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The finding of Alzheimer type II astrocytes, in addition to the pathognomonic combination of laminar cerebrocortical necrosis and eosinophil infiltration, in the brains of pigs is reported for the first time in cases of indirect salt poisoning following water deprivation.

The development of leukemia as a consequence of vector-mediated genotoxicity in gene therapy trials for X-linked severe combined immunodeficiency (SCID-X1) has prompted substantial research effort into the design and safety testing of integrating vectors. An important element of vector design is the selection and evaluation of promoter-enhancer elements with sufficient strength to drive reliable immune reconstitution, but minimal propensity for enhancer-mediated insertional mutagenesis. In this study, we set out to explore the effect of promoter-enhancer selection on the efficacy and safety of human immunodeficiency virus-1-derived lentiviral vectors in γc-deficient mice. We observed incomplete or absent T- and B-cell development in mice transplanted with progenitors expressing γc from the phosphoglycerate kinase (PGK) and Wiscott–Aldrich syndrome (WAS) promoters, respectively. In contrast, functional T- and B-cell compartments were restored in mice receiving an equivalent vector containing the elongation factor-1-α (EF1α) promoter; however, 4 of 14 mice reconstituted with this vector subsequently developed lymphoma. Extensive analyses failed to implicate insertional mutagenesis or γc overexpression as the underlying mechanism. These findings highlight the need for detailed mechanistic analysis of tumor readouts in preclinical animal models assessing vector safety, and suggest the existence of other ill-defined risk factors for oncogenesis, including replicative stress, in gene therapy protocols targeting the hematopoietic compartment.

All MPS-VI cats treated thus far with weekly intravenous enzyme replacement therapy (IV ERT) with recombinant human N -acetylgalactosamine-4-sulphatase (rhASB) from 3 months of age onwards developed circulating anti-rhASB antibodies. In view of this, the possibility of inducing immune tolerance by using a short-course tolerisation regimen was tested. Starting at 4 months of age, MPS-VI ( n = 5) and unaffected cats ( n = 2) received cyclosporine and azathioprine over a 22-day period plus weekly IV ERT with 0.1 mg/kg rhASB. After a 4-week resting period, these cats were administered weekly IV ERT with 1 mg/kg rhASB until 11 or 17 months of age. Four unaffected cats ( n = 4) received weekly IV ERT only. Health, growth and seroconversion were regularly monitored. Four out of five MPS-VI cats tolerated rhASB well, as indicated by negligible or low antibody titres and absence of hypersensitivity reactions. One MPS-VI cat exhibited elevated antibody titres and hypersensitivity reactions during some IV treatments. The two unaffected cats that received the tolerisation regimen remained seronegative, however, only half of the unaffected cats not submitted to this regimen seroconverted. Only minor side-effects were attributed to the short-course of cyclosporine and azathioprine. Two MPS-VI cats also well-tolerated four weekly intrathecal injections of rhASB and consequently exhibited less oligosaccharide fragments in cerebrospinal fluid and less vacuolation within their dura mater. These data indicate that a relatively high rate of immunotolerance towards rhASB can be achieved in MPS-VI cats with a short-course tolerisation regimen ultimately permitting removal of lysosomal storage within the dura mater with the use of intrathecal therapy.

Sheep, particularly lambs, with high circulating levels of Clostridium perfringens type D epsilon toxin develop severe neurologic signs and often die suddenly. On microscopic examination, in the brain, there is microvascular endothelial injury and diffuse vasogenic edema. The aquaporin (AQP) family of membrane water-channel proteins, especially AQP-4, is important in the regulation of water balance in the brain and facilitates reabsorption of excess fluid. In rats given epsilon toxin, generalized cerebral edema was demonstrated by marked albumin extravasation and was correlated with widespread upregulation of AQP-4 in astrocytes. These results suggest that AQP-4 has a role in the clearance of edema fluid from brains damaged by this clostridial toxin.

To study immediate early gene, c-fos, expression as a marker of neural stress after whole of gestation exposure of the fetal mouse brain to mobile telephone-type radiofrequency fields.Using a purpose-designed exposure system at 900 MHz, pregnant mice were given a single, far-field, whole body exposure at a specific absorption rate of 4 W/kg for 60 min/day from day 1 to day 19 of gestation. Pregnant control mice were sham-exposed or freely mobile in a cage without further restraint. Immediately prior to parturition on gestational day 19, fetal heads were collected, fixed in 4% paraformaldehyde and paraffin embedded. Any stress response in the brain was detected by c-fos immunohistochemistry in the cerebral cortex, basal ganglia, thalamus, hippocampus, midbrain, cerebellum and medulla.c-fos expression was of limited, but consistent, neuroanatomical distribution and there was no difference in immunoreactivity between exposed and control brains.In this animal model, no stress response was detected in the fetal brain using c-fos immunohistochemistry after whole of gestation exposure to mobile telephony.

Background: Irinotecan is a common cytotoxic agent used in advanced colorectal cancers. However, a major clinical problem with this cytotoxic is that it causes gastrointestinal mucositis manifest by severe diarrhoea. To date there is no established single dose of irinotecan in rats to enable determination of changes occurring following administration. Therefore, the primary aim of this study was to determine a single dose of irinotecan that induced reproducible gastrointestinal mucositis in DA rats. The secondary aim was to determine if the presence of tumour altered the development of mucositis. Methods: Eighty-eight rats were divided into two groups, 44 received tumours and 44 remained tumour naïve. These were randomized to receive a single dose of irinotecan at 150, 200, 250 or 300 mg/kg. Two control groups of rats received either no treatment or 2 doses of 150 mg/kg irinotecan, shown previously to induce reproducible gastrointestinal mucositis. Rats were monitored closely for incidence and severity of diarrhoea, and mortality, before being killed 48 and 144 h following treatment. Results: Rats administered 250 and 300 mg/kg of irinotecan all developed diarrhoea, and this was associated with high mortality rates (up to 100%). Necropsies revealed that many of these rats had duodenal perforations and fatty lysis consistent with peritonitis. The lower doses of 150 and 200 mg/kg irinotecan also caused diarrhoea, but were not associated with high mortality rates. Histopathological examination confirmed small and large intestinal damage in all rats that received irinotecan, regardless of dose. Tumour-bearing rats had worse diarrhoea and higher mortality compared to tumour-naïve rats. Conclusions: We find that a single dose of 200 mg/kg irinotecan causes reproducible gastrointestinal mucositis as measured by levels of diarrhoea, and small and large intestinal histology. Importantly this dose has a low mortality. The response to irinotecan is more pronounced in tumour-bearing rats.

Summary Aims To study the effect of acute exposure to global system for mobile communication radiofrequency fields on immediate early gene, c-fos , expression in the brain. Methods Using a purpose-designed exposure system at 900 MHz, mice were given a single, far-field, whole body exposure for 60 minutes at a specific absorption rate of 4W/kg. Control mice were sham-exposed or freely mobile in a cage without further restraint. c-fos protein expression was detected immunohistochemically in perfusion-fixed brains. Results Activation of c-fos in exposed and sham-exposed brains was comparable, but was greatly increased compared with freely moving controls. Conclusion These results suggest that the majority of the acute genomic response detected by c-fos expression was due to immobilisation rather than irradiation.

The aim of these studies was to assess and quantitate the effects of cyclosporin-A (CyA) on brain APP messenger RNA and neuronal perikaryal APP antigen expression following controlled focal head impact in sheep. Impact results in a significant increase in both APP mRNA and neuronal perikaryal APP antigen expression. Post-traumatic administration of CyA (intrathecal 10 mg/kg) resulted in a reduction in APP mRNA and neuronal perikaryal antigen expression. At 2 h postinjury, CyA treatment caused a statistically significant (p0.05) 1.3 +/- 0.1-fold decrease in APP mRNA in the central gray matter of impacted sheep compared to untreated impacted sheep. A more profound reduction in APP mRNA synthesis (1.6 +/- 0.2 fold) was evident at 6 h (p0.05). The mean percentage brain area with APP immunoreactive neuronal perikarya at 6 h post-injury was 94.5% in untreated impacted animals, 10.0% in CyA-treated impacted animals, 5.5% in untreated nonimpacted animals, and 6% in CyA-treated non-impacted controls. These results demonstrate that CyA has a downregulatory effect on increased APP expression caused by TBI.

Cortical neuronal and glial c-fos immunoreactivity has been demonstrated in experimental and human brain injury. c-fos is one of the immediate early genes important in signal transduction linking environmental stimuli to the cellular genome. c-fos immunoreactivity was semi-quantitated in a head impact sheep model using a grid system applied to standard coronal brain sections obtained from 12 impacted and 4 control sheep. Substantial glial and neuronal c-fos immunoreactivity was present in the pericontusional (penumbra) region, but was absent or minimal in the core of the contusion. Apart from these focal changes, c-fos immunoreactivity was diffusely distributed, with greater involvement in the cerebrum on the side of impact. In the cerebellum, Bergmann glia showed prominent c-fos immunoreactivity, while Purkinje cells were consistently immunonegative. c-fos immunoreactivity varied in different regions of the brain (focal and diffuse patterns) in this ovine head impact model.

Objective To assess the effect of impact with a nonpene-trating captive bolt pistol in pigs by studying the resulting traumatic brain injury (TBI) and to compare the pathological changes with those found previously in the brains of sheep using a similar experimental paradigm. Procedure The unrestrained heads of six, anaesthetised, 7- to 8-week-old, Large White pigs were impacted in the temporal region with a nonpenetrating captive bolt pistol. Four hours postimpact, brains were perfusion-fixed with 4% paraformaldehyde. Coronal sections from six levels along the brain were cut and stained with haematoxylin and eosin and immunohistochemically for amyloid precursor protein, a sensitive marker of axonal injury (AI) in the brain after trauma. Results TBI in pigs was characterised only by very mild AI, whereas AI in sheep after captive bolt impact to the same head region was much more severe and widely distributed and often associated with vascular damage such as contusions, subarachnoid and intraparenchymal haemorrhage. Conclusions TBI in pigs was much less severe than in sheep after non-penetrating mechanical impact of similar magnitude, confirming the importance of interspecies differences in determining an appropriate physical method of euthanasia.

Animal models have played a critical role in elucidating the complex pathogenesis of traumatic brain injury, the major cause of death and disability in young adults in Western countries. This review discusses how different types of animal models are useful for the study of neuropathologic processes in traumatic, blunt, nonmissile head injury.

Summary 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 immuno-histochemistry 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.

Objective To determine the severity and distribution of structural changes in the brains of adult sheep stunned by penetrating captive bolt. Procedure The unconstrained heads of ten, anaesthetised, unhorned, 2-year-old Merino sheep were impacted at the summit of the head with a penetrating captive bolt pistol. Six sheep were ventilated and four received no respiratory support. Two hours after impact, brains from the six ventilated sheep were perfusion-fixed with 4% paraformaldehyde. Sixteen whole, serial coronal sections from each brain were stained with haematoxylin and eosin and immunohistochemi-cally for amyloid precursor protein, a sensitive marker of axonal and neuronal reaction in the brain after trauma. Pathological changes in these brains were then quantified by morphometric analysis. Results Structural change in all impacted brains was a mixture of focal injury around the wound track and more widely distributed damage in the cerebral hemispheres, cerebellum and brainstem, but varied considerably in severity between individual sheep. All nonventilated sheep died rapidly following respiratory arrest. Conclusions After penetrating captive bolt stunning, damage to the central reticular formation, axonal connections, and the cortical mantle is the likely reason for failure of respiratory control and traumatic loss of consciousness.

A total of 120 E mu-Pim1 heterozygous mice and 120 wild-type mice were exposed for 1 h/day 5 days/week at each of the four exposure levels in "Ferris-wheel" exposure systems for up to 104 weeks to GSM-modulated 898.4 MHz radiation at SARs of 0.25, 1.0, 2.0 and 4.0 W/kg. In addition, 120 heterozygous and 120 wild-type mice were sham-exposed; there was also an unrestrained negative control group. Four exposure levels were used to investigate whether a dose-response effect could be detected. Independent verification confirmed that the exposures in the current study were nonthermal. There was no significant difference in the incidence of lymphomas between exposed and sham-exposed groups at any of the exposure levels. A dose-response effect was not detected. The findings showed that long-term exposures of lymphoma-prone mice to 898.4 MHz GSM radiofrequency (RF) radiation at SARs of 0.25, 1.0, 2.0 and 4.0 W/kg had no significant effects when compared to sham-irradiated animals. A previous study (Repacholi et al., Radiat. Res. 147, 631-640, 1997) reported that long-term exposure of lymphoma-prone mice to one exposure level of 900 MHz RF radiation significantly increased the incidence of non-lymphoblastic lymphomas when compared to sham-irradiated animals.

Broadly neutralizing antibodies (bNAbs) are a consistent protective immune correlate in human immunodeficiency virus (HIV) patients as well as in passive immunotherapy studies. The inability to elicit bNAbs is the core reason underlining the repeated failures in traditional HIV vaccine research. Rare monoclonal bNAbs against HIV, however, have been produced. The significance of producing and studying more monoclonal bNAbs against HIV is underlined by its capability of defining critical epitopes for antigen designs aimed at the development of a serum-neutralizing HIV vaccine. In this regard, traditional antigen preparations have failed. There is a need to clearly advocate the concept, and systematic study, of more sophisticated 'designer antigens' (DAGs), which carry epitopes that can lead to the elicitation of bNAbs. Using an extremely efficient cell-to-cell HIV infection model for the preparation of HIV prefusion intermediates, we have investigated a novel and systematic approach to produce (not screen for) potential bNAbs against HIV. We have established the concept and the experimental system for producing formaldehyde-fixed HIV DAGs that carry temperature-arrested prefusion intermediates. These prefusion intermediates are structures on the cell surface after viral attachment and receptor engagement but before fully functional viral entry. Using defined HIV prefusion DAGs, we have produced monoclonal antibodies (mAbs) specific to novel epitopes on HIV prefusion intermediates. These mAbs do not react with the static/native surface HIV or cellular antigens, but react with the DAGs. This is a paradigm shift from the current mainstream approach of screening elite patients' bNAbs.

The heads of anaesthetized lambs aged 4–5 weeks were subjected to impact (temporal, frontal or occipital) of constant strength with a humane stunner. Two hours later, the brains were perfusion-fixed with 4% paraformaldehyde and serial whole coronal slices processed by routine methods. Sections were stained with haematoxylin and eosin or labelled with a monoclonal antibody to amyloid precursor protein, a sensitive marker of axonal injury and neuronal reaction. Microscopical evaluation of axonal, neuronal and vascular damage was performed with a quantitative grid system. Frontal impact produced the greatest damage, followed by occipital then temporal impact. An unusual lesion found in the majority of lambs subjected to impact was multifocal necrosis of the cerebellar granular layer. The findings should assist clinicians in evaluating the probable outcome of traumatic head injury in domestic animals.

Amyloid precursor protein (APP), a membrane spanning glycoprotein which plays an important role in neuronal growth and synaptic plasticity, is increased after traumatic brain injury (TBI) and has been used as a sensitive marker of neuronal damage in an adult sheep head impact model. We hypothesised that APP expression would similarly be increased in lambs, suggesting that in the immature injured brain APP is also upregulated as an acute phase response to trauma. Ten anaesthetised and ventilated 4-5 week old Merino lambs sustained a left temporal head impact from a humane stunner. At 2 h after impact, there was widespread and intense neuronal cell body APP immunoreactivity which was more widely distributed than axonal APP. APP messenger RNA (mRNA) expression was also markedly increased with a distribution similar to that of APP antigen. These results demonstrate that APP antigen and mRNA are sensitive early indicators of TBI in paediatric cases.

Vasogenic cerebral oedema (VCO) was induced in Hooded Wistar rats by intraperitoneal injection of Clostridium perfringens type D epsilon prototoxin. Animals were killed, 1 h to 14 d postinjection, by perfusion fixation under general anaesthesia. VCO was detected by the presence of endogenous albumin in the brain, visualised by immunocytochemistry. As early as 1 h postinjection, albumin was detected in the walls of cerebral micro vessels. Maximal diffuse leakage within the neural parenchyma was seen at 24 and 48 h and immunoreactivity was still present at 4 d. At 7 d only few foci were seen, and at 14 d albumin distribution was similar to that in controls. Ultrastructural assessment of the microvessels showed swelling of many astrocytic processes and abnormalities of the endothelial cells varying from swelling with loss of cytoplasmic organelles to cells showing increased electron density. Immunostaining for the endothelial barrier antigen (EBA) showed strongly immunoreactive vessels throughout normal brains. Experimental animals showed partial reduction in EBA expression, most evident at 24 and 48 h, with gradual recovery to normal by 14 d. The exact role that EBA plays in the intact BBB remains obscure.

In a blunt, nonmissile, head impact model of traumatic brain injury in 4-5-week-old Merino lambs, multiple foci of internal granular layer necrosis were found in all 10 impacted animals. This lesion has not previously been reported after human or animal head injury. Temporal lobe impact contusions, predomi- nantly microscopic (8/10) and contralateral contusions (2/10), parenchymal (10/10) and subarachnoid (10/10) hemorrhage, and widely distributed axonal injury were also observed. Although the precise pathogenesis of this focal granule cell necrosis and often attendant red cell change in Purkinje cells was unclear, an ischemic etiology due to trauma-related vascular damage is postulated. 1 its role in pediatric head- injured patients has been less studied. 5 Accordingly, a pre- viously described adult ovine closed head injury model 6 (an impact-generated rotational acceleration of a relatively large gyrencephalic brain in an unconstrained head) was modified to study axonal injury in lambs as an experimental model of pediatric head injury. 4 In this model, all 10 head-impacted lambs showed numerous foci of internal granular layer ne- crosis. This cerebellar lesion was not present in nonimpacted control lambs or in similarly head-injured adult sheep. 6 Anesthetized and ventilated 4-5-week-old Merino lambs were placed in a sphinx position with their heads supported to permit free rotational and lateral movement of the head after impact. A humane stunner (model MKL, Karl Schermer & Co., Karlsruhe, Germany) was aligned with the left temporal fossa, and a constant charge (Schermer No. 11) was used to impact these animals. This mechanical device, approved for humane euthanasia of livestock, 3 uses a charge to propel a captive bolt with an attached, heavy, mushroom-shaped head against the skull. Continuous physiological monitoring for mean arterial blood pressure, intracranial pressure, cerebral blood flow, and blood gases ensured that no hypoxic or hypotensive episodes supervened during the experimental period. Two hours after impact, the brain was perfusion fixed with 4% paraformalde- hyde and then postfixed in formalin for 7 days. Brains were sectioned at 5-mm coronal intervals, embedded in paraffin, and cut and stained with hematoxylin and eosin (HE). Selected small areas of cerebellum were also removed from paraffin blocks, embedded in plastic, cut at 1 m, and stained with toluidine blue. Four control lambs were not impacted but were otherwise subjected to the same experimental protocol. At necropsy, two lambs had fractures of the outer table of the skull, two lambs exhibited depressed fractures, and in all other lambs the inner and outer tables of the skull remained intact. Eight lambs had microscopic impact contusions, and two had contralateral contusions. Microscopically, widely distrib- uted (multifocal) amyloid precursor protein-positive axonal in- jury was found in impacted and contralateral hemispheres and bilaterally in brain stem and cerebellum. Parenchymal and sub- arachnoid hemorrhage was also similarly distributed. Acute multifocal internal granular layer necrosis occurred in all 10 impacted lambs. These necrotic foci were usually small, well circumscribed, and round to ovoid in shape (Fig. 1) or sometimes more extensive and irregular in outline. They were randomly distributed with no discernible predilection for the crests or sulci of the cerebellar folia and often in- volved the full thickness of the granular layer. The granule cell nuclei were markedly condensed, and the cytoplasm ei- ther greatly reduced and paler staining or the hyperchromatic nuclei were disposed in a nonstaining matrix without visible cytoplasm. Necrotic foci had a remarkably similar appear- ance in routinely stained paraffin-embedded sections 2 hours after impact, were well demarcated from adjacent normal compact granular layer (Fig. 1), and sometimes contained hemorrhage. In semithin sections stained with toluidine blue (Fig. 2), damaged granule cell nuclei were condensed with still discernible coarse chromatin clumps or pyknotic, small, and uniformly hyperchromatic. The granule cell cytoplasm was expanded, clear, and poorly delimited. Contiguous Pur- kinje cells showed ischemic (red cell) change with pyknotic nuclei and shrunken, angular, and hypereosinophilic cyto- plasm, exhibited less severe cytoplasmic shrinkage and eo- sinophilia than frankly necrotic neurons, or were apparently unaffected. In plastic-embedded sections, necrotic Purkinje cells were shrunken and darkly stained (Fig. 2), with often fine cytoplasmic vacuolation. There was sometimes edema- tous distension of the layer dissecans, and a few normally inconspicuous Bergmann's glia had swollen, vesicular nuclei and slightly enlarged, clear cytoplasm. Small blood vessels in and immediately adjacent to these necrotic foci in the granular layer were frequently occluded by tightly packed erythrocytes, whereas vessels in the surrounding unaffected parenchyma were normally perfused or contained a few re- sidual unflushed erythrocytes. In all control lambs, the cer- ebellum was microscopically normal and there were no other neuropathologic changes.

This paper reports neuronal changes in rat brains subacutely intoxicated with Copyright Clostridium perfringens type D epsilon toxin. Neuronal damage was characterized by either (1) progressive cytoplasmic vacuolation leading to necrosis, or (2) shrunken hyperchromatic cells with nuclear pyknosis. The neuronal injury was also often bilaterally symmetrical, particularly in the brainstem. These findings suggest that, after gaining access to brain tissue by producing an increase in vascular permeability, epsilon toxin later exerts a directly cytotoxic effect on neurons.

Sir,We previously published a paper in Pathology examining vascular permeability changes in the fetal brain after whole of gestation exposure of pregnant mice to mobile communication radiofrequency...

exposure to hypoxia and doxorubicin. Cell death, as measured by flow cytometric analysis of Annexin V and Propidium Iodide staining, was reduced in the presence of each type of CM. Conclusions: Conditioned media from MSCs had both mitogenic and anti-apoptotic effects on cardiomyocytes and endothelial cells, with STRO-1Bright CM performing especially well. This further supports the evidence that paracrine actions contribute to the cardiac reparative properties of MSCs. doi:10.1016/j.hlc.2007.06.071

Alpha-mannosidosis is a lysosomal storage disorder resulting from deficient activity of lysosomal alpha-mannosidase. It has been described previously in humans, cattle, and cats, and is characterized in all of these species principally by neuronal storage leading to progressive mental deterioration. Two guinea pigs with stunted growth, progressive mental dullness, behavioral abnormalities, and abnormal posture and gait, showed a deficiency of acidic alpha-mannosidase activity in leukocytes, plasma, fibroblasts, and whole liver extracts. Fractionation of liver demonstrated a deficiency of lysosomal (acidic) alpha-mannosidase activity. Thin layer chromatography of urine and tissue extracts confirmed the diagnosis by demonstrating a pattern of excreted and stored oligosaccharides almost identical to that of urine from a human alpha-mannosidosis patient. Widespread neuronal vacuolation was observed throughout the CNS, including the cerebral cortex, hippocampus, thalamus, cerebellum, midbrain, pons, medulla, and the dorsal and ventral horns of the spinal cord. Lysosomal vacuolation also occurred in many other visceral tissues and was particularly severe in pancreas, thyroid, epididymis, and peripheral ganglion. Axonal spheroids were observed in some brain regions, but gliosis and demyelination were not observed. Ultrastructurally, most vacuoles in both the CNS and visceral tissues were lucent or contained fine fibrillar or flocculent material. Rare large neurons in the cerebral cortex contained fine membranous structures. Skeletal abnormalities were very mild. Alpha-mannosidosis in the guinea pig closely resembles the human disease and will provide a convenient model for investigation of new therapeutic strategies for neuronal storage diseases, such as enzyme replacement and gene replacement therapies.

Whether radiofrequency (RF) fields are carcinogenic is controversial; epidemiological data have been inconclusive and animal tests limited. The aim of the present study was to determine whether long-term exposure to pulse-modulated RF fields similar to those used in digital mobile telecommunications would increase the incidence of lymphoma in E mu-Pim1 transgenic mice, which are moderately predisposed to develop lymphoma spontaneously. One hundred female E mu-Pim1 mice were sham-exposed and 101 were exposed for two 30-min periods per day for up to 18 months to plane-wave fields of 900 MHz with a pulse repetition frequency of 217 Hz and a pulse width of 0.6 ms. Incident power densities were 2.6-13 W/m2 and specific absorption rates were 0.008-4.2 W/kg, averaging 0.13-1.4 W/kg. Lymphoma risk was found to be significantly higher in the exposed mice than in the controls (OR = 2.4. P = 0.006, 95% CI = 1.3-4.5). Follicular lymphomas were the major contributor to the increased tumor incidence. Thus long-term intermittent exposure to RF fields can enhance the probability that mice carrying a lymphomagenic oncogene will develop lymphomas. We suggest that such genetically cancer-prone mice provide an experimental system for more detailed assessment of dose-response relationships for risk of cancer after RF-field exposure.

The hepatotoxicity of tunicamycin was studied in 8 to 10-week-old guinea pigs. Acute hepatic damage was produced consistently in guinea pigs given a single dose of 400 micrograms/kg of tunicamycin and observed at intervals up to 72 h post-injection. Significant elevations occurred in serum levels of liver enzymes and ammonia, while concentrations of serum proteins were lowered. A periportal pattern of hepatocellular damage, with death of many hepatocytes, was consistently observed by 72 h. Severe vacuolation of hepatocytes resulted from lipid accumulation and dilatation of cisternae of rough endoplasmic reticulum, and bile ductule hyperplasia was also observed. Swollen hepatocyte cytoplasm protruded into many hepatic blood vessels, and detached portions of hepatocytes produced emboli in pulmonary and cerebral capillaries, thus contributing to capillary occlusion in the brains of treated guinea pigs.

The distribution, severity and frequency of brain lesions produced in mice by the administration of Clostridium perfringens Type D epsilon toxin were examined by light microscopy. The granular layer of the cerebellum was the area most frequently affected in mice given single doses of toxin. Sequential changes in brain morphology were examined from 1 h to 7 days after injection of toxin. Lesions progressed from an initial vasogenic oedema to malacic foci which commonly were focal and bilaterally symmetrical, with a predilection for white matter. The topographical distribution of these malacic areas is discussed.

Mice were given lethal and sublethal doses of Clostridium perfringens Type D epsilon toxin and the early morphological changes in perfusion-fixed intoxicated brains were examined from 30 min to 6 h post-inoculation. The initial ultrastructural finding was swelling of astrocytes, especially the perivascular extensions of these cells. Astrocytes in the cerebellum appeared to be particularly sensitive to this toxin. These changes were quickly followed by evidence of severe endothelial damage, with the endothelial cytoplasm becoming attenuated, vacuolated and very electron-dense. A pathogenetic sequence of events leading to malacia, derived from ultrastructural observations, is proposed.

Axonal injury (AI), an important determinant of clinical outcome after traumatic head injury in adults, has been little studied in child neurotrauma. In this experimental model, 10 anaesthetised, ventilated and physiologically monitored 4–5 week old lambs sustained a temporal impact to an unconstrained head. Examination of the brain 2 hours post-impact using amyloid precursor protein immunostaining, a sensitive marker of axonal damage, revealed widespread multifocal AI in the ipsilateral and contralateral hemispheres, brain stem and cerebellum in all cases. Impact contusions (predominantly microscopic) ( 8 10 ), contralateral contusions ( 2 10 ), parenchymal haemorrhage ( 10 10 ) and focal subarachnoid haemorrhage ( 10 10 ) were also present. Unusual multifocal cerebellar granular layer necrosis occurred in all impacted lambs. The pattern of AI in these lamb brains 2 hours post-impact was similar to that found in adult sheep using the same experimental paradigm.

// Melissa R. Pitman 1 , Jason A. Powell 1, 3 , Carl Coolen 1 , Paul A.B. Moretti 1 , Julia R. Zebol 1 , Duyen H. Pham 1 , John W. Finnie 4, 5 , Anthony S. Don 6 , Lisa M. Ebert 1, 3 , Claudine S. Bonder 1, 2, 3 , Briony L. Gliddon 1 , Stuart M. Pitson 1, 2, 3, * 1 Centre for Cancer Biology, University of South Australia and SA Pathology, Adelaide, SA 5000, Australia 2 School of Molecular and Biomedical Science, University of Adelaide, SA 5005, Australia 3 School of Medicine, University of Adelaide, SA 5005, Australia 4 School of Veterinary Science, University of Adelaide, SA 5005, Australia 5 SA Pathology, Hanson Institute Centre for Neurological Diseases, Adelaide, SA 5000, Australia 6 Prince of Wales Clinical School, University of New South Wales, Sydney, NSW 2052, Australia Correspondence to: Stuart M. Pitson, e-mail: stuart.pitson@unisa.edu.au Keywords: Apoptosis, in silico docking, molecular modeling, small molecule inhibitor, sphingosine kinase Received: September 05, 2014 Accepted: January 25, 2015 Published: March 11, 2015 ABSTRACT The dynamic balance of cellular sphingolipids, the sphingolipid rheostat, is an important determinant of cell fate, and is commonly deregulated in cancer. Sphingosine 1-phosphate is a signaling molecule with anti-apoptotic, pro-proliferative and pro-angiogenic effects, while conversely, ceramide and sphingosine are pro-apoptotic. The sphingosine kinases (SKs) are key regulators of this sphingolipid rheostat, and are attractive targets for anti-cancer therapy. Here we report a first-in-class ATP-binding site-directed small molecule SK inhibitor, MP-A08, discovered using an approach of structural homology modelling of the ATP-binding site of SK1 and in silico docking with small molecule libraries. MP-A08 is a highly selective ATP competitive SK inhibitor that targets both SK1 and SK2. MP-A08 blocks pro-proliferative signalling pathways, induces mitochondrial-associated apoptosis in a SK-dependent manner, and reduces the growth of human lung adenocarcinoma tumours in a mouse xenograft model by both inducing tumour cell apoptosis and inhibiting tumour angiogenesis. Thus, this selective ATP competitive SK inhibitor provides a promising candidate for potential development as an anti-cancer therapy, and also, due to its different mode of inhibition to other known SK inhibitors, both validates the SKs as targets for anti-cancer therapy, and represents an important experimental tool to study these enzymes.