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Complement C3 activation contributes to COVID-19 pathology, and C3 targeting has emerged as a promising therapeutic strategy. We provide interim data from ITHACA, the first randomized trial evaluating a C3 inhibitor, AMY-101, in severe COVID-19 (PaO2/FiO2 ≤ 300 mmHg). Patients received AMY-101 ( n = 16) or placebo ( n = 15) in addition to standard of care. AMY-101 was safe and well tolerated. Compared to placebo (8 of 15, 53.3%), a higher, albeit nonsignificant, proportion of AMY-101–treated patients (13 of 16, 81.3%) were free of supplemental oxygen at day 14. Three nonresponders and two placebo-treated patients succumbed to disease-related complications. AMY-101 significantly reduced CRP and ferritin and restrained thrombin and NET generation. Complete and sustained C3 inhibition was observed in all responders. Residual C3 activity in the three nonresponders suggested the presence of a convertase-independent C3 activation pathway overriding the drug’s inhibitory activity. These findings support the design of larger trials exploring the potential of C3-based inhibition in COVID-19 or other complement-mediated diseases.

The current dynamics of the COVID-19 pandemic have become a serious concern with the emergence of a series of mutant variants of the SARS-CoV-2 virus. Unlike the previous strain, it is reported that the descendants are associated with increased risk of transmission yet causing less impact in terms of hospital admission, the severity of illness, or mortality. Moreover, the vaccine efficacy is also not believed to vary among the population depending on the variants of the virus and ethnicity. It has been determined that the mutations recorded in the spike gene and protein of the newly evolved viruses are specificallyresponsible for this transformation in the behavior of the virus and its disease condition. Hence, this study aimed to compare the immunogenic profiles of the spike protein from the latest variants of the SARS-CoV-2 virus concerning the probability of COVID-19 severity. Genome sequences of the latest SARS-CoV-2 variants were obtained from GISAID and NCBI repositories. The translated protein sequences were run against T-cell and B-cell epitope prediction tools. Subsequently, antigenicity, immunogenicity, allergenicity, toxicity, and conservancy of the identified epitopes were ascertained using various prediction servers. Only the non-allergic and non-toxic potential epitopes were matched for population relevance by using the Human Leucocyte Antigen population registry in IEDB. Finally, the selected epitopes were validated by docking and simulation studies. The evaluated immunological parameters would concurrently reveal the severity of COVID-19, determining the infection rate of the pathogen. Our immunoinformatics approach disclosed that spike protein of the five variants was capable of forming potential T and B-cell epitopes with varying immune responses. Although the Wuhan strain showed a high number of epitope/HLA combinations, relatively less antigenicity and higher immunogenicity results in poor neutralizing capacity, which could be associated with increased disease severity. Our data demonstrate that increased viral antigenicity with moderate to high immunogenicity, and several potential epitope/HLA combinations in England strain, the USA, India, and South Africa variants, could possess a high neutralizing ability. Therefore, our findings reinforce that the newly circulating variants of SARS-CoV-2 might be associated with more infectiousness and less severe disease condition despite their greater viremia, as reported in the recent COVID-19 cases, whichconsequently determine their increased epidemiological fitness.

In order to develop a new type of improved wound dressing, we combined the wound healing properties of nanotitania with the advantageous dressing properties of nanocellulose to create three different hybrid materials. The hemocompatibility of the synthesized hybrid materials was evaluated in an in vitro human whole blood model. To our knowledge, this is the first study of the molecular interaction between hybrid nanotitania and blood proteins. Two of the hybrid materials prepared with 3 nm colloidal titania and 10 nm hydrothermally synthesized titania induced strong coagulation and platelet activation but negligible complement activation. Hence, they have great potential as a new dressing for promoting wound healing. Unlike the other two, the third hybrid material using molecular ammonium oxo-lactato titanate as a titania source inhibited platelet consumption, TAT generation, and complement activation, apparently via lowered pH at the surface interface. It is therefore suitable for applications where a passivating surface is desired, such as drug delivery systems and extracorporeal circuits. This opens the possibility for a tailored blood response through the surface functionalization of titania.

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-β2glycoprotein I (anti-β2GPI) 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-β2GPI 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-β2GPI performed better than conventional triple positivity. Future studies need to address if aPS/PT can replace LA, as this would simplify clinical procedures.

Objective The role of antiphospholipid antibodies (aPL) during apparently normal pregnancy is still unclear. IgA aPL are prevalent in populations of African origin. Our aim was to measure all isotypes of anticardiolipin (anti-CL) and anti–β2 glycoprotein I (anti-β2GPI) in healthy pregnant and non-pregnant women of different ethnicities. Methods Healthy Sudanese pregnant women ( n = 165; 53 sampled shortly after delivery), 96 age-matched Sudanese female controls and 42 healthy pregnant and 249 non-pregnant Swedish women were included. IgA/G/M anti-CL and anti-β2GPI were tested at one time point only with two independent assays in Sudanese and serially in pregnant Swedes. IgA anti-β2GPI domain 1 and as controls IgA/G/M rheumatoid factor (RF), IgG anti–cyclic citrullinated peptide 2 (anti-CCP2) and anti–thyroid peroxidase (anti-TPO) were investigated in Sudanese females. Results Pregnant Sudanese women had significantly higher median levels of IgA anti-CL, IgA anti-β2GPI ( p 2GPI (both assays; p 2GPI occurrence was increased among Sudanese pregnant women compared with national controls. No corresponding increase during pregnancy was found for IgA anti-β2GPI domain 1 antibodies. Both IgG anti-CL and IgG control autoantibodies decreased during and directly after pregnancy among Sudanese. Serially followed Swedish women showed no changes in IgA aPL, whereas IgG/M anti-CL decreased. Conclusions IgA aPL are increased in Sudanese but not in Swedish women, without corresponding increase in IgA domain 1. Whether due to ethnicity and/or environmental influences the occurrence of IgA aPL during Sudanese pregnancies, and its clinical significance, is yet to be determined.

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-β2 glycoprotein I (β2GPI) were analysed with two independent assays. IgA anti-β2GPI 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-β2GPI in Sudanese patients. Thromboses in Swedes were associated with IgG/M aPL. Fetal loss was associated with aPL in both cohorts. Conclusions IgA anti-β2GPI 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.

Zwitterionic material-based polymer brush significantly prevents protein adsorption and cell adhesion, which leads to the blood compatibility. However, zwitterionic polymer itself is difficult to be modified further, for the blood compatibility since the charged balance is impaired after the modification. In this research, chemically modifiable mixed charge polymer brush is designed, without impairing its characteristics. Condensed mixed charge polymer brush will work like zwitterionic material because neighbouring opposite charge is reported to be important in the zwitterionic material. Cationic polymer brush with primary amine group, which is based on 2-aminoethyl methacrylate (AEMA), was prepared and modified by succinic anhydride to obtain carboxylic group induced poly(AEMA). The ratio of primary amine group and carboxylic group was optimized to obtain the polyampholyte brush. The blood compatibility was evaluated by measuring coagulation/complement activation, protein adsorption and cell adhesion induced by the polymer. Our designed cationic-based polyampholyte brush prevented coagulation/complement activation comparable to poly(2-methacryloyloxyethyl phosphorylcholine) brush, based on intra-monomer interaction, because condensed mix charge works like zwitterion.

Pentraxin 3 (PTX3) is a locally produced multifunctional protein involved in inflammation, matrix deposition, and immunity. As patients with seropositive rheumatoid arthritis (RA) have a more severe disease course and higher risk of joint destruction than seronegative patients, the aim of the present study was to examine differences in PTX3 in synovial fluid (SF) (and serum) in seropositive compared to seronegative RA, and other local markers of inflammation and destruction.Ninety-seven RA patients with knee effusion were included. Serum and SF levels of PTX3, as well as serum levels of anti-citrullinated protein antibody and rheumatoid factor of immunoglobulin A and M subclasses, and markers of inflammation and potential destruction in SF: white blood cell counts, tumour necrosis factor, interleukin-6, vascular endothelial growth factor, metalloproteinase 3, and cartilage oligomeric matrix protein, were analysed. In addition, a radiographic knee examination was performed.Seropositive patients had significantly higher PTX3 levels in SF than seronegative patients, whereas there was no difference for serum levels. SF-PTX3 levels correlated with disease activity and with local inflammatory markers, especially polymorphonuclear cells, and with autoantibody levels. There was no correlation between PTX3 levels in serum and SF.The correlation of disease activity and autoantibody levels with SF-PTX3 levels in antibody-positive patients suggests a role for PTX3 in the inflammatory process specifically in seropositive RA joints, and supports the hypothesis that seropositive and seronegative RA are different disease entities. Polymorphonuclear granulocytes may be an important source of PTX3 in RA SF.

Background Recent studies have shown divergent associations between rheumatoid arthritis (RA)-associated autoantibodies and RA onset phenotype. Whereas some studies report more inflammation in seronegative patients [1, 2], others report that rheumatoid factor (RF) associates with high baseline disease activity [3]. Objectives To define how individual RA-associated autoantibodies associate with individual disease activity score (DAS28, DAS28CRP) components at the time of RA diagnosis. Methods Sixteen individual ACPA reactivities, anti-CCP2, IgA-, IgG- and IgM-RF were analyzed centrally in baseline sera from 1600 RA patients classified according to the 1987 ACR criteria. All antibody cut-offs were determined as the 98th percentile among controls. The results were related to CRP, ESR, number of swollen and tender joints, DAS28, DAS28CRP, global disease activity (visual analogue scale), and health assessment questionnaire obtained at baseline. Results Individually, anti-CCP2, IgA RF and IgM RF associated with low counts of swollen and tender joints, with strongest association to anti-CCP2, and with high ESR. The association to low joint counts was stronger to number of individual ACPA reactivities than to anti-CCP2 levels (table). In multiple regression adjusted for age, sex and inclusion year, ACPA number associated strictly with low joint counts whereas IgM RF level associated with increased inflammatory markers, especially ESR (table). Also among IgM RF negative patients, occurrence of ACPA associated with low swollen and tender joint counts. Contrary, among ACPA negative patients, IgM showed no association to elevated ESR. The effect of RF on ESR increased with the number of individual ACPA reactivities present, most prominently for IgM RF (figure). Conclusion In early RA, ACPA associate with low counts of affected joints and IgM RF associates with elevated ESR in an ACPA-dependent manner. The latter finding relates to in vitro studies showing enhanced inflammatory effect of ACPA immune complexes with addition of RF[4, 5]. Future RA studies relating autoantibodies to disease activity may benefit from evaluating the impact of individual antibodies as well as distinct DAS components separately. References [1] Barra L, et al. J Rheumatol 2014;41:2361. [2] Nordberg LB, et al. Ann Rheum Dis 2017;76:341. [3] Aletaha D, et al. Arthritis Res Ther 2015;17:229. [4] Laurent L, et al. Ann Rheum Dis 2015;74:1425. [5] Sokolove J, et al. Arthritis Rheumatol 2014;66:813. Disclosure of Interests Eleftheria Pertsinidou: None declared, Vivek Anand Manivel: None declared, Lars Klareskog Grant/research support from: Yes, but not for the presented study., Lars Alfredsson: None declared, Linda Mathsson Employee of: employed by Thermo Fisher Scientific, Monika Hansson: None declared, Martin Cornillet: None declared, Guy Serre: None declared, Rikard Holmdahl: None declared, Karl Skriner: None declared, Per-Johan Jakobsson: None declared, Helga Westerlind: None declared, Johan Askling Grant/research support from: Karolinska Institutet (JA) has or has had research agreements with the following pharmaceutical companies, mainly in the context of the ATRIS national safety monitoring programme for rheumatology biologicals: Abbvie, BMS, MSD, Eli Lilly, Pfizer, Roche, Samsung Bioepis, and UCB., Consultant for: Karolinska Institutet has received remuneration for JA participating in ad boards arranged by Lilly, Novartis, and Pfizer., Saedis Saevarsdottir Employee of: Part-time employee at deCODE Genetics/Amgen Inc, working on genetic research unrelated to this project., Johan Ronnelid: None declared

In the alternative pathway (AP) an amplification loop is formed, which is strictly controlled by various fluid-phase and cell-bound regulators resulting in a state of homeostasis. Generation of the "C3b-like" C3(H

Career situation of first and presenting author Young investigator. Introduction Separate studies have previously observed an association between exposure to physical workload (PW) and risk of developing rheumatoid arthritis (RA). Levels of antibodies against collagen type II (CII), which are associated with HLA-DRB*01 and *03 are transiently elevated at the time of RA diagnosis. PW might impose mechanical stress on the articular cartilage where CII is the main constituent, leading to immune activation and production of anti-CII antibodies. Objectives We hypothesize that exposure to PW would associate more strongly with anti-CII positive RA than with anti-CII negative RA. Methods Data involving 2916 incident RA cases and 5130 controls from the Swedish Epidemiological Investigations in Rheumatoid Arthritis (EIRA) population-based case-control study were analysed. Information on exposure to PW was collected through questionnaires. The odds ratios (OR) with 95% CI of developing anti-CII positive RA or anti-CII negative RA was calculated using logistic regression. Results The OR observed for the association between different types of PW and anti-CII positive RA ranged from 1.2 (0.8–1.8) to 1.7 (1.5–3.1). The OR for the association between PW and anti-CII negative RA ranged from 1.3 (1.1–1.4) to 1.6 (1.4–1.8). No statistically significant difference was observed between the OR for anti-CII positive RA and anti-CII negative RA (all p-values>0.10). Moreover, stratification for HLA-DRB*01 and *03 did not yield any statistical differences. Conclusions We found no evidence suggesting an association between PW and elevated anti-CII levels at the time of diagnosis. Acknowledgements We thank the investigators and participants from the EIRA study. The authors also thank Marie-Louise Serra and Lena Nise for data collection. Disclosure of Interest None declared.

Background:Antiphospholipid antibodies (aPL) of IgA isotype are prevalent in SLE patients of African-American, Afro-Caribbean and South-African origin, but data from North Africa are lackingObjectives:To compare and determine clinical significance of aPL isotypes in Sudanese and Swedish SLE patients using nation-based cutoffsMethods:Consecutive Sudanese (n=115) and Swedish (n=340) SLE patients and 106 and 318 age- and sex-matched national controls were included. All patients fulfilled the 1982 ACR SLE classification criteria. IgA/G/M anti-cardiolipin and anti-β2GPI were analyzed with two independent assays (Fluorescence Enzyme Immuno Assay (FEIA) from Thermo Fisher Scientific and Particle-based Multi-Analyte Technology (PMAT) from Inova Diagnostics). IgA anti-β2GPI domain1 (D1) was investigated with chemiluminescence (CIA), Inova Diagnostics. APS-related events were obtained from patients’ records. Manufacturers’, 95thand 99thpercentile cutoffs, based on national controls calculated by non-parametric methods, were usedResults:Sudanese patients had higher levels and prevalence of IgA aPL using manufacturers’ cutoffs (Table 1 and Figure 1). IgA/IgG aPL were also higher among Sudanese controls, But, Swedish patients were more often positive for IgA anti-β2GPI with both assays when national cutoffs were applied (Table 1). Occurrence of IgA anti-D1 did not differ between the countries. Venous thromboses were less common among Sudanese patients and no aPL, including IgA anti-β2GPI associated with thrombosis in Sudanese patients. Thrombosis in Swedes associated with IgG/IgM aPL. Fetal loss associated with aPL in both cohorts most evident after national adjustmentsTable 1.FEIASudann=92FEIASweden n=291PPMAT/CIASudann=93PMAT/CIASweden n=333PIgA CL median/IQR6.5/5.24.0/4.1194/185148/112IgG CL4.1/5.13.0/4.50.0002205/137190/215.10.09IgM CL2.7/6.83.7/5.40.02214/251.5193/35020.2IgA β2GPI6.3/14.22.7/3.8212/347118/152IgG β2GPI4.2/2.91.8/2.3158/110.7145.2/147.50.04IgM β2GPI2.0/3.01.4/2.20.006146.2/189.7132.5/235.40.09IgA B2GP1 D11542/1253.51683/1518.10.045IgA CL common cutoff n(%)9(9.8)17(5.8)0.232(35.2)75(22.7)0.02IgG CL4(4.3)21(6.7)0.414(15)72(22.1)0.1IgM CL3(3.2)14(4.5)0.610(11.4)43(13.1)0.6IgA β2GPI32(34.8)43(14.9)38(41.8)76(23)0.0004IgG β2GPI10(10.9)44(14.1)0.45(5.4)50(15.3)0.01IgM β2GPI10(10.9)27(8.7)0.512(13.6)42(12.9)0.8IgA B2GP1 D16(6.4)38(11.4)0.2IgA CL 95thcutoffn(%)9(9.8)54(18.6)0.04736(39.6)121(26.6)0.6IgG CL8(8.7)42(13.5)0.224(25.8)103(31.6)0.3IgM CL8(8.7)39(12.5)0.316(18.2)47(14.4)0.4IgA β2GPI18(19.6)100(34.7)0.00620(22)125(37.8)0.005IgG β2GPI2(2.1)67(21.5)19(20.4)83(25.5)0.3IgM β2GPI14(15.22)57(18.3)0.520(22.7)57(17.5)0.3IgA B2GP1 D125(26.9)69(20.7)0.2Figure 1.Conclusion:IgA anti-β2GPI levels were generally higher among Sudanese compared to Swedish SLE patients and controls, but after national adjustments more Swedish than Sudanese patients were positive. IgA anti-D1 was not increased in Sudanese patients. Previous studies on populations of African-origin, which demonstrate high prevalence of IgA aPL positivity, should be re-evaluated using similar cutoff approachAcknowledgments: :We thank Maryam Poorafshar at Thermo Fischer Scientific and Silvia Casas and Michael Mahler at Inova Diagnostics for help with analysesDisclosure of Interests:None declared

ANTI-COLLAGEN TYPE II ANTIBODIES ARE ASSOCIATED WITH EARLY INFLAMMATION IN MALAYSIAN RHEUMATOID ARTHRITIS PATIENTS WITH THREE DIFFERENT ETHINICITIES

Anti-Collagen Type Ii Antibodies Are Associated With An Acute Onset Rheumatoid Arthritis Phenotype And Prognosticate Lower Degree Of Inflammation

Granulocyte Reactivity is Associated with Cartilage Destruction in Patients with Anti-collagen Antibodies

The fibrillar protein collagen type II (CII) is essentially confined to hyaline cartilage in diarthrodial joints. Antibodies against CII (anti-CII) were previously described in 3%–27% of rheumatoid arthritis (RA) patients, and Kim et al described anti-CII to be associated with elevated levels of C reactive protein (CRP) and erythrocyte sedimentation rate (ESR) in a heterogeneous group of RA patients with 2–432 months of disease duration.1 Contrary to anticitrullinated protein antibodies, anti-CII are not detected before RA onset.2 We have shown that anti-CII levels are highest at the time of RA diagnosis and thereafter decline, and that elevated anti-CII levels at diagnosis associate with elevated CRP, ESR, swollen joint count, disease activity score and radiological destruction at the time of diagnosis but not later, thus representing an acute onset RA phenotype.3–5 It is plausible that production of pro-inflammatory cytokines by macrophages …

Objectives Cathepsin S and cathepsin L are endosomal proteolytic enzymes involved in the degradation of extracellular matrixes, angiogenesis and antigen presentation. Cathepsins could thus play several roles in the disease process of RA. The aim of this study was to examine differences in cathepsin S and cathepsin L levels in serum and SF of RA patients with and without ACPA and RF. Methods In this study 121 patients with RA and clinical signs of knee synovitis were recruited. Patient characteristics were collected and matched samples of serum and SF were analysed for cathepsin S, cathepsin L, ACPA, IgA and IgM RF, CRP and MMP3. Results SF levels of cathepsin L, cathepsin S and MMP3 were significantly higher than in serum. Serum levels of both cathepsins were significantly higher in patients with ACPA, IgM-RF and IgA-RF compared with patients without these antibodies. SF levels of both cathepsins correlated with DAS28 and CRP in ACPA- and RF-positive but not in seronegative patients. Conclusion The differences in cathepsin S and cathepsin L between RA patients with and without autoantibodies indicate that these cathepsins have a specific role in the disease process of seropositive RA. In this phenotype, cathepsin serum levels may reflect the autoimmune activity, whereas the levels in SF may reflect the local inflammatory and matrix degrading process in the joint.

Background RA patients with anti-collagen type II (CII) antibodies have a distinct acute onset phenotype, associated with cytokine induction by surface-bound anti-CII immune complexes (IC). Mononuclear cells and PMN are accumulated both in the synovial fluid and tissue, in close proximity to hyaline cartilage where anti-CII IC might form. Therefore we wanted to investigate to what extent these two cell types cooperate in anti-CII IC induced responses. Methods Healthy donor granulocytes (PMN) and PBMC were stimulated individually or together (cocultures) with surface-bound IC (anti-CII IC, tetanus toxoid (TT)/anti-TT IC, directly bound IgG), GM-CSF or LPS. Blocking and neutralising studies were performed with antibodies against TLR4, FcgγRIIa, FcgγRIII and GM-CSF. Supernatant cytokine and chemokine levels were analysed with ELISA or ALBIA. Results Anti-CII IC induced cytokine production by PBMC, whereas PMN alone produced negligible cytokine levels. TNF-aα production was downregulated in all coculture systems compared to PBMC cultures, whereas levels of CXCL8, RANTES and MCP-1 were specifically upregulated in anti-CII IC-stimulated cocultures. The CXCL8 upregulation was dependent on anti-CII IC, as CXCL8 production was downregulated in cocultures stimulated with other IC or LPS. The increase of CXCL8 in anti-CII IC stimulated cocultures was totally dependent on TLR4, partly on PMN enzymes, FcgγRIIa, FcgγRIII, and density of anti-CII in IC. Like anti-CII IC, GM-CSF induced coculture-dependent CXCL8 enhancement, and GM-CSF neutralisation diminished the anti-CII IC-dependent CXCL8 enhancement. Conclusion In anti-CII-positive RA patients, PMN amplify accumulation of inflammatory cells by inducing chemokines. This mechanism is dependent on TLR4, PMN enzymes, GM-CSF and the joint-specific autoantigen CII. Local TLR4 blockade, PMN enzyme inhibition or GM-CSF neutralisation might be used to suppress acute joint inflammation in early RA.

Interleukin-2 (IL-2) is an immunoregulatory cytokine whose biological effects are mediated through interaction with specific receptors on the surface of target cells. Due to its presumed role in generating a normal immune response, IL-2 is being evaluated for the treatment of a variety of tumors, in addition to infectious diseases. Main drawback of human IL-2 is that the molecule is relatively unstable. Therefore, with the objective of increasing the stability of the molecule, site directed mutagenesis of human IL-2 gene was carried out. Early studies indicated that mutations at three Cysteine residues (58, 105, 125) which are in the active sites of human IL-2 resulted in the reduced stability as well as the biological activity of the molecule. Therefore, mutations were carried out at the positions of amino acid other than the receptor binding sites at 111Valine to Arginine, 117Lysine to Glutamine and 133 Threonine to Asparagine of the human sequence by comparing it with the bovine sequence which has higher stability than the human counterpart, using SWISS PDB tool. To understand the biological activity of the mutated IL-2, energy minimization studies were carried out using SWISS-PDB. Docking studies were performed to check the reliability of the results using HEX DOCK, ARGUS LAB and PATCH DOCK between the IL-2 receptor and its mutated Ligand. These docking results also confirmed that the reliability of these mutated IL-2 gene. Stability, half life and ADME characteristics of these mutants can be studied in a detailed manner in the in vivo studies.

Anti-type II collagen immune complex-induced granulocyte reactivity is associated with joint erosions in ra patients with anti-collagen antibodies

Background and Objectives Reports from Nigeria claim that rheumatoid arthritis (RA) in Western Africa has relatively low disease activity with a 29 occurrence of hand erosions No data are published on RA in Sudan and our aim was to collect a first Sudanese RA cohort for comparative studies. Materials and Methods 264 consecutive patients (87% females) with RA according to the 1987 ACR criteria attending two rheumatology centres in Khartoum between December 2008 and September 2010 were included. Samples analysed in Uppsala for anti-CCP and IgM RF. Results The mean age at inclusion was 48 years (range 14–80). Median disease duration was 3.8 years. ESR data was available for 113 patients, with a mean of 60.3 mm/1 h (range 10–140). Mean blood haemoglobin was 12.1 g/L. On clinical examination, 26% (68/264) had Z deformity, 14% (38/264) had Swan neck deformity and 9% (25/264) had Boutonniere deformity. X-rays of hands were available for 86 patients, with 49/86 (57%) showing erosions. 40% were treated with methotrexate, 7% with sulfasalazine 3% azathioprine, 2% with leflunomide and 2% with hydroxychloroquine in monotherapy. 41 (16%) were treated with steroids + DMARD monotherapy, 48 (18%) with DMARD combinations. Three % were treated with steroids only, and 9% with NSAIDs only. 52% were anti-CCP2 positive and 51% were IgM RF positive, corresponding to 97.6% specificity compared to the Sudanese healthy controls. Compared to Swedish RA patients (Ronnelid et al , ARD 2012) Sudanese patients had 270% higher mean ESR (55 versus 21 mm/h; p Conclusions RA as presented in an outpatient clinic in Khartoum is severe and with earlier RA onset than in Sweden. Sudanese patients show significantly higher ESR levels than Swedish patients, more Sudanese than Nigerian RA patients have radiological erosions, and the number of patients with classical hand deformities is substantial. Blood haemoglobin levels are rather well preserved. Immunological and genetic characterisation is now underway.

IgA Rheumatoid Factor is more Predominant than anti-CCP in Sudanese Rheumatoid Arthritis Patients, whereas IgG RF is a Strong Prognostic Marker and Associated with Early Onse

Background and Objectives We have earlier shown that surface-bound immune complexes containing anti-type II collagen antibodies (anti-CII IC) from rheumatoid arthritis (RA) patients and anti-CII IC stimulate monocyte proinflammatory cytokine production, associated with an acute onset RA phenotype. Anti-CII IC in joint cartilage are exposed to cells in the synovial fluid (SF). Neutrophil granulocytes are the major cell type in SF, where they co-localise with mononuclear cells (MNC). The objective was to investigate whether also granulocytes respond to anti-CII IC, and whether such a response was dependent on interaction with other cells in SF. Materials and Methods An anti-CII RA serum together with human native collagen (CII) was used to create surface-bound anti-CII IC. Heparinised blood from 8 healthy donors was separated into neutrophil granulocytes (>95% purity) and MNC. For each donor, the granulocyte cell fractions as well as co-cultures (granulocytes + MNC) (0.5 × 10E6/ml of each cell fraction) was cultured on anti-CII IC as well as on negative control IC prepared with normal human serum on CII and in a positive control IC system with purified IgG coated onto plastic. After 18 hours, cells were harvested for the measurement of CD11b, CD66b, CD16 and CD32 on granulocytes by flow cytometry, and supernatant levels of TNF and IL-8 was measured by ELISA. Results In granulocyte cultures both anti-CII IC and control IC induced significant up-regulation of CD11b and CD66b, and significant down-regulation of CD16 and CD32. When the granulocytes were co-cultured with MNC, there was a significant increase in CD11b up-regulation and CD16 down-regulation than granulocytes, with no effect on CD32 and CD66b. In the co-culture system, the anti-CII IC-induced production of IL-8 was significantly increased, but no such difference was noted for TNF. Isolated granulocyte fractions produced very low levels of TNF and IL-8 after IC stimulation. Conclusions Isolated granulocytes respond to RA anti-CII IC in a model system mimicking IC in RA cartilage. The granulocyte responses depend on interaction with MNC. Our anti-CII dependent RA phenotype is a human counterpart to collagen antibody-induced arthritis. Strong granulocyte reactivity to anti-CII IC might therefore be related to the Ncf1 gene involved in NADPH activity important in collagen-induced arthritis models.