Single-cell RT-PCR, in situ hybridization histochemical, and immunohistochemical studies of substance P and enkephalin co-occurrence in striatal projection neurons in rats

Single-cell RT-PCR studies in 3-4-week-old rats have raised the possibility that as many as 20% of striatal projection neurons may be a unique type that contains both substance P (SP) and enkephalin (ENK). We used single-cell RT-PCR, retrograde labeling, in situ hybridization histochemistry, and immunolabeling to characterize the abundance of this cell type, its projection target(s), and any developmental changes in its frequency. We found by RT-PCR that 11% of neurons containing either SP or ENK contained both in 4-week-old rats, while in 4-month-old rats SP/ENK colocalization was only 3%. SP-only neurons tended to co-contain dynorphin and ENK-only neurons neurotensin, while SP/ENK neurons tended to contain dynorphin. Single-cell RT-PCR showed SP/ENK co-occurrence in 4-week-old rats to be no more common among striatal neurons retrogradely labeled from the substantia nigra than among those retrogradely labeled from globus pallidus. Double-label in situ hybridization showed SP/ENK perikarya to be scattered throughout striatum, making up 8% of neurons containing either SP or ENK at 4 weeks, but only 4% at 4 months. Immunolabeling showed that presumptive striatal terminals in globus pallidus externus, globus pallidus internus and substantia nigra pars reticulata that colocalized SP and ENK were scarce. Terminals colocalizing SP and ENK were, however, abundant in the substantia nigra pars compacta. Thus, SP-only and ENK-only neurons make up the vast majority of striatal projection neurons in rats, the frequency of SP/ENK colocalizing striatal neurons is low in adult rats (3-4%), and SP/ENK colocalizing neurons primarily project to SNc but do not appear to be confined to striosomes.     

Acute fulminant myocarditis in children and adolescents: the role of mechanical circulatory assist

We report children and adolescents in fulminant myocarditis undergoing prolonged circulatory support with different assist devices. Between 1994 and 2004, seven children and adolescents (aged 7-18 years, mean age 13.5 years) were treated with VADs (5 Thoratec, 1 Medos, 1 Novacor) for circulatory support. Three patients underwent left ventricular support; biventricular support was necessary in four patients. Four patients (three left VADs, one bi-VAD) could be successfully bridged to heart transplantation after a mean support time of 163 days (56-258 days). One 7-year-old girl (Medos-BVAD) died after a support time of 11 days because of irreversible multiorgan failure. One 18-year-old patient was successfully weaned from Thoratec BVAD after 66 days with complete recovery of left ventricular function. As good markers, atrial and brain natriuretic peptides were found which reached normal values after recovery of myocardial function. A 15-year-old girl is still on the device. In children or adolescents with irreversible shock in fulminant myocarditis with an anticipated mortality of 100%, both successful bridging to heart transplantation and successful bridging to recovery are possible. Young patients with fulminant myocarditis should be rapidly transferred to a clinic with a mechanical circulatory support program to offer this life-saving option.     

Molecular characterization of BCL6 breakpoints in primary diffuse large B-cell lymphomas of the central nervous system identifies GAPD as novel translocation partner

Primary central nervous system lymphomas (PCNSL) constitute diffuse large B-cell lymphomas arising in and remaining confined to the brain. Little information is available on cytogenetic changes in PCNSL, and recurrent chromosomal translocations have not yet been identified. Fluorescence in situ hybridization (FISH) of a series of 13 PCNSL from immunocompetent patients revealed 3 cases with signal patterns of a BCL6-specific probe suggesting a breakpoint in this oncogene locus in chromosome band 3q27. Here, we describe cloning of the translocation breakpoints by long-distance inverse polymerase chain reaction (LDI-PCR) in 2 of these tumors. Both breakpoints affected the first intron of BCL6. In one PCNSL, the HSPCA (HSP90A) gene in 14q32.31 was identified as BCL6 partner. In the second lymphoma, the gene encoding glyceraldehyde-3-phosphate dehydrogenase (GAPD) on 12p13.31 was detected as a hitherto unknown partner of BCL6. Our results suggest translocation-mediated BCL6 oncogene activation as a so far unknown pathogenetically relevant mechanism in PCNSL.     

Estimating mental workload through event-related fluctuations of pupil area during a task in a virtual world

Monitoring mental load for optimal performance has become increasingly central with the recently evolving need to cope with exponentially increasing amounts of data. This paper describes a non-intrusive, objective method to estimate mental workload in an immersive virtual reality system, through analysis of frequencies of pupil fluctuations. We tested changes in mental workload with a number of task-repetitions, level of predictability of the task and the effect of prior experience in predictable task performance, on mental workload of unpredictable task performance. Two measures were used to calculate mental workload: the ratio of Low Frequency to High Frequency components of pupil fluctuations, and the High Frequency alone, all extracted from the Power Spectrum Density of pupil fluctuations. Results show that mental workload decreases with a number of repetitions, creating a mode in which the brain acts as an automatic controller. Automaticity during training occurs only after a minimal number of repetitions, which once achieved, resulted in further improvements in the performance of unpredictable motor tasks, following training in a predictable task. These results indicate that automaticity is a central component in the transfer of skills from highly predictable to low predictable motor tasks. Our results suggest a potentially applicable method to brain–computer-interface systems that adapt to human mental workload, and provide intelligent automated support for enhanced performance.     

Lymphoma stem cells: enough evidence to support their existence?

While leukemia-originating stem cells are critical in the initiation and maintenance of leukemias, the existence of similar cell populations that may generate B-cell lymphoma upon mutation remains uncertain. Here we propose that committed lymphoid progenitor/precursor cells with an active V-D-J recombination program are the initiating cells of follicular lymphoma and mantle cell lymphoma when targeted by immunoglobulin (IG)- gene translocations in the bone marrow. However, these pre-malignant lymphoma-initiating cells cannot drive complete malignant transformation, requiring additional cooperating mutations in specific stem-cell programs to be converted into the lymphoma-originating cells able to generate and sustain lymphoma development. Conversely, diffuse large B-cell lymphoma and sporadic Burkitt’s lymphoma derive from B lymphocytes that acquire translocations through IG-hyper-mutation or class-switching errors within the germinal center. Although secondary reprogramming mutations are generally required, some cells such as centroblasts or memory B cells that have certain stem cell-like features, or lymphocytes with MYC rearrangements that deregulate self-renewal pathways, may bypass this need and directly function as the lymphoma-originating cells. An alternative model supports an aberrant epigenetic modification of gene sets as the first occurring hit, which either leads to retaining stem-cell features in hematopoietic stem or progenitor cells, or reprograms stemness into more committed lymphocytes, followed by secondary chromosomal translocations that eventually drive lymphoma development. Isolation and characterization of the cells that are at the origin of the different B-cell non-Hodgkin’s lymphomas will provide critical insights into the disease pathogenesis and will represent a step towards the development of more effective therapies.     

Timing-dependent plasticity in human primary somatosensory cortex

Animal experiments suggest that cortical sensory representations may be remodelled as a consequence of changing synaptic efficacy by timing-dependent associative neuronal activity. Here we describe a timing-based associative form of plasticity in human somatosensory cortex. Paired associative stimulation (PAS) was performed by combining repetitive median nerve stimulation with transcranial magnetic stimulation (TMS) over the contralateral postcentral region. PAS increased exclusively the amplitude of the P25 component of the median nerve-evoked somatosensory-evoked potential (MN-SSEP), which is probably generated in the superficial cortical layers of area 3b. SSEP components reflecting neuronal activity in deeper cortical layers (N20 component) or subcortical regions (P14 component) remained constant. PAS-induced enhancement of P25 amplitude displayed topographical specificity both for the recording (MN-SSEP versus tibial nerve-SSEP) and the stimulation (magnetic stimulation targeting somatosensory versus motor cortex) arrangements. Modulation of P25 amplitude was confined to a narrow range of interstimulus intervals (ISIs) between the MN pulse and the TMS pulse, and the sign of the modulation changed with ISIs differing by only 15 ms. The function describing the ISI dependence of PAS effects on somatosensory cortex resembled one previously observed in motor cortex, shifted by ∼7 ms. The findings suggest a simple model of modulation of excitability in human primary somatosensory cortex, possibly by mechanisms related to the spike-timing-dependent plasticity of neuronal synapses located in upper cortical layers.     

Effect of high hydrostatic pressure on biological properties of extracellular bone matrix proteins

In orthopedic surgery, sterilization of bone used for reconstruction of osteoarticular defects caused by malignant tumors is carried out in various ways. At present, to devitalize tumor-bearing osteochondral segments, extracorporal irradiation or autoclaving is mainly used but both methods have substantial disadvantages, for instance, loss of biomechanical and biological integrity of the bone. In particular, after reimplantation, integration of the implant at the autograft-host junction is often impaired due to alteration of osteoinductivity as a result of its irradiation or autoclaving. As an alternative approach, high hydrostatic pressure (HHP) treatment of bone is suggested, a new technology which is in the preclinical testing stage, with the aim to inactivate tumor cells but leaving the biomechanical properties of bone, cartilage, and tendons intact. We investigated the influence of HHP on the major extracellular matrix (ECM) proteins, fibronectin (FN), vitronectin (VN), and type I collagen (Col-I), present in bone tissue, which are accountable for the biological properties within the bone. FN, VN, and Col-I were subjected to HHP < or = 600 MPa prior to coating of cell culture plates with these matrix proteins. Thereafter, the capacity of HHP-pretreated FN, VN, and Col-I to affect cell proliferation, cell adherence, and spreading of human primary osteoblast-like cells and the human osteosarcoma cell line Saos-2, was tested. Interestingly, even at HHP < or = 600 MPa, all three ECM proteins retained their biological properties because no significant changes were observed between HHP-treated and non-treated FN, VN, and Col-I regarding their biological properties to affect cell adherence, spreading, and proliferation. These data encourage further exploration of the potential of HHP to sterilize tumor-affected bone segments prior to reimplantation. While during this treatment eukaryotic cells including tumor cells will be irreversibly impaired, the bone's biomechanical properties and the biological properties of the ECM proteins FN, VN, and Col-I, respectively, are preserved.