Breast milk is the primary source of nutrition and hydration for the newborn infant but also plays an important role in the child’s first immune defense. Additionally, several breast milk factors have been implicated in immune-related health outcomes later in life, including immunoglobulins, cytokines, chemokines, growth factors and, more recently, non-coding RNA (ncRNA) species. In this systematic review, we provide a comprehensive summary of the current literature on endogenous ncRNAs found in human breast milk. Thirty (30) relevant studies were identified and, whilst the majority studies focused on microRNAs (miRNAs), there is evidence that breast milk contains high quantities of RNA which also include long-coding RNAs, circular RNAs, as well as other short RNAs and fragmented tRNA and rRNAs. Among studies investigating miRNAs, miR-148a-3p, miR-30a/d-5p, miR-22-3p, miR-146b-5p, miR-200a/c-3p, and the 5p end of the let-7 miRNAs were commonly reported among the top 10 miRNAs in the cell, lipid, and skim milk fractions of breast milk. Methodological difference and small sample sizes limit the possibility of conclusively identifying which maternal and infant characteristics affect the miRNA profile. The highly expressed miRNAs were generally reported to be similar across lactational stage, milk fraction, maternal and infant characteristics, or infant growth and health. All the same, individual studies identify potential differences in miRNA expression levels which should be confirmed by future studies. Stability, uptake, and physiological functions of miRNAs were also considered in several studies. Breast milk miRNAs are relatively resistant to a range of harsh conditions and uptake experiments suggest that extracellular vesicles containing miRNAs and circular RNAs can be taken up by intestinal epithelial cells. Although the evidence regarding the functional effect of breast milk miRNAs is limited, the predicted functions range from metabolic and biosynthetic processes to signaling pathways, cellular adhesion, communication, growth, and differentiation. Finally, this systematic review highlights some of the methodological challenges and knowledge gaps which can help direct future research in this field. In particular, it is important to further investigate the bioavailability of miRNAs in different milk fractions, and to characterize other ncRNAs which are largely unstudied.Systematic Review RegistrationPROSPERO https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=138989, identifier CRD42020138989.

Cyclophosphamide (CPA) is a chemotherapeutic agent that is primarily activated in the liver by cytochrome P4502B6 (CYP2B6) and then transported to the tumor via blood flow. To prevent deleterious secondary effects, P450-based gene-directed enzyme prodrug therapy (GDEPT) consists of expressing CYP2B6 in tumor cells before CPA treatment. Given the relatively low affinity of CYP2B6 for CPA, the aim of our work was to modify CYP2B6 to increase its catalytic efficiency (V max / K m ) to metabolize CPA into 4Ј-OH CPA. A molecular model of CYP2B6 was built, and four residues in close contact with the substrate were subjected to mutagenesis. Canine CYP2B11 exhibiting a particularly low K m to CPA, the amino acids exclusively present in the CYP2B11 substrate recognition sequences were substituted in human CYP2B6. All mutants (n ϭ 26) were expressed in Saccharomyces cerevisiae and their enzymatic constants (K m , V max ) evaluated using CPA as substrate. Five mutants exhibited a 2-to 3-fold higher catalytic efficiency than wild-type CYP2B6. A double mutant, comprising the two most effective mutations, showed a 4-fold increase in K m /V max . Molecular dynamic simulations of several mutants were found to be consistent with the observed modifications in catalytic efficiency. Finally, expression of the CYP2B6 114V/477W double mutant, contrary to wt CYP2B6, allowed switching of a resistant human head and neck cancer cell line (A-253) into a sensitive cell line toward CPA. Thus, we were able to obtain a new efficient CYP2B6 mutant able to metabolize CPA, an important step in the GDEPT strategy for human cancer treatment.

Introduction There is a need for a fast, efficient and safe way to induce tolerance in patients with severe allergic rhinitis. Intralymphatic immune therapy has been shown to be effective. Methods Patients with severe birch and timothy allergy were randomized and received three doses of 0.1 ml of birch and 5‐grass allergen extracts (10,000 SQ units/ml, ALK‐Abelló), or birch and placebo or 5‐grass and placebo by ultrasound‐guided injections into inguinal lymph nodes at monthly intervals. Rhinoconjunctivitis total symptom score, medication score and rhinoconjunctivitis quality of life questionnaire were evaluated before treatment and after each birch and grass pollen season during three subsequent years. Circulating proportions of T helper subsets and allergen‐induced cytokine and chemokine production were analysed by flow cytometry and Luminex. Results The three groups reported fewer symptoms, lower use of medication and improved quality of life during the birch and grass pollen seasons each year after treatment at an almost similar rate independently of treatment with one or two allergens. Mild local pain was the most common adverse event. IgE levels to birch decreased, whereas birch‐induced IL‐10 secretion increased in all three groups. IgG4 levels to birch and timothy and skin prick test reactivity remained mainly unchanged. Conjunctival challenge tests with timothy extract showed a higher threshold for allergen. In all three groups, regulatory T cell frequencies were increased 3 years after treatment. Conclusions Intralymphatic immunotherapy with one or two allergens in patients with grass and birch pollen allergy was safe, effective and may be associated with bystander immune modulatory responses. Clinical Trial Registration: EudraCT (2013‐004726‐28).

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Breast milk is an essential source of nutrition and hydration for the infant. In addition, this highly complex biological fluid contains numerous immunologically active factors such as microorganisms, immunoglobulins, cytokines and microRNAs (miRNAs). Here, we set out to predict the function of the top 10 expressed miRNAs in human breast milk, focusing on their relevance in oral tolerance development and allergy prevention in the infant. The top expressed miRNAs in human breast milk were identified on basis of previous peer-reviewed studies gathered from a recent systematic review and an updated literature search. The miRNAs with the highest expression levels in each study were used to identify the 10 most common miRNAs or miRNA families across studies and these were selected for subsequent target prediction. The predictions were performed using TargetScan in combination with the Database for Annotation, Visualization and Integrated Discovery. The ten top expressed miRNAs were: let-7-5p family, miR-148a-3p, miR-30-5p family, miR-200a-3p + miR-141-3p, miR-22-3p, miR-181-5p family, miR-146b-5p, miR-378a-3p, miR-29-3p family, miR-200b/c-3p and miR-429-3p. The target prediction identified 3,588 potential target genes and 127 Kyoto Encyclopedia of Genes and Genomes pathways; several connected to the immune system, including TGF-b and T cell receptor signaling and T-helper cell differentiation. This review highlights the role of breast milk miRNAs and their potential contribution to infant immune maturation. Indeed, breast milk miRNAs seem to be involved in several pathways that influence oral tolerance development.