A comprehensive ovine model of blood transfusion.

BACKGROUND: The growing awareness of transfusion-associated morbidity and mortality necessitates investigations into the underlying mechanisms. Small animals have been the dominant transfusion model but have associated limitations. This study aimed to develop a comprehensive large animal (ovine) model of transfusion encompassing: blood collection, processing and storage, compatibility testing right through to post-transfusion outcomes. MATERIALS AND METHODS: Two units of blood were collected from each of 12 adult male Merino sheep and processed into 24 ovine-packed red blood cell (PRBC) units. Baseline haematological parameters of ovine blood and PRBC cells were analysed. Biochemical changes in ovine PRBCs were characterized during the 42-day storage period. Immunological compatibility of the blood was confirmed with sera from potential recipient sheep, using a saline and albumin agglutination cross-match. Following confirmation of compatibility, each recipient sheep (n = 12) was transfused with two units of ovine PRBC. RESULTS: Procedures for collecting, processing, cross-matching and transfusing ovine blood were established. Although ovine red blood cells are smaller and higher in number, their mean cell haemoglobin concentration is similar to human red blood cells. Ovine PRBC showed improved storage properties in saline-adenine-glucose-mannitol (SAG-M) compared with previous human PRBC studies. Seventy-six compatibility tests were performed and 17·1% were incompatible. Only cross-match compatible ovine PRBC were transfused and no adverse reactions were observed. CONCLUSION: These findings demonstrate the utility of the ovine model for future blood transfusion studies and highlight the importance of compatibility testing in animal models involving homologous transfusions.

Treatment with quercetin and 3',4'-dihydroxyflavonol inhibits platelet function and reduces thrombus formation in vivo.

Flavonols are polyphenolic compounds with reported cardiovascular benefits and have been shown to exhibit antiplatelet properties in vitro. While some studies have shown inhibition of platelet aggregation following dietary supplementation with flavonol rich foods, few studies have assessed the ability of flavonols to inhibit platelet mediated thrombus generation in vivo. Furthermore, the duration of benefit and the influence of different dosing regimens remain unclear. In this study we investigate the ability of two structurally related flavonols; quercetin (Que) and 3',4'-dihydroxyflavonol (DiOHF) to inhibit platelet aggregation, platelet granule exocytosis and vessel occlusion in a well characterized mouse model of platelet mediated arterial thrombosis. We investigated the effect of a single 6 mg/kg intravenous bolus and daily 6 mg/kg intraperitoneal doses over seven consecutive days. Carotid artery blood flow after injury was better maintained in mice treated with both Que and DiOHF when compared to the vehicle for both dosage regimens. This improved blood flow corresponded to inhibition of platelet aggregation and platelet dense granule exocytosis following chemical stimulation of PAR4. We therefore provide evidence of inhibition of platelet-mediated arterial thrombosis by flavonols in vivo, and demonstrate that this effect persists for at least 24 h after the last intraperitoneal dose. These data suggest a potential clinical role for flavonols as anti-platelet therapy.

Next Generation Sequencing of Human Platelet Antigens for Routine Clinical Investigations and Donor Screening.

Human platelet antigen (HPA) genotyping is performed in a number of clinical scenarios, including characterization of immune-mediated thrombocytopenia and provision of HPA-matched platelets. Current gold-standard methods for HPA genotyping utilize single nucleotide variant (SNV) based approaches. This review aims to ascertain if next generation sequencing (NGS) has reasonable grounds to replace SNV-based genotyping for HPA systems. A systematic review was conducted following a comprehensive literature search in accordance with the Preferred Reporting Items for Systematic Review and Meta-Analysis guidelines. Studies were subjected to screening based on a defined set of inclusion/exclusion criteria. Study quality, characteristics and results were extracted and a meta-analysis was performed to assess the concordance of HPA genotyping results between NGS and the SNV-based comparators for HPA-1,-2,-3,-4,-5,-15. In total, 3374 potentially eligible articles were identified, only 6 of which were included in the meta-analysis. The pooled proportion agreement for the overall concordance of the 6 included studies was shown to be 0.998, 95%CI [0.995, 0.999], P < .001. The discrepancies between HPA genotypes obtained by the two platforms were due to allele dropout in real-time PCR, thus discordant results were in favor of NGS over SNV-based comparators. Currently available platforms for NGS are not without their limitations, including high upfront and ongoing costs, data management and storage, accurate variant calling and availability of appropriately trained staff. Despite the high level of concordance between NGS and current gold-standard methods, these significant challenges mean that NGS is currently not viable as a stand-alone technique for HPA typing.

Evolution of the Fermi surface of BiTeCl with pressure.

We report measurements of Shubnikov-de Haas oscillations in the giant Rashba semiconductor BiTeCl under applied pressures up to ∼2.5 GPa. We observe two distinct oscillation frequencies, corresponding to the Rashba-split inner and outer Fermi surfaces. BiTeCl has a conduction band bottom that is split into two sub-bands due to the strong Rashba coupling, resulting in two spin-polarized conduction bands as well as a Dirac point. Our results suggest that the chemical potential lies above this Dirac point, giving rise to two Fermi surfaces. We use a simple two-band model to understand the pressure dependence of our sample parameters. Comparing our results on BiTeCl to previous results on BiTeI, we observe similar trends in both the chemical potential and the Rashba splitting with pressure.

Pressure-induced superconductivity in the giant Rashba system BiTeI.

At ambient pressure, BiTeI exhibits a giant Rashba splitting of the bulk electronic bands. At low pressures, BiTeI undergoes a transition from trivial insulator to topological insulator. At still higher pressures, two structural transitions are known to occur. We have carried out a series of electrical resistivity and AC magnetic susceptibility measurements on BiTeI at pressure up to ∼40 GPa in an effort to characterize the properties of the high-pressure phases. A previous calculation found that the high-pressure orthorhombic P4/nmm structure BiTeI is a metal. We find that this structure is superconducting with T c values as high as 6 K. AC magnetic susceptibility measurements support the bulk nature of the superconductivity. Using electronic structure and phonon calculations, we compute T c and find that our data is consistent with phonon-mediated superconductivity.

Enzymic synthesis of steroid sulphates. XI. Study of the oestrogen binding site of oestrogen sulphotransferase by affinity labelling with 4-mercuri-17beta-oestradiol.

Oistrogen sulphotransferase (3"-phosphoadenylylsulphate: oestrone sulphotransferase, EC 2.8.2.4) contains asingle sulphydryl group thought to be at, or near, the oestrogen-binding site. 4-mercuri-17beta-oestradiol, the activity of the enzyme decreased with increasing concentration of the oestrogen derivative. However, some 40% of the activity remained when all the sulphydryl had reacted to form mercaptide. Formation of mercaptide was only marginally decreased in the presence of the substrate 17beta-oestradiol. Other steroids, such as 11-deoxycorticosterone and testosterone, which are non-substrates for the enzyme, were more effective than 17beta-oestradiol in inhibiting mercaptide formation. Bovine serum albumin also reacted with 4-mercure-17beta-oestradiol and the effects of various steroids on mercaptide formation by the affinity label closely paralleled those found for the enzyme. 2t is concluded that the single sulphydryl group in the enzyme is not directly involved in the binding of oestrogen at the active site but is perhaps in closer proximity to a second site capable of binding certain non-substrate steroids.

Evaluation of pre- and postoperative chemotherapy for resectable adenocarcinoma of the esophagus or gastroesophageal junction.

Thirty-five consecutive patients with resectable adenocarcinoma of the esophagus or gastroesophageal junction were treated with two preoperative and three or four postoperative chemotherapy courses consisting of etoposide, fluorouracil, and cisplatin (EFP) to evaluate the rate of curative resection, clinical and pathologic response, toxic effects, and survival. One hundred thirty-seven courses with a median number of five courses (range, one to six) were administered. Preoperative EFP resulted in 17 (49%) major responses, including six patients who did not have carcinoma cells in the repeat endoscopic biopsy specimens and cytologic brushings. Among 32 patients who had surgery, 25 (78%) had curative resection, one patient had a complete pathologic response, and one had microscopic carcinoma in the resected specimen. Six patients had microscopic carcinoma at the resection margins and received postoperative radiotherapy. At a median follow-up of 20 months, the projected survival of 35 patients is 23 months (range, 6 to 33+). Fifteen patients died of their carcinomas, and 15 patients were alive (median follow-up, 20+ months; range, 15+ to 33+ months) with no evidence of relapse. There were no deaths related to chemotherapy, surgery, or radiotherapy. EFP-induced toxic reactions were moderate. Our data suggest that multiple courses of EFP are feasible. Future strategies for this disease should consider prolonged chemotherapy with regimens that result frequently in pathologic complete responses.

Early tumor response to intraarterial or intravenous administration of carboplatin to treat naturally occurring lower urinary tract carcinoma in dogs.

BACKGROUND: Survival times and tumor responses associated with malignant neoplasia of the lower urinary tract are poor despite the vast array of current treatments. Therefore, the evaluation of alternative treatments, such as intraarterial administration of chemotherapy (IAC) should be considered. OBJECTIVE: To describe a technique for superselective catheterization for IAC and to evaluate initial tumor response by ultrasonography after both IAC and intravenous administration of chemotherapy (IVC). ANIMALS: Client-owned dogs with lower urinary tract neoplasia treated with either IVC (n = 15) or IAC (n = 11). METHODS: Retrospective study. An arterial approach via the carotid or femoral artery was utilized to obtain superselective access and administer chemotherapy in the IAC cases. Medical record review was performed, data were recorded, and recorded variables were evaluated statistically. RESULTS: Intraarterial chemotherapy was successfully administered in all cases. There was a significantly greater decrease in longest unidimensional measurement in the IAC group as compared to the IVC group (P = .013). The IAC group was also significantly more likely to have a tumor response as assessed by modified RECIST guidelines (P = .049). Dogs in the IAC group were significantly less likely to develop anemia (P = .001), lethargy (P = .010) and anorexia (P = .024). CONCLUSION AND CLINICAL IMPORTANCE: This study demonstrated the feasibility and efficacy of performing IAC for lower urinary tract neoplasia. Further investigation is necessary as the follow-up time was short and the impact on long-term outcome and survival was not determined.

Atomic force microscopy and scanning tunnelling microscopy: refining techniques for studying biomolecules.

The scanning tunnelling microscope and the atomic force microscope offer the prospect of real-time, nanometre-scale imaging of biomolecules and biosurfaces under physiological environments. Much effort is therefore being made to establish these techniques as routine biophysical tools. The considerable recent progress that has been made in biotechnological applications is reviewed, highlighting specific examples of the applications of this new and exciting method of analysis.

Isoelectric focusing of IgA and IgM in composite acrylamide-agarose gels.

A convenient method for isoelectric focusing of intact polymeric IgA and IgM is described. This technique employed composite gels containing 1.0% acrylamide and 0.75% agarose which exhibited minimal electroendosmotic properties. The spectrotypes obtained with mouse IgA myeloma proteins, a human IgA myeloma and rabbit secretory IgA preparations were compared in three gel systems: 5% acrylamide, 0.8% agarose and the composite gel. With respect to resolution of component bands, the composite gel was superior to the other two systems. Hapten binding studies with MOPC-315 IgA and a rabbit secretory IgA anti-DNP antibody indicated that the focused IgA molecules retained their binding site integrity in the composite gel. The pI ranges obtained with microscale sucrose isoelectric focusing and composite gel system showed good correspondence, with the latter exhibiting enhanced resolution. Studies with MOPC-104E IgM revealed improved resolution in the composite gel when compared to the agarose system. Comparison of pI ranges for IgA and IgM immunoglobulins obtained in the present study with those reported previously suggest that IgA spectrotypes are confined to an acidic pI range (3.4--6.4), whereas IgM spectrotypes are not (4.3--8.8).

A scanning tunnelling microscopy comparison of passive antibody adsorption and biotinylated antibody linkage to streptavidin on microtiter wells.

An antiferritin antibody was either, (a) passively adsorbed to microwells or (b) biotinylated and immobilised to streptavidin coated microwells. Scanning tunnelling microscope (STM) imaging of these well surfaces coated with a platinum (95%) carbon (5%) coating (Pt/C) conductive layer showed a randomly oriented array of antibodies for passive adsorption whereas for biotin-streptavidin immobilisation there was a more uniform and even distribution of antibodies on the well surface. On further incubation with ferritin STM imaging showed that for passive adsorption approximately 5% of the surface was functional, while for the biotinylated antibody it was greater than 60%. The images presented in this paper show graphically the loss of functionality that occurs using passive adsorption and, conversely, the preservation of antibody functionality using the biotin-streptavidin linkage for antibody immobilisation. These results correlate well with the work of others in the field.

Appearance of hyperostosis frontalis interna on indium-111 leukocyte scans: potential diagnostic pitfall.

The appearance of hyperostosis frontalis interna on an [111In]leukocyte scan is reported. Recognition of the potential for normal accumulation of 111In-labeled white blood cells within this common process involving the skull is necessary to avoid misdiagnosis.

Hemobilia associated with hepatic artery aneurysms: scintigraphic detection with technetium-99m-labeled red blood cells.

Biliary tract bleeding is an unusual cause of upper gastrointestinal hemorrhage. Rupture of hepatic artery aneurysms is one of the least common etiologies of hemobilia. Both cholescintigraphy and [99mTc]red blood cell scintigraphy are useful in the diagnosis of this rare disorder. The combination of focal obstruction on cholescintigraphy and intermittent visualization of the major bile ducts on red blood cell scintigraphy should suggest the diagnosis of hemobilia and prompt an angiogram to determine the cause.

Thyroid uptake and imaging with iodine-123 at 4-5 hours: replacement of the 24-hour iodine-131 standard.

A study was carried out to determine the suitability of utilizing a 4 to 5 hr interval from administration of iodine-123 to imaging and uptake measurement as a replacement for the 24-hr standard originally established with iodine-131. In 55 patients who underwent scintigraphy at 4 and 24 hr, there was no discrepancy between paired images. In 55 patients who had uptake measured at 4 and 24 hr and in 191 patients who had uptake measured at 5 and 24 hr, the early measurements proved equal or better discriminants of euthyroid from hyperthyroid patients. In our institutions, these findings and the logistical advantages of completing the exam in 4-5 hr led us to abandon the 24-hr study in the majority of patients.

Protein C is responsible for the rapid inactivation of factor Va following blood clotting in vitro.

When whole blood is allowed to clot in vitro, factor V is rapidly activated to factor Va which is subsequently inactivated. We developed two monoclonal anti-protein C antibodies, one of which inhibits protein C activation and the other inhibits protein C activity. The addition of either antibody to blood before clotting in vitro significantly inhibited the inactivation of factor Va, confirming the essential role of protein C in mediating the rapid inactivation of factor Va.

A Ser162-->Leu mutation within glycoprotein (GP) IIIa (integrin beta3) results in an unstable alphaIIbbeta3 complex that retains partial function in a novel form of type II Glanzmann thrombasthenia.

Platelets from Glanzmann thrombasthenia patient BL express approximately 30% of the normal alphaIIbbeta3 content and support fibrin-mediated clot retraction, but fail to bind fibrinogen or aggregate following cellular activation. BL platelets bind neither activation-dependent nor activation-independent ligands. DNA sequence analysis of BL platelet mRNA revealed a homozygous C583-->T point mutation in a conserved region of beta3, resulting in a Ser162Leu amino acid substitution. This mutation appears to produce destabilizing effects on the alphaIIbbeta3 complex, as evidenced by the fact that (1) the BL alphaIIbbeta3 complex exhibited altered sedimentation velocity through sucrose gradients, (2) alphaIIb and beta3 was not recognized by complex-dependent monoclonal antibodies or co-precipitated by integrin subunit-specific antibodies, and (3) biosynthesis and trafficking of the alphaIIbbeta3Leu162 complex was delayed relative to that of the wild-type control. Taken together, these data implicate the region encompassing Ser162 in the stabilization and ligand binding properties of the alphaIIbbeta3 complex.

Scanning tunneling microscopy imaging of the tumour associated antigenic 20 amino acid human polymorphic epithelial mucin core peptide fragment.

Human polymorphic epithelial mucins (PEM) are high molecular weight glycoproteins that are associated with breast cancer. Recent structural studies have identified that the protein core of PEM contains a 20 amino acid tandem repeat that has elements of secondary structure which coincide with the epitopes for a number of tumour reactive antibodies. In our continuing structural studies we have now investigated the use of the scanning tunneling microscope (STM) to directly image the conformation of the twenty amino acid PEM core peptide. High resolution STM images reveal that the peptide has an overall topography similar to that predicted by molecular modelling. The images identify directly that the free peptide is conformationally non-restricted and can adopt a number of discrete conformations in the solid state.

Staging of non-small cell bronchogenic carcinoma. Relationship of the clinical evaluation to organ scans.

Organ scans are generally performed on patients with bronchogenic carcinoma only when clinical evaluation is suspicious for metastases. However, it is not clear whether the clinical abnormalities will direct attention to the single organ which should be scanned, or if all three organs (bone, brain, liver) should be evaluated if any clinical abnormality is present. We investigated the use of triple organ radionuclide scanning and computerized tomography (CT) of the brain in the initial staging of patients with non-small cell bronchogenic carcinoma with no obvious metastases. Of 122 patients with newly diagnosed lung cancer, 53 met our criteria for further study. Thirty-three (62 percent) of these had at least one clinical abnormality suggestive of metastasis. Bone scanning detected metastases in seven (21 percent) and head CT in two additional patients (6 percent). Brain and liver scanning had no yield. In only five of these nine patients did the clinical abnormality direct attention to the organ with detectable metastases. Twenty of the 53 (38 percent) patients had a negative routine clinical evaluation, yet bone scanning showed metastases in three (15 percent). We concluded that clinical abnormalities are not specific for the organ in which metastases may be detected, so three scans (bone, liver, CT of the brain) should be obtained if there is any suspicion of metastasis based on history, physical examination, and laboratory tests. The value of bone scanning in clinically normal patients deserves further study.

Studies to show that with podophyllotoxin the early replicative stages of herpes simplex virus type 1 depend upon functional cytoplasmic microtubules.

The antiviral activity of podophyllotoxin against herpes simplex type 1 virus (HSV-1) grown in Vero cells was studied by a simple microtitration assay. Antiviral effects were induced at similar concentrations as direct cellular toxicity, as characterised by a time-dependent loss of cell monolayer. Podophyllotoxin-mediated toxicity arises from cytoplasmic microtubular, and hence cytoskeletal, decay. Some degree of selectivity was seen for inhibition of virus replication over direct cellular toxicity. Podophyllotoxin acted against an early viral process, as an antiviral effect was still seen if drug was removed 2 h after infection. Similar effects were seen with colchicine, a classical tubulin-binding compound, but not with bromovinyldeoxyuridine. Podophyllotoxin was capable of inducing a cytoprotective effect in Vero cells, as pre-treatment of cells abrogated virus growth for up to 90 min after removal of drug. This is coincident with the repolymerisation of cellular microtubules and re-formation of the cytoskeleton. We conclude that HSV-1 relies upon a functional cellular cytoskeleton for efficient completion of an early replicative event. Such a process may be the transport of viral material to the nucleus or inhibition of the formation of intranuclear viral 'replication factories', bodies containing cytoskeletal fragments constructed after viral infection.

Evaluation of continuous-infusion alpha-difluoromethylornithine therapy for colorectal carcinoma.

A total of 32 evaluable patients with measurable advanced colorectal carcinoma were treated with continuous-infusion alpha-difluoromethylornithine (DFMO) at a median daily dose of 8 g/m2 (range, 6-14 g/m2). DFMO was infused over 24 h daily for 28 days, followed by a rest period of 7 days. Of the 32 patients, 14 had received no prior chemotherapy. A total of 65 courses was given, with the median being 2 (range, 1-9 courses). None of the patients achieved a partial or complete response; however, 3 patients achieved a minor response and 14 had stable disease. The frequent toxic effects of DFMO included thrombocytopenia (which was dose-limiting), malaise, nausea, vomiting, reversible hearing loss, and diarrhea. Our data suggest that continuous-infusion DFMO therapy is feasible and results in only mild gastrointestinal toxicity. Although DFMO proved to be ineffective as a single agent in this trial, it could probably best be used in combination with cytotoxic agents known to enhance its antitumor activity in a preclinical setting.