A chymotrypsinlike protease with an Mr of 95,000 was extracted from Treponema denticola ATCC 35405 and was partially purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteolytic activity was detected in an electrophoretogram containing polyacrylamide that was conjugated to bovine serum albumin. A single band of activity was detected when the T. denticola extract was solubilized and electrophoresed in the presence of sodium dodecyl sulfate. No activity was found in extracts of Treponema vincentii. The enzyme hydrolyzed transferrin, fibrinogen, alpha 1-antitrypsin, immunoglobulin A, immunoglobulin G, gelatin, bovine serum albumin, and a synthetic peptide containing phenylalanine. It did not degrade collagen or synthetic substrates containing arginine or proline. For the hydrolysis of azocoll, the pH optimum of the enzyme was 7.5. Heating at temperatures above 50 degrees C destroyed the activity. Reducing agents and the chelators EDTA and ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid increased the enzyme activity, while phenylmethylsulfonyl fluoride, L-1-tosylamide-2-phenylethyl chloromethyl ketone, sulfhydryl reagents, and human serum reduced activity. The ability of the enzyme to hydrolyze a number of humoral proteins suggests that it may be involved in spirochete invasiveness and tissue destruction.

Herpes simplex virus (HSV) entry into cells requires the binding of glycoprotein D (gD) to one of several cell surface receptors. The crystal structure of gD bound to one of these receptors, HveA/HVEM, reveals that the core of gD comprises an immunoglobulin fold flanked by a long C-terminal extension and an N-terminal hairpin loop. HveA is a member of the tumor necrosis factor receptor family and contains four cysteine-rich domains (CRDs) characteristic of this family. Fourteen amino acids within the gD N-terminal loop comprise the entire binding site for HveA. To determine the contribution of each gD contact residue to virus entry, we constructed gD molecules mutated in these amino acids. We determined the abilities of the gD mutants to bind receptors, facilitate virus entry, and mediate cell-cell fusion. Seven of the gD mutants exhibited wild-type levels of receptor binding and gD function. Results from the other seven gD mutants revealed three critical regions at the gD-HveA interface. (i) Several gD residues that participate in an intermolecular beta-sheet with HveA were found to be crucial for HveA binding and entry into HveA-expressing cells. (ii) Two gD residues that contact HveA-Y23 contributed to HveA binding but were not required for mediating entry into cells. HveA-Y23 fits into a crevice on the surface of gD and was previously shown to be essential for gD binding. (iii) CRD2 was previously shown to contribute to gD binding, and this study shows that one gD residue that contacts CRD2 contributes to HveA binding. None of the gD mutations prevented interaction with nectin-1, another gD receptor. However, when cotransfected with the other glycoproteins required for fusion, two gD mutants gained the ability to mediate fusion of cells expressing nectin-2, a gD receptor that interacts with several laboratory-derived gD mutants but not with wild-type gD. Thus, results from this panel of gD mutants as well as those of previous studies (A. Carfi, S. H. Willis, J. C. Whitbeck, C. Krummenacher, G. H. Cohen, R. J. Eisenberg, and D. C. Wiley, Mol. Cell 8:169-179, 2001, and S. A. Connolly, D. J. Landsburg, A. Carfi, D. C. Wiley, R. J. Eisenberg, and G. H. Cohen, J. Virol. 76:10894-10904, 2002) provide a detailed picture of the gD-HveA interface and the contacts required for functional interaction. The results demonstrate that of the 35 gD and HveA contact residues that comprise the gD-HveA interface, only a handful are critical for complex formation.

Immunological memory is characterized by heightened immunoglobulin (Ig) G antibody production caused in part by enhanced plasma cell formation conferred by conserved transmembrane and cytoplasmic segments in isotype-switched IgG B cell receptors. We tested the hypothesis that the IgG tail enhances intracellular B cell antigen receptor (BCR) signaling responses to antigen by analyzing B cells from Ig transgenic mice with IgM receptors or chimeric IgMG receptors containing the IgG tail segment. The IgG tail segment enhanced intracellular calcium responses but not tyrosine or extracellular signal-related kinase (ERK) phosphorylation. Biochemical analysis and crosses to CD22-deficient mice established that IgG tail enhancement of calcium and antibody responses, as well as marginal zone B cell formation, was not due to diminished CD22 phosphorylation or inhibitory function. Microarray profiling showed no evidence for enhanced signaling by the IgG tail for calcium/calcineurin, ERK, or nuclear factor kappaB response genes and little evidence for any enhanced gene induction. Instead, almost half of the antigen-induced gene response in IgM B cells was diminished 50-90% by the IgG tail segment. These findings suggest a novel "less-is-more" hypothesis to explain how switching to IgG enhances B cell memory responses, whereby decreased BCR signaling to genes that oppose marginal zone and plasma cell differentiation enhances the formation of these key cell types.

It has been suggested that epidermal Langerhans cells (LC) bearing immunoglobulin E (IgE) may be involved in the genesis of atopic disease. The identity of the IgE receptor(s) on LC remained unclear, although it represents a crucial point in understanding cellular events linked to the binding of allergens to LC via IgE. In this report, we demonstrate that epidermal LC express the high affinity receptor for the Fc fragment of IgE (Fc epsilon RI) which has, so far, only been described on mast cells and basophils. Epidermal LC react with antibodies specific for the alpha subunit of the tetrameric (alpha, beta, 2 gamma) Fc epsilon RI. Specific transcripts for Fc epsilon RI alpha and Fc epsilon RI gamma were detected in LC and correspond to those of human basophils and of the human basophil cell line KU812. Furthermore, human basophils, KU812 cells, and LC express the putative beta subunit. Thus human LC express the complete structure of Fc epsilon RI. This finding opens new perspectives in the putative functional role of this structure on antigen-presenting cells.

Laboratory confirmation of pertussis by culture, PCR, or detection of antibody increase in paired sera is hampered by low sensitivity in the later stages of the disease. Therefore, we investigated whether, and at which level, concentrations of immunoglobulin G (IgG) antibodies against pertussis toxin (PT), IgG-PT, in a single serum sample are indicative of active or recent pertussis. IgG-PT, measured by enzyme-linked immunosorbent assay in units per milliliter, was analyzed in 7,756 sera collected in a population-based study in The Netherlands, in the sera of 3,491 patients with at least a fourfold increase of IgG-PT, in paired sera of 89 patients with positive cultures and/or PCR results, and in the sera of 57 patients with clinically documented pertussis with a median follow-up of 1.4 years. We conclude that, independently of age, IgG-PT levels of at least 100 U/ml are diagnostic of recent or active infection with Bordetella pertussis. Such levels are present in less than 1% of the population and are reached in most pertussis patients within 4 weeks after disease onset and persist only temporarily.

Most outbreaks of Rift Valley fever (RVF) occur in remote locations after floods. To determine environmental risk factors and long-term sequelae of human RVF, we examined rates of previous Rift Valley fever virus (RVFV) exposure by age and location during an interepidemic period in 2006. In a randomized household cluster survey in 2 areas of Ijara District, Kenya, we examined 248 residents of 2 sublocations, Gumarey (village) and Sogan-Godud (town). Overall, the RVFV seropositivity rate was 13% according to immunoglobulin G ELISA; evidence of interepidemic RVFV transmission was detected. Increased seropositivity was found among older persons, those who were male, those who lived in the rural village (Gumarey), and those who had disposed of animal abortus. Rural Gumarey reported more mosquito and animal exposure than Sogan-Godud. Seropositive persons were more likely to have visual impairment and retinal lesions; other physical findings did not differ.

The active vitamin D metabolite 1,25-dihydroxyvitamin D3 [1,25-D3] is thought to promote many of its actions through interaction with a specific intracellular receptor. The discovery of such receptors in monocytes and activated lymphocytes has led investigators to evaluate the role of the hormone on the immune system. The sterol inhibits lymphocyte proliferation and immunoglobulin production in a dose-dependent fashion. At a molecular level, 1,25-D3 inhibits the accumulation of mRNA for IL-2, IFN-gamma, and GM-CSF. At a cellular level, the hormone interferes with T helper cell (Th) function, reducing Th-induction of immunoglobulin production by B cells and inhibiting the passive transfer of cellular immunity by Th-clones in vivo. The sterol promotes suppressor cell activity and inhibits the generation of cytotoxic and NK cells. Class II antigen expression on lymphocytes and monocytes is also affected by the hormone. When given in vivo, 1,25-D3 has been particularly effective in the prevention of autoimmune diseases such as experimental autoimmune encephalomyelitis and murine lupus but its efficacy has been limited by its hypercalcemic effect. Synthetic vitamin D3 analogues showing excellent 1,25-D3-receptor binding but less pronounced hypercalcemic effects in vivo have recently enhanced the immunosuppressive properties of the hormone in autoimmunity and transplantation.

The development of hemolytic alloantibodies and erythrocyte autoantibodies complicates transfusion therapy in thalassemia patients. The frequency, causes, and prevention of this phenomena among 64 transfused thalassemia patients (75% Asian) were evaluated. The effect of red blood cell (RBC) phenotypic differences between donors (mostly white) and Asian recipients on the frequency of alloimmunization was determined. Additional transfusion and patient immune factors were examined. 14 (22%) of 64 patients (75% Asian) became alloimmunized. A mismatched RBC phenotype between the white population, comprising the majority of the donor pool, and that of the Asian recipients, was found for K, c, S, and Fyb antigens, which accounts for 38% of the alloantibodies among Asian patients. Patients who had a splenectomy had a higher rate of alloimmunization than patients who did not have a splenectomy (36% vs 12.8%; P =.06). Erythrocyte autoantibodies, as determined by a positive Coombs test, developed in 25% or 16 of the 64 patients, thereby causing severe hemolytic anemia in 3 of 16 patients. Of these 16, 11 antibodies were typed immunoglobulin G [IgG], and 5 were typed IgM. Autoimmunization was associated with alloimmunization and with the absence of spleen (44% and 56%, respectively). Transfused RBCs had abnormal deformability profiles, more prominent in the patients without a spleen, which possibly stimulated antibody production. Transfusion of phenotypically matched blood for the Rh and Kell (leukodepleted in 92%) systems compared to blood phenotypically matched for the standard ABO-D system (leukodepleted in 60%) proved to be effective in preventing alloimmunization (2.8% vs 33%; P =.0005). Alloimmunization and autoimmunization are common, serious complications in Asian thalassemia patients, who are affected by donor-recipient RBC antigen mismatch and immunological factors.

Genetic defects affecting the humoral immune response and especially the production of antibodies of the immunoglobulin G (IgG) isotype result in a heightened susceptibility to infections. Studies over the last years have demonstrated the crucial role of Fc-receptors for IgG (FcγRs) widely expressed on innate immune effector cells in mediating the protective function of IgG. During the last years, additional ligands interacting with FcγRs as well as additional receptors binding to IgG glycosylation variants have been identified. In this review, we discuss how the interaction of these different ligands with classical and novel Fcγ-receptors influences the immune response and which strategies microorganisms have developed to prevent them.

Influenza vaccines capable of inducing cross-reactive or heterotypic immunity could be an important first line of prevention against a novel subtype virus. Influenza virus-like particles (VLPs) displaying functional viral proteins are effective vaccines against replication-competent homologous virus, but their ability to induce heterotypic immunity has not been adequately tested. To measure VLP vaccine efficacy against a known influenza pandemic virus, recombinant VLPs were generated from structural proteins of the 1918 H1N1 virus. Mucosal and traditional parenteral administrations of H1N1 VLPs were compared for the ability to protect against the reconstructed 1918 virus and a highly pathogenic avian H5N1 virus isolated from a fatal human case. Mice that received two intranasal immunizations of H1N1 VLPs were largely protected against a lethal challenge with both the 1918 virus and the H5N1 virus. In contrast, mice that received two intramuscular immunizations of 1918 VLPs were only protected against a homologous virus challenge. Mucosal vaccination of mice with 1918 VLPs induced higher levels of cross-reactive immunoglobulin G (IgG) and IgA antibodies than did parenteral vaccination. Similarly, ferrets mucosally vaccinated with 1918 VLPs completely survived a lethal challenge with the H5N1 virus, while only a 50% survival rate was observed in parenterally vaccinated animals. These results suggest a strategy of VLP vaccination against a pandemic virus and one that stimulates heterotypic immunity against an influenza virus strain with threatening pandemic potential.

The gene (spg) for an immunoglobulin G (IgG)-binding protein from a Streptococcus clinical isolate of Lancefield group G was cloned and expressed in Escherichia coli. The complete nucleotide sequence of the gene and 5'-flanking sequences was determined. The DNA sequence includes an open reading frame which encodes a hypothetical protein of 448 amino acid residues (Mr = 47,595). The 5' end of this open reading frame encodes a sequence resembling a typical secretion signal sequence, and the remainder of the encoded protein has features reminiscent of staphylococcal protein A and of streptococcal M6 protein, including repeated sequences and a similar C-terminal structure. Aside from this C-terminal structure, the encoded protein has little direct amino acid sequence homology to either protein A or M6 protein. In E. coli, the cloned gene directs the synthesis of a protein which binds to immunoglobulins, including rabbit immunoglobulin, goat IgG, and human IgGG described by Björck and Kronvall (L. Björck and G. Kronvall, J. Immunol. 133:969-974, 1984), a type III Fc receptor from a group G streptococcus.

The generation of reactive oxygen species (ROS) by the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex plays a critical role in the antimicrobial functions of the phagocytic cells of the immune system. The catalytic core of this oxidase consists of a complex between gp91(phox), p22(phox), p47(phox), p67(phox), p40(phox), and rac-2. Mutations in each of the phox components, except p40(phox), have been described in cases of chronic granulomatous disease (CGD), defining their essential role in oxidase function. We sought to establish the role of p40(phox) by investigating the NADPH oxidase responses of neutrophils isolated from p40(phox-/-) mice. In the absence of p40(phox), the expression of p67(phox) is reduced by approximately 55% and oxidase responses to tumor necrosis factor alpha/fibrinogen, immunoglobulin G latex beads, Staphylococcus aureus, formyl-methionyl-leucyl-phenylalanine, and zymosan were reduced by approximately 97, 85, 84, 75, and 30%, respectively. The defect in ROS production by p40(phox-/-) neutrophils in response to S. aureus translated into a severe, CGD-like defect in the killing of this organism both in vitro and in vivo, defining p40(phox) as an essential component in bacterial killing.

The currently available human vaccine for anthrax, derived from the culture supernatant of Bacillus anthracis, contains the protective antigen (PA) and traces of the lethal and edema factors, which may contribute to adverse side effects associated with this vaccine. Therefore, an effective expression system that can provide a clean, safe, and efficacious vaccine is required. In an effort to produce anthrax vaccine in large quantities and free of extraneous bacterial contaminants, PA was expressed in transgenic tobacco chloroplasts by inserting the pagA gene into the chloroplast genome. Chloroplast integration of the pagA gene was confirmed by PCR and Southern analysis. Mature leaves grown under continuous illumination contained PA as up to 14.2% of the total soluble protein. Cytotoxicity measurements in macrophage lysis assays showed that chloroplast-derived PA was equal in potency to PA produced in B. anthracis. Subcutaneous immunization of mice with partially purified chloroplast-derived or B. anthracis-derived PA with adjuvant yielded immunoglobulin G titers up to 1:320,000, and both groups of mice survived (100%) challenge with lethal doses of toxin. An average yield of about 150 mg of PA per plant should produce 360 million doses of a purified vaccine free of bacterial toxins edema factor and lethal factor from 1 acre of land. Such high expression levels without using fermenters and the immunoprotection offered by the chloroplast-derived PA should facilitate development of a cleaner and safer anthrax vaccine at a lower production cost. These results demonstrate the immunogenic and immunoprotective properties of plant-derived anthrax vaccine antigen.

The unique clinical characteristics of primary progressive multiple sclerosis (PPMS) pose particular diagnostic difficulties, both in excluding other causes of progressive syndromes and in confirming the diagnosis of MS, which is not adequately addressed by current diagnostic criteria. This article presents new diagnostic criteria developed by a group of investigators on the basis of a review of their considerable experience with PPMS. (We conclude that at least 1 year of clinical progression must be documented before a diagnosis of PPMS is made.) Three levels of diagnostic certainty have been defined-definite, probable, and possible--based on clinical findings, abnormal cerebrospinal fluid, abnormalities on magnetic resonance imaging (MRI) of the brain and spinal cord, and evoked potentials. In definite PPMS, evidence of intrathecal synthesis of immunoglobulin G together with one of the following three MRI criteria is required: (1) nine brain lesions, (2) two spinal cord lesions, or (3) four to eight brain lesions and one spinal cord lesion. Preliminary testing of these criteria was carried out on a cohort of 156 patients participating in a European natural history study of PPMS: 64% fulfilled the criteria for definite PPMS, 35% for probable PPMS, and only 1% for possible PPMS. These criteria now require prospective validation in a cohort of newly diagnosed patients and by postmortem examination.

OBJECTIVE

Bronchopulmonary dysplasia (BPD) of preterm neonates is associated with an increased recruitment of inflammatory cells into the airways. To evaluate further the role of inflammation in the pathogenesis of BPD, tracheobronchial aspirate fluid of neonates with birth weight < 1200 g (n = 59) was sequentially analyzed in a prospective study.

METHODS

Tracheobronchial aspirate fluid was assessed for chemotactic activity, neutrophil cell count, concentrations of elastase-alpha 1-proteinase inhibitor and activity of free elastase, concentrations of chemoattractants (complement component C5-derived anaphylatoxin, leukotriene B4, interleukin-8), and albumin concentrations as well as alpha 1-proteinase inhibitor activity. The secretory component for immunoglobulin A was used as reference protein. Only specimens without evidence of microbiological colonization were studied.

RESULTS

In neonates with prolonged respiratory disease (BPD-risk neonates, n = 24, fraction of inspired oxygen>> or = 0.3 and/or peak inspiratory pressure>> or = 16 cm H2O at day 10 postnatal age, birth weight 892 +/- 36 g, gestational age 27.2 +/- 0.3 weeks) chemotactic activity, cell count, concentrations of the chemoattractants complement component C5-derived anaphylatoxin, leukotriene B4, interleukin-8, as well as levels of elastase-alpha 1-proteinase inhibitor were significantly higher at day 10 and/or day 15 of postnatal age compared with neonates without chronic pulmonary disease (total n = 35; day 10, n = 11; day 15, n = 8). There was no difference in free elastolytic activity. Concentrations of albumin as well as alpha 1-proteinase inhibitor activity were higher in BPD-risk patients on day 15, indicating an increased pulmonary leak.

CONCLUSIONS

We conclude that preterm neonates at risk for the development of BPD show an enhanced inflammatory reaction in the lungs and an associated increase in pulmonary microvascular permeability. We speculate that inflammation may play an important role in the pathogenesis of BPD.

BACKGROUND

Despite available treatment with intravenous immunoglobulin (IVIg), morbidity and mortality are considerable in patients with Guillain-Barré syndrome (GBS). Our aim was to assess whether methylprednisolone, when taken with IVIg, improves outcome when compared with IVIg alone.

METHODS

We did a double-blind, placebo-controlled, multicentre, randomised study, to which we enrolled patients who were unable to walk independently and who had been treated within 14 days after onset of weakness with IVIg (0.4 g/kg bodyweight per day) for 5 days. We assigned 233 individuals to receive either intravenous methylprednisolone (500 mg per day; n=116) or placebo (n=117) for 5 days within 48 h of administration of first dose of IVIg. Because age is an important prognostic factor, we split treatment groups into two age-groups-ie, younger than age 50 years, or 50 years and older. Our primary outcome was an improvement from baseline in GBS disability score of one or more grades 4 weeks after randomisation. Analysis was by intention to treat.

RESULTS

We analysed 225 patients. GBS disability scores increased by one grade or more in 68% (76 of 112) of patients in the methylprednisolone group and in 56% (63 of 113) of controls (odds ratio [OR] 1.68, 95% CI 0.97-2.88; p=0.06). After adjustment for age and degree of disability at entry, treatment OR was 1.89 (95% CI 1.07-3.35; p=0.03). Side-effects did not differ greatly between groups.

CONCLUSIONS

We noted no significant difference between treatment with methylprednisolone and IVIg and IVIg alone. Because of the relevance of prognostic factors and the limited side-effects of methylprednisolone, the potential importance of combination treatment with the drug and IVIg, however, warrants further investigation.

The antigen of a monoclonal antibody that is specific for cells of human carcinoma of the colon is a monosialoganglioside as determined by the direct binding of antibody to thin-layer chromatograms of total lipid extracts of tissues. Binding of antibody to chromatograms is detected by autoradiography after the application of iodine-125-labeled F(ab')2 of rabbit immunoglobulin G antibodies to mouse immunoglobulins.

In a cohort study of 56 convalescent patients with severe acute respiratory syndrome (SARS), titers of immunoglobulin G (IgG) antibodies and neutralizing antibodies (NAbs) against SARS-associated coronavirus were assessed at regular intervals (at 1, 4, 7, 10, 16, and 24 months after the onset of disease) by use of enzyme-linked immunosorbent assay and neutralization assay. IgG antibody and NAb titers were highly correlated, peaking at month 4 after the onset of disease and decreasing thereafter. IgG antibodies remained detectable in all patients until month 16, and they became undetectable in 11.8% of patients at month 24. The finding that NAbs remained detectable throughout follow-up is reassuring in terms of protection provided against reinfection; however, NAb titers decreased markedly after month 16.

Immunohistochemical localization of the androgen receptor (AR) was performed in reproductive tissues, submaxillary gland, pituitary, and brain of the rat and in human prostate. AR was visualized using either of two polyclonal antibodies raised against peptides with sequences derived from rat and human AR. Tissue sections of 6-8 microns, frozen in isopentane and fixed in paraformaldehyde, were stained using immunoglobulin G fractions of immune, preimmune, and peptide-adsorbed immune sera in the avidin-biotin peroxidase procedure. AR was prominent in nuclei of acinar epithelial cells of epididymis, ventral prostate, seminal vesicle, and ductus deferens from the intact rat. Androgen withdrawal, 3 days after castration, resulted in the loss of receptor immunostaining, which was restored within 15 min of androgen administration. Stromal cell staining was absent or weak in the ventral prostate of intact rats, but was more evident in the epididymis. AR was confined to nuclei of cells within and bordering the interstitial compartment of the testis, including Sertoli cells, peritubular myoid cells, and interstitial cells, and was undetectable in germ cells. Submaxillary gland epithelial cells and a population of rat anterior pituitary cells showed strong nuclear staining of AR. In rat brain, AR was present in the medial preoptic, arcurate, and ventromedial nuclei of the hypothalamus, the medial nucleus of the amygdala, the CA-1 hippocampus, and the cortex. AR was prominent in acinar epithelial cells in human benign prostatic hyperplasia and was also present in stroma of fibromuscular benign hyperplasia. Heterogeneous staining was observed in stromal and epithelial cells of prostatic adenocarcinoma. The results of these studies indicate that AR can be detected immunohistochemically in a variety of tissues and cell types using antipeptide polyclonal antibodies. The presence of AR in tissues correlated with their known androgen responsiveness.

Infections caused by human parvovirus B19 are known to be controlled mainly by neutralizing antibodies. To analyze the immune reaction against parvovirus B19 proteins, four cell lines secreting human immunoglobulin G monoclonal antibodies (MAbs) were generated from two healthy donors and one human immunodeficiency virus type 1-seropositive individual with high serum titers against parvovirus. One MAb is specific for nonstructural protein NS1 (MAb 1424), two MAbs are specific for the unique region of minor capsid protein VP1 (MAbs 1418-1 and 1418-16), and one MAb is directed to major capsid protein VP2 (MAb 860-55D). Two MAbs, 1418-1 and 1418-16, which were generated from the same individual have identity in the cDNA sequences encoding the variable domains, with the exception of four base pairs resulting in only one amino acid change in the light chain. The NS1- and VP1-specific MAbs interact with linear epitopes, whereas the recognized epitope in VP2 is conformational. The MAbs specific for the structural proteins display strong virus-neutralizing activity. The VP1- and VP2-specific MAbs have the capacity to neutralize 50% of infectious parvovirus B19 in vitro at 0.08 and 0.73 microgram/ml, respectively, demonstrating the importance of such antibodies in the clearance of B19 viremia. The NS1-specific MAb mediated weak neutralizing activity and required 47.7 micrograms/ml for 50% neutralization. The human MAbs with potent neutralizing activity could be used for immunotherapy of chronically B19 virus-infected individuals and acutely infected pregnant women. Furthermore, the knowledge gained regarding epitopes which induce strongly neutralizing antibodies may be important for vaccine development.

B1 cells are an important cell population for the production of natural antibodies and for antibacterial immunoglobulin responses. Here we identified the mouse protein Siglec-G as a B1 cell inhibitory receptor. Siglec-G was expressed in a B cell-restricted way, with large amounts present in B1 cells. When overexpressed, Siglec-G inhibited B cell receptor-mediated calcium signaling. Siglec-G-deficient mice had massive expansion of the B1a cell population, which began early in development and was B cell intrinsic. Siglec-G-deficient mice had higher titers of natural IgM antibodies but not a higher penetrance of IgG autoantibodies. Siglec-G-deficient B1 cells showed a strongly enhanced calcium signaling. Our results demonstrate that Siglec-G-dependent negative regulation exists in B1 cells, which may explain the naturally muted signaling response of B1 cells.

Systemic sclerosis is an autoimmune disease characterized by immunological and vascular abnormalities. Autoantibodies against intracellular antigens are associated with particular clinical features of the disease, whereas autoantibodies against cell surface antigens may be pathogenic by inducing endothelial cell damage, considered the primary event in the pathogenesis of the disease. Latent human cytomegalovirus infection may contribute to progression of systemic sclerosis through its ability to infect endothelial cells; however, direct links between human cytomegalovirus infection and systemic sclerosis are still lacking. Molecular mimicry is one of the mechanisms that account for the link between infection and autoimmunity. Here we have identified an immunodominant peptide using systemic sclerosis serum screening of a random peptide library; such peptide shares homology with autoantigens and with the human cytomegalovirus late protein UL94 (ref. 9). Immunoglobulin G antibodies against the peptide affinity-purified from the sera of patients with systemic sclerosis specifically recognized the viral product and autoantigens; moreover, such antibodies induced endothelial cell apoptosis through specific interaction with the cell surface integrin-NAG-2 protein complex. Our results provide evidence that antibodies against human cytomegalovirus cause apoptosis of endothelial cells, considered the initial pathogenic event of systemic sclerosis, and indicate a previously unknown mechanism for the etiological link between human cytomegalovirus infection and autoimmunity.

Hendra virus (HeV) and Nipah virus (NiV) are closely related emerging viruses comprising the Henipavirus genus of the Paramyxovirinae, which are distinguished by their ability to cause fatal disease in both animal and human hosts. These viruses infect cells by a pH-independent membrane fusion event mediated by their attachment (G) and fusion (F) glycoproteins. Previously, we reported on HeV- and NiV-mediated fusion activities and detailed their host-cell tropism characteristics. These studies also suggested that a common cell surface receptor, which could be destroyed by protease, was utilized by both viruses. To further characterize the G glycoprotein and its unknown receptor, soluble forms of HeV G (sG) were constructed by replacing its cytoplasmic tail and transmembrane domains with an immunoglobulin kappa leader sequence coupled to either an S-peptide tag (sG(S-tag)) or myc-epitope tag (sG(myc-tag)) to facilitate purification and detection. Expression of sG was verified in cell lysates and culture supernatants by specific affinity precipitation. Analysis of sG by size exclusion chromatography and sucrose gradient centrifugation demonstrated tetrameric, dimeric, and monomeric species, with the majority of the sG released as a disulfide-linked dimer. Immunofluorescence staining revealed that sG specifically bound to HeV and NiV infection-permissive cells but not to a nonpermissive HeLa cell line clone, suggesting that it binds to virus receptor on host cells. Preincubation of host cells with sG resulted in dose-dependent inhibition of both HeV and NiV cell fusion as well as infection by live virus. Taken together, these data indicate that sG retains important native structural features, and we further demonstrate that administration of sG to rabbits can elicit a potent cross-reactive neutralizing antibody response against infectious HeV and NiV. This HeV sG glycoprotein will be exceedingly useful for structural studies, receptor identification strategies, and vaccine development goals for these important emerging viral agents.

Hendra virus (HeV) and Nipah virus (NiV) are closely related emerging viruses comprising the Henipavirus genus of the Paramyxovirinae. Each has a broad species tropism and can cause disease with high mortality in both animal and human hosts. These viruses infect cells by a pH-independent membrane fusion event mediated by their attachment (G) and fusion (F) envelope glycoproteins (Envs). Seven Fabs, m101 to -7, were selected for their significant binding to a soluble form of Hendra G (sG) which was used as the antigen for panning of a large naïve human antibody library. The selected Fabs inhibited, to various degrees, cell fusion mediated by the HeV or NiV Envs and virus infection. The conversion of the most potent neutralizer of infectious HeV, Fab m101, to immunoglobulin GGGG as measured by immunoprecipitation and less with binding to sG as measured by enzyme-linked immunosorbent assay and Biacore. m101 and m102 competed with the ephrin-B2, which we recently identified as a functional receptor for both HeV and NiV, indicating a possible mechanism of neutralization by these antibodies. The m101, m102, and m103 antibodies competed with each other, suggesting that they bind to overlapping epitopes which are distinct from the epitopes of m106 and m107. In an initial attempt to localize the epitopes of m101 and m102, we measured their binding to a panel of 11 G alanine-scanning mutants and identified two mutants, P185A and Q191 K192A, which significantly decreased binding to m101 and one, GG. These results suggest that m101 to -7 are specific for HeV or NiV or both and exhibit various neutralizing activities; they are the first human monoclonal antibodies identified against these viruses and could be used for treatment, prophylaxis, and diagnosis and as research reagents and could aid in the development of vaccines.