Increase In Nitroblue Tetrazolium Reduction Research Articles

Bioconversion of chitin waste through Stenotrophomonas maltophilia for production of chitin derivatives as a Seabass enrichment diet

Marine wastes pose a great threat to the ecosystem leading to severe environmental hazards and health issues particularly the shellfish wastes. The shellfish waste which contains half of the amount of chitin can be efficiently transformed into useful products. Various approaches for the hydrolysis of chitin like physical, chemical, and enzymatic processes are there. Still, the use of enzyme chitinase is well documented as an effective and eco-friendly method. The present study summarizes the isolation of chitinase enzyme producing bacteria from different shrimp waste disposal sites in Parangipettai (India), and the possible use of an enzyme hydrolyzate as an immunostimulant to Asian Seabass (Lates calcarifer). The potential chitinase-producing bacteria were identified by 16S rRNA gene sequencing as Stenotrophomonas maltophilia. After purification, the chitinase specific activity was 5.01 (U/ml) and the protein content was 72 mg and the recovery rate was 48.06%. The optimum pH and temperature for the chitinolytic activity were 6.5 and at 35–50 °C, respectively. The animal experiment trial was done with our feed supplements which included 0.0 (control), 0.5%, 1% and 2% of chitin degraded product. All the supplementary feed had an optimal 42% (w/w) of crude protein. The feed protein level was 41–43% on average and gross energy was 13–17 kcal/g and the feed was observed to exhibit a significantly higher (p < 0.05) survival rate, condition factor, specific growth rates, and body weight gain was also found to be promising compared to other fishes fed with control diet only. The red blood cells (RBC) and white blood cell (WBC) counts were found to increase significantly after being challenged with infection in animals fed with chitin derivatives from 1st week to 3rd week when compared to the control. The hematocrit (Hct) values were low on the 2nd and 3rd week in infected fish fed with chitin derivatives. This low level was due to infection lyses of the red blood cells and increased nitro blue tetrazolium reduction. The control diet-fed fish showed 70% mortality but the chitin derivative supplemented fishes showed only 20% mortality post-infection. The results of the study encompass that the use of chitin-derivate enriched feed further is taken into large-scale approaches thereby benefitting the aquaculture sector.

Involvement of calreticulin in cell proliferation, invasion and differentiation in diallyl disulfide-treated HL-60 cells.

Diallyl disulfide (DADS) has shown potential as a therapeutic agent in various cancers. Previously, calreticulin (CRT) was found to be downregulated in differentiated HL-60 cells treated with DADS. The present study investigated the role of CRT proteins in DADS-induced proliferation, invasion and differentiation in HL-60 cells. The present study demonstrated that DADS treatment significantly changed the morphology of HL-60 cells and caused the significant time-dependent downregulation of CRT. Small interfering RNA (siRNA)-mediated knockdown of CRT expression significantly inhibited proliferation, decreased invasion ability, increased the expression of cluster of differentiation (CD)11b and reduced the expression of CD33 in DADS-treated HL-60 cells. DADS also significantly affected cell proliferation, invasion and differentiation in CRT-overexpressed HL-60 cells. Nitroblue tetrazolium (NBT) reduction assays showed decreased NBT reduction activity in the CRT overexpression group and increased NBT reduction in the CRT siRNA group. Following treatment with DADS, the NBT reduction abilities in all groups were increased. In conclusion, the present study clearly demonstrates the downregulation of CRT during DADS-induced differentiation in HL-60 cells and indicates that CRT is involved in cell proliferation, invasion and differentiation in DADS-treated HL-60 cells.

Effect of smoking on neutrophil oxidative metabolism

Alterations in neutrophil function by tobacco products may play a central role in the pathogenesis of periodontal diseases and several smoking-related systemic diseases. The aim of the study was to evaluate the effect of smoking on neutrophil oxidative metabolism. The study included 17 smoking men free of systemic diseases who were referred for treatment of various odontological diseases to outpatient department of Kaunas University of Medicine Hospital. The age of subjects varied from 22 to 43 years. All subjects answered the questions about smoking habits. Clinical examination included assessment of oral hygiene status according to the OHI-s index and periodontal status according to Russell and Ramfjord indices. To evaluate the oxidative metabolism of neutrophils, luminol- and liucigenin-dependent chemiluminescence and nitroblue tetrazolium test were used. After smoking, extracellular liucigenin-dependent chemiluminescence response was higher as compared to the response before smoking, but total (intra- and extracellular) luminol-dependent chemiluminescence response was the same both before and after smoking. Exposure of neutrophils to smoking caused a significant increase in nitroblue tetrazolium reduction. The release of reactive oxygen species in neutrophils exposed to smoking may alter the pathogenic processes in periodontal diseases.

Quantitative Analysis of Gene Expression for Vascular Endothelial Growth Factor and Its Application.

Vascular endothelial growth factor (VEGF) is a crucial mediator of angiogenesis, and plays an important role in pathogenesis of leukemia. However, the importance of VEGF in differentiation or apoptosis of leukemic cells remains to be evaluated. A competitor DNA fragment template of VEGF gene mimic was constructed with the method of gene recombinant technologies, and a competitive quantitative reverse transcriptase-polymerase chain reaction (cQRT-PCR) for analyzing VEGF gene expression was performed to assess the regulation of VEGF gene expression in the process of all-trans retinoic acid (ATRA)-induced differentiation of an acute promyelocytic leukemia cell line NB4. In construction of a standard curve from which the amount of target cDNA was derived, serial dilutions of the target were co-amplified with a constant amount of mimic, and the intensities of bands corresponding to the target and the mimic were measured. CD11b antigen and nitroblue tetrazolium (NBT) reduction rate of NB4 cells were also assayed at different time points. cQRT-PCR was a sensitive and reliable tool for analysis of VEGF gene expression, with a detectable range from 1 to 2 times 10 the fifth power molecules. The number of VEGF gene transcripts detected by means of cQRT-PCR assay was 42.3, 12.6, 3.6, and less than 1.0 times 10 the fifth powder per microgram of NB4 total RNA at 0, 12, 24 and 48 hours after ATRA treatment, respectively. The rapid down-regulation of VEGF gene expression during ATRA-induced NB4 cell differentiation was accompanied by an upregulation of CD11b expression and an increased NBT reduction rate. In conclusion, cQRT-PCR could be used as an efficient method of qualitative analysis of VEGF gene expression. ATRA significantly depresses VEGF expression and its antileukemic effect can be brought through the two ways of differentiation induction and angiogenesis inhibition.

Reactive oxygen species produced by NADPH oxidase are involved in pollen tube growth

Tip-localized reactive oxygen species (ROS) were detected in growing pollen tubes by chloromethyl dichlorodihydrofluorescein diacetate oxidation, while tip-localized extracellular superoxide production was detected by nitroblue tetrazolium (NBT) reduction. To investigate the origin of the ROS we cloned a fragment of pollen specific tobacco NADPH oxidase (NOX) closely related to a pollen specific NOX from Arabidopsis. Transfection of tobacco pollen tubes with NOX-specific antisense oligodeoxynucleotides (ODNs) resulted in decreased amount of NtNOX mRNA, lower NOX activity and pollen tube growth inhibition. The ROS scavengers and the NOX inhibitor diphenylene iodonium chloride (DPI) inhibited growth and ROS formation in tobacco pollen tube cultures. Exogenous hydrogen peroxide (H2O2) rescued the growth inhibition caused by NOX antisense ODNs. Exogenous CaCl2 increased NBT reduction at the pollen tube tip, suggesting that Ca2+ increases the activity of pollen NOX in vivo. The results show that tip-localized ROS produced by a NOX enzyme is needed to sustain the normal rate of pollen tube growth and that this is likely to be a general mechanism in the control of tip growth of polarized plant cells.

Reactive oxygen species produced by NADPH oxidase are involved in pollen tube growth

Tip-localized reactive oxygen species (ROS) were detected in growing pollen tubes by chloromethyl dichlorodihydrofluorescein diacetate oxidation, while tip-localized extracellular superoxide production was detected by nitroblue tetrazolium (NBT) reduction. To investigate the origin of the ROS we cloned a fragment of pollen specific tobacco NADPH oxidase (NOX) closely related to a pollen specific NOX from Arabidopsis. Transfection of tobacco pollen tubes with NOX-specific antisense oligodeoxynucleotides (ODNs) resulted in decreased amount of NtNOX mRNA, lower NOX activity and pollen tube growth inhibition. The ROS scavengers and the NOX inhibitor diphenylene iodonium chloride (DPI) inhibited growth and ROS formation in tobacco pollen tube cultures. Exogenous hydrogen peroxide (H2O2) rescued the growth inhibition caused by NOX antisense ODNs. Exogenous CaCl2 increased NBT reduction at the pollen tube tip, suggesting that Ca2+ increases the activity of pollen NOX in vivo. The results show that tip-localized ROS produced by a NOX enzyme is needed to sustain the normal rate of pollen tube growth and that this is likely to be a general mechanism in the control of tip growth of polarized plant cells.

Detection of superoxide anion generation by equine spermatozoa

To identify the generation of the superoxide anion by equine spermatozoa. Multiple ejaculates collected from 3 Thoroughbred stallions. Induced superoxide production by reduced nicotinamide adenine dinucleotides (NAD[P]H; ie, reduced nicotinamide adenine dinucleotide [NADH] and reduced nicotinamide adenine dinucleotide phosphate [NADPH]) was measured by use of a nitroblue tetrazolium (NBT) reduction assay on whole spermatozoa and a cytochrome c reduction assay on isolated membrane fractions of spermatozoa. Localization of superoxide generation was determined by use of NBT cytochemistry. A dose-dependent increase in NBT reduction was found in the presence of NADPH, which was inhibited by superoxide dismutase (SOD). The flavoprotein inhibitor, diphenyleneiodonium (DPI; 5 or 15 microM), significantly decreased NBT reduction. Cytochrome c reduction by plasma membranes of spermatozoa was significantly higher in the presence of NADPH than in its absence. Cytochemical staining of equine spermatozoa in the presence of NADPH and NADH revealed diaphorase labeling in the spermatozoon midpiece and head. This staining was inhibited by DPI and SOD. Results of our study indicate that superoxide generation is associated with a membrane-associated NAD(P)H oxidase present in equine spermatozoa, although mitochondrial generation of superoxide is also detected. This oxidase may play a role in cell signaling or may also contribute to cytopathic effects associated with oxidative stress in equine spermatozoa.

Leucocyte mobilization and function in vitro of blood and exudative leucocytes after inguinal herniotomy.

The effect of surgical trauma on leucocyte mobilization in vivo and the function of blood and exudative leucocytes was studied in 15 patients after inguinal herniotomy. Mobilization of leucocytes to skin windows with chambers was reduced 48 h after anaesthesia (P less than 0.01) compared with 21 healthy volunteers. Blood leucocyte chemotaxis was reduced during the period of anaesthesia (P less than 0.05). Serum independent phagocytosis was increased from 24 h after anaesthesia (P less than 0.01) but without a corresponding increase in the serum dependent phagocytosis. The oxidative metabolic activity of resting leucocytes in terms of Nitro Blue Tetrazolium (NBT) reduction decreased 24-48 h after anaesthesia without any change in NBT reduction during phagocytosis. In contrast, exudative leucocytes exhibited reduced phagocytosis independent of serum factors (P less than 0.05) and increased NBT reduction by resting leucocytes. It is concluded that a diminished accumulation of leucocytes as inflammatory lesions occurs postoperatively and that changes in the function of circulating leucocytes do not always reflect the changes in function of the exudative leucocyte.

Superoxide generation links protein kinase C activation to impaired ATP-sensitive K+ channel function after brain injury.

BACKGROUND AND PURPOSE--Endothelin-1, in concentrations similar to that present in cerebrospinal fluid after fluid percussion brain injury (FPI), increases superoxide anion (O2-) production. Endothelin-1 also contributes to altered cerebral hemodynamics after FPI through impairment of ATP-sensitive K+ (KATP) channel function through protein kinase C (PKC) activation. Generation of O2- additionally occurs after FPI. Nitric oxide and cGMP elicit pial artery dilation through KATP channel activation. The present study was designed to determine whether PKC activation generates O2-, which, in turn, could link such activation to impaired KATP channel function after FPI. METHODS--Injury of moderate severity (1.9 to 2.1 atm) was produced by the lateral FPI technique in anesthetized newborn pigs equipped with a closed cranial window. Superoxide dismutase-inhibitable nitroblue tetrazolium (NBT) reduction was determined as an index of O2- generation. RESULTS--Phorbol 12, 13-dibutyrate (10(-6) mol/L), a PKC activator, increased superoxide dismutase-inhibitable NBT reduction from 1+/-1 to 37+/-5 pmol/mm2. Staurosporine (10(-7) mol/L), a PKC antagonist, blocked the NBT reduction after phorbol 12,13-dibutyrate and blunted the NBT reduction observed after FPI (1+/-1 to 15+/-2 versus 1+/-1 to 5+/-1 pmol/mm2 after FPI in the absence versus presence of staurosporine). Exposure of the cerebral cortex to a xanthine oxidase O2--generating system increased NBT reduction in a manner similar to FPI and blunted pial artery dilation to the KATP channel agonists cromakalim and calcitonin gene-related peptide, the nitric oxide releasers sodium nitroprusside and S-nitroso-N-acetylpenicillamine, and the cGMP analogue 8-bromo-cGMP (10+/-1% and 21+/-1% versus 4+/-1% and 9+/-1% for 10(-8) and 10(-6) mol/L cromakalim before and after activated oxygen-generating system exposure). CONCLUSIONS--These data show that PKC activation increases O2- production and contributes to such production observed after FPI. These data also show that an activated system that generates an amount of O2- similar to that observed with FPI blunted pial artery dilation to KATP channel agonists and nitric oxide/cGMP. These data suggest, therefore, that O2- generation links PKC activation to impaired KATP channel function after FPI.

Detection of urinary tract infections by reduction of nitroblue tetrazolium: Technical Note

Nitroblue tetrazolium (NBT) reduction to formazan has been used as a marker for nitric oxide synthase (NOS). Since inducible NOS activity is elevated in urine from patients with urinary tract infections (UTIs), we investigated the accuracy of NBT reduction as an early predictor of UTIs and quantified the relationship between inducible NOS and NBT. Urine samples from 434 patients were screened for the presence of UTIs with leukocyte-esterase and nitrite dipsticks and with NBT reduction. The rapid screening results from each test were compared to urine culture results. In addition, NBT reduction parameters were measured in urine pellet at 595 nm after incubation with one of four factors: NOS cofactors, NOS inhibitors, NADH, or superoxide dismutase/catalase. As a urine screening test for UTIs, NBT reduction was more sensitive with a higher negative predictive accuracy than the nitrite dipstick. NBT reduction also was more specific with a higher positive predictive accuracy and negative predictive accuracy than the leukocyte-esterase dipstick. In infected urine pellet, both NADPH, a NOS cofactor, and NADH increased NBT reduction. Superoxide dismutase/catalase decreased NBT reduction. Although NOS may not be the only NBT reducing enzyme, rapid, visible reduction of NBT is induced in urine from patients with UTIs.

Reaction of Human Alveolar Macrophages to Exposure toAspergillus fumigatusand Inert Particles

In vitrointeraction of human alveolar macrophages (AM) with heat-killed conidia fromAspergillus fumigatusand inert silica particles of similar size, about 3 μm, was studied. The conidia were phagocytized significantly faster by AM than were the control particles partly due to the faster rate of attachment but especially due to the faster rate of ingestion. Quantitative nitroblue tetrazolium (NBT) reduction by AM, reflecting their release of oxygen radicals, was increased by a factor of 2 to 3 in response to the conidia during phagocytosis. The silica particles induced a moderate but significant increase in NBT reduction. Conidia, but not silica particles, showed a considerable percentage (around 8%) of phagolysosomes with neutral pH after 3 h and a smaller percentage (around 1%) after 24 h of incubation. The pH of phagolysosomes with conidia tended to be higher after 3 h, but was significantly lower after 24 h than the pH of phagolysosomes with silica particles. Despite the markedly increased oxidative metabolism there was no increase in cytokine production [interleukins (IL) 6 and 8 and tumor necrosis factor α (TNF-α)] after exposure to conidia. The silica particles induced a significant decrease in IL-6 and IL-8 production and a tendency toward decreased production of TNF-α. The occurrence of phagolysosomes with neutral pH suggests unsealed phagolysosomes from which not only oxygen metabolites but also enzymes might escape from the cell. Lung damage may thus be the result of repeated or long-term exposure toAspergillus conidia.

Enhancement of Activity of la,25-Dihydroxyvitamin D3 for Growth Inhibition and Differentiation Induction of Human Myelomonocytic Leukemia Cells by Tretinoin Tocoferil, an α-Tocopherol Ester of All-trans Retinoic Acid

Tretinoin tocoferil is an alpha-tocopherol ester of all-trans retinoic acid (RA) and safely used in the treatment of skin ulcer. Tretinoin tocoferil inhibited proliferation of human promyelocytic leukemia HL-60 cells and induced granulocytic differentiation of the cells, but less than RA. alpha-Tocopherol did not affect differentiation of HL-60 cells, but at high concentrations enhanced its nitroblue tetrazolium (NBT)-reducing activity and expression of surface antigen CD11b, which are markers of myelomonocytic differentiation induced by RA. Tretinoin tocoferil increased NBT reduction in HL-60 cells treated with RA. It also enhanced the differentiation of HL-60 cells induced by dimethyl sulfoxide, phorbol-12-myristate 13-acetate or 1alpha,25-dihydroxyvitamin D3 (VD3). In combination with a low concentration of VD3, it induced the NBT-reducing activity of human monoblastic U937 cells very effectively. Moreover, it enhanced the differentiation of human myelomonocytic ML-1, THP-1, P39/TSU, and P31/FUJ cells induced by VD3. In combination with VD3, it synergistically inhibited the proliferation of HL-60, U937, ML-1, THP-1, P39/TSU, and P31/FUJ cells and decreased the effective concentration of VD3 to a 10(-10) mol/L level. Because tretinoin tocoferil was reported to induce neither retinoid-related toxicity nor teratogenicity, the therapeutic advantage of the use of it in treatment of myelomonocytic leukemia is suggested.

In vitro of modulation of bovine blood neutrophils and mononuclear cells by oxytetracycline

SUMMARY The effect of oxytetracycline (otc) on bovine blood mononuclear cells and neutrophil functions was examined in vitro. Neutrophil functions tested include respiratory burst, peroxidase, and antibacterial activities. Neutrophils were treated with otc (10 to 1,500 μg/ml) before exposure to either opsonized zymosan or bacteria. A dose-response inhibition antibacterial activity to high concentrations of otc (500 to 1,000 μg/ml) was observed. Beginning at a concentration 15 μg/ml, otc treatment of neutrophil lysates resulted in decreased peroxidase activity. A dose response was not observed. In contrast, respiratory burst, measured by nitroblue tetrazolium dye reduction, increased after otc exposure, but only at high concentrations (500 and 1,000 μg/ml) of otc. Mitogen-induced proliferation of blood mononuclear cells cocultured with otc and concanavalin A, phytohemagglutinin-P, or pokeweed mitogen was inhibited at an otc concentration of 100 μg/ml at 48 and 72 hours of culture. These results indicate that blood mononuclear cells are more sensitive to the inhibitory effects of otc than are neutrophils. Furthermore, the otc-mediated inhibition of neutrophil antimicrobial activity is inversely related to the increase in nitroblue tetrazolium reduction. This suggests that otc is uncoupling the hexose monophosphate shunt from production of secreted oxygen radicals. These results also suggest that the peroxidase enzyme system has a large biological reserve capacity.

Generation of reactive oxygen metabolites by the haemocytes of the mussel Mytilus edulis

Generation of superoxide anion by stimulated haemocytes of Mytilus edulis was demonstrated using dihydrorhodamine 123 and quantified using reduction of nitroblue tetrazolium (NBT). In the presence of zymosan or phorbol myristate acetate, there was an increased reduction of NBT to formazan. The addition of superoxide dismutase (SOD) and iodoacetamide to the incubation medium resulted in a significant reduction in deposition of reduced formazan. Incubation of haemocytes with the SOD inhibitor diethyldithiocarbamate (DDC) gave rise to a small but significant increase in NBT reduction. The production of hydrogen peroxide by haemocytes was quantified using horseradish peroxidase-dependent oxidation of phenol red. The presence of SOD in the incubation medium together with zymosan resulted in a significant increase in H 2O 2 production. Haemocytes incubated with DDC prior to the assay or with sodium nitroprusside during the assay showed a decrease in H 2O 2 production with increasing concentration of the inhibitor.

Oxido-reductive functions of Entamoeba histolytica in relation to virulence.

Entamoeba histolytica can reduce nitro-blue tetrazolium (NBT) in Hank's balanced salt solution to almost the same extent as in Eagle's minimal medium. Further, this reduction was stimulated only to a minor degree by glucose, pyruvate and DL-serine, substrates known to support respiratory activity (O2 uptake) in E. histolytica. However, both NADH and NADPH increased NBT reduction several-fold, the effect being greater with NADPH. A sizeable proportion of this endogenous dye-reducing capability (in Hank's solution) was associated with low-speed sediments obtainable from amoebic homogenates, which also shared the bulk of 125I labelling (when the homogenates were prepared after surface labelling with Na 125I). Conversion of the dye to formazan was strongly inhibited by -SH blocking agents, but was not influenced by rotenone and antimycin A. The activity was also inhibited by H2O2, but stimulated by catalase. Superoxide dismutase only slightly curtailed NBT reduction in intact cells, but inhibited it in homogenates in a concentration-dependent manner to a maximal extent of 33%. Almost the same degree of curtailment of this activity was induced by anaerobic conditions. Both concanavalin A (Con A) and phorbol myristate acetate stimulated the activity in intact cells, though the effect of Con A was nullified by alpha-methyl mannoside. Both NBT-reducing capability and alcohol dehydrogenase activities were higher in the more virulent IP:106 strain, and they increased with time in cultures of NIH:200 in a cholesterol-enriched environment.

Restoration of Phagocyte Function by Interferon-γ in X-Linked Chronic Granulomatous Disease Occurs at the Level of a Progenitor Cell

AbstractPhagocytes from X-linked chronic granulomatous disease (X-CGD) patients are deficient in their ability to generate superoxide because of a defective gene that encodes a heavy chain of cytochrome b, a critical component in the superoxide-generating pathway. Previously we have shown that a single in vivo treatment of selected X-CGD patients with interferon-γ (INF-γ) resulted 14 days later in near-normal levels of superoxide generation by phagocytes. The effect persisted for 28 days. This prolonged effect suggested that the lymphokine affected progenitor cells. In this study, we examined progenitor-derived colonies from the peripheral blood from this unusual X-CGD kindred. Progenitor-derived colonies examined before treatment were unable to generate superoxide as visualized by lack of nitro blue tetrazolium (NBT) reduction compared with normal controls. By contrast, colonies derived 7 days after a single INF-γ injection were able to generate superoxide as shown by increased NBT reduction. Colonies harvested 21 days after treatment contained only rare cells capable of NBT reduction. Our results indicate that INF-γ can reprogram the myeloid progenitor cells to express a partially corrected phenotype. This corrected phenotype is later expressed in daughter cells.

Restoration of phagocyte function by interferon-gamma in X-linked chronic granulomatous disease occurs at the level of a progenitor cell

Phagocytes from X-linked chronic granulomatous disease (X-CGD) patients are deficient in their ability to generate superoxide because of a defective gene that encodes a heavy chain of cytochrome b, a critical component in the superoxide-generating pathway. Previously we have shown that a single in vivo treatment of selected X-CGD patients with interferon-gamma (INF-gamma) resulted 14 days later in near-normal levels of superoxide generation by phagocytes. The effect persisted for 28 days. This prolonged effect suggested that the lymphokine affected progenitor cells. In this study, we examined progenitor-derived colonies from the peripheral blood from this unusual X-CGD kindred. Progenitor-derived colonies examined before treatment were unable to generate superoxide as visualized by lack of nitro blue tetrazolium (NBT) reduction compared with normal controls. By contrast, colonies derived 7 days after a single INF-gamma injection were able to generate superoxide as shown by increased NBT reduction. Colonies harvested 21 days after treatment contained only rare cells capable of NBT reduction. Our results indicate that INF-gamma can reprogram the myeloid progenitor cells to express a partially corrected phenotype. This corrected phenotype is later expressed in daughter cells.

Spontaneous NBT reduction by monocytes as a marker of disease activity in children with histiocytosis

In an attempt to define a biological marker of monocyte hyperactivation in the course of infantile histiocytosis, the spontaneous nitroblue tetrazolium (NBT) reduction assay was applied to monocytes from 13 children with Langerhans cell histiocytosis (LCH), familial haemophagocytic lymphohistiocytosis (FHL), juvenile xanthogranuloma or malignant histiocytosis. Significant increase in NBT reduction was observed in the patients with both active LCH and FHL in comparison with control subjects, who were either healthy or affected by different conditions. A close relationship between spontaneous reduction rate and clinical condition of the patients was evident in patients tested at diagnosis, during remission and during the course of disease reactivation. Interleukin-1 (IL-1) production by monocytes was also evaluated: the patients with LCH and FHL displayed a significant increase in in vitro IL-1 production by lipopolysaccharide-stimulated monocytes. In our experience the spontaneous NBT reduction assay was a sensitive, quite specific, low-cost and reproducible test for the evaluation of children with histiocytosis. Useful information may be obtained at diagnosis but also during the clinical course of disease by using this marker of monocyte spontaneous activation.

Spontaneous NBT reduction by monocytes as a marker of disease activity in children with histiocytosis

SummaryIn an attempt to define a biological marker of monocyte hyperactivation in the course of infantile histiocytosis, the spontaneous nitroblue tetrazolium (NBT) reduction assay was applied to monocytes from 13 children with Langerhans cell histiocytosis (LCH), familial haemophagocytic lymphohistiocytosis (FHL), juvenile xanthogranuloma or malignant histiocytosis. Significant increase in NBT reduction was observed in the patients with both active LCH and FHL in comparison with control subjects, who were either healthy or affected by different conditions. A close relationship between spontaneous reduction rate and clinical condition of the patients was evident in patients tested at diagnosis, during remission and during the course of disease reactivation. Interleukin‐1 (IL‐1) production by monocytes was also evaluated: the patients with LCH and FHL displayed a significant increase in in vitro IL‐1 production by lipopolysaccharide‐stimulated monocytes. In our experience the spontaneous NBT reduction assay was a sensitive, quite specific, low‐cost and reproducible test for the evaluation of children with histiocytosis. Useful information may be obtained at diagnosis but also during the clinical course of disease by using this marker of monocyte spontaneous activation.

Induction of cytidine deaminase in HL-60 myeloid leukemic cells by 5-AZA-2′-deoxycytidine

5-Aza-2′-deoxycytidine (5-AZA-CdR), a potent inhibitor of DNA methylation and an active antileukemic agent, produced an induction of cytidine deaminase activity in human HL-60 myeloid leukemic cells. This increase in enzyme activity correlated with the induction of differentiation of the HL-60 leukemic cells as shown by an increase in nitroblue tetrazolium reduction, loss of clonogenicity and decrease in DNA synthesis. The concentration of 5-AZA-CdR that produced these effects was in the range of 0.1–1.0 μM. The increase in cytidine deaminase activity became apparent starting at 48 h from the start of the 5-AZA-CdR treatment and at 72 h was about 6-fold greater than the non-treated cell. Since cytidine deaminase is the major enzyme involved in the catabolism of cytosine nucleoside analogues, these results may be relevant in the evaluation of the clinical response of leukemic patients treated with 5-AZA-CdR.