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Background: Patients with hereditary antithrombin deficiency (HAD) have an increased risk of venous thromboembolism (VTE). The American Thrombosis and Hemostasis Network (ATHN) 12: HAD Pilot Project established a registry to collect data on patients with HAD., Objectives: To inform current practice and serve as a platform to design a multicenter global registry for patients with HAD., Methods: The HAD registry was designed in 2020 to identify 100 patients with HAD receiving care at ATHN-affiliated centers. Demographics, type of HAD, thrombotic events, risk factors, anticoagulants, and antithrombin (AT) concentrate administration were recorded., Results: Ninety-four patients were included; 65% were females; 51% had type 1 HAD. Mean age at diagnosis was 26 years (SD, 18 years); 61% had VTE: 55% deep vein thrombosis and 27% pulmonary embolisms. Eight patients had arterial thrombosis. Recurrent thrombosis occurred in 58.6% of patients (44.8%) despite anticoagulation. The main risk factor for thrombosis in females was estrogen. Direct oral anticoagulants were prescribed in 30%, heparin in 34%, and warfarin in 32%. There were 139 pregnancies. Low-molecular-weight heparin was administered in 33% and AT concentrate in 19% and 11% prior to and after delivery, respectively. Twelve patients developed thrombosis in pregnancy. Seventy-nine patients underwent 239 surgeries or procedures, mainly gastrointestinal and vascular. Overall, 35% of participants received AT concentrate without adverse events., Conclusion: In ATHN 12, VTE was the predominant manifestation, frequently recurrent. There was a trend toward using direct oral anticoagulants. Low-molecular-weight heparin was administered in one-third of pregnancies and AT concentrate in one-fifth without adverse events. These data should encourage prospective studies to optimize the management of these patients., Competing Interests: Declaration of competing interests M.T.D.: participation on Pharmacosmos Advisory Board and member of American Society of Hematology Panel on Practice Guidelines for Anticoagulation in COVID-19. E.S.: no conflict of interest. J.C.R.: has received honorarium from F. Hoffman-La Roche AG, Genentech, Novo Nordisk, Octapharma, Pfizer, Sanofi, and Takeda. M.D.T.: received consulting fees from Pfizer, Sanofi, Sobi-Swedish Orphan, Biovitrum, Takeda Amgen, Octapharma Inc, Genentech, BioMarin, UCB Biosciences Dova Pharmaceuticals, and Novo Nordisk and payment/honoraria for lectures, presentations, speakers’ bureaus, manuscript writing, or educational event from Sobi-Swedish Orphan Biovitrum, Genentech, Amgen, Novartis, BioMarin, and Sanofi. J.S.: no conflict of interest. J.C.: no conflict of interest. M.R.: consulting for Catalyst Biosciences, CSL Behring, Genentech, Hema Biologics, Kedrion, Novo Nordisk, Pfizer, Sanofi, Takeda, and uniQure and leadership or fiduciary role in Foundation for Women and Girls with Blood Disorders; Partners in Bleeding Disorders, Thrombosis and Hemostasis Societies of North America., (Copyright © 2024 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

Hemophilia is a rare genetic bleeding disorder historically associated with high morbidity and mortality. Some individuals with hemophilia suffer associated chronic joint disease, chronic pain, and other physical and mental health challenges. In the last 50 years, a better understanding of the pathophysiology of the disease has resulted in extraordinary therapeutic advances leading to enhanced quality of life and increased life expectancy. We present an illustrated review of the evolution of hemophilia treatment from the development of non-factor therapies to gene therapy., Competing Interests: Dr. Michael D. Tarantino reports consulting fees from Pfizer, Sanofi, Sobi-Swedish Orphan Biovirtum, Takeda, Amgen, INC, Octapharma, Genentech, BioMarin, UCB Biosciences, Dova Pharmaceuticals, Novo Nordisk, and payment honoraria from Sobi, Genentech, Amgen, Novartis, BioMarin, and Sanofi. Dr. Jonathan C. Roberts reports grants from Takeda and Genetech, consulting fees from CSL Behring, F. Hoffman-La Roche AG, Sanofi, HEMA Biologics, Novartis, Novo Nordisk, Pfizer, Takeda, and Genentech. Dr. María G. Español reports participation on Sevenfact Advisory Board from HEMA biologics and payment honoraria from Octapharma., (© 2023 The Author(s).)

Von Willebrand disease (VWD) is an inherited bleeding disorder caused by quantitative or qualitative deficiencies in von Willebrand factor (VWF). People with VWD may experience excessive, recurrent or prolonged bleeding, particularly during menstruation, childbirth, surgery or following trauma. However, many VWD patients are undiagnosed, and therefore inadequately treated. Reasons for the underdiagnosis of VWD include its relatively mild symptoms, complex diagnosis, lack of awareness among non-specialist healthcare providers and the general population, and a lack of prioritisation of disorders disproportionately affecting females. The vwdtest.com platform was launched as part of a global initiative to raise awareness and improve diagnosis of VWD. Besides providing VWD-specific educational resources, the website includes an online bleeding self-assessment tool and offers diagnostic support for individuals, and their providers, who have a score suggestive of a bleeding disorder. vwdtest.com helps to address these unmet needs, especially in regions with limited access to educational and diagnostic resources., Competing Interests: Declaration of Competing Interest FFCM has received grant/research support from Bayer and Octapharma, and honoraria or consultation fees from Bayer, Octapharma and Sanofi. ABF has received research support from Baxalta/Shire/Takeda, CSL-Behring, and honoraria or consultation fees from Baxalta/Takeda, CSL Behring, Grifols, Kedrion, LFB and Octapharma. AS has no competing interests to declare. CMM has received research support from Baxter/Takeda, CSL Behring and Grifols and honoraria or consultation fees from CSL Behring, LFB, Octapharma and Takeda. She has participated in advisory boards for Takeda and CSL Behring. JCR has disclosed receiving research support from Takeda and Genentech. He has participated in advisory boards for Sanofi Genzyme, Takeda, Octapharma, Novo Nordisk, Pfizer, and CSL Behring. He is on speaker bureaus for Sanofi Genzyme, Novo Nordisk, Octapharma, and Takeda. AD has no competing interests to declare. JJ received an unrestricted educational grant from Octapharma. EB has received grant/research support from Bayer, Baxalta/Takeda, CSL Behring and Sobi, and honoraria or consultation fees from Bayer, Baxalta/Takeda, CSL Behring and Octapharma., (Copyright © 2022. Published by Elsevier Ltd.)

Background: Extremely premature neonates have increased risk for bleeding, perhaps the most devastating version of which being intraventricular hemorrhage (IVH). Limited data are available for coagulation parameters in this vulnerable population., Objectives: We conducted a prospective cohort study characterizing coagulation laboratory parameters in extremely premature neonates 23-30 weeks gestational age (GA) and determined coagulation parameters and clinical risk factors associated with IVH., Patients/methods: One hundred twenty neonates 23-30 weeks GA were enrolled, and umbilical cord blood samples were obtained and processed at the time of birth. Coagulation parameters including prothrombin time (PT), international normalized ratio (INR), activated partial thromboplastin time (APTT), and activity assays for factors II, VII, IX, X, XIII, and XIII subunit A antigen were performed by standard methods. Clinical risk factors were analyzed for association with IVH., Results: Of the enrolled neonates, 29 (24.2%) experienced IVH. Persistent pulmonary hypertension (PPHN) independently predicted IVH risk with odds ratio (OR) 5.3 (95% confidence interval [CI] 1.1-24.3), P = .0338; and chronic lung disease (CLD) approached significance with OR 2.3 (95% CI 0.9-5.5), P = .0659. Coagulation parameters were evaluated for association with IVH, and there was no significant difference among coagulation tests in neonates with or without IVH or per GA. Reduced factor XIII subunit A showed significant association with death, P = .003., Conclusions: We present a large, prospective study of laboratory coagulation parameters in extremely premature neonates, including factor X, factor XIII, and factor XIII subunit A not previously described in this population. These findings may impact clinical practice and should encourage additional study in this vulnerable population., (© 2022 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis.)

The effort for combating the COVID-19 pandemic around the world has resulted in a huge amount of data, e.g., from testing, contact tracing, modelling, treatment, vaccine trials, and more. In addition to numerous challenges in epidemiology, healthcare, biosciences, and social sciences, there has been an urgent need to develop and provide visualisation and visual analytics (VIS) capacities to support emergency responses under difficult operational conditions. In this paper, we report the experience of a group of VIS volunteers who have been working in a large research and development consortium and providing VIS support to various observational, analytical, model-developmental, and disseminative tasks. In particular, we describe our approaches to the challenges that we have encountered in requirements analysis, data acquisition, visual design, software design, system development, team organisation, and resource planning. By reflecting on our experience, we propose a set of recommendations as the first step towards a methodology for developing and providing rapid VIS capacities to support emergency responses., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: Goal Attainment Scaling for Hemophilia (GOAL-Hēm) is a novel, hemophilia-specific, validated patient engagement tool and patient-reported outcome instrument., Objective: We evaluated the degree to which the language of GOAL-Hēm was patient-centric and the content valuable and relevant for people with hemophilia (PWH) and/or their caregivers., Patients/methods: Patients and caregivers participated in one of three investigations: an online survey, one-on-one patient interviews, or a focus group. The survey and interviews assessed the clarity and relevance of the GOAL-Hēm menu items. Interviews were semistructured, audio recorded, and transcribed verbatim. Feedback from interviews was coded as "clear," "unclear," "remove," or "add." The focus group explored participants' experience of GOAL-Hēm and elicited recommendations for implementation. Quotations from focus group and interview transcripts were indexed and charted to emergent themes for analysis., Results: Participants comprised 19 adults with hemophilia and 19 caregivers of children with hemophilia (survey, n  = 20; interview, n  = 12; focus group, n  = 6). After their feedback, 32% (15/48) of goals were retained unchanged. Further feedback resulted in the removal of 45% (286/635) of the goal descriptors, and 30% (193/635) of the retained descriptors were modified. Three new (total = 38) goals and 42 descriptors (total = 368) were added to the menu. Thematic analysis indicated that participants were enthusiastic about patient-centric language, empowered through the goal-setting process, and recognized GOAL-Hēm could measure clinically meaningful change., Conclusion: By listening closely to patients and caregivers, we refined GOAL-Hēm to better capture the experiences of PWH, enhance content validity, and augment implementation strategies. Incorporating the patient voice is integral to developing patient-centered outcome measures., (© 2022 The Authors. Research and Practice in Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis (ISTH).)

Dual energy X-ray absorptiometry (DXA) was evaluated for its accuracy in predicting total lean, fat and bone in beef carcass sides and primal cuts. Left carcass sides (n = 316) were broken down into primal cuts, scanned using DXA and then dissected to fat, lean and bone. The DXA estimates for bone, lean and fat from the primals (n = 237) were used to calibrate partial least squares regression (PLSR) models for predicting tissue weights. Models were validated using 79 additional carcass sides, which were broken into primals, scanned using DXA, and subsequently dissected to fat, lean and bone. Models were highly accurate for predicting tissue weights for the entire carcass side (lean R 2  = 0.991, fat R 2  = 0.985 and bone R 2  = 0.941) and within most primal cuts. Results suggest DXA technology can be utilized to accurately predict carcass tissue composition for whole carcass sides and within most primals., (Crown Copyright © 2018. Published by Elsevier Ltd. All rights reserved.)

Pork bellies (n = 198) were scanned with dual energy X-ray absorptiometry (DXA). Visible and near-infrared reflectance (Vis-NIR) spectra were collected from the lean (latissimus dorsi), subcutaneous fat and intermuscular fat layers. Belly-flop angle and subjective belly scores were collected as measures of pork belly softness. Vis-NIR spectra from a single fat layer could explain between 72.7 and 81.1% of the variation in pork belly softness (43.6-72.4% in validation set). The combination of the lean and subcutaneous layers improved the calibration model fit to 79.7-99.9% (66.3-71.5% in validation set). The DXA estimates explained 62.3% of variation in pork belly softness (65.2% in validation set). Results indicated that DXA and NIR technologies could potentially be utilized for pork belly softness sorting in the pork industry., (Copyright © 2018. Published by Elsevier Ltd.)

Consumption of central nervous system tissue (CNST) from cattle with bovine spongiform encephalopathy (BSE) is thought to cause the human neurological disease, variant Creutzfeldt-Jacob disease. To identify points of cross-contamination of beef carcasses with CNST, 55 young beef cattle were slaughtered and processed through a federally inspected multispecies abattoir. The objectives of this study were to evaluate CNST spread following the placement of a plug in the penetration site of the skull after captive bolt stunning, to evaluate cross-contamination of carcasses before and after splitting, to compare the effects of hot water pasteurization (84°C for 10 s) versus cold water wash (10°C for 30 s) for reducing CNST on the carcass, and to examine other possible sources of cross-contamination in the abattoir. Results indicated that the use of a plastic plug reduced CNST contamination near the bolt penetration site. This study also confirmed that carcass splitting resulted in an increase in CNST contamination at various areas of the carcass. Hot water pasteurization appeared to be an effective means of removing CNST contamination from carcasses in most of the areas sampled.

Approximately 20% to 25% of patients with von Willebrand disease (VWD) have a qualitative defect of the von Willebrand factor (VWF) protein activities. Variant VWD typically is classified as type 1C, 2A, 2B, 2M, or 2N depending on the VWF activity defect. Traditionally, diagnosis has relied on multiple clinical laboratory assays to assign VWD phenotype. We developed an enzyme-linked immunosorbent assay (ELISA) to measure the various activities of VWF on a single plate and evaluated 160 patient samples enrolled in the Zimmerman Program for the Molecular and Clinical Biology of von Willebrand Disease with type 2 VWD. Using linear discriminate analysis (LDA), this assay was able to identify type 1C, 2A, 2B, 2M, or 2N VWD with an overall accuracy of 92.5% in the patient study cohort. LDA jackknife analysis, a statistical resampling technique, identified variant VWD with an overall accuracy of 88.1%, which predicts the assay's performance in the general population. In addition, this assay demonstrated correlation with traditional clinical laboratory VWF assays. The VWF multiplex activity assay may be useful as a same-day screening assay when considering the diagnosis of variant VWD in an individual patient., (© 2016 by The American Society of Hematology.)

Background: Low levels of HDL-C are an independent cardiovascular risk factor associated with increased premature cardiovascular death. However, HDL-C therapies historically have been limited by issues relating to immunogenicity, hepatotoxicity and scalability, and have been ineffective in clinical trials., Objective: We examined the feasibility of using injectable acoustic microspheres to locally deliver human ApoA-I DNA plasmids in a pre-clinical model and quantify increased production of HDL-C in vivo., Methods: Our novel site-specific gene delivery system was examined in naïve rat model and comprised the following steps: (1) intravenous co-administration of a solution containing acoustically active microspheres (Optison™, GE Healthcare, Princeton, New Jersey) and human ApoA-I plasmids; (2) ultrasound verification of the presence of the microspheres within the liver vasculature; (3) External application of locally-directed acoustic energy, (4) induction of microsphere disruption and in situ sonoporation; (4) ApoA-I plasmid hepatic uptake; (5) transcription and expression of human ApoA-I protein; and (6) elevation of serum HDL-C., Results: Co-administration of ApoA-I plasmids and acoustic microspheres, activated by external ultrasound energy, resulted in transcription and production of human ApoA-I protein and elevated serum HDL-C in rats (up to 61%; p-value < 0.05)., Conclusions: HDL-C was increased in rats following ultrasound directed delivery of human ApoA-I plasmids by microsphere sonoporation. The present method provides a novel approach to promote ApoA-I synthesis and nascent HDL-C elevation, potentially permitting the use of a minimally-invasive ultrasound-based, gene delivery system for treating individuals with low HDL-C., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

There are two major clinical subsets of pemphigus vulgaris (PV)-mucosal PV (mPV) and mucocutaneous PV (mcPV). The mPV subset exhibits anti-human desmoglein (Dsg) 3 autoantibodies that fail to recognize murine Dsg3 (mDsg3); thus, passive transfer experiments of mPV IgG into wild-type (WT) mice have been unsuccessful at inducing disease. We therefore generated a fully humanized Dsg3 (hDSG3) murine model utilizing a hDsg3 transgenic animal crossed to the mDsg3 knockout line. Expression of hDsg3 in the mucosa rescues the mDsg3 knockout phenotype. Well-characterized mPV sera bind mucosal epithelia from the hDsg3 mice, but not mucosal tissues from WT mice, as detected by indirect immunofluorescence (IF). The majority of mPV sera preferentially recognize hDsg3 compared with mDsg3 by immunoprecipitation as well. Passive transfer of mPV IgG into adult hDsg3 mice, but not WT mice, induces suprabasilar acantholysis in mucosal tissues, thus confirming the pathogenicity of mPV anti-hDsg3 IgG in vivo. Human anti-hDsg3 antibodies are detected in perilesional mucosa as well as in sera of recipient mice by IF. These findings suggest that the Dsg3 epitopes targeted by pathogenic mPV IgG are human specific. This hDsg3 mouse model will be invaluable in studying the clinical transition from mPV to mcPV.

Modeling human body response to dynamic loading events and developing biofidelic human surrogate systems require accurate material properties over a range of loading rates for various human organ tissues. This work describes a technique for measuring the shear properties of soft biomaterials at high rates of strain (100-1000 s(-1)) using a modified split Hopkinson pressure bar (SHPB). Establishing a uniform state of stress in the sample is a fundamental requirement for this type of high-rate testing. Input pulse shaping was utilized to tailor and control the ramping of the incident loading pulse such that a uniform stress state could be maintained within the specimen from the start of the test. Direct experimental verification of the stress uniformity in the sample was obtained via comparison of the force measured by piezoelectric quartz force gages on both the input and the output sides of the shear specimen. The technique was demonstrated for shear loading of silicone gel biosimulant materials and porcine brain tissue. Finite element simulations were utilized to further investigate the effect of pulse shaping on the loading rate and rise time. Simulations also provided a means for visualization of the degree of shear stress and strain uniformity in the specimen during an experiment. The presented technique can be applied to verify stress uniformity and ensure high quality data when measuring the dynamic shear modulus of soft biological simulants and tissue., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Background: Ketamine has been used to model cognitive and behavioral symptoms of schizophrenia. Current hypotheses state that inadequate glutamatergic transmission in schizophrenia leads to a deficiency in gamma-aminobutyric acid (GABA)ergic inhibitory mechanisms and treatment with a GABA type A receptor subunits alpha2/alpha3 (GABA(Aalpha2/3)) modulator improved working memory performance in a preliminary study in patients. Here, we used ketamine to impair spatial working memory and disrupt behavior to examine the capacity for the GABA(Aalpha2/3) agonist 7-(1,1-dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2-fluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine (TPA023) to reverse these symptoms., Methods: Rhesus monkeys received TPA023 (.7, 2.0, and 5 mg/kg; by mouth) or vehicle 45 minutes before ketamine (1.0-1.7 mg/kg; intramuscular) or saline in a semirandomized Latin square design. Behavioral observations were acquired at approximately 5 minutes, and spatial delayed response performance was tested at 15 minutes postinjection., Results: Ketamine produced a profound impairment in spatial working memory in association with the emergence of hallucinatory-like behaviors. TPA023 at all doses blocked ketamine's cognitive-impairing ability but did not influence the behavioral symptoms., Conclusions: Acute GABA(Aalpha2/3) agonist administration reverses the working memory deficits induced by ketamine in primates. This finding indicates that the consequences of N-methyl-D-aspartate deficiency on the function of prefrontal circuits involved in working memory can be completely overcome by acute enhancement of GABA signaling., (Copyright 2010 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)

The efficacy of a series of 2-aryl/alkyl selenazolidine-4(R)-carboxylic acids (SCAs) in reducing NNK [4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone]-induced lung adenomas in female A/J mice, a model for tobacco-related lung tumorigenesis, has been investigated. With selenazolidines in the diet for 1 month prior to carcinogen administration and during the subsequent 4 months of tumor development, 2-butylSCA, 2-cyclohexylSCA, 2-phenylSCA and 2-oxoSCA were chemopreventive, significantly reducing mean lung tumor numbers from the 10.9 of unsupplemented controls to 4.7, 5.3, 2.8 and 4.7, respectively. When selenazolidine supplementation began three days after carcinogen administration (i.e., post-initiation), 2-butylSCA, 2-cyclohexylSCA, and 2-oxoSCA were chemopreventive. In both regimens, selenocystine was also chemopreventive. In the post-initiation protocol, but with intervention at a precancerous stage (13 days), whole genome expression analysis of lung RNA identified six gene transcripts that weakly correlated with the efficacy of tumor reduction by the four selenocompounds at 4 months. None of these genes were among those identified to be influenced by chemopreventive selenium compounds in human lung cancer cell lines. When supplementation was for 1 month-prior until 3 days-after carcinogen administration, 2-butylSCA, and 2-phenylSCA were chemopreventive but selenocystine was ineffective. Both 2-butylSCA and 2-phenylSCA retained their chemopreventive activity (44% and 40% tumor number reduction, respectively), when the supplementation was shortened and restricted to a pre-initiation period (days -9 to -2). With supplementation spanning 2 days-prior until 3 days-after NNK, reductions in tumor numbers by 2-phenylSCA (26%) and 2-butylSCA (17%) did not achieve statistical significance. Thus, several 2-aryl/alkyl selenazolidines possess chemopreventive activity against NNK-induced lung tumors, and variously demonstrate pre-initiation and post-initiation efficacy.

Novel selenazolidines, designed as l-selenocysteine prodrugs and potential cancer chemopreventive agents, were examined for their ability to affect the transcription of murine hepatic chemoprotective enzymes. Compounds investigated were selenazolidine-4(R)-carboxylic acid (SCA) and six 2-substituted derivatives that cover a C log P range of -0.512 to -3.062. Their biological effects were compared with those of L-selenocystine. Gene transcripts were examined 24 h after a single dose, administered i.p. and i.g., and covered a range of chemoprotective enzymes; alpha, mu and pi class glutathione transferases (Gsts), UDP-glucuronosyltransferases (Ugts) 1a1, 1a6, 1a9, and 2b5, glutathione peroxidase 1 (Gpx), thioredoxin reductase (Tr), NAD(P)H-quinone oxidoreductase 1 (Nqo), and microsomal epoxide hydrolase (Meh). When given i.g., 2-butyl SCA (BSCA) resulted in elevations in alpha, mu and pi class Gsts, Ugt1a6, Tr, and Gpx, and 2-phenyl SCA (PhSCA) elevated GstP, Ugt1a9, Tr, Gpx (3 kb), and Meh. Other derivatives with C log P values both lower [2-(2'-hydroxy)phenyl SCA (PhOHSCA) and 2-methyl SCA (MSCA)] and higher [2-cyclohexyl SCA (ChSCA) and 2-oxo SCA (OSCA)] than BSCA and PhSCA elevated far fewer transcripts; PhOHSCA (Ugt1a1, Gpx), MSCA (Ugt1a1, Meh), ChSCA (Ugt1a1, Ugt1a9), and OSCA (Ugt1a6, Ugt1a9, GstM). When given i.p., the most pervasive transcript changes were parallel increases in Nqo and Tr transcripts which occurred with BSCA, PhSCA, MSCA, and OSCA. PhSCA also increased GstP, and PhOHSCA increased Ugt1a1 and Ugt1a6 levels. Unique among the compounds, PhSCA reduced the transcript levels of GstA, and the 1.6 kb transcript of Gpx although only when given i.p. Neither l-selenocystine nor SCA affected the level of any transcript and no compound altered the amount of Ugt2b5 mRNA. Despite chemical similarity and common ability to potentially serve as a source of l-selenocysteine, each selenazolidine compound appeared to elicit a unique pattern of mRNA responses and by either route of administration, there was no correlation between the magnitude of response of any gene and the calculated C log P values of the organoselenium compounds.

Selenite and organoselenium compounds have been examined at supranutritional levels for their ability to influence the activity and mRNA levels of chemoprotective enzymes in the livers of selenium-sufficient mice and the changes compared to those elicited by oltipraz. Compounds investigated included novel selenocysteine prodrugs that have previously been evaluated for their ability to reduce the tumorigenicity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in mice. Following seven daily doses (i.g.), all compounds except 2-methylselenazolidine-4(R)-carboxylic acid (MSCA) increased thioredoxin reductase activity (43-92%) but only for 2-oxoselenazolidine-4(R)-carboxylic acid (OSCA) was there an accompanying increase in mRNA. No compound enhanced glutathione peroxidase activity, although sodium selenite significantly elevated the mRNA of this enzyme. Oltipraz was an efficacious inducer of both thioredoxin reductase and glutathione peroxidase mRNAs. Sodium selenite, selenazolidine-4(R)-carboxylic acid (SCA), and OSCA elevated NAD(P)H-quinone oxidoreductase mRNA but only for OSCA was the elevation in mRNA accompanied by an increase in enzyme activity. L-Selenocystine significantly increased this activity without increasing mRNA levels. Sodium selenite, L-selenocystine, L-selenomethionine, and Se-methyl-L-selenocysteine all enhanced glutathione S-transferase activity. The increased activity with sodium selenite was accompanied by increases in mRNAs of Gst alpha, Gst mu and Gst pi classes, while for L-selenocystine and Se-methyl-L-selenocysteine, only an elevation in the mRNA for the Gst alpha class was observed. Gst alpha and Gst mu class mRNAs were elevated by OSCA without a significant elevation in enzyme activity. SCA and MSCA both elevated a Gst pi mRNA and MSCA elevated Gst mu in addition. By comparison, oltipraz only significantly elevated the mRNA of Gst mu, adding to the conclusion that across the entire study, no selenium compound appears to be acting purely through the antioxidant response typified by oltipraz. Despite their chemical similarity, the three cysteine prodrugs, SCA, MSCA, and OSCA, each produced its own unique pattern of effects on protective enzymes and none was identical to the pattern elicited by sodium selenite, L-selenocystine, L-selenomethionine, and Se-methyl-L-selenocysteine. The study also shows that after 7 days of administration, there was only occasional concordance between elevations in mRNA and enzyme activity for any selenium compound and for any protective enzyme, there was no response in common for all selenium compounds.

Compliance-gaining strategies refer to subtle differences in ways people use language when their goal is to influence someone else's behavior. This stands in contrast to other kinds of persuasion aimed only at influencing others beliefs and attitudes. We have developed a new method of coding what physicians say when they are trying to influence patients' behaviors. This method applies theory and methods from the fields of interpersonal influence, linguistics and social psychology. We tested the reliability of this new method by randomly selecting 37 audiotaped medical interviews collected for an unrelated study [J. Gen. Int. Med., 9 (1994) 402] and having three coders independently identify physician compliance-gaining utterances and then independently apply one of 57 codes to each utterance. These codes also were categorized on two underlying dimensions reflecting whether the physician (1) framed the compliance-gaining utterance in a direct or indirect way, and (2) did or did not give a justification for that direct or indirect request. Reliability among coders and coders agreement with the final utterance identification and coding decisions, measured as per cent agreement among coders and/or, where appropriate, by Cohen's kappa were good to excellent. Most physicians' strategies were indirect and incomplete. For female patients, physicians used significantly more strategies, including more indirect strategies, complete strategies, "prescriptions" and "demands". For male patients, physicians used a greater percent of direct strategies, including "procedural demands". This method provides a reliable and promising new technique for observing naturally occurring physician compliance-gaining speech.

We have previously reported the synthesis and characterization of two new classes of selenazolidine-4(R)-carboxylic acids (2-oxo and 2-methyl-SCAs) (OSCA and MSCA, respectively), as well as the "parent" compound, selenazolidine-4(R)-carboxylic acid (SCA, selenaproline). These compounds were designed as prodrugs of L-selenocysteine with potential application in cancer chemoprevention or other clinical uses. We will be exploring the chemopreventive activity of the new compounds in the well-established A/J mouse model of tobacco-induced lung carcinogenesis. The objectives of the present study were to investigate several fundamental biochemical endpoints after selenazolidine administration compared with other selenium-containing agents. Groups of mice were fed either AIN-76A diet alone or the diet supplemented with the following selenium compounds (ppm Se): sodium selenite (5), L-selenomethionine (3.75), L-selenocystine (15), Se-methyl-L-selenocysteine (3), MSCA (5, 10, or 15), OSCA (5, 10, or 15), or SCA (5, 10, or 15). After 28 days of supplementation, toxicity of the selenazolidines was not evident, as measured by outward appearance and behavior, body and organ weight changes, and histological evaluation of liver and lung tissue. Select treatment groups showed significant increases in selenium levels in blood and tissues. Increased activity of selenium-dependent glutathione peroxidase (GPx) in blood and liver illustrated that the selenazolidines provided a source of biologically-available selenium.

A method for the separation and quantitation of several important biological thiolamines is described. The procedure employs a C18 reversed-phase HPLC system to separate the dinitrophenyl derivatives of reduced and oxidized glutathione and cysteine and relies on an internal standard, Nepsilon-methyllysine, to minimize experimental error. The method was validated in three matrices (water, HepG2 cell lysates, and mouse liver homogenates) using several criteria. The detector response was linear for the dinitrophenyl derivatives of glutathione, glutathione disulfide, cysteine, and cystine in the concentrations ranging from 10 to 50 nmol/ml. Inter- and intra-day variation, percent recovery in the biological matrices, and limits of detection and quantitation were determined. For the most accurate determination, it is essential that standard curves be produced daily and in the same matrix as that being analyzed.

Methods not only for characterizing but also for quantitating melanin subtypes from the two types of melanin found in hair--eumelanin and pheomelanin--have been established. In relation to testing for drugs of abuse in hair, these methods will allow for correction of drug binding to specific melanin subtypes and will serve to improve drug measurement in hair. 5,6-Dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA) make up the majority of the eumelanin polymer while benzothiazene units derived from 2-cysteinyl-S-Dopa (2-CysDopa) and 5-cysteinyl-S-Dopa (5-CysDopa) compose the majority of the pheomelanin polymer. Our results show that: (1) pyrrole-2,3-dicarboxylic acid (PDCA) and pyrrole-2,3,5-tricarboxylic acid (PTCA), markers for DHI and DHICA units, respectively, are produced in 0.37 and 4.8% yields, respectively, when melanins are subjected to alkaline hydrogen peroxide degradation, (2) 3-aminotyrosine (3AT) and 4-amino-3-hydroxyphenylalanine (AHP), markers for 2-CysDopa and 5-CysDopa, respectively, are produced in 16 and 23% yield, respectively, when subjected to hydriodic acid hydrolysis, and (3) that black human hair contains approximately 99% eumelanin and 1% pheomelanin, brown and blond hair contain 95% eumelanin and 5% pheomelanin; and red hair contains 67% eumelanin and 33% pheomelanin. These data will allow deeper investigation into the relationship between melanin composition and drug incorporation into hair.

Hypoxemia during bronchoscopy occurs frequently. It can usually be managed by supplemental oxygen and bronchodilators or, in some cases, occasionally stopping the procedure. Benzocaine spray is commonly used as a topical anesthetic agent during bronchoscopy. However, it has been associated with the development of methemoglobinemia. The following is a case report of hypoxia during bronchoscopy from benzocaine-induced methemoglobinemia and its management.

The synthetic porphyrins, N-benzyl-5,10,15,20-tetrakis (4-carboxyphenyl) porphine (N-bzHTCPP) and N-4-nitrobenzyl-5-(4-carboxyphenyl)-10,15,20-tris(4-sulfophenyl) porphine (N-bzHCS3P), represent excellent radiocopper chelating agents that may find utility in antibody-mediated diagnosis and/or therapy. N-bzHCS3P was conjugated to an anti-renal cell carcinoma (RCC) antibody, A6H, and labeled with copper-67. 67CuCS3P-A6H was studied for its biodistribution in human RCC xenograft-bearing nude mice, along with the radiolabeled free porphyrins. The porphyrins resulted in tumor:blood ratios in the range of 3 to 4 after 48 h. The radiolabeled antibody achieved a tumor:blood ratio of over 16 after 45 h, indicating accumulation at the desired site. However, unwanted localization also occurred in the liver and spleen, which will have to be rectified before realizing the full potential of this approach.

The radiation protective effect of thiol compounds is unequivocal and their use is only limited by their toxic effects. We used the principle of alpha alkylation, which renders amino acids unmetabolizable, to reduce the toxicity of homocysteine. This product, alpha-methyl-homocysteine thio-lactone, was tested for toxicity and radiation protective effect along with known protectors L-cysteine, cysteamine and WR 1065 in cell culture using V79-4 Chinese hamster lung cells. The three-day growth curve assays, useful to measure overall effects on cell growth, revealed lowest toxicity for alpha-methyl-homocysteine thiolactone (GL-2). Clonogenic survival tests, used to evaluate the retention of reproductive integrity, were carried out and revealed that GL-2 had no adverse effects in this test system. Radiation protection tests showed that GL-2 exhibited protective activity against radiation induced lethality above that seen with cysteine and cysteamine, but below WR 1065. However, GL-2 showed little or no negative effects toward the cell itself, in direct contrast to WR 1065. Our findings show a potentially important tool and principle to reduce toxicity of radiation protectors with analogous structures.

Phthalate esters are widely used in the manufacture of plastics and have been shown to cause testicular toxicity, purportedly, by targeting the Sertoli cell alone. Recent evidence, however, indicates that a paracrine control exists between Sertoli and Leydig cells and the breakdown of one component of this relationship is therefore detrimental to normal function. However, no data that explore the influence of testicular toxins on Leydig cell structure and function have been published hitherto. The preliminary studies reported here were initiated to test the hypothesis that phthalate intoxication may adversely alter Leydig cell structural and functional integrity. Four phthalate esters, namely, di(2-ethylhexyl) phthalate (DEHP, di-n-pentyl phthalate (DPP)., di-n-octyl phthalate (DOP), and diethyl phthalate (DEP) were investigated in vivo and their monoesters (MEHP, MPP, MOP, and MEP, respectively) in vitro for indications of Leydig cell toxicity in the rat. Rats were dosed by oral gavage with 2 g phthalate diester/kg/day in corn oil vehicle for 2 days, while Leydig cell primary cultures were incubated with 1,000 microM monoester for 2 hr. Light and electron microscopy were undertaken to determine the type and degree of any changes. Phthalate esters exerted a direct effect on Leydig cell structure and function (as determined by testosterone output) with correlation of the in vitro and in vivo effects of MEHP (DEHP) and MOP (DOP). No effects on Leydig cell structure or function were seen with MPP (DPP), although Sertoli cell cytoplasmic rarefaction and vacuolation were observed in vivo. DEP produced Leydig cell ultrastructural alterations in vivo. We conclude that individual phthalate esters may exert effects on both Sertoli and Leydig cells or one cell type alone.

There is little consistency in the literature regarding the procedures for sample preparation employed for the measurement of glutathione (GSH) in biological tissues. Most procedures use an acid to homogenize tissue samples to precipitate proteins from the mixture and to minimize oxidative changes. Others employ 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) in the homogenization, which serves only to block thiol groups. The present studies were undertaken to critically compare the two methods of sample preparation on resulting GSH values determined by the Tietze method in numerous organs of mice. It was found that kidney, liver, and pancreas GSH levels were seriously underestimated when DTNB was used instead of acid to prepare the tissue samples. This discrepancy was eliminated when the animals were pretreated with AT-125, confirming the participation of gamma-glutamyltranspeptidase, the enzyme responsible for the first step in the degradation of GSH. GSH added to kidney homogenates in DTNB was degraded rapidly and continuously in a time-dependent fashion. In contrast, GSH added to acid homogenates produced stable GSH values up to 8 h after sample preparation in most cases. Storage of acid homogenates at -70 degrees C for 12 months gave results identical to original measurements, within 10% error, for 9 of 10 samples tested.

Potential immunological influences on peripheral catecholamine secretion were investigated by measuring epinephrine secretion from chromaffin cells in vitro in response to cell-free conditioned media from mononuclear cells. Chromaffin cells were isolated from bovine adrenals whereas mononuclear cells were isolated from bovine spleen tissue or whole bovine blood. In secretion experiments epinephrine release and epinephrine remaining in cells was determined such that secretion was expressed as % of total cell content. After 90 minutes exposure to conditioned media, 22.8 +/- 1.1% of content was released compared to 1.7 +/- 0.2% with RPMI media. Secretion after filtration (< 3,000 MW cutoff) was 21.6 +/- 0.9% whereas after boiling and boiling in acid, secretion was 10.2 +/- 0.2 and 4.3 +/- 0.1% respectively. Dialysis (< 3,000 MW cutoff) reduced the 90 min conditioned media-stimulated epinephrine secretion from 22.5 +/- 3.8% to 2.3 +/- 0.3%. Neither atropine nor hexamethonium blockade altered the conditioned media-stimulated epinephrine secretion. These results suggest that mononuclear cells produce a low molecular weight substance--most likely a peptide--that contributes to the stimulation of epinephrine secretion.

RibCys, a thiazolidine prodrug of L-cysteine synthesized by the condensation of the sulfhydryl-containing amino acid with the aldose monosaccharide D-ribose, successfully elevated glutathione (GSH) levels in numerous organs of tumor-bearing CDF1 mice. GSH content was assayed 1,2,4,8 and 16 h after RibCys administration (8 mmol/kg, i.p.); various organs achieved maximal GSH content at different time points. GSH in the liver was elevated 1.5-fold compared to untreated controls at the 16-h time point. Kidney GSH also was maximal at 16 h and achieved 1.6-times control values. GSH in muscle achieved 2.5 times the levels in control animals, while the bladder was elevated 2.1-fold, and the heart 1.8-fold. Other tissues tested (spleen, pancreas, lung) showed a 1.1- to 1.2-fold increase in GSH content. GSH in implanted L1210 tumors was also elevated only 1.2-fold. These data suggest the possibility of protecting organs other than the liver from toxic insults that require the intervention of GSH for detoxication and may allow such protection without compromising the utility of chemotherapy.

Some recent liquid phases for gas-liquid chromatography with improved thermostability have been evaluated for their use in the gas-liquid chromatographic-mass spectrometric (GC-MS) analysis of the complex mixtures of sterol trimethylsilyl ethers obtained, often in small amounts, from marine organisms. A combination of Dexsil 300 GC, SE-30 "ultraphase" and Silar 5CP is recommended for use in this technique because of the excellent separating efficiency and the very low substrate bleed. GC-MS analysis of the sterol trimethylsilyl ethers obtained from the cockle Cerastoderma edule has established the identity and relative proportions of the eleven sterols present.

Methods were developed to label antibodies with copper-67, a potentially useful medical radioisotope, using the porphyrin chelating agent N-benzyl-5,10,15,20-tetrakis(4-carboxyphenyl) porphine. The porphyrin was activated for coupling using either (1) 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide HCl and N-hydroxysuccinimide or (2) 1,1'-carbonyldiimidazole. The coupling reactions were optimized as a function of activation time, coupling time, coupling pH, and reagent concentrations to achieve maximum coupling to IgG monomer. Sodium dodecylsulfate polyacrylamide gel electrophoresis was used to determine coupling yields. After purification by gel filtration, the antibody-porphyrin conjugates were labeled with copper-67 in aqueous solution. The coupling protocols were used to label antibodies from several species, demonstrating the general utility of these methods. Characterization of the conjugates indicated that the porphyrin label was attached randomly to the IgG molecule. Antigen binding capacities after conjugation were unaltered or slightly lowered as determined by a competitive ELISA.