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We describe a comprehensive screening campaign of warheads, linked to a hydroxamate chelating anchor, for the modification of Cys165 within the BoNT/A protease. Engaging thorough enzyme kinetics, we detail a remarkable proximity-driven covalent bond with an epoxide warhead, a weak electrophile; yet, one that possessed superior irreversible inhibition, and pharmacological properties, when compared to intrinsically higher reactive warheads. This directed, selective covalent bond was contingent upon the crucial hydroxamate-Zn2+ chelating interaction as exemplified by examining non-chelating compounds. We discuss previous approaches using non-target selective cysteine-reactive warheads to modify the BoNT/A protease of which none present any therapeutic potential – our bifunctional strategy allows the use of intrinsically less reactive warheads to intercept the cysteine, which will allow for less off-target modifications of such inhibitors. Moreover, we also broach that this bifunctional approach is not a one-off strategy that we believe can be broadly translated to other metalloproteases that possess non-catalytic, yet, nucleophilic residues within the enzymes catalytic sphere.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), is a disease with world wide consequences, affecting nearly a third of the world's population. The established vaccine for TB, an attenuated strain of Mycobacterium bovis Calmette Guerin (BCG), has existed since 1921. Lactoferrin, an iron-binding protein found in mucosal secretions and granules of neutrophils was hypothesized to be an ideal adjuvant to enhance the efficacy of the BCG vaccine, specifically because of previous reports of lactoferrin enhancement of IL-12 production from macrophages infected with BCG. Different vaccination protocols were investigated for generation of host protective responses against MTB infection using lactoferrin admixed to the BCG vaccine. Resulting effects demonstrate that BCG/lactoferrin increased host protection against MTB infection by decreasing organ bacterial load and reducing lung histopathology; significant reduction in tissue CFUs and pathology were observed post-challenge compared to those seen with BCG alone. Addition of lactoferrin to the vaccine led to reduced pathological damage upon subsequent infection with virulent MTB, with positive results demonstrated when admixed in oil-based vehicle (incomplete Freund's adjuvant, IFA) or when given with BCG in saline. The observed post-challenge results paralleled increasing production of IFN-gamma and IL-6, but only limited changes to proinflammatory mediators TNF-alpha or IL-1beta from BCG-stimulated splenocytes. Overall, these studies indicate that lactoferrin is a useful and effective adjuvant to improve efficacy of the BCG vaccine, with potential to reduce related tissue damage and pulmonary histopathology.

Context.—St John's wort induces the activity of the cytochrome P450 enzyme system causing treatment failure because of increased metabolism of many drugs. Procainamide is metabolized by a different pathway to N-acetyl procainamide.Objective.—To study St John's wort–procainamide interaction using a mouse (Swiss Webster) model.Design.—One group of mice (group A, 4 mice in each group) was fed St John's wort each day for 2 weeks (last dose 1 day before administration of procainamide); another group (group B) received the same dose of St John's wort for 1 week. The third group (group C) received only a single dose 1 hour before administration of procainamide, and the control group (group D) received no St John's wort. All groups later received a single oral dose of procainamide. Blood was drawn 1, 4, and 24 hours after administration of procainamide and concentrations in serum of procainamide as well as N-acetyl procainamide were measured using immunoassays.Results.—The procainamide concentrations 1 hour after administration was highest in group C (mean, 11.59 μg/mL) followed by group A (9.92 μg/mL), whereas group B (7.44 μg/mL) and control group D (7.36 μg/mL) showed comparable values. The concentration in group C was significantly greater than the control group D (P = .03, 2-tailed independent t test). N-Acetyl procainamide concentrations and estimated half-life of procainamide among groups were comparable. In a separate experiment when mice were fed purified hypericin, the active component of St John's wort, a significant increase in bioavailability (53%) of procainamide was observed compared with the control group.Conclusions.—St John's wort has an acute effect to increase bioavailability of procainamide but has no effect on its metabolism.

Summary Setting M. tuberculosis (MTB) lose virulence during prolonged culture on artificial media. This loss of virulence is associated with a change in colony morphology. Several studies suggested that trehalose 6,6′ dimycolate (TDM or cord factor), contributes to colony morphology. Objective To investigate the role of TDM in colony morphology of MTB using clinical isolates selected to have colony morphology typical of virulent or attenuated organisms. Design Use immunohistochemical and physical chemical methods to assess the presence and distribution of TDM in rapidly growing pellicles of MTB. Results TDM forms an insoluble crystalline monolayer at the air–water interfaces that is more rigid than that formed by any other biologic amphiphile and is strong enough to support a spreading pellicle of MTB. The surface of young pellicles of the isolate with virulent morphology displayed the regular linear pattern characteristic of monolayers of TDM. TDM was also identified in the open spaces of pellicles of MTB by immunohistochemistry. MTB with morphology of attenuated organisms had neither of these properties. Conclusion These data suggest that the characteristic morphology of colonies of virulent MTB is due to TDM released from the surface of the organisms.

Trehalose 6,6'-dimycolate (TDM) is the most abundant, most granulomagenic, and most toxic lipid extractable from the surface of virulent Mycobacterium tuberculosis (MTB). We further examined its toxicity, which requires activation by oily surfaces. Injections of MTB and/or TDM into sensitized mice induced caseating granulomas that centered on oil droplets. If large doses of MTB were injected in saline, caseating granulomas developed in adipose tissue, but MTB with surface TDM removed induced only acute inflammation that did not persist. Variations in protocols produced several variants of caseating granulomas, each with characteristics of human tuberculosis. In each instance, MTB were localized in fat cells or oil drops during initiation of caseating granulomas suggesting that necrosis was caused by activation of the toxicity of TDM toxicity. Evidence extending these findings to the lung was derived from the observation that in sensitized mice, as in humans, tuberculosis development stimulates accumulation of lipid selectively in alveoli. MTB preferentially associated with lipid droplets in developing necrotic foci in late-stage murine tuberculosis. This supports the hypothesis that pulmonary tuberculosis sequesters MTB in a protected environment that accumulates lipid until it is able to activate the toxicity of TDM and initiate necrosis that results in caseating granulomas.

The development of pulmonary granulomatous lesions during mycobacterial infection is a complex phenomenon, in part caused by responses elicited towards the surface glycolipid trehalose 6,6'-dimycolate (TDM; cord factor). The molecular mechanisms underlying granuloma formation following challenge with TDM are not yet completely understood. The present study defines pathologic differences in acute response to Mycobacterium tuberculosis TDM in C57BL/6 mice and mice lacking the C5a receptor (C5aR-/-). Mice were intravenously injected with TDM prepared in water-in-oil-in-water emulsion and examined for histologic response and changes in proinflammatory cytokines and chemokines in lung tissue. Control C5a receptor-sufficient mice demonstrated a granulomatous response that peaked between days 4 and 7. Increased production of macrophage inflammatory protein-1 alpha (MIP-1alpha), interleukin-1beta (IL-1beta) and CXC chemokine KC (CXCL1) correlated with development of granulomas, along with modest change in tumor necrosis factor-alpha (TNF-alpha). In contrast, the C5aR-/- mice revealed markedly exacerbated inflammatory response. The receptor-deficient mice also demonstrated a lack of coherent granulomatous response, with severe oedema present and instances of lymphocytic cuffing around pulmonary vessels. Lung weight index was increased in the C5aR-/- mice, correlating with increased MIP-1alpha, KC, IL-1beta and TNF-alpha over that identified in the congenic C5aR-sufficient controls. Correlate experiments performed in C5-deficient (B10.D2-H2d H2-T18c Hco/oSnJ) mice revealed similar results, leading to the conclusion that C5 plays a significant role in mediation of chemotactic and activation events that are the basis for maturation of granulomatous responses to TDM.

The present study defines pathologic differences in acute and hypersensitive responses to Mycobacterium tuberculosis glycolipid trehalose-6,6'-dimycolate (TDM, cord factor) in normal BALB/c mice and those deficient in group II CD1 molecule CD1d1. Mice immunized against TDM demonstrate hypersensitive responses, yet the mechanisms for TDM presentation remain elusive. Mice lacking CD1d (CD1D(-/-)) demonstrate dysregulated granulomatous response to TDM, compared with CD1D(+/-) heterozygous controls. Because CD1d-restricted T cells can regulate macrophage immune functions at mucosal surfaces, we hypothesized that CD1D(-/-) mice would show deficient TDM-induced hypersensitive pulmonary granulomatous response in which T cells play a central role. Control CD1D(+/+) mice sensitized and subsequently challenged with TDM demonstrated aggressive inflammation defined by monocytic lesions contained by CD3(+) lymphocytic cuffing. CD1D(-/-) mice demonstrated distinctly different pathologies, with edema present concurrent with extended, nonfocal mononuclear cell-based granulomatous reactions. Furthermore, CD1D(-/-) mice did not demonstrate destructive lesions, and CD3(+) lymphocytes were only loosely organized in proximity to reactive pathology. The CD1d-deficient mice demonstrated rapid increases in proinflammatory mRNAs, with significant differences in interferon-gamma (IFN-gamma) compared to the wild-type group. IFN-gamma, interleukin-6 (IL-6), and IL-12 proteins were significantly elevated in the CD1D(-/-) group compared with wild-type mice (p0.05) 2 days after TDM challenge. However, by 7 days postadministration, similar production for all cytokines and proinflammatory molecules examined was present in both groups of mice. These experiments provide evidence for a role for CD1d in mediation of pathology during hypersensitive responses to the mycobacterial glycolipid TDM.

Background: Ginseng is a widely used herbal product in China, other Asian countries, and in the Unites States. There is a traditional belief that ginseng stimulates immune functions. In this study, the innate effects of Asian and Siberian ginsengs on cytokines and chemokines produced by cultured macrophages were examined. Materials and methods: The effects of Asian and Siberian ginseng on cytokines and chemokines produced by cultured macrophages were examined. Mouse macrophages (J774A.1) were incubated with Asian or Siberian ginseng at varying concentrations (1, 10, 100, and 1000 μg/ml) for 24 h and then harvested for RNA isolation. The expression levels of IL-1β, IL-12, TNF-α, MIP-1 α, and MIP-2 mRNA were measured by quantitative PCR. Results: Our data showed that Asian ginseng induced a statistically significant increase in IL-12 expression at both mRNA and protein levels. However, the minor twofold increase is probably biologically insignificant. No significant increase of IL-12 by Siberian ginseng was observed at any dose level studied. No significant change in IL-1β, IL-15, TNF-α, or MIP-1α mRNA was observed by either Asian or Siberian ginseng treatment. Conclusions: Our data showed statistically significant differential regulation of IL-12 by Asian ginseng. Siberian ginseng did not show a statistically significant increase. We conclude that both Asian ginseng and Siberian ginseng cannot significantly stimulate innate macrophage immune functions that influence cellular immune responses. Therefore, contrary to the popular belief, Asian and Siberian ginseng may not stimulate immune function.

HbhA ofMycobacterium tuberculosisis a multifunctional binding protein, binding to both sulfated sugars such as heparin and to human complement component C3. HbhA may therefore interact with host molecules and/or host cells duringM. tuberculosisinfection and play a role in the pathogenesis of this bacterium. The purpose of this study was to use allelic exchange to create anM. tuberculosisstrain deficient in expression of HbhA to determine whether this protein's C3-binding activity plays a role in the pathogenesis ofM. tuberculosis. An in-frame, 576-bp unmarked deletion in thehbhAgene was created usingsacBas a counterselectable marker. Southern blotting and PCR analyses confirmed deletion ofhbhAin the ΔhbhAmutant. The ΔhbhAmutant strain grew at a rate similar to that of the parent in broth culture and in J774.A1 murine macrophage-like cells but was deficient in growth compared to the parent strain in the lungs, liver, and spleen of infected mice. In addition, the ΔhbhAmutation did not reduce binding ofM. tuberculosisto human C3 or to J774.A1 cells in the presence or absence of serum, suggesting that in the absence of HbhA, other molecules serve as C3-binding molecules on theM. tuberculosissurface. Taken together, these data indicate that HbhA is important in the infectivity ofM. tuberculosis, but its ability to bind C3 is not required for mycobacterial adherence to macrophage-like cells. Using the ΔhbhAmutant strain, a secondM. tuberculosisC3-binding protein similar in size to HbhA was identified as HupB, but the role of HupB as a C3-binding protein in intact organisms remains to be determined.

Background : Chinese medicines are freely available without prescription and are widely used by the general population. Chan Su and Dan Shen are both indicated for the treatment of cardiac diseases. Severe toxicity from Chan Su has been reported. We studied the possibility of removing Chan Su and Dan Shen from human sera using activated charcoal and equilibrium dialysis, and also examined the potential benefit of preventing absorption of these agents from the G.I. tract in the mouse model. Methods : For in vitro studies, drug-free serum pools were supplemented with Chan Su or Dan Shen and then either treated with activated charcoal (10 and 25 mg/ml), or passed through a column packed with activated charcoal. Serum pools supplemented with Chan Su or Dan Shen were also subjected to equilibrium dialysis against phosphate buffer (pH 7.4) using dialysis membrane with molecular cut-off of 25,000 Da. Removal of Chan Su or Dan Shen from the serum was monitored by measuring the apparent digoxin concentration using the fluorescence polarization immunoassay (FPIA) for digoxin (Abbott Laboratories). Results : We observed the fast and effective removal of both Chan Su and Dan Shen from the serum by activated charcoal. We also observed significant removal of both Chan Su and Dan Shen when the serum pools containing these Chinese medicines were passed through columns packed with activated charcoal. Although equilibrium dialysis was also effective in removing these Chinese medicines from the serum, 24 h was required for complete removal of Dan Shen activity, and for Chan Su, complete removal was not achieved even after 24 h. In our in vivo model, we observed significantly less digoxin activity in the group of mice that received activated charcoal compared to the control group. Conclusions : Activated charcoal is effective in preventing absorption of these Chinese medicines from the G.I. tract and can also remove these agents from the serum.

Background: Danshen, a traditional Chinese medicine used in the management of cardiovascular diseases, is available without prescription in the US. Because Danshen is used to treat cardiovascular diseases, we studied the potential interference of Danshen with serum digoxin measurement using various immunoassays. Methods: Blood was collected 1 day before and then 1 and 2 h after feeding mice with Danshen. The apparent digitoxin activities were measured by the fluorescence polarization immunoassay (FPIA). We also added microliter amounts of Danshen extract to digoxin pools prepared from patients receiving digoxin. The digoxin concentrations were measured using the fluorescence polarization immunoassay (FPIA), microparticle enzyme immunoassay (MEIA) and chemiluminescent assay (CLIA). The observed values were compared with original values. We also fed mice with Danshen. Results: We observed measurable digoxin-like immunoreactivity in sera of mice after feeding with Danshen. We also observed falsely lower digoxin concentrations (negative interference) when MEIA was used for digoxin measurement. However, serum digoxin concentrations were falsely elevated with FPIA. We observed no interference of Danshen in serum digoxin measurement using the CLIA. Conclusions: Danshen appears to contain digoxin-like immunoreactivity but does not interfere with serum digoxin measurement when CLIA was used.

Serum cortisol levels were evaluated in mice following intravenous administration of purified mycobacterial glycolipid trehalose 6,6′-dimycolate (TDM). C57BL/6 mice develop lung granulomas in response to TDM, while A/J mice are deficient in this process. Administration of TDM to C57BL/6 mice led to a rapid reduction in serum cortisol, concurrent with initiation of the granulomatous response and cytokine and chemokine mRNA induction. Cortisol levels were lowest on day 5 after TDM administration, but there was significant production of IL-6, TNF-α and IL-1β messages. Granuloma formation and full immune responsiveness to TDM were only apparent upon a sufficient decrease in levels of systemic cortisol. Treatment of the C57BL/6 mice with hydrocortisone abolished inflammatory responses. Histologically nonresponding A/J mice exhibited higher constitutive serum cortisol and demonstrated different kinetics of cortisol reduction upon administration of TDM. A/J mice demonstrated hyperplastic morphology in the suprarenal gland with a high degree of vacuolization in the medullary region and activation of cells in the zona fasciculata and zona reticularis. The A/J mice were dysregulated with respect to cytokine responses thought to be necessary during granuloma formation. The high constitutive serum cortisol in the A/J mice may therefore contribute to pulmonary immunoresponsiveness and the establishment of an environment counterproductive to the initiation of granulomatous responses. The identification of a mycobacterial glycolipid able to influence serum cortisol levels is unique and is discussed in relation to immunopathology during tuberculosis disease.

The biologic effects of the mycobacterial glycolipid trehalose-6,6'-dimycolate (TDM) include granuloma formation and macrophage activation and are dependent on physical conformation. In mice, the group II CD1 surface molecule CD1d has been implicated in glycolipid presentation. The importance of CD1d interactions in pathology has yet to be established. We hypothesized that mice lacking CD1d (CD1D(-/-)) would demonstrate dysregulated granulomatous response to TDM, compared with CD1D(+/-) heterozygous controls. Mice were intravenously injected with TDM-coated polystyrene-divinylbenzene beads and examined for histologic response and for changes in inflammatory cytokine and chemokine mRNA. Control CD1D heterozygous mice demonstrated a granulomatous response, which peaked at day 5. Increased mRNA for tumor necrosis factor-alpha (TNF-alpha) and macrophage inflammatory protein-1alpha (MIP-1alpha) correlated with development of granulomas, with very little change in interleukin-1beta (IL-1beta) and monocyte chemoattractant protein-1 (MCP-1). In contrast, the CD1D(-/-) mice revealed markedly different responses. Five days after administration, severe pulmonary hemorrhage was induced. The relative size of inflammation surrounding coated bead in the CD1D(-/-) mice was nearly double that induced in the CD1D(+/-) mice. CD1D(-/-) mice also demonstrated elevated mRNA for both inflammatory cytokines and chemokines by day 1 after administration, significantly earlier than responses seen in the heterozygous controls.

Trehalose-6,6'-dimycolate (TDM), or cord factor, is a mycobacterial cell wall component that induces granuloma formation and proinflammatory cytokine production in vivo and in vitro. The purpose of this work was to better understand the mechanisms by which TDM promotes lung granuloma formation. This was accomplished by characterizing cytokine mRNA expression during TDM-induced alveolitis culminating in cohesive granuloma development. A single intravenous injection of TDM given to C57BL/6 mice produced lung granulomas that peaked in number 5 days after challenge and were nearly resolved by 14 days. mRNA in whole lung preparations was quantitated by bioluminescent RT-PCR. Tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and IL-6 were significantly elevated during granuloma development and decreased during granuloma resolution. There were no detectable changes in mRNA for interferon-y (IFN-y), IL-2, IL-4, IL-5, IL-10, and IL-12(p40). The level of TNF-alpha protein extracted from lung minces highly correlated with morphologic indices of granulomatous inflammation, indicating that it may be an important modulator of the inflammatory intensity induced by TDM. TDM may interact specifically with macrophages in vivo, as evidenced by induction of TNF-alpha, IL-1beta, and IL-6, but not IFN-gamma, protein in bone marrow-derived macrophages from C57BL/6 mice. TDM may therefore play an important role early in macrophage activation during the host granulomatous response to mycobacteria.

Amikacin is an aminoglycoside which is used in the treatment of infection from gram negative bacteria. Amikacin is also used synergistically with penicillin against gram positive cocci. Amikacin cannot be delivered orally probably due to efflux of drug by P-glycoprotein pump in the brush border of intestine. We studied the possibility of delivering amikacin orally in mice using a copolymer (CRL-1605) as a vehicle. This copolymer inhibits P—glycoprotein pump. Two different doses of amikacin were used (500 mg/kg and 100 mg/kg). The concentration of polymer used was 132 mg/kg. The liquid formulation was fed to mice by gavage and serum amikacin concentrations were estimated after one hour and two hours using flourescence polarization immunoassay. We observed a two fold increase in serum amikacin concentration when amikacin was orally delivered in the presence of CRL-1605 compared to controls (amikacin alone). We conclude that gastrointestinal absorption of amikacin is significantly increased in the presence of CRL-1605 in mice.

Recurrent Clostridium difficile infection (rCDI) affects 10% to 25% of patients with initial CDI (iCDI). Initiation of new therapies that reduce recurrences rests on identifying patients at high risk for rCDI at iCDI onset.To develop a predictive model for rCDI based on factors present at iCDI onset.Retrospective cohort study.Large urban academic medical center.All adult patients with an inpatient iCDI from January 1, 2003 to December 31, 2009.None.Positive toxin assay for C difficile with no C difficile infection in the previous 60 days constituted iCDI. Repeat positive toxin within 42 days of stopping iCDI treatment defined rCDI. Three demographic, 13 chronic, and 12 acute disease characteristics, and 7 processes of care prior to or at the onset of iCDI, were assessed for association with rCDI. A logistic regression model to identify predictors for rCDI was developed and cross-validated.Among the 4196 patients enrolled, 425 (10.1%) developed rCDI. Six factors (case status as community-onset healthcare-associated, ≥2 hospitalizations in the prior 60 days, new gastric acid suppression, fluoroquinolone and high-risk antibiotic use at the onset of iCDI, age) predicted rCDI in multivariate analyses, whereas intensive care unit stay appeared protective. The model achieved moderate discrimination (C statistic 0.643) and calibration (Brier score 0.089). Its negative predictive value was 90% or higher across a wide range of risk.Among patients hospitalized with rCDI, multiple factors present at the onset of iCDI increased the risk for rCDI. Recognizing patients at high-risk for rCDI can help clinicians tailor early treatment and prevention.

We evaluated an adjuvant, TiterMax, as an alternative to complete Freund's adjuvant (CFA) for producing antisera in animals. TiterMax, consists of a microparticulate stabilized water-in-oil emulsion of a metabolizable oil, squalene, with the adjuvant block copolymer CRL89-41. This paper reports two evaluations of TiterMax versus CFA and other commercially available adjuvants. In the first study, mice were immunized with a hapten, trinitrophenol, conjugated to hen egg albumin (TNP-HEA) in one of several adjuvants: TiterMax, CFA, Adjuvax, Ribi adjuvant system (RAS), Alhydrogel or Lipovant. TiterMax induced higher longer lasting titers with fewer injections than any of the other adjuvants. The magnitude of the response to TNP varied with species and route of immunization. In the second study, CFA, TiterMax, Adjuvax and RAS were compared in rabbits, mice and goats. Animals were immunized with luteinizing hormone-releasing hormone (LHRH) conjugated to BSA in each adjuvant using comparable protocols. TiterMax induced titers against the peptide equivalent to CFA in all three species. The inflammatory responses induced by TiterMax were mild and transient compared with those induced by CFA. These data suggest that TiterMax is an effective alternative to CFA in many situations.

Non-ionic block copolymers and lipopolysaccharides are both effective immunological adjuvants which are thought to act via distinct mechanisms. We hypothesized that they might produce synergistic effects when used together. We prepared a series of lipopolysaccharide (LPS) preparations ranging from the smallest precursor, lipid X through complete LPS with O-polysaccharide chains. Three preparations with reduced toxicity, monophosphoryl lipid A, partially hydrolysed Ra-LPS and LPS of Rhodopseudomonas sphaeroides were also utilized. All LPS preparations except the smallest were effective adjuvants for inducing early antibody responses to trinitrophenyl-conjugated hen egg albumin (TNP-HEA) when injected in squalane-in-water emulsions with copolymer L141. Only the larger LPS preparations induced sustained antibody responses. By itself, emulsions of copolymer L141 induced a predominant IgG1 antibody isotype response with lesser amounts of IgG2a and IgG2b. Surprisingly, all of the LPS preparations tested increased the proportion of IgG2 isotypes even though some had little effect on overall titres. The detoxified Ra-LPS (Ra-detox) was the most effective preparation for both increasing antibody titres and inducing the desirable IgG2a and IgG2b isotypes. These results demonstrate that the combination of LPS and block polymer adjuvants can produce synergistic effects without unacceptable toxicities.

DanShen is a traditional Chinese medicine indicated for cardiovascular diseases. The potential interference of DanShen with serum digoxin measurement was investigated using a new enzyme-linked chemiluminescent immunosorbent (ECLIA) digoxin assay. Aliquots of drug-free serum were supplemented with ethyl acetate extract of DanShen (4 different brands studied), and apparent digoxin concentrations were measured by the ECLIA as well as fluorescence polarization immunoassay (FPIA) and a turbidimetric assay for comparison. Mice were also fed 4 DanShen preparations and apparent digoxin concentrations were subsequently measured. In another experiment, serum pools containing digoxin were further supplemented with DanShen extracts and digoxin concentrations were measured again by all 3 assays. No apparent digoxin concentration was observed when aliquots of drug-free serum pools were supplemented with DanShen and digoxin concentrations were measured by the ECLIA or the turbidimetric assay. In contrast, significant apparent digoxin concentrations were observed using FPIA, and the highest apparent digoxin concentration was observed with brand 4 of DanShen extract. Similarly, when mice were fed with this herb, significant apparent digoxin concentrations were also observed using FPIA, but neither ECLIA nor turbidimetric assay showed any apparent digoxin concentration. When aliquots of digoxin pool were further supplemented with various DanShen extract, the apparent digoxin concentrations were significantly increased when FPIA was used. In contrast, digoxin concentrations in the presence of DanShen extract compared well with the digoxin concentration of the original pool when ECLIA or turbidimetric assay was used. We conclude that DanShen does not interfere with serum digoxin measurement using a more recently released ECLIA digoxin assay.

Asian and Siberian ginsengs contain glycosides with structural similarities to digoxin. We studied potential interference of ginseng in 5 digoxin immunoassays in 3 Asian (2 liquid extracts, 1 capsule) and 3 Siberian ginseng preparations (1 liquid extract, 2 capsules). With the fluorescence polarization immunoassay (FPIA), we observed apparent digoxin activity in 1 Asian liquid preparation and in the liquid extract and 1 capsule form of Siberian ginseng. In mice fed ginseng, we observed digoxin activities in the serum (Asian, 0.48-0.68 ng/mL [0.6-0.9 nmol/L]; Siberian, 0.20-0.47 ng/mL [0.3-0.6 nmol/L]), indicating that such interferences also occur in vivo. Serum pools prepared from samples from patients receiving digoxin and then supplemented with Asian or Siberian ginseng showed falsely increased digoxin values using the FPIA (e.g., for Asian ginseng, 1.54 ng/mL [2.0 nmol/L] vs control value, 1.10 ng/mL [1.4 nmol/L]) and falsely decreased values using the microparticle enzyme immunoassay (MEIA; 0.73 ng/mL [0.9 nmol/L] vs control value, 1.04 ng/mL [1.3 nmol/L]). Digoxin-like immunoreactive substances (DLISs) showed synergistic effects with ginsengs in interfering with the FPIA and MEIA for digoxin. No interference was observed with 3 other digoxin assays, even in the presence of elevated DLISs.

Improved nontoxic adjuvants, especially adjuvants capable of inducing cell-mediated immunity (CMI), are needed for research in immunology and for development of human and veterinary vaccines. Bovine Lactoferrin, an effector molecule shown to directly participate in host defense, was assessed at various concentrations as an adjuvant component for induction of DTH responses to sheep red blood cells (SRBC). Subcutaneous immunization with Lactoferrin enhanced delayed type hypersensitivity (DTH) in CBA mice in a dose-dependent fashion; DTH responses were most significantly increased when sensitization was accomplished using Lactoferrin at 50 microg/dose and 250 microg/dose. Furthermore, Lactoferrin admixed with suboptimal dose of SRBC enhanced DTH responses by over 17-fold. Peritoneal cells collected from mice intraperitoneally injected with a 100 microg/dose of Lactoferrin demonstrated modest, but significant, production of TNF-alpha, IL-12 and MIP-1alpha when cultured in vitro, compared to saline-injected controls. J774A.1 murine macrophages stimulated with Lactoferrin resulted in increased TNF-alpha protein production, and upregulated IL-12 and IL-15 mRNA. Levels of message for chemokines MIP-1alpha and MIP-2 were also increased in a dose-dependent way. Taken together, these results indicate that Lactoferrin as an adjuvant may stimulate macrophages to generate a local environment likely to push immune responses towards development and maintenance of CMI.

Chan Su, a Chinese medicine prepared from the skin glands of Chinese toads, is used in the treatment of cardiovascular diseases. Severe toxicity and even death has been reported from overdose with Chan Su. The cardiotonic effect of Chan Su is attributed to bufadienolides, which also have apparent digitoxin activity. We demonstrated that these components of Chan Su could be neutralized by digibind, both in vitro and in vivo. For in vitro experiments, we supplemented drug-free serum pools with aqueous extract of Chan Su. Then, to aliquots of serum pool containing Chan Su, various amounts of digibind (10, 25 or 50 microg/ml of serum) were added. After incubation, total and free digitoxin concentrations (in the protein-free ultrafiltrate) were measured using the fluorescence polarization immunoassay (FPIA) and a FLX/TDx analyzer. For in vivo experiments, mice were fed with Chan Su by gavage. After 45 min, 200 microg of digibind was administered by injection. Fifteen minutes after injection, blood was collected for analysis of total and free apparent digitoxin activities. We observed complete removal of apparent digitoxin activity from protein-free ultrafiltrate both in vitro and in vivo by digibind, indicating that digibind successfully binds Chan Su. We conclude that digibind neutralizes Chan Su, and measuring the free digitoxin concentrations can monitor such an effect.

Traditional Chinese medicines are readily available without prescription from herbal drug stores. One such Chinese medicine, Chan Su, which is prepared from the skin gland of toads, has cardiotonic effect due to bufadienolides. Here we report rapid detection of the presence of Chan Su in blood using the fluorescence polarization immunoassay for digitoxin. In our study mice were fed with a dose of 75 mg/kg of Chan Su and blood was drawn before, and 1 and 2 h after feeding. We observed significant digitoxin-like immunoreactivity in the sera. For example in one mouse the digitoxin-like immunoreactivity was undetectable before feeding with Chan Su, but was 19.7 ng/ml 1 h and 8.8 ng/ml 2 h afterwards. The apparent half-life of Chan Su is approximately 1 h in mice. In another experiment, we studied protein binding of Chan Su by measuring total and free Chan Su concentrations (ultrafiltrate prepared by using Centrifree Micropartition Filter, molecular weight cutoff: 30000 Da). Chan Su was strongly bound to serum proteins. We observed higher free fraction in uremic sera and sera from patients with liver disease. We identified albumin as one of the proteins that bind Chan Su in serum.

The relationship among organism growth, immunopathology, and survival was studied in C57BL/6 and A/J mice acutely infected with Mycobacterium tuberculosis (MTB) (Erdman). Although organisms grew at similar rates in the lungs of both mouse strains, A/J mice died prior to 14 days after infection, whereas C57BL/6 mice survived twice as long. The lungs of A/J mice exhibited necrotizing interstitial inflammation and widely distributed acid-fast bacilli without granuloma formation. In contrast, the lungs of C57BL/6 mice had relatively mild interstitial inflammation, which was replaced by focal granulomas, and acid-fast bacilli were primarily within granulomas. MTB induced similar granulomas for A/J and C57BL/6 mice in spleen and liver. In the lung, the A/J mice produced only transient messages for interferon-y (IFN-y), interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), IL-10, and inducible nitric oxide synthase (iNOS). The C57BL/6 mice, in contrast, produced a delayed but sustained response in the lung correlating with granuloma onset and characterized by high induction of IL-6, IFN-gamma, IL-1beta, IL-10, and TNF-alpha. Responses in the liver and spleen were also evaluated. These results demonstrate that histopathology and cytokine response to MTB infection varies among organs in mice. Increased survival during acute infection may, therefore, depend on the ability to contain organisms within granulomas in the lung.

Humoral response against sporozoites is not effective in protecting individuals from getting malaria. Reduction in the infectivity of sporozoites has not been quantified for most anti-sporozoite vaccines tested. Quantification requires animal models providing predictable prepatent periods, e.g., time elapsed between sporozoite inoculation and detection of parasitemia, to be used as an indicator of activity against sporozoites. A delay in prepatent period from vaccinated animals would therefore reflect a protective effect in reducing the number of parasites. We report the vaccination of rhesus monkeys with a synthetic peptide reproducing part of the repeated region of the circumsporozoite protein of Plasmodium cynomolgi. This peptide was conjugated to the carrier protein diphtheria toxoid and injected with four adjuvant formulations that differed only by the type of emulsion or immunomodulator. Because all five control animals had a synchronous prepatent period after challenge with live sporozoites, it was possible to quantify the protective efficacy for each vaccine formulation, even though all monkeys developed parasitemia. Sporozoite elimination correlated with the immunomodulator and the type of emulsion. Such elimination was related neither to antibody titer against the immunizing peptide or the whole sporozoite, nor to antibody isotype induced by the vaccine formulation.

The adjuvant activity of block copolymers varies with the lengths of the chains of polyoxypropylene (POP) and polyoxyethylene (POE). This project evaluated the adjuvant activity of new copolymers with long polyoxypropylene chains in mice immunized with TNP-hen egg albumin in 2% squalone-in-water emulsions. Two of the new copolymers, L141 and L180.5, not only stimulated higher antibody titres to TNP than older preparations, but also induced a higher percentage of the IgG2 isotypes. The smaller older copolymers, L101 and L121, induced higher absolute levels of IgG3 antibody and relative increases in IgG1. Incorporation of immunomodulators (cell wall skeletons, monophosphoryl lipid A or threonyl muramyl dipeptide) increased mean IgG titres but also increased variability of responsiveness among individuals.

Avian myeloblastosis virus is the prototype virus for the myb oncogene. The virus was originally purified by Hall and colleagues between 1938 and 1941 by serial passage in chicken embryos (Hall et al. 1941; Pollard and Hall 1942; Hall and Pollard 1943). In retrospect, it is clear that the original tumor tissue contained a mixture of viruses including Marek’s Disease Virus, and avian leukosis virus. This contamination of virus stocks was a common problem in most of the early work due to the widespread distribution of these viruses in chicken flocks; so that even purified stocks became contaminated with other virus types when transferred to other laboratories (Johnson 1941). The strain A virus which Hall isolated induced a characteristic leukemia leading to death of the chicken in an average of 14 days post inoculation in about 80% of birds inoculated as embryos (Hall and Pollard 1943). The virus also caused similar disease in quail, turkey, pheasant, and duck (Pollard and Hall 1942; Hall and Pollard 1943), and appears to be identical to that induced by the current BAI Strain A propagated and studied for many years by Beard and colleagues (Beard 1963).