Anti-histone and anti-nucleosome rather than anti-dsDNA antibodies associate with IFN-induced biomarkers in Sudanese and Swedish Systemic Lupus Erythematosus patients.

OBJECTIVES: In SLE, anti-dsDNA can co-occur with autoantibodies against other chromatin components, like histones and nucleosomes. These antibodies induce type-1 interferon production, a hallmark of SLE. We measured antinuclear antibody (ANA) sub-specificities and investigated their associations to inflammatory biomarkers including interferon-regulated chemokines. METHODS: We included 93 Sudanese and 480 Swedish SLE patients and matched controls (N = 104 + 192). Autoantibodies targeting ANA-subspecificites: dsDNA, Sm, Sm/U1RNPcomplex, U1RNP, SSA/Ro52, SSA/Ro60, SSB/La, ribosomal P, PCNA and histones were quantified in all subjects, anti-nucleosome only in the Swedish patients, with a bead-based multiplex immunoassay. Levels of 72 plasma biomarkers were determined with Proximity Extension Assay technique or ELISA. RESULTS: Among Sudanese patients, the investigated antibodies significantly associated with 9/72 biomarkers. Anti-histone antibodies showed the strongest positive correlations with MCP-3 and S100A12 as well as with interferon I-inducible factors MCP-1 and CXCL10. Anti-dsDNA antibodies associated with CXCL10 and S100A12, but in multivariate analyses, unlike anti-histone, associations lost significance.Among Swedish patients, MCP-1, CXCL10, SA100A12 also demonstrated stronger associations to anti-histone and anti-nucleosome antibodies, compared with anti-dsDNA and other ANA sub-specificities. In multiple regression models, anti-histone/nucleosome retained the strongest associations. When excluding anti-histone or anti-nucleosome positive patients, the associations between MCP-1/CXCL10 and anti-dsDNA were lost. In contrast, when excluding anti-dsDNA positive patients, associations with anti-histone and anti-nucleosome remained significant. CONCLUSION: In two cohorts of different ethnical origin, autoantibodies targeting chromatin correlate stronger with IFN-induced inflammatory biomarkers than anti-dsDNA or other ANA sub-specificities. Our results suggest that anti-histone/nucleosome autoantibodies may be main drivers of type-1 interferon activity in SLE.

Associations with thrombosis are stronger for antiphosphatidylserine/prothrombin antibodies than for the Sydney criteria antiphospholipid antibody tests in SLE.

OBJECTIVES: Antiphosphatidylserine/prothrombin complex antibodies (aPS/PT) are risk factors for thrombosis, yet further validation of their clinical relevance in different ethnic groups is required. We investigated the performance of aPS/PT of IgA/G/M isotypes among Sudanese and Swedish systemic lupus erythematosus (SLE) patients. METHODS: Consecutive SLE patients/matched controls from Sudan (n = 91/102) and Sweden (n = 332/163) were included. All patients fulfilled the 1982 ACR SLE classification criteria. IgA/G/M of aPS/PT, anti-cardiolipin and anti-ß2glycoprotein I (anti-ß2GPI) were tested in both cohorts, and lupus anticoagulant (LA) also in the Swedish cohort. Clinical antiphospholipid syndrome-related events and atherosclerosis, measured as carotid plaques were assessed for associations. Univariate and multivariate analyses adjusting for cardiovascular risk factors were performed. RESULTS: Sudanese SLE patients had higher levels of IgM aPS/PT, but using national cut-offs, the frequency of positivity was similar to Swedish patients for all isotypes. Among Swedish patients, all isotypes of aPS/PT associated with venous thromboembolism (VTE), while only IgA aPS/PT associated with arterial thrombosis (AT). aPS/PT antibodies associated strongly with LA and they were, independently, the best predictor for VTE. Double positivity for aPS/PT and anti-ß2GPI associated with higher VTE risk than the conventional triple positivity. Carotid plaques did not associate with any antiphospholipid antibody. CONCLUSIONS: IgA aPS/PT associated with AT, and the association of IgG/M aPS/PT with VTE outperforms LA and criteria antiphospholipid antibodies in Swedish SLE patients. Furthermore, double positivity for aPS/PT and anti-ß2GPI performed better than conventional triple positivity. Future studies need to address if aPS/PT can replace LA, as this would simplify clinical procedures.

Accumulation of antinuclear associated antibodies in circulating immune complexes is more prominent in SLE patients from Sudan than Sweden.

The role of anti-nuclear autoantibody (ANA) specificities in immune complexes (IC) formation has been studied to a limited extent in SLE, and not at all in African SLE patients. We compared ANA in IC from Sudanese and Swedish SLE patients. We included 93 Sudanese and 332 Swedish SLE patients fulfilling the 1982 ACR criteria. IC were captured using C1q-coated beads. ANA specificities were quantified in sera and IC. Results were related to modified SLEDAI. Whereas serum levels of anti-Sm, anti-dsDNA and anti-ribosomal P were higher in Swedish patients, IC levels of most ANA specificities were higher among Sudanese patients. This difference was especially prominent for anti-chromatin antibodies, which remained after adjustment for age, disease duration and treatment. Total levels of C1q-binding IC correlated with levels of specific ANA in IC, with highest correlations for anti-chromatin antibodies among Sudanese patients. Whereas occurrence of anti- SSA/Ro60, anti-histone and anti-U1RNP in both serum and IC associated with high SLEDAI score, anti-dsDNA in IC but not in serum associated with high SLEDAI. ANA, especially antibodies targeting chromatin, accumulate more in IC from Sudanese SLE patients. If the autoantibody fraction forming IC is pathogenically important, this might explain the generally described severe SLE in black populations.

High IgA antiphospholipid autoantibodies in healthy Sudanese explain the increased prevalence among Sudanese compared to Swedish systemic lupus erythematosus patients.

OBJECTIVES: IgA antiphospholipid antibodies (aPL) are prevalent in systemic lupus erythematosus (SLE) patients of African American, Afro-Caribbean and South African origin. Nevertheless, data from North Africa are lacking, and most studies use manufacturer-suggested cut-offs based on Caucasian controls. Therefore, we compared aPL isotypes in Sudanese and Swedish SLE patients using nation-based cut-offs. METHODS: Consecutive SLE patients and age- and sex-matched controls from Sudan (N = 115/106) and Sweden (N = 340/318) were included. All patients fulfilled the 1982 American College of Rheumatology SLE classification criteria. Antiphospholipid syndrome-related events were obtained from patients' records. IgA/G/M anticardiolipin and anti-ß2 glycoprotein I (ß2GPI) were analysed with two independent assays. IgA anti-ß2GPI domain 1 (D1) was also investigated. Manufacturers' cut-offs and the 95th and 99th percentile cut-offs based on national controls were used. RESULTS: Sudanese patients and controls had higher levels and were more often positive for IgA aPL than Swedes when using manufacturers' cut-offs. In contrast, using national cut-offs, the increase in IgA aPL among Sudanese patients was lost. Occurrence of IgA anti-D1 did not differ between the countries. Venous thromboses were less common among Sudanese patients and did not associate with aPL. No clinical associations were observed with IgA anti-ß2GPI in Sudanese patients. Thromboses in Swedes were associated with IgG/M aPL. Fetal loss was associated with aPL in both cohorts. CONCLUSIONS: IgA anti-ß2GPI prevalence was higher among Sudanese compared to Swedish patients when manufacturers' cut-offs were used. This situation was reversed when applying national cut-offs. Anti-D1 was not increased in Sudanese patients. Previous studies on populations of African origin, which demonstrate a high prevalence of IgA aPL positivity, should be re-evaluated using a similar cut-off approach.

Sudanese and Swedish patients with systemic lupus erythematosus: immunological and clinical comparisons.

OBJECTIVE: SLE is known to have an aggressive phenotype in black populations, but data from African cohorts are largely lacking. We therefore compared immunological and clinical profiles between Sudanese and Swedish patients using similar tools. METHODS: Consecutive SLE patients from Sudan (n = 115) and Sweden (n = 340) and from 106 Sudanese and 318 Swedish age- and sex-matched controls were included. All patients fulfilled the 1982 ACR classification criteria for SLE. Ten ANA-associated specificities and C1q-binding immune complexes (CICs) were measured. Cut-offs were established based on Sudanese and Swedish controls, respectively. Disease activity was measured with a modified SLEDAI and organ damage with the SLICC Damage Index. In a nested case-control design, Swedish and Sudanese patients were matched for age and disease duration. RESULTS: Females constituted 95.6% and 88.1% of Sudanese and Swedish patients, respectively (P = 0.02), with younger age at inclusion (33 vs 47.7 years; P < 0.0001) and shorter disease duration (5 vs 14 years; P < 0.0001) among Sudanese patients. Anti-Sm antibodies were more frequent in Sudanese patients, whereas anti-dsDNA, anti-histone and CICs were higher in Swedish patients. In the matched analyses, there was a trend for higher SLEDAI among Swedes. However, Sudanese patients had more damage, solely attributed to high frequencies of cranial/peripheral neuropathy and diabetes. CONCLUSION: While anti-Sm is more common in Sudan than in Sweden, the opposite is found for anti-dsDNA. Sudanese patients had higher damage scores, mainly because of neuropathy and diabetes. Sudanese patients were younger, with a shorter SLE duration, possibly indicating a more severe disease course with impact on survival rates.

Occurrence of anti-CCP2 and RF isotypes and their relation to age and disease severity among Sudanese patients with rheumatoid arthritis.

OBJECTIVE: Anti-cyclic citrullinated peptide 2 antibodies (anti-CCP2) and rheumatoid factor (RF) in rheumatoid arthritis (RA) has been extensively assessed in industrialized countries. We investigated the diagnostic and prognostic impact of anti-CCP2 and RF isotypes in a Sudanese cross-sectional RA cohort. METHODS: Consecutive RA patients (n = 281) diagnosed according to the 1987 ACR criteria were included 2008-2010. Anti-CCP2 and RF isotypes (IgA, IgM, and IgG) were measured by enzyme immunoassay in 262 patients, with reference intervals aligned to the same diagnostic specificity as for anti-CCP2 (97.6%) using national controls. RESULTS: IgA RF was the predominant RA-associated autoantibody (56%), followed by IgM RF and anti-CCP2 (both 52%) and IgG RF (49%). In receiver operator characteristic analysis, IgA RF also showed the largest area under the curve. Patients with IgG RF were younger and had 8 years lower median age of disease onset compared to antibody negative patients (p < 0.0001). IgG RF was the only marker associated with a high number of involved joints (p = 0.028), and together with anti-CCP2 were the strongest markers for finger deformities (p = 0.016 and p = 0.012), respectively. No statistical differences were found for disease duration, ESR and Hb levels, and occurrence of erosions/osteopenia for any of the investigated autoantibodies. CONCLUSION: Whereas IgA RF showed the best diagnostic performance, IgG RF associated with low age of RA onset, high number of involved joints, and finger deformities. These findings indicate that RA-associated antibodies other than conventional IgM RF and anti-CCP2 might be informative in non-Caucasian RA populations.

The genetically determined production of the alarmin eosinophil-derived neurotoxin is reduced in visceral leishmaniasis.

Visceral leishmaniasis (VL) is the most severe form of leishmaniasis. Recent findings indicate that dendritic cells have a key role in the defense against the Leishmania parasite and that the activity of this cell may be modified by the eosinophil secretory protein eosinophil-derived neurotoxin (EDN). We hypothesized that the interactions between dendritic cells and EDN might be of importance in the disease development. Cellular content of EDN was analyzed by ELISA. The single-nucleotide polymorphisms at positions 405, 416, and 1122 in the EDN gene were analyzed by real-time PCR with TaqMan® reagents. The study cohorts comprised 239 Sudanese subjects (65 healthy controls and 174 with VL) and 300 healthy Swedish controls. The eosinophil content of EDN was lower in VL as compared with controls (p < 0.0001). The EDN405 (G>C) genotype distribution was similar among Swedish and Sudanese controls, whereas VL subjects had a higher prevalence of the EDN405-GG genotype (p < 0.0001). The content of EDN in the eosinophils was closely linked to the EDN405 polymorphism (p = 0.0002). Our findings suggest that the predisposition to acquire VL is related to the genetic polymorphism of the EDN gene and the reduced production by the eosinophil of this gene product.

Active Rheumatoid Arthritis in Central Africa: A Comparative Study Between Sudan and Sweden.

OBJECTIVE: To compare clinical characteristics and treatment between simultaneously investigated Sudanese and Swedish outpatients with rheumatoid arthritis (RA). METHODS: Outpatients with RA from Sudan (n = 281) and Sweden (n = 542) diagnosed according to the 1987 American College of Rheumatology criteria were recruited between December 2008 and September 2010 and compared concerning clinical presentation, treatment, and laboratory findings, including immunoglobulin M with rheumatoid factor (IgM-RF). RESULTS: Sudanese patients had lower inclusion age (median 49 vs 68 yrs), disease duration (48 vs 107 mos), and disease onset age (43 vs 56 yrs) as compared with Swedish patients (p < 0.0001 for all). When stratified concerning the age of inclusion, Swedish patients between 41-50 years had, however, a significantly lower age of onset, with a similar trend for all age groups above 30 years. The female preponderance was higher among Sudanese patients (89.3% vs 72.5%, p < 0.0001), and smoking was nonexistent among Sudanese female patients (p < 0.0001). Erythrocyte sedimentation rate levels and number of tender joints were significantly higher among Sudanese patients. The proportion of IgM-RF positivity was lower among Sudanese patients with RA (52.4% vs 75.5%, p < 0.0001). Higher proportions of Sudanese patients with RA were treated with methotrexate (MTX) and disease-modifying antirheumatic drug combinations, but none of them used biologics. Sudanese patients used lower doses of MTX and sulfasalazine (p < 0.0001) and higher doses of prednisolone (p < 0.0001) than Swedish patients. CONCLUSION: Sudanese patients with RA have significantly higher disease activity and are often IgM-RF-seronegative. Together with reports from Uganda and Cameroon, our data indicate a cluster of highly active and often seronegative RA in central Africa.

General false positive ELISA reactions in visceral leishmaniasis. Implications for the use of enzyme immunoassay analyses in tropical Africa.

Leishmaniasis is a neglected disease in tropical countries. Clinical and laboratory features may mimic autoimmune diseases and this can complicate the Leishmania diagnosis. Due to our previous investigation for false anti-CCP2 reactivity in Leishmania-infected subjects and our interest in immunity against the joint-specific collagen type II (CII) in rheumatoid arthritis (RA) we investigated the same cohort for anti-CII antibodies. We found elevated anti-CII reactivity in Leishmania-infected patients as compared to controls. When anti-CII OD values were compared with BSA-blocked control plates we found higher reactivity against BSA than in CII-coated plates in many Leishmania-infected patients. The percentage of such false positive anti-CII reactions increased with inflammatory activity, and was found in almost all Leishmania patients with highly active inflammatory disease, but was as low in Sudanese healthy controls as well as among Swedish RA patients. The correlation coefficients between false positive anti-CII and anti-CCP2 measured with a commercial ELISA were highest for patients with the most inflammatory disease but non-significant for Sudanese controls and Swedish RA patients, arguing that our findings may have general implications for ELISA measurements in leishmaniasis. ELISA investigations in areas endemic for leishmaniasis might benefit from individual-specific control wells for each serum sample. This approach might also be applicable to other geographical areas or patient groups with high incidence of inflammatory and infectious diseases.

Activity and turnover of eosinophil and neutrophil granulocytes are altered in visceral leishmaniasis.

Visceral leishmaniasis (VL) is a health issue in Sudan. Our aim was to investigate the involvement of eosinophils and neutrophils in VL by serum and plasma measurements of eosinophil cationic protein (ECP) and myeloperoxidase (MPO) and some key cytokines and chemokines. Blood was collected from 125 VL patients and 181 healthy Sudanese controls from the same rural area. Results showed reduced eosinophil and neutrophil counts in the VL group (P=0.0001 and P=0.002, respectively). Serum-ECP levels were higher in the controls (P<0.0001), while plasma MPO levels were higher in the VL group (P<0.0001). Levels of IL-5, granulocyte macrophage-colony stimulating factor (GM-CSF) and IL-17 were increased among the VL group (P<0.0001, P=0.017 and P=0.03, respectively), whereas eotaxin and IL-8 levels were reduced (P<0.0001 and P=0.002, respectively). Positive correlations were found between IL-8 and ECP/MPO (P<0.0001). We conclude that eosinophil and neutrophil turnover and activity are increased in subjects in rural areas of Sudan. In VL the turnover was further increased, but the relatively low secretory activity of eosinophils and neutrophils in VL may relate to the reduced production and availability of the chemokines eotaxin and IL-8. The combined assay of ECP and MPO in serum and plasma provides further insight into the mechanisms of eosinophil and neutrophil involvement in disease and constitutes a novel approach to the study of disease processes.

Profiling of copy number variations (CNVs) in healthy individuals from three ethnic groups using a human genome 32 K BAC-clone-based array.

To further explore the extent of structural large-scale variation in the human genome, we assessed copy number variations (CNVs) in a series of 71 healthy subjects from three ethnic groups. CNVs were analyzed using comparative genomic hybridization (CGH) to a BAC array covering the human genome, using DNA extracted from peripheral blood, thus avoiding any culture-induced rearrangements. By applying a newly developed computational algorithm based on Hidden Markov modeling, we identified 1,078 autosomal CNVs, including at least two neighboring/overlapping BACs, which represent 315 distinct regions. The average size of the sequence polymorphisms was approximately 350 kb and involved in total approximately 117 Mb or approximately 3.5% of the genome. Gains were about four times more common than deletions, and segmental duplications (SDs) were overrepresented, especially in larger deletion variants. This strengthens the notion that SDs often define hotspots of chromosomal rearrangements. Over 60% of the identified autosomal rearrangements match previously reported CNVs, recognized with various platforms. However, results from chromosome X do not agree well with the previously annotated CNVs. Furthermore, data from single BACs deviating in copy number suggest that our above estimate of total variation is conservative. This report contributes to the establishment of the common baseline for CNV, which is an important resource in human genetics.

Circulating immune complexes (IC) and IC-induced levels of GM-CSF are increased in sudanese patients with acute visceral Leishmania donovani infection undergoing sodium stibogluconate treatment: implications for disease pathogenesis.

Infection with Leishmania donovani is associated with IL-10 as well as with GM-CSF. Immune complexes (IC) exert important functions by stimulation of monocytes/macrophage-mediated production of pro- and anti-inflammatory cytokines in rheumatic diseases. In this investigation, we have explored IC-induced cytokine production during Leishmania infection. Sera from 43 patients with visceral leishmaniasis (VL), 17 patients with post-kala-azar dermal leishmaniasis, and 20 healthy Sudanese controls were precipitated with polyethylene glycol (PEG). The PEG precipitates were added to serum-free PBMC for 20 h,whereupon supernatant levels of IL-1beta, IL-6, IL-10, IL-1 receptor antagonist protein, TNF-alpha, TNF receptor p75, and GM-CSF were investigated using ELISA. Circulating levels of C1q-binding IC were also measured in the serum samples. PEG precipitates from Leishmania-infected patients induced significantly higher levels of GM-CSF (p = 0.0037) and IL-10 (p < 0.0001), as well as of IL-6 (p < 0.0001) and IL-1 receptor antagonist (p = 0.0238) as compared with PEG precipitates from controls. Patients with acute VL as well as VL patients receiving sodium stibogluconate treatment displayed significantly increased levels of PEG precipitate-induced GM-CSF. The induction of GM-CSF by circulating IC was especially prominent in acute VL patients receiving sodium stibogluconate treatment; ANOVA revealed significant interaction between disease activity and treatment for PEG precipitate-induced levels of GM-CSF (disease activity, p = 0.0006; treatment, p = 0.0005; interaction, p = 0.0046). Parallel associations were determined for C1q-binding immune complexes, but not for any cytokine other than GM-CSF. The importance of IC-induced GM-CSF in leishmaniasis warrants further study.

Cystatin C as a marker of immune complex-associated renal impairment in a Sudanese population with visceral leishmaniasis.

Renal function was studied in visceral leishmaniasis (VL) and post-kala-azar dermal leishmaniasis (PKDL) by means of the specific marker cystatin C and related to circulating immune complexes and cytokine production. Forty patients with VL (23 with sub-acute disease and 17 with acute disease), 17 patients with PKDL, and 22 healthy controls were included. Cystatin C, but not creatinine, was significantly raised in VL (P = 0.004). The highest levels of cystatin C were found in those with acute disease (P < 0.0001). In VL, cystatin C levels were positively correlated to circulating immune complexes and production of granulocyte-macrophage colony stimulating factor (GM-CSF), but negatively correlated to aspartate aminotransferase and lactate dehydrogenase. We conclude that cystatin C is a superior marker of glomerular function in leishmaniasis and that immune complex deposition and GM-CSF are two functions that most likely are causally involved in the mechanisms leading to glomerular dysfunction in leishmaniasis.