Benjamin D. Schwartz

Collagen-induced arthritis in the BB rat. Prevention of disease by treatment with CTLA-4-Ig.

Antigen-specific T cell activation requires two independent signalling events, one mediated through T cell receptor engagement by the antigen-presenting cell-expressed peptide/class II major histocompatibility complex, and the second through the cognate interactions of costimulatory molecules expressed on the T cell and antigen-presenting cell. There is evidence from in vitro and in vivo experimental systems suggesting that the CD28/B7 costimulatory pathway is crucial for induction of maximal T cell proliferation and T helper-B cell collaboration for IgG production. This pathway can be blocked by CTLA-4-Ig, a soluble form of CTLA-4 which binds with high avidity to the CD28 ligands, B7-1 and B7-2. Here, we show that CTLA-4-Ig treatment prevents clinical and histological manifestations of disease in a collagen-induced arthritis model of rheumatoid arthritis in the diabetes resistant BB/Wor rat, when therapy is initiated before immunization with bovine type II collagen (BIIC). Anti-BIIC antibody titers are reduced in CTLA-4-Ig-treated rats compared to diseased control animals. Histologically, joints from CTLA-4-Ig-treated animals show no histological abnormalities, in contrast to control antibody-treated animals, which show complete erosion of the articular cartilage and bone. Despite the efficacy of CTLA-4-Ig in preventing clinical and histological signs of arthritis and reducing antibody responses to BIIC, delayed type hypersensitivity responses to collagen 18 d or more after CTLA-4-Ig treatment ends are similar in CTLA-4-Ig-treated and untreated rats, suggesting that the prolonged disease suppression observed does not result from induction of T cell anergy.

Peptide characterization with a sulfoethyl aspartamide column.

A strong cation-exchange (SCX) high-performance liquid chromatography column (sulfoethyl aspartamide, 200 x 4.6 mm) was used to analyze more than 50 peptides, ranging in length from 5 to 20 residues. These data show that the elution positions of the peptides increase monotonically with the number of positively charged residues. [A 60-min linear gradient of 0 to 100% eluent B at 1 ml/min was used, where eluent A is 5 mM phosphate (pH 3.0)-acetonitrile (75:25) and eluent B is eluent A + 0.5 M sodium chloride.] A comparison of SCX with a standard C18 reversed-phase (RP) column [60-min linear gradient of 0 to 60% B at 1 ml/min, where eluent A is 0.1% trifluoroacetic acid (TFA), and eluent B is 0.095% TFA-acetonitrile (10:90)] further demonstrates the utility of SCX in peptide characterization. SCX separated an (Arg)3-containing peptide from the Arg-deleted peptide while RP could not. In addition, SCX and RP resolved the methionine oxidation products of ACTH (4-10) (RP: Met [O] less than Met [O2] less than Met; SCX; Met [O] less than Met less than Met [O2]), suggesting a mixed-mode mechanism for the ion-exchange system. Finally, SCX separated the sulfated and non-sulfated forms of cholecystokinin (26-33) and Leu-enkephalin as well as the N-terminal acetylated forms of neurotensin (8-13) and angiotensinogen (1-14) from the respective unmodified peptides.

Polymorphism in the promoter region of HLA-DRB genes

Polymorphism is a hallmark of the molecules encoded within the MHC of humans and other mammals. Recently, evidence of polymorphism has also been shown to exist in the transcriptional regulatory regions of HLA-DQB genes. In this article, we report that polymorphism exists also in the promoter region of HLA-DRB genes. The sequence of the regulatory region of DRB genes from five homozygous DR B-cell lines, each of a distinct DR haplotype, revealed a number of differences, some of which are in the critical class II boxes that are generally conserved in class II promoters. The major differences occurred in a comparison of DR4 to the other DR haplotypes. These data suggest the existence of another important source of HLA class II polymorphism that may play a role in susceptibility to HLA-associated autoimmune disease.

Strong-cation-exchange sulfoethyl aspartamide chromatography for peptide mapping of Staphylococcus aureus V8 protein digests

In two recent reports (D. L. Crimmins, J. Gorka, R. S. Thoma, and B. D. Schwartz (1988) J. Chromatogr. 443, 63-71; A. J. Alpert and P. C. Andrews (1988) J. Chromatogr. 443, 85-96) a sulfoethyl aspartamide column was shown to efficiently analyze peptides less than 25 residues in length which differ in the number of nominal positive charges at pH 3.0. In particular, the elution order for a series of distinct peptides ranging in nominal charge from +1 to +7 was found to be monotonic in nature indicating that separation was primarily via a cation-exchange mechanism. The present study employs this chromatographic system to isolate and characterize major fragments of proteolytic digests. Six commercially available proteins of known sequence (myoglobin, beta-casein, concanavalin A, carbonic anhydrase, lentil lectin, and enolase) were digested with Staphylococcus aureus V8 to generate peptide fragments. The resulting mixture was chromatographed on a sulfoethyl aspartamide column to isolate major fragments which were then subjected to amino acid analysis and N-terminal sequencing. With complete proteolysis (i.e., peptide fragments terminating in either an aspartic or a glutamic acid) separation of the fragments should result from the sum of histidine, lysine, and arginine residues contained in each fragment. Most of the peptide fragments eluted at the expected time on the sulfoethyl aspartamide column. Those fragments with anomalous behavior resulted from incomplete cleavage or cleavage at nonacidic residues or were greater than 35 residues in length. Each proteolytic digest was also analyzed by standard reverse-phase C4 chromatography to compare the peptide maps for these two distinct chromatographic modes.

Chemical characteristics of Ia antigens

The discovery that maj or histocompatibility complex (MHC)-linked genes regulate the ability of an animal to respond to an immunologic challenge [4, 35] stimulated intense investigation into the mechanisms by which these immune response (Ir) genes exert their influence. As part of the investigation, an effort was made to identify and analyse products of Ir genes. Since it was possible to reciprocally immunize animals which differed virtually only by the genetic region (the immune response or I region) containing the Ir genes, the use of serological techniques to detect Ir gene products seemed an appropriate starting point [17,/8, 25, 28, 46]. The antisera raised by these reciprocal immunizations reacted primarily with lymphocyte cell surface antigens which were termed I region associated or Ia antigens. While it is not yet evident whether the Ia antigens are in fact determined by the Ir genes themselves, or by genes closely linked to the Ir genes, it is clear that these antigens play a critical role in the regulation of the immune response. Evidence for this contention was derived from several in vivo and in vitro functional studies. Ia antigens have been shown to be necessary for successful collaboration of immunocompetent cells [32, 44]. In addition anti-Ia antisera can inhibit antigen-elicited genetically controlled immune responses as measured by in vitro T cell proliferation [54, 59], can inhibit the induction of mixed lymphocyte reactions (MLR) [27, 37], can inhibit the induction of in vitro antibody responses [24, 40] and can react with antigen specific helper and suppressor factors derived from T cells [38, 65]. Thus, though identity of Ia antigens and Ir gene products has not been established, the Ia antigens have nonetheless been subjected to extensive biochemical analysis in an effort to elucidate the physiology of these antigens, and their relationship to Ir genes. This review will summarize the information presently available regarding the structure and chemistry of the Ia antigens in mice and guinea pigs.

Automated, isocratic separation of phenylthiohydantoin-amino acids by tandem reverse phase high-pressure liquid chromatography columns

Abstract Quantitative separation of the phenylthiohydantoin derivatives of all 20 naturally occurring amino acids is described utilizing two tandem C 18 columns and an isocratic elution buffer system. The separation was performed in 35 min. In conjunction with a Spectra-Physics SP8000 liquid chromatograph programmed for “peak-picking” sample collection and autoinjection, unattended, overnight analysis of multiple sequencer steps can be performed.

Facile analysis and purification of deblocked N-terminal pyroglutamyl peptides with a strong cation-exchange sulfoethyl aspartamide column

A high-performance strong cation-exchange Sulfoethyl Aspartamide column was used to analyze and purify five N-terminal pyroglutamyl peptides after treatment with Pyroglutamate Aminopeptidase. The resulting deblocked N-1 peptides possess an increased positive charge and are therefore retained to a greater extent by the column. Salt gradient elution in a pH 3 mobile phase was then used to recover the desired peptides and the purified deblocked peptides were directly subjected to N-terminal sequence analysis. The same digests were also chromatographed on a C18 reversed-phase column using standard trifluoroacetic acid-acetonitrile gradient elution. The elution order for the parent peptide and the N-1 peptide on the reversed-phase column was reversed from that on the Sulfoethyl Aspartamide column and the resolution of the two peptides obtained on the reversed-phase column was less than that observed on the cation-exchange column. In addition, the Sulfoethyl Aspartamide column was shown to be useful to monitor the extent of N-terminal glutamine cyclization formed during peptide purification and storage.

Diversity and regulation of expression of human leukocyte antigen class II molecules

The initiation of an immune response requires that a foreign antigen be degraded, and that one of the degradative fragments be presented in the context of a human leukocyte antigen (HLA) class II molecule to an antigen-specific helper T cell. The success of this process is maximized by the diversity of the class II molecules possessed by a given person. The expression of the HLA class II molecules is highly selective. Aberrant expression has been postulated to be responsible for autoimmune disease. The interaction of inducible tissue-specific transacting factors with cis-acting genetic elements adjacent to the coding portion of the class II genes is responsible for both normal and aberrant expression of class II molecules. The cDNA encoding one such transacting factor that binds to the cis-acting element known as the Y box has been cloned.

Invariant proteins associated with guinea-pig Ia antigens☆

Analysis of guinea-pig Ia immunoprecipitates by two-dimensional gel electrophoresis demonstrated the specific association of Ia molecules with several types of invariant proteins. These include a 33,000 mol. wt basic protein homologous to murine invariant chain (Ii), and a set of 34,000-36,000 mol. wt proteins more acidic than Ii (acidic invariant chain). Two 23,000-25,000 mol. wt non-polymorphic proteins with pIs of 6.0 and 6.5 were also observed in association with Ia, as was a basic protein of mol. wt 42,000. Pulse/chase studies using [35S]methionine demonstrated that Ii, but not acidic invariant chain, was associated with newly synthesized Ia molecules. The amount of 35S-Ii decreased greatly throughout the chase period. 35S-acidic invariant chain was clearly present in Ia precipitates by 30 min after Ia synthesis, but was not detected 4 hr after synthesis. Only acidic invariant chain was associated with mature Ia antigens bound by the lectin Ricinus communis I. Our results indicate that guinea-pig invariant proteins are differentially bound by Ia molecules during maturation of Ia alpha- and beta-chains, and suggest that acidic invariant chain could be a processed form of Ii.

Histoplasmosis in non-endemic regions

Abstract In the past seven years it has become increasingly recognized that histoplasmosis is a frequent cause of pulmonary disease in endemic areas. It is the purpose of this report to show that histoplasmosis is also a common cause of pulmonary involvement in non-endemic regions. Roentgenographically, histoplasmosis may simulate many forms of pulmonary disease. It is necessary, therefore, to perform skin tests with tuberculin, histoplasmin and coccidioidin routinely in the diagnostic work-up of pulmonary conditions if one is to detect histoplasmosis. The complement fixation test for histoplasmosis is of relatively minor importance in detecting inactive cases of this disease. It need only be performed in those individuals reacting to the histoplasmin skin test, in whom a positive serologic reaction can serve as confirmatory evidence of the presence of histoplasmosis. We believe that cases of histoplasmosis will be discovered in increasing numbers in nonendemic regions, in proportion to the awareness of its varied manifestations and the zeal with which the search is made.