Molecular and structural basis of anti-DNA antibody specificity for pyrrolated proteins.

Anti-DNA antibodies (Abs), serological hallmarks of systemic lupus erythematosus (SLE) and markers for diagnosis and disease activity, show a specificity for non-nucleic acid molecules, such as N-pyrrolated proteins (pyrP) containing Nε-pyrrole-L-lysine (pyrK) residues. However, the detailed mechanism for the binding of anti-DNA Abs to pyrP remains unknown. In the present study, to gain structural insights into the dual-specificity of anti-DNA Abs, we used phage display to obtain DNA-binding, single-chain variable fragments (scFvs) from SLE-prone mice and found that they also cross-reacted with pyrP. It was revealed that a variable heavy chain (VH) domain is sufficient for the recognition of DNA/pyrP. Identification of an antigenic sequence containing pyrK in pyrP suggested that the presence of both pyrK and multiple acidic amino acid residues plays important roles in the electrostatic interactions with the Abs. X-ray crystallography and computer-predicted simulations of the pyrK-containing peptide-scFv complexes identified key residues of Abs involved in the interaction with the antigens. These data provide a mechanistic insight into the molecular basis of the dual-specificity of the anti-DNA Abs and provide a basis for therapeutic intervention against SLE.

An Emerging Role for Anti-DNA Antibodies in Systemic Lupus Erythematosus.

Anti-DNA antibodies are hallmark autoantibodies produced in systemic lupus erythematosus (SLE), but their pathogenetic role is not fully understood. Accumulating evidence suggests that some anti-DNA antibodies enter different types of live cells and affect the pathophysiology of SLE by stimulating or impairing these cells. Circulating neutrophils in SLE are activated by a type I interferon or other stimuli and are primed to release neutrophil extracellular traps (NETs) on additional stimulation. Anti-DNA antibodies are also involved in this process and may induce NET release. Thereafter, they bind and protect extracellular DNA in the NETs from digestion by nucleases, resulting in increased NET immunogenicity. This review discusses the pathogenetic role of anti-DNA antibodies in SLE, mainly focusing on recent progress in the two research fields concerning antibody penetration into live cells and NETosis.

Increased binding of anti-dsDNA antibodies to short oligonucleotides modified with topoisomerase I reveals a potential new enzyme function independent from DNA relaxation.

OBJECTIVE: Topoisomerase I (topo I) is a highly conserved enzyme which is known to reduce torsional stress at double-stranded (ds) DNA. Torsional stress induced by supercoiling of dsDNA requires either very long dsDNA existing in genomic DNA or circulation as presented in plasmid DNA. To enable DNA relaxation, topo I induce a transient single-strand break followed by stress-relieving rotation of the released DNA strand. Our group found by serendipity that the topo I inhibitor irinotecan is able to suppress murine systemic lupus erythematosus (SLE), an autoimmune disease which is characterized by the existence of pathogenic anti-dsDNA antibodies (abs). As a possible mechanism we demonstrated in the absence of immunosuppression an increased binding of anti-dsDNA abs to long genomic or circulated plasmid dsDNA modified with topo I. RESULTS: Here we show that this effect requires active site tyrosine of topo I which is known to facilitate DNA relaxation activity. Moreover, topo I enhanced anti-dsDNA abs binding to short linear oligonucleotides down to a size of 42 bp. Since oligonucleotides of such length are devoid of torsional stress and relaxation respectively, our results suggest a new and unknown function for the enzyme topo I.

Anti-Ro/SSA and/or anti-La/SSB antibodies are associated with adverse endometrial status.

PROBLEM: Anti-Ro/SSA and/or anti-La/SSB (anti-SSA/SSB) antibodies impair pregnancy outcomes, including embryo implantation and pregnancy maintenance. Optimal endometrial immune status is essential for successful pregnancy. However, whether these antibodies affect endometrial immune status is still unclear. Menstrual blood can be collected non-invasively, differs from peripheral blood, and can reflect the endometrial immune status. We herein focused on changes in subsets of natural killer (NK) cells and T cells in menstrual blood. METHODS OF STUDY: Menstrual blood samples from anti-SSA/SSB antibody-positive (n = 18) and anti-SSA/SSB antibody-negative control (n = 8) women were collected, and the profile of lymphocyte subsets was analyzed. The phenotypes of menstrual blood CD49a- and CD49a+ NK cells were compared, and the abundance of NK and CD49a+ NK cells in menstrual blood of the two groups was assessed. Additionally, CD4+T and CD8+T cells were investigated for their ability to secret functional cytokines. RESULTS: Menstrual blood contains a large number of (mostly CD49a+) NK cells, which exhibited a more exhausted phenotype with greater expression of the immune checkpoint molecules programmed cell death protein 1 and Tim-3 compared to CD49a- conventional NK cells. CD8+T cells in menstrual blood from anti-SSA/SSB antibody-positive women produced a stronger response after stimulation, accompanied by increased interferon-γ, tumor necrosis factor-α, and granzyme B secretion (P < 0.05, separately). CONCLUSION: Menstrual blood cell composition differs between anti-SSA/SSB antibody-positive women and normal controls, especially in terms of CD49a+ NK cells and CD8+T cells, unbalancing the immune cell composition and inflammatory uterine microenvironment and possibly contributing to adverse pregnancy outcomes.

The association between autoantibody types and salivary gland hypofunction in patients with primary Sjögren's syndrome.

BACKGROUND: This study analyzed the association between autoantibody types and salivary gland hypofunction in patients with primary Sjögren's syndrome (pSS). METHODS: A retrospective analysis was performed on patients who visited the Department of Orofacial Pain and Oral Medicine at Yonsei University Dental Hospital from January 1, 2010 to May 31, 2021, and who were diagnosed with pSS. Out of 191 patients who fulfilled the 2016 American College of Rheumatology/European League Against Rheumatism classification criteria, 50 were positive for both anti-Ro/SSA and anti-La/SSB, whereas 97 had anti-Ro/SSA but not anti-La/SSB antibodies. Forty-four patients for whom neither anti-Ro/SSA nor anti-La/SSB antibodies were found were diagnosed with Sjögren's syndrome by minor salivary gland biopsy. RESULTS: The anti-Ro/SSA antibody-positive group showed higher rheumatoid factor (RF) levels than the anti-Ro/SSA antibody-negative group. The anti-La/SSB antibody-positive group showed lower unstimulated whole saliva (UWS), stimulated whole saliva (SWS), higher erythrocyte sedimentation rate and RF level than the anti-La/SSB antibody-negative group. In addition, the group with both anti-Ro/SSA and anti-La/SSB antibodies showed lower UWS than the group with only anti-Ro/SSA antibodies. However, there were no significant differences in UWS or SWS after taking pilocarpine, and C-reactive protein. CONCLUSIONS: UWS and SWS were lower when a patient was positive for anti-La/SSB, showing that anti-La/SSB is more likely to be involved in salivary gland hypofunction than anti-Ro/SSA in patients with pSS. Therefore, performing laboratory tests, including anti-La/SSB, helps predict the prognosis of salivary gland function in patients with suspected pSS.

A Small Step, a Giant Leap: Somatic Hypermutation of a Single Amino Acid Leads to Anti-La Autoreactivity.

The anti-La mab 312B, which was established by hybridoma technology from human-La transgenic mice after adoptive transfer of anti-human La T cells, immunoprecipitates both native eukaryotic human and murine La protein. Therefore, it represents a true anti-La autoantibody. During maturation, the anti-La mab 312B acquired somatic hypermutations (SHMs) which resulted in the replacement of four aa in the complementarity determining regions (CDR) and seven aa in the framework regions. The recombinant derivative of the anti-La mab 312B in which all the SHMs were corrected to the germline sequence failed to recognize the La antigen. We therefore wanted to learn which SHM(s) is (are) responsible for anti-La autoreactivity. Humanization of the 312B ab by grafting its CDR regions to a human Ig backbone confirms that the CDR sequences are mainly responsible for anti-La autoreactivity. Finally, we identified that a single amino acid replacement (D > Y) in the germline sequence of the CDR3 region of the heavy chain of the anti-La mab 312B is sufficient for anti-La autoreactivity.

Ultrasonographic characteristics of major salivary glands in anti-centromere antibody-positive primary Sjögren's syndrome.

PURPOSE: To investigate salivary gland ultrasonography (SGUS) findings in primary Sjögren's syndrome (pSS) patients positive for the anti-centromere antibody (ACA) and compare these with those in ACA-negative pSS patients. METHODS: We analyzed demographic, clinical, laboratory, and SGUS data of pSS patients who fulfilled the 2002 American-European Consensus Group classification criteria for pSS. SGUS findings of four major salivary glands (bilateral parotid and submandibular glands) were scored in five categories and compared between ACA-positive and ACA-negative pSS patients. Linear regression analysis was performed to elucidate the factors associated with SGUS score. RESULTS: In total, 121 pSS patients were enrolled (19, ACA-positive). The ACA-positive patients were older (67.0 vs 58.0 years, P = 0.028), whereas anti-Ro/SSA and anti-La/SSB positivity was more prevalent in the ACA-negative group (89.2% vs 21.1%, P < 0.001, and 47.1% vs 10.5%, P = 0.007, respectively). The total SGUS and hypoechoic area scores were lower in ACA-positive patients (16.0 vs 23.0, P = 0.027, and 4.0 vs 7.0, P = 0.004, respectively). In univariate regression analysis, being positive for unstimulated salivary flow rate (USFR < 1.5 ml/15 min), anti-Ro/SSA, and rheumatoid factor were positively associated whereas ACA positivity was negatively associated with the SGUS score. In multivariate regression analysis, being positive for USFR, anti-Ro/SSA, and rheumatoid factor showed significant association with the SGUS score. CONCLUSIONS: ACA-positive pSS patients showed a lower SGUS score than ACA-negative patients, which was especially prominent in the hypoechoic area component.

The Binding Mechanisms of Antibodies to DNA from Healthy Subjects and Patients with Systemic Lupus Erythematosus: The Role of Monogamous Bivalency and Fc Dependence.

Abs to DNA (anti-DNA) are a unique population of Abs that bind structural determinants on the DNA molecule. In systemic lupus erythematosus (SLE), anti-DNA Abs bind to conserved antigenic determinants, with the phosphodiester backbone being the most likely. In contrast, otherwise healthy subjects (HS) express anti-DNA that bind selectively to nonconserved sites on certain bacterial and viral DNA. As shown previously, SLE anti-DNA bind by a mechanism termed Fc-dependent monogamous bivalency. In this mechanism, both Fab sites interact with determinants on the same extended DNA molecule, reflecting the low affinity of each Fab site; the requirement for the Fc region suggests some contribution of the C region to increase avidity. In this study, we investigated whether anti-DNA from HS also bind to bacterial DNA by Fc-dependent monogamous bivalency. For this purpose, we compared the activity of intact IgG with Fab and F(ab')2 fragments prepared from the plasmas of SLE patients and HS using ELISAs with DNA from calf thymus or Micrococcus luteus These studies showed that Fab fragments from all plasmas tested, both SLE and HS, failed to bind significantly to DNA compared with intact IgG. By contrast, some, but not all, F(ab')2 preparations from both SLE patients and HS showed binding to M. luteus DNA; F(ab')2 fragments from SLE plasmas, however, did not bind significantly to calf thymus DNA. Together, these findings suggest that although anti-DNA Abs, whether from SLE or HS, bind by monogamous bivalency, binding to bacterial DNA does not require the Fc region.

TRIM21/Ro52 - Roles in Innate Immunity and Autoimmune Disease.

TRIM21 (Ro52/SSA1) is an E3 ubiquitin ligase with key roles in immune host defence, signal transduction, and possibly cell cycle regulation. It is also an autoantibody target in Sjögren's syndrome, systemic lupus erythematosus, and other rheumatic autoimmune diseases. Here, we summarise the structure and function of this enzyme, its roles in innate immunity, adaptive immunity and cellular homeostasis, the pathogenesis of autoimmunity against TRIM21, and the potential impacts of autoantibodies to this intracellular protein.

Anti-Ro60 and anti-Ro52/TRIM21: Two distinct autoantibodies in systemic autoimmune diseases.

As iconic and important diagnostic autoantibodies, anti-Ro60 and anti-Ro52/tri-partite motif-containing 21 (TRIM21) make a common appearance in a number of systemic autoimmune disorders such as systemic lupus erythematosus (SLE). These autoantibodies often co-exist together; yet despite their close relationship, there is no evidence that they are physically linked and probably reflect a convergence of separate processes of failed immunological tolerance. Confusingly, they are sometimes classed together as the "SSA" or "Ro" autoantibody system without clear distinction between the two. In this Short Communication, we discuss the diagnostic merits for separate detection and reporting of these two autoantibodies, and discuss avenues for future research. Indeed, further insight into their fascinating origins and pathogenic roles in autoimmunity will surely shed light on how we can prevent and treat devastating autoimmune disorders.

Hydroxychloroquine alleviates the neurotoxicity induced by anti-ribosomal P antibodies.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by a wide spectrum of autoantibodies, among which anti-ribosomal P (anti-P) antibodies are considered to be closely related to the neuropsychiatric SLE (NPSLE). Hydroxychloroquine (HCQ) has been proven to be effective against a variety of autoimmune diseases and is an essential drug for the treatment of SLE. In this study, we investigated the effects of anti-ribosomal P (anti-P) antibodies on neural cells and determined whether hydroxychloroquine (HCQ) influenced the anti-P antibodies-induced changes. The results showed that the binding of anti-P antibodies with mouse neuroblastoma- 2a (N2a) cells and rat primary neurons resulted in elevated intracellular calcium levels, inducing decreased cell viability and cell apoptosis. These inhibitory effects were alleviated by HCQ in a concentration-dependent manner by reducing the intracellular calcium levels and modulating the expression of apoptotic proteins. In summary, our study demonstrates that anti-P antibodies induce neural cell damage. HCQ could ease the damage effects and may play a neuroprotective role in NPSLE.

[A case of dermatomyositis positive for anti-nuclear matrix protein 2 antibody without dermatologic symptoms].

We report a 47-year-old woman who presented with progressive myalgia, weakness in the proximal limbs, and dysphagia for a month and a half. No skin rash was observed on admission. Examination of MRI data suggested inflammatory changes in the proximal limbs and trunk muscles. Biopsy specimens from the left biceps muscle showed no perifascicular atrophy, but immunohistochemical staining revealed the presence of myxovirus resistance protein A (MxA) in myofibers, strongly suggesting dermatomyositis (DM). In addition, her serum was positive for anti-nuclear matrix protein 2 (anti-NXP-2) antibody, which is reportedly useful as a marker of DM without skin lesions. Her symptoms gradually improved upon intravenous methylprednisolone pulse therapy in conjunction with oral prednisolone, oral tacrolimus, and intravenous immunoglobulin therapy. Our findings suggest that in cases where inflammatory muscle disease is suspected, anti-NXP-2 antibody analyses should be considered for precise diagnosis, even if there are no dermatological symptoms.

Harnessing SLE Autoantibodies for Intracellular Delivery of Biologic Therapeutics.

Intracellular delivery of therapeutic antibodies is highly desirable but remains a challenge for biomedical research and the pharmaceutical industry. Approximately two-thirds of disease-associated targets are found inside the cell. Difficulty blocking these targets with available drugs creates a need for technology to deliver highly specific therapeutic antibodies intracellularly. Historically, antibodies have not been believed to traverse the cell membrane and neutralize intracellular targets. Emerging evidence has revealed that anti-DNA autoantibodies found in systemic lupus erythematosus (SLE) patients can penetrate inside the cell. Harnessing this technology has the potential to accelerate the development of drugs against intracellular targets. Here, we dissect the mechanisms of the intracellular localization of SLE antibodies and discuss how to apply these insights to engineer successful cell-penetrating antibody drugs.

The Emerging Role of Renal Tubular Epithelial Cells in the Immunological Pathophysiology of Lupus Nephritis.

Systemic lupus erythematosus (SLE) is a systemic, autoimmune disease that can involve virtually any organ of the body. Lupus nephritis (LN), the clinical manifestation of this disease in the kidney, is one of the most common and severe outcomes of SLE. Although a key pathological hallmark of LN is glomerular inflammation and damage, tubulointerstitial lesions have been recognized as an important component in the pathology of LN. Renal tubular epithelial cells are resident cells in the tubulointerstitium that have been shown to play crucial roles in various acute and chronic kidney diseases. In this context, recent progress has been made in examining the functional role of tubular epithelial cells in LN pathogenesis. This review summarizes recent advances in our understanding of renal tubular epithelial cells in LN, the potential role of tubular epithelial cells as biomarkers in the diagnosis, prognosis, and treatment of LN, and the future therapeutic potential of targeting the tubulointerstitium for the treatment of patients with LN.

Anti-nucleosome antibodies increase the risk of renal relapse in a prospective cohort of patients with clinically inactive systemic lupus erythematosus.

An important goal in the management of systemic lupus erythematosus (SLE) is the prediction of relapses. This study assesses whether anti-nucleosome antibodies (anti-NCS) increase the risk of renal relapse in inactive SLE. A prospective cohort of 115 patients with inactive SLE (M-SLEDAI ≤ 2) were followed for 12 months to assess the development of relapse (increase of M-SLEDAI ≥ 4) and specific renal flare (renal SLEDAI ≥ 4). At baseline, we identified potential risk factors for relapse, including anti-NCS. At baseline, 18 (16%) of the 115 patients with inactive SLE were anti-NCS positive. At the 12-month follow-up, anti-NCS-positive patients had a higher incidence of renal relapse compared to anti-NCS-negative patients (38.9% vs 13.4%, respectively). In Cox regression analysis, after adjusting for age, disease duration, anti-dsDNA, and immunosuppressive drugs, the presence of anti-NCS positivity at baseline increased the risk of renal relapse (HR: 5.31, 95% CI 2.03-13.92). Nevertheless, there were no differences in the incidence of other relapses in anti-NCS-positive versus anti-NCS-negative. Our results indicate that in inactive SLE, anti-NCS determination can be useful for identifying patients with a higher risk of developing renal relapse. Interestingly, this study identified that continued use of oral immunosuppressive therapy in patients with inactive SLE can reduce the risk of renal relapse.

Two of a kind? Immunological and clinical risk factors differ between recurrent implantation failure and recurrent miscarriage.

Recurrent miscarriage (RM) and recurrent implantation failure (RIF) are unsolved challenges in reproductive medicine. Whether RIF patients share the same risk factors as RM patients is a matter of debate. Besides clinical factors, immune alterations are discussed in both conditions. The scope of this study was to compare the prevalence of clinical and immunological risk factors in a large cohort of RM and RIF patients. Between 11/2011 and 02/2019, 613 RM and 185 RIF patients were included. A screening for anatomical malformations, endocrine, autoimmune, prothrombotic and parental chromosomal disorders was performed. The immune status was assessed using flow cytometry analysis of peripheral lymphocyte subpopulations and uterine natural killer cells (uNK cells) using immunohistochemistry. RM patients showed a higher rate of intrauterine adhesions and elevated antinuclear antibodies ≥ 1:160 (p < 0.05). A higher prevalence of submucous fibroids and increased factor VIII levels were observed in RIF patients (p < 0.05). The prevalence of an antiphospholipid syndrome (APLS) was low and did not differ between the two groups. RIF patients had higher numbers of peripheral regulatory T-cells (p < 0.05). Significant more RIF patients were diagnosed with elevated uNK cells (p < 0.05). Differences in clinical and immunological risk factors of RM and RIF patients reflect different entities. Lower Tregs in RM and higher uNK cells in RIF patients might be related to the previous exposure of the immune system to fetal cells. The low prevalence of an APLS indicates a potential overestimation of this factor in the pathophysiology of RM and RIF.

Injection of CD40 DNA vaccine ameliorates the autoimmune pathology of non-obese diabetic mice with Sjögren's syndrome.

BACKGROUND: Overexpression of CD40 has been reported in patients with primary Sjögren's syndrome (pSS). The increased CD40 expression promote autoimmune response and enhance inflammation in pSS. The aim of this study is to block CD40-CD154 interaction with CD40 DNA vaccine to slow the disease progression of SS in non-obese diabetic (NOD) mice. METHODS: Female NOD mice were treated with CD40 DNA vaccine, empty vector and normal saline. The salivary flow rate was measured, whereas lymphocytes infiltration in the salivary glands was assessed by histopathology. Expression of CD40 and B220 in salivary were examined by immunohistochemistry. Splenic lymphocyte phenotypes were analyzed by flow cytometry. CD40, IL-1ß, TNF-α and IL-6 levels in the salivary glands were detected by PCR. Serum anti-CD40 antibody was measured by ELISA. Serum anti-nuclear antibody (ANA) was monitored by immunofluorescence. RESULTS: NOD mice treated with CD40 DNA vaccine showed higher levels of anti-CD40 antibody compared with the controls. The expression of CD40 in the salivary glands of NOD mice in CD40 DNA vaccine group was decreased. The infiltration of lymphocytes was reduced in the salivary glands and saliva secretion was increased in the treatment group. The expression level of TNF-α and IL-6 in salivary glands were declined. The splenic dendritic cell and plasma cell populations were reduced and the level of ANA was decreased in NOD mice with CD40 DNA vaccine treatment. CONCLUSIONS: CD40 DNA vaccine inhibits the immune response and reduce inflammation in epithelial tissues SS in non-obese diabetic (NOD) mice, suggesting that CD40 DNA vaccine could be a new therapeutic approach in treatment of pSS.

Drug-induced Bullous Pemphigoid and Lupus Erythematosus Occurring under Anti-TNF-α and IL-6 Therapy in a Patient with Rheumatoid Arthritis.

A 65-year-old Japanese woman, who was diagnosed with rheumatoid arthritis and Sjögren's syndrome with various autoantibodies including anti-DNA antibody, developed bullous pemphigoid (BP) and hematological abnormalities like lupus erythematosus after adalimumab therapy. The discontinuation of adalimumab resolved those disorders but polyarthritis thereafter relapsed. The introduction of abatacept was not effective, but tocilizumab was found to be effective for polyarthritis, however, thereafter both bullous disease and severe pancytopenia developed. Discontinuation of tocilizumab was effective, but polyarthritis again developed, and baricitinib resolved it. There is an increasing number of reports of drug-induced BP and lupus erythematosus, and biologics might trigger an alteration in the pathophysiological/clinical course of rheumatic disorder.

Antinuclear antibodies in follicular fluid may reduce efficacy of in vitro fertilization and embryo transfer by invading endometrium and granular cells.

PROBLEM: The mechanism(s) by which antinuclear antibodies (ANA) induce implantation failure are not clear, and little information regarding the function of autoantibodies in reproductive tissues is available. METHODS OF STUDY: A total of 380 patients who underwent in vitro fertilization and embryo transfer (IVF-ET) were divided into control, serum positive, and follicular fluid (FF) positive groups based on the results of indirect immunofluorescence assay for ANA in the serum and FF. Immunofluorescence assay was performed to evaluate the existence of ANA in granular cells and endometrial tissues. Presence in FF of soluble apoptotic markers, including Bcl-2, Caspase-3, cleaved PARP, Cytochrome C, GAPDH, and p53, was assessed using magnetic bead based assays. RESULTS: The patients in the FF positive group had the lowest numbers of retrieved oocytes, fertilizations, and high-quality embryos. The fertilization rate, and the proportion of two pronuclear (2PN) embryos in patients in the FF positive group were significantly lower than those in the other two groups. The FF positive group also had the lowest clinical pregnancy rate, and the highest early miscarriage rate. Granulosa cells and endometrial tissues in patients in the FF positive group were ANA positive. High levels of BCL-2, Caspase-3, Cytochrome C, GAPDH, and p53 were found in the FF of patients in the FF positive group. CONCLUSIONS: Antinuclear antibodies in FF and endometrial tissues may cause imbalanced apoptosis, resulting in poor IVF-ET treatment outcomes. Local autoimmunity and cell apoptosis in reproductive tissues could be considered new therapeutic targets for improving IVF-ET treatment efficacy.

In vivo cutaneous antinuclear antibody positivity in palisaded neutrophilic and granulomatous dermatitis.

Palisaded neutrophilic and granulomatous dermatitis (PNGD) is commonly associated with underlying systemic inflammatory and neoplastic diseases, infections, and drug reactions. In vivo cutaneous antinuclear antibodies (ANA) have been described in skin biopsies from patients with known autoimmune disorders, but not previously reported in the setting of PNGD. We present two patients with systemic lupus erythematosus (SLE) and histopathologically confirmed PNGD. Direct immunofluorescence (DIF) studies revealed in vivo cutaneous ANA positivity in both patients. DIF findings in the skin mirrored serum autoantibody results. ANA positivity in skin specimens is reported as highly predictive of systemic connective tissue diseases (SCTD), although specific testing is not currently recommended as part of the laboratory work-up or diagnostic criteria for these disorders. In this case report, positive ANA results in skin biopsies of PNGD reflect the serological findings and clinical evidence of SLE in both patients. In vivo cutaneous ANA positivity is an interesting and supportive finding in PNGD in the setting of SCTD.