A fraction containing kaempferol-3,4'-dimethylether from Larrea divaricata Cav. induces macrophage activation on mice infected with Candida albicans.

Larrea divaricata Cav. is a plant growing in South America. Both the infusion and a derived fraction (F1) of L. divaricata have proved to have immunomodulatory properties. Moreover, F1 can activate macrophages obtained from mice infected with Candida albicans. In this work, F1 was administrated to infected animals, and the state and type of activation of resident macrophages were studied. Results showed that F1 was able to activate macrophages obtained from infected mice by both classical and alternative pathways, probably by inducing a translocation of nuclear factor kappa-B. F1 increases not only the lysosomal activity of macrophages but also the production of phagosomal superoxide anion as a consequence of the activation of the Nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) complex. F1 induced an increase in the macrophage capacity to kill the fungus, which was reflected in a decrease in the levels of colonization of organs. A main flavonoid, kaempferol-3,4'-dimethylether, was identified in F1 by HPLC. This compound increased in vitro production of nitric oxide in heat-killed C. albicans-stimulated macrophages. The flavonoid could thus be considered one of the responsible molecules mediating the overall effects of F1 on the immune system in infected animals.

Macrophages activation by a purified fraction, free of nordihydroguaiaretic acid (NDGA), from Larrea divaricata Cav. as a potential novel therapy against Candida albicans.

Larrea divaricata Cav. (jarilla) is a plant with well-documented applications in Argentinean folk medicine. In order to determine if the treatment with a purified fraction named F1 was capable to maintain a state of priming of macrophages after 15 days of mice infection with Candida albicans. Infected and uninfected mice were used. The effect of F1 on: cytosolic protein levels, apoptosis, phagocytosis, reactive oxygen species production, nitric oxide (NO), cell activity, lysosomal activity and the tissue fungal burden were studied. The results showed that F1 increased macrophages yeast phagocytosis and reactive oxygen species and NO production. All these effects were related to a decrease of cell activity and possible apoptosis. In conclusion, it was observed that F1 could induce a state of long-term activation of macrophages, since we observed increased activity of macrophages 15 days after infection, and it could be related to the elimination of C. albicans. These data may suggest that F1 fraction could be useful against disseminated candidiasis in patients and further studies on this field are desirable.

Macrophage secretions modulate the steroidogenesis of polycystic ovary in rats: effect of testosterone on macrophage pro-inflammatory cytokines.

AIMS: The macrophage secretions' effect on ovarian steroidogenesis is investigated in a polycystic ovary syndrome rat model (PCO rat). The influence of testosterone environment on the expression of macrophage pro-inflammatory cytokines that participate in ovarian steroidogenesis is studied. MAIN METHODS: PCO rats were induced by estradiol valerate. Spleen macrophages were cultured with and without testosterone (10(-6) M) and their secretions were used to stimulate ovaries from PCO and control rats. Ovarian hormones released and ovary mRNA levels of P450 aromatase and 3ß-hydroxysteroid dehydrogenase were measured by radioimmunoassay and RT-PCR, respectively. The tumor necrosis factor alpha (TNFα) and nitric oxide (NO) levels in macrophage culture medium, along with the TNFα, interleukin (IL)-6, IL-10 and androgen receptors (AR) mRNA levels in macrophage cells were determined. KEY FINDINGS: Macrophages from PCO rats released more TNFα and NO, expressed higher TNFα and IL-6, lower AR, and no change in IL-10 mRNA levels than control macrophages. TNFα, IL-6 and AR changes were greater after macrophage testosterone treatment. Macrophage secretions from PCO rats stimulated androstenedione and decreased estradiol release and ovarian mRNA P450 aromatase expression in PCO rats compared to macrophage secretions from control rats. These effects were greater when macrophages from PCO rats were treated with testosterone. Ovarian progesterone response was unchanged. SIGNIFICANCE: The differential steroidogenic ability of macrophage secretions from PCO rats is associated to the in vitro testosterone environment. Testosterone, probably acting on macrophage AR, induces a greater release of TNFα, modifying ovarian response by increasing androstenedione and slightly decreasing estradiol without affecting progesterone.

Neutralization of Pseudomonas aeruginosa enzymatic activity by antibodies elicited with proteins of Larrea divaricata Cav.

Larrea divaricata Cav. (Jarilla) is a bush widely used in folk therapy for the treatment of several pathologies. Partially purified proteins of crude extract (JPCE) cross-react with proteins of Gram-negative bacteria, including Pseudomonas aeruginosa, which is an opportunistic pathogen that causes several intrahospitalary infections. This bacterium produces many proteins with enzymatic activity, including hemolysins and proteases that play a major role in acute infection caused by this bacterium. The aim of our work was to investigate if antibodies against with L. divaricata neutralize the hemolytic and proteolytic activity of P. aeruginosa. The hemolytic activity of soluble cellular proteins was inhibited 100% and extracellular proteins (EP) showed an inhibition between 44 and 95% when both bacterial fractions were treated with anti-JPCE serum. Also, in EP the neutralization was directed towards the active site of the hemolysin. When protease activity of extracellular products was tested, bands of 217, 155, 121, 47 and 27 kDa were observed in native zymograms. Neutralization between 55 and 70% of the bands of 217, 155 and 121 kDa was observed when EP were treated with anti-JPCE serum. In conclusion, our data clearly demonstrate that antibodies elicited with L. divaricata' proteins are able to neutralize the hemolytic and proteolytic activity of P. aeruginosa cellular and extracellular proteins. Our study constitutes the first report that associates the immunogenicity of plant proteins and bacterial proteins with enzymatic activity. These findings could be relevant in the development of alternatives therapies for patients suffering intrahospitalary opportunistic infections with P. aeruginosa.

In vivo effect of three fractions of Larrea divaricata Cav. (jarilla) on the innate immune system: macrophage response against Candida albicans.

Larrea divaricata Cav. (jarilla) is a plant with well-documented applications in folk medicine in Argentina. In this study, we aimed to evaluate functional parameters of peritoneal macrophages isolated from mice injected with three fractions (F1, F2 and F3) of L. divaricata. The response of macrophages against Candida albicans was evaluated. Cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test, apoptosis was evaluated using Giemsa, acridine orange/ethidium bromide and ladder assay, oxidative burst was assayed using nitroblue tetrazolium test and nitrite production using Griess assay. Cell stimulation and their ability to kill C. albicans in vitro were measured. The number and cell viability were similar to controls. However, we found that F1 induces pre-activation of macrophages, and this pre-activation is enhanced by C. albicans. The effects exerted by F1 make it more important than F2 and F3 for the treatment of disseminated candidiasis in patients with immunodeficiency diseases such as AIDS and chronic granulomatous disease, among others.

In vitro immunomodulatory effects of fractions obtained from aqueous extracts of Larrea divaricata Cav (Jarilla) on mouse peritoneal macrophages.

BACKGROUND AND AIM: Larrea divaricata Cav. (Zygophyllaceae) is a plant widely used in Argentina. MATERIAL AND METHODS: We isolated different fractions of L. divaricata aqueous extract containing minor amounts of NDGA, and we analyzed these fractions on mouse macrophages. RESULTS: We showed that a fraction without NDGA was capableof activating macrophages, principally through the production of mitochondrial anion superoxide and H(2)O(2). This could be important in the defense of infections. Moreover, this fraction decreased NO level suggesting an anti-inflammatory action. CONCLUSION: These results indicate that NDGA was not the compound responsible for the immunomodulatory action exerted by the aqueous extract from L. divaricata.

Modulatory effect of hydrogen peroxide on tumoral lymphocytes proliferation.

BW 5147 (murine lymphoma cell line). We analyzed the effect of H2O2 in cell proliferation testing nitric oxide and apoptosis. Enzymes involved in the regulation of H2O2 levels as superoxide dismutase (SOD) and peroxidase (PER) were analyzed. H2O2 exerted a biphasic effect. The inhibitory effect of H2O2 was related to the activation of the ERK and P38 pathway, NO production and apoptosis. The high proliferation was associated with a low level of H2O2 related to a low SOD and a high PER activities. Drugs capable of producing an increase in H2O2 levels could be used in cancer.

Cross-reaction between proteins of Larrea divaricata Cav. (jarilla) and proteins of Gram-negative bacteria.

Larrea divaricata is an abundant plant of northwest of Argentina used to treat different pathologies. We aimed to characterize the immunogenicity of proteins from a partially purified crude aqueous extract (JPCE) of jarilla. We evaluated the cross reaction between JPCE and whole cell-bacterial proteins (W-CBP) of Escherichia coli, Pseudomonas aeruginosa, Proteus vulgaris, and Klebsiella pneumoniae using a mouse anti-JPCE serum. Protein profiles of JPCE and W-CBP were analyzed. For JPCE, 18 bands were observed in a 20-176 kDa range. Levels of IgG against JPCE and W-CBP were determined. Bacterial proteins showed a strong reaction with the anti-JPCE serum. Plant proteins could be used as immune stimulants.

Larrea divaricata Cav (Jarilla): production of superoxide anion, hydrogen peroxide and expression of zymosan receptors.

Larrea divaricata is a plant widely used in folk medicine in Argentina. This work aimed to study the mechanisms of decoction activity on the release of oxygen reactive species. Decoction increased the binding of zymosan-FITC and superoxide production. Cadmium decreased the superoxide production as well as malonate and barbital. Decoction decreased the release of hydrogen peroxide. Decoction increased the reduction of MTT but not when malonate and barbital were included. Together, decoction increased the expression of dectin-1 leading to increased superoxide production. It is possible that decoction increases the activity of peroxidase, and decreases the Cu, Zn-superoxide dismutase.

In vivo immunomodulatory effects of aqueous extracts of Larrea divaricata Cav.

Several medicinal plants are considered immunomodulatory as they display a variety of anti-inflammatory, antimicrobial and antitumoral effects. Larrea divaricata Cav. (jarilla) (Zygophyllaceae) is a plant widely used in popular medicine to treat tumors, infections, and inflammatory diseases. So far, the immunostimulating activities of Larrea divaricata have not been studied in vivo. In this work, we used healthy mice to assess the immunomodulatory potential of aqueous extracts of Larrea divaricata Cav. We found that Decoction (D) and Infusion (I) from Larrea divaricata Cav showed any acute hepatotoxic activity. Only D at 0.5 mg/kg increased the carrageenan-induced inflammation. Macrophages harvested from treated mice showed no signs of apoptosis. These cells showed a significant increase in NO and TNF-alpha release and exhibited the strongest expression of iNOS. Decoction also increased the phagocytosis of zymosan and the binding of LPS-FITC. The expression of CD14, TLR4 and CR3 was lower in macrophages of mice treated than in controls. Thus, Larrea divaricata was able to prime Mphi in vivo and to induce full activation in vitro. Our finding contribute to characterize the biological activity of Larrea divaricata and to understand the ability of these extracts to enhance immune responses.

Early effects triggered by Larrea divaricata Cav. on murine macrophages at apoptotic concentrations.

Decoction and infusion of Larrea divaricata were tested at apoptotic concentrations (1 and 4 mg/ml) on peritoneal murine macrophages. Consistent changes were observed after incubation with 4 mg/ml decoction. Phagocytosis of zymosan, lysosomal enzyme activity, nitric oxide production, TNF-alpha release, and expression of CD14, TLR4, and CR3 increased significantly. Decoction at 1 and 4 mg/ml increased the binding of LPS-FITC. Apoptosis triggered by L. divaricata decoction is consequence of cell activation. The effects are independent of nordihydroguaiaretic acid. This "activation and death" could be the mechanism of L. divaricata to exert the antituberculosis effect known in folk medicine.

Actividad antifungica de extractos de plantas usadas en medicina popular en Argentina / Antifungal activity of plant extracts used in folk medicine in Argentina

Los hongos pueden causar enfermedades en humanos, especialmente en pacientes inmunosuprimidos. En este estudio, extractos de 10 plantas utilizadas en medicina popular en Argentina fueron ensayadas para estudiar la actividad antifúngica in vitro contra 4 cepas de hongos. De todas las plantas testeadas, solo 4 mostraron actividad antifúngica: Larrea divaricata Cav, Gnaphalium gaudichaudianum D.C, Baccharis trimera Less y Schinus terebenthifolius.

Fungi may cause very serious diseases in humans specially in immunosuppressed patients. In this study, extracts of 10 plants used in popular medicine in Argentine were assayed for in vitro antifungal activity against 4 fungal strains. Out of all the plants tested, Larrea divaricata Cav, Gnaphalium gaudichaudianum D.C, Baccharis trimera Less and Schinus terebenthifolius proved to have antifungal activity.

Activation and apoptosis of mouse peritoneal macrophages by extracts of Larrea divaricata Cav. (jarilla).

Two aqueous extracts, decoction and infusion from Larrea divaricata Cav. (Zygophyllaceae) were investigated for immunomodulating activity on peritoneal macrophages (MPhi). Both extracts reduced significantly the cell viability assessed with the MTT assay at 1 and 4 mg/ml (decoction) and 0.8-4 mg/ml (infusion). Apoptotic morphology showed that at 1 and 4 mg/ml both infusion and decoction triggered an increment of the apoptosis. Pretreatment of MPhi with decoction increased significantly the phagocytosis of zymosan and Candida albicans. The production of NO was estimated as nitrite using the Griess reagent. A slight but significant increase in NO release was observed after the incubation of both extracts (0.2 mg/ml) with LPS during 48 h. As shown in western blot data MPhi cultured with infusion and LPS exhibited the stronger expression of iNOS compared with untreated cells. Both extracts (0.2 mg/ml) increased the binding of LPS-FITC to cells compared with untreated ones. The addition of Staphylococcus aureus blocked completely the binding of LPS-FITC to cells. L. divaricata stimulated the MPhi activation at 0.2 mg/ml whereas it showed a clear pro-apoptotic activity at higher concentrations. The dual effects of L. divaricata are relevant considering the use of this plant to activate the immune system.

Prevalence of some bacteria yeasts and molds in meat foods in San Luis, Argentina.

In this work we evaluate the microbiological quality and the hygiene degree of meat foods consumed in the city of San Luis. A total of 515 meat food samples (315 from fresh sausages, 100 from hamburgers and 100 from ground beef) were processed, being the most of them non-industrial products. The microbiological quality was determined by counts of total mesophilic bacteria, coliforms, Escherichia coli, molds and yeasts, and Clostridium perfringens. The number of total mesophilic aerobes was within the 10(6) cfu/g limit set by the Argentinaan Alimentary Code (AAC). Two hundred seventy six samples exhibited E. coli levels between 10(1) and 10(3) cfu/g. The 58.26% of the samples with E. coli counts above > 10(1) cfu/g came from hamburgers and fresh sausages exceeding the AAC limits. Counts of molds and yeasts ranged between 10(3) and 10(6) cfu/g. From a total of 515 samples, 126 exhibited C. perfringens, out of which 80 (64.08%) gave counts > 10(2)/g, exceeding the limits set by the AAC. Out of these 80 samples, C. perfringens counts were above 10(5) cfu/g in 12 of them, and E. coli was also detected in 48 samples (38.10%). The samples with counts > 10(5) C. perfringens/g are potentially responsible for alimentary intoxication. The results obtained indicate the need to improve the processing and handling conditions of these products.

Prevalence and characterization of Clostridium perfringens from spices in Argentina.

Spices can present high microbial counts and Clostridium perfringens, Bacillus cereus, Salmonella and Shigella, among others have been isolated from spices. C. perfringens is an important pathogen agent causing, among other diseases, enteritis in humans caused by C. perfringens enterotoxin (CPE) which causes human food poisoning and enterotoxemia in domestic animals. The aims of the present work were (i) to establish the hygienic sanitary quality of some spices in San Luis, Argentina; (ii) to determine the presence of C. perfringens in these spices by means of the most probable number (MPN) and count on plate methods; (iii) to characterize the enterotoxigenic strains of C. perfringens by PCR and immunological methods such as reverse passive latex agglutination (RPLA) and (iv) to type by PCR C. perfringens strains isolated. A total of 115 samples of spices, 67 of which were purchased in local retail stores and 48 domestically collected were analysed. Total aerobe counts on tryptone glucose yeast extract agar medium of the 115 samples were between <10 and 10(6) CFU/g. The colifecal counts using Mac Conkey broth of the 115 samples were <4-10(3)CFU/g, with 28 samples (24.34%) exceeding the limit established by the Spanish Alimentary Code (10 CFU/g) while 2 samples (1.73%) had a sulfite reducing anaerobe load above standard limits. A total of 14 C. perfringens strains (12.17%) were isolated and characterized from 115 samples by the standard biochemical tests. Four of which (28.60%) turned out to be enterotoxigenic by PCR and RPLA. In order to type C. perfringens strains based on their main toxins, the 14 strains were analysed by PCR. All strains belonged to type A. All RPLA positive strains were cpe(+) by PCR. The percentage of enterotoxigenic strains was more elevated that those reported in other studies for this type of sample. These results indicate that sanitary conditions in different production stages of species must be improved to reduce health hazards. The high percentage (24.34%) of samples with colifecal values above standard limits is an indication of deficient sanitary conditions. These results suggest the need to provide legislation on the sanitary and hygienic quality of spices in our country.

Enterotoxin Production in Two-Culture Media by Yersinia enterocolitica Isolated from Sausages, Porcine Ceca and Tongues in San Luis, Argentina.

Twenty-six strains of Yersinia enterocolitica isolated from fresh sausages, bovine tongues, and porcine ceca and tongues were evaluated for the production of heat-stable enterotoxin (ST, infant mouse test) in trypticase soy broth (TSB) and casamino acids broth (CAB). The gut weight/body weight ratios obtained with the two media were similar. Sixteen strains produced ST in both media; nine strains did not. Strain 23 produced ST in CAB but not in TSB.

Incidence of Clostridium perfringens in Fresh Sausages in Argentina.

A simple iron-milk medium was used for the isolation and enumeration (MPN) if Clostridium perfringens from fresh sausages. Samples were diluted (log 10) in 0.1% peptone water, aliquots seeded and incubated at 45°C for 16-18 h, and cultures with clots and stormy fermentation were considered positive. Confirmation of C. perfringens was made by subculturing in tryptose-sulfite-neomycin agar under anaerobic conditions. Suspect colonies were identified by standard procedures, including nitrate reduction, gelatin liquefaction, lipase and lecithinase production, starch hydrolysis, hemolysis and reverse CAMP test. From 136 samples studied, 110 were positive. Twenty-six, 48, 25, 7, 3, and 1 samples contained, respectively 10, 102, 103, 104, 105, and 109 C. perfringens per g of food. Three per cent of samples contained enough bacteria to produce food poisoning. The frequency of distribution approximately follows the Poisson equation with a µ=1.793.

Identificación de Clostridium chauvoei por la prueba de coaglutinción / Identification of clostridium chauvoei by coagglutination test

El método de Coaglutinación estafilocócica (COA) para la identificación de clostridium chavoei fue estudiado con cuatro inmunosueros obtenidoa a partir de las siguientes preparaciones antigénicas (cultivos de 12h de la cepa 5078 de C. chavoei: a) células tratadas con formol al 0.5% (CF); b) células calentadas a ebullición 2h (CO) (ambas preparaciones con una concentración de 1.05 x 10**9 células/ml); c) extrato de veronal (EV); células lavadas y lisofilizadas se extrajeron con regulador veronal pH 8.50; y d) suspensión flagelar (F): al cultivo lavado dos veces con solución fisiológica se desflageló por agitación manual, se centrifugó a 3.500 x g y 16.000 x g durante 20 min. Los flagelos fueron separados por filtración en membrana filtrante (poro 0.2µm) observándoselos por impregnación argéntica. Lotes de 3 conejos se inocularon con 5 dosis de cada antígeno (0.2; 0.4; 0.8; 1.6 y 3.2ml) por vía IV. Para la preparación de la proteína A se siguió la técnica modificada de Kronvall. La cepa Cowan I de Staphylococcus aureus se estabilizó adiconando 0.1 ml de cada antisuero a 1 ml de suspensión. Como testigo se sensibilizaton estafilococos con suero normal de conejo. La COA se realizó con las cepas de C. chauvoei 5078, Chl y Ch2, con líquidos biológicos de ratones inoculados con la cepa 5078, con las cepas de C. septicum 3606 y S9 y una cepa de C. perfringens. Las lecturas se realizaron entre 2 y 8 min y se informaron de 0 a 4 +. La prueba fue específica para C. chauvoei y no dio reacciones cruzadas con C. spticum ni C. perfringens. La COA fue más efectiva cuando se utilizaron los antisueros obtenidos con EV (los antígenos estarían más purificados) y con F. En este caso se deberían usar los antígenos flagelares f y g, aunque escparían las mutantes inmóviles. El antisuero CF mostró una COS inferior a las anteriores, pero podría emplearse porque la preparación del antígeno es más sencilla. La COA con anti-CO fue menorm dudosa o negativa, el calor destruye componentes antigénicos. La COA puede ser un valioso elemento para el diagnóstico de C. Chauvoei