Calreticulin mutant mice develop essential thrombocythemia that is ameliorated by the JAK inhibitor ruxolitinib.

Mutations of calreticulin (CALR) are detected in 25-30% of patients with essential thrombocythemia (ET) or primary myelofibrosis and cause frameshifts that result in proteins with a novel C-terminal. We demonstrate that CALR mutations activated signal transducer and activator of transcription 5 (STAT5) in 293T cells in the presence of thrombopoietin receptor (MPL). Human megakaryocytic CMK11-5 cells and erythroleukemic F-36P-MPL cells with knocked-in CALR mutations showed increased growth and acquisition of cytokine-independent growth, respectively, accompanied by STAT5 phosphorylation. Transgenic mice expressing a human CALR mutation with a 52 bp deletion (CALRdel52-transgenic mice (TG)) developed ET, with an increase in platelet count, but not hemoglobin level or white blood cell count, in association with an increase in bone marrow (BM) mature megakaryocytes. CALRdel52 BM cells did not drive away wild-type (WT) BM cells in in vivo competitive serial transplantation assays, suggesting that the self-renewal capacity of CALRdel52 hematopoietic stem cells (HSCs) was comparable to that of WT HSCs. Therapy with the Janus kinase (JAK) inhibitor ruxolitinib ameliorated the thrombocytosis in TG mice and attenuated the increase in number of BM megakaryocytes and HSCs. Taken together, our study provides a model showing that the C-terminal of mutant CALR activated JAK-STAT signaling specifically downstream of MPL and may have a central role in CALR-induced myeloproliferative neoplasms.

TET2 is essential for survival and hematopoietic stem cell homeostasis.

Ten-Eleven-Translocation 2 (TET2) is an enzyme that catalyzes the conversion of 5-methylcytosine into 5-hydroxymethylcytosine (5-hmC) and thereby alters the epigenetic state of DNA; somatic loss-of-function mutations of TET2 are frequently observed in patients with diverse myeloid malignancies. To study the function of TET2 in vivo, we analyzed Ayu17-449 (TET2(trap)) mice, in which a gene trap insertion in intron 2 of TET2 reduces TET2 mRNA levels to about 20% of that found in wild-type (WT) mice. TET2(trap/trap) mice were born at Mendelian frequency but died at a high rate by postnatal day 3, indicating the essential role of TET2 for survival. Loss of TET2 results in an increase in the number of hematopoietic stem cells (HSCs)/progenitors in the fetal liver, and TET2(trap/trap) HSCs exhibit an increased self-renewal ability in vivo. In competitive transplantation assays, TET2(trap/trap) HSCs possess a competitive growth advantage over WT HSCs. These data indicate that TET2 has a critical role in survival and HSC homeostasis.

Anaesthetists' role in computer keyboard contamination in an operating room.

To store anaesthetic records in computers, anaesthetists usually input data while still wearing dirty wet gloves. No studies have explored computer contamination in the operating room (OR) or anaesthetists' awareness of the importance of handwashing or hand hygiene. We investigated four components of keyboard contamination: (1) degree of contamination, (2) effect of cleaning with ethyl alcohol, (3) bacterial transmission between gloves and keyboards by tapping keys, and (4) frequency of anaesthetists' performing hand hygiene. Most of the bacteria on keyboards were coagulase-negative staphylococci and Bacillus spp.; however, meticillin-resistant Staphylococcus aureus was also found. Cleaning keyboards with ethyl alcohol effectively reduced bacterial counts. Wet contaminated gloves and keyboards transmitted meticillin-susceptible Staphylococcus epidermidis from one to the other more readily than dry contaminated gloves and keyboards. Only 17% of anaesthetists performed hand hygiene before anaesthesia, although 64% or 69% of anaesthetists performed hand hygiene after anaesthesia or before lunch. To prevent cross-contamination, keyboards should be routinely cleaned according to the manufacturer's instructions and disinfected once daily, or, when visibly soiled with blood or secretions. Moreover, anaesthetists should be aware that they could spread microbes that might cause healthcare-associated infection in the OR. Anaesthetists should perform hand hygiene before and after anaesthesia and remove gloves after each procedure and before using the computer.

[A case of a traumatic anterior cerebral artery aneurysm following the penetration of the skull base by an iron rod].

A 61-year-old male fell from a position 1 m high when building a house. An iron rod, which protruded upward from a solid base in cement, penetrated this patient's neck 15 cm to the head and was successfully extracted by himself. On admission, he complained of headache and vomiting. General examination disclosed nasal bleeding, intraoral bleeding, and L figured skin laceration in the left side of his neck at the level of the thyroid cartilage. Mild disorientation (JCS2) was noted. Otolaryngological examination disclosed hyperemia on the left side of the vocal cord as well as at the dome of the superior pharynx. Plain skull film disclosed pneumocephalus and that a piece of bone fragment of the planum sphenoidale had penetrated the brain. CT demonstrated air in the subarachnoid space, ventricular hemorrhage, intracerebral hematoma in the right frontal lobe, and subarachnoid hemorrhage in the anterior interhemispheric fissure. CAG detected neither cerebral vascular abnormalities nor cerebral aneurysm. While staying in our department, he developed mild fever and CSF rhinorrhea. The diagnosis of bacterial meningitis was made from the CSF finding and was well controlled with conservative therapy. CSF rhinorrhea stopped spontaneously with conservative treatment. Sagittal MRI continuously demonstrated contusional hematoma in the base of the right frontal lobe just above the fractured planum sphenoidale and genu of the corpus callosum following the course of the intracranially invading iron rod. The right CAG on Day 10 demonstrated vasospasm on the A1 and a 1 cm sized saccular cerebral aneurysm at the proximal right fronto-polar artery. CAG on Day 17 again showed the persistent presence of the aneurysm. For the purpose of preventing delayed rupture of the aneurysm, radical surgical treatment was planned. Microsurgical dissection disclosed that the aneurysm was located just behind the elevated fracture of the planum sphenoidale. Severe arachnoid adhesion was noted around the aneurysm. The aneurysm was successfully clipped with preservation of the parent artery without inducing new neurological deficits. From the general, otolaryngological, neuroradiological, and operative findings, this aneurysm was diagnosed as a traumatic cerebral artery aneurysm following the penetration of the skull base by the iron rod. The CAG performed at 8 months postoperatively demonstrated the patency of the parent artery and that there was no recurrence of the aneurysm. An unusual case of a traumatic cerebral artery aneurysm following the penetration of the skull base by an iron rod was thus reported.

Stimulus effects of the medial pontine reticular formation and the mesencephalic locomotor region upon medullary reticulospinal neurons in acute decerebrate cats.

In acute decerebrate cats, medial pontine reticular formation (mPRF) and the mesencephalic locomotor region (MLR) were stimulated and their stimulus effects upon 250 medullary reticulospinal neurons (RSNs) were studied. One hundred and twenty-six RSNs were mono- and disynaptically activated. From the response patterns of the RSNs, they were divided into the mPRF-activated RSNs (n = 67) and the MLR-activated RSNs (n = 59). The former group of RSNs was located in the nucleus reticularis gigantocellularis (NRGc), while the latter group of RSNs was distributed in both the NRGc and the nucleus reticularis magnocellularis (NRMc). The activity of MLR-excited 12 RSNs was suppressed with the preceding mPRF stimulation. These RSNs were mainly located in the NRMc. Most mPRF-excited RSNs increased their discharge rates during mPRF-evoked suppression of postural muscle tone, and most MLR-excited RSNs increased their discharge rates during MLR-evoked locomotion. With mPRF stimulation, MLR-evoked locomotion was suppressed with cessation of MLR-excited RSNs activity. These results suggest that mPRF stimulation suppresses the activity of the locomotor rhythm generating system at the levels of not only the spinal cord but also the medullary output cells.

Neuroanatomical and neurophysiological bases of postural control.

At this stage of our study, we do not know yet the relative contribution of each pathway that descends from the cerebral cortex, the cerebellar nuclei, and the SN to the control of pontomedullary reticuloreticular and reticulospinal neurons. Nor do we know the resultant activation patterns of reticulospinal neurons and those of axial and proximal motoneurons in relation to postural fixation and equilibrium. In dealing with the physiology of posture, such as the maintenance or change of body position, we are dealing with the emergent properties of the total system that are not described by the sum of the properties of its parts. Learning how a system subcomponent behaves in a carefully defined and constrained set of circumstances gives us no guarantee that the subcomponent will behave according to the same rules if the circumstances were changed (59). Further behavioral studies in animal and human subjects, especially using natural posture and movements, plus studies aimed at elucidating the fine architecture of the target system, including identification of acting neurotransmitters at each synaptic level of a system subcomponent, are now needed to confirm existing hypotheses (10, 37, 47) and elaborate new ones related to the control of posture and movement.

Multi-segmental and generalized suppression of postural muscle tone evoked by stimulating the dorsal tegmental field and the medial pontine reticular formation in acute decerebrate cats.

In acute decerebrate cat, stimulation of the dorsal tegmental field (DTF) of the caudal pons along its midline and the medial pontine reticular formation (mPRF) evokes generalized suppression of postural muscle tone. In this study, the effects of stimulation of the DTF area were compared with those evoked by stimulating the mPRF unilaterally or bilaterally. By stimulating the mPRF bilaterally, simultaneous suppression of tonic activities in the neck, lumbar back, forelimb and hindlimb muscles, bilaterally, was evoked as that evoked by stimulating the DTF area alone.

Extracellular levels of serotonin in the medial pontine reticular formation in acute decerebrate cats with a microdialysis technique.

Extracellular levels of serotonin (5-HT) and 5-hydroxyindolacetic acid (5-HIAA) were measured in the medial pontine reticular formation of acute decerebrate cats. The mean basal levels of 5-HT and 5-HIAA were 26 fmol/20 microliters and 15 pmol/20 microliters. Perfusion of the dialysis probe with high K+ and Ca(2+)-free Ringer solution for 60 min resulted in 4.8-8.5 x increase and 25-48% decrease in the extracellular levels of 5-HT, respectively, in comparison to the basal 5-HT levels. Perfusion with TTX-added Ringer solution for 60 min resulted in a consistent decrease in the extracellular levels of 5-HT.

Extracellular levels of serotonin in the medial pontine reticular formation in relation to sleep-wake cycle in cats: a microdialysis study.

Extracellular levels of endogenous serotonin (5-hydroxytryptamine; 5-HT) and its major metabolite, 5-hydroxyindoleacetic acid (5-HIAA) were measured in the medial pontine reticular formation (medial PRF) of intact cats. A microdialysis probe was inserted through a guide cannula into the medial PRF. At least 12 h after the probe insertion, in vivo brain microdialysis was initiated. The perfusion rate was 1 microliters/min, and perfusate fractions at regular intervals of 20 min were collected. Changes in extracellular 5-HT levels were compared across sleep-wake states of the animals, such as waking (W), slow wave sleep (SWS) and rapid eye movement (REM) sleep. To assess sleep-wake states, EEG, EMG, EOG and PGO waves were simultaneously recorded in parallel with microdialysis of the medial PRF. Extracellular 5-HT levels were highest (20-28 fmol/20 microliters) during W. As the animals entered SWS, 5-HT levels decreased to about 90% of those during W. The state of REM sleep usually interrupted SWS for 3-8 min. During the longer periods of REM sleep, during the 20 min periods in which the perfusates were collected, we observed the lowest 5-HT levels (60-50%).

Pontine-induced generalized suppression of postural muscle tone in a reflexively standing acute decerebrate cat.

In a reflexively standing acute decerebrate cat, the pontine tegmentum was electrically stimulated and the effects on postural muscle tone and locomotor movements evoked by stimulation of the mesencephalic locomotor region (MLR) were studied. A stimulating microelectrode was placed systematically at 1-mm increments throughout the pons (H - 2 to H - 10) at levels ranging from P0.0 to P6.5 dorsoventrally and mediolaterally from LR0 to L4 or R4. Another stimulating microelectrode was placed in the physiologically identified MLR. Stimuli delivered to the dorsomedial regions of the pontine tegmentum (P3 to P4, LR1.5 to 2.5, H - 4 to H - 6) resulted in simultaneous and bilateral suppression of tonic activities in the neck, lumbar back, forelimb and hindlimb muscles. The pontine inhibitory sites corresponded to the medial area of the central tegmental field (FTC) and the central area of the gigantocellular tegmental field (FTG), bilaterally. Effects of pontine induced suppression on those muscles were stimulus frequency and stimulus intensity-dependent and the effects persisted even after termination of the stimulation. With concomitant pontine stimulation, MLR-evoked locomotor movements were suppressed along with prolongation of the forelimb and hindlimb step cycles.

Characterization of very virulent Marek's disease viruses isolated in Japan.

Pathogenicity of two isolates of Marek's disease virus (MDV), MS1 and MS2, from chickens was examined in two genetically different strains of chickens, MD-susceptible P-2 chickens and less susceptible PDL-1 chickens. The isolates induced an early mortality syndrome unassociated with lymphoproliferative lesions in P-2 chickens. There were no significant differences in pathogenicity between our isolates and the Md/5 strain of very virulent MDV (vvMDV) in both P-2 and PDL-1 chickens. Protective indices of turkey herpes virus (HVT) vaccine against challenge with MS1 or MS2 in P-2 chickens were 54% and 28%, respectively, whereas HVT gave more than 80% protection in PDL-1 chickens. These results indicate that the two isolates could be classified as vvMDV. In contrast, a bivalent vaccine composed of HVT and serotype 2 MDV, and CVI988 vaccine gave good protection against challenge with the isolates in P-2 chickens; however, the best protection was given by the CBI988 vaccine. This is the first report of isolation of vvMDV in Japan.