Epidemiology of Salmonella enterica subspecies enterica serotypes, isolated from imported, farmed and feral poultry in the Cayman Islands.

Non-typhoidal Salmonellae (NTS) are common foodborne pathogens throughout the world causing acute gastroenteritis. Compared to North America and Europe, there is little information on NTS in the Caribbean. Here we investigated the prevalence and characteristics of NTS present in the local poultry of the Cayman Islands to determine the public health risk. In total, we collected 156 samples. These were made up of boot swabs of 31 broiler farms and 31 layer farms (62 samples), paper bedding from 45 imported chick boxes, and 49 pooled cecum samples from feral chickens, each sample representing 10 individual chickens. Salmonella was isolated using the ISO 6579 protocol and isolates were characterized using Whole Genome Sequencing (WGS) analysis. Eighteen Salmonella isolates were obtained and comprised six S. enterica subspecies enterica serotypes and one subspecies houtenae serotype. Serotypes were: S. Kentucky (n = 9), S. Saintpaul (n = 5), S. Javiana (n = 1), S. Senftenberg (n = 1), S. Poona (n = 1) and S. Agona (n = 1). S. Kentucky strains were all ST152 and clonally related to poultry strains from the United states. S. Saintpaul ST50 strains showed clonality to North American strains. Over half of the strains (n = 11) contained resistance genes to at least two antibiotic groups and five strains were MDR, mainly those from imported day-old chicks. The blaCMY-2 gene was found in S. Kentucky from day-old chicks. Strains from feral poultry had no acquired AMR genes. While serotypes from feral poultry have been identified in human infections, they pose minimal risk due to their low virulence.

Determination of minimum individual cow colostral immunoglobulin G concentration required to provide adequate total immunoglobulin G mass in pooled colostrum fed to Jersey calves.

OBJECTIVE: Quantify the minimum individual cow colostral immunoglobulin G (IgG) concentration required for pooling to achieve adequate transfer of passive immunity in calves. ANIMALS: 201 Jersey cows. METHODS: Colostrum was collected from 28 pools and heat treated before being fed to calves or stored. Parity, total number of cows contributing to the pool, individual cow colostral volume contributions, and total volume of each colostrum pool were recorded. Colostrum IgG concentrations in individual and pooled (pre- and post-heat treatment) samples were analyzed by radial immunodiffusion and Brix refractometry. Colostral IgG concentration of ≥ 50g/L was considered the current recommended dairy industry standard for acceptable colostrum quality. Multivariable models were performed to determine factors affecting pooled colostral IgG concentrations. The minimum colostral IgG concentration required for pooling to achieve the recommended total mass of at least 200g IgG to be fed to a calf was calculated. RESULTS: Total pool volume and the number of cows contributing to the pool were significant factors affecting IgG concentration. Colostrum pools from ≤ 7 cows, with a minimum pool IgG concentration of 70.4 g/L (22.9% Brix) or colostrum pool volume ≤ 40 L, with a minimum pool IgG concentration of 66.2 g/L (21.8% Brix) achieved the recommended total mass of at least 200g IgG in 4L of colostrum. CLINICAL RELEVANCE: When feeding pooled colostrum, IgG concentrations higher than the industry standard of 50 g/L is recommended to reduce the risk of failure of transfer of passive immunity in calves.

Orthopoxvirus Zoonoses-Do We Still Remember and Are Ready to Fight?

The eradication of smallpox was an enormous achievement due to the global vaccination program launched by World Health Organization. The cessation of the vaccination program led to steadily declining herd immunity against smallpox, causing a health emergency of global concern. The smallpox vaccines induced strong, humoral, and cell-mediated immune responses, protecting for decades after immunization, not only against smallpox but also against other zoonotic orthopoxviruses that now represent a significant threat to public health. Here we review the major aspects regarding orthopoxviruses' zoonotic infections, factors responsible for viral transmissions, as well as the emerging problem of the increased number of monkeypox cases recently reported. The development of prophylactic measures against poxvirus infections, especially the current threat caused by the monkeypox virus, requires a profound understanding of poxvirus immunobiology. The utilization of animal and cell line models has provided good insight into host antiviral defenses as well as orthopoxvirus evasion mechanisms. To survive within a host, orthopoxviruses encode a large number of proteins that subvert inflammatory and immune pathways. The circumvention of viral evasion strategies and the enhancement of major host defenses are key in designing novel, safer vaccines, and should become the targets of antiviral therapies in treating poxvirus infections.

Ectromelia Virus Affects the Formation and Spatial Organization of Adhesive Structures in Murine Dendritic Cells In Vitro.

Ectromelia virus (ECTV) is a causative agent of mousepox. It provides a suitable model for studying the immunobiology of orthopoxviruses, including their interaction with the host cell cytoskeleton. As professional antigen-presenting cells, dendritic cells (DCs) control the pericellular environment, capture antigens, and present them to T lymphocytes after migration to secondary lymphoid organs. Migration of immature DCs is possible due to the presence of specialized adhesion structures, such as podosomes or focal adhesions (FAs). Since assembly and disassembly of adhesive structures are highly associated with DCs' immunoregulatory and migratory functions, we evaluated how ECTV infection targets podosomes and FAs' organization and formation in natural-host bone marrow-derived DCs (BMDC). We found that ECTV induces a rapid dissolution of podosomes at the early stages of infection, accompanied by the development of larger and wider FAs than in uninfected control cells. At later stages of infection, FAs were predominantly observed in long cellular extensions, formed extensively by infected cells. Dissolution of podosomes in ECTV-infected BMDCs was not associated with maturation and increased 2D cell migration in a wound healing assay; however, accelerated transwell migration of ECTV-infected cells towards supernatants derived from LPS-conditioned BMDCs was observed. We suggest that ECTV-induced changes in the spatial organization of adhesive structures in DCs may alter the adhesiveness/migration of DCs during some conditions, e.g., inflammation.

Differential Activation of Splenic cDC1 and cDC2 Cell Subsets following Poxvirus Infection of BALB/c and C57BL/6 Mice.

Conventional dendritic cells (cDCs) are innate immune cells that play a pivotal role in inducing antiviral adaptive immune responses due to their extraordinary ability to prime and polarize naïve T cells into different effector T helper (Th) subsets. The two major subpopulations of cDCs, cDC1 (CD8α+ in mice and CD141+ in human) and cDC2 (CD11b+ in mice and CD1c+ in human), can preferentially polarize T cells toward a Th1 and Th2 phenotype, respectively. During infection with ectromelia virus (ECTV), an orthopoxvirus from the Poxviridae family, the timing and activation of an appropriate Th immune response contributes to the resistance (Th1) or susceptibility (Th2) of inbred mouse strains to the lethal form of mousepox. Due to the high plasticity and diverse properties of cDC subpopulations in regulating the quality of a specific immune response, in the present study we compared the ability of splenic cDC1 and cDC2 originating from different ECTV-infected mouse strains to mature, activate, and polarize the Th immune response during mousepox. Our results demonstrated that during early stages of mousepox, both cDC subsets from resistant C57BL/6 and susceptible BALB/c mice were activated upon in vivo ECTV infection. These cells exhibited elevated levels of surface MHC class I and II, and co-stimulatory molecules and showed enhanced potential to produce cytokines. However, both cDC subsets from BALB/c mice displayed a higher maturation status than that of their counterparts from C57BL/6 mice. Despite their higher activation status, cDC1 and cDC2 from susceptible mice produced low amounts of Th1-polarizing cytokines, including IL-12 and IFN-γ, and the ability of these cells to stimulate the proliferation and Th1 polarization of allogeneic CD4+ T cells was severely compromised. In contrast, both cDC subsets from resistant mice produced significant amounts of Th1-polarizing cytokines and demonstrated greater capability in differentiating allogeneic T cells into Th1 cells compared to cDCs from BALB/c mice. Collectively, our results indicate that in the early stages of mousepox, splenic cDC subpopulations from the resistant mouse strain can better elicit a Th1 cell-mediated response than the susceptible strain can, probably contributing to the induction of the protective immune responses necessary for the control of virus dissemination and for survival from ECTV challenge.

Sex-differential non-specific effects of adjuvanted and non-adjuvanted rabies vaccines versus placebo on all-cause mortality in dogs (NERVE-Dog study): a study protocol for a randomized controlled trial with a nested case-control study.

BACKGROUND: It has been proposed that childhood vaccines in high-mortality populations may have substantial impacts on mortality rates that are not explained by the prevention of targeted diseases, nor conversely by typical expected adverse reactions to the vaccines, and that these non-specific effects (NSEs) are generally more pronounced in females. The existence of these effects, and any implications for the development of vaccines and the design of vaccination programs to enhance safety, remain controversial. One area of controversy is the reported association of non-live vaccines with increased female mortality. In a previous randomized controlled trial (RCT), we observed that non-live alum-adjuvanted animal rabies vaccine (ARV) was associated with increased female but not male mortality in young, free-roaming dogs. Conversely, non-live non-adjuvanted human rabies vaccine (NRV) has been associated with beneficial non-specific effects in children. Alum adjuvant has been shown to suppress Th1 responses to pathogens, leading us to hypothesize that alum-adjuvanted rabies vaccine in young dogs has a detrimental effect on female survival by modulating the immune response to infectious and/or parasitic diseases. In this paper, we present the protocol of a 3-arm RCT comparing the effect of alum-adjuvanted rabies vaccine, non-adjuvanted rabies vaccine and placebo on all-cause mortality in an owned, free-roaming dog population, with causal mediation analysis of the RCT and a nested case-control study to test this hypothesis. METHODS: Randomised controlled trial with a nested case-control study. DISCUSSION: We expect that, among the placebo group, males will have higher mortality caused by higher pathogen loads and more severe disease, as determined by haematological parameters and inflammatory biomarkers. Among females, we expect that there will be no difference in mortality between the NRV and placebo groups, but that the ARV group will have higher mortality, again mediated by higher pathogen loads and more severe disease. We anticipate that these changes are preceded by shifts in key serum cytokine concentrations towards an anti-inflammatory immune response in females. If confirmed, these results will provide a rational basis for mitigation of detrimental NSEs of non-live vaccines in high-mortality populations.

Non-specific effects of veterinary vaccines: a systematic review.

The benefits of vaccines have been centred on their specific effects on subsequent infections by target pathogens. Recent studies, however, have opened up new insights into additional effects of vaccines known as non-specific effects (NSEs) or heterologous effects of vaccines. While several articles have reviewed epidemiological and immunological evidence for NSEs of vaccines in humans, similar works on veterinary vaccines are scarce. The objective of this paper was to review the findings of published studies on NSEs of vaccines developed or repurposed for use in animals. In total 8412 titles were retrieved from PubMed and CABI databases on the 30th of April 2021. After the final stage of screening, 45 eligible articles were included in the review. Data from these articles were summarised and presented here. In general, most of the vaccines studied in the reviewed articles have beneficial NSEs against multiple pathogens and disease conditions. There were, however, fewe studies reporting detrimental NSEs from both non-live and live vaccines which is in contrast to the currently existing evidence of beneficial NSEs of live vaccines and detrimental NSEs of non-live vaccines. This review may be used as a complement for future review of RCT studies of NSEs of vaccines in animals and provide a useful addition to the evolving understanding of the NSEs of vaccines.

Nonlinear Mixed-Effect Pharmacokinetic Modeling and Distribution of Doxycycline in Healthy Female Donkeys after Multiple Intragastric Dosing-Preliminary Investigation.

Doxycycline (DXC) is a broad-spectrum antibacterial antimicrobial administered to horses for the treatment of bacterial infections which may also affect donkeys. Donkeys have a different metabolism than horses, leading to differences in the pharmacokinetics of drugs compared to horses. This study aimed to describe the population pharmacokinetics of DXC in donkeys. Five doses of DXC hyclate (10 mg/kg) were administered via a nasogastric tube, q12 h, to eight non-fasted, healthy, adult jennies. Serum, urine, synovial fluid and endometrium were collected for 72 h following the first administration. Doxycycline concentration was measured by competitive enzyme immunoassay. Serum concentrations versus time data were fitted simultaneously using the stochastic approximation expectation-maximization algorithm for nonlinear mixed effects. A one-compartment model with linear elimination and first-order absorption after intragastric administration, best described the available pharmacokinetic data. Final parameter estimates indicate that DXC has a high volume of distribution (108 L/kg) as well as high absorption (10.3 h-1) in donkeys. However, results suggest that oral DXC at 10 mg/kg q12 h in donkeys would not result in a therapeutic concentration in serum, urine, synovial fluid or endometrium by comparison to the minimum inhibitory concentration of common equine pathogens. Further studies are recommended to identify appropriate dosage and dosing intervals of oral DXC in donkeys.

Identification and Antimicrobial Resistance of Dermatophilus congolensis from Cattle in Saint Kitts and Nevis.

Dermatophilosis is a form of dermatitis caused by the bacterium Dermatophilus congolensis. The disease usually presents as localized purulent dermatitis, crusty hair masses or widespread matting of the hair. This condition is most common in domestic ruminants; but it can also affect other wild animals and humans. Antimicrobial therapy is used in many regions to treat clinical dermatophilosis with varying results. In this study, we aimed to assess the antimicrobial susceptibility of D. congolensis isolates. Fifty-two isolates were obtained from animals showing clinical signs of the disease at farms in St. Kitts. The isolates were then confirmed as D. congolensis by phenotypic tests, PCR and MALDI-TOF Mass Spectrometry. Furthermore, minimum inhibitory concentrations (MIC) of 16 antimicrobial agents were determined, using the broth microdilution method. Although most antimicrobials showed MICs in line with published values, the tetracycline results displayed a clear bimodal distribution over the tested range, with most isolates showing low MICs and 6 isolates much higher values (+/- 100-fold increase). These results indicate the presence of acquired tetracycline resistance in D. congolensis on the island of St. Kitts. Whether the current observation has implications for efficacy of treating the disease must be confirmed in further research.

Comprehensive Molecular Dissection of Dermatophilus congolensis Genome and First Observation of tet(Z) Tetracycline Resistance.

Dermatophilus congolensis is a bacterial pathogen mostly of ruminant livestock in the tropics/subtropics and certain temperate climate areas. It causes dermatophilosis, a skin disease that threatens food security by lowering animal productivity and compromising animal health and welfare. Since it is a prevalent infection in ruminants, dermatophilosis warrants more research. There is limited understanding of its pathogenicity, and as such, there is no registered vaccine against D. congolensis. To better understanding the genomics of D. congolensis, the primary aim of this work was to investigate this bacterium using whole-genome sequencing and bioinformatic analysis. D. congolensis is a high GC member of the Actinobacteria and encodes approximately 2527 genes. It has an open pan-genome, contains many potential virulence factors, secondary metabolites and encodes at least 23 housekeeping genes associated with antimicrobial susceptibility mechanisms and some isolates have an acquired antimicrobial resistance gene. Our isolates contain a single CRISPR array Cas type IE with classical 8 Cas genes. Although the isolates originate from the same geographical location there is some genomic diversity among them. In conclusion, we present the first detailed genomic study on D. congolensis, including the first observation of tet(Z), a tetracycline resistance-conferring gene.

Draft Genome Sequences of 40 Dermatophilus congolensis Isolates from Bovine Dermatophilosis Cases in St. Kitts and Nevis.

Dermatophilus congolensis causes dermatophilosis in cattle, mainly in tropical climates. Despite the economic losses caused by this bacterium, its pathogenic factors are less well understood. We report draft genomes of D. congolensis strains isolated during a dermatophilosis outbreak in cattle in St. Kitts and Nevis. Some isolates contain tet(Z), which is responsible for resistance to tetracyclines.

Disrupting Neurons and Glial Cells Oneness in the Brain-The Possible Causal Role of Herpes Simplex Virus Type 1 (HSV-1) in Alzheimer's Disease.

Current data strongly suggest herpes simplex virus type 1 (HSV-1) infection in the brain as a contributing factor to Alzheimer's disease (AD). The consequences of HSV-1 brain infection are multilateral, not only are neurons and glial cells damaged, but modifications also occur in their environment, preventing the transmission of signals and fulfillment of homeostatic and immune functions, which can greatly contribute to the development of disease. In this review, we discuss the pathological alterations in the central nervous system (CNS) cells that occur, following HSV-1 infection. We describe the changes in neurons, astrocytes, microglia, and oligodendrocytes related to the production of inflammatory factors, transition of glial cells into a reactive state, oxidative damage, Aß secretion, tau hyperphosphorylation, apoptosis, and autophagy. Further, HSV-1 infection can affect processes observed during brain aging, and advanced age favors HSV-1 reactivation as well as the entry of the virus into the brain. The host activates pattern recognition receptors (PRRs) for an effective antiviral response during HSV-1 brain infection, which primarily engages type I interferons (IFNs). Future studies regarding the influence of innate immune deficits on AD development, as well as supporting the neuroprotective properties of glial cells, would reveal valuable information on how to harness cytotoxic inflammatory milieu to counter AD initiation and progression.

Cathepsins in Bacteria-Macrophage Interaction: Defenders or Victims of Circumstance?

Macrophages are the first encounters of invading bacteria and are responsible for engulfing and digesting pathogens through phagocytosis leading to initiation of the innate inflammatory response. Intracellular digestion occurs through a close relationship between phagocytic/endocytic and lysosomal pathways, in which proteolytic enzymes, such as cathepsins, are involved. The presence of cathepsins in the endo-lysosomal compartment permits direct interaction with and killing of bacteria, and may contribute to processing of bacterial antigens for presentation, an event necessary for the induction of antibacterial adaptive immune response. Therefore, it is not surprising that bacteria can control the expression and proteolytic activity of cathepsins, including their inhibitors - cystatins, to favor their own intracellular survival in macrophages. In this review, we summarize recent developments in defining the role of cathepsins in bacteria-macrophage interaction and describe important strategies engaged by bacteria to manipulate cathepsin expression and activity in macrophages. Particularly, we focus on specific bacterial species due to their clinical relevance to humans and animal health, i.e., Mycobacterium, Mycoplasma, Staphylococcus, Streptococcus, Salmonella, Shigella, Francisella, Chlamydia, Listeria, Brucella, Helicobacter, Neisseria, and other genera.

Participation of Endosomes in Toll-Like Receptor 3 Transportation Pathway in Murine Astrocytes.

TLR3 provides immediate type I IFN response following entry of stimulatory PAMPs into the CNS, as it is in HSV infection. The receptor plays a vital role in astrocytes, contributing to rapid infection sensing and suppression of viral replication, precluding the spread of virus beyond neurons. The route of TLR3 mobilization culminating in the receptor activation remains unexplained. In this research, we investigated the involvement of various types of endosomes in the regulation of the TLR3 mobility in C8-D1A murine astrocyte cell line. TLR3 was transported rapidly to early EEA1-positive endosomes as well as LAMP1-lysosomes following stimulation with the poly(I:C). Later, TLR3 largely associated with late Rab7-positive endosomes. Twenty-four hours after stimulation, TLR3 co-localized with LAMP1 abundantly in lysosomes of astrocytes. TLR3 interacted with poly(I:C) intracellularly from 1 min to 8 h following cell stimulation. We detected TLR3 on the surface of astrocytes indicating constitutive expression, which increased after poly(I:C) stimulation. Our findings contribute to the understanding of cellular modulation of TLR3 trafficking. Detailed analysis of the TLR3 transportation pathway is an important component in disclosing the fate of the receptor in HSV-infected CNS and may help in the search for rationale therapeutics to control the replication of neuropathic viruses.

Role of Cadherins in Cancer-A Review.

Cadherins play an important role in tissue homeostasis, as they are responsible for cell-cell adhesion during embryogenesis, tissue morphogenesis, differentiation and carcinogenesis. Cadherins are inseparably connected with catenins, forming cadherin-catenin complexes, which are crucial for cell-to-cell adherence. Any dysfunction or destabilization of cadherin-catenin complex may result in tumor progression. Epithelial mesenchymal transition (EMT) is a mechanism in which epithelial cadherin (E-cadherin) expression is lost during tumor progression. However, during tumorigenesis, many processes take place, and downregulation of E-cadherin, nuclear ß-catenin and p120 catenin (p120) signaling are among the most critical. Additional signaling pathways, such as Receptor tyrosine kinase (RTK), Rho GTPases, phosphoinositide 3-kinase (PI3K) and Hippo affect cadherin cell-cell adhesion and also contribute to tumor progression and metastasis. Many signaling pathways may be activated during tumorigenesis; thus, cadherin-targeting drugs seem to limit the progression of malignant tumor. This review discusses the role of cadherins in selected signaling mechanisms involved in tumor growth. The clinical importance of cadherin will be discussed in cases of human and animal cancers.

Potential use of a canine whole blood culture system to evaluate the immune response to Leptospira.

In susceptible hosts, protection from Leptospira infection is mediated by the innate immune response at the point of entry and humoral immunity. Thus, identifying and segregating the initial host response at the representative host-pathogen interface is needed to understand the typical outcomes of Leptospira infection, clearance, persistence, or disease. An in vitro whole blood culture system to study the overall immune response using pathogenic and non-pathogenic Leptospira strains was explored in this study. Using an ELISA, increased IL-8, TNF alpha, and IL-1 in blood samples stimulated with pathogenic and nonpathogenic Leptospira compared to unstimulated controls were detected. In RT2 Profiler PCR Array assays, consistent upregulation of 22 genes and downregulation of 25 genes were observed. Few of the notable upregulated genes included BPI, CCL3, CXCL2, IL-6, IL-8, TLR1, TLR2, TLR6, and TNF and downregulated genes included, LBP, LYZ, MPO, MYD88. IFNß was upregulated in samples treated with pathogenic Leptospira and IL-1ß was upregulated in samples treated with nonpathogenic Leptospira. Toll- like Receptor signaling and expression of pattern recognition receptors were two of the five prominent canonical pathways observed. Individual deconvolution of each of the specific and significant pathways observed in this study may improve the understanding of the pathogenesis of this important zoonotic agent. The use of this system in conjunction with whole transcriptome analysis in a larger population, may unveil the robust nature of host/Leptospira interaction.

Rabies Vaccination of 6-Week-Old Puppies Born to Immunized Mothers: A Randomized Controlled Trial in a High-Mortality Population of Owned, Free-Roaming Dogs.

To achieve global elimination of human rabies from dogs by 2030, evidence-based strategies for effective dog vaccination are needed. Current guidelines recommend inclusion of dogs younger than 3 months in mass rabies vaccination campaigns, although available vaccines are only recommended for use by manufacturers in older dogs, ostensibly due to concerns over interference of maternally-acquired immunity with immune response to the vaccine. Adverse effects of vaccination in this age group of dogs have also not been adequately assessed under field conditions. In a single-site, owner-blinded, randomized, placebo-controlled trial in puppies born to mothers vaccinated within the previous 18 months in a high-mortality population of owned, free-roaming dogs in South Africa, we assessed immunogenicity and effect on survival to all causes of mortality of a single dose of rabies vaccine administered at 6 weeks of age. We found that puppies did not have appreciable levels of maternally-derived antibodies at 6 weeks of age (geometric mean titer 0.065 IU/mL, 95% CI 0.061-0.069; n = 346), and that 88% (95% CI 80.7-93.3) of puppies vaccinated at 6 weeks had titers ≥0.5 IU/mL 21 days later (n = 117). Although the average effect of vaccination on survival was not statistically significant (hazard ratio [HR] 1.35, 95% CI 0.83-2.18), this effect was modified by sex (p = 0.02), with the HR in females 3.09 (95% CI 1.24-7.69) and the HR in males 0.79 (95% CI 0.41-1.53). We speculate that this effect is related to the observed survival advantage that females had over males in the unvaccinated group (HR 0.27; 95% CI 0.11-0.70), with vaccination eroding this advantage through as-yet-unknown mechanisms.

Cell Surface Expression of Endosomal Toll-Like Receptors-A Necessity or a Superfluous Duplication?

Timely and precise delivery of the endosomal Toll-like receptors (TLRs) to the ligand recognition site is a critical event in mounting an effective antimicrobial immune response, however, the same TLRs should maintain the delicate balance of avoiding recognition of self-nucleic acids. Such sensing is widely known to start from endosomal compartments, but recently enough evidence has accumulated supporting the idea that TLR-mediated signaling pathways originating in the cell membrane may be engaged in various cells due to differential expression and distribution of the endosomal TLRs. Therefore, the presence of endosomal TLRs on the cell surface could benefit the host responses in certain cell types and/or organs. Although not fully understood why, TLR3, TLR7, and TLR9 may occur both in the cell membrane and intracellularly, and it seems that activation of the immune response can be initiated concurrently from these two sites in the cell. Furthermore, various forms of endosomal TLRs may be transported to the cell membrane, indicating that this may be a normal process orchestrated by cysteine proteases-cathepsins. Among the endosomal TLRs, TLR3 belongs to the evolutionary distinct group and engages a different protein adapter in the signaling cascade. The differently glycosylated forms of TLR3 are transported by UNC93B1 to the cell membrane, unlike TLR7, TLR8, and TLR9. The aim of this review is to reconcile various views on the cell surface positioning of endosomal TLRs and add perspective to the implication of such receptor localization on their function, with special attention to TLR3. Cell membrane-localized TLR3, TLR7, and TLR9 may contribute to endosomal TLR-mediated inflammatory signaling pathways. Dissecting this signaling axis may serve to better understand mechanisms influencing endosomal TLR-mediated inflammation, thus determine whether it is a necessity for immune response or simply a circumstantial superfluous duplication, with other consequences on immune response.

Deficiency of Selected Cathepsins Does Not Affect the Inhibitory Action of ECTV on Immune Properties of Dendritic Cells.

Ectromelia virus (ECTV), an orthopoxvirus, undergoes productive replication in conventional dendritic cells (cDCs), resulting in the inhibition of their innate and adaptive immune functions. ECTV replication rate in cDCs is increased due to downregulation of the expression of cathepsins - cystein proteases that orchestrate several steps during DC maturation. Therefore, this study was aimed to determine if downregulation of cathepsins, such as B, L or S, disrupts cDC capacity to induce activating signals in T cells or whether infection of cDCs with ECTV further weakens their functions as antigen-presenting cells. Our results showed that cDCs treated with siRNA against cathepsin B, L and S synthesize similar amounts of pro-inflammatory cytokines and exhibit comparable ability to mature and stimulate alloreactive CD4+ T cells, as untreated wild type (WT) cells. Moreover, ECTV inhibitory effect on cDC innate and adaptive immune functions, observed especially after LPS treatment, was comparable in both cathepsin-silenced and WT cells. Taken together, the absence of cathepsins B, L and S has minimal, if any, impact on the inhibitory effect of ECTV on cDC immune functions. We assume that the virus-mediated inhibition of cathepsin expression in cDCs represents more a survival mechanism than an immune evasion strategy.

Mitochondrial Heat Shock Response Induced by Ectromelia Virus is Accompanied by Reduced Apoptotic Potential in Murine L929 Fibroblasts.

Poxviruses utilize multiple strategies to prevent activation of extrinsic and intrinsic apoptotic pathways for successful replication. Mitochondrial heat shock proteins (mtHsps), especially Hsp60 and its cofactor Hsp10, are engaged in apoptosis regulation; however, until now, the influence of poxviruses on mtHsps has never been studied. We used highly infectious Moscow strain of ectromelia virus (ECTV) to investigate the mitochondrial heat shock response and apoptotic potential in permissive L929 fibroblasts. Our results show that ECTV-infected cells exhibit mostly mitochondrial localization of Hsp60 and Hsp10, and show overexpression of both proteins during later stages of infection. ECTV infection has only moderate effect on the electron transport chain subunit expression. Moreover, increase of mtHsp amounts is accompanied by lack of apoptosis, and confirmed by reduced level of pro-apoptotic Bax protein and elevated levels of anti-apoptotic Bcl-2 and Bcl-xL proteins. Taken together, we show a positive relationship between increased levels of Hsp60 and Hsp10 and decreased apoptotic potential of L929 fibroblasts, and further hypothesize that Hsp60 and/or its cofactor play important roles in maintaining protein homeostasis in mitochondria for promotion of cell survival allowing efficient replication of ECTV.