Concentrated Supernatant Research Articles

THE ANTI-ASPERGILLUS POTENTIAL OF OPTIMIZED BIOSYNTHESIZED REDUCED GRAPHENE OXIDE/SILVER NANOCOMPOSITE USING ESCHERICHIA COLI D8 (MF062579)

Conventional antifungal agents have failed to treat several infectious diseases of many Aspergillus strains. These strains have been linked to the production of high-potency mycotoxins, which cause mould diseases on fruits and vegetables and have negative health consequences. The current study aimed to develop new effective nanomaterials using efficient methods that were stable and antifungal. The current work has been carried out to use the free cell supernatant of Escherichia coli D8 (MF062579) in the biosynthesis of reduced graphene oxide/silver nanocomposite (rGO/AgNC). At room temperature, the nanocomposite was prepared by an economical and simple one-step approach. Different parameters were optimized in the biofabrication of rGO/AgNC such as silver nitrate concentration, bacterial supernatant concentration, pH value, and temperature. Ultraviolet-visible spectroscopy (UV-Vis), Fourier transform infrared spectroscopy (FT-IR), transmission electron microscopy (TEM), X-ray diffraction (XRD), and Zeta analyses were used to evaluate the optimized rGO/AgNC. The biosynthesis process was performed within minutes in the incidence of solar irradiation. The mean size of silver nanoparticles (AgNPs) was estimated to be 9-18 nm. The biogenesis of spherical-shaped, well-dispersed AgNPs was validated by TEM images. AgNPs have a positive potential value, as seen by the zeta potential graph. rGO/AgNC had a harmful effect on the ultrastructure of rGO/AgNC-treated Aspergillus including membranes damage, malformation, and complete lysis of fungal cells in addition to enzymatic inhibition in lactate dehydrogenase activity. This study states a well-designed approach to develop a new antimicrobial agent, rGO/AgNC, against pathogenic human and phytopathogenic Aspergillus spp. in addition to a probable mechanism for the nanomaterial’s antimicrobial action.

Genomic and pathogenic characterization of RTX toxin producing Rodentibacter sp. that is closely related to Rodentibacter haemolyticus.

Rodentibacter spp. are opportunistic pathogens that are often isolated from the upper respiratory tracts of laboratory rodents. In particular, R. pneumotropicus and R. heylii require considerable caution in rodent colonies, as they cause lethal pneumonia in rodents. A new species, R. haemolyticus, has recently been classified in the genus, and a very closely related strain, Rodentibacter sp. strain JRC, has been isolated in Japan. This study focused on strain JRC by performing genomic and pathogenic analyses. Draft genome sequencing of strain JRC identified several genes coding for putative virulent proteins, including hemolysin and adhesin. Furthermore, we found a new RTX (repeats-in-structural toxin) toxin gene in the genome, which was predicted to produce a critical virulence factor (RTXIA) similar to Enterobacteriaceae. The concentrated culture supernatant containing RTX toxin (RTXIA) showed cytotoxicity toward RAW264.7 cells. Pre-incubation with anti-CD11a attenuated the cytolysis, suggesting that the concentrated culture supernatant containing RTXIA is cell surface LFA-1 mediated cytolysin. Experimental infection of strain JRC intranasally with 5 female BALB/c-Rag2-/- mice showed 60% lethality and was not significantly different from those of R. pneumotropicus ATCC 35149T using the log-rank test. Combined with our finding that RTXIA has an almost identical amino acid sequence (98% identity) to that of R. haemolyticus 1625/19T, these results strongly suggest that RTXIA-producing strain JRC (and related R. haemolyticus) is pathogenic to immunodeficient rodents, and both agents should be excluded in laboratory rodent colonies.

Toll-like Receptor 7 (TLR7) Is Expressed in Adipocytes and the Pharmacological TLR7 Agonist Imiquimod and Adipocyte-Derived Cell-Free Nucleic Acids (cfDNA) Regulate Adipocyte Function.

Endosome-localized Toll-like receptors (TLRs) 3 and 9 are expressed and functionally active in adipocytes. The functionality and role of TLR7 in adipocyte biology and innate immunity of adipose tissue (AT) is poorly characterized. We analyzed TLR7 mRNA and protein expression in murine 3T3-L1 and primary adipocytes, in co-cultures of 3T3-L1 adipocytes with murine J774A.1 monocytes and in human AT. The effects of TLR7 agonists imiquimod (IMQ) and cell-free nucleic acids (cfDNA) on adipokine concentration in cell-culture supernatants and gene expression profile were investigated. We found that TLR7 expression is strongly induced during adipocyte differentiation. TLR7 gene expression in adipocytes and AT stroma-vascular cells (SVC) seems to be independent of TLR9. IMQ downregulates resistin concentration in adipocyte cell-culture supernatants and modulates gene expression of glucose transporter Glut4. Adipocyte-derived cfDNA reduces adiponectin and resistin in cell-culture supernatants and potentially inhibits Glut4 gene expression. The responsiveness of 3T3-L1 adipocytes to imiquimod is preserved in co-culture with J774A.1 monocytes. Obesity-related, adipocyte-derived cfDNA engages adipocytic pattern recognition receptors (PRRs), modulating AT immune and metabolic homeostasis during adipose inflammation.

Effect of calcium peroxide pretreatment on anaerobic digestion of primary and secondary sludge of A2/O process

In order to evaluate comprehensively the impact of calcium peroxide (CP) on anaerobic digestion (AD) of primary sludge (P-sludge) and secondary sludge (S-sludge) of anaerobic/anoxic/aerobic process, a series of batch experiments were conducted in the lab scale. Results showed that CP pretreatment could kill Fecal Coliform completely in 72.0 h and promote the solubilization of S-sludge more significantly than P-sludge. Compared with the control, the maximum CH4 production potential and the maximum specific CH4 production rate of P-sludge AD only increased by 11.2 % and 6.10 %, respectively, but those of S-sludge AD increased by 40.7 % and 58.4 % after the same pretreatment, indicating that CP pretreatment was more conducive to methane production of S-sludge. Moreover, the increase of alkalinity and initial acetate concentration of S-sludge was greater than that of P-sludge after pretreatment according to the process analysis of hydrolysis. Notably, CP pretreatment reduced the PO43−-P concentration and chromaticity in the AD supernatant of both sludges, which was beneficial to the subsequent treatment of biogas slurry. In addition, CP pretreatment increased the content of total nutrient and decreased the content of harmful heavy metals in both biogas residues. Conclusively, compared with P-sludge, CP pretreatment is more suitable for S-sludge AD. This work firstly provides a comprehensive evaluation of CP pretreatment to waste sludge produced in anaerobic/anoxic/aerobic process of wastewater treatment plant (WWTP), which is of great significance in enriching the pretreatment technology of waste sludge and promoting the application of CP pretreatment in WWTP.

Basic calcium phosphate crystals induce the expression of extracellular matrix remodelling enzymes in tenocytes.

Basic calcium phosphate (BCP) crystals contribute to several syndromes associated with tendon disease, including acute calcific tendinitis and Milwaukee shoulder syndrome. Interactions between BCP crystals and tenocytes (tendon cells) may contribute to these clinical syndromes. This study aimed to determine the direct effects of BCP crystals on tenocyte function and viability. In vitro assays were used to assess changes in human tenocytes cultured with BCP crystals. Real-time PCR was used to determine changes in the expression of tendon-related genes and extracellular matrix remodelling enzymes (MMPs; a disintegrin and metalloproteases, ADAMTS; and tissue inhibitor of metalloproteinases, TIMPs). ELISA was used to measure protein concentrations in tenocyte supernatants. MTT and alamarBlue™ assays were used to determine changes in cell viability. BCP crystals upregulated tenocyte gene expression of MMP-1, MMP-3, ADAMTS-4 and TIMP-1 after 24 h. Time-course experiments showed expression peaked at 8 h for TIMP-1 and 48 h for MMP-1 and ADAMTS-4. Cyclooxygenase (COX)-1 gene expression was upregulated after 48 h. Tenocytes did not alter expression of scleraxis and tendon collagens, and expression of pro-inflammatory cytokines was not induced with BCP crystals. BCP crystals increased tenocyte release of prostaglandin E2 (PGE2) and MMP-1 protein after 24 h. However, neither COX-1 inhibition nor COX-2 inhibition led to consistent change in BCP crystal-induced tenocyte gene expression of extracellular matrix remodelling enzymes. BCP crystals had no effect on tenocyte viability. BCP crystals induce extracellular matrix remodelling enzymes, but not inflammatory cytokines, in tenocytes.

Effects of Macrophage Inflammatory Protein 1 Alpha and Beta Chemokine Concentrations in the Progression of Endometriosis

Objective: The aim of this study was to understand the pathophysiologic mechanisms of macrophage inflammatory protein 1 alpha (MIP1a) and beta (MIP1b) chemokines in endometriosis, and to understand the immuno-pathophysiology underlying the progression of this disease. Methods: Analyses and conclusions outlined in this study were based on in vitro experiments conducted using supernatants collected from cultured lymphocytes taken from women with endometriosis and from healthy donors. The study group included 30 patients meeting the outlined inclusion criteria who were diagnosed at various clinical stages of endometriosis following a laparoscopic procedure and subsequent histopathological examination. The control group were 50 patients with infertility but without endometriosis. Blood samples were taken and the resulting buffy coat was used to establish lymphocyte cultures. After plating the cells, phytohaemagglutinin (PHA) was added and after 24 hours of incubation, supernatants were collected by centrifugation and subjected to analysis. Results: MIP1a and MIP1b levels in PHA-stimulated lymphocyte cultures from women with endometriosis were elevated when compared to controls, and this difference was highly statistically significant for both cytokines (p = 0.00001 and p = 0.000026, respectively). Additionally, we analyzed the correlation between the occurrence of endometriosis and PHA-stimulated chemokine concentration in lymphocyte culture supernatants. Both MIP1a and MIP1b exhibited a statistical significance with the presence of endometriosis (p = 0.00001). Conclusions: In this study, we observed a significant increase in secretion of selected chemokine factors in in vitro cultures of lymphocytes from women diagnosed with endometriosis. This feature may indicate an essential role for these chemotactic factors in the pathogenesis of endometriosis. To fully understand the influence of MIP1a and MIP1b on disease progression, it will be necessary in future studies to determine chemokine concentrations at each stage of endometriosis.

Spleen tyrosine kinase mediates innate and adaptive immune crosstalk in SARS‐CoV‐2 mRNA vaccination

Durable cell‐mediated immune responses require efficient innate immune signaling and the release of pro‐inflammatory cytokines. How precisely mRNA vaccines trigger innate immune cells for shaping antigen specific adaptive immunity remains unknown. Here, we show that SARS‐CoV‐2 mRNA vaccination primes human monocyte‐derived macrophages for activation of the NLRP3 inflammasome. Spike protein exposed macrophages undergo NLRP3‐driven pyroptotic cell death and subsequently secrete mature interleukin‐1β. These effects depend on activation of spleen tyrosine kinase (SYK) coupled to C‐type lectin receptors. Using autologous cocultures, we show that SYK and NLRP3 orchestrate macrophage‐driven activation of effector memory T cells. Furthermore, vaccination‐induced macrophage priming can be enhanced with repetitive antigen exposure providing a rationale for prime‐boost concepts to augment innate immune signaling in SARS‐CoV‐2 vaccination. Collectively, these findings identify SYK as a regulatory node capable of differentiating between primed and unprimed macrophages, which modulate spike protein‐specific T cell responses.

Evaluation of selected hematological, biochemical and oxidative stress parameters in stored canine CPDA-1 whole blood

Blood transfusions are mainly given to intensive care patients; therefore, additional complications that could arise from storage lesions in preserved blood should be avoided. It has been shown that human stored red blood cells are subject to changes that are considered to be a number of interdependent processes involving metabolic disarrangement and oxidative stress. The aim of our study was to determine alterations in selected hematological and biochemical parameters and to assess whether and when oxidative stress is a significant phenomenon in stored dog CPDA-1 whole blood. Ten ½ unit bags of whole blood donated from dogs and preserved with CPDA-1 (anticoagulant containing citrate, phosphate, dextrose and adenine) were stored for 5 weeks. Each week, a 9 ml sample was drawn aseptically to measure hematological parameters, selected metabolites, free hemoglobin content, osmotic fragility, antioxidant enzyme activity, total antioxidant capacity, malondialdehyde concentration and protein carbonyl content.The results revealed an MCV decrease in the first week of storage and then a gradual increase; osmotic fragility decreased at that time and remained low throughout the study period. Leukodepletion became significant in the fourth week of storage. The free hemoglobin concentration continuously increased, with the greatest changes observed in the last two weeks of storage. The total antioxidant capacity changed in a reverse manner. Superoxide dismutase and glutathione peroxidase activities decreased from week 0 to week 3, and catalase activity tended to decrease over time. The highest malondialdehyde concentrations in blood supernatant were measured in the first week of storage, and the carbonyl concentration increased after 35 days.Hematological changes and oxidative stress are already present in the first week of storage, resulting in depletion of the antioxidant system and subsequent accumulation of oxidation products as well as erythrocyte hemolysis, which are most pronounced at the end of the storage period.

Photorhabdus and Xenorhabdus as Symbiotic Bacteria for Bio-Control Housefly (Musca domestica L.)

<b>Background and Objective:</b> The housefly poses a threat to the public health of humans and domestic animals since it can carry and transmit pathogens. Despite there are many attempts to control this insect, most of them depend on conventional pesticides. Thus, the current study aimed to evaluate the efficacy of whole-cell suspension, cell-free supernatant and crude cells of the symbiotic bacteria <i>Photorhabdus</i> sp. and <i>Xenorhabdus</i> sp., as bio-control agents for housefly stages. <b>Materials and Methods:</b> The <i>Photorhabdus</i> sp. and <i>Xenorhabdus</i> sp., were isolated from the entomopathogenic nematodes, <i>Heterorhabditis indica</i> and <i>Steinernema feltiae</i>, respectively. The phenotypic, as well as the enzymatic characterizations of both bacteria, were determined. In addition, histopathological changes of the alimentary canal of <i>M. domestica</i> adults treated with whole-cell suspensions (at 3×10<sup>8 </sup>cells mL<sup></sup><sup>1</sup>) of both bacteria were carefully examined using transmission electron microscopy. <b>Results:</b> The results showed that both symbiotic bacteria significantly suppressed larvae, pupae and adults of <i>M. domestica</i>, particularly when they were applied as whole-cell suspensions. For example, the highest concentration of whole-cell suspension, cell-free supernatant and crude cells of <i>Photorhabdus</i> sp., induced larval mortalities by 94.7, 64.0 and 45.3%, while those of <i>Xenorhabdus</i> sp., induced larval mortalities by 58.7, 46.7 and 30.7% at 96 hrs, respectively. The results also showed that whole-cell suspensions of both symbiotic bacteria caused severe histopathological changes in the ultrastructure of the treated adults' alimentary canal. <b>Conclusion:</b> Both symbiotic bacteria can be effectively used, particularly the whole-cell suspension, as bio-control agents against the housefly either in the larval or adult stage.

Circulating Exosomal miR-493-3p Affects Melanocyte Survival and Function by Regulating Epidermal Dopamine Concentration in Segmental Vitiligo

Circulating exosomal microRNAs have been used as potential biomarkers for various disorders. However, to date, the microRNA expression profile of circulating exosomes in patients with segmental vitiligo (SV) has not been identified. Thus, we aimed to identify the expression profile of circulating exosomal microRNAs and investigate their role in the pathogenesis of SV. Our study identified the expression profile of circulating exosomal microRNAs in SV and selected miR-493-3p as a candidate biomarker whose expression is significantly increased in circulating exosomes and perilesions in patients with SV. Circulating exosomes were internalized by human primary keratinocytes and increased dopamine secretion invitro. Furthermore, miR-493-3p overexpression in keratinocytes increased dopamine concentration in the culture supernatant, which led to a significant increase in ROS and melanocyte apoptosis as well as a decrease in melanocyte proliferation and melanin synthesis in the coculture system by targeting HNRNPU. We also confirmed that HNRNPU could bind to and regulate COMT, a major degradative enzyme of dopamine. Hence, circulating exosomal miR-493-3p is a biomarker for SV, and the miR-493-3p/HNRNPU/COMT/dopamine axis may contribute to melanocyte dysregulation in the pathogenesis of SV.

In-Vitro Cell-Induced Corrosion by Macrophages on Cobalt-Chromium-Molybdenum Alloy.

Patients have received cobalt-chromium-molybdenum (CoCrMo) implants for their joint replacement for decades. There have been reports of inflammatory cell-induced corrosion (ICIC) of these implants from retrieval studies. The goal of this study is to see if we could recreate ICIC invitro and whether electrocautery damage to alloy surfaces may hasten this process. Murine macrophages were cultured on CoCr disks with and without damage from a monopolar electrocautery. Culture medium was replaced every 12hours and supernatant was collected every 4 days. After 30 days, cells were removed, counted, and digested. The metal concentrations in the supernatant and within cells were assessed using inductively coupled plasma spectrometry for comparison. The Co supernatant concentration was higher in the undamaged disks with activated macrophages. Higher concentrations of Co and Mo were found in the supernatant of the undamaged disks vs the electrocautery (EC) corrosion damaged disks. There was a significantly higher intracellular Co and Mo concentration with activated cells on CoCrMo disks vs the control group and no difference compared to EC damaged disk group. Scanning electron microscopy displayed microscopic pitting on the surfaces exposed to macrophages without EC damage. We found that macrophages could reproduce findings of ICIC pits on the surface of CoCrMo alloy and that the addition of EC damage to the surface did not increase the process. The clinical significance of these findings should be further investigated to determine if this could explain a small number of poor total knee arthroplasty reported outcomes.

Wild-Blueberry Volatile and Phenolic Fractions Mitigate LPS-Induced Inflammatory Responses in Whole Blood of Children

Abstract Objectives The anti-inflammatory effects of berries have been attributed to their polyphenol content. In vitro models of activated murine macrophages have shown that volatiles in berries exert anti-inflammatory effects as well. However, human data in this area are scarce. To address this limitation we measured the effects of wild-blueberry phenolic (WBB-P) and WBB volatile (WBB-V) fractions in LPS-treated whole blood (WB) of children. Methods Fasting WB was collected (n = 19), and treated with high and low doses of either WBB-P (2 μg/mL or 0.02 μg/mL), WBB-V (2 ng/mL or 0.02 ng/mL), or culture medium (RPMI) as control and incubated for 1 hour at 37°C. To evaluate potential anti-inflammatory effects of WBB molecules, lipopolysaccharide (LPS, 0.2 μg/mL or 1 μg/mL) was added to the wells, and samples incubated for 48 hours. Interleukin (IL)-1β, IL-6, IL-10 and TNF-α supernatant concentrations were measured using enzyme-linked immunosorbent assay. One-factor ANOVA analysis with participant ID as random effect was used to compare cytokine concentrations (ng/ml) of WBB fraction treatments against their respective RPMI + LPS controls. Results Children (12.0 ± 0.6 years old) were predominantly white (79%), and with a BMI percentile of 51 ± 35. At both LPS levels, IL-6 concentrations were lower in the high-dose WBB-P and WBB-V treatments compared to the control group. LPS-High: WBB-P: 22.2 ± 6.7 vs. RPMI: 28.2 ± 8.3 (p = 0.005); WBB-V: 24.4 ± 7.2 vs. RPMI: 28.2 ± 8.3 (p = 0.013). LPS-low: WBB-P: 20.4 ± 5.5 vs. RPMI: 26.6 ± 7.7 (p = 0.001); WBB-V: 20.6 ± 6.5 vs. RPMI: 26.6 ± 7.7 (p < 0.0001). At the lowest LPS level, TNF-α concentration was 23% lower in the high-dose WBB-V treatment compared to the control group (WBB-V: 0.45 ± 0.06 vs. RPMI: 0.55 ± 0.12, p = 0.003). IL-1β, and IL-10 were not modified by WBB fraction treatments. Conclusions These preliminary results indicate that both WBB-P and WBB-V fractions mitigate inflammatory responses in LPS treated WB of children. Blueberry fractions do not impact IL-1β or IL-10 secretion by immune cells in WB. Funding Sources Center for Childhood Obesity Prevention NIH-NIGMS 5P20GM109096. USDA-ARS 6026–51,000-012–06S. The Arkansas Center for Advancing Pediatric Therapeutics NIH 8UG1OD024945.

Cold storage alters the immune characteristics of platelets and potentiates bacterial-induced aggregation.

Cold-stored platelets are currently under clinical evaluation and have been approved for limited clinical use in the United States. Most studies have focused on the haemostatic functionality of cold-stored platelets; however, limited information is available examining changes to their immune function. Two buffy-coat-derived platelet components were combined and split into two treatment arms: room temperature (RT)-stored (20-24°C) or refrigerated (cold-stored, 2-6°C). The concentration of select soluble factors was measured in the supernatant using commercial ELISA kits. The abundance of surface receptors associated with immunological function was assessed by flow cytometry. Platelet aggregation was assessed in response to Escherichia coli and Staphylococcus aureus, in the presence and absence of RGDS (blocks active conformation of integrin α2 β3 ). Cold-stored platelet components contained a lower supernatant concentration of C3a, RANTES and PF4. The abundance of surface-bound P-selectin and integrin α2 β3 in the activated conformation increased during cold storage. In comparison, the abundance of CD86, CD44, ICAM-2, CD40, TLR1, TLR2, TLR4, TLR3, TLR7 and TLR9 was lower on the surface membrane of cold-stored platelets compared to RT-stored components. Cold-stored platelets exhibited an increased responsiveness to E. coli- and S. aureus-induced aggregation compared to RT-stored platelets. Inhibition of the active conformation of integrin α2 β3 using RGDS reduced the potentiation of bacterial-induced aggregation in cold-stored platelets. Our data highlight that cold storage changes the in vitro immune characteristics of platelets, including their sensitivity to bacterial-induced aggregation. Changes in these immune characteristics may have clinical implications post transfusion.

FC 112: HLA-Specific Memory B Cells and Paternal HLA Molecular Mismatch in Female Transplant Candidates Sensitized by Pregnancy

Abstract BACKGROUND AND AIMS Access to kidney transplantation (KT) may be limited by HLA sensitization. It is clinically relevant to know both level of HLA-immunization and potential sensitizing events in the history transplant candidates [1–3]. Pregnancy is a frequent sensitizing event in female transplant candidates. Women exposed to foreign paternal HLA can generate specific HLA antibodies (HLA-Ab) and memory B cells (mBc) [4, 5], which can be re-stimulated upon antigen reencounter after TR. We aimed to study the existence of mBc able to produce HLA-Ab in female KT candidates with previous pregnancies, to compare them with HLA-Ab in their serum and to analyse them in the context of molecular HLA mismatch between mother and father. METHOD We selected eight HLA sensitized women awaiting KT, who fulfilled these criteria: minimum of one successful pregnancy before KT (12–44 years before) from a single partner, HLA-Ab (HLA class I or II) and availability of a DNA sample from the father of their children. All women in the study cohort and the father of their children were HLA typed by next-generation sequencing (NGS®) for HLA–A, B, C, DRB1, DRB3/4/5, DQB1/A1 and DPB1. We used peripheral blood mononuclear cells cryopreserved before KT. We cultured them with a polyclonal stimulation cocktail consisting of a Toll-like receptor 7/8 agonist and a proleukin for 10 days. We examined concentrated culture supernatants (SN) derived from activated cells, purified IgG and identified HLA-Abs using single antigen beads (SAB) on Luminex. Then, we compared the HLA-Ab profiles of SN and serum. We also studied the paternal molecular HLA incompatibilities with the HLAMatchmaker software. RESULTS After 10-day polyclonal stimulation, mBc increased from 29.90% (day 0) to 65.29% (day 10) (Figure 1), and antibody-secreting cells (ASC) from 0.08% (day 0) to 29.37% (day 10). SAB analysis revealed 211 HLA-Abs in serum and 116 HLA-Abs in SN: 98/211 (46.5%) of total HLA-Ab were serum-exclusive [78/98 (52.9%) class I and 25/98 (34.3%) class II] and only 3/116 (2.59%) HLA-Ab were SN-exclusive, all class II. Finally, we found 113/327 (34.6%) HLA-Ab shared by serum and SN: 65/113 (57.5%) class I and 48/113 (42.5%) class II. Seven of eight women had Pregnancy-Induced Antibodies (PIA) in serum (20 HLA-Abs: 10 HLA class I and 10 HLA class II) and 4/8 women had PIA in SN (8 HLA-Abs: 2 HLA class I and 6 HLA class II) (Table 1). The HLAMatchmaker analysis showed that antibodies were reacting against incompatible paternal epitopes: in 6/8 women for HLA class I and 5/8 women for class HLA II in serum, and in 2/8 women for HLA class I and 4/8 women for HLA class II in SN. CONCLUSION We describe the study of the memory B cell compartment and the specific HLA-Abs produced by them with Luminex technology in KT female candidates with previous pregnancies. We found that the HLA-Abs secreted by these memory B cells followed a restricted pattern in number and intensity compared with serum HLA-Ab. The study of paternal HLA molecular mismatch incompatibilities explains many of these HLA-Abs decades after pregnancies. The entire value of this tool for risk stratification in HLA-sensitized patients awaiting TK is currently a matter of study.

Benefits of lumican on human bone health: clinical evidence using bone marrow aspirates.

Background/AimsLumican, a small leucine-rich proteoglycan, has shown osteoprotective effects by synchronously stimulating bone formation and suppressing bone resorption. To clarify the role of lumican in human bone metabolism, the association between lumican concentrations and osteoporosis-related phenotypes was evaluated using bone marrow (BM) samples directly reflecting local microenvironments.MethodsBM aspirates were obtained from 77 patients during hip surgery for either fragility hip fractures (HF) (n = 29) or osteoarthritis (n = 48) and centrifuged. Concentrations of lumican and biochemical bone markers in BM supernatants were measured using enzyme linked immunosorbent assays.ResultsAfter considering confounders, lumican concentrations in BM supernatants were 16.9% lower in patients with HF than in controls, with each increase in the standard deviation of lumican concentration being associated with a 61% lower likelihood of HF. The odds ratios for HF decreased linearly with increasing lumican tertiles in BM, with the odds of having fragility HF markedly lower in participants in the highest than in the lowest lumican tertile. Higher lumican level correlated significantly with higher femur neck bone mineral density and higher bone-specific alkaline phosphatase levels, but not with tartrate-resistant acid phosphatase 5b concentrations, in BM supernatants.ConclusionsThese data clinically validate previous in vitro and animal experiments showing the beneficial roles of lumican for bone homeostasis and suggest that lumican may contribute to a reduction in fracture risk in humans mainly through its stimulation of bone formation.

Antimicrobial and Antibiofilm Effect of Inulin-Type Fructans, Used in Synbiotic Combination with Lactobacillus spp. Against Candida albicans.

There is great interest in the search for new alternatives to antimicrobial drugs, and the use of prebiotics and probiotics is a promising approach to this problem. This study aimed to assess the effect of inulin-type fructans, used in synbiotic combinations with Lactobacillus paracasei or Lactobacillus plantarum, on the production of short-chain fatty acids and antimicrobial activity against Candida albicans. The inhibition assay using the L. paracasei and L. plantarum supernatants resulting from the metabolization of inulin-type fructans displayed growth inhibition and antibiofilm formation against C. albicans. Inhibition occurred at concentrations of 12.5, 25, and 50% of the L. paracasei supernatant and ata concentration of 50% of the L. plantarum supernatant. The analysis of short-chain fatty acids by gas chromatography showed that lactic acid was the dominating produced metabolite. However, acetic, propionic, and butyric acids were also detected in supernatants from both probiotics. Therefore, the synbiotic formulation of L. paracasei or L. plantarum in the presence of inulin-type fructans constitutes with anticandidal effect is a possible option to produce antifungal drugs or antimicrobial compounds.

CD163+ M2 Macrophages Promote Fibrosis in IgG4-Related Disease Via Toll-like Receptor 7/Interleukin-1 Receptor-Associated Kinase 4/NF-κB Signaling.

ObjectiveIgG4‐related disease (IgG4‐RD) is a fibro‐inflammatory condition that can affect multiple organs. We previously demonstrated that TLR7‐transgenic C57BL/6 mice showed elevated serum IgG1 levels and inflammation with fibrosis in the salivary glands (SGs), lungs, and pancreas. Moreover, we observed extensive Toll‐like receptor 7 (TLR‐7)–positive CD163+ M2 macrophage infiltration in SGs from IgG4‐RD patients. We undertook this study to examine the fibrotic mechanism via the TLR‐7 pathway.MethodsGene expression in SGs from human TLR7‐transgenic mice and IgG4‐RD patients was analyzed using DNA microarrays. We extracted the common up‐regulated TLR‐7–related genes in SGs from TLR7‐transgenic mice and IgG4‐RD patients. Finally, we investigated the interaction between CD163+ M2 macrophages and fibroblasts before and after stimulation with the TLR‐7 agonist loxoribine.ResultsIn TLR7‐transgenic mice and IgG4‐RD patients, IRAK3 and IRAK4 were significantly overexpressed. Real‐time polymerase chain reaction validated the up‐regulation of only IRAK4 in IgG4‐RD patients compared with the other groups (P < 0.05). Interleukin‐1 receptor–associated kinase 4 (IRAK4) was strongly detected in and around germinal centers in SGs from patients with IgG4‐related dacryoadenitis and sialadenitis alone. Double immunofluorescence staining showed that IRAK4‐positive cells were mainly colocalized with CD163+ M2 macrophages in SGs (P < 0.05). After stimulation with loxoribine, CD163+ M2 macrophages exhibited significantly enhanced expression of IRAK4 and NF‐κB and increased supernatant concentrations of fibrotic cytokines. Finally, we confirmed that the number of fibroblasts was increased by culture with the supernatant of CD163+ M2 macrophages following stimulation with loxoribine (P < 0.05).ConclusionCD163+ M2 macrophages promote fibrosis in IgG4‐RD by increasing the production of fibrotic cytokines via TLR‐7/IRAK4/NF‐κB signaling.

Effectiveness of NLRP3 Inhibitor as a Non-Hormonal Treatment for ovarian endometriosis

BackgroundEndometriosis is a complex syndrome characterized by an estrogen-dependent chronic inflammatory process that affects 10% of women of reproductive age. Ovarian endometriosis (OE) is the most common lesion in endometriosis and may cause infertility, in addition to dysmenorrhea. Hormonal treatments, which are the conventional treatment methods for endometriosis, suppress ovulation and hence are not compatible with fertility. The inflammasome is a complex that includes Nod-like receptor (NLR) family proteins, which sense pathogen-associated molecular patterns and homeostasis-altering molecular processes. It has been reported that the nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain-containing (NLRP) 3 inflammasome, which contributes to the activation of interleukin-1 beta (IL-1β), might be related to the progression of endometriosis. Therefore, the aim of the present study was to evaluate non-hormonal therapies for OE, such as inhibitors of the NLRP3 inflammasome.MethodsThe expression of NLRP3 was measured in the eutopic endometrium (EM) of patients with and without endometriosis and OE samples, as well as stromal cells derived from the endometrium of patients with and without endometriosis and OE samples (endometrial stromal cells with endometriosis [ESCs] and cyst-derived stromal cells [CSCs]). The effects of an NLRP3 inhibitor (MCC950) on ESCs and CSCs survival and IL-1β production were evaluated. We then administered MCC950 to a murine model of OE to evaluate its effects on OE lesions and ovarian function.ResultsNLRP3 gene and protein expression levels were higher in OE and CSCs than in EM and ESCs, respectively. MCC950 treatment significantly reduced the survival of CSCs, but not that of ESCs. Moreover, MCC950 treatment reduced the co-localization of NLRP3 and IL-1β in CSCs, as well as IL-1β concentrations in CSCs supernatants. In the murine model, MCC950 treatment reduced OE lesion size compared to phosphate-buffered saline treatment (89 ± 15 vs. 49 ± 9.3 mm3 per ovary; P < 0.05). In the MCC950-treated group, IL-1β and Ki67 levels in the OE-associated epithelia were reduced along with the oxidative stress markers of granulosa cells.ConclusionsThese results indicated that NLRP3/IL-1β is involved in the pathogenesis of endometriosis and that NLRP3 inhibitors may be useful for suppressing OE and improving the function of ovaries with endometriosis.

Development of a bead-based multiplex assay to quantify bovine interleukin-10, tumor necrosis factor-α, and interferon-γ concentrations in plasma and cell culture supernatant

The quantification of cytokines can improve our understanding of immune response and inflammation dynamics in dairy cows. Bead-based assays provide a sensitive, high-throughput platform, allowing for simultaneous quantification of multiple cytokines within a wide linear detection range. Our objective was to develop a multiplex bead-based assay using monoclonal antibodies for simultaneous quantification of bovine tumor necrosis factor (TNF)-α, IL-10, and IFN-γ in plasma and peripheral blood mononuclear cell (PBMC) culture supernatants. Recombinant cytokine standards produced in mammalian cells were used to determine the lower limit of detection and the linear detection range for each cytokine. The lower limit of detection was 110 pg/mL for IL-10, 95 pg/mL for TNF-α, and 20 pg/mL for IFN-γ. The linear quantification range was 110 to 241,000 pg/mL for IL-10, 95 to 620,000 pg/mL for TNF-α, and 20 to 130,000 pg/mL for IFN-γ. All 3 monoclonal capture and detection antibodies were specific for their respective cytokine analyte when using the recombinant IL-10, TNF-α, and IFN-γ standards. Intraassay and interassay coefficients of variation (CV) were <10% and <12%, respectively, for all analytes and samples matrices. Next, concentrations of native cytokines were determined in PBMC culture supernatants (n = 4) and in plasma from whole-blood samples (n = 6) with or without stimulation with Escherichia coli lipopolysaccharide or a mix of phorbol myristate acetate (PMA) and ionomycin. Peak concentrations of all 3 cytokines were secreted from PBMC after PMA/ionomycin stimulation (TNF-α, 8 h, range: 39,266–506,422 pg/mL; IL-10, 18 h, range: 15,770–63,415 pg/mL; IFN-γ 18 h, range: 189,977–492,659 pg/mL). In contrast, the highest concentrations in plasma from whole-blood stimulation were observed for IL-10 and TNF-α after LPS stimulation (TNF-α, 4 h, range: 1,764–13,460 pg/mL; IL-10, 24 h, range: 2,401–6,371 pg/mL), whereas PMA and ionomycin induced the highest secretion of IFN-γ (18 h, range: 53–20,215 pg/mL). In conclusion, the multiplex assay can quantify native IL-10, TNF-α, and IFN-γ across a broad concentration range in bovine plasma and cell culture supernatant, thereby providing a novel tool to evaluate inflammatory profiles in cattle and especially in dairy cows with inflammatory conditions. The existing multiplex assay can be expanded in the future by adding bead assays for additional bovine cytokines.

The therapeutic mechanism of Shenyuan Gan in lipopolysaccharide-induced neuroinflammation in BV2 microglial cells

ObjectiveTo study the therapeutic effects of Shenyuan Gan (参远苷, SYG) on the inflammatory response in BV2 microglial cells induced by lipopolysaccharide (LPS). MethodsThe cytotoxicity of SYG to BV2 microglial cells was evaluated using a Cell Counting Kit-8 (CCK-8) assay, and the effect of SYG concentrations on LPS-induced BV2 microglial cells was studied. The morphological changes were observed using an optical microscope. The nitric oxide (NO) concentration in cell culture supernatant was determined using Griess reagent. The expression of cytokines and inflammatory mediators were also measured by an enzyme-linked immunosorbent assay (ELISA). Western blot analysis was used to determine the levels of inducible NO synthase (iNOS), nuclear factor-kappa B (NF-κB) p65, alpha inhibitor of NF-κB (IκB-α), phosphorylation-IκB-α (p-IκB-α), NOD-like receptor 3 (NLRP3), and caspase-1 expression. Moreover, the expression of iNOS, NLRP3, and ionized calcium binding adapter molecule 1 (Iba1) was also observed using immunofluorescent staining. ResultsSYG had a low cytotoxic effect on BV2 microglial cells and could significantly decr-ease LPS-induced morphological changes of BV2 microglial cells (P < 0.05). ELISA results showed that SYG significantly inhibited the LPS-induced increase in interleukin (IL)-1β and IL-6 in BV2 microglia cells (P < 0.05), and Western blot analysis showed that the phosphorylation levels of iNOS, NF-κB p65, and IκB-α as well as NLRP3 and caspase-1 expression were also significantly decreased, and IκB-α expression was increased after SYG treatment (P < 0.05, compared with the LPS-treated group). The immunofluorescence results were consistent with the Western blot results, and Iba1 staining indicated that the cell morphology tended to be resting. These results indicate that SYG has a certain inhibitory effect on LPS-induced inflammation in BV2 microglial cells. ConclusionSYG can inhibit LPS-induced release of inflammatory factors in BV2 microglial cells by affecting the phosphorylation levels of NF-κB p65 and IκB-α. SYG is a valuable candidate for treating neuroinflammation-related diseases.