A. E. G. K. Von Dem Borne

Soluble Fc gamma receptor III in human plasma originates from release by neutrophils.

FcRIII (the CD16-antigen), a low affinity receptor for IgG, is expressed by neutrophils, natural killer lymphocytes, and macrophages. We have developed a sensitive radioimmunoassay to quantify FcRIII. A soluble form of FcRIII was identified in human plasma. Immunoprecipitation of FcRIII from plasma showed that the plasma form of FcRIII has an identical electrophoretic mobility as the FcRIII expressed by neutrophils. Moreover, the plasma form of FcRIII exhibited the same polymorphism as does the neutrophil FcRIII. The neutrophil expresses the phosphatidylinositol-linked form of FcRIII, encoded by the gene FcRIII-1. Because it is not known whether this gene is also active in nonhematopoietic cells, we analyzed patients with an acquired clonal disorder of their hematopoietic cells, paroxysmal nocturnal hemoglobinuria (PNH). PNH patients appeared to have a strongly reduced expression of FcRIII on their neutrophils. The concentration of FcRIII in the plasma of these patients was also reduced, indicating that plasma FcRIII originates from neutrophils. A patient deficient in FcRIII-1 but with a normal expression of FcRIII-2 had no soluble FcRIII in her plasma, also indicating that plasma FcRIII originates from neutrophils. The electrophoretic mobility of the protein backbone of plasma FcRIII and FcRIII released by activated neutrophils was identical, whereas deglycosylated FcRIII obtained from a lysate of neutrophils migrated slower. This indicates that plasma FcRIII originates from activation-induced release by neutrophils. Stimulation of neutrophils or neutrophil cytoplasts (closed membrane vesicles filled with cytoplasm) with low concentrations of FMLP (10(-9)-10(-8) M) or phorbol myristate acetate (1-10 ng/ml) induced a dose-dependent release of FcRIII. The plasma concentration of FcRIII was relatively constant (range 40-280% of the mean). Soluble FcRIII was also detected in inflamed joint fluids of arthritis patients, suggesting that FcRIII is also released by activated neutrophils in vivo.

The VS and V blood group polymorphisms in Africans: a serologic and molecular analysis

BACKGROUND: VS and V are common red cell antigens in persons of African origin. The molecular background of these Rh system antigens is poorly understood.

Subsets of CD34+ Cells and Rapid Hematopoietic Recovery After Peripheral-Blood Stem-Cell Transplantation

PURPOSE To study whether there is a relationship between transplanted cell dose and rate of hematopoietic recovery after peripheral-blood stem-cell (PBSC) transplantation, and to obtain an indication whether specific subsets of CD34+ cell populations contribute to rapid recovery of neutrophils or platelets. PATIENTS AND METHODS Based on data from 59 patients, we calculated for each day after PBSC transplantation the dose of CD34+ cells that resulted in rapid recovery of either neutrophils or platelets in the majority (> 70%) of patients. Using dual-color flow cytometry, subsets of peripheral-blood CD34+ cells were quantified and the numbers of CD34+ cells belonging to each of the reinfused subsets correlated with hematopoietic recovery following high-dose chemotherapy. RESULTS The calculated threshold values with a high probability of engraftment showed a steep dose-effect relationship between CD34+ cell dose and time to recovery of both neutrophils or platelets. Predominantly CD34+ cells with the phenotype of myeloid precursors were mobilized. A minority of CD34+ cells expressed the erythroid and megakaryocytic lineage-associated antigens and a low but distinct population of CD34+ cells expressed antigens associated with multipotent stem cells. Analysis showed that the number of CD34+CD33- cells (r = -.74, P < .05), as well as the number of CD34+CD41+ cells (r = -.81, P < .005), correlated significantly better with time to neutrophil and platelet recovery, respectively, than with the total number of CD34+ cells (r = -.55 and r = -.56, respectively). CONCLUSION The numbers of CD34+CD33- cells and CD34+CD41+ cells may help to predict short-term repopulation capacity of PBSCs, especially when relatively low numbers of CD34+ cells per kilogram are reinfused.

NH2-terminal globular domain of human platelet glycoprotein Ib alpha has a methionine 145/threonine145 amino acid polymorphism, which is associated with the HPA-2 (Ko) alloantigens.

The glycoprotein (GP) Ib/IX complex, a prominent platelet GP complex, is the primary receptor for vWF. Previously, we have established that an antigenic polymorphism of platelets, the HPA-2 or Ko alloantigen system, is located on the 45-kD amino-terminal globular domain of GPIb alpha. With the polymerase chain reaction, we have amplified two segments of the GPIb alpha gene coding for the first 382 amino acids of two HPA-2a and two HPA-2b homozygous individuals. Nucleotide sequence analysis revealed as the only difference a C-T polymorphism at position 434 of the coding region for the mature protein. This base change results in a substitution of threonine (ACG) in HPA-2a (Kob) to methionine (ATG) in HPA-2b (Koa) at amino acid position 145. The C-T polymorphism is reflected in a difference in restriction enzyme recognition, resulting in an Aha 2-site in the HPA-2b allele and a SfaN1 site in the HPA-2a allele. Restriction fragment length polymorphism analysis of the amplified DNA of 3 HPA-2(a-,b+), 2 HPA-2(a+,b+), and 11 HPA-2(a+,b-) donors showed that these restriction sites were associated with the HPA-2 alleles. DNA-typing for the HPA-2 alloantigen system on genomic DNA obtained from a small number of cells may be applied for determining the genotype of a fetus from an immunized mother or of severely thrombocytopenic patients.

Anti-neutrophil cytoplasmic autoantibodies in patients with symptomatic HIV infection.

Antibodies against cytoplasmic antigens of neutrophils, producing perinuclear (p‐ANCA) as well as cytoplasmic staining with central accentuation (c‐ANCA), have been described in non‐HIV‐infected patients with specific pathology such as glomerulonephritis and vasculitis. Here, we report on a patient with a vasculitis‐like syndrome and a positive ANCA‐test who appeared to he infected by HIV. Further analysis revealed that ANCA, p‐ANCA as well as c‐ANCA without central accentuation can be demonstrated in the serum of HIV+ individuals. In a cross‐sectional study on individuals indifferent stages of HIV infection, we found that the occurrence of ANCA was limited to the symptomatic stages of HIV infection: p‐ANCA was found in one out of 10 ARC patients and in two out of II AIDS patients with malignancies (AIDS‐MAL), but not in AIDS patients with opportunistic infections (AIDS‐OI). c‐ANCA was found in four of the ARC patients, in two of the 14 AIOS‐OI patients and in two AIOS‐MAL patients. The presence of ANCA was not related to the degree of hypergammaglobulinaemia nor to specific symptomatology. ANCA containing sera from HIV+ individuals did not read with HEp2 cells nor with cytoplasmic antigens of lymphocytes, natural killer (NK) cells or eosinophils. Five out of the 11 (two p‐ANCA and three c‐ANCA) sera reacted weakly with cytoplasmic antigens of moncytes. All sera reacted with karyoplasts but not with cytoplasts prepared from neutrophils. These results suggest that HIV‐ANCA might be directed against myeloid cell‐specific granule constituents. However, sandwich‐ELISAs with MoAbs against granule antigens that are frequently the target antigens of ANCA in HIV individuals were negative. Also immunoprcecipitation and immunoblotting, using lysates of neutrophil granules, did not allow further identification of the target antigens of HIV‐ANCA.

Relevance of classic anti-neutrophil cytoplasmic autoantibody (C-ANCA)-mediated inhibition of proteinase 3-α1-antitrypsin complexation to disease activity in Wegener's granulomatosis

In the sera of patients with Wegeners granulomatosis (WG), C‐ANCA can be detected that are directed against proteinase 3 (PR3). We have previously observed that C‐ANCA interfere with PR3 proteolytic activity and with complexation of PR3 with its major physiologic inhibitor, α1antitrypsin (α1AT). In the present study we investigated whether this inhibitory effect of C‐ANCA on PR3‐α1 AT complexation correlates with clinical activity of WG. Serial serum samples of eight consecutive patients with histologically proven relapses of WG were tested. At the moment of relapse all sera revealed inhibitory activity towards PR3‐α‐1 AT complexation (median 22%, range 10–59%). Disease activity score (r=0.87, P<0.02) and C‐rcactive protein (CRP) levels (r= 0.66. P<0.1) correlated with C‐ANCA inhibition of PR3‐α1 AT complexation, while they did not correlate with the C‐ANCA titre detected by indirect immunofluorcsccnce (IIF) nor with IgG anti‐PR3 antibody level measured by ELISA. The inhibitory effect of C‐ANCA on PR3‐α1 AT complexation had risen significantly at the moment of relapse compared with values 3 months (P<0.05) and 6 months (P<0.01) before relapse. Eight patients with established WG and positive for C‐ANCA but without clinical evidence of relapse served as controls. In this group no inhibitory effect of C‐ANCA on PR3‐α1AT complexation was observed in 7/8 patients sera. Sera of one control patient contained moderate C‐ANCA inhibitory activity towards PR3‐α1AT complexalion, which remained at a constant level during the 6 months period of observation. Thus, disease activity in WG appears to be more closely related to C‐ANCA inhibitory activity towards PR3‐α1AT complexation.

Increased mean platelet volume in septicaemia.

An increased mean platelet volume (MPV), measured by the Coulter counter model S plus, was found in 13 of 25 patients with proven septicaemia but in none of 25 patients with localised bacterial infection and negative blood cultures. The increase in MPV was found both in patients with normal and low platelet counts and was not related to a particular micro-organism. Patients who responded favourably to antibiotic treatment all had normal MPVs after one week of treatment. However, 9 of 11 patients with a prolonged course of their infection due to endocarditis or abdominal abscesses had raised MPVs after seven days of treatment, and four patients who died of infection in the first week all had increased MPVs on the day of their death. An increased MPV in a patient with bacterial infection possibly indicates that the infection has become invasive--that is, that septicaemia has occurred. A persistent rise or further increase indicates that treatment is inadequate.

Neutrophil cytoplasmic antibodies (p-ANCA) in ulcerative colitis.

AIMS--To study ulcerative colitis associated neutrophil cytoplasmic antibodies (p-ANCA) in respect of class and subclass distribution, antigen specificity, and (sub)cellular localisation of the antigen(s) to which these antibodies are directed. METHODS--p-ANCA positivity was determined using the standard indirect immunofluorescence test (IIFT). The immunoglobulin (Ig) subclass distribution of p-ANCA was investigated using monoclonal antibodies directed against IgG1, IgG2, IgG3, and IgG4. Intracellular antigen localisation studies were performed on (fractionated) neutrophils using antigen-specific antibodies. RESULTS--In contrast to vasculitis associated ANCA, ulcerative colitis p-ANCA are mainly of IgG1 and IgG3 subclass and lack IgG4. Ulcerative colitis p-ANCA are myeloid specific. IIFT data indicate that the related antigen(s) seem(s) to be located not in the cytosol, but in the granules (most likely the azurophil granules) of the neutrophil. CONCLUSIONS--p-ANCA in ulcerative colitis have a different immunoglobulin subclass distribution than the ANCA of systemic necrotising vasculitis and necrotising and crescentic glomerulonephritis. This may point to differences in immune regulation between these diseases. Both cathepsin G and lactoferrin are recognised by a subpopulation of ulcerative colitis p-ANCA. In our series, eight out of 36 (22%) of ulcerative colitis associated p-ANCA react with lactoferrin and seven (19.5%) other sera with cathepsin G. None of them recognised both antigens. The main target antigen(s) of ulcerative colitis p-ANCA still remain(s) to be identified.

Soluble Fc gamma receptor III (CD 16) and eicosanoid concentrations in gut lavage fluid from patients with inflammatory bowel disease: reflection of mucosal inflammation.

BACKGROUND--Activated neutrophils cause tissue injury in inflammatory bowel disease (IBD). Upon activation, they shed soluble Fc gamma IIIb receptors (sFc gamma RIIIb). The subsequent inflammatory response is modulated by several mediators, including neutrophil derived leukotriene B4 (LTB4), thromboxane B2 (TXB2), and prostaglandin E2 (PGE2). The aim of this study was to determine the value of gut lavage sFc gamma RIII and eicosanoid measurements for the assessment of mucosal inflammation in IBD. METHODS--A total of 18 patients with active IBD, 10 ulcerative colitis (UC), and eight Crohns disease (CD), and 12 control patients underwent whole gut lavage. Disease activity, endoscopic appearance, and histopathology were graded. Samples were processed for the determination of sFc gamma RIIIb, LTB4, PGE2, and TXB2. RESULTS--Soluble Fc gamma RIIIb concentrations were increased in both IBD groups. Significant correlations were seen between sFc gamma RIIIb and LTB4 values with histology scores. Mean eicosanoid lavage fluid concentrations in control patients were 14.1 pg/ml for LTB4, 5.6 pg/ml for PGE2, and 397 pg/ml for TXB2. Concentrations of all eicosanoids in IBD patients were significantly increased: LTB4 in UC: mean 73.2 pg/ml, in CD: 96.4 pg/ml (both p < 0.01 v controls). PGE2 in UC: 20.2 pg/ml, in CD: 43.4 pg/ml (p < 0.01). TXB2 in UC: 719.3 pg/ml, in CD: 180.6 pg/ml (both p < 0.05). CONCLUSIONS--Whole gut lavage fluid analysis is an effective method to study mucosal eicosanoid production. Soluble Fc gamma RIIIb concentrations in gut lavage fluid closely correlate with histological signs of mucosal inflammation and with lavage LTB4 concentration. These data suggest that lavage Fc gamma RIIIb assessment may be used as a simple assay to estimate mucosal neutrophil infiltration in IBD.

Corynebacterium CDC group JK (Corynebacterium jeikeium) sepsis in haematological patients : a report of three cases and a systematic literature review

We describe 3 patients with Corynebacterium jeikeium sepsis in neutropenic phase during treatment for acute myeloid leukaemia. Fever was the first symptom. All had a central venous catheter which was removed. Two patients developed subcutaneous nodules containing pus when the neutrophil count recovered; 1 had intracutaneous and pulmonary lesions. They were treated with vancomycin and recovered when the neutrophil count started to rise. A review of 80 neutropenic patients with C. jeikeium sepsis reported in the literature, together with our 3 cases indicates that risk factors for infection are the presence of a central venous catheter, being an adult male or postmenopausal female, profound and prolonged neutropenia and exposure to multiple antibiotics. Skin lesions are reported in 48% and pulmonary lesions in 36% of the patients. The overall mortality is 34% but in patients with recovery of the bone marrow only 5%. Therefore haematopoietic growth factors should be considered in neutropenic patients with C. jeikeium infection.