Pathophysiology of idiopathic dystonia: findings from genetic animal models

Dystonia is a common movement disorder which is thought to represent a disease of the basal ganglia. However, the pathogenesis of the idiopathic dystonias, i.e. the neuroanatomic and neurochemical basis, is still a mystery. Research in dystonia is complicated by the existence of various phenotypic and genotypic subtypes of idiopathic dystonia, probably related to heterogeneous dysfunctions.In neurological diseases in which no obvious neuronal degeneration can be found, such as in idiopathic dystonia, the identification of a primary defect is difficult, because of the large number of chemically distinct, but functionally interrelated, neurotransmitter systems in the brain.The variable response to pharmacological agents in patients with idiopathic dystonia supports the notion that the underlying biochemical dysfunctions vary in the subtypes of idiopathic dystonia. Hence, in basic research it is important to clearly define the involved type of dystonia.Animal models of dystonias were described as limited. However, over the last years, there has been considerable progress in the evaluation of animal models for different types of dystonia.Apart from animal models of symptomatic dystonia, genetic animal models with inherited dystonia which occurs in the absence of pathomorphological alterations in brain and spinal cord are described.This review will focus mainly on genetic animal models of different idiopathic dystonias and pathophysiological findings. In particular, in the case of the mutant dystonic (dt) rat, a model of generalized dystonia, and in the case of the genetically dystonic hamster (dt^sz), a model of paroxysmal dystonic choreoathetosis has been used, as these show great promise in contributing to the identification of underlying mechanisms in idiopathic dystonias, although even a proper animal model will probably never be equivalent to a human disease.Several pathophysiological findings from animal models are in line with clinical observations in dystonic patients, indicating abnormalities not only in the basal ganglia and thalamic nuclei, but also in the cerebellum and brainstem. Through clinical studies and neurochemical data several similarities were found in the genetic animal models, although the current data indicates different defects in dystonic animals which is consistent with the notion that dystonia is a heterogenous disorder.Different supraspinal dysfunctions appear to lead to manifestation of dystonic movements and postures. In addition to increasing our understanding of the pathophysiology of idiopathic dystonia, animal models may help to improve therapeutic strategies for this movement disorder.     

贵州省2004年分离到的脊髓灰质炎病毒分子生物学特征

目的研究贵州省2004年分离到的脊髓灰质炎(脊灰)病毒分子生物学特征。方法对贵州省2004年分离到的所有脊灰病毒,用聚合酶链反应-限制性酶切片段长度多态性分析(PCR-RFLP)和酶联免疫吸附试验(ELISA)法进行了型内鉴定,并对型内鉴定异常株进行了VP1区的序列测定。结果贵州省在2004年急性弛缓性麻痹(AFP)病例及接触者、流动人口和健康儿童的粪便标本中,共有95例分离到脊灰病毒。其中Ⅰ型22例,Ⅱ型26例,Ⅲ型21例,混合型19例,脊灰病毒混合非脊灰肠道病毒7例。经用PCR-RFLP和ELISA方法进行型内鉴定,共有16株病毒与疫苗株病毒存在差异,其中3株脊灰病毒与疫苗株病毒在PCR-RFLP图谱上有差异[其中1株同时为双反应(DRV)],3株ELISA结果为DRV,11株ELISA结果为非疫苗类似株(NSL)。在这些型内鉴定异常株病毒中,Ⅰ型13株,Ⅱ型3株。对这16株脊灰病毒进行VP1区序列测定,发现9株Ⅰ型疫苗衍生脊灰病毒(VDPV)和1株Ⅱ型VDPV。结论根据对贵州省2004年从95例AFP病例及接触者、流动人口、健康儿童分离的脊灰病毒的血清定型结果和型内鉴定结果及对13株Ⅰ型和8株Ⅱ型脊灰病毒VP1区核苷酸序列测定证实,脊灰减毒活疫苗病毒在人群的循环导致疫苗病毒神经毒力恢复突变。通过AFP病例监测系统及时发现了Ⅰ型VDPV的循环和Ⅱ型VDPV。对2004年下半年脊灰病毒基因特点的分析,提示贵州省已经阻断了Ⅰ型VDPV的循环。     

High rate of hematological responses to sorafenib in FLT3‐ITD acute myeloid leukemia relapsed after allogeneic hematopoietic stem cell transplantation

Relapse represents the most significant cause of failure of allogeneic hematopoietic stem cell transplantation (HSCT) for FLT3‐ITD‐positive acute myeloid leukemia (AML), and available therapies are largely unsatisfactory. In this study, we retrospectively collected data on the off‐label use of the tyrosine kinase inhibitor sorafenib, either alone or in association with hypomethylating agents and adoptive immunotherapy, in 13 patients with post‐transplantation FLT3‐ITD‐positive AML relapses. Hematological response was documented in 12 of 13 patients (92%), and five of 13 (38%) achieved complete bone marrow remission. Treatment was overall manageable in the outpatient setting, although all patients experienced significant adverse events, especially severe cytopenias (requiring a donor stem cell boost in five patients) and typical hand‐foot syndrome. None of the patients developed graft‐vs.‐host disease following sorafenib alone, whereas this was frequently observed when this was given in association with donor T‐cell infusions. Six patients are alive and in remission at the last follow‐up, and four could be bridged to a second allogeneic HSCT, configuring a 65 ± 14% overall survival at 100 d from relapse. Taken together, our data suggest that sorafenib might represent a valid treatment option for patients with FLT3‐ITD‐positive post‐transplantation relapses, manageable also in combination with other therapeutic strategies.     

Channel Interaction and Current Level Affect Across-Electrode Integration of Interaural Time Differences in Bilateral Cochlear-Implant Listeners

Sensitivity to interaural time differences (ITDs) is important for sound localization. Normal-hearing listeners benefit from across-frequency processing, as seen with improved ITD thresholds when consistent ITD cues are presented over a range of frequency channels compared with when ITD information is only presented in a single frequency channel. This study aimed to clarify whether cochlear-implant (CI) listeners can make use of similar processing when being stimulated with multiple interaural electrode pairs transmitting consistent ITD information. ITD thresholds for unmodulated, 100-pulse-per-second pulse trains were measured in seven bilateral CI listeners using research interfaces. Consistent ITDs were presented at either one or two electrode pairs at different current levels, allowing for comparisons at either constant level per component electrode or equal overall loudness. Different tonotopic distances between the pairs were tested in order to clarify the potential influence of channel interaction. Comparison of ITD thresholds between double pairs and the respective single pairs revealed systematic effects of tonotopic separation and current level. At constant levels, performance with double-pair stimulation improved compared with single-pair stimulation but only for large tonotopic separation. Comparisons at equal overall loudness revealed no benefit from presenting ITD information at two electrode pairs for any tonotopic spacing. Irrespective of electrode-pair configuration, ITD sensitivity improved with increasing current level. Hence, the improved ITD sensitivity for double pairs found for a large tonotopic separation and constant current levels seems to be due to increased loudness. The overall data suggest that CI listeners can benefit from combining consistent ITD information across multiple electrodes, provided sufficient stimulus levels and that stimulating electrode pairs are widely spaced.     

Acoustic Cues for Sound Source Distance and Azimuth in Rabbits, a Racquetball and a Rigid Spherical Model

There are numerous studies measuring the transfer functions representing signal transformation between a source and each ear canal, i.e., the head-related transfer functions (HRTFs), for various species. However, only a handful of these address the effects of sound source distance on HRTFs. This is the first study of HRTFs in the rabbit where the emphasis is on the effects of sound source distance and azimuth on HRTFs. With the rabbit placed in an anechoic chamber, we made acoustic measurements with miniature microphones placed deep in each ear canal to a sound source at different positions (10–160 cm distance, ±150° azimuth). The sound was a logarithmically swept broadband chirp. For comparisons, we also obtained the HRTFs from a racquetball and a computational model for a rigid sphere. We found that (1) the spectral shape of the HRTF in each ear changed with sound source location; (2) interaural level difference (ILD) increased with decreasing distance and with increasing frequency. Furthermore, ILDs can be substantial even at low frequencies when distance is close; and (3) interaural time difference (ITD) decreased with decreasing distance and generally increased with decreasing frequency. The observations in the rabbit were reproduced, in general, by those in the racquetball, albeit greater in magnitude in the rabbit. In the sphere model, the results were partly similar and partly different than those in the racquetball and the rabbit. These findings refute the common notions that ILD is negligible at low frequencies and that ITD is constant across frequency. These misconceptions became evident when distance-dependent changes were examined.     

4D microstructural changes in dentinal tubules during acid demineralisation

ObjectiveDental erosion is a common oral condition caused by chronic exposure to acids from intrinsic/extrinsic sources. Repeated acid exposure can lead to the irreversible loss of dental hard tissues (enamel, dentine, cementum). Dentine can become exposed to acid following severe enamel erosion, crown fracture, or gingival recession. Causing hypersensitivity, poor aesthetics, and potential pulp involvement. Improving treatments that can restore the structural integrity and aesthetics are therefore highly desirable. Such developments require a good understanding of how acid demineralisation progresses where relatively little is known in terms of intertubular dentine (ITD) and peritubular dentine (PTD) microstructure. To obtain further insight, this study proposes a new in vitro method for performing demineralisation studies of dentine.MethodsAdvanced high-speed synchrotron X-ray microtomography (SXM), with high spatial (0.325 μm) and temporal (15 min) resolution, was used to conduct the first in vitro, time-resolved 3D (4D) study of the microstructural changes in the ITD and PTD phases of human dentine samples (∼0.8 × 0.8 × 5 mm) during 6 h of continuous acid exposure.ResultsDifferent demineralisation rates of ITD (1.79 μm/min) and PTD (1.94 μm/min) and their progressive width-depth profiles were quantified, which provide insight for understanding the mechanisms of dentine demineralisation.SignificanceInsights obtained from morphological characterisations and the demineralisation process of ITD and PTD during acid demineralisation would help understand the demineralisation process and potentially aid in developing new therapeutic dentine treatments. This method enables continuous examination of relatively large volumes of dentine during demineralisation and also demonstrates the potential for studying the remineralisation process of proposed therapeutic dentine treatments.     

Bone marrow stroma‐mediated resistance to FLT3 inhibitors in FLT3‐ITD AML is mediated by persistent activation of extracellular regulated kinase

A consistent pattern of response has been observed when FMS‐like tyrosine kinase 3 (FLT3) tyrosine kinase inhibitors (TKIs) have been used as monotherapy to treat patients with relapsed or refractory FLT3‐ internal tandem duplication (ITD) acute myeloid leukaemia (AML). Circulating blasts are cleared from the peripheral blood, while bone marrow blasts are either unaffected or are cleared from the marrow at a much slower rate. We used an in vitro model of FLT3‐ITD AML blasts co‐cultured with normal human bone marrow stromal cells to investigate the basis for this dichotomous response pattern to FLT3 inhibitors. We have found that in blasts on stroma, potent FLT3 inhibition predominantly results in cell cycle arrest rather than apoptosis. The anti‐apoptotic effect is mediated through a combination of direct cell‐cell contact and soluble factors. The addition of exogenous FLT3 ligand (FL) augments the protection, primarily by shifting the 50% inhibitory concentration for FLT3 inhibition upwards. Cytokine‐activated extracellular regulated kinase (ERK), rather than STAT5, appears to be the most important downstream signalling protein mediating the protective effect, and inhibition of MEK significantly abrogates stromal‐mediated resistance. These findings explain the phenomenon of peripheral blood versus bone marrow blast responses and suggest that the combination of potent FLT3 inhibition and MEK inhibition is a promising strategy for the treatment of FLT3‐ITD AML.     

Ponatinib may overcome resistance of FLT3-ITD harbouring additional point mutations, notably the previously refractory F691I mutation

Fms-like tyrosine kinase (FLT3) mutations are the most frequent mutations in patients with acute myeloid leukaemia (AML) that confer a poor prognosis. Constitutively active FLT3-ITD (internal tandem duplications) mutations define a promising target for therapeutic approaches using small molecule inhibitors. However, several point mutations of the FLT3 tyrosine kinase domain (FLT3-TKD) have been identified to mediate resistance towards FLT3 tyrosine kinase inhibitors (FLT3-TKI), including secondary mutations of FLT3. We investigated the cellular effects of the recently characterised FLT3-TKI ponatinib (AP24534) on murine myeloid cells transfected with FLT3-ITD with or without additional point mutations of the FLT3-TKD including the (so far) multi-resistant F691I mutation. Ponatinib effectively induced apoptosis not only in the parental FLT3-ITD cell line but also in all stably transfected subclones harbouring additional FLT3-TKD point mutations (N676D, F691I or G697R). These observations correlated with a strong inhibition of FLT3-ITD and its downstream targets STAT5, AKT and ERK1/2 upon ponatinib incubation, as determined by Western blotting. We conclude that ponatinib represents a promising FLT3-TKI that should be further investigated in clinical trials. The targeted therapy of FLT3-ITD-positive AML with ponatinib might be associated with a lower frequency of secondary resistance caused by acquired FLT3-TKD mutations.     

Binaural responses in the auditory midbrain of chicken (Gallus gallus)

The auditory midbrain is the location in which neurons represent binaural acoustic information necessary for sound localization. The external nucleus of the midbrain inferior colliculus (IC) of the barn owl is a classic example of an auditory space map, but it is unknown to what extent the principles underlying its formation generalize to other, less specialized animals. We characterized the spiking responses of 139 auditory neurons in the IC of the chicken (Gallus gallus) in vivo, focusing on their sensitivities to the binaural localization cues of interaural time (ITD) and level (ILD) differences. Most units were frequency‐selective, with best frequencies distributed unevenly into low‐frequency and high‐frequency (> 2 kHz) clusters. Many units showed sensitivity to either ITD (65%) or ILD (66%) and nearly half to both (47%). ITD selectivity was disproportionately more common among low‐frequency units, while ILD‐only selective units were predominantly tuned to high frequencies. ILD sensitivities were diverse, and we thus developed a decision tree defining five types. One rare type with a bell‐like ILD tuning was also selective for ITD but typically not frequency‐selective, and thus matched the characteristics of neurons in the auditory space map of the barn owl. Our results suggest that generalist birds such as the chicken show a prominent representation of ITD and ILD cues in the IC, providing complementary information for sound localization, according to the duplex theory. A broadband response type narrowly selective for both ITD and ILD may form the basis for a representation of auditory space.     

儿童白血病患者Flt-3/ITD突变分析及其临床意义

背景与目的:Flt-3跨膜区内部串联重复(Flt-3 intemal tandem duplication,Flt-3/ITD)突变是近年来发现的Flt-3基因最常见的一种突变类型,是急性髓系白血病(acute myeloid leukaemia,AML)中发生率最高且与预后相关的突变.本研究旨在探讨Flt-3/ITD突变与儿童白血病发生的关系及其临床意义.方法:采用聚合酶链反应(polymerase chain reaction,PCR)联合序列测定,检测302例儿童白血病患者骨髓Flt-3/ITD突变情况,其中包括AML 122例、急性淋巴细胞白血病(acute lymphoblastic leukemia,ALL)124例、幼年型慢性粒细胞白血病(juvenile chronic myelogenous leukemia,JCML)17例和骨髓异常增生综合征(myelodysplastic syndromes,MDS)39例.结果:122例AML患者中98例(80.33%)Flt-3阳性;21例(17.21%)发生Flt-3/ITD突变,分别为M0 3例、M1 2例、M2 4例、M4 8例及M5 4例,突变率分别为42.86%(3/7)、22.22%(2/9)、12.90%(4/31)、44.44%(8/18)和15.38%(4/26).此外,124例ALL中72例Flt-3阳性,阳性率为58.06%,其中2例发现Flt-3/ITD突变,突变率1.61%.测序及Blast比对分析显示,外显子11区均有ITD,各例ITD的复制区域不同,长短不等(24～95 bp).39例MDS和17例JCML患者中均未检测到Flt-3/ITD突变.临床资料显示,21例Flt-3/ITD突变的AML患者中有19例在短期内死亡,这19例的中位生存时间为13.5个月(0～47个月),死亡率为90.48%,与无Flt-3/ITD突变患者相比有显著性差异(P＜0.05).Flt-3/ITD突变阳性患者外周血中性粒细胞平均比率与Flt-3/ITD突变阴性患者无显著性差异(P＞0.05).染色体核型分析显示,Flt-3/ITD的AML患者中3例存在有染色体异位,分别为t(11;12)(p15;q13)、t(6;9)(p23;q23)、inv16(q21;q23).结论:Flt-3/ITD突变多发现于AML,罕见于ALL,未见于MDS和JCML.该突变与儿童白血病特别是儿童AML的发生及进展有关.Flt-3/ITD可以作为判断AML预后的重要标志之一.