Extracts of Talaromyces purpureogenus Strains from Apis mellifera Bee Bread Inhibit the Growth of Paenibacillus spp. In Vitro.

Honey bees coexist with fungi that colonize hive surfaces and pollen. Some of these fungi are opportunistic pathogens, but many are beneficial species that produce antimicrobial compounds for pollen conservation and the regulation of pathogen populations. In this study, we tested the in vitro antimicrobial activity of Talaromyces purpureogenus strains isolated from bee bread against Paenibacillus alvei (associated with European foulbrood disease) and three Aspergillus species that cause stonebrood disease. We found that methanol extracts of T. purpureogenus strains B18 and B195 inhibited the growth of P. alvei at a concentration of 0.39 mg/mL. Bioactivity-guided dereplication revealed that the activity of the crude extracts correlated with the presence of diketopiperazines, a siderophore, and three unknown compounds. We propose that non-pathogenic fungi such as Talaromyces spp. and their metabolites in bee bread could be an important requirement to prevent disease. Agricultural practices involving the use of fungicides can disrupt the fungal community and thus negatively affect the health of bee colonies.

Crude Extracts of Talaromyces Strains (Ascomycota) Affect Honey Bee (Apis mellifera) Resistance to Chronic Bee Paralysis Virus.

Viruses contribute significantly to the global decline of honey bee populations. One way to limit the impact of such viruses is the introduction of natural antiviral compounds from fungi as a component of honey bee diets. Therefore, we examined the effect of crude organic extracts from seven strains of the fungal genus Talaromyces in honey bee diets under laboratory conditions. The strains were isolated from bee bread prepared by honey bees infected with chronic bee paralysis virus (CBPV). The antiviral effect of the extracts was also quantified in vitro using mammalian cells as a model system. We found that three extracts (from strains B13, B18 and B30) mitigated CBPV infections and increased the survival rate of bees, whereas other extracts had no effect (B11 and B49) or were independently toxic (B69 and B195). Extract B18 inhibited the replication of feline calicivirus and feline coronavirus (FCoV) in mammalian cells, whereas extracts B18 and B195 reduced the infectivity of FCoV by ~90% and 99%, respectively. Our results show that nonpathogenic fungi (and their products in food stores) offer an underexplored source of compounds that promote disease resistance in honey bees.

Combination treatment of rat adjuvant-induced arthritis with methotrexate, probiotic bacteria Enterococcus faecium, and selenium.

The effects of the probiotic bacteria Enterococcus faecium (EF) and selenium were studied on methotrexate (MTX) treatment in rats with adjuvant arthritis. Arthritic rats were treated orally with the following substances: lyophilized EF (15 mg/kg/day, 5 days a week), sodium selenite pentahydrate (SSe; 0.050 mg/kg containing 0.015 mg/kg selenium, 5 days a week), MTX (0.6 mg/kg/week), and their combinations for the period of 50 days from adjuvant application. Levels of serum albumin, serum nitrite/nitrate concentrations, hind paw swelling, arthrogram scores, whole-body bone mineral density (BMD), and bone erosions were evaluated as markers of inflammation and destructive changes associated with arthritis. Long-term preventive treatment with low-dose MTX significantly inhibited the markers of both inflammation and arthritis. EF or SSe when administered singly or in combination had no significant effect on the given parameters in arthritic rats. EF, but not SSe, potentiated the beneficial effects of MTX, which resulted in a more significant reduction of hind paw swelling, arthrogram scores, and whole-body BMD decrease. EF also had a tendency to improve the effect of MTX on serum albumin and nitrite/nitrate concentrations. Our results indicate that EF may increase the preventive effect of MTX treatment in rat adjuvant arthritis by improving its anti-inflammatory and antiarthritic effects.

The effects of Enterococcus faecium and selenium on methotrexate treatment in rat adjuvant-induced arthritis.

The effects of probiotic bacteria Enterococcus faecium (EF) and selenium were studied on methotrexate (MTX) treatment in rats with adjuvant arthritis (AA). Arthritic rats were preventive treated orally with the following substances: lyophilized EF (15mg/kg/day, 5 days a week); sodium selenite pentahydrate (SSe, 0.050mg/kg containing 0.015 mg/kg selenium, 5 days a week); MTX (0.6 mg/kg/week), and their combinations for the period of 50 days from adjuvant application. Levels of serum albumin, serum nitrite/nitrate concentrations, hind paw swelling, arthrogram scores, whole body bone mineral density (BMD), and bone erosions were evaluated as markers of inflammation and destructive changes associated with arthritis. Long-term preventive treatment with low-dose MTX significantly inhibited the markers of both inflammation and arthritis. EF or SSe when administered singly or in combination had no significant effect on given parameters in arthritic rats. EF but not SSe potentiated the beneficial effects of MTX, which resulted in a more significant reduction of hind paw swelling, arthrogram scores and whole body BMD decrease. EF had a tendency to improve also the effect of MTX on serum albumin and nitrite/nitrate concentrations. Our results indicate that EF may increase the preventive effect of MTX treatment in rat AA by improving its anti-inflammatory and anti-arthritic effects.

Cyclosporine blood level monitoring. Cross-reactivity of anti-cyclosporine A monoclonal with its sulphate metabolite: an in vitro study.

Cyclosporine is metabolized in the liver by the Cytochrom P450 system. Many of the metabolites have been identified and thoroughly studied. However the sulphate metabolite is the least known. The metabolite is hydrophilic and thus it is not easily detectable by HPLC and it is not available in pure form for analysis. We have isolated the metabolite from transplant patients receiving CYA as part of their immunosuppressive therapy. The amino acid sequence was determined and the molecule was synthesised in our laboratories. We have tested the molecule in vitro using cell culture to determine its activity. 1/2 ml of blood+1/2 ml of RPMI was incubated with the following concentration of either CSA or CSS: 0, 25, 50, 100, 250, 500, 750, 1000, 1250, 1500, 2000 ng/ml for 2 and 1/2h at 37 degrees C with 5% CO2. The blood was then stimulated with Ionomycine and PMA (phorpol 12 myristate 13-acetate) for additional 2h. Supernatant was collected and assayed for the concentration of the following cytokines: IL-1a, IL-2, Interferon (IFNa), IL-6, IL-12, TNFa. The cells were used to evaluate the expression of cell activation marker CD69. Blood was assayed for CYA concentration using Abbott TDx assay. Results the level of the metabolite was actually higher than the parent compound indicating that the monoclonal antibodies detected the S form preferentially. The results with other CYA monoclonal was also similar. These results suggest that blood level monitoring of CYA many not be accurate as all the monoclonal antibodies currently used cross react with the metabolites.

Effects of the peptide mycotoxin destruxin E on insect haemocytes and on dynamics and efficiency of the multicellular immune reaction.

Destruxins (DTXs) are cyclic peptide toxins secreted by the entomopathogenic fungus Metarhizium anisopliae var. anisopliae. The effects of DTX E, the most active compound of this family on haemocytes, the immunocompetent insect cells, and on the dynamics and efficacy of the multicellular defense of insect hosts have been investigated. Ultrastructural alterations have been observed in circulating plasmatocytes and granular haemocytes, and in attached haemocytes of Galleria mellonella larvae treated with a toxic dose of DTX E (LC50). These changes appear as a consequence of disturbances induced in the cellular calcium balance. An effect on the cell surface of granulocytes was also noted in cells incubated with the toxin and FITC-Con A, even when the concentration of DTX was as low as 0.005 microg/ml. Morphological studies of haemocytic capsules formed in vivo revealed disturbances of the multicellular defense mechanism after toxin treatment However, an attempt to establish if these changes were significant was unsuccessful. In contrast, comparative assays regarding the effect of toxin treatment on the efficacy of the antifungal effect of encapsulation has given conclusive results. The germination of injected Aspergillus niger spores became slightly but significantly increased, and when the granuloma were incubated the fungus escaped more easily from the haemocytic envelope. These results are discussed in terms of significance of the contribution of DTXs to the fungal infection process. It is suggested that the fungal peptides may intervene during the disease by a true immune-inhibitory effect occurring at doses which do not cause paralysis or any general sign of toxicity (e.g., 0.8 microg/g of body weight).