Differential expression of immunomodulatory galectin-1 in peripheral leukocytes and adult tissues and its cytosolic organization in striated muscle.

Galectin-1 (Gal-1) is important in immune function and muscle regeneration, but its expression and localization in adult tissues and primary leukocytes remain unclear. To address this, we generated a specific monoclonal antibody against Gal-1, termed alphahGal-1, and defined a sequential peptide epitope that it recognizes, which is preserved in human and porcine Gal-1, but not in murine Gal-1. Using alphahGal-1, we found that Gal-1 is expressed in a wide range of porcine tissues, including striated muscle, liver, lung, brain, kidney, spleen, and intestine. In most types of cells, Gal-1 exhibits diffuse cytosolic expression, but in cells within the splenic red pulp, Gal-1 showed both cytosolic and nuclear localization. Gal-1 was also expressed in arterial walls and exhibited prominent cytosolic and nuclear staining in cultured human endothelial cells. However, human peripheral leukocytes and promyelocytic HL60 cells lack detectable Gal-1 and also showed very low levels of Gal-1 mRNA. In striking contrast, Gal-1 exhibited an organized cytosolic staining pattern within striated muscle tissue of cardiac and skeletal muscle and colocalized with sarcomeric actin on I bands. These results provide insights into previously defined roles for Gal-1 in inflammation, immune regulation and muscle biology.

Lactobacillus rhamnosus blocks inflammatory signaling in vivo via reactive oxygen species generation.

Uncontrolled inflammatory responses in the immature gut may play a role in the pathogenesis of many intestinal inflammatory syndromes that present in newborns or children, such as necrotizing enterocolitis (NEC), idiopathic inflammatory bowel diseases (IBD), or infectious enteritis. Consistent with previous reports that murine intestinal function matures over the first 3 weeks of life, we show that inflammatory signaling in the neonatal mouse gut increases during postnatal maturation, with peak responses occurring at 2-3 weeks. Probiotic bacteria can block inflammatory responses in cultured epithelia by inducing the generation of reactive oxygen species (ROS), which inhibit NF-kappaB activation through oxidative inactivation of the key regulatory enzyme Ubc12. We now report for the first time that the probiotic Lactobacillus rhamnosus GG (LGG) can induce ROS generation in intestinal epithelia in vitro and in vivo. Intestines from immature mice gavage fed LGG exhibited increased GSH oxidation and cullin-1 deneddylation, reflecting local ROS generation and its resultant Ubc12 inactivation, respectively. Furthermore, prefeeding LGG prevented TNF-alpha-induced intestinal NF-kappaB activation. These studies indicate that LGG can reduce inflammatory signaling in immature intestines by inducing local ROS generation and may be a mechanism by which probiotic bacteria can prevent NEC in premature infants or reduce the severity of IBD in children.

Mouse models of IgG- and IgM-mediated hemolysis.

Well-characterized mouse models of allo-immune antibody-mediated hemolysis would provide a valuable approach for gaining greater insight into the pathophysiology of hemolytic transfusion reactions. To this end, mouse red blood cells (mRBCs) from human glycophorin A transgenic (hGPA-Tg) donor mice were transfused into non-Tg recipients that had been passively immunized with IgG or IgM hGPA-specific monoclonal antibodies (mAbs). In this novel murine "blood group system," mRBCs from hGPA-Tg mice are "antigen positive" and mRBCs from non-Tg mice are "antigen negative." Passive immunization of non-Tg mice with the IgG1 10F7 and IgG3 NaM10-2H12 anti-hGPA mAbs each induced rapid clearance of incompatible transfused hGPA-Tg-mRBCs in a dose-response manner. Using various knockout mice as transfusion recipients, both the complement system and activating Fcgamma receptors were found to be important in the clearance of incompatible mRBCs by each of these IgG mAbs. In addition, the IgM E4 anti-hGPA mAb induced complement-dependent intravascular hemolysis of transfused incompatible hGPA-Tg-mRBCs accompanied by gross hemoglobinuria. These initial studies validate the relevance of these new mouse models for addressing important questions in the field of transfusion medicine.

Crystallographic analysis of the NNA7 Fab and proposal for the mode of human blood-group recognition.

The NNA7 Fab antibody fragment recognizes the human N-type blood-group antigen comprised of the N-terminal glycopeptide of glycophorin A (GPA). A mutant form of this Fab fragment, NNA7-G91S, exhibits markedly reduced antigen binding. To provide insight into how these Fab fragments recognize this glycopeptide antigen, the crystal structures of NNA7 and NNA7-G91S were solved and refined to 1.83 and 1.97 A resolution, respectively. Both molecules are composed of the same heavy (H) chain Fd fragment, but each contains a slightly different light (L) chain owing to the G91S substitution. Specifically, the G91S mutation pushes the backbone of the neighboring H chain away from complementarity-determining region 3 (CDR3) of the L-chain variable region, allowing an additional glycerol cryoprotectant molecule to enter the antigen-combining site near the putative location of O-linked glycosylation. Each Fab fragment also possesses a well defined 2-(N-morpholino)ethanesulfonic acid (MES) molecule trapped in its antigen-combining site, as well as a crystallographic symmetry-related molecule comprising an amino-acid sequence that is virtually identical to the N-terminus of GPA. The MES molecule interacts with the H-chain CDR in a manner reminiscent of antibody-carbohydrate complexes. These results suggest a model for recognition of the glycopeptide antigen that accounts for the deleterious effect of the G91S substitution.

Purification, crystallization and X-ray diffraction analysis of a recombinant Fab that recognizes a human blood-group antigen.

The NNA7 Fab fragment recognizes the human glycopeptide N blood-group antigen and has a high affinity for N-type glycophorin A (GPA). To provide insight into how antibodies recognize glycopeptide antigens, soluble Fab fragments were expressed in Escherichia coli, purified and crystallized using the hanging-drop vapor-diffusion method at 293 K. The best crystals were obtained from solutions of PEG monomethyl ether 5000 containing 4-8 mM yttrium chloride (YCl3). This rare-earth ion, which could be substituted with various lanthanides, changed the habit of crystals from multinucleated rods with a diffraction limit of 4.25 A resolution to a diamond-shaped morphology that grew as single crystals and diffracted X-rays to 1.75 A resolution. Data were collected that indicated that the crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 57.9, b = 77.1, c = 118.1 A and one Fab fragment per asymmetric unit. A molecular-replacement solution has been obtained and 86% of the molecule was fitted by use of an automated refinement procedure (ARP).