Animal unit hygienic conditions influence mouse intestinal microbiota and contribute to T-cell-mediated colitis.

Inflammatory bowel disease (IBD) is a group of chronic inflammatory disorders of the gastrointestinal tract with worldwide increasing incidence. Recent studies indicate that certain species of intestinal bacteria are strongly associated with IBD. Helper T lymphocytes are not only the key players in mediating host defense against a wide variety of pathogens but also contribute to pathogenesis of many immune-related diseases. Here, using the T cell transfer model of colitis, we observed that the mice maintained in a specific-pathogen free (SPF) unit after receiving naïve CD4+ T cells developed mild disease. The same mice developed different degrees of disease when they were maintained in a conventional animal facility (non-SPF), where some pathogens were detected during routine health monitoring. Consistently, increased circulating inflammatory cytokines as well as Th1 and Th17 cells were detected in mice housed in non-SPF units. 16S rRNA sequencing of feces samples enabled us to identify changes in the microbiota composition of mice kept in different facilities. Our data indicate that environmental factors influence gut microbiota composition of mice, leading to development of colitis in a T-cell-dependent manner. In conclusion, changes in environmental conditions and microbial status of experimental animals appear to contribute to progression of colitis.

A real-time health notification system aimed at enhancing the interaction between animal care staff and researchers promotes animal welfare.

Regardless of the microbiological status of an animal facility, research animals may experience health problems, leading to pain, suffering and distress. Simple and efficient tools are needed to collect data systematically, allowing researchers to react and resolve animals' health issues. We have developed a real-time notification method for recording clinical observations, which caretakers can input into the ELLI record-keeping system, accompanied by a picture or video. A browser-based interface system sends alerts using a three-tier scale (+, 120 hours; ++, 72 hours; +++, 24 hours) by email and/or SMS. The percentage of animal health notifications for rodents was 1.31% in 2016, 1.33% in 2017 and 1.58% in 2018, with 34-44% for coat and skin conditions (wounds, bites and scratches). All other notifications, including environment and behaviour, procedure-specific indicators (weight loss, bleeding and abnormal secretions) and other abnormalities such as eye and teeth malformations, ranged from 5% to 10% during the three-year period. Researchers displayed good compliance by reacting to the notifications within the expected time frame. Most health notifications concerned genetically modified (GM) animals without a predetermined harmful phenotype, regardless of being on project licence or maintenance licence. Health notification records may be useful retrospectively not only to review the health and welfare issues of new GM lines but also to evaluate the actual severity of procedures. The health notification system described here provides valuable information to the veterinarian and the animal welfare body by helping to address specific health conditions and to improve animal welfare and implement the 3Rs.

In vivo Induction of Functional Inhibitory IgG Antibodies by a Hypoallergenic Bet v 1 Variant.

Allergic sensitization to the major allergen Bet v 1 represents the dominating factor inducing a vast variety of allergic symptoms in birch pollen allergic patients worldwide, including the pollen food allergy syndrome. In order to overcome the huge socio-economic burden associated with allergic diseases, allergen-specific immunotherapy (AIT) as a curative strategy to manage the disease was introduced. Still, many hurdles related to this treatment exist making AIT not the patients' first choice. To improve the current situation, the development of hypoallergen-based drug products has raised attention in the last decade. Herein, we investigated the efficacy of the novel AIT candidate BM4, a hypoallergenic variant of Bet v 1, to induce treatment-relevant cross-reactive Bet v 1-specific IgG antibodies in two different mammals, Wistar rats and New Zealand White rabbits. We further analyzed the cross-reactivity of BM4-induced Wistar rat antibodies with the birch pollen-associated food allergens Mal d 1 and Cor a 1, and the functional capability of the induced antibodies to act as IgE-blocking IgG antibodies. Enzyme-linked immunosorbent assay (ELISA) was used to determine the titers of rat IgG1, IgG2a, IgG2b, and IgE, as well as rabbit IgG and IgE antibodies. To address the functional relevance of the induced IgG antibodies, the capacity of rat sera to suppress binding of human IgE to Bet v 1 was investigated by using an inhibition ELISA and an IgE-facilitated allergen-binding inhibition assay. We found that the treatment with BM4 induced elevated Bet v 1-specific IgG antibody titers in both mammalian species. In Wistar rats, high BM4-specific IgG1, IgG2a, and IgG2b titers (104 to 106) were induced, which cross-reacted with wild-type Bet v 1, and the homologous allergens Mal d 1 and Cor a 1. Rat allergen-specific IgG antibodies sustained upon treatment discontinuation. Sera of rats immunized with BM4 were able to significantly suppress binding of human IgE to the wild-type allergens and CD23-mediated human IgE-facilitated Bet v 1 binding on B cells. By contrast, treatment-induced IgE antibody levels were low or undetectable. In summary, BM4 induced a robust IgG immune response that efficiently blocked human IgE-binding to wild-type allergens, underscoring its potential therapeutic value in AIT.

Safety Study of Single-Dose Intravenously Administered DOTA-Siglec-9 Peptide in Sprague Dawley Rats.

The peptide-based radioactive compound [68Ga]Ga-DOTA-Siglec-9 is a novel agent for imaging of inflammation with positron emission tomography. The drug target of [68Ga]Ga-DOTA-Siglec-9 is vascular adhesion protein 1. Previous studies have obtained promising results with [68Ga]Ga-DOTA-Siglec-9 in experimental animals. However, before taking this novel imaging agent into clinical trials, safety and toxicological studies need to be performed with the nonradioactive precursor compound DOTA-Siglec-9. This extended single-dose toxicity study was designed to provide information on the major toxic effects of DOTA-Siglec-9 and to indicate possible target organs after a single intravenous (iv) injection in rats. The study was performed using 60 adult Hsd: Sprague Dawley rats and included a control group and a treatment group to investigate the toxicity of DOTA-Siglec-9 solution at a final concentration of 0.2 mg/mL after a single iv injection of 582 µg/kg. The maximum dose tested was 1,000-fold the clinical dose on a mg/kg basis as indicated in European Medicines Agency International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use guideline M3(R2). The planned human clinical dose is approximately 0.582 µg of DOTA-Siglec-9 per kg of body mass. This study demonstrates that iv administration of DOTA-Siglec-9 at a dose of 582 µg/kg was well tolerated in rats and did not produce toxicologically significant adverse effects.

Resembling breast milk: influence of polyamine-supplemented formula on neonatal BALB/cOlaHsd mouse microbiota.

Infant microbiota is influenced by numerous factors, such as delivery mode, environment, prematurity and diet (breast milk or formula). In addition to its nutritional value, breast milk contains bioactive substances that drive microbial colonisation and support immune system development, which are usually not present in infant formulas. Among these substances, polyamines have been described to be essential for intestinal and immune functions in newborns. However, their effect on the establishment of microbiota remains unclear. Therefore, the aim of the present study was to ascertain whether an infant formula supplemented with polyamines has an impact on microbial colonisation by modifying it to resemble that in breast-fed neonatal BALB/c mice. In a 4 d intervention, a total of sixty pups (14 d old) were randomly assigned to the following groups: (1) breast-fed group; (2) non-enriched infant formula-fed group; (3) three different groups fed an infant formula enriched with increasing concentrations of polyamines (mixture of putrescine, spermidine and spermine), following the proportions found in human milk. Microbial composition in the contents of the oral cavity, stomach and small and large intestines was analysed by quantitative PCR targeted at fourteen bacterial genera and species. Significantly different (P< 0·05) microbial colonisation patterns were observed in the entire gastrointestinal tract of the breast-fed and formula-fed mice. In addition, our findings demonstrate that supplementation of polyamines regulates the amounts of total bacteria, Akkermansia muciniphila, Lactobacillus, Bifidobacterium, Bacteroides-Prevotella and Clostridium groups to levels found in the breast-fed group. Such an effect requires further investigation in human infants, as supplementation of an infant formula with polyamines might contribute to healthy gastrointestinal tract development.

Infant formula supplemented with polyamines alters the intestinal microbiota in neonatal BALB/cOlaHsd mice.

Polyamines play a critical role in the development of intestinal and immune systems during the infant breastfeeding period, but the effect of polyamines on the microbiota has not been reported. The aim of our study was to characterize the impact on the colonization pattern in neonatal BALB/cOlaHsd mice after supplementing an infant formula (IF) with a mixture of putrescine (PUT), spermidine (SPD) and spermine (SPM). A total of 48 pups (14 days old) were randomly assigned to 4-day intervention groups as follows: breast-fed (unweaned) pups (n=12); weaned pups (n=12) fed an infant formula (IF); weaned pups (n=12) fed an IF enriched with a low concentration of PUT, SPD and SPM (2.10, 22.05 and 38.00 µg/day, respectively); and weaned pups (n=12) fed with IF enriched with a high concentration of PUT, SPD and SPM (8.40, 88.20 and 152.00 µg/day, respectively) of polyamines in accordance with normal proportions found in human milk. Microbiota composition was analyzed by fluorescent in situ hybridization (FISH) with flow cytometry detection. Microbiota changes in formula-fed mice were significantly greater following supplementation with polyamines (P<.01). Bifidobacterium group bacteria, Akkermansia-like bacteria and Lactobacillus-Enterococcus group levels were higher in the groups fed infant formula supplemented with polyamines, resulting in even higher numbers of bacteria than in the breastfed pups. Our findings indicate that infant formulas enriched with polyamines may interact with gut microbiota, suggesting that further studies in human infants are required to assess the impact of polyamines on both growth and microbiota levels.