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Background: The presence of bacteria in bile is an important factor in the formation of pigment gallstones. The bile of healthy people is sterile and bacteria in the biliary system come from endogenous infection from the gut. Yet, the route of bacterial translocation into the bile duct is still unclear. Theoretically, two routes exist: one is through the intestinal barrier and the other is by direct reflux from the sphincter of Oddi. This study was undertaken to explore the relationship between the effectiveness of intestinal barrier and the formation of pigment gallstones in hamsters., Methods: Thirty-two hamsters were divided into an experimental and a control group, with 16 hamsters in each group. A low protein and high cellulose diet was given for 6 weeks to induce the formation of pigment gallstones in the experimental group (PS) and a normal diet was given to the control group (CON). Morphological changes, changes in the levels of serum endotoxin and diamine oxidase, and changes in the numbers of B lymphocytes, plasma cells and secretory immunoglobin A (sIgA) in the intestinal mucosa were assessed after 6 weeks., Results: Four hamsters died during lithogenesis and body weight decreased in the PS group. Pigment gallstones were found in 11 hamsters at the end of the experiment, giving a lithogenesis rate of 91.67%. The serum endotoxin level before and after gallstone formation in the PS group was 0.2960+/-0.1734 U/ml and 8.2964+/-4.6268 U/ml, respectively (P<0.05). The blood diamine oxidase level before and after gallstone formation in the PS group was 2.6333+/-0.8037 U/ml and 3.3642+/-0.9545 U/ml, respectively (P<0.05). The numbers of B lymphocytes, plasma cells and sIgA in the intestinal mucosa in the PS group were 71.56+/-2.89, 68.65+/-2.09 and 27.56+/-1.07, respectively, and were significantly decreased compared with the corresponding values in the CON group (94.25+/-3.69, 93.47+/-3.98 and 42.57+/-1.96, respectively, P<0.05)., Conclusions: A low protein and high cellulose diet can markedly reduce intestinal barrier function and facilitate the formation of pigment gallstones. The decrease of intestinal barrier function may take part in the formation of pigment gallstones.

Immunity to Francisella tularensis is largely mediated by T lymphocytes but an important role of B lymphocytes in early stage of infection was previously uncovered. We wanted to find out if F. tularensis is able to infect B cells and/or influence them by direct contact. To investigate this possibility we infected B cell lines from mouse (A20) or humans (Ramos RA-1), or primary mouse spleen cells, with F. tularensis LVS and F. tularensis FSC200 in vitro. In all cases, we detected bacteria on the cell surface and inside the B cells using transmission electron microscopy. More than 20% cells were infected by microbes after 24h. The number of bacteria, determined by CFU, increased about 1 log during 24h. Infection with live bacteria led to apoptosis of Ramos cells and mouse CD19(+) spleen cells. Approximately 30% of cells were apoptotic after 24h and 70% after 48 h, independently of the F. tularensis strain, while only 10% of non-infected cell were apoptotic at either time point. Apoptosis was confirmed by Western blot using anti-PARP antibodies. Thus, this study demonstrates unique phenomenon - namely, the ability of the intracellular pathogen F. tularensis to invade and induce apoptosis in B cells.

Johne's disease (JD) is characterized by a protracted period of subclinical infection. Infected cows may remain in the subclinical state until stressors such as parturition and lactation invoke more clinical signs of disease. The objective of this study was to evaluate changes in the percentages of CD4(+), CD8(+), and gammadelta T-cells, B-cells, monocytes, as well as the expression of the activation marker, CD5, on these cell subpopulations in the peripheral blood of dairy cows naturally infected with Mycobacterium avium subsp. paratuberculosis (MAP) during the periparturient period. Peripheral blood mononuclear cells (PBMCs) were collected from 3 wk pre- to 4 wk post-calving and freshly isolated or cultured for 7d. Day 7 cultures were infected with live MAP at a 10:1 MOI (bacteria to adherent PBMC), and cultures were incubated for an additional 24h. Fluorescent antibody labeling of lymphocyte subsets and monocytes was conducted and analyzed with flow cytometry. Freshly isolated PBMCs from subclinical cows expressed a greater (P<0.05) percentage of CD8(+) and gammadelta T-cells compared with clinical cows. The percentage of CD4(+) T-cells increased (P<0.08) in clinical cows as parturition approached. During the postpartum period, clinical cows had greater (P<0.05) CD4:CD8 ratios compared with subclinical and control cows. After 8d, uninfected PBMCs from clinical cows had greater (P<0.05) percentages of CD14(+) cells compared with subclinical cows. When infected with live MAP, there was no effect of infection group or parturition on cell subpopulations. In fresh PBMCs, clinical cows expressed lower percentages of CD4(+)CD5(bright) and CD8(+)CD5(bright) compared with control cows, but greater percentages of CD5(dim) cells for all lymphocyte subsets. These results suggest changes in the percentages of lymphocyte subsets, monocytes, and CD5 markers are modulated by both infection status and the periparturient period.

The spleen plays a pivotal role in the immune defense against encapsulated bacteria such as Streptococcus pneumoniae. Murine splenic marginal zone macrophages express the C-type lectin SIGNR1, which is crucial for the capture of S. pneumoniae from blood. In this study, we demonstrate that SIGNR1 is able to interact in vitro with the juxtaposing marginal zone B cell population, which is responsible for the production of the early IgM response against the S. pneumoniae-epitope phosphorylcholine. Strikingly, SIGNR1-deficient mice display a reduction in the marginal zone B cell population. In addition, ex vivo B cell stimulation assays demonstrate a decrease in phosphorylcholine specificity in the splenic B cell population derived from SIGNR1-deficient mice, whereas the total IgM response is unaffected. Therefore, we hypothesize that antigens are presented by SIGNR1 expressed by marginal zone macrophages to the developing marginal zone B cell population thereby influencing the repertoire of this B cell population, which is pivotal for the early immune response against encapsulated bacteria such as S. pneumoniae.

Periodontal disease is a chronic inflammatory disease in the oral cavity, which culminates in alveolar bone loss. Porphyromonas gingivalis is a consensus periodontal pathogen that has been implicated in adult forms of periodontitis. We previously demonstrated that IL-10-deficient mice exhibit a hyperinflammatory phenotype and are highly susceptible to P. gingivalis-induced periodontitis, indicating an important anti-inflammatory effect of IL-10 in suppressing bone loss. In this study, we analyzed the pathway(s) by which IL-10 deficiency leads to severe P. gingivalis-induced periodontitis. Because Stat3 is essential in IL-10 signaling, immune cell-specific Stat3-deficient mice were subjected to P. gingivalis infection to identify the key IL-10-responsive cells in preventing periodontitis. Myeloid cell-specific Stat3-deficient mice exhibited increased periodontal bone loss (p < 0.001), whereas T cell- and B cell-specific Stat3 mice were resistant, suggesting that macrophages (MP) and/or polymorphonuclear leukocytes are the key target cells normally suppressed by IL-10. Myeloid cell-specific Stat3-deficient mice exhibited elevated gingival CD40L gene expression in vivo compared with wild-type controls (p < 0.01), and Stat3-deficient MPs exhibited vigorous P. gingivalis-stimulated IL-12 production in vitro and induced elevated Ag-specific T cell proliferation compared with wild-type MPs (p < 0.01). Of importance, both IL-12p40/IL-10 and T cell/IL-10 double-deficient mice were resistant to P. gingivalis-induced periodontitis, demonstrating roles for both IL-12p40 and T cells in pathogenesis in a hyperinflammatory model of disease. These data demonstrate that P. gingivalis-induced periodontitis in IL-10-deficient mice is dependent upon IL-12p40-mediated proinflammatory T cell responses.

In immunocompetent humans, cat scratch disease (CSD) is elicited by the Gram-negative bacterium Bartonella henselae and is characterized by a benign regional lymphadenopathy, the pathogenesis of which is poorly understood. Here, we describe a novel mouse model of Bartonella-induced CSD-like disease that allowed us to investigate the mechanisms leading to lymphadenopathy in vivo. In wild-type mice, a subcutaneous inoculation of either viable or inactivated B. henselae led to a strong swelling of the draining lymph node, which was long-lasting despite the rapid elimination of the bacteria. Carboxyfluorescein- and bromodesoxyuridine-labeling experiments showed that lymph node enlargement resulted from modified immigration and enhanced proliferation of lymphocytes, preferentially of B cells. A comparative analysis of B. henselae and the rodent pathogen B. grahamii in wild-type versus interferon-alpha/beta-receptor I chain-deficient mice revealed that interferon-alpha/beta is not only differentially induced by these two Bartonella species but also exerts an inhibitory effect on the development of lymphadenopathy both in vitro and in vivo. These data demonstrate that the lymphadenopathy of human CSD can be reproduced and studied in a mouse model and provide the first insights into the underlying immunological mechanisms.

Rapid waning of anti-polysaccharide bactericidal Ab and vaccine effectiveness is observed following infant immunization with the serogroup C meningococcal (MenC) glycoconjugate vaccine. This is despite the demonstrable presence of immunological memory. Persistence of functional Ab, therefore, appears to be the key determinant of MenC conjugate vaccine effectiveness. Ab persistence is thought to depend in the short term on the survival of plasma cells generated during priming and in the longer term on the production of new Ab secreting cells from memory B cells. In this study, we found a strong association between the level of MenC-specific Ab and the frequency of memory B cells measured at 5 mo of age (1 mo after 3-dose primary immunization with MenC conjugate vaccine), and the persistence of functional Ab at one year of age. These findings suggest that these two parameters are good markers of B cell responses to priming and can be used as predictors of long term humoral immunity induced by glycoconjugate vaccines received in early infancy.

The aim of this work was to discover if Mycoplasma fermentans, which is known to infect B cells, could be the cause of the raised ecto-5'-nucleotidase observed in the synovial fluid of rheumatoid arthritis patients. The ecto-5'-nucleotidase activity in the patients' serum has been shown to correlate with the erythrocyte sedimentation rate and DNA from the mycoplasma has been found in the synovial fluid. B lymphoblastoid cell lines were exposed to 16 strains of Mycoplasma fermentans and their ecto-5'-nucleotidase, CD73, was measured both biochemically and by mouse antibodies to human ecto 5'-nucleotidase using the fluorescence activated cell sorter. The type strain, PG 18, did not grow with the B cells. Some of the mycoplasma strains (9/15) increased the cellular ecto-5'-nucleotidase activity from twice to 17 fold, and usually showed 5'-nucleotidase activity themselves. At least one strain, M106, induced human 5'-nucleotidase on the normally 5'-nucleotidase negative Daudi and Raji Burkitt's lymphoma cell lines, and increased sevenfold the 5'-nucleotidase on the monocyte/macrophage cell line THP-1. Growing the cells in aged medium increased the level of mycoplasma infection. This mycoplasma-induced enzyme showed a conformational change and an increase in activity with a glycosylation change involving mannose groups. The other group of strains, mostly of respiratory or cell culture origin, usually did not have any 5'-nucleotidase of their own and decreased the B-cell enzyme activity by about half. Electron microscopy and flow cytometry showed that the strain M106 was filamentous and could be found inside the B-cells. The 5'-nucleotidase-inducing strains of M. fermentans may be important in the aetiology of rheumatoid arthritis.

Mycobacterium abscessus is an emerging rapidly growing mycobacterium that causes tuberculous-like lesions in humans. We studied the immune control of this organism in C57BL/6 mice challenged intravenously with 10(7) CFU. Bacteria were eliminated from both the spleen and the liver within 90 days, and liver histology showed organized granulomatous lesions. A T- and B-cell requirement was investigated by challenging Rag2-/-, Cd3epsilon-/-, and muMT-/- mice. Rag2-/- and Cd3epsilon-/- mice were significantly impaired in the ability to clear M. abscessus from the liver and spleen, and muMT-/- mice were significantly impaired in the ability to clear M. abscessus from the liver, suggesting that infection control was primarily T cell dependent in the spleen and both T and B cell dependent in the liver. The liver granulomatous response was similar to that of wild-type controls in muMT-/- mice but completely absent in Cd3epsilon-/- and Rag2-/- mice. We studied the involvement of gamma interferon (IFN-gamma) and tumor necrosis factor (TNF) by challenging C57BL/6 mice deficient in the IFN-gamma receptor (Ifngr1-/-) and in TNF (Tnf-/-). Ifngr1-/- mice were significantly impaired in M. abscessus control both in the spleen and in the liver, and granulomas were profoundly altered. The effect was even more substantial in Tnf-/- mice; they failed to control M. abscessus infection in the liver and died within 20 to 25 days after infection with many hepatic inflammatory foci and major lesions of ischemic necrosis in the liver and kidney. These features were not observed with the closely related species M. chelonae. T-cell immunity, IFN-gamma, and TNF are central factors for the control of M. abscessus in C57BL/6 mice, as they are for the control of pathogenic slowly growing mycobacteria.

The human immune response during coccidioidomycosis is intimately involved with the development of delayed-type hypersensitivity and cellular immunity. Sixty percent of those infected have no symptoms and benign outcome is generally associated with a specific cellular immune response to coccidioidal antigens. We have recently teased out the human pulmonary granulomatous response during coccidioidomycosis and noted that there are perigranulomatous clusters of lymphocytes consisting predominantly of B lymphocytes and CD4(+) T lymphocytes. In other work, we have found that the mannose receptor as well as the toll-like receptors TLR2 and TLR4 may have a role in recognizing glycosylated coccidioidal antigens. In addition, the IL-12 receptor axis appears to be operative during antigen recognition and IL-12p40 may be the active moiety. Finally, peripheral blood mononuclear cells from persons with disseminated coccidioidomycosis are able to respond to coccidioidal antigen when it is presented by a mature monocyte-derived IL-4-generated dendritic cell (DC). These observations could be useful in the development of a human vaccine against coccidiodomycosis.

Background & Aims: Recognition of infection leads to induction of adaptive immunity through activation of antigen-presenting cells (APCs). Among APCs, dendritic cells (DCs) have the unique capacity to deliver antigens from the periphery to T cells in secondary lymphoid organs., Methods: We analyzed molecular mechanisms of the Helicobacter pylori-induced APC activation in vitro and investigated the influence of Myd88 signaling on the phenotype of adaptive immunity to H pylori in a murine infection model., Results: The adaptor protein Myd88 mediates Toll-like receptor (TLR), interleukin (IL)-1, and IL-18 signaling. DCs from wild-type, IL-1R(-/-), and IL-18(-/-) mice responded to H pylori with secretion of proinflammatory cytokines and up-regulation of major histocompatibility complex II and costimulatory molecules. In Myd88(-/-) DCs these processes were impaired profoundly, showing that TLR-dependent H pylori-sensing affects DC activation. Analysis of the H pylori-specific DC transcriptome revealed that large parts of the bacteria-induced transcriptional changes depended on Myd88 signaling, comprising numerous genes involved in crucial steps of immune regulation, such as DC maturation/differentiation, antigen uptake/presentation, and effector cell recruitment/activation. The impaired ability of Myd88(-/-) DCs, B cells, and macrophages to mount a proinflammatory response to H pylori in vitro was reflected in vivo by reduced gastric inflammation and increased bacterial colonization in Myd88-deficient mice. Furthermore, Helicobacter-specific IgG2c/IgG1 ratios were reduced in Myd88(-/-) animals, suggesting the involvement of the Myd88-dependent pathway in the instruction of adaptive immunity toward a T helper cell type 1 phenotype., Conclusions: A principal pathway by which DCs sense H pylori and become activated is the TLR-dependent signaling cascade. In vivo, Myd88 signaling affects adaptive immunity to the bacterium.

Bacteria colonize the intestine shortly after birth and thereafter exert several beneficial functions, including induction of protective immunoglobulin A (IgA) antibodies. The distal intestine contains IgA(2), which is more resistant to bacterial proteases than is IgA(1). The mechanism by which B cells switch from IgM to IgA(2) remains unknown. We found that human intestinal epithelial cells (IECs) triggered IgA(2) class switching in B cells, including IgA(1)-expressing B cells arriving from mucosal follicles, through a CD4(+) T cell-independent pathway involving a proliferation-inducing ligand (APRIL). IECs released APRIL after sensing bacteria through Toll-like receptors (TLRs) and further increased APRIL production by activating dendritic cells via thymic stromal lymphopoietin. Our data indicate that bacteria elicit IgA(2) class switching by linking lamina propria B cells with IECs through a TLR-inducible signaling program requiring APRIL. Thus, mucosal vaccines should activate IECs to induce more effective IgA(2) responses.

Our intestine is colonized by an impressive community of bacteria, that has profound effects on the immune functions. The relationship between gut microbiota and the immune system is one of reciprocity: bacteria have important contribution in nutrient processing and education of the immune system and conversely, the immune system, particularly gut-associated lymphoid tissues (GALT) plays a key role in shaping the repertoire of gut microbiota. In this review we discuss new insights into the role of IgA in the maintenance of immune homeostasis and the reciprocal interactions between gut B cells and intestinal bacteria.

The differential expression of homing receptors (HR) and complementary vascular addressins was studied in T and B lymphocytes from ovine tonsils and draining lymph nodes (LN) in uninfected and Brucella melitensis-infected sheep. In uninfected sheep, CD4+CD25+ T cells expressed proportionally more L-selectin and beta1 integrin than beta7 integrin in pharyngeal and palatine tonsils and in parotid LN (PLN), retropharyngeal LN (RLN) and the peripheral prescapular LN (PSLN). In contrast, memory CD4+CD45RA- T cells expressed an equivalent proportion of the three HR in PLN and PSLN, whereas beta1 and beta7 integrins were proportionally more expressed than L-selectin in pharyngeal tonsil. beta7 integrin was proportionally more expressed than beta1 integrin or L-selectin in palatine tonsils, RLN and the mucosal mesenteric LN (MLN). beta1 integrin was proportionally more expressed in IgG+ and IgA+ cells than beta7 integrin and L-selectin in tonsils, PLN and RLN. The main endothelial addressin expressed on venules in both pharyngeal and palatine tonsils, the PLN and RLN, as well as in the PSLN, was the peripheral PNAd, while in the MLN it was MAdCAM-1. Conjunctival infection by Brucella resulted in an increase of CD4+CD25+ and CD4+CD45RA- T cell subsets, which was associated to modifications of HR expression. CD4+CD45RA- T cells expressed proportionally more beta1 and beta7 integrins than L-selectin in regional PLN and RLN, but also in PSLN. The infection induced an increase of IgG+ and IgA+ cell percentages expressing beta1 integrin in all LN, and also beta7 integrin in the RLN. PNAd continued to be expressed on venules of tonsils and draining LN after Brucella infection, and MAdCAM-1 was also weakly expressed on RLN venules. These results suggest that lymphocyte trafficking through tonsils and draining LN could involve L-selectin/PNAd interactions, as well as beta1 or beta7 integrin, possibly in interaction with VCAM-1 or MAdCAM-1. The homing of antigen-specific lymphocytes in these tissues could be modulated after conjunctival infection with Brucella, which induces the recruitment of lymphocytes that express both beta1 and/or beta7 integrin in regional and more distant LN.

Some infectious agents produce molecules capable of interacting specifically with the immunoglobulin heavy- or light-chain variable regions, independently of the conventional-binding site. They are referred to as B cell superantigens (SAgs) and include protein A of Staphylococcus aureus (S. aureus), gp120 of HIV-1, and protein L of Peptostreptococcus magnus (P. magnus). In contrast to conventional antigens, B cell superantigens interact with conserved framework regions of immunoglobulins and can target a large proportion of B cells. In experimental models, they have been demonstrated to deplete B cell subsets responsible for innate functions, namely B-1a and marginal zone (MZ) B cells. As a result, the interactions of these superantigens with host cells impair the humoral immune response. In addition to providing clues toward understanding host-pathogen interactions and microbial pathogenesis, B cell superantigens represent potential therapeutic agents that could be used to specifically modulate expansion of B cell subsets in diseased subjects. In systemic autoimmune diseases, for example, there is activation and expansion of B cells that secrete pathogenic autoantibodies. Their depletion results in clinical improvement in both experimental animals and patients. Currently, attempts are being made to specifically deplete pathogenic autoantibody-producing B cells. Since B-1a and MZ B cells have been found to be expanded in autoimmune disorders, B cell superantigens, used alone or in combination with other biological agents, may have beneficial effects in autoimmune disease management.

The present paradigm dictates that phagocytosis is accomplished mainly by 'professional' phagocytes (such as macrophages and monocytes), whereas B cells lack phagocytic capabilities. Here we demonstrate that B cells from teleost fish have potent in vitro and in vivo phagocytic activities. Particle uptake by B cells induced activation of 'downstream' degradative pathways, leading to 'phagolysosome' formation and intracellular killing of ingested microbes. Those results indicate a previously unknown function for B cells in the innate immunity of these primitive animals. A considerable proportion of Xenopus laevis B cells were also phagocytic. Our findings support the idea that B cells evolved from an ancestral phagocytic cell type and provide an evolutionary framework for understanding the close relationship between mammalian B lymphocytes and macrophages.

The objective of the study was to analyze changes in peripheral blood leukocyte subsets in cases of naturally occurring exudative epidermitis (EE) in pigs. Five of ten piglets developed the chronic clinical form of EE 2-5 days after weaning (PW). Blood samples were obtained at 7, 14 and 21 days from both normal and clinically affected piglets for routine haematology and for the determination of CD45, CD21, CD4, CD8 and gammadeltaTCR cell markers by flow cytometry. When compared with clinically normal piglets EE affected pigs showed significantly decreased values of monocytes at 14 and 21 days PW, and increased numbers of neutrophils and leukocytes at 21 days PW. The EE affected pigs also had an early significant CD4(+) and CD8(high+) T lymphocyte proliferative response at 7 days PW. However affected pigs had a significantly reduced number of B (CD21(+)) and gammadeltaTCR(+) T lymphocytes in blood at 21 days PW. Although all values remained within the normal range, the significant differences in some peripheral blood leukocyte subsets between the two groups of piglets suggest that the generalised cutaneous infection with Staphylococcus hyicus is severe enough to induce a systemic inflammatory and immune responses.

The fungus, Histoplasma capsulatum, produces a persistent infection. Reactivation histoplasmosis is largely a result of impaired immunity, but the perturbations associated with escape of the fungus from host defenses remain ill-defined. We analyzed a murine model of reactivation to elucidate the host defects that permit reactivation. C57BL/6 mice were infected intranasally and, 42 days later, they were depleted of CD4(+) and CD8(+) cells. Elimination of these cells, but not either alone, produced a persistent infection over several weeks. Neutralization of IFN-gamma, TNF-alpha, or both did not induce reactivation. Endogenous IL-10 exacerbated reactivation. Depletion of T cells in B cell(-/-) mice induced a markedly higher burden in organs when compared with wild type. However, the infection remained persistent. Elimination of CD4(+) cells alone or neutralization of cytokines increased the fungal load. The persistent infection was not dependent on gammadelta T cells or NK cells. Elimination of Thy-1.2(+) cells in mice given mAb to CD4 and CD8 transformed reactivation into a progressive, lethal infection in B cell(-/-) and wild-type mice, but the tempo of progression was accelerated in the former. The data reveal the complex control by the host to prevent reactivation of this fungus.

Helicobacter pylori infection of the human stomach causes chronic gastritis that can lead to gastric cancer. Because activated lymphocytes persist in the gastric mucosa, and because a high multiplicity of infection (MOI) of H. pylori is needed to induce apoptosis in vitro, we speculated that resistance of lymphocytes to apoptosis is an important feature of the immune response to H. pylori. Freshly isolated mouse splenocytes underwent substantial spontaneous apoptosis and displayed a biphasic response to H. pylori, in which low MOI (1-10) markedly inhibited apoptosis, whereas high MOI (> or =75) potentiated apoptosis. Low MOI reduced mitochondrial membrane depolarization, caspase-3 and caspase-9 activation, and cytochrome c release and increased Bcl-2 levels. Low MOI also induced cellular proliferation. When cells were subjected to fluorescence-activated cell sorting after coculture with H. pylori, CD19+ B cells were found to be protected from apoptosis and undergoing proliferation at low MOI, whereas CD3+ T cells did not exhibit this pattern. The protective effect of low MOI on apoptosis persisted even when B cells were isolated before activation. Immunophenotyping showed that all B-cell subsets examined were protected from apoptosis at low MOI. Additionally, gastric infection with H. pylori resulted in protection of splenic B cells from spontaneous apoptosis. Our results suggest that the low levels of H. pylori infection that occur in vivo are associated with B-cell survival and proliferation, consistent with their potential to evolve into mucosa-associated lymphoid tissue lymphoma.

Studies of microbial superantigens that target large clonal sets of B cells through conserved antigen-receptor-variable-region sites are providing new insights into the mechanisms of B-cell activation-induced cell death. These investigations have shown differences between the clonal regulation of follicular B cells (B2 cells) and the innate-like marginal-zone B cells and B1 cells, and have also shown how B-cell superantigens can affect specialized host defences against infection. Agents designed to emulate the properties of B-cell superantigens might also provide new approaches for the treatment of B-cell-mediated autoimmune and neoplastic diseases.

Non-Hodgkin lymphomas develop from nodal and extranodal lymphoid tissues. A distinct subset of extranodal lymphomas arising from B cells of the marginal zone (MZ) of mucosa-associated lymphoid tissue (MALT) or spleen has been individualized. Growing evidence indicates that MZ lymphomas are associated with chronic antigenic stimulation by microbial pathogens and/or autoantigens. The list of microbial species associated with MZ lymphoproliferations has grown longer with molecular investigations and now comprises at least 5 distinct members: H. pylori, C. jejuni, B. burgdorferi, C. psittaci, and hepatitis C virus (HCV), which have been associated with gastric lymphoma, immunoproliferative small intestinal disease, cutaneous lymphoma, ocular lymphoma, and spleen lymphoma, respectively. A pathophysiologic scenario involving chronic and sustained stimulation of the immune system leading to lymphoid transformation has emerged. It defines a distinct category of infection-associated lymphoid malignancies, in which the infectious agent does not directly infect and transform lymphoid cells, as do the lymphotropic oncogenic viruses Epstein-Barr virus (EBV), human herpesvirus 8 (HHV8), and human T-lymphotropic virus 1 (HTLV-1), but rather indirectly increases the probability of lymphoid transformation by chronically stimulating the immune system to maintain a protracted proliferative state.

Autoantibodies are removed from the repertoire at two checkpoints during B cell development in the bone marrow and the periphery. Despite these checkpoints, up to 20% of the antibodies expressed by mature naive B cells in healthy humans show low levels of self-reactivity. To determine whether self-reactive antibodies are also part of the antigen-experienced memory B cell compartment, we analyzed recombinant antibodies cloned from single circulating human IgM+ memory B cells. Cells expressing antibodies specific for individual bacterial polysaccharides were expanded in the IgM+ memory compartment. In contrast, B cells expressing self-reactive and broadly bacterially reactive antibodies were removed from the repertoire in the transition from naive to IgM+ memory B cell. Selection against self-reactive antibodies was implemented before the onset of somatic hypermutation. We conclude that a third checkpoint selects against self-reactivity during IgM+ memory B cell development in humans.

We have previously shown that zidovudine plus sulfamethoxazole-trimethoprim exposure decreases immune cell populations in the bone marrow of healthy mice by inducing apoptosis. The hypothesis of the current work was that this toxicity would have an adverse impact on the immune response. To determine this, BALB/c mice were treated with zidovudine, sulfamethoxazole-trimethoprim, the combination of both drugs, or vehicle only (control) via oral gavage for 21 days. On day 4 after dosing completion, the mice were infected intratracheally with 1x10(7) Pneumocystis murina organisms. Immune cell populations (in lung digest, bronchoalveolar lavage fluid, tracheobronchial lymph node, and bone marrow samples), the lung Pneumocystis burden, and serum Pneumocystis-specific antibody titers were determined at days 6, 10, and 20 postinfection. While total bone marrow cellularity was recovered by day 6 postinfection in the combination exposure group, B-cell numbers did not recover until 10 days postinfection, primarily due to the persistent depletion of the late pre-B-cell phenotype. The numbers of CD4+ and CD8+ T cells, as well as the numbers of total B cells and activated B cells in tracheobronchial lymph nodes, were decreased at days 10 and 20 as a result of zidovudine plus sulfamethoxazole-trimethoprim exposure compared to the numbers in the control group. No significant differences in lung lavage or lung digest cell populations were observed. There was a trend of a delay in Pneumocystis clearance in the combination treatment group, and Pneumocystis-specific serum immunoglobulin G titers were reduced at day 20 postinfection. Together, these data indicate that the combination of zidovudine and sulfamethoxazole-trimethoprim adversely affects the humoral immune response to Pneumocystis.

Besides various gastroduodenal diseases, Helicobacter pylori infection may be involved in autoimmune disorders like rheumatoid arthritis (RA) or idiopathic thrombocytopenic purpura. Such autoimmune disorders are often associated with autoreactive antibodies produced by B-1 cells, a subpopulation of B lymphocytes. These B-1 cells are mainly located in the pleural cavity or mucosal compartment. The existence of H. pylori urease-specific immunoglobulin A (IgA)-producing B cells in the mucosal compartment and of their specific IgM in the sera of acutely infected volunteers suggests the possibility that urease stimulates mucosal innate immune responses. Here, we show for the first time that purified H. pylori urease predominantly stimulates the B-1-cell population rather than B-2 cells, which produce antigen-specific conventional antibodies among splenic B220(+) B cells. The fact that such stimulation of B-1 cells was not affected by the addition of polymyxin B indicates that the effect of purified H. pylori urease was not due to the contamination with bacterial lipopolysaccharide. Furthermore, the production of various B-1-cell-related autoreactive antibodies such as IgM-type rheumatoid factor, anti-single-stranded DNA antibody, and anti-phosphatidyl choline antibody was observed when the splenic B cells were stimulated with purified H. pylori urease in vitro. These findings suggest that H. pylori components, urease in particular, may be among the environmental triggers that initiate various autoimmune diseases via producing autoreactive antibodies through the activation of B-1 cells. The findings shown here offer important new insights into the pathogenesis of autoimmune disorders related to H. pylori infection.

Helicobacter pylori causes various gastroduodenal diseases including gastric MALT lymphoma, but the mechanism underlying H. pylori-induced carcinogenesis is not known. The alternative pathway for NF-kappaB activation, which involves the processing of NF-kappaB2/p100 to p52, has been implicated in lymphocyte survival, attenuated apoptosis, and secondary lymphoid tissue development. In this study, we investigated H. pylori-induced activation of NF-kappaB through the alternative pathway in B lymphocytes. In immunoblot and EMSA, H. pylori induced NF-kappaB2/p100 processing to p52 and subsequent nuclear accumulation in IM-9 (human B cell line) cells and human peripheral blood B cells, but not in AGS (human gastric cancer cell line) cells. The activation of the alternative pathway was LPS-dependent but not cag pathogenicity island-dependent. Alternative pathway activation by H. pylori was associated with attenuated apoptosis. The expression levels of B lymphocyte chemoattractant, EBI-1 ligand chemokine, and stromal cell-derived factor-1alpha mRNAs were up-regulated in cocultured human B cells and in infected human gastric mucosa. In the infected mucosa, NF-kappaB2/p100 and p52 were detected immunohistochemically in the cytoplasm and nuclear compartments of lymphocytes, but not in epithelial cells. In summary, H. pylori activates the alternative NF-kappaB pathway in B lymphocytes. The effects on chemokine production and antiapoptosis mediated by H. pylori-induced processing of NF-kappaB2/p100 to p52 may drive lymphocytes to acquire malignant potential.

The plague is caused by the bacterium Yersinia pestis. Plague bacteria are thought to inject effector Yop proteins into host cells via the type III pathway. The identity of the host cells targeted for injection during plague infection is unknown. We found, using Yop beta-lactamase hybrids and fluorescent staining of live cells from plague-infected animals, that Y. pestis selected immune cells for injection. In vivo, dendritic cells, macrophages, and neutrophils were injected most frequently, whereas B and T lymphocytes were rarely selected. Thus, it appears that Y. pestis disables these cell populations to annihilate host immune responses during plague.

Gamma interferon (IFN-gamma)-activated macrophages use an alternative processing mechanism to present Salmonella antigens to CD8(+) T lymphocytes. This pathway involves processing of antigen in a vacuolar compartment followed by secretion and loading of antigenic peptides to major histocompatibility complex class I (MHC-I) molecules on macrophage cell surface and bystander cells. In this study, we have shown that B lymphocytes are not able to process Salmonella antigens using this alternative pathway. This is due to differences in Salmonella enterica serovar Typhimurium-containing vacuoles (SCV) when comparing late endosomal-lysosomal processing compartments in B lymphocytes to those in macrophages. The IFN-gamma-activated IC21 macrophage cell line and A-20 B-cell line were infected with live or dead Salmonella enterica serovar Typhimurium. The SCV in B cells were in a late endosomal-lysosomal compartment, whereas SCV in macrophages were remodeled to a non-characteristic late endosomal-lysosomal compartment over time. Despite the difference in SCV within macrophages and B lymphocytes, S. enterica serovar Typhimurium survives more efficiently within the IFN-gamma-activated B cells than in activated macrophage cell lines. Similar results were found during in vivo acute infection. We determined that a lack of remodeling of late endosomal-lysosomal compartments by live Salmonella infection in B lymphocytes is associated with the inability to use the alternative MHC-I antigen-processing pathway, providing a survival advantage to the bacterium. Our data also suggest that the B lymphocyte late endosome-lysosome environment allows the expression of Salmonella virulence mechanisms favoring B lymphocytes in addition to macrophages and dendritic cells as a reservoir during in vivo infection.

B cells are divided into two categories: conventional or B-2B cells and B-1B cells, the latter of which are distinguished by their different ontogeny. B cell lymphoma 1 (BCL1), the first-reported case of a spontaneously developed mouse B-lymphoma, expresses CD5, surface IgM, Mac-1, CD43 and low level of B220, and is likely to have B-1a cell origin. However, antigens recognized by IgM produced by the BCL1 cells (BCL1-IgM) have not been identified. Here, we demonstrate that BCL1-IgM reacts with Escherichia coli (E. coli). Our initial finding that several recombinant proteins expressed in E. coli bound to BCL1-B20 prompted us to examine the possibility that BCL1 cells may bind E. coli. Indeed, BCL1 cells bound fluorescein-labeled E. coli. To elucidate the structure on the BCL1 cells responsible for E. coli-binding, we produced a monoclonal antibody capable of inhibiting BCL1 binding to E. coli. The antibody recognizes an idiotypic epitope on the BCL1-IgM. Moreover, polyclonal antibody against IgM and secreted BCL1-IgM purified from the supernatants inhibited BCL1 binding to E. coli. Finally, transfection of non-lymphoid cells with cDNA of heavy and light chains of BCL1-IgM conferred the cells ability to bind E. coli. These results clearly indicate that BCL1-IgM bind E. coli and suggest that BCL1 lymphoma is a typical B-1 cell-derived lymphoma, characterized not only by the surface phenotype, but also by the reactivity of its IgM with commensal bacteria.

Objective: To investigate the expression of CD158b on peripheral blood lymphocytes after kidney transplantation., Methods: Sixty two kidney transplant patients were divided into two groups (normal group and rejection group) according to pathologic results and clinical situation. Blood samples were assessed for percentage of CD3+; CD19+; CD3-CD16/56+; CD3+CD158b+; CD19+CD158b+, and CD3-CD16/56+CD158b+ subsets., Results: The percentages of CD3+ cells preop as well as at 1 and 7 postoperative and the day acute rejection happened were 60.06 +/- 4.67, 40.43 +/- 4.11, 31.67 +/- 4.04, and 39.21 +/- 5.20, respectively. The percentages of CD3-CD16/56+ were 21.65 +/- 1.79, 33.84 +/- 5.45, 38.10 +/- 4.86, and 39.53 +/- 4.80, respectively. The percentages of CD3+CD158b+ were 1.46 +/- 0.31, 1.88 +/- 0.70, 2.03 +/- 1.04, and 0.65 +/- 0.12, respectively. The percentages of CD3-CD16/56+CD158b+ were 5.87 +/- 1.24, 3.57 +/- 0.57, 2.82 +/- 0.45, and 1.60 +/- 0.33, respectively., Conclusions: The percentage of CD3+ cells in the normal and the rejection groups decreased significantly. The percentages of CD158b+T cells decreased significantly after acute rejection. The percentage of CD158b+NK cells decreased significantly after kidney transplantation, decreasing gradually after acute rejection. The percentage of CD158b+ total T cells decreased significantly following acute rejection. The percentage of CD3-CD16/56+CD158b+ of total NK cells decreased significantly after kidney transplantation and after acute rejection. Because few factors interfere with the expression of CD158b on NK cells, monitoring of this marker may be accurate and sensitive.

A fractal analysis is presented for the detection of pathogens such as Franscisela tularensis, Yersinia pestis (the bacterium that causes plague), Bacillus anthracis, Venezuelan equine encephalitis (VEE) virus, Vavcinia virus, and Escherichia coli using a cellular analysis and notification of antigens risks and yields (CANARY) biosensor [T.H. Rider, M.S. Petrovic, F.E. Nargi, J.D Harper, E.D. Schwoebel, R.H. Mathews, D.J. Blanchard, L.T Bortolin, A.M. Young, J. Chen, M.A. Hollis, A cell-based sensor for rapid identification of pathogens, Science 301 (2003, 11 July) 213-215, T.H. Rider, M.S. Petrovic, F.E. Nargi, J.D. Harper, E.D. Schwoebel, R.H. Mathews, D.J. Blanchard, L.T. Bortolin, A.M. Young, J. Chen, M.A. Hollis, A cell-based sensor for rapid identification of pathogens, Science 301 (2003, 11 July) 213-215. Science Online, www.sciencemag.org/cgi/content/full/031/5630/213/DC1]. In general, the binding and dissociation rate coefficients may be adequately described by either a single- or a dual-fractal analysis. An attempt is made to relate the binding rate coefficient to the degree of heterogeneity (fractal dimension value) present on the biosensor surface. Binding and dissociation rate coefficient values obtained are presented. Due to the dilute nature of the analyte(s) present, in some cases, a triple-fractal analysis is required to adequately describe the binding kinetics. It should be noted, and this is not entirely unexpected, that there is a lot of variation in the original experimental data when dilute concentrations of the analyte were analyzed by the CANARY biosensor [T.H. Rider, M.S. Petrovic, F.E. Nargi, J.D Harper, E.D. Schwoebel, R.H. Mathews, D.J. Blanchard, L.T Bortolin, A.M. Young, J. Chen, M.A. Hollis, A cell-based sensor for rapid identification of pathogens, Science 301 (2003, 11 July) 213-215, Science Online, www.sciencemag.org/cgi/content/full/031/5630/213/DC1]. The data analyzed in this manuscript appears smoother since only discrete points at different time intervals were analyzed. The kinetics aspects along with the affinity values presented are of interest and should along with the rate coefficients presented for the binding and the dissociation phase be of significant interest in help designing better biosensors for an application area that is bound to gain increasing importance in the future.

The IL-12/IFN-gamma axis is crucial for protective immunity to Mycobacterium in humans and mice. Our goal was to analyze the relative contribution of various human blood cell subsets and molecules to the production of, or response to IL-12 and IFN-gamma. We designed an assay for the stimulation of whole blood by live M. bovis Bacillus Calmette-Guerin (BCG) alone, or BCG plus IL-12 or IFN-gamma, measuring IFN-gamma and IL-12 levels. We studied patients with a variety of specific inherited immunodeficiencies resulting in a lack of leukocytes, or T, B, and/or NK lymphocytes, or polymorphonuclear cells, or a lack of expression of key molecules such as HLA class II, CD40L, NF-kappaB essential modulator (NEMO), and IL-1 receptor-associated kinase-4 (IRAK-4). Patients with deficiencies in IL-12p40, IL-12 receptor beta1 chain (IL-12Rbeta1), IFN-gammaR1, IFN-gammaR2, and STAT-1 were used as internal controls. We showed that monocytes were probably the main producers of IL-12, and that NK and T cells produced similar amounts of IFN-gamma. NEMO and IRAK-4 were found to be important for IL-12 production and subsequent IFN-gamma production, while a lack of CD40L or HLA class II had no major impact on the IL-12/IFN-gamma axis. The stimulation of whole blood by live BCG thus triggers the IL-12/IFN-gamma axis by an IRAK-4- and NEMO-dependent, non-cognate interaction between monocytes, NK, and T lymphocytes.

Despite the fact that, in a number of countries, vaccination programmes are extensively used to control Salmonella infection in poultry, information on the immune mechanisms, especially the cellular response, is still needed. The aim of the study was to characterise the B cell and macrophage response in caecum (IgA+, IgM+, IgG+ cells, macrophages), bursa of Fabricius (IgM+ cells, macrophages), and spleen (IgM+ cells) of chicks after oral administration of a non-attenuated Salmonella (S.) typhimurium wild-type strain (infection) or an attenuated commercial live S. typhimurium vaccine strain (immunisation) to day-old chicks as compared to non-treated control birds using immunohistochemistry and image analysis. In caecum, higher counts of IgM-secreting cells were detected in infected animals compared with the controls from day 5 until day 12 of age. In contrast, in treated groups, IgA-secreting cells were found in higher numbers only between day 8 and 12 of age. Infected birds showed a higher number of IgA+ cells in spleen and bursa of Fabricius compared to the controls. In the bursa of Fabricius of immunised and infected birds, a depletion of strongly stained IgM+ cells and macrophages was established between day 5 and 9 indicating a possibly special and independent role of this organ during the immunological reaction against Salmonella organisms. The results suggest that IgM- and IgA-secreting cells are of importance in the caecal immune response of chickens against Salmonella strains. Immunised chickens always showed a weaker immune reaction compared to infected animals. Present findings regarding the B cell reaction within avian caeca prove a participation of both humoral and cellular immunity in defence against Salmonella strains. Immunohistochemical examination of the cellular response (B cells and macrophages) in relevant organs of chickens may be an important tool to evaluate the immunogenic characteristics of potential Salmonella live vaccine candidates.

Intestinal bacteria are required for development of gut-associated lymphoid tissues (GALT), which mediate a variety of host immune functions, such as mucosal immunity and oral tolerance. In rabbits, the intestinal microflora are also required for developing the preimmune Ab repertoire by promoting somatic diversification of Ig genes in B cells that have migrated to GALT. We studied the mechanism of bacteria-induced GALT development. Bacteria were introduced into rabbits in which the appendix had been rendered germfree by microsurgery (we refer to these rabbits as germfree-appendix rabbits). We then identified specific members of the intestinal flora that promote GALT development. The combination of Bacteroides fragilis and Bacillus subtilis consistently promoted GALT development and led to development of the preimmune Ab repertoire, as shown by an increase in somatic diversification of VDJ-C micro genes in appendix B cells. Neither species alone consistently induced GALT development, nor did Clostridium subterminale, Escherichia coli, or Staphylococcus epidermidis. B. fragilis, which by itself is immunogenic, did not promote GALT development; hence, GALT development in rabbits does not appear to be the result of an Ag-specific immune response. To identify bacterial pathways required for GALT development, we introduced B. fragilis along with stress-response mutants of B. subtilis into germfree-appendix rabbits. We identified two Spo0A-controlled stress responses, sporulation and secretion of the protein YqxM, which are required for GALT development. We conclude that specific members of the commensal, intestinal flora drive GALT development through a specific subset of stress responses.

Marek's disease virus (MDV) is classified as an oncogenic lymphotropic herpesvirus of chickens. MDV productively and cytolytically infects B, alphabetaT and gammadeltaT lymphocytes and latently infects T-helper lymphocytes. The aims of this study were to identify whether MDV infects macrophages in vivo and, if so, whether quantitative differences in macrophage infection are associated with MDV strain virulence. Chickens were infected with either virulent MDV (HPRS-16) or 'hypervirulent' MDV (C12/130). Flow cytometry with monoclonal antibodies recognizing MDV pp38 antigen and leukocyte antigens was used to identify MDV lytically infected cells. Macrophages from HPRS-16- and C12/130-infected chickens were pp38(+). It is demonstrated that macrophages are pp38(+) because they are infected and not because they have phagocytosed MDV antigens, as assessed by confocal microscopy using antibodies recognizing MDV antigens of the three herpesvirus kinetic classes: infected cell protein 4 (ICP4, immediate early), pp38 (early) and glycoprotein B (gB, late). Spleen macrophages from MDV-infected chickens were ICP4(+), pp38(+) and gB(+), and ICP4 had nuclear localization denoting infection. Finally, MDV pp38(+) macrophages had high inherent death rates, confirming cytolytic MDV infection, although production of virus particles has not been detected yet. These results have two fundamental implications for understanding MDV pathogenesis: (i) MDV evolved to perturb innate, in addition to acquired, immunity and (ii) macrophages are excellent candidates for transporting MDV to primary lymphoid organs during the earliest stages of pathogenesis.

Immunity to Listeria monocytogenes is largely mediated by T lymphocytes. Recently, B lymphocytes or their secreted products are implicated to provide immunity against L. monocytogenes infection. To understand whether L. monocytogenes can infect and kill B cells as a possible strategy to initiate an infection, we examined the effects of L. monocytogenes on a human B lymphoma (Ramos RA-1) and mouse primary B cells in vitro. L. monocytogenes infection resulted in significantly (p

Various leukocytes are involved in the reaction to counter Salmonella infection in chicken. The various leukocyte types react differently after an infection, since some clear the infection while others may cause dissemination of Salmonella throughout the chicken. Therefore, we investigated in vitro the entry and survival of Salmonella enterica serotype Enteritidis in chicken cell lines of various cell types, including two macrophage cell lines, HD11 and MQ-NCSU (NCSU), two B-cell lines LSCC-1104-X5 (1104) and LSCC-RP9 (RP9), and a T-cell line MDCC-MSB-1 (MSB-1). The macrophages were able to internalize high numbers of S. Enteritidis. In contrast and as expected, cells of the T-cell line MSB-1 and the B-cell line RP9 internalized bacteria at a much lower level. After S. Enteritidis entered the macrophages, the number of intracellular S. Enteritidis decreased over time, so that after 48h no more than 20% of the bacteria, which had entered, survived intracellularly. In contrast to macrophages, the number of S. Enteritidis in cells of the T-cell line MSB-1 and the B-cell line RP9 increased rapidly within 12h post-inoculation. Thereafter the number of intracellular S. Enteritidis decreased only slowly. In conclusion, all three different cell types were able to control and to start clearing S. Enteritidis, although macrophages were far more effective compared to T- and B-cells. However, none of the cell lines were able to clear S. Enteritidis fully within 48h. These results suggest that the three cell types play an important but different role in the dissemination and elimination of S. Enteritidis throughout the animal.

Cesarean-derived piglets were reared for 5 wk under germfree conditions or monoassociated with a benign Escherichia coli (G58-1) or a enterohemorrhagic strain (933D) derived from O157:H7, and immunized i.p. with the T-dependent (TD) Ags fluorescein-labeled (FL) keyhole limpet hemocyanin or trinitrophenylated (TNP) keyhole limpet hemocyanin and the type 2 T-independent Ags TNP-Ficoll or FL-Ficoll. Only colonized piglets showed an increase in serum IgG, IgA, and IgM and had serum Abs to FL, TNP, and colonizing bacteria. While serum Abs to FL or TNP appeared following colonization alone, secondary responses were restricted to piglets immunized using TD carriers. While animals colonized with 933D had significantly higher total serum IgG and IgM levels and specific IgG Abs than those colonized with G58-1, no differences were seen in serum IgA levels, B cell diversification in the ileal Peyer's patches, and specific activity (ELISA activity per micrograms of Ig) of pre-boost serum IgG and IgM anti-TNP and anti-FL Abs. Serum IgA Abs to TNP, FL, or bacteria were not detected. Ag-driven responses, as measured by an increase in specific Ab activity, were only observed in secondary responses to TD Ags and to colonizing, pathogenic E. coli. We propose that germline-encoded, isotype-switched B cells in newborn piglets differentiate to Ab-secreting cells 1) after stimulation by bacteria-activated APCs or 2) through direct stimulation by bacterial products. We further propose that Ag-driven systemic responses require both bacterial colonization and TD Ags translocated to the peritoneum.

Objective: We previously reported that B cells lacking the RP105 molecule, which proved to be highly activated B cells, are increased in the peripheral blood of patients with systemic lupus erythematosus (SLE). In the present study, we attempted to determine whether RP105-negative B cells obtained from SLE patients would be capable of producing autoantibodies as well as immunoglobulins., Methods: RP105-positive and RP105-negative B cells, sorted by cell sorter, were cultured for 5 days without stimulation, or were stimulated with Staphylococcus aureus Cowan 1 strain (SAC) or recombinant interleukin-6 (IL-6). For the assay of autoantibodies, RP105-positive and RP105-negative B cells were cultured separately for 10 days with anti-CD3 antibody-stimulated T cells. The production of immunoglobulins and autoantibodies was determined by enzyme-linked immunosorbent assay., Results: We demonstrated that RP105-negative B cells, but not RP105-positive B cells, obtained from SLE patients could spontaneously produce IgG and IgM in vitro until day 5. SAC and IL-6 enhanced production of IgG and IgM by RP105-negative B cells but failed to induce such production by RP105-positive B cells. The latter cells, however, when cocultured with activated T cells in the presence of IL-10, produced IgG, although the amount was very small compared with that produced by RP105-negative B cells. Most important, under these conditions, anti-double-stranded DNA antibodies were produced only by the RP105-negative B cells obtained from SLE patients., Conclusion: These data indicate that RP105-negative B cells, constituting a subset of B cells in SLE patients, are highly activated and may be responsible for the production of autoantibodies as well as polyclonal immunoglobulins.

Background: Identification of differentially expressed genes between normal and diseased states is an area of intense current medical research that can lead to the discovery of new therapeutic targets. However, isolation of differentially expressed genes by subtraction often suffers from unreported contamination of the resulting subtraction library with clones containing DNA sequences not from the original RNA samples., Materials and Methods: Subtraction using cDNA representational difference analysis (RDA) was performed on human B cells from normal or common variable immunodeficiency patients. The material remaining after the subtraction was cloned and individual clones were sequenced. The sequence of one clone with similarity to integrases (ILG1, integrase-like gene-1) was used to obtain the full length cDNA sequence and as a probe for the presence of this sequence in RNA or genomic DNA samples., Results: After five rounds of cDNA RDA, 23.3% of the clones from the resulting subtraction library contained Escherichia coli DNA. In addition, three clones contained the sequence of a new integrase, ILG1. The full length cDNA sequence of ILG1 exhibits prokaryotic, but not eukaryotic, features. At the DNA level, ILG1 is not similar to any known gene. At the protein level, ILG1 has 58% similarity to integrases from the cryptic P4 bacteriophage family (S clade). The catalytic domain of ILG1 contains the conserved features found in site-specific recombinases. The critical residues that form the catalytic active site pocket are conserved, including the highly conserved R-H-R-Y hallmark of these recombinases. Interestingly, ILG1 was not present in the original B cell populations. By probing genomic DNA, ILG1 could only be detected in the E. coli TOP10F' strain used in our laboratory for molecular cloning, but not in any of its precursor strains, including TOP10. Furthermore, bacteria cultured from the mouth of the laboratory worker who performed cDNA RDA were also positive for ILG1., Conclusions: In the course of our studies using cDNA RDA, we have isolated and identified ILG1, a likely active site-specific recombinase and new member of the bacteriophage P4 family of integrases. This family of integrases is implicated in the horizontal DNA transfer of pathogenic genes between bacterial species, such as those found in pathogenic strains of E. coli, Shigella, Yersinia, and Vibrio cholera. Using ILG1 as a marker of our laboratory E. coli strain TOP10F', our evidence suggests that contaminating bacterial DNA in our subtraction experiment is due to this laboratory bacterial strain, which colonized exposed surfaces of the laboratory worker. Thus, identification of differentially expressed genes between normal and diseased states could be dramatically improved by using extra precaution to prevent bacterial contamination of samples.

The potential sequelae of intestinal infection with Yersinia enterocolitica include reactive arthritis, erythema nodosum, Reiter's syndrome and other autoimmune diseases. The role of the immune response in the pathogenesis of these diseases has not been fully defined, but autoimmune manifestations may be a consequence of the increase in autoantibodies as a result of polyclonal B-cell activation induced by Yersinia. We investigated the effects of Y. enterocolitica O:3 derivatives on B lymphocyte activation in vivo. Groups of five specific pathogen free (SPF) Swiss mice were inoculated with bacterial cell extract, Yersinia outermembrane proteins (Yops) or lipopolysaccharide (LPS) obtained from Y. enterocolitica O:3 and their immunoglobulin-secreting spleen cells were detected by isotype-specific protein A plaque assay. The presence of specific anti-Yersinia antibodies and autoantibodies was determined in mouse sera by ELISA. In all experiments a marked increase in the number of secretory cells of different isotypes was observed as early as the third day after inoculation. IgG and IgM anti-Yersinia antibodies were detected in the sera of all inoculated mice, and autoantibodies against myosin in the sera of those inoculated with bacterial cell extract. The sera from animals stimulated with LPS reacted with myelin, actin and laminin, while the sera from mice inoculated with Yops reacted with myelin, thyroglobulin and cardiolipin. These results suggest that SPF Swiss mice inoculated with any one of the Y. enterocolitica derivatives tested exhibited polyclonal activation of B lymphocytes as a result of stimulation by various bacterial components and not only LPS stimulation.

Helicobacter pylori infection has been implicated in the development of gastrointestinal malignancy in adults and children. The histopathological processes that lead to such development are unknown. We compared the immune cell repertoire of mucosal lymph follicles in children with H. pylori infection to B cell type mucosal associated lymphoid tissue (MALT)-lymphoma of adults. The B and T cell populations residing within the lymph follicles and/or within B cell type MALT lymphoma were characteriZed by an immunohistochemical technique, utilizing B and T cell markers including: CD3, CD4, CD8 (T cells); CD20, CD40, GD74, BLA36, CD80, CD86 (B cells). Stain intensity was compared between the samples. T cell repertoire was observed within the lymph follicles, but not in the B cell MALT-lymphoma specimens. No significant difference was observed between the staining of CD40, CD74, CD8, and BLA36. The B cell markers, CD80 and CD86, were found within the centrocytic zone of the lymph follicle. In the B cell repertoire, no significant difference was observed between the lymph follicles of children with H. pylori infection and the adult MALT-lymphoma specimens except in CD20. B and T cells were in close anatomical proximity, enabling them to interact and exchange immunological information.

Objective: To determine whether defective T cell apoptosis is associated with the development of a chronic arthritis subsequent to mycoplasma infection, and to determine whether deletion of T cells can prevent the development of this arthritis., Methods: B6 wild-type (B6-+/+), B6-lpr/lpr, and B6-gld/gld mice were infected with Mycoplasma pulmonis. The severity of lymphocytic infiltration and joint damage was evaluated, and the degree of recovery of viable mycoplasma from the spleen and joints was determined. Antigen-presenting cells derived from Fas mutant lpr mice (lpr-APC) were transfected ex vivo with an adenovirus (Ad) vector to yield lpr-APC expressing high levels of Fas ligand (lpr-APC-AdFasL), which in turn were transferred intraperitoneally into M pulmonis-infected B6-gld/gld mice. The development of arthritis subsequent to M pulmonis infection and the induction of apoptosis of cells within the synovial tissue and lymph nodes of lpr-APC-AdFasL-treated B6-gld/gld mice were determined., Results: Infection of B6-lpr/lpr and B6-gld/gld mice with M pulmonis resulted in an acute-phase inflammation of the synovium that later developed into a chronic erosive arthritis. Similar infection of B6-+/+ mice resulted only in an acute joint inflammatory response that resolved. Chronic arthritis in B6-gld/gld mice and B6-lpr/lpr was not due to persistent infection, since there were no differences in the rates of clearance of M pulmonis from the joints of B6-gld/gld or B6-lpr/lpr mice compared with B6-+/+ mice. Treatment of infected B6-gld/gld mice with lpr-APC-AdFasL resulted in a significantly decreased incidence of chronic arthritis that was associated with a decrease in lymph node T cells, but not with apoptosis of synovial T cells or fibroblasts., Conclusion: Defective Fas/FasL-mediated apoptosis of T cells is an important factor that rendered arthritis-resistant B6 mice susceptible to the development of a chronic erosive arthritis subsequent to mycoplasma infection. In vivo lpr-APC-AdFasL cell-gene therapy is a safe and effective method for inhibiting the development of this arthritis.

Phagocytosis of Opa+ Neisseria gonorrhoeae (gonococcus, GC) by neutrophils is in part dependent on the interaction of Opa proteins with CGM1a (CEACAM3/CD66d) antigens, a neutrophil-specific receptor. However, the signaling pathways leading to phagocytosis have not been characterized. Here we show that interaction of OpaI bacteria with neutrophils or CGM1a-transfected DT40 cells induces calcium flux, which correlates with phagocytosis of bacteria. We identified an immunoreceptor tyrosine-based activation motif (ITAM) in CGM1a, and showed that the ability of CGM1a to transduce signals and mediate phagocytosis was abolished by mutation of the ITAM tyrosines. We also demonstrated that CGM1a-ITAM-mediated bacterial phagocytosis is dependent on Syk and phospholipase C activity in DT40 cells. Unexpectedly, the activation of the CGM1a-ITAM phagocytic pathway by Opa+ GC results in induction of cell death.

The most recent therapeutic approaches can improve the outcome of B-cell neoplasia. By PCR analysis we amplify tumor specific DNA sequences of clonal IgH rearrangement from a limited number of malignant cells against a background of normal B cells. Recently described PCR based techniques for tracking minimal residual disease (MRD) in B lymphoproliferative disorders have given promising but discordant results, with significant variations in the sensitivity and specificity of the procedures. We have developed a three step single strand conformational polymorphism polymerase chain reaction (SSCP-PCR) strategy which is able to detect clonal malignant cells in B lymphoproliferative disorders at a frequency as low as 1 in 10(6) cells. Since this method is simple, rapid, reliable and as specific as ASO-PCR, it could be especially useful in monitoring patients affected by B lymphoproliferative disorders in complete haematological and immunophenotypic remission.

In vitro studies have demonstrated direct interactions between Borrelia burgdorferi and human B and T cells. However, largely because disseminated infections typically occur at very low density, little is known about associations between spirochetes and mammalian host cells in vivo. To assess whether spirochetes interact directly with lymphocytes in mammals, we developed a mouse model for lymphotropism. By repeatedly coincubating spirochetes with primary mouse lymphocytes that were immobilized by adherence to immunomagnetic beads, we were able to preferentially enrich cultures for or against bacteria with constitutive affinity for murine B and T cells. Populations of lymphotropically enriched, stock infectious, and lymphotropically depleted spirochetes were injected intradermally into mice. Lymphocytes were then purified from the blood and spleens of challenged mice and placed into spirochetal culture medium. Cultures of B. burgdorferi were obtained from primary lymphocyte preparations from mice challenged with each of the three populations of spirochetes. Recovery of lymphocyte-associated bacteria occurred within 1 h of challenge with enriched bacteria. Lymphocyte preparations from mice challenged with stock infectious and lymphotropically depleted bacteria produced cultures after 1 day postchallenge. All lymphocyte preparations were culture negative after 1 week. These results demonstrate that lymphotropic B. burgdorferi is infectious in mice and suggest that associations between spirochetes and lymphocytes occur in vivo. The results also suggest that factors involved in lymphocytic binding may be inducible in vivo. Thus, this system provides a model for studying the role of such interactions in mammalian infections.

Bacterial type III secretion system drives the translocation of virulence factors into the cystosol of host target cells. In phagocytes and in Epstein-Barr virus immortalized B lymphocytes, NADPH oxidase generates O(-2) through an electron transfer chain the activity of which depends on the assembly of three, p67(phox), p47(phox) and p40(phox) cytosolic activating factors with Rac 1/2 and a membrane redox component, cytochrome b(558). In p67(phox) deficient chronic granulomatous disease (CGD) patients, p67-phox is missing and NADPH oxidase activity is abolished. ExoS is a virulence factor of Pseudomonas aeruginosa which is secreted via the type III secretion system: it was fused with p67(phox). Pseudomonas aeruginosa synthesized and translocated the hybrid ExoS-p67(phox) fusion protein into the cytosol of B lymphocytes via the type III secretion system. Purified ExoS-p67(phox) hybrid protein was as efficient as normal recombinant p67(phox) in cell-free reconstitution of NADPH oxidase activity. Therefore, ExoS-p67(phox) was transferred via the type III secretion system of Pseudomonas aeruginosa into the cytosol of B lymphocytes from a p67(phox)-deficient CGD patient and functionally reconstituted NADPH oxidase activity. In the complementation process, ExoS acted as a molecular courier for protein delivery: the reconstitution of an active NADPH oxidase complex suggests type III secretion system to be a new approach for cellular therapy., (Copyright 2000 Academic Press.)

Infection by Helicobacter pylori leads to injury of the gastric epithelium and a cellular infiltrate that includes CD4+ T cells. H. pylori binds to class II MHC molecules on gastric epithelial cells and induces their apoptosis. Because urease is an abundant protein expressed by H. pylori, we examined whether it had the ability to bind class II MHC and induce apoptosis in class II MHC-bearing cells. Flow cytometry revealed the binding of PE-conjugated urease to class II MHC+ gastric epithelial cell lines. The binding of urease to human gastric epithelial cells was reduced by anti-class II MHC Abs and by staphylococcal enterotoxin B. The binding of urease to class II MHC was confirmed when urease bound to HLA-DR1-transfected COS-1 (1D12) cells but not to untransfected COS-1 cells. Urease also bound to a panel of B cell lines expressing various class II MHC alleles. Recombinant urease induced apoptosis in gastric epithelial cells that express class II MHC molecules, but not in class II MHC- cells. Also, Fab from anti-class II MHC and not from isotype control Abs blocked the induction of apoptosis by urease in a concentration-dependent manner. The adhesin properties of urease might point to a novel and important role of H. pylori urease in the pathogenesis of H. pylori infection.

Human granulocytic ehrlichiosis (HGE) is a febrile tick-borne illness caused by a recently discovered intracellular bacterium remarkable for its tropism for professionally phagocytic neutrophils. Monoclonal antibodies against the P-selectin binding domain of the leukocyte P-selectin glycoprotein ligand, PSGL-1, prevented HGE cell binding and infection, as did enzymatic digestion of PSGL-1. Furthermore, simultaneous neoexpression in nonsusceptible cells of complementary DNAs for both PSGL-1 and its modifying alpha-(1,3) fucosyltransferase, Fuc-TVII, allowed binding and infection by HGE. Thus, the HGE bacterium specifically bound to fucosylated leukocyte PSGL-1. Selectin mimicry is likely central to the organism's unique ability to target and infect neutrophils.

The attenuated Salmonella typhimurium PhoPc strain is avirulent but immunogenic via the oral route in mice and is attenuated in survival in macrophage cell lines. In this study, the fate of PhoPc bacteria expressing green fluorescent protein was investigated in murine Peyer's patches. The survival of PhoPc was monitored after orogastric inoculation of BALB/c mice. Bacteria persisted for several weeks in the Peyer's patches and were also recovered from the mesenteric lymph nodes and spleen. Confocal microscopy analysis identified dendritic cells as the Peyer's patch cell type that internalized PhoPc expressing green fluorescent protein at early time points. In addition, live PhoPc were found in Peyer's patch dendritic cells and not in B cells 3 days after orogastric inoculation. Taken together, these results provide strong evidence that PhoPc is internalized and survives within Peyer's patch dendritic cells. As these cells are potent antigen-presenting cells, these data could explain the immunogenicity of S. typhimurium vaccine strains in vivo.