Relative CTLA-4, PTPN-22, and interleukin 37 mRNA expressions in patients with lupus nephritis.

OBJECTIVES: Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by T-cell immune-dysregulation and loss of tolerance to self-antigens. CTLA-4 and PTPN-22 are involved in the inhibition of T-lymphocytes activation. IL-37 is an anti-inflammatory cytokine that suppresses innate immunity. The relative expression of CTLA-4, IL-37 and PTPN-22 were evaluated as negative regulators of immune response in SLE patients, lupus nephritis (LN) and disease activity. METHODS: Real-Time PCR was performed to determine relative CTLA-4, IL-37, and PTPN-22 mRNA expressions in fifty-eight SLE patients, who were divided into two groups: 29 SLE patients without nephritis and 29 patients with LN, versus fifty controls. RESULTS: There was a significantly high-expression of CTLA-4 and IL-37 genes in SLE patients compared to controls (p=0.005; 0.018 respectively). There was no difference in relative PTPN-22 mRNA expression between the SLE patients and controls. Relative CTLA-4 mRNA expression decreased in LN patients (p=0.044), however, relative IL-37 mRNA over-expressed in LN patients (p=0.001) compared to those without LN. There was a significant over-expression of relative IL-37 andPTPN-22 mRNA in active SLE patients. But, there was a non-significant difference in CTLA-4 expression with disease activity. Regression analysis revealed patients with relative IL-37 mRNA over-expression had two times more to develop lupus nephritis (OR=1.906, 95% CI=1.218-2.983, p=0.005). CONCLUSIONS: Relative IL-37mRNA expression was elevated in SLE patients and associated with renal involvement and disease activity. It could be considered as a new promising predicting tool for LN. Relative PTPN-22 mRNA expression was correlated with disease activity only in SLE patients.

Long Non-coding RNA Genes Polymorphisms H19 (rs2251375) and MALAT1 (rs3200401) Association with Rheumatoid Arthritis and Their Correlation with Disease Activity in a Cohort of Egyptian Patients: A Pilot Study.

Rheumatoid arthritis (RA) is a chronic, progressive, inflammatory, autoimmune disease that could be disabling throughout its course. It affects people in their most reproductive years with relatively high morbidity and mortality. Long non-coding RNAs became one of the epigenetic mechanisms to prove a link to RA pathogenesis and development, including H19 and MALAT1 genes. These two genes' expressions had proved to increase in multiple diseases, attracting attention to their polymorphisms and their possible risk role. Assess the association between H19 SNP (rs2251375) and MALAT1 SNP (rs3200401) and the susceptibility of RA and its disease activity. In this pilot study, 200 hundred subjects (100 RA patients and 100 healthy controls) were investigated for a possible link between the polymorphisms H19 SNP (rs2251375) and MALAT1 SNP (3200401) and RA susceptibility and disease activity. RA-related investigations and clinical assessment were done. Real-time PCR genotyping of both SNPs was done using TaqMan® MGB probes. There was no association between the SNPs and risk of developing RA. However, both SNPs had a significant association with high disease activity. H19 SNP (rs2251375) heterozygous genotype CA had an association with elevated levels of ESR (p = 0.04) and higher DAS28-ESR score (p = 0.03). MALAT1 (rs3200401) C allele had an association with elevated ESR (p = 0.001), DAS28-ESR (p = 0.03), and DAS28-CRP (p = 0.007), while CC genotype had an association with DAS28-CRP (p = 0.015). Linkage disequilibrium and haplotyping of the alleles of both SNPs were analyzed as both genes are present on chromosome 11, but no significant association was found between any of the combinations of the alleles (p > 0.05), denoting that (rs2251375) and (rs3200401) are not in linkage disequilibrium. There is no association between H19 SNP (rs2251375) and MALAT1 SNP (rs3200401) and the susceptibility of RA. However, there is an association between H19 SNP (rs2251375) genotype CA and MALAT1 SNP (rs3200401) genotype CC with RA high disease activity.

Assessment of Angiopoietin-2 Single Nucleotide Polymorphism in Patients with Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is an inflammatory autoimmune disease that destroys joint cartilage and causes disability. Synovial inflammation, with angiogenesis, is an early event in the progression of the disease. Angiopoietin 2 (ANGPT2) is a cytokine with both inflammatory and angiogenic effects. Many genes can influence RA susceptibility and disease activity. The aim is to assess the relationship between ANGPT2 gene polymorphism (rs3020221) and RA. The study was a case-control study that included 212 RA patients and 238 age-and gender-matched healthy volunteers. RA disease activity was assessed using the Disease Activity Score 28 index. Erythrocyte sedimentation rate, C-reactive protein, rheumatoid factor, and antibody to cyclic citrullinated peptide were measured. ANGPT2 rs3020221 C > T SNP genotyping was done using real-time polymerase chain reaction (PCR). The TT genotype was more frequently represented in RA patients than in healthy controls (18.9% and 7.1%, respectively, p < 0.001) and increased the chance of developing RA four-fold, as compared to other genotypes (OR = 4.00, 95% CI = 2.09-7.63) (p < 0.001). The CT genotype was associated with elevated levels of the inflammatory markers ESR and CRP in RA patients (p = 0.012 and 0.037, respectively) as well as the DAS28 ESR Score (p < 0.001). The presence of the T allele either under the dominant model (for genotypes CT and TT) or the recessive model (for the genotype TT) predicts RA disease. Assessment of ANGPT2 gene polymorphism is useful to predict the patients with susceptibility to RA. The presence of T allele increased the risk of developing RA disease by two folds.

The Association of Angiopoietin-2 1064 C/T Rs3020221 Gene Polymorphism with Knee Osteoarthritis.

Osteoarthritis (OA) is a common type of arthritis, affecting millions of people around the world. Angiopoietin-2 (Angpt-2) has a role in the development of chronic inflammatory diseases. We aimed to assess the serum Angpt-2 levels in knee OA patients and to investigate the association of Angpt-2 gene polymorphism(rs3020221 C/T) with knee OA susceptibility and severity. Angiopoietin-2(rs3020221C/T) gene polymorphism was identified in 254 knee OA patients and 227 healthy controls using real-time polymerase chain reaction. Serum Angpt-2 was measured using ELISA. The Arabic version of the Western Ontario and McMaster Universities Osteoarthritis (WOMAC) Index and Kellgren-Lawrence (KL) grading score were used to assess the clinical and radiological severity of OA and their relationship with Angpt-2(rs3020221C/T) gene polymorphism was investigated. Serum Angpt-2 levels were significantly higher in knee OA patients than in the controls (P = .001). OA patients with C/T genotype had a four times greater risk of developing OA than other genotypes (OR = 4.39, 95% CI = 2.85-6.76). Additionally, the T allele presented more in OA patients 224/508 (44%) with two times risk of developing OA (OR = 1.86, 95% CI = 1.43-2.43, p = .001). Angpt-2 SNP (rs3020221C/T) genotype C/T was significantly associated with elevated serum Angpt-2 levels (14.15 ± 5.62 ng/ml). The serum Angpt-2 levels are significantly elevated in OA patients and Angpt-2 gene polymorphism (rs3020221 C/T) may be a risk factor for OA development and both are associated with the severity of knee OA. Carriers of the C/T genotype have a significantly higher serum Angpt-2 levels and a greater risk of developing OA.

Lab-scale Preparation of Recombinant Human Insulin-like Growth Factor-1 in Escherichia coli and its Potential Safety on Normal Human Lung Cell Line.

BACKGROUND: Insulin-like growth factor-1 (IGF-1) is structurally similar to insulin and acts as an endocrine hormone secreted by the liver. OBJECTIVE: Production of recombinant human IGF-1 (rhIGF-1) in Escherichia coli (E.coli) and evaluation of its proliferation stimulatory activity. METHODS: hIGF-1 gene cloned into pBSK (+) simple vector was transformed into TOP 10 chemically competent cells of E. coli. Polymerase chain reaction (PCR) was achieved using specific hIGF-1 gene primers to confirm the successful transformation. To express the rhIGF-1 in E. coli (Rosetta (DE3) pLysS); the hIGF-1 gene was cloned into the pET-15b expression vector and then the recombinant pET-15b/IGF-1 vector was transformed into a chemically prepared competent expression bacterial cells; Rosetta (DE3) pLysS. The rhIGF-1 was expressed as insoluble aggregates called inclusion bodies (IBs) using a 2 mM Isopropyl ß-D-1-thiogalactopyranoside (IPTG) inducer. IBs were solubilized in a denatured form using 6 M guanidinium hydrochloride (GdmCl), followed by in vitro protein refolding using the rapid dilution method. The refolded hIGF-1 was purified using the HiTrap- ANX anion exchange column. Western blot and ELISA using rabbit polyvalent anti-hIGF- 1 were performed to confirm the protein antigenic identity. Cell proliferation activity of rhIGF-1 was testified on normal human lung cell line (WI-38). RESULTS: rhIGF-1 was purified from the HiTrap-ANX column at a concentration of 300 µg/ml. Western blot showed a single 7.6 kDa band obtained in the induced Rosetta (DE3) pLYsS. ELISA confirmed the molecular identity of the rhIGF-1 epitope, the concentration of purified rhIGF-1 obtained from the ELISA standard curve using rhIGF-1 reference protein as a standard was 300 µg/ml, and activity on WI-38 cells was 2604.17I U/mg. CONCLUSION: Biologically active native rhIGF-1 protein was successfully expressed. Patents related to the preparation of IGF-1 were mentioned along the text.

Interleukin 16 polymorphism and susceptibility of rheumatoid arthritis disease in Egyptian population.

Rheumatoid arthritis (RA) is a systemic and multiple-stage disorder characterized by chronic inflammation with extensive synovitis. The genetic and environmental factors are associated with the risk for RA development. In RA, the induced IL-16 may play a role in initiating, sustaining and increasing the inflammatory response and development of synovitis, nevertheless IL-16's actual role in RA pathogenesis must be studied further. This study intended to investigate the association of IL-16 gene polymorphism and RA disease, to determine the genetic role of IL-16 polymorphism and predict the risk of RA development and clinical disease activity. One hundred and Fifty RA patients and 150 apparently healthy control subjects were included in this case-control study. RA disease activity and functional status were evaluated for all RA patients. IL-16 gene polymorphism (SNP rs11556218 T/G) was genotyped using real-time polymerase chain reaction. The difference in IL-16 (rs11556218 T/G) genotype frequencies between RA patients and controls was not statistically significant. However, the G allele was frequently presented in RA patients as compared to controls (p=0.047). Moreover, G allele carriers had two times more risk to develop RA disease than T allele carriers (OR=2.598; 95%CI=1.078-6.825) with dominant genetic association. Alternatively, the G/G genotype was associated with high CDAI, RADAS-5 and HAQ disability index in comparing to other genotypes (T/T-T/G). In conclusion, there was an association between allele G of IL-16 polymorphism (rs11556218 T/G) and risk of RA disease development. In addition, there was an association between genotype G/G and increased clinical disease activity and health disability.

Attitudes of dental and chiropractic students towards a shared learning programme-An interprofessional learning model.

INTRODUCTION: Interprofessional learning (IPL) is the first stage towards the goal of interprofessional collaborative care. To enhance IPL experience, the School of Dentistry, International Medical University developed an IPL model based on the core competencies and the learning outcomes for dental and chiropractic students in their second and fourth year, respectively. The model was based on experiential learning and adult learning theories in addition to Miller's framework for clinical competencies. METHODS: The programme was developed as a student-centred, collaborative approach to achieve the learning outcomes for dental and chiropractic students. Second-year dental students (n = 46) and chiropractic students (n = 23) in their fourth year participated in the programme. The focus of the programme was to address the prevention of work-related musculoskeletal disorders (WMSDs) amongst dental students and to provide the chiropractic students with the opportunity to assess and identify risk factors for WMSDs in the dental setting. The readiness for interprofessional learning scale (RIPLS) questionnaire was completed prior to the interprofessional education programme and once again afterwards to determine dental and chiropractic students' awareness of roles and responsibilities of the other profession, and their attitudes to interprofessional education and teamwork. RESULTS: Dental and chiropractic students showed similar levels of readiness for shared learning. The results of this study suggest that the IPL programme contributed to the development of the students' positive perceptions towards the positive professional identity and the roles of other healthcare professionals. CONCLUSION: This study provides initial support for the integrated interprofessional learning experiences within the school. The results of the study will shape future curricula changes to further strengthen interprofessional education and subsequent interprofessional collaborative care.

New antimicrobial and collagen crosslinking formulated dentin adhesive with improved bond durability.

OBJECTIVE: Here we describe a novel formulation, based on quaternary ammonium (QA) and riboflavin (RF), which combines antimicrobial activities and protease inhibitory properties with collagen crosslinking without interference to bonding capabilities, was investigated. METHODS: Experimental adhesives modified with different fractions of dioctadecyldimethyl ammonium bromide quaternary ammonium and riboflavin (QARF) were formulated. Dentine specimens were bonded to resincomposites with control or the experimental adhesives to be evaluated for bond strength, interfacial morphology, micro-Raman analysis, nano-CT and nano-leakage expression. In addition, the antibacterial and biocompatibilities of the experimental adhesives were investigated. The endogenous proteases activities and their molecular binding-sites were studied. RESULTS: Modifying the experimental adhesives with QARF did not adversely affect micro-tensile bond strength or the degree of conversion along with the demonstration of anti-proteases and antibacterial abilities with acceptable biocompatibilities. In general, all experimental adhesives demonstrated favourable bond strength with increased and improved values in 1% QARF adhesive at 24 h (39.2 ± 3.0 MPa) and following thermocycling (34.8 ± 4.3 MPa). SIGNIFICANCE: It is possible to conclude that the use of QARF with defined concentration can maintain bond strength values when an appropriate protocol is used and have contributed in ensuring a significant decrease in microbial growth of biofilms. Incorporation of 1% QARF in the experimental adhesive lead to simultaneous antimicrobial and anti-proteolytic effects with low cytotoxic effects, acceptable bond strength and interfacial morphology.

Association of autoimmune regulator gene polymorphism with susceptibility to rheumatoid arthritis in Egyptian population.

The autoimmune regulator (AIRE) gene controls autoimmunity via its transcript AIRE protein that suppresses naïve T cells during central selection. The role of AIRE polymorphism in rheumatoid arthritis (RA) autoimmunity remains elusive. This study aimed to investigate the association of two selected SNPs, namely, rs760426 and rs2075876, with RA susceptibility in the Suez Canal Zone population. The study population included 100 RA patients, and the control group included 100 healthy subjects who were age- and sex-matched to the RA group. SNP genotyping was performed using real-time polymerase chain reaction-based allelic discrimination assay, the odds ratio was defined to assess the strength of the association. For rs760426, combining genotypes data revealed a significant increase for A/G genotype in the RA cases (47%, n = 47) than in the control group (27%, n = 27) in both co-dominant and over-dominant models (P = 0.013 and 0.003 respectively). In addition, rs760426 correlated to duration of RA (P = 0.031) and anti-cyclic citrullinated peptide antibody (P = 0.021). For rs2075876, there was a significant increase in the A/A genotype in RA patients compared with control subjects. In the co-dominant model, the frequency of A/A was 14% and 7% respectively (P = 0.02). In contrast to rs760426, rs2075876 associated with the risk of increased body mass index (P = 0.014) and the positivity of rheumatoid factor (RF) (P = 0.043). The frequency of minor alleles, G allele in rs760426 SNP, and A allele in rs2075876 was higher in RA patients than in control. The haplotype frequency of both G and A alleles in rs760426 and rs2075876 receptively was 11% in RA group with statistically significant difference (P = <0.001) between RA patients and healthy control. SNPs rs760426 and rs2075876 in the AIRE gene may contribute to the risk for RA susceptibility. These two polymorphisms were associated with variable risk factors and predictive biomarkers for RA. The mutant allele (G) of rs760426 SNP has significant indication of poor prognosis.

Functionally graded biomimetic biomaterials in dentistry: an evidence-based update.

Design and development of novel therapeutic strategies to regenerate lost tissue structure and function is a serious clinical hurdle for researchers. Traditionally, much of the research is dedicated in optimising properties of scaffolds. Current synthetic biomaterials remain rudimentary in comparison to their natural counterparts. The ability to incorporate biologically inspired elements into the design of synthetic materials has advanced with time. Recent reports suggest that functionally graded material mimicking the natural tissue morphology can have a more exaggerated response on the targeted tissue. The aim of this review is to deliver an overview of the functionally graded concept with respect to applications in clinical dentistry. A comprehensive understanding of spatiotemporal arrangement in fields of restorative, prosthodontics, periodontics, orthodontics and oral surgery is presented. Different processing techniques have been adapted to achieve such gradients ranging from additive manufacturing (three dimensional printing/rapid prototyping) to conventional techniques of freeze gelation, freeze drying, electrospinning and particulate leaching. The scope of employing additive manufacturing technique as a reliable and predictable tool for the design and accurate reproduction of biomimetic templates is vast by any measure. Further research in the materials used and refinement of the synthesis techniques will continue to expand the frontiers of functionally graded membrane based biomaterials application in the clinical domain.

Novel riboflavin/VE-TPGS modified universal dentine adhesive with superior dentine bond strength and self-crosslinking potential.

OBJECTIVE: To modify a universal dentine adhesive with different concentrations of riboflavin and D-Alpha 1000 Succinate polyethylene (VE-TPGS) as a chemical enhancer and to assess the micro-tensile bond strength (24h/12 months), determine resin penetration, measurement of intermolecular interactions and cytotoxicity. MATERIALS AND METHODS: An experimental adhesive system based on bis-GMA, HEMA and hydrophobic monomer was doped with RF0.125 (RF - Riboflavin) or RF/VE-TPGS (0.25/0.50) and submitted to µTBS evaluation. Resin dentine slabs were prepared and examined using SEM and TEM. Adhesion force was analysed on ends of AFM cantilevers deflection. Quenched peptide assays were performed using fluorescence scanner and wavelengths set to 320nm and 405nm. Cytotoxicity was assessed using human peripheral blood mononuclear cell line. Molecular docking studies were carried out using Schrödinger small-molecule drug discovery suite 2018-2. Data from viable cell results was analyzed using one-way ANOVA. Bond strength values were analysed by two-way ANOVA. Nonparametric results were analyzed using a Kruskal-Wallis test at a 0.05 significance level. RESULTS: RF/VE-TPGS0.25 groups showed highest bond strength results after 24-h storage in artificial saliva (p<0.05). RF/VE-TPGS0.50 groups showed increased bond strength after 12-months of ageing. RF/VE-TPGS modified adhesives showed appreciable presence of a hybrid layer. Packing fraction indicated solid angle profiles describing well sized density and topology relations for the RF/VE-TPGS adhesives, in particular with the RF/VE-TPGS0.50 specimens. Qualitative analysis of the phenotype of macrophages was prominently CD163+ in the RF/VE-TPGS0.50. Both the compounds showed favourable negative binding energies as expressed in terms of 'XP GScore'. CONCLUSION: New formulations based on the incorporation of RF/VE-TPGS in universal adhesives may be of significant potential in facilitating penetration, distribution and uptake of riboflavin within the dentine surface.

Impact of chronic schistosomiasis and HBV/HCV co-infection on the liver: current perspectives.

Schistosomiasis is a public health problem in many countries. Its prevalence is increasing annually; the current infection rate is one in 30 individuals. The WHO reported that at least 206.4 million people all over the world required preventive treatments for schistosomiasis in 2016. Chronic schistosomiasis, hepatitis B virus (HBV) and hepatitis C virus (HCV) co-infection are common in countries where schistosomiasis is endemic. The effects of the hepatotropic virus co-infection may modify the Th2-dominated granulomatous phase of schistosomal infection. These viruses induce a strong-specific T cell response, with infiltration of large numbers of specific interferon-γ-producing CD8+ cells into the liver. The outcome of liver diseases depends on the underlying causes, host immune response and concomitant infections. Co-infection of schistosomiasis with HBV/HCV infection causes advanced liver disease and worsens the outcome, especially with higher viral load titers, which increase the mortality rate through an increased incidence of liver cirrhosis and hepatocellular carcinoma. The exposure risk for HBV in patients with HCV and schistosomiasis was two and half times greater than that in CHC patients without schistosomiasis. Finally, chronic schistosomiasis and HBV/HCV co-infection have serious effects on liver pathology. Co-infection accelerates the progression of liver disease and leads to advanced liver diseases and liver failure.

Co-Blend Application Mode of Bulk Fill Composite Resin.

Objective: To evaluate the effect of a new application method of bulk-fill flowable composite resin material on bond-strength, nanoleakage, and mechanical properties of dentine bonding agents. MATERIALS AND METHODS: Sound extracted human molars were randomly divided into: manufacturer's instructions (MI), manual blend 2 mm (MB2), and manual blend 4 mm (MB4). Occlusal enamel was removed and flattened, dentin surfaces were bonded by Prime & Bond universal (Dentsply and Optibond FL, Kerr). For the MI group, adhesives were applied following the manufacturer's instructions then light-cured. For MB groups, SDR flow+ bulk-fill flowable composite resin was applied in 2- or 4-mm increment then manually rubbed by a micro brush for 15 s with uncured dentine bonding agents and the mixture was light-cured. Composite buildup was fabricated incrementally using Ceram.X One, Dentsply nanohybrid composite resin restorative material. After 24-h water storage, the teeth were sectioned to obtain beams of about 0.8 mm2 for 24-h and thermocycled micro-tensile bond strength at 0.5 mm/min crosshead speed. Degree of conversion was evaluated with micro-Raman spectroscopy. Contraction gaps at 24 h after polymerization were evaluated and atomic force microscopy (AFM) nano-indentation processes were undertaken for measuring the hardness across the interface. Depth of resin penetration was studied using a scanning electron microscope (SEM). Bond strength data was expressed using two-way ANOVA followed by Tukey's test. Nanoindentation hardness was separately analyzed using one-way ANOVA. RESULTS: Factors "storage F = 6.3" and "application F = 30.11" significantly affected the bond strength to dentine. For Optibond FL, no significant difference in nanoleakage was found in MI/MB4 groups between baseline and aged specimens; significant difference in nanoleakage score was observed in MB2 groups. Confocal microscopy analysis showed MB2 Optibond FL and Prime & Bond universal specimens diffusing within the dentine. Contraction gap was significantly reduced in MB2 specimens in both adhesive systems. Degree of conversion (DC) of the MB2 specimens were numerically more compared to MS1 in both adhesive systems. CONCLUSION: Present study suggests that the new co-blend technique might have a positive effect on bond strengths of etch-and-rinse adhesives to dentine.