Seroprevalence of Brucella antibodies in harbor seals in Alaska, USA, with age, regional, and reproductive comparisons.

Populations of harbor seal Phoca vitulina in the Gulf of Alaska have dramatically declined during the past 4 decades. Numbers of seals in Glacier Bay, in southeast Alaska, USA, have also declined despite extensive protection. Causes of the declines and slow recovery are poorly understood. Brucellosis is a zoonotic disease that adversely affects reproduction in many domestic species. We measured the seroprevalence of Brucella antibodies in 554 harbor seals in 3 Alaska locations: Prince William Sound (PWS), Glacier Bay (GB), and Tracy Arm Fords Terror (TAFT) Wilderness Area. Objectives included testing for regional, sex, age, and female reproductive state differences in Brucella antibody seroprevalence, persistence in titers in recaptured seals, and differences in titers between mother seals and their pups. Overall, 52% of adults (AD), 53% of subadults (SA), 77% of yearlings (YRL), and 26% of <5 mo old pups were seropositive. Matched mother-pup samples were consistent with dependent pups acquiring maternal passive immunity to Brucella. Results show higher seroprevalence (64%) for AD and SA seals in the depressed and declining populations in PWS and GB than in TAFT (29%). Lactating females were less likely to be seropositive than other AD females, including pregnant females. Further research is needed to seek evidence of Brucella infection in Alaskan harbor seals, identify effects on neonatal viability, and assess zoonotic implications for Alaska Natives who rely on harbor seals for food.

Northern elephant seal platelets: analysis of shape change and response to platelet agonists.

Blood platelets have a vital role in the maintenance of normal mammalian hemostasis. Rapid pressure changes and temperatures lower than 20 degrees C cause activation of human and terrestrial mammal platelets. Elephant seals are routinely subjected to pressures as high as 150 atm, yet do not suffer from the thrombotic effects of platelet activation associated with rapid decompression. We examined the ultrastructure of Northern elephant seal (Mirounga angustirostris) platelets and their functional and morphological response to various platelet agonists. Unstimulated elephant seal platelets are discoid cells, with a microtubule coil, randomly dispersed alpha and dense granules, and glycogen granules. There are well-defined areas of membranous invaginations that indicate the presence of an open canalicular system (OCS). Aggregometry was used to determine the response of elephant seal platelets to various platelet agonists. Dose-dependent curves were generated for thrombin, collagen, and adenosine diphosphate (ADP). Platelet response to thrombin was dose-dependent and was maximal at 2.5 U/ml. Platelets collected in sodium citrate had blunted responses to both ADP and collagen. ADP stimulation caused only reversible, primary activation (shape change) at > or = 5 microM, while platelets did not aggregate in response to any concentration of collagen. Platelets collected in sodium heparin did respond fully to both to ADP and collagen. There was small, reversible shape change in response to ristocetin, but no response to epinephrine. Decreased sensitivity of elephant seal platelets to agonists may be a protective mechanism developed in response to rapid pressure changes and cold temperatures associated with adaptation to an extreme environment.

Urokinase plasminogen activator system gene expression is increased in human breast carcinoma and its bone metastases--a comparison of normal breast tissue, non-invasive and invasive carcinoma and osseous metastases.

The urokinase plasminogen activator (uPA) system has been widely associated with the development of breast carcinoma. However, the role of the urokinase pathway in the development of osseous breast cancer metastases has been largely overlooked. We studied the expression of uPA, urokinase plasminogen activator receptor (uPAR)- and plasminogen activator inhibitor type-1 (PAI-1) in human breast carcinomas and their bone metastases, using in situ hybridisation. Studies were performed using paraffin-embedded tissue from 13 ductal carcinomas, 23 invasive ductal carcinomas, five normal breasts and 25 bone metastases. The majority of the tumours examined expressed low to moderate levels of uPA mRNA and low to high levels of uPAR and PAI-1 mRNA, which was predominantly localised to the epithelial tumour cells. There was slight over-expression of uPA and PAI-1 mRNA and a marked increase in uPAR mRNA expression in the malignant tumours compared with benign tissue. Overall, uPAR and PAI-1 mRNA expression was found to be more variable than uPA mRNA, suggesting a possible role of the receptor and inhibitor in the regulation of uPA activity. Increased alpha1(I) procollagen (COL) and osteopontin (OPN) mRNA expression was detected, primarily in the stromal cells, in malignant tumours compared with the benign tissue. The increased expression of the components of the uPA system on the epithelial tumour cells may account for the activation of the proteolytic cascade that occurs during breast cancer metastasis to bone. Furthermore, the over-expression of COL and OPN suggests a possible interaction between these matrix proteins and the uPA system.

Animal models for studies on cold-induced platelet activation in human beings.

When human platelets are chilled below about 20 degrees C, they spontaneously activate, a phenomenon that limits their storage lifetime. We have previously shown that this activation in chilled human platelets is associated with passage through a lipid phase transition. Because animal models are necessary for Investigating methods for cold storage of platelets, it is essential to determine whether such phase transitions and chilling-induced activation are found in these models. In this study we examined platelets from some commonly used animal models-pigs, rhesus monkeys, mice, dogs, and rabbits. Using Fourier transform infrared spectroscopy (FTIR), we detected the thermotropic membrane phase transition in Intact platelets and assessed the morphologic response of the platelets to chilling. Statistical analysis of both FTIR and shape change show that of the animal models tested, pig platelets are most similar to human platelets. These studies suggest that pigs and pig platelets are the models of choice for the study of cold-induced platelet activation in human beings.

Lipid phase separation correlates with activation in platelets during chilling.

When human platelets are chilled below 22 degrees C, they spontaneously activate, a phenomenon that severely limits their storage life. It has previously been proposed that there is a correlation between cold-induced platelet activation and passage of the membranes through a liquid-crystalline to gel phase transition. Because animal models are essential for developing methods for cold storage of platelets, it is necessary to investigate such a correlation in animal platelets. In this work, horse platelets were used as a model, and it was found that cold-induced morphological activation is related to the lipid phase transition. Using fluorescence microscopy with the lipophilic fluorescent dye 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (Dil-C18), and Fourier transform infrared spectroscopy (FTIR), it was found that lipid phase separation occurs during cooling and low temperature storage. Furthermore, removal of cholesterol from the plasma membrane also induced a phase separation, possibly between specific phospholipid classes. Steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) and trimethylammonium-DPH (TMA-DPH) were compared in cells and multilamellar vesicles (MLV) composed of platelet lipids. Cholesterol depletion led to a decrease in the fluorescence anisotropy of the two probes, which can be explained by changes in the order of the phospholipid molecules. In addition, the lipid composition and fatty acid profile of the cellular phospholipids were determined. Based of the similarities between horse and human platelets, it is suggested that horse platelets may be used as a model for studying cold-stored platelets. The results are discussed in relation to the possible role of phase separation during cell signalling.

Platelet-erythrocyte adhesion in sickle cell disease.

BACKGROUND: The abnormal adherence of sickle red blood cells (sRBC) to other cell types likely contributes to vaso-occlusion. Increased numbers of platelet-erythrocyte aggregates (PEA) and platelet activation have been described in sickle cell disease. The present study was undertaken to determine the contribution, if any, of the extracellular matrix protein thrombospondin to the adhesion of sRBC and platelets. METHODS: Platelet activation and PEA were measured using fluorescent-labeled monoclonal antibodies and flow cytometry. Platelet red-cell adhesion was measured by a gravity sedimentation assay. Erythrocyte-bound thrombospondin (TSP) was determined by enzyme-linked immunoabsorbant assay (ELISA). RESULTS: Our studies demonstrate significant platelet activation and adhesion of sRBC to platelets in sickle cell disease. Thrombospondin was detected on sRBC. There was variable inhibition of sRBC-platelet adhesion by antibodies to CD36 (thrombospondin receptor) and antibodies to thrombospondin. CONCLUSIONS: Thrombospondin on sRBC may mediate, at least in part, sRBC-platelet adhesion in sickle cell disease. The study of heterotypic cell-cell interactions is important in understanding the pathogenesis of vaso-occlusion in sickle cell disease.