Does equipment change impact blood contamination with irinotecan and its two major metabolites in a centralized cytotoxic pharmacy unit?

BACKGROUND: Antineoplastic drugs exposure is a major problem for caregivers' health. The aim of this study is to assess blood contamination with irinotecan and its two metabolites in a centralized pharmacy unit for cytotoxic drug preparations workers before and after protective equipment changes. METHODS: The study took place in a university hospital centralized pharmacy unit for cytotoxic drug and was performed in two parts, before (Round 1: R1) and after equipment changes (Round 2: R2). Collection of pharmacy staff blood samples was performed in UHPLC-MS/MS. Plasma and red blood cell irinotecan and its metabolites (SN38; APC) were determined with a validated analytical method detection test. RESULTS: A total of 15/36 (41.6%) assays were positive in R1 and 16/72 (22.2%) in R2 with a significant decrease between periods (P = 0.035). For plasma dosages, no difference between the two periods was found (P = 0.71); respectively 4/18 (22.2%) assays were positive in R1 and 6/36 (16.6%) in R2. For red blood cells dosages, a significant decrease between periods was found (P = 0.01); respectively 11/18 (61%) were positive in R1 and 10/36 (27.8%) in R2. CONCLUSIONS: These dosages make it possible to have the very first evaluation for plasma and red blood cell contamination with irinotecan and its metabolites in the context of equipment changes, both at individual and collective levels. This work would help to protect health workers from the potential risks represented by these molecules, especially by revealing a contamination of workers in order to objectify the results of exposure.

Low dose Rituximab for pre-emptive treatment of Epstein Barr virus reactivation after allogenic hematopoietic stem cell transplantation.

INTRODUCTION: The most used preemptive therapy for Epstein Barr virus reactivation post allogeneic hematopoietic stem cell (HSCT) transplant is Rituximab, 375 mg/m2, once weekly until EBV viremia negativity. There is no data suggesting such a high dose. OBJECTIVE: We hypothesized that a lower dose of Rituximab would be as efficient with less toxicity. PATIENTS: In a retrospective, monocentric study, we analyzed 16 consecutive patients treated preemptively with low dose Rituximab for EBV reactivation post HSCT. Patients were treated with low Rituximab dose of 100 mg/m² weekly. Success was defined by a decrease of EBV viremia of 1 log10 and below 1000 UI/ml, and the absence of post-transplant lymphoproliferative disorder (PTLD). RESULTS: Success rate was 93.4% (15/16). One (1/16, 6%) PTLD was diagnosed after preemptive therapy, despite a negative viremia. CONCLUSION: A low dose of Rituximab of 100 mg/m² per injection for pre-emptive therapy of EBV reactivation post HSCT is safe and effective for preventing PTLD. Prospective, randomized, multicentric trials with larger number of patient are needed to determine the best rituximab dose.

Cloning and characterization of chitinases from interior spruce and lodgepole pine.

Chitinases have been implicated in the defence of conifers against insects and pathogens. cDNA for six chitinases were cloned from interior spruce (Picea glauca x engelmannii) and four from lodgepole pine (Pinus contorta). The cloned interior spruce chitinases were annotated class I PgeChia1-1 and PgeChia1-2, class II PgeChia2-1, class IV PgeChia4-1, and class VII PgeChia7-1 and PgeChia7-2; lodgepole pine chitinases were annotated class I PcChia1-1, class IV PcChia4-1, and class VII PcChia7-1 and PcChia7-2. Chitinases were expressed in Escherichia coli with maltose-binding-protein tags and soluble proteins purified. Functional characterization demonstrated chitinolytic activity for the three class I chitinases PgeChia1-1, PgeChia1-2 and PcChia1-1. Transcript analysis established strong induction of most of the tested chitinases, including all three class I chitinases, in interior spruce and lodgepole pine in response to inoculation with bark beetle associated fungi (Leptographium abietinum and Grosmannia clavigera) and in interior spruce in response to weevil (Pissodes strobi) feeding. Evidence of chitinolytic activity and inducibility by fungal and insect attack support the involvement of these chitinases in conifer defense.

Diversity and decay ability of basidiomycetes isolated from lodgepole pines killed by the mountain pine beetle.

When lodgepole pines (Pinus contorta Douglas ex Louden var. latifolia Engelm. ex S. Watson) that are killed by the mountain pine beetle (Dendroctonus ponderosae) and its fungal associates are not harvested, fungal decay can affect wood and fibre properties. Ophiostomatoids stain sapwood but do not affect the structural properties of wood. In contrast, white or brown decay basidiomycetes degrade wood. We isolated both staining and decay fungi from 300 lodgepole pine trees killed by mountain pine beetle at green, red, and grey stages at 10 sites across British Columbia. We retained 224 basidiomycete isolates that we classified into 34 species using morphological and physiological characteristics and rDNA large subunit sequences. The number of basidiomycete species varied from 4 to 14 species per site. We assessed the ability of these fungi to degrade both pine sapwood and heartwood using the soil jar decay test. The highest wood mass losses for both sapwood and heartwood were measured for the brown rot species Fomitopsis pinicola and the white rot Metulodontia and Ganoderma species. The sap rot species Trichaptum abietinum was more damaging for sapwood than for heartwood. A number of species caused more than 50% wood mass losses after 12 weeks at room temperature, suggesting that beetle-killed trees can rapidly lose market value due to degradation of wood structural components.

Characterization of microsatellite loci in the fungus, Grosmannia clavigera, a pine pathogen associated with the mountain pine beetle.

The largest forest pest epidemic in Canadian history caused by the mountain pine beetle (MPB) and its fungal associates has killed over 15 million hectares of forest. Sixty simple sequence repeat regions were identified from Grosmannia clavigera, an MPB associated fungus. Eight loci genotyped in 53 isolates from two populations in British Columbia, Canada revealed three to 10 alleles per locus and gene diversities of 0 to 0.79. All but two of these loci showed length polymorphism in Leptographium longiclavatum, a related MPB fungal associate. These microsatellites will be useful in population genetic studies of these fungi.

Genetic Diversity and the Presence of Two Distinct Groups in Ophiostoma clavigerum Associated with Dendroctonus ponderosae in British Columbia and the Northern Rocky Mountains.

ABSTRACT The sapstaining fungal pathogen Ophiostoma clavigerum is associated with the mountain pine beetle (Dendroctonus ponderosae), which is currently the most destructive forest pest in North America. The genetic diversity of O. clavigerum populations collected from five sites in Canada and two sites in the United States was estimated with amplified fragment length polymorphism (AFLP) analysis. Genomic DNA from 170 O. clavigerum isolates was digested with EcoRI and PstI and amplified with six primer sets. A total of 469 AFLP markers consisting of 243 monomorphic and 226 polymorphic loci were scored. The overall genetic diversity of the O. clavigerum population was low (Hs = 0.0531) and the differentiation of the seven O. clavigerum populations was moderate (Phi = 0.143). Genetic distances among the populations were not significantly correlated with geographic distance (r = 0.3235, P = 0.074). Two genetically distinct groups in the O. clavigerum populations were shown by unique AFLP profiles and the unweighted pair group method with arithmetic averages. Further work to characterize biological differences between the two groups will be needed to confirm whether cryptic species are present in the O. clavigerum population.

A spontaneous albino mutant of Ceratocystis resinifera results from a point mutation in the polyketide synthase gene, PKS1.

We characterized a spontaneous albino mutant of Ceratocystis resinifera. Compared with the wild-type progenitor strain, the albino mutant had a reduced linear growth on culture medium, but its growth on lodgepole pine sapwood was unaffected. The albino mutant did not produce any coloured pigment on agar media or wood. However, upon exposure to exogenous scytalone, an intermediate metabolite of the melanin pathway, the production of a brownish melanin was restored. This suggests that the albino phenotype resulted from a mutation affecting the melanin synthesis pathway, upstream of the scytalone synthesis step. Melanin production was restored in the mutant by transforming it with a wild-type copy of the Ceratocystis resinifera polyketide synthase gene, PKS1. The complemented transformants produced melanin, indicating that the PKS1 gene was defective in the albino mutant. Sequence analysis revealed that the PKS1 allele found in the albino contained a single point mutation that resulted in an amino acid change from serine to proline at the 3' end of the beta-ketoacyl synthase motif.

A second reductase gene involved in melanin biosynthesis in the sap-staining fungus Ophiostoma floccosum.

Two different DNA sequences amplified by PCR from Ophiostoma floccosum show high homology to reductase genes of the fungal melanin biosynthetic pathway and suggest that two reductases may participate in this pathway in O. floccosum. Previously, one reductase gene, THN1 had been isolated from O. floccosum and proved to be involved in melanin biosynthesis. The second reductase gene, THN2, encodes a protein of 284 amino acids and is the longest fungal melanin reductase so far reported. The protein sequences deduced from the two THN genes show 44% identity. The products of THN1 and THN2 belong to two different groups of melanin reductases based on a comparative analysis of sequences from several fungal species. We confirmed the function of the THN2 gene by complementing dihydoxynaphthalene (DHN)-melanin-deficient, non-pathogenic mutants of Colletotrichum lagenarium and Magnaporthe grisea that lack the 1,3,8-trihydroxynaphthalene reductase. THN2 transcripts were detected in these transformants. These melanin-producing transformants showed mycelial and appressorium melanization. C. lagenarium transformants that formed melanin exhibited pathogenicity. This suggests that the cloned THN2 gene product functions as the 1,3,8-trihydroxynaphthalene reductase in the DHN melanin biosynthetic pathway in both C. lagenarium and M. grisea.

A scytalone dehydratase gene from Ophiostoma floccosum restores the melanization and pathogenicity phenotypes of a melanin-deficient Colletotrichum lagenarium mutant.

Scytalone dehydratase is involved in the production of fungal dihydroxynaphthalene (DHN) melanin. We have isolated and characterized OSD1, a gene encoding scytalone dehydratase from the sap-staining fungus Ophiostoma floccosum by PCR-based cloning. Sequence analysis suggests that the OSD1 gene encodes a protein of 216 amino acids with a molecular weight of 24.2 kDa that shows 51-70% sequence identity to other scytalone dehydratases. The cloned OSD1 contains two introns of 76 bp and 63 bp in length, and is the longest scytalone dehydratase gene sequence so far reported. Transformation of a DHN melanin-deficient, non-pathogenic, mutant of Colletotrichum lagenarium with the OSD1 gene restored melanin production and pathogenicity. The ability of the mutant to produce the OSD1 gene product was confirmed by RT-PCR analysis. These data show that the cloned OSD1 gene product can function in the DHN melanin biosynthetic pathway in C. lagenarium.

Gas chromatography-mass spectrometry method for the simultaneous determination of wood extractive compounds in quaking aspen.

We have developed a rapid gas chromatography-mass spectrometry (GC-MS) method for the detailed compositional analysis of 70 underivatized wood extractive components present in quaking aspen (Populus tremuloides Michx.). Forty-four compounds were unequivocally identified by retention time and mass spectral comparison with standards. An additional 26 chromatographic peaks were assigned to broad chemical classes using retention time and mass spectra features. The results were compared to the respective tert.-butyldimethylsilyl derivatized wood extractives profile, and it was determined that derivatization was unnecessary for the GC-MS analysis of the target compounds.

Phylogenetic relationship of Ophiostoma piliferum to other sapstain fungi based on the nuclear rRNA gene.

The nuclear rRNA gene of Ophiostoma piliferum was analyzed to understand its phylogenetic relationships to other sapstain fungi. Phylograms based on nucleotide sequences of the rRNA gene showed that the relationships between O. piliferum and other Ophiostoma species varied depending on the regions of the rRNA gene analyzed. Intraspecies variation in O. piliferum was found in the internal transcribed spacer regions, and the variation was related to the geographic origin of O. piliferum strains. A useful molecular marker for differentiating O. piliferum from other sapstain Ophiostoma species was generated by the HaeIII restriction fragment length polymorphism of the 26S rRNA gene.

Rapid detection of Ophiostoma piceae and O. quercus in stained wood by PCR.

A rapid, sensitive, and simple method was developed to detect the sapstain fungi Ophiostoma piceae and O. quercus in stained wood. By using microwave heating for DNA extraction and PCR with internal transcribed spacer-derived-specific primers, detection was feasible within 4 h, even with DNA obtained from a single synnema. This method can easily be extended for the detection of other wood-inhabiting fungi.

Isolation and characterization of a subtilisin-like serine proteinase secreted by the sap-staining fungus Ophiostoma piceae.

Identification of key enzymes of sapstain fungi which cause wood discoloration is necessary for targeted inhibition strategies. Therefore proteinases involved in the nitrogen pathway have been characterized. The sap-staining fungus Ophiostoma piceae strain 387N produced proteolytic enzymes when grown on wood and protein-supplemented media. Proteolytic activity in culture filtrates was inhibited by PMSF and EDTA. The major protein in culture filtrates was a proteinase with a pI of 5.6 and a molecular weight of 33 kDa. This was the major proteinase produced by O. piceae and it was purified from culture filtrates by hydrophobic interaction chromatography. The proteinase was susceptible to autolytic degradation during chromatographic separations when ammonium sulfate was not present. When azocoll was used as a substrate, the proteolytic activity of the purified proteinase was determined to be optimal at pH 7-9 and 40 degrees C. Similar pH and temperature optima were obtained using succinyl-ala-ala-pro-phe-p-nitroanilide as the substrate. The N-terminal sequence of the protein showed a high degree of homology with fungal alkaline serine proteinases classified as subtilisin class II enzymes. Agreement in inhibition patterns and electrophoretic and catalytic properties suggested the secretion of the same proteinase during growth on wood. Understanding the role of this proteinase during fungal colonization is an important step toward disrupting fungal growth on wood.

Characterization of the cleavage specificity of a subtilisin-like serine proteinase from Ophiostoma piceae by liquid chromatography/mass spectrometry and tandem MS.

A proteinase secreted by the sapstaining fungus Ophiostoma piceae is thought to be necessary for the primary retrieval of nitrogen from wood proteins. By using mass spectrometry (MS) techniques, we have established the cleavage specificity of this subtilisin-like serine proteinase. This work demonstrated the potential of MS in determining cleavage specificities of newly isolated proteinases in a relatively short time frame, and determined that the O. piceae proteinase showed a substrate specificity similar to that of proteinase K. Primary cleavage of the insulin B-chain occurred between Leu15 and Tyr16. In addition numerous secondary cleavage sites occurred after hydrophobic, polar, and charged amino acids indicating a broad specificity.

Factors affecting autolysis of a subtilisin-like serine proteinase secreted by Ophiostoma piceae and identification of the cleavage site.

The extracellular serine proteinase secreted by Ophiostoma piceae was degraded by autoproteolysis under certain conditions. At elevated temperatures the mature protein of 33 kDa was rapidly degraded without any accumulation of protein breakdown products. Glycerol, calcium ions and ammonium sulfate raised the heat stability of the enzyme, increasing its half-life at 45 degrees C from 1.9 min to 9.4 min, 40.4 min and 2 h, respectively. Thermal unfolding of the proteinase also occurred at higher temperatures in the presence of calcium ions and ammonium sulfate. Under conditions of heating, altered pH or partial depletion of protein-bound ions by EDTA, the structure of the proteinase was more susceptible to proteolysis. The major hydrolytic fragments of 19 kDa and 14 kDa, had N-terminal sequences of Ala1-Tyr2-Thr3-Thr4-Gln5-Thr6-Gly7-Ala8-Pro9-and Ser170-Glu171-Pro172-Se173-Val174-X-Thr 176-Val177-Gly178-Ala179-, respectively. Since the former sequence was identical to the N-terminus of the native protein, the major autoproteolytic cleavage site for a class II subtilase appeared to be the N-side of Ser170 using numbering based on the sequence of proteinase K. The secondary structure of the proteinase from O. piceae was similar to that of proteinase K based on circular dichroic spectra in the far ultraviolet region. This cleavage site was in a similar region to that identified for class I subtilases, which was located in an outer exposed loop of the tertiary structure of subtilisin-like serine proteinases.

Biological degradation of resin acids in wood chips by wood-inhabiting fungi.

Resin acids in many pulp mill effluents are primary sources of toxicity to fish. Inconsistent biological detoxification of chlorinated and nonchlorinated resin acids in secondary treatment of pulp mill effluents is a continuing source of concern. An alternative approach to effluent detoxification is to remove or modify the toxic compounds present in wood chips prior to pulping. Results from experiments in which lodgepole pine sapwood chips were inoculated with several fungal candidates indicate that the total resin acid content can be reduced by up to 67% after fungal growth. Such a treatment could be an efficient and environmentally acceptable way for deresinating wood chips and so decreasing the toxicity of pulp mill effluents.

Extracellular lipase production by a sapwood-staining fungus, Ophiostoma piceae.

The extracellular lipase production of a sapwood-staining fungus, Ophiostoma piceae, grown in liquid media, was optimally active at pH 5.5 and 37°C. Although glucose, fructose, sucrose, starch and dextrin, as carbon sources for growth gave similar mycelial yields, which were higher than those obtained with arabinose, galactose or raffinose, the cells growing on those carbohydrates produced little extracellular lipase. However, both high biomass and lipase activity were obtained when plant oils (olive, soybean, corn, sunflower seed, sesame, cotton seed or peanut) were used as carbon sources. Among the nitrogen sources examined, Casamino acids gave the best growth, whereas (NH4)2SO4 gave the best lipase production. The highest lipase productivity seen was obtained in a medium with olive oil as carbon source and a combination of (NH4)2SO4and peptone as nitrogen source.

A comparison of two xylanases from the thermophilic fungi Thielavia terrestris and Thermoascus crustaceus.

Two thermophilic xylanases (xylanase II from Thielavia terrestris 255B and the 32-kDa xylanase from Thermoascus crustaceus 235E) were studied to determine if they had different and complementary modes of action when they hydrolysed various types of xylans. Partial amino acid sequencing showed that these two enzymes belonged to different families of beta-1,4-glycanases. Xylanase II achieved faster solubilization of insoluble xylan whereas the 32-kDa xylanase was more effective in producing xylose and short xylo-oligomers. An assessment of the combined hydrolytic action of the two xylanases did not reveal any co-operative action. The sugars released when the two thermophilic xylanases were used together were almost identical to those released when the 32-kDa xylanase acted alone. The two xylanases were able to remove about 12% of the xylan remaining in an aspen kraft pulp. This indicated that either one of these thermophilic enzymes may be useful for enhancing the bleaching of kraft pulps.

Monoclonal antibodies to Gliocladium roseum, a potential biological control fungus of sap-staining fungi in wood.

Immunological probes were developed to discriminate between a potential biological control fungus and sap-staining fungi present in wood. This paper describes the production of monoclonal antibodies to isolated cell wall fragments of the biological control fungus Gliocladium roseum. Two monoclonals, designated 6A5 and 3F12, were characterized. Their specificity was assessed by ELISA, by immunogold silver staining light microscopy, by immunogold electron microscopy, and by immunoblotting. Monoclonal 6A5 specifically recognized G. roseum and closely related species and did not react with any of 21 sap-staining fungi tested. Monoclonal 3F12 recognized most of the biological control fungi tested and also showed reactivity with two of the 21 sap-staining fungi. Both monoclonals appeared to recognize carbohydrate epitopes of the cell wall in G. roseum. Although the antibodies were produced against the cell wall of fungus grown in liquid culture, they also detected specific fungi in wood and, therefore, can be used for studies of wood colonization by fungi and for investigations of the interactions between different fungi growing on wood.

Purification and characterization of a xylanase from the thermophilic ascomycete Thelavia terrestris 255B.

Thielavia terrestris 255B, a thermophilic ascomycete, produced two major forms of xylanase with pIs of 4.6 (xylanase I) and 6.1 (xylanase II). The latter enzyme could be purified to greater than 99% homogeneity using anion-exchange chromatography and gel filtration. Xylanase II had a mol wt of 25.7 kDa (SDS-PAGE) and a pH and a temperature optimum of 3.6-4.0 and 60-65 degrees C, respectively. The ratio of the enzyme's activity against xylan and carboxymethylcellulose was 500-1000 to 1, indicating a possible application of this enzyme in biobleaching processes. The amino acid sequence of this protein is being determined, and initial data suggest that the enzyme belongs to a group of low-mol wt xylanases that have been isolated from both bacteria and fungi.