Jose Luis Rodriguez-Sanchez

Identification of protein components reactive with anti-PM/Scl autoantibodies.

The PM/Scl antigen from mammalian cells has been characterized as a nucleolar and nucleoplasmic molecular complex containing at least 16 polypeptides ranging in molecular weight from 110 to 20 kD. Of these polypeptides. we have found those of68, 39 and 20 kD to be in a phosphorilated form. Whereas the entire complex was precipitated by all the anti‐PM/Scl sera tested, in immunoblots the antibodies specifically recognized determinants on the 110‐kD protein. This protein was immunoprecipitated more preferentially from nuclcoli extracts than from total cell extracts. Moreover, this protein disappeared from the immunoprecipitates when treated with DNAse. Likewise, the immunoblol reaction of the specific antibodies with the 110‐kD protein was abolished by treatment of the extracts with DNAse and trypsin, and was resistant when extracts were treated with RNAsc. Affinity‐purified antibodies from this protein selectively stained the nucleoli and the nucleoplasm of the mammalian cells. Moreover, when the cultured cells used in immunofluorescence were treated with DNAse, the affinity purified antibodies from the 110‐kD protein gave negative fluorescence. However, when whole anti‐PM/Scl sera were used, a nucleolar and nucleoplasmic staining was found. We conclude that the 110‐kD protein has at least one of the autoimmunogenic epilopes of the PM/Sel antigen, recognized by all anti‐PM/Scl sera tested. Other epitopes differing in their DNAse sensitivity may also be present in the PM/Scl antigen.

Cancer-Associated Myositis and Anti-p155 Autoantibody in a Series of 85 Patients With Idiopathic Inflammatory Myopathy

A new autoantibody against a 155-kDa protein has been described in patients with myositis. We conducted a study to determine the occurrence and types of cancer occurring in a cohort of patients with polymyositis (PM) or dermatomyositis (DM) and analyzed the value of this autoantibody as a serologic marker of cancer-associated myositis (CAM). Serum samples from all patients were examined by protein immunoprecipitation assays with HeLa cells to determine the presence of a 155-kDa protein band. HLA-DRB1 and DQA1 typing was performed by polymerase chain reaction-reverse sequence specific oligonucleotide. Statistical analyses were carried out with the Mann Whitney U and Fisher exact tests. Associations were determined using odds ratios (ORs) with 95% confidence intervals (CI). Eighty-five patients with myositis (20 PM and 65 DM) were included. CAM was detected in 16 patients (19%), 14 with DM. The shawl sign rash was significantly more frequent in patients with CAM than in those without (p < 0.01). Adenocarcinoma was the most frequent type of cancer (87.5%). Anti-p155 autoantibody was found in 1 of the 20 (5%) patients with PM and in 15 of the 65 (23%) patients with DM. A relationship between anti-p155 and CAM was found in DM patients (OR, 23; 95% CI, 5.23-101.2). The HLA-DQA1*0102 allele was not found in any of the anti-p155-positive patients. The prevalence of CAM in our cohort was 19%. Autoantibody against p155 was highly related to CAM and could be a reliable marker of cancer in patients with DM. Abbreviations CAM = cancer-associated myositis, CI = confidence interval, DM = dermatomyositis, OR = odds ratio, PM = polymyositis.

Distinctive response of naïve lymphocytes from cord blood to primary activation via TCR

Umbilical cord blood (UCB) is now being considered an alternative to bone marrow for restoring hematopoiesis after myeloablative therapy. The lower risk of acute and chronic graft‐versus‐host disease in patients who received UCB cells seems related to the nature of UCB–T cells. Phenotypically, UCB–CD3+ cells are mostly naive (CD45RA+) and represent a transitional population between thymocytes and adult T cells. We examined the immune reactivity of highly purified, negatively selected CD4+CD45RA+ cells by mimicking activation via T cell receptor (TCR). All experiments included the extensively characterized adult peripheral blood (APB) cells as reference. On the contrary to APB, naive UCB–CD4+ cells were able to proliferate with anti‐CD3 stimulation alone. With addition of interleukin (IL)‐2 or costimulatory signal, both populations reached similar proliferation. Forty‐eight hours after anti‐CD3 stimulation, CD4+CD45RA+ from UCB, but not APB, showed characteristic blastic morphology and significant expression of CD25 on the surface. A low concentration of IL‐2 was detected at 24 h by anti‐CD3‐stimulated UCB CD4+CD45RA+, which rapidly disappeared. By 72 h after activation, CD4+CD45RA+ UCB cells showed extensive apoptosis, whereas CD4+CD45RA+ APB cells showed low levels of apoptosis. Using RNase protection assay, we observed that CD95L levels were significantly higher in naive CD4+ cells from UCB than from APB after activation. However, neutralizing Fas‐Fc protein was unable to inhibit anti‐CD3‐induced apoptosis, suggesting that this was a CD95‐independent mechanism. These results indicate that UCB–CD4+CD45RA+ cells are able to start proliferating as a result of early IL‐2 production after TCR engagement alone, but probably, as a result of the consumption of this IL‐2, they undergo cell death.

Role of the STAT1 pathway in apoptosis induced by fludarabine and JAK kinase inhibitors in B-cell chronic lymphocytic leukemia

Signal transducers and activators of transcription (STAT) proteins comprise a family of transcription factors that have been implicated in tumoral transformation, especially in hematological malignancies. Because of this, the JAK/STAT pathway is attractive as a therapeutic target in these tumors. In the present study, we analyzed the ability of fludarabine and two JAK kinase inhibitors, AG490 and WHI-P131, to block STAT1 activation and induce apoptosis on B-cell chronic lymphocytic leukemia (B-CLL) cells. All drugs were able to induce a high percentage of apoptosis on B-CLL cells from all patients studied. However, only AG490 and WHI-P131 were able to strongly suppress the STAT1 activation of B-CLL cells. In conclusion, our data show that JAK kinase inhibitors, such as AG490 and WHI-P131 are able to inhibit the STAT1 pathway on B-CLL cells and are strong inductors of apoptosis on these cells.

Description of a Novel Panallergen of Cross-Reactivity between Moulds and Foods

The present investigation is undertaken to demonstrate a novel cross-reactivity between aeroallergens (moulds fungi imperfecti) and allergens from foods (spinach and mushroom Agaricus bisporus). We have performed a dual study in vivo and in vitro, in a population of atopic patients. Data from in vivo tests performed with spinach and mushroom have been statistically analysed. To the in vitro assays, mushroom and spinach extracts have been obtained, and sera from moulds allergic patients analysed by means of IgE–immunoblott assays. Inhibition experiments have been also performed to study a possible relation between proteins. Statistical analysis of data showed a relation between allergenicity to moulds (Alternaria alternata, Cladosporium herbarum and/or Aspergillus fumigatus), and positive skin prick tests with mushroom and/or spinach. The immunoblotts performed showed that seven moulds allergic patients had a strong recognition of a protein with a molecular weight of about 30 kD present both in spinach and mushroom extracts, and by means of inhibition assays we could determine that these two proteins were related. This study demonstrates the existence of a new allergen responsible for cross reactivity between moulds and two frequently consumed foods, mushroom and spinach. We conclude that a novel cross-reactive allergen between aeroallergens and foods has been identified.

Anti-cyclic citrullinated peptide and anti-keratin antibodies in patients with idiopathic inflammatory myopathy

OBJECTIVE To investigate the prevalence of anti-cyclic citrullinated peptide (anti-CCP) and anti-keratin antibodies (AKAs) in a cohort of patients with idiopathic inflammatory myopathy. METHODS In a cross-sectional study, we determined the presence of anti-CCP and AKAs by ELISA and IIF, respectively, in a cohort of 90 consecutive patients with idiopathic inflammatory myopathy. Associations between anti-CCP and clinical manifestations or other autoantibodies were determined with the chi-square and Mann-Whitney U-tests. Radiographs of hands were retrospectively evaluated. Serum autoantibody profile was determined in all patients. RESULTS Twelve patients were positive to anti-CCP (13.3%); in eight cases values were moderate-high. AKAs were not detected in any patient. Comparison between patients positive and negative to anti-CCP did not show clinical or biological differences. Arthritis joint erosions or positive status to anti-synthetase antibodies were not more frequent in patients with anti-CCP antibodies. Prevalence of RF was the only variable significantly associated with the presence of these antibodies (P = 0.043). CONCLUSIONS High titres of anti-CCP can occasionally be found in patients with inflammatory myopathy. Therefore, a possible diagnosis of RA should be considered with caution in these patients.

Identification of the autoantigen HB as the barrier-to-autointegration factor.

The HB autoantigen, a 10-kDa DNA-binding protein recognized by autoantibodies only when bound to DNA, was identified by two-dimensional electrophoresis. Silver-stained protein spots corresponding to the antigen were excised from two-dimensional electrophoresis gels, digested with trypsin, and analyzed by matrix-assisted laser desorption/ionization-reflectron time of flight and nano-electrospray ionization-ion trap/mass spectrometry. Data base search identified the HB antigen as the barrier-to-autointegration factor, a cellular protein implicated in the cellular cycle that blocks autointegration and promotes intermolecular integration of retrovirus such as the Moloney murine leukemia and the human immunodeficiency type 1 virus. The physicochemical characteristics described for these proteins, their ability to bind double-stranded DNA but not single-stranded DNA, and their nuclear localization confirm that HB and barrier-to-autointegration factor are the same protein.

Different strains of donor parental lymphoid cells induce different models of chronic graft-versus-host disease in murine (Balb/c × A/J)F1 hybrid hosts

The induction of a chronic graft-versus-host (cGVH) disease in (Balb/c x A/J)F1 mice by the intravenous injection of either Balb/c or A/J parental lymphoid cells led to the development of two different models of disease. In this paper we compared the clinical aspects and the antigen specificities which recognized the autoantibodies developed by the animals of these two models of cGVH disease. Renal disease, alopecia, and purpura lesions were common in both models, although their frequency and intensity varied between groups. The models were differentiated by two main characteristics. When donor cells were of Balb/c origin, a joint disease similar to rheumatoid arthritis developed in 50% of the animals, and when donor cells were of A/J origin, 25% of the animals developed edema of the front feet, occasionally with loss of the nails, similar to that of scleroderma. Differences among the autoantibodies found in the sera of these two groups of mice were also observed. After the injection of Balb/c lymphoid cells, rheumatoid factors reactive with human and murine IgG were characteristically present (69 and 75%, respectively) and a statistically significant correlation was found between high titers of rheumatoid factor and arthritis (P less than 0.001). Antinuclear antibodies (ANAs) were present in all animals. Anti-dsDNA and anti-histones were positive in 50 and 25%, respectively. Anti-snRNP were detected at a low titer in 35% of the animals. When donor cells were of A/J origin, ANAs were also present in all mice. Anti-dsDNA, anti-histones, and anti-snRNPs antibodies were present in 90, 15, and 65%, respectively. The most outstanding characteristics among anti-snRNPs were the high titers of anti-U1 and anti-U3 detected in 50 and 30%, respectively. Rheumatoid factors reactive with human and murine IgG were positive in 15 and 42% of animals, respectively, but no significant correlation was found between these factors and disease. Our results indicate that the graft-versus-host disease induced in the same F1 strain of mice can be manifested in different forms of connective tissue disease, depending on whether the cells come from one or the other of the parental strains. Furthermore, in this paper the occurrence of rheumatoid factors in mice with cGVH is described for the first time.

The two isoforms of the 90-kDalton nucleolus organizer region autoantigen (upstream binding factor) bind with different avidity to DNA modified by the antitumor drug cisplatin

It has been previously described that some proteins containing HMG boxes are able to bind more strongly to DNA modified with cis-diamminedichloroplatinum (II) (cisplatin) than to unmodified DNA. In the present study, we analyzed the interaction of cisplatin-modified DNA with the human autoantigen NOR-90 (UBF), a transcription factor that contains several HMG boxes. Using autoantibodies against NOR-90 to perform ELISA and immunoprecipitation, it was confirmed that NOR-90 (UBF) was able to bind cisplatin-modified DNA more avidly than unmodified DNA or trans-diamminedichloroplatinum(II) (transplatin) modified DNA. Moreover, by Southwestern, we observed that the 97 kDalton isoform of NOR-90 (UBF1) was able to bind cisplatin-modified DNA more strongly than the 94 kDalton isoform (UBF2); binding of unmodified DNA or transplatin-modified DNA was not detected with either isoform. Sera containing autoantibodies against NOR-90 did not inhibit, but increased the binding of NOR-90 to cisplatin-modified DNA.

HK-ATPase Expression in the Susceptible BALB/c and the Resistant DBA/2 Strains of Mice to Autoimmune Gastritis

Neonatal thymectomy (NTx) in mice induces a group of alterations in the immune system homeostasis that results in the development of a variety of organ-specific autoimmune diseases such as gastritis, thyroiditis, oophoritis and orchitis. Given the importance of self-antigen expression in thymus for the control of autoreactive cells and generation of regulatory cells, we have compared the expression of parietal cell antigen in two strains of mice with the same H-2: BALB/c (susceptible to develop gastritis after NTx) and DBA/2 (resistant). We detected mRNA of HK-ATPase α and β chains in day 1 thymi of both strains. Fifty percent of BALB/c mice presented mRNA levels similar to DBA/2. However, lower mRNA levels were found in the remaining BALB/c mice that may correspond to those that would develop AIG after NTx. Since the presence of the antigen in periphery is also necessary for the induction of regulatory cells, we have compared both strains observing in day 1 stomachs from resistant DBA/2 strain, a significantly higher content of positive cells for HK-ATPase subunits than stomachs from susceptible BALB/c strain. Also, the presence of antinuclear Abs in NTx BALB/c mice makes this model a useful experimental system for analyzing the responsible mechanisms breaking the non-specific self-tolerance.