Innate Lymphocyte Mechanisms in Skin Diseases.

Innate lymphocyte populations are emerging as key effectors in tissue homeostasis, microbial defense, and inflammatory skin disease. The cells are evolutionarily ancient and carry conserved principles of function, which can be achieved through shared or unique specific mechanisms. Recent technological and treatment advances have provided insight into heterogeneity within and between individuals and species. Similar pathways can extend through to adaptive lymphocytes, which softens the margins with innate lymphocyte populations and allows investigation of nonredundant pathways of immunity and inflammation that might be amenable to therapeutic intervention. Here, we review advances in understanding of innate lymphocyte biology with a focus on skin disease and the roles of commensal and pathogen responses and tissue homeostasis.

CD1 lipidomes reveal lipid-binding motifs and size-based antigen-display mechanisms.

The CD1 system binds lipid antigens for display to T cells. Here, we solved lipidomes for the four human CD1 antigen-presenting molecules, providing a map of self-lipid display. Answering a basic question, the detection of >2,000 CD1-lipid complexes demonstrates broad presentation of self-sphingolipids and phospholipids. Whereas peptide antigens are chemically processed, many lipids are presented in an unaltered form. However, each type of CD1 protein differentially edits the self-lipidome to show distinct capture motifs based on lipid length and chemical composition, suggesting general antigen display mechanisms. For CD1a and CD1d, lipid size matches the CD1 cleft volume. CD1c cleft size is more variable, and CD1b is the outlier, where ligands and clefts show an extreme size mismatch that is explained by uniformly seating two small lipids in one cleft. Furthermore, the list of compounds that comprise the integrated CD1 lipidome supports the ongoing discovery of lipid blockers and antigens for T cells.

Unconventional human CD61 pairing with CD103 promotes TCR signaling and antigen-specific T cell cytotoxicity.

Cancer remains one of the leading causes of mortality worldwide, leading to increased interest in utilizing immunotherapy strategies for better cancer treatments. In the past decade, CD103+ T cells have been associated with better clinical prognosis in patients with cancer. However, the specific immune mechanisms contributing toward CD103-mediated protective immunity remain unclear. Here, we show an unexpected and transient CD61 expression, which is paired with CD103 at the synaptic microclusters of T cells. CD61 colocalization with the T cell antigen receptor further modulates downstream T cell antigen receptor signaling, improving antitumor cytotoxicity and promoting physiological control of tumor growth. Clinically, the presence of CD61+ tumor-infiltrating T lymphocytes is associated with improved clinical outcomes, mediated through enhanced effector functions and phenotype with limited evidence of cellular exhaustion. In conclusion, this study identified an unconventional and transient CD61 expression and pairing with CD103 on human immune cells, which potentiates a new target for immune-based cellular therapies.

Staphylococcal phosphatidylglycerol antigens activate human T cells via CD1a.

Expressed on epidermal Langerhans cells, CD1a presents a range of self-lipid antigens found within the skin; however, the extent to which CD1a presents microbial ligands from bacteria colonizing the skin is unclear. Here we identified CD1a-dependent T cell responses to phosphatidylglycerol (PG), a ubiquitous bacterial membrane phospholipid, as well as to lysylPG, a modified PG, present in several Gram-positive bacteria and highly abundant in Staphylococcus aureus. The crystal structure of the CD1a-PG complex showed that the acyl chains were buried within the A'- and F'-pockets of CD1a, while the phosphoglycerol headgroup remained solvent exposed in the F'-portal and was available for T cell receptor contact. Using lysylPG and PG-loaded CD1a tetramers, we identified T cells in peripheral blood and in skin that respond to these lipids in a dose-dependent manner. Tetramer+CD4+ T cell lines secreted type 2 helper T cell cytokines in response to phosphatidylglycerols as well as to co-cultures of CD1a+ dendritic cells and Staphylococcus bacteria. The expansion in patients with atopic dermatitis of CD4+ CD1a-(lysyl)PG tetramer+ T cells suggests a response to lipids made by bacteria associated with atopic dermatitis and provides a link supporting involvement of PG-based lipid-activated T cells in atopic dermatitis pathogenesis.

Broad and strong memory CD4+ and CD8+ T cells induced by SARS-CoV-2 in UK convalescent individuals following COVID-19.

The development of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines and therapeutics will depend on understanding viral immunity. We studied T cell memory in 42 patients following recovery from COVID-19 (28 with mild disease and 14 with severe disease) and 16 unexposed donors, using interferon-γ-based assays with peptides spanning SARS-CoV-2 except ORF1. The breadth and magnitude of T cell responses were significantly higher in severe as compared with mild cases. Total and spike-specific T cell responses correlated with spike-specific antibody responses. We identified 41 peptides containing CD4+ and/or CD8+ epitopes, including six immunodominant regions. Six optimized CD8+ epitopes were defined, with peptide-MHC pentamer-positive cells displaying the central and effector memory phenotype. In mild cases, higher proportions of SARS-CoV-2-specific CD8+ T cells were observed. The identification of T cell responses associated with milder disease will support an understanding of protective immunity and highlights the potential of including non-spike proteins within future COVID-19 vaccine design.

Orphan nuclear receptors-induced ALT-associated PML bodies are targets for ALT inhibition.

Orphan nuclear receptors (NRs), such as COUP-TF1, COUP-TF2, EAR2, TR2 and TR4, are implicated in telomerase-negative cancers that maintain their telomeres through the alternative lengthening of telomeres (ALT) mechanism. However, how telomere association of orphan NRs is involved in ALT activation remains unclear. Here, we demonstrate that telomeric tethering of orphan NRs in human fibroblasts initiates formation of ALT-associated PML bodies (APBs) and features of ALT activity, including ALT telomere DNA synthesis, telomere sister chromatid exchange, and telomeric C-circle generation, suggesting de novo ALT induction. Overexpression of orphan NRs exacerbates ALT phenotypes in ALT cells, while their depletion limits ALT. Orphan NRs initiate ALT via the zinc finger protein 827, suggesting the involvement of chromatin structure alterations for ALT activation. Furthermore, we found that orphan NRs and deficiency of the ALT suppressor ATRX-DAXX complex operate in concert to promote ALT activation. Moreover, PML depletion by gene knockout or arsenic trioxide treatment inhibited ALT induction in fibroblasts and ALT cancer cells, suggesting that APB formation underlies the orphan NR-induced ALT activation. Importantly, arsenic trioxide administration abolished APB formation and features of ALT activity in ALT cancer cell line-derived mouse xenografts, suggesting its potential for further therapeutic development to treat ALT cancers.

The levels of soluble CD137 are increased in tuberculosis patients and associated with disease severity and prognosis.

Tuberculosis (TB) was the leading cause of death from a single infectious agent before the coronavirus pandemic. Therefore, it is important to search for severity biomarkers and devise appropriate therapies. A total of 139 pulmonary TB (PTB) patients and 80 healthy controls (HCs) were recruited for plasma soluble CD137 (sCD137) detection through ELISA. Moreover, pleural effusion sCD137 levels were measured in 85 TB patients and 36 untreated lung cancer patients. The plasma cytokine levels in 64 patients with PTB and blood immune cell subpopulations in 68 patients with PTB were analysed via flow cytometry. Blood sCD137 levels were higher in PTB patients (p = 0.012) and correlated with disease severity (p = 0.0056). The level of sCD137 in tuberculous pleurisy effusion (TPE) was markedly higher than that in malignant pleurisy effusion (p = 0.018). Several blood cytokines, such as IL-6 (p = 0.0147), IL-8 (p = 0.0477), IP-10 (p ≤ 0.0001) and MCP-1 (p = 0.0057), and some laboratory indices were significantly elevated in severe PTB (SE) patients, but the percentages of total lymphocytes (p = 0.002) and cytotoxic T cells (p = 0.036) were significantly lower in SE patients than in non-SE patients. In addition, the sCD137 level was negatively correlated with the percentage of total lymphocytes (p = 0.0008) and cytotoxic T cells (p = 0.0021), and PTB patients with higher plasma sCD137 levels had significantly shorter survival times (p = 0.0041). An increase in sCD137 is a potential biomarker for severe TB and indicates a poor prognosis.

Hydroxytryptophan biosynthesis by a family of heme-dependent enzymes in bacteria.

Hydroxytryptophan serves as a chemical precursor to a variety of bioactive specialized metabolites, including the human neurotransmitter serotonin and the hormone melatonin. Although the human and animal routes to hydroxytryptophan have been known for decades, how bacteria catalyze tryptophan indole hydroxylation remains a mystery. Here we report a class of tryptophan hydroxylases that are involved in various bacterial metabolic pathways. These enzymes utilize a histidine-ligated heme cofactor and molecular oxygen or hydrogen peroxide to catalyze regioselective hydroxylation on the tryptophan indole moiety, which is mechanistically distinct from their animal counterparts from the nonheme iron enzyme family. Through genome mining, we also identify members that can hydroxylate the tryptophan indole ring at alternative positions. Our results not only reveal a conserved way to synthesize hydroxytryptophans in bacteria but also provide a valuable enzyme toolbox for biocatalysis. As proof of concept, we assemble a highly efficient pathway for melatonin in a bacterial host.

Sodium-Glucose Cotransporter-2 Inhibitors and the Risk for Dialysis and Cardiovascular Disease in Patients With Stage 5 Chronic Kidney Disease.

BACKGROUND: No studies have reported the long-term outcomes of initiating sodium-glucose cotransporter-2 inhibitors (SGLT2is) in patients with estimated glomerular filtration rates less than 20 mL/min/1.73 m2 to predialysis. OBJECTIVE: To compare the risk for dialysis, cardiovascular events, and death between SGLT2i users and nonusers in patients with type 2 diabetes (T2D) and stage 5 chronic kidney disease (CKD). DESIGN: Target trial emulation study. SETTING: Taiwan's National Health Insurance Research Database (NHIRD). PARTICIPANTS: By applying sequential target trial emulation principle, 23 854 SGLT2i users and 23 892 SGLT2i nonusers were selected from the NHIRD for patients with T2D and stage 5 CKD from 1 May 2016 to 31 October 2021. MEASUREMENTS: Conditional Cox proportional hazards models were used to compare the risks for dialysis, hospitalization for heart failure, acute myocardial infarction (AMI), diabetic ketoacidosis (DKA), acute kidney injury (AKI), and all-cause mortality between SGLT2i users and nonusers. RESULTS: In the intention-to-treat model, compared with no SGLT2i use, SGLT2i use was associated with lower risks for dialysis (hazard ratio [HR], 0.34 [95% CI, 0.27 to 0.43]), hospitalization for heart failure (HR, 0.80 [CI, 0.73 to 0.86]), AMI (HR, 0.61 [CI, 0.52 to 0.73]), DKA (HR, 0.78 [CI, 0.71 to 0.85]), and AKI (HR, 0.80 [CI, 0.70 to 0.90]), but there was no difference in the risk for all-cause mortality (HR, 1.11 [CI, 0.99 to 1.24]). The Kaplan-Meier curves and subgroup analyses also showed that initiation of an SGLT2i in stage 5 CKD was associated with a lower risk for long-term dialysis than no SGLT2i use. LIMITATION: This result may not apply to patients without T2D. CONCLUSION: This emulated target trial showed that SGLT2i use was associated with a lower risk for dialysis, cardiovascular events, DKA, and AKI than no SGLT2i use in patients with T2D and stage 5 CKD. PRIMARY FUNDING SOURCE: National Health Research Institutes, Taiwan.

Discovery of a Bacterial Hydrazine Transferase That Constructs the N-Aminolactam Pharmacophore in Albofungin Biosynthesis.

Structural motifs containing nitrogen-nitrogen (N-N) bonds are prevalent in a large number of clinical drugs and bioactive natural products. Hydrazine (N2H4) serves as a widely utilized building block for the preparation of these N-N-containing molecules in organic synthesis. Despite its common use in chemical processes, no enzyme has been identified to catalyze the incorporation of free hydrazine in natural product biosynthesis. Here, we report that a hydrazine transferase catalyzes the condensation of N2H4 and an aromatic polyketide pathway intermediate, leading to the formation of a rare N-aminolactam pharmacophore in the biosynthesis of broad-spectrum antibiotic albofungin. These results expand the current knowledge on the biosynthetic mechanism for natural products with N-N units and should facilitate future development of biocatalysts for the production of N-N-containing chemicals.

Newly identified disorder of copper metabolism caused by variants in CTR1, a high-affinity copper transporter.

The high-affinity copper transporter CTR1 is encoded by CTR1 (SLC31A1), a gene locus for which no detailed genotype-phenotype correlations have previously been reported. We describe identical twin male infants homozygous for a novel missense variant NM_001859.4:c.284 G > A (p.Arg95His) in CTR1 with a distinctive autosomal recessive syndrome of infantile seizures and neurodegeneration, consistent with profound central nervous system copper deficiency. We used clinical, biochemical and molecular methods to delineate the first recognized examples of human CTR1 deficiency. These included clinical phenotyping, brain imaging, assays for copper, cytochrome c oxidase (CCO), and mitochondrial respiration, western blotting, cell transfection experiments, confocal and electron microscopy, protein structure modeling and fetal brain and cerebral organoid CTR1 transcriptome analyses. Comparison with two other critical mediators of cellular copper homeostasis, ATP7A and ATP7B, genes associated with Menkes disease and Wilson disease, respectively, revealed that expression of CTR1 was highest. Transcriptome analyses identified excitatory neurons and radial glia as brain cell types particularly enriched for copper transporter transcripts. We also assessed the effects of Copper Histidinate in the patients' cultured cells and in the patients, under a formal clinical protocol. Treatment normalized CCO activity and enhanced mitochondrial respiration in vitro, and was associated with modest clinical improvements. In combination with present and prior studies, these infants' clinical, biochemical and molecular phenotypes establish the impact of this novel variant on copper metabolism and cellular homeostasis and illuminate a crucial role for CTR1 in human brain development. CTR1 deficiency represents a newly defined inherited disorder of brain copper metabolism.

Phasic dopamine signals are reduced in the spontaneously hypertensive rat and increased by methylphenidate.

The spontaneously hypertensive rat (SHR) is a selectively bred animal strain that is frequently used to model attention-deficit hyperactivity disorder (ADHD) because of certain genetically determined behavioural characteristics. To test the hypothesis that the characteristically altered response to positive reinforcement in SHRs may be due to altered phasic dopamine response to reward, we measured phasic dopamine signals in the SHRs and Sprague Dawley (SD) rats using in vivo fast-scan cyclic voltammetry. The effects of the dopamine reuptake inhibitor, methylphenidate, on these signals were also studied. Phasic dopamine signals during the pairing of a sensory cue with electrical stimulation of midbrain dopamine neurons were significantly smaller in the SHRs than in the SD rats. Over repeated pairings, the dopamine response to the sensory cue increased, whereas the response to the electrical stimulation of dopamine neurons decreased, similarly in both strains. However, the final amplitude of the response to the sensory cue after pairing was significantly smaller in SHRs than in the SD rats. Methylphenidate increased responses to sensory cues to a significantly greater extent in the SHRs than in the SD rats, due largely to differences in the low dose effect. At a higher dose, methylphenidate increased responses to sensory cues and electrical stimulation similarly in SHRs and SD rats. The smaller dopamine responses may explain the reduced salience of reward-predicting cues previously reported in the SHR, whereas the action of methylphenidate on the cue response suggests a potential mechanism for the therapeutic effects of low-dose methylphenidate in ADHD.

Costimulatory capacity of CD137 mAbs on T cells depends on elaborate CRD structures but not on blocking ligand-receptor binding.

CD137 is mainly a costimulatory receptor of CD8+ T cells. Two representative CD137 antibodies, utomilumab, and urelumab, show different costimulatory capacities in clinical trials. Balancing the antitumor effect and systemic toxicity of T cells activated by CD137 signaling is a challenge that requires clinical consideration. In this study, a panel of specific anti-human CD137 monoclonal antibodies (mAbs) were prepared and their affinities, isotypes, CD137-CRD (cysteine-rich domain) binding regions, cross-reactivity to mouse and rhesus CD137, inhibition of ligand-receptor binding and costimulatory activities were analyzed. The results showed that anti-human CD137 mAbs had high cross-reactivity with rhesus CD137. MAbs fell into three clusters according to their different binding regions of the CD137 extracellular domain. They bound to CRDI+CRDII, CRDIII or CRDIV+STP. CRDIII-binding mAbs had the strongest blocking activity. Highly costimulatory CD137 mAbs showed stronger abilities to promote CD8+ T-cell proliferation. However, the costimulatory capacity of mAbs on T cells was not closely related to their ability to block CD137L-CD137 binding and may be controlled by more elaborate CRD conformational structures. This study provides additional information for the development of next-generation CD137 mAbs to meet clinical needs.

T-cell subsets and cytokines are indicative of neoadjuvant chemoimmunotherapy responses in NSCLC.

PURPOSE: Neoadjuvant PD-1 blockade combined with chemotherapy is a promising treatment for resectable non-small cell lung cancer (NSCLC), yet the immunological mechanisms contributing to tumor regression and biomarkers corresponding to different pathological responses remain unclear. METHODS: Using dynamic and paired blood samples from NSCLC patients receiving neoadjuvant chemoimmunotherapy, we analyzed the frequencies of CD8 + T-cell and Treg subsets and their dynamic changes during neoadjuvant treatment through flow cytometry. Cytokine profiles and function-related gene expression of CD8 + T cells and Tregs were analyzed through flow cytometry and mRNA-seq. Infiltrating T-cell subsets in resected tissues from patients with different pathological responses were analyzed through multiplex immunofluorescence. RESULTS: Forty-two NSCLC patients receiving neoadjuvant chemoimmunotherapy were enrolled and then underwent surgical resection and pathological evaluation. Nineteen patients had pCR (45%), 7 patients had MPR (17%), and 16 patients had non-MPR (38%). In patients with pCR, the frequencies of CD137 + CD8 + T cells (P = 0.0475), PD-1 + Ki-67 + CD8 + T cells (P = 0.0261) and Tregs (P = 0.0317) were significantly different from those of non-pCR patients before treatment. pCR patients usually had low frequencies of CD137 + CD8 + T cells, PD-1 + Ki-67 + CD8 + T cells and Tregs, and their AUCs were higher than that of tissue PD-L1 expression. Neoadjuvant chemoimmunotherapy markedly improved CD8 + T-cell proliferation and activation, especially in pCR patients, as the frequencies of CD137 + CD8 + (P = 0.0136) and Ki-67 + CD8 + (P = 0.0391) T cells were significantly increased. The blood levels of cytokines such as IL-2 (P = 0.0391) and CXCL10 (P = 0.0195) were also significantly increased in the pCR group, which is consistent with the high density of activated cytotoxic T cells at the tumor site (P < 0.0001). CONCLUSION: Neoadjuvant chemoimmunotherapy drives CD8 + T cells toward a proliferative and active profile. The frequencies of CD137 + CD8 + T cells, PD-1 + Ki-67 + CD8 + T cells and Tregs at baseline might predict the response to neoadjuvant chemoimmunotherapy in NSCLC patients. The increase in IL-2 and CXCL10 might reflect the chemotaxis and enrichment of cytotoxic T cells at the tumor site and a better response to neoadjuvant chemoimmunotherapy.

Passive immunotherapy for Alzheimer's disease: challenges & future directions.

Passive immunotherapy with specific antibodies targeting Amyloid ß (Aß) peptide or tubulin-associated unit (tau) protein has emerged as a promising therapeutic approach in Alzheimer's disease (AD). However, in a recent phase III clinical study, Sperling et al. (N Engl J Med 10.1056/NEJMoa2305032, 2023) reported that solanezumab, a monoclonal antibody targeting Aß peptide, failed to slow cognitive decline in AD patients. Previously, three other anti-Aß antibodies, bapineuzumab, crenezumab, and gantenerumab, have also failed to show similar beneficial effects. In addition, three humanized antibodies targeting tau protein failed in their phase II trials. However, other anti-Aß antibodies, such as lecanemab (a humanized mAb binds to soluble Aß protofibrils), donanemab (a humanized mAb binds to insoluble, N-terminal truncated form of Aß peptides) and aducanumab (a human mAb binds to the aggregated form of Aß), have been shown to slow the decline of cognitive functions in early stage AD patients. The specific targets used in passive immunotherapy in these clinical trials may explain the divergent clinical outcomes. There are several challenges and limitations of passive immunotherapy using anti-Aß antibodies and long term longitudinal studies are needed to assess their efficacy, side effects and cost effectiveness in a wider spectrum of subjects, from pre-dementia to more advanced dementia. A combination therapeutic approach using both anti-Aß antibodies and other pharmaceutical agents should also be explored.

Therapeutic efficacy of humanized monoclonal antibodies targeting dengue virus nonstructural protein 1 in the mouse model.

Dengue virus (DENV) which infects about 390 million people per year in tropical and subtropical areas manifests various disease symptoms, ranging from fever to life-threatening hemorrhage and even shock. To date, there is still no effective treatment for DENV disease, but only supportive care. DENV nonstructural protein 1 (NS1) has been shown to play a key role in disease pathogenesis. Recent studies have shown that anti-DENV NS1 antibody can provide disease protection by blocking the DENV-induced disruption of endothelial integrity. We previously demonstrated that anti-NS1 monoclonal antibody (mAb) protected mice from all four serotypes of DENV challenge. Here, we generated humanized anti-NS1 mAbs and transferred them to mice after DENV infection. The results showed that DENV-induced prolonged bleeding time and skin hemorrhage were reduced, even several days after DENV challenge. Mechanistic studies showed the ability of humanized anti-NS1 mAbs to inhibit NS1-induced vascular hyperpermeability and to elicit Fcγ-dependent complement-mediated cytolysis as well as antibody-dependent cellular cytotoxicity of cells infected with four serotypes of DENV. These results highlight humanized anti-NS1 mAb as a potential therapeutic agent in DENV infection.

Sanitization of hydroponic farming facilities in Singapore: what, why, and how.

This study performed microbial analysis of nutrient film technique (NFT) hydroponic systems on three indoor farms in Singapore (the "what"). To justify the necessity of sanitizing hydroponic systems, strong biofilm-forming bacteria were isolated from the facility and investigated for their influence on Salmonella colonization on polyvinyl chloride (PVC) coupons in hydroponic nutrient solutions (the "why"). Finally, sanitization solutions were evaluated with both laboratory-scale and field-scale tests (the "how"). As a result, the microbiome composition in NFT systems was found to be highly farm specific. The strong biofilm formers Corynebacterium tuberculostearicum C2 and Pseudoxanthomonas mexicana C3 were found to facilitate the attachment and colonization of Salmonella on PVC coupons. When forming dual-species biofilms, the presence of C2 and C3 also significantly promoted the growth of Salmonella (P < 0.05). Compared with hydrogen peroxide (H2O2) and sodium percarbonate (SPC), sodium hypochlorite (NaOCl) exhibited superior efficacy in biofilm removal. At 50 ppm, NaOCl reduced the Salmonella Typhimurium, C2, and C3 counts to <1 log CFU/cm2 within 12 h, whereas neither 3% H2O2 nor 1% SPC achieved this effect. In operational hydroponic systems, the concentration of NaOCl needed to achieve biofilm elimination increased to 500 ppm, likely due to the presence of organic matter accumulated during crop cultivation and the greater persistence of naturally formed multispecies biofilms. Sanitization using 500 ppm NaOCl for 12 h did not impede subsequent plant growth, but chlorination byproduct chlorate was detected at high levels in the hydroponic solution and in plants in the sanitized systems without rinsing. IMPORTANCE: This study's significance lies first in its elucidation of the necessity of sanitizing hydroponic farming systems. The microbiome in hydroponic systems, although mostly nonpathogenic, might serve as a hotbed for pathogen colonization and thus pose a risk for food safety. We thus explored sanitization solutions with both laboratory-scale and field-scale tests. Of the three tested sanitizers, NaOCl was the most effective and economical option, whereas one must note the vital importance of rinsing the hydroponic systems after sanitization with NaOCl.

Enhancement of NETosis by ACE2-cross-reactive anti-SARS-CoV-2 RBD antibodies in patients with COVID-19.

BACKGROUND: High levels of neutrophil extracellular trap (NET) formation or NETosis and autoantibodies are related to poor prognosis and disease severity of COVID-19 patients. Human angiotensin-converting enzyme 2 (ACE2) cross-reactive anti-severe acute respiratory syndrome coronavirus 2 spike protein receptor-binding domain (SARS-CoV-2 RBD) antibodies (CR Abs) have been reported as one of the sources of anti-ACE2 autoantibodies. However, the pathological implications of CR Abs in NET formation remain unknown. METHODS: In this study, we first assessed the presence of CR Abs in the sera of COVID-19 patients with different severity by serological analysis. Sera and purified IgG from CR Abs positive COVID-19 patients as well as a mouse monoclonal Ab (mAb 127) that can recognize both ACE2 and the RBD were tested for their influence on NETosis and the possible mechanisms involved were studied. RESULTS: An association between CR Abs levels and the severity of COVID-19 in 120 patients was found. The CR Abs-positive sera and IgG from severe COVID-19 patients and mAb 127 significantly activated human leukocytes and triggered NETosis, in the presence of RBD. This NETosis, triggered by the coexistence of CR Abs and RBD, activated thrombus-related cells but was abolished when the interaction between CR Abs and ACE2 or Fc receptors was disrupted. We also revealed that CR Abs-induced NETosis was suppressed in the presence of recombinant ACE2 or the Src family kinase inhibitor, dasatinib. Furthermore, we found that COVID-19 vaccination not only reduced COVID-19 severity but also prevented the production of CR Abs after SARS-CoV-2 infection. CONCLUSIONS: Our findings provide possible pathogenic effects of CR Abs in exacerbating COVID-19 by enhancing NETosis, highlighting ACE2 and dasatinib as potential treatments, and supporting the benefit of vaccination in reducing disease severity and CR Abs production in COVID-19 patients.

Engineered extracellular vesicles carrying let-7a-5p for alleviating inflammation in acute lung injury.

BACKGROUND: Acute lung injury (ALI) is a life-threatening respiratory condition characterized by severe inflammation and lung tissue damage, frequently causing rapid respiratory failure and long-term complications. The microRNA let-7a-5p is involved in the progression of lung injury, inflammation, and fibrosis by regulating immune cell activation and cytokine production. This study aims to use an innovative cellular electroporation platform to generate extracellular vesicles (EVs) carring let-7a-5p (EV-let-7a-5p) derived from transfected Wharton's jelly-mesenchymal stem cells (WJ-MSCs) as a potential gene therapy for ALI. METHODS: A cellular nanoporation (CNP) method was used to induce the production and release of EV-let-7a-5p from WJ-MSCs transfected with the relevant plasmid DNA. EV-let-7a-5p in the conditioned medium were isolated using a tangential flow filtration (TFF) system. EV characterization followed the minimal consensus guidelines outlined by the International Society for Extracellular Vesicles. We conducted a thorough set of therapeutic assessments, including the antifibrotic effects using a transforming growth factor beta (TGF-ß)-induced cell model, the modulation effects on macrophage polarization, and the influence of EV-let-7a-5p in a rat model of hyperoxia-induced ALI. RESULTS: The CNP platform significantly increased EV secretion from transfected WJ-MSCs, and the encapsulated let-7a-5p in engineered EVs was markedly higher than that in untreated WJ-MSCs. These EV-let-7a-5p did not influence cell proliferation and effectively mitigated the TGF-ß-induced fibrotic phenotype by downregulating SMAD2/3 phosphorylation in LL29 cells. Furthermore, EV-let-7a-5p regulated M2-like macrophage activation in an inflammatory microenvironment and significantly induced interleukin (IL)-10 secretion, demonstrating their modulatory effect on inflammation. Administering EVs from untreated WJ-MSCs slightly improved lung function and increased let-7a-5p expression in plasma in the hyperoxia-induced ALI rat model. In comparison, EV-let-7a-5p significantly reduced macrophage infiltration and collagen deposition while increasing IL-10 expression, causing a substantial improvement in lung function. CONCLUSION: This study reveals that the use of the CNP platform to stimulate and transfect WJ-MSCs could generate an abundance of let-7a-5p-enriched EVs, which underscores the therapeutic potential in countering inflammatory responses, fibrotic activation, and hyperoxia-induced lung injury. These results provide potential avenues for developing innovative therapeutic approaches for more effective interventions in ALI.

Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Is Associated With Recurrence and Survival of Resectable Non-Small Cell Lung Cancer (NSCLC): A Retrospective Study.

INTRODUCTION: Curative lung resection remains the key therapeutic strategy for early-stage non-small cell lung cancer (NSCLC). However, a proportion of patients still experience variable outcomes and eventually develop recurrence or die from their disease. Proprotein convertase subtilisin/kexin type 9 (PCSK9) has been identified as a deleterious factor that inhibits tumor cells apoptosis and leads to reduction of lymphocyte infiltration. However, there has been no research on the predicted role of PCSK9 as an immunohistochemical biomarker with survival in resectable NSCLC. METHODS: One hundred sixty-three patients with resectable NSCLC were retrospectively reviewed, and PCSK9 expression of resected NSCLC was analyzed by immunohistochemistry using tissue microarrays. RESULTS: PCSK9 was associated with recurrence (42.1% relapsed in the PCSK9lo group versus 57.9% relapsed in the PCSK9hi group, P = 0.006) and survival status (39.6% dead in PCSK9lo group versus 60.4% dead in PCSK9hi group, P = 0.004) in patients with resectable NSCLC. Moreover, resectable NSCLC patients with higher PCSK9 expression in tumor tissue experienced poorer disease-free survival (median disease-free survival: 10.5 versus 25.2 mo, hazard ratio = 1.620, 95% confidence interval: 1.124-2.334) and overall suvrival (median overall suvrival: 20.0 versus 54.1 mo, hazard ratio = 1.646, 95% confidence interval: 1.101-2.461) compared to those with lower PCSK9 expression. CONCLUSIONS: High PCSK9 expression of tumor was correlated with recurrence and worse survival status of resectable NSCLC in our retrospective study, which indicated that PCSK9 in NSCLC may be an immunohistochemical biomarker of poor prognosis for patients with resectable NSCLC. Further large-scale prospective studies are warranted to establish these results.