mRNA-LNP vaccine-induced CD8+ T cells protect mice from lethal SARS-CoV-2 infection in the absence of specific antibodies.

The role of CD8+ T cells in SARS-CoV-2 pathogenesis or mRNA-LNP vaccine-induced protection from lethal COVID-19 is unclear. Using mouse-adapted SARS-CoV-2 virus (MA30) in C57BL/6 mice, we show that CD8+ T cells are unnecessary for the intrinsic resistance of female or the susceptibility of male mice to lethal SARS-CoV-2 infection. Also, mice immunized with a di-proline prefusion-stabilized full-length SARS-CoV-2 Spike (S-2P) mRNA-LNP vaccine, which induces Spike-specific antibodies and CD8+ T cells specific for the Spike-derived VNFNFNGL peptide, are protected from SARS-CoV-2 infection-induced lethality and weight loss, while mice vaccinated with mRNA-LNPs encoding only VNFNFNGL are protected from lethality but not weight loss. CD8+ T cell depletion ablates protection in VNFNFNGL but not in S-2P mRNA-LNP-vaccinated mice. Therefore, mRNA-LNP vaccine-induced CD8+ T cells are dispensable when protective antibodies are present but essential for survival in their absence. Hence, vaccine-induced CD8+ T cells may be critical to protect against SARS-CoV-2 variants that mutate epitopes targeted by protective antibodies.

Multiple and Consecutive Genome Editing Using i-GONAD and Breeding Enrichment Facilitates the Production of Genetically Modified Mice.

Genetically modified (GM) mice are essential tools in biomedical research. Traditional methods for generating GM mice are expensive and require specialized personnel and equipment. The use of clustered regularly interspaced short palindromic repeats (CRISPR) coupled with improved-Genome editing via Oviductal Nucleic Acids Delivery (i-GONAD) has highly increased the feasibility of producing GM mice in research laboratories. However, genetic modification in inbred mouse strains of interest such as C57BL/6 (B6) is still challenging because of their low fertility and embryo fragility. We have successfully generated multiple novel GM mouse strains in the B6 background while attempting to optimize i-GONAD. We found that i-GONAD reduced the litter size in superovulated pregnant females but did not impact pregnancy rates. Natural mating or low-hormone dose did not increase the low fertility rate observed in superovulated B6 females. However, diet enrichment had a positive effect on pregnancy success. We also optimized breeding conditions to increase the survival of small litters by co-housing i-GONAD-treated pregnant B6 females with synchronized pregnant FVB/NJ companion mothers. Thus, GM mice generation was increased by an enriched diet and shared pup rearing with highly fertile females such as FVB/NJ. In the present study, we generated 16 GM mice using a CRISPR/Cas system to target individual and multiple loci simultaneously or consecutively. We also compared homology-directed repair efficiency using different methods for LoxP insertion for conditional knockout mouse production. We found that a two-step serial LoxP insertion, in which each LoxP sequence was inserted individually in different i-GONAD procedures, was a low-risk high-efficiency method for generating floxed mice.

Design and preclinical evaluation of a universal SARS-CoV-2 mRNA vaccine.

Because of the rapid mutations of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), an effective vaccine against SARS-CoV-2 variants is needed to prevent coronavirus disease 2019 (COVID-19). T cells, in addition to neutralizing antibodies, are an important component of naturally acquired protective immunity, and a number of studies have shown that T cells induced by natural infection or vaccination contribute significantly to protection against several viral infections including SARS-CoV-2. However, it has never been tested whether a T cell-inducing vaccine can provide significant protection against SARS-CoV-2 infection in the absence of preexisting antibodies. In this study, we designed and evaluated lipid nanoparticle (LNP) formulated mRNA vaccines that induce only T cell responses or both T cell and neutralizing antibody responses by using two mRNAs. One mRNA encodes SARS-CoV-2 Omicron Spike protein in prefusion conformation for induction of neutralizing antibodies. The other mRNA encodes over one hundred T cell epitopes (multi-T cell epitope or MTE) derived from non-Spike but conserved regions of the SARS-CoV-2. We show immunization with MTE mRNA alone protected mice from lethal challenge with the SARS-CoV-2 Delta variant or a mouse-adapted virus MA30. Immunization with both mRNAs induced the best protection with the lowest viral titer in the lung. These results demonstrate that induction of T cell responses, in the absence of preexisting antibodies, is sufficient to confer protection against severe disease, and that a vaccine containing mRNAs encoding both the Spike and MTE could be further developed as a universal SARS-CoV-2 vaccine.

Evaluation of a combination of Citrus aurantium var. Dulcis essential oil and albendazole for the treatment of sheep gastrointestinal nematodes.

Citrus fruits are consumed all over the world and their by-products are used for animal feed and essential oils production. This study aimed to evaluate the in vitro and in vivo activity of Citrus aurantium var. Dulcis essential oil (CaEO) combined with ABZ against benzimidazole resistant Haemonchus contortus. In vitro egg hatching assays (EHA) were performed using CaEO and ABZ to estimate the effective concentration to achieve 50% egg death (EC50) values and calculate the test essential oil and drug combinations using a simplex-centroid mixture design. These concentrations were used for a second round of EHAs. Sixteen sheep were randomly allocated into two groups and treated with ABZ and the combination of CaEO and ABZ, and faecal egg count reduction tests were performed. In the first round of EHA, CaEO and ABZ showed EC50 values of 0.57 and 0.0048 mg mL-1, respectively. The H. contortus strain used in the study was shown to be highly benzimidazole resistant, with only 1.5% of parasites having susceptible ß-tubulin SNP genotypes. The ABZ reduced the shedding of nematode eggs by 78%, however, its combination with CaEO reduced faecal egg counts by only 9%. The present study is important to highlight the interferences of natural products in anthelmintic metabolism and consequently in drug efficacy.

Resistance To Poxvirus Lethality Does Not Require the Necroptosis Proteins RIPK3 or MLKL.

Receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like pseudokinase (MLKL) are proteins that are critical for necroptosis, a mechanism of programmed cell death that is both activated when apoptosis is inhibited and thought to be antiviral. Here, we investigated the role of RIPK3 and MLKL in controlling the Orthopoxvirus ectromelia virus (ECTV), a natural pathogen of the mouse. We found that C57BL/6 (B6) mice deficient in RIPK3 (Ripk3-/-) or MLKL (Mlkl-/-) were as susceptible as wild-type (WT) B6 mice to ECTV lethality after low-dose intraperitoneal infection and were as resistant as WT B6 mice after ECTV infection through the natural footpad route. Additionally, after footpad infection, Mlkl-/- mice, but not Ripk3-/- mice, endured lower viral titers than WT mice in the draining lymph node (dLN) at three days postinfection and in the spleen or in the liver at seven days postinfection. Despite the improved viral control, Mlkl-/- mice did not differ from WT mice in the expression of interferons or interferon-stimulated genes or in the recruitment of natural killer (NK) cells and inflammatory monocytes (iMOs) to the dLN. Additionally, the CD8 T-cell responses in Mlkl-/- and WT mice were similar, even though in the dLNs of Mlkl-/- mice, professional antigen-presenting cells were more heavily infected. Finally, the histopathology in the livers of Mlkl-/- and WT mice at 7 dpi did not differ. Thus, the mechanism of the increased virus control by Mlkl-/- mice remains to be defined. IMPORTANCE The molecules RIPK3 and MLKL are required for necroptotic cell death, which is widely thought of as an antiviral mechanism. Here we show that C57BL/6 (B6) mice deficient in RIPK3 or MLKL are as susceptible as WT B6 mice to ECTV lethality after a low-dose intraperitoneal infection and are as resistant as WT B6 mice after ECTV infection through the natural footpad route. Mice deficient in MLKL are more efficient than WT mice at controlling virus loads in various organs. This improved viral control is not due to enhanced interferon, natural killer cell, or CD8 T-cell responses. Overall, the data indicate that deficiencies in the molecules that are critical to necroptosis do not necessarily result in worse outcomes following viral infection and may improve virus control.

Interferon partly dictates a divergent transcriptional response in poxvirus-infected and bystander inflammatory monocytes.

Inflammatory monocytes (iMOs) and B cells are the main targets of the poxvirus ectromelia virus (ECTV) in the lymph nodes of mice and play distinct roles in surviving the infection. Infected and bystander iMOs control ECTV's systemic spread, preventing early death, while B cells make antibodies that eliminate ECTV. Our work demonstrates that within an infected animal that survives ECTV infection, intrinsic and bystander infection of iMOs and B cells differentially control the transcription of genes important for immune cell function and, perhaps, cell identity. Bystander cells upregulate metabolism, antigen presentation, and interferon-stimulated genes. Infected cells downregulate many cell-type-specific genes and upregulate transcripts typical of non-immune cells. Bystander (Bys) and infected (Inf) iMOs non-redundantly contribute to the cytokine milieu and the interferon response. Furthermore, we uncover how type I interferon (IFN-I) or IFN-γ signaling differentially regulates immune pathways in Inf and Bys iMOs and that, at steady state, IFN-I primes iMOs for rapid IFN-I production and antigen presentation.

Decontamination of wastewater containing contaminants of emerging concern by electrooxidation and Fenton-based processes - A review on the relevance of materials and methods.

In recent years, there has been an increasingly growing interest regarding the use of electrochemical advanced oxidation processes (EAOPs) which are considered highly promising alternative treatment techniques for addressing environmental issues related to pollutants of emerging concern. In EAOPs, electrogenerated oxidizing agents, such as hydroxyl radical (HO•), can react non-selectively with a wide range of organic compounds, degrading and mineralizing their structures to unharmful molecules like CO2, H2O, and inorganic ions. To this date, a broad spectrum of advanced electrocatalysts have been developed and applied for the treatment of compounds of interest in different matrices, specifically aiming at enhancing the degradation performance. New combined methods have also been employed as alternative treatment techniques targeted at circumventing the major obstacles encountered in Fenton-based processes, such as high costs and energy consumption, which still contribute significantly toward inhibiting the large-scale application of these processes. First, some fundamental aspects of EAOPs will be presented. Further, we will provide an overview of electrode materials which have been recently developed and reported in the literature, highlighting different anode and cathode structures employed in EAOPs, their main advantages and disadvantages, as well as their contribution to the performance of the treatment processes. The influence of operating parameters, such as initial concentrations, pH effect, temperature, supporting electrolyte, and radiation source, on the treatment processes were also studied. Finally, hybrid techniques which have been reported in the literature and critically assess the most recent techniques used for evaluating the degradation efficiency of the treatment processes.

Anthelmintic evaluation and essential oils composition of Hyptis dilatata Benth. and Mesosphaerum suaveolens Kuntze from the Brazilian Amazon.

Hyptis dilatata and Mesosphaerum suaveolens belong to Lamiaceae, are aromatic and medicinal subshrubs with antiparasitic potential and occurrence in the Amazon Region. The essential oils of both species were extracted, analyzed, and identified by GC and GC-MS and then evaluated their anthelmintic activities against the nematode Caenorhabditis elegans. Primary constituents of the samples of H. dilatata oils were limonene (72.6%), myrcene (11.5%), and p-cymene (10.3%) to PAMA19 sample, and camphor (25.5%), α-pinene (25.4%), 1,8-cineole (18.8%), ß-pinene (12.0%), and limonene (5.9%) to PAMA108 sample, while in the oil of M. suaveolens, PAMA131 sample, predominated bicyclogermacrene (23.5%, 1,8-cineole (23.0%), germacrene D (17.2%), and (E)-caryophyllene (10.4%). The sample oil of H. dilatata (PAMA108) exhibited the lower anthelmintic inhibitory concentration, with an IC50 value of 2.09 mg/mL for C. elegans Bristol N2 strain, while the oils of H. dilatata (PAMA19) and M. suaveolens (PAMA131) showed an IC50 up of 4 mg/mL for C. elegans IVR15 and Bristol N2 strains. These results suggest that the H. dilatata and M. suaveolens oils constituents' combination can be helpful as a nematicidal agent due to their synergistic action.

Mechanisms of Antiviral Cytotoxic CD4 T Cell Differentiation.

Cytotoxic CD4 T lymphocytes (CD4-CTL) are important in antiviral immunity. For example, we have previously shown that in mice, CD4-CTL are important to control ectromelia virus (ECTV) infection. How viral infections induce CD4-CTL responses remains incompletely understood. We demonstrate here that not only ECTV but also vaccinia virus and lymphocytic choriomeningitis virus induce CD4-CTL, though the response to ECTV is stronger. Using ECTV, we also demonstrate that in contrast to CD8-CTL, CD4-CTL differentiation requires constant virus replication and ceases once the virus is controlled. We also show that major histocompatibility complex class II molecules on CD11c+ cells are required for CD4-CTL differentiation and for mousepox resistance. Transcriptional analysis indicated that antiviral CD4-CTL and noncytolytic T helper 1 (Th1) CD4 T cells have similar transcriptional profiles, suggesting that CD4-CTL are terminally differentiated classical Th1 cells. Interestingly, CD4-CTL and classical Th1 cells expressed similar mRNA levels of the transcription factors ThPOK and GATA-3, necessary for CD4 T cell linage commitment, and Runx3, required for CD8 T cell development and effector function. However, at the protein level, CD4-CTL had higher levels of the three transcription factors, suggesting that further posttranscriptional regulation is required for CD4-CTL differentiation. Finally, CRISPR/Cas9-mediated deletion of Runx3 in CD4 T cells inhibited CD4-CTL but not classical Th1 cell differentiation in response to ECTV infection. These results further our understanding of the mechanisms of CD4-CTL differentiation during viral infection and the role of posttranscriptionally regulated Runx3 in this process. IMPORTANCE While it is well established that cytotoxic CD4 T cells (CD4-CTLs) directly contribute to viral clearance, it remains unclear how CD4-CTL are induced. We now show that CD4-CTLs require sustained antigen presentation and are induced by CD11c-expressing antigen-presenting cells. Moreover, we show that CD4-CTLs are derived from the terminal differentiation of classical T helper 1 (Th1) subset of CD4 cells. Compared to Th1 cells, CD4-CTLs upregulate protein levels of the transcription factors ThPOK, Runx3, and GATA-3 posttranscriptionally. Deletion of Runx3 in differentiated CD4 T cells prevents induction of CD4-CTLs but not classical Th1 cells. These results advance our knowledge of how CD4-CTLs are induced during viral infection.

IFN-ß Acts on Monocytes to Ameliorate CNS Autoimmunity by Inhibiting Proinflammatory Cross-Talk Between Monocytes and Th Cells.

IFN-ß has been the treatment for multiple sclerosis (MS) for almost three decades, but understanding the mechanisms underlying its beneficial effects remains incomplete. We have shown that MS patients have increased numbers of GM-CSF+ Th cells in circulation, and that IFN-ß therapy reduces their numbers. GM-CSF expression by myelin-specific Th cells is essential for the development of experimental autoimmune encephalomyelitis (EAE), an animal model of MS. These findings suggested that IFN-ß therapy may function via suppression of GM-CSF production by Th cells. In the current study, we elucidated a feedback loop between monocytes and Th cells that amplifies autoimmune neuroinflammation, and found that IFN-ß therapy ameliorates central nervous system (CNS) autoimmunity by inhibiting this proinflammatory loop. IFN-ß suppressed GM-CSF production in Th cells indirectly by acting on monocytes, and IFN-ß signaling in monocytes was required for EAE suppression. IFN-ß increased IL-10 expression by monocytes, and IL-10 was required for the suppressive effects of IFN-ß. IFN-ß treatment suppressed IL-1ß expression by monocytes in the CNS of mice with EAE. GM-CSF from Th cells induced IL-1ß production by monocytes, and, in a positive feedback loop, IL-1ß augmented GM-CSF production by Th cells. In addition to GM-CSF, TNF and FASL expression by Th cells was also necessary for IL-1ß production by monocyte. IFN-ß inhibited GM-CSF, TNF, and FASL expression by Th cells to suppress IL-1ß secretion by monocytes. Overall, our study describes a positive feedback loop involving several Th cell- and monocyte-derived molecules, and IFN-ß actions on monocytes disrupting this proinflammatory loop.

Resistance to lethal ectromelia virus infection requires Type I interferon receptor in natural killer cells and monocytes but not in adaptive immune or parenchymal cells.

Type I interferons (IFN-I) are antiviral cytokines that signal through the ubiquitous IFN-I receptor (IFNAR). Following footpad infection with ectromelia virus (ECTV), a mouse-specific pathogen, C57BL/6 (B6) mice survive without disease, while B6 mice broadly deficient in IFNAR succumb rapidly. We now show that for survival to ECTV, only hematopoietic cells require IFNAR expression. Survival to ECTV specifically requires IFNAR in both natural killer (NK) cells and monocytes. However, intrinsic IFNAR signaling is not essential for adaptive immune cell responses or to directly protect non-hematopoietic cells such as hepatocytes, which are principal ECTV targets. Mechanistically, IFNAR-deficient NK cells have reduced cytolytic function, while lack of IFNAR in monocytes dampens IFN-I production and hastens virus dissemination. Thus, during a pathogenic viral infection, IFN-I coordinates innate immunity by stimulating monocytes in a positive feedback loop and by inducing NK cell cytolytic function.

Combination of synthetic anthelmintics and monoterpenes: Assessment of efficacy, and ultrastructural and biophysical properties of Haemonchus contortus using atomic force microscopy.

The resistance of Haemonchus contortus to synthetic anthelmintics is of increasing concern; and different strategies are being evaluated to improve parasite control. The present study investigated the in vitro effects of combinations of synthetic compounds and monoterpenes. Additionally, the chemical association of the best combinations and their impact on the ultrastructural and biophysical properties of H. contortus eggs was evaluated. We assessed the efficacy of the monoterpenes, carvacrol, thymol, r-carvone, s-carvone, citral, and p-cymene and the anthelmintics, albendazole and levamisole using the egg hatch test (EHT) and the larval migration inhibition test (LMIT), respectively. The minimum effective concentrations of the monoterpenes, according to the EHT (efficacy ranging from 4.4%-11.8%) and LMIT (efficacy ranging from 5.6%-7.4%), were used in combination with different concentrations of synthetic compounds, and the IC50 and synergism rate (SR) were calculated. Fourier-transform infrared spectroscopy (FTIR) was used to analyze the chemical association between the best combinations as revealed by the in vitro tests (albendazole and levamisole with r-carvone or s-carvone). Atomic force microscopy (AFM) was used to assess the ultrastructural and biophysical properties of H. contortus eggs treated with the albendazole and r-carvone combination. Among the monoterpenes, the highest efficacies were exhibited by carvacrol (IC50 = 185.9 µg/mL) and thymol (IC50 = 187.0 µg/mL), according to the EHT, and s-carvone and carvacrol (IC50 = 1526.0 and 1785.3 µg/mL, respectively), according to the LMIT. According to the EHT, albendazole showed a slight statistically significant synergism in combination with r-carvone (SR = 3.8) and s-carvone (SR = 3.0). According to the LMIT, among the monoterpenes, r-carvone (SR = 1.7) and s-carvone (SR = 1.7) showed an increase in efficacy with levamisole; however, this was not statistically significant. The FTIR spectra of albendazole and levamisole, in association with r-carvone and s-carvone, indicated the presence of chemical interactions between the synthetic and natural molecules, contributing to the possible synergistic effects of these associations. Eggs treated with albendazole and r-carvone showed an increase in roughness and a decrease in height, suggesting that the treatment induced damage to the egg surface and an overflow of its internal contents. Overall, the combination of albendazole with r-carvone and s-carvone was efficacious against H. contortus, demonstrating a chemical association between the compounds; the significant changes in the egg ultrastructure justify this efficacy.

Nemabiome metabarcoding reveals differences between gastrointestinal nematode species infecting co-grazed sheep and goats.

Our current understanding of differences in the epidemiology of gastrointestinal nematode (GIN) species in co-grazed sheep and goats is inadequate with reference to the development of sustainable control strategies. The next-generation metabarcoding sequencing method referred to as the 'nemabiome' allows some of these differences to be explored to describe the intensity of co-infecting GIN species. We applied this platform to study sheep and goats that were co-grazed on Guinea grass pasture in northeastern Brazil. Co-grazed goats and sheep were treated with a monepantel anthelmintic, then exposed to the same gastrointestinal nematode species. Overall, there were differences in the prevalence of GIN species identified in the sheep and goats; Trichostrongylus colubriformis and Teladorsagia circumcincta predominated in goat kids, while Haemonchus contortus predominated in adult does, ewes and lambs once burdens became re-established after anthelmintic treatment. Description of the pattern of re-infection following anthelmintic treatment was prevented by the unpredicted poor efficacy of 2.5 mg/kg and 5 mg/kg, respectively, of monepantel against O. columbianum and T. circumcincta in lambs, and T. circumcincta adult does. Differences in drug efficacy between host age and species groups may be important when considering sustainable GIN control strategies for co-grazed animals. The aggregated FECs of the adult does and goat kids representing re-established GIN burdens, were higher than those of the co-grazed adult ewes and lambs. This implies that there are inherent differences in GIN species adaptation to the two naïve small ruminant host species, and shows the need for better understanding of the factors giving rise to this situation associated with exposure to infective larvae and host responses. At the start of the study, the adult does were co-infected with several GIN species, with the highest intensity of T. circumcincta, contrasting with the situation in the adult ewes, in which H. contortus predominated. However, once burdens became re-established after treatment, H. contortus predominated in both adult does and ewes. This demonstrates the potential for host burdens of H. contortus to establish and predominate after anthelmintic treatment when burdens of co-infecting GIN species are low.

Recombinogenic, genotoxic, and cytotoxic effects of azathioprine using in vivo assays.

Azathioprine (Aza) is a purine antimetabolite immunosuppressant that is widely employed for immunosuppressive therapy in post-transplant recipients or patients with autoimmune diseases. Chronic use of immunosuppressants might produce several side effects, including a high rate of neoplasms in these patients. Considering that genotoxic effects are associated with an increased risk of developing cancer, the aim of this study was to examine the recombinogenic, genotoxic, and cytotoxic effects of Aza using Somatic Mutation and Recombination Test (SMART) in Drosophila melanogaster, as well as comet and micronucleus assays in mouse bone marrow cells. Further, the adverse effects of Aza were determined in mouse hepatic and renal tissues using histopathological analysis. Data demonstrated that Aza induced significant increased genotoxicity in D. melanogaster and mouse bone marrow cells at all concentrations tested. Homologous recombination was the predominant genotoxic event noted for the first time to be initiated by Aza in SMART. In histopathological analysis, Aza did not show any marked toxic activity in mouse hepatic and renal tissues. Therefore, the high rate of neoplasms reported in patients with long-term use of Aza may be attributed, at least partially, to the genotoxic action of this drug.

Reflexões multidisciplinares da Ciência Aberta em vivências de Discente e Docentes

O movimento em prol da Ciência Aberta (CA) abarca diferentes escolas de pensamento e diferentes iniciativas, defendendo não somente o acesso aberto às publicações e aos dados, mas também uma maior transparência no processo de pesquisa. Entretanto percebe-se uma resistência do pesquisador à adesão às práticas de CA. Este artigo sintetiza argumentos pró e contra ao movimento da CA percebidos a partir de reflexões desenvolvidas no âmbito de disciplina eletiva de curso de pós-graduação stricto sensu interdisciplinar. Os resultados da discussão realizada por discentes e docentes são apresentadas em oito categorias: reprodutibilidade, acesso aberto, dados abertos, transparência, assimetria entre setor público e privado, integridade, avaliação por pares aberta e preprints, sistema de avaliação da produção científica e políticas de Ciência Aberta. Em síntese, por um lado o uso das práticas de CA parece ter potência para revelar dilemas sobre integridade e promover a abertura de dados de pesquisa, o que pode "assombrar" pesquisadores. Por outro lado, a possibilidade de mitigar riscos à qualidade das evidências científicas, fortalecer a reprodutibilidade e a credibilidade na ciência favorece a disseminação destas práticas e de seu debate nos ambientes formativos do pesquisador contribuindo para o distensionamento ao uso de práticas de CA.

Defective early innate immune response to ectromelia virus in the draining lymph nodes of aged mice due to impaired dendritic cell accumulation.

It is known that aging decreases natural resistance to viral diseases due to dysfunctional innate and adaptive immune responses, but the nature of these dysfunctions, particularly in regard to innate immunity, is not well understood. We have previously shown that C57BL/6J (B6) mice lose their natural resistance to footpad infection with ectromelia virus (ECTV) due to impaired maturation and recruitment of natural killer (NK) cells to the draining popliteal lymph node (dLN). More recently, we have also shown that in young B6 mice infected with ECTV, the recruitment of NK cells is dependent on a complex cascade whereby migratory dendritic cells (mDCs) traffic from the skin to the dLN, where they produce CCL2 and CCL7 to recruit inflammatory monocytes (iMOs). In the dLN, mDCs also upregulate NKG2D ligands to induce interferon gamma (IFN-γ) expression by group 1 innate lymphoid cells (G1-ILCs), mostly NK in cells but also some ILC1. In response to the IFN-γ, the incoming uninfected iMOs secret CXCL9 to recruit the critical NK cells. Here, we show that in aged B6 mice, the trafficking of mDCs to the dLN in response to ECTV is decreased, resulting in impaired IFN-γ expression by G1-ILCs, reduced accumulation of iMOs, and attenuated CXCL9 production by iMOs, which likely contributes to decrease in NK cell recruitment. Together, these data indicate that defects in the mDC response to viral infection during aging result in a reduced innate immune response in the dLN and contribute to increased susceptibility to viral disease in the aged.

Inhibition kinetics of digestive proteases for Anticarsia gemmatalis.

Anticarsia gemmatalis Hübner, 1818 (Lepidoptera) is a major pest of soybean in the Brazil. It is known that the reduction of proteolytic activity by the ingestion of protease inhibitors reduces digestion and larval development of the insects. Control via inhibition of the digestive enzymes necessitates deeper knowledge of the enzyme kinetics and the characterization of the inhibition kinetics of these proteases, for better understanding of the active centers and action mechanisms of this enzyme. Trypsin-like proteases found in the gut of Anticarsia gemmatalis were purified in a p-aminobenzamidine agarose column. Kinetic characterization showed KM 0.503 mM for the L-BApNA substrate; Vmax= 46.650 nM s-1; Vmax/[E]= 9.256 nM s-1 mg L-1 and Vmax/[E]/KM= 18.402 nM s-1 mg L-1 mM. The Ki values for the inhibitors benzamidine, berenil, SKTI and SBBI were 11.2 µM, 32.4 µM, 0.25 nM and 1.4 nM, respectively, and all revealed linear competitive inhibition. The SKTI showed the greatest inhibition, which makes it a promising subject for future research to manufacture peptide mimetic inhibitors.

Larval development and proteolytic activity of Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae) exposed to different soybean protease inhibitors.

Anticarsia gemmatalis represents a relevant factor for lowering soybean and other legume crop productivities. Protease inhibitors affect protein degradation and reduce the availability of amino acids, impairing the development and survival of insect pests. To evaluate the possible use of proteinaceous protease inhibitors in the management of this pest, the activities of midgut proteases and the growth and development of A. gemmatalis larvae exposed to soybean Bowman-Birk trypsin-chymotrypsin inhibitor (SBBI) and soybean Kunitz trypsin inhibitor (SKTI) were determined. The survival curves obtained using Kaplan-Meier estimators indicated that SKTI and SBBI stimulated larval survival. However, the development of A. gemmatalis was delayed, and prepupal weight decreased in the presence of both inhibitors. The results showed that SKTI and SBBI inhibited the trypsin-like and total proteolytic activities of larvae on the 12th day after eclosion. On the 15th day after eclosion, larvae exposed to SKTI increased the activities of trypsin and total proteases. Although SKTI and SBBI did not affect the survival of the insect, they had effects on midgut proteases in a stage wherein A. gemmatalis fed voraciously, increased the larval cycle, and decreased prepupal weight. These findings provide baseline information about the potential of proteinaceous protease inhibitors to manage the velvetbean caterpillar, avoiding chemical pesticides.

Chronic Lymphocytic Choriomeningitis Infection Causes Susceptibility to Mousepox and Impairs Natural Killer Cell Maturation and Function.

Chronic viral infections. like those of humans with cytomegalovirus, human immunodeficiency virus (even when under antiretroviral therapy), and hepatitis C virus or those of mice with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13), result in immune dysfunction that predisposes the host to severe infections with unrelated pathogens. It is known that C57BL/6 (B6) mice are resistant to mousepox, a lethal disease caused by the orthopoxvirus ectromelia virus (ECTV), and that this resistance requires natural killer (NK) cells and other immune cells. We show that most B6 mice chronically infected with CL13 succumb to mousepox but that most of those that recovered from acute infection with the LCMV Armstrong (Arm) strain survive. We also show that B6 mice chronically infected with CL13 and those that recovered from Arm infection have a reduced frequency and a reduced number of NK cells. However, at steady state, NK cells in mice that have recovered from Arm infection mature normally and, in response to ECTV, get activated, become more mature, proliferate, and increase their cytotoxicity in vivo Conversely, in mice chronically infected with CL13, NK cells are immature and residually activated, and following ECTV infection, they do not mature, proliferate, or increase their cytotoxicity. Given the well-established importance of NK cells in resistance to mousepox, these data suggest that the NK cell dysfunction caused by CL13 persistence may contribute to the susceptibility of CL13-infected mice to mousepox. Whether chronic infections similarly affect NK cells in humans should be explored.IMPORTANCE Infection of adult mice with the clone 13 (CL13) strain of lymphocytic choriomeningitis virus (LCMV) is extensively used as a model of chronic infection. In this paper, we show that mice chronically infected with CL13 succumb to challenge with ectromelia virus (ECTV; the agent of mousepox) and that natural killer (NK) cells in CL13-infected mice are reduced in numbers and have an immature and partially activated phenotype but do respond to ECTV. These data may provide additional clues why humans chronically infected with certain pathogens are less resistant to viral diseases.

Loss of Resistance to Mousepox during Chronic Lymphocytic Choriomeningitis Virus Infection Is Associated with Impaired T-Cell Responses and Can Be Rescued by Immunization.

It is well established that chronic viral infections can cause immune suppression, resulting in increased susceptibility to other infectious diseases. However, the effects of chronic viral infection on T-cell responses and vaccination against highly pathogenic viruses are not well understood. We have recently shown that C57BL/6 (B6) mice lose their natural resistance to wild-type (WT) ectromelia virus (ECTV) when chronically infected with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13). Here we compared the T-cell response to ECTV in previously immunologically naive mice that were chronically infected with CL13 or that were convalescent from acute infection with the Armstrong (Arm) strain of LCMV. Our results show that mice that were chronically infected with CL13 but not those that had recovered from Arm infection have highly defective ECTV-specific CD8+ and CD4+ T-cell responses to WT ECTV. These defects are at least partly due to the chronic infection environment. In contrast to mice infected with WT ECTV, mice chronically infected with CL13 survived without signs of disease when infected with ECTV-Δ036, a mutant ECTV strain that is highly attenuated. Strikingly, mice chronically infected with CL13 mounted a strong CD8+ T-cell response to ECTV-Δ036 and survived without signs of disease after a subsequent challenge with WT ECTV. Our work suggests that enhanced susceptibility to acute viral infections in chronically infected individuals can be partly due to poor T-cell responses but that sufficient T-cell function can be recovered and resistance to acute infection can be restored by immunization with highly attenuated vaccines.IMPORTANCE Chronic viral infections may result in immunosuppression and enhanced susceptibility to infections with other pathogens. For example, we have recently shown that mice chronically infected with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13) are highly susceptible to mousepox, a disease that is caused by ectromelia virus and that is the mouse homolog of human smallpox. Here we show chronic CL13 infection severely disrupts the expansion, proliferation, activation, and cytotoxicity of T cells in response due at least in part to the suppressive effects of the chronic infection milieu. Notably, despite this profound immunodeficiency, mice chronically infected with CL13 could be protected by vaccination with a highly attenuated variant of ECTV. These results demonstrate that protective vaccination of immunosuppressed individuals is possible, provided that proper immunization tools are used.