Frontal sinus depth at four landmarks in breeds of dog typically affected by sinonasal aspergillosis.

The objective of this study was to assess whether the frontal sinuses in dogs with aspergillosis and of breeds typically affected by this condition were deeper at a more caudal location. CT scans of the head performed at the Small Animal Teaching Hospital, University of Liverpool, between April 2007 and March 2009 for dogs diagnosed with aspergillosis (group 1) and unaffected dogs of similar breeds (group 2) were selected for study. Sinus depth was measured at four standardised locations from reconstructed images of these CT scans. Data were compared for differences in sinus depth between groups and between landmarks. No significant difference was found between measurements within individual dogs or for each of the various landmarks between groups. Difference in depth of the sinuses between landmarks was significant (P<0.001). Sinus depth was significantly greater at the more caudal landmarks and was shallowest at the previously recommended landmark for sinus entry. In 54 per cent of dogs, the frontal sinus depth measured less than or equal to 2 cm at one or more of the landmarks. Sinus entry at the deepest point will reduce the risk of accidentally damaging underlying structures. This may be approximately 1 cm caudal, in breeds of dog that typically develop aspergillosis, to a previously suggested landmark.

Recent trends in the intrinsic water-use efficiency of ringless rainforest trees in Borneo.

Stable carbon isotope (δ(13)C) series were developed from analysis of sequential radial wood increments from AD 1850 to AD 2009 for four mature primary rainforest trees from the Danum and Imbak areas of Sabah, Malaysia. The aseasonal equatorial climate meant that conventional dendrochronology was not possible as the tree species investigated do not exhibit clear annual rings or dateable growth bands. Chronology was established using radiocarbon dating to model age-growth relationships and date the carbon isotopic series from which the intrinsic water-use efficiency (IWUE) was calculated. The two Eusideroxylon zwageri trees from Imbak yielded ages of their pith/central wood (±1 sigma) of 670 ± 40 and 759 ± 40 years old; the less dense Shorea johorensis and Shorea superba trees at Danum yielded ages of 240 ± 40 and 330 ± 40 years, respectively. All trees studied exhibit an increase in the IWUE since AD 1960. This reflects, in part, a response of the forest to increasing atmospheric carbon dioxide concentration. Unlike studies of some northern European trees, no clear plateau in this response was observed. A change in the IWUE implies an associated modification of the local carbon and/or hydrological cycles. To resolve these uncertainties, a shift in emphasis away from high-resolution studies towards long, well-replicated time series is proposed to develop the environmental data essential for model evaluation. Identification of old (greater than 700 years) ringless trees demonstrates their potential in assessing the impacts of climatic and atmospheric change. It also shows the scientific and applied value of a conservation policy that ensures the survival of primary forest containing particularly old trees (as in Imbak Canyon and Danum).

Promoter trapping identifies real genes in C. elegans.

Promoter trapping involved screening uncharacterized fragments of C. elegans genomic DNA for C. elegans promoter activity. By sequencing the ends of these DNA fragments and locating their genomic origin using the available genome sequence data, promoter trapping has now been shown to identify real promoters of real genes, exactly as anticipated. Developmental expression patterns have thereby been linked to gene sequence, allowing further inferences on gene function to be drawn. Some expression patterns generated by promoter trapping include subcellular details. Localization to the surface of particular cells or even particular aspects of the cell surface was found to be consistent with the genes, now associated with these patterns, encoding membrane-spanning proteins. Data on gene expression patterns are easier to generate and characterize than mutant phenotypes and may provide the best means of interpreting the large quantity of sequence data currently being generated in genome projects.

Lectin staining of peritoneal mesothelial cells in vitro.

A survey of lectin-binding specificities present on rodent and human mesothelial cells propagated and maintained in tissue culture was made using fluorescein isothiocynate conjugated (FITC) lectins. Rodent and human cells exhibited cell-associated fluorescence following exposure to the FITC-lectins from C. ensiformis, T. vulgaris, A. hypogaea, E. cristagalli and B. simplicifolia, but not with lectins from G. max and D. biflorus. Rodent cells were also positive for FITC-M. pomifera lectin binding. Human, but not rodent, cells were positive for FITC-T. purpureas lectin binding. Exposure of rabbit mesothelial cells in vitro to FITC-lectins that bound to the cell surface resulted in the appearance of discrete loci of putatively intracellular fluorescence. Exposure of cells to ferritin-labelled T. vulgaris lectin at 37 degrees C for as little as 7.5 minutes resulted in the appearance of ferritin-size particles in intracellular vesicles. These results demonstrate 1. the presence of lectin-binding sites in and on peritoneal mesothelial cells from rodents and humans and 2. a possible role of such sites in mediating the entry of lectin-like endogenous molecules into the vacuolar apparatus of these cells.

In vivo and in vitro characterization of mesothelial lipid inclusions.

The nature of intracytoplasmic lipid inclusions found in cultured rabbit and rat peritoneal mesothelial cells was examined by ultrastructural and biochemical techniques. Transmission electron microscopy also demonstrated extracellular release of these lipid bodies. Differential fixation with tannic acid revealed 2 types of inclusions, lamellated (lamellar bodies) and nonlamellated (homogeneous). The lamellar bodies were found near or in the Golgi apparatus and on the cell surface where occasionally they were observed in exocytotic pouches. The homogeneous inclusions were the predominant species being found primarily intracellularly. Lipid bodies obtained from the culture media over the cells displayed on electron microscopy the same morphological characteristics as those seen intracellularly. Exposure of confluent cultures of mesothelial cells to the vital lipid stain Nile Red caused the appearance of intensely fluorescent droplets in or on the cells at wave lengths consistent with staining for phosphatidylcholine-rich vesicles. Incubation of the cells with (14C)-choline and subsequent analysis of phospholipid formation revealed high rates of (14C)-phosphatidylcholine addition to both intra- and extracellular lipid pools. Taken together, mesothelial cells exhibit lipid bodies similar in ultrastructure to the surfactant containing organelles of Type II pneumocytes.

Autosomal recessive polycystic kidney disease: characterization of human peritoneal and cystic kidney cells in vitro.

Renal cystic epithelia and peritoneal mesothelia from two humans with autosomal recessive polycystic kidney disease (ARPKD) were grown in culture. Cystic epithelial and mesothelial cells formed continuous monolayers in vitro. By electron microscopy, cystic renal cells exhibited a single apical cilium and numerous short, stubby microvilli, both in situ and in vitro. Mesothelial cells exhibited intra- and extracellular membrane-limited, lipid-filled vesicles and surface microvilli. Cystic kidney cells in vitro stained positive for lectins from Cancanavalia ensiformis (concanavalin A), Triticum vulgaris, Erythrina cristagalli, Ulex europeaus, and Arachis hypogaea. Immunocytochemical and lectin staining revealed the renal and peritoneal cells to be of collecting tubule and mesothelial origin, respectively. Both cell types showed large depositions of glycogen granules in the cytoplasm during propagation in certain culture media; in kidney cells, dibutyryl cyclic adenosine monophosphate (cAMP) abolished glycogen depositions. Glycogen deposition also was observed in liver tissue obtained by needle biopsy from one patient. No bacteria were cultured from nor endotoxin detected in the renal cyst fluid. Relative to serum, the cyst fluids contained low sodium, potassium, and chloride levels. Thus, cultured ARPKD cells demonstrate a number of characteristics that are different from cells derived from the autosomal dominant form of renal cystic disease (ADPKD).

A simple and sensitive bioassay for the detection of toxic materials using a unicellular green alga.

A simple assay for phytotoxicity, using small scale cultures of Chlorella vulgaris, is described. Growth after 3 days' incubation in 0.5 cm(3) medium is assessed by DMSO-acetone extraction and chlorophyll determination. The method is sensitive enough to detect phytotoxicity in small samples of material and this is illustrated by the inhibitory effects of microgram additions of copper, cadmium and zinc.

Expression of von Willebrand factor in plasma and platelets of cats.

Immunochemical methods that are used to assess von Willebrand factor in human beings and dogs were used to assess von Willebrand factor in 3 cat species. Our findings indicated that the expression and multimeric composition of von Willebrand factor in plasma and platelets of cats were similar to those reported in human beings and dogs. We suggest that these methods may be used to evaluate von Willebrand disease in members of the cat family used in this study.

Isolation and propagation in vitro of peritoneal mesothelial cells.

Mesothelial cells lining the peritoneal cavity are the primary site of molecular exchange during peritoneal dialysis, a life support system for over 50,000 patients worldwide. In this study, techniques are described for the isolation and propagation in culture of peritoneal mesothelial cells from rats and rabbits. For comparison, mesothelial cells were also obtained from the serosal surface of human colonic tissue. By electron microscopy the cultured cells were found to exhibit microvilli, a well-developed endoplasmic reticulum and golgi apparatus, micropinocytotic vesicles, and lipid-filled intracellular vesicles. Immunochemical probes revealed the expression by these cells in vitro of cytokeratin, fibronectin, vimentin, and keratin, but not von Willebrand factor. Mesothelial cells from rat, rabbit, and human exhibited contact inhibition, but differences in growth rates and dependence on supplements to the growth media. This work provides a multispecies comparison of the behavior of mesothelial cells in vitro for the purpose of developing an experimental system for the study of mesothelial cell biology and the role of these cells in peritoneal dialysis.

An undetectable source of technical error that could lead to false negative results in enzyme linked immunosorbent assay of antibodies to HIV-1.

Since the institution of routine testing for antibodies to Human Immunodeficiency Virus (HIV) using the enzyme-linked immunosorbent assay (ELISA), the specificity and sensitivity of this assay system has received significant scrutiny. During previous use of this methodology, we have quantified rates of false biological positive results using commercial kit assays in a normal donor population. In this study, we have identified a potential source for false negative results. Using multiple lots of two different commercial ELISA kits, the absorbance readings at the test end point could not differentiate between normal non-reactive donor samples and blanks containing no sample. These results occur using normal donor samples, even though the assays could distinguish between blank wells and the manufacturers' "normal controls", provided with the assay. Our findings suggest that a technical pipetting error is presently undetectable, either visually or by statistical methods, and could permit an untested, potentially HIV-1 positive, unit to be released into the transfusable blood supply. A possible solution is suggested.

Canine platelet von Willebrand factor: quantification and multimeric analysis.

Washed canine platelets were shown to express a significant level of von Willebrand factor (vWf). Canine platelet vWf differed from canine plasma vWf by the absence of satellite bands associated with each multimer when resolved by SDS-agarose gel electrophoresis. Expression and multimeric composition of canine platelet vWf was quite similar to that of human platelet vWf. Quantification in both lysed, washed canine platelets and in releasate of washed canine platelets yielded estimates of platelet vWf at approximately 2% of circulating vWf in this species, with approximately 15% of this being released into the fluid phase on activation. This contrasts with findings in humans, in which approximately 10%-25% of circulating vWf is compartmentalized in platelets. The difference in relative levels of canine and human platelet vWf could not be accounted for by differences in platelet ultrastructure. The decreased relative level may account for reports that canine platelets contain no vWf.

Canine von Willebrand factor expresses a multimeric composition similar to human von Willebrand factor.

Canine von Willebrand factor (vWf) was compared to human vWf. Antisera raised against human vWf or canine vWf cross-reacted with both heterologous proteins and reactions of partial identity were seen using crossed immunoelectrophoresis. Similar patterns of multimerization were obtained for vWf from both canine and human sources using the enzyme-linked immunoelectrotransfer blot method. However, the canine protein displayed an altered electrophoretic mobility. The molecular weight of the vWf monomer was estimated by SDS-PAGE and found to be indistinguishable from that of human vWf monomer. Canine vWf is decreased in animals with clinically evident hypothyroidism and in heterozygous "carriers" of von Willebrand's disease (vWd) that display no clinical symptoms of vWd. Results expand the concept that von Willebrand's disease in dogs may be a useful model for study of vWd in humans, and that immunochemical methods established for studies of human vWf appear appropriate for studies of canine vWf.

A second discriminator for biological false positive results in enzyme-linked immunosorbent assays for antibodies to human immunodeficiency virus (HTLV-III/LAV).

Two commercially available enzyme-linked immunosorbent assays (ELISA) were compared in screening a large population of volunteer blood donors. One ELISA utilized the human T-lymphotropic virus, Type III (HTLV-III) grown on National Institutes of Health T-lymphocyte cell line, H-9, as antigen source; the second used lymphadenopathy associated virus (LAV) grown on Pasteur Institutes' T-lymphocyte cell line, CEM-F. Biological false positives (BFP) occurred at a rate of approximately 0.5 percent using each antigen source. However, distinct populations of BFP donors were detected when the two antigen sources were compared. Results indicate that at least two separate sets of antigens are recognized in ELISA by our normal population and result in BFP. Sequential utilization of tests using these distinct sources adds a second discriminator to identification of BFP, with the potential for decreasing the requirement for Western blot analysis.

Endothelial cell synthesis of von Willebrand antigen II, von Willebrand factor, and von Willebrand factor/von Willebrand antigen II complex.

von Willebrand antigen II (vW AgII) and von Willebrand factor (vWf) are immunochemically distinct proteins that are deficient in the plasma and platelets of patients with severe von Willebrand's disease. Normal human umbilical vein endothelial cells were cultured in the presence of [35S]methionine. Crossed immunoelectrophoresis of endothelial cell supernates and detergent-solubilized endothelial cells demonstrated specific incorporation of the [35S]methionine into vW AgII. Furthermore, when endothelial cells were lysed in the presence of proteolytic inhibitors, a second, less anodal peak was identified on crossed immunoelectrophoresis. This peak represented a complex of vW AgII and vWf and demonstrated a reaction of complete identity with the vW AgII immunoprecipitate. When plasma, serum, or platelets were evaluated by crossed immunoelectrophoresis, this "complex" peak was not present. When antibodies to vWf, fibronectin, or fibrinogen were present in the first dimension of crossed immunoelectrophoresis, only the antibodies to vWf removed the complex. Radioiodinated polyclonal and monoclonal antibodies to vWf also localized vWf to this complex. Under reducing conditions, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [35S]methionine-labeled immunoprecipitates indicated that the molecular weight of vW AgII is 98,000 and that vWf was present as two species of 220,000 and 260,000 mol wt, respectively. Immunofluorescent microscopy of endothelial cells demonstrated colocalization of vW AgII and vWf in endothelial cells with intense immunostaining of the same subcellular granules.

Correlation between circulating levels of von Willebrand's antigen II and von Willebrand factor: discrimination between type I and type II von Willebrand's disease.

Classification of the subtypes of von Willebrand's disease (vWd) has been based on a quantitative deficiency or an abnormal multimeric composition of von Willebrand factor (vWf). Although the co-deficiency of a second protein, von Willebrand's antigen II (vW AgII), had been previously recognized, its concentration in a relatively large number of normal individuals or patients with well-defined vWd variants had not been studied. The plasma from patients with type I, IIA, IIB, IIC, and III (severe) vWd was evaluated, and the concentrations of vW AgII and vWf were determined. Although patients with type I and III vWd had reduced levels of both proteins, the plasma vW AgII concentration was normal in patients with type II vWd. Analysis of the results indicates that type I and type II variants can be discriminated with greater than 80% accuracy by comparison of results of these two antigenic assays. The normal levels of vW AgII in type II variants suggest a possible difference in the pathophysiology of type I and type II vWd.

A study of a Caucasian family with variant von Willebrand's disease in association with vascular telangiectasia and haemoglobinopathy.

A family was identified which carries multi-haematological disorders including Type IIA von Willebrand's disease, vascular telangiectasia, and a haemoglobinopathy (haemoglobin S trait). In the affected individuals, the von Willebrand's disease varies in its expression from an asymptomatic form to a severe form especially in those patients with telangiectasia. Some patients have vascular telangiectasia in the mucous membranes of the mouth and lips. In two patients endoscopy disclosed telangiectasia in the mucous membranes of the gastrointestinal tract. All of the patients who had telangiectasia also had von Willebrand's disease. An incidental finding was the presence of an abnormal haemoglobin (haemoglobin S) in some family members. The pattern of inheritance of the haemoglobinopathy was unrelated to the inheritance pattern of von Willebrand's disease. The presence of haemoglobin S did not interfere with the aggregation of platelets in response to ristocetin.

The effect of DDAVP on plasma levels of von Willebrand antigen II in normal individuals and patients with von Willebrand's disease.

The infusion of 1-deamino-(8-D-arginine)-vasopressin (DDAVP) causes not only an elevation in factor VIII-related antigen (FVIIIR:Ag), but also a marked elevation of plasma von Willebrand antigen II (vWAgII). vWAgII reaches a peak concentration at 60 min and is elevated 3-8-fold over basal levels in normal individuals and individuals with type I, IIA, and IIB von Willebrand's disease. As the mechanism of hemostatic alteration brought about by DDAVP might be due to release of endothelial cell proteins, endothelial cell cultures were performed. The cultures demonstrated synthesis and secretion of vWAgII, as evidenced by the incorporation of 35S-methionine into the vWAgII molecule. Thus, vWAgII, like FVIIIR:Ag, is an endothelial cell protein.

Variant von Willebrand's disease and pregnancy.

The clinical course and coagulation profile of a pregnant patient with variant von Willebrand's disease were followed from the second trimester through puerperium. The clinical course was characterized by a normal delivery and absence of abnormal bleeding or need for replacement therapy. The coagulation profile demonstrated an increase in factor VIII procoagulant activity, factor-VIII-related antigen, and platelet aggregation activity in response to ristocetin prior to delivery. Postpartum, these factors decreased to prepregnancy values with distinctly different patterns. Factor VIII procoagulant activity continued to rise for 5 days after delivery and then decreased with a half-life of approximately 6 days. Factor-VIII-related antigen began to decrease just prior to delivery, displaying a half-life or approximately 6 days. Ristocetin cofactor activity, however, dropped immediately postpartum and displayed a half-life of approximately 6 hr. The ristocetin cofactor activity was associated with factor-VIII-related antigen, which displayed a significantly smaller molecular weight than does normal factor-VIII-related antigen. Larger aggregates of factor-VIII-related antigen. Larger aggregates of factor-VIII-related antigen did not appear during the pregnancy, and ristocetin cofactor activity could not be demonstrated in fragments of less than 0,8 x 10(6).