Kostmann disease and other forms of severe congenital neutropenia.

Congenital neutropenia with autosomal recessive inheritance was first described by the Swedish paediatrician Rolf Kostmann who coined the term 'infantile genetic agranulocytosis'. The condition is now commonly referred to as Kostmann disease. These patients display a maturation arrest of the myelopoiesis in the bone marrow and reduced neutrophil numbers and suffer from recurrent, often life-threatening infections. The molecular mechanism underlying congenital neutropenia has been intensively investigated, and mutations in genes that impinge on programmed cell death have been identified. The present review provides an overview of these studies.

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.

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.

Early recognition of reverse pseudohyperkalemia in heparin plasma samples during leukemic hyperleukocytosis can prevent iatrogenic hypokalemia.

OBJECTIVES: To investigate the cause of apparent hyperkalemia in leukemic heparin plasma. DESIGN AND METHODS: Lithium heparin plasma and serum samples from a patient with chronic lymphocytic leukemia (CLL) with hyperleukocytosis were transported by either a pneumatic tube system or manual transport and analyzed either immediately or after 4 h. RESULTS: Pneumatic tube transported samples resulted in higher plasma potassium levels than manually transported samples. Serum potassium was lower than plasma potassium, confirming the suspicion of "reverse" pseudohyperkalemia. Letting the pneumatic tube transported samples stand on the bench for 4 h before centrifugation surprisingly resulted in decreased or unchanged plasma potassium. CONCLUSIONS: The reverse pseudohyperkalemia in heparin plasma samples from a CLL patient was caused by pneumatic tube transport. Our results suggest extracellular leakage of potassium, followed by active transport of potassium into intact leukemic cells. This is the first Swedish case of reverse pseudohyperkalemia in a CLL patient, where clinical suspicion of false hyperkalemia and awareness of the phenomenon lead to a rapid laboratory diagnosis. The demonstration of reverse pseudohyperkalemia prevented potentially dangerous medical interventions, such as potassium lowering treatment.

Normal levels of constitutive and death receptor-mediated apoptosis of peripheral blood neutrophils from patients with chronic idiopathic neutropenia.

To investigate the role of neutrophil apoptosis in the pathogenesis of chronic neutropenia, we examined constitutive and death receptor-mediated apoptosis ex vivo of peripheral blood neutrophils obtained from six chronic idiopathic neutropenia (CIN) patients and six healthy adult blood donors. Apoptosis was quantified based on phosphatidylserine externalization and caspase-3 activation in freshly isolated neutrophils or after overnight cultivation of neutrophils in the absence or presence of pro- or anti-apoptotic factors, including the pan-caspase inhibitor, zVAD-fmk. Neutrophils from CIN patients receiving treatment with granulocyte colony-stimulating factor appeared to be more prone to constitutive apoptosis than cells from untreated patients; however, further investigations in larger cohorts of patients are needed to validate these pilot studies. Overall, the level of neutrophil apoptosis was similar in patient and control groups, thus supporting the notion that the underlying defect in these neutropenia patients lies elsewhere, such as in the bone marrow microenvironment.

The proximal promoter of the human cathepsin G gene conferring myeloid-specific expression includes C/EBP, c-myb and PU.1 binding sites.

Cathepsin G is a hematopoietic serine protease stored in the azurophil granules of neutrophil granulocytes. The mRNA of cathepsin G is transiently expressed during the promyelocyte stage of neutrophil maturation. The protease plays several roles in inflammatory actions of neutrophils, such as bactericidal effects. A human cathepsin G gene fragment of 6 kb directs a promyelocyte-specific expression in transgenic mice, indicating the presence of necessary cis-acting elements. However, neither the precise architecture of the promoter, nor the trans-acting factors responsible for its activation, have been characterized. In the present work, 2.6 kb upstream of the translation start site of the human cathepsin G gene was cloned. When transfected to monoblast-like U937 or to acute promyelocytic leukemia NB4 cells, both expressing endogenous cathepsin G, the initial 360 bp upstream of the translation start were sufficient to direct a strong expression of a luciferase reporter gene. No expression was observed in erythroid K562 control cells. Further deletions revealed three major regulatory regions containing the consensus binding-sites for the transcription factors C/EBP, c-myb and PU.1. Moreover, a GC-rich region, similar to a cis-element in the proteinase 3 promoter, was identified. Direct binding of the trans-factors C/EBPalpha, C/EBPepsilon, c-myb and PU.1 to the promoter was shown by chromatin immunoprecipitation. The functional significance of the cis-elements was verified by site-directed mutagenesis. Mutations of the putative PU.1 site moderately decreased the activity of the promoter in monoblastic U937 cells, but not in promyelocytic NB4 cells. Separate mutations of the putative C/EBP binding site, c-myb-binding site or the GC-rich element resulted in a dramatically reduced transcriptional activity in both cell lines, suggesting cooperation between corresponding trans-factors.

Biosynthetic profiles of neutrophil serine proteases in a human bone marrow-derived cellular myeloid differentiation model.

BACKGROUND AND OBJECTIVES: Human leukocyte elastase, proteinase 3 and cathepsin G are neutrophil granule proteins belonging to the hematopoietic serine protease superfamily. In addition to their established roles in inflammation, they have recently been implicated as regulators of granulopoiesis and mediators of apoptosis. We set out to characterize the individual biosynthetic profiles of these proteins in a neutrophil differentiation model. DESIGN AND METHODS: CD34+CD38+ hematopoietic progenitor cells from 21 healthy human bone marrow donors were cultured in vitro in the presence of recombinant human granulocyte colony-stimulating factor (G-CSF). Biosynthetic radiolabeling was performed in cells from 13 subjects after various periods of differentiation induction. Following protein extraction, the proteins were specifically immunoprecipitated from cell lysates and media and run in gel electrophoresis. Biosynthetic profiles of azurophil granule proteins, in particular members of the neutrophil serine protease family, were examined during myeloid differentiation. RESULTS: The onset of synthesis of myeloperoxidase, lysozyme, leukocyte elastase, and proteinase 3 occurred early after differentiation induction with G-CSF, while synthesis of cathepsin G, azurocidin, and bactericidal/permeability-increasing protein was detected somewhat later. Cathepsin G and proteinase 3 were retained intracellularly relatively efficiently, while leukocyte elastase and lysozyme were secreted to a greater extent. Cell morphology and positive immunocytochemistry for lactoferrin as well as flow cytometric analysis of selected surface antigens confirmed neutrophil-like maturation. INTERPRETATION AND CONCLUSIONS: We demonstrate that azurophil granule proteins, including proforms of human leukocyte elastase, proteinase 3 and cathepsin G, are constitutively secreted to various degrees during in vitro myeloid differentiation of human hematopoietic progenitor cells, in addition to being stored intracellularly in active forms. These findings suggest protein-specific sorting mechanisms and may have implications for the regulation of granulopoiesis.