PubMed Health⌕ Search

Biomedical subjects

A H Kang

Publications and source records attributed to A H Kang.

At least 19 recordsLinked to original sources
PubMed1992

Characterization of a tolerogenic T cell epitope of type II collagen and its relevance to collagen-induced arthritis.

A synthetic peptide representing sequences of type II collagen, (CII 245-270), has previously been used to induce tolerance and suppress arthritis in DBA/1 mice. To determine important residues, a series of peptides, each containing one or two site-directed substitutions, was generated. Mononuclear cells from DBA/1 mice immunized with CII were cultured in the presence of each peptide and the T cell response determined by measuring IFN-gamma in culture supernatant fluids. Substitutions within the region CII 260-270 led to significant decreases in IFN-gamma responses, identifying this sequence as a T cell epitope. To determine the effects of substitutions within this epitope on arthritis, substituted peptides were administered to neonatal mice as tolerogens. Five site-directed substitutions, four of which included the insertion of a residue found in type I collagen to replace its type II counterpart, abrogated the ability of the peptides to induce tolerance and suppress arthritis. These substitutions were located at residues 260, 261, 263, 264, and 266. Two patterns of T cell reactivity were observed. Peptides containing individual substitutions at positions 261, 264, or 266 were capable of generating a significant T lymphokine response, although those containing substitutions at residues 260 or 263 were ineffective Ag. Systematic analysis of the fine structures of T cell determinants important for autoimmune arthritis can lead to strategies for therapeutic intervention.

Amino Acid Sequence↗
PubMed1992

Collagen-induced arthritis in rats. Examination of the epitope specificities of circulating and cartilage-bound antibodies produced by outbred and inbred rats using cyanogen bromide-derived peptides purified from heterologous and homologous type II collagens.

To determine the number and location of antibody binding epitopes on type II collagen, outbred and inbred rats were immunized with chick, bovine, human, and rat type II collagen (CII, BII, HII, and RII); all sera were assayed for reaction with a panel of CB peptides purified and renatured from the immunizing collagen and from RII. Antibody reaction patterns (profiles) varied among individual outbred rats but were essentially constant over time and changed little after boosting. The strongest antibody reactions were to CB11, CB9-7, and CB12 followed by CB8, CB10, and CB6. Antibody profiles varied depending on the species of collagen used for immunization and the strain of rat immunized. Except for CB10, where antibodies were largely specific for heterologous collagens, antibodies reactive with all other CB peptides cross-reacted strongly with renatured rat CB peptides. Sera from inbred BB rats immunized with BII, CII, or HII reacted best with CB11, unlike antisera to RII that reacted strongly with CB9-7. Inbred LEW, COP, WKY, F344, and BUF rats immunized with BII reacted strongest with CB9-7 and variably with CB11 and CB12. BBxLEW F1 hybrid rats reacted almost equally with CB11 and CB9-7 producing an antibody profile intermediate to those elicited in the parent strains. Finally, antibodies reactive with rat CB11, CB9-7, and CB12 could be eluted from normal rat cartilage incubated in anti-BII serum; antibody eluate profiles generally paralleled the profile produced by the sera applied to cartilage. Taken together, these findings indicate that multiple antibody-reactive epitopes on type II collagen may be instrumental in the initiation of collagen-induced arthritis in rats, particularly shared or cross-reactive epitopes located within CB11, CB9-7, CB12, and CB8.

Animals↗
PubMed1992

Induction of arthritis with monoclonal antibodies to collagen.

mAb were developed from DBA/1 mice immunized with chick type II collagen. A total of 69 IgG antibodies was isolated and characterized. The majority (36%) reacted with a CNBr-derived peptide CB11 previously identified as containing a major immunogenic and arthritogenic epitope(s). Seven of the antibodies reactive with CB11 crossreacted strongly with mouse type II collagen. These were administered to DBA/1 mice in an attempt to induce arthritis. Individual antibodies were able to induce mild lesions consisting of minimal synovial proliferation but not overt arthritis. However, a combination of antibodies induced severe arthritis with marked destruction of articular cartilage. The minimal effective combination consisted of three antibodies. Arthritis developed within 48 to 72 h after injection of the antibodies and persisted for the duration of the observation period of 3 wk. Antibody levels were measured at intervals and persisted for the 3 wk observation period although at diminishing levels. Competitive binding assays demonstrated that each of the effective antibodies bound independently suggesting that some spatial or quantitative relationship was important possibly related to their ability to activate complement.

Animals↗
PubMed1991

Regulation of extracellular matrix production by chemically synthesized subfragments of type I collagen carboxy propeptide.

The complete COOH-propeptide of human alpha 1(I) procollagen was chemically synthesized as a series of overlapping subfragments which were then tested for their effect on extracellular matrix protein production by subconfluent human lung fibroblasts (HFL-1). One peptide (R11; residues 197-241) stimulated production of both collagen and fibronectin by 6-8-fold while a second peptide with a partial overlap with R11 (R9; residues 182-216) enhanced collagen accumulation. The peptide R12 (residues 197-216), which has a sequence common to both R9 and R11, also stimulated collagen production, suggesting that this 20-residues peptide alone contains the required structure for activity. The other synthetic peptides, R1-R13, were inactive in their ability to alter collagen or fibronectin production. Consistent with previously published data, the COOH-terminal peptide, R14, inhibited extracellular matrix production [Aycock, R.A., Raghow, R., Stricklin, G.P., Seyer, J.M., & Kang, A.H. (1986) J. Biol. Chem. 261, 14355-14360]. Both R9 and R11 preferentially stimulated production of collagen types I and III and fibronectin in dose-dependent manner. Elevated collagen and fibronectin production was evident at 4-h posttreatment, and maximal enhancement was seen at 8 h after exposure to peptides. Interestingly, subconfluent cultures of HFL-1 fibroblasts responded vigorously to the stimulatory action of R9 and R11 while confluent cells failed to show any response. Steady-state levels of messenger RNAs encoding type I procollagen and fibronectin were not measurably altered by treatment with R9 or R11, suggesting that the regulation of procollagens and fibronectin by these peptides involves posttranscriptional mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

Immunity to type XI collagen in mice. Evidence that the alpha 3(XI) chain of type XI collagen and the alpha 1(II) chain of type II collagen share arthritogenic determinants and induce arthritis in DBA/1 mice.

To determine whether native bovine type XI collagen (BXI) is arthritogenic, five strains of inbred mice were immunized with BXI/CFA. Arthritis was not observed in any of these strains, though it was prevalent in DBA/1 and B10.RIII controls immunized with bovine type II collagen (BII). Antisera from BXI-immunized mice reacted with mouse type XI collagen (MsXI), weakly with the alpha-chains of BXI, and minimally with mouse type II collagen (MsII). However, antisera to BII reacted with MsII and MsXI, indicating antibodies to conformation-independent epitopes shared by alpha 1(II) and alpha 3(XI). Mice immunized with BXI containing a small amount of BII developed arthritis much like those immunized with BII; sera from these mice reacted with MsXI and MsII. Delayed-type hypersensitivity responses differed from IgG responses, i.e., BXI elicited responses to alpha 1(XI), alpha 2(XI), alpha 3(XI), and alpha 1(II); BII, to alpha 3(XI) and alpha 1(II) exclusively. To determine whether alpha 1(XI), alpha 2(XI), alpha 3(XI), and alpha 1(II) are arthritogenic, DBA/1J mice were immunized with each alpha-chain. Arthritis was seen in mice injected with alpha 3(XI) or alpha 1(II). Sera to both alpha-chains reacted similarly with MsII and peptide fragment alpha 1(II)-CB11. Epitope mapping using polyclonal and mAb to type II collagen revealed that all polyclonal and 11 of 14 mAb reacted with alpha 3(XI) and alpha 1(II), whereas three mAb reacted only with alpha 1(II). In conclusion, BXI is immunogenic but not arthritogenic in five strains of mice, whereas alpha 3(XI) and alpha 1(II) are arthritogenic and immunogenic in DBA/1 mice and share greater than or equal to 11 epitopes recognized by autoantibody.

Animals↗
PubMed1991

Genomic organization of the human procollagen alpha 1(II) collagen gene.

The nucleotide sequence of the human procollagen alpha 1(II) collagen gene extending from within the first intron through exon 15, and part of the 15th intron has been determined. This sequence analysis (7056 bases) identifies the intron/exon organization of the region of this gene encoding the N-propeptide and part of the triple-helical domain. Structural comparison of this with the genes of other human fibrillar collagens shows considerable diversity in terms of size and number of introns and exons that encodes the N-propeptide domain. Although the genomic structure of the human procollagen alpha 1(II) gene is quite different from the rat procollagen alpha 1(II) gene, the nucleotide coding sequences are 89% identical.

Base Sequence↗
PubMed1991

In vitro methylation of the promoter and enhancer of Pro alpha 1(I) collagen gene leads to its transcriptional inactivation.

We created pCOL-KT, a plasmid construct in which the promoter/enhancer of human Pro alpha 1(I) gene is linked to the chloramphenicol acetyl transferase reporter gene. The Pro alpha 1(I) promoter/enhancer in pCOL-KT was methylated in vitro and tested for transcriptional activity by transient expression analysis. Methylation of the construct with bacterial methylases reduced transcriptional activity about 25-fold. Site-specific methylation of eight potential canonical sites of eukaryotic methylation within the promoter greatly reduced transcriptional activity. Chromatin conformation of the transfected pCOL-KT DNA was analyzed by nuclease sensitivity. Although both methylated and unmethylated transfected DNA had increased susceptibility to DNase I compared with the endogenous gene, the methylated transfected DNA showed increased resistance to nuclease when compared with unmethylated transfected DNA, indicating that the methylation of the DNA alters the chromatin conformation. We also tested the ability of a human rhabdomyosarcoma cell line that does not express type I collagen to support transcription from an exogenously added Pro alpha 1(I) promoter/enhancer. The transformed cell line is able to support transcription from the Pro alpha 1(I) promoter/enhancer. Treatment of the transformed cell line with 5-azacytidine, a potent inhibitor of DNA methylation, resulted in transcriptional activation of the Pro alpha 1(I) gene. These findings, along with the extreme methylation sensitivity of the Pro alpha 1(I) promoter and enhancer, suggest that DNA methylation may be an important mechanism of transcriptional inactivation of interstitial collagen genes.

Base Sequence↗
PubMed1991

Kupffer cells from carbon tetrachloride-injured rat livers produce chemotactic factors for fibroblasts and monocytes: the role of tumor necrosis factor-alpha.

Conditioned media from cultured Kupffer and mononuclear macrophagic cells obtained 48 hr after CCl4 administration to rats contains chemotactic factors for human skin fibroblasts and human monocytes. The chemotactic mediator for fibroblasts was approximately 17 kD and was more prominent at early stages of culture. It induced a dose-dependent chemotactic response in fibroblasts. Although the conditioned medium from cultured Kupffer cells of normal rats also contained detectable biological activity, it was significantly less than that in conditioned medium from cultured Kupffer cells from CCl4-treated rats. The activity obtained after purification by high-performance liquid chromatography was completely ablated by incubation with tumor necrosis factor-alpha antibody. Transforming growth factor-beta antibody diminished biological activity by 20%. Human recombinant tumor necrosis factor-alpha and transforming growth factor-beta used in the assay as control showed significant chemotactic activity. The chemotactic activity present in whole normal conditioned medium was only present after 24 and 48 hr of culture. Furthermore, this activity was not neutralized by human recombinant tumor necrosis factor-alpha or transforming growth factor-beta antibodies. Incubation of whole 6-hr conditioned medium with human recombinant tumor necrosis factor-alpha and transforming growth factor-beta antibodies demonstrated and confirmed that tumor necrosis factor-alpha plays a major role in inducing the chemotactic response. On acidification of this supernatant, we found a notable increase in the biological response that could be neutralized by transforming growth factor-beta antibody. Thus tumor necrosis factor-alpha and transforming growth factor-beta may sequentially provide important signals for fibroblast and monocyte recruitment in vivo at initial stages of liver injury.

Animals↗
PubMed1991

Stimulation of phospholipase A2 activity in human platelets by trypsin and collagen.

Type I collagen enhanced human platelet phospholipase A2 activity whether added to platelet-rich plasma or washed platelets. The stimulatory effect of type I collagen on platelet membrane phospholipase A2 activity was also observed in a cell-free system utilizing platelet membranes. The release of arachidonic acid was enhanced by types I and III but not by type V collagen. The activation of platelet phospholipase A2 by type I collagen was inhibited by soybean trypsin inhibitor and mimicked by trypsin. However, type I collagen addition was not associated with any detectable changes in platelet membrane proteins while trypsin altered many proteins. These results point to acid soluble phospholipase A2 activity of platelets as an enzyme activated by type I collagen.

Blood Platelets↗
PubMed1991

The specialized centers of research in rheumatoid arthritis. Recent progress and prospects for future advances.

Specialized Centers of Research (SCOR) in arthritis are interdisciplinary research programs to investigate disease pathogenesis as well as advance diagnosis and treatment. A recent meeting of investigators from the three SCOR programs in rheumatoid arthritis demonstrated progress in several important research areas. Because of the multiplier effects of SCOR programs, new investigators have been enlisted into arthritis research as issues related to this disease become a focus of investigation throughout universities and medical centers. Continued progress by the SCOR programs should provide new targets for therapeutic intervention as well as strategies for monitoring disease activity.

Academic Medical Centers↗
PubMed1991

Interleukin 1 stimulates phosphatidylinositol kinase activity in human fibroblasts.

IL-1 mediates multiple cellular immune and inflammatory responses, but little is known of the intracellular biochemical mechanisms involved in IL-1 actions. We studied the effects of IL-1 on phosphatidylinositol (PtdIns) metabolism and confirmed reports indicating that IL-1 does not stimulate increased PtdIns turnover; however, we observed the accumulation of PtdIns-4-phosphate (PtdInsP) in response to IL-1. Using a fibroblast membrane preparation, we were able to detect stimulated PtdInsP accumulation within 10 s of IL-1 addition. Increased PtdInsP accumulation was due to stimulated PtdIns kinase activity, not the inhibition of PtdInsP hydrolysis by phospholipase(s). PtdIns kinase activity was magnesium dependent, increased as a function of IL-1 concentration, and specifically phosphorylated the D4 position of inositol. Stimulated PtdIns kinase activity could be detected at 10(-12) M IL-1 in fibroblast membranes, a concentration within the physiological range for IL-1 action; half-maximal activity was reached at approximately 10(-10) M IL-1. Heat denaturation of IL-1 or treatment of IL-1 with anti-IL-1 antibody abrogated the IL-1 effect. These findings demonstrate the direct, IL-1-mediated, stimulation of PtdIns kinase. IL-1-stimulated PtdIns kinase activity represents an important physiological regulatory effect by IL-1 as it could control the synthesis and/or maintenance of phosphorylated derivatives of PtdIns which comprise only a very small pool of substrates for the generation of the second messengers inositol 1,4,5-triphosphate and diacylglycerol.

1-Phosphatidylinositol 4-Kinase↗
PubMed1990

Sphingosine potentiates IL-1-mediated prostaglandin E2 production in human fibroblasts.

IL-1 stimulates PGE2 production in human fibroblasts by stimulating arachidonic acid (AA) mobilization and cyclooxygenase synthesis. Cyclooxygenase is the first enzyme in the pathway that converts AA to PGE2. To examine the role of protein kinase C (PKC) in IL-1-mediated PGE2 production, we treated cells with PMA, which stimulated PGE2 production suggesting a positive role for PKC activation in the regulation of PGE2 synthesis. Therefore, we tested the effect of sphingosine, a PKC inhibitor, on IL-1-induced PGE2 production. Alone, sphingosine had little effect on PGE2 production. However, when sphingosine was added with IL-1, or IL-1 was added to sphingosine-pretreated cells, PGE2 production increased severalfold, suggesting that the inhibition of PKC results in enhanced IL-1-mediated PGE2 production; structural analogs of sphingosine did not potentiate the IL-1 effect. In cells made deficient in PKC by prolonged exposure to PMA, IL-1-mediated PGE2 production was enhanced compared with normal cells, further suggesting that functional PKC is not required for, and may down-modulate, IL-1-mediated PGE2 production. These findings also suggest that PMA and IL-1 stimulate PGE2 synthesis via fundamentally different pathways. In separate studies on the effect of IL-1 on AA mobilization, we found that IL-1 induced an increase in phospholipase A2 (PLA2) activity and that cycloheximide blocked the increase, suggesting the requirement for new protein synthesis. We also found that the PLA2 activity increased as a result of IL-1 exposure was further stimulated by sphingosine. Thus, in addition to its primary effects on the cell, which are likely mediated via PKC, we present evidence suggesting that sphingosine may also play a role in potentiating an IL-1-induced PLA2 activity, resulting in increased availability of AA for conversion to PGE2.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1990

Human HLA-DR beta gene hypervariable region homology in the biobreeding BB rat: selection of the diabetic-resistant subline as a rheumatoid arthritis research tool to characterize the immunopathologic response to human type II collagen.

Collagen arthritis (CA), an autoimmune model of rheumatoid arthritis (RA), has been studied in various animals. However, it has not been studied in an animal with a genetic background relevant to RA. We selected rats from a diabetic-resistant (DR) subline of the diabetic BB rat because they have an autoimmune disease-prone background, but not the immunodeficiencies of the diabetic BB rat, and the third hypervariable region (HVRIII) of the BB RT1.D beta gene appeared to encode a nucleotide sequence of the human HLA DR beta gene, which has been reported to be associated with susceptibility to RA. We synthesized oligonucleotide primers flanking the RT1.D beta HVRIII, cloned polymerase chain reaction-amplified DNA into M13mp18, and confirmed the presence of the susceptibility sequence (SS) (RRRAA) by the dideoxy sequencing method in a colony of DR BB/Wor-UTM rats. When immunized with human type II collagen (CII) in incomplete Freunds adjuvant (IFA), arthritis developed rapidly by day 10 with 100% incidence. Light and electron microscopy revealed an unusually severe and aggressive, bidirectional pattern of cartilage resorption by synovial and subchondral mononuclear and multinucleated inflammatory cells. These findings coincided with a predominant humoral response to the cyanogen bromide (CB) 11 fragment of the human CII molecule by the pathogenic IgG2a isotype. This study provides further support to the role of CA as a relevant RA model, the specific roles of the CB11 fragment as a major site of arthritogenic epitopes, and of antibody mechanisms in the pathogenesis of CA. Furthermore, the identification of an RA SS in an immune response gene of the DR BB rat presents a novel opportunity to determine with an animal model the role of other antigens as well as this SS in RA.

Amino Acid Sequence↗
PubMed1990

Collagen-platelet interaction: type XI collagen-induced platelet aggregation.

Type XI collagen in its native fibrillar but not in soluble monomeric form mediates human platelet aggregation and release of adenosine triphosphate in a dose-dependent manner. Its action is inhibited by aspirin. Type XI collagen also increased radiolabelled phosphate incorporation into protein bands with molecular weights of 42 KDa and 22 KDa, respectively. In contract, these events were not observed in platelets incubated with type IX collagen. These results suggest that the fibrillar type XI collagen has the same ability as other types of collagen to induce human platelet aggregation.

Adenosine Triphosphate↗
PubMed1990

A factor from damaged rat kidney stimulates collagen biosynthesis by mesangial cells.

Rats were administered CCl4, a well-defined nephrotoxin, for 20 weeks to produce glomerular sclerosis. Tubular degeneration and necrosis with interstitial fibrosis was clearly evident by histological examination. Kidneys were homogenized in phosphate-buffered saline and a collagen synthesis-stimulating factor was isolated by Sephadex G-50 gel filtration. The 5 kDa component stimulated both type I and type IV procollagen synthesis by mesangial cells and type I procollagen synthesis by rat skin fibroblasts. In each cell type, 2-6-fold increases in procollagen protein production or cell proliferation was noted. The steady-state levels of mRNA encoding for procollagen alpha 1(I) and procollagen alpha 1(IV) chains in mesangial cells were determined by by hybridization to their corresponding cDNA clones. The type I procollagen mRNA was elevated 1.4-fold compared to a 1.6-fold increase in mRNA encoding for type IV procollagen. The similar properties and chemical characteristics of this fibrogenic factor with a factor from fibrotic liver suggests they are the same and that a common endogenous collagen synthesis stimulator may be present in fibrosing organs, thus providing a driving force for collagen over-production.

Animals↗
PubMed1990

Comparison and accuracy of methodologies employed for analysis of hydropathy, flexibility and secondary structure of proteins.

Various methods have been developed recently for predicting antigenic determinants in a protein by analyzing its hydropathicity, flexibility or secondary structure. The predicted results by these methods were compared directly to the observed structure of the proteins to determine how accurate they were. Regression and correlation of the predicted hydropathicity versus the observed solvent accessibility and the predicted chain flexibility versus actual mobility were determined. Three separate methods of hydropathic analysis had correlation coefficients of 0.52, 0.63 and 0.57 respectively, while a published method for chain flexibility analysis had a 0.19 correlation coefficient. Similarly, agreement between the observed and the predicted secondary structures by two methods was also determined. The limitations of these techniques were clearly defined.

Amino Acid Sequence↗
PubMed1990

Regulation of TGF beta gene expression in rat liver intoxicated with carbon tetrachloride.

Transforming growth factor beta (TGF beta) has been implicated as having a central role in the postinflammatory tissue regeneration and fibrosis. To test its potential involvement in events that follow hepatotoxin-mediated liver regeneration and fibrosis, we quantitated changes in the steady-state levels of TGF beta mRNA in parenchymal and nonparenchymal cells at various times after an acute treatment with CCl4, and also compared TGF beta gene expression in these two cell types from livers subjected to chronic CCl4 treatment. The parenchymal and nonparenchymal cells from normal liver contained undetectable amounts of TGF beta mRNA. In contrast, we could readily detect TGF beta specific transcripts in both the parenchymal and nonparenchymal cells after acute injury. Nonparenchymal cells from acutely injured liver contained fivefold greater amounts of TGF beta mRNA, which peaked at 48 h and declined thereafter. In chronically treated rat livers (1, 2, 3, and 7 wk after the initiation of CCl4 treatment), increased expression of TGF beta mRNA was found only in nonparenchymal cells obtained after 2-3 wk of treatment. Strikingly large elevations in the steady-state levels of beta-actin mRNA in CCl4-treated liver were also observed, which may be related to the known regenerative processes associated with acute liver toxicity. Changing dynamics of TGF beta gene expression, therefore, appear to be an important attribute of regenerating liver after acute or chronic CCl4 toxicity.

Actins↗