(SWR x SJL)F1 mice: a new model of lupus-like disease.

During the study of autoimmune models we found that (SWR x SJL)F1 mice (both parental strains with the V beta a phenotype) spontaneously produced immunoglobulin G (IgG) antibodies directed against Sm/U1 small nuclear ribonucleoproteins (snRNPs). In some of these females, the presence of these autoantibodies was found as early as 10 wk of age. Their frequency increased with age i.e., 70% at 40 wk. At that time, only 10% of males developed anti-Sm/U1snRNP antibodies. Anti-Sm/U1snRNP antibodies from positive mice generally recognized the peptides BB', D, 70 kD, and A from RNPs. These polypeptides are known to bear the autoantigenic epitopes that are recognized by human sera containing anti-Sm and anti-U1snRNP antibodies. Reactivity of IgG antibodies with the octapeptide sequence PPPGMRPP was also found in 30% of anti-Sm/U1snRNP positive (SWR x SJL)F1 mice that precipitated BB' peptides. This octapeptide has been described as the most immunoreactive linear epitope in systemic lupus erythematosus (SLE) patients with anti-Sm and anti-U1snRNP antibodies. Approximately 30% of anti-Sn/U1snRNP positive females, later produced anti-dsDNA antibodies. This fact was accompanied by the development of proteinuria due to glomerulonephritis mediated by immunocomplexes. In addition to the specific autoimmune response, (SWR x SJL)F1 females also showed other immunologic abnormalities such as hypergammaglobulinemia, and an approximately twofold increase in spleen cell number compared with control mice. These results indicate that (SWR x SJL)F1 females develop clinical and serological abnormalities similar to those observed in human SLE and constitute a novel model for the study of the genetic mechanisms that result in autoimmunity.

Bullous pemphigoid and multiple sclerosis: a report of two cases with ELISA test.

We report two patients with longstanding multiple sclerosis (MS) who developed vesicles and bullae consistent with the diagnosis of bullous pemphigoid (BP). Both patients showed linear IgG at the dermal-epidermal junction, located on the epidermal side of patients' skin previously treated with 1M NaCl. In the two cases, the ELISA test was positive for the extracellular fragment of BP 180. However, the indirect immunofluorescence test (IIF) was repeatedly negative. Therapy either with prednisone plus dapsone or prednisone alone was initiated and the disease was controlled after 23 and 15 months of therapy, in patients 1 and 2, respectively. However, the first patient had a flare-up 2 months after treatment was stopped. The association of MS and BP has been described previously in 35 cases. We compare our two cases with the 25 patients previously reported in detail in the literature. We emphasize the role of the ELISA test in establishing the diagnosis of BP.

Effect of enzymatic treatments on RNP and Sm antigenic reactivities--I. Loss of RNP but increase of Sm antigenic reactivity after RNase treatment of nuclear extract.

Changes of RNP and Sm antigenic reactivities of a nuclear extract after enzymatic treatments were studied and quantified by the ELISA test. After RNase treatment of the nuclear extract, about a 300% increase of the Sm antigenic reactivity and more than a 95% decrease of RNP antigenic reactivity was found. Data from RNP-depleted nuclear extracts and column fractionation show that the increase in Sm antigenic reactivity after RNase treatment mainly comes from the RNP-Sm complex.

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.

Thyroglobulin autoantibody levels below the cut-off for positivity can interfere with thyroglobulin measurement.

Antithyroglobulin antibodies can interfere with the measurement of thyroglobulin yielding spuriously high or low levels depending on the method used. Interference is unrelated to the antibody concentration and can occur at very low concentrations. We report a patient in whom antithyroglobulin antibodies below the cut-off for positivity nearly led to an incorrect diagnosis of thyrotoxicosis factitia.

[Comparative immunopathologic study of oral aphthae, non-involved mucosa from aphthae patients, healthy mucosa and trauma-induced ulcers]. / Estudio inmunopatológico comparativo de aftas orales, mucosas no afectas por úlceras de pacientes aftosos, mucosas sanas y úlceras inducidas de forma traumática.

The presence of IgG, IgA, IgM, C1q, C3c, fibrinogen and properdin has been studied using direct immunofluorescence technique. The same study has been performed in clinically normal mucosa of patients with aphthous ulcers, normal mucosa of healthy volunteers and traumatically induced ulcers. We have also studied the possible presence of reactive serous immunoglobulins with normal mucosa by indirect immunofluorescence. Our findings do not support previous studies which claimed the presence of vasculitis induced by the presence of immunoreactants in the vessel walls.

Correlation of immunological markers with graft vasculopathy development in heart transplantation.

This study examined the imbalance between T effector cells (Th1 defined as CD3+ interferonγ+) and T regulatory cells (Treg defined as CD4+CD25(high)FoxP3+) as a valuable albeit limited marker of cardiac allograft vasculopathy (CAV) after heart transplantation (HTx). CAV remains, with neoplasms, the most important cause of death in patients surviving the first year after HTx. It is an immune-mediated pathology, although nonimmune factors may also play a role. The process included concentric fibrous intima hyperplasia that narrows the entire length of the affected arteries. Coronary angiography is the usual method of diagnosis. Because a transplanted heart is a denervated organ, CAV is not diagnosed until the disease reaches an advanced stage, in which case transplantation is the only option for treatment. Although the host's immune response against an allogeneic graft is the major cause of endothelial dysfunction, the objective of this study was to detect anti-allogeneic responses on peripheral blood, seeking to identify signs of CAV before classical methods to predict outcomes in HTx recipients. CD3, CD4, CD8, CD19, CD56, Th1, and the Treg mononuclear cell populations were studied in 37 de novo and 20 long-term (more than 3 years) HTx patients as well as 20 healthy volunteers using flow cytometry. A progressive increase in CD8 and Th1 percentages and decrease in the CD4 population were detected during follow-up. Although Th1 changes also reflect processes not related to CAV receiver operating characteristics analysis of Th1/Treg ratio showed an area under the curve of 0.976, with an estimated sensitivity of 100% and specificity of 90%. The positive prediction value was 58.8% and the negative prediction value, 100%. These results prove that the Th1/Treg ratio was an important marker to following host immune response after HTx. The results confirm the need to test other T lymphocyte subsets.

Assessment of immunological markers as mediators of graft vasculopathy development in heart transplantation.

Heart transplantation (HT) remains the treatment of choice for patients with end-stage heart failure. Cardiac allograft vasculopathy (CAV), a diffuse form of coronary atherosclerosis, is the major cause of death after the first year of HT. CAV is thought to be multifactorial in origin. Although nonimmune factors may play a role in CAV development, it is primarily an immune-mediated disease. CAV is diagnosed by routine annual coronary angiography, and usually when diagnosed, the disease is advanced. There is a need to develop noninvasive surrogate markers for early detection. For this purpose, careful immune monitoring and graft histologic assessment are mandatory. The main objective of this study was the assessment of immunologic markers as mediators of CAV development in HT. Flow cytometry was performed to assess peripheral blood mononuclear cell populations forming CD3, CD4, CD8, CD19, CD56, Th1 (CD3+IFNγ+) or Treg (CD4+CD25(high)FoxP3+) markers among 20 de novo HT recipients. The control group included 13 patients who were more than 2 years post-HT (four with and nine without CAV) as well as 20 healthy subjects. CAV-related events over 2 years' follow-up correlated with the Th1/Treg ratio. An increased Th1 lymphocyte percentage was detected over the follow-up. Patients with medium and high Th1/Treg ratios showed higher acute rejection scores as well as greater incidences of CAV. These results indicated that the Th1/Treg ratio may represent a valuable marker to monitor allospecific T-cell responses in peripheral blood. Changes in the Th1/Treg ratio may help in the early detection of patients at risk for CAV. More studies with longer follow-up are needed to confirm these preliminary results.

Therapy of paraneoplastic pemphigus with Rituximab: a case report and review of literature.

Paraneoplastic pemphigus (PNP) is an autoimmune blistering disease with poor prognosis when associated with malignant neoplasm. We report the case of a patient with PNP associated with a CD20+ non-Hodgkin follicular lymphoma who was treated with Rituximab plus corticosteroids and short courses of cyclosporin. One and a half years after Rituximab therapy, oral ulcerations had cleared and oral methylprednisolone was slowly tapered down without further recurrences. In the course of the disease, the patient developed sepsis due to Listeria monocytogenes and viral infections by human herpes virus 1 and 3. At the end-stage of the disease she developed a cutaneous infection from Mycobacterium chelonae. The patient died 2 years and 7 months after the onset of PNP. Rituximab may be useful for PNP therapy, but further studies are necessary to confirm this hypothesis.

Purification of hnRNP from HeLa cells with a monoclonal antibody and its application in ELISA: detection of autoantibodies.

HnRNP antigen from HeLa cells was purified using a monoclonal antibody (383 IgM) that recognizes heterogeneous nuclear ribonucleoprotein (hnRNP). From extracts of HeLa cells radiolabelled with 32P, this antibody immunoprecipitates relatively large RNAs of heterogeneous size which are synthesized in the presence of actinomycin D at doses which suppress synthesis of ribosomal RNAs (characteristic features of heterogeneous nuclear RNA). In immunoblots, 383 IgM binds to seven polypeptides: one of approximately 23,000 daltons, three between 30,000 and 43,000 daltons which correspond to the known hnRNP polypeptides called A1, A2 and C1, one of approximately 50,000 daltons, and a doublet of approximately 120,000 daltons. These proteins comigrate through sucrose density gradients suggesting that they are physically associated. Thus, 383 IgM appears to define an epitope that is shared among a number of the protein components of hnRNP. This antibody has been used to design a simple and fast protocol which allows the determination of autoantibodies from human sera by ELISA.

Autoantibodies against a serine tRNA-protein complex implicated in cotranslational selenocysteine insertion.

We describe an autoantibody specificity present in a subgroup of patients with a severe form of autoimmune chronic active hepatitis. These antibodies precipitate a 90-nucleotide RNA from human whole cell extracts and recognize a 48-kDa polypeptide in immunoblotting assays. The RNA is a UGA suppressor serine tRNA that carries selenocysteine (tRNA[Ser]Sec)), as shown by sequence analysis. The protein does not appear to be seryl-tRNA synthetase; rather, it is an excellent candidate for a factor involved in cotranslational selenocysteine incorporation in human cells.

Anti-golgi complex autoantibodies in a patient with Sjögren syndrome and lymphoma.

During routine immunofluorescence studies of the serum of a patient with Sjögren's syndrome and lymphoma we detected antibodies giving a cytoplasmic pattern which did not correspond to previously described patterns found for autoantibodies. Using different cells and tissues as substrates for indirect immunofluorescence, including rat liver, rat small bowel, rat testicle, human thyroid, guinea-pig plasma cells and cultured human fibroblasts, the cytoplasmic structure to which these autoantibodies are directed seems to be the golgi complex, a conclusion supported by histochemical studies. Furthermore, these antibodies were absorbed by isolated golgi vesicles. The autoantibodies are of IgG and IgA classes, and the antigen(s) with which they react is(are) resistant to treatment with DNAse and RNAse. None of the sera from 50 normal individuals, seven patients with Sjögren's syndrome (five of them primary and two associated with rheumatoid arthritis; none of them with lymphoma), 25 patients with mixed connective tissue disease, 10 patients with systemic lupus erythematosus and five patients with progressive systemic sclerosis, had antibodies directed against this cytoplasmic specificity, as determined by indirect immunofluorescence. This is the first time that autoantibodies directed to the golgi complex are reported. The significance of this finding awaits further descriptions in patients with a clinical picture similar to the one reported here.

The role of BALB/c donor CD8+ lymphocytes in graft-versus-host disease in (BALB/c x A/J)F1 (CAF1) mice.

To investigate the role of donor T lymphocyte subsets in the development of chronic graft-vs-host disease (GVHD) induced in (BALB/c x A/J)F1 (CAF1) mice by injecting BALB/c lymphoid cells, we analyzed the effect that CD8+ cell removal from donor inoculum has on the manifestation of the disease. Compared with age- and sex-matched CAF1 mice injected with whole lymphocyte inoculum, CAF1 mice injected with CD8(+)-depleted inoculum exhibited: 1) a higher incidence and exacerbation of nephritis by immunocomplexes; 2) higher (five- to sevenfold) spontaneous IL-4 production; 3) higher frequency titer and precocity of anti-dsDNA, anti-histone, and IgM and IgG rheumatoid factors; 4) a dramatic change in the frequency and titer of anti-U1 small nuclear ribonucleoprotein Abs; and 5) a markedly decreased engraftment (10- to 15-fold) on BALB/c donor lymphocytes. In contrast, rheumatoid arthritis-like disease, a later clinical manifestation of the GVHD in CAF1 + BALB/c model, is not present in the CD8(+)-depleted model (CAF1 + CD8-BALB/c). Considered together, these data suggest that CD8+ donor T lymphocytes play an important role in the degree of chimerism, modulation of the response to autoantigens, and clinical aspects developed in the GVHD model presented here.

Autoantibodies to a transfer RNA-associated protein in a murine model of chronic graft versus host disease.

We established chronic graft vs host disease (GVHD) in (C57BL/10 x DBA/2)F1 mice with an injection of lymphoid cells from the parental DBA/2 strain. In addition to Abs earlier reported, of the 20 animals studied 13 developed Abs against transfer RNA/protein particles. Ten of the 13 sera immunoprecipitated a similar-sized RNA that co-migrated in PAGE with isoleucine tRNA. In immunoblots against proteins affinity purified using anti-isoleucyl-tRNA synthetase prototype serum, 7 of the 10 sera reacted with a polypeptide of 76 kDa that was similar in size to a protein recognized by a human anti-isoleucyl-tRNA synthetase serum. Three of 10 sera significantly and specifically inhibited isoleucyl-tRNA synthetase enzyme activity and one inhibited lysyl-tRNA synthetase activity. These data suggest that the autoantibodies to tRNA-associated proteins that develop in GVHD mice may react with amino acyl-tRNA synthetases, particularly those belonging to the multienzyme complex. Such autoantibodies are associated with myositis in humans, and these mice showed evidence compatible with myositis that appeared to be a manifestation of their GVHD. No previous example of spontaneous development of antisynthetases in animals has been described. We also demonstrated the presence of Abs against the NOR:90 nucleolar Ag as a new target in chronic GVHD. We conclude that chronic GVHD in mice provides a model for the study of the autoimmune responses that characterize human diseases such as mixed connective tissue disease, scleroderma, SLE, and myositis with a wider autoantibody response than that described so far.

Coexistence of two antisynthetases in a patient with the antisynthetase syndrome.

We describe the immunologic findings in a patient with the antisynthetase syndrome characterized by prominent arthritis, lung fibrosis, and subclinical myositis. At disease onset and during the followup, this patient's serum showed 2 different subsets of antisynthetase autoantibodies: anti-Jo-1, which reacted with histidyl-transfer RNA (tRNA) synthetase by immunoblot and inhibited its enzymatic function; and anti-0J, which immunoprecipitated the multi-enzyme complex of synthetases, and reacted with lysyl-tRNA synthetase by immunoblot. This is the first report of anti-Jo-1 and another antisynthetase antibody being found together in the same patient.

Repeat direct immunofluorescence (DIF) test, using, 1 M NaCl treated skin, in the subepidermal autoimmune bullous diseases that contain IgG at the dermal epidermal junction.

Knowledge of autoimmune bullous diseases has greatly increased with the recognition of new entities, and the use of the direct immunofluorescence (DIF) using 1 molar per liter of sodium chloride (1 M NaCl) treated skin has been proposed. To estimate the frequency with which the different DIF patterns are present, we performed a systematic study of the skin or oral mucosa samples in which linear deposits of IgG at the basement membrane zone were detected by routine DIF in the last 6 years. The DIF tests were done on 56 samples before and after splitting the epidermis from the dermis with 1M NaCl. In 40 biopsies (72%) IgG was found on either the epidermal side or on both sides after 1M NaCl split. These cases corresponded to bullous pemphigoid (n=33), herpes gestationis (n=5) and cicatricial pemphigoid (n=2). In 6 cases (10.7%), IgG deposits were observed only on the floor, five corresponding to bullous pemphigoid and one to bullous pemphigoid-like eruption induced by amoxicillin. Repeat direct immunofluorescence using 1M NaCl split skin indicates that at least 12% of patients who were initially diagnosed as bullous pemphigoid, may in fact suffer a different entity, requiring other techniques to achieve the right diagnosis. This test can be a useful routine screening for autoimmune bullous diseases.

Detection of antibodies to small nuclear ribonucleoproteins and small cytoplasmic ribonucleoproteins using unlabeled cell extracts.

RNA molecules immunoprecipitated with sera from patients who have rheumatic diseases can be readily detected in polyacrylamide gels by using ethidium bromide and silver stains. With these stains, we found that RNA patterns characteristic of a broad range of specific small nuclear ribonucleoproteins and small cytoplasmic ribonucleoproteins were recognizable. The stains correctly identified antibodies to ribonucleoproteins in 33 (92%) of 36 patient sera selected for study because of known antibody specificities. The silver stain method detected antibodies to ribonucleoproteins in 25 (76%) of 33 patients with classic systemic lupus erythematosus, a frequency that approximated the frequency observed in the Lerner-Steitz assay, which is based on autoradiography. This approach considerably simplifies the latter radioimmunoassay with minimal loss of precision and sensitivity.