Granulocyte Respiratory Burst Research Articles

Immunotoxicity of stainless-steel nanoparticles obtained after 3D printing

This study aims to investigate the in vitro effects of nanoparticles (NPs) produced during the selective laser melting (SLM) of 316 L stainless steel metal powder on the immune response in a human blood model. Experimental data did not reveal effect on viability of 316 L NPs for the tested doses. Functional immune assays showed a significant immunosuppressive effect of NPs. There was moderate stimulation (117%) of monocyte phagocytic activity without significant changes in phagocytic activity and respiratory burst of granulocytes. A significant dose-dependent increase in the levels of the pro-inflammatory cytokine TNF-a was found in blood cultures treated with NPs. On the contrary, IL-8 chemokine levels were significantly suppressed. The levels of the pro-inflammatory cytokine IL-6 were reduced by only a single concentration of NPs. These new findings can minimise potential health risks and indicate the need for more research in this area.

Respiratory Burst and TNF-α Receptor Expression of Neutrophils after Sepsis and Severe Injury-Induced Inflammation in Children.

Systemic inflammatory response syndrome (SIRS) is defined as the systemic host response to infection or a non-infectious factor. The purpose of this study was to evaluate the involvement of reactive oxygen species (ROS) in severe inflammation and to assess the discrimination strength of the neutrophil BURSTTEST assay regarding its etiology in three groups of patients (sepsis, burns, and bone fractures) who met the SIRS criteria. The neutrophil activation (respiratory burst of granulocytes as well as p55 and p75 tumor necrosis factor (TNF-α) receptor expression) was evaluated twice using flow cytometry, and the results were compared with healthy controls and among SIRS subjects. A decreased oxygen metabolism in neutrophils after E. coli stimulation and increased TNF-α receptor expression were found in septic and burned patients on admission, while ROS production augmented and TNF-α receptor expression diminished with the applied therapy. The significant differences in neutrophil respiratory burst intensity among septic and burned patients and those with sepsis and bone fractures were found (however, there were not any such differences between patients with thermal and mechanical injuries). This study indicates that the neutrophil BURSTTEST evaluation might be a clinically reliable marker for differentiating the SIRS etiology.

REGULATORY INFLUENCE OF BLOOD MONOCYTES ON THE POPULATION COMPOSITION OF GRANULOCYTES AND THE STATE OF THEIR RESPIRATORY BURST IN THE WIDESPREAD PURULENT PERITONITIS

The aim of the study was to investigate the regulatory effect of monocytes and their subpopulations on the population composition of granulocyte leukocytes and the state of their respiratory burst in widespread purulent peritonitis (WPP). The study involved 24 patients aged 30-65 with acute surgical diseases and injuries of abdominal organs complicated by WPP. As a control 25 relatively healthy people of the same age range were examined. A study of the population composition of monocytes and granulocyte leukocytes in blood was performed using a two-platform technology on the hematological analyzer Sysmex XE-5000 (Sysmex Inc., USA) and FC-500 flow cytometer (Beckman Coulter, USA) using the Cytodiff antibody kit (Beckman Coulter, USA). A study of the monocytes number expressing HLA-DR- and CD64-receptor was performed by flow cytometry using direct immunofluorescence of whole peripheral blood. The respiratory burst state of neutrophilic granulocytes was studied by chemiluminescence analysis on a 36-channel chemiluminescence analyzer BLM-3607 (MedBioTech, Russia). As indicators of chemiluminescence were used luminol and lucigenin. The enhancement of chemiluminescence induced by zymosan was evaluated by the ratio of the area of the induced chemiluminescence to the spontaneous area and was defined as the activation index. It has been established that the immune-inflammatory process in WPP is characterized by a decrease in the number of classical monocytes in the peripheral blood and an increase in the content of non-classical monocytes. In WPP in peripheral blood the level of monocytes expressing HLA-DR receptors decreases. The change in the ratio of monocytes subpopulations characterizes the increase in the role of the proinflammatory fraction in the WPP pathogenesis. Changes in the population composition of granulocytes in the blood in patients with WPP also characterize the development of an acute inflammatory process. In this case, there is a decrease in the number of basophils in the peripheral blood, which, apparently, is determined by the presence of an allergic component in WPP and, accordingly, their migration to the inflammation area. In patients with WPP activation of a respiratory burst of granulocytes of blood was detected, the intensity of which is determined by the synthesis of primary and secondary active oxygen species. The results of the correlation analysis made it possible to establish that in WPP the regulatory role of non-classical monocytes increases aimed at stimulating the inflammatory processes (an increase in the number of mature and immature forms of neutrophils and stimulation of the activity of a respiratory explosion of granulocytes). The revealed features of the regulatory effect of monocytes on the population composition and the intensity of the respiratory burst of granulocytes can be used in the development of immunotherapeutic methods aimed at reducing the activity of the inflammatory process in WPP.

Effect of 28-day oral administration of silver nanocolloid on the peripheral blood leukocytes in mice.

Silver nanoparticles, which have found a wide range of applications owing to their antimicrobial properties, are also recommended as dietary supplements in alternative medicine. Studies on rodents confirm that nanosilver is absorbed from the digestive tract into the bloodstream, which implies its possible interactions with leukocytes. The objective of the experiment discussed herein has been to determine the effect of 28-day oral administration of different doses (0.25, 2.5, 25 ppm) of commercial silver nanocolloid on hematological parameters, percentages of particular lymphocyte populations and activity of the peripheral blood leukocytes in mice. All the tested colloid doses decreased the counts of monocytes in the animals' blood and induced phenotypic modifications among lymphocytes: an increase in CD4+/CD8+ T cell distribution, a decrease in NK and NKT cell distribution (doses of 0.25 and 2.5 ppm) and an increased CD4+:CD8+ ratio (25 ppm). Silver nanocolloid also affected the activity of cells, depressing the proliferation of lymphocytes (0.25 ppm) and stimulating phagocytosis as well as the respiratory burst of granulocytes and monocytes (all doses). The results verify the influence of orally administered silver colloid on the peripheral blood leukocytes, at the same time implying the potential risk of developing an inappropriate immune response of an organism exposed to prolonged administration of this substance.

Lessons from Anaplasma phagocytophilum: Chromatin Remodeling by Bacterial Effectors

Bacterial pathogens can alter global host gene expression via histone modifications and chromatin remodeling in order to subvert host responses, including those involved with innate immunity, allowing for bacterial survival. Shigella flexneri, Listeria monocytogenes, Chlamydia trachomatis, and Anaplasma phagocytophilum express effector proteins that modify host histones and chromatin structure. A. phagocytophilum modulates granulocyte respiratory burst in part by dampening transcription of several key phagocyte oxidase genes. The A. phagocytophilum protein AnkA localizes to the myeloid cell nucleus where it binds AT-rich regions in the CYBB promoter and decreases its transcription. AT-rich regions of DNA are characteristic of matrix attachment regions (MARs) which are critical for chromatin structure and transcription. MAR-binding proteins, such as SATB1, interact with histone modifying enzymes resulting in altered gene expression. With A. phagocytophilum infection, histone deacetylase 1 (HDAC1) expression is increased and histone H3 acetylation is decreased at the CYBB promoter, suggesting a role for AnkA in altering host epigenetics and modulating gene transcription, at this, and perhaps other loci. This review will focus on how bacterial pathogens alter host epigenetics, by specifically examining A. phagocytophilum AnkA cis-regulation of CYBB transcription and epigenetic changes associated with infection.

Humoral and cell-mediated immunity following vaccination with synthetic Candida cell wall mannan derived heptamannoside–protein conjugate: Immunomodulatory properties of heptamannoside–BSA conjugate

Chemically defined glycoprotein conjugate composed of synthetically prepared mannan‐derived heptamannoside with terminal β-1,2-linked mannose residue attached to the α-1,3-linked mannose residues and BSA as carrier protein (M7–BSA conjugate) was analysed for the capacity to induce protective humoral immunity and appropriate alteration cellular immunity. To identify protective antigenic structure of Candida cell wall mannan M7–BSA conjugate was used for BALB/c mice immunization. The obtained results were compared with placebo group and with heat‐inactivated C. albicans whole cells immunization. The administration route of M7–BSA conjugate secondary booster injection significantly affected the intensity of humoral immune response and the specificity of produced antibodies. All prepared sera were able to elevate candidacidal activity of polymorphonuclear leukocytes (PMN) in cooperation with complement. Moreover, polyclonal sera obtained after secondary subcutaneous (s.c.) booster injection of M7–BSA conjugate were able to induce candidacidal activity of PMN also in complement independent manner. M7–BSA conjugate immunization induced increases of phagocytic activity and respiratory burst of granulocytes, caused a raise of the proportion of CD3+ T lymphocytes and increased the CD4+/CD8+ T lymphocyte ratio. We observed also an increasing proportion of CD4+CD25+ T cells compared to immunization with heat inactivated whole C. albicans cells, which in turn promoted an increase of the CD8+CD25+ cell proportion. Immunization with M7–BSA conjugate induced Th1, Th2 and Th17 immune responses as indicated by the elevation of relevant cytokines levels. These data provide some insights on the immunomodulatory properties of oligomannosides and contribute to the development of synthetic oligosaccharide vaccines against fungal diseases.

Impact of Human Granulocyte and Monocyte Isolation Procedures on Functional Studies

One of the first lines of defense against infection is the activation of the innate immune system. It is becoming clear that autoimmune diseases, such as rheumatoid arthritis and Crohn's disease, may be caused by disturbed innate immunity, and relating granulocyte and monocyte functions to the patient genotype has become an important part of contemporary research. Although it is essential to move this field forward, a systematic study comparing the efficacy and suitability for functional studies of the various available protocols for the isolation of these immune cells has not been performed. Here, we compare human granulocyte functionality under three enrichment protocols: (i) Ficoll density gradient centrifugation, (ii) anti-CD15 antibody-conjugated microbeads (positive selection), and (iii) Polymorphoprep. Primary monocytes were isolated in parallel using (i) anti-CD14 magnetic microbeads, (ii) non-monocyte depletion by antibody-conjugated magnetic microbeads (negative selection), (iii) RosetteSep antibody cocktail, and (iv) the classical adherence protocol. The best results in terms of purity and cell functionality were obtained with positive selection by magnetic microbeads for both human granulocytes and monocytes. Whereas phagocytosis of Escherichia coli bacteria was identical in all isolation procedures tested, the granulocyte respiratory burst was higher in positively selected cells. In addition, different granulocyte enrichment procedures affect cell surface receptor expression to different extents. In toto, we propose that positive selection of granulocytes and monocytes be adopted as the procedure of choice for studies of human granulocyte and monocyte functions but caution investigators to be aware of possible alterations in cell phenotypes with different isolation procedures.

The Flavonoid Luteolin Inhibits Fcγ-Dependent Respiratory Burst in Granulocytes, but Not Skin Blistering in a New Model of Pemphigoid in Adult Mice

Bullous pemphigoid is an autoimmune blistering skin disease associated with autoantibodies against the dermal-epidermal junction. Passive transfer of antibodies against BP180/collagen (C) XVII, a major hemidesmosomal pemphigoid antigen, into neonatal mice results in dermal-epidermal separation upon applying gentle pressure to their skin, but not in spontaneous skin blistering. In addition, this neonatal mouse model precludes treatment and observation of diseased animals beyond 2–3 days. Therefore, in the present study we have developed a new disease model in mice reproducing the spontaneous blistering and the chronic course characteristic of the human condition. Adult mice were pre-immunized with rabbit IgG followed by injection of BP180/CXVII rabbit IgG. Mice pre-immunized against rabbit IgG and injected 6 times every second day with the BP180/CXVII-specific antibodies (n = 35) developed spontaneous sustained blistering of the skin, while mice pre-immunized and then treated with normal rabbit IgG (n = 5) did not. Blistering was associated with IgG and complement C3 deposits at the epidermal basement membrane and recruitment of inflammatory cells, and was partly dependent on Ly-6G-positive cells. We further used this new experimental model to investigate the therapeutic potential of luteolin, a plant flavonoid with potent anti-inflammatory and anti-oxidative properties and good safety profile, in experimental BP. Luteolin inhibited the Fcγ-dependent respiratory burst in immune complex-stimulated granulocytes and the autoantibody-induced dermal-epidermal separation in skin cryosections, but was not effective in suppressing the skin blistering in vivo. These studies establish a robust animal model that will be a useful tool for dissecting the mechanisms of blister formation and will facilitate the development of more effective therapeutic strategies for managing pemphigoid diseases.

Influence of tourniquet application on venous blood sampling for serum chemistry, hematological parameters, leukocyte activation and erythrocyte mechanical properties

Venous blood sampling is usually performed using a tourniquet to help locate and define peripheral veins to achieve successful and safe venipuncture. Despite widespread usage of tourniquets for venipuncture by medical and laboratory staff, very few are aware of the effects of tourniquet application on laboratory parameters. In addition, definitive guidelines regarding when and how to use a tourniquet for blood sampling are lacking. The aim of the present study was to define the optimal sampling time after tourniquet removal to avoid adverse impact on laboratory analytes. Blood oxygen and carbon dioxide partial pressure, pH, oxyhemoglobin saturation (satO(2)), hematological parameters, serum electrolyte concentrations, erythrocyte, deformability and aggregation, leukocyte activation and nitrite/nitrate concentrations obtained 180 s after tourniquet release were compared with baseline values for 10 healthy subjects. Blood gases, hematological parameters and serum electrolyte levels were not affected by the application and removal of a tourniquet. However, there were significant decreases in erythrocyte deformability at 90, 120, 180 s, and increases in erythrocyte aggregation at 5 and 30 s following removal of the tourniquet. A significant increase in granulocyte respiratory burst at 60 s was observed, confirming leukocyte activation due to application of the tourniquet. There were no significant alterations of blood nitrite/nitrate levels. Our blood sampling technique which mimicked the application and release of a tourniquet indicated unaltered values for routine blood gases, hematological testing and serum electrolyte levels. Conversely, hemorheological measurements can be affected. Therefore, it is strongly recommended that tourniquet application should be avoided during blood sampling or, if this is not possible, the procedure should be well standardized and details of the sampling method should be reported.

Post-160-km Race Illness Rates and Decreases in Granulocyte Respiratory Burst and Salivary IgA Output are Not Countered by Quercetin Ingestion

Post-160-km Race Illness Rates and Decreases in Granulocyte Respiratory Burst and Salivary IgA Output are Not Countered by Quercetin Ingestion

Post-160-km Race Illness Rates and Decreases in Granulocyte Respiratory Burst and Salivary IgA Output are Not Countered by Quercetin Ingestion

This study measured the influence of the flavonoid quercetin on immune changes and incidence rates of upper respiratory tract infections in ultramarathoners competing in the 160-km Western States Endurance Run. Sixty-three runners were randomized to quercetin and placebo groups, and under double-blinded methods ingested 1000 mg/day quercetin for 3 wks before, during, and 2 wks after the race. Thirty-nine of the 63 subjects (n = 18 for quercetin, n = 21 for placebo) finished the race and provided blood and saliva samples the morning before the race and 15 - 30 min postrace. Upper respiratory tract infections were assessed during the week before and the 2-wk period after the race using an illness symptom checklist. Race times did not differ significantly between quercetin and placebo groups. Significant pre- to postrace decreases were measured for natural killer cells (43 %), granulocyte respiratory burst activity (55 %), and salivary IgA output (48 %), and increases for neutrophil (288 %) and monocyte (211 %) cell counts, with no significant group differences. Postrace illness rates did not differ between groups. In conclusion, quercetin supplementation for 3 wks before and 2 wks after the Western States Endurance Run had no effect on illness rates, perturbations in leukocyte subset counts, or decreases in granulocyte respiratory burst activity and salivary IgA.

The Effect of Ageing on Modulation of Immune Response

P-338 Introduction: In the context of a cross-sectional molecular epidemiology study, the possible immunomodulatory effects of ageing were examined. While the effects of ageing on basic leukocyte subsets have been examined, studies aimed at more detailed information on delicate balance of expression of signal molecules and lymphocyte immune functions have been limited. Methods: Immune parameters were examined in 140 older (born 1932–1937) and 140 younger people (born 1977–1982). Immune function assays–proliferative response of lymphocytes to mitogens and antigens and phagocytic activity and respiratory burst of neutrophils were performed. Analysis of activation marker CD69 and costimulatory molecule CD28 on leukocytes was evaluated using flow cytometry. Results: Proliferative activity of T-lymphocytes in vitro in cell cultures derived from older people was significantly decreased (23530±1107) in comparison with younger people (49302±2044; p<0.001). Significant age-related differences in T-cell function were recorded equally in population of men and women (p<0.001, p<0.001). Regardless the age, women had lower cell response in comparison to men (women 34789±1858 vs. men 39722±2078; p<0.01). Older people had significantly suppressed also T-dependent B-cell response in total population (p<0.01) and group of women (older women 4713±224 vs. younger women 5792±313; p<0.01). Moreover, basal cell proliferation was significantly diminished in older people (2049± 94) in comparison with younger population (2602±115; p<0.001). Significant differences were seen in women (older 1999±101 vs. younger 2541±162; p<0.01 as well as men group (older 2180±218 vs. younger 2666±164; p<0.01). Surprisingly, increased percentage of phagocytic activity of monocytes, granulocytes and respiratory burst of granulocytes was found in older population (p<0.001) in comparison with younger group. Decrease in basal values of expression of activation marker CD69 on leukocytes was observed in older population and in smokers (p<0.01). Older people had also significantly decreased expression of costimulatory molecule CD28 on lymphocytes in all groups; on granulocytes in older people and women (p<0.05). Discussion and Conclusions: Our findings showed marked changes in proliferative function of lymphocytes that may contribute to modulation of immune response to antigen. Decreased expression of activation marker CD69 on cells may be first signal of early activation impairment. It is known, that combining of receptor CD28 with ligand CD95 may protect receptor-dependent cell apoptosis. Depletion in expression of costimulatory molecule CD28 may modulate increased cell susceptibility to apoptosis in older population.

Glycosyl‐phosphatidyl‐inositol‐defective granulocytes from paroxysmal nocturnal haemoglobinuria patients show increased bacterial ingestion but reduced respiratory burst induction

Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by the emergence of a GPI-defective clonal hematopoiesis. Its clinical features are hemolytic anemia, cytopenia, and thrombosis. Circulating monocytes and granulocytes are largely GPI-defective in PNH patients. This study aims to investigate the granulocyte functional properties in PNH. We analyzed bacterial-dependent intracellular ingestion and the consequent activation of oxidative burst in GPI-defective granulocytes from four neutropenic PNH patients. Our data show a significant increase in the ability of GPI-defective granulocytes to ingest opsonized bacteria. In addition, an impaired respiratory burst effectiveness in response to two independent bacterial stimuli, the N-formyl-MetLeuPhe (fMLP) synthetic bacterial peptide and E. coli, was revealed. The occurrence of neutropenia and the severe impairment of oxidative burst, occurring in chronic granulomatosis disease, were unable to significantly affect phagocytosis. Thus, additional mechanisms, able to differentially affect ingestion ability and respiratory burst effectiveness, have to be hypothesized. The reduced burst effectiveness of GPI-defective granulocytes was maintained after treatment with phorbol 12-myristate 13-acetate, a pharmacological stimulus able to extensively recruit and to trigger intracellular protein kinase C (PKC). Moreover, blocking of PKC has been observed to severely affect granulocyte respiratory burst with a mild effect on the phagocytosis. These data suggest a role for a modulation of intracellular PKC in the pathogenesis of the impaired granulocyte oxidative burst.

Conditional haploinsufficiency of NCF1 (encoding p47 phox), a signaling gene with a heterozygous phenotype potentially subject to natural selection

Even a minor degree of haploinsufficiency could eventually reduce the frequency of an autosomal immunodeficiency disease. Searching for such a condition, we have re-examined the phenotype of mice +/− for the NCF1 gene encoding p47 phox and humans +/− for NCF1 and NCF2 using a procedure that allowed the respiratory burst of granulocytes and macrophages to be measured simultaneously. The mice showed significant haploinsufficiency in granulocytes but not in macrophages (i.e. conditional haploinsufficiency). Our human data were obtained from blister cells, and were too scattered to allow a firm conclusion. In view of recent re-evaluation of the role of the respiratory burst these findings are compatible with the view that haploinsufficiency occurs particularly among rate-limiting genes that operate in regulatory/signaling pathways.

Activation of the Cation Channel Long Transient Receptor Potential Channel 2 (LTRPC2) by Hydrogen Peroxide: A SPLICE VARIANT REVEALS A MODE OF ACTIVATION INDEPENDENT OF ADP-RIBOSE

LTRPC2 is a cation channel recently reported to be activated by adenosine diphosphate-ribose (ADP-ribose) and NAD. Since ADP-ribose can be formed from NAD and NAD is elevated during oxidative stress, we studied whole cell currents and increases in the intercellular free calcium concentration ([Ca(2+)](i)) in long transient receptor potential channel 2 (LTRPC2)-transfected HEK 293 cells after stimulation with hydrogen peroxide (H(2)O(2)). Cation currents carried by monovalent cations and Ca(2+) were induced by H(2)O(2) (5 mm in the bath solution) as well as by intracellular ADP-ribose (0.3 mm in the pipette solution) but not by NAD (1 mm). H(2)O(2)-induced currents developed slowly after a characteristic delay of 3-6 min and receded after wash-out of H(2)O(2). [Ca(2+)](i) was rapidly increased by H(2)O(2) in LTRPC2-transfected cells as well as in control cells; however, in LTRPC2-transfected cells, H(2)O(2) evoked a second delayed rise in [Ca(2+)](i). A splice variant of LTRPC2 with a deletion in the C terminus (amino acids 1292-1325) was identified in neutrophil granulocytes. This variant was stimulated by H(2)O(2) as the wild type. However, it did not respond to ADP-ribose. We conclude that activation of LTRPC2 by H(2)O(2) is independent of ADP-ribose and that LTRPC2 may mediate the influx of Na(+) and Ca(2+) during oxidative stress, such as the respiratory burst in granulocytes.

Ortho-substituted polybrominated biphenyls activate respiratory burst in granulocytes from humans

The in vivo consequences of exposure to polychlorinated biphenyls (PCB) have been reported to involve reduced phagocytic function, which could be related to increased susceptibility to infections. Though less abundant in the environment, polybrominated biphenyls (PBB) have similar toxicological properties as PCB. In this respect the effect of different PBBs on human granulocytes was elucidated. Ortho-substituted PBBs activated respiratory burst, measured by the chemiluminescence assay, and elevated intracellular calcium. The most active polybrominated congener 2,2′,5-TBB increased chemiluminescence in a concentration-dependent manner, and ED 50 was approximately 10 μM. PBBs stimulated elevation of intracellular [Ca 2+] in human granulocytes. The [Ca 2+]i was elevated from 50 to 250 nM. The respiratory burst due to stimulation by PBBs was inhibited by U73122, ethanol (1%), wortmannin, and bisindolylmaleimide and by the elimination of extracellular calcium in the same way as shown previously for PCBs, indicating that PBB act by the same mechanisms.

Chemically de-acetylated 2′,7′-dichlorodihydrofluorescein diacetate as a probe of respiratory burst activity in mononuclear phagocytes

2′,7′-dichlorodihydrofluorescein diacetate (H 2DCFDA) is a fluorogenic probe commonly used to detect cellular production of reactive oxygen species (ROS), for example in the respiratory burst of granulocytes and mononuclear phagocytes. This method depends on the de-acetylation of H 2DCFDA by cellular esterases, to form the oxidant-sensitive compound, 2′,7′-dichlorodihydrofluorescein (H 2DCF). Importantly, however, not all cells possess sufficient esterase activity to produce the H 2DCF needed for accurate measurement of ROS. In this study, we used chemically de-acetylated probe (H 2DCF) to assess the phorbol-ester-triggered respiratory burst of rainbow trout macrophages, which, like some mammalian mononuclear phagocytes, appear to have low probe-esterase activity. We compared this approach to the use of intact H 2DCFDA and the cytochrome c reduction assay. The H 2DCF and cytochrome c reduction assays gave similar portrayals of the kinetics of the macrophage respiratory burst, while H 2DCFDA did not. We therefore recommend the use of H 2DCF over H 2DCFDA for quantification of the production of reactive oxygen species. Additionally, we stress the need to test reaction buffers or culture media used with H 2DCF(DA) for their ability to oxidize the probe directly or indirectly. As an example, we have observed that tyrosine combined with ubiquitous metal contaminants of physiological buffers can result in high levels of oxidation, which may be incorrectly interpreted as cellular activity.

Genetic studies of three Japanese patients with p22-phox-deficient chronic granulomatous disease: detection of a possible common mutant CYBA allele in Japan and a genotype-phenotype correlation in these patients.

Chronic granulomatous disease (CGD) is a disorder caused by defects in the NADPH oxidase responsible for superoxide generation in phagocytes. Cytochrome b558, an essential component of this enzyme, is a heterodimer formed by a 91 kDa glycoprotein (gp91-phox) and a 22 kDa polypeptide (p22-phox). Mutations in the p22-phox gene (CYBA) locus in 16q24 result in one of the rare autosomal recessive forms of CGD. We performed mutation analysis in three female CGD patients suspected of having this form of the disease and found two novel mutations in CYBA. Whereas patient 1 with severe phenotype had a homozygous nonsense mutation in exon 1 (C-35 --> T, Gln-3 --> stop), patients 2 and 3 with mild phenotype shared the same homozygous missense mutation in exon 2 (G-98 --> A, Gly-24 --> Arg). None of the parents of patients 2 and 3 is related. Therefore, this mutation could be a hot-spot or a common mutation in the Japanese population. Patients 2 and 3, but not patient 1, were demonstrated to have detectable p22-phox expression and significant granulocyte respiratory burst (ROB) activity. In this study, we were able to demonstrate an excellent correlation between genotype, p22-phox expression, ROB activity and clinical phenotype in these patients.

CNS granulomatosis in a child with chronic granulomatous disease

Chronic granulomatous disease (CGD) is a disease in which a granulomatous process involves various organ systems and in which recurrent infections are seen. The basic defect is the absence of the granulocyte respiratory burst. CNS involvement is rare. We present a report of a child with CGD and CNS involvement, presenting with hydrocephalus. Candida was identified in the granulomata. The patient responded well to a CSF shunt and antifungal therapy.

Cytokine Priming of the Granulocyte Respiratory Burst in Myelodysplastic Syndromes

Increased susceptibility to infections in patients with myelodysplastic syndromes (MDS) is thought to be due to neutropenia as well as functional abnormalities of neutrophils. In the present study we examined the effect of two different stimulants (NLP, PMA) and three cytokines (αTNF, G-CSF and GM-CSF), both singly and in combination on granulocyte (RB) in 25 MDS patients compared to seven healthy controls. Single fMLP and PMA-stimulation showed similar results for both groups. Preincubation with cytokines enhanced fMLP-stimu-lated RB in most MDS patients and controls, but in patients to a significantly lesser extent when compared to the control group (p ≤ 0,05). Combinations of αTNF + GM-CSF and αTNF + G-CSF were highly synergistic in priming NLP-stimulated burst in both groups. But again, as with the single cytokine priming this effect was markedly reduced in MDS patients compared to controls (p ≤ 0,05). A specific priming defect for one of the cytokines or a cytokine combination could not be demonstrated. Serum αTNF levels were measured in 18 and neu-trophil alkaline phosphatase (NAP) index in 23 patients. Results did not correlate with variations of the RB in MDS patients.We conclude that reduced aTNF, GM-CSF and G-CSF priming of granulocyte RB is a frequent finding in MDS and may contribute to the enhanced susceptibility to bacterial infections.