Dietary modulation of avian coccidiosis

During the past several years, our laboratory has been investigating the anticoccidial activities of various natural products that have potential use as dietary supplements for coccidiosis control. Sources of fats containing high concentrations of n-3 fatty acids such as menhaden oil and flaxseed oil and flaxseed, when added to starter rations and fed to chicks from one day of age, effectively reduce lesions caused by the caecal parasite Eimeria tenella, but not lesions caused by Eimeria maxima. Our results are consistent with reports of effects of diets high in n-3 fatty acids on other protozoan parasites which suggest that the state of oxidative stress induced by these diets in the cells of both host and parasites is responsible for their parasitic actions. Artemisinin, a naturally occurring (Artemisia annua) endoperoxide and effective antimalarial significantly lowers lesions from E. tenella when given at low levels as a feed additive. The mechanism of its action is also considered to involve induction of oxidative stress. Diets supplemented with 8 p.p.m. gamma-tocopherol (abundant in flaxseeds) or with 1% of the spice tumeric, reduce mid-small intestinal lesion scores and improve weight gains during E. maxima infections. These compounds may exert their anticoccidial activity because they are effective antioxidants. Betaine, a choline analogue found in high concentrations in sugar beets, improves nutrient utilisation by animals under stress. When provided as a dietary supplement at a level of 0.15% it has enhanced the anticoccidial activity of the ionophore, salinomycin. Betaine may act as an osmoprotectant whereby it improves the integrity and function of the infected intestinal mucosa. In in vivo studies, betaine plus salinomycin significantly inhibit invasion of both E. tenella and E. acervulina. However, subsequent development of E. acervulina is inhibited more effectively with this combination treatment than development of E. tenella.     

Dietary modulation of carcinoma development in a mouse model for human familial adenomatous polyposis

Familial adenomatous polyposis (FAP) is caused by a dominant mutation in the adenomatous polyposis coli (APC) gene. Individuals with FAP progressively develop adenomas and carcinomas of the colon and rectum. We developed a mouse model for this disorder by genetically modifying the Apc gene. The resulting mice Apc1638 progressively develop neoplasms in the colon and remainder of the gastrointestinal tract. In this study when Apc1638 mice were fed a Western-style diet, they developed an increased incidence of the end point of carcinomas and number of invasive tumors. The findings therefore demonstrated dietary modulation of carcinoma incidence in mice with a targeted mutation providing a model for the study of gene-environment interactions in cancer.     

Dietary interventions of human carcinogenesis

The diet is a complex mixture that is associated with approximately 30% of human cancer in the U.S. Extensive laboratory studies indicate that the diet is composed of many mutagens/carcinogens as well as antimutagens/anticarcinogens. Overwhelming evidence from epidemiological studies indicates that a diverse diet that is high in fruits and vegetables and low in certain fats, along with moderate caloric intake and exercise, is most closely associated with reduced cancer risk. Dietary intervention studies using complex food items (fruits, vegetables, and fats) support these epidemiological observations; dietary interventions using single compounds (vitamins, antioxidants, etc.) have generally not. Estimates suggest that appropriate dietary changes could reduce the percentage of deaths due to prostate, colorectal, pancreatic, and breast cancer by >/=50%.     

Leukotriene activity modulation in asthma

Leukotrienes constitute a class of inflammatory mediators synthesised from arachidonic acid, a product of cell membrane metabolism. Synthesis occurs in the 5-lipoxygenase enzyme pathway, which produces several species of leukotrienes, each with characteristic biological activities. With regard to asthma, the leukotrienes are particularly important because of their ability to directly and potently mediate bronchoconstriction; in addition, they specifically stimulate the secretion of mucus into the airways and the extravasation of fluids and proteins into the airway tissues, both of which contribute to airway obstruction. A number of antileukotriene agents have been developed with the goal of modulating the inflammatory process in various disease states. These agents fall into 2 general classes: leukotriene receptor antagonists and leukotriene synthesis inhibitors. Results of antileukotriene agents in preclinical and clinical trials indicate that antileukotriene agents attenuate the response to challenges with inhaled leukotrienes, cold air, exercise, aspirin and allergen; in addition, they have shown efficacy in clinical asthma and have not been associated with serious adverse effects. Although results to date indicate that these medications are well tolerated and effective in the treatment of asthma, the recent approval by the FDA of 2 antileukotriene agents will give physicians further insight into how patients with asthma respond to them.     

Does warfarin enhance platelet activity?

Salmonella infection can cause appendicitis by direct invasion of the appendix, or can mimic appendicitis by causing mild inflammation of the appendix, ileum, or lymph nodes. Clinical presentation and radiologic and laboratory evaluation may not distinguish the extent of underlying pathology. This case of a child with an atypical presentation of Salmonella who underwent diagnostic laparotomy illustrates the overlap of enteric infections and acute appendicitis. A literature review confirms the variety of clinical scenarios of patients with suspected appendicitis and Salmonella-positive cultures. We conclude that enteric infection should be considered in children with atypical presentations of appendicitis, and that the knowledge that Salmonella can progress to appendicitis should guide management if signs and symptoms of appendicitis develop.     

Dietary fatty acid modulation of events associated with mouse skin tumor promotion

Increasing levels of dietary corn oil have been correlated with inhibition of 12-O-tetradecanoylphorbol-13-acetate-(TPA) promoted skin tumorigenesis in mice (Leyton et al. Cancer Res. 51, 907-915, 1991). This study was undertaken to assess the effects of dietary corn oil on several events associated with tumor promotion. Three semipurified diets containing 15% (wt/wt) total fat with increasing levels of linoleate (0.8%, 4.5%, and 8.4%) supplied by corn oil were fed to mice for at least four weeks. Although incorporation of linoleate into epidermal phosphatidylcholine increased with increasing amounts of dietary corn oil, the elongated desaturated product of linoleate, arachidonate, was similar or decreased slightly in mice fed the three diets. Minimal activity of delta 6-desaturase, the rate-limiting enzyme in the conversion of linoleate to arachidonic acid, was found in the epidermis compared with the liver, suggesting that linoleate is not converted to arachidonic acid in the skin. Subcellular distribution of protein kinase C was altered in mice fed 0.8% linoleate, where 69% of protein kinase C activity was in the cytosol compared with 78% and 74% for groups fed 4.5% and 8.4% linoleate, respectively. Activation of partially purified protein kinase C isolated from mouse epidermis by linoleate was significantly lower (p < 0.01) than that isolated by arachidonic acid. TPA-induced vascular permeability was significantly greater (p < 0.05), whereas hyperplasia 48 hours after TPA treatment was significantly lower, in mice fed the 8.4% linoleate diet. However, TPA induction of ornithine decarboxylase activity did not appear to be significantly modified by dietary linoleate. These data suggest that cellular processes associated with carcinogenesis are affected by the level of dietary linoleate.     

Membrane microviscosity and human platelet function

An increased sensitivity to epinephrine-induced aggregation has been observed both in platelets obtained from patients with type IIa hyperlipoproteinemia and in normal platelets following incubation with cholesterol-rich lecithin dispersions. We have reported previously that the membrane fraction of platelets is enriched with cholesterol relative to phospholipid under each of these conditions. To further explore the effect of cholesterol on platelet membranes, we have examined the fluidity (microviscosity) of whole platelets and platelet subcellular fractions using a hydrophobic fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene (DPH), under conditions in which the cholesterol-to-phospholipid mole ratio (C/PL) of platelets was varied by incubation with various cholesterol-lecithin sonicated dispersions. The C/PL of platelets directly influenced the rotational diffusion of DPH, as indicated by changes in fluorescence polarization. This was reflected in an increase in microviscosity at 37 degrees C (ETA37) from 2.84 P in normal platelets to 4.06 P in platelets with a 118% increase in C/PL. Conversely, platelets with a 43% decrease in C/PL had a 13% decrease in eta37. A strong correlation (r = 0.94) existed between C/PL and eta37 throughout this entire range. However, C/PL had no effect on the excited-state fluorescence lifetime of DPH. Both C/PL and eta37 were lower in isolated platelet membranes than in the platelet granule fraction. When platelets were incubated for 20 h with cholesterol-rich dispersions, there was an increase in C/PL and eta37 in both the membrane and granule fractions. However, this occurred more rapidly in membranes so that, at 5 h (a time when an increased sensitivity of whole platelets to epinephrine is evident), membrane C/PL had increased 55% and eta37 had increased 42%, whereas granule C/PL and eta37 had changed minimally. Cholesterol-rich platelets and subcellular fractions had a lower fusion (or flow) activation energy for viscosity (deltaE), reflecting a higher degree of order, and the converse was true in cholesterol-poor platelets. Moreover, a strong negative correlation existed between the percent change in deltaE and the percent change in eta37 induced either by cholesterol incorporation or depletion. These data demonstrate that cholesterol influences the fluidity and the degree of order within the hydrophobic core of platelet membranes. Changes induced in these physical properties by an excess of cholesterol relative to phospholipid may underlie the abnormal reception or transmission of the aggregation stimulus in cholesterol-rich platelets.     

Dietary soy isoflavones inhibit in-vivo constrictor responses of coronary arteries to collagen-induced platelet activation

In effusion cytology the distinction of reactive mesothelia from metastatic carcinoma cells may be a diagnostic challenge. Immunocytochemistry using antibodies suitable to detect epithelial cells must be considered carefully due to limited sensitivity and specificity of these antibodies. Efficient results in histological differential diagnosis of malignant mesothelioma versus lung-adenocarcinoma applying a novel antiserum against the calcium binding protein calretinin inspirated us to investigate the value of anti-calretinin antibody in effusion cytology combined with an epithelial marker. Cytoslides prepared by cytocentrifugation from 42 malignant and 65 reactive effusion specimens were immunostained using antibodies against calretinin and the epithelial marker Ber-EP4. Positive immunoreaction for calretinin in normal and reactive mesothelial cells was noted in 93% of the cases, whereas immunoreaction for calretinin was completely negative in the metastatic cells in 95% of the malignant effusions. Metastatic carcinoma cells were detected with anti-Ber-EP4 in 83% of malignant effusions. Non-specific positive reactions for Ber-EP4 in single mesothelial cells were observed in 16% of all cases and, moreover, frequently with macrophages or neutrophilic granulocytes. Our results demonstrate high sensitivity and specificity of anti-calretinin antibody for mesothelial cells in effusion specimens. They support its application to improve the diagnostic reliability of epithelial markers, especially because anti-calretinin antibody could be helpful in the assessment of false positive and false negative reactions of epithelial markers.     

Growth modulation of human tumor cells by a growth-inhibiting activity derived from tumorigenic V79 Chinese hamster cells

A growth-inhibiting activity was identified in supernatants of the neoplastic V79 Chinese hamster cell line based on its ability to inhibit the proliferation of the same cell line. The partially purified activity, provisionally termed "growth inhibiting factor" (GIF) activity, inhibited the growth of a wide variety of human tumor cells, but not various normal human fibroblasts. This species-nonspecific activity was reversible, saturable, and highly potent in tumorigenic cell lines, and was noted in both monolayer culture and in soft agar. The inhibitory activity of GIF was also exhibited in a chemically defined serum-free medium supplemented with insulin and transferrin. GIF activity was stable to acid, heat, trypsin, and dithiothreitol but sensitive to alpha-chymotrypsin. The pattern of growth modulation by GIF on V79 cells was apparently different from those exhibited by bifunctional peptides such as transforming growth factor-beta, tumor necrosis factor-alpha, and interleukin-1-alpha. In addition, GIF activity cannot be ascribed to these cytokines based on the physicochemical and immunologic properties. Although GIF has yet to be purified to homogeneity, these data suggest that GIF might be a novel growth regulator which has a critical role in regulating growth of V79 cells. The growth modulation of tumor cells by this tumor-derived growth inhibiting activity suggested the presence of an autocrine growth regulatory mechanism even in tumor cells.     

Role of surface glycoproteins in human platelet function

Glycoproteins present at the external surface of cells probably play specific roles in cellular function. Increasing evidence suggests that the glycoproteins span the plasma membrane with the bulk of the bound carbohydrate asymmetrically distributed on the outer surface. Micellar association of glycoproteins in membranes leads to pore formation and functional roles in transport through the membrane, while surface glycoproteins have been shown to be enzymes, to determine cell specificity and contribute to the cell surface change. The platelet plasma membrane contains 3 major glycoproteins, glycoproteins I, II and III as characterized in order of their decreasing molecular weight. Glycoprotein I appears to have the highest sialic acid content and to give rise to a platelet specific acidic macroglycopeptide on trypsin digestion. Specific glycoprotein abnormalities in the platelets of patients with Glanzmann's thrombasthenia suggest that the glycoproteins play a role in the mechanism of platelet aggregation. A much reduced content of glycoprotein I in the platelets of 2 patients with the Bernard Soulier syndrome may be associated with their defective adhesion to subendothelium and indicates a possible relationship on the platelet surface with the von Willebrand factor protein. Preliminary evidence suggests that in common with other plasma membranes the platelet membrane has a fluid structure and that the organization of the glycoproteins on the platelet surface is extremely sensitive to stimuli and susceptible to change.     

Heterogeneity of antibody response to human platelet transfusion

To study the antibody response to human platelet transfusions, nine thrombocytopenia patients with bone marrow failure were given 6 U (3X10(11)) of random platelet concentrates twice a week. Before transfusion, none of the patients had preexisting antibodies detectable with lymphocytotoxicity, platelet aggregation, or capillary leukoagglutination techniques. After receiving 18-78 U of platelets, they became refractory to further transfusions of random platelets and alloantibodies were detectable. Two patterns of antibody response could be identified. In three patients, the sera were not lymphocytotoxic with a panel of standard cells in which all the known HLA antigens in the first and second series were represented at least once. Yet, they caused platelet aggregation with 30, 24, and 60%, respectively, of a donor population studied. The aggregating activities were inhibited by antihuman IgG but not by antihuman IgA or antihuman IgM antiserum. The aggregating antibodies could be absorbed out with donor platelets but not lymphocytes or granulocytes. Antibodies from two of these patients aggregated platelets of their respective siblings matched for both HLA haplotypes. Transfusion of platelets from these two siblings did not increase the platelet count while platelets obtained from aggregation-negative donors did. The sera from the remaining six patients were lymphocytotoxic with 15-100% of the panel of standard cells. They also had aggregating antibodies, which could be absorbed out by both platelets and lymphocytes, suggesting that they were HLA antibodies. These data suggest that the development of platelet-specific antibodies may play an important role in the immunological rejection of isologous platelets, and should be considered in the selection of donors for patients who are refractory to platelets from random donors.     

Triflavin potentiates the antiplatelet activity of platelet activating factor receptor antagonist on activated neutrophil-induced platelet aggregation

In this study, specific platelet activating factor (PAF) receptor antagonist ginkgolide B (BN52021) was tested for its antiplatelet activity in zymosan activated polymorphonuclear neutrophil-induced platelet aggregation. Triflavin was also tested for its antiplatelet activity compared with PAF receptor antagonist. Triflavin, an Arg-Gly-Asp-containing disintegrin purified from venom peptide inhibited platelet aggregation by interfering with the interaction of fibrinogen with the glycoprotein IIb/IIIa complex. Furthermore, we also report an efficient high resolution method for quantitative analysis of PAF using high-performance capillary electrophoresis (HPCE). The supernatant of polymorphonuclear neutrophils after their activation by opsonized zymosan induces the aggregation of washed rabbit platelets. In rabbit platelets, BN52021 (100-1000 microM) only partially inhibited activated polymorphonuclear neutrophil-induced platelet aggregation, and its maximal inhibition was estimated to be about 79%. Triflavin also partially inhibited platelet aggregation about 82% induced by activated polymorphonuclear neutrophils. Furthermore, after treatment with a combination of triflavin (0.26 microM) with various concentrations of BN52021 (4-1000 microM), the inhibitory effect of platelet aggregation was almost completely. This inhibition was greater than that produced by the individual drugs alone. These results indicate that a combination of glycoprotein IIb/IIIa complex and PAF receptor antagonist could completely inhibit activated polymorphonuclear neutrophil-induced platelet aggregation. In addition, the amount of PAF released from zymosan (6 mg/ml)-activated polymorphonuclear neutrophils was accurately calculated about 11.8+/-1.5 ng/10(6) cells, and did not further increase even at a high concentration of zymosan (10 mg/ml). These results suggest that PAF play a major role in the interaction between platelets and polymorphonuclear neutrophils. This interaction may be important in the pathogenesis of thrombosis and inflammatory diseases. Our present findings support the hypothesis that combination therapy with glycoprotein IIb/IIIa complex antagonists and PAF receptor antagonists may represent a new approach to the treatment of ischemic disorders.     

Opioid and GABA modulation of accumbens-evoked ventral pallidal activity

The purpose of this work has been to establish the pattern of prenatal growth and normal development of the digestive tract and annex glands during goat embryonic stages. 21 embryos with ages ranging from 14 to 34 days (1.69 to 5.90 cm CR) as determined by registering the mating time, were obtained by cesarean section. This material was histologically processed to obtain complete serial sections of the stomatodaeum, foregut, midgut, hindgut and cloaca. In this work, it is chronologically described the morphogenesis and histogenesis of the mouth, hypophysis, pharynx and its derivatives, stomach, intestine, liver, pancreas and cloaca. The results obtained establish chronological comparisons with the development of the lamb and gives information on the unitary origin of the gastric compartments in ruminants.     

Pharmacological modulation of human cardiac Na+ channels

Pharmacological modulation of human sodium current was examined in Xenopus oocytes expressing human heart Na+ channels. Na+ currents activated near -50 mV with maximum current amplitudes observed at -20 mV. Steady-state inactivation was characterized by a V1/2 value of -57 +/- 0.5 mV and a slope factor (k) of 7.3 +/- 0.3 mV. Sodium currents were blocked by tetrodotoxin with an IC50 value of 1.8 microM. These properties are consistent with those of Na+ channels expressed in mammalian myocardial cells. We have investigated the effects of several pharmacological agents which, with the exception of lidocaine, have not been characterized against cRNA-derived Na+ channels expressed in Xenopus oocytes. Lidocaine, quinidine and flecainide blocked resting Na+ channels with IC50 values of 521 microM, 198 microM, and 41 microM, respectively. Use-dependent block was also observed for all three agents, but concentrations necessary to induce block were higher than expected for quinidine and flecainide. This may reflect differences arising due to expression in the Xenopus oocyte system or could be a true difference in the interaction between human cardiac Na+ channels and these drugs compared to other mammalian Na+ channels. Importantly, however, this result would not have been predicted based upon previous studies of mammalian cardiac Na+ channels. The effects of DPI 201-106, RWJ 24517, and BDF 9148 were also tested and all three agents slowed and/or removed Na+ current inactivation, reduced peak current amplitudes, and induced use-dependent block. These data suggest that the alpha-subunit is the site of interaction between cardiac Na+ channels and Class I antiarrhythmic drugs as well as inactivation modifiers such as DPI 201-106.     

Priming reveals attentional modulation of human motion sensitivity

Although recent fMRI and single unit recording studies have shown that attention modulates neural activity in motion sensitive areas of extrastriate cortex, these approaches cannot reveal qualitative or quantitative effects of attention on perception of motion. To investigate this, we asked observers to select one of two orthogonal directions in a brief, transparent dot display (prime) and then measured their sensitivity to global directional motion in a second uni-directional dot display (probe) presented a short time later. When probe direction matched the attended prime direction, sensitivity was degraded. But, when probe direction matched the ignored prime direction, sensitivity was enhanced, even though both components were of equal physical strength. Sensitivity was unchanged for directions opposite to either previously seen direction. Neither sensory adaptation nor opponent direction mechanisms can account for these data. Rather, processes initiated by visual selection must underlie these dramatic changes in motion sensitivity.     

Gemfibrozil enhances platelet activity in patients with combined hyperlipoproteinemia

We have examined the pharmacokinetic interaction between isradipine and lovastatin in six male and six female, healthy, normotensive, human subjects after a single dose and after treatment for 5 days. The isradipine plasma concentrations were determined by a radioimmunoassay and the lovastatin serum concentrations by gas chromatography-mass spectrometry (GC/MS) and by the inhibition of the 3-hydroxy-3-methylglutaric-coenzyme reductase activity. We found that the apparent serum concentrations of lovastatin were 4- to 6-fold higher in the reductase-inhibition assay than the GC/MS assay, suggesting that the bulk of the reductase inhibition is due to active metabolites. The peak and the time-to-peak concentrations were unaffected by the treatments, either after the first dose or after continued administration. In male subjects, after repeated doses of isradipine, the lovastatin area under the time-concentration curves (AUCs) decreased by 40% as determined by the GC/MS assay (P < .001) and 20% as determined by the reductase-inhibition assay (P < .0022). In the female subjects, isradipine treatment decreased the lovastatin AUCs as determined by the GC/MS assay, but this was not statistically significant due to a high variance. Furthermore, in the female subjects, isradipine had no effect on the lovastatin AUCs as determined by the reductase-inhibition assay. Because the lovastatin peak and the time-to-peak concentrations were unaffected by isradipine treatment, the decreased lovastatin AUCs were probably not due to altered intestinal absorption. More likely, because lovastatin has a high hepatic clearance, the decreased AUCs seen after isradipine treatment could be due to increases in the clearance of lovastatin secondary to increased hepatic blood flow.     

Structure/function correlation of spermine-analogue-induced modulation of peptidyltransferase activity

The typing of human platelet antigens (HPA) can be useful in many clinical situations such as neonatal alloimmune thrombocytopenia, post-transfusion purpura, and platelet transfusion refractoriness. The fluorescent-based single-strand conformation polymorphism (F-SSCP) technique is a fast and convenient way to perform HPA genotyping. Universal sequences from phage M13 were introduced at both ends of specific PCR-products by using 5'-tailed primers. A short second round of PCR with universal primers coupled to Cy-5 enabled the PCR-products to be fluorescently labelled. F-SSCP was performed by gel electrophoresis on an automated fluorescent DNA analyser. Genotyping of the three major HPA systems carried by the GP IIb-IIIa complex showed the F-SSCP technique to be accurate and reliable. A single gel procedure has been sufficient to detect HPA genetic polymorphisms tested to date. Neither restriction enzyme, radioactive material, nor any other hazardous chemicals such as ethidium bromide were required. This technique enabled us to genotype HPA-1, -3 and -4 alleles easily and to diagnose materno-fetal incompatibility in a rare alloantigenic system. F-SSCP is a promising technique for the detection of new mutations and/or DNA polymorphisms on a large scale.