Substrate specificity of antinuclear antibodies in scleroderma.

Studies of antinuclear antibodies (ANA) were carried out in 39 cases of systemic scleroderma and for comparison in 19 cases of systemic lupus erythematosus (SLE) and 4 of mixed connective tissue disease (MCTD) using indirect immunofluorescence (IF) methods under standard conditions. The results on three different substrates--monkey esophagus, guineapig lip and rat liver--are reported. In 48.7% of scleroderma cases ANA showed a substrate specificity. The highest percentage of positive results in scleroderma was obtained on monkey esophagus (97.4%) and the lowest on rat liver (61.5%). In SLE and MCTD, in contrast, only about 13% of the sera displayed such specificity. If only sera with substrate specificity are considered, the positive results on monkey esophagus and rat liver are 94.7% and 21.1%, respectively. Titers of sera reacting positively on 2 or 3 substrates were mostly in agreement, although some sera both in systemic scleroderma and SLE showed higher titers on monkey esophagus. The IF pattern was usually the same regardless of the substrate, Tests for ANA in scleroderma should be performed on at least 2 substrates simultaneously.     

Bullous pemphigoid antibodies. Human skin as a substrate for indirect immunofluorescence assay

Human skin, the target organ for bullous pemphigoid (BP) antibodies, is thought to be a less sensitive substrate for the indirect immunofluorescence assay of BP antibodies than monkey or guinea pig esophagus. To examine the reasons for this puzzling phenomenon, we compared the titers of BP antibodies obtained when human skin, monkey, and guinea pig esophagus were used as substrates. We found the titers of BP antibodies obtained with human skin from sites commonly involved in BP (flexor arm, flexor thigh, popliteal fossa) were as high and usually higher than those obtained with monkey and guinea pig esophagus. In contrast, much lower titers were obtained with human skin from sites rarely involved in the disease (scalp, face, extensor arm). These findings suggest that human skin as a substrate is at least as sensitive as monkey or guinea pig esophagus for the indirect immunofluorescence assay of BP antibodies when the skin is obtained from regions on the body commonly involved in BP.     

The clinical relevance of serum antinuclear antibodies in Japanese patients with systemic sclerosis

BACKGROUND: Systemic sclerosis (SSc) is a connective tissue disorder with excessive fibrosis of the skin and various internal organs. Although SSc is a heterogeneous disease, it has been reported that the particular antinuclear antibodies (ANA) are often indicative of clinical features, disease course and overall severity. OBJECTIVE: To clarify the association of clinical and prognostic features with serum ANA in Japanese patients with SSc. METHODS: We studied 203 Japanese patients diagnosed with SSc, who visited our hospital during the period 1983-2005. Six SSc-related ANA were identified using indirect immunofluorescence, double immunodiffusion and immunoprecipitation assays. RESULTS: Patients with SSc were classified into six ANA-based subgroups and a group without ANA. As expected, antitopoisomerase I antibody (Ab, n=64), anti-RNA polymerases (RNAP) Ab (n=12) and anti-U3 RNP Ab (n=5) were associated with diffuse cutaneous SSc, whereas anticentromere Ab (ACA, n=75), anti-Th/To Ab (n=7) and anti-U1 RNP Ab (n=10) were frequently detected in patients with limited cutaneous SSc. Clinical features of the ANA-negative group (n=10) were heterogeneous. Consistent with previous findings in Caucasian and/or black African patients, antitopoisomerase I Ab was associated with the involvement of vascular and pulmonary fibrosis, leading to decreased survival rate. However, no patients with anti-RNAP Ab developed renal crisis and the frequency of isolated pulmonary hypertension in patients with ACA, anti-Th/To Ab or anti-U3 RNP Ab was similar to that in other ANA-based subgroups. CONCLUSION: These results indicate that the clinical relevance of SSc-related ANA in Japanese patients differs in some aspects from that in Caucasian and/or black African patients.     

Autoantibodies in psoriasis as predictors for loss of response and anti-infliximab antibody induction

Background Infliximab is successfully used to treat psoriasis and psoriatic arthritis. However, some patients lose therapeutic response after several cycles. Antibodies to infliximab (infliximab‐Abs) are induced during treatment in a subgroup of patients and are thought to be associated with loss of response (LOR). Routine screening for infliximab‐Abs is expensive and not regularly performed. A reliable and affordable method for identifying patients who are at risk for LOR to infliximab is desirable. Objectives To analyse the development of antinuclear antibodies (ANA)/antidouble‐stranded DNA antibodies (anti‐dsDNA) over time in patients with psoriasis receiving infliximab. To analyse if there is an association between ANA titres/anti‐dsDNA concentrations, infliximab‐Ab status and LOR. Methods A retrospective data analysis of 29 patients with psoriasis receiving infliximab was carried out. ANA titres and anti‐dsDNA concentrations were regularly monitored in these patients and sera were tested for infliximab‐Abs by enzyme‐linked immunosorbent assay. Results Median ANA titres increased from 1 : 80 [interquartile range (IQR) 0 to 1 : 320, n = 29] pretreatment, to 1 : 1280 (IQR 1 : 640 to 1 : 1920, n = 15) after infusion 10, and 1 : 1920 (IQR 1 : 1280 to 1 : 2560, n = 10) after infusion 20. Infliximab‐Abs were found in 21% of patients. Infliximab‐Ab‐positive patients and patients with LOR had significantly higher pretreatment anti‐dsDNA concentrations and higher pretreatment ANA titres than infliximab‐Ab‐negative and responsive patients, respectively. Conclusions The results of this study suggest a role for autoantibodies in the identification of patients with psoriasis at higher risk of developing infliximab‐Abs and of LOR under treatment with infliximab.     

Rat bladder epithelium: a sensitive substrate for indirect immunofluorescence of bullous pemphigoid

Serological diagnosis of bullous pemphigoid is based on immunoblotting or indirect immunofluorescence on normal human salt-split skin. These methods are expensive or time-consuming and not available as a routine test in all laboratories. We used rat bladder epithelium as substrate for indirect immunofluorescence and compared it with other substrates and with immunoblotting. Twenty-nine bullous pemphigoid sera were studied on rat bladder epithelium, monkey oesophagus, salt-split skin and with immunoblotting on human keratinocyte cultures. Indirect immunofluorescence on rat bladder epithelium proved to be more sensitive (72%) than on monkey oesophagus alone (45%) and less sensitive than on salt-split skin (97%). Rat bladder epithelium, when tested on 41 sera of a control group, showed a very high specificity: 2/41 (95%). In combination with immunoblotting on keratinocyte extracts, indirect immunofluorescence on rat bladder epithelium allowed 93% of sera to be recognized, a value close to the salt-split skin alone. Rat bladder epithelium appears to be a more sensitive substrate than monkey oesophagus for the diagnosis of bullous pemphigoid and, although less specific, it is easier and faster than using salt-split skin, which remains indispensable to distinguish bullous pemphigoid from epidermolysis bullosa acquisita.     

Distribution of monkey esophagus antigens reactive with IgA-class antibodies in the sera of dermatitis herpetiformis patients

Summary Antibodies of IgA class reactive to the lining of the smooth muscle bundles, i.e., endomysium (EmA), are present in the sera of patients with dermatitis herpetiformis and coeliac disease. These antibodies can be detected on monkey esophagus, a substrate of choice for pemphigus and pemphigoid antibodies. The amount of antigen reactive with IgA-EmA is greatest in the lower portion of the esophagus and decreases towards the uppermost part.This work was supported in part by the Summerhill Foundation and by the Polish Academy of Medicine     

Desmosomal antigens are not recognized by the majority of pemphigus autoimmune sera

Sera from 7 patients with pemphigus vulgaris and both mouse and rabbit antisera against bovine epidermal desmosomes contained antibodies that bound to cell surface components of the spinous layer of bovine epidermis. The antidesmosomal sera showed significant binding to purified desmosomal proteins in an enzyme-linked immunosorbent assay (ELISA). Two of 7 pemphigus sera bound to desmosomal protein-coated microtiter plates at low dilution titers. Two of 6 normal human sera also bound to desmosomal protein-coated microtiter plates at titers comparable to those of the pemphigus sera. Indirect immunofluorescent labeling of frozen sections of monkey esophagus revealed striking differences in the distribution of pemphigus antigens and desmosomal constituents. Pemphigus antisera produced rather uniform fluorescence around the borders of spinous cells of the esophageal epithelium, while anti-desmosomal antibodies bound in a punctate pattern. Anti-desmosomal antibodies labeled cells of the basal layer in a strongly punctate pattern. Only 1 pemphigus serum appreciably labeled basal cells. Two of 3 anti-desmosomal antisera bound avidly in the upper differentiating layers of the epithelium. Pemphigus antibodies did not. Pemphigus sera that reacted with desmosomal proteins in ELISA were absorbed by affinity chromatography on immobilized desmosomal proteins. This treatment did not alter the immunofluorescent labeling patterns produced by these sera. From these results we conclude that the pemphigus autoantibodies studied here bind to epithelial cell surface antigens which are distinguishable from the structural components of desmosomes.     

Serum soluble nucleosome and the broad family of antinucleosome antibodies are associated with organ and tissue damage in systemic lupus erythematosus in a Chinese population

Nucleosomes and the broad family of antinucleosome antibodies (ANAs; anti-double-stranded DNA, antihistone and antinucleosome antibodies) may contribute to the pathogenesis of systemic lupus erythematosus (SLE). We collected clinical information on 90 patients with SLE and 73 healthy volunteers and measured serum levels of the ANA family using a double-sandwich ELISA. The results showed that the levels of serum nucleosomes of patients with SLE was significantly lower and the levels of ANA were significantly higher than healthy controls. Negative correlations between serum nucleosomes and ANA, and positive correlations between individual ANAs were found. Patients with SLE with positive ANA had a significantly higher frequency of renal disorders than those with negative ANA. Determination of serum nucleosomes and ANAs contributes to SLE monitoring.     

In vitro effects of pemphigus antibodies on skin

Acantholysis occurring in rhesus monkey skin explants cultured on sera of 9 pemphigus patients was found to be largely dependent on the titre of intercellular antibody, and not on the participation of complement. Skin explants cultured on normal human sera and pemphigoid sera failed to give rise to intercullular staining or to develop lesions. Six of eight 'negative' pemphigus sera with intercellular antibody titres of less than 20 on skin (and titres ranging from 20 to 160 on monkey esophagus) reacted with the skin explants as revealed by direct immunofluorescence with an anti-IgG conjugate. The binding of antibodies from 3 of these 6 reactive sera resulted in some pathological changes in the explants. At least two of these 3 'negative' sera came from pemphigus patients with skin lesions.     

Circulating IgA anti-basement membrane antibodies in linear dermatitis herpetiformis (Duhring): immunofluorescence and immunoelectronmicroscopic studies.

IgA deposits were observed by direct immunofluorescence in linear distribution along the basement membrane zone in a case of dermatitis herpetiformis (Duhring). In addition, in the serum of the same patient circulating IgA antibasement membrane zone antibodies were detected by indirect immunofluorescence, utilizing normal human skin and monkey esophagus as substrates. The ultrastructural localization of in vivo-bound IgA and circulating IgA antibasement membrane zone antibodies fixed to substrate tissue in vitro was found to be in the uppermost strata of the dermis below the basal lamina.     

Clinical significance and correlation of antinuclear antibodies and anti-DNA antibodies.

The clinical significance and correlation of antinuclear antibodies (ANA) and anti-DNA antibodies was studied using 142 ANA positive sera from different patients having various diseases. High titers of ANA were found briefly in systemic lupus erythematosus and sometimes in scleroderma or mixed connective tissue disease. The peripheral pattern of ANA was seen exclusively in systemic lupus erythematosus and occasionally in mixed connective tissue disease. Anti-DNA antibodies could be found in systemic lupus erythematosus, discoid lupus erythematosus, scleroderma, chronic active hepatitis, but a high titer of anti-DNA (over 60 unit/ml) was present only in patients with systemic lupus erythematosus, especially those having lupus nephritis. There was little correlation between ANA and anti-DNA antibodies.     

Diagnosis of systemic lupus erythematosus. Importance of antinuclear antibody titers and peripheral staining patterns.

Titers and patterns of antinuclear antibodies (ANA) in sera from 134 normal blood donors, 20 patients with rheumatoid arthritis, 15 patients with systemic scleroderma, and 32 patients with diagnosed or suspected systemic lupus erythematosus (SLE) were studied. The difference between the findings with sera of patients with SLE and normal subjects in terms of high (greater than 160) titers of ANA was greater than in terms of peripheral staining patterns. However, in comparing sera from patients with SLE with sera from patients with other connective tissue diseases, greater differences were found in the incidence of peripheral patterns of ANA compared to differences in the frequency of high ANA titers. Maximum specificity in the diagnosis of SLE was achieved when both titers and patterns of ANA were considered.     

Pemphigus foliaceus associated with absence of intercellular antigens in lower layers of epidermis.

A patient with pemphigus foliaceus was found to lack normal intercellular (IC) antigens in the lower layers of the epidermis. This was evidenced by the inability of her own IC antibodies, or of those from other patients with pemphigus vulgaris, to bind to the IC substance in the lower layers of her epidermis; whereas these same antibodies reacted to IC antigens in all layers of normal allogeneic skin and monkey and guinea pig esophagus. Lack of IC antigens in the lower layers of the epidermis may account for the subcorneal location of bullae in some patients with pemphigus foliaceus.     

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.     

Antinuclear antibodies in patients with polymorphic light eruption: a long-term follow-up study

BACKGROUND: Previous studies have shown elevated titres of antinuclear antibodies (ANA) in 2.9-19% of patients with polymorphic light eruption (PLE). A diagnosis of lupus erythematosus (LE) was finally established in some of these ANA-positive patients. OBJECTIVES: To investigate whether the presence of ANA in patients with PLE merely represents an epiphenomenon or is associated with an increased risk of eventual progression to LE. METHODS: We identified 472 patients with PLE who had received prophylactic photo(chemo)therapy between 1986 and 2003 and were routinely tested for the presence of ANA. All ANA-positive (ANA titre of>or=1:80) patients were asked to attend for a follow-up examination comprising a medical history, complete skin inspection and a detailed laboratory analysis including ANA and antibodies against extractable nuclear antigens. RESULTS: Of all the patients, 55 (11.7%) were found to be ANA positive on one or several occasions, and three (0.6%) also had antibodies to SS-A/Ro. Thirty-nine (71%) of all ANA-positive patients including all Ro+ subjects were available for follow-up after a median follow-up period of 8 years (interquartile range 5-11.5). Twenty-five patients showed persistence of ANA positivity with a median titre of 1:160 (range 1:80-1:640), whereas in 14 patients ANA titres had returned to normal levels. None of the patients revealed additional clinical, histopathological or laboratory abnormalities suggestive of LE. CONCLUSIONS: After a median follow-up period of 8 years none of the ANA-positive patients developed LE. Our findings indicate that PLE is a benign disease without tendency to progress to LE.     

The prevalence of antinuclear antibodies in patients with apparent polymorphic light eruption

Polymorphic light eruption (PLE) is a very common photosensitive disorder, the most important differential diagnosis of which is lupus erythematosus (LE). One-hundred and forty-two patients with PLE were screened for circulating antinuclear (ANA), Ro and La antibodies over a 2-year period. Results were negative in 66 patients. Sixty-two patients had low-titre ANA of various patterns, ranging from trace to 1/80 without evidence of LE although one later developed subacute cutaneous LE. Fourteen had more significant findings, six with ANA ranging from 1/160 to 1/1280 but no anti-Ro antibodies, four with ANA ranging from 1/160 to 1/1280 and also with anti-Ro antibodies and four patients with anti-Ro antibodies but low-titre ANA, one of whom later developed discoid LE. Three of these 14 patients fulfilled the American Rheumatism Association criteria for the diagnosis of systemic LE, but it was not certain in any of the patients whether the PLE-like rash represented cutaneous LE or coincidental PLE. However the overall 10% incidence of definite or possible LE in patients with suspected PLE suggests that all PLE patients should be screened for LE.     

用Hep-2细胞作抗原检测抗核抗体——FANA试验和FLISA

本文提出在酶标板小孔内培养Hep-2细胞的方法制作抗原,用于免疫酶联吸附检测法(ELISA)测定血清抗核抗体(ANA),并用同一抗原作FANA试验对两种方法检测ANA的敏感性进行比较,共检测血清22份(SLE8份、其他疾病9份,正常对照5份)。检测结果表明二种试验方法的特异性一致,ELISA检测血清ANA的敏感性比FANA试验显著提高,该方法特异性强、敏感性高、简便、更适用于微量法。     

Diagnostic features of pemphigus vulgaris in patients with bullous pemphigoid. Molecular analysis of autoantibody profile

BACKGROUND: The simultaneous presence of features of pemphigus vulgaris (PV) in patients with bullous pemphigoid (BP) has previously been reported in the literature. OBJECTIVE: The purpose of this retrospective study is to present 13 patients with an initial diagnosis of BP, who subsequently demonstrated coexistent serological features of both BP and PV. METHODS: The following information on each patient was documented, at the time of initial diagnosis: clinical profile on presentation, histology, direct immunofluorescence, indirect immunofluorescence (IIF) using monkey esophagus as substrate, salt-split skin (SSS) and an immunoblot assay. Since all 13 patients failed to respond to conventional systemic therapy, intravenous immunoglobulin (IVIg) was used as an alternative treatment modality. Prior to initiating IVIg therapy, in all 13 patients, serological studies were performed. In addition to IIF using monkey esophagus, an immunoblot assay and SSS, an enzyme-linked immunosorbent assay (ELISA) was performed to detect antibodies to desmogleins. These different assays were done to identify pathological autoantibodies typical of BP and PV. A control group of 25 healthy normal individuals, 37 patients with BP, 17 patients with PV and 12 patients with pemphigus foliaceus were used for comparison of serological studies. RESULTS: At the time of initial presentation, histological and immunopathological studies confirmed the diagnosis of BP in all 13 patients. Prior to the initiation of IVIg therapy, results of IIF using monkey esophagus as substrate demonstrated high levels of anti-intercellular cement substance (anti-ICS) or antikeratinocyte cell surface antibody. Sera of all 13 patients on SSS bound to the epidermal side of the split. In an immunoblot, using bovine gingival lysate as substrate, sera of 6 patients bound to both a 230-kD (BP Ag1) and 180-kD protein (BP Ag2), while 7 sera bound to only a 230-kD protein. All 13 patients had high levels of antibodies to desmoglein 3 on ELISA. In a pilot experiment, the anti-ICS antibody in sera from 6 random patients was found to be predominantly of the IgG4 subclass. Use of IVIg resulted in an effective clinical response and the maintenance of a prolonged clinical remission. CONCLUSION: In patients with BP, who are nonresponsive to conventional therapy, the presence of two autoimmune diseases or a dual diagnosis should be considered.     

Juvenile linear scleroderma associated with serologic abnormalities

We investigated 24 juvenile cases of linear scleroderma for the presence of systemic disease and serologic abnormalities. Thirteen of 24 patients had antinuclear antibodies (ANA) at titers of 1:40 or greater. Rheumatoid factor (titers greater than or equal to 1:20) was detected in seven of 17 patients tested, five of whom also had ANA. Two of five patients with ANA and rheumatoid factor had systemic diseases, such as nephritis and Raynaud's phenomenon. One patient with ANA developed typical dermatomyositis. Consequently, patients with linear scleroderma may be at some risk for developing systemic collagen-vascular diseases. On initial presentation, patients with linear scleroderma should give a complete history and receive a thorough physical examination as well as undergo laboratory evaluations for the presence of ANA and rheumatoid factor. Long-term observation with periodic reevaluation is appropriate for many members of this group.     

SLE脑损害危险因素的临床初步分析

为初步探讨发生SLE脑损害（NPLE）的危险因素，对17例NPLE患者及29例无脑损害的SLE（非NPLE）患者进行临床表现及实验室检查结果方面的统计学比较，并分析NPLE与各临床特点之间的相关性。结果显示，NPLE病组发热、血小板减少、抗Sm抗体阳性、脑脊液（CSF）抗核抗体（ANA）阳性、CSF蛋白增加及氯化物减少的发生率显著高于非NPLE组（P＜0．05），而盘状红斑发生率则显著低于非NPLE组（P＜0．05）；发热、血小板减少、抗Sm抗体阳性、CSF ANA阳性、CSF蛋白增加及氯化物减少可能是发生NPLE的危险因素，盘状红斑及抗Ro抗体阳性可能是NPLE的保护性因素。