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J Bux

Publications and source records attributed to J Bux.

At least 37 records · Page 2Linked to original sources
PubMed2000

Serum granulocyte colony-stimulating factor levels are not increased in patients with autoimmune neutropenia of infancy.

OBJECTIVE: To assess the role of granulocyte colony-stimulating factor (G-CSF) in autoimmune neutropenia (AIN). DESIGN: Serum G-CSF levels were measured in 57 children with AIN. Two different G-CSF-dependent assays were used: a solid-phase "sandwich" enzyme-linked immunosorbent assay and a proliferation assay. Sera from healthy persons and from patients with severe congenital neutropenia were used for negative and positive controls. RESULTS: The median G-CSF level in healthy persons (n = 13) was low, 45.6 pg/mL (range <39 to 141 pg/mL). The median G-CSF level in patients with AIN (n = 57) was very similar, 45.5 pg/mL (range <39 to 2500 pg/mL). Forty-five (79%) of 57 patients with AIN had levels within the range of the control group. Seven (12%) had marginally increased G-CSF levels (141 to 400 pg/mL), and only 5 (9%) had levels higher than 400 pg/mL. The G-CSF levels measured by enzyme-linked immunosorbent assay correlated well with levels measured by the proliferation assay, thus demonstrating that antibodies present in patient sera did not affect the biologic activity of G-CSF. CONCLUSION: G-CSF production in AIN is not increased despite the low neutrophil count, similar to thrombopoietin in immune thrombocytopenic purpura.

Autoimmune Diseases↗
PubMed2000

Molecular genetics of granulocyte polymorphisms.

BACKGROUND AND OBJECTIVES: Granulocyte (neutrophil) antibodies have been implicated in transfusion-related acute lung injury (TRALI) and immune neutropenias. In the last decade enormous efforts have been made to characterize the involved alloantigens. MATERIALS AND METHODS: Review of the literature. RESULTS: The NA1, NA2, and SH antigens have been identified as polymorphic forms of the neutrophil Fc gamma receptor IIIb encoded by three alleles. The antigens MART and OND have been located on leucocyte adhesion molecules (beta 2 integrins) and were found to be due to single nucleotide mutations in the alpha M (CD11b) and alpha L (CD11a) subunits. Recently, we succeeded in throwing light on the primary structure of the NB1 antigen. On the basis of these findings a new nomenclature has been introduced for human neutrophil alloantigens (HNA nomenclature). Glycoprotein location has made the development of antigen-specific immunoassays possible and antigen characterization on the molecular level now allows genotyping by DNA techniques. CONCLUSION: Considerable progress has been made in the characterization of granulocyte antigens. Further studies will improve our diagnostic tools and will facilitate the prevention and management of transfusion reactions and immune neutropenias.

Gene Frequency↗
PubMed1999

The use of allele-specific recombinant Fc gamma receptor IIIb antigens for the detection of granulocyte antibodies.

The Fcgamma receptor IIIb (FcgammaRIIIb) for the Fc domain of IgG is expressed exclusively on neutrophils. The FcgammaRIIIb bears allotypic polymorphisms referred to as NA1, NA2, and SH, which are known for their frequent involvement in alloimmune and autoimmune neutropenias as well as in transfusion reactions. The bactericidal capacity of isolated neutrophils is easily activatable, and activation results in self-desintegration, thus preventing storage of neutrophils. As a result, only freshly isolated granulocytes can be used for antibody screening, often making it impossible to use typed panel cells. To provide a readily available source of typed panel cells, we therefore established stable mammalian cells expressing recombinant NA1, NA2, and SH antigens. We isolated mRNA from typed neutrophils and then transcribed it in cDNA. The cDNA that codes for the different forms of the FcgammaRIIIb was amplified by polymerase chain reaction and was subsequently subcloned into the mammalian expression vector pcDNA3. Chinese hamster ovary (CHO) cells were transfected with allele-specific constructs, and stable cell lines expressing FcgammaRIIIb were selected by flow cytometry. Because human sera show high background fluorescence with transfectants in flow cytometry, the monoclonal antibody-specific isolation of granulocyte antigens (MAIGA) assay was performed. By MAIGA assay, we tested 14 well-characterized human alloantibodies directed against the antigens NA1, NA2, and SH; 5 FcgammaRIIIb-specific isoantibodies; and 12 FcgammaRIIIb-reactive autoantibodies. Except one NA1- and one SH-specific alloantibody, all other antibodies could be identified by the use of CHO transfectants. In contrast to neutrophils, fixed CHO cells can be stored at 4 degrees C for at least 4 weeks or stored frozen for a longer period. This longer shelf life of the transfected CHO cells compared with isolated neutrophils will simplify the detection of the clinically most important FcgammaRIIIb-reactive alloantibodies and autoantibodies.

Alleles↗
PubMed1999

Serum G-CSF levels are not increased in patients with antibody-induced neutropenia unless they are suffering from infectious diseases.

By the use of a G-CSF-specific ELISA we determined the serum granulocyte-colony stimulating factor (G-CSF) levels in 63 patients with antibody-induced neutropenia including neonatal immune neutropenia, autoimmune neutropenia, and drug-induced immune neutropenia. In the sera of 20 patients, elevated G-CSF levels of 60-1006 pg/ml (normal <39 pg/ml) were observed. These patients suffered from infectious diseases at the time of blood collection. G-CSF levels normalized after successful antibiotic treatment, indicating that increased G-CSF production in patients with immune neutropenia may be primarily the result of infection and not of neutropenia.

Adult↗
PubMed1998

Diagnosis and clinical course of autoimmune neutropenia in infancy: analysis of 240 cases.

Primary autoimmune neutropenia (AIN) is caused by granulocyte-specific autoantibodies and occurs predominantly in infancy. Clinical presentation and diagnosis have not been well established, resulting in burdening diagnostic investigations and unnecessary treatment with granulocyte colony-stimulating factor (G-CSF). In the present study, clinical, laboratory, and immunologic data of 240 infants with primary AIN were evaluated. Suspected association with parvovirus B19 infection was investigated using serologic and DNA-based methods. Primary AIN was mainly diagnosed at the age of 5 to 15 months but was observed as early as day 33 of life. In 90% of the cases, AIN was associated with benign infections despite severe neutropenia. Spontaneous remission, shown by 95% of the patients, usually occurred within 7 to 24 months. Autoantibodies in the patient's sera were not always present, and screening had to be repeated several times until antibody detection succeeded. About 35% of the autoantibodies showed preferential binding to granulocytes from NA1 and NA2 homozygous donors. Bone marrow was typically normocellular or hypercellular, with a variably diminished number of segmented granulocytes. A significant association with parvovirus B19 infection was not found. Symptomatic treatment with antibiotics was sufficient in most patients. Eighty-nine percent of the patients received antibiotics (cotrimoxazole) for prophylaxis of infections. For severe infections or for surgical preparation, G-CSF, corticosteroids, and intravenous IgG were administered, resulting in increased neutrophil counts in 100%, 75%, and 50% of the patients treated, respectively. In combination with the detection of granulocyte-specific antibodies, the typical clinical picture allowed diagnosis of AIN without burdening investigations. Treatment with G-CSF was found to be a reliable alternative to temporarily increase the neutrophil count.

Adrenal Cortex Hormones↗
PubMed1997

Characterization of a new alloantigen (SH) on the human neutrophil Fc gamma receptor IIIb.

Polymorphic structures of the neutrophil Fc gamma receptor IIIb (Fc gamma RIIIb) result in alloantibody formation that causes alloimmune neonatal neutropenia and transfusion reactions. Alloantigens located on Fc gamma RIIIb include the antigens NA1 and NA2. In four cases of alloimmune neonatal neutropenia, granulocyte-specific alloantibodies directed against a thus far unknown antigen were detected by granulocyte agglutination and immunofluorescence tests in the maternal sera. By the use of the monoclonal antibody-specific immobilization of granulocyte antigens (MAIGA) assay, the new antigen, termed SH, was located on the Fc gamma RIIIb. Nucleotide sequence analysis of the Fc gamma RIIIb coding region from a SH(+) individual showed a single-base C-->A mutation at position 266, which results in an Ala78Asp amino acid substitution. A family study confirmed that this nucleotide difference is inherited, and corresponds to the SH phenotype. Serologic typing of 309 randomly selected individuals showed an antigen frequency of 5% in the white population. The same frequency was found by genotyping, for which a technique based on polymerase chain reaction (PCR) using sequence-specific primers (PCR-SSP) was developed. Typing of all SH(+) individuals for NA1 and NA2, and PCR-restriction fragment length polymorphism analysis of the NA-specific PCR products from five SH(+) individuals using the SH-specific endonuclease SfaN 1 showed that SH antigen is very probably the result of an additional mutational event in the NA2 form of the Fc gamma RIIIB gene. Immunochemical studies also demonstrated that the SH determinants reside on the 65- to 80-kD NA2 isoform of the Fc gamma RIIIb. Our findings show the existence of an additional polymorphism of the Fc gamma RIIIb, which can result in alloantibody formation causing alloimmune neonatal neutropenia.

Adult↗
PubMed1997

Report on the second international granulocyte serology workshop.

BACKGROUND: The methods of granulocyte antibody and antigen detection have improved considerably since the First International Granulocyte Serology Workshop. STUDY DESIGN AND METHODS: Thirteen laboratories participated in the Second International Granulocyte Serology Workshop. The study was designed to meet five goals: 1) establishment of antigen-typed granulocyte panels, 2) determination of the proficiency of granulocyte antibody detection by laboratory and by technique, 3) identification of granulocyte antibodies present in uncharacterized sera, 4) genotyping of NA antigens by DNA-based techniques, and 5) collection and exchange of information to standardize granulocyte antibody screening. RESULTS: NA1-, NA2- and NB1-specific antibodies were detected by more than two-thirds of the participants with the granulocyte immunofluorescence test (GIFT) but by fewer than two-thirds with the granulocyte agglutination test (GAT). The determination of the NB2-, 5b-, and Fc gamma RIIIb-specific antibodies was the most problematic. Ninety percent of the participants were not able to identify NB2 antibodies in sera with suspected NB2 specificity. The 5b antibodies were only detected by laboratories that performed the GAT. Isoantibodies to Fc gamma RIIIb were identified when the monoclonal antibody-specific immobilization of granulocyte antigens (MAIGA) assay or an Fc gamma RIIIb-deficient panel cell was available. In 4 of 11 uncharacterized sera, the presence of NA1, NA2, and Fc gamma RIIIb antibodies could be confirmed by the MAIGA assay. In polymerase chain reaction with sequence-specific primers (PCR-SSP), the NA genotypes of all DNA samples were correctly determined. CONCLUSION: A combination of the GIFT and GAT is still the best means of antibody detection. The MAIGA assay allows identification of isoantibodies to Fc gamma RIIIb or alloantibodies to NA antigens. The NA genotype can be reliably determined by PCR-SSP. Cell panels should cover NA1, NA2, NB1, 5b, and, if possible, SH.

Antigens, CD↗
PubMed1996

Transfusion-related acute lung injury due to HLA-A2-specific antibodies in recipient and NB1-specific antibodies in donor blood.

Transfusion-related acute lung injury (TRALI) is a hazardous but little-known complication of blood transfusion, characterized by non-cardiogenic lung oedema after blood transfusion. Leucoagglutinating antibodies in the donor plasma are considered to play a central role in the pathogenesis of TRALI but no recommended procedure currently exists for their detection, and most of them have not yet been well characterized. Serum samples of two patients who have developed TRALI within 30 min of blood transfusion and the sera of the involved blood donors were investigated for leucocyte antibodies by granulocyte immunofluorescence, granulocyte agglutination and lymphocytotoxicity assays using typed test cells. Suspected specificities of the detected antibodies were confirmed by a luminoimmunoblot assay and the antigen capture assay MAIGA. One case was associated with granulocyte agglutinating anti-HLA-A2 antibodies in the recipient's (i.e. patient's) own blood and the other with donor-related non-agglutinating antibodies directed against the granulocyte-specific antigen NB1. Leucocyte incompatibility between donor and recipient was shown in both cases by crossmatching and typing of the incompatible cells for the appropriate antigen. The results show that TRALI is associated not only with donor- but also with recipient-related leucocyte antibodies. In addition to leucoagglutinating antibodies, non-agglutinating granulocyte-specific antibodies can be also involved. For immunodiagnosis, sera from both must be investigated by a combination of granulocyte and lymphocyte (HLA) antibody screening tests and leucocyte incompatibility verified by crossmatching.

GPI-Linked Proteins↗
PubMed1995

NA gene frequencies in the German population, determined by polymerase chain reaction with sequence-specific primers.

BACKGROUND: The granulocyte antigens NA1 and NA2 are often targets of granulocyte antibodies causing immune neutropenia. Currently, NA typing relies on the properties of the typing sera or antibodies and the techniques used. Therefore, the technique of polymerase chain reaction with sequence-specific primers (PCR-SSP) was adapted for DNA-based NA typing and was used for determining the NA gene frequencies in the German population. STUDY DESIGN AND METHODS: The genomic DNA of 160 unrelated healthy individuals was typed for NA1 and NA2 by PCR-SSP. In 60 granulocyte samples, the NA phenotype was additionally determined by the antigen capture assay and the granulocyte immunofluorescence test (GIFT) and correlated with the genotyping results. RESULTS: Results of the antigen capture assay and PCR-SSP correlated precisely, whereas nine individuals were typed heterozygous only by GIFT. The gene frequencies were 0.35 for NA1 and 0.65 for NA2. CONCLUSION: The NA2 gene is more frequent in the German population than the NA1 gene, as determined by genotyping using PCR-SSP. In contrast to GIFT, which showed an error rate for NA typing of 15 percent, PCR-SSP and the antigen-capture assay are more reliable methods of NA typing of granulocytes.

Base Sequence↗
PubMed1995

Genotyping of the granulocyte-specific NA antigens from small quantities of blood or serum.

To avoid the well-known shortcomings of phenotyping granulocytes for the NA antigens using NA-specific human sera, a DNA-based method to determine the NA genotype was developed. Genomic DNA was isolated from blood cells or serum, amplified by polymerase chain reaction (PCR), immobilized on nylon membrane and genotyped using digoxigenin-labeled, sequence-specific oligonucleotides (SSO). The genotyping results of whole blood samples from 54 and of serum from 20 individuals correlated perfectly with our phenotyping using the antigen capture assay MAIGA. In three cases with the phenotype "NA-null" no hybridization of the NA-specific oligonucleotides occurred. These data show that SSO is a reliable method for NA genotyping especially if only small volumes of blood or even only serum probes are available.

Base Sequence↗
PubMed1995

Evidence that the granulocyte-specific antigen NC1 is identical with NA2.

The neutrophil-specific antigen NC1 is defined by an antibody in the serum of a mother who gave birth to a child with alloimmune neonatal neutropenia. NC1 has been reported to be associated with the neutrophil-specific antigen NA2, but the precise relation of NC1 and NA2 remained unclear. Therefore, we investigated the serum using the antigen capture assay MAIGA and the granulocyte (GIFT) and lymphocyte (LIFT) immunofluorescence tests. In GIFT, no NA association was observed. In LIFT, serum antibodies bound preferably to lymphocytes with the HLA antigens HLA-B7 and cross-reacting antigens. In MAIGA, an antibody specific for the NA2 variant of the granulocyte Fc gamma-receptor III was observed. The NA2 specificity was confirmed by testing granulocytes from 40 further different donors. This indicates that the NC1 and NA2 antigens are identical. A positive GIFT result but a negative one in LIFT using cells of an NA2-negative typed individual suggest the presence of an additional, non-NA2-specific granulocyte antibody.

Antibodies, Monoclonal↗
PubMed1994

[Transfusion associated acute pulmonary insufficiency. Diagnostic confirmation by the demonstration of granulocytic antibodies].

In two patients, a 50-year-old woman (case 1) and a 2-year-old girl (case 2) acute shortness of breath requiring artificial ventilation developed 3-5 hours after infusion of two erythrocyte concentrates in case 1 and unfiltered platelet-enriched plasma (20 ml/kg) in case 2. The chest radiograph showed diffuse infiltrations in the lungs of both patients. After administration of catecholamines and respirator therapy, extubation was possible in the first patient after two days, in the second after five days. In neither case had there been any evidence of hypervolaemia, heart failure or infection to explain the lung findings. However, the serum of case 1 and the serum of the platelet donor had antibodies against granulocytes. The granulocyte-compatibility test (between patient serum and donor granulocytes) was positive. This confirmed the clinical suspicion of transfusion-related acute lung injury (TRALI). However, demonstration of antibodies was not only important for the diagnosis, but made it possible to recognize a blood donor whose serum contained antibodies against granulocytes which can provoke TRALI.

Catecholamines↗
PubMed1994

Alloimmune neonatal neutropenia resulting from immunization to a high-frequency antigen on the granulocyte Fc gamma receptor III.

BACKGROUND: Alloimmune neonatal neutropenia is mainly caused by NA- or NB1-specific alloantibodies. An antibody in the serum of a Turkish mother who had given birth to an infant with alloimmune neonatal neutropenia showed no NA or NB specificity and was therefore investigated further. STUDY DESIGN AND METHODS: The number of antibody-binding sites was calculated by determination of elutable IgG from granulocytes using a quantitative sandwich enzyme-linked immunosorbent assay. Complement activation was tested by immunofluorescence (C3d) and cytotoxicity tests. The antigen was identified using the antigen-capture assay, monoclonal antibody-specific immobilization of granulocyte antigens, and a modified immunoprecipitation method based upon biotinylation of proteins and visualization by luminescence (luminoimmunoprecipitation). Family study and determination of antigen frequency were done by immunofluorescence and agglutination tests. RESULTS: A noncytotoxic, granulocyte-specific alloantibody that recognized the Fc gamma receptor III, independent of the NA phenotype, was detected, and 242,000 binding sites per cell were calculated. Of granulocytes from 150 randomly selected German blood donors, the alloantibody bound to all. The maternal cells were typed NA1/NA2- and NB1-positive. CONCLUSION: These data reveal the presence of a previously unrecognized, high-frequency epitope on the granulocyte Fc gamma receptor III. Luminoimmunoprecipitation proved to be a simple, nonradioactive technique that was useful in identifying the molecule involved.

Antigens↗
PubMed1994

Typing of the granulocyte-specific NA antigens by restriction fragment length polymorphism analysis.

An RNA-based method has been developed to genotype donors for the granulocyte-specific alloantigens NA1 and NA2. mRNA was isolated from granulocytes, reversely transcribed into cDNA and amplified using an Fc-gamma-receptor III-1 sequence-specific primer in the polymerase chain reaction (PCR). PCR products were analysed by restriction fragment length polymorphism (RFLP) using the restriction endonuclease Taq I, which provided a distinct restriction fragment pattern corresponding to the NA alleles. 17 donors were typed by PCR-RFLP and the results were in close accordance with those obtained by serological phenotyping by granulocyte immunofluorescence and the antigen capture assay MAIGA.

Antigens, Surface↗
PubMed1994

[RNA typing of granulocyte NA antigens by means of PCR-RFLP analysis].

An RNA-based method was developed for genotyping of the granulocyte-specific alloantigens NA1 and NA2, mRNA was isolated from neutrophils and reversely transcribed into cDNA. To avoid coamplification of Fc-gamma receptor III-2 in the polymerase chain reaction (PCR), a Fc-gamma receptor III-1 sequence-specific primer was employed. Restriction analysis using Taq I led to NA-specific restriction fragment length polymorphism (RFLP) of PCR-amplified cDNA. The results of the PCR-RFLP typing were in good concordance with serological phenotyping by granulocyte immunofluorescence test and the antigen capture assay MAIGA.

Granulocytes↗