Last data update: Apr 22, 2024. (Total: 46599 publications since 2009)
Records 1-5 (of 5 Records) |
Query Trace: Shakamuri P [original query] |
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Development of a new peptide-bead coupling method for an all peptide-based Luminex multiplexing assay for detection of Plasmodium falciparum antibody responses
Wakeman BS , Shakamuri P , McDonald MA , Weinberg J , Svoboda P , Murphy MK , Kariuki S , Mace K , Elder E , Rivera H , Qvarnstrom Y , Pohl J , Shi YP . J Immunol Methods 2021 499 113148 Using a recombinant protein antigen for antibody testing shows a sum of antibody responses to multiple different immune epitopes existing in the protein antigen. In contrast, the antibody testing to an immunogenic peptide epitope reflects a singular antibody response to the individual peptide epitope. Therefore, using a panel of peptide epitopes provides an advantage for profiling multiple singular antibody responses with potential to estimate recent malaria exposure in human infections. However, transitioning from malaria immune epitope peptide-based ELISA to an all peptide bead-based multiplex Luminex assay presents some challenges including variation in the ability of different peptides to bind beads. The aim of this study was to develop a peptide coupling method while demonstrating the utility of these peptide epitopes from multiple stage antigens of Plasmodium falciparum for measuring antibodies. Successful coupling of peptide epitopes to beads followed three steps: 1) development of a peptide tag appended to the C-terminus of each peptide epitope consisting of beta-alanine-lysine (x 4)--cysteine, 2) bead modification with a high concentration of adipic acid dihydrazide, and 3) use of the peptide epitope as a blocker in place of the traditional choice, bovine serum albumin (BSA). This new method was used to couple 12 peptide epitopes from multiple stage specific antigens of P. falciparum, 1 Anopheles mosquito salivary gland peptide, and 1 Epstein-Barr virus peptide as an assay control. The new method was applied to testing of IgG in pooled samples from 30 individuals with previously repeated malaria exposure in western Kenya and IgM and IgG in samples from 37 U.S. travelers with recent exposure to malaria. The new peptide-bead coupling method and subsequent multiplex Luminex assay showed reliable detection of IgG to all 14 peptides in Kenyan samples. Among 37 samples from U.S. travelers recently diagnosed with malaria, IgM and IgG to the peptide epitopes were detected with high sensitivity and variation. Overall, the U.S. travelers had a much lower positivity rates of IgM than IgG to different peptide epitopes, ranging from a high of 62.2% positive for one epitope to a low of only 5.4% positive for another epitope. In contrast, the travelers had IgG positive rates from 97.3% to 91.9% to various peptide epitopes. Based on the different distribution in IgM and IgG positivity to overall number of peptide epitopes and to the number of pre-erythrocytic, erythrocytic, gametocytic, and salivary stage epitopes at the individual level, four distinct patterns of IgM and IgG responses among the 37 samples from US travelers were observed. Independent peptide-bead coupling and antibody level readout between two different instruments also showed comparable results. Overall, this new coupling method resolves the peptide-bead coupling challenge, is reproducible, and can be applied to any other immunogenic peptide epitopes. The resulting all peptide bead-based multiplex Luminex assay can be expanded to include other peptide epitopes of P. falciparum, different malaria species, or other diseases for surveillance, either in US travelers or endemic areas. |
Citrullination-Resistant LL-37 Is a Potent Antimicrobial Agent in the Inflammatory Environment High in Arginine Deiminase Activity.
Bryzek D , Golda A , Budziaszek J , Kowalczyk D , Wong A , Bielecka E , Shakamuri P , Svoboda P , Pohl J , Potempa J , Koziel J . Int J Mol Sci 2020 21 (23) LL-37, the only member of the mammalian cathelicidin in humans, plays an essential role in innate immunity by killing pathogens and regulating the inflammatory response. However, at an inflammatory focus, arginine residues in LL-37 can be converted to citrulline via a reaction catalyzed by peptidyl-arginine deiminases (PAD2 and PAD4), which are expressed in neutrophils and are highly active during the formation of neutrophil extracellular traps (NETs). Citrullination impairs the bactericidal activity of LL-37 and abrogates its immunomodulatory functions. Therefore, we hypothesized that citrullination-resistant LL-37 variants would retain the functionality of the native peptide in the presence of PADs. To test this hypothesis, we synthetized LL-37 in which arginine residues were substituted by homoarginine (hArg-LL-37). Bactericidal activity of hArg-LL-37 was comparable with that of native LL-37, but neither treatment with PAD4 nor exposure to NETs affected the antibacterial and immunomodulatory activities of hArg-LL-37. Importantly, the susceptibilities of LL-37 and hArg-LL-37 to degradation by proteases did not significantly differ. Collectively, we demonstrated that citrullination-resistant hArg-LL-37 is an attractive lead compound for the generation of new agents to treat bacterial infections and other inflammatory diseases associated with enhanced PAD activity. Moreover, our results provide a proof-of-concept for synthesis of therapeutic peptides using homoarginine. |
Prescriber perceptions of fluoroquinolones, extended-spectrum cephalosporins, and Clostridioides difficile infection
Szymczak JE , Muller BM , Shakamuri NS , Hamilton KW , Gerber JS , Laguio-Vila M , Dumyati GK , Fridkin SK , Guh AY , Reddy SC , Lautenbach E . Infect Control Hosp Epidemiol 2020 41 (8) 1-7 BACKGROUND: Fluoroquinolones (FQs) and extended-spectrum cephalosporins (ESCs) are associated with higher risk of Clostridioides difficile infection (CDI). Decreasing the unnecessary use of FQs and ESCs is a goal of antimicrobial stewardship. Understanding how prescribers perceive the risks and benefits of FQs and ESCs is needed. METHODS: We conducted interviews with clinicians from 4 hospitals. Interviews elicited respondent perceptions about the risk of ESCs, FQs, and CDI. Interviews were audio recorded, transcribed, and analyzed using a flexible coding approach. RESULTS: Interviews were conducted with 64 respondents (38 physicians, 7 nurses, 6 advance practice providers, and 13 pharmacists). ESCs and FQs were perceived to have many benefits, including infrequent dosing, breadth of coverage, and greater patient adherence after hospital discharge. Prescribers stated that it was easy to make decisions about these drugs, so they were especially appealing to use in the context of time pressures. They described having difficulty discontinuing these drugs when prescribed by others due to inertia and fear. Prescribers were skeptical about targeting specific drugs as a stewardship approach and felt that the risk of a negative outcome from under treatment of a suspected bacterial infection was a higher priority than the prevention of CDI. CONCLUSIONS: Prescribers in this study perceived many advantages to using ESCs and FQs, especially under conditions of time pressure and uncertainty. In making decisions about these drugs, prescribers balance risk and benefit, and they believed that the risk of CDI was acceptable in compared with the risk of undertreatment. |
Citrullination Alters the Antiviral and Immunomodulatory Activities of the Human Cathelicidin LL-37 During Rhinovirus Infection.
Casanova V , Sousa FH , Shakamuri P , Svoboda P , Buch C , D'Acremont M , Christophorou MA , Pohl J , Stevens C , Barlow PG . Front Immunol 2020 11 85 Human rhinoviruses (HRV) are the most common cause of viral respiratory tract infections. While normally mild and self-limiting in healthy adults, HRV infections are associated with bronchiolitis in infants, pneumonia in immunocompromised patients, and exacerbations of asthma and COPD. The human cathelicidin LL-37 is a host defense peptide (HDP) with broad immunomodulatory and antimicrobial activities that has direct antiviral effects against HRV. However, LL-37 is known to be susceptible to the enzymatic activity of peptidyl arginine deiminases (PAD), and exposure of the peptide to these enzymes results in the conversion of positively charged arginines to neutral citrullines (citrullination). Here, we demonstrate that citrullination of LL-37 reduced its direct antiviral activity against HRV. Furthermore, while the anti-rhinovirus activity of LL-37 results in dampened epithelial cell inflammatory responses, citrullination of the peptide, and a loss in antiviral activity, ameliorates this effect. This study also demonstrates that HRV infection upregulates PAD2 protein expression, and increases levels of protein citrullination, including histone H3, in human bronchial epithelial cells. Increased PADI gene expression and HDP citrullination during infection may represent a novel viral evasion mechanism, likely applicable to a wide range of pathogens, and should therefore be considered in the design of therapeutic peptide derivatives. |
Carbon nanoparticles inhibit the antimicrobial activities of the human cathelicidin LL-37 through structural alteration
Findlay F , Pohl J , Svoboda P , Shakamuri P , McLean K , Inglis NF , Proudfoot L , Barlow PG . J Immunol 2017 199 (7) 2483-2490 Host defense peptides, also known as antimicrobial peptides, are key elements of innate host defense. One host defense peptide with well-characterized antimicrobial activity is the human cathelicidin, LL-37. LL-37 has been shown to be upregulated at sites of infection and inflammation and is regarded as one of the primary innate defense molecules against bacterial and viral infection. Human exposure to combustion-derived or engineered nanoparticles is of increasing concern, and the implications of nanomaterial exposure on the human immune response is poorly understood. However, it is widely acknowledged that nanoparticles can interact strongly with several immune proteins of biological significance, with these interactions resulting in structural and functional changes of the proteins involved. This study investigated whether the potent antibacterial and antiviral functions of LL-37 were inhibited by exposure to, and interaction with, carbon nanoparticles, together with characterizing the nature of the interaction. LL-37 was exposed to carbon black nanoparticles in vitro, and the antibacterial and antiviral functions of the peptide were subsequently assessed. We demonstrate a substantial loss of antimicrobial function when the peptide was exposed to low concentrations of nanomaterials, and we further show that the nanomaterial-peptide interaction resulted in a significant change in the structure of the peptide. The human health implications of these findings are significant, as, to our knowledge, this is the first evidence that nanoparticles can alter host defense peptide structure and function, indicating a new role for nanoparticle exposure in increased disease susceptibility. |
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