Tyrosine kinase inhibitor insensitivity of non-cycling CD34+ human acute myeloid leukaemia cells with FMS-like tyrosine kinase 3 mutations.

The efficacy of tyrosine kinase (TK) inhibitors on non-cycling acute myeloid leukaemia (AML) cells, previously shown to have potent tumourigenic potential, is unknown. This pilot study describes the first attempt to characterize non-cycling cells from a small series of human FMS-like tyrosine kinase 3 (FLT3) mutation positive samples. CD34+ AML cells from patients with FLT3 mutation positive AML were cultured on murine stroma. In expansion cultures, non-cycling cells were found to retain CD34+ expression in contrast to dividing cells. Leukaemic gene rearrangements could be detected in non-cycling cells, indicating their leukaemic origin. Significantly, the FLT3-internal tandem duplication (ITD) mutation was found in the non-cycling fraction of four out of five cases. Exposure to the FLT3-directed inhibitor TKI258 clearly inhibited the growth of AML CD34+ cells in short-term cultures and colony-forming unit assays. Crucially, non-cycling cells were not eradicated, with the exception of one case, which exhibited exquisite sensitivity to the compound. Moreover, in longer-term cultures, TKI258-treated non-cycling cells showed no growth impairment compared to treatment-naive non-cycling cells. These findings suggest that non-cycling cells in AML may constitute a disease reservoir that is resistant to TK inhibition. Further studies with a larger sample size and other inhibitors are warranted.

XBP1s levels are implicated in the biology and outcome of myeloma mediating different clinical outcomes to thalidomide-based treatments.

Immunoglobulin production by myeloma plasma cells depends on the unfolded protein response for protein production and folding. Recent studies have highlighted the importance of IRE1alpha and X box binding protein 1 (XBP1), key members of this pathway, in normal B-plasma cell development. We have determined the gene expression levels of IRE1alpha, XBP1, XBP1UNSPLICED (XBP1u), and XBP1SPLICED (XBP1s) in a series of patients with myeloma and correlated findings with clinical outcome. We show that IRE1alpha and XBP1 are highly expressed and that patients with low XBP1s/u ratios have a significantly better overall survival. XBP1s is an independent prognostic marker and can be used with beta2 microglobulin and t(4;14) to identify a group of patients with a poor outcome. Furthermore, we show the beneficial therapeutic effects of thalidomide in patients with low XBP1s/u ratios. This study highlights the importance of XBP1 in myeloma and its significance as an independent prognostic marker and as a predictor of thalidomide response.

Non-genetic mechanisms communicating antibiotic resistance:rethinking strategies for antimicrobial drug design

Introduction: Infections by multidrug-resistant bacteria are of great concern worldwide. In many cases, resistance is not due to the presence of specific antibiotic-modifying enzymes, but rather associated with a general impermeability of the bacterial cell envelope. The molecular bases of this intrinsic resistance are not completely understood. Moreover, horizontal gene transfers cannot solely explain the spread of intrinsic resistance among bacterial strains. Areas covered: This review focuses on the increased intrinsic antibiotic resistance mediated by small molecules. These small molecules can either be secreted from bacterial cells of the same or different species (e.g., indole, polyamines, ammonia, and the Pseudomonas quinolone signal) or be present in the bacterial cell milieu, whether in the environment, such as indole acetic acid and other plant hormones, or in human tissues and body fluids, such as polyamines. These molecules are metabolic byproducts that act as infochemicals and modulate bacterial responses toward antibiotics leading to increasing or decreasing resistance levels. Expert opinion: The non-genetic mechanisms of antibiotic response modulation and communication discussed in this review should reorient our thinking of the mechanisms of intrinsic resistance to antibiotics and its spread across bacterial cell populations. The identification of chemical signals mediating increased intrinsic antibiotic resistance will expose novel critical targets for the development of new antimicrobial strategies.

Potential strategies for the eradication of multi-drug resistant Gram-negative bacterial infections

Antimicrobial resistance is one of the leading threats to society. The increasing burden of multidrug-resistant Gram-negative infection is particularly concerning as such bacteria are demonstrating resistance to nearly all currently licensed therapies. Various strategies have been hypothesized to treat multidrug-resistant Gram-negative infections including: targeting the Gram-negative outer membrane; neutralization of lipopolysaccharide; inhibition of bacterial efflux pumps and prevention of protein folding. Silver and silver nanoparticles, fusogenic liposomes and nanotubes are potential strategies for extending the activity of licensed, Gram-positive selective, antibiotics to Gram-negatives. This may serve as a strategy to fill the current void in pharmaceutical development in the short term. This review outlines the most promising strategies that could be implemented to solve the threat of multidrug-resistant Gram-negative infections

Can the protein costs of bacterial resistance be offset by altered feeding behaviour?

1. Mounting an immune response is likely to be costly in terms of energy and nutrients, and so it is predicted that dietary intake should change in response to infection to offset these costs. The present study focuses on the interactions between a specialist grass-feeding caterpillar species, the African armyworm Spodoptera exempta, and an opportunist bacterium, Bacillus subtilis.
2. The main aims of the study were (i) to establish the macronutrient costs to the insect host of surviving a systemic bacterial infection, (ii) to determine the relative importance of dietary protein and carbohydrate to immune system functions, and (iii) to determine whether there is an adaptive change in the host's normal feeding behaviour in response to bacterial challenge, such that the nutritional costs of resisting infection are offset.
3. We show that the survival of bacterially infected larvae increased with increasing dietary protein-to-carbohydrate (P:C) ratio, suggesting a protein cost associated with bacterial resistance. As dietary protein levels increased, there was an increase in antibacterial activity, phenoloxidase (PO) activity and protein levels in the haemolymph, providing a potential source for this protein cost. However, there was also evidence for a physiological trade-off between antibacterial activity and phenoloxidase activity, as larvae whose antibacterial activity levels were elevated in response to immune activation had reduced PO activity.
4. When given a choice between two diets varying in their P:C ratios, larvae injected with a sub-lethal dose of bacteria increased their protein intake relative to control larvae whilst maintaining similar carbohydrate intake levels. These results are consistent with the notion that S. exempta larvae alter their feeding behaviour in response to bacterial infection in a manner that is likely to enhance the levels of protein available for producing the immune system components and other factors required to resist bacterial infections (‘self-medication’).

A complete lipopolysaccharide inner core oligosaccharide is required for resistance of Burkholderia cenocepacia to antimicrobial peptides and bacterial survival in vivo

Burkholderia cenocepacia is an important opportunistic pathogen of patients with cystic fibrosis. This bacterium is inherently resistant to a wide range of antimicrobial agents, including high concentrations of antimicrobial peptides. We hypothesized that the lipopolysaccharide (LPS) of B. cenocepacia is important for both virulence and resistance to antimicrobial peptides. We identified hldA and hldD genes in B. cenocepacia strain K56-2. These two genes encode enzymes involved in the modification of heptose sugars prior to their incorporation into the LPS core oligosaccharide. We constructed a mutant, SAL1, which was defective in expression of both hldA and hldD, and by performing complementation studies we confirmed that the functions encoded by both of these B. cenocepacia genes were needed for synthesis of a complete LPS core oligosaccharide. The LPS produced by SAL1 consisted of a short lipid A-core oligosaccharide and was devoid of O antigen. SAL1 was sensitive to the antimicrobial peptides polymyxin B, melittin, and human neutrophil peptide 1. In contrast, another B. cenocepacia mutant strain that produced complete lipid A-core oligosaccharide but lacked polymeric O antigen was not sensitive to polymyxin B or melittin. As determined by the rat agar bead model of lung infection, the SAL1 mutant had a survival defect in vivo since it could not be recovered from the lungs of infected rats 14 days postinfection. Together, these data show that the B. cenocepacia LPS inner core oligosaccharide is needed for in vitro resistance to three structurally unrelated antimicrobial peptides and for in vivo survival in a rat model of chronic lung infection.

Capsule polysaccharide mediates bacterial resistance to antimicrobial peptides

The innate immune system plays a critical role in the defense of areas exposed to microorganisms. There is an increasing body of evidence indicating that antimicrobial peptides and proteins (APs) are one of the most important weapons of this system and that they make up the protective front for the respiratory tract. On the other hand, it is known that pathogenic organisms have developed countermeasures to resist these agents such as reducing the net negative charge of the bacterial membranes. Here we report the characterization of a novel mechanism of resistance to APs that is dependent on the bacterial capsule polysaccharide (CPS). Klebsiella pneumoniae CPS mutant was more sensitive than the wild type to human neutrophil defensin 1, beta-defensin 1, lactoferrin, protamine sulfate, and polymyxin B. K. pneumoniae lipopolysaccharide O antigen did not play an important role in AP resistance, and CPS was the only factor conferring protection against polymyxin B in strains lacking O antigen. In addition, we found a significant correlation between the amount of CPS expressed by a given strain and the resistance to polymyxin B. We also showed that K. pneumoniae CPS mutant bound more polymyxin B than the wild-type strain with a concomitant increased in the self-promoted pathway. Taken together, our results suggest that CPS protects bacteria by limiting the interaction of APs with the surface. Finally, we report that K. pneumoniae increased the amount of CPS and upregulated cps transcription when grown in the presence of polymyxin B and lactoferrin.

Chemical Communication of Antibiotic Resistance by a Highly Resistant Subpopulation of Bacterial Cells

The overall antibiotic resistance of a bacterial population results from the combination of a wide range of susceptibilities displayed by subsets of bacterial cells. Bacterial heteroresistance to antibiotics has been documented for several opportunistic Gram-negative bacteria, but the mechanism of heteroresistance is unclear. We use Burkholderia cenocepacia as a model opportunistic bacterium to investigate the implications of heterogeneity in the response to the antimicrobial peptide polymyxin B (PmB) and also other bactericidal antibiotics. Here, we report that B. cenocepacia is heteroresistant to PmB. Population analysis profiling also identified B. cenocepacia subpopulations arising from a seemingly homogenous culture that are resistant to higher levels of polymyxin B than the rest of the cells in the culture, and can protect the more sensitive cells from killing, as well as sensitive bacteria from other species, such as Pseudomonas aeruginosa and Escherichia coli. Communication of resistance depended on upregulation of putrescine synthesis and YceI, a widely conserved low-molecular weight secreted protein. Deletion of genes for the synthesis of putrescine and YceI abrogate protection, while pharmacologic inhibition of putrescine synthesis reduced resistance to polymyxin B. Polyamines and YceI were also required for heteroresistance of B. cenocepacia to various bactericidal antibiotics. We propose that putrescine and YceI resemble "danger" infochemicals whose increased production by a bacterial subpopulation, becoming more resistant to bactericidal antibiotics, communicates higher level of resistance to more sensitive members of the population of the same or different species.

Multidrug transporter proteins of the major facilitator superfamily: not only for drug efflux

Multidrug resistance in prokaryotes is due primarily to efflux of offending antimicrobials from the cell by representatives of several different families of integral membrane transporter proteins. Clearly, in evolutionary terms, these proteins did not arise specifically to pump human-made antimicrobials out of the cell and thereby confer resistance. Despite this, often only their role in antibiotic resistance is characterised and highlighted.
In recent years, however, a transition from the traditional anthropocentric perception of antibiotic resistance mechanisms in microorganisms has occurred, with naturally produced antimicrobials now generally regarded as physiologically important signalling molecules or sources of nutrition for bacteria rather than antimicrobial agents, and bacterial multidrug efflux proteins not merely as a defensive response to antimicrobials but as important players in fundamental physiological processes such as cellular homeostasis.
This emerging perspective supports the notion that a better understanding of the complexities of infection and multidrug resistance in bacteria can be achieved via a more detailed understanding of those physiological processes. In this chapter, we review the ‘true’ physiological roles of multidrug efflux proteins of the largest non-ATP-hydrolysing family of membrane transporters, the major facilitator superfamily, and explore the evidence for their function in processes such as pH and metal homeostasis, import and export of metabolites and biofilm formation

The resistance of polyvinylpyrrolidone–Iodine–poly(e-caprolactone) blends to adherence of Escherichia coli

In this study, the resistance of biodegradable biomaterials, composed of blends of poly(e-caprolactone) (PCL) and the polymeric antimicrobial complex, polyvinylpyrrolidone–iodine (PVP-I) to the adherence of a clinical isolate of Escherichia coli is described. Blends of PCL composed of a range of high (50,000 g mol1) to low (5000 g mol1) molecular weight ratios of polymer and either
devoid of or containing PVP-I (1% w/w) were prepared by solvent evaporation. Following incubation (4 h), there was no relationship between m. wt. ratio of PCL in ?lms devoid of PVP-I and adherence ofE. coli. Conversely, microbial adherence to PCL containing PVP-I decreased as the ratio of high:low m. wt. polymer was decreased and was approximately 1000 fold lower than that to comparator ?lms devoid of PVP-I. Following periods of immersion of PVP-I containing PCL ?lms under sink conditions in phosphate buffered saline, subsequent adherence of E. coli was substantially reduced for 2 days (40:60 m. wt. ratio) and 6 days (100:0 m. wt. ratio). Concurrent exposure of PCL and E. coli to sub-minimum inhibitory concentrations (sub-MIC) of PVP-I signi?cantly reduced microbial adherence to the biomaterial; however, the molecular weight ratio of PCL did not affect this outcome. Pretreatment of PCL with similar sub-MIC of PVP-I prior to inclusion within the microbial adherence assay signi?cantly decreased the subsequent adherence of E. coli. Greatest reduction in adherence was observed following treatment of PCL (40:60 m. wt. ratio) with 0.0156% w/w PVP-I. In conclusion, this study has illustrated the utility of PVP-I as a suitable therapeutic agent for incorporation within PCL as a novel biomaterial. Due to the combined antimicrobial and biodegradable properties, these biomaterials offer a promising strategy for the reduction in medical device related infection. © 2004 Elsevier Ltd. All rights reserved.

Characterisation and evaluation of novel surfactant bacterial anti-adherent coatings for endotracheal tubes designed for the prevention of ventilator-associated pneumonia.

It is accepted that ventilator-associated pneumonia is a frequent cause of morbidity and mortality in intensive care patients. This study describes the physicochemical properties of novel surfactant coatings of the endotracheal tube and the resistance to microbial adherence of surfactant coated endotracheal tube polyvinylchloride (PVC). Organic solutions of surfactants containing a range of ratios of cholesterol and lecithin (0:100, 25:75, 50:50, 75:25, dissolved in dichloromethane) were prepared and coated onto endotracheal tube PVC using a multiple dip-coating process. Using modulated temperature differential scanning calorimetry it was confirmed that the binary surfactant systems existed as physical mixtures. The surface properties of both surfactant-coated and uncoated PVC, following treatment with either pooled human saliva or phosphate-buffered saline (PBS), were characterised using dynamic contact angle analysis. Following treatment with saliva, the contact angles of PVC decreased; however, those of the coated biomaterials were unaffected, indicating different rates and extents of macromolecular adsorption from saliva onto the coated and uncoated PVC. The advancing and receding contact angles of the surfactant-coated PVC were unaffected by sonication, thereby providing evidence of the durability of the coatings. The cell surface hydrophobicity and zeta potentials of isolates of Staphylococcus aureus and Pseudomonas aeruginosa, following treatment with either saliva or PBS, and their adherence to uncoated and surfactant-coated PVC (that had been pre-treated with saliva) were examined. Adherence of S. aureus and Ps. aeruginosa to surfactant-coated PVC at each successive time period (0.5, 1, 2, 4, 8 h) was significantly lower than to uncoated PVC, the extent of the reduction frequently exceeding 90%. Interestingly, the microbial anti-adherent properties of the coatings were dependent on the lecithin content. Based on the impressive microbial anti-adherence properties and durability of the surfactant coating on PVC following dip coatings, it is proposed that these systems may usefully reduce the incidence of ventilator-associated pneumonia when employed as luminal coatings of the endotracheal tube.

A new monoclonal antibody, P2A8(6), that specifically recognizes a novel epitope on the multidrug resistance-associated protein 1 (MRP1), but not on MRP2 nor MRP3

Multidrug resistance (NIDR) is a major problem in the chemotherapeutic treatment of cancer. Overexpression of the multidrug resistance-associated protein 1 (MRP1), is associated with NIDR in certain tumors. A number of MRP1-specific MAbs, which facilitate both clinical and experimental investigations of this protein, are available. To add to this panel of existing antibodies, we have now generated an additional MRP1-specific monoclonal antibody (MAb), P2A8(6), which detects a unique heat stable epitope on the MRP1 molecule. Female Wistar rats were immunized via footpad injections with a combination of two short synthetic peptides corresponding to amino acids 235-246 (peptide A) and 246-260 (peptide B) of the MRP1 protein. Immune reactive B cells were then isolated from the popliteal lymph nodes for fusion with SP2/O-Ag14 myeloma cells. Resultant hybridoma supernatants were screened for MRP1-specific antibody production. Antibody P2A8(6) was characterized by Western blotting and immunocytochemistry on paired multidrug resistant (MRP1 overexpressing) and sensitive parental cell lines. The antibody detects a protein of 190 kDa in MRP1-expressing cell lines but not in MRP2- or MRP3-transfected cell lines. P2A8(6) stains drug-selected and MRP1-transfected cell lines homogeneously by immunocytochemistry and recognizes MRP1 by immunohistochemistry on formalin-fixed paraffin wax-embedded tissue sections. Peptide inhibition studies confirm that P2AS(6) reacts with peptide B (amino acids 246-260), therefore recognizing a different epitope from that of all currently available MRP1 MAbs. This new MAb, chosen for its specificity to the MRP1 protein, may be a useful addition to the currently available range of MRP1-specific MAbs.