Characterization, bio-uptake and toxicity of polymer-coated silver nanoparticles and their interaction with human peripheral blood mononuclear cells

Silver nanoparticles (AgNPs) are widely used in medical applications due to their antibacterial and antiviral properties. Despite the extensive study of AgNPs, their toxicity and their effect on human health is poorly understood, as a result of issues such as poor control of NP properties and lack of proper characterization. The aim of this study was to investigate the combined characterization, bio-uptake, and toxicity of well-characterized polyvinylpyrrolidone (PVP)-coated AgNPs in exposure media during exposure time using primary human cells (peripheral blood mononuclear cells (PBMCs)). AgNPs were synthesized in-house and characterized using a multimethod approach. Results indicated the transformation of NPs in RPMI medium with a change in size and polydispersity over 24 h of exposure due to dissolution and reprecipitation. No aggregation of NPs was observed in the RPMI medium over the exposure time (24 h). A dose-dependent relationship between PBMC uptake and Ag concentration was detected for both AgNP and AgNO3 treatment. There was approximately a two-fold increase in cellular Ag uptake in the AgNO3 vs the NP treatment. Cytotoxicity, using LDH and MTS assays and based on exposure concentrations was not significantly different when comparing NPs and Ag ions. Based on differential uptake, AgNPs were more toxic after normalizing toxicity to the amount of cellular Ag uptake. Our data highlights the importance of correct synthesis, characterization, and study of transformations to obtain a better understanding of NP uptake and toxicity. Statistical analysis indicated that there might be an individual variability in response to NPs, although more research is required.

DksA controls the response of the Lyme disease spirochete Borrelia burgdorferi to starvation

ABSTRACTThe pathogenic spirocheteBorrelia burgdorferisenses and responds to diverse environmental challenges, including changes in nutrient availability, throughout its enzootic cycle inIxodesspp. ticks and vertebrate hosts. This study examined the role of DnaK suppressor protein (DksA) in the transcriptional response ofB. burgdorferito starvation. Wild-type anddksAmutantB. burgdorferistrains were subjected to starvation by shifting mid-logarithmic phase cultures grown in BSK II medium to serum-free RPMI medium for 6 h under microaerobic conditions (5% CO2, 3% O2). Microarray analyses of wild-typeB. burgdorferirevealed that genes encoding flagellar components, ribosomal proteins, and DNA replication machinery were downregulated in response to starvation. DksA mediated transcriptomic responses to starvation inB. burgdorferias thedksA-deficient strain differentially expressed only 47 genes in response to starvation compared to the 500 genes differentially expressed in wild-type strains. Consistent with a role for DksA in the starvation response ofB. burgdorferi, fewer CFUs were observed fordksAmutant after prolonged starvation in RPMI medium compared to wild-typeB. burgdorferi. Transcriptomic analyses revealed a partial overlap between the DksA regulon and the regulon of RelBbu, the guanosine tetraphosphate and guanosine pentaphosphate [(p)ppGpp] synthetase that controls the stringent response; the DksA regulon also included many plasmid-borne genes. Additionally, thedksAmutant strain exhibited constitutively elevated (p)ppGpp levels compared to the wild-type strain, implying a regulatory relationship between DksA and (p)ppGpp. Together, these data indicate that DksA along with (p)ppGpp direct the stringent response to effectB. burgdorferiadaptation to its environment.IMPORTANCEThe Lyme disease bacteriumBorrelia burgdorferimust sense and respond to diverse environments as it cycles between its tick vectors and various vertebrate hosts.B. burgdorferimust withstand prolonged periods of starvation while it resides in unfedIxodesticks. In this study, the regulatory protein DksA is shown to play a pivotal role controlling the transcriptional responses ofB. burgdorferito starvation. The results of this study suggest that DksA gene regulatory activity impactsB. burgdorferimetabolism, virulence gene expression, and the ability of this bacterium to complete its natural life cycle.

In vitro anti-Candida albicans activity of new thiatriazole derivative agents

Purpose: We tested the antifungal activity of N,N-phenyl-1,2,3,4-thiatriazole-5-yl-2,4-b-resorcyl-carbothioamide (PTR), of n-3-(1,2,4-dithiazole-5-thione)--resorcylcarbothioamide (DTRTA), of N,N-phenyl-1,2,3,4-thiatriazol-5-yl-2,4-b-resorcyl-carbothioamide (PHARA) against Candida albicans strains in vitro. Materials and methods: We synthesized PTR, DTRTA and PHARA at the Department of Chemistry University of Agriculture in Lublin. We tested the selected three samples with the lowest value of MIC - PTR, DTRTA and PHARA. A reference strain of C. albicans ATCC 10231 and 250 strains of C. albicans isolated from the patients was used. The enzymatic activity of the yeast-like fungi was performed by API ZYM test (bioMériux). Results: The mean MIC C. albicans ATCC 10231 on Sabouraud’s Medium was 12.5 mg/L and YNB Medium and RPMI medium - 6.25 mg/L. The mean MIC C. albicans on Sabouraud’s Medium - exposure to PTR - 19.77 mg/L; exposure to DTRTA -21.06 mg/L, exposure to PHARA - 21.54 mg/L; on YNB Medium - exposure to PTR - 17.79 mg/L; exposure to DTRTA - 16.23 mg/l, exposure to PHARA - 18.92 mg/L and RPMI Medium - exposure to PTR - 12.73 mg/L; exposure to DTRTA -10.93 mg/l, exposure to PHARA - 10.65 mg/L. The reference C. albicans strain ATCC 10231 had 5 enzymes inhibited - after exposure to PTR inhibited the enzymatic activity of 13 enzymes, exposure to DTRTA inhibited the enzymatic activity of 10 enzymes and exposure to PHARA inhibited the enzymatic activity of 13 enzymes. The C. albicans isolates had 3 enzymes inhibited - after exposure to PTR - 5 enzymes was inhibited, exposure to DTRTA - 9 enzymes was inhibited and exposure to PHARA - 4 enzymes was inhibited. Conclusion: The synthesized compounds PTR, DTRA and PHARA exert a moderate antifungal activity against the C. albicans strains in vitro.

Stimulation of Bovine Whole-Blood Samples Cultured in Media Supplemented with Recombinant Interleukin-7 (IL-7) and IL-12 Extends the Life Span of the Gamma Interferon Assay To Detect Mycobacterium bovis-Infected Cattle

The gamma interferon (IFN-γ) assay is widely used to measure cell-mediated immune (CMI) response for the early detection of tuberculosis infection. Processing whole-blood samples for CMI-based diagnostics is time sensitive and usually must occur within 8 h of collection to ensure optimal assay performance. In this study, we developed and tested a modified protocol, in which whole-blood samples fromMycobacterium bovis-infected cattle were diluted 1:1 in RPMI medium containing 0.3% fetal bovine serum (FBS) added or not to recombinant mouse interleukin-7 (rmIL-7) or rmIL-12, alone or in combination, and stored at 4°C. At 3 and 6 days postcollection, the diluted blood samples were adjusted to 10% FBS, dispensed into culture trays, stimulated with a bovine purified protein derivative fromM. bovis, and incubated at 37°C in 5% CO2in air. Plasma was removed and assayed for an IFN-γ response using bovine IFN-γ enzyme-linked immunosorbent assay (ELISA) (Bovigam). The results were then compared with those obtained from the conventional procedure. The IFN-γ responses of the samples stored up to 6 days postcollection in the supplemented RPMI medium were similar to those observed in the samples processed within 8 h after sampling, indicating that lymphocyte vitality and response were preserved. The addition of rmIL-7 and rmIL-12, alone or in combination, to culture medium can enhance lymphocyte survival and thus extends the time limit within which the IFN-γ assay can be applied as a diagnostic tool in bovine tuberculosis surveillance and eradication.

High-Throughput Microfluidic Method To Study Biofilm Formation and Host-Pathogen Interactions in Pathogenic Escherichia coli

ABSTRACTBiofilm formation and host-pathogen interactions are frequently studied using multiwell plates; however, these closed systems lack shear force, which is present at several sites in the host, such as the intestinal and urinary tracts. Recently, microfluidic systems that incorporate shear force and very small volumes have been developed to provide cell biology models that resemblein vivoconditions. Therefore, the objective of this study was to determine if the BioFlux 200 microfluidic system could be used to study host-pathogen interactions and biofilm formation by pathogenicEscherichia coli. Strains of various pathotypes were selected to establish the growth conditions for the formation of biofilms in the BioFlux 200 system on abiotic (glass) or biotic (eukaryotic-cell) surfaces. Biofilm formation on glass was observed for the majority of strains when they were grown in M9 medium at 30°C but not in RPMI medium at 37°C. In contrast, HRT-18 cell monolayers enhanced binding and, in most cases, biofilm formation by pathogenicE. coliin RPMI medium at 37°C. As a proof of principle, the biofilm-forming ability of a diffusely adherentE. colimutant strain lacking AIDA-I, a known mediator of attachment, was assessed in our models. In contrast to the parental strain, which formed a strong biofilm, the mutant formed a thin biofilm on glass or isolated clusters on HRT-18 monolayers. In conclusion, we describe a microfluidic method for high-throughput screening that could be used to identify novel factors involved inE. colibiofilm formation and host-pathogen interactions under shear force.

Cryptococcus neoformans-Cryptococcus gattii Species Complex: an International Study of Wild-Type Susceptibility Endpoint Distributions and Epidemiological Cutoff Values for Fluconazole, Itraconazole, Posaconazole, and Voriconazole

ABSTRACTEpidemiological cutoff values (ECVs) for theCryptococcus neoformans-Cryptococcus gattiispecies complex versus fluconazole, itraconazole, posaconazole, and voriconazole are not available. We established ECVs for these species and agents based on wild-type (WT) MIC distributions. A total of 2,985 to 5,733 CLSI MICs forC. neoformans(including isolates of molecular type VNI [MICs for 759 to 1,137 isolates] and VNII, VNIII, and VNIV [MICs for 24 to 57 isolates]) and 705 to 975 MICs forC. gattii(including 42 to 260 for VGI, VGII, VGIII, and VGIV isolates) were gathered in 15 to 24 laboratories (Europe, United States, Argentina, Australia, Brazil, Canada, Cuba, India, Mexico, and South Africa) and were aggregated for analysis. Additionally, 220 to 359 MICs measured using CLSI yeast nitrogen base (YNB) medium instead of CLSI RPMI medium forC. neoformanswere evaluated. CLSI RPMI medium ECVs for distributions originating from at least three laboratories, which included ≥95% of the modeled WT population, were as follows: fluconazole, 8 μg/ml (VNI,C. gattiinontyped, VGI, VGIIa, and VGIII), 16 μg/ml (C. neoformansnontyped, VNIII, and VGIV), and 32 μg/ml (VGII); itraconazole, 0.25 μg/ml (VNI), 0.5 μg/ml (C. neoformansandC. gattiinontyped and VGI to VGIII), and 1 μg/ml (VGIV); posaconazole, 0.25 μg/ml (C. neoformansnontyped and VNI) and 0.5 μg/ml (C. gattiinontyped and VGI); and voriconazole, 0.12 μg/ml (VNIV), 0.25 μg/ml (C. neoformansandC. gattiinontyped, VNI, VNIII, VGII, and VGIIa,), and 0.5 μg/ml (VGI). The number of laboratories contributing data for other molecular types was too low to ascertain that the differences were due to factors other than assay variation. In the absence of clinical breakpoints, our ECVs may aid in the detection of isolates with acquired resistance mechanisms and should be listed in the revised CLSI M27-A3 and CLSI M27-S3 documents.