Quantitative determination of Fcgamma receptor genes by means of fluorescence-based real-time polymerase chain reaction.

The granulocyte antigens HNA-1a, -1b and -1c reside on the FcgammaIIIb receptor, which is exclusively expressed on neutrophils. They are involved in autoimmune and alloimmune neutropenias as well as in severe transfusion reactions. Recent family studies show the HNA-1c antigen to be inheritably linked to HNA-1a, resulting in individuals carrying three genes. Using sequence-specific primers, the HNA antigens can be discerned in a polymerase chain reaction (PCR), but not the number of coding FCGR3B genes present in each individual. Therefore a real-time kinetic PCR method using the LightCycler technique was established to ascertain the amount of FCGR3B genes in each individual. For this purpose, the FCGR3B genes of four HNA-1a,-1b,-1c (+), one HNA-1b,1c (+) and one HNA-1b (+) individual were quantified. In addition, two families, in which the mother was FCGR3B deficient, were analyzed. Quantification showed two of the four HNA-1a,-1b and-1c (+) individuals to have three genes and the other two to have only two genes. The HNA-1b,-1c (+) individual had also only two genes. As expected, the HNA-1b donor was typed for two genes. No FCGR3B genes could be detected in the FCGR3B-deficient mothers of either family; their husbands however, carried two FCGR3B genes. Accordingly, quantitative PCR showed the offspring of both families to have only one FCGR3B gene. Quantitative PCR with the Light Cycler has been revealed to be a fast and reliable method for the determination of FCGR3B genes present. FCGR3B quantification confirms the idea of the HNA-1c antigen to be inherited not only linked to HNA-1a, but also to be passed down on its own.

Molecular basis of the neutrophil glycoprotein NB1 (CD177) involved in the pathogenesis of immune neutropenias and transfusion reactions.

The human granulocyte alloantigen NB1, recently clustered as CD177, is heterogenously expressed on neutrophils of 88-97% of healthy individuals. Since its molecular nature has remained unknown, we isolated NB1 glycoprotein from granulocyte lysate by immunoaffinity chromatography. MALDI-TOF mass spectrometry identified a 50,556 Da glycoprotein which was reduced to 43,069 Da after removal of N-linked carbohydrates. Following N-terminal amino acid sequencing and NB1-specific primer construction, rapid amplification of cDNA ends PCR yielded a 1,614-bp cDNA for NB1. COS-7 cells transfected with the cDNA expressed immunoreactive NB1 glycoprotein. A 1,311-bp sequence was identified to be the entire coding region. The 5' and 3' untranslated regions consist of 27 bp and 276 bp, respectively. The open reading frame codes for 437 amino acids of which the first 21 form the signal peptide. The remaining 416 residues form a N-terminal extracellular protein with two cysteine-rich domains, three N-linked glycosylation sites and short transmembrane and cytoplasmic segments including a glycosyl-phosphatidylinositol attachment (omega) site. Database searches revealed homology to Ly-6 (uPAR) domain, suggesting that NB1 belongs to urokinase plasminogen activator receptor/CD59/Ly-6 snake toxin superfamily.

HNA-1a, HNA-1b, and HNA-1c (NA1, NA2, SH) frequencies in African and American Blacks and in Chinese.

The granulocyte antigens HNA-1a, -1b, and -1c (formerly named NA1, NA2 and SH) which reside on the neutrophil FcgammaReceptor IIIb (FcgammaRIIIb) play a major role in immune neutropenias and pulmonary transfusion reactions. In an attempt to shed some light on the origin and history of these antigens we typed the DNA of Blacks from South Africa (n=99), and Ghana (n=27), of 56 African Americans, and of 138 Chinese from Taiwan for HNA-1a,-1b, and -1c antigens using polymerase chain reaction with sequence-specific primers (PCR-SSP). In African and American Blacks, the HNA-1b antigen was more frequent than HNA-1a (77 vs. 67% and 77 vs. 59%, respectively). In contrast, in Chinese HNA-1a was more frequent than HNA-1b (91 vs. 54%). We observed 3 individuals with FcgammaRIIIB deficiency among the 126 tested African Blacks indicating a higher frequency of FcgammaRIIIB deficiency in Blacks than the reported 0.1% in Europeans. In addition, the frequency of HNA-1c in African and American Blacks (38 and 23%, respectively) was higher than the reported 5% in Europeans. Among the 57 HNA-1c (+) Blacks, all were HNA-1b (+) but only 26 were HNA-1a (+) supporting the idea that the HNA-1c antigen is the result of an additional point mutation in the allele coding for HNA-1b. Recently, HNA-1a, -1b, and -1c (+) Europeans have been reported to have three distinct FcgammaRIIIB genes. Among 26 Blacks who had been typed HNA-1a,b,c (+) by PCR-SSP we identified only 7 having three FcgammaRIIIB genes by DNA sequencing. When we sequenced the DNA of 6 HNA-1a,b,c (+) Europeans we found 4 of the individuals had three FcyRIIIB genes. Therefore, we assume that in Africa the point mutation occurred first in the HNA-1b allele resulting in the HNA-1c allele and the FcgammaRIIIB gene duplication took place later.

Use of diagnostic categories in urinary cytology in comparison with the bladder tumour antigen (BTA) test in bladder cancer patients.

In recent years the use of diagnostic categories for extragenital cytology has increasingly been discussed as an approach to improve the quality of reports. Diagnostic categories reflect the adequacy of the materials for interpretation and the presence or absence of cancer cells. There is a tendency to add intermediate groups as qualifying probably malignant cases or findings associated with a serious cancer risk. Since 1971 we have added one of the following to the final diagnosis in all cases: unsatisfactory for cytological diagnosis, negative for cancer, repeat test suggested, suspicious of cancer, and positive for cancer. To evaluate whether diagnostic categories are useful for comparison of cytological results with those of an alternative test, cytological data were compared with the results of the Bard bladder tumour antigen (BTA) test in voided urine from 119 patients (76 with and 43 without bladder cancer). The diagnostic categories enabled us to calculate sensitivities and specificities of cytology based on different thresholds or decision levels. The BTA test had significantly higher sensitivity (79%) and lower specificity (60%) than urinary cytology with three different thresholds in cytology results (sensitivities: 16-43%, specificities: 81-100%). The present findings suggest that diagnostic categories improve comparison of cytologic results with those of alternative screening and diagnostic aids such as the BTA test.

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.

Immunohistochemical localization of the murine transferrin receptor (TfR) on blood-tissue barriers using a novel anti-TfR monoclonal antibody.

A novel monoclonal antibody (mAb), 8D3 (IgG2a), that specifically recognizes the murine transferrin receptor (TfR) was produced by immunizing a Lewis rat with a polyoma middle T oncogene-transformed endothelioma cell line. The 8D3 mAb was obtained by immunohistochemical screening for exclusive staining of vessels forming a blood-brain barrier (BBB), but not of other vessels. The anti-TfR mAb 8D3 recognizes the TfR also in FACS analysis and in western blots and should prove to be useful for affinity purification of the TfR. Whereas 8D3 brightly stains BBB-forming vessels in the central nervous system of mice, it does not stain the fenestrated capillaries within the choroid plexus and the circumventricular organs. In testis, where the blood-tissue barrier is located at the level of the Sertoli cells, the 8D3 mAb specifically stains Sertoli cells but not endothelial cells. Finally, in vitro, 8D3 does not interfere with iron uptake of lymphocytes as it does not influence their proliferation. Taken together, 8D3 represents a versatile new tool to study the tissue distribution of the murine TfR and TfR-mediated transcytosis across tissue barriers in the mouse.

Circulatory clearance of transfused antibody-sensitized red cells in an entirely allogenic rabbit model.

BACKGROUND: Antibodies against histocompatibility antigens on phagocytes abrogate their Fc gamma-receptor-mediated functions in vitro. Studies were carried out to determine whether this phenomenon also exists in vivo. METHODS: An allogenic blood group antibody was generated in rabbits. Red cells sensitized with this antibody were internalized in vitro by rabbit phagocytes. Another allogenic antibody specific for major histocompatibility complex (MHC) antigens of rabbits was produced. Plasma containing this antibody was infused into rabbits with phagocytes expressing the corresponding MHC antigens. Thereafter, the sensitized rabbit red blood cells were transfused into the rabbits. RESULTS: The following 3 phases of the circulatory clearance of transfused sensitized red cells could be observed when MHC antibodies were not given prior to the transfusion: 1. initial rapid clearance of the cells (t/2 = 1.7-3.3 min), 2. release of the cells back into the circulation after 0.5-24 h and 3. terminal slow clearance which was, however, faster than that with unsensitized cells. Two independent experiments carried out on the same recipient out of the 3 recipients analysed showed that the prior treatment of the recipient with MHC-alloantibodies extended the circulatory half-life of the sensitized red cells during the terminal phase from t/2 = 1 day to t/2 = 3 and 4 days, respectively. CONCLUSION: Antibodies against MHC antigens on phagocytes can reduce the Fc gamma-receptor-mediated immune elimination of sensitized red cells. This corresponds to the observation that many cases of morbus haemolyticus neonatorum show unexpectedly reduced levels of foetal red cell destruction and a good clinical outcome if mothers not only produce antibodies to Rh (D) but also to foetal MHC antigens.

Comparison of polymerase chain reaction and conventional culture for the detection of legionellas in hospital water samples.

A detection system for Legionella spp. based on the polymerase chain reaction (PCR) was used to assess the diagnostic value of PCR for the surveillance of contamination of man-made water systems by legionellas. A previously-published primer system was chosen to amplify a fragment of the 5S-ribosomal gene of Legionella spp. A total of 78 water samples from various sources were examined by PCR and culture on MWY Legionella selective agar. Fifty-seven of 78 water samples were positive by both test systems (73%), nine were positive by PCR only (11.5%), another nine were positive by culture but negative by PCR (11.5%), and three were negative by both techniques (3.8%). The PCR was inhibited when large amounts of rust were present in the samples. Culture failed to detect legionellas in samples that contained large numbers of other bacteria capable of overgrowing the legionellas. These results show that PCR is a rapid and sensitive technique for the detection of legionella contamination in water samples and that PCR and culture complement each other in monitoring of environmental water samples.

[Clinical importance of granulocyte-specific antibodies]. / Klinische Bedeutung von granulozytenspezifischen Antikörpern.

Clinical and laboratory data of 184 patients with immune neutropenia were evaluated. They suffered from autoimmune neutropenia (n = 165), alloimmune neonatal neutropenia (n = 18) and from transfusion-associated lung injury (n = 1). Autoimmune neutropenia was predominantly found in patients below 3 years. Patients were usually affected by benign bacterial infections. The peripheral blood count showed normal or diminished leukocyte counts with median absolute neutrophil counts of 285 cells/microliters. Bone marrow examination revealed in 60% of the cases a hypercellular marrow with a shift to the left. In 36% the bone marrow was normal and in 4% a hypocellular marrow was found. Spontaneous remission occurred in all newborns and, so far, in 4 patients with autoimmune neutropenia. Symptomatic treatment of the infections was sufficient in most of the patients.

Autoimmune neutropenia: clinical and laboratory studies in 143 patients.

Clinical and laboratory data of 143 patients with primary or secondary autoimmune neutropenia (AIN) were evaluated. Primary AIN was found predominantly in children below 3 years, whereas secondary AIN was more frequent in patients 40-60 years of age. Female patients with primary AIN were slightly more prevalent (54%) than male patients (46%). The peripheral blood count showed normal or diminished leukocyte counts with median absolute neutrophil counts of 250 cells/microliters. In 38% of the patients neutropenia was accompanied by monocytosis. Bone marrow examination revealed in 95% a normo- or hypercellular marrow with a marked reduction of mature neutrophils in 56% of the specimens. Twenty-three percent of the sera showed specificity for the NA1 antigen. Patients were usually affected by benign bacterial infections of the skin and of the upper respiratory tract, as well as by recurrent otitis media. Infections were treated symptomatically, and only six patients required continuous administration of antibiotics. Remission of neutropenia during treatment occurred in three of six patients treated with intravenous immunoglobulin G and in three of four patients who received steroid therapy. Except for one patient neutropenia relapsed after discontinuation of therapy. During a follow-up of 6-36 months, spontaneous remission has been observed in four patients.

[Serologic characterization of granulocyte-specific antibodies in neonatal alloimmune neutropenias]. / Serologische Charakterisierung von granulozytenspezifischen Antikörpern bei neonatalen Alloimmunneutropenien.

Serological properties of nine granulocyte specific alloantibodies in maternal sera and sera of the newborns were investigated by granulocyte immunofluorescence, granulocyte agglutination and granulocyte cytotoxicity. In the immunofluorescence assay the reactions were more prominent than in the agglutination assay. Only one antibody yielded a positive reaction in the cytotoxicity assay. Four NA2-, two NA1- and one NB1-antibody could be identified. The immunofluorescence assay showed a mixed pattern with the NB1-antibody, indicating a heterogenous distribution of the NB1-antigen on granulocytes of some donors. In titration studies with heterozygous and homozygous donors a gene-doses-effect was demonstrable.

Ex vivo antigen preparation for the serological detection of drug-dependent antibodies in immune haemolytic anaemias.

Two patients with severe, intravascular haemolysis due to drug-dependent antibodies are described. The antibodies were directed against presumptive metabolites of buthiazide (International Non-proprietary Name, butizide) and nomifensine. Their detection was possible only in the presence of ex vivo antigens (i.e. fresh serum of volunteers after ingestion of the drugs) while in vitro antigen preparations yielded inconclusive results. Both antibodies lysed normal red cells in the presence of ex vivo antigens and complement. The buthiazide-related antibody was IgG (subclass IgG1), the nomifensine-related antibody was IgM. We conclude that the use of ex vivo antigens is of great importance in the serological evaluation of cases with suspected drug-dependent immune haemolysis.