For this study we specifically optimized the virulence of a bacteriophage of our collection towards this clinical strain and studied its efficacy both in vitro and in vivo. A clinical strain of P. aeruginosa isolated from a CF patient was used to evaluate bacteriophage curative and preventive treatments on a lung infection model. Both treatments successfully rescued mice from lethal infections, underlying the potential use of bacteriophages to combat lung pathogens. We also showed that pro-inflammatory and cytotoxicity markers, as well as histology observations, were all concordant with these results for survival. Moreover, we observed that bacterial debris released during bacteriophage treatments were not pro-inflammatory, as production of cytokines was not strongly stimulated in either the curative or the preventive treatments. A pro-inflammatory response is often used as an argument against phage therapy. Four days after administration of the preventative treatment that consisted of intranasal bacteriophage solution only, the lung concentrations of IL-6 and KC were low. The main cause of bacterial destruction can therefore be attributed to the bacteriophage itself. However, the cytokine concentrations were still significantly higher than those measured 24 h after administration of a PBS MK-1775 control solution, and partial involvement of a weak immune response stimulated by the bacteriophage solution cannot thus be ruled out. Indeed, the quality of the bacteriophage preparation, particularly the elimination of endotoxins, influenced the extent of the immune response following pre-infection treatments. This is in contrast to post-infection treatments, where the immune system has already been primed by bacteria when the bacteriophages were administered. Humans are constantly exposed to bacterial viruses an estimated 1031 are on the Earth —but little is known about the immunological consequences of this. Recently, a metagenomic analysis of viral DNA present in the lungs of CF patients identified over 100 different viral genomes, and this was considered to be a low diversity compared with other environments. Nevertheless, the role of the immune system in shaping viral diversity has yet to be deeply studied. Furthermore, the immune response to therapeutic bacteriophages has not been extensively investigated because most of the available data were obtained from model viruses which have little interest as therapeutic agents. Bacteriophage treatments of P. aeruginosa chronic lung infections in animal models have still not been investigated, and no specific procedure has been developed to isolate bacteriophages with higher infectivity against chronic clinical strains. However, several lines of evidence are encouraging. First, some bacteriophages are known to possess hydrolases that degrade bacterial exopolysaccharides. Second, several in vitro biofilm models have shown that bacteriophages can access and infect bacteria grown under these conditions.

In the study by Davison et al, transport status explained only 1.6% of the variation in volume status. It can be hypothesized that fluid overload is induced by not adapting the dwell time appropriately to the transport status of the patient. Although it has been stated that removal of salt can be impaired in patients on APD, hydration status in patients on APD and CAPD was comparable in the multivariate analysis in the EuroBCM cohort, just as in previous observations. Of note, in one of these studies, the number of cycles per night was limited, so the dwell time was probably long enough to allow diffusive sodium transport. To maintain fluid balance, fast transporters need short dwells, to avoid negative ultrafiltration, and implementing APD might be of value in this patient category. On the other hand, slow transporters need long dwells to avoid sodium sieving, and APD with short cycles might be detrimental in this patient group. Johnson et al recently reported that APD was associated with better survival in fast, but with worse survival in low transporters, an observation that is compatible with this paradigm. As Davison et al, we found a negative association between serum albumin and overhydration. As this is a cross-sectional cohort, it is however impossible to determine whether low albumin is a consequence or a cause of overhydration. In the EuroBCM study cohort, polyglucose use was associated with less overhydration and more underhydration in some countries, whereas the opposite was true in other countries, pointing to PF-4217903 potential underlying differences in practice related to the use of polyglucose. In a subcohort of the EuroBCM trial, excluding countries were alternative PD solutions and APD are not liberally available due to logistical reasons, we observed a neutral impact both of the solution type and the PD modality on fluid overload, just as it was found in the cohort of Davison et al. This study is a cross sectional study, and as such, no causal relations can be drawn. However, our observations can generate some interesting hypotheses on the association between practices and hydration status. It would be interesting e.g. to study the impact on hydration status and residual renal function using a prospective protocol where implementation of polyglucose, dwell length and use of APD vs CAPD is guided by BCM based assessment of fluid overload. Another limitation is the rather crude evaluation of fluid output using patient charts as a reference, which might induce inaccuracies. However, this is the way fluid output is measured in real life. Of special interest for a future prospective study in this regard is the potential impact of bag overfill on the overestimation of ultrafiltration and fluid overload. It can be that the overestimation of real ultrafiltration by neglecting overfill can lead to overhydration, as it gives the patient and the physician the false feeling of adequate ultrafiltration.

Many physicians estimate hydration status by using clinical parameters, such as edema, weight gain or blood pressure. Although there was a direct correlation between systolic blood pressure and tissue hydration, a substantial proportion of patients did not comply with this paradigm. A number of patients had systolic hypertension, despite normohydration or even tissue underhydration. These are probably patients who suffer from vascular stiffness. Further dehydration of these patients in an attempt to normalize blood pressure might be dangerous, as it might abruptly compromise coronary perfusion. A number of patients had a low or normal blood pressure, despite being fluid overloaded. It is conceivable that many of these patients suffer congestive heart failure. Normotension in these patients NVP-BKM120 distributor should not be seen as equivalent to euvolemia, as also reported in HD patients. In many studies on fluid overload, attention is focused on fluid output, neglecting that fluid status is a balance of fluid output and input. In the EuroBCM study, there was a very weak association between fluid overload and diuresis, but this association disappeared in the multivariate analysis. Davison et al found a small influence of residual GFR, but not of peritoneal ultrafiltration or daily urine output, on volume status. Wiggins et al demonstrated that total fluid output one month after the initiation of PD was not associated with patient survival. All these point out that in studies on fluid status, both fluid input and output should be considered. In addition, and maybe even more of importance, clinicians should be aware that patients can be overhydrated because of dietary incompliance, despite having substantial residual diuresis. Dietary intake of fluid and salt should thus be conisdered when managing fluid overloaded patients. In our BCM cohort, the use of high hypertonic bags was associated with fluid overload. It is tempting to attribute this observation to bias by indication. However, an alternative potential hypothesis could be that the strategy of using hypertonic bags is not effective in returning patients back to euvolemia for a sustained period of time, as it can lead to dysregulation of glycemic control, and thus to hyperosmolarity and thirst. Sustained exposure to hypertonic exchanges can also negatively impact on the peritoneal membrane function, leading to further detrimental consequences on fluid balance. Further studies in this regard are warranted. This is compatible with the negative impact of high initial peritoneal fluid removal : it is likely that those with a high fluid output achieved this at the expense of increased use of hypertonic bags, thus damaging the peritoneal membrane in the long term. There was an association between peritoneal membrane transport characteristics and tissue hydration, as already demonstrated by others.

We are examining the long-term effects of bacterial infection on host cell characters. We observed a dose-dependent increase in the efficiency of ExoS54-Cre-mediated recombination, which peaked around 30% at a MOI of 50 for R26R-EYFP mESC. The subsequent decline in recombination is clearly not due to insufficient protein delivery, but more likely (+)-JQ1 distributor resultant of excess protein translocation or bacterial cytotoxicity. Deletion of the P. aeruginosa type III secreted exotoxins resulted in a considerable decrease in cytotoxicity, as compared to that of wild-type PAK-J strain, which allowed cells to remain viable and undergo gene expression changes after infection. However, P. aeruginosa possesses additional virulence factors that contribute to cytotoxicity. Efforts are currently underway to further reduce the toxicity of this strain in an attempt to enhance cell viability and the efficacy of protein delivery. In addition to reduction of cytotoxicity, we are also in the process of engineering a strain which is more sensitive to antibiotics. Currently, gentamicin and ciprofloxacin are used to eradicate any residual bacteria after infection. While bacterial survival assays have indicated that these conditions are sufficient to destroy lingering intracellular and extracellular bacteria, it will be more convenient to infect with a strain that is sensitive to antibiotics commonly used in cell culture, such as penicillin and streptomycin. Alternatively, an auxotrophic mutant can also be utilized in which specific nutrient withdrawal will result in inhibition of the bacterial growth. These studies serve as a foundation for the bacterial delivery of transcription factors to efficiently modulate concentration-dependent and temporal activation of gene expression to direct cell fate switch without jeopardizing genomic integrity which is critical for future clinical translation. The ability of few exogenous transcription factors to completely redirect endogenous gene expression is epitomized by the discovery of nuclear reprogramming. Induction of pluripotency, while almost exclusively achieved by transgene expression, has been documented to occur with recombinant protein transduction as well, albeit at extremely low efficiency . Having demonstrated the ability to deliver large quantities of nuclear targeting protein directly into eukaryotic cells, efforts are currently underway to harness the power of the type III secretion system to deliver nuclear reprogramming factors to efficiently induce pluripotency and lineage specific differentiation. Antimicrobial peptides, an innate immune component ubiquitous among plants and animals, are variously active against a wide range of pathogens, such as gram-positive bacteria, gramnegative bacteria, fungi and protozoa. They are therefore proposed as one of the most likely substitutes for common antibiotics, to confront an increasingly serious threat to human health caused by antibiotic-resistant bacterial infection.

To replace many of the current methods that have several pitfalls such as inefficient, mutagenic and clinically inapplicable. To illustrate the utility of this system, we have generated a strain of P. aeruginosa with Cycloheximide diminished cytotoxicity to deliver Cre, a well characterized protein which functions only in the nucleus to interact with DNA and exerts its recombinase activity. The availability of numerous Cre-dependent reporters has conveniently allowed us to observe the effects of Cre delivery to both differentiated and pluripotent cells. The ability of bacterially delivered ExoS54-Cre to induce high levels of b-galactosidase activity in TE26 cells indicates that the process of type III secretion and the presence of the ExoS54 signal sequence do not interfere with the ability of this protein to properly localize to the nucleus of host cells or exert its biological function. Our experimental data support the notion that Cre-mediated DNA recombination can only occur when cells are in S phase or when the target loxP sites are freely available, as evident from the observation that the LacZ positive recombinant TE26 cells increase in proportion to the S phase cells during the Cre delivery by the bacteria. For TE26 cells, one full cell cycle takes around 34 hours to complete under our culture condition and a transition time from G2 to S phase takes about 20 hours, thus to achieve close to 100% efficiency of recombination, the injected Cre protein needs to be stable for a time period when all of the cells go through the S phase, which is at least 20 hours for TE26 cell. Apparently, the intracellular stability of the injected ExoS54-Cre is much shorter than the required 20 hours, presumably due to proteasome mediated breakdown. It is possible to achieve 100% Cre-mediated recombination by combining several approaches, including an increase of S-phase cell fraction by synchronization, an increase of the half life of the translocated Cre by utilizing a proteosome inhibitor, and successive infections for Cre to encounter S phase for every cell in the population. While embryonic stem cells are notoriously sensitive, the presence of EYFP positive colonies after bacterial delivery of ExoS54-Cre indicates that these cells are susceptible to P. aeruginosa infection, but neither infection nor protein delivery significantly affects the cellular morphology of R26R-EYFP mESCs. This finding is in accordance with the results of a recent study which demonstrates that mESC infection with other T3SS expressing bacteria, such as Shigella flexneri, does not alter pluripotency, as evaluated by Oct4 expression levels. Given the inherent difficulty of manipulating embryonic stem cells, we have achieved relatively high delivery efficiency in a short time with a single infection. The infection efficiency may further be increased with multiple rounds of protein delivery. We are currently investigating the proficiency of repeated protein delivery to modify gene expression.