Assessment of flow-mediated dilatation in the superficial femoral artery using a sustained shear stress stimulus via calf plantar-flexion exercise.

NEW FINDINGS: What is the central question of this study? The aim was to establish the ability of a newly designed leg exercise technique to produce sustained elevations in shear rate that stimulate flow-mediated dilatation (FMD) in the superficial femoral artery and to determine the repeat trial stability of the FMD response. What is the main finding and its importance? Calf plantar-flexion exercise can be used to increase shear stress and stimulate FMD in the superficial femoral artery. However, the magnitude of FMD varied systematically when multiple trials were repeated in short succession. The superficial femoral artery (SFA) is susceptible to vascular disease, and a technique to assess flow-mediated dilatation (FMD) in this vessel in response to a sustained shear stress stimulus could provide important information about endothelial function. The aim of this study was to establish the ability of a newly designed SFA leg exercise-FMD (LEX-FMD) technique to produce sustained elevations in shear rate, which stimulate FMD, and to determine the repeat trial stability of the FMD response. The SFA FMD stimulated by reactive hyperaemia (RH) and calf plantar-flexion exercise (LEX) was assessed via ultrasound in 19 healthy men (n = 10) and women (n = 9). The two experimental visits included either four trials of LEX-FMD or four trials of RH-FMD. The shear stress stimulus was estimated as the shear rate (blood velocity/SFA diameter). Results are expressed as the means ± SD. The LEX steady-state shear rate was consistent between trials (P = 0.176), whereas the RH shear rate area under the curve was higher in trial 1 versus trials 2-4 (P < 0.05). The %RH-FMD (four-trial mean 4.9 ± 2.5%) and absolute RH-FMD were not significantly different between trials (P = 0.465 and P = 0.359, respectively). Both %LEX-FMD and absolute LEX-FMD were higher during trial 3 (4.8 ± 3.4%) than trial 1 (3.6 ± 2.7%; P = 0.026 and P = 0.026, respectively). The magnitude of RH-FMD and LEX-FMD did not differ (P = 0.241). These results indicate that calf plantar-flexion exercise can be used to increase shear stress and stimulate FMD in the SFA. However, although SFA RH-FMD was stable across four trials, LEX-FMD varied systematically when multiple trials were repeated in rapid succession.

Impaired brachial artery endothelial function in young healthy women following an acute painful stimulus.

INTRODUCTION: Impaired endothelial function has been observed during and immediately following an acutely painful stimulus. However, the extent to which this persists following pain dissipation is unclear. PURPOSE: To determine whether painful ischemic handgrip exercise (pain task) causes impaired flow-mediated dilation (FMD) after the sensation of pain and hemodynamic responses have abated. A second purpose was to determine whether the response to pain differed with a predisposition to magnify, ruminate, and feel helpless about pain (pain catastrophizing status). METHODS: Brachial artery FMD stimulated by reactive hyperemia was assessed via ultrasound in 18 (9 high catastrophizing) healthy, women (20 ± 1 years) before and 15 min after a 3 min pain task. The shear stress stimulus for FMD was estimated as shear rate (blood velocity/brachial artery diameter). RESULTS (MEAN ± SD): None of the variables were significantly impacted by pain catastrophizing status and are presented pooled across group. The pain task increased pain ratings [1 ± 1-6 ± 3 (0-10 scale) (p < 0.001)], mean arterial pressure (MAP) (p < 0.001) and heart rate (HR) (p < 0.001), all returning to pre-pain levels ≤2-min post-pain task (pre-pain vs. 2-min post-pain: pain rating p = 1.000; MAP p = 0.142; HR p = 0.992). The shear rate stimulus was not different between pre- and post-pain task FMD tests (p = 0.200). FMD decreased post-pain task (10.8 ± 4.6 vs. 7.0 ± 2.7 %, p < 0.001). CONCLUSION: These results indicate that, regardless of pain catastrophizing status, painful ischemic handgrip exercise has a deleterious impact on endothelial function that persists after the pain sensation and hemodynamic responses have abated.

CMV enhancer-promoter is preferentially active in exocrine cells in vivo.

The CMV enhancer-promoter sequence is often used as a transcriptional regulatory element in vector systems. We have used this control element to drive expression of GFP in a lentivirus vector transgene in pigs and chickens. Promoted as a 'universal' enhancer/promoter element capable of transcriptional activity in a number of cells in vitro, CMV-GFP transgene expression in vivo is preferentially observed in exocrine cells. This expression profile validates the use of this transcriptional control sequence to target expression to exocrine cells in gene transfer strategies.

Somatic cell nuclear transfer.

Cloning by nuclear transfer from adult somatic cells is a remarkable demonstration of developmental plasticity. When a nucleus is placed in oocyte cytoplasm, the changes in chromatin structure that govern differentiation can be reversed, and the nucleus can be made to control development to term.

Embryo development and establishment of pregnancy after embryo transfer in pigs: coping with limitations in the availability of viable embryos.

Embryo transfer and pregnancy maintenance strategies in pigs were evaluated with reference to situations in which limited numbers of viable embryos or micromanipulated embryos are available, such as pig cloning. Development of embryos with compromised zona pellucida was compared with development of embryos with intact zona pellucida. Micromanipulation had no effect on blastocyst production rates after development in vivo or in vitro, but development in vivo improved the number of embryos reaching the blastocyst stage. Transfer of embryos with compromised zona pellucida resulted in live piglets. Several hormone treatments to maintain pregnancy were tested in a model in which three embryos were transferred into unmated recipient gilts, compared with transfer of three embryos into mated recipients. None of the hormonal treatments resulted in pregnancy rates of more than 25% at term and no more than 9% of transferred embryos survived, in comparison with 50% of the mated recipients successfully carrying 25% of transferred embryos. Lastly, the developmental potential of parthenogenetic embryos was assessed and 62% of transferred embryos resulted in pregnancies, none of which continued beyond day 55 of gestation. After co-transfer of three fertilized embryos with 55-60 parthenogenetic embryos into each of six recipients, two live piglets were delivered. The results from the present study indicate that transfer of zona pellucida compromised embryos can yield litters of normal piglets. In addition, it was demonstrated in a model system involving the transfer of three fertilized embryos into mature gilts that hormonal pregnancy maintenance strategies support a low proportion of embryos to term. Lastly, the present study shows for the first time a comparably effective but novel alternative for pregnancy maintenance in the pig involving the co-transfer of parthenote embryos.

Systemic mycosis due to Aspergillus deflectus in a dog.

A 4-year-old, entire female, German Shepherd Dog was referred with a 3-month history of right foreleg lameness that partially responded to nonsteroidal anti-inflammatory and antimicrobial therapy. The bitch lost weight, was polydipsic and had reduced exercise tolerance. On referral, the animal was in poor condition, pyrexic and exhibited moderate pain on full extension of the right shoulder. Blood, urine and joint fluid were obtained and radiographs were taken of the right shoulder and chest. The bitch was lymphopaenic, hyperfibrinogenaemic, hyperglobulinaemic, mildly azotaemic, mildly proteinuric and isosthenuric. Branching fungal hyphae were present in the urine. On radiography, the thorax contained a large ventral mediastinal mass and the humeral head had extensive areas of radiolucency. An aspirate from the right humeroscapular joint exhibited branched fungal hyphae and numerous neutrophils and macrophages. A diagnosis of disseminated mycosis was made and euthanasia was performed. At necropsy, numerous caseating granulomas were present, especially in the kidneys, adrenal glands, heart and lymph nodes. Extensive osteomyelitis involved the head of the right humerus, the sternebrae and the fifth intervertebral disc. Fungal hyphae were detected in sections of granulomas in all affected organs and a diagnosis of disseminated fungal granulomatosis was made. Aspergillus deflectus was readily isolated from affected lymph nodes, but confirming its identity as A deflectus using standard procedures proved difficult. The identity of the fungus was finally confirmed by sequencing part of the 185 rRNA of the isolate. This is the first report in Australia of a disseminated mycosis caused by A deflectus. Previously, the involvement of A deflectus as a cause of disseminated mycosis was limited to 5 cases from the West Coast of the USA, four of which occurred in German Shepherd Dogs.

Reduced levels of connexin43 in cervical dysplasia: inducible expression in a cervical carcinoma cell line decreases neoplastic potential with implications for tumor progression.

Loss of gap junctional intercellular communication (GJIC) has been linked to aberrant proliferation and an enhanced neoplastic phenotype. Many human tumors, including the cervical carcinoma line HeLa, have been reported to be deficient in expression of the gap junction protein connexin43 (Cx43) and GJIC. To determine if this is an early event in carcinogenesis, we utilized immunohistochemistry to screen a series of cervical biopsy samples and demonstrated a major reduction in Cx43 expression in dysplastic regions compared to normal epithelia. To determine whether this loss influences the neoplastic behavior of cervical carcinoma cells, we have constructed HeLa cell lines in which Cx43 expression can be induced in response to doxycycline. This approach allows for the discrimination of Cx43-mediated effects from those due to pre-existing clonal heterogeneity. Cx43 induction in these cells led to assembly of functional junctions but did not alter growth control in vitro as measured by logarithmic growth, saturation density or focus formation when in co-culture with growth-controlled fibroblasts. However, Cx43 induction decreased two indices of neoplasia: it reduced anchorage-independent growth and attenuated the growth rate of tumor xenografts. These results indicate that established HeLa cell lines are unresponsive to Cx43-mediated signals which are thought to mediate growth control of non-transformed cells, however, Cx43 expression can still reduce aspects of the neoplastic phenotype of these cells, indicating that loss of connexin signaling in dysplastic cells may contribute to their neoplastic progression.

Mechanical property evaluation of pressable restorative ceramics.

OBJECTIVE: The aim of this study was to evaluate the flexure strength under static and cyclic loading and the fracture toughness under static loading of six restorative ceramic materials. Specifically the intent was to compare four leucite (K2O.Al2O(3).4SiO2) strengthened feldspathic (pressable) porcelains (OA, OI, E, and FP) to a low fusing feldspathic porcelain (F) and an experimental (EC) lithium disilicate containing ceramic. METHODS: All materials were tested as a control in air and distilled water (without aging) and after three months aging in air or distilled water to determine flexure strength and fracture toughness. A staircase approach was used to determine the cyclic flexure strength. EDS and SEM analysis were conducted on the polished and fracture surfaces. RESULTS: The mean flexure strength for the controls in air and water (without aging or cyclic loading) ranged from 67 to 99 MPa, except the experimental ceramic that was twice as strong with a mean flexure strength of 191-205 MPa. For the mean fracture toughness, the range was 1.1-1.9 MPa/m0.5 with the experimental ceramic being 2.7 MPa/m0.5. The effect of testing in water and aging for three months caused a moderate reduction in the mean flexure strength (6-17%), and a moderate to severe reduction in the mean fracture toughness (5-39%). The largest decrease (15-60%) in mean flexure strength was observed when the samples were subjected to cyclic loading. SIGNIFICANCE: The lithium disilicate containing ceramic had a significantly higher flexure strength and fracture toughness when compared to the four pressable leucite strengthened ceramics and the low fusing conventional porcelain. All of the leucite containing pressable ceramics did provide an increase in mean flexure strength (17-19%) and mean fracture toughness (3-64%) over the conventional feldspathic porcelain. Further, the influence of testing environment and loading conditions implies that these ceramic materials in the oral cavity might be susceptible to cyclic fatigue, resulting in a significant decrease in the survival time of all-ceramic restorations.

Correlation between growth control, neoplastic potential and endogenous connexin43 expression in HeLa cell lines: implications for tumor progression.

A HeLa cell line, obtained from the ATCC, was cloned and found to exhibit a spectrum of in vitro and in vivo growth characteristics as well as variable expression of endogenous connexin43 (Cx43), a widely expressed gap junction protein implicated in growth control. The majority of clones expressed functional Cx43, which contrasted with previous studies reporting that HeLa cells are completely negative for Cx43 mRNA/protein expression. This endogenous Cx43 expression correlated with increased growth control: Cx43-positive clones exhibited a decreased saturation density and a diminished growth capacity when in co-culture with growth-controlled normal cells in constrast to Cx43-negative clones. Endogenous Cx43 expression was negatively correlated with neoplastic potential as evidenced by attenuated anchorage-independent growth and decreased tumorigenicity in immunodeficient mice. Treatment of Cx43-negative cells with 5-aza-2'-deoxycytidine resulted in expression of Cx43, suggesting gene silencing via DNA methylation. These results support the concept of growth control via junctionally transmitted signals and suggest an epigenetic mechanism for tumor cells to circumvent this control during carcinogenesis. Moreover, the heterogeneous nature of this cell line and the ease of connexin43 gene induction suggest caution in the interpretation of results involving gene transfection using noninducible gene expression systems.

Comparative effects of all-trans beta-carotene vs. 9-cis beta-carotene on carcinogen-induced neoplastic transformation and connexin 43 expression in murine 10T1/2 cells and on the differentiation of human keratinocytes.

9-cis beta-Carotene was extracted from a commercial extract of the algae Dunaliella salina (Betatene), and its actions on proliferation and gene expression were examined in murine 10T1/2 cells and human HaCaT keratinocytes. The 9-cis isomer was less active than all-trans beta-carotene in reducing proliferation and in upregulating expression of connexin 43 in 10T1/2 cells. However, it had comparable ability to suppress carcinogen-induced neoplastic transformation. When tested in HaCaT cells in organotypic culture, it was less active in inducing connexin 43 expression and suppressing expression of keratin K1. In this assay the all-trans isomer was highly active at 10(-6) M, whereas 10(-5) M 9-cis beta-carotene was required to produce a comparable effect. Only small reductions in expression of the basal keratin 5 were seen. All-trans and 9-cis retinoic acids, potential metabolites of beta-carotene isomers, were studied in the same systems. In contrast to the carotenoids, the 9-cis isomer of retinoic acid was approximately 10-fold more active in suppressing neoplastic transformation and inducing connexin 43 expression in both cell types than the all-trans isomer. The retinoic acid isomers were about equipotent in suppressing K1 expression. Cellular levels of 9-cis beta-carotene were approximately 3.5-fold lower than levels of all-trans beta-carotene, suggesting that part, but not all, of this decreased activity of the 9-cis isomer was due to decreased cell uptake. Thus 9-cis beta-carotene is consistently less active than the all-trans isomer; that 9-cis retinoic acid is, in general, much more potent than the all-trans isomer suggests little or no conversion from the carotenoid to the retinoid under these culture conditions.

Thermal and cyclic loading of endodontic posts.

The intent of this study was to determine pullout (shear) strength of carbon and stainless steel serrated posts from teeth. The tooth-posts groups were controls, thermal-cycled, load-cycled (load applied at either a 45 degrees angle or along the long axis of the post), and thermal- and load-cycled. Human extracted third molars were embedded in denture acrylic. Post space was prepared and the posts cemented with a resin cement according to manufacturer's instructions. The testing was in tension at a loading rate of 2 mm/min. The pullout (shear) strengths (MPa+/-SD) for the carbon posts were: controls 6.1+/-3.3; load-cycled 4.3+/-2.2; and thermal-cycled 4.2+/-2.2. The thermal-cycled and then load-cycled carbon posts all fractured during testing and were unable to be evaluated for shear strength. The pullout (shear) strengths (MPa+/-SD) for the stainless steel posts were: controls 6.6+/-2.9; load-cycled 7.1+/-3.2; thermal-cycled 4.8+/-2.0; and thermal load-cycled 6.8+/-3.5. The statistical analysis indicated no significant difference in the pullout (shear) strength between any of the post groups tested. Two different operators then prepared specimens using carbon posts abraded with 50 microm alumina and non-abraded carbon posts. The alumina abrasion caused a decrease in the post bond strength with the alumina particles impinging on the carbon fibers resulting in their fracture. The significant difference between bond strengths was attributed to each operator's years of clinical experience.

Primary closure of fasciotomy incisions with a skin-stretching device in patients with burn and trauma.

Closure of fasciotomy wounds is often a clinical problem after successful management of compartment syndrome. Commonly, split-thickness skin grafts or regional composite grafts are used for fasciotomy closure. However, functional and cosmetic results would be improved if primary reapproximation of these wounds were more practical. The main obstacle that must be overcome is excessive tension on the wound edges. A recently developed skin-stretching device (Sure-Closure, Life Medical Sciences, Princeton, N.J.) allows large tissue defects to be closed with approximation of the wound edges. In this report we describe two patients in whom closure of the fasciotomy incisions was successfully accomplished with the skin-stretching device. These patients included an 11-month-old girl with a circumferential burn of the left arm, and a 42-year-old woman involved in a motor vehicle accident who sustained frostbite and crush injury to her left upper extremity without bone fractures. The skin-stretching device produced excellent functional and cosmetic wound closure results and eliminated the need for additional operative procedures.

Laparoscopic intracorporeal harvest of jejunal tissue for autologous transplantation.

Our goal in this study was to assess the feasibility and safety of performing an intracorporeal laparoscopic jejunal harvest. The initial technique was developed and refined in a pig and then a dog model. In the animal studies, careful dissection of the jejunal flap with its feeding vessel was accomplished along with an intracorporeal anastomosis. The laparoscopic dissection was facilitated by temporarily anchoring the jejunal flap to the anterior abdominal wall and transilluminating the mesentery. We present the first case report of a patient who underwent a laparoscopic jejunal harvest, intracorporeal small bowel anastomosis, and a microvascular anastomosis in the neck for reconstruction of the laryngopharynx.

Hormonal induction of lactation in non-pregnant ewes.

Exogenous hormone treatments designed to induce nonpregnant ewes to lactate at levels sufficient to rear orphan lambs were investigated in three breeds (Blackface, Dorset and Greyface) and in a total of 14 groups. The hormone treatments consisted of an induction phase lasting 1-6 weeks during which most ewes received daily subcutaneous injections of 75 mg of progesterone and 100 microg of oestradiol-17beta, and a trigger phase lasting 1 or 2 weeks during which 5 mg of oestradiol-17beta and/or 10 mg of dexamethasone were given daily and the daily dose of progesterone was reduced to zero. From the end of the trigger phase for at least 2 weeks, milk production was determined three times daily by hand milking after prior intravenous injections of 5 IU of oxytocin. The linear dimensions of the udder were measured in each ewe at 5-6 day intervals throughout the induction and trigger phases. Udder sizes increased in response to hormone treatment in all cases, and the rate of increase was usually 1.75-11.3 times greater during the trigger phase than during the induction phase. Accumulation of milk in the udder during the trigger phase resulted in mean milk yields of 212-763 ml on the first day of milking, which were higher than the mean yields of 130-354 ml on the second day. Thereafter the mean daily milk yields increased progressively to reach 579-1301 ml after 14 days of milking. Group comparisons revealed the following: an induction phase of at least 4 weeks duration was required to ensure that all hormone-treated ewes produced 800 ml or more of milk/day by 14 days of milking; during the trigger phase, oestradiol-17beta alone was a more effective lactogenic agent than dexamethasone alone, and dexamethasone apparently hindered the lactogenic actions of oestradiol-17beta when both hormones were given together; extending the duration of the trigger phase from 1 to 2 weeks did not improve subsequent milk yields; and there were no significant breed differences in milk yield responses to similar hormone treatments. It is concluded that a 4-6 week induction phase followed by a 1-week trigger phase using the progesterone and oestradiol-17beta doses noted above but excluding dexamethasone would induce in most nonpregnant ewes lactation at levels sufficient to rear orphan lambs. Compared to the compositions of normal colostrum and milk, the milk from some of the present ewes had lower dry matter contents, fat concentrations and immunoglobulin-G concentrations and generally similar lactose concentrations. These differences were not considered to be sufficient to jeopardize the survival of lambs reared by such ewes.

Successful fostering of orphan lambs by non-pregnant ewes induced to lactate using exogenous hormone treatment.

Six nonpregnant ewes were induced to lactate by an exogenous hormone treatment lasting 6 weeks. At the end of the treatment one alien lamb was adopted by each ewe. Each ewe was initially indifferent to the alien lamb and required only mild restraint to allow the lambs to suck successfully. Maternal interest subsequently increased and strong ewe-lamb bonding was evident by 80 minutes (n=1), 10 hours (n=4) or 24 hours (n=1) in different cases. The lambs required supplementary milk for 4-6 days while milk production by the ewes increased to adequate levels. Thereafter they were reared entirely by the ewes. Another four nonpregnant ewes were induced to lactate by a similar hormone treatment and were then hand-milked thrice daily for 23 weeks. One alien lamb was then removed after 10-30 minutes. The ewes were then injected subcutaneously with 5 mg of oestradiol-17beta and were placed in fostering stocks. After a further 3-5 hours the lambs were re-introduced. Strong ewe-lamb bonding occurred during the subsequent 10 hours in all cases. All 10 lambs were reared successfully by these ewes until at least 3 months of age, when observations stopped. It was concluded firstly that nonpregnant ewes induced to lactate artificially will adopt and rear orphan lambs successfully, provided that additional milk is given to meet shortfalls during the first 4-6 days; and secondly that a 5 mg injection of oestradiol-17beta may facilitate fostering of lambs by ewes in established lactation whether the lactation is induced artificially or not.

Clinicopathological investigation of primary, uncomplicated maedi-visna virus infection.

Maedi-visna virus infection in a flock of sheep in Scotland was associated with respiratory disease, neurological disease, mastitis and lameness. The major clinical signs were dyspnoea (particularly on exercise), progressive fore- and hindlimb ataxia and balance defects, mammary induration and multilimb lameness, occasionally with enlarged carpal joints. Pathological examinations revealed lesions in the lungs, central nervous system, mammary glands and joints which were consistent with those induced by maedi-visna virus. The was no clinical or pathological evidence of concurrent sheep pulmonary adenomatosis, and pulmonary bacterial infections, when they occurred, were superimposed on the lesions due to maedi-visna virus.

The conservation of dinucleotide microsatellites among mammalian genomes allows the use of heterologous PCR primer pairs in closely related species.

The high degree of polymorphism displayed by DNA microsatellites makes them useful as DNA markers in linkage studies. A search of the DNA sequence databases revealed that the locations of dinucleotide microsatellites are often conserved among mammalian species, enabling the prediction of the presence of DNA microsatellites using comparative genetic data. In closely related species such as cattle and sheep, this conservation was close enough to allow PCR primers designed for use in one species to be used to analyze microsatellite length polymorphism in the other. A total of 48 sets of primer pairs, flanking bovine microsatellites and giving polymorphic PCR products in that species, were tested with template DNA from sheep, horses, and humans. Specific products were obtained in 27 cases (56%) with ovine DNA, 20 of which (42%) showed polymorphisms. With equine DNA, 3 (6.2%) gave specific but monomorphic products, while no specific products were obtained using human DNA. The ability to use heterologous PCR primers, coupled with comparative mapping information will facilitate the use of DNA microsatellites in gene mapping studies in closely related species such as cattle and sheep, rat and mouse, or primates.