Comparative Proteomics of Human and Macaque Milk Reveals Species-Specific Nutrition during Postnatal Development.

Milk has been well established as the optimal nutrition source for infants, yet there is still much to be understood about its molecular composition. Therefore, our objective was to develop and compare comprehensive milk proteomes for human and rhesus macaques to highlight differences in neonatal nutrition. We developed a milk proteomics technique that overcomes previous technical barriers including pervasive post-translational modifications and limited sample volume. We identified 1606 and 518 proteins in human and macaque milk, respectively. During analysis of detected protein orthologs, we identified 88 differentially abundant proteins. Of these, 93% exhibited increased abundance in human milk relative to macaque and include lactoferrin, polymeric immunoglobulin receptor, alpha-1 antichymotrypsin, vitamin D-binding protein, and haptocorrin. Furthermore, proteins more abundant in human milk compared with macaque are associated with development of the gastrointestinal tract, the immune system, and the brain. Overall, our novel proteomics method reveals the first comprehensive macaque milk proteome and 524 newly identified human milk proteins. The differentially abundant proteins observed are consistent with the perspective that human infants, compared with nonhuman primates, are born at a slightly earlier stage of somatic development and require additional support through higher quantities of specific proteins to nurture human infant maturation.

Heterologous expression and purification of biologically active domains 3 and 4 of human polymeric immunoglobulin receptor and its interaction with choline binding protein A of Streptococcus pneumoniae.

Streptococcus pneumoniae, one of the common causes of pneumonia, colonises the epithelium via the interaction between a choline binding protein of S. pneumoniae and the human polymeric immunoglobulin receptor (pIgR). One of the functions of pIgR is to mediate the transcytosis of polymeric immunoglobulins from the basolateral to the apical surface of epithelial cells. S. pneumoniae invades human epithelial cells by exploiting the transcytosis machinery. Due to an increase in the prevalence of antibiotic resistant strains of S. pneumoniae, and the limitations and expense of the vaccines available, extensive research may provide insights into the potential of new therapeutic regimes. This study investigated the potential of pIgR domains as an alternative non-antibiotic immune therapy for treating pneumonia. The aim was to determine the binding affinity of recombinant D3D4 protein, the domains of pIgR responsible for binding S. pneumoniae, to recombinant R1R2 repeat domains of choline binding protein A of S. pneumoniae. Biologically active recombinant D3D4 was produced in Escherichia coli using a gel filtration chromatography refolding method, a novel approach for the refolding of pIgR domains, after the purification of inclusion bodies using nickel affinity chromatography. Surface Plasmon resonance (SPR) spectroscopy showed that purified recombinant D3D4 binds recombinant R1R2 with an equilibrium dissociation constant (KD) of 3.36×10(-7)M.

Radiomarcaje y estudios de biodistribución de nanopartículas poliméricas como adyuvantes para la vacunación oftálmica frente a la brucelosis / Radiolabeling and biodistribution studies of polymeric nanoparticles as adjuvants for ocular vaccination against brucellosis

Objetivo. Optimizar el radiomarcaje con 99mTc de nanopartículas de Gantrez® manosiladas y cargadas con el antígeno de Brucella ovis (Man-NP-HS) y llevar a cabo estudios de biodistribución en un ratón tras la administración de las nanopartículas por vía ocular. Material y métodos. Las Man-NP-HS se obtuvieron por el método de desplazamiento del disolvente. Se purificaron, liofilizaron y caracterizaron. A continuación, se marcaron con 74MBq de 99mTcO4− previamente reducido con una disolución ácida de cloruro de estaño, trabajando en ausencia de oxígeno y con un pH final de 4. El rendimiento del marcaje se evaluó mediante TLC. Los estudios de biodistribución se llevaron a cabo en ratones tras la administración oftálmica de la formulación y de un control de 99mTcO4− libre. Para ello, se sacrificaron los animales a las 2 y a las 24h tras la administración ocular y se contaron los órganos en un contador gamma. Resultados. Se obtuvo un rendimiento de marcaje superior al 90%. Los estudios de biodistribución de 99mTc-Man-NP-HS permitieron detectar la actividad concentrada en la mucosa nasal y ocular y el tracto gastrointestinal tanto a las 2 como a las 24h frente a la biodistribución de 99mTcO4− libre que permaneció concentrado en la piel alrededor del ojo y en el tracto gastrointestinal. Conclusión. Los estudios de biodistribución de 99mTc-Man-NP-HS tras la administración oftálmica han permitido demostrar su biodistribución en las mucosas y el tracto gastrointestinal, característica indispensable como sistema de liberación de antígenos a través de la mucosa ocular. Esto, junto con su elevada respuesta inmune, efectiva protección y no virulencia, convierte a estas nanopartículas en una vacuna ideal antibrucelosis (AU)

Purpose. To optimize radiolabeling with 99mTc of mannosylated Gantrez® nanoparticles loaded with the Brucella Ovis antigen (Man-NP-HS) and to carry out biodistribution studies in mice after ocular administration of the nanoparticles. Material and methods. Man-NP-HS nanoparticles were prepared by the solvent displacement method. They were purified, lyophilized and characterized. Following this, they were radiolabeled with 74 MBq of 99mTcO4− previously reduced with an acidic stannous chloride solution, working in absence of oxygen and at a final pH of 4. Radiolabeling yield was evaluated by TLC. Biodistribution studies were carried out in mice after ocular administration of the formulation and control of free 99mTcO4−. To do so, the animals were humanely killed at 2 and 24hours after the ocular administration and activity in organs was measured in a Gamma counter. Results. Radiolabeling yield obtained was greater than 90%. Biodistribution studies of 99mTc-Man-NP-HS showed radioactivity accumulated at 2 and 24hours in nasal and ocular mucosa and gastrointestinal tract, in contrast to biodistribution of free 99mTcO4− that remained concentrated in the skin around the eye and gastrointestinal tract. Conclusion. Biodistribution studies of 99mTc-Man-NP-HS after ocular instillation have made it possible to demonstrate its biodistribution in nasal mucosa and gastrointestinal tract. This characteristic is essential as an antigenic delivery system throughout the ocular mucosa. This, together with its elevated immune response, effective protection and intrinsic avirulence make them a suitable anti-Brucella vaccine candidate (AU)

The IgA/IgM receptor expressed on a murine B cell lymphoma is poly-Ig receptor.

T560, a mouse B lymphoma that originated in gut-associated lymphoid tissue, expresses receptors that bind dimeric IgA and IgM in a mutually inhibitory manner but have little affinity for monomeric IgA. Evidence presented in this paper indicates that the receptor is poly-Ig receptor (pIgR) known in humans and domestic cattle to bind both IgA and IgM. The evidence includes the demonstration that binding of IgM is J chain dependent, and that pIg-precipitated receptor has an appropriate Mr of 116-120 kDa and can be detected on immunoblots with specific rabbit anti-mouse pIgR. Overlapping RT-PCR performed using template mRNA from T560 cells and oligonucleotide primer pairs designed from the published sequence of mouse liver pIgR indicate that T560 cells express mRNA virtually identical with that of the epithelial cell pIgR throughout its external, transmembrane, and intracytoplasmic coding regions. Studies using mutant IgAs suggest that the Calpha2 domain of dimeric IgA is not involved in high-affinity binding to the T560 pIgR. Inasmuch as this mouse B cell pIgR binds IgM better than IgA, it is similar to human pIgR and differs from rat, mouse, and rabbit epithelial cell pIgRs that bind IgA but not IgM. Possible explanations for this difference are discussed. All clones of T560 contain some cells that spontaneously secrete both IgG2a and IgA, but all of the IgA recoverable from the medium and from cell lysates is monomeric; it cannot be converted to secretory IgA by T560 cells.

Cellubrevin is present in the basolateral endocytic compartment of hepatocytes and follows the transcytotic pathway after IgA internalization.

The endocytic compartment of polarized cells is organized in basolateral and apical endosomes plus those endocytic structures specialized in recycling and transcytosis, which are still poorly characterized. The complexity of the various populations of endosomes has been demonstrated by the exquisite repertoire of endogenous proteins. In this study we examined the distribution of cellubrevin in the endocytic compartment of hepatocytes, since its intracellular location and function in polarized cells are largely unknown. Highly purified rat liver endosomes were isolated from estradiol-treated rats, and the early/sorting endosomal fraction was further subfractionated in a multistep sucrose density gradient, and studied. Analysis of dissected endosomal fractions showed that cellubrevin was located in early/sorting endosomes, with Rab4, annexins II and VI, and transferrin receptor, but in a specific subpopulation of these early endosomes with the same density range as pIgA and Raf-1. Interestingly, only in those isolated endosomal fractions, endosomes enriched in transcytotic structures (of livers loaded with IgA), the polymeric immunoglobulin receptor specifically co-immunoprecipitated with cellubrevin. In addition, confocal and immuno-electron microscopy identification of cellubrevin in tubular structures underneath the sinusoidal plasma membrane together with the re-organization of cellubrevin, in the endocytic compartment, after the IgA loading, strongly suggest the involvement of cellubrevin in the transcytosis of pIgA.

Secretory component, the receptor for polymeric immunoglobulin, has nothing to do with beta-galactosyltransferase in human milk.

Secretory component (SC) in external secretions is a soluble form of the polymeric immunoglobulin-receptor that is expressed on the cell membrane of mucosal epithelial cells. beta-(1-4)galactosyl transferase (beta-GT) is an enzyme that transfers galactose to non-reducing N-acetylglucosamine residues on various glycoproteins and is present in a soluble form in secretions as well as in a membrane-bound form. beta-GT is considered to have affinity for glycoproteins, including IgA in secretion. It has been claimed that these two proteins are related to or identical with each other. In the present study, we defined that the SC and the beta-GT are each independent molecules by the following facts; (1) both molecules are separable either by antibody-affinity chromatography, conventional ion-exchange or molecular exclusion chromatography, (2) conventionally purified SC from human milk contained neither enzymatic activity or antigenic determinants of the beta-GT, (3) recombinant beta-GT does not show reactivity with antibodies to SC, and (4) the SC showed no reactivity with antibody to beta-GT.

Calmodulin binds to the basolateral targeting signal of the polymeric immunoglobulin receptor.

We have identified a major calmodulin (CaM)-binding protein in rat liver endosomes using 125I-CaM overlays from two-dimensional protein blots. Immunostaining of blots demonstrates that this protein is the polymeric immunoglobulin receptor (pIgR). We further investigated the interaction between pIgR and CaM using Madin-Darby canine kidney cells stably expressing cloned wild-type and mutant pIgR. We found that detergent-solubilized pIgR binds to CaM-agarose in a Ca(2+)-dependent fashion, and binding is inhibited by the addition of excess free CaM or the CaM antagonist W-13 (N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide), suggesting that pIgR binding to CaM is specific. Furthermore, pIgR is the most prominent 35S-labeled CaM-binding protein in the detergent phase of Triton X-114-solubilized, metabolically labeled pIgR-expressing Madin-Darby canine kidney cells. CaM can be chemically cross-linked to both solubilized and membrane-associated pIgR, suggesting that binding can occur while the pIgR is in intact membranes. The CaM binding site is located in the membrane-proximal 17-amino acid segment of the pIgR cytoplasmic tail. This region of pIgR constitutes an autonomous basolateral targeting signal. However, binding of CaM to various pIgR mutants suggests that CaM binding is not necessary for basolateral targeting. We suggest that CaM may be involved in regulation of pIgR transcytosis and/or signaling by pIgR.