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.     

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.     

Binding of antibodies to the extractable nuclear antigens SS-A/Ro and SS-B/La is induced on the surface of human keratinocytes by ultraviolet light (UVL): implications for the pathogenesis of photosensitive cutaneous lupus

Autoantibodies to the non-histone nucleoprotein antigens SS-A/Ro, SS-B/La, and RNP are highly associated with photosensitive cutaneous lupus erythematosus (LE). In order to better understand the potential mechanisms of ultraviolet (UV) light on photosensitivity in patients with cutaneous LE, we designed immunopathologic in vitro and in vivo experiments to evaluate the effects of UV on the binding of such autoantibodies to the surface of human keratinocytes, one major target of immunologic damage in photosensitive LE. Short-term 2% paraformaldehyde fixation of suspensions of cultured human keratinocytes previously incubated with monospecific antiserum probes enabled the detection of ENA expression on the cell surface by flow-cytometry analysis. UVB light (280-320 nm) induced the binding of monospecific antibody probes for SS-A/Ro and SS-B/La on keratinocytes in a dose-dependent pattern with maximal induction observed at the dose of 200 mJ/cm2 UVB. Binding of SS-A/Ro, SS-B/La, and RNP antibody was augmented strongly, but binding of anti-Sm was very weak. In contrast, UVA (320-400 nm) light had no effect on the induction of binding of these antibody probes. Identical results were seen by standard immunofluorescence techniques. Hydroxyurea-treated keratinocytes showed similar induction of those antigens by UVB irradiation, which suggested that ENA expression on cultured keratinocytes by UVB were cell-cycle independent. Tunicamycin, an inhibitor of glycosylation of proteins, reduced UVB light effect on the SS-A/Ro and SS-B/La antigen's expression. These in vitro FACS analyses revealed that ENA augmentation on the keratinocyte cell surface was dose dependent, UVB dependent, glycosylation dependent, and cell-cycle independent. In vivo ENA augmentation on the keratinocyte surface was examined in suction blister epidermal roofs. Specific antibody probes for SS-A/Ro, SS-B/La, RNP, and Sm bound to human keratinocytes in intact suction blister epidermis following UVL irradiation in vivo. Using three different protocols, we have demonstrated that antibodies to SS-A/Ro, SS-B/La, and U1RNP bind to UVL-irradiated human keratinocytes. We speculate that this antibody binding is an important inducer of antibody dependent keratinocyte damage in photosensitive cutaneous lupus.     

间接血凝法和对流免疫电泳法测定抗ENA抗体

本文分析和比较了被动间接血凝法(PHA)和对流免疫电泳(CIE)检测抗可提取性抗原(ENA)抗体的结果.发现SLE患者中35,700(60/68)抗RNP抗体阳性,39.9(67/168)抗Sm抗体阳性.15例MCTD中抗RNP抗体均阳性10000 PHA法检测抗Sm抗体敏感,而CIE法检测抗RNP抗体敏感.在PHA法中56℃加热一小时处理ENA和RNase消化ENA结果相似.     

免疫印迹法检测SLE患者血清ENA抗体与临床关系的研究

用免疫印迹法检测８５例红斑狼疮患者ＥＮＡ多肽抗体，阳性率为６８．２４％，５８例ＳＬＥ患者Ｓｍ抗体为３６．２１％，ＲＮＰ抗体为３４．４８％，ＳＳＡ抗体为２２．４１％，ＳＳＢ抗体为８．６２％，核糖体抗体为１７．２４％。试验结果表明ＥＮＡ抗体与ＳＬＥ病情活动无关。     

抗Sm抗体阳性系统性红斑狼疮临床特征探讨

为了探讨抗Ｓｍ抗体阳性的系统性红斑猥亵疮（ＳＬＥ）的临床特征,对４９例抗Ｓｍ抗体阳性ＳＬＥ患者的临床资料进行了分析,并与８５例抗Ｓｍ抗体阴性ＳＬＥ患者的临床资料进行比较。结果显示抗Ｓｍ抗体阳性ＳＬＥ患者的病程较短,光敏感、关节痛和雷诺氏现象较多见（Ｐ〈０．０１）或Ｐ〈０．０５）,白细胞减少、蛋白尿、补体Ｃ３下降的发生我高（Ｐ〈０．０１或Ｐ〈０．０５）。说明抗Ｓｍ抗体阳性ＳＬＥ患者的临床表现明显,易     

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.     

Unilateral nail dystrophy after C4 complete spinal cord injury

Summary Fifty-five patients with biopsy-proven cutaneous lupus erythematosus (LE) were identified in whom a prospective and retrospective review of the clinical and laboratory data allowed subclassification into systemic (SLE). subacute (SCLE). or discold (DLE) variants. In addition to conventional direct immunofluorescence. an indirect immunolluorescent technique. using a monoclonal antibody, was employed to assess deposition of the membranolytic attack complex (C_5b?9) in skin lesions. Deposition of C_5b?9 within the epidermis correlated with a diagnosis of SCLE with or without antibodies to Ro and was seen in SLE patients with antibodies to extractable nuclear antigens Ro. La, Sm. and RNP. and in DLE patients with positive antinuclear antibodies and/or extracutaneous manifestations. In the SLE group, vascular C_5b?9 deposition was present in six patients. Of these, tour had circulating lupus anticoagulant, one had lymphocytic vasculitis, and two had antibodies to Ro. In two patients with SLE there was keratinocyte decoration for immunoglobulin G but not for C_5b?9, in the absence of seropositivity for antibodies to Ro. La. Sm. and ribonucleoprotein (RNP). The immunohistological examination of skin lesions using a monoclonal antibody to C_5b?9 is a valuable adjunct in the subclassification of LE. The presence of C_5b?9 within skin lesions of patients with LE implies a pathogenic role for complement-mediated pore formation.     

猪胸腺ENA中Sm与RNP抗原活性的研究

本文报道了从猪胸腺制备的ENA中Sm,RNP抗原活性的有关研究工作.通过CIE法与从兔胸腺丙酮粉提取的ENA比较,发现SLE等结缔组织病患者血清中抗Sm、抗RNP抗体的检出率,用猪胸腺ENA虽高于用兔胸腺ENA,但两者并无显著性差异.通过对流免疫参比电泳和琼脂糖双向免疫扩散试验证实所检出的抗体为抗Sm或抗RNP抗体.SDS-PAGE平板电泳分析,发现猪ENA和兔ENA的成份一致,其主要成分的分子量为24kd.各批猪ENA,抗原活性相对稳定.制备的猪ENA使用方便,不同温度条件下保存较长时间不失抗原活性.     

Large speckle-like thready and thready antinuclear antibody patterns as markers for different clinical presentations in lupus erythematosus

Fifty-one patients with lupus erythematosus were studied retrospectively. They were chosen on the basis of their antinuclear antibody (ANA) immunofluorescent pattern. Only those with the thready or the large speckle-like thready patterns were studied. Autoantibody profiles consisting of ANA, anti-single-stranded deoxyribonucleic acid (ssDNA) antibody, and anti-extractable nuclear antigen (ENA) antibody determinations were obtained. The patients with the thready ANA pattern and anti-ENA (Sm) antibodies had a significantly higher incidence of pulmonary, joint, and renal involvement than the anti-ENA negative patients with the large speckle-like thready pattern. There was also a significantly higher incidence of Raynaud's phenomenon in patients with the thready pattern than in those with the large speckle-like thready pattern. Photosensitivity was seen significantly more frequently in the patients with the large speckle-like thready pattern than in those with the thready pattern.     

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     

A prospective immunofluorescence study of immune deposits in the skin of primary Sj?gren's syndrome

There are conflicting opinions concerning the epidermal immunofluorescence pattern in primary Sj?gren's syndrome. In a prospective study of 12 patients we found a characteristic pattern of epidermal nuclear/cytoplasmic IgG deposits in 8 (67%). This pattern was associated with the presence of antibodies against SSA/Ro and SSB/La in the serum and was also found in 2 out of 5 LE patients with monospecific antibodies against SSA/Ro. There is a resemblance to the pattern of dust-like particles described in the diseased skin of patients with subacute cutaneous LE. In one patient with primary Sj?gren's syndrome, IgG deposits were confined to epidermal cell nuclei (in vivo ANA). Instead of antibodies against SSA/Ro or SSB/La, this particular patient had nRNP-antibodies. From this study, we conclude that the epidermal IgG deposits in primary Sj?gren's syndrome may represent antibody binding to the sites within epidermal cells where the respective antigens are located.     

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.     

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.     

活化淋巴细胞的ENA免疫原性分析

目的 为了进一步分析活化淋巴细胞可浸出核抗原(ENA)的免疫原性。方法 按照Sharp法提取了正常淋巴细胞和活化淋巴细胞的ENA,分别免疫同系BALB/C小鼠。用ELISA法检测免疫血清中抗dsDNA抗体的动态变化,并进行了免疫血清的ENA多肽谱分析,用间接免疫荧光法观察抗核抗体的荧光核型,并用直接免疫荧光法观察了免疫小鼠的肾脏病理。结果 活性ENA免疫的小鼠血清中可检测出抗核抗体,包括抗ENA抗体和抗dsDNA抗体,抗核抗体核型有颗粒型、均质型、周边型及核仁型;免疫小鼠的肾脏切片可观察到肾小球有IgG类免疫复合物沉积。而正常ENA免疫的小鼠血清中始终检测不出抗核抗体、抗ENA抗体、抗dsDNA抗体,免疫小鼠的肾脏切片亦未见IgG类免疫复合物沉积。结论 活化淋巴细胞ENA亦具有免疫原性,可驱动抗核抗体生成,引起SLE样综合征。     

Antinuclear antibodies as immunologic markers for a benign subset and different clinical characteristics of scleroderma

Antinuclear antibody (ANA) test results were correlated with the clinical status of 56 patients with systemic scleroderma. Three groups were identified. (1) The speckled pattern represented a benign clinical subset. Acrosclerosis, Raynaud's phenomenon, calcinosis, and esophageal dysmotility characterized this group. None of these patients had pulmonary, renal, or cardiac disease. (2) Two patterns and ANA-negative test results were associated with a different incidence of certain clinical characteristics. The thready pattern was associated with pulmonary involvement. Diffuse skin involvement and Raynaud's phenomenon were found with the nucleolar pattern. Patients with ANA-negative test results had the most severe disease, including renal failure. (3) Two patterns were not associated with different clinical characteristics. These were the small speckle-like thready pattern and the homogeneous pattern. This study supports the theory that ANA patterns may be used as immunologic markers for different clinical characteristics of patients with scleroderma as they have already been used in lupus erythematosus.     

The presence of anti–basement membrane zone antibodies in the sera of patients with non–bullous lupus erythematosus

We performed indirect immunofluorescence (IF) studies using 1 mol/1 sodium chloride split skin to determine whether or not a positive IF is specific to patients with bullous lupus erythematosus (LE). We examined the sera from 21 patients with systemic LE (SLE), three of which were obtained from two SLE patients and one subacute cutaneous LE (SCLE) patient with bullous eruptions. As a comparison, we also studied the sera from patients with discoid LE (DLE,n= 7). SCLE (n= 1), systemic sclerosis (SSc, n= 20), bullous pemphigoid (n= 2) and normal individuals (n= 10). Sera from 16 SLE, four DLE and two SSc revealed a linear deposition of IgG isotype antibody at the epidermal side and/or the dermal side on indirect IF of split skin. The sera from three patients with bullous eruption and from 12 patients of SLE, SCLE, DLE without bullous eruption or SSc were further analysed by immunoblotting using five defined antigens, i.e, dermal extract, epidermal extract, three fusion proteins of 230 kDa bullous pemphigoid antigen (BPAG), 180 kDa BPAG, and human epidermolysis bullosa acquisita (EBA) antigen. Two SLE sera as well as one of the SCLE and the DLE serum reacted with 230 kDa BPAG in epidermal extract, and one of the SCLE and the DLE serum also reacted with the fusion protein of 180 kDa BPAG, No serum reacted with the dermal extract or the fusion protein of 230 kDa BPAG or EBA antigen. There was no consistent correlation between split–skin IF results and immunoblotting results. These results may suggest that even non–bullous LE patients often have autoantibodies to the basement membrane zone antigens, most of which are less pathogenic. Although we rarely examine the sera from non–bullous LE patients, we should keep this phenomenon in mind to avoid overestimating the results of split–skin test and immunoblotting.     

用对流免疫电泳法检测SLE患者抗ENA抗体

本文报告了用对流免疫电泳(CIE)法检测58例SLE患者抗ENA抗体的结果,将CIE所得的阳性血清,分别与含抗Sm,RNP和La抗体的标准对照血清作参比电泳进行核对.抗Sm抗体阳性者20例,阳性率为32.75%.抗RNP抗体阳性者15例.阳性率为25.86%,抗La抗体阳性者1例、还在2例患者中分别检测到与抗RNP抗体和抗Sm抗体在免疫学上性质不相同的二个抗体.最后作者讨论了临床表现与抗 Sm抗体和抗RNP抗体的关系.     

Evaluation of the past history of chilblain in cases of systemic lupus erythematosus (SLE) and its similar diseases

We sent out a questionnaire to 47 patients of SLE and its similar diseases as to their past histories of chilblain. The results of the patients were compared to those of 141 cases of control. Although the percentage of cases who had revealed chilblain frequently (40.0%) and the age of chilblain onset (mean: 10.8 years old) in SLE group were not significantly different from those of control (28.4% and 12.4 years old, respectively), the chilblain which SLE patients developed had some characteristics compared to that of control, (1) higher incidence of chilblain episodes, (2) longer duration until cure, (3) more liability that chilblain leads to erosion or ulceration and (4) frequent occurrence of chilblain in the other seasons than winter. Especially SLE patients with the characteristic of (4) had higher association rates of Raynaud's phenomena, chilblain LE and livedo, suggesting disorder of peripheral circulation. It was also revealed that females are generally more liable to develop chilblain than males (females: 40.0%, males: 13.1%). Those results suggest some important relationship between chilblain and LE lesions. It is supposed that chilblain with the characteristics described above may possibly be transformed into LE lesions.     

Clinicopathological evaluation of in vivo epidermal nuclear fluorescence

Background.  Nuclear fluorescence in keratinocytes is an occasional phenomenon, often present in autoimmune diseases, especially in connective-tissue disease (CTD); however, its clinical significance remains unclear.
Aim.  To investigate the profile of patients with positive nuclear staining on direct immunofluorescence (DIF) of skin samples.
Methods.  A retrospective analysis of 28 patient records from our immunodermatology laboratory was performed between May 2003 and June 2006. Inclusion criteria were the presence of autoantibodies (IgG, IgA or IgM) or complement (C3) binding keratinocyte nuclei on DIF.
Results.  The most prevalent diseases related to the nuclear keratinocyte DIF staining were systemic lupus erythematosus ( n  = 9), mixed CTD ( n  = 3), overlap syndrome ( n  = 3), Sjögren's syndrome ( n  = 1), and CREST (calcinosis, Raynaud's phenomenon, oesophageal dysmotility, sclerodactyly and telangiectasia) syndrome ( n  = 1). Serum antinuclear antibody (ANA) was positive in 20 of 28 patients, with titres varying from 1 : 160 to 1 : 1280. Of the 20 patients with positive anti-nuclear antibodies (ANA), 17 were positive for anti-extractable nuclear antigen antibodies, 12 had anti-SSA/Ro, 11 had anti-SSB/La and 8 had anti-ribonucleoprotein. Eight patients were negative for ANA. Positive predictive value of in vivo ANA for systemic CTDs was 75%.
Conclusion.  The present data suggest that in vivo ANA evaluation is an additional and feasible auxiliary tool for diagnosing CTDs.