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The ixodicidal activity of the methanolic extracts of Artemisia ludoviciana (Astereceae), Cordia boissieri (Boraginaceae) and Litchi chinensis (Sapindaceae) against two field populations of Rhipicephalus (Boophilus) microplus from the state of Nuevo Leon (NL) and Veracruz (VER) was evaluated. The extract of L. chinensis in the concentration of 150 mg/ml showed efficacies of 100% and 99% against engorged females and mortalities of 98% and 99% against larvae. C. boissieri in the same concentration showed efficacies of 71% and 37% against engorged adults and mortalities of 33.04% and 10.33% against larvae and A. ludoviciana had efficacies of 94% and 83% in adults and mortalities of 89.39% and 89.21% against larvae in both populations respectively. The enzymatic activity of Acetylcholinesterase (AChE), Carboxylesterase (CaE), Glutathione-S-Transferase (GST) and Alkaline Phosphatase (ALP) was measured in both populations of ticks. As a result, a significant difference between both populations was shown, being the VER population the one that exhibited a higher enzymatic activity (p ≤ 0.05). It can be concluded that the methanolic extract of the seed of L. chinensis shows potential ixodicidal activity and can be used as an alternative source of tick control, however, prior characterization, toxicity and formulation studies are necessary.

No presente trabalho, a atividade ixodicida de extratos metanólicos de Artemisia ludoviciana (Astereceae), Cordia boissieri (Boraginaceae) e Litchi chinensis (Sapindaceae) contra Rhipicephalus (Boophilus) microplus foi avaliada em duas populações de campo nos estados de Nuevo León (NL) y Veracruz (VER). O extrato de L. chinensis na concentração de 150 mg/ml, apresentou eficácia de 100% e 99% contra fêmeas ingurgitadas, e taxas de mortalidade de 98% e 99% contra larvas. C. boissieri, na mesma concentração, apresentou eficácia de 71% e 37% contra adultas ingurgitados, e taxas de mortalidade de 33,04% e 10,33% contra larvas e A. ludoviciana apresentou eficácias de 94% e 83% em adultos e mortalidade de 89,39% e 89,21% contra larvas em ambas as populações respectivamente. Por outro lado, a atividade enzimática da Acetilcolinesterase (AChE), Carboxilesterase (CaE), Glutationa-S-Transferase (GST) e Fosfatase Alcalina (ALP) foi medida em ambas as populações de carrapatos. Como resultado, foi apresentada uma diferença significativa entre as populações, sendo a população VER a que apresentou maior atividade enzimática (p ≤ 0,05). Assim, pode-se concluir que o extrato metanólico da semente de L. chinensis apresenta potencial atividade ixodicida e pode ser utilizado como fonte alternativa de controle de carrapatos, porém estudos prévios de caracterização, toxicidade e formulação são necessários.

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RESUMO

The ixodicidal activity of the methanolic extracts of Artemisia ludoviciana (Astereceae), Cordia boissieri (Boraginaceae) and Litchi chinensis (Sapindaceae) against two field populations of Rhipicephalus (Boophilus) microplus from the state of Nuevo Leon (NL) and Veracruz (VER) was evaluated. The extract of L. chinensis in the concentration of 150 mg/ml showed efficacies of 100% and 99% against engorged females and mortalities of 98% and 99% against larvae. C. boissieri in the same concentration showed efficacies of 71% and 37% against engorged adults and mortalities of 33.04% and 10.33% against larvae and A. ludoviciana had efficacies of 94% and 83% in adults and mortalities of 89.39% and 89.21% against larvae in both populations respectively. The enzymatic activity of Acetylcholinesterase (AChE), Carboxylesterase (CaE), Glutathione-S-Transferase (GST) and Alkaline Phosphatase (ALP) was measured in both populations of ticks. As a result, a significant difference between both populations was shown, being the VER population the one that exhibited a higher enzymatic activity (p ≤ 0.05). It can be concluded that the methanolic extract of the seed of L. chinensis shows potential ixodicidal activity and can be used as an alternative source of tick control, however, prior characterization, toxicity and formulation studies are necessary.

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Bacterial infection impairs the healing process, promoting the chronicity of inflammation and wounds. Because antibiotics fail to eradicate bacteria, especially in biofilm form, new therapeutic modalities may be required. In the present study, the effectiveness of bacteriotherapy with Lactobacillus plantarum on infected chronic venous ulcers was investigated and its effects on interleukin (IL)-8 production by cells from the ulcer bed and neutrophils isolated from peripheral blood that were previously challenged in vitro with Pseudomonas aeruginosa and L. plantarum were studied. Topical application of L. plantarum culture to lesions (25-60 cm(2)) of 14 diabetic and 20 non-diabetic patients induced debridement, granulation tissue formation and total healing after 30 days in 43% diabetics and in 50% non-diabetics. No significant differences between the groups were observed. The cells from ulcer beds collected after treatment with L. plantarum for 10 days showed a decrease in the percentage of polymorphonuclear, apoptotic and necrotic cells and an enhancement of IL-8 production. IL-8 production by isolated neutrophils from these patients was compared with that in diabetics without ulcers, as well as normal subjects under basal conditions, and after infection of polymorphonuclear cells with P. aeruginosa preincubated either with or without L. plantarum. The basal values in diabetic and ulcer patients were higher than normal (p <0.001) and were increased by P. aeruginosa infection in normal, diabetics (p <0.001) and non-diabetics with ulcers (p <0.01). Preincubation with L. plantarum decreased IL-8 production in patients with ulcers non-diabetic and diabetic (p <0.001). Lactobacillus plantarum treatment reduced wound bacterial load, neutrophils, apoptotic and necrotic cells, modified IL-8 production and induced wound healing.

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We study the differences between Pseudomonas aeruginosa from an infected wound (clinical strain) and a colony isolated from it. We assessed the in vitro inhibition of these P. aeruginosa biofilms by DNase and filtrate of Lactobacillus plantarum cultures (acid=AF and neutralize=NF) with crystal violet technique. Inhibition by AF was greatest than DNase for clinical and isolated strain (p<0.001) and greatest than NF for clinical (p<0.05) and isolated strain (p<0.001). Using a burn model in mice, we compared the infection producing by clinical and isolated strains in planktonic and biofilm form. Deaths were quantified and the infection was assessed by determining CFU/g of tissue in the lesion, spleen and liver. The infections with planktonic bacteria tended to become systemic and more deadly than biofilm infections. All infected wounds required the same healing period (30 days). These findings were independent of the origin of the bacteria (clinical or colony isolated strain).

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In a previous study we determined that by-products of Lactobacillus plantarum inhibited pathogenicity of Pseudomonas aeruginosa and is effective in the treatment of infected wounds. This study assesses the cytotoxic activity of acetic acid (AA), supernatants of L. plantarum and P. aeruginosa, with and without signal acyl-homoserine-lactones (AHL), and mixtures of both bacterial supernatants on human neutrophils. Cytotoxicity was determined through viability using trypan blue, apoptosis by Annexin V, necrosis by propidium iodide and intracellular pH by SNARF-1. We found that supernatants of L. plantarum caused less cytotoxicity than AA at the same extracellular pH (p<0.05). P. aeruginosa induced a remarkable drop in intracellular pH, which was independent of extracellular pH. This intracellular acidity was correlated with a significant decrease in viability and was higher than supernatants of AHL producing P. aeruginosa (p<0.05). When supernatants were mixed, the quantity of AHL diminished (p<0.001) and the cytotoxic effect induced by P.aeruginosa was ameliorated by L. plantarum supernatant (p<0.001 vs p<0.01). These results are in agreement with the inflammatory in vivo assays determined by intradermal inoculations in Balb/c mice. Our findings will be useful for the formulation of effective and inexpensive products to resolve infected chronic wounds in our hospitals.

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This study evaluated the ability of the probiotic organism Lactobacillus plantarum to inhibit the pathogenic activity of Pseudomonas aeruginosa, both in vitro and in vivo, and investigated the mechanisms involved in such protection. L. plantarum whole cultures, culture filtrates (acid filtrate and neutralised acid filtrate) and isolated, washed cells were tested in vitro for their effects on the production of the P. aeruginosa quorum-sensing signal molecules, acyl-homoserine-lactones (AHLs), and two virulence factors controlled by these signal molecules, elastase and biofilm. All were inhibited by L. plantarum cultures and filtrates, but not by isolated, washed cells. The acid L. plantarum growth medium itself had some inhibitory activity, but the greatest activity was exerted by the whole culture. To test the in-vivo activity of L. plantarum, a burned-mouse model was used in which burns infected with P. aeruginosa were treated with L. plantarum at 3, 4, 5, 7 and 9 days post-infection. Samples from skin, liver and spleen taken after 5, 10 and 15 days demonstrated inhibition of P. aeruginosa colonisation by L. plantarum. There was also an improvement in tissue repair, enhanced phagocytosis of P. aeruginosa by tissue phagocytes, and a decrease in apoptosis at 10 days. These results indicate that L. plantarum and/or its by-products are potential therapeutic agents for the local treatment of P. aeruginosa burn infections.

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Yogurt consumption has been associated with decreased risk of colon cancer. The apoptosis induced by yogurt as a mechanism involved in the inhibition of an experimental intestinal tumor induced by 1,2 dimethylhydrazine (DMH) in BALB/c mice, has been studied. An imbalance between apoptosis and mitosis or cell proliferation is believed to underlie colon cancer development and progression. We determined the effect of yogurt feeding in the induction of apoptosis and the relationship between mitosis and apoptosis in mice injected with the carcinogen DMH and in mice injected with DMH and fed with a diet supplemented with yogurt. The percentage of mitotic and apoptotic cells was analyzed every 2 weeks from week 8 to 16. They were analyzed on histological slices from the large intestine by colchicine, or Tunel test for mitosis and apoptosis determination, respectively. An increase in the mitosis during the first 4 weeks of tumor development with apoptosis negative and severe dysphasia, was observed in animals treated with the carcinogen. In those animals that received DMH and were given yogurt, a moderate cell proliferation with a significant increase in the number of apoptotic cells was determined. The increase in the apoptotic activity was also observed in the group of animals that received only a diet supplemented with yogurt. The results suggest that yogurt would influence the balance between mitosis and apoptosis, which was modified during the carcinogenesis process, by increasing cellular apoptosis. Dietary supplementation of yogurt may play a role in modulating cell proliferation or apoptosis during the development of colorectal carcinoma.

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Health claims of lactic acid bacteria (LAB) used in functional foods and pharmaceutical preparations are based on the capacity of these microorganisms to stimulate the host immune system. In this study, the antigenic effect of LAB (Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus) on the gut immune system of BALB/c mice was evaluated. A dose-dependent increase of the Bcl2 protein was observed with all LAB assayed. Furthermore, the analysis of cytokine-producing cells in the lamina propria of gut showed that TNFalpha and INFgamma values, determined in macrophages cultured from Peyer patches, were enhanced for all the LAB assayed. An important increase of interleukins IL-10 and IL-4 was observed mainly in mice fed with Lactobacillus delbrueckii ssp. bulgaricus or Lactobacillus casei, while a significant induction of IL-2 and IL-12 was only observed with L. acidophilus (P<0.01). These effects were dose dependent. The role of produced cytokines in the balance Th1/Th2 was determined by a systemic antibody response against parenterally injected ovoalbumin. L. casei, L. delbrueckii ssp. bulgaricus and L. acidophilus enhanced the IgG1 response favouring Th2 balance, while L. acidophilus also increased the IgG2a response inducing Th1 balance. S. thermophilus did not influence the balance Th1/Th2. Our studies showed that lactic acid bacteria induce distinct mucosal cytokine profiles showing different adjuvant capacity among them. Thus, selection of probiotic strain with immunological properties must be well defined to influence cytokine expression that favour the claimed immune response.

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In order to grow, solid tumors need to develop new blood vessels. Neoplastic cells secrete growth factors that stimulate angiogenesis and tumor growth. Since Carrageenan acts as in vitro blocking agent which interferes with growth factor-receptor binding, we tested its action in vivo in order to analyze its growth inhibition capability in an experimental murine fibrosarcoma model. Indomethacin was used as a non-steroidal anti-inflammatory agent to neutralize the inflammatory action of Carrageenan. A murine fibrosarcoma was induced with methylcholanthrene in Balb/c mice and maintained by serial passage of tumor cells in mice of the same strain. Tumor volume was evaluated measuring two dimensions and applying the formula V = 0.4 x d2 x D. The mice with tumors were separated into groups; one of them was used as control and the other ones were treated with Indomethacin, Carrageenan and Carrageenan-Indomethacin. Tumor volume was compared between groups using the Student t Test. We demonstrated that Carrageenan and Indomethacin inhibit tumor growth. The inhibitory action of Carrageenan is significantly higher than the antitumoral effect of either Indomethacin or Carrageenan-Indomethacin.

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The induction of the gut mucosal immune response is dependent on the antigen interacting with the M cells of Peyer's patches and with the immune cells associated with this lymphoid organ. In previous studies we showed that the mucosal immunostimulation by LAB varied depending upon the strain being studied. Some of them increased the inflammatory immune response and others enhanced the level of secretory antibody (S-IgA). Our aim was to determine the pathway of the internalisation of LAB strains in the intestine, to provide a basis for understing the different behaviour exhibited by them. The presence of LAB on Peyer's patches or in the immune cells associated with the villi of small intestine was determined using fluorescein-labelled bacteria and by transmission electron microscopy. Mice were dosed orally by intubation, with 101 cells of labelled single strains of Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus. For electron microscopy studies unlabelled bacteria were used. Histological preparations of the small and large intestine were performed 1 hour after administration of the bacteria. Fluorescent bacteria of L. casei and S. thermophilus were found only in Peyer's patches while L. delbrueckii ssp. bulgaricus and L. acidophilus were observed in Peyer's patches and in the small intestine. L. acidophilus was also found in the large intestine. We confirmed these findings by electron microscopy. We determined that for L. casei the pathway of internalisation was via the M and FAE cells of Peyer's patches, while for S. thermophilus, L. acidophilus and L. delbrueckii ssp. bulgaricus the interaction with the immune cells of Peyer's patches was through the follicle associated epithelium (FAE). L. delbrueckii ssp. bulgaricus and L. acidophilus also interacted with the epithelial cells of the small intestine and L. acidophilus with epithelial cells of the large intestine. These results suggest that the different effects of LAB on the mucosal immunostimulation, are related to the different pathways of gut internalisation used to take contact with the immune cells associated with the lamina propria intestinal.

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In order to grow, solid tumors need to develop new blood vessels. Neoplastic cells secrete growth factors that stimulate angiogenesis and tumor growth. Since Carrageenan acts as in vitro blocking agent which interferes with growth factor-receptor binding, we tested its action in vivo in order to analyze its growth inhibition capability in an experimental murine fibrosarcoma model. Indomethacin was used as a non-steroidal anti-inflammatory agent to neutralize the inflammatory action of Carrageenan. A murine fibrosarcoma was induced with methylcholanthrene in Balb/c mice and maintained by serial passage of tumor cells in mice of the same strain. Tumor volume was evaluated measuring two dimensions and applying the formula V = 0.4 x d2 x D. The mice with tumors were separated into groups; one of them was used as control and the other ones were treated with Indomethacin, Carrageenan and Carrageenan-Indomethacin. Tumor volume was compared between groups using the Student t Test. We demonstrated that Carrageenan and Indomethacin inhibit tumor growth. The inhibitory action of Carrageenan is significantly higher than the antitumoral effect of either Indomethacin or Carrageenan-Indomethacin.

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The effect of yogurt on the inhibition of colon tumours induced by 1,2-dimethylhydrazine in BALB/c mice has been studied, and the hypothesis examined that yogurt induces a great reduction in the inflammatory immune response and inhibits tumour growth. Mice were assigned to five experimental groups: a control group fed with a conventional balanced diet, and four other test groups that received yogurt supplements for 2, 5, 7 or 10 consecutive days. At the end of each feeding period, mice were given subcutaneous injections of 1,2-dimethylhydrazine (20 mg/kg) once a week for 8 weeks. After tumour induction, yogurt was given again for 2, 5, 7 or 10 consecutive days each 10 d for 20 weeks. By week 20, 70% of the animals in the control group had developed colorectal tumours. From week 8, there was a considerable infiltration of mononuclear cells into the lamina propria of the large intestine. There was an increase in the number of IgG-producing cells and a slight increase in the IgA-secreting cells, and of CD8+ but not CD4+ T lymphocytes, a high level of beta-glucuronidase activity in the intestinal fluid and leucocytosis with neutrophilia in the blood. However, in the test groups given yogurt tumour growth was inhibited, the effect being more evident with 7 or 10 d treatment. The inflammatory immune response as measured by the characteristics we assessed was also reduced, with an increase in the IgA-secreting cells and in CD4+ T lymphocytes. The blood count was similar to that of normal animals and no colorectal tumours were observed in week 20. We suggest that one of the mechanisms by which yogurt exerts antitumour activity is through its immunomodulator activity, by reducing the inflammatory immune response, which was markedly increased when the carcinogen was administered.

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The effect of giving yogurt supplements to BALB/c mice on the various gut-associated lymphoid cells was studied. Animals were fed for 2, 5, 7 and 10 consecutive days. The different lymphoid cell types were identified and counted by haematoxylin-eosin staining of histological slices. The numbers of cells secreting IgA, IgG and IgM and the numbers of T lymphocytes were determined by direct immunofluorescence. The degree of activation of the intestinal macrophages in the small intestine was assessed by measuring the beta-glucuronidase (EC 3.2.1.31) released into the intestinal fluid, and also by a histochemical method. Throughout the feeding period, there were no histological alterations in the gut, but there was marked cell infiltration, mainly of plasma cells and lymphocytes. The number of macrophages on the small intestine increased significantly after feeding for 2 d, while the beta-glucuronidase activity was only slightly higher that of the controls. After a 7 d feeding period, the number of IgA secreting cells increased, while the values for cells secreting IgM and IgG and for T lymphocytes remained similar to those of the controls. The effect of giving yogurt on lymphoid cells associated with the large intestine was mainly on the numbers of IgA secreting B cells and T lymphocytes, with a marked increase during the whole feeding period in the latter type of cell. Since giving yogurt mainly enhanced the IgA secreting B cells in both small and large intestines, this increase would strengthen the host's defence mechanisms in the intestinal mucosa. Although the number of macrophages was increased, there was no enhancement in their activity, which might have harmed the host by producing an inflammatory response.

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An indirect immunofluorescence assay was carry out to determine the IgM and IgG antibody responses to yeast and mycelial forms of Candida albicans in mice injected with a 5 x 5(5) and 5 x 10(7) live cells suspensions. Prior adsorption of the serum samples with heat-killed blastospores enabled us to follow the specific antimycelial response which were detected considerably later than expected. Slow level of antibodies were obtained within an infection of 5 x 10(5) cell for both antibody classes and for yeast and mycelial forms. When a 5 x 10(7) cell dose was used for inoculation, maximum titers of antibodies to blastospores and mycelium in non-adsorbed sera appeared almost simultaneously (days 15 and 13, respectively). When serum samples from mice infected with the same dose were previously adsorbed with blastospores, the antimycelium antibodies for both types of Igs, were detected delayed during the infection course. In this case the higher titer for IgG appeared on day 33 and on day 23 for IgM. We suggest that the high titer obtained with the blastospore forms for the 5 x 10(7) cell dose may be due to a major immunogenicity of this forms, for to induce an immune response in the host, or that the delay in the antimycelium antibodies detection could be due to that a blastospore form is the predominant in the infection early stages. Implications of this fact for pathogenesis are discussed.

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Growth of a methylcholanthrene-induced fibrosarcoma in BALB/c mice was accompanied by an increase in the activation state of tumour-associated macrophages (TAM), as measured by their FcIgG receptor expression, phagocytic index and beta-glucuronidase levels. All of these parameters were markedly higher in TAM than in peritoneal macrophages (PM) derived from the same animal. On the other hand, PM from tumour-bearing mice showed lower activation parameters than PM from normal animals. We also studied the effect on tumour development of three inhibitors of prostaglandin synthesis: indomethacin, piroxicam and aspirin. Intraperitoneal administration of these drugs during 8 d was followed by the regression of palpable tumours. Indomethacin (90 mg/d) induced 45% regression, while with piroxicam (two 400 mg/d doses and six 200 mg/d doses) and aspirin (1 mg/d) 32% and 30% regressions, respectively, were observed. The growth rate of nonregressing tumours, which had reached different volumes by the end of the treatment, was delayed to a similar extent by the three anti-inflammatory non-steroidal drugs (NSAID). With respect to TAM, the treatment did not induce any significant change in their activation state, though both piroxicam and indomethacin increased slightly the TAM number. In contrast, NSAID administration was followed by a remarkable increase in the activation parameters of PM when compared with PM from tumour-bearing mice receiving no treatment. Indeed, these parameters were in some cases higher than those of PM from normal mice. The leukocytosis (60,000/microliters) with neutrophilia (80%) induced by tumour growth on peripheral blood leukocytes (PBL) was reversed by the treatment to values close to normal, in parallel with the reduction of tumour size. A drop in haematocrit was also noted which was most probably a consequence of tumour growth rather than of the treatment. This study reveals that the three NSAID tested have a remarkable antitumour activity, which correlates with the restoration of PM activity and PBL values.

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In this investigation is demonstrated that the nurse needs, in order to carry out a successful work, to learn about the composition of the population of the area under her care. In Patana, a Maisí municipality with 165 inhabitants, 93 male and 72 females, prevailing ninth grade school level and an initial low hygienic culture level, which is considered as regular at the present time, intestinal parasitism and malnutrition are the main diseases in the zone. It is well demonstrated that there is a wide relationship between hygienic conditions at home and community and the incidence of the fore-mentioned diseases and that the educational work performed by the nurse in the area under her care exerts a positive influence, but the support of grassroot mass organizations is required.

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