Comparative anatomy and phylogenetic distribution of the mammalian cecal appendix.

A recently improved understanding of gut immunity has merged with current thinking in biological and medical science, pointing to an apparent function of the mammalian cecal appendix as a safe-house for symbiotic gut microbes, preserving the flora during times of gastrointestinal infection in societies without modern medicine. This function is potentially a selective force for the evolution and maintenance of the appendix, and provides an impetus for reassessment of the evolution of the appendix. A comparative anatomical approach reveals three apparent morphotypes of the cecal appendix, as well as appendix-like structures in some species that lack a true cecal appendix. Cladistic analyses indicate that the appendix has evolved independently at least twice (at least once in diprotodont marsupials and at least once in Euarchontoglires), shows a highly significant (P < 0.0001) phylogenetic signal in its distribution, and has been maintained in mammalian evolution for 80 million years or longer.

Increased levels of IgE and autoreactive, polyreactive IgG in wild rodents: implications for the hygiene hypothesis.

To probe the potential role of Th1 versus Th2 reactivity underlying the hygiene hypothesis, intrinsic levels of Th1-associated and Th2-associated antibodies in the serum of wild rodents were compared with that in various strains of laboratory rodents. Studies using rat lung antigens as a target indicated that wild rats have substantially greater levels of autoreactive, polyreactive immunoglobulin G (IgG), but not autoreactive, polyreactive IgM than do laboratory rats, both on a quantitative and qualitative basis. Increased levels of serum IgG and IgE were observed in both wild rats and wild mice relative to their laboratory-raised counterparts, with the effect being most pronounced for IgE levels. Further, wild rats had greater intrinsic levels of both Th1- and Th2-associated IgG subclasses than did lab rats. The habitat (wild versus laboratory raised) had a more substantial impact on immunoglobulin concentration than did age, strain or gender in the animals studied. The presence in wild rodents of increased intrinsic, presumably protective, non-pathogenic responses similar to both autoimmune (autoreactive IgG, Th1-associated) and allergic (IgE, Th2-associated) reactions as well as increased levels of Th1-associated and Th2-associated IgG subclasses points toward a generally increased stimulation of the immune system in these animals rather than a shift in the nature of the immunoreactivity. It is concluded that, at least to the extent that feedback inhibition is a controlling element of immunoreactivity, an overly hygienic environment may affect the threshold of both types of immune responses more so than the balance between the different responses.

Biophysical characteristics of anti-Gal(alpha)1-3Gal IgM binding to cell surfaces: implications for xenotransplantation.

BACKGROUND: Natural antibodies directed against cell surface carbohydrates are thought to be vital to host defense and to initiate the rejection of xenografts and ABO-incompatible allografts. The biophysical properties underlying the association and dissociation of these antibodies from cell surfaces is incompletely understood. We investigated those properties for the binding of Galalpha1-3Gal antibodies to porcine endothelial cell surfaces, because such interactions might be relevant to the clinical application of xenotransplantation. RESULTS AND CONCLUSIONS: The initial rate of binding of anti-Galalpha1-3Gal antibodies to endothelial cells was found to depend on antibody concentration, antibody diffusion, and antigen concentration. The presence of an intact glycocalyx had a greater impact on antibody binding than mobility of antigen in cell membranes. Disruption of glycocalyx increased the amount of antibody bound at equilibrium by more than 50%. Although the binding of anti-Galalpha1-3Gal antibodies to cell surfaces could be inhibited by soluble Galalpha1-3Gal, once bound, some anti-Galalpha1-3Gal could not be dissociated by competitive inhibitors of binding or by denaturation of the bound Ig with chaotropic reagents, but could be dissociated by reduction of disulfide bonds, suggesting that attachment to cell surfaces was, at least in part, by means other than specific reaction with the epitope.

Naturally occurring anti-alpha-galactosyl antibodies: relationship to xenoreactive anti-alpha-galactosyl antibodies.

Antibodies produced by an individual without a known history of sensitization to the relevant antigen are called "natural" antibodies. Some natural antibodies, called xenoreactive antibodies, react with the cells of foreign species. Most xenoreactive antibodies in humans and higher primates bind to a nonreducing terminal galactose expressed by pigs and other lower mammals. Although human natural antibodies which bind to one or more of a variety of terminal alpha-galactosyl structures have been identified previously, the antigen recognized by anti-alpha-galactosyl antibodies on the cells of foreign species is thought to be exclusively Galalpha1-3Gal. Thus, anti-alpha-galactosyl antibodies which do not react with Galalpha1-3Gal are thought to be nonxenoreactive. Here, we identify natural antibodies in human serum which bind to Galalpha1-6Hexosepyrranosides but not Galalpha1-3Gal, indicating that these antibodies are not xenoreactive. Various lower mammals were found to have natural anti-Galalpha1-2Gal antibodies in their sera, suggesting that at least some anti-Galalpha1-2Gal antibodies might not be xenoreactive and indicating, surprisingly, that anti-alpha-galactosyl antibodies are much more phylogenetically disperse than previously known. Also surprising was the finding that some natural antibodies which bind to Galalpha1-3Gal in vitro do not bind to porcine xenografts. These studies show that naturally occurring anti-alpha-galactosyl antibodies in mammalian serum include antibodies with a greater variety of reactivities than previously thought, only some of which would bind to a porcine xenograft. Further, these studies show that the methods used to detect anti-alpha-galactosyl antibodies of relevance in xenotransplantation must be carefully evaluated to avoid detection of anti-alpha-galactosyl antibodies which would not bind to a porcine organ and which therefore are not involved in xenograft rejection.

Immunochemical properties of anti-Gal alpha 1-3Gal antibodies after sensitization with xenogeneic tissues.

In antigen-driven immune responses to proteins, antibodies of low avidity and limited complement fixing capacity undergo affinity maturation to yield antibodies of higher avidity which fix complement to a greater extent. The products of antigen-driven responses to carbohydrates are less defined. To investigate the evolution of natural antibodies against carbohydrates following sensitization, we studied natural antibodies specific for Gal alpha 1-3Gal in patients sensitized to that antigen as a result of perfusion of their blood through porcine livers for the treatment of hepatic failure. The natural antibodies against Gal alpha 1-3Gal, which occur in all unsensitized individuals, were predominantly IgM and IgG2, with average functional avidities of 5 x 10(-9) and 2 x 10(-8) M, respectively. After sensitization, the classes of anti-Gal alpha 1-3Gal included IgM, IgG2, and IgG1, and the average functional avidities of IgM and IgG were 3 x 10(-9) and 2 x 10(-9) M, respectively. The activation of complement by anti-Gal alpha 1-3Gal per microgram of Ab, measured by the fixation of C3bi on porcine cells, increased after sensitization owing to changes in subclass and avidity. Deposition of C3bi correlated with the concentrations of IgG1 and IgM but not IgG2 against Gal alpha 1-3Gal. Consistent with this finding, purified IgG1, but not IgG2, anti-Gal alpha 1-3Gal fixed complement on porcine cells. These results demonstrate that the properties of anticarbohydrate antibodies evolve after sensitization to increase complement fixation on potential targets. These properties may result from the altered costimulation of the humoral response to Gal alpha 1-3Gal due to sensitization.

Differential recognition by proteins of alpha-galactosyl residues on endothelial cell surfaces.

The binding of proteins to cell surface carbohydrates contributes to cell-cell interactions in development, immunity, and various physiologic processes. Such interactions are presumably dictated not only by the chemical structure of the carbohydrate but also reflect the properties of the protein and the microenvironment in which the protein-carbohydrate interaction occurs. To explore the factors influencing the recognition of cell surface carbohydrates by proteins, the extent to which three classes of proteins--anti-Gal alpha 1-3Gal IgM found in higher primates, Griffonia simplicifolia type I lectin, isolectin B4 (GS-IB4), and alpha-galactosidase--interact with Gal alpha 1-3Gal on porcine cell surfaces was tested. Although the Gal alpha 1-3Gal residues expressed on porcine endothelial cells and recognized by anti-Gal alpha 1-3Gal IgM and GS-IB4 were both sensitive to cleavage by alpha-galactosidase, the sites recognized by GS-IB4 were more sensitive to cleavage than sites recognized by anti-Gal alpha 1-3Gal IgM. Cross-blocking studies on porcine cell surfaces revealed that a significant proportion of anti-Gal alpha 1-3Gal IgM bound to sites not recognized by GS-IB4; however, anti-Gal alpha 1-3Gal IgM and GS-IB4 recognized the same sites on solubilized membrane proteins and model compounds. These results suggest that features of the cell surface such as the three-dimensional arrangement of Gal alpha 1-3Gal and characteristics of the protein such as size and valency play critical roles in specific interactions on cell surfaces.

Fate of antigen in xenotransplantation: implications for acute vascular rejection and accommodation.

Antigen down-modulation plays a critical role in xenotransplants involving humoral responses against the Forssman antigen and may play a role in the long-term survival of ABO-incompatible allografts. The present study investigates the fate of porcine antigens in pig-to-primate xenotransplantation. Human antibodies bound to the glycocalyx of cultured porcine aortic endothelial cells as judged by electron microscopy and were shed from the cell surface in a complex with fibronectin, a glycoprotein that is found in the apical membrane glycocalyx of cultured cells. Antibody was shed in a metabolically dependent process with a t(1/2) of 2 to 3 hours. However, the amount of antigen on the cell surface did not change appreciably within 24 hours, suggesting that antigen modulation did not occur. Over the ensuing days, antigen expression decreased, although the change was always less than 50% of baseline. Changes in antigen expression were due for the most part to changes in expression of alpha-galactosyl residues. Consistent with results obtained in vitro, antigen expression in porcine organ transplants remained at approximately the baseline level as determined by immunofluorescence analysis of IgM binding to graft endothelium. If, as these results suggest, antigen is not down-modulated in pig-to-primate xenotransplantation, then therapies aimed at prolonged xenograft survival must focus on antibody or genetic manipulation of antigen expression.

Specificity of xenoreactive anti-Gal alpha 1-3Gal IgM for alpha-galactosyl ligands.

The transplantation of organs from lower animals such as pigs into humans is prevented by a severe rejection reaction initiated by complement fixing xenoreactive natural antibodies. Most anti-pig xenoreactive natural antibodies in humans are thought to recognize Gal alpha 1-3Gal beta 1-4GlcNAc and are also thought to recognize, albeit less avidly, Gal alpha 1-6Glc. Gal alpha 1-6Glc has been used as a ligand for purification of 'anti-Gal alpha 1-3Gal antibodies' and as a therapeutic or reagent to prevent the binding of these antibodies to porcine organs or cells. We tested the specificity of anti-Gal alpha 1-3Gal IgM for Gal alpha 1-6Glc and related saccharides. Based on inhibition of binding of xenoreactive anti-Gal alpha 1-3Gal IgM to porcine cells by soluble saccharides, anti-Gal alpha 1-3Gal IgM in a human serum was found to consist of a mixture of antibodies which have a similar affinity for Gal alpha 1-3Gal but varying affinities for Gal alpha 1-6Glc and other structures. Twenty to 40% of the anti-Gal alpha 1-3Gal IgM from the population tested did not recognize Gal alpha 1-6Glc. The binding of anti-Gal alpha 1-3Gal IgM to Gal alpha 1-6Glc varied widely from individual to individual, some samples lacking almost entirely anti-Gal alpha 1-3Gal IgM which bound to Gal alpha 1-6Glc.

Training psychologists to function as competent scientists in criminal forensics.

Psychologists planning to work in the criminal forensic area can benefit from a structured training experience, with learning objectives designed to bridge the gap between clinical practice and the legal system.

A novel ribonucleoprotein complex defined by monoclonal antibodies to NIH 3T3 cells transfected with human pancreatic adenocarcinoma DNA.

Three monoclonal antibodies elicited to NIH 3T3 cells transfected with DNA from a human pancreatic adenocarcinoma cell line recognized a novel ribonucleoprotein complex. Minimally, this ribonucleoprotein complex contained a Mr 240,000 protein (by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and two RNA species with apparent sizes of 1.5 and 3.0 kilobases (by formaldehyde agarose gel electrophoresis). In addition to a cytoplasmic and nuclear subcellular localization, the RNA antigen was secreted from human tumor cell lines and NIH 3T3 cells transfected with pancreatic tumor DNA (inhibitable by monensin) and was apparently not a viral or Mycoplasma contaminant. The ribonucleoprotein antigen was detected in some normal tissues by immunoperoxidase but was not found in or secreted from in vitro cultured normal human fibroblasts, nontransfected or spontaneously transformed NIH 3T3 cells, or normal peripheral blood leukocytes.