High expression of neutrophil and monocyte CD64 with simultaneous lack of upregulation of adhesion receptors CD11b, CD162, CD15, CD65 on neutrophils in severe COVID-19.

BACKGROUND AND AIMS: The pronounced neutrophilia observed in patients with coronavirus disease 2019 (COVID-19) infections suggests a role for these leukocytes in the pathology of the disease. Monocyte and neutrophil expression of CD64 and CD11b have been reported as early biomarkers to detect infections. The aim of this study was to study the expression of receptors for IgG (CD64) and adhesion molecules (CD11b, CD15s, CD65, CD162, CD66b) on neutrophils and monocytes in patients with severe COVID-19 after admission to an intensive care unit (ICU). METHODS: The expression of receptors was analyzed using flow cytometry. EDTA blood from 23 patients with confirmed COVID-19 infection was sampled within 48 h of admission to the ICU. Leukocytes were labeled with antibodies to CD11b, CD15s, CD65s, CD162, CD64, and CD66b. Expression of receptors was reported as mean fluorescence intensity (MFI) or the percentage of cells expressing receptors. RESULTS: Results are presented as comparison of COVID-19 patients with the healthy group and the receptor expression as MFI. Neutrophil receptors CD64 (2.5 versus 0.5) and CD66b (44.5 versus 34) were increased and CD15 decreased (21.6 versus 28.3) when CD65 (6.6 versus 4.4), CD162 (21.3 versus 21.1) and CD11b (10.5 versus 12) were in the same range. Monocytes receptors CD64 (30.5 versus 16.6), CD11b (18.7 versus 9.8), and CD162 (38.6 versus 36.5) were increased and CD15 decreased (10.3 versus 17.9); CD65 were in the same range (2.3 versus 1.96). CONCLUSION: Monocytes and neutrophils are activated during severe COVID-19 infection as shown by strong upregulation of CD64. High monocyte and neutrophil CD64 can be an indicator of a severe form of COVID19. The adhesion molecules (CD11b, CD162, CD65, and CD15) are not upregulated on otherwise activated neutrophils, which might lead to relative impairment of tissue migration. Low adhesion profile of neutrophils suggests immune dysfunction of neutrophils. Monocytes maintain upregulation of some adhesion molecules (CD11b, CD162) suggesting the persistence of an increased ability to migrate into tissues, even during a severe stage of COVID-19. Future research should focus on CD64 and CD11b kinetics in the context of prognosis.

Data set on sedimentology, palaeoecology and chronology of middle to late pleistocene deposits on the Taimyr Peninsula, Arctic Russia.

This Data in Brief paper contains data (including images) from Quaternary sedimentary successions investigated along the Bol'shaya Balakhnya River and the Luktakh-Upper Taimyra-Logata river system on southern Taimyr Peninsula, NW Siberia (Russia). Marine foraminifera and mollusc fauna composition, extracted from sediment samples, is presented. The chronology (time of deposition) of the sediment successions is reconstructed from three dating methods; (i) radiocarbon dating of organic detritus (from lacustrine/fluvial sediment) and molluscs (marine sediment) as finite ages (usually <42 000 years) or as non-finite ages (>42 000-48 000 years) on samples/sediments beyond the radiocarbon dating limit; (ii) Electron Spin Resonance (ESR) dating on marine molluscs (up to ages >400 000 years); (iii) Optically Stimulated Luminescence (OSL) dating, usually effective up to 100-150 0000 years. Terrestrial Cosmogenic Nuclide (TCN) exposure dating has been applied to boulders resting on top of moraine ridges (Ice Marginal Zones). See (Möller et al., 2019) (doi.org/10.1016/j.earscirev.2019.04.004) for interpretation and discussion of all data.

HNL (Human Neutrophil Lipocalin) and a multimarker approach to the distinction between bacterial and viral infections.

OBJECTIVES: The distinction between bacterial and viral causes of acute infections is a major clinical challenge. In this report we investigate the diagnostic performance in this regard of nine candidate biomarkers together with HNL (Human Neutrophil Lipocalin). METHODS: Blood was obtained from patients with symptoms of infectious (n = 581). HNL was measured in whole blood (B-HNL) after pre-activation with the neutrophil activator fMLP or in plasma (P-HNL). Azurocidin also known as heparin-binding protein (HBP), Calprotectin, PMN-CD64, CRP (C-reactive protein), IP-10 (Interferon γ-induced Protein 10 kDa), PCT (Procalcitonin), TK1 (Thymidine kinase 1), TRAIL (TNF-related apoptosis-inducing ligand) were measured in plasma/serum. Area under the ROC (receiver operating characteristics) curve (AuROC) was used for the evaluation of the clinical performance of the biomarkers. RESULTS: Side-by-side comparisons of the ten biomarkers showed large difference in the AuROC with B-HNL being the superior biomarker (0.91, 95% CI 0.86-0.95) and with the other nine biomarkers varying from AuROC of 0.63-0.79. The combination of B-HNL with IP-10 and/or TRAIL increased the diagnostic performance further to AuROCs of 0.94-0.97. The AuROCs of the combination of CRP with IP-10 and/or TRAIL were significantly lower than combinations with B-HNL 0.87 (95% CI 0.83-0.91). CONCLUSION: The diagnostic performance of whole blood activated HNL was superior in the distinction between bacterial or viral infections. The addition of IP-10 and/or TRAIL to the diagnostic algorithm increased the performance of B-HNL further. The rapid analysis of HNL, reflecting bacterial infections, together with biomarkers reflecting viral infections may be the ideal combination of diagnostic biomarkers of acute infections.

Postglacial relative sea level change and glacier activity in the early and late Holocene: Wahlenbergfjorden, Nordaustlandet, Svalbard.

Sediment cores from Kløverbladvatna, a threshold lake in Wahlenbergfjorden, Nordaustlandet, Svalbard were used to reconstruct Holocene glacier fluctuations. Meltwater from Etonbreen spills over a threshold to the lake, only when the glacier is significantly larger than at present. Lithological logging, loss-on-ignition, ITRAX scanning and radiocarbon dating of the cores show that Kløverbladvatna became isolated from Wahlenbergfjorden c. 5.4 cal. kyr BP due to glacioisostatic rebound. During the Late Holocene, laminated clayey gyttja from lacustrine organic production and surface runoff from the catchment accumulated in the lake. The lacustrine sedimentary record suggests that meltwater only spilled over the threshold at the peak of the surge of Etonbreen in AD 1938. Hence, we suggest that this was the largest extent of Etonbreen in the (mid-late) Holocene. In Palanderbukta, a tributary fjord to Wahlenbergfjorden, raised beaches were surveyed and organic material collected to determine the age of the beaches and reconstruct postglacial relative sea level change. The age of the postglacial raised beaches ranges from 10.7 cal. kyr BP at 50 m a.s.l. to 3.13 cal. kyr BP at 2 m a.s.l. The reconstructed postglacial relative sea level curve adds valuable spatial and chronological data to the relative sea level record of Nordaustlandet.

The genetically determined production of the alarmin eosinophil-derived neurotoxin is reduced in visceral leishmaniasis.

Visceral leishmaniasis (VL) is the most severe form of leishmaniasis. Recent findings indicate that dendritic cells have a key role in the defense against the Leishmania parasite and that the activity of this cell may be modified by the eosinophil secretory protein eosinophil-derived neurotoxin (EDN). We hypothesized that the interactions between dendritic cells and EDN might be of importance in the disease development. Cellular content of EDN was analyzed by ELISA. The single-nucleotide polymorphisms at positions 405, 416, and 1122 in the EDN gene were analyzed by real-time PCR with TaqMan® reagents. The study cohorts comprised 239 Sudanese subjects (65 healthy controls and 174 with VL) and 300 healthy Swedish controls. The eosinophil content of EDN was lower in VL as compared with controls (p < 0.0001). The EDN405 (G>C) genotype distribution was similar among Swedish and Sudanese controls, whereas VL subjects had a higher prevalence of the EDN405-GG genotype (p < 0.0001). The content of EDN in the eosinophils was closely linked to the EDN405 polymorphism (p = 0.0002). Our findings suggest that the predisposition to acquire VL is related to the genetic polymorphism of the EDN gene and the reduced production by the eosinophil of this gene product.

Human Neutrophil Lipocalin in Activated Whole Blood Is a Specific and Rapid Diagnostic Biomarker of Bacterial Infections in the Respiratory Tract.

The distinction between bacterial and viral causes of infections of the respiratory tract is a major but important clinical challenge. We investigated the diagnostic performance of human neutrophil lipocalin (HNL) in respiratory tract infections compared to those of C-reactive protein (CRP) and procalcitonin (PCT). Patients were recruited from the emergency department and from a primary care unit (n = 162). The clinical diagnosis with regard to bacterial or viral cause of infection was complemented with objective microbiological/serological testing. HNL was measured in whole blood after preactivation with the neutrophil activator formyl-methionine-leucine-phenylalanine (fMLP) (B-HNL), and CRP and PCT were measured in plasma. Head-to-head comparisons of the three biomarkers showed that B-HNL was a superior diagnostic means to distinguish between causes of infections, with areas under the concentration-time curve (AUCs) of receiver operating characteristic (ROC) analysis for HNL of 0.91 (95% confidence interval [CI], 0.83 to 0.96) and 0.92 (95% CI, 0.82 to 0.97) for all respiratory infections and for upper respiratory infections, respectively, compared to 0.72 (95% CI, 0.63 to 0.80) and 0.68 (95% CI, 0.56 to 0.79) for CRP, respectively (P = 0.001). In relation to major clinical symptoms of respiratory tract infections (cough, sore throat, stuffy nose, and signs of sinusitis), AUCs varied between 0.88 and 0.93 in those patients with likely etiology (i.e., etiology is likely determined) of infection, compared to 0.63 and 0.71 for CRP, respectively, and nonsignificant AUCs for PCT. The diagnostic performance of B-HNL is superior to that of plasma CRP (P-CRP) and plasma PCT (P-PCT) in respiratory tract infections, and the activity specifically reflects bacterial challenge in the body. The rapid and accurate analysis of HNL by point-of-care technologies should be a major advancement in the diagnosis and management of respiratory infections with respect to antibiotic treatment.

Differential neutrophil chemotactic response towards IL-8 and bacterial N-formyl peptides in term newborn infants.

BACKGROUND: A prerequisite for an effective innate immunity is the migrative ability of neutrophils to respond to inflammatory and infectious agents such as the intermediate interleukin (IL)-8 and the end-target formyl-methionyl-leucyl-phenylalanine (fMLP) chemoattractants. The aim was to study the chemotactic capacity of neutrophils from newborn infants and adults in response to IL-8 and the bacterial peptide fMLP. METHODS: In the under-agarose cell migration assay, isolated leukocytes from healthy adults and from cord blood of healthy term newborn infants were studied with dose responses towards IL-8 and fMLP. The same number of leukocytes (1 × 105 cells), with the same distribution of neutrophils and monocytes, were analyzed in neonates and adults. Chemotaxis was distinguished from randomly migrating neutrophils, and the neutrophil pattern of migration, i.e. the migration distance and the number of migrating neutrophils per distance, was evaluated. RESULTS: In comparison to adults, fewer neutrophils from newborn infants migrated towards IL-8 and for a shorter distance (P < .01, respectively). The number of neutrophils migrating to different gradients of fMLP, the distance they migrated, and the correlation between the number and the distance were the same for neonates and adults. Random migration did not differ in any instance. CONCLUSION: Chemotaxis of neutrophils from newborn infants was as co-ordinated as neutrophils from adults in response to fMLP, whereas the response to IL-8 was reduced. The differential response of neutrophils from neonates to intermediate and end-target chemoattractants could indicate a reduced infectious response.

Human Neutrophil Lipocalin as a Superior Diagnostic Means To Distinguish between Acute Bacterial and Viral Infections.

The distinction between causes of acute infections is a major clinical challenge. Current biomarkers, however, are not sufficiently accurate. Human neutrophil lipocalin (HNL) concentrations in serum or whole blood activated by formyl-methionine-leucine-phenylalanine (fMLP) were shown to distinguish acute infections of bacterial or viral cause with high accuracy. The aim was therefore to compare the clinical performance of HNL with currently used biomarkers. Seven hundred twenty-five subjects (144 healthy controls and 581 patients with signs and symptoms of acute infections) were included in the study. C-reactive protein (CRP), the expression of CD64 on neutrophils, procalcitonin (PCT), and blood neutrophil counts were measured by established techniques, and HNL concentrations were measured in whole-blood samples after activation with fMLP. All tested biomarkers were elevated in bacterial as opposed to viral infections (P < 0.001). CRP, PCT, and CD64 expression in neutrophils was elevated in viral infections compared to healthy controls (P < 0.001). In the distinction between healthy controls and patients with bacterial infections, the areas under the receiver operating characteristic (ROC) curves were >0.85 for all biomarkers, whereas for the distinction between bacterial and viral infections, only HNL concentration in fMLP-activated whole blood showed an area under the ROC curve (AUROC) of >0.90 and superior clinical performance. The clinical performance of HNL in fMLP-activated whole blood was superior to current biomarkers and similar to previous results of HNL in serum. The procedure can be adopted for point-of-care testing with response times of <15 min.

Human neutrophil lipocalin in fMLP-activated whole blood as a diagnostic means to distinguish between acute bacterial and viral infections.

UNLABELLED: The distinction between causes of acute infections is a major clinical challenge. Current biomarkers, however, are not sufficiently accurate. Human neutrophil lipocalin (HNL) in serum distinguishes acute infections with high accuracy, but in the emergency setting the assay time should be <15-20min, which excludes the use of serum samples. The aim was therefore to develop a novel rapid assay principle and test its clinical performance. METHODS: Serum and neutrophils obtained from 84 infected and 20 healthy subjects were used in the experimental study. 725 subjects (144 healthy controls and 581 patients with signs and symptoms of acute infections) were included in the clinical study. HNL was measured in EDTA-plasma by ELISA or in heparinized whole blood after fMLP activation by a prototype point-of-care assay. RESULTS: Increased release of HNL from neutrophils after activation with fMLP was seen already after 5 min incubation. The release of HNL from purified neutrophils after 15 min incubation with fMLP was significantly correlated to the HNL concentrations in serum obtained from the same patient (r = 0.74, p < 0.001). In the distinction between healthy controls and patients with bacterial infections, the areas under the ROC-curves were 0.95 (95% CI 0.91-0.97) and 0.88 (95% CI 0.84-0.91) for HNL in fMLP-activated whole blood and EDTA-plasma, respectively, (p < 0.001) and in the distinction between bacterial and viral infections 0.91 (95% CI 0.86-0.95) and 0.76 (95% CI 0.70-0.81), respectively (p < 0.001). CONCLUSION: The clinical performance of HNL in fMLP-activated whole blood was superior to HNL in EDTA-plasma and similar to HNL in serum. The procedure can be adopted for point-of-care testing with response times of <15 min.

Neutrophil CD64 expression - comparison of two different flow cytometry protocols on EPICs MCL and the Leuko64(™) assay on a Celldyn Sapphire haematology analyser.

OBJECTIVE: To evaluate the Trillium Diagnostics Leuko64(™) assay on Abbott Celldyn Sapphire haematology analyser compared to two flow cytometry protocols on Beckman Coulter EPICS MCL flow cytometer. MATERIALS AND METHODS: CD64 expression on neutrophils was determined by two flow cytometry protocols and by a commercial assay on an automatic haematology analyser. The inclusion of study subjects was based on elevated procalcitonin (PCT) values, identifying patients where a systemic infection was suspected. Healthy blood donors were used as a reference group. RESULTS: Statistically significant correlations between the Trillium Diagnostics Leuko64(™) assay and the flow cytometry methods were found when measuring neutrophil CD64 expression. CONCLUSIONS: The good correlation between a reference method and an automated haematology analyser method for CD64 expression on neutrophils supports introduction of the latter assay for routine use as an independent biomarker of bacterial infection and inflammation.

Anti-type II collagen immune complex-induced granulocyte reactivity is associated with joint erosions in RA patients with anti-collagen antibodies.

INTRODUCTION: Rheumatoid arthritis (RA) patients with autoantibodies against collagen type II (CII) are characterized by acute RA onset with elevated inflammatory measures and early joint erosions as well as increased production of tumor necrosis factor-α (ΤΝF-α) by peripheral blood mononuclear cells (PBMC) stimulated by anti-CII immune complexes (IC) in vitro. Polymorphonuclear granulocytes (PMN) are abundant in RA synovial fluids, where they might interact directly with anti-CII IC in the articular cartilage, but no studies have investigated PMN responses towards anti-CII IC. The aim was to investigate whether PMN react towards anti-CII IC, and to what extent such reactivity might relate to the clinical acute onset RA phenotype associated with elevated levels of anti-CII. METHODS: PMN and PBMC isolated from healthy donors were stimulated with IC made with a set of 72 baseline patient sera (24 anti-CII positive, 48 anti-CII negative) chosen from a clinically well-characterized RA cohort with two-year radiological follow-up with Larsen scoring. PMN expression of cluster of differentiation (CD)11b, CD66b, CD16 and CD32 was measured by flow cytometry, whereas PMN production of myeloperoxidase (MPO) and interleukin (IL)-17, and PBMC production of ΤΝF-α was measured with enzyme linked immunosorbent assay. RESULTS: PMN expression of CD11b, CD66b and MPO, and PBMC production of ΤΝF-α were upregulated whereas PMN expression of CD16 and CD32 were downregulated by anti-CII IC. CD16, CD66b, and MPO production correlated to serum anti-CII levels (Spearman's ρ = 0.315, 0.675 and 0.253, respectively). CD16 was associated with early joint erosions (P = 0.024, 0.034, 0.046 at baseline, one and two years) and CD66b was associated with changes in joint erosions (P = 0.017 and 0.016, at one and two years compared to baseline, respectively). CD66b was associated with baseline C-reactive protein and PBMC production of ΤΝF-α was associated with baseline erythrocyte sedimentation rate, in accordance with our earlier findings. No clinical associations were observed for MPO or IL-17. CONCLUSION: PMN responses against anti-CII IC are more closely associated with early joint erosions than are PBMC cytokine responses. PMN reactivity against anti-CII IC may contribute to joint destruction in newly diagnosed RA patients with high levels of anti-CII.

The production of the eosinophil proteins ECP and EPX/EDN are regulated in a reciprocal manner.

Previous studies showed that the biological activity and the eosinophil content of eosinophil cationic protein (ECP, RNase 3) are determined by single-nucleotide polymorphisms (SNPs) in the ECP (RNase3) gene. In this study, we report the prevalence of a common SNP in the eosinophil protein x/eosinophil-derived neurotoxin (EPX/EDN, RNase2) and the association with the cellular contents of EPX/EDN and ECP. The genes were sequenced and the EPX/EDN405(G>C) rs2013109 SNPs were also determined by TaqMan 5'nuclease allelic discrimination assay. ECP and EPX/EDN in purified eosinophils or in whole blood extracts were analysed by sensitive immunoassays. The study included 379 non-allergic and allergic subjects. The genotype prevalence of the EPX/EDN405(G>C) polymorphism was GG 59%, GC 36% and CC 6%. The cellular contents of ECP and EPX/EDN were related in a reciprocal fashion with the sums of the protein contents being constant. The contents were associated with the ECP562(G>C) rs2233860 and EPX/EDN405(G>C) gene polymorphisms. The cellular content of eosinophil peroxidase (EPO) was not associated with the ECP and EPX/EDN genotypes. The prevalence of the EPX/EDN405(G>C) genotypes and the contents of the proteins were similar in non-allergic and allergic subjects.The production and storage of the two ancestral proteins, ECP and EPX/EDN likely share common regulatory mechanisms, which result in opposing productions of the two proteins.

Effect of simulated extracorporeal circulation and glyceryl-tri-nitrate on leukocyte activation.

OBJECTIVES: During extracorporeal circulation (ECC), a mechanical pump and an oxygenator replace the functions of the heart and lungs. The aim of this study is to test the effect of the nitric oxide donor glyceryl-tri-nitrate on activation markers of the innate immune system during simulated ECC. DESIGN: Whole blood concentrations of selected leukocyte adhesion molecules, complement system components and myeloperoxidase (MPO) were measured in an in vitro system of simulated ECC. RESULTS: Simulated ECC stimulated the expression of monocyte LPS-receptor CD14 and C3b-receptor CD35. Glyceryl-tri-nitrate significantly reduced the expression of leukocyte Fcγ receptor CD32 over time, compared to control. Simulated ECC increased the concentrations of MPO, terminal complement complex, and complement component C3a. Addition of glyceryl-tri-nitrate did not significantly affect these changes. CONCLUSIONS: Simulated ECC induces the increased expression of some leukocyte markers. Glyceryl-tri-nitrate addition significantly reduces the expression of some leukocyte activation markers.

Effect of glyceryl trinitrate on staphylococcus aureus growth and leukocyte activation during simulated extracorporeal circulation.

BACKGROUND: Previously, nitric oxide has been shown to possess antimicrobial effects. In this study, we aim to test the effect of glyceryl trinitrate (GTN) on Staphylococcus aureus growth during simulated extracorporeal circulation (SECC) and also to examine the effect of S. aureus, alone and in combination with GTN, on activation markers of the innate immune system during SECC. METHODS: In an in vitro system of SECC, we measured GTN-induced changes in markers of leukocyte activation in whole blood caused by S. aureus infestation, as well as the effect of GTN on S. aureus growth. RESULTS: GTN had no effect on S. aureus growth after 240 minutes SECC. Staphylococcus aureus reduced the expression of granulocyte Fcγ-receptor CD32 but stimulated the expression of monocyte CD32. Staphylococcus aureus stimulated expression of some leukocyte adhesion key proteins, activation marker CD66b, lipopolysaccharide-receptor CD14, and C3b-receptor CD35. Staphylococcus aureus and GTN addition induced significant increases in monocyte CD63 (lysosomal granule protein) levels. CONCLUSION: GTN does not affect S. aureus growth during SECC and has no effect on SECC-induced leukocyte activation.

Eosinophil associated genes in the inflammatory bowel disease 4 region: correlation to inflammatory bowel disease revealed.

AIM: To study the association between inflammatory bowel disease (IBD) and genetic variations in eosinophil protein X (EPX) and eosinophil cationic protein (ECP). METHODS: DNA was extracted from ethylene diamine tetraacetic acid blood of 587 patients with Crohn's disease (CD), 592 with ulcerative colitis (UC) and 300 healthy subjects. The EPX405 (G > C, rs2013109), ECP434 (G > C, rs2073342) and ECP562 (G > C, rs2233860) gene polymorphisms were analysed, by the 5'-nuclease allelic discrimination assay. For determination of intracellular content of EPX and ECP in granulocytes, 39 blood samples was collected and extracted with a buffer containing cetyltrimethylammonium bromide. The intracellular content of EPX was analysed using an enzyme-linked immunosorbent assay. The intracellular content of ECP was analysed with the UniCAP(®) system as described by the manufacturer. Statistical tests for calculations of results were χ(2) test, Fisher's exact test, ANOVA, Student-Newman-Keuls test, and Kaplan-Meier survival curve with Log-rank test for trend, the probability values of P < 0.05 were considered statistically significant. RESULTS: The genotype frequency for males with UC and with an age of disease onset of ≥ 45 years (n = 57) was for ECP434 and ECP562, GG = 37%, GC = 60%, CC = 4% and GG = 51%, GC = 49%, CC = 0% respectively. This was significantly different from the healthy subject's genotype frequencies of ECP434 (GG = 57%, GC = 38%, CC = 5%; P = 0.010) and ECP562 (GG = 68%, GC = 29%,CC = 3%; P = 0.009). The genotype frequencies for females, with an age of disease onset of ≥ 45 years with CD (n = 62), was for the ECP434 and ECP562 genotypes GG = 37%, GC = 52%, CC = 11% and GG = 48%, GC = 47% and CC = 5% respectively. This was also statistically different from healthy controls for both ECP434 (P = 0.010) and ECP562 (P = 0.013). The intracellular protein concentration of EPX and ECP was calculated in µg/10(6) eosinophils and then correlated to the EPX 405 genotypes. The protein content of EPX was highest in the patients with the CC genotype of EPX405 (GG = 4.65, GC = 5.93, and CC = 6.57) and for ECP in the patients with the GG genotype of EPX405 (GG = 2.70, GC = 2.47 and CC = 1.90). ANOVA test demonstrated a difference in intracellular protein content for EPX (P = 0.009) and ECP (P = 0.022). The age of disease onset was linked to haplotypes of the EPX405, ECP434 and ECP562 genotypes. Kaplan Maier curve showed a difference between haplotype distributions for the females with CD (P = 0.003). The highest age of disease onset was seen in females with the EPX405CC, ECP434GC, ECP562CC haplotype (34 years) and the lowest in females with the EPX405GC, ECP434GC, ECP562GG haplotype (21 years). For males with UC there was also a difference between the highest and lowest age of the disease onset (EPX405CC, ECP434CC, ECP562CC, mean 24 years vs EPX405GC, ECP434GC, ECP562GG, mean 34 years, P = 0.0009). The relative risk for UC patients with ECP434 or ECP562-GC/CC genotypes to develop dysplasia/cancer was 2.5 (95%CI: 1.2-5.4, P = 0.01) and 2.5 (95%CI: 1.1-5.4, P = 0.02) respectively, compared to patients carrying the GG-genotypes. CONCLUSION: Polymorphisms of EPX and ECP are associated to IBD in an age and gender dependent manner, suggesting an essential role of eosinophils in the pathophysiology of IBD.

Activity and turnover of eosinophil and neutrophil granulocytes are altered in visceral leishmaniasis.

Visceral leishmaniasis (VL) is a health issue in Sudan. Our aim was to investigate the involvement of eosinophils and neutrophils in VL by serum and plasma measurements of eosinophil cationic protein (ECP) and myeloperoxidase (MPO) and some key cytokines and chemokines. Blood was collected from 125 VL patients and 181 healthy Sudanese controls from the same rural area. Results showed reduced eosinophil and neutrophil counts in the VL group (P=0.0001 and P=0.002, respectively). Serum-ECP levels were higher in the controls (P<0.0001), while plasma MPO levels were higher in the VL group (P<0.0001). Levels of IL-5, granulocyte macrophage-colony stimulating factor (GM-CSF) and IL-17 were increased among the VL group (P<0.0001, P=0.017 and P=0.03, respectively), whereas eotaxin and IL-8 levels were reduced (P<0.0001 and P=0.002, respectively). Positive correlations were found between IL-8 and ECP/MPO (P<0.0001). We conclude that eosinophil and neutrophil turnover and activity are increased in subjects in rural areas of Sudan. In VL the turnover was further increased, but the relatively low secretory activity of eosinophils and neutrophils in VL may relate to the reduced production and availability of the chemokines eotaxin and IL-8. The combined assay of ECP and MPO in serum and plasma provides further insight into the mechanisms of eosinophil and neutrophil involvement in disease and constitutes a novel approach to the study of disease processes.

Associations of ECP (eosinophil cationic protein)-gene polymorphisms to allergy, asthma, smoke habits and lung function in two Estonian and Swedish sub cohorts of the ECRHS II study.

BACKGROUND: The Eosinophil Cationic Protein (ECP) is a potent multifunctional protein. Three common polymorphisms are present in the ECP gene, which determine the function and production of the protein. The aim was to study the relationship of these ECP gene polymorphisms to signs and symptoms of allergy and asthma in a community based cohort (The European Community Respiratory Health Survey (ECRHS)). METHODS: Swedish and Estonian subjects (n = 757) were selected from the larger cohort of the ECRHS II study cohort. The prevalence of the gene polymorphisms ECP434(G>C) (rs2073342), ECP562(G>C) (rs2233860) and ECP c.-38(A>C) (rs2233859) were analysed by DNA sequencing and/or real-time PCR and related to questionnaire-based information of allergy, asthma, smoking habits and to lung functions. RESULTS: Genotype prevalence showed both ethnic and gender differences. Close associations were found between the ECP434(G>C) and ECP562(G>C) genotypes and smoking habits, lung function and expression of allergic symptoms. Non-allergic asthma was associated with an increased prevalence of the ECP434GG genotype. The ECP c.-38(A>C) genotypes were independently associated to the subject being atopic. CONCLUSION: Our results show associations of symptoms of allergy and asthma to ECP-genotypes, but also to smoking habits. ECP may be involved in impairment of lung functions in disease. Gender, ethnicity and smoking habits are major confounders in the evaluations of genetic associations to allergy and asthma.

The FMCA-GM assays, high throughput non-clonogenic alternatives to CFU-GM in preclinical hematotoxicity testing.

One of the most common dose limiting adverse effects in cancer treatment is myelotoxicity. The aim of this study was to develop an in vitro method for measuring potential myelotoxic properties of a drug candidate in a high throughput setting. Human CD34(+) progenitor cells from umbilical cord blood were plated in 384-well microplates with drugs in liquid culture, supplemented with specific cytokines for the granulocytopoietic-macrophage lineage. After 7 or 14 days of proliferation and differentiation the cells were analyzed using the automated non-clonogenic fluorometric microculture cytotoxicity assay (FMCA). Two types of assays setups were evaluated, the FMCA-GM7 where cells were exposed to drugs directly after thawing and cytotoxicity measured on day 7 in contrast to the FMCA-GM14 where the cells were cultured 7 days prior to plating and drug exposure, with viability analysis on day 14 of differentiation. Drug sensitivity was similar in both assays and method validation was performed using 24 drugs with known myelotoxic profile (acyclovir, bortezomib, busulfan, carboplatin, chloramphenicol, chlorpromazine, cisplatin, cytarabine, clozapine, doxorubicin, erlotinib, etoposide, 5-fluorouracil, fludarabine, gefitinib, gemcitabine, hydroxyurea, imatinib, lomustine, melphalan, sorafenib, sunitinib, taxol and 6-thioguanine). The 50% inhibitory concentrations (IC(50)) from the FMCA-GM7 and the FMCA-GM14 correlated highly (r = 0.83) and (r = 0.82), respectively, with IC(50) from the established clonogenic assay (CFU-GM), obtained from the literature. The current data suggests that the FMCA-GM could offer a simple and robust alternative to the CFU-GM assay in preclinical hematotoxicity studies.

Tissue localization and the establishment of a sensitive immunoassay of the newly discovered human phospholipase B-precursor (PLB-P).

Human phospholipase B-precursor (PLB-P) is a newly identified and purified protein from human neutrophils. The precise function of PLB-P in vivo is not yet known. Its existence in neutrophils and the enzymatic activity against phospholipids imply a role in the defence against invading microorganisms and in the generation of lipid mediators of inflammation. We describe here the generation of specific antibodies against PLB-P, the tissue localizations of PLB-P and the establishment of an accurate, specific, and reproducible radioimmunoassay (RIA). A survey of normal and malignant tissues showed strong immunostaining of PLB-P in neuronal and myeloid cells and in adrenal glands. Elevated levels were found in sera of patients with influenza A infection i.e. >1 microg/L and in gut fluids of patients with inflammatory bowel disease i.e. >20 microg/L. The levels correlated to markers of neutrophil activation, suggesting a neutrophil origin of PLB-P in these conditions. The antibodies and the assay will be useful in the future basic and clinical investigations of PLB-P.