Characterization of a canine long-term T cell line (DLC 01) established from a dog with Sézary syndrome and producing retroviral particles.

The canine DLC 01 cell line derives from a lymph node of a dog with Sézary syndrome. The DLC 01 cell phenotype is CD4-, CD8+, CD45+, DQ+, similar to that of original cells after treatment with dimethylsulfoxide or phorbol myristate. Canine cutaneous T cell lymphoma are usually CD4-, CD8+ in contrast to their human counterparts which are CD4+, CD8-. Therefore, the DLC 01 cell line appears to be a unique model to study the mechanism of all surface molecule expression in vitro. Viral particles with retrovirus type-C morphology were found in ultrathin sections of DLC 01 cell pellets. Retroviral particles are spontaneously produced after the 50th cell passage or after induction with 0.5% dimethylsulfoxide. This is the first description of a dog lymphoid cell line spontaneously growing and producing a retrovirus. It was found to share several features in common with feline and murine leukemia viruses.

Comparative studies on naturally occurring antikeratin antibodies in human sera.

Comparative studies on the specificity of the so-called antiepidermal antibodies (Abs) found in human sera were performed by immunoblotting, enzyme-linked immunosorbent assay (ELISA), and immunoelectron microscopy (IEM). After a screening test by indirect immunofluorescence (IF), sera obtained from patients with various diseases and controls could be classified in 5 different groups according to the IF patterns on the epidermis: sera reactive with: (1) the stratum corneum (SC); (2) the upper layer (U-Cyt); (3) the whole epidermis (G-Cyt); (4) basal cells (B-Cyt); and (5) negative ones. By immunoblotting, all the 23 IF-positive sera were found to bind to one or more keratin bands, and did not show any reactivity with epidermal Nonidet P-40 soluble proteins. SC-Abs were mainly directed against a 67 kD Keratin band, whereas U-Cyt- and G-Cyt-Abs bound to both 58-56 kD and 67-63 kD keratins. B-Cyt-Abs reacted strongly with 63 kD Keratins and slightly with a 50 kD band. Antikeratin Abs were detected by immunoblotting even in the IF-negative sera. The ELISA study showed that sera with high IF titers contained high levels of antikeratin Abs. In the IEM study using sera containing U-Cyt- or B-Cyt-Abs, 2 distinct reaction patterns were demonstrated: U-Cyt-Abs stained tonofilaments of suprabasal keratinocytes, while B-Cyt-Abs characteristically reacted with those of basal cells. Moreover, SC-, U-Cyt-, and G-Cyt-Abs were absorbed out by insoluble epidermal proteins, and B-Cyt-Abs were decreased in titer after the absorption test. The present study provides strong evidence that most, though not all, human antiepidermal Abs are directed against different keratin polypeptides, and that antikeratin Abs commonly occur in almost all human sera.

Enzyme-linked immunosorbent assay for anti-histone antibodies and their presence in systemic lupus erythematosus sera.

We describe an enzyme-linked immunosorbent assay (ELISA) with adsorption of histones (total and fractions) on glass beads and saturation of excess sites with sheep serum. The anti-histone antibodies are detected with peroxidase conjugate and developed with Trinder's reagent which has great stability. This very sensitive method detects anti-histone antibodies in 53% of SLE patients and in virtually no other diseases. Positive reactions are observed only with total histones and fractions H1 and H2b.

Non-specific binding of heat-aggregated IgG to histone detected by ELISA.

Anti-histone antibodies are currently detected by micro ELISA in systemic lupus erythematosus sera from humans, mice and dogs. Here we show that the control-heated sera may bind non-specifically to the whole histones and histone fractions. The heated immunoglobulins binding to histones are mainly IgG and to a lesser extent IgA, but never IgM. These false positive ELISA reactions occurred only with aggregated IgG which binds to histones via Fc; IgM rheumatoid factor prevented their fixation. Immune complexes do not seem to interfere significantly in the detection of anti-histone antibodies with the ELISA test.

Use of immunoblotting to characterize the mitochondrial antigens recognized by anti-mitochondrial autoantibodies.

Sera with anti-mitochondrial autoantibodies detected by indirect immunofluorescence and/or enzyme-linked immunosorbent assay (ELISA) were examined by immunoblotting against pig heart mitochondria. Seven types of reactions were defined, according to the pattern of the labelled bands. Type I sera reacted with 12 bands located within four zones. The most intensively labelled bands were located at 70, 67, 58, 63 and 43 kDa. Other types gave decreasing band numbers. When beef heart mitochondria were used, sera belonging to each of the above types had a profile of labelled bands which sometimes differed from those obtained with pig heart mitochondria. When the chloroform extracted F1-ATPase from beef heart mitochondria was used to prepare the immunoblots, primary biliary cirrhosis (PBC) sera with anti-mitochondria antibodies reacted with all the bands although zone A bands were less labelled. Rat liver mitochondria gave seven bands with type I sera among which the 57 and 35 kDa bands were specific for rat liver mitochondria, as shown by absorption tests. Sera of PBC patients were also tested in immunoblotting against rat liver subcellular fractions including mitoplasts, submitochondrial particles, inner membrane, outer membrane, matrix proteins and inter-membrane proteins. Antigenic bands of A and B zones were localized in the inner membrane and/or in the matrix proteins and the 35 kDa band in inter-membrane proteins. The outer membrane gave no reaction. The most frequent anti-mitochondrial autoantibody types in PBC were type II, then I, whilst for chronic active hepatitis type III was the most common. Type V was only seen in a patient suffering from a typical PBC. Some sera from patients with syphilis, collagenous colitis or progressive systemic sclerosis labelled one or two bands distinct from those labelled by the PBC sera. Sera from patients with drug-induced hepatitis with endoplasmic reticulum antibodies and with systemic lupus erythematosus were generally found negative by immunoblotting.

Naturally occurring antibodies reacting with lipoic acid: screening method, characterization and biochemical interest.

The development of a covalent enzyme-linked immunoassay (CELIA) using lipoic acid covalently bound to modified polystyrene microplates has permitted the detection, in the sera of normal BALB/c mice, of natural antibodies reacting with lipoic acid (LA). Hybridomas producing monoclonal anti-LA antibodies were obtained from splenocytes of non-immune BALB/c mice. Two of them, of IgM isotype, recognized LA but failed to react with dihydrolipoic acid (DHLA, the reduced form of LA), suggesting that the integrity of the dithiolane ring was of importance for antibody recognition. They did not give positive reactions with other disulfide linked biological molecules such as oxidized glutathione or cystine. Anti-LA antibodies, coated on polystyrene microplates, were used for the detection of free LA in a competitive assay based on peroxidase-LA conjugate.

Intracellular localization of thymosin alpha 1 by immunoelectron microscopy using a monoclonal antibody.

Distribution of thymosin alpha 1 in normal mice (OF1) or autoimmune mice (NZB) was investigated using immunocytochemical techniques on sections of GMA- and Epon-embedded mouse thymuses. A monoclonal antibody directed against synthetic thymosin alpha 1 was used. With the immunofluorescence assay, patchy staining of thymosin alpha 1 was found in the cytoplasm of epithelial cells of the subcapsullary and medullary zones of OF1 thymus. In NZB thymus, the fluorescent pattern was less precisely localized. At the electron microscopic level, immunolabeling of Epon-embedded ultra-thin sections revealed ferritin in some vacuoles of epithelial cells. Ferritin labeling in OF1 thymus was found in several small vacuoles of the same cell, but was present in large, dense vacuoles in NZB thymus. These differences might reflect differences in the secretory process of thymic hormone.

Lack of reactivity of anti-human immunodeficiency virus (HIV) P17/18 antibodies against alpha 1 thymosin and of anti-alpha 1 thymosin monoclonal antibody against P17/18 protein.

The blood rate of alpha 1 thymosin is increased during HIV infection, despite the thymus involution. Anti-alpha 1 thymosin antibodies inhibit HIV replication in vitro. A homology between alpha 1 thymosin and the HIV P17/18 core protein exists and would explain a cross-antigenicity. We have studied the interaction between anti P17/18 antibodies from HIV patients and alpha 1 thymosin and between an anti-alpha 1 thymosin monoclonal antibody and the P17/18 protein. We were unable to confirm any cross-reactivity. During acquired immune deficiency syndrome, a major involution of the thymus appears with a severe depletion of thymocytes and epithelial cells. Certain thymic functions are missing, as corroborated by the reduction of the hormone thymulin in the blood. At the same time, the blood rate of the 2 other hormones (partly of thymic origin), alpha 1 thymosin and beta 4 thymosin is increased. One of the theories explaining this discordance is that patients with acquired immunodeficiency syndrome produce molecules which have a cross antigenicity with these thymic hormones. Sarin et al. have recorded a 50% homology between the C-terminal part (last 18 aminoacids) of alpha 1 thymosin and the part between the 92nd and the 109th aminoacids of the HIV P17/18 protein. The cross reactivity between this P17/18 protein and alpha 1 thymosin would explain the high rates of alpha 1 thymosin found in the radio-immunoassay of sera from patients infected with HIV. Another result of this cross-reactivity is the ability of alpha 1 thymosin antibodies to inhibit HIV replication in the H9 permissive cell line.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of histamine on the induction of experimental autoimmune hemolytic anemia on OF1, BALB/c and SJL mice.

Erythrocyte autoantibodies can be elicited in mice by injection of rat red blood cells (RBC) which are cross-reactive with mouse RBC. In this report, we show that histamine injected in vivo delayed the production of autoantibodies among BALB/c and OF1 mice. In contrast, the induction of autoantibodies in SJL mice was not affected by histamine. Furthermore, histamine did not affect the production of anti-rat RBC. Cimetidine (anti-H2) but not diphenhydramine (anti-H1) inhibited the histamine effect.

Monoclonal antibodies against an epitope common to desmin and some keratins.

Hybridomas between spleen cells from autoimmune MRL/n mice and Sp2/0-Ag 14 myeloma cell line, are obtained by electric field-mediated fusion. A monoclonal antibody directed against a common antigenic determinant of desmin and some keratins has been obtained and characterized.

Generation of hybridoma antibodies to double-stranded DNA from non-autoimmune BALB/c strain: studies on anti-idiotype.

A hybridoma obtained between normal spleen cells from BALB/c mice (a non-autoimmune strain) and SP2-O-Ag 14 myeloma cell line was designated as HB2. These hybrid cells produced an IgM kappa-anti-ds-DNA antibody, but their specificity was limited to some polydeoxyribonucleotides such as natural ds-DNA from calf thymus, poly dG-poly dC, poly d(GC) and poly d(GC)-poly d(GC). In contrast, poly dA-poly dT, poly d(AT) were not recognized. The configuration of the nucleic acid helix plays a small role if any, in the building of the epitopes recognized by the hybridoma HB2 antibodies, while the presence of G and C appeared to be essential. These epitopes could not be found on ss- and ds-polyribonucleotides. B cells able to produce anti-ds-DNA antibodies are therefore present in non-autoimmune BALB/c mice, but not enough to produce the corresponding antibodies at a detectable level in the serum. Following immunization of BALB/c mice with hybridoma HB2 monoclonal antibodies, anti-idiotype antibodies were obtained which also recognized idiotopes present in the serum from both murine MRL/1 and human systemic lupus erythematosus (SLE).

Identification of FTS (facteur thymique serique) on thymus ultrathin sections using monoclonal antibodies.

Using a new immunoelectromicroscopical technique, this study confirms the localization of the 'facteur thymique sérique' (FTS) in the reticulo-epithelial cells of mouse thymus. The use of anti-FTS monoclonal antibodies on ultrathin sections for electron microscopy reveals FTS in cytoplasmic vacuoles, labelling density depending on the density of the vacuolar content. The successful application of this technique opens the way to its use for double-immunolabelling.

Heterogeneity of ribosomal autoantibodies from human, murine and canine connective tissue diseases.

Antiribosomal auto-antibodies (anti-Rib.Ab) have been studied in connective tissue diseases (human, dog and mouse) by immunoblotting after one-dimensional (1D) or two-dimensional (2D) gel electrophoresis of rat ribosomes. Anti-Rib.Ab could be found in systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and other connective tissue diseases (progressive systemic sclerosis, PSS; Sjögren syndrome, SjS; mixed connective tissue disease, MCTD; and dermatomyositis, DM with the frequencies 41.7%, 54.6% and 33%, respectively. Immunoblotting after 1D gel electrophoresis showed the great heterogeneity of ribosomal proteins recognized by the anti-Rib.Ab. In the SLE, however, the most frequent antibodies stained bands of the 40S subunit: 30 kDa (34% of positive sera), 19.5 kDa (24.5%) and 43 kDa (17%). In RA, the 25-kDa band of the 60S subunit was the most common (54% of positive sera). In the other human connective tissue diseases, there was no particular predominance. In the MRL/1, anti-Rib.Ab were very frequent (92.6%). The 43-kDa band of the 40S subunit was found in 100% of positive sera. Seventeen out of nineteen dogs with SLE gave positive results on immunoblot, and all of them stained the 43-kDa band of the 40S subunit. 2D gel electrophoresis gave identification of Po, L7, L5, Sb, S19, S13 and L2 proteins in SLE, S3 and SjS, L35a and L37a in RA, and L7, S6 and/or L7a in MRL/1.

Canine monocytes express a receptor specific for murine Fc gamma 2a and Fc gamma 3 immunoglobulins.

Murine monoclonal antibodies (mAb) of IgM and IgG subclasses that do not bind to canine cells have been used to detect the expression of Fc receptors on canine peripheral blood mononuclear cells and the cell line MAXEY-DH82. Murine Ig of IgG2a and IgG3 isotypes bound specifically to canine peripheral blood monocytes and to MAXEY-DH82, whereas Ig of IgG1, IgG2b, and IgM did not. No other cell types, including resting and activated peripheral blood lymphocytes, expressed this canine Fc receptor (cFcR) specific for murine IgG2a and IgG3. MAXEY-DH82 was used to characterize this Fc gamma 2a/gamma 3 receptor. Binding of murine IgG2a and IgG3 is trypsin sensitive and partially suppressed by phosphatidyl inositol-phospholipase C (PI-PLC) treatment, indicating that this cFc gamma 2a/gamma 3 receptor is a lipid-anchored protein. Preincubation of MAXEY-DH82 with canine sera or canine IgG prevented the binding of murine gamma 2a/gamma 3 Ig, demonstrating that this receptor is a cFc receptor for canine IgG. The cFc gamma R expressed on canine monocytes and on MAXEY-DH82 is probably the analog of the murine and human Fc gamma RI. The specific expression of this analog by canine cells could be used in identifying and purifying canine monocytes.

Antinuclear antibodies detected by indirect immunofluorescence on HEp2 cells and by immunoblotting in patients with systemic sclerosis.

The HEp2 cell cultures appeared highly sensitive in detecting the antinuclear antibodies (ANAb) in systemic sclerosis, principally anticentromere antibodies of the CREST syndrome. The immunoblotting used with either complex cellular extracts from HeLa and rabbit thymus or purified nuclear components (high mobility group (HMG) proteins and histones) is able to identify precisely the ANAb targets and to contribute to diagnosis. With nuclear extracts of HeLa cells, the sera from 75.8% of CREST syndrome subjects stained 18 and 22 kD proteins. Corresponding antibodies were also detected in 72.7% of these patients, on HEp2 centromers by indirect immunofluorescence. With the same extracts, 33.3% of sera from diffuse sclerosis/acrosclerosis patients contain antibodies staining 86, 73, 32 and 30kD. These sera also stain 77, 66 and 63kD from thymus extracts. Corresponding antibodies will be the anti-SCL-70 antibodies defined by double immunodiffusion. The anti-HMG antibodies were infrequent in systemic sclerosis, rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) and consequently without interest for diagnosis. The anti-whole histones antibodies which are less frequent in diffuse sclerosis/acrosclerosis (35.7%) than in SLE (41.3%) recognize especially H1 and H2A in the first diseases, H1 and H2B in SLE and H1 and H3 in RA.

Abnormalities of lymphocyte subsets in canine systemic lupus erythematosus.

Canine systemic lupus erythematosus (SLE) is a disease clinically very similar to its human counterpart. But so far, no study has reported an accurate evaluation of the lymphocyte subsets in the canine disease. Here, we present a study in which lymphocyte subsets have been evaluated in the peripheral blood of 20 dogs suffering from spontaneous systemic lupus erythematosus (SLE) in active and inactive phases, before and during treatment with prednisone and levamisole. 22 healthy dogs have been used as a control population. We show that canine SLE in active phases is associated with a several lymphopenia (1050 +/- 520 10(6) cells/l versus 2130 +/- 1 020 10(6) cells/l in controls). A striking finding is the imbalance of the CD4 and CD8 subsets (respectively 56.7 +/- 10.7% and 10.9 +/- 3.8% of CD4+ and CD8+ lymphocytes versus 40.5 +/- 11.5% and 18 +/- 4.4% in controls) and a strong activation of T-cells in active phases (64.1 +/- 16.9% of 2B3+ cells versus 46.5 +/- 16.7%). Moreover, we observed a persistence of the T subset imbalance during spontaneous evolution. In contrast, the treatment induced in dogs showing a good response the correction of CD4/CD8 ratio and no clinical manifestations, whereas in low responders no such improvements were observed. Thus, this work suggests that the main immunological imbalance seen in SLE could be associated with defective suppressor cells and provides further evidence of similarity of human and dog SLE.

Autoantibodies directed against ribosomal proteins in systemic lupus erythematosus and rheumatoid arthritis: a comparative study.

To assess the specificity of autoantibodies (aAbs) directed against the ribosomal P-proteins (RPPaAbs) in patients with systemic lupus erythematosus (SLE) and to investigate aAbs directed to other ribosomal proteins, 100 SLE, 100 rheumatoid arthritis (RA), 25 thyroiditis and 20 blood-donors were analyzed in a comparative study using an immunoblotting technique. Forty-eight percent of SLB sera contained aAbs directed against the ribosomal proteins of the 60 S subunit compared to 9% for RA, 5% for blood donors and 0% for thyroiditis. RPPaAbs were only found in SLE (25%) and aAbs directed to a 31 kDa and/or a 28 kDa protein of the 60 S subunit were found with a statistically higher frequency for SLE compared to RA (p < 0.0001). aAbs directed to proteins of the 40 S subunit were present in 63% of the SLE sera compared to 42% for RA, 4% for thyroiditis and 5% for blood donors. The number of positive sera was not statistically different between SLE and RA but a much more intense reactivity was observed for SLE sera. These data shows that the aAbs against the ribosomal proteins, especially the P-proteins along with the 28 and 31 kDa proteins of the 60 S subunit proteins, can be considered as useful biological markers for t he diagnosis of SLE inclinical practice.

Autoantibodies and human immunodeficiency viruses infection: a case-control study.

OBJECTIVE: To determine the prevalence of organ-specific and non-specific autoantibodies in HIV-infected patients. DESIGN: A multicentric collaborative case-control study including 105 HIV patients and 100 sex- and age-matched HIV-negative healthy volunteers. METHODS: Antinuclear, anti-ds DNA, anti-histone, anti-Sm, rheumatoid factor(IgM), anti-beta 2 glycoprotein 1, antineutrophil cytoplasmic, anti-LKM1, anti-LCA1, anti-gastric parietal cell, antiplatelet, anti-intermediate filament, anti-mitotic spindle apparatus, anti-Golgi, anti-ribosome and anti-thyroid autoantibodies were screened in six European laboratories. RESULTS: Only IgG and IgM anticardiolipin, IgG antiplatelet, anti-smooth muscle and anti-thyroglobulin antibodies were statistically more frequent in HIV patients. There was no correlation with the numbers of CD4+ cells except in the case of anti-smooth muscle antibodies. We were unable to find specific autoantibodies such as anti-ds DNA, anti-Sm, AMA, anti-LKM1, anti-LCA1 or anti-beta 2 GP1 antibodies in these patients. CONCLUSIONS: Our results indicate that the autoantibody profile of HIV infections is comparable to those of other chronic viral infections. HIV does not seem to be more autoimmunogenic than other viruses.

Specificities of antinuclear antibodies detected in dogs with systemic lupus erythematosus.

Five hundred and eighty dogs with at least one clinical sign compatible with a systemic lupus erythematosus (SLE) were entered in a prospective study aimed at evaluating the prevalence of antinuclear antibodies (ANAb). SLE was diagnosed in 38 of these dogs (group A) which fulfilled at least four American Rheumatism Association (ARA) criteria; of these, sixteen had ANAb titers greater than or equal to 4096. The 23 dogs which met three or two ARA criteria (group B) had an ANAb geometric mean titer (GMT) of 259. Dogs (group C) with only 1 criterium had an ANAb GMT of 75. Anti-ds-DNA Ab were present in 6 dogs from group A (16%), and 2 dogs from group B (9%). Anti-histone Ab were present among dogs from group A, B and C with frequencies of 81%, 67% and 26%, respectively. Among dogs from group A, the ANAb titers and the levels of anti-histone Ab correlated positively when individual sera were considered. Antibodies against the soluble nuclear antigen (SNA) were detected in 74%, 39% and 13% of the dogs from groups A, B and C, respectively. Antibodies initially described in human SLE also exist in SLE dogs. Anti-Sm Ab were found in 24% of dogs in group A. With anti-RNP Ab the frequency was still lower (10%). However, two other types of anti-SNA Ab against RNAse and trypsin-resistant antigens, not found in human "reference sera", were often detected. The first type (anti-type 1 Ab) was found in 26% and 9% of group A and group B, The first type (anti-type 1 Ab) was found in 26% and 9% of group A and group B, respectively; the second type (anti-type 2 Ab) is less frequent, and was found in 13% and 17% of group A and B, respectively. It appears that testing for anti-Sm, anti-type 1 and anti-histone Ab should be performed in order to improve the diagnosis of SLE in dogs.

Lymphocyte subset abnormalities in German shepherd dog pyoderma (GSP).

Peripheral blood lymphocyte subpopulations were studied in 12 German shepherd dogs suffering from deep pyoderma (GSP). Twelve other healthy but matched dogs were used as controls. GSP was found to be associated with an imbalance in the CD4 and CD8 subsets (respectively 37.3 +/- 8.7% and 28.6 +/- 6.6%, as compared to 47.5 +/- 8.8% and 19.3 +/- 4.0% in the controls). The activation markers were not affected by GSP. Moreover, analysis of the B-cell populations showed a striking decrease in the level of CD21 cells (5.5 +/- 3.3% of CD21+ lymphocytes, compared to 12.2 +/- 6.0 in the controls). This study suggests that the immunological imbalance observed in GSP may be associated with defective helper cells, and provides further evidence that dogs suffering from GSP are not immunologically normal reactors.