Immunophenotypic differentiation patterns of normal hematopoiesis in human bone marrow: reference patterns for age-related changes and disease-induced shifts.

BACKGROUND: The abundance of monoclonal antibodies (mAb) and the routine use of quadruple stainings in flow cytometry allow stepwise analysis of bone marrow (BM) samples that are suspected for abnormal hematopoiesis. A screening phase that precedes lineage-specific classification phases should be sufficient to assess whether the BM has a normal or abnormal composition, as well as to identify the abnormal differentiation lineage. METHODS: For a quick and easy flow cytometric screening of BM samples, we selected six quadruple immunostainings that cover multiple differentiation stages of the B-cell, monocytic, granulocytic, and erythroid lineages: TdT/CD20/CD19/CD10 and CD45/CD34/CD19/CD22 for B cells, CD34/CD117/CD45/CD13.33 for precursor granulocytic and precursor monocytic cells (myelo/monoblasts), CD14/CD33/CD45/CD34 for monocytic cells, CD16/CD13/CD45/CD11b for granulocytic cells, and CD71/CD235a/CD45/CD117 for erythroid cells. RESULTS: The six quadruple immunostainings reveal specific staining patterns in normal BM, which allow the recognition of various subpopulations of the respective lineages. These staining patterns can be used as a frame of reference for recognition of normal and abnormal BM development. Examples of normal (age-related) variations in these otherwise stable staining patterns are presented together with several abnormal differentiation patterns. CONCLUSIONS: Although alternative immunostainings can be used (e.g., including NK- and T-cell markers), we feel that the selected six stainings represent a comprehensive and easy screening phase for quick identification of shifts in the composition of the studied differentiation lineages, reflecting age-related changes or disease-induced BM abnormalities.

Fcgamma receptor IIa, IIIa, and IIIb polymorphisms in German patients with systemic lupus erythematosus: association with clinical symptoms.

BACKGROUND: Receptors for IgG play an important part in immune complex clearance. Several studies have identified polymorphisms of receptors for the Fc fragment of IgG (FcgammaR) as genetic factors influencing susceptibility to disease or disease course of systemic lupus erythematosus (SLE). OBJECTIVE: To examine these possibilities by evaluating a panel of clinical parameters in a cohort of 140 German patients with SLE for correlations with the FcgammaRIIa, IIIa, and IIIb polymorphisms in an explorative study. METHODS: 140 German patients with SLE according to American College of Rheumatology (ACR) criteria and 187 German controls were genotyped for the FcgammaRIIa, IIIa, and IIIb polymorphisms. Associations between FcgammaR genotypes, combined genotypes and clinical as well as laboratory features were analysed. RESULTS: No significant skewing of any of the three FcgammaR polymorphisms was seen in the German SLE cohort studied. Various clinical and serological parameters were found more frequently and at younger age in homozygous patients with the genotypes IIA-R/R131 or IIIA-F/F158 than in patients with IIA-H/H131 or IIIA-V/V158. These effects were even more pronounced in patients with the low binding combined phenotypes of the FcgammaRIIa, IIIa (double negative phenotypes) and FcgammaRIIa, IIIa, and IIIb (triple negative phenotypes). In patients with the double negative IIA and IIIA genotypes significantly higher frequencies of nephritis (63% v 33%) and proteinuria according to ACR criteria (58% v 11%), anaemia (84% v 55%), and anticardiolipin antibodies (63% v 22%) were found than in patients with the double positive genotypes. Patients with the IIA-R/R131 genotype and the double negative homozygous genotype had an earlier incidence of clinical symptoms, haematological and immunological abnormalities. Accordingly, SLE is diagnosed earlier in these patients, the difference reaching statistical significance only in the double negative v the double positive genotype (26.3 v 39.5 years) and the IIIA-F/F158 genotype v the rest (26.7 v 32.0 years). Most relevant is the fact that a higher median disease activity (ECLAM score) was demonstrated, both in the IIA-R/R131 homozygous (3.3 v 2.7) and the double negative (3.4 v 2.3) patients, reaching statistical significance in the first group. CONCLUSION: The results of this explorative study support the view that the FcgammaRIIa/IIIa and IIIb polymorphisms constitute factors influencing clinical manifestations and the disease course of SLE but do not represent genetic risk factors for the occurrence of SLE. Higher frequencies of clinical symptoms, haematological and immunological abnormalities as well as an earlier onset of clinical symptoms, haematological and immunological markers of active disease were found in patients with the IIA-R/R131 genotype and the double negative and triple negative genotypes.

Relevance of Fcgamma receptor and interleukin-10 polymorphisms for meningococcal disease.

The contribution of individual Fcgamma receptor (FcgammaR) subclasses to meningococcal phagocytosis was studied. In addition, functional FcgammaR polymorphisms were determined in 50 patients with meningococcal disease (MD), in 183 first-degree relatives of MD patients, and in 239 healthy control subjects, to study the association of FcgammaR genotypes with disease. Efficient internalization of opsonized Neisseria meningitidis serogroup B was mediated via multiple FcgammaR subclasses on phagocytes. Accordingly, a low-efficiency combination of FcgammaRIIa-R/R131, FcgammaRIIIa-F/F158, and FcgammaRIIIb-NA2/2 genotypes was increased significantly in relatives of patients with MD, compared with healthy control subjects (P<.05; odds ratio, 2.6; 95% confidence interval, 1.1-6.3). FcgammaRIIa and FcgammaRIIIa genotype distributions differed between patients with sepsis and those with meningitis. Combined genotypes of FcgammaRIIa and interleukin-10 -1082, which was previously reported as being associated with MD outcome, were distributed randomly in control subjects but not in relatives of patients with MD (P<.01). These data provide further evidence for the association of polymorphic genes on chromosome 1 and MD.

The Fcgamma receptor genotype as a risk factor for generalized early-onset periodontitis in Japanese patients.

BACKGROUND: Genetic polymorphisms of immunoglobulin G (IgG) Fc receptors (FcgammaR) were recently shown to be associated with recurrence rates of adult periodontitis (AP). The purpose of this study was to evaluate whether FcgammaR polymorphisms are also associated with generalized early-onset periodontitis (G-EOP) in Japanese patients. METHODS: Thirty-eight Japanese patients with G-EOP and 83 Japanese patients with AP were identified according to established clinical criteria, including measurements of probing depth, clinical attachment level, and alveolar bone level. FcgammaR genotypes for 3 bi-allelic polymorphisms were determined in these G-EOP and AP patients and 104 race-matched healthy controls by means of allele-specific polymerase chain reactions. RESULTS: There was a significant difference in the distribution of FcgammaRIIIb genotypes between G-EOP patients and healthy controls (P = 0.02). Additionally, a significant over-representation of FcgammaRIIIb-NA2 allele was observed in G-EOP patients as compared to AP patients and controls (P= 0.02, P= 0.009, respectively). Moreover, we found a strong association between G-EOP and the composite genotype comprising FcgammaRIIIb-NA2 and FcgammaRIIIa-158F (G-EOP versus controls: odds ratio 2.4, 95% CI 1.0-6.0, chi2 = 4.13, P= 0.04). CONCLUSIONS: This study indicates that the FcgammaRIIIb-NA2 allele and possibly FcgammaRIIIa-158F could be associated with susceptibility to G-EOP in Japanese patients.

Relevance of IgG receptor IIIb (CD16) polymorphism to handling of Porphyromonas gingivalis: implications for the pathogenesis of adult periodontitis.

Polymorphonuclear neutrophils (PMNs) are essential in host defense against periodontopathic bacteria such as Porphyromonas gingivalis. The uptake of immunoglobulin G (IgG)-opsonized bacteria via IgG Fc receptors (Fc gamma R) on PMN constitutes a central defense mechanism in periodontium. Fc gamma RIIIb is the most abundantly expressed Fc gamma R on PMN and is functionally polymorphic. The Fc gamma RIIIb-NA1 and IIIb-NA2 allotypes interact differently with IgG1- and IgG3-opsonized particles. We recently showed recurrence rates of adult periodontitis (AP) to be higher in patients carrying at least 1 Fc gamma RIIIb-NA2 allele. In this study we evaluated the functional relevance of the Fc gamma RIIIb polymorphism to anti-P. gingivalis PMN effector functions. Our results showed Fc gamma RIIIb-NA2-carrying PMN from both patients with AP and healthy controls to be less efficient in phagocytosis and induction of oxidative burst upon interaction with IgG1- and IgG3-opsonized P. gingivalis. These functional differences between Fc gamma RIIIb-NA1 and IIIb-NA2 were observed in the presence of CD32-blocking antibody fragments, but not upon blocking CD16. Moreover, PMNs from AP patients exhibited increased Fc gamma RIIIb-allelic differences in IgG3-induced oxidative burst compared to control PMNs. These results support the concept that Fc gamma RIIIb heterogeneity may influence the clinical course of AP.

Functional human monoclonal antibodies of all isotypes constructed from phage display library-derived single-chain Fv antibody fragments.

We have constructed a series of eukaryotic expression vectors that permit the rapid conversion of single chain (sc) Fv antibody fragments, derived from semi-synthetic phage display libraries, into intact fully human monoclonal antibodies (mAb) of each isotype. As a model, a scFv fragment specific for sheep red blood cells (SRBC) was isolated from a semi-synthetic phage antibody (Ab) display library, and used to produce human mAbs of IgM, IgG1-IgG4, IgA1, IgA2m(1) and IgE isotype in vitro in stably transfected cells. N-terminal protein sequence analysis of purified immunoglobulin heavy (H) and light (L) chains revealed precise proteolytic removal of the leader peptide. Biochemical analysis of purified recombinant human mAbs demonstrated that properly glycosylated molecules of the correct molecular size were produced. The IgG and IgA mAbs retained SRBC-binding activity, interacted with different Fc receptor-transfectants, and induced complement-mediated hemolysis and Ab-dependent phagocytosis of SRBC by neutrophils in a pattern consistent with the immunoglobulin (Ig) H chain isotype. We conclude that in vitro produced recombinant human mAbs constructed from phage display library-derived scFv fragments mirror their natural counterparts and may represent a source of mAbs for use in human therapy.

Heparin-induced thrombocytopenia: new insights into the impact of the FcgammaRIIa-R-H131 polymorphism.

Heparin-induced thrombocytopenia (HIT), a severe complication of heparin treatment, can be associated with new thrombotic complications. HIT antibodies activate platelets via the platelet Fcgamma-receptor (FcgammaRIIa), which carries a functionally relevant polymorphism (FcgammaRIIa-R-H131). The effect of this polymorphism on the clinical manifestations of HIT is controversial. We determined prospectively the FcgammaRIIa-R-H131 genotypes in 389 HIT patients, in 351 patients with thrombocytopenia or thrombosis due to causes other than HIT and without detectable HIT antibodies, and in 256 healthy blood donors. For this purpose, a novel nested sequence-specific primer-polymerase chain reaction (SSP-PCR) was developed. FcgammaRIIa-R/R131 was found to be overrepresented in the HIT patients (27%) compared with the control groups (non-HIT patients [21%] and blood donors [20%]). In a subgroup of 122 well-characterized HIT patients, the genotype distribution in patients presenting with thrombocytopenia only was compared with that of patients who developed thromboembolic complications. The frequency of FcgammaRIIa-R/R131 among patients with thrombotic events was significantly elevated (37% v 17%; P = .036). Our results indicate that genotype distribution can be correlated to the clinical outcome of patients with HIT. We speculate that the reduced clearance of immune complexes in patients with the FcgammaRIIa-R/R131 allotype causes prolonged activation of endothelial cells and platelets, thus increasing the risk for thrombotic complications.

Fcgamma receptor IIa polymorphism in Caucasian patients with systemic lupus erythematosus: association with clinical symptoms.

OBJECTIVE: The class II human leukocyte Fcy receptor for IgG (FcgammaRIIa) occurs in 2 codominantly expressed allelic forms (R131 and H131). Cells expressing IIa-H131 interact much more effectively with complexed IgG2 and IgG3 than do cells with IIa-R131. This might be linked to variability in immune complex handling, and therefore related to disease pathogenesis. The present study examines these possibilities in a cohort of Caucasian patients with systemic lupus erythematosus (SLE). METHODS: One hundred eight Caucasian patients were diagnosed with SLE according to the American College of Rheumatology criteria. The SLE patients and 187 Caucasian controls were genotyped for the FcgammaRIIa polymorphism, and associations between FcgammaRIIa genotypes, selected HLA haplotypes, and clinical as well as laboratory features were analyzed. RESULTS: No significant skewing of the FcgammaRIIa polymorphism was observed in the SLE cohort. Various clinical and serologic parameters were found more frequently or at a younger age in patients homozygous for the genotype IIa-R/R131 compared with those with the genotype IIa-H/H131. In patients with the genotype IIa-R/R131, significantly higher frequencies of proteinuria, hemolytic anemia, anti-nuclear RNP antibodies, and hypocomplementemia were found. The only clinical symptom observed more frequently in patients homozygous for IIa-H/H131 was livedo. Patients with the IIa-R/R131 genotype were significantly younger at disease onset and had an earlier incidence of arthritis, sicca syndrome, nephritis, lymphadenitis, hematologic abnormalities, immunologic abnormalities, lupus anticoagulant, cryoglobulinemia, and hypocomplementemia. HLA-DR3 was found in 41.7% of SLE patients, but was not associated with clinical symptoms, serologic abnormalities, or the homozygous genotypes of the FcgammaRIIa, although an association with a significantly later onset of SLE was found. CONCLUSION: The FcgammaRIIa polymorphism constitutes an additional factor that might influence the clinical manifestations and course of SLE, but does not represent a genetic risk factor for the occurrence of SLE.

Relevance of immunoglobulin G Fc receptor polymorphism to recurrence of adult periodontitis in Japanese patients.

Polymorphonuclear neutrophil (PMN) phagocytic function has been shown to be impaired in some patients with periodontitis. PMN constitutively express members of two immunoglobulin G receptor (Fc gammaR) classes: Fc gammaRIIa (CD32) and Fc gammaRIIIb (CD16). Both receptors exhibit genetically determined structural and functional biallelic polymorphisms, which have been shown to influence PMN phagocytic function. In this study, we assessed the relevance of these Fc gammaR polymorphisms to susceptibility to adult periodontitis and recurrence rate. The distribution of Fc gammaRIIa and Fc gammaRIIIb genotypes of 100 Japanese patients with adult periodontitis during follow-up was compared to the distribution of genotypes in 105 race-matched healthy controls. No significant skewing of distributions of Fc gammaRIIa and Fc gammaRIIIb genotypes was observed between patients and controls. Notably, however, a significant overrepresentation of the Fc gammaRIIIb-NA2 allotype was found in patients with disease recurrence (P < 0.05; odds ratio, 4.29; 95% confidence interval, 1.19 to 16.24). Moreover, the annual rate of recurrence was significantly higher in patients with the Fc gammaRIIIb-NA2/NA2 and Fc gammaRIIIb-NA1/NA2 genotypes than in Fc gammaRIIIb-NA1/NA1 individuals (P < 0.05). Fc gammaRIIa-R/R131 individuals also exhibited higher recurrence rates, though the difference was not statistically significant (P = 0.06). These results suggest that the Fc gammaRIIIb-NA2 allotype represents a risk factor for recurrence of adult periodontitis.

Clinical relevance of Fc gamma receptor polymorphisms.

Human IgG receptors are very heterogeneous and we currently distinguish three Fc gamma receptor classes specifying at least 12 receptor isoforms. On top of this complexity, Fc gamma R are further found to differ between different individuals. Polymorphisms have been identified for all three Fc gamma R classes. The best-studied ones represent allelic variation of Fc gamma RIIa (CD32) and Fc gamma RIIIb (CD16). The Fc gamma RIIa polymorphism is now considered to be a heritable risk factor for autoimmune and infectious diseases, and support for a relevant role of the IIIb polymorphism has also been obtained. A detailed analysis of the exact contribution of each of these Fc gamma R polymorphisms in relation to previously implicated risk factors should unravel the pathophysiological importance of Fc gamma R polymorphisms in the near future.

Antigen topography is critical for interaction of IgG2 anti-red-cell antibodies with Fc gamma receptors.

IgG antibodies to the Rh D polypeptide on red cells are normally IgG1 or IgG3, whereas antibodies produced in response to carbohydrate antigens such as the A and B blood groups are predominantly IgG2. The consequences of this isotype restriction for the immune destruction of red cells were investigated. Human IgG2 anti-D and IgG2 anti-A were isolated by affinity purification from an unusual anti-D serum (DEL) and anti-A sera, respectively. These antibodies were compared with IgG1 and IgG3 monoclonal anti-D in in vitro functional assays of the interaction between IgG-coated red cells (EA-IgG) and cells bearing IgG Fc receptors (Fc gamma R). Dimeric IgG2 anti-D bound efficiently to cell lines transfected with Fc gamma RIIa-H131, an allotypic form of Fc gamma RIIa which readily interacts with IgG2, IgG1 and IgG3. Unexpectedly, however, -D- phenotype red cells coated with IgG2 anti-D did not form rosettes with these cells, whereas EA-IgG2 anti-A and EA-IgG1 and EA-IgG3 anti-D effectively formed rosettes with these transfectants at the same sensitization level (100,000 molecules IgG/red cell). In antibody-dependent cell-mediated cytotoxicity (ADCC) assays, lysis of EA-IgG2 anti-A was mediated via Fc gamma RIIa, whereas lysis of EA-IgG1 and EA-IgG3 anti-D was mediated via Fc gamma RI or Fc gamma RIII; EA-IgG2 anti-D was inactive in all functional assays. These experiments suggest that both IgG subclass and antigen structure affect functional IgG-Fc gamma R interactions. The topography of the Rh D antigen, an integral membrane protein, ensures that anti-D is bound near the lipid bilayer surrounded by the glycocalyx. This may sterically hinder access of Fc gamma RIIa-H131 to the Fc gamma R recognition site on the relatively inflexible IgG2 anti-D, but not to that of IgG1 or IgG3 anti-D. In contrast, IgG2 bound to the A antigen on glycoproteins is not so constrained. The topography of the D and A antigens may thus determine whether functional interactions of red-cell-bound IgG2 anti-D and IgG2 anti-A with cells bearing Fc gamma receptors can occur.

Skewed distribution of IgG Fc receptor IIa (CD32) polymorphism is associated with renal disease in systemic lupus erythematosus patients.

OBJECTIVE: Fc gamma receptors of class IIa (Fc gamma RIIa) occur in 2 allelic forms, with either a low (IIa-R131) or a high (IIa-H131) affinity for complexed IgG2 and IgG3. This polymorphism might have implications for the handling of immune complexes. Therefore, we determined the distribution of the Fc gamma RIIa allotypes in patients with systemic lupus erythematosus (SLE), with or without a history of lupus nephritis. METHODS: We studied 95 unrelated white European patients with SLE, as defined by the American College of Rheumatology criteria, 50 of whom had a history of lupus nephritis, and 69 healthy white European control subjects. Fc gamma RIIa allotypes were determined by immunophenotyping of blood monocytes. RESULTS: It was found that lupus nephritis was significantly associated with the "low affinity" Fc gamma RIIa R/R131 allotype and with the R131 allele, compared with healthy controls. No significant association was found upon comparison of groups with and without nephritis. CONCLUSION: SLE patients with a history of lupus nephritis have an abnormal distribution of Fc gamma RIIa allotypes. Fc gamma RIIa may well play a role in the pathogenesis of lupus nephritis, since IIa-R/R131 SLE patients seem to have a higher incidence of developing this complication.

Functional analysis of human Fc gamma RII (CD32) isoforms expressed in B lymphocytes.

The low affinity IgG receptor Fc gamma RII (CD32) represents the most widely distributed class of human Fc gamma R. To analyze the biologic functions of different Fc gamma RII isoforms, we stably transfected Fc gamma RIIb1, IIb1*, IIb2, IIa, and a IIa tail- mutant to the mouse IIA1.6 B lymphoma cell line. Of these, Fc gamma RIIb1* represents a receptor variant that is identical to IIb1 except for a single amino acid difference in the cytoplasmic tail (amino acid position 11) where a tyrosine (IIb1) is replaced by an aspartic acid (IIb1*). Evaluation of capping ability showed the Fc gamma RIIb1 molecules to cap effectively, which was even more apparent with IIb1*. None of the Fc gamma RIIa, IIa tail-, or IIb2 isoforms capped significantly. Internalization of Fc gamma R-antibody complexes proved very efficient for both the Fc gamma RIIa and IIb2 isoforms, whereas the IIb1 molecules internalized moderately compared with IIb1*, which internalized less efficiently. Notably, human IgG aggregates were internalized effectively by Fc gamma RIIa and moderately by IIb2. Neither Fc gamma RIIb1 nor IIb1* proved capable of internalizing such IgG aggregates. Cross-linking of the different Fc gamma R molecules showed Fc gamma RIIa capable of triggering increases in [Ca2+]i. Fc gamma R expressed on B cells were able to down-regulate [Ca2+]i on co-cross-linking with slgG. Notably, all three Fc gamma RIIb receptors proved active in this respect, in contrast to Fc gamma RIIa. The cell distribution of these Fc gamma RII isoforms was analyzed in a panel of human B cell lines to complement the IIA1.6 B cell model. Fc gamma RIIa was found expressed both at message and protein levels in all tested human B cell lines. In the pre-B cell lines evaluated, no Fc gamma RIIb molecules were detectable, whereas both Fc gamma RIIb1 and IIb2 molecules were found present in more mature B cell lines. These data support both a complex expression pattern of Fc gamma RII isoforms in B cell lines and functional differences between these B cell molecules.

Interaction of a human Fc gamma RIIb1 (CD32) isoform with murine and human IgG subclasses.

A group of Fc receptor molecules, classified CD32, recognize the Fc moiety of IgG with low affinity. We report the isolation and identification of different hFc gamma RIIb cDNA clones, amongst which are cDNA clones encoding hFc gamma RIIb1 and hFc gamma RIIb2. Two hFc gamma RIIb1 encoding cDNA clones (pIP9 and pIP14) were isolated, which differed by three nucleotides, probably because of allelic variation. The nucleotide differences result in one amino acid change between the allelic hFc gamma RIIb1 variants. This substitution is located at amino acid position 11 of the cytoplasmic tail; a tyrosine in hFc gamma RIIb1 (clone pIP9) was replaced by an aspartic acid in clone pIP14 (encoding hFc gamma RIIb1*). A complication in studying ligand specificity of Fc receptors is the potential co-expression of different classes, subclasses, or polymorphic forms of FcR on the same cell. We therefore used murine fibroblasts transfected with cDNA clone pIP14, encoding a hFc gamma RIIb1* isoform, as our model system. These fibroblasts were found to interact with erythrocytes sensitized with mIgG2a and mIgG2b in rosetting assays performed at 4 and 37 degrees C. Interestingly, hFc gamma RIIb1* transfectants bound mIgG1 sensitized erythrocytes only weakly at 4 degrees C, whereas profound binding was observed at 37 degrees C. The ligand specificity for human (h) IgG isotypes was found to be hlgG3 > or = hlgG1 > hlgG4 > hlgG2, as determined at 4 degrees C with hlgG dimeric complexes. However, when assayed at 37 degrees C, the binding of hlgG2 dimers increased significantly. Next, we evaluated whether these transfectants were capable of supporting anti-CD3 induced T cell proliferation.(ABSTRACT TRUNCATED AT 250 WORDS)

On the interaction of IgG subclasses with the low affinity Fc gamma RIIa (CD32) on human monocytes, neutrophils, and platelets. Analysis of a functional polymorphism to human IgG2.

An allotypic form of the low affinity IgG Fc receptor Fc gamma RIIa (CD32), termed low responder (LR) because of its weak reactivity with mouse (m) IgG1, interacts efficiently with human (h) IgG2. Fc gamma RIIaLR is the first known human FcR that binds this IgG subclass. In this study, we analyzed the role of Fc gamma RIIa in binding of stable hIgG-subclass dimers, and in induction of T cell mitogenesis using chimeric anti-CD3 mAb. We demonstrate that the functional polymorphism to hIgG2 is expressed on the majority of Fc gamma R-bearing peripheral blood cells: monocytes, neutrophils, and platelets. We were able to assess Fc gamma RII-mediated IgG-binding without interference of other Fc gamma R-classes, by blockade of Fc gamma RI on monocytes, and by using neutrophils of an individual deficient for the Fc gamma RIIIB gene. This study indicates as subclass specificity: hIgG3 >hIgG1,hIgG2 >> hIgG4 for Fc gamma RIIaLR and hIgG3,hIgG1 >> hIgG2 > hIgG4 for Fc gamma RIIaHR. Comparing the serum hIgG levels of individuals homozygous for the two fc gamma RIIa allotypic forms, we observed significantly lower hIgG2 serum levels in individuals expressing the hIgG2-binding LR allotypic form. This observation may implicate that Fc gamma RIIa regulates hIgG subclass production or turnover in man.

A single amino acid in the second Ig-like domain of the human Fc gamma receptor II is critical for human IgG2 binding.

The low-affinity human Fc gamma RIIa is encoded by a single gene with allelic variation, defined by low-responder and high-responder alleles (LR and HR). The HR Fc gamma RIIa transcript interacts strongly with murine (m) IgG1 complexes, in contrast to the LR Fc gamma RIIa. Furthermore, the transcripts can be discriminated by mAb 41H16, which recognizes an epitope expressed on the HR Fc gamma RIIa molecule. We report that this receptor is also polymorphic in its reactivity with human (h) IgG2. Binding studies using well-defined hIgG dimers revealed that LR Fc gamma RIIa molecules can efficiently bind hIgG2, in contrast to HR Fc gamma RIIa. Previous work of others showed one amino acid difference between the allelic forms of Fc gamma RII. We, however, found a second amino acid difference between both allelic forms. In this study, hybrid Fc gamma RIIa molecules were constructed to determine the epitope for mAb 41H16 and the binding domain for mIgG1 and hIgG2 complexes. Our data point to the importance of the amino acid at position 131, located in the second Ig-like domain of Fc gamma RIIa. When an arginine residue is present at amino acid position 131, the receptor is recognized by mAb 41H16. Furthermore, the receptor can bind mIgG1-sensitized indicator E, but binds hIgG2 dimers only weakly. When a histidine residue is present at this amino acid position, hIgG2 dimers do bind efficiently to Fc gamma RII, whereas mIgG1-sensitized E and mAb 41H16 exhibit a strongly diminished binding.

Measurement of the humoral immune response against Streptococcus pneumoniae type 14-derived antigens by an ELISA and ELISPOT assay based on biotin-avidin technology.

A Streptococcus pneumoniae type 14-specific ELISA and ELISPOT assay have been developed based on the use of biotinylated type 14 capsular polysaccharide (S14PS-biotin). A major advantage of this application over other methods is the use of 10-100-fold less antigen than that reported in the literature for other similar assays. Moreover, the prepared biotinylated polysaccharides are very stable and it is possible to use the same procedures for other pneumococcal polysaccharide antigens (e.g., S6BPS) with no major changes necessary in the ELISA and ELISPOT protocols. Furthermore, a simple thin layer chromatography method has been developed as a method for quality control of the biotinylated polysaccharide. Immunization with the thymus-independent antigen S14PS resulted in the induction of IgM spot-forming cells (SFC) and antibodies while S14PS-protein conjugates induced a thymus-dependent response. The immune response to the conjugates was enhanced by the addition of the adjuvant Quil A resulting in high levels of both IgG SFC and antibodies at day 14 after immunization. The developed assays are reliable and reproducible tools for studying the humoral immune response against Streptococcus pneumoniae type 14 capsular polysaccharide derived antigens.

Surface hydrophobicity and opsonic requirements of coagulase-negative staphylococci in suspension and adhering to a polymer substratum.

The opsonic requirements for phagocytosis in suspension of 38 clinical isolates of coagulase-negative staphylococci recovered from neonates with septicemia were found to be related to the degree of surface hydrophobicity of these strains. Sixteen isolates were adequately opsonized only in the presence of complement; this group was significant more hydrophobic (p less than 0.001) than the 22 strains not requiring complement for efficient uptake in suspension. Evidence showed that hydrophobic groups present on the bacterial surface may interfere with IgG opsonization. In contrast, IgG sufficed as an opsonin without complement being required for the efficient uptake of these hydrophobic strains when adhering to a polymer surface.

Modulation of adherence of coagulase-negative staphylococci to Teflon catheters in vitro.

The mechanism of adherence of Staphylococcus epidermidis to commercially available catheters was studied in vitro in a quantitative assay employing 3H-labelled bacteria. It was found that adherence to Teflon catheters was significantly related to the degree of hydrophobicity of the strains. When hydrophobic groups were removed from Staphylococcus epidermidis by pepsin treatment, adhesion was almost completely abolished. Preincubation of catheters in human serum also caused a 80-90% reduction of adherence. Preincubation of Staphylococcus epidermidis in serum similarly decreased adhesion. This effect of serum was mainly due to albumin, while IgG and fibronectin were less effective. Culture of Staphylococcus epidermidis in subinhibitory concentrations (0.5 MIC) of cephalothin, clindamycin and vancomycin resulted in a 30-80% reduction in adhesion.

Opsonic defense to Staphylococcus epidermidis in the premature neonate.

The determinants of opsonic defense to Staphylococcus epidermidis were studied in 47 premature newborns. Opsonic activity for S. epidermidis in serum from premature newborns proved to be proportional to gestational age (r = .664, P less than .001). The level of IgG antibodies to staphylococcal peptidoglycan in neonatal sera was similarly proportional to gestational age (r = .604, P less than .001). However, all opsonic activity of premature neonatal serum proved to be heat labile, i.e., dependent on activation of complement. Thus, no heat-stable, IgG-dependent opsonic activity to S. epidermidis was detected in any of the preterm sera, despite the presence of IgG antibodies to peptidoglycan. Further studies with purified IgG isolated from paired sera from term neonates and their mothers revealed that at similar concentrations the opsonic activity to S. epidermidis of neonatal, transplacentally derived IgG was only 26% of the activity of maternal IgG, a finding that may explain the absence of heat-stable opsonic activity in preterm newborns.