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Backgroundː Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), within few months of being declared as a global pandemic by WHO, the number of confirmed cases has been over 75 million and over 1.6 million deaths since the start of the Pandemic and still counting, there is no consensus on factors that predict COVID-19 case progression despite the diversity of studies that reported sporadic laboratory predictive values predicting severe progression. We review different biomarkers to systematically analyzed these values to evaluate whether are they are correlated with the severity of COVID-19 disease and so their ability to be a predictor for progression. Methods The current meta-analysis was carried out to identify relevant articles using eight different databases regarding the values of biomarkers and risk factors of significance that predict progression of mild or moderate cases into severe and critical cases. We defined the eligibility criteria using a PICO model. Results Twenty-two relevant articles were selected for meta-analysis the following biomarkers C-reactive protein, interleukin-6, LDH, neutrophil, %PD-1 expression, D-dimer, creatinine, AST and Cortisol all recorded high cut-off values linked to severe and critical cases while low lymphocyte count, and low Albumin level were recorded. Also, we meta- analyzed age and comorbidities as a risk factors of progression as hypertension, Diabetes and chronic obstructive lung diseases which significantly correlated with cases progression (p Conclusions ː The current meta-analysis is the first step for analysing and getting cut-off references values of significance for prediction COVID-19 case progression. More studies are needed on patients infected with SARS-CoV-2 and on a larger scale to establish clearer threshold values that predict progression from mild to severe cases. In addition, more biomarkers testing also help in building a scoring system for the prediction and guiding for proper timely treatment.

Lung cancer is a leading cause of cancer-associated death in all over the globe. This study was undertaken to determine the expression and interaction of membrane-bound receptors CD74 and CD44 in human lung adenocarcinoma cells and their associated signaling was also attempted. Levels of CD74 and CD44 were studied in human lung adenocarcinoma-evolved cells A549 and H460. CD74-mediated downstream signaling was studied by the nuclear-transcription-factor NF-κB and prostaglandin E (PGE) production. Flow-cytometric analysis showed that both CD74 and CD44 were perfectly expressed in A549 cells. Importantly, Western immunoblotting showed that A549 cells expressed only two isoforms of CD74 at 33 and 35 kDa but isoform at 41 kDa was absent. These results were verified in H460 cells. Confocal microscopy showed CD74 and CD44 was colocalized but heterotypic interaction between them was missing in both A549 and H460 cells. Activation of NF-κB and production of PGE in human lung cancer cells were comparable with other cancer cells. In conclusion, this is the first study that shows A549 and H460 cells expressed two distinctive isoforms of CD74 but isoform at 41 kDa was absent. Due to the absence of this isoform, the direct physical interaction between them CD74 and CD44 was lacking. Furthermore, the data also demonstrated that lacking of direct physical interaction between CD74 and CD44 had no effect on NF-κB activation and PGE production indicating that CD74-mediated downstream signaling occurs either through coreceptors or indirect interaction with CD44 in human lung cancer cells. Abbreviation: CD: cluster of differentiation; SCLC: small cell lung cancer; NSCLC: nonsmall cell lung cancer; SCC: squamous cell carcinoma; ADC: adenocarcinoma; LCC: large cell carcinoma.

Background: Screening of blood products is considered a mandatory protocol implemented in health care facilities in order to reduce the onset of transfusion-transmitted infections (TTIs). This study was aimed to determine the sero-prevalence of ABO and Rh blood groups and their associated TTIs among blood donors in the Central Region of Saudi Arabia. Methods: This was retrospective study performed on the blood donors’ records from March 2017 to December 2018 at Buraidah Central Hospital Blood Bank. Study was conducted on a total of 4590 blood donors. ABO and Rh typing was performed.The blood samples were also screened serologically for hepatitis B surface antigen (HBsAg), anti-hepatitis B core total antibodies (anti-HBc total), hepatitis C virus (HCV), human immunodeficiency viruses (HIV), human T-lymphotrophic virus-1 (HTLV-1) and veneral disease research laboratory test(VDRL) for syphilis. Results: Out of 4590 blood donors, O positive blood group was found to be highest (42%), followed by A positive (23.4%), B positive (20.9%), O negative (5.45%), AB positive (3.4%), A negative (2.8%), B negative (2.1%) and AB negative (0.5%). Moreover, total number of Rh-negative donors was significantly lowered as compared with Rh-positive. Seroreactive tests were found to be positive in only 1.002% of all studied donors and mainly found in male donors. Among TTI, anti-HBc total was the highest (0.784%), followed by HBsAg, HCV, VDRL and TPHA. Whereas all tested donors were found to be negative for HIV infections. Conclusions: The information collected for the frequency of ABO blood phenotypic groups has a vital significance in establishing a simple blood group database. This study clearly determined significantly lower rate of seropositive TTIs among the studied blood donors but still steps are needed to improve the knowledge and to prevent the seropositive occurrence of TTIs. Keywords: Blood group, TTIs, Anti-HBc total, HBsAg, HCV, VDRL, KSA

Anti-DNA antibodies are now considered as a universal diagnostic feature for the patients with systemic lupus erythematosus (SLE) but the mechanism(s) involved in the generation of these autoantibodies remains to be investigated. Bisphenol A (BPA) is a synthetic phenol extensively used in the manufacturing of polycarbonated plastics. Upon mixing in the diet, it causes several health hazards. This study was undertaken to investigate the contribution of BPA induced DNA damage in SLE patients. Human DNA was modified by BPA in-vitro and the binding characteristics of SLE circulating immunoglobulin Gs (SLE-IgGs) with BPA damaged DNA (BPA-DNA) were screened and compared with the IgGs from normal healthy humans (NH-IgGs). Immunogenicity of BPA-DNA was determined by immunisation in rabbits. DNA from SLE patients (SLE-DNA) or healthy humans (NH-DNA) were isolated and their binding specificity with rabbit anti-BPA-DNA-IgGs was studied. Treatment of human DNA with BPA caused extensive damaged. Circulating SLE-IgGs showed strong recognition of BPA-DNA. BPA-DNA induced high titre antibodies in rabbits. Rabbit anti-BPA-DNA-IgGs showed strong cross reaction with isolated DNA from SLE patients. In short, we concluded that the structural alterations in DNA by BPA, generate neo-epitopes that may be a factor responsible for the induction of anti-DNA autoantibodies in SLE.

Bronchial asthma (BA), atopic dermatitis (AD), and allergic rhinitis (AR) are well known atopic disorders with complex etiologies. This study was undertaken to investigate the role of filaggrin, eosinophil major basic protein (MBP) and leukotriene B4 (LTB4) in patients with BA, AD, and AR. Sera from 1,246 patients with different atopic disorders and 410 normal healthy controls were collected and were evaluated for filaggrin, MBP and LTB4 by specific sandwich ELISAs, whereas immunoglobulin E (IgE) was used as a positive control for atopic patients. Serum analysis showed that filaggrin levels were remarkably high in patients with AD and in patients with multiple (mixed) atopic disorders (p < 0.001), whereas its levels in BA and AR patients were low but much higher than in normal human sera (p < 0.01). MBP levels were also high in AR, BA and mixed atopic patients, whereas AD patients showed no increase of MBP (p > 0.05). In contrast, LTB4 level was found to be significantly low in all tested atopic patients groups as compared to the levels of LTB4 present in normal human sera (p < 0.001). In conclusion, these findings support an association between filaggrin, MBP or LTB4 and atopic disorders. Our data strongly suggest that filaggrin, MBP or LTB4 might be useful in elucidating the mechanisms involved in the pathogenesis of these atopic disorders.

BACKGROUND: The cluster of differentiation (CD) 74 is known for its immunological functions and its elevated level was reported in various cancer cells. AIM: The aim of the present study was to investigate the expression and potential roles of CD74 in the proliferative and apoptotic activity of breast cancer. METHODS: Expression of CD74, macrophage migration inhibitory factor (MIF) and CD44 was assayed in CAMA-1 and MDA-MB-231 cell lines using flow cytometry. CD74 was knocked down using CD74 siRNA-transfection in CAMA-1, and MDA-MB-231 cells and proliferation and apoptosis were determined in the transfected breast cancer cells. RESULTS: The data showed that CD74, MIF and CD44 were expressed in breast cancer cell lines and were associated with cell proliferation and apoptosis. Correlation analysis revealed that CD74 was positively correlated and colocalised with MIF on the cell-surface of CAMA-1 and MDA-MB-231. The knockdown of CD74 significantly reduced CAMA-1 and MDA-MB-231 cell proliferation and increased the level of apoptotic cells. CONCLUSION: We concluded that the interactions of CD74 with MIF and CD74 with CD44 could be a potential tumour marker for breast cancer cells. Moreover, the level of co-expression of MIF and CD74 or CD44 could be a surrogate marker for the efficacy of anti-angiogenic drugs, particularly in breast cancer tumours. In short, the study revealed the potential roles of CD74 in the proliferation and apoptosis of breast cancer which may serve as a potential therapeutic target for breast cancer.

Abnormal post-transcriptional modulations in inflammatory genes by microRNAs (miRNAs) play a crucial role in human disorders including arthritis. In this study, we determined the effect of hsa-miR-125b-5p on interleukin (IL)-1β induced inflammatory genes in human osteoarthritic (OA) chondrocytes. Bioinformatics algorithms showed 3′untranslated region (3′UTR) of TRAF6 mRNA (NM_004620.3) has perfectly matched ‘seed-sequence’ for hsa-miR-125b-5p. Treatment of cells with IL-1β up-regulates TRAF6 mRNA and down-regulates hsa-miR-125b-5p expression. This negative correlation between TRAF6 and hsa-miR-125b-5p was verified by transfection with miR-125b mimic (pre-miR-125b). Moreover, transfection with miR-125b mimic caused marked inhibition of IL-1β-induced phosphorylation of p38-MAPK, JNK-MAPKs and ERK-MAPKs and also suppressed the nuclear levels of NF-κBp50, NF-κBp65 and inhibited the activation of IκBα. Furthermore, transfected chondrocytes with miR-125b mimic in the presence of IL-1β also showed marked inhibition in the secretion of several proinflammatory cytokines, chemokines and growth factors including IL-6, IL-8, INF-γ, TGF-β1, IGFBP-1 and PGDF-BB. Importantly, this transfection also significantly inhibited IL-1β- induced MMP-13 expression/production. In short, this study concludes that hsa-miR-125b-5p acts as a negative co-regulator of inflammatory genes including MMP-13 via targeting TRAF6/MAPKs/NF-κB pathway in human OA chondrocytes.

Inducible nitric oxide synthase (iNOS) expression is associated with the pathogenesis of osteoarthritis (OA). This study was undertaken to investigate whether interleukin-1β (IL-1β)-mediated induction of iNOS can be regulated by microRNA-26a-5p (hsa-miR-26a-5p) in OA. Bioinformatics approaches show that 3'UTR of iNOS mRNA contained the 'seed-matched-sequence' for hsa-miR-26a-5p. IL-1β-induced expression of iNOS correlated with the down-regulation of miR-26a-5p in human OA chondrocytes. hsa-miR-26a-5p directly suppressed the luciferase activity of 3'UTR-iNOS reporter clone. Transfection with pre-miR-26a-5p induced marked silencing of iNOS expression. Activation of NF-κB pathway down-regulated the expression of hsa-miR-26a-5p and induced iNOS expression. In short, this is the first report that shows hsa-miR-26a-5p is a direct regulator of iNOS expression in human chondrocytes. hsa-miR-26a-5p may be an important regulator of human cartilage homeostasis and a new target for OA therapy.

Background Allergic reactions have been implicated as contributions in a number of atopic disorders, including atopic dermatitis (AD), allergic rhinitis (AR) and bronchial asthma (BA). However, the potential for filaggrin protein, eosinophil major basic protein (MBP) and immunoglobulin E (IgE) to elicit allergic response or to contribute to atopic disorders remains largely unexplored in pediatric patients. This study was undertaken to investigate the status and contribution of filaggrin protein, eosinophil MBP and total IgE in pediatric patients with AD, AR and BA. Methods Sera from 395 pediatric patients of AD, AR or BA with varying levels of disease activity according to the disease activity index and 410 age-matched non-atopic healthy controls were evaluated for serum levels of atopic markers, including filaggrin, eosinophil MBP and IgE. Results Serum analysis showed that filaggrin levels were remarkably high in pediatric patients with AD, followed by BA and AR, whereas its levels were low in non-atopic pediatric controls. Eosinophil MBP levels in sera of atopic patients were significantly high as compared with their respective controls, but its levels were highest in AR patients, followed by AD and BA. Total IgE in sera of AD patients was markedly high, followed by AR and BA patients, whereas its levels were low in non-atopic pediatric controls. Interestingly, not only was an increased number of subjects positive for filaggrin protein, eosinophil MBP or total IgE, but also their levels were statistically significantly higher among those atopic patients whose disease activity scores were higher as compared with atopic patients with lower disease activity scores. Conclusions These findings strongly support a role of filaggrin protein, eosinophil MBP and IgE in the onset of allergic reactions in pediatric patients with AD, AR and BA. The data suggest that filaggrin, eosinophil MBP or IgE might be useful in evaluating the progression of AD, AR or BA and in elucidating the mechanisms involved in the pathogenesis of these pediatric disorders.

Objectives: To investigate the role of reactive-oxygen-species (ROS) induced epitopes on human-serum-albumin (HSA) and thyroid antigens in psoriasis autoimmunity. Methods: This study was performed in the College of Medicine, Qassim University, Buraidah, Saudi Arabia between May 2014 and February 2015. The study was designed to explore the role of ROS-induced epitopes in psoriasis autoimmunity. Singlet-oxygen (or ROS)-induced epitopes on protein (ROS-epitopes-albumin) was characterized by in-vitro and in-vivo. Thyroid antigens were prepared from rabbit thyroid, and thyroglobulin was isolated from thyroid extract. Immunocross-reactions of protein-A purified anti-ROS-epitopes-HSA-immunoglobulin G (IgGs) with thyroid antigen, thyroglobulin, and their oxidized forms were determined. Binding characteristics of autoantibodies in chronic plaque psoriasis patients (n=26) against ROS-epitopes-HSA and also with native and oxidized thyroid antigens were screened, and the results were compared with age-matched controls (n=22). Results: The anti-ROS-epitopes-HSA-IgGs showed cross-reactions with thyroid antigen, thyroglobulin and with their oxidized forms. High degree of specific binding by psoriasis IgGs to ROS-epitopes-HSA, ROS-thyroid antigen and ROS-thyroglobulin was observed. Immunoglobulin G from normal-human-controls showed negligible binding with all tested antigens. Moreover, sera from psoriasis patients had higher levels of carbonyl contents compared with control sera. Conclusion: Structural alterations in albumin, thyroid antigens by ROS, generate unique neo-epitopes that might be one of the factors for the induction of autoantibodies in psoriasis. Saudi Med J 2015; Vol. 36 (12): 1408-1419 doi: 10.15537/smj.2015.12.12612

Vitiligo is a common pigmentary disorder, the precise etiology of which remains obscure. Tyrosinase, a key enzyme involved in melanin synthesis, has now been implicated as an autoantigen for vitiligo patients, but it is not clear how this prevalent protein becomes antigenic in vitiligo.To investigate the status and contribution of oxidized tyrosinase in vitiligo and to explore whether oxidized tyrosinase has a role in disease progression.Tyrosinase was modified by reactive-oxygen-species (ROS). Binding characteristics of antibodies in vitiligo patients (n=25) with varying disease duration (DD) and disease severity were screened against ROS-modified tyrosinase (ROS-tyrosinase) by immunoassays and their results were compared with healthy controls (n=23).The ROS caused extensive alterations in conformation and function of tyrosinase. Protein-A purified IgGs from vitiligo patients (Vt-IgG) showed strong binding to ROS-tyrosinase in comparison with IgGs from healthy controls (p0.001). Interestingly, not only was there an increased number of subjects positive for anti-ROS-tyrosinase-IgGs, but also the levels of these IgGs were significantly higher among vitiligo patients, whose DD were ≥10 years as compared to patients with short DD (10 years). In addition, a significant correlation was observed between the levels of anti-ROS-tyrosinase-IgGs and the patients' ages or with disease severity. Experimentally induced anti-ROS-tyrosinase-IgGs show reactivity with tyrosinase from vitiligo patients. Furthermore, vitiligo patients had lower levels of tyrosinase activity compared with healthy controls. Not only these, levels of carbonylation were also higher among vitiligo patients whose DD were ≥10 years as compared to patients with DD10 years.This is the first study to demonstrate the role of oxidized tyrosinase in vitiligo. Our novel results support an association between oxidized tyrosinase and vitiligo autoimmunity. The stronger antibodies response to oxidized tyrosinase in vitiligo patients with higher DD or with severe patients suggests that oxidized tyrosinase may be a useful biomarker in evaluating the progression of vitiligo and in elucidating the mechanisms of disease pathogenesis.

Alopecia areata (AA) is a non-scarring hair loss disorder that ranges in severity from patchy loss of scalp hair (AA patchy persistent; AAP) to loss of all scalp and body hair (alopecia universalis; AU). The cause of AA is unknown but most evidences support that AA has an autoimmune etiology, where free radicals play an important role. This study was undertaken to investigate the role of nitric oxide (NO) modified erythrocytes superoxide dismutase (eSOD) in AA. Data revealed that NO-induced damage in eSOD caused alteration in hydrophobic or aromatic amino acids and protein carbonyl contents. NO-specific quencher, carboxyl-PTIO further reiterates NO-modifications. Specificity of antibodies from AA patients (n=26) was analyzed toward NO-modified eSOD (NO-eSOD) and their results were compared with healthy controls (n=30). Protein-A purified IgG of AA patients (AA-IgG) showed strong binding to NO-eSOD in comparison with IgG from controls. In addition, AA-IgG from patients with AU recognized NO-eSOD in a greater extent as compared to AA-IgG from patients with AAP. Furthermore, AU patients' sera contained higher levels of NO or carbonyl contents and lower levels of SOD activity compared with AAP patients' or control sera. In conclusion, this is the first study to demonstrate the role of NO-modified-eSOD in AA. Our novel results conclude that perturbations in SOD by NO presenting unique neo-epitopes that might be one of the factors for the antigen driven antibodies induction in AA. Preferential binding of NO-eSOD by AA-IgG pointed out the likely role of NO-eSOD in the initiation/progression of AA.

Lipid peroxidative-mediated reactions have been implicated in alopecia areata (AA). However, the potential for lipid peroxidation to elicit an autoimmune response or to contribute in disease pathogenesis remain unexplored. This study was undertaken to investigate the status/contribution of lipid oxidative-by-product malondialdehyde modified DNA (MDA-DNA) in AA.DNA was modified by MDA. Binding characteristics of AA circulating immunoglobulin G (AA-IgG) MDA-modified DNA were screened. Immunogenicity of MDA-DNA was probed by inducing antibodies in rabbits. DNA samples from AA patients were isolated (DNA-AA) and their immune reactions with rabbit anti-MDA-DNA-IgGs were evaluated.Our data show that MDA caused extensive DNA damage. Protein-A purified IgG from 45% of AA patients showed strong binding to MDA-DNA in comparison with native DNA (p0.05). MDA-DNA was found to be highly immunogenic in rabbits. Rabbit anti-MDA-DNA-IgG showed binding with isolated DNA from AA patients.This is the first study to demonstrate the role of MDA-modified DNA in AA patients. Our novel results conclude that perturbations in DNA by MDA present unique neo-epitopes that might be one of the factors for the antigen driven antibodies induction in AA. These results provide an important insight into the immunological mechanisms occurring in AA.

Background and objective Recent evidence has demonstrated that mitochondria possess their own nitric oxide synthase (mtNOS) and can produce endogenous reactive-nitrogen-species (RNS) including peroxynitrite (ONOO–). This study was undertaken to investigate the role of mitochondrial DNA (mtDNA) damage by ONOO– in systemic lupus erythematosus (SLE) autoimmunity. Methods MtDNA was isolated from fresh goat liver and modified by ONOO–, generated by synergistic action of nitric oxide (NO) and superoxide (O–2) donors. Modifications occurring in mtDNA were characterized by physicochemical techniques. SLE patients ( n = 50) with varying disease activity according to the SLE Disease Activity Index (SLEDAI) and healthy controls ( n = 34) were evaluated for antibodies to native and ONOO–-modified mtDNA by immunoassays. Gel retardation assays were performed to cross-examine the immunoassay results using affinity-purified SLE immunoglobulin G (IgG). Nitrosative stress in SLE patients was studied by measuring nitrotyrosine and inducible NO synthase (iNOS). Results The ONOO– caused extensive damage to mtDNA as evident by ultraviolet (UV) hyperchromicity and loss of florescence intensity. Thermal melting studies, agarose gel electrophoresis and nuclease S1 digestibility clearly indicate structural perturbation in mtDNA by ONOO–. Quenching studies with specific NO or O–2 quenchers confirmed that the damaging agent was ONOO–. SLE autoantibodies exhibited enhanced binding with ONOO–-mtDNA as compared to their native analog. Interestingly, not only was there an increased number of subjects positive for ONOO–-mtDNA, but also the levels of anti-ONOO–-mtDNA antibodies were statistically significantly higher among SLE patients whose SLEDAI scores were ≥ 20 as compared with SLE patients with lower SLEDAI scores (SLEDAI Conclusions Our novel results provide an important insight into the immunological basis of anti-DNA autoantibody generation in SLE. Our data conclude that modification of mtDNA by ONOO– causes structural perturbations, resulting in the generation of neo-epitopes, and making it a potential immunogen in SLE. The mtDNA modified by ONOO– may be useful in evaluating the progression of SLE and in elucidating the mechanisms of disease pathogenesis.

Protein modifications by 4-hydroxy-2-nonenals (HNE) are involved in various diseases. Histones are DNA protective nucleoprotein, which adopt different structures under oxidative stress. This study was undertaken to test the role of HNE-modified-histone-H2A (HNE-H2A) in systemic lupus erythematosus (SLE). Our data revealed that HNE-mediated-lipid peroxidation in histone-H2A caused alteration in histidine, lysine and cystein residues. In addition, protein carbonyl contents were also high in HNE-H2A. HNE-specific quencher, L-carnosine further reiterates HNE-modifications. Specificity of autoantibodies from SLE patients (n=48) were analyzed towards HNE-H2A and their results were compared with sex- and age-matched controls (n=36). SLE autoantibodies show preferential binding to HNE-H2A in comparison with histone-H2A (p

Background: Acne vulgaris is a multifactorial skin disorder of unknown etiology. Free radical-mediated reactions have been implicated but their role in eliciting this response and contributing to disease progress remains unexplored. This study was undertaken to investigate the status and contribution of oxidative/nitrosative stress in patients with acne vulgaris. Methods: Sera from 50 acne vulgaris with varying levels of disease activity (mild, moderate, and severe) according to the Global Acne Grading System (GAGS) and 40 age- and sex-matched controls were evaluated for serum levels of oxidative/nitrosative stress markers, including protein oxidation, lipid peroxidation and nitric oxide (NO), superoxide dismutase (SOD), and glutathione (GSH). Results: Serum analysis showed significantly higher levels of carbonyl contents, malondialdehyde (MDA) and NO, in acne patients compared with healthy controls (P < 0.05). Interestingly, not only there were an increased number of subjects positive for carbonyl contents, but also the levels of these oxidants were significantly increased with the increase of the disease activity (P < 0.05). In addition, a significant correlation was observed between the levels of carbonyl contents and the GAGS scores (r = 0.341, r = 0.355, and r = 0.299, respectively). Furthermore, sera from acne patients had lower levels of SOD and GSH compared with healthy control sera. Conclusion: These findings support an association between oxidative/nitrosative stress and acne. The stronger response observed in serum samples from patients with higher GAGS scores suggests that markers of oxidative/nitrosative stress may be useful in evaluating the progression of acne and in elucidating the mechanisms of disease pathogenesis. J. Clin. Lab. Anal. 27:45‐ 52, 2013. C � 2012 Wiley Periodicals, Inc.

The role of reactive oxygen species (ROS)-damaged human serum albumin (HSA) in lung cancer (LC) patients is investigated. The binding characteristics of LC antibodies with native and ROS-damaged HSA are assessed. Smoking and non-smoking LC patients (n=40) are examined by a direct binding enzyme-linked immunosorbent assay (ELISA) and the results are compared with healthy age- and gender-matched smoking and non-smoking subjects (n=38). A high degree of specific binding in approximately 70% of cancer immunoglobulin G (IgG) towards ROS-damaged HSA was observed, compared to results with its native analogue (P

Background: Osteoarthritis (OA) is a progressive joint disorder, which remains the leading cause of chronic disability in aged people. Nuclear factor-kappa B (NF)-κB is a major cellular event in OA and its activation by interleukin-1β (IL-1β) plays a critical role in cartilage breakdown in these patients. Objective: In this study, we examined the effect of lactoferrin on NF-κB activation, cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) production in stimulated human articular chondrocytes. Materials and Methods: Human chondrocytes were derived from OA articular cartilage and treated with camel lactoferrin and then stimulated with IL-1β. Gene expression was determined by TaqMan assays and protein expression was studied by Western immunoblotting. NF-κB activity and PGE2 levels were determined by ELISA based assays. NF-κB activity was also determined by treatment of chondrocytes with NF-κB specific inhibitor Bay 11–7082. Results: Lactoferrin inhibited IL-1β-induced activation and nuclear translocation of NF-κB p65 in human OA chondrocytes. Lactoferrin also inhibited mRNA/protein expression of COX-2 and production of PGE2. Moreover, Bay 11–7082 also inhibited IL-1β-induced expression of COX-2 and production of PGE2. The inhibitory effect of lactoferrin on the IL-1β induced expression of COX-2 or production of PGE2 was mediated at least in part via suppression of NF-κB activation. Conclusions: Our data determine camel lactoferrin as a novel inhibitor of IL-1β-induced activation of NF-κB signaling events and production of cartilage-degrading molecule PGE2 via inhibition of COX-2 expressions. These results may have important implications for the development of novel therapeutic strategies for the prevention/treatment of OA and other degenerative/inflammatory diseases. SUMMARY Lactoferrin shows anti-arthritic activity in IL-1β stimulated primary human chondrocytes.Lactoferrin inhibits IL-1β-induced NF-κB activation.Lactoferrin inhibits production of cartilage degrading PGE2 via inhibition of COX-2 expression. Abbreviations Used: OA: Osteoarthritis IL-1β: Interleukin-1 beta NF-κB: Nuclear factor-kappa B COX-2: cyclooxygenase-2 PGE2: prostaglandin E2

Osteoarthritis (OA) is a most common form of arthritis worldwide leading to significant disability. MicroRNAs (miRNAs) are non‐coding RNAs involved in various aspects of cartilage development, homoeostasis and pathology. Several miRNAs have been identified which have shown to regulate expression of target genes relevant to OA pathogenesis such as matrix metalloproteinase (MMP)‐13, cyclooxygenase (COX)‐2, etc. Epigallocatechin‐3‐O‐gallate (EGCG), the most abundant and active polyphenol in green tea, has been reported to have anti‐arthritic effects, however, the role of EGCG in the regulation of miRNAs has not been investigated in OA. Here, we showed that EGCG inhibits COX‐2 mRNA/protein expression or prostaglandin E2 (PGE 2) production via up‐regulating microRNA hsa‐miR‐199a‐3p expression in interleukin (IL)‐1β‐stimulated human OA chondrocytes. This negative co‐regulation of hsa‐miR‐199a‐3p and COX‐2 by EGCG was confirmed by transfection of OA chondrocytes with anti‐miR‐199a‐3p. Transfection of OA chondrocytes with anti‐miR‐199a‐3p significantly enhanced COX‐2 expression and PGE 2 production (P < 0.001), while EGCG treatment significantly inhibited anti‐miR‐199a‐3p transfection‐induced COX‐2 expression or PGE 2 production in a dose‐dependent manner. These results were further re‐validated by co‐treatment of these transfection OA chondrocytes with IL‐1β and EGCG. EGCG treatment consistently up‐regulated the IL‐1β‐decreased hsa‐miR‐199a‐3p expression (P < 0.05) and significantly inhibited the IL‐1β‐induced COX‐2 expression/PGE 2 production (P < 0.05) in OA chondrocytes transfected with anti‐hsa‐miR‐199a‐3p. Taken together, these results clearly indicate that EGCG inhibits COX‐2 expression/PGE 2 production via up‐regulation of hsa‐miR‐199a‐3p expression. These novel pharmacological actions of EGCG on IL‐1β‐stimulated human OA chondrocytes provide new suggestions that EGCG or EGCG‐derived compounds inhibit cartilage breakdown or pain by up‐regulating the expression of microRNAs in human chondrocytes.

Reactive oxygen species (ROS) plays an important role in carcinogenesis and hydroxyl radical (OH) contributes to the structural changes in DNA that characterize the cancer like phenotype. The role of hydroxyl radical (OH) damaged thymidine 5'-monophosphate (TMP) in cancer patients has been investigated in the present study. TMP was conjugated to bovine serum albumin (BSA), and then TMP-BSA conjugate was modified by hydroxyl radicals. Cancer patients (n = 99) were screened by direct binding ELISA for the detection of antibodies against native and ROS-modified conjugates and the results were compared with healthy age-matched controls (n = 29). High degree of specific binding by cancer serum antibodies towards ROS modified TMP-BSA conjugate, in comparison to unmodified conjugate (p

The role of advanced glycation end products (AGEs)-damaged immunoglobulin G (AGE-IgG) in type 1 diabe- tes has been investigated in the present study. IgG was isolated from the normal humans and was subjected to in vitro gly- cation with glucose. The AGEs caused extensive damaged to IgG. The AGE-IgG was found to be highly immunogenic in rabbits as compared to native IgG. The binding characteristics of circulating autoantibodies in type 1 diabetes mellitus (DM) patients against native and AGE-IgG were assessed. Type 1 DM patients (n=31) were examined by ELISA and their results were compared with healthy age-matched human controls (n=22). High degree of specific binding by 61.3 % of DM sera autoantibodies towards AGE-IgG was observed, in comparison to its native analog (p< 0.05). Sera from those type 1 DM patients having smoking history, high aging with high degree of disease showed substantially stronger binding to AGE-IgG over native IgG in particular. IgG from type 1 DM patients (DM-IgG) contained higher levels of carbonyls as compared to normal human subjects (normal-IgG) (p

Objectives The role of hydroxyl radical ( OH) damaged Immunoglobulin G (IgG) in rheumatoid arthritis (RA) has been investigated. Design and methods The study was hypothesized that oxidative by-products, like OH-damage IgG, help to initiate autoimmunity in RA. To test this hypothesis, IgG was modified by OH. Immunogenicity of native and modified IgG was probed by inducing polyclonal antibodies in rabbits. Autoantibodies from 77 RA sera were screened by direct binding and competition ELISA. Results The OH caused extensive damage to IgG. The OH-IgG was found to be highly immunogenic in rabbits as compare to native IgG. High degree of specific binding by 72.7% RA sera autoantibodies towards OH-IgG was observed, in comparison to its native analogue (p Conclusion The OH modification of IgG causes perturbations, resulting in the generation of neo-epitopes, and making it a potential immunogen. The IgG modified with the ·OH may be one of the factors for the induction of circulating RA autoantibodies.

Peroxynitrite is a reactive oxidant produced from nitric oxide ((.)NO) and superoxide anion (O(2)(.-)). It is produced by the body in response to environmental toxins, stress, ultraviolet light, ischemia/reperfusion, inflammation, etc. In vivo, peroxynitrite is formed in macrophages, endothelial cells, platelets, leukocytes, and neurons. It reacts with a variety of biomolecules including proteins, lipids, and DNA. We have investigated the photochemical addition of lysine to native DNA in view of its potential importance in the photo-cross-linking of histones to DNA in chromatin. Lysine-and arginine-rich histone H1 in nucleosome on modification by physical, chemical, or environmental agents forms histone-DNA adducts. We have characterized the photoadducts by absorption, fluorescence, and chromatographic methods. The UV absorption spectra of the DNA-lysine photoadduct showed hyperchromism, indicating structural distortions in DNA either due to single-strand breaks or opening of the double helix at the site of lysine conjugation. On peroxynitrite treatment, the melting temperature (T(m)) of the DNA-lysine adduct increased by 15 degrees C compared to the native DNA-lysine adduct. A decrease in the fluorescence intensity of the DNA-lysine photoadduct with respect to the modified adduct was observed. The gel filtration profile of the peroxynitrite-modified adduct was also found to be different from that of the native DNA and DNA-lysine photoadduct. Hence, the peroxynitrite-modified photoadduct may have important implications in toxicology, mutagenesis, and carcinogenesis.

Glycated human serum albumin (HSA) is known to be involved in the pathogenesis of several diseases, and we have therefore investigated possible alterations in HSA on glycation. HSA was incubated for 5 and 20 weeks independently with constant glucose concentration at 37 degrees C under aerobic conditions. Biochemical, spectral, electrophoretic, circular dichroism spectropolarimetric, and thermodynamic analyses confirmed that the structure and stability of HSA is significantly affected on glucose modification. Glycated HSA-AGE-20w showed appreciable elevation (15.8%) in beta-sheet structure and decrease in alpha-helix (10.4%) and random coil (5.7%) structures. Slight changes have also been observed in turns (3.2%) of HSA-AGE-20w. Quenching studies with antioxidants diethylene triaminepentaacetic acid and superoxide dismutase showed inhibition in glycation to the extent of 50-65 and 30-40%, respectively. The novelty of present study is that glycation of HSA can cause induction of secondary and tertiary structure changes that may generate thermodynamically more stable high molecular weight aggregates having remarkably increased beta-sheet structure than its non-glycated form. This may interfere with the normal function of HSA, thus contributing to diabetic complications.

Introduction: Atopic dermatitis (AD) is a chronic inflammatory skin disorder. Immunological/inflammatory reactions are reported to play a role in AD but their role in disease activity has not been fully investigated. This study was done to investigate the role of immunoglobulin E (IgE), interleukin (IL)-18 and IL-12 in AD patients with different disease severities. Materials and Methods: Sera from 50 AD infants with varying levels of disease activity according to the scoring index of atopic dermatitis (SCORAD) index and 30 age-matched healthy controls were evaluated for serum levels of IgE, IL-18 and IL-12/p40. Results: Serum analysis showed higher levels of IgE, IL-18 or IL12/p40 in AD patients compared with controls. Interestingly, not only was there an increased number of subjects positive for IgE, IL-18 or IL-12/p40, but also the levels of these parameters were higher among AD patients whose SCORAD scores were higher. In addition, a significant correlation was observed between the levels of these parameters and SCORAD scores. Conclusion: These findings support an association between IgE, IL-18 or IL-12/p40 and AD. The stronger response observed in serum samples from patients with higher SCORAD scores suggest that IgE, IL-18 and IL-12/p40 may be useful in evaluating the progression of AD and in elucidating the mechanisms of disease pathogenesis.

Vitiligo is a pigmentation disorder of unknown aetiology, but it has been reported in association with other autoimmune diseases including type 1 diabetes mellitus (T1D). Vitiligo and T1D share a common theory of autoimmunity, but still an inflammatory link between them remains to be investigated. This study investigates the status and contribution of the inflammatory markers tumour necrosis factor-α (TNFα), interleukin (IL)-6 and IL-1 in patients with vitiligo, T1D and vitiligo-associated T1D (Vt-T1D). The data showed that sera from Vt-T1D patients (n = 21) had higher levels of TNFα, IL-6 and IL-1β compared with vitiligo patients (n = 39), T1D patients (n = 37) or controls (n = 42). Interestingly, serum levels of IL-6 were found to be significantly higher in Vt-T1D patients compared with the levels of TNFα and IL-1β. These data also showed that IL-6 was high in Vt patients as compared to the levels of TNFa and L-1β, whereas in T1D patients, IL-6 and TNFα were almost the same but were higher than IL-1β. In conclusion, this is the first study to show an inflammatory link between vitiligo and T1D. The data conclude that IL-6 plays an important role in the pathogenesis of Vt-T1D patients and is likely to gain favour as a therapeutic target in these patients.

Present study probes the role of peroxynitrite (ONOO(-))-modified thymidine-5'-monophosphate (TMP) in SLE patients with different disease activity scores according to the SLE Disease Activity Index (SLEDAI). Serum analysis showed significant increased number of subjects positive for anti-ONOO(-)-TMP-protein antibodies in SLE patients with different SLEDAI scores. Interestingly, the levels of these antibodies were significantly higher among SLE patients, whose SLEDAI scores were ≥20. In addition, a significant correlation was observed between the levels of anti-ONOO(-)-TMP-protein antibodies and the SLEDAI score (r = 0.595, p < 0.0001). In short, this study shows a positive association between anti-ONOO(-)-TMP-protein antibodies and SLEDAI. The stronger response observed in patients with higher SLEDAI scores suggests that anti-ONOO(-)-TMP-protein antibodies may be useful in evaluating the progression of SLE and in elucidating the mechanisms of disease pathogenesis.

This study was undertaken to investigate the status and contribution of oxidized catalase in systemic lupus erythematosus (SLE) and to explore whether oxidized catalase has a role in disease progression.Catalase (CAT) was modified by reactive oxygen species (ROS). Sera from 50 SLE patients with varying levels of disease activity according to SLE Disease-Activity-Index (SLEDAI) and 45 age- and sex-matched healthy controls were evaluated for antibodies against oxidized CAT.Serum analysis showed significantly higher level of anti-oxidized-CAT-antibodies in SLE patients compared with controls. Interestingly, not only was there an increased number of subjects positive for anti-oxidized-CAT-antibodies, but also the levels of these antibodies were significantly higher among SLE patients, whose SLEDAI scores were ≥ 10 as compared with lower SLEDAI scores (10). In addition, significant correlation was observed between the levels of anti-oxidized-CAT-antibodies and SLEDAI score (r = 0.796). Furthermore, sera from SLE patients had lower levels of CAT activity compared with control sera.These findings support an association between oxidized CAT and SLE. The stronger response observed in serum samples from patients with higher SLEDAI scores suggests that oxidized CAT may be a useful biomarker in evaluating the progression of SLE and in elucidating the mechanisms of disease pathogenesis.

This study was undertaken to investigate the role of peroxynitrite (ONOO(-)) modified thymine-5'-monophosphate (TMP) in the generation of anti-DNA autoantibodies in patients with systemic lupus erythematosus (SLE). TMP was exposed to ONOO(-) in vitro and challenged in vivo. TMP and ONOO(-)-modified-TMP were found to be nonimmunogenic in rabbits. TMP-linked-BSA and ONOO(-)-modified-TMP-BSA induced high titer antibodies. Induced antibodies against ONOO(-)-TMP-BSA show crossreactions with nucleic acids conformers. A high degree of specific binding by SLE autoantibodies with ONOO(-)-TMP-BSA was observed. Our novel results provide an important insight into the immunological basis of anti-DNA autoantibodies generation in SLE.

Epithelial and endothelial tight junctions (TJs) act as a physical barrier against the diff usion of material between adjacent cells. Cell-cell barrier failure is associated with the loss of cellular diff erentiation and increased antigen exposure. Th ese events play an important role in the pathogenesis of a number of diseases, including infl ammatory bowel diseases, asthma, cystic fi brosis, and cancer. At the molecular level, the TJ barrier can be attributed to a complex array of proteins, including claudins, a diverse family of transmembrane proteins that form ion and size-selective aqueous pores. Th e stability of claudin-based junctions is maintained by an array of protein scaff olds, with linkages to the intercellular actin network. Recent studies have determined the detrimental eff ects of proinfl ammatory cytokines on TJ-dependent barrier properties. Proinfl ammatory cytokines, such as interferon gamma and tumor necrosis factor alpha, and interleukins, remodel TJs through the regulation of both claudin and TJ scaff old proteins. Additionally, proinfl ammatory cytokines regulate actin cytoskeletal dynamics, through the myosin light chain intercellular signaling cascade, resulting in structural and functional changes in TJs. Conversely, anti-infl ammatory cytokines such as IL-10 and transforming growth factor beta attenuate pro-infl ammatory stimuli. While it remains unclear whether TJ dysfunction is causative or symptomatic of detrimental infl ammatory stimuli, investigations into the molecular mechanisms involved have increased our understanding of TJ regulation. Th is chapter will review recent advances in our understanding of the mechanisms of cytokine modulation of TJ structure and barrier function.

Objective Role of reactive oxygen species (ROS) modified human Immunoglobulin G (IgG) in systemic lupus erythematosus (SLE) has been investigated. Methods Human IgG was modified by hydroxyl-radicals. Immunogenicity of native and modified human IgG was probed by inducing polyclonal antibodies in rabbits. Cross-reactions of induced antibodies with nucleic acid, chromatin, different blood proteins and their ROS modified conformers were determined by competitive inhibition ELISA. The binding characteristics of circulating autoantibodies in SLE patients (n = 72) against native and modified IgG were screened by direct binding and competition ELISA and the results were compared with healthy age-matched controls (n = 39). Results Induced antibodies against ROS-modified human IgG exhibited diverse antigen binding characteristics. Native DNA, native chromatin and their ROS-modified conformers were found to be effective inhibitors of induced antibody-immunogen interaction. Induced antibodies against native human IgG showed negligible binding to the above mentioned nucleic acid antigens. SLE sera (48.6%) showed strong binding to ROS-human IgG in comparison with its native analogue ( P < 0.01). Normal human sera (NHS) showed negligible binding with either antigen ( P > 0.05). Conclusion ROS-induced modifications in human IgG present neo-epitopes, and make it a potential immunogen. The induced antibodies against ROS-modified human IgG resembled the diverse antigen-binding characteristics of naturally occurring SLE anti-DNA autoantibodies. ROS-modified IgG may be one of the factors for the induction of circulating SLE autoantibodies.

Objective. To investigate whether advanced glycation end products (AGEs) induce the expression of IL-6 and IL-8 through the receptor for AGEs (RAGE)-activated pathways in human OA chondrocytes. Methods. OA chondrocytes were stimulated with AGE-modified BSA (AGE-BSA). Gene expression of IL-6 and IL-8 was quantified by TaqMan assays and the production was determined using ELISAs. Immunoblotting was used to analyse the activation of mitogen-activated protein kinases (MAPKs) and the degradation of IκBα. Activation of NF-κB was determined using an ELISA. Pharmacological studies to elucidate the involved pathways were executed using transfection with small interfering RNAs (siRNAs), inhibitors of MAPKs and NF-κB. Results. AGE-BSA induced the expression of IL-6 and IL-8 in OA chondrocytes, which was inhibited by pre-treatment with soluble RAGE (sRAGE) or RAGE knockdown by siRNAs. Treatment with SB202190 (p38-MAPK inhibitor) or PD98059 (ERK inhibitor) inhibited AGE-BSA-induced IL-6 and IL-8 expression. However, SP600125 (JNK inhibitor) had no effect on AGE-BSA-induced IL-6 expression but inhibited the expression of IL-8. Treatment with NF-κB inhibitors suppressed AGE-BSA-induced IL-6 and IL-8 expression. Conclusions. This is the first study to demonstrate that AGEs induce the expression of IL-6 and IL-8 in OA chondrocytes. A novel finding of our studies is that in OA chondrocytes, AGE-BSA-induced expression of IL-6, but not of IL-8, was independent of the JNK pathway. Activation of NF-κB was an absolute requirement for both IL-6 and IL-8 expression. These results demonstrate that AGE-BSA-induced expression of IL-6 and IL-8 via RAGE is mediated through different MAPK signalling pathways in OA and possibly in other degenerative diseases.

Objective Aberrant posttranscriptional regulation of matrix metalloproteinases (MMPs) by microRNA has emerged as an important factor in human diseases. The aim of this study was to determine whether the expression of MMP-13 in human osteoarthritis (OA) chondrocytes is regulated by microRNA. Methods Chondrocytes were stimulated with interleukin-1β (IL-1β) in vitro. Total RNA was prepared using TRIzol reagent. Polymerase chain reaction (PCR)–based arrays were used to determine the expression profile of 352 human microRNA. Gene expression was quantified using TaqMan assays, and microRNA targets were identified using bioinformatics. Transfection with reporter construct and microRNA mimic was used to verify suppression of target messenger RNA (mRNA). Gene expression of argonaute and Dicer was determined by reverse transcription–PCR, and expression of protein was determined by immunoblotting. The role of activated MAP kinases (MAPKs) and NF-κB was evaluated using specific inhibitors. Results In IL-1β–stimulated OA chondrocytes, 42 microRNA were down-regulated, 2 microRNA were up-regulated, and the expression of 308 microRNA remained unchanged. In silico analysis identified a sequence in the 3′-untranslated region (3′-UTR) of MMP-13 mRNA complementary to the seed sequence of microRNA-27b (miR-27b). Increased expression of MMP-13 correlated with down-regulation of miR-27b. Overexpression of miR-27b suppressed the activity of a reporter construct containing the 3′-UTR of human MMP-13 mRNA and inhibited the IL-1β–induced expression of MMP-13 protein in chondrocytes. NF-κB and MAPK activation down-regulated the expression of miR-27b. Conclusion Our data demonstrated the expression of miR-27b in both normal and OA chondrocytes. Furthermore, IL-1β–induced activation of signal transduction pathways associated with the expression of MMP-13 down-regulated the expression of miR-27b. Thus, miR-27b may play a role in regulating the expression of MMP-13 in human chondrocytes.

Hydroxyl radical-mediated in vitro modification of human serum albumin (HSA) showed 59.2% hyperchromicity at lambdamax, 30% loss of alpha helical structure and 71.4% loss of tryptophan fluorescence. The reactive oxygen species (ROS)-modified HSA was highly immunogenic in rabbits as compare to native HSA. The antibody binding was inhibited to the extent of 97% with the immunogen as inhibitor, indicating the induction of immunogen specific antibodies. Experimentally induced antibodies against modified HSA exhibited diverse antigen binding characteristics. Native plasmid DNA, ROS-modified plasmid DNA and ROS-chromatin were found to be an effective inhibitor of induced antibody-immunogen interaction. Induced antibodies against native HSA showed negligible binding to the above mentioned nucleic acid antigens. Band shift assay reiterated the recognition towards nucleic acid antigens. Thus, the induced antibodies against *OH modified HSA resembled the diverse antigen-binding characteristics of naturally occurring systemic lupus erythematosus (SLE) anti-DNA autoantibodies.

The role of hydroxyl radical (.OH) damaged human serum albumin (HSA) in type 1 diabetes has been investigated in the present study. Hydroxyl radical induced modification on HSA has been studied by UV absorption spectroscopy, ANS fluorescence and carbonyl estimation. Hydroxyl radical modified HSA was found to be highly immunogenic in rabbits as compared to native HSA. The binding characteristics of circulating autoantibodies in type 1 diabetes patients against native and modified HSA were assessed. Diabetes patients (n=31) were examined by direct binding ELISA and the results were compared with healthy age-matched controls (n=22). High degree of specific binding by 54.8% of patients sera towards .OH modified HSA, in comparison to its native analogue (p