An Automated Approach to Assess Relative Galectin-Glycan Affinity Following Glycan Microarray Analysis.

Numerous studies have highlighted the utility of glycan microarray analysis for the elucidation of protein-glycan interactions. However, most current glycan microarray studies analyze glycan binding protein (GBP)-glycan interactions at a single protein concentration. While this approach provides useful information related to a GBP's overall binding capabilities, extrapolation of true glycan binding preferences using this method fails to account for printing variations or other factors that may confound relative binding. To overcome this limitation, we examined glycan array binding of three galectins over a range of concentrations to allow for a more complete assessment of binding preferences. This approach produced a richer data set than single concentration analysis and provided more accurate identification of true glycan binding preferences. However, while this approach can be highly informative, currently available data analysis approaches make it impractical to perform binding isotherms for each glycan present on currently available platforms following GBP evaluation. To overcome this limitation, we developed a method to directly optimize the efficiency of assessing association constants following multi-GBP concentration glycan array analysis. To this end, we developed programs that automatically analyze raw array data (kdMining) to generate output graphics (kaPlotting) following array analysis at multiple doses. These automatic programing methods reduced processing time from 32.8 h to 1.67 min. Taken together, these results demonstrate an effective approach to glycan array analysis that provides improved detail and efficiency when compared to previous methods.

Innate immune Galectin-7 specifically targets microbes that decorate themselves in blood group-like antigens.

Adaptive immunity can target a nearly infinite range of antigens, yet it is tempered by tolerogenic mechanisms that limit autoimmunity. Such immunological tolerance, however, creates a gap in adaptive immunity against microbes decorated with self-like antigens as a form of molecular mimicry. Our results demonstrate that the innate immune lectin galectin-7 (Gal-7) binds a variety of distinct microbes, all of which share features of blood group-like antigens. Gal-7 binding to each blood group expressing microbe, including strains of Escherichia coli, Klebsiella pneumoniae, Providencia alcalifaciens, and Streptococcus pneumoniae, results in loss of microbial viability. Although Gal-7 also binds red blood cells (RBCs), this interaction does not alter RBC membrane integrity. These results demonstrate that Gal-7 recognizes a diverse range of microbes, each of which use molecular mimicry while failing to induce host cell injury, and thus may provide an innate form of immunity against molecular mimicry.

Immune-mediated alopecias and their mechanobiological aspects.

Alopecia is a non-specific term for hair loss clinically diagnosed by the hair loss pattern and histological analysis of patient scalp biopsies. The immune-mediated alopecia subtypes, including alopecia areata, lichen planopilaris, frontal fibrosing alopecia, and central centrifugal cicatricial alopecia, are common, significant forms of alopecia subtypes. For example, alopecia areata is the most common autoimmune disease with a lifetime incidence of approximately 2% of the world's population. In this perspective, we discuss major results from studies of immune-mediated alopecia subtypes. These studies suggest the key event in disease onset as the collapse in immune privilege, which alters the hair follicle microenvironment, e.g., upregulation of major histocompatibility complex molecules and increase of cytokine production, and results in immune cell infiltration, inflammatory responses, and damage of hair follicles. We note that previous studies have established that the hair follicle has a complex mechanical microenvironment, which may regulate the function of not only tissue cells but also immune cell infiltrates. This suggests a potential for mechanobiology to contribute to alopecia research by adding new methods, new approaches, and new ways of thinking, which is missing in the existing literature. To fill this a gap in the alopecia research space, we develop a mechanobiological hypothesis that alterations in the hair follicle microenvironment, specifically in the mechanically responsive tissues and cells, partially due to loss of immune privilege, may be contributors to disease pathology. We further focus our discussion on the potential for applying mechanoimmunology to the study of T cell infiltrates in the hair follicle, as they are considered primary contributors to alopecia pathology. To establish the connection between the mechanoimmunological hypothesis and immune-mediated alopecia subtypes, we discuss what is known about the role of T cells in immune-mediated alopecia subtypes, using the most extensively studied AA as our model.

Evaluation of the Bactericidal Activity of Galectins.

Over a century ago, Karl Landsteiner discovered that blood group antigens could predict the immunological outcome of red blood cell transfusion. While the discovery of ABO(H) blood group antigens revolutionized transfusion medicine, many questions remain regarding the development and regulation of naturally occurring anti-blood group antibody formation. Early studies suggested that blood group antibodies develop following stimulation by bacteria that express blood group antigens. While this may explain the development of anti-blood group antibodies in blood group-negative individuals, how blood group-positive individuals protect themselves against blood group-positive microbes remained unknown. Recent studies suggest that several members of the galectin family specifically target blood group-positive microbes, thereby providing innate immune protection against blood group antigen-positive microbes regardless of the blood group status of an individual. Importantly, subsequent studies suggest that this unique form of immunity may not be limited to blood group expressing microbes, but may reflect a more generalized form of innate immunity against molecular mimicry. As this form of antimicrobial activity represents a unique and unprecedented form of immunity, we will examine important considerations and methodological approaches that can be used when seeking to ascertain the potential antimicrobial activity of various members of the galectin family.

Galectin-9 recognizes and exhibits antimicrobial activity toward microbes expressing blood group-like antigens.

While adaptive immunity recognizes a nearly infinite range of antigenic determinants, immune tolerance renders adaptive immunity vulnerable to microbes decorated in self-like antigens. Recent studies suggest that sugar-binding proteins galectin-4 and galectin-8 bind microbes expressing blood group antigens. However, the binding profile and potential antimicrobial activity of other galectins, particularly galectin-9 (Gal-9), has remained incompletely defined. Here, we demonstrate that while Gal-9 possesses strong binding preference for ABO(H) blood group antigens, each domain exhibits distinct binding patterns, with the C-terminal domain (Gal-9C) exhibiting higher binding to blood group B than the N-terminal domain (Gal-9N). Despite this binding preference, Gal-9 readily killed blood group B-positive Escherichia coli, whereas Gal-9N displayed higher killing activity against this microbe than Gal-9C. Utilization of microarrays populated with blood group O antigens from a diverse array of microbes revealed that Gal-9 can bind various microbial glycans, whereas Gal-9N and Gal-9C displayed distinct and overlapping binding preferences. Flow cytometric examination of intact microbes corroborated the microbial glycan microarray findings, demonstrating that Gal-9, Gal-9N, and Gal-9C also possess the capacity to recognize distinct strains of Providencia alcalifaciens and Klebsiella pneumoniae that express mammalian blood group-like antigens while failing to bind related strains that do not express mammalian-like glycans. In each case of microbial binding, Gal-9, Gal-9N, and Gal-9C induced microbial death. In contrast, while Gal-9, Gal-9N, and Gal-9C engaged red blood cells, each failed to induce hemolysis. These data suggest that Gal-9 recognition of distinct microbial strains may provide antimicrobial activity against molecular mimicry.

Full-Length Galectin-3 Is Required for High Affinity Microbial Interactions and Antimicrobial Activity.

While adaptive immunity enables the recognition of a wide range of microbial antigens, immunological tolerance limits reactively toward self to reduce autoimmunity. Some bacteria decorate themselves with self-like antigens as a form of molecular mimicry to limit recognition by adaptive immunity. Recent studies suggest that galectin-4 (Gal-4) and galectin-8 (Gal-8) may provide a unique form of innate immunity against molecular mimicry by specifically targeting microbes that decorate themselves in self-like antigens. However, the binding specificity and antimicrobial activity of many human galectins remain incompletely explored. In this study, we defined the binding specificity of galectin-3 (Gal-3), the first galectin shown to engage microbial glycans. Gal-3 exhibited high binding toward mammalian blood group A, B, and αGal antigens in a glycan microarray format. In the absence of the N-terminal domain, the C-terminal domain of Gal-3 (Gal-3C) alone exhibited a similar overall binding pattern, but failed to display the same level of binding for glycans over a range of concentrations. Similar to the recognition of mammalian glycans, Gal-3 and Gal-3C also specifically engaged distinct microbial glycans isolated and printed in a microarray format, with Gal-3 exhibiting higher binding at lower concentrations toward microbial glycans than Gal-3C. Importantly, Gal-3 and Gal-3C interactions on the microbial microarray accurately predicted actual interactions toward intact microbes, with Gal-3 and Gal-3C displaying carbohydrate-dependent binding toward distinct strains of Providentia alcalifaciens and Klebsiella pneumoniae that express mammalian-like antigens, while failing to recognize similar strains that express unrelated antigens. While both Gal-3 and Gal-3C recognized specific strains of P. alcalifaciens and K. pneumoniae, only Gal-3 was able to exhibit antimicrobial activity even when evaluated at higher concentrations. These results demonstrate that while Gal-3 and Gal-3C specifically engage distinct mammalian and microbial glycans, Gal-3C alone does not possess antimicrobial activity.

Leukemia Cutis With Histopathologic and Immunophenotypic Features Resembling S100-Negative CD1a-Positive Cutaneous Histiocytosis.

ABSTRACT: S100-negative CD1a-positive cutaneous histiocytosis is an exceedingly rare histiocytosis that is defined histopathologically by a dense dermal infiltrate of ovoid mononuclear cells with grooved nuclei and ample cytoplasm with variable nuclear atypia and mitoses that are immunohistochemically positive for CD1a and negative for S100 and CD207 (langerin). The histogenesis of S100-negative CD1a-positive histiocytosis is unclear, and its precursor cell has yet to be characterized. Although all cases thus far have been described as benign and occasionally self-resolving, the clinical course and outcome of this disease are not fully understood. This case expands the spectrum of disease associated with S100-negative CD1a-positive histiocytosis given its malignant course.

The Sweet-Side of Leukocytes: Galectins as Master Regulators of Neutrophil Function.

Among responders to microbial invasion, neutrophils represent one of the earliest and perhaps most important factors that contribute to initial host defense. Effective neutrophil immunity requires their rapid mobilization to the site of infection, which requires efficient extravasation, activation, chemotaxis, phagocytosis, and eventual killing of potential microbial pathogens. Following pathogen elimination, neutrophils must be eliminated to prevent additional host injury and subsequent exacerbation of the inflammatory response. Galectins, expressed in nearly every tissue and regulated by unique sensitivity to oxidative and proteolytic inactivation, appear to influence nearly every aspect of neutrophil function. In this review, we will examine the impact of galectins on neutrophils, with a particular focus on the unique biochemical traits that allow galectin family members to spatially and temporally regulate neutrophil function.

Anemia induces gut inflammation and injury in an animal model of preterm infants.

BACKGROUND: While very low birth weight (VLBW) infants often require multiple red blood cell transfusions, efforts to minimize transfusion-associated risks have resulted in more restrictive neonatal transfusion practices. However, whether restrictive transfusion strategies limit transfusions without increasing morbidity and mortality in this population remains unclear. Recent epidemiologic studies suggest that severe anemia may be an important risk factor for the development of necrotizing enterocolitis (NEC). However, the mechanism whereby anemia may lead to NEC remains unknown. STUDY DESIGN AND METHODS: The potential impact of anemia on neonatal inflammation and intestinal barrier disruption, two well-characterized predisposing features of NEC, was defined by correlation of hemoglobin values to cytokine levels in premature infants and by direct evaluation of intestinal hypoxia, inflammation and gut barrier disruption using a pre-clinical neonatal murine model of phlebotomy-induced anemia (PIA). RESULTS: Increasing severity of anemia in the preterm infant correlated with the level of IFN-gamma, a key pro-inflammatory cytokine that may predispose an infant to NEC. Gradual induction of PIA in a pre-clinical model resulted in significant hypoxia throughout the intestinal mucosa, including areas where intestinal macrophages reside. PIA-induced hypoxia significantly increased macrophage pro-inflammatory cytokine levels, while reducing tight junction protein ZO-1 expression and increasing intestinal barrier permeability. Macrophage depletion reversed the impact of anemia on intestinal ZO-1 expression and barrier function. CONCLUSIONS: Taken together, these results suggest that anemia can increase intestinal inflammation and barrier disruption likely through altered macrophage function, leading to the type of predisposing intestinal injury that may increase the risk for NEC.

Risk Factors Associated with Blood Exposure for Sporadic Hepatitis E in Dhaka, Bangladesh.

Fecal contamination of drinking water is associated with large hepatitis E virus (HEV) outbreaks of genotypes 1 and 2 in endemic areas. Sporadic transmission of HEV genotypes 3 and 4 in high-income countries has been associated with exposure to blood and animal contact. The objective of the study was to identify the risk factors for hepatitis E and the genotype(s) causing sporadic hepatitis E in Dhaka, Bangladesh. We selected, from a diagnostic center in Dhaka between November 2008 and November 2009, cases presenting with jaundice and anti-HEV IgM antibodies and age-matched controls were defined as those with no history of jaundice and normal blood test results. Serum samples were tested for HEV RNA using real-time reverse transcriptase polymerase chain reaction followed by a sequencing and phylogenetic analysis. A total of 109 cases and 109 controls were enrolled. The cases were more likely to be male (adjusted matched odds ratios [mOR] 2.2, 95% CI: 1.2-3.9; P = 0.01), or have reported contact with another person's blood or blood product, or contact with blood-contaminated sharp instruments (adjusted mOR 2.1, 95% CI: 1.1-4.1; P = 0.03) than controls. There were no significant differences between the cases and the controls in terms of reported high-risk sexual intercourse, consumption of undercooked meat, or contact or drinking fecally-contaminated water. The sera from three cases carried HEV RNA, all belonging to genotype 1. Findings from this study suggest that contact with human blood and sharing sharp instruments may transmit sporadic hepatitis E in Dhaka, Bangladesh. Efforts to prevent the transmission of blood-borne pathogens may also prevent sporadic HEV transmission in this endemic setting.

Antigen Density Dictates Immune Responsiveness following Red Blood Cell Transfusion.

Although RBC transfusion can result in the development of anti-RBC alloantibodies that increase the probability of life-threatening hemolytic transfusion reactions, not all patients generate anti-RBC alloantibodies. However, the factors that regulate immune responsiveness to RBC transfusion remain incompletely understood. One variable that may influence alloantibody formation is RBC alloantigen density. RBC alloantigens exist at different densities on the RBC surface and likewise exhibit distinct propensities to induce RBC alloantibody formation. However, although distinct alloantigens reside on the RBC surface at different levels, most alloantigens also represent completely different structures, making it difficult to separate the potential impact of differences in Ag density from other alloantigen features that may also influence RBC alloimmunization. To address this, we generated RBCs that stably express the same Ag at different levels. Although exposure to RBCs with higher Ag levels induces a robust Ab response, RBCs bearing low Ag levels fail to induce RBC alloantibodies. However, exposure to low Ag-density RBCs is not without consequence, because recipients subsequently develop Ag-specific tolerance. Low Ag-density RBC-induced tolerance protects higher Ag-density RBCs from immune-mediated clearance, is Ag specific, and occurs through the induction of B cell unresponsiveness. These results demonstrate that Ag density can potently impact immune outcomes following RBC transfusion and suggest that RBCs with altered Ag levels may provide a unique tool to induce Ag-specific tolerance.

Variability in the performance characteristics of IgG anti-HEV assays and its impact on reliability of seroprevalence rates of hepatitis E.

Hepatitis E is a major public health problem in developing countries and is increasingly being recognized as a cause of substantial sporadic viral hepatitis infections in industrialized countries. Variable rates of hepatitis E seroprevalence have been reported from the same geographic regions depending on the assay used. In this study, we evaluated the performance characteristics of four assays which included two commercial assays, Wantai HEV-IgG ELISA kit (Wantai, China), and DS-EIA-ANTI-HEV-G kit (DSI, Italy), one NIH-developed immunoassay (NIH-55 K, Kuniholm et al. [2009] Journal of Infectious Diseases 200:48-56), previously used in several major seroprevalence studies and one in-house Western blot assay (CDC-WB). The limit of detection of IgG anti-HEV is 100 mIU/mL for Wantai assay, 200 mIU/mL for CDC-WB assay, 1000 mIU/mL for DSI assay, and 40 mIU/mL for NIH-55 K assay. Pairwise concordance between the four assays ranged from 56% to 87%. The concordance among all four assays was observed in 52% of the samples, while the concordance among three assays was observed in 37% of the samples. These data show a wide discordance between various IgG anti-HEV assays and warrant a comprehensive evaluation of all the assays using well characterized global serum reference panels.

Key regulators of galectin-glycan interactions.

Protein-ligand interactions serve as fundamental regulators of numerous biological processes. Among protein-ligand pairs, glycan binding proteins (GBPs) and the glycans they recognize represent unique and highly complex interactions implicated in a broad range of regulatory activities. With few exceptions, cell surface receptors and secreted proteins are heavily glycosylated. As these glycans often represent highly regulatable post-translational modifications, alterations in glycosylation can fundamentally impact GBP recognition. Among GBPs, galectins in particular appear to engage a diverse set of glycan determinants to impact a broad range of biological processes. In this review, we will explore factors that impact galectin activity, including the effect of glycan modification on galectin-glycan interactions.

Innate immunity against molecular mimicry: Examining galectin-mediated antimicrobial activity.

Adaptive immunity provides the unique ability to respond to a nearly infinite range of antigenic determinants. Given the inherent plasticity of the adaptive immune system, a series of tolerance mechanisms exist to reduce reactivity toward self. While this reduces the probability of autoimmunity, it also creates an important gap in adaptive immunity: the ability to recognize microbes that look like self. As a variety of microbes decorate themselves in self-like carbohydrate antigens and tolerance reduces the ability of adaptive immunity to react with self-like structures, protection against molecular mimicry likely resides within the innate arm of immunity. In this review, we will explore the potential consequences of microbial molecular mimicry, including factors within innate immunity that appear to specifically target microbes expressing self-like antigens, and therefore provide protection against molecular mimicry.