Hydrational and intrinsic compressibilities of globular proteins

To evaluate further bacterial DNA immunization as a model to study antigen drive in the anti-DNA response, the specificity of induced monoclonal anti-DNA antibodies was characterized. A panel of IgM and IgG monoclonal anti-DNA antibodies was produced from spleen cells of BALB/c mice immunized with single-stranded DNA from E. coli complexed to methylated bovine serum albumin in complete Freund's adjuvant. The binding of these antibodies to DNA and non-DNA antigens was tested by ELISA to assess their range of polyspecificity. These monoclonal antibodies were found to bind to nucleic acid as well as non-nucleic acid antigens, such as beta-galactosidase, cardiolipin, Ro, La and Sm. These studies demonstrate that anti-DNA antibodies from normal mice, although induced by bacterial DNA, may display a broad range of antigen recognition and thus resemble lupus anti-DNA antibodies, many of which are polyspecific, in their pattern of cross-reactivity.     

Radiodiagnosis of elbow and shoulder--questions by the clinician

Anti-double stranded(ds) DNA antibody is one of markers of systemic lupus erythematosus (SLE). Two human monoclonal anti-DNA antibody-producing cell lines were established from two SLE patients. One cell line secreted IgG isotype antibody (KSUG) and the other secreted IgM isotype antibody (KSUN). The light chains of the two immunoglobulins were lambda chains. The nucleotide sequences for the immunoglobulin variable region genes of the two antibodies were determined and compared to germline sequences. The heavy and lambda light chains of KSUG were VH3 family and V lambda IIIb, respectively. The heavy and lambda light chains of KSUN were VH4 family and V lambda IX, respectively. Antibody KSUG, IgG isotype, showed somatic mutations, whereas KSUN, IgM isotype, used the germline gene without mutation. These findings reconfirm the current paradigms that IgM anti-DNA antibodies are produced by utilizing germline genes whereas IgG anti-DNA antibodies are produced by somatic mutations.     

Independently derived IgG anti-DNA autoantibodies from two lupus-prone mouse strains express a VH gene that is not present in most murine strains

The origin and structure of two clonally unrelated IgG anti-DNA autoantibodies from lupus-prone MRL/Ipr mice were examined. One of these antibodies, H241, binds dsDNA and glomeruli and deposits in the kidneys of normal mice, whereas the other, H102, binds only ssDNA and does not deposit in kidneys. The VH genes of these two antibodies were almost identical to each other and were frequently expressed in anti-DNA antibodies derived from lupus-prone mice. Six other clonally unrelated anti-DNA antibodies from the literature or from data banks expressed nearly identical VH genes (< or = 4 nucleotide differences) and eight others had nearly identical protein sequences (< or = 3 amino acid differences). Analysis of the germ line with oligonucleotide probes from the CDR regions suggests that all 10 autoantibodies are derived from a single member of the J558 gene family, which is present only in mice with the j haplotype for the J558 gene family. The amount of somatic mutation in these VH genes appears to be low, suggesting that some V, N, and D gene combinations can generate high affinity IgG anti-DNA auto-antibodies with little or no somatic mutation. Unusual reading frames, D-D fusions, and inversions were common in the IgG antibodies and may have been co-selected. Although the N and D regions of one IgM and all five IgG autoantibodies contained Arg residues, the presence of Arg residues was not correlated with binding to dsDNA or with pathogenicity. These results suggest that differences in the Ag-binding properties and the pathogenicity of these antibodies are determined by the CDR3 region and the L chain.     

Autoreactivity of human VH domains from cDNA libraries: analysis with a bacterial expression system

Previous studies showed that VH domains of several anti-DNA Abs can bind DNA in the absence of VL. In the current work, we tested the VH autoreactive potential more generally, examining VH domains that did not come from known autoantibodies. Using a bacterial expression system, we produced 11 fusion proteins, each containing a VH domain and a B domain of staphylococcal protein A. The VH domains were coded in cDNA libraries from circulating B cells of healthy young adult humans. Thus, binding properties of the Ig molecules from which they came were unknown. The B cells had not been stimulated in vitro. Seven cDNA clones combined the frequently expressed VH3-23 gene segment with varied DH and JH segments. The other clones contained unmutated VH3-7, VH3-9, VH3-53, and VH4-39 segments. We compared these bacterial expression products with single-chain Fv, VH and VL domains of IgM mAb 18/2, a VH3-23-encoded, DNA-binding autoantibody. Submicromolar concentrations of 5 of the 11 VH domains bound to ssDNA. Those and one more also bound to immobilized poly(dT), and two bound to circular plasmid dsDNA. Soluble poly(dT) was the most potent inhibitor in competitive ELISA. Seven of the VH domains also bound to immobilized nuclear ribonucleoprotein, four to histone and none to thyroglobulin. Two interacted with the matrix of a Sephacryl S-100 column. The polyreactive autoantigen-binding properties of these VH domains raise the question of whether these properties may play a role in the formation of the VH repertoire of circulating B cells.     

Lasers in pediatric surgery

OBJECTIVE: To characterize immunologic specificity and possible antiidiotype activity of IgG anti-F(ab')2 in normal subjects as well as in patients with active and inactive systemic lupus erythematosus (SLE). METHODS: IgG anti-F(ab')2 and anti-double-stranded DNA (anti-dsDNA) were affinity isolated from immunoadsorption columns of F(ab')2 and dsDNA linked to Sepharose 4B. Affinity-purified IgG anti-F(ab')2 (APAF) and affinity-isolated IgG anti-dsDNA (APAD) were tested by enzyme-linked immunosorbent assay (ELISA) for other cross-reacting specificities including anti-Sm, anti-Sm/RNP, and anti- Crithidia binding. Anti-DNA specificity of APAF and APAD was assayed by S1 nuclease treatment of heat-denatured DNA. Rabbit antiidiotypic antisera were prepared by immunization with APAF and APAD from normal subjects and SLE patients and absorption with insolubilized human Cohn fraction II (Fr II). VL and VH regions of 5 monoclonal IgM antibodies with anti-F(ab')2/anti-DNA specificity generated by Epstein-Barr virus B cell stimulation were sequenced by polymerase chain reaction and characterized for VH and VL subgroup. APAF and APAD were also examined by high-resolution electron microscopy for possible ring forms indicative of antiidiotypic V-region interactions. RESULTS: APAF from normal subjects, representing 0.08-0.18% of serum IgG, showed striking relative concentrations of both anti-F(ab')2 and anti-DNA, as well as anti-Sm and anti-Sm/RNP ELISA reactivity. Both APAF and APAD reacting with F(ab')2 or dsDNA on the ELISA plate could be cross-inhibited by F(ab')2 or DNA in solution. Anti-DNA reactivity in normal APAF and APAD was much more sensitive to S1 nuclease treatment than similar fractions from SLE patients. Neither APAF nor APAD from controls produced positive antinuclear immunofluorescence or positive Crithidia staining, whereas these were strongly positive using SLE APAF and APAD. Absorbed rabbit antisera against normal or SLE APAF and APAD showed strong ELISA reactivity against both APAF and APAD, but no residual reactivity with normal Fr II. VL and VH sequencing of monoclonal human IgM antibodies showing both anti-F(ab')2 and anti-DNA reactivity showed relative VH3, V kappa 1 or VH1, V kappa 3 restriction. No evidence of ring forms or V-region "kissing" dimers was obtained when normal or SLE APAD or APAF was examined by high-resolution electron microscopy. CONCLUSION: IgG anti-F(ab')2 in both normal subjects and SLE patients represents a polyreactive Ig subfraction with concomitant anti-DNA, anti-Sm, and anti-Sm/RNP specificities. Anti-DNA reactivity in SLE is qualitatively different from that in normal APAD and APAF since normal APAD and APAF anti-DNA is much more sensitive to S1 nuclease digestion of denatured dsDNA. APAF and APAD share distinct V-region antigens which may be related to prominent VH3 or VH1 antigenic components. No evidence for in vivo complexing of anti-DNA and anti-F(ab')2 as ring forms or antiidiotype-IgG complexes was observed during ultrastructural studies. In both normal individuals and SLE patients, APAF may represent a small polyreactive IgG subfraction which also contains antinuclear and anti-DNA specificities.     

Anti-DNA, anti-deoxyribonucleoprotein and rheumatoid factor measured by ELISA in patients with systemic lupus erythematosus, Sj?gren's syndrome and rheumatoid arthritis

Serum concentrations of anti-DNA and anti-deoxyribonucleoprotein (NP) antibodies were measured in parallel by standardized ELISA methods with a polyvalent anti-immunoglobulin conjugate in patients with systemic lupus erythematosus (SLE), Sj?gren's syndrome (SS) and rheumatoid arthritis (RA). High levels of these antibodies predominated in systemic lupus erythematosus. While an appreciable incidence of antibodies also occurred in SS and RA, they were mostly at lower levels. By using heavy chain-specific anti-immunoglobulin conjugates, IgG antibodies to both DNA and NP were found in SLE more frequently and at higher levels than were IgM antibodies. In contrast, IgM antibodies to DNA and NP predominated in SS and RA. The immunoglobulin class of the anti-DNA and anti-NP responses in a given SLE patient were not infrequently different. For example, a patient might show a very high IgG but low IgM anti-DNA value, with the reverse being true for anti-NP. IgG anti-DNA antibodies were significantly associated with depressions of C3. During changes in SLE serology, normalization of DNA binding by Farr radioimmunoassay and/or complement was most frequently associated with normalization of the IgG anti-DNA antibody concentrations. In patients simultaneously possessing elevated levels of anti-DNA, anti-NP and rheumatoid factor (RF), absorption with aggregated human IgG usually decreased only the RF activity. In some, however, such absorption decreased all three antibody values simultaneously. The latter findings support observations that some RF possess antinuclear properties.     

Expression of monovalent fragments derived from a human IgM autoantibody in E. coli. The input of the somatically mutated CDR1/CDR2 and of the CDR3 into antigen binding specificity

A hybridoma producing a polyspecific human monoclonal IgM antibody (named CB03) has been derived from a fusion of mouse myeloma cells with human spleen lymphocytes obtained from an autoimmune patient suffering from chronic idiopathic thrombocytopenia. The antibody was found to be encoded by somatically mutated VHI and VlambdaIII genes. To study the input of mutated complementarity regions (CDRs) into antibody specificity, the antigen binding features of the purified complete IgM antibody were compared with (i) a Fab fragment by hot tryptic digestion and (ii) recombinant monovalent fragments expressed in E. coli. In detail, vectors were constructed encoding for (i) rFab03 and single chain Fv03 fragments containing the VH and VL genes connected by a linker sequence, (ii) scFc1.1. fragments containing the VH germline equivalent and the CB03 wild-type CDR3 region, and (iii) scFv fragments containing the CDR1 and CDR2 in germline configuration and the CDR3 expressed in the CB253 human fetal B cell hybridoma producing a polyspecific IgM antibody. The expression vectors contained at the 3' end either a (His)6 motif allowing purification on Ni(2+)-agarose or a c-myc tag for specifically detecting the expression products by a murine monoclonal antibody. Western blotting and ELISA analyses of the expression products indicate: (i) recombinant Fab fragments were found in the bacterial periplasm in extremely low amounts (1-10 micrograms from 1 litre bacterial culture), (ii) scFv fragments were obtained in suitable amounts from bacterial periplasm (800-1000 micrograms/l), (iii) the monovalent recombinant fragments as well as the Fab obtained by tryptic digestion reflected the polyspecific antigen binding features of the complete IgM antibody, but did bind to the antigens with much lower affinity, and (iv) the CDR3 was found to be of critical importance for the antigen binding pattern of this particular IgM. We discuss the expression of recombinant scFv fragments in E. coli as a suitable method in studying the role of the somatic mutation in autoantibody generation.     

Clinical utility of immunoglobulin heavy chain gene rearrangement identification for tumour cell detection in multiple myeloma

To clarify the cellular origin of de novo CD5+ diffuse large B-cell lymphoma (CD5+ DLBL), particularly in comparison with other CD5+ B-cell neoplasms such as chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL), we analyzed the nucleotide sequence of the Ig heavy chain variable region (IgVH) genes of de novo CD5+ DLBL cases. All 4 cases examined had extensive somatic mutations in contrast with CLL or MCL. The VH gene sequences of de novo CD5+ DLBL displayed 86.9% to 95.2% homology with the corresponding germlines, whereas those of simultaneously analyzed CLL and MCL displayed 97.6% to 100% homology. The VH family used was VH3 in 1 case, VH4 in 2 cases, and VH5 in 1 case. In 2 of 4 examined cases, the distribution of replacement and silent mutations over the complementarity determining region and framework region in the VH genes was compatible with the pattern resulting from the antigen selection. Clinically, CD5+ DLBL frequently involved a variety of extranodal sites (12/13) and lymph node (11/13). Immunophenotypically, CD5+ DLBL scarcely expressed CD21 and CD23 (3/13 and 2/13, respectively). These findings indicate that de novo CD5+ DLBL cells are derived from a B-1 subset distinct from those of CLL or MCL.     

Effect of acute and chronic arecoline treatment on cerebral metabolism and blood flow in the conscious rat

To investigate the structural contribution of the light chain of anti-DNA antibodies to fine specificity, the VKappa genes of two monoclonal anti-DNA antibodies, termed H241 and H102, were cloned and sequenced. H102 and H241 are independently derived from MRL-lpr/lpr mice and differ in their fine specificity: H241 binds dsDNA and normal glomeruli in vitro and deposits in the kidney in vivo, whereas H102 binds only ssDNA and does not deposit in the kidney. Both are encoded by nearly identical VH genes but different N and D regions. Our previous results have demonstrated that the VH gene for H102 and H241 encodes eight other anti-DNA antibodies that also differed in fine specificity. This suggested that the gene product encoded by the VH 102/241 gene, may have intrinsic affinity for DNA, but is unlikely to determine fine specificity or nephritogenicity. In the present study we examined whether the VKappa gene might account for the difference in nephritogenicity. The complete nucleotide and deduced amino acid sequence of VK 102 and VK241 revealed that they are very dissimilar to each other (< 60% homology). VK 241 defined a new member of the VKappa gene family and was moderately homologous to two other VK genes encoding anti-DNA antibodies and to one VK gene encoding an anti-histone antibody all from lupus strains of mice. In addition, sequence diversity in the VK CDR1 region and position 96 of the CDR3 region was observed that may be of significance in determining fine specificity. VK 102 was highly homologous to two other VKappa genes, VKs17.2 and VK C8.5, both encoding anti-DNA antibodies and members of the VK20 gene family. It was striking that all three members of the VK 20 gene family code for DNA reactivity. This suggests that certain VKappa genes may also be used to repeatedly code for anti-DNA reactivity.     

Somatic diversification and affinity maturation of IgM and IgG anti-DNA antibodies in murine lupus

Molecular events occurring during the process of generation of pathogenic immunoglobulin (Ig)G anti-DNA antibodies in systemic lupus erythematosus (SLE) were studied using a newly established method. We analyzed the Ig variable (V) region gene sequence and DNA-binding activity of IgM and IgG anti-DNA monoclonal antibodies (mAb) from individual SLE-prone (NZB x NZW) F1 mice. The first event appeared to be clonal selection and expansion of IgM anti-DNA clones, in which several clones had intraclonal V gene mutations. Although the number of mutations was small, the mutated IgM clones were associated with an increase in DNA-binding activity. The somatic mutations located in complementarity-determining regions (CDR) and in framework regions (FR) of V genes were apparently related to changes in DNA-binding activity. IgG anti-DNA clones that progressively increased in number with aging had numerous somatic mutations in the V region genes and there was a pair of clones which showed an intraclonal accumulation of mutations, in association with increase in the DNA-binding activity. All these findings show that somatic mutations associated with affinity maturation of the V region begin immediately before isotype-switching from IgM to IgG of the clones that have been selected and expanded, in an antigen-driven manner and/or by other forces. We propose that further accumulations of intraclonal somatic hypermutation, in association with selection and expansion of high affinity IgG clones, may lead to formation of highly pathogenic anti-DNA antibodies.     

Molecular basis for the interaction between human IgM and staphylococcal protein A

To examine the relationship between VH gene usage and reactivity of immunoglobulins, we cloned B cells from peripheral blood from adults and from human neonatal cord blood by EBV transformation. Nearly one-third of the B cell clones from both sources produced IgM reactive with staphylococcal protein A (SPA). None of such IgM reacted with other antigens, except for the crude extract of Staphylococcus aureus. All of 22 B cell clones producing IgM reactive with SPA expressed VH3 genes, while none of the control 15 clones secreting IgM nonreactive with SPA expressed VH3. The IgM proteins reactive with SPA could be clearly divided into two subjects based on the differential binding avidity to solid-phase SPA. Both kappa and lambda light chains were used in each subset of SPA-reactive IgM. Sequence analysis of the PCR products from seven VH3-IgM clones revealed that the VH3 genes were used in nearly germline configuration. The D and J gene usage was diverse. Comparison of amino acid sequences between antibodies with high and low avidity to SPA suggests that the differential avidity is related to amino acid sequence differences in the complementarity determining region 2 and framework region 3. The high frequencies of B cells committed to the production of SPA-reactive IgM in normal blood and the restricted use of VH3 heavy chain genes in nearly germline configuration in these cells support the notion that SPA behaves like a superantigen toward human B cells.     

Quantitative assessment of IgM antibodies towards an immunodominant B-cell epitope within the preS2 domain of HBV in the natural course and during combined prednisone/interferon alpha 2b treatment of chronic hepatitis B virus infection

A direct binding enzyme-linked immunosorbent assay (ELISA) was established for quantitative determination of serum IgM antibodies towards a synthetic peptide corresponding to a selected segment (14-21) of the preS2-gene product containing an immunodominant linear B-cell epitope. The prevalence of IgM anti-preS2 (14-21) antibody titers > 1,000 for hepatitis B e antigen (HBeAg)-positive patients with chronic hepatitis B virus (HBV) infection was 38% (22/58) and 10% (2/21) for HBeAg-negative subjects (P < 0.005). IgM anti-preS2 (14-21) reactivity was detected during the clinical course of chronic HBV infection and IgM anti-peptide antibody titers declined and disappeared before spontaneous HBe/anti-HBe seroconversion. Recombinant interferon (IFN)-alpha 2b with an antecedent short course of corticosteroids was administered to eight Chinese patients with chronic HBV infection. The IgM anti-preS2 (14-21) reactivity was monitored consecutively during treatment and patients were followed for more than 1 year. A close association between the presence of pretreatment IgM anti-preS2 (14-21) in serum and the capacity to respond favorably to the combined prednisone/IFN-alpha 2b therapy was detected. The IgM anti-preS2 (14-21) titers decreased during treatment with subsequent loss of detectable antibodies 8-16 weeks after the initiation of therapy. This decrease was concomitant with an alanine aminotransferase (ALT) augmentation preceding the disappearance of HBV-DNA and anti-HBe seroconversion. Long-term remission was not observed in treated patients who lacked detectable levels of pretreatment IgM anti-preS2 (14-21) in the circulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

The J558 VH CDR3 region contributes little to antibody avidity; however, it is the recognition element for cognate T cell control of the alpha(1-->3) dextran-specific antibody response

Analysis of the humoral immune response of BALB/c mice to alpha(1-->3) dextran (Dex) reveals novel aspects of T cell-mediated control of 'type 2 thymus-independent' responses against polysaccharide antigens. The IgM and IgG antibody response, dominated by the J558 idiotype (Id), is controlled by Id-specific T cells. These regulatory T cells, for which the T cell clone 178-4 Ts with characterized TCR alpha and beta chain sequences is the prototype, expand in all BALB/c mice upon immunization with Dex. They suppress in a cognate interaction the expansion of J558 Id-bearing B cells, committed for production of IgG antibodies. Furthermore they provide a gate which precludes variability in the VH CDR3 region of IgG antibodies appearing occasionally in the periphery. The VH CDR3 region is the recognition element of 178-4 Ts analogous T cells but contributes little to affinity for the antigen. For recognition by 178-4 Ts cells not even minimal sequence deviations of the J558 Id peptide are allowed. The tight germline programmed complementarity between J558 Id-bearing Dex-specific B and J558 Id-specific 178-4 Ts analogous T cells leaves little room on both sides for ontogenetic variability.     

Heterogeneity of anti-phospholipid and anti-endothelial cell antibodies

The role of the anti-phospholipid antibodies (APLA) and anti-endothelial cell antibodies (AECA) in the pathogenesis of anti-phospholipid syndrome (APS) is unclear. Differences in the reported involvement of APLA may be due, in part, to the polyclonal nature of these antibodies and the use of serum and serum fractions for analysis. To circumvent this issue, we generated monoclonal antibodies (MAB) from three patients with APS and two healthy controls. We then compared the antigen binding patterns and the heavy chain variable region (VH) DNA sequences of the MAB derived from patients with APS to those from healthy controls. The results of this study indicate that APLA and AECA comprise a highly heterogeneous population of antibodies with respect to the antigens they recognize, as well as VH gene usage. MAB derived from patients with APS do not differ from those derived from normal individuals based on either antigen recognition or VH gene usage. These results suggest the importance of additional predisposing factors in the pathogenesis of APS.     

Anti-idiotype monoclonal antibodies specific for the MOPC167 anti-phosphocholine transgene-encoded antibody

Four rat x mouse hybridomas secreting monoclonal anti-idiotypic (anti-Id) antibodies (MAb) specific for the transgene-encoded antibody of the 207-4 transgenic mouse line, which carries the VH1/V kappa 24 gene segments of the IgA, phosphocholine-(PC) specific MOPC167 myeloma, were developed from a fusion of Ag8-X63.653 mouse cells with spleen cells from a rat immunized with MOPC167 and HPCM27 anti-PC antibodies. The anti-Id MAb were shown by ELISA to be specific for PC-binding proteins of VH1/V kappa 24 H and L chains of various isotypes. They did not bind VH1/V kappa 22, VH1/V kappa 8, or VH1/V kappa 1 PC-binding proteins or other IgA or IgM myeloma proteins. Analysis by flow cytometry demonstrated that these MAb bind to the transgene-encoded membrane immunoglobulin (sIgM) as expressed on > 95% of the B220 positive 207-4 spleen cells. All four MAb were able to inhibit the binding of MOPC167 to PC conjugated to bovine serum albumin. Differences in fine specificity of binding were demonstrated by differential staining of spleen cells of the 216-7 mu kappa delta Mem MOPC167 transgenic mice. In these mice endogenous H chains associate with the transgene encoded L chain to form MOPC167 crossreactive idiotopes. Two of the MAb, 28-4-3 and 28-6-20, stained significant numbers of cells, while MAb 28-5-15 did not bind to 216-7 cells. Three of the MAb, 28-5-15, 28-6-20, and 28-4-3, when conjugated to Sepharose beads, were able to induce DNA synthesis in cultures of 207-4 transgenic spleen cells. None of the MAb were able to induce an antibody response in vivo. These MAb should prove useful in staining PC-transgenic B cells for flow cytometry studies and in defining early cellular events in the activation of idiotype positive B cells by anti-Id antibodies.     

A single predominantly expressed polymorphic immunoglobulin VH gene family, related to mammalian group, I, clan, II, is identified in cattle

In order to understand the generation of antibody diversity in cattle, seven cDNAs, from heterohybridomas secreting bovine IgM and IgG1 antibodies, were cloned and structurally analyzed for rearranged bovine VDJ genes. All of the seven bovine VH genes, together with four available bovine VH gene sequences, shared a high nucleotide sequence homology (84.2-93.5%). Based upon the criteria of nucleic acid homology > or =80%, all of the bovine VH gene sequences isolated from the expressed antibody repertoire constitute a single VH gene family, which we have designated as bovine VH1 (Bov VH1). An analysis of 44 bovine IgM-secreting mouse x cattle heterohybridomas, originating from polyclonally-activated PBLs from bovine leukemia virus-infected cattle, revealed that all of these expressed Bov VH1 (100%) based upon DNA sequencing and Northern dot blot. The bovine VH genes showed highest DNA sequence similarity, ranging between 81.5 and 87.6%, with a single sheep VH gene family (related to human VH4) and are, thus, closest to the VH genes from another ruminant species. The Bov VH1 gene family is most homologous to the murine VH Q-52 (71.8-78%) and human VH4 (67.4-69.8%) gene families, which belong to mammalian group, I, clan, II, VH genes. The CDR3 length of rearranged bovine VDJ genes is characteristically long (15-23 amino acids). The bovine JH gene segments were most homologous to human JH4 (82.1-87.2%) and JH5 (84.6-89.7%) genes, suggesting the existence of at least two JH gene segments. An analysis of CDRs provides evidence that somatic hypermutations contribute significantly to the generation of antibody diversity in cattle. Southern blot analysis of BamH I, EcoR I and Hind III digested genomic DNA from four cattle breeds (Holstein, Jersey, Hereford and Charolais) revealed three RFLP patterns; the genomic complexity of Bov VH1 ranged between 13 and 15 genes. These observations provide evidence for polymorphism at the bovine Ig-VH locus, similar to that seen in mice and humans.     

Elevated carbohydrate-deficient transferrin predicts prolonged intensive care unit stay in traumatized men

Antibodies to DNA are believed to play an important role in systemic lupus erythematosus (SLE). High affinity IgG antibodies which show marked specificity for double stranded DNA (dsDNA) are particularly closely linked to the occurrence and severity of tissue damage. Sequence analysis of mouse and human monoclonal antibodies has previously suggested that mutations in the complementarity determining regions (CDRs) play a major role in determining these binding properties. In many cases such mutations increase the overall number of basic residues in the CDRs. To further elucidate the role played by such mutations it is important to develop methods of expressing cloned autoantibody cDNA in the form of functional whole immunoglobulin molecules. We describe a system in which autoantibody VH and VL cDNA from monoclonal human anti-DNA antibodies, B3 and WRI176 were cloned into separate vectors which allowed their expression as whole heavy and whole light chains respectively. By cotransfecting mammalian cells with pairs of heavy and light chain vectors it was possible to produce whole IgG molecules from each of the four possible VH/VL combinations. Only antibody produced by homologous VH and VL pairs bound DNA, suggesting that in these autoantibodies both chains are important in conferring this property.     

Structure and binding properties of monoclonal antibodies to core histones from autoimmune mice

Histones are frequent targets of self-reactive antibodies during autoimmune syndromes. We report the specificities and V region genes of three IgG anti-histone MAbs obtained from autoimmune mice. Each of the MAbs, named LG2-1, LG2-2 and BWA3, is directed against a different determinant located in the basic amino-terminal domain of core histones. LG2-1 reacts with a peptide from histone H3 (residues 30-45), LG2-2 recognizes the amino-terminus of H2B (residues 1-13) and BWA3 binds an epitope corresponding to a region of high sequence similarity between H2A and H4 (residues 1-20 and 1-29, respectively). The analysis of their V region sequences indicates that the H chain CDRs of these MAbs are remarkable for the presence of negatively charged amino acid residues that may play a role in the binding to cationic histones. The H chain importance in conferring reactivity to histones is corroborated by the observation that each of the VH gene segments of these MAbs is very similar to VH genes of previously described murine anti-histone antibodies.     

The double edged sword of the immune response: mutational analysis of a murine anti-pneumococcal, anti-DNA antibody

Anti-double-stranded (ds) DNA antibodies are not only an important diagnostic marker for SLE, but also play an important role in tissue injury. Microbial antigen may be a stimulus for the production of these antibodies. We isolated 99D.7E, an IgG2b monoclonal antibody from a nonautoimmune BALB/c mouse that is cross-reactive with both dsDNA and phosphorylcholine, the dominant hapten on the pneumococcal cell wall. While partially protective against a bacterial challenge, 99D.7E is also pathogenic to the kidney. To identify those molecular motifs that confer on anti-PC antibodies the potential for autoreactivity, we created a panel of 99D.7E mutants with single amino acid substitutions in the heavy chain, and examined the changes in antigen binding and renal deposition. Our results support the hypothesis that charge and affinity for dsDNA are not adequate predictors of the pathogenicity of anti-DNA antibodies. Differential renal damage from anti-dsDNA antibodies may be due to differences in fine specificity, rather than differential affinity for dsDNA. Importantly, high affinity IgG antibodies cross-reactive with bacterial and self antigen exist and can display pathogenic potential, suggesting that defects in peripheral regulation of B cells, activated by foreign antigen but cross-reactive with self antigen, might lead to autoimmune disorders.