Proximal sodium reabsorption: An independent determinant of blood pressure response to salt.

The purpose of this study was to evaluate the contribution of renal sodium handling by the proximal tubule as an independent determinant of blood pressure responsiveness to salt in hypertension. We measured blood pressure (BP), renal hemodynamics, and segmental renal sodium handling (with lithium used as a marker of proximal sodium reabsorption) in 38 hypertensive patients and 27 normotensive subjects (15 young and 12 age-matched) on a high and low sodium diet. In control subjects, changing the diet from a low to a high sodium content resulted in no change in BP and increases in glomerular filtration rate (P<0.05), renal plasma flow (P<0.05), and fractional excretion of lithium (FE(Li), P<0.01). In hypertensive patients, comparable variations of sodium intake induced an increase in BP with no change in renal hemodynamics and proximal sodium reabsorption. When analyzed by tertiles of their BP response to salt, salt-insensitive hypertensive patients of the first tertile disclosed a pattern of adaptation of proximal sodium reabsorption comparable to that of control subjects, whereas the most salt-sensitive patients of the third tertile had an inverse pattern with a high FE(Li) on low salt and a lower FE(Li) on high salt, suggesting an inappropriate modulation of proximal sodium reabsorption. The BP response to salt correlated positively with age (r=0.34, P=0.036) and negatively with the changes in FE(Li) (r=-0.37, P=0.029). In a multivariate analysis, the changes in FE(Li) were significantly and independently associated with the salt-induced changes in BP. These results suggest that proximal sodium reabsorption is an independent determinant of the BP response to salt in hypertension.

The effect of endocrine responsiveness on high-risk breast cancer treated with dose-intensive chemotherapy: results of International Breast Cancer Study Group Trial 15-95 after prolonged follow-up.

BACKGROUND: The role of adjuvant dose-intensive chemotherapy and its efficacy according to baseline features has not yet been established. PATIENTS AND METHODS: Three hundred and forty-four patients were randomized to receive seven courses of standard-dose chemotherapy (SD-CT) or three cycles of dose-intensive epirubicin and cyclophosphamide (epirubicin 200 mg/m(2) plus cyclophosphamide 4 mg/m(2) with filgrastim and progenitor cell support). All patients were assigned tamoxifen at the completion of chemotherapy. The primary end point was disease-free survival (DFS). This paper updates the results and explores patterns of recurrence according to predicting baseline features. RESULTS: At 8.3-years median follow-up, patients assigned DI-EC had a significantly better DFS compared with those assigned SD-CT [8-year DFS percent 47% and 37%, respectively, hazard ratio (HR) 0.76; 95% confidence interval 0.58-1.00; P = 0.05]. Only patients with estrogen receptor (ER)-positive disease benefited from the DI-EC (HR 0.61; 95% confidence interval 0.39, 0.95; P = 0.03). CONCLUSIONS: After prolonged follow-up, DI-EC significantly improved DFS, but the effect was observed only in patients with ER-positive disease, leading to the hypothesis that efficacy of DI-EC may relate to its endocrine effects. Further studies designed to confirm the importance of endocrine responsiveness in patients treated with dose-intensive chemotherapy are encouraged.

Combination of lentivector immunization and low-dose chemotherapy or PD-1/PD-L1 blocking primes self-reactive T cells and induces anti-tumor immunity.

In the last two decades, anti-cancer vaccines have yielded disappointing clinical results despite the fact that high numbers of self/tumor-specific T cells can be elicited in immunized patients. Understanding the reasons behind this lack of efficacy is critical in order to design better treatment regimes. Recombinant lentivectors (rLVs) have been successfully used to induce antigen-specific T cells to foreign or mutated tumor antigens. Here, we show that rLV expressing a murine nonmutated self/tumor antigen efficiently primes large numbers of self/tumor-specific CD8(+) T cells. In spite of the large number of tumor-specific T cells, however, no anti-tumor activity could be measured in a therapeutic setting, in mice vaccinated with rLV. Accumulating evidence shows that, in the presence of malignancies, inhibition of T-cell activity may predominate overstimulation. Analysis of tumor-infiltrating lymphocytes revealed that specific anti-tumor CD8(+) T cells fail to produce cytokines and express high levels of inhibitory receptors such as programmed death (PD)-1. Association of active immunization with chemotherapy or antibodies that block inhibitory pathways often leads to better anti-tumor effects. We show here that combining rLV vaccination with either cyclophosphamide or PD-1 and PD-L1 blocking antibodies enhances rLV vaccination efficacy and improves anti-tumor immunity.

Long-term results of a multicenter SAKK trial on high-dose ifosfamide and doxorubicin in advanced or metastatic gynecologic sarcomas.

BACKGROUND: Dose intensive chemotherapy has not been tested prospectively for the treatment of gynecologic sarcomas. We investigated the antitumor activity and toxicity of high-dose ifosfamide and doxorubicin, in the context of a multidisciplinary strategy for the treatment of advanced and metastatic, not pretreated, gynecologic sarcomas. PATIENTS AND METHODS: Thirty-nine patients were enrolled onto a phase I-II multicenter trial of ifosfamide, 10 g/m2 as a continuous infusion over 5 days, plus doxorubicin intravenously, 25 mg/m2/day for 3 days with Mesna and granulocyte-colony-stimulating factor every 21 days. Salvage therapy was allowed after chemotherapy. RESULTS: Among the 37 evaluable patients, the tumor was locally advanced (n = 11), with concomitant distant metastases (n = 5) or with distant metastases only (n = 21). After a median of three (range 1-7) chemotherapy cycles, six patients experienced a complete response and 12 a partial response for an overall response rate of 49% (95% CI 32% to 66%). The response rate was higher in poorly differentiated tumors (62%) compared with moderately well differentiated ones (18%), but was not different according to histology subtypes. Eleven patients had salvage therapy, either immediately following chemotherapy (n = 7) or at time of progression (n = 4). With a median follow-up time of 5 years, the median overall survival was 30.5 months. Hematological toxicity was as expected neutropenia, thrombopenia and anemia > or = grade 3 at 50%, 34% and 33% of cycles respectively. No toxic death occurred. CONCLUSIONS: High-dose ifosfamide plus doxorubicin is an active regimen for all subtypes of gynecological sarcomas. Its toxicity was manageable in a multicentric setting. The prolonged survival might be due to the multidisciplinary strategy that was possible in one-third of the patients.

Efficacy and safety of a phospholipid emulsion (GR270773) in Gram-negative severe sepsis: results of a phase II multicenter, randomized, placebo-controlled, dose-finding clinical trial.

OBJECTIVE: To assess the survival benefit and safety profile of low-dose (850 mg/kg) and high-dose (1350 mg/kg) phospholipid emulsion vs. placebo administered as a continuous 3-day infusion in patients with confirmed or suspected Gram-negative severe sepsis. Preclinical and ex vivo studies show that lipoproteins bind and neutralize endotoxin, and experimental animal studies demonstrate protection from septic death when lipoproteins are administered. Endotoxin neutralization correlates with the amount of phospholipid in the lipoprotein particles. DESIGN: A three-arm, randomized, blinded, placebo-controlled trial. SETTING: Conducted at 235 centers worldwide between September 2004 and April 2006. PATIENTS: A total of 1379 patients participated in the study, 598 patients received low-dose phospholipid emulsion, and 599 patients received placebo. The high-dose phospholipid emulsion arm was stopped, on the recommendation of the Independent Data Monitoring Committee, due to an increase in life-threatening serious adverse events at the fourth interim analysis and included 182 patients. MEASUREMENTS AND MAIN RESULTS: A 28-day all-cause mortality and new-onset organ failure. There was no significant treatment benefit for low- or high-dose phospholipid emulsion vs. placebo for 28-day all-cause mortality, with rates of 25.8% (p = .329), 31.3% (p = .879), and 26.9%, respectively. The rate of new-onset organ failure was not statistically different among groups at 26.3%, 31.3%, 20.4% with low- and high-dose phospholipid emulsion, and placebo, respectively (one-sided p = .992, low vs. placebo; p = .999, high vs. placebo). Of the subjects treated, 45% had microbiologically confirmed Gram-negative infections. Maximal changes in mean hemoglobin levels were reached on day 10 (-1.04 g/dL) and day 5 (-1.36 g/dL) with low- and high-dose phospholipid emulsion, respectively, and on day 14 (-0.82 g/dL) with placebo. CONCLUSIONS: Treatment with phospholipid emulsion did not reduce 28-day all-cause mortality, or reduce the onset of new organ failure in patients with suspected or confirmed Gram-negative severe sepsis.

Acute respiratory syndrome after inhalation of waterproofing sprays: a posteriori exposure-response assessment in 102 cases

Waterproofing agents are widely used to protect leather and textiles in both domestic and occupational activities. An outbreak of acute respiratory syndrome following exposure to waterproofing sprays occurred during the winter 2002-2003 in Switzerland. About 180 cases were reported by the Swiss Toxicological Information Centre between October 2002 and March 2003, whereas fewer than 10 cases per year had been recorded previously. The reported cases involved three brands of sprays containing a common waterproofing mixture, that had undergone a formulation change in the months preceding the outbreak. A retrospective analysis was undertaken in collaboration with the Swiss Toxicological Information Centre and the Swiss Registries for Interstitial and Orphan Lung Diseases to clarify the circumstances and possible causes of the observed health effects. Individual exposure data were generated with questionnaires and experimental emission measurements. The collected data was used to conduct numeric simulation for 102 cases of exposure. A classical two-zone model was used to assess the aerosol dispersion in the near- and far-field during spraying. The resulting assessed dose and exposure levels obtained were spread on large scales, of several orders of magnitude. No dose-response relationship was found between exposure indicators and health effects indicators (perceived severity and clinical indicators). Weak relationships were found between unspecific inflammatory response indicators (leukocytes, C-reactive protein) and the maximal exposure concentration. The results obtained disclose a high interindividual response variability and suggest that some indirect mechanism(s) predominates in the respiratory disease occurrence. Furthermore, no threshold could be found to define a safe level of exposure. These findings suggest that the improvement of environmental exposure conditions during spraying alone does not constitute a sufficient measure to prevent future outbreaks of waterproofing spray toxicity. More efficient preventive measures are needed prior to the marketing and distribution of new waterproofing agents.

Substance P-induced skin inflammation is not modulated by a single dose of sitagliptin in human volunteers.

Substance P (SP), an undecapeptide belonging to the tachykinin family, is released during the activation of sensory nerves, and causes vasodilation, edema and pain through activation of tissular Neurokinin 1 receptors. SP proinflammatory effects are terminated by angiotensin converting enzyme (ACE) and neutral endopeptidase (NEP), while the aminopeptidase dipeptidylpeptidase IV (DPPIV) can also play a role. The aim of this randomized, crossover, double-blind study was to assess the cutaneous vasoreactivity (flare and wheal reaction, burning pain sensation) to intradermal injection of ascending doses of SP in six volunteers receiving a single therapeutic dose of the DPPIV inhibitor sitagliptin or a matching placebo. Cutaneous SP challenges produced the expected, dose-dependent flare and wheal response, while eliciting mild to moderate local pain sensation with little dose dependency. However, no differences were shown in the responses observed under sitagliptin compared with placebo, while the study would have been sufficiently powered to detect a clinically relevant increase in sensitivity to SP. The results of this pilot study are in line with proteolytic cleavage of SP by ACE and NEP compensating the blockade of DPPIV to prevent an augmentation of its proinflammatory action.

TLR3 and Rig-like receptor on myeloid dendritic cells and Rig-like receptor on human NK cells are both mandatory for production of IFN-gamma in response to double-stranded RNA.

Cross-talk between NK cells and dendritic cells (DCs) is critical for the potent therapeutic response to dsRNA, but the receptors involved remained controversial. We show in this paper that two dsRNAs, polyadenylic-polyuridylic acid and polyinosinic-polycytidylic acid [poly(I:C)], similarly engaged human TLR3, whereas only poly(I:C) triggered human RIG-I and MDA5. Both dsRNA enhanced NK cell activation within PBMCs but only poly(I:C) induced IFN-gamma. Although myeloid DCs (mDCs) were required for NK cell activation, induction of cytolytic potential and IFN-gamma production did not require contact with mDCs but was dependent on type I IFN and IL-12, respectively. Poly(I:C) but not polyadenylic-polyuridylic acid synergized with mDC-derived IL-12 for IFN-gamma production by acting directly on NK cells. Finally, the requirement of both TLR3 and Rig-like receptor (RLR) on mDCs and RLRs but not TLR3 on NK cells for IFN-gamma production was demonstrated using TLR3- and Cardif-deficient mice and human RIG-I-specific activator. Thus, we report the requirement of cotriggering TLR3 and RLR on mDCs and RLRs on NK cells for a pathogen product to induce potent innate cell activation.

Drug concentration response relationships in normal volunteers after oral administration of losartan, an angiotensin II receptor antagonist.

The aim of this study was to investigate the relationships between plasma concentrations of losartan, an orally active angiotensin II inhibitor, its active metabolite EXP3174, and angiotensin II blockade. Six healthy subjects received single oral doses of 40, 80, or 120 mg losartan and placebo at 1-week intervals in a crossover study. Angiotensin II blockade was assessed by the blood pressure response to exogenous angiotensin II before and after losartan administration. EXP3174 reached higher plasma concentrations and was eliminated more slowly than its parent compound; its levels paralleled the profile of angiotensin II blockade closer than losartan. Inhibition of the pressure response was dose dependent. The Hill-shaped relationship between response and EXP3174 concentration (or time-integrated variables) approached a plateau with 80 mg. The dose-dependent increase in plasma renin and angiotensin II exhibited a considerable individual scatter. We conclude that losartan produces a dose-dependent, effective angiotensin II blockade that is largely determined by the active metabolite EXP3174.

Cell-permeable peptides induce dose- and length-dependent cytotoxic effects.

We have explored the threshold of tolerance of three unrelated cell types to treatments with potential cytoprotective peptides bound to Tat(48-57) and Antp(43-58) cell-permeable peptide carriers. Both Tat(48-57) and Antp(43-58) are well known for their good efficacy at crossing membranes of different cell types, their overall low toxicity, and their absence of leakage once internalised. Here, we show that concentrations of up to 100 microM of Tat(48-57) were essentially harmless in all cells tested, whereas Antp(43-58) was significantly more toxic. Moreover, all peptides bound to Tat(48-57) and Antp(43-58) triggered significant and length-dependent cytotoxicity when used at concentrations above 10 microM in all but one cell types (208F rat fibroblasts), irrespective of the sequence of the cargo. Absence of cytotoxicity in 208F fibroblasts correlated with poor intracellular peptide uptake, as monitored by confocal laser scanning fluorescence microscopy. Our data further suggest that the onset of cytotoxicity correlates with the activation of two intracellular stress signalling pathways, namely those involving JNK, and to a lesser extent p38 mitogen-activated protein kinases. These responses are of particular concern for cells that are especially sensitive to the activation of stress kinases. Collectively, these results indicate that in order to avoid unwanted and unspecific cytotoxicity, effector molecules bound to Tat(48-57) should be designed with the shortest possible sequence and the highest possible affinity for their binding partners or targets, so that concentrations below 10 microM can be successfully applied to cells without harm. Considering that cytotoxicity associated to Tat(48-57)- and Antp(43-58) bound peptide conjugates was not restricted to a particular type of cells, our data provide a general framework for the design of cell-penetrating peptides that may apply to broader uses of intracellular peptide and drug delivery.

Enhanced cytotoxicity and decreased CD8 dependence of human cancer-specific cytotoxic T lymphocytes after vaccination with low peptide dose.

In mice, vaccination with high peptide doses generates higher frequencies of specific CD8+ T cells, but with lower avidity compared to vaccination with lower peptide doses. To investigate the impact of peptide dose on CD8+ T cell responses in humans, melanoma patients were vaccinated with 0.1 or 0.5 mg Melan-A/MART-1 peptide, mixed with CpG 7909 and Incomplete Freund's adjuvant. Neither the kinetics nor the amplitude of the Melan-A-specific CD8+ T cell responses differed between the two vaccination groups. Also, CD8+ T cell differentiation and cytokine production ex vivo were similar in the two groups. Interestingly, after low peptide dose vaccination, Melan-A-specific CD8+ T cells showed enhanced degranulation upon peptide stimulation, as assessed by CD107a upregulation and perforin release ex vivo. In accordance, CD8+ T cell clones derived from low peptide dose-vaccinated patients showed significantly increased degranulation and stronger cytotoxicity. In parallel, Melan-A-specific CD8+ T cells and clones from low peptide dose-vaccinated patients expressed lower CD8 levels, despite similar or even stronger binding to tetramers. Furthermore, CD8+ T cell clones from low peptide dose-vaccinated patients bound CD8 binding-deficient tetramers more efficiently, suggesting that they may express higher affinity TCRs. We conclude that low peptide dose vaccination generated CD8+ T cell responses with stronger cytotoxicity and lower CD8 dependence.

Response curves of deterministic and probabilistic cellular automata in one and two dimensions

One of the most important problems in the theory of cellular automata (CA) is determining the proportion of cells in a specific state after a given number of time iterations. We approach this problem using patterns in preimage sets - that is, the set of blocks which iterate to the desired output. This allows us to construct a response curve - a relationship between the proportion of cells in state 1 after niterations as a function of the initial proportion. We derive response curve formulae for many two-dimensional deterministic CA rules with L-neighbourhood. For all remaining rules, we find experimental response curves. We also use preimage sets to classify surjective rules. In the last part of the thesis, we consider a special class of one-dimensional probabilistic CA rules. We find response surface formula for these rules and experimental response surfaces for all remaining rules.

A Bayesian approach for dose-escalation in a phase I clinical trial incorporating pharmacodynamic endpoints

Bayesian decision procedures have already been proposed for and implemented in Phase I dose-escalation studies in healthy volunteers. The procedures have been based on pharmacokinetic responses reflecting the concentration of the drug in blood plasma and are conducted to learn about the dose-response relationship while avoiding excessive concentrations. However, in many dose-escalation studies, pharmacodynamic endpoints such as heart rate or blood pressure are observed, and it is these that should be used to control dose-escalation. These endpoints introduce additional complexity into the modeling of the problem relative to pharmacokinetic responses. Firstly, there are responses available following placebo administrations. Secondly, the pharmacodynamic responses are related directly to measurable plasma concentrations, which in turn are related to dose. Motivated by experience of data from a real study conducted in a conventional manner, this paper presents and evaluates a Bayesian procedure devised for the simultaneous monitoring of pharmacodynamic and pharmacokinetic responses. Account is also taken of the incidence of adverse events. Following logarithmic transformations, a linear model is used to relate dose to the pharmacokinetic endpoint and a quadratic model to relate the latter to the pharmacodynamic endpoint. A logistic model is used to relate the pharmacokinetic endpoint to the risk of an adverse event.

A double-blind placebo-controlled study to establish the bifidogenic dose of inulin in healthy humans

Objective: To evaluate the bifidogenic efficacy of two inulin doses in healthy human adults. Design: A double-blind, placebo-controlled, crossover human study. Setting: Food Microbial Sciences Unit, The University of Reading, Reading, UK. Subjects: Thirty healthy volunteers, 15 men, 15 women ( age range 19-35). Interventions: Subjects consumed a chocolate drink containing placebo ( maltodextrin, 8 g/day), 5 g/day inulin and 8 g/day inulin for a 2-week treatment period. Each treatment was followed by a 1-week washout at the end of which volunteers progressed to the next treatment. Faecal samples were obtained at the start of the study ( baseline) and at the end of each treatment and washout period. Fluorescent in situ hybridization was used to monitor populations of Bifidobacterium genus, Bacteroides - Prevotella, Lactobacillus - Enterococcus and Clostridium perfringens - histolyticum subgroup. Results: Bifidobacterial levels increased significantly upon ingestion of both the low ( 9.78 +/- 0.29 log(10) cells/g faeces, P < 0.05) and the high inulin dose ( 9.79 +/- 0.38 log(10) cells/g faeces, P < 0.05) compared to placebo ( 9.64 +/- 0.23 log(10) cells/g faeces). Conclusions: Both inulin doses exhibited a bifidogenic effect but a higher volunteer percentage responded to the high dose. A dose response effect was not observed but the magnitude of increase in bifidobacteria levels depended on their initial numbers. The higher the initial concentrations the smaller was the increase upon ingestion of the active treatments. Sponsorship: Financial support for the completion of this project was provided by Sensus ( Roosendaal, The Netherlands).