DEFINING THE BIOCHEMICAL AND BIOPHYSICAL CHARACTERISTICS OF ANTI-HLA-C*07:02 ALLOANTIBODIES IN SOLID ORGAN TRANSPLANTATION

Ph.DDOCTOR OF PHILOSOPHY (SOM

Cloning and biochemical characterization of CG16936, a putative glutathione transferase in Drosophila Melanogaster / Tanusya Murali

CG16936, which was classified as DmGSTE12 was studied in this project. This study cloned and expressed CG16936 and the resulting recombinant CG16936 has been reactive towards 1,2-dichloro-4-nitrobenze (DCNB) (0.4324 μmol/min/mg) but of no activities towards, 1-chloro-2,4-nitrobenzene, trans-2-hexenal, p-nitrobenzyl chloride (NBC), ethacryanic acid (EA), trans-4-phenyl-butene-2-one (PBO), hexa-2,4-dienal, trans,trans-hepta-2,4-dienal, 1,2-epoxy-3-p-nitrophenoxy propane (EPNP), bromosulfophthalein (BSP) and trans-oct-2-enal. In previous work, it was shown that CG16936 was expressed in response to odorant treatment to fruit flies. Our work investigated the direct contribution of the recombinant CG16936 towards conjugation of odorants Glutathione (GSH). No evidence of conjugated product yielded when the recombinant protein acted upon GSH and trans-2-hexenal on thin layer chromatography. The absence of enzymatic activity hence suggested the inability of CG16936 to conjugate t-2-hexenal to GSH. Upon exposure to benzaldehyde, expression of CG16936 was demonstrated unchanged. The evidences suggested that CG16936 could have assumed other physiological function other than detoxification of odorant and its direct involvement in oxidative stress

Cumulative iodinated contrast exposure for computed tomography during acute kidney injury and major adverse kidney events

10.1007/s00330-020-07428-xEUROPEAN RADIOLOGY3153258-326

Hybrid structural modeling of alloantibody binding to human leukocyte antigen with rapid and reproducible cross-linking mass spectrometry

10.1016/j.crmeth.2023.100569CELL REPORTS METHODS3

Electronic health records accurately predict renal replacement therapy in acute kidney injury

Abstract Background Electronic health records (EHR) detect the onset of acute kidney injury (AKI) in hospitalized patients, and may identify those at highest risk of mortality and renal replacement therapy (RRT), for earlier targeted intervention. Methods Prospective observational study to derive prediction models for hospital mortality and RRT, in inpatients aged ≥18 years with AKI detected by EHR over 1 year in a tertiary institution, fulfilling modified KDIGO criterion based on serial serum creatinine (sCr) measures. Results We studied 3333 patients with AKI, of 77,873 unique patient admissions, giving an AKI incidence of 4%. KDIGO AKI stages at detection were 1(74%), 2(15%), 3(10%); corresponding peak AKI staging in hospital were 61, 20, 19%. 392 patients (12%) died, and 174 (5%) received RRT. Multivariate logistic regression identified AKI onset in ICU, haematological malignancy, higher delta sCr (sCr rise from AKI detection till peak), higher serum potassium and baseline eGFR, as independent predictors of both mortality and RRT. Additionally, older age, higher serum urea, pneumonia and intraabdominal infections, acute cardiac diseases, solid organ malignancy, cerebrovascular disease, current need for RRT and admission under a medical specialty predicted mortality. The AUROC for RRT prediction was 0.94, averaging 0.93 after 10-fold cross-validation. Corresponding AUROC for mortality prediction was 0.9 and 0.9 after validation. Decision tree analysis for RRT prediction achieved a balanced accuracy of 70.4%, and identified delta-sCr ≥ 148 μmol/L as the key factor that predicted RRT. Conclusion Case fatality was high with significant renal deterioration following hospital-wide AKI. EHR clinical model was highly accurate for both RRT prediction and for mortality; allowing excellent risk-stratification with potential for real-time deployment

Switching Heavy Chain Constant Domains Denatures the Paratope 3D Architecture of Influenza Monoclonal Antibodies

Several human monoclonal Abs for treating Influenza have been evaluated in clinical trials with limited success despite demonstrating superiority in preclinical animal models including mice. To conduct efficacy studies in mice, human monoclonal Abs are genetically engineered to contain mouse heavy chain constant domain to facilitate the engagement of Fc-receptors on mouse immune effector cells. Although studies have consistently reported discrepancies in Ab effectiveness following genetic engineering, the structural and mechanistic basis for these inconsistencies remain uncharacterized. Here, we use homology modeling to predict variable region (VR) analogous monoclonal Abs possessing human IgG1, mouse IgG1, and mouse IgG2a heavy chain constant domains. We then examine predicted 3D structures for variations in the spatial location and orientation of corresponding paratope amino acid residues. By structurally aligning crystal structures of Fabs in complex with hemagglutinin (HA), we show that corresponding paratope amino acid residues for VR-analogous human IgG1, mouse IgG1, and mouse IgG2a monoclonal Abs interact differentially with HA suggesting that their epitopes might not be identical. To demonstrate that variations in the paratope 3D fine architecture have implications for Ab specificity and effectiveness, we genetically engineered VR-analogous human IgG1, human IgG4, mouse IgG1, and mouse IgG2a monoclonal Abs and explored their specificity and effectiveness in protecting MDCK cells from infection by pandemic H1N1 and H3N2 Influenza viruses. We found that VR-analogous monoclonal Abs placed on mouse heavy chain constant domains were more efficacious at protecting MDCK cells from Influenza virus infection relative to those on human heavy chain constant domains. Interestingly, mouse but not human heavy chain constant domains increased target breadth in some monoclonal Abs. These data suggest that heavy chain constant domain sequences play a role in shaping Ab repertoires that go beyond class or sub-class differences in immune effector recruitment. This represents a facet of Ab biology that can potentially be exploited to improve the scope and utilization of current therapeutic or prophylactic candidates for influenza

Serial Quantification of Urinary Protein Biomarkers to Predict Drug-induced Acute Kidney Injury

10.2174/1389200220666190711114504CURRENT DRUG METABOLISM208656-66

Defining the structural basis for human leukocyte antigen reactivity in clinical transplantation

The current state-of-the-art technology employed to assess anti-human leukocyte antigen antibodies (Anti-HLA Ab) for donor-recipient matching and patient risk stratification in renal transplantation is the single antigen bead (SAB) assay. However, there are limitations to the SAB assay as it is not quantitative and due to variations in techniques and reagents, there is no standardization across laboratories. In this study, a structurally-defined human monoclonal alloantibody was employed to provide a mechanistic explanation for how fundamental alloantibody biology influences the readout from the SAB assay. Performance of the clinical SAB assay was evaluated by altering Anti-HLA Ab concentration, subclass, and detection reagents. Tests were conducted in parallel by two internationally accredited laboratories using standardized protocols and reagents. We show that alloantibody concentration, subclass, laboratory-specific detection devices, subclass-specific detection reagents all contribute to a significant degree of variation in the readout. We report a significant prozone effect affecting HLA alleles that are bound strongly by the test alloantibody as opposed to those bound weakly and this phenomenon is independent of complement. These data highlight the importance for establishing international standards for SAB assay calibration and have significant implications for our understanding of discordance in previous studies that have analyzed its clinical relevance.Published versio

Defining the structural basis for human alloantibody binding to human leukocyte antigen allele HLA-A(star)11:01

10.1038/s41467-019-08790-1NATURE COMMUNICATIONS10