Content validity of the Rodeo-SCAT.

The purpose of this study was to establish the content validity of the Rodeo SCAT for the sport of rodeo and bull riding. The study design was comprised of expert consensus and content validation. A modified Ebel procedure was employed to content validate the rodeo SCAT. Content validation using this method includes experts agreeing on the importance of each item that comprises the rodeo SCAT. This 3-stage process involved: 1) face validation by a local committee: 2) initial expert consensus measurement via distance; and 3) a face-to-face discussion for items that did not originally achieve 80% consensus of the group. Experts were chosen from the Canadian Professional Rodeo Sport Medicine Team (Canada) and the Justin Sports Medicine Team (USA). 27 out of a total possible 68 items achieved 80% consensus in the second stage. In the third stage, 4 of the 68 items were removed with consensus from the expert group. All remaining items achieved 80% consensus for inclusion. In summary, the rodeo SCAT is content valid and thus, appropriate for use in the sport of rodeo context or environment.

Prevalence of food allergy in Taiwan: a questionnaire-based survey.

AIM: Food allergy is common in children and adults, and could be potentially fatal in minor groups. It is important for physicians to identify the prevalence of food allergies and to recognise common food allergens to make precise diagnosis and choose correct therapeutic approaches. METHODS: We used a nationwide, cross-sectional, random questionnaire-based survey to estimate the self-reported and expert-screened prevalence of food allergies and to identify the common food allergens in Taiwan. In this study, the perceptional diagnosis of food allergies was screened by physicians according to descriptions of convincing symptoms and medical recordings; in the meantime, non-allergic adverse reactions to foods, including food intolerance or food avoidance, were clarified. RESULTS: A total of 30 018 individuals who met the inclusion criteria was evaluated, and 6.95% of them were diagnosed as victims of food allergies. The prevalence was 3.44% in children under 3 years of age, 7.65% in children aged 4-18 years and 6.40% in adults respectively. About 77.33% of the food allergy population had experienced recurrent allergic attacks. Systemic reactions happened about 4.89% in food allergies group. The most commonly reported food allergen in Taiwan is seafood, including shrimp, crab, fish and mollusc. In addition, mango, milk, peanuts and eggs were also important food allergens in the general population; while milk, shellfish, peanuts and eggs were common in children. CONCLUSIONS: Less than 10% of the Taiwan population suffers from food allergy with different allergic symptoms to variable food allergens in different age groups.

Thymic peptide modulates glutathione redox cycle and antioxidant enzymes in macrophages.

The effect of a 6-kDa thymic peptide (TP) on the oxidative burst of the murine macrophage cell line J774 was determined. TP was incubated with J774 cells at 37 degrees C and 5% CO2 for 18 h, oxidative burst was triggered by zymosan, and chemiluminescence was amplified by luminol and measured in an automated luminometer. TP exhibited a concentration-dependent suppression of oxidative burst. To study the mechanisms involved in TP's suppression of oxidative burst, its effect on the glutathione redox cycle and antioxidant enzymes was investigated. J774 cells were incubated with varying concentrations of TP for 18 h, washed, resuspended in phosphate-buffered saline, and sonicated to obtain cell lysate. Biochemical assays were performed with the lysate. TP was shown to increase the level of glutathione, and activities of glutathione-peroxidase and glutathione-reductase, indicating its ability to modulate the glutathione redox cycle. The activity of antioxidant enzyme superoxide dismutase was enhanced significantly by TP treatment while catalase activity remained unaffected. These results suggest that TP possesses an antioxidant property and therefore may be involved in the regulation of free radical mediated reactions.

Tumor-specific T-lymphocyte cytotoxicity enhanced by low dose of C. parvum.

Three routes of immunotherapy with Corynebacterium parvum (CP) on an ascitic Friend virus-induced leukemia were evaluated. Only the intraperitoneal route, which provided optimal contact between CP and tumor cells, showed prolonged mean survival time. Greatest effectiveness was obtained with multiple injections of CP at weekly intervals and with small initial tumor load. Of particular interest was that lower dosages of CP (5 and 25 micrograms) gave longer protection than dosages of 50 and 250 micrograms. Using in vitro 125I-iododeoxyuridine release assay, these lower dosages were shown to selectively enhance the cytotoxicity associated with T lymphocytes, whereas higher dosages appeared to primarily augment the activity of phagocytes. Moderate natural killer cell activity was observed with both the lower and higher dosages of CP. Data from this study indicate that route of administration, dosage of CP, and size of tumor burden are crucial variables determining optimal response to immunotherapy.

Chemiluminescence in a macrophage cell line modulated by biological response modifiers.

We have studied a murine macrophage cell line, J774, and found these cells capable of a zymosan-triggered chemiluminescent oxidative burst. Such activity was enhanced by preincubation with Corynebacterium parvum (CP), bacillus Calmette-Guerin, and lipopolysaccharide (LPS). Under similar conditions, CP and LPS were shown to enhance J774-mediated tumor cell lysis. We have also demonstrated that murine interferon alpha + beta rendered J774 cells more sensitive to the actions of CP and LPS. These results indicate that J774 cells may be useful for the in vitro evaluation of biological response modifiers as well as the study of oxygen radical production by macrophages.

S-allyl cysteine inhibits activation of nuclear factor kappa B in human T cells.

Reactive oxygen species are involved in signal transduction pathways leading to nuclear factor kappa B (NF-kappa B) activation which has been implicated in the regulation of gene transcription. We recently reported that a garlic compound, S-allyl cysteine (SAC), protects bovine pulmonary artery endothelial cells from oxidant injury induced by hydrogen peroxide (H2O2). In this study we determined the effects of SAC on NF-kappa B activation in human T lymphocytes (Jurkat cells) induced by tumor necrosis factor alpha (TNF- alpha) and H2O2. Activated NF-kappa B in nuclear extracts was measured by an electrophoretic mobility shift assay using 32P-labeled probe. SAC consistently exhibited a dose-dependent inhibition of NF-kappa B activation induced by both TNF-alpha and H2O2. Supershift with specific antibodies to NF-kappa B subunits confirmed that the inducible retarded bands observed in the EMSA and p65-p50 heterodimer of the NF-kappa B/Rel protein. Our data suggest that SAC may act via antioxidant mechanisms to block NF-kappa B activation in Jurkat cells.

Ginkgo biloba attenuates oxidative stress in macrophages and endothelial cells.

The action of Ginkgo biloba extract (GBE) as an antioxidant was studied using various models of oxidative stress in macrophages and vascular endothelial cells. GBE was incubated with murine macrophages (J774) at 37 degrees C and 5% CO2 for 1 h; oxidative burst was triggered by zymosan. The intensity of fluorescence was measured directly in 96-well plates using a computerized microplate fluorometer at 485 nm excitation and 530 nm emission. GBE exhibited both time- and concentration-dependent suppression of oxidative burst. Confluent monolayers of bovine pulmonary artery endothelial cells (PAEC) were preincubated with different concentrations of GBE for 16 h, washed, and then exposed to an organic oxidant tert-butyl hydroperoxide (tBHP) for 2 h. Lipid peroxidation products of PAEC were determined by measuring thiobarbituric acid-reactive substances (TBARS). Cell injury was assessed by measuring the release of intracellular lactate dehydrogenase (LDH), and cell viability was determined by the methylthiazol tetrazolium (MTT) assay. tBHP increased production of TBARS in PAEC. Preincubation with GBE inhibited the increase of TBARS induced by tBHP. GBE protected biomembranes from oxidative injury by decreasing intracellular LDH leakage from PAEC. MTT assay showed that GBE minimized loss of cell viability induced by oxidative injury. The extensive antioxidant effect of GBE may be valuable to the prevention and treatment of various disorders related to free radical-induced pathology.

An automated micro-fluorometric assay for monitoring oxidative burst activity of phagocytes.

A micro-fluorometric assay using 2',7'-dichlorofluorescein diacetate (DCFH-DA) to monitor oxidative burst (OB) in phagocytes has been developed. This assay is based on the oxidation of nonfluorescent DCFH-DA to highly fluorescent 2',7'-dichlorofluorescein (DCF) both intracellularly and extracellularly. A murine macrophage cell line, J774, and a human monocytic cell line, Mono Mac 6, were used as models. The cells were harvested from tissue culture flasks, washed, counted and adjusted to desired concentrations. They were then dispensed into a 96-well flat-bottom tissue culture plate. After adding DCFH-DA and an agent eliciting OB, the plates were incubated in 5% CO2 at 37 degrees C for various periods. The intensity of fluorescence was measured directly in the wells of the tissue culture plate with the cells in situ using a computerized microplate fluorometer at 485 nm excitation and 530 nm emission. This assay provided a rapid measurement of oxidative burst of phagocytes. The automated micro-fluorometric assay may be suitable for screening the immunomodulating activities of various biological and pharmacological substances.

A rapid and simple microfluorometric phagocytosis assay.

A microfluorometric method for phagocytosis study has been developed using fluorescein conjugated Escherichia coli K-12 particles. This technique is based on the uptake of fluorescent particles and quenching of extracellular fluorescence at the end of the assay. A murine macrophage cell line, J774, was used as a phagocyte model. The cells were harvested from tissue culture flasks and adjusted to 1 x 10(6) cells/ml. They were then dispensed into a 96-well tissue culture plate, 100 microliters/well, and incubated at 37 degrees C in 5% CO2 for 1 h to allow cells to adhere to the bottom of the wells. The culture medium was aspirated and 100 microliters of fluorescent E. coli particles suspended in Hanks' buffer were added. The plates were further incubated for various time periods. Buffer solution in the wells was removed by aspiration. Extracellular fluorescence was then quenched by adding 100 microliters of trypan blue (250 micrograms/ml, pH 4.4). The dye was removed after 1 min. The intensity of fluorescence associated with intracellular fluorescent particles was measured directly in the wells using a computerized microplate fluorometer at 485 nm excitation and 530 nm emission. This assay provided a rapid and objective measurement of phagocytosis activity. Using a cultured cell line and a 96-well microtiter plate format, this assay can facilitate the screening of a large number of various biological and pharmacological substances for their modulating effects on phagocytosis.

A simple fluorometric assay for the determination of cell numbers.

A sensitive fluorometric assay was developed for the enumeration of cells in microtiter plates. This assay is based on the fluorescence enhancement of propidium iodide (PI) upon binding with double-stranded nucleic acids. This fluorochrome is compatible with a wide range of reagents commonly used in the laboratory, thus washing the cells before staining is not necessary. PI, together with Triton-X 100 and EDTA, was added directly to the cell culture. After 16-18 h incubation at room temperature, intensity of fluorescence was determined with a micro-plate fluorometer. This quick and simple method is sensitive for as little as 1.95 x 10(3) mononuclear leukocytes, and provides a linear correlation (r = 0.999) between cell number and fluorescence up to 1 x 10(6) cells. Since PI has a large Stokes shift with excitation wavelength at common visible range and emission wavelength far out in the red region of the spectrum, it allows simultaneous detection of DNA and other fluorescent compounds such as calcein and fluorescein. This assay may prove to be a valuable alternative for cell number determination.

Effect of allixin, a phytoalexin produced by garlic, on mutagenesis, DNA-binding and metabolism of aflatoxin B1.

Allixin, a phytoalexin isolated from garlic, was examined for its effects on aflatoxin B1(AFB1)-induced mutagenesis using Salmonella typhimurium TA100 as the bacterial tester strain and rat liver S9 fraction as the metabolic activation system. The effects of allixin on the binding of [3H]AFB1 to calf thymus DNA and on the formation of metabolites of [3H]AFB1 were also determined. Allixin showed a dose-related inhibition of Histidine+ revertants induced by AFB1. Allixin at 75 micrograms/ml inhibited [3H]AFB1 binding to calf thymus DNA and reduced formation of AFB1-DNA adducts. In addition, allixin exhibited a concentration-dependent inhibition of the formation of organosoluble metabolites and the glutathione conjugates of [3H]AFB1. The data indicate that the effect of allixin on AFB1-induced mutagenesis and binding of metabolites to DNA may be mediated through an inhibition of microsomal P-450 enzymes. Allixin may thus be useful in the chemoprevention of cancer.

Chinese medicinal herbs modulate mutagenesis, DNA binding and metabolism of benzo[a]pyrene 7,8-dihydrodiol and benzo[a]pyrene 7,8-dihydrodiol-9,10-epoxide.

Oldenlandia diffusa(OD) and Scutellaria barbata (SB) have been used in traditional Chinese medicine for treating liver, lung and rectal tumors. In this study, the effects of aqueous extracts of these two herbs on benzo[a]pyrene 7,8-dihydrodiol. (BaP 7,8-DHD) and benzo[a]pyrene 7,8-dihydrodiol-9,10-epoxide (BPDE)-induced mutagenesis using Salmonella typhimurium TA100 as the bacterial tester strain and rat liver 9000 x g supernatant (S9) as the metabolic activation system were assessed. We also determined the effects of these two herbs on BaP 7,8-DHD and BPDE binding to calf thymus DNA. Organosoluble metabolites of BaP 7,8-DHD and water-soluble conjugates of BaP 7,8-DHD and BPDE were analyzed by high-performance liquid chromatography (HPLC) and alumina column liquid chromatography. Mutagenesis assays revealed that these two herbs produced a significant concentration-dependent inhibition of histidine-independent (His+) revertants induced by BaP 7,8-DHD and BPDE. OD and SB also inhibited BPDE-induced mutagenesis in a concentration-dependent manner in the absence of S9. SB had a greater inhibitory effect than OD. SB significantly inhibited BaP 7,8-DHD and BPDE binding to DNA while OD significantly enhanced DNA binding of both compounds. OD and SB inhibited the formation of organosoluble metabolites of BaP 7,8-DHD and decreased the formation of water-soluble conjugates of BaP 7,8-DHD and BPDE. However, the fraction of the total radioactivity in the water-soluble conjugates present as sulfate and glutathione was increased by OD and SB. Glucuronide fraction was decreased. The results of this study affirm our previous work suggesting that these two Chinese medicinal herbs possess antimutagenic properties and further suggest that they act as blocking agents through a scavenging mechanism.

Modulation of cytochrome P-450IA1-mediated mutagenicity, DNA binding and metabolism of benzo[a]pyrene by Chinese medicinal herbs.

Oldenlandia diffusa (OD) and Scutellaria barbata (SB) have been used in traditional Chinese medicine for treating liver, lung and rectal tumors. We previously showed that they inhibited mutagenesis, DNA binding and metabolism of benzo[a]pyrene (BaP) and aflatoxin B1 (AFB1) bioactivated by Aroclor 1254-induced rat hepatic S9. The purpose of this study was to investigate the effects of OD and SB on the cytochrome P-450IA1-mediated mutagenicity of BaP in Salmonella typhimurium TA100 using beta-naphthoflavone (beta NF)-induced rat hepatic S9. We also determined the effects of OD and SB on cytochrome P-450IA1-linked ethoxyresorufin O-deethylase (EROD) activity in beta NF-induced hepatic microsomes. In addition, we studied the effects of these two herbs on BaP metabolite binding to calf thymus DNA and using high performance liquid chromatography (HPLC) we investigated the effects of OD and SB on the metabolism of BaP by beta NF-induced S9. Our experimental results showed that OD and SB inhibited the mutagenicity of BaP in the presence of either non-induced or beta NF-induced S9. SB significantly inhibited BaP binding to DNA. These effects correlated with the inhibition of cytochrome P-450IA1-linked EROD activity in beta NF-induced microsomes and with an inhibition of beta NF-induced S9 mediated metabolism of [3H]BaP as determined by HPLC. These results suggest that OD and SB may possess antimutagenic activity by inhibiting P-450IA-mediated metabolism of BaP.

Pycnogenol protects neurons from amyloid-beta peptide-induced apoptosis.

Neuronal apoptosis is one of the pathological features of Alzheimer's disease (AD). Morphological pathology reveals that neuronal apoptosis is associated with senile plaques containing amyloid-beta peptide (Abeta) in AD brains. Reactive oxygen species (ROS) has been proposed to be involved in the apoptotic mechanism of Abeta-mediated neurotoxicity. In the present study, using a rat pheochromocytoma (PC12) cell line, we investigated the effect of Pycnogenol (PYC), a potent antioxidant and ROS scavenger, on Abeta(25-35)-induced apoptosis and ROS generation. We used vitamin E, a known antioxidant agent, to verify the effect of PYC. Abeta(25-35)-induced apoptosis in PC12 cells was demonstrated by: (1) a dose-dependent loss of cell viability; (2) a time- and dose-dependent increase in the apoptotic cells; (3) an induction of DNA fragmentation; and (4) an increase in caspase-3 activity and cleavage of poly (ADP-ribose) polymerase (PARP). Our data showed that a significant increase in ROS formation preceded apoptotic events after PC12 cells were exposed to Abeta(25-35). We further found that PYC not only suppressed the generation of ROS but also attenuated caspase-3 activation, DNA fragmentation, PARP cleavage, and eventually protected against Abeta-induced apoptosis. Vitamin E also suppressed cell death and caspase-3 activation induced by Abeta(25-35). Taken together, these results suggest that ROS may be involved in Abeta-induced apoptosis in PC12 cells. They further suggest that PYC can reduce apoptosis, possibly by decreasing free radical generation in PC12 cells.

Herpes zoster during varicella.

A 4-year-old boy had varicella infection. Two days later vesicular lesions clustered within the left 10th thoracic dermatome. Varicella-zoster virus IgM antibody in serum was positive. He was diagnosed with varicella infection combined with herpes zoster. This is the first case report in the medical literature.

Modulation of hematoporphyrin derivative-sensitized phototherapy with corynebacterium parvum in murine transitional cell carcinoma.

The interaction of photodynamic therapy (PDT) with hematoporphyrin derivative (Hpd) and immunotherapy with Corynebacterium parvum (CP) was studied in a murine transitional cell carcinoma (MBT-2) model. C3H/He mice were transplanted subcutaneously in the hind limb with 2.5 X 10(5) tumor cells. One day after transplantation, mice were randomized into groups to receive saline (control), PDT, CP 25 micrograms, CP 250 micrograms, CP 25 micrograms + PDT, and CP 250 micrograms + PDT. PDT was administered by intraperitoneal (IP) injection of Hpd (12.5 micrograms/g body weight), followed twenty-four hours later by photoirradiation. CP was given intralesionally at the same time as IP injection of Hpd (24 hours before photoirradiation). A low dose of CP (25 micrograms) was shown to enhance the effect of PDT while PDT reduced the benefit obtained with high dose of CP (250 micrograms). In a second series of experiments, CP (250 micrograms) treatment after photoirradiation was shown to give significantly greater benefit than CP treatment before photoirradiation. The study thus indicates that the effectiveness of combined immunophototherapy is dependent on the sequence of the combination and its intricate relationship with the dosage of CP. The enhancement of PDT by low dose of CP in this model suggests the usefulness of this combined immunophototherapy in enhancing tumor control and in lessening deleterious side effects.

Inhibition of dexamethasone-induced cytochrome P450-mediated mutagenicity and metabolism of aflatoxin B1 by Chinese medicinal herbs.

Oldenlandia diffusa (OD) and Scutellaria barbata (SB) have been used in traditional Chinese medicine for treating liver, lung and rectal tumours. We previously showed that they inhibited mutagenesis, DNA binding and metabolism of aflatoxin B1 (AFB1) and benzo(a)pyrene (BaP) bioactivated by Aroclor 1254-induced rat S9. The purpose of this study was to investigate the effects of OD and SB on the mutagenicity of AFB1 in Salmonella typhimurium TA100 using dexamethasone (DXM)-induced rat hepatic S9, on cytochrome P450-linked aminopyrine N-demethylase (APND) activity in DXM-induced hepatic microsomes and on the metabolism of AFB1 by DXM-induced S9 using high-performance liquid chromatography (HPLC). The experimental results showed that OD and SB consistently inhibited the mutagenicity of AFB1 bioactivated by either non-induced or DXM-induced S9. These effects correlated with the inhibition of cytochrome P450-linked APND activity in DXM-induced microsomes and with an inhibition of DXM-induced S9 mediated metabolism of [3H]AFB1 as determined by HPLC. Since DXM treatment has been associated with an induction of the CYP3 enzyme family, these results suggest that OD and SB may possess antimutagenic and antitumorigenic activity towards AFB1 through an inhibition of CYP3-mediated metabolism of AFB1.

Antioxidant effects of fructosyl arginine, a Maillard reaction product in aged garlic extract.

The amino-carbonyl (Maillard) reaction of amino acids with sugars is a nonenzymatic browning reaction that takes place during the processing, cooking, and storage of foods. Maillard reaction products (MRPs) have been shown to possess interesting chemical and biological properties including antimutagenic and antioxidant activity. In this study, we determined the antioxidant effects of fructosyl arginine (Fru-Arg), a MRP in aged garlic extract. Low density lipoprotein (LDL) was incubated with Cu(2+) at 37 degrees C and 5% CO(2) for 24 hours, which resulted in an increase of thiobarbituric acid reactive substances (TBARS) indicating lipid peroxidation. Coincubation of Cu(2+) with Fru-Arg and LDL resulted in a significant inhibition of TBARS formation. Pulmonary artery endothelial cells (PAEC) were exposed to 0.1 mg/mL oxidized LDL (Ox-LDL) at 37 degrees C and 5% CO(2) for 24 hours. Lactate dehydrogenase (LDH) release, as an index of cell membrane damage, and TBARS were measured. Ox-LDL caused an increase of LDH release and TBARS formation. Pretreatment of PAEC with Fru-Arg inhibited these changes. Murine macrophages were incubated with Ox-LDL, and the release of peroxides was measured using a fluorometric assay. Ox-LDL caused an increased release of peroxides. Coincubation of macrophages with Fru-Arg and Ox-LDL inhibited the release of peroxides dose-dependently. In a cell free system, Fru-Arg was shown to scavenge hydrogen peroxide. These data suggest that Fru-Arg is a potent antioxidant, and thus may be useful for the prevention of atherosclerosis and other disorders associated with oxidative stress.

Neuropeptide Y receptor subtypes.

Neuropeptide Y (NPY) is an amidated 36-amino acid peptide with a wide distribution in the central and peripheral nervous system. It can evoke numerous physiological responses by activating specific receptors. Studies using NPY analogs in various model systems and cell types demonstrate different orders of ligand potency and receptor binding affinity. These studies suggest the existence of multiple subtypes of NPY receptors. NPY has been described to bind to at least three different receptors, Y1, Y2 and Y3. NPY has also been shown to interact with sigma receptor in vivo and in vitro. There are indications that more subtypes might exist. Ligand binding studies reveal that Y1, Y2 and Y3 receptors are all G-protein coupled. It is not yet confirmed whether the sigma receptor that interacts with NPY is G-protein coupled. Some studies show that NPY receptors may interact with other classical receptors, including alpha- and beta-adrenoceptors and cholinergic receptors. In the case of alpha- and beta-adrenoceptors, the receptor-receptor interaction is possibly via a pertussis toxin-sensitive G-protein. NPY receptors are coupled to various signal transduction mechanisms including inhibition of adenylate cyclase, and stimulation or inhibition of increases in intracellular Ca2+. Specific links between individual NPY receptor subtype and a particular signal transduction pathway are not established.

Thymic peptide protects vascular endothelial cells from hydrogen peroxide-induced oxidant injury.

Oxidant injury of the vascular endothelium is considered an early event in the pathogenesis of atherosclerosis. In this study, the antioxidant effect of a 6-kDa thymic peptide (TP), isolated from calf thymus, was investigated in vitro using vascular endothelial cells. Confluent monolayers of bovine pulmonary artery endothelial cells (PAEC) were preincubated with different concentrations of TP for 24 h, washed, and then exposed to hydrogen peroxide (H2O2) for 2 or 4 h. Cell injury was assessed by measuring cell viability with methylthiazol tetrazolium (MTT) assay, and by determining the release of intracellular lactate dehydrogenase (LDH). Lipid peroxidation products of PAEC were monitored as malondialdehyde (MDA) with a thiobarbituric acid fluorometric assay. H2O2 (120 or 240 microM) incubated with PAEC decreased cell viability, increased LDH release, and elevated MDA production. Preincubation of PAEC with TP (25-150 micrograms/ml) before H2O2 exposure significantly increased cell viability, decreased LDH release, and reduced MDA production. These results demonstrate that TP can protect vascular endothelial cells from oxidant injury. The data thus suggest that TP may be useful for the prevention and/or treatment of atherosclerosis, and further suggest that immune modulating agents may directly or indirectly influence the functions of vascular endothelium.