particular, methionine-starved pneumococci showed a massive accumulation of many metabolites such as glutamine, glutamic acid, lactate, and cyclic AMP (cAMP). In the meantime, methionine-starved pneumococci showed a lower intracellular pH and prolonged survival. Isotope tracing revealed that pneumococci depend predominantly on amino acid uptake to replenish intracellular glutamine but cannot convert glutamine to methionine. Further genetic and biochemical analyses strongly suggested that glutamine is involved in the formation of a "prosurvival" metabolic state by maintaining an appropriate intracellular pH, which is accomplished by the enzymatic release of ammonia from glutamine. Methionine starvation-induced intracellular pH reduction and glutamine accumulation also occurred to various extents under the limitation of other amino acids. These findings have uncovered a new metabolic mechanism of bacterial adaptation to amino acid limitation and perhaps other stresses, which may be used as a potential therapeutic target for infection control. IMPORTANCE Bacteria are known to cope with amino acid starvation by halting growth and prolonging survival via the stringent response signaling system. Previous investigations have allowed us to understand how the stringent response regulates many aspects of macromolecule synthesis and catabolism, but how amino acid starvation promotes bacterial survival at the metabolic level remains largely unclear. This paper reports our systematic profiling of the methionine starvation-induced metabolome in S. pneumoniae. To the best of our knowledge, this represents the first reported bacterial metabolome under amino acid starvation. These data have revealed that the significant accumulation of glutamine and lactate enables S. pneumoniae to form a "prosurvival" metabolic state with a lower intracellular pH, which inhibits bacterial growth for prolonged survival. Our findings have provided insightful information on the metabolic mechanisms of pneumococcal adaptation to nutrient limitation during the colonization of the human upper airway.

Two-Component Response Regulator OmpR Regulates Mucoviscosity through Energy Metabolism in Klebsiella pneumoniae.

Hypermucoviscosity is a hallmark of hypervirulent Klebsiella pneumoniae (hvKP). However, the molecular basis of its regulation is largely unknown. We hypothesize that hypermucoviscosity is modulated via two-component signal transduction systems (TCSs). In-frame deletion mutants of all 33 response regulators of hvKP ATCC43816 were generated using CRISPR/CAS and evaluated for their impacts on hypermucoviscosity. The response regulator OmpR is required for hypermucoviscosity in vitro and virulence in vivo in a mouse pneumonia model. The ΔompR mutant lost its mucoidy but retained its capsule level and comparable rmpADC expression, so transcriptomic analysis by RNA-Seq was performed to identify differentially expressed genes (DEGs) in ΔompR mutant. The top 20 Gene Ontology terms of 273 DEGs belong to purine ribonucleotide triphosphate biosynthetic and metabolic process, transmembrane transport, and amino acid metabolism. Among the overexpressed genes in the ΔompR mutant, the atp operon encoding F-type ATP synthase and the gcvTHP encoding glycine cleavage system were characterized further as overexpression of either operon reduced the mucoviscosity and increased the production of ATP. Furthermore, OmpR directly bound the promoter region of the atp operon, not the gcvTHP, suggesting that OmpR regulates the expression of the atp operon directly and gcvTHP indirectly. Hence, the loss of OmpR led to the overexpression of F-type ATP synthase and glycine cleavage system, which altered the energetic status of ΔompR cells and contributed to the subsequent reduction in the mucoviscosity. Our study has uncovered a previously unknown regulation of bacterial metabolism by OmpR and its influence on hypermucoviscosity. IMPORTANCE Hypermucoviscosity is a critical virulent factor for Klebsiella pneumoniae infections, and its regulation remains poorly understood at the molecular level. This study aims to address this knowledge gap by investigating the role of response regulators in mediating hypermucoviscosity in K. pneumoniae. We screened 33 response regulators and found that OmpR is essential for hypermucoviscosity and virulence of K. pneumoniae in a mouse pneumonia model. Transcriptomic analysis uncovered that genes involved in energy production and metabolism are highly upregulated in the ΔompR mutant, suggesting a potential link between bacterial energy status and hypermucoviscosity. Overexpression of those genes increased production of ATP and reduced mucoviscosity, recapitulating the ΔompR mutant phenotype. Our findings provide new insights into the regulation of K. pneumoniae hypermucoviscosity by a two-component signal transduction system, highlighting the previously unknown role of OmpR in regulating bacterial energy status and its influence on hypermucoviscosity.

Identification of the mouse Kupffer cell receptors recognizing pneumococcal capsules by affinity screening.

Kupffer cells (KCs) are the major sentinels to guard the bloodstream by recognizing diverse microbial ligands of blood-borne pathogens. Here, we establish a protocol for identifying the KC receptors recognizing the capsular polysaccharides (CPSs) of low-virulence Streptococcus pneumoniae in a mouse model. This protocol includes preparation of CPS-coated microspheres and KC membrane proteins, affinity pulldown of CPS-binding proteins, and functional validation of the CPS receptors. This protocol provides a platform to investigate the receptor-ligand interactions between KCs and encapsulated bacteria. For complete details on the use and execution of this protocol, please refer to An et al. (2022).1.

Transforming growth factor-ß1 attenuates inflammation and lung injury with regulating immune function in ventilator-induced lung injury mice.

Ventilator-induced lung injury (VILI) is a lung injury induced or aggravated by mechanical ventilation. Transforming growth factor (TGF)-ß1 is a cytokine that mediates immune function, enabling inflammatory attenuation and tissue repair. Here, we hypothesized that it plays an important role in the attenuation of VILI and inflammation. Ventilation with high tidal volume was performed on C57BL/6 mice to establish a VILI model. After 4 h of ventilation, mice were sacrificed (end of ventilation [EOV]) or extubated for resuscitation at 4 h (post-ventilation 4 h [PV4h]), 8 h (PV8h) and 24 h post-ventilation (PV1d). Recombinant mouse TGF-ß1 (rTGF-ß1) and the neutralization antibody of TGF-ß1 (nTAb) were used in vivo to examine the effect of TGF-ß1 on immune function and inflammatory attenuation in VILI mice. Lung injury was exacerbated at the same trend as the interleukin (IL)-1ß level, peaking at PV1d, whereas IL-6 and tumor necrosis factor (TNF)-α levels gradually reduced. Most active phagosomes, swollen round mitochondria, and cavitating lamellar bodies were observed at PV4h. The CD4+ T cells were significantly increased from PV4h to PV1d, and the CD8a + T cells were higher in the PV4h and PV1d groups; furthermore, the mice in the PV8h group showed highest proportion of CD4+CD8a+ T cells and CD4+/CD8a+ ratio. CD19 + and CD5 + CD19 + B cells in VILI mice began to increase at PV1d. The pulmonary expression of latent and monomer TGF-ß1 increased at PV4h and PV8h. Treatment of rTGF-ß1 only induced high expression of latent and monomer TGF-ß1 at EOV to decrease pulmonary levels of IL-1ß, IL-6, and TNF-α; however, lung injury attenuated from EOV to PV1d. TGF-ß1 induced the delayed elevation of CD4+/CD8a+ T cells ratio and activation of pulmonary CD4+CD8a+ double-positive T cells under certain conditions. Elastic fibers and celluloses, although relatively less proteoglycan, were observed with the overexpression of TGF-ß1 at PV4h and PV8h. In conclusion, TGF-ß1 attenuates the inflammatory response and lung injury of VILI via immune function regulation.

Inhibition of PD-1 Alters the SHP1/2-PI3K/Akt Axis to Decrease M1 Polarization of Alveolar Macrophages in Lung Ischemia-Reperfusion Injury.

Polarization of alveolar macrophages (AMs) into the M1 phenotype contributes to inflammatory responses and tissue damage that occur during lung ischemia-reperfusion injury (LIRI). Programmed cell death factor-1 (PD-1) regulates polarization of macrophages, but its role in LIRI is unknown. We examined the role of PD-1 in AM polarization in models of LIRI in vivo and in vitro. Adult Sprague-Dawley rats were subjected to ischemia-reperfusion with or without pretreatment with a PD-1 inhibitor, SHP1/2 inhibitor, or Akt activator. Lung tissue damage and infiltration by M1-type AMs were assessed. As an in vitro complement to the animal studies, rat alveolar macrophages in culture were subjected to oxygen/glucose deprivation and reoxygenation. Levels of SHP1/2 and Akt proteins were evaluated using Western blots, while levels of pro-inflammatory cytokines were measured using enzyme-linked immunosorbent assays. Injury upregulated PD-1 both in vivo and in vitro. Inhibiting PD-1 reduced the number of M1-type AMs, expression of SHP1 and SHP2, and levels of inflammatory cytokines. At the same time, it partially restored Akt activation. Similar results were observed after inhibition of SHP1/2 or activation of the PI3K/Akt pathway. PD-1 promotes polarization of AMs to the M1 phenotype and inflammatory responses through the SHP1/2-PI3K/Akt axis. Inhibiting PD-1 may be an effective therapeutic strategy to limit LIRI.