Caroline M. Broughton

Clinical use of streptolysin-O to facilitate antisense oligodeoxyribonucleotide delivery for purging autografts in chronic myeloid leukaemia

Antisense oligodeoxyribonucleotides (ODN) targeted against the breakpoint in BCR-ABLmRNA will specifically decrease BCR-ABL mRNA, provided cells are first permeabilised with Streptolysin-O (SL-O). We used 18-mer chimeric methylphosphonodiester: phosphodiester linked (4–9-4) ODN complementary to 9 bases either side of the BCR-ABL junction to purge harvests ex vivo in three CML patients who remained completely Ph positive after multiple chemotherapy courses. After CD34+ cell selection and SL-O permeabilisation, harvests were purged with 20 μM ODN. After purging, all individual CFU-GM colonies grown from the two b3a2 breakpoint cases remained positive for BCR-ABL mRNA. In contrast, all 24 colonies grown from the b2a2 breakpoint case were BCR-ABL mRNA negative. Patients were conditioned with busulphan 16 mg/kg. The initial post-transplant course was uneventful, although the time to return to 0.5 × 109/l neutrophils was slow at 25–51 days. Both chronic phase patients remain in haematological remission at +724 and +610 days, although each has cytogenetic evidence of relapse. The b2a2 accelerated phase patient died of myeloid blast transformation at day +91. The present SL-O-facilitated ODN purging strategy appears to be without significant toxicity, and offers considerable improvements in ODN delivery to the cytosol.

Staphylococcus aureus with reduced glycopeptide susceptibility in Liverpool, UK

OBJECTIVES To investigate if colonization with heterogeneous glycopeptide-intermediate Staphylococcus aureus (hGISA) is associated with hGISA bacteraemia. METHODS Isolates of methicillin-resistant S. aureus (MRSA) from blood cultures and from swabs to detect MRSA colonization were screened for reduced susceptibility to glycopeptides by an agar incorporation method. Isolates detected by this screen were tested for glycopeptide resistance by MacroEtest, standard MIC Etest methods and population analysis profile-AUC (PAP-AUC) analysis. S. aureus isolates with and without reduced glycopeptide susceptibility were characterized by PFGE and spa typing. RESULTS MRSA isolates with reduced susceptibility to glycopeptides, as identified by the MacroEtest method, were detected in the colonization screens of 86 of 2550 MRSA-positive patients. The isolates were confirmed by Etest MIC and PAP-AUC analysis as hGISA. A total of 82/86 of the hGISA colonizing isolates were EMRSA-16 by PFGE; the remainder were EMRSA-15. Bacteraemia with hGISA was identified in five patients during the study period; two isolates were EMRSA-16 and three were EMRSA-15. hGISA colonization could not be linked to hGISA bacteraemia and hGISA bacteraemia could not be linked to hGISA colonization. Four of the five hGISA bacteraemias developed following teicoplanin therapy for a central venous catheter-associated MRSA bacteraemia. CONCLUSIONS Laboratory strategies to reduce morbidity associated with hGISA should focus on testing for hGISA in bacteraemic (rather than colonizing) MRSA isolates in patients with recurrent S. aureus bacteraemia following glycopeptide exposure.

In vivo development of heterogeneous glycopeptide-intermediate Staphylococcus aureus (hGISA), GISA and daptomycin resistance in a patient with meticillin-resistant S. aureus endocarditis

We report a patient who developed a meticillin-resistant Staphylococcus aureus (MRSA) central venous catheter infection complicated by infective endocarditis. The patient was initially treated with glycopeptides, which led to the development of heterogeneous glycopeptide resistance, the detection of which required the use of a macro Etest screening test. Subsequently, the causative strain, confirmed by PFGE as a UK epidemic MRSA-15, was treated with daptomycin, and again resistance developed in vivo. The development in vivo of resistance to both these agents suggests that the resistance mechanisms may be associated. We suggest that the clinician managing MRSA infection should anticipate daptomycin resistance when reduced glycopeptide susceptibility is detected.

Preclinical studies of streptolysin-O in enhancing antisense oligonucleotide uptake in harvests from chronic myeloid leukaemia patients

Antisense oligodeoxyribonucleotides (ODN) have been shown to produce a sequence-specific cleavage of BCR-ABL mRNA. They may therefore have clinical potential for purging harvests from chronic myeloid leukaemia (CML) patients, prior to autografting. Whilst ODN are highly effective in cell-free systems, their uptake into intact cells is very poor. We have previously reported that reversible permeabilisation of CML cell lines with Streptolysin-O (SL-O) can dramatically increase intracytoplasmic and nuclear ODN uptake. In this study, we examined whether SL-O permeabilisation could be used to enhance ODN uptake into bone marrow (BM) and peripheral blood stem cell (PBSC) harvests from CML patients, without undue toxicity. All 19 harvests studied were from patients in stable chronic phase of CML. Samples studied were either fresh BM harvests following leucoconcentration, fresh PBSC collections, or from previously cryopreserved harvests. Cells were permeabilised by SL-O to load them with fluorescein-labelled ODN. The proportion of permeabilised and viable cells was assessed by fluorescein uptake and propidium iodide exclusion, respectively, by flow cytometry. The effect of SL-O on ODN uptake and cell toxicity was unpredictable on simple mononuclear fractions of harvests. In contrast, SL-O consistently significantly enhanced ODN uptake in samples which were first selected for CD34-positive cells, and this was achieved without either direct toxicity or inhibition of CFU-GM growth. The SL-O concentration required for optimal permeabilisation varied considerably from case to case, in line with previous data on cell lines. PBSC harvests positively selected for CD34-positive cells tended to achieve superior permeabilisation to CD34 positively selected BM harvests. SL-O can be used to safely enhance the intracellular uptake of antisense ODN. This is best achieved on harvests which are first selected for CD34-positive cells.

Competitive Inhibition Flow Analysis Assay for the Non-Culture-Based Detection and Serotyping of Pneumococcal Capsular Polysaccharide

ABSTRACT Traditional confirmation procedures for the identification of a pneumococcal serotype require an isolate. Non-culture-based confirmation protocols are available. Some of these confirm only the presence of pneumococci, and others are capable of identifying a limited number of serotypes. The increased use of pneumococcal polysaccharide and conjugate vaccines, especially in high-risk patient groups, and the likely increase in the number of serotypes included in future versions of the conjugate vaccines have necessitated the need for improved enhanced surveillance in order to assess their impact on public health. Since 2006, a multiplexed assay has been used at the Health Protection Agency of the United Kingdom for the detection of 14 pneumococcal serotypes which requires pneumococcal serotype-specific monoclonal antibodies (MAbs). We have developed a microsphere competitive inhibition method capable of detecting 23 pneumococcal capsular polysaccharide serotypes in cerebrospinal fluid (CSF) and urine and serotyping pneumococcal suspensions, utilizing an international reference serum, 89-SF. The assay was shown to be reproducible and specific for homologous polysaccharide. Validation of the assay was performed with a selection of MAbs specific for pneumococcal capsular polysaccharide serotypes, which confirmed the specificity of the assay. Analysis of pneumolysin PCR-positive CSF samples in the competitive inhibition assay determined a serotype for 89% of the samples. The assay developed here is well suited to large-scale epidemiologic studies because the assay is simple, robust, and rapid and utilizes readily available resources.

Invasive Streptococcus pneumoniae in Children, Malawi, 2004–2006

Of 176 invasive Streptococcus pneumoniae isolates from children in Malawi, common serotypes were 1 (23%), 6A/B (18%), 14 (6%), and 23F (6%). Coverage with the 7-valent pneumococcal conjugate vaccine (PCV) was 39%; PCV10 and PCV13 increased coverage to 66% and 88%, respectively. We found chloramphenicol resistance in 27% of isolates and penicillin nonsusceptibility in 10% (by using meningitis breakpoints); all were ceftriaxone susceptible.

Molecular status of individual CFU-GM colonies derived from chemotherapy-mobilised peripheral blood stem cells in chronic myeloid leukaemia

Following chemotherapy in chronic myeloid leukaemia (CML), some peripheral blood (PB) cells may be Philadelphia (Ph) chromosome negative. The BCR‐ABL mRNA status of residual Ph+ progenitors is not known. We examined the BCR‐ABL mRNA status of individual colony‐forming‐unit granulocyte‐macrophage (CFU‐GM) colonies derived from PB harvested following chemotherapy. Seven patients were treated with 200 mg/m2/day cytarabine and 20 mg/m2/day Idarubicin and followed by Lenograstim. PB collections commenced daily when the white blood cell count reached 0.6 × 109/l and continued until at least 5 × 108/kg nucleated cells were obtained. CD34+ cells, Ph status, and CFU‐GM were estimated at each harvest. For each patient, up to 24 individual CFU‐GM colonies were analysed for BCR‐ABL status. Two cases were BCR‐ABL negative on all colonies and completely Ph−, and another case was BCR‐ABL positive in all colonies and completely Ph+. In contrast, in two patients all colonies were BCR‐ABL negative, despite virtually complete Ph+ metaphases. The final assessible case had five of nine BCR‐ABL negative colonies, despite 94% Ph+ metaphases. After chemotherapy priming, the PB may contain Ph+ CFU‐GM that do not express BCR‐ABL. Genes Chromosom. Cancer 18:292–298, 1997.

Glycopeptide and daptomycin resistance in community-associated MRSA in the UK

Methicillin-resistant Staphylococcus aureus (MRSA) infections are commonly treated with glycopeptides. When glycopeptide resistance occurs, one of the few remaining therapeutic options is daptomycin. We describe the in vivo development of both glycopeptide and daptomycin resistance in a patient from the UK with community-associated (CA) MRSA infective endocarditis. A 35-year-old man presented to our hospital in Liverpool, UK, in 2008. A diagnosis of tricuspid valve infective endocarditis was made, secondary to intravenous drug use. Both MRSA and group G haemolytic streptococcus grew from the blood cultures. Antimicrobial therapy was initiated with teicoplanin and rifampicin, but after 3 weeks of treatment MRSA was cultured again from the patient’s blood, suggesting that resistance may have developed. Glycopeptide minimum inhibitory concentration (MIC) testing was therefore performed. The MIC to teicoplanin was determined to be 8 mg/L, identifying the MRSA isolate as glycopeptide-resistant S. aureus (GRSA). Daptomycin therapy was prescribed, but 1 week later, after an initial clinical improvement, the patient suffered cerebral emboli. Blood cultures again recovered MRSA, leading to daptomycin MIC testing. Daptomycin resistance was confirmed with an MIC to daptomycin of 4 mg/L. Linezolid, ciprofloxacin and gentamicin were then administered to the patient (Fig. 1). The patient clinically responded to this treatment, and mitral valve replacement was subsequently completed. A total of 6 weeks of oral ciprofloxacin and linezolid were prescribed post-operatively, and the patient was well upon review in January 2010. This patient was initially infected with a glycopeptidesusceptible MRSA, with initial teicoplanin and vancomycin MICs of B2 mg/L [MICs by Etest method (bioMerieux, Marcy l’Etoile, France)]. However, following treatment with teicoplanin, the isolate became glycopeptide resistant, with teicoplanin and vancomycin MICs of 8 and 3 mg/L, respectively. The MIC breakpoint for glycopeptide resistance is[2 mg/L according to the European Committee on Antimicrobial Susceptibility testing (EUCAST 2010: http://www.eucast.org). The development of glycopeptide resistance was associated with that of daptomycin resistance. The daptomycin MIC increased from a range of 0.19–0.25 to 1.5–3 mg/L, and this occurred prior to in vivo exposure to daptomycin (EUCAST MIC breakpoint for daptomycin resistance is [1 mg/L). Daptomycin therapy was associated with a further rise in the daptomycin MIC to 4 mg/L (Table 1). The MRSA isolate was confirmed as CA-MRSA [1] based on its susceptibility to ciprofloxacin [2], positivity for the Panton–Valentine Leukocidin (PVL) gene [3] and spa type t127 [4]. SCCmec typing, which is also used to differentiate CA-MRSA and health care-associated MRSA (HA-MRSA) is not valid in the UK as HA-MRSA and CA-MRSA are both commonly SCCmec type IV [5]. Pulsed field gel electrophoresis (PFGE) confirmed the A. Kirby (&) C. M. Broughton Institute of Infection and Global Health, Department of Clinical Infection, Microbiology and Immunology, University of Liverpool, 8th Floor Duncan Building, Daulby Street, Liverpool L69 3GA, UK e-mail: amk@liv.ac.uk

Expression of c- myc is not critical for cell proliferation in established human leukemia lines

BackgroundA study was undertaken to resolve preliminary conflicting results on the proliferation of leukemia cells observed with different c-myc antisense oligonucleotides.ResultsRNase H-active, chimeric methylphosphonodiester / phosphodiester antisense oligodeoxynucleotides targeting bases 1147–1166 of c-myc mRNA downregulated c-Myc protein and induced apoptosis and cell cycle arrest respectively in cultures of MOLT-4 and KYO1 human leukemia cells. In contrast, an RNase H-inactive, morpholino antisense oligonucleotide analogue 28-mer, simultaneously targeting the exon 2 splice acceptor site and initiation codon, reduced c-Myc protein to barely detectable levels but did not affect cell proliferation in these or other leukemia lines. The RNase H-active oligodeoxynucleotide 20-mers contained the phosphodiester linked motif CGTTG, which as an apoptosis inducing CpG oligodeoxynucleotide 5-mer of sequence type CGNNN (N = A, G, C, or T) had potent activity against MOLT-4 cells. The 5-mer mimicked the antiproliferative effects of the 20-mer in the absence of any antisense activity against c-myc mRNA, while the latter still reduced expression of c-myc in a subline of MOLT-4 cells that had been selected for resistance to CGTTA, but in this case the oligodeoxynucleotide failed to induce apoptosis or cell cycle arrest.ConclusionsWe conclude that the biological activity of the chimeric c-myc antisense 20-mers resulted from a non-antisense mechanism related to the CGTTG motif contained within the sequence, and not through downregulation of c-myc. Although the oncogene may have been implicated in the etiology of the original leukemias, expression of c-myc is apparently no longer required to sustain continuous cell proliferation in these culture lines.