Tools for the Precision Medicine Era: How to Develop Highly Personalized Treatment Recommendations From Cohort and Registry Data Using Q-Learning.

Q-learning is a method of reinforcement learning that employs backwards stagewise estimation to identify sequences of actions that maximize some long-term reward. The method can be applied to sequential multiple-assignment randomized trials to develop personalized adaptive treatment strategies (ATSs)-longitudinal practice guidelines highly tailored to time-varying attributes of individual patients. Sometimes, the basis for choosing which ATSs to include in a sequential multiple-assignment randomized trial (or randomized controlled trial) may be inadequate. Nonrandomized data sources may inform the initial design of ATSs, which could later be prospectively validated. In this paper, we illustrate challenges involved in using nonrandomized data for this purpose with a case study from the Center for International Blood and Marrow Transplant Research registry (1995-2007) aimed at 1) determining whether the sequence of therapeutic classes used in graft-versus-host disease prophylaxis and in refractory graft-versus-host disease is associated with improved survival and 2) identifying donor and patient factors with which to guide individualized immunosuppressant selections over time. We discuss how to communicate the potential benefit derived from following an ATS at the population and subgroup levels and how to evaluate its robustness to modeling assumptions. This worked example may serve as a model for developing ATSs from registries and cohorts in oncology and other fields requiring sequential treatment decisions.

Statin use and asthma control in patients with severe asthma.

OBJECTIVES: We hypothesised that severe asthmatics taking a statin drug, in addition to inhaled corticosteroids/long-acting ß-agonist inhaler therapy, would have better asthma symptom control and improved lung function compared to their controls. STUDY DESIGN: A retrospective, cross-sectional study of 165 patients with severe asthma seen from 2001-2008. Hierarchical linear and logistic regression models were used for modelling fitting. SETTING: University of California, Davis Medical Center (Sacramento, California, USA). Academic, single-centre, severe asthma subspecialty clinic. PARTICIPANTS: 612 screened, 223 eligible and 165 adult patients were included in the final study (N=165; 31 statin users and 134 non-users). PRIMARY AND SECONDARY OUTCOME MEASURES: The primary endpoint was asthma control as measured by the Asthma Control Test (ACT). The secondary endpoints included lung function, symptoms and the need for corticosteroid burst and peripheral eosinophil count. RESULTS: At baseline, statin users compared to non-users were older, had lower lung function (FEV1% predicted, FEV1, forced vital capacity and FEF25-75%) and had a higher prevalence of comorbid conditions. Statin use was associated with more aspirin and ipratropium inhaler use than in non-users. Patients in both groups were obese (body mass index ≥ 30). Statin users had better asthma symptom control compared to non-users (higher adjusted mean ACT score by 2.2±0.94 points, p<0.02). Median statin use was for 1 year. There were no statistically significant differences in lung function, corticosteroid or rescue bronchodilator use or peripheral eosinophilia between the two groups. CONCLUSIONS: In our severe asthma referral population, statin users already taking inhaled controller therapy achieved better asthma control compared to non-users. The implications of this study is that patients with severe asthma could potentially benefit from added statin treatment. Because our study population was on average obese, the obese severe asthmatic may be a viable asthma subphenotype for further studies. Prospective randomised clinical trials evaluating the safety and efficacy of statins in severe asthma are warranted.

Limited analytical capacity for cyanotoxins in developing countries may hide serious environmental health problems: simple and affordable methods may be the answer.

In recent years, the international demand for commodities has prompted enormous growth in agriculture in most South American countries. Due to intensive use of fertilizers, cyanobacterial blooms have become a recurrent phenomenon throughout the continent, but their potential health risk remains largely unknown due to the lack of analytical capacity. In this paper we report the main results and conclusions of more than five years of systematic monitoring of cyanobacterial blooms in 20 beaches of Montevideo, Uruguay, on the Rio de la Plata, the fifth largest basin in the world. A locally developed microcystin ELISA was used to establish a sustainable monitoring program that revealed seasonal peaks of extremely high toxicity, more than one-thousand-fold greater than the WHO limit for recreational water. Comparison with cyanobacterial cell counts and chlorophyll-a determination, two commonly used parameters for indirect estimation of toxicity, showed that such indicators can be highly misleading. On the other hand, the accumulated experience led to the definition of a simple criterion for visual classification of blooms, that can be used by trained lifeguards and technicians to take rapid on-site decisions on beach management. The simple and low cost approach is broadly applicable to risk assessment and risk management in developing countries.

Nitric oxide synthase enzymes in the airways of mice exposed to ovalbumin: NOS2 expression is NOS3 dependent.

OBJECTIVES AND DESIGN: The function of the airway nitric oxide synthase (NOS) isoforms and the lung cell types responsible for its production are not fully understood. We hypothesized that NO homeostasis in the airway is important to control inflammation, which requires upregulation, of NOS2 protein expression by an NOS3-dependent mechanism. MATERIALS OR SUBJECTS: Mice from a C57BL/6 wild-type, NOS1(-/-), NOS2(-/-), and NOS3(-/-) genotypes were used. All mice strains were systemically sensitized and exposed to filtered air or ovalbumin (OVA) aerosol for two weeks to create a subchronic model of allergen-induced airway inflammation. METHODS: We measured lung function, lung lavage inflammatory and airway epithelial goblet cell count, exhaled NO, nitrate and nitrite concentration, and airway NOS1, NOS2, and NOS3 protein content. RESULTS: Deletion of NOS1 or NOS3 increases NOS2 protein present in the airway epithelium and smooth muscle of air-exposed animals. Exposure to allergen significantly reduced the expression of NOS2 protein in the airway epithelium and smooth muscle of the NOS3(-/-) strain only. This reduction in NOS2 expression was not due to the replacement of epithelial cells with goblet cells as remaining epithelial cells did not express NOS2. NOS1(-/-) animals had significantly reduced goblet cell metaplasia compared to C57Bl/6 wt, NOS2(-/-), and NOS3(-/-) allergen-exposed mice. CONCLUSION: The airway epithelial and smooth muscle cells maintain a stable airway NO concentration under noninflammatory conditions. This "homeostatic" mechanism is unable to distinguish between NOS derived from the different constitutive NOS isoforms. NOS3 is essential for the expression of NOS2 under inflammatory conditions, while NOS1 expression contributes to allergen-induced goblet cell metaplasia.

Compensatory evolution of gene regulation in response to stress by Escherichia coli lacking RpoS.

The RpoS sigma factor protein of Escherichia coli RNA polymerase is the master transcriptional regulator of physiological responses to a variety of stresses. This stress response comes at the expense of scavenging for scarce resources, causing a trade-off between stress tolerance and nutrient acquisition. This trade-off favors non-functional rpoS alleles in nutrient-poor environments. We used experimental evolution to explore how natural selection modifies the regulatory network of strains lacking RpoS when they evolve in an osmotically stressful environment. We found that strains lacking RpoS adapt less variably, in terms of both fitness increase and changes in patterns of transcription, than strains with functional RpoS. This phenotypic uniformity was caused by the same adaptive mutation in every independent population: the insertion of IS10 into the promoter of the otsBA operon. OtsA and OtsB are required to synthesize the osmoprotectant trehalose, and transcription of otsBA requires RpoS in the wild-type genetic background. The evolved IS10 insertion rewires expression of otsBA from RpoS-dependent to RpoS-independent, allowing for partial restoration of wild-type response to osmotic stress. Our results show that the regulatory networks of bacteria can evolve new structures in ways that are both rapid and repeatable.

Diarrhea and parasitosis in Salta, Argentina.

BACKGROUND: Salta city is the capital of the province with the same name located in the northwest of Argentina. Its great growth over the last decade was not organized and the population expanded to occupy places where water and sanitation were not yet available. Although the Arenales River, crossing the city, receives the impact of point and non-point source pollution, the water is used for many purposes, including domestic in the poorest areas, industrial, and recreational with children as the main users. According to the World Health Organization, an estimated 24% of the global disease burden and 23% of all deaths can be attributed to environmental factors. In particular, an estimated 94% of the diarrheal burden of disease is attributable to environment, and is associated with risk factors such as unsafe drinking water and poor sanitation and hygiene. Chronic diarrhea can be caused by an infection or other etiologies; however, most of the times the etiological agent is not identified. METHODOLOGY: All the cases of diarrhea and parasitosis reported during 2005 in four public health centers of the city of Salta were classified by gender and age, analyzed, and represented geographically to show areas of higher morbidity rates, which were probably related to environmental factors. RESULTS: Water, poor sanitation, and pollution are candidate risk factors. Diarrhea cases showed seasonality, with the highest incidence during late spring and summer, while parasitosis was persistent throughout the year. CONCLUSION: Our spatial analysis permitted us to detect the regions of higher incidence of diarrhea and parasitosis during 2005 in the area of study.

Arginase enzymes in isolated airways from normal and nitric oxide synthase 2-knockout mice exposed to ovalbumin.

Arginase has been suggested to compete with nitric oxide synthase (NOS) for their common substrate, l-arginine. To study the mechanisms underlying this interaction, we compared arginase expression in isolated airways and the consequences of inhibiting arginase activity in vivo with NO production, lung inflammation, and lung function in both C57BL/6 and NOS2 knockout mice undergoing ovalbumin-induced airway inflammation, a mouse model of asthma. Arginases I and II were measured by western blot in isolated airways from sensitized C57BL/6 mice exposed to ovalbumin aerosol. Physiological and biochemical responses - inflammation, lung compliance, airway hyperreactivity, exhaled NO concentration, arginine concentration - were compared with the responses of NOS2 knockout mice. NOS2 knockout mice had increased total cells in lung lavage, decreased lung compliance, and increased airway hyperreactivity. Both arginase I and arginase II were constitutively expressed in the airways of normal C57BL/6 mice. Arginase I was up-regulated approximately 8-fold in the airways of C57BL/6 mice exposed to ovalbumin. Expression of both arginase isoforms were significantly upregulated in NOS2 knockout mice exposed to ovalbumin, with about 40- and 4-fold increases in arginases I and II, respectively. Arginine concentration in isolated airways was not significantly different in any of the groups studied. Inhibition of arginase by systemic treatment of C57BL/6 mice with a competitive inhibitor, Nomega-hydroxy-nor-l-arginine (nor-NOHA), significantly decreased the lung inflammatory response to ovalbumin in these animals. We conclude that NOS2 knockout mice are more sensitive to ovalbumin-induced airway inflammation and its sequelae than are C57BL/6 mice, as determined by increased total cells in lung lavage, decreased lung compliance, and increased airway hyperreactivity, and that these findings are strongly correlated with increased expression of both arginase isoforms in the airways of the NOS2 knockout mice exposed to ovalbumin.

Enzyme-linked immunosorbent assay for screening dioxin soil contamination by uncontrolled combustion during informal recycling in slums.

Uncontrolled combustion due to garbage recycling is a widespread activity among slum dwellers in distressed economy countries and has been indicated as a major source of dioxin contamination. However, because of the high cost and complexity of gas chromatography/high-resolution mass spectrometry (GC-HRMS) analysis, the magnitude of the problem remains largely unknown. The present study describes a first approach toward the use of a dioxin antibody-based enzyme-linked immunosorbent assay (ELISA) as the basis for a sustainable, simple, and low-cost monitoring program to assess the toxicological impact of uncontrolled combustion in slums. A panel of 16 samples was analyzed by GC-HRMS and ELISA on split extracts. Close to 20% of the analyzed samples showed dioxin concentrations up to almost twice the guidance level for residential soil in several countries, pointing out the need for performing a large-scale monitoring program. Despite the potential for variations in dioxin congener distribution due to the mixed nature of the incinerated material, there was a good correlation between the toxic equivalents as determined by GC-HRMS and ELISA. Furthermore, an interlaboratory ELISA validation showed that the capacity to perform the dioxin ELISA was successfully transferred between laboratories. It was concluded that the ELISA method performed very well as a screening tool to prioritize samples for instrumental analysis, which allows cutting down costs significantly.

Arginases I and II in lungs of ovalbumin-sensitized mice exposed to ovalbumin: sources and consequences.

Arginase gene expression in the lung has been linked to asthma both in clinical studies of human patients and in the well-studied mouse model of ovalbumin-induced airway inflammation. Arginase is thought to regulate NO levels in the lung by its ability to divert arginine, the substrate for nitric oxide synthases that produce citrulline and NO, into an alternative metabolic pathway producing ornithine and urea. In the present study arginase I and arginase II concentrations were measured in isolated microdissected airway preparations from sensitized Balb/c mice exposed to ovalbumin aerosol. We found that arginase II was constitutively expressed in the airways of normal mice, whereas arginase I was undetectable in normal airways, while its expression was increased in airways of mice exposed to ovalbumin. The expression of arginase I strongly correlated with the presence of lung inflammation, as quantified by differential cell counts in lung lavage, suggesting that most, or all, of the arginase I in lungs of mice exposed to ovalbumin is present in the inflammatory cells rather than in the airway epithelium. There was also a significant correlation between increased expression of arginase I in the isolated airways and decreased lung compliance. On the other hand, while we found arginase II expression to also be significantly increased in airways from mice exposed to ovalbumin as compared with normal airways, the relative increase was much less than that observed for arginase I, suggesting that there was a smaller contribution of inflammatory cells to the arginase II content of the airways in mice exposed to ovalbumin. There was no apparent correlation between the content of arginase in isolated airways and exhaled NO concentration in the expired air from mice exposed to ovalbumin. However, there was a correlation between exhaled NO concentration from mice exposed to ovalbumin and the lymphocyte content of the lung lavage. The concentration of arginine found in isolated airways from Balb/c mice exposed for 2 weeks to ovalbumin was about half of the value found in isolated microdissected airways from normal mice. Treatment of mice systemically with an arginase inhibitor significantly increased the amount of NO produced, as measured as the amount of nitrite+nitrate (NOx) in lung lavage supernatant prepared from mice exposed to ovalbumin. Our results are consistent with the hypothesis that the response of the lung to ovalbumin challenge includes an adaptive response in the large airways regulating the concentration of arginine within cells of the airway epithelium and subepithelial layer, by shunting of arginine into the metabolic pathway for increased synthesis of NO.

Detecting Abrupt Changes in a Piecewise Locally Stationary Time Series.

Non-stationary time series arise in many settings, such as seismology, speech-processing, and finance. In many of these settings we are interested in points where a model of local stationarity is violated. We consider the problem of how to detect these change-points, which we identify by finding sharp changes in the time-varying power spectrum. Several different methods are considered, and we find that the symmetrized Kullback-Leibler information discrimination performs best in simulation studies. We derive asymptotic normality of our test statistic, and consistency of estimated change-point locations. We then demonstrate the technique on the problem of detecting arrival phases in earthquakes.

Influence of sample collection and storage on the detection of platelet factor 4-heparin antibodies.

Heparin-induced thrombocytopenia is a life threatening thrombotic disorder caused by antibodies to platelet factor 4 (PF4) and heparin. Commercial immunoassays are frequently used for the detection of PF4-heparin antibodies, and several studies have reported that higher antibody titers are more frequently associated with adverse events. It is not known if conditions involving sample preparation and/or storage affect the operational characteristics of PF4-heparin immunoassays. We compared the detection of PF4-heparin antibodies from 48 patient samples collected concordantly in serum separator tubes or tubes containing EDTA or sodium citrate. We also examined the effects of extended sample storage on whole blood collected in serum separator, EDTA, or citrate tubes at 4 degrees C for up to 96 hours on antibody detection. We noted that serum or plasma anticoagulated with sodium citrate or EDTA yielded comparable results. In addition, we could not demonstrate any significant sample deterioration after storage at 4 degrees C in any medium for up to 4 days. These findings suggest that PF4-heparin antibodies are largely insensitive to the effects of sample preparation and storage.

Differentiation of the roles of NO from airway epithelium and inflammatory cells in ozone-induced lung inflammation.

Mice lacking inducible nitric oxide synthase (NOS2-/-) are more susceptible to ozone-induced lung inflammation and injury than their isogenic wild-type (NOS2+/+) counterparts, demonstrating an apparent protective effect for NOS2 in murine lungs. We hypothesized that nitric oxide (NO) generated from either NOS2 in the airway epithelial cells or the bone-marrow-derived inflammatory cells was responsible for the protective effect of NOS2. To test this hypothesis, we prepared chimeric mice by killing their endogenous bone marrow cells by whole body irradiation followed by bone marrow transplantation from a heterologous donor mouse. We exposed C57BL/6 (NOS2+/+), NOS2-/-, and chimeric NOS2 mice (NOS2-/+, NOS2+/-) to 1 ppm of ozone for 3 consecutive nights. NOS2-/- mice were more severely injured after exposure to ozone than C57BL/6 mice, including a more robust inflammatory cell influx (4.14 x 10(5) +/- 2.19 x 10(5) vs. 2.78 x 10(5) +/- 1.36 x 10(5) cells respectively; P = 0.036) and greater oxidation of total protein sulfhydryls (R-SH) in their blood plasma. Chimeric NOS2-/+ mice, which had bone marrow from NOS2-/- mice transplanted into C57BL/6 recipients, had a significantly greater response to ozone (increased numbers of neutrophils in lung lavage and decreased concentrations of exhaled NO) as compared to the reciprocal chimeric strain (NOS2+/-). We conclude that NOS2 has a protective effect against acute lung injury caused by ozone inhalation, which may be mediated, in part, by NO generated by NOS2 from inflammatory cells, predominantly neutrophils, recruited into the lung.

ELISA as an affordable methodology for monitoring groundwater contamination by pesticides in low-income countries.

The traditional instrumental technology for pesticide residue analysis is too expensive and labor-intense to meet the regional needs concerning environmental monitoring. ELISA methodology was used for a pilot scale study of groundwater quality in an agricultural region a few kilometers southwest of Montevideo, the capital city of Uruguay. The study spanned 2 years and examined concentrations (detection limits are given in [ppb]) of two triazine herbicides (simazine [0.3] and atrazine [0.4]) and the carbamate insecticide carbaryl [10] and its major metabolite 1-naphthol [17]. In general, pesticide concentrations were below detection limits in the samples tested and in all cases were well below the maximum contaminant levels set by the U.S. EPA. 1-Naphthol was detected frequently by ELISA, but the assay may have tended to systematically overestimate this analyte. To our knowledge, this is the first study of its type in Uruguay and perhaps the first systematic approach to monitoring for organic pesticides in groundwater water sources in the temperate region of South America.