Anti-Blood Group Antibodies in Intravenous Immunoglobulin May Complicate Interpretation of Antibody Titers in ABO-Incompatible Transplantation.

Patients receiving ABO-incompatible (ABOi) kidney transplants are treated before and after transplant with combination therapy, such as intravenous immunoglobulin (IVIG) and therapeutic plasma exchange, to prevent allograft rejection by reducing anti-A and anti-B titers. Although generally considered safe, it is well known that commercial IVIG products contain detectable anti-A and anti-B, which can be associated with hemolysis. Different preparative manufacturing techniques during the production of IVIG affect ABO antibody levels in IVIG preparations; therefore, some manufacturers now use new methods to reduce anti-A/B levels at the preproduction stage. The variations in implementing these strategies creates the potential for significant variation in antibody titers between products and, in some cases, even between lots of the same IVIG product. We report a case of persistently elevated anti-A titers in an ABOi kidney transplant recipient associated with elevated ABO antibody titers present in the preparation of IVIG used at our facility.

Altered generation of induced regulatory T cells in the FVB.mdr1a-/- mouse model of colitis.

The FVB.mdr1a(-/-) mouse, lacking the small molecule pump P-glycoprotein (P-gp), is a commonly used model for the study of spontaneous T cell-mediated colitis. In addition, MDR1 polymorphisms and P-gp deficiency in humans have been linked to the development of ulcerative colitis. We now demonstrate that mice with P-gp deficiency have decreased levels of Foxp3(+) regulatory T cells (Tregs) in the intestinal lamina propria. This decrease is not due to either increased Treg apoptosis, altered Treg trafficking, or enhanced Treg plasticity to become Foxp3(+)IL-17(+) cells. Instead, P-gp deficiency appears to restrict the development of induced Treg cells (iTregs), as fewer Foxp3(+) iTregs developed from naive FVB.mdr1a(-/-) T cells both upon transforming growth factor-ß (TGF-ß) treatment in vitro and after adoptive transfer into FVB.rag2(-/-) recipients. Rather, in vitro TGF-ß treatment results in a IL-17(+)CD4(+) T cell. This failure of iTregs to develop explains the decrease in Foxp3(+) Tregs in the FVB.mdr1a(-/-) intestine, representing a need to investigate this novel disease mechanism in human inflammatory bowel disease patients with MDR1 polymorphisms.

A role for IL-1 alpha in inducing pathologic inflammation during bacterial infection.

Infection with pathogenic microbes often results in a significant inflammatory response. A cascade of proinflammatory cytokines including tumor necrosis factor alpha (TNF-alpha) and IL-1 initiates this response. Although there is a clear role for IL-1 during infection, little is known to distinguish the role of IL-1 alpha from that of IL-1 beta during this process. With the use of Yersinia enterocolitica as a model enteric pathogen, we have identified a specific role for IL-1 alpha in inducing pathologic inflammation during bacterial infection. Depletion of IL-1 alpha in mice infected with wild-type Y. enterocolitica results in significantly decreased intestinal inflammation. Furthermore, a bacterial mutant that does not induce IL-1 alpha expression but induces normal levels of IL-1 beta, TNF-alpha, and IFN-gamma, causes greatly reduced intestinal inflammation and is attenuated by LD(50) analysis in the C57BL/6 mouse model. These results demonstrate a distinct and unrecognized role for IL-1 alpha in inducing intestinal inflammation that cannot be compensated for by the endogenous levels of IL-1 beta, TNF-alpha, or IFN-gamma that are produced in response to Y. enterocolitica. Additionally, these results suggest that IL-1 alpha-induced inflammation is a major contributor to the pathology of yersiniosis.

Development of intestinal intraepithelial lymphocytes, NK cells, and NK 1.1+ T cells in CD45-deficient mice.

The transmembrane protein tyrosine phosphatase CD45 is differentially required for the development and function of B, T, and NK cells, with mice partially deficient for CD45 having a significant inhibition of T cell, but not NK or B cell, development. CD45-mediated signaling has also been implicated in the development of intrathymic, but not extrathymic, intestinal intraepithelial T lymphocytes (iIELs) in the CD45ex6(-/-) mouse. As NK1.1(+) CD3(+) (NK-T) cells can also develop through extrathymic pathways, we have investigated the role of CD45 in NK-T cell development. In mice with a complete absence of CD45 expression (CD45ex9(-/-)) the NK-T cell population was maintained in the iIEL compartment, but not in the spleen. Functionally, CD45-deficient NK-T cells were unable to secrete IL-4 in response to TCR-mediated signals, a phenotype similar to that of CD45-deficient iIELs, in which in vitro cytokine production was dramatically reduced. Using the CD45ex9(-/-) mouse strain, we have also demonstrated that only one distinct population of NK-T cells (CD8(+)) appears to develop normally in the absence of CD45. Interestingly, although an increase in cytotoxic NK cells is seen in the absence of CD45, these NK calls are functionally unable to secrete IFN-gamma. In the absence of CD45, a significant population of extrathymically derived CD8alphaalpha(+) iIELs is also maintained. These results demonstrate that in contrast to conventional T cells, CD45 is not required during the development of CD8(+) NK-T cells, NK cells, or CD8alphaalpha(+) iIELs, but is essential for TCR-mediated function and cytokine production.

Spontaneous and continuous cyclooxygenase-2-dependent prostaglandin E2 production by stromal cells in the murine small intestine lamina propria: directing the tone of the intestinal immune response.

The mechanisms allowing the gastrointestinal immune system to avoid an inappropriate inflammatory response to nonpathogenic luminal Ags are poorly understood. We have previously described a role for cyclooxygenase (COX)-2-dependent arachidonic acid metabolites produced by the murine small intestine lamina propria in controlling the immune response to a dietary Ag. To better understand the role of COX-2-dependent arachidonic acid metabolites produced by the lamina propria, we examined the pattern of expression and the cellular source of COX-2 and COX-2-dependent PGE(2). We now demonstrate that non-bone marrow-derived lamina propria stromal cells have basal COX-2 expression and that COX-2-dependent PGE(2) production by these cells is spontaneous and continuous. The other mucosal and nonmucosal lymphoid compartments examined do not share this phenotype. In contrast to the majority of descriptions of COX-2 expression, COX-2 expression by lamina propria stromal cells is not dependent upon exogenous stimuli, including adhesion, LPS signaling via Toll-like receptor 4, or the proinflammatory cytokines TNF-alpha, IFN-gamma, and IL-1 beta. These findings, in conjunction with the known immunomodulatory capacities of PGs, suggest that COX-2 expression by the small intestine lamina propria is a basal state contributing to the hyporesponsiveness of the intestinal immune response.

The gastrointestinal ecosystem: a precarious alliance among epithelium, immunity and microbiota.

The gastrointestinal (GI) tract is a complex ecosystem generated by the alliance of GI epithelium, immune cells and resident microbiota. The three components of the GI ecosystem have co-evolved such that each relies on the presence of the other two components to achieve its normal function and activity. Experimental systems such as cell culture, germ-free animal models and intestinal isografts have demonstrated that each member of the GI ecosystem can follow a predetermined developmental pathway, even if isolated from the other components of the ecosystem. However, the presence of all three components is required for full physiological function. Genetic or functional alterations of any one component of this ecosystem can result in a broken alliance and subsequent GI pathology. A more detailed understanding of the interactions among microbiota, GI epithelium and the immune system should provide insight into multiple human disease states.

Bacterial invasion augments epithelial cytokine responses to Escherichia coli through a lipopolysaccharide-dependent mechanism.

One mechanism of initiating innate host defenses against uropathogenic Escherichia coli (UPEC) is the production of cytokines by bladder epithelial cells; however, the means by which these cells recognize bacterial pathogens is poorly understood. Type 1 pili, expressed by the majority of UPEC, have been shown to have a critical role in inducing the expression of IL-6 in bladder epithelial cells after exposure to E. coli. In this study, we demonstrate that type 1 pili are not sufficient to activate IL-6 production by bladder epithelial cells. Instead, it was shown that bacterial invasion mediated by type 1 pili augments bladder epithelial responses to E. coli via an LPS-dependent mechanism, leading to the production of IL-6. RNA transcripts for the LPSR Toll-like receptor 4 (TLR4) was detected in cultured bladder epithelial cells. The in vivo role of TLR4 was assessed using C3H/HeJ mice, which express a dominant negative form of TLR4. After infection with UPEC, C3H/HeJ mice have large foci of intracellular bacteria that persist within the bladder epithelium in the absence of any notable inflammatory response. These results indicate that LPS is required for bacterial invasion to enhance host responses to E. coli within the bladder.

Altered enteroendocrine cell expression in T cell receptor alpha chain knock-out mice.

Mice lacking T cell receptor alpha chain (TCRalpha(-/-)) develop inflammation of the colon. We have examined the effect of this inflammation on the colonic epithelium by studying markers of epithelial cuff, enteroendocrine, and immune cell differentiation. Using immunohistochemical techniques, colons were compared in normal C57/BL6 and murine TCR alpha(-/-) mice aged 2 and 3 weeks and 3-11 months. TCR alpha(-/-) mice aged 3-11 months had histologic evidence of inflammation with increased expression of CD45, CD4+, CD8+, and B220+ cells and a decrease in expression of IgA+ cells. There was a decrease in the number of cholecystokinin, serotonin, and neurotensin enteroendocrine expressing cells in the colon of TCR alpha(-/-) mice. These changes were not present in 2-3-week-old suckling/weaning mice. In contrast, peptide tyrosine tyrosine (PYY), glucagon-like peptide-1, and gastrin expression did not change and small intestinal enteroendocrine cells remained unaltered. The change in colonic enteroendocrine cell expression appears to be a specific response, since only a subset of these cells was altered, and the epithelium was intact by histologic analysis. The absence of functional T cells in TCR alpha(-/-) colon has a marked effect on differentiation of a specific subpopulation of enteroendocrine cells, prior to loss of integrity of the epithelium.

Abnormal immune function in vivo in a murine model of lysosomal storage disease.

Lysosomal storage diseases are a class of inborn errors of metabolism that lead to widespread disease in multiple tissues. The murine model of mucopolysaccharidosis type VII (MPS VII) closely parallels the human syndrome and has been extensively used to investigate the natural history and therapeutic strategies for lysosomal storage diseases in general. Here we demonstrate a previously undescribed immune defect in the MPS VII mouse. Although the normal populations of cells are present in lymph nodes of these mice, MPS VII mice show a blunted T cell proliferative response and decreased antibody production after immunization with antigens. One mechanism of this defect is ineffective processing of protein antigens, as responses to peptide antigens are normal. This phenotype is presumably caused by the lysosomal disorder, as the defect can be corrected in vivo by direct enzyme replacement therapy. These findings have implications for the use of this animal model, and may have clinical significance for other, more-common lysosomal storage diseases.

Cyclooxygenase-2-dependent arachidonic acid metabolites are essential modulators of the intestinal immune response to dietary antigen.

Intestinal inflammatory diseases are mediated by dysregulated immune responses to undefined luminal antigens. Feeding hen egg-white lysozyme to mice expressing a transgenic T-cell receptor that recognizes hen egg-white lysozyme peptide 46-61 resulted in no intestinal pathology; however, simultaneous administration of cyclooxygenase-2 inhibitors and dietary hen egg-white lysozyme resulted in increased proliferation of lamina propria mononuclear cells and crypt epithelial cells, crypt expansion and villus blunting. Lamina propria mononuclear cells produce high levels of cyclooxygenase-2-dependent arachidonic acid metabolites, which act as immunomodulators in the immune response to dietary antigen. These findings establish that cyclooxygenase-2-dependent arachidonic acid metabolites are essential in the development and maintenance of intestinal immune homeostasis.

Cellular immune responses are essential for the development of Helicobacter felis-associated gastric pathology.

The bacteria Helicobacter pylori is a major human pathogen that infects over half of the world's population. Infection initiates a series of changes in the gastric mucosa, beginning with atrophic gastritis and leading in some patients to peptic ulcer disease, mucosa-associated lymphomas, and gastric adenocarcinoma. Although this cascade of events clearly occurs, little is known about the role of the host immune response in disease progression. We have utilized the C57BL/6 Helicobacter felis mouse model to critically analyze the role of the adaptive immune response in the development of Helicobacter-associated gastric pathology. Infection of B and T cell-deficient RAG-1-/- mice or T cell-deficient TCRbetadelta-/- mice with H. felis resulted in high levels of colonization, but no detectable gastric pathology. Conversely, infection of B cell-deficient microMT mice resulted in severe gastric alterations identical with those seen in immunocompetent C57BL/6-infected mice, including gastric mucosal hyperplasia and intestinal metaplasia. These results demonstrate that the host T cell response is a critical mediator of Helicobacter-associated gastric pathology, and that B cells and their secreted Abs are not the effectors of the immune-mediated gastric pathology seen after H. felis infection. These results indicate that in addition to specific Helicobacter virulence factors, the host immune response is an important determinant of Helicobacter-associated disease.

Helicobacter pylori attaches to NeuAc alpha 2,3Gal beta 1,4 glycoconjugates produced in the stomach of transgenic mice lacking parietal cells.

Helicobacter pylori infection of the human stomach is associated with altered acid secretion, loss of acid-producing parietal cells, and, in some hosts, adenocarcinoma. We have used a transgenic mouse model to study the effects of parietal cell ablation on H. pylori pathogenesis. Ablation results in amplification of the presumptive gastric epithelial stem cell and its immediate committed daughters. The amplified cells produce sialylated oncofetal carbohydrate antigens that function as receptors for H. pylori adhesins. Attachment results in enhanced cellular and humoral immune responses. NeuAc alpha 2,3Gal beta 1,4 glycoconjugates may not only facilitate persistent H. pylori infection in a changing gastric ecosystem, but by promoting interactions with lineage progenitors and/or initiated cells contribute to tumorigenesis in patients with chronic atrophic gastritis.

Molecular basis for the enterocyte tropism exhibited by Salmonella typhimurium type 1 fimbriae.

Salmonella typhimurium exhibits a distinct tropism for mouse enterocytes that is linked to their expression of type 1 fimbriae. The distinct binding traits of Salmonella type 1 fimbriae is also reflected in their binding to selected mannosylated proteins and in their ability to promote secondary bacterial aggregation on enterocyte surfaces. The determinant of binding in Salmonella type 1 fimbriae is a 35-kDa structurally distinct fimbrial subunit, FimHS, because inactivation of fimHS abolished binding activity in the resulting mutant without any apparent effect on fimbrial expression. Surprisingly, when expressed in the absence of other fimbrial components and as a translational fusion protein with MalE, FimHS failed to demonstrate any specific binding tropism and bound equally to all cells and mannosylated proteins tested. To determine if the binding specificity of Salmonella type 1 fimbriae was determined by the fimbrial shaft that is intimately associated with FimHS, we replaced the amino-terminal half of FimHS with the corresponding sequence from Escherichia coli FimH (FimHE) that contains the receptor binding domain of FimHE. The resulting hybrid fimbriae bearing FimHES on a Salmonella fimbrial shaft exhibited binding traits that resembled that of Salmonella rather than E. coli fimbriae. Apparently, the quaternary constraints imposed by the fimbrial shaft on the adhesin determine the distinct binding traits of S. typhimurium type 1 fimbriae.

Epithelial attachment alters the outcome of Helicobacter pylori infection.

Genetically defined in vivo models are needed to assess the importance of target cell attachment in bacterial pathogenesis. Gastric colonization by Helicobacter pylori in human populations is common and persistent, and has various outcomes including peptic ulcers and cancer. The impact of attachment on the course of infection was examined in transgenic mice expressing a human receptor for H. pylori in their gastric epithelium. Persistent infection by a clinical isolate occurred at comparable microbial densities in transgenic and nontransgenic littermates. However, microbial attachment in transgenic mice resulted in production of autoantibodies to Lewisx carbohydrate epitopes shared by bacteria and acid-secreting parietal cells, chronic gastritis, and parietal cell loss. This model should help identify bacterial and host genes that produce attachment-related pathology.

Expression of thin aggregative fimbriae promotes interaction of Salmonella typhimurium SR-11 with mouse small intestinal epithelial cells.

The factors that mediate binding of Salmonella typhimurium to small intestinal epithelial cells have not been fully characterized. In this paper we demonstrate that elimination of production of thin aggregative fiber by a transposon insertion within the gene encoding the subunit protein of the fiber reduced binding of S. typhimurium SR-11 to a conditionally immortalized proximal small intestinal epithelial cell line established from transgenic mice. This binding defect could be overcome by transcomplementation with a wild-type allele. The conditionally immortalized cell line should prove useful in identifying the epithelial cell receptor for bacterial attachment since expression of its bacterial binding activity can be induced by manipulating the line's proliferative status.

Enzyme-based antigen localization and quantitation in cell and tissue samples (Midwestern assay).

Quantitation of antigen concentration in cell and tissue samples typically requires antigen extraction, which precludes antigen localization in the same sample. Similarly, antigen immunolocalization in fixed cells or tissue sections provides limited information about antigen concentration. We have developed a rapid and sensitive assay for simultaneous antigen localization and quantitation in cell and tissue samples that does not involve antigen extraction, radioactive materials, or image analysis. Fixed cells and/or tissue sections are used with antigen-specific enzyme-linked probes to generate soluble reaction products that are spectrophotometrically quantifiable and deposited reaction products that are microscopically localizable. The amount of soluble reaction product is dependent on several variables, including antigen concentration, probe specificity and sensitivity, sample size, and enzyme reaction time. These variables can be experimentally controlled so that soluble reaction product is proportional to antigen concentration in the sample. This assay was used in multiple applications including detection of Ki-67 nuclear antigen immunoreactivity in human brain tumors, in which it showed a clear relationship with visually determined Ki-67 cell labeling indexes. This assay, termed the Midwestern assay, should be applicable to a wide variety of antigens in both clinical and research samples.

Moluccella laevis lectin, a marker for cellular differentiation programs in mouse gut epithelium.

We have assembled a system for testing the hypothesis that changes in glycoconjugate production represent markers for defining developmental, spatial, and environmental influences on the proliferation and differentiation programs of various mouse gut epithelial cell lineages. Multilabel immunohistochemical methods were used to survey the interactions of purified lectins with 1) normal fetal, neonatal, and adult FVB/N mouse gut, 2) gastric and intestinal isografts harvested at various developmental stages, and 3) transgenic mouse models of intestinal epithelial cell hyperplasia, dysplasia, and/or neoplasia. As a demonstration of the system's utility, we used the recently purified, alpha-N-acetyl-D-galactosamine-specific, Moluccella laevis lectin (MLL). In the adult FVB/N mouse stomach, MLL only recognizes glycoconjugates produced by a population of nonproliferating neck and prezymogenic cells that occupy a pivotal point in the complex, migration-associated differentiation program of the zymogenic cell lineage. In the developing FVB/N stomach, MLL binds to members of the zymogenic and pit lineages even before morphogenesis of gastric units is completed. Expression of MLL epitopes in pit cells is restricted to the period before the gastric epithelium has completed its morphoregulatory program. Analysis of gastric isografts indicates that these lineage- and developmental stage-specific patterns of glycoconjugate accumulation are not influenced by normal luminal contents. In the adult FVB/N intestine, MLL binding can be used to operationally define variations in the differentiation programs of 1) members of the enteroendocrine and goblet cell lineages during their migration along the crypt-to-villus axis and 2) cells comprising the follicle-associated epithelium overlying Peyer's patches. Accumulation of MLL epitopes in villus-associated enterocytes does not appear to be affected when these cells are induced to reenter the cell cycle by simian virus 40 large T antigen (SV40 TAg). MLL reactivity is not diminished when enterocytes begin to dedifferentiate as a result of production of SV40 TAg, human K-rasVal12, and a dominant negative human p53 mutant. The lack of change in MLL binding properties may reflect the brief residence time of enterocytes on the villus. These results indicate that glycoconjugate production represents a very useful tool for studying gut epithelial cell biology. Preliminary studies suggest that this is also true in the human gut.

Use of transgenic mice to study regulation of gene expression in the parietal cell lineage of gastric units.

The mechanisms that regulate cell lineage-specific and differentiation-dependent patterns of gene expression in the gastric units of the stomach are largely unknown. Transgenic mice were generated in order to identify cis-acting sequences that determine the zymogenic cell-specific pattern of expression of the mouse intrinsic factor (InF) gene and the parietal cell-specific pattern of expression of the mouse H+/K(+)-ATPase beta-subunit gene. Portions of the 5'-nontranscribed domains of each gene were linked to the human growth hormone (hGH) gene beginning at its nucleotide +3. RNA blot hybridization studies combined with multilabel immunocytochemical surveys using a panel of lineage-specific antibodies and lectins indicated that nucleotides -1035 to +24 of the mouse H+/K(+)-ATPase beta-subunit gene direct a pattern of reporter production which recapitulates the parietal cell-specific and developmental patterns of expression of the endogenous gene. Analysis of three mosaic founders containing H+/K(+)-ATPase beta-subunit-1035 to +24/hGH+3 revealed that they had monophenotypic gastric units: a given unit contained either a wholly hGH-positive or a wholly hGH-negative population of parietal cells. These latter findings provide very strong evidence that gastric units are monoclonal, i.e. they are supplied by stem cells having one genotype. Although some, but not all, parietal cells are apparently derived from the same committed progenitor as zymogenic cells, virtually all parietal cells in a given gastric unit, but none of its zymogenic cells, express InF-1029 to +55/hGH+3. This suggests that InF-1029 to +55 may contain cis-acting sequences which allow parietal cell expression in other species (e.g. humans) but lack additional elements which normally function in mice to suppress InF expression in this lineage. The absence of hGH in zymogenic cells also means that the transcriptional regulatory environments of parietal and zymogenic cells derived from the same precursor are distinguishable by InF-1029 to +55. H+/K(+)-ATPase beta-subunit-1035 to +24 and InF-1029 to +55 are the only two sequences reported to date that are able to direct foreign gene expression exclusively to a gastric epithelial cell lineage in transgenic mice. This ability to deliver gene products to parietal cells can now be exploited to identify factors that control their normal proliferation and differentiation programs and/or to specifically alter their biological properties.