The landscape of innate and adaptive immune cell subsets in patients with adult-onset Still's disease.

OBJECTIVE: Adult-onset Still's disease (AOSD) is a systemic autoinflammatory disorder. The understanding of the changes in adaptive immune cells and the crosstalk between innate and adaptive immune systems in AOSD is limited. This study aimed to examine the peripheral immune cell composition and inflammatory protein levels in AOSD patients. METHODS: Twenty-nine active AOSD patients were enrolled. Flow cytometry was used to analyze the cell populations in peripheral blood. Antibody chips were utilized to detect the protein expression profile in serum. RESULTS: In active AOSD patients, there was an increase in the percentage of classical and non-classical monocytes among peripheral blood mononuclear cells. The proportion of natural killer (NK) cells decreased, with an increase in CD56dim NK1 cells and a decrease in CD56bright NK2 cells compared with healthy controls (HC). The percentage of naïve central memory T cells was decreased, while the percentage of effector and effector memory T cells was increased among adaptive lymphocytes. The proportion of naïve B and memory B cells was decreased, while plasma cells were increased in AOSD patients, indicating activation of the adaptive immune system. Additionally, the serum levels of 40 proteins were elevated in AOSD patients, primarily involved in cytokine-cytokine receptor interaction, inflammatory response, and regulation of MAPK cascade. CONCLUSION: Our findings showed the activation of the innate and adaptive immune system in AOSD. The protein-protein interaction analysis suggested potential communication between innate and adaptive cell subsets. These findings provide new insights into the pathogenesis of the disease and the development of targeted therapies.

The Simultaneous Detection of Multiple Antibiotics in Milk and Pork Based on an Antibody Chip Biosensor.

In the modern farming industry, the irrational or illegal use of veterinary drugs leads to residues in animal-derived food, which can seriously threaten human health. Efficient detection of low concentrations of drug residues in animal products in a short time is a key challenge for analytical methods. This study proposes to use an antibody chip biosensor for rapid and automated analysis of cephalosporins, aminoglycosides, and sulfonamide antibiotics in pork and milk. 3D polymer slides were applied for the preparation of antibody chips. Ovalbumin (OVA) or bovine serum albumin (BSA) conjugates of the haptens were immobilized as spots on disposable chips. Monoclonal antibodies (mAbs) against cefalexin, ceftiofur, gentamicin, neomycin, and sulfonamides allowed the simultaneous detection of the respective analytes. Antibody binding was detected by a second antibody labeled with Cy3-generating fluorescence, which was scanned a with chip scanner. The limits of detection (LOD) for all the analytes were far below the respective maximum residue limits (MRLs) and ranged from 0.51 to 4.3 µg/kg. The average recoveries of all the analytes in each sample were in the range of 81.6-113.6%. The intra- and inter-assay CV was less than 12.9% and showed good accuracy and precision for all the antibiotics at the MRL level. The sample pretreatment method is simple, and the results are confirmed to be accurate by LC-MS/MS; therefore, this method is valuable for the quality control of animal-derived food.

Advances in multiplex molecular detection technologies for harmful algae.

As the eutrophication of natural water bodies becomes more and more serious, the frequency of outbreaks of harmful algal blooms (HABs) mainly formed by harmful algae also increases. HABs have become a global ecological problem that poses a serious threat to human health and food safety. Therefore, it is extremely important to establish methods that can rapidly detect harmful algal species for early warning of HABs. The traditional morphology-based identification method is inefficient and inaccurate. In recent years, the rapid development of molecular biology techniques has provided new ideas for the detection of harmful algae and has become a research hotspot. The current molecular detection methods for harmful algal species mainly include fluorescence in situ hybridization, sandwich hybridization, and quantitative PCR (qPCR), but all of these methods can only detect single harmful algal species at a time. The establishment of methods for the simultaneous detection of multiple harmful algal species has become a new trend in the development of molecular detection technology because various harmful algal species may coexist in the natural water environment. The established molecular techniques for multiple detections of harmful algae mainly include gene chip, multiplex PCR, multiplex qPCR, massively parallel sequencing, antibody chip, and multiple isothermal amplification. This review mainly focuses on the principles, advantages and disadvantages, application progress, and application prospects of these multiple detection technologies, aiming at providing effective references not only for the fisheries but also for economic activities, environment, and human health.

[Inflammatory mechanism of hippocampal tissue injury induced by PM2.5 in nasal drip in mice].

Objective: To investigate the inflammatory mechanism of nasal instillation of fine particulate matter (PM2.5)on hippocampal tissue injury in mice.Methods: Thirty C57BL/6J mice were randomly divided into 3 groups(n=10):control group, low-dose group, high-dose group. The nasal instillation doses of PM2.5 in the low-dose group and the high-dose group were 1.5 mg/kg BW and 7.5 mg/kg BW, respectively, and the control group was given saline with an equal volume. Saline was sprayed once every other time for 12 times. The serum levels of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) and interleukin-6 (IL-6) were determined by ELISA method. HE staining and electron microscopy were used to observe the pathological changes and ultrastructure of lung tissue and hippocampus. The inflammatory cytokine levels in hippocampus were detected by antibody chip technique. Results: There was no significant effect of PM2.5 nasal instillation on serum TNF-α, IL-1ß and IL-6 levels (P＞0.05), and there was no obvious pathological changes in lung tissue structure. In hippocampus, low-dose and high-dose PM2.5 exposure could lead to disordered neuronal arrangement in the hippocampal CA3 region, and there were neurological changes around the neuron cells and ultrastructural changes such as edema around small blood vessels. Compared with the control group, the levels of inflammatory cytokines such as CX3CL1, CSF2 and TECK in the low-dose group were increased significantly (P ＜0.05), while sTNFR1 was decreased significantly (P＜0.05); the inflammatory factors CX3CL1, CSF2, and TCA-3 were significantly increased in the high-dose group (P＜0.05), while leptin, MIG, and FASLG were significantly decreased (P＜0.05). Conclusion: Nasal instillation of PM2.5 can induce tissue damage in the hippocampus of mice, and its mechanism of action may be the olfactory brain pathway. The increasing of TNF-α and IL-6 and the decreasing of sTNFR1 and FASLG may be involved in inflammatory mechanisms.

Chip-based serum proteomics approach to reveal the potential protein markers in the sub-acute stroke patients receiving the treatment of Ginkgo Diterpene Lactone Meglumine Injection.

ETHNOPHARMACOLOGICAL RELEVANCE: Ginkgo biloba L. is a kind of traditional Chinese medicinal material with a long history. Its main active ingredients, ginkgolides, can be used for the treatment of stroke and other cardio-cerebrovascular diseases. Ginkgo Diterpene Lactone Meglumine Injection (GDLI), a modernized TCM, has attracted much attention because of its neuroprotective and anti-inflammatory properties. AIM OF THE STUDY: To uncover the effects of GDLI on ischemic stroke patients, as well as the underlying biomarkers involved in sub-acute stroke. MATERIALS AND METHODS: We used a state-of-the-art targeted proteomics chip to investigate the association between numerous serum proteins (1101 proteins) and the sub-acute phase post-ischemic stroke. Then, the relative proteins of anti-apoptosis, anticoagulant, and neuroprotection of GDLI were verified in animal models. RESULTS: Compared with the serum from healthy volunteers, we identified 15 up-regulated proteins and 26 down-regulated proteins (FC ≥ 1.5) involved in inflammatory response, immune response, and nervous system development in the sub-acute ischemic stroke. The pro-inflammatory proteins, such as IL17, MSP-R, G-CSF-R, TLR3, MIP-3ß, TNFRSF19, and TNFRSF12, were significantly increased in serum, illustrating that the chronic inflammatory state was evident in the sub-acute stage of ischemic stroke. However, the common pro-inflammatory proteins, such as IL-1ß, IL-6, IL-8, TNF-α, IFN-γ, and IL-10, known to be up-regulated in acute stroke, had close or lightly lower levels than healthy humans (FC ≥ 1.5, P > 0.05). And some cytokines (IL3, CCL13, TNFRSF3, IL10 R beta, HLA-A, IL-1 F8/FIL1 eta, TNFRSF8, CCL18) were also markedly down-regulated in the sub-acute phase of stroke. These proteins are highly associated with the onset of stroke-induced immunosuppression and post-stroke infection. Moreover, we noticed that Ginkgo Diterpene Lactone Meglumine Injection (GDLI) treatment for 14 days was helpful to the recovery of patients in the subacute period. After the treatment of GDLI, it was observed that several inflammatory cytokines (i.e. IL-17 and IL-28A), chemokine (i.e. CCL14), and Coagulation Factor III were reduced. Meanwhile, the anti-inflammatory cytokines (IL-10 R alpha, GREMLIN, and Activin C) and neurotrophic factors (Neurturin and IGFBP2) were found to be up-regulated in stroke patients through self-control observation. Finally, we identified the IGFBP2 as a novel marker in the animal models. CONCLUSIONS: In summary, the potential markers in sub-acute stroke patients were highly different from known protein markers in the acute phase of ischemic stroke. The serum protein IGFBP2 could be novel biomarkers for the treatment of GDLI in sub-acute stroke patients. Our present findings provide an innovative insight into the novel treatment of GDLI in ischemic stroke therapy.

A Microarray Immunoassay for Serum Thyrotropin and Thyroglobulin Using Antibodies Immobilized on Track-Etched Membranes.

Serum thyroglobulin (Tg) and thyroid stimulating hormone (TSH) measurements have evolved as important analytes for monitoring the prognosis of patients with differentiated thyroid cancer, post-thyroidectomy. Individual analyte immunoassay is the current practice in clinical pathology, but the simultaneous assay for all relevant analytes for a given disease, can reduce assay costs, improve patient compliance and give the clinician more information for an unequivocal diagnosis. Microarray immunoassay (MI) can achieve this goal and, hence, we have developed and validated a immuno-radiometric MI for quantitation of serum TSH and Tg by using highly micro-porous polycarbonate (PC) track-etched membranes (TEM) to immobilize the monoclonal anti-TSH and polyclonal anti-Tg antibodies in ~1 mm diameter spots. Non-competitive immunoassays were performed using mixture of 125I labeled monoclonal anti-TSH and anti-Tg antibodies. Phosphorimager was used to quantify the bound radioactivity. TSH and Tg were detected with detection limit of 0.07 µIU/ml and 0.13 ng/ml respectively, which is lower than the clinically required cut-off level. The assay showed: acceptable intra-assay precision within 20 % and recovery in the range of 76-111.2 %. MI compared well with the established immunoradiometric assay (IRMA) with r = 0.98, p < 0.01 (n = 41). No cross-reactivity was seen between the immobilized antibodies. Although two hormones are addressed in this report, MI using PC TEM and isotopic/non-isotopic tracers has the potential for highly automated multiplexed analysis.

Expression levels of cytokines and chemokines increase in human peripheral blood mononuclear cells stimulated by activation of the Toll-like receptor 5 pathway.

Recognition of pathogen-associated molecular patterns by Toll-like receptors (TLRs) activates innate and adaptive immune responses. Among the 11 members of the human TLR family, TLR-5 is known to play an important role in the defense against bacterial invasion by binding to flagellin, a conserved component of bacteria. Previous studies have demonstrated that the activation of TLR-5 induces the expression of interleukin (IL)-10, IL-12 and interferon-ß. However, the aim of the present study was to analyze the expression of a wider range of immune-related molecules upon stimulation with a TLR-5 agonist. Following isolation from healthy volunteers, peripheral blood mononuclear cells (PBMCs) were stimulated with flagellin, a TLR-5 agonist. At 4 h after stimulation, quantitative polymerase chain reaction (PCR) and an antibody chip array were conducted to determine the mRNA expression levels of immune molecules and the protein secretion of immune molecules in the supernatant, respectively. The PCR results revealed that activation of TLR-5 significantly influenced the expression of a number of important molecules. In addition, the antibody chip array demonstrated the induction (IL-8) and inhibition [monocyte chemoattractant protein (MCP)-1, MCP-3 and macrophage inflammatory protein-1α) of protein secretion following TLR-5 stimulation. Therefore, the present study demonstrated the importance of TLR-5 in regulating the biological function of PBMCs. In the future, research should focus on the roles of the candidate molecules in TLR-5-mediating functions.

Toll-like receptor 4 agonist, lipopolysaccharide, increases the expression levels of cytokines and chemokines in human peripheral blood mononuclear cells.

Toll-like receptors (TLRs) are members of the pattern recognition receptor family and are essential in the innate immune response. In total, 11 TLRs exist in humans, which are expressed in a variety of cells, including peripheral blood cells. TLR4 plays a significant role in the defense against gram-negative pathogens by recognizing the lipopolysaccharide (LPS) molecules in these bacteria. The aim of the present study was to detect the expression level variation of a number of major immune molecules in peripheral blood mononuclear cells (PBMCs) stimulated by LPS, in order to identify candidate genes involved in the biological functions mediated by TLR4. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis and an antibody chip were performed in the current study. The RT-qPCR results revealed a marked enhancement in the expression levels of various molecules, including cytokines, chemokines, growth factors and protein kinases. In addition, the antibody chip identified the increased secretion of crucial proinflammatory molecules in the supernatants collected from LPS-treated PBMCs. In conclusion, a large number of molecules were found to be involved in TLR4-mediated functions.