Changes in the expression of α-tocopherol-related genes in liver and mammary gland biopsy specimens of peripartum dairy cows.

Blood α-tocopherol (α-Toc) concentrations decline gradually throughout the prepartum period, reaching the nadir after calving in dairy cows. The 6 α-Toc-related molecules [α-Toc transfer protein (TTPA); afamin; scavenger receptor class B, Type I; ATP-binding cassette transporter A1; tocopherol-associated protein (SEC14L2); and cytochrome P450 family 4, subfamily F, polypeptide 2 (CYP4F2)] are expressed in liver and other peripheral tissues. These molecules could regulate α-Toc transport, blood concentrations, and metabolism of α-Toc. Therefore, the aim of this study was to evaluate the changes in the expression of α-Toc-related genes in liver and mammary gland tissues of dairy cows around calving, which have remained elusive until now. In experiment (Exp.) 1, 28 multiparous Holstein cows were used (from -5 to 6 wk relative to parturition) to monitor the changes in dietary α-Toc intake, blood concentrations of α-Toc, and lipoproteins; in Exp. 2, 7 peripartum Holstein cows were used (from -4 to 4 wk relative to parturition) for liver tissue biopsy; and in Exp. 3, 10 peripartum Holstein cows were used (from -8 to 6 wk relative to parturition) to carry out the mammary gland tissue biopsy and milk sampling. In Exp. 1, the serum α-Toc concentrations declined gradually with decreasing amount of α-Toc intake and plasma high-density lipoprotein concentrations toward calving time. However, in the early lactation period after calving, serum α-Toc concentrations remained at a lower concentration despite the recovery of α-Toc intake and plasma high-density lipoprotein concentrations. In Exp. 2, just after calving, the TTPA, SEC14L2, afamin, and albumin mRNA expression levels in the liver were temporarily downregulated, and the hepatic mRNA levels of endoplasmic reticulum stress-induced unfolded protein response markers and acute-phase response marker increased at calving. In Exp. 3, the concentrations of α-Toc in colostrum were greater than those in precolostrum (samples were collected at wk -1 relative to parturition) and mature milk. The expression of TTPA, SEC14L2, and CYP4F2 mRNA in bovine mammary gland tissue was detected. However, TTPA and SEC14L2 mRNA expressions showed the opposite trends: the expression levels of TTPA mRNA peaked whereas SEC14L2 mRNA reached a nadir at calving. These results indicate that the expression of α-Toc-related genes involved in specific α-Toc transfer and metabolism in the liver and mammary gland are altered during calving. Moreover, these changes might be associated with the maintenance of lower serum α-Toc concentrations after calving.

Effects of altering total mixed ration conservation method when feeding dry-rolled versus steam-flaked hulled rice on lactation and digestion in dairy cows.

We evaluated the effects of different methods of conserving the total mixed ration (TMR) and processing hulled rice (Oryza sativa L.) on intake, digestion, ruminal fermentation, lactation performance, and nitrogen utilization in dairy cows. Eight multiparous Holstein cows (126 ± 19 d in milk) were used in a replicated 4 × 4 Latin square design with a 2 × 2 factorial arrangement of dietary treatments. The experimental diets used fresh TMR and ensiled TMR containing either dry-rolled (DR) hulled rice or steam-flaked (SF) hulled rice. The fresh TMR was prepared every morning. The ensiled TMR was prepared by baling fresh TMR and then sealing it with a bale wrapper; this was stored outdoors at 10 to 30°C for >4 mo. The method of grain processing did not affect the dry matter (DM) intake. The DM intake tended to be greater for ensiled TMR than for fresh TMR. Apparent total-tract DM digestibility and milk yield were increased by feeding ensiled TMR instead of fresh TMR and by replacing DR with SF. An interaction effect between the TMR conservation method and the grain processing method was detected for DM digestibility and milk yield; replacing DR with SF increased the DM digestibility and milk yield in cows fed fresh TMR, but this did not affect the cows fed ensiled TMR. The milk fat and lactose contents did not differ among dietary treatments. The milk protein concentration was higher for the cows fed SF processed hulled rice than those fed DR, but it was not influenced by the TMR conservation method. The ruminal total volatile fatty acid concentration was higher for the cows fed ensiled TMR compared with those fed fresh TMR, but it was not affected by the grain processing method. The molar proportion of acetate was decreased and propionate was increased by feeding ensiled TMR instead of fresh TMR and by replacing DR with SF. The concentrations of rumen ammonia N and plasma urea N were higher for the cows fed ensiled TMR than fresh TMR and were lower for SF than DR. Feeding ensiled TMR instead of fresh TMR increased the cows' urinary N excretion and decreased the retention N. Replacing DR with SF decreased the urinary N excretion, increased the milk N secretion, and then improved the nitrogen efficiency. These results show that feeding ensiled TMR instead of fresh TMR has an adverse effect on N utilization, but it increases digestion and milk production. Replacing DR with SF also increases digestion, milk yield, and milk protein content, and the improvement of milk yield by replacing DR with SF was prominent in the cows fed fresh TMR.

Oxidation resistance 1 is essential for protection against oxidative stress and participates in the regulation of aging in Caenorhabditis elegans.

Human oxidation resistance 1 (OXR1) functions in protection against oxidative damage and its homologs are highly conserved in eukaryotes examined so far, but its function still remains uncertain. In this study, we identified a homolog (LMD-3) of human OXR1 in the nematode Caenorhabditis elegans (C. elegans). The expressed LMD-3 was able to suppress the mutator phenotypes of E. coli mutMmutY and mutT mutants. Purified LMD-3 did not have enzymatic activity against 8-oxoG, superoxide dismutase (SOD), or catalase activities. Interestingly, the expression of LMD-3 was able to suppress the methyl viologen or menadione sodium bisulfite-induced expression of soxS and sodA genes in E. coli. The sensitivity of the C. elegans lmd-3 mutant to oxidative and heat stress was markedly higher than that of the wild-type strain N2. These results suggest that LMD-3 protects cells against oxidative stress. Furthermore, we found that the lifespan of the C. elegans lmd-3 mutant was significantly reduced compared with that of the N2, which was resulted from the acceleration of aging. We further examined the effects of deletions in other oxidative defense genes on the properties of the lmd-3 mutant. The deletion of sod-2 and sod-3, which are mitochondrial SODs, extended the lifespan of the lmd-3 mutant. These results indicate that, in cooperation with mitochondrial SODs, LMD-3 contributes to the protection against oxidative stress and aging in C. elegans.

Effect of grass hay intake on fiber digestion and digesta retention time in the hindgut of horses.

Eight Thoroughbred horses were used to examine the effects of grass hay intake on the fiber digestion and the retention time of digesta in the total gastrointestinal tract and the hindgut segments. The horses were randomly assigned to 2 groups and offered 2.0 (high intake [HI]) or 1.3 kg DM/(100 kg BW • d; low intake [LI]) of timothy hay in equal amounts every 3 h for 17 d. The digestibility and total tract mean retention time of digesta (tMRT) in the total gastrointestinal tract were measured from d 11 to d 15. To measure the mean retention time of digesta in each hindgut segment (sMRT), the horses were euthanized on d 17, after being fed a series of hay markers that were labeled with different rare earth elements. The digesta were collected from the ileum, cecum, right ventral colon (RVC), left ventral colon (LVC), left dorsal colon, right dorsal colon (RDC), and small colon (SC) to measure fiber digestibility, sMRT, VFA concentration, and fibrolytic activities. The digestibility of NDF and ADF in the total gastrointestinal tract was lower (P < 0.01) for HI than for LI. The cumulative disappearance of NDF and ADF from the ileum to the LVC attained 90% of total tract digestion regardless of hay intake. Values did not differ according to treatments in the ileum and the cecum but were lower (P < 0.01) for HI than for LI in the postcecal segments. This was caused by the smaller increase in the cumulative NDF and ADF disappearance from the cecum to the RVC for HI than for LI. The solid phase tMRT was shorter (P = 0.04) for HI than for LI. Although sMRT in the RDC and SC were shorter (P = 0.04 and P < 0.01, respectively) for HI than for LI, no differences were observed in the other hindgut segments. There was no difference between treatments in the total VFA concentration, VFA proportions, or the specific activity of carboxymethyl cellulase and xylanase in each hindgut segment. The fibrolytic activity for the cecum and RVC were greatest and decreased beyond the LVC. A clear relation between mean retention time and fiber digestion was observed in the total gastrointestinal tract, but the segments where a difference in sMRT was detected because of the intake were not the same as those with the fiber digestibility depression. Although the reduction in total tract fiber digestibility with increased feed intake was clearly shown to occur between the cecum and RVC, which were the main segments for fiber digestion, it could be explained neither by the sMRT nor by the fibrolytic activity in these segments.

Effect of substituting brown rice for corn on lactation and digestion in dairy cows fed diets with a high proportion of grain.

The effects of the substitution of brown rice (Oryza sativa L.; BR) for corn (Zea mays L.) in ensiled total mixed ration (TMR) that had a high proportion of grain on feed intake, lactation performance, ruminal fermentation, digestion, and N utilization were evaluated. Nine multiparous Holstein cows (51 ± 9 d in milk) were used in a replicated 3 × 3 Latin square design with 3 dietary treatments: a diet containing 0, 20, or 40% steam-flaked BR and 40, 20, or 0% steam-flaked corn (dry matter basis). Cows were fed ad libitum an ensiled TMR consisting of 40.7% alfalfa silage, 11.8% grass silage, 7.1% soybean meal, and 40.0% steam-flaked grain (dry matter basis). The ensiled TMR was prepared by baling fresh TMR, and then sealed by a bale wrapper and stored outdoors at 5 to 30 °C for over 6 mo. Dry matter intake and milk yield were lower for cows fed 40% BR than for cows fed 40% corn. The ruminal pH and total volatile fatty acid concentrations were not affected by dietary treatment. The ruminal ammonia-N concentration decreased as the percentage of BR in the diets was elevated. The proportion of acetate decreased, and that of propionate and butyrate increased with the increasing levels of BR. Plasma urea-N concentrations was lower and glucose and insulin concentrations were higher for cows fed 40% BR than for cows fed 40% corn. The whole-tract apparent digestibility of dry matter, organic matter, and starch increased, and the digestibility of neutral detergent fiber and acid detergent fiber decreased with the increasing BR level in the diet, with no dietary effect on crude protein digestion. As a proportion of N intake, the urinary N excretion was lower and the retention of N was higher for cows fed 40% BR than for cows fed 40% corn, with no dietary effect observed on N secretion in milk and fecal N excretion. These results show that substituting BR for corn decreases urinary N losses and improves N utilization, but causes adverse effects on milk production when cows are fed high-grain diets at 40% of dietary dry matter.

Short communication: effect of grazing on the concentrations of total sialic acid and hexose in bovine milk.

Sialic acid, which is located at the terminal end of glycoconjugates, is believed to have important biological functions. Its concentration in bovine milk varies depending on lactation stage and season. However, it remains unclear whether dietary factors, especially fresh forage, affect the total sialic acid concentration in milk. The purpose of the present study was to investigate the effect of grazing on the concentrations of total sialic acid and hexose in bovine milk. Six healthy dairy cows were used in a crossover design (3 cows fed fresh forage and 3 cows fed grass silage) for 2 wk. Individual milk samples were collected at 2 consecutive milkings (morning and evening) at 0, 1, 3, 5, 8, 11, and 14 d of the experimental period, and 2 consecutive samples in each cow were combined on each sampling day in proportion of the morning and evening milk yields. No differences in body weight, milk yield, or milk composition were observed between the 2 groups during the experimental period. The hexose concentration in milk did not differ between these groups during the experimental period. Conversely, the total sialic acid concentration in the milk of each grazing cow significantly increased at 11 and 14 d of the experimental period compared with that at 0 d. In the grass silage group, the total sialic acid concentration at the end of the experimental period tended to be lower than that at 0 d, but the decrease was not significant. These results indicate that grazing management could have increased the concentration of sialoglycoconjugates in milk. This suggests that grazing may increase the biological function of milk because it is thought that sialic acid is significant in many ways.

Detection of Paracoccidioides brasiliensis gp43 gene in sputa by loop-mediated isothermal amplification method.

The fungus Paracoccidioides brasiliensis is the pathogen of paracoccidioidomycosis (PCM), a systemic mycosis prevalent in Latin America. The loop-mediated isothermal amplification method (LAMP) was used in this study to detect the presence of P. brasiliensis in sputa samples from patients with chronic PCM, suspected PCM, and a negative control. The target P. brasiliensis gp43 gene was amplified in less than 4 hr in 11 of 18 sputa samples tested. The LAMP method had the advantage of speed and simplicity compared with the classic diagnostic methods such as the histopathological test or biological material culture and did not require sophisticated technical apparatus. It would be an important aid in cases where immediate treatment would mean patient survival, especially in immune-suppressed patients.

Continuous remission in an infant with chest wall malignant rhabdoid tumor after relapse.

Malignant rhabdoid tumor (MRT) is a highly aggressive tumor that occurs in infancy or childhood. The prognosis, especially in infants, is very poor. Here we report the long-term survival of a 5-month-old boy with MRT that arose from the chest wall. After total resection of the tumor, the patient was given 4 cycles of doxorubicin, vincristine, and cyclophosphamide, alternating with ifosfamide and etoposide. After 18 months off therapy, he had a local recurrence at the same site. After a second total resection, he was given additional chemotherapy with 30.6-Gy local irradiation. No further recurrence has been observed for 5 years since the second complete remission. Currently, he is alive and well at 7.5 years post-onset. Our experience in this case suggests a fundamental strategy of successful treatment of this highly malignant pediatric tumor: (1) complete resection of the localized tumor, (2) intensive multiagent chemotherapy for the minimal disseminated disease, and (3) radiotherapy for local control of the disease.

Investigation of the possible association between nosocomial candiduria and candidaemia.

This study aimed to determine whether candiduria is associated with the occurrence of nosocomial candidaemia. In the case-control part of the study, 115 cases (nosocomial candidaemia) and 115 controls (nosocomial bacteraemia) were similar in age, severity of condition and time of hospitalisation. There was a significant association of candidaemia with candiduria (OR 9.79; 95% CI 2.14-44.76). In the microbiology part of the study, 23 pairs of Candida-positive urine and blood cultures were obtained from 23 patients. In ten (43%) cases, the urine and blood culture isolates belonged to different species, and molecular typing showed a difference in two of the 13 cases yielding the same species from both specimens. Overall, there was a significant association between candiduria and candidaemia, but the Candida isolates from urine and blood were different for 52% of the patients. Thus, the data indicated that the urinary tract was probably not a source for the candidaemia.

Diagnosis of candidemia by polymerase chain reaction and blood culture: prospective study in a high-risk population and identification of variables associated with development of candidemia.

The diagnosis of candidemia is important for prompt initiation of antifungal therapy. Two hundred twenty-five patients at high risk for candidemia who had blood cultures drawn and were hospitalized for more than 15 days were followed-up prospectively over a 2-year period. Polymerase chain reaction (PCR) and whole-blood cultures monitored by the automated BactAlert system (Organon Teknika, Durham, NC, USA) were used to detect candidemia in all patients hospitalized in high-risk areas for more than 15 days. DNA was extracted and amplified using ITS5 and ITS4 base pair primers, and the PCR products were sequenced for identification of Candida spp. A blood culture positive for Candida was considered the gold standard for diagnosis of candidemia. Variables associated with the development of candidemia diagnosed by positive blood culture were also evaluated in the patients. The overall mortality rate was 26.1%. Mortality in candidemic patients was 41.9% and in noncandidemic patients 22.5% (p = 0.009). PCR sensitivity and specificity were 72.1 and 91.2%, respectively. Positive and negative predictive values were 65.9 and 93.2%, respectively. The logistic regression of the multivariate analysis showed that parenteral nutrition (p < 0.0001), fever (p = 0.01), neutropenia (p = 0.04), and an indwelling urinary catheter (p = 0.02) were significant variables associated with the development of candidemia. The PCR technique in conjunction with DNA sequencing was a helpful tool in the diagnosis of candidemia.

Evaluation of Fusarium solani hyphae and conidia susceptibility to amphotericin B and itraconazole: study of a clinical case.

Fusarium species are hyaline moulds belonging to the hyalohyphomycosis group that are usually found in the soil and plants. This organism has emerged as a cause of disseminated invasive disease. The correlation between in vitro value and clinical efficacy is low and many patients remain unresponsive to treatment despite in vitro susceptibility. We determined growth control for Fusarium solani using the BioCell-Tracer system that measures the growth rate of a single fungal hypha, and the effect of different concentrations of amphotericin B and itraconazole. The MIC for these two drugs was also determined by a broth microdilution technique, using RPMI 1640. Different MICs for amphotericin B were obtained by the two different methods. This paper describes a case of infection due to Fusarium solani in an allogeneic bone marrow transplanted patient, the microbiological diagnostic, antifungal susceptibility tests for conidia and hypha and clinical correlation.

Antifungal susceptibility and pathogenic potential of environmental isolated filamentous fungi compared with colonizing agents in immunocompromised patients.

Infection is a major cause of morbidity and mortality in bone marrow transplant recipients and in patients with hematological malignancies. The source of infection is almost always endogenous flora or the hospital environment. The present study evaluated bone marrow transplant recipients and patients with hematological malignancies colonized and/or infected with filamentous fungi. During 1 year, environmental air samples were also taken from the bone marrow transplant unit by a modification of gravity air-setting plate (GASP) methodology. Fusarium spp. were the most prevalent genus in the fall and Cladosporium spp. in the winter. Clinically isolated strains grew better at 37 degrees C than environmental strains. According to NCCLS M-38P methods, environmental Aspergillus strains showed higher MICs to miconazol and itraconazol, and clinical Fusarium strains were less susceptible to fluconazole.

IL-4-producing CD8(+) T cells may be an immunological hallmark of chronic GVHD.

Chronic graft-versus-host disease (cGVHD) occurs in approximately 60-80% of those who survive over 100 days after allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, the pathophysiology of cGVHD is poorly understood. To gain more insight into the immunological mechanism of cGVHD, we examine cytokine production of peripheral blood T cells from 19 patients in the chronic phase of allo-HSCT. The percentage of IFN-gamma-producing CD8(+) T cells among CD8(+) T cells was significantly higher in patients with or without cGVHD than in normal control subjects (P<0.001). On the other hand, the percentage of IL-4-producing CD8(+) T cells among CD8(+) T cells was significantly higher in patients with cGVHD (mean 3.3%; range 1.3-8.2%) than in patients without cGVHD (mean 1.2%; range 0.8-1.7%) and normal control subjects (mean 1.1%; range 0.1-1.6%) (both P<0.001). By contrast, the percentage of IL-4-producing CD4(+) T cells was not different among patients with and without cGVHD and normal controls. These findings suggest that IL-4-producing CD8(+) T cells may be an immunological marker of cGVHD.

Antigenuria and antigenaemia in experimental murine paracoccidioidomycosis.

In this study, Swiss mice were experimentally infected with Paracoccidoides brasiliensis (Pb18) and we investigated the levels of gp43 in urine and plasma, anti-gp43 and IgG-gp43 immune complexes in plasma. These levels were correlated with the histopathological findings. Blood and urine samples were collected from mice at 7, 28, 56 and 84 days after intravenous inoculation of 10(5) yeast cells, and analysed by ELISA. The results showed increased levels of soluble gp43 in the plasma in all periods, and anti-gp43 IgG and immune complexes after day 28. High gp43 levels were detected in the urine, except for day 28, coincident with the presence of compact granulomas in lungs. All the infected mice showed fungal cells in the lungs, with initial granulomatous lesions at day 7, dissemination of lesions to other organs at day 56, and granulomas lacking the surrounding mononuclear cells infiltration, especially at days 56 and 84. Our results suggest that gp43 diffuses passively into the urine, and the determination of gp43 levels in urine samples may be a non-invasive alternative method for diagnosis and follow up of PCM. Further studies are needed to determine if the cellular immune response correlate with decreased urine gp43 levels.

Oral Candida flora from Brazilian human immunodeficiency virus-infected patients in the highly active antiretroviral therapy era.

One of the main opportunistic fungal infections amongst immunocompromised individuals is oral candidosis, which has been found in up to 90% of human immunodeficiency virus (HIV)-infected patients. This study employed yeasts isolated from the saliva and oral cavities of 114 HIV-infected patients living in Campinas, São Paulo. Of the isolates, 57.8% were identified as Candida albicans and 42.1% as non-C. albicans. The latter isolates were subsequently identified as C. krusei (7.5%), C. lusitaniae (5.2%), C. tropicalis (4.6%), C. parapsilosis (4.6%), C. glabrata (2.8%), C. kefyr (1.7%), C. guilliermondii (1.7%), C. intermedia (1.1%), C. norvegensis (0.5%), and Rhodotorula rubra (1.7%). Susceptibility of the isolates to amphotericin B, fluconazole, miconazole, and itraconazole was also determined by a microdilution method adopted by the National Committee for Clinical Laboratory Standards. The isolates demonstrated various susceptibilities to the antifungal agents. In particular 29 C. albicans and 13 non-C. albicans isolates showed low susceptibility to FLCZ (> 64 micro g/ml). This study revealed huge diversity of Candida species, in particular the increasing emergence of non-C. albicans associated with the oral flora of HIV-infected patients.

Oral Candida flora from Brazilian human immunodeficiency virus-infected patients in the highly active antiretroviral therapy era

One of the main opportunistic fungal infections amongst immunocompromised individuals is oral candidosis, which has been found in up to 90 percent of human immunodeficiency virus (HIV)-infected patients. This study employed yeasts isolated from the saliva and oral cavities of 114 HIV-infected patients living in Campinas, São Paulo. Of the isolates, 57.8 percent were identified as Candida albicans and 42.1 percent as non-C. albicans. The latter isolates were subsequently identified as C. krusei (7.5 percent), C. lusitaniae (5.2 percent), C. tropicalis (4.6 percent), C. parapsilosis (4.6 percent), C. glabrata (2.8 percent), C. kefyr (1.7 percent), C. guilliermondii (1.7 percent), C. intermedia (1.1 percent), C. norvegensis (0.5 percent), and Rhodotorula rubra (1.7 percent). Susceptibility of the isolates to amphotericin B, fluconazole, miconazole, and itraconazole was also determined by a microdilution method adopted by the National Committee for Clinical Laboratory Standards. The isolates demonstrated various susceptibilities to the antifungal agents. In particular 29 C. albicans and 13 non-C. albicans isolates showed low susceptibility to FLCZ (> 64 æg/ml). This study revealed huge diversity of Candida species, in particular the increasing emergence of non-C. albicans associated with the oral flora of HIV-infected patients.

Blood dendritic cells are decreased in acute graft-versus-host disease.

The recipients of allogeneic hematopoietic stem cell transplantation (allo-HSCT) often develop acute graft-versus-host disease (aGVHD), which is closely related to morbidity and mortality. However, the essential part of the immune responses elicited in aGVHD remains largely unknown. We attempt to determine if peripheral blood dendritic cells (PBDCs) are altered in aGVHD, and find that the number of PBDCs (both myeloid and lymphoid DCs) is significantly decreased. Immunohistochemical staining of the biopsied skin from patients with aGVHD demonstrates that a number of fascin(+) cells with dendritic projections infiltrate the dermis of the skin. Based on these findings, we hypothesize that the PBDCs are recruited to the affected tissues and may thus play important roles in immune responses elicited in aGVHD.

Phaeohyphomycosis caused by Chaetomium globosum in an allogeneic bone marrow transplant recipient.

Bone marrow transplant recipients are highly susceptible to opportunistic fungal infections. This is the report, of the first case of a Chaetomium systemic infection described in Brazil. A 34 year-old patient with chronic myeloid leukemia underwent an allogeneic sibling matched bone marrow transplant. Seven months later, he developed systemic infection with enlargement of the axillary and cervical lymph nodes. Culture of the aspirates from both lymph nodes yielded Chaetomium globosum. The infection was successfully treated with amphotericin B. The increasing population of immunosupressed patients requires a careful microbiologic investigation for uncommon fungal infections.

Synergistic antifungal effect of fluconazole and human polymorphonuclear leukocytes on Paracoccidioides brasiliensis: effect of interferon-gamma and granulocyte-macrophage colony-stimulating factor.

To better understand the in vivo efficacy of fluconazole (FCZ), we investigated the possible synergy of fungistatic FCZ with human polymorphonuclear leukocytes (PMN) against Paracoccidioides brasiliensis (Pb). The effect of interferon-gamma (IFN-gamma) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in this system was also studied. For this purpose, FCZ, PMN, PMN + FCZ, PMN + IFN-gamma, PMN + IFN-gamma + FCZ, PMN + GM-CSF and PMN + GM-CSF + FCZ were co-cultured with Pb and the cfu of Pb was measured. The antifungal effect of FCZ on yeast cells of Pb was concentration-dependent. At 0.1 microg ml(-1), FCZ had no effect on the growth of Pb. At 0.2 microg ml(-1) FCZ showed a growth-inhibitory effect on three isolates of Pb in a long-term (120 h) assay, and at 0.6 microg ml(-1) or higher FCZ was fungicidal. Fungistatic concentration of FCZ (0.4 microg ml(-1)) acted synergistically with fungistatic PMN for killing isolate Bt-4 during the first 24 h of co-culture. Moreover, IFN-gamma and GM-CSF substantially enhanced the synergistic antifungal effect of PMN and FCZ. These findings provide a better understanding of why FCZ is more efficacious in in vivo models of paracoccidioidomycosis than is predicted by in vitro susceptibility tests.