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 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-κB 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-α induced intestinal NF-κB 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 severity of IBD in children.

The binding properties of Pseudomonas aeruginosa agglutinin-I (PA-IL) with glycoproteins (gps) and polysaccharides were studied by both the biotin/avidin-mediated microtiter plate lectin-binding assay and the inhibition of agglutinin-glycan interaction with sugar ligands. Among 36 glycans tested for binding, PA-IL reacted best with two glycoproteins containing Galalpha1-->4Gal determinants and a human blood group ABO precursor equivalent gp, but this lectin reacted weakly or not at all with A and H active gps or sialylated gps. Among the mammalian disaccharides tested by the inhibition assay, the human blood group Pkactive Galalpha1-->4Gal, was the best. It was 7.4-fold less active than melibiose (Galalpha1-->6Glc). PA-IL has a preference for the alpha-anomer in decreasing order as follows: Galalpha1-->6 >Galalpha1-->4 >Galalpha1-->3. Of the monosaccharides studied, the phenylbeta derivatives of Gal were much better inhibitors than the methylbeta derivative, while only an insignificant difference was found between the Galalpha anomer of methyl- and p -NO2-phenyl derivatives. From these results, it can be concluded that the combining size of the agglutinin is as large as a disaccharide of the alpha-anomer of Gal at nonreducing end and most complementary to Galalpha1-->6Glc. As for the combining site of PA-IL toward the beta-anomer, the size is assumed to be less than that of Gal; carbon-6 in the pyranose form is essential, and hydrophobic interaction is important for binding.

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.

Previous study on the binding properties of a lectin isolated from Codium fragile subspecies tomentosoides (CFT) indicates that this lectin recognizes the GalNAc alpha1--> sequence at both reducing and nonreducing ends. In this study, the carbohydrate specificity of CFT was further characterized by quantitative precipitin (QPA) and inhibition of lectin-enzyme binding assays. Of the glycoforms tested for QPA, all asialo-GalNAc alpha1--> containing glycoproteins reacted well with the lectin. Asialo hamster and ovine submandibular glycoproteins, which contain almost exclusively Tn (GalNAc alpha1-->Ser/Thr) residues as carbohydrate side chains, and Streptococcus type C polysaccharide completely precipitated the lectin added, while the GalNAc beta1-->containing Tamm-Horsfall Sd(a+) glycoprotein and its asialo product were inactive. Among the oligosaccharides tested for inhibiting lectin-glycoprotein interaction, GalNAc alpha1-->3GalNAc beta1-->3Gal alpha1-->4Gal beta1--> 4Glc(Fp) and Gal beta1-->3GalNAc alpha1-->benzyl (T alpha) were the best, and about 125-fold more active than GalNAc. They were about 3.3, 6.6, and 43 times more active than Tn containing glycopeptides, GalNAc alpha1-->3(LFuc alpha1--> 2)Gal(Ah) and Gal beta1-->3GalNAc(T), respectively. From the present and previous results, it is concluded that the combining site of CFT is probably of a groove type that recognizes from GalNAc alpha1--> to pentasaccharide(Fp). The carbohydrate specificity of this lectin can be constructed and summarized in decreasing order by lectin determinants as follows: Fp and T alpha > Tn cluster > Ah >> I/II.

Well-characterized mouse models of alloimmune 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 IntroductionHemolytic transfusion reactions (HTRs) are dangerous complications of blood transfusions. IgM-mediated HTRs (IgM-HTRs) occur acutely, are usually due to ABO incompatibility, typically cause intravascular hemolysis, and can lead to shock, renal failure, coagulopathy, and death. 1 Although infrequent, IgM-HTRs have a high mortality rate. IgG-mediated HTRs (IgG-HTRs) are more common, but usually less severe, than IgM-HTRs; they can occur acutely or be delayed, typically cause extravascular hemolysis, and occasionally result in renal failure, coagulopathy, and death. 2 Symptomatic immune-mediated red blood cell (RBC) destruction also occurs in autoimmune hemolytic anemia (AIHA), 3 blood group incompatible transplantation, 4 and immune thrombocytopenic purpura (ITP) treated with Rh-immune globulin. 5 Although various different modalities are used to treat HTRs, there is no definitive evidence regarding efficacy. Given the sporadic nature of HTRs, designing human trials to evaluate treatment options is difficult. Therefore, to study the mechanisms underlying HTRs, to evaluate the efficacy of existing treatments, and to develop new treatments, a relevant, inexpensive, and tractable animal model is required. 6 A mouse model would be ideal for this purpose, 7 given the abundance of reagents and relevant knockout (KO) and transgenic (Tg) animals. Although mouse blood group polymorphisms exist, 8 and transfused incompatible mouse RBC (mRBCs) are rapidly cleared, 9,10 no one has exploited these findings to develop a model of HTRs. The availability of a Tg mouse expressing human glycophorin A (hGPA; gene symbol designation, GYPA) on mRBCs 11 offers an approach using a well-described glycoprotein target and well-characterized reagents.We describe initial studies validating mouse models of IgG-and IgM-mediated alloimmune hemolysis. Thus, mRBCs from hGPA-Tg donors were transfused into non-Tg recipients that were passively immunized with IgG or IgM hGPA-specific monoclonal antibodies (mAbs). The clearance of the transfused mRBCs was quantified and the roles of complement and Fc␥ receptors were evaluated. Materials and methods AntibodiesPurified mAbs NaM10-2H12 (IgG3), NaM26-3F4 (IgG1), and NaM10-6G4 (IgG2a), which recognize peptide epitopes on the M and N forms of hGPA, 12 were purchased from EFS (Nantes, France). Hybridomas producing mAb 6A7, an IgG1 specific for a sialic acid-dependent epitope on M-type hGPA, 13-15 10F7, an IgG1 recognizing a nonpolymorphic epitope on hGPA, 13,14 and J11d, a rat IgM recognizing CD24 on mRBCs, 16,17...