Interleukin-8 and neutrophil extracellular traps in children with lupus nephritis and vitamin C deficiency.

BACKGROUND: Vitamin C is a potent scavenger of reactive oxygen species, which induce neutrophil extracellular trap (NET) formation. NETs are a major source of autoantigens and are involved in systemic lupus erythematosus (SLE) pathogenesis. We determined vitamin C status and evaluated NET formation and inflammatory cytokines in children with lupus nephritis. METHODS: Serum vitamin C was measured in 46 patients (82.6% females, mean age 14.5 ± 0.3 years). Vitamin C levels < 0.3 mg/dL indicated vitamin C deficiency. Patients were divided into two groups according to serum vitamin C levels: normal and low (< 0.3 mg/dL). We compared NET formation and levels of SLE-related cytokines, including interleukin (IL)-8, IL-10, and tumor necrosis factor-α (TNF-α), between groups. NET formation was determined through measurement of serum citrullinated histone 3 levels and mRNA expression of peptidyl arginine deiminase-4 and assessment of the percentage of neutrophils with NETs by immunofluorescence. RESULTS: Nine patients (19.6%) had vitamin C deficiency. Kidney pathology assessment at disease onset revealed that histological activity index and number of kidney biopsies containing crescentic glomeruli were higher in vitamin C-deficient patients, but chronicity index was not. NET formation and serum IL-8 were more prominent in vitamin C-deficient patients. Serum IL-8 levels were 12.9 ± 5.2 pg/mL in low vitamin C group and 5.2 ± 0.9 pg/mL in normal vitamin C group (p = 0.03). Serum IL-10 and TNF-α were similar between groups. CONCLUSIONS: Our study demonstrated correlation among vitamin C deficiency, increased NET formation, and IL-8 upregulation in children with lupus nephritis. A prospective study is required to evaluate cause‒effect relationships of vitamin C status, NET formation and IL-8 expression.

Differential Gene Expression Involved in Bone Turnover of Mice Expressing Constitutively Active TGFß Receptor Type I.

Transforming growth factor beta (TGF-ß) is ubiquitously found in bone and plays a key role in bone turnover. Mice expressing constitutively active TGF-ß receptor type I (Mx1;TßRICA mice) are osteopenic. Here, we identified the candidate genes involved in bone turnover in Mx1;TßRICA 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 TßRI-associated bone loss.

Lupus exacerbation in ovalbumin-induced asthma in Fc gamma receptor IIb deficient mice, partly due to hyperfunction of dendritic cells.

BACKGROUND: Although allergy might be another factor that exacerbates lupus as demonstrated by several epidemiologic studies, the direct correlation between lupus activities and allergy is still in question. OBJECTIVE: To explore the correlation between allergic reaction and lupus activities. METHODS: The allergic asthma model using ovalbumin (OVA) administration in wildtype (WT) and Fc gamma receptor IIb deficient (FcgRIIb-/-) mice (a lupus-prone model) together with in vitro experiments on bone marrow-derived dendritic cells (DCs) were performed. RESULTS: At 2-weeks-post OVA, both WT and FcgRIIb-/- mice demonstrated similar allergic reaction as indicated by an elevation of IgE and IL-4 in serum with asthma-liked lung histology (lung weight, inflammatory score, and bronchial thickness) with increased spleen weight. Apoptosis in the lungs and spleens (activated caspase 3 immunohistochemistry) was detected only in OVA-administered FcgRIIb-/- mice. Surprisingly, OVA-administered FcgRIIb-/- mice, demonstrated active lupus nephritis, as indicated by anti-dsDNA, proteinuria, and renal immune complex deposition (immunohistochemistry analysis) implying an impact of allergy on lupus activities. Meanwhile, serum creatinine and gut permeability defect (FitC-dextran assay and endotoxemia) were not different between the FcgRIIb-/- mice with OVA versus with control. In parallel, FcgRIIb-/- DCs were more susceptible to activations by OVA and lipopolysaccharide (LPS) than WT DCs as demonstrated by CD80 with major histocompatibility complex II (MHC II) using flow cytometry analysis. CONCLUSION: OVA-induced allergy in FcgRIIb-/- mice exacerbated lupus activity, possibly due to hyper-responsiveness of FcgRIIb-/- DCs over WT from the loss of inhibitory FcgRIIb. The proper control of allergy might be beneficial for lupus.

Enhanced lupus progression in alcohol-administered Fc gamma receptor-IIb-deficiency lupus mice, partly through leaky gut-induced inflammation.

Alcohol can induce a leaky gut, with translocation of microbial molecules from the gut into the blood circulation. Although the contribution of inflammation to organ-mediated damage in lupus has been previously demonstrated, the mechanistic roles of alcohol consumption in lupus activation are not known. Herein, we tested the effects of 10-week lasting alcohol administration on organ damages and immune responses in 8-week-old lupus-prone Fc gamma receptor IIb-deficient (FcγRIIb-/- ) mice. Our study endpoints were evaluation of systemic inflammation and assessment of fecal dysbiosis along with endotoxemia. In comparison with alcohol-administered wild-type mice, FcγRIIb-/- mice demonstrated more prominent liver damage (enzyme, histological score, apoptosis, malondialdehyde oxidant) and serum interleukin(IL)-6 levels, despite a similarity in leaky gut (fluorescein isothiocyanate-dextran assay, endotoxemia and gut occludin-1 immunofluorescence), fecal dysbiosis (microbiome analysis) and endotoxemia. All alcohol-administered FcγRIIb-/- mice developed lupus-like characteristics (serum anti-dsDNA, proteinuria, serum creatinine and kidney injury score) with spleen apoptosis, whereas control FcγRIIb-/- mice showed only a subtle anti-dsDNA. Both alcohol and lipopolysaccharide (LPS) similarly impaired enterocyte integrity (transepithelial electrical resistance), and only LPS, but not alcohol, upregulated the IL-8 gene in Caco-2 cells. In macrophages, alcohol mildly activated supernatant cytokines (tumor necrosis factor-α and IL-6), but not M1 polarization-associated genes (IL-1ß and iNOS), whereas LPS prominently induced both parameters (more prominent in FcγRIIb-/- macrophages than wild type). There was no synergy in LPS plus alcohol compared with LPS alone in both enterocytes and macrophages. In conclusion, alcohol might exacerbate lupus-like activity partly through a profound inflammation from the leaky gut in FcγRIIb-/- mice.

The leaky gut and the gut microbiome in sepsis - targets in research and treatment.

Both a leaky gut (a barrier defect of the intestinal surface) and gut dysbiosis (a change in the intestinal microbial population) are intrinsic to sepsis. While sepsis itself can cause dysbiosis, dysbiosis can worsen sepsis. The leaky gut syndrome refers to a status with which there is an increased intestinal permeability allowing the translocation of microbial molecules from the gut into the blood circulation. It is not just a symptom of gastrointestinal involvement, but also an underlying cause that develops independently, and its presence could be recognized by the detection, in blood, of lipopolysaccharides and (1→3)-ß-D-glucan (major components of gut microbiota). Gut-dysbiosis is the consequence of a reduction in some bacterial species in the gut microbiome, as a consequence of intestinal mucosal immunity defect, caused by intestinal hypoperfusion, immune cell apoptosis, and a variety of enteric neuro-humoral-immunity responses. A reduction in bacteria that produce short-chain fatty acids could change the intestinal barriers, leading to the translocation of pathogen molecules, into the circulation where it causes systemic inflammation. Even gut fungi might be increased in human patients with sepsis, even though this has not been consistently observed in murine models of sepsis, probably because of the longer duration of sepsis and also antibiotic use in patients. The gut virobiome that partly consists of bacteriophages is also detectable in gut contents that might be different between sepsis and normal hosts. These alterations of gut dysbiosis altogether could be an interesting target for sepsis adjuvant therapies, e.g., by faecal transplantation or probiotic therapy. Here, current information on leaky gut and gut dysbiosis along with the potential biomarkers, new treatment strategies, and future research topics are mentioned.

Lacticaseibacillus rhamnosus dfa1 Attenuate Cecal Ligation-Induced Systemic Inflammation through the Interference in Gut Dysbiosis, Leaky Gut, and Enterocytic Cell Energy.

Despite an uncommon condition, the clinical management of phlegmon appendicitis (retention of the intra-abdominal appendiceal abscess) is still controversial, and probiotics might be partly helpful. Then, the retained ligated cecal appendage (without gut obstruction) with or without oral Lacticaseibacillus rhamnosus dfa1 (started at 4 days prior to the surgery) was used as a representative model. At 5 days post-surgery, the cecal-ligated mice demonstrated weight loss, soft stool, gut barrier defect (leaky gut using FITC-dextran assay), fecal dysbiosis (increased Proteobacteria with reduced bacterial diversity), bacteremia, elevated serum cytokines, and spleen apoptosis without kidney and liver damage. Interestingly, the probiotics attenuated disease severity as indicated by stool consistency index, FITC-dextran assay, serum cytokines, spleen apoptosis, fecal microbiota analysis (reduced Proteobacteria), and mortality. Additionally, impacts of anti-inflammatory substances from culture media of the probiotics were demonstrated by attenuation of starvation injury in the Caco-2 enterocyte cell line as indicated by transepithelial electrical resistance (TEER), inflammatory markers (supernatant IL-8 with gene expression of TLR4 and NF-κB), cell energy status (extracellular flux analysis), and the reactive oxygen species (malondialdehyde). In conclusion, gut dysbiosis and leaky-gut-induced systemic inflammation might be helpful clinical parameters for patients with phlegmon appendicitis. Additionally, the leaky gut might be attenuated by some beneficial molecules from probiotics.

Lipopolysaccharide Tolerance Enhances Murine Norovirus Reactivation: An Impact of Macrophages Mainly Evaluated by Proteomic Analysis.

Because of endotoxemia during sepsis (a severe life-threatening infection), lipopolysaccharide (LPS) tolerance (the reduced responses to the repeated LPS stimulation) might be one of the causes of sepsis-induced immune exhaustion (the increased susceptibility to secondary infection and/or viral reactivation). In LPS tolerance macrophage (twice-stimulated LPS, LPS/LPS) compared with a single LPS stimulation (N/LPS), there was (i) reduced energy of the cell in both glycolysis and mitochondrial activities (extracellular flux analysis), (ii) decreased abundance of the following proteins (proteomic analysis): (a) complex I and II of the mitochondrial electron transport chain, (b) most of the glycolysis enzymes, (c) anti-viral responses with Myxovirus resistance protein 1 (Mx1) and Ubiquitin-like protein ISG15 (Isg15), (d) antigen presentation pathways, and (iii) the down-regulated anti-viral genes, such as Mx1 and Isg15 (polymerase chain reaction). To test the correlation between LPS tolerance and viral reactivation, asymptomatic mice with and without murine norovirus (MNV) infection as determined in feces were tested. In MNV-positive mice, MNV abundance in the cecum, but not in feces, of LPS/LPS mice was higher than that in N/LPS and control groups, while MNV abundance of N/LPS and control were similar. Additionally, the down-regulated Mx1 and Isg15 were also demonstrated in the cecum, liver, and spleen in LPS/LPS-activated mice, regardless of MNV infection, while N/LPS more prominently upregulated these genes in the cecum of MNV-positive mice compared with the MNV-negative group. In conclusion, defects in anti-viral responses after LPS tolerance, perhaps through the reduced energy status of macrophages, might partly be responsible for the viral reactivation. More studies on patients are of interest.

Lipopolysaccharide Impedes Bone Repair in FcγRIIB-Deficient Mice.

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-ß, 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.

Accelerated Bone Loss in Transgenic Mice Expressing Constitutively Active TGF-ß Receptor Type I.

Transforming growth factor beta (TGF-ß) 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-ß receptor type I under the control of Mx1-Cre (Mx1;TßRICA 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 TßRI 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;TßRICA femurs. Interestingly, Gli1, Wnt3a, Sp7, Alpl, Ptch1, Ptch2, and Shh mRNA expression were reduced whereas Tnfsf11/Tnfrsf11b ratio was increased in Mx1;TßRICA mandibles. Similarly, osteoclast-related genes were increased in Mx1;TßRICA osteoclasts whereas osteoblast-related genes were reduced in Mx1;TßRICA osteoblasts. Western blot analysis indicated that SMAD2 and SMAD3 phosphorylation was increased in Mx1;TßRICA osteoblasts, and SMAD3 phosphorylation was increased in Mx1;TßRICA osteoclasts. CTSK was increased while RUNX2 and PTCH1 was decreased in Mx1;TßRICA mice. Microindentation analysis indicated decreased hardness in Mx1;TßRICA mice. Our study indicated that Mx1;TßRICA mice were osteopenic by increasing osteoclast number and decreasing osteoblast number, possibly by suppressing Hedgehog signaling pathways.

Less Severe Sepsis in Cecal Ligation and Puncture Models with and without Lipopolysaccharide in Mice with Conditional Ezh2-Deleted Macrophages (LysM-Cre System).

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-1ß and expression of genes in pro-inflammatory M1 macrophage polarization (IL-1ß 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.

Immunosuppressive Polymeric Nanoparticles Targeting Dendritic Cells Alleviate Lupus Disease in Fcgr2b-/- Mice by Mediating Antigen-Specific Immune Tolerance.

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.

Less Severe Polymicrobial Sepsis in Conditional mgmt-Deleted Mice Using LysM-Cre System, Impacts of DNA Methylation and MGMT Inhibitor in Sepsis.

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-1ß), 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.

The Regulatory Roles of Ezh2 in Response to Lipopolysaccharide (LPS) in Macrophages and Mice with Conditional Ezh2 Deletion with LysM-Cre System.

The responses of macrophages to lipopolysaccharide (LPS) might determine the direction of clinical manifestations of sepsis, which is the immune response against severe infection. Meanwhile, the enhancer of zeste homologue 2 (Ezh2), a histone lysine methyltransferase of epigenetic regulation, might interfere with LPS response. Transcriptomic analysis on LPS-activated wild-type macrophages demonstrated an alteration of several epigenetic enzymes. Although the Ezh2-silencing macrophages (RAW264.7), using small interfering RNA (siRNA), indicated a non-different response to the control cells after a single LPS stimulation, the Ezh2-reducing cells demonstrated a less severe LPS tolerance, after two LPS stimulations, as determined by the higher supernatant TNF-α. With a single LPS stimulation, Ezh2 null (Ezh2flox/flox; LysM-Crecre/-) macrophages demonstrated lower supernatant TNF-α than Ezh2 control (Ezh2fl/fl; LysM-Cre-/-), perhaps due to an upregulation of Socs3, which is a suppressor of cytokine signaling 3, due to the loss of the Ezh2 gene. In LPS tolerance, Ezh2 null macrophages indicated higher supernatant TNF-α and IL-6 than the control, supporting an impact of the loss of the Ezh2 inhibitory gene. In parallel, Ezh2 null mice demonstrated lower serum TNF-α and IL-6 than the control mice after an LPS injection, indicating a less severe LPS-induced hyper-inflammation in Ezh2 null mice. On the other hand, there were similar serum cytokines after LPS tolerance and the non-reduction of serum cytokines after the second dose of LPS, indicating less severe LPS tolerance in Ezh2 null mice compared with control mice. In conclusion, an absence of Ezh2 in macrophages resulted in less severe LPS-induced inflammation, as indicated by low serum cytokines, with less severe LPS tolerance, as demonstrated by higher cytokine production, partly through the upregulated Socs3.

Less Severe Lipopolysaccharide-Induced Inflammation in Conditional mgmt-Deleted Mice with LysM-Cre System: The Loss of DNA Repair in Macrophages.

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-1ß) 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.

Determination of the number of times for reuse of hemodialysis dialyzer through macrophage responses against dialyzer-eluted protein, a proposed method.

BACKGROUND: Although the number of times dialyzer-reuse in hemodialysis is currently determined by the total volume of the dialyzer, the determination by macrophage activation using dialyzer-eluted protein might predict systemic inflammation. OBJECTIVE: The pro-inflammatory activities of the proteins from 5- and 15-times reused dialyzers were tested as a proof of concept experiment. METHODS: Accumulated proteins in dialyzers were eluted by the roller pump (the recirculation of 100 mL of buffer in a dialyzer with a roller pump at 15 mL/min for 2 h) or infusion procedures (infusion of 100 mL buffer in a dialyzer for 2 h) using chaotropic or potassium phosphate buffers (KPB) before the activation on macrophages cell lines (THP-1-derived human macrophages or RAW264.7 murine macrophages). RESULTS: The concentrations of dialyzer-eluted protein from both methods were not different and the infusion procedure was further used. The eluted proteins (by both buffers) from 15-times-reused dialyzers reduced cell viability, increased supernatant cytokines (TNF-α and IL-6), and upregulated pro-inflammatory genes (IL-1ß and iNOS) in either THP-1-derived or RAW264.7 macrophages (higher responses in RAW264.7 cells) compared with the new dialyzer. Meanwhile, the 5-times-reused dialyzer protein did not reduce cell viability but enhanced some of these pro-inflammatory macrophage markers. CONCLUSIONS: Due to the simpler preparation of KPB over chaotropic buffer with an easier protocol of RAW264.7 over THP-1-derived macrophages, the responses of RAW264.7 against dialyzer-eluted protein with infusion method using KPB buffer were proposed for determination of the number of times dialyzer reuse in hemodialysis.

TMAO reductase, a biomarker for gut permeability defect induced inflammation, in mouse model of chronic kidney disease and dextran sulfate solution-induced mucositis.

BACKGROUND: The excretion of trimethylamine N-oxide (TMAO) (uremic toxin) into the intestine might be enhanced, due to the limited renal elimination in chronic kidney disease (CKD), possibly induced TMAO reductase (a TMAO-neutralizing enzyme) in gut bacteria. Detection of TMAO reductase in serum could be used as a biomarker of gut permeability defect. OBJECTIVE: To explore the correlation between serum TMAO reductase, gut leakage, and systemic inflammation in CKD. METHODS: Mouse models of gut leakage; including 5/6 nephrectomy-induced chronic kidney disease (CKD), a model without colitis, and 1.5% dextran sulfate solution (DSS), a colitis model, were performed. In parallel, serum samples from patients with chronic hemodialysis (n = 48) and the healthy control (n = 20) were analyzed. RESULTS: Gut-leakage (FITC-dextran, endotoxemia, and reduced intestinal tight junction protein) was detected in both CKD and DSS models. While TMAO reductase and TMAO were elevated in the serum of both mouse models and patients, TMAO reductase correlated with TMAO, gut- leakage, and serum IL-6 only in mice but not in patients. Notably, endotoxemia was used as a surrogate marker of gut leakage in patients. In patients, TMAO reductase and TMAO did not correlate with serum IL-6 and vascular complications using the ankle-brachial index and cardio-ankle vascular index. CONCLUSIONS: Serum TMAO reductase was elevated in CKD mice and patients with CKD. Serum TMAO reductase was correlated with TMAO and gut-leakage only in mice but not in patients. Further studies in patients are needed to determine the benefit of serum TMAO reductase in patients with CKD.

Social restriction versus herd immunity policies in the early phase of the SARS-CoV-2 pandemic: A mathematical modelling study.

BACKGROUND: Two main strategies to cope with the coronavirus disease 2019 (COVID-19) pandemic-lockdown (social restriction) and non-lockdown (herd immunity plan)-have been implemented in several countries. OBJECTIVE: This study aims to statistically compare the outcomes of the two strategies, represented by data from Thailand and Sweden, respectively. METHODS: Data for COVID-19 pandemic control from Thailand, representing social restriction, versus data from Sweden, representing the herd immunity plan, collected from January 13 to May 31, 2020, were analyzed by using the SIR (susceptible, infectious, recovered) model. RESULTS: The SIR model analysis demonstrated a beneficial effect of each model on the attenuation of the mortality rate, with lower mortality in social restriction and shorter overall pandemic duration in the herd immunity plan. However, the herd immunity plan demonstrated a higher mortality rate than social restriction (46.9% versus 1.9%) despite the later entry of the virus in Sweden. When the SIR model was used for predicting the COVID-19 status, Sweden was shown to likely end its COVID-19 epidemic earlier than Thailand (268 vs. 368 days). With the nonlinear estimation, at least one log difference between total confirmed cases versus active cases could be used as an indicator for relaxation of the lockdown policy in Thailand. CONCLUSIONS: Both the social restriction and herd immunity plans are beneficial for COVID-19 pandemic control in terms of the amelioration of pandemic mortality. The cumulative number of total recovered cases might be a potential parameter that could be used for determining the policy direction for COVID-19 control.

Lactobacillus rhamnosus L34 attenuates chronic kidney disease progression in a 5/6 nephrectomy mouse model through the excretion of anti-inflammatory molecules.

BACKGROUND: Although pathogenic gut microbiota causes gut leakage, increases translocation of uremic toxins into circulation and accelerates CKD progression, the local strain of Lactobacillus rhamnosus L34 might attenuate gut leakage. We explored the effects of L34 on kidney fibrosis and levels of gut-derived uremic toxins (GDUTs) in 5/6 nephrectomy (5/6Nx) mice. METHODS: At 6 weeks post-5/6Nx in mice, either L34 (1 × 106 CFU) or phosphate buffer solution (as 5/6Nx control) was fed daily for 14 weeks. In vitro, the effects of L34-conditioned media with or without indoxyl sulfate (a representative GDUT) on inflammation and cell integrity (transepithelial electrical resistance; TEER) were assessed in Caco-2 (enterocytes). In parallel, the effects on proinflammatory cytokines and collagen expression were assessed in HK2 proximal tubular cells. RESULTS: At 20 weeks post-5/6Nx, L34-treated mice showed significantly fewer renal injuries, as evaluated by (i) kidney fibrosis area (P < 0.01) with lower serum creatinine and proteinuria, (ii) GDUT including trimethylamine-N-oxide (TMAO) (P = 0.02) and indoxyl sulfate (P < 0.01) and (iii) endotoxin (P = 0.03) and serum TNF-α (P = 0.01) than 5/6Nx controls. Fecal microbiome analysis revealed an increased proportion of Bacteroidetes in 5/6Nx controls. After incubation with indoxyl sulfate, Caco-2 enterocytes had higher interleukin-8 and nuclear factor κB expression and lower TEER values, and HK2 cells demonstrated higher gene expression of TNF-α, IL-6 and collagen (types III and IV). These indoxyl sulfate-activated parameters were attenuated with L34-conditioned media, indicating the protective role of L34 in enterocyte integrity and renal fibrogenesis. CONCLUSION: L34 attenuated uremia-induced systemic inflammation by reducing GDUTs and gut leakage that provided renoprotective effects in CKD.

Halogenated Baicalein as a Promising Antiviral Agent toward SARS-CoV-2 Main Protease.

The coronavirus disease pandemic is a constant reminder that global citizens are in imminent danger of exposure to emerging infectious diseases. Therefore, developing a technique for inhibitor discovery is essential for effective drug design. Herein, we proposed fragment molecular orbital (FMO)-based virtual screening to predict the molecular binding energy of potential severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease inhibitors. The integration of quantum mechanical approaches and trajectory analysis from a microsecond molecular dynamics simulation was used to identify potential inhibitors. We identified brominated baicalein as a potent inhibitor of the SARS-CoV-2 main protease and confirmed its inhibitory activity in an in vitro assay. Brominated baicalein did not demonstrate significant toxicity in either in vitro or in vivo studies. The pair interaction energy from FMO-RIMP2/PCM and inhibitory constants based on the protease enzyme assay suggested that the brominated baicalein could be further developed into novel SARS-CoV-2 protease inhibitors.

Gut Barrier Damage and Gut Translocation of Pathogen Molecules in Lupus, an Impact of Innate Immunity (Macrophages and Neutrophils) in Autoimmune Disease.

The gut barrier is a single cell layer that separates gut micro-organisms from the host, and gut permeability defects result in the translocation of microbial molecules from the gut into the blood. Despite the silent clinical manifestation, gut translocation of microbial molecules can induce systemic inflammation that might be an endogenous exacerbating factor of systemic lupus erythematosus. In contrast, circulatory immune-complex deposition and the effect of medications on the gut, an organ with an extremely large surface area, of patients with active lupus might cause gut translocation of microbial molecules, which worsens lupus severity. Likewise, the imbalance of gut microbiota may initiate lupus and/or interfere with gut integrity which results in microbial translocation and lupus exacerbation. Moreover, immune hyper-responsiveness of innate immune cells (macrophages and neutrophils) is demonstrated in a lupus model from the loss of inhibitory Fc gamma receptor IIb (FcgRIIb), which induces prominent responses through the cross-link between activating-FcgRs and innate immune receptors. The immune hyper-responsiveness can cause cell death, especially apoptosis and neutrophil extracellular traps (NETosis), which possibly exacerbates lupus, partly through the enhanced exposure of the self-antigens. Leaky gut monitoring and treatments (such as probiotics) might be beneficial in lupus. Here, we discuss the current information on leaky gut in lupus.