Targeting expression to megakaryocytes and platelets by lineage-specific lentiviral vectors.

Essentials Platelet phenotypes can be modified by lentiviral transduction of hematopoietic stem cells. Megakaryocyte-specific lentiviral vectors were tested in vitro and in vivo for restricted expression. The glycoprotein 6 vector expressed almost exclusively in megakaryocytes. The platelet factor 4 vector was the strongest but with activity in hematopoietic stem cells. SUMMARY: Background Lentiviral transduction and transplantation of hematopoietic stem cells (HSCs) can be utilized to modify the phenotype of megakaryocytes and platelets. As the genetic modification in HSCs is transmitted onto all hematopoietic progenies, transgene expression from the vector should be restricted to megakaryocytes to avoid un-physiologic effects by ectopic transgene expression. This can be achieved by lentiviral vectors that control expression by lineage-specific promoters. Methods In this study, we introduced promoters of megakaryocyte/platelet-specific genes, namely human glycoprotein 6 (hGP6) and hGP9, into third generation lentiviral vectors and analyzed their functionality in vitro and in vivo in bone marrow transplantation assays. Their specificity and efficiency of expression was compared with lentiviral vectors utilizing the promoters of murine platelet factor 4 (mPf4) and hGP1BA, both with strong activity in megakaryocytes (MKs) used in earlier studies, and the ubiquitously expressing phosphoglycerate kinase (hPGK) and spleen focus forming virus (SFFV) enhancer/promoters. Results Expression from the mPf4 vector in MKs and platelets was the strongest similar to expression from the viral SFFV promoter, however, the mPf4 vector, also exhibited considerable off-target expression in hematopoietic stem and progenitor cells. In contrast, the newly generated hGP6 vector was highly specific to megakaryocytes and platelets. The specificity was also retained when reducing the promoter size to 350 bp, making it a valuable new tool for lentiviral expression in MKs/platelets. Conclusion MK-specific vectors express preferentially in the megakaryocyte lineage. These vectors can be applied to develop murine models to study megakaryocyte and platelet function, or for gene therapy targeting proteins to platelets.

[Uroliths of cats in Switzerland from 2002 to 2009]. / Urolithen bei Katzen in der Schweiz von 2002 bis 2009.

INTRODUCTION: In this study data on composition of uroliths collected from cats and epidemiologic data of affected cats in Switzerland from 2002 to 2009 are summarised. Of 884 stones analysed 50% (n=441) were composed of calcium oxalate, 45% (n=398) of struvite, 3% (n=18) of ammonium urate, 1% (n=12) were mixed stones, 1% (n=9) were composed of silica, 3 stones were solidified blood, 2 consisted of cystine and 1of xanthine. 40% of the ureteral stones were composed of struvite. Domestic cats had significantly less calcium oxalate stones compared to British Shorthair or Persian cats. Cats with calcium oxalate stones were older and cats with struvite stones were younger than other affected cats. Female and male cats were equally affected with stones. Compared to studies from other countries, in Switzerland silica stones occurred more often and ureteral stones were more often composed of Struvite. The present study shows that occurrence and prevalence of urinary calculi of cats from Switzerland exhibited only slight differences to studies from other countries.

INTRODUCTION: Dans le présent travail, on résume les données relatives à la composition des calculs urinaires chez les chats en Suisse ainsi que les données épidémiologiques de ces animaux entre 2002 et 2009. Parmi les 884 calculs analysés, 50% (n=441) étaient composés d'oxalate de calcium, 45% (n=398) de struvite, 3% (n=18) d'urate d'ammonium, 1% (n=12) étaient des calculs mixtes 1% (n=9) se composaient de silicate, 3 calculs étaient formé de sang aggloméré, 2 étaient composés de cystine et un de xanthine. 40% des calculs urétraux se composaient de struvite. Les chats de maison avaient significativement moins de calculs d'oxalate de calcium que les British Shorthair ou les Persans. Les chats présentant des calculs d'oxalate de calcium étaient plus âgés et ceux présentant des struvites plus jeunes que tous les autres chats atteints. Les femelles et les mâles souffraient de calculs de façon équivalente. En comparaison avec des études menées dans d'autres pays, les calculs de silicate étaient plus fréquents en Suisse et les calculs urétraux se composaient plus souvent de struvite. Cette étude démontre que la survenue et la fréquence de calculs urinaires chez les chats en Suisse ne présente que peu de différence avec les études faites dans d'autres pays.

Anxiety modulates cognitive deficits in a perinatal glutathione deficit animal model of schizophrenia.

In this study, we investigated long-term repercussion of early glutathione deficit by l-buthionine-(S,R)-sulfoximine (BSO) injections as a rat model of schizophrenia. BSO rats were tested through various behavioral tasks requiring animals to take into account previously delivered information. We showed that relative to controls, BSO rats (1) were less active and more anxious in an Elevated Plus Maze test, allowing us to split them into two subgroups with high and low anxiety levels; (2) demonstrated normal abilities of behavioral flexibility tested with a rat-adapted version of the Wisconsin Card Sorting Test (WCST), with even higher abilities in anxious BSO rats suggesting reduced interference of previously acquired rules; (3) did not forage normally in radial arm mazes and mainly used clockwise strategies; (4) exhibited a lack of habituation during a startle response task; and (5) showed a normal prepulse inhibition of the startle response (PPI) and a normal conditioned taste aversion (CTA). All these results indicate that early glutathione deficit provokes persistent changes in adulthood and improves the validity of this animal model of schizophrenia. They further suggest difficulties binding temporally separated events (WCST), except when the salience of this information is very strong (CTA). We propose that the transient glutathione deficit during cerebral development could alter a "cognitive binding" process in interaction with the emotional state that could possibly account for the disruption of integrative function that characterizes schizophrenia.

[Uroliths of dogs in Switzerland from 2003 to 2009]. / Urolithen von Hunden in der Schweiz von 2003 bis 2009.

Information on composition of uroliths collected between 2003 and 2009 from dogs in Switzerland and epidemiologic data of affected dogs are summarised in this paper. Of 490 stones analysed 44% were composed of calcium oxalate, 330% of struvite, 80% of silica, 7% of urate, 3% of cystine, 3% were mixed stones and 1% each were calcium phosphate and xanthine stones. Compared to other dogs, Norwich Terriers, Norfolk Terriers, Miniature Schnauzers, Miniature Pinscher and Yorkshire Terriers had a significantly increased risk to suffer from calcium oxalate stones, Dalmatians and Continental Bulldogs from urate stones and English Bulldogs from cystine stones. No breed had an increased risk of struvite or silica stones. Stones composed of silica were more prevalent in Switzerland compared to other countries and were more common in the eastern part than in the western part of Switzerland. This study shows that there are differences in occurrence and prevalence of uroliths between Switzerland and surveys of other countries.

Do computers teach better? A media comparison study for case-based teaching in radiology.

A prospective study was performed to better define the role of computers in teaching radiology to medical students. Two hundred twenty-five 3rd-year students were randomly assigned to one of four groups and exposed to 10 radiology cases as well as to a voluntary weekly radiology lecture. Group A used computer-based cases with interactive elements; group B used computer-based cases without interactive elements; group C used paper-based cases with interactive elements; and group D was not exposed to the cases and served as a control group. On a multiple-choice question test, groups A, B, and C showed significant improvement (+11.2%, +15.1%, and +13.0%, respectively), whereas group D did not (+0.6%). On an image interpretation test, group A showed the most improvement (+15.7% [P <.001]), followed by group B (+15.1% [P <.01]) and group C (+10.2% [P <.05]); group D showed no significant improvement (+8.5%). No significant differences in the learning outcome were found between the two interactive groups (computer based and paper based). Computer-based teaching with case studies (with or without interactivity) improves students' problem-solving ability in radiology.

A behavioral study of alpha-1b adrenergic receptor knockout mice: increased reaction to novelty and selectively reduced learning capacities.

Knockout mice lacking the alpha-1b adrenergic receptor were tested in behavioral experiments. Reaction to novelty was first assessed in a simple test in which the time taken by the knockout mice and their littermate controls to enter a second compartment was compared. Then the mice were tested in an open field to which unknown objects were subsequently added. Special novelty was introduced by moving one of the familiar objects to another location in the open field. Spatial behavior and memory were further studied in a homing board test, and in the water maze. The alpha-1b knockout mice showed an enhanced reactivity to new situations. They were faster to enter the new environment, covered longer paths in the open field, and spent more time exploring the new objects. They reacted like controls to modification inducing spatial novelty. In the homing board test, both the knockout mice and the control mice seemed to use a combination of distant visual and proximal olfactory cues, showing place preference only if the two types of cues were redundant. In the water maze the alpha-1b knockout mice were unable to learn the task, which was confirmed in a probe trial without platform. They were perfectly able, however, to escape in a visible platform procedure. These results confirm previous findings showing that the noradrenergic pathway is important for the modulation of behaviors such as reaction to novelty and exploration, and suggest that this is mediated, at least partly, through the alpha-1b adrenergic receptors. The lack of alpha-1b adrenergic receptors in spatial orientation does not seem important in cue-rich tasks but may interfere with orientation in situations providing distant cues only.

Auditory cues support place navigation in rats when associated with a visual cue.

Rats, like other crepuscular animals, have excellent auditory capacities and they discriminate well between different sounds [Heffner HE, Heffner RS, Hearing in two cricetid rodents: wood rats (Neotoma floridana) and grasshopper mouse (Onychomys leucogaster). J Comp Psychol 1985;99(3):275-88]. However, most experimental literature concerning spatial orientation almost exclusively emphasizes the use of visual landmarks [Cressant A, Muller RU, Poucet B. Failure of centrally placed objects to control the firing fields of hippocampal place cells. J Neurosci 1997;17(7):2531-42; and Goodridge JP, Taube JS. Preferential use of the landmark navigational system by head direction cells in rats. Behav Neurosci 1995;109(1):49-61]. To address the important issue of whether rats are able to achieve a place navigation task relative to auditory beacons, we designed a place learning task in the water maze. We controlled cue availability by conducting the experiment in total darkness. Three auditory cues did not allow place navigation whereas three visual cues in the same positions did support place navigation. One auditory beacon directly associated with the goal location did not support taxon navigation (a beacon strategy allowing the animal to find the goal just by swimming toward the cue). Replacing the auditory beacons by one single visual beacon did support taxon navigation. A multimodal configuration of two auditory cues and one visual cue allowed correct place navigation. The deletion of the two auditory or of the one visual cue did disrupt the spatial performance. Thus rats can combine information from different sensory modalities to achieve a place navigation task. In particular, auditory cues support place navigation when associated with a visual one.

Molecular and prognostic classification of advanced melanoma: a multi-marker microcontamination assay of peripheral blood stem cells.

The presence or absence of melanoma cells in human peripheral blood has recently been shown to be associated with disease prognosis, including overall survival. The detection of tyrosinase mRNA-positive circulating melanoma cells by reverse transcription-polymerase chain reaction (RT-PCR) has been limited to disseminated tumours expressing measurable amounts of this melanocyte-specific enzyme. To biologically classify both melanotic and amelanotic melanomas and to evaluate the clinical and prognostic relevance of tumour cell microcontamination, we examined autologous peripheral blood stem cell (PBSC) harvests from patients with advanced malignant melanoma prior to dose-escalated chemotherapy. To assay heterogeneous melanoma cell antigen expression, we developed a highly sensitive RT-PCR using four melanoma- and one tumour-associated antigen as molecular markers. Expression of the melanocyte-associated transcripts of tyrosinase, MART1/Melan-A, tyrosinase-related protein-1 (TRP-1) and tyrosinase-related protein-2 (TRP-2) as well as the tumour-specific transcript of MAGE-3 was analysed by RT-PCR in PBSC harvests from 31 patients. Seven of the 31 PBSC harvests tested positive for one or more molecular markers: two patients for tyrosinase only, and one patient for MAGE-3 only, one patient for tyrosinase and MAGE-3, one for tyrosinase and MART1/Melan-A, and two patients for tyrosinase, MART1/Melan-A, TRP-2 and MAGE-3. mRNA-positive patients exhibited a significantly impaired overall survival (P = 0.0032), with a median survival of 3 months as opposed to 10 months in PBSC mRNA-negative patients. In conclusion, the use of this multiple-marker microcontamination assay allowed for molecular and prognostic classification of advanced malignant melanoma.

The place preference task: a new tool for studying the relation between behavior and place cell activity in rats.

This study describes a task that combines random searching with goal directed navigation. The testing was conducted on a circular elevated open field (80 cm in diameter), with an unmarked target area (20 cm in diameter) in the center of 1 of the 4 quadrants. Whenever the rat entered the target area, the computerized tracking system released a pellet to a random point on the open field. Rats were able to learn the task under light and in total darkness, and on a stable or a rotating arena. Visual information was important in light, but idiothetic information became crucial in darkness. Learning of a new position was quicker under light than in total darkness on a rotating arena. The place preference task should make it possible to study place cells (PCs) when the rats use an allothetic (room frame) or idiothetic (arena frame) representation of space and to compare the behavioral response with the PCs' activity.

Postnatal intracerebroventricular administrations of NGF alter spatial memory in adulthood.

The present work assessed the effects of intracerebroventricular injections (2x5 mg/2.5 ml) of recombined human nerve growth factor (rhNGF) at postnatal days 2 and 3 upon the development of spatial learning capacities in rats. The treated rats were trained at the age of 22 days to escape onto an invisible platform at a fixed position in space in a Morris navigation task. For half of the subjects, the training position was also cued, a procedure aimed at facilitating escape and reducing attention to the distant spatial cues. At the age of 2 months all the rats were retrained in the same task. Treatment effects were found in both immature and adult rats. The injection of NGF induced a slight alteration of the immature rats' performance. In contrast, a marked impairment of spatial abilities was shown in the 2-month-old rats. The most consistent effects were a significant increase in the escape latency and a decrease bias towards the training platform area during probe trials. The reduction of spatial memory was particularly marked if the subjects had been trained in a cued condition. Taken together, these experiments reveal that an acute pharmacological treatment that leads to transient modifications during early development might induce a behavioural change long after treatment. Thus, the development and the maintenance of an accurate spatial representation are tightly related to the development of brain structures that could be altered by precocious NGF administrations.

Enhanced visuospatial memory following intracerebroventricular administration of nerve growth factor.

The present work assessed the effects of intracerebroventricular injections of rh recombined human nerve growth factor (rh NGF) (5 micrograms/2.5 microl) at postnatal days 12 and 13 upon the development of spatial learning capacities. The treated rats were trained at the age of 22 days to escape onto an invisible platform at a fixed position in space in a Morris navigation task. For half of the subjects, the training position was also cued, a procedure aimed at facilitating escape and at reducing attention to the distant spatial cues. Later, at the age of 6 months, all the rats were trained in a radial-arm maze task. Treatment effects were found in both immature and adult rats. The injection of NGF improved the performance in the Morris navigation task in both training conditions. There was a significant reduction in the escape latency and an increased bias toward the training platform quadrant during probe trials. The most consistent effect was the precocious development of an adult-like spatial memory. In the radial-arm maze, the NGF-treated rats made significantly fewer reentries than vehicle rats and this effect was particularly marked in the treated female rats. Taken together, these experiments reveal that the development and the maintenance of an accurate spatial representation are tightly related to the development of brain structures facilitated by the action of NGF. Moreover, these experiments demonstrate that an acute pharmacological treatment that leads to a transient modification in the choline acetyltransferase activity can induce a behavioral change long after the treatment.

Olfactory traces and spatial learning in rats.

We conducted an experiment to assess the use of olfactory traces for spatial orientation in an open environment in rats, Rattus norvegicus. We trained rats to locate a food source at a fixed location from different starting points, in the presence or absence of visual information. A single food source was hidden in an array of 19 petri dishes regularly arranged in an open-field arena. Rats were trained to locate the food source either in white light (with full access to distant visuospatial information) or in darkness (without any visual information). In both cases, the goal was in a fixed location relative to the spatial frame of reference. The results of this experiment revealed that the presence of noncontrolled olfactory traces coherent with the spatial frame of reference enables rats to locate a unique position as accurately in darkness as with full access to visuospatial information. We hypothesize that the olfactory traces complement the use of other orientation mechanisms, such as path integration or the reliance on visuospatial information. This experiment demonstrates that rats can rely on olfactory traces for accurate orientation, and raises questions about the establishment of such traces in the absence of any other orientation mechanism. Copyright 1998 The Association for the Study of Animal Behaviour.

Indications, management, and complications of temporary inferior vena cava filters.

PURPOSE: We describe the results of a preliminary prospective study using different recently developed temporary and retrievable inferior vena cava (IVC) filters. METHODS: Fifty temporary IVC filters (Günther, Günther Tulip, Anthéor) were inserted in 47 patients when the required period of protection against pulmonary embolism (PE) was estimated to be less than 2 weeks. The indications were documented deep vein thrombosis (DVT) and temporary contraindications for anticoagulation, a high risk for PE, and PE despite DVT prophylaxis. RESULTS: Filters were removed 1-12 days after placement and nine (18%) had captured thrombi. Complications were one PE during and after removal of a filter, two minor filter migrations, and one IVC thrombosis. CONCLUSION: Temporary filters are effective in trapping clots and protecting against PE, and the complication rate does not exceed that of permanent filters. They are an alternative when protection from PE is required temporarily, and should be considered in patients with a normal life expectancy.

[Recurrent abdominal colic, myalgia and mononeuritis multiplex]. / Rezidivierende abdominelle Koliken, Myalgien und Mononeuritis multiplex.

The case of a 30-year-old male, who presented with a three months history of fever, night sweats, weight loss and myalgia is reported. Subsequently abdominal cramps, bloody diarrhea and mononeuropathy multiplex developed. An abdominal and renal angiogram showed changes of vascular structures diagnostic for polyarteritis nodosa. An immunosuppressive treatment (Prednisolon 100 mg/day and Cyclophosphamid 200 mg/day) was started. However, diffuse peritonitis as the aftermath of bowel infarction, which comprised the total length of the jejunum and the proximal parts of the ileum, developed at the third week of this treatment. Despite immediate surgical resection of the ischemic bowel septic complications occurred and the patient died.

Septal lesions impair the acquisition of a cued place navigation task: attentional or memory deficit?

These experiments were designed to analyze how medial septal lesions reducing the cholinergic innervation in the hippocampus might affect place learning. Rats with quisqualic lesions of the medial septal area (MS) were trained in a water maze and on a homing table where the escape position was located at a spatially fixed position and further indicated by a salient cue suspended above it. The lesioned rats were significantly impaired in reaching the cued escape platform during training. In addition rats, did not show any discrimination of the training sector during a probe trial in which no platform or cue was present. This impairment remained significant during further training in the absence of the cue. When the cued escape platform was located at an unpredictable spatial location, the MS-lesioned rats showed no deficit and spent more time under the cue than control rats during the probe trial. On the homing board, with a salient object in close proximity to the escape hole, the MS rats showed no deficit in escape latencies, although a significant reduction in spatial memory was observed. However, this was overcome by additional training in the absence of the cue. Under these conditions, rats with septal lesions were prone to develop a pure guidance strategy, whereas normal rats combined a guidance strategy with a memory of the escape position relative to more distant landmarks. The presence of a salient cue appeared to decrease attention to environmental landmarks, thus reducing spatial memory. These data confirm the general hypothesis that MS lesions reduce the capacity to rely on a representation of the relation between several landmarks with different salience.

Olfactory cues potentiate learning of distant visuospatial information.

The influence of proximal olfactory cues on place learning and memory was tested in two different spatial tasks. Rats were trained to find a hole leading to their home cage or a single food source in an array of petri dishes. The two apparatuses differed both by the type of reinforcement (return to the home cage or food reward) and the local characteristics of the goal (masked holes or salient dishes). In both cases, the goal was in a fixed location relative to distant visual landmarks and could be marked by a local olfactory cue. Thus, the position of the goal was defined by two sets of redundant cues, each of which was sufficient to allow the discrimination of the goal location. These experiments were conducted with two strains of hooded rats (Long-Evans and PVG), which show different speeds of acquisition in place learning tasks. They revealed that the presence of an olfactory cue marking the goal facilitated learning of its location and that the facilitation persisted after the removal of the cue. Thus, the proximal olfactory cue appeared to potentiate learning and memory of the goal location relative to distant environmental cues. This facilitating effect was only detected when the expression of spatial memory was not already optimal, i.e., during the early phase of acquisition. It was not limited to a particular strain.

Protein-induced increase in urinary dopamine in normal and diabetic rats: role of catecholamine precursors.

Feeding and protein intake increase renal dopamine excretion (UDAV). Here the contribution of amino acids (AA), L-tyrosine (Tyr), and L-phenylalanine (Phe) to UDAV in conscious normal rats and in animals with streptozotocin (STZ)-induced (60 mg/kg) diabetes mellitus was investigated. Feeding a standard chow (17.3% protein) increased UDAV in normal rats over twofold compared with the fasted state, but the effect was completely abolished by feeding a low-protein (LP, 0.03%) diet. In STZ rats, UDAV was equal to that of normal rats during the fasted periods but was higher in fed animals, resulting most likely from the higher protein intake of STZ rats. In another series, rats on LP diet were given AA solutions (7, 14, and 21 g.kg-1.24 h-1) by gastric tube, which dose dependently increased UDAV to 67.3 +/- 4.3, 91.1 +/- 5.0, and 129 +/- 17 nmol.kg-1.day-1, respectively, compared with tap water as vehicle control (H2O, 55.6 +/- 7.0 nmol.kg-1.day-1). In rats kept without access to chow, administration of AA including Phe and Tyr (AAPT) increased UDAV twofold compared with H2O, whereas AA solution without Tyr and Phe did not change UDAV. Tyr or Phe alone increased UDAV to the same extent as observed in AAPT. Higher doses of Tyr further increased UDAV dose dependently but with saturation characteristics. UDAV of the animals that were in a slightly negative sodium balance was not correlated to renal sodium excretion. It is concluded that, in conscious rats, the increase in UDAV in response to feeding 1) depends on the supply of catecholamine precursors solely, 2) is dose dependent and saturable, and 3) is not affected by experimental diabetes mellitus.

Spatial learning by rats across visually disconnected environments.

Two spatial tasks were designed to test specific properties of spatial representation in rats. In the first task, rats were trained to locate an escape hole at a fixed position in a visually homogeneous arena. This arena was connected with a periphery where a full view of the room environment existed. Therefore, rats were dependent on their memory trace of the previous position in the periphery to discriminate a position within the central region. Under these experimental conditions, the test animals showed a significant discrimination of the training position without a specific local view. In the second task, rats were trained in a radial maze consisting of tunnels that were transparent at their distal ends only. Because the central part of the maze was non-transparent, rats had to plan and execute appropriate trajectories without specific visual feedback from the environment. This situation was intended to encourage the reliance on prospective memory of the non-visited arms in selecting the following move. Our results show that acquisition performance was only slightly decreased compared to that shown in a completely transparent maze and considerably higher than in a translucent maze or in darkness. These two series of experiments indicate (1) that rats can learn about the relative position of different places with no common visual panorama, and (2) that they are able to plan and execute a sequence of visits to several places without direct visual feed-back about their relative position.

Integration of olfactory information in a spatial representation enabling accurate arm choice in the radial arm maze.

The aim of the present study was to determine whether and how rats can use local olfactory cues for spatial orientation. Rats were trained in an eight-arm radial maze under different conditions as defined by the presence or absence of supplementary olfactory cues marking each arm, the availability of distant visuospatial information, and the illumination of the maze (light or darkness). The different visual conditions were designed to dissociate among the effects of light per se and those of visuospatial cues, on the use of olfactory cues for accurate arm choice. Different procedures with modifications of the arrangement of olfactory cues were used to determine if rats formed a representation of the spatial configuration of the olfactory cues and if they could rely on such a representation for accurate arm choice in the radial maze. The present study demonstrated that the use of olfactory cues to direct arm choice in the radial arm maze was critically dependent on the illumination conditions and implied two different modes of processing of olfactory information according to the presence or the absence of light. Olfactory cues were used in an explicit manner and enabled accurate arm choice only in the absence of light. Rats, however, had an implicit memory of the location of the olfactory cues and formed a representation of the spatial position of these cues, whatever the lighting conditions. They did not memorize the spatial configuration of the olfactory cues per se but needed these cues to be linked to the external spatial frame of reference.