Autoantibodies associated with primary biliary cholangitis are common among patients with systemic lupus erythematosus even in the absence of elevated liver enzymes.

Knowledge of concomitant autoimmune liver diseases (AILD) is more detailed in primary Sjögren's syndrome (pSS) compared to systemic lupus erythematosus (SLE). Herein, the prevalence of autoantibodies associated with autoimmune hepatitis (AIH) and primary biliary cholangitis (PBC) was investigated in stored sera from patients with SLE (n = 280) and pSS (n = 114). Antibodies against mitochondria (AMA), liver-kidney microsomal (LKM) antigen, smooth muscle (SMA) and anti-nuclear antibodies (ANA) were analysed with immunofluorescence microscopy. In addition, AILD-associated autoantibodies were tested with immunoblot. Prior to sampling, eight SLE (2·9%) and three pSS (2·6%) cases were diagnosed with AILD. Among SLE-cases without known AILD (n = 272), 26 (9·6%) had PBC-associated autoantibodies, 15 (5·5%) AIH-associated autoantibodies (excluding ANA) and one serological overlap. Most subjects with PBC-associated autoantibodies had liver enzymes within reference limits (22 of 27, 81%) or mild laboratory cholestasis (two of 27, 7·4%), while one fulfilled the diagnostic PBC-criteria. AMA-M2 detected by immunoblot was the most common PBC-associated autoantibody in SLE (20 of 272, 7·4%). The prevalence of SMA (4·4%) was comparable with a healthy reference population, but associated with elevated liver enzymes in four of 12 (25%), none meeting AIH-criteria. The patient with combined AIH/PBC-serology had liver enzymes within reference limits. Among pSS cases without known AILD (n = 111), nine (8·1%) had PBC-associated, 12 (10·8%) AIH-associated autoantibodies and two overlapped. PBC-associated autoantibodies were found as frequently in SLE as in pSS but were, with few exceptions, not associated with laboratory signs of liver disease. Overall, AILD-associated autoantibodies were predominantly detected by immunoblot and no significant difference in liver enzymes was found between AILD autoantibody-negative and -positive patients.

Longitudinal anti-nuclear antibody (ANA) seroconversion in systemic lupus erythematosus: a prospective study of Swedish cases with recent-onset disease.

Serum immunoglobulin (Ig)G anti-nuclear antibodies (ANA) detected by indirect immunofluorescence (IF) microscopy remains a hallmark of systemic lupus erythematosus (SLE). Whether or not IF-ANA status varies over time is controversial. We therefore designed a prospective study with longitudinal follow-up of patients with recent-onset SLE. The study population consisted of 54 recently diagnosed SLE cases, all meeting the 1982 American College of Rheumatology (ACR) and/or the 2012 Systemic Lupus International Collaborating Clinics (SLICC) criteria. Clinical follow-up data, including disease activity, organ damage and sera, were collected from clinical onset of SLE and onwards, in most cases yearly (0-96 months). IF-ANA was analysed on human epithelial cells-2 (HEp-2) cells and categorized regarding staining patterns. Using an addressable laser bead assay (FIDIS™ Connective profile), we measured IgG-ANA fine specificities against Ro52/SSA, Ro60/SSA, Sjögren's syndrome type B antigen (La/SSB), Smith antigen (Sm), Smith antigen/ribonucleoprotein (Sm/RNP), U1 RNP (U1RNP), dsDNA, ribosomal-P protein and histone. At baseline, all patients were judged ANA-positive at an abnormal titre corresponding to the 95th percentile of healthy blood donors, but seven of 54 patients (13%) lost ANA-positivity over time. Homogeneous (AC-1; 46%) and speckled (AC-4 or 5; 31%) were the most frequently observed patterns at inclusion, whereas 7% switched pattern at least once during follow-up. Established associations between ANA fine specificities and clinical data were confirmed. Levels of anti-Sm/RNP, but not of anti-dsDNA, correlated with clinical disease activity [modified SLE disease activity 2000 (mSLEDAI-2K)]. Our data indicate that a considerable proportion of Swedish patients with SLE lose ANA-positivity over time, whereas consistent staining patterns were frequent. The clinical and mechanistic relevance of ANA seroconversion remains uncertain. Further prospective evaluations in larger SLE populations with more diverse ethnicities are warranted.

Neurofilament light chain in cerebrospinal fluid and prediction of disease activity in clinically isolated syndrome and relapsing-remitting multiple sclerosis.

BACKGROUND AND PURPOSE: Improved biomarkers are needed to facilitate clinical decision-making and as surrogate endpoints in clinical trials in multiple sclerosis (MS). We assessed whether neurodegenerative and neuroinflammatory markers in cerebrospinal fluid (CSF) at initial sampling could predict disease activity during 2 years of follow-up in patients with clinically isolated syndrome (CIS) and relapsing-remitting MS. METHODS: Using multiplex bead array and enzyme-linked immunosorbent assay, CXCL1, CXCL8, CXCL10, CXCL13, CCL20, CCL22, neurofilament light chain (NFL), neurofilament heavy chain, glial fibrillary acidic protein, chitinase-3-like-1, matrix metalloproteinase-9 and osteopontin were analysed in CSF from 41 patients with CIS or relapsing-remitting MS and 22 healthy controls. Disease activity (relapses, magnetic resonance imaging activity or disability worsening) in patients was recorded during 2 years of follow-up in this prospective longitudinal cohort study. RESULTS: In a logistic regression analysis model, NFL in CSF at baseline emerged as the best predictive marker, correctly classifying 93% of patients who showed evidence of disease activity during 2 years of follow-up and 67% of patients who did not, with an overall proportion of 85% (33 of 39 patients) correctly classified. Combining NFL with either neurofilament heavy chain or osteopontin resulted in 87% overall correctly classified patients, whereas combining NFL with a chemokine did not improve results. CONCLUSIONS: This study demonstrates the potential prognostic value of NFL in baseline CSF in CIS and relapsing-remitting MS and supports its use as a predictive biomarker of disease activity.

Circulating cartilage oligomeric matrix protein in juvenile idiopathic arthritis.

OBJECTIVES: Raised serum cartilage oligomeric matrix protein (sCOMP) has been reported to predict erosive disease in early rheumatoid arthritis (RA). In juvenile idiopathic arthritis (JIA), subnormal sCOMP levels have been associated with ongoing inflammation and growth retardation. In this study we aimed to assess sCOMP, C-reactive protein (CRP), and insulin-like growth factor (IGF)-1 in children/adolescents with JIA and in referents. METHOD: We enrolled 52 JIA patients at planned outpatient visits and 54 inpatients with ongoing infection ('infection referents'). A total of 120 referents testing negative for immunoglobulin (Ig)E-mediated allergy ('IgE referents') served as controls. All serum samples were analysed for COMP, IGF-1, and CRP. RESULTS: The average sCOMP level was highest among the IgE referents and lowest among the infection referents. In the JIA patients, the level of sCOMP was not associated with the level of CRP or with clinical signs of disease activity. CONCLUSIONS: The results of this study do not support routine clinical analysis of sCOMP levels in patients with JIA.

Cerebrospinal fluid levels of neurofilament and tau correlate with brain atrophy in natalizumab-treated multiple sclerosis.

BACKGROUND AND PURPOSE: Brain atrophy is related to clinical deterioration in multiple sclerosis (MS) but its association with intrathecal markers of inflammation or neurodegeneration is unclear. Our aim was to investigate whether cerebrospinal fluid (CSF) markers of inflammation or neurodegeneration are associated with brain volume change in natalizumab-treated MS and whether this change is reflected in non-lesional white matter metabolites. METHODS: About 25 patients with natalizumab-treated MS were followed for 3 years with assessment of percentage brain volume change (PBVC) and absolute quantification of metabolites with proton magnetic resonance spectroscopy (1 H MRS). Analyses of inflammatory [interleukin 1ß (IL-1ß), IL-6, C-X-C motif chemokine 8 (CXCL8), CXCL10, CXCL11, C-C motif chemokine 22] and neurodegenerative [neurofilament light protein (NFL), glial fibrillary acidic protein, myelin basic protein, tau proteins] markers were done at baseline and 1-year follow-up. RESULTS: The mean decline in PBVC was 3% at the 3-year follow-up, although mean 1 H MRS metabolite levels in non-lesional white matter were unchanged. CSF levels of NFL and tau at baseline correlated negatively with PBVC over 3 years (r = -0.564, P = 0.012, and r = -0.592, P = 0.010, respectively). CONCLUSIONS: A significant 3-year whole-brain atrophy was not reflected in mean metabolite change of non-lesional white matter. In addition, our results suggest that CSF levels of NFL and tau correlate with brain atrophy development and may be used for evaluating treatment response in inflammatory active MS.

Regional brain shrinkage over two years: individual differences and effects of pro-inflammatory genetic polymorphisms.

We examined regional changes in brain volume in healthy adults (N=167, age 19-79years at baseline; N=90 at follow-up) over approximately two years. With latent change score models, we evaluated mean change and individual differences in rates of change in 10 anatomically-defined and manually-traced regions of interest (ROIs): lateral prefrontal cortex (LPFC), orbital frontal cortex (OF), prefrontal white matter (PFw), hippocampus (Hc), parahippocampal gyrus (PhG), caudate nucleus (Cd), putamen (Pt), insula (In), cerebellar hemispheres (CbH), and primary visual cortex (VC). Significant mean shrinkage was observed in the Hc, CbH, In, OF, and PhG, and individual differences in change were noted in all regions, except the OF. Pro-inflammatory genetic variants modified shrinkage in PhG and CbH. Carriers of two T alleles of interleukin-1ß (IL-1ß C-511T, rs16944) and a T allele of methylenetetrahydrofolate reductase (MTHFR C677T, rs1801133) polymorphisms showed increased PhG shrinkage. No effects of a pro-inflammatory polymorphism for C-reactive protein (CRP-286C>A>T, rs3091244) or apolipoprotein (APOE) ε4 allele were noted. These results replicate the pattern of brain shrinkage observed in previous studies, with a notable exception of the LPFC, thus casting doubt on the unique importance of prefrontal cortex in aging. Larger baseline volumes of CbH and In were associated with increased shrinkage, in conflict with the brain reserve hypothesis. Contrary to previous reports, we observed no significant linear effects of age and hypertension on regional brain shrinkage. Our findings warrant further investigation of the effects of neuroinflammation on structural brain change throughout the lifespan.

Time to secondary progression in patients with multiple sclerosis who were treated with first generation immunomodulating drugs.

BACKGROUND: It is currently unknown whether early immunomodulatory treatment in relapsing-remitting MS (RRMS) can delay the transition to secondary progression (SP). OBJECTIVE: To compare the time interval from onset to SP in patients with RRMS between a contemporary cohort, treated with first generation disease modifying drugs (DMDs), and a historical control cohort. METHODS: We included a cohort of contemporary RRMS patients treated with DMDs, obtained from the Swedish National MS Registry (disease onset between 1995-2004, n = 730) and a historical population-based incidence cohort (onset 1950-64, n = 186). We retrospectively analyzed the difference in time to SP, termed the "period effect" within a 12-year survival analysis, using Kaplan-Meier and Cox regression analysis. RESULTS: We found that the "period" affected the entire severity spectrum. After adjusting for onset features, which were weaker in the contemporary material, as well as the therapy initiation time, the DMD-treated patients still exhibited a longer time to SP than the controls (hazard ratios: men, 0.32; women, 0.53). CONCLUSION: Our results showed there was a longer time to SP in the contemporary subjects given DMD. Our analyses suggested that this effect was not solely driven by the inclusion of benign cases, and it was at least partly due to the long-term immunomodulating therapy given.

IgA antibodies against tissue transglutaminase, endomysium and gliadin in idiopathic polyneuropathy.

OBJECTIVES: To study the prevalence of antibodies of IgA class against tissue transglutaminase (tTG), endomysium (EMA) and gliadin (AGA) in patients with chronic idiopathic axonal polyneuropathy (CIAP) and to characterize the patients clinically and neurophysiologically. METHODS: Of 182 patients, 126 patients agreed to blood sampling. Sera were analysed by ELISAs detecting anti-tTG and AGA, whereas EMA was analysed by indirect immunofluorescence (IF) microscopy. Gastrointestinal symptoms were assessed by data from medical records and patient interviews. RESULTS: Nine of 126 patients (7%) were seropositive in at least one test (five with positive anti-tTG and/or EMA and four with positive AGA only). One patient with elevated levels of all specificities had laboratory signs of malabsorption and gastrointestinal complaints with abdominal pain and diarrhoea. CONCLUSIONS: Elevated levels of IgA-AGA were slightly more frequent in patients with CIAP (4%) compared to 2.5% in 1866 healthy blood donors. Highly specific serological markers indicative of coeliac disease (CD) (anti-tTG and EMA) were somewhat more common in our patients with CIAP (4%) than expected from normal reference values and from studies of the prevalence of CD in the general population. Even though these findings may indicate a relationship, the aetiological importance is unclear.

Transcriptional characteristics of CD4+ T cells in multiple sclerosis: relative lack of suppressive populations in blood.

BACKGROUND: Multiple sclerosis (MS) is hypothetically caused by autoreactive Th1 and Th17 cells, whereas Th2 and regulatory T cells may confer protection. The development of Th subpopulations is dependant on the expression of lineage-specific transcription factors. OBJECTIVE: The aim of this study was to assess the balance of CD4(+)T cell populations in relapsing-remitting MS. METHODS: Blood mRNA expression of TBX21, GATA3, RORC, FOXP3 and EBI3 was assessed in 33 patients with relapsing-remitting MS and 20 healthy controls. In addition, flow cytometry was performed to assess T lymphocyte numbers. RESULTS: In relapsing-remitting MS, diminished expression of FOXP3 (Treg) was found (p < 0.05), despite normal numbers of CD4(+)CD25(hi)Treg. Immunoregulatory EBI3 and Th2-associated GATA3 ([a-z]+) was also decreased in MS (p < 0.005 and p < 0.05, respectively). Expression of TBX21 (Th1) and RORC (Th17) did not differ between patients and controls. Similar changes were observed when analysing beta-interferon treated (n = 12) or untreated (n = 21) patients. Analysis of transcription factor ratios, comparing TBX21/GATA3 and RORC/FOXP3, revealed an increase in the RORC/FOXP3 ratio in patients with relapsing-remitting MS (p < 0.005). CONCLUSION: Our findings indicate systemic defects at the mRNA level, involving downregulation of beneficial CD4(+)phenotypes. This might play a role in disease development by permitting activation of harmful T cell populations.

Antibodies against deamidated gliadin peptides identify adult coeliac disease patients negative for antibodies against endomysium and tissue transglutaminase.

BACKGROUND: This study was done to evaluate the diagnostic utility of antibodies against deamidated gliadin peptides compared to traditional markers for coeliac disease. AIM: To evaluate diagnostic utility of antibodies against deamidated gliadin peptide (DGP). METHODS: Sera from 176 adults, referred for endoscopy without previous analysis of antibodies against tissue transglutaminase (tTG) or endomysium (EmA), were retrospectively analysed by ELISAs detecting IgA/IgG antibodies against DGP or a mixture of DGP and tTG, and compared with IgA-tTG and EmA. Seventy-nine individuals were diagnosed with coeliac disease. RESULTS: Receiver operating characteristic analyses verified the manufacturers' cut-off limits except for IgA/IgG-DGP/tTG. In sera without IgA deficiency, the sensitivity was higher for IgA/IgG-DGP (0.85-0.87) compared with IgA-tTg (0.76) and EmA (0.61). All tests showed high specificity (0.95-1.00). Eighteen coeliac disease-sera were negative regarding IgA-tTG, nine of which were positive for IgA/IgG-DGP. Sera from coeliac disease-patients >70 years were more often negative for IgA-tTG (50%) and IgA/IgG-DGP (36%) than younger patients (15% and 8% respectively) (P < 0.01). Three of the four IgA-deficient patients were positive in the IgA/IgG-DGP assay. CONCLUSIONS: In this study of patients unselected regarding IgA-tTg/EmA, thus unbiased in this respect, IgA/IgG-DGP identified adult coeliac disease patients negative for antibodies against endomysium and tissue transglutaminase. Serology is often negative in elderly patients with coeliac disease; a small bowel biopsy should therefore be performed generously before coeliac disease is excluded.

Natalizumab treatment in multiple sclerosis: marked decline of chemokines and cytokines in cerebrospinal fluid.

Natalizumab exerts impressive therapeutic effects in patients with multiple sclerosis (MS). The proposed main mode of action is reducing transmigration of leukocytes into the CNS, but other immunological effects may also be operative. Cytokines and chemokines are involved in the regulation of inflammatory responses and may reflect the disease process in MS. The objective of this study was to evaluate the effects of natalizumab treatment on cytokine and chemokine profiles systemically and intrathecally in multiple sclerosis. We used luminex to analyse a panel of cytokines (IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, TNF-alpha, IFN-gamma, GM-CSF) and chemokines (CXCL9, CXCL10, CXCL11, CCL17, CCL22) in blood and cerebrospinal fluid (CSF) from 31 patients with relapsing MS before and after one year of natalizumab treatment. There was a marked decline in CSF levels of cytokines and chemokines, thus including pro-inflammatory cytokines (IL-1beta, IL-6 and IL-8) as well as chemokines associated with both Th1 (CXCL9, CXCL10, CXCL11) and Th2 (CCL22). Circulating plasma levels of some cytokines (GM-CSF, TNF-alpha, IL-6 and IL-10) also decreased after one year of treatment. This is the first study to show that natalizumab treatment is associated with a global decline in cytokine and chemokine levels at a protein level. This finding was most pronounced in CSF, in line with the reduced transmigration of cells into CNS, whereas reduction in plasma levels indicates other possible mechanisms of natalizumab treatment.

Elevated number of cells secreting transforming growth factor beta in Guillain-Barré syndrome.

We used ELISPOT and cell ELISA to study secretion of IL-4, IFN-gamma, TGF-beta, IL-6, and TNF-alpha by circulating mononuclear cells during the course of Guillain-Barré syndrome (GBS). Compared to healthy controls, patients with GBS had higher numbers of TGF-beta-secreting cells and the number of individuals with myelin-peptide-induced IL-4 and TGF-beta secretion was higher in the GBS group. No significant differences were seen concerning the predominantly pro-inflammatory cytokines IFN-gamma, IL-6 or TNF-alpha. Our findings indicate a down-regulatory role for TGF-beta and IL-4 in GBS.

Transfer of myelin-specific cells deviated in vitro towards IL-4 production ameliorates ongoing experimental allergic neuritis.

A causal role of IL-4 (Th2) production for recovery in experimental allergic neuritis (EAN) was indicated by experiments where Th1-like autoreactive cell populations, taken from the induction phase of the disease, were deviated to extensive secretion of IL-4 in a selective fashion, by ex vivo stimulation with autoantigen in the presence of IL-4. The deviated cells were adoptively transferred to EAN rats at a time just prior to the onset of clinical signs. This treatment ameliorated EAN compared with sham treatment. This therapeutic approach, with generation of autoreactive IL-4-secreting cells ex vivo followed by subsequent adoptive transfer, may become a new selective treatment of organ-specific autoimmune diseases since, in contrast to previous attempts, it is done in a physiological and technically easy way.

APC stimulated by CpG oligodeoxynucleotide enhance activation of MHC class I-restricted T cells.

Oligonucleotides containing unmethylated CpG motifs (cytosine-phosphorothioate-guanine oligodeoxynucleotide (CpG ODN)) are potent immunostimulatory agents capable of enhancing the Ag-specific Th1 response when used as immune adjuvants. We evaluated the cellular mechanisms responsible for this effect. Development of a CTL response was enhanced when mice were immunized with peptide-pulsed dendritic cells (DCs) treated with CpG ODN. However, in vitro, CpG ODN had no direct effect on highly purified T cells. In vitro, CpG ODN treatment of peptide- or protein-pulsed DCs enhanced the ability of the DCs to activate class I-restricted T cells. The presence of helper T cells enhanced this effect, indicating that treatment with CpG ODN does not obviate the role of T cell help. The enhanced ability of CpG ODN-treated DCs to activate T cells was present but blunted when DCs derived from IL-12 knockout mice were used. Fixation of Ag-pulsed, CpG ODN-treated DCs limited their ability to activate T cells. In contrast, fixation had little effect on DC activation of T cells when DCs were not exposed to CpG ODN. This indicates that production of soluble factors by DCs stimulated with CpG ODN plays a particularly important role in their ability to activate class I-restricted T cells. We conclude that CpG ODN enhances the development of a cellular immune response by stimulating APCs such as DCs, to produce IL-12 and other soluble factors.

CpG oligodeoxynucleotides enhance monoclonal antibody therapy of a murine lymphoma.

Bacterial DNA and synthetic oligodeoxynucleotides containing unmethylated cytosine-guanine dinucleotides known as cytosine phosphorothioate guanine oligodeoxynucleotides (CpG ODN) can activate various immune-cell subsets, including cells that participate in antibody-dependent cell-mediated cytotoxicity (ADCC). Studies have shown that CpG ODN enhance the efficacy of antitumor monoclonal antibody (MoAb) therapy in the 38C13 murine B-cell lymphoma. We performed a series of in vivo experiments using this tumor model to better characterize combination therapy with MoAb and CpG ODN. CpG ODN enhanced the efficacy of MoAb therapy of lymphoma in a dose-dependent manner. This effect was seen whether the CpG ODN was given before or after the MoAb therapy, but was decreased when CpG ODN was given more than 2 days after MoAb therapy. Three doses of CpG ODN and MoAb were more effective than single doses. There was no obvious toxicity with multiple dosing. These studies confirm that immunostimulatory CpG ODN enhance the efficacy of MoAb therapy, and that multiple courses of combination therapy with CpG ODN can serve as an effective therapy for lymphoma. Further exploration of this potentially potent combination of treatments, including clinical evaluation, is indicated.

Immunostimulatory CpG oligodeoxynucleotides enhance the immune response to vaccine strategies involving granulocyte-macrophage colony-stimulating factor.

Immunostimulatory oligodeoxynucleotides containing the CpG motif (CpG ODN) can activate various immune cell subsets and induce production of a number of cytokines. Prior studies have demonstrated that both CpG ODN and granulocyte-macrophage colony-stimulating factor (GM-CSF) can serve as potent vaccine adjuvants. We used the 38C13 murine lymphoma system to evaluate the immune response to a combination of these two adjuvants. Immunization using antigen, CpG ODN, and soluble GM-CSF enhanced production of antigen-specific antibody and shifted production towards the IgG2a isotype, suggesting an enhanced TH1 response. This effect was most pronounced after repeat immunizations with CpG ODN and antigen/GM-CSF fusion protein. A single immunization with CpG ODN and antigen/GM-CSF fusion protein 3 days before tumor inoculation prevented tumor growth. CpG ODN enhanced the production of interleukin-12 by bone marrow-derived dendritic cells and increased expression of major histocompatibility complex class I and class II molecules, particularly when cells were pulsed with antigen/GM-CSF fusion protein. We conclude that the use of CpG ODN in combination with strategies involving GM-CSF enhances the immune response to antigen and shifts the response towards a TH1 response and that this approach deserves further evaluation in tumor immunization approaches and other conditions in which an antigen-specific TH1 response is desirable.

Recovery from Guillain-Barré syndrome is associated with increased levels of neutralizing autoantibodies to interferon-gamma.

Guillain-Barré syndrome (GBS) is an immune-mediated demyelinating disease of peripheral nerves that is often preceded by an infection and is usually self-restricted. The Th1 cytokine interferon-gamma (IFN-gamma) is thought to be disease-promoting in organ-specific autoimmune diseases. We report the spontaneous induction of IFN-gamma and a mechanism involving the generation of neutralizing autoantibodies (Aabs) to IFN-gamma that may regulate the disease. Numbers of cells spontaneously secreting IFN-gamma in peripheral blood were augmented in GBS, in particular at the peak of clinical disease, and decreased during recovery. This decrease was associated with elevated serum concentrations of IgG Aabs to IFN-gamma. These Aabs specifically bound to IFN-gamma and neutralized its effects in a biological assay. Aabs to IFN-gamma are proposed to be another important regulatory mechanism in IFN-gamma-driven GBS.