Adaptive treatment and robust control.

A control theory perspective on determination of optimal dynamic treatment regimes is considered. The aim is to adapt statistical methodology that has been developed for medical or other biostatistical applications to incorporate powerful control techniques that have been designed for engineering or other technological problems. Data tend to be sparse and noisy in the biostatistical area and interest has tended to be in statistical inference for treatment effects. In engineering fields, experimental data can be more easily obtained and reproduced and interest is more often in performance and stability of proposed controllers rather than modeling and inference per se. We propose that modeling and estimation should be based on standard statistical techniques but subsequent treatment policy should be obtained from robust control. To bring focus, we concentrate on A-learning methodology as developed in the biostatistical literature and H∞ -synthesis from control theory. Simulations and two applications demonstrate robustness of the H∞ strategy compared to standard A-learning in the presence of model misspecification or measurement error.

Campylobacter infection associated with consumption of duck liver pâté: a retrospective cohort study in the setting of near universal exposure.

SUMMARY: A retrospective cohort study was performed following several reported cases of gastrointestinal illness after a catered event. The attack rate was 45/77 (58·4%) by clinical case definition, with four individuals confirmed to have Campylobacter. There was near universal exposure to most foodstuffs served; consumption of duck liver pâté [relative risk (RR) 2·53, 95% confidence interval (CI) 1·05-6·10], mixed leaf salad (RR 2·91, 95% CI 1·22-6·92) and table water (RR undefined, P < 0·01) were associated with illness in univariate analysis, with only the latter associated in the final multivariable model (P < 0·001). Samples of cooked duck liver pâté subsequently prepared using identical methods at the venue were contaminated with Campylobacter jejuni and C. coli; water sampling was negative. Making inferences about causation in the presence of near universal exposures in this study required consideration of the limitations of statistical analysis, with the most compelling evidence of the causal role of inadequately prepared duck liver pâté provided by environmental investigation.

Implantation of bone marrow-derived mesenchymal stem cells demonstrates improved outcome in horses with overstrain injury of the superficial digital flexor tendon.

REASONS FOR PERFORMING STUDY: Mesenchymal stem (progenitor; stromal) cell (MSC) therapy has gained popularity for the treatment of equine tendon injuries but without reports of long-term follow-up. OBJECTIVES: To evaluate the safety and reinjury rate of racehorses after intralesional MSC injection in a large study of naturally occurring superficial digital flexor tendinopathy and to compare these data with those published for other treatments. METHODS: Safety was assessed clinically, ultrasonographically, scintigraphically and histologically in a cohort of treated cases: 141 client-owned treated racehorses followed-up for a minimum of 2 years after return to full work. Reinjury percentages were compared to 2 published studies of other treatments with similar selection criteria and follow-up. The number of race starts, discipline, age, number of MSCs injected and interval between injury and treatment were analysed. RESULTS: There were no adverse effects of the treatment with no aberrant tissue on histological examination. The reinjury percentage of all racehorses with follow-up (n = 113) undergoing MSC treatment was 27.4%, with the rate for flat (n = 8) and National Hunt (n = 105) racehorses being 50 and 25.7%, respectively. This was significantly less than published for National Hunt racehorses treated in other ways. No relationship between outcome and age, discipline, number of MSCs injected or injury to implantation interval was found. CONCLUSIONS: Whilst recognising the limitations of historical controls, this study has shown that MPC implantation is safe and appears to reduce the reinjury rate after superficial digital flexor tendinopathy, especially in National Hunt racehorses. POTENTIAL RELEVANCE: This study has provided evidence for the long-term efficacy of MSC treatment for tendinopathy in racehorses and provides support for translation to human tendon injuries.

Antibiotic resistance in faecal bacteria isolated from horses receiving virginiamycin for the prevention of pasture-associated laminitis.

Enterococcus faecium, a major cause of potentially life-threatening hospital-acquired human infections, can be resistant to several antimicrobials, such that streptogramin quinupristin-dalfopristin (Q/D) is one of the few antibiotics still effective. Consequently use of the streptogramin virginiamycin as an animal growth promoter was banned in the EU in 1999 as some believed this contributed to the emergence of Q/D resistant E. faecium. Virginiamycin is advocated for preventing equine pasture-associated laminitis, but its effect on equine faecal bacterial Q/D resistance has not been determined. Faecal samples were obtained from horses receiving virginiamycin, horses co-grazing and horses not exposed to virginiamycin. Streptogramin resistant E. faecium were cultured from 70% (21/30) of animals treated with virginiamycin, 75% (18/24) of co-grazing animals and 69% (11/16) of animals not exposed. ermB and vatD genes were detected using real time PCR in 63% and 66% of animals treated with virginiamycin, 75% and 71% of co-grazing animals and 63% and 69% of animals not exposed. Antimicrobial resistance genes were present only in samples which had cultured Q/D resistant E. faecium. There was no significant difference between groups with respect to antimicrobial resistance. The gene load of vatD was significantly (p=0.04) greater in unexposed animals compared to those treated with virginiamycin. The use of virginiamycin to prevent pasture-associated laminitis does not appear to be related to an increased Q/D resistance frequency. However, in view of the high frequency of resistance within all groups, the horse is a reservoir of Q/D resistant genes and clones that potentially could be transferred transiently to humans.

Maturational alterations in gap junction expression and associated collagen synthesis in response to tendon function.

Energy-storing tendons including the equine superficial digital flexor tendon (SDFT) contribute to energetic efficiency of locomotion at high-speed gaits, but consequently operate close to their physiological strain limits. Significant evidence of exercise-induced microdamage has been found in the SDFT which appears not to exhibit functional adaptation; the degenerative changes have not been repaired by the tendon fibroblasts (tenocytes), and are proposed to accumulate and predispose the tendon to rupture during normal athletic activity. The anatomically opposing common digital extensor tendon (CDET) functions only to position the digit, experiencing significantly lower levels of strain and is rarely damaged by exercise. A number of studies have indicated that tenocytes in the adult SDFT are less active in collagen synthesis and turnover than those in the immature SDFT or the CDET. Gap junction intercellular communication (GJIC) is known to be necessary for strain-induced collagen synthesis by tenocytes. We postulate therefore that expression of GJ proteins connexin 43 and 32 (Cx43; Cx32), GJIC and associated collagen expression levels are high in the SDFT and CDET of immature horses, when the SDFT in particular grows significantly in cross-sectional area, but reduce significantly during maturation in the energy-storing tendon only. The hypothesis was tested using tissue from the SDFT and CDET of foetuses, foals, and young adult Thoroughbred horses. Cellularity and the total area of both Cx43 and Cx32 plaques/mm(2) of tissue reduced significantly with maturation in each tendon. However, the total Cx43 plaque area per tenocyte significantly increased in the adult CDET. Evidence of recent collagen synthesis in the form of levels of neutral salt-soluble collagen, and collagen type I mRNA was significantly less in the adult compared with the immature SDFT; procollagen type I amino-propeptide (PINP) and procollagen type III amino-propeptide (PIIINP) levels per mm(2) of tissue and PINP expression per tenocyte also decreased with maturation in the SDFT. In the CDET PINP and PIIINP expression per tenocyte increased in the adult, and exceeded those in the adult SDFT. The level of PINP per mm(2) was greater in the adult CDET than in the SDFT despite the higher cellularity of the latter tendon. In the adult SDFT, levels of PIIINP were greater than those of PINP, suggesting relatively greater synthesis of a weaker form of collagen previously associated with microdamage. Tenocytes in monolayers showed differences in Cx43 and Cx32 expression compared with those in tissue, however there were age- and tendon-specific phenotypic differences, with a longer time for 50% recovery of fluorescence after photobleaching in adult SDFT cells compared with those from the CDET and immature SDFT. As cellularity reduces following growth in the SDFT, a failure of the remaining tenocytes to show a compensatory increase in GJ expression and collagen synthesis may explain why cell populations are not able to respond to exercise and to repair microdamage in some adult athletes. Enhancing GJIC in mature energy-storing tendons could provide a strategy to increase the cellular synthetic and reparative capacity.

Real-time RT-PCR detection of Bovine Viral Diarrhoea virus in whole blood using an external RNA reference.

A novel two-step real-time RT-PCR assay using SYBR Green I was developed for the detection of acute Bovine Viral Diarrhoea virus (BVDV) infection in whole blood from cattle. During infection animals experience a characteristic transient leucopenia and the number of cells per volume of blood changes over time; so quantitation of viral load by reference to a cellular housekeeping gene is not ideal as this may hide significant animal to animal variation. Therefore, to facilitate comparison of different samples, an external RNA reference was used for normalisation whereby each sample was spiked with the RNA virus, Canine Enteric Coronavirus (CECov), prior to RNA extraction, for comparative purposes. Real-time RT-PCR was carried out with two primer sets designed to amplify either a 156 bp region of the BVDV 5'-UTR or a 280 bp region of the CECov nucleocapsid protein gene. Linearity and efficiency of the assay was established and the method assessed using samples from BVDV-challenged calves. Viral RNA was quantified on days 6 and 14 post-challenge by real-time RT-PCR. Infectious virus isolation by traditional cell culture was negative after day 7. This study demonstrates encouraging results for rapid, sensitive and reliable detection of acute BVDV infection and provides an alternative real-time RT-PCR method for use on whole blood samples or samples where suitable housekeeping genes are not available.

Immune responses to non-structural protein 3 (NS3) of bovine viral diarrhoea virus (BVDV) in NS3 DNA vaccinated and naturally infected cattle.

Immune responses to non-structural protein 3 (NS3) of bovine viral diarrhoea virus (BVDV) were investigated. cDNA encoding NS3 from type 1a BVDV was used to vaccinate five calves, another five calves remained unvaccinated. Three weeks after final vaccination animals were challenged intranasally with heterologous type 1a BVDV. Anti-NS3 antibodies were detected in only one animal post-vaccination. Partial protection from virus challenge was observed in the vaccinates. Virus was not isolated from nasal mucosa of two vaccinates, and virus clearance from nasal mucosa was faster in the vaccinates compared to the controls. While elevated rectal temperatures were evident in both groups 7 days post-challenge, the mean increase in the controls was twice that observed in the vaccinates. In conclusion, NS3 DNA vaccination induced humoral immunity in one calf, and prevented fever and virus establishment in the nasal mucosa in 2/5 calves, demonstrating the efficacy of NS3 vaccination, which may benefit future development of pestivirus and flavivirus vaccines.

Immune cell subpopulations in regenerated splenic tissue in rats.

BACKGROUND: Asplenic patients have an increased risk of infections. Operations such as autotransplantation or splenic artery ligation have been suggested to ensure retention of functional splenic tissue after splenectomy, but their protective value is unclear. Immune responses, such as production of antibody, remain impaired in humans and animals even when such tissue is present, and phagocytosis is less efficient than by normal spleen tissue. In the present study the cellular composition of regenerated tissue is determined. METHODS: Splenic tissue was obtained from rats 6-9 months after splenic autotransplantation, splenic artery ligation or sham operation. The lymphocyte and macrophage subpopulations were labelled using a panel of monoclonal antibodies and analysed by flow cytometry. RESULTS: Both the total number of cells and the number of cells per gram of tissue were significantly reduced. There was a substantial reduction in the percentage of some of the cells examined (CD4+ and CD8+ T lymphocytes subsets), but not all (B lymphocytes, ED1+ and ED2+ macrophages, OX2+ and OX6+ cells). CONCLUSIONS: The reduction in the T lymphocyte subsets in regenerated splenic tissue compared with the normal spleen might explain the immunological dysfunction which persists after splenic autotransplantation. The reduction in the number of macrophages may be responsible for the alteration in phagocytic efficiency of regenerated splenic tissue.

Usefulness of magnetic resonance imaging for evaluating great-vessel anatomy after arterial switch operation for D-transposition of the great arteries.

Magnetic resonance imaging (MRI) produces high-resolution images of great-vessel anatomy in pediatric patients. In this study seven patients, aged 6 to 27 months were evaluated by using gated MRI and two-dimensional echocardiography 4 to 26 months after arterial switch operation for D-transposition of the great arteries. Measurements were taken at the right and left ventricular outflow tracts, beneath the semilunar valves, at the midaortic sinuses, at the anastomotic sites of the main pulmonary artery and the aorta, at the origin of the branch pulmonary arteries, and at the distal pulmonary arteries 1 cm beyond the bifurcation. Concordant results were obtained with both imaging techniques from all sides with the exception of the left pulmonary artery and the right pulmonary artery. With MRI, four patients had significant narrowing at the right pulmonary artery origin and six patients had narrowing at the left pulmonary artery origin. With two-dimensional echocardiogram, two patients had narrowing at the right pulmonary artery origin and four patients had narrowing at the left pulmonary artery origin. The measured pulmonary artery intraluminal diameters in these patients were consistently smaller when assessed by MRI versus two-dimensional echocardiography. To verify these results, five of seven patients underwent cardiac catheterization to provide physiologic correlation before reoperation; the MRI results were found to be significantly closer to the actual catheterization measurements. We conclude that MRI is a sensitive imaging technique for evaluation of great-vessel anatomy in patients after arterial switch operation for D-transposition of the great arteries. It is particularly useful in the evaluation of the branch pulmonary artery anatomy.

Estrogen and progesterone regulate secretion of insulin-like growth factor binding proteins by human breast cancer cells.

We examined the effects of estradiol and progesterone agonist, promegestone (R5020), on secretion of insulin-like growth factors (IGF) and binding proteins (IGFBPs) by T-47D human breast cancer cells cultured in a serum-free defined medium. Estrogen, IGF-I and IGF-II promoted and R5020 inhibited growth under these conditions. Cells secreted IGFs and four IGFBPs. The amounts of all IGFBPs in cell-conditioned media were increased by estradiol and reduced by R5020, which also abolished the stimulatory effect of estradiol on IGFBPs without inducing an IGFBP protease. Therefore estrogen and progesterone may alter growth of breast cancers by regulating tumour secretion of IGFBPs and hence change carcinoma responsiveness to IGFs.

Ovarian and hemolymph ecdysteroid titers during vitellogenesis in Macrobrachium rosenbergii.

Changes in ovarian and hemolymph ecdysteroid concentration and composition during vitellogenesis have been investigated in the freshwater prawn Macrobrachium rosenbergii. Free ecdysteroids (20-hydroxyecdysone and ecdysone) in hemolymph increased in concentration during vitellogenesis from zero at stage 0 to 1.5 ng/ml at stage I to 7.3 ng/ml in mature, stage IV animals. 20-Hydroxyecdysonoic acid (1.2 ng/ml) was detected in the stage IV hemolymph. Ovarian-free ecdysteroid concentration, expressed as nanograms per gram of tissue, fell during vitellogenesis from 83.2 ng/g at stage 0, non-pigmented tissue to 14.2 ng/g at stage IV tissue, being minimal at stage I (6.3 ng/g). However, expression of ovarian free ecdysteroid content as nanograms per ovary revealed a rise from 7.7 ng/ovary at stage 0, nonpigmented tissue to 28.3 ng/ovary at stage IV, again being minimal at stage I (2.0 ng/ovary). 20-Hydroxyecdysonoic acid and ecdysonoic acid were identified at ovarian stages II-III and stage IV.

Innovation through collaboration.

In light of the Canadian Nurses Association's position that the baccalaureate degree would be the requirement for entry to practice by the year 2000, plus evidence of a rapidly changing health care system, changing client characteristics, and on-going economic constraints, administrators of nursing programs in Edmonton recognized the need for a more process-oriented curriculum to prepare nurses to be more capable of facing future challenges. Not surprisingly, limited funds and differing human and material resources meant they could not complete a major curriculum change individually. Collaboration proved to be the key that increased access to baccalaureate nursing education in Alberta.

Progesterone metabolism in vitro in the decapod crustacean, Penaeus monodon.

Thin-layer chromatography (TLC) and reversed-phase high-performance liquid chromatography (RP-HPLC) with on-line detection of radioactive steroids were applied to identify metabolites of [4-14C]progesterone incubated in vitro with prawn ovary. There was extensive metabolism of progesterone by stage II (vitellogenic) ovary of Penaeus monodon. The most abundant metabolites were 5 alpha-pregnane derivatives together with two minor metabolites, 20 alpha-hydroxypregn-4-en-3-one and 1,4-pregnadiene-3,20-dione. In contrast, a much lower level of progesterone metabolism was observed in stage 0 (immature) ovary of this species. The hepatopancreas, gill, and abdominal muscle of P. monodon all metabolised [4-14C]progesterone to varying degrees, generating materials similar to those produced by the ovary. A comparative study of progesterone metabolism in stage II ovary of Nephrops norvegicus indicated that one metabolite, 20 alpha-hydroxypregn-4-en-3-one, was produced.

Inhibition of T47D human breast cancer cell growth by the synthetic progestin R5020: effects of serum, estradiol, insulin, and EGF.

The mechanism of the antiproliferative effects of progestins on human breast cancer cells is not known. In view of the ability of estrogen to stimulate human breast cancer cell production of peptide growth factors, and since previous studies have suggested that the inhibitory action of progestins is dependent on estrogen-stimulated growth, the present study examined the interaction of growth factors and the synthetic progestin R5020 on the proliferation of T47D human breast cancer cells. In this study, the concentrations of estradiol, insulin, and EGF for optimal stimulation of T47D cell growth in 3% dextran-charcoal treated fetal bovine serum (DCC-FBS) were determined to be 1 nM, 100 nM, and 1 nM, respectively. Furthermore, incubation with these optimal concentrations of estradiol, insulin, and EGF in various combinations produced additive effects on T47D cell proliferation, suggesting that these agents act, at least in part, by different mechanisms. In contrast, in a chemically defined medium (DM), both estradiol and EGF were unable to stimulate T47D cell proliferation. In the case of estradiol, the inability to demonstrate stimulation of T47D cell growth in DM was not due to down-regulation of the estrogen receptor. R5020 inhibited the growth of T47D cells, although its effect was more marked in the presence of 3% DCC-FBS than in DM. Stimulation of T47D cell growth by either estradiol or insulin in 3% DCC-FBS was effectively inhibited by R5020. In contrast, growth of T47D cells stimulated by EGF in the absence of estradiol was not markedly inhibited by R5020, the growth being comparable to that of untreated control cells. These findings suggest that the inhibitory effect of R5020 on T47D cell proliferation is dominant over the action of some, but not all, breast cancer mitogens.

Identification of free and conjugated ecdysteroids in cercariae of the schistosome Trichobilharzia ocellata.

Extracts of cercariae of the avian schistosome Trichobilharzia ocellata were analysed for the presence of ecdysteroids by radioimmunoassay, high-performance liquid chromatography monitoring fractions by radioimmunoassay, and gas chromatography/mass spectrometry (selected ion monitoring). Both free ecdysteroids and polar conjugated ecdysteroids were detected in the cercarial extracts. The free ecdysteroid fraction, as well as the hydrolysed polar conjugated ecdysteroid fraction, contained both ecdysone and 20-hydroxyecdysone in approximately equal amounts. The amount of ecdysteroids detected is comparable to those found in other platyhelminths. A possible role for the ecdysteroids in the development of the parasite and/or the interactions between the parasite and its intermediate host, the freshwater snail Lymnaea stagnalis, is discussed.

A new directly moulded patellar-tendon-bearing socket.

Silicone rubbers and casting tapes individually have previously been used in the manufacture of sockets (Swanson, 1972; Sweitzer, 1973; Ruder, 1977; Graves, 1980; Aqualite, 1982). The authors believe that the present combination of these materials to manufacture a directly moulded socket with a complete silicone rubber lining of variable thickness has not previously been described. The new socket, after addition of the modular components, allows fitting of an aligned below-knee prosthesis within three hours. The socket (Fig. 1.) is made directly on the below-knee stump, can be completed with experience in an hour and does not require the use of specialized equipment. The socket consists of an outer supportive Scotchflex layer inside which is a lining of soft smooth biocompatible silicone rubber of deliberately variable thickness to allow pressure tolerant areas to accept more load and pressure sensitive areas to accept less load (Fig.2). The thicker areas of silicone are produced by applying carefully cut Plastazote pads to the pressure sensitive areas. The thickness and extent of the pads is individually assessed according to the estimated sensitivity of the particular area (Fig. 3). The Scotchflex socket is then manufactured directly on the below-knee stump with these pads applied. The pads are then removed prior to insertion of a semi-liquid silicone rubber. Thus, when the socket with the liquid silicone rubber is re-applied to the stump, the space produced by the pads is filled by the rubber which then sets at room temperature. In this way a layer of variable thickness is produced.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibody-dependent cellular cytotoxicity in blood monocytes and alveolar macrophages in patients with lung cancer.

Antibody-dependent cellular cytotoxicity (ADCC), mediated by blood monocytes (MNC) and pulmonary alveolar macrophages (PAM) obtained from 31 patients with lung cancer and 13 control subjects, was determined. The ADCC of the PAM obtained from patients with lung cancer was significantly less (40% reduction) than that of the control group. This finding was demonstrated over the range of effector-to-target cell ratios. The ADCC of blood MNC from the cancer patients was not different from that of control subjects and in both groups the ADCC of blood MNC was significantly greater than that of lung macrophages. Comparison of PAM ADCC in smokers and nonsmokers within the control group suggested that the lower activity in cancer patients was not simply an effect of smoking.

The effect of tamoxifen on the growth of human malignant melanoma in vitro.

The effect of tamoxifen on the growth of malignant melanoma was investigated using human cell lines and single-cell suspensions prepared from patients' tumours cultured in soft agar. Tamoxifen stimulated both [3H]-thymidine incorporation and cell numbers in all of the cell lines tested. Cytoplasmic oestrogen receptor (ER) was detected in one of the responding lines and progesterone receptor (PR) in another. Tumour colony formation in soft agar culture was satisfactorily established from tumour cell suspensions from 13 of 21 patients, only one of which had detectable cytoplasmic ER. Greater than 50% reduction in colony formation with 5 X 10(-7) M tamoxifen occurred in two tumours, neither of which contained ER. These results indicate that tamoxifen has the potential to either retard or accelerate the growth of human malignant melanoma.

A method for socket duplication using Silcoset R 105.

A technique for duplicating an existing comfortable socket has been in use for two years. The method, using Silcoset R 105, provides the prosthetist with a re-usable positive mould thus enabling the manufacture of two prostheses of comparable fit.

Monocyte antibody-dependent cellular cytotoxicity in splenectomized subjects.

Monocyte antibody-dependent cellular cytotoxicity (ADCC) was determined in normal subjects by using human A1 erythrocytes and immune human anti-A1 antiserum. The experimental data were fitted to a mathematical model and the values for maximal cytotoxicity and monocyte numbers required to produce 15% specific cytotoxicity (MD15) were subsequently computed and compared with the values for these two parameters estimated from the dose response curves. The values for both were significantly associated, and the mathematical model permitted precise quantitation of cytotoxicity in instances where this was impossible by standard methods. Maximal monocyte ADCC was significantly reduced in a group of splenectomized subjects, whereas the MD15 was increased. These findings emphasize the possible influences of the method of quantitation of ADCC and provide one explanation for the apparent conflict of published data.