Increased P-selectin gene expression in the liver vasculature and its role in the pathophysiology of neutrophil-induced liver injury in murine endotoxin shock.

We studied the role of P-selectin, an adhesion molecule known to be important for neutrophil localization to sites of inflammation, in a model of inflammatory liver injury. Male C3Heb/FeJ (ET-sensitive) mice treated with 700 mg/kg galactosamine and 100 microg/kg Salmonella abortus equi endotoxin (Gal/ET), murine tumor necrosis factor alpha (TNF-alpha, 15 microg/kg), or interleukin-1 (IL-1, 13-23 microg/kg), showed increased P-selectin mRNA levels in the liver. In contrast, C3H/HeJ (ET-resistant) mice responded only to cytokines with P-selectin mRNA formation. Whereas no P-selectin expression was detectable in control livers, there was temporary staining of endothelium in large blood vessels but not in sinusoids between 3 and 5 h after ET, TNF-alpha, or IL-1 treatment. Severe liver injury induced by Gal/ET at 7 h was not inhibited by an anti-P-selectin antibody in C3Heb/FeJ mice or in P-selectin-deficient animals. Sequestration of neutrophils in sinusoids, i.e. those neutrophils that have been identified as critical for the injury, was not affected by the antibody or in P-selectin-deficient mice. However, the temporary margination in portal and post-sinusoidal venules was reduced by 75% in anti-P-selectin antibody-treated animals and by 51% in P-selectin-deficient mice. We conclude that hepatic P-selectin gene transcription in vivo involves cytokines. However, blocking P-selectin neither attenuated sinusoidal neutrophil sequestration nor prevented neutrophil-induced liver injury during endotoxin shock but attenuated neutrophil margination in larger vessels. Thus, our data demonstrate similarities and fundamental differences in the requirement for adhesion molecules to localize neutrophils in the liver vasculature compared to other organs during an inflammatory response.

Remodeling and neointimal formation in the carotid artery of normal and P-selectin-deficient mice.

BACKGROUND: Inflammatory reactions such as leukocyte activation with platelet adherence and release of inflammatory mediators occur after percutaneous transluminal coronary angioplasty and may play a role in restenosis. Vascular remodeling with neointimal formation was studied in normal C57Bl/J6 and P-selectin-deficient mice. METHODS AND RESULTS: The left common carotid artery was ligated just proximal to the carotid bifurcation. Four weeks later, left carotids and contralateral controls were snap-frozen. Computer-aided morphometry was performed to measure ratios of neointimal to medial area (NI/M) in 10 sections per animal as a measure of the thickness of the neointimal lesion. For normal mice, NI/M was 1.13+/-0.2 (n=20), whereas NI/M was reduced by 76% to 0.27+/-0.1 (n= 19) in P-selectin knockout mice. Vascular constriction (as measured by the length of external elastic lamina) was the same in both groups, but the circumference of the lumen in knockout mice was 26% larger. Also, normal and P-selectin-deficient mice were killed at 3 and 7 days after ligation (n=6 for each group per time point). Histological staining and immunostaining for CD45 showed no inflammatory cell presence in P-selectin knockout mice. However, in normal mice, leukocyte infiltration was observed in the adventitia, media, and developing neointima. Also, P-selectin immunostaining was observed in media and developing neointima of normal mice. CONCLUSIONS: These data suggest that P-selectin is involved in processes leading to cell migration and proliferation associated with vascular remodeling, presumably by mediating leukocyte recruitment and the interaction between platelets and leukocytes.

Cardiac rehabilitation/exercise in patients with implantable cardioverter defibrillators.

There have been no specific exercise or daily activity guidelines determined for patients with automatic implantable cardioverter defibrillators. Two patients, one with a Ventritex Cadence Model V-100 defibrillator and one with a CPI Ventak Model 1550 defibrillator were enrolled in monitored cardiac rehabilitation. One patient had symptoms of syncope and cardiodefibrillation during a vigorous short walk prior to cardiac rehabilitation and became fearful of any activity. Stress testing on this patient was terminated early because his atrial fibrillation rate approached the defibrillization rate. A low dose of beta blockade was added to his regimen. He underwent repeat stress testing and was placed in cardiac rehabilitation. This patient had no further shocks, and it was assumed that his shock was due to high atrial fibrillation rates. The second patient experienced recurrent shocks on amiodarone, propafenone, and mexiletine with presyncope. However, stress testing did not disclose abnormalities. The patient was fearful of any activity and was placed in cardiac rehabilitation. During an average of 26 sessions of cardiac rehabilitation, no symptoms have been noted, and the patients have returned to a more normal lifestyle. Specific exercise and lifestyle criteria should be given to patients with cardioverter defibrillators. Stress testing with monitored exercise can develop such a program. Larger numbers of patients need to be studied.

In vitro and in vivo inhibition of rat vascular smooth muscle cell migration and proliferation by a 2-aminochromone U-86983.

Vascular smooth muscle cell migration and proliferation are the primary events that govern neointimal thickening and thus they determine the extent to which delayed restenosis occurs after percutaneous transluminal coronary angioplasty. In this study, the in vitro and in vivo smooth muscle cell antichemotactic and antiproliferative properties of a 2-aminochromone, 2-(4-morpholinyl)-8-(3-pyridinylmethoxy)-4H-1-benzopyran-4-one (U-86983), were examined. Migration and proliferation of early-passage rat vascular smooth muscle cells were inhibited by U-86983 in a concentration-dependent manner (IC50S, approximately 10 microM and 3.5 microM, respectively). Longer-term studies showed that the proliferation of smooth muscle cells was inhibited by U-86983 for at least 7 days and was fully reversible on removal of the drug. In addition, the effect of U-86983 on neointimal formation was examined in rats subjected to left common carotid artery balloon dilatation injury. Continual (2-week) i.v. administration of U-86983 (216 mg kg-1 day-1) resulted in a mean plasma drug concentration of 2.39 micrograms/ml (blood level, approximately 3.5 microM) and a 42% (P = .003) reduction in the neointima/media ratio of the injured artery. In agreement with the in vitro reversibility results, administration of U-86983 for only 2, 4 or 7 days did not affect significantly the neointimal thickness measured at 14 days, which indicated that the stimuli for smooth muscle cell migration and/or proliferation were still present 1 week after injury.(ABSTRACT TRUNCATED AT 250 WORDS)

Osteoarthritis of the knee: effects on gait, strength, and flexibility.

This study examined the differences in gait mechanics, isokinetic knee strength, and flexibility between a group of adults with symptomatic osteoarthritis (OA) of the knee (n = 15) and an age-, mass-, and gender-matched group of control subjects (n = 15). Both groups performed under similar environmental conditions. Our results suggest that patients with symptomatic OA of the knee have poorer flexibility in both the affected and unaffected legs and demonstrate significantly less (p less than .05) knee angular velocity and, to a lesser extent, knee range of motion during gait. They have an increased loading rate in the unaffected leg after heel strike, exert less peak vertical force during pushoff, and are significantly weaker in both the dominant and nondominant legs compared to adults with no lower extremity disease.

A method for cultivating morphologically undifferentiated embryonic stem cells from porcine blastocysts.

Variable conditions were tested to determine an in-vitro cultivation method for the formation of morphologically undifferentiated embryonic stem cells from the inner cell mass (ICM) derived outgrowth of porcine blastocysts. Although all 16 Day-9 embryos failed to form colonies, 14 such colonies were obtained from a total of 69 Day-10 embryos when they were co-cultivated with porcine uterine fibroblast (PUF) cells over a 6-day period. The best results were obtained in Dulbecco's modified Eagle medium (DMEM) with 10% fetal calf serum and 10% porcine serum supplemented with bovine insulin and beta-mercaptoethanol, in which six out of seven embryos formed adequate ICM-derived colonies. Since murine fibroblasts were not found to be suitable feeder cells in this procedure, an endocrine synergistic interaction, which promotes embryonic attachment and colony formation, between porcine blastocysts and PUF cells is hypothesized. Continued propagation of the ICM-derived cells was not dependent on these factors; a total of seven cell lines were obtained after three to five subsequent passages on murine feeder-layers that resembled morphologically undifferentiated embryonic cells.

Production of transgenic pigs harbouring a rat phosphoenolpyruvate carboxykinase-bovine growth hormone fusion gene.

Over the past 5 years, reports detailing the production of transgenic pigs have focussed on enhanced growth performance. Phenotypic side-effects observed in pigs harbouring chimaeric constructs containing metallothionein or Moloney murine leukaemia virus transcriptional activators fused to growth hormone (GH) structural genes have been attributed to chronic overexpression of GH. In an effort to regulate a transgene product more effectively, a liver specific 460 bp 5' flanking sequence of the rat phosphoenolpyruvate carboxykinase (PEPCK) gene was ligated to a BamHI site of the first exon of the genomic bovine GH (bGH) structural gene. Following micro-injection of the PEPCK/bGH construct into 1- and 2-cell pig zygotes. 124 offspring were produced of which 7 pigs were determined to be transgenic by dot-blot and Southern analysis. The PEPCK gene expression, in terms of tissue and developmental specificity, appears similar to that observed in PEPCK/bGH transgenic mice. Germ-line transmission was identified in 1 of 3 mated founders. Dramatic influences on backfat thickness were observed including a 41% reduction in backfat depth when compared to non-transgenic sex-matched littermate control pigs. Both the regulation and characterization of gene expression in PEPCK/bGH transgenic pigs are under investigation.

A study of the source of estradiol-17 beta in the bull.

A study was designed to determine if the bull testes secretes estradiol-17 beta, as has been reported for several other species. Two experiments were conducted. In the first experiment, five Angus-sired crossbred bulls were fitted with catheters in the spermatic and jugular veins and sampled every 15 min for six hours. One was bled from three cannula, the third being in the spermatic artery. In the second experiment, these same bulls were castrated and jugular vein blood was collected at timed intervals for two hours. Plasma samples were assayed for estradiol-17 beta (E2) and testosterone (T) using highly specific radioimmunoassays. This experiments gave evidence that: a. Concentrations of E2 were significantly lower (P less than .05) in the jugular vein than the spermatic vein in each of the five bulls, although the mean concentration for all bulls was not great for either vein nor was the difference great between the two veins. In confirmation of past studies, T concentration in the jugular vein was much lower than in the spermatic vein in each bull as was overall mean. b. Removal of the testes caused E2 to decrease during the 25 min post-castration but the difference was not significant (P greater than .05), whereas T decreased 4-fold (P less than .01). During the next 95 min., the concentrations of both hormones increased 3- and 6-fold, respectively, as did cortisol concentration. It is concluded that the bull testes secretes E2, but the secretion is minor to that of T and that another source of both hormones can be the adrenal gland, such as during stress.(ABSTRACT TRUNCATED AT 250 WORDS)

Residue levels of endogenous estrogens in beef tissues.

Endogenous estrogen concentrations in edible tissues of cows, bulls and steers were compared with those of steers with estrogen implants. Concentrations are expressed as pg estrogen/g of tissue, wet weight. In addition, depletion rates of estradiol-17 beta (E2 beta) estrone (E1) and estradiol-17 alpha (E2 alpha) from these tissues and blood plasma were determined. In muscle, the main estrogen was E2 beta, regardless of sexual status (nonpregnant cow, bull or steer); however, concentrations approached the lower limits of analytical sensitivity. In liver and kidney, E2 beta and E1 were equimolar (25 to 40 pg/g for each) in heifers and steers, whereas in kidney fat, concentrations of E1 exceeded those of E2 beta. Concentrations of E1 in fat were slightly higher in bulls than in cows or steers. The predominant estrogen in fat during pregnancy was E1, with concentrations 150 times greater than those of nonpregnant heifers or nonimplanted steers and 75 times the concentration found in steers with E2 beta implants. In kidney of pregnant cows, E1 rose 40-fold and in liver 10-fold over that of implanted steers. Concentrations of E2 alpha were low and depleted rapidly after withdrawal of an E2 beta implant. Tissue depletion studies of the three estrogens demonstrated that E1 disappeared from plasma, fat, liver and kidney more slowly than E2 beta or E2 alpha. Depletion of E2 beta from the tissue can be manipulated as shown by the faster rate of depletion in implanted steers than in nonimplanted steers. Because E1 is cleared from fat more slowly than E2, and E1 concentrations are higher, this estrogen-tissue combination should be used to monitor estrogen implants as anabolic agents.

Fluorinated phenytoin anticonvulsant analogs.

Six ring-fluorinated phenytoin analogs were synthesized, and their anticonvulsant activity in the maximal electroshock seizure and subcutaneous pentylenetetrazol assays was determined. 5-(4-Fluorophenyl)-5-phenylhydantoin, 5-(3-fluorophenyl)-5-phenylhydantoin, and 5,5-bis(4-fluorophenyl)hydantoin were active in the maximal electroshock seizure assay. The compounds were much less potent than phenytoin but showed an extremely long duration of action.