We have determined the complete nucleotide sequence of the C57BL/6 allele of the mouse immunoglobulin gamma 2a chain gene. A comparison with the BALB/c gamma 2a gene for 1912 nucleotides reveals that the two alleles exhibit extensive divergence, since there are 138 single-base-pair differences and 8 insertions or deletions. We have compared the two gamma 2a alleles with the two corresponding gamma 2b alleles, which differ in only 12 positions. It appears that among the 134 differences between the two gamma 2a alleles, 70 are at positions where gamma 2a and gamma 2b are identical in the BALB/c haplotype and 54 are at positions where gamma 2a and gamma 2b are identical in the C57BL/6 haplotype. All these results suggest that nonreciprocal gene conversion between nonallelic genes can introduce sequence homogeneity in linked genes and can generate extensive divergence and polymorphism in allelic genes. We suggest that the gamma 2a and gamma 2b gene ancestors freely diverged after duplication, and that the conversion events were promoted by a deletion shortening the distance between the two loci.

Monoclonal antibodies against purified fimbriae from this organism blocked its adherence to buccal epithelial cells. Three clones of monoclonal antibodies against these fimbriae were selected for use. The isotype of the three was IgGimmunoglobulin G (IgG) antibodies inhibited bacterial adherence to the human buccal epithelial cells, but had no effect on bacterial haemagglutination to various animal and human erythrocytes. The papain-cleaved Fab fragment, which did not allow cell to cell cross-linking, also inhibited adherence of B. gingivalis 381 but did not interfere with haemagglutination. Thus the fimbriae of B. gingivalis 381 may be responsible for adherence to epithelial cells, which supports the notion that a different type of fimbria or a lectin-like protein may be acting as haemagglutinin in this bacterium.

Triggering of mast cells and basophils by immunoglobulin E (IgE) and antigen induces various biochemical signals, including tyrosine kinase activation, which lead to cell degranulation and the release of mediators of the allergic reaction. The high-affinity receptor for IgE (Fc epsilon RI) responsible for initiating these events is a complex structure composed of an IgE-binding alpha-chain, a beta-chain and a homodimer of gamma-chains. It has been assumed that beta and gamma, which have extensive cytoplasmic domains, play an important but undefined role in coupling Fc epsilon RI to signal transduction mechanisms. Here we show that Fc epsilon RI engagement induces immediate in vivo phosphorylation on beta (tyrosine and serine) and gamma (tyrosine and threonine) by at least two different non-receptor kinases. We take advantage of unique features of this receptor system to demonstrate that the phosphorylation signal is restricted to activated receptors and is immediately reversible upon receptor disengagement by undefined phosphatases. Rapid phosphorylation and dephosphorylation may be a general mechanism to couple and uncouple activated receptors to other effector molecules. This could be particularly relevant to other multimeric receptors containing Fc epsilon RI gamma-chains or the related zeta and eta chains such as the T-cell antigen receptor (TCR) and the low-affinity receptor for immunoglobulin G (Fc gamma RIII, CD16).

Two proteins which are capable of dispersing cell clusters of Staphylococcus aureus have been purified from a S. aureus FDA209P culture supernatant. Both of them were found to have bacteriolytic activity. From the elution profile of column chromatography and Western blot (immunoblot) analysis, one of them was identified as a 51-kDa endo-beta-N-acetylglucosaminidase (GL). The other was a 62-kDa protein on the basis of sodium dodecyl sulfate gel electrophoresis. Analysis of the peptidoglycan fragments following treatment with the 62-kDa protein indicated that this protein is an N-acetylmuramyl-L-alanine amidase (AM). In vitro studies of cluster dispersion activities using S. aureus mutant strains Lyt66 or S. aureus Wood46 grown as clusters demonstrated that these two enzymes act synergistically to disperse clusters into single cells. Antiserum against the 51-kDa GL cross-reacted with the 62-kDa AM, and S. aureus FDA209P grown in the presence of anti-51-kDa-GL immunoglobulin G induced giant clusters. Clusters induced by anti-51-kDa GL and by Cibacron blue F3G-A were dispersed by coincubation with the 51-kDa GL and the 62-kDa AM. Western blot analysis demonstrated that the 51-kDa GL and the 62-kDa AM were missing in culture supernatants of S. aureus Lyt66, Wood46, and RUSAL2 (Tn551 autolysin-defective mutant), which grow in clusters. These results strongly suggest that the 51-kDa GL and 62-kDa AM are involved in cell separation of daughter cells after cell division.

Helminthiases, which are highly prevalent in areas where malaria is endemic, have been shown to modulate or suppress the immune response to unrelated antigens or pathogens. In this study, we established a murine model of coinfection with a gastrointestinal nematode parasite, Heligmosomoides polygyrus, and the blood-stage malaria parasite Plasmodium chabaudi AS in order to investigate the modulation of antimalarial immunity by concurrent nematode infection. Chronic infection with the nematode for 2, 3, or 5 weeks before P. chabaudi AS infection severely impaired the ability of C57BL/6 mice to control malaria, as demonstrated by severe mortality and significantly increased malaria peak parasitemia levels. Coinfected mice produced significantly lower levels of gamma interferon (IFN-gamma) during P. chabaudi AS infection than mice infected with malaria alone. Concurrent nematode infection also suppressed production of type 1-associated, malaria-specific immunoglobulin G2a. Mice either infected with the nematode alone or coinfected with the nematode and malaria had high transforming growth factor beta1 (TGF-beta1) levels, and concurrent nematode and malaria infections resulted in high levels of interleukin-10 in vivo. Splenic CD11c(+) dendritic cells (DC) from mice infected with malaria alone and coinfected mice showed similarly increased expression of CD40, CD80, and CD86, but DC from coinfected mice were unable to induce CD4(+) T-cell proliferation and optimal IFN-gamma production in response to the malaria antigen in vitro. Importantly, treatment of nematode-infected mice with an anthelmintic drug prior to malaria infection fully restored protective antimalarial immunity and reduced TGF-beta1 levels. These results demonstrate that concurrent nematode infection strongly modulates multiple aspects of immunity to blood-stage malaria and consequently impairs the development of protective antimalarial immunity.

BACKGROUND

Respiratory syncytial virus (RSV) is the most common cause of lower respiratory tract infection in infants. MEDI-493 (palivizumab) is a humanized monoclonal antibody to the fusion protein of RSV and is active in animal models for prevention of pulmonary RSV replication.

OBJECTIVE

To describe the safety, tolerance, immunogenicity and pharmacokinetics of repeat intravenous doses of MEDI-493 in premature infants or infants with bronchopulmonary dysplasia.

METHODS

Phase I/II multicenter, randomized, double blind, placebo-controlled, dose escalation trial.

METHODS

Infants born prematurely (< or = 35 weeks of gestation) who were < or = 6 months of age and infants with bronchopulmonary dysplasia who were < or = 24 months of age were eligible for study participation. STUDY AGENTS: Participants received 3, 10 or 15 mg/kg MEDI-493 or 0.9% saline intravenously every 30 days for up to five doses.

RESULTS

MEDI-493 was safe and well-tolerated and did not induce a specific anti-MEDI-493 response. The mean half-life of 20 days was comparable with that of other immunoglobulin G preparations. Mean trough serum concentrations 30 days after Infusion 1 were 6.8, 36.1 and 60.6 microg/ml for the 3-, 10- and 15-mg/kg dose groups, respectively. After Infusion 2 the trough concentrations were 11.9, 45.2 and 70.7 microg/ml. After subsequent doses the mean trough values ranged from 14 to 18 microg/ml in those given 3 mg/kg and were>> 40 microg/ml for patients who received 10 or 15 mg/kg MEDI-493 (46 to 72 microg/ml and 88 to 96 microg/ml, respectively).

CONCLUSIONS

MEDI-493 was safe and well-tolerated in this high risk pediatric population. Mean serum concentrations of MEDI-493 that have been shown to produce a 2-log reduction in pulmonary RSV titer in cotton rats were maintained when 10 or 15 mg/kg MEDI-493 was given every 30 days to pediatric patients at high risk for serious RSV disease. Monthly doses of 15 mg/kg maintained concentrations of>> 40 microg/ml for the majority of patients.

IL-21 is a type I cytokine that like IL-2, IL-4, IL-7, IL-9, and IL-15 shares the common cytokine receptor gamma chain, gamma(c). IL-21 is produced by activated CD4(+) T cells, NKT cells, and Th17 cells and has pleiotropic actions on a range of lymphoid lineages. IL-21 regulates immunoglobulin production and drives B cell terminal differentiation into plasma cells, cooperatively expands CD8(+) T cells and drives Th17 differentiation, has inhibitory effects on antigen presentation by dendritic cells, and can be pro-apoptotic for B and NK cells. Moreover, IL-21 has potent anti-tumor effects and is implicated in the development of autoimmune diseases. Regulating IL-21 actions in vivo therefore has clinical potential for a range of diseases and is an area of active investigation.

Two distinct patterns of neutralization were identified by comparing the neutralization curves of monoclonal antibodies (MAbs) directed at the two surface proteins, VP4 and VP7, of rhesus rotavirus. VP7-specific MAbs were able to neutralize virus efficiently, and slight increases in antibody concentration resulted in a sharp decline in infectivity. On the other hand, MAbs to VP4 proved much less efficient at neutralizing rhesus rotavirus, and the fraction of infectious virus decreased gradually throughout a wide range of antibody concentrations. MAbs directed at VP8*, the smaller trypsin cleavage fragment of VP4, were shown to efficiently prevent binding of radiolabeled virions to MA104 cell monolayers, to an extent and at concentrations comparable to those required for neutralization of infectivity. Conversely, MAbs recognizing VP7 or the larger VP4 trypsin cleavage product, VP5*, showed little or no inhibitory effect on virus binding to cells. All MAbs studied were able to neutralize rotavirus that was already bound to the surface of cells. The MAbs directed at VP8*, but not those recognizing VP5* or VP7, were shown to mediate release of radiolabeled virus from the surface of the cells. With MAbs directed at VP7, papain digestion of virus-bound antibody molecules led to an almost complete recovery of infectivity. Neutralization could be fully restored by incubation of virus-Fab complexes with anti-mouse immunoglobulin G antiserum. Neutralization with MAbs directed at VP8* proved insensitive to digestion with papain as well as to the addition of anti-immunoglobulin antibodies.

Synaptic inhibition is a central factor in the fine tuning of neuronal activity in the central nervous system. Symptoms consistent with reduced inhibition such as stiffness, spasms and anxiety occur in paraneoplastic stiff person syndrome with autoantibodies against the intracellular synaptic protein amphiphysin. Here we show that intrathecal application of purified anti-amphiphysin immunoglobulin G antibodies induces stiff person syndrome-like symptoms in rats, including stiffness and muscle spasms. Using in vivo recordings of Hoffmann reflexes and dorsal root potentials, we identified reduced presynaptic GABAergic inhibition as an underlying mechanism. Anti-amphiphysin immunoglobulin G was internalized into neurons by an epitope-specific mechanism and colocalized in vivo with presynaptic vesicular proteins, as shown by stimulation emission depletion microscopy. Neurons from amphiphysin deficient mice that did not internalize the immunoglobulin provided additional evidence of the specificity in antibody uptake. GABAergic synapses appeared more vulnerable than glutamatergic synapses to defective endocytosis induced by anti-amphiphysin immunoglobulin G, as shown by increased clustering of the endocytic protein AP180 and by defective loading of FM 1-43, a styryl dye used to label cell membranes. Incubation of cultured neurons with anti-amphiphysin immunoglobulin G reduced basal and stimulated release of γ-aminobutyric acid substantially more than that of glutamate. By whole-cell patch-clamp analysis of GABAergic inhibitory transmission in hippocampus granule cells we showed a faster, activity-dependent decrease of the amplitude of evoked inhibitory postsynaptic currents in brain slices treated with antibodies against amphiphysin. We suggest that these findings may explain the pathophysiology of the core signs of stiff person syndrome at the molecular level and show that autoantibodies can alter the function of inhibitory synapses in vivo upon binding to an intraneuronal key protein by disturbing vesicular endocytosis.

We have used a quantitative assay that measures independent rate constants for phagocytosis and killing of Staphylococcus aureus to investigate the involvement of superoxide and myeloperoxidase in bacterial killing by human neutrophils. To inhibit superoxide-dependent processes, superoxide dismutase was cross-linked to immunoglobulin G and the conjugate was attached to the surface of S. aureus via protein A in its cell wall. Myeloperoxidase was inhibited with azide, and myeloperoxidase-deficient neutrophils were used. Adding the NADPH oxidase inhibitor diphenyleneiodonium, to prevent superoxide production, decreased the killing rate to 25%, indicating that oxidative killing mechanisms predominate in this system. The rate constant for killing of S. aureus with superoxide dismutase attached was 70% of that for control bacteria linked to inactivated enzyme. Superoxide dismutase had no effect in the presence of diphenyleneiodonium. The rate of killing was decreased to 33% in the presence of azide and to 40% with myeloperoxidase-deficient neutrophils. Superoxide dismutase had no effect in the presence of azide. On the assumption that the oxidative and nonoxidative components of killing can be considered separately, the oxidative rate was decreased by almost half by superoxide dismutase and was about six times lower when myeloperoxidase was inactive. We conclude that myeloperoxidase-dependent processes are strongly favored by human neutrophils as their prime mechanism of oxidative killing of S. aureus and that superoxide makes a direct contribution to killing. Our results also suggest that superoxide acts in conjunction with a myeloperoxidase-dependent pathway.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer mortality worldwide and ranks second in China. The prognosis of HCC remains dismal mainly because of its late diagnosis, especially in patients with coexisting chronic liver diseases. To identify serum biomarkers for HCC, sera from 20 healthy volunteers, 20 hepatitis B virus (HBV) infected patients and 20 HCC patients were selected for screening study and same number of sera into the same three groups were used for validation study. A strategy including sonication, albumin and immunoglobulin G (IgG) depletion and desalting was optimized for screening differentially expressed proteins of low abundance in serum. By 2-DE image analysis and MALDI-TOF-MS/MS identification, eight proteins including heat-shock protein 27 (HSP27), alpha-fetoprotein (AFP), alpha-1 antitrypsin, clusterin, caeruloplasmin, haptoglobin alpha2 chain, tranferrin and transthyretin were found significantly changed among the healthy, HBV and HCC groups. Further validation study by Western blot showed the detection of HSP27 in 90% HCC sera and two HBV sera, but in none of normal sera. Thus, 2-DE based serum proteome analysis can be useful in the screening of serum biomarkers for HCC and HSP27 could aid in the diagnosis of HCC though further validation is needed.

This study describes the clinicopathologic features of 5 patients who developed a fulminant Epstein-Barr virus (EBV)-positive clonal T-cell lymphoproliferative disorder (LPD) after acute EBV infection. One additional patient developed a similar disorder in the setting of long-standing chronic active EBV infection. Detailed immunophenotyping, in situ hybridization for EBV early RNA-1 (EBER1) and polymerase chain reaction (PCR) analyses for immunoglobulin (Ig) heavy chain and T-cell receptor (TCR)-gamma gene rearrangements were performed on paraffin-embedded tissue from all patients. In addition, EBV strain typing and detection of the characteristic 30-bp deletion of the latent membrane protein-1 (LMP-1) gene were performed by PCR. Controls included 8 cases of uncomplicated infectious mononucleosis (IM). Patients included 4 males and 2 females with a median age of 18 years (2-37 years). Three patients were Mexican, 2 were white, and 1 was of Asian descent. All presented with fever, hepatosplenomegaly, and pancytopenia; 5 were previously healthy, but had a clinical history of a recent viral-like upper respiratory illness (1 week to 2 months), and 1 patient had documented chronic active EBV infection for 7 years. Serologic data for EBV were incomplete but titers were either negative or only modestly elevated in 3 cases. In 1 case serology was consistent with severe chronic active EBV infection. In the remaining 2 cases serologic studies were not performed. All patients died within 7 days to 8 months of presentation with T-cell LPD. On histologic examination, the liver and spleen showed prominent sinusoidal and portal lymphoid infiltrates of CD3(+), beta F1(+), EBER1(+) T cells lacking significant cytologic atypia. Two cases were CD4(+), 2 cases were CD8(+), and 2 cases had admixed CD4(+) and CD8(+) cells without clear subset predominance. All were TIA-1(+), CD56(-). Only rare B cells were noted. Marked erythrophagocytosis was present. Molecular analysis revealed identical T-cell clones in 2 or more sites (liver, spleen, lymph node) in 5 cases. All patients carried type A EBV; 4 cases had wild-type EBV-LMP, and 2 showed the 30-bp deletion. This fulminant T-cell LPD after acute/chronic EBV infection is characterized by hepatosplenomegaly, often without significant lymphadenopathy, fever, liver failure, pancytopenia, and erythrophagocytosis indicative of a hemophagocytic syndrome. EBV serology may be misleading, with lack of elevated titers. The presence of an EBER1(+) T-cell infiltrate with scant B cells should alert one to this diagnosis. Although cytologic atypia is minimal, studies for T-cell clonality confirm the diagnosis. (Blood. 2000;96:443-451)

BACKGROUND

Necrotizing and crescentic glomerulonephritis related to antineutrophil cytoplasmic autoantibodies (ANCA) is typically referred to as "pauci-immune"; however, it is not unusual for renal biopsies in such cases to exhibit some immune complex deposition within glomeruli on immunofluorescence and/or electron microscopic study. The composition and intraglomerular localization of such deposits in ANCA-glomerulonephritis has not been widely studied, and their potential pathologic and clinical significance is not clear, although a possible synergistic effect between immune complexes and ANCA in producing more severe glomerulonephritis is suggested by some human and animal studies.

METHODS

Electron micrographs from 126 renal biopsies showing necrotizing/crescentic glomerulonephritis characterized by positive ANCA serology [C-ANCA, anti-proteinase 3 (anti-PR3), or anti-myeloperoxidase (MPO)] or necrotizing arteritis in the absence of known ANCA results were examined for the presence, quantity, and location of electron-dense deposits. The presence or absence of such deposits was correlated with histologic findings (fraction of glomeruli with crescents and segmental necrotizing lesions, mesangial and endocapillary hypercellularity), immunofluorescence findings, and clinical data, including serum creatinine and 24-hour urine protein levels at the time of biopsy.

RESULTS

Sixty-eight (54%) of these biopsies showed glomerular immune complex deposits on electron microscopy; 87% of the latter also showed positive immunofluorescence findings for at least one immunoglobulin or complement component, although staining was relatively mild in most instances (< or =2+ on a 0 to 4+ scale in all but eight cases). Nearly half of biopsies negative for deposits by electron microscopy also showed positive immunofluorescence findings, though even more so than in cases with deposits on electron microscopy the intensity of immunofluorescence staining in these biopsies was typically very weak (trace or trace to 1+ in most cases, none >2+). Hypercellularity within the glomerular tuft was seen in 50% of biopsies with deposits on electron microscopy but only 14% of those without deposits; in each group this was usually mild and mesangial. Notably, the presence of deposits on electron microscopy was associated with a higher median level of proteinuria (3.2 versus 1.3 g/24 hours, P < 0.0001) and a higher median percentage of glomeruli with crescents (62.5% versus 44.0%, P= 0.06).

CONCLUSIONS

Immune complex deposits were found on electron microscopy in just over half of renal biopsies with crescentic glomerulonephritis associated with positive ANCA serology and/or necrotizing arteritis. Clinical correlations suggest that these immune complex deposits may somehow potentiate the effect of ANCA in producing glomerulonephritis.

OBJECTIVE

Osteoclasts are terminally differentiated cells of the monocyte/macrophage lineage that resorb bone matrix. Bone destruction in rheumatoid arthritis is mainly attributable to the abnormal activation of osteoclasts, and studies on activation of osteoclasts by the immune system have led to the new research field called osteoimmunology. This interdisciplinary field is very important to biologic research and to the treatment of diseases associated with the bone and immune systems.

RESULTS

The T-cell-mediated regulation of osteoclast differentiation is dependent on cytokines and membrane-bound factors expressed by T cells. The cross-talk between receptor activator of nuclear factor-kappaB ligand and interferon-gamma has been shown to be crucial for the regulation of osteoclast formation in arthritic joints. Recent studies indicate that an increasing number of immunomodulatory factors are associated with the regulation of bone metabolism: nuclear factor of activated T cells c1 has been shown to be the key transcription factor for osteoclastogenesis, the activation of which requires calcium signaling induced by the immunoglobulin-like receptors.

CONCLUSIONS

New findings in osteoimmunology will be instrumental in the development of strategies for research into the treatment of various diseases afflicting the skeletal and immune systems.

BACKGROUND

Immunoglobulin A nephropathy (IgAN) is the most common cause of chronic renal failure among primary glomerulonephritis patients. The best treatment for IgAN remains poorly defined. We planned a long-term, prospective, open-label, multicentre, centrally randomized controlled trial to assess whether the combination of prednisone and ramipril was more effective than ramipril alone in patients with proteinuric IgAN.

METHODS

Ninety-seven biopsy-proven IgAN patients with moderate histologic lesions, 24-h proteinuria>> or =1.0 g and estimated glomerular filtration rate (eGFR)>> or = 50 ml/min/ 1.73 m(2) were randomly allocated to receive a 6-month course of oral prednisone plus ramipril (combination therapy group) or ramipril alone (monotherapy group) for the total duration of follow-up. The primary outcome was the progression of renal disease defined as the combination of doubling of baseline serum creatinine or end-stage kidney disease (ESKD). The secondary outcomes were the rate of renal function decline defined as the eGFR slope over time, and the reduction of 24-h proteinuria.

RESULTS

After a follow-up of up to 96 months, 13/49 (26.5%) patients in the monotherapy group reached the primary outcome compared with 2/48 (4.2%) in the combination therapy group. The Kaplan-Meier analysis showed a significantly higher probability of not reaching the combined outcome in the combination therapy group than in the monotherapy group (85.2% versus 52.1%; log-rank test P = 0.003). In the multivariate analysis, baseline serum creatinine and 24-h proteinuria were independent predictors of the risk of primary outcome; treatment with prednisone plus ramipril significantly reduced the risk of renal disease progression (hazard ratio 0.13; 95% confidence interval 0.03-0.61; P = 0.01). The mean rate of eGFR decline was higher in the monotherapy group than in the combination therapy group (-6.17 +/- 13.3 versus -0.56 +/- 7.62 ml/min/ 1.73 m(2)/year; P = 0.013). Moreover, the combined treatment reduced 24-h proteinuria more than ramipril alone during the first 2 years.

CONCLUSIONS

Our results suggest that the combination of corticosteroids and ramipril may provide additional benefits compared with ramipril alone in preventing the progression of renal disease in proteinuric IgAN patients in the long-term follow-up.

Immunglobulin G (IgG) sialylation represents a key checkpoint that determines the engagement of pro- or anti-inflammatory Fcγ receptors (FcγR) and the direction of the immune response. Whether IgG sialylation influences osteoclast differentiation and subsequently bone architecture has not been determined yet, but may represent an important link between immune activation and bone loss. Here we demonstrate that desialylated, but not sialylated, immune complexes enhance osteoclastogenesis in vitro and in vivo. Furthermore, we find that the Fc sialylation state of random IgG and specific IgG autoantibodies determines bone architecture in patients with rheumatoid arthritis. In accordance with these findings, mice treated with the sialic acid precursor N-acetylmannosamine (ManNAc), which results in increased IgG sialylation, are less susceptible to inflammatory bone loss. Taken together, our findings provide a novel mechanism by which immune responses influence the human skeleton and an innovative treatment approach to inhibit immune-mediated bone loss.

Toll-like receptor 7 (TLR7) is critical to the induction of antiviral immunity, but TLR7 dosage is also a key pathogenic factor in systemic lupus erythematosus (SLE), an autoimmune disease with strong female bias. SLE prevalence is also elevated in individuals with Klinefelter syndrome, who carry one or more supernumerary X chromosomes, suggesting that the X chromosome complement contributes to SLE susceptibility. TLR7 is encoded by an X chromosome locus, and we examined here whether the TLR7 gene evades silencing by X chromosome inactivation in immune cells from women and Klinefelter syndrome males. Single-cell analyses of TLR7 allelic expression demonstrated that substantial fractions of primary B lymphocytes, monocytes, and plasmacytoid dendritic cells not only in women but also in Klinefelter syndrome males express TLR7 on both X chromosomes. Biallelic B lymphocytes from women displayed greater TLR7 transcriptional expression than the monoallelic cells, correlated with higher TLR7 protein expression in female than in male leukocyte populations. Biallelic B cells were preferentially enriched during the TLR7-driven proliferation of CD27+ plasma cells. In addition, biallelic cells showed a greater than twofold increase over monoallelic cells in the propensity to immunoglobulin G class switch during the TLR7-driven, T cell-dependent differentiation of naive B lymphocytes into immunoglobulin-secreting cells. TLR7 escape from X inactivation endows the B cell compartment with added responsiveness to TLR7 ligands. This finding supports the hypothesis that enhanced TLR7 expression owing to biallelism contributes to the higher risk of developing SLE and other autoimmune disorders in women and in men with Klinefelter syndrome.

Although unrelated hematopoietic cell transplantation (HCT) is curative for many hematologic malignancies, complications and relapse remain challenging obstacles. Natural killer (NK) cells, which recover quickly after transplantation, produce cytokines and express killer immunoglobulin-like receptors (KIRs) that regulate their cytotoxicity. Some clinical trials based on a KIR ligand mismatch strategy are associated with less relapse and increased survival, but results are mixed. We hypothesized that T cells in the graft may affect NK cell function and KIR expression after unrelated transplantation and that these differences correlate with clinical outcomes. NK cell function was evaluated using 77 paired samples from the National Marrow Donor Program Research Repository. Recipient NK cells at 100 days after both unmanipulated bone marrow (UBM) and T-cell depleted (TCD) transplants were compared with NK cells from their healthy donors. NK cells expressed fewer KIRs and produced more interferon gamma (IFN-gamma) after UBM compared to TCD transplants. Multivariate models showed that increased NK cell IFN-gamma production correlated with more acute graft-versus-host disease (GVHD), and decreased KIR expression correlated with inferior survival. These results support the notion that T cells in the graft affect NK cell reconstitution in vivo. Understanding these mechanisms may result in strategies to improve clinical outcomes from unrelated HCT.

Sjögren's syndrome is a systemic autoimmune disease characterized by lymphocytic infiltrations of lacrimal and salivary glands. SS patients produce a variety of autoantibodies, including RF and ANA. Genetic factors, including HLA-DR3, predispose to primary SS. In contrast to normal SGs, the SS SG epithelial cells express high levels of HLA-DR antigens. This class II gene expression on the target organ may represent the structural basis for HLA-associated disease susceptibility. The glands are infiltrated with CD4+ T cells that can produce cytokines, including IL-2 and interferon-gamma. B cells within the SG produce autoantibodies, including RF. These SG B cells frequently use the VKIIIb subgroup of kappa light chain, a feature that SS patients share with Waldenstrom's macroglobulinemia patients. B cells undergo small clonal expansions that can be detected on Southern blot as immunoglobulin gene rearrangements, and SS patients have a markedly increased risk of developing non-Hodgkin's B-cell lymphoma involving the SGs and cervical lymph nodes. Due to accessibility of the SG for biopsy and the characteristic patterns of autoantibody production, SS provides an opportunity to study the target organ for autoimmune destruction and the transition from autoimmunity to lymphoma.

BACKGROUND

Multiple sclerosis is an autoimmune disorder characterised by the repeated occurrence of demyelinating lesions within the central nervous system. Uncontrolled studies and experimental evidence suggest beneficial effects of repeated administration of intravenous immunoglobulin (IVIg) by immunomodulating mechanisms and induction or remyelination. We aimed to investigate the efficacy of IVIg in a randomised double-blind multicentre study.

METHODS

Patients with relapsing-remitting multiple sclerosis were randomly assigned a monthly dose of IVIg (0.15-0.2 g/kg bodyweight) or placebo. Duration of treatment was 2 years. The primary outcome measures were the effect of treatment on clinical disability-measured by the absolute change in Kurtzke's expanded disability status scale (EDSS) score- and the proportion of patients with improved, stable, or worse clinical disability >> or = 1.0 grade on EDSS score).

RESULTS

Of the 243 patients screened, 150 met our eligibility criteria and were randomly assigned to IVIg or placebo. Before the start of treatment two patients in the placebo group dropped out, so there were 75 patients in the IVIg group and 73 in the placebo group. Intention-to-treat analysis showed that IVIg treatment had a beneficial effect on the course of clinical disability. The EDSS score decreased in the IVIg-treated patients and increased in the placebo group (-0.23 [95% CI -0.43 to -0.03] vs 0.12 [-0.13 to 0.37], p = 0.008). In the IVIg group, the numbers of patients with improved, stable, or worse clinical disability were 23 (31%), 40 (53%), and 12 (16%) compared with ten (14%), 46 (63%), and 17 (23%) in the placebo group. Side-effects were reported in three (4%) IVIg-treated patients and in four (5%) placebo-group patients, but were not directly linked to study medication.

CONCLUSIONS

Monthly IVIg is an effective and well-tolerated treatment for patients with relapsing-remitting multiple sclerosis.

To study the effect of wall protein A on bacterial opsonization, phagocytosis of 10 strains of Staphylococcus aureus with high and low protein A contents was measured. Those strains that contained the highest concentrations of protein A were phagocytized by human neutrophils at a slower rate than strains with little or no protein A when normal human serum and purified immunoglobulin G (IgG) were used as opsonic sources. When IgG-deficient serum was used as an source, however, protein A-rich strains were phagocytized more rapidly than protein A-deficient strains. Extracellular (purified) protein A decrease the opsonic activity of all sera tested including IgG-deficient serum. It is proposed that when IgG is not present in the opsonic medium, cell wall protein A is capable of activating complement at the bacterial surface and thereby opsonization is promoted.

Magic-angle spinning (MAS) solid-state NMR (SSNMR) techniques have emerged in recent years for solving complete structures of uniformly labeled proteins lacking macroscopic order. Strategies used thus far have relied primarily on semiquantitative distance restraints, analogous to the nuclear Overhauser effect (NOE) routinely used in solution NMR. Here, we present a complementary approach for using relative orientations of molecular fragments, determined from dipolar line shapes. Whereas SSNMR distance restraints typically have an uncertainty of approximately 1 A, the tensor-based experiments report on relative vector (pseudobond) angles with precision of a few degrees. By using 3D techniques of this type, vector angle (VEAN) restraints were determined for the majority of the 56-residue B1 immunoglobulin binding domain of protein G [protein GB1 (a total of 47 HN-HN, 49 HN-HC, and 12 HA-HB restraints)]. By using distance restraints alone in the structure calculations, the overall backbone root-mean-square deviation (bbRMSD) was 1.01 +/- 0.13 A (1.52 +/- 0.12 A for all heavy atoms), which improved to 0.49 +/- 0.05 A (1.19 +/- 0.07 A) on the addition of empirical chemical shift [torsion angle likelihood obtained from shift and sequence similarity (TALOS)] restraints. VEAN restraints further improved the ensemble to 0.31 +/- 0.06 A bbRMSD (1.06 +/- 0.07 A); relative to the structure with distances alone, most of the improvement remained (bbRMSD 0.64 +/- 0.09 A; 1.29 +/- 0.07 A) when TALOS restraints were removed before refinement. These results represent significant progress toward atomic-resolution protein structure determination by SSNMR, capabilities that can be applied to a large range of membrane proteins and fibrils, which are often not amenable to solution NMR or x-ray crystallography.

Human autoimmune diseases thought to arise from the combined effects of multiple susceptibility genes include systemic lupus erythematosus (SLE) and autoimmune diabetes. Well-characterised polygenic mouse models closely resembling each of these diseases exist, and genetic evidence links receptors for the Fc portion of immunoglobulin G (FcR) with their pathogenesis in mice and humans [1] [2] [3]. FcRs may be activatory or inhibitory and regulate a variety of immune and inflammatory processes [4] [5]. FcgammaRII (CD32) negatively regulates activation of cells including B cells and macrophages [6]. FcgammaRII-deficient mice are prone to immune-mediated disease [7] [8] [9]. The gene encoding FcgammaRII, Fcgr2, is contained in genetic susceptibility intervals in mouse models of SLE such as the New Zealand Black (NZB) contribution to the (NZB x New Zealand White (NZW)) F1 strain [1] [10] [11] and the BXSB strain [12], and in human SLE [1] [2] [3]. We therefore sequenced Fcgr2 and identified a haplotype defined by deletions in the Fcgr2 promoter region that is present in major SLE-prone mouse strains (NZB, BXSB, SB/Le, MRL, 129 [13]) and non-obese diabetic (NOD) mice but absent in control strains (BALB/c, C57BL/6, DBA/2, C57BL/10) and NZW mice. The autoimmune haplotype was associated with reduced cell-surface expression of FcgammaRII on macrophages and activated B cells and with hyperactive macrophages resembling those of FcgammaRII-deficient mice, and is therefore likely to play an important role in the pathogenesis of SLE and possibly diabetes.

The aryl hydrocarbon receptor complex heterodimeric transcription factor, comprising the basic helix-loop-helix-Per-ARNT-Sim (bHLH-PAS) domain aryl hydrocarbon receptor (AHR) and aryl hydrocarbon receptor nuclear translocator (ARNT) proteins, mediates the toxic effects of TCDD (2,3,7,8 tetrachlorodibenzo-p-dioxin). The molecular events underlying TCDD-inducible gene activation, beyond the activation of the AHRC, are poorly understood. The SRC-1/NCoA-1, NCoA-2/GRIP-1/TIF-2, and p/CIP/AIB/ACTR proteins have been shown to act as mediators of transcriptional activation. In this report, we demonstrate that SRC-1, NCoA-2, and p/CIP are capable of independently enhancing TCDD-dependent induction of a luciferase reporter gene by the AHR/ARNT dimer. Furthermore, injection of anti-SRC-1 or anti-p/CIP immunoglobulin G into mammalian cells abolishes the transcriptional activity of a TCDD-dependent reporter gene. We demonstrate by coimmunoprecipitation and by a reporter gene assay that SRC-1 and NCoA-2 but not p/CIP are capable of interacting with ARNT in vivo after transient transfection into mammalian cells, while AHR is capable of interacting with all three coactivators. We confirm the interactions of ARNT and AHR with SRC-1 with immunocytochemical techniques. Furthermore, SRC-1, NCoA-2, and p/CIP all associate with the CYP1A1 enhancer region in a TCDD-dependent fashion, as demonstrated by chromatin immunoprecipitation assays. We demonstrate by yeast two-hybrid, glutathione S-transferase pulldown, and mammalian reporter gene assays that ARNT requires its helix 2 domain but not its transactivation domain to interact with SRC-1. This indicates a novel mechanism of action for SRC-1. SRC-1 does not require its bHLH-PAS domain to interact with ARNT or AHR, but utilizes distinct domains proximal to its p300/CBP interaction domain. Taken together, these data support a role for the SRC family of transcriptional coactivators in TCDD-dependent gene regulation.