Prescribed safer supply during dual public health emergencies: a qualitative study examining service providers perspectives on early implementation.

BACKGROUND: Within North America and worldwide, drug related overdoses have increased dramatically over the past decade. COVID-19 escalated the need for a safer supply to replace unregulated substances and to reduce toxicity and overdoses. Service providers play an integral role in the delivery of safer supply but there is little empirical evidence that conceptualizes effective safer supply from their perspectives. This study explored early implementation and impacts of a safer supply program, capturing the perspectives of an interdisciplinary team of service providers on tensions and issues encountered in the development of the SAFER program. METHODS: Using a community-based participatory approach, we conducted individual interviews with program providers (n = 9). The research team was composed of researchers from a local drug user organization, a local harm reduction organization, and academic researchers. The Consolidated Framework for Implementation Research (CFIR) informed the interview guide. Data was analyzed using thematic analysis. RESULTS: There are six themes describing early implementation: (1) risk mitigation prescribing as context for early implementation; (2) developing SAFER specific clinical protocols; (3) accessibility challenges and program innovations; (4) interdisciplinary team and wraparound care; (5) program tensions between addiction medicine and harm reduction; (6) the successes of safer supply and future visions. CONCLUSION: Early implementation issues and tensions included prescriber concerns about safer supply prescribing in a highly politicized environment, accessibility challenges for service users such as stigma, encampment displacement, OAT requirements, program capacity and costs, and tensions between addiction medicine and harm reduction. Navigating these tensions included development of clinical protocols, innovations to reduce accessibility challenges such as outreach, wraparound care, program coverage of medication costs and prescribing safer supply with/without OAT. These findings contribute important insights for the development of prescribed safer supply programs.

A concept mapping study of service user design of safer supply as an alternative to the illicit drug market.

BACKGROUND: Within North America and worldwide, drug-related overdoses have increased dramatically over the past decade. COVID-19 escalated the need for a safer supply of illicit substances to reduce overdoses with hopes of replacing substances obtained from the illicit drug market. Drug users1 should be at the centre of program and policy decisions related to the development and implementation of safer supply. Yet, there is little empirical research that conceptualizes effective safer supply from their perspectives. METHOD: Within a community based participatory approach to research, we conducted a concept mapping study to foreground the perspectives of drug users and develop a conceptual model of effective safer supply. Our team was composed of researchers from a local drug user organization, a local harm reduction organization, and academic researchers. The focused prompt developed by the team was: "Safe supply would work well if…" Sixty-three drug users participated in three rounds of focus groups as part of the concept mapping process, involving brainstorming, sorting, rating and naming of themes. RESULTS: The concept mapping process resulted in six clusters of statements: 1) Right dose and right drugs for me; 2) Safe, positive and welcoming spaces; 3) Safer supply and other services are accessible to me; 4) I am treated with respect; 5) I can easily get my safer supply; and 6) Helps me function and improves my quality of life (as defined by me). The statements within each cluster describe key components central to an effective model of safer supply as defined by drug users. CONCLUSION: The results of this study provide insights into key components of effective safer supply to inform planning and evaluation of future safer supply programs informed by drug user perspectives.

Optimising perioperative care for hip and knee arthroplasty in South Africa: a Delphi consensus study.

BACKGROUND: A structured approach to perioperative patient management based on an enhanced recovery pathway protocol facilitates early recovery and reduces morbidity in high income countries. However, in low- and middle-income countries (LMICs), the feasibility of implementing enhanced recovery pathways and its influence on patient outcomes is scarcely investigated. To inform similar practice in LMICs for total hip and knee arthroplasty, it is necessary to identify potential factors for inclusion in such a programme, appropriate for LMICs. METHODS: Applying a Delphi method, 33 stakeholders (13 arthroplasty surgeons, 12 anaesthetists and 8 physiotherapists) from 10 state hospitals representing 4 South African provinces identified and prioritised i) risk factors associated with poor outcomes, ii) perioperative interventions to improve outcomes and iii) patient and clinical outcomes necessary to benchmark practice for patients scheduled for primary elective unilateral total hip and knee arthroplasty. RESULTS: Thirty of the thirty-three stakeholders completed the 3 months Delphi study. The first round yielded i) 36 suggestions to preoperative risk factors, ii) 14 (preoperative), 18 (intraoperative) and 23 (postoperative) suggestions to best practices for perioperative interventions to improve outcomes and iii) 25 suggestions to important postsurgical outcomes. These items were prioritised by the group in the consecutive rounds and consensus was reached for the top ten priorities for each category. CONCLUSION: The consensus derived risk factors, perioperative interventions and important outcomes will inform the development of a structured, perioperative multidisciplinary enhanced patient care protocol for total hip and knee arthroplasty. It is anticipated that this study will provide the construct necessary for developing pragmatic enhanced care pathways aimed at improving patient outcomes after arthroplasty in LMICs.

The MMP3 gene in musculoskeletal soft tissue injury risk profiling: A study in two independent sample groups.

Matrix metalloproteinase-3 (MMP3) is a mediator of matrix remodelling and a proposed susceptibility locus in the genetic profile of musculoskeletal soft tissue injuries. Therefore, this study aimed to validate the MMP3 gene as a risk marker for these injuries by conducting a case control genetic association study in two independent samples groups. Three previously investigated MMP3 variants (rs679620, rs591058 and rs650108) in addition to the functional promoter variant (rs3025058) were genotyped in 195 Australian control participants and 79 Australian individuals with chronic Achilles tendinopathy. Similarly, 234 South African individuals with acute anterior cruciate ligament ruptures and 232 matched control participants were also analysed. Based on high linkage with the previously associated MMP3 variant rs679620, rs3025058 was inferred and found to be associated with increased risk for Achilles tendinopathy within the South African group (P = 0.012; OR: 2.88; 95% CI: 1.4 to 6.1). Lastly, the 6A-G-C-G haplotype, constructed from the investigated variants, was significantly associated with reduced risk for Achilles tendinopathy (29% CON vs. 20% TEN, P = 0.037) in the Australian group. In conclusion, a signal surrounding MMP3 is apparent with respect to Achilles tendinopathy. However, whether the investigated variants are contributing to injury susceptibility or whether they are merely linked to the risk conferring variants mapping elsewhere within the MMP gene cluster on chromosome 11, still requires refining.

First Report of Alfalfa mosaic virus and Soybean dwarf virus on Soybean in North Dakota.

Soybean (Glycine max L.) is the major oilseed crop in North Dakota, with production concentrated in the eastern half of the state. Only one virus, Soybean mosaic virus, has been reported from soybean in North Dakota (4). In July and August of 2010, 200 soybean fields from 25 counties were surveyed for Alfalfa mosaic virus (AMV) and Soybean dwarf virus (SbDV). AMV and SbDV have been detected infecting soybean in multiple Midwestern states and are reported to reduce yields in soybean (1,3). Each field was sampled with a grid pattern across the area with at least 8 km between fields. From each field, leaves were collected from 20 plants without regard for symptoms along a transect of approximately 170 m. Leaves from each field were bulked and sap was extracted in phosphate buffer and stored at -80°C until tested using double-antibody sandwich (DAS)-ELISA with positive controls and reagents and protocols from Agdia Inc. (Elkhart, IN). Using DAS-ELISA, AMV was detected in eight of the 200 soybean fields. For sequence-based virus detection, total RNA was extracted from all field samples using a Qiagen RNeasy Plant Mini Kit (Germantown, MD), pooled, depleted of ribosomal RNA (RiboZero Epicentre, Madison, WI), reverse transcribed, sequenced using an Illumina HiSeq2000 (San Diego, CA), and compared to all available viral amino acid and nucleotide sequences. The analysis detected AMV and SbDV sequences in the pool of 200 fields. The presence of AMV and SbDV was confirmed by quantitative real-time reverse transcription (qRT)-PCR (1,3). For AMV, total RNA extracted from bulked leaves from each of the 200 fields was tested using AMVspecific primers (5'-ATGCTACCCAGGCATGTATATTT-3' and 5'-GCTGCATCTTTCGCCAGAA-3') and a FAM-labeled minor-groove binding TaqMan probe (5'-TGGACGTTACCCCCGGA-3'). One field sample from Cass county positive for AMV by ELISA was also positive for AMV by qRT-PCR, confirming the presence of AMV in the field sample. For SbDV, an RNA pool representing all 200 fields, subpools, and individual field samples was analyzed by qRT-PCR (1) and DAS-ELISA. One field sample from Grand Forks County tested positive for SbDV by qRT-PCR and DAS-ELISA, confirming the presence of SbDV in the field sample. Because leaf samples were collected and pooled prior to analysis, the symptom phenotypes of individual field plants could not be correlated with positive ELISA or qRT-PCR results. AMV was reported by the American Phytopathological Society Virus Working Group (2007 to 2008) to be widely prevalent in North Dakota, but we found no peer-reviewed reports of verified AMV identification on any crop in the state. To our knowledge, this is the first confirmed report of AMV and SbDV infecting soybean in North Dakota. Serious infestations by the soybean aphid, Aphis glycines, requiring chemical control, have occurred in recent years in North Dakota. Because A. glycines is a vector for both viruses (1,2), the distribution, incidence, and agronomic impact of AMV and SbDV could be affected in years when A. glycines infestations are high. In addition, AMV is seedborne in soybean and may cause seed mottling, a concern for the food-grade soybean industry where production is primarily for export. References: (1) V. D. Damsteegt et al. Plant Dis. 95:945, 2011 (2) J. H. Hill et al. Plant Dis. 85:561, 2001. (3) H. A. Hobbs et al. Plant Health Progress doi:10.1094/PHP-2010-0827-01-BR, 2010. (4) B. D. Nelson and L. L. Domier. Plant Dis. 93:760, 2009.

Targeted mydriasis strategies for diabetic retinopathy screening clinics.

PURPOSE: To examine factors necessitating pupil dilation to achieve gradable diabetic screening photographs using a digital non-mydriatic camera and to establish techniques to predict the need for dilation and to validate them. METHODS: Prospective clinic-based cross-sectional study with follow-up validation study. The participants' involved consecutive patients attending the diabetic retinopathy screening clinic at a University Hospital. Best corrected visual acuity, age, sex, pupil size, mean spherical equivalent, cataract grade and the requirement for dilation to achieve gradable photographs in 90 patients were recorded. Data analysis using principal component analysis and multivariate analysis of variance derived a set of equations to predict the requirement for dilation. The predictive powers of these equations were validated in an independent group of 51 patients. RESULTS: Smaller pupil size, denser nuclear colour, older age, poorer best-corrected visual acuity, cortical lens opacity and posterior subcapsular lens opacity were associated with the need for dilation (P<0.001 in all). Single variables used in isolation had a poorer predictive value than combining variables. Dilating patients with either a pupil size>3.75 mm or age>59 years correctly allocates 83 and 78% of patients, respectively to dilation or not. Combining pupil size with age produces a decision table that improves the predictive value to 84%. In the validation study this table had a predictive value of 80%. CONCLUSION: We have produced and validated criteria based on a range of clinical variables for application in a clinical setting that allows for the development of targeted mydriasis.

Sequence diversity of readthrough proteins of Soybean dwarf virus isolates from the Midwestern United States.

The amino acid sequence diversity of readthrough proteins (RTPs) of 24 dwarfing isolates of Soybean dwarf virus (SbDV) from Wisconsin and Illinois was analyzed. The RTP, a minor component of viral capsids, has a significant role in specificity of aphid transmission of luteovirids. Among the isolates, nucleotide sequence identities ranged from 95 to 100%. The predicted amino acid sequences differed at 56 amino acid positions in the 54 kDa RTD compared to only five positions in the 22 kDa CP. Phylogenetic analysis of both amino acid and nucleotide sequences showed three distinct clusters of SbDV isolates.

First Report of Soybean dwarf virus in Soybean in Northern Illinois.

Soybean dwarf virus (SbDV), a member of the Luteoviridae, is transmitted persistently by colonizing aphids and causes significant yield losses in soybean (Glycine max L.) in Japan. In the United States, SbDV is endemic in red and white clover (Trifolium pratense L. and T. repens L.) (1,3). Even so, SbDV has been detected in soybean only in Virginia (2) and Wisconsin (4). A study conducted in Illinois during 2001 and 2002 detected SbDV in clover but not soybean (3). During August of 2006, two surveys for virus diseases in soybean were conducted in Illinois. In the first survey, 30 soybean leaf samples were collected without regard for symptoms from each of 10 fields in each of five northern Illinois counties (Carroll, Jo Daviess, Ogle, Stephenson, and Winnebago). In the second survey, 10 random soybean leaf samples and 10 samples with virus-like symptoms were collected from each of 30 soybean rust sentinel plots spread throughout Illinois. Total RNA was extracted from pools of 90 to 100 plants and analyzed by quantitative real-time reverse transcriptase (QRT)-PCR using a fluorescently labeled minor groove binding probe (VIC-5'-AGCATATCCAAAGACGC-3'-MGBNFQ, nt 2358-2374) and flanking primers (5'-TGGCTATTATAGAATGGTGCGTAAAC-3', nt 2327-2351; and 5'-GCCATGGAAATGAGGGAATG-3', nt 2395-2376). From the first survey, pools from Carroll, Jo Daviess, and Ogle were positive for SbDV. Analysis of individual leaf samples from positive pools by double-antibody sandwich-ELISA (Agdia, Elkhart, IN) showed that one sample in each county was positive for SbDV. On the basis of the number of randomly sampled plants, the incidence of SbDV infection in northern Illinois was approximately 0.3%. In the second survey, SbDV was detected in one pool containing symptomatic plants from five soybean rust sentinel plots. Further QRT-PCR analysis showed that the sentinel plot in Bureau County was positive for SbDV. Because of the sampling protocols used, it was not possible to determine symptom phenotypes of SbDV-positive samples. Sequence analysis of the combined coat protein (CP) and readthrough domain (RTD) encoding region (nt 3019-5094) of SbDV isolates from Bureau (GenBank Accession No. EU095847) and Carroll (GenBank Accession No. EU095846) counties showed that the predicted amino acid sequences were 96 and 95% identical to a Japanese dwarfing isolate of SbDV (GenBank Accession No. AB038150), respectively. The predicted CP amino acid sequences of the Illinois isolates were identical and RTD amino acid sequences differed at six positions. To our knowledge, this is the first report of infection of soybean plants in Illinois with SbDV. References: (1) V. D. Damsteegt et al. Phytopathology 89:374, 1999. (2) A. Fayad et al. Phytopathology (Abstr.) 90(suppl.):S132, 2000. (3) B. Harrison et al. Plant Dis. 89:28, 2005. (4) A. Phibbs et al. Plant Dis. 88:1285, 2004.

Green Stem Disorder of Soybean.

Green stem disorder of soybean (Glycine max) is characterized by delayed senescence of stems with normal pod ripening and seed maturation. Three different field research approaches were designed to determine the relationship of green stem disorder to Bean pod mottle virus (BPMV) and other potential factors that may be involved in causing this disorder. The first research approach surveyed green stem disorder and BPMV in individual plants monitored in several commercial soybean fields during three growing seasons. Leaf samples from maturing plants (growth stage R6) were tested by enzyme-linked immunosorbent assay (ELISA) for BPMV. The percentage of monitored plants infected with BPMV at growth stage R6 in some fields was higher than the incidence of green stem disorder at harvest maturity. Many plants infected with BPMV did not develop green stem disorder, and conversely, many plants that had green stem disorder were not infected with BPMV. According to a chi-square test of independence, the data indicated that green stem disorder was independent of BPMV infection at growth stage R6 (P = 0.98). A second research approach compared green stem disorder incidence in an identical set of soybean entries planted in two locations with different levels of natural virus infection. Despite differences in virus infection, including BPMV incidence, 20 of 24 entries had similar green stem disorder incidence at the two locations. A third research approach completed over two growing seasons in field cages showed that green stem disorder developed without BPMV infection. BPMV infection did not increase green stem disorder incidence in comparison to controls. Bean leaf beetle, leaf hopper, or stinkbug feeding did not have an effect on the incidence of green stem disorder. The cause of the green stem disorder remains unknown.

Occurrence of Seed Coat Mottling in Soybean Plants Inoculated with Bean pod mottle virus and Soybean mosaic virus.

Soybean seed coat mottling often has been a problematic symptom for soybean growers and the soybean industry. The percentages of seed in eight soybean lines with seed coat mottling were evaluated at harvest after inoculating plants during the growing season with Bean pod mottle virus (BPMV), Soybean mosaic virus (SMV), and both viruses inside an insect-proof cage in the field. Results from experiments conducted over 2 years indicated that plants infected with BPMV and SMV, alone or in combination, produced seed coat mottling, whereas noninoculated plants produced little or no mottled seed. BPMV and SMV inoculated on the same plants did not always result in higher percentages of mottled seed compared with BPMV or SMV alone. There was significant virus, line, and virus-line interaction for seed coat mottling. The non-seed-coat-mottling gene (Im) in Williams isoline L77-5632 provided limited, if any, protection against mottling caused by SMV and none against BPMV. The Peanut mottle virus resistance gene Rpv1 in Williams isoline L85-2308 did not give any protection against mottling caused by SMV, whereas the SMV resistance gene Rsv1 in Williams isoline L78-379 and the resistance gene or genes in the small-seeded line L97-946 gave high levels of protection against mottling caused by SMV. The correlations (r = 0.77 for year 2000 and r = 0.89 for year 2001) between virus infection of the parent plant and seed coat mottling were significant (P = 0.01), indicating that virus infection of plants caused seed coat mottling.

Distribution of Leaf-Feeding Beetles and Bean pod mottle virus (BPMV) in Illinois and Transmission of BPMV in Soybean.

Bean leaf beetles (BLB; Cerotoma trifurcata) were collected in soybean (Glycine max) fields in 58 and 99 Illinois counties surveyed during the 2000 and 2001 growing seasons, respectively. In 2000, BLB counts were highest in the central portion of the state. BLB counts were lower the following year, but were more uniformly distributed throughout the state. BLB tested positive for Bean pod mottle virus (BPMV) in 37 of 41 counties assayed in 2000. In 2001, BLB tested positive for BPMV in 86 of 99 counties sampled. In 2000 and 2001, western corn rootworm (WCR; Diabrotica virgifera virgifera) adults were abundant in soybean fields only in east central Illinois. WCR adults tested positive for BPMV in 21 of 21 east central Illinois counties in 2000 and 20 of 24 sampled in 2001. BPMV was detected in soybean plants in 38 of 46 counties sampled in 2000. Field-collected WCR adults transmitted BPMV to potted soybean plants at low rates either directly from BPMV-infected soybean fields or with prior feeding on BPMV-infected plants. This is the first report of the distribution of BLB, WCR adults, and BPMV in Illinois and of BPMV transmission by adult WCR.

Prevention of morphological changes in alginate microcapsules for islet xenotransplantation.

BACKGROUND: Alginate microcapsule swelling, which occurs as a result of increased hydrophilicity owing to the Ca++ that remains after rapid chelation of the inner alginate core, is a problem in encapsulation. We have previously shown that exchange of the residual divalent Ca++ with the monovalent Na+ through the use of 6 mmol/L Na2SO4 decreases swelling in chelated alginate-polylysine-alginate microcapsules, and this process enhances their durability. The purpose of the present study was to examine the morphology of Na2SO4-treated microcapsules in long-term incubation with the use of serum-supplemented culture medium. METHODS: Spherical beads of purified alginate (3%) that were gelled with 1.1% CaCl2 were first coated with polylysine, and then with 0.24% alginate. After rapid chelation of the inner alginate core with 55 mmol/L sodium citrate, the capsules were either incubated for 30 minutes in 6 mmol/L Na2SO4 or left untreated (control). Each group of capsules was then placed in a flask containing Ham's culture medium supplemented with 20% porcine serum and incubated at 37 degrees C. RESULTS: The diameters of Na2SO4-treated capsules only increased modestly from a mean +/- SD of 635 +/- 22.08 to 684.53 +/- 17.86 microm (P<0.0001) by day 7, with no further increases thereafter. In contrast, control capsules showed a steady increase in their mean diameters, which changed from 639.55 +/- 21.44 to 735.48 +/- 108.85 microm (P < 0.0001) by day 66. In addition, whereas treated capsules remained spherical, control capsules showed progressive polymorphism. CONCLUSION: We have developed a new method of making more durable and stable microcapsules that can be used for islet cell xenotransplantation.

Mutations in ATP-cassette binding proteins G5 (ABCG5) and G8 (ABCG8) causing sitosterolemia.

Sitosterolemia is an autosomal recessive disorder caused by mutations in two adjacent genes encoding coordinately regulated ATP binding cassette (ABC) half transporters (ABCG5 and ABCG8). In this paper we describe three novel mutations causing sitosterolemia: 1) a frameshift mutation (c.336-337insA) in ABCG5 that results in premature termination of the protein at amino acid 197; 2) a missense mutation that changes a conserved residue c.1311C>G; N437K) in ABCG5 and 3) a splice site mutation in ABCG8 (IVS1-2A>G). This study expands the spectrum of the ABCG5 and ABCG8 mutations that cause sitosterolemia. Nine nonsynonymous polymorphisms are also reported: I523V, C600Y, Q604E, and M622V in ABCG5; and D19H, Y54C, T400K, A632V, and Y641F in ABCG8.

High-density lipoprotein binding to scavenger receptor-BI activates endothelial nitric oxide synthase.

Atherosclerosis is the primary cause of cardiovascular disease, and the risk for atherosclerosis is inversely proportional to circulating levels of high-density lipoprotein (HDL) cholesterol. However, the mechanisms by which HDL is atheroprotective are complex and not well understood. Here we show that HDL stimulates endothelial nitric oxide synthase (eNOS) in cultured endothelial cells. In contrast, eNOS is not activated by purified forms of the major HDL apolipoproteins ApoA-I and ApoA-II or by low-density lipoprotein. Heterologous expression experiments in Chinese hamster ovary cells reveal that scavenger receptor-BI (SR-BI) mediates the effects of HDL on the enzyme. HDL activation of eNOS is demonstrable in isolated endothelial-cell caveolae where SR-BI and eNOS are colocalized, and the response in isolated plasma membranes is blocked by antibodies to ApoA-I and SR-BI, but not by antibody to ApoA-II. HDL also enhances endothelium- and nitric-oxide-dependent relaxation in aortae from wild-type mice, but not in aortae from homozygous null SR-BI knockout mice. Thus, HDL activates eNOS via SR-BI through a process that requires ApoA-I binding. The resulting increase in nitric-oxide production might be critical to the atheroprotective properties of HDL and ApoA-I.

Sequence diversity in genes of lipid metabolism.

Elevated plasma lipoprotein levels play a crucial role in the development of coronary artery disease. Genetic factors strongly influence the levels of plasma lipoproteins, but the genes and sequence variations contributing to the most common forms of dyslipidemias are not known. We used GeneChip probe arrays to resequence the coding regions of 10 key genes of lipid metabolism. The sequences of these genes were analyzed in 80 dyslipidemic individuals. Fourteen nonsynonymous and twenty-two synonymous single nucleotide changes were identified that could be confirmed by conventional sequencing. Seven of the fourteen nonsynonymous sequence variants were polymorphisms with allele frequency >1% in the general population. The remaining seven were not found in normolipidemic controls (25 Caucasians and 25 African-Americans). The relationship between nonsynonymous sequence variations and various dyslipidemias was explored in association and family studies. No evidence was found for coding sequence variations in any of the 10 genes contributing to dyslipidemia. Only a single sequence variation, a missense mutation in the low density lipoprotein receptor gene, co-segregated with hyperlipidemia in the proband's family. This study illustrates some of the difficulties associated with identifying sequence variations contributing to complex traits.

Autosomal recessive hypercholesterolemia caused by mutations in a putative LDL receptor adaptor protein.

Atherogenic low density lipoproteins are cleared from the circulation by hepatic low density lipoprotein receptors (LDLR). Two inherited forms of hypercholesterolemia result from loss of LDLR activity: autosomal dominant familial hypercholesterolemia (FH), caused by mutations in the LDLR gene, and autosomal recessive hypercholesterolemia (ARH), of unknown etiology. Here we map the ARH locus to an approximately 1-centimorgan interval on chromosome 1p35 and identify six mutations in a gene encoding a putative adaptor protein (ARH). ARH contains a phosphotyrosine binding (PTB) domain, which in other proteins binds NPXY motifs in the cytoplasmic tails of cell-surface receptors, including the LDLR. ARH appears to have a tissue-specific role in LDLR function, as it is required in liver but not in fibroblasts.

Discovery of 4-[3-(trans-3-dimethylaminocyclobutyl)-1H-indol-5-ylmethyl]-(4S)-oxazolidin-2-one (4991W93), a 5HT(1B/1D) receptor partial agonist and a potent inhibitor of electrically induced plasma extravasation.

Utilizing a pharmacophoric model of binding of 3-(2-aminoethyl)indoles to 5HT(1B/1D) receptors, we identified the 3-aminocyclobutyl group as a potential ethylamine isostere. A novel multidimensional chemometric approach was used to predict the intrinsic activity (degree of agonism) at the receptor. A qualitative model for pharmacokinetic properties was then used to guide the synthesis toward molecules likely to have oral bioavailability in humans. A novel synthetic route to 3-(3-dimethylaminocyclobutyl)indoles was developed. Analogues showed generally lower intrinsic activity at 5HT(1B/1D) receptors than their ethylamine counterparts. 4-[3-(trans-3-Dimethylaminocyclobutyl)-1H-indol-5-ylmethyl]-(4S)-oxazolidin-2-one (4991W93, 1) was identified as a partial agonist against 5HT(1B/1D) receptors, with low intrinsic activity. This molecule also has significant activity against 5HT(1F) receptors but is selective over other 5HT receptors. In addition this compound was found to be an exceptionally potent inhibitor of electrically induced plasma extravasation. Compound 1 may have utility in the treatment and prophylaxis of migraine.

A reliable method for isolation of viable porcine islet cells.

HYPOTHESIS: Mechanical injury and oxidative stress caused by reoxygenation of isolated porcine islet cells result in their unresponsiveness to glucose stimulation. DESIGN: Adult pigs (weighing 25-30 kg) were anesthetized, and following intra-arterial infusion of ice-cold University of Wisconsin solution, a complete pancreatectomy was performed. The pancreatic duct was cannulated for infusion of digestion medium containing collagenase type P, 1.5 mg/mL; deoxyribonuclease I, 10 000 U; and a water-soluble analogue of vitamin E (Trolox), 1 mmol/L. After 20-minute incubations on ice, and at 37 degrees C, the pancreas was hand shaken for 1 minute, followed by filtration and separation on an automatic cell separator (COBE 2991). Islet cells, identified by dithizone staining, were perifused at 37 degrees C. RESULTS: The mean +/- SEM yield of intact purified islet cells (50-200 microm in diameter), and mostly present in clusters, was 2398 +/- 143 cells per gram (n = 12). Glucose stimulation caused a significant increase in biphasic insulin secretion in the perifusion experiments. CONCLUSION: We have developed a simple, reproducible, and reliable procedure for isolating intact and viable porcine islet cells suitable for xenotransplantation.