Multimerin is found in the alpha-granules of resting platelets and is synthesized by a megakaryocytic cell line.

In this report, we describe the intracellular localization of multimerin in platelets and its biosynthesis by Dami cells, a megakaryocytic cell line. Immunoelectron microscopy was used to examine frozen thin sections of resting and activated platelets. Multimerin was localized within the platelet alpha-granule in an eccentric position. Within activated platelets, multimerin was found in the surface-connected open cannalicular system and on the external plasma membrane. Light microscopic immunocytochemistry demonstrated multimerin in normal megakaryocytes and in Dami cells after stimulation with PMA. Confirmation of multimerin biosynthesis by Dami cells was obtained by metabolic labeling studies. Both platelet and Dami cell multimerin demonstrated several subunit sizes on reduced SDS-PAGE. However, peptide mapping confirmed structural homology between the different multimerin subunits. Glycosidase digestion demonstrated that multimerin is heavily glycosylated with mainly complex, N-linked carbohydrate. In contrast to the multimerin isolated from platelets, cultured Dami cells secreted mainly smaller multimers of the protein. Biosynthesis of multimerin by a megakaryocytic cell line supports endogenous biosynthesis by megakaryocytes as the origin of this platelet alpha-granule protein.

Impact of 5-HT3 receptor blockade on colonic haemodynamic responses to ischaemia and reperfusion in the rat.

5-HT(3) receptor antagonists are clinically available for treating patients with irritable bowel syndrome (IBS) but their use is restricted because of a link with some episodes of ischaemic colitis. However, the role of 5-HT3 receptors in regulating colonic blood flow has not been systematically investigated. Thus, we examined acute and chronic treatment with alosetron, a potent and selective antagonist of the 5-HT3 receptor, on baseline colonic blood flow and haemodynamic responses during occlusion and reactive hyperaemia in the pentobarbitone-anaesthetized rat. Colonic haemodynamics were assessed using ultrasonic recordings of superior mesenteric blood flow (MBF) and laser Doppler recordings of colonic vascular perfusion (VP). Blood pressure (BP) was also monitored and in some experiments tissue oxygen was detected polarographically. Alosetron (10, 30, 100 microg kg(-1), i.v.) had no effect on baseline haemodynamics nor responses to nitric oxide synthase inhibition with N(omega)-nitro-l-arginine methyl ester (l-NAME) (16 mg kg(-1)). Arterial occlusion (5 min) reduced MBF (-98.6 +/- 0.6%) and VP (-70.7 +/- 5.4%) followed by a post-occlusion reactive hyperaemia (MBF = +94.5 +/- 19.1%; VP = +60.0 +/- 22.3%) the magnitude of which was unchanged following acute (30 microg kg(-1)) or chronic alosetron administration (0.5 mg kg(-1) twice daily, 5 days). Alosetron did not significantly alter baseline colonic blood flow in the anaesthetized rat; nor did it interfere with vascular control mechanisms activated during occlusion and reactive hyperaemia.

Activation of the cannabinoid 2 (CB2) receptor inhibits murine mesenteric afferent nerve activity.

Abstract Cannabinoid 2 (CB2) receptors have both antinociceptive and antihypersensitivity effects, although the precise mechanisms of action are still unclear. In this study, the modulatory role of CB2 receptors on the mesenteric afferent response to the endogenous immunogenic agent bradykinin (BK) was investigated. Mesenteric afferent recordings were obtained from anaesthetized wild-type and CB2(-/-) mice using conventional extracellular recording techniques. Control responses to BK were obtained in all experiments prior to administration of either CB2 receptor agonist AM1241, or AM1241 plus the CB2 receptor antagonist AM630. Bradykinin consistently evoked activation of mesenteric afferents (n = 32). AM1241 inhibited the BK response in a dose dependent manner. In the presence of AM630 (10 mg kg(-1)), however, AM1241 (10 mg kg(-)1) had no significant effect on the BK response. Moreover, AM1241 had also no significant effect on the BK response in CB2(-/-) mice. Activation of the CB2 receptor inhibits the BK response in mesenteric afferents, demonstrating that the CB2 receptor is an important regulator of neuroimmune function. This may be a mechanism of action for the antinociceptive and antihypersensitive effects of CB2 receptor agonists.

A novel use of reticulated hyaluronic acid (Healaflow) for hypotony eyes in patients with uveitis.

Persistent ocular hypotony is a complex and ongoing challenge faced in ophthalmology. It can result in early ocular phthisis and associated visual decline, pain and deformity. We present the first case series, in which repeated intracameral injections of highly reticulated hyaluronic acid (Healaflow) have successfully prevented the complications of ocular hypotony in the long term. We believe it is a viable management option that can bring about a significant improvement to the quality of life in this subgroup of patients while avoiding frequent intervention.

MNK1 and MNK2 mediate adverse effects of high-fat feeding in distinct ways.

The MAP kinase-interacting kinases (MNK1 and MNK2) are non-essential enzymes which are activated by MAP kinases. They are implicated in controlling protein synthesis. Here we show that mice in which the expression of either MNK1 or MNK2 has been knocked out (KO) are protected against adverse effects of high-fat feeding, and in distinct ways. High-fat diet (HFD)-fed MNK2-KO show less weight gain than wild-type animals, and improved glucose tolerance, better insulin sensitivity and markedly diminished adipose tissue inflammation. This suggests MNK2 plays a role in adipogenesis and/or lipogenesis and in macrophage biology. MNK1-KO/HFD mice show better glucose tolerance and insulin sensitivity, but gain weight and show similar adipose inflammation to WT animals. These data suggest MNK1 participates in mediating HFD-induced insulin resistance. Our findings reveal distinct roles for the MNKs in a novel area of disease biology, metabolic dysfunction, and suggests they are potential new targets for managing metabolic disease.

Heterogeneity of heparin lots associated with thrombocytopenia and thromboembolism.

Thromboembolic complications may develop in patients with heparin-associated thrombocytopenia, presumably due to the formation of platelet aggregates. An unexpectedly high incidence of pulmonary embolism following coronary artery bypass surgery occurred during a brief period of time at a single institution, and all of these cases were found to be associated with thrombocytopenia. All patients tested during thrombocytopenia (five of five) had an increase in platelet-associated antibody. Serum samples from all five patients tested caused normal platelets to aggregate in vitro in the presence of one specific lot of beef lung heparin, which was in use in the operating room at the time; none of six other lots of beef lung heparin mediated in vitro platelet aggregation. Heparinase digestion of the heparin abolished the aggregating activity. It is concluded that thrombocytopenia and platelet activation caused by heparin may vary greatly even among different lots of heparin prepared from the same source.

Choline acetyltransferase (ChAT) immunoreactivity in paraffin sections of normal and diseased intestines.

There is increasing interest in localizing nerves in the intestine, especially specific populations of nerves. At present, the usual histochemical marker for cholinergic nerves in tissue sections is acetylcholinesterase activity. However, such techniques are applicable only to frozen sections and have uncertain specificity. Choline acetyltransferase (ChAT) is also present in cholinergic nerves, and we therefore aimed to establish a paraffin section immunocytochemical technique using an anti-ChAT antibody. Monoclonal anti-choline acetyltransferase (1.B3.9B3) and a biotin-streptavidin detection system were used to study the distribution of ChAT immunoreactivity (ChAT IR) in paraffin-embedded normal and diseased gastrointestinal tracts from both rats and humans. Optimal staining was seen after 6-24 hr of fixation in neutral buffered formalin and overnight incubation in 1 microgram/ml of 1.B3.9B3, with a similar distribution to that seen in frozen sections. In the rat diaphragm (used as a positive control), axons and motor endplates were ChAT IR. Proportions of ganglion cells and nerve fibers in the intramural plexi of both human and rat gastrointestinal tracts were also ChAT IR, as well as extrinsic nerve bundles in aganglionic segments of Hirschsprung's disease. Mucosal cholinergic nerves, however, were not visualized. In addition, non-neuronal cells such as endothelium, epithelium, and inflammatory cells were ChAT IR. We were able to localize ChAT to nerves in formalin-fixed, paraffin-embedded sections. The presence of ChAT IR in non-neuronal cells indicates that this method should be used in conjunction with other antibodies. Nevertheless, it proves to be a useful technique for studying cholinergic neuronal distinction in normal tissues and pathological disorders.

Treatment of steatorrhoea in cystic fibrosis: a comparison of enteric-coated microspheres of pancreatin versus non-enteric-coated pancreatin and adjuvant cimetidine.

Enteric-coated microspheres of pancreatin were compared with non-enteric-coated pancreatin combined with cimetidine taken 40 min before meals in the treatment of patients with cystic fibrosis. Fourteen adults with steatorrhoea due to cystic fibrosis were investigated in an open, randomized crossover study, over two consecutive 28-day treatment periods. Lipase intake was adjusted to each patient's previous requirements and was the same during both months; they were instructed to continue with their normal diet. Patients collected faeces for 72 h at the end of each month and completed diary cards daily throughout. Bowel actions were less frequent on enteric-coated microspheres of pancreatin than on non-enteric-coated pancreatin/cimetidine (1.7 vs. 2.4/day; P less than 0.001) and stool character was improved (P less than 0.001). Mean daily faecal weight was similar on enteric-coated microspheres of pancreatin to that on the combination (254 g vs. 291 g; N.S.), whereas daily faecal fat excretion tended to be less on enteric-coated microspheres of pancreatin (21 g vs. 27 g; N.S.), and percentage fat absorption tended to be greater (81% vs. 73%; N.S.). Mean body weight increased by 0.3 kg on enteric-coated microspheres of pancreatin and fell by 0.1 kg on the combination (N.S.). These data indicate that enteric-coated microspheres of pancreatin are at least as effective as non-enteric-coated pancreatin with cimetidine in the treatment of steatorrhoea in cystic fibrosis.

An immunohistochemical study of pleomorphic adenomas of the salivary gland: glial fibrillary acidic protein-like immunoreactivity identifies a major myoepithelial component.

An immunohistochemical study of 34 pleomorphic adenomas of the major salivary glands demonstrated phenotypic differences among the various morphologic regions in these tumors. The phenotypes expressed were comparable to those of normal salivary gland cells. In the normal glands, myoepithelial cells were immunoreactive for glial fibrillary acidic protein (GFAP), S-100 protein, and keratin; acinic cells exhibited strong, predominantly nuclear S-100 staining and weaker keratin staining; intercalated ducts had both cytoplasmic and nuclear S-100 positivity; and several epithelial antigens were observed throughout the ductal system. In the tumors, the presence of classic epithelial markers (including carcinoembryonic antigen, epithelial membrane antigen, secretory component, and keratin) in the luminal cells of ducts and the intense immunoreactivity with GFAP (with weaker keratin and S-100 staining) in periductal and stromal cells indicated distinct epithelial and myoepithelial differentiation. Solid epithelioid areas consisted phenotypically of intercalated duct/acinic cells and/or myoepithelial cells, the former exhibiting predominant nuclear S-100 positivity. The presence of GFAP-like immunoreactivity in normal myoepithelial cells strongly supports the extensive involvement of this cell in pleomorphic adenomas. The spectrum of phenotypes expressed adds weight to existing evidence for pleomorphism rather than a mixed origin of this tumor. The combination of keratin, S-100, and GFAP immunostaining is particularly useful in identifying the component cells in pleomorphic adenomas of the salivary glands.

Novel cellular interactions and networks involving the intestinal immune system and its microenvironment.

The interactions we have described enable the intestine to respond appropriately to antigenic challenge in an effective and coordinated way. This is of vital importance when one considers the dual role of the intestine as a first line of defence against harmful microorganisms and as the route by which the animal obtains nutrition. Under normal circumstances, these interactions select for an appropriate cell phenotype by providing a network of interactions that contribute to intestinal homeostasis. If there is dysfunction of any component, then other cells will be affected. For example, if down-regulation of the mucosal immune response is not effective, damage to the epithelium, nerves and muscle may occur during an inflammatory response. Similarly, if the integrity of the epithelium is disrupted, damage to the elements of the mucosal immune system may occur. This model would suggest that these interactions must be considered if one wishes to adequately explain diseases such as IBD and design innovative therapeutic regimens. Future interdisciplinary research will shed light on the web of interactions occurring in the intestinal environment and provide a novel view of the respective contributions of the immune system and its local environment to cell differentiation, function and regulation.

Pegfilgrastim, a sustained-duration form of filgrastim, significantly improves neutrophil recovery after autologous marrow transplantation in rhesus macaques.

Daily administration of filgrastim decreases the duration of severe neutropenia in the clinical setting. A sustained-duration form of filgrastim, pegfilgrastim, significantly reduces scheduling protocols to a single injection per chemotherapy cycle while maintaining therapeutic efficiency. We examined the ability of a single injection of pegfilgrastim to significantly improve neutrophil recovery following autologous bone marrow transplantation (AuBMT) in rhesus macaques. On day 1, postmyeloablation (920 cGy x-irradiation) and AuBMT, animals received either 0.1% autologous serum for 18 consecutive days (n=13), or single doses of pegfilgrastim via the subcutaneous (s.c.) or intravenous (i.v.) route (300 or 100 micro g/kg), or a single dose of filgrastim at 300 micro g/kg via the s.c. or i.v. route, or filgrastim at 10 micro g/kg via the s.c. route (n=4) on a daily basis (range=days 12-17). Pharmacokinetic parameters and neutrophil recovery were assessed. A single dose of pegfilgrastim via the i.v. or s.c. route was as effective as daily filgrastim administration, resulting in significant improvement of neutrophil recovery after myeloablation and ABuMT. Effective pegfilgrastim plasma concentrations were maintained in neutropenic animals until after the onset of hematopoietic recovery. Enhanced pharmacokinetics in AuBMT cohorts are consistent with self-regulating, neutrophil-mediated clearance.

Feulgen microdensitometry and analysis of S-phase cells in cervical tumour biopsies.

Tissue specimens from the cervical tumours of 70 patients undergoing radiotherapy were examined by Feulgen microdensitometry. Twenty-five of the 70 specimens were also subjected to in-vitro tritiated thymidine autoradiography to determine the proportion of DNA synthesising cells they contained. A spectrum of frequency distributions of nuclear DNA content was obtained from Feulgen microdensitometry, but by inspecting the data the basic DNA content of malignant cells could be established in most cases. Fifty-nine per cent of the tumours were 'diploid', 10% 'tetraploid', 13% 'diploid to tetraploid', and the remaining 18% 'aneuploid'. Grafical analysis of DNA frequencies from 48 'diploid' or 'tetraploid' tumours enabled the proportion of DNA synthesising (S) cells to be estimated by frequency distribution analysis. Estimates of the S component ranged from nil to 30%, were log normally distributed, and comparable to direct measurements of cells in S determined by autoradiography for 25 cases (range 1.2-28.7%). For all paired data the mean difference was 1.2 +/- 1.45% (confidence limits), suggesting that overall Feulgen microdensitometry analysis may be an equally valid technique in providing cellular kinetic information with human tumour material.

Nerve remodelling during intestinal inflammation.

The intestinal mucosa contains a dense nerve network and many inflammatory cells, and these may interact through the exchange of regulatory molecules. Evidence suggests that intestinal mucosal mast cells are innervated, and it is known that the density of this cell type changes significantly in nematode-infected rats. Recent data indicates that rat jejunal mucosal nerves remodel after Nippostrongylus brasiliensis infection, with degenerative and regenerative phases during the acute and recovery stages of inflammation. Seven weeks postinfection there is a net increase in the density and number per villus of mucosal nerves. These changes suggest that mucosal nerves exhibit structural plasticity in inflamed tissues, which must impact on interactions between the enteric nervous system and other mucosal elements in disease.

Electrical stimulation of the vagus nerve modulates the histamine content of mast cells in the rat jejunal mucosa.

Mast cells are best known for their participation in allergic reactions. However, a number of recent studies suggest that mast cells are subject to nervous control. In the gut mucosa, mast cells are intimately associated with nerves, and the psychologically conditioned release of RMCP II (a mucosal mast cell-derived mediator) has been reported. These data suggest the potential for CNS regulation of intestinal mucosal mast cells. In this study, we stimulated the cervical vagi and found an increased histamine content in mucosal mast cells, without apparent degranulation. Furthermore, these changes could be prevented by subdiaphragmatic vagotomy. These data support the potential for intestinal mucosal mast cell regulation by the central nervous system and suggest modulation of mast cells without degranulation.

Effect of truncal vagotomy and capsaicin on mast cells and IgA-positive plasma cells in rat jejunal mucosa.

Immunocompetent cells, including mast cells and plasma cells (PC), in the intestinal mucosa are closely apposed to nerve fibres. Recent work has shown that vagal afferent nerves penetrate the jejunal mucosa and contact intestinal mucosal mast cells (IMMC); and that electrical stimulation of the vagus results in increased IMMC histamine content. To determine if the vagus nerve exerts a trophic effect on immunocompetent cells in the gut mucosa, the effects of truncal vagotomy and neonatal capsaicin treatment on IMMC and IgA containing PC in the lamina propria of rat jejunum were investigated. Three weeks after vagotomy, microdensitometric assessment of Alcian blue stained sections revealed 25% fewer IMMC in vagotomized animals than in controls (P < 0.05). Three months after neonatal capsaicin administration 28% fewer IMMC were found in treated rat jejunum, compared with littermate controls (P < 0.05). Three weeks post-surgery, IgA-PC densities were increased in both vagotomized animals (that also underwent pyloroplasty) and pyloroplasty controls, compared to animals subjected to laparotomy only. The proportion of lamina propria areas remained stable, indicating that the observations reflected real reductions in the numbers of IMMC. We also determined the densities of B-50 (a nerve growth-associated protein, also called GAP-43) immunoreactive nerve fibres in the lamina propria, as well as nerve profile areas, three weeks after vagotomy, and these parameters were unchanged. Taken together, these findings support the hypothesis that the vagus exerts a trophic effect on IMMC; and that capsaicin-sensitive nerves also affect the IMMC population. These data add to the growing body of evidence for a functional connection between IMMC and the nervous system.

Joint pathology and behavioral performance in autoimmune MRL-lpr Mice.

Young autoimmune MRL-lpr mice perform more poorly than age-matched controls in tests of exploration, spatial learning, and emotional reactivity. Impaired behavioral performance coincides temporally with hyperproduction of autoantibodies, infiltration of lymphoid cells into the brain, and mild arthritic-like changes in hind paws. Although CNS mechanisms have been suggested to mediate behavioral deficits, it was not clear whether mild joint pathology significantly affected behavioral performance. Previously we observed that 11-week-old MRL-lpr mice showed a trend for disturbed performance when crossing a narrow beam. The first aim of the present study was to test the significance of this trend by increasing the sample size and, second, to examine the possibility that arthritis-like changes interfere with performance in brief locomotor tasks. For the purpose of the second goal, 18-week-old mice that differ widely in severity of joint disease were selectively taken from the population and tested in beam walking and swimming tasks. It was expected that the severity of joint inflammation would be positively correlated with the degree of locomotor impairment. The larger sample size revealed that young MRL-lpr mice perform significantly more poorly than controls on the beam-walking test, as evidenced by more foot slips and longer traversing time. However, significant correlation between joint pathology scores and measures of locomotion could not be detected. The lack of such relationship suggests that mild joint pathology does not significantly contribute to impaired performance in young, autoimmune MRL-lpr mice tested in short behavioral tasks.

Retroviral transfer of genes into canine hematopoietic progenitor cells.

Amphotropic retroviral vectors containing either the bacterial neomycin phosphotransferase gene or a mutant dihydrofolate reductase gene (DHFR*) were used to infect canine hematopoietic progenitor cells. Successful transfer and expression of both genes in canine hematopoietic progenitor cells has been achieved as measured by the ability of the viruses to confer resistance to either methotrexate (MTX) or the aminoglycoside G418, respectively. Gene transfer was achieved using helper-free retroviral vectors. The rate of gene expression in canine granulocyte/macrophage colony-forming units (CFU-GM) after cocultivation for 24 hours with virus-producing packaging cells ranged from 6-25%. Autologous marrow cocultivated for 24 hours with virus-producing packaging cells was transplanted into six dogs after lethal total body irradiation. All dogs showed engraftment within two weeks and four dogs survived for 5-7 months without adverse effects. One dog that had been given marrow infected with a DHFR* virus and that received MTX as in vivo selection after marrow transplantation and survived, showed 0.1 and 0.03% MTX-resistant CFU-GM at weeks 3 and 5. The efficiency of gene transfer into canine CFU-GM has been increased threefold by culturing marrow cells for six days in long-term marrow culture after 24 hour cocultivation with virus producing packaging cells.

Chordoma: cytologic and immunocytochemical study of four cases.

We present the cytologic features and the immunocytochemical profile of four cases of chordoma on fine-needle aspiration biopsies. The physaliferous cells in signet-ring, pearl-like formations and the trabecular arrangement with rounded contours are distinctive. Other cell types and cellular arrangements are also described. The negative immunoreactivity of carcinoembryonic antigen (CEA) and the positive staining pattern for neuron-specific enolase (NSE), S-100 protein, epithelial membrane antigen (EMA), and keratin provide a profile that, in the appropriate clinical setting, can be useful in the differential diagnosis of chordoma from similar-appearing neoplasms in small biopsies and fine-needle aspirates.

Peripheral nerve sheath neoplasms in canadian slaughter cattle.

Two thousand four hundred and fifty-six bovine neoplasms were submitted during a ten year period of which 238 (9.7%) were neurofibromas. The neoplasms were mainly in the heart but also in the thorax, mediastinum and in some visceral organs. On gross examination 41 of the 99 cardiac neurofibromas were suspected to be Cysticercus bovis. Immunohistochemistry for S-100 protein and neuron specific enolase staining was used to confirm the diagnosis in several cases.