Transendothelial migration of hematopoietic progenitor cells. Role of chemotactic factors.

There is increasing evidence that hematopoietic stem cell mobilization and homing is regulated not only by adhesion molecules and cytokines, but also by chemotactic factors that support transendothelial migration across the bone marrow sinusoidal endothelium. Many receptors for chemotactic mediators belong to the family of G protein-coupled seven-transmembrane receptors (7-TMR). Signaling via G proteins, particularly Gi proteins, results in a chemotactic response of the cells towards a gradient of the corresponding ligand. Recent studies have provided evidence for expression of several 7-TMR on immature hematopoietic progenitor cells, which potentially mediate chemotactic effects: chemokine receptors (e.g., CXCR4, receptor for stromal cell-derived factor-1), receptors for lipid mediators (e.g., the cysteinyl leukotriene receptor cysLT1 and the peripheral cannabinoid receptor cb2), and receptors for neuroendocrine hormones (e.g., the somatostatin receptor sst2). From these studies it can be concluded that migration of hematopoietic progenitor and stem cells is controlled by a variety of chemotactic factors rather than by a single chemokine (e.g., SDF-1). Trafficking of immature hematopoietic cells may require combined and interactive regulatory functions of these mediators.

Chemotaxis and transendothelial migration of CD34(+) hematopoietic progenitor cells induced by the inflammatory mediator leukotriene D4 are mediated by the 7-transmembrane receptor CysLT1.

Recent studies suggest that bone marrow (BM)-derived chemotactic mediators such as chemokines play key roles in hematopoietic stem cell trafficking. Lipid mediators, particularly leukotrienes, are involved in leukocyte chemotaxis during inflammation but have also been detected in the normal BM. Therefore, the effects of leukotrienes on hematopoietic progenitor cells were analyzed. Cysteinyl leukotrienes, particularly leukotriene D4 (LTD4), induced strong intracellular calcium fluxes and actin polymerization in mobilized and BM CD34(+) progenitors. Chemotaxis and in vitro transendothelial migration of CD34(+) and more primitive CD34(+)/CD38(-) cells were 2-fold increased by LTD4 at an optimum concentration of 25 to 50 nM. Accordingly, CD34(+) cells expressed the 7-transmembrane LTD4 receptor CysLT1 by reverse transcriptase-polymerase chain reaction and Western blot. Effects of LTD4 were suppressed by the CysLT1 receptor antagonist MK-571 and reduced by pertussis toxin. In contrast, LTB4 induced strong responses only in mature granulocytes. LTD4-induced calcium fluxes were also observed in granulocytes but were not reduced by MK-571, suggesting that these effects were mediated by other receptors (eg, CysLT2) rather than by CysLT1. In addition, expression of 5-lipoxygenase, the key enzyme of leukotriene biosynthesis, was detected in both hematopoietic progenitor cells and mature leukocytes. The study concludes that the functionally active LTD4 receptor CysLT1 is preferentially expressed in immature hematopoietic progenitor cells. LTD4 released in the BM might regulate progenitor cell trafficking and could also act as an autocrine mediator of hematopoiesis. This would be a first physiologic effect of cysteinyl leukotrienes apart from the many known pathophysiologic actions related to allergy and inflammation. (Blood. 2001;97:3433-3440)

Functional response of leukaemic blasts to stromal cell-derived factor-1 correlates with preferential expression of the chemokine receptor CXCR4 in acute myelomonocytic and lymphoblastic leukaemia.

The chemokine stromal cell-derived factor-1 (SDF-1) that is released by bone marrow (BM) stromal cells and contributes to stem cell homing may also play a role in the trafficking of leukaemic cells. We analysed SDF-1-induced intracellular calcium fluxes in leukaemic blasts from the peripheral blood of patients with newly diagnosed acute myeloid leukaemia (AML) and lymphoblastic leukaemia (B-lineage ALL), determined the effect of BM stromal cell-conditioned medium on in vitro transendothelial migration (TM) and measured expression of the SDF-1 receptor, CXCR4, by flow cytometry. AML FAB M1/2 blasts did not show calcium fluxes and TM was not stimulated. In myelomonocytic AML (M4/5), however, SDF-1 induced significant calcium fluxes and TM was increased twofold by the conditioned medium. M3 and M4 blasts with eosinophilia (M4eo) showed intermediate activity and M6 blasts showed no functional activity. In ALL, strong calcium fluxes and increased TM (2.5-fold) were observed. Accordingly, expression of CXCR4 was low in undifferentiated (M0) AML, myeloid (M1/2) AML and erythroid (M6) AML, but high [mean fluorescence (MF) > 50] in promyelocytic (M3) AML, myelomonocytic (M4/5) AML and B-lineage ALL. We conclude that, in AML, SDF-1 is preferentially active in myelomonocytic blasts as a result of differentiation-related expression of CXCR4. Functional activity of SDF-1 and high expression of CXCR4 in B-lineage ALL is in accordance with the previously described activity of SDF-1 in early B cells. SDF-1 may contribute to leukaemic marrow infiltration, as suggested by increased CXCR4 expression and migratory response in BM-derived blasts compared with circulating cells.

Expression and secretion of vascular endothelial growth factor-A by cytokine-stimulated hematopoietic progenitor cells. Possible role in the hematopoietic microenvironment.

In the hematopoietic microenvironment, bone marrow endothelial cells may play an important role in trafficking and maintenance of progenitor and stem cells due to adhesive interactions and paracrine secretion of hematopoietic growth factors. However, it is unknown whether progenitors in turn modulate endothelial proliferation and function. We analyzed mRNA expression (Northern blot) and release of vascular endothelial growth factor-A (VEGF-A), which specifically acts on endothelial cells, by cytokine-stimulated peripheral blood-derived CD34+ hematopoietic progenitor cells. While unstimulated CD34+ cells expressed VEGF-A mRNA weakly without cytokine release in vitro, incubation for 24 hours with a single cytokine (e.g., kit ligand [KL]) resulted in increased VEGF-A mRNA expression and significant secretion of VEGF-A into the supernatant. The amount of VEGF released was substantially augmented by incubation with a combination of cytokines (e.g., KL, IL-3, GM-CSF, G-CSF), or by exposure to hematopoietic cytokines for a longer time period. In addition, we show that VEGF induced the release of hematopoietic growth factors (GM-CSF) by bone marrow endothelial cells and that in vitro stromal cell-derived factor-1 (SDF-1) driven transendothelial progenitor cell migration was increased by the presence of VEGF, which might be due to pore formation (increased endothelial fenestration). In vivo, release of VEGF by progenitor cells may result in a paracrine loop supporting proliferation of both endothelium and progenitors and may facilitate transendothelial migration during cytokine-induced progenitor cell mobilization.

Identification of an alpha-helical epitope region on the PM/Scl-100 autoantigen with structural homology to a region on the heterochromatin p25beta autoantigen using immobilized overlapping synthetic peptides.

The polymyositis-scleroderma overlap syndrome (PM/Scl) autoantigen is a nucleolar multiprotein particle, presumably participating in the maturation of 5.8S rRNAs. The major target antigens of this particle are two polypeptides with apparent molecular masses of 100 and 75 kDa. In this study we identified the major linear epitopes along the PM/Scl-100 protein sequence by probing overlapping oligopeptides with anti-PM/Scl autoantisera. A major epitope region was identified between amino acids 231 and 245 of the PM/Scl-100 polypeptide. Mutational analysis of the corresponding peptide LDVPPALADFIHQQR by glycine-walk followed by immunodetection of the resulting peptides indicated that amino acids 234, 237, 240, and 241 of the PM/Scl-100 autoantigen are essential for binding of the corresponding antibodies. These results allow us to propose a local alpha-helical secondary structure for the PM/Scl-100 major epitope region. A homology search with the peptide LDVPPALADFIHQQR against the Swiss-Model three-dimensional database reveals some topological homology of the PM/Scl-100 major epitope region with the heterochromatin modifier protein p25beta, a known autoantigen recognized by antibodies from a subset of scleroderma patients.

Overexpression of the chemokine receptor CXCR4 in B cell chronic lymphocytic leukemia is associated with increased functional response to stromal cell-derived factor-1 (SDF-1).

The chemokine receptor CXCR4 and its ligand stromal cell-derived factor-1 (SDF-1) play an important role in trafficking of normal lymphocytes, monocytes, as well as hematopoietic stem- and progenitor cells. SDF-1 constitutively produced by bone marrow stromal cells acts as a chemoattractant supporting the homing of stem cells and may also contribute to the tropism of malignant cells for the bone marrow. Low-grade lymphoproliferative disorders, particularly B cell chronic lymphocytic leukemia (B-CLL), are characterized by the presence of bone marrow infiltration. Therefore, we analyzed expression of the chemokine receptor CXCR4 in B-CLL, and investigated the functional effect of SDF-1 on the malignant cells. By flow cytometry, CXCR4 was consistently expressed on circulating CLL cells at a fluorescence intensity four-fold greater than that of normal B cells, and three-fold greater than that of CD19+/CD5+ cells from the normal bone marrow. CXCR4 was functionally active as demonstrated by a rapid flux of intracellular free calcium in response to SDF-1, which was significantly reduced by the partially blocking CXCR4 antibody 12G5. Moreover, transendothelial migration of B-CLL cells in vitro was stimulated by conditioned medium from bone marrow stromal cells due to its content of SDF-1, as suggested by reduced migration after addition of the CXCR4 antibody 12G5. In accordance with the CXCR4 overexpression, migration of CLL cells was more efficiently stimulated by recombinant SDF-1 compared to migration of normal B cells. We conclude that CXCR4 is overexpressed and functionally active in B-CLL, and may therefore contribute to the tropism of B-CLL cells for the bone marrow stroma.

Expression of vascular endothelial growth factor (VEGF) and its receptors in human neuroblastoma.

Angiogenic factors may play a role in the biology of neuroblastoma, a well vascularised tumour, which frequently spreads haematogenously. Therefore, we analysed expression of vascular endothelial growth factor (VEGF) in six human neuroblastoma cell lines and five primary neuroblastomas. High VEGF levels (1-3 ng/10(6) cells/day) were found in the supernatant of all cell lines examined (SK-N-LO, SK-N-SH, LS, SH-SY5Y, IMR-32, Kelly). VEGF peptide was also detected in tissue homogenates from four of five primary tumours. Reverse transcription-polymerase chain reaction (RT-PCR) revealed that VEGF165 is the major isoform produced by neuroblastomas. In addition, all cell lines and primary tumours expressed the mitogenic VEGF receptor FLK-1, whilst the non-mitogenic receptor FLT-1 was less frequently positive, suggesting that the tyrosine kinase FLK-1 is involved in malignant transformation of neuroblastoma cells. However, neutralising antibodies to VEGF did not inhibit growth of neuroblastoma cell lines, which argues against a role of VEGF as an autocrine growth factor, at least for cell lines in vitro. We conclude that neuroblastoma cells produce VEGF, which may contribute to tumour vascularisation, growth and invasion.

Regulation of transendothelial migration of hematopoietic progenitor cells.

Transendothelial migration of hematopoietic progenitor cells occurs in the bone marrow during mobilization and homing, and may therefore play a key role in the trafficking of hematopoietic stem cells. We hypothesize that adhesion molecules, chemokines, and paracrine cytokines are involved in this multifactorial process. As suggested in several studies, downregulation of adhesion molecules (e.g., integrins) may contribute to mobilization of progenitors due to a decreased avidity to bone marrow stromal and endothelial cells, which express the corresponding ligands. Using an in vitro model of transendothelial migration, we have shown that only a small number of more mature, committed progenitors migrates spontaneously under the control of adhesion molecules of the beta-2-integrin family and their corresponding endothelial/stromal ligands. However, transendothelial migration of progenitors in vitro is substantially enhanced by the chemokine stromal cell-derived factor-1 (SDF-1), which is constitutively produced by bone marrow stromal cells. More primitive progenitors also respond to this chemokine. In addition, the ligand for SDF-1, the chemokine receptor CXCR-4, is expressed in greater levels on bone marrow CD34+ cells as compared to mobilized progenitors, suggesting that downregulation of chemokine receptors occurs during progenitor mobilization. Indeed, bone marrow CD34+ cells migrate more avidly in response to SDF-1 than mobilized progenitors. Paracrine cytokines may also play a role in hematopoietic stem cell trafficking, since growth factor-stimulated hematopoietic cells produce cytokines that act on endothelial cells (e.g., vascular endothelial growth factor, VEGF), modifying their proliferation, motility, permeability, and fenestration. We conclude that transendothelial migration of hematopoietic progenitor cells is regulated by adhesion molecules, paracrine cytokines, and chemokines. Cytotoxic therapy as well as exogenously administered hematopoietic growth factors may affect adhesion molecule expression, the local cytokine and chemokine milieu, and chemokine receptor expression, which indirectly results in mobilization of hematopoietic stem cells.

Identification of major linear epitopes on the sp100 nuclear PBC autoantigen by the gene-fragment phage-display technology.

Approximately 20-30% of sera from patients suffering from primary biliary cirrhosis contain autoantibodies against a nuclear protein termed sp100. By indirect cytoimmunofluorescence it was shown that the sp100 autoantigen is distributed in up to 20 dot-like structures per nucleus co-localizing with the so-called nuclear bodies. In western blots these sera react with a protein with an apparent molecular mass of 100kDa. By screening expression libraries with affinity-purified anti-sp100 antibodies we isolated a full-length sp100 cDNA whose sequence exactly matched the previously published sp100 sequence and encodes a protein of 481 amino acids with a deduced molecular mass of 53 kDa. In an attempt to determine immunoreactive regions on the sp100 antigen with the recently developed gene-fragment phage-display technology we were able to identify a stretch of sixteen amino acids (IKKEKPFSNSKVECQA) at position 296-311 as a major antigenic region (antigenic region 1) on the sp100-autoantigen. A second antigenic region (antigenic region 2) of twenty amino acids in length could be identified between amino acids 332-351 (EGSTDVDEPLEVFISAPRSE). By using immobilized synthetic peptides and various sp100-positive PBC patient sera the corresponding epitopes could be shown to be centered around epitope cores of six amino acids (SNSKVE, antigenic region 1) and nine amino acids (EPLEVFISA, antigenic region 2) respectively.

The chemokine receptor CXCR-4 is expressed on CD34+ hematopoietic progenitors and leukemic cells and mediates transendothelial migration induced by stromal cell-derived factor-1.

The chemokine stromal cell-derived factor-1 (SDF-1) and its receptor CXCR-4 (fusin, LESTR) are likely to be involved in the trafficking of hematopoietic progenitor and stem cells, as suggested by the reduced bone marrow hematopoiesis in SDF-1-deficient mice and the chemotactic effect of SDF-1 on CD34+ progenitor cells. Migration of leukemic cells might also depend on the expression of chemokine receptors. Therefore, we analyzed expression of CXCR-4 on mobilized normal CD34+ progenitors and leukemic cells. In addition, SDF-1-induced transendothelial migration across a bone marrow endothelial cell layer was assessed in vitro. By flow cytometry, CXCR-4 was found to be expressed in significant amounts on circulating CD34+ hematopoietic progenitor cells, including more primitive subsets (CD34+/CD38- and CD34+/Thy-1+ cells). In accordance with the immunofluorescence data, CD34+ progenitors efficiently migrated across endothelium in response to SDF-1 containing conditioned medium from the stromal cell line MS-5. Leukemic blasts (mostly CD34+) from patients with acute myeloblastic leukemia (AML) expressed variable amounts of CXCR-4, which was functionally active, as demonstrated by a positive correlation between the SDF-1-induced transendothelial migration and the cell surface density of CXCR-4 (r = 0.97). Also recombinant SDF-1beta induced migration of CXCR-4-positive leukemic blasts. The effect of both conditioned medium and recombinant SDF-1 was inhibited by a CXCR-4 blocking antibody. In contrast, CD34+ leukemic cell lines (KG1, KG1a, Kasumi-1, MOLM-1) expressed low levels or were negative for CXCR-4, and did not migrate. By reverse transcriptase-polymerase chain reaction (RT-PCR), however, basal levels of CXCR-4 mRNA were also detected in all leukemic cell lines. We conclude that CXCR-4 is expressed on CD34+ cells including more primitive, pluripotent progenitors, and may therefore play a role in the homing of hematopoietic stem cells. CXCR-4 expressed in variable amounts on primary AML leukemic cells is functionally active and may be involved in the trafficking of malignant hematopoietic cells.

Antibodies against a peptide sequence located in the linker region of the HMG-1/2 box domains in sera from patients with juvenile rheumatoid arthritis.

OBJECTIVE: To extend our work on the mapping of B cell epitopes on nucleosomal high mobility group (HMG) proteins in the sera of patients with juvenile rheumatoid arthritis (JRA). METHODS: Seventy-seven pauciarticular-onset JRA serum samples from antinuclear antibody (ANA)-positive patients and 42 polyarticular-onset JRA patient sera found to react with HMG-2 by immunoblotting were used in this study. To identify B cell epitopes on HMG-2, recombinant HMG-2 protein fragments were used in enzyme-linked immunosorbent assay (ELISA) and in competition ELISA experiments with a set of overlapping synthetic peptides. Fine epitope mapping was achieved by oligopeptide synthesis, followed by immunoblotting. RESULTS: Pauciarticular, but not polyarticular, JRA patient sera were found to recognize a lysine-rich major epitope (KKGKKKDP), which is located in the linker region of the HMG box domains of the HMG-2 nonhistone chromosomal protein. No significant immunoreactions were observed in sera from ANA-negative JRA patients and in sera from children with nonrheumatic diseases, indicating that this epitope seems to be specific for pauciarticular-onset JRA. CONCLUSION: In addition to our previous finding that JRA sera will react with a defined epitope on HMG-17, pauciarticular JRA patient sera were also found to recognize a defined epitope on the HMG-2 protein, thus suggesting the importance of this epitope in the etiology of JRA.

Mapping of epitopes recognized by PM/Scl autoantibodies with gene-fragment phage display libraries.

Sera from patients suffering from the polymyositis/scleroderma overlap syndrome (PM/Scl) recognize two antigenically non-related proteins with apparent molecular masses of 100 kDa and 75 kDa respectively. The two proteins are part of a particle termed PM/Scl localized in the granular component of the nucleolus. The predominant immunoreactivity of the PM/Scl sera was shown to be directed against the 100 kDa protein. The cDNA of the 100 kDa protein has been cloned recently and its immunogenic regions have been partially mapped using recombinant proteins. Thus far the localization of antigenic determinants on polypeptides has been done by expressing defined cDNA fragments in bacteria or by synthesizing overlapping short peptides and probing their immunoreactivity with antibodies. Here we present an alternative approach to localize autoimmune epitopes using sera containing polyclonal antibodies and gene-fragment phage display libraries. For epitope fine mapping of the PM/Scl-100 protein random fragments of the corresponding cDNA were cloned into the PIII protein of fUSE-5. These gene-fragment phage display libraries were incubated with affinity purified anti-PM/Scl-100 antibodies to enrich for epitope-displaying phages. All PM/Scl sera tested recognized 23 consecutive amino acids (229-251) encoded by four overlapping fUSE-5 clones, suggesting that a major epitope is contained within the 23 amino acids. In addition a minor epitope was localized in a region of 21 amino acids (775-795) encoded by two overlapping fUSE-5 clones since only three out of the seventeen sera reacted with this amino acid sequence. Additional fine mapping of the major epitope was done using synthetic oligopeptides. Thus, a stretch of 16 amino acids at position 229-244 could be identified as a major epitope on the deduced PM/Scl-100 amino acid sequence.

A member of the mouse LRR transcript family with homology to the human Sp100 gene.

A previously isolated cDNA sequence with homology to the long-range repeat (LRR) cluster in chromosome 1 of the house mouse, Mus musculus, was identified as derived from a 1.3 kb polyadenylated RNA. This transcript belongs to a family of polyadenylated RNAs which are synthesized from a multicopy gene included in the LRR copies. The representation of the 1.3 kb transcript in genomic DNA was studied in lambda and cosmid clones from the LRR cluster. Two different types of LRRs were detected with respect to the arrangement of coding regions. In the type-1 arrangement, the sequence is split into five exons, and in the type-2 arrangement, into six exons. The respective exons with their flanking regions were sequenced. The analysis of splice signals revealed that LRR copies with a type-1 arrangement are presumably the source of the 1.3 kb transcript. The 1.3 kb transcript has sequence homology to a human gene encoding Sp100, a nuclear antigen recognized by autoantibodies from patients suffering from some autoimmune diseases including primary biliary cirrhosis. Mouse exons II and III exhibit 71% homology at the nucleotide level and 56% homology at the amino acid level to the human Sp100 cDNA. We mapped the human Sp100 gene to chromosome 2. This location corroborates the assumption that the human Sp100 gene and the mouse LRR gene are homologous, as the human chromosome 2 contains the segment which is homologous to the mouse LRR region.

Sera from JRA patients contain antibodies against a defined epitope in chromosomal protein HMG-17.

Autoantibodies against the nonhistone nucleosomal protein HMG-17 have been detected in a high percentage of ANA-positive patients with pauciarticular-onset JRA4. Here we report on the epitope mapping of the HMG-17 autoantigen with a set of overlapping and nested synthetic peptides spanning the entire amino acid sequence of the human HMG-17 protein. Competition ELISA experiments defined a proline and lysine rich octapeptide PKPEPKPK as the major epitope recognized by more than 70% of the HMG-17 positive JRA sera. Point mutations introduced in the autoimmune peptide determined the amino acid residues important for autoantibody recognition. Computer based sequence comparison shows close homology between the HMG-17 autoimmune epitope and certain infectious organisms, supporting the possibility that molecular mimicry is an important factor in the etiology of JRA.

Antineutrophil cytoplasmic autoantibodies (ANCA) recognizing a recombinant myeloperoxidase subunit.

Sera from 76 patients with diverse vasculitis, systemic lupus erythematosus (SLE) and hydralazine-induced lupus (HiL) were analysed by ELISA for their reactivity with native, reduced or urea-denatured MPO, respectively, as well as with bacterially expressed heavy and light subunits of MPO. All sera (n = 20) recognizing native MPO showed a positive reaction with reduced MPO, while 12 recognized the denatured protein. Most of the linear epitopes are located in the light subunit, since 9 MPO-positive sera recognized significantly the bacterially expressed, denatured light subunit, while only one serum recognized the bacterially expressed heavy subunit purified under denaturing conditions.

Cloning and characterization of the cDNA coding for a polymyositis-scleroderma overlap syndrome-related nucleolar 100-kD protein.

About 50% of patients with the polymyositis-scleroderma overlap syndrome are reported to have autoantibodies to a nucleolar particle termed PM/Scl. The particle consists of several polypeptides of which two proteins of 75 and 100 kD have been identified as the major antigenic components. Here we report on the cDNA cloning and partial epitope mapping of the 100-kD autoantigen from human placenta and HeLa lambda gt11 libraries. The deduced amino acid sequence encodes a protein of 885 amino acid residues with a molecular mass of 100.8 kD. Rabbit antibodies raised against a recombinant protein fragment reacted in immunofluorescence and immunoblotting in the same manner as human autoantibodies directed against the nucleolar 100-kD protein. Sequence analysis shows close homology to a consensus sequence of 12 amino acids from serine/threonine kinases, suggesting a possible function for this autoantigen. A major antigenic region is found to be located within the NH2-terminal third of the polypeptide.

Autoantibodies to the chromosomal protein HMG-17 in juvenile rheumatoid arthritis.

OBJECTIVE: To determine the antibody profiles in sera from patients with juvenile rheumatoid arthritis (JRA). METHODS: Immunoblotting using nuclear extracts and recombinant high-mobility group (HMG) nonhistone chromosomal proteins. RESULTS: Antibodies directed against HMG-17 were found in 47% of antinuclear antibody (ANA)-positive patients with pauciarticular-onset JRA and in 16% of ANA-positive patients with polyarticular-onset JRA. HMG-17 values of 6% and 8%, respectively, were detected in ANA-negative patients with JRA and in those with nonrheumatic diseases. CONCLUSION: There is evidence for a high prevalence of anti-HMG-17 antibodies in sera of patients with pauciarticular-onset JRA.

Autoantibodies to nonhistone chromosomal proteins HMG-1 and HMG-2 in sera of patients with juvenile rheumatoid arthritis.

IgG antibodies against the high mobility group (HMG) nonhistone chromosomal proteins HMG-1 and/or HMG-2 were detected in the sera of 49 (39%) of 126 antinuclear antibody (ANA)-positive patients with juvenile rheumatoid arthritis (JRA), by immunoblotting. Clinical diagnosis classified these patients in 2 major groups, 105 with pauciarticular-onset JRA and 21 with polyarticular-onset JRA. Anti-HMG-1 and/or anti-HMG-2 antibodies were found in 8 (25%) of 32 pauciarticular-onset JRA patients with uveitis and in 34 (47%) of 73 patients without uveitis, whereas anti-HMG-1 and/or anti-HMG-2 antibodies were found in 4 (24%) of 17 children with polyarticular-onset JRA without uveitis. Among 53 sera from ANA-negative JRA patients, 3 (6%) were positive for anti-HMG-1 and/or anti-HMG-2 antibodies, whereas no reactivity to HMG-1 or HMG-2 proteins was observed in 48 sera from age-matched children with nonrheumatic diseases.

A monoclonal antibody directed against the head region of vimentin.

The production and identification of a monoclonal antibody directed against an epitope in the aminoterminal head region of vimentin is described. Enzyme-linked immunosorbent assay, protein blotting and indirect immunofluorescence were used. The wide range of cross-reactivity within cytoskeletal proteins observed for this antibody gives evidence for a determinant in an evolutionarily conserved region. Computer comparison of aminoacid sequences of the immunoreactive proteins and biochemical cleavage of vimentin provide possible clues to some antigenic determinants.

Autoimmune sera recognize a 100 kD nuclear protein antigen (sp-100).

Autoimmune sera from patients suffering from undifferentiated connective tissue diseases (UCTD), Sjögren's syndrome (SS), primary biliary cirrhosis (PBC) and other disorders were found to contain antibodies that produce a distinctive nuclear spot pattern with HEp-2 cells in immunofluorescence studies. These spots which vary in size and number, are spread over the whole nucleus with the exception of the nucleoli. This pattern is easily distinguishable from the staining patterns of anti-centromere, anti-RNP, anti-nucleolar and anti-Scl-70 antibodies. In cells of non-human origin this pattern is discerned only at high serum concentrations. Immunoblotting experiments with a soluble protein fraction from HeLa nuclei revealed that the antigenic target common to all sera is a polypeptide of 100 kD with a pI value of about 5.2. The correlation between immunofluorescence and immunoblotting data was confirmed by affinity-purification of sp-100 specific autoantibodies followed by immunofluorescence experiments.