Lactoferrin in a Context of Inflammation-Induced Pathology.

Much progress has been achieved to elucidate the function of lactoferrin (LTF), an iron-binding glycoprotein, in the milieu of immune functionality. This review represents a unique examination of LTF toward its importance in physiologic homeostasis as related to development of disease-associated pathology. The immunomodulatory nature of this protein derives from its unique ability to "sense" the immune activation status of an organism and act accordingly. Underlying mechanisms are proposed whereby LTF controls disease states, thereby pinpointing regions of entry for LTF in maintenance of various physiological pathways to limit the magnitude of tissue damage. LTF is examined as a first line mediator in immune defense and response to pathogenic and non-pathogenic injury, as well as a molecule critical for control of oxidative cell function. Mechanisms of interaction of LTF with its receptors are examined, with a focus on protective effects via regulation of enzyme activities and reactive oxygen species production, immune deviation, and prevention of cell apoptosis. Indeed, LTF serves as a critical control point in physiologic homeostasis, functioning as a sensor of immunological performance related to pathology. Specific mediation of tissue pathophysiology is described for maintenance of intestinal integrity during endotoxemia, elicited airway inflammation due to allergens, and pulmonary damage during tuberculosis. Finally, the role of LTF to alter differentiation of adaptive immune function is examined, with specific recognition of its utility as a vaccine adjuvant to control subsequent lymphocytic reactivity. Overall, it is clear that while the ability of LTF to both sequester iron and to direct reactive oxygen intermediates is a major factor in lessening damage due to excessive inflammatory responses, further effects are apparent through direct control over development of higher order immune functions that regulate pathology due to insult and injury. This culminates in attenuation of pathological damage during inflammatory injury.

CHO expressed recombinant human lactoferrin as an adjuvant for BCG.

Lactoferrin (LF), an iron binding protein with immune modulatory activities, has adjuvant activity to enhance vaccine efficacy. Tuberculosis (TB) is a pulmonary disease caused by the pathogen Mycobacterium tuberculosis (MTB). Progressive TB disease is clinically defined by damaging pulmonary pathology, a result of inflammation due to immune reactivity. The current vaccine for TB, an attenuated strain of Mycobacterium bovis, Bacillus Calmette Guerin (BCG), has only limited efficacy to prevent adult pulmonary TB. This study examines a Chinese hamster ovary (CHO) expressed recombinant human LF (rHLF) to boost efficacy of the BCG vaccine and delay early pathology post infectious challenge. C57BL/6 mice were immunized with BCG, or BCG admixed with either rHLF or bovine LF (bLF; internal control), or remained unvaccinated. Mice were then aerosol challenged with Erdman MTB. All vaccinated mice demonstrated decreased organ bacterial load up to 19 weeks post infection compared with non-vaccinated controls. Furthermore, mice receiving bLF or rHLF supplemented BCG vaccines showed a modest decrease in lung pathology developed over time, compared to the BCG vaccine alone. While mice vaccinated with BCG/rHLF demonstrated increased general lung inflammation at day 7, it occurred without noticeable increase in pro-inflammatory cytokines. At later times, decreased pathology in the rHLF groups correlated with decreased inflammatory cytokines. Splenic recall to BCG antigens showed BCG/rHLF vaccination increased production of IFN-γ, IL-6, and GM-CSF compared to naïve, BCG, and BCG/bLF groups. Analysis of T cell stimulating functions of bone marrow derived macrophages and dendritic cells treated with BCG/bLF or BCG/rHLF showed decreases in IL-10 production when co-cultured with sensitized CD4 and CD8 T cells, compared to those cultured with macrophages/dendritic cells treated with BCG without LF. These results indicate that addition of rHLF to the BCG vaccine can modulate development of host pathology early post infectious challenge, most likely through host immune regulation affecting hypersensitive responses.

Effects of CHO-expressed recombinant lactoferrins on mouse dendritic cell presentation and function.

Lactoferrin (LF), a natural iron-binding protein, has previously demonstrated effectiveness in enhancing the Bacillus Calmette-Guérin (BCG) tuberculosis vaccine. This report investigates immune modulatory effects of Chinese hamster ovary (CHO) cell-expressed recombinant mouse and human LFs on mouse bone marrow-derived dendritic cells (BMDCs), comparing homologous and heterologous functions. BCG-infected BMDCs were cultured with LF, and examined for class II presentation molecule expression. Culturing of BCG-infected BMDCs with either LF decreased the class II molecule-expressing population. Mouse LF significantly increased the production of IL-12p40, IL-1ß and IL-10, while human LF-treated BMDCs increased only IL-1ß and IL-10. Overlaying naïve CD4 T-cells onto BCG-infected BMDCs cultured with mouse LF increased IFN-γ, whereas the human LF-exposed group increased IFN-γ and IL-17 from CD4 T cells. Overlay of naïve CD8 T cells onto BCG-infected BMDCs treated with mouse LF increased the production of IFN-γ and IL-17, while similar experiments using human LF only increased IL-17. This report is the first to examine mouse and human recombinant LFs in parallel experiments to assess murine DC function. These results detail the efficacy of the human LF counterpart used in a heterologous system to understand LF-mediated events that confer BCG efficacy against Mycobacterium tuberculosis challenge.

Rapid detection of the active cardiac glycoside convallatoxin of lily of the valley using LOCI digoxin assay.

OBJECTIVES: To explore the luminescent oxygen channeling technology-based digoxin immunoassay (LOCI digoxin assay) for rapid detection of lily of the valley extract and convallatoxin. The potential in vitro binding of convallatoxin with Digibind was also evaluated. METHODS: Aliquots of a drug-free serum pool and a digoxin serum pool were supplemented with lily of the valley extract or convallatoxin, and then apparent digoxin concentrations were measured using the LOCI digoxin assay. Mice were administered lily of the valley extract or 50 µg of convallatoxin, and digoxin concentrations in serum specimens were measured 1 and 2 hours after gavage. Aliquots of a serum pool supplemented with convallatoxin or lily of the valley extract were further supplemented with various concentrations of Digibind and free apparent digoxin concentrations were measured. RESULTS: Apparent digoxin concentrations were observed when aliquots of a drug-free serum pool were supplemented with convallatoxin or lily of the valley extract, and also with convallatoxin or herbal extract. Bidirectional interference of convallatoxin and lily of the valley extract with serum digoxin measurement using the LOCI assay was also observed. Digibind was capable of binding convallatoxin in vitro. CONCLUSIONS: LOCI digoxin assay can be used for rapid detection of convallatoxin, and Digibind can bind convallatoxin in vitro.

Bidirectional (negative/positive) interference of oleandrin and oleander extract on a relatively new Loci digoxin assay using Vista 1500 analyzer.

BACKGROUND: Oleander interferes with serum digoxin measurements using various immunoassays. The potential interference of oleander and its active ingredient, oleandrin, with a relatively new homogenous sequential chemiluminescent digoxin assay based on luminescent oxygen channeling technology (LOCI digoxin assay, Siemens Diagnostics) has not been previously reported. METHODS: Aliquots of a digoxin-free serum pool were supplemented with increasing concentrations of oleandrin, or with oleander extract, followed by measuring the apparent digoxin concentrations using the LOCI digoxin assay using Vista 1500 analyzer. Mice were fed oleandrin or oleander extract, and their blood digoxin levels at 1 and 2 h were measured with the LOCI digoxin assay. In addition, two digoxin serum pools were prepared by combining sera of patients receiving digoxin; aliquots of both pools were supplemented with oleandrin or oleander extract and digoxin concentrations were again measured. Attempts to overcome this interference were made by measuring free digoxin concentration using a third digoxin pool. RESULTS: Significant apparent digoxin concentrations were observed after supplementing aliquots of the drug-free serum pool with oleandrin or oleander extract. Mice fed with oleandrin or oleander extract also showed apparent digoxin levels 1 and 2 h after feeding. Digoxin values were also falsely lower or elevated (bidirectional interference) when aliquots of digoxin serum pools were further supplemented with oleandrin or oleander extract depending on concentration; this interference was not eliminated by free digoxin monitoring. CONCLUSIONS: Oleandrin interferes with LOCI digoxin assay.

Novel spot tests for detecting the presence of zinc sulfate in urine, a newly introduced urinary adulterant to invalidate drugs of abuse testing.

OBJECTIVES: To find a suitable method for detecting zinc sulfate in adulterated urine. METHODS: Two rapid spot tests to detect the presence of zinc sulfate in urine were developed. RESULTS: Addition of 3 to 4 drops of 1N sodium hydroxide solution to approximately 1 mL of urine containing zinc sulfate led to the formation of a white precipitate, which was soluble in excess sodium hydroxide. In the second spot test, addition of 3 to 4 drops of 1% sodium chromate solution to 1 mL of urine containing zinc sulfate followed by the addition of 4 to 5 drops of 1N sodium hydroxide led to formation of a yellow precipitate (zinc chromate). Detection limit of these visual spot tests was 10 mg/mL of zinc sulfate in urine. Twenty drug-free urine specimens and urine containing high amounts of sugar or reducing substances were tested with no false-positive spot test results observed. However, if lead is present in high amounts in urine, it may cause false-positive spot test results. When aliquots of urine controls for drugs of abuse testing were supplemented with different amounts of zinc sulfate, false-negative drug test results were observed except for amphetamine. Zinc sulfate also falsely reduced measured urine alcohol level in urine. CONCLUSIONS: Zinc sulfate can invalidate urine drug and alcohol testing but can be detected using the novel spot tests developed.

Influence of oral lactoferrin on Mycobacterium tuberculosis induced immunopathology.

The ability of lactoferrin to provide protection and decrease immunopathology in infectious diseases was evaluated using an aggressive aerosol model of Mycobacterium tuberculosis (MTB) infection. C57BL/6 mice were challenged with MTB strain Erdman and treated with 0.5% bovine lactoferrin added to the drinking water starting at day 0 or day 7 post-infection. Mice were sacrificed at three weeks post-challenge and evaluated for organ bacterial burden, lung histopathology, and ELISpot analysis of the lung and spleen for immune cell phenotypes. Mice given tap water alone had lung log10 colony forming units (CFUs) of 7.5 ± 0.3 at week 3 post-infection. Lung CFUs were significantly decreased in mice given lactoferrin starting the day of infection (6.4 ± 0.7), as well as in mice started therapeutically on lactoferrin at day 7 after established infection (6.5 ± 0.4). Quantitative immunohistochemistry using multispectral imaging demonstrated that lung inflammation was significantly reduced in both groups of lactoferrin treated mice, with decreased foamy macrophages, increased total lymphocytes, and increased numbers of CD4+ and CD8+ cells. ELISpot analysis showed that lactoferrin treated mice had increased numbers of CD4 + IFN-γ+ and IL-17 producing cells in the lung, cells that have protective functions during MTB infection. Lactoferrin alone did not alter the proliferation of MTB in either broth or macrophage culture, but enhanced IFN-γ mediated MTB killing by macrophages in a nitric oxide dependent manner. These studies indicate that lactoferrin may be a novel therapeutic for the treatment of tuberculosis, and may be useful in infectious diseases to reduced immune-mediated tissue damage.

Comparing efficacy of BCG/lactoferrin primary vaccination versus booster regimen.

Lactoferrin is an iron binding glycoprotein possessing multiple immune modulatory activities, including ability to affect macrophage cytokine production, promote maturation of T- and B-lymphocyte and immature dendritic cells, and enhance the ability of macrophages and dendritic cells to stimulate antigen-specific T-cells. These characteristics of lactoferrin suggested that it could function as an effective adjuvant enhance efficacy of the BCG, the current vaccine for tuberculosis disease. Admix of lactoferrin to the BCG vaccine promoted host protective responses that surpasses activity of the BCG vaccine alone as determined by decreasing pulmonary pathology upon challenge with virulent Mycobacterium tuberculosis (MTB). This study builds on previous reports by examining the effectiveness of the lactoferrin adjuvant comparing primary vaccination versus an immunization schedule with a booster administered at 8 weeks. BCG/lactoferrin vaccinating, given once or twice, demonstrated an improvement in pulmonary disease compared to both the BCG vaccinated and non-immunized groups. The splenic recall profiles showed a difference in cytokine production induced by mycobacterial antigen from splenocytes isolated from mice immunized with BCG/lactoferrin once or twice. Production of IL-17 is increased in the BCG/lactoferrin 2× group compared to the primary vaccinated group. Both BCG/lactoferrin vaccinated group exhibited increase production of IFN-γ compared to the non-immunized group and decreased production of IL-10 compared to the group vaccinated with only BCG. This study illustrates that the adjuvant activity of lactoferrin to enhance BCG efficacy occurs whether the vaccination regimen is a single delivery or combined with a booster, leading to enhanced host protection and decreased disease manifestation.

Effect of Chinese medicines Chan Su, Asian ginseng, Siberian ginseng, and American ginseng on a new digoxin immunoassay based on luminescent oxygen channeling technology.

BACKGROUND: Chan Su, Asian ginseng, Siberian ginseng, and American ginseng are known to interfere with various digoxin immunoassays. Recently, a homogeneous sequential chemiluminescent assay for digoxin based on the luminescent oxygen channeling technology (LOCI digoxin) for application on the Dimension and Vista platform has been introduced into the market. The effects of interference by Chan Su and various ginsengs on this new immunoassay have not yet been reported. MATERIALS AND METHODS: Aliquots of a drug-free serum pool were supplemented with Chan Su, Asian ginseng, Siberian ginseng, and American ginseng representing the expected in vivo concentrations after normal usage and cases of overdose. Serum digoxin concentrations were measured using the LOCI digoxin assay on the Vista 1500 analyzer. We also prepared 3 digoxin pools from patients receiving digoxin. Two digoxin pools were supplemented with these traditional medicines to investigate their effect on serum digoxin measurements. Mice were fed Chan Su extract to determine the potential of in vivo derived interfering factors. The possibility of eliminating interference of Chan Su on serum digoxin measurement was also investigated, by measuring free digoxin concentration after supplementing aliquots of the third digoxin pool with various amounts of Chan Su extract. RESULTS: A clinically significant interference by Chan Su with serum digoxin measurement was observed using the LOCI digoxin assay. The various ginsengs demonstrated negligible effects. In addition, apparent digoxin concentrations were observed in sera of mice after feeding them with Chan Su; the half-life of digoxin-like immunoreactive components was approximately 1 hour. Moreover, serum digoxin concentrations were significantly elevated in the presence of Chan Su, whereas the various ginsengs exhibited no effect. Monitoring free digoxin can only partly eliminate the interference of Chan Su in serum digoxin measurement. CONCLUSIONS: Chan Su interferes with serum digoxin measurement using the LOCI Digoxin, whereas the ginsengs demonstrated no measurable interference at clinically relevant concentrations.

Rapid detection of oleander poisoning by Dimension Vista digoxin assay (Flex Reagent Cartridge).

Oleander poisoning can be detected by digoxin immunoassays and for last two decades the fluorescence polarization immunoassay (FPIA) has been used for rapid detection of oleander poisoning in clinical laboratories. Recently, Abbott Laboratories (Abbott Park, IL) discontinued this assay. Therefore, we explored the possibility of using another digoxin assay (Dimension Vista Flex Reagent Cartridge, Tina Quant, EMIT 2000 and old FPIA assay for comparison) for rapid detection of oleander poisoning. When aliquots of drug-free serum pools were supplemented with pure oleandrin or oleander extract, we observed the highest apparent digoxin values using Dimension Vista digoxin assay (Flex Reagent Cartridge). We also observed significant apparent digoxin values in vivo in sera of mice both 1 and 2 hr after feeding with oleander extract. When a serum pool prepared from patients taking digoxin was further supplemented with various amounts of oleander extract, the highest falsely elevated digoxin values were observed with Dimension Vista digoxin assay. Monitoring free digoxin using Dimension Vista digoxin assay (Flex Reagent Cartridge) did not eliminate this interference. Digibind neutralized digoxin-like factors of oleander extract and such effect can be monitored by observing significant reduction in apparent free digoxin levels in the presence of Digibind as measured in the protein-free ultrafiltrate using Dimension Vista digoxin assay (Flex Reagent Cartridge).

Lactoferrin enhances efficacy of the BCG vaccine: comparison between two inbred mice strains (C57BL/6 and BALB/c).

The current vaccine for tuberculosis (TB), an attenuated strain of Mycobacterium bovis Bacillus Calmette Guerin (BCG), is effective to prevent childhood onset of the disease, but its efficacy is reduced in adults. One strategy to improve the existing vaccine is to develop more effective adjuvants. Lactoferrin, an iron-binding glycoprotein possessing immune modulatory activities, is a promising adjuvant candidate. The studies presented here examine the effect of lactoferrin to enhance efficacy of the BCG vaccine using a vaccination/challenge protocol (8 weeks boost and challenge at 12 weeks post-boost) that focuses on reduction in development of pathological changes to lung tissue. C57BL/6 and BALB/c mice vaccinated with BCG/lactoferrin exhibited protection upon Mycobacterium tuberculosis (MTB) challenge, showing reduced pulmonary disease pathology and decreased organ bacterial load. In addition, BCG/lactoferrin-treated macrophages isolated from BALB/c mice, which express a relative reduced T(H)1 phenotypic response to MTB antigens compared to the C57BL/6 mouse, were able to activate a higher percentage of IFN-gamma-producing CD4+ splenocytes. Overall, lactoferrin stands as an adjuvant capable of enhancing efficacy of the BCG vaccine through induction of T(H)1 immune responses, even in hosts typically demonstrative of reduced T(H)1 responsiveness to BCG antigens.

Lactoferrin modulation of BCG-infected dendritic cell functions.

Lactoferrin, an 80-kDa iron-binding protein with immune modulating properties, is a unique adjuvant component able to enhance efficacy of the existing Mycobacterium bovis Bacillus Calmette Guerin (BCG) vaccine to protect against murine model of tuberculosis. Although identified as having effects on macrophage presentation events, lactoferrin's capability to modulate dendritic cells (DCs) function when loaded with BCG antigens has not been previously recognized. In this study, the potential of lactoferrin to modulate surface expression of MHC II, CD80, CD86 and CD40 from bone marrow-derived dendritic cells (BMDCs) was examined. Generally, lactoferrin decreased pro-inflammatory cytokines [tumor necrosis factor (TNF)-alpha, IL-6 and IL-12p40] and chemokines [macrophage inflammatory protein (MIP)-1alpha and MIP-2] and increased regulatory cytokine, transforming growth factor-beta1 and a T-cell chemotatic factor, monocyte chemotactic protein-1, from uninfected or BCG-infected BMDCs. Culturing BCG-infected BMDCs with lactoferrin also enhanced their ability to respond to IFN-gamma activation through up-regulation of maturation markers: MHC I, MHC II and the ratio of CD86:CD80 surface expression. Furthermore, lactoferrin-exposed BCG-infected DCs increased stimulation of BCG-specific CD3(+)CD4(+) splenocytes, as defined by increasing IFN-gamma production. Finally, BCG-/lactoferrin-vaccinated mice possessed an increased pool of BCG antigen-specific IFN-gamma producing CD3(+)CD4(+)CD62L(-) splenocytes. These studies suggest a mechanism in which lactoferrin may exert adjuvant activity by enhancing DC function to promote generation of antigen-specific T cells.

A novel recombinant human lactoferrin augments the BCG vaccine and protects alveolar integrity upon infection with Mycobacterium tuberculosis in mice.

Lactoferrin, an iron binding glycoprotein, possesses multiple immune modulatory activities, including the ability to promote antigen specific cell-mediated immunity. Previous studies showed that adding bovine lactoferrin to the BCG vaccine (an attenuated strain of Mycobacterium bovis Bacillus Calmette Guerin) resulted in increased host protective responses upon subsequent challenge with virulent Erdman Mycobacterium tuberculosis (MTB) in mice. The studies outlined here investigate utility of a novel recombinant human lactoferrin to enhance the BCG vaccine and protect against alveolar injury during experimental MTB infection in mice. Sialylated and non-sialylated forms of the recombinant human lactoferrin (rhLF), glycoengineered in yeast (Pichia pastoris) and expressing humanized N-glycosylation patterns, were examined for their ability to enhance efficacy of the BCG vaccine in a murine TB model system. Results indicated that the sialylated form of the recombinant human lactoferrin generated increased antigen specific recall responses to BCG antigens. Furthermore, augmented protection was demonstrated using the sialylated lactoferrin adjuvant with BCG, resulting in significant reduction in associated pathology following challenge with virulent organisms.

Interaction of white and pink grapefruit juice with acetaminophen (paracetamol) in vivo in mice.

Grapefruit juice increases bioavailability of a number of drugs because of inhibition of the P-glycoprotein pump and inhibition of intestinal cytochrome P450 3A4 enzyme. However, interaction between acetaminophen (also known as paracetamol in many parts of the world) and grapefruit juice has never been reported. The interaction of grapefruit juice with acetaminophen was examined in an in vivo mouse model. BALB/c mice were fed 200 microL of white grapefruit juice or pink grapefruit juice by oral gavage (three mice in each group) followed by oral delivery of 10, 50, or 100 mg/kg acetaminophen 1 hour later. Blood was withdrawn from the retro-orbital venous plexus at 1 hour and 2 hours after feeding with acetaminophen. The concentrations of acetaminophen in sera of mice were determined by fluorescence polarization immunoassay. White grapefruit juice increased concentrations of acetaminophen in mice both 1 hour and 2 hours after feeding compared to controls. In contrast, pink grapefruit juice increased acetaminophen concentrations 2 hours after feeding compared to controls. Because acetaminophen is almost completely absorbed these effects seems to be related to increased elimination half-life of acetaminophen because of interaction with grapefruit juice.

Immune modulation of macrophage pro-inflammatory response by goldenseal and Astragalus extracts.

Goldenseal (Hydrastis canadenisis) is a native American medicinal plant used as an immune stimulant. Astragalus (Astragalus membranaceus) is a widely used herbal product in China, other Asian countries, and the United States as an immune stimulant to be taken on first clinical signs of infection. In this study, the innate effects of goldenseal and Astragalus on pro-inflammatory cytokines produced by cultured macrophages were examined using two different commercial preparations of goldenseal and Astragalus. Both goldenseal and Astragalus were found to exhibit little to no direct effect on stimulation of mouse macrophages (J774A.1 cells), with only Astragalus able to affect production of tumor necrosis factor (TNF)-alpha when used in high concentrations. However, both goldenseal and Astragalus were able to modify responses from lipopolysaccharide-stimulated macrophages, with identified immunomodulatory effects to reduce production of TNF-alpha, interleukin (IL)-6, IL-10, and IL-12 in a dose-dependent manner. The results obtained indicate that both goldenseal and Astragalus exhibit abilities to modulate macrophage responses during stimulation. Therefore, it is hypothesized that their historical use as therapeutic agents may be due to reduction in the pro-inflammatory response that indirectly leads to limiting of clinical symptoms during infection. Both products differ in their immune stimulatory patterns, offering insight into differential use and therapeutic potential of these products to regulate macrophage immune responses and activation events.

Rapid detection of oleander poisoning by Digoxin III, a new Digoxin assay: impact on serum Digoxin measurement.

We studied the potential for detecting oleander with a new immunoassay (Digoxin III, Abbott Laboratories, Abbott Park, IL) by comparing results with those from the fluorescence polarization immunoassay (FPIA) and Digoxin II assay (Abbott). In aliquots of drug-free serum pools supplemented with pure oleandrin or oleander extract, we observed apparent digoxin values using all 3 immunoassays, but values obtained by the Digoxin III were higher than obtained by the other assays. We also observed significant apparent digoxin values in vivo in serum samples of mice 1 and 2 hours after feeding oleander extract. The average half-life of digoxin-like factors was 1.1 hours. In a serum pool (prepared from patients taking digoxin) supplemented with oleander extract, the observed digoxin values were falsely lowered when measured by the Digoxin II but falsely elevated when measured by the Digoxin III and FPIA. Monitoring free digoxin using the Digoxin III cannot eliminate this interference. Digibind neutralized digoxin-like factors of oleander extract; the effect can be monitored by observing a significant reduction in apparent free digoxin levels in the presence of Digibind as measured in protein-free ultrafiltrate using the Digoxin III. The Digoxin III is highly sensitive for measuring oleander.

Effect of Chinese medicines Chan Su and Lu-Shen-Wan on serum digoxin measurement by Digoxin III, a new digoxin immunoassay.

Chan Su and Lu-Shen-Wan are Chinese medicines that crossreact with digoxin immunoassays. Recently, Abbott Laboratories released a new digoxin immunoassay, Digoxin III. We studied potential interference of Chan Su and Lu-Shen-Wan with the Digoxin III assay by comparing results obtained by using Digoxin II and fluorescence polarization immunoassay, also manufactured by Abbott Laboratories. Aliquots of a drug-free serum pool were supplemented with aqueous extract of Chan Su or Lu-Shen-Wan and apparent digoxin concentrations were measured using all three digoxin assays. Significant crossreactivity of Chan Su and Lu-Shen-Wan was observed with the new Digoxin III assay. Moreover, when mice were fed with Chan Su or Lu-Shen-Wan, significant apparent digoxin concentrations were also observed in the sera of mice using the Digoxin III assay indicating that such interferences are also present in vivo. When serum pools prepared from patients receiving digoxin were further supplemented with Chan Su or Lu-Shen-Wan extract, falsely elevated digoxin values were observed with both Digoxin III and fluorescence polarization immunoassay, but digoxin values were falsely lowered using the Digoxin II assay. For example, when one aliquot of Digoxin Serum Pool 1 containing 0.94 ng/mL of digoxin was supplemented with 5.0 microg/mL of Chan Su extract, the digoxin concentration was falsely elevated to 6.60 ng/mL as measured by the Digoxin III assay and 6.99 as measured by the fluorescence polarization immunoassay assay. In contrast, the observed digoxin value was falsely lowered to 0.72 ng/mL using the Digoxin II assay. Interference of Chan Su and Lu-Shen-Wan in the Digoxin III assay cannot be eliminated by monitoring free digoxin concentrations. Digibind neutralizes digoxin-like immunoreactive components of Chan Su and such effect can be monitored by measuring apparent free digoxin concentrations using the Digoxin III assay. We conclude the both Chan Su and Lu-Shen-Wan significantly interfere with serum digoxin measurements by the new Digoxin III assay.

Lactoferrin enhanced efficacy of the BCG vaccine to generate host protective responses against challenge with virulent Mycobacterium tuberculosis.

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.

Effect of Indian Ayurvedic medicine Ashwagandha on measurement of serum digoxin and 11 commonly monitored drugs using immunoassays: study of protein binding and interaction with Digibind.

CONTEXT: Ashwagandha, a popular Ayurvedic medicine, is now available in the United States. Alkaloids found in this herb have structural similarity with digoxin. OBJECTIVE: To study potential interference of Ashwagandha with serum digoxin measurement by immunoassays. Potential interference was also investigated with immunoassays for 11 other commonly monitored drugs. In addition, interaction of components of Ashwagandha with the Fab fragment of antidigoxin antibody (Digibind) was investigated. DESIGN: Two different brands of liquid extract and 1 dry powdered form of Ashwagandha were used for this investigation. Aliquots of drug-free serum were supplemented with various concentrations of Ashwagandha and apparent digoxin concentrations were measured by 3 digoxin immunoassays. Mice were fed with Ashwagandha and apparent digoxin concentrations were measured 1 and 3 hours after feeding. Potential interference of Ashwagandha with immunoassays of 11 other drugs was also investigated. Interaction of components of Ashwagandha with Digibind was studied in vitro. RESULTS: Significant apparent digoxin concentrations were observed both in vitro and in vivo using the fluorescence polarization immunoassay of digoxin, whereas the Beckman and the microparticle enzyme immunoassay digoxin assay demonstrated minimal interference. Immunoassays of 11 other drugs tested were unaffected. When Ashwagandha extract was added to a serum pool containing digoxin, falsely elevated digoxin value was observed with fluorescence polarization immunoassay, but values were falsely lowered when measured by the microparticle enzyme immunoassay. Digibind neutralized digoxin-like immunoreactive components of Ashwagandha in vitro. CONCLUSIONS: Components of Ashwagandha interfered with serum digoxin measurements using immunoassays. Digibind neutralized free digoxin-like immunoreactive components of Ashwagandha.

Drug-herb interaction: effect of St John's wort on bioavailability and metabolism of procainamide in mice.

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 microg/mL) followed by group A (9.92 microg/mL), whereas group B (7.44 microg/mL) and control group D (7.36 microg/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.