The speckled epidermal nuclear immunofluorescence of mixed connective tissue disease seems to develop as an in vitro phenomenon

The pathogenesis of speckled epidermal nuclear immunofluorescence in patients with mixed connective tissue disease (MCTD) was studied by reproducing this reaction in guinea-pigs, using serum samples containing high litre antibody to ribonucleoprotein (RNP). Immunofluorescence studies on specimens obtained from guinea-pig skin into which scrum samples containing high titre RNP antibody had been injected intradermally, revealed positive epidermal nuclear staining for IgG. This speckled immunofluorescence was demonstrable immediately after injection and remained so for 24 or 48 h. The pattern of fluorescence was similar in all cases, and there was no penetration of RNP antibody through the cell membrane. The epidermal nudear fluorescence was not detected with sera at a dilution of 1:100 or more. These results provide strong evidence that the epidermal nuclear immunofluorescence observed in patients with high titre antibody to RNP develops as an in vitro phenomenon.     

Anti-U1RNP antibodies in patients with localized scleroderma

Abstract Antibodies to U1 ribonucleoproteins (RNP) have been detected in serum from patients with various autoimmune diseases. However, the presence of anti-U1RNP antibodies in patients with localized scleroderma has not been reported. In this study, we examined the frequency of anti-U1RNP antibodies using immunoprecipitation of U small nuclear RNAs and determined the antigen specificity by immunoblotting. Of 70 serum samples from patients with localized scleroderma, 2 (3%) immunoprecipitated U1 small nuclear RNA. Indirect immunofluorescence using HEp-2 cells as substrate showed coarse speckled nuclear fluorescence without nucleolar staining in both of the samples positive for anti-U1RNP antibodies. In addition, the presence of anti-U1RNP antibodies in each serum sample was confirmed by immunodiffusion against HeLa cell extracts. Immunoblotting analysis showed anti-70 kDa antibodies in each serum sample. This reaction against 70 kDa protein in the patients with localized scleroderma was analogous to that in patients with systemic sclerosis or mixed connective tissue disease. Both patients with positive serum were diagnosed as having linear scleroderma, but neither had evidence of Raynaud’s phenomenon or sclerodactyly. These results indicate that the presence of anti-U1RNP antibodies is one of the serological abnormalities in localized scleroderma, and that the mechanism of induction of anti-U1RNP antibodies in patients with localized scleroderma might be similar to that in patients with systemic sclerosis and mixed connective tissue disease. Received: 12 February 2001 / Revised: 27 April 2001 / Accepted: 11 July 2001     

Direct immunofluorescent findings in scleroderma syndromes

Cutaneous direct immunofluorescent findings were examined in 78 patients who had either vascular scleroderma (group 1, 52 patients) or scleroderma with features of myositis or lupus erythematosus (group 2, 26 patients). Group 2 had higher antinuclear antibody levels, erythrocyte sedimentation rates, serum IgG concentrations, frequency of positive LE clot test, and rheumatoid factor activity. Ninety-two percent of group 1 (48 patients) had negative direct immunofluorescent findings, whereas 77% of group 2 (20 patients) had positive findings at the basement membrane or in the blood vessels (or both). The 6 patients in group 2 who had negative immunofluorescent findings were all on systemic steroid therapy. Of the 17 patients in group 2 who had tests for antibody to extractable nuclear antigen, only 3 had high-titer antibody to ribonucleoprotein--a pattern characteristic of mixed connective tissue disease. Direct cutaneous immunofluorescence is proposed as a means of identifying those patients with scleroderma who may be steroid-responsive.     

The significance of the 'dust-like particles' pattern of immunofluorescence. A study of 66 cases

Subacute cutaneous lupus erythematosus (SCLE) is a marker of a unique subset of lupus erythematosus patients. A 'dust-like particles' direct immunofluorescence (DIF) staining pattern, which consists of fine granular particles of immunoglobulin(s) scattered through the epidermis and the cellular infiltrates of the dermis, was reported to be specific for SCLE. In this study, we assessed the real specificity of this staining pattern, which had not yet been evaluated. We systematically searched for the dust-like particles staining pattern among the 4374 skin biopsy specimens submitted for direct cutaneous immunofluorescence during a 7-year period (1989–96). The corresponding patient records were reviewed. Dust-like particles were observed in 66 samples originating from 60 patients. Only 53% of the patients had SCLE. The remaining patients had systemic lupus erythematosus with visceral involvement (17%), discoid lupus erythematosus (3%), mixed connective tissue disease (2%), Sjögren syndrome (2%) and other diseases. Eighty-five per cent of the patients had connective tissue disease. Seventy-seven per cent of the patients were positive for antinuclear antibodies, but only 36% were positive for anti-Ro (SSA) antibodies. This study shows that the dust-like particles staining pattern is not specific for SCLE, but is highly suggestive of connective tissue disease. The nature of the antigen responsible for the immunoglobulin deposition and the prognostic significance of this DIF pattern remain to be established.     

Mixed connective tissue disease syndrome.

Fifteen patients with epidermal nuclear staining on direct immunofluorescence of normal skin and high titer serum antibody to ribonuclease-sensitive extractable nuclear antigen (ENA) had diffuse nonscarring and focal alopecia, abnormal pigmentation, swollen hands with sclerodactyly, and chronic cutaneous lupus erythematosus (LE) as the most common dermatologic features. Direct immunofluorescence of normal, unexposed skin revealed a particulate ('speckled') epidermal nuclear staining pattern in all 15 patients and subepidermal immunoglobulin deposits in 5. Ribonucleoprotein antibodies in high titer are associated with this characteristic type of epidermal nuclear staining. These findings provide easily detectable markers for a less aggressive subset of LE characterized by distinctive clinical and laboratory features consistent with mixed connective tissue disease.     

Clinical significance of antinuclear matrix antibody in serum from patients with anti-U1RNP antibody

Abstract Serum containing anti-U1RNP antibodies reacts with the nuclear matrix, the relatively insoluble component of the cell nucleus, in addition to U1RNP. In this study, we determine the serum titer and clinical correlations of antinuclear matrix antibodies in samples from patients with anti-U1RNP antibodies. The patients with anti-U1RNP antibodies were classified as having mixed connective tissue disease (MCTD, 15 patients), systemic sclerosis (SSc, 12 patients), systemic lupus erythematosus (SLE, 7 patients), and undifferentiated CTD (UCTD, 9 patients). Antinuclear matrix antibodies were detected using indirect immunofluorescence staining on HCl-treated HEp-2 cells. The antinuclear matrix antibody titer was significantly higher in serum from patients with MCTD or SSc than in serum from patients with SLE or UCTD. The antinuclear matrix antibody titer was significantly increased in serum from patients with sclerodactyly, pitting scars, contracture of the phalanges, and decreased carbon monoxide diffusion capacity. Thus, a higher titer of antinuclear matrix antibodies in serum from patients with anti-U1RNP antibodies may be associated with a clinical diagnosis of MCTD or SSc rather than a diagnosis of SLE or UCTD. Received: 6 July 1999 / Revised: 11 October 1999 / Accepted: 27 October 1999     

Clinicopathological significance of cutaneous epidermal nuclear staining by direct immunofluorescence

Epidermal antinuclear antibody (ANA) staining was noted during routine direct immunofluorescence (DIF) of skin biopsies from 22 cases at St John's Dermatology Centre over a 2-year period. We have reviewed the clinical, serological and immunopathological features of these patients. They comprised 13 cases of lupus erythematosus (LE), 3 dermatomyositis, 1 morphoea, 1 systemic sclerosis, 1 CREST syndrome, 1 mixed connective tissue disorder and 1 probable cutaneous sarcoidosis. Five (38.4%) patients with LE had moderate to severe oral mucosal involvement. Epidermal nuclear staining (ENS) was seen following IgG deposition in 21 cases and IgA in only 1 case. Complement C3 staining was an additional feature in 1 patient. Circulating ANA was absent in 7 cases at the time of biopsy, confirming that this pattern of staining does not occur as a result of tissue con-lamination during processing. The presence of ENS by IMF corroborates a diagnosis of a connective tissue disorder, and our results suggest that it may also be associated with oral involvement in E.E.     

The prognostic value of nailfold capillary changes for the development of connective tissue disease in children and adolescents with primary raynaud phenomenon: a follow-up study of 250 patients

To assess the prognostic value of capillaroscopy findings for the development of connective tissue disease in children and adolescents with Raynaud phenomenon, we followed up a group of 250 (mean age 15 years) for 1 to 6 years after the first capillaroscopy was performed. Every 6 months they were screened for signs and symptoms of connective tissue disease. Analysis was performed on capillary changes registered 6 months before the development of connective tissue disease. Capillary changes were classified into three types: normal, nonspecific, and sclerodermatous. At the end of the follow-up period, 191 (76%) subjects had primary Raynaud phenomenon, 27 (10.8%) were diagnosed as having undifferentiated connective tissue disease, and 32 (12.8%) fulfilled the criteria for a diagnosis of a specific connective tissue disease. Systemic lupus erythematosus was found in nine (3.6%) patients, rheumatoid arthritis in 10 (4%) patients (six of them with juvenile onset rheumatoid arthritis), and scleroderma spectrum disorders in 13 (5.2%). The mean time for the evolution of Raynaud phenomenon into undifferentiated connective tissue disease or a form of the disease was 2 years. Most of the subjects with primary Raynaud phenomenon (173/191, 91%), undifferentiated connective tissue disease (22/27, 81%), juvenile onset rheumatoid arthritis/rheumatoid arthritis (7/10, 70%), and systemic lupus erythematosus (6/9, 67%) had normal capillary findings. Nonspecific capillary changes occurred in 3 of 10 (30%) patients with rheumatoid arthritis, 2 of 9 (22%) with systemic lupus erythematosus, 4 of 27 (15%) with undifferentiated connective tissue disease, and 18 of 191 (9%) with primary Raynaud phenomenon. Of all the subjects, only 10 (4%) showed sclerodermatous disease type capillary changes 6 months before the expression of a particular disease: eight (62%) of these developed scleroderma spectrum disorders, one expressed systemic lupus erythematosus, and one had undifferentiated connective tissue disease. We concluded that there were no specific capillary changes predictive for future development of systemic lupus erythematosus, juvenile onset rheumatoid arthritis/rheumatoid arthritis, and undifferentiated connective tissue disease in children and adolescents with Raynaud phenomenon. Most of our study subjects with Raynaud phenomenon who developed these diseases had normal capillary findings or nonspecific changes. Children and adolescents who developed scleroderma spectrum disorders showed a sclerodermatous type of capillary changes 6 months before the expression of the disease, indicating that this type of capillary changes in children and adolescents with Raynaud phenomenon highly correlated with further development of scleroderma spectrum disorders.     

Anticytoskeletal antibodies in systemic autoimmune diseases

Aim To investiate the presence of antinuclear and anticytoskeletal autoantibodies in patients with systemic autoimmune and connective tissue diseases. Setting 646 sera of 322 patients with systemic sclerosis, systemic lupus eryhtematosus, mixed connective tissue disease, rheumatoid arthritis with or without secondary Sjögren's syndrome, and localized scleroderma, and 127 healthy controls were tested. Methods ‘Routine’ indirect immunofluorescence technique on HEp-2 cells. Results Antinuclear antibodies were found in 45–91.1% of the sera. Anti-intermediate filament antibodies were detected in 24 patients. Eight of the 24 cases had another (second) organ-specific autoimmune disease, mainly chronic active hepatitis or autoimmune thyroid disease.     

In-vivo epidermal nuclear reactions: a selective process

Epidermal in-vivo nuclear reactions of IgG occur primarily in patients with mixed connective tissue disease or systemic lupus erythematosus and have been associated with high titres of circulating antibodies to ribonucleoprotein (RNP). This study was carried out to examine whether these epidermal nuclear reactions are true or simply an excision artefact. We observed the epidermal nuclear reactions for IgG only and not for other immunoglobulins in both in-vivo and in-vitro organ-culture studies, despite the presence of antinuclear antibodies (ANA) of all immunoglobulin classes. The association of the in-vitro epidermal nuclear reactions with serum RNP antibodies, although not absolute was statistically significant. The absorption of the serum with extractable nuclear antigen (ENA) preparation diminished the nuclear reactivity on tissue explants. In addition, the penetration of ANA into the nuclei of skin explants was both time and temperature dependent and was inhibited by sodium azide and by oligomycin. We conclude that the epidermal nuclear staining reactions observed by direct immunofluorescence on skin biopsies is selective and that the penetration of IgG into the epidermal cell nuclei is an active process and not an artefact.     

免疫印迹技术检测七种结缔组织病自身抗体

以兔胸腺可提取性核抗原（ＥＮＡ）的免疫印迹技术（ＩＢＴ），对１７７例结缔组织病患者及３２例正常人血清进行了７种自身抗体的测定。抗Ｓｍ抗体见于５０％的ＳＬＥ患者，敏感性较高；抗ＲＮＰ抗体可在多种结缔组织病中测出，在ＳＬＥ中阳性率较高；抗Ｒｏ（ＳＳＡ）、抗Ｌａ（ＳＳＢ）抗体见于部分ＳＬＥ患者；抗核糖体抗体仅见于ＳＬＥ，可能为ＳＬＥ的又一标记。本文ＩＢＴ具有广谱、快速、精确及稳固等优点，对提高结缔组织病的诊断和鉴别诊断水平有重要意义。     

Acid lysosomal hydrolases in systemic sclerosis and other connective tissue diseases

The activities of five lysosomal hydrolases were determined fluorometrically in the serum of patients with systemic sclerosis (PSS), systemic lupus erythematosus (SLE), dermatomyositis (DM), rheumatoid arthritis (RA), or Raynaud's disease (RD). In PSS the β-galactosidase activity was significantly increased compared with controls and the other connective tissue diseases. The β-N-acetyl-glucosaminidase was significantly increased in PSS, SLE and DM. In PSS both enzymes were more active in the early stage of the disease than later. These changes of enzyme pattern seem to be a relatively reliable marker for the differential diagnosis of PSS compared to other connective tissue diseases, especially for RD, in which the β-galactosidase activity was significantly decreased. Further work is required to determine whether these polysaccharide-degrading acid hydrolases play a role in the pathogenesis of PSS.     

Mixed connective tissue disease characterized by speckled epidermal nuclear IgG deposition in normal skin

Four African female patients are described, who presented with the features of systemic sclerosis. Overlapping features of lupus erythematosus or dermatomyositis were present in three cases but were not prominent. Direct immunofluorescence of uninvolved skin revealed a particulate (or speckled) epidermal nuclear staining, with specificity for IgG. In view of the reported association between this finding and mixed connective tissue disease, these patients were treated with corticosteroids and marked improvement occurred in all cases. The usefulness of this investigation in making the distinction between systemic sclerosis and mixed connective tissue disease and in indicating a potentially effective form of therapy is discussed.     

皮肌炎/临床无肌病性皮肌炎患者血清中CADM-140抗体的检测和临床意义

目的 对皮肌炎（DM）/临床无肌病性皮肌炎（CADM）患者进行CADM-140抗体检测,探讨CADM-140抗体与临床特征间的联系。方法 采集38例DM（22例）/CADM（16例）患者血清,另外采集46例伴有肺间质病变的其他结缔组织病患者血清,包括8例多发性肌炎、15例系统性红斑狼疮、5例系统性硬化病、6例干燥综合征、6例混合性结缔组织病、6例特发性肺纤维化和5例正常对照者。以重组黑素瘤分化相关基因5（rMDA-5）为底物,通过ELISA检测患者血清中CADM-140抗体,比较CADM-140抗体阳性与阴性患者的临床特征。 结果 ①16例CADM和22例DM患者血清CADM-140抗体阳性例数分别为7例和2例,CADM患者阳性率（43.8%）显著高于DM（9.1%）（P ＜ 0.05）,46例伴有肺间质病变的其他结缔组织病患者及5例正常人均阴性;②CADM-140抗体阳性患者皮肤溃疡和坏死的发生率为8/9,红细胞沉降率为（40.8 ± 23.1） mm/1 h,CADM-140抗体阴性组分别为6.9%和（22.5 ± 16.8） mm/1 h,两组比较,P ＜ 0.01和 ＜ 0.05;CADM-140抗体阳性患者乳酸脱氢酶水平显著高于阴性组（分别为328.3 ± 104.2和241.1 ± 100.3 IU/L,P ＜ 0.05）,而肌酸激酶显著低于阴性组（分别为156.3 ± 260.8和1806.2 ± 3737.1 IU/L,P ＜ 0.05）;两组间抗核抗体阳性率和恶性肿瘤发生率的差异无统计学意义;③CADM-140抗体阳性患者不仅肺间质病变发生率显著高于阴性组（分别为9/9和48.3%,P ＜ 0.01）,而且急进型肺间质病变发生率也显著高于阴性组（分别为5/9和0,P ＜ 0.05）。阳性组肺高分辨率CT评分（122.9 ± 54.8）显著高于阴性组（70.0 ± 59.8）（P ＜ 0.05）。结论 通过检测CADM-140抗体不仅可以判断DM/CADM是否合并肺间质病变,还可能是伴发急进型肺间质病变的血清学标记,动态观察血清CADM-140抗体水平也许有助于预测肺间质病变病程。     

Immunofluorescence studies in progressive systemic sclerosis (scleroderma) and mixed connective tissue disease

Immunofluorescence (IF) investigations of the skin were performed in thirty patients with progressive systemic sclerosis (scleroderma) and eight patients with mixed connective tissue disease (MCTD). The results show that speckled epidermal nuclear immunoglobulin deposition occurs not only in MCTD but also in true scleroderma. Granular IgM deposition at the dermo-epidermal junction of light-exposed skin was detected in both groups of patients, but six of eight MCTD patients also showed a granular IgM band in non-exposed skin. Antinuclear antibodies (ANA) were demonstrated in the sera of 96% and 100% of patients with scleroderma and MCTD respectively. The pattern of nuclear IF staining in scleroderma included dense fine speckles, large coarse speckles, threads, nucleolar and centromere staining. In MCTD, by contrast, the ANA staining pattern consisted of threads. The significance of ANA titres and immunological specificities for the in vivo reaction of serum ANA with epidermal nuclear antigens is discussed.     

Observations on the in vivo reaction of antinuclear antibodies with epidermal cells

In evaluating the normal skin of five patients with mixed connective tissue disease, four patients were noted to have both basement membrane zone and speckled epidermal nuclear immunofluorescence. The positive epidermal nuclear reaction was found to be associated with IgG, and no evidence of complement involvement was seen. In vitro testing demonstrated that normal control skin incubated with high-titred antisera to nuclear ribonucleoprotein reporduced the findings observed in direct staining, but incubation with high-titred antisera to other nuclear antigens such as Sm and single-stranded deoxyribonucleic acid did not cause positive epidermal nuclear staining. The association of antibodies to ribonucleoprotein and speckled epidermal nuclear immunofluorescence is discussed. Also considered is the possibility of other factors affecting nuclear membrane permeability.     

Nucleolar, homogeneous and peripheral fluorescence of epidermal nuclei in direct immunofluorescence: 4 cases (author's transl)]

Epidermal nucleolar IgG deposition on direct immunofluorescence of covered normal skin was found in two patients with scleroderma and high serum concentrations of antibody to nucleolar antigen. Epidermal homogeneous and peripheral IgG deposition was also observed in two patients with systemic lupus erythematosus, antinuclear antibody of homogeneous staining pattern, but without antibody to extractable nuclear antigen. Epidermal nuclear IgG deposition in a speckled, nucleolar, homogeneous or peripheral pattern appears to correlate with high titer serum antinuclear antibody giving on immunofluorescence the same staining pattern. These immunopathologic findings cannot be considered as being specific of a subset of connective tissue disease.     

Spectrum of autoantibodies other than anti‐desmoglein in pemphigus patients

Background Pemphigus is a life‐threatening autoimmune blistering disease mediated by autoantibodies against adhesion molecule of the skin. Its concurrence with systemic and organ‐specific autoimmune disease was described in case reports. Objectives To evaluate the presence of a broad spectrum of organ‐specific and non‐organ‐specific autoantibodies other than anti‐desmoglein antibodies in pemphigus patients. Patients and methods Serum samples were obtained from 105 pemphigus foliaceus (PF) patients, 51 pemphigus vulgaris (PV) patients and 50 controls. Both indirect immunofluorescence assay and ELISA were used to assess the presence of autoantibodies related to connective tissue diseases, autoimmune hepatitis, vasculitis, rheumatoid arthritis, coeliac disease, diabetes and thyroiditis. Results Significant difference was observed between the three groups for anti‐thyroglobulin antibodies in the pemphigus foliaceus group (18% vs. 4%, P = 0.03). A significantly higher occurrence of IgM anti‐cardiolipin (P = 0.03), IgG anti‐reticulin (P = 0.01) and IgG anti‐gliadin antibodies (P = 0.008) were observed in the PV group. Cases with more than four autoantibodies were frequently positives for both anti‐desmoglein 1 and anti‐desmoglein 3. Conclusion Autoantibodies other than anti‐desmoglein antibodies are not rare in pemphigus patients. Clinical and serological follow‐up of pemphigus patients with positive autoantibodies are needed to clarify their impact in disease evolution.     

Speckled (particulate) epidermal nuclear IgG deposition in normal skin. Correlation of clinical features and laboratory findings in 46 patients with a subset of connective tissue disease characterized by antibody to extractable nuclear antigen.

Clinical and laboratory findings were correlated from 46 patients with IgG localization in epidermal nuclei in a speckled (particulate) pattern on direct immunofluorescence of normal skin. Cutaneous manifestations included lupus erythematosus (LE), swollen hands or sclerodactyly, alopecia, vasculitis, and dyspigmentation. Systemic manifestations included arthritis or arthralgia, Raynaud's phenomenon, serositis, vascular headaches, mild renal disease, myositis, and sicca syndrome. High titer (mean = 1:142, 800) serum antibody to extractable nuclear antigen (ENA) was found in 81%. Eighty-six percent had antibody to an RNase-sensitive antigenic component of ENA (ribonucleoprotein or RNP); 14% had antibody to an RNase-resistant ENA termed Sm. Deposition of IgG in a speckled pattern in epidermal nuclei is an immunopathologic marker for a subset of connective tissue disease characterized by antibody to ENA. Those with Sm specificity had systemic LE (SLE); Those with RNP specificity had Raynaud's phenomenon usually associated with overlapping features of SLE, scleroderma, and/or dermatomyositis.     

间接血凝法在测定SLE患者血清中抗ds-DNA抗体的意义

抗核抗体(ANA)的测定已较普遍地应,用于临床诊断,由于它的非特异性对诊断系统型红斑性狼疮(SLE)仍有一定的局限性.抗双链去氧核糖核酸(ds-DNA,以下简称DNA)抗体的测定对诊断SLE有较大的特异性^(1,2),目前已用作ANA侧定后进二步明确诊断、了解病情特别是反映狼疮性肾炎的有用实验室指标之^③.测定坑DNA抗体的方法很多,考虑到方法的简便,易于推广,我们采用了间接血凝法.