Morphological and molecular characterization of Trichuris muris (Nematoda: Trichuridae): studies from two commensal rodent species.

In this paper we re-describe Trichuris muris based on morphological data following isolation from two commensal rodent species, Mus musculus from Mexico and Rattus rattus from Argentina. Furthermore, we provide a molecular characterization based on mitochondrial (cytochrome c oxidase subunit 1 mitochondrial gene) and nuclear (internal transcribed spacer 2 region) markers in order to support the taxonomic identification of the studied specimens of T. muris from M. musculus. We distinguished T. muris from 29 species of Trichuris found in American rodents based on morphological and biometrical features, such as the presence of a spicular tube, length of spicule, size of proximal and distal cloacal tube and non-protrusive vulva. We suggest that spicular tube patterns can be used to classify Trichuris species in three groups. Considering that the diagnosis among the species of this genus is mainly based on morphometry, this proposal represents a relevant contribution. We provide molecular studies on two markers, making this the first contribution for T. muris in the Americas. This study makes an important contribution to the integrative taxonomy of cosmopolitan nematode species, and its correct determination from the parasitological study of commensal rodents.

Occurrence of Ancylostoma Caninum from a Gray Fox Urocyon Cinereoargenteus in Southeastern Mexico.

The hookworm Ancylostoma caninum is a common nematode of wild and domestic canids worldwide. In Mexico, there are few records of helminths in wild canids, especially in the southeastern region. The aim of the present study was to examine the helminths from a gray fox Urocyon cinereoargenteus in southeastern Mexico. A road-killed female gray fox found in Merida, Yucatan, Mexico, was examined for helminths. Only nematodes were found in the intestine of the gray fox and identified using morphological studies and molecular analysis of 28S rRNA gene fragments. The characteristics exhibited by the nematode specimens were in accordance with descriptions of A. caninum: e. g. oral opening with a pair of prominent chitinous plates bearing three pairs of ventral teeth, lateral rays with a common trunk, dorsal ray divided into two branches with each branch terminating in three digitations. BLAST analysis of the 28S sequence showed similarity and coverage values of 99.8 % and 100 %, respectively, with a sequence of A. caninum from the domestic dog Canis familiaris in Australia. The genetic distance between the Australian specimen and the Yucatan specimen of A. caninum was 0.1 %, that is, they were only different in a single nucleotide. The gray fox examined in this study was found close to a rural community where A. caninum has been recorded from domestic dogs, which could be the source of infection. Our study increases the distribution of this nematode parasitizing the gray fox in Mexico and provides the first nucleotide sequence of A. caninum from the gray fox.

Morphological and Molecular Identification of Nematodes in The Tayra Eira Barbara from Campeche, Mexico.

The tayra Eira barbara is a Neotropical mustelid considered as an endangered species by Mexican environmental authorities. Despite the considerable information available on the biology and ecology of E. barbara, little is known about its helminth fauna. Here, we provided new records of nematodes from a road-killed tayra in Calakmul, Campeche, Mexico. The species identification of nematodes was based on morphological studies and molecular analysis of fragments of the 28S gene. The tayra specimen was infected by three nematodes: Molineus sp., Physalopterinae gen. sp. and Angiostrongylus vasosum. To our knowledge, this study is the first to report the natural infection of E. barbara with Molineus sp. and Physalopterinae gen. sp. Our study provides the first nucleotide sequences of nematodes parasitizing E. barbara providing a starting point against which future studies may be compared.

Patterns of helminth infections in Rattus rattus and Mus musculus from two Mayan communities in Mexico.

The black rat Rattus rattus and the house mouse Mus musculus are two commensal rodent species that harbour and shed zoonotic pathogens, including helminths. The aim of this survey was to study the helminth community and the patterns of infections in R. rattus and M. musculus from two Mayan communities in Mexico. Gastrointestinal helminths were isolated from 322 M. musculus and 124 R. rattus, including Gongylonema neoplasticum, Hassalstrongylus aduncus, Hassalstrongylus musculi, Hydatigera taeniaeformis metacestode, Hymenolepis diminuta, Nippostrongylus brasiliensis, Oligacanthorhynchidae gen. sp., Syphacia muris, Syphacia obvelata, Rodentolepis microstoma and Trichuris muris. The overall richness of helminths was seven in R. rattus and six in M. musculus. The results of generalized linear models showed that juvenile rodents had lower probabilities of being infected with G. neoplasticum, H. taeniaeformis and H. musculi than adult rodents. A positive association between the prevalence of S. muris and rat abundance was found. The intensity of infection with S. muris was higher in the rainy season than in the dry season; the opposite result was found for H. musculi infection. Male R. rattus harboured more S. muris specimens. The intensity of infection with T. muris was inversely associated with mouse abundance. The presence of the zoonotic H. diminuta, as well as H. taeniaeformis and R. microstoma in rodent populations indicates that there is risk of transmission, and that their entire life cycle occurs in the study area.

A survey of zoonotic pathogens carried by house mouse and black rat populations in Yucatan, Mexico.

The house mouse (Mus musculus) and the black rat (Rattus rattus) are reservoir hosts for zoonotic pathogens, several of which cause neglected tropical diseases (NTDs). Studies of the prevalence of these NTD-causing zoonotic pathogens, in house mice and black rats from tropical residential areas are scarce. Three hundred and two house mice and 161 black rats were trapped in 2013 from two urban neighbourhoods and a rural village in Yucatan, Mexico, and subsequently tested for Trypanosoma cruzi, Hymenolepis diminuta and Leptospira interrogans. Using the polymerase chain reaction we detected T. cruzi DNA in the hearts of 4·9% (8/165) and 6·2% (7/113) of house mice and black rats, respectively. We applied the sedimentation technique to detect eggs of H. diminuta in 0·5% (1/182) and 14·2% (15/106) of house mice and black rats, respectively. Through the immunofluorescent imprint method, L. interrogans was identified in 0·9% (1/106) of rat kidney impressions. Our results suggest that the black rat could be an important reservoir for T. cruzi and H. diminuta in the studied sites. Further studies examining seasonal and geographical patterns could increase our knowledge on the epidemiology of these pathogens in Mexico and the risk to public health posed by rodents.

Infection levels of intestinal helminths in two commensal rodent species from rural households in Yucatan, Mexico.

The aim of the present study was to calculate the prevalence and intensity of intestinal helminths in the house mouse (Mus musculus) and the black rat (Rattus rattus) trapped in rural households of Yucatan, Mexico. Sampling was conducted during the rainy season from October to December 2011 and the dry season from January to March 2012. A total of 154 M. musculus and 46 R. rattus were examined, with 84.2% of M. musculus being infected with helminths compared with a significantly lower prevalence of 52.2% in R. rattus (P< 0.01). Adult M. musculus were more likely to be infected with helminths (89%) than subadults (63%) (P< 0.01). Four helminth species were identified: Taenia taeniaeformis larvae, Nippostrongylus brasiliensis, Syphacia muris and Trichuris muris. Nippostrongylus brasiliensis was present more frequently in M. musculus than in R. rattus (P< 0.01) and in adult mice compared to subadults (P< 0.01). Trichuris muris was present only in adult mice. This is the first report of N. brasiliensis, S. muris and T. muris in Yucatan, Mexico, as well as the first to report the presence of N. brasiliensis in M. musculus from Mexico. The helminth fauna of commensal rodents present in households appears to constitute a low potential health risk to local inhabitants; however, it would be advisable to conduct further studies to better understand the public health risk posed by these rodent intestinal helminths.

Effects on platelet function of an EP3 receptor antagonist used alone and in combination with a P2Y12 antagonist both in-vitro and ex-vivo in human volunteers.

EP3 receptor antagonists may provide a new approach to the treatment of atherothrombotic disease by blocking the ability of prostaglandin E2 (PGE2) to promote platelet function acting via EP3 receptors. DG-041 is an EP3 antagonist in the early stage of clinical development. Here, we quantitated effects on platelet function of DG-041 in-vitro and ex-vivo after administration to man when given alone and concomitantly with clopidogrel or clopidogrel and aspirin. With its unique mechanism of action, it was anticipated that DG-041 would potentiate inhibition of platelet function when given in combination with clopidogrel without materially increasing bleeding time. Initially, in-vitro studies were performed to determine inhibitory effects of DG-041 (3 µM) used alone or in combination with the P2Y12 antagonist cangrelor (1 µM), both without and with aspirin (100 µM). Platelet aggregation and P-selectin expression were measured in whole blood (n = 10) following stimulation with the thromboxane A2 (TXA2) mimetic U46619 (0.3 or 1 µM) in combination with either the EP3 agonist sulprostone (0.1 µM), or PGE2 (1 µM). DG-041 alone partially inhibited platelet function in-vitro, as did cangrelor. Addition of both DG-041 and cangrelor in combination provided significantly greater inhibition. An ex-vivo study was then performed using the same experimental approaches. This clinical study was a prospective, randomised, blinded (for DG-041/matching placebo), blocked, crossover study designed to compare the effects of DG-041, clopidogrel, or the combination of DG-041 with either clopidogrel or clopidogrel and aspirin. Healthy volunteers (n = 42) were randomly assigned to receive no background treatment, clopidogrel (300 mg loading dose plus 75 mg daily) or clopidogrel and aspirin (75 mg daily) for 10 days alongside DG-041 (200 mg twice daily) or placebo for 5 days, crossed over to placebo or DG-041 for the next 5 days. Platelet effects and bleeding time were measured at baseline, days 5 and 10. DG-041 partially inhibited platelet function ex-vivo, as did clopidogrel, while administration of both DG-041 and clopidogrel provided significantly greater inhibition. Administration of DG-041 alone did not increase bleeding time, and did not significantly affect the increased bleeding time seen with clopidogrel or clopidogrel with aspirin. Using these experimental approaches, the antiplatelet effects of DG-041 and a P2Y12 antagonist used alone and in combination can be determined both in-vitro and ex-vivo. Results show inhibitory effects of DG-041 on platelet function acting via EP3 receptor blockade, confirmed to be additional to those brought about by P2Y12 blockade. In both in-vitro and ex-vivo studies, aspirin neither promoted nor negated the effects of the other drugs.

The occurrence of the larval cestode Cysticercus fasciolaris in rodent populations from the Cuxtal ecological reserve, Yucatan, Mexico.

Cysticercus fasciolaris is the larval stage of the cestode Taenia taeniaeformis, whose definitive hosts are mainly cats. This larval stage uses a wide variety of small rodents, and occasionally birds and humans, as intermediate hosts. In the Yucatan, there are no reports of the presence of this cestode in animal populations. The aim of this study was to evaluate the occurrence of C. fasciolaris in rodent populations from the Cuxtal ecological reserve, Yucatan, Mexico. Trapping of rodents was conducted from October 2009 to April 2010 in 40 households in Molas, in which Sherman traps were placed both inside and outside backyards. Rodents were dissected to inspect the liver for the presence of the worm. To determine risk factors associated with infection, univariate analysis was performed using sex, age, species, trapping site, and season as independent variables. Variables with a P value <  0.2 were analysed using a logistic regression model. In this study, 411 individuals of six rodent species were trapped; Mus musculus was the most abundant (78%), followed by Rattus rattus (13%) and the wild species Peromyscus yucatanicus, Ototylomys phyllotis, Heteromys gaumeri and Reithrodontomys gracilis (9%). Only 7.5% (n = 31) of M. musculus and R. rattus were infected with C. fasciolaris (demonstrated by the presence of liver cysts) with a prevalence of 9.0% and 3.5%, respectively. Both adults and male mice were 4.33 and 3.46 (OR values) times more likely to have C. fasciolaris than juveniles and females respectively. We can conclude that in the Cuxtal Reserve, Yucatan, Mexico, the prevalence of C. fasciolaris is higher in M. musculus, and that adult males had a higher probability of infection. Wild species, mainly P. yucatanicus, were not found to be infected with the cestode, but its presence in the backyards of households could result in a potential risk of acquiring this infection.

Measurement of platelet P-selectin for remote testing of platelet function during treatment with clopidogrel and/or aspirin.

There is great interest in assessing the efficacy of treatment with clopidogrel and aspirin in patients with cardiovascular disease using procedures that can be used in a remote setting. Here we have established methods to assess the effects of clopidogrel and aspirin on platelets based on measurements of platelet P-selectin. Platelets were stimulated in whole blood by adding the combination of adenosine diphosphate and the TXA(2) mimetic U46619 (ADP/U4, designed to assess P2Y(12) inhibition) or the combination of arachidonic acid and epinephrine (AA/Epi, designed to assess COX-1 inhibition). The stimulated samples were then fixed using a fixative solution that provides stability for at least 9 days, and sent to a central laboratory for analysis of P-selectin by flow cytometry. Measurements were performed in blood from healthy volunteers and patients with cardiovascular disease. The inhibitory effects of clopidogrel and aspirin were assessed ex vivo and the effects of the direct acting P2Y(12) antagonist cangrelor and aspirin were assessed in vitro. Measurements of platelet aggregation were also performed for comparison. In healthy volunteers clopidogrel ex vivo and cangrelor in vitro markedly inhibited P-selectin expression induced by ADP/U4. Aspirin did not inhibit and did not interfere with the effects of clopidogrel or cangrelor using this test. There was very little overlap of results obtained in the absence and presence of clopidogrel or cangrelor. In contrast, over half of 42 patients with cardiovascular disease did not respond well to clopidogrel treatment, although cangrelor was still effective. Aspirin markedly inhibited P-selectin expression induced by AA/Epi. Clopidogrel had much less effect and did not interfere with the effects of aspirin. There was no overlap of results obtained in the absence and presence of aspirin. Aspirin provided near-complete inhibition in 29 of 30 patients with cardiovascular disease. Aggregometry measurements agreed well with the P-selectin data obtained ex vivo following both clopidogrel and aspirin treatment. It is concluded that measurements of P-selectin performed on fixed blood samples following platelet stimulation in whole blood in a remote setting can be used effectively to monitor the effects of clopidogrel and aspirin.

Acyl derivatives of coenzyme A inhibit platelet function via antagonism at P2Y1 and P2Y12 receptors: a new finding that may influence the design of anti-thrombotic agents.

We have performed a detailed investigation of the effects on platelet function of coenzyme A (CoA) and several acyl-CoAs. Platelet aggregation was measured by turbidimetry and by platelet counting; platelet shape change was measured using light scattering; P-selectin, Ca2+ mobilization and vasodilator-stimulated phosphoprotein (VASP) phosphorylation were measured by flow cytometry. The compounds investigated inhibited ADP-induced platelet aggregation; those with saturated acyl groups containing 16-18 carbons were most effective. The effects of palmitoyl-CoA (16:0) were studied in depth. It inhibited platelet shape change and Ca2+ mobilization brought about by ADP (but not other agonists) indicating antagonism at P2Y(1) receptors, and also inhibited ADP-induced P-selectin expression. Effects of palmitoyl-CoA on the platelet aggregation and Ca2+ mobilization induced by several different agonists and agonist combinations were compared with those of MRS 2179 (a P2Y(1) antagonist) and AR-C69931 (a P2Y(12) antagonist), and were consistent with palmitoyl-CoA acting mainly at P2Y(1) but also with partial antagonism at P2Y(12) receptors. Antagonism at P2Y(12) receptors was confirmed in studies of VASP-phosphorylation. Palmitoyl-CoA did not act as an antagonist at P2X(1) receptors. The results are discussed in relation to the possibility that acyl-CoAs may contribute as endogenous modulators of platelet function and might serve as lead compounds for the design of novel antithrombotics.

Quantitation of platelet aggregation and microaggregate formation in whole blood by flow cytometry.

Platelet aggregation and microaggregate formation were measured in samples of stirred whole blood by flow cytometry. Blood samples were stirred in a multi-sample agitator with ADP, fixed and labelled with a platelet-specific CD42a-FITC fluorescent antibody. The blood was then diluted and applied directly to a flow cytometer. Platelets were identified using a gating procedure based on their expression of CD42a and then quantified. Aggregation was monitored as a fall in the number of single platelets. Both reversible and irreversible aggregation responses to ADP were determined and these were found to correlate directly with aggregation responses determined using a well-established single platelet counting technique using the Ultra-Flo 100 Whole Blood Platelet Counter. We found from flow cytometry that ADP-induced aggregation was coupled with a transient formation of platelet microaggregates over the initial 60 s following ADP addition. Assessment of single platelet loss by flow cytometry was found to be a reliable way of monitoring aggregation responses and provided new information on rapid microaggregate formation in ADP-stimulated blood.

Photoelectron spectroscopic and electronic structure studies of CH(2)O bonding and reactivity on ZnO surfaces: steps in the methanol synthesis reaction.

Adsorption of CH(2)O on ZnO(0001) has been investigated using XPS, NEXAFS, variable-energy photoelectron spectroscopy (PES), and density functional theory (DFT) calculations. CH(2)O is chemisorbed on the (0001) surface at 130 K. Its C1s XPS peak position at 292.7 eV and NEXAFS sigma shape resonance at 302.6 eV are consistent with an eta(1) bound surface geometry. Geometry optimized DFT calculations also indicate that CH(2)O is bound to the Zn(II) site in an eta(1) configuration through its oxygen atom. The variable-energy PES of the eta(1) bound CH(2)O/ZnO(0001) complex exhibits four valence band features at 21.2, 16.4, 13.8, and 10.7 eV below the vacuum level providing an experimental and theoretical description of this surface interaction. Annealing the ZnO(0001)/CH(2)O surface complex to 220 K decomposes the chemisorbed CH(2)O, producing formyl (291.5 eV), methoxide (290.2 eV), and formate (293.6 eV) intermediates. Thus this reaction coordinate involves the conversion of an oxygen bound formaldehyde to a carbon bound formyl species on ZnO(0001). Only formate is formed on the ZnO(100) surface. DFT is used to explore surface intermediates and the transition state in the methanol synthesis reaction (MSR). The bonding interactions of H(2), CO, CH(3)O(-), HCO(-), and trans-HCOH to the ZnO(0001) surface are elucidated using geometry optimization. H(2) was found to be heterolytically cleaved on the ZnO(0001) surface, and carbon monoxide, formyl, and methoxide are calculated to be eta(1) bound. These results are consistent with observed metal oxide surface reactivity where heterolytic bond cleavage is dominant. The oxygen atom in the bound formyl was found to be activated for attack by a proton. This results in the planar eta(1) bound trans-HCOH surface species. The transition state in the gas phase rearrangement of trans-HCOH to formaldehyde was calculated to have a barrier of 31 kcal/mol. The correlation diagram for this rearrangement in the gas phase indicates that configuration interaction at the crossing of two levels helps to lower the barrier. A transition state calculation was also performed for this rearrangement on the ZnO(0001) surface. The surface transition state geometry is significantly different than the gas phase. The surface geometry is no longer planar (23 degrees dihedral angle) and is displaced parallel to the surface. Interaction with the Zn(II) site at the crossing of surface bound CH(2)O and trans-HCOH levels further lowers the barrier to rearrangement relative to gas phase by 9 kcal/mol. The rearrangement of trans-HCOH (carbon bound) to CH(2)O (oxygen bound) on ZnO(0001) was calculated to be the overall barrier of the MSR reaction.

Interactions of platelets with Synthocytes, a novel platelet substitute.

A platelet substitute, Synthocytes, is being developed for the prevention and treatment of thrombocytopenia. Synthocytes are composed of fibrinogen adsorbed on heat stabilised albumin microcapsules of defined size. The purpose of this study was to perform experiments in vitro to investigate the capacity of Synthocytes to interact with platelets, one of the means through which Synthocytes may contribute to haemostatic plug formation in vivo. Synthocytes were found to interact with platelets as shown by platelet aggregation assays and measurements of [(14)C]5HT release from platelets in whole blood and platelet-rich plasma. Platelet-Synthocytes co-aggregate formation was demonstrated directly using flow cytometry and the presence of activated platelets in these co-aggregates was demonstrated using an antibody to P-selectin. Synthocytes enhanced platelet responsiveness to conventional aggregating agents such as ADP. Indeed, antagonists of the action of ADP on platelets inhibited the direct effects of Synthocytes on platelets in whole blood, as did a GPIIb/IIIa antagonist. Enhancement of annexin V binding was also observed, indicative of increased pro-coagulant activity. Experiments performed with control microcapsules (lacking fibrinogen) confirmed the importance of fibrinogen in the interactions that occurred. The results suggest that fibrinogen on the surface of Synthocytes can interact with GPIIb/IIIa on platelets to induce platelet activation, secretory activity and aggregation, and that ADP contributes to this process. This initial interaction renders platelets more susceptible to the stimulatory effects of other platelet-activating agents. It is considered likely that in the clinical setting of thrombocytopenia any interaction between Synthocytes and residual platelets that are present may contribute to primary haemostasis.

The central role of the P(2T) receptor in amplification of human platelet activation, aggregation, secretion and procoagulant activity.

Adenosine diphosphate (ADP) is an important platelet agonist and ADP released from platelet dense granules amplifies responses to other agonists. There are three known subtypes of ADP receptor on platelets: P2X(1), P2Y(1) and P(2T) receptors. Sustained ADP-induced aggregation requires co-activation of P2Y(1) and P(2T) receptors. AR-C69931MX, a selective P(2T) receptor antagonist and novel antithrombotic agent, was studied to characterize further the function of the P(2T) receptor. The roles of the P2Y(1) receptor and thromboxane A(2) were assessed using the selective P2Y(1) antagonist A2P5P and aspirin respectively. Aggregation was measured by whole blood single-platelet counting and platelet-rich plasma turbidimetry, using hirudin anticoagulation. Dense granule release was estimated using ([14)C]-5-hydroxytryptamine (HT)-labelled platelets. Ca(2+) mobilization, P-selectin expression, Annexin V binding and microparticle formation were determined by flow cytometry. P(2T) receptor activation amplified ADP-induced aggregation initiated by the P2Y(1) receptor, as well as amplifying aggregation, secretion and procoagulant responses induced by other agonists, including U46619, thrombin receptor-activating peptide (TRAP) and collagen, independent of thromboxane A(2) synthesis, which played a more peripheral role. P(2T) receptor activation sustained elevated cytosolic Ca(2+) induced by other pathways. These studies indicate that the P(2T) receptor plays a central role in amplifying platelet responses and demonstrate the clinical potential of P(2T) receptor antagonists.

2H-Thieno[3,2-e]- and [2,3-e]-1,2-thiazine-6-sulfonamide 1,1-dioxides as ocular hypotensive agents: synthesis, carbonic anhydrase inhibition and evaluation in the rabbit.

Novel non-chiral 2H-thieno[3,2-e]- and [2,3-e]-1,2-thiazine-6-sulfonamide 1,1-dioxides were synthesized for evaluation as potential candidates for the treatment of glaucoma. All of the compounds prepared were potent high affinity inhibitors of human carbonic anhydrase II, Ki < 0.5 nM. Additionally, inhibition of recombinant human carbonic anhydrase IV was determined for selected compounds; these were shown to be moderate to potent inhibitors of this isozyme with IC50 values ranging from 4.25 to 73.6 nM. Of the compounds evaluated for their ability to lower intraocular pressure in naturally hypertensive Dutch-belted rabbits, 5a, 17a3, 17b1, 17b2, 17h2 and 17i1 showed significant efficacy (> 20% decrease) in this model following topical ocular administration.

Effects of cytochalasin H, a potent inhibitor of cytoskeletal reorganisation, on platelet function.

Platelets contain a well-developed and dynamic cytoskeleton composed mainly of actin and actin-associated proteins. Upon platelet activation there is rapid polymerisation of actin and a marked reorganisation of the platelet cytoskeleton. Cytochalasins are agents that interfere with the polymerisation of actin, and it has recently been discovered that cytochalasin H (CyH) is particularly effective as an inhibitor of the cytoskeletal reorganisation that occurs in platelets following activation by adenosine diphosphate (ADP). Here we have used CyH to inhibit platelet cytoskeletal reorganisation and to determine its effects on various aspects of platelet function. Experiments were performed in hirudinized platelet-rich plasma (PRP) or whole blood obtained from human volunteers. PRP was treated with 10 microM CyH or vehicle, then activated by ADP. The effect of CyH on cytoskeletal reorganisation was determined by SDS-PAGE of the Triton X-100 insoluble cytoskeletons and quantitated by densitometry. Platelet aggregation and aggregate stability in PRP were measured by monitoring changes in light absorbance; aggregation was measured in whole blood via platelet counting. Shape change, P-selectin expression and changes in intracellular calcium were measured using flow cytometry. CyH prevented the normal incorporation of actin, alpha-actinin and actin-binding protein into the cytoskeleton that occurred following ADP activation, and incorporation of myosin was markedly reduced. Aggregation was only partially inhibited but, more dramatically, the rate of disaggregation following addition of certain agents that interfere with fibrinogen binding to glycoprotein IIb/IIIa on the surface of platelets was markedly increased. The ADP-induced shape change was also inhibited. CyH had no effect on calcium mobilisation. Curiously, expression of P-selectin was potentiated by CyH, suggesting a modulatory role of the cytoskeleton in platelet secretory activity. The results suggest that cytoskeletal reorganisation plays an important role in platelet shape change and aggregation and contributes in a major way to the stability of the aggregates that form.

A mouse fibroblast line cycles between monolayer and spheroid forms, regulates Met and HGF expression, and releases an attachment and growth-promoting substance.

A subline of mesoderm-derived mouse NIH3T3 fibroblasts was selected for its ability to proliferate in serum-free media. This cell line (SFDH) grows as a monolayer at low density and spontaneously forms dense, multicellular spheroids at high density. Spheroid formation can also be induced by the addition of dexamethasone, polybrene, or heparin. Spheroids eventually detach from the substrate, but will reattach and re-form monolayers when transferred to fresh culture vessels and media, repeating the cycle again upon reaching high density. Thin section analysis of spheroids shows morphologically-distinct regions of cells, including an attenuated outer surface and a cuboidal interior with occasional lumen-like areas. Over time in culture, spheroids express increasing levels of met, the Met ligand-SF/HGF and cytokeratin, an epithelial marker, in comparison to monolayers. Both monolayer and spheroid-derived cells are rapidly tumorigenic in nude mice. Media conditioned by SFDH cells contain factors that stimulate growth and attachment of a variety of tumorigenic and non-tumorigenic cell lines, inducing cells to divide in serum-free media for up to 14 days when plated on tissue culture-treated and nontreated plastic surfaces pre-coated with SFDH conditional media. The growth-stimulating activity fractionates as a single peak over a sepharose column in the presence of 6 m urea, and sediments as a high molecular weight complex. Growth-stimulating activity can be neutralized by several antisera specific for hepatocyte growth factor, and the same sera recognize a novel approximately 37 kD protein in active supernatants. The cyclic, continuous nature of alternating monolayer and spheroid forms makes this cell line appropriate for studying changing gene expression patterns in progressive cell-cell/cell-matrix interactions.

A whole blood assay of inhibition of platelet aggregation by glycoprotein IIb/IIIa antagonists: comparison with other aggregation methodologies.

We have used a whole blood single-platelet counting assay (WB-SPC) that is sensitive to microaggregation for monitoring GPIIb/IIIa antagonists and have compared this with other methodologies. In vitro effects of the GPIIb/IIIa antagonist fradafiban on ADP-induced platelet aggregation were determined using WBSPC and PRP turbidimetry, comparing citrate and hirudin anticoagulation. Fradafiban was a more potent inhibitor of aggregation assessed by PRP turbidimetry compared to WBSPC. Citrate showed only a trend towards enhancing fradafiban potency (p = 0.087). Citrated blood from 8 patients with unstable angina, randomised to receive oral lefradafiban (the oral prodrug of fradafiban) or placebo, was studied before and during treatment using WBSPC, PRP turbidimetry, impedance aggregometry and Rapid Platelet Function Assay (RPFA, Accumetrics). RPFA, PRP turbidimetry and WBSPC measurements correlated well. Impedance aggregometry responses were oversensitive to GPIIb/IIIa blockade. WBSPC was most discriminating at high levels of inhibition and offered a rapid means of monitoring GPIIb/IIIa antagonist effect within the therapeutic range of inhibition.