Monocyte Assay Research Articles

RNA Sequencing Analysis of Monocytes Exposed to Airway Fluid From Children With Pediatric Acute Respiratory Distress Syndrome.

Monocytes are plastic cells that assume different polarization states that can either promote inflammation or tissue repair and inflammation resolution. Polarized monocytes are partially defined by their transcriptional profiles that are influenced by environmental stimuli. The airway monocyte response in pediatric acute respiratory distress syndrome (PARDS) is undefined. To identify differentially expressed genes and networks using a novel transcriptomic reporter assay with donor monocytes exposed to the airway fluid of intubated children with and at-risk for PARDS. To determine differences in gene expression at two time points using the donor monocyte assay exposed to airway fluid from intubated children with PARDS obtained 48-96 hours following initial tracheal aspirate sampling. In vitro pilot study carried out using airway fluid supernatant. Academic 40-bed PICU. Fifty-seven children: 44 children with PARDS and 13 children at-risk for PARDS. None. We performed bulk RNA sequencing using a transcriptomic reporter assay of monocytes exposed to airway fluid from intubated children to discover gene networks differentiating PARDS from at-risk for PARDS and those differentiating mild/moderate from severe PARDS. We also report differences in gene expression in children with PARDS 48-96 hours following initial tracheal aspirate sampling. We found that interleukin (IL)-10, IL-4, and IL-13, cytokine/chemokine signaling, and the senescence-associated secretory phenotype are upregulated in monocytes exposed to airway fluid from intubated children with PARDS compared with those at-risk for PARDS. Signaling by NOTCH, histone deacetylation/acetylation, DNA methylation, chromatin modifications (B-WICH complex), and RNA polymerase I transcription and its associated regulatory apparatus were upregulated in children with PARDS 48-96 hours following initial tracheal aspirate sampling. We identified gene networks important to the PARDS airway immune response using bulk RNA sequencing from a monocyte reporter assay that exposed monocytes to airway fluid from intubated children with and at-risk for PARDS. Mechanistic investigations are needed to validate our findings.

A-152 Enhancing PNH Diagnosis with Multicolor Flow Cytometry Immunophenotyping Protocol in a Medical Center in Taiwan

Abstract Background Paroxysmal Nocturnal Hemoglobinuria (PNH) is a rare hematopoietic stem cell disorder resulting from mutations in the X-linked PIG-A gene, leading to an inability to produce GPI-anchored proteins on RBCs and WBCs. PNH presents in three variants: classic (hemolytic), associated with specified bone marrow disorders, and subclinical. Flow cytometric analysis stands as the gold standard for PNH detection, crucial in diagnosis, monitoring, and clinical management. Recognizing potential variations in PNH testing among labs, it is essential to establish a precise and consistent protocol for accurate diagnosis and patient management. This report aims to demonstrate the effectiveness of a PNH immunophenotyping protocol adhering to ICCS/ESCCA PNH consensus guidelines. Methods A total of 501 samples were analyzed from Jan 1st, 2021 to Dec 31st, 2023. All the acquisitions were accomplished by a 3-laser and 10-PMT Navios EX (Beckman Coulter) flow cytometry. A typical combination of anti-CD235a-FITC (clone KC16) and anti-CD59-PE (clone MEM43) antibodies were adopted to identify the PNH clone size in the RBC tube. To achieve higher sensitivities in PNH granulocyte and PNH monocyte detection, a single tube, 6-color mixture of FLAER, monoclonal antibodies including CD14-APC, CD24-PE, CD33-APC-H7, CD15-PacB, and CD45-KrO, to immunostaining simultaneously was developed. An unstained control was run after the full-stained tube to determine the position of threshold for positivity, and to exam the potential carryover into the test sample. Results RBC and granulocyte assays can detect PNH phenotypes at the 0.009% level or higher with more than 50,000 cells of gated cells acquired, while the monocyte assay can detect at >=0.069% with at least 20,000 cells acquired. Analysis was conducted on five hundred and one patients to investigate potential PNH clones in their blood cells. Among them, 45 patients were identified as having classic PNH, while PNH clones were not detected in 168 cases, constituting 33.5% of total cases. Minor PNH clone (<1.0%) in single cell lineage were observed in 144 cases, making up 28.7% of all cases. Minor PNH clones found in more than one cell lineages were found in 155 cases (30.9% of 501). Additionally, minor PNH clones were identified across RBCs, granulocytes, and monocytes, with percentages of 32.9%, 23.8%, and 43.9%, respectively of all patients. Notably, monocyte clones exceeded granulocyte clones in 23.0% of cases with WBC PNH clones, irrespective of clone sizes. Furthermore, we investigated seven patients exhibiting persistent minor PNH clones in 2 to 3 cell lineages. Among them, five had aplastic anemia or myelodysplastic syndromes, while one underwent evaluation for young stroke and one was investigated for venous thromboembolism. Conclusions Flow cytometry labs aim not only to identify classic PNH patients but also to consistently detect minor or small clones in individuals with bone marrow disorders or subclinical conditions. Our findings are compatible with international reports, underscoring the importance of routinely examining patients with minor clones. Such assays serve as essential tools for ongoing monitoring, facilitating PNH diagnosis and management by physicians.

Technical, gating and interpretation recommendations for the partitioning of circulating monocyte subsets assessed by flow cytometry.

The monocyte subset partitioning by flow cytometry, known as "monocyte assay," is now integrated into the new classifications as a supporting criterion for CMML diagnosis, if a relative accumulation of classical monocytes above 94% of total circulating monocytes is observed. Here we provide clinical flow cytometry laboratories with technical support adapted for the most commonly used cytometers. Step-by-step explanations of the gating strategy developed on whole peripheral blood are presented while underlining the most common difficulties. In a second part, interpretation recommendations of circulating monocyte partitioning from the dedicated French working group "CytHem-LMMC" are shared as well as the main pitfalls, including false positive and false negative cases. The particular flow-defined inflammatory profile is described and the usefulness of the nonclassical monocyte specific marker, namely slan, highlighted. Examples of reporting to the physician with frequent situations encountered when using the monocyte assay are also presented.

ELN IMDS Flow Working Group Validation of the Monocyte Assay for Chronic Myelomonocytic Leukemia Diagnosis By Flow Cytometry

▪IntroductionIt was proposed that peripheral blood (PB) monocyte subset analysis evaluated by flow cytometry, hereafter referred to as “monocyte assay”, could rapidly and efficiently distinguish chronic myelomonocytic leukemia (CMML) from other causes of monocytosis by highlighting an increase in the classical monocyte (cMo) fraction above 94%. However, the robustness of this assay required a large multicenter validation.MethodsPB and/or bone marrow (BM) samples from adult patients displaying monocytosis were assessed with the “monocyte assay” by ten ELN iMDS Flow working group centers (6 equipped with BD FACSCanto™ II (BD Biosciences), 3 with Navios™ (Beckman Coulter) and one with BD™ LSRII (BD Biosciences)) with harmonized protocols. The corresponding files were reanalyzed in a blind fashion by a skilled operator and the cMo (CD14 ++CD16 -) percentages obtained by both analyses were compared. Information regarding age, gender, complete blood count, marrow cytomorphology, cytogenetics and molecular analysis was collected. Confirmed diagnoses were collected when available as well as follow-up for CMML patients.ResultsThe comparison between cMo percentages from 267 PB files provided by the 10 centers and the centralized cMo percentages showed a good global significant correlation (r=0.88; p<0.0001; FigA) with no bias (FigB). Confirmed diagnoses were available for 212 files, namely 101 CMML according to the WHO criteria, 99 reactive monocytosis, and 12 MPN with monocytosis. A phenotype in favor of CMML, either classical with accumulation of cMo ≥94% or a bulbous aspect (FigC), was observed respectively in 81 and 14 patients. Hence, a total of 95 out of the 101 CMML patients translated into a sensitivity of 94% (FigD). Assessment of C reactive protein counts were available in seven of the 14 patients with the characteristic bulbous profile and correlated with an inflammatory state, showing a median of 93.0 [7.0-157.4] mg/L. Conversely, a phenotype not in favor of CMML (FigC) was observed in 83 of the 99 patients with reactive monocytosis and in 10 of 12 patients with MPN with monocytosis, leading to a 84% specificity (FigD). We established a Receiver Operator Curve (ROC) and again obtained a 94% cut-off value of cMo with an area under the ROC curve (AUC) of 0.865 (FigE).The second aim of this multicenter study was to assess the feasibility of the monocyte assay on 117 BM samples provided by 7 out of the 10 ELN centers, 43 of which being paired to PB samples. The comparison between cMo percentages provided by the 7 centers and the centralized cMo percentages showed a lower global significant correlation compared to PB samples (r=0.74; p<0.0001; FigF) with a slight underestimation of cMo percentage by the participating centers (FigG). The comparison between PB and BM samples cMo% obtained by centralized reanalysis showed an excellent global correlation (r=0.93; p<0.0001; FigH) with a higher percentage in the marrow (FigI). Seventy-nine files were associated to a confirmed diagnosis, as expected mostly CMML (n=69), only seven reactive monocytosis and three MPN with monocytosis. Thus, we determined a sensitivity of the “monocyte assay” on BM samples of 87% (a phenotype in favor of CMML being observed in 60 out of the 69 CMML with 6 bulbous aspect profiles) and a specificity of 80% (a phenotype not in favor of CMML being observed in 5 of the 7 patients with reactive monocytosis and in 3 of the 3 patients with MPN with monocytosis).ConclusionsThis ELN multicenter study demonstrates the robustness of the monocyte assay with only limited variability of cMo percentages, validates the 94% cutoff value, confirms its high sensitivity and specificity in PB and finally, also confirms the possibility of its use in BM samples. [Display omitted] DisclosuresKern: MLL Munich Leukemia Laboratory: Other: Part ownership.

Multicentric Validation of the “Monocyte Assay” for Chronic Myelomonocytic Leukemia Diagnosis By Flow Cytometry

Introduction:Accumulation of classical monocytes (MO1:CD14++CD16-) ≥94% of total peripheral blood monocytes analyzed by flow cytometry (FCM) was demonstrated to be a specific and sensitive tool that distinguishes chronic myelomonocytic leukemia (CMML) from reactive monocytosis (Selimoglu-Buet D et al, Blood 2015). The 94% threshold was further validated in an independent study (Talati C et al, Blood 2017) and is increasingly used as a diagnostic marker of CMML. Since this “monocyte assay” has been largely adopted by French diagnosis laboratories, we evaluated its performance in a multicentric evaluation.Methods: A short survey was sent by personalized emails between July and September 2016 to 48 French laboratories performing routinely FCM diagnosis of myeloid neoplasms. Afterwards, a few selected centers were asked to send their raw data files as well as the corresponding MO1 percentage evaluation in order to perform a centralized blind analysis using Kaluza® Software (Beckman Coulter®). Biological and clinical data were also provided when available.Results: We obtained a comprehensive reply to the survey with a 100% rate of response. Thirty of 48 centers (63%) use routinely the “monocyte assay”: apart from the three centers that participated to the initial study and keep using it, 27 centers spontaneously implemented it, most often following the initial publication and/or a meeting presentation. Eighteen out of 27 (67%) centers use at least the published exclusion panel for non-monocyte mature blood cells (i.e. CD2 and CD56 for T lymphocytes and NK cells, CD24 for B lymphocytes and granulocytes). Ten centers use at least one exclusion marker while two use no one. Nine centers perform 1 to 2 tests per month, fifteen 3 to 6, three 6 to 10 and three more than 10 tests. All the surveyed cytometrists consider the “monocyte assay” useful for CMML diagnosis. Among the 18 centers that do not use the “monocyte assay”, 8 intend to develop it soon, the 10 remaining centers not excluding a possible implementation.In a second step, five centers (named A to E) were selected according to their frequency of use and their antibody panel, excluding the three centers which were part of the initial study. They provided 329 FCM raw data files with the corresponding MO1 percentage. All these files were reanalyzed in a blind fashion. This centralized analysis compared to the MO1 percentage determined by the centers showed an excellent correlation (r=0.93; p<0.0001, Figure 1A) without any major bias (Center A: r = 0.93; B: r=0.97; C: r=0.89; D: r=0.93, E: r=0.93; p<0.0001). As the cut-off value of 94% MO1 is the most relevant parameter in the “monocyte assay”, we compared the patients with MO1 percentage ≥94% by centralized analysis (i.e. suspected of being diagnosed as CMML) and found an excellent concordance between both analyses (matching for 110 analyses/ 115 (95.7%), Figure 1B). Conversely, of the 214 patients with a fraction of MO1 <94% by the centralized analysis, 201 (93.5%) displayed MO1 percentage <94% in the original center. In most of the 18 divergent cases, the difficulty in drawing MO1 gate was related to an insufficient number of classical monocytes analyzed. Hence, we decided to exclude all the files showing MO1 event number below 10,000 (Figure 1C), thereby improving the correlation (r=0.94) and the concordance for the 245 remaining files (total matching files: 95%). Among these latter files, we collected clinical data for 84 patients for whom an overt diagnosis was made, namely 46 CMML, 24 reactive monocytosis and 14 non-CMML malignancies. MO1 accumulation ≥94% determined by centralized analysis was observed in 43 of the 46 CMML (Figure 1D), indicating a sensitivity of 93.5% in accordance with previous results. A specificity of 86.8% was calculated as 5 of the 38 “non-CMML” patients displayed MO1 <94% determined by centralized analysis. Interestingly, using these data we established a new Receiver Operator Curve (ROC) and obtained yet again a 94% cut-off value.Conclusion: This study demonstrates a strong adhesion of the French laboratories to the “monocyte assay” for CMML diagnosis set-up in 2015, with 30 user centers so far. We also demonstrate that this FCM test requires the acquisition of a minimum of 10,000 events. Finally, we observed a low variability of the MO1 percentage quantification between centers and validated the published 94% cut-off value with a 93.5% sensitivity and a 86.8% specificity. [Display omitted] DisclosuresFenaux:Celgene: Honoraria, Research Funding; Astex: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Astex: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Janssen: Honoraria, Research Funding. Wagner Ballon:Alexion: Consultancy, Honoraria.

Use of Sysmex Hematology Analyzer based Hematopoietic Progenitor Cell (XN-HPC) Count in Allogeneic Peripheral Blood Stem Cell Transplant Setting; Is It Substantially Reliable?

Allogeneic stem cell transplantation remains the only curative option for various benign and malignant hematological disorders. The procedure of allogeneic stem cell transplantation entails the administration of adequate doses of hematopoietic progenitor cells, aiming for complete and sustained hematopoietic reconstitution. Hematopoietic progenitors are obtained either through a bone marrow harvest or from GCSF-mobilized peripheral blood of the identified donor. Over the years various modalities have been utilized to establish the adequacy of harvested stem cell product e.g. Colony-forming unit– granulocyte monocyte (CFU-GM) assay, total mononuclear cell count (MNC), and CD34+ cell count

High Diagnostic Utility of Flow Cytometry Based Peripheral Blood Monocyte Subset Analysis, CD56 and CD2 Expression in Chronic Myelomonocytic Leukemia (CMML)

High Diagnostic Utility of Flow Cytometry Based Peripheral Blood Monocyte Subset Analysis, CD56 and CD2 Expression in Chronic Myelomonocytic Leukemia (CMML)

Detection of Dysferlin Gene Pathogenic Variants in the Indian Population in Patients Predicted to have a Dysferlinopathy Using a Blood-based Monocyte Assay and Clinical Algorithm: A Model for Accurate and Cost-effective Diagnosis.

Background:Limb-girdle muscular dystrophy (LGMD) is the most common adult-onset class of muscular dystrophies in India, but a majority of suspected LGMDs in India remain unclassified to the genetic subtype level. The next-generation sequencing (NGS)-based approaches have allowed molecular characterization and subtype diagnosis in a majority of these patients in India.Materials and Methods:(I) To select probable dysferlinopathy (LGMD2B) cases from other LGMD subtypes using two screening methods (i) to determine the status of dysferlin protein expression in blood (peripheral blood mononuclear cell) by monocyte assay (ii) using a predictive algorithm called automated LGMD diagnostic assistant (ALDA) to obtain possible LGMD subtypes based on clinical symptoms. (II) Identification of gene pathogenic variants by NGS for 34 genes associated with LGMD or LGMD like muscular dystrophies, in cases showing: absence of dysferlin protein by the monocyte assay and/or a typical dysferlinopathy phenotype, with medium to high predictive scores using the ALDA tool.Results:Out of the 125 patients screened by NGS, 96 were confirmed with two dysferlin variants, of which 84 were homozygous. Single dysferlin pathogenic variants were seen in 4 patients, whereas 25 showed no variants in the dysferlin gene.Conclusion:In this study, 98.2% of patients with absence of the dysferlin protein showed one or more variants in the dysferlin gene and hence has a high predictive significance in diagnosing dysferlinopathies. However, collection of blood samples from all over India for protein analysis is expensive. Our analysis shows that the use of the “ALDA tool” could be a cost-effective alternative method. Identification of dysferlin pathogenic variants by NGS is the ultimate method for diagnosing dysferlinopathies though follow-up with the monocyte assay can be useful to understand the phenotype in relation to the dysferlin protein expression and also be a useful biomarker for future clinical trials.

Establishing a Proteomics-Based Monocyte Assay To Assess Differential Innate Immune Activation Responses.

Innate immune cells are complex systems that can be simultaneously activated in a variety of ways. Common methods currently used to estimate the response of innate immune cells to stimuli are usually biased toward a single mode of activation. The aim of this study was to assess the possibility of designing an assay based on unbiased proteome analysis that would be capable of predicting the complex response of the innate immune system to various challenges. Monocytes were used as representative cells of the innate immune system. The underlying hypothesis was that their proteome response to different activating molecules would reflect the immunogenicity of these molecules. To identify the main modes of response, we treated the human monocytic THP-1 cell line with nine different stimuli. Differentiation and activation were determined to be the two major modes of monocyte response, with PMA causing the strongest differentiation and Pam3CSK4 causing the strongest proinflammatory activation. The established assay was applied to characterize the monocyte response to epidermal growth factor peptide containing isoaspartate, which induced differentiation but not proinflammatory activation. Because of its versatility, robustness, and specificity, this new assay is likely to find a niche among the more established immunological methods.

Food Polyphenols Fail to Cause a Biologically Relevant Reduction of COX-2 Activity.

Epidemiologic studies show a correlation between the dietary intake of food polyphenols and beneficial health effects. Several in vitro studies indicate that the anti-inflammatory potential of polyphenols is, at least in part, mediated by a modulation of the enzymes of the arachidonic acid cascade, such as the prostaglandin forming cyclooxygenases (COXs). Evidence that this mode of action can be transferred to the situation in vivo is scarce. This study characterized effects of a subset of polyphenols on COX–2 expression and activity in vitro and compared the potency with known drugs. Next, the in vivo relevance of the observed in vitro effects was tested. Enzyme assays and incubations of polyphenols with the cancer cell line HCA–7 and lipopolysaccharide (LPS) stimulated primary monocytes support the hypothesis that polyphenols can effect COX–2 expression and activity in vitro. The effects were most pronounced in the monocyte assay for wogonin, apigenin, resveratrol and genistein with IC50 values of 1.5 μM, 2.6 μM, 2.8 μM and 7.4 μM. However, these values are 100- to 1000-fold higher in comparison to those of the known pharmaceuticals celecoxib, indomethacin and dexamethasone. In an animal model of LPS induced sepsis, pretreatment with polyphenols (i. p. 100 mg/kg bw) did not result in decreased plasma or tissue prostaglandin levels, whereas the positive control celecoxib effectively attenuated LPS induced prostaglandin formation. These data suggest that despite the moderate potency in vitro, an effect of polyphenols on COX–2 during acute inflammation is unlikely, even if a high dose of polyphenols is ingested.

G.P.287: Genetic and epigenetic determinants of low dysferlin expression in monocytes

Dysferlinopathies are autosomal recessive inherited muscular dystrophies caused by mutations in the gene DYSF. Dysferlin is primarily expressed in skeletal muscle, cardiac muscle and peripheral blood monocytes. Dysferlin expression in skeletal muscle and monocytes strongly correlate in the healthy and disease states. We evaluated the efficiency of the monocyte assay to detect carriers and to determine the carrier frequency of dysferlinopathies in the general population. We enrolled 149 healthy volunteers and collected peripheral blood samples for protein analysis. While eighteen of these individuals with protein levels in the range of 40–64% were predicted to be carriers by the monocyte assay, subsequent DYSF sequencing analysis in 14 of these 18 volunteers detected missense variants in only four. Analysis of DNA methylation patterns at DYSF locus showed no changes in DNA methylation levels at CpG island and shores between samples. Our results suggest that: (1) dysferlin expression can also be regulated by factors outside of the dysferlin gene, but not related to DNA methylation; (2) carrier frequency and therefore the number of affected individuals could be higher than previously estimated; and (3) although reliable for evaluating dysferlinopathies, results of the monocyte assay can not be used when determining carrier status and molecular analysis of DYSF must be performed.

Genetic and epigenetic determinants of low dysferlin expression in monocytes.

Dysferlinopathies are autosomal recessive inherited muscular dystrophies caused by mutations in the gene DYSF. Dysferlin is primarily expressed in skeletal muscle, cardiac muscle, and peripheral blood monocytes. Expression in skeletal muscle and monocytes strongly correlates in healthy and disease states. We evaluated the efficiency of the monocyte assay to detect carriers and to determine the carrier frequency of dysferlinopathies in the general population. We enrolled 149 healthy volunteers and collected peripheral blood samples for protein analysis. While 18 of these individuals with protein levels in the range of 40%-64% were predicted to be carriers by the monocyte assay, subsequent DYSF sequencing analysis in 14 of 18 detected missense variants in only four. Analysis of DNA methylation patterns at the DYSF locus showed no changes in methylation levels at CpG islands and shores between samples. Our results suggest that: (1) dysferlin expression can also be regulated by factors outside of the dysferlin gene, but not related to DNA methylation; (2) carrier frequency and therefore the number of affected individuals could be higher than previously estimated; and (3) although reliable for evaluating dysferlinopathies, the monocyte assay cannot be used to determine the carrier status; for this, a molecular analysis of DYSF must be performed.

Identification of Tetrahydropyrido[4,3-d]pyrimidine Amides as a New Class of Orally Bioavailable TGR5 Agonists.

Takeda G-protein-coupled receptor 5 (TGR5) represents an exciting biological target for the potential treatment of diabetes and metabolic syndrome. A new class of high-throughput screening (HTS)-derived tetrahydropyrido[4,3-d]pyrimidine amide TGR5 agonists is disclosed. We describe our effort to identify an orally available agonist suitable for assessment of systemic TGR5 agonism. This effort resulted in identification of 16, which had acceptable potency and pharmacokinetic properties to allow for in vivo assessment in dog. A key aspect of this work was the calibration of human and dog in vitro assay systems that could be linked with data from a human ex vivo peripheral blood monocyte assay that expresses receptor at endogenous levels. Potency from the human in vitro assay was also found to correlate with data from an ex vivo human whole blood assay. This calibration exercise provided confidence that 16 could be used to drive plasma exposures sufficient to test the effects of systemic activation of TGR5.

Increased Cellular and Circulating Biomarkers of Oxidative Stress in Nascent Metabolic Syndrome

Metabolic syndrome (MetS) confers a greater risk for both cardiovascular disease (CVD) and diabetes. Oxidative stress (OS) could contribute to this excess risk. However, there are few data examining both cellular and circulating biomarkers of OS in MetS uncomplicated by diabetes and CVD. The aim of the study was to evaluate both cellular and circulating biomarkers of OS in MetS uncomplicated by diabetes or CVD. At an academic medical center, we compared MetS (n=43) vs. control subjects (n=33). Fasting blood was collected for monocyte isolation and assay of OS biomarkers. Monocyte nicotinamide adenine dinucleotide phosphate oxidase activity (p22 phox and p47), superoxide anion release, oxidized-low-density lipoprotein (Ox-LDL), nitrotyrosine, and nuclear factor erythroid 2-related factor were measured. There was significantly increased release of superoxide from the monocytes (basal and after activation) of MetS compared with controls adjusted for body mass index. Body mass index-adjusted plasma levels of Ox-LDL and nitrotyrosine were significantly increased in MetS. There was a linear trend between biomarkers of oxidative stress and increasing number of features of MetS. Also, there was a significant increase in nicotinamide adenine dinucleotide phosphate oxidase membrane expression of p22 phox and p47 phox in MetS. The major cellular antioxidant defense, nuclear factor erythroid 2-related factor was significantly decreased. There were significant correlations between homeostasis model assessment insulin resistance index and both Ox-LDL and nitrotyrosine and superoxide and Ox-LDL. Collectively, nascent MetS is associated with increased OS as evidenced by both circulating and cellular biomarkers, and this could contribute to the risk for both diabetes and CVD.

Enamel Matrix Derivative Exhibits Anti‐Inflammatory Properties in Monocytes

Enamel matrix derivative (EMD) is used clinically to promote periodontal tissue regeneration with variable efficacy. EMD application results in significantly higher frequencies of sites without clinical signs of inflammation; additionally, patients receiving EMD therapy report significantly less post-treatment discomfort. However, there are few reports that focus on defining the biologic mechanisms for the observed anti-inflammatory effects of EMD. The aim of this study was to evaluate the influence of EMD on inflammatory-associated markers using an in vitro monocyte assay. Rat monocytes were exposed to lipopolysaccharide (LPS; 100 ng/ml from Escherichia coli or Actinobacillus actinomycetemcomitans) along with EMD (0, 50, 100, or 200 microg/ml). Levels of tumor necrosis factor-alpha (TNF-alpha) and prostaglandin E(2) (PGE(2)) in conditioned media were analyzed by enzyme-linked immunosorbent assay. In addition, the effects of exogenous PGE(2) on TNF-alpha production from LPS-stimulated monocytes were determined. LPS-stimulated monocytes exposed to EMD exhibited a decrease in TNF-alpha production (0.10- to 0.52-fold) and an increase in PGE(2) production (1.31- to 2.71-fold) compared to controls not treated with EMD. Exogenously applied PGE(2) decreased TNF-alpha production by LPS-stimulated monocytes in a dose-dependent manner, and EMD treatment enhanced this PGE(2)-mediated inhibition of TNF-alpha production. In addition to EMD's published role in inducing proliferation, migration, adhesion, mineralization, and differentiation of periodontal ligament cells, our results indicated that EMD modulates two inflammation-associated factors, TNF-alpha and PGE(2), in monocytes.

Inhibitory Effect of Known Antioxidants and of Press Juice from Herring (Clupea harengus) Light Muscle on the Generation of Free Radicals in Human Monocytes

Reactive oxygen species (ROS) can cause oxidative stress, which has been linked to various diseases. It has been suggested that antioxidant-rich foods can reduce such oxidative stress. However, the lack of suitable model systems to screen for in vivo effects of food-derived antioxidants has prevented a clear consensus in this area. In this study, the aim was to use a single-cell model system (human monocyte) to evaluate whether certain pure antioxidants and complex muscle extracts (herring light muscle press juice, PJ) could prevent ROS formation under in vivo like conditions. ROS were excreted from the monocytes upon stimulation with phorbol myristate acetate and were then detected as isoluminol-enhanced chemiluminescence (CL). Adding 2000 units of catalase and 50 units of superoxide dismutase to the monocytes model lowered the CL response by 35 and 86%, respectively. Ascorbate (14.1 mM) lowered the response by 99%, alpha-tocoperhol (188 microM) by 37%, and Trolox (50 microM) by almost 100%. Crude herring PJ gave a dose-dependent reduction in the CL response. At 10, 100, and 1000 times dilution, the PJ reduced the CL signal by 93, 60.5, and 10.6%. PJ fractionated into low molecular weight (LMW) (<1000 Da) and high molecular weight (>3500 Da) fractions decreased the CL response by 52.9 and 71.4%, respectively, at a 100-fold dilution. Evaluation of the PJ samples in the oxygen radical absorbance capacity test indicated that proteins may be the primary radical scavenging compounds of PJ, whereas the ROS-preventing effect obtained from the LMW fraction may also be attributed to other mechanisms. Thus, this study proved that the monocyte assay can be a useful tool for studying whether food-derived antioxidants can limit ROS production under physiologically relevant conditions. It also showed that herring contains numerous aqueous compounds demonstrating antioxidative effects in the monocyte model system.

Functional assessment of bovine monocytes isolated from peripheral blood

Bovine monocytes were isolated from the peripheral blood of cattle by adherence of peripheral blood mononuclear cells to plastic. Three in vitro methods were modified to evaluate bovine monocyte function. These methods were: (1) ingestion of 125I-iododeoxyuridine labeled Staphylococcus aureus; (2) antibody-dependent cell-mediated cytotoxicity; (3) luminol-dependent and native chemiluminescence. Description of monocyte isolation and assay development is discussed in the text. Assays to evaluate monocyte function are useful for assessing immune status of the animal.

A Retrospective Study of Red Cell Maternal Antibodies by Chemiluminescence

AbstractPlasma samples from 109 patients with maternal IgG alloantibodies were investigated using a chemiluminescence (CL) assay, a functional test, to predict which antibodies were clinically significant. The CL assay was able to distinguish between those patients who were unaffected or mildly affected requiring only phototherapy, and those patients with moderate or severe haemolytic disease of the fetus or newborn (HDN) requiring transfusion therapy. The CL result was compared with the anti‐D quantification result, the number of IgG molecules bound per red cell and, in 80% of the cases, the monocyte monolayer assay. If mothers carrying Rh‐negative fetuses were ignored, then the CL assay correctly predicted the outcome for 93.4% of all cases (including those other than D), while the AutoAnalyzer and monocyte assay predicted correctly 92.7% (of the anti‐D patients) and 81.5%, respectively. Greater than 80% of patients with severe or moderate HDN had both IgG1+IgG3 subclasses in the maternal plasma, while those infants who were unaffected or only mildly affected had a greater chance of having IgG1 only (44%) in the maternal plasma, IgG3 only (27%) or both subclasses (29%).

A Retrospective Study of Red Cell Maternal Antibodies by Chemiluminescence

Plasma samples from 109 patients with maternal IgG alloantibodies were investigated using a chemiluminescence (CL) assay, a functional test, to predict which antibodies were clinically significant. The CL assay was able to distinguish between those patients who were unaffected or mildly affected requiring only phototherapy, and those patients with moderate or severe haemolytic disease of the fetus or newborn (HDN) requiring transfusion therapy. The CL result was compared with the anti-D quantification result, the number of IgG molecules bound per red cell and, in 80% of the cases, the monocyte monolayer assay. If mothers carrying Rh-negative fetuses were ignored, then the CL assay correctly predicted the outcome for 93.4% of all cases (including those other than D), while the AutoAnalyzer and monocyte assay predicted correctly 92.7% (of the anti-D patients) and 81.5%, respectively. Greater than 80% of patients with severe or moderate HDN had both IgGl+lgG3 subclasses in the maternal plasma, while those infants who were unaffected or only mildly affected had a greater chance of having IgGl only (44%) in the maternal plasma, IgG3 only (27%) or both subclasses (29%).

Azole phenoxy hydroxyureas as selective and orally active inhibitors of 5-lipoxygenase.

Azole phenoxy hydroxyureas are a new class of 5-lipoxygenase (5-LO) inhibitors. Structure-activity relationship studies have demonstrated that electronegative substituents on the 2-phenyl portion of the oxazole tail increased the ex vivo potency of these inhibitors. Similar substitutions on the thiazole analogs had only minor contribution to the ex vivo activity. The trifluoromethyl-substituted oxazole 24 was the best compound of the oxazole series in both the ex vivo (6 h pretreated rats) and in vivo (3 h pretreated rats) RPAR assay with ED50 values of approximately 1 and 3.6 mg/kg, respectively, but was weakly active in the allergic guinea pig assay. Oxazole 50 was equally active in both the RPAR and guinea pig in vivo models and was similar to zileuton. The unsubstituted thiazole 52 was the best compound of the thiazole series, by inhibiting the leukotriene B4 biosynthesis in the RPAR assay (3 h pretreated rats) by 99%, at an oral dose of 10 mg/kg, and the bronchoconstriction in the allergic guinea pig by 50%, at an intravenous dose of 10 mg/kg. Oxazole 24 demonstrated high and selective 5-LO inhibitory activity in the in vitro assays, with IC50 values ranging from 0.08 microM in mouse macrophages to 0.8 microM in human peripheral monocytes to 1.2 microM in human whole blood. This activity was selective for 5-LO, as concentrations up to 15 microM in mouse macrophages did not affect prostaglandin formation. Oxazole 59 was the most active inhibitor in the human monocyte assay with an IC50 value of 7 nM.