Automatic analysis of the micronucleus test in primary human lymphocytes using image analysis.

The in vitro micronucleus test (MNT) is a well-established test for early screening of new chemical entities in industrial toxicology. For assessing the clastogenic or aneugenic potential of a test compound, micronucleus induction in cells has been shown repeatedly to be a sensitive and a specific parameter. Various automated systems to replace the tedious and time-consuming visual slide analysis procedure as well as flow cytometric approaches have been discussed. The ROBIAS (Robotic Image Analysis System) for both automatic cytotoxicity assessment and micronucleus detection in human lymphocytes was developed at Novartis where the assay has been used to validate positive results obtained in the MNT in TK6 cells, which serves as the primary screening system for genotoxicity profiling in early drug development. In addition, the in vitro MNT has become an accepted alternative to support clinical studies and will be used for regulatory purposes as well. The comparison of visual with automatic analysis results showed a high degree of concordance for 25 independent experiments conducted for the profiling of 12 compounds. For concentration series of cyclophosphamide and carbendazim, a very good correlation between automatic and visual analysis by two examiners could be established, both for the relative division index used as cytotoxicity parameter, as well as for micronuclei scoring in mono- and binucleated cells. Generally, false-positive micronucleus decisions could be controlled by fast and simple relocation of the automatically detected patterns. The possibility to analyse 24 slides within 65h by automatic analysis over the weekend and the high reproducibility of the results make automatic image processing a powerful tool for the micronucleus analysis in primary human lymphocytes. The automated slide analysis for the MNT in human lymphocytes complements the portfolio of image analysis applications on ROBIAS which is supporting various assays at Novartis.

Best practices for heart failure: a focused review.

Heart failure (HF) is a progressive condition and is associated with high patient mortality rates and frequent hospitalizations. This article provides an overview of clinical, self-management support, and care transitions best practices for HF care in the home care sector. Clinicians armed with competencies in HF management are positioned to meet the 3-part aim of healthcare reform: improved health, better care, and lower cost.

How can we improve our understanding of cardiovascular safety liabilities to develop safer medicines?

Given that cardiovascular safety liabilities remain a major cause of drug attrition during preclinical and clinical development, adverse drug reactions, and post-approval withdrawal of medicines, the Medical Research Council Centre for Drug Safety Science hosted a workshop to discuss current challenges in determining, understanding and addressing 'Cardiovascular Toxicity of Medicines'. This article summarizes the key discussions from the workshop that aimed to address three major questions: (i) what are the key cardiovascular safety liabilities in drug discovery, drug development and clinical practice? (ii) how good are preclinical and clinical strategies for detecting cardiovascular liabilities? and (iii) do we have a mechanistic understanding of these liabilities? It was concluded that in order to understand, address and ultimately reduce cardiovascular safety liabilities of new therapeutic agents there is an urgent need to: • Fully characterize the incidence, prevalence and impact of drug-induced cardiovascular issues at all stages of the drug development process. • Ascertain the predictive value of existing non-clinical models and assays towards the clinical outcome. • Understand the mechanistic basis of cardiovascular liabilities; by addressing areas where it is currently not possible to predict clinical outcome based on preclinical safety data. • Provide scientists in all disciplines with additional skills to enable them to better integrate preclinical and clinical data and to better understand the biological and clinical significance of observed changes. • Develop more appropriate, highly relevant and predictive tools and assays to identify and wherever feasible to eliminate cardiovascular safety liabilities from molecules and wherever appropriate to develop clinically relevant and reliable safety biomarkers.

Review series: Examples of chronic care model: the home-based chronic care model: redesigning home health for high quality care delivery.

Individuals with chronic obstructive pulmonary disease (COPD) face significant challenges due to frequent distressing dyspnea and deficits related to activities of daily living. Individuals with COPD are often hospitalized frequently for disease exacerbations, negatively impacting quality of life and healthcare expenditure burden. The home-based chronic care model (HBCCM) was designed to address the needs of patients with chronic diseases. This model facilitates the re-design of chronic care delivery within the home health sector by ensuring patient-centered evidence-based care. This HBCCM foundation is Dr. Edward Wagner s chronic care model and has four additional areas of focus: high touch delivery, theory-based self management, specialist oversight and the use of technology. This article will describe this model in detail and outline how model use for patients with COPD can bring value to stakeholders across the health care continuum.

Theory-based telehealth and patient empowerment.

Health care technology holds great potential to improve the quality of health care delivery. One effective technology is remote patient monitoring, whereby patient data, such as vital signs or symptom reports, are captured from home monitoring devices and transmitted to health care professionals for review. The use of remote patient monitoring, often referred to as telehealth, has been widely adopted by health care providers, particularly home care agencies. Most agencies have invested in telehealth to facilitate the early identification of disease exacerbation, particularly for patients with chronic diseases such as heart failure and diabetes. This technology has been successfully harnessed by agencies to reduce rehospitalization rates through remote data interpretation and the provision of timely interventions. We propose that the use of telehealth by home care agencies and other health care providers be expanded to empower patients and promote disease self-management with resultant improved health care outcomes. This article describes how remote monitoring, in combination with the application of salient adult learning and cognitive behavioral theories and applied to telehealth care delivery and practice, can promote improved patient self-efficacy with disease management. We present theories applicable for improving health-related behaviors and illustrate how theory-based practices can be implemented in the field of home care. Home care teams that deliver theory-based telehealth function as valuable partners to physicians and hospitals in an integrated health care delivery system.

Telehealth infection control: a movement toward best practice.

As healthcare transitions to the home environment, there is an increasing need to prevent and control infections traced to telehealth equipment. Background information about infection control is presented, and the need for a survey of agency cleaning policies is described. Findings from 31 agencies using telehealth equipment are described followed by suggested Best Practice Guidelines for the cleaning of telehealth equipment.

Depression revealed: the need for screening, treatment, and monitoring.

Depression is common among both the elderly and those with chronic conditions. Unidentified and inadequately treated depression in home health patients has serious and costly consequences. The authors argue that many negative consequences can be avoided by careful management. They discuss the importance of adding a structured depression tool to routine assessments when indicated, present a 3-step approach for depression screening and monitoring, and describe 1 method for tool selection based on measurement soundness and utility.

Selective block of IKs plays a significant role in MAP triangulation induced by IKr block in isolated rabbit heart.

INTRODUCTION: The role of IKr (rapidly-activating delayed rectifier K(+) current) block in triangulation of monophasic action potentials (MAP) and in development of torsade de pointes (TdP) arrhythmia is known. Combined IKr and IKs (slowly-activating delayed rectifier K(+) current) block has been demonstrated to promote TdP. The aim of this study was to describe a possible implication of IKs block in MAP triangulation. METHODS: Four contact electrodes were placed on the epicardium of the left ventricle of Langendorff-perfused rabbit hearts to record monophasic action potentials (MAP), with an IKr blocker d,l-sotalol (3 to 100 microM, n=6) or a non-selective IKr blocker, quinidine (1 to 30 microM, n=6). Their effects were assessed with or without a specific IKs blocker chromanol 293B (20 microM, n=6), on MAP duration at 30, 60 and 90% of repolarization (APD30, 60 and 90, respectively) and MAP triangulation (APD90-APD30) at 1 and 0.2 Hz. RESULTS: D,L-sotalol increased significantly APD90 and triangulation with reverse use-dependency for concentrations > or =10 microM. Quinidine markedly prolonged APD90 and triangulation with reverse use-dependency at concentrations > or =3 microM. Chromanol 293B alone had no effects on APD, but when combined with D,L-sotalol or quinidine (i) increased APD prolonging effects, (ii) lowered values of pro-arrhythmic concentrations, (iii) increased incidence and length of D,L-sotalol- or quinidine-induced Early Afterdepolarizations (EADs) and TdP. All these events were primarily due to an important slowing of final repolarization, i.e. a marked increased triangulation. CONCLUSION: IKs, even of low amplitude in rabbits, plays a key role in ventricular repolarization. IKs is involved in prolonged MAP duration mainly by triangulation and subsequent increased drug arrhythmogenicity. Therefore drug affinity for IKs must be evaluated with IKr studies as part of preclinical drug cardiac safety assessment.

Relevance of in vitro SCREENIT results for drug-induced QT interval prolongation in vivo: a database review and analysis.

INTRODUCTION: The use of an isolated rabbit heart model (SCREENIT) to predict drug-induced QTc prolongation in animals was assessed using hERG and telemetry data. PURPOSE: We compiled data from (i) hERG assay (IC50s), (ii) SCREENIT assay (APD60) and (iii) in vivo non-rodent telemetry studies (QTc interval) and evaluated the reliability of APD60 to fit with IC50s and QTc prolongation using the ratio to free plasma level (FPL). Eighty-two compounds were separated into three classes based on hERG IC50s (class I: IC50s< or =1 microM, n=7; class II: IC50s>1 microM to < or =10 microM, n=15; class III: IC50s>10 microM, n=60). RESULTS: Three class I compounds did not prolong QTc at the FPL equivalent to their IC50s (43% hERG false positives). There were no false positives in SCREENIT. Six class II compounds prolonged the QTc interval. Results showed 40% hERG false negatives and no SCREENIT false negatives. Nine compounds had no effect on QTc, and two prolonged APD60 at an equivalent concentration/FPL (13% false positives). Three class III compounds prolonged QTc at an FPL lower than maximum SCREENIT concentrations (5% false negatives). Four other compounds generated SCREENIT false positive results (7%). CONCLUSION: SCREENIT increased the predictability of preclinical results for QTc prolongation without generating any false positive results in class I (13% in class II). Making decisions without isolated heart data increases the risk for eliminating efficient drugs displaying hERG inhibition.

Home-based chronic care. An expanded integrative model for home health professionals.

The Chronic Care Model (CCM) developed by is an influential and accepted guide for the care of patients with chronic disease. Wagner acknowledges a current healthcare focus on acute care needs that often circumvents chronic care coordination. He identifies the need for a "division of labor" to assist the primary care physician with this neglected function. This article posits that the role of chronic care coordination assistance and disease management fits within the purview of home healthcare and should be central to home health chronic care delivery. An expanded Home-Based Chronic Care Model (HBCCM) is described that builds on Wagner's model and integrates salient theories from fields beyond medicine. The expanded model maximizes the potential for disease self-management success and is intended to provide a foundation for home health's integral role in chronic disease management.

Patient education. Timeless principles of learning: a solid foundation for enhancing chronic disease self-management.

The use of evidence-based principles of learning can contribute to the empowerment of patients as they adopt self-management skills aligned with healthy behaviors. This article, jointly written by a nurse and an educator, describes these timeless principles and how home care clinicians and patients benefit from their use.

Diagnostic quality of biopsy specimens: comparison between a conventional biopsy forceps and multibite forceps.

BACKGROUND: The endoscopic biopsy is a prerequisite for histopathologic diagnosis. Various types of forceps are used to obtain tissue specimens. The aim of this study was to assess and compare the diagnostic quality of biopsy specimens obtained with a conventional forceps and a Multibite forceps. METHODS: In a prospective, partially blinded, and randomized trial that included 250 patients referred for diagnostic upper and/or lower endoscopy, 510 biopsy specimens obtained with the Multibite forceps were compared with 520 specimens obtained with a conventional forceps. An experienced, blinded pathologist evaluated the specimens for diameter, depth of specimen, artifacts, anatomic orientation, vitality, general histologic quality, and diagnostic quality. Statistical analysis was performed by using the Fisher exact test. A p value of < 0.05 was regarded as significant. RESULTS: There were no statistically significant differences between the specimens obtained with the 2 forceps. The p values for the evaluated parameters were as follows: diameter 0.45, depth of specimen 0.56, artifacts 1.0, pathoanatomic orientation 0.40, vitality 0.45, and histologic diagnostic quality 0.53. CONCLUSION: The quality of biopsy specimens obtained with the Multibite forceps is comparable with that of specimens taken with a conventional forceps. Use of the Multibite forceps saves time in that 4 specimens can be obtained in 1 pass in situations in which a large number of specimens are needed or when the potential for transmission of infection is of concern.

Influence of cytotoxicity and compound precipitation on test results in the alkaline comet assay.

We use the comet assay as part of our genotoxicity screening battery for newly synthesized drug candidates. A dataset of more than 250 tests carried out with 75 drug candidates of various chemical classes was analyzed to elucidate the influence of cytotoxicity and compound precipitation on DNA migration in the comet assay. Using a V79 Chinese hamster cell line, 38 of the compounds were negative and 37 were positive in the comet assay. The reproducibility of test results between repeat experiments was 85%. Data on 72 tests with a negative call in which the compounds were tested up to highly cytotoxic concentrations demonstrated that cytotoxicity, as determined by Trypan blue dye exclusion and occurrence of cells with completely fragmented chromatin, did not lead to false positive test results. The majority (64.2%) of compounds with a positive call induced elevated DNA migration in the absence of excessive cytotoxicity. Compound precipitation was observed in 84 tests. In 88.1% of these cases, the test result at the precipitating concentration did not differ from that found at the highest soluble concentration. Half of the remaining 11.9% of contrary results (most of them weak effects) were not reproducible in the respective repeat experiment, indicating no or only a negligible influence of precipitation on test results. The data indicate that using V79 cells, the comet assay specifically detects genotoxic effects and is not confounded by cytotoxicity or compound precipitation under the conditions used.

Diamond stents for palliation of malignant bile duct obstruction: a prospective multicenter evaluation.

BACKGROUND AND STUDY AIMS: Various types of self-expandable metal stents have been introduced for biliary drainage in patients with malignant jaundice, showing prolonged patency compared with plastic endoprostheses. However, there has only been prolonged experience with a meaningful number of patients using the Wallstent. We evaluated the Diamond stent, a self-expanding uncoated biliary metal stent, in a prospective uncontrolled multicenter setting. PATIENTS AND METHODS: The eligibility criterion was obstructive jaundice due to inoperable malignant disease. Between August 1995 and January 2000, 126 patients, who received a total of 134 Diamond stents in four European centers, were followed prospectively. RESULTS: Technical and clinical success rates were 96 % and 98 %, respectively. No major procedure-related complications occurred. The 30-day mortality rate was 13 %. Stent occlusion occurred in 28 patients (22 %). Overall median stent patency was 477 days; overall median survival was 173 days. Stent occlusion, confirmed by endoscopic retrograde cholangiopancreatography, was successfully treated with plastic stents in all patients. Cost analysis revealed estimated costs of 3440 euros per patient for palliative treatment with the Diamond stent. CONCLUSIONS: The Diamond stent compares favorably with other biliary metal stents for patients requiring biliary drainage of malignant jaundice.

Use of the alkaline comet assay for industrial genotoxicity screening: comparative investigation with the micronucleus test.

We evaluated the suitability of the alkaline comet assay as a screening test in industrial routine testing of new chemicals. Thirty-six pharmaceutical compounds with unknown genotoxic potential were tested comparatively in the comet assay and micronucleus test (MNT) using V79 Chinese hamster cells. The comparison of results is generally based on at least two independent experiments, each with two replicate cultures at a minimum of three concentrations. We found a high degree of concordance between results of the comet assay and MNT. All compounds with negative MNT results were also negative in the comet assay. All positive compounds in the comet assay were also positive in the MNT. However, 16 of 38 positive MNT results were negative in the comet assay. Some of the contrary findings may be due to aneugenic effects, which are detected in the MNT but not in the comet assay. However, the majority of the contrary results may be a consequence of cytotoxicity, which can induce elevated micronucleus frequencies but may not lead to positive effects in the comet assay. Additional data of 39 compounds tested in the Ames test and the comet assay were compared. Four of these compounds that were Ames positive were also positive in the comet assay. However, the comet assay also detected 16 compounds that were negative in the Ames test. We believe that the comet assay in vitro is a useful, fast screening system in mammalian cells that can be used in a test battery during drug development.

Intralaboratory validation of alternative endpoints in the murine local lymph node assay for the identification of contact allergic potential: primary ear skin irritation and ear-draining lymph node hyperplasia induced by topical chemicals.

We validated a two-tiered murine local lymph node assay (LLNA) with a panel of standard contact (photo)allergens and (photo)irritants with the aim of improving the discrimination between contact (photo) allergenic potential and true skin (photo)irritation potential. We determined ear weights to correlate chemical-induced skin irritation with the ear-draining lymph node (LN) activation potential. During tier I LLNAs, a wide range of concentrations were applied on three consecutive days to the dorsum of both ears. Mice were exposed to UVA light immediately after topical application to determine the photoreactive potential of some test chemicals. Mice were killed 24 h after the last application to determine ear and LN weights and LN cell counts. It was possible to classify the tested chemicals into three groups according to their threshold concentrations for LN activation and skin irritation: (1) chemicals with a low LN activation potential and no or very low skin irritation potential; (2) chemicals with a marked LN activation potential higher than a distinct skin irritation potential; and (3) chemicals with LN activation potential equal to or lower than their skin irritation potential. Group 1 consisted only of contact allergens, indicating that LN activation in the absence of skin irritation points to a contact allergenic activity. Since groups 2 and 3 comprised irritants and contact allergens, a tier II LLNA protocol was used to finally differentiate between true irritants and contact allergens. Briefly, mice were pretreated with mildly to moderately irritating concentrations of the chemical to the shaved back and after 12 days were challenged on the ears as described above in order to elicit a contact allergenic response in the ear skin and the ear-draining LN. With this approach, tier II LLNAs have to be conducted only in cases for which skin irritation potential is in the range of LN activation potential and no structure-activity relationship data indicating a contact allergenic hazard are available.

Automatic analysis of slides processed in the Comet assay.

In recent years the Comet assay (or single cell gel electrophoresis assay) has been established as a rapid and sensitive method for the detection of DNA damage. For early genotoxicity screening of new chemical entities in industrial toxicology, the Comet assay is more and more used for assessment of the DNA damaging potential of a test compound. In order to increase compound screening throughput, we have established an image analysis system for fully automated measurement of microscope slides processed in the Comet assay. For the comparative investigation various cell types, such as V79 Chinese hamster cells, mouse lymphoma cells and human leukocytes, were treated with several test compounds. Using tail moment as the quantitative parameter for comet formation, we show a very high correlation between our automatic image analysis system and a commercially available, interactive system (Comet Assay II of Perceptive Instruments). The possibility of analyzing 50 samples within 1 day and the high reproducibility of results make automated image processing a powerful tool for automatic analysis of slides processed in the Comet assay.

Cytokine expression profiles during murine contact allergy: T helper 2 cytokines are expressed irrespective of the type of contact allergen.

We have investigated the cytokine response pattern following sensitisation (induction) of BALB/c mice with different chemicals (dinitrochlorobenzene, dinitrofluorobenzene, oxazolone, glutaraldehyde, formaldehyde, trimellitic anhydride, croton oil) and elicitation (challenge) of contact allergy in sensitised animals. The results of our investigations showed that different chemicals induced both T helper (Th) 1 cytokines [interleukin (IL) 2, interferon beta (IFNgamma) [corrected] and Th2 cytokines (IL-4, IL-10) at different stages during murine contact allergy. We also confirmed our previous findings that IL-4 and IL-10 release were up-regulated during the challenge phase regardless the contact allergen used, whereas the release of IFNgamma [corrected] did not show a clear preference for being up- or down-regulated. In our hands, the increased expression of Th2 cytokines after challenge exposure to contact allergens appeared as a stable marker of secondary contact allergenic responses. Quantitative differences in the expression of IL-4 were observed between different contact allergens. The present results clearly indicate that skin sensitisers were able to elicit cytokine response patterns, which could not be related to a clear-cut Th1 or Th2 type of cytokine response. Furthermore, dermal application of contact allergens produced different kinetics of cytokine secretion upon induction and challenge. In our hands, the co-expression of Th1 and Th2 type cytokines appeared as a universal consequence of dermal application of contact allergens to responsive mice. Our results indicate that co-expression of Th1 and Th2 cytokines during contact allergy is an important feature of murine contact allergy in responsive mice and that chemicals differ in their potency to induce the expression of these cytokines. Furthermore, the results do not support the view that different chemicals induce Th1 or Th2 cytokines in a mutually exclusive manner depending on their preference for inducing either contact or respiratory allergy. The results are expected to renew the discussion about the usefulness of the Th1/Th2 paradigm in certain areas of immunotoxicology.

[Generalized intestinal CMV infection with protein-losing syndrome in ulcerative colitis]. / Generalisierte intestinale CMV-Infektion mit Eiweissverlustsyndrom bei Colitis ulcerosa.

Infection by cytomegalovirus (CMV) in immunocompetent patients is rare, and if it occurs it is most often associated with ulcerative colitis. This case illustrates a CMV infection in a patient with an ulcerative colitis combined with CMV-induced protein losing enteropathy, a condition reported in immunocompetent individuals in only a very few cases worldwide. It demonstrates the importance of differentiating between a flare-up of ulcerative colitis and CMV colitis. The indication for antiviral therapy is discussed. A 76-years-old patient with a 23-year history of leftsided ulcerative colitis presented with acute pancolitis sparing the rectum. He showed no evidence of impaired host defence, nor has he ever had taken immunosuppressive drugs. Disseminated primary CMV infection involving of the colon, the oesophagus and the small intestine with protein losing enteropathy was diagnosed on the basis of histology, culture and serology. In view of the long duration of the illness and the highly active infection, antiviral therapy with ganciclovir was given and led to a dramatical improvement of all disease manifestations. The patient subsequently remained in remission from ulcerative colitis for three years.

Cytomegalovirus-induced transient protein-losing hypertrophic gastropathy in an immunocompetent adult.

Transient protein-losing hypertrophic gastropathy with similarity to Ménétrier's disease is described. Acute infection with cytomegalovirus (CMV) could be shown to play a causative role. Immunodeficiency was ruled out. The 34-year-old patient had complete resolution of the disease without antiviral treatment. To our knowledge the present report is the first case of CMV-associated protein-losing hypertrophic gastropathy in an immunocompetent adult. To date, a similar disorder has only been described in children. CMV infection should be considered in patients with acute and symptomatic protein loss of gastrointestinal origin.