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Biomedical subjects

M M Newkirk

Publications and source records attributed to M M Newkirk.

52 records · Page 3Linked to original sources
PubMed1992

Effect of ultraviolet irradiation on selected host cell proteins including Ro/SS-A and Epstein-Barr virus in cultured lymphoblastoid cell lines.

Although systemic lupus erythematosus (SLE) and Sjögren's syndrome (SS) are distinct collagen vascular illnesses, they share certain features. Both have clinical manifestations involving skin and mucous membranes and characteristically have high titers of circulating autoantibodies to the cellular components Ro/SS-A, calreticulin/Ro, 52 kDa Ro and La/SS-B. Viruses have been postulated to be involved in the pathogenesis of both diseases. Sensitivity to sun is a cardinal feature of SLE, and UV light may be involved in its pathogenesis. Using human B-lymphoblastoid cell lines, the effect of the resident Epstein-Barr virus on the expression of the above cellular components was investigated by flow cytometry. Sublethal irradiation with ultraviolet B light appeared to diminish EBV antigen expression (gp350/220) during the first 48 to 72 hours in culture, whereas there was no change in the expression of MHC class I or immunoglobulin host cell proteins, and an apparent increase in the expression of host cell autoantigens. The virus appeared to be more sensitive to UVB-induced damage yet did appear to be able to undergo repair. No direct correlation could be made between the presence of the virus and the increase in autoantigen expression. La/SS-B and/or 52 kDa Ro antigen(s) were found to be present in the cytoplasm of the B lymphoblastoid cells at a higher base level in EBV-infected cell lines than in the EBV-negative cell lines.

Amino Acid Sequence↗
PubMed1992

Complete amino acid sequence of the variable regions of two highly related heavy and light chains of human IgG (SFL) and IgM (RIV) rheumatoid factors.

The complete amino acid structure of the variable regions of two monoclonal human rheumatoid factors (RF), antibodies that bind to the Fc portion of IgG, is presented. Although these RFs are of different isotypes, IgG (SFL) and IgM (RIV), they are highly related. They probably derive from the same KIII light chain variable region used, but the heavy chains are derived from genes of the VHIII family that are probably evolutionarily related. An analysis of the level of somatic mutation reveals that antigen selection was probably involved in the maturation of these clones. These antibodies, although highly related, are not merely IgM to IgG switch variants which occurred independently in different individuals. Little is currently known about the structure of IgG RFs and this study indicates that the level of somatic mutation of SFL is similar to other autoantibodies or antiviral antibodies of the IgG isotype.

Amino Acid Sequence↗
PubMed1992

Binding characteristics of human hybridoma IgG and IgM rheumatoid factors: influence of IgG isotype and carbohydrate content.

The interaction of human monoclonal rheumatoid factors (RF) with the Fc portion of IgG is complex. We have investigated the influence of the nature of the antigen (Fc) on the binding of hybridoma-generated RF derived from patients with rheumatoid arthritis or systemic lupus erythematosus. For IgM RF, the interaction is strongly influenced by the primary structure of the Fc with little or no effect of the carbohydrate, which is positioned at amino acid 297 in the gamma-2 domain. In contrast, our preliminary data suggest that IgG RF binding is affected both by the primary structure and the nature of the carbohydrate of the Fc. These results suggest that the antigen selection events which lead to the induction and production of IgG RF are likely to be different from those that induce IgM RF production.

Carbohydrate Metabolism↗
PubMed1991

Complications of immunosuppression associated with weekly low dose methotrexate.

Complications of immunosuppression are thought to be rare with the use of low dose pulse methotrexate (MTX) for nonneoplastic conditions. We describe 4 complications of immunosuppression observed in a group of 41 patients who had received MTX for at least 6 months, during a 2-year period. We report the first case of a reversible lymphoproliferative disorder similar to that reported with immunosuppressive therapy associated with organ transplantation. Two cases of disseminated herpes zoster and 1 case with Pneumocystis carinii pneumonia are described. As the indications for the use of low dose MTX broaden and older patients with other comorbid diseases are included, our experience suggests that complications of immunosuppression with prolonged use of MTX may be seen more commonly.

Aged↗
PubMed1990

Importance of the IgG isotype, not the state of glycosylation, in determining human rheumatoid factor binding.

We investigated the influence of carbohydrate on the binding of human rheumatoid factors (RF) to the Fc fragment of IgG. The monoclonal RF studied were derived from the serum of patients with mixed cryoglobulinemia or from hybridomas generated from patients with rheumatoid arthritis (RA) and systemic lupus erythematosus. Polyclonal RF were derived from patients with RA. The carbohydrate located on the Fc fragment, regardless of whether it contained different amounts of mannose or reduced amounts of galactose, or was removed, did not affect the binding of the RF. In contrast, the isotype of the Fc was found to be critical. Two groups of hybridoma RF could be delineated. One group bound preferentially to IgG1 and/or IgG2, and a second group (primarily from patients with RA) bound preferentially to IgG3 and/or IgG4. Our results indicate that the isotype of the Fc fragment, and not the extent of galactosylation, influences the binding of the RF.

Arthritis, Rheumatoid↗
PubMed1990

Molecular cloning, expression, and chromosome 19 localization of a human Ro/SS-A autoantigen.

Ro/SS-A antibodies are found in a number of human autoimmune disorders including Sjogren's syndrome and several systemic lupus erythematosus-related disorders. These heterogeneous autoantibodies are known to recognize several distinct cellular antigens. With synthetic oligonucleotides corresponding to amino acid sequence information we have isolated a full-length cDNA clone which encodes a human Ro ribonucleoprotein autoantigen. The 1,890-base pair clone contains an open reading frame that encodes a 417-amino acid, 48-kD polypeptide that migrates aberrantly at 60 kD by SDS-PAGE. Rabbit antibodies raised against this protein's recently described amino-terminal epitope react with a previously identified 52-kD human Ro protein and immunoprecipitate the human cytoplasmic RNAs. Ultraviolet light cross-linking studies suggest that this Ro protein binds each of the four major human cytoplasmic RNAs. The deduced amino acid sequence is 63% homologous to an Onchocerca volvulus antigen. Southern filter hybridization analysis indicates that this gene is not highly polymorphic and exists as a single copy in the human genome. Chromosomal localization studies place this gene on the short arm of chromosome 19 near the gene encoding the low density lipoprotein receptor.

Amino Acid Sequence↗
PubMed1989

A major autoepitope is present on the amino terminus of a human SS-A/Ro polypeptide.

A human SS-A/Ro antigen is present on the polypeptide component of a particle composed of hyRNA and a 60 kD protein. We have now purified the Wil-2 cell 60 kilo dalton (kD) SS-A/Ro protein and determined its amino-terminal amino acid sequence. A synthetic peptide corresponding to residues 7 to 24 of this sequence (RoSP7-24) exhibited enzyme-linked immunosorbent assay (ELISA) binding activity with immunodiffusion-defined, monospecific anti-SS-A/Ro sera. In addition, ELISA binding of monospecific anti-SS-A/Ro sera to native SS-A/Ro antigen was partially inhibited (35%) by KLH-RoSP7-24. Sera from patients known to frequently produce precipitating anti-SS-A/Ro antibody (subacute cutaneous lupus erythematosus [SCLE], 56 patients; Sjögren's syndrome [SS], 41 patients; mothers of infants with neonatal LE [NLE], 10 individuals; infants with congenital heart block [CHB], 5 patients) were tested for reactivity to RoSP7-24 in ELISA. Overall, 38% of SCLE sera, 36% of SS sera, 50% of maternal NLE sera and 20% of CHB infant sera had anti-RoSP7-24 binding levels greater than 2 standard deviations above the mean of that of normal individuals. Of the sera which had anti-SS-A/Ro detected by double immunodiffusion and/or counterimmunoelectrophoresis, 68% of SCLE patients, 71% of SS patients, 55% of NLE mothers and 20% of CHB infants had significantly elevated RoSP7-24 ELISA binding levels. These findings strongly suggest that a major autoepitope of native human SS-A/Ro resides on the amino terminal portion of the Wil-2 SS-A/Ro 60 kD polypeptide.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Antinuclear↗
PubMed1989

Ro/SS-A and the pathogenic significance of its antibodies.

Ro/SS-A is a soluble cellular protein to which antibodies are frequently directed in patients with Sjögren's syndrome (SS) and systemic lupus erythematosus (SLE)-related disorders. The pathogenic significance of these antibodies is uncertain. Our effort to further characterize the immunological and structural nature of Ro/SS-A suggests that the native human Ro/SS-A molecule is not glycosylated and consists of two disulfide linked domains. The amino terminus of the smaller domain contains a major epitope reactive with anti-Ro/SS-A sera. We have isolated and characterized a complementary DNA (cDNA) clone which encodes the Ro/SS-A polypeptide. The Ro/SS-A gene does not appear to be highly polymorphic and exists as a single copy on the short arm of chromosome 19. The isolation of the Ro/SS-A cDNA should allow for a more complete characterization of Ro/SS-A epitopes which should help further elucidate the importance of Ro/SS-A antibodies in the pathogenesis of disease.

Antibodies, Antinuclear↗
PubMed1988

Reductive methylation and 13C NMR studies of the lysyl residues of fd gene 5 protein. Lysines 24, 46, and 69 may be involved in nucleic acid binding.

We have examined the role of lysyl residues in the binding of fd gene 5 protein to a nucleic acid polymer. The lysyl residues of the protein were chemically modified to form N epsilon, N epsilon-dimethyllysyl derivatives containing 13C-enriched methyl groups. The 13C NMR spectrum of the modified protein was studied as a function of pH and salt concentration. Differences in the local magnetic environment of the six dimethyllysyl amino groups allowed all six 13C resonances to be resolved for samples in the pH range 8.5-9.0 at less than 50 mM ionic strength. One of the dimethylamino resonances was split at low pH, indicating that the two methyl groups were nonequivalent and that the corresponding lysyl residue (either Lys-3 or Lys-7) might be involved in an ion-pairing interaction. Specific lysyl residues were protected from methylation when the protein was bound to poly(rU). The level of protection of individual lysyl residues was quantitated using peptide mapping and sequencing of gene 5 protein labeled with 3H and 14C radioactive labels. Lysines 24, 46, and 69 showed significant protection (33-52%) from methylation in the protein-polynucleotide complex, suggesting that these 3 residues form part of the nucleic acid-binding site. The alpha-amino group of Met-1 was relatively unreactive in both the free and bound protein, which indicated that the amino terminus is not as exposed in solution as in the crystal structure (Brayer, G.D., and McPherson, A. (1983) J. Mol. Biol. 169, 565-596).

Amino Acid Sequence↗
PubMed1988

Complete protein sequences of the variable regions of the cloned heavy and light chains of a human anti-cytomegalovirus antibody reveal a striking similarity to human monoclonal rheumatoid factors of the Wa idiotypic family.

The complete amino acid and nucleotide sequences of the variable regions of the heavy and light polypeptide chains of a human neutralizing IgGl anti-cytomegalovirus (CMV) antibody reveal a striking homology to IgM rheumatoid factors (RFs) of the Wa idiotypic family. The anti-CMV antibody and Wa RFs have in common VKIIIb, JKl, and VHIa gene segments but use different DH and JH gene segments. The anti-CMV antibody does not have RF activity and does not express the Wa idiotype. The Wa RFs do not have anti-CMV activity. A subset of Wa RFs, however, and the anti-CMV antibody do share several idiotypes on the VHIa and VKIIIb polypeptides. Since there are major differences in the antigen binding characteristics and some of the other expressed idiotypes, these data suggest that the D and J region amino acids are crucial to such specificities. Although the use of such highly homologous gene segments in different immune responses is well-documented in murine systems, these data represent the first such example in the human.

Amino Acid Sequence↗
PubMed1988

Molecular characterization of human Ro/SS-A antigen. Amino terminal sequence of the protein moiety of human Ro/SS-A antigen and immunological activity of a corresponding synthetic peptide.

The Ro/SS-A antigen was purified from an Epstein-Barr virus-transformed human B lymphoblastoid cell line. The amino terminal amino acid sequence of the 60-kD polypeptide bearing this antigenic epitope was determined to be: (formula; see text) A peptide composed of residue 6-19 was synthesized by the solid-phase method. Immunodiffusion-defined monospecific autoimmune sera to Ro/SS-A reacted with this synthetic peptide in ELISA, whereas autoantibodies with other specificities such as anti-La/SS-B and anti-Sm, as well as normal human sera, were not reactive. In addition, rabbit anti-peptide 6-19 antisera reacted specifically with native human Ro/SS-A antigen in ELISA. Furthermore, this synthetic peptide inhibited the binding of rabbit anti-peptide antiserum to native human Ro/SS-A. An additional synthetic peptide corresponding to residues 7-24 partially inhibited the binding of a patient anti-Ro/SS-A serum to native Ro/SS-A. These results suggest that the amino terminal portion of the molecule represents a major epitope of Ro/SS-A. The determination of the amino acid sequence of Ro/SS-A and the availability of synthetic peptide(s) bearing this antigen should provide additional approaches to further characterize the autoimmune response to this antigen.

Amino Acid Sequence↗
PubMed1987

Complete amino acid sequences of variable regions of two human IgM rheumatoid factors, BOR and KAS of the Wa idiotypic family, reveal restricted use of heavy and light chain variable and joining region gene segments.

Evidence derived from the complete amino acid sequences of the variable regions of both the heavy and light chains of two members (BOR and KAS) of the Wa idiotypic family of human rheumatoid factors suggests that not only are the light chains of these molecules derived from possibly one variable region gene segment, but the heavy chain variable regions are all derived from the VHI subgroup of human V region genes. These molecules exhibit a surprising conservation in the size of D region, and all use the JH4 gene element. This restriction in use of VL, VH, D, and JH suggests all of these elements may play a crucial role in either antigen binding and/or expression of the crossreactive idiotype.

Amino Acid Sequence↗
PubMed1987

A new protocol to digest human IgM with papain that results in homogeneous Fab preparations that can be routinely crystallized.

A method has been developed for the routine production of Fab fragments from human IgM in high yield. After the IgM is purified at physiological pH, it is digested with papain in the presence of cysteine at room temperature for 16 hours. The Fab fragments are purified initially by gel filtration and then by ion exchange chromatography. The yield of Fab has been 60-80%. Some heterogeneity in the size of the Fabs from the different monoclonal IgMs has been observed. Fab fragments from four different IgM rheumatoid factors (RF) have been crystallized after such digestion and purification, in a variety of conditions including phosphate buffer alone or with the precipitating agents ammonium sulfate, polyethylene glycol or methylpentanediol. This modified papain digestion method has also been used for another non-RF monoclonal human IgM with equally good yield. Biological activity can be detected in the purified Fab fragment indicating that this procedure does not destroy the native conformation of the molecule.

Amino Acid Sequence↗
PubMed1983

Estimation of polymeric IgA in human serum: an assay based on binding of radiolabeled human secretory component with applications in the study of IgA nephropathy, IgA monoclonal gammopathy, and liver disease.

Binding of 125I-human secretory component (SC) to human polymeric immunoglobulin A (pIgA) was employed to measure quantitatively the pIgA present in human sera. Interference by IgM in some sera was prevented by removal of IgM with glutaraldehyde polymerized anti-IgM antibodies. 125I-SC complexed to pIgA was measured by precipitation with anti-IgA antibodies and the quantity of pIgA in human serum was estimated by comparing the quantity of 125I-SC bound by several dilutions of human serum to that bound by standard quantities of human monoclonal pIgA proteins. The assay was specific for pIgA because heat-aggregated monomeric IgA or hypogammaglobulinemic serum did not bind 125I-SC greater than a precipitate formed with human monoclonal IgG and anti-IgG. Moreover, analysis of a series of IgA myeloma sera indicated no correlation between the IgA content of the serum and the quantity of pIgA measured. The quantity of pIgA found in 30 normal human sera was 0.13 +/- 0.08 mg/ml (1S.D.), which consisted of 11.3 +/- 5.3% (1 SD) of the total IgA. Patients with IgA monoclonal gammopathy were most often found to have predominantly monomeric IgA. Patients with IgA nephropathy also showed an elevation of pIgA, but this appeared to be a consequence of an overt IgA elevation. IgA nephropathy patients with elevated serum IgA in fact showed a significant elevation of monomeric IgA. Selective elevation of pIgA was observed in patients with primary biliary cirrhosis and alcoholic liver disease. A comparison of this assay with other assays to measure pIgA is discussed.

Aged↗
PubMed1975

Fate of ingested hepatitis B antigen in blood-sucking insects.

The fate of ingested hepatitis B antigen (HBsAg) in two mosquito species and two Hemiptera species was compared with the rate of blood meal digestion by these insects. In both mosquito species HBsAg was detected by radioimmunoassay for only a few hours after ingestion, disappearing well before the time of blood meal digestion. Production of a protease by the mosquito midgut may have been responsible for destruction of the antigen. In contrast, in the bedbug HBsAg remained detectable throughout a 5-week testing period. Moreover, titers rose during the last week, when blood meal digestion was complete, suggesting possible replication of the antigen. At no time was antigen detected in eggs or feces of any species tested, but juvenile bedbugs fed HBsAg when in the fourth or fifth instar stage still contained antigen after molting. These studies suggest that bedbugs may potentially be a more dangerous source of hepatitis B transmission than mosquitoes.

Animals↗