The stability of immunoglobulin a in human milk and saliva stored on filter paper at ambient temperature

Objectives: Immunoglobulin A dominates mucosal surfaces and is a biomarker of interest in populations with a high disease burden. The objectives of this work are to describe an ELISA for IgA and test the stability of storage on filter paper for human milk and saliva collection to be used in remote field locations. Methods: A two‐site sandwich ELISA for IgA was developed. To test filter paper storage capabilities under field conditions, 248 matched whole and dried human milk filter paper samples and 251 matched whole and dried saliva samples were collected from northern Kenyan women. Whole samples were frozen in liquid nitrogen while dried samples were stored at ambient temperature for up to 8 weeks. Recovered dried IgA levels were compared to whole IgA levels and adjusted for time stored at ambient temperature. Results: The lower limit of quantification for this assay is 10.1 ng/ml. Linearity of dilution for human milk and saliva samples was excellent. High and low‐control coefficient of variation values across plates were 9.1 (341.8 ng/ml) and 9.4% (132.5 ng/ml). IgA was detected in all whole and dried samples. There is a moderate concordance between dried and whole samples ( R 2 = 0.62). There is a small but significant effect of time stored, with a loss of ∼1 μg/ml per day ( P = 0.0052). Conclusions: This IgA assay is a cost‐effective alternative to commercial secretory IgA kits. Human milk and saliva can be stored on filter paper for up to 8 weeks. Am. J. Hum. Biol., 2011. © 2011 Wiley‐Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/87148/1/21218_ftp.pd

Menstrual Blood as a Potential Source of Endometrial Derived CD3+ T Cells

Studies of T cell-mediated immunity in the human female genital tract have been problematic due to difficulties associated with the collection of mucosal samples. Consequently, most studies rely on biopsies from the lower female genital tract or remnant tissue from hysterectomies. Availability of samples from healthy women is limited, as most studies are carried out in women with underlying pathologies. Menstruation is the cyclical sloughing off of endometrial tissue, and thus it should be a source of endometrial cells without the need for a biopsy. We isolated and phenotyped T cells from menstrual and peripheral blood and from endometrial biopsy-derived tissue from healthy women to determine the types of T cells present in this compartment. Our data demonstrated that T cells isolated from menstrual blood are a heterogeneous population of cells with markers reminiscent of blood and mucosal cells as well as unique phenotypes not represented in either compartment. T cells isolated from menstrual blood expressed increased levels of HLA-DR, αEβ7 and CXCR4 and reduced levels of CD62L relative to peripheral blood. Menstrual blood CD4+ T cells were enriched for cells expressing both CCR7 and CD45RA, markers identifying naïve T cells and were functional as determined by antigen-specific intracellular cytokine production assays. These data may open new avenues of investigation for cell mediated immune studies involving the female reproductive tract without the need for biopsies

Humoral Immune Response to Keyhole Limpet Haemocyanin, the Protein Carrier in Cancer Vaccines

Keyhole limpet haemocyanin (KLH) appears to be a promising protein carrier for tumor antigens in numerous cancer vaccine candidates. The humoral immune response to KLH was characterized at the single-cell level with ELISPOT combined with separations of cell populations according to their expression of homing receptors (HRs). The analysis of HR expressions is expected to reveal the targeting of the immune response in the body. Eight orally primed and four nonprimed volunteers received KLH-vaccine subcutaneously. Circulating KLH-specific plasmablasts were found in all volunteers, 60 KLH-specific plasmablasts/106 PBMC in the nonprimed and 136/106 in the primed group. The proportion of L-selectin+ plasmablasts proved high and integrin α4β7+ low. KLH serving as protein carrier in several vaccines, the homing profile of KLH-specific response may be applicable to the cancer antigen parts in the same vaccines. The present data reflect a systemic homing profile, which appears advantageous for the targeting of immune response to cancer vaccines

The N-Glycans Determine the Differential Blood Clearance and Hepatic Uptake of Human Immunoglobulin (Ig)a1 and Iga2 Isotypes

Human immunoglobulin (Ig)A exists in blood as two isotypes, IgA1 and IgA2, with IgA2 present as three allotypes: IgA2m(1), IgA2m(2), and IgA2m(n). We now demonstrate that recombinant, chimeric IgA1 and IgA2 differ in their pharmacokinetic properties. The major pathway for the clearance of all IgA2 allotypes is the liver. Liver-mediated uptake is through the asialoglycoprotein receptor (ASGR), since clearance can be blocked by injection of excess galactose-Ficoll ligand and suppressed in ASGR-deficient mice. In contrast, only a small percentage of IgA1 is cleared through this pathway. The clearance of IgA1 lacking the hinge region with its associated O-linked carbohydrate was more rapid than that of wild-type IgA1. IgA1 and IgA2 that are not rapidly eliminated by the ASGR are both removed through an undefined ASGR-independent pathway with half-lives of 14 and 10 h, respectively. The rapid clearance of IgA2 but not IgA1 through the liver may in part explain why the serum levels of IgA1 are greater than those of IgA2. In addition, dysfunction of the ASGR or altered N-linked glycosylation, but not O-glycans, that affects recognition by this receptor may account for the elevated serum IgA seen in liver disease and IgA nephropathy

Plasma-Derived Polyreactive Secretory-Like IgA and IgM Opsonizing Salmonella enterica Typhimurium Reduces Invasion and Gut Tissue Inflammation through Agglutination.

Due to the increasing emergence of antibiotic-resistant strains of enteropathogenic bacteria, development of alternative treatments to fight against gut infections is a major health issue. While vaccination requires that a proper combination of antigen, adjuvant, and delivery route is defined to elicit protective immunity at mucosae, oral delivery of directly active antibody preparations, referred to as passive immunization, sounds like a valuable alternative. Along the gut, the strategy suffers, however, from the difficulty to obtain sufficient amounts of antibodies with the appropriate specificity and molecular structure for mucosal delivery. Physiologically, at the antibody level, the protection of gastrointestinal mucosal surfaces against enteropathogens is principally mediated by secretory IgA and secretory IgM. We previously demonstrated that purified human plasma-derived IgA and IgM can be associated with secretory component to generate biologically active secretory-like IgA and IgM (SCIgA/M) that can protect epithelial cells from infection by Shigella flexneri in vitro. In this study, we aimed at evaluating the protective potential of these antibody preparations in vivo. We now establish that such polyreactive preparations bind efficiently to Salmonella enterica Typhimurium and trigger bacterial agglutination, as observed by laser scanning confocal microscopy. Upon delivery into a mouse ligated intestinal loop, SCIgA/M-mediated aggregates persist in the intestinal environment and limit the entry of bacteria into intestinal Peyer's patches via immune exclusion. Moreover, oral administration to mice of immune complexes composed of S. Typhimurium and SCIgA/M reduces mucosal infection, systemic dissemination, and local inflammation. Altogether, our data provide valuable clues for the future appraisal of passive oral administration of polyreactive plasma-derived SCIgA/M to combat infection by a variety of enteropathogens

Surface expression of secretory component and HLA class II DR antigen on glandular epithelial cells from human endometrium and two endometrial adenocarcinoma cell lines

The expression of secretory component (SC) by human glandular endometrial cells cultured in vitro was significantly increased by estradiol in the medium. Interferon-gamma and interleukin-4 stimulated the expression of SC only in the presence of estrogen. Tumor necrosis factor-alpha plus estrogen also caused a significant increase in the number of cells expressing SC. HLA class II antigen DR was detected on few glandular epithelial cells of human endometrium cultured in control medium, whereas interferon-gamma and interleukin-4, but not tumor necrosis factor-alpha, caused significant increases in the expression of DR. Estrogen in the culture medium did not significantly affect DR expression. The human endometrial adenocarcinoma cell lines, HEC and RL-95, expressed SC in approximately 50 and 20% of the cells. Also, approximately 20% of the RL-95 cells stained for DR antigen. Interferon-gamma did not influence the degree of expression of either surface marker of the two cell lines. Cells of both lines bound polymeric IgA and IgM but showed little to no binding of monomeric IgA, IgG, or an IgM previously shown not to bind SC.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44844/1/10875_2004_Article_BF00919384.pd

Differential glycosylation of envelope gp120 is associated with differential recognition of HIV-1 by virus-specific antibodies and cell infection

Background: HIV-1 entry into host cells is mediated by interactions between the virus envelope glycoprotein (gp120/gp41) and host-cell receptors. N-glycans represent approximately 50% of the molecular mass of gp120 and serve as potential antigenic determinants and/or as a shield against immune recognition. We previously reported that N-glycosylation of recombinant gp120 varied, depending on the producer cells, and the glycosylation variability affected gp120 recognition by serum antibodies from persons infected with HIV-1 subtype B. However, the impact of gp120 differential glycosylation on recognition by broadly neutralizing monoclonal antibodies or by polyclonal antibodies of individuals infected with other HIV-1 subtypes is unknown. Methods: Recombinant multimerizing gp120 antigens were expressed in different cells, HEK 293T, T-cell, rhabdomyosarcoma, hepatocellular carcinoma, and Chinese hamster ovary cell lines. Binding of broadly neutralizing monoclonal antibodies and polyclonal antibodies from sera of subtype A/C HIV-1-infected subjects with individual gp120 glycoforms was assessed by ELISA. In addition, immunodetection was performed using Western and dot blot assays. Recombinant gp120 glycoforms were tested for inhibition of infection of reporter cells by SF162 and YU.2 Env-pseudotyped R5 viruses. Results: We demonstrated, using ELISA, that gp120 glycans sterically adjacent to the V3 loop only moderately contribute to differential recognition of a short apex motif GPGRA and GPGR by monoclonal antibodies F425 B4e8 and 447-52D, respectively. The binding of antibodies recognizing longer peptide motifs overlapping with GPGR epitope (268 D4, 257 D4, 19b) was significantly altered. Recognition of gp120 glycoforms by monoclonal antibodies specific for other than V3-loop epitopes was significantly affected by cell types used for gp120 expression. These epitopes included CD4-binding site (VRC03, VRC01, b12), discontinuous epitope involving V1/V2 loop with the associated glycans (PG9, PG16), and an epitope including V3-base-, N332 oligomannose-, and surrounding glycans-containing epitope (PGT 121). Moreover, the different gp120 glycoforms variably inhibited HIV-1 infection of reporter cells. Conclusion: Our data support the hypothesis that the glycosylation machinery of different cells shapes gp120 glycosylation and, consequently, impacts envelope recognition by specific antibodies as well as the interaction of HIV-1 gp120 with cellular receptors. These findings underscore the importance of selection of appropriately glycosylated HIV-1 envelope as a vaccine antigen