Expression of activated M-Ras in hemopoietic stem cells initiates leukemogenic transformation, immortalization and preferential generation of mast cells.

Cultures of purified hemopoietic stem cells transduced with an activated mutant of M-Ras contained abnormal cells that, despite the presence of only low levels of growth factors, generated large, dense colonies of macrophages and blast cells. Cells from these colonies survived and grew continuously in the absence of growth factors and generated clonal cell-lines that were mainly composed of well-differentiated mast cells, with a low frequency of undifferentiated cells. When transplanted into sublethally irradiated syngeneic mice, four out of four such clones gave rise to a systemic mastocytosis and mast-cell leukemia. However, the donor clones also generated low percentages of cells with the morphological and cell-surface characteristics of erythrocytes, granulocytes, monocytes and T- and B-lymphocytes. These data indicate that signals downstream of activated M-Ras are sufficient to transform hemopoietic stem cells, and while preserving their capacity to generate other cell-lineages in vivo, result in preferential generation of mast cells.

Listeria Rhombencephalitis Complicating Anti-TNF Treatment during an Acute Flare of Crohn's Colitis.

Patients with Crohn's disease often require the use of immunosuppressant drugs to control disease activity. Such medication includes steroids, azathioprine, and biologic therapy. These suppress the immune response, and the patient is more susceptible to infection. We present a case of a 69-year-old gentleman with a history of Crohn's colitis who had ongoing symptoms of diarrhoea in spite of standard treatment. Biologic therapy was considered to be the next step, and screening for infection was undertaken prior to use. Three days following anti-TNF treatment, he became drowsy, and examination revealed pyrexia, slurred speech, and nystagmus. Investigation revealed presence of Listeria rhombencephalitis. He demonstrated poor neurological recovery. Listeria monocytogenes is an infection commonly associated with food sources. Some patients develop a self-limiting diarrhoeal illness, but in the immunosuppressed population, the clinical features may be more sinister. Cotrimoxazole prophylaxis is already recommended for those on triple immunosuppression. We propose the early initiation of this treatment, including where biologic use is anticipated. In those on multiple immunosuppressants, a diet similar to that followed in pregnancy may minimise risk of acquiring this infection. Clinicians must always have a high index of suspicion for opportunistic infection in such immunocompromised patients.

Dual metalloprotease inhibitors: mercaptoacetyl-based fused heterocyclic dipeptide mimetics as inhibitors of angiotensin-converting enzyme and neutral endopeptidase.

A series of 7,6- and 7,5-fused bicyclic thiazepinones and oxazepinones were generated and incorporated as conformationally restricted dipeptide surrogates in mercaptoacyl dipeptides. These compounds are potent inhibitors of angiotensin-converting enzyme (ACE) and neutral endopeptidase (NEP) both in vitro and in vivo. Compound 1a, a 7,6-fused bicyclic thiazepinone, demonstrated excellent blood pressure lowering in a variety of animal models characterized by various levels of plasma renin activity and significantly potentiated urinary sodium, ANP, and cGMP excretion in a cynomolgus monkey assay. On the basis of its potency and duration of action, compound 1a (BMS-186716) was advanced into clinical development for the treatment of hypertension and congestive heart failure.

Biphenylsulfonamide endothelin antagonists: structure-activity relationships of a series of mono- and disubstituted analogues and pharmacology of the orally active endothelin antagonist 2'-amino-N- (3,4-dimethyl-5-isoxazolyl)-4'-(2-methylpropyl)[1, 1'-biphenyl]-2-sulfonamide (BMS-187308).

Substitution at the ortho position of N-(3,4-dimethyl-5-isoxazolyl) benzenesulfonamide led to the identification of the biphenylsulfonamides as a novel series of endothelin-A (ETA) selective antagonists. Appropriate substitutions on the pendant phenyl ring led to improved binding as well as functional activity. A hydrophobic group such as isobutyl or isopropoxyl was found to be optimal at the 4'-position. Introduction of an amino group at the 2'-position also led to improved analogues. Combination of the optimal 4'-isobutyl substituent with the 2'-amino function afforded an analogue (20, BMS-187308) with improved ETA binding affinity and functional activity. Compound 20 also has good oral activity in inhibiting the pressor effect caused by an ET-1 infusion in rats. Doses of 10 and 30 micromol/kg iv 20 attenuated the pressor responses due to the administration of exogenous ET-1 to conscious monkeys, indicating that the compound inhibits the in vivo activity of endothelin-1 in nonhuman primates.

The inhibitory effect of GR32191, a thromboxane receptor blocking drug, on human platelet aggregation, adhesion and secretion.

GR32191, a potent selective thromboxane receptor antagonist, has been shown to inhibit completely prostaglandin endoperoxide and thromboxane A2 (TxA2)-induced platelet aggregation, [14C]-serotonin secretion and beta-thromboglobulin secretion. Deposition of human platelets onto damaged rabbit aorta in vitro is reduced in the presence of GR32191 which appears to inhibit aggregation of platelets but not direct adhesion of platelets to subendothelium. The effects of non-prostanoid platelet activating agents whose mode of action requires the biosynthesis of TxA2 are also inhibited by GR32191. Prostanoids which inhibit platelet function, such as prostacyclin or PGD2, retain their inhibitory properties in the presence of GR32191 which does not inhibit phospholipase A2, prostaglandin cyclooxygenase, thromboxane synthase, 12-lipoxygenase or cAMP phosphodiesterase activity. The inhibitory action of GR32191 on platelet aggregation, mural thrombus formation and platelet protein storage granule secretion suggests that it has potential in treating thrombotic disease in man.

Ewing's sarcoma of bone: clinicopathologic aspects of 303 cases from the Intergroup Ewing's Sarcoma Study.

Clinicopathologic features of 303 cases of Ewing's sarcoma of bone collected by the Intergroup Ewing's Sarcoma Study group are described. Ewing's sarcoma was found to arise predominantly in the inferior segment of the skeleton in young white patients from 10 to 15 years of age. Of the several recognized histologic patterns, the filagree pattern--an organoid pattern consisting of bicellular strands of tissue separated by filmy vascular stroma--was associated with the lowest survival rates. Light microscopic evidence of cytoplasmic glycogen in tumor cells was found to be useful in but not requisite for diagnoses. The most important predictor of clinical behavior, regardless of treatment modality, was the site of primary involvement. Survival among patients with primary tumors in pelvic bones was lowest, whereas survival among patients with lesions in distal bones of the extremities was highest.

Pharmacokinetics and antiviral activity of a novel isonucleoside, BMS-181165, against simian varicella virus infection in African green monkeys.

A novel nucleoside analog BMS-181165 with potent activity against varicella-zoster virus was tested for efficacy in a simian varicella virus infection in African green monkeys. BMS-181165 was effective in preventing the development of a rash, decreasing the development of viremia and preventing death in infected monkeys when administered orally at 4, 16 or 64 mg/kg/day. The compound is well orally absorbed in monkeys, between 44 to 50% oral bioavailability, and may prove of value in therapy of varicella-zoster infections in humans.

Effect of GR32191 and other thromboxane receptor blocking drugs on human platelet deposition onto de-endothelialized arteries.

A range of thromboxane A2 receptor blocking (TxRB) drugs, prostacyclin and aspirin have been assessed as inhibitors of human platelet deposition onto rabbit and human de-endothelialized arteries in vitro. Platelet deposition was quantified by measuring the radioactivity associated with de-endothelialized arteries following superfusion with 111indium-labelled human platelets reconstituted in blood. Using rabbit aorta, all of the compounds tested produced a similar maximum inhibition (approximately 70%) of platelet deposition; from scanning EM studies the residual deposition appeared to represent a monolayer of adhered platelets. The potency of the TxRB's for inhibiting deposition was GR32191 greater than or equal to GR36246 greater than SQ29,548 greater than ICI185282 greater than or equal to AH23848 much greater than BM13.177 consistent with their TxRB potency on human platelets. Using human umbilical arteries, the TxRB's achieved a smaller maximum inhibition of deposition (approximately 50%) than did prostacyclin or the fibrinogen receptor blocking peptide Gly-Arg-Gly-Asp-Ser (GRGDS) (60-75%). In addition, using human umbilical arteries, the structurally-related TxRB's GR32191 and GR36246 exhibited a greater than 1000-fold enhancement in potency as inhibitors of platelet deposition over that seen in the rabbit aorta. In preliminary experiments, GR32191 also displayed a similar high potency on human cerebral arteries. In contrast, the structurally unrelated compounds SQ29,548, ICI185282 and BM13.177 exhibited similar potencies on human umbilical arteries to those observed on the rabbit aorta; aspirin and prostacyclin also displayed similar potencies on the two preparations. The enhanced effect of GR32191 and GR36246 on human umbilical arteries therefore appears unrelated to their action as TxRB's on human platelets although the mechanism of this unique action is at present unknown. However, if these drugs exhibited a similar high potency for preventing mural thrombus formation in vivo in man, they may represent a major advance in the treatment of occlusive vascular disease.

Temporal and spatial partitioning of water resources among eight woody species in a Hawaiian dry forest.

Lowland dry forests are unique in Hawaii for their high diversity of tree species compared with wet forests. We characterized spatial and temporal partitioning of soil water resources among seven indigenous and one invasive dry forest species to determine whether the degree of partitioning was consistent with the relatively high species richness in these forests. Patterns of water utilization were inferred from stable hydrogen isotope ratios (δD) of soil and xylem water, zones of soil water depletion, plant water status, leaf phenology, and spatial patterns of species distribution. Soil water δD values ranged from -20‰ near the surface to -48‰ at 130 cm depth. Metrosideros polymorpha, an evergreen species, and Reynoldsia sandwicensis, a drought-deciduous species, had xylem sap δD values of about -52‰, and appeared to obtain their water largely from deeper soil layers. The remaining six species had xylem δD values ranging from -33 to -42‰, and apparently obtained water from shallower soil layers. Xylem water δD values were negatively correlated with minimum annual leaf water potential and positively correlated with leaf solute content, an integrated measure of leaf water deficit. Seasonal patterns of leaf production ranged from dry season deciduous at one extreme to evergreen with near constant leaf expansion rates at the other. Species tapping water more actively from deeper soil layers tended to exhibit larger seasonality of leaf production than species relying on shallower soil water sources. Individuals of Myoporum sandwicense were more spatially isolated than would be expected by chance. Even though this species apparently extracted water primarily from shallow soil layers, as indicated by its xylem δD values, its nearly constant growth rates across all seasons may have been the result of a larger volume of soil water available per individual. The two dominant species, Diospyros sandwicensis and Nestegis sandwicensis, exhibited low leaf water potentials during the dry season and apparently drew water mostly from the upper portion of the soil profile, which may have allowed them to exploit light precipitation events more effectively than the more deeply rooted species. Character displacement in spatial and temporal patterns of soil water uptake was consistent with the relatively high diversity of woody species in Hawaiian dry forests.

Drug delivery matrix containing native protein precipitates suspended in a poloxamer gel.

Sustained delivery systems can achieve more constant blood levels of protein therapeutics than those obtained with bolus doses, leading to improved drug efficacy and fewer adverse side effects. Several different polymeric delivery systems have been studied, including poloxamers, which are unique because they can be prepared in aqueous buffers that are compatible with proteins. Poloxamers are nontoxic block copolymers of poly(ethylene oxide) and poly(propylene oxide). Certain poloxamers exhibit reversible thermal gelation. Thus, a solution of protein and poloxamer prepared at low temperatures and injected extravascularly will form a gel as it warms to body temperature. Subsequently, the protein is released slowly from the gel. To date, however, poloxamer gel delivery systems have been limited to relatively low protein concentrations (i.e., < or = 0.4 mg/mL) that produce a completely soluble protein and an optically clear gel. Much higher concentrations of other protein drugs might be needed to obtain an efficacious sustained dose. In the current in vitro study we found that a poloxamer 407 (22% wt/wt) matrix could be prepared containing tens of milligrams/mililiter of the model proteins alpha-chymotrypsin and lactate dehydrogenase. Under these conditions the protein forms a homogeneous suspension. Warming through the poloxamer 407 transition temperature (ca. 18 degrees C) results in a gel that retains a homogeneous distribution of protein precipitates for several days at 37 degrees C. Infrared spectroscopy documented that the precipitated proteins in the suspension have native secondary structure. Furthermore, the fully active protein can be recovered completely when the gel is dissolved in excess buffer. Finally, at the higher protein concentrations used to form the suspensions in poloxamer 407, protein stability during incubation at 37 degrees C was greatly improved over that seen at lower protein concentrations.

Controlling deamidation rates in a model peptide: effects of temperature, peptide concentration, and additives.

The rate of deamidation of the Asn residue in Val-Tyr-Pro-Asn-Gly-Ala (VYPNGA), a model peptide, was determined at pH 9 (400 mM Tris buffer) as a function of temperature and peptide concentration. Over the temperature range 5-65 degrees C, deamidation followed Arrhenius behavior, with an apparent activation energy of 13.3 kcal/mol. Furthermore, increasing the peptide concentration slows the rate of deamidation. Self-stabilization with respect to deamidation has not been reported previously. The rate of deamidation was also determined in the presence of sucrose and poloxamer 407 (Pluronic F127). In both cases, the rate of deamidation was retarded by up to 40% at 35 degrees C. In aqueous solutions containing poloxamer 407, the degree of stabilization is independent of formation of a reversible thermosetting gel. With sucrose, maximum reduction in the deamidation rate was attained with as little as 5% (w/v). Addition of sucrose results in a greater conformational preference for a type II beta-turn structure, which presumably is less prone to intramolecular cyclization and subsequent deamidation.

Increased acquisition of a complex appetitive task after MSH and MIF.

After daily injections of melanocyte stimulating hormone (MSH), MSH-release inhibiting factor (MIF), or diluent albino rats ran a 12 choice Warden maze for a palatable food reward. Rats receiving the hormones had shorter latencies and made fewer errors than controls during learning but, unlike results with simple tasks, there were no differences during extinction. The results demonstrated that both MSH and MIF-I could facilitate the acquisition of an appetitive task which seemed of sufficient complexity to emphasize differences in performance.

Neonatal treatment with TRH affects development, learning, and emotionality in the rat.

Rats that received injections of TRH from Days 2-7 postnatally ran faster to a palatable food reward than controls at 33-37 days of age. Rats treated with TRH were less emotional and more active in an open field both as pups and as adults. There were few notable differences in developmental measures except that the teeth erupted earlier in control animals. Weights of glands and brain parts showed that ovaries, testes, pineal gland, and hypothalamus were heavier in treated animals. The results indicated that TRH given early in life may affect adult behavior by reducing emotionally.

Carbon uptake, growth and resource-use efficiency in one invasive and six native Hawaiian dry forest tree species.

Photosynthetic gas exchange, nitrogen- and water-use efficiency, leaf water potential and seasonal patterns of leaf production were studied in seven, dominant dry-forest species from the island of Lana'i, Hawaii, including the rapidly colonizing, non-native Schinus terebinthifolius (Raddi). We evaluated whether unique physiological characteristics of the invasive species explain its capacity to rapidly invade dry forests throughout the Hawaiian Islands. Apparent anomalies in stable carbon isotope data (delta13C) relative to other results led us to study effects of environmental conditions and physiological performance during leaf expansion on delta13C. Species that expanded all their foliage at the beginning of the wet season had more negative leaf delta13C values during the dry season than species with continuous leaf expansion. Among species, S. terebinthifolius had a strong seasonal pattern of leaf production and the most negative delta13C (-29 per thousand). With respect to almost every trait measured, S. terebinthifolius fell at an end of the range of values for the native species. Rapid growth of S. terebinthifolius in this ecosystem may be partially explained by its high maximum CO2 assimilation rates (15 micromol m-2 s-1), low leaf mass per area, high photosynthetic nitrogen-use efficiency per unit leaf mass or area and large decrease in stomatal conductance during the dry season. Relative to the native species, the invasive species exhibited striking phenotypic plasticity, including high rates of stem growth and water and CO2 uptake during the wet season, and maintenance of leaves and high leaf water potentials, as a result of reduced water loss, during the dry season, enabling it to utilize available resources effectively.

A study of Klebsiella pneumoniae infection in the uterus of the mare.

Two experiments incorporating 13 mares were conducted for the purpose of producing and monitoring intrauterine infection with Klebsiella pneumoniae. In the pilot study, the infection was produced with strains of K pneumoniae type 68 and type 10 isolated from the genital tract of stallions with a history of breeding problems. In the principal study, K pneumoniae type 68 was used to produce the infection. Tampons and guarded culture swabs were used to obtain uterine samples in the pilot study. In comparing the efficacies of isolation of K pneumoniae with the tampons and isolation with standard guarded culture swab, the tampon proved to be a more reliable means with which to isolate K pneumoniae and was used in the principal study. In both studies, inoculated mares became infected and remained infected at least until the postinoculation estrous cycle was initiated or was completed. Some of the inoculated mares remained infected through more than one estrous cycle. The numbers of K pneumoniae decreased in the uterus of mares after completing the estrous cycle after inoculation. Klebsiella pneumoniae was not demonstrable in frozen tissue sections of uterine biopsy specimens stained by fluorescent antibody technique. Postinoculation sera antibody titers to K pneumoniae, as determined, using the capsule swelling technique, were no higher than 1:8.

Influence of density and location on degradation of sustained-release boluses given to cattle.

To better understand the factors involved in erosion of sustained-release ruminal boluses, studies were conducted to determine the relationship between density of the bolus and location in the forestomachs and the influence of these factors on bolus erosion. Boluses with densities of 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, and 2.4 g/cm3 were produced from inert materials and administered to 6 fistulated Hereford heifers. A minimum density of 1.6 g/cm3 was required to prevent regurgitation from the ruminoreticulum and a minimum of 2.0 g/cm3 for retention in the reticulum. Release of oxytetracycline from 2 boluses placed in the reticulum or in the rumen was measured in 12 fistulated Hereford heifers. Boluses retained in the reticulum had higher rates of release (P less than 0.05) than those in the rumen. The smaller reticular compartment apparently caused greater contact between individual boluses and resulted in more rapid erosion.

Transmission of Anaplasma marginale Theiler by Dermacentor andersoni Stiles and Dermacentor variabilis (Say).

Cattle were inoculated with a Virginia isolate of Anaplasma marginale Theiler and served as an infective source for laboratory-reared Dermacentor andersoni Stiles and D variabilis (Say) nymphs. Following a molt and subsequent feeding of adult ticks on susceptible cows, transstadial transmission of A marginale occurred from cattle with parasitemias ranging from undetectable (in a carrier cow) to a peak of 27% (in an acutely ill cow). Homogenates of gut tissue from unfed, incubated, and feeding adult ticks that were infected as nymphs were all infective; however, the prepatent period varied according to treatment of ticks after they had molted to the adult stage. Prepatent periods of infections in calves resulting from injection of homogenated tissue from unfed adult ticks was 29 and 34 days longer than the prepatent periods that developed in calves exposed to homogenated tissue from ticks that were feeding or incubated at 37 C for 3 days. Transovarial transmission did not occur when F1 larvae, hatched from eggs produced by female ticks known to be infective, fed on susceptible cattle.

Chemoprophylaxis of vector-borne anaplasmosis with sustained-release boluses.

Sustained-release oxytetracycline boluses (20%) delivering 145 to 150 mg/bolus/day were administered to complement-fixation test-negative cattle at dosages of 2.0, 2.5, and 3.0 mg/kg of body weight. Subsequent challenge exposure of cattle with Dermacentor andersoni Stiles infected with Anaplasma marginale Theiler indicated that treatment with the dosage of 2.5 mg/kg prevented clinical anaplasmosis and that treatment with the dosage of 3.0 mg/kg prevented disease infection.

Effective/simple variance trending.

This article describes a simple, effective process that provides facilitated variance tracking while meeting the Joint Commission on Accreditation of Healthcare Organizations' requirement for Multidisciplinary Care Plans.