Molecular detection of Hepatozoon spp. in non-hematophagous bats in Brazil

Even though Hepatozoon spp. has been molecularly detected in several wild animals in Brazil, there is no report on the occurrence of Hepatozoon spp. DNA in bats in Brazil. This study aimed at detecting Hepatozoon, in addition to ectoparasites, in non-hematophagous bats sampled in central-western Brazil using blood smears, hematoxylin-eosin (HE)-staining liver/spleen preparations and molecular and phylogenetic techniques. A total of 135 spleen, 127 liver, and 133 blood samples were collected from 135 non-hematophagous bats from 12 different species in two different sites in Campo Grande, Mato Grosso do Sul state, in the Brazilian Cerrado region. Spleen and blood DNA samples were submitted to two conventional PCR protocols for Hepatozoon spp. based on 18S rRNA. No Hepatozoon spp. gamonts or meronts were observed in blood smears and HE-stained-liver preparations, respectively. While none of the spleen samples was positive for Hepatozoon spp. in the PCR assays, 5 (3 %) blood samples contained 18S rRNA Hepatozoon DNA, including 2/37 (5 %) Artibeus lituratus, 2/32 (6 %) A. planirostris, and 1/23 (4 %) Platyrrhinus lineatus. Out of 5 bats positive for Hepatozoon spp., 3 were parasitized by either Macronyssidae/Spinturnicidae mites or Streblidae flies. BLAST analysis showed that the sequences detected in bats had >99 % identity with Hepatozoon sequences detected in amphibians and reptiles from Brazil, including Hepatozoon caimani detected in Caiman crocodilus. The phylogenetic inferences estimated by the Maximum Likelihood and Bayesian methods clustered the Hepatozoon sequences detected in Brazilian bats with those detected in reptiles and amphibians.     

The stabilizing effect of gelatin on dilutions of various tuberculins

Adsorption of highly diluted tuberculin to the inner surface of containers is particularly pronounced for acid-precipitated PPD, and certain such preparations are routinely issued with the non-ionic detergent Tween 80 as a stabilizing agent. It has been shown, however, that Tween 80, besides its anti-adsorptive effect, also has a depressive in vivo effect, especially on tuberculin reactions that would have been weak even for a test without Tween. The authors have shown in a previous report that gelatin (0.1%) also has an anti-adsorptive effect, without apparently modifying the reaction in vivo.     

Relative antifertility activity of three vaginal contraceptive products in the rabbit: relationship to in vitro data.

Three types of vaginal contraceptive preparations were used to investigate the feasibility of utilizing the rabbit as a model for the study of vaginal contraceptives. DELFEN^* Contraceptive Foam, DELFEN Contraceptive Cream and PRECEPTIN^* Contraceptive Gel and corresponding vehicle preparations were administered intravaginally to rabbits. Following mating, the reduction in mean implant number was used as an index of contraceptive potency. Dose response curves indicated that the contraceptive potency rank was DELFEN Contraceptive Foam>DELFEN Contraceptive Cream>PRECEPTIN Contraceptive Gel (ED50s = 0.017, 0.39 and 2.8 ml, respectively). The ED50s for the corresponding vehicles were 12.7, 2.1 and 4.5 ml. The relationships of these in vivo data to in vitro end points were also studied using rabbit and human semen. In a titration test, one gram of DELFEN Contraceptive Foam, DELFEN Contraceptive Cream or PRECEPTIN Contraceptive Gel immobilized sperm contained in 121, 25 and 10 ml of rabbit semen, respectively, whereas sperm contained in 1.6, 8.7 and 6.4 ml were immobilized by the corresponding vehicles. Human sperm were found to be more susceptible than rabbit sperm to immobilizing agents contained in these products. The rabbit does appear to provide a model for the in vivo evaluation of relative potencies of vaginal contraceptive preparations.     

Bionomics and control of Culex pipiens fatigans Wied. in Ceylon

The climatic and housing conditions which favour the breeding of Culex pipiens fatigans in Ceylon, and its life-cycle and resting-habits, are described. The results of the treatment of breeding-places (mainly catch-pits) and the interior of houses with various insecticides of specific action on larvae or adults, in different preparations and concentrations, are given, and suggestions are made for the control of the mosquito on this island. The fundamental measure for successful control is efficient sanitation, but until this can be achieved, application of larvicides seems to be the method of choice.     

The roles of tobacco,alcohol, and diet in the etiology of upper alimentary and respiratory tract cancers

This paper reviews the epidemiological evidence associating alcohol and tobacco consumption as well as diet as risk factors for upper alimentary and respiratory tract cancer. The possible association of alcohol-related nutritional deficiencies (especially zinc and vitamin A) in this process is discussed. Experimental studies on the metabolism and carcinogenicity of N-nitrosopyrrolidine (NPYR) and N′-nitrosonornicotine (NNN), two cyclic nitrosamines which are potential environmental carcinogens, are presented, and it is shown that microsomes isolated from hamsters consuming ethanol are able to perform the initial metabolic step (α-hydroxylation) at greater rates than similar preparations from control animals. We conclude that increased attention should be paid to the possibility that alcohol-related deficiencies in micronutrients are a contributing factor in the etiology of upper alimentary and respiratory tract cancers.     

Concentration and separation of erythrocytes infected with Plasmodium falciparum by gradient centrifugation.

Concentration of infected erythrocytes was achieved in cell suspensions derived from long-term culture of Plasmodium falciparum growing asynchronously in human erythrocytes. This new procedure involves the slow centrifugation (at 33 g) of erythrocyte suspensions through 5% Ficoll solutions. Mature asexual erythrocytic forms are preferentially retained in the gradient solution (top fraction). After further gradient centrifugation of these parasitized cells, the concentration of mature forms is increased 15- to 31-fold and a mature form parasitaemia of 71-81% is obtained in the final erythrocyte suspension. Furthermore, at least 75% of the total number of the mature forms can be retrieved by this method. Parasitized cells that are not retained in the gradient are sedimented to the bottom of the tube (bottom fraction) and consist predominantly of ring forms. Parasites from both the top and bottom fractions are viable and have been used to initiate short-term synchronous cultures. By providing purified parasite preparations, this simple procedure will facilitate immunological, chemotherapeutic, and biochemical studies with P. falciparum.     

Refining HPV 16 L1 purification from E. coli: Reducing endotoxin contaminations and their impact on immunogenicity

HPV 16 L1 capsomeres purified from Escherichia coli represent a promising and potentially cost-effective alternative to the recently licensed VLP-based vaccines for the prevention of cervical cancer. However, recombinant protein preparations from bacteria always bear the risk of contaminating endotoxins which are highly toxic in humans and therefore have to be eliminated from vaccine preparations. In this study, we measured the LPS concentration at various stages of the purification of HPV 16 L1 from E. coli and determined that it enhances the immunogenicity of HPV 16 VLPs and capsomeres. We confirmed the immunogenicity of the L1 capsomeres in TLR4^−/− mice without the enhancing effect of the LPS and then elaborated a suitable protocol using Triton X-114 phase separation for the removal of LPS without any significant protein loss or influence on the structural integrity of the particles. The LPS-free capsomeres purified from E. coli induced neutralizing L1-specific antibodies. Our results demonstrate the excellent potential of capsomeres as an economically interesting alternative vaccine to prevent cervical cancer that could be made available in developing countries.     

Growth of protozoa in tissue culture. III. Trypanosoma cruzi

The technique used for cultivation of exoerythrocytic forms of P. gallinaceum has been applied to the growth of Trypanosoma cruzi also. A suspension of trypanosomes from the blood of an infected mouse was added to cultures of rat embryo. During the first 2 days great numbers of trypanosomes were present in the culture fluid, not particularly associated with the cells. Then they gradually became fewer until by about the 12th day they were rare or absent. Later small numbers reappeared in the culture fluid and persisted there until the end of the culture. Stained preparations made after. 6 days or more showed great numbers of intracellular parasites in all stages between the rounded leishmanoid form and the mature trypanosome form. The parasite occurred in cardiac muscle fibres, in macrophages and in elongated cells with processes (probably reticulo-endothelial cells). Cultures have been maintained for 59 days. As described by Meyer (1942) T. cruzi could also be grown in cultures from the brain of chick embryo.This technique offers favourable conditions for study of the intracellular development of T. cruzi and of the action of drugs upon it.     

The effectiveness of low dose slow release aspirin as an antiplatelet agent.

An open, randomized, parallel group study of three different aspirin preparations was carried out. The objective of this study was to compare their ability to inhibit the production of thromboxane A2 (measured as thromboxane B2 [TXB2]) from platelets. Three aspirin preparations were studied: Acetard 300 mg, Acetard 100 mg and Platet 100 mg. The study was conducted in 45 healthy adult volunteers and treatment continued once daily for 7 days. The results of the TXB2 assay show that the administration of all three treatment preparations produced a rapid drop in TXB2 levels of all volunteers. The baseline TXB2 level was reduced by 95% for all groups by day 3. Analysis by day showed a significant difference (P < 0.05) between treatments at day 1, with Acetard 100 mg having higher TXB2 levels than the other two groups. Analysis of changes from baseline showed a significant reduction (P < 0.05) in TXB2 levels at Days 1 to 14 for all three groups. Platelet aggregation also showed a significant reduction, being reduced to 10% of control at 7 days. It then reverted back to baseline by 28 days for all treatment groups. In conclusion, low dose aspirin is very effective as an antiplatelet agent and in a slow release form loses none of its patency.     

Comparison of the effects of three different (-)-hydroxycitric acid preparations on food intake in rats: response

A response to Louter-van de Haar J, Wielinga PY, Scheurink AJ, Nieuwenhuizen AG: Comparison of the effects of three different (-)-hydroxycitric acid preparations on food intake in rats. Nutr Metabol 2005, 2:23     

Preparations from Various Organs of Sea Buckthorn (Elaeagnus rhamnoides (L.) A. Nelson) as Important Regulators of Hemostasis and Their Role in the Treatment and Prevention of Cardiovascular Diseases

Numerous studies on the chemical composition of various organs of sea buckthorn (Elaeagnus rhamnoides (L.) A. Nelson) have found the plant to be a rich source of vitamins, phenolic compounds, amino acids, fatty acids, and micro- and macro-elements. Furthermore, other studies on preparations from various organs have found them to have significant anti-cancer, anti-ulcer, and hepatoprotective properties, as well as various antibacterial and antiviral activities. This paper reviews the current literature concerning the effect of different sea buckthorn preparations, i.e., extracts and fractions with various chemical contents, on hemostasis, and their positive role in the treatment and prevention of cardiovascular diseases. It also sheds new light on the mechanisms involved in their action on hemostasis both in vivo and in vitro. For these studies, biological materials, including blood platelets, plasma, and blood, were isolated from healthy subjects and those with cardiovascular risk factors. In addition, it describes the cardioprotective potential of commercial products from different organs of sea buckthorn.     

Influence of Tilia tomentosa Moench Extract on Mouse Small Intestine Neuromuscular Contractility

Functional gastrointestinal disorders (FGIDs) are characterized by abdominal pain, bloating and bowel disturbances. FGID therapy is primarily symptomatic, including treatment with herbal remedies. Flower extract of Tilia tomentosa Moench (TtM) is occasionally used as an anti-spasmodic in popular medicine. Since its effect on intestinal response is unknown, we evaluated the influence of TtM extract on small intestine contractility. Ileal preparations from C57BL/6J mice were mounted in organ baths to assess changes in muscle tension, following addition of TtM extract (0.5–36 μg/mL) or a vehicle (ethanol). Changes in contractile response to receptor- and non-receptor-mediated stimuli were assessed in ileal preparations pretreated with 12 μg/mL TtM. Alterations in the enteric nervous system neuroglial network were analyzed by confocal immunofluorescence. Increasing addition of TtM induced a marked relaxation in ileal specimens compared to the vehicle. Pretreatment with TtM affected cholinergic and tachykininergic neuromuscular contractions as well as K⁺-induced smooth muscle depolarization. Following incubation with TtM, a significant reduction in non-adrenergic non-cholinergic-mediated relaxation sensitive to Nω-Nitro-L-arginine methyl ester hydrochloride (pan-nitric oxide synthase inhibitor) was found. In vitro incubation of intestinal specimens with TtM did not affect the myenteric plexus neuroglial network. Our findings show that TtM-induced intestinal relaxation is mediated by nitric oxide pathways, providing a pharmacological basis for the use of TtM in FGIDs.     

Clinical trials of artemisinin and its derivatives in the treatment of malaria in China

Since 1979 several derivatives of artemisinin have been synthesized and studied in China. Artemisinin suppositories, artesunate (oral or parenteral), intramuscular artemether and dihydroartemisinin tablets have all proved rapidly effective. In all, 2352 patients (2150 with Plasmodium falciparum and 202 with P. vivax) have been included in clinical trials from our centre. All preparations have been well tolerated. These drugs have now replaced chloroquine and quinine for the treatment of malaria in China.     

Sublethal physiological effects of chlorinated paraffins on the flounder, Platichthys flesus L.

Exposure of flounder, Platichthys flesus L., to two different chlorinated paraffin preparations, Witachlor 149 and Hülz 70C, caused sublethal effects on hematology and intermediary metabolism, together with changed activities in xenobiotic and steroid metabolizing enzymes, especially in female fish. The changes observed must be considered important and urge for further studies as the chlorinated paraffins are used in steadily increasing amounts and constitute a possible ecological threat.     

The rapid isolation from human blood of concentrated,white-cell-free preparations of Plasmodium falciparum

The sucrose gradient centrifugation method has been applied to representative samples of human (Gambian) blood infected with ring, trophozoite, schizont and gametocyte stages of P. falciparum in order to assess quantitatively the efficiency of recovery, white cell removal and the degree of enrichment of the infected cell fraction. Maximal white cell removal was 90%. (a.v.) Average infected cell recoveries varied with the level of white cell contamination, namely 47% (2–5% WBC), 34% (1–2% WBC) and 24% (< 1% WBC); infected cells were enriched 2 · 9-fold on average, and up to 22-fold in the case of gametocytes.Preliminary attempts to prepare free parasites by nitrogen cavitation of infected cells showed that disruption took place at much lower pressures than those required to break normal red cells.Gel diffusion analyses showed that the most highly purified infected cell preparations retained the full precipitinogenic spectrum of the original crude preparation.     

A comparison of the effectiveness of two freeze-dried BCG vaccines against Mycobacterium leprae in mice

It has previously been shown that BCG vaccination affords mice protection against Mycobacterium leprae and most of this work was carried out using fresh liquid preparations of a strain originating from S. R. Rosenthal some years ago. In the present study, the effectiveness of the Japanese and Glaxo freeze-dried BCG vaccines was tested since such preparations would make it possible to administer vaccine of standard viability anywhere in the world, including leprosy-endemic areas. The Japanese and Glaxo vaccines, and the usual fresh liquid preparations, were administered in equivalent amounts to mice, which were then challenged with Myco. leprae. All the vaccines provided distinct protection. It was not possible, however, to say which vaccine was most effective because their optimal activities were not manifested at comparable times.     

Chitin synthetase as a target for the design of anti-fungal agents

In Saccharomyces cerevisiae cells permeabilized with toluene—ethanol, the activity of chitin synthetase, an important target for anti-fungal agents was compared with that of particulate extracts of the enzyme. The two preparations gave very similar results with regard to enzyme kinetics: chitin synthetase exhibited an allosteric behavior that could be shifted to hyperbolic Michaelis—Menten kinetics by the addition of 42 mM N-acetylglucosamine; the K_m of chitin synthetase for UDP-N-acetylglucosamine was 0.5 mM for both preparations. Polyoxin D strongly inhibited chitin synthetase in both preparations, whereas amphotericin B did not inhibit the enzymatic activity. Diflubenzuron exhibits an original mode of action, inhibiting the activation of chitin synthetase zymogen by trypsin, although it is not, by itself, a trypsin inhibitor. An analog of diflubenzuron, which is not active in vivo on insects, did not show any activity in our system either. Therefore, the permeabilized cells described herein are an interesting biological preparation for the screening of potential anti-fungal agents which target chitin synthetase.     

Effectiveness of locally inoculated antirabies serum and gamma-globulin in rabies infection of mice

In an attempt to determine the specific local action of antirabies serum and antirabies gamma-globulin preparations in vivo, experiments were carried out on mice infected with fixed rabies virus in the pad of the foot and then given inoculations of either of these preparations in the same and in the opposite foot at various intervals thereafter.     

Efficacy of novel lipid-formulated whole bacterial cell vaccines against Mycobacterium avium subsp. paratuberculosis in sheep

Mycobacterium avium subsp. paratuberculosis [MAP], the causative agent of enteric Johne's disease, incurs significant economic losses to the livestock industry. Prophylactic vaccination can be employed as a control means, however mineral oil-based vaccines currently in practice have limited efficacy, produce strong antibody responses that confound serological diagnostic testing, and cause severe injection site reactions. In the present study, the safety and efficacy of a commercial mineral oil-adjuvanted vaccine (Gudair™) was compared with novel parenteral-route vaccines in sheep; these comprised live or heat-killed (HK) whole cell preparations of MAP strain 316F, formulated into a food-grade lipid vaccine delivery matrix. Subcutaneous administration of lipid-formulated live or HK 316F-induced significantly fewer adverse injection site reactions than Gudair™; adverse injection site reactions were eliminated altogether by intraperitoneal (i.p.) injection of lipid-formulated live 316F. Injections of lipid-formulated 316F-induced significant peripheral blood cell-mediated immune (CMI) responses in the absence of antibody, while Gudair™-induced strong antibody and CMI reactivity. Vaccinated and non-vaccinated control sheep were challenged via oral inoculation of a virulent MAP isolate, and disease progress was monitored for 16 months, followed by necropsy. All vaccine regimes reduced the overall pathological grading of biopsied intestinal tract (IT) tissues; among these, only Gudair™ promoted a significant reduction in the incidence of histopathological IT lesions, while only i.p. injection of lipid-formulated live 316F significantly reduced the incidence of gross IT lesions. All lipid-formulated vaccines (but not Gudair™) significantly reduced the incidence of bacteriological culture-confirmed MAP infection. This study identifies a new vaccination strategy against Johne's disease in sheep using conventional MAP vaccine strains formulated in a metabolisable lipid delivery matrix.     

Clearance of Tritrichomonas foetus in experimentally infected heifers protected with vaccines based on killed-T. foetus with different adjuvants

Tritrichomonas foetus is a flagellated protozoan that causes a sexually transmitted disease in cattle. Trichomonosis is characterized by early abortions, subfertility and a significant decrease in productivity. Vaccine preparations containing whole T. foetus can reduce the time of residence of the pathogen in the host cervix after experimental infection. Here, T. foetus vaccines prepared with different adjuvants were tested, in parallel with a commercial vaccine, for their efficacy to clear the infection. The median time for clearance of infection was 69 days in non-immunized animals, 55 days in animals treated with aluminum hydroxide, 41 days with oil-in-water or saponin based vaccines or with a commercial vaccine and 27 days in animals treated with saponin plus aluminum hydroxide. A slight increase in the risk of T. foetus clearance from the genital tract was found with the saponin based vaccine (hazard ratio, 2.52; 95% confidence interval, 1.03–6.17) or the commercial vaccine (hazard ratio, 2.61; 95% confidence interval, 1.07–6.38). A significant increase in the risk of T. foetus clearance was found with the combination of saponin plus aluminum hydroxide based vaccine (hazard ratio, 5.12; 95% confidence interval, 2.04–12.83).