混合性结缔组织病皮肤表皮细胞核染色的价值

采用 IIF法对 2 32例 CTD患者的外观正常皮肤进行了免疫荧光研究。结果发现 10 0 %的MCTD患者的表皮细胞核有 S型 Ig G沉积 ,并显著高于 SL E患者 ( 6.6% ) ,同时血清伴有高滴度、单一的抗 RNP抗体和高滴度的 S型 ANA。因此 ,S型 Ig G ENS和高滴度单一抗 RNP抗体之间具有高度的相关性。我们认为 S型 Ig G ENS可作为 MCTD的免疫病理学特征 ,其对 MCTD具有重要的辅助诊断价值     

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.     

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     

In vitro complement activation by antinuclear antibodies in the epidermis from patients with systemic sclerosis

Skin biopsies from twenty-one systemic sclerosis patients with significant serum fluorescent antinuclear antibody (FANA) titres (more than 1:40) were examined by direct immunofluorescence (DIF) and by in vitro immunofluorescent complement (C) activation (C + DIF). Eight patients showed epidermal nuclear ANA deposits by DIF. In vitro complement activation was achieved in epidermal nuclei from all eight patients. In addition, one patient with diffuse scleroderma revealed a dense speckled, and two patients with the CREST syndrome a discrete speckled, epidermal nuclear staining pattern by C + DIF. The latter two patients showed high titres of anticentromere antibody in their sera. We consider that the C + DIF findings of these two patients with the CREST syndrome reflect binding of serum anticentromere antibodies to their antigens in vivo.     

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     

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.     

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.     

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.     

Mixed immunofluorescence findings in mixed connective tissue disease

An adult female with mixed connective tissue disease is described who had non-scarring alopecia, telangiectases, Raynaud's phenomenon and swollen hands. No clinical signs of SLE and only modest signs of myositis were present. High levels of RNP antibodies were detected in serum and direct immunofluorescence showed IgG staining of epidermal nuclei with a speckled pattern. Elution techniques performed on circulating lymphoid cells showed mixed features of both LE and scleroderma, namely antibodies to basal zone, to epidermal basal cells and to endothelial cells in mid-dermis.     

Epidermal nuclear immunofluorescence: serological correlations supporting an in vivo reaction

Epidermal nuclear deposits of immunoglobulins (Ig) were studied by direct immunofluorescence in three groups of patients: ten scleroderma (SD, systemic sclerosis), seven dermatomyositis (DM) and seven systemic lupus erythematosus (SLE). Each patient had skin biopsies taken from three different sites (nailfold, forearm, buttock) on the same day that a serum sample was also obtained. Epidermal nuclear deposits were observed in nine of twenty-four patients (five SD, two DM, two SLE). A high serum ANA titre correlated significantly with the presence of epidermal nuclear Ig deposits. The nucleolar epidermal nuclear pattern was limited to the SD group, four of ten patients showing this pattern. Two of nine patients with positive results in the nailfold and forearm had negative findings in the buttock, supporting the view that deposition of Ig in the epidermal nuclei occurs in vivo.     

Cutaneous immunofluorescence study of erythema multiforme: correlation with light microscopic patterns and etiologic agents

Direct immunofluorescence microscopy was positive in 88% of forty-one skin biopsy specimens from thirty-four patients with the clinical diagnosis of erythema multiforme. The most common finding, present in 67% of positive specimens, was the cytoid body, or fluorescent keratinocyte, which stained most often with IgM (homogeneously) or with C3 (speckled). Other findings included basement membrane zone (BMZ) fluorescence, primarily with fibrinogen and C3, and vascular fluorescence, most commonly C3 in a granular pattern. Correlation of direct immunofluorescent and light microscopic findings revealed that (1) the fluorescent keratinocyte was prevalent only in epidermal and mixed patterns, correlating with the eosinophilic necrotic keratinocyte by light microscopy, and (2) vascular fluorescence was most prominent in dermal forms. Herpes simplex-associated erythema multiforme showed exclusively a mixed histologic pattern, whereas the drug-related form was primarily epidermal.     

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.     

Use of 1M NaCl split skin in the indirect immunofluorescence of the linear IgA bullous dermatoses

We compared 1M NaCl split skin with intact skin as substrates for detection of circulating IgA anti-basement membrane (BMZ) antibodies in linear IgA dermatosis (LAD). The sera of 63 patients with LAD including 27 adults and 36 with chronic bullous dermatosis of childhood (CBDC) were examined. 62% of patients overall had circulating IgA anti-BMZ antibodies detectable on intact skin. 73% of patients had circulating antibodies detectable on lM NaCl split skin as an additional 7 sera were positive. This was a statistically significant increase (p<0.01). The sera were mostly positive at a higher titre on the split skin when compared with intact skin. On routine indirect immunofluorescence (IIF) all positive sera produced linear fluorescence on the epidermal side of the split. Twenty serum samples were incubated with split skin overnight; 4 of these specimens exhibited linear fluorescence on the epidermal and dermal sides of the split after this prolonged incubation. These findings suggest that 1M NaCl split skin is a more sensitive substrate for detection of circulating IgA anti-BMZ antibodies in LAD, that these antibodies are heterogeneous and that the target antigen has an epidermal component.     

直接免疫荧光检查在诊断及鉴别硬皮病与混合结缔组织病上的应用

50例PSS、10例MCTD患者前臂伸侧皮肤活检DIF检查发现:10%PSS与60% MCTD出现有表皮细胞核Ig着色,MCTD均为IgG大斑点型,PSS则以均质为主,无1例为IgG大斑点型.10%PSS与30%MCTD患者BMZ出现Ig带状沉积.作者结论为:(1)在患有手部皮肤不典型硬化、雷诺氏现象、关节炎的患者,若DIF出现表皮细胞核大斑点IgG着色,即可诊断为MCTD.(2)BMZ带状Ig沉积不能做为区分二者的主要依据.     

Bullous pemphigoid: serum antibody titre and antigen specificity

Abstract 2 antigens have been identified as possible targets for autoantibody depositions in bullous pemphigoid: a 230-kD protein (BP230) and a 180-kD protein (BP180). We studied the relationship of these 2 antigens with the immunofluorescence determined serum antibody titre. 2 groups of bullous pemphigoid patients were selected on the basis of immunoblot-determined antibody specificity. One group (13 patients) had antibody specificity for BP230 and not for BP180, while the other group (9 patients) had antibody specificity for BP180 and not for BP230. The immunofluorescence titres of the circulating antibodies determined on monkey oesophagus substrate displayed, for the BP230-specific group, a mean of 1:1102. The maximal observed titre was 1:5120. The mean titre in the BP180-specific group was only 1:29, with a highest titre of only 1:160. This result suggests that in routine indirect immunofluorescence of bullous pemphigoid sera, the contribution of the BP180-specific antibodies to the total anti-epidermal basement membrane zone antibody titre is relatively much lower than that of the BP230-specilic antibodies. Thus, at high dilutions, only the BP230-speeific antibodies contribute to the overall indirect immunfluorescence titre.     

Anticentromere antibodies (ACA): clinical distribution and disease specificity

Sera from 3528 patients with autoimmune disease, and non-autoimmune disease, and 500 normal individuals were studied For the presence of anticentromere antibodies (ACA) by indirect immunofluorescence on HEP-2 cells. Sixty-seven specimens were identified showing discrete speckled staining: 55 (82.1%), 11 (16.4%), and one (1.5%), were from patients with autoimmune disease, non-autoimmune disease and normal control subjects, respectively. These ACA were present frequently in CREST syndrome (55%), Raynaud's disease (29.6%) and primary biliary cirrhosis (30%). Only 16.4% of the antibody positive patients carried a clinical diagnosis of CREST, which means that ACA are not specific for CREST syndrome. High antibody titre persisted irrespective of whether or not the patients had active disease. The ACA were present infrequently in Sjögren's syndrome, systemic lupus erythematosus, rheumatoid arthritis, immune thrombocytopenic purpura, Graves' disease, immune haemolytic anaemia, and vitiligo. Sera from 107 patients with various other autoimmune diseases were negative for ACA.     

Autoantibodies to nucleolar antigens in systemic scleroderma: clinical correlations

Indirect immunofluorescence(IIF) and double immunodiffusion (DID) were performed on the sera of 64 patients who had a nucleolar immunofluorescence pattern on HEp-2 cells. Forty-nine of the sera were from 296 patients with systemic scleroderma (SSc) and 15 sera were from 214 patients with systemic lupus erythematosus (SLE). A homogeneous nucleolar staining pattern was found in 45 of the 64 sera (70.3%), a clumpy fluorescence associated with fibrillarin antibody in 14 (21.8%) and a speckled pattern was found in five of the sera (7.8%). There was a clear correlation between the sera which showed a homogeneous nucleolar staining pattern with symptoms of the polymyositis/scleroderma overlap syndrome that differed from SSc with concomitant myositis. The clumpy pattern was mainly associated with diffuse scleroderma and the speckled pattern with limited scleroderma (previously called acrosclerosis).     

Anti-Ro/SSA antibodies. Association with a particulate (large speckledlike thread) immunofluorescent nuclear staining pattern

Twenty-one patients with clinical and histopathologic evidence of subacute cutaneous lupus erythematosus and one patient with Sj?gren's syndrome and vasculitis had anti-Ro/SSA (Sj?gren's syndrome A) antibodies as demonstrated by double immunodiffusion assay using a saline extract of human spleen as the source of antigen. These serum samples were negative when tested for other nuclear antigens, including native DNA, Sm, and RNP. When tested for antinuclear antibodies (ANAs) by immunofluorescence, only ten of 22 serum samples were ANA positive on mouse liver substrate, while 18 of 22 had a positive ANA when HEp-2 tumor cells were utilized. With imprints of human spleen as test substrate, all 22 serum samples yielded a positive ANA result and a particulate (large speckledlike thread) staining pattern. Absorption of two of these serum samples with human spleen extract containing Ro/SSA antigen inhibited both the particulate staining pattern using human spleen imprints and the anti-Ro/SSA precipitin line by double immunodiffusion. These studies suggest that anti-Ro/SSA antibodies and antibodies producing the particulate nuclear staining pattern on human spleen imprints are either one and the same or closely paralleling antibody systems.     

Antibodies eluted from lymphoid cell membrane. Occurrence in certain varieties of scleroderma.

By means of acid elution two antibodies could be removed successfully from the circulating lymphocytes of 11 patients with certain varieties of scleroderma. One was specifically directed against nuclear antigen(s) of endothelial cells (NEC) of the dermal blood vessels, and another against nuclear antigen(s) of epidermal basal cells (NBC) of the involved and uninvolved skin of the patients. In two cases of acroscleroderma, the eluates failed to react with either endothelial or basal cells of involved or uninvolved skin. In none of 20 healthy controls involved in this study could an antibody be eluted from the circulating lymphocytes. The aforementioned antibodies do not bind complement in vitro and do occur in the serum of four patients. Circulating antinuclear antibody (speckled type) was detectable in two cases of scleroderma.     

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.