Inhibitory effect of cyclic terpenes (limonene, menthol, menthone and thymol) on Fusarium verticillioides MRC 826 growth and fumonisin B1 biosynthesis.

The minimum inhibitory concentration (MIC) of cyclic terpenes (limonene, menthol, menthone and thymol) against Fusarium verticillioides MRC 826 was assessed by using the semisolid agar antifungal susceptibility (SAAS) technique. Limonene, menthol, menthone and thymol were evaluated at final concentrations of 25, 50, 75, 150, 200, 250, 500 and 1000 microL/L of culture medium. Limonene and thymol showed the highest inhibitory effects on F. verticillioides development. Thus, the effects of monoterpenes on fumonisin B1 (FB1) biosynthesis were evaluated by using corn grain (Zea mays) as substrate. The monoterpenes were inserted on maize 1 day before inoculation with a conidial suspension of F. verticillioides to give final concentrations of 75 ppm. At this concentration, thymol was the most active inhibitor on FB1 biosynthesis.

Subchronic mycotoxicoses in rats. Histopathological changes and modulation of the sphinganine to sphingosine (Sa/So) ratio imbalance induced by Fusarium verticillioides culture material, due to the coexistence of aflatoxin B1 in the diet.

Mycotoxicoses are diseases caused by consumption of diets contaminated with mycotoxins, a special class of fungal secondary metabolites. Fumonisin B1 (FB1) and aflatoxin B1 (AFB1), the main toxins synthesized by toxicogenic stocks of Fusarium spp. and Aspergillus spp., respectively, can coexist in grains and in its by-products. We investigated a probable synergism of a fumonisins-containing Fusarium verticillioides culture material and AFB1 in the induction of hepatocyte apoptosis in rats subchronically fed on a mixture of them. Furthermore, the possibility of modifications in the fumonisins-induced Sa/So ratio imbalance in tissues and urine from rats poisoned with this mycotoxin, due to the presence of AFB1 in the diet, was evaluated. The co-exposure to fumonisins and AFB1 produced a higher liver toxicity, with respect to their individual administration, inducing apoptosis and mitotic hepatocytes. There was an inversion of the typical Sa/So ratio in rats fed on the culture material as well as in those subjected to a diet co-contamined with fumonisins and AFB1. Moreover, the later had a synergistic effect in the induction of Sa/So variations in kidneys. Therefore, the mixture of fumonisins and AFB1 induced toxic responses which could not be considered a sum of the effects caused individually by these mycotoxins.

Immunobiological effects of AFB1 and AFB1-FB1 mixture in experimental subchronic mycotoxicoses in rats.

Maize co-contamination with aflatoxin B1 (AFB1) and fumonisin B1 (FB1) is frequently found in several countries. Although the alterations on nutritional and immunologic parameters induced by these mycotoxins, when administered individually, are partially characterised, little is known about the effects induced in animals by a subchronic administration of both toxins mixtures. We have studied the nutritional and immunological alterations induced in rats fed during 90 days with a diet without mycotoxins, containing 40 ppb AFB1, and with a diet containing a mixture of 40 ppb AFB1 and 100 ppm FB1. Animals fed with the mixture of toxins obtained lower body weight than the control ones. The mitogenic response of spleen mononuclear cells (SMC) in vivo was higher in animals fed with AFB1. In in vitro studies, lower proliferations of SMC pre-exposed to AFB1 and to the mixture of toxins were detected. The SMC of animals fed with AFB1 produced lower levels of IL-2, higher of IL-4 and equal levels of IL-10. The SMC of animals fed with both toxins produced higher levels of IL-4, lower of IL-10 and equal levels of IL-2. The SMC preincubated with an AFB1-FB1 mixture produced higher concentrations of IL-4, lower of IL-10 and equal levels of IL-2. The peritoneal macrophages of animals that consumed AFB1 released less H(2)O(2), while animals fed with the mixture of toxins produced higher levels. In in vitro studies, macrophages pre-exposed to the mixture of toxins released less H(2)O(2). These results show different immunobiological effects produced by a mixture of mycotoxins in comparison to the individual action of the same toxins.

Experimental subchronic mycotoxicoses in mice: individual and combined effects of dietary exposure to fumonisins and aflatoxin B1.

We have used a murine model of subchronic mycotoxicoses produced by ingestion of mycotoxins. The five groups of animals studied were fed for 30, 60 and 90 days, respectively, with commercial diet (CD), experimental control diet (ECD), experimental with fumonisin B1 diet (EFD) and experimental with mixtures of mycotoxins diet (EMD). The animals fed EFD and EMD showed a significant increase in feed consumption/day with respect to the animals fed ECD (P < 0.005 for both groups). The biochemical measurements showed significant differences at 90 days in those animals fed EAD exhibiting a marked decrease in the values of alkaline phosphatase (ALP) and cholesterol (P < 0.05), along with a significant increase in calcium (P < 0.01). Differences in the decrease of the parameters studied were observed in mice fed EFD for triglycerides, cholesterol and calcium (P < 0.05 for all of them). The activity of aspartate transaminase (AST) increased significantly in animals fed EMD (P < 0.01). The tissue specimens at 60 days showed lesions in the livers of the animals fed EAD and EFD. At 90 days, and in those fed EAD, EFD and EMD, the lesions were intensified in the liver at 60 days in 80, 90 and 100% of the animals, respectively.

Possibility of in-hospital infection by Cryptococcus neoformans in patients with AIDS.

The objective of the present work was to carry out a survey of soil samples taken from different areas of a hospital of infectious disease located in the city of Córdoba, where three AIDS patients were hospitalized during different periods in the same ward. The three of them returned with meningeal cryptococcosis between three or five months after having been discharged. Cryptococcus neoformans was isolated in 8/10 samples collected outside the hospital, near the pigeon house. The samples collected from the AIDS patients ward and its surroundings were negative. These findings suggest that the patients may have been infected by the fungus during their first stay in hospital.

Subchronic mycotoxicoses in Wistar rats: assessment of the in vivo and in vitro genotoxicity induced by fumonisins and aflatoxin B(1), and oxidative stress biomarkers status.

Some evidence suggests that fumonisin B(1) (FB(1)), a worldwide toxic contaminant of grains produced by Fusarium verticillioides, exhibits an oxidative stress mediated genotoxicity. We studied the DNA damage (by the alkaline comet and the micronucleus tests) and biomarkers of cellular oxidative stress (malondialdehyde, MDA; catalase, CAT; and superoxide dismutase, SOD) in spleen mononuclear cells of male Wistar rats subchronically (90 days) fed on a control experimental diet (CED) or poisoned with experimental diets contaminated with a culture material containing 100 ppm of FB(1) (FED), with 40 ppb of aflatoxin B(1) (a common toxic co-contaminant in cereals, AFB(1)ED), and with a mixture of both toxins (MED). The DNA damage was found in 13.7%, 81.7%, 98.0% and 99.3% (comet assay) and in 2.8%, 7.0%, 10.8% and 8.8% (micronucleus technique) in groups CED, FED, AFB(1)ED and MED, respectively. The MDA levels as well as the CAT and SOD activities were increased in all the poisoned animals. A similar behavior was observed in cells exposed in vitro to the toxins. These data support the hypothesis of an oxidative stress mediated genotoxicity induced by FB(1). Furthermore, the extent of DNA damage assessed by the comet assay suggests a possible protective effect of the fumonisins-AFB(1) mixtures in vitro against the genotoxicity induced individually by the toxins.

Biochemical basis for the killing of Cryptococcus neoformans by rat peritoneal cells.

The biochemical basis of peritoneal cell cytotoxicity for Cryptococcus neoformans was studied by measuring the killing of the yeast by peritoneal resident cells and peritoneal exudate cells obtained from normal and proteose-peptone-injected animals, respectively. Both cell populations killed C. neoformans to an equivalent extent after 3 h incubation. Exudate cells showed anti-cryptococcal activity from the first hour of incubation, while no killing was observed with resident cells before 3 h. Both cell populations triggered a respiratory burst in response to opsonized C. neoformans as indicated by the fact that killing of the yeast was inhibited by scavengers of reactive oxygen intermediates (ROI). C. neoformans susceptibility to H2O2 and hydroxyl radicals in cell-free systems is demonstrated by incubating a yeast suspension with different concentrations of H2O2 and Fenton's reagents, respectively. These results suggest that oxygen metabolites play an active role in C. neoformans killing.

Immunosuppression in experimental cryptococcosis in rats. Induction of thymic suppressor cells.

The presence of the microorganism, cortical hyperplasia and germinal centers was detected in the thymus of rats infected with 10(7) viable Cryptococcus neoformans cells and immunized at 7 days afterwards with 2.5 mg (0.1 ml) of human serum albumin (HSA) incorporated to complete Freund's adjuvant (CFA) (Group 2). There was no modification of the glandular structure in the thymus of the animals only immunized with HSA-CFA (Group 1). The weight of the thymus of group 2, animals infected and immunized, was increased compared with the weight of the thymus of group 1 animals, this became evident by the increase of the thymic index (TI) (p less than 0.005). This rate was obtained calculating the thymus weight/total body weight ratio x 1000. Thymic cells (10(7) cells in 1 ml) obtained from both groups of animals were transferred to normal syngeneic rats of the same sex. The recipient rats were immunized with HSA-CFA 24 h later and 14 days after the transference, the response of delayed-type hypersensitivity (DTH) was studied in them. It could be observed that the thymic cells coming from group 2 animals were capable of suppressing significatively the afferent pathway of the DTH response to HSA when compared with the response of the animals that received cells coming from group 1 rats (p less than 0.0001).

Aromatic plants essential oils activity on Fusarium verticillioides Fumonisin B(1) production in corn grain.

The minimum inhibitory concentration (MIC) of Origanum vulgare, Aloysia triphylla, Aloysia polystachya and Mentha piperita essential oils (EOs) against Fusarium verticillioides M 7075 (F. moniliforme, Sheldon) were assessed, using the semisolid agar antifungal susceptibility (SAAS) technique. O. vulgare, A. triphylla, A. polystachya and M. piperita EOs were evaluated at final concentrations of 10, 20, 40, 50, 100, 200, 250, 500, 1000 and 1500 epsilonl per litre (epsilonl/l) of culture medium. A. triphylla and O. vulgare EOs showed the highest inhibitory effects on F. verticillioides mycelial development. This inhibition was observed at 250 and 500 epsilonl/l for EOs coming from Aloysia triphylla and O. vulgare, respectively. Thus, the effects of EOs on FB(1) production were evaluated using corn grain (Zea mays) as substrate. The EOs were inserted on the 5th, 10th, 15th and 20th day of maize postinoculation with a conidia suspension of F. verticillioides. O. vulgare and A. triphylla were applied to give final concentrations of 30 ppm and 45 ppm, respectively. Different effects were observed in the toxicogenicity at the 20th day treatment. The O. vulgare EO decreased the production level of FB(1) (P < 0.01) while A. triphyla EO increased it (P < 0.001) with respect to those obtained in the inoculated maize, not EOs treated. Results obtained in the present work indicate that fumonisin production could be inhibited or stimulated by some constituents of EOs coming from aromatic plants. Further studies should be performed to identify the components of EOs with modulatory activity on the growth and fumonisins production of Fusarium verticillioides.

Apoptosis induction by glucuronoxylomannan of Cryptococcus neoformans.

We studied the ability of glucuronoxylomannan (GXM), the major constituent of Cryptococcus neoformans capsular polysaccharide, to induce apoptosis in lymphocytes from normal rats. Spleen mononuclear cells (Smc) from normal rats treated with GXM for 24 h exhibited, in comparison with controls, an increased hypodiploidy in the DNA profile after staining with propidium iodide, as well as increased ladder-type DNA fragmentation in agarose gel electrophoresis and a high number of positive cells in the terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) reaction. Furthermore, increased hypodiploidy in the DNA profile was also observed in Smc expressing T-cell receptor (TCR +). We also studied the induction of apoptosis in lungs and spleens from rats in the immunosuppressor period of disseminated cryptococcosis. TUNEL labeling of lungs and spleens from rats obtained 14 days after infection with C. neoformans showed a large number of apoptotic cells. Our results provide strong cytometric, molecular and morphological evidence that apoptosis could be a previously unrecognized immunosuppressive property of GXM in vitro. This programmed cell death may be involved in the immunosuppression observed during C. neoformans infection.

Immunosuppression in experimental cryptococcosis in rats. Induction of efferent T suppressor cells to a non-related antigen.

Using a rat model, we have previously demonstrated that infection with Cryptococcus neoformans can trigger the production of a series of suppressor cells that specifically inhibit the cell-mediated immune response to a non-related antigen, human serum albumin (HSA), that has been injected 7 days after the infection. We previously determined that the cryptococcal infection induces afferent suppressor or suppressor induction cells (Ts1) to HSA. The primary objective of the present study was to investigate the suppressor cells involved in the efferent phase of delayed-type hypersensitivity (DTH) response to HSA in rats infected with C. neoformans and immunized with the non-related antigen and determine the role that the Ts1 cell plays in the induction of that cell. For this purpose, the spleen mononuclear (SpM) cells containing the Ts1 or SpM cells from immunized non-infected rats (used as donor controls) were transferred to two groups of syngeneic naive recipients (first recipients). Later, the SpM cells from both groups of animals were transferred to rats immunized with HSA (second recipients). The efferent limb of the DTH response to HSA was suppressed in the recipients that received SpM cells from donors injected with Ts1 cells. Additional HSA antigen was not required for induction of these efferent suppressor cells. Furthermore, we here show that these cells are resistant to treatment with cyclophosphamide (Cy), and that they can activate another suppressor population. The latter are Cy sensitive and are present in the immune recipient.

Immunosuppression in experimental cryptococcosis: variation of splenic and thymic populations and expression of class II major histocompatibility complex gene products.

Previous studies from our laboratory have shown that infection with Cryptococcus neoformans can trigger the production of a series of suppressor cells that specifically inhibit the cell-mediated immune response to a nonrelated antigen, the human serum albumin (HSA). In the present study, we determined the variation of thymus and spleen cell populations in rats infected with C. neoformans and immunized with HSA-CFA at the time when suppressor activity was demonstrated. At 21 days postinfection, the number and the percentage of CD4+CD8+ cells were significantly increased in the thymus together with a minor imbalance in other thymocytes subsets. The study of two class II molecules encoded within the major histocompatibility complex, IA and IE, showed that the total number of class II IA-positive cells was increased in the glands of animals infected when compared to the glands of animals only immunized, while the corresponding percentages were lower than those in control rats. On the contrary a significant increase in both the number and the percentage of IE phenotype was observed in the thymus of infected rats, compared to the animals that were only immunized and used as a control. The IE/IA phenotype ratio within each group was increased in rats injected with the fungus. The study of spleen populations revealed an increase in CD4+ and CD8+ cells and a decrease in the B cells. The IE antigen was increased in the spleen of infected animals. The IA molecule expression showed no difference between the infected animals and the control groups. The IE/IA phenotypes ratio was mildly increased in the spleen of infected rats. These findings reveal that the cryptococcal infection can render an important imbalance in the thymus and spleen T-cell compartment together with a significant increase in the expression of the IE molecule at the time when the suppressor activity was demonstrated in this model.

Immunosuppression in experimental cryptococcosis in rats. Induction of afferent T suppressor cells to a non-related antigen.

To demonstrate the nature of the suppressor cells elicited in rats infected with Cryptococcus neoformans and immunized with human serum albumin (HSA), spleen mononuclear (SpM) cells were fractionated through a nylon wool column. The adherent and non-adherent populations were collected and transferred to syngeneic rats. In all cases, the non-adherent or T-enriched cells adoptively transferred suppression to HSA, however, the suppressive effects of the non-adherent cells were never as great as those of the unpassed population of SpM cells. The fractions adherent to nylon wool also diminished the delayed-type hypersensitivity response to HSA although this was not significant, but glass-adherent cells did exhibit significant suppressor activity. Immunized, non-infected rats were used as donor controls. Furthermore, we showed that the T-enriched-cells are sensitive to treatment with low doses of cyclophosphamide and that they bind HSA. These data indicate that immune suppression of the induction of the delayed-type hypersensitivity response to HSA in cryptococcosis can occur as a result of infection with C. neoformans, and that at least one mechanism involved is the induction of adherent and non-adherent suppressor cells. Characterization of the non-adherent cells indicates that they are Ts1 cells.

Experimental coccidioidomycosis: effects of cyclophosphamide in immunologic responses.

Rats were infected with Coccidioides immitis and injected with cyclophosphamide three days pre or post infection. Administration of the drug before the infection caused enhancement of DTH response and decrease of the colony forming units (CFU). Conversely, injection of the drug three days post infection produced contrary effects, indicating that a normal T-cell function is essential as a defense mechanism in C. immitis infection.

Modulation of I-A and I-E expression in macrophages by T-suppressor cells induced in Cryptococcus neoformans infected rats.

When the I-A and I-E expressions were assessed in peritoneal macrophages from Cryptococcus neoformans infected animals, a significant decrease in the former was observed when compared with normal macrophages (p < 0.001) whereas a significant increase in the I-E expression was observed when compared with controls (p < 0.005). On the other hand, when studying the in vitro action of Ts cells on the macrophages, it was observed that the I-A expression was significantly reduced in macrophages upon contact with Ts cells. Similar results were obtained when Ts cells were replaced by a soluble factor. In contrast, the I-E expression was significantly increased by in vitro action of the Ts cell or its soluble factor. Indomethacin partially restored I-A and I-E expression in the macrophages to control levels.

Immunosuppression in experimental cryptococcosis in rats: modification of macrophage functions by T suppressor cells. Macrophages functions in cryptococcosis.

The influence of lymphocytes on the modulation of macrophage functions in altered immune states induced by Cryptococcus neoformans infection in rats has been investigated. In this report we observed a decrease of 'in vitro' phagocytic activity by peritoneal cells (PC) from rats that received T suppressor cells induced by cryptococcal infection, against both the same microorganism that stimulated this suppressor population (p less than 0.05) and another non-pathogenic primary yeast (Candida tropicalis), (p less than 0.02). The microbicide function of the PC from these animals present a significant decrease in challenge by C. tropicalis (p less than 0.002) when compared with PC from animals transferred with T normal cells. The transference of T suppressor cells induced by cryptococcal infection in animals immunized with human serum albumin-complete Freund's adjuvant (HSA-CFA) produces a significant alteration of the phagocytosis to HSA-human red cells (HSA-HRC) when compared with the phagocytosis observed in animals that received T normal cells or the phagocytosis of normal animals (p less than 0.001). We could also observe that the DTH to HSA studied during 30 days was negative in rats transferred with PC sensitizated with HSA and treated with suppressor T cells, when compared with the DTH response of animals transferred with PC-HSA cocultured with normal cells (p less than 0.05 21st day). The data presented in this paper illustrated that following infection of rats with C. neoformans there is a change in some population of accessory cells behavior reflected by the modification of several functions, such as phagocytosis, lytic activity and antigen presentation.

Inhibition of I-A expression in rat peritoneal macrophages due to T-suppressor cells induced by Cryptococcus neoformans.

The expression of I-A antigen in rat peritoneal cells was significantly reduced during infection with Cryptococcus neoformans. When studying the in vitro action of T-suppressor cells induced by the fungus, or a soluble factor from the T-suppressor cells, a significant decrease in I-A expression by the peritoneal cells was observed. This expression was partially restored by indomethacin.

Non-specific immunosuppression in experimental cryptococcosis in rats.

The delayed type hypersensitivity response to human serum albumin (HSA) of rats infected intraperitoneally with 10(7) viable C. neoformans cells, and 7 days after, immunized with human serum albumin was significantly diminished (p less than 0.05) when compared with the response observed in rats immunized with human serum albumin and non infected. The spleen mononuclear cells from suppressed rats transferred to normal syngeneic recipients of the same sex suppress the afferent phase of the response (p less than 0.02) suggesting that cells present in the spleen might be one of the responsible mechanisms for the suppression to non-related antigens in infected animals.