Tubulointerstitial damage is a common histopathological feature of acute and chronic renal diseases and a prognostic indicator of renal function outcome. Monocytes infiltrating the interstitium, through the release of cytokines and/or growth factors, may play a key role in the pathogenesis of tubulointerstitial damage. Monocyte chemotactic peptide-1 (MCP-1) is a specific and powerful chemoattractant and activating factor for monocytes. This study investigated MCP-1 expression and its correlation with monocyte infiltration and tubulointerstitial damage in biopsies of patients with acute interstitial nephritis (AIN) and a chronic glomerulonephritis, namely immunoglobulin. A nephropathy (IgAN), often characterized by tubulointerstitial involvement. Six patients with AIN and 20 patients with IgAN, nine with mild (G1 to 2) and 11 with moderate to severe histologic lesions (G3 to 5), were studied. MCP-1 gene and protein expression were evaluated by in situ hybridization and immunohistochemistry. Infiltrating CD68-positive cells were identified as monocytes. MCP-1, weakly expressed in normal kidneys, was clearly upregulated in AIN biopsies. The gene and the protein expression were primarily localized in tubular and glomerular parietal epithelial cells, as well as in infiltrating mononuclear cells. In IgAN, a striking increase in MCP-1 mRNA and protein expression was observed only in the biopsies with moderate to severe lesions, with a pattern of expression similar to AIN. The MCP-1 expression strictly correlated with monocyte infiltrates and tubulointerstitial damage. In addition, the urinary excretion of this chemokine was studied in 17 IgAN patients. MCP-1 protein concentration was higher, compared with healthy subjects, in IgAN patients, especially in the G3 to 5 group, and directly correlated with the renal MCP-1 gene expression. In conclusion, these data suggest that production of MCP-1 in the tubulointerstitial compartment may play a key role in modulating monocytes influx and, consequently, tubulointerstitial damage.

We examined the roles of the Drosophila Gq alpha proteins (DGq) in the phototransduction pathway. The DGq proteins immunolocalized to the ocelli and all eight retinular photoreceptor cell rhabdomeres. An affinity-purified anti-DGq alpha immunoglobulin blocked the light-dependent GTP hydrolysis activity associated with Drosophila head membranes in vitro, suggesting that rhodopsin stimulated DGq. Dominantly active DGq1 mutants exhibited a light-independent GTPase activity and abnormal electrophysiological light responses, such as reduced retinal sensitivity and slow response kinetics compared with wild-type flies. Dominant DGq2 mutants exhibited a light-independent GTPase activity with normal electrophysiological light responses. Retinas of double mutants of DGq1, but not DGq2, with the light-dependent retinal degeneration mutant rdgB degenerated even in the dark. DGq1 stimulation of rdgB retinal degeneration in the dark was norpA-dependent. These results indicate that DGq1 mediates the stimulation by light-activated rhodopsin of the norpA-encoded phospholipase C in the visual transduction cascade.

The effect of academic stress on immune function, as measured by the rate of secretion of salivary secretory immunoglobulin A (s-IgA), was studied prospectively in 64 first-year dental school students. Perceived stress and s-IgA secretion rate were measured five times--during an initial low-stress period, three high-stress periods coinciding with major examinations, and a final low-stress period. The s-IgA secretion rate was significantly lower in high-stress than low-stress periods for the whole group. In addition, personality characteristics differentiated patterns of s-IgA secretion rates. Students characterised by a great need to establish and maintain warm personal relationships secreted more s-IgA at each point than did all other subjects. The s-IgA secretion rates of those with a high inhibited need for power continued to decline through the final low-stress period rather than recovering as in all other subjects.

Bacteria can invade the biliary tract by ascending from the duodenum and via the hematogenous route from the hepatic portal venous blood. The sphincter of Oddi, situated at the junction of the biliary tract and the upper gastrointestinal tract, forms an effective mechanical barrier to duodenal reflex and ascending bacterial infection. Conversely, Kupffer cells and the tight junctions between hepatocytes help prevent bacteria and toxic metabolites from entering the hepatobiliary system from the portal circulation. The continuous flushing action of bile and the bacteriostatic effects of bile salts keeps the biliary tract sterile under normal conditions. Secretory immunoglobulin A (sIgA), the predominant immunoglobulin in the bile, and mucus excreted by the biliary epithelium probably function as antiadherence factors, preventing microbial colonization. When barrier mechanisms break down, as in surgical or endoscopic sphincterotomy and with insertion of biliary stents, pathogenic bacteria enter the biliary system at high concentrations and take up residence on any foreign bodies. Intrabiliary pressure is a key factor in the development of cholangitis. Chronic biliary obstruction raises the intrabiliary pressure. This adversely influences the defensive mechanisms such as the tight junctions, Kupffer cell functions, bile flow, and sIgA production in the system, resulting in a higher incidence of septicemia and endotoxemia in these patients. Knowledge of biliary defense against infection is still quite primitive. Unclear are the roles of sIgA in the bile, mechanism of bacterial adhesion to the biliary epithelium, Kupffer cell function in biliary obstruction, and the antimicrobial activity of bile salts.

Immunoglobulin G (IgG) molecules are glycosylated in CH2 at Asn297; the N-linked carbohydrates attached there have been shown to contribute to antibody (Ab) stability and various effector functions. The carbohydrate attached to the IgG constant region is a complex biantennary structure. Alterations in the structure of oligosaccharide have been associated with human diseases such as rheumatoid arthritis and osteoarthritis. To study the effects of altered carbohydrate structure on Ab effector function, we have used gene transfection techniques to produce mouse-human chimeric IgG1 Abs in the Chinese hamster ovary (CHO) cell line Lec 1, which is incapable of processing the high-mannose intermediate through the terminal glycosylation steps. We also produced IgG1 Abs in Pro-5, the wild-type CHO cell line that is the parent of Lec 1. The Pro-5-produced Ab (IgG1-Pro-5) was similar to IgG1-My 1, a myeloma-produced IgG1 Ab of the same specificity, in its biologic properties such as serum half-life, ability to effect complement-mediated cytolysis, and affinity for Fc gamma RI. Although the Lec 1-produced Ab, IgG1-Lec 1, was properly assembled and retained antigen specificity, it was incapable of complement-mediated hemolysis and was substantially deficient in complement consumption, C1q binding, and C1 activation. IgG1-Lec 1 also showed reduced but significant affinity for Fc gamma R1 receptors. The in vivo half-life of IgG1-Lec 1 was shorter than that of either the myeloma- or Pro-5-produced counterpart, with more being cleared during the alpha-phase and with more rapid clearance during the beta-phase. Clearance of IgG1-Lec 1 could be inhibited by the administration of yeast-derived mannan. Thus the uptake of IgG1-Lec 1 appears to be accelerated by the presence of terminally mannosylated oligosaccharide. Therefore, certain Ab functions as well as the in vivo fate of the protein are dramatically affected by altered carbohydrate structure. Expression of Igs in cell lines with defined glycosylation mutations is shown to be a useful technique for investigating the contribution of carbohydrate structure to Ab function.

Milk is traditionally considered an ideal source of the basic elemental nutrients required by infants. More detailed examination is revealing that milk represents a more functional ensemble of components with benefits to both infants and mothers. A comprehensive peptidomics method was developed and used to analyze human milk yielding an extensive array of protein products present in the fluid. Over 300 milk peptides were identified originating from major and many minor protein components of milk. As expected, the majority of peptides derived from β-casein, however no peptide fragments from the major milk proteins lactoferrin, α-lactalbumin, and secretory immunoglobulin A were identified. Proteolysis in the mammary gland is selective-released peptides were drawn only from specific proteins and typically from only select parts of the parent sequence. A large number of the peptides showed significant sequence overlap with peptides with known antimicrobial or immunomodulatory functions. Antibacterial assays showed the milk peptide mixtures inhibited the growth of Escherichia coli and Staphylococcus aureus . The predigestion of milk proteins and the consequent release of antibacterial peptides may provide a selective advantage through evolution by protecting both the mother's mammary gland and her nursing offspring from infection.

We studied 10 patients who had arthritis of the knee joint, but no other signs of rheumatic disease. The clinical diagnosis of osteoarthritis was corroborated by arthroscopic evidence of characteristic cartilage degeneration. Signs of inflammation were confined to areas of the synovial membrane that lay near the cartilage; thus, the major part of the joint cavity was not affected. The intensity of the synovial inflammation varied within the areas involved, but was always most pronounced in regions rimming the cartilage. Biopsy samples selected from regions of intensely inflamed synovium contained foci of T lymphocytes, which were bordered by immunoglobulin-carrying B lymphocytes and plasma cells, as well as strongly HLA-DR positive dendritic-like cells adjoined to alpha Leu-3a+ T helper lymphocytes. In tissue samples taken from macroscopically noninflamed areas, only a few infiltrating lymphocytes were seen. Thus, the inflammatory synovial changes found in osteoarthritis appear to be anatomically restricted and of varied intensity but, when present, are microscopically indistinguishable from the changes that have been previously described as indicative of rheumatoid arthritis.

Immunoglobulin A (IgA) is commonly recognized as the most prevalent antibody (Ab) at mucosal sites with an important role in defense by shielding mucosal surfaces from invasion by pathogens. However, its potential to both actively dampen excessive immune responses or to initiate potent proinflammatory cellular processes is less well known. Interestingly, either functional outcome is mediated through interaction with the myeloid IgA Fc receptor FcαRI (CD89). Monomeric interaction of IgA with FcαRI triggers inhibitory signals that block activation via other receptors, whereas multimeric FcαRI crosslinking induces phagocytosis, reactive oxygen species production, antigen presentation, Ab-dependent cellular cytotoxicity, and cytokine release. Thus, FcαRI acts as a regulator between anti- and proinflammatory responses of IgA. As such, the biology of FcαRI, and its multifaceted role in immunity will be the focus of this review.

Multiple myeloma (MM) is the most common form of plasma cell dyscrasia, characterized by a marked heterogeneity of genetic lesions and clinical course. It may develop from a premalignant condition (monoclonal gammopathy of undetermined significance, MGUS) or progress from intramedullary to extramedullary forms (plasma cell leukemia, PCL). To provide insights into the molecular characterization of plasma cell dyscrasias and to investigate the contribution of specific genetic lesions to the biological and clinical heterogeneity of MM, we analysed the gene expression profiles of plasma cells isolated from seven MGUS, 39 MM and six PCL patients by means of DNA microarrays. MMs resulted highly heterogeneous at transcriptional level, whereas the differential expression of genes mainly involved in DNA metabolism and proliferation distinguished MGUS from PCLs and the majority of MM cases. The clustering of MM patients was mainly driven by the presence of the most recurrent translocations involving the immunoglobulin heavy-chain locus. Distinct gene expression patterns have been found to be associated with different lesions: the overexpression of CCND2 and genes involved in cell adhesion pathways was observed in cases with deregulated MAF and MAFB, whereas genes upregulated in cases with the t(4;14) showed apoptosis-related functions. The peculiar finding in patients with the t(11;14) was the downregulation of the alpha-subunit of the IL-6 receptor. In addition, we identified a set of cancer germline antigens specifically expressed in a subgroup of MM patients characterized by an aggressive clinical evolution, a finding that could have implications for patient classification and immunotherapy.

Rotavirus infections are a leading cause of severe, dehydrating gastroenteritis in children <5 years of age. Despite the global introduction of vaccinations for rotavirus over a decade ago, rotavirus infections still result in >200,000 deaths annually, mostly in low-income countries. Rotavirus primarily infects enterocytes and induces diarrhoea through the destruction of absorptive enterocytes (leading to malabsorption), intestinal secretion stimulated by rotavirus non-structural protein 4 and activation of the enteric nervous system. In addition, rotavirus infections can lead to antigenaemia (which is associated with more severe manifestations of acute gastroenteritis) and viraemia, and rotavirus can replicate in systemic sites, although this is limited. Reinfections with rotavirus are common throughout life, although the disease severity is reduced with repeat infections. The immune correlates of protection against rotavirus reinfection and recovery from infection are poorly understood, although rotavirus-specific immunoglobulin A has a role in both aspects. The management of rotavirus infection focuses on the prevention and treatment of dehydration, although the use of antiviral and anti-emetic drugs can be indicated in some cases.

(<em>A</em>bstractText>The gastrointestinal tract is one of the most microbiologically active ecosystems that plays a crucial role in the working of the mucosal immune system (MIS). In this ecosystem, the consumed probiotics stimulate the immune system and induce a network of signals mediated by the whole bacteria or their cell wall structure. This review is aimed at describing the immunological mechanisms of probiotics and their beneficial effects on the host.</<em>A</em>bstractText>(<em>A</em>bstractText>Once administered, oral probiotic bacteria interact with the intestinal epithelial cells (IECs) or immune cells associated with the lamina propria, through Toll-like receptors, and induce the production of different cytokines or chemokines. Macrophage chemoattractant protein 1, produced by the IECs, sends signals to other immune cells leading to the activation of the MIS, characterized by an increase in <em>immunoglobulin</em> <em>A</em>+ cells of the intestine, bronchus and mammary glands, and the activation of T cells. Specifically, probiotics activate regulatory T cells that release IL-10. Interestingly, probiotics reinforce the intestinal barrier by an increase of the mucins, the tight junction proteins and the Goblet and Paneth cells. <em>A</em>nother proposed mechanism of probiotics is the modulation of intestinal microbiota by maintaining the balance and suppressing the growth of potential pathogenic bacteria in the gut. Furthermore, it has been demonstrated that long-term probiotics consumption does not affect the intestinal homeostasis. The viability of probiotics is crucial in the interaction with IECs and macrophages favoring, mainly, the innate immune response. Macrophages and Dendritic cells (DCs) play an important role in this immune response without inducing an inflammatory pattern, just a slight increase in the cellularity of the lamina propria. Besides, as part of the machinery that probiotics activate to protect against different pathogens, an increase in the microbicidal activity of peritoneal and spleen macrophages has been reported. In malnutrition models, such as undernourishment and obesity, probiotic was able to increase the intestinal and systemic immune response. Furthermore, probiotics contribute to recover the histology of both the intestine and the thymus damaged in these conditions. Probiotic bacteria are emerging as a safe and natural strategy for allergy prevention and treatment. Different mechanisms such as the generation of cytokines from activated pro-T-helper type 1, which favor the production of IgG instead of IgE, have been proposed. Key Messages: Probiotic bacteria, their cell walls or probiotic fermented milk have significant effects on the functionality of the mucosal and systemic immune systems through the activation of multiple immune mechanisms.</<em>A</em>bstractText>

BACKGROUND

Phthalates are ubiquitous in the environment, but concentrations in multiple media from breast-feeding U.S. women have not been evaluated.

OBJECTIVE

The objective of this study was to accurately measure and compare the concentrations of oxidative monoester phthalate metabolites in milk and surrogate fluids (serum, saliva, and urine) of 33 lactating North Carolina women.

METHODS

We analyzed serum, saliva, urine, and milk for the oxidative phthalate metabolites mono(3-carboxypropyl) phthalate, mono(2-ethyl-5-carboxypentyl) phthalate (MECPP), mono(2-ethyl-5-hydroxyhexyl) phthalate, and mono(2-ethyl-5-oxohexyl) phthalate using isotope-dilution high-performance liquid chromatography tandem mass spectroscopy. Because only urine lacks esterases, we analyzed it for the hydrolytic phthalate monoesters.

RESULTS

We detected phthalate metabolites in few milk (< 10%) and saliva samples. MECPP was detected in>> 80% of serum samples, but other metabolites were less common (3-22%). Seven of the 10 urinary metabolites were detectable in>> or = 85% of samples. Monoethyl phthalate had the highest mean concentration in urine. Metabolite concentrations differed by body fluid (urine>> serum>> milk and saliva). Questionnaire data suggest that frequent nail polish use, immunoglobulin A, and fasting serum glucose and triglyceride levels were increased among women with higher concentrations of urinary and/or serum phthalate metabolites; motor vehicle age was inversely correlated with certain urinary phthalate concentrations.

CONCLUSIONS

Our data suggest that phthalate metabolites are most frequently detected in urine of lactating women and are less often detected in serum, milk, or saliva. Urinary phthalate concentrations reflect maternal exposure and do not represent the concentrations of oxidative metabolites in other body fluids, especially milk.

The human leukocyte antigens (HLA) are implicated in the genetic susceptibility to a large number of diseases. Some of the diseases associated with HLA class II are related to specific amino acids or epitopes of the domain of the HLA class II molecule that is distal to the membrane. In man, selective immunoglobulin A deficiency is the most common immunodeficiency, frequently resulting in recurrent sino-pulmonary infections and gastro-intestinal disorders. Associations have been described with HLA class I, and to a lesser extent with different class II alleles, which might indicate that they share some common feature. Here we study 95 IgA-D patients and find positive associations with three DR-DQ haplotypes and a strong negative association with a fourth haplotype. Comparison of the sequences of the polymorphic amino-terminal domain of the DQ beta chain showed that the three 'susceptibility' haplotypes all had a neutral alanine or valine at position 57. The 'protective' allele had the negatively charged aspartic acid at this position (Asp57). Codon 57 of the HLA-DQ beta chain has been implicated in the susceptibility to insulin-dependent diabetes mellitus. Our data suggest that the same amino acid position could possibly also influence susceptibility and resistance to selective immunoglobulin A deficiency.

Most reports dealing with vaccines against botulinum toxin have focused on the injection route of administration. This is unfortunate, because a mucosal vaccine is likely to be more efficacious for patients and pose fewer risks to health care workers and to the environment. Therefore, efforts were made to generate a mucosal vaccine that provides protection against the botulinum serotypes that typically cause human illness (serotypes A, B, and E). This work demonstrated that carboxy-terminal peptides derived from each of the three serotypes were able to bind to and penetrate human epithelial barriers in vitro, and there was no cross inhibition of membrane binding and transcytosis. The three polypeptides were then tested in vivo as a trivalent vaccine that could be administered to mice by the intranasal route. The results indicated that the mucosal vaccine evoked high secretory titers of immunoglobulin A (IgA), as well as high circulating titers of IgG and IgA, and it also evoked a high level of resistance to challenge with toxin. The immunoglobulin responses and the levels of resistance to challenge were increased by coadministration of adjuvants, such as chitosan and vitamin E. At least three mechanisms were identified to account for the antibody-induced resistance: (i) blockade of toxin absorption across epithelial cells, (ii) enhanced clearance of toxin from the circulation, and (iii) blockade of toxin action at the neuromuscular junction. These results are a compelling demonstration that a mucosal vaccine against multiple serotypes of botulinum toxin has been identified.

OBJECTIVE

To report that antibodies to synaptic proteins may occur in association with slow, progressive cognitive decline.

METHODS

A total of 24 patients with progressive cognitive dysfunction of unclear etiology were examined for onconeuronal and synaptic receptor antibodies. The effect of serum was examined in cultures of dissociated mouse hippocampal neurons.

RESULTS

Seven patients had immunoglobulin A (IgA), but no immunoglobulin G (IgG), antibodies against NMDA receptor (NMDAR). Anti-NMDAR IgA positive patients' serum, but not serum from control individuals, caused dramatic decrease of the levels of NMDAR and other synaptic proteins in neurons, along with prominent changes in NMDAR-mediated currents. These effects correlated with the titer of IgA NMDAR antibodies and were reversed after removing patients' serum from the culture media. When available, comprehensive clinical assessment and brain metabolic imaging showed neurologic improvement after immunotherapy.

CONCLUSIONS

A subset of patients with slowly progressive cognitive impairment has an underlying synaptic autoimmunity that decreases the density of NMDAR and other synaptic proteins, and alters synaptic currents. This autoimmunity can be demonstrated examining patients' serum and CSF for NMDAR IgA antibodies, identifying possible candidates for immunotherapy.

The importance of locally and systemically formed antibodies of various classes for protection against experimental cholera has been studied in mice immunized with cholera toxin. Groups of mice were given various numbers of peroral or intravenous immunizations, or a combination of both. Serum antibodies and antibodies synthesized by spleen and small intestine in vitro during tissue culture were measured by the enzyme-linked immunosorbent assay, and protective immunity against intestinal toxin challenge was determined by means of a small-bowel loop assay. Regression analyses showed a close correlation between the magnitude of intestinal synthesis of specific immunoglobulin A (IgA) antibodies and protection (r = 0.98), whereas neither the local formation of IgG or IgM nor the production of antitoxin antibodies of any immunoglobulin class by spleen showed any significant correlation with protection. The serum titers of IgG and IgM antibodies did not show any such relation, whereas the level of specific IgA in serum, probably mainly derived from the intestine, correlated significantly (r = 0.90).

Ten new patients with ataxia telangiectasia-like disorder (ATLD) from three unrelated Saudi Arabian families have been identified aged 5-37 representing the largest cohort of ATLD patients ever identified. They presented with an early-onset, slowly progressive, ataxia plus ocular apraxia phenotype with an absence of tumor development, even in the oldest patient. Extra-neurological features such as telangiectasia, raised alpha-fetoprotein and reduced immunoglobulin levels were absent. No translocations were found in the two investigated patients, and the presence of microcephaly was noted in four out of eight ascertained patients. All patients are homozygous for a novel missense mutation (630G->>C, W210C) of the MRE11 gene. The cellular consequences of this amino acid change, localized in the nuclease domain of the Mre11 protein, have been determined in fibroblast cultures established from two individuals. They showed high constitutive levels of Mre11 and Rad50 proteins compared with cells from normal individuals but a very low level of the Nbs1 protein. After exposure to ionizing radiation, a dose-dependent defect in ataxia telangiectasia mutated (ATM)-serine 1981, p53-serine 15 and Chk2 phosphorylation, and p53 stabilization were noted, together with a failure to form Mre11 foci and enhanced radiation sensitivity. Formation of gammaH2AX foci was similar to that seen in normal fibroblasts under the experimental conditions examined. These results emphasize the importance of functional interactions among the three proteins of the Mre11-Rad50-Nbs1 complex and lend support to a role of this complex as a sensor of DNA double-strand breaks, acting upstream of ATM.

Infections due to Shiga toxin-producing Escherichia coli (STEC) are responsible for severe diarrheal disease in humans and livestock, and these bacteria have recently emerged as a leading cause of renal failure in children. In this study, we have examined medium- and temperature-dependent production of secreted proteins from a STEC O26 serotype strain. Growth of bacteria in Luria broth led to the detection of secreted polypeptides of 104, 55, 54, and 37 kDa (p104, p55, p54, and p37, respectively). When grown in serum-free tissue culture medium, only p104, p37 and two additional polypeptides of 25 and 22 kDa (p25 and p22) were present in supernatant fluids. Production of these polypeptides was growth temperature dependent and induced in cultures grown at 37 degrees C. N-terminal amino acid sequencing revealed that p104 was homologous to the secreted p110 of enteropathogenic Escherichia coli (EPEC), and both proteins belong to a family of secreted proteins in pathogenic bacteria of which the immunoglobulin A protease of Neisseria gonorrhoeae is the prototype. The N-terminal amino acid sequences of p55 and p54 were unique to the STEC strain, while p37 and p25 were found to be highly homologous to the similarly sized EspA and EspB proteins, previously detected in culture supernatants of EPEC. Molecular cloning and sequencing of STEC espB alleles from two different serotypes showed that the encoded polypeptides were about 80% homologous. A monoclonal antibody raised against STEC EspB also cross-reacted with its EPEC analog and allowed us to demonstrate medium- and temperature-dependent production of this important virulence factor in STEC and EPEC strains of differing serotypes.

Formalin fixation followed by routine paraffin embedding allows the demonstration of intracellular immunoglobulin by a sandwich technique using either peroxidase or fluorescein isothyocyanate labelled antibody conjugates. The results compared favourably with those obtained using either the fresh frozen or the Sainte-Marie method for preserving tissue immunoglobulins. The formalin-paraffin method with peroxidase-labelled antibody has advantages for routine use. Morphology is excellent, preparations are permanent, and retrospective studies of stored paraffin-embedded tissue are possible. Some of the problems of labelled antibody studies are discussed.

Streptococcus pneumoniae (pneumococcus) remains a significant health threat worldwide, especially to the young and old. While some of the biomolecules involved in pneumococcal pathogenesis are known and understood in mechanistic terms, little is known about the molecular details of bacterium/host interactions. We report here the solution structure of the 'repeated' adhesion domains (domains R1 and R2) of the principal pneumococcal adhesin, choline binding protein A (CbpA). Further, we provide insights into the mechanism by which CbpA binds its human receptor, polymeric immunoglobulin receptor (pIgR). The R domains, comprised of 12 imperfect copies of the leucine zipper heptad motif, adopt a unique 3-alpha-helix, raft-like structure. Each pair of alpha-helices is antiparallel and conserved residues in the loop between Helices 1 and 2 exhibit a novel 'tyrosine fork' structure that is involved in binding pIgR. This and other structural features that we show are conserved in most pneumococcal strains appear to generally play an important role in bacterial adhesion to pIgR. Interestingly, pneumococcus is the only bacterium known to adhere to and invade human cells by binding to pIgR.

Mucopolysaccharidosis (MPS) IIIB is a lysosomal storage disease with severe neurological manifestations due to alpha-N-acetylglucosaminidase (NaGlu) deficiency. The mechanism of neuropathology in MPS IIIB is unclear. This study investigates the role of immune responses in neurological disease of MPS IIIB in mice. By means of gene expression microarrays and real-time quantitative reverse transcriptase-polymerase chain reaction, we demonstrated significant up-regulation of numerous immune-related genes in MPS IIIB mouse brain involving a broad range of immune cells and molecules, including T cells, B cells, microglia/macrophages, complement, major histocompatibility complex class I, immunoglobulin, Toll-like receptors, and molecules essential for antigen presentation. The significantly enlarged spleen and lymph nodes in MPS IIIB mice were due to an increase in splenocytes/lymphocytes, and functional assays indicated that the T cells were activated. An autoimmune component to the disease was further suggested by the presence of putative autoantigen or autoantigens in brain extracts that reacted specifically with serum IgG from MPS IIIB mice. We also demonstrated for the first time that immunosuppression with prednisolone alone can significantly slow the central nervous system disease progression. Our data indicate that immune responses contribute greatly to the neuropathology of MPS IIIB and should be considered as an adjunct treatment in future therapeutic developments for optimal therapeutic effect.

T cell receptors (TCRs) and immunoglobulins (Igs) derive a large fraction of their repertoire from diversity generated at the junctions of the V, D, and J coding segments. This diversity is derived both from the random deletion of nucleotides from the ends of coding regions and from the subsequent addition of nontemplated N region nucleotides. While the vast majority of TCRs and Igs from adult mice have N regions, less than 5% of both TCR-gamma/delta and Ig from fetal and neonatal mice have N regions. This study analyzed the ontogeny of junctional diversity of TCR-alpha/beta. Genomic DNA or C beta-primed cDNA was prepared from thymocytes of mice at varying stages in ontogeny, and the rearranged V beta 8 or V beta 5 sequences were amplified by polymerase chain reactions. Sequencing of the V beta-D beta-J beta junctions showed few N regions early in ontogeny, although the fraction of sequences with N regions exceeded that previously reported for Ig and for TCR-gamma/delta. N regions were found in 13% of V beta junctional sequences from day 18-19 fetal thymocytes, 33% of sequences from newborn thymocytes, 76% of sequences from day 4 postnatal thymocytes, and 88% of sequences from 5-wk-old thymocytes. In addition, nonrandom usage of the D beta and J beta segments was observed in both fetal and adult TCR sequences. While the usage of each of the six J beta 2 segments was different, the same pattern of usage was seen regardless of whether D beta 1 or D beta 2 was used, suggesting that a factor controlling the rate of usage of each J segment is intrinsic to the J gene itself. Since TCRs derive so much of their diversity from N regions, the relative paucity of N regions in fetal alpha/beta T cells would create a fetal TCR-alpha/beta repertoire that would be quite different from, and smaller than, the adult repertoire. The lack of N regions might be predicted to limit the range of affinities of TCR-MHC + peptide interactions, which may have important consequences for positive and negative selection of fetal and newborn T cells.

Beside the immunoglobulin (Ig) heavy and light chains the murine B cell receptor of the IgM class contains a heterodimer of two transmembrane proteins (IgM-alpha and Ig-beta). By N-terminal sequencing of IgM-alpha and Ig-beta we have identified the genes encoding these proteins as mb-1 and B29, respectively. Both genes are B cell specific and have been previously cloned from B minus T cell subtractive cDNA libraries. We have constructed expression vectors of the two genes and demonstrate that expression of the mb-1 and B29 genes can influence the surface expression of IgM in micron-transfected myeloma cells. From the known sequences of the IgM-alpha and Ig-beta proteins and from the results of previous transfection experiments with various vectors expressing the mu chain we have developed a structural model of the B cell antigen receptor of class IgM which we compare with that of the T cell antigen receptor.

Vascular cell adhesion molecule-1 (VCAM-1) is induced on endothelial cells by inflammatory cytokines, and binds mononuclear leukocytes through the integrin very late antigen-4 (VLA-4) (alpha 4 beta 1). This adhesion pathway has been implicated in a diverse group of physiological and pathological processes, including B cell development, leukocyte activation and recruitment to sites of inflammation, atherosclerosis, and tumor cell metastasis. The major form of VCAM-1 (VCAM-7D) has seven extracellular immunoglobulin (Ig)-like domains, of which the three NH2-terminal domains (domains 1-3) are similar in amino acid sequence to domains 4-6. By functional analysis of VCAM-7D relative to VCAM-6D (a minor 6-domain form of VCAM-1 in which domain 4 is deleted because of alternative splicing), and chimeric constructs between VCAM-1 and its structural relative intercellular adhesion molecule-1 (ICAM-1), we show that either the first or the homologous fourth domain of VCAM-1 is required for VLA-4-dependent adhesion. Either of these binding sites can function in the absence of the other. When both are present, cell binding activity is increased relative to monovalent forms of the molecule. The homologous binding regions appear to have originated by internal duplication of a portion of a monovalent ancestral gene, before the mammalian radiation. Thus VCAM-1 exemplifies evolution of a functionally bivalent cell-cell adhesion molecule by intergenic duplication. We have also produced a new class of anti-VCAM-1 monoclonal antibodies that block domain 4-dependent adhesion, and demonstrate that both binding sites participate in the adhesion function of VCAM-1 on endothelial cells in vitro. Therefore both sites must be blocked in clinical, animal, or in vitro studies depending on the use of anti-VCAM-1 antibodies to inactivate the VCAM-1/VLA-4 adhesion pathway.