Lactic acid bacteria that activate immune gene expression in Caenorhabditis elegans can antagonise Campylobacter jejuni infection in nematodes, chickens and mice.

BACKGROUND: Campylobacter jejuni is the major micro-bacillary pathogen responsible for human coloenteritis. Lactic acid bacteria (LAB) have been shown to protect against Campylobacter infection. However, LAB with a good ability to inhibit the growth of C. jejuni in vitro are less effective in animals and animal models, and have the disadvantages of high cost, a long cycle, cumbersome operation and insignificant immune response indicators. Caenorhabditis elegans is increasingly used to screen probiotics for their anti-pathogenic properties. However, no research on the use of C. elegans to screen for probiotic candidates antagonistic to C. jejuni has been conducted to date. RESULTS: This study established a lifespan model of C. elegans, enabling the preselection of LAB to counter C. jejuni infection. A potential protective mechanism of LAB was identified. Some distinct LAB species offered a high level of protection to C. elegans against C. jejuni. The LAB strains with a high protection rate reduced the load of C. jejuni in C. elegans. The transcription of antibacterial peptide genes, MAPK and Daf-16 signalling pathway-related genes was elevated using the LAB isolates with a high protection rate. The reliability of the lifespan model of C. elegans was verified using mice and chickens infected with C. jejuni. CONCLUSIONS: The results showed that different LAB had different abilities to protect C. elegans against C. jejuni. C. elegans provides a reliable model for researchers to screen for LAB that are antagonistic to C. jejuni on a large scale.

Dirty mice join the immunologist's toolkit.

The microbiota is a driving force that influences host physiological functions. In this review, we discuss some of the methods that have been used in the pursuit of relevant host-microbiota interactions that control immune fitness and disease susceptibility, with a focus on dirty mice which have been recently incorporated in the immunologist's toolkit.

Mite Burden and Immunophenotypic Response to Demodex musculi in Swiss Webster, BALB/c, C57BL/6, and NSG Mice.

Detection methods for Demodex musculi were historically unreliable, and testing was rarely performed because its prevalence in laboratory mice was underestimated. Although infestations are unapparent in most mouse strains, D. musculi burdens are higher and clinical signs detected in various immunodeficient strains. The parasite's influence on the immune system of immunocompetent mice is unknown. We characterized mite burden (immunocompetent and immunodeficient strains) and immunologic changes (immunocompetent strains only) in naïve Swiss Webster (SW; outbred), C57BL/6NCrl (B6; Th1 responder), BALB/cAnNCrl (BALB/c; Th2 responder) and NOD. Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG; immunodeficient) mice after exposure to Demodex-infested NSG mice. Infested and uninfested age-matched mice of each strain (n = 5) were euthanized 14, 28, 56, and 112 d after exposure. Mite burden was determined through PCR analysis and skin histopathology; B-cell and CD4+ and CD8+ T-cell counts and activation states (CD25 and CD69) were evaluated by using flow cytometry; CBC counts were performed; and serum IgE levels were measured by ELISA. Mite burden and PCR copy number correlated in NSG mice, which had the highest mite burden, but not in immunocompetent strains. Infested immunocompetent animals developed diffuse alopecia by day 112, and both BALB/c and C57BL/6 mice had significantly increased IgE levels. These findings aligned with the skewed Th1 or Th2 immunophenotype of each strain. BALB/c mice mounted the most effective host response, resulting in the lowest mite burden of all immunocompetent strains at 112 d after infestation without treatment. Clinically significant hematologic abnormalities were absent and immunophenotype was unaltered in immunocompetent animals. Topical treat- ment with imidacloprid-moxidectin (weekly for 8 wk) was effective at eradicating mites by early as 7 d after treatment. IgE levels decreased substantially in infested BALB/c mice after treatment. These findings demonstrate a need for D. musculi surveillance in mouse colonies, because the infestation may influence the use of infested mice in select studies.

New Insights into the Immune System Using Dirty Mice.

The mouse (Mus musculus) is the dominant organism used to investigate the mechanisms behind complex immunological responses because of their genetic similarity to humans and our ability to manipulate those genetics to understand downstream function. Indeed, our knowledge of immune system development, response to infection, and ways to therapeutically manipulate the immune response to combat disease were, in large part, delineated in the mouse. Despite the power of mouse-based immunology research, the translational efficacy of many new therapies from mouse to human is far from ideal. Recent data have highlighted how the naive, neonate-like immune system of specific pathogen-free mice differs dramatically in composition and function to mice living under barrier-free conditions (i.e., "dirty" mice). In this review, we discuss major findings to date and challenges faced when using dirty mice and specific areas of immunology research that may benefit from using animals with robust and varied microbial exposure.

Effects of whey protein hydrolysate on growth promotion and immunomodulation in mouse pups in artificial rearing system.

This study aimed to investigate the effects of whey protein hydrolysate (WPH) on the growth and immunity of mouse pups in artificial rearing (AR) system. Mouse pups were reared in the AR system with artificial milk including 5% WPH (AR with WPH) or not (AR without WPH), and the remaining pups were reared by their mother (dam) for 14 days after birth. The body weight change and body weight gain rates in the AR with WPH group were significantly higher than those observed in the AR without WPH group and similar to those in the dam group. Moreover the feed and protein efficiencies in the AR with WPH group were significantly higher than those of the AR without WPH group. In addition, the supplement of WPH in the AR system was shown to significantly elevate the number of CD3+ CD8+ , B220+ CD19+ , IA/IE+ CD11c+ , and CD11b+ in the thymocyte and/or splenocyte, and the thymus weight. Furthermore, MALDI-TOF/MS analysis identified the amino acid sequences corresponding to some peptides, and indicated that VRTPEVDDE had the highest relative intensity among the peptides from tested WPH. Therefore, WPH would be required to not only promote growth, but also exert immunomodulatory activities in mouse pups in AR system.

Generation and evaluation of anti-mouse IgG IgY as secondary antibody.

In order to evaluate the possibility of using IgY as the secondary antibody in immunoassay, specific IgY (1: 128,000) was generated by immunizing hens with mouse serum IgG purified by protein A column. IgY was extracted from egg yolk by polyethylene glycol 6000 (PEG-6000), and further purified using protein M affinity chromatography column. The purified IgY was conjugated with horseradish peroxidase (HRP) and fluorescein isothiocyanate (FITC), in that order. The reactivity of conjugated antibodies was evaluated by ELISA, Western blot and Immunofluorescence, demonstrating that the obtained IgY was able to conjugate with enzymes, react with mouse primary IgG antibody, and subsequently amplify the antigen-antibody signals in different immune reaction conditions, in a comparable secondary effect to conventional goat anti-mouse IgG antibody. The obtained conjugated antibodies showed high stability in broad pH ranges (4-10; >70%) and high thermostability at 37 °C for 84 h (>85%). Despite the need to further consider and evaluate the industrial standardization and production process, our data provided the primary evidence that conjugated IgY antibodies can be used as a secondary antibody for broad immunological analysis.

Avoidance of allergens as an environmental method in the prevention of inhaled allergy symptoms.

Asthma and other inhaled allergies are some of the most common paediatric diseases. The association of exposure to allergens with induction and exacerbation of symptoms has been proven. The majority of allergens are permanently or periodically suspended in the air, which leads to impaired quality of life for sensitive patients. Therefore, many methods of prevention and therapy of allergic diseases have been developed. The method of allergen exposure avoidance is often the first and the most significant measure. The present research has been conducted to evaluate, based on scientific data, which measures have the most reliable evidence of effectiveness. Environmental allergen avoidance methods, despite limited evidence supporting their clinical efficacy, are listed as the main therapeutic approaches in most recommendations. The significance of the holistic approach is also emphasised: only simultaneous introduction of several avoidance methods can bring possibly beneficial effects for the patient.

High-resolution 3D imaging and topological mapping of the lymph node conduit system.

The conduit network is a hallmark of lymph node microanatomy, but lack of suitable imaging technology has prevented comprehensive investigation of its topology. We employed an extended-volume imaging system to capture the conduit network of an entire murine lymph node (comprising over 280,000 segments). The extensive 3D images provide a comprehensive overview of the regions supplied by conduits, including perivascular sleeves and distinctive "follicular reservoirs" within B cell follicles, surrounding follicular dendritic cells. A 3D topology map of conduits within the T-cell zone showed homogeneous branching, but conduit density was significantly higher in the superficial T-cell zone compared with the deep zone, where distances between segments are sufficient for T cells to lose contact with fibroblastic reticular cells. This topological mapping of the conduit anatomy can now aid modeling of its roles in lymph node function, as we demonstrate by simulating T-cell motility in the different T-cell zones.

Single-cell transcriptomics of the naked mole-rat reveals unexpected features of mammalian immunity.

The immune system comprises a complex network of specialized cells that protects against infection, eliminates cancerous cells, and regulates tissue repair, thus serving a critical role in homeostasis, health span, and life span. The subterranean-dwelling naked mole-rat (NM-R; Heterocephalus glaber) exhibits prolonged life span relative to its body size, is unusually cancer resistant, and manifests few physiological or molecular changes with advancing age. We therefore hypothesized that the immune system of NM-Rs evolved unique features that confer enhanced cancer immunosurveillance and prevent the age-associated decline in homeostasis. Using single-cell RNA-sequencing (scRNA-seq) we mapped the immune system of the NM-R and compared it to that of the short-lived, cancer-prone mouse. In contrast to the mouse, we find that the NM-R immune system is characterized by a high myeloid-to-lymphoid cell ratio that includes a novel, lipopolysaccharide (LPS)-responsive, granulocyte cell subset. Surprisingly, we also find that NM-Rs lack canonical natural killer (NK) cells. Our comparative genomics analyses support this finding, showing that the NM-R genome lacks an expanded gene family that controls NK cell function in several other species. Furthermore, we reconstructed the evolutionary history that likely led to this genomic state. The NM-R thus challenges our current understanding of mammalian immunity, favoring an atypical, myeloid-biased mode of innate immunosurveillance, which may contribute to its remarkable health span.

Mapping Viral Susceptibility Loci in Mice.

Genetic alleles that contribute to enhanced susceptibility or resistance to viral infections and virally induced diseases have often been first identified in mice before humans due to the significant advantages of the murine system for genetic studies. Herein we review multiple discoveries that have revealed significant insights into virus-host interactions, all made using genetic mapping tools in mice. Factors that have been identified include innate and adaptive immunity genes that contribute to host defense against pathogenic viruses such as herpes viruses, flaviviruses, retroviruses, and coronaviruses. Understanding the genetic mechanisms that affect infectious disease outcomes will aid the development of personalized treatment and preventive strategies for pathogenic infections.

Genetic background and immunological status influence B cell repertoire diversity in mice.

The relationship between the immune repertoire and the physiopathological status of individuals is essential to apprehend the genesis and the evolution of numerous pathologies. Nevertheless, the methodological approaches to understand these complex interactions are challenging. We performed a study evaluating the diversity harbored by different immune repertoires as a function of their physiopathological status. In this study, we base our analysis on a murine scFv library previously described and representing four different immune repertoires: i) healthy and naïve, ii) healthy and immunized, iii) autoimmune prone and naïve, and iv) autoimmune prone and immunized. This library, 2.6 × 109 in size, is submitted to high throughput sequencing (Next Generation Sequencing, NGS) in order to analyze the gene subgroups encoding for immunoglobulins. A comparative study of the distribution of immunoglobulin gene subgroups present in the four libraries has revealed shifts in the B cell repertoire originating from differences in genetic background and immunological status of mice.

PTH Immunoassay Interference Due to Human Anti-Mouse Antibodies in a Subject With Obesity With Normal Parathyroid Function.

CONTEXT: Immunoassay interference has been most often found with prolactin measurement. However, only few data exist on immunoassay interference for other hormones. CASE DESCRIPTION: A 36-year-old woman with obesity (body mass index, 31 kg/m2) had regularly attended our endocrine unit for type 2 diabetes therapy. When she was included as a control subject in a study for obesity management, detailed laboratory testing was performed, including PTH. In the absence of clinical symptoms, she presented with normal calcium, phosphate, and vitamin D levels. However, the PTH levels were >5000 ng/L. These results were obtained using the Roche Elecsys electrochemiluminescence assay. Repeated measurements with this assay (mouse antibody) led to the same findings. However, using an Euroimmun assay (goat antibody), the exact PTH values were measured at 18.0 ng/L. After pretreatment with a heterophilic antibody blocking reagent, the results of the Roche assay had decreased to a normal level. This phenomenon was explained by the detection of human anti-mouse antibodies in the proband's serum. CONCLUSIONS: In cases of prolactin immunoassay interference, endogenous antibodies will bind to the hormone in vivo, resulting in complexes of a high molecular weight that are less efficiently cleared by the kidneys and, thus, accumulate in the blood. In contrast, the PTH values >5000 ng/L detected in our subject most likely had resulted from the specific interference of the human anti-mouse antibodies present in the proband's serum with the assay antibody, resulting in artificial stimulation of the Roche assay detection system ex vivo.

Development and characterization of a canine-specific anti-CD94 (KLRD-1) monoclonal antibody.

Natural killer (NK) cells are non-T, non-B lymphocytes are part of the innate immune system and function without prior activation. The human NK cell surface determinant, CD94, plays a critical role in regulation of NK cell activity as a heterodimer with NKG2 subclasses. Canine NK cells are not as well defined as the human and murine equivalents, due in part to the paucity of reagents specific to cell surface markers. Canines possess NK/NKT cells that have similar morphological characteristics to those found in humans, yet little is known about their functional characteristics nor of cell surface expression of CD94. Here, we describe the development and function of a monoclonal antibody (mAb) to canine (ca) CD94. Freshly isolated canine CD94+ cells were CD3+/-, CD8+/-, CD4-, CD21-, CD5low, NKp46+, and were cytotoxic against a canine target cell line. Anti-caCD94 mAb proved useful in enriching NK/NKT cells from PBMC for expansion on CTAC feeder cells in the presence of IL-2 and IL-15. The cultured cells were highly cytolytic with co-expression of NKp46 and reduced expression of CD3. Transmission electron microscopy revealed expanded CD94+ lymphocytes were morphologically large granular lymphocytes with large electron dense granules. Anti-caCD94 (mAb) can serve to enrich NK/NKT cells from dog peripheral blood for ex vivo expansion for HCT and is a potentially valuable reagent for studying NK/NKT regulation in the dog.

Recommended housing densities for research mice: filling the gap in data-driven alternatives.

Space recommendations for mice made in the Guide for Care and Use of Laboratory Animals have not changed since 1972, despite important improvements in husbandry and caging practices. The 1996 version of the Guide put forth a challenge to investigators to produce new data evaluating the effects of space allocation on the well-being of mice. In this review, we summarize many studies published in response to this challenge. We distinguish between studies using ventilated or nonventilated caging systems and those evaluating reproductive performance or general well-being of adult mice. We discuss how these studies might affect current housing density considerations in both production and research settings and consider gaps in mouse housing density research. Additionally, we discuss reliable methods used to monitor and quantify general well-being of research mice. Collectively, this large body of new data suggests that husbandry practices dictating optimal breeding schemes and space allocation per mouse can be reconsidered. Specifically, these data demonstrate that prewean culling of litters has no benefit, trio breeding is an effective production strategy without adversely affecting pup survival and well-being, and housing of adult mice at densities of up to twice current Guide recommendations does not compromise well-being for most strains.-Svenson, K. L., Paigen, B. Recommended housing densities for research mice: filling the gap in data-driven alternatives.

Clonal and atypical Toxoplasma strain differences in virulence vary with mouse sub-species.

The severe virulence of Toxoplasma gondii in classical laboratory inbred mouse strains contradicts the hypothesis that house mice (Mus musculus) are the most important intermediate hosts for its transmission and evolution because death of the mouse before parasite transmission equals death of the parasite. However, the classical laboratory inbred mouse strains (Mus musculus domesticus), commonly used to test Toxoplasma strain differences in virulence, do not capture the genetic diversity within Mus musculus. Thus, it is possible that Toxoplasma strains that are severely virulent in laboratory inbred mice are avirulent in some other mouse sub-species. Here, we present insight into the responses of individual mouse strains, representing strains of the genetically divergent Mus musculus musculus, Mus musculus castaneus and Mus musculus domesticus, to infection with individual clonal and atypical Toxoplasma strains. We observed that, unlike M. m. domesticus, M. m. musculus and M. m. castaneus are resistant to the clonal Toxoplasma strains. For M. m. musculus, we show that this is due to a locus on chromosome 11 that includes the genes that encode the interferon gamma (IFNG)-inducible immunity-related GTPases (Irgs) that can kill the parasite by localising and subsequently vesiculating the parasitophorous vacuole membrane. However, despite the localization of known effector Irgs to the Toxoplasma parasitophorous vacuole membrane, we observed that some atypical Toxoplasma strains are virulent in all the mouse strains tested. The virulence of these atypical strains in M. m. musculus could not be attributed to individual rhoptry protein 5 (ROP5) alleles, a secreted parasite pseudokinase that antagonises the canonical effector Irgs and is indispensable for parasite virulence in laboratory inbred mice (M. m. domesticus). We conclude that murine resistance to Toxoplasma is modulated by complex interactions between host and parasite genotypes and may be independent of known effector Irgs on murine chromosome 11.

The Toll Pathway in the Central Nervous System of Flies and Mammals.

Toll receptors, first identified to regulate embryogenesis and immune responses in the adult fly and subsequently defined as the principal sensors of infection in mammals, are increasingly appreciated for their impact on the homeostasis of the central as well as the peripheral nervous systems. Whereas in the context of immunity, the fly Toll and the mammalian TLR pathways have been researched in parallel, the expression pattern and functionality have largely been researched disparately. Herein, we provide data on the expression pattern of the Toll homologues, signaling components, and downstream effectors in ten different cell populations of the adult fly central nervous system (CNS). We have compared the expression of the different Toll pathways in the fly to the expression of TLRs in the mouse brain and discussed the implications with respect to commonalities, differences, and future perspectives.

A comparative study on effects of static electric field and power frequency electric field on hematology in mice.

With the development of the ultra high voltage transmission technology, the voltage level of transmission line rised. Accordingly, the strength of electric field in the vicinity of transmission line increased, thus possible health effects from electric field have caused many public attentions. In this study, in order to compare effects induced by static electric field (SEF) and power frequency electric field (PFEF) on immune function, Institute of Cancer Research (ICR) mice were exposed to 35 kV/m SEF (0 Hz) and PFEF (50 Hz),respectively. Several indicators of white blood cell, red blood cell as well as hemoglobin in peripheral blood were tested after exposure of 7, 14 and 21 days, respectively. There was no significant difference in any indicators under SEF exposure of 35 kV/m for 7d, 14d and 21d between experimental group and control group. Under the PFEF exposure of 35 kV/m, white blood cell count significantly reduced after exposure of 7d, 14d and 21d. Meanwhile, red blood cell count significantly reduced after exposure of 7d, and returned to normal level through the compensatory response of organism after exposure of 14d and 21d. Hemoglobin concentration significantly decreased only after exposure of 21d. Based on tested results of hematological indicators, SEF exposure of 35 kV/m did not affect immune functions in mice but PFEF exposure of 35 kV/m could cause a decline of immune function. This difference of effects from SEF and PFEF on immune function was possibly caused by the difference of the degree of molecular polarization and ion migration in organism under exposure of two kinds of electric fields.

IL-27 regulates the number, function and cytotoxic program of antiviral CD4 T cells and promotes cytomegalovirus persistence.

The role of IL-27 in antiviral immunity is still incompletely understood, especially in the context of chronic viruses that induce a unique environment in their infected host. Cytomegalovirus (CMV) establishes a persistent, tissue localized infection followed by lifelong latency. CMV infects the majority of people and although asymptomatic in healthy individuals, can cause serious disease or death in those with naïve or compromised immune systems. Therefore, there is an urgent need to develop a protective CMV vaccine for people at-risk and identifying key regulators of the protective immune response towards CMV will be crucial. Here we studied mouse CMV (MCMV) in IL-27 receptor deficient animals (Il27ra-/-) to assess the role of IL-27 in regulating CMV immunity. We found that IL-27 enhanced the number of antiviral CD4 T cells upon infection. However, in contrast to a well-established role for CD4 T cells in controlling persistent replication and a positive effect of IL-27 on their numbers, IL-27 promoted MCMV persistence in the salivary gland. This coincided with IL-27 mediated induction of IL-10 production in CD4 T cells. Moreover, IL-27 reduced expression of the transcription factor T-bet and restricted a cytotoxic phenotype in antiviral CD4 T cells. This is a highly intriguing result given the profound cytotoxic phenotype of CMV-specific CD4 T cells seen in humans and we established that dendritic cell derived IL-27 was responsible for this effect. Together, these data show that IL-27 regulates the number and effector functions of MCMV-specific CD4 T cells and could be targeted to enhance control of persistent/latent infection.

Immune Responses Induced by HSP60 DNA Vaccine against Toxoplasma gondii Infection in Kunming Mice.

Toxoplasma gondii can infect all the vertebrates including human, and leads to serious toxoplasmosis and considerable veterinary problems. T. gondii heat shock protein 60 (HSP60) is associated with the activation of antigen presenting cells by inducing initial immune responses and releasing inflammatory cytokines. It might be a potential DNA vaccine candidate for this parasite. A pVAX-HSP60 DNA vaccine was constructed and immune responses was evaluated in Kunming mice in this study. Our data indicated that the innate and adaptive immune responses was elicited by successive immunizations with pVAX-HSP60 DNA, showing apparent increases of CD3e+CD4+ and CD3e+CD8a+ T cells in spleen tissues of the HSP60 DNA-immunized mice (24.70±1.23% and 10.90±0.89%, P<0.05) and higher levels of specific antibodies in sera. Furthermore, the survival period of the immunized mice (10.53±4.78 day) were significantly prolonged during the acute T. gondii infection. Decrease of brain cysts was significant in the experimental group during the chronic infection (P<0.01). Taken together, TgHSP60 DNA can be as a vaccine candidate to prevent the acute and chronic T. gondii infections.