Microsatellite-primed PCR and random primer amplification polymorphic DNA for the identification and epidemiology of dermatophytes.

This study demonstrates the capacity of the one-step polymerase chain reaction (PCR) fingerprinting method using the microsatellite primers (GACA)4 or (GTG)5 (MSP-PCR) to identify six of the most frequent dermatophyte species causing cutaneous mycosis. PCR with (GACA)4 was a suitable method to recognise Microsporum canis, Microsporum gypseum, Trichophyton rubrum and Trichophyton interdigitale among 82 Argentinian clinical isolates, producing the most simple and reproducible band profiles. In contrast, the identification of Trichophyton mentagrophytes and Trichophyton tonsurans was achieved using PCR with (GTG)5. In this way, the sequential application of PCR using (GACA)4 and (GTG)5 allowed the successful typification of clinical isolates which had not been determined by mycological standard techniques. In this work, the intraspecies variability among 33 clinical isolates of M. canis was detected using random amplification of polymorphic DNA (RAPD-PCR) with the primers OPI-07 and OPK-20. The genetic variations in the isolates of M. canis were not associated with clinical features of lesions or pet ownership, but a geographical restriction of one genotype was determined with OPK-20, suggesting a clonal diversity related to different ecological niches in certain geographical areas. The results of this work demonstrate that the detection of intraspecies polymorphisms in M. canis by RAPD-PCR may be applied in future molecular epidemiological studies to identify endemic strains, the route of infection in an outbreak or the coexistence of different strains in a single infection.

Immunization with crude antigens plus aluminium hydroxide protects cattle from Fasciola hepatica infection.

The ability of total homogenate (TH) of Fasciola hepatica conjugated with aluminium hydroxide (alum) or Freund's complete adjuvant (FCA) to protect cattle against experimental fasciolosis was evaluated. Compared with the infected group, the immunized animals with alum-TH and FCA-TH presented a significant reduction in fluke burden (85.9% and 96.8%, respectively), a higher percentage of short-sized worms, a marked reduction in the released eggs in faeces (89% and 57%, respectively), as well as an increased production of specific antibodies before infection. The alum-TH immunized group also showed a significant increase in the antigen-specific proliferation of peripheral blood mononuclear cells (PBMC) as early as 4 weeks before infection. Although both immunized groups (alum-TH and FCA-TH) were able to develop an efficient protective immune response to metacercarial challenge, an earlier PBMC response, lower hepatic damage and less effect on weight gain were found in alum-immunized animals. Therefore, alum is a good candidate for future immunization against bovine fasciolosis.

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.

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.

Immunobiological effects of fumonisin B1 in experimental subchronic mycotoxicoses in rats.

Fumonisin B1 (FB1), the principal secondary metabolite produced by the fungus Fusarium verticillioides (Gibberella fujikuroi mating population A), is a potent toxin that can be found in fungus-contaminated corn and corn-based food products. We have investigated the immunobiological effects of subchronic dietary exposure to FB1 in male Wistar rats. Animals were fed with diets containing 0 (control) or 100 ppm of FB1 for 12 weeks. The total FB1 intake on day 90 was 810 mg/kg of body weight. Food consumption, body weight, and body weight gain on day 90 were reduced in animals exposed to FB1. Histopathologic changes consisted of histiocytic perivascular infiltrate and an increased number of Kupffer cells in the liver, necrosis and apoptosis of tubular epithelial cells in the kidney, and increased mitotic figures and lymphocytic infiltrate in the small intestine. Serum enzyme alkaline phosphatase was significantly elevated in rats fed FB1, while triglyceride levels decreased compared to controls. Treatment with FB1 in vivo or in vitro did not have a significant effect on mitogen-induced proliferation of spleen mononuclear cells. However, increased levels of interleukin-4 (IL-4) and decreased levels of IL-10 were released by these cells in culture compared to controls. FB1 in vivo or in vitro decreased the hydrogen peroxide (H(2)O(2)) released by peritoneal macrophages, while no changes in levels of superoxide anion produced by total peritoneal cells were detected. The results from the present work demonstrate that subchronic FB1 intake could affect the small intestine and alter the interleukin profile and some main functions of macrophages in antitumor activity.

Cytokines involved in the immunosuppressor period in experimental fasciolosis in rats.

The aim of this study was to evaluate the kinetics of the cytokines interferon-gamma, interleukin-2, interleukin-10 and interleukin-4 produced by spleen mononuclear cells stimulated by Con A during an experimental infection in rats with Fasciola hepatica. The proliferative response to Con A of Spm cells from rats infected with F. hepatica was significantly decreased on day 7 post-infection (P<0.006) and simultaneously an increase of interferon-gamma, interleukin-10 and interleukin-4 production along with a decrease of interleukin-2 by spleen mononuclear cells were observed. Interleukin-4 and interleukin-10 were involved in ablating cellular proliferation in vitro, as the addition of neutralising antibodies to either cytokine reversed the proliferative block. The addition of exogenous recombinant interleukin-2 also restored the proliferative response by spleen mononuclear cells obtained 7 days after infection from infected rats. At the same time, we found an increase in interleukin-10 production by peritoneal cells (in close contact with the flukes) and decreased nitric oxide levels. In addition, histological studies on the liver on day 7 after infection showed the presence of parasite inside migratory tunnels in the parenchyma, and polymorphonuclear leukocytes, predominantly eosinophils, around the parasite. The transient suppression in proliferative response mediated by cytokines interleukin-4 and interleukin-10 in the spleen, and diminution of nitric oxide production in the peritoneum could be mechanisms to evade the protective immune response during the first stages of liver penetration by the parasite.

Mechanisms for induction of immunosuppression during experimental cryptococcosis: role of glucuronoxylomannan.

In previous work we have demonstrated that spleen mononuclear (Spm) cells from rats obtained 14 days after infection with Cryptococcus neoformans showed a diminution in proliferative response to Concanavalin A (Con A). In this study we further investigate some characteristics of the Spm cell population involved in the immunosuppressor phenomenon induced by C. neoformans. We observed that unstimulated Spm cells expressing T-cell receptor (TCR+) from infected rats were reduced in number after 96 h of culture. When the Spm cells from infected rats were stimulated with Con A, increased production of IL-10, reduced levels of IL-2, and decreased CD11a surface expression were shown. These immunosuppressor phenomena were also observed when the capsular polysaccharide, glucuronoxylomannan (GXM), was added to cultures of Spm cells from normal rats. However, GXM had a more pronounced effect in reducing the number of cells surviving in culture than that observed during infection and produced an increase in IL-4 production by Con-A-stimulated Spm cells. Addition of anti-IL-10 monoclonal antibody to cultures restored the lymphoproliferation of Spm cells from infected animals, indicating that IL-10 production is a suppressor mechanism of cell-mediated immunity during experimental infection. The results presented here indicate that at least two mechanisms mediate the nonspecific suppression in this model of cryptococcosis: IL-10 production and diminution of the number of T cells. GXM could be involved, since it has a pronounced effect in the reduction of Spm cells in vitro.

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.

Involvement of nitric oxide in protecting mechanism during experimental cryptococcosis.

In the present study we investigated the role of nitric oxide (NO) in the effector mechanisms of host defense against Cryptococcus neoformans in vivo. Our results showed an increase of NO produced by the peritoneal macrophages from 14-days infected rats compared with normal rats. These cells were capable of killing C. neoformans to a greater extent than macrophages from noninfected rats (80% vs 20%, respectively). The killing of C. neoformans by infected cells was efficiently inhibited (80% to 35%, P < 0.001) by adding aminoguanidine (AG) to the cultures. We observed that in vivo administration of AG to the infected animals efficiently inhibited the metabolism producing NO and failed to affect that of normal animals. When the NO synthase (NOS) was inhibited in vivo in the infected animals, a marked increase of the fungi charge in the organs was observed with respect to the normal animals treated with AG. We also observed that the course of the infection is drastically modified after the inhibition of NO production because all the animals infected and treated with AG died from cryptococcosis before 20 days postinfection (p.i.). These results indicate that NO is a crucial molecule in the effector mechanisms in this infection model.

Potential role for excretory-secretory forms of glutathione-S-transferase (GST) in Fasciola hepatica.

The excretory-secretory antigen of Fasciola hepatica (ESA) is involved in the suppressive phenomena of cellular immune responses in rats. The ESA can depress the proliferative response of spleen mononuclear cells and inhibit nitric oxide (NO) production by peritoneal cells. In the present study we identified ESA proteins of ca 24 kDa, which shared significant sequence homology to glutathione-S-transferase (GST) obtained from homogenates of F. hepatica adults, other helminths and different mammals. When the dimeric form of these proteins ca 48 kDa was cultured with rat spleen cells, a significant decrease of proliferative response to Con A was detected, starting from 20 micrograms/ml of ESA protein (P < 0.03). We also observed a significant inhibition of nitrite production by incubation with the dimeric form in normal peritoneal macrophages (P < 0.04). These results indicated that the GST secreted by the parasite could be involved in evasion of the parasite from the host immune response.

Fasciola hepatica-induced immune suppression of spleen mononuclear cell proliferation: role of nitric oxide.

The aim of the present study was to evaluate the proliferative response of spleen mononuclear cells (Spm) to mitogens in rats infected with Fasciola hepatica and its correlation with Spm and peritoneal cell (PC) nitric oxide (NO) production on Days 1, 3, 7, 14, 30, and 60 postinfection. In addition, histological changes in the liver were also studied. The proliferative response to Con A of F. hepatica-infected Spm was significantly decreased on Day 7 postinfection (P < 0.01). However, a pronounced increase of the proliferative response was detected from Day 3 until Day 60 when Spm were stimulated with LPS. In order to determine whether NO levels were modified during F. hepatica infections, we quantified nitrite in Spm and PC supernatants in cultures. Our results indicate a profound decrease of nitrite production by LPS-stimulated PC on the first and second weeks postinfection, and an increase in the levels of this mediator on LPS-stimulated Spm at the same postinfection time. The F. hepatica excretory-secretory antigen (ESA) was in part involved in the decrease of nitrite production by LPS-stimulated PC. A mechanism to avoid an immune response during the first stages of liver penetration could explain the transient suppression observed in Spm proliferative responses. On the other hand, the decrease in NO production by rat-infected PC could also be one of the strategies of the parasite to avoid the potential killing effect of NO during peritoneal migration.

Lack of involvement of nitric oxide in the macrophage-mediated inhibition of spleen cell proliferation during experimental cryptococcosis.

We investigated the proliferative response to mitogens of spleen mononuclear (Spm) cells from Cryptococcus neoformans-infected rats. We determined reactive oxygen intermediates (ROI) and nitric oxide (NO) production by peritoneal and Spm cells, and evaluated the correlation of the proliferative response with NO and ROI production. The proliferative response of Spm cells from infected rats dramatically decreased at 14 and 21 days postinfection (PI). The unresponsiveness of Spm cells from 14-day infected rats was not abrogated by the addition of L-NAME and AG, indicating that NO is not involved in the antiproliferative response of experimental cells. When SOD, catalase, and indomethacin were added to the cultures, the suppression was still observed, indicating that ROI and prostaglandins are not involved in the unresponsiveness of lymphocytes. The proliferative response of lymphocytes from 14-day infected rats was significantly improved when cultures were made in the presence of Con A and exogenous IL-2. Additionally, a purified T-rich fraction from infected rats cultured with control macrophages recovered the normal proliferative response. This result indicates that macrophages from infected rats mediate the unresponsiveness of lymphocytes, probably by reducing the ability of lymphocytes to secrete IL-2.

Inhibition of spleen cell proliferative response to mitogens by excretory-secretory antigens of Fasciola hepatica.

The effect of Fasciola hepatica excretory-secretory antigen (ESA) on the proliferative response of spleen mononuclear (SpM) cells of normal rats to stimulation with mitogens has been examined. When ESA was added to normal SpM cells, there was a decrease in the proliferative response to concanavalin A (Con A) or lipo-polysaccharide (LPS) in a dose-dependent manner. The addition of indomethacin, which blocks prostaglandin synthesis, or N omega-nitro-L-arginine methyl ester (L-NAME) a specific inhibitor of nitric oxide (NO) synthase, had no effect on the ability of ESA to suppress the proliferative response to Con A. However, supplementation of the culture media with catalase, which degrades hydrogen peroxide (H2O2) or superoxide dismutase (SOD) to remove superoxide anion (O2), resulted in a restoration of proliferation to Con A. When LPS was used as mitogenic stimulus no inhibitor added to the culture restored the proliferation. These results suggest that H2O2 and O2- are involved in the suppressor phenomenon induced by ESA in the T-cell proliferative events.

Modification of accessory activity of peritoneal cells from Fasciola hepatica infected rats.

The effect of Fasciola hepatica excretory-secretory antigen (ESA) was studied in the modulation of the accessory functions of peritoneal cells (PC) of rats infected with the parasite. PC rats infected with F. hepatica 7 and 14 days previously showed a marked decrease in phagocytic activity against Candida tropicalis (P < 0.007 and P < 0.004, respectively). The same effect was observed when the assay was carried out with PC from animals injected 7 days before with ESA (P < 0.001) including PC previously treated in vitro with ESA. To investigate the effect of ESA on the antigen presenting ability, PC of animals infected before transfer to syngeneic normal rats were stimulated in vitro with either F. hepatica whole antigen (FhWA), ESA or human serum albumin (HSA). Delayed type hypersensitivity (DTH) response to the different antigens studied over a 35 day period was negative in rats transferred with sensitised PC. When these animals were immunised with the corresponding antigen the DTH response became positive. A similar result was obtained in PC receptors from ESA inoculated rats and in vitro stimulated with FhWA or HSA. These data suggest that the alterations observed in the functions of peritoneal cells may, in part be due to the effect of the F. hepatica ESA.

Involvement of excretion-secretion products from Fasciola hepatica inducing suppression of the cellular immune responses.

Normal rats i.p. injected with Fasciola hepatica excretor-secretor antigen (ESA) induced a population of spleen mononuclear (SpM) cells, which suppressed the delayed type hypersensitivity (DTH) response to parasite antigens as well as to non-related antigens (human serum albumin) by adoptive transfer. A similar effect was observed when the cell transfer was performed with SpM cells non-adherent to nylon wool. The DTH was not modified by cells transfer adherent to nylon wool in syngeneic receptor animals. The observed suppression depended on the concentration and inoculation moment of the antigen; 1.8 mg of protein ESA being enough to suppress the DTH response at the different days studied, before and after immunization with whole F. hepatica antigens. A marked suppression was observed when ESA was injected on day 7 pre-immunization. On the other hand, inoculation of ESA treated with 0.01 M sodium periodate (carbohydrate oxidant) diminished the suppressor effect found after the native ESA inoculation, indicating participation of ESA glucidic components in induced suppression. Inoculation of ESA fractions obtained from polyacrylamide gel elution with different MW range, showed that components between 12 and 23 kDa actively induced suppression to the DTH response to parasite antigens.

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.

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.

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.

Serological, electrophoretic and biological properties of Fasciola hepatica antigens.

Fasciola hepatica somatic antigen, its partially purified fractions and excretion-secretion products were investigated as to serological, electrophoretic and biological properties. In a Sephadex G-100 column (SG-100), Fasciola hepatica total antigen (FhTA) gave 5 fractions, and SDS-PAGE analysis showed they were glycoproteins ranging from 14 to 94 kDa molecular weight (MW). When these fractions were analyzed by enzyme-linked immunotransfer blot (EITB) and immunodiffusion in gel (ID) with serum from immunized rats with FhTA, the presence of different antigenic components was revealed. In the SDS-PAGE of excretor-secretor antigen (ESA), it was possible to observe peptides from 12 to 22 kDa, which were also present in FhTA. When the FhTA, its fractions and the ESA were analyzed by EITB with the immune rat serum (IRS), it was observed that only some fractions of the SG-100 shared antigens with the FhTA and ESA. Moreover, DTH and ITH responses were studied in FhTA immunized rats challenged with these different antigen components, revealing that the protein/carbohydrate ratio is important for inducing DTH response. The ESA was the most active component in the DTH and ITH response.

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.