We show clear evidence for direct infection of various human epithelial cells by Epstein-Barr virus (EBV) in vitro. The successful infection was achieved by using recombinant EBV (Akata strain) carrying a selective marker gene but without any other artificial operations, such as introduction of the known EBV receptor (CD21) gene or addition of polymeric immunoglobulin A against viral gp350 in culture. Of 21 human epithelial cell lines examined, 18 became infected by EBV, as ascertained by the detection of EBV-determined nuclear antigen (EBNA) 1 expression in the early period after virus exposure, and the following selection culture easily yielded a number of EBV-infected clones from 15 cell lines. None of the human fibroblasts and five nonhuman-derived cell lines examined was susceptible to the infection. By comparison, cocultivation with virus producers showed approximately 800-fold-higher efficiency of infection than cell-free infection did, suggesting the significance of direct cell-to-cell contact as a mode of virus spread in vivo. Most of the epithelial cell lines infectable with EBV were negative for CD21 expression at the protein and mRNA levels. The majority of EBV-infected clones established from each cell line invariably expressed EBNAAs, rightward transcripts from the BamHI-A region of the virus genome, and latent membrane protein (LMP) 2A, but not the other EBNAs or LMP1. This restricted form of latent viral gene expression, which is a central issue for understanding epithelial oncogenesis by EBV, resembled that seen in EBV-associated gastric carcinoma and LMP1-negative nasopharyngeal carcinoma. The results indicate that direct infection of epithelial cells by EBV may occur naturally in vivo, and this could be mediated by an unidentified, epithelium-specific binding receptor for EBV. The EBV convertants are viewed, at least in terms of viral gene expression, as in vitro analogs of EBV-associated epithelial tumor cells, thus facilitating analysis of an oncogenic role(s) for EBV in epithelial cells.

Lymphocytes are most reliably subdivided on the basis of their receptors for antigen at the cell surface. Three subtypes of lymphocytes are well defined: B cells that bear surface immunoglobulin and make antibody, CD4+T cells with CD3 alpha beta receptors specific for antigen associated with class II major histocompatibility complex molecules, and CD8+T cells with CD3 alpha beta receptors specific for antigen associated with class I MHC molecules. These T cells are responsible for known forms of cell-mediated immunity. The discovery of a third rearranging T-cell specific gene called gamma (refs 1 and 2) has revealed the presence of a new class of T cells bearing a new receptor type, CD3 gamma delta (refs 3-7). To date, neither the function nor the specificity of cells bearing this receptor has been determined. Because gamma delta T cells are the main lymphocyte of epidermis, it was proposed that such cells could be important in surveillance of all epithelia. We have isolated intraepithelial lymphocytes from murine small intestine, and shown that they predominantly or exclusively express CD3 gamma delta receptors. Unlike the epidermal lymphocytes, these cells also express CD8, and they use a different V lambda gene to form their receptor. This strongly suggests that gamma delta T cells home in a very specific manner to epithelia, where they presumably mediate their function.

The candidate oncogene bcl-3 was discovered as a translocation into the immunoglobulin alpha-locus in some cases of B-cell chronic lymphocytic leukaemias. The protein Bcl-3 contains seven so-called ankyrin repeats. Similar repeat motifs are found in a number of diverse regulatory proteins but the motifs of Bcl-3 are most closely related to those found in I kappa B proteins in which the ankyrin repeat domain is thought to be directly involved in inhibition of NF-kappa B activity. No biological function has yet been described for Bcl-3, but it was noted recently that Bcl-3 interferes with DNA-binding of the p50 subunit of NF-kappa B in vitro. Here we demonstrate that Bcl-3 can aid kappa B site-dependent transcription in vivo by counteracting the inhibitory effects of p50/NF-kappa B homodimers. Bcl-3 may therefore aid activation of select NF-kappa B-regulated genes, including those of the human immunodeficiency virus.

The role of antibodies (Abs) in the development of chronic colitis in T cell receptor (TCR)-alpha-/- mice was explored by creating double mutant mice (TCR-alpha-/- x immunoglobulin (Ig)mu-/-), which lack B cells. TCR-alpha-/- x Ig mu-/- mice spontaneously developed colitis at an earlier age, and the colitis was more severe than in TCR-alpha-/- mice. Colitis was induced in recombination-activating gene-1 (RAG-1-/-) mice by the transfer of mesenteric lymph node (MLN) cells from TCR-alpha-/- x Ig mu-/- mice. When purified B cells from TCR-alpha-/- mice were mixed with MLN cells before cell transfer, colitis did not develop in RAG-1-/- mice. Administration of the purified Ig from TCR-alpha-/- mice and a mixture of monoclonal autoAbs reactive with colonic epithelial cells led to attenuation of colitis in TCR-alpha-/- x Ig mu-/- mice. Apoptotic cells were increased in the colon, MLN, and spleen of TCR-alpha-/- x Ig mu-/- mice as compared to Ig mu-/- mice and TCR-alpha-/- mice. Administration of the purified Ig from TCR-alpha-/- mice into TCR-alpha-/- x Ig mu-/- mice led to decrease in the number of apoptotic cells. These findings suggest that although B cells are not required for the initiation of colitis, B cells and Igs (autoAbs) can suppress colitis, presumably by affecting the clearance of apoptotic cells.

Molecular cloning has recently established that the 15;12 chromosome translocations in murine plasmacytomas fuse DNA from chromosome 15 to the immunoglobulin heavy (H) chain locus, usually within the switch recombination region near the alpha constant region gene. We show here that the incoming DNA bears the cellular gene (c-myc) homologous to the oncogene (v-myc) of avian retrovirus MC29. In human Burkitt lymphomas bearing an 8;14 translocation, c-myc was also rearranged, apparently (in at least two cases) to an H chain switch recombination region (mu or alpha), and both products of a reciprocal chromosome exchange were detectable. Both the murine and human c-myc genes contain two exons homologous to v-myc, and additional 5' and 3' murine genomic segments (apparent exons) were defined by hybridization to c-myc mRNAs. In plasmacytomas, chromosome breakpoints fall near or within the 5' exon and apparently disrupt the normal c-myc transcriptional unit, because plasmacytoma c-myc mRNAs differ from the mRNA in lines without c-myc rearrangement. The translocated gene presumably has lost its normal 5' regulatory sequences and may well encode an altered myc polypeptide. We propose that altered expression of the c-myc gene, induced by translocation to an immunoglobulin locus, is a critical oncogenic event for these B lymphoid tumors. Two events may be required, because the plasmacytoma oncogene capable of transforming fibroblasts is not c-myc.

Progression to destructive insulitis in nonobese diabetic (NOD) mice is linked to the failure of regulatory cells, possibly involving T helper type 2 (Th2) cells. Natural killer (NK) T cells might be involved in diabetes, given their deficiency in NOD mice and the prevention of diabetes by adoptive transfer of <em>alpha</em>/beta double-negative thymocytes. Here, we evaluated the role of NK T cells in diabetes by using transgenic NOD mice expressing the T cell antigen receptor (TCR) <em>alpha</em> chain V<em>alpha</em>14-J<em>alpha</em>281 characteristic of NK T cells. Precise identification of NK1.1(+) T cells was based on out-cross with congenic NK1.1 NOD mice. All six transgenic lines showed, to various degrees, elevated numbers of NK1.1(+) T cells, enhanced production of interleukin (IL)-4, and increased levels of serum <em>immunoglobulin</em> E. Only the transgenic lines with the largest numbers of NK T cells and the most vigorous burst of IL-4 production were protected from diabetes. Transfer and cotransfer experiments with transgenic splenocytes demonstrated that V<em>alpha</em>14-J<em>alpha</em>281 transgenic NOD mice, although protected from overt diabetes, developed a diabetogenic T cell repertoire, and that NK T cells actively inhibited the pathogenic action of T cells. These results indicate that the number of NK T cells strongly influences the development of diabetes.

The <em>alpha</em>-gal epitope (Gal<em>alpha</em>1-3Galbeta1-(3)4GlcNAc-R) is abundantly synthesized on glycolipids and glycoproteins of non-primate mammals and New World monkeys by the glycosylation enzyme <em>alpha</em>1,3galactosyltransferase (<em>alpha</em>1,3GT). In humans, apes and Old World monkeys, this epitope is absent because the <em>alpha</em>1,3GT gene was inactivated in ancestral Old World primates. Instead, humans, apes and Old World monkeys produce the anti-Gal antibody, which specifically interacts with <em>alpha</em>-gal epitopes and which constitutes approximately 1% of circulating <em>immunoglobulins</em>. Anti-Gal has functioned as an immunological barrier, preventing the transplantation of pig organs into humans, because anti-Gal binds to the <em>alpha</em>-gal epitopes expressed on pig cells. The recent generation of <em>alpha</em>1,3GT knockout pigs that lack <em>alpha</em>-gal epitopes has resulted in the elimination of this immunological barrier. Anti-Gal can be exploited for clinical use in cancer immunotherapy by targeting autologous tumour vaccines to APC, thereby increasing their immunogenicity. Autologous intact tumour cells from haematological malignancies, or autologous tumour cell membranes from solid tumours are processed to express <em>alpha</em>-gal epitopes by incubation with neuraminidase, recombinant <em>alpha</em>1,3GT and with uridine diphosphate galactose. Subsequent immunization with such autologous tumour vaccines results in in vivo opsonization by anti-Gal IgG binding to these <em>alpha</em>-gal epitopes. The interaction of the Fc portion of the vaccine-bound anti-Gal with Fcgamma receptors of APC induces effective uptake of the vaccinating tumour cell membranes by the APC, followed by effective transport of the vaccinating tumour membranes to the regional lymph nodes, and processing and presentation of the tumour-associated antigen (TAA) peptides. Activation of tumour-specific T cells within the lymph nodes by autologous TAA peptides may elicit an immune response that in some patients will be potent enough to eradicate the residual tumour cells that remain after completion of standard therapy. A similar expression of <em>alpha</em>-gal epitopes can be achieved by transduction of tumour cells with an adenovirus vector (or other vectors) containing the <em>alpha</em>1,3GT gene, thus enabling anti-Gal-mediated targeting of the vaccinating transduced cells to APC. Intratumoral delivery of the <em>alpha</em>1,3GT gene by various vectors results in the expression of <em>alpha</em>-gal epitopes. Such expression of the xenograft carbohydrate phenotype is likely to induce anti-Gal-mediated destruction of the tumour lesion, similar to rejection of xenografts by this antibody. Opsonization of the destroyed tumour cell membranes by anti-Gal IgG further targets them to APC, thus converting the tumour lesion, treated by the <em>alpha</em>1,3GT gene, into an in situ autologous tumour vaccine.

Mucosal antibody defense depends on a complex cooperation between local B cells and secretory epithelia. Mucosa-associated lymphoid tissue gives rise to B cells with striking J-chain expression that are seeded to secretory effector sites. Such preferential homing constitutes the biological basis for local production of polymeric immunoglobulin A (pIgA) and pentameric IgM with high affinity to the epithelial pIg receptor that readily can export these antibodies to the mucosal surface. This ultimate functional goal of mucosal B-cell differentiation appears to explain why the J chain is also expressed by IgG- and IgD-producing plasma cells (PCs) occurring at secretory tissue sites; these immunocytes may be considered as 'spin-offs' from early effector clones that through class switch are on their way to pIgA production. Abundant evidence supports the notion that intestinal PCs are largely derived from B cells initially activated in gut-associated lymphoid tissue (GALT). Nevertheless, insufficient knowledge exists concerning the relative importance of M cells, major histocompatibility complex class II-expressing epithelial cells, and professional antigen-presenting cells for the uptake, processing, and presentation of luminal antigens in GALT to accomplish the extensive and sustained priming and expansion of mucosal B cells. Likewise, it is unclear how the germinal center reaction in GALT so strikingly can promote class switch to IgA and expression of J chain. Although B-cell migration from GALT to the intestinal lamina propria is guided by rather well-defined adhesion molecules and chemokines/chemokine receptors, the cues directing preferential homing to different segments of the gut require better definition. This is even more so for the molecules involved in homing of mucosal B cells to secretory effector sites beyond the gut, and in this respect, the role of Waldever's ring (including the palatine tonsils and adenoids) as a regional inductive tissue needs further characterization. Data suggest a remarkable compartmentalization of the mucosal immune system that must be taken into account in the development of effective local vaccines to protect specifically the airways, eyes, oral cavity, small and large intestines, and urogenital tract.

Microglial activation and adaptive immunity have been implicated in the neurodegenerative processes in Parkinson disease. It has been proposed that these responses may be triggered by modified forms of alpha-synuclein (alpha-SYN), particularly nitrated species, which are released as a consequence of dopaminergic neurodegeneration. To examine the relationship between alpha-SYN, microglial activation, and adaptive immunity, we used a mouse model of Parkinson disease in which human alpha-SYN is overexpressed by a recombinant adeno-associated virus vector, serotype 2 (AAV2-SYN); this overexpression leads to slow degeneration of dopaminergic neurons. Microglial activation and components of the adaptive immune response were assessed using immunohistochemistry; quantitative polymerase chain reaction was used to examine cytokine expression. Four weeks after injection, there was a marked increase in CD68-positive microglia and greater infiltration of B and T lymphocytes in the substantia nigra pars compacta of the AAV2-SYN group than in controls. At 12 weeks, CD68 staining declined, but B- and T-cell infiltration persisted. Expression of proinflammatory cytokines was enhanced, whereas markers of alternative activation (i.e. arginase I and interleukins 4 and 13) were not altered. Increased immunoreactivity for mouse immunoglobulin was detected at all time points in the AAV2-SYN animals. These data show that overexpression of alpha-SYN alone, in the absence of overt neurodegeneration, is sufficient to trigger neuroinflammation with both microglial activation and stimulation of adaptive immunity.

High frequency of erythrocyte (red blood cell [RBC]) genetic disorders such as sickle cell trait, thalassemia trait, homozygous hemoglobin C (Hb-C), and glucose-6-phosphate dehydrogenase (G6PD) deficiency in regions with high incidence of Plasmodium falciparum malaria and case-control studies support the protective role of those conditions. Protection has been attributed to defective parasite growth or to enhanced removal of the parasitized RBCs. We suggested enhanced phagocytosis of rings, the early intraerythrocytic form of the parasite, as an alternative explanation for protection in G6PD deficiency. We show here that P falciparum developed similarly in normal RBCs and in sickle trait, beta- and alpha-thalassemia trait, and HbH RBCs. We also show that membrane-bound hemichromes, autologous immunoglobulin G (IgG) and complement C3c fragments, aggregated band 3, and phagocytosis by human monocytes were remarkably higher in rings developing in all mutant RBCs considered except alpha-thalassemia trait. Phagocytosis of ring-parasitized mutant RBCs was predominantly complement mediated and very similar to phagocytosis of senescent or damaged normal RBCs. Trophozoite-parasitized normal and mutant RBCs were phagocytosed similarly in all conditions examined. Enhanced phagocytosis of ring-parasitized mutant RBCs may represent the common mechanism for malaria protection in nonimmune individuals affected by widespread RBC mutations, while individuals with alpha-thalassemia trait are likely protected by a different mechanism.

Vertebrate T cells express either an alpha beta or gamma delta T cell receptor (TCR). The developmental relatedness of the two cell types is unresolved. alpha beta + T cells respond to specific pathogens by collaborating with immunoglobulin-producing B cells in distinct lymphoid organs such as the spleen and Peyer's patches. The precise influence of alpha beta + T cells on B cell development is poorly understood. To investigate the developmental effects of alpha beta + T cells on B cells and gamma delta + T cells, mice homozygous for a disrupted TCR alpha gene were generated. The homozygotes showed elimination of alpha beta + T cells and the loss of thymic medullae. Despite this, gamma delta + T cells developed in normal numbers, and there was an increase in splenic B cells.

The events of B-cell differentiation can be reconstructed in part through an analysis of the organisation of heavy-chain gene segments in differentiated B cells. A mouse immunoglobulin alpha heavy-chain gene is composed of at least three noncontiguous germ-line DNA segments--a VH gene segment, a JH gene segment associated with the Cmu gene segment, and the C alpha gene segment. These gene segments are joined together by two distinct types of DNA rearrangements--a V-J joining and a CH switch.

beta-O-linked N-acetylglucosamine (O-GlcNAc) is an abundant posttranslational modification of resident nuclear and cytoplasmic proteins in eukaryotes. Increasing evidence suggests that O-GlcNAc plays a regulatory role in numerous cellular processes. Here we report on the production and characterization of a highly specific mouse monoclonal antibody, MAb CTD110.6, that specifically reacts with O-GlcNAc. The antibody recognizes O-GlcNAc in beta-O-glycosidic linkage to both serine and threonine. We could detect no cross-reactivity with alpha-linked Ser/Thr-O-GlcNAc, alpha-linked Ser-O-linked N-acetylgalactosamine (O-GalNAc), or N-linked oligosaccharides on ovalbumin and immunoglobulin G. The monosaccharide GlcNAc, but not GalNAc, abolishes immunoreactivity, further demonstrating specificity toward O-GlcNAc. Furthermore, galactose capping of O-GlcNAc sites also inhibits CTD110.6 immunoreactivity. Enrichment of GlcNAc-containing glycoproteins using the lectin wheat germ agglutinin dramatically enriches for CTD110.6-reactive proteins. The antibody reacts with a large number of proteins from cytoplasmic and nuclear extracts and readily detects in vivo changes in O-GlcNAc modification. These studies demonstrate that CTD110.6 is highly specific toward O-GlcNAc, with no cross-reactivity toward similar carbohydrate antigens or toward peptide determinants.

Mucosal surfaces are protected specifically by secretory immunoglobulin A (SIgA) and SIgM generated through external translocation of locally produced dimeric IgA and pentameric IgM. Their active transport is mediated by the epithelial polymeric Ig receptor (pIgR), also called the transmembrane secretory component. Paracellular passive external transfer of systemic and locally produced antibodies also provides mucosal protection, making the biological importance of secretory immunity difficult to assess. Here we report complete lack of active external IgA and IgM translocation in pIgR knockout mice, indicating no redundancy in epithelial transport mechanisms. The knockout mice were of normal size and fertility but had increased serum IgG levels, including antibodies to Escherichia coli, suggesting undue triggering of systemic immunity. Deterioration of their epithelial barrier function in the absence of SIgA (and SIgM) was further attested to by elevated levels of albumin in their saliva and feces, reflecting leakage of serum proteins. Thus, SIgA did not appear to be essential for health under the antigen exposure conditions of these experimental animals. Nevertheless, our results showed that SIgA contributes to maintenance of mucosal homeostasis. Production of SIgA might therefore be a variable in the initiation of human immunopathology such as inflammatory bowel disease or gluten-sensitive enteropathy.

The role of endogenous gamma interferon (IFN-gamma) in protective immunity against blood-stage Plasmodium chabaudi AS malaria was studied using IFN-gamma gene knockout (GKO) and wild-type (WT) C57BL/6 mice. Following infection with 10(6) parasitized erythrocytes, GKO mice developed significantly higher parasitemia during acute infection than WT mice and had severe mortality. In infected GKO mice, production of interleukin 12 (IL-12) p70 and tumor necrosis factor alpha in vivo and IL-12 p70 in vitro by splenic macrophages was significantly reduced compared to that in WT mice and the enhanced nitric oxide (NO) production observed in infected WT mice was completely absent. WT and GKO mice had comparable numbers of total nucleated spleen cells and B220(+) and Mac-1(+) spleen cells both before and after infection. Infected WT mice, however, had significantly more F4/80(+), NK1.1(+), and F4/80(+)Ia(+) spleen cells than infected GKO mice; male WT had more CD3(+) cells than male GKO mice. In comparison with those from WT mice, splenocytes from infected GKO mice had significantly higher proliferation in vitro in response to parasite antigen or concanavalin A stimulation and produced significantly higher levels of IL-10 in response to parasite antigen. Infected WT mice produced more parasite-specific immunoglobulin M (IgM), IgG2a, and IgG3 and less IgG1 than GKO mice. Significant gender differences in both GKO and WT mice in peak parasitemia levels, mortality, phenotypes of spleen cells, and proliferation of and cytokine production by splenocytes in vitro were apparent during infection. These results thus provide unequivocal evidence for the central role of endogenous IFN-gamma in the development of protective immunity against blood-stage P. chabaudi AS.

The human serum immunoglobulins IgG and IgA1 are produced in bone marrow and both interact with specific cellular receptors that mediate biological events. In contrast to IgA1, the glycosylation of IgG has been well characterized, and its interaction with various Fc receptors (Fc Rs) has been well studied. In this paper, we have analyzed the glycosylation of IgA1 and IgA1 Fab and Fc as well as three recombinant IgA1 molecules, including two N-glycosylation mutants. Amino acid sequencing data of the IgA1 Fc O-glycosylated hinge region indicated that O-glycans are located at Thr228, Ser230, and Ser232, while O-glycan sites at Thr225 and Thr236 are partially occupied. Over 90% of the N-glycans in IgA1 were sialylated, in contrast to IgG, where < 10% contain sialic acid. This paper contains the first report of Fab glycosylation in IgA1, and (in contrast to IgG Fab, which contains only N-linked glycans) both N- and O-linked oligosaccharides were identified. Analysis of the N-glycans attached to recombinant IgA1 indicated that the Cα 2 N-glycosylation site contained mostly biantennary glycans, while the tailpiece site, absent in IgG, contained mostly triantennary structures. Further analysis of these data suggested that processing at one Fc N-glycosylation site affects the other. Neutrophil Fcα R binding studies, using recombinant IgA1, indicated that neither the tailpiece region nor the N-glycans in the C alpha 2 domain contribute to IgA1-neutrophil Fcα R binding. This contrasts with IgG, where removal of the Fc N-glycans reduces binding to the Fcγ R. The primary sequence and disulfide bond pattern of IgA1, together with the crystal structures of IgG1 Fc and mouse IgA Fab and the glycan sequencing data, were used to generate a molecular model of IgA1. As a consequence of both the primary sequence and S-S bond pattern, the N-glycans in IgA1 Fc are not confined within the inter-α-chain space. The accessibility of the Cα 2 N-glycans provides an explanation for the increased sialylation and galactosylation of IgA1 Fc over that of IgG Fc N-glycans, which are confined in the space between the two Cγ 2 domains. This also suggests why in contrast to IgG Fc, the IgA1 N-glycans are not undergalactosylated in rheumatoid arthritis.

We have studied somatic cell hybrids between P3x63Ag8 mouse myeloma cells deficient in hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) and either human peripheral lymphocytes or human lymphoblastoid or myeloma cells for the production of human immunoglobulin chains and for the expression of enzyme markers assigned to each of the different human chromosomes. Human chromosome 14 was the only human chromosome present in all independent hybrids producing mu, gamma, and alpha human heavy chains. In two of the independent hybrids that produced human heavy chains, human chromosome 14 was the only human chromosome present in the hybrid cells. Loss of human chromosome 14 from these hybrids resulted in the concomitant loss of their ability to produce human immunoglobulin heavy chains. In view of these results, we conclude that the genes for human immunoglobulin heavy chains are located on human chromosome 14 in immunoglobulin-producing human cells.

Although altered cytokine homeostasis has been implicated in the pathogenesis of alcoholic liver disease, the relationship between cytokines and metabolic consequences of alcoholic liver disease is unknown. We, therefore, sought to correlate circulating concentrations of tumor necrosis factor-alpha, interleukin-1 and interleukin-6 to clinical and biochemical parameters of liver disease in chronic alcoholic patients. We used an enzyme-linked immunosorbent assay to measure plasma tumor necrosis factor and interleukin-1 and a bioassay to measure serum interleukin-6 in three groups of alcoholic men as follows: (a) actively drinking alcoholic men without evidence of chronic liver disease, (b) nondrinking alcoholic men with stable cirrhosis and (c) patients with acute alcoholic hepatitis. Mean cytokine concentrations were elevated in cirrhotic patients and alcoholic hepatitis patients compared with controls and alcoholic patients without liver disease. Tumor necrosis factor-alpha and interleukin-1 alpha concentrations remained elevated for up to 6 mo after diagnosis of alcoholic hepatitis, whereas interleukin-6 normalized in parallel with clinical recovery. Concentrations of all three cytokines were correlated with biochemical parameters of liver injury and hepatic protein synthesis plus serum immunoglobulin concentrations. We could not demonstrate a relationship between cytokine concentrations and peripheral endotoxemia. Percentages of peripheral blood monocytes that reacted with monoclonal antibodies to CD25 (interleukin-2 receptor) and human lymphocyte antigen-DR were similar for alcoholic patients and controls. These data suggest that tumor necrosis factor-alpha and interleukin-1 alpha are related to some of the metabolic consequences of both acute and chronic alcohol-induced liver disease, whereas interleukin-6 is related to abnormalities seen in acute liver injury.

The Research Group on Progressive Renal Diseases conducted a national survey, in Japan, of cases of primary glomerulonephritis (GN) in 1985 and 1993. The results of the survey, reported here, revealed a high prevalence and relatively poor prognosis for immunoglobulin A nephropathy (IgAN). Using immunofluorescent microscopy, 47.2% of 1,063 patients were diagnosed as having IgAN; 62.8% of patients had diffuse mesangial proliferative GN, and focal mesangial proliferative GN was observed in 23.0%. Nearly 70% of the patients had no clinical symptoms, and the IgAN was detected by routine physical examination. The mean period of observation was 11.8 +/- 6.3 years. Renal survival rates for the 502 cases of IgAN, in which the start of dialysis and renal-related death were end points, were 96%, 85%, 75%, and 61% at 5, 10, 15, and 20 years, respectively, from the time of the detection of the earliest known renal abnormalities. Renal survival rates of patients with diffuse mesangial proliferative GN were 96%, 83%, 75%, and 59% at 5, 10, 15, and 20 years, respectively. At the end of the observation period, 20% of patients had improved, 45.8% showed no change, 13.5% had deteriorated, and 20.4% had renal-related death. The risk factors for renal failure by logistic multivariate analysis were serum creatinine concentration>> or =1.4 mg/dL (relative risk, 3.5) and levels of urinary protein>> or = +(dipstick) (relative risk, 6.4), determined at the time of biopsy. These parameters can be useful for assessing prognosis during the relatively advanced stages of this disease. It is important to note that a relatively high percentage of patients with IgAN progressed to end-stage renal failure even when their histologic findings comprised only minor glomerular abnormalities or focal proliferative changes.

We have employed a germfree mouse model to study the development and persistence of a humoral mucosal immune response to a gram-negative murine commensal organism, Morganella morganii. M. morganii bacteria rapidly colonize the gut, resulting in hypertrophy of Peyer's patches (PP), including germinal center reactions (GCR), and the development of specific immunoglobulin A (IgA) responses detected in vitro in PP fragment cultures and by ELISPOT assays of lamina propria cells. The GCR peaks 14 days after infection and begins to wane thereafter. Upon colonization, the organisms successfully translocate to the mesenteric lymph node and spleen, but the number of translocating bacteria begins to drop with the onset of a specific IgA response. A clonal B-cell microculture technique was used to determine the frequency of specific IgA plasmablasts and IgA memory cells. The frequencies of preplasmablasts were seen to be higher in the earlier stages of germinal center development, whereas the frequencies of antigen-specific memory cells appeared to remain at a relatively constant level even after 193 days postmonoassociation. We suggest that a successful secretory IgA response can attenuate chronic stimulation of GCR even though the bacteria persist in the gut. The observed developing hyporesponsiveness to a chronically present commensal organism may be relevant to the use of bacterial vectors for mucosal immunization.

Gram-negative flagellin, a Toll-like receptor 5 (TLR5) agonist, is a potent inducer of innate immune effectors such as cytokines and nitric oxide. In the lung, flagellin induces a localized and transient innate immune response characterized by neutrophil infiltration and the production of cytokines and chemokines. In view of the extraordinary potency of flagellin as an inducer of innate immunity and the contribution of innate responses to the development of adaptive immunity, we evaluated the efficacy of recombinant Salmonella flagellin as an adjuvant in an acellular plague vaccine. Mice immunized intranasally or intratracheally with the F1 antigen of Yersinia pestis and flagellin exhibited dramatic increases in anti-F1 plasma immunoglobulin G (IgG) titers that remained stable over time. In contrast, control mice had low or undetectable antibody responses. The IgG1/IgG2a ratio of antibody titers against F1 in immunized mice is consistent with a Th2 bias. However, no significant antigen-specific IgE production was detected. Interferons, tumor necrosis factor alpha, and interleukin-6 were not essential for the adjuvant effects of flagellin. Preexisting antiflagellin antibodies had no significant effect on the adjuvant activity of flagellin. Importantly, intranasal immunization with flagellin and the F1 antigen was protective against intranasal challenge with virulent Y. pestis CO92, with 93 to 100% survival of immunized mice. Lastly, vaccination of cynomolgus monkeys with flagellin and a fusion of the F1 and V antigens of Y. pestis induced a robust antigen-specific IgG antibody response.

STAT5 and interleukin 7 (IL-7) signaling are thought to control B lymphopoiesis by regulating the expression of key transcription factors and by activating variable (V(H)) gene segments at the immunoglobulin heavy-chain (Igh) locus. Using conditional mutagenesis to delete the gene encoding the transcription factor STAT5, we demonstrate that the development of pro-B cells was restored by transgenic expression of the prosurvival protein Bcl-2, which compensated for loss of the antiapoptotic protein Mcl-1. Expression of the genes encoding the B cell-specification factor EBF1 and the B cell-commitment protein Pax5 as well as V(H) gene recombination were normal in STAT5- or IL-7 receptor alpha-chain (IL-7Ralpha)-deficient pro-B cells rescued by Bcl-2. STAT5-expressing pro-B cells contained little or no active chromatin at most V(H) genes. In contrast, rearrangements of the immunoglobulin-kappa light-chain locus (Igk) were more abundant in STAT5- or IL-7Ralpha-deficient pro-B cells. Hence, STAT5 and IL-7 signaling control cell survival and the developmental ordering of immunoglobulin gene rearrangements by suppressing premature Igk recombination in pro-B cells.

25-Hydroxycholesterol is produced in mammalian tissues. The function of this oxysterol is unknown. Here we describe a central role for 25-hydroxycholesterol in regulating the immune system. In initial experiments, we found that stimulation of macrophage Toll-like receptors (TLR) induced expression of cholesterol 25-hydroxylase and the synthesis of 25-hydroxycholesterol. Treatment of naïve B cells with nanomolar concentrations of 25-hydroxycholesterol suppressed IL-2-mediated stimulation of B cell proliferation, repressed activation-induced cytidine deaminase (AID) expression, and blocked class switch recombination, leading to markedly decreased IgA production. Consistent with these findings, deletion of the mouse cholesterol 25-hydroxylase gene caused an increase in serum IgA. Conversely, inactivation of the CYP7B1 oxysterol 7alpha-hydroxylase, which degrades 25-hydroxycholesterol, decreased serum IgA. The suppression of IgA class switching in B cells by a macrophage-derived sterol in response to TLR activation provides a mechanism for local and systemic negative regulation of the adaptive immune response by the innate immune system.

Prevention of infections by vaccination remains a compelling goal to improve public health. Most infections involve the mucosae, but the development of vaccines against many of these pathogens has yet to be successful. Mucosal vaccines would make immunization procedures easier, be better suited for mass administration, and most efficiently induce immune exclusion - a term coined for non-inflammatory antibody shielding of internal body surfaces - mediated principally by secretory immunoglobulin A (SIgA). The exported antibodies are polymeric, mainly IgA dimers (pIgA) - produced by local plasma cells stimulated by antigens that target the mucosae. SIgA was early shown to be complexed with an epithelial glycoprotein - the secretory component (SC). In 1974, a common SC-dependent transport of pIgA and pentameric IgM was proposed. From the basolateral surface, pIg-SC complexes are taken up by endocytosis and finally extruded into the lumen. Membrane SC is now referred to as polymeric Ig receptor (pIgR). In 1980, it was shown to be synthesized as a larger transmembrane protein - first cloned from rabbit and then from human. Mice deficient for pIgR showed that this is the only receptor responsible for epithelial transport of IgA and IgM. In the gut, induction of B cells occurs in gut-associated lymphoid tissue, particularly the Peyer's patches, but also in mesenteric lymph nodes. Plasma cell differentiation is accomplished in the lamina propria to which the memory/effector cells home. The airways also receive such cells from nasopharynx-associated lymphoid tissue - but by different homing receptors. Such compartmentalization is a challenge for development of mucosal vaccines.