ABSTRACT
OBJECTIVE: Adult-onset Still's disease (AOSD) and secondary hemophagocytic lymphohistiocytosis (sHLH) are both hyperferritinemic cytokine storm syndromes that can be difficult to distinguish from each other in hospitalized patients. The objective of this study was to compare the inflammatory markers ferritin, D-dimer, C-reactive protein (CRP), and soluble CD25 (sCD25) in patients with AOSD and sHLH. These four markers were chosen as they are widely available and represent different aspects of inflammatory diseases: macrophage activation (ferritin); endothelialopathy (D-dimer); interleukin-1/interleukin-6/tumour necrosis factor elevation (CRP) and T cell activation (sCD25). METHODS: This was a single-center retrospective study. Patients diagnosed by the Hematology service at Vancouver General Hospital for AOSD or sHLH from 2009 to 2023 were included. RESULTS: There were 16 AOSD and 44 sHLH patients identified. Ferritin was lower in AOSD than HLH (median 11 360 µg/L vs. 29 020 µg/L, p = .01) while D-dimer was not significantly different (median 5310 mg/L FEU vs. 7000 mg/L FEU, p = .3). CRP was higher (median 168 mg/L vs. 71 mg/L, p <.01) and sCD25 was lower (median 2220 vs. 7280 U/mL, p = .004) in AOSD compared to HLH. The combined ROC curve using CRP >130 mg/L and sCD25< 3900 U/mL to distinguish AOSD from HLH had an area under the curve (AUC) of 0.94 (95% confidence interval 0.93-0.97) with sensitivity 91% and specificity 93%. CONCLUSIONS: These findings suggest that simple, widely available laboratory tests such as CRP and sCD25 can help clinicians distinguish AOSD from HLH in acutely ill adults with extreme hyperferritinemia. Larger studies examining a wider range of clinically available inflammatory biomarkers in a more diverse set of cytokine storm syndromes are warranted.
ABSTRACT
The motility of microglia involves intracellular signaling pathways that are predominantly controlled by changes in cytosolic Ca2+ and activation of PI3K/Akt (phosphoinositide-3-kinase/protein kinase B). In this letter, we develop a novel biophysical model for cytosolic Ca2+ activation of the PI3K/Akt pathway in microglia where Ca2+ influx is mediated by both P2Y purinergic receptors (P2YR) and P2X purinergic receptors (P2XR). The model parameters are estimated by employing optimization techniques to fit the model to phosphorylated Akt (pAkt) experimental modeling/in vitro data. The integrated model supports the hypothesis that Ca2+ influx via P2YR and P2XR can explain the experimentally reported biphasic transient responses in measuring pAkt levels. Our predictions reveal new quantitative insights into P2Rs on how they regulate Ca2+ and Akt in terms of physiological interactions and transient responses. It is shown that the upregulation of P2X receptors through a repetitive application of agonist results in a continual increase in the baseline [Ca2+], which causes the biphasic response to become a monophasic response which prolongs elevated levels of pAkt.
Subject(s)
Microglia , Proto-Oncogene Proteins c-akt , Microglia/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Adenosine Triphosphate/metabolism , Calcium/metabolism , Receptors, Purinergic/metabolismABSTRACT
Glucagon-like peptide receptor (GLP-1R) agonists (e.g., semaglutide, liraglutide, etc.) are efficient treatment options for people with type 2 diabetes and obesity. The manufacturing method to produce semaglutide, a blockbuster GLP-1 drug on the market, involves multistep synthesis. The large peptide has a hydrophobic fatty acid side chain that makes it sparingly soluble, and its handling, purification, and large-scale production difficult. The growing demand for semaglutide that the manufacturer is not capable of addressing immediately triggered a worldwide shortage. Thus, we have developed a potential alternative analogue to semaglutide by replacing the hydrophobic fatty acid with a hydrophilic human complex-type biantennary oligosaccharide. Our novel glycoGLP-1 analogue was isolated in an â¼10-fold higher yield compared with semaglutide. Importantly, our glycoGLP-1 analogue possessed a similar GLP-1R activation potency to semaglutide and was biologically active in vivo in reducing glucose levels to a similar degree as semaglutide.
Subject(s)
Glucagon-Like Peptide 1 , Glycosylation , Humans , Animals , Glucagon-Like Peptide 1/metabolism , Glucagon-Like Peptide 1/chemistry , Glucagon-Like Peptides/pharmacology , Glucagon-Like Peptides/chemistry , Glucagon-Like Peptides/analogs & derivatives , Glucagon-Like Peptide-1 Receptor/agonists , Glucagon-Like Peptide-1 Receptor/metabolism , Hypoglycemic Agents/pharmacology , Hypoglycemic Agents/chemistry , Hypoglycemic Agents/chemical synthesis , Male , Blood Glucose/drug effects , Blood Glucose/metabolism , Protein Engineering , MiceABSTRACT
A quarter of a century ago, designer peptide drugs finally broke through the glass ceiling. Despite the resistance by big pharma, biotechnology companies managed to develop injectable peptide-based drugs, first against orphan or other small volume diseases, and later for conditions affecting large patient populations such as type 2 diabetes. Even their lack of gastrointestinal absorption could be utilized to enable successful oral dosing against chronic constipation. The preference of peptide therapeutics over small molecule competitors against identical medical conditions can be achieved by careful target selection, intrachain and terminal amino acid modifications, appropriate conjugation to stability enhancers and chemical space expansion, innovative delivery and administration techniques and patient-focused marketing strategies. Unfortunately, however, pharmacoeconomical considerations, including the strength of big pharma to develop competing small molecule drugs, have somewhat limited the success of otherwise smart peptide-based therapeutics. Yet, with increasing improvement in peptide drug modification and formulation, these are continuing to gain significant, and growing, acceptance as desirable alternatives to small molecule compounds.
ABSTRACT
Exenatide was the first marketed GLP-1 receptor agonist for the treatment of type 2 diabetes. Modification to the chemical structure or the formulation has the potential to increase the stability of exenatide. We introduced human complex-type sialyloligosaccharide to exenatide at the native Asn28 position. The synthesis was achieved using both solid phase peptide synthesis (SPPS) and Omniligase-1-mediated chemoenzymatic ligation. The results demonstrate that glycosylation increases the proteolytic stability of exenatide while retaining its full biological activity.
Subject(s)
Diabetes Mellitus, Type 2 , Humans , Exenatide , Diabetes Mellitus, Type 2/drug therapy , Hypoglycemic Agents , Glycosylation , Peptide Hydrolases , Glucagon-Like Peptide-1 Receptor/agonists , VenomsABSTRACT
Astrocytes display a highly complex, spongiform morphology, with their fine terminal processes (leaflets) exercising dynamic degrees of synaptic coverage, from touching and surrounding the synapse to being retracted from the synaptic region. In this paper, a computational model is used to reveal the effect of the astrocyte-synapse spatial relationship on ionic homeostasis. Specifically, our model predicts that varying degrees of astrocyte leaflet coverage influences concentrations of K+, Na+ and Ca2+, and results show that leaflet motility strongly influences Ca2+ uptake, as well as glutamate and K+ to a lesser extent. Furthermore, this paper highlights that an astrocytic leaflet that is in proximity to the synaptic cleft loses the ability to form a Ca2+ microdomain, whereas when the leaflet is remote from the synaptic cleft, a Ca2+ microdomain can form. This may have implications for Ca2+-dependent leaflet motility.
Subject(s)
Astrocytes , Synapses , Astrocytes/metabolism , Synapses/metabolism , Glutamic Acid/metabolism , Homeostasis , Calcium SignalingABSTRACT
The day is rapidly approaching where current antibiotic therapies will no longer be effective due to the development of multi-drug resistant bacteria. Antimicrobial peptides (AMPs) are a promising class of therapeutic agents which have the potential to help address this burgeoning problem. Proline-rich AMPs (PrAMPs) are a sub-class of AMPs, that have multiple modes of action including modulation of the bacterial protein folding chaperone, DnaK. They are highly effective against Gram-negative bacteria and have low toxicity to mammalian cells. Previously we used an in silico approach to identify new potential PrAMPs from the DRAMP database. Four of these peptides, antibacterial napin, attacin-C, P9, and PP30, were each chemically assembled and characterized. Together with synthetic oncocin as a reference, each peptide was then assessed for antibacterial activity against Gram-negative/Gram-positive bacteria and for in vitro DnaK modulation activity. We observed that these peptides directly modulate DnaK activity independently of eliciting or otherwise an antibiotic effect. Based on our findings, we propose a change to our previously established PrAMP definition to remove the requirement for antimicrobial activity in isolation, leaving the following classifiers: >25% proline, modulation of DnaK AND/OR the 70S ribosome, net charge of +1 or more, produced in response to bacterial infection AND/OR with pronounced antimicrobial activity.
ABSTRACT
Antimicrobial peptides (AMPs) are host defense peptides, and unlike conventional antibiotics, they possess potent broad spectrum activities and, induce little or no antimicrobial resistance. They are attractive lead molecules for rational development to improve their therapeutic index. Our current studies examined dimerization of the de novo designed proline-rich AMP (PrAMP), Chex1-Arg20 hydrazide, via C-terminal thiol addition to a series of bifunctional benzene or phenyl tethers to determine the effect of orientation of the peptides and linker length on antimicrobial activity. Antibacterial assays confirmed that dimerization per se significantly enhances Chex1-Arg20 hydrazide action. Greatest advantage was conferred using perfluoroaromatic linkers (tetrafluorobenzene and octofluorobiphenyl) with the resulting dimeric peptides 6 and 7 exhibiting potent action against Gram-negative bacteria, especially the World Health Organization's critical priority-listed multidrug-resistant (MDR)/extensively drug-resistant (XDR) Acinetobacter baumannii as well as preformed biofilms. Mode of action studies indicated these lead PrAMPs can interact with both outer and inner bacterial membranes to affect the membrane potential and stress response. Additionally, 6 and 7 possess potent immunomodulatory activity and neutralise inflammation via nitric oxide production in macrophages. Molecular dynamics simulations of adsorption and permeation mechanisms of the PrAMP on a mixed lipid membrane bilayer showed that a rigid, planar tethered dimer orientation, together with the presence of fluorine atoms that provide increased bacterial membrane interaction, is critical for enhanced dimer activity. These findings highlight the advantages of use of such bifunctional tethers to produce first-in-class, potent PrAMP dimers against MDR/XDR bacterial infections.
ABSTRACT
OBJECTIVE: Methotrexate (MTX) is often the primary medication to treat various rheumatic diseases (RDs) because of its low cost and its demonstrated efficacy in controlling disease activity. However, a concern has been the potential for hepatic fibrosis associated with long-term MTX usage. This study investigated the association between cumulative MTX intake and development of liver fibrosis by utilizing noninvasive transient elastography (FibroScan). METHODS: All patients with inflammatory arthritis treated with MTX were offered screening with FibroScan. A certified technician measured liver stiffness after patients adhered to a fast. Relevant clinical information was obtained by patient survey and medical records review. The population was divided into quartiles based on participants' cumulative dosage of MTX. RESULTS: Five hundred twenty patients with RD were included in this study. The prevalence of stages F3 or F4 liver fibrosis was 13.3% in the control group and 12.7% in the entire sample. Compared with subgroup 1 (control with cumulative MTX exposure of ≤ 499 mg), MTX subgroups 2 to 4 were not significantly correlated with higher FibroScan scores (P = 0.82, 0.59, and 0.18, respectively). In multivariable linear regression analysis, statistically significant factors for liver stiffness were BMI, waist circumference, male sex, and age. CONCLUSION: No significant correlation between the cumulative MTX dosage and liver stiffness, even at high MTX doses, was observed. The analyses showed significant correlations between the FibroScan score and BMI. These findings were reassuring in that current rheumatology practice appears to be safe and effective in screening for liver fibrosis in patients on long-term low-dose MTX therapy.
Subject(s)
Arthritis, Rheumatoid , Elasticity Imaging Techniques , Arthritis, Rheumatoid/drug therapy , Humans , Liver Cirrhosis/diagnostic imaging , Liver Cirrhosis/drug therapy , Male , Methotrexate/adverse effectsABSTRACT
The World Health Organisation has deemed several multi-drug resistant (MDR) nosocomial bacterial pathogens to be of significant threat to human health. A stark increase in morbidity, mortality and the burden to healthcare systems around the world can be attributed to the development of resistance in these bacteria. Accordingly, alternative antimicrobial agents have been sought as an attractive means to combat MDR pathogens, with one such example being antimicrobial peptides (AMPs). Given the reported activity of AMPs, including Pardaxin, MSI-78, dermaseptin-PC (DMPC) and Cecropin B, it is important to understand their activities and modes of action against bacteria for further AMP design. In this study, we compared these AMPs against a panel of nosocomial bacterial pathogens, followed by detailed mechanistic studies. It was found that Pardaxin (1-22) and MSI-78 (4-20) displayed the most pronounced antimicrobial activity against the tested bacteria. The mechanistic studies by membrane permeability and molecular dynamics simulation further confirmed the strong membrane interaction and structure of Pardaxin (1-22) and MSI-78 (4-20), which contributed to their potent activity. This study demonstrated a structure and activity guidance for further design of Pardaxin (1-22) and MSI-78 (4-20) as therapeutics against MDR pathogens. The different effects of DMPC (1-19) and Cecropin B (1-21) on membrane integrity and phospholipid membrane interactions provided critical information for the rational design of next-generation analogues with specificity against either Gram-negative or Gram-positive bacteria.
Subject(s)
Antimicrobial Peptides , Cross Infection , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Cross Infection/drug therapy , Drug Resistance, Multiple, Bacterial , Gram-Positive Bacteria , Humans , Microbial Sensitivity TestsABSTRACT
The life and work of Robert Charles Sheppard (1932-2019), Bob Sheppard informally among friends, is outlined. He was a leading pioneer of solid phase peptide synthesis and made the most significant and fundamental European contribution to the art of peptide synthesis since Emil Fischer.
Subject(s)
Solid-Phase Synthesis TechniquesABSTRACT
The role of insulin and insulin-like peptides (ILPs) in vertebrate animals is well studied. Numerous ILPs are also found in invertebrates, although there is uncertainty as to the function and role of many of these peptides. We have identified transcripts with similarity to the insulin family in the tentacle transcriptomes of the sea anemone Oulactis sp. (Actiniaria: Actiniidae). The translated transcripts showed that these insulin-like peptides have highly conserved A- and B-chains among individuals of this species, as well as other Anthozoa. An Oulactis sp. ILP sequence (IlO1_i1) was synthesized using Fmoc solid-phase peptide synthesis of the individual chains, followed by regioselective disulfide bond formation of the intra-A and two interchain disulfide bonds. Bioactivity studies of IlO1_i1 were conducted on human insulin and insulin-like growth factor receptors, and on voltage-gated potassium, sodium, and calcium channels. IlO1_i1 did not bind to the insulin or insulin-like growth factor receptors, but showed weak activity against KV1.2, 1.3, 3.1, and 11.1 (hERG) channels, as well as NaV1.4 channels. Further functional studies are required to determine the role of this peptide in the sea anemone.
Subject(s)
Insulin/chemistry , Insulin/genetics , Sea Anemones/metabolism , Amino Acid Sequence , Animals , Circular Dichroism , Gene Expression Profiling/methods , Gene Expression Regulation , Insulin/metabolism , Peptides/chemistry , Peptides/genetics , Peptides/metabolismABSTRACT
Regulation of cytosolic calcium (Ca2+) dynamics is fundamental to microglial function. Temporal and spatial Ca2+ fluxes are induced from a complicated signal transduction pathway linked to brain ionic homeostasis. In this paper, we develop a novel biophysical model of Ca2+ and sodium (Na+) dynamics in human microglia and evaluate the contribution of purinergic receptors (P2XRs) to both intracellular Ca2+ and Na+ levels in response to agonist/ATP binding. This is the first comprehensive model that integrates P2XRs to predict intricate Ca2+ and Na+ transient responses in microglia. Specifically, a novel compact biophysical model is proposed for the capture of whole-cell patch-clamp currents associated with P2X4 and P2X7 receptors, which is composed of only four state variables. The entire model shows that intricate intracellular ion dynamics arise from the coupled interaction between P2X4 and P2X7 receptors, the Na+/Ca2+ exchanger (NCX), Ca2+ extrusion by the plasma membrane Ca2+ ATPase (PMCA), and Ca2+ and Na+ leak channels. Both P2XRs are modelled as two separate adenosine triphosphate (ATP) gated Ca2+ and Na+ conductance channels, where the stoichiometry is the removal of one Ca2+ for the hydrolysis of one ATP molecule. Two unique sets of model parameters were determined using an evolutionary algorithm to optimise fitting to experimental data for each of the receptors. This allows the proposed model to capture both human P2X7 and P2X4 data (hP2X7 and hP2X4). The model architecture enables a high degree of simplicity, accuracy and predictability of Ca2+ and Na+ dynamics thus providing quantitative insights into different behaviours of intracellular Na+ and Ca2+ which will guide future experimental research. Understanding the interactions between these receptors and other membrane-bound transporters provides a step forward in resolving the qualitative link between purinergic receptors and microglial physiology and their contribution to brain pathology.
Subject(s)
Calcium/metabolism , Microglia/metabolism , Models, Biological , Receptors, Purinergic P2X/physiology , Adenosine Triphosphate/metabolism , Algorithms , Cell Membrane/physiology , Humans , Hydrolysis , Membrane Potentials , Sodium/metabolism , Sodium-Calcium Exchanger/metabolismABSTRACT
The growing epidemic of diabetes means that there is a need for therapies that are more efficacious, safe, and convenient. Here, we report the efficient synthesis of a novel disulfide dimer of human insulin tethered at the N-terminus of its B-chain through placement of a cysteine residue. The resulting peptide was shown to bind to both the insulin receptor isoform B and insulin-like growth factor-1 receptor with comparable affinity to native insulin. In in vivo insulin tolerance tests, the dimer was equipotent to Actrapid insulin and possessed a sustained duration of action greater than that of Actrapid and Glargine. While the secondary structure of our dimeric insulin was similar to that of insulin, it was more resistant to proteolysis. More importantly, our analogue was produced in quantitative yield from a monomeric thiol insulin scaffold. Our results suggest that this dimer has significant potential to address the clinical needs in the treatment of diabetes.
Subject(s)
Insulin/chemistry , Animals , Binding, Competitive , Humans , Kinetics , Male , Mice , Mice, Inbred C57BLABSTRACT
The current commercially available glucagon formulations for the treatment of severe hypoglycemia must be reconstituted immediately prior to use, owing to the susceptibility of glucagon to fibrillation and aggregation in an aqueous solution. This results in the inconvenience of handling, misuse, and wastage of this drug. To address these issues, we synthesized a glycosylated glucagon analogue in which the 25th residue (Trp) was replaced with a cysteine (Cys) and a Br-disialyloligosaccharide was conjugated at the Cys thiol moiety. The resulting analogue, glycoglucagon, is a highly potent full agonist at the glucagon receptor. Importantly, glycoglucagon exhibits markedly reduced propensity for fibrillation and enhanced thermal and metabolic stability. This novel analogue is thus a valuable lead for producing stable liquid glucagon formulations that will improve patient compliance and minimize wastage.
Subject(s)
Glucagon , Hypoglycemia , Cysteine , HumansABSTRACT
Oral dental infections are one of the most common diseases affecting humans, with caries and periodontal disease having the highest incidence. Caries and periodontal disease arise from infections caused by oral bacterial pathogens. Current misuse and overuse of antibiotic treatments have led to the development of antimicrobial resistance. However, recent studies have shown that cationic antimicrobial peptides are a promising family of antibacterial agents that are active against oral pathogenic bacteria and also possess less propensity for development of antimicrobial resistance. This timely Review has a focus on two primary subjects: (i) the oral bacterial pathogens associated with dental infections and (ii) the current development of antimicrobial peptides targeting oral pathogens.
Subject(s)
Antimicrobial Cationic Peptides , Microbiota , Anti-Bacterial Agents/pharmacology , Antimicrobial Cationic Peptides/pharmacology , Bacteria , HumansABSTRACT
Neurotransmitter dynamics within neuronal synapses can be controlled by astrocytes and reflect key contributors to neuronal activity. In particular, Glutamate (Glu) released by activated neurons is predominantly removed from the synaptic space by perisynaptic astrocytic transporters EAAT-2 (GLT-1). In previous work, we showed that the time course of Glu transport is affected by ionic concentration gradients either side of the astrocytic membrane and has the propensity for influencing postsynaptic neuronal excitability. Experimental findings co-localize GABA transporters GAT-3 with EAAT-2 on the perisynaptic astrocytic membrane. While these transporters are unlikely to facilitate the uptake of synaptic GABA, this paper presents simulation results which demonstrate the coupling of EAAT-2 and GAT-3, giving rise to the ionic-dependent reversed transport of GAT-3. The resulting efflux of GABA from the astrocyte to the synaptic space reflects an important astrocytic mechanism for modulation of hyperexcitability. Key results also illustrate an astrocytic-mediated modulation of synaptic neuronal excitation by released GABA at the glutamatergic synapse.
ABSTRACT
Increasing antibiotic resistance in Gram-negative bacteria has mandated the development of both novel antibiotics and alternative therapeutic strategies. Evidence of interplay between several gastrointestinal peptides and the gut microbiota led us to investigate potential and broad-spectrum roles for the incretin hormone, human glucose-dependent insulinotropic polypeptide (GIP) against the Enterobacteriaceae bacteria, Escherichia coli and Erwinia amylovora. GIP had a potent disruptive action on drug efflux pumps of the multidrug resistant bacteria E. coli TG1 and E. amylovora 1189 strains. The effect was comparable to bacterial mutants lacking the inner and outer membrane efflux pump factor proteins AcrB and TolC. While GIP was devoid of direct antimicrobial activity, it has a potent membrane depolarizing effect, and at low concentrations, it significantly potentiated the activity of eight antibiotics and bile salt by reducing MICs by 4-8-fold in E. coli TG1 and 4-20-fold in E. amylovora 1189. GIP can thus be regarded as an antimicrobial adjuvant with potential for augmenting the available antibiotic arsenal.