Efficacy of iclaprim against wild-type and thymidine kinase-deficient methicillin-resistant Staphylococcus aureus isolates in an in vitro fibrin clot model.

Iclaprim is a novel diaminopyrimidine antibiotic that is active against methicillin-resistant Staphylococcus aureus (MRSA). However, it is known that the activity of diaminopyrimidines against S. aureus is antagonized by thymidine through uptake and conversion to thymidylate by thymidine kinase. Unlike with humans, for whom thymidine levels are low, thymidine levels in rodents are high, thus precluding the accurate evaluation of iclaprim efficacy in animal models. We have studied the bactericidal activity of iclaprim against an isogenic pair of MRSA isolates, the wild-type parent AW6 and its thymidine kinase-deficient mutant AH1252, in an in vitro fibrin clot model. Clots, which were aimed at mimicking vegetation structure, were made from human or rat plasma containing either the parent AW6 or the mutant AH1252, and they were exposed to homologous serum supplemented with iclaprim (3.5 microg/ml), trimethoprim-sulfamethoxazole (TMP-SMX; 8/40 microg/ml), vancomycin (40 microg/ml), or saline, each of which was added one time for 48 h. In rat clots, iclaprim and TMP-SMX were bacteriostatic against the parent, AW6. In contrast, they were bactericidal (> or = 3 log10 CFU/clot killing of the original inoculum) against the mutant AH1252. Vancomycin was the most active drug against AW6 (P < 0.05), but it showed an activity similar those of iclaprim and TMP-SMX against AH1252. In human clots, iclaprim was bactericidal against both AW6 and AH1252 strains and was as effective as TMP-SMX and vancomycin (P > 0.05). Future studies of animals using simulated human kinetics of iclaprim and thymidine kinase-deficient MRSA, which eliminate the thymidine-induced confounding effect, are warranted to support the use of iclaprim in the treatment of severe MRSA infections in humans.

Cancer cell-autonomous contribution of type I interferon signaling to the efficacy of chemotherapy.

Some of the anti-neoplastic effects of anthracyclines in mice originate from the induction of innate and T cell-mediated anticancer immune responses. Here we demonstrate that anthracyclines stimulate the rapid production of type I interferons (IFNs) by malignant cells after activation of the endosomal pattern recognition receptor Toll-like receptor 3 (TLR3). By binding to IFN-α and IFN-β receptors (IFNARs) on neoplastic cells, type I IFNs trigger autocrine and paracrine circuitries that result in the release of chemokine (C-X-C motif) ligand 10 (CXCL10). Tumors lacking Tlr3 or Ifnar failed to respond to chemotherapy unless type I IFN or Cxcl10, respectively, was artificially supplied. Moreover, a type I IFN-related signature predicted clinical responses to anthracycline-based chemotherapy in several independent cohorts of patients with breast carcinoma characterized by poor prognosis. Our data suggest that anthracycline-mediated immune responses mimic those induced by viral pathogens. We surmise that such 'viral mimicry' constitutes a hallmark of successful chemotherapy.

Novel micelle carriers for cyclosporin A topical ocular delivery: in vivo cornea penetration, ocular distribution and efficacy studies.

Cornea transplantation is one of the most performed graft procedures worldwide with an impressive success rate of 90%. However, for "high-risk" patients with particular ocular diseases in addition to the required surgery, the success rate is drastically reduced to 50%. In these cases, cyclosporin A (CsA) is frequently used to prevent the cornea rejection by a systemic treatment with possible systemic side effects for the patients. To overcome these problems, it is a challenge to prepare well-tolerated topical CsA formulations. Normally high amounts of oils or surfactants are needed for the solubilization of the very hydrophobic CsA. Furthermore, it is in general difficult to obtain ocular therapeutic drug levels with topical instillations due to the corneal barriers that efficiently protect the intraocular structures from foreign substances thus also from drugs. The aim of this study was to investigate in vivo the effects of a novel CsA topical aqueous formulation. This formulation was based on nanosized polymeric micelles as drug carriers. An established rat model for the prevention of cornea graft rejection after a keratoplasty procedure was used. After instillation of the novel formulation with fluorescent labeled micelles, confocal analysis of flat-mounted corneas clearly showed that the nanosized carriers were able to penetrate into all corneal layers. The efficacy of a 0.5% CsA micelle formulation was tested and compared to a physiological saline solution and to a systemic administration of CsA. In our studies, the topical CsA treatment was carried out for 14 days, and the three parameters (a) cornea transparency, (b) edema, and (c) neovascularization were evaluated by clinical observation and scoring. Compared to the control group, the treated group showed a significant higher cornea transparency and significant lower edema after 7 and 13 days of the surgery. At the end point of the study, the neovascularization was reduced by 50% in the CsA-micelle treated animals. The success rate of cornea graft transplantation was 73% in treated animals against 25% for the control group. This result was as good as observed for a systemic CsA treatment in the same animal model. This new formulation has the same efficacy like a systemic treatment but without the serious CsA systemic side effects. Ocular drug levels of transplanted and healthy rat eyes were dosed by UPLC/MS and showed a high CsA value in the cornea (11710 ± 7530 ng(CsA)/g(tissue) and 6470 ± 1730 ng(CsA)/g(tissue), respectively). In conclusion, the applied formulation has the capacity to overcome the ocular surface barriers, the micelles formed a drug reservoir in the cornea from, where a sustained release of CsA can take place. This novel formulation for topical application of CsA is clearly an effective and well-tolerated alternative to the systemic treatment for the prevention of corneal graft rejection.

Prediction of sustained antihypertensive efficacy of chronic captopril therapy: relationships to immediate blood pressure response and control plasma renin activity

The blood pressure (BP) lowering effect of the orally active angiotensin converting enzyme inhibitor, captopril (SQ14225), was studied in 59 hypertensive patients maintained on a constant sodium intake. Within 2 hours of the first dose of captopril BP fell from 171/107 to a maximum low of 142/92 mm Hg (p less than 0.001), and after 4 to 8 days to treatment BP averaged 145/94 mm Hg (p less than 0.001). The magnitude of BP drop induced by captopril was significantly correlated to baseline plasma renin activity (PRA) both during the acute phase (r = -0.38, p less than 0.01) and after the 4 to 8-day interval (r = -0.33, p less than 0.01). Because of considerable scatter in individual data, renin profiling was not precisely predictive of the immediate or delayed BP response of separate patients. However, the BP levels achieved following the initial dose of captopril were closely correlated to BP measured after 4 to 8 days of therapy, and appeared to have greater predictive value than control PRA of the long-term efficacy of chronic captopril therapy despite marked BP changes occurring in some patients during the intermediate period. Because of these intermediate BP changes, addition of a diuretic to enhance antihypertensive effectiveness of angiotensin blockade should be restrained for several days after initiation of captopril therapy.

iNKT/CD1d-antitumor immunotherapy significantly increases the efficacy of therapeutic CpG/peptide-based cancer vaccine.

BACKGROUND: Therapeutic cancer vaccines aim to boost the natural immunity against transformed cancer cells, and a series of adjuvants and co-stimulatory molecules have been proposed to enhance the immune response against weak self-antigens expressed on cancer cells. For instance, a peptide/CpG-based cancer vaccine has been evaluated in several clinical trials and was shown in pre-clinical studies to favor the expansion of effector T versus Tregs cells, resulting in a potent antitumor activity, as compared to other TLR ligands. Alternatively, the adjuvant activity of CD1d-restricted invariant NKT cells (iNKT) on the innate and adaptive immunity is well demonstrated, and several CD1d glycolipid ligands are under pre-clinical and clinical evaluation. Importantly, additive or even synergistic effects have been shown upon combined CD1d/NKT agonists and TLR ligands. The aim of the present study is to combine the activation and tumor targeting of activated iNKT, NK and T cells. METHODS: Activation and tumor targeting of iNKT cells via recombinant α-galactosylceramide (αGC)-loaded CD1d-anti-HER2 fusion protein (CD1d-antitumor) is combined or not with OVA peptide/CpG vaccine. Circulating and intratumoral NK and H-2Kb/OVA-specific CD8 responses are monitored, as well as the state of activation of dendritic cells (DC) with regard to activation markers and IL-12 secretion. The resulting antitumor therapy is tested against established tumor grafts of B16 melanoma cells expressing human HER2 and ovalbumin. RESULTS: The combined CD1d/iNKT antitumor therapy and CpG/peptide-based immunization leads to optimized expansion of NK and OVA-specific CD8 T cells (CTLs), likely resulting from the maturation of highly pro-inflammatory DCs as seen by a synergistic increase in serum IL-12. The enhanced innate and adaptive immune responses result in higher tumor inhibition that correlates with increased numbers of OVA-specific CTLs at the tumor site. Antibody-mediated depletion experiments further demonstrate that in this context, CTLs rather than NK cells are essential for the enhanced tumor inhibition. CONCLUSIONS: Altogether, our study in mice demonstrates that αGC/CD1d-antitumor fusion protein greatly increases the efficacy of a therapeutic CpG-based cancer vaccine, first as an adjuvant during T cell priming and second, as a therapeutic agent to redirect immune responses to the tumor site.

Enhancing efficacy of anticancer vaccines by targeted delivery to tumor-draining lymph nodes.

The sentinel or tumor-draining lymph node (tdLN) serves as a metastatic niche for many solid tumors and is altered via tumor-derived factors that support tumor progression and metastasis. tdLNs are often removed surgically, and therapeutic vaccines against tumor antigens are typically administered systemically or in non-tumor-associated sites. Although the tdLN is immune-suppressed, it is also antigen experienced through drainage of tumor-associated antigens (TAA), so we asked whether therapeutic vaccines targeting the tdLN would be more or less effective than those targeting the non-tdLN. Using LN-targeting nanoparticle (NP)-conjugate vaccines consisting of TAA-NP and CpG-NP, we compared delivery to the tdLN versus non-tdLN in two different cancer models, E.G7-OVA lymphoma (expressing the nonendogenous TAA ovalbumin) and B16-F10 melanoma. Surprisingly, despite the immune-suppressed state of the tdLN, tdLN-targeting vaccination induced substantially stronger cytotoxic CD8+ T-cell responses, both locally and systemically, than non-tdLN-targeting vaccination, leading to enhanced tumor regression and host survival. This improved tumor regression correlated with a shift in the tumor-infiltrating leukocyte repertoire toward a less suppressive and more immunogenic balance. Nanoparticle coupling of adjuvant and antigen was required for effective tdLN targeting, as nanoparticle coupling dramatically increased the delivery of antigen and adjuvant to LN-resident antigen-presenting cells, thereby increasing therapeutic efficacy. This work highlights the tdLN as a target for cancer immunotherapy and shows how its antigen-experienced but immune-suppressed state can be reprogrammed with a targeted vaccine yielding antitumor immunity.

Safety and efficacy in gamma knife radiosurgery for trigeminal neuralgia due to megadolichbasilar artery compression: a prospective series of 29 consecutive patients

Purpose: To analyse prospectively the long-term results of Gamma Knife surgery (GKS) in patients with trigeminal neuralgia secondary to megadolichobasilar artery (MBA). Methods: Between December 1992 and November 2010, 33 consecutive patients presenting with ITN secondary to MBA were operated by GKS and followed prospectively in Timone University Hospital. The follow up is at least of 1 year in 29 patients. The median age was 74.90 years (range 51 to 90). The GKS typically was performed using MR and CT imaging guidance and a single 4 mm isocenter. The median of the prescription dose (at the 100%) was 90 Gy (range 80 to 90). The target was placed on the cisternal portion of the Vth nerve. Clinical and dosimetric parameters were analyzed. GKS was the first surgical procedure in 23 patients (79.31%). Results: The median follow- up period was 46.12 months (range 12.95 to 157.93). All the 29 patients (100%) were initially pain free in a median time of 13.5 days (range 0 to 240). The probability of remaining pain free at 0.5, 1, 2 years was 93.1%, 79.3% and 75.7% respectively, reaching at this time the flat part of the curve. Seven patients (24.13%) experienced a recurrence with a median delay of 10.75 months (range 3.77 to 12.62). The actuarial rate of recurrence was not higher than in our population with essential TN although atypical pain was associated with a much higher risk of recurrence (HR= 6.92, p= 0.0117). The hypoesthesia actuarial rates at 0.5 years was 4.3% and at 1 year reach 13% and remains stable till 12 years with a median delay of onset of 7 (5, 12) months. Female patients had a statistically much lower probability of developing a facial numbness (p of 0.03). No patient reported a bothersome hypoesthesia. Conclusion: Retrogaserian, high dose GKS, turned out to be very safe with only 13.04% hypoesthesia, which was never disabling (0%), while achieving high quality pain control. The majority of the patients demonstrated a prolonged effect of radiosurgery in absence of any trigeminal nerve disturbance.

Relationship of imatinib-free plasma levels and target genotype with efficacy and tolerability

Imatinib has revolutionised the treatment of chronic myeloid leukaemia (CML) and gastrointestinal stromal tumours (GIST). Using a nonlinear mixed effects population model, individual estimates of pharmacokinetic parameters were derived and used to estimate imatinib exposure (area under the curve, AUC) in 58 patients. Plasma-free concentration was deduced from a model incorporating plasma levels of alpha(1)-acid glycoprotein. Associations between AUC (or clearance) and response or incidence of side effects were explored by logistic regression analysis. Influence of KIT genotype was also assessed in GIST patients. Both total (in GIST) and free drug exposure (in CML and GIST) correlated with the occurrence and number of side effects (e.g. odds ratio 2.7+/-0.6 for a two-fold free AUC increase in GIST; P<0.001). Higher free AUC also predicted a higher probability of therapeutic response in GIST (odds ratio 2.6+/-1.1; P=0.026) when taking into account tumour KIT genotype (strongest association in patients harbouring exon 9 mutation or wild-type KIT, known to decrease tumour sensitivity towards imatinib). In CML, no straightforward concentration-response relationships were obtained. Our findings represent additional arguments to further evaluate the usefulness of individualizing imatinib prescription based on a therapeutic drug monitoring programme, possibly associated with target genotype profiling of patients.

Efficacy of an eradication protocol in case of Pseudomonas aeruginosa primo-infections in children with cystic fibrosis

Introduction: Patients with cystic fibrosis (CF) are more susceptible to pathogens like P. aeruginosa (PA). PA primo-­‐infections require particular attention, as with failure in eradication, there is accelerated lung deterioration. The main aim of this study is to assess the rate of PA eradication according to our particular protocol with inhaled tobramycin and oral ciprofloxacin, as there is no consensus in the literature on what eradication protocol the best is. Methods: Retrospective single centre study with data analysis from June 1st 2007 to June 1st 2011 of patients who had primo-­‐infections exclusively treated by 3 x 28 days of inhaled tobramycin and oral ciprofloxacin for the first and last 21 days. Success in eradication is defined by ≥ 3 negative bacteriologies for 6 months after the beginning of the protocol. If ≥ 1 bacteriology is positive, we consider the eradication as a failure. Results: Out of 41 patients, 18 were included in our analysis. 7 girls (38.9%) and 11 boys (61.1%) followed the eradication protocol. Boys had 12 primo-­‐infections and girls had 8. Among these 20 primo-­‐infections, 16 (80%) had an all-­‐overall success in eradication and 4 (20%) a failure. No significant statistical difference for age between these groups (t-­‐test = 0.07, p = 0.94), neither for FEV1% (t-­‐test = 0.96, p = 0.41) nor BMI (t-­‐test = 1.35, p = 0.27). Rate of success was 100% for girls and 66.6% for boys. Conclusion: Our protocol succeeded in an overall eradication rate of 80%, without statistical significant impact on FEV1 % and BMI values. However, there is a sex difference with eradication rates in girls (100%) and boys (66.6%). A sex difference has not yet been reported in the literature. This should be evaluated in further studies.