RasGRP1 overexpression in T-ALL increases basal nucleotide exchange on Ras rendering the Ras/PI3K/Akt pathway responsive to protumorigenic cytokines.

Ras GTPases are activated by RasGEFs and inactivated by RasGAPs, which stimulate the hydrolysis of RasGTP to inactive RasGDP. GTPase-impairing somatic mutations in RAS genes, such as KRAS(G12D), are among the most common oncogenic events in metastatic cancer. A different type of cancer Ras signal, driven by overexpression of the RasGEF RasGRP1 (Ras guanine nucleotide-releasing protein 1), was recently implicated in pediatric T-cell acute lymphoblastic leukemia (T-ALL) patients and murine models, in which RasGRP1 T-ALLs expand in response to treatment with interleukins (ILs) 2, 7 and 9. Here, we demonstrate that IL-2/7/9 stimulation activates Erk and Akt pathways downstream of Ras in RasGRP1 T-ALL but not in normal thymocytes. In normal lymphocytes, RasGRP1 is recruited to the membrane by diacylglycerol (DAG) in a phospholipase C-γ (PLCγ)-dependent manner. Surprisingly, we find that leukemic RasGRP1-triggered Ras-Akt signals do not depend on acute activation of PLCγ to generate DAG but rely on baseline DAG levels instead. In agreement, using three distinct assays that measure different aspects of the RasGTP/GDP cycle, we established that overexpression of RasGRP1 in T-ALLs results in a constitutively high GTP-loading rate of Ras, which is constantly counterbalanced by hydrolysis of RasGTP. KRAS(G12D) T-ALLs do not show constitutive GTP loading of Ras. Thus, we reveal an entirely novel type of leukemogenic Ras signals that is based on a RasGRP1-driven increased in flux through the RasGTP/GDP cycle, which is mechanistically very different from KRAS(G12D) signals. Our studies highlight the dynamic balance between RasGEF and RasGAP in these T-ALLs and put forth a new model in which IL-2/7/9 decrease RasGAP activity.

Endothelin-1 stimulates human colonic myofibroblast contraction and migration.

BACKGROUND: Although the contractile, migratory, and proliferative responses of subepithelial myofibroblasts to injury have been postulated to be important events in intestinal wound healing, contractile force generation and migration by these cells has not been investigated previously, and the signals that regulate proliferation by these cells are poorly understood. AIMS: The primary aim of this study was to test the hypothesis that the inflammatory mediator endothelin-1 modulates contraction, migration, and proliferation of intestinal myofibroblasts. We also sought to examine the signal transduction pathways which might underlie these putative effects. METHODS: Contraction, migration, proliferation, cytosolic [Ca(2+)], and myosin phosphorylation were measured in human colonic subepithelial myofibroblasts in the absence and presence of endothelin receptor agonists and antagonists. RESULTS: Endothelin-1, but not interleukin 1 alpha, interleukin 6, interleukin 8, interleukin 10, or tumour necrosis factor alpha, induced a rapid and robust generation of contractile force, which was associated with an increase in cytosolic [Ca(2+)] and myosin phosphorylation. Inhibition of rho associated kinase reduced endothelin-1 stimulated myosin phosphorylation and contractile force development. Endothelin-1 stimulated migration with a dose-response relationship similar to that observed for contraction. Endothelin A and B receptors mediated contraction while migration was mediated predominantly through endothelin B receptors. Platelet derived growth factor and serum, but not endothelin-1, induced proliferation. CONCLUSIONS: Endothelin-1 stimulates colonic subepithelial myofibroblast contraction and migration via endothelin receptor mediated myosin phosphorylation. These results support an important role for subepithelial myofibroblasts in the injury response of the gut and consequently intestinal wound repair.

RhoA/rho-associated kinase mediates fibroblast contractile force generation.

The intracellular signals governing contractile force generation by non-muscle cells remain uncertain. Our aim was to test the hypothesis that the rhoA/rho-associated kinase signaling pathway is a principal mediator of contractile force generation in non-muscle cells. We measured myosin II regulatory light chain (MLC) phosphorylation and directly quantitated force generation by chicken embryo fibroblasts in the absence and presence of selective inhibitors of rhoA, and its downstream effector, rho-associated kinase. Inactivation of rhoA, with C3 transferase, inhibited serum-stimulated MLC phosphorylation and contractile force generation. Y-27632, an inhibitor of rho-associated kinase, reduced basal contractile tension, and inhibited both serum and endothelin-1 stimulated MLC phosphorylation and contractile force generation. The results of this study provide novel evidence indicating that the rhoA/rho-associated kinase signaling pathway is a principal mediator of MLC phosphorylation and consequent contractile force generation by non-muscle cells.

Current trends in removable prosthodontics.

BACKGROUND: This article discusses trends in the demographics and treatment of the edentulous patient. It is clear that there still is a tremendous need for removable-prosthodontic services today. While the basic process of making dentures has changed little over the past several decades, new materials and techniques can help laboratories and clinicians provide functional, esthetic restorations that offer exceptional value to patients. Implant treatment is a tremendous adjunct to removable prosthodontics in the treatment of edentulous patients, but it is not within the financial reach of all dental patients. CLINICAL IMPLICATIONS: The clinical skills required to deliver excellent complete denture care are also paramount to successful implant prosthodontics (fixed and removable) and esthetic dentistry. Even so, the opportunities to develop these skills and the interest appear to be decreasing at the same time that the need is projected to increase. In service to our patients, the profession must examine this trend closely.

Continuous fiberoptic PCO2 monitoring indicates poorer gastric perfusion during supraceliac aortic clamping than conventional gastric tonometry in humans: a pilot study.

OBJECTIVE: To compare two monitors of gastric perfusion intraoperatively--continuous fiberoptic carbon dioxide partial pressure (PCO2) sensor and conventional gastric tonometer. DESIGN: Prospective, unblinded study. SETTING: University teaching hospital. PARTICIPANTS: Adult patients undergoing major abdominal surgery. INTERVENTIONS: A fiberoptic probe (Biomedical Sensors, Pfizer, High Wycombe, England) capable of continuous PCO2 measurement and adapted to fit into the saline-filled balloon of a tonometric orogastric tube (Tonometrics, Instrumentarium Corp, Helsinki, Finland) was placed in the patients. The fiberoptic probe was attached to a Paratrend 7 machine (Biomedical Sensors, Pfizer) providing continuous intragastric PCO2 data. A second tonometric orogastric tube was passed and used according to the manufacturer's instructions, yielding intermittent PCO2 values. MEASUREMENTS AND MAIN RESULTS: Twelve patients provided 79 data pairs: 33 without aortic clamp, 23 infraceliac aortic clamp, and 23 supraceliac aortic clamp. Data are presented as mean +/- SD, and analysis of variance was used for comparison (p < 0.01); bias and precision were also calculated. Intramusosal PCO2 and PCO2 gradient were significantly higher, and intramucosal pH was significantly lower from continuous fiberoptic measurement in the supraceliac clamp group. In the no-clamp and infraceliac clamp groups, the differences between the 2 methods of measurement were not significant. CONCLUSION: Conventional gastric tonometry overestimated perfusion during conditions of compromised gastric blood flow (supraceliac aortic clamp).

Caffeine- or halothane-induced contractures of masseter muscle are similar to those of vastus muscle in normal humans.

BACKGROUND: Skinned fibers from normal human masseter muscle have greater caffeine and calcium sensitivity than skinned fibers from vastus muscle. We examined sensitivity to caffeine and halothane in fresh, cut muscle bundles (non-skinned) from human masseter muscle. METHODS: Masseter bundles (caffeine, n=25, halothane, n=19) excised from 10 humans under general anesthesia had tension measured in 37 degrees C baths during the addition of caffeine (0.5, 1, 2, 4, 8, 32 mM) or 3% halothane. Results were compared to those of our previous studies (1989, 1997, 25 patients) of vastus bundles (caffeine, n=71, halothane, n=63) using the same protocol, technicians, and equipment. RESULTS: Baseline force in the caffeine test was 2.10+/-1.57 for masseter, and 2.02+/-1.68 and 1.82+/-1.29 respectively for vastus muscle. Force at 32 mM caffeine concentration was 11.2+/-9.9 g for masseter, 11.0+/-5.4 and 13.5+/-7.5 g for vastus. Concentration-response curves were virtually identical. In the halothane group, neither baseline values (masseter 1.47+/-1.30, vastus 1.91+/-1.32 and 2.15+/-1.71) nor contractures in response to 3% halothane were different. Most bundles had no contracture in response to 3% halothane; 3 masseter bundles and 2 vastus bundles (1989) developed contractures of less than 0.05 g. Three vastus bundles (1997) developed contractures >0.2 g. CONCLUSION: Contracture responses of intact cut masseter and vastus bundles (non-skinned) do not differ with respect to caffeine and halothane. Responses of skinned fibers might demonstrate greater sensitivity under certain conditions, but they do not reflect those of intact cut bundles.

Differential regulation of human blood monocyte and alveolar macrophage inflammatory cytokine production by nitric oxide.

BACKGROUND: Nitric oxide (NO) has been associated with airway inflammation in asthma. Our previous work suggests that NO functions in an anti-inflammatory capacity through downregulation of stimulated cytokine secretion by normal human alveolar macrophages. Functional differences between alveolar macrophages and blood monocytes are thought to be related to maturation. OBJECTIVE: The purpose of this study was to determine the effect of NO on stimulated cytokine production by monocytes from asthmatics and normal healthy controls. METHODS: Monocytes and alveolar macrophages were obtained from normal volunteers (n = 13) and asthmatics with atopy (n = 7). Monocyte and alveolar macrophage cultures were stimulated with 0.5 microgram/mL lipopolysaccharide +/- 1.0 mM DETA NONOate (releases NO in culture with t1/2 = 20 hours at 37 degrees C) and incubated for 24 hours. Cell-free supernatants were collected and assayed by ELISA for tumor necrosis factor-alpha (TNF) and granulocyte macrophage colony stimulating factor (GM-CSF). RESULTS: Nitric oxide did not inhibit TNF production in monocytes of asthmatics and normals (mean +/- SEM % TNF stimulation = 19.6 +/- 9.7). Similar to previous results, NO did inhibit alveolar macrophages (% TNF suppression = 60.6 +/- 4.4). To determine whether this differential effect of NO on the two cell populations was related to maturation, monocytes were matured by culture for 7 days. The in vitro matured monocytes demonstrated 51.7 +/- 7.9% suppression of TNF. For each cell population, the responses of the asthmatics and healthy controls were not different. The differential effect is not cytokine specific since similar results were obtained with GM-CSF. CONCLUSION: These results demonstrate a differential effect of NO on monocyte and alveolar macrophages cytokine regulation and this effect may be related to the state of maturation.

Managing latex allergy in patients and health care workers.

The new and important problem of latex allergy deserves the attention of all health care professionals and institutions. Latex products we use every day may cause serious consequences for patients and coworkers. All providers must develop a plan for protecting allergic patients and staff from latex exposure, and for managing allergic reactions should they occur.

Shared IgE-binding sites among separated components of natural rubber latex.

BACKGROUND: IgE-mediated allergy to proteins present in natural rubber latex is a well-recognized problem. Latex contains a complex mixture of proteins ranging in molecular weight from 6 to > 200 kD. OBJECTIVE: The aim of this study was to determine whether shared allergenic sites exist on separate latex components. METHODS: Following electrophoresis, latex components at 14, 24, and 46 kD were electroeluted from SDS-polyacrylamide gels and used in ELISA inhibition and immunoblot inhibition studies of human latex-specific IgE antibodies. RESULTS: A minimum of 4 major allergenic sites (for convenience labeled A through D) were found to exist in 3 components of nonammoniated latex. Minimally, 2 are present in the 14-kD component (A, B) and 3 in the 24-kD component (A-C). The 46-kD fraction has 3 or more antigenic sites (A, C, D) but lacks one (B) that is present in both the 14- and 24-kD components. CONCLUSIONS: Four different IgE-binding moieties were detected among three latex protein components (14, 24 and 46 kD). Some of these allergenic sites were shared by two or more components. Recovery of these and others from fragmented latex components will allow identification of their amino acid composition and their availability will ultimately lead to better diagnostic and therapeutic options for patients with latex allergy.

Latex hypersensitivity in emergency medical service providers.

BACKGROUND: Emergency medical service providers have a high degree of exposure to latex products. Patients utilizing emergency medical services can be allergic to latex products used during rescue efforts. OBJECTIVE: To determine the prevalence of latex hypersensitivity among emergency medical service providers. METHODS: Study questionnaires were distributed to a group of emergency medical service providers. Skin prick testing was performed using latex, common aeroallergens, and food extracts. Patch testing was done using latex and individual rubber additives. Serum latex-specific IgE was also measured. RESULTS: A total of 93 completed surveys were collected. Average exposure to latex in the work environment was 8.2 years. Eighty-four (90%) used latex gloves routinely at work. Of those, thirteen (14%) gave history of reaction to latex gloves. Sixty-two percent were not aware of the possibility of latex allergy in themselves or their patients. Forty-one (44%) had skin testing. Of those, four (10%) had positive prick tests for at least one of the four latex preparations used. Five had positive skin tests to avocado extract without supporting clinical history. Two had positive skin tests to banana, one with supporting clinical history for banana allergy. No food cross-reactivity with latex was demonstrated. Latex-specific serum correlated with prick skin test results. No positive reactions were noted with patch testing. CONCLUSIONS: A significant percentage of emergency medical service providers were not aware of the occupational risk of latex allergy or the potential risk in their patients. A positive prick skin test for latex was present in 4 of 41 (10%), representing one-third of those who reported symptoms from latex exposure.

A guinea pig model of hypersensitivity to allergenic fractions of natural rubber latex.

Allergy to natural rubber latex is a growing medical problem with life-threatening aspects. The aim of this study was to learn if guinea pigs could serve as a suitable model for immediate-type hypersensitivity to latex. Guinea pigs were immunized either with whole non-ammoniated latex extract, or with one of nine SDS-PAGE-separated components. Other animals were injected with electroeluted latex components localized on gel at 14, 24 and a cluster around 45 kD. Before and after immunization, sera from the animals were examined by ELISA, immunoblots, passive cutaneous anaphylaxis (PCA) and passive systemic anaphylaxis. Latex-specific antibodies were detected by ELISA and immunoblots in sera from all immunized animals. PCA assays showed that the guinea pigs had homocytotropic antibodies dilutable to 1:250. PCA was abolished when sera from animals immunized with allergens in alum were heated at 56 degrees C for 30 min indicating the antibodies were of the E isotype. Passive systemic anaphylaxis was induced in 4 of 10 guinea pigs. The results show that guinea pigs are capable of making antibodies to latex protein components that mediate dermal and systemic anaphylaxis, paralleling the spectrum of clinical and laboratory findings of humans with immediate-type clinical latex hypersensitivity.

Latex hypersensitivity: its prevalence among dental professionals.

Reports of hypersensitivity to latex are growing among oral health care workers, who have a high degree of exposure to latex products. The authors undertook a study to determine the prevalence of latex hypersensitivity among oral health care workers in a hospital dental practice. Among the 34 people who participated in the study, 12 percent had positive results in a skin prick test for latex. This suggests that the true prevalence rate of immediate hypersensitivity to latex in this group of oral health care workers is similar to that in other health care workers who use latex gloves frequently.

Femoral and lateral femoral cutaneous nerve block for muscle biopsies in children.

Retrospective chart review (1978-1993) of 179 children less than age 18 (10.0 +/- 3.8 SD yrs) undergoing muscle biopsy for determination of susceptibility to malignant hyperthermia provided data. One hundred and forty-six patients received femoral and lateral femoral cutaneous nerve blocks as their primary anaesthetic. We examined age, weight, duration of surgery, time to discharge from hospital, choice and dosage of local anaesthetics, choice and dosage of sedation, postoperative pain medications, and complications. All children receiving this form of anaesthesia remained outpatients. Between 1978 and 1985 procaine (10 mg.kg-1) with hyaluronidase or 2-chloroprocaine (12 mg.kg-1) provided nerve blockade; after 1985, lignocaine (6.8 mg.kg-1), or a combination of lignocaine or mepivacaine and 2-chloroprocaine, were the preferred agents. More recently the combination of 2-chloroprocaine and bupivacaine has been popular. Three patients required admission to the recovery room postoperatively, due to heavy sedation. Forty-three children (29%) received pain medication during recovery. Femoral and lateral femoral cutaneous block anaesthesia with light to moderate sedation is well tolerated in children undergoing anterior thigh procedures.

Prolonged duration of succinylcholine in patients receiving anticonvulsants: evidence for mild up-regulation of acetylcholine receptors?

Succinylcholine (SCh) normally causes a small increase in serum potassium concentration, but certain conditions may predispose to severe hyperkalaemia. This is due to "up-regulation" of skeletal muscle acetylcholine receptors (AChR), which also results in resistance to non-depolarizing muscle relaxants (NDMR). Anticonvulsant therapy causes NDMR resistance because of sub-clinical blockade, and diminished release, of acetylcholine. We studied nine patients chronically receiving anticonvulsants (phenytoin and/or carbamazepine) and nine control patients. Anaesthesia was induced typically with thiopentone or propofol; isoflurane and N2O were used for maintenance. The ulnar nerve was supramaximally stimulated and mechanical twitch height was measured with a force transducer at the adductor pollicis, before and after SCh 1 mg.kg-1, until return to baseline height. Plasma potassium concentration was measured before and at three, five, and ten minutes following SCh. Mean maximum potassium rise was 0.2 mEq.L-1 in each group. The time for return to baseline twitch height was 14.3 +/- 2.3 min (mean +/- SD) in the anticonvulsant group and 10.0 +/- 1.6 min in the control group, P = 0.001. The recovery index (time for 25% to 75% recovery) was 2.6 +/- 0.9 min in the anticonvulsant group and 1.4 +/- 0.3 min in the control group, P < 0.01. The normal potassium response coupled with prolonged duration suggests a hypersensitivity to SCh that is consistent with an anticonvulsant-induced mild up-regulation of AChR.