Rare autoantibodies to cellular antigens in systemic lupus erythematosus.

OBJECTIVE: A high number of antinuclear antibody specificities can be detected in systemic lupus erythematosus (SLE). Some of them are related to a distinct clinical subset of disease, independently of their frequency. The aim of our study was to investigate, in a cohort of SLE patients, the prevalence and the clinical relevance of autoantibodies to cellular antigens less frequently found in SLE. METHODS: Antinuclear antibodies were detected by indirect immunofluorescence on HEp-2 cells while counterimmunoelectrophoresis was used to detect anti-ENA antibodies in 540 patients with SLE, classified according to ACR and SLICC criteria. Clinical and serological features were collected from clinical charts. RESULTS: A total of 319 (58.9%) out of 540 sera were positive for anti-ENA antibodies. Anti-Ro/SSA was found in 235 sera, 50 of which also contained anti-La/SSB. Anti-U1RNP were detected in 67, anti-Sm in 46 and anti-ribosomal P protein in 13 sera. In a multivariate analysis anti-Sm was associated with discoid lupus (p = 0.045) and photosensitivity (p = 0.037), anti-U1RNP with malar rash and Raynaud's phenomenon (p = 0.01 and p = 0.0004, respectively) and anti-Ro/SSA with malar rash, oral ulcers, xerostomia, xerophthalmia and rheumatoid factor (p = 0.029, p = 0.01, p = 0.031, p = 0.002 and p = 0.028, respectively). Other anti-ENA antibodies were found in 50 positive sera (15.6%). Anti-Ki antibodies were detected in 31, anti-Ku in 8, anti-centromere in 5, isolated anti-La/SSB, anti-PCNA and anti-Topo I in 3 each and anti-Jo-1 in 2 sera. About half of these antibodies (27 out of 50) were detected as the single anti-ENA specificity in serum. At multivariate analysis anti-Ki was significantly associated with male gender while anti-Ku with African ethnicity (p = 0.017 and p < 0.0001, respectively). No sign of muscular or pulmonary involvement was found in anti-Jo-1-positive patients whilst features of systemic sclerosis were detectable in two out of three anti-Topo I. CONCLUSIONS: Our study shows that antibodies to cellular antigens more rarely found in SLE are detectable in more than 15% of patients with anti-ENA antibodies. Most of them are found as single anti-ENA specificity. Anti-Ki and anti-Ku are found in a subset of disease, characterized by male gender and African origin, respectively. Clinical features of scleroderma were found only in patients with anti-Topo I.

Prevalence and clinical associations of anti-Ku antibodies in systemic autoimmune diseases.

We retrospectively analysed the prevalence and clinical features associated to anti-Ku antibodies in patients affected by different autoimmune diseases. Anti-Ku antibodies are detected in 147 sera out of 7239 anti-ENA positive sera (2%). They are found in 2% of patients with systemic sclerosis (SSc) (8 out of 379), 1.8% of systemic lupus erythematosus (SLE) (7 out of 372) and 1.8% of undifferentiated connective tissue disease (UCTD) (9 out of 496) and more rarely in Sjögren Syndrome and rheumatoid arthritis. Most of anti-Ku positive patients were affected by UCTD and overlap syndromes, including polymyositis, SSc and SLE. Interstitial lung disease, myositis, articular symptoms, Raynaud's phenomenon and sicca represents the main clinical features detected in our cohort. The rate and severity of pulmonary disease is similar to those found in other SSc patients. Isolated anti-Ku were detected in about 47% of sera. No clinical differences were observed between these patients and subjects with multiple anti-nuclear specificities. However, anti-Ku are usually detected in association with other serological markers in SLE and Sjögren Syndrome, while they occurred isolated in SSc and polymyositis.

Isotype switching and titer variation of anti-Ro/SSA antibodies over time in 100 patients with undifferentiated connective tissue disease (UCTD).

OBJECTIVE: To correlate the clinical course of the disease with the titer, the isotype profile and the switch of the anti-Ro/SSA antibodies in a cohort of patients affected by UCTD. METHODS: One hundred selected patients with anti-Ro/SSA antibodies detected by counterimmunoelectrophoresis (CIE), and affected by UCTD with a mean follow-up of 7.6 years (SD 4.8 yrs.), were studied. The titer of IgA, IgG and IgM anti-Ro/SSA antibodies was determined in two different sera, obtained at the time of diagnosis and at the last visit, by ELISA with Ro/SSA recombinant proteins as substrate. RESULTS: Thirty-five patients evolved from UCTD to a different connective tissue disease, while 65 showed a stable disease. Anti-Ro/SSA antibodies were detected in 91% and 97% of the patients, at baseline and during follow-up, respectively. IgG dominates the anti-Ro response. The titer of IgA, IgM and IgG anti-Ro/SSA did not differ significantly between the two groups of patients with UCTD. An increasing trend of IgG and IgA anti-Ro/SSA titer could be detected in patients evolving in primary Sjögren's Syndrome (pSS), but only the increase of IgG anti-Ro/SSA was significant (p=0.0235). CONCLUSION: IgG dominates the anti-Ro/SSA response in patients with UCTD. No substantial change of the antibody isotype against Ro/SSA peptides could be observed during follow-up. The titer of IgG anti-Ro/SSA significantly raised in the group of patients evolving in pSS.

Anti-Ro/SSA antibodies in rheumatoid arthritis: clinical and immunologic associations.

OBJECTIVE: To assess the prevalence of anti-Ro/SSA in RA and to analyse clinical and serological features of anti-Ro/SSA positive patients with RA. METHODS: 195 consecutive patients affected by RA were studied by counterimmunoelectrophoresis and ELISA for the detection of anti-Ro/SSA antibodies. Anti-Ro were found in 12 patients, with a prevalence of 6%. These 12 patients were pooled with other 15 patients known to have anti-Ro/SSA antibodies and RA, in order to evaluate their clinical and laboratory features. RESULTS: Anti-Ro positive patients showed a common pattern of joint involvement at onset and a comparable progression of disease compared to anti-Ro negative subjects. In addition, extra-articular manifestations (such as xerophthalmia, xerostomia, scleritis, oral ulcers and amyloidosis) and peculiar autoantibody profile (hypergammaglobulinemia, anti-dsDNA and AMA) were found significantly associated to anti-Ro/SSA positivity. Even though DMARDs withdrawals were more frequently detected in anti-Ro/SSA patients, especially when using gold salts, no statistical difference between the two groups was detected. In addition, anti-TNFalpha treatment did not cause further progression of autoimmunity neither on laboratory nor on clinical ground. CONCLUSION: Anti-Ro/SSA can be detected in about 6% of patients affected by RA. These patients presented a peculiar clinical picture characterised by extra-articular manifestations some of which are known to be anti-Ro/SSA correlated, while others are more disease-specific (amyloidosis, episcleritis). Anti-Ro/SSA are significantly associated with other autoantibodies not specific for RA such as anti-dsDNA and AMA. Treatment with anti-TNF drugs did not cause further progression of autoimmunity neither on laboratory nor on clinical ground.

Clinical and serological features of 35 patients with anti-Ki autoantibodies.

The objective of this study was to analyse clinical and serological associations of anti-Ki antibodies. Thirty-five patients with anti-Ki antibodies, detected by CIE, selected from laboratory routine, were studied. All patients were affected by autoimmune diseases: SLE and pSS were the most frequent diagnoses. The cohort was constituted by 27 female and eight males. Main clinical features were skin involvement (60%), xerophtalmia (48.6%), Raynaud's phenomenon (43%), photosensitivity (34%), xerostomia (31.4%). CNS involvement was present in four (11.4%) and renal disease in seven cases (20%). ANA, anti-dsDNA and RF were detected in 100%, 60% and 34.5%. In SLE, anti-Ki was detected in 6% of cases, more frequently in males compared to other SLE patients without anti-Ki (P < 0.004). Nineteen anti-Ki positive patients affected by SLE showed more frequently malar rash and multiple autoantibody specificities compared to 16 anti-Ki positive patients with other diseases (P = 0.044 and P = 0.0003, respectively). Our study confirms a preferential occurrence of anti-Ki antibodies in patients with sicca and skin involvement. Malar rash and multiple ANA specificities were significantly associated with SLE compared to other diseases in our study. Anti-Ki were detected in 6% of patients with SLE with a significant prevalence in males.

Undifferentiated connective tissue disease with antibodies to Ro/SSa: clinical features and follow-up of 148 patients.

OBJECTIVE: To evaluate the clinical and serologic profile, the rate of progression to well defined CTD and the possible predictors of disease evolution in patients affected by UCTD with antibodies anti-RoISSA. METHODS: 148 patients diagnosed as UCTD were retrospectively evaluated. Antibodies to SSA/Ro were determined by counter-immunoelectrophoresis and ELISA. RESULTS: Thirty-six patients (24.3%) developed a well-defined CTD after a mean follow-up of 4.5 years. Most patients developed primary Sjögren's syndrome (SS) (50%) or systemic lupus erythematosus (SLE) (30.5%). Leukopenia and xerophthalmia developed more frequently in the group of patients evolving to defined CTDs (p < 0.0032 and p < 0.0063). Leukopenia independently predicted the evolution in CTD by multivariate regression analysis (p < 0.019). Anti-dsDNA predicted the evolution in SLE (p < 0.0207), while the presence of additional anti-ENA specificity to anti-Ro/SSA was not associated with the outcome. CONCLUSION: 24.3% of patients with UCTD and antibodies to Ro/SSA can progress in a relatively short period of time to well-defined CTDs. The development of primary SS could be predicted by xerophthalmia and SLE by the appearance of anti-dsDNA antibodies.

Chilblain lupus erythematosus is associated with antibodies to SSA/Ro.

Chillblain Lupus Erythematosus (CL) of Hutchinson is a subtype of Lupus Erythematosus characterized by erythematous lesions symmetrically distributed on the face, nose, fingers and toes, knees and heels. The lesions are induced by cold, damp climates. A number of patients affected by CL eventually develop features of Systemic Lupus Erythematosus (SLE). We report here 7 patients, all but one affected by SLE, with chilblain cutaneous lesions on their hands, feet and face. The onset of CL preceded the diagnosis of SLE, from 1 to 10 years in 3 cases, it was concurrent in one case and was subsequent in the other 2 cases. Six out of the seven patients referred typical Raynaud's phenomenon and one had acrocyanosis. CL lesions developed and were aggravated by the cold during autumn and winter, they improved during summer. Skin biopsy performed in 5 patients from the lesions showed, on histology, a typical pattern of alterations with granular deposits at the dermo-epidermal junction on direct immunofluorescence. Laboratory findings showed: ANA and anti-SSA/Ro were detected in all the patients, anti-SSA/Ro were isolated in 4 patients and associated with anti-Sm in one case, anti-U1 RNP in one case and with anti-Sm and anti-RNP in a third case. Complement consumption was observed in 5 patients, anti-dsDNA in the six patients with SLE, hypergammaglobulinemia in 4 and rheumatoid factor in one. The fine specificity of anti-SSA/Ro as determined by immunoblotting using a human spleen extract as a substrate, showed: anti-60kD and anti-52 kD in two sera, anti-60kD isolated in 2 sera, anti-52kD isolated in one serum (from the patient without SLE) while 2 sera did not blotted. In conclusion, our study confirms the previous report of anti-SSA/Ro antibodies in association with CL. This clinical and serologic association widens the spectrum of cutaneous disease that is associated with antibodies to SSA/Ro to include conditions such as to SCLE, hypergammaglobulinemic purpura and neonatal lupus.

DNA typing of maternal HLA in congenital complete heart block: comparison with systemic lupus erythematosus and primary Sjögren's syndrome.

OBJECTIVE: To investigate which maternal HLA allele or haplotype is primarily associated with isolated congenital complete heart block (CCHB) in offspring. METHODS: HLA class II typings were assessed by line probe assay and polymerase chain reaction-sequence-specific oligonucleotide probe methods, and HLA class I by the microlymphocytotoxicity test, in 13 Italian anti-Ro-positive mothers of children with CCHB and 41 anti-Ro-positive mothers with healthy children (20 mothers with systemic lupus erythematosus [SLE] and 21 with Sjögren's syndrome [SS]). Anti-Ro antibodies were studied by immunoblot. RESULTS: HLA-DRB1*03011 and DRB1*03011; DQA1*0501;DQB1*0201 were more frequent in mothers of infants with CCHB than in mothers who had SLE, but not in mothers who had SS and whose children were healthy. Mothers of infants with CCHB were either HLA-B5/35, B17, or B44 positive and had a higher prevalence of B44;DRB11;DQA1*0501;DQB1*0301 and isolated anti-52-kd antibodies, which were absent in SS and SLE controls. CONCLUSION: Mothers of infants with CCHB presented a strong genetic similarity to mothers who had SS, except for HLA class I phenotype. HLA-DRB1*03011;DQA1*0501;DQB1*0201 seemed not to be primary CCHB-associated genes, but were involved in an SS-like anti-Ro/La response. The combined presence of HLA-DRB1*03011 and anti-52-kd SSA/Ro antibodies conveyed the highest risk of giving birth to an affected child.

Chilblain lupus erythematosus is associated with antibodies to SSA/Ro.

Chilblain lupus erythematosus (CL) of Hutchinson is a subtype of lupus erythematosus (LE) characterized by erythematous lesions induced by cold, damp climates. A number of patients affected by CL eventually develop features of systemic lupus erythematosus (SLE). We report here 9 patients with chilblain cutaneous lesions, 6 of them were affected by SLE and 2 by SCLE. The onset of CL preceded the diagnosis of LE, from 1 to 10 years in 3 cases, it was concurrent in one case and was subsequent in the remaining 4 cases. Raynaud's phenomenon and photosensitivity were other prominent clinical features in patients with CL. Nailfold capillaroscopy revealed pathological changes in every patient examined. ANA and anti-SSA/Ro antibodies were detected in all nine patients. Anti-SSB/La were detected in 2 cases, anti-Sm in one case, and anti-Sm and anti-RNP in a one case. Antibodies to dsDNA and complement consumption were found in the six patients with SLE. The fine specificity of anti-SSA/Ro was determined by immunoblotting: anti-60kD and anti-52 kD were detected in three sera, anti-60kD alone in 5 sera, while one serum did not blot. In conclusion, the present study suggests that chilblain LE is associated with SSA/Ro autoantibodies, as is SCLE, hypergammaglobulinemic purpura and neonatal lupus erythematosus.

High prevalence of peripheral neuropathy in hepatitis C virus infected patients with symptomatic and asymptomatic cryoglobulinaemia.

BACKGROUND: Fifty per cent of patients with chronic hepatitis C, show detectable cryoglobulinaemia, even though most of them do not show cryoglobulinaemia related symptoms. Peripheral neuropathy is present in most of the patients with symptomatic cryoglobulinaemia, where it may be the first clinical manifestation. The prevalence of peripheral neuropathy in patients with hepatitis C and cryoglobulinaemia is unknown. AIMS: To assess the prevalence of peripheral neuropathy in HCV infected patients with symptomatic or asymptomatic detectable cryoglobulinaemia. PATIENTS AND METHODS: Eighty-nine patients with HCV infection and detectable cryoglobulinaemia underwent electrophysiological studies. RESULTS: Electrophysiological evidence of peripheral neuropathy was found in 37% and was significantly associated with: the presence of cryoglobulinaemia syndrome, older age, higher rheumatoid factor reactivity and immunoglobulin M levels and reduced complement C4 activity. However, electrophysiological evidence of peripheral neuropathy was unrelated to cryocrit levels and type of cryoglobulinaemia and was found in 23/68 patients without any symptoms of cryoglobulinaemia other than pain and paresthesia. CONCLUSIONS: These findings suggest that peripheral neuropathy is frequent in patients with hepatitis C and detectable cryoglobulins. Neuropathy was found to be present in 1/3 of patients without other cryoglobulinaemia-related symptoms, thus a direct or indirect role of HCV, independent of cryoglobulinaemia, in the pathogenesis of nerve damage cannot be ruled out.

Determination of antibodies to extractable nuclear antigens by commercial kits: a multicenter study.

Several enzyme immunoassays for serum antibodies to extractable nuclear antigen have recently become available. The aim of this study was to evaluate the results obtained with: (1) the same kit under different conditions; (2) different enzyme immunoassays; (3) Western blot and enzyme immunoassays. Twenty-five sera from patients with autoimmune disorders were tested in five different laboratories by one Western blot and four enzyme immunoassay commercial kits. The different methods produced comparable qualitative results. However, semiquantitative evaluation, based on a cut-off value (index), yielded different results due both to laboratory conditions and to the kits employed. Standardization of commercial products and methods should be improved so that the results of different laboratories can be compared and large-scale and follow-up studies conducted. Western blot analysis could also be useful to analyze complex reactivities, although greater experience is necessary to interpret these results correctly.

Isolated congenital complete heart block: longterm outcome of mothers, maternal antibody specificity and immunogenetic background.

OBJECTIVE: To assess the longterm outcome of mothers of children with isolated congenital complete heart block (CCHB), and the maternal specific immunoblot pattern and HLA antigens. METHODS: Fifteen mothers of 16 children with isolated CCHB were investigated; their followup extended up to 15.8 years on average after the index delivery. Anti-Ro and La antibodies were detected by counterimmunoelectrophoresis and ELISA; anti-Ro antibodies were studied by immunoblot. HLA typing was done using a microcytotoxicity test. RESULTS: One mother has systemic lupus erythematosus (SLE) before the index delivery. The other mothers developed only minor symptoms (arthralgia, dry eyes and photosensitivity) resembling primary Sjögren's syndrome more than classic lupus. All 15 mothers were anti-Ro and 9 were also anti-La positive, a mean of 12.5 years after the index delivery. Eight mothers reacted with the 52 kDa SSA(Ro) component, and 2 also with the 60 kDa SSA(Ro) component. The prevalence of the DR3 antigen and of the B44/DR5, DR3/DQ2 and A1/Cw7/B8/DR3/DQ2 haplotypes was significantly increased. CONCLUSION: The longterm outcome for the mothers of children with CCHB is more reassuring than generally assumed. All the mothers were anti-Ro positive by sensitive ELISA: Reactivity to the denaturated 52 kDa SSA(Ro) component seems characteristic of these mothers, who presented a particular immunogenetic background.

Isolated congenital complete heart block: longterm outcome of children and immunogenetic study.

OBJECTIVE: To assess the longterm cardiologic and immunologic outcome of children with isolated congenital complete heart block (CCHB) and their HLA antigens. METHODS: Sixteen children with isolated CCHB were investigated. HLA typing was done using a microcytotoxicity test. RESULTS: Three patients died (18.7%), one in utero (35 weeks), one 2 days after birth, and one at 6 years of age. The mean age of the 13 living children is now 18.3 years (range 2-34). Eight (50%) have been permanently paced for symptoms. No patient developed clinical symptoms or serological abnormalities suggesting immune disease. The A31 antigen was more prevalent, but one pair of HLA identical twins was observed, and only one had CCHB. CONCLUSION: Patients with isolated CCHB have significant cardiac mortality, and after a long followup many of them are paced to control symptoms, but in our small sample those who survive the perinatal period mostly lead a normal life. The longterm immunological outcome of these children seems good. CCHB is not related to a specific HLA pattern in affected children.

Deforming arthropathy of the hands in systemic lupus erythematosus is associated with antibodies to SSA/Ro and to SSB/La.

Thirteen patients with systemic lupus erythematosus and deforming arthropathy (DA) of the hands were compared with 111 patients with SLE without deforming arthropathy. Clinical features were comparable in the two groups. Patients fulfilling criteria for mixed connective tissue disease (MCTD) were not included in the present study. A higher prevalence of antibodies to SSA/Ro (P < 0.0125) and SSB/La (P < 0.004) were found in the SLE-DA group. The detection of antibodies to SSA/Ro and SSB/La was even more strictly associated with DA in SLE antibodies to SSA/Ro alone (P < 0.002). Regarding the fine specificity of anti-SSA/Ro, a prevalent response to the 52 kD protein of the Ro antigen was found. We conclude that patients with SLE developing deformities of the hands belong to a subset of patients with circulating antibodies to SSA/Ro, particularly to the 52 kD component, and to SSB/La.

Noradrenaline, atrial natriuretic peptide, bombesin and neurotensin in myocardium and blood of rats in congestive cardiac failure.

Rats given monocrotaline develop severe right ventricular hypertrophy often accompanied by ascites and pleural effusions. In rats with right ventricular hypertrophy and no serous effusions ("hypertrophy" group), ventricular concentrations of noradrenaline were reduced but ventricular contents were unchanged. Atrial concentrations of noradrenaline were unaffected. Those with more severe right ventricular hypertrophy and serous effusions ("failure" group) had greatly reduced concentrations of noradrenaline in all four chambers, particularly on the right side; the right and left ventricular contents of noradrenaline were also diminished. The distributions of ir-ANP, ir-bombesin and ir-neurotensin in the normal rat heart are presented. ANP concentration fell to 33% in the right atrium and 46% in the left atrium of "failure" animals and to 57% in the right atrium of "hypertrophy" animals. Right ventricular content of ANP, normally low, increased more than two-fold in both groups, the concentration remaining unchanged. Left ventricular content of ANP decreased in the "failure" group. Concentrations of bombesin and neurotensin fell in both ventricles of both groups. Ventricular contents of bombesin did not change, but ventricular contents of neurotensin decreased, especially on the right side. Plasma ANP rose nearly six-fold while plasma bombesin and neurotensin fell in the "failure" group. Plasma peptide concentrations were unchanged in the "hypertrophy" group. The studies show the utility of the monocrotaline model in distinguishing between the effects of hypertrophy and those associated specifically with the syndrome of congestive cardiac failure.

Immunoreactivity to various peptides in the human carotid body.

Human carotid bodies, removed at routine necropsies, have been subjected to radioimmunoassay for various peptides. Average levels of immunoreactivity, expressed in pm/g, were: met-enkephalin 612, leu-enkephalin 162, bombesin 73, neurotensin 67, VIP 9 and substance P 16. No alpha-hANP immunoreactivity could be detected.