Reconstitution of immune cell populations in multiple sclerosis patients after autologous stem cell transplantation.

Multiple sclerosis is an inflammatory T cell-mediated autoimmune disease. In a Phase II clinical trial, high-dose immunosuppressive therapy combined with autologous CD34+ haematopoietic stem cell transplant resulted in 69·2% of subjects remaining disease-free without evidence of relapse, loss of neurological function or new magnetic resonance imaging (MRI) lesions to year 5 post-treatment. A combination of CyTOF mass cytometry and multi-parameter flow cytometry was used to explore the reconstitution kinetics of immune cell subsets in the periphery post-haematopoietic cell transplant (HSCT) and the impact of treatment on the phenotype of circulating T cells in this study population. Repopulation of immune cell subsets progressed similarly for all patients studied 2 years post-therapy, regardless of clinical outcome. At month 2, monocytes and natural killer (NK) cells were proportionally more abundant, while CD4 T cells and B cells were reduced, relative to baseline. In contrast to the changes observed at earlier time-points in the T cell compartment, B cells were proportionally more abundant and expansion in the proportion of naive B cells was observed 1 and 2 years post-therapy. Within the T cell compartment, the proportion of effector memory and late effector subsets of CD4 and CD8 T cells was increased, together with transient increases in proportions of CD45RA-regulatory T cells (Tregs ) and T helper type 1 (Th1 cells) and a decrease in Th17·1 cells. While none of the treatment effects studied correlated with clinical outcome, patients who remained healthy throughout the 5-year study had significantly higher absolute numbers of memory CD4 and CD8 T cells in the periphery prior to stem cell transplantation.

Radiographer workforce role expansion to improve patient safety related to nasogastric tube placement for feeding in adults.

AIM: To determine whether the active involvement of radiographers in nasogastric tube (NGT) management at a large multisite healthcare institution can contribute to risk reduction regarding feeding through misplaced NGTs. MATERIALS AND METHODS: Despite national guidance in the National Health Service advising on safe practice to confirm NGT position, a number of "never events" (feeding through misplaced NGT) continue to occur due to misinterpretation of the check radiograph. Practice change was introduced, including all plain film radiographers providing contemporaneous comments on NGT position on the check radiograph. The success of the system was assessed to determine the accuracy of radiographer comments against the reference standard of the radiologist report to see whether the system has helped reduce the number of "never events". RESULTS: During the first 27 months post-implementation, 4,675 check NGT radiography examinations were analysed. Two hundred and twenty-seven examinations were excluded due to absent or incomplete radiographer comments. The accuracy of the radiographer comments was 98.5% (95% confidence interval [CI]: 97.7-99.5%), sensitivity 97.4% (95% CI: 96.3-98.3%), specificity 98.9% (95% CI: 98.5-99.2%), positive predictive value 96.8% (95% CI: 95.6-97.7%), and negative predictive value 99.1% (95% CI: 98.8-99.4%). CONCLUSION: After focused training, radiographer comments are a safe, sustainable, and workable solution offering an effective solution for image misinterpretation issues relating to NGT "never events". This should be considered for wider implementation in healthcare.

CREB: a major mediator of neuronal neurotrophin responses.

Neurotrophins regulate neuronal survival, differentiation, and synaptic function. To understand how neurotrophins elicit such diverse responses, we elucidated signaling pathways by which brain-derived neurotrophic factor (BDNF) activates gene expression in cultured neurons and hippocampal slices. We found, unexpectedly, that the transcription factor cyclic AMP response element-binding protein (CREB) is an important regulator of BDNF-induced gene expression. Exposure of neurons to BDNF stimulates CREB phosphorylation and activation via at least two signaling pathways: by a calcium/calmodulin-dependent kinase IV (CaMKIV)-regulated pathway that is activated by the release of intracellular calcium and by a Ras-dependent pathway. These findings reveal a previously unrecognized, CaMK-dependent mechanism by which neurotrophins activate CREB and suggest that CREB plays a central role in mediating neurotrophin responses in neurons.

Dendrites are more spiny on mature hippocampal neurons when synapses are inactivated.

Dendrites of CA1 pyramidal neurons in mature rat hippocampal slices were exposed to different levels of synaptic activation. In some slices, synaptic transmission was blocked with glutamate receptor antagonists, sodium and calcium channel blockers and/or a nominally calcium-free medium with high magnesium. In other slices, synapses were activated with low-frequency control stimulation or repeated tetanic stimulation. In slices with blocked synaptic transmission, dendrites were spinier than in either of the activated states. Thus, mature neurons can increase their numbers of spines, possibly compensating for lost synaptic activity.

How multiple-synapse boutons could preserve input specificity during an interneuronal spread of LTP.

A model is proposed whereby the spread of long-term potentiation (LTP) between potentiated and neighboring neurons is initiated by a retrograde signal that is restricted to the synaptic clefts of the potentiated neurons. Next, a change, such as enhanced release of neurotransmitter, occurs in the presynaptic boutons that are associated with potentiated synapses. This change affects all synapses that are located on the potentiated boutons, and leads to LTP at synapses on neighboring neurons that share multiple-synapse boutons with the initially potentiated neurons. In this model, restricting the retrograde signal to the potentiated synaptic clefts ensures the axonal-input specificity of LTP, and the induction of the secondary LTP requires the same cellular mechanisms as those of induction of the primary LTP.

Quantal analysis and synaptic anatomy--integrating two views of hippocampal plasticity.

The excitatory synapses onto CA1 pyramidal cells have become a model system for understanding the activity-dependent changes in synapses that underlie learning and memory. Here we examine physiological and anatomical results that are relevant to understanding the mechanisms of synaptic transmission and plasticity at these synapses. Three main points are discussed. First, quantal analysis indicates a large heterogeneity of postsynaptic efficacies for different synapses on the same cell. Reconstructions from electron microscopy show that synapse size is also highly heterogeneous. Reasons for suspecting a relationship between synaptic size and efficacy are discussed. Second, physiological evidence indicates that the changes during long-term potentiation are both pre- and postsynaptic. Similarly, several lines of anatomical evidence suggest that plasticity affects the structure of both the pre- and postsynaptic elements. The detailed registration of structures across the synapse and the physical linkage between pre- and postsynaptic elements suggest a 'structural unit hypothesis' for coordinating pre- and postsynaptic modifications. Third, quantal analysis indicates that stimulation of a single axon can release multiple quanta. Anatomical evidence shows that cell pairs can be connected by multiple synapses, suggesting that multiple quanta may be released at independent sites. These results raise the possibility that one component of synaptic plasticity is mediated by changes in the number of functional synaptic sites.

Structure, development, and plasticity of dendritic spines.

Dendritic spines are distinguished by their shapes, subcellular composition, and synaptic receptor subtypes. Recent studies show that actin-dependent movements take place in spine heads, that spines emerge from stubby and shaft synapses after dendritic filopodia disappear, and that spines can form without synaptic activation, are maintained by optimal activation, and are lost with excessive activation or during degeneration.

Dense core vesicles resemble active-zone transport vesicles and are diminished following synaptogenesis in mature hippocampal slices.

Large dense core vesicles (approximately 100 nm) contain neuroactive peptides and other co-transmitters. Smaller dense core vesicles (approximately 80 nm) are known to contain components of the presynaptic active zone and thought to transport and deliver these components during developmental synaptogenesis. It is not known whether excitatory axons in area CA1 contain such dense core vesicles, and whether they contribute to synaptic plasticity of mature hippocampus. Serial section electron microscopy was used to identify dense core vesicles in presynaptic axons in s. radiatum of area CA1 in adult rat hippocampus. Comparisons were made among perfusion-fixed hippocampus and hippocampal slices that undergo synaptogenesis during recovery in vitro. Dense core vesicles occurred in 26.1+/-3.6% of axonal boutons in perfusion fixed hippocampus, and in only 17.6+/-4.5% of axonal boutons in hippocampal slices (P<0.01). Most of the dense core vesicle positive boutons contained only one dense core vesicle, and no reconstructed axonal bouton had more than a total of 10 dense core vesicles in either condition. Overall the dense core vesicles had average diameters of 79+/-11 nm. These small dense core vesicles were usually located near nonsynaptic membranes and rarely occurred near the edge of a presynaptic active zone. Their size, low frequency, locations, and decrease following recuperative synaptogenesis in slices are novel findings that merit further study with respect to small dense core vesicle content and possible contributions to synapse assembly and plasticity in the mature hippocampus.

Influence of SNP*SNP interaction on BMI in European American adolescents: findings from the National Longitudinal Study of Adolescent Health.

BACKGROUND: Adolescent obesity is predictive of future weight gain, obesity and adult onset severe obesity (body mass index [BMI] ≥40 kg m(-2) ). Despite successful efforts to identify Single Nucleotide Polymorphisms (SNPs) influencing BMI, <5% of the 40-80% heritability of the phenotype has been explained. Identification of gene-gene (G-G) interactions between known variants can help explain this hidden heritability as well as identify potential biological mechanisms affecting weight gain during this critical developmental period. OBJECTIVE: We have recently shown distinct genetic effects on BMI across the life course, and thus it is important to examine the evidence for epistasis in adolescence. METHODS: In adolescent participants of European descent from wave II of the National Longitudinal Study of Adolescent Health (Add Health, n = 5072, ages 12-21, 52.5% female), we tested 34 established BMI-related SNPs for G-G interaction effects on BMI z-score. We used mixed-effects regression, assuming multiplicative interaction models adjusting for age, sex and geographic region, with random effects for family and school. RESULTS: For 28 G-G interactions that were nominally significant (P < 0.05), we attempted to replicate our results in an adolescent sample from the Childhood European American Cohort from Philadelphia. In the replication study, one interaction (PRKD1-FTO) was significant after correction for multiple testing. CONCLUSIONS: Our results are suggestive of epistatic effects on BMI during adolescence and point to potentially interactive effects between genes in biological pathways important in obesity.

The interaction between physical activity and obesity gene variants in association with BMI: Does the obesogenic environment matter?

Little is known about how obesity susceptibility single nucleotide polymorphisms (SNPs) interact with moderate to vigorous physical activity (MVPA) in relation to BMI during adolescence, once obesogenic neighborhood factors are accounted for. In race stratified models, including European (EA; N=4977), African (AA; N=1726), and Hispanic Americans (HA; N=1270) from the National Longitudinal Study of Adolescent to Adult Health (1996; ages 12-21), we assessed the evidence for a SNPxMVPA interaction with BMI-for-age Z score, once accounting for obesogenic neighborhood factors including physical activity amenities, transportation and recreation infrastructure, poverty and crime. Eight SNPxMVPA interactions with suggestive significance (p<0.10; three in each EA, and AA, two in HA) were observed showing attenuation on BMI-for-age Z score in adolescents with ≥5 versus <5 bouts/week MVPA, except for rs10146997 (near NRXN3). Findings were robust to the inclusion of neighborhood-level variables as covariates. These findings suggest that any attenuation from MVPA on a genetic susceptibility to obesity during adolescence is likely not operating through obesogenic neighborhood factors.

Three-dimensional relationships between hippocampal synapses and astrocytes.

Recent studies show that glutamate transporter-mediated currents occur in astrocytes when glutamate is released from hippocampal synapses. These transporters remove excess glutamate from the extracellular space, thereby facilitating synaptic input specificity and preventing neurotoxicity. Little is known about the position of astrocytic processes at hippocampal synapses. Serial electron microscopy and three-dimensional analyses were used to investigate structural relationships between astrocytes and synapses in stratum radiatum of hippocampal area CA1 in the mature rat in vivo and in slices. Only 57 +/- 11% of the synapses had astrocytic processes apposed to them. Of these, the astrocytic processes surrounded less than half (0.43 +/- 22) of the synaptic interface. Other studies suggest that astrocytes extend processes toward higher concentrations of glutamate; thus the presence of astrocytic processes at particular hippocampal synapses might signal which ones are releasing glutamate. The distance between nearest neighboring synapses was usually (approximately 95%) <1 microgram. Astrocytic processes occurred along the extracellular path between 33% of the neighboring synapses, neuronal processes occurred along the path between another 66% of the neighboring synapses, and only 1% of the synapses were close enough such that neither astrocytic nor neuronal processes occurred between them. These morphological arrangements suggest that the glutamate released at approximately two-thirds of hippocampal synapses might diffuse to other synapses, unless neuronal glutamate transporters are more effective than previously reported. The findings also suggest that physiological recordings made from hippocampal astrocytes do not uniformly sample the glutamate released from all hippocampal synapses.

Three-dimensional comparison of ultrastructural characteristics at depressing and facilitating synapses onto cerebellar Purkinje cells.

Cerebellar Purkinje cells receive two distinctive types of excitatory inputs. Climbing fiber (CF) synapses have a high probability of release and show paired-pulse depression (PPD), whereas parallel fiber (PF) synapses facilitate and have a low probability of release. We examined both types of synapses using serial electron microscopic reconstructions in 15-d-old rats to look for anatomical correlates of these differences. PF and CF synapses were distinguishable by their overall ultrastructural organization. There were differences between PF and CF synapses in how many release sites were within 1 microm of a mitochondrion (67 vs 84%) and in the degree of astrocytic ensheathment (67 vs 94%). However, the postsynaptic density sizes for both types of synapses were similar (0.13-0.14 microm(2)). For both types of synapses, we counted the number of docked vesicles per release site to test whether this number determines the probability of release and synaptic plasticity. PF and CF synapses had the same number of anatomically docked vesicles (7-8). The number of docked vesicles at the CF does not support a simple model of PPD in which release of a single vesicle during the first pulse depletes the anatomically docked vesicle pool at a synapse. Alternatively, only a fraction of anatomically docked vesicles may be release ready, or PPD could result from multivesicular release at each site. Similarities in the number of docked vesicles for PF and CF synapses indicate that differences in probability of release are unrelated to the number of anatomically docked vesicles at these synapses.

Systemic embolization complicating right ventricular myocardial infarction.

We describe a patient in whom multiple episodes of pulmonary and systemic embolization occurred in the setting of right ventricular infarction. The mechanism of paradoxical embolization was determined based on transesophageal echocardiographic findings that included right atrial spontaneous contrast, appendage thrombosis, patent foramen ovale with right-to-left atrial shunting, and absence of left ventricular thrombosis. Recurrent thromboembolization was prevented by percutaneous placement of a "clam-shell" occluder across the patent foramen. This clinical scenario may be more common than previously believed and is best detected with transesophageal echocardiography. Early identification of this condition can lead to prompt treatment and prevention of subsequent thromboembolic morbidity.

Mineral spirits inhalation associated with hemolysis, pulmonary edema, and ventricular fibrillation.

A previously healthy 42-year-old woman developed severe dyspnea, chest discomfort, and malaise several hours after prolonged exposure to concentrated vapors from mineral spirits. On the way to the hospital, she sustained a cardiopulmonary arrest; on arrival several minutes later, she was found to be in ventricular fibrillation and was resuscitated. Her hospital course included slowly resolving cardiac abnormalities, amnesia, noncardiogenic pulmonary edema, abrupt hemolytic anemia, sustained rhabdomyolysis, and other metabolic abnormalities. It is highly probable that this syndrome represented acute and near-lethal toxicity caused by the inhalational exposure to the petroleum distillate known as mineral spirits. It is important that physicians be aware of this syndrome in order to recognize it on presentation and to warn patients of the risk of such toxic exposure.

Three-dimensional analysis of the structure and composition of CA3 branched dendritic spines and their synaptic relationships with mossy fiber boutons in the rat hippocampus.

This paper is the third in a series to quantify differences in the composition of subcellular organelles and three-dimensional structure of dendritic spines that could contribute to their specific biological properties. Proximal apical dendritic spines of the CA3 pyramidal cells receiving synaptic input from mossy fiber (MF) boutons in the adult rat hippocampus were evaluated in three sets of serial electron micrographs. These CA3 spines are unusual in that they have from 1 to 16 branches emerging from a single dendritic origin. The branched spines usually contain subcellular organelles that are rarely found in adult spines of other brain regions including ribosomes, multivesicular bodies (MVB), mitochondria, and microtubules. MVBs occur most often in the spine heads that also contain smooth endoplasmic reticulum, and ribosomes occur most often in spines that have spinules, which are small nonsynaptic protuberances emerging from the spine head. Most of the branched spines are surrounded by a single MF bouton, which establishes synapses with multiple spine heads. The postsynaptic densities (PSDs) occupy about 10-15% of the spine head membrane, a value that is consistent with spines from other brain regions, with spines of different geometries, and with immature spines. Individual MF boutons usually synapse with several different branched spines, all of which originate from the same parent dendrite. Larger branched spines and MF boutons are more likely to synapse with multiple MF boutons and spines, respectively, than smaller spines and boutons. Complete three-dimensional reconstructions of representative spines with 1, 6, or 12 heads were measured to obtain the volumes, total surface areas, and PSD surface areas. Overall, these dimensions were larger for the complete branched spines than for unbranched or branched spines in other brain regions. However, individual branches were of comparable size to the large mushroom spines in hippocampal area CA1 and in the visual cortex, though the CA3 branches were more irregular in shape. The diameters of each spine branch were measured along the cytoplasmic path from the PSD to the origin with the dendrite, and the lengths of branch segments over which the diameters remained approximately uniform were computed for subsequent use in biophysical models. No constrictions in the segments of the branched spines were thin enough to reduce charge transfer along their lengths.(ABSTRACT TRUNCATED AT 400 WORDS)

Three-dimensional organization of cell adhesion junctions at synapses and dendritic spines in area CA1 of the rat hippocampus.

Recent work has emphasized the role of adhesion molecules in synaptic plasticity, including long-term potentiation in the hippocampus. Such adhesion molecules are concentrated in junctions that are characterized by dense thickenings on both sides of the junction and are called puncta adhaerentia (PA). Reconstruction from serial electron microscopy was used to determine the location and size of PA in the stratum radiatum of hippocampal area CA1, where many of the previous functional studies have been performed. PAs were found at the edges of synapses on 33% of dendritic spines. The areas occupied by PA were variable across different types of synapses, occupying 0.010+/-0.005 microm2 at macular synapses and 0.034+/-0.031 microm2 at perforated synapses. Another zone, called a vesicle-free transition zone (VFTZ), was identified. Like the PA, this zone also had no presynaptic vesicles and was located at the edges of synapses; however, unlike the PA, the presynaptic thickening was less than the postsynaptic thickening. Together, 45% of spine synapses had PA and/or VFTZ occupying 23+/-11% of the total junctional area between axons and spines. PA also occurred at nonsynaptic sites involving neuronal as well as glial elements. Most (64%) of these PAs occurred between nonsynaptic portions of dendritic spines and neighboring astrocytic processes. Smooth endoplasmic reticulum was often apposed to one or both sides of the synaptic and the nonsynaptic PA. These findings provide further data as a structural basis for understanding the roles of cell adhesion junctions in hippocampal synaptic function and plasticity.

Critical assessment of the involvement of perforations, spinules, and spine branching in hippocampal synapse formation.

Several studies propose that long-term enhancement of synaptic transmission between neurons results from the enlargement, perforation, and splitting of synapses and dendritic spines. Unbiased analyses through serial electron microscopy were used to assess the morphological basis for synapse spilitting in hippocampal area CA1. Few perforated synapses and almost no split (i.e., branched) spines occurred at postnatal day 15, an age of high synaptogenesis; thus, synapse splitting is unlikely to be important during development. The synapse splitting hypothesis predicts an intermediate stage of branched spines with both heads sharing the same presynaptic bouton. Ninety-one branched dendritic spines were traced through serial sections, and the different branches never synapsed with the same presynaptic bouton. Projections from spines, called "spinules," have been thought to extend from perforations in the postsynaptic density (PSD), thereby dividing the presynaptic bouton. Forty-six spinules were traced, and only 13% emerged from perforations in the PSD. Most spinules emerged from the edges of nonperforated PSDs, or from spine necks, where they extended into boutons that were not presynaptic to the spine. In summary, these morphological characteristics are inconsistent with synapse and spine splitting. An alternative is discussed whereby perforated synapses and spinules are transient components of synaptic activation, and branched spines appear from synapses forming in close proximity to one another.

Ultrastructural study of cholecystokinin-immunoreactive cells and processes in area CA1 of the rat hippocampus.

We used light and electron microscopic immunocytochemical methods to examine the structure of neuronal perikarya and processes containing cholecystokinin-like immunoreactivity (CCK-IR) in area CA1 of the rat hippocampus. The morphology of stained perikarya, their positions within all laminae, and the orientation of their dendrites indicate that CCK-IR is located in interneurons. These cells were seen in the electron microscope to have deeply folded nuclei and to receive both symmetric and asymmetric synaptic junctions on their cell somata and dendritic shafts. Their dendrites are essentially spine-free, but form bulges at the site of some asymmetric synaptic junctions. Axonal varicosities containing CCK-IR make symmetric synaptic junctions with cell somata and dendritic shafts of both pyramidal and non-pyramidal neurons. In addition, CCK-IR varicosities form symmetric junctions with unstained non-pyramidal neurons and with CCK-IR cells, suggesting either recurrent innervation of one cell on itself or interaction between interneurons. The presence of CCK-IR varicosities and synaptic junctions on pyramidal cells is in agreement with physiological data which indicate that CCK has a direct postsynaptic action. The observation of CCK-IR varicosities forming synaptic junctions on non-pyramidal cells suggests that CCK might also modify the response of interneurons.

The development of long-term potentiation in hippocampus and neocortex.

The development of long-term potentiation (LTP), an enduring alteration in synaptic efficacy following afferent activation, was examined in CA1 hippocampus and primary visual cortex of rat. Both regions show little LTP prior to postnatal day 5, demonstrate a maximal potentiated response around postnatal day 15, and a subsequent decline to adult levels. These results are discussed with respect to the underlying mechanism of action and behavioral significance of these critical-period phenomena.

Variation in the number, location and size of synaptic vesicles provides an anatomical basis for the nonuniform probability of release at hippocampal CA1 synapses.

Synaptic vesicles, synaptic clefts and postsynaptic areas were measured in three dimensional reconstructions at representative axonal boutons in hippocampal area CA1. Both docked and non-docked vesicles were counted and measured. Small boutons on thin spines had about 2-6 docked vesicles from a pool of more than 200 vesicles. Medium-sized boutons on medium-sized mushroom-shaped dendritic spines contained about 13-16 docked vesicles from a pool of more than 450 vesicles. A large bouton synapsing with a large mushroom-shaped dendritic spine had two clusters of vesicles totaling more than 1000 vesicles. The postsynaptic density was segmented into two discrete zones under the two clusters of vesicles and 36 docked vesicles were distributed over its surfaces. Two multiple-synapse boutons contained more than 500 vesicles with 2-12 docked vesicles observed at each of the two postsynaptic densities on each bouton. This nonuniform number of docked vesicles provides an anatomical basis for the non-uniform probability of release that occurs across hippocampal synapses of different sizes. In addition, the volume of each synaptic vesicle was determined to be 0.4-5.2% of the total volume of the reconstructed synaptic clefts into which they presumably release their contents. However, since each vesicle contains more than 10 times the concentration of glutamate needed to saturate the postsynaptic receptors, these data also support the hypothesis that release a single synaptic vesicle will activate all of the postsynaptic receptors.