A Direct MS-Based Approach to Profile Human Milk Secretory Immunoglobulin A (IgA1) Reveals Donor-Specific Clonal Repertoires With High Longitudinal Stability

Recently, a mass spectrometry-based approach was introduced to directly assess the IgG1 immunoglobulin clonal repertoires in plasma. Here we expanded upon this approach by describing a mass spectrometry-based technique to assess specifically the clonal repertoire of another important class of immunoglobulin molecules, IgA1, and show it is efficiently and robustly applicable to either milk or plasma samples. Focusing on two individual healthy donors, whose milk was sampled longitudinally during the first 16 weeks of lactation, we demonstrate that the total repertoire of milk sIgA1 is dominated by only 50-500 clones, even though the human body theoretically can generate several orders of magnitude more clones. We show that in each donor the sIgA1 repertoire only changes marginally and quite gradually over the monitored 16-week period of lactation. Furthermore, the observed overlap in clonal repertoires between the two individual donors is close to non-existent. Mothers provide protection to their newborn infants directly by the transfer of antibodies via breastfeeding. The approach introduced here, can be used to visualize the clonal repertoire transferred from mother to infant and to detect changes in-time in that repertoire adapting to changes in maternal physiology.

Induced Polyspecificity of Human Secretory Immunoglobulin A Antibodies: Is It Possible to Improve Their Ability to Bind Pathogens?

<b><i>Introduction:</i></b> As has been shown previously, various protein-modifying agents can change the antigen-binding properties of immunoglobulins. However, induced polyspecificity of human secretory immunoglobulin A (sIgA) has not been previously characterized in detail. <b><i>Methods:</i></b> In the present study, human secretory immunoglobulin A (IgA) was exposed to buffers with acidic pH, to free heme, or to pro-oxidative ferrous ions, and the antigen-binding behavior of the native and modified IgA to viral and bacterial antigens was compared using Western blotting and enzyme-linked immunosorbent assay. The ability of these agents to modulate the antigen-binding properties of human sIgA toward a wide range of pathogen peptides was investigated using an epitope microarray. <b><i>Results:</i></b> We have shown that acidic pH, heme, and pro-oxidative ferrous ions influenced the binding of secretory IgA in opposite directions (either increasing or decreasing); however, the strongest effect was observed when using buffers with low pH. This fraction had the highest number of affected reactivities; most of them were increased and most of the new ones were toward common pathogens. <b><i>Conclusions:</i></b> Thus, it was shown that all investigated treatments can alter to some degree the antigen-binding of secretory IgA, but acidic pH has the most potentially beneficial effect by increasing binding to a largest number of common pathogens’ antigens.

Exogenous and Endogenous Triggers Differentially Stimulate Pigr Expression and Antibacterial Secretory Immunity in the Murine Respiratory Tract

Purpose Transport of secretory immunoglobulin A (SIgA) through the airway epithelial cell barrier into the mucosal lumen by the polymeric immunoglobulin receptor (pIgR) is an important mechanism of respiratory mucosal host defense. Identification of immunomodulating substances that regulate secretory immunity might have therapeutic implications with regard to an improved immune exclusion. Thus, we sought to analyze secretory immunity under homeostatic and immunomodulating conditions in different compartments of the murine upper and lower respiratory tract (URT&LRT). Methods Pigr gene expression in lung, trachea, and nasal-associated lymphoid tissue (NALT) of germ-free mice, specific pathogen-free mice, mice with an undefined microbiome, as well as LPS- and IFN-γ-treated mice was determined by quantitative real-time PCR. IgA levels in bronchoalveolar lavage (BAL), nasal lavage (NAL), and serum were determined by ELISA. LPS- and IFN-γ-treated mice were colonized with Streptococcus pneumoniae and bacterial CFUs were determined in URT and LRT. Results Respiratory Pigr expression and IgA levels were dependent on the degree of exposure to environmental microbial stimuli. While immunostimulation with LPS and IFN-γ differentially impacts respiratory Pigr expression and IgA in URT vs. LRT, only prophylactic IFN-γ treatment reduces nasal colonization with S. pneumoniae. Conclusion Airway-associated secretory immunity can be partly modulated by exposure to microbial ligands and proinflammatory stimuli. Prophylactic IFN-γ-treatment modestly improves antibacterial immunity in the URT, but this does not appear to be mediated by SIgA or pIgR.

Thawing rate and heating temperature effects on immunoglobulin A and lysozyme activity in human milk

BackgroundThe rate of infants receiving frozen HM is increasing, allowing critically ill preterm infants and infants with working mothers to benefit from the advantages of their mother's milk. The effects of thawing and warming on secretory immunoglobulin A (SIgA) and lysozyme activity in frozen human milk (HM) should be investigated to identify optimal methods for preserving immune factors in frozen HM.MethodsForty mothers that delivered full-term infants provided milk that was frozen and stored at -18°C two months before analyses. We compared the methods involving placing the container in a 4°C refrigerator overnight (slow thawing, ST) and placing it in a container of warm water (rapid thawing, RT). Additionally, we investigated the effect of warming temperature using room temperature (25°C) and physiological temperature (37°C). SIgA concentrations and lysozyme activities in the milk samples were determined by ELISA kits and fluorometric lysozyme activity assay kits, respectively. Data were analyzed by paired t-tests.ResultsSIgA concentrations and lysozyme activity were reduced by 16.5-52.1% and 16.8-39.3% in frozen HM compared to fresh HM, respectively. Significantly higher SIgA concentrations were maintained with slow thawing and warming at 37°C than with rapid thawing and warming at 25°C (p <0.001). Greater lysozyme activity was retained at 25°C with slow thawing than with rapid thawing (p <0.001) and more was preserved at 25°C than at 37°C with slow thawing (p <0.01).ConclusionsThawing HM overnight in the refrigerator before warming has the potential to preserve SIgA levels and lysozyme activity better than thawing immediately after removal from the freezer. Broader temperatures ranges should be analyzed to determine the temperature that minimizes HM SIgA and lysozyme activity losses.Trial registrationNot applicable

THE USE OF IMMUNOLOGICAL INDICATORS IN ORDER TO FORM AN IMMUNOCOMPROMISED GROUP FOR VACCINATION AGAINST PNEUMOCOCCAL INFECTION

Pneumococci (Streptococcus pneumoniae) are one of the leading causes of morbidity and mortality among people over 60 years of age and workers in some professional groups. According to the medical literature, the frequency of invasive forms of pneumococcal infection among people of working age is 3.8 per 100,000 population. Increased susceptibility to colonization of the respiratory tract and subsequent morbidity may be due to concomitant pathology, exposure to immunocompromising, including harmful production factors. It should be noted that the source of the pathogen is not only sick people, but also healthy carriers. The level of asymptomatic colonization in the adult population is 5-7%, and in families with children increases to 30%. Vaccination is a way to effectively prevent respiratory diseases caused by this infection. The purpose of our study is to substantiate immunological indications for the formation of immunocompromised groups among workers exposed to the aerogenic factor at work for subsequent vaccination against pneumococcal infection. Results: It has been shown that low bactericidal activity of neutrophils (NBT-test) and a high level of secretory immunoglobulin can be used as a marker of immunodeficiency in workers of a ferrous metallurgy enterprise. When a doctor assesses the immune status of workers, he needs to take into account the presence of diseases that are part of the groups of immunological syndrome complexes (infectious-inflammatory, autoimmune, allergic, immunoproliferative) and the composition of industrial aerosols