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Anti-thymocyte globulin (ATG) based Graft versus Host disease (GVHD) prophylaxis is widely used for unrelated donor allogeneic hematopoietic cell transplantation (HCT) although optimal dose remains unclear. Registry-based and single-center retrospective analyses have suggested superior outcomes with PTCy-based GVHD prophylaxis regimens when compared to ATG-based in mismatched unrelated donor (MMUD) allogeneic HCT. However, these studies used doses of ATG ≥ 5mg/kg. Outcomes following MMUD allogeneic HCT with lower dose ATG-based GVHD prophylaxis regimens have not been extensively studied. Thus, we analyzed outcomes of HLA 9/10 MMUD allogeneic HCTs using lower dose ATG-based regimens at our centre.

INTRODUCTION

The Candida albicansfitness test is a whole cell screening platform that utilizes a mixed-pool of C. albicansmutants, each of which carries a heterozygous deletion of a particular gene. In the presence of an antifungal inhibitor, a subset of these mutants exhibits a growth phenotype of hypersensitivity or hyposensitivity. Collectively these mutants reflect aspects of the mechanism of action of the compound in question. In the course of screening natural products a culture of Streptomycessp. MS-1-4 was discovered to produce a compound, dretamycin, which yielded a fitness profile exhibiting significant hypersensitivity of the DRE2heterozygote and hyposensitivity of the DIP5heterozygote. Herein we report the production, isolation, and structure elucidation of dretamycin.

We developed a murine model of CNS disease to obtain a better understanding of the pathogenesis of CNS involvement in pre-B-cell acute lymphoblastic leukemia (ALL). Semiquantitative proteomic discovery–based approaches identified unique expression of asparaginyl endopeptidase (AEP), intercellular adhesion molecule 1 (ICAM1), and ras-related C3 botulinum toxin substrate 2 (RAC2), among others, in an invasive pre-B-cell line that produced CNS leukemia in NOD-SCID mice. Targeting RAC2 significantly inhibited in vitro invasion and delayed disease onset in mice. Induced expression of RAC2 in cell lines with low/absent expression of AEP and ICAM1 did not result in an invasive phenotype or murine CNS disease. Flow cytometric analysis identified an enriched population of blast cells expressing ICAM1/lymphocyte function associated antigen-1 (LFA-1)/CD70 in the CD10+/CD19+fraction of bone marrow aspirates obtained from relapsed compared with normal controls and those with primary disease. CD10+/CD19+fractions obtained from relapsed patients also express RAC2 and give rise to CNS disease in mice. Our data suggest that combinations of processes are involved in the pathogenesis of CNS disease in pre-B-cell ALL, support a model in which CNS disease occurs as a result of external invasion, and suggest that targeting the processes of adhesion and invasion unique to pre-B cells may prevent recurrences within the CNS.

Although survival of children with acute lymphoblastic leukaemia has improved greatly in the past two decades, the outcome of those who relapse has remained static. We investigated the outcome of children with acute lymphoblastic leukaemia who relapsed on present therapeutic regimens.

Thiazolyl peptides are a class of highly rigid trimacrocyclic compounds consisting of varying but large numbers of thiazole rings. The need for new antibacterial agents to treat infections caused by resistant bacteria prompted a reinvestigation of this class, leading to the previous isolation of thiazolyl peptides, namely, thiazomycin (5) and thiazomycin A (6), congeners of nocathiacins (1−4). Continued chemical screening led to the isolation of six new thiazolyl peptide congeners (8−13), of which three had truncated structures lacking an indole residue. From these, compound 8showed activity similar to thiazomycin. Two compounds (9and 10) showed intermediate activities, and the three truncated compounds (11−13) were essentially inactive. The discovery of the truncated compounds revealed the minimal structural requirements for activity and suggested probable biosynthetic pathways for more advanced compounds. The isolation, structure elucidation, antibacterial activity, and proposed biogenesis of thiazomycins are herein described.

Several protocols for bacterial isolation and techniques for aerobic plate counting rely on the use of a spiral plater to deposit concentration gradients of microbial suspensions onto a circular agar plate to isolate colony growth. The advantage of applying a gradient of concentrations across the agar surface is that the original microbiological sample can be applied at a single concentration rather than as multiple serial dilutions. The spiral plater gradually dilutes the sample across a compact area and therefore saves time preparing dilutions and multiple agar plates. Commercial spiral platers are not automated and require manual sample loading. Dispensing of the sample volume and rate of gradients are often very limited in range. Furthermore, the spiral sample application cannot be used with rectangular microplates. Another limitation of commercial spiral platers is that they are useful only for dilute, filtered suspensions and cannot plate suspensions of coarse organic particles therefore precluding the use of many kinds of microorganism-containing substrata. An automated agar plate spreader capable of processing 99 rectangular microplates in unattended mode is described. This novel instrument is capable of dispensing discrete volumes of sample in a linear pattern. It can be programmed to dispense a sample suspense at a uniform application rate or across a decreasing concentration gradient.

The importance of pneumocandin B0 as the fermentation-derived starting material for the antifungal drug candidate, MK-991, along with the identification of our production strain as Z. arboricola (ATCC 20868) as CBS prompted a search for other strains of Z. arboricola or Zalerion species with improved titres or that might produce natural pneumocandin analogues. Analysis of morphology, secondary metabolites profiles, and DNA fingerprinting demonstrated that ATCC 20868 was not congeneric with Z. arboricola. Ribosomal DNA sequences were compared among Zalerion species and pneumocandin-producing fungi and with rDNA sequences in GenBank. No good matches with sequences in GenBank were obtained for Z. arboricola or Z. maritimum, but for Z. varium, P. carpinea and ATCC 20868, relevant similarities were observed with ITS1 sequences from fungi of Leotiales. ATCC 20868 was phylogenetically more akin to P. carpinea, another pneumocandin producer, than initially suspected. The closest relative of ATCC 20868 seemed to be Hymenoscyphus monotropae. We conclude that the genus Zalerion is artificial; its species bear no phylogenetic relation among themselves. ATCC 20868 and Z. varium were related to fungi of the Leotiales. We propose a new anamorph genus and species, Glarea lozoyensis, to accommodate ATCC 20868.

Abstract: The effects of exposing seawater adapted cichlid fish, tilapia(Sarotherodon mossambicus Peters) to sublethal concentrations of toluene on the activities of lactate dehydrogenase, succinate dehydrogenase and acetylcholinesterase were studied. The activity of lactate dehydrogenase increased while those of succinate dehydrogenase and acetylcholinesterase were inhibited in most tissues of the exposed fish. The alterations in the dehydrogenases suggested that some changes in carbohydrate metabolism may have occurred. Acetylcholinesterase inhibition in brain and other tissues indicated impairment of nervous function in toluene-intoxicated tilapia.

Chromosome replication in the asymmetrically dividing bacteria Caulobacter crescentus is discontinuous with the new, motile swarmer cell undergoing an obligatory presynthetic gap period (G1 period) of 60 min before the initiation of DNA synthesis and stalk formation. To examine the regulation of the cell division cycle at the molecular level, we have cloned the DNA chain elongation gene dnaC from a genomic DNA library constructed in cosmid vector pLAFR1-7. To ensure that the cloned sequence corresponded to dnaC, we isolated the gene by genetic complementation of the temperature-sensitive allele dnaC303 on DNA fragment that contained a Tn5 insertion element tightly linked by transduction to dnaC. The size of the dnaC gene was estimated to be 1,500 bp or less based on the pattern of complementation by subcloned restriction and BAL 31 deletion fragments. Nuclease S1 assays were used to map the transcription start site and to determine the pattern of dnaC expression in the cell cycle. Large amounts of the dnaC transcript began to accumulate only in the late G1 period of the swarmer cell and then peaked early during chromosome replication. We confirmed that the gene is periodically transcribed by monitoring the rate of beta-galactosidase synthesis directed by a dnaC promoter-lacZ fusion in a synchronous cell culture. dnaC is the first C. crescentus cell cycle gene whose regulation has been reported, and the discontinuous pattern of its expression suggests that the DNA synthetic period in these dimorphic bacteria is regulated in part by the stage-specific expression of DNA replication genes.

Measles virus (MV) mRNA transcription and replication are thought to be controlled by cis-acting sequence elements contained within the terminal MV genomic noncoding nucleotides. To validate these promoter and regulatory signal assignments, cDNAs were constructed allowing synthesis of RNAs corresponding to a MV genome in which all coding and intercistronic regions were replaced by the chloramphenicol acetyl transferase (CAT) coding sequence. Transcript production by T7 polymerase starting and ending precisely with the MV genome terminal residues was achieved by fusing the T7 polymerase promoter and the hepatitis delta virus genome ribozyme followed by tandem T7 polymerase termination sequences to the MV genomic 5′ and 3′ ends, respectively. Transfaction of these negative polarity transcripts, mimicking natural defective interfering RNAs of the internal deletion type, into MV-infected 293 cells gave rise to CAT activity which could be serially transferred and massively amplified together with progeny helper virus in fresh cells. Transfer was blocked only by antibodies able to neutralize MV infectivity, indicating that the chimeric RNA not only was encapsidated, transcribed, and replicated, but also packaged into virions. Sequence analyses confirmed that both the expected chimeric antigenome and mRNA products were transcribed and replicated with fidelity during serial passage. Minor changes introduced in the transcription promoter markedly compromised function. This system now can be exploited to examine MV genomic cis-acting regulatory elements arid extended to the development of full-length MV cDNAs.

We previously reported that lysine inhibits in vivo homocitrate synthesis in the lysine bradytroph, Penicillium chrysogenumL1, and that such feedback inhibition could explain the known lysine inhibition of penicillin formation. In the present study, it was found that dialyzed cell-free extracts of mutant L1converted [1-14C]acetate to homocitrate. This homocitrate synthase activity was extremely labile but could be stabilized by high salt concentrations. The pH optimum of the reaction was 6.9, and the Kmwas 5.5 mM with respect to α-ketoglutarate. The reaction was also dependent upon the presence of Mg2+, adenosine 5′-triphosphate, and coenzyme A. Surprisingly, the activity in these crude extracts was not inhibited by lysine. Benzylpenicillin at a high concentration (20 mM) partially inhibited the enzyme, an effect that was enhanced by lysine. Casein hydrolysate also partially inhibited the enzyme.

Lysine-regulatory mutants of Penicillium chrysogenumwhich excrete lysine were found to be deficient in production of penicillin. A revertant recovered the ability to produce penicillin.

An efficient method for isolation of auxotrophs of Penicillium chyrysogenuminvolving mutagenesis with ethyl methanesulfonate followed by enrichment with sodium pentachlorophenate was developed. The auxotroph frequencies obtained were 30 to 40%.

Polyethylene glycol conjugated L-asparaginase (PEG-ASNase) is used in varying doses between 1000–2500 U/m2 in the therapy of ALL and associated with a wide variation in pharmacokinetics. UKALL 2003 used intramuscular PEG-ASNase at 1000 U/m2.Patients enrolled in the UKALL 2003 trial (ISRCTN: 07355119), were consented for trough asparaginase activity analysis during therapy. National Cancer Institute (NCI) standard risk (SR) patients with rapid early response and non-high risk (HR) cytogenetics (Lancet Oncol. 2010;11:429) received a 3 drug induction (Dexamethasone, Vincristine & PEG-ASNase). All other patients received additional Daunorubicin. PEG-ASNase was given on days 4 and 18 of induction, and at least once post induction. Trough plasma asparaginase activity was measured by the indooxine method (Anal. Biochem. 2002;309:117). The lower limit of assay detection was 34 U/L. Adequate asparaginase activity was defined as a trough level of > 100 U/L. IgG and IgM antibodies to PEG-ASNase and native asparaginase were measured by indirect ELISA. Asparaginase activity was correlated with defined risk factors, minimal residual disease (MRD) at day 28 of induction and development of anti-asparaginase antibodies using the chi-squared test.Between July 2008 to July 2011, 482 patients aged 1–25 years from 27 centres were recruited. Numbers of samples assayed in induction were 335 & 371 after first and second doses respectively. Overall, 86% (n=606/706) of samples had adequate activity during induction time points. There was > 10 fold variation in activity levels (Figure 1). Three hundred and nine out of 706 samples had activity > 3 times the therapeutic threshold, while 51/100 samples with inadequate activity had no detectable drug levels (median: below detection limit, range: < 34–99 U/L). Thus, increasing the dose of PEG-ASNase in induction is unlikely to benefit patients with inadequate activity. Compared to SR patients, NCI HR patients had a higher incidence of inadequate asparaginase activity in induction (p=0.002). Inadequate asparaginase activity correlated with high MRD (≥ 10−4) in SR patients (p=0.045), especially those with good risk cytogenetics (p=0.012), and in particular the high hyperdiploid subgroup (p=0.03). Inadequate asparaginase activity during induction did not correlate with MRD in HR patients (p=0.699), possibly because these patients received in addition Daunorubicin (Table 1).Results of serial asparaginase activity (at least one time point each in induction and post induction), measured in 282 patients are summarised in Table 2. Antibodies were detected in 18 of 81 patients tested. All had anti-PEG and 7 in addition also had anti-asparaginase antibodies. While all antibody positive patients had inadequate asparaginase activity at one time point, 17 had adequate activity prior to antibody detection, suggesting immune-mediated drug inactivation at re-exposure. Antibodies were not detected in 14/15 patients who had inadequate activity at first exposure, so the mechanism here remains unclear.The reported incidence of asparaginase toxicity in this study was 6.6% (n=32/482). This included hypersensitivity (n=17/482) that was almost exclusively seen in HR patients (n=16/17), thrombosis (n=10/482) and pancreatitis (n=5/482).Intramuscular PEG-ASNase given fortnightly at 1000 U/m2 during induction provides adequate asparaginase activity in 86% of patients. Monitoring asparaginase activity may benefit patients who receive 3 drug induction and improve the resolution of the current prognostic classification.Off Label Use: PEG-asparaginase. Essink:medac: Employment. Kuehnel:medac: Employment.

Abstract 2573

Despite improvement in frontline therapy in childhood acute lymphoblastic leukemia (ALL), central nervous system (CNS) relapse remains a significant clinical problem. The ALLR3 trial (ISCRTN 45724312) was designed specifically to address this issue with the use of drugs known to penetrate the CNS. The trial incorporated a randomization between Mitoxantrone and Idarubicin during induction. Mitoxantrone showed an early benefit in all patients resulting in closure of the randomization in December 2007 (ASH Annual Meeting Abstracts, Nov 2009; 114:3390). Subsequently all patients now receive Mitoxantrone. Here we report on the outcome of patients with isolated CNS relapse (iCNSr) or combined CNS relapse (involvement of CNS and bone marrow, cCNSr).CNS involvement was defined as ≥5 WBC/μl with morphological evidence of blasts in the cerebrospinal fluid (CSF). Combined relapse (cCNSr) was defined as CNS disease with ≥ 5% blasts in the bone marrow. Time to relapse was classified as, Very Early: within 18 months of first diagnosis; Early: after 18 months of first diagnosis but within 6 months of stopping therapy and Late: more than 6 months after stopping therapy. All patients received 3 blocks of chemotherapy. Subsequently, allogenic stem cell transplant (allo-SCT) was offered to all very early relapses (iCNSr & cCNSr), early iCNSr (irrespective of immunophenotype), all T-cell cCNSr (irrespective of time to relapse) and early or late pre-B cCNSr that had a minimal residual disease level of ≥ 104 at the end of induction. All other patients were offered chemotherapy and cranial radiotherapy.Of a total of 330 relapsed patients, 102 (31%) had CNS involvement. Of these 63 (62%) had iCNSr and 39 (38%) had cCNSr. The incidence of CNS disease was higher in males (M:F, CNS relapses 2.5:1 vs all relapses 1.5:1). CNS relapses had a higher proportion of T-cell disease (pre B:T CNS relapses 3.6:1 vs all relapses 7.8:1]. The number of patients presenting in very early, early and late phases were 19 (19%), 55 (54%) and 28 (27%) respectively. All late iCNSr patients were males. Almost all late relapses (iCNSr and cCNSr) (27/28) were of a pre B phenotype. At the end of induction phase, 91/102 (89%) achieved complete remission (CR) and 82/102 (80%) remained in CR after 3 blocks of chemotherapy. The estimated 3-year overall survival (OS) and progression free survival (PFS) for all patients with CNS disease was 45.5% (95%CI 32.9, 58.0) & 43.4% (95%CI 32.0, 54.7) respectively. There were no significant differences in survival with respect to site of the disease (combined vs isolated), gender or immunophenotype (pre B vs T). As shown in Table 1, CNS relapse patients who received Mitoxantrone had a significantly improved outcome when compared to those who received Idarubicin. This was most evident in those who had i) iCNSr, ii) pre-B phenotype and iii) allo-SCT, when analyzed on an intention to treat basis. This represents a considerable improvement in outcome compared to the results obtained in these sub-groups of patients in the previous UK ALLR2 study (Roy A et.al. Br. J. Haem. 2005;130:67-75).Mitoxantrone is highly effective in children with relapsed pre B ALL who have CNS involvement. As there were no other differences between patients treated on Mitoxantrone or Idarubicin, effective systemic therapy is as important as CNS directed therapy, if not more, in treating patients with CNS relapse.Off Label Use: Most drugs used in this protocol are off label as the majority of drugs used in childhood ALL are not liscensed for use in children.

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