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Abstract Although the association between gut dysbiosis (imbalance of the microbiota) in systemic lupus erythematosus (SLE) is well-known, the simultaneous exploration in gut dysbiosis in fecal and different intestinal sections before and after lupus onset (at 2, 4, 6, 8, and 10 months old) resulting from the loss of inhibitory Fc gamma receptor IIb (FcGIIb) and pristane induction have never been conducted. Anti-dsDNA (an important lupus autoantibody) and proteinuria developed as early as 6 months old in both models, with higher levels in FcGRIIb deficient (FcGRIIb-/-) mice. Compared to the healthy control at 2 and 4 months, the lupus mice (both FcGRRIIb-/- and pristane) and healthy mice at 6 months old demonstrated an alteration as indicated by the Shannon alpha diversity index, highlighting influences of lupus- and age-induced dysbiosis, respectively. Non-metric multidimensional scaling (NMDS) revealed that the fecal microbiota of FcGRIIb-/- mice were distinct from the age-matched healthy control at all timepoints (at 6 month, p

Abstract Although the role of low-density granulocytes (LDGs), neutrophils in the peripheral blood mononuclear cell (PBMC) fraction, and neutrophil extracellular traps (NETs) in assessing lupus disease severity is acknowledged, data specific to childhood-onset lupus remains scarce. This study analyzed 46 patients with childhood-onset systemic lupus erythematosus (82.6% females, mean age 14.5 ± 0.3 years), including 26 cases with normal complement levels and 20 with low complement levels, along with 20 healthy adult volunteers. Key parameters that distinguished healthy volunteers from lupus patients and differentiated between lupus patients with low and normal complement were serum interferon (IFN)-α, serum citrullinated histone 3 (CitH3), and extracellular traps (ETs) in LDGs. However, NETs (assessed by nuclear staining morphology), LDG abundance, and other parameters (such as endotoxemia, cytokines, and double-stranded (ds) DNA) did not show such differentiation. When lipopolysaccharide (LPS) was administered to LDGs in the PBMC fraction, it induced ETs in both low and normal complement groups, indicating the inducible nature of ETs. In adult healthy volunteers, activation by recombinant IFN-α or dsDNA in isolated neutrophils induced LDGs and NETs (identified using immunofluorescent staining for CitH3, myeloperoxidase, and neutrophil elastase) at 45 min and 3 h post-stimulation, respectively. Additionally, approximately half of the LDGs underwent late apoptosis at 3 h post-stimulation, as determined by flow cytometry analysis. Activation by IFN-α or dsDNA in LDGs also led to a more pronounced expression of CD66b, an adhesion molecule, compared to regular-density neutrophils, suggesting higher activity in LDGs. In conclusion, IFN-α and/or dsDNA in serum may transform regular-density neutrophils into LDGs before progressing to NETosis and apoptosis, potentially exacerbating lupus severity through cell death-induced self-antigens. Therefore, LDGs and ETs in LDGs could provide deeper insights into the pathophysiology of childhood-onset lupus.

Abstract The complexity of systemic lupus erythematosus (SLE) arises from intricate genetic and environmental interactions, with STING playing a pivotal role. This study aims to comprehend the function of STING using the pristane-induced lupus (PIL) model in Sting missense mutant mice (Goldenticket or Sting Gt ), which contrasts with previous research using Sting knockout mice. Investigating two-month-old Sting Gt mice over six months post-PIL induction, we observed a profound reduction in autoimmune markers, including antinuclear and anti-dsDNA antibodies, germinal center B cells, and plasma cells, compared to their wild-type counterparts. A pivotal finding was the marked decrease in IL-17-producing T cells. Notably, the severity of lupus nephritis and pulmonary hemorrhages was significantly diminished in Sting Gt mice. These findings demonstrate that different genetic approaches to interfere with STING signaling can lead to contrasting outcomes in SLE pathogenesis, which highlights the need for a nuanced understanding of the role of STING in drug development for SLE. In summary, the loss of Sting function in Goldenticket mutant mice rescued autoimmune phenotypes in PIL. This study reveals the critical nature of STING in SLE, suggesting that the method of STING modulation significantly influences disease phenotypes and should be a key consideration in developing targeted therapies.

Abstract Gut microbiota manipulation may reverse metabolic abnormalities in obesity. Our previous studies demonstrated that inulin supplementation significantly promoted Bifidobacterium and fat-free mass in obese children. We aimed to study gut-muscle axis from inulin supplementation in these children. In clinical phase, the plasma samples from 46 participants aged 7–15 years, were analyzed for muscle biomarkers before and after 6-month inulin supplementation. In parallel, the plausible mechanism of muscle production via gut-muscle axis was examined using macrophage cell line. Bifidobacterium was cultured in semi-refined medium with inulin used in the clinical phase. Cell-free supernatant was collected and used in lipopolysaccharide (LPS)-induced macrophage cell line to determine inflammatory and anti-inflammatory gene expression. In clinical phase, IL-15 and creatinine/cystatin C ratio significantly increased from baseline to the 6th month. In vitro study showed that metabolites derived from Bifidobacterium capable of utilizing inulin contained the abundance of SCFAs. In the presence of LPS, treatment from Bifidobacterium + inulin downregulated TNF-α, IL-6, IL-1β, and iNOS, but upregulated FIZZ-1 and TGF-β expression. Inulin supplementation promoted the muscle biomarkers in agreement with fat-free mass gain, elucidating by Bifidobacterium metabolites derived from inulin digestion showed in vitro anti-inflammatory activity and decreased systemic pro-inflammation, thus promoting muscle production via gut-muscle axis response. Clinical Trial Registry number: NCT03968003.

Abstract The absence of stimulator of interferon genes (STING) in 129.B6.Fcgr2b-deficient mice rescue lupus phenotypes. The administration of a STING inhibitor (ISD017) into the young 129.B6.Fcgr2b-deficient mice prevents lupus nephritis development. This study mainly aimed to evaluate the effects of STING inhibition (ISD107) on established SLE in mice to prove that ISD017 could be a good therapeutic drug to reverse the already set-up autoimmunity and kidney impairment. Twenty-four-week-old Fcgr2b-deficient mice were treated with cyclophosphamide (25 mg/kg, intraperitoneal, once per week), ISD017 (10 mg/kg, intraperitoneal, three times per week), or control vehicle for 8 weeks, and were analyzed for phenotypes. Both ISD017 and cyclophosphamide treatment increased long-term survival and reduced the severity of glomerulonephritis in Fcgr2b-deficient mice. While cyclophosphamide reduced activated B cells (B220+GL-7+), ISD017 decreased activated T cells (CD4+CD69+) and neutrophils (Ly6c+Ly6g+) in Fcgr2b-deficient mice. In addition, ISD017 reduced IL-1β and interferon-inducible genes. In summary, ISD017 treatment in symptomatic 129.B6.Fcgr2b-deficient mice reduced the severity of glomerulonephritis and increased long-term survival. ISD017 worked comparably to cyclophosphamide for treating lupus nephritis in 129.B6.Fcgr2b-deficient mice. ISD017 reduced activated T cells and neutrophils, while cyclophosphamide targeted activated B cells. These results suggested that STING inhibitors can potentially be a new therapeutic drug for treating lupus.

Background: Anti-SARS-CoV-2 and immunomodulatory drugs are important for treating clinically severe patients with respiratory distress symptoms. Alpha- and gamma-mangostins (AM and GM) were previously reported as potential 3C-like protease (3CLpro) and Angiotensin-converting enzyme receptor 2 (ACE2)-binding inhibitors in silico. Objective: We aimed to evaluate two active compounds, AM and GM, from Garcinia mangostana for their antivirals against SARS-CoV-2 in live virus culture systems and their cytotoxicities using standard methods. Also, we aimed to prove whether 3CLpro and ACE2 neutralization were major targets and explored whether any additional targets existed. Methods: We tested the translation and replication efficiencies of SARS-CoV-2 in the presence of AM and GM. Initial and subgenomic translations were evaluated by immunofluorescence of SARS-CoV-2 3CLpro and N expressions at 16 h after infection. The viral genome was quantified and compared with the untreated group. We also evaluated the efficacies and cytotoxicities of AM and GM against four strains of SARS-CoV-2 (wild-type B, B.1.167.2, B.1.36.16, and B.1.1.529) in Vero E6 cells. The potential targets were evaluated using cell-based anti-attachment, time-of-drug addition, in vitro 3CLpro activities, and ACE2-binding using a surrogated viral neutralization test (sVNT). Moreover, additional targets were explored using combinatorial network-based interactions and Chemical Similarity Ensemble Approach (SEA). Results: AM and GM reduced SARS-CoV-2 3CLpro and N expressions, suggesting that initial and subgenomic translations were globally inhibited. AM and GM inhibited all strains of SARS-CoV-2 at EC50 of 0.70–3.05 μM, in which wild-type B was the most susceptible strain (EC50 0.70–0.79 μM). AM was slightly more efficient in the variants (EC50 0.88–2.41 μM), resulting in higher selectivity indices (SI 3.65–10.05), compared to the GM (EC50 0.94–3.05 μM, SI 1.66–5.40). GM appeared to be more toxic than AM in both Vero E6 and Calu-3 cells. Cell-based anti-attachment and time-of-addition suggested that the potential molecular target could be at the post-infection. 3CLpro activity and ACE2 binding were interfered with in a dose-dependent manner but were insufficient to be a major target. Combinatorial network-based interaction and chemical similarity ensemble approach (SEA) suggested that fatty acid synthase (FASN), which was critical for SARS-CoV-2 replication, could be a target of AM and GM. Conclusion: AM and GM inhibited SARS-CoV-2 with the highest potency at the wild-type B and the lowest at the B.1.1.529. Multiple targets were expected to integratively inhibit viral replication in cell-based system.

Neutrophil dysfunction is a form of immune suppression in patients with β-thalassemia (Beta-thal), although data on this are limited. In this study, blood from patients and healthy volunteers was analyzed. Flow cytometry analysis demonstrated an increase in immature neutrophils (CD16− CD62L+) and aged (senescent) neutrophils (CD16+ CD62L−) in Beta-thal patients compared to healthy volunteers. The Beta-thal neutrophils demonstrated less prominent chemotaxis and phagocytosis than healthy neutrophils at the baseline. With phorbol myristate acetate (PMA) or lipopolysaccharide (LPS) stimulations, some of the indicators, including the flow cytometry markers (CD11b, CD62L, CD66b, CD63, apoptosis, and reactive oxygen species) and neutrophil extracellular traps (NETs; detected by anti-citrullinated histone 3 immunofluorescence), were lower than the control. Additionally, low-density neutrophils (LDNs), which are found in the peripheral blood mononuclear cell (PBMC) fraction, were observed in Beta-thal patients but not in the control group. The expression of CD11b, CD66b, CD63, arginase I, and ROS in LDNs was higher than the regular normal-density neutrophils (NDNs). The proliferation rate of CD3+ T cells isolated from the PBMC fraction of healthy volunteers was higher than that of the cells from patients with Beta-thal. The incubation of red blood cell (RBC) lysate plus ferric ions with healthy NDNs transformed the NDNs into the aged neutrophils (decreased CD62L) and LDNs. In conclusion, iron overload induces neutrophil diversity along with some dysfunctions.

Candida glabrata is an important pathogen causing invasive infection associated with a high mortality rate. One mechanism that causes the failure of Candida eradication is an increase in regulatory T cells (Treg), which play a major role in immune suppression and promoting Candida pathogenicity. To date, how C. glabrata induces a Treg response remains unclear. Dendritic cells (DCs) recognition of fungi provides the fundamental signal determining the fate of the T-cell response. This study investigated the interplay between C. glabrata and DCs and its effect on Treg induction. We found that C. glabrata β-glucan was a major component that interacted with DCs and consequently mediated the Treg response. Blocking the binding of C. glabrata β-glucan to dectin-1 and complement receptor 3 (CR3) showed that CR3 activation in DCs was crucial for the induction of Treg. Furthermore, a ligand–receptor binding assay showed the preferential binding of C. glabrata β-glucan to CR3. Our data suggest that C. glabrata β-glucan potentially mediates the Treg response, probably through CR3-dependent activation in DCs. This study contributes new insights into immune modulation by C. glabrata that may lead to a better design of novel immunotherapeutic strategies for invasive C. glabrata infection.

Summary: Background: Repurposed drugs with host-directed antiviral and immunomodulatory properties have shown promise in the treatment of COVID-19, but few trials have studied combinations of these agents. The aim of this trial was to assess the effectiveness of affordable, widely available, repurposed drugs used in combination for treatment of COVID-19, which may be particularly relevant to low-resource countries. Methods: We conducted an open-label, randomized, outpatient, controlled trial in Thailand from October 1, 2021, to June 21, 2022, to assess whether early treatment within 48-h of symptoms onset with combinations of fluvoxamine, bromhexine, cyproheptadine, and niclosamide, given to adults with confirmed mild SARS-CoV-2 infection, can prevent 28-day clinical deterioration compared to standard care. Participants were randomly assigned to receive treatment with fluvoxamine alone, fluvoxamine + bromhexine, fluvoxamine + cyproheptadine, niclosamide + bromhexine, or standard care. The primary outcome measured was clinical deterioration within 9, 14, or 28 days using a 6-point ordinal scale. This trial is registered with ClinicalTrials.gov (NCT05087381). Findings: Among 1900 recruited, a total of 995 participants completed the trial. No participants had clinical deterioration by day 9, 14, or 28 days among those treated with fluvoxamine plus bromhexine (0%), fluvoxamine plus cyproheptadine (0%), or niclosamide plus bromhexine (0%). Nine participants (5.6%) in the fluvoxamine arm had clinical deterioration by day 28, requiring low-flow oxygen. In contrast, most standard care arm participants had clinical deterioration by 9, 14, and 28 days. By day 9, 32.7% (110) of patients in the standard care arm had been hospitalized without requiring supplemental oxygen but needing ongoing medical care. By day 28, this percentage increased to 37.5% (21). Additionally, 20.8% (70) of patients in the standard care arm required low-flow oxygen by day 9, and 12.5% (16) needed non-invasive or mechanical ventilation by day 28. All treated groups significantly differed from the standard care group by days 9, 14, and 28 (p

Summary: This study investigated the potential of using SARS-CoV-2 viral concentrations in dust as an additional surveillance tool for early detection and monitoring of COVID-19 transmission. Dust samples were collected from 8 public locations in 16 districts of Bangkok, Thailand, from June to August 2021. SARS-CoV-2 RNA concentrations in dust were quantified, and their correlation with community case incidence was assessed. Our findings revealed a positive correlation between viral concentrations detected in dust and the relative risk of COVID-19. The highest risk was observed with no delay (0-day lag), and this risk gradually decreased as the lag time increased. We observed an overall decline in viral concentrations in public places during lockdown, closely associated with reduced human mobility. The effective reproduction number for COVID-19 transmission remained above one throughout the study period, suggesting that transmission may persist in locations beyond public areas even after the lockdown measures were in place.

Abstract Although cell-free DNA (cfDNA) is an emerging sepsis biomarker, the use of cfDNA, especially as diagnostic and prognostic indicators, has surprisingly not been systemically analyzed. Data of adult patients with sepsis that conducted cfDNA measurement within 24 h of the admission was collected from PubMed, ScienceDirect, Scopus, and Cochrane Library until October 2022. The Quality in Prognosis Studies (QUIPS) and Quality Assessment in Diagnostic Studies-2 (QUADAS-2) tools were used to reduce the risk of biased assessment. The mean difference (MD) of cfDNA concentration and the standardized mean difference (SMD) between populations was calculated using Review Manager (RevMan) version 5.4.1 package software. Pooled analysis from 18 included studies demonstrated increased serum cfDNA levels in sepsis when compared with healthy control (SMD = 1.02; 95% confidence interval (CI) 0.46–1.57) or non-sepsis patients in the intensive care unit (ICU) (SMD = 1.03; 95% CI 0.65–1.40), respectively. Meanwhile, a slight decrease in the statistical value was observed when compared with non-sepsis ICU patients with SIRS (SMD = 0.74; 95% 0.41–1.06). The lower cfDNA levels were also observed in sepsis survivors compared to the non-survivors (SMD at 1.43; 95%CI 0.69–2.17) with the pooled area under the receiver operating characteristic curve (AUC) of 0.76 (95% CI 0.64–0.87) for the mortality prediction. Levels of cfDNA showed a pooled sensitivity of 0.81 (95% CI 0.75–0.86) and specificity of 0.72 (95% CI 0.65–0.78) with pooled diagnostic odd ratio (DOR) at 25.03 (95% CI 5.48–114.43) for the identification of sepsis in critically ill conditions. The cfDNA levels were significantly higher in patients with sepsis and being a helpful indicator for the critically ill conditions of sepsis. Nevertheless, results of the test must be interpreted carefully with the context of all clinical situations.

Abstract Because of the high incidence of Pseudomonas aeruginosa biofilms-related nosocomial infections, venoms from common Thai snakes were tested. Although venoms from king cobra (Ophiophagus hannah; OH) and green pit viper (Trimeresurus albolabris) showed the broadest antibacterial spectrum, OH venom demonstrated more profound anti-biofilm activities against P. aeruginosa. Additionally, purified l-amino acid oxidase from OH venom (OH-LAAO), using a three-step chromatography and protein identification, reduced biofilm mass as indicated by the downregulation of several genes, including the genes for biofilm synthesis (algD and pslB) and biofilm regulators (algU, gacA, and siaD). Moreover, OH-LAAO disrupted Pseudomonas-preformed biofilms via upregulation of several genes for biofilm dispersion (nbdA, bdlA, and dipA) and biofilm degradation (endA and pslG), resulting in a reduction of the biofilm biomass. Due to the antimicrobial effects and anti-biofilm activities (reduced production plus increased dispersion) neutralized by catalase, a hydrogen peroxide (H2O2)-degrading enzyme, the enhanced H2O2 by OH venom might be one of the anti-biofilm mechanisms. Hence, OH-LAAO was proposed as a novel agent against Pseudomonas biofilms for either treatment or prevention. More studies are interesting.

BackgroundMost of the current bacteriophages (phages) are mostly isolated from environments. However, phages isolated from feces might be more specific to the bacteria that are harmful to the host. Meanwhile, some phages from the environment might affect non-pathogenic bacteria for the host.MethodsHere, bacteriophages isolated from mouse feces were intratracheally (IT) or intravenously (IV) administered in pneumonia mice caused by Pseudomonas aeruginosa at 2 hours post-intratracheal bacterial administration. As such, the mice with phage treatment, using either IT or IV administration, demonstrated less severe pneumonia as indicated by mortality, serum cytokines, bacteremia, bacterial abundance in bronchoalveolar lavage fluid (BALF), and neutrophil extracellular traps (NETs) in lung tissue (immunofluorescence of neutrophil elastase and myeloperoxidase).ResultsInterestingly, the abundance of phages in BALF from the IT and IV injections was similar, supporting a flexible route of phage administration. With the incubation of bacteria with neutrophils, the presence of bacteriophages significantly improved bactericidal activity, but not NETs formation, with the elevated supernatant IL-6 and TNF-α, but not IL-1β. In conclusion, our findings suggest that bacteriophages against Pseudomonas aeruginosa can be discovered from feces of the host.ConclusionsThe phages attenuate pneumonia partly through an enhanced neutrophil bactericidal activity, but not via inducing NETs formation. The isolation of phages from the infected hosts themselves might be practically useful for future treatment. More studies are warranted.

Given the rise of multidrug-resistant (MDR) Pseudomonas aeruginosa infections, alternative treatments are needed. Anti-pseudomonal phage therapy shows promise, but its clinical application is limited due to the development of resistance and a lack of biofilm penetration. Recently, adjuvants like CaEDTA have shown the ability to enhance the effectiveness of combined antimicrobial agents. Here, we tested a phage-adjuvant combination and demonstrated the effectiveness of intranasally inhaled phage (KKP10) + CaEDTA in addition to ceftazidime/avibactam (CZA) for chronic P. aeruginosa lung infections. The results emphasize that intranasal inhalation of phage along with CaEDTA can successfully re-sensitize MDR P. aeruginosa to CZA in a triple combination treatment. This promising approach shows potential as a therapy for chronic respiratory tract infections.

Although vaccine administration by microneedles has been demonstrated, delivery reliability issues have prevented their implementation. Through an ex vivo porcine skin experiment, we show visual evidence indicating that detachable dissolvable microneedles (DDMN) can deposit cargo into the dermis with insignificant loss of cargo to the stratum corneum. Using ovalbumin (OVA), a model antigen vaccine, as a cargo, the ex vivo experiments yielded a delivery efficiency of 86.08 ± 4.16 %. At room temperature, OVA could be stabilized for up to 35 days in DDMN made from hyaluronic acid and trehalose. The DDMN matrix could improve the denaturation temperature of the OVA from around 70–120 °C to over 150 °C, as demonstrated by differential scanning calorimetric analysis. In vivo delivery of OVA antigen into the mice's skin via DDMN elicited 10 times higher specific antibody responses compared to conventional intramuscular injection. We envision DDMN as an effective, precise dosing, intradermal vaccine delivery system that may require no cold-chain, offers a dose-sparing effect, and can be administered easily.

Transforming growth factor beta (TGF-β) is ubiquitously found in bone and plays a key role in bone turnover. Mice expressing constitutively active TGF-β receptor type I (Mx1;TβRICA mice) are osteopenic. Here, we identified the candidate genes involved in bone turnover in Mx1;TβRICA mice using RNA sequencing analysis. A total of 285 genes, including 87 upregulated and 198 downregulated genes, were differentially expressed. According to the KEGG analysis, some genes were involved in osteoclast differentiation (Fcgr4, Lilrb4a), B cell receptor signaling (Cd72, Lilrb4a), and neutrophil extracellular trap formation (Hdac7, Padi4). Lilrb4 is related to osteoclast inhibition protein, whereas Hdac7 is a Runx2 corepressor that regulates osteoblast differentiation. Silencing Lilrb4 increased the number of osteoclasts and osteoclast marker genes. The knocking down of Hdac7 increased alkaline phosphatase activity, mineralization, and osteoblast marker genes. Therefore, our present study may provide an innovative idea for potential therapeutic targets and pathways in TβRI-associated bone loss.

Abstract High rates of new cervical cancer cases and deaths occur in low- and middle-income countries yearly, and one reason was found related to limitation of regular cervical cancer screening in local and low-resource settings. HPV has over 150 types, yet certain 14–20 high-risk and 13–14 low-risk types are common, and, thus, most conventional HPV nucleic acid assays, for examples, Cobas 4800 HPV test (Roche Diagnostics, New Jersey, USA) and REBA HPV-ID (Molecules and Diagnostics, Wonju, Republic of Korea) were developed to cover these types. We thereby utilized bioinformatics combined with recent isothermal amplification technique at 35–42 °C to firstly describe multiplex recombinase polymerase amplification assay that is specific to these common 20 high-risk and 14 low-risk types, and also L1 and E6/E7 genes that target different stages of cervical cancer development. Multiplex primer concentrations and reaction incubation conditions were optimized to allow simultaneous two gene detections at limit of detection of 1000 copies (equivalent to 2.01 fg) for L1 and 100 copies (0.0125 fg) for E6/E7, respectively. The assay was validated against urogenital and other pathogens, normal flora, and human control. In 130 real clinical sample tests, the assay demonstrated 100% specificity, 78% diagnostic accuracy, and 75% sensitivity compared with REBA HPV-ID test, and is much more rapid (15–40 min), less expensive (~ 3–4 USD/reaction) and does not require instrumentation (35–42 °C reaction condition so hand holding or tropical temperature is possible). Hence, the developed novel assay provides alternative screening tool for potential local screening. Furthermore, as this assay uses safe chemical reagents, it is safe for users.

The prevalence of obesity is increasing, and the coexistence of obesity and systemic lupus erythematosus (lupus) is possible. A high-fat diet (HFD) was orally administered for 6 months in female 8-week-old Fc gamma receptor IIb deficient (FcgRIIb−/−) lupus or age and gender-matched wild-type (WT) mice. Lupus nephritis (anti-dsDNA, proteinuria, and increased creatinine), gut barrier defect (fluorescein isothiocyanate dextran), serum lipopolysaccharide (LPS), serum interleukin (IL)-6, liver injury (alanine transaminase), organ fibrosis (liver and kidney pathology), spleen apoptosis (activated caspase 3), and aorta thickness (but not weight gain and lipid profiles) were more prominent in HFD-administered FcgRIIb−/− mice than the obese WT, without injury in regular diet-administered mice (both FcgRIIb−/− and WT). In parallel, combined palmitic acid (PA; a saturated fatty acid) with LPS (PA + LPS) induced higher tumor necrotic factor-α, IL-6, and IL-10 in the supernatant, inflammatory genes (inducible nitric oxide synthase and IL-1β), reactive oxygen species (dihydroethidium), and glycolysis with reduced mitochondrial activity (extracellular flux analysis) when compared with the activation by each molecule alone in both FcgRIIb−/− and WT macrophages. However, the alterations of these parameters were more prominent in PA + LPS-administered FcgRIIb−/− than in the WT cells. In conclusion, obesity accelerated inflammation in FcgRIIb−/− mice, partly due to the more potent responses from the loss of inhibitory FcgRIIb against PA + LPS with obesity-induced gut barrier defect.

Although current regimens of immunosuppressive drugs are effective in renal transplant recipients, long-term renal allograft outcomes remain suboptimal. For many years, the diagnosis of renal allograft rejection and of several causes of renal allograft dysfunction, such as chronic subclinical inflammation and infection, was mostly based on renal allograft biopsy, which is not only invasive but also possibly performed too late for proper management. In addition, certain allograft dysfunctions are difficult to differentiate from renal histology due to their similar pathogenesis and immune responses. As such, non-invasive assays and biomarkers may be more beneficial than conventional renal biopsy for enhancing graft survival and optimizing immunosuppressive drug regimens during long-term care. This paper discusses recent biomarker candidates, including donor-derived cell-free DNA, transcriptomics, microRNAs, exosomes (or other extracellular vesicles), urine chemokines, and nucleosomes, that show high potential for clinical use in determining the prognosis of long-term outcomes of kidney transplantation, along with their limitations.

BackgroundEvidence has demonstrated inferior humoral immune responses after SARS-CoV-2 vaccination in kidney transplant recipients compared to the general population. However, data on cellular immune responses in this population have not been established.MethodsWe searched the MEDLINE, Scopus, and Cochrane databases and included studies reporting cellular immune response rates in kidney transplant recipients after receiving SARS-CoV-2 vaccines. Studies that reported factors associated with cellular immune responders or non-responders were also included (PROSPERO: CRD42022375544).ResultsFrom a total of 1,494 articles searched, 53 articles were included in the meta-analysis. In all, 21 studies assessed cellular immune response by interferon-γ enzyme-linked immunosorbent spot (IFN-γ ELISPOT), 22 studies used interferon-γ release assay (IGRA), and 10 studies used flow cytometric analysis. The pooled response rate after two doses (standard regimen) and three doses of vaccination was 47.5% (95%CI 38.4-56.7%) and 69.1% (95%CI 56.3-80.6%) from IFN-γ ELISPOT, 25.8% (95%CI 19.7-32.4%) and 14.7% (95%CI 8.5-22.2%) from IGRA, and 73.7% (95%CI 55.2-88.8%) and 86.5% (95%CI 75.3-94.9%) from flow cytometry, respectively. Recipients with seroconversion were associated with a higher chance of having cellular immune response (OR 2.58; 95%CI 1.89-3.54). Cellular immune response in kidney transplant recipients was lower than in dialysis patients (OR 0.24; 95%CI 0.16-0.34) and the general population (OR 0.10; 95%CI 0.07-0.14). Age and immunosuppressants containing tacrolimus or corticosteroid were associated with inferior cellular immune response.ConclusionCellular immune response after SARS-CoV-2 vaccination in kidney transplant recipients was lower than in dialysis patients and the general population. Age, tacrolimus, and corticosteroid were associated with poor response. Cellular immune response should also be prioritized in vaccination studies.Systematic review registrationhttps://www.crd.york.ac.uk/prospero/, identifier CRD42022375544.

Summary: Equitable SARS-CoV-2 surveillance in low-resource communities lacking centralized sewers is critical as wastewater-based epidemiology (WBE) progresses. However, large-scale studies on SARS-CoV-2 detection in wastewater from low-and middle-income countries is limited because of economic and technical reasons. In this study, wastewater samples were collected twice a month from 186 urban and rural subdistricts in nine provinces of Thailand mostly having decentralized and non-sewered sanitation infrastructure and analyzed for SARS-CoV-2 RNA variants using allele-specific RT-qPCR. Wastewater SARS-CoV-2 RNA concentration was used to estimate the real-time incidence and time-varying effective reproduction number (Re). Results showed an increase in SARS-CoV-2 RNA concentrations in wastewater from urban and rural areas 14–20 days earlier than infected individuals were officially reported. It also showed that community/food markets were “hot spots” for infected people. This approach offers an opportunity for early detection of transmission surges, allowing preparedness and potentially mitigating significant outbreaks at both spatial and temporal scales.

Summary: Developing an effective therapy to overcome carbapenemase-positive Klebsiella pneumoniae (CPKp) is an important therapeutic challenge that must be addressed urgently. Here, we explored a Ca-EDTA combination with aztreonam or ceftazidime-avibactam in vitro and in vivo against diverse CPKp clinical isolates. The synergy testing of this study demonstrated that novel aztreonam-Ca-EDTA or ceftazidime-avibactam-Ca-EDTA combination was significantly effective in eliminating planktonic and mature biofilms in vitro, as well as eradicating CPKp infections in vivo. Both combinations revealed significant therapeutic efficacies in reducing bacterial load in internal organs and protecting treated mice from mortality. Conclusively, this is the first in vitro and in vivo study to demonstrate that novel aztreonam-Ca-EDTA or ceftazidime-avibactam-Ca-EDTA combinations provide favorable efficacy and safety for successful eradication of carbapenemase-producing Klebsiella pneumoniae planktonic and biofilm infections.

It is unclear how the immune system controls the transition from latent tuberculosis (TB) infection (LTBI) to active pulmonary infection (PTB). Here, we applied mass spectrometry cytometry time-of-flight (CyTOF) analysis of peripheral blood mononuclear cells to compare the immunological landscapes in patients with high tuberculous bacillary load PTB infections and LTBI. A total of 32 subjects (PTB [n = 12], LTBI [n = 17], healthy volunteers [n = 3]) were included. Participants with active PTBs were phlebotomized before administering antituberculosis treatment, whereas participants with LTBI progressed to PTB at the time of household screening. In the present study, CyTOF analysis identified significantly higher percentages of mucosal-associated invariant natural killer T (MAIT NKT) cells in subjects with LTBI than in those with active PTB and healthy controls. Moreover, 6 of 17 (35%) subjects with LTBI progressed to active PTB (LTBI progression) and had higher proportions of MAIT NKT cells and early NKT cells than those without progression (LTBI non-progression). Subjects with LTBI progression also showed a tendency toward low B cell levels relative to other subject groups. In conclusion, MAIT NKT cells were substantially more prevalent in subjects with LTBI, particularly those with progression to active PTB.

Although macrophage depletion is a possible emerging therapeutic strategy for osteoporosis and melanoma, the lack of macrophage functions can lead to inappropriate microbial control, especially the regulation of intestinal microbiota. Cecal ligation and puncture (CLP) sepsis was performed in regular mice and in mice with clodronate-induced macrophage depletion. Macrophage depletion significantly increased the mortality and severity of sepsis-CLP mice, partly through the increased fecal Ascomycota, especially Kazachstania pintolopesii, with polymicrobialbacteremia (Klebsiella pneumoniae, Enterococcus faecalis, and Acinetobacter radioresistens). Indeed, macrophage depletion with sepsis facilitated gut dysbiosis that directly affected gut permeability as yeast cells were located and hidden in the colon crypts. To determine the interactions of fungal molecules on bacterial abundance, the heat-kill lysate of fungi (K. pintolopesii and C. albicans) and purified (1→3)-β-d-glucan (BG; a major component of the fungal cell wall) were incubated with bacteria that were isolated from the blood of macrophage-depleted mice. There was enhanced cytokine production of enterocytes (Caco-2) after the incubation of the lysate of K. pintolopesii (isolated from sepsis mice), the lysate of C. albicans (extracted from sepsis patients), and BG, together with bacterial lysate. These data support a possible influence of fungi in worsening sepsis severity. In conclusion, macrophage depletion enhanced K. pintolopesii in feces, causing the overgrowth of fecal pathogenic bacteria and inducing a gut permeability defect that additively worsened sepsis severity. Hence, the fecal fungus could be spontaneously elevated and altered in response to macrophage-depleted therapy, which might be associated with sepsis severity.

Abstract The global prevalence of colistin-resistant Klebsiella pneumoniae (ColRkp) facilitated by chromosomal and plasmid-mediated Ara4N or PEtN-remodeled LPS alterations has steadily increased with increased colistin usage for treating carbapenem-resistant K. pneumoniae (CRkp). Our study demonstrated the rising trend of ColRkp showing extensively and pandrug-resistant characteristics among CRkp, with a prevalence of 28.5%, which was mediated by chromosomal mgrB, pmrB, or phoQ mutations (91.5%), and plasmid-mediated mcr-1.1, mcr-8.1, mcr-8.2 alone or in conjunction with R256G PmrB (8.5%). Several genetic alterations in mgrB (85.1%) with increased expressions of Ara4N-related phoPQ and pmrK were critical for establishing colistin resistance in our isolates. In this study, we discovered the significant associations between extensively drug-resistant bacteria (XDR) and pandrug-resistant bacteria (PDR) ColRkp in terms of moderate, weak or no biofilm-producing abilities, and altered expressions of virulence factors. These ColRkp would therefore be very challenging to treat, emphasizing for innovative therapy to combat these infections. Regardless of the underlying colistin-resistant mechanisms, colistin-EDTA combination therapy in this study produced potent synergistic effects in both in vitro and in vivo murine bacteremia, with no ColRkp regrowth and improved animal survival, implying the significance of colistin-EDTA combination therapy as systemic therapy for unlocking colistin resistance in ColRkp-associated bacteremia.

Abstract Overcoming colistin-resistant Acinetobacter baumannii (CoR-AB) has become a major concern due to the lack of effective antibiotics. This study aimed to explore the prevalence of CoR-AB clinical isolates in Thailand, their mechanisms of resistance, and test the efficacy of colistin plus sulbactam against CoR-AB isolates. The colistin resistance rate among carbapenem-resistant A. baumannii was 15.14%. The mcr gene or its variants were not detected in CoR-AB isolates by PCR screening. The lipid A mass spectra of CoR-AB isolates showed the additional [M–H]− ion peak at m/z = 2034 that correlated to the phosphoethanolamine (pEtN) addition to lipid A (N = 27/30). The important amino acid substitutions were found at position S14P, A138T, A227V in PmrB that are associated with overexpression of the pEtN transferase (PmrC) and contributed the pEtN addition. The lipopolysacccharide production genes (lpxACD) were not related to lipid A mass spectra. A colistin plus sulbactam combination exhibited the synergy rate at 86.7% against CoR-AB isolates compare to sulbactam (85.89% resistance) or colistin (15.14% resistance) alone. The excellent synergistic activity of colistin plus sulbactam combination has the potential for the treatment of CoR-AB infections.

Abstract Because macrophage dysfunction from some emerging therapies might worsen gut-derived sepsis, cecal ligation and puncture (CLP) sepsis are performed in mice with clodronate-induced macrophage depletion. Macrophage depletion (non-sepsis) increased fecal Ascormycota, with a subtle change in bacterial microbiota, that possibly induced gut-barrier defect as Candida pintolopesii and Enterococcus faecalis were identified from blood. Sepsis in macrophage-depleted mice was more severe than sepsis control as indicated by mortality, cytokines, organ injury (liver, kidney, and spleen), gut-leakage (FITC-dextran), fecal Proteobacteria, and blood organisms (bacteria and fungi). Lysate of C. pintolopesii or purified (1 → 3)-β-d-glucan (BG; a major component of fungal cell wall) enhanced growth of Klebsiella pneumoniae and Escherichia coli that were isolated from the blood of macrophage-depleted CLP mice implying a direct enhancer to some bacterial species. Moreover, the synergy of LPS and BG on enterocytes (Caco-2) (Transepithelial electrical resistance) and neutrophils (cytokines) also supported an influence of gut fungi in worsening sepsis. In conclusion, macrophage depletion enhanced sepsis through the selectively facilitated growth of some bacteria (dysbiosis) from increased fecal fungi that worsened gut-leakage leading to the profound systemic responses against gut-translocated LPS and BG. Our data indicated a possible adverse effect of macrophage-depleted therapies on enhanced sepsis severity through spontaneous elevation of fecal fungi.

The impacts of metabolomic changes (reduced short-chain-fatty acids; SCFAs) in uremic condition is not fully understood. Once daily Candida gavage with or without probiotics (different times of administration) for 1 week prior to bilateral nephrectomy (Bil Nep) in 8-week-old C57BL6 mice as the possible models more resemble human conditions were performed. Candida-administered Bil Nep mice demonstrated more severe conditions than Bil Nep alone as indicated by mortality (n = 10/group) and other 48 h parameters (n = 6-8/group), including serum cytokines, leaky gut (FITC-dextran assay, endotoxemia, serum beta-glucan, and loss of Zona-occludens-1), and dysbiosis (increased Enterobacteriaceae with decreased diversity in microbiome analysis) (n = 3/group for fecal microbiome) without the difference in uremia (serum creatinine). With nuclear magnetic resonance metabolome analysis (n = 3-5/group), Bil Nep reduced fecal butyric (and propionic) acid and blood 3-hydroxy butyrate compared with sham and Candida-Bil Nep altered metabolomic patterns compared with Bil Nep alone. Then, Lacticaseibacillus rhamnosus dfa1 (SCFA-producing Lacticaseibacilli) (n = 8/group) attenuated the model severity (mortality, leaky gut, serum cytokines, and increased fecal butyrate) of Bil Nep mice (n = 6/group) (regardless of Candida). In enterocytes (Caco-2 cells), butyrate attenuated injury induced by indoxyl sulfate (a gut-derived uremic toxin) as indicated by transepithelial electrical resistance, supernatant IL-8, NFκB expression, and cell energy status (mitochondria and glycolysis activities by extracellular flux analysis). In conclusion, the reduced butyrate by uremia was not enhanced by Candida administration; however, the presence of Candida in the gut induced a leaky gut that was attenuated by SCFA-producing probiotics. Our data support the use of probiotics in uremia.

Abstract Low fluid intake, low urinary citrate excretion, and high oxidative stress are main causative factors of calcium oxalate (CaOx) nephrolithiasis. HydroZitLa contains citrate and natural antioxidants and is developed to correct these three factors simultaneously. Antioxidants theoretically can prolong the lifespan of organisms. In this study, we preclinically investigated the antilithogenic, lifespan-extending and anti-aging effects of HydroZitLa in HK-2 cells, male Wistar rats, and Caenorhabditis elegans. HydroZitLa significantly inhibited CaOx crystal aggregation in vitro and reduced oxidative stress in HK-2 cells challenged with lithogenic factors. For experimental nephrolithiasis, rats were divided into four groups: ethylene glycol (EG), EG + HydroZitLa, EG + Uralyt-U, and untreated control. CaOx deposits in kidneys of EG + HydroZitLa and EG + Uralyt-U rats were significantly lower than those of EG rats. Intrarenal expression of 4-hydroxynonenal in EG + HydroZitLa rats was significantly lower than that of EG rats. The urinary oxalate levels of EG + HydroZitLa and EG + Uralyt-U rats were significantly lower than those of EG rats. The urinary citrate levels of EG + HydroZitLa and EG + Uralyt-U rats were restored to the level in normal control rats. In C. elegans, HydroZitLa supplementation significantly extended the median lifespan of nematodes up to 34% without altering feeding ability. Lipofuscin accumulation in HydroZitLa-supplemented nematodes was significantly lower than that of non-supplemented control. Additionally, HydroZitLa inhibited telomere shortening, p16 upregulation, and premature senescence in HK-2 cells exposed to lithogenic stressors. Conclusions, HydroZitLa inhibited oxidative stress and CaOx formation both in vitro and in vivo. HydroZitLa extended the lifespan and delayed the onset of aging in C. elegans and human kidney cells. This preclinical evidence suggests that HydroZitLa is beneficial for inhibiting CaOx stone formation, promoting longevity, and slowing down aging.

Macrophage polarization requires different energy sources and metabolic processes. Therefore, cell energy interference to alter macrophage functions has been proposed as a treatment for severe inflammatory diseases, including sepsis. In this study, targeting cell energy using BAM15 (a mitochondrial uncoupling agent) in human THP-1 and mouse RAW264.7 macrophages prominently interfered with M1 but not M2 polarization. Free BAM15 (BAM15) and BAM15-loaded PLGA particles (BAM15 particles) reduced the inflammatory response of M1 macrophages and enhanced the expression of M2 signature genes with the restoration of mitochondrial activity (extracellular flux analysis) in RAW264.7 cells. Furthermore, BAM15 particles but not BAM15 showed specific effects on the inflammatory response of macrophages but not neutrophils, and the particles were actively captured by splenic and liver macrophages in vivo. Administration of BAM15 and BAM15 particles attenuated the severity of sepsis in LPS-induced sepsis mice. Interestingly, BAM15 particles but not BAM15 alleviated LPS-induced liver injury by reducing hepatic inflammation. Our findings substantiate the superior efficacy of macrophage-targeted therapy using a BAM15 particle-delivery system and provide further support for clinical development as a potential therapy for severe inflammatory diseases.

Chronic inflammation contributes to the development of skeletal disorders in patients with systemic lupus erythematosus (SLE). Activation of the host immune response stimulates osteoclast activity, which in turn leads to bone loss. Regenerating bone in the inflammatory microenvironments of SLE patients with critical bone defects remains a great challenge. In this study, we utilized lipopolysaccharide (LPS) to imitate locally and systemically pathogenic bacterial infection and examined the bone regeneration performance of LPS-associated mandibular and tibial bone regeneration impairment in FcγRIIB−/− mice. Our results indicated that a loss of FcγRIIB alleviates bone regeneration in both mandibles and tibiae. After LPS induction, FcγRIIB−/− mice were susceptible to impaired fracture healing in tibial and mandibular bones. LPS decreased the mineralization to collagen ratio in FcγRIIB−/− mice, indicating a mineralization defect during bone repair. An osteoblast-associated gene (Col1a1) was attenuated in FcγRIIB-deficient mice, whereas Bglap, Hhip, and Creb5 were further downregulated with LPS treatment in FcγRIIB−/− mice compared to FcγRIIB−/− mice. Alpl and Bglap expression was dcreased in osteoblasts derived from bone chips. An osteoclast-associated gene, Tnfsf11/Tnfrsf11 ratio, ewas increased in LPS-induced FcγRIIB−/− mice and in vitro. Furthermore, systemic LPS was relatively potent in stimulating production of pro-inflammatory cytokines including TNF-α, IL-6, and MCP-1 in FcγRIIB−/− mice compared to FcγRIIB−/− mice. The levels of TNF-α, IFN-β, IL-1α, and IL-17A were increased, whereas IL-10 and IL-23 were decreased in FcγRIIB−/− mice treated locally with LPS. These findings suggest that both local and systemic LPS burden can exacerbate bone regeneration impairment, delay mineralization and skeletal repair, and induce inflammation in SLE patients.

Because of gut-barrier defect (gut-leakage) after acute kidney injury (AKI) and higher abundance of Candida albicans in human intestines compared with mouse guts, Candida administration in renal ischemia reperfusion injury (I/R) mice possibly more closely resemble patients with AKI than non-Candida model. Fungi in feces were detectable only in mice with Candida administration. Candida renal-I/R mice, when compared with non-Candida I/R, demonstrated more profound injuries, including (i) gut-leakage; FITC-dextran assay and serum (1→3)-β-D-glucan (BG), (ii) systemic inflammation (serum cytokines), and (iii) neutrophil extracellular traps (NETs); gene expression of peptidyl arginase 4 (PAD4) and IL-1β, nuclear morphology staining by 4′,6-diamidino-2-phenylindole (DAPI) and co-staining of myeloperoxidase (MPO) with neutrophil elastase (NE) in peripheral blood neutrophils. Although renal excretory function (serum creatinine) and renal histology score were nondifferent between renal-I/R mice with and without Candida, prominent renal NETs (PAD4 and IL-1β expression with MPO and NE co-staining) was demonstrated in Candida renal-I/R mice. Additionally, neutrophil activation by lipopolysaccharide (LPS) plus BG (LPS + BG), when compared with LPS alone, caused (i) NETs formation; dsDNA, DAPI-stained nuclear morphology and MPO with NE co-staining, (ii) inflammatory responses; Spleen tyrosine kinase (Syk) and NFκB expression, and (iii) reduced cell energy status (maximal respiratory capacity using extracellular flux analysis). Also, LPS + BG-activated NETs formation was inhibited by a dectin-1 inhibitor, supporting an impact of BG signaling. In conclusion, Candida-renal I/R demonstrated more prominent serum BG and LPS from gut translocation that increased systemic inflammation and NETs through TLR-4 and dectin-1 activation. The influence of gut fungi in AKI should be concerned.

BK polyomavirus (BKV) mainly causes infection in uroepithelial and renal tubular epithelial cells of either immunocompetent or immunocompromised hosts. Despite asymptomatic or mild clinical features in immunocompetent hosts with BK infection, serious complications are frequently found in immunocompromised patients, especially patients with kidney transplantation. Accordingly, BKV-associated nephropathy (BKVN) demonstrates a wide range of clinical manifestations, including ureteric stenosis and hemorrhagic cystitis. In addition, BKV re-infection in post-kidney transplantation is also a main cause of kidney allograft dysfunction and graft loss. Since the direct anti-BKV is unavailable, immune response against BKV infection is the main mechanism for organism control and might be a novel strategy to treat or suppress BKV. As such, the innate immunity, consisting of immune cells and soluble molecules, does not only suppress BKV but also enhances the subsequent adaptive immunity to eradicate the virus. Furthermore, the re-activation of BKV in BKVN of kidney-transplanted recipients seems to be related to the status of innate immunity. Therefore, this review aims to collate the most recent knowledge of innate immune response against BKV and the association between the innate immunity status of kidney-transplanted recipients and BKV re-activation.

IntroductionType I interferon (IFN) plays a vital role in the pathogenesis of systemic lupus erythematosus. Cyclic GMP AMP synthase (cGAS) is a cytosolic DNA sensor that recognizes dsDNA and creates cGAMP to activate STING-mediated type I IFN production. The activation of STING induces lupus disease in Fcgr2b deficient mice through the differentiation of dendritic cells. In contrast, Cgas-deficient mice could be generated more autoantibody production and proteinuria in pristane-induced lupus (PIL). These data suggested that the other dsDNA sensors could be involved in lupus development mechanisms.MethodsThis study aimed to identify the cGAS-mediated mechanisms contributing to lupus pathogenesis in PIL. The Cgas-deficient and WT mice were induced lupus disease with pristane and subsequently analyzed autoantibody, histopathology, and immunophenotypes. The lung tissues were analyzed with the expression profiles by RT-PCR and western blot. The bone marrow-derived macrophages were stimulated with inflammasome activators and observed pyroptosis.ResultsThe Cgas-/- mice developed more severe pulmonary hemorrhage and autoantibody production than WT mice. The activated dendritic cells, IFN-g-, and IL-17a-producing T helper cells, and infiltrated macrophages in the lung were detected in Cgas-/- mice higher than in WT mice. We observed an increase in expression of Aim2, Casp11, and Ifi16 in the lung and serum IL-1a but IL-1b in pristane-injected Cgas-/- mice. The rise of Caspase-11 in the lung of pristane-injected Cgas-/- mice suggested noncanonical inflammasome activation. The activation of AIM2 and NLRP3 inflammasomes in bone marrow-derived macrophages (BMDMs) enhanced the number of dead cells in Cgas-/- mice compared with WT mice. Activation of the inflammasome significantly induced pyroptosis in Cgas-/- BMDMs. The dsDNA level, but not mitochondrial DNA, increased dramatically in pristane-injected Cgas-/- mice suggesting the dsDNA could be a ligand activating inflammasomes. The cGAS agonist-induced BMDM activation in the Cgas-/- mice indicated that the activation of DNA sensors other than cGAS enhanced activated macrophages.ConclusionThese findings suggested that cGAS hampers the unusual noncanonical inflammasome activation through other DNA sensors.

Abstract Development of an effective therapy to overcome colistin resistance in Klebsiella pneumoniae, a common pathogen causing catheter-related biofilm infections in vascular catheters, has become a serious therapeutic challenge that must be addressed urgently. Although colistin and EDTA have successful roles for eradicating biofilms, no in vitro and in vivo studies have investigated their efficacy in catheter-related biofilm infections of colistin-resistant K. pneumoniae. In this study, colistin resistance was significantly reversed in both planktonic and mature biofilms of colistin-resistant K. pneumoniae by a combination of colistin (0.25–1 µg/ml) with EDTA (12 mg/ml). This novel colistin-EDTA combination was also demonstrated to have potent efficacy in eradicating colistin-resistant K. pneumoniae catheter-related biofilm infections, and eliminating the risk of recurrence in vivo. Furthermore, this study revealed significant therapeutic efficacy of colistin-EDTA combination in reducing bacterial load in internal organs, lowering serum creatinine, and protecting treated mice from mortality. Altered in vivo expression of different virulence genes indicate bacterial adaptive responses to survive in hostile environments under different treatments. According to these data discovered in this study, a novel colistin-EDTA combination provides favorable efficacy and safety for successful eradication of colistin-resistant K. pneumonia catheter-related biofilm infections.

Abstract Patients with systemic lupus erythematosus (SLE) have increased inflammatory cytokines, leading to periodontitis and alveolar bone loss. However, the mechanisms driving this phenomenon are still unknown. Here, we have identified novel therapeutic targets for and mediators of lupus-mediated bone loss using RNA-sequencing (RNA-seq) in a FcγRIIB -/- mouse model of lupus associated osteopenia. A total of 2,710 upregulated and 3,252 downregulated DEGs were identified. The GO and KEGG annotations revealed that osteoclast differentiation, bone mineralization, ossification, and myeloid cell development were downregulated. WikiPathways indicated that Hedgehog, TNFα NF-κB and Notch signaling pathway were also decreased. We identified downregulated targets, Sufu and Serpina12, that have important roles in bone homeostasis. Sufu and Serpina12 were related to Hedgehog signaling proteins, including Gli1, Gli2, Gli3, Ptch1, and Ptch2. Gene knockdown analysis demonstrated that Sufu, and Serpina12 contributed to osteoclastogenesis and osteoblastogenesis, respectively. Osteoclast and osteoblast marker genes were significantly decreased in Sufu-deficient and Serpina12-deficient cells, respectively. Our results suggest that alterations in Hedgehog signaling play an important role in the pathogenesis of osteopenia in FcγRIIB -/- mice. The novel DEGs and pathways identified in this study provide new insight into the underlying mechanisms of mandibular bone loss during lupus development.

Transforming growth factor beta (TGF-β) is a key factor mediating the intercellular crosstalk between the hematopoietic stem cells and their microenvironment. Here, we investigated the skeletal phenotype of transgenic mice expressing constitutively active TGF-β receptor type I under the control of Mx1-Cre (Mx1;TβRICA mice). μCT analysis showed decreased cortical thickness, and cancellous bone volume in both femurs and mandibles. Histomorphometric analysis confirmed a decrease in cancellous bone volume due to increased osteoclast number and decreased osteoblast number. Primary osteoblasts showed decreased ALP and mineralization. Constitutive TβRI activation increased osteoclast differentiation. qPCR analysis showed that Tnfsf11/Tnfrsf11b ratio, Ctsk, Sufu, and Csf1 were increased whereas Runx2, Ptch1, and Ptch2 were decreased in Mx1;TβRICA femurs. Interestingly, Gli1, Wnt3a, Sp7, Alpl, Ptch1, Ptch2, and Shh mRNA expression were reduced whereas Tnfsf11/Tnfrsf11b ratio was increased in Mx1;TβRICA mandibles. Similarly, osteoclast-related genes were increased in Mx1;TβRICA osteoclasts whereas osteoblast-related genes were reduced in Mx1;TβRICA osteoblasts. Western blot analysis indicated that SMAD2 and SMAD3 phosphorylation was increased in Mx1;TβRICA osteoblasts, and SMAD3 phosphorylation was increased in Mx1;TβRICA osteoclasts. CTSK was increased while RUNX2 and PTCH1 was decreased in Mx1;TβRICA mice. Microindentation analysis indicated decreased hardness in Mx1;TβRICA mice. Our study indicated that Mx1;TβRICA mice were osteopenic by increasing osteoclast number and decreasing osteoblast number, possibly by suppressing Hedgehog signaling pathways.

Despite the known influence of DNA methylation from lipopolysaccharide (LPS) activation, data on the O6-methylguanine-DNA methyltransferase (MGMT, a DNA suicide repair enzyme) in macrophages is still lacking. The transcriptomic profiling of epigenetic enzymes from wild-type macrophages after single and double LPS stimulation, representing acute inflammation and LPS tolerance, respectively, was performed. Small interfering RNA (siRNA) silencing of mgmt in the macrophage cell line (RAW264.7) and mgmt null (mgmtflox/flox; LysM-Crecre/−) macrophages demonstrated lower secretion of TNF-α and IL-6 and lower expression of pro-inflammatory genes (iNOS and IL-1β) compared with the control. Macrophage injury after a single LPS dose and LPS tolerance was demonstrated by reduced cell viability and increased oxidative stress (dihydroethidium) compared with the activated macrophages from littermate control mice (mgmtflox/flox; LysM-Cre−/−). Additionally, a single LPS dose and LPS tolerance also caused mitochondrial toxicity, as indicated by reduced maximal respiratory capacity (extracellular flux analysis) in the macrophages of both mgmt null and control mice. However, LPS upregulated mgmt only in LPS-tolerant macrophages but not after the single LPS stimulation. In mice, the mgmt null group demonstrated lower serum TNF-α, IL-6, and IL-10 than control mice after either single or double LPS stimulation. Suppressed cytokine production resulting from an absence of mgmt in macrophages caused less severe LPS-induced inflammation but might worsen LPS tolerance.

The O6-methylguanine-DNA methyltransferase (MGMT) is a DNA suicide repair enzyme that might be important during sepsis but has never been explored. Then, the proteomic analysis of lipopolysaccharide (LPS)-stimulated wild-type (WT) macrophages increased proteasome proteins and reduced oxidative phosphorylation proteins compared with control, possibly related to cell injury. With LPS stimulation, mgmt null (mgmtflox/flox; LysM-Crecre/-) macrophages demonstrated less profound inflammation; supernatant cytokines (TNF-α, IL-6, and IL-10) and pro-inflammatory genes (iNOS and IL-1β), with higher DNA break (phosphohistone H2AX) and cell-free DNA, but not malondialdehyde (the oxidative stress), compared with the littermate control (mgmtflox/flox; LysM-Cre-/-). In parallel, mgmt null mice (MGMT loss only in the myeloid cells) demonstrated less severe sepsis in the cecal ligation and puncture (CLP) model (with antibiotics), as indicated by survival and other parameters compared with sepsis in the littermate control. The mgmt null protective effect was lost in CLP mice without antibiotics, highlighting the importance of microbial control during sepsis immune modulation. However, an MGMT inhibitor in CLP with antibiotics in WT mice attenuated serum cytokines but not mortality, requiring further studies. In conclusion, an absence of mgmt in macrophages resulted in less severe CLP sepsis, implying a possible influence of guanine DNA methylation and repair in macrophages during sepsis.

Oxyresveratrol (ORV) is one of the novel antioxidants having been extensively studied in recent years. One of the main sources of ORV is Artocarpus lakoocha, which has been used in traditional medicine in Thailand for decades. However, the role of ORV in skin inflammation has not been clearly demonstrated. Therefore, we investigated the anti-inflammatory effects of ORV on dermatitis model. The effect of ORV was examined on human immortalized and primary skin cells exposed to bacterial components including peptidoglycan (PGN) and lipopolysaccharide (LPS) and 2,4-Dinitrochlorobenzene (DNCB)-induced dermatitis mouse model. PGN and LPS were used to induce inflammation on immortalized keratinocytes (HaCaT) and human epidermal keratinocytes (HEKa). We then performed MTT assay, Annexin V and PI assay, cell cycle analysis, real-time PCR, ELISA and Western blot in these in vitro models. H&E staining, immunohistochemistry (IHC) staining with CD3, CD4 and CD8 markers were used to evaluate the effects of ORV in in vivo model of skin inflammation using BALB/c mice. Pretreatment of HaCaT and HEKa cells with ORV inhibited pro-inflammatory cytokine production through inhibition of NF-κB pathway. In DNCB-induced dermatitis mouse model, ORV treatment reduced lesion severity, and skin thickness and numbers of CD3, CD4 and CD8 T cells in the sensitized skin of mice. In conclusion, it has been demonstrated that ORV treatment can ameliorate inflammation in the in vitro models of skin inflammation and in vivo models of dermatitis, suggesting a therapeutic potential of ORV for treatment of skin diseases particularly eczema.

Introduction: SARS-CoV-2 RNA is excreted in feces of most patients, therefore viral load in wastewater can be used as a surveillance tool to develop an early warning system to help and manage future pandemics. Methods: We collected wastewater from 24 random locations at Bangkok city center and 26 nearby suburbs from July to December 2020. SARS-CoV-2 RNA copy numbers were measured using real-time polymerase chain reaction (PCR). Results: SARS-CoV-2 RNA was detected in wastewater from both the city center and suburbs. Except for July, there were no significant differences in copy numbers between the city center and suburbs. Between October and November, a sharp rise in copy number was observed in both places followed by two to three times increase in December, related to SARS-CoV-2 cases reported for same month. Conclusions: Our study provided the first dataset related to SARS-CoV-2 viral RNA in the wastewater of Bangkok. Our results suggest that wastewater could be used as a complementary source for detecting viral RNA and predicting upcoming outbreaks and waves.

Controlof immune responses through the immunometabolism interference is interesting for sepsis treatment. Then, expression of immunometabolism-associated genes and BAM15, a mitochondrial uncoupling agent, was explored in a proinflammatory model using lipopolysaccharide (LPS) injection. Accordingly, the decreased expression of mitochondrial uncoupling proteins was demonstrated by transcriptomic analysis on metabolism-associated genes in macrophages (RAW246.7) and by polymerase chain reaction in LPS-stimulated RAW246.7 and hepatocytes (Hepa 1–6). Pretreatment with BAM15 at 24 h prior to LPS in macrophages attenuated supernatant inflammatory cytokines (IL-6, TNF-α, and IL-10), downregulated genes of proinflammatory M1 polarization (iNOS and IL-1β), upregulated anti-inflammatory M2 polarization (Arg1 and FIZZ), and decreased cell energy status (extracellular flux analysis and ATP production). Likewise, BAM15 decreased expression of proinflammatory genes (IL-6, TNF-α, IL-10, and iNOS) and reduced cell energy in hepatocytes. In LPS-administered mice, BAM15 attenuated serum cytokines, organ injury (liver enzymes and serum creatinine), and tissue cytokines (livers and kidneys), in part, through the enhanced phosphorylated αAMPK, a sensor of ATP depletion with anti-inflammatory property, in the liver, and reduced inflammatory monocytes/macrophages (Ly6C +ve, CD11b +ve) in the liver as detected by Western blot and flow cytometry, respectively. In conclusion, a proof of concept for inflammation attenuation of BAM15 through metabolic interference-induced anti-inflammation on macrophages and hepatocytes was demonstrated as a new strategy of anti-inflammation in sepsis.

Because studies on all fecal organisms (bacteria, fungi, and viruses) in sepsis are rare and bacteriophages during sepsis might have adapted against gut bacteria with possible pathogenicity, cecal ligation and puncture (CLP; a sepsis mouse model) was evaluated. In fecal bacteriome, sepsis increased Bacteroides and Proteobacteria but decreased Firmicutes, while fecal virome demonstrated increased Podoviridae when compared with sham feces. There was no difference in the fungal microbiome (predominant Ascomycota in both sham and CLP mice) and the abundance of all organisms between sepsis and control groups. Interestingly, the transfers of feces from CLP mice worsened sepsis severity when compared with sham fecal transplantation, as evaluated by mortality, renal injury (serum creatinine and histology), liver damage (liver enzyme and histology), spleen apoptosis, serum cytokines, endotoxemia, and bacteremia. In contrast, the transfers of fecal viral particles from sepsis mice, but not from sham mice, attenuated inflammation in CLP sepsis possibly through the decrease in several fecal pathogenic bacteria (such as Proteobacteria, Gammaproteobacteria, and Prevotellaceae) as evaluated by fecal microbiome analysis. Perhaps the isolation of favorable bacteriophages in sepsis feces and increased abundance ex vivo before oral treatment in a high concentration are beneficial.

Despite a well-known association between gut barrier defect (leaky gut) and several diseases, data on translocation of pathogen molecules, including bacterial DNA (blood bacteriome), lipopolysaccharide (LPS), and serum (1→3)-β-D-glucan (BG), from the gut to the blood circulation (gut translocation) in dengue are still less studied. Perhaps, dengue infection might induce gut translocation of several pathogenic molecules that affect the disease severity. At the enrollment, there were 31 dengue cases in febrile and critical phases at 4.1 ± 0.3 days and 6.4 ± 1.1 days of illness, respectively, with the leaky gut as indicated by positive lactulose-to-mannitol excretion ratio. With blood bacteriome, the patients with critical phase (more severe dengue; n = 23) demonstrated more predominant abundance in Bacteroidetes and Escherichia spp. with the lower Bifidobacteria when compared with the healthy control (n = 5). Meanwhile, most of the blood bacteriome results in dengue with febrile stage (n = 8) were comparable to the control, except for the lower Bifidobacteria in dengue cases. Additionally, endotoxemia at the enrollment was demonstrated in five (62.5%) and 19 (82.6%) patients with febrile and critical phases, respectively, while serum BG was detectable in two (25%) and 20 (87%) patients with febrile and critical phases, respectively. There were higher peripheral blood non-classical monocytes and natural killer cells (NK cells) at the enrollment in patients with febrile phage than in the cases with critical stage. Then, non-classical monocytes (CD14-CD16+) and NK cells (CD56+CD16-) increased at 4 and 7 days of illness in the cases with critical and febrile stages, respectively, the elevation of LPS and/or BG in serum on day 7 was also associated with the increase in monocytes, NK cells, and cytotoxic T cells. In summary, enhanced Proteobacteria (pathogenic bacteria from blood bacteriomes) along with increased endotoxemia and serum BG (leaky gut syndrome) might be collaborated with the impaired microbial control (lower non-classical monocytes and NK cells) in the critical cases and causing more severe disease of dengue infection.

Abstract Obesity, a major healthcare problem worldwide, induces metabolic endotoxemia through the gut translocation of lipopolysaccharides (LPS), a major cell wall component of Gram-negative bacteria, causing a chronic inflammatory state. A combination of several probiotics including Lactobacillus acidophilus 5 (LA5), a potent lactic acid-producing bacterium, has previously been shown to attenuate obesity. However, data on the correlation between a single administration of LA5 versus microbiota alteration might be helpful for the probiotic adjustment. LA5 was administered daily together with a high-fat diet (HFD) for 8 weeks in mice. Furthermore, the condition media of LA5 was also tested in a hepatocyte cell-line (HepG2 cells). Accordingly, LA5 attenuated obesity in mice as demonstrated by weight reduction, regional fat accumulation, lipidemia, liver injury (liver weight, lipid compositions, and liver enzyme), gut permeability defect, endotoxemia, and serum cytokines. Unsurprisingly, LA5 improved these parameters and acidified fecal pH leads to the attenuation of fecal dysbiosis. The fecal microbiome analysis in obese mice with or without LA5 indicated; (i) decreased Bacteroidetes (Gram-negative anaerobes that predominate in non-healthy conditions), (ii) reduced total fecal Gram-negative bacterial burdens (the sources of gut LPS), (iii) enhanced Firmicutes (Gram-positive bacteria with potential benefits) and (iv) increased Verrucomycobia, especially Akkermansia muciniphila, a bacterium with the anti-obesity property. With LA5 administration, A. muciniphila in the colon were more than 2,000 folds higher than the regular diet mice as determined by 16S rRNA. Besides, LA5 produced anti-inflammatory molecules with a similar molecular weight to LPS that reduced cytokine production in LPS-activated HepG2 cells. In conclusion, LA5 attenuated obesity through (i) gut dysbiosis attenuation, partly through the promotion of A. muciniphila (probiotics with the difficulty in preparation processes), (ii) reduced endotoxemia, and (iii) possibly decreased liver injury by producing the anti-inflammatory molecules.

Despite a previous report on less inflammatory responses in mice with an absence of the enhancer of zeste homologue 2 (Ezh2), a histone lysine methyltransferase of epigenetic regulation, using a lipopolysaccharide (LPS) injection model, proteomic analysis and cecal ligation and puncture (CLP), a sepsis model that more resembles human conditions was devised. As such, analysis of cellular and secreted protein (proteome and secretome) after a single LPS activation and LPS tolerance in macrophages from Ezh2 null (Ezh2flox/flox; LysM-Crecre/−) mice (Ezh2 null) and the littermate control mice (Ezh2fl/fl; LysM-Cre−/−) (Ezh2 control) compared with the unstimulated cells from each group indicated fewer activities in Ezh2 null macrophages, especially by the volcano plot analysis. Indeed, supernatant IL-1β and expression of genes in pro-inflammatory M1 macrophage polarization (IL-1β and iNOS), TNF-α, and NF-κB (a transcription factor) were lower in Ezh2 null macrophages compared with the control. In LPS tolerance, downregulated NF-κB compared with the control was also demonstrated in Ezh2 null cells. In CLP sepsis mice, those with CLP alone and CLP at 2 days after twice receiving LPS injection, representing sepsis and sepsis after endotoxemia, respectively, symptoms were less severe in Ezh2 null mice, as indicated by survival analysis and other biomarkers. However, the Ezh2 inhibitor improved survival only in CLP, but not LPS with CLP. In conclusion, an absence of Ezh2 in macrophages resulted in less severe sepsis, and the use of an Ezh2 inhibitor might be beneficial in sepsis.

Dendritic cells (DCs) are the most potent antigen-presenting cells that have multifaceted functions in the control of immune activation and tolerance. Hyperresponsiveness and altered tolerogenicity of DCs contribute to the development and pathogenesis of system lupus erythematosus (SLE); therefore, DC-targeted therapies aimed at inducing specific immune tolerance have become of great importance for the treatment of SLE. This study developed a new nanoparticle (NP) containing a biodegradable PDMAEMA-PLGA copolymer for target-oriented delivery to DCs in situ. PDMAEMA-PLGA NPs provided sustained drug release and exhibited immunosuppressive activity in FLT3L and GM-CSF-derived bone marrow in conventional DCs (BM-cDCs). PDMAEMA-PLGA NPs improved dexamethasone capability to convert wild-type and Fcgr2b-/- BM-cDCs from an immunogenic to tolerogenic state, and BM-cDCs treated with dexamethasone-incorporated PDMAEMA-PLGA NPs (Dex-NPs) efficiently mediated regulatory T cell (Treg) expansion in vitro. Dex-NP therapy potentially alleviated lupus disease in Fcgr2b-/- mice by mediating Foxp3+ Treg expansion in an antigen-specific manner. Our findings substantiate the superior efficacy of DC-targeted therapy using the PDMAEMA-PLGA NP delivery system and provide further support for clinical development as a potential therapy for SLE. Furthermore, PDMAEMA-PLGA NP may be a versatile platform for DC-targeted therapy to induce antigen-specific immune tolerance to unwanted immune responses that occur in autoimmune disease, allergy, and transplant rejection.