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Mouse monoclonal antibodies (MAbs) specific for the lipopolysaccharide (LPS) core types R1, R2, and R3 of Escherichia coli and a cross-reactive MAb that binds to the LPS core of almost all E. coli were used in ELISA to determine the frequency of cores resembling R1, R2, and R3 in strains of E. coli isolated from clinical samples (blood and urine specimens) and from the feces of asymptomatic individuals. Of the 180 wild-type isolates, 123 were assigned to R1 core type, 14 to R2, and 18 to R3. Twenty-five wild-type E. coli isolates could not be assigned to a particular core type and may have either an R4 or K12 core or a previously unrecognized core type. R1 core type was associated with O types 1, 4, 6, 8, and 18 and with K1 or K5 capsules. R3 was associated with O15.O75 isolates could be of either R1 or R2 core type

Cell surface mucins configure the cell surface by presenting extended protein backbones that are heavily O-glycosylated. The glycopeptide structures establish physicochemical properties at the cell surface that enable and block the formation of biologically important molecular complexes. Some mucins, such as MUC1, associate with receptor tyrosine kinases and other cell surface receptors, and engage in signal transduction in order to communicate information regarding conditions at the cell surface to the nucleus. In that context, the MUC1 cytoplasmic tail (MUC1CT) receives phosphorylation signals from receptor tyrosine kinases and serine/threonine kinases, which enables its association with different signaling complexes that conduct these signals to the nucleus and perhaps other subcellular organelles. We have detected the MUC1CT at promoters of over 500 genes, in association with several different transcription factors, and have shown that promoter occupancy can vary under different growth factor conditions. However, the full biochemical nature of the nuclear forms of MUC1 and its function at these promoter regions remain undefined. I will present evidence that nuclear forms of the MUC1CT include extracellular and cytoplasmic tail domains. In addition, I will discuss evidence for a hypothesis that the MUC1CT possesses a novel catalytic function that enables remodeling of the transcription factor occupancy of promoters, and thereby engages in regulation of gene expression.

The cytokine production profile, focusing on interleukin-4 (IL-4), interferon-gamma (IFN-gamma) and interleukin-10 (IL-10) of human CD4+, CD8+ and CD4- CD8- alpha beta T cells cloned from the peripheral blood of healthy individuals was compared. Solid-phase anti-CD3 stimulation of CD4- CD8- alpha beta T cell clones from one individual revealed a significantly increased frequency of IL-4-producing clones (81%), compared to CD4+ T cells (24%) or CD8+ (28%). All five CD4- CD8- alpha beta T-cell clones from two other individuals also produced IL-4. Clones that produced IFN-gamma with undetectable IL-4 production, thus being of the 'classical' Th1 phenotype, were infrequent in CD4- CD8- alpha beta T-cell clones (19%) compared to CD4+ (71%), and CD8+ clones (72%) cloned in OKT3, and CD4+ cells cloned in phytohaemaglutinin A (77%). Unlike previously reported studies with gamma delta cells, the alpha beta CD4- CD8- T cells produced IL-10 at appreciable frequency (38%) in PHA generated clones. The supernatants from anti-CD3 stimulated CD4- CD8- alpha beta T-cell clones contained sufficient IL-4 to activate B cells, enhancing CD23 and surface immunoglobulin M (IgM) expression and co-stimulating B-cell proliferation. These findings suggest that the function of CD4- CD8- alpha beta T cells is distinct from that of most CD4+ or CD8+ T cells.

There is increasing evidence that TNF-alpha is a cytokine of major importance in the pathogenesis of rheumatoid arthritis. Since TNF-alpha mediates its effects via high affinity receptors, we were interested in investigating their expression and function in cells from rheumatoid tissue. Synovial fibroblasts derived from rheumatoid synovial tissue are stimulated by TNF-alpha to proliferate and release cytokines, prostaglandins, proteases and protease inhibitors. We have evaluated through which receptor stimulation of DNA synthesis and the release of the proinflammatory agents, IL-6, IL-8 and PGE2 are induced. It was found that rheumatoid synovial fibroblasts express both the p55 and p75 TNF receptor, in a ratio of 4:1. TNF-alpha-stimulated synovial fibroblast DNA synthesis and the release of IL-6, IL-8 and PGE2 was inhibited by antagonist monoclonal antibodies against either the p55 or the p75 TNF receptor, although the blockade of the p55 TNF receptor had a more potent effect than inhibition of the p75 TNF receptor alone. Similarly, specific monoclonal antibodies, agonistic for either the p55 or p75 TNF receptor stimulated synovial fibroblast DNA synthesis, as well as IL-6, IL-8 and PGE2 release. Both p55 and p75 TNF receptors on dermal and gingival fibroblasts were also involved in TNF-alpha-mediated DNA synthesis and IL-6, IL-8 and PGE2 release, although differences in the levels of DNA synthesis and release of inflammatory cytokines and PGE2 were observed between the three fibroblast types.

BACKGROUND: Mice with inactivated tumor necrosis factor (TNF) and lymphotoxin alpha (LT alpha) genes have profound abnormalities of the immune system including lymphocytosis, lack of lymph nodes, undifferentiated spleen, hypoimmunoglobulinaemia, and defective Ig class switch. Here, we asked whether this phenotype is due to incompetent lymphohemopoietic progenitors or to a defective environment. MATERIALS AND METHODS: Lethally irradiated TNF-LT alpha-deficient and wild-type mice received bone marrow cells from either TNF-LT alpha-deficient or wild-type mice. The reconstitution and transfer of the phenotype was followed by morphological and functional analyses. RESULTS: Bone marrow cells from wild-type mice restored the synthesis of TNF and LT alpha, corrected the splenic microarchitecture, normalized the lymphocyte counts in the circulation, and repopulated the lamina propria with IgA-producing plasma cells of TNF-LT alpha-deficient mice. Furthermore, the formation of germinal centers in the spleen and the defective Ig class switch in response to a T-cell dependent antigen was corrected, while no lymph nodes were formed. Conversely, the TNF-LT alpha phenotype could be transferred to wild-type mice by bone marrow transplantation after lethal irradiation. CONCLUSIONS: These data demonstrate that most TNF- and LT alpha-producing cells are bone marrow derived and radiosensitive, and that the immunodeficiency due to TNF-LT alpha deletion can be corrected to a large extent by normal bone marrow cell transplantation. The genotype of the donor bone marrow cells determines the functional and structural phenotype of the TNF-LT alpha-deficient adult murine host, with the exception of lymph node formation. These findings may have therapeutic implications for the restoration of genetically defined immunodeficiencies in humans.

The cytokine production profile, focusing on interleukin-4 (IL-4), interferon-gamma (IFN-gamma) and interleukin-10 (IL-10) of human CD4+, CD8+ and CD4- CD8- alpha beta T cells cloned from the peripheral blood of healthy individuals was compared. Solid-phase anti-CD3 stimulation of CD4- CD8- alpha beta T cell clones from one individual revealed a significantly increased frequency of IL-4-producing clones (81%), compared to CD4+ T cells (24%) or CD8+ (28%). All five CD4- CD8- alpha beta T-cell clones from two other individuals also produced IL-4. Clones that produced IFN-gamma with undetectable IL-4 production, thus being of the 'classical' Th1 phenotype, were infrequent in CD4- CD8- alpha beta T-cell clones (19%) compared to CD4+ (71%), and CD8+ clones (72%) cloned in OKT3, and CD4+ cells cloned in phytohaemaglutinin A (77%). Unlike previously reported studies with gamma delta cells, the alpha beta CD4- CD8- T cells produced IL-10 at appreciable frequency (38%) in PHA generated clones. The supernatants from anti-CD3 stimulated CD4- CD8- alpha beta T-cell clones contained sufficient IL-4 to activate B cells, enhancing CD23 and surface immunoglobulin M (IgM) expression and co-stimulating B-cell proliferation. These findings suggest that the function of CD4- CD8- alpha beta T cells is distinct from that of most CD4+ or CD8+ T cells.

There is increasing evidence that TNF-alpha is a cytokine of major importance in the pathogenesis of rheumatoid arthritis. Since TNF-alpha mediates its effects via high affinity receptors, we were interested in investigating their expression and function in cells from rheumatoid tissue. Synovial fibroblasts derived from rheumatoid synovial tissue are stimulated by TNF-alpha to proliferate and release cytokines, prostaglandins, proteases and protease inhibitors. We have evaluated through which receptor stimulation of DNA synthesis and the release of the proinflammatory agents, IL-6, IL-8 and PGE2 are induced. It was found that rheumatoid synovial fibroblasts express both the p55 and p75 TNF receptor, in a ratio of 4:1. TNF-alpha-stimulated synovial fibroblast DNA synthesis and the release of IL-6, IL-8 and PGE2 was inhibited by antagonist monoclonal antibodies against either the p55 or the p75 TNF receptor, although the blockade of the p55 TNF receptor had a more potent effect than inhibition of the p75 TNF receptor alone. Similarly, specific monoclonal antibodies, agonistic for either the p55 or p75 TNF receptor stimulated synovial fibroblast DNA synthesis, as well as IL-6, IL-8 and PGE2 release. Both p55 and p75 TNF receptors on dermal and gingival fibroblasts were also involved in TNF-alpha-mediated DNA synthesis and IL-6, IL-8 and PGE2 release, although differences in the levels of DNA synthesis and release of inflammatory cytokines and PGE2 were observed between the three fibroblast types.

Bone marrow transplantation (BMT) is currently used to treat patients with severe haematological disorders. One of its major complications is acute graft versus host disease (aGVHD). In this report we show that, during the early stages of lymphoid cell repopulation, an excess of suppressor T cell activity and a deficit of helper T cell activity are easily demonstrated by functional studies in healthy BMT recipients. On the contrary, in patients with active signs of aGVHD, a loss of suppressor T cells, as assessed by coculturing recipient T cells with donor B cells in an in vitro pokeweed mitogen (PWM) dependent Ig secreting assay, is detected. The loss of suppressor T cells in these patients is revealed also by the analysis of peripheral blood lymphocytes with the OKT5 suppressor cytotoxic monoclonal antibody (MoAb) but not with the OKT8 suppressor cytotoxic MoAb. Our data demonstrate that in several BMT recipients, during active aGVHD, a suppressor T cell deficit can be demonstrated both functionally and phenotypically.

A state of immunosuppression or immunodeficiency has been described after surgical trauma. Cellular immune functions are more heavily affected. At present the relationship between these abnormalities and the increased incidence of infections in surgical patients has not been clarified. The activation of the humoral compartment and the appearance of lymphoblastoid B cells spontaneously secreting IgG and IgA have been observed in surgical patients. These cells are a marker of a recent antigenic exposure. In this study the kinetics of appearance of this B cell subset and the relationship between spontaneous Ig secretion and DNA synthesis have been analysed in six cholecystectomized patients. A peak of spontaneous IgG and IgA secretion is evident 5 and 7 days after the intervention. In some patients (two out of six) the appearance of lymphoblastoid B cells is cyclical. A second wave of spontaneous Ig secretion becomes evident 14 days after surgery. Double immunofluorescent staining of peripheral blood lymphocytes for BrdU and cytoplasmic Ig (cIg) was employed to demonstrate that a fraction of lymphoblastoid B cells is actively proliferating and that other cells negative for cIg but active in DNA synthesis appear in the circulation. These data confirm the signs of activation observed after elective surgery in otherwise healthy subjects.

This paper evaluates the cellular immune system of 16 cadaver kidney transplant recipients more than three years after transplantation. The number and percent of active E rosette forming cells, of E rosette forming cells and of IgG and IgM bearing lymphocytes were determined; the blastogenic response of peripheral blood lymphocytes to PHA and PWM was studied in homologous and autologous serum. Significant alterations of the tests performed were found in patients who had undergone kidney transplant, suggesting a pathogenetic role of chronic immunosuppressive therapy but not excluding other possible factors.

Cellular responses to LPS, the major lipid component of the outer membrane of Gram-negative bacteria, are enhanced markedly by the LPS-binding protein (LBP), a plasma protein that transfers LPS to the cell surface CD14 present on cells of the myeloid lineage. LBP has been shown previously to potentiate the host response to LPS. However, experiments performed in mice with a disruption of the LBP gene have yielded discordant results. Whereas one study showed that LBP knockout mice were resistant to endotoxemia, another study did not confirm an important role for LBP in the response of mice challenged in vivo with low doses of LPS. Consequently, we generated rat mAbs to murine LBP to investigate further the contribution of LBP in experimental endotoxemia. Three classes of mAbs were obtained. Class 1 mAbs blocked the binding of LPS to LBP; class 2 mAbs blocked the binding of LPS/LBP complexes to CD14; class 3 mAbs bound LBP but did not suppress LBP activity. In vivo, class 1 and class 2 mAbs suppressed LPS-induced TNF production and protected mice from lethal endotoxemia. These results show that the neutralization of LBP accomplished by blocking either the binding of LPS to LBP or the binding of LPS/LBP complexes to CD14 protects the host from LPS-induced toxicity, confirming that LBP is a critical component of innate immunity.

To investigate the roles of tumor necrosis factor (TNF) and lymphotoxin (LT)-alpha in the development and function of the immune system, the Tnf and Ltalpha genes were simultaneously inactivated in mice by homologous recombination. These mutant mice are highly susceptible to Listeria monocytogenes infection and resistant to endotoxic shock induced by the combined administration of D-galactosamine (D-GaIN) and lipopolysaccharide (LPS). Their splenic microarchitecture is disorganized, characterized by the loss of the clearly defined marginal zone, ill defined T and B cell areas, and absence of MAdCAM-1 and reduced ICAM-1, VCAM-1 and Mac-1 expression. They are devoid of peripheral lymph nodes and Peyer's patches, and show a strong reduction of IgA+ plasma cells in the intestinal lamina propria. The alymphoplasia is accompanied by a marked B lymphocytosis and reduced basal lg levels. Ig depositions in the renal glomerulus and a strong up-regulation of MHC class I antigen expression on endothelial cells of different tissues are observed. The primary humoral immune response towards sheep red blood cells reveals a defective IgG isotype switch, while that against vesicular stomatitis virus is normal. The cytotoxic T cell responses are attenuated, although still effective, against vaccinia, lymphocytic choriomeningitis virus (LCMV-ARM) and LCMV-WE. In conclusion, the combined inactivation of Tnf and Ltalpha confirms their essential role in the normal development and function of the immune system.