Investigation of the epitope specificities of antibodies to islet β-cell autoantigens

Glutamic acid decarboxylase-65 (GAD-65) and the tyrosine phosphatase-like protein IA-2 are major targets of autoimmunity in type I diabetes mellitus (type I DM), stiff-man syndrome (SMS) and autoimmune polyendocrine syndrome (APS). In this study, the precise epitopes in GAD-65 of three different mouse monoclonal antibodies (MoAbs) (N-terminal MoAb within amino acid residues 4-17. C-terminal MoAb within amino acid residues 572-585 and GAD-6). human monoclonal antibody (b96.11 huAb) and polyclonal antibodies (SMS patients' sera) were investigated. The precise epitopes in IA-2 of two different MoAbs (76B and 76F) were also investigated. These precise epitope investigations were performed using two different phage-displayed random peptide libraries with different characteristics (T7 phage library gene X C9C and linear 9-mers, and M13 filamentous phage library gene III C7C and linear 12-mers and gene VIII 5C4C4). Sequencing of N-terminal and C-terminal MoAb reactive peptides which were obtained from the successful biopanning using M13 gene III linear 12-mers phage library and were selected by high affinity binding in immnuo-blotting assay using nitro-cellulose membranes and in capture ELISA revealed that the most significant motif recognised was P-G-x-x-x-W-S-F and F-L-I-x-E-I/V/L-D-x-L respectively which showed conservative substitutions and may correspond to the position 4-10 amino acids (aa) of GAD-65 (P-G-S-G-F-W-S-F) and to the position 573-581 aa of GAD-65 (F-L-I-E-E-I-E-R-L), respectively. To further define the N-terminal MoAb epitope, sequencing of N-terminal MoAbs reactive peptides which were obtained from the successful biopanning using M13 gene VIII 5C4C4 phage Iibrary and were selected by high affinity binding in immnuo-blotting assay using nitro-cellulose membrane and in capture ELISA. revealed a motif of S-T-P which does not correspond to 4-17 aa of GAD-65 and does not overlap with the previous motif of the N-terminal MoAb. i.c. P-G-S-G-F-W-S-F (4-10 aa) of GAD-65. Therefore, the M13 pVIlI 5C4C4 worked with N-terminal MoAb by expressing a relevant sequence for the N-terminal MoAb but which is unlike its epitope in GAD-65. To further define the N-terminal MoAb epitope sequencing of N-terminal MoAb reactive peptides, which were obtained from the successful biopanning using T7 gene X linear 9-mers phage library and were selected by high affinity binding in immnuo-blotting assay using nitro-cellulose membrane revealed a motif of P-X-X-G which may correspond to 4-7 aa of GAD-65 (P-G-S-G) which overlaps with the previous motif P-G-S-G-F-W-S-F (4-10aa). Sequencing of GAD-6 MoAb reactive peptides which were obtained from the successful biopanning using T7 gene X C9C phage library and were selected by high affinity binding in immnuo-blotting assay using nitro-cellulose membrane and in ELISA, revealed two different motifs of R/K-L/ A/I-x-K and M-x-x-A which showed conservative substitutions and may correspond to the position 525-52X and of GAD-65 (R-L-S-K) and to the position 523-526 aa of GAD-65 (M-S-R-L) respectively, which overlap with each other. To further define the GAD-6 epitope sequencing of GAD-6 reactive peptides which were obtained from the successful biopanning using T7 gene X linear 9-mers phage library and were selected by high affinity binding in immnuo-blotting assay using nitro-cellulose membrane revealed a motif of R-x-x-K, which may correspond to 525-528 aa of GAD-65 (R-L-S-K) and overlaps with the previous motif of the GAD-6 selected from T7 gene X C9C phage library. To further define the GAD-6 epitope sequencing of GAD-6 reactive peptides which were obtained from the successful biopanning using M13 gene VIII 5C4C4 phage library and were selected by high affinity binding in immnuo-blotting assay using nitro-cellulose membrane and in capture ELISA revealed a motif or M-x-x-A which may correspond to 523-526 aa of GAD-65 (M-S-R-L), and overlaps with the previous motif selected from T7 gene X C9C phage library. Thus, the overall motif or GAD-6 may correspond to 523-528 aa of GAD-65 (M-S-R-L-S-K). To further define the GAD-6 epitope, sequencing or GAD-6 reactive peptides which were obtained from the successful biopanning using MI3 gene III C7C and linear 12-mers phage libraries, did not show a clear motif and did not show reactivity with GAD-6 by capture ELISA. A possible explanation for this is that the peptides which are specific to GAD-6 are not present in these M13 pIII phage libraries. Sequencing of b96.11 huAb reactive peptides, which were obtained from the successful biopanning using M13 gene III linear 12-mers phage library and were selected by moderate affinity binding in immnuo-blotting assay using nitro-cellulose membrane revealed two different motifs of IV-T/S-A/G/L-T/S-A\L and S-T/S-G/A/L/I which showed conservative substitutions and may correspond to the position 332-336 aa of GAD-65 (V-S-A-T-A) and to the position 338-340 aa of GAD-65 (T-T-V). respectively. Thus the overall motif of b96.11 might correspond to 332-340 aa of GAD-65. In a capture ELISA system for the detection of GAD-65 specific antibodies, b78 huAb bound slightly better with GAD-6 (rather than N-terminal MoAb ) as the capture MoAb but b96.11 bound much better with GAD-6 as the capture MoAb. This might suggest that GAD-6 does interfere with b78 huAb binding to GAD-65 more than it does with b96.11 huAb. However it must also suggest that the GAD-6 and b78 huAb epitopes are not directly overlapping. Sequencing of SMS sera reactive peptides which were obtained from the successful biopanning using M13 gene III linear 12-mers phage library and were selected by moderate affinity binding in immnuo-blotting assay using nitro-cellulose membrane revealed two different motifs of L/A-A-x-T/S-R/H/K and of T/S-T-V-I/L-F-E-L/G/I/V/A-H/K-L/G-x-K/R which showed conservative substitutions and may correspond to the position 371-375 aa of GAD-65 (L-L-M-S-R) and to the position 463-472 aa of GAD-65 (T-T-G-F-E-A-H-V-D-K). respectively. as public epitopes of SMS patients' sera. Sequencing of 76B and 76F MoAbs reactive peptides, which were obtained from the successful biopanning using T7 gene X C9C and M 13 gene III linear 12-mers phage libraries and were selected by high affinity binding in immnuo-blotting assay using nitro-cellulose membrane and in ELISA, revealed that the most significant motif recognised was D-x-K-P-L-S and F-x-Y-Q, respectively which may correspond to the position 477-482 aa of IA-2 (D-Q-K-P-L-S) and to the position 626-629 aa of IA-2 (F-E-Y-Q) respectively. The studies described in this thesis have shown that the epitope mapping of different antibodies on GAD-65 and IA-2 may help to understand the relationship between antigenicity and structure in these autoantigens which are targets in type I DM and related disorders (e.g. SMS and APS)

Investigation of the epitope specificities of antibodies to islet β-cell autoantigens

Glutamic acid decarboxylase-65 (GAD-65) and the tyrosine phosphatase-like protein IA-2 are major targets of autoimmunity in type I diabetes mellitus (type I DM), stiff-man syndrome (SMS) and autoimmune polyendocrine syndrome (APS). In this study, the precise epitopes in GAD-65 of three different mouse monoclonal antibodies (MoAbs) (N-terminal MoAb within amino acid residues 4-17. C-terminal MoAb within amino acid residues 572-585 and GAD-6). human monoclonal antibody (b96.11 huAb) and polyclonal antibodies (SMS patients' sera) were investigated. The precise epitopes in IA-2 of two different MoAbs (76B and 76F) were also investigated. These precise epitope investigations were performed using two different phage-displayed random peptide libraries with different characteristics (T7 phage library gene X C9C and linear 9-mers, and M13 filamentous phage library gene III C7C and linear 12-mers and gene VIII 5C4C4). Sequencing of N-terminal and C-terminal MoAb reactive peptides which were obtained from the successful biopanning using M13 gene III linear 12-mers phage library and were selected by high affinity binding in immnuo-blotting assay using nitro-cellulose membranes and in capture ELISA revealed that the most significant motif recognised was P-G-x-x-x-W-S-F and F-L-I-x-E-I/V/L-D-x-L respectively which showed conservative substitutions and may correspond to the position 4-10 amino acids (aa) of GAD-65 (P-G-S-G-F-W-S-F) and to the position 573-581 aa of GAD-65 (F-L-I-E-E-I-E-R-L), respectively. To further define the N-terminal MoAb epitope, sequencing of N-terminal MoAbs reactive peptides which were obtained from the successful biopanning using M13 gene VIII 5C4C4 phage Iibrary and were selected by high affinity binding in immnuo-blotting assay using nitro-cellulose membrane and in capture ELISA. revealed a motif of S-T-P which does not correspond to 4-17 aa of GAD-65 and does not overlap with the previous motif of the N-terminal MoAb. i.c. P-G-S-G-F-W-S-F (4-10 aa) of GAD-65. Therefore, the M13 pVIlI 5C4C4 worked with N-terminal MoAb by expressing a relevant sequence for the N-terminal MoAb but which is unlike its epitope in GAD-65. To further define the N-terminal MoAb epitope sequencing of N-terminal MoAb reactive peptides, which were obtained from the successful biopanning using T7 gene X linear 9-mers phage library and were selected by high affinity binding in immnuo-blotting assay using nitro-cellulose membrane revealed a motif of P-X-X-G which may correspond to 4-7 aa of GAD-65 (P-G-S-G) which overlaps with the previous motif P-G-S-G-F-W-S-F (4-10aa). Sequencing of GAD-6 MoAb reactive peptides which were obtained from the successful biopanning using T7 gene X C9C phage library and were selected by high affinity binding in immnuo-blotting assay using nitro-cellulose membrane and in ELISA, revealed two different motifs of R/K-L/ A/I-x-K and M-x-x-A which showed conservative substitutions and may correspond to the position 525-52X and of GAD-65 (R-L-S-K) and to the position 523-526 aa of GAD-65 (M-S-R-L) respectively, which overlap with each other. To further define the GAD-6 epitope sequencing of GAD-6 reactive peptides which were obtained from the successful biopanning using T7 gene X linear 9-mers phage library and were selected by high affinity binding in immnuo-blotting assay using nitro-cellulose membrane revealed a motif of R-x-x-K, which may correspond to 525-528 aa of GAD-65 (R-L-S-K) and overlaps with the previous motif of the GAD-6 selected from T7 gene X C9C phage library. To further define the GAD-6 epitope sequencing of GAD-6 reactive peptides which were obtained from the successful biopanning using M13 gene VIII 5C4C4 phage library and were selected by high affinity binding in immnuo-blotting assay using nitro-cellulose membrane and in capture ELISA revealed a motif or M-x-x-A which may correspond to 523-526 aa of GAD-65 (M-S-R-L), and overlaps with the previous motif selected from T7 gene X C9C phage library. Thus, the overall motif or GAD-6 may correspond to 523-528 aa of GAD-65 (M-S-R-L-S-K). To further define the GAD-6 epitope, sequencing or GAD-6 reactive peptides which were obtained from the successful biopanning using MI3 gene III C7C and linear 12-mers phage libraries, did not show a clear motif and did not show reactivity with GAD-6 by capture ELISA. A possible explanation for this is that the peptides which are specific to GAD-6 are not present in these M13 pIII phage libraries. Sequencing of b96.11 huAb reactive peptides, which were obtained from the successful biopanning using M13 gene III linear 12-mers phage library and were selected by moderate affinity binding in immnuo-blotting assay using nitro-cellulose membrane revealed two different motifs of IV-T/S-A/G/L-T/S-A\L and S-T/S-G/A/L/I which showed conservative substitutions and may correspond to the position 332-336 aa of GAD-65 (V-S-A-T-A) and to the position 338-340 aa of GAD-65 (T-T-V). respectively. Thus the overall motif of b96.11 might correspond to 332-340 aa of GAD-65. In a capture ELISA system for the detection of GAD-65 specific antibodies, b78 huAb bound slightly better with GAD-6 (rather than N-terminal MoAb ) as the capture MoAb but b96.11 bound much better with GAD-6 as the capture MoAb. This might suggest that GAD-6 does interfere with b78 huAb binding to GAD-65 more than it does with b96.11 huAb. However it must also suggest that the GAD-6 and b78 huAb epitopes are not directly overlapping. Sequencing of SMS sera reactive peptides which were obtained from the successful biopanning using M13 gene III linear 12-mers phage library and were selected by moderate affinity binding in immnuo-blotting assay using nitro-cellulose membrane revealed two different motifs of L/A-A-x-T/S-R/H/K and of T/S-T-V-I/L-F-E-L/G/I/V/A-H/K-L/G-x-K/R which showed conservative substitutions and may correspond to the position 371-375 aa of GAD-65 (L-L-M-S-R) and to the position 463-472 aa of GAD-65 (T-T-G-F-E-A-H-V-D-K). respectively. as public epitopes of SMS patients' sera. Sequencing of 76B and 76F MoAbs reactive peptides, which were obtained from the successful biopanning using T7 gene X C9C and M 13 gene III linear 12-mers phage libraries and were selected by high affinity binding in immnuo-blotting assay using nitro-cellulose membrane and in ELISA, revealed that the most significant motif recognised was D-x-K-P-L-S and F-x-Y-Q, respectively which may correspond to the position 477-482 aa of IA-2 (D-Q-K-P-L-S) and to the position 626-629 aa of IA-2 (F-E-Y-Q) respectively. The studies described in this thesis have shown that the epitope mapping of different antibodies on GAD-65 and IA-2 may help to understand the relationship between antigenicity and structure in these autoantigens which are targets in type I DM and related disorders (e.g. SMS and APS)

In Silico Analysis of Differentially Expressed Genes in Colorectal Carcinoma

Background: Colorectal carcinoma (CRC) is a primary cause of morbidity and mortality worldwide. Resistance to therapy contributes to poor patient prognosis. The aim of our study is to identify the key proteins and interaction networks implicated in CRC which may serve as possible therapeutic targets and help in overcoming therapy resistance.Methods: The microarray dataset of 58 cases and 62 controls was used to identify Differentially Expressed Genes (DEGs).After constructing protein-protein interaction networks , Cytoscape analysis was done to identify the hub proteins. Based on sub graph centrality, between-ness and degree (≥10), hub proteins were selected for further literature search and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis.Results: A total of 85 up-regulated genes and 95 down-regulated genes of CRC patients were selected based on criteria of P>0.05 and fold change>2.0. The PPI analysis revealed STAT3, HNRNPA2B1, RBM8A, RBM25, ATM, HIST1H2BK, SRSF5 and HNRNPDLas hub proteins. On the basis of criteria set for cytoscape analysis, STAT3 and HNRNPA2B1 were identified as key hub proteins. KEGG pathway analysis revealed vital role of STAT3 in carcinogenesis.Conclusion: In addition of HNRNPA2B1 activation by STAT3, cross talk of STAT3 with other oncogenic signaling pathways signifies its role in colorectal carcinogenesis. Our study highlights thatSTAT3may be a possible therapeutic target which may help in overcoming the dilemma of resistance to drug treatment in advanced cases.Keywords: STAT3, drug resistance, targeted therapy, bioinformatics    

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Seroprevalence of Toscana and sandfly fever Sicilian viruses in humans and livestock animals from western Saudi Arabia

International audienceHigh seroprevalence rates of several phleboviruses have been reported in domestic animals and humans in sandfly-infested regions. Sandfly Fever Sicilian virus (SFSV) and Toscana virus (TOSV) are two of these viruses commonly transmitted by Phlebotomus sandflies. While SFSV can cause rapidly resolving mild febrile illness, TOSV could involve the central nervous system (CNS), causing diseases ranging from aseptic meningitis to meningoencephalitis. Sandfly-associated phleboviruses have not been investigated before in Saudi Arabia and are potential causes of infection given the prevalence of sandflies in the country. Here, we investigated the seroprevalence of SFSV and TOSV in the western region of Saudi Arabia in samples collected from blood donors, livestock animals, and animal handlers. An overall seroprevalence of 9.4% and 0.8% was found in humans for SFSV and TOSV, respectively. Seropositivity was significantly higher in non-Saudis compared to Saudis and increased significantly with age especially for SFSV. The highest seropositivity rate was among samples collected from animal handlers. Specifically, in blood donors, 6.4% and 0.7% tested positive for SFSV and TOSV nAbs, respectively. Animal handlers showed higher seroprevalence rates of 16% and 1% for anti-SFSV and anti-TOSV nAbs, respectively, suggesting that contact with livestock animals could be a risk factor. Indeed, sera from livestock animals showed seropositivity of 53.3% and 4.4% in cows, 27.5% and 7.8% in sheep, 2.2% and 0.0% in goats, and 10.0% and 2.3% in camels for SFSV and TOSV, respectively. Together, these results suggest that both SFSV and TOSV are circulating in the western region of Saudi Arabia in humans and livestock animals, albeit a

Hematological and biochemical parameters among obese students at the PSAU, Alkharj, KSA

Management of obesity represents a global problem that challenges the provision of healthcare services in most countries. Saudi Arabia ranked number 29 on a 2007 list of countries with 6% of its population being overweight (BMI > 25).In a university setting, we studied hematological parameters (including whole blood counts, haemoglobin and platelets), the presence of basophilia, iron levels and lipid profiles in obese students, and also in non-obese student controls. We found a significant increase in whole blood count in obese compared to healthy individuals, and also found a high level of basophilia compared to healthy controls.  We also report that the obese student group suffered from low iron levels, and also a reduced total iron binding capacity, as compared to healthy controls. Levels of cholesterol and triglycerides was significantly higher in obese students compared to healthy controls. This study can be interpreted that universities across the Kingdom, and beyond, should consider targeting obesity management in their students to try to reduce the prevalence of obesity and associated disorders, and to support such healthcare programs by offering a variety of environmental, physical exercise and nutritional interventions

Relationship between obesity and immunological parameters among students at the PSAU University-Alkharj, KSA

Obesity represents a major worldwide health problem, all aspects of which have not fully defined, nor fully understood.  In the current study, we investigated a population of university students in terms of the relationship between incidence of obesity in individuals (n=171),within this larger cohort (n=500), with the comorbidities that these high BMI individuals also carried. We also report important statistical differences in blood levels each of cardiac-related protein (CRP)(p=0.002), IL-6(p=0.005), &leptin(p=0.02), when we related the blood values with individual student BMIs which were used as a measure of obesity

Relationship Between Obesity and Immunological Parameters Among Students at the PSAU University-Alkharj, KSA

Obesity represents a major worldwide health problem, all aspects of which have not fully defined, nor fully understood.  In the current study, we investigated a population of university students in terms of the relationship between incidence of obesity in individuals (n=171),within this larger cohort (n=500), with the comorbidities that these high BMI individuals also carried. We also report important statistical differences in blood levels each of cardiac-related protein (CRP)(p=0.002), IL-6(p=0.005), &leptin(p=0.02), when we related the blood values with individual student BMIs which were used as a measure of obesity