Multiplex Assay Research Articles

Detection of Francisellaceae and the differentiation of main European F. tularensis ssp. holarctica strains (Clades) by new designed qPCR assays

BackgroundThe zoonotic and highly infectious pathogen Francisella tularensis is the etiological agent of tularemia. Tularemia in humans is mainly caused by F. tularensis subspecies tularensis and holarctica, but Francisella species like F. novicida, F. philomiragia, F. hispaniensis and others are known to cause tularemia-like infections in immunocompromised humans. In addition to these Francisella species, further genera of the family Francisellaceae have been described, such as Allofrancisella, Parafrancisella and Pseudofrancisella, but less is known about the distribution and putative virulence of these genera. The methods currently available were not made for a fast and easy detection of all these strains and genera of Francisellaceae.ResultsWe developed a multiplex quantitative real-time PCR assay that can accurately detect all genera of Francisellaceae, including Francisella, Francisella-like endosymbionts, Allofrancisella, Parafrancisella and Pseudofrancisella. In addition, we developed a qPCR assay to differentiate the major clades (B.4, B.6 and B.12 [B.71 and B.72]) of F. tularensis ssp. holarctica strains. Both primer sets were shown to work on isolated DNA out of human and tick samples.ConclusionSince the developed qPCRs are able to detect all genera of Francisellaceae tested, an easy and fast identification of opportunistic Francisella strains causing tularemia-like symptoms in humans or animals is possible now. The application of these qPCR assays will thus improve the capability for clinical diagnostics and molecular typing during epidemiological investigations.

A Pilot Study on the Impact of Smoking on Adipose Derived Stem Cell Function in Burn Patients.

Adipose-derived stem cells (ADSCs) have an important role in the modulation of burned tissue repair through the release of paracrine factors that stimulate the wound healing response. In this study, we tested the hypothesis that smoking status alters the profile of paracrine factors secreted from ADSCs isolated from damaged adipose tissue. Adipose tissue was collected from adult patients (N=8) with severe burn injuries (>20% total body surface area) at the index operation. ADSCs were extracted and cultured in vitro. Supernatants were harvested 30 hours after plating and used for cytokine determinations by Multiplex assay. Fluorescence activated single cell sorting (FACS) confirmed their phenotype with markers CD 90, CD 166, and CD 73. Univariate analyses were performed to compare the two cohorts (Smokers vs non smokers). Higher amounts of anti-inflammatory cytokines IL-4 (p=0.03) and IL-10 (p=0.04) and pro-inflammatory cytokines TNF-alpha (p=0.03), IL-8, and IFN-gamma (p=0.03) were detected in burn patients who were current everyday smokers when compared to nonsmokers, or former smokers. No significant differences in supernatant concentrations of IL-17, IL-1 beta, TGF-alpha, IL-6, and IL-13 were observed (p>0.05). Mortality was higher in the smoker group when compared to non-smokers. The results from this study suggest that smoking status in patients with a major burn injury may alter the paracrine factors secreted from ADSCs, and on-going studies will increase sample size and refine experimental approach. Furthermore, these results support the need for studies examining the systemic effects of smoking status of patients suffering burn injuries impacts the wound healing.

Targeted sputum sequencing for rapid and broad drug resistance of Mycobacterium tuberculosis.

Rapid detection of drug resistance in Mycobacterium tuberculosis (Mtb) from clinical samples facilitates the timely provision of optimal treatment regimens for tuberculosis (TB) patients. In November, 2023, the WHO released its second catalogue of resistance-conferring mutations in Mtb. Utilizing this information, we developed a single 17-plex PCR assay covering 16 key resistance genes and modified thermo-protection buffer to amplify 30 kbp DNA directly from sputum samples for nanopore sequencing. We implemented our protocol using rapid barcoding for sequencing with both a Flongle and a MinION flow cell. The single multiplex PCR assay was successfully validated on clinical sputum samples using the thermo-protection buffer. The protocol was applied to both Flongle and MinION flow cells, analyzing 12 and 40 samples, respectively. Data analysis suggested that optimal performance could be achieved by processing 6 and 12 samples with similar microscope staining scores on these two platforms. This approach facilitated rapid antimicrobial resistance (AMR) predictions directly from sputum on the day of collection or the following day, with a cost of less than $35 per sample. Compared to AMR predictions based on whole-genome sequencing (WGS) using Mykrobe and TBProfiler, our amplicon-based analysis tool, ARapidTb, demonstrated superior resistance detection capabilities. When analyzing publicly available nanopore WGS datasets for 442 isolates, ARapidTb achieved agreement rates of 95.8% and 98.0%, outperforming Mykrobe (89.4% and 98.3%) and TBProfiler (75.6% and 89.8%). Our study significantly reduces the time required for drug resistance detection, enabling quicker initiation of appropriate treatments and potentially improving patient outcomes and TB management.

The High-Affinity IL-2 Receptor Affects White Matter Damage after Cerebral Ischemia by Regulating CD8 + T Lymphocyte Differentiation.

IL-2/IL-2R inhibition improved the prognosis of ischemic stroke by regulating T cells, while the respective contribution of T cells with high/medium/low-affinity IL-2 receptors remained unclear. Single-cell RNA sequencing data of ischemic brain tissue revealed that most of the high-affinity IL-2R would be expressed by CD8 + T cells, especially by a highly-proliferative subset. Interestingly, only the CD8 + T cells with high-affinity IL-2R infiltrated ischemic brain tissues, highly expressing 32 genes (including Cdc20, Cdca3/5, and Asns) and activating 7 signaling pathways (including the interferon-alpha response pathway, a key mediator in the proliferation, migration, and cytotoxicity of CD8 + T cells). Its interaction with endothelial cells and the ligand-receptor interaction analysis also suggested an augmented brain infiltration after cerebral ischemia. In IL-2Rα KO mice, who would have no high- or low-affinity IL-2R in CD8 + T cells, the RNA-seq, qPCR, immunofluorescence, and multiplex assays found that the expression of CD8b, CD122, CD132, and Vcam-1 was upregulated in the acute phase of cerebral ischemia, with decreasing H2-k1 positive cells and increasing Vcam-1 and CD8b positive cells in brain tissue. However, inflammation pathways in brain were inhibited and peripheral inflammatory cytokine levels were reduced, indicating that CD8 + T cells changed into an anti-inflammatory phenotype. The IL-2Rα KO mice after cerebral ischemia also performed better in behavioral tests and had more favorable results in diffusion tensor imaging, electrophysiology, and MBP testing. Our findings suggested that the CD8 + T cells with high-affinity IL-2R, as well as IL-2Rα, might be targeted to improve the clinical management of ischemic stroke.

Molecular identification and antibiotic clearance of Mycoplasma arginini and Mycoplasma orale from cell cultures infected with Rickettsia or Ehrlichia species.

Mycoplasma (Class: Mollicutes) contamination in cell cultures is a universal concern for research laboratories. Some estimates report contamination in up to 35% of continuous cell lines. Various commercial antibiotic treatments can successfully decontaminate clean cell lines in vitro; however, in vitro decontamination of bacterial cultures remains challenging. Intracellular bacteria like those in the genera Rickettsia and Ehrlichia require cell culture for primary isolation and propagation and are thus vulnerable to contamination with mycoplasmas. Some analyses have reported successful antibiotic clearance of contaminating mycoplasmas in Rickettsia cultures; however, many of these studies do not identify the contaminating mycoplasma species and often include only a few isolates. To our knowledge, there are no published studies reporting decontamination of mycoplasmas from Ehrlichia cultures. In this study, we developed a specific multiplex assay to identify two of the most common mycoplasma culture contaminants, Mycoplasma arginini and Mycoplasma orale, in cell cultures infected with Rickettsia or Ehrlichia species. We further describe the successful in vitro decontamination of M. arginini, M. orale, and co-contaminations with both mycoplasmas from multiple Rickettsia and Ehrlichia cultures using daptomycin and clindamycin.IMPORTANCEMycoplasma contamination is a frequent problem in bacterial cell culture. These prolific organisms thrive in the extracellular environment in vitro and can persist in cell lines indefinitely without treatment. Historically, mycoplasma-contaminated Rickettsia cultures were cleared of contaminants by inoculating laboratory mice and re-isolating mycoplasma-free Rickettsia from brain endothelial cells. However, this method requires the sacrifice of live animals and is not always effective. Mycoplasma clearance via mouse inoculation requires a patent infection of murine central nervous system endothelial cells, which may not occur with some mildly pathogenic or nonpathogenic rickettsial species. In vitro antibiotic treatment represents an alternate method to eliminate contaminating mycoplasmas from rickettsial cultures. This method requires minimal adjustment of laboratories that already maintain rickettsial cultures and is not dependent on the use of laboratory animals. As such, the comprehensive strategy for Mycoplasma arginini and Mycoplasma orale elimination presented here can improve laboratory efficiency for in vitro research with intracellular bacteria.

Long-term cultivation of retinal pigment epithelium cells on nanofiber scaffolds.

The retinal pigment epithelium (RPE) plays an important role in the pathogenesis of age-related macular degeneration (AMD) and other retinal degenerative diseases. The introduction of healthy RPE cell cultures into the subretinal space offers a potential treatment strategy. The aim of this study was the long-term culture and characterisation of RPE cells on nanofiber scaffolds. Nanofiber scaffolds consisting of polycaprolactone (PCL) and collagen were prepared by electrospinning. Porcine RPE cell cultures were maintained on PCL scaffolds, PCL-collagen scaffolds, and controls at the bottom of 24-well plates. Cell culture analysis was performed by immunohistochemistry, while the release of inflammatory cytokines IL-6, IL-8, TNF-α, and PDGF-β was measured by ELISA and multiplex assays. Ultrastructural features were examined by transmission electron microscopy. The observation period averaged 42.7 weeks for controls, 38.7 weeks for PCL scaffold cultures, and 36.1 weeks for PCL-collagen scaffold cultures, with cell number and morphology remaining stable. TNF-α levels in the supernatants were minimal, IL-6 levels were consistently low, and IL-8 levels decreased from initially high to lower levels over time. RPE cells were stably cultured on nanofiber scaffolds for extended periods of time. The long-term physiological properties of RPE cells, including phagocytic ability and visual cycle enzyme activity, need to be further investigated before clinical application. In addition, controlling the expression of inflammatory mediators is a major challenge. Despite these hurdles, overcoming them is critical given the increasing prevalence of retinal degenerative diseases.

Multiplex one-step RT‒qPCR assays for simultaneous detection of AMDV, MEV and CDV

BackgroundAleutian mink disease, mink viral enteritis and canine distemper are known as the three most serious diseases that cause great economic loss in the mink industry. In clinical practice, aleutian mink disease virus (AMDV), mink enteritis virus (MEV) and canine distemper virus (CDV) are common mixed infections, and they have similar clinical clinical signs, such as diarrhoea. Therefore, a rapid and accurate differential diagnosis method for use on mink ranches is essential for the control of these three pathogens. Here, we developed multiplex one-step real-time quantitative PCR (RT‒qPCR) assays for the simultaneous detection and quantification of AMDV, MEV and CDV by using three primers and probes based on the conserved NS1, VP2 and N genes, respectively.ResultsThe results showed that the established method can not cross-react with other mink pathogens, with a detection sensitivity of 25 copies/µL and a coefficient of variation less than 3.51%. Moreover, the interference experiment showed that the presence of AMDV, MEV and CDV templates at different concentrations would not interfere with the detection results. Furthermore, two hundred clinical samples of mink with diarrhoea were simultaneously analysed using multiplex RT‒qPCR and single RT‒qPCR, the Kappa values were all greater than 0.921, indicating that there was a high degree of coincidence between the two detection methods.ConclusionsIn conclusion, multiplex RT‒qPCR exhibited high specificity, sensitivity, and reproducibility, indicating that this method can be used as a reliable and specific tool for the differential detection and quantification of AMDV, MEV and CDV.

Multiplex basophil activation tests for allergy diagnosis: present and future applications

The basophil activation test (BAT) has become a major cellular in vitro test for evaluating the allergenic activity of specific IgEs. The impact of the BAT is due to the ability of blood basophil granulocytes to present IgE on the high-affinity FcεRI receptor and to mirror the mast cell response that elicits an acute allergic reaction. The BAT proved to be able to identify allergic patients at risk of reacting to a low dose of the allergen and/or developing life-threatening reactions and thus can significantly improve the current management of allergic patients. However, to improve the diagnostic utility for identifying the allergenic activity of different genuinely sensitizing allergens and lower the procedure and labour requirements, developing a multiplex BAT approach incorporating multiple allergen components would be highly anticipated. Recently, the novel multiplex BAT was described utilizing two major innovative steps. The first step was the fluorescent labeling of allergens. The second step was applying fluorescently labeled allergens in flow cytometry assessment to analyze the activation of basophil subpopulations gated according to the binding of different allergens or to evaluate the fluorescence intensity of multiple allergens on the surface of basophils. These novel cellular multiplex approaches will advance our understanding of IgE-mediated responses. Integration of multiplex BAT, in addition to multiplex IgE assays into practice, will personalize the measurement of allergenic IgE activity and can help estimate the likelihood of clinical relevance and risks for multiple allergens when testing individual allergic patients.

Detection and prognostic relevance of PLA2R epitopes in idiopathic membranous nephropathy: a simultaneous quantitative multiplex suspension array detection method

Abstract Objective This study aimed to develop a multiplex suspension assay for the simultaneous quantitative detection of anti-cysteine-rich domain (anti-CysR)/C-type lectin domain 1 (CTLD1)/C-type lectin domain 6–8 (CTLD678)-IgG4 antibodies of M-type phospholipase A2 receptor (PLA2R) in the serum samples of patients to evaluate the clinical application value of PLA2R epitope spreading in disease prognosis. Methods CysR, CTLD1, and CTLD678 antigen domains were coupled to three types of microspheres. The optimal dilution ratio of biotinylated anti-human IgG4 was identified, and a multiplex suspension assay was evaluated in terms of linearity, sensitivity, precision, specificity, and recovery rate. Lastly, the relationship between epitope spreading and the severity of idiopathic membranous nephropathy (IMN) was evaluated. Results The content of all three epitopes could be detected simultaneously within two hours. The intra-assay precision ranged from 4.74% to 9.48% , and the inter-assay precision was between 5.13% and 13.92% . Specific experiments showed that the human IgA antibody did not cause a cross-reaction. The recovery rates ranged between 90% and 100%. The cut-off values of epitopes CysR, CTLD1 and CTLD678 between healthy individuals and patients were 6.30, 12.38, and 10.06 Ru/mL, respectively. Among them, the positive rates of epitopes CysR, CTLD1 and CTLD678 were 100%, 34%, and 75%, respectively. In addition, group 3 (CTLD1 response) accounted for 73% of the twelve patients who were not in remission. Meanwhile, When the concentration of CTLD1 exceeds 42.76 Ru/mL, the patient's prognosis may be poorer. Conclusion A multiplex suspension assay was developed for the simultaneous quantitative detection of anti-CysR/CTLD1/CTLD678-IgG4 antibodies. The epitope migration sequence of PLA2R molecules during disease progression may not follow a simple linear rule. Among them, the epitope CTLD1 is likely to exert the most significant influence on patient remission.

Concurrent Viewing of H&E and Multiplex Immunohistochemistry in Clinical Specimens

Background/Objectives: Performing hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) on the same specimen slide provides advantages that include specimen conservation and the ability to combine the H&E context with biomarker expression at the individual cell level. We previously used invisible deposited chromogens and dual-camera imaging, including monochrome and color cameras, to implement simultaneous H&E and IHC. Using this approach, conventional H&E staining could be simultaneously viewed in color on a computer monitor alongside a monochrome video of the invisible IHC staining, while manually scanning the specimen. Methods: We have now simplified the microscope system to a single camera and increased the IHC multiplexing to four biomarkers using translational assays. The color camera used in this approach also enabled multispectral imaging, similar to monochrome cameras. Results: Application is made to several clinically relevant specimens, including breast cancer (HER2, ER, and PR), prostate cancer (PSMA, P504S, basal cell, and CD8), Hodgkin’s lymphoma (CD15 and CD30), and melanoma (LAG3). Additionally, invisible chromogenic IHC was combined with conventional DAB IHC to present a multiplex IHC assay with unobscured DAB staining, suitable for visual interrogation. Conclusions: Simultaneous staining and detection, as described here, provides the pathologist a means to evaluate complex multiplexed assays, while seated at the microscope, with the added multispectral imaging capability to support digital pathology and artificial intelligence workflows of the future.

Resurgence of common respiratory viruses and mycoplasma pneumoniae after ending the zero-COVID policy in Shanghai

China has adhered to policies of zero-COVID for almost three years since the outbreak of COVID-19, which has remarkably affected the circulation of respiratory pathogens. However, China has begun to end the zero-COVID policies in late 2022. Here, we reported a resurgence of common respiratory viruses and Mycoplasma pneumoniae with unique epidemiological characteristics among children after ending the zero-COVID policy in Shanghai, China, 2023. Children hospitalized with acute respiratory tract infections were enrolled from January 2022 to December 2023. Nine common respiratory viruses and 2 atypical bacteria were detected in respiratory specimens from the enrolled patients using a multiplex PCR-based assay. The data were analyzed and compared between the periods before (2022) and after (2023) ending the zero-COVID policies. A total of 8550 patients were enrolled, including 6170 patients in 2023 and 2380 patients in 2022. Rhinovirus (14.2%) was the dominant pathogen in 2022, however, Mycoplasma pneumoniae (38.8%) was the dominant pathogen in 2023. Compared with 2022, the detection rates of pathogens were significantly increased in 2023 (72.9% vs. 41.8%, p < 0.001). An out‐of-season epidemic of respiratory syncytial virus was observed during the spring and summer of 2023. The median age of children infected with respiratory viruses in 2023 was significantly greater than that in 2022. Besides, mixed infections were more frequent in 2023 (23.8% vs. 28.9%, p < 0.001). China is now facing multiple respiratory pathogen epidemics with changing seasonality, altered age distribution, and increasing mixed infection rates among children in 2023. Our finding highlights the need for public health interventions to prepare for the respiratory pathogen outbreaks in the post-COVID-19 era.

The Effect of Peri-Implant Therapy on the Expression of Th17-Related Cytokines in Patients with Peri-Implant Mucositis and Peri-Implantitis: A Prospective Longitudinal Study

Background/Objectives: Cytokines related to the Th17 response have been associated with peri-implant diseases; however, the effect of peri-implant therapy on their modulation remains underexplored. To evaluate the effect of peri-implant therapy on the expression of cytokines related to the Th17 response in the peri-implant crevicular fluid (PICF) (GM-CSF, IFN-γ, IL-1β, IL-4, IL-6, IL-10, IL-12 (p70), IL-17A, IL-21, IL-23, and TNF-α) of partially edentulous patients with peri-implant disease (PID). Methods: Thirty-seven systemically healthy individuals presenting with peri-implant mucositis (PIM) (n = 20) or peri-implantitis (PI) (n = 17) were treated and evaluated at baseline (T0) and three months after therapy (T1). Clinical parameters (probing depth (PD), clinical attachment level (CAL), plaque index, and bleeding on probing index (BoP), were evaluated. The PIM group underwent non-surgical therapy, while the PI group received a surgical approach. PICF was collected with absorbent paper strips and analyzed with a multiplex assay. Results: Eighty-eight implants were treated in 37 patients (56 in the PIM group and 32 in the PI group). After therapy, significant reductions in PD, CAL, plaque index, and BoP were observed in the PIM group (p &lt; 0.05). In the PI group, significant reductions in PD, CAL, and BoP were noted (p &lt; 0.05). The PIM group showed a significant reduction of IL-17A and TNF-α after therapy, while the PI group showed a significant reduction of IL-1β, IL-6, and TNF-α (p &lt; 0.05). Conclusions: The peri-implant therapy for patients with PID reduced the expression of cytokines related to the Th17 response in PICF.

A Multiplex Polymerase Chain Reaction Assay for the Detection of Herpes Simplex Virus, Cytomegalovirus, and Varicella-Zoster Virus in Cerebrospinal Fluid

Viral meningitis poses a significant clinical challenge due to its rapid onset and potential progression to life-threatening encephalitis. Early detection of treatable viral pathogens such as Herpes simplex virus (HSV), Cytomegalovirus (CMV), and Varicella-zoster virus (VZV) is essential for initiating appropriate therapies. However, multiplex PCRs for the rapid and simultaneous detection of these pathogens are scarce due to the complex PCR design and the elaborate validation process using cerebrospinal fluid samples. In this study, we established and validated a novel multiplex PCR assay for detecting HSV, CMV, and VZV in cerebrospinal fluid samples and implemented the assay on a fully automated platform.

Sample-to-answer microfluidic device towards the point-of-need detection of Staphylococcus aureus enterotoxin genes in ruminant milk.

Milk is commonly screened both for indicators of animal disease and health, but also for foodborne hazards. Included in these analyses is the detection of Staphylococcus aureus, that can produce an enterotoxin, causing staphylococcal food poisoning (SFP), which often leads to sudden onset of significant gastrointestinal symptoms in humans. Epidemiological data on SFP are limited, particularly in low- and middle-income countries. Many conventional assays for the detection of staphylococcal enterotoxins rely on the detection of the genes coding for them, either directly in food samples or after bacterial culture. Currently, many of the nucleic acid-based methods used require specific expertise and equipment, whilst bacterial culture takes 24-48 hours; both are contributory factors that limit efforts either during food safety emergencies or routine screening. Here we present the development of a "sample-to-answer" isothermal nucleic acid loop-mediated amplification (LAMP) assay in a microfluidic device for the detection of Staphylococcus aureus enterotoxin genes in ruminant milk. A multiplex LAMP assay targeting two of the most prevalent S. aureus enterotoxin-encoding genes (A and B) was integrated into a microfluidic device combining simple 1 : 10 dilution for sample preparation and a lateral flow assay for easy readout. We achieved a limit of detection of 104 colony forming units per ml in spiked cow and goat milk samples, an order of magnitude more sensitive than the European recommendation for the maximum allowable presence of coagulase-positive staphylococci in raw milk. The assay showed no cross-reactivity in detecting other tested non-enterotoxigenic S. aureus strains or associated foodborne pathogens. The test integrated the simplicity of use of microfluidic devices with the sensitivity, specificity and rapidity of a nucleic acid-based assay, and a simple lateral flow readout to provide an appropriate device to ensure the safety of milk for human consumption. To illustrate its potential for point-of-need practical applications, the test was performed in agricultural settings in rural Turkey in a limited feasibility exercise.

Phenotype-Led Identification of IL-10 Upregulators in Human CD4+ T-cells and Elucidation of Their Pharmacology as Highly Selective CDK8/CDK19 Inhibitors.

Therapeutics promoting the endogenous production of IL-10 have the potential to restore homeostasis in inflammatory disorders such as inflammatory bowel disease (IBD). Here we describe the identification of a series of IL-10 upregulators based on a pyrimidyl-piperidine scaffold through a high throughput phenotypic CD4+ T-cell multiplex assay. In vitro optimization of the initial hit yielded a lead with good potency and an in vitro clearance profile, compound 3-7, which additionally demonstrated efficacy in a murine endotoxin challenge PK-PD mechanistic model. Target deconvolution efforts identified compound 3-7 as a highly selective CDK8/19 inhibitor, and crystallographic studies unveiled its binding mode to the CDK8/Cyclin-C complex, characterized by an unusual water-mediated hydrogen bond to the kinase hinge region.

Olive leaf extract (OLE) reduces mast cell-mediated allergic inflammation.

Mast cell-mediated reactions promote various allergic disease, including anaphylaxis, allergic rhinitis, asthma, and atopic dermatitis. Different data demonstrated an intricate relationship between the use of antihistaminic drugs, the onset of side effects, and the development of resistance, underscoring the importance to find novel therapeutic approaches to treat allergic diseases. Olive leaf extract (OLE), is a by-product of the olive tree rich in bioactive compounds, known for its numerous therapeutic properties, including antioxidant, anti-tumoral and antidiabetic effects. In this study, we investigated the effect of OLE on the mast-cell-mediated allergic inflammation using human mast cells HMC-1.2. OLE reduced histamine and β-Hexosaminidase release from HMC cells activated by phorbol 12-myristate 13-acetate and calcium ionophore A23187 (PMACI) through modulation of calcium signal. Moreover, OLE decreased the PMACI-stimulated gene expression of proinflammatory cytokines such as tumor necrosis factor-a (TNF-α), interleukin-8 (IL-8) and interleukin-6 (IL-6) in human mast cells. This result was confirmed by multiplex assay in which the pre-treatment with OLE reduced the effective secretion of TNF-α, IL-6 and IL-8. These effects were correlated to ROS reduction and modulation of both mitochondrial mass and membrane potential. Finally, the inhibitory effect of OLE was nuclear factor (NF)-kB dependent as demonstrated by both activity assay and Western Blot analysis. Taken together, our results demonstrated that OLE inhibits mast-cell-derived allergic inflammation modulating mast cells degranulation, proinflammatory cytokines release and NF-kB activation. Therefore, OLE could represent a novel potential therapeutic approach for the treatment of mast cell-associated disorders.

Evaluation of the efficacy of herbal (neem) and chemical (chlorhexidine) chips as local drug delivery agents in the treatment of chronic periodontitis: A clinical and microbiological study

Context: Herbal remedies can be effective in controlling periodontal disease. Neem is gaining attention as a reliable alternative to pharmaceuticals. Aims: The purpose of this study was to compare the efficacy of chlorhexidine (CHX) and neem chips (NCs) when used with scaling and root planing (SRP). Settings and Design: The study design involves interventional prospective study. Methods: A total of 45 periodontal pockets were divided into three groups of 15 sites each. Included both males and females of age group 35–55 years. Group I–SRP alone, Group II–SRP and CHXC, and Group III–SRP and NC. Parameters evaluated were plaque index (PI), gingival index (GI), probing pocket depth (PPD), and clinical attachment level (CAL). A qualitative multiplex polymerase chain reaction assay aided in detecting red complex organisms. Statistical Analysis Used: Evaluated at baseline, 10th day, and 1 month. Kruskal–Wallis test and Mann–Whitney U-test were used for the analysis. Results: There was a noticeable variation in the PPD at the start between the SRP, NC, and CHX groups. However, no significant differences were observed in other aspects such as the PI, GI, CAL, and the presence of Porphyromonas, Treponema, and Tannerella bacteria at the baseline, 10th day, and 30th day. Conclusions: All groups improved clinically, but no statistical difference was found in all clinical and microbiological parameters from baseline to day 30. Trial registration: https://ctri.nic.in/Clinicaltrials/login.php.Date registered 21/10/2020.reg no.CTRI/2020/10/028532.

IL-8 Downregulation Mediates the Beneficial Effects of Infection-Induced Fever on Breast Cancer Prognosis.

Previous studies have reported that infection-induced fever is associated with improved breast cancer prognosis, potentially through the modulation of cytokines. However, the key cytokines and the underlying mechanisms through which fever exerts its anti-tumor effects remain unclear. A total of 794 breast cancer patients were recruited between 2008 and 2017, with follow-up extending until October 31st, 2023. Infection-induced fever was assessed using questionnaires, while a multiplex assay evaluated a panel of 27 cytokines. The mediation effects of various cytokines were analyzed through model-based causal mediation analysis. Additionally, we explored modifications to these mediation effect by examining interactions among the cytokines themselves as well as their interactions with infection-induced fever. Bioinformatic analyses were conducted to elucidate the biological pathways mediating infection-induced fever. The relationship between infection-induced fever and improved breast cancer prognosis was mediated by a decrease in interleukin-8 (IL-8) levels. Furthermore, our findings revealed that the downregulation of IL-8, which mediates the beneficial effects of fever, was antagonized by IL-2, IL12p70 and IL-7. By intersecting the biological pathways influenced by IL-8, alongside those affected by IL-2, IL12p70, or IL-7, we found that these latter cytokines antagonized the mediation effects of IL-8 via regulating critical pathways such as neutrophil degranulation, extracellular matrix organization and asparagine N-linked glycosylation. Infection-induced fever may improve breast cancer prognosis through IL-8 downregulation and the mediation mechanisms may be involved in neutrophil degranulation, extracellular matrix organization and asparagine N-linked glycosylation. Such findings not only provide valuable insights into effectively managing febrile responses for breast cancer patients, but also underscore the therapeutic potential of cytokines in breast cancer patients.

MARKERS OF INFLAMMATION ARE MODIFIED BY SEX, FRAILTY AND EXERCISE IN A HEAD-DOWN TILT BEDREST STUDY

Abstract Prolonged bedrest can induce physiological stress thereby exacerbating frailty and inflammation. We explored whether Head-Down Tilt (HDT) bedrest with or without exercise would alter inflammatory markers implicated in frailty. Twenty 55-65-year-old males (baseline frailty index (FI)=0.02±0.02) and females (FI=0.03±0.03) were subjected to HDT bedrest (2-weeks). Half were randomly assigned to a high-intensity interval/aerobic/strength training exercise intervention for 60 minutes/day (Control: n=4 males, 5 females; Exercise n=5 males, 6 females). FI and serum inflammatory cytokines were measured (multiplex assay) during bedrest (days 0,1,3,7,14), and recovery (days 0,4,32). Data were analyzed with linear mixed models. Proinflammatory marker levels (interleukin (IL)-6, IL-8) increased during bedrest in all groups but were higher in males than females (IL-6: ß=8.87, IL-8: ß=12.93; p&amp;lt; 0.01). IL-8 declined during recovery in all groups, although IL-8 and IL-6 remained higher in males (IL-6: ß=9.32, IL-8: ß=2.85, p&amp;lt; 0.01). The anti-inflammatory cytokine IL-10 was higher in males than females during bedrest, declined in recovery and remained highest in males. During bedrest proinflammatory cytokines (IL-1ß, IL-12) increased as frailty increased in all participants (IL- ß: ß=0.01, IL-12: ß=0.02, p&amp;lt; 0.05) whereas monocyte chemoattractant protein-1 levels declined (ß =-20.54, p&amp;lt; 0.05). The anti-inflammatory protein IL-1ra increased with frailty throughout bedrest in all, especially those who exercised (ß=3.15, p&amp;lt; 0.01). In older adults, inflammatory markers increased during bedrest, declined during recovery and were higher in males than females. Some inflammatory markers increased in parallel with frailty during bedrest. Exercise also increased IL-1ra levels, which may help reduce inflammation in older adults during prolonged bedrest.

Single priming and a booster dose of 10-valent and 13-valent pneumococcal conjugate vaccine (PCV) maintains suppression of vaccine serotype colonization in South African children at 3, 4, and 5 years of age: a single-centre, open-labelled, randomized trial

ABSTRACT Background Surveillance on nasopharyngeal Streptococcus pneumoniae carriage in older children would be informative in determining whether a single priming and booster dose of pneumococcal conjugate vaccine (PCV) provides durable protection against pneumococcal disease compared with traditional dosing schedules. Methods and objectives We report on the secondary study objective to evaluate overall, vaccine-serotype (VT), and non-vaccine serotype (NVT) S. pneumoniae colonization at 3, 4, and 5 years of age in children who were randomized to receive 10-valent or 13-valent PCV formulations at 6 (6w + 1) or 14 (14w + 1) weeks compared with a two-dose primary series (2 + 1), with all children receiving a booster dose at 9 months of age, using a multiplex nanofluidic qPCR assay. Results The prevalence of overall, VT, or NVT at 5 years of age between the 2 + 1 compared with the 6w + 1 or 14w + 1 groups for both PCV10 and PCV13 did not differ. Conclusion Although inconclusive, our findings suggest that a reduced 1 + 1 PCV dosing schedule is unlikely to increase breakthrough cases of VT pneumococcal disease in older children, which can inform decision-making on transitioning to a 1 + 1 schedule in South Africa. Clinical trial registration: The trial is registered at www.clinicaltrials.gov (identifier is NCT04275284).