Antiphospholipid antibodies, cocaine use, and stroke in a young woman.

Stroke, which is unusual in young adults, has been linked to recreational drug use and the antiphospholipid syndrome (aPS). Cocaine use has been related to stroke in this population, and aPS is a known cause of thrombotic neurologic events. We report a patient with positive lupus anticoagulant and anticardiolipin antibodies as well as a history of cocaine use immediately preceding the acute onset of multiple neurologic deficits. The findings in this case suggest a relationship between the use of cocaine, the aPS, and stroke in young adults.

Monoclonal antibodies specific for novel murine cell surface markers define subpopulations of germinal center cells.

A panel of mAbs has been generated which selectively, but not exclusively, recognizes populations of cells within germinal centers of immunized mice. All four mAbs stain B cell populations as defined by flow cytometry. The mAbs FH9.5 and C3.5 also stain T cell subsets (CD4+ and CD8+, respectively). Following density gradient centrifugation of spleen cells from immunized mice the majority of FH9.5+ and C3.5+ B cells are found in the low density, activated fractions. The cells bearing the epitope(s) recognized by the C6C3 and the A6A2 mAbs are less frequent, and from flow cytometric analysis the cells stained with these mAbs are B cells and myeloid cells. The surface markers defined by the four mAbs are not induced following mitogen stimulation of small resting B cells suggesting that these molecules are not general activation markers. Cell lines from a variety of hematopoietic lineages expressing the four markers have been identified. The cell surface molecule immunoprecipitated by the FH9.5 mAb is a polypeptide of 23-28 kDa. The C3.5 antigen is an 85- to 95-kDa protein. These mAbs will be useful in elucidating the complex events involved in B cell differentiation and maturation which occur within germinal centers.

Idiotope structure and genetic diversity in anti-streptococcal group A carbohydrate antibodies.

Three cross-reactive idiotopes(Id), termed IdX, IdI-1, and Id5, that are present on free L chains from murine anti-group A streptococcal carbohydrate antibodies have been mapped; these Id distinguish between products of three homologous V kappa genes. For each determinant, sequence analysis of anti-streptococcal group A carbohydrate antibody V domains yielded small numbers of amino acids invariably associated with Id expression. Flow micro-fluorimetry was used to isolate three IdI-1- spontaneous mutants of the IdI-1+ hybridoma GAC 39; all had single amino acid changes in the L chain at position 60 and 77, all retained other Id, and all bound group A carbohydrate. Computer modeling was used to examine spatial relationships between Id. A number of the conserved Id5 and IdX residues cluster in the L chain framework region 1 around the first back loop connecting strands of the beta pleated sheets, and overlap at residue 15 (Id5, proline; IdX, leucine). This overlap accords with the mutually exclusive expression of Id5 and IdX. The IdI-1 loss variants have mutations of residues 60 or 77 on adjacent back loops, approximately 7.5 and 14 A from residue 15. Competitive inhibition of anti-IdX and anti-IdI-1 binding to antibodies expressing both Id can be attributed to steric hindrance. The framework back loops may be favored sites for cross-reactive Id expressed by products of a single V region gene. IdI-3a, an individual Id not associated with use of a particular gene segment, has been localized in part to residue 31 (hypervariable region 1) of the H chain.

Antigen induced rheumatoid factors: characteristics of monoclonal rheumatoid factors produced after protein and carbohydrate immunization.

We generated a panel of monoclonal rheumatoid factors (MRF) from BALB/c mice immunized with ovalbumin, dextran or group-A-carbohydrate. Individual MRF were analyzed in terms of their binding to the four isotypes of murine IgG, isotypes of human IgG, and rabbit IgG, the idiotypes they express, and the VH gene families they employ. We found that antigen induced rheumatoid factors could be divided into three different families based on their isotypic specificity for murine IgG: an IgG1 binding family, and IgG3 binding family, and a family of MRF that bound all four murine isotypes. Rheumatoid factors belonging to all three families were isolated from mice immunized with carbohydrate antigens. The rheumatoid factors isolated from protein immunized mice all belonged to the IgG1 binding family. We were able to define two cross-reactive idiotypes among MRF, one expressed by a subgroup of the IgG1 binding family, and a second cross-reactive idiotype expressed by some members of the pan-binding family. We determined VH gene use in five of six MRF belonging to the IgG1 binding family and four of four members of the pan-binding family. Four of the IgG1 binding rheumatoid factors and three of the pan-binding rheumatoid factors utilize the J558 VH gene family. Rheumatoid factors produced after carbohydrate antigen immunization, as compared with those generated by protein immunization, are diverse in their isotypic specificity, and show a greater ability to bind heterologous IgG.

A potential new radiopharmaceutical for parathyroid imaging: radiolabeled parathyroid-specific monoclonal antibody--I. Evaluation of 125I-labeled antibody in a nude mouse model system.

Radioiodinated BB5-G1, a parathyroid-specific monoclonal antibody, and its F(ab')2 and Fab fragments were characterized using a nude mouse model system. Blood clearance studies indicated that the most slowly clearing species was the 125I-BB5-G1 intact antibody, while the most rapidly clearing one was the 125I-Fab fragment. 125I-F(ab')2 retained its capacity to localize in the human parathyroid tissue implants with the uptake at 24 h being similar to that observed with the intact antibody. Poor localization was observed with the Fab fragment. These results suggest that BB5-G1 or its F(ab')2 fragment may be useful for parathyroid imaging.

A potential new radiopharmaceutical for parathyroid imaging: radiolabeled parathyroid-specific monoclonal antibody--II. Comparison of 125I- and 111In-labeled antibodies.

The biodistributions of 111In-BB5-G1 and 111In-F(ab')2 were compared with the biodistributions of the corresponding 125I-labeled molecules. For BB5-G1 intact antibody, the relative uptake of the 111In- and 125I-labeled molecules in human parathyroid tissue implants was similar at 24 h, but by 96 h the uptake of the 111In-BB5-G1 %ID/g was four times greater than that observed with the 125I-labeled antibody. For the F(ab')2 fragments, the relative parathyroid uptake of the two preparations was similar at all times tested. The uptake by the clearance organs was significantly higher when the 111In-labeled molecules were used. Imaging results suggest that 111In-BB5-G1 or 111In-F(ab')2 may be a useful radiopharmaceutical for parathyroid radioimmunodetection.

Genetics and primary structure of V kappa gene segments encoding antibody to streptococcal group A carbohydrate. Comparison of V kappa gene structure with idiotope expression.

Two gene segments, V kappa-25-39 and V kappa-25-47, that encode antibody to streptococcal group A carbohydrate in A/J mice were found to be more than 95% homologous in nucleotide sequence in both coding and noncoding regions. It was previously shown that V kappa-25-39 encodes immunoglobulins that express the IdX and IdI-1 idiotopes, whereas V kappa-25-47 encodes IdX+, and IdI-1- immunoglobulins. V kappa gene segments that were clearly allelic to V kappa-25-47 are used to encode IdX+, IdI-1- anti-group A carbohydrate antibodies by C.B20 mice and likely by C57BL/6 mice. Murine strains that are deficient in IdI-1 idiotope expression were investigated by Southern blotting with a 5' probe from V kappa-25-39. Two IdI-1-deficient strains, CE/J and C58/J, had a grossly altered V kappa gene segment structure compared with the A/J prototype. In contrast, the IdI-1-deficient strain, C57BL/6, was indistinguishable from A/J with the 5'V kappa-25-39 probe, indicating that more subtle genetic changes account for the loss of IdI-1 expression in C57BL/6 mice. The evolution of V kappa-25-39 and V kappa-25-47 gene segments was deduced by comparison with the homologous V kappa 24B gene segment of Mus pahari. V kappa-25-39 and V kappa-25-47 likely have recently duplicated once in A/J and related strains of laboratory mice and may have duplicated again in CE/J mice. Thus, individual members of the V kappa 24 gene family, to which V kappa-25-39 and V kappa-25-47 belong, are preserved while the number of gene copies expands or contracts. This fact is strong evidence that evolutionary forces have maintained the V kappa 24 gene family, all of which encode antibody specific for carbohydrate found in bacterial pathogens.

Antigen-induced rheumatoid factors. Protein and carbohydrate antigens induce different rheumatoid factor responses.

Rheumatoid factors, autologous IgM anti-IgG, were produced after immunization with protein or carbohydrate antigens. After immunization with either type of antigen, the kinetics of the rheumatoid factor response reflected the kinetics of the dominant IgG isotype in the anti-antigen response. Secondary immunization with protein antigens induced an IgM rheumatoid factor response which was consistently greater than that seen after carbohydrate immunization, and almost exclusively specific for the IgG1 isotype. In contrast, primary or hyperimmunization with carbohydrate antigens gave rise to a more heterogeneous response dominated by IgM anti-IgG3, with lesser amounts of IgM anti-IgG2b and anti-IgG1. Direct immunization with immune complexes gave similar results, as complexes composed of IgG1 induced exclusively IgM anti-IgG1, whereas those complexes made up of IgG3 gave rise to IgM rheumatoid factors binding IgG3 and IgG2b. Rheumatoid factor production, with isotypic specificity defined by the immunizing antigen, appears to be a natural consequence of immunization with a variety of protein and carbohydrate antigens.

Low-temperature culture of human islets or in vivo treatment with L3T4 antibody produces a marked prolongation of islet human-to-mouse xenograft survival.

In previous studies we have shown that rejection of islet xenografts transplanted between closely related species (rat to mouse) can be prevented by destruction or alteration of antigen-presenting cells in the donor islets and temporary immunosuppression of the recipients. Relatively few studies have been reported on the survival of islet xenografts transplanted between widely discordant species. In the present study, isolated human islets were transplanted beneath the renal capsule of B6 mice made diabetic by the injection of streptozotocin. The effect of culturing the human islets at 24 degrees C for 7 days with or without the administration of anti-L3T4 for 7 days after transplantation was determined. A marked prolongation of the mean survival time was obtained with low-temperature culture alone (greater than 40.2 +/- 9.9 days), with anti-L3T4 alone (greater than 45.2 +/- 6.3 days), and with the combination of these regimens (greater than 51.9 +/- 5.1 days) as compared to controls (7.5 +/- 1.1 days). The surprising finding was the marked effect of low-temperature culture alone on prolonging human islet xenograft survival because this treatment of the donor islets had no effect on the survival of rat islet xenografts. Intact human islets were present in approximately equal to 80% of the recipients after returning to a diabetic state, whereas xenografts of rat islets were completely destroyed. The findings indicate that complete rejection of islets across this widely discordant species barrier is slower than across a closely related barrier and may be occurring by a different rejection process.

Subpopulations of B lymphocytes in germinal centers.

With two new monoclonal antibodies and flow cytometry, we defined three subpopulations among B cells expressing binding sites for peanut agglutinin (i.e., B cells of the germinal center). On monoclonal antibody (5B5) binds globotriaosyl ceramide. The B lymphocytes binding 5B5 have binding sites for peanut agglutinin on the surface and express only small amounts of sIgD and sIgM. When tested against a panel of B cell lines, only Burkitt's lymphoma cells were 5B5+. Moreover, the 5B5+ cells have larger average sizes and a large fraction of proliferating cells. The other monoclonal antibody (HK23) binds a 90,000 protein. Lymphocytes binding HK23 are 5B5- and include T cells and a subpopulation of B cells. In contrast to 5B5+ cells, the HK23+ and peanut agglutinin positive B cells express a large amount of sIgM. These two subpopulations of germinal centers are distinct from the germinal center B cell subpopulation expressing the CD23 (Blast-2) antigen. The CD23+ B cells are 5B5- and express an intermediate level of HK23 antigen. In addition, CD23+ B cells are highly variable in number, whereas the proportions of HK23+ and 5B5+ cells are relatively stable.

Modulation of the murine immune response to streptococcal group A carbohydrate by immunization with monoclonal anti-idiotope.

Using monoclonal anti-idiotopes with previously defined specificities for the variable (V) domain of HGAC 39, a monoclonal antibody against streptococcal group A carbohydrate (GAC), we have studied the effect of anti-idiotope on an anticarbohydrate immune response. Anti-IdI-3a and anti-IdI-3b are anti-idiotopes which recognize binding site-associated determinants, whereas anti-IdX recognizes a framework-associated determinant on the HGAC 39 V kappa domain. Each of three anti-idiotopes elicited a specific idiotope response, as measured by inhibition radioimmunoassay, in A/J and C57BL/6J mice. A single immunization with conjugated anti-IdI-3a elicited an idiotope(+), GAC-binding(+) response in C57BL/6J and (BALB/c X CBA/N)F1 male mice, but not in A/J or (CBA/N X BALB/c)F1 male, X-linked immunodeficient mice. When C57BL/6J mice immunized initially with anti-idiotope were further treated with group A vaccine, those receiving anti-IdX had the greatest increase in anti-GAC activity. Stimulation of an anticarbohydrate response with anti-idiotope may therefore be enhanced by selecting anti-idiotopes against both binding site- and framework-associated determinants.

Syrinx 2A: an improved lambda phage vector designed for screening DNA libraries by recombination in vivo.

The Syrinx 2A phage and pi AN13 plasmid were designed for screening of DNA libraries by homologous recombination in vivo. Syrinx 2A carries multiple cloning sites and a recently identified lambda gene, rap (recombination adept with plasmid), required for efficient phage-plasmid recombination. We describe a rapid, reliable, and technically easy method to screen Syrinx 2A libraries, expand the resulting phage-plasmid cointegrates, and subclone plasmid in as little as 2 days. Recombination screening allows one specific member of a closely related multigene family to be isolated selectively.

Construction of an extended three-dimensional idiotope map by electron microscopic analysis of idiotope-anti-idiotope complexes.

A three-dimensional map of the positions of four idiotypic determinants (idiotopes or Ids) and an isotypic determinant was derived by transmission electron microscopy of negatively stained immune complexes. Each complex was composed of a monoclonal Id-expressing IgG and one or two varieties of monoclonal anti-Id (or anti-isotype) Fab fragment or IgG. Data from the various combinations of Id and anti-Id (and anti-isotype) were used to construct a low-resolution three-dimensional model that revealed not only the approximate locations of Ids on the surface of the antibody variable domains but also details of the geometry of Id-anti-Id interactions not otherwise available. The Ids were shown to be dispersed over the variable domains, extending from the complementarity-determining region to near the variable-constant switch region. Thus, immunoelectron microscopy is a useful complement to serologic, biochemical, and genetic strategies for the topographical analysis of immunoglobulin Ids or other epitopes. This same approach should be of broader applicability in the study of epitopes and receptor sites on other macromolecules.

Molecular dissection of the murine antibody response to streptococcal group A carbohydrate.

Monoclonal antibodies (mAb) to streptococcal group A carbohydrate (GAC) are encoded by a minimum of two VH, four JH, four V kappa, three J kappa, one V lambda, and one J lambda gene segments. The IdX, IdI-1, and Id5 idiotypic determinants are expressed by anti-GAC mAb and are found on free kappa chains. Each pattern of these determinants is encoded by a distinct V kappa gene segment, apparently without the requirement for a particular J kappa, VH, or JH gene segment, or somatic mutation. In contrast, the binding site-associated idiotypic determinant IdI-3a does not correlate with any single V or J gene segment.

Ir gene-controlled response to haptenated hen ovomucoid: isotypic specificity and dominant nonresponsiveness.

We have examined the isotypic pattern of the response of mice to the Ir gene-controlled antigen, dinitrophenyl-ovomucoid (DNP-OM). H-2 kappa mice are high responders (HR); (H-2b,d mice are low responders (LR). The isotype patterns of HR and LR strains differ both quantitatively and qualitatively. In the primary response to doses of 20-100 micrograms DNP-OM, HR strains produce IgM and IgG antibodies, whereas LR strains produce only IgM. Background genes modify the kinetics of the IgGl primary response in HR strains, but no background was found which allowed an IgG response in a LR strain. In secondary responses, priming with 0.2 microgram DNP-OM increases secondary responses in HR strains, and decreases them in LR strains. Control of this response maps to I-A, and is not altered by the bm 12 I-Ab mutation. The LR phenotype is dominant in (HR X LR)F1 mice.

Interaction of IgG3 anti-streptococcal group A carbohydrate (GAC) antibody with streptococcal group A vaccine: enhancing and inhibiting effects of anti-GAC, anti-isotypic, and anti-idiotypic antibodies.

Enhancement of binding of one monoclonal antibody to an antigen in the presence of a second monoclonal antibody (specific for an independent epitope on the same antigen) has been observed for several antigen-antibody systems involving primarily protein, or glycoprotein, antigens. We have analyzed the interaction between radiolabeled IgG3 kappa anti-streptococcal group A carbohydrate (GAC) antibody (125I-HGAC 39) and streptococcal group A vaccine (GAV; traditionally used to elicit anti-GAC antibody) in the absence and presence of unlabeled anti-GAC antibodies, anti-isotypic antibodies, or anti-idiotypic antibodies, respectively. A variety of significant enhancing or inhibiting effects on the binding of 125I-HGAC 39 to solid-phase GAV (GAVsp) were noted. First, high concentrations of IgG3 anti-GAC antibodies specifically inhibit binding of 125I-HGAC 39 to GAVsp, but the presence of lower concentrations of IgG3 anti-GAC antibodies is associated with markedly increased (up to 300 to 400%) binding of 125I-HGAC 39 to GAVsp. In contrast, with the concentrations used, IgM anti-GAC antibodies only inhibit binding of 125I-HGAC 39 to GAVsp. A monoclonal anti-gamma 3 antibody (2E.6) also enhances binding (up to 700%) of 125I-HGAC 39 to GAVsp, whereas another high-affinity anti-isotypic antibody, anti-C kappa (187.1), only inhibits binding of 125I-HGAC 39 to GAVsp. In a similar manner, an antiidiotypic antibody (anti-IdX) specific for a framework idiotope located near the C kappa domain inhibits the interaction between 125I-HGAC 39 and GAVsp. Evidence is presented to suggest that neither anti-C kappa nor anti-IdX blocks the HGAC 39 paratope, and therefore, the inhibition of binding mediated by these antibodies must be on some other basis. An alternative explanation for this effect, on the basis of the impairment of functional bivalency of 125I-HGAC 39, is discussed. Finally, anti-idiotypic antibodies (anti-IdI-3a and anti-IdI-1) that bind closer to the antigen-binding site of HGAC 39 inhibit binding of 125I-HGAC 39 to GAVsp in a manner that is most readily interpreted as competition for the GAC-binding site (or nearby sites) on the HGAC 39 variable domain. These effects are shown to require specific immunologic recognition of either GAVsp or 125I-HGAC 39.

Human parathyroid antigen: characterization and localization with monoclonal antibodies.

A cell surface antigen on human parathyroid cells was identified by monoclonal antibodies. The antigen, called parathyroid antigen (PTA), is found on both of two major polypeptides (190 kDa and 160 kDa) apparently associated exclusively with parathyroid cells. To determine whether PTA was a suitable target for in vivo imaging, 125I-labeled anti-PTA was injected into nude mice bearing human parathyroid xenografts. IgG1 anti-PTA antibody showed excellent radiolocalization of the grafts, whereas IgM anti-PTA, containing the same variable domains as the IgG1 antibody, showed little specific binding. These results suggest that antibody isotype is an important parameter for the in vivo immunoscintigraphy of specific cellular antigens.

Analysis of anti-streptococcal group A carbohydrate idiotope levels in sera: correlation of magnitude of expression with idiotope position and VK haplotype.

We have employed monoclonal anti-idiotopes to map the corresponding idiotopes on the variable domain of a prototype antibody specific for streptococcal group A carbohydrate (GAC). Idiotope variability, as assessed by direct and competitive binding assays, previously was shown to correlate with idiotope position; determinants farther from the binding site were less variable. We now describe a relationship between idiotope position and idiotope concentration in normal and GAC-immune sera from mice of several inbred and recombinant inbred strains. Employing sera as inhibitors in a competitive radioimmunoassay, we demonstrate that idiotopes farther from the binding site (more proximal) tend to be present at higher levels in GAC-hyperimmune sera. Only the most proximal idiotope was detected in normal serum, and this idiotope was present in normal sera from all 12 strains tested. Finally, significant interstrain differences in patterns of idiotope expression were observed, and some of these differences appear to be correlated with allelic differences at immunoglobulin structural gene loci.

A new polymorphic determinant on HLA-DQ molecules.

In man, the immune response genes are located within the HLA-D/DR region, and the gene products, the Ia antigens, are expressed on B lymphocytes, monocytes, and a percentage of null cells and activated T lymphocytes. We recently identified a human Ia antigen, K19, which appeared to be limited in its expression to B lymphocytes, and to be preferentially expressed on the more mature cells within this population. This work was facilitated by a monoclonal antibody. HK-19, which recognized a monomorphic determinant of this Ia molecule. We now report the characterization of a second monoclonal antibody, HK-13, which recognized the same molecule as HK-19, but only on cells from some individuals. The greater affinity of HK-13 allowed more complete characterization of the K19/K13 molecule. This characterization included cytofluorography, two-dimensional gel electrophoresis, tryptic peptide mapping, and partial N-terminal amino acid sequencing, and indicated that K19 and K13 were epitopes on HLA-DQ (DC) molecules. The pattern of reactivity of HK-13 on a panel of typing cells did not correlate with any of the known HLA-DQ polymorphic determinants. Thus, HK-13 is a new polymorphic determinant of the HLA-DQ series.