Antibiotic innovation may contribute to slowing the dissemination of multiresistant Streptococcus pneumoniae: the example of ketolides

Background

Despite increasingly frequent bacterial resistance to antibiotics, antibacterial innovation is rare. Ketolides constitute one of the very few new antibiotic classes active against Streptococcus pneumoniae developed during the last 25 years. Their mechanism of action resembles that of macrolides, but they are unaffected by common resistance mechanisms. However, cross-resistance to ketolides has been observed in some macrolide-resistant strains. We examined how new antibiotic exposure may affect overall pneumococcal resistance patterns in the population. The aims of this study were to assess the potential dissemination of newly emerged resistances and to control the selection of strains already multiresistant to existing antimicrobials.

Methodology/Principal Findings

We developed an age-structured population model for S. pneumoniae transmission in a human community exposed to heptavalent vaccine, and β-lactams, macrolides and ketolides. The dynamics of intra-individual selection of resistant strains under antibiotic exposure and interindividual transmission were simulated, with antibiotic-specific resistance mechanisms defining the path to co-resistances and cross-resistances, and parameters concerning the French situation. Results of this simulation study suggest that new antibiotic consumption could markedly slow the diffusion of multiresistant strains. Wider use was associated with slower progression of multiresistance. When ketolides were prescribed to all ages, resistance to them reached 10% after >15 years, while it took >40 years when they were prescribed only to adults. In the scenario according to which new antibiotics totally replaced former antimicrobials, the β-lactam resistance rate was limited at 70%.

Conclusions

In a context of widespread vaccination and rational use of antibiotics, innovative antibiotic, prescribed to all age groups, may have an added impact on multiresistant-strain dissemination in the population.     

Signaling signatures and functional properties of anti-human CD28 superagonistic antibodies

Superagonistic CD28 antibodies (CD28SAs) activate T lymphocytes without concomitant perturbation of the TCR/CD3-complex. In rodents these reagents induce the preferential expansion of regulatory T cells and can be used for the treatment of autoimmune diseases. Unexpectedly, the humanized CD28 superagonist TGN1412 caused severe and life threatening adverse effects during a recently conducted phase I clinical trail. The underlying molecular mechanisms are as yet unclear. We show that TGN1412 as well as the commercially available CD28 superagonist ANC28.1 induce a delayed but extremely sustained calcium response in human naïve and memory CD4⁺ T cells but not in cynomolgus T lymphocytes. The sustained Ca⁺⁺-signal was associated with the activation of multiple intracellular signaling pathways and together these events culminated in the rapid de novo synthesis of high amounts of pro-inflammatory cytokines, most notably IFN-γ and TNF-α. Importantly, sustained transmembranous calcium flux, activation of Src-kinases as well as activation of PI3K were found to be absolutely required for CD28SA-mediated production of IFN-γ and IL-2. Collectively, our data suggest a molecular basis for the severe side effects caused by TGN1412 and impinge upon the relevance of non-human primates as preclinical models for reagents that are supposed to modify the function of human T cells.     

Early Detection of Erlotinib Treatment Response in NSCLC by 3′-Deoxy-3′-[18F]-Fluoro-L-Thymidine ([18F]FLT) Positron Emission Tomography (PET)

Background

Inhibition of the epidermal growth factor receptor (EGFR) has shown clinical success in patients with advanced non-small cell lung cancer (NSCLC). Somatic mutations of EGFR were found in lung adenocarcinoma that lead to exquisite dependency on EGFR signaling; thus patients with EGFR-mutant tumors are at high chance of response to EGFR inhibitors. However, imaging approaches affording early identification of tumor response in EGFR-dependent carcinomas have so far been lacking.

Methodology/Principal Findings

We performed a systematic comparison of 3′-Deoxy-3′-[¹⁸F]-fluoro-L-thymidine ([¹⁸F]FLT) and 2-[¹⁸F]-fluoro-2-deoxy-D-glucose ([¹⁸F]FDG) positron emission tomography (PET) for their potential to identify response to EGFR inhibitors in a model of EGFR-dependent lung cancer early after treatment initiation. While erlotinib-sensitive tumors exhibited a striking and reproducible decrease in [¹⁸F]FLT uptake after only two days of treatment, [¹⁸F]FDG PET based imaging revealed no consistent reduction in tumor glucose uptake. In sensitive tumors, a decrease in [¹⁸F]FLT PET but not [¹⁸F]FDG PET uptake correlated with cell cycle arrest and induction of apoptosis. The reduction in [¹⁸F]FLT PET signal at day 2 translated into dramatic tumor shrinkage four days later. Furthermore, the specificity of our results is confirmed by the complete lack of [¹⁸F]FLT PET response of tumors expressing the T790M erlotinib resistance mutation of EGFR.

Conclusions

[¹⁸F]FLT PET enables robust identification of erlotinib response in EGFR-dependent tumors at a very early stage. [¹⁸F]FLT PET imaging may represent an appropriate method for early prediction of response to EGFR TKI treatment in patients with NSCLC.     

Dynamic loading of deformable porous media can induce active solute transport

Active solute transport mediated by molecular motors across porous membranes is a well-recognized mechanism for transport across the cell membrane. In contrast, active transport mediated by mechanical loading of porous media is a non-intuitive mechanism that has only been predicted recently from theory, but not yet observed experimentally. This study uses agarose hydrogel and dextran molecules as a model experimental system to explore this mechanism. Results show that dynamic loading can enhance the uptake of dextran by a factor greater than 15 over passive diffusion, for certain combinations of gel concentration and dextran molecular weight. Upon cessation of loading, the concentration reverts back to that achieved under passive diffusion. Thus, active solute transport in porous media can indeed be mediated by cyclical mechanical loading.     

Phosphoinositide 3-kinases gamma and delta, linkers of coordinate C5a receptor-Fcgamma receptor activation and immune complex-induced inflammation

Fcgamma receptors (FcgammaR) and the C5a receptor (C5aR) are key effectors of the acute inflammatory response to IgG immune complexes (IC). Their coordinated activation is critical in IC-induced diseases, although the significance of combined signaling by these two different receptor classes in tissue injury is unclear. Here we used the mouse model of the passive reverse lung Arthus reaction to define their requirements for distinct phosphoinositide 3-kinase (PI3K) activities in vivo. We show that genetic deletion of class IB PI3Kgamma abrogates C5aR signaling that is crucial for FcgammaR-mediated activation of lung macrophages. Thus, in PI3Kgamma(-/-) mice, IgG IC-induced FcgammaR regulation, cytokine release, and neutrophil recruitment were blunted. Notably, however, C5a production occurred normally in PI3Kgamma(-/-) mice but was impaired in PI3Kdelta(-/-) mice. Consequently, class IA PI3Kdelta deficiency caused resistance to acute IC lung injury. These results demonstrate that PI3Kgamma and PI3Kdelta coordinate the inflammatory effects of C5aR and FcgammaR and define PI3Kdelta as a novel and essential element of FcgammaR signaling in the generation of C5a in IC disease.     

Mitochondrial adaptations to steatohepatitis induced by a methionine- and choline-deficient diet

Nonalcoholic fatty liver disease (NAFLD) has become common liver disease in Western countries. There is accumulating evidence that mitochondria play a key role in NAFLD. Nevertheless, the mitochondrial consequences of steatohepatitis are still unknown. The bioenergetic changes induced in a methionine- and choline-deficient diet (MCDD) model of steatohepatitis were studied in rats. Liver mitochondria from MCDD rats exhibited a higher rate of oxidative phosphorylation with various substrates, a rise in cytochrome oxidase (COX) activity, and an increased content in cytochrome aa3. This higher oxidative activity was associated with a low efficiency of the oxidative phosphorylation (ATP/O, i.e., number of ATP synthesized/natom O consumed). Addition of a low concentration of cyanide, a specific COX inhibitor, restored the efficiency of mitochondria from MCDD rats back to the control level. Furthermore, the relation between respiratory rate and protonmotive force (in the nonphosphorylating state) was shifted to the left in mitochondria from MCDD rats, with or without cyanide. These results indicated that, in MCDD rats, mitochondrial ATP synthesis efficiency was decreased in relation to both proton pump slipping at the COX level and increased proton leak although the relative contribution of each phenomenon could not be discriminated. MCDD mitochondria also showed a low reactive oxygen species production and a high lipid oxidation potential. We conclude that, in MCDD-fed rats, liver mitochondria exhibit an energy wastage that may contribute to limit steatosis and oxidative stress in this model of steatohepatitis.     

Divalent metal ions promote the formation of the 5'-splice site recognition complex in a self-splicing group II intron

Group II introns are ribozymes occurring in genes of plants, fungi, lower eukaryotes, and bacteria. These large RNA molecular machines, ranging in length from 400 to 2500 nucleotides, are able to catalyze their own excision from pre-mRNA, as well as to reinsert themselves into RNA or sometimes even DNA. The intronic domain 1 contains two sequences (exon binding sites 1 and 2, EBS1 and EBS2) that pair with their complementary regions at the 3′-end of the 5′-exon (intron binding sites 1 and 2, IBS1 and IBS2) such defining the 5′-splice site. The correct recognition of the 5′-splice site stands at the beginning of the two steps of splicing and is thus crucial for catalysis. It is known that metal ions play an important role in folding and catalysis of ribozymes in general. Here, we characterize the specific metal ion requirements for the formation of the 5′-splice site recognition complex from the mitochondrial yeast group II intron Sc.ai5γ. Circular dichroism studies reveal that the formation of the EBS1 · IBS1 duplex does not necessarily require divalent metal ions, as large amounts of monovalent metal ions also promote the duplex, albeit at a 5000 times higher concentration. Nevertheless, micromolar amounts of divalent metal ions, e.g. Mg²⁺ or Cd²⁺, strongly promote the formation of the 5′-splice site. These observations illustrate that a high charge density independent of the nature of the ion is needed for binding EBS1 to IBS1, but divalent metal ions are presumably the better players.     

Comparing soluble and co-immobilized catalysts for 2-ketoaldose production by pyranose 2-oxidase and auxiliary enzymes

The tri-enzyme system pyranose 2-oxidase (P2O), laccase, and catalase was used to study major parameters in the homogeneous and heterogeneous application of a multi-component enzymatic machinery. P2O oxidizes aldoses to 2-ketosugars, which are interesting intermediates in carbohydrate chemistry, and concomitantly reduces oxygen or alternative electron acceptors. The enzyme was immobilized on eleven agarose or acrylic resins using various coupling methods. The binding capacity was determined and an acrylic carrier with the most suitable properties selected for detailed studies. As P2O shows higher turnover numbers with the electron acceptor 1,4-benzoquinone than with oxygen, the use of this alternative electron acceptor was enabled by employing laccase for the continuous reoxidation of hydroquinone. The laccase regeneration system was found to increase the specific productivity up to 3-fold. Catalase was used to disproportionate the formed hydrogen peroxide in close proximity to the oxygen consuming enzymes and applied in different amounts to adjust the hydrogen peroxide concentration, which was found to be the main reason for enzyme deactivation under turnover conditions. In contrast to homogeneous catalysis, the specific productivity of heterogeneous catalysts under the applied experimental conditions was limited primarily by oxygen transfer, an effect significantly reduced by the laccase regeneration system.     

O-phospho-L-serine: a modulator of bone healing in calcium-phosphate cements

Bone substitution materials are seen as an alternative to autogenous bone transplants in the reconstruction of human bone structures. The aim of the present animal study was to evaluate the clinical handling and the conditions of bone healing after the application of a phosphoserine and collagen-I-modified calcium-phosphate cement (Biozement D). The application of phosphoserine is supposed to influence the texture of the extracellular matrix. Standardised bone defects were created in the lower jaw of 10 adult minipigs. These defects were reconstructed with a pasty calcium-phosphate cement mixture. After a healing time of 4 months, the animals were sacrificed. The mandibles of all animals were resected and non-decalcified histological sections of the areas of interest were prepared. The experiment was evaluated by means of qualitative histology and histomorphometry. The hydroxyapatite cement entirely hardened intraoperatively. Modelling and handling of the cement was facile and the margin fit to the host bone was excellent. Histology showed that resorption started in the periphery and proceeded exceptionally fast. The bony substitution, especially in phosphoserine-endowed cements, was very promising. After a healing period of 4 months, phosphoserine cements showed a bone regeneration of nearly two-thirds of the defect sizes. In the applied animal experiment, the newly developed hydroxyapatite collagen-I cement is well suited for bone substitution due to its easy handling, its excellent integration and good resorption characteristics. The addition of phosphoserine is very promising in terms of influencing resorption features and bone regeneration.