Jim Manavis

Stalning af amyloid percursor protein to study axonal damage in mild head Injury

The most common definition of cerebral concussion is that of a transient loss of neurological function without macroscopic or microscopic abnormality in the brain. However, some patients have persistent symptoms and subtle neuropsychological deficits, particularly affecting memory. We have studied five patients aged 59-89 years who sustained mild concussive head injury and died of other causes (2-99 days post-injury). Immunostaining with an antibody to amyloid precursor protein, a marker of fast axonal transport, showed multifocal axonal injury in all five. All had axonal damage in the fornices, which are important in memory function.

Elevated Serum Levels of Stromal-Derived Factor-1α Are Associated with Increased Osteoclast Activity and Osteolytic Bone Disease in Multiple Myeloma Patients

Multiple myeloma (MM) is an incurable plasma cell (PC) malignancy able to mediate massive destruction of the axial and craniofacial skeleton. The aim of this study was to investigate the role of the potent chemokine, stromal-derived factor-1α (SDF-1α) in the recruitment of osteoclast precursors to the bone marrow. Our studies show that MM PC produce significant levels of SDF-1α protein and exhibit elevated plasma levels of SDF-1α when compared with normal, age-matched subjects. The level of SDF-1α positively correlated with the presence of multiple radiological bone lesions in individuals with MM, suggesting a potential role for SDF-1α in osteoclast precursor recruitment and activation. To examine this further, peripheral blood–derived CD14+ osteoclast precursors were cultured in an in vitro osteoclast-potentiating culture system in the presence of recombinant human SDF-1α. Although failing to stimulate an increase in TRAP+, multinucleated osteoclast formation, our studies show that SDF-1α mediated a dramatic increase in both the number and the size of the resorption lacunae formed. The increased osteoclast motility and activation in response to SDF-1α was associated with an increase in the expression of a number of osteoclast activation–related genes, including RANKL, RANK, TRAP, MMP-9, CA-II, and Cathepsin K. Importantly, the small-molecule CXCR4-specific inhibitor, 4F-Benzoyl-TE14011 (T140), effectively blocked osteoclast formation stimulated by the myeloma cell line, RPMI-8226. Based on these findings, we believe that the synthesis of high levels of SDF-1α by MM PC may serve to recruit osteoclast precursors to local sites within the bone marrow and enhance their motility and bone-resorbing activity. Therefore, we propose that inhibition of the CXCR4-SDF-1α axis may provide an effective means of treatment for MM-induced osteolysis.

FUS mutations in amyotrophic lateral sclerosis: clinical, pathological, neurophysiological and genetic analysis

Objective FUS gene mutations were recently identified in familial amyotrophic lateral sclerosis (ALS). The present studies sought to define the clinical, post-mortem and neurophysiological phenotypes in ALS families with FUS mutations and to determine the frequency of FUS mutations in familial and sporadic ALS. Methods FUS was screened for mutations in familial and sporadic ALS cases. Clinical, post-mortem and neurophysiological features of large families with FUS mutations are described. Results and conclusions FUS mutations were evident in 3.2% (4/124) of familial ALS, representing the second most common gene abnormality to be described in familial ALS after SOD1. No mutations were present in 247 sporadic ALS cases. The clinical presentation in 49 affected patients was consistent with a predominantly lower motor neuron disorder, supported by post-mortem findings. Upper motor neuron involvement varied, with Wallerian degeneration of corticospinal tracts present in one post-mortem case but absent in a second case from the same family. Features of cortical hyperexcitability demonstrated upper motor neuron involvement consistent with other forms of familial and sporadic ALS. One case presented with frontotemporal dementia (FTD) indicating that this may be a rare presenting feature in families with FUS mutation. Ubiquitin-positive cytoplasmic skein-like inclusions were present in lower motor neurons, but in contrast to sporadic ALS, no TDP-43 pathology was evident. Mutation-specific clinical features were identified. Patients with a R521C mutation were significantly more likely to develop disease at a younger age, and dropped-head syndrome was a frequent feature. Reduced disease penetrance was evident among most affected families.

Early Expression and Cellular Localization of Proinflammatory Cytokines Interleukin-1β, Interleukin-6, and Tumor Necrosis Factor-α in Human Traumatic Spinal Cord Injury

Study Design. Post-traumatic inflammatory response was studied in 11 human cases of acute spinal cord contusion injury. Objectives. To examine the inflammatory cellular response and the immunocytochemical expression and localization of interleukin-1β, internleukin-6, and tumor necrosis factor-&agr;in human spinal cord after contusion injury. Summary of Background Data. The post-traumatic inflammatory response plays an important role in secondary injury mechanisms after spinal cord injury, and inter-leukin-1β, internleukin-6, and tumor necrosis factor-&agr; are key inflammatory mediators. Methods. The study group comprised 11 patients with spinal cord contusion injury and 2 normal individuals. Histologic and immunocytochemical assessments were undertaken to evaluate the inflammatory cellular response and the immunoexpression of interleukin-1β, internleukin-6, and tumor necrosis factor-&agr; in the injured human spinal cord. The cellular sources of interleukin-1β, internleukin-6, and tumor necrosis factor-&agr; were elucidated by immunofluorescence double-labeled confocal imaging. Results. Increased immunoreactivity of interleukin-1β, internleukin-6, and tumor necrosis factor-&agr;was detected in neurons 0.5 hour after injury, and in neurons and microglia 5 hours after injury, but the expression of these proinflammatory cytokines was short-lived and declined sharply to baseline by 2 days after injury. In the inflammatory cellular response, as early as 0.5 hour after spinal cord injury, activated microglia were detected, and axonal swellings and axons were surrounded by microglial processes. Numerous neutrophils appeared in the injured cord 1 day after injury, and then their number declined dramatically, whereas macrophages progressively increased after day 1. Conclusions Endogenous cells (neurons and microglia) in the human spinal cord, not the blood-borne leukocytes, contribute to the early production of interleukin-1β, interleukin-6, and tumor necrosis factor-&agr; in the post-traumatic inflammatory response, and microglia are involved the early response to traumatic axonal injury.

Severity-dependent expression of pro-inflammatory cytokines in traumatic spinal cord injury in the rat.

The post-traumatic inflammatory response in acute spinal cord contusion injury was studied in the rat. Mild and severe spinal cord injury (SCI) was produced by dropping a 10 g weight from 3 and 12 cm at the T12 vertebral level. Increased immunoreactivity of TNF-alpha in mild and severe SCI was detected in neurons at 1 h post-injury, and in neurons and microglia at 6 h post-injury, with a less significant increase in mild SCI. Expression was short-lived and declined sharply by 1 d post-injury. RT-PCR showed an early significant up-regulation of IL-1 beta, IL-6 and TNF-alpha mRNAs, maximal at 6 h post-injury with return to control levels by 24 h post-injury, the changes being less statistically significantly in mild SCI. Western blot showed early transient increases of IL-1 beta, IL-6 and TNF-alpha proteins in severe SCI but not mild SCI. Immunocytochemical, western blotting and RT-PCR analyses suggest that endogenous cells (neurons and microglia) in the spinal cord, not blood-borne leucocytes, contribute to IL-1 beta, IL-6 and TNF-alpha production in the post-traumatic inflammatory response and that their up-regulation is greater in severe than mild SCI.

Electrophysiological, Electroanatomical, and Structural Remodeling of the Atria as Consequences of Sustained Obesity

BACKGROUND Obesity and atrial fibrillation (AF) are public health issues with significant consequences. OBJECTIVES This study sought to delineate the development of global electrophysiological and structural substrate for AF in sustained obesity. METHODS Ten sheep fed ad libitum calorie-dense diet to induce obesity over 36 weeks were maintained in this state for another 36 weeks; 10 lean sheep with carefully controlled weight served as controls. All sheep underwent electrophysiological and electroanatomic mapping; hemodynamic and imaging assessment (echocardiography and dual-energy x-ray absorptiometry); and histology and molecular evaluation. Evaluation included atrial voltage, conduction velocity (CV), and refractoriness (7 sites, 2 cycle lengths), vulnerability for AF, fatty infiltration, atrial fibrosis, and atrial transforming growth factor (TGF)-β1 expression. RESULTS Compared with age-matched controls, chronically obese sheep demonstrated greater total body fat (p < 0.001); LA volume (p < 0.001); LA pressure (p < 0.001), and PA pressures (p < 0.001); reduced atrial CV (LA p < 0.001) with increased conduction heterogeneity (p < 0.001); increased fractionated electrograms (p < 0.001); decreased posterior LA voltage (p < 0.001) and increased voltage heterogeneity (p < 0.001); no change in the effective refractory period (ERP) (p > 0.8) or ERP heterogeneity (p > 0.3). Obesity was associated with more episodes (p = 0.02), prolongation (p = 0.01), and greater cumulative duration (p = 0.02) of AF. Epicardial fat infiltrated the posterior LA in the obese group (p < 0.001), consistent with reduced endocardial voltage in this region. Atrial fibrosis (p = 0.03) and TGF-β1 protein (p = 0.002) were increased in the obese group. CONCLUSIONS Sustained obesity results in global biatrial endocardial remodeling characterized by LA enlargement, conduction abnormalities, fractionated electrograms, increased profibrotic TGF-β1 expression, interstitial atrial fibrosis, and increased propensity for AF. Obesity was associated with reduced posterior LA endocardial voltage and infiltration of contiguous posterior LA muscle by epicardial fat, representing a unique substrate for AF.

Upregulation of Amyloid Precursor Protein Messenger RNA in Response to Traumatic Brain Injury: An Ovine Head Impact Model

There is evidence that the amyloid precursor protein (APP) plays an important role in neuronal growth and synaptic plasticity and that its increased expression following traumatic brain injury represents an acute phase response to trauma. We hypothesized that the previously described increased APP expression in response to injury (Van den Heuvel et al., Acta Neurochir. Suppl. 71, 209-211) is due to increased mRNA expression and addressed this by examining the expression of APP mRNA and APP within neuronal cell bodies over time in an ovine head impact model. Twenty-five anesthetized and ventilated 2-year-old Merino ewes sustained a left temporal head impact using a humane stunner and 9 normal sheep were used as nonimpact controls. Following postimpact survival periods of 15, 30, 45, 60, and 120 min, brains were perfusion fixed in 4% paraformaldehyde and examined according to standard neuropathological protocol. APP mRNA and antigen expression were examined in 5-microm sections by nonisotopic in situ hybridization and APP immunocytochemistry. The percentage of brain area with APP immunoreactivity within neuronal cell bodies in the impacted animals increased with time from a mean of 7.5% at 15 min to 54.5% at 2 h. Control brains showed only very small numbers of weakly APP-positive neuronal cell bodies ranging from 2 to 14% (mean 7%). Increased expression of APP mRNA was first evident in impacted hemispheres at 30 min after impact and progressively increased over time to involve neurons in all sampled regions of the brain, suggesting increased transcription of APP. In contrast, APP mRNA was undetectable in tissue from nonimpacted sheep. These data show that APP mRNA and antigen expression are sensitive early indicators of neuronal injury with widespread upregulation occurring as early as 30 min after head impact.

Correlation of mismatch repair genes immunohistochemistry and microsatellite instability status in HNPCC-associated tumours

Aim: The aim of this study was to assess the performance of immunohistochemistry using antibodies for MLH1, MSH2, MSH6 and PMS2 mismatch repair gene proteins against microsatellite instability (MSI) testing. Methods: Tumour samples included in this study were derived from referred patients for screening for hereditary non‐polyposis colorectal cancer (HNPCC) and patients who had resections for colorectal cancer that were examined at our institution. MSI was assessed at nine loci (BAT25, BAT26, BAT40, D2S123, D10S197, D17S579, D18S34, D5S346 and D17S250) in all cases. Immunohistochemistry for MLH1 and MSH2 was performed in all cases. Staining for MSH6 and PMS2 was performed in selected cases only. Results: There were 742 tumours including 661 colorectal lesions and 81 extracolonic tumours of the HNPCC spectrum. Among the 555 MSI‐negative tumours, 554 showed an intact protein expression. Amongst the 187 MSI‐positive tumours, 126 showed abnormal expression of MLH1 gene protein, 41 showed abnormal expression of MSH2 gene, three showed abnormal expression of MSH6 only, one showed abnormal expression of PMS2 gene protein only and one case showed abnormal expression of all four proteins. Conclusion: Immunohistochemistry offers an alternative method for assessment of MSI status which is fast and relatively inexpensive compared with MSI testing. We achieved a sensitivity rate of 92% and specificity of 99.8% for immunohistochemistry testing assessed against the MSI testing. It has to be accepted that a small fraction of MSI‐positive cases will be missed by testing with immunohistochemistry alone.

Human Adult Dental Pulp Stem Cells Enhance Poststroke Functional Recovery Through Non-Neural Replacement Mechanisms

Human adult dental pulp stem cells (DPSCs), derived from third molar teeth, are multipotent and have the capacity to differentiate into neurons under inductive conditions both in vitro and following transplantation into the avian embryo. In this study, we demonstrate that the intracerebral transplantation of human DPSCs 24 hours following focal cerebral ischemia in a rodent model resulted in significant improvement in forelimb sensorimotor function at 4 weeks post‐treatment. At this time, 2.3 ± 0.7% of engrafted cells had survived in the poststroke brain and demonstrated targeted migration toward the stroke lesion. In the peri‐infarct striatum, transplanted DPSCs differentiated into astrocytes in preference to neurons. Our data suggest that the dominant mechanism of action underlying DPSC treatment that resulted in enhanced functional recovery is unlikely to be due to neural replacement. Functional improvement is more likely to be mediated through DPSC‐dependent paracrine effects. This study provides preclinical evidence for the future use of human DPSCs in cell therapy to improve outcome in stroke patients.

Effect of long-term mobile communication microwave exposure on vascular permeability in mouse brain

Aims: To study the effect of long‐term exposure to global system for mobile communication (GSM) radiofrequency fields on vascular permeability in murine brains. Methods: Using a purpose‐designed exposure system at 900 MHz, mice were given a 60‐minute far‐field, whole body exposure on each of 5 days per week for 104 weeks at specific absorption rates (SAR) of 0.25, 1.0, 2.0 and 4.0 W/kg. Control mice were sham‐exposed or permitted free movement in a cage to evaluate any stress‐related effects. Albumin immunohistochemistry was used to detect increased vascular permeability and the efficacy of the vascular tracer was confirmed with a positive control group exposed to a clostridial toxin known to increase vascular permeability in the brain. Results: In all exposed and control groups, albumin extravasation was minimal, often leptomeningeal, and was deemed insignificant as a maximum of three capillaries or venules in a given brain showed leakage from the very many blood vessels present in the three coronal brain sections. Conclusions: These results suggest that prolonged exposure to mobile telephone‐type radiation produces negligible disruption to blood‐brain barrier integrity at the light microscope level using endogenous albumin as a vascular tracer.Abbreviations: BBB, blood-brain barrier; GSM, global system for mobile communication; RF, radiofrequency; SAR, specific absorption rate.