Hepatobiliary circulation and dominant urinary excretion of homogentisic acid in a mouse model of alkaptonuria.

Altered activity of specific enzymes in phenylalanine-tyrosine (phe-tyr) metabolism results in incomplete breakdown of various metabolite substrates in this pathway. Increased biofluid concentration and tissue accumulation of the phe-tyr pathway metabolite homogentisic acid (HGA) is central to pathophysiology in the inherited disorder alkaptonuria (AKU). Accumulation of metabolites upstream of HGA, including tyrosine, occurs in patients on nitisinone, a licenced drug for AKU and hereditary tyrosinaemia type 1, which inhibits the enzyme responsible for HGA production. The aim of this study was to investigate the phe-tyr metabolite content of key biofluids and tissues in AKU mice on and off nitisinone to gain new insights into the biodistribution of metabolites in these altered metabolic states. The data show for the first time that HGA is present in bile in AKU (mean [±SD] = 1003[±410] µmol/L; nitisinone-treated AKU mean [±SD] = 45[±23] µmol/L). Biliary tyrosine, 3(4-hydroxyphenyl)pyruvic acid (HPPA) and 3(4-hydroxyphenyl)lactic acid (HPLA) are also increased on nitisinone. Urine was confirmed as the dominant elimination route of HGA in untreated AKU, but with indication of biliary excretion. These data provide new insights into pathways of phe-tyr metabolite biodistribution and metabolism, showing for the first time that hepatobiliary excretion contributes to the total pool of metabolites in this pathway. Our data suggest that biliary elimination of organic acids and other metabolites may play an underappreciated role in disorders of metabolism. We propose that our finding of approximately 3.8 times greater urinary HGA excretion in AKU mice compared with patients is one reason for the lack of extensive tissue ochronosis in the AKU mouse model.

On fragmenting, densely mineralised acellular protrusions into articular cartilage and their possible role in osteoarthritis.

High density mineralised protrusions (HDMP) from the tidemark mineralising front into hyaline articular cartilage (HAC) were first described in Thoroughbred racehorse fetlock joints and later in Icelandic horse hock joints. We now report them in human material. Whole femoral heads removed at operation for joint replacement or from dissection room cadavers were imaged using magnetic resonance imaging (MRI) dual echo steady state at 0.23 mm resolution, then 26-µm resolution high contrast X-ray microtomography, sectioned and embedded in polymethylmethacrylate, blocks cut and polished and re-imaged with 6-µm resolution X-ray microtomography. Tissue mineralisation density was imaged using backscattered electron SEM (BSE SEM) at 20 kV with uncoated samples. HAC histology was studied by BSE SEM after staining block faces with ammonium triiodide solution. HDMP arise via the extrusion of an unknown mineralisable matrix into clefts in HAC, a process of acellular dystrophic calcification. Their formation may be an extension of a crack self-healing mechanism found in bone and articular calcified cartilage. Mineral concentration exceeds that of articular calcified cartilage and is not uniform. It is probable that they have not been reported previously because they are removed by decalcification with standard protocols. Mineral phase morphology frequently shows the agglomeration of many fine particles into larger concretions. HDMP are surrounded by HAC, are brittle, and show fault lines within them. Dense fragments found within damaged HAC could make a significant contribution to joint destruction. At least larger HDMP can be detected with the best MRI imaging ex vivo.

Developing Decision-Making Expertise in Professional Sports Staff: What We Can Learn from the Good Judgement Project.

Success within performance sports is heavily dependent upon the quality of the decisions taken by educated and experienced staff. Multi-disciplinary teams (MDTs) typically collate voluminous data, and staff typically undergo extensive and rigorous technical and domain-specific training. Although sports professionals operate in sometimes volatile, uncertain, complex and ambiguous decision-making environments, a common assumption seems to be that education and experience will automatically lead to enhanced and effective decision-making capabilities. Accordingly, there are few formal curriculums, in coaching or sports science contexts, focussed on translating the extensive research on judgement and decision-making expertise to professional sports staff. This article aims to draw on key research findings to offer insights and practical recommendations to support staff working within professional performance contexts. Through this distillation, we hope to enhance understanding of the factors underpinning effective decision-making in dynamic, high-stakes professional sporting environments. Broadly, the conclusions of this research demonstrate that decision-making efficacy is enhanced through application of three specific strategies: (i) Design of more engaging professional cultures harnessing the power of collectives encouraging diverse opinions and perspectives, and fostering and promoting collaborative teamwork, (ii) education specifically targeting debiasing training, designed to counter the most common cognitive pitfalls and biases and, (iii) the implementation of evaluation strategies integrating rigorous testing and real-time feedback.

Ochronosis in a murine model of alkaptonuria is synonymous to that in the human condition.

OBJECTIVE: Alkaptonuria (AKU) is a rare genetic disease which results in severe early onset osteoarthropathy. It has recently been shown that the subchondral interface is of key significance in disease pathogenesis. Human surgical tissues are often beyond this initial stage and there is no published murine model of pathogenesis, to study the natural history of the disease. The murine genotype exists but it has been reported not to demonstrate ochronotic osteoarthropathy consistent with the human disease. Recent anecdotal evidence of macroscopic renal ochronosis in a mouse model of tyrosinaemia led us to perform histological analysis of tissues of these mice that are known to be affected in human AKU. DESIGN: The homogentisate 1,2-dioxygenase Hgd(+/)(-)Fah(-)(/)(-) mouse can model either hereditary tyrosinaemia type I (HT1) or AKU depending on selection conditions. Mice having undergone Hgd reversion were sacrificed at various time points, and their tissues taken for histological analysis. Sections were stained with haematoxylin eosin (H&E) and Schmorl's reagent. RESULTS: Early time point observations at 8 months showed no sign of macroscopic ochronosis of tissues. Macroscopic examination at 13 months revealed ochronosis of the kidneys. Microscopic analysis of the kidneys revealed large pigmented nodules displaying distinct ochre colouration. Close microscopic examination of the distal femur and proximal fibula at the subchondral junctions revealed the presence of numerous pigmented chondrocytes. CONCLUSIONS: Here we present the first data showing ochronosis of tissues in a murine model of AKU. These preliminary histological observations provide a stimulus for further studies into the natural history of the disease to provide a greater understanding of this class of arthropathy.

The role of calcified cartilage and subchondral bone in the initiation and progression of ochronotic arthropathy in alkaptonuria.

OBJECTIVE: Alkaptonuria is a genetic disorder of tyrosine metabolism, resulting in elevated circulating concentrations of homogentisic acid. Homogentisic acid is deposited as a polymer, termed ochronotic pigment, in collagenous tissues, especially cartilages of weight-bearing joints, leading to a severe osteoarthropathy. We undertook this study to investigate the initiation and progression of ochronosis from the earliest detection of pigment through complete joint failure. METHODS: Nine joint samples with varying severities of ochronosis were obtained from alkaptonuria patients undergoing surgery and compared to joint samples obtained from osteoarthritis (OA) patients. Samples were analyzed by light and fluorescence microscopy, 3-dimensional scanning electron microscopy (SEM), and the quantitative backscattered electron mode of SEM. Cartilage samples were mechanically tested by compression to determine Young's modulus of pigmented, nonpigmented, and OA cartilage samples. RESULTS: In alkaptonuria samples with the least advanced ochronosis, pigment was observed intracellularly and in the territorial matrix of individual chondrocytes at the boundary of the subchondral bone and calcified cartilage. In more advanced ochronosis, pigmentation was widespread throughout the hyaline cartilage in either granular composition or as blanket pigmentation in which there is complete and homogenous pigmentation of cartilage matrix. Once hyaline cartilage was extensively pigmented, there was aggressive osteoclastic resorption of the subchondral plate. Pigmented cartilage became impacted on less highly mineralized trabeculae and embedded in the marrow space. Pigmented cartilage samples were much stiffer than nonpigmented or OA cartilage as revealed by a significant difference in Young's modulus. CONCLUSION: Using alkaptonuria cartilage specimens with a wide spectrum of pigmentation, we have characterized the progression of ochronosis. Intact cartilage appears to be resistant to pigmentation but becomes susceptible following focal changes in calcified cartilage. Ochronosis spreads throughout the cartilage, altering the mechanical properties. In advanced ochronosis, there is aggressive resorption of the underlying calcified cartilage leading to an extraordinary phenotype in which there is complete loss of the subchondral plate. These findings should contribute to better understanding of cartilage-subchondral interactions in arthropathies.

Screening for diabetic retinopathy: a comparative trial of photography and scanning laser ophthalmoscopy.

AIMS: To evaluate the sensitivity and specificity of wide-field scanning laser ophthalmoscopy (WSLO) in the detection of referable diabetic eye disease, and to compare its performance with digital retinal photography. METHODS: Patients enrolled into the study underwent non-mydriatic WSLO imaging, then single- and dual-field mydriatic digital retinal photography, and examination with slit lamp biomicroscopy, the reference standard. Grading of retinopathy was performed in a masked fashion. RESULTS: A total of 380 patients (759 eyes) were recruited to the study. Technical failure rates for dilated single-field retinal photography, dual-field retinal photography and undilated WSLO were 6.3, 5.8 and 10.8%, respectively (0.005 < p < 0.02 for photography vs. WSLO). The respective indices for screening sensitivity were 82.9, 82.9 and 83.6% (p > 0.2). Specificity was 92.1, 91.1 and 89.5%, respectively (p > 0.2). CONCLUSIONS: Sensitivity and specificity for WSLO were similar to retinal photography. The technical failure rate was greater for the WSLO used in this study.

PPAR agonists modulate human osteoclast formation and activity in vitro.

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear steroid hormone superfamily and exist in three isoforms: PPARalpha, beta and gamma, each with specific functions. In this study, we have investigated the expression of PPARs by human osteoclast precursors and osteoclasts generated in vitro. In addition, the effects of fibrates and isoform-specific PPAR agonists on osteoclast formation and resorption in vitro were determined. Human peripheral blood mononuclear cells (PBMCs) were stimulated with human recombinant RANKL and M-CSF to generate osteoclasts. RNA was extracted at days 0, 7, 14 and 21 and RT-PCR for all three PPAR isoforms demonstrated their expression throughout this culture period. To determine the effect on osteoclast formation, PPAR agonists (10(-8) M to 10(-5) M) were added from the beginning of the culture until day 14 and the number of multinucleated osteoclasts counted. The effect of PPAR agonists on osteoclast function was similarly determined by treating mature, multinucleated osteoclasts cultured on dentine wafers with PPAR agonists (10(-8) M to 10(-5) M) for 7 days and quantifying resorption. Bezafibrate and fenofibrate, which non-discriminately activate all PPAR isoforms, significantly inhibited the formation of multinucleated osteoclasts from PBMC in vitro. Bezafibrate treatment of mature osteoclast resulted in 50% inhibition (at 10(-8) M and 10(-7) M) of resorption, yet fenofibrate had no significant effect. Activation of individual PPARs with isoform-specific agonist (GW9578, L165041 and ciglitizone which preferentially activate PPARalpha, beta and gamma respectively) resulted in significant dose-dependent inhibition of multinucleated osteoclast formation. Divergent effects on osteoclast resorption were observed; GW9578 had no significant effect on resorption, whereas ciglitizone and L165041 dose-dependently inhibited and stimulated resorption, respectively. These data show for the first time expression of all three PPAR isoforms throughout the development and maturation period of osteoclasts generated from human PBMCs. In addition, we demonstrate that isoform-specific PPAR agonists have strong effects on multinucleation and highly variable effects on bone resorption. In conclusion, this study highlights the potential of PPARs as therapeutic targets in diseases with accelerated osteoclast formation and resorption.

Functional evidence for a colorectal cancer tumor suppressor gene at chromosome 8p22-23 by monochromosome transfer.

Chromosome 8p is considered, from loss of heterozygosity analysis, to be a strong candidate for the location of a tumor suppressor gene inactivated in colorectal cancer. We have found a 53% (27 of 51) rate of allelic loss at the LPL locus on 8p22, with the smallest region of overlap of deletions including the region D8S258 to D8S277. Using microcell-mediated monochromosome 8 transfer into three colorectal cancer cell lines, SW480, SW620 and HT29, we have demonstrated a reduction of tumorigenicity in SW620 hybrids. Partial deletions of chromosome 8 in some SW620/8 hybrids further delineate the critical region(s) to 8p22-23. Hybrids of the colorectal cancer cell lines SW480 and HT29 containing chromosome 8 did not show suppression of tumorigenesis, but the H29/8 hybrid showed total suppression of soft agar clonicity. This indicates an alternate pathway of mutational progression in these three lines, despite the fact that SW480 was derived from the same patient as SW620.

Frequent loss of heterozygosity and three critical regions on the short arm of chromosome 8 in ovarian adenocarcinomas.

Many chromosomal regions undergo loss of heterozygosity (LOH) in ovarian adenocarcinomas but few of the target regions have been finely mapped. One of the chromosome arms likely to harbour one or more tumour suppressor genes inactivated in ovarian cancer is the short arm of chromosome 8 which is frequently deleted in many other solid tumours. We have examined a large panel of microsatellite markers on 8p for LOH in 53 ovarian adenocarcinomas. LOH was observed in 27 tumours (51%), with a significant trend towards a higher frequency of LOH in more advanced tumours. Detailed examination of nine tumours with partial deletions defined three regions of overlap, two in 8p23 and one in 8p22, which suggests that there might be as many as three tumour or metastasis suppressor genes on 8p which are inactivated during ovarian tumorigenesis. LOH on 8p was significantly associated with 9p LOH which suggests that inactivation of target genes on these chromosomes may be cooperative events.

Plant chromosome homology: hypotheses relating rendezvous, recognition and reciprocal exchange.

Many higher eukaryotes have dispersed repetitive DNA and multiple instances of segmental duplications. As well, many plants and lower animals are polyploids. Thus restricting reciprocal genetic exchange to truly homologous chromosomes is likely a multi-step process. We propose the following sequence of events. First the ability to form a synaptonemal complex (SC) prematurely (i.e. before homology checking/recognition) is precluded by the organization of chromosomes during premeiotic S phase. Next rough alignment is accomplished regionally by having key allelic transcription units brought to the same transcription center. Once rough alignment is accomplished, close alignment can occur in conjunction with homology checking/recognition. Successful homology checking produces changes that now permit SC formation within the region of the check. Some organisms (with challenges to true homology such as dispersed repetitive DNA and segmental duplications) may require that, for a region to be competent to form an SC, successful homology checks must occur at both ends of the region. Successful early SC formation may provide an environment in which recombination intermediates can be earmarked for resolution into crossovers. Later in prophase I SC formation can occur nonhomologously, if two unsynapsed chromosomal axes meet.

Automated routine HLA-B27 typing by flow cytometry.

An increasing demand for HLA-B27 typing as one of the tests used in the diagnosis of ankylosing spondylitis had led us to develop a rapid, automated, flow cytometric assay using whole blood and an HLA-B27 specific monoclonal antibody FD705. This article shows the data from 2093 samples tested during a 2 year period of routine HLA-B27 typing. 21.6% were clearly HLA-B27 positive whilst 73.2% were HLA-B27 negative, the remaining 5.2% required further testing before assignment of HLA-B27 status. Additional work was carried out on blood samples from individuals positive for the newly described subtype HLA-B2708.

Detection of kidney reactive antibodies at crossmatch in renal transplant recipients.

We have investigated retrospectively sera from 70 renal allograft recipients for the presence of antibodies binding to a preanastomosis biopsy of the donor kidney by an indirect immunoperoxidase technique. All recipients had a negative T cell lymphocytotoxic crossmatch. A positive immunoperoxidase crossmatch for kidney-reactive antibodies was seen in 13/70 (18.6%) recipients--6 reacting with endothelium, 4 with epithelium, and 3 with both cell types. Neither the presence of these antibodies nor their pattern of staining correlated with recipient graft outcome. Following transplantation endothelial reactive antibodies developed in 15/43 (35%) patients, whereas tubular epithelial antibodies occurred in 5/43 (12%). The antibodies were persistent and accompanied graft failure in 10/14 (71%) patients, while transient antibodies were only associated with graft failure in 2/12 (17%) recipients. Development of lymphocytotoxic antibodies did not correlate with the presence of renal reactive antibodies or eventual graft outcome. Smooth muscle and antinuclear antibodies in recipient sera prior to transplantation were associated with improved graft survival. Eluates of 8/14 rejected grafts confirmed the presence of renal reactive antibodies, with patterns of staining similar to those observed in recipient sera. Antibodies in 7/8 recipients were shown by absorption studies to have HLA class I and/or II specificity, the remaining recipient having proximal tubular brush border antibodies.

Interactions between cytokines and eicosanoids: a study using human peritoneal macrophages.

To examine the interactions between the main pro-inflammatory cytokines and eicosanoids produced by human inflammatory cells, human peritoneal macrophages (hp-M phi) were isolated from ascitic fluid of patients with portal hypertension. Interactions between interleukin-1 beta (IL-1 beta), IL-6, tumor necrosis factor alpha (TNF-alpha), leukotriene B4 (LTB4) and prostaglandin E2 (PGE2) were studied by addition or inhibition of several cytokines and eicosanoids: human recombinant IL-1 beta (hrIL-1 beta) addition, LTB4 addition and 5-lipoxygenase inhibition (6-hydroxy-2-(4-sulfamoylbenzylamino)-4,5,7-trimethylbenzothiaz ole hydrochloride (E6080)), PGE2 addition and cyclooxygenase inhibition (indomethacin). In hp-M phi hrIL-1 beta stimulated the LTB4 production, while the PGE2 production was inhibited. HrIL-1 beta had no significant effect on IL-6 production in hp-M phi. LTB4 did not regulate IL-1 beta and IL-6 production. Increasing PGE2 down regulated the TNF-alpha production, but did not effect the IL-1 beta and IL-6 production.

A comparison of serological, cellular and DNA-RFLP methods for HLA matching in the selection of related bone marrow donors.

Serological, cellular and DNA-RFLP (restriction fragment length polymorphism) methods of determining HLA compatibility between 10 leukaemic patients and potential related bone marrow donors were systematically compared. DR beta/DQ alpha/DQ beta/DNA-RFLP typing of these families gave results in agreement with those obtained by serological methods (matching for HLA-A, -B and -DR), supported by mixed lymphocyte culture (MLC) data, indicating the validity and accuracy of DNA-RFLP matching in transplantation. However, a significant minority of four leukaemic patients plus two healthy individuals were not clearly HLA-DR typable by serology, but all such individuals were easily typable by DNA-RFLP. These results were supported by MLC data, where available. In addition, all data were in agreement with previously reported correlations between DNA-RFLPs and HLA-DR serology, allowing unambiguous assignment of HLA-DR types where these were previously in doubt. These results demonstrate the value of DNA-RFLP HLA class II DR and DQ typing in leukaemic patients requiring marrow transplantation who are not clearly typable by traditional methods and suggest that this approach should constitute an important element of future HLA matching programmes for bone marrow transplantation.

M2 delta-to-oxalate pi* conjugation in oxalate-bridged complexes containing M-M quadruple bonds.

The electronic structures of oxalate-bridged, quadruply-bonded dimolybdenum and ditungsten compounds have been investigated by a variety of computational methods employing density function theory (gradient corrected and time-dependent) which reveal the consequences of strong mixing of M2 delta and oxalate pi orbitals within extended chains and cyclic structures.

Sex identification of elk (Cervus elaphus canadensis), moose (Alces alces), and white-tailed deer (Odocoileus virginianus) using the polymerase chain reaction.

We have developed a PCR-based protocol to determine the gender of tissue samples originating from elk (Cervus elaphus canadensis), moose (Alces alces) and white-tailed deer (Odocoileus virginianus). The technique simultaneously amplifies a conserved region of the sex-determining gene on the Y-chromosome (Sry) and a region of the Fragile X mental retardation gene (Fmr-1). The multiplex nature of this protocol allows the determination of gender using the Sry marker with the Fmr-1 marker providing an internal control. This technique is applicable to the enforcement of the validation tag system for game species. Data are provided from a wildlife investigation in Ontario.

Application of DNA fingerprinting to enforcement of hunting regulations in Ontario.

DNA fingerprinting has been used in investigations of 40 cases of infractions of hunting regulations involving white-tailed deer (Odocoileus virginianus) and moose (Alces alces) in Ontario. In most of these cases, individual-specific DNA fingerprints obtained with the Jeffrey's 33.15 multilocus probe were used to link the animal remains found at the illegal kill site to blood and tissue samples of the dead animal associated with a suspect. DNA fingerprints from 27 white-tailed deer and 19 moose were obtained in order to establish the level of band-sharing in DNA fingerprints among unrelated individuals in each species. We also determined the levels of band-sharing among animals from the same region and calculated the probability of two individuals sharing the same DNA fingerprint. Details are presented from cases in which the evidence was presented and accepted by Ontario courts.

Forensic application of repetitive DNA markers to the species identification of animal tissues.

Highly repetitive DNA markers have been used for determining the species origin of animal tissues in cases of illegal commercialization and poaching of game animals. This approach has been used in cases involving white-tailed deer (Odocoileus virginianus), moose (Alces alces) and black bear (Ursus americanus). Digesting the DNA with various restriction enzymes, agarose electrophoresis and staining with ethidium bromide revealed unique banding patterns for each species. These patterns have been used to distinguish meat from game animal species from commercial sources of meat and organs. Data are presented from two Ontario court cases that demonstrate the application of the procedure.

Postexposure rabies prophylaxis and preexposure rabies vaccination failure in domestic animals.

OBJECTIVES: To determine the effectiveness of postexposure rabies prophylaxis (PEX) recommendations, as mandated by the state of Texas, and to investigate PEX and preexposure rabies vaccination failures. DESIGN: Retrospective study. ANIMALS: 1,345 unvaccinated domestic animals that had received PEX and 6 animals that had preexposure rabies vaccination failure. PROCEDURE: Zoonotic incident case report forms from 1979 through 1994 were reviewed for information about unvaccinated domestic animals that received PEX after exposure to a rabid animal, according to state protocol; the reports were also reviewed for information about preexposure rabies vaccination failures. From 1979 through 1987, the PEX protocol was to immediately vaccinate the animal against rabies, isolate it for 6 months, and administer a booster vaccination 1 month prior to release from isolation. From 1988 through 1994, the protocol was to immediately vaccinate the animal against rabies, isolate it for 90 days, and give booster vaccinations during the third and eighth weeks of the isolation period. RESULTS: From 1979 through 1987, 713 animals received PEX; 2 failures were recorded. From 1988 through 1994, 632 animals received PEX; 3 failures were recorded. From 1991 through 1994, 6 preexposure rabies vaccination failures were recorded. CLINICAL IMPLICATIONS: An effective PEX schedule for domestic animals includes immediate rabies vaccination, with a minimum of 1 booster vaccination, and 90 days' strict isolation.

Postexposure rabies prophylaxis protocol for domestic animals and epidemiologic characteristics of rabies vaccination failures in Texas: 1995-1999.

OBJECTIVE: To determine whether postexposure rabies prophylaxis (PEP) in domestic animals, as mandated by the state of Texas, has continued to be effective and to evaluate PEP and preexposure rabies vaccination failures from 1995 through 1999. DESIGN: Retrospective study. ANIMALS: 830 unvaccinated domestic animals (621 dogs, 78 horses, 71 cats, and 60 cattle) that received PEP and 4 animals (3 dogs and 1 horse) that had preexposure rabies vaccination failure. PROCEDURE: Zoonotic incident case reports from 1995 through 1999 were reviewed for information regarding unvaccinated domestic animals that received PEP according to state protocol after exposure to a rabid animal; reports were also reviewed for information regarding preexposure rabies vaccination failures. The PEP recommendations were to immediately vaccinate the animal against rabies, isolate the animal for 90 days, and administer booster vaccinations during the third and eighth weeks of the isolation period. Rabies vaccines used in the PEP protocol were administered via the route prescribed by the USDA. RESULTS: From 1995 through 1999, 830 animals received PEP; 4 failures were recorded. Additionally, 4 preexposure rabies vaccination failures were recorded. CONCLUSIONS AND CLINICAL RELEVANCE: Results of this study indicate that an effective PEP protocol for unvaccinated domestic animals exposed to rabies includes immediate vaccination against rabies, a strict isolation period of 90 days, and administration of booster vaccinations during the third and eighth weeks of the isolation period. This PEP schedule has proven to be effective for control of rabies in domestic animals.