Identification of four genes responsible for antimicrobial resistance of MEL-B against S. aureus.

There is increasing interest in the antimicrobial activity of mannosylerythritol lipids-B (MEL-B) against Gram-positive bacteria such as Staphylococcus aureus (S. aureus). However, the specific molecules involved in MEL-B's antimicrobial action against S. aureus have not been identified. This study utilized the Nebraska transposon mutant library (NTML), which contains 1920 mutants, each lacking three-quarters of the genes found in S. aureus. The NTML was screened to identify mutants resistant to MEL-B. Four mutants (Accession Number: SAUSA300_0904, SAUSA300_0752, SAUSA300_0387, and SAUSA300_2311) largely unaffected by incubation with MEL-B, indicating MEL-B resistance. Despite the strong binding of MEL-B to these mutants, the four molecules encoded by the deleted genes (yjbI, clpP, pbuX, or brpS) in each mutant were not directly recognized by MEL-B. Given that these molecules are not localized on the outer surface of S. aureus and that the antibacterial activity of MEL-B against S. aureus is facilitated by the effective transfer of two antibacterial fatty acids (caprylic acid and myristoleic acid) to S. aureus via ME, the deletion of each of the four molecules may alter the peptidoglycan structure, potentially inhibiting the effective transfer of these antimicrobial fatty acids into S. aureus.

The impact of high-IgE levels on metabolome and microbiome in experimental allergic enteritis.

BACKGROUND: The pathological mechanism of the gastrointestinal forms of food allergies is less understood in comparison to other clinical phenotypes, such as asthma and anaphylaxis Importantly, high-IgE levels are a poor prognostic factor in gastrointestinal allergies. METHODS: This study investigated how high-IgE levels influence the development of intestinal inflammation and the metabolome in allergic enteritis (AE), using IgE knock-in (IgEki) mice expressing high levels of IgE. In addition, correlation of the altered metabolome with gut microbiome was analysed. RESULTS: Ovalbumin-sensitized and egg-white diet-fed (OVA/EW) BALB/c WT mice developed moderate AE, whereas OVA/EW IgEki mice induced more aggravated intestinal inflammation with enhanced eosinophil accumulation. Untargeted metabolomics detected the increased levels of N-tau-methylhistamine and 2,3-butanediol, and reduced levels of butyric acid in faeces and/or sera of OVA/EW IgEki mice, which was accompanied with reduced Clostridium and increased Lactobacillus at the genus level. Non-sensitized and egg-white diet-fed (NC/EW) WT mice did not exhibit any signs of AE, whereas NC/EW IgEki mice developed marginal degrees of AE. Compared to NC/EW WT mice, enhanced levels of lysophospholipids, sphinganine and sphingosine were detected in serum and faecal samples of NC/EW IgEki mice. In addition, several associations of altered metabolome with gut microbiome-for example Akkermansia with lysophosphatidylserine-were detected. CONCLUSIONS: Our results suggest that high-IgE levels alter intestinal and systemic levels of endogenous and microbiota-associated metabolites in experimental AE. This study contributes to deepening the knowledge of molecular mechanisms for the development of AE and provides clues to advance diagnostic and therapeutic strategies of allergic diseases.

Genetic aspects of immunoglobulins and cyclophilin A in milk as potential indicators of mastitis resistance in Holstein cows.

Mastitis is one of the most frequent and costly diseases affecting dairy cattle. Natural antibodies (immunoglobulins) and cyclophilin A (CyPA), the most abundant member of the family of peptidyl prolyl cis/trans isomerases, in milk may serve as indicators of mastitis resistance in dairy cattle. However, genetic information for CyPA is not available, and knowledge on the genetic and nongenetic relationships between these immune-related traits and somatic cell score (SCS) and milk yield in dairy cattle is sparse. Therefore, we aimed to comprehensively evaluate whether immune-related traits consisting of 5 Ig classes (IgG, IgG1, IgG2, IgA, and IgM) and CyPA in the test-day milk of Holstein cows can be used as genetic indicators of mastitis resistance by evaluating the genetic and nongenetic relationships with SCS in milk. The nongenetic factors affecting immune-related traits and the effects of these traits on SCS were evaluated. Furthermore, the genetic parameters of immune-related traits according to health status and genetic relationships under different SCS environments were estimated. All immune-related traits were significantly associated with SCS and directly proportional. Additionally, evaluation using a classification tree revealed that IgA, IgG2, and IgG were associated with SCS levels. Genetic factor analyses indicated that heritability estimates were low for CyPA (0.08) but moderate for IgG (0.37), IgA (0.44), and IgM (0.44), with positive genetic correlations among Ig (0.25-0.96). We also evaluated the differences in milk yield and SCS of cows between the low and high groups according to their sires' estimated breeding value for immune-related traits. In the high group, IgA had a significantly lower SCS in milk at 7 to 30 d compared with that in the low group. Furthermore, the Ig in milk had high positive genetic correlations between healthy and infected conditions (0.82-0.99), suggesting that Ig in milk under healthy conditions could interact with those under infected conditions, owing to the genetic ability based on the level of Ig in milk. Thus, Ig in milk are potential indicators for the genetic selection of mastitis resistance. However, because only the relationship between immune-related traits and SCS was investigated in this study, further study on the relationship between clinical mastitis and Ig in milk is needed before Ig can be used as an indicator of mastitis resistance.

Roles of mannosylerythritol lipid-B components in antimicrobial activity against bovine mastitis-causing Staphylococcus aureus.

Mannosylerythritol lipid-B (MEL-B), which comprises ester-bonded hydrophilic ME and hydrophobic fatty acids, is a bio-surfactant with various unique properties, including antimicrobial activity against most gram-positive bacteria. The gram-positive Staphylococcus aureus is a causative pathogen of dairy cattle mastitis, which results in considerable economic loss in the dairy industry. Here, we demonstrate the efficacy of MEL-B as a disinfectant against bovine-derived S. aureus and elucidate a mechanism of action of MEL-B in the inhibition of bacterial growth. The growth of bovine mastitis causative S. aureus BM1006 was inhibited when cultured with MEL-B above 10 ppm. The activity of MEL-B required fatty acids (i.e., caprylic and myristoleic acids) as ME, the component of MEL-B lacking fatty acids, did not inhibit the growth of S. aureus even at high concentrations. Importantly, ME-bound fatty acids effectively inhibited the growth of S. aureus when compared with free fatty acids. Specifically, the concentrations of ME-bound fatty acids and free caprylic and myristoleic acids required to inhibit the growth of S. aureus were 10, 1442, and 226 ppm, respectively. The involvement of ME in the antimicrobial activity of MEL-B was confirmed by digestion of MEL-B with alkali, which dissociated ME and fatty acids. These results indicated that a mechanism of action of MEL-B in inhibiting the growth of S. aureus could be explained by the effective transporting of antimicrobial fatty acids to the bacterial surface via hydrophilic ME.

In vivo emergence of beige-like fat in chickens as physiological adaptation to cold environments.

While it has been hypothesized that brown adipocytes responsible for mammalian thermogenesis are absent in birds, the existence of beige fat has yet to be studied directly. The present study tests the hypothesis that beige fat emerges in birds as a mechanism of physiological adaptation to cold environments. Subcutaneous neck adipose tissue from cold-acclimated or triiodothyronine (T3)-treated chickens exhibited increases in the expression of avian uncoupling protein (avUCP, an ortholog of mammalian UCP2 and UCP3) gene and some known mammalian beige adipocyte-specific markers. Morphological characteristics of white adipose tissues of treated chickens showed increased numbers of both small and larger clusters of multilocular fat cells within the tissues. Increases in protein levels of avUCP and mitochondrial marker protein, voltage-dependent anion channel, and immunohistochemical analysis for subcutaneous neck fat revealed the presence of potentially thermogenic mitochondria-rich cells. This is the first evidence that the capacity for thermogenesis may be acquired by differentiating adipose tissue into beige-like fat for maintaining temperature homeostasis in the subcutaneous fat 'neck warmer' in chickens exposed to a cold environment.

Interorgan Metabolism, Nutritional Impacts, and Safety of Dietary L-Glutamate and L-Glutamine in Poultry.

L-glutamine (Gln) is the most abundant amino acid (AA) in the plasma and skeletal muscle of poultry, and L-glutamate (Glu) is among the most abundant AAs in the whole bodies of all avian tissues. During the first-pass through the small intestine into the portal circulation, dietary Glu is extensively oxidized to CO2, but dietary Gln undergoes limited catabolism in birds. Their extra-intestinal tissues (e.g., skeletal muscle, kidneys, and lymphoid organs) have a high capacity to degrade Gln. To maintain Glu and Gln homeostasis in the body, they are actively synthesized from branched-chain AAs (abundant AAs in both plant and animal proteins) and glucose via interorgan metabolism involving primarily the skeletal muscle, heart, adipose tissue, and brain. In addition, ammonia (produced from the general catabolism of AAs) and α-ketoglutarate (α-KG, derived primarily from glucose) serve as substrates for the synthesis of Glu and Gln in avian tissues, particularly the liver. Over the past 20 years, there has been growing interest in Glu and Gln metabolism in the chicken, which is an agriculturally important species and also a useful model for studying some aspects of human physiology and diseases. Increasing evidence shows that the adequate supply of dietary Glu and Gln is crucial for the optimum growth, anti-oxidative responses, productivity, and health of chickens, ducklings, turkeys, and laying fowl, particularly under stress conditions. Like mammals, poultry have dietary requirements for both Glu and Gln. Based on feed intake, tissue integrity, growth performance, and health status, birds can tolerate up to 12% Glu and 3.5% Gln in diets (on the dry matter basis). Glu and Gln are quantitatively major nutrients for chickens and other avian species to support their maximum growth, production, and feed efficiency, as well as their optimum health and well-being.

Gut microbiota development in mice is affected by hydrogen peroxide produced from amino acid metabolism during lactation.

The development of gut microbiota during infancy is an important event that affects the health status of the host; however, the mechanism governing it is not fully understood. l-Amino acid oxidase 1 (LAO1) is a flavoprotein that catalyzes the oxidative deamination of particular l-amino acids and converts them into keto acids, ammonia, and H2O2. Our previous study showed that LAO1 is present in mouse milk and exerts protection against bacteria by its production of H2O2. The data led us to consider whether LAO1, H2O2, or both could impact infant gut microbiota development via mother's milk consumption in mice. Different gut microbiota profiles were observed in the wild-type (WT) and LAO1-knockout mouse pups. The WT pups' microbiota was relatively simple and composed of only a few dominant bacteria, such as Lactobacillus, whereas the lactating knockout pups had high microbiota diversity. Cross-fostering experiments indicated that WT milk (containing LAO1) has the ability to suppress the diversity of microbiota in pups. We observed that the stomach content of pups fed WT milk had LAO1 proteins and the ability to produce H2O2. Moreover, culture experiments showed that Lactobacillus was abundant in the feces of pups fed WT milk and that Lactobacillus was more resistant to H2O2 than Bifidobacterium and Escherichia. Human breast milk produces very little H2O2, which could be the reason for Lactobacillus not being dominant in the feces of breast-fed human infants. In mouse mother's milk, H2O2 is generated from the process of free amino acid metabolism, and H2O2 may be a key player in regulating the initial acquisition and development of gut microbiota, especially growth of Lactobacillus, during infancy.-Shigeno, Y., Zhang, H., Banno, T., Usuda, K., Nochi, T., Inoue, R., Watanabe, G., Jin, W., Benno, Y., Nagaoka, K. Gut microbiota development in mice is affected by hydrogen peroxide produced from amino acid metabolism during lactation.

Lipopolysaccharide (LPS)-binding protein stimulates CD14-dependent Toll-like receptor 4 internalization and LPS-induced TBK1-IKKϵ-IRF3 axis activation.

Toll-like receptor 4 (TLR4) is an indispensable immune receptor for lipopolysaccharide (LPS), a major component of the Gram-negative bacterial cell wall. Following LPS stimulation, TLR4 transmits the signal from the cell surface and becomes internalized in an endosome. However, the spatial regulation of TLR4 signaling is not fully understood. Here, we investigated the mechanisms of LPS-induced TLR4 internalization and clarified the roles of the extracellular LPS-binding molecules, LPS-binding protein (LBP), and glycerophosphatidylinositol-anchored protein (CD14). LPS stimulation of CD14-expressing cells induced TLR4 internalization in the presence of serum, and an inhibitory anti-LBP mAb blocked its internalization. Addition of LBP to serum-free cultures restored LPS-induced TLR4 internalization to comparable levels of serum. The secretory form of the CD14 (sCD14) induced internalization but required a much higher concentration than LBP. An inhibitory anti-sCD14 mAb was ineffective for serum-mediated internalization. LBP lacking the domain for LPS transfer to CD14 and a CD14 mutant with reduced LPS binding both attenuated TLR4 internalization. Accordingly, LBP is an essential serum molecule for TLR4 internalization, and its LPS transfer to membrane-anchored CD14 (mCD14) is a prerequisite. LBP induced the LPS-stimulated phosphorylation of TBK1, IKKϵ, and IRF3, leading to IFN-ß expression. However, LPS-stimulated late activation of NF-κB or necroptosis were not affected. Collectively, our results indicate that LBP controls LPS-induced TLR4 internalization, which induces TLR adaptor molecule 1 (TRIF)-dependent activation of the TBK1-IKKϵ-IRF3-IFN-ß pathway. In summary, we showed that LBP-mediated LPS transfer to mCD14 is required for serum-dependent TLR4 internalization and activation of the TRIF pathway.

Staphylococcus aureus-specific IgA antibody in milk suppresses the multiplication of S. aureus in infected bovine udder.

BACKGROUND: Bovine mastitis caused by Staphylococcus aureus (S. aureus) is extremely difficult to control and new methods for its prevention and management are required. Nasal vaccines may prevent initial bovine mastitis infection caused by S. aureus. However, limited information is available regarding induction of mucosal immune response through nasal immunization with antigen and its suppression of S. aureus multiplication during bovine mastitis. This study sought to investigate whether induction of immunoglobulin A (IgA) in milk by nasal immunization could suppress multiplication of S. aureus in the bovine udder. RESULTS: Nasal immunization with formalin-killed S. aureus conjugated with a cationic cholesteryl-group-bearing pullulan-nanogel was performed. Anti-S. aureus-specific IgA antibodies were significantly more abundant in the milk of immunized cows than in non-immunized animals (P < 0.05). S. aureus counts in the quarter were negative in both non-immunized and nasal-immunized cows 1 week after mock infusion. In S. aureus-infused quarters, S. aureus multiplication was significantly suppressed in immunized compared with non-immunized cows (P < 0.05). Furthermore, a significant negative correlation was found between S. aureus-specific IgA antibodies and S. aureus counts in infused quarters of both non-immunized and nasal-immunized cows (r = - 0.811, P < 0.01). CONCLUSION: In conclusion, the present study demonstrates that S. aureus-specific IgA antibodies in milk successfully suppressed the multiplication of S. aureus in infected bovine udders. Although the exact mechanism explaining such suppressive effect remains to be elucidated, nasal vaccines that can induce humoral immunity may help prevent initial infection with S. aureus and the onset of bovine mastitis.

Identification of a novel mechanism of action of bovine IgG antibodies specific for Staphylococcus aureus.

Staphylococcus aureus is a major pathogen that causes subclinical mastitis associated with huge economic losses to the dairy industry. A few vaccines for bovine mastitis are available, and they are expected to induce the production of S. aureus-specific antibodies that prevent bacterial adherence to host cells or promote opsonization by phagocytes. However, the efficacy of such vaccines are still under debate; therefore, further research focusing on improving the current vaccines by seeking additional mechanisms of action is required to reduce economic losses due to mastitis in the dairy industry. Here, we generated S. aureus-specific bovine IgG antibodies (anti-S. aureus) that directly inhibited bacterial growth in vitro. Inhibition depended on specificity for anti-S. aureus, not the interaction between Protein A and the fragment crystallizable region of the IgG antibodies or bacterial agglutination. An in vitro culture study using S. aureus strain JE2 and its deletion mutant JE2ΔSrtA, which lacks the gene encoding sortase A, revealed that the effect of anti-S. aureus was sortase-A-independent. Sortase A is involved in the synthesis of cell-wall-associated proteins. Thus, other surface molecules, such as membrane proteins, cell surface polysaccharides, or both, may trigger the inhibition of bacterial growth by anti-S. aureus. Together, our findings contribute insights into developing new strategies to further improve the available mastitis vaccine by designing a novel antigen on the surface of S. aureus to induce inhibitory signals that prevent bacterial growth.

Nanoformulations of Rilpivirine for Topical Pericoital and Systemic Coitus-Independent Administration Efficiently Prevent HIV Transmission.

Vaginal HIV transmission accounts for the majority of new infections worldwide. Currently, multiple efforts to prevent HIV transmission are based on pre-exposure prophylaxis with various antiretroviral drugs. Here, we describe two novel nanoformulations of the reverse transcriptase inhibitor rilpivirine for pericoital and coitus-independent HIV prevention. Topically applied rilpivirine, encapsulated in PLGA nanoparticles, was delivered in a thermosensitive gel, which becomes solid at body temperature. PLGA nanoparticles with encapsulated rilpivirine coated the reproductive tract and offered significant protection to BLT humanized mice from a vaginal high-dose HIV-1 challenge. A different nanosuspension of crystalline rilpivirine (RPV LA), administered intramuscularly, protected BLT mice from a single vaginal high-dose HIV-1 challenge one week after drug administration. Using transmitted/founder viruses, which were previously shown to establish de novo infection in humans, we demonstrated that RPV LA offers significant protection from two consecutive high-dose HIV-1 challenges one and four weeks after drug administration. In this experiment, we also showed that, in certain cases, even in the presence of drug, HIV infection could occur without overt or detectable systemic replication until levels of drug were reduced. We also showed that infection in the presence of drug can result in acquisition of multiple viruses after subsequent exposures. These observations have important implications for the implementation of long-acting antiretroviral formulations for HIV prevention. They provide first evidence that occult infections can occur, despite the presence of sustained levels of antiretroviral drugs. Together, our results demonstrate that topically- or systemically administered rilpivirine offers significant coitus-dependent or coitus-independent protection from HIV infection.

Immunoregulatory effects triggered by immunobiotic Lactobacillus jensenii TL2937 strain involve efficient phagocytosis in porcine antigen presenting cells.

BACKGROUND: Immunobiotic Lactobacillus jensenii TL2937 modulates porcine mononuclear phagocytes from Peyer's patches (PPMPs) and induces a differential production of pro- and anti-inflammatory cytokines in response to Toll-like receptor (TLR)-4 activation. In view of the important role played by phagocytosis in the activation of antigen presenting cells (APCs), the aim of the present work was to examine the interaction of TL2937 with porcine PPMPs focusing on phagocytosis. In addition, this study aimed to investigate whether the effects of L. jensenii TL2937 in porcine blood monocyte-derived dendritic cells (MoDCs) are similar to those found in PPMPs considering that MoDCs do not recapitulate all functions of mucosal APCs. RESULTS: Studies showed a high ability of porcine CD172a(+) PPMPs to phagocytose L. jensenii TL2937. Interestingly, our results also revealed a reduced capacity of the non-immunomodulatory L. plantarum TL2766 to be phagocytosed by those immune cells. Phagocytosis of L. jensenii TL2937 by porcine PPMPs was partially dependent on TLR2. In addition, we demonstrated that TL2937 strain was able to improve the expression of IL-1ß, IL-12 and IL-10 in immature MoDCs resembling the effect of this immunobiotic bacterium on PPMPs. Moreover, similarly to PPMPs those immunomodulatory effects were related to the higher capacity of TL2937 to be phagocytosed by immature MoDCs. CONCLUSIONS: Microbial recognition in APCs could be effectively mediated through ligand-receptor interactions that then mediate phagocytosis and signaling. For the immunobiotic strain TL2937, TLR2 has a partial role for its interaction with porcine APCs and it is necessary to investigate the role of other receptors. A challenge for future research will be advance in the full understanding of the molecular interactions of immunobiotic L. jensenii TL2937 with porcine APCs that will be crucial for the successful development of functional feeds for the porcine host. This study is a step in that direction.

Cyclophilin A is a new M cell marker of bovine intestinal epithelium.

Microfold (M) cells in the follicle-associated epithelium (FAE) of Peyer's patches contribute to the mucosal immune response by the transcytosis of microorganisms. The mechanism by which M cells take up microorganisms, and the functional proteins by which they do this, are not clear. In order to explore one such protein, we developed a 2H5-F3 monoclonal antibody (2H5-F3 mAb) through its binding to bovine M cells, and identified the antibody reactive molecule as cyclophilin A (Cyp-A). The localization patterns of Cyp-A were very similar to the localization pattern of cytokeratin (CK) 18-positive M cells. Cyp-A was identified at the luminal surface of CK18-positive M cells in bovine jejunal and ileal FAE. The membranous localization of Cyp-A in the bovine intestinal cell line (BIE cells) increased as cells differentiated toward M cells, as determined by flow cytometry analysis. Additionally, BIE cells released Cyp-A to the extracellular space and the differentiation of BIE cells to M cells increased the secretion of Cyp-A, as determined by western blotting. Accordingly, Cyp-A may be localized in M cells in the small intestinal epithelium of cattle. The rise of the membranous localization and secretion of Cyp-A by differentiation toward M cells indicates that Cyp-A has an important role in the function of M cells. While Cyp-A of the M cell membrane may contribute to the uptake of viruses with peptidyl-prolyl cis-trans isomerase activity, in the extracellular space Cyp-A may work as a chemokine and contribute to the distribution of immuno-competent cells.

NFIL3-deficient mice develop microbiota-dependent, IL-12/23-driven spontaneous colitis.

NFIL3 is a transcription factor that regulates multiple immunologic functions. In myeloid cells, NFIL3 is IL-10 inducible and has a key role as a repressor of IL-12p40 transcription. NFIL3 is a susceptibility gene for the human inflammatory bowel diseases. In this article, we describe spontaneous colitis in Nfil3(-/-) mice. Mice lacking both Nfil3 and Il10 had severe early-onset colitis, suggesting that NFIL3 and IL-10 independently regulate mucosal homeostasis. Lymphocytes were necessary for colitis, because Nfil3/Rag1 double-knockout mice were protected from disease. However, Nfil3/Rag1 double-knockout mice adoptively transferred with wild-type CD4(+) T cells developed severe colitis compared with Rag1(-/-) recipients, suggesting that colitis was linked to defects in innate immune cells. Colitis was abrogated in Nfil3/Il12b double-deficient mice, identifying Il12b dysregulation as a central pathogenic event. Finally, germ-free Nfil3(-/-) mice do not develop colonic inflammation. Thus, NFIL3 is a microbiota-dependent, IL-10-independent regulator of mucosal homeostasis via IL-12p40.

Vaginal memory T cells induced by intranasal vaccination are critical for protective T cell recruitment and prevention of genital HSV-2 disease.

UNLABELLED: Protective immunity against genital pathogens causing chronic infections, such as herpes simplex virus 2 (HSV-2) or human immunodeficiency virus, requires the induction of cell-mediated immune responses locally in the genital tract. Intranasal immunization with a thymidine kinase-deficient (TK(-)) mutant of HSV-2 effectively induces HSV-2-specific gamma interferon (IFN-γ)-secreting memory T cell production and protective immunity against intravaginal challenge with wild-type HSV-2. However, the precise mechanism by which intranasal immunization induces protective immunity in the distant genital mucosa more effectively than does systemic immunization is unknown. Here, we showed that intranasal immunization with live HSV-2 TK(-) induced the production of effector T cells and their migration to, and retention in, the vaginal mucosa, whereas systemic vaccination barely established a local effector T cell pool, even when it induced the production of circulating memory T cells in the systemic compartment. The long-lasting HSV-2-specific local effector T cells induced by intranasal vaccination provided superior protection against intravaginal wild-type HSV-2 challenge by starting viral clearance at the entry site earlier than with intraperitoneal immunization. Intranasal immunization is an effective strategy for eliciting high levels of cell-mediated protection of the genital tract by providing long-lasting antigen (Ag)-specific local effector T cells without introducing topical infection or inflammation. IMPORTANCE: Intranasal (i.n.) vaccines against sexually transmitted diseases that are caused by viruses such as herpes simplex virus 2 (HSV-2) have long been in development, but no vaccine candidate is currently available. Understanding the cellular mechanisms of immune responses in a distant vaginal mucosa induced by i.n. immunization with HSV-2 will contribute to designing such a vaccine. Our study demonstrated that i.n. immunization with an attenuated strain of HSV-2 generated long-lasting IFN-γ-secreting T cells in vaginal mucosa more effectively than systemic immunization. We found that these vaginal effector memory T cells are critical for the early stage of viral clearance at natural infection sites and prevent severe vaginal inflammation and herpes encephalitis.

Extracellular cyclophilin A possesses chemotaxic activity in cattle.

Cyclophilin A (CyPA) was originally discovered in bovine thymocytes as a cytosolic binding protein of the immunosuppressive drug cyclosporine A. Recent studies have revealed that in mice and humans, CyPA is secreted from cells in injured or infected tissues and plays a role in recruiting inflammatory cells in those tissues. Here we found that in cattle abundant level of extracellular CyPA was observed in tissues with inflammation. To aid in investigating the role of extracellular CyPA in cattle, we generated recombinant bovine CyPA (rbCyPA) and tested its biological activity as an inflammatory mediator. When bovine peripheral blood cells were treated with rbCyPA in vitro, we observed that rbCyPA reacts with the membranous surface of granulocytes, monocytes and lymphocytes. Chemotaxis analysis showed that the granulocytes migrate toward rbCyPA and the migration is inhibited by pre-treatment with an anti-bovine CyPA antibody. These results indicate that, as for mice and humans, extracellular CyPA possesses chemotactic activity to recruit inflammatory cells (e.g., granulocytes) in cattle, and could thus be a potential therapeutic target for the treatment of inflammation.

Uptake through glycoprotein 2 of FimH(+) bacteria by M cells initiates mucosal immune response.

The mucosal immune system forms the largest part of the entire immune system, containing about three-quarters of all lymphocytes and producing grams of secretory IgA daily to protect the mucosal surface from pathogens. To evoke the mucosal immune response, antigens on the mucosal surface must be transported across the epithelial barrier into organized lymphoid structures such as Peyer's patches. This function, called antigen transcytosis, is mediated by specialized epithelial M cells. The molecular mechanisms promoting this antigen uptake, however, are largely unknown. Here we report that glycoprotein 2 (GP2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for mucosal antigens. Recombinant GP2 protein selectively bound a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane. Consistently, these bacteria were colocalized with endogenous GP2 on the apical plasma membrane as well as in cytoplasmic vesicles in M cells. Moreover, deficiency of bacterial FimH or host GP2 led to defects in transcytosis of type-I-piliated bacteria through M cells, resulting in an attenuation of antigen-specific immune responses in Peyer's patches. GP2 is therefore a previously unrecognized transcytotic receptor on M cells for type-I-piliated bacteria and is a prerequisite for the mucosal immune response to these bacteria. Given that M cells are considered a promising target for oral vaccination against various infectious diseases, the GP2-dependent transcytotic pathway could provide a new target for the development of M-cell-targeted mucosal vaccines.

Human breast milk and antiretrovirals dramatically reduce oral HIV-1 transmission in BLT humanized mice.

Currently, over 15% of new HIV infections occur in children. Breastfeeding is a major contributor to HIV infections in infants. This represents a major paradox in the field because in vitro, breast milk has been shown to have a strong inhibitory effect on HIV infectivity. However, this inhibitory effect has never been demonstrated in vivo. Here, we address this important paradox using the first humanized mouse model of oral HIV transmission. We established that reconstitution of the oral cavity and upper gastrointestinal (GI) tract of humanized bone marrow/liver/thymus (BLT) mice with human leukocytes, including the human cell types important for mucosal HIV transmission (i.e. dendritic cells, macrophages and CD4⁺ T cells), renders them susceptible to oral transmission of cell-free and cell-associated HIV. Oral transmission of HIV resulted in systemic infection of lymphoid and non-lymphoid tissues that is characterized by the presence of HIV RNA in plasma and a gradual decline of CD4⁺ T cells in peripheral blood. Consistent with infection of the oral cavity, we observed virus shedding into saliva. We then evaluated the role of human breast milk on oral HIV transmission. Our in vivo results demonstrate that breast milk has a strong inhibitory effect on oral transmission of both cell-free and cell-associated HIV. Finally, we evaluated the effect of antiretrovirals on oral transmission of HIV. Our results show that systemic antiretrovirals administered prior to exposure can efficiently prevent oral HIV transmission in BLT mice.

Specific expression of apolipoprotein A-IV in the follicle-associated epithelium of the small intestine.

BACKGROUND: Peyer's patches (PPs), which are covered by specialized follicle-associated epithelium (FAE) including M cells, play a central role in immune induction in the gastrointestinal tract. This study is to investigate a new molecule to characterize PPs. METHODS: We generated a monoclonal antibody (mAb 10-15-3-3) that specifically reacts to the epithelium of PPs and isolated lymphoid follicles. Target antigen was analyzed by immunoprecipitation and mass spectrometry. Localization and expression of target antigen were evaluated by immunofluorescence, in situ hybridization and real-time PCR. RESULTS: Immunoprecipitation and mass spectrometry revealed that mAb 10-15-3-3 recognized apolipoprotein A-IV (ApoA-IV), a well-known lipid transporter; this finding was confirmed by the specific reactivity of mAb 10-15-3-3 to cells transfected with the murine ApoA-IV gene. Immunofluorescence using mAb 10-15-3-3 showed intestinal localization of ApoA-IV, in which strong expression of the ApoA-IV protein occurred throughout the entire intestinal epithelium during developing period before weaning but was restricted to the FAE in adult mice. In support of these findings, in situ hybridization showed strong expression of the ApoA-IV gene throughout the entire intestinal epithelium during developing period before weaning, but this expression was restricted to the FAE predominantly and the tips of villi to a lesser extent in adult mice. Deficiency of ApoA-IV had no effect on the organogenesis of PP in mice. CONCLUSIONS: Our current results reveal ApoA-IV as a novel FAE-specific marker especially in the upper small intestine of adult mice.