Alternative Non-Immune F(ab')2-Mediated Immunoglobulin Binding to Group C and G Streptococci

We tested 140 bacterial strains representing 19 different species for binding or purified radiolabelled F(ab')₂ fragments prepared by pepsin digestion of polyclonal and monoclonal human IgG. Both polyclonal and monoclonal F(ab')₂ fragments showed positive binding to group C and G streptococci with maximum uptake levels of 50% and 85%. Binding was obtained both with fresh bacteria and with organisms stabilized by heat treatment. F(ab')₂ fragments of two human IgG1 myeloma proteins with anti-staphylolysin specificity showed a similar binding pattern. IgG present in normal human serum inhibited the uptake of F(ab')₂ fragments, whereas albumin and fibrinogen and purified Fc fragments prepared by papain digestion of polyclonal IgG and monoclonal IgG1 did not show such capacity. Fourteen human myeloma proteins representing IgA, IgM and the four IgG subclasses were tested for inhibiting capacity. Reactivity was noted with at least one myeloma protein within each IgG subclass but not with IgA or with IgM monoclonal proteins. Normal rabbit serum was as inhibitory as normal human serum, whereas dog serum was less reactive. These data demonstrate that group C and G streptococci carry a heat-stable surface component interacting with the F(ab')₂ portion of the IgG molecule. The results suggest that the reactive site on the immunoglobulin molecule may reside in the more constant part of the variable domain. This new reactivity is different from the previously known non-immune reaction involving the IgG Fc portion. This alternative non-immune reactivity is analogous to but distinct from the alternative protein A reaction in Staphylococcus aureus .     

Mitogenic Properties of Fab and F(ab'')2 Fragments of Rabbit Anti-Human β2-Microglobulin for Human Lymphocytes

Bivalent F(ab')2 fragments and monovalent Fab fragments of rabbit anti-human beta2-microglobulin (anti-beta2m) stimulated DNA synthesis in human lymphocytes. Mitogenicity of anti-beta2m antibodies can therefore be ascribed to the antigen-binding site and not to the Fc portion of the molecule. The mitogenic response to F(ab')2, and sometimes Fab, fragments of anti-beta2m IgG was comparable to that obtained with original IgG antibodies when tested at the same protein concentration. Since Fab monomers of anti-beta2m can cause lymphocyte activation, 'cross-linking' of hypothetical beta2-microblobulin-containing lymphocyte receptors does not seem necessary for activation. F(ab')2, as well as Fab, fragments of anti-beta2m blocked the cytotoxic effect of anti-beta2m IgG, showing that the fragments did indeed react with beta2-microblobulin on the cell surface. F(ab')2 dimers, but not Fab monomers, of anti-beta2m were capable of inhibiting the cytotoxic effect of an anti-HLA-A2 antiserum. The mitogenic activity of both anti-beta2m IgG and Fab monomers of such antibodies disappeared after absorption with highly purified beta2-microblobulin. The mitogenic effect of anti-beta2m IgG was inhibited to a minor extent by exposure of cells to high concentrations of pooled multispecific anti-HLA antibodies. This effect was probably nonspecific.     

On the Interaction between β2-Microglobulin and Group A Streptococci

beta 2-microglobulin (beta 2m) was found to interact with many group A streptococcal strains. The interaction appeared to require multipoint attachment, since monomeric beta 2m in solution showed no binding, whereas both beta 2m monomers bound to liposomes, and beta 2m in aggregates showed affinity for the bacteria. Aggregated HLA antigens (-A, -B and -C) and aggregated beta 2m exhibited the same binding patterns when tested in binding experiments with various group A streptococcal strains. Furthermore, beta 2m aggregates in excess completely blocked the binding of aggregated HLA antigens, thereby demonstrating that beta 2m is able to interact with streptococcal surface structures also when it is part of the HLA antigen complex. M protein-positive group A streptococcal strains bound significantly more beta 2m than M protein-negative variants of these strains. Purified M 12 protein partly inhibited the binding of radiolabelled beta 2m aggregates to whole streptococci, and in gel filtration and affinity chromatography experiments, the M 12 protein interacted with beta 2m. These various data suggest that the interaction between beta 2m and group A streptococci could be mediated by M protein. Lipoteichoic acid (LTA) is a constituent of the streptococcal cell wall that has been reported to form complexes with M protein at the bacterial cell surface. However, LTA did not influence the interaction between beta 2m and streptococci, suggesting that the binding of beta 2m to streptococcal M protein represents a pure protein-protein interaction. In vivo such an interaction could be established between infecting streptococci and host cells. Among 45 strains of different M types large differences in beta 2m binding were recorded, whereas among 60 strains of the classical nephritogenic M types 12 and 49, all were highly beta 2m-reactive, which points towards a role for beta 2m in streptococcal pathogenicity.     

Binding of β2-Microglobulin by Heterologous Sera

Purified human, rat or guinea-pig beta 2-microglobulin (beta 2m) was mixed with sera from guinea-pig, rat, mouse, rabbit, horse, goat, cow, rhesus monkey or man. The mixtures were incubated at 37 degrees C for various lengths of time. When the sera were separated by gel-chromatography on Sephadex G-200, beta 2m was traced not only in 'free' form but also in fractions with higher molecular weights. Evidence is presented suggesting that heterologous beta 2m binds to beta 2m-containing molecules in sera by exchange with the homologous counterpart.     

Three New Fragments, F(ab)2, F(c)2, and Fab/c, Obtained by Papain Proteolysis of Normal Human IgG.

Three new fragments, each with a molecular weight of about 100,000, were isolated after papain proteolysis of normal monomer human IgG. The fragments isolated were as follows: F(c)₂ fragment with Fc determinants only, Fab/c fragment with both Fc and Fab determinants, and F(ab') fragment with only Fab determinants. The F(c)₂ fragment appeared to be a dimer of Fc stabilized by disulphide bonds, whilst the F(ab)₂ fragment consisted of Fab subunits mainly held together by non-covalent forces. The Fab/c fragment is probably a single Fab fragment and the Fc fragment held together by an unbroken heavy chain.     

Human Anti-F(ab'')2 Antibodies and Pepsin Agglutinators React with Fv Determinants

Affinity-purified human IgG anti-tetanus antibody was subjected to papain and then pepsin digestion, and residual fragments retaining antibody activity were re-isolated by adsorption and elution from Sepharose-tetanus toxoid columns. Both Fab' and Fv fragments isolated by gel filtration showed strong reactivity with anti-F(ab')2 antibodies. Failure of tetanus toxoid to completely block reactivity of anti-F(ab')2 antibody with anti-tetanus Fv fragments indicates that these antibodies react with framework antigens within variable antibody regions.     

Increased expression of collagen receptors: α1β1 and α2β1 integrins on blood eosinophils in bronchial asthma

BACKGROUND: Eosinophils are one of the major effector cells in bronchial asthma. Their infiltration of airways correlates with the asthma severity. Recruitment and activation of eosinophils are partially mediated by integrins alpha4beta1 and alpha4beta7. Collagens type I and IV constitute important components of extracellular matrix and vascular basement membrane, respectively. Therefore, collagen-binding integrins (alpha1beta1 and alpha2beta1) may also play a role in eosinophil lung infiltration. OBJECTIVE: To evaluate the possible presence of alpha1beta1 and alpha2beta1 integrins on peripheral blood eosinophils from asthmatic subjects. METHODS: Collagen receptors were studied on eosinophils separated by immunomagnetic CD16-negative method from healthy donors (n=13) and patients with moderate persistent atopic bronchial asthma (n=15). Surface receptor identification was performed by flow cytometry and cell adhesion assay. RESULTS: Eosinophils isolated from the patients showed increased expression of both alpha1beta1 and alpha2beta1 integrins as compared with healthy controls. Moreover, adhesive function of eosinophils to collagen type IV was inhibited by snake venom disintegrins: viperistatin and obtustatin. These disintegrins contain KTS active motif and are specific inhibitors of alpha1beta1 integrin. CONCLUSION: We demonstrated for the first time that collagen receptors: alpha1beta1 and alpha2beta1 integrins are overexpressed on the surface of peripheral blood eosinophils of asthmatic subjects. Further studies may reveal potential application of KTS-disintegrins or their structural analogs for therapy of bronchial asthma.     

Serial Measurements of Pregnancy-specific β1-glycoprotein (β1SP1) in Patients with Trophoblastic Disease

A radioimmunoassay for pregnancy-specific-β₁-glycoprotein (Bohn. 1971) has been established with a limit of detection of 2 μMg/litre in serum. The assay has been used to measure serial levels of pregnancy-specific-β₁-glycoprotein (β₁SP₁) in the serum of patients with choriocarcinoma and teratoma for comparison with measurements of the β-subunit of human chorionic gonadotrophin. The value of the assay for β₁SP₁, in the management of these patients is discussed.     

Specific contribution of human T-type calcium channel isotypes (α1G, α1H and α1I) to neuronal excitability

The glutamine transporter SN1 has recently been identified as one of the major glutamine transporters in hepatocytes and brain astrocytes. It appears to be the molecular correlate of system N amino acid transport. Two different transport mechanisms have been proposed for this transporter. These are an electroneutral mechanism, in which glutamine uptake is coupled to an exchange of 1Na⁺ and 1H⁺, or an electrogenic mechanism coupled to the exchange of 2Na⁺ against 1H⁺. This study was performed to solve these discrepancies and to investigate the reversibility of the transporter. When SN1 was expressed in Xenopus laevis oocytes, glutamine uptake was accompanied by a cotransport of 2–3 Na⁺ ions as determined by ²²Na⁺ fluxes. However, at the same time a rapid release of intracellular Na⁺ was observed indicating an active exchange of Na⁺ ions. The driving force of the proton electrochemical gradient was equivalent to that of the sodium electrochemical gradient. Acidification of the extracellular medium caused the transporter to run in reverse and to release glutamine. Determination of accumulation ratios at different driving forces were in agreement with an electroneutral 1Na⁺-glutamine cotransport-1H⁺ antiport. Inward currents that were observed during glutamine uptake were much smaller than expected for a stoichiometric cotransport of charges. A slippage mode in the transporter mechanism and pH-regulated endogenous oocyte cation channels are likely to contribute to the observed currents.     

Biochemical and Biosynthetic Studies of a Crystallizable Human γ1 Heavy-Chain Disease Protein

We have studied the structure of a crystallizable gamma 1 heavy-chain disease protein that lacks the entire VH and C gamma 1 domains. The protein starts within the hinge region at aspartic acid 221 (Eu numbering). The native protein is a disulphide-linked dimer with an apparent molecular weight of 52,000, consistent with the biochemical data obtained on the whole protein and its cyanogen bromide fragments. The carbohydrate content of this protein was 6.8%. As shown by biosynthesis experiments intracytoplasmic gamma chains synthesized by neoplastic cells had an apparent molecular weight similar to that of the serum heavy-chain disease protein. These data are compared with those obtained for other gamma 1 heavy-chain disease proteins beginning in the hinge region, and the mechanisms leading to those abnormal Ig products are discussed.     

Adhesion of Activated T Lymphocytes to Vascular Smooth Muscle Cells and Dermal Fibroblasts is Mediated by β1- and β2-Integrins

Several studies during recent years have demonstrated the potential for vascular smooth muscle cells (SMC) and dermal fibroblasts to participate in immune interactions such as antigen presentation and alloreactivity. The molecular interactions mediating lymphocyte adhesion to these mesenchymal cells have, however, not previously been characterized in detail. In the present study we demonstrate ICAM-1 (CD54) expression by cultured human SMC and its up-regulation by IL-1, IFN-gamma, and bacterial lipopolysaccharide. Monoclonal antibodies were used to define the molecular interactions in the adhesion of 51Cr-labelled T lymphoblasts to adherent SMC and fibroblasts. ICAM-1 appeared to mediate adhesion of T lymphocytes by binding to the beta 2-integrin CD11a/CD18 (LFA-1) expressed by the lymphoblasts. We present evidence for the involvement of at least three different mechanisms in the adhesion of activated T lymphocytes to cultured fibroblasts. It was found that beta 2-integrin-mediated interaction could only account for less than half of the binding activity. The remaining adhesion was partly mediated by beta 1-integrins, presumably via VLA-5 since an anti-VLA-5 antibody and an RGD-containing peptide blocked adhesion to the same degree. However, antibodies to beta 1-, beta 2-, and beta 3-integrin subunits added together only inhibited adhesion by approximately 50%. The residual adhesion could be blocked by inhibition of cell metabolism and was increased by stimulation of the lymphocytes with phorbol ester, suggesting involvement of other, as yet undefined, adhesion molecules. The molecular interactions between lymphocytes and mesenchymal cells demonstrated in this study may have implications in several inflammatory conditions such as vasculitis, atherosclerosis, and connective tissue diseases.     

Vaccination with β2-Microglobulin-Deficient Dendritic Cells Protects Against Growth of β2-Microglobulin-Deficient Tumours

Defects in cell surface expression of major histocompatibility complex class I antigen molecules are common in tumour cells. We have previously described the generation of adaptive immunity to tumour cells deficient in the transporter associated with antigen processing molecule. In this study, we demonstrate enhanced in vivo protection against growth of β₂-microglobulin-deficient tumour cells in syngeneic C57Bl/6 mice, following vaccination with β₂-microglobulin-deficient dendritic cells. In vitro analysis suggested that vaccinated mice produced CD3⁺ cells, which could induce apoptosis in syngeneic β₂-microglobulin-deficient tumour and non-malignant cells. Further investigation of target cell recognition suggested that also tumour cells lacking expression of classical major histocompatibility complex class I heavy chains and functional transporter associated with antigen processing molecules were recognized by CD3⁺ effector cells from vaccinated mice. Histopathological examination of organs from vaccinated mice showed no significant vaccination-induced pathology. The present findings point to a new possible strategy to counteract the growth of major histocompatibility complex class I-deficient tumour cells.