Mark Fidock - Academia.edu (original) (raw)

Papers by Mark Fidock

Journal of Immunological Methods

Eosinophil-derived neurotoxin (EDN) is a surrogate biomarker of eosinophil activation and has con... more Eosinophil-derived neurotoxin (EDN) is a surrogate biomarker of eosinophil activation and has considerable potential as a precision medicine biomarker in diseases where eosinophils may play a causative role. Clinical data for EDN have been generated using different quantitative immunoassays, but comparisons between these individual data sets are challenging as no internationally recognised EDN standards or orthogonal methods exist. In this study we aimed to compare commercial EDN assays from ALPCO, MBL, LSBio and CUSABIO for sample commutability. Firstly, we analytically validated the ALPCO enzyme linked immunosorbent assay (ELISA) and demonstrated appropriate analytical characteristics, including an intra/inter-assay precision coefficient-of-variation of between 1.9 and 6.8%. EDN purified from blood proved to be a good quality control material, whereas recombinant EDN, expressed in E.coli, did not react in the ALPCO immunoassay. Using healthy and asthma patient serum samples we confirmed that the ALPCO assay correlated well with the MBL assay, with a coefficient of determination (R2) of 0.92. However, the results from LSBio and CUSABIO assays were not commutable to the other assays.

Journal of Immunological Methods

Myeloperoxidase (MPO) is predominantly expressed by neutrophils and is an important enzyme used b... more Myeloperoxidase (MPO) is predominantly expressed by neutrophils and is an important enzyme used by the immune system for the neutralisation of bacteria and other microorganisms. The strong oxidative activity of MPO has been linked to pro-inflammatory responses in surrounding cells and tissues with implication in the pathophysiology of cardiovascular, neuroscience and inflammatory diseases. This broad disease association has made MPO an attractive biomarker and therapeutic target. Here we describe the construction and validation of a single combined MPO activity and protein concentration assay using commercially available reagents. This method offers the investigative laboratory the ability to generate results from blood plasma samples in a single analytical run using the same sample aliquot.

Antiviral Therapy, 2012

Background: This study presents preclinical data of a novel interferon (IFN)-α8 fusion protein, P... more Background: This study presents preclinical data of a novel interferon (IFN)-α8 fusion protein, PF-04849285, and compares it with IFN-α2 and pegylated IFN-α2; the latter being the current standard of care for HCV. Methods: The antiviral properties were evaluated in vitro using the HCV replication assay (replicon) and the general encephalomyocarditis virus assay. The binding affinity to both IFNR-subunits was assessed using surface plasmon resonance. Ex vivo experiments using cynomolgus monkey and human blood were used for the evaluation of induction of IFN-inducible biomarkers (interferon inducible protein 10 [IP-10], 2′-5′-oligoadenylate synthetase [OAS2] and interleukin-6 [IL-6]). The molecule was tested intravenously and subcutaneously in cynomolgus monkey in a single dose study for two weeks at 0.01, 1, 5 and 20 mg/kg. Each route and dose combination was given to a single male animal, blood samples were collected for evaluation of biomarkers and pharmacokinetics. The compound was also tested in cynomolgus monkey in a multiple dose study for four weeks, with a twice-a-week dosing prior to a three-week wash-out period for toxicokinetics, pharmacokinetics, and biomarker evaluation at 20, 50 or 100 mg/kg subcutaneously and 20 mg/kg intravenously. Results: The molecule is 10× more potent than the pegylated IFN-α2a, with potency similar to the unmodified IFN-α2a. No unanticipated findings were observed in cynomolgus monkey when dosed up to 20 mg/kg, >10,000fold margin over the anticipated efficacious human dose. Conclusions: The biomarker and toxicological findings were consistent with a potent IFN molecule. The potency and pharmacokinetic properties of the molecule are consistent with dosing at least twice daily with the potential for monthly dosing.

Handbook of Biomarkers and Precision Medicine, 2019

Handbook of Biomarkers and Precision Medicine, 2019

Three distinct genes encode alpha(1)-adrenoceptors. Although homodimers of each subtype have been... more Three distinct genes encode alpha(1)-adrenoceptors. Although homodimers of each subtype have been reported, certain but not all combinations of heterodimers of the alpha(1)-adrenoceptors appear to form. Key studies in this field are reviewed and the approaches that have been applied to monitoring the selectivity and the basis of alpha(1)-adrenoceptor dimerization are discussed.

We have cloned cDNAs encoding three human alpha-1 adrenergic receptor (AR) subtypes and character... more We have cloned cDNAs encoding three human alpha-1 adrenergic receptor (AR) subtypes and characterized pharmacological properties of the expressed receptor protein. A number of significant sequence corrections have been identified and compared with previously published data, at both nucleotide and amino acid levels; the most major differences occur for the human alpha-1a/dAR. Pharmacological characterization was performed simultaneously using six cloned alpha-1AR subtypes (human and rat alpha-1a/d, human and hamster alpha-1b, human and bovine alpha-1c) stably expressed in rat-1 fibroblasts at approximately equal receptor concentrations (1-2 pmol/mg of total protein). In general, human alpha-1AR subtypes have similar pharmacology compared to their rat, hamster and bovine homologs, although a few minor species differences important for alpha-1AR classification are noted. In addition, much lower inactivation (approximately 20%) by the alkylating agent chloroethylclonidine is noted in th...

Using combinations of bioluminescence resonance energy transfer, time-resolved fluorescence reson... more Using combinations of bioluminescence resonance energy transfer, time-resolved fluorescence resonance energy transfer and the functional complementation of pairs of inactive receptor-G protein fusion proteins, the human alpha(1A-1)-adrenoceptor was shown to form homodimeric/oligomeric complexes when expressed in human embryonic kidney (HEK) 293 cells. Saturation bioluminescence resonance energy transfer studies indicated the alpha(1A-1)-adrenoceptor homodimer interactions to be high affinity and some 75 times greater than interactions between the alpha(1A-1)-adrenoceptor and the delta opioid peptide receptor. Only a fraction of the alpha(1A-1)-adrenoceptors was at the plasma membrane of HEK293 cells at steady state. However, dimers of alpha(1A-1)-adrenoceptors were also present in intracellular membranes, and the dimer status of those delivered to the cell surface was unaffected by the presence of agonist. Splice variation can generate at least three forms of the human alpha(1A-1)-a...

Handbook of Biomarkers and Precision Medicine

Clinical & Experimental Allergy, 2011

Mast cells are specialized secretory cells releasing multiple inflammatory mediators when activat... more Mast cells are specialized secretory cells releasing multiple inflammatory mediators when activated. Activation requires antigen/IgE cross-linking of FcɛRI receptors, initiating a complex intracellular signalling cascade. Ceramide kinase (CERK) is a novel lipid kinase implicated in several inflammatory cellular signalling processes. We sought to investigate a role for CERK in FCɛRI/IgE-mediated mast-cell activation. The rat and human mast cell-lines RBL-2H3 and LAD-2, respectively, were stimulated via FcɛRI or with the active product of CERK [ceramide-1-phosphate (C-1P)]. Multiple end-points were measured by enzyme-linked immunosorbent assay; histamine (pre-formed early-phase mediator), prostaglandin D₂ (PGD₂ - rapidly metabolized early-phase mediator) and interleukin (IL) -13 (de novo transcribed late-phase mediator). We demonstrated that C-1P alone induced release of histamine and PGD₂ and was additive to antigen-mediated activation. C-1P did not stimulate IL-13 by a statistically significant amount. Using a specific inhibitor of CERK, antigen-mediated release of histamine and PGD₂ was significantly inhibited. Finally, we identified that, for histamine, CERK was downstream of spleen tyrosine kinase, phosphoinositol-3 kinases and phospholipase C, but upstream of c-jun N-terminal kinase (JNK); while for PGD₂ CERK was positioned upstream of JNK, mitogen-activated protein kinase kinase and cyclooxygense. We have identified a differential role for CERK in mast-cell activation and begun to elucidate its position in the mast cell-signalling cascade, thereby suggesting a model by which CERK may be mediating its effects. This type of study is essential for complete understanding of activation pathways that may eventually be used to identify new targets for drug discovery in inflammatory diseases.

Biochemical Society Transactions, 2004

Three distinct genes encode α1-adrenoceptors. Although homodimers of each subtype have been repor... more Three distinct genes encode α1-adrenoceptors. Although homodimers of each subtype have been reported, certain but not all combinations of heterodimers of the α1-adrenoceptors appear to form. Key studies in this field are reviewed and the approaches that have been applied to monitoring the selectivity and the basis of α1-adrenoceptor dimerization are discussed.

Eosinophils are recruited to sites of inflammation via the action of a number of chemical mediato... more Eosinophils are recruited to sites of inflammation via the action of a number of chemical mediators, including PAF, leukotrienes, eotaxins, ECF-A and histamine. Although many of the cell-surface receptors for these mediators have been identified, histamine-driven chemotaxis has not been conclusively attributed to any of the three known histamine receptor subtypes, suggesting the possibility of a 4th histamine-responsive receptor on eosinophils. We have identified and cloned a novel G protein-coupled receptor (GPCR), termed Pfi-013, from an IL-5 stimulated eosinophil cDNA library which is homologous to the human histamine H3 receptor, both at the sequence and gene structure level. Expression data indicates that Pfi-013 is predominantly expressed in peripheral blood leukocytes, with lower expression levels in spleen, testis and colon. Ligand-binding studies using Pfi-013 expressed in HEK-293Gα15 cells, demonstrates specific binding to histamine with a Kd of 3.28 ± 0.76 nM and possesse...

PLOS ONE

There is a growing body of evidence for the utility of eosinophil-derived neurotoxin (EDN) as a b... more There is a growing body of evidence for the utility of eosinophil-derived neurotoxin (EDN) as a biomarker in asthma, including association with eosinophilic airway inflammation, assessment of disease severity and potential for predicting pathogenic risks, including exacerbations. However, to interpret any biomarker data with confidence, it is first important to understand the preanalytical factors and biological variation that may affect its reliable measurement and results interpretation. In this study we defined the healthy serum EDN reference range for men and women as 1.98 to 26.10 ng/mL, with no significant gender differences. Smoking did not impact the mean EDN levels and no circadian rhythm was identified for EDN, unlike blood eosinophils (EOS) where levels peaked at 00:00h. EDN expression in different cell types was investigated and shown to occur primarily in eosinophils, indicating they are likely to be the main cellular repository for EDN. We also confirm that the quantif...

Journal of Immunological Methods

Eosinophil-derived neurotoxin (EDN) is a surrogate biomarker of eosinophil activation and has con... more Eosinophil-derived neurotoxin (EDN) is a surrogate biomarker of eosinophil activation and has considerable potential as a precision medicine biomarker in diseases where eosinophils may play a causative role. Clinical data for EDN have been generated using different quantitative immunoassays, but comparisons between these individual data sets are challenging as no internationally recognised EDN standards or orthogonal methods exist. In this study we aimed to compare commercial EDN assays from ALPCO, MBL, LSBio and CUSABIO for sample commutability. Firstly, we analytically validated the ALPCO enzyme linked immunosorbent assay (ELISA) and demonstrated appropriate analytical characteristics, including an intra/inter-assay precision coefficient-of-variation of between 1.9 and 6.8%. EDN purified from blood proved to be a good quality control material, whereas recombinant EDN, expressed in E.coli, did not react in the ALPCO immunoassay. Using healthy and asthma patient serum samples we confirmed that the ALPCO assay correlated well with the MBL assay, with a coefficient of determination (R2) of 0.92. However, the results from LSBio and CUSABIO assays were not commutable to the other assays.

Journal of Immunological Methods

Myeloperoxidase (MPO) is predominantly expressed by neutrophils and is an important enzyme used b... more Myeloperoxidase (MPO) is predominantly expressed by neutrophils and is an important enzyme used by the immune system for the neutralisation of bacteria and other microorganisms. The strong oxidative activity of MPO has been linked to pro-inflammatory responses in surrounding cells and tissues with implication in the pathophysiology of cardiovascular, neuroscience and inflammatory diseases. This broad disease association has made MPO an attractive biomarker and therapeutic target. Here we describe the construction and validation of a single combined MPO activity and protein concentration assay using commercially available reagents. This method offers the investigative laboratory the ability to generate results from blood plasma samples in a single analytical run using the same sample aliquot.

Antiviral Therapy, 2012

Background: This study presents preclinical data of a novel interferon (IFN)-α8 fusion protein, P... more Background: This study presents preclinical data of a novel interferon (IFN)-α8 fusion protein, PF-04849285, and compares it with IFN-α2 and pegylated IFN-α2; the latter being the current standard of care for HCV. Methods: The antiviral properties were evaluated in vitro using the HCV replication assay (replicon) and the general encephalomyocarditis virus assay. The binding affinity to both IFNR-subunits was assessed using surface plasmon resonance. Ex vivo experiments using cynomolgus monkey and human blood were used for the evaluation of induction of IFN-inducible biomarkers (interferon inducible protein 10 [IP-10], 2′-5′-oligoadenylate synthetase [OAS2] and interleukin-6 [IL-6]). The molecule was tested intravenously and subcutaneously in cynomolgus monkey in a single dose study for two weeks at 0.01, 1, 5 and 20 mg/kg. Each route and dose combination was given to a single male animal, blood samples were collected for evaluation of biomarkers and pharmacokinetics. The compound was also tested in cynomolgus monkey in a multiple dose study for four weeks, with a twice-a-week dosing prior to a three-week wash-out period for toxicokinetics, pharmacokinetics, and biomarker evaluation at 20, 50 or 100 mg/kg subcutaneously and 20 mg/kg intravenously. Results: The molecule is 10× more potent than the pegylated IFN-α2a, with potency similar to the unmodified IFN-α2a. No unanticipated findings were observed in cynomolgus monkey when dosed up to 20 mg/kg, >10,000fold margin over the anticipated efficacious human dose. Conclusions: The biomarker and toxicological findings were consistent with a potent IFN molecule. The potency and pharmacokinetic properties of the molecule are consistent with dosing at least twice daily with the potential for monthly dosing.

Handbook of Biomarkers and Precision Medicine, 2019

Handbook of Biomarkers and Precision Medicine, 2019

Three distinct genes encode alpha(1)-adrenoceptors. Although homodimers of each subtype have been... more Three distinct genes encode alpha(1)-adrenoceptors. Although homodimers of each subtype have been reported, certain but not all combinations of heterodimers of the alpha(1)-adrenoceptors appear to form. Key studies in this field are reviewed and the approaches that have been applied to monitoring the selectivity and the basis of alpha(1)-adrenoceptor dimerization are discussed.

We have cloned cDNAs encoding three human alpha-1 adrenergic receptor (AR) subtypes and character... more We have cloned cDNAs encoding three human alpha-1 adrenergic receptor (AR) subtypes and characterized pharmacological properties of the expressed receptor protein. A number of significant sequence corrections have been identified and compared with previously published data, at both nucleotide and amino acid levels; the most major differences occur for the human alpha-1a/dAR. Pharmacological characterization was performed simultaneously using six cloned alpha-1AR subtypes (human and rat alpha-1a/d, human and hamster alpha-1b, human and bovine alpha-1c) stably expressed in rat-1 fibroblasts at approximately equal receptor concentrations (1-2 pmol/mg of total protein). In general, human alpha-1AR subtypes have similar pharmacology compared to their rat, hamster and bovine homologs, although a few minor species differences important for alpha-1AR classification are noted. In addition, much lower inactivation (approximately 20%) by the alkylating agent chloroethylclonidine is noted in th...

Using combinations of bioluminescence resonance energy transfer, time-resolved fluorescence reson... more Using combinations of bioluminescence resonance energy transfer, time-resolved fluorescence resonance energy transfer and the functional complementation of pairs of inactive receptor-G protein fusion proteins, the human alpha(1A-1)-adrenoceptor was shown to form homodimeric/oligomeric complexes when expressed in human embryonic kidney (HEK) 293 cells. Saturation bioluminescence resonance energy transfer studies indicated the alpha(1A-1)-adrenoceptor homodimer interactions to be high affinity and some 75 times greater than interactions between the alpha(1A-1)-adrenoceptor and the delta opioid peptide receptor. Only a fraction of the alpha(1A-1)-adrenoceptors was at the plasma membrane of HEK293 cells at steady state. However, dimers of alpha(1A-1)-adrenoceptors were also present in intracellular membranes, and the dimer status of those delivered to the cell surface was unaffected by the presence of agonist. Splice variation can generate at least three forms of the human alpha(1A-1)-a...

Handbook of Biomarkers and Precision Medicine

Clinical & Experimental Allergy, 2011

Mast cells are specialized secretory cells releasing multiple inflammatory mediators when activat... more Mast cells are specialized secretory cells releasing multiple inflammatory mediators when activated. Activation requires antigen/IgE cross-linking of FcɛRI receptors, initiating a complex intracellular signalling cascade. Ceramide kinase (CERK) is a novel lipid kinase implicated in several inflammatory cellular signalling processes. We sought to investigate a role for CERK in FCɛRI/IgE-mediated mast-cell activation. The rat and human mast cell-lines RBL-2H3 and LAD-2, respectively, were stimulated via FcɛRI or with the active product of CERK [ceramide-1-phosphate (C-1P)]. Multiple end-points were measured by enzyme-linked immunosorbent assay; histamine (pre-formed early-phase mediator), prostaglandin D₂ (PGD₂ - rapidly metabolized early-phase mediator) and interleukin (IL) -13 (de novo transcribed late-phase mediator). We demonstrated that C-1P alone induced release of histamine and PGD₂ and was additive to antigen-mediated activation. C-1P did not stimulate IL-13 by a statistically significant amount. Using a specific inhibitor of CERK, antigen-mediated release of histamine and PGD₂ was significantly inhibited. Finally, we identified that, for histamine, CERK was downstream of spleen tyrosine kinase, phosphoinositol-3 kinases and phospholipase C, but upstream of c-jun N-terminal kinase (JNK); while for PGD₂ CERK was positioned upstream of JNK, mitogen-activated protein kinase kinase and cyclooxygense. We have identified a differential role for CERK in mast-cell activation and begun to elucidate its position in the mast cell-signalling cascade, thereby suggesting a model by which CERK may be mediating its effects. This type of study is essential for complete understanding of activation pathways that may eventually be used to identify new targets for drug discovery in inflammatory diseases.

Biochemical Society Transactions, 2004

Three distinct genes encode α1-adrenoceptors. Although homodimers of each subtype have been repor... more Three distinct genes encode α1-adrenoceptors. Although homodimers of each subtype have been reported, certain but not all combinations of heterodimers of the α1-adrenoceptors appear to form. Key studies in this field are reviewed and the approaches that have been applied to monitoring the selectivity and the basis of α1-adrenoceptor dimerization are discussed.

Eosinophils are recruited to sites of inflammation via the action of a number of chemical mediato... more Eosinophils are recruited to sites of inflammation via the action of a number of chemical mediators, including PAF, leukotrienes, eotaxins, ECF-A and histamine. Although many of the cell-surface receptors for these mediators have been identified, histamine-driven chemotaxis has not been conclusively attributed to any of the three known histamine receptor subtypes, suggesting the possibility of a 4th histamine-responsive receptor on eosinophils. We have identified and cloned a novel G protein-coupled receptor (GPCR), termed Pfi-013, from an IL-5 stimulated eosinophil cDNA library which is homologous to the human histamine H3 receptor, both at the sequence and gene structure level. Expression data indicates that Pfi-013 is predominantly expressed in peripheral blood leukocytes, with lower expression levels in spleen, testis and colon. Ligand-binding studies using Pfi-013 expressed in HEK-293Gα15 cells, demonstrates specific binding to histamine with a Kd of 3.28 ± 0.76 nM and possesse...

PLOS ONE

There is a growing body of evidence for the utility of eosinophil-derived neurotoxin (EDN) as a b... more There is a growing body of evidence for the utility of eosinophil-derived neurotoxin (EDN) as a biomarker in asthma, including association with eosinophilic airway inflammation, assessment of disease severity and potential for predicting pathogenic risks, including exacerbations. However, to interpret any biomarker data with confidence, it is first important to understand the preanalytical factors and biological variation that may affect its reliable measurement and results interpretation. In this study we defined the healthy serum EDN reference range for men and women as 1.98 to 26.10 ng/mL, with no significant gender differences. Smoking did not impact the mean EDN levels and no circadian rhythm was identified for EDN, unlike blood eosinophils (EOS) where levels peaked at 00:00h. EDN expression in different cell types was investigated and shown to occur primarily in eosinophils, indicating they are likely to be the main cellular repository for EDN. We also confirm that the quantif...