Percentage Of NBT-positive Cells Research Articles

Immune state correlates with histopathological level and reveals molluscan health in populations of Modiolus kurilensis by integral health index (IHI).

Quantitative analysis of the histopathological and immune parameters of bivalve Modiolus kurilensis collected from water areas with different level of ecotoxicological stress was performed. Significant differences between samples from polluted and non-polluted sites were revealed for total haemocyte count; percentage of agranulocytes; size and internal complexity of agranulocytes and granulocytes; phagocytic activity; percentage of NBT-positive cells; hemolytic activity and plasma protein concentration; percentage of the optical density of haemolymph major polypeptide bands at 55 kDa, 78 kDa, and 124 kDa; concretion coverage area in the kidney tubules; thickness of the tubular basement membrane; nephrocyte shape; and karyopyknosis of the kidneys; and hypervacuolisation; necrosis; karyopyknosis; haemocyte infiltration; fibrosis; and invasion of the digestive gland. Analysis of the global histopathological condition index based on the weighted indices also revealed that both the digestive gland and kidneys showed significantly greater histopathological changes in the bivalves collected from polluted water. Bivalve histopathology is an established tool in aquatic toxicology. However, it reflects a morphological picture of change, which, as a rule, can be clearly recorded only at the later stages of pathology, and in some cases, indicates an adaptation to stressors within the physiological norm. In this respect, a promising and highly sensitive biomarker of the functional state of bivalves, in terms of norm and pathology as well as their habitat, is the evaluation of immune status in combination with morphological changes. However, the use of different methods and scales of assessment and the diagnosis of biomarkers, characterised by different profiles of the stress response, makes it difficult to compare the results of different studies. We propose a reliable and powerful system for assessing the physiological state of bivalve molluscs, expressed in the integral health index (IHI) and based on the standardisation of the numerical values for all parameters that have significant differences between animals collected from impacted and non-impacted water areas. In our study, IHI calculated in three variants (for histopathological parameters, for immunological parameters, and in combination) showed the most significant differences in each of the cases, but the strongest difference (-4.07) was in calculating the total IHI, which included both the immune and histopathological parameters (p = 0.00005).

Polycomb Group Protein BMI1 In Leukemia Development and Progression:Blocking the Cell Differentiation

AbstractAbstract 2150BMI1 is the most frequently altered PcG group protein observed in cancer and is useful in predicting prognosis as a biomarker in some malignant blood diseases including myelodysplastic syndromes (MDS) and chronic myelogenous leukemia (CML). However, the role and effect of BMI1 in these diseases is not clear. We have found that BMI1 expression in MDS CD34+ cells (n=13) was correlated with IPSS (R2=0.514,P=0.013). The MDS patient with overexpressed BMI1 shortly after diagnosis displayed a disease progression: one case of MDS-RA progressed to MDS-RAEB and two cases of MDS-RAEB transformed to AML, in company with more serious cytopenia in one or more lineages of bone marrow cells.A similar situation was found in CML. As the CML-CP patients (n=181) accelerated to CML-BP (n=27),the hemoglobin level as well as WBC and Plt counts markedly decreased (p<0.05), whereas the BMI1 transcriptional level in CML-BP (n=12) was much higher than that in CML-CP (n=26) detected by qRT-PCR (p<0.05). To further explore the role of BMI1 in malignant myeloid transformation, we cloned BMI1 cDNA ORF and transfected it into K562 by MSCV retroviral vector. The BMI1 protein expression in transfectant subclones was about three-folds of the untransfected control. NBT assay demonstrated that the differentiation induced by 20nm/L TPA for 72 hours in transfected cell were less than that in control cells. The percentage of NBT positive cells in K562, K562-MSCV, K562- BMI1SC1 and K562-BMI1SC2 were 51±7%, 60±8%, 27±6%, 33±7% (n=3), respectively. In addition, the immunophenotype of CD11b, CD13, CD15, CD33, CD34, CD64 and HLA-DR in TPA-treated cells detected by FCM found that only the mean fluorescence intensity of CD15 in transfected cells was obviously lower than that in control. The mean value of CD15 in K562, K562-MSCV, K562-BMI1SC1, K562-BMI1SC2 were 3.38, 3.25, 2.42, 2.54, respectively. The benzidine staining showed BMI1 transfected K562 treated by 40mm/L sodium butyrate for 72 hours had slower erythroid differentiation compared to that in control cells. The benzidine positive cell in K562, K562-MSCV, K562-BMI1SC1, K562-BMI1SC2 were 28±4%, 31±5%, 10±4%, 9±5% (n=3), respectively. We also found that the mean fluorescence intensity of GPA and CD71 were lower in BMI1 transfected cell compared to control after sodium butyrate treatment. The mean value of GPA in K562, K562-MSCV, K562-BMI1SC1, K562-BMI1SC2 were 36.4, 32.1, 20.6, 16.2 and the mean of CD71 were 3.07, 3.13, 2.8, 1.32, respectively. These in vitro results suggested that overexpression of BMI1 can inhibit cell myelomonocytoid and erythroid differentiation. In summary, we conclude that polycomb group BMI1 may contribute to clonal myeloid transformation by blocking multiple lineage differentiation and potentiating the stemness of the leukemia-initiating cells via possible epigenetic mechanisms. Disclosures:No relevant conflicts of interest to declare.

Modulation of Cellular Immune Response by Orbifloxacin in Noninfected and E. coli-Infected Mice

The studies were conducted on noninfected and Escherichia (E) coli-infected mice treated with orbifloxacin administered orally 10 times at 24-hr intervals at a dose of 2.5 mg/kg. Orbifloxacin did not change the activity of peritoneal macrophages in noninfected mice. Administration of orbifloxacin in E. coli-infected mice modulated the effects of infection on the percentage of phagocyting macrophages, the percentage of NBT-positive cells, and nitric oxide production. Orbifloxacin did not affect the synthesis and release of interleukin-1 by macrophages. Orbifloxacin exerted a modulating effect on the subsets of lymphocytes in thymus, spleen, and mesenteric lymph node cells in noninfected and E. coli-infected mice.

NBT reactivity correlates to the distribution of PMNs between rat pulmonary and systemic circulation.

The rat systemic and pulmonary vascular transit times of radiolabeled polymorphonuclear granulocytes (experimental population referred to as "Polys") and mononuclear leukocytes (experimental population referred to as "Monos") (111In) in relation to those of erythrocytes (51Cr) were measured under physiological conditions. Results were also expressed as "circulating" pool and "marginated" pool of the two leukocyte fractions, and it was investigated whether the degree of spontaneous granulocyte activation, measured with the nitro blue tetrazolium (NBT) reduction test, was of importance for the retention of Polys in the lungs. The measured mean vascular transit was similar for Monos and Polys and several times slower than that of the erythrocytes in the pulmonary (17.4 times) and the systemic (4.1 times) vascular beds. The percentage of NBT-positive cells was < 10% in most animals, indicating a low level of spontaneous granulocyte activation. Increasing levels of NBT-positive cells correlated with a redistribution of granulocytes from the systemic marginated cell pool to the pulmonary marginated cell pool. Microscopic evaluation of sections from embedded lung tissue biopsies, obtained after intravital staining of the leukocytes, confirmed the isotope data on pulmonary transit.

New-type inducers of differentiation of HL-60 leukemia cells suppress c-myc expression.

New-type inducers of differentiation of human promyelocytic leukemia cells HL-60, i.e., 4- (5, 6, 7, 8-tetrahydro-5, 5, 8, 8-tetramethyl-2-naphthalenylcarbamoyl) benzoic acid (Am 80) and (E) -4- [3- (3, 5-di-tert-butylphenyl) -3-oxo-l-propenyl] benzoic acid (Ch 55), suppress c-myc expression as all-trans-retinoic acid (RA) does. The decreased level of c-myc expression was detected within 5-14 h after the start of treatment, and preceded morphological differentiation and functional differentiation measured in terms of nitroblue tetrazolium (NBT) reduction. Treatment of HL-60 cells with 0.1 μm Am 80, Ch 55 or RA for 96 h caused more than 90% suppression of c-myc expression and around 10-fold increase in the percentage of NBT-positive cells.

PMN heterogeneity: long-term stability of fluorescent membrane potential responses to the chemoattractant N-formyl-methionyl-leucyl- phenylalanine in healthy adults and correlation with respiratory burst activity

Polymorphonuclear neutrophils (PMNs) were isolated from 24 healthy adults 20 to 61 years of age and the proportion of cells that demonstrated depolarization responses to the synthetic chemotaxin N- formyl-methionyl-leucyl-phenylalanine (FMLP) were enumerated using the lipophilic fluorescent cyanine dye 3,3′-di-pentyl-oxacarbocyanine [di-O- C(5)(3)] and flow cytometry. The membrane potential responses were correlated to the cells' respiratory burst capabilities as measured by nitroblue tetrazolium (NBT) reduction and/or superoxide production in response to FMLP; 40.2% +/- 15.1% (mean +/- SD) of cells depolarized to FMLP. The mean SE for duplicate determinations 1 hour apart was 3.8% (range 0.4% to 13.6%, n = 15). There was no correlation between the percentage of depolarizing PMNs and the yield of cells, the percentage of immature cells, or the circulating WBC count. There was no difference in the average age of men (34.9 +/- 9.9 years, n = 11) v women (33.8 +/- 8.5, n = 13) (mean +/- SD) studied, or in the percentage of depolarizing PMNs when men and women were compared (42.2 +/- 10.6% v 43.1 +/- 13.3%, respectively). However, there was a significant increase in the percentage of depolarizing PMNs with increasing age of women (r = .61, P less than .025) but not men (r = .03, P greater than .05). Analysis of variance revealed significantly greater person-to-person variability in the membrane potential response than in day-to-day variability in the same person (P less than .0005). The percentage of depolarizing PMNs in response to FMLP was significantly correlated with the percentage of NBT-positive cells from both purified PMNs and from whole blood (r = .849, P less than .0005, r = .857, P less than .05, respectively), and with the amount of superoxide produced, expressed as a percentage of that amount produced by cytochalasin B (cyto-B)-pretreated cells (r = .565, P less than .01). The data indicate that PMNs from healthy adults demonstrate a heterogeneous membrane potential response to the chemotaxin FMLP that correlates with the cells' oxidative responsiveness and that intersubject differences can be detected. In addition, the proportion of responsive PMNs increases with increasing age in women.

PMN Heterogeneity: Long-Term Stability of Fluorescent Membrane Potential Responses to the Chemoattractant N-Formyl-Methionyl-Leucyl-Phenylalanine in Healthy Adults and Correlation With Respiratory Burst Activity

Polymorphonuclear neutrophils (PMNs) were isolated from 24 healthy adults 20 to 61 years of age and the proportion of cells that demonstrated depolarization responses to the synthetic chemotaxin N-formyl-methionyl-leucyl-phenylalanine (FMLP) were enumerated using the lipophilic fluorescent cyanine dye 3,3'-di-pentyl-oxacarbocyanine [di-O-C(5)(3)] and flow cytometry. The membrane potential responses were correlated to the cells' respiratory burst capabilities as measured by nitroblue tetrazolium (NBT) reduction and/or superoxide production in response to FMLP; 40.2% +/- 15.1% (mean +/- SD) of cells depolarized to FMLP. The mean SE for duplicate determinations 1 hour apart was 3.8% (range 0.4% to 13.6%, n = 15). There was no correlation between the percentage of depolarizing PMNs and the yield of cells, the percentage of immature cells, or the circulating WBC count. There was no difference in the average age of men (34.9 +/- 9.9 years, n = 11) v women (33.8 +/- 8.5, n = 13) (mean +/- SD) studied, or in the percentage of depolarizing PMNs when men and women were compared (42.2 +/- 10.6% v 43.1 +/- 13.3%, respectively). However, there was a significant increase in the percentage of depolarizing PMNs with increasing age of women (r = .61, P less than .025) but not men (r = .03, P greater than .05). Analysis of variance revealed significantly greater person-to-person variability in the membrane potential response than in day-to-day variability in the same person (P less than .0005). The percentage of depolarizing PMNs in response to FMLP was significantly correlated with the percentage of NBT-positive cells from both purified PMNs and from whole blood (r = .849, P less than .0005, r = .857, P less than .05, respectively), and with the amount of superoxide produced, expressed as a percentage of that amount produced by cytochalasin B (cyto-B)-pretreated cells (r = .565, P less than .01). The data indicate that PMNs from healthy adults demonstrate a heterogeneous membrane potential response to the chemotaxin FMLP that correlates with the cells' oxidative responsiveness and that intersubject differences can be detected. In addition, the proportion of responsive PMNs increases with increasing age in women.

Hodgkin's disease derived cell lines HDLM-2 and L-428: Comparison of morphology, immunological and isoenzyme profiles

The cell lines HDLM-2 and L-428 were established from the pleural effusions of two patients with Hodgkin's disease. We studied and compared phenotypic characteristics of HDLM-2 and L-428 cells before and during induction of differentiation with 12- O-tetradecanoylphorbol 13-acetate (TPA) using a number of parameters. TPA-treated HDLM-2 and L-428 cultures did not show adhesion to plastic surface or aggregation of cells; the cells did not develop pseudopodia and were not phagocytic. Only a slight increase in the percentage of NBT-positive cells was observed for L-428 cells. TPA led to a cessation of cell proliferation and to a dose-dependent decrease in the number of viable cells in both cell lines. In HDLM-2 and L-428, treatment with TPA induced distinct morphological changes indicative of a partial differentiation along the myeloid cell lineage. In addition, the production and expellation of benzidine-positive, unnucleated particles were observed in HDLM-2 and L-428 cells. The induced isoenzyme profiles of carboxylic esterase and acid phosphatase resembled those found in myelomonocytic leukemia cell lines. Both cell lines were negative for immunological markers of the T- and B-cell lineages, but reacted with several markers associated with the myelomonocytic cell lineages. HDLM-2 cells produced a factor which could induce differentiation in 12 leukemia cell lines. The overall results suggest that Hodgkin and Sternberg-Reed cells constitute a unique cell type and might be derived from cells of the myelomonocytic cell lineage.

Phagocytic activity and bactericidal capacity of polymorphonuclear leukocytes in children with recurrent otitis media

In 19 children with recurrent otitis media the phagocytic activity of blood granulocytes was studied and indirectly also their bactericidal capacity by means of the NBT test and myeloperoxidase activity. The mean value of granulocytes containing latex particles was 44.32% in the patients group, which differed significantly from the mean value established in controls: 66.54%. The mean phagocytic number in the patients group, 1.82 was also significantly diminished in comparison to controls: 2.41. The average number of nitroblue tetrazolium (NBT)-positive cells in patients, 86.74% as well the mean myeloperoxidase index, 2.52 did not differ from the mean control values, 92% and 2.42, respectively. In two patients a lower percentage of NBT-positive cells was determined. They are probably heterozygous carriers of an enzyme deficiency related to a depressed bactericidal activity of phagocytes. Patients with recurrent otitis media should be tested on possible phagocytic dysfunctions which may be helpful in understanding the pathogenesis of infections and also in identifying infants at particular risk of otitis media.

歯周病患者の白血球の Nitroblue tetrazolium 還元試験の検討

Sixty patients with various degrees of periodontal disease were studied to determine whether measurement of the reduction of nitroblue tetrazolium dye (NBT) by polymorphonuclear neutrophils was of value in diagnosis in periodontal diseaseThe following results were obtained.1. A significant correlation was found between the percentage of NBT-positive cells and the salivary leukocyte count which is the parameter of periodontal inflammation (p<0.01).2. The percentage of NBT-positive cells, however, did not show a signifcant correlation with Dunning and Leach's Gingival-Bone count.3. The rate of NBT-positive cells in blood from the gingiva was higher than that in venous blood. It was testified that this was due to contamination of blood with dental plaque when blood was taken from the gingiva.The neutrophils were stimulated both by the phagocytosis of oral microorganisms and by endotoxin.

Nitroblue-tetrazolium reduction test in pregnancy

NBT reduction test was performed in 121 pregnant women, 40 postpartum women, and 97 nonpregnant women of childbearing age. In the first and third trimesters the percentage of NBT positive cells was within the normal limits. During the weeks 14 to 27 (second trimester) of pregnancy there was a significant increase (P < 0.005) in NBT reduction, with a peak during weeks 18 to 24 (fifth and sixth months). Twenty-four hours after delivery we found a second rise and two days later the values returned to normal. It has been suggested that the increase in NBT reduction during pregnancy and 24 hours after delivery is in correlation with the penetration of trophoblasts in the maternal circulation which takes place at the same time. We conclude that the NBT reduction test cannot serve as an aid to the differentiation between bacterial and viral infection in the second trimester of pregnancy.

Nitroblue tetrazolium dye test as an aid in thedifferential diagnosis of febrile disorders

Nitroblue tetrazolium (NBT) dye tests were performed upon blood obtained at time of admission or during the course of hospitalization from 247 febrile patients and 20 afebrile individuals. Detailed clinical and laboratory diagnostic evaluation permitted subsequent placement of 181 of the febrile patients into 8 specific groups. Knowledge of only percentage and absolute number of NBT-positive cells permitted reclassification into 4 groups as follows: (1) normal subjects, (2) individuals with viral infection, noninfectious fevers, or partially treated bacterial infection, (3) untreated bacterial infection, and (4) bacterial infection unresponsive to therapy provided. There was generally no correlation between percentage of NBT-positive cells and either white blood cell counts or body temperature recorded at the time blood for the NBT test was obtained. Discriminant analysis was used to prepare a nomogram which permits classification of patients prospectively into one of the 4 groups, if the percentage and absolute number of NBT-positive cells are known.