Fluralaner systemic treatment of chickens results in mortality in Triatoma gerstaeckeri, vector of the agent of Chagas disease.

BACKGROUND: Chagas disease remains a persistent vector-borne neglected tropical disease throughout the Americas and threatens both human and animal health. Diverse control methods have been used to target triatomine vector populations, with household insecticides being the most common. As an alternative to environmental sprays, host-targeted systemic insecticides (or endectocides) allow for application of chemicals to vertebrate hosts, resulting in toxic blood meals for arthropods (xenointoxication). In this study, we evaluated three systemic insecticide products for their ability to kill triatomines. METHODS: Chickens were fed the insecticides orally, following which triatomines were allowed to feed on the treated chickens. The insecticide products tested included: Safe-Guard® Aquasol (fenbendazole), Ivomec® Pour-On (ivermectin) and Bravecto® (fluralaner). Triatoma gerstaeckeri nymphs were allowed to feed on insecticide-live birds at 0, 3, 7, 14, 28 and 56 days post-treatment. The survival and feeding status of the T. gerstaeckeri insects were recorded and analyzed using Kaplan-Meier curves and logistic regression. RESULTS: Feeding on fluralaner-treated chickens resulted 50-100% mortality in T. gerstaeckeri over the first 14 days post-treatment but not later; in contrast, all insects that fed on fenbendazole- and ivermectin-treated chickens survived. Liquid chromatography tandem mass spectrometry (LC-QQQ) analysis, used to detect the concentration of fluralaner and fenbendazole in chicken plasma, revealed the presence of fluralaner in plasma at 3, 7, and 14 days post-treatment but not later, with the highest concentrations found at 3 and 7 days post-treatment. However, fenbendazole concentration was below the limit of detection at all time points. CONCLUSIONS: Xenointoxication using fluralaner in poultry is a potential new tool for integrated vector control to reduce risk of Chagas disease.

Immunomodulatory Effects of Saccharomyces cerevisiae Fermentation Product Supplementation on Immune Gene Expression and Lymphocyte Distribution in Immune Organs in Broilers.

A study was conducted to evaluate the molecular and cellular immunomodulatory effects of a Saccharomyces cerevisiae fermentation product (Original XPC, Diamond V) in broilers. Our lab has previously demonstrated that broilers fed XPC generate faster and stronger antigen-specific humoral immune responses to Newcastle disease virus (NDV) vaccination. This study aims at investigating the mechanism behind this increased immunocompetence. One-day-old broilers were randomly assigned to one of two treatments: 1.25 kg/ton S. cerevisiae fermentation product (XPC treatment group) or control diet. Birds were vaccinated against NDV on day 1 (B1 strain) and day 21 (LaSota strain) post-hatch. Innate and adaptive immune-related gene expression profiles in central (thymus and bursa of Fabricius) and peripheral (spleen) immune organs were investigated at 14 and 28 days of age by qPCR array. Fold changes larger than 1.2 (P < 0.05) between treated and control were considered significant. Lymphocyte subpopulations in central and peripheral immune organs and blood leukocytes were analyzed by flow cytometry at 14, 21, 28, and 42 days of age. In the spleen, Th1 immune responses and antiviral genes, such as IFN-γ, and its downstream genes signal transducer and activator of transcription (STAT4) and NFκB, were significantly upregulated in the treated group by 14 days of age. In the thymus, genes belonging to different functional groups were influenced at different time points. Cytokine genes associated with lymphocyte maturation, differentiation, and proliferation, such as IL-1R, IL-2, and IL-15 were significantly upregulated in the treated group by 28 days of age. Genes preferentially expressed in the medulla of the thymus and mature thymocytes, such as Myxovirus resistance gene 1, interferon regulatory factor-1, interferon regulatory factor-7, and STAT1, were upregulated in the birds supplemented with XPC. Birds supplemented with XPC had significantly higher percentages of CD3+, CD4+, and CD8+ T-cells in the thymus at day 28 of age, indicating production of more mature T-cells, which was consistent with gene expression results. Results suggest that XPC supplementation primes broilers to become more immunocompetent, without compromising growth performance.

Microneedle-mediated immunization of an adenovirus-based malaria vaccine enhances antigen-specific antibody immunity and reduces anti-vector responses compared to the intradermal route.

Substantial effort has been placed in developing efficacious recombinant attenuated adenovirus-based vaccines. However induction of immunity to the vector is a significant obstacle to its repeated use. Here we demonstrate that skin-based delivery of an adenovirus-based malaria vaccine, HAdV5-PyMSP142, to mice using silicon microneedles induces equivalent or enhanced antibody responses to the encoded antigen, however it results in decreased anti-vector responses, compared to intradermal delivery. Microneedle-mediated vaccine priming and resultant induction of low anti-vector antibody titres permitted repeated use of the same adenovirus vaccine vector. This resulted in significantly increased antigen-specific antibody responses in these mice compared to ID-treated mice. Boosting with a heterologous vaccine; MVA-PyMSP142 also resulted in significantly greater antibody responses in mice primed with HAdV5-PyMSP142 using MN compared to the ID route. The highest protection against blood-stage malaria challenge was observed when a heterologous route of immunization (MN/ID) was used. Therefore, microneedle-mediated immunization has potential to both overcome some of the logistic obstacles surrounding needle-and-syringe-based immunization as well as to facilitate the repeated use of the same adenovirus vaccine thereby potentially reducing manufacturing costs of multiple vaccines. This could have important benefits in the clinical ease of use of adenovirus-based immunization strategies.

Immunity against heterosubtypic influenza virus induced by adenovirus and MVA expressing nucleoprotein and matrix protein-1.

Alternate prime/boost vaccination regimens employing recombinant replication-deficient adenovirus or MVA, expressing Influenza A virus nucleoprotein and matrix protein 1, induced antigen-specific T cell responses in intradermally (ID) vaccinated mice; with the strongest responses resulting from Ad/MVA immunization. In BALB/C mice the immunodominant response was shifted from the previously identified immunodominant epitope to a novel epitope when the antigen was derived from A/Panama/2007/1999 rather than A/PR/8. Alternate immunization routes did not affect the magnitude of antigen-specific systemic IFN-γ response, but higher CD8(+) T-cell IFN-γ immune responses were seen in the bronchoalveolar lavage following intransal (IN) boosting after intramuscular (IM) priming, whilst higher splenic antigen-specific CD8(+) T cell IFN-γ was seen following IM boosting. Partial protection against heterologous influenza virus challenge was achieved following either IM/IM or IM/IN but not ID/ID immunization. These data may be of relevance for the design of optimal immunization regimens for human influenza vaccines, especially for influenza-naïve infants.

Coated microneedle arrays for transcutaneous delivery of live virus vaccines.

Vaccines are sensitive biologics that require continuous refrigerated storage to maintain their viability. The vast majority of vaccines are also administered using needles and syringes. The need for cold chain storage and the significant logistics surrounding needle-and-syringe vaccination is constraining the success of immunization programs. Recombinant live viral vectors are a promising platform for the development of vaccines against a number of infectious diseases, however these viruses must retain infectivity to be effective. Microneedles offer an effective and painless method for delivery of vaccines directly into skin that in the future could provide solutions to current vaccination issues. Here we investigated methods of coating live recombinant adenovirus and modified vaccinia virus Ankara (MVA) vectors onto solid microneedle arrays. An effective spray-coating method, using conventional pharmaceutical processes, was developed, in tandem with suitable sugar-based formulations, which produces arrays with a unique coating of viable virus in a dry form around the shaft of each microneedle on the array. Administration of live virus-coated microneedle arrays successfully resulted in virus delivery, transcutaneous infection and induced an antibody or CD8(+) T cell response in mice that was comparable to that obtained by needle-and-syringe intradermal immunization. To our knowledge, this is the first report of successful vaccination with recombinant live viral vectored vaccines coated on microneedle delivery devices.

Microneedle array design determines the induction of protective memory CD8+ T cell responses induced by a recombinant live malaria vaccine in mice.

BACKGROUND: Vaccine delivery into the skin has received renewed interest due to ease of access to the immune system and microvasculature, however the stratum corneum (SC), must be breached for successful vaccination. This has been achieved by removing the SC by abrasion or scarification or by delivering the vaccine intradermally (ID) with traditional needle-and-syringes or with long microneedle devices. Microneedle patch-based transdermal vaccine studies have predominantly focused on antibody induction by inactivated or subunit vaccines. Here, our principal aim is to determine if the design of a microneedle patch affects the CD8(+) T cell responses to a malaria antigen induced by a live vaccine. METHODOLOGY AND FINDINGS: Recombinant modified vaccinia virus Ankara (MVA) expressing a malaria antigen was percutaneously administered to mice using a range of silicon microneedle patches, termed ImmuPatch, that differed in microneedle height, density, patch area and total pore volume. We demonstrate that microneedle arrays that have small total pore volumes induce a significantly greater proportion of central memory T cells that vigorously expand to secondary immunization. Microneedle-mediated vaccine priming induced significantly greater T cell immunity post-boost and equivalent protection against malaria challenge compared to ID vaccination. Notably, unlike ID administration, ImmuPatch-mediated vaccination did not induce inflammatory responses at the site of immunization or in draining lymph nodes. CONCLUSIONS/SIGNIFICANCE: This study demonstrates that the design of microneedle patches significantly influences the magnitude and memory of vaccine-induced CD8(+) T cell responses and can be optimised for the induction of desired immune responses. Furthermore, ImmuPatch-mediated delivery may be of benefit to reducing unwanted vaccine reactogenicity. In addition to the advantages of low cost and lack of pain, the development of optimised microneedle array designs for the induction of T cell responses by live vaccines aids the development of solutions to current obstacles of immunization programmes.

Repertoire-based selection into the marginal zone compartment during B cell development.

Marginal zone (MZ) B cells resemble fetally derived B1 B cells in their innate-like rapid responses to bacterial pathogens, but the basis for this is unknown. We report that the MZ is enriched in "fetal-type" B cell receptors lacking N regions (N(-)). Mixed bone marrow (BM) chimeras, made with adult terminal deoxynucleotidyl transferase (TdT)(+/+) and TdT(-/-) donor cells, demonstrate preferential repertoire-based selection of N(-) B cells into the MZ. Reconstitution of irradiated mice with adult TdT(+/+) BM reveals that the MZ can replenish N(-) B cells in adult life via repertoire-based selection and suggest the possibility of a TdT-deficient precursor population in the adult BM. The mixed chimera data also suggest repertoire-based bifurcations into distinct BM and splenic maturation pathways, with mature "recirculating" BM B cells showing a very strong preference for N(+) complementarity-determining region (CDR) 3 compared with follicular B cells. Because the T1 and MZ compartments are both the most enriched for N(-) H-CDR3, we propose a novel direct T1-->MZ pathway and identify a potential T1-MZ precursor intermediate. We demonstrate progressive but discontinuous repertoire-based selection throughout B cell development supporting multiple branchpoints and pathways in B cell development. Multiple differentiation routes leading to MZ development may contribute to the reported functional heterogeneity of the MZ compartment.

Immune modulation by cadmium and lead in the acute reporter antigen-popliteal lymph node assay.

Immune modulation by heavy metals may cause serious adverse health effects in humans, although the mechanisms involved are not well understood. Both cadmium and lead are important environmental and occupational toxins. Therefore, in the current study, the costimulatory/adjuvant effects and the T-cell-activating potential of these metals (i.e., CdCl2 and PbCl2), are examined. These immune-modulating properties are critical in the development of conditions such as allergy, hypersensitivity, and autoimmunity. Using the direct popliteal lymph node assay (PLNA) and reporter antigen-popliteal lymph node assay (RA-PLNA) both metals were examined individually for immunotoxicity. Mercury (i.e., HgCl2) was included for comparative purposes as its effects in the RA-PLNA are well documented. Seven days following a single footpad injection containing metal and/or RA (trinitrophenyl-ovalbumin [TNP-OVA] or TNP-Ficoll), BALB/c mice were sacrificed and the popliteal lymph nodes (PLNs) removed. PLN cellularity, TNP-specific antibody-secreting cells (ASCs), and lymphocyte subsets were assessed. All three metals strongly stimulated T- and B-cell proliferation and ASC production following coinjection with the RA TNP-OVA. In each case, ASC production was skewed towards the IgG1 isotype. In addition, all three metals induced IgG production to TNP-Ficoll (although relatively weakly in the case of Cd). These results show that each of these metals can provide adjuvant signals to promote lymphocyte proliferation and enhance adaptive immune responses to unrelated antigens. Skewing of immune responses towards T helper type 2 responses suggests that each of these metals can enhance allergic and hypersensitivity reactions to environmental antigens. Furthermore, the induction of IgG responses to TNP-Ficoll, a T-cell-independent antigen, indicates that each of these metals can activate neoantigen-specific T cells. T-cell activation by metals can lead to metal hypersensitivity and has been implicated in the development of autoimmunity. This is the first report of immune modulation by CdCl2 and PbCl2 in the RA-PLNA.

Tipping the balance towards tolerance: the basis for therapeutic immune modulation by gold?

Gold salts have long been used in the treatment of rheumatoid arthritis. However, the basis for their therapeutic immune-modulating properties has never been satisfactorily explained. Furthermore, treatments are often marred by the development of adverse immune reactions such as hypersensitivity and even exacerbation of autoimmunity. We would like to propose a hypothesis to explain the basis for both the beneficial and adverse immune-modulating effects of gold in the treatment of rheumatoid arthritis. If accepted, this hypothesis will allow for the development of safer and more effective treatments with gold salts. The principle underlying this hypothesis also has broader implications for how immune hypersensitivity and tolerance are perceived.

Radical mediators and mitogen-activated protein kinase signaling in oxygen-dependent radiosensitivity of human tumor cell lines.

Oxygen enhancement of tumor radiosensitivity is attributed to DNA damage by reactive oxygen species. The mechanism remains unclear but may involve mitochondria as major sources of oxygen and nitrogen radicals as well as central effectors of energy homeostasis and apoptosis. Here we used dihydrorhodamine and 2',7'-dichlorodihydrofluorescein to compare mitochondrial and total cell generation, respectively, of reactive oxygen or nitrogen species in cells irradiated at 5 Gy. Irradiation in the presence of oxygen selectively stimulated mitochondrial radical production in HeLa and MeWo cells, but in MCF7 cells radical production was more generalized. In all three cell lines oxygen impaired cell proliferation as measured by resazurin reduction 7 days after irradiation. Antioxidants N-acetylcysteine, ascorbic acid, and melatonin largely prevented dye oxidation during normoxic irradiation yet had no effect on oxygen-dependent irradiation injury. However, NO synthase inhibitor N(G)-monomethyl-L-arginine protected HeLa and MCF7 though not MeWo cells, consistent with their different levels of constitutive NO generation. SB203580 inhibition of p38 MAPK appreciably protected HeLa and marginally protected MCF7 cells against oxygen-dependent irradiation injury, while the less specific JNK/SAPK inhibitor SP600125 and ERK inhibitor U0126 had no effect. None of the inhibitors affected MeWo radiosensitivity. Therefore oxygen-enhanced radiosensitivity in these tumor cell lines does not depend on extensive production of oxygen radicals and is cell-type dependent. NO mediates oxygen-dependent injury in HeLa and MCF7 cells, by p38-dependent and MAPK-independent mechanisms, respectively. In MeWo cells this oxygen-enhanced radiosensitivity is independent of both NO and MAPK signaling.

UV Irradiation of Shell Eggs: Effect on Populations of Aerobes, Molds, and Inoculated Salmonella typhimurium.

The effects were investigated of 254-nm UV radiation on populations of Salmonella typhimurium , aerobes, and molds on the shells of eggs. In the first experiment, the CFU of attached S. typhimurium cells on unwashed clean shell eggs were determined after 0, 1, 3, 5, and 7 min of UV treatment (620 µW/cm2) on both ends of the egg. All UV treatments significantly reduced S. typhimurium CFU (P < .01). UVtreatment (620 µW/cm2) in 1-min alternating light and dark cycles for 5 min (three light and two dark) was compared to 0, 3, and 5 min of UV treatment. No significant differences in microbial populations were observed among light and dark cycles and the other UV treatments. In a subsequent experiment, the same UV treatments were utilized to evaluate photoreactivation. After UV exposure, eggs were exposed to 1 h of fluorescent light or I h of darkness or cultured immediately. S. typhimurium CFU were significantly (P < .01) reduced by the UV treatments. However, no significant differences between microbial populations exposed to UV treatment and UV radiation plus photoreactivation were detected. For studies of aerobic bacteria and molds, different UV treatment times (0, 15, and 30 min) at the intensity of 620 µW/cm2 and different intensities (620, 1350, and 1720 µW/cm2) for 15 min were evaluated. Mold CFU per egg were either 0 or 1 for all UV treatments and a 99% reduction of CFU of aerobic bacteria per egg were observed for all UV treatments. It appears from these studies that UV light can significantly reduce populations of S. typhimurium , aerobes, and molds on shell eggs.