A genome screen in multiple sclerosis reveals susceptibility loci on chromosome 6p21 and 17q22.

The population prevalence of multiple sclerosis is 0.1%; however, the risk of the disease in the siblings of affected individuals is very much higher at 3-5%. The importance of genetic factors in accounting for this increased risk is confirmed by the results of twin and adoption studies. Despite the evidence for a strong genetic effect, a weak major histocompatibility complex (MHC) association is the only consistently observed feature in the genetics of multiple sclerosis. Other candidates have been proposed, including genes encoding the immunoglobulin heavy chain, T cell receptor beta chain and APOC2, but none has yet been confirmed. Evidence for linkage and association to the myelin basic protein gene has been reported in a genetically isolated Finnish population, but it has not been possible to reproduce these results in other populations. We used a two-stage approach to search the human genome for the genes causing susceptibility to multiple sclerosis. Two principal regions of linkage are identified, chromosomes 17q22 and 6p21 (MHC). Our results are compatible with genetic models involving epistatic interaction between these and several additional genes.

Frailty as an indicator of postoperative complications following surgical excision of non-melanoma skin cancer on the head and neck.

INTRODUCTION: Non-melanoma skin cancer (NMSC) predominantly affects those aged over 90 years, with 85% of lesions arising on the head and neck, where surgical excision remains the treatment of choice. Frailty is a measure of physiologic age and can be used as a predictor of adverse treatment outcomes. The aim of this study was to determine if the Rockwood Frailty Index is predictive of complications following excision of NMSC. METHODS: Data were collected prospectively for patients who underwent an excision of a suspected NMSC from the head or neck across a two-month period. Details of the patient, lesion and procedure were recorded alongside ASA grade and Rockwood's Frailty score. Postoperative complications were recorded four weeks later. RESULTS: There was a total of 125 patients: 74 (60%) male, 51 (40%) female; mean age was 78 (±9.8) years. Of the excised sites, 61% were closed primarily, 26% with a full thickness skin graft (FTSG), 13% with a local flap. Frailty ranged from 1 to 7 (median = 4). ASA ranged from 1 to 4 (median = 3). A total of 21 (17%) patients reported postoperative complications. Within this group, the median frailty and ASA grades were 5 and 3. Both frailty and ASA were positively significantly associated with age (p ≤ 0.001). There was no significant difference between the frailty or ASA grades of patients that experienced complications and those who did not. Patients who had a FTSG were significantly more likely to experience complications (p ≤ 0.05). CONCLUSIONS: Frailty is not predictive of postoperative complications following excision of NMSC on the head and neck. Postoperative complications are significantly more associated with FTSG.

Evaluation of novel renal biomarkers with a cisplatin model of kidney injury: gender and dosage differences.

A number of novel urinary biomarkers have been identified and partially qualified for use as markers for renal injury in rats. We use two novel multiplex assays to quantify biomarker concentration in multiple urine collections made prior to and following administration of cisplatin, a common nephrotoxicant, to rats. We investigate the correlation of the magnitude of biomarker changes with the severity of histopathological observations and explore the relationship of these to both dose and sex. The novel biomarkers evaluated are urinary albumin, alpha glutathione s-transferase (α-GST), glutathione S-transferase-yb1 (GSTYb1), lipocalin-2, kidney injury molecule-1 (KIM-1), osteopontin, and renal papillary antigen 1 (RPA-1) and plasma cystatin C, alongside the traditional biomarkers of plasma urea, creatinine, and urinary n-acetyl-beta-d-glucosaminidase (NAG), total protein, and glucose. We show for all time points, and for almost all doses, that male rats consistently had either more severely graded or a higher incidence of histologically observed lesions than females; that changes in urinary glucose, total urinary protein, NAG, and the novel urinary biomarkers albumin, osteopontin, and KIM-1 are clearly temporally associated; and that changes are related to the severity of injury. We also found that receiver operating characteristic curve analysis and area under the curve are significantly higher than urea or creatinine for all new biomarkers except aGST, GSTYb1, cystatin c, and total protein in both sexes.

Prognostic indicators and outcomes following surgical management of metastatic cutaneous squamous cell carcinoma of the head and neck.

Metastatic cutaneous SCC carries a poor prognosis with five-year survival of 25%-57%. The aim of this study is to examine the outcomes following surgery with adjuvant therapy for management of metastatic cSCC in a UK-based population. This is a retrospective review of patients with metastatic cSCC of the head and neck who underwent primary surgery at a regional center during a six-year period. Overall and disease specific survival were calculated using Kaplan-Meier and log-rank tests. Results were reported as hazard ratios (HR) with 95% confidence intervals. Forty-five patients met the inclusion criteria. The mean time to discovery of metastases was 9.3 months (range, 0-40 months). Only two patients (4%) had discovery of metastases after two years, with none after 3.3 years. The overall five5-year survival was 31% (95% CI 15%to 48%) with two-year survival at 48% (95% CI 31%to 63%). The median OS survival was 722 days (95% CI 607to 1359). Patients aged >80 years had a decreased OS. This is the largest UK based study documenting the overall and disease specific survival associated with metastatic cutaneous SCC of the head and neck. Our overall survival is comparable to similar studies, but remains poor. Total number of involved nodes, and lymph node ratio were not statistically significant.

The novel sodium glucose transporter 2 inhibitor dapagliflozin sustains pancreatic function and preserves islet morphology in obese, diabetic rats.

AIMS: To investigate whether glucose lowering with the selective sodium glucose transporter 2 (SGLT2) inhibitor dapagliflozin would prevent or reduce the decline of pancreatic function and disruption of normal islet morphology. METHODS: Female Zucker diabetic fatty (ZDF) rats, 7-8 weeks old, were placed on high-fat diet. Dapagliflozin (1 mg/kg/day, p.o.) was administered for ∼33 days either from initiation of high-fat diet or when rats were moderately hyperglycaemic. Insulin sensitivity and pancreatic function were evaluated using a hyperglycaemic clamp in anaesthetized animals (n = 5-6); ß-cell function was quantified using the disposition index (DI) to account for insulin resistance compensation. Pancreata from a matched subgroup (n = 7-8) were fixed and ß-cell mass and islet morphology investigated using immunohistochemical methods. RESULTS: Dapagliflozin, administered from initiation of high-fat feeding, reduced the development of hyperglycaemia; after 24 days, blood glucose was 8.6 ± 0.5 vs. 13.3 ± 1.3 mmol/l (p < 0.005 vs. vehicle) and glycated haemoglobin 3.6 ± 0.1 vs. 4.8 ± 0.26% (p < 0.003 vs. vehicle). Dapagliflozin improved insulin sensitivity index: 0.08 ± 0.01 vs. 0.02 ± 0.01 in obese controls (p < 0.03). DI was improved to the level of lean control rats (dapagliflozin 0.29 ± 0.04; obese control 0.15 ± 0.01; lean 0.28 ± 0.01). In dapagliflozin-treated rats, ß-cell mass was less variable and significant improvement in islet morphology was observed compared to vehicle-treated rats, although there was no change in mean ß-cell mass with dapagliflozin. Results were similar when dapagliflozin treatment was initiated when animals were already moderately hyperglycaemic. CONCLUSION: Sustained glucose lowering with dapagliflozin in this model of type 2 diabetes prevented the continued decline in functional adaptation of pancreatic ß-cells.

Cellular and histopathological changes in the infrapatellar fat pad in the monoiodoacetate model of osteoarthritis pain.

OBJECTIVE: The infrapatellar fat pad (IPFP) has been identified as a source of anterior knee pain. Fibrosis and marked inflammatory infiltrate in the IPFP of patients with arthritis of the knee and reduction in pain post knee replacement in patients following resection of the IPFP have been observed. We have investigated changes in the IPFP of rats undergoing the monoiodoacetate (MIA) model of degenerative joint disease, a model that exhibits some histopathological similarities to osteoarthritis (OA). METHODS: Rats were injected intra-articularly with MIA and the development of weight bearing asymmetry was followed for 21 days as compared to vehicle-injected animals. In addition, IPFPs were removed from both ipsilateral and contralateral joints. Both inflammatory infiltrate and histopathological changes were analysed. RESULTS: MIA injection caused marked weight bearing asymmetry. Ipsilateral IPFP wet weights were significantly increased on days 1 and 3 in MIA-treated animals. MIA treatment also resulted in significant increases in IPFP total white blood cells and monocytes on days 1, 3, and 7 and neutrophils on days 1 and 3. This was supported by histopathological findings at early time points which progressed to adipocyte necrosis, IPFP fibrosis, patellar cartilage and subchondral bone necrosis with synovial hyperplasia at later timepoints. CONCLUSIONS: The current study clearly demonstrated that marked inflammatory changes in the IPFP occur during the early stage of the MIA model of OA which may contribute to the pain observed at this early stage. The role of the IPFP in later stages of the model needs to be further explored.

Reduced expression of the KATP channel subunit, Kir6.2, is associated with decreased expression of neuropeptide Y and agouti-related protein in the hypothalami of Zucker diabetic fatty rats.

The link between obesity and diabetes is not fully understood but there is evidence to suggest that hypothalamic signalling pathways may be involved. The hypothalamic neuropeptides, pro-opiomelanocortin (POMC), neuropeptide Y (NPY) and agouti-related protein (AGRP) are central to the regulation of food intake and have been implicated in glucose homeostasis. Therefore, the expression of these genes was quantified in hypothalami from diabetic Zucker fatty (ZDF) rats and nondiabetic Zucker fatty (ZF) rats at 6, 8, 10 and 14 weeks of age. Although both strains are obese, only ZDF rats develop pancreatic degeneration and diabetes over this time period. In both ZF and ZDF rats, POMC gene expression was decreased in obese versus lean rats at all ages. By contrast, although there was the expected increase in both NPY and AGRP expression in obese 14-week-old ZF rats, the expression of NPY and AGRP was decreased in 6-week-old obese ZDF rats with hyperinsulinaemia and in 14-week-old rats with the additional hyperglycaemia. Therefore, candidate genes involved in glucose, and insulin signalling pathways were examined in obese ZDF rats over this age range. We found that expression of the ATP-sensitive potassium (K(ATP)) channel component, Kir6.2, was decreased in obese ZDF rats and was lower compared to ZF rats in each age group tested. Furthermore, immunofluorescence analysis showed that Kir6.2 protein expression was reduced in the dorsomedial and ventromedial hypothalamic nuclei of 6-week-old prediabetic ZDF rats compared to ZF rats. The Kir6.2 immunofluorescence colocalised with NPY throughout the hypothalamus. The differences in Kir6.2 expression in ZF and ZDF rats mimic those of NPY and AGRP, which could infer that the changes occur in the same neurones. Overall, these data suggest that chronic changes in hypothalamic Kir6.2 expression may be associated with the development of hyperinsulinaemia and hyperglycaemia in ZDF rats.

Mechanisms of non-genotoxic carcinogenesis.

Until recently, the mechanism of carcinogenesis has been regarded as a two-stage phenomenon involving damage to the genetic material, which initiates the process, followed by a cell-division stimulus, which promotes the development of the tumour. However, exposure to some chemicals has been shown to result in carcinogenesis without involvement of the initiation step. The mechanism of non-genotoxic carcinogenesis is not fully understood, but is believed to involve stimulation of cell division with a consequent increased probability of a mutation occurring spontaneously. In this article, Ian Shaw and Huw Jones review the theories of non-genotoxic carcinogenesis with reference to specific examples of known non-genotoxic carcinogens.

A high-resolution radiation hybrid map of the region surrounding the Gorlin syndrome gene.

We have constructed a set of hybrid cell lines by irradiation of GM 64063 (human chromosome 9q only on hamster background) and fusion to hamster A23 Tk-, 109 independent lines were tested by PCR with 24 markers from chromosome 9q. The marker density is highest in the 9q22.3-q31 region containing the gene for Gorlin syndrome, a familial predisposition to basal cell carcinoma. The resolution of our map in this region is significantly higher than other published maps and will enable accurate placing of new markers and genes within the 9q22.3-q3.1 region. This is important since yeast artificial chromosomes from the region are likely to contain deletions.

Blood-brain barrier integrity and host responses in experimental metastatic brain tumours.

The effect of brain implants of Walker 256 carcinosarcoma tumour cells on the integrity of the blood-brain barrier was examined in rats using labelled albumin, horseradish peroxidase and trypan blue. Barrier integrity was intact within 1 hour of implantation but was gradually reduced within the tumour after 3.5 days. This was related to alterations in the fine structure of the tumour capillaries. Dissociated tight junctions were apparent within the tumour centre, but no fenestrated endothelium or gap junctions were observed by electron microscopy.

A screen of candidates from peaks of linkage: evidence for the involvement of myeloperoxidase in multiple sclerosis.

We tested 11 microsatellite markers for evidence of transmission distortion in 744 trio families with multiple sclerosis. Ten of the markers lie within or near to candidate genes selected on the basis that they map within the regions of potential linkage identified in our previously reported linkage genome screen, while the eleventh is an anonymous marker which had previously shown modest evidence for transmission distortion in our sibling pair families. Only the marker related to the myeloperoxidase (MPO) gene revealed tentative evidence for linkage disequilibrium and further work on this gene is clearly needed in order to resolve the status of this region in conferring susceptibility to multiple sclerosis.

No evidence for association of multiple sclerosis with the complement factors C6 and C7.

Four genome screens in multiple sclerosis have been completed and each has identified evidence for linkage in the pericentromeric region of chromosome 5. This region encodes a number of candidate genes including those for the complement components C6, C7 and C9. We have used a multiplexed oligoligation assay (OLA) to test single nucleotide polymorphisms (SNPs) from the C6 and C7 genes for evidence of association with multiple sclerosis in our sibling pair families. There was no statistically significant difference in the allele frequencies of these polymorphisms in the index cases from our families when compared with locally derived controls. No evidence for transmission distortion was seen with any of the polymorphisms, or with the haplotype built from the three SNPs from the C7 gene. Despite offering themselves as potential candidates these complement genes appear not to confer susceptibility to multiple sclerosis.

Phenobarbital-induced hepatocellular proliferation: anti-bromodeoxyuridine and anti-proliferating cell nuclear antigen immunocytochemistry.

We report modifications to immunocytochemical detection procedures for proliferating cell nuclear antigen (PCNA) which permit its identification in liver samples previously fixed for BrdU immunocytochemistry. Both methods have been used for the assessment of phenobarbital-induced cell proliferation in rat liver. The difficulties associated with the hitherto unsuccessful application of PCNA immunocytochemical methods to tissues fixed in formalin for BrdU visualization were overcome by epitope unmasking with acid hydrolysis, extension of primary antiserum (PC10) incubation, and employment of streptavidin-ABC-HRP. BrdU delivery via osmotic minipumps for 48 hr before euthanasia, followed by fixation in cold formalin for 14 days, yielded reliable and reproducible hepatocellular labeling and a peak of cell proliferation in all lobes on Day 3 (i.e., labeling during Days 1-3) of dosing with 80 mg/kg/day phenobarbital. Labeling indices (LI) of both control and phenobarbital-treated liver were lower in the left and right median lobes as compared with the lateral lobes. In sections of the left lateral lobe from the same liver, PCNA immunocytochemistry revealed a peak of proliferative activity (about one third of the maximum LI generated by BrdU incorporation) on Day 1. These findings, together with the advantages and disadvantages of both techniques, are discussed in the context of their applications to different investigative requirements.

Assessment of the labeling index of cohorts of the anterior pituitary cell population in phenobarbital-treated male rats by a double immunohistochemical technique for bromodeoxyuridine and pituitary hormones.

A previous study demonstrated that administration of phenobarbital for up to 7 days to male AP Wistar rats caused alterations in labeling indices (LIs) of several different tissues as determined by immunohistochemical visualization of bromodeoxyuridine (BrdU) incorporation into S-phase nuclei. The pivotal role of the pituitary gland in the function of the endocrine system and changes in circulating hormone levels that result from administration of xenobiotics prompted our consideration of the possible changes in LIs of individual cohorts of the anterior pituitary cell population that may occur as a specific functional adaptation during phenobarbital administration. We evaluated the LIs of individual anterior pituitary cell cohorts by modifying a double immunohistochemical staining method for bromodeoxyuridine and pituitary hormones using a sequential peroxidase-anti-peroxidase (PAP)/alkaline phosphatase-anti-alkaline phosphatase (APAAP) method employing diaminobenzidine and New Fuchsin chromogens, respectively. The method was robust and reproducible. Differences were noted in individual anterior pituitary cohort LIs between control and phenobarbital-treated groups, although no statistically significant difference was evident. We conclude that no detectable effects on individual cohort LIs were induced by treatment with phenobarbital for up to 7 days and that any functional adaptation to treatment was associated with increased hormone release. We believe that the visualization, identification, and quantitation of replicating cells in specific hormone-positive cohorts of the anterior pituitary cell population provide opportunities for understanding the influence of xenobiotics and disease processes on pituitary function.

Comparative effects of anti-platelet agents as adjuncts to tissue plasminogen activator in a dog model of occlusive coronary thrombosis.

1. This study compares a cyclo-oxygenase inhibitor (aspirin), a 5-HT2 antagonist (ZM170809) and a combined thromboxane synthase inhibitor/receptor antagonist (ZD1542) as adjuncts to tissue plasminogen activator (rt-PA). 2. Application of an anodal current (332 +/- 4.1 microA) to the stenosed left circumflex coronary artery of 20 anaesthetized dogs produced a stable platelet-rich occlusive thrombus. 3. After initial i.v. administration of recombinant human tissue type plasminogen activator (rt-PA, 3 mg bolus +2 mg kg-1 h-1 for 30 min) thrombolysis occurred in 15 out of 20 dogs. All 15 dogs reoccluded. 4. The second i.v. administration of rt-PA in the presence of either aspirin, ZM170809, ZD1542 or saline resulted in thrombolysis in all 20 dogs. 5. Both the combined thromboxane synthase inhibitor/receptor antagonist (ZD1542) and 5-HT2 antagonist (ZM170809) significantly (P < 0.05) reduced the time taken to lyse the thrombus compared with the saline group. The times were 14.4 +/- 2.7 min, 18.0 +/- 3.9 min and 36.8 +/- 6.2 min for ZD1542, ZM170809 and saline respectively. 6. Aspirin did not offer any additional benefit to using rt-PA alone. The times to thrombolysis were 36.8 +/- 8.4 min for aspirin and 36.8 +/- 6.2 min for the saline group. 7. The number of dogs in which the circumflex coronary artery reoccluded within 60 min of terminating the second infusion of rt-PA were five for saline, four for aspirin, two for ZD1542 and two for ZM170809. 8. These results indicate that both ZD1542 and ZM170809 are more effective adjuncts than aspirin in thrombolysis and may provide an improvement in current clinical practice.

Anti-inflammatory activity of c(ILDV-NH(CH2)5CO), a novel, selective, cyclic peptide inhibitor of VLA-4-mediated cell adhesion.

1. Small, N- to C-terminal cyclized peptides containing the leucyl-aspartyl-valine (LDV) motif from fibronectin connecting segment-1 (CS-1) have been investigated for their effects on the adhesion of human T-lymphoblastic leukaemia cells (MOLT-4) to human plasma fibronectin in vitro mediated by the integrin Very Late Antigen (VLA)-4 (alpha4beta1, CD49d/CD29). 2. Cyclo(-isoleucyl-leucyl-aspartyl-valyl-aminohexanoyl-) (c(ILDV-NH(CH2)5CO)) was approximately 5 fold more potent (IC50 3.6+/-0.44 microM) than the 25-amino acid linear CS-1 peptide. Cyclic peptides containing two more or one less methylene groups had similar potency to c(ILDV-NH(CH2)5CO) while a compound containing three less methylene groups, c(ILDV-NH(CH2)2CO), was inactive at 100 microM. 3. c(ILDV-NH(CH2)5CO) had little effect on cell adhesion mediated by two other integrins, VLA-5 (alpha5,beta1, CD49e/CD29) (K562 cell adhesion to fibronectin) or Leukocyte Function Associated molecule-1 (LFA-1, alphabeta2, CD11a/CD18) (U937 cell adhesion to Chinese hamster ovary cells transfected with intercellular adhesion molecule-1) at concentrations up to 300 microM. 4. c(ILDV-NH(CH2)5CO) inhibited ovalbumin delayed-type hypersensitivity or oxazolone contact hypersensitivity in Balb/c mice when dosed continuously from subcutaneous osmotic mini-pumps (0.1-10 mg kg(-1) day(-1)). Maximum inhibition (approximately 40%) was similar to that caused by the monoclonal antibody PS/2 (7.5 mg kg(-1) i.v.) directed against the alpha4 integrin subunit. 5. c(ILDV-NH(CH2)5CO) also inhibited oxazolone contact hypersensitivity when dosed intravenously 20 h after oxazolone challenge (1-10 mg kg(-1)). Ear swelling was reduced at 3 h and 4 h but not at 1 h and 2 h post-dose (10 mg kg(-1)). 6. Small molecule VLA-4 inhibitors derived from c(ILDV-NH(CH2)5CO) may be useful as anti-inflammatory agents.

Characterisation of Candida albicans infections of haematogenous and mucosal origin in mice lacking the interferon gamma receptor protein.

Mice harbouring a null deletion mutation in the IFNgamma receptor gene were used to study the role of IFNgamma responsiveness during experimental systemic candidiasis of mucosal or haematogenous origin. After intravenous (i.v.) or intranasal (i.n.) challenge with Candida albicans the progression of infection and concomitant cellular and antibody anti-C. albicans immune responses were analysed. During the week following i.v. challenge, the rate of C. albicans multiplication in kidneys, liver and spleen was faster in IFNgammaR (-/-) than IFNgammaR (+/+) mice. As a result, IFNgammaR (-/-) mice perished earlier than IFNgammaR (+/+) mice when challenged with equal numbers of live yeast cells. However, the overall susceptibility of the two mouse strains, in terms of survival against different C. albicans challenge doses over a 60-day period, was similar. No differences were found in the cellular anti-C. albicans response generated by i.v. challenge in both mouse strains. In contrast the kinetics and strength of the serum anti-C. albicans antibody responses were markedly different. Significantly stronger, predominantly IgG2a antibody responses accompanied the eventual control of C. albicans infection in IFNgammaR (-/-) mice. Following intranasal challenge, there was no difference in the rate of C. albicans clearance from the lungs of IFNgammaR (-/-) and IFNgammaR (+/+) mice. However, 48 h after challenge, large, conspicuous abscesses appeared in the lungs, liver, kidneys and spleen of IFNgammaR (-/-) mice. These abscesses were characterised by the presence of C. albicans and abundant neutrophilic infiltrates, but very few macrophages. No such abscesses developed in i.n. challenged IFNgammaR (+/+) mice. In both mouse strains, i.n. challenge induced strong systemic anti-C. albicans cellular responses, but relatively low titre systemic antibody responses. Mucosal anti-C. albicans antibody responses were detected in IFNgammaR (+/+), but not IFNgammaR (-/-) mice. Splenic adherent macrophages obtained from IFNgammaR (-/-) mice exhibited a significantly lower candidacidal activity than those of IFNgammaR (+/+) mice, and as expected, were not responsive to IFNgamma. In summary, these data suggest that IFNgamma has a role in limiting C. albicans multiplication during the early stages of infection, as well as in preventing the development of C. albicans-associated abscesses. Activation of macrophages by IFNgamma might be pivotal in mediating this role.

The role of ultrastructural investigations in neurotoxicology.

The ways in which ultrastructural approaches have been applied to the investigation of xenobiotic-induced toxicity of the nervous system have been briefly reviewed. These approaches have been grouped in 3 broad areas, viz. morphology, function and composition. Firstly, morphological approaches permit the visualisation of changes in intercellular relationships, the identification of the subcellular target(s) of a xenobiotic substance and the discrimination between what may appear ostensibly to be identical cellular responses to one or more chemically distinct toxins. Secondly, functional approaches using, e.g. cytochemistry, ion precipitation, immunocytochemistry and autoradiography provide indications of metabolic state, the identity or the intra- or extracellular location of the "reactive species". Thirdly, those approaches, viz. electronprobe X-ray microanalysis and electron energy loss spectroscopy which provide information of the elemental composition of cells and tissues permit an assessment of the subcellular distribution and compartmentalisation of endogenous substances and toxic or therapeutic xenobiotics. In concert, ultrastructural approaches possess the ability to contribute unique information on the effects of exposure of cells of the nervous system to toxic substances and so direct further investigation towards an understanding of the mechanism of action.

Neuropathological changes in rat brain following oral administration of 2-chloropropionic acid.

The agrochemical intermediate, L-2-Chloropropionic acid (L-2-CPA) and D-2-chloropropionic acid (D-CPA), when administered separately by oral gavage to rats, produced extensive cerebellar granule cell necrosis (> 80%) characterised by varying degrees of nuclear condensation and nuclear karyorrhexis. In contrast a few necrotic Purkinje cells (< 5%) were observed. Purkinje cell damage consisted of cytoplasmic and nuclear shrinkage and hyperchromasia. Karyorrhexis was not seen in Purkinje cells. Extensive vacuolation (edema) was present both in the cerebellar granular layer and Purkinje cell layer. Astrogliosis (hypertrophy and hyperplasia) was seen at lesion sites and activity was recognised by positive glial fibrillary acidic protein (GFAP) staining. Proliferative activity of astrocytes at lesion sites was confirmed by positive proliferating cell nuclear antigen (PCNA) immunostaining. Astrogliosis was focused exclusively in the necrotic granule cell layer. A single oral dose of 750 mg/kg of either stereoisomer or three consecutive daily doses of 250mg/kg L-2-CPA. produced the lesion. Cerebellar water content increased with time in parallel with the edematous response noted by neuropathological examination. The earliest onset of the lesion was observed at 36 hours appearing more extensive at 48 and 72 hours post-dosing. No other major neuropathological changes were detected in the brain, spinal cord, spinal ganglia, Gasserian ganglia, peripheral nerves and voluntary muscle following L-2-CPA administration. In conclusion, both stereoisomers of CPA were found to be selective neurotoxicants of the rat central nervous system.

Allylamine toxicity in embryonic myocardial myocyte reaggregate cultures.

This study investigated the response of chick myocardial myocyte reaggregates (MMR) to allylamine, which is specifically toxic to the cardiovascular system, and its putative toxic metabolite, acrolein. Toxicity was assessed by determining cellular ATP content, spontaneous beating and ultrastructural alterations. Both allylamine and acrolein caused a dose-dependent loss of beating and depletion of cellular ATP content. Treatment with either 10 or 100 mum-allylamine produced marked alterations in reaggregate tissue morphology at all times, which was characterized by widespread myocyte necrosis and a reduction in tissue compactness. Exposure of aggregates to acrolein produced generalized tissue destruction following administration of either 10 or 100 mum-acrolein. The concentration of allylamine required to cause loss of MMR beating was greatly increased when incubations were carried out in serum-free medium. It is suggested that the presence of benzylamine oxidase in serum contributes to the toxicity of allylamine to MMR, by metabolizing allylamine to acrolein. This proposal is supported by the finding that the related amines n-methylallylamine and diallylamine had no effect on spontaneous beating, which may be because they are very poor substrates for benzylamine oxidase. The data presented demonstrate that allylamine is highly toxic to MMR, and we believe that further investigations into the role of serum in allylamine toxicity would improve our understanding of the biochemical basis of its action.