IgG Antinuclear Antibodies Research Articles

Deletion of the Mitochondrial Membrane Protein Fam210b Is Associated with the Development of Systemic Lupus Erythematosus.

Mitochondrial dysfunction has been increasingly recognized as a trigger for systemic lupus erythematosus (SLE). Recent bioinformatics studies have suggested Fam210b as a significant candidate for the classification and therapeutic targeting of SLE. To experimentally prove the role of Fam210b in SLE, we constructed Fam210b knockout (Fam210b-/-) mice using the CRISPR-Cas9 method. We found that approximately 15.68% of Fam210b-/- mice spontaneously developed lupus-like autoimmunity, which was characterized by skin ulcerations, splenomegaly, and an increase in anti-double-stranded DNA (anti-dsDNA) IgG antibodies and anti-nuclear antibodies(ANA). Single-cell sequencing showed that Fam210b was mainly expressed in erythroid cells. Critically, the knockout of Fam210b resulted in abnormal erythrocyte differentiation and development in the spleens of mice. Concurrently, the spleens exhibited an increased number of CD71+ erythroid cells, along with elevated levels of reactive oxygen species (ROS) in the erythrocytes. The co-culture of CD71+ erythroid cells and lymphocytes resulted in lymphocyte activation and promoted dsDNA and IgG production. In summary, Fam210b knockout leads to a low probability of lupus-like symptoms in mice through the overproduction of ROS in CD71+ erythroid cells. Thus, Fam210b reduction may serve as a novel key marker that triggers the development of SLE.

Different course and management of khat-induced autoimmune hepatitis: report on three cases

Khat is a plant that is commonly used for its stimulating effects and is chewed for its psychoactive properties. It creates feelings of euphoria that are similar to when taking amphetamines. There is an association between khat and liver injury, but the mechanism is not well known. We present three cases of khat-induced liver injury. All cases have elevated IgG and either positive antinuclear antibodies (ANA) or anti-smooth muscle antibody (ASMA); each case has a different course and requires different management. One case improved only by stopping khat, one required a short course of steroids and the last case required treatment such as that for autoimmune hepatitis (AIH).

A105 CHRONIC DIARRHOEA AND ELEVATED LIVER ENZYMES IN A PATIENT WITH AUTOIMMUNE POLYGLANDULAR SYNDROME TYPE 1

Abstract Background Autoimmune polyendocrine syndrome type 1 (APS1) is a rare inherited autoimmune disease, characterized by primary adrenocortical insufficiency and hypoparathyroidism. Pathologic mutations in the autoimmune regulator (AIRE) gene, which participates in the regulation of T-cell self-tolerance can lead to APS-1. Defective function of AIRE encourages production of multiple anti-cytokine and organ-specific autoantibodies, leading to severe autoimmune disease across multiple organs. Autoimmune enteropathy is one of the autoimmune conditions associated with APS-1. Symptoms include chronic diarrhoea and weight loss. Extraintestinal manifestations can include haemolytic anaemia and hepatitis. Purpose To report on a patient with APS-1 who exhibited exacerbations of acute-on-chronic diarrhoea and transaminitis. Method We describe the case of a 14-year-old female, the first child of healthy, first-cousin consanguineous parents, with a history of APS type 1 with a homozygous disease-causing variant in the AIRE gene (c.415C>T, p.139*), hypoparathyroidism and Addison’s disease who presented with worsening diarrhoea and unintentional weight loss (15kg) in the past year. Upper and lower endoscopies performed 10 months and 2 months prior to this admission revealed chronic gastritis and patchy active colitis, without features of a specific etiology. Enteroendocrine cells were present. Celiac disease, inflammatory bowel disease, eosinophilic gastroenteritis, and IPEX syndrome were clinically and histologically ruled out. Result(s) On admission, the patient was having five watery stools per day with associated hypocalcemia and hypokalemia which were felt to be more resistant to treatment than should be explained by her Addison’s disease alone. Albumin was preserved (46 g/L). A stool PCR test for Adenovirus was positive, though serum adenovirus PCR was negative. ALT was 231 U/L and AST was 283 U/L. IgG level was elevated (17.7 g/L), anti-nuclear antibody (ANA) was positive (1:320), and anti-smooth muscle antibody (ASMA) was also positive (1:160). It was therefore suspected that he had autoimmune enteropathy and autoimmune hepatitis, and a liver biopsy was performed as well as upper and lower endoscopies. Liver biopsy demonstrated only minimal macrosteatosis (less than 5%) but no evidence of hepatitis. No abnormal endoscopic findings were observed. Histology showed no typical features of autoimmune enteropathy. Enteroendocrine cells were noted in both small and large intestinal biopsies. Her elevated liver enzymes and diarrhea were resolved spontaneously over a 2-week period. Conclusion(s) We present a patient with APS-1 with acute-on-chronic diarrhoea and transaminitis with elevated IgG and positive ANA and ASMA. Endoscopy and liver biopsy did not reveal evidence of autoimmune enteropathy or autoimmune hepatitis. This case raises the importance of maintaining a broad differential diagnosis when diarrhoea is encountered in APS-1 as this may occur for reasons outside of autoimmune enteroendocrine cell loss. Please acknowledge all funding agencies by checking the applicable boxes below None Disclosure of Interest None Declared ENDOSCOPY, TECHNOLOGY & IMAGING

Antinuclear antibodies produced in HLA-DR transgenic humanized mice developed chronic graft-versus-host disease

BackgroundChronic graft versus host disease (GVHD) has been reported in humanized mice after the implantation of human hematopoietic stem cells (hu-HSC). As such, humanized mice have been applied to a mouse model of chronic GVHD; however, B-cell activation and autoantibody production did not occur, and the clinical features of chronic GVHD were not sufficiently reproduced. The purpose of this study was to establish an improved humanized mouse model with chronic GVHD using HLA-DR transgenic NOD/Shi-scid, IL-2RγKO (NOG) mice. MethodsCD34-positive cells were isolated from blood extracted from HLA-DRB1∗0405-positive umbilical cords using magnetic cell isolation. Then these were transplanted into NOG-Iab KO, HLA-DR 0405 Tg mice aged 8–16 weeks. GVHD symptoms were observed 26 weeks after transplantation. Histological findings of the skin, lung, liver, and spleen were compared with those of non-humanized mice. Antinuclear antibodies (ANA) were measured by indirect immunofluorescence using sera isolated 26 weeks after transplantation. ResultsAlthough GVHD symptoms were not observed in humanized (hu-HSC) NOG-Iab KO, HLA-DR 0405 Tg mice during the observation period, histological findings of human T-cell infiltration were observed in the skin, liver, and lung, suggesting that GVDH was present; human tingible body macrophages or clusters of BCL-6-positive human B-cells were observed in the spleen. Furthermore, human IgG ANA with peripheral or homogeneous staining patterns were also detected in the sera. ConclusionHu-HSC NOG-Iab KO, HLA-DR 0405 Tg mice differed from conventional models in terms of B-cell activation and ANA production. This study is the first to report on B-cell activation and autoantibody production in humanized mice with chronic GVHD, suggesting that hu-HSC NOG-Iab KO, HLA-DR 0405 Tg mice could be applied to a new humanized mouse model of chronic GVHD.

Distribution of IgG subclass anti-nuclear antibodies (ANAs) in systemic lupus erythematosus.

Our study purpose was to detect the distribution of anti-nuclear antibody (ANA) IgG subclasses in patients with systemic lupus erythematosus (SLE) and to evaluate their influence on the inflammatory process in SLE. We determined the serum levels of ANA IgG subclasses from 70 SLE patients, 25 patients with other autoimmune diseases (OAD), and 25 healthy controls using ELISA. The serum level of total ANA IgG and the avidity of ANA IgG, dsDNA IgG, and dsDNA IgG subclasses were analysed by ELISA. The results indicated that levels of four ANA IgG subclasses (IgG1, IgG2, IgG3 and IgG4) and total IgG were significantly higher in SLE patients than in OAD patients and healthy controls (p < 0.001). Moreover, the level of each ANA IgG subclass and the prevalence of high-avidity IgG ANAs (HA IgG ANAs) were significantly higher in the active cases than in the inactive cases of SLE and LN. Furthermore, level of ANA IgG subclasses decreased as level of dsDNA IgG subclasses decreased in 30 patients with SLE. In comparison, ANA IgG3 was significantly effective in high-dose prednisone combined with hydroxychloroquine (p = 0.025). Additionally, it revealed that level of dsDNA IgG had a significant influence on four ANA IgG subclasses, especially on ANA IgG3 (β coefficient = 0.649, p < 0.001). Level of ANA IgG3 was also positively related to the serum level of dsDNA IgG (r = 0.729, p < 0.001) and RAI of HA IgG ANAs (r = 0.504, p < 0.001). However, the level of ANA IgG4 was positively related to the serum level of albumin (r = 0.572, p < 0.001) and RAI of HA IgG ANAs (r = 0.549, p < 0.001). Moreover, the results revealed that cutaneous and renal involvement were mainly associated with the ANA IgG1 and IgG4 subclasses. Although, arthritic involvement was mainly associated with ANA IgG3. First, we demonstrated that the ANA IgG subclasses were diagnostic tools in SLE patients. Furthermore, HA IgG ANAs might affect the distribution of ANA IgG3 and IgG4. Moreover, ANA IgG3 might play a particular role in the activity of SLE disease and therapy. Therefore, an altered ANA IgG subclass distribution might be a risk factor influencing the inflammatory process in SLE.

Autoantibodies in outbred Swiss Webster mice following exposure to gold and mercury

Exposure to heavy metals may have toxic effects on several human organs causing morbidity and mortality. Metals may trigger or exacerbate autoimmunity in humans. Inbred mouse strains with certain H-2 haplotypes are susceptible to xenobiotic-induced autoimmunity; and their immune response to metals such as mercury, gold, and silver have been explored. Serum antinuclear antibodies (ANA), polyclonal B-cell activation, hypergammaglobulinemia and tissue immune complex deposition are the main features of metal-induced autoimmunity in inbred mice. However, inbred mouse strains do not represent the genetic heterogeneity in humans. In this study, outbred Swiss Webster (SW) mice exposed to gold or mercury salts showed immune and autoimmune responses. Intramuscular injection of 22.5 mg/kg.bw aurothiomalate (AuTM) induced IgG ANA in SW mice starting after 5 weeks that persisted until week 15 although with a lower intensity. This was accompanied by elevated serum levels of total IgG antibodies against chromatin and total histones. Exposure to gold led to development of serum IgG autoantibodies corresponding to H1 and H2A histones, and dsDNA. Both gold and mercury induced polyclonal B-cell activation. Eight mg/L mercuric chloride (HgCl2) in drinking water, caused IgG antinucleolar antibodies (ANoA) after 5 weeks in SW mice accompanied by immune complex deposition in kidneys and spleen. Serum IgG antibodies corresponding to anti-fibrillarin, and anti-PM/Scl-100 antibodies, were observed in mercury-exposed SW mice. Gold and mercury trigger systemic autoimmune response in genetically heterogeneous outbred SW mice and suggest them as an appropriate model to study xenobiotic-induced autoimmunity.

Assessment of a high-avidity IgG ANAs for the diagnosis and activity prediction of systemic lupus erythematosus.

Our aim was to investigate the prevalence value of a high-avidity antinuclear antibody (ANA) of the IgG isotype (HA IgG ANA) compared with that of ANAs of other isotypes in patients with systemic lupus erythematosus (SLE) and to assess the associations of HA IgG ANA with the activity of SLE and lupus nephritis. We retrospectively analyzed clinical and laboratory data from subjects. Blood samples were acquired from 101 SLE patients, 67 patients with other autoimmune diseases, and 65 healthy donors. The levels of HA IgG ANA and other isotype ANAs were measured by indirect immunofluorescence (IIF). The prevalence and diagnosis value of HA IgG ANA and other antibodies in SLE patient were tested. The advantage of HA IgG ANA compared with HA anti-dsDNA antibodies IgG (HA dsDNA IgG) was verified by ELISA. We monitored the relative avidity indexes (RAIs) of HA IgG ANA and HA dsDNA IgG at 3 time points after the start of treatment in the same individuals with SLE. The prevalence of HA IgG ANA was significantly higher in active cases than in inactive cases of SLE and LN, which is consistent with data for IgG ANAs, anti-dsDNA IgG antibodies, low C3 levels, low C4 levels, and anti-C1q antibodies. HA IgG ANA showed moderate sensitivity and specificity (80% and 81.3%) for discriminating active and inactive SLE cases. However, HA IgG ANA showed no significant differences among the different clinical manifestations of SLE. Compared with that of HA dsDNA IgG, the RAI of HA IgG ANA was positively related to SLEDAI scores after treatment at 0, 1, and 3months (r = 0.6813, p = 0.0026; r = 0.5972, p = 0.0114; r = 0.4817, p = 0.0474). First, we demonstrated that HA IgG ANA was a reliable diagnostic tool in SLE patients. Furthermore, HA IgG ANA was supposed to be more appropriate for identifying the activity of SLE compared with HA dsDNA IgG. In summary, HA IgG ANA may be a new biomarker for diagnosing SLE and identifying SLE activity. Key Points • We first introduced the concept of a "high-avidity IgG ANA (HA IgG ANA)" that could distinguish between the early stage of SLE and SLE that had been active for some time. • The relative avidity indexes (RAIs) of HA IgG ANA and HA dsDNA IgG were presented and applied here to evaluate the avidities of antibodies involved in SLE. • In our study, we confirmed the value of HA IgG ANA in diagnosing SLE. In addition, HA IgG ANA was more appropriate for identifying the activity of SLE than was HA dsDNA IgG. • In conclusion, HA IgG ANA could be a potential biomarker for the assessment of the prognosis of SLE activity.

Patterns of ANA+ B cells for SLE patient stratification.

IgG antinuclear antibodies (ANAs) are a dominant feature of several autoimmune diseases. We previously showed that systemic lupus erythematosus (SLE) is characterized by increased ANA+ IgG plasmablasts/plasma cells (PCs) through aberrant IgG PC differentiation rather than an antigen-specific tolerance defect. Here, we aimed to understand the differentiation pathways resulting in ANA+ IgG PCs in SLE patients. We demonstrate distinct profiles of ANA+ antigen-experienced B cells in SLE patients, characterized by either a high frequency of PCs or a high frequency of IgG+ memory B cells. This classification of SLE patients was unrelated to disease activity and remained stable over time in almost all patients, suggesting minimal influence of disease activity. A similar classification applies to antigen-specific B cell subsets in mice following primary immunization with T-independent and T-dependent antigens as well as in lupus-prone mouse models (MRL/lpr and NZB/W). We further show that, in both lupus-prone mice and SLE patients, the classification correlates with the serum autoantibody profile. In this study, we identified B cell phenotypes that we propose reflect an extrafollicular pathway for PC differentiation or a germinal center pathway, respectively. The classification we propose can be used to stratify patients for longitudinal studies and clinical trials.

Systemic translocation of Staphylococcus drives autoantibody production in HIV disease

BackgroundIncreased autoreactive antibodies have been reported in HIV disease; however, the mechanism accounting for autoantibody induction in HIV remains unknown.ResultsHerein, we show that seasonal influenza vaccination induces autoantibody production (e.g., IgG anti-nuclear antibody (ANA) and anti-double-stranded DNA antibody (anti-dsDNA)) in some viral-suppressed antiretroviral therapy (ART)-treated HIV+ subjects, but not in healthy controls. These autoantibodies were not derived from antigen-specific B cells but from activated “bystander” B cells analyzed by single-cell assay and by study of purified polyclonal ANAs from plasma. To explore the mechanism of autoantibody generation in HIV+ subjects, plasma level of microbial products, gene expression profile of B cells, and B cell receptor (BCR) repertoires were analyzed. We found that autoantibody production was associated with increased plasma level of microbial translocation; the patients with high autoantibodies had skewed B cell repertoires and upregulation of genes related to innate immune activation in response to microbial translocation. By analyzing circulating microbial 16S rDNA in plasma, the relative abundance of Staphylococcus was found to be associated with autoantibody production in HIV+ subjects. Finally, we found that injection of heat-killed Staphylococcus aureus promoted germinal center B cell responses and autoantibody production in mice, consistent with the notion that autoantibody production in HIV+ patients is triggered by microbial products.ConclusionsOur results showed that translocation of Staphylococcus can promote B cell activation through enhancing germinal center response and induces autoantibody production. It uncovers a potential mechanism linking microbial translocation and autoimmunity in HIV+ disease and provides a strong rationale for targeting Staphylococcus to prevent autoantibody production.

Loss of an IgG plasma cell checkpoint in patients with lupus

IgG antinuclear antibodies (ANAs) are a feature of several autoimmune diseases. These antibodies arise through defects in central or peripheral tolerance checkpoints. The specific checkpoints breached in patients with autoimmune disease are not fully understood. We sought to study whether autoreactive plasma cells in lupus models and patients with systemic lupus erythematosus (SLE) arise as a consequence of defective antigen-specific selection or a global enhancement of IgG plasma cell differentiation. Weoptimized and validated a novel technique to detect naturally occurring ANA+ B cells and plasma cells. We observed a major checkpoint for generation of ANA+ IgG+ plasma cells in both nonautoimmune mice and healthy human subjects. Interestingly, we observed increased numbers of ANA+ IgG+ plasma cells despite normal tolerance checkpoints in immature and naive B cells of lupus-prone MRL/lpr and NZB/W mice, as well as patients with SLE. This increase was due to increased numbers of total IgG+ plasma cells rather than lack of selection against ANA+ plasma cells. Using a method that permits quick and accurate quantification of autoreactive B cells and plasma cells invivo within a native B-cell repertoire in mice and human subjects, we demonstrate the importance of a checkpoint that restricts the generation of IgG plasma cells and protects against IgG ANAs. Our observations suggest a fundamentally revised understanding of SLE: that it is a disease of aberrant B-cell differentiation rather than a defect in antigen-specific B-cell tolerance.

Positive Antinucleolar Antibodies on Direct Immunofluorescence of Lesional Skin of a Patient With Limited Systemic Sclerosis

To the Editor: Limited systemic sclerosis (SSc) is an autoimmune disease characterized by fibrosis of the skin and internal organs. Circulant anticentromere antibody is usually positive.1 Antinucleolar antibodies are positive less frequently (15%–40%),2 having prognostic significance on the overall course of the patients. We present a case of limited SSc with negative serologic test for extractable nuclear antigens in which direct immunofluorescence revealed positive antinuclear antibodies (ANA) with nucleolar pattern. A 54-year-old man was referred to the dermatology department because of a 5-year history of multiple asymptomatic telangiectasias on the face and neckline. Clinical examination revealed the presence of sclerodactyly and multiple scars of ulcers in the fingertips that appeared when the patient worked in a frigorific chamber. Capillaroscopy showed predominance of areas of angiogenesis and some small avascular areas. The patient complained about mild interphalangeal arthralgias without inflammatory changes. Unspecific gastrointestinal symptoms and dyspnea were also present. High-resolution chest computed tomography showed mild pulmonary fibrosis predominantly in peripheral areas and bases of the lungs. Echocardiography confirmed the clinical suspicion of pulmonary hypertension, and bosentan therapy was initiated. Circulant ANA resulted positive at 1:1280 dilution. The determinations of extractable nuclear antigens, antibodies against parietal cells, against smooth muscle, and against double-stranded DNA were all negative. A skin biopsy of the neckline area with telangiectasia revealed lymphocytic perifolliculitis, with no other alterations. Direct immunofluorescence study (DIF) performed on lesional skin showed the presence of IgG ANA with a nucleolar pattern (Fig. 1).FIGURE 1.: Direct immunofluorescence on a skin biopsy from an area with telangiectasia in the patient's neckline. IgG resulted positive with a nucleolar pattern (×100).Circulating autoantibodies are found in as many as 95% of patients with SSc and carry greater weight in the newly proposed ACR/EULAR classification system. Centromere (CEN) proteins (P), topoisomerase I, and RNA polymerase III autoantibodies closely reflect patterns of organ involvement and disease progression.3 Circulating anti-Th/To type antinucleolar antibodies are detectable mainly by immunoprecipitation, which is not performed routinely in most clinical laboratories. Its positivity is related to limited skin involvement and frequent fibrosis of internal organs (interstitial lung disease, pericarditis, and renal involvement). Frequency of negative circulating antibodies with positive DIF findings is largely unknown because the last is not routinely performed. In the study by Kulthanan et al,4 a subgroup of patients with SSc presented positive DIF findings in 77% of the cases with the dermoepidermal junction and epidermal nuclei as the most common site, and of these patients, 45% presented negative serum ANA. Shibeshi et al5 reported a DIF positivity rate of 25,7% in 63 patients with SSc, with only one case with no circulating autoantibodies at all, and another with positive serum ANA but no characteristic antibodies of SSc. Antinucleolar pattern has been rarely reported in DIF of patients with SSc. Only 6 cases were found in the literature, all of them with high ANA titles. As these cases date from the 1980s, only circulating anti-ribonucleoprotein was performed, resulting positive in one of the patients.6,7 Our patient had ANA in peripheral blood, but more specific antibodies were not detected (antinucleolar, Scl70, or anticentromere). However, a skin biopsy with DIF was allowed to demonstrate the fixation of antibodies with a nucleolar pattern. Nucleolar pattern ANA in DIF of a skin biopsy is an unusual finding. We believe this technique can help to confirm clinical diagnosis and assess prognosis on individual patients.

自身免疫性多腺体综合征IV型：胸腺瘤、白癜风、重症肌无力、系统性红斑狼疮、寻常型天疱疮及普秃并发一例

On November 30th, 2017, a 62-year-old female patient was admitted to the hospital because of multiple depigmented patches all over the body for 16 years, as well as erythema and blisters on the scalp, trunk and upper extremities for more than 8 years, which had been aggravated for 1 year. The patient underwent surgical resection of thymoma in 1996, and developed myasthenia gravis in 2004. Skin examination showed scattered erythemas of various sizes on the scalp, face, chest and upper extremities, with crusts on the surface of some erythemas and positive Nikolsky′s sign. Hypopigmented macules mingled with depigmentated macules of various sizes were scattered all over the body. A large area of depigmentation was observed on both hands, and the nail plates were atrophic. Hairs, eyebrows, eyelashes, axillary and pubic hairs were abscent. Laboratory examination showed the presence of homogeneous IgG antinuclear antibody with a titer of 1∶640, anti-double stranded DNA antibody (276 IU/ml) , lupus anticoagulant (1.62) , anti-Dsg1 antibody (> 150 U/ml) and anti-Dsg3 antibody (91 U/ml) . Histopathological examination of dorsal blister tissues and hematoxylin-eosin staining showed fissured blisters and acantholytic cells in the epidermis, monolayer basal cells on the basilar membrane, and perivascular infiltration of a few lymphocytes in the superficial dermis. The patient successively developed thymoma, vitiligo, myasthenia gravis, systemic lupus erythematosus, pemphigus vulgaris and alopecia universalis, so a diagnosis of autoimmune polyglandular syndrome type Ⅳ was made. Since autoimmune-mediated endocrinopathies are prone to occur successively, changes of related hormones and antibodies in the patient should be monitored for early diagnosis and treatment of newly emerging autoimmune diseases. Key words: Thymoma; Vitiligo; Pemphigus; Myasthenia gravis; Lupus erythematosus, systemic; Alopecia; Autoimmune polyglandular syndrome

Different biochemical patterns in type II and type III mixed cryoglobulinemia in HCV positive patients

BackgroundReversible cryoprecipitability of proteins is observed as a concomitant feature of immune complex formation. Mixed cryoglobulinemia (MC) is systemic vasculitis, associated with mixed IgM and IgG cryoglobulins (CGs) showing rheumatoid factor (RF) activity. It is frequently associated with hepatitis C virus (HCV). This study investigates the presence of IgG RF and anti-nuclear antibodies (ANA) in cryoprecipitates of patients with type III and type II MC, to understand the biochemical patterns associated with different types of MC to a greater degree. MethodsSera from 70 HCV untreated patients with type III or type II MC were tested by immunofixation for IgG3 and through ELISA for IgG RF. Cryoprecipitates were analysed for ANA by indirect immunofluorescence to identify specific patterns. ResultsAfter stratification according to MC type, the ANA patterns between type II and type III MC were statistically different. IgG3 levels and IgG-RF positivity were significantly higher in type III cryoprecipitate. We observed a higher positivity of IgG3 and a significant difference between the liver fibrosis stage, ANA and IgG-RF in the cryoprecipitate. ConclusionResults show a combination of biochemical markers and autoantibodies associated to mixed cryoglobulinemia; these findings could be further investigated in order to ascertain their usefulness in assessing the risk for the development of mixed cryoglobulinemia.

CLINICAL SIGNIFICANCE OF N-TERMINAL FRAGMENT OF NATRIURETIC PEPTIDE IN PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS WHO DO NOT RECEIVE PATHOGENETIC THERAPY

Objective: to determine the level of the N-terminal fragment of brain natriuretic peptide progenitor (NT-proBNP) in patients with systemic lupus erythematous (SLE) prior to immunosuppressive therapy and its possible association with inflammatory markers, traditional risk factors (TRFs) for cardiovascular diseases (CVD), and transthoracic echocardiographic (EchoCG) parameters.Subjects and methods. The investigation enrolled 28 SLE patients fulfilled the 1997 ACR criteria, including 23 (82%) women (median age, 28.5 [25.0; 32.0] years), who had no clinical signs of CVD and received no immunosuppressive therapy. A control group consisted of 27 age-and sex-matched healthy donors. Disease activity was assessed by SLEDAI-2K; irreversible damages were measured using SLICC. The median duration of SLE was 21.0 [5.0; 60.0] months, the scores of SLEDAI-2K and SLICC/DI were 11 [8; 19] and 0 [0; 0], respectively. The investigators estimated the concentration of Creactive protein (CRP), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), carried out EchoCG, and assessed TRFs. The serum concentration of NT-proBNP was determined by electrochemiluminescence (Roche Diagnostics, Switzerland). The normal range for NT-proBNP was ≤125.0 pg/ml. Results and discussion. The patients with SLE had elevated levels of NT-proBNP compared with the controls: 160.7 [88.6; 335.4] and 55.2 [36.6; 70.3] pg/ml, respectively (p &lt; 0.001). The patients were divided into two groups: 1) 18 (64%) patients had a NT-proBNP concentration of &gt; 125.0 pg/ml; 2) 10 (36%) patients had no more than this level. As compared with Group 2, Group 1 had the elevated values of IgG anti-cardiolipin (aCL) antibodies (p &lt; 0.01), creatinine (p &lt; 0.05), left ventricular (LV) end-systolic dimension (ESD) (p &lt; 0.05) and decreases in LV ejection fraction (EF) (p &lt; 0.01), glomerular filtration rate (GFR) (p &lt; 0.05), and concentration of anti-Ro antibodies (p &lt; 0.05). In all the patients (n = 5 (18%)) with LV diastolic dysfunction (DD), the NT-proBNP level was much higher than normal; its median was 799.2 [276.6; 1777.0] pg/ml, but no statistically significant differences were found in the frequency of LV DD between the groups (p = 0.066). In the patients with SLE, the NT-proBNP level correlated positively with that of creatinine (r = 0.480; p &lt; 0.01), uric acid (r = 0.427; p &lt; 0.05), IgG aCL (r = 0.710; p &lt; 0.001), anti-double-stranded DNA (anti-dsDNA) antibodies (r = 0.395; p &lt; 0.05), antinuclear antibodies (ANA) (r = 0.256; p &lt; 0.05), LV ESD (r = 0.442; p &lt; 0.05), pulmonary artery systolic pressure (r = 0.486; p &lt; 0.05) and negatively with hemoglobin level (r = -0.493; p &lt; 0.01), C4 complement component (r = -0.475; p &lt; 0.05), GFR (r = -0.58; p &lt; 0.01), and EF (r = -0.505; p &lt; 0.01). The level of NT-proBNP was ascertained to be unassociated with the clinical manifestations of SLE (skin, mucosae, kidneys, nervous system damage, as well as arthritis, serositis, and hematological disorders) and markers of inflammation (CRP, IL-6, TNF-α).Conclusion. The NT-proBNP concentration in the patients with SLE was significantly higher than in the control group (p &lt; 0.001), more than 60% of the untreated SLE patients had elevated NT-proBNP values (&gt;125.0 pg/ml). The higher level of NT-proBNP is associated with the immunological parameters of SLE activity (elevated values of IgG aCL, anti-dsDNA, and ANA as well as C4 hypocomplementemia) and with the markers that reflect deterioration in renal and myocardial functions. There was no relationship of NT-proBNP levels to the clinical manifestations of SLE and the markers of inflammation (CRP, IL-6, and TNF-α) and TRFs.

Identification of novel plasma cell subsets in a mouse model of SLE

Abstract Systemic lupus erythematosus (SLE) is an autoimmune disease driven by the secretion of anti-nuclear antibodies (ANA) from a subset of terminally differentiated B cells, called plasma cells (PC). PC persist in the bone marrow for the lifetime of the host, posing challenges for the development of therapies for SLE and other autoimmune diseases. The mechanisms of long-term persistence of PC are poorly understood. Utilizing the congenic New Zealand Black Autoimmunity 2 (NBA2) mouse strain, which develops IgG ANA similar to SLE, our lab has identified novel subsets of mature bone marrow PC. which are highly heterogeneous in both phenotype and function. We have characterized these populations of PC based on the expression of CD93 and CXCR3. While CD93+ PC are largely quiescent, CXCR3+ PC are highly proliferative in the bone marrow. In addition, CXCR3+ PC are highly enriched for IgG secretion, while CD93+ PC are enriched for IgA secretion. CD93+ PC are detected in the bone marrow of B6 mice at early time points after immunization with KLH, whereas CXCR3+ PC are nearly absent in the bone marrow yet are present in the spleen. In contrast, significant numbers of both CD93+ and CXCR3+ PC are found in both the spleen and bone marrow of NBA2 mice. Characterization of transcription factors reveals that all PC populations express high levels of BLIMP-1 and IRF-4, demonstrating their commitment to a mature PC fate. These data suggest that distinct subpopulations of PC may be responsible for the pathogenesis of SLE. Current research is focused on elucidating the mechanism by which distinct PC subsets give rise to ANA.

Predominant role for activation-induced cytidine deaminase in generating IgG anti-nucleosomal antibodies of murine SLE

Serum IgG anti-nuclear antibodies (ANA) directed to complexes of DNA and histones are a hallmark of systemic lupus erythematosus (SLE) and reflect a failure in lymphocyte self-tolerance. A prior study utilizing spontaneously autoimmune B6.Nba2 mice deficient in terminal deoxynucleotidyl transferase (TdT) and with heterozygous deficiencies in Jh and Igk loci underscored the importance of somatic hypermutation (SHM) as a major generator of SLE-associated ANA. This interpretation had to be qualified because of severely limited opportunities for receptor editing and restricted VHCDR3 diversity. Therefore, we performed the converse study using mice that carried functional Tdt genes and wild type Jh and Igk loci but that could not undergo SHM. Analyses of ANA and ANA-producing hybridomas from B6.Nba2 Aicda−/− mice revealed that few animals produced high titers of the prototypical ANA directed to complexes of histones and DNA, that this response was delayed and that those cells that did produce such antibody exhibited limited clonal expansion, unusual Jk use and only infrequent dual receptor expression. This, together with the additional finding of an intrinsic propensity for SHM to generate Arg codons selectively in CDRs, reinforce the view that most IgG autoimmune clones producing prototypical anti-nucleosome antibodies in wild type mice are created by SHM.

The development of autoimmune features in aging mice is closely associated with alterations of the peripheral CD4⁺ T-cell compartment.

Some signs of potential autoimmunity, such as the appearance of antinuclear antibodies (ANAs) become prevalent with age. In most cases, elderly people with ANAs remain healthy. Here, we investigated whether the same holds true for inbred strains of mice. Indeed, we show that most mice of the C57BL/6 (B6) strain spontaneously produced IgG ANA at 8-12 months of age, showed IgM deposition in kidneys and lymphocyte infiltrates in submandibular salivary glands. Despite all of this, the mice remained healthy. ANA production is likely CD4(+) T-cell dependent, since old (40-50 weeks of age) B6 mice deficient for MHC class II do not produce IgG ANAs. BM chimeras showed that ANA production was not determined by age-related changes in radiosensitive, hematopoietic progenitor cells, and that the CD4(+) T cells that promote ANA production were radioresistant. Thymectomy of B6 mice at 5 weeks of age led to premature alterations in T-cell homeostasis and ANA production, by 15 weeks of age, similar to that in old mice. Our findings suggest that a disturbed T-cell homeostasis may drive the onset of some autoimmune features.

Production of IgG autoantibody requires expression of activation-induced deaminase in early-developing B cells in a mouse model of SLE.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the presence of pathogenic IgG antinuclear antibodies. Pathogenic IgG autoantibody production requires B-cell activation, leading to the production of activation-induced deaminase (AID) and class switching of IgM genes to IgG. To understand how and when B cells are activated to produce these IgG autoantibodies, we studied cells from 564Igi, a mouse model of SLE. 564Igi mice develop a disease profile closely resembling that found in human SLE patients, including the presence of IgG antinucleic acid Abs. We have generated 564Igi mice that conditionally express an activation-induced cytidine deaminase transgene (Aicda(tg) ), either in all B cells or only in mature B cells. Here, we show that class-switched pathogenic IgG autoantibodies were produced only in 564Igi mice in which AID was functional in early-developing B cells, resulting in loss of tolerance. Furthermore, we show that the absence of AID in early-developing B cells also results in increased production of self-reactive IgM, indicating that AID, through somatic hypermutation, contributes to tolerance. Our results suggest that the pathophysiology of clinical SLE might also be dependent on AID expression in early-developing B cells.

Immunological evaluation of β-thalassemia major patients receiving oral iron chelator deferasirox.

To determine the immune abnormalities and occurrence of infections in transfusion-dependent β-thalassemia major patients receiving oral iron chelator deferasirox (DFX). An observational study. Hematology Clinics, King Khalid University Hospital, Riyadh, Saudi Arabia, from July to December 2010. Seventeen patients with β-thalassemia major (12 females, median age 26 years) receiving deferasirox (DFX) for a median duration of 27 months were observed for any infections and had their immune status determined. Immune parameters studied included serum immunoglobulins and IgG subclasses, serum complement (C3 and C4) and anti-nuclear antibody (ANA) level, total B and T-lymphocytes, CD4+ and CD8+ counts, CD4+/CD8+ ratio, and natural killer (NK) cells. Immunological parameters of the patients were compared with age, gender, serum ferritin level and splenectomy status. Lymphocyte subsets were also compared with age and gender matched normal controls. A considerable reduction in serum ferritin was achieved by DFX from a median level of 2528 to 1875 μmol/l. Serum IgG levels were increased in 7 patients. Low C4 levels were found in 9 patients. Total B and T-lymphocytes were increased in 14 patients each, while CD4+, CD8+ and NK cells were increased in 13, 12 and 11 patients respectively. Absolute counts for all lymphocyte subsets were significantly higher compared to the normal controls (p ² 0.05 for all parameters). Raised levels of IgG were associated with older age, female gender, splenectomized status and higher serum ferritin levels but this did not reach statistical significance except for the higher ferritin levels (p=0.044). Increased tendency to infections was not observed. Patients with β-thalassemia major receiving DFX exhibited significant immune abnormalities. Changes observed have been described previously, but could be related to DFX. The immune abnormalities were not associated with increased tendency to infections.

Ly108 expression distinguishes subsets of invariant NKT cells that help autoantibody production and secrete IL-21 from those that secrete IL-17 in lupus prone NZB/W mice

Lupus is a systemic autoimmune disease characterized by anti-nuclear antibodies in humans and genetically susceptible NZB/W mice that can cause immune complex glomerulonephritis. T cells contribute to lupus pathogenesis by secreting pro-inflammatory cytokines such as IL-17, and by interacting with B cells and secreting helper factors such as IL-21 that promote production of IgG autoantibodies. In the current study, we determined whether purified NKT cells or far more numerous conventional non-NKT cells in the spleen of NZB/W female mice secrete IL-17 and/or IL-21 after TCR activation in vitro, and provide help for spontaneous IgG autoantibody production by purified splenic CD19+ B cells. Whereas invariant NKT cells secreted large amounts of IL-17 and IL-21, and helped B cells, non-NKT cells did not. The subset of IL-17 secreting NZB/W NKT cells expressed the Ly108loCD4−NK1.1− phenotype, whereas the IL-21 secreting subset expressed the Ly108hiCD4+NK1.1− phenotype and helped B cells secrete a variety of IgG anti-nuclear antibodies. α-galactocylceramide enhanced the helper activity of NZB/W and B6.Sle1b NKT cells for IgG autoantibody secretion by syngeneic B cells. In conclusion, different subsets of iNKT cells from mice with genetic susceptibility to lupus can contribute to pathogenesis by secreting pro-inflammatory cytokines and helping autoantibody production.