Somatic CALR mutations in myeloproliferative neoplasms with nonmutated JAK2.

BACKGROUND: Somatic mutations in the Janus kinase 2 gene (JAK2) occur in many myeloproliferative neoplasms, but the molecular pathogenesis of myeloproliferative neoplasms with nonmutated JAK2 is obscure, and the diagnosis of these neoplasms remains a challenge. METHODS: We performed exome sequencing of samples obtained from 151 patients with myeloproliferative neoplasms. The mutation status of the gene encoding calreticulin (CALR) was assessed in an additional 1345 hematologic cancers, 1517 other cancers, and 550 controls. We established phylogenetic trees using hematopoietic colonies. We assessed calreticulin subcellular localization using immunofluorescence and flow cytometry. RESULTS: Exome sequencing identified 1498 mutations in 151 patients, with medians of 6.5, 6.5, and 13.0 mutations per patient in samples of polycythemia vera, essential thrombocythemia, and myelofibrosis, respectively. Somatic CALR mutations were found in 70 to 84% of samples of myeloproliferative neoplasms with nonmutated JAK2, in 8% of myelodysplasia samples, in occasional samples of other myeloid cancers, and in none of the other cancers. A total of 148 CALR mutations were identified with 19 distinct variants. Mutations were located in exon 9 and generated a +1 base-pair frameshift, which would result in a mutant protein with a novel C-terminal. Mutant calreticulin was observed in the endoplasmic reticulum without increased cell-surface or Golgi accumulation. Patients with myeloproliferative neoplasms carrying CALR mutations presented with higher platelet counts and lower hemoglobin levels than patients with mutated JAK2. Mutation of CALR was detected in hematopoietic stem and progenitor cells. Clonal analyses showed CALR mutations in the earliest phylogenetic node, a finding consistent with its role as an initiating mutation in some patients. CONCLUSIONS: Somatic mutations in the endoplasmic reticulum chaperone CALR were found in a majority of patients with myeloproliferative neoplasms with nonmutated JAK2. (Funded by the Kay Kendall Leukaemia Fund and others.).

Gene expression profiling reveals clear differences between EBV-positive and EBV-negative posttransplant lymphoproliferative disorders.

Posttransplant patients are at risk of developing a potentially life-threatening posttransplantation lymphoproliferative disorder (PTLD), most often of diffuse large B cell lymphoma (DLBCL) morphology and associated with Epstein-Barr Virus (EBV) infection. The aim of this study was to characterize the clinicopathological and molecular-genetic characteristics of posttransplant DLBCL and to elucidate whether EBV(+) and EBV(-) posttransplant DLBCL are biologically different. We performed gene expression profiling studies on 48 DLBCL of which 33 arose posttransplantation (PT-DLBCL; 72% EBV+) and 15 in immunocompetent hosts (IC-DLBCL; none EBV+). Unsupervised hierarchical analysis showed clustering of samples related to EBV-status rather than immune status. Except for decreased T cell signaling these cases were inseparable from EBV(-) IC-DLBCL. In contrast, a viral response signature clearly segregated EBV(+) PT-DLBCL from EBV(-) PT-DLBCL and IC-DLBCL cases that were intermixed. The broad EBV latency profile (LMP1+/EBNA2+) was expressed in 59% of EBV(+) PT-DLBCL and associated with a more elaborate inflammatory response compared to intermediate latency (LMP1+/EBNA2-). Inference analysis revealed a role for innate and tolerogenic immune responses (including VSIG4 and IDO1) in EBV(+) PT-DLBCL. In conclusion we can state that the EBV signature is the most determining factor in the pathogenesis of EBV(+) PT-DLBCL.

How to improve housing conditions of laboratory animals: the possibilities of environmental refinement.

Housing systems for captive animals have often been designed on the basis of economic and ergonomic considerations, such as equipment, costs, space, workload, ability to observe the animals and to maintain a certain degree of hygiene, with little or no consideration for animal welfare. Environmental refinement can be defined as any modification in the environment of captive animals that seeks to enhance the physical and psychological well-being of the animals by providing stimuli which meet the animals' species-specific needs. This article provides an overview of environmental factors that influence the well-being of captive animals with specific reference to the needs of the most common laboratory species. It is important to evaluate environmental refinement in terms of the benefit to the animal, by assessing the use of and preference for certain enrichment, the effect on behaviour, and the performance of species-typical behaviour on physiological parameters. It is also necessary to evaluate the impact of refinement on scientific outcome, including whether and how statistical power is affected. Communication and team work between animal welfare scientists, animal research scientists, institutional animal welfare officers, veterinarians and animal ethics committees, animal facility management and personnel, are essential for success.

Surgical successes and failures of invasive video-EEG monitoring in the presurgical evaluation of epilepsy.

Invasive monitoring with intracranial electrodes continues to play a critical role in the presurgical evaluation of patients with medically intractable epilepsy. Intracranial monitoring helps in localizing the epileptogenic zone and can be used to delineate eloquent cortical areas adjacent to this zone. In this review we analyzed surgical successes and failures of invasive video-electroencephalography (EEG) monitoring. Thorough understanding of all potential complications is of paramount importance not only for detection and successful management of intractable epilepsy but also for medicolegal purposes, as patients and their relatives need to be fully informed about the possible risks associated with invasive monitoring. A mortality rate between 0.5% and 2.8% has been reported. Cerebrospinal fluid (CSF) leaks and infections are the most frequent complications, with an incidence ranging from 0-31.3% and from 0-17.4%, respectively. The incidence of intracranial hemorrhage is reported to be up to 14% with subdural hematomas being the most prevalent. Epidural hematomas are less frequent and encountered in up to 2.6% of cases. Intraparenchymal hematomas are even less frequent and are typically associated with the placement of depth electrodes. In 47-98% of cases, invasive video-EEG monitoring results into resective surgery. Invasive video-EEG monitoring is a reasonably safe and effective method to help delineate the epileptogenic zone and its relation to eloquent cortex.

Housing environment influences the need for pain relief during post-operative recovery in mice.

The impact of invasive experimental procedures on perceived stress and pain may be dependent on both physical and social environmental conditions. The aim of this study was to evaluate the effects of a physically and a socially enriched environment on the need for pain relief following painful experimental procedures. A non-invasive method to administer analgesics post-operatively is by means of self-administration which is a feasible objective method to measure perceived pain during the post-operative recovery period. In the present study eight groups of mice housed in different conditions underwent the surgical procedure of caecal manipulation or only exposure to anaesthesia. After surgery the mice were given the choice to self-administer an analgesic available in one of their water bottles during two post-operative weeks. It was shown that socially enriched mice drank i.e. self-administered, less from the analgesic containing water than the non-enriched and socially deprived groups. Mice that underwent operation self-administered more analgesic than mice that received only anaesthesia without operation. The findings indicate that the recovery environment can contribute positively to attenuate the need for pain relief in animals submitted to invasive procedures.

Short-term effects of a disturbed light-dark cycle and environmental enrichment on aggression and stress-related parameters in male mice.

In the laboratory setting, environmental factors have a major influence on the well-being of laboratory animals. The present study shows the importance of a semi-natural light-dark cycle. In this experiment one cohort of mice was kept with a continuous lighting for one week. After the first week the artificial light-dark cycle was 12:12 with lights on at 07:00 h. The second cohort of mice was kept with this 12:12 h light-dark cycle from the start. Half of each cohort received environmental enrichment. In order to analyse corticosterone levels, urine samples were collected. To measure agonistic behaviour, the behaviour of the mice was recorded on videotape immediately after cage cleaning. A significant difference in corticosterone levels between cohorts was found during disturbed lighting, but not after lighting conditions were reset to 12:12 h. In the first test week, mice subjected to disturbed lighting also showed a significantly shorter agonistic latency than control mice. This difference had disappeared when in the second test week all mice experienced 12:12 h lighting. No effects of enriched housing were found. This experiment has shown that disturbed lighting for socially-housed male mice caused physiological and behavioural changes indicative of stress, not only leading to much higher levels of corticosterone but also to shorter agonistic latency within the groups.

Long-term effects of husbandry procedures on stress-related parameters in male mice of two strains.

In socially unstable groups of male laboratory mice, individuals may experience a chronic stress situation. Previous experiments have shown that the transfer of specific olfactory cues during cage cleaning, and the provision of nesting material decrease aggression and stress in group-housed male mice. In this study, the combined effect of these husbandry procedures were tested for their long-term effect on stress in groups of moderately aggressive (BALB/c) and severely aggressive (CD-1) male mice. The physiological and behavioural stress-related parameters used were body weight, food and water intake, spleen and thymus weight, adrenal tyrosine hydroxylase activity, urine corticosterone levels and behaviour in a cage emergence test. Long-term provision of nesting material and its transfer during cage cleaning was found to influence several stress-related physiological parameters. Mice housed in cages enriched with nesting material had lower urine corticosterone levels and heavier thymuses, and they consumed less food and water than standard-housed mice. Furthermore, marked differences were found between strains. CD-1 mice were less anxious in the cage emergence test, weighed more, ate and drank more, and had heavier thymuses but lighter spleens and lower corticosterone levels than BALB/c mice. We conclude that the long-term provision of nesting material, including the transfer of nesting material during cage cleaning, reduces stress and thereby enhances the welfare of laboratory mice.

Preference for social contact versus environmental enrichment in male laboratory mice.

Due to their aggressive nature, male mice are less frequently used than female mice in biomedical research. When aggressive males are being used, individual housing is common practice. The question arises whether this is an acceptable housing for a social species. The present study was designed to gain more insight into the nature of inter-male social contact and into the potential of a form of environmental enrichment (nesting material) to compensate for the lack of social contact. In a series of tests, we analysed whether male mice of different ages preferred to spend time (1) near a familiar cage mate versus an empty cage, or (2) near to a familiar cage mate versus direct contact with nesting material (tissues). Dwelling time in each of the test cages and sleeping sites was recorded, as was the behaviour of the test mice. Results indicated that when other conditions were similar, male mice preferred to sleep in close proximity to their familiar cage mate. Furthermore, the need to engage in active social behaviour increased with age. Tissues were used to a large extent for sleeping and sleep-related behaviour. It is concluded that single housing in order to avoid aggression between male mice is a solution with evident negative consequences for the animals. When individual housing is inevitable due to excessive aggressive behaviour, the presence of nesting material could partly compensate for the deprivation of social contact.

The importance of learning young: the use of nesting material in laboratory rats.

Unlike mice, adult laboratory rats do not spontaneously build nests when nesting material is offered. As a result, nesting material is often regarded as unsuitable environmental enrichment for laboratory rats. Wild rats and pet rats, however, have been observed to build complex nests from nesting material at hand. It was hypothesized that nest building in rats is an acquired behaviour, rather than genetically predisposed. To test this hypothesis, the progeny of three Wistar rats provided with nesting material (Kleenex tissues) during pregnancy and three standard-housed rats were divided in 34 same-sex couples with access to nesting material: (1) from the age of 8 weeks (n=7); (2) from weaning (n=8); (3) from birth (n=17). The latter were subdivided into two groups after weaning, one provided with Kleenex tissues (n=9), the other with Enviro-dri (n=8). At the age of 12 weeks, all couples were provided with both types of nesting material for one week. Amount, shape, and soiling of the nesting material were scored on weekdays, and behaviour of the rats was scored twice weekly. Results indicated that the older the rats were when first provided with nesting material, the greater the amount of nesting material eaten and soiled, and the nests built were less elaborate. Overall, Enviro-dri was eaten less than Kleenex tissues. It is concluded that rats need to learn the proper use of nesting material. When provided from birth, nesting material is a suitable type of environmental enrichment for laboratory rats.

Male management: Coping with aggression problems in male laboratory mice.

In a laboratory environment, aggressive interactions between male mice may exceed normal levels leading to negative effects both on the well-being of the animals and on the validity of experimental results. In this paper we review results from the literature and our own research with regard to coping with excessive aggressive behaviour in male laboratory mice. Based on this review practical recommendations concerning the housing and care of male laboratory mice are formulated. In short, it is recommended to avoid individual housing, to transfer odour cues from the nesting area during cage cleaning and to apply nesting material as environmental enrichment. Furthermore, group size should be optimized to three animals per cage. Further research, in particular into the effects of frequency, duration, type and severity of disturbances during an experiment on the degree of aggression, is recommended.

Modulation of aggression in male mice: influence of group size and cage size.

Aggression in group-housed male mice is known to be influenced by both cage size and group size. However, the interdependency of these two parameters has not been studied yet. In this study, the level of aggression in groups of three, five, or eight male BALB/c mice housed in cages with a floor size of either 80 or 125 cm(2)/animal was estimated weekly after cage cleaning for a period of 14 weeks. Furthermore, urine corticosterone levels, food and water intake, body weight, and number of wounds were measured weekly. At the end of the experiment, tyrosine hydroxylase (TH) activity, testosterone levels, and weight of spleen, thymus, testes, and seminal vesicles were determined. Results indicate a moderate increase of intermale aggression in larger cages when compared to the smaller cages. Aggression in groups of eight animals was considerably higher than in groups of three animals. The increase of agonistic behavior was observed both in dominant and subordinate animals. Physiological parameters indicate differences in stress levels between dominant and subordinate animals. It is concluded that aggressive behavior in group-housed male BALB/c mice is best prevented by housing the animals in small groups of three to five animals, while decreasing floor size per animal may be used as a temporary solution to decrease high levels of aggression in an existing social group.

Validation of a new system for the automatic registration of behaviour in mice and rats.

A newly developed behaviour registration system, Laboratory Animal Behaviour Observation, Registration and Analysis System (LABORAS) for the automatic registration of different behavioural elements of mice and rats was validated. The LABORAS sensor platform records vibrations evoked by animal movements and the LABORAS software translates these into the corresponding behaviours. Data obtained by using LABORAS were compared with data from conventional observation methods (observations of videotapes by human observers). The results indicate that LABORAS is a reliable system for the automated registration of eating, drinking, grooming, climbing, resting and locomotion of mice during a prolonged period of time. In rats, grooming, locomotion and resting also met the pre-defined validation criteria. The system can reduce observation labour and time considerably.

Analgesics in mice used in cancer research: reduction of discomfort?

During the last decades, an increase is apparent in the use of analgesics for laboratory animals in situations where this was previously considered unnecessary. Mice with advanced tumours often show clear signs of discomfort which may be a result of chronic pain or a result of general ill-being. The syngeneic murine tumour model most frequently used in our experiments was used to investigate whether this discomfort can be reduced with an analgesic. Twenty DBA/2 mice bearing SL2 lymphoma were given 0.5 mg/kg buprenorphine (Temgesic) in food gel twice daily, 20 tumour-bearing mice were given control food gel at the same times. Indicators of well-being were monitored daily. These included behavioural parameters such as exploration, grooming, and posture; food and water consumption and fur quality. All mice showed a clear increase of discomfort with time: explorative behaviours and grooming decreased, while sitting in hunched posture increased. Food and water consumption and fur quality also decreased. Major significant differences between the buprenorphine treated group and the control group were not apparent. In conclusion, we could not document a positive effect or buprenorphine on discomfort in mice as evaluated by our scoring system. It remains possible that pain itself was not the primary cause of the discomfort in mice bearing these tumours, or that the analgesic effect of buprenorphine was insufficient under these circumstances.

Nesting material as environmental enrichment has no adverse effects on behavior and physiology of laboratory mice.

Environmental enrichment may improve the quality of life of captive animals by altering the environment of animals so that they are able to perform more of the behavior that is within the range of the animal's species-specific repertoire. When enrichment is introduced into an animal's environment, it is important to evaluate the effect of the enrichment program and to assess whether the animal continues to use the enrichment in the long-term. Groups of mice were housed under either standard or enriched conditions for several weeks. Nesting material which was highly preferred in previous studies was used as enrichment. During the period of differential housing several behavioral parameters (behavioral tests and handling) and physiological parameters (urine and plasma corticosterone, food and water intake, body and adrenal weight) were monitored to determine the impact of environmental enrichment. Observations were made to determine whether or not the mice continued to use the enrichment. The results indicated that throughout the study all mice used the nesting material to build nests and that mice from enriched conditions weighed more than mice housed under standard conditions, although the latter consumed more food. No major differences for behavioral and physiological parameters were found between the groups of mice housed under different conditions. Therefore it is not likely that supply of nesting material will jeopardize the outcome of experiments.

Preferences for nesting material as environmental enrichment for laboratory mice.

Behavioural and psychological needs of laboratory animals generally cannot adequately be met in standard laboratory cages. Environmental enrichment, which provides a more structured environment can enhance the well-being of laboratory animals. They may perform more of their species-specific behaviour and may control their environment in a better way. An easily applicable form of enrichment for laboratory mice is nesting material. Six different types of nesting materials were evaluated in a preference test with male and female animals of two strains [C57BL/6J or BALB/c, n = 48]. No significant differences in preference were found between the strains or between the sexes. All mice showed a clear preference for cages with tissues or towels as compared to paper strips or no nesting material, and for cages with cotton string or wood-wool as compared to wood shavings or no nesting material. Paper-derived materials were preferred over wood-derived materials, although the results also suggest that the nature (paper or wood) of the nesting material is less important than its structure, which determines the nestability of the material. Nesting material may be a relatively simple method to contribute to the well-being of laboratory mice.

Behavior of conduct disordered children in interaction with each other and with normal peers.

This study investigated the behavior of children with conduct disorder or oppositional defiant disorder (CD/ODD) in interaction with each other and with normal control (NC) children in a semi-standardized setting over a period of 25 minutes. This short time turned out to be sufficient to demonstrate the behavioral manifestations of CD/ODD in children's interactions with peers. In addition, the role of the interactional partner on antisocial behaviour of CD/ODD children became apparent.