Sex-specific cortical, hippocampal and thalamic whole genome transcriptome data from controls and a G72 schizophrenia mouse model.

OBJECTIVES: The G72 mouse model of schizophrenia represents a well-known model that was generated to meet the main translational criteria of isomorphism, homology and predictability of schizophrenia to a maximum extent. In order to get a more detailed view of the complex etiopathogenesis of schizophrenia, whole genome transcriptome studies turn out to be indispensable. Here we carried out microarray data collection based on RNA extracted from the retrosplenial cortex, hippocampus and thalamus of G72 transgenic and wild-type control mice. Experimental animals were age-matched and importantly, both sexes were considered separately. DATA DESCRIPTION: The isolated RNA from all three brain regions was purified, quantified und quality controlled before initiation of the hybridization procedure with SurePrint G3 Mouse Gene Expression v2 8  ×  60 K microarrays. Following immunofluorescent measurement und preprocessing of image data, raw transcriptome data from G72 mice and control animals were extracted and uploaded in a public database. Our data allow insight into significant alterations in gene transcript levels in G72 mice and enable the reader/user to perform further complex analyses to identify potential age-, sex- and brain-region-specific alterations in transcription profiles and related pathways. The latter could facilitate biomarker identification and drug research and development in schizophrenia research.

Sex- and region-specific cortical and hippocampal whole genome transcriptome profiles from control and APP/PS1 Alzheimer's disease mice.

A variety of Alzheimer's disease (AD) mouse models has been established and characterized within the last decades. To get an integrative view of the sophisticated etiopathogenesis of AD, whole genome transcriptome studies turned out to be indispensable. Here we carried out microarray data collection based on RNA extracted from the retrosplenial cortex and hippocampus of age-matched, eight months old male and female APP/PS1 AD mice and control animals to perform sex- and brain region specific analysis of transcriptome profiles. The results of our studies reveal novel, detailed insight into differentially expressed signature genes and related fold changes in the individual APP/PS1 subgroups. Gene ontology and Venn analysis unmasked that intersectional, upregulated genes were predominantly involved in, e.g., activation of microglial, astrocytic and neutrophilic cells, innate immune response/immune effector response, neuroinflammation, phagosome/proteasome activation, and synaptic transmission. The number of (intersectional) downregulated genes was substantially less in the different subgroups and related GO categories included, e.g., the synaptic vesicle docking/fusion machinery, synaptic transmission, rRNA processing, ubiquitination, proteasome degradation, histone modification and cellular senescence. Importantly, this is the first study to systematically unravel sex- and brain region-specific transcriptome fingerprints/signature genes in APP/PS1 mice. The latter will be of central relevance in future preclinical and clinical AD related studies, biomarker characterization and personalized medicinal approaches.

[The application of real-world evidence in drug regulatory decision-making]. / Die Anwendung von Real-world-Evidenz in Entscheidungsprozessen der Arzneimittelregulation.

Drug regulation is a system to support and protect public health. Drugs with market access must be effective, safe and of high quality. Therefore, drug regulatory decision-making by the competent authorities is made on a scientific basis. Real-world evidence (RWE) from real-world data (RWD) has so far predominantly been taken into account in a supportive manner in drug regulatory decision-making with regard to drug safety after marketing authorisation. The extensive potential of RWE for regulatory decision-making processes along the entire product life cycle has been increasingly used and further examined in recent years.This article provides an overview of current applications of RWE in drug regulatory decision-making processes. The potentials of RWE along with the hurdles to be addressed are described and examples of current projects on RWE research for drug regulation are given. The work is based on current international literature as well as examples from international and European initiatives and regulatory practice, which aim to support an increased use of RWD/RWE in regulatory decision-making processes. In order to be able to utilise the potential of RWE even more in the future, it is important to make relevant RWD sources more readily available through research projects and initiatives, to further develop evaluative methods and to establish the significance of RWE.

Whole genome transcriptome data from the WT cortex and hippocampus of female and male control and APP/PS1 Alzheimer's disease mice.

A variety of Alzheimer disease (AD) mouse models has been established and characterized within the last decades. These models are generated to meet the principal criteria of AD isomorphism, homology and predictability to a maximum extent. To get an integrative view of the sophisticated etiopathogenesis of AD, whole genome transcriptome data analysis turns out to be indispensable. Here, we present a microarray-based transcriptome data collection based on RNA extracted from the retrosplenial (RS) cortex and the hippocampus of APP/PS1 AD mice and control animals. Experimental animals were age matched and importantly, both sexes were considered separately. Isolated RNA was purified, quantified und quality controlled prior to the hybridization procedure with SurePrint G3 Mouse Gene Expression v2 8 × 60K microarrays. Following immunofluorescent measurement und preprocessing/extraction of image data, raw transcriptome data were uploaded including differentially expressed gene candidates and related fold changes in APP/PS1 AD mice and controls. Our data allow further insight into alterations in gene transcript levels in APP/PS1 AD mice compared to controls and enable the reader/user to carry out complex transcriptome analysis to characterize potential age-, sex- and brain-region-specific alterations in e.g., neuroinflammatory, immunological, neurodegenerative and ion channel pathways.

Cav3 T-Type Voltage-Gated Ca2+ Channels and the Amyloidogenic Environment: Pathophysiology and Implications on Pharmacotherapy and Pharmacovigilance.

Voltage-gated Ca2+ channels (VGCCs) were reported to play a crucial role in neurotransmitter release, dendritic resonance phenomena and integration, and the regulation of gene expression. In the septohippocampal system, high- and low-voltage-activated (HVA, LVA) Ca2+ channels were shown to be involved in theta genesis, learning, and memory processes. In particular, HVA Cav2.3 R-type and LVA Cav3 T-type Ca2+ channels are expressed in the medial septum-diagonal band of Broca (MS-DBB), hippocampal interneurons, and pyramidal cells, and ablation of both channels was proven to severely modulate theta activity. Importantly, Cav3 Ca2+ channels contribute to rebound burst firing in septal interneurons. Consequently, functional impairment of T-type Ca2+ channels, e.g., in null mutant mouse models, caused tonic disinhibition of the septohippocampal pathway and subsequent enhancement of hippocampal theta activity. In addition, impairment of GABA A/B receptor transcription, trafficking, and membrane translocation was observed within the septohippocampal system. Given the recent findings that amyloid precursor protein (APP) forms complexes with GABA B receptors (GBRs), it is hypothesized that T-type Ca2+ current reduction, decrease in GABA receptors, and APP destabilization generate complex functional interdependence that can constitute a sophisticated proamyloidogenic environment, which could be of potential relevance in the etiopathogenesis of Alzheimer's disease (AD). The age-related downregulation of T-type Ca2+ channels in humans goes together with increased Aß levels that could further inhibit T-type channels and aggravate the proamyloidogenic environment. The mechanistic model presented here sheds new light on recent reports about the potential risks of T-type Ca2+ channel blockers (CCBs) in dementia, as observed upon antiepileptic drug application in the elderly.

Evaluation of the EMPAR study population on the basis of metabolic phenotypes of selected pharmacogenes.

The impact of genetic variability of pharmacogenes as a possible risk factor for adverse drug reactions is elucidated in the EMPAR (Einfluss metabolischer Profile auf die Arzneimitteltherapiesicherheit in der Routineversorgung/English: influence of metabolic profiles on the safety of drug therapy in routine care) study. EMPAR evaluates possible associations of pharmacogenetically predicted metabolic profiles relevant for the metabolism of frequently prescribed cardiovascular drugs. Based on a German study population of 10,748 participants providing access to healthcare claims data and DNA samples for pharmacogenetic assessment, first analyses were performed and evaluated. The aim of this first evaluation was the characterization of the study population with regard to general parameters such as age, gender, comorbidity, and polypharmacy at baseline (baseline year) as well as important combinations of cardiovascular drugs with relevant genetic variants and predicted metabolic phenotypes. The study was registered in the German Clinical Trials Register (DRKS) on July 6, 2018 (DRKS00013909).

The Janus-like Association between Proton Pump Inhibitors and Dementia.

Early pharmacoepidemiological studies suggested that Proton Pump Inhibitors (PPIs) might increase the risk of Alzheimer's Disease (AD) and non-AD related dementias. These findings were supported by preclinical studies, specifically stressing the proamyloidogenic and indirect anticholinergic effects of PPIs. However, further large-scale pharmacoepidemiological studies showed inconsistent results on the association between PPIs and dementia. Pharmacodynamically, these findings might be related to the LXR/RXR-mediated amyloid clearance effect and anti-inflammatory action of PPIs. Further aspects that influence PPI effects on AD are related to patient- specific pharmacokinetic and pharmacogenomic characteristics. In conclusion, a personalized (individualized) medicinal approach is necessary to model and predict the potential harmful or beneficial effects of PPIs in AD and non-AD-related dementias in the future.

N-Nitrosodimethylamine-Contaminated Valsartan and the Risk of Cancer­A Longitudinal Cohort Study Based on German Health Insurance Data.

BACKGROUND: N-Nitrosodimethylamine (NDMA), classified as a probable human carcinogen, has been found as a contaminant in the antihypertensive drug valsartan. Potentially carcinogenic effects associated with the consumption of NDMAcontaminated valsartan have not yet been analyzed in large-scale cohort studies. We therefore carried out the study reported here to explore the association between NDMA-contaminated valsartan and the risk of cancer. METHODS: This cohort study was based on longitudinal routine data obtained from a large German statutory health insurance provider serving approximately 25 million insurees. The cohort comprised patients who had filled a prescription for valsartan in the period 2012-2017. The endpoint was an incident diagnosis of cancer. Hazard ratios (HR) for cancer in general and for certain specific types of cancer were calculated by means of Cox regression models with time-dependent variables and adjustment for potential confounders. RESULTS: A total of 780 871 persons who had filled a prescription for valsartan between 2012 and 2017 were included in the study. There was no association between exposure to NDMA-contaminated valsartan and the overall risk of cancer. A statistically significant association was found, however, between exposure to NDMA-contaminated valsartan and hepatic cancer (adjusted HR 1.16; 95% confidence interval [1.03; 1.31]). CONCLUSION: These findings suggest that the consumption of NDMA-contaminated valsartan is associated with a slightly increased risk of hepatic cancer; no association was found with the risk of cancer overall. Close observation of the potential long-term effects of NDMA-contaminated valsartan seems advisable.

Influence of metabolic profiles on the safety of drug therapy in routine care in Germany: protocol of the cohort study EMPAR.

INTRODUCTION: Pre-emptive testing of pharmacogenetically relevant single-nucleotide polymorphisms can be an effective tool in the prevention of adverse drug reactions and therapy resistance. However, most of the tests are not used as standard in routine care in Germany because of lacking evidence for the clinical and economical benefit and their impact on the usage of healthcare services. We address this issue by investigating the influence of pharmacogenetic profiles on the use of healthcare services over an extended period of several years using routine care data from a statutory health insurance company. The goal is to provide clinical evidence whether pre-emptive pharmacogenetic testing of metabolic profiles in routine care in Germany is beneficial and cost-effective. METHODS AND ANALYSIS: The EMPAR (Einfluss metabolischer Profile auf die Arzneimitteltherapiesicherheit in der Routineversorgung) study is a non-interventional cohort study conducted to analyse pharmacogenetic risk factors that are important for drug therapy by means of endpoints relevant for healthcare. The analysis is based on pharmacogenetic profiles and statutory health insurance data. We perform pharmacogenetic, pharmacoepidemiological and pharmacoeconomic analyses using health care utilisation scores and machine learning techniques. Therefore, we aim to include about 10 000 patients (≥18 years) insured by the health insurance provider Techniker Krankenkasse. The study focuses on patients with prescriptions of anticoagulants and prescriptions of cholesterol-lowering drugs. Also, a screening for special pharmacogenetic characteristics will be performed in patients with at least one Y57.9! diagnosis (Complication of medical and surgical care: drug or medicament, unspecified). Outcomes include the utilisation of health insurance services, the incidence of incapacity for work and costs for drugs and treatment. ETHICS AND DISSEMINATION: The protocol was approved by the Ethics Committee of the Medical Faculty, University of Bonn (Lfd. Nr. 339/17). The results of this research project will be published in scientific open access journals and at conferences. TRIAL REGISTRATION NUMBER: German Clinical Trials Register, DRKS00013909.

Enrichment designs using placebo nonresponders.

Enrichment designs that select placebo nonresponders have gained much attention during the last years in areas with high placebo response rates, eg, in depression. Proposals were made that re-randomize patients who did not respond to placebo during a first study phase as the sequential parallel design (SPD). This design uses in a second phase an enriched patient population where the treatment effect is expected to be more pronounced. This may be problematic if an effect in the overall population is claimed. Proposals were made to combine the treatment effects in the overall population from study phase 1 and the enriched population from study phase 2, alleviating but not solving the issue of a potential selection bias. This paper shows how this bias corresponding to the effect difference between the overall population and the enriched population depends on the variability of a potential subject-by-treatment interaction. Sample sizes are given, which lead to a significant result in the combining test with a given probability if actually the average effect in the overall population is zero. If, on the other hand, no subject-by-treatment interaction is given, the enrichment is shown to be inefficient. We conclude that enrichment designs using placebo nonresponders are not able to claim a positive average effect in the overall population if a subject-by-treatment interaction cannot be excluded. It cannot be used to demonstrate positive efficacy in the overall population in a pivotal phase III trial but may be used in early phases to demonstrate varying treatment effects between patients.

Glucocorticoid Therapy is Associated with a Lower Risk of Dementia.

BACKGROUND: Recent evidence indicates an important role for neuroinflammation in the pathological cascade of Alzheimer's disease (AD), and neuroinflammation is increasingly being recognized as a potential therapeutic target. OBJECTIVE: To assess the impact of glucocorticoids on the risk of developing dementia. METHODS: We used health insurance data of the largest German health insurer from 2004-2013 with a baseline sample of 176,485 persons aged 50 years and older to study the association of glucocorticoid treatment and incidence of dementia. Cox proportional-hazard models were calculated adjusting for sex, age, and comorbidities known to be major risk factors for dementia and were given as hazard ratios (HR) with 95% confidence intervals (CI). We further stratified glucocorticoid treatment by route of application and treatment duration. RESULTS: Of the 176,485 dementia-free persons, 19,938 were diagnosed with dementia by the end of 2013. The risk of suffering from dementia was significantly lower for glucocorticoid users compared to non-users (HR = 0.81, CI = 0.78-0.84). The lowest risk was found among users of inhaled glucocorticoid (HR = 0.65, CI = 0.57-0.75), followed by nasal (HR = 0.76, CI = 0.66-0.87), other (HR = 0.84, CI = 0.80-0.88), and oral users (HR = 0.83, CI = 0.78-0.88). We found no difference in risk reduction between long- and short-term-users. CONCLUSION: Longitudinal German health insurance data indicate that the use of glucocorticoids is associated with a lower risk of dementia. Prospective clinical trials will be necessary to determine whether glucocorticoids can have a positive impact on neuroinflammation and thus protect persons against dementia.

Perspective of elderly patients on chronic use of potentially inappropriate medication - Results of the qualitative CIM-TRIAD study.

Although potentially inappropriate medication (PIM) is associated with risk of harm due to adverse effects, it is frequently prescribed for elderly patients. The aim of this qualitative multi-center study was to gain insight into contextual factors that might lead to chronic PIM use. We conducted semi-structured interviews with elderly patients with or without chronic PIM use (patient interviews: n = 52). Patients were between 86 and 96 years old. The participants were recruited from the AgeCoDe study. Interviews were audiotaped and transcribed verbatim. The transcripts of the interviews were analysed using qualitative content analysis. Deductive and inductive categories were determined. We found contextual factors related to the patient and related to patient-general practitioner (GP) communication that might lead to chronic PIM use (i.e., positive features of PIM, maintaining characteristics of medication intake, barriers to deprescribe PIM, external actors supporting PIM intake, system-related factors). Besides certain health-related behaviours (e.g., own obligation to report to GP) and medication-related attitudes and knowledge (e.g., awareness of side effects and interaction of medicines), patient-GP-interactions that were characterised by mutual agreements on drugs (e.g., concerning dosage or discontinuation of a drug) might be advantageous to reduce the probability of chronic PIM use. The results might assist in the development of guidelines and educational programs aiming to reduce PIM use in the elderly.

Hyperuricemia and dementia - a case-control study.

BACKGROUND: There is evidence that uric acid may have antioxidant and neuroprotective effects and might therefore alter the risk for neurodegenerative diseases such as dementia. So far, the relation between serum uric acid (SUA) levels or hyperuricemia and dementia remains elusive. Most studies focused on the disease or SUA levels. Effects of anti-hyperuricemic treatment have not been considered yet. This study investigated the association between hyperuricemia and dementia taking into account anti-hyperuricemic treatment. METHODS: We used longitudinal German public health insurance data and analyzed the association between hyperuricemia with and without different treatment options and dementia in a case-control design. Applying logistic regression the analysis was adjusted for several potential confounders including various comorbidities and polypharmacy. RESULTS: We identified 27,528 cases and 110,112 matched controls of which 22% had a diagnosis of hyperuricemia or gout and 17% received anti-hyperuricemic drugs. For patients with a diagnosis of hyperuricemia we found a slightly reduced risk for dementia (adjusted odds ratio [OR] 0.94, 95% confidence interval [CI] 0.89 to 0.98). The risk reduction was more pronounced for patients treated with anti-hyperuricemic drugs (adjusted OR 0.89, 95% CI 0.85 to 0.94, for regular treatment). CONCLUSIONS: Our results showed a slight reduction for dementia risk in patients with hyperuricemia, both with and without anti-hyperuricemic treatment.

Informal caregivers' perspectives on health of and (potentially inappropriate) medication for (relatively) independent oldest-old people - a qualitative interview study.

BACKGROUND: Oldest-old persons frequently receive potentially inappropriate medication. Medication use takes place under the patients' informal caregivers' influence. We explored informal caregivers' perspectives on medication of (relatively) independent oldest-old persons to identify starting points for safer medication prescription/handling. METHODS: In this exploratory qualitative interview study we interviewed 45 informal caregivers of 45 oldest-old persons (23 with potentially inappropriate medication/22 without potentially inappropriate medication). Interviews were recorded, transcribed and content analyzed (deductive/inductive coding). RESULTS: Interviewees had little knowledge about/influence on oldest-old persons' medication, but declared to monitor oldest-old persons' needs for assistance. They were unaware of the concept of potentially inappropriate medication but sometimes sensitive to substance dependency. Most informal caregivers were satisfied with the oldest-old persons' medication and viewed medication as increasing the patients' quality of life. Inadequate communication was found between informal caregivers and general practitioners. CONCLUSIONS: Influence of informal caregivers on (relatively) independent oldest-old persons' medication seems low. Stakeholders need to be aware that there is a transitional period where independency of oldest-old persons decreases and support needs increase which may be missed by (in-)formal caregivers or concealed by oldest-old persons. Monitoring patients' medication competencies; measures supporting communication between informal caregivers and health care professionals; provision of educational and support resources for informal caregivers and the acceptance of oldest-old persons' increasing assistance needs may increase medication safety.

Association of joint replacement surgery with incident dementia diagnosis in German claims data.

ABSTRACTBackground:Cognitive decline is an important complication of joint replacement surgeries in senior people. METHODS: We determined incidence rates of dementia diagnosis following endoprosthetic joint replacement surgery (upper and lower extremities). The observation period covered up to 28 quarters using German claims data comprising 154,604 cases 65 years and older. Effects were controlled for cerebrovascular and vascular risk factors, age, sex, the presence of a diagnosis of delirium, and regular prescription of sedative or analgesic drugs (SAD). RESULTS: The rate of incident dementia diagnoses in people without joint replacement surgery was 21.34 per 1,000 person years, compared with 80.76 incident cases when joint replacement surgery was conducted during the quarter of the incident dementia diagnosis; rates declined to 21.77 incident cases 7 and more quarters after joint replacement surgery had taken place. This pattern was maintained when controlling for delirium diagnosis and regular prescription of SAD. Among 10,563 patients with at least one joint replacement surgery, patients with a diagnosis of delirium in the quarter of the surgery were at increased risk of a dementia diagnosis compared to patients without such a diagnosis (HR=2.00, p < 0.001). CONCLUSION: In people surviving the high-risk phase for dementia immediately after surgery, long-term risk of dementia may reach the level of those without surgery. These findings encourage consequent perioperative management to reduce the risk of dementia as well as prospective studies of potentially beneficial effects of joint replacement surgery on mid- to long-term recovery of mobility and cognition in geriatric patients.

Use of Antiepileptic Drugs and Dementia Risk-an Analysis of Finnish Health Register and German Health Insurance Data.

OBJECTIVES: To evaluate the association between regular antiepileptic drug (AED) use and incident dementia. DESIGN: Case-control analysis. SETTING: Finnish public health register and German health insurance data. PARTICIPANTS: Individuals with dementia of any type (German data, N=20,325) and Alzheimer's disease (AD; Finnish data, N=70,718) were matched with up to four control persons without dementia. MEASUREMENTS: We analyzed the association between regular AED use and dementia. To address potential protopathic bias, a lag time of 2 years between AED use and dementia diagnosis was introduced. Odds ratios (ORs) were calculated by applying conditional logistic regression, adjusted for potential confounding factors such as comorbidities and polypharmacy. RESULTS: Regular AED use was more frequent in individuals with dementia than controls. Regular use of AEDs was associated with a significantly greater risk of incident dementia (adjusted OR=1.28, 95% confidence interval (CI)=1.14-1.44) and AD (adjusted OR=1.15, 95% CI=1.09-1.22) than no AED use. We also detected a trend toward greater risk of dementia with higher exposure. When AEDs with and without known cognitive adverse effects (CAEs) were compared, a significantly greater risk of dementia was observed for substances with known CAEs (dementia: OR=1.59, 95% CI=1.36-1.86; AD: OR=1.19, 95% CI=1.11-1.27). CONCLUSION: AEDs, especially those with known CAEs, may contribute to incident dementia and AD in older persons.

The Impact of Antipsychotic Drugs on Long-term Care, Nursing Home Admission, and Death in Dementia Patients.

Background: Behavioral and psychological symptoms of dementia are commonly treated with antipsychotic drugs (APDs), which have been associated with adverse health effects. We examine the effect of APDs on long-term care (LTC), nursing home (NH) admission, and death of dementia patients. Methods: We used health claims data of the largest German health insurer from 2004 to 2010 and followed newly-diagnosed dementia patients aged 60 years and older into LTC, NH, and until death. Cox proportional hazards models were estimated to explore whether the risk of these outcomes differed between patients receiving haloperidol, melperone, risperidone, or quetiapine. Results: In a cohort of 6,930 dementia patients who were initially free of LTC dependency, APD users generally faced a twofold increased risk of LTC relative to nonusers. Quetiapine was the exception, showing a comparatively lower risk (HR = 1.64; CI = 1.35-1.98). Among 9,950 dementia patients initially living in private homes, the risk of moving into a NH was generally increased by about 50% among APD users relative to nonusers. Risk of death (N = 10,921) was significantly higher for haloperidol-, melperone-, and risperidone- but not for quetiapine users (HR = 0.91; CI = 0.78-1.08). The excess mortality associated with haloperidol and melperone was greater among patients living in private households. Conclusions: In our study, APDs appeared to accelerate adverse health outcomes in German dementia patients. Differentiating between the effect of antipsychotic drug use among dementia patients residing in private households and in NHs, we found that excess mortality for haloperidol and melperone users was higher in private settings.

Mutational profiling in the peripheral blood leukocytes of patients with systemic mast cell activation syndrome using next-generation sequencing.

Mast cell activation syndrome (MCAS) and systemic mastocytosis (SM) are two clinical systemic mast cell activation disease variants. Few studies to date have investigated the genetic basis of MCAS. The present study had two aims. First, to investigate whether peripheral blood leukocytes from MCAS patients also harbor somatic mutations in genes implicated in SM using next-generation sequencing (NGS) technology and a relatively large MCAS cohort. We also addressed the question, whether some of the previously as somatic reported mutations are indeed germline mutations. Second, to identify germline mutations of relevance to MCAS pathogenesis. Here, mutation frequency in the present MCAS cohort was compared to that in public- and in-house databases in the case of frequent variants, and co-segregation was investigated in multiply affected families in the case of rare variants (allele frequency < 1%). MCAS diagnoses were assigned according to current criteria. Twenty five candidate genes were selected on the basis of published findings for SM. NGS was performed using a 76kbp custom designed Agilent SureSelect Target Enrichment and an Illumina Hiseq2000 2x100bp sequencing run. NGS revealed 67 germline mutations. No somatic mutations were detected. None of the germline mutations showed unequivocal association with MCAS. Failure to detect somatic mutations was probably attributable to the dilution of mutated mast cell DNA in normal leukocyte DNA. The present exploratory association findings suggest that some of the detected germline mutations may be functionally relevant and explain familial aggregation. Independent replication studies are therefore warranted.