Whole Blood Cultures Research Articles

An efficient and simple method for enriching metaphase cells for dicentric chromosome assay.

As compared to peripheral blood mononuclear cell (PBMC) culture, a lower mitotic index (MI) is seen in whole blood (WB) culture, but WB can be directly used for culture in dicentric chromosome assay (DCA). The purpose of this study is to develop a simple protocol for metaphase enrichment to improve the metaphase frequency of WB culture. Fixed cells were obtained after performing WB and PBMC cultures for DCA after conventional fixation. An additional low-speed centrifugation of 200×g for 1min was performed, separating the fixed cells of WB culture into a pellet and a supernatant fraction. The additional low-speed centrifugation enriched metaphase frequency and provided an MI comparable to the PBMC culture in the pellet fraction. Our study suggests that it is possible to increase the number of metaphase cells on slides using the slow centrifugation method, which could contribute to the efficiency of chromosome aberration analysis in biodosimetry.

Increased dicentric chromosome in peripheral lymphocytes is related to acute skin toxicity induced by radiotherapy in cancer patients

ObjectiveTo investigate the impacts of radiotherapy (RT) on dicentric chromosomes in peripheral blood lymphocytes of cancer patients, in order to explore the relationship between dicentric chromosomes and RT-induced adverse reactions. MethodsA total of 33 cancer patients after postoperative RT in a tertiary hospital from October 2021 to May 2022 were enrolled in this study. These patients were grouped according to the grade of acute skin and marrow toxicities determined based on the scoring criteria for acute morbidity developed by the Radiation Therapy Oncology Group (RTOG). Peripheral blood samples were collected from each patient before and after RT, followed by whole-blood lymphocyte culture and chromosome analysis. Dicentric chromosomes were searched automatically using a high-throughput chromosome analysis system and were then confirmed manually. Finally, the relationships of the frequency of dicentric chromosomes (also referred to as the dic frequency) with acute skin and marrow toxicities were assessed. ResultsAfter RT, the mean counts of white blood cells (WBCs), lymphocytes, and platelets significantly decreased, while the red blood cells (RBCs) and hemoglobin notably increased (P ​< ​0.001), while the dic frequency was elevated remarkably, significantly higher in patients with higher-grade (>1) acute skin toxicities compared to those with lower-grade (=1) acute skin toxicities (Z ​= ​−1.985, P ​= ​0.047). However, no significant relationship was observed between the dic frequency and acute marrow toxicity after RT (P ​> ​0.05). Logistic regression showed that radiosensitivity denoted by the post-RT dic frequency in peripheral blood lymphocytes produced insignificant impacts on the severity of RT-induced acute skin toxicities (P ​= ​0.060). ConclusionThe elevated dic frequency in peripheral blood lymphocytes of cancer patients with higher-grade acute skin toxicities suggests enhanced radiosensitivity of peripheral blood lymphocytes, which is associated with the occurrence of RT-induced adverse reactions.

An antibody to IL-1 receptor 7 protects mice from LPS-induced tissue and systemic inflammation.

Interleukin-18 (IL-18), a pro-inflammatory cytokine belonging to the IL-1 Family, is a key mediator ofautoinflammatory diseases associated with the development of macrophage activation syndrome (MAS).High levels of IL-18 correlate with MAS and COVID-19 severity and mortality, particularly in COVID-19patients with MAS. As an inflammation inducer, IL-18 binds its receptor IL-1 Receptor 5 (IL-1R5), leadingto the recruitment of the co-receptor, IL-1 Receptor 7 (IL-1R7). This heterotrimeric complex subsequentlyinitiates downstream signaling, resulting in local and systemic inflammation. We reported earlier the development of a novel humanized monoclonal anti-human IL-1R7 antibody whichspecifically blocks the activity of human IL-18 and its inflammatory signaling in human cell and wholeblood cultures. In the current study, we further explored the strategy of blocking IL-1R7 inhyperinflammation in vivo using animal models. We first identified an anti-mouse IL-1R7 antibody that significantly suppressed mouse IL-18 andlipopolysaccharide (LPS)-induced IFNg production in mouse splenocyte and peritoneal cell cultures. Whenapplied in vivo, the antibody reduced Propionibacterium acnes and LPS-induced liver injury and protectedmice from tissue and systemic hyperinflammation. Importantly, anti-IL-1R7 significantly inhibited plasma,liver cell and spleen cell IFNg production. Also, anti-IL-1R7 downregulated plasma TNFa, IL-6, IL-1b,MIP-2 production and the production of the liver enzyme ALT. In parallel, anti-IL-1R7 suppressed LPSinducedinflammatory cell infiltration in lungs and inhibited the subsequent IFNg production andinflammation in mice when assessed using an acute lung injury model. Altogether, our data suggest that blocking IL-1R7 represents a potential therapeutic strategy to specificallymodulate IL-18-mediated hyperinflammation, warranting further investigation of its clinical application intreating IL-18-mediated diseases, including MAS and COVID-19.

Abstract 2730: Beyond antibodies and CAR-T: Topologically-engineered, super-dimeric antibody-like molecules with dual Fc domains for trispecific, bivalent targeting of CD19, CD20, and Fcgamma receptors

Abstract Background: Depletion of B cells has resulted in major therapeutic benefits in autoimmune diseases and hematological malignancies. Several FDA-approved B cell-depleting antibody-based therapies are available, including antibody-drug conjugates, T cell engagers, CAR-T cells, and antibody variants with enhanced effector functions through Fc engineering. However, a significant unmet need remains in both oncology and autoimmune disease. All currently approved B cell-depleting therapies target a single B cell antigen, increasing potential for incomplete depletion and emergence of escape mutants that give rise to relapse and treatment failure. Methods: Here we describe trispecific antibody-like molecules generated using GEM-DIMER technology. In addition to bivalent target binding of both CD19 and CD20, the GEM-DIMER candidates are designed to have two Fc domains to enable powerful effector functions via cooperative binding of Fcgamma receptors. The approach is based on incorporation of a super-dimerization domain into the hinge region of an immunoglobulin heavy chain, enabling the combination of all of the components of two IgG antibodies into a single molecule. Using this approach, we generated GEM-DIMER candidates from rituximab and FMC63, the parental anti-CD19 antibody for anti-CD19 scFv used in approved CAR-T cell therapies. Results: CD19/CD20-targeting GEM-DIMER molecules demonstrated binding to both CD19 and CD20 with affinities comparable to the individual parent antibodies. Importantly, CD19/CD20-targeting GEM-DIMER molecules demonstrated robust depletion of human B cells in overnight cultures of whole blood. Moreover, antibody-dependent cellular cytotoxicity (ADCC) was demonstrated in co-cultures of the human B cell lymphoma cell line Raji and human peripheral blood mononuclear cells (PBMC). As expected, due to the presence of dual Fc domains, CD19/CD20-targeting GEM-DIMER molecules exhibited enhanced binding to Fcgamma receptors, further explaining the robust effector functions observed. Conclusions: CD19/CD20-targeting GEM-DIMER molecules are promising candidates to provide efficient depletion of both CD19+ and CD20+ cells, providing potential for broad and deep depletion of B cells with reduced risk of emergence of antigen escape variants. These data support the advancement of CD19/CD20-targeting GEM-DIMER molecules in multiple indications where depletion of CD19+ and/or CD20+ B cells is needed. Preparations for clinical investigation are ongoing. Citation Format: Daniel J. Capon, Nelson L. Chan, Larisa Troitskaya, Marina Fomin, Ursula Edman, Brendon Frank, Benjamin Z. Capon, Brian Law, Steven J. Chapin, Gavin M. Lewis, Malcolm L. Gefter, Juha Punnonen. Beyond antibodies and CAR-T: Topologically-engineered, super-dimeric antibody-like molecules with dual Fc domains for trispecific, bivalent targeting of CD19, CD20, and Fcgamma receptors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2730.

Rapid Phenotypic Antibiotic Susceptibility Profiling of Clinical Escherichia coli and Klebsiella pneumoniae Blood Cultures.

Bloodstream infections (BSI) are defined by the presence of viable bacteria or fungi, accompanied by systemic signs of infection. Choosing empirical therapy based solely on patient risk factors and prior antibiotic susceptibility test (AST) may lead to either ineffective treatment or unnecessarily broad-spectrum antibiotic exposure. In general, Clinical & Laboratory Standards Institute guideline-approved ASTs have a turnaround time of 48-72 h from sample to answer, a period that may result in a critical delay in the appropriate selection of therapy. Therefore, reducing the time required for AST is highly advantageous. We have previously shown that our novel rapid AST method, MAPt (Micro-Agar-PCR-test), accurately identifies susceptibility profiles for spiked bioterrorism agents like Bacillus anthracis, Yersinia pestis and Francisella tularensis directly from whole-blood and blood culture samples, even at low bacterial levels (500 CFU/mL). This study evaluated the performance of MAPt on routine bloodstream infection (BSI), focusing on Escherichia coli and Klebsiella pneumoniae isolates from clinical cultures, including resistant strains to some of the six tested antibiotics. Notably, MAPt yielded results exceeding 95% agreement with the standard hospital method within a significantly shorter timeframe of 6 h. These findings suggest significant potential for MAPt as a rapid and reliable BSI management tool.

Different immunological responses following immunization with two mRNA vaccines

IntroductionImmunological responses were investigated following immunization with two mRNA vaccines: BNT162b2 and mRNA-1273. MethodsNeutralizing antibody (NAb) was assayed before, 2–4 weeks after, and 3 and 6 months after the primary immunization, and the same time-points after booster dose with 6- or 8-months interval. Whole-blood culture was stimulated with spike antigen, and cytokine production was assayed. ResultsNAb was detected after primary immunization, NAb titers began to decrease three months after primary immunization with BNT162b2, lower than those after mRNA-1273, and elevated after booster immunization. The NAb level was 1/2 lower against δ variant, and 1/16 lower against omicron variant in comparison with that against α variant. Cytokine production following immunization with mRNA-1273 was maintained within three months at higher levels of Th1 (TNF-α), Th2 (IL-4 and IL-5), and inflammatory cytokines (IL-6 and IL-17) than that following immunization with BNT162b2, reflecting prominent levels of NAb following immunization with mRNA-1273. Cytokine production decreased six months after primary immunization in both vaccine recipients and was enhanced following booster doses. During the omicron outbreak, medical staff members in the outpatient office experienced asymptomatic infection, with a greater than 4-fold increase in NAb titers against omicron variant even after booster immunization. Asymptomatic infection enhanced the production of Th2 and inflammatory cytokines. ConclusionmRNA-1273 induced stronger NAb responses with wide-range cross-reactive antibodies against δ and omicron variants. mRNA-1273 induced higher levels of Th1, Th2, and inflammatory cytokines than BNT162b2 did, reflecting higher levels of NAb against variant strains.

Using whole blood cultures in interferon gamma release assays to detect Mycobacterium tuberculosis complex infection in Asian elephants (Elephas maximus).

Elephants are susceptible to Mycobacterium tuberculosis (M. tb) complex (MTBC) infections. Diagnosis of tuberculosis (TB) in elephants is difficult, and most approaches used for human TB diagnosis are not applicable. An interferon gamma release assay (IGRA) to diagnose TB in Asian elephants (Elephas maximus) using peripheral blood mononuclear cells (PBMCs) has been previously developed. Although the assay is shown to be valid in determining MTBC infection status, the laborious PBMC isolation process makes it difficult to use. In this study, we simplified the method by using whole blood cultures (WC) as the starting material. Using PBMC cultures for IGRA, the MTBC infection status of 15 elephants was first confirmed. Among these animals, one has been previously confirmed for M. tb infection by both TB culture and PCR and the other was confirmed for MTBC infection in this study by droplet digital PCR (ddPCR) method. WC for IGRA consisted of an unstimulated sample, a mitogen stimulated sample, and sample stimulated with recombinant M. tb antigens, ESAT6 and CFP10. Using WC for IGRA in the 15 enrolled elephants, the results showed that 7 out of 15 samples yielded MTBC infection positive status that were completely concordant with those from the results using PBMCs. To test this method, WC for IGRA were applied in another elephant cohort of 9 elephants. The results from this cohort revealed a perfect match between the results from PBMC and WC. Responses to ESAT6 or CFP10 by PBMC and WC were not completely concordant, arguing for the use of at least two M. tb antigens for stimulation. Given the ease of sample handling, smaller blood sample volumes and equivalent efficacy relative to the PBMC approach, using WC for IGRA provides a novel, rapid, and user-friendly TB diagnostic method for determining the MTBC infection in elephants.

Blood transcriptome responses to PFOA and GenX treatment in the marsupial biomedical model Monodelphis domestica.

Introduction: Perfluoroalkyl and poly-fluoroalkyl substances (PFASs) are widely used in industrial and consumer products. Due to their environmental persistence and bioaccumulation, PFASs can be found in the blood of humans and wild animals all over the world. Various fluorinated alternatives such as GenX have been developed to replace the long-chain PFASs, but there is limited information about their potential toxicity. Methods:The current study developed blood culture protocols to assess the response to toxic compounds in the marsupial, Monodelphis domestica. After whole-blood culture conditions were tested and optimized, changes in gene expression in response to PFOA and GenX treatment were assessed. Results: More than 10,000 genes were expressed in the blood transcriptomes with and without treatment. Both PFOA and GenX treatment led to significant changes in the whole blood culture transcriptomes. A total of 578 and 148 differentially expressed genes (DEGs) were detected in the PFOA and GenX treatment groups, 32 of which overlapped. Pathway enrichment analysis revealed that DEGs involved in developmental processes were upregulated after PFOA exposure, while those enriched for metabolic and immune system processes were downregulated. GenX exposure upregulated genes associated with fatty acid transport pathways and inflammatory processes, which is consistent with previous studies using rodent models. Discussion: To our knowledge, this study is the first to investigate the effect of PFASs in a marsupial model. The findings provide supportive evidence for significant transcriptomic alterations, suggesting that this mammalian model may provide a mechanism for exploring the potential toxicity of PFOA and GenX.

PATHOLOGICAL IMMUNITY CHANGES IN COMORBID PATIENTS WITH EXACERBATION OF BRONCHIAL ASTHMA

The aim of the study – to investigate immunological changes in patients with exacerbation of bronchial asthma with different severity and controllability depending on the presence of comorbid pathology. Materials and methods of research. The work is based on the analysis of data from a comprehensive clinical and laboratory examination of 190 patients with exacerbation of asthma. The relative content of T–lymphocytes and their main subpopulations was determined on a flow cytofluorimeter FACS Calibur (Canada) by phenotyping lymphocytes with monoclonal antibodies (BECKMAN COULTER, USA) to surface membrane differentiation antigens (CD): CD3+19– (pan T-cells), CD4+–8+– (T−helpers/inducers), CD4–8+ (cytotoxic T lymphocytes), CD3–16+ (natural killers). To identify the imbalance of immunoregulatory subpopulations of T cells, the immunoregulatory index was calculated – the ratio of the number of CD4+8– Lf and CD4-8+ Lf. To calculate the absolute content in the peripheral blood of individual populations of Lf, leukogram indicators determined on the hematological analyzer ABH-miscros60, France were used. The proliferative activity of lymphocytes was evaluated in cultures of whole blood in the reaction of blast transformation of these cells with morphological accounting of the results. Statistical processing of the obtained data was carried out using licensed software products included in the Microsoft Office Professional 2007 package, license Russian Academic OPEN No Level № 43437596 in Excel. Conclusions. In moderate asthma, the frequency of pathological changes in the T-system of immunity did not depend on the presence of comorbid pathology, as a rule. In severe asthma, an increase in the content of pan–T cells occurred 1.2 times more often than in moderate asthma, but this was not observed in people with concomitant pathology. In this group, the frequency of increasing the content of T–helpers and decreasing the content of cytotoxic T – cells in patients with severe asthma without concomitant diseases was 1.3 times more frequent than in patients with comorbid pathology. No pathological changes in the T–system were detected in patients with controlled asthma. In partially controlled asthma, these changes were registered 1.5 times more often than in controlled asthma, and in its uncontrolled course, the prevalence of pathological changes in the T–system of immunity was much higher than in the two previous groups. In patients with concomitant diseases, these changes, as a rule, had a higher frequency than in patients without comorbid pathology.

Metabolic and immune/inflammatory alterations induced by a triathlon under extreme conditions.

PurposeTo investigate the effects of triathlon racing under extreme conditions on metabolic and immune/inflammatory responses.MethodsThirteen amateur athletes participated in an extreme triathlon competition (swim – 3.8 km; cycling – 180 km; running – 4 2 km; with a 3,700 m accumulated altitude). Blood samples were collected on three different occasions: pre-competition (baseline), immediately post-competition (IM), and 12 h post-competition (12 h) to evaluate glycemic and lipid profiles, leukocytes count, and cytokines levels in plasma and in whole-blood cell culture supernatant stimulated or not with LPS.ResultsDecreased glucose and triglycerides levels, increased LDL, and a significant leukocytosis were observed at IM and 12 h compared to baseline. In addition, higher serum levels of IL-6, IL-8, and IL-10 were found at IM than in baseline and post-12 h. Whereas increased IL-12p40 levels were observed for 12 h compared to baseline. At baseline, in LPS-stimulated cell culture, IL-6, IL-8, and IL-12p70 were higher, while IL-12p40 levels were lower than non-stimulated cell culture. At IM, IL-12p40 levels were unchanged, while higher levels of other cytokines were found in LPS-stimulated cell culture compared to non-stimulated cell culture. The 12 h results showed higher levels of IL-6, IL-8, and IL-10 in LPS-stimulated cell culture than in non-stimulated cell culture. Additionally, a significant negative correlation between circulating glucose levels and IL-6 was found.ConclusionThe triathlon competition's performance under extreme conditions has remarkable impacts on the lipid profile and systemic immune/inflammatory responses. For the first time, significant alterations in the cytokine responses of whole blood cell culture to LPS-stimulation in baseline, IM, and 12h were demonstrated.

Estimation of the effect of lithium salts on cytokine production by blood cells in in vitro experiments

Aim. To study the effects of lithium salts on production of cytokines by immunocompetent cells in the whole-blood culture of patients with alcohol dependence and affective disorders.Materials and methods. The study materials were blood samples from 25 patients with alcohol dependence (AD) and 12 patients with bipolar disorder (BD). Blood diluted 1:1 with complete RPMI-1640 medium (Gibco, UK) was added to the wells of the culture plate, then new lithium salts (succinate, fumarate, pyruvate, ascorbate) and a reference salt – lithium carbonate at a final concentration of 1.2 mmol / l per lithium ion – were added. In parallel, control samples without lithium salts were tested; the samples were incubated for a day. The concentration of cytokines (interferon (IFN) γ, interleukin (IL)-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-17A, tumor necrosis factor (TNF) α) was determined in the culture supernatants on the MAGPIX multiplex analyzer (Luminex, USA) (Center for Collective Use “Medical Genomics”, Tomsk NRMC) using the Human Cytokine / Chemokine Magnetic Bead Panel (Merck, Germany).Results. All lithium salts had a unidirectional effect on the production of cytokines by immunocompetent cells (ICC), except for lithium ascorbate and IL-8. The concentrations of cytokines in the supernatants of loaded and control samples (spontaneous production) were comparable, which indicates an absence of stimulating or suppressing effects of salts on the functional activity of ICC under the experimental conditions. The effect of lithium ascorbate as an IL-8 inducer was detected: the production of IL-8 induced by lithium ascorbate was 2.3–2.5 times higher than its spontaneous production.Conclusion. The obtained results, as well as the previously revealed antioxidant and cytoprotective properties of new lithium salts, confirmed that they are promising for development of pharmacological agents with combined action.

Mycobacterium leprae Induces Neutrophilic Degranulation and Low-Density Neutrophil Generation During Erythema Nodosum Leprosum.

Erythema Nodosum Leprosum (ENL) is a recurrent acute inflammatory complication of leprosy affecting up to 50% of all Borderline Lepromatous and Lepromatous Leprosy (BL/LL) patients. Although ENL is described as an immune reaction mediated by neutrophils, studies demonstrating the direct role of neutrophils in ENL are still rare. One subpopulation of low-density neutrophils (LDNs), present within the fraction of peripheral blood mononuclear cells (PBMC), has been associated with the pathogenesis and severity of diseases like sepsis, lupus, and tuberculosis. We herein analyzed LDNs and high-density neutrophils (HDNs) in terms of frequency, phenotype, and morphology. Serum levels of MMP-9 (a neutrophilic degranulation marker) were evaluated by ELISA; and LDNs were generated in vitro by stimulating healthy-donor, whole-blood cultures. PBMC layers of ENL patients presented segmented/hypersegmented cells that were morphologically compatible with neutrophils. Immunofluorescence analyses identified LDNs in ENL. Flow cytometry confirmed the elevated frequency of circulating LDNs (CD14−CD15+) in ENL patients compared to healthy donors and nonreactional Borderline Tuberculoid (BT) patients. Moreover, flow cytometry analyses revealed that ENL LDNs had a neutrophilic-activated phenotype. ENL patients under thalidomide treatment presented similar frequency of LDNs as observed before treatment but its activation status was lower. In addition, Mycobacterium leprae induced in vitro generation of LDNs in whole blood in a dose-dependent fashion; and TGF-β, an inhibitor of neutrophilic degranulation, prevented LDNs generation. MMP-9 serum levels of BL/LL patients with or without ENL correlated with LDNs frequency at the same time that ultrastructural observations of ENL LDNs showed suggestive signs of degranulation. Together, our data provide new insights into the knowledge and understanding of the pathogenesis of ENL while enriching the role of neutrophils in leprosy.

Investigation of IL-2 and IFN-γ to EBV Peptides in Stimulated Whole Blood among Multiple Sclerosis Patients and Healthy Individuals

Introduction: Epstein-Barr virus (EBV), a double-stranded DNA virus, has 2 phases of lytic and latent infection in host cells. After infecting B lymphocytes, EBV becomes persistent in these cells. In healthy individuals, T lymphocytes play a key role in killing EBV-infected B cells. Statistical studies have shown that symptomatic EBV infection increases the risk of MS. Methods: This study intended to measure the immune system’s response against the different components of EBV, focusing particularly on T lymphocytes’ reaction. Consequently, the mRNA level of IL-2 and IFN-γ, liable for impressing autoimmune diseases and as indicators of T-cell function, was compared in EBNA1- and BRLF1-treated whole blood (WB) cultures of 10 healthy individuals and 10 MS patients using real-time RT-PCR. Results: The analysis of the results demonstrated a significant increased level of IL-2 in MS patients than healthy subjects after exposure to both peptides. Also, the mRNA level of IFN-γ increased in MS patients in EBNA1-treated WB culture. Conclusion: According to the study’s results, EBV peptides can reactivate immune cells, especially T lymphocytes, and may indirectly induce inflammation and develop MS; however, it seems that long-time exposure to these peptides has reducing effect on T-cell function and faces the control of infected B lymphocytes with difficulties.

The Cytokinesis-Block Micronucleus Assay on Human Isolated Fresh and Cryopreserved Peripheral Blood Mononuclear Cells.

The cytokinesis-block micronucleus (CBMN) assay is a standardized method used for genotoxicity studies. Conventional whole blood cultures (WBC) are often used for this assay, although the assay can also be performed on isolated peripheral blood mononuclear cell (PBMC) cultures. However, the standardization of a protocol for the PBMC CBMN assay has not been investigated extensively. The aim of this study was to optimize a reliable CBMN assay protocol for fresh and cryopreserved peripheral blood mononuclear cells (PBMCS), and to compare micronuclei (MNi) results between WBC and PBMC cultures. The G0 CBMN assay was performed on whole blood, freshly isolated, and cryopreserved PBMCS from healthy human blood samples and five radiosensitive patient samples. Cells were exposed to 220 kV X-ray in vitro doses ranging from 0.5 to 2 Gy. The optimized PBMC CBMN assay showed adequate repeatability and small inter-individual variability. MNi values were significantly higher for WBC than for fresh PBMCS. Additionally, cryopreservation of PBMCS resulted in a significant increase of MNi values, while different cryopreservation times had no significant impact. In conclusion, our standardized CBMN assay on fresh and cryopreserved PBMCS can be used for genotoxicity studies, biological dosimetry, and radiosensitivity assessment.

Genetic and Functional Characterization of Toll-Like Receptor Responses in Immunocompetent Patients With CMV Mononucleosis.

Background: Human cytomegalovirus (CMV) modulates both innate and adaptive immune responses. However, limited data are available on the role of receptors of innate immunity, such as toll-like receptors (TLRs) in contributing to antiviral responses and inflammation.Objectives: The aim of this translational study was to characterize TLR responses in immunocompetent patients with primary and symptomatic CMV infection.Study Design: The study population consisted of 40 patients suffering from CMV mononucleosis and 124 blood donors included as controls. We evaluated the association between TLR2, 3, 4, 7 and 9 gene single nucleotide polymorphism (SNP) and susceptibility to symptomatic CMV infection in immunocompetent adults. Additionally, functional TLR-mediated cytokine responses in supernatants of short-term cultures of whole blood from patients with CMV mononucleosis and blood donors were evaluated.Results: TLR2 and TLR7/8 responses were altered in CMV infected patients as compared to healthy donors and were associated with the release of higher levels of the pro-inflammatory cytokines IL-6 and TNF-α, but not of the anti-inflammatory mediator IL-10. The analysis on the TLR SNPs indicated no difference between patients with CMV infection and the control group.Conclusions: No variation in the TLR2,3,4,7 and 9 genes was associated to the development of symptomatic CMV infection in immunocompetent adults. Nevertheless, TLR-mediated responses in CMV-infected patients appeared to be skewed toward a pro-inflammatory profile, which may contribute to the development of inflammatory symptoms during the CMV mononucleotic syndrome.

Hypercapnia exacerbates the disruption of the blood‑brain barrier by inducing interleukin‑1β overproduction in the blood of hypoxemic adult rats.

Refractory hypoxemia is the main symptom of acute respiratory distress syndrome (ARDS). Low tidal volume ventilation is routinely applied in clinical practice to correct hypoxemia, which aims to prevent ventilator-induced lung injury. However, this ventilation strategy inevitably leads to hypercapnia. Our previous study demonstrated that hypercapnia aggravated cognitive impairment in hypoxemic rats; however, the underlying mechanism remains unclear. The aim of the present study was to investigate whether hypercapnia exacerbates the blood-brain barrier (BBB) disruption through inducing interleukin (IL)-1β overproduction in the blood of hypoxemic rats. The BBB permeability in a rat model of hypercapnia/hypoxemia was evaluated. The levels of IL-1β in the blood of rats and human whole-blood cultures were assessed. The expression of IL-1 receptor 1 (IL-1R1), phosphorylated IL-1R1-associated kinase (p-IRAK-1) and tight junctional proteins in cerebral vascular endothelial cells was examined in vitro and in vivo. In addition, IL-1Ra, an IL-1 receptor antagonist, was used to determine whether hypercapnia affects tight junctional protein expression in hypoxic cerebral vascular endothelial cells through inducing IL-1β overproduction. It was observed that hypercapnia alone did not disrupt the BBB, but aggravated the damage to the BBB integrity in hypoxemic rats. Hypercapnia increased IL-1β expression in the blood of hypoxemic rats as well as in hypoxic human whole-blood cultures. IL-1R1 and p-IRAK-1 expression was increased, while that of tight junctional proteins was reduced by hypercapnia in hypoxemic cerebral vascular endothelial cells in vitro and in vivo. Additionally, the expression of tight junctional proteins was markedly increased following treatment with IL-1Ra. These results suggest that hypercapnia-induced IL-1β overproduction in the hypoxemic blood may decrease tight junctional protein expression in cerebrovascular endothelial cells via the IL-1R1/p-IRAK-1 pathway, further disrupting BBB integrity, and eventually resulting in increased BBB permeability.

Treatment with 7% and 10% CO2 enhanced expression of IL-1β, TNF-α, and IL-6 in hypoxic cultures of human whole blood.

ObjectiveThis study investigated whether hypercapnia influenced the inflammatory response of hypoxic blood.MethodsHuman whole blood was cultured with 0.2% oxygen (O2) and treated with 5%, 7%, or 10% carbon dioxide (CO2). Interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and IL-6 were evaluated in whole blood cultures. Reactive oxygen species (ROS) production and expression levels of caspase-1 and IL-1β were evaluated in THP-1 monocytic cells.ResultsIL-1β, TNF-α, and IL-6 levels were higher in the hypoxia + 7% CO2 group than in the hypoxia + 5% CO2 group. The hypoxia + 10% CO2 group had the highest IL-1β, TNF-α, and IL-6 levels, compared with the hypoxia + 7% CO2 and hypoxia + 5% CO2 groups. Expression levels of IL-1β, TNF-α, and IL-6 were significantly negatively correlated with pH levels in the cell culture medium. Treatment with 7% and 10% CO2 increased the production of ROS and the expression of caspase-1 and IL-1β in hypoxia-activated THP-1 cells.ConclusionsHigh levels of CO2 treatment increased expression levels of IL-1β, TNF-α, and IL-6 in hypoxic whole blood cultures. High levels of CO2-induced ROS overproduction and NLRP3 inflammasome activation in monocytes may comprise a target to mitigate the inflammatory response of hypoxic blood.

The Impact of Acute Ingestion of a Ketone Monoester Drink on LPS-Stimulated NLRP3 Activation in Humans with Obesity

Activation of the NOD-like receptor pyrin-domain containing 3 (NLRP3) inflammasome is associated with chronic low-grade inflammation in metabolic diseases such as obesity. Mechanistic studies have shown that β-hydroxybutyrate (OHB) attenuates activation of NLRP3, but human data are limited. In a randomized, double-blind, placebo-controlled crossover trial (n = 11) we tested the hypothesis that acutely raising β-OHB by ingestion of exogenous ketones would attenuate NLRP3 activation in humans with obesity. Blood was sampled before and 30 min post-ingestion of a ketone monoester drink ((R)-3-hydroxybutyl (R)-3-hydroxybutyrate, 482 mg/kg body mass) or placebo. A 75 g oral glucose load was then ingested, and a third blood sample was obtained 60 min following glucose ingestion. NLRP3 activation was quantified by assessing monocyte caspase-1 activation and interleukin (IL)-1β secretion in ex vivo lipopolysaccharide (LPS)-stimulated whole-blood cultures. LPS-stimulated caspase-1 activation increased following glucose ingestion (main effect of time; p = 0.032), with no differences between conditions. IL-1β secretion did not differ between conditions but was lower 60 min post-glucose ingestion compared to the fasting baseline (main effect of time, p = 0.014). Plasma IL-1β was detectable in ~80% of samples and showed a decrease from fasting baseline to 60 min in the ketone condition only (condition × time interaction, p = 0.01). In individuals with obesity, an excursion into hyperglycemia following ingestion of a glucose load augments LPS-induced activation of caspase-1 in monocytes with no apparent impact of raising circulating β-OHB concentration via ingestion of exogenous ketones. Exogenous ketone supplementation may impact plasma IL-1β, but these findings require confirmation in studies with larger sample sizes.

Toxoplasma gondii protects from IgE sensitization and induces Th1/Th2 immune profile.

Toxoplasma gondii (T gondii) infection has been associated with protection against allergy and autoimmune diseases. We investigated the effects of T gondii infection on cytokine and antibody responses in atopic and nonatopic Brazilian subjects. We have measured in whole-blood cultures, Th1 (IFN-γ and IL-12), Th2 (IL-5) and regulatory cytokine IL-10 in blood cells unstimulated and stimulated with pokeweed mitogen or T gondii soluble tachyzoites antigen (STAg) or Dermatophagoides pteronyssinus antigen. A significant negative association was found between high levels of anti-dust mite IgE and T gondii seropositivity (OR=0.46; 95%CI=0.25-0.85). STAg stimulation induced a mixed profile of Th1 and Th2 cytokines (IFN-γ, IL-12 and IL-5) in Tg-positive atopic individuals compared with Tg-negative atopic individuals (P<.0001, P=.033 and P=.003, respectively). In contrast, IL-10 production was not different between these groups. No association was found between T gondii infection and asthma. We hypothesized that the protective effect on atopy might be related to the strong Th1 immune response to T gondii found on the seropositive subjects. From our knowledge, this is the first study to investigate the association between atopy and T gondii infection in Brazilian subjects, analysing the cellular immune responses.

Patients with enthesitis related arthritis show similar monocyte function pattern as seen in adult axial spondyloarthropathy

BackgroundAxial SpA and Enthesitis related arthritis (ERA) patients show strong HLA-B27 association, gut dysbiosis, high toll like receptor (TLR)2 and 4 expression on monocytes, pro-inflammatory cytokine production and elevated levels of TLR4 endogenous ligands [tenascin-c (TNC) and myeloid related protein (MRP)8/14] in serum. Hence, we aimed to understand if these diseases have similar or different monocyte response.MethodsFifty adult axial SpA, 52 ERA patients and 25 healthy controls (HC) were enrolled. Cytokine-producing monocyte frequency before and after stimulation with lipopolysaccharide (LPS), peptidoglycan (PG), TNC or MRP8 were measured in whole blood (WB) and synovial fluid mononuclear cells (SFMC) by flow cytometry. Also, IL-6, TNF, MMP3, TNC and MRP8/14 levels were measured in unstimulated and TLR ligand stimulated WB cultures supernatant by ELISA. Finally, the mRNA expression levels of TNF and IL-6 were measured post stimulation with LPS, TNC and MRP8.ResultsAt baseline, ERA and axial SpA patients showed similar TNF-α producing monocyte frequency which was higher than HC. MRP8 simulation led to increased TNF-α producing monocyte frequency in ERA than axial SpA. TNC and MRP8 stimulation led to similar IL-6 producing monocyte frequency in axial SpA and ERA patients. Baseline TNF and IL-6 producing monocyte frequency also modestly correlated with disease activity scores. TNF and IL-6 producing monocyte frequency increased in response to TLR stimulation in SFMC from both patients.In culture supernatants, axial SpA and ERA patients showed similar TNF production at baseline. MRP8 and TNC stimulation led to higher TNF production from ERA. Baseline IL-6 and MMP3 production was higher in ERA while TLR stimulation led to similar IL-6 and MMP3 production from axial SpA and ERA. TNC stimulation led to higher MMP3 production in ERA. mRNA expression in response to TLR stimulation was observed to be similar in axial SpA and ERA.TNC production was higher in ERA at baseline, while MRP8/14 production was higher in axial SpA than ERA post stimulation.ConclusionERA patients have similar monocyte response to exogenous and endogenous TLR ligands as patients with axial SpA. This suggests that differences between pediatric and adult-onset SpA are minimal and they may have a common pathogenesis.