Stacking nonenzymatic circuits for high signal gain Chen, Xi; Briggs, Neima; McLain, Jeremy R. ...
Proceedings of the National Academy of Sciences - PNAS,
04/2013, Letnik:
110, Številka:
14
Journal Article
Recenzirano
Odprti dostop
Signal amplification schemes that do not rely on protein enzymes show great potential in areas as abstruse as DNA computation and as applied as point-of-care molecular diagnostics. Toehold-mediated ...strand displacement, a programmable form of dynamic DNA hybridization, can be used to design powerful amplification cascades that can achieve polynomial or exponential amplification of input signals. However, experimental implementation of such amplification cascades has been severely hindered by circuit leakage due to catalyst-independent side reactions. In this study, we systematically analyzed the origins, characteristics, and outcomes of circuit leakage in amplification cascades and devised unique methods to obtain high-quality DNA circuits that exhibit minimal leakage. We successfully implemented a two-layer cascade that yielded 7,000-fold signal amplification and a two-stage, four-layer cascade that yielded upward of 600,000-fold signal amplification. Implementation of these unique methods and design principles should greatly empower molecular programming in general and DNA-based molecular diagnostics in particular.
Background Limited therapeutic options exist for coronavirus disease 2019 (COVID-19). COVID-19 convalescent plasma (CCP) is a potential therapeutic, but there is limited data for patients with ...moderate-to-severe disease. Research question What are outcomes associated with administration of CCP in patients with moderate-to-severe COVID-19 infection? Study design and methods We conducted a propensity score-matched analysis of patients with moderate-to-severe COVID-19. The primary endpoints were in-hospital mortality. Secondary endpoints were number of days alive and ventilator-free at 30 days; length of hospital stay; and change in WHO scores from CCP administration (or index date) to discharge. Of 151 patients who received CCP, 132 had complete follow-up data. Patients were transfused after a median of 6 hospital days; thus, we investigated the effect of convalescent plasma before and after this timepoint with 77 early (within 6 days) and 55 late (after 6 days) recipients. Among 3,217 inpatients who did not receive CCP, 2,551 were available for matching. Results Early CCP recipients, of whom 31 (40%) were on mechanical ventilation, had lower 14-day (15% vs 23%) and 30-day (38% vs 49%) mortality compared to a matched unexposed cohort, with nearly 50% lower likelihood of in-hospital mortality (HR 0.52, 95% CI 0.28-0.96; P = 0.036). Early plasma recipients had more days alive and ventilator-free at 30 days (+3.3 days, 95% CI 0.2 to 6.3 days; P = 0.04) and improved WHO scores at 7 days (-0.8, 95% CI: -1.2 to -0.4; P = 0.0003) and hospital discharge (-0.9, 95% CI: -1.5 to -0.3; P = 0.004) compared to the matched unexposed cohort. No clinical differences were observed in late plasma recipients. Interpretation Early administration of CCP improves outcomes in patients with moderate-to-severe COVID-19, while improvement was not observed with late CCP administration. The importance of timing of administration should be addressed in specifically designed trials.
Current iterations of the hygiene hypothesis suggest an adaptive role for helminth parasites in shaping the proper maturation of the immune system. However, aspects of this hypothesis are based on ...assumptions that may not fully account for realities about human helminth infections. Such realities include evidence of causal associations between helminth infections and asthma or inflammatory bowel disease as well as the fact that helminth infections remain widespread in the United States, especially among populations at greatest risk for inflammatory and autoimmune diseases.
Human whipworm (Trichuris trichiura) infects approximately 1 in 15 people worldwide, representing the leading infectious cause of colitis and subsequent, inflammatory bowel disease (IBD). Current ...control measures focused on mass deworming have had limited success due to low drug efficacies. Vaccination would be an ideal, cost-effective strategy to induce protective immunity, leading to control of infection and transmission. Here we report the identification of whey acidic protein, a whipworm secretory protein, as a strong immunogen for inducing protective efficacy in a surrogate mouse T. muris infection model. The recombinant WAP protein (rTm-WAP49), as well as a single, highly conserved repeat within WAP (fragment 8) expressed as an Na-GST-1 fusion protein (rTm-WAP-F8+Na-GST-1), generate a strong T helper type 2 (Th2) immune response when delivered as subcutaneous vaccines formulated with Montanide ISA 720. Oral challenge with T. muris infective eggs following vaccination led to a significant reduction in worm burden of 48% by rTm-WAP49 and 33% by rTm-WAP-F8+Na-GST-1. The cellular immune correlates of protection included significant antigen-specific production of Th2 cytokines IL-4, IL-9, and IL-13 by cells isolated from the vaccine-draining inguinal lymph nodes, parasite-draining mesenteric lymph nodes, and spleen in mice vaccinated with either rTm-WAP49 or rTm-WAP-F8+Na-GST-1. The humoral immune correlates included a high antigen-specific ratio of IgG1 to IgG2a, without eliciting an IgE-mediated allergic response. Immunofluorescent staining of adult T. muris with WAP antisera identified the worm's pathogenic stichosome organ as the site of secretion of native Tm-WAP protein into the colonic mucosa. Given the high sequence conservation for the WAP proteins from T. muris and T. trichiura, the results presented here support the WAP protein to be further evaluated as a potential human whipworm vaccine candidate.
Ascariasis remains the most common helminth infection in humans. As an alternative or complementary approach to global deworming, a pan-anthelminthic vaccine is under development targeting Ascaris, ...hookworm, and Trichuris infections. As16 and As14 have previously been described as two genetically related proteins from Ascaris suum that induced protective immunity in mice when formulated with cholera toxin B subunit (CTB) as an adjuvant, but the exact protective mechanism was not well understood.
As16 and As14 were highly expressed as soluble recombinant proteins (rAs16 and rAs14) in Pichia pastoris. The yeast-expressed rAs16 was highly recognized by immune sera from mice infected with A. suum eggs and elicited 99.6% protection against A. suum re-infection. Mice immunized with rAs16 formulated with ISA720 displayed significant larva reduction (36.7%) and stunted larval development against A. suum eggs challenge. The protective immunity was associated with a predominant Th2-type response characterized by high titers of serological IgG1 (IgG1/IgG2a > 2000) and high levels of IL-4 and IL-5 produced by restimulated splenocytes. A similar level of protection was observed in mice immunized with rAs16 formulated with alum (Alhydrogel), known to induce mainly a Th2-type immune response, whereas mice immunized with rAs16 formulated with MPLA or AddaVax, both known to induce a Th1-type biased response, were not significantly protected against A. suum infection. The rAs14 protein was not recognized by A. suum infected mouse sera and mice immunized with rAs14 formulated with ISA720 did not show significant protection against challenge infection, possibly due to the protein's inaccessibility to the host immune system or a Th1-type response was induced which would counter a protective Th2-type response.
Yeast-expressed rAs16 formulated with ISA720 or alum induced significant protection in mice against A. suum egg challenge that associates with a Th2-skewed immune response, suggesting that rAS16 could be a feasible vaccine candidate against ascariasis.
Advances in rapid diagnostics for bloodstream infections Briggs, Neima; Campbell, Sheldon; Gupta, Shaili
Diagnostic microbiology and infectious disease,
January 2021, 2021-01-00, 20210101, Letnik:
99, Številka:
1
Journal Article
Recenzirano
Septicemia from bloodstream infections (BSI) is the second largest cause of inpatient mortality and the single most expensive condition for US hospitals to manage. There has been an explosive ...development of commercial diagnostic systems to accelerate the identification and antimicrobial susceptibility testing (AST) of causative pathogens. Despite adoption of advanced technologies like matrix-assisted laser desorption imaging–time-of-flight mass spectrometry and multiplex polymerase chain reaction for rapid identification, clinical impact has been variable, in part due to the persistent need for conventional AST as well as prescriber understanding of these rapidly evolving platforms. Newer technologies are expanding on rapid detection of genotypic determinants of resistance, but only recently has rapid phenotypic AST been available. Yet, improved outcomes with rapid diagnostic platforms are still most evident in conjunction with active antimicrobial stewardship. This review will outline key advancements in rapid diagnostics for BSI and the role of antimicrobial stewardship in this new era.
Liposomal amphotericin B (LAmB) is used in the treatment of opportunistic fungal and parasitic infections, including leishmaniasis. Given its lack of known teratogenicity in pregnancy, LAmB is a ...preferred agent for treatment for these patients. However, significant gaps remain in determining optimal dosing regimens for LAmB in pregnancy. We describe the use of LAmB for a pregnant patient with mucocutaneous leishmaniasis (MCL) using a dosing strategy of 5 mg/kg/day for days 1-7 using ideal body weight followed by 4 mg/kg weekly using adjusted body weight. We reviewed the literature for LAmB dosing strategies, particularly dosing weight, in pregnancy. Of the 143 cases identified in 17 studies, only one reported a dosing weight, in which ideal body weight was used. Five Infectious Diseases Society of America guidelines in total discussed the use of amphotericin B in pregnancy but no guidelines included recommendations for dosing weight. This review describes our experience in using ideal body weight for dosing LAmB in pregnancy for the treatment of MCL. Use of ideal body weight may minimize risk of adverse effects to the fetus compared to the use of total body weight while maintaining efficacy for treatment of MCL in pregnancy.
Abstract
Soil-transmitted helminth (STH) infections, specifically Ascaris, Trichuris, and Hookworm, compose three of the most prevalent Neglected Tropical Diseases, infecting over a billion people ...worldwide. Recurrent childhood STH infections have been shown to cause stunted physical growth, reduced physical fitness, and decreased school performance. Ascaris and Trichuris are of notable interest, given their abrupt decline from a peak prevalence and intensity in pre-adolescence to adulthood, with no known published works evaluating the possible underlying immunological mechanism. We used modern molecular techniques to (a) determine the burden of disease and (b) evaluate the immune profile of 30 adolescents and 51 adults between the ages 13-45, in rural Honduras, endemic for Ascaris and Trichuris. Using quantitative PCR of DNA extracted from stool we quantified the burden of disease of both Ascaris and Trichuris. Positive samples were stratified into groups based on the degree of infection, and controlled for with samples positive for 6 other common gastrointestinal parasites. Each group was immunologically characterized for serum Th1, Th2, and Th17 cytokines and associated antibodies. Results from this pilot study identify the basic serum immune profile associated with protection against Ascaris and Trichuris and serve as the foundation for a second, more comprehensive study that will guide vaccine antigen selection against each parasite by mimicking endemic host protective responses.
T cells genetically modified to stably express immunoreceptors are being assessed for therapeutic potential in clinical trials. T cells expressing a chimeric antigen receptor (CAR) are endowed with a ...new specificity to target tumor-associated antigen (TAA) independent of major histocompatibility complex. Our approach to nonviral gene transfer in T cells uses ex vivo numeric expansion of CAR T cells on irradiated artificial antigen presenting cells (aAPC) bearing the targeted TAA. The requirement for aAPC to express a desired TAA limits the human application of CARs with multiple specificities when selective expansion through coculture with feeder cells is sought. As an alternative to expressing individual TAAs on aAPC, we expressed 1 ligand that could activate CAR T cells for sustained proliferation independent of specificity. We expressed a CAR ligand (designated CARL) that binds the conserved IgG4 extracellular domain of CAR and demonstrated that CARL aAPC propagate CAR T cells of multiple specificities. CARL avoids technical issues and costs associated with deploying clinical-grade aAPC for each TAA targeted by a given CAR. Using CARL enables 1 aAPC to numerically expand all CAR T cells containing the IgG4 domain, and simplifies expansion, testing, and clinical translation of CAR T cells of any specificity.
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