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.     

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.     

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.     

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.     

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     

The titers and complement-activating abilities of anticentromere antibody in systemic sclerosis, other connective tissue diseases, and other related conditions

The anticentromere antibody is considered to be a useful serologic marker for the CREST syndrome. But this antibody also appears in other related conditions less frequently. We classified 29 patients with anticentromere antibodies into 3 groups: (1) 16 patients with systemic sclerosis or Raynaud's phenomenon alone; (2) 7 patients with other connective tissue diseases; (3) 6 patients with other conditions. Ig class reactivities and complement-fixing abilities of anticentromere antibody were measured by the indirect immunofluorescence test. The whole Ig titers were high (1025 or more) in all patients belonging to group 1. However, the properdin-fixing anticentromere antibody titers of these patients were relatively low (256 or less). In contrast, the patients in group 2 and 3 were shown to have higher C3- and properdin-activating abilities which were determined by the ratios of the titers of C3- and properdin-fixing anticentromere antibody to the IgG titers although the whole Ig titers of these patients were widely distributed. These data suggest that the patients who have low whole Ig titers and/or high properdin-fixing titers do not belong to the scleroderma spectrum and that the patients without clinical features of scleroderma have high C3- and properdin-activating abilities.     

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.     

Anticentromere-protein-B-DNA complex activities in anticentromere antibody-positive patients

Centromere protein B (CENP-B), which is an alphoid DNA binding protein, is the target antigen in autoimmune disease patients (often those with scleroderma). In this study, we analysed activities of anti-CENP-B-DNA complex in anticentromere antibody (ACA)-positive patients using DNA immunoprecipitation with purified CENP-B. The activities correlated with ACA titres and were closely associated with Raynaud's phenomenon. Patients with CREST symptoms (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasia) showed higher activities than those with no symptoms. Our results suggest that autoimmune responses to native CENP-B may have an important role in the pathogenesis of scleroderma.     

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.     

An organ culture model for the study of pemphigus acantholysis

An in vitro model for the study of pemphigus acantholysis has been developed. The histological changes of pemphigus vulgaris were reproduced in vitro in organ culture by growing normal human skin in the presence of pemphigus vulgaris or pemphigus foliaceus sera. At 24 h a suprabasilar split was noted and at 72 h extensive suprabasilar acantholysis developed. Direct immunofluorescent tests demonstrated that pemphigus antibody became bound to the epidermal intercellular space antigen(s) during the first 6-12 h. As acantholysis increased the presence of tissue-fixed antibody decreased. The fixation of the pemphigus antibody to the skin prior to the development of acantholysis provides strong evidence for the pathogenetic role of this antibody in the production of acantholysis. The data suggest that complement is not required in this model for the production of the acantholytic changes of pemphigus since heating the serum for 30 min at 56 degrees C did not destroy the acantholytic activity and no complement (C3) could be detected by DIF of organ culture explants.     

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.     

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.     

Urticaria. An immunofluorescence and histopathology study.

Fourteen randomly chosen patients with "garden variety" urticaria were studied for the presence of vasculitis and immunoglobulins and complement. Results of direct immunofluorescence (DIF) of the involved skin were negative, although two patients had immunoglobulins and complement demonstrable in the cytoplasm of the epidermal cells. Results of DIF of uninvolved skin were also predominantly negative. Findings from serum samples tested by indirect immunofluorescence (IIF) were negative, except for one positive in low titer (1:10, the basement membrane zone). Serum C3 and C4 levels were normal in five patients, both levels were low in two, and the C4 level was low in one patient. No skin-reactive immunoglobulins were found in these three patients by DIF or IIF. The ESR was measured and found to be elevated in four patients. Results of immunofluorescence proved negative in these cases. Of the 12 patients studied by hematoxylineosin staining to determine histology, none exhibited vasculitis. We believe that vasculitis with antigen-antibody reactions is not the rule in "garden variety" urticaria.     

Positive direct immunofluorescence and autoantibody profiles in psoriasis patients

Psoriasis and systemic lupus erythematosus are common, but their coexistence is thought to be infrequent. Each of these diseases has a broad clinical spectrum, so that diagnosis may not be straightforward. This study aimed to investigate the following immunological parameters in psoriasis patients: (i) direct immunofluorescence (DIF); (ii) antinuclear antibody; (iii) anti-double-stranded DNA (dsDNA); (iv) anti-Ro; and (v) anti-nuclear ribonucleoprotein (nRNP). Of 300 cases, comprising 189 men (62.9%) and 111 women (37.1%), 17 (5.7%; 10 men, seven women) were positive for at least one immunological parameter. Nine of 300 (3%; seven male, two female) biopsy specimens from sun-exposed psoriatic lesions demonstrated bright continuous bands of granular IF along the dermoepidermal junction with immunoreactant immunoglobulin (Ig)G, IgM, C3 and fibrinogen. The intensity of IF at the dermoepidermal junction was graded 3+ and 2+. Three cases demonstrated IgM, two had IgG, two fibrinogen and six cases showed C3. Three cases demonstrated more than one immunoreactant. One case demonstrated C3 at the vessels. No specimen demonstrated IgA deposition. Three hundred sera were obtained from patients with psoriasis, of which five demonstrated elevated antinuclear antibody (ANA) titer; dilution titer varied from 1:80-640. Three had a homogeneous pattern and two had a speckled pattern. None had a peripheral pattern. Five (1.7%) of 300 demonstrated anti-Ro, two had negative ANA, and three were positive ANA, two of which were speckled and the other homogenous. Anti-dsDNA, anti-Sm and anti-nRNP were not detected. Ten patients had positive DIF but negative ANA, while five had positive ANA; all had negative DIF results. Thus, the incidence of psoriasis and lupus erythematosus coexistence is low and a baseline immunological screening test for psoriasis might not prove worthwhile.     

Clinical distribution of anticentromere antibody in Japanese patients

Serum samples from 401 subjects were screened for the presence of anticentromere antibody using HEp-2 cells as the substrate for an indirect immunofluorescence method. Anticentromere antibody was found in 16 cases; 8 out of 62 patients with systemic sclerosis, 3 out of 7 patients with primary Raynaud's disease, 2 out of 41 patients with systemic lupus erythematosus, 1 out of 54 asymptomatic relatives of systemic sclerosis and 2 out of 50 patients with miscellaneous diseases. In systemic sclerosis, the patients with anticentromere antibody were limited in CREST (calcinosis, Raynaud's phenomenon, esophageal involvement, sclerodactylia and telangiectasia) variant. As reported previously by many investigators, anticentromere antibody is considered as a useful immunologic marker for CREST variant of systemic sclerosis, although this antibody is widely distributed in other conditions with less frequency.     

Scl 70 antibody—a specific marker of systemic sclerosis

Scl 70 antibodies were tested for in 107 patients with systemic sclerosis: 68 with acrosclerosis and 39 with diffuse scleroderma. Anticentromere antibodies (ACA) and other antinuclear antibodies (ANA) were tested for by indirect immunofluorescence on HEp-2 cells. Positive results for Scl 70 antibodies were obtained in 77% of cases of diffuse scleroderma and 44% of acrosclerosis. ACA and Scl 70 antibodies were found to be mutually exclusive. If acrosclerosis cases positive for anticentromere antibodies are excluded, the percentage of acrosclerosis cases positive for Scl 70 was 63%. ACA were found to be a marker of a benign, abortive subset of acrosclerosis with almost no cutaneous involvement (CREST), whereas Scl 70 did not discriminate between acrosclerosis and diffuse scleroderma. On HEp-2 cells Scl 70 positive sera gave a characteristic, fine speckled, almost homogeneous nuclear staining pattern.     

Generalized morphea-like progressive systemic sclerosis with anticentromere antibodies and eosinophilic cellulitis

A 67-year-old woman without any history of exposure to organic solvents suffered from Raynaud's phenomenon, sclerodactylia, contracture of finger joints, diffuse pigmentation, pulmonary fibrosis, and generalized morphea-like eruptions on the trunk; she was diagnosed as generalized morphea-like progressive systemic sclerosis. She had a high titer of anticentromere antibody in her serum without any symptoms of CREST syndrome. She also had eosinophilic cellulitis on her extremities, which subsided within 6 months, and seemed to be due to a hypersensitivity reaction to mosquito bites. The occurrence of these two diseases together in our case may suggest some similarities in their pathogenesis.     

系统性红斑狼疮患者皮损中C_3沉积与病情活动的关系

目的探讨系统性红斑狼疮(SLE)患者皮损中C3的沉积与血清中抗体的阳性率、补体C3水平及病情活动之间的关系。方法用直接免疫荧光法(DIF)检测皮损部位的免疫反应物;常规方法检测患者血清中自身抗体及补体;根据临床表现和实验室检查指标,进行系统性红斑狼疮病情活动性评分(SLEDAI)。结果 65例SLE患者中,37例(56.92%)真表皮交界处有一种或几种抗体和/或补体沉积,即DIF阳性,将65例患者按皮损中C3的沉积情况分成3组:DIF阴性组28例(43.08%),C3沉积组21例(32.31%)和仅有其他抗体沉积而无C3沉积组16例(24.62%)。各组间血清抗体ANA,dsDNA,SSA,RNP和Sm的阳性率差异均无统计学意义(P>0.05)。而C3沉积组的血清C3水平明显低于其余两组(P<0.05),且C3沉积组的病情活动性明显高于其余两组(P<0.05)。结论 C3单独及与其他抗体在皮损部位的沉积,比DIF阴性组和仅有其他抗体而无C3沉积组具有更低的血清C3水平及更高的病情活动性。     

Relevance of complement fixing antinuclear antibodies

Background Connective tissue diseases (CTDs) are a heterogeneous group of disorders defined by the association of a variety of clinical manifestations with immunologic and other laboratory findings. Overlap of syndromes and aberrant findings appear rather frequently. Methods Sera of eight antinuclear antibody (ANA) negative, cases of subacute cutaneous lupus erythematosus (SCLE) with antibodies to Ro (SS-A) and a ninth case with clinical and laboratory signs of Sjögren’s syndrome and systemic lupus erythematosus (SLE) were tested for complement (C′) fixing antinuclear antibodies (C-ANAs). The ninth case was examined in depth by direct immunofluorescence (DIF) and a two-step “C + DIF” test of biopsies for C′ fixation to in vivo bound ANAs, as well as serum tests for C-ANA, ANA, and SCLE markers. Results Sera of five of the eight ANA negative, Ro(SS-A) positive SCLE cases had C-ANAs. The ninth case, a 50-year-old woman with clinical and laboratory signs of Sjögren’s syndrome and SLE, gave a strong positive C + DIF reaction in the skin biopsy for in vivo bound ANAs that fix C′, but negative ANAs and C-ANAs in routine serum tests; they revealed antimitochondrial antibodies. Serum tests on normal skin, however, revealed weak ANA and strong C-ANA reactions with in vitro fixed C′. Conclusions ANA negative cases of SCLE or Sjögren’s syndrome may have C-ANAs. A case with Sjögren’s syndrome and signs of SLE had both in vivo and in vitro C′ fixing ANAs. C-ANA tests can aid in the identification of such cases.     

Reactivation of human herpesvirus (HHV) family members other than HHV-6 in drug-induced hypersensitivity syndrome

BACKGROUND: Drug-induced hypersensitivity syndrome (DIHS) is characterized by a severe multiorgan hypersensitivity reaction that usually appears after a 3-6-week exposure to certain drugs, including anticonvulsants. There are some reports showing that serum IgG levels often decrease at the early stage of DIHS. Reactivation of human herpesvirus (HHV)-6 has been reported in patients with DIHS, and some other DIHS patients showed reactivation of cytomegalovirus (CMV) or Epstein-Barr virus (EBV). OBJECTIVES: To determine whether reactivation of HHV-6, HHV-7, CMV and/or EBV occurs in patients with DIHS. METHODS: Titres of IgG and IgM antibodies to HHV-6 and HHV-7 were determined using an indirect immunofluorescence antibody assay on admission and at various times after admission. Anti-CMV IgG and IgM antibody titres and anti-EBV capsid antigen IgG, IgA, IgM, and EBV nuclear antigen and EBV early antigen IgG titres were determined by enzyme immunoassay. Polymerase chain reaction (PCR) procedures for HHV-6, HHV-7, CMV and EBV DNAs were performed using serum samples. IgG antibody titres to HHV-6, HHV-7, CMV and EBV were increased after the onset in seven, six, seven and two of seven patients, respectively. IgG antibody titres to HHV-6 and HHV-7 were elevated simultaneously 21-38 days after the onset. RESULTS: IgG antibody titres to CMV and EBV were elevated 10-21 days after the elevation of HHV-6 and HHV-7 antibody titres. PCR showed that HHV-6, HHV-7, CMV and EBV DNAs became positive in six, five, seven and two of seven patients, respectively. HHV-6 and HHV-7 DNAs were detected 21-35 days after the onset, and CMV DNA was detected 10-21 days after detection of HHV-6 and HHV-7 DNAs. CONCLUSION: The present study suggests that in addition to HHV-6 reactivation, reactivation of HHV-7, CMV and/or EBV may also occur following drug eruption in some patients with DIHS.