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Objectives: The aim of this study was to systematically evaluate adverse drug reactions (ADRs) in children consulting at the pediatric emergency unit during a 6-month period., Method: The regional pharmacovigilance center (CRPV) and the department of clinical pharmacology prospectively and systematically recorded all potential ADRs among patients younger than 18 years of age in the pediatric emergency unit reported at the daily staff meetings. All cases were then screened and validated by the CRPV. For validated cases, preventability, seriousness, and off-label use were evaluated., Results: During the study period, from 1 March to 1 September 2005, 90 children presented potential adverse drug events. ADRs were confirmed in 43 patients, 19 females and 24 males. Thirty-four patients (79%) were under the age of 5. According to the European definition, 14 patients (33%) had serious ADRs. One anaphylactic shock after amoxicillin injection; antimalarial prophylaxis misuse leading to convulsive status epilepticus, convulsion, and coma after hepatitis B and MMR vaccines were deemed life-threatening. Three ADRs were considered avoidable. Antibiotics and vaccines were the most common possible cause of ADRs (76%). Skin reactions (n=27), fever (n=8), and gastric disorders (n=5) were the most common clinical manifestations., Conclusions: Because ADRs were reported by clinicians on a voluntary basis, serious ADRs were probably reported more systematically. Compared to a similar period without active monitoring, active drug monitoring of ADRs doubled the number of confirmed cases 43 vs 17, p<0.001. Close collaboration between the pharmacovigilance center, pharmacologists, and clinicians is necessary and seems feasible for improving the monitoring of ADRs in children.

Periodontitis is an inflammatory disease of the supporting structures of the teeth caused by, among other pathogens, Prevotella intermedia. Many strains of P. intermedia are resistant to killing by the human complement system, which is present at up to 70% of serum concentration in gingival crevicular fluid. Incubation of human serum with recombinant cysteine protease of P. intermedia (interpain A) resulted in a drastic decrease in bactericidal activity of the serum. Furthermore, a clinical strain 59 expressing interpain A was more serum-resistant than another clinical strain 57, which did not express interpain A, as determined by Western blotting. Moreover, in the presence of the cysteine protease inhibitor E64, the killing of strain 59 by human serum was enhanced. Importantly, we found that the majority of P. intermedia strains isolated from chronic and aggressive periodontitis carry and express the interpain A gene. The protective effect of interpain A against serum bactericidal activity was found to be attributable to its ability to inhibit all three complement pathways through the efficient degradation of the alpha-chain of C3 -- the major complement factor common to all three pathways. P. intermedia has been known to co-aggregate with P. gingivalis, which produce gingipains to efficiently degrade complement factors. Here, interpain A was found to have a synergistic effect with gingipains on complement degradation. In addition, interpain A was able to activate the C1 complex in serum, causing deposition of C1q on inert and bacterial surfaces, which may be important at initial stages of infection when local inflammatory reaction may be beneficial for a pathogen. Taken together, the newly characterized interpain A proteinase appears to be an important virulence factor of P. intermedia.

The periodontal pathogen Porphyromonas gingivalis is highly resistant to the bactericidal activity of human complement, which is present in the gingival crevicular fluid at 70% of serum concentration. All thirteen clinical and laboratory P. gingivalis strains tested were able to capture the human complement inhibitor C4b-binding protein (C4BP), which may contribute to their serum resistance. Accordingly, in serum deficient of C4BP, it was found that significantly more terminal complement component C9 was deposited on P. gingivalis. Moreover, using purified proteins and various isogenic mutants, we found that the cysteine protease high molecular weight arginine-gingipain A (HRgpA) is a crucial C4BP ligand on the bacterial surface. Binding of C4BP to P. gingivalis appears to be localized to two binding sites: on the complement control protein 1 domain and complement control protein 6 and 7 domains of the alpha-chains. Furthermore, the bacterial binding of C4BP was found to increase with time of culture and a particularly strong binding was observed for large aggregates of bacteria that formed during culture on solid blood agar medium. Taken together, gingipains appear to be a very significant virulence factor not only destroying complement due to proteolytic degradation as we have shown previously, but was also inhibiting complement activation due to their ability to bind the complement inhibitor C4BP.

Time-dependent density functional theory has been used to calculate the one-photon and two-photon absorption spectra of free-base porphyrin, a substituted zinc porphyrin, and a zinc porphyrin dimer, in order to assess the validity of the method to reproduce the large increase in the two-photon absorption (TPA) cross-section for the dimer. Three hybrid functionals with varying amounts of exact exchange were tested, and the calculated one-photon absorption spectra for each of the molecular systems were shown to be in qualitative agreement with the measured spectra. All three functionals predict a large enhancement in the TPA cross-section for the dimer relative to the monomer, in agreement with experimental results. However, because of the sensitivity of the resonance enhancement factor to small differences in the relevant state energies, quantitative prediction of the TPA cross-section by this method is still a challenge.

Background: This multicenter prospective study was designed to evaluate the performance characteristics of a new commercially available enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to Trypanosoma cruzi in blood donors, the ORTHO T. cruzi ELISA Test System (Ortho-Clinical Diagnostics)., Study Design and Methods: Assay specificity was evaluated among 40,665 serum and ethylenediaminetetraacetate (EDTA) plasma specimens from volunteer blood donors and 481 T. cruzi antibody-negative specimens from a high-risk population. Sensitivity was evaluated among 106 T. cruzi-infected subjects identified by parasite detection, among 93 radioimmunoprecipitation assay (RIPA)-positive specimens from high-risk subjects, and 662 specimens presumed positive for the presence of T. cruzi antibodies by serologic methods. Also assessed were the equivalence of serum and plasma as specimen sources, performance equivalence of automated and semiautomated processing methods, nonspecific reactivity in specimens from other disease states or clinical conditions, and assay precision., Results: Assay specificity was 99.998 percent in volunteer blood donors and 99.4 percent among high-risk subjects. Sensitivity was 100 percent among specimens positive by parasite detection, or by serologic methods, and 98.9 percent among RIPA-positive specimens from high-risk subjects. No differences were demonstrated between serum and plasma or between semiautomated and automated processing methods. Cross-reactivity was observed with known positive leishmaniasis specimens. Total inter- and intraassay variability was less than 10 percent with both the automated and the semiautomated methods., Conclusion: The ORTHO T. cruzi ELISA Test System is an effective, qualitative assay for screening blood donors for immunoglobulin G antibodies to T. cruzi. The assay was licensed for donor screening by the FDA in December 2006.

Gingipains are cysteine proteases produced in large quantities by Porphyromonas gingivalis which together constitute important virulence factors in the pathogenesis of periodontal disease by that organism. Described is this unit is an efficient procedure for the purification of gingipains from the growth medium of P. gingivalis strain HG66, along with detailed protocols for growth of the organism and basic characterization of the purified proteases. The purification procedure consists of acetone precipitation followed by gel filtration to separate high-molecular-mass gingipains (Kgp and HRgpA) from RgpB. Kgp and HRgpA are further separated on Arg-Sepharose by the virtue of differential elution from the affinity matrix with lysine (Kgp) and arginine (HRgpA) eluant. Obtained from these procedures, the gingipains are stable and can be stored at -80 degrees C for years without loss of activity., ((c) 2007 by John Wiley & Sons, Inc.)

We report one- and two-photon absorption excitation energies and cross sections for a series of 7-aminocoumarins using time-dependent density functional theory with various basis sets and functionals, including exchange-correlation functionals using the Coulomb-attenuating method, to evaluate their performance in the gas phase and in solvents. Except for the results of one functional, the computed one-photon excitation energies and transition dipole moments are in good agreement with experiment. The range of errors obtained from various functionals is discussed in detail. The relationship of donor and acceptor groups with the one- and two-photon resonances and intensities is also discussed.

The mature 507-residue RgpB protein belongs to an important class of extracellular outer membrane-associated proteases, the gingipains, from the oral pathogen Porphyromonas gingivalis that has been shown to play a central role in the virulence of the organism. The C termini of these gingipains along with other outer membrane proteins from the organism share homologous sequences and have been suggested to function in attachment of these proteins to the outer membrane. In this report, we have created a series of truncated and site-directed mutants of the C terminus from a representative member of this class, the RgpB protease, to investigate its role in the maturation of these proteins. Truncation of the last two residues (valyl-lysine) from the C terminus is sufficient to create an inactive version of the protein that lacks the posttranslational glycosylation seen in the wild type, and the protein remains trapped behind the outer membrane. Alanine scanning of the last five residues revealed the importance of the C-terminal motif in mediating correct posttranslational modification of the protein. This result may have a wider implication in a novel secretory pathway in distinct members of the Cytophaga-Flavobacterium-Bacteroidetes phylum.

A series of one-photon absorption spectra for fluorene-based donor-pi-acceptor molecules is presented and spectroscopically assigned, based upon the results obtained from time-dependent density functional theory. The computed excitation energies were generally shown to be in good agreement with experiment, particularly when compared to results from measurements carried out in a nonpolar solvent, which were available for some molecules. The computed oscillator strengths may resolve discordant experimental values in some cases, for example, for AF-380, AF-270, and AF-295. However, a quantitative comparison between computed and observed oscillator strengths is complicated by band overlapping. Thus, the computed extinction coefficients obtained by summing over the Gaussian bands are useful in such cases.

Linear and quadratic response time-dependent density functional theories have been applied to calculate the photophysical properties of donor-pi-acceptor molecules which are known to have large nonlinear absorption. The linear absorption and two-photon absorption spectra predicted using hybrid functionals, including the Coulomb-attenuated model, with continuum solvation models are reported and compared to experiment and to previous theoretical predictions. While the quadratic response with these functionals overestimated the TPA cross sections relative to experiment when a Gaussian linewidth function was used, a fairly good agreement was obtained when a Lorentzian linewidth function was applied. In addition, the comparison of the TPA cross sections calculated by the sum over states with those calculated by the two-state approximation indicates the importance of the higher energy states in TPA, particularly in nondegenerate experiments.

The efficient clearance of microbes by neutrophils requires the concerted action of reactive oxygen species and microbicidal components within leukocyte secretory granules. Rubrerythrin (Rbr) is a nonheme iron protein that protects many air-sensitive bacteria against oxidative stress. Using oxidative burst-knockout (NADPH oxidase-null) mice and an rbr gene knockout bacterial strain, we investigated the interplay between the phagocytic oxidative burst of the host and the oxidative stress response of the anaerobic periodontal pathogen Porphyromonas gingivalis. Rbr ensured the proliferation of P. gingivalis in mice that possessed a fully functional oxidative burst response, but not in NADPH oxidase-null mice. Furthermore, the in vivo protection afforded by Rbr was not associated with the oxidative burst responses of isolated neutrophils in vitro. Although the phagocyte-derived oxidative burst response was largely ineffective against P. gingivalis infection, the corresponding oxidative response to the Rbr-positive microbe contributed to host-induced pathology via potent mobilization and systemic activation of neutrophils. It appeared that Rbr also provided protection against reactive nitrogen species, thereby ensuring the survival of P. gingivalis in the infected host. The presence of the rbr gene in P. gingivalis also led to greater oral bone loss upon infection. Collectively, these results indicate that the host oxidative burst paradoxically enhances the survival of P. gingivalis by exacerbating local and systemic inflammation, thereby contributing to the morbidity and mortality associated with infection.

Objective: To determine whether sodium butyrate (NaB), a major short-chain fatty acid produced in the human gut by bacterial fermentation of dietary fiber, enhances transforming growth factor (TGF)-beta signaling and potentiates its tumor suppressor activity in the gut., Summary Background Data: The molecular mechanisms by which dietary fiber decreases the risk of colon cancers are poorly characterized. TGF-beta is an important tumor suppressor in the gut and has many similar biologic activities as NaB. Therefore, we hypothesized that the chemo-preventive effects of NaB are mediated in part by enhancing TGF-beta signaling and its tumor suppressor function in the gut., Methods: The effects of NaB on Smad3 expression in rat intestinal epithelial (RIE-1) cells and 6 human colon cancer cell lines were examined. The effects of NaB on TGF-beta-induced Smad3 phosphorylation and plasminogen activator inhibitor-1 (PAI-1) and cyclooxygenase-2 (COX-2) gene expression were also examined in RIE-1 cells. Finally, the effects of NaB and TGF-beta on anchorage-independent growth were examined in Akt-transformed RIE-1 cells., Results: NaB induced Smad3 in RIE-1 cells and in 4 human colon cancer cell lines. NaB enhanced TGF-beta-induced Smad3 phosphorylation and potentiated TGF-beta-induced PAI-1 expression. NaB and TGF-beta synergistically inhibited anchorage-independent growth of Akt-transformed RIE-1 cells., Conclusions: These results demonstrate that NaB induces Smad3 and potentiates TGF-beta signaling and its tumor suppressor activity in gut epithelial cells. Our data reveal a novel molecular mechanism that may explain in part the beneficial effects of dietary fiber in decreasing the risk of colon cancers.

Inorganic sulfate is essential for numerous functions in mammalian physiology. In the present study, we characterized the functional properties of the rat Na+-sulfate cotransporter NaS2 (rNaS2), determined its tissue distribution, and identified its gene (slc13a4) structure. Expression of rNaS2 protein in Xenopus oocytes led to a Na+-dependent transport of sulfate that was inhibited by phosphate, thiosulfate, tungstate, selenate, oxalate, and molybdate, but not by citrate, succinate, or DIDS. Transport kinetics of rNaS2 determined a K(M) for sulfate of 1.26 mM. Na+ kinetics determined a Hill coefficient of n=3.0+/-0.7, suggesting a Na+:SO4 (2-) stoichiometry of 3:1. rNaS2 mRNA was highly expressed in placenta, with lower levels found in the brain and liver. slc13a4 maps to rat chromosome 4 and contains 17 exons, spanning over 46 kb in length. This gene produces two alternatively spliced transcripts, of which the transcript lacking exon 2 is the most abundant form. Its 5' flanking region contains CAAT- and GC-box motifs and a number of putative transcription factor binding sites, including GATA-1, SP1, and AP-2 consensus sequences. This is the first study to characterize rNaS2 transport kinetics, define its tissue distribution, and resolve its gene (slc13a4) structure and 5' flanking region.

A quinoidal porphyrin has been synthesised with such a curved pi-system that pi-pi stacking leads to the formation of cyclic trimer aggregates in the crystal, which pack to generate cylindrical channels with an internal diameter of 1.0 nm.

We report a comprehensive time-dependent density functional theory (TDDFT) study of one-photon and two-photon absorption (OPA and TPA, respectively) spectra for donor-pi-acceptor molecules. The calculated excitation energies were generally shown to be in good agreement with experiment, particularly when compared to results from measurements carried out in a nonpolar solvent, although the oscillator strengths were overestimated in some cases. Calculated TPA cross sections applying the two-state approximation were shown to be highly dependent on the form of the line-shape function used. Although a good agreement with experimental TPA spectra was generally observed, the wide range in the experimentally measured values and lack of systematic experimental data on solvent effects limited a detailed comparison as yet.

Paired subgingival plaque samples representing the most-diseased and least-diseased sites were collected from 34 adult patients with diagnosed chronic periodontitis. The percentage of Porphyromonas gingivalis relative to the total anaerobic and gram-negative bacterial load at each site was determined by real-time PCR. Based on variations in the noncatalytic C terminus of the Lys-gingipain (Kgp), it was reasoned that DNA sequence variation in the 3'-coding region of the kgp gene might determine functional biotypes. Perusal of the available sequence information in GenBank indicated three such forms of the kgp gene corresponding to P. gingivalis strains HG66, 381, and W83. Analysis of patient samples revealed the presence of a fourth genotype (W83v) that showed duplication of a sequence recognized by the W83 reverse primer. The four biotypes, HG66, 381, W83, and W83v, were present in the study group in the ratio 8:11:6:5, respectively. Each subject was colonized by one predominant biotype, and only three patients were colonized by a trace amount of a second biotype.

The orb-weaving spider Nephila edulis incorporates into its web a band of decaying animal and plant matter. While earlier studies demonstrate that larger spiders utilise these debris bands as caches of food, the presence of plant matter suggests additional functions. When organic and plastic items were placed in the webs of N. edulis, some of the former but none of the latter were incorporated into the debris band. Using an Y-maze olfactometer, we show that sheep blowflies Lucilia cuprina are attracted to recently collected debris bands, but that this attraction does not persist over time. These data reveal an entirely novel foraging strategy, in which a sit-and-wait predator attracts insect prey by utilising the odours of decaying organic material. The spider's habit of replenishing the debris band may be necessary to maintain its efficacy for attracting prey.

The hypothalamic neuropeptide hormone GnRH is the central regulator of reproductive function. GnRH stimulates the synthesis and release of the gonadotropins LH and FSH by the gonadotropes of the anterior pituitary through activation of the G-protein-coupled GnRH receptor. In this study, we investigated the role of translational control of hormone synthesis by the GnRH receptor in the novel gonadotrope cell line LbetaT2. Using immunohistochemical and RIA studies with this model, we show that acute GnRH-induced synthesis and secretion of LH are dependent upon new protein synthesis but not new mRNA synthesis. We examined the response to GnRH and found that activation of cap-dependent translation occurs within 4 h. LHbeta promoter activity was also examined, and we found no increases in LHbeta promoter activity after 6 h of GnRH stimulation. Additionally, we show that increased phosphorylation of translation initiation proteins, 4E-binding protein 1, eukaryotic initiation factor 4E, and eukaryotic initiation factor 4G, occur in a dose- and time-dependent manner in response to GnRH stimulation. Quantitative luminescent image analysis of Western blots shows that 10 nm GnRH is sufficient to cause a maximal increase in factor phosphorylation, and maximal responses occur within 30 min of stimulation. Further, we demonstrate that the MAPK kinase inhibitor, PD 98059, abolishes the GnRH-mediated stimulation of a cap-dependent translation reporter. More specifically, we demonstrate that PD 98059 abolishes the GnRH-mediated stimulation of a downstream target of the ERK pathway, MAPK-interacting kinase. Based on these findings, we conclude that acute GnRH stimulation of LbetaT2 cells increases translation initiation through ERK signaling. This may contribute to the acute increases in LHbeta subunit production.

Background: The FDA recommended new travel deferrals in May 2002 to prevent the potential transmission of variant CJD (vCJD). The predicted impact of such deferrals on the blood supply was controversial., Study Design and Methods: We distributed anonymous, self-administered questionnaires to donors over 2 weeks in April 2002. Two thousand surveys were allocated to each of five geographic groupings of blood centers in the western and central US. Results were analyzed with chi-squared tests and logistic regression., Results: Of 9800 surveys, 7405 (76%) were returned and analyzed. Overall, 257 (3.47%; 95% CI, 3.05-3.89) donors responding to the survey met the expanded May and November 2002 FDA vCJD travel criteria for deferral. Deferral rates ranged from 1 percent or less in Northern and Southern centers, 3 to 4 percent in San Francisco, California, and Scottsdale, Arizona, to over 13 percent in El Paso, Texas (p < 0.0001). Service at a European military base was the most common reason for deferral, whereas in San Francisco most deferrals were for UK and European travel or residence. The vCJD deferral was also associated with ages 30 through 49 years and with male gender. During June through September 2002, observed deferral rates were 1.6 percent at the same centers., Conclusions: Predicted deferrals due the new vCJD travel criteria depend upon the number of military donors at a blood center, as well as on the age and gender of donors. Actual deferrals were lower than predicted by an anonymous survey.

The gingipains have been implicated in the pathogenicity of Porphyromonas gingivalis, a major etiologic agent of chronic periodontitis. Mature gingipains often present as a membrane-bound glycosylated proteinase-adhesin complex comprising multiple adhesin domains (HA1 to -4) and a catalytic domain. Using recombinant adhesin domains, we were able to show that patients with chronic periodontitis produce significantly more immunoglobulin G reactive with gingipain domains than a corresponding group with healthy periodontium. Titers were predominantly directed toward the carbohydrate epitopes shared between the gingipains and the lipopolysaccharide of P. gingivalis with little recognition of the peptide backbone of the catalytic domains. Distribution of titers to peptide epitopes of the adhesin domains was as follows: HA4 approximately HA1 > HA3 >> HA2. No correlation was observed between markers of disease severity and titers to individual adhesins within the disease group. Posttreatment titers showed no change or a decrease in titers for the majority of patients except for titers to the HA2 domain which showed marked increases in a few responding patients. Since the HA2 domain is important in hemoglobin binding and acquisition of essential porphyrin, boosting titers of antibodies to this domain may have the potential to control the growth of this organism.

The porphyrin requirements for growth recovery of Porphyromonas gingivalis in heme-depleted cultures are investigated. In addition to physiologically relevant sources of heme, growth recovery is stimulated by a number of noniron porphyrins. These data demonstrate that, as for Haemophilus influenzae, reliance on captured iron and on exogenous porphyrin is manifest as an absolute growth requirement for heme. A number of outer membrane proteins including some gingipains contain the hemoglobin receptor (HA2) domain. In cell surface extracts, polypeptides derived from HA2-containing proteins predominated in hemoglobin binding. The in vitro porphyrin-binding properties of a recombinant HA2 domain were investigated and found to be iron independent. Porphyrins that differ from protoporphyrin IX in only the vinyl aspect of the tetrapyrrole ring show comparable effects in competing with hemoglobin for HA2 and facilitate growth recovery. For some porphyrins which differ from protoporphyrin IX at both propionic acid side chains, the modification is detrimental in both these assays. Correlations of porphyrin competition and growth recovery imply that the HA2 domain acts as a high-affinity hemophore at the cell surface to capture porphyrin from hemoglobin. While some proteins involved with heme capture bind directly to the iron center, the HA2 domain of P. gingivalis recognizes heme by a mechanism that is solely porphyrin mediated.

Acetaminophen (APAP)-induced hepatocellular necrosis can be prevented by treatment with peroxisome proliferators. This protection is associated with lowered protein arylation and glutathione depletion in mice. Peroxisome proliferators have been shown to activate nuclear receptors. These receptors, termed peroxisome proliferator activated receptors (PPARs), can also be activated by free fatty acids. This study was designed to determine if treatment with the PPAR activator docosahexaenoic acid (DHA) would also lower APAP toxicity. Male CD-1 mice received 250 mg DHA/kg or 500 mg clofibrate (CFB)/kg, i.p., for 5 d. Controls received corn oil vehicle, i.p. After overnight fasting, mice received 800 mg APAP/kg, p.o. At 24 h after APAP, hepatotoxicity was evident in control mice by elevated plasma sorbitol dehydrogenase activity (SDH) and histologic evidence of hepatic degeneration and necrosis. As expected, CFB pretreatment significantly decreased this. Similarly, DHA protected against APAP-induced hepatotoxicity at 24 h after challenge. However, treatment with DHA did not increase hepatic glutathione prior to APAP, as previously shown with CFB. Interestingly, DHA did not increase palmitoyl coenzyme A (CoA) oxidase activity or other biochemical parameters associated with peroxisome proliferation after 5 d of treatment at 250 mg/kg. No significant alterations in microsomal APAP glucuronidation or cytochrome P-450-mediated bioactivation were detected either. Collectively, these results show that DHA also prevents APAP-induced hepatotoxicity at 24 h after challenge. However, the association between resistance against APAP-induced liver injury, PPAR activation, and peroxisome proliferation is not clearly understood.

Previous studies have shown that triggering multiple myeloma (MM) cells via CD40 induces IL-6-mediated autocrine growth as well as increased expression of cell surface adhesion molecules including CD11a, CD11b, CD11c, and CD18. In this study, we generated the 5E2 mAb which targets an antigen that is induced upon CD40 ligand (CD40L) activation of MM cells. Immunofluorescence, immunoprecipitation, and protein sequencing studies identified the target antigen of 5E2 mAb as the 86-kD subunit of the Ku autoantigen. We demonstrate that increased cell surface expression of Ku on CD40L-treated cells is due to migration of Ku from the cytoplasm to the cell surface membrane. Moreover, cell surface Ku on CD40L-treated MM cells mediates homotypic adhesion of tumor cells, as well as heterotypic adhesion of tumor cells to bone marrow stromal cells and to human fibronectin; and 5E2 mAb abrogates IL-6 secretion triggered by tumor cell adherence to bone marrow stromal cells. These data suggest that CD40L treatment induces a shift of Ku from the cytoplasm to the cell surface, and are the first to show that Ku functions as an adhesion molecule. They further suggest that cell surface Ku may play a role in both autocrine and paracrine IL-6-mediated MM cell growth and survival.

The defining feature of autoimmune disease is the presence of specific autoreactive lymphocytes. Systemic lupus erythematosus (SLE), for example, is characterized by a discrete set of antibodies directed to nuclear antigens; these include autoantibodies to DNA and snRNPs that are diagnostic for SLE. The murine model of SLE, the MRL-lpr/lpr mouse, likewise, has a similar autoantibody profile. To understand how SLE-associated autoantibodies are regulated in healthy individuals and to identify mechanisms underlying their expression in autoimmunity, we have developed a transgenic (tg) model system using multiple sets of tgs. The development of B cells bearing these tgs has been studied in BALB/c and MRL-lpr/lpr autoimmune backgrounds, and the relative fates of anti-ssDNA and anti-dsDNA tg B cells when they are a part of a diverse as well as monoclonal B cell repertoire have been evaluated.

The current model of T cell activation requires two signals. The first signal is specific, requiring T cell receptor recognition and binding to MHC/Antigen presented by an antigen-presenting cell. The second signal is nonspecific, resulting from the binding of B7 ligand on the antigen-presenting cell with its receptor, CD28, on the T cell. If both signals are provided, the T cell will proliferate and secrete cytokines. Recently, it has been shown that CTLA4, another receptor for B7 that is upregulated following T cell after activation, can deliver an inhibitory signal, downregulating T cell proliferation. The B7 family of ligands has two family members, B7-1 and B7-2. They both bind to CD28 and CTLA4, but they differ in their binding affinity, structure, and temporal expression. Considerable research has been done on the CD28/B7 costimulatory pathway. Different ways of manipulating this pathway could provide insights into the mechanism and treatment of opposing pathological states. Blocking the CD28/B7 pathway could result in immunosuppression, with implications for the treatment of autoimmune diseases, organ transplantation, and graft vs. host disease. Activating the CD28/B7 pathway could be useful for including the immune system to recognize and eliminate tumors that evade the immune system. Finally, the CD28/B7 pathway could be involved with maintaining immune tolerance, as recent studies suggest the preferential binding of the B7-CTLA4 pathway results in the down-regulation of the responding T cells. Thus, the B7/CD28/CTLA4 pathway has the ability to both positively and negatively regulate immune responses.

Both anti-single-stranded (ss) and anti-double-stranded (ds) DNA antibodies are associated with the autoimmune disease systemic lupus erythematosus (SLE), but only anti-dsDNA antibodies are considered one of the diagnostic criteria. Using Ig transgenes coding for anti-DNA we have determined the fate of anti-dsDNA B cells in a non-autoimmune environment. In a Rag-2 wild-type background, B cells expressing the anti-dsDNA Ig transgenes are present in the spleen but dsDNA specificity is disrupted due to expression of endogenous L chains. In a Rag-2-deficient background where co-expression of endogenous Ig is blocked, splenic B cells expressing only the anti-dsDNA transgene Ig are present, indicating that endogenous Ig expression is not required for bone marrow export. The anti-dsDNA B cells that persist are profoundly crippled in that they are unable to proliferate to lipopolysaccharide or anti-Ig stimulation. Furthermore, these anti-dsDNA Ig transgene B cells show a decreased lifespan relative to non-transgene BALB/c B cells. Persistence of anti-dsDNA B cells in the periphery of non-autoimmune mice raises the possibility that their appearance in the context of SLE is due to their reactivation by T cell help.

Anti-DNA Abs are prevalent in the serum of systemic lupus erythematosus (SLE) patients and in the MRL-lpr/lpr mouse model of SLE, but are generally absent in normal individuals. We have studied the regulation of anti-ssDNA B cells in a non-autoimmune (BALB/c) background by using Ig transgenes (Tgs) encoding anti-DNA Abs. In one case, they are present with other non-DNA-binding B cells (the VH3H9 Tg with endogenous light chains); in the other, they are present as an essentially monospecific population (VH3H9/Vkappa8). We have previously observed that serum anti-ssDNA levels in these Tg mice were no higher than those of non-Tg mice, despite the fact that anti-ssDNA B cells dominate the peripheral B cell repertoire. These results suggested that the anti-ssDNA Tg B cells present are functionally inactivated. In this paper, we isolate B cells from VH3H9/Vkappa8 Tg mice to show that this is indeed the case and go on to further define this state. We demonstrate that VH3H9/Vkappa8 Tg B cells have diminished Ig secretion in response to both T-independent and T-dependent stimuli compared with non-Tg controls. VH3H9/Vkappa8 Tg B cells also show suboptimal proliferation in response to anti-IgM F(ab)'2 fragments and LPS, and are phenotypically distinct in expressing decreased total surface Ig. Despite their functional defects, however, VH3H9/Vkappa8 Tg B cells have an in vivo turnover rate comparable to non-Tg B cells, suggesting that they are long lived.

Mice homozygous for the lpr mutation have B and T cell defects and develop autoantibodies, suggesting that lpr plays a role in their genesis. The lpr defect has been identified as a mutation in the apoptosis-associated Fas receptor (FasR) gene. To begin to define the role of FasR in B cells, we have surveyed FasR expression on B-lineage cells from early progenitors in the bone marrow through their maturation in the periphery. Contrary to some reports, we found that FasR is expressed on B cells at all stages of their development and is highest on germinal center B cells. FasR is not expressed on lpr!lpr-derived cells. These data are consistent with the idea that lpr/lpr mice have an intrinsic B cell defect that may be manifested in developing as well as peripheral B cells. An unexpected finding is that B-1 (CD5) B cells do not constitutively express FasR: FasR becomes detectable on B-1 B cells only after activation.

The goal of this study was to examine the in vivo site of autoantibody production in normal and autoimmune-prone mice. B cells were identified in tissue sections with IgM- and IgG2a-specific riboprobes that readily distinguished resting cells from antibody-forming cells (AFC). In normal mice, the few identifiable IgG2a-secreting cells were found in the red pulp. By contrast, in Ipr mice exceedingly high numbers of IgG2a and autoantibody-producing cells were found deep within the T cell-rich periarteriolar lymphoid sheaths (PALS). This unusual anatomic location of autoantibody-secreting B cells is unique to Fas dysregulated strains, since IgG2-producing cells in MRL/+ and (SWR x NZB)F1 mice were found predominantly in the red pulp or outer PALS, similar to normal mice. Furthermore, analysis of spleens from Ipr and non-Ipr anti-DNA immunoglobulin transgenic mice revealed dramatic accumulation of Tg+ cells in the inner PALS only in Ipr mice. These data suggest that in the absence of Fas, autoreactive B cells accumulate in T cell-rich zones, and this anatomic feature may contribute to autoantibody production.

We have used an Ig transgene (VH3H9) that increases the frequency of anti-DNA autoantibodies to address whether the production of antinuclear Abs in systemic lupus erythematosus is the consequence of a breakdown of B cell tolerance. We have shown that nonautoimmune mice regulate anti-DNA B cells, and that lupus-prone MRL-lpr/lpr mice are defective in this regulation. Here we show that a subset of anti-DNA B cells, namely those that stain nuclei in a homogeneous fashion, not only fail to be deleted in MRL-lpr/lpr mice, but undergo preferential clonal expansion. In addition, we describe a surprising finding: the VH3H9 transgene is less efficient at inhibiting endogenous heavy chain gene rearrangement on the autoimmune-prone MRL-lpr/lpr genetic background than on the nonautoimmune BALB/c background.

Anti-DNA antibodies, specifically those that stain nuclei in a homogenous nuclear (HN) fashion, are diagnostic of systemic lupus erythematosus (SLE) and the MRL-lpr/lpr SLE murine model. We have used a heavy chain transgene that increases the frequency of anti-HN antibodies to address whether their production in SLE is the consequence of a defect in B cell tolerance. Anti-HN B cells were undetectable in nonautoimmune-prone transgenic mice, but in MRL-lpr/lpr transgenic mice their Ig was evident in the sera and they were readily retrievable as hybridomas. We conclude that nonautoimmune animals actively delete anti-HN-specific B cells, and that MRL-lpr/lpr mice are defective in this process possibly because of the lpr defect in the fas gene.

Many studies have reported age-related changes in immune responses that could be due to alterations in lymphoid cell numbers or functions. Here we report the results of studies using immunofluorescent staining and in vitro assays of cellular function to compare the expression of cell surface antigens on lymphocytes from mice up to 2 years of age. No significant changes were observed in the frequencies of spleen cells bearing class I or class II major histocompatibility complex (MHC) antigens, surface immunoglobulin, or Thy-1, Ly-1, Ly-2, or L3T4 antigens. However, the densities (per cell) of both class I and class II MHC antigens were increased significantly on cells from aged as compared to young mice, whereas the densities of the other cell surface antigens studied were unchanged or slightly decreased. The increased levels of MHC antigen expression in old relative to young mice were shown to be functionally significant regarding immunological stimulation. These data suggest that T-cell clones silent in young individuals may be activated in comparable situations in older animals, leading to immunological alterations perhaps including increased autoreactivity.

Phagocytic cells from non-lactating bovine mammary glands have the capacity to secrete hydrogen peroxide when exposed to the soluble membrane stimulant phorbol myristate acetate (PMA). Unfractionated cell suspensions, containing mainly neutrophils and macrophages, and cell monolayers enriched for macrophages secreted hydrogen peroxide. A correlation was observed between the amount of hydrogen peroxide secreted, the antibacterial activity of the cells and the number of neutrophils present in the cell suspensions. Pre-exposure of cells to PMA significantly impaired their antibacterial activity against Staphylococcus aureus suggesting the importance of oxygen metabolism in the bactericidal capacity of these cells.

Macrophages were isolated from the mammary glands of non-lactating (dry) cows and their ability to phagocytose and kill staphylococci in vitro assessed. Normal bovine serum enhanced the uptake of staphylococci and was required for optimal killing in the bactericidal test. Dry gland secretion interfered with uptake. Secretions taken progressively into the dry period became more inhibitory. The phagocytic ability of macrophages was significantly less than that of neutrophils present in the same gland preparation when tested in the presence of dry gland secretion. A marked variation in the antibacterial activity of macrophages from different cows was noted.