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D L Robbins

Publications and source records attributed to D L Robbins.

At least 19 recordsLinked to original sources
PubMed2004

Vibrational spectra of germanium-carbon clusters. II. GeC(7) and GeC(9).

Experimental and theoretical studies of a novel family of germanium-carbon clusters (Ge(n)C(m)) that were initiated with our earlier identification of the GeC(3)Ge cluster have now been extended to the GeC(7) and GeC(9) chains. The new clusters, which were formed by laser ablation and trapped in solid Ar at approximately 10 K, have been identified using Fourier-transform infrared (FTIR) measurements coupled with density-functional theory (DFT) calculations. The nu(1)(sigma) vibrational fundamental of linear GeC(7) has been identified at 2063.6 cm(-1), and an absorption at 1928.3 cm(-1) has been assigned to the nu(4)(sigma) fundamental of linear GeC(9). FTIR measurements of the isotopic shifts for the assignments are in good agreement with the DFT predictions.

Journal Article↗
PubMed2001

Mutational analysis of immunoglobulin germline derived Vlambda4A light chains in rheumatoid arthritis.

In order to better characterize the expression of a family of light chains previously expressed in IgM rheumatoid factors, we studied the usage and somatic mutational pattern of the Vlambda4A light chain gene in rheumatoid arthritis (RA) patients. We sequenced 11 different transcripts of Vlambda4A from the synovial tissue of three different RA patients. For comparison, we found 8 rearranged transcripts of Vlambda4A from 4 normal peripheral blood lymphocyte libraries and 1 rearranged transcript from a non-RA con-A-resistant hybridoma in GenBank. A previously undescribed polymorphism of Vlambda4A was noted. Furthermore, conserved replacement mutations in the complementary determining regions, common silent mutations around these replacement mutations, and two subsets of mutated sequences were detected in multiple RA patients. These mutation patterns also correlated with observed consistencies in the rearrangements of the Vlambda4A/Jlambda junction. These data suggest that there is clonal expansion of Vlambda4A light chains in the RA synovium in response to a RA-specific antigen or as the result of an idiotypic response in RA.

Aged↗
PubMed2001

Mutational analysis of immunoglobulin germline derived Vlambda4B light chains in rheumatoid arthritis.

OBJECTIVE: We investigated the somatic mutational pattern of a specific Vlambda light chain variable region (V) gene in rheumatoid arthritis (RA) patients. The Vlambda4B light chain was chosen because of its location on the lambda locus and because of its previously observed use in IgM rheumatoid factors. METHODS: We sequenced 13 different mRNA transcripts of Vlambda4B from the synovium of three different RA patients. These were compared to 31 identifiable Vlambda4B sequences from GenBank, which were obtained from the PBL of patients without RA. RESULTS: A subset of Vlambda4B had a high rate of mutation, especially in the framework regions within the RA synovium. Furthermore, a set of codons within the first complementary determining region of Vlambda4B displayed frequent replacement mutations but did not possess any silent mutations. CONCLUSION: The hypermutation of RA synovial-derived Vlambda4B sequences, especially in the framework areas, may contribute to or may be the result of altered mutational mechanisms and/or prolonged B cell life.

Aged↗
PubMed1999

The specificity of synovial IgM rheumatoid factors (RF) for genetically engineered IgG antibodies is not affected by the method used to immortalize RF-producing B cells.

Previously, we have shown that some rheumatoid factors (RFs) produced by Epstein-Barr virus (EBV)-transformed B cells from patients with rheumatoid arthritis (EBV-RA-RF) appear to be disease-specific autoantibodies that bind differently to defined epitopes on genetically engineered IgG antibodies, compared with RFs expressed by patients with Waldenstrom's macroglobulinaemia (Wmac-RFs) and healthy immunized donors (HID-RFs). To exclude the possibility that EBV transformation is responsible for these differences, we have now studied 15 other monoclonal IgM RFs from patients with RA that were produced by heterohybridoma-B-cell fusion (HRA-RFs). These HRA-RFs show the same gross specificity profiles for IgG as do their EBV-RA-RF counterparts. However, when the specificities of the HRA-RF and EBV-RA-RF panels were combined and compared with those RFs from patients with Wmac or HID, significant differences in binding specificity were again observed. Hybrid IgG3/4 antibodies made by exon shuffles between the IgG3 and IgG4 wild-type genes, and families of IgG variant antibodies made by site-directed mutagenesis, were used to map the fine specificity of HRA-RFs. The fine specificity of HRA-RFs were also similar to those of EBV-RA-RFs. These studies demonstrate that the method used for immortalizing IgM, RF-producing B cells from RA patients does not influence the specificity of the RFs obtained. Furthermore, some RFs expressed in RA have distinct and unique specificities, and may therefore represent disease-specific autoantibodies.

Animals↗
PubMed1998

Detection system for reaction-rate analysis in a low-volume proteinase-inhibition assay.

High-throughput screening of large combinatorial chemical libraries in biochemical assays will benefit from reduced reagent volume and increased speed of measurement. Standard assays typically are performed in 96-well microtiter plates having 200-microL well volumes and up to an hour of incubation time. In this paper, we demonstrate a technique for precise and rapid measurement of the progress of an enzymatic reaction and its inhibition with reduced volume and time (for this work, the assay was mixed at the 200-microL level and detected in 2-microL volumes with minutes of total assay time). Directly measuring the enzyme activity in the small volume format yields a precise value for the median inhibitory concentration (IC50) of an inhibitor compound. The model assay is the endoproteolytic cleavage of a small fluorogenic peptide by human neutrophil collagenase (MMP-8). The fluorogenic peptide was labeled at one end with a UV/blue fluorophore (N-methylanthranilyl) and at the other end with a quencher (dinitrophenol). To generate inhibition data, a hydroxamate peptide analog inhibitor of collagenase, actinonin, was included in the reaction. The experiments were performed using ultraviolet laser illumination (325 nm wavelength) and parallel fluorescence detection by a cooled, charge-coupled-device camera system to increase sensitivity and speed. The assay volume was reduced to 2 microL for data collection, and the total time for mixing, incubation, and measurement was less than 6 min. For comparison to a standard format, the same assay was performed in a 96-well microtiter plate in 200 microL using 30 min of incubation and measurement in a microtiter plate fluorimeter. Median inhibitory concentrations (IC50) for actinonin of 73 +/- 16 and 100 +/- 14 nM were obtained in the 2- and 200-microL assays, respectively. One concern with assay miniaturization and increases in throughput is a potential loss of precision and accuracy. Laser excitation and parallel detection of fluorescence is a promising approach for increased speed and reduced cost without loss of precision for proteinase inhibition assays.

Fluorescence↗
PubMed1998

Effect of anticardiolipin/beta2-glycoprotein I complexes on production of thromboxane A2 by platelets from patients with the antiphospholipid syndrome.

OBJECTIVE: Antiphospholipid antibodies (aPL) reactive with anionic phospholipids and beta2-glycoprotein I (beta2-GPI) are found in the sera of patients with autoimmune diseases. Clinically, aPL/beta2-GPI complexes are associated with arterial and venous thrombosis, fetal loss, and thrombocytopenia, i.e., the antiphospholipid syndrome (APS). The mechanism of thrombosis is not known. We hypothesized that aPL/beta2-GPI complexes could perturb the platelet membrane and increase production of thromboxane A2 (TXA2, a proaggregatory prostanoid). METHODS: We isolated an IgG fraction containing anticardiolipin antibody (aCL) and the plasma cofactor, beta2-GPI, from a patient with a high titer of aCL and thrombotic cerebrovascular disease. We then examined the effect of aCL, beta2-GPI, and the aCL/beta2-GPI complex on platelet TXB2 (a stable metabolite of TXA2) biosynthesis in vitro from 7 healthy controls. We also measured in vitro platelet TXB2 biosynthesis in 7 patients with APS and in 8 controls. RESULTS: We found: (1) significantly increased in vitro TXB2 production by platelets from controls after incubation with aCL/beta2-GPI complexes; (2) moderately increased TXB2 production by aCL alone; (3) no increase in TXB2 production by beta2-GPI alone; and (4) significantly increased 11-dehydro-TXB2, a metabolite of TXB2 production in vivo, in the urine of patients with APS compared with controls. CONCLUSION: These data suggest that aCL/beta2-GPI complexes play a role in activating platelets to produce TXA2, which could contribute to the prothrombotic state found in patients with APS.

Adult↗
PubMed1997

Analysis of the molecular basis of synovial rheumatoid factors in rheumatoid arthritis.

The objective of this study was to better understand the molecular basis of IgM rheumatoid factor in rheumatoid arthritis (RA). We recently generated 10 different monoclonal IgM RF (mRF) molecules isolated from the synovium of a single patient with RA. The heavy (H) and light chain (L) variable region (V) genes of these 10 mRFs were cloned and sequenced. Six mRFs used kappa light chains and 4 mRFs used lambda light chains. Of particular interest, 8 of 10 heavy chains used the JH4 joining region gene, and all five VH4 heavy chains used the DK4 diversity region gene with the JH4. Four of the VH4 clones used the same germline gene, likely representing a novel but closely related germline gene to VH4.18, and may be clonally related because of the extensive homology in their heavy chain sequence. Two VH4 clones shared the same light chain gene, VkappaIIIb kv325 (99% homology) and the same JK4 joining region gene, while three VH4 clones used two different light chain genes, an uncommon Vkappa4 and a Vlambda4 gene, respectively. In this RA patient, there was recurrent utilization of VH4-DK4-21/10-JH4 genes and a recurring association with gene elements Vkappa3 and Vlambda4. Recurring usage of Vkappa3 (kv325) and Vlambda4 (lv418) gene elements may result from a light chain editing process whereby immature autoreactive B cells encountering self-antigen attempt, and often succeed, in altering their specificities through secondary Ig light chain gene rearrangement. Moreover, the oligoclonality of these RFs suggest clonal relatedness secondary to an antigen-driven response.

Antibodies, Monoclonal↗
PubMed1996

Isolation and purification of anticardiolipin antibody from plasma of a patient with antiphospholipid syndrome: induced generation of platelet thromboxane A2 synthesis.

Antiphospholipid antibodies, particularly anticardiolipin antibodies (aCL) are autoantibodies frequently detected in the serum of patients with systemic lupus erythematosus (SLE) and the primary antiphospholipid antibody syndrome (PAPS). These patients commonly suffer from thrombosis, recurrent fetal loss and thrombocytopenia. Since platelet aggregation is pivotal in the genesis of thrombosis, we tested the hypothesis that perturbation of platelet membrane by aCL/beta 2-glycoprotein (aCL/beta 2GP) complex could trigger the biosynthesis of TXA2, a proaggregatory metabolite of AA. The preincubation of 14C-arachidonic acid (14C-AA)-labeled platelet pellets (14C-PP) from normal individuals with aCL alone followed by incubation with thrombin, resulted in a moderate increase in platelet thromboxane B2 (14C-TXB2) biosynthesis when compared to controls (without aCL). Similar incubations with beta 2GP-I alone resulted in negligible 14C-TXB2 biosynthesis. In contrast, the preincubations of normal 14C-PP with aCL/beta 2GP-I complex resulted in marked thrombin-induced TXB2 biosynthesis, underscoring the requirement of beta 2GP-I in aCL-induced platelet TXB2 biosynthesis. Taken together, these results are consistent with the view that aCL/beta 2GP-I platelet interactions do play a role, at least in part, in platelet hyperactivity and thrombosis in antiphospholipid antibody syndrome.

Adult↗
PubMed1996

Immunologic mechanisms in common rheumatologic diseases.

Rheumatoid arthritis and seronegative spondyloarthropathies are rheumatologic diseases that likely are caused by inflammatory reactions occurring in genetically predisposed individuals mounting an immune response to the antigen. Understanding the immunopathology of these diseases provides insight into their etiology, pathogenesis, and a rationale for therapies targeting immune component interactions. Although the antigen in rheumatoid arthritis is not known, several bacterial antigens have been associated with seronegative spondyloarthropathies. These antigens result in an interaction between the human leukocyte antigen-B27 restricted CD8 positive T lymphocytes and the antigen presenting cell, producing an inflammatory response. Rheumatoid factors are autoantibodies directed against the fragment crystallizable portion of the immunoglobulin G. Rheumatoid factor immunoglobulin G immune complexes contribute to the inflammatory events in the rheumatoid joint, and may play an important role in antigen presentation. A novel antigen capture enzyme linked immunosorbent assay was developed that mimicked B cell surface expressed rheumatoid factor. Conversely, a direct binding enzyme linked immunosorbent assay mimicked secreted rheumatoid factor. Comparison of rheumatoid binding enzyme linked immunosorbent assays showed that the physical state of rheumatoid factor can affect binding characteristics. The state of glycosylation of immunoglobulin G may contribute to its antigenic structure. These physical characteristics may be important in rheumatoid factor's pathogenic role in rheumatoid arthritis.

Antigens, CD↗
PubMed1996

Antigen binding differences between secreted and cell surface expressed rheumatoid factor derived from inflamed synovium.

OBJECTIVE: Rheumatoid factor (RF) is the predominant autoantibody in rheumatoid arthritis (RA). but its role in the pathogenesis of RA remains unclear. We hypothesized that surface RF (sRF) expressed on antigen presenting B cells (B-APC) might have different binding specificities than secreted RF. METHODS: We examined RF binding in a novel RF antigen capture enzyme linked immunoassay (ACE) that mimicked sRF binding, and compared it with a direct binding enzyme linked immunoassay (DBE) that mimicked secreted RF. RESULTS: Significant differences in binding characteristics by the same rheumatoid synovial cell (RSC) derived monoclonal RF (mRF) were observed between the ACE and the DBE. For example, several mRF that demonstrated the classical Ga binding pattern (binding to IgG1, 2, and 4) in the DBE showed considerable binding to selected IgG3 proteins in the ACE; and several mRF that bound only to rabbit IgG in DBE bound to human IgG in the ACE. CONCLUSION: These RF reactivity differences may be attributed to conformational modifications in the RF and IgG molecules that expose different epitopes or altered binding sites depending on the physical state of the antibody and/or antigen and may be important pathogenically.

Animals↗
PubMed1994

Chemical and serologic characterization of human lambda VIII light chains.

The primary structural features and serologic properties of a newly recognized human lambda light (L) chain V region subgroup (V lambda VIII) were elucidated through study of two monoclonal L chains, Bence Jones proteins HAG and BIV. The V region amino acid sequences of these components were highly homologous to each other and to that deduced from the prototypic V lambda VIII cDNA, Humla8f10, which encodes the L chains of the IgM lambda rheumatoid factor HAF10. Proteins HAG and BIV could be classified as members of the V lambda VIII subgroup and distinguished from L chains of the V lambda I, V lambda II, V lambda III, V lambda IV, and V lambda VI subgroups on the basis of amino acid sequence. In addition to distinctive residues found within the V lambda gene-encoded portion of the molecules, L chains HAG, BIV, and HAF10 contained remarkably different second complementarity-determining regions (CDR2) that consisted of 11 residues, rather than the seven typically found among members of the other five V lambda subgroups. This elongated structure would presumably impart to the ligand-binding site of lambda VIII molecules a markedly different canonical structure compared with those of lambda I, lambda II, lambda III, lambda IV, and lambda VI L chains. By using Bence Jones protein HAG as an immunogen, we obtained polyclonal and monoclonal anti-V lambda VIII subgroup-specific Abs that were used to identify and quantify lambda VIII-related molecules in normal and pathologic states. Among the Ig lambda components present in the serum of normal individuals, approximately 3% had lambda VIII L chains, a frequency comparable to that found among monoclonal Ig lambda proteins or surface(s) Ig lambda+ cells obtained from patients with malignant plasma cell- or B cell-related disorders, respectively. In contrast, lambda VIII L chains were detected on approximately 19% of monoclonal IgM lambda rheumatoid factors produced by B cell lines established from PBLs or synovial cells from patients with rheumatoid arthritis. The results of our studies provide new information on the structural and immunochemical features of lambda VIII L chains and the possible functional importance of the human V lambda VIII subgroup.

Amino Acid Sequence↗
PubMed1994

Preferential utilization of a novel V lambda 3 gene in monoclonal rheumatoid factors derived from the synovial cells of rheumatoid arthritis patients.

OBJECTIVE: To further our understanding about the molecular genetics of rheumatoid factor (RF) in rheumatoid arthritis (RA). METHODS: The heavy and light chain variable region (V) genes of 5 new human monoclonal IgM RFs were cloned and sequenced using the polymerase chain reaction and the dideoxynucleotide termination method. RESULTS: The results reveal the recurrent usage in two RA patients of a novel V lambda 3 germline gene, designated Humlv3c93. Specifically, in 2 of 3 RFs (C93 and D53) from one patient, the light chains in the V lambda gene-encoded region were identical to each other and to the light chain of an RF (H4) from another patient. Serologically, the light chains of these 3 RFs were classified as members of the V lambda 3b sub-subgroup. Each of the RFs was encoded by a different VH gene. Both C93 and D53 bound specifically with human and rabbit IgG, whereas H4 was monospecific for rabbit IgG. CONCLUSION: Since the lv3c93 gene is not homologous to any reported V lambda sequence from natural autoantibodies, it is possible that lv3c93 may represent a disease-specific RF-related V lambda gene. Moreover, the amino acid sequence CSGGSCY in the third complementarity-determining regions of 2 of the RF heavy chains is encoded by the DLR2 gene segment and has been found previously in 2 other RA-derived RFs, and thus may play a significant role in antigen binding.

Amino Acid Sequence↗
PubMed1994

IgG3 reactive rheumatoid factor in rheumatoid arthritis: etiologic and pathogenic considerations.

Rheumatoid factor (RF) is a polyclonal autoantibody directed against the Fc portion of IgG. Although the role of RF in patients with rheumatoid arthritis (RA) is unclear, immune complexes that form between RF and IgG can activate the classical complement (C) pathway, leading to pathogenic outcomes involving inflammatory events and tissue damage. The specificity of serum RF and RF produced by rheumatoid synovial cells (RSC) is different. Serum RF has specificity for rabbit IgG and human IgG subclasses IgG1, 2, and 4, but binds poorly to IgG3. The affinity of serum RF for IgG Fc is low, having an association constant of 10(4)-10(5) M-1. RSC RF, however, has specificity for human IgG and high avidity for IgG3. Because of this greater specificity and avidity for IgG3, and because RSC RF may be pathogenically more important than serum RF, an important role for IgG3-reactive RF in RA may exist. Binding of RF to IgG may be dependent on the allotype and glycosylation of IgG. Infectious agents present in RA patients may directly or indirectly induce the production of certain RF. In this communication, we review and expand on several observations examining the role of IgG3-reactive RF in RA including: 1) binding differences between RF derived from RSC and serum; 2) glycosylation characteristics of IgG and its interaction with RF; 3) apparent allotype dependent binding of IgG3-reactive RF; and 4) possible relationship between infectious agents and the production of IgG3-reactive RF. Taken together, these observations suggest an important role for IgG3-reactive RF in better understanding the etiology and pathogenesis of RA.

Amino Acid Sequence↗
PubMed1993

Molecular analysis of rheumatoid factors derived from rheumatoid synovium suggests an antigen-driven response in inflamed joints.

OBJECTIVE: Understanding the molecular genetic basis for rheumatoid factor (RF) production is necessary to a better understanding of the etiology and pathogenesis of rheumatoid arthritis (RA). We sought to define the genetic basis of RF in RA. METHODS: The heavy and light chain variable region genes encoding 4 human monoclonal RF were cloned and sequenced using the polymerase chain reaction and the dideoxynucleotide chain-termination method. RESULTS: The heavy and light chains of the C6 RF and the light chain of the G9 RF were encoded by 3 new RF-related Ig V-region genes. The heavy and light chains of D5 and G4 RFs were identical; most of their mutations caused amino acid substitutions. CONCLUSIONS: The RF-related Ig V-region gene repertoire is large and is still expanding. The data from D5 and G4 strongly suggest that these 2 RFs arise in an antigen-driven response in rheumatoid synovium. The presumed germline V genes for C6 may represent disease-specific RF-related V genes.

Adult↗
PubMed1993

Allotypic dependency of the specificity and avidity of human monoclonal IgM rheumatoid factors derived from rheumatoid synovial cells.

OBJECTIVE: To better understand the genetic derivation and pathogenicity of rheumatoid factor (RF) molecules in rheumatoid arthritis (RA), we have focused our studies on rheumatoid synovial cells (RSC). METHODS: Five monoclonal human IgM rheumatoid factor (mRF)-secreting hybridomas were produced from the RSC of an RA patient. Fine subclass specificities and avidities of these RSC mRFs were compared with several paraprotein monoclonal IgM RFs using direct binding (reactivity) and competitive inhibition (specificity and avidity) enzyme-linked immunosorbent assays. RESULTS: The following observations were made: 1) RSC mRF had greater avidity for IgG than did paraprotein mRF; 2) 4 of the 5 RSC RF were highly avid for IgG3; and, 3) the avidity of RSC RF binding for IgG3 was highest for IgG molecules expressing the G3m(5) allotype. CONCLUSION: We conclude that RSC RF have different specificities and avidities than do paraprotein RF. This may suggest an antigen-driven process in RA synovium, with the production of higher-avidity IgG3m(5)-specific RSC RF, which could have special pathogenetic importance.

Adult↗
PubMed1993

Rheumatoid arthritis: new science, new treatment.

Rheumatoid arthritis (RA) is a chronic systemic inflammatory disease that occurs two to four times as often in women as in men and increases in incidence with advancing age. It affects synovial-lined joints and can also affect the pulmonary, cardiac, nervous, integumentary, and reticuloendothelial systems. RA is manifested clinically by malaise and fatigue, followed by a symmetric pattern of joint inflammation characterized by pain and stiffness. RA most likely occurs in the setting of a genetically predisposed individual, triggered by infectious agents or endogenous antigens. Many of the newer treatments being studied involve blocking cytokine-mediated interactions between cells of the synovium.

Aged↗
PubMed1992

A systematic approach to defining the germline gene counterparts of a mutated autoantibody from a patient with rheumatoid arthritis.

OBJECTIVE: To define the germline counterparts of potentially highly mutated autoantibodies in disease states. METHODS: We developed a systematic approach by first characterizing a rearranged Ig gene and its upstream flank, and then designing suitable primers to amplify specifically the putative germline counterpart. RESULTS: We identified and characterized the germline counterpart of a rheumatoid factor heavy chain variable region. CONCLUSION: We showed unequivocally that the heavy chain of a rheumatoid factor, derived from synovial tissue, has 4 replacement mutations from the corresponding germline gene. The technique allows quick assessment of the degree of somatic mutation in many autoantibodies, and thus can help to elucidate the underlying mechanisms for the induction and sustained production of such antibodies in patients.

Amino Acid Sequence↗