Murine splenic dendritic cells (DCs) can be divided into two subsets based on CD8alpha expression, but the specific role of each subset in stimulation of T cells is largely unknown. An important function of DCs is the ability to take up exogenous antigens and cross-present them in the context of major histocompatibility complex (MHC) class I molecules to CD8(+) T cells. We previously demonstrated that, when cell-associated ovalbumin (OVA) is injected into mice, only the CD8(+) DC subset cross-presents OVA in the context of MHC class I. In contrast to this selectivity with cell-associated antigen, we show here that both DC subsets isolated from mice injected with OVA/anti-OVA immune complexes (OVA-IC) cross-present OVA to CD8(+) T cells. The use of immunoglobulin G Fc receptor (Fc(gamma)R) common gamma-chain-deficient mice revealed that the cross-presentation by CD8(-) DCs depended on the expression of gamma-chain-containing activating FcgammaRs, whereas cross-presentation by CD8(+) DCs was not reduced in gamma-chain-deficient mice. These results suggest that although CD8(+) DCs constitutively cross-present exogenous antigens in the context of MHC class I molecules, CD8(-) DCs only do so after activation, such as via ligation of Fc(gamma)Rs. Cross-presentation of immune complexes may play an important role in autoimmune diseases and the therapeutic effect of antitumor antibodies.

Monocytes and macrophages, which may play a central role in the pathogenesis of infection with human immunodeficiency virus type 1 (HIV-1), express the CD4 molecule and Fc receptors (FcR) for immunoglobulin G (IgG). To explore the possibility that FcR mediate HIV-1 infection of monocytes, studies were conducted with the human monocytic cell line U937. These cells were exposed to HIV-1 complexed with various concentrations of serum from HIV-1 antibody-positive individuals and monitored for HIV-1 replication. Serum samples from antibody-negative normal individuals did not affect virus yields. High concentrations of antibody-positive sera showed virus-neutralizing activity; however, cells infected with HIV-1 in the presence of antibody-positive sera at subneutralizing concentrations significantly enhanced virus replication. This infection enhancement was blocked by heat-aggregated gamma-globulin. Moreover, the IgG fraction from an HIV-1 antibody-positive serum enhanced HIV-1 infection at the same serum dilution equivalents. In contrast, IgG-F(ab')2 did not enhance HIV-1 infection but showed neutralizing activity with HIV-1. These results are compatible with the concept of FcR-mediated infection enhancement and suggest that this immunological response to HIV-1, instead of protecting the host, potentially facilitates the infection.

PspC is one of three designations for a pneumococcal surface protein whose gene is present in approximately 75% of all Streptococcus pneumoniae strains. Under the name SpsA, the protein has been shown to bind secretory immunoglobulin A (S. Hammerschmidt, S. R. Talay, P. Brandtzaeg, and G. S. Chhatwal, Mol. Microbiol. 25:1113-1124, 1997). Under the name CbpA, the protein has been shown to interact with human epithelial and endothelial cells (C. Rosenow et al., Mol. Microbiol. 25:819-829, 1997). The gene is paralogous to the pspA gene in S. pneumoniae and was thus called pspC (A. Brooks-Walter, R. C. Tart, D. E. Briles, and S. K. Hollingshead, Abstracts of the 97th General Meeting of the American Society for Microbiology 1997). Sequence comparisons of five published and seven new alleles reveal that this gene has a mosaic structure, and modular domains have contributed to gene diversity during evolution. Two major clades exist: clade A alleles are larger and contain an extra module that is shared with many pspA alleles; clade B alleles are smaller and lack this pspA-like domain. All alleles have a proline-rich domain and a choline-binding repeat domain that show 0% divergence from similar domains in the PspA protein. Immunization of a rabbit with a recombinant clade B PspC molecule produced antiserum that cross-reacted with both PspC and PspA from 15 pneumococcal isolates. The cross-reactive antibodies afforded cross-protection in a mouse model system. Mice immunized with PspC were protected against challenge with a strain that expressed PspA but not PspC. The PspA- and PspC-cross-reactive antibodies were directed to the proline-rich domain present in both molecules.

BACKGROUND

The relative efficacy of plasma exchange (PE) and intravenous immunoglobulin (IVIg) for the treatment of Guillain-Barré syndrome has not been established. We compared PE with IVIg, and with a combined regimen of PE followed by IVIg, in an international, multicentre, randomised trial of 383 adult patients with Guillain-Barré syndrome.

METHODS

The patients were randomly assigned PE (five 50 mL/kg exchanges over 8-13 days), IVIg (Sandoglobulin, 0.4 g/kg daily for 5 days), or the PE course immediately followed by the IVIg course. The inclusion criteria were severe disease (aid needed for walking) and onset of neuropathic symptoms within the previous 14 days. Patients were followed up for 48 weeks.

RESULTS

Four patients were excluded because they did not meet the randomisation criteria. All the remaining 379 patients were assessed for the major outcome criterion-change on a seven-point disability grade scale-by an observer unaware of treatment assignment, 4 weeks after randomisation. At that time, the mean improvement was 0.9 (SD 1.3) in the 121 PE-group patients, 0.8 (1.3) in the 130 IVIg-group patients, and 1.1 (1.4) in the 128 patients who received both treatments (intention-to-treat analysis). None of the differences between the groups for this major outcome criterion was significant. The difference between PE alone and IVIg alone was so small that a 0.5 grade difference was excluded at the 95% level of confidence. There was no significant difference between any of the treatment groups in the secondary outcome measures: time to recovery of unaided walking, time to discontinuation of ventilation, and trend describing the recovery from disability up to 48 weeks. There was a non-significant trend towards a more favourable outcome on some outcome measures with combined treatment.

CONCLUSIONS

In treatment of severe Guillain-Barré syndrome during the first 2 weeks after onset of neuropathic symptoms, PE and IVIg had equivalent efficacy. The combination of PE with IVIg did not confer a significant advantage.

BACKGROUND

CC chemokine receptor-7 (CCR7), which plays a critical role in the migration of activated dendritic cells to regional lymph nodes via afferent lymphatic vessels, is also expressed by human breast and melanoma cell lines. Because neoplastic cells also enter lymphatic vessels before metastasis to the lymph nodes, we investigated whether CCR7 expression enhances metastasis of B16 murine melanoma cells to regional lymph nodes.

METHODS

B16 cells were transduced with a retroviral vector containing CCR7 complementary DNA (CCR7-B16 cells) or with vector alone (pLNCX2-B16 control cells). The functional assay for CCR7 protein was Ca(2+) flux stimulated by the chemokine CCL21, a CCR7-specific ligand produced by lymphatic endothelial cells. B16 tumor cells were injected into the footpad of mice. Tumor cell metastasis to draining lymph nodes was assessed by measuring messenger RNA (mRNA) for tyrosinase-related protein-1 (TRP), a melanocyte-specific enzyme, with real-time, quantitative reverse transcription-coupled polymerase chain reaction. All statistical tests were two-sided.

RESULTS

One week after injection into the footpad, 701-fold (95% confidence interval [CI] = 64- to 1336-fold) more TRP mRNA was detected in draining lymph nodes from CCR7-B16 cell-injected mice than in those from control cell-injected mice. Three weeks after footpad injection, 58% (11 of 19) of the draining lymph nodes from CCR7-B16 cell-injected mice and 5% (one of 19) of those from control mice showed gross metastases (P<.001). CCR7-B16 cells isolated from lymph node metastases retained functional CCR7 expression. Lymph node metastasis of CCR7-B16 cells was blocked by neutralizing anti-CCL21 antibodies (metastasis in none of five lymph nodes) but not by control immunoglobulin G (three of five). Enhanced metastasis of CCR7-B16 cells was specific for a lymphatic route because both CCR7-B16 and control cells co-injected intravenously metastasized to the lung at the same frequency.

CONCLUSIONS

Expression of a single chemokine receptor gene, CCR7, increased B16 cell metastasis to draining lymph nodes, suggesting that cancer cells may co-opt normal mechanisms of lymph node homing during metastasis.

Tumor-specific clonal immunoglobulin expressed by B-cell lymphomas (idiotype [Id]) can serve as a target for active immunotherapy. We have previously described the vaccination of 4 patients with follicular lymphoma using dendritic cells (DCs) pulsed with tumor-derived Id protein and now report on 35 patients treated using this approach. Among 10 initial patients with measurable lymphoma, 8 mounted T-cell proliferative anti-Id responses, and 4 had clinical responses--2 complete responses (CRs) (progression-free [PF] for 44 and 57 months after vaccination), 1 partial response (PR) (PF for 12 months), and 1 molecular response (PF for 75+ months). Subsequently, 25 additional patients were vaccinated after first chemotherapy, and 15 of 23 (65%) who completed the vaccination schedule mounted T-cell or humoral anti-Id responses. Induction of high-titer immunoglobulin G anti-Id antibodies required coupling of Id to the immunogenic carrier protein keyhole limpet hemocyanin (Id-KLH). These antibodies could bind to and induce tyrosine phosphorylation in autologous tumor cells. Among 18 patients with residual tumor at the time of vaccination, 4 (22%) had tumor regression, and 16 of 23 patients (70%) remain without tumor progression at a median of 43 months after chemotherapy. Six patients with disease progression after primary DC vaccination received booster injections of Id-KLH protein, and tumor regression was observed in 3 of them (2 CRs and 1 PR). We conclude that Id-pulsed DC vaccination can induce T-cell and humoral anti-Id immune responses and durable tumor regression. Subsequent boosting with Id-KLH can lead to tumor regression despite apparent resistance to the primary DC vaccine.

We cloned overlapping DNA segments that encompass the region from the immunoglobulin JH segments to the C gamma 3 gene of BALB/c mouse. We have now cloned the entire region (about 200 kilobases) of the constant-region gene family of the immunoglobulin heavy chain, the organization of which is 5'-JH-6.5 kb-C mu-4.5 kb-C delta-55 kb-C gamma 3-34 kb-C gamma 1-21 kb-C gamma 2b-15 kb-C gamma 2a-14 kb-C epsilon-12 kb-C alpha-3'. Using these cloned DNAs, we have characterized several structural features of the constant-region gene loci. There are no other J region segments except for those at the 5' side of the C mu gene. The S region is 5' to each CH gene except for the C delta gene, and the nucleotide sequences of the S region share some homology. There is no reasonably conserved pseudogene. There are at least two species of reiterated sequences scattered in these loci. Cloning and Southern blot hybridization analyses indicate that the general organizations of the heavy-chain gene loci of BALB/c and C57BL/6 mice, which have many different serological markers, are fundamentally similar but different in the lengths of S regions. Restriction enzyme cleavage maps of the whole constant-region gene loci were constructed with respect to eight restriction endonucleases.

The capacity of Staphylococcus aureus to form biofilms on host tissues and implanted medical devices is one of the major virulence traits underlying persistent and chronic infections. The matrix in which S. aureus cells are encased in a biofilm often consists of the polysaccharide intercellular adhesin (PIA) or poly-N-acetyl glucosamine (PNAG). However, surface proteins capable of promoting biofilm development in the absence of PIA/PNAG exopolysaccharide have been described. Here, we used two-dimensional nano-liquid chromatography and mass spectrometry to investigate the composition of a proteinaceous biofilm matrix and identified protein A (spa) as an essential component of the biofilm; protein A induced bacterial aggregation in liquid medium and biofilm formation under standing and flow conditions. Exogenous addition of synthetic protein A or supernatants containing secreted protein A to growth media induced biofilm development, indicating that protein A can promote biofilm development without being covalently anchored to the cell wall. Protein A-mediated biofilm formation was completely inhibited in a dose-dependent manner by addition of serum, purified immunoglobulin G, or anti-protein A-specific antibodies. A murine model of subcutaneous catheter infection unveiled a significant role for protein A in the development of biofilm-associated infections, as the amount of protein A-deficient bacteria recovered from the catheter was significantly lower than that of wild-type bacteria when both strains were used to coinfect the implanted medical device. Our results suggest a novel role for protein A complementary to its known capacity to interact with multiple immunologically important eukaryotic receptors.

C-reactive protein (CRP) is an ancient highly conserved molecule and a member of the pentraxin family of proteins. CRP is secreted by the liver in response to a variety of inflammatory cytokines. Levels of CRP increase very rapidly in response to trauma, inflammation, and infection and decrease just as rapidly with the resolution of the condition. Thus, the measurement of CRP is widely used to monitor various inflammatory states. CRP binds to damaged tissue, to nuclear antigens and to certain pathogenic organisms in a calcium-dependent manner. The function of CRP is felt to be related to its role in the innate immune system. Similar to immunoglobulin (Ig)G, it activates complement, binds to Fc receptors and acts as an opsonin for various pathogens. Interaction of CRP with Fc receptors leads to the generation of proinflammatory cytokines that enhance the inflammatory response. Unlike IgG, which specifically recognizes distinct antigenic epitopes, CRP recognizes altered self and foreign molecules based on pattern recognition. Thus, CRP is though to act as a surveillance molecule for altered self and certain pathogens. This recognition provides early defense and leads to a proinflammatory signal and activation of the humoural, adaptive immune system.

Attenuated Salmonella enterica serovar Typhimurium expressing recombinant antigens from other pathogens elicits primarily a Th1-type dominant immune response to both recombinant and Salmonella antigens. The immunogenicity and appropriate subcellular location of the recombinant antigen in the Salmonella vaccine strain may contribute to augmenting immune responses by facilitating adequate exposure of recombinant antigen to antigen-presenting cells for processing. To allow for secretion from gram-negative bacteria and overexpression of antigen, a DNA fragment encoding a highly antigenic alpha-helical region of PspA (pneumococcal surface protein A) was subcloned downstream from the beta-lactamase signal sequence in the multicopy Asd(+) pYA3493 vector to create pYA3494. pYA3493 was derived from a class of Asd(+) vectors with reduced expression of Asd to minimize selective disadvantage and enhance immunization of expressed recombinant antigens. The S. enterica serovar Typhimurium vaccine strain was constructed by the introduction of deletion mutations Delta crp-28 and Delta asdA16. Approximately 50% of the recombinant PspA (rPspA) expressed in a Salmonella strain harboring pYA3494 was detected in the combined supernatant and periplasmic fractions of broth-grown recombinant Salmonella. After a single oral immunization in BALB/c mice with 10(9) CFU of the recombinant Salmonella vaccine strain carrying pYA3494, <em>immunoglobulin</em> <em>G</em> (Ig<em>G</em>) antibody responses were stimulated to both the heterologous antigen rPspA and Salmonella lipopolysaccharide (LPS) and outer membrane proteins (OMPs). About half, and even more at later times after immunization, of the antibodies induced to rPspA were Ig<em>G</em>1 (indicating a Th2-type response), whereas 60 to 70% of the antibodies to LPS and 80 to 90% of those to OMPs were Ig<em>G</em>2a (indicating a Th1-type response). A sublethal infection with Streptococcus pneumoniae WU2 boosted PspA antibody levels and maintained Ig<em>G</em>2a/Ig<em>G</em>1 ratios similar to those seen before the challenge. Oral immunization with Salmonella-PspA vaccine protected 60% of immunized mice from death after intraperitoneal challenge with 50 times the 50% lethal dose of virulent S. pneumoniae WU2.

We have engineered the Fc region of a human immunoglobulin G (IgG) to generate a mutated antibody that modulates the concentrations of endogenous IgGs in vivo. This has been achieved by targeting the activity of the Fc receptor, FcRn, which serves through its IgG salvage function to maintain and regulate IgG concentrations in the body. We show that an IgG whose Fc region was engineered to bind with higher affinity and reduced pH dependence to FcRn potently inhibits FcRn-IgG interactions and induces a rapid decrease of IgG levels in mice. Such FcRn blockers (or 'Abdegs,' for antibodies that enhance IgG degradation) may have uses in reducing IgG levels in antibody-mediated diseases and in inducing the rapid clearance of IgG-toxin or IgG-drug complexes.

Epstein-Barr virus (EBV) latency III infection converts B lymphocytes into lymphoblastoid cell lines (LCLs) by expressing EBV nuclear and membrane proteins, EBNAs, and latent membrane proteins (LMPs), which regulate transcription through Notch and tumor necrosis factor receptor pathways. The role of NF-kappa B in LMP1 and overall EBV latency III transcriptional effects was investigated by treating LCLs with BAY11-7082 (BAY11). BAY11 rapidly and irreversibly inhibited NF-kappa B, decreased mitochondrial membrane potential, induced apoptosis, and altered LCL gene expression. BAY11 effects were similar to those of an NF-kappa B inhibitor, Delta N-I kappa B alpha, in effecting decreased JNK1 expression and in microarray analyses. More than 80% of array elements that decreased with Delta N-I kappa B alpha expression decreased with BAY11 treatment. Newly identified NF-kappa B-induced, LMP1-induced, and EBV-induced genes included pleckstrin, Jun-B, c-FLIP, CIP4, and I kappa B epsilon. Of 776 significantly changed array elements, 134 were fourfold upregulated in EBV latency III, and 74 were fourfold upregulated with LMP1 expression alone, whereas only 28 were more than fourfold downregulated by EBV latency III. EBV latency III-regulated gene products mediate cell migration (EBI2, CCR7, RGS1, RANTES, MIP1 alpha, MIP1 beta, CXCR5, and RGS13), antigen presentation (major histocompatibility complex proteins and JAW1), mitogen-activated protein kinase pathway (DUSP5 and p62Dok), and interferon (IFN) signaling (IFN-gamma R alpha, IRF-4, and STAT1). Comparison of EBV latency III LCL gene expression to immunoglobulin M (IgM)-stimulated B cells, germinal-center B cells, and germinal-center-derived lymphomas clustered LCLs with IgM-stimulated B cells separately from germinal-center cells or germinal-center lymphoma cells. Expression of IRF-2, AIM1, ASK1, SNF2L2, and components of IFN signaling pathways further distinguished EBV latency III-infected B cells from IgM-stimulated or germinal-center B cells.

Optimal T-cell activation and T-cell expansion require triggering by T-cell antigen receptors and co-stimulatory signals provided by accessory cells. A major co-stimulatory pathway involves crosslinking the CD28 molecule on T cells by its ligands CD80 or CD86 expressed on antigen-presenting cells. But recent studies on CD28-deficient mice have indicated that CD28 is not required for all T-cell responses and that additional T-cell co-stimulatory pathways exist. Here we describe a novel glycoprotein, of relative molecular mass 70,000 (M(r) 70K), designated SLAM, that belongs to the immunoglobulin gene superfamily, which is involved in T-cell stimulation. SLAM is constitutively expressed on peripheral-blood CD45ROhigh memory T cells, T-cell clones, immature thymocytes, and a proportion of B cells, and is rapidly induced on naive T cells after activation. Engagement of SLAM enhances antigen-specific proliferation and cytokine production by T cells carrying the CD4 antigen (CD4+). Particularly, the production of interferon-gamma (IFN-gamma) is strongly upregulated, even in T helper type 2 (Th2) CD4+ T-cell clones, whereas no induction of interleukin (IL)-4 or IL-5 production was observed in Th1 clones. In addition, the engagement of SLAM induces directly the proliferation of CD4+ T-cell clones and preactivated T cells, in the absence of any other stimuli, and without CD28 involvement. Thus SLAM is a novel receptor on T cells that, when engaged, potentiates T-cell expansion in a CD28-independent manner and induces a Th0/Th1 cytokine production profile.

Autoantibodies and immune complexes are major pathogenic factors in autoimmune injury, responsible for initiation of the inflammatory cascade and its resulting tissue damage. This activation results from the interaction of immunoglobulin (Ig)G Fc receptors containing an activation motif (ITAM) with immune complexes (ICs) and cytotoxic autoantibodies which initiates and propagates an inflammatory response. In vitro, this pathway can be interrupted by coligation to FcgammaRIIB, an IgG Fc receptor containing an inhibitory motif (ITIM). In this report, we describe the in vivo consequences of FcgammaRII deficiency in the inflammatory response using a mouse model of IC alveolitis. At subthreshold concentrations of ICs that fail to elicit inflammatory responses in wild-type mice, FcgammaRII-deficient mice developed robust inflammatory responses characterized by increased hemorrhage, edema, and neutrophil infiltration. Bronchoalveolar fluids from FcgammaRII-/- stimulated mice contain higher levels of tumor necrosis factor and chemotactic activity, suggesting that FcgammaRII deficiency lowers the threshold of IC stimulation of resident cells such as the alveolar macrophage. In contrast, complement- and complement receptor-deficient mice develop normal inflammatory responses to suprathreshold levels of ICs, while FcRgamma-/- mice are completely protected from inflammatory injury. An inhibitory role for FcgammaRII on macrophages is demonstrated by analysis of FcgammaRII-/- macrophages which show greater phagocytic and calcium flux responses upon FcgammaRIII engagement. These data reveal contrasting roles for the cellular receptors for IgG on inflammatory cells, providing a regulatory mechanism for setting thresholds for IC sensitivity based on the ratio of ITIM to ITAM FcgammaR expression. Exploiting the FcgammaRII inhibitory pathway could thus provide a new therapeutic approach for modulating antibody-triggered inflammation.

The partial control of viremia during acute human immunodeficiency virus type 1 (HIV-1) infection is accompanied by an HIV-1-specific cytotoxic T-lymphocyte (CTL) response and an absent or infrequent neutralizing antibody response. The control of HIV-1 viremia has thus been attributed primarily, if not exclusively, to CTL activity. In this study, the role of antibody in controlling viremia was investigated by measuring the ability of plasma or immunoglobulin G from acutely infected patients to inhibit primary strains of HIV-1 in the presence of natural-killer (NK) effector cells. Antibody that inhibits virus when combined with effector cells was present in the majority of patients within days or weeks after onset of symptoms of acute infection. Furthermore, the magnitude of this effector cell-mediated antiviral antibody response was inversely associated with plasma viremia level, and both autologous and heterologous HIV-1 strains were inhibited. Finally, antibody from acutely infected patients likely reduced HIV-1 yield in vitro both by mediating effector cell lysis of target cells expressing HIV-1 glycoproteins and by augmenting the release of beta-chemokines from NK cells. HIV-1-specific antibody may be an important contributor to the early control of HIV viremia.

OBJECTIVE

To explore the increased incidence of intravenous immunoglobulin- (IVIG) resistance among San Diego County patients with Kawasaki disease (KD) in 2006 and to evaluate a scoring system to predict IVIG-resistant patients with KD.

METHODS

We performed a retrospective review of patients with KD treated within 10 days of fever onset. With multivariate analysis, independent predictors of IVIG-resistance were combined into a scoring system.

RESULTS

In 2006, 38.3% of patients with KD in San Diego County were IVIG-resistant, a significant increase over previous years. IVIG-resistance was not associated with a particular brand or lot of IVIG. Resistant patients were diagnosed earlier, had higher percent bands, and higher concentrations of C-reactive protein, alanine aminotransferase, and gamma-glutamyl transferase. They also had lower platelet counts and age-adjusted hemoglobin concentrations and were more likely to have aneurysms (P = .0008). A scoring system developed to predict IVIG-resistant patients using illness day, percent bands, gamma-glutamyl transferase, and age-adjusted hemoglobin had a sensitivity of 73.3% and specificity of 61.9%.

CONCLUSIONS

An unexplained increase in IVIG-resistance was noted among patients with KD in San Diego County in 2006. Scoring systems based on demographic and laboratory data were insufficiently accurate to be clinically useful in our ethnically diverse population.

To learn if Chlamydia trachomatis causes in young infants a distinctive penumonia characterized by chronic, afebrile course, diffuse lung involvement and elevated serum immunoglobulins G and M, 47 black infants four to 24 weeks of age were examined for nasopharyngeal shedding of C. trachomatis and serum immunofluorescent antibody to lymphogranuloma venereum Type I. Nasopharyngeal C. trachomatis was found in 18 of 20 with the pneumonia syndrome, two of 15 with various other illnesses and 10 of 12 with inclusion conjunctivitis but without lower respiratory illness. Chlamydial antibody titers of infants with the pneumonia syndrome were significantly elevated (geometric mean-1, pneumonia vs. conjunctivitis = 24,833 vs. 1024 P less than 0.001). No other commonly recognized respiratory pathogens were consistently associated with the pneumonia syndrome. We believe these findings demonstrate an association between the distinctive pneumonia syndrome and C. trachomatis. This, in turn, is a particular facet of a more general event consisting of frequent colonization of the respiratory tract by C. trachomatis in natally acquired infection.

Sarcoidosis is a disease of unknown etiology characterized by noncaseating epithelioid granulomas, oligoclonal CD4(+) T cell infiltrates, and immune complex formation. To identify pathogenic antigens relevant to immune-mediated granulomatous inflammation in sarcoidosis, we used a limited proteomics approach to detect tissue antigens that were poorly soluble in neutral detergent and resistant to protease digestion, consistent with the known biochemical properties of granuloma-inducing sarcoidosis tissue extracts. Tissue antigens with these characteristics were detected with immunoglobulin (Ig)G or F(ab')(2) fragments from the sera of sarcoidosis patients in 9 of 12 (75%) sarcoidosis tissues (150-160, 80, or 60-64 kD) but only 3 of 22 (14%) control tissues (all 62-64 kD; P = 0.0006). Matrix-assisted laser desorption/ionization time of flight mass spectrometry identified Mycobacterium tuberculosis catalase-peroxidase (mKatG) as one of these tissue antigens. Protein immunoblotting using anti-mKatG monoclonal antibodies independently confirmed the presence of mKatG in 5 of 9 (55%) sarcoidosis tissues but in none of 14 control tissues (P = 0.0037). IgG antibodies to recombinant mKatG were detected in the sera of 12 of 25 (48%) sarcoidosis patients compared with 0 of 11 (0%) purified protein derivative (PPD)(-) (P = 0.0059) and 4 of 10 (40%) PPD(+) (P = 0.7233) control subjects, suggesting that remnant mycobacterial catalase-peroxidase is one target of the adaptive immune response driving granulomatous inflammation in sarcoidosis.

B lymphocyte antigen receptors, membrane immunoglobulins (mIg), function in focusing and internalization of antigen for subsequent presentation to T cells and in transmembrane transduction of signals leading to cell activation, anergy, or deletion. Until quite recently, the ability of this receptor to transduce signals in spite of a virtual lack of cytoplasmic structure, left a significant gap in our understanding of how it is coupled to cytoplasmic signal propagators. Studies conducted during the past five years have defined a mIg-associated protein complex homologous to the CD3 complex associated with the T cell antigen receptor. Components of this disulfide linked heterodimeric complex, Ig-alpha and Ig-beta, contain an approximately 26 residue sequence motif termed ARH1, also known as TAM, which binds to cytoplasmic effectors, including src-family tyrosine kinases, and contains all structural information needed for signal transduction. Receptor associated src-family kinases which are activated following receptor cross-linking, also associate with downstream effectors, including phospholipase C gamma (PLC gamma), p21ras. GTPase activating protein (GAP), phosphatidylinositol 3-kinase (PI3-k) and microtubule associate protein kinase (MAPk2). In some cases, these associations are induced by receptor cross-linking and lead directly to effector activation. The current literature indicates that these interactions may occur in sequence and culminate in the activation of three major pathways of signal propagation including those mediated by PLC gamma, p21ras and PI3-k. This chapter reviews various molecular aspects of the B cell antigen receptor complex, including extended structure of the complex, and receptor-effector interactions and their biologic consequences. Finally, an integrated model of antigen receptor signaling is presented.

Conjugated and unlabeled peroxidase antibody methods have proven to be quite satisfactory in localizing sites of antigen-antibody reaction. The use of avidin-biotin-peroxidase complex (ABC), as well as protein A, can contribute significantly to the field of immunohistochemistry. The sensitivity and specificity of several immunohistochemical methods is compared. In general, the ABC method produced the most intense staining and the least background staining of any method tested. The unlabeled antibody (peroxidase-antiperoxidase: PAP) method also yielded satisfactory results, but it was less intense than the ABC method. In comparison to the PAP method, the indirect conjugated method presented slightly inferior staining intensities and significantly higher background staining. Protein A techniques produced a range of staining sensitivities similar to or inferior to the PAP technique. The main disadvantage in using protein A is that it reacts with intrinsic immunoglobulin (Ig) G, thus producing an intense background. Therefore, its use is not recommended on tissues that have either abundant immunoglobulins in their interstitium or numerous IgG-containing plasma cells.

Localization of beta-2 integrins in normal and Alzheimer disease temporal cortex was studied immunohistochemically. Resting microglia were found to express constitutively CD11a (LFA-1), CD11b (Mac-1, CR3), CD11c (P150, 95; CR4), and CD18 (beta-2). They were also found to express constitutively leukocyte common antigen and the immunoglobulin receptor Fc gamma RI. The intensity of expression of each of these antigens was enhanced on reactive microglia in Alzheimer disease tissue. HLA-DR was detected on only a few microglia in control tissue, but was intensely expressed on large numbers of reactive microglia in Alzheimer tissue. These data are consistent with a leukocyte origin and a phagocytic role for microglia. They provide further evidence of an inflammatory response of brain tissue in Alzheimer disease. The microglia were found to make up 9-12% of the total glial population in gray matter and 7.5-9% in white matter.

1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) is a secosteroid hormone that renders dendritic cells (DCs) tolerogenic, favoring the induction of regulatory T cells. Induction of DCs with tolerogenic properties by 1,25(OH)(2)D(3) is associated with increased selective expression of immunoglobulin-like transcript 3 (ILT3), suggesting its involvement in the immunoregulatory properties of this hormone. Here we show an in vivo correlate of the increased ILT3 expression on DCs in healing psoriatic lesions following topical treatment with the 1,25(OH)(2)D(3) analog calcipotriol. Analysis of DC subsets reveals a differential regulation of ILT3 expression by 1,25(OH)(2)D(3), with a marked up-regulation in myeloid DCs but no effect on its expression by plasmacytoid DCs. A regulatory role for ILT3 expressed on DCs is indicated by the increased interferon-gamma (IFN-gamma) secretion promoted by anti-ILT3 addition to cultures of DCs and T cells, but this effect is blunted in 1,25(OH)(2)D(3)-treated DCs, suggesting ILT3-independent mechanisms able to regulate T-cell activation. Although ILT3 expression by DCs is required for induction of regulatory T cells, DC pretreatment with 1,25(OH)(2)D(3) leads to induction of CD4(+)Foxp3(+) cells with suppressive activity irrespective of the presence of neutralizing anti-ILT3 monoclonal antibody (mAb), indicating that ILT3 expression is dispensable for the capacity of 1,25(OH)(2)D(3)-treated DCs to induce regulatory T cells.

The characteristic electroencephalographic patterns within the hippocampus are theta and sharp waves. Septal neurons are believed to play an essential role in the rhythm generation of the theta pattern. The present study examined the physiological consequences of complete and selective damage of septohippocampal cholinergic neurons on hippocampal theta activity in rats. A selective immunotoxin against nerve growth factor receptor bearing cholinergic neurons (192 immunoglobulin G-saporin), [Wiley R. G. et al. (1991) Brain Res. 562, 149-153] was infused into the medial septal area (0.11-0.42 microgram). Hippocampal electrical activity was monitored during trained wheel running, drinking and the paradoxical phase of sleep, as well as following cholinomimetic treatment. A moderate dose of toxin (0.21 microgram) eliminated the septohippocampal cholinergic projection, as evidenced by a near total absence of choline acetyltransferase-immunoreactive neurons in the medial septum and the vertical limb of the diagonal band, and by the absence of acetylcholinesterase-positive fibers in the dorsal hippocampus. In the same rats, parvalbumin immunoreactivity, a reliable marker for septohippocampal GABAergic neurons, [Freund T. F. (1989) Brain Res. 478, 375-381], remained unaltered. In addition, retrograde transport of the tracer fluorogold demonstrated that the parvalbumin cell population preserved its axonal projection to the hippocampus. Following toxin treatment, the power of hippocampal theta, but not its frequency, decreased in a dose-dependent manner. Reduction of theta power occurred between three and seven days after the toxin treatment and remained unaltered thereafter up to eight weeks. A dose which eliminated all septohippocampal cholinergic neurons (0.21 microgram) left a small but significant theta peak in the power spectra during wheel running, paradoxical phase of sleep and intraseptal infusion of carbachol (5 micrograms). Peripheral administration of physostigmine (1 mg/kg) induced only slow (1.5-2.0 Hz) rhythmic waves. No changes were observed in the gamma (50-100 Hz) band. These findings indicate that the integrity of the septohippocampal GABAergic projection is sufficient to maintain some hippocampal theta activity. We hypothesize that cholinergic neurons serve to increase the population phase-locking of septal cells and thereby regulate the magnitude of hippocampal theta.

Using a single vector targeting strategy, we have generated mice with a combined deficiency of interleukin (IL)-4 and IL-13 to clarify their roles in T helper type 2 (Th2) cell responses. Using immunological challenges normally characterized by a Th2-like response, we have compared the responses of the double-deficient mice with those generated by wild-type, IL-4-deficient, and IL-13-deficient mice. Using a pulmonary granuloma model, induced with Schistosoma mansoni eggs, we demonstrate that although eosinophil infiltration, immunoglobulin E, and IL-5 production are reduced in the IL-4-deficient mice and IL-13-deficient mice, they are abolished only in the combined absence of both cytokines. Furthermore, IL-4/13-deficient animals are severely impaired in their ability to expel the gastrointestinal nematode Nippostrongylus brasiliensis. Unexpectedly, N. brasiliensis-infected IL-4/13-deficient mice developed elevated IL-5 and eosinophilia, indicating that compensatory mechanisms exist for the expression of IL-5, although serum IgE remained undetectable. IL-4/13-deficient mice default to a Th1-like phenotype characterized by the expression of interferon gamma and the production of IgG2a and IgG2b. We conclude that IL-4 and IL-13 cooperate to initiate rapid Th2 cell-driven responses, and that although their functions overlap, they perform additive roles.