Specific Pathogen-free Animals Research Articles

CXXC5 variant in an immunodeficient patient with a progressive loss of hematopoietic cells

Partial loss of function variant in CXXC5 identified in an immunodeficient patient with progressive bone marrow failure, corrected by bone marrow transplant.

Specific Pathogen-Free Animals for Civilian and Military Trauma: a Cautionary Note in the Translation of New Drug Therapies.

Specific-pathogen free (SPF) animals were introduced into biomedical research in the early 1960s to reduce the incidence of disease into experimental design. The goal was to provide animals with selected microbiota compatible with sustained health. Sixty years later, SPF status has become a variable itself in biomedical research. Alterations in the gut microbiome-host relationship can profoundly influence basic physiology, immune/inflammatory function, susceptibility to infection and disease, and behavior. In addition, it can influence the translational success of a drug or technology from animal models to humans. We discuss this aspect of SPF status in animal models used for military or civilian trauma and shock research. Currently, there is a broad spectrum of SPF exclusion and inclusion criteria which vary from one supplier or animal husbandry facility. If translation to humans is the end-game of trauma research, we recommend replicating a gut microbiome similar to the wild-type for optimal success. We further suggest that at the end of each publication a URL access be provided on Animal Microbial/Pathogen Exclusion Status that a study was based upon. This may help address the differences in results within a single laboratory or between laboratories around the world and improve translation success.

Development of a DIVA ELISA for diagnosis of Aujeszky's disease using recombinant gE fused to thioredoxin as antigen

The major control methods for Aujeszky's Disease (AD) involve SHV1 gE gene-deleted vaccines and ELISA for detection of specific gE antibodies in infected animals, distinguishing infected animals from vaccinated animals (DIVA). This work aimed to develop a DIVA ELISA recombinant gE (gErec) for AD diagnosis using recombinant gE fused to thioredoxin protein. The analytical sensitivity and specificity were assessed with World Organisation for Animal Health (OIE) AD serum and sera from specific pathogen free (SPF), vaccinated SPF and AD-vaccinated SPF animals. The OIE serum reacted up to the recommended limit of detection and the other sera presented negative results. The cut-off point, diagnostic sensitivity and diagnostic specificity were determined by receiver operating curve analysis. This cut-off value corresponded to a diagnostic sensitivity of 97.60% and diagnostic specificity of 96.42%. Furthermore, two other cut-off points were chosen to discuss the ELISAgErec as a screening test in AD-endemic and free areas.

Reprint of: Manipulation of microbiota reveals altered callosal myelination and white matter plasticity in a model of Huntington disease

Structural and molecular myelination deficits represent early pathological features of Huntington disease (HD). Recent evidence from germ-free (GF) animals suggests a role for microbiota-gut-brain bidirectional communication in the regulation of myelination. In this study, we aimed to investigate the impact of microbiota on myelin plasticity and oligodendroglial population dynamics in the mixed-sex BACHD mouse model of HD. Ultrastructural analysis of myelin in the corpus callosum revealed alterations of myelin thickness in BACHD GF compared to specific-pathogen free (SPF) mice, whereas no differences were observed between wild-type (WT) groups. In contrast, myelin compaction was altered in all groups when compared to WT SPF animals. Levels of myelin-related proteins were generally reduced, and the number of mature oligodendrocytes was decreased in the prefrontal cortex under GF compared to SPF conditions, regardless of genotype. Minor differences in commensal bacteria at the family and genera levels were found in the gut microbiota of BACHD and WT animals housed in standard living conditions. Our findings indicate complex effects of a germ-free status on myelin-related characteristics, and highlight the adaptive properties of myelination as a result of environmental manipulation.

Identification of CD34 <sup>+</sup>ABCA3 <sup>+</sup> Cells as the Origin of Lung Adenocarcinoma Through the Interaction between Lung Progenitor Club and Lgr6 Cells

Targeting cancer stem cells (CSCs) results in evolved phenotypes with increased malignancy, leading to therapy failure. Here, we propose a new therapeutic strategy: investigating “transitional” cells that represent the stage between normal lung stem cells and lung CSCs. We used Gprc5a knockout mice, the first animal model of spontaneous lung adenocarcinoma that served as an incubator for transitional cancerous cells. We found CD34+ABCA3+ cells with a CSC-like phenotype within the Club cell population in mice that had not yet developed tumors, and confirmed the self-renewal and tumor initiation capacities of this subset in vitro, in vivo and clinical samples. Mechanistically, the generation of CD34+ABCA3+ cells was associated with the interaction between Club and Lgr6 cells and the subsequent activation of the ECM1-α10β1-ABCA3 axis. Thus, the present study unveils a new kind of lung cancer-initiating cell (LCIC) and provides potential markers for the early diagnosis of lung cancer. Funding Statement: This study was supported by grants from the National Natural Science Foundation of China (No. 81601985 for H. Yin). Declaration of Interests: The authors declare no conflicts of interest Ethics Approval Statement: Human tissue samples were obtained from the Fudan University Shanghai Cancer Center with approval of the hospital ethical committee .Mice were maintained according to a protocol approved by the Fudan University School of Medicine Animal Care and Use Committee in the specific pathogen-free animal facility at the university

Extracellular vesicles derived from injured vascular tissue promote the formation of tertiary lymphoid structures in vascular allografts.

Tertiary lymphoid structures (TLS) accumulate at sites of chronic injury where they function as an ectopic germinal center, fostering local autoimmune responses. Vascular injury leads to the release of endothelial‐derived apoptotic exosome‐like vesicles (ApoExo) that contribute to rejection in transplanted organs. The purpose of the study was to evaluate the impact of ApoExo on TLS formation in a model of vascular allograft rejection. Mice transplanted with an allogeneic aortic transplant were injected with ApoExo. The formation of TLS was significantly increased by ApoExo injection along with vascular remodeling and increased levels of antinuclear antibodies and anti‐perlecan/LG3 autoantibodies. ApoExo also enhanced allograft infiltration by γδT17 cells. Recipients deficient in γδT cells showed reduced TLS formation and lower autoantibodies levels following ApoExo injection. ApoExo are characterized by proteasome activity, which can be blocked by bortezomib. Bortezomib treated ApoExo reduced the recruitment of γδT17 cells to the allograft, lowered TLS formation, and reduced autoantibody production. This study identifies vascular injury‐derived extracellular vesicles (ApoExo), as initiators of TLS formation and demonstrates the pivotal role of γδT17 in coordinating TLS formation and autoantibody production. Finally, our results suggest proteasome inhibition with bortezomib as a potential option for controlling TLS formation in rejected allografts.

Interactions Between Gut Microbiota and Acute Restraint Stress in Peripheral Structures of the Hypothalamic-Pituitary-Adrenal Axis and the Intestine of Male Mice.

The gut microbiota play an important role in shaping brain functions and behavior, including the activity of the hypothalamus-pituitary-adrenocortical (HPA) axis. However, little is known about the effect of the microbiota on the distinct structures (hypothalamus, pituitary, and adrenals) of the HPA axis. In the present study, we analyzed the influence of the microbiota on acute restraint stress (ARS) response in the pituitary, adrenal gland, and intestine, an organ of extra-adrenal glucocorticoid synthesis. Using specific pathogen-free (SPF) and germ-free (GF) male BALB/c mice, we showed that the plasma corticosterone response to ARS was higher in GF than in SPF mice. In the pituitary, stress downregulated the expression of the gene encoding CRH receptor type 1 (Crhr1), upregulated the expression of the Fkbp5 gene regulating glucocorticoid receptor sensitivity and did not affect the expression of the proopiomelanocortin (Pomc) and glucocorticoid receptor (Gr) genes. In contrast, the microbiota downregulated the expression of pituitary Pomc and Crhr1 but had no effect on Fkbp5 and Gr. In the adrenals, the steroidogenic pathway was strongly stimulated by ARS at the level of the steroidogenic transcriptional regulator Sf-1, cholesterol transporter Star and Cyp11a1, the first enzyme of steroidogenic pathway. In contrast, the effect of the microbiota was significantly detected at the level of genes encoding steroidogenic enzymes but not at the level of Sf-1 and Star. Unlike adrenal Sf-1, the expression of the gene Lrh-1, which encodes the crucial transcriptional regulator of intestinal steroidogenesis, was modulated by the microbiota and ARS and this effect differed between the ileum and colon. The findings demonstrate that gut microbiota have an impact on the response of the pituitary, adrenals and intestine to ARS and that the interaction between stress and the microbiota during activation of glucocorticoid steroidogenesis differs between organs. The results suggest that downregulated expression of pituitary Pomc and Crhr1 in SPF animals might be an important factor in the exaggerated HPA response of GF mice to stress.

Conventional and Specific-Pathogen Free Rats Respond Differently to Anesthesia and Surgical Trauma

Specific-pathogen free (SPF) animals were introduced in the 1960s to minimize disease and infection as variables in biomedical research. Our aim was to examine differences in physiological response in rat colonies bred and housed in a conventional versus SPF facility, and implications for research. Sprague-Dawley rats were anesthetized and catheterized for blood and pressure monitoring, and electrocardiogram (ECG) leads implanted. Hematology was assessed, and coagulation profile using rotational thromboelastometry. Health screening was outsourced to Cerberus Sciences. SPF rats had significantly lower pulse pressure (38% decrease), arrhythmias and prolonged QTc (27% increase) compared to conventional rats. No arrhythmias were found in conventional rats. SPF rats had significantly higher white cell, monocyte, neutrophil and lymphocyte counts, and were hyperfibrinolytic, indicated by EXTEM maximum lysis >15%. Independent assessment revealed similar pathogen exclusion between colonies, with the exception of Proteus in SPF animals. Returning to a conventional facility restored normal host physiology. We conclude that SPF animals displayed an abnormal hemodynamic, hematological and hemostatic phenotype in response to anesthesia and surgery, and provide a number of recommendations to help standardize research outcomes and translation.

Decontamination of genetically modified mice strains by embryo transfer for obtaining SPF colonies in a Brazilian animal facility

The introduction of new strains of mice in specific pathogen-free (SPF) animal facilities should be performed carefully to avoid breaking sanitary barriers. To meet this need, animals should be rederived to reduce infection risk and thus avoid research interference caused by loss of animal health status and welfare. The objective of this study was to implement mice embryo transfer in the laboratory mouse facility of the Department of Immunology at the Institute of Biomedical Sciences/University of São Paulo, Brazil. Embryo transfers were performed to rederive genetically modified mouse strains with undefined sanitary status, received from different research and educational institutions. Fertilized eggs at two-cell stage were obtained by natural means and transferred into the oviducts of SPF pseudo-pregnant female mice. All surgical procedures were performed under aseptic conditions. A total of 625 embryos were transferred into therecipients. 148 pups were born, of which 140 were reared. Viruses, bacteria and intestinal protozoa were eliminated using this technique. The improvement in the microbiological status of mice allowed their expansion in our SPF facility. With these results, we can stimulate the use of embryo transfer technique between rodent facilities in Brazil and thus encourage the distribution of better models to our scientific community.

Characteristics of the intestinal flora of specific pathogen free chickens with age

Specific pathogen-free (SPF) experimental animals are recognized as standard laboratory animals in the fields of biomedical, animal husbandry and veterinary research and production. Intestinal flora plays a critical role in nutrient absorption, improving health and protecting the host from pathogens. We therefore explored the variation and maintenance of intestinal flora in SPF chicks in order to better understand the composition of intestinal microflorain SPF chickens, and provide reference for the study of intestinal flora of SPF experimental animals. Five chicks were randomly selected at each of 14, 28, and 42 days, and ceca were removed for DNA extraction. The Illumina Miseq platform was used for microbiome analysis of the V3–V4 region of the 16S rRNA gene. During the course of chick gut microbiome development, we observed major changes in diversity, especially between day 14 and day 28. Firmicutes, Proteobacteria, and Bacteroidetes were the main bacterial taxa, and Firmicutes increased significantly with age. The genus with the highest relative abundance was Lactobacillus, followed by Faecalibacterium. In addition, while abundance of Ruminococcaceae spp., Ruminococcus, and Blautia increased with age, Lactobacillus, Enterobacteriaceae spp., and Oscillospira decreased with age. Interestingly, the abundance of Faecalibacterium first increased and then decreased over time. The characteristics of SPF chicken gut flora at different ages establish a basis for the regulation of intestinal flora in the early stage of brooding, and also provide a theoretical foundation for controlling and preventing infections and poultry diseases in newborn chickens.

Porcine Hemagglutinating Encephalomyelitis Virus: A Review.

The porcine hemagglutinating encephalomyelitis virus (PHEV) is classified as a member of genus Betacoronavirus, family Coronaviridae, sub-family Cornavirinae, and order Nidovirales. PHEV shares the same genomic organization, replication strategy, and expression of viral proteins as other nidoviruses. PHEV produces vomiting and wasting disease (VWD) and/or encephalomyelitis, being the only known neurotropic coronavirus affecting pigs. First clinical outbreak was reported in 1957 in Ontario, Canada. Although pigs are the only species susceptible to natural PHEV infections, the virus displays neurotropism in mice and Wistar rats. Clinical disease, morbidity, and mortality is age-dependent and generally reported only in piglets under 4 weeks old. The primary site of replication of PHEV in pigs is the respiratory tract, and it can be further spread to the central nervous system through the peripheral nervous system via different pathways. The diagnosis of PHEV can be made using a combination of direct and indirect detection methods. The virus can be isolated from different tissues within the acute phase of the clinical signs using primary and secondary pig-derived cell lines. PHEV agglutinates the erythrocytes of mice, rats, chickens, and several other animals. PCR-based methods are useful to identify and subsequently isolate animals that are actively shedding the virus. The ability to detect antibodies allows producers to know the status of first-litter gilts and evaluate their risk of tier offspring to infection. PHEV is highly prevalent and circulates subclinically in most swine herds worldwide. PHEV-related disease is not clinically relevant in most of the swine-producing countries, most likely because of dams are immune to PHEV which may confer passive immunity to their offspring. However, PHEV should be considered a major source of economic loss because of the high mortality on farms with high gilt replacement rates, specific pathogen-free animals, and gnotobiotic swine herds. Thus, in the absence of current PHEV vaccines, promoting virus circulation on farms with early exposure to gilts and young sows could induce maternal immunity and prevent disease in piglets.

Specific pathogen-free (SPF) animal status as a variable in biomedical research: Have we come full circle?

In this commentary, we discuss the pros and cons of using specific pathogen-free (SPF) animals in biomedical research, and present individual cases where altering the gut microbiome has dramatically changed the animal's basic physiology, immune/inflammatory functions and susceptibility to infection and disease. We argue that SPF manipulation of the microbiome-host relationship has itself become a confounding variable in biomedical research, which could have major implications to human translation.

Manipulation of microbiota reveals altered callosal myelination and white matter plasticity in a model of Huntington disease

Structural and molecular myelination deficits represent early pathological features of Huntington disease (HD). Recent evidence from germ-free (GF) animals suggests a role for microbiota-gut-brain bidirectional communication in the regulation of myelination. In this study, we aimed to investigate the impact of microbiota on myelin plasticity and oligodendroglial population dynamics in the mixed-sex BACHD mouse model of HD. Ultrastructural analysis of myelin in the corpus callosum revealed alterations of myelin thickness in BACHD GF compared to specific-pathogen free (SPF) mice, whereas no differences were observed between wild-type (WT) groups. In contrast, myelin compaction was altered in all groups when compared to WT SPF animals. Levels of myelin-related proteins were generally reduced, and the number of mature oligodendrocytes was decreased in the prefrontal cortex under GF compared to SPF conditions, regardless of genotype. Minor differences in commensal bacteria at the family and genera levels were found in the gut microbiota of BACHD and WT animals housed in standard living conditions. Our findings indicate complex effects of a germ-free status on myelin-related characteristics, and highlight the adaptive properties of myelination as a result of environmental manipulation.

Facts, truths and myths about SPF shrimp in Aquaculture

AbstractShrimp domestication and genetic improvement programmes began in late 1980s, in the United States of America, under the United States Marine Shrimp Farming Program (USMSFP), using the Pacific whiteleg shrimp Penaeus vannamei. The USMSFP was based on proven concepts from the livestock and poultry industries and began with establishing a specific pathogen‐free (SPF) shrimp stock. The original shrimp stock was obtained using rigorous screening of captured wild shrimp for selection of individuals naturally free of major shrimp pathogens. Although the concept of SPF animals was well defined for terrestrial animals, it was relatively new for aquaculture, and it took some time to be adopted by the aquaculture community. In the early 1990s, parallel to USMSFP, several other programmes on genetic improvement of shrimp were also initiated in Latin America. Subsequently, several new terminologies and products, such as specific pathogen resistant (SPR) shrimp, specific pathogen tolerant (SPT) shrimp and even ‘all pathogen exposed’ (APE) shrimp, entered the shrimp industry vocabulary and became commercial. This led to confusion in the shrimp industry about the meaning, relationship and significance of these new terms with respect to SPF. This position paper attempts to clarify these concepts, provide science‐based definitions, reconfirms the importance of developing, maintaining and using domesticated, specific pathogen‐free (SPF) shrimp stocks (which may also achieve SPR and/or SPT status) to reduce the risk of disease outbreaks and increase production and profit. The same principles would apply to development of domesticated SPF stocks for other species used in aquaculture. The paper also discusses the difficulties of confirming and certifying SPF status due to the presence of endogenous viral elements (EVEs) and calls for internationally agreed science and evidence‐based technical guidelines for producing healthy shrimp.

Mouse Models Of Helicobacter Infection And Gastric Pathologies.

Helicobacter pylori is a gastric pathogen that is present in half of the global population and is a significant cause of morbidity and mortality in humans. Several mouse models of gastric Helicobacter infection have been developed to study the molecular and cellular mechanisms whereby H. pylori bacteria colonize the stomach of human hosts and cause disease. Herein, we describe protocols to: 1) prepare bacterial suspensions for the in vivo infection of mice via intragastric gavage; 2) determine bacterial colonization levels in mouse gastric tissues, by polymerase chain reaction (PCR) and viable counting; and 3) assess pathological changes, by histology. To establish Helicobacter infection in mice, specific pathogen-free (SPF) animals are first inoculated with suspensions (containing ≥105 colony-forming units, CFUs) of mouse-colonizing strains of either Helicobacter pylori or other gastric Helicobacter spp. from animals, such as Helicobacter felis. At the appropriate time-points post-infection, stomachs are excised and dissected sagittally into two equal tissue fragments, each comprising the antrum and body regions. One of these fragments is then used for either viable counting or DNA extraction, while the other is subjected to histological processing. Bacterial colonization and histopathological changes in the stomach may be assessed routinely in gastric tissue sections stained with Warthin-Starry, Giemsa or Haematoxylin and Eosin (H&E) stains, as appropriate. Additional immunological analyses may also be undertaken by immunohistochemistry or immunofluorescence on mouse gastric tissue sections. The protocols described below are specifically designed to enable the assessment in mice of gastric pathologies resembling those in human-related H. pylori diseases, including inflammation, gland atrophy and lymphoid follicle formation. The inoculum preparation and intragastric gavage protocols may also be adapted to study the pathogenesis of other enteric human pathogens that colonize mice, such as Salmonella Typhimurium or Citrobacter rodentium.

Microbiota affects the expression of genes involved in HPA axis regulation and local metabolism of glucocorticoids in chronic psychosocial stress

The commensal microbiota affects brain functioning, emotional behavior and ACTH and corticosterone responses to acute stress. However, little is known about the role of the microbiota in shaping the chronic stress response in the peripheral components of the hypothalamus-pituitary-adrenocortical (HPA) axis and in the colon. Here, we studied the effects of the chronic stress-microbiota interaction on HPA axis activity and on the expression of colonic corticotropin-releasing hormone (CRH) system, cytokines and 11β-hydroxysteroid dehydrogenase type 1 (11HSD1), an enzyme that determines locally produced glucocorticoids. Using specific pathogen-free (SPF) and germ-free (GF) BALB/c mice, we showed that the microbiota modulates emotional behavior in social conflicts and the response of the HPA axis, colon and mesenteric lymph nodes (MLN) to chronic psychosocial stress. In the pituitary gland, microbiota attenuated the expression of Fkbp5, a gene regulating glucocorticoid receptor sensitivity, while in the adrenal gland, it attenuated the expression of genes encoding steroidogenesis (MC2R, StaR, Cyp11a1) and catecholamine synthesis (TH, PNMT). The pituitary expression of CRH receptor type 1 (CRHR1) and of proopiomelanocortin was not influenced by microbiota. In the colon, the microbiota attenuated the expression of 11HSD1, CRH, urocortin UCN2 and its receptor, CRHR2, but potentiated the expression of cytokines TNFα, IFNγ, IL-4, IL-5, IL-6, IL-10, IL-13 and IL-17, with the exception of IL-1β. Compared to GF mice, chronic stress upregulated in SPF animals the expression of pituitary Fkbp5 and colonic CRH and UCN2 and downregulated the expression of colonic cytokines. Differences in the stress responses of both GF and SPF animals were also observed when immunophenotype of MLN cells and their secretion of cytokines were analyzed. The data suggest that the presence of microbiota/intestinal commensals plays an important role in shaping the response of peripheral tissues to stress and indicates possible pathways by which the environment can interact with glucocorticoid signaling.

Prevalence of lapine rotavirus, astrovirus, and hepatitis E virus in Canadian domestic rabbit populations

Lapine rotavirus and astrovirus have been associated with disease in rabbits, and there is strong evidence of zoonotic transmission of lapine hepatitis E virus (HEV). Outbreaks of enteritis are common on commercial meat farms, resulting in poor welfare, high rabbit mortality, and significant financial losses for rabbit producers. Currently, none of these viruses are routinely tested by diagnostic laboratories. In this study, we assessed the prevalence of rotavirus, astrovirus, and HEV RNA in 205 pooled and individual fecal samples from healthy Canadian laboratory, companion, shelter and commercial meat rabbit populations. Viral RNA were extracted and amplified via RT-PCR using virus-specific primers. Positive samples from the first cohort of samples tested were sequenced and aligned to previously identified viruses to confirm the products. Almost 45% (13/29) of the surveyed commercial rabbit farms were astrovirus-positive. Three commercial meat rabbit samples were positive for rotavirus, and either astrovirus or HEV RNA was also detected. Three companion rabbit samples also tested positive for lapine HEV. Samples from specific pathogen-free laboratory animals were negative for all viruses. Sequencing results showed highest identity to rotavirus A strain 30–96, lapine astrovirus strain 2208 and lapine HEV strain CMC-1. These results permit a better understanding of the prevalence of rotavirus, astrovirus, and hepatitis E virus in Canadian domestic rabbit populations, and continued screening for viruses may help to reduce risk of zoonotic agent transmission as well as providing a better understanding of potential causative agents of rabbit enteritis.

Application of 1H NMR spectroscopy to the metabolic phenotyping of rodent brain extracts: A metabonomic study of gut microbial influence on host brain metabolism

1H NMR Spectroscopy has been applied to determine the neurochemical profiles of brain extracts from the frontal cortex and hippocampal regions of germ free and normal mice and rats. The results revealed a number of differences between germ free (GF) and conventional (CV) rats or specific pathogen-free (SPF) mice with microbiome-associated metabolic variation found to be both species- and region-dependent. In the mouse, the GF frontal cortex contained lower amounts of creatine, N-acetyl-aspartate (NAA), glycerophosphocholine and lactate, but greater amounts of choline compared to that of specific pathogen free (SPF) mice. In the hippocampus, the GF mice had greater creatine, NAA, lactate and taurine content compared to those of the SPF animals, but lower relative quantities of succinate and an unidentified lipid-related component. The GF rat frontal cortex contained higher relative quantities of lactate, creatine and NAA compared to the CV animals whilst the GF hippocampus was characterized by higher taurine and phosphocholine concentrations and lower quantities of NAA, N-acetylaspartylglutamate and choline compared to the CV animals. Of note is that, in both rat and mouse brain extracts, concentrations of hippocampal taurine were found to be greater in the absence of an established microbiome. The results provide further evidence that brain biochemistry can be influenced by gut microbial status, specifically metabolites involved in energy metabolism demonstrating biochemical dialogue between the microbiome and brain.

Influence of Size and Shape on the Anatomical Distribution of Endotoxin-Free Gold Nanoparticles.

The transport and the delivery of drugs through nanocarriers is a great challenge of pharmacology. Since the production of liposomes to reduce the toxicity of doxorubicin in patients, a plethora of nanomaterials have been produced and characterized. Although it is widely known that elementary properties of nanomaterials influence their in vivo kinetics, such interaction is often poorly investigated in many preclinical studies. The present study aims to evaluate the actual effect of size and shape on the biodistribution of a set of gold nanoparticles (GNPs) after intravenous administration in mice. To this goal, quantitative data achieved by inductively coupled plasma mass spectrometry and observational results emerging from histochemistry (autometallography and enhanced dark-field hyperspectral microscopy) were combined. Since the immune system plays a role in bionano-interaction we used healthy immune-competent mice. To keep the immune surveillance on the physiological levels we synthesized endotoxin-free GNPs to be tested in specific pathogen-free animals. Our study mainly reveals that (a) the size and the shape greatly influence the kinetics of accumulation and excretion of GNPs in filter organs; (b) spherical and star-like GNPs showed the same percentage of accumulation, but a different localization in liver; (c) only star-like GNPs are able to accumulate in lung; (d) changes in the geometry did not improve the passage of the blood brain barrier. Overall, this study can be considered as a reliable starting point to drive the synthesis and the functionalization of potential candidates for theranostic purposes in many fields of research.

Sensing of the microbiota by NOD1 in mesenchymal stromal cells regulates murine hematopoiesis

AbstractThe microbiota is known to influence the generation of hematopoietic progenitors, although the pathways underlying this process are still poorly understood. NOD1 and NOD2 are intracellular sensors for both Gram-positive and Gram-negative bacteria, but their role in steady-state hematopoiesis has never been characterized. We observed that stimulation with NOD1 or NOD2 ligand had no effect on the survival/proliferation of hematopoietic precursors. Nonetheless, NOD1, but not NOD2, ligand induced expression of multiple hematopoietic cytokines (interleukin-7 [IL-7], Flt3L, stem cell factor [SCF], ThPO, and IL-6) from bone marrow mesenchymal stromal cells (MSCs) in vitro. Moreover, in vivo administration of NOD1 ligand to germ-free mice restored the numbers of hematopoietic stem cells and precursors in bone marrow as well as serum concentrations of IL-7, Flt3L, SCF, and ThPO to the levels displayed by specific pathogen-free control animals. Based on these findings, we propose that NOD1 signaling in MSCs serves as an important pathway underlying the requirement for microbiota in the maintenance of steady-state hematopoiesis. This function is distinct from that triggered by lipopolysaccharide in both its broad effects on multiple progenitors and specific targeting of MSCs as cytokine producing intermediates.