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Biomedical subjects

D A Ingram

Publications and source records attributed to D A Ingram.

At least 19 recordsLinked to original sources

Hyperactivation of p21(ras) and the hematopoietic-specific Rho GTPase, Rac2, cooperate to alter the proliferation of neurofibromin-deficient mast cells in vivo and in vitro.

Mutations in the NF1 tumor suppressor gene cause neurofibromatosis type I (NF1), a disease characterized by the formation of cutaneous neurofibromas infiltrated with a high density of degranulating mast cells. A hallmark of cell lines generated from NF1 patients or Nf1-deficient mice is their propensity to hyperproliferate. Neurofibromin, the protein encoded by NF1, negatively regulates p21(ras) activity by accelerating the conversion of Ras-GTP to Ras-GDP. However, identification of alterations in specific p21(ras) effector pathways that control proliferation in NF1-deficient cells is incomplete and critical for understanding disease pathogenesis. Recent studies have suggested that the proliferative effects of p21(ras) may depend on signaling outputs from the small Rho GTPases, Rac and Rho, but the physiologic importance of these interactions in an animal disease model has not been established. Using a genetic intercross between Nf1(+/)- and Rac2(-)(/)- mice, we now provide genetic evidence to support a biochemical model where hyperactivation of the extracellular signal-regulated kinase (ERK) via the hematopoietic-specific Rho GTPase, Rac2, directly contributes to the hyperproliferation of Nf1-deficient mast cells in vitro and in vivo. Further, we demonstrate that Rac2 functions as mediator of cross-talk between phosphoinositide 3-kinase (PI-3K) and the classical p21(ras)-Raf-Mek-ERK pathway to confer a distinct proliferative advantage to Nf1(+/)- mast cells. Thus, these studies identify Rac2 as a novel mediator of cross-talk between PI-3K and the p21(ras)-ERK pathway which functions to alter the cellular phenotype of a cell lineage involved in the pathologic complications of a common genetic disease.

Animals↗

Neurofibromin GTPase-activating protein-related domains restore normal growth in Nf1-/- cells.

Members of the Ras superfamily of signaling proteins modulate fundamental cellular processes by cycling between an active GTP-bound conformation and an inactive GDP-bound form. Neurofibromin, the protein product of the NF1 tumor suppressor gene, and p120GAP are GTPase-activating proteins (GAPs) for p21(Ras) (Ras) and negatively regulate output by accelerating GTP hydrolysis on Ras. Neurofibromin and p120GAP differ markedly outside of their conserved GAP-related domains (GRDs), and it is therefore unknown if the respective GRDs contribute functional specificity. To address this question, we expressed the GRDs of neurofibromin and p120GAP in primary cells from Nf1 mutant mice in vitro and in vivo. Here we show that expression of neurofibromin GRD, but not the p120GAP GRD, restores normal growth and cytokine signaling in three lineages of primary Nf1-deficient cells that have been implicated in the pathogenesis of neurofibromatosis type 1 (NF1). Furthermore, utilizing a GAP-inactive mutant NF1 GRD identified in a family with NF1, we demonstrate that growth restoration is a function of NF1 GRD GAP activity on p21(Ras). Thus, the GRDs of neurofibromin and p120GAP specify nonoverlapping functions in multiple primary cell types.

Animals↗

Genetic and biochemical evidence that haploinsufficiency of the Nf1 tumor suppressor gene modulates melanocyte and mast cell fates in vivo.

Neurofibromatosis type 1 (NF1) is a common autosomal-dominant disorder characterized by cutaneous neurofibromas infiltrated with large numbers of mast cells, melanocyte hyperplasia, and a predisposition to develop malignant neoplasms. NF1 encodes a GTPase activating protein (GAP) for Ras. Consistent with Knudson's "two hit" model of tumor suppressor genes, leukemias and malignant solid tumors in NF1 patients frequently demonstrate somatic loss of the normal NF1 allele. However, the phenotypic and biochemical consequences of heterozygous inactivation of Nf1 are largely unknown. Recently neurofibromin, the protein encoded by NF1, was shown to negatively regulate Ras activity in Nf1-/- murine myeloid hematopoietic cells in vitro through the c-kit receptor tyrosine kinase (dominant white spotting, W). Since the W and Nf1 locus appear to function along a common developmental pathway, we generated mice with mutations at both loci to examine potential interactions in vivo. Here, we show that haploinsufficiency at Nf1 perturbs cell fates in mast cells in vivo, and partially rescues coat color and mast cell defects in W(41) mice. Haploinsufficiency at Nf1 also increased mast cell proliferation, survival, and colony formation in response to Steel factor, the ligand for c-kit. Furthermore, haploinsufficiency was associated with enhanced Ras-mitogen-activated protein kinase activity, a major downstream effector of Ras, via wild-type and mutant (W(41)) c-kit receptors. These observations identify a novel interaction between c-kit and neurofibromin in vivo, and offer experimental evidence that haploinsufficiency of Nf1 alters both cellular and biochemical phenotypes in two cell lineages that are affected in individuals with NF1. Collectively, these data support the emerging concept that heterozygous inactivation of tumor suppressor genes may have profound biological effects in multiple cell types.

Animals↗

I.v. immunoglobulin reduces circulating proinflammatory cytokines in Guillain-Barré syndrome.

BACKGROUND: Treatment with human i.v. immunoglobulin (IVIg) modifies the course of Guillain-Barré syndrome (GBS), but its specific mode of action is unknown. Cellular interactions mediated through the release of cytokines play a role in the pathogenesis of GBS and may be regulated by IVIg therapy. OBJECTIVE: To delineate possible immunoregulatory mechanisms of IVIg in patients with GBS. METHODS: Circulating levels of the proinflammatory cytokines, tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta, were assayed in 21 patients with GBS before and serially after IVIg therapy. Comparisons were made with serum concentration of the anti-inflammatory cytokines, soluble TNF-alpha receptor and IL-10. Serial measurements were also performed in 12 untreated patients with relatively mild disease and 7 patients treated by plasma exchange. RESULTS: Circulating levels of TNF-alpha and IL-1beta decreased after treatment with IVIg but remained relatively high in untreated patients and in those treated by plasma exchange. Clinical improvement in patients treated with IVIg was associated with a reduction in unbound TNF-alpha during the acute phase of the illness. Circulating levels of anti-inflammatory cytokines were not affected by IVIg treatment. CONCLUSION: Data presented here suggest a novel mechanism of action of IVIg that involves selective modulation of circulating proinflammatory cytokines.

Adult↗

Paraneoplastic painful ulnar neuropathy.

A 58-year-old woman developed painful, bilateral ulnar neuropathy in conjunction with small cell lung carcinoma and high serum titer of anti-Hu antibody. An incidental stage I plasma cell dyscrasia, with immunoglobulin G kappa monoclonal protein, was also present. Electropysiological assessment excluded a generalized neuropathy, and nerve biopsy showed marked loss of myelinated and small unmyelinated fibers, without inflammatory changes or amyloid deposition. High titers of circulating anti-Hu antibody can be associated with symptoms resembling a paraneoplastic mononeuropathy.

Antibodies↗

Circulating tumor necrosis factor-alpha correlates with electrodiagnostic abnormalities in Guillain-Barré syndrome.

Autoimmune damage to peripheral nerves, mediated by activated T lymphocytes and macrophages, underlies the pathogenesis of inflammatory demyelination in Guillain-Barré syndrome. Both T lymphocytes and macrophages secrete tumor necrosis factor-alpha, a cytokine that exerts toxic effects on myelin, Schwann cells, and endothelial cells. The reportedly high serum levels of this cytokine in patients with Guillain-Barré syndrome may reflect the degree of immune activation rather than a direct pathogenic effect. We compared serum levels of tumor necrosis factor-alpha, interleukin-1 beta, and soluble interleukin-2 receptor with well-established electrodiagnostic criteria for primary demyelination in 23 patients with Guillain-Barré syndrome, to assess the relationship between these cytokines and peripheral myelin damage. High serum levels of tumor necrosis factor-alpha were associated with prolonged distal motor latencies and slowed motor conduction velocities, prolonged or absent F-wave responses, and reduced amplitude of distal compound muscle action potentials. No significant correlation was observed between electrodiagnostic criteria for primary demyelination and serum levels of interleukin-1 beta or soluble interleukin-2 receptor. These findings suggest a putative role of tumor necrosis factor-alpha in the pathogenesis of peripheral nerve demyelination in Guillain-Barré syndrome.

Adult↗

Upregulation of neuropeptides and neuropeptide receptors in a murine model of immune inflammation in lung parenchyma.

The lung is richly supplied with peptidergic nerves that store and secrete substance P (SP), vasoactive intestinal peptide (VIP), and other neuropeptides known to potently modulate leukocyte function in vitro and airway inflammation in vivo. To investigate and characterize neuromodulation of immune responses compartmentalized in lung parenchyma, neuropeptide release and expression of neuropeptide receptors were studied in lungs of antigen-primed C57BL/6 mice after intratracheal challenge with sheep erythrocytes. The concentrations of cytokines in bronchoalveolar lavage (BAL) fluid rose early and peaked on day 1 for interleukin (IL)-2, interferon gamma, and IL-10; days 1 to 2 for IL-6; and day 3 for IL-4, whereas the total number and different types of leukocytes in BAL fluid peaked subsequently on days 4 to 6 after i.t. antigen challenge. Immunoreactive SP and VIP in BAL fluid increased maximally to nanomolar concentrations on days 1 to 3 and 2 to 7, respectively in lungs undergoing immune responses. The high-affinity SP receptor (NK-1 R), and VIP types I (VIPR1) and II (VIPR2) receptors were localized by immunohistochemistry to surface membranes of mononuclear leukocytes and granulocytes in perivascular, peribronchiolar, and alveolar inflammatory infiltrates during immune responses. As quantified by reverse transcription-polymerase chain reaction, significant increases were observed in levels of BAL lymphocyte mRNA encoding NK-1 R (days 2 to 4), VIPR1 (days 2 to 4), and VIPR2 (days 4 to 6), and in alveolar macrophage mRNA encoding NK-1 R (days 2 to 6) and VIPR1 (days 2 to 4), but not VIPR2. Systemic treatment of mice with a selective, nonpeptide NK-1 R antagonist reduced significantly the total numbers of leukocytes, lymphocytes, and granulocytes retrieved by BAL on day 5 of the pulmonary immune response. The results indicate that SP and VIP are secreted locally during pulmonary immune responses, and are recognized by leukocytes infiltrating lung tissue, and thus their interaction may regulate the recruitment and functions of immune cells in lung parenchyma.

Animals↗

Activation of complement by Fluosol attributable to the pluronic detergent micelle structure.

Fluosol, a complex mixture of perfluorocarbons with a high oxygen-carrying capacity emulsified with the detergent pluronic F-68 and various lipids, recently was approved for adjuvant therapy to reduce myocardial ischemia during coronary angioplasty. Anaphylactoid reactions after Fluosol infusion through activation of the complement pathway have been reported in some patients. We examined the mechanism of complement activation by Fluosol. In vitro, incubation of both dog and human plasma with Fluosol for 1 h caused a significant reduction in total hemolytic complement levels (CH50). None of the individual components of Fluosol tested activated complement. A reduction in CH50 levels similar to that observed with Fluosol was obtained after incubation of dog or human plasma with the detergent pluronic F-68 in combination with either perfluorocarbon. In vivo, a bolus injection of the detergent and perfluorocarbon fully mimicked the anaphylactoid reaction of Fluosol previously observed in dogs, with transient profound hypotension, tachycardia, and reduction in CH50 levels occurring < or = 5 min. To investigate further the mechanism by which the pluronic/perfluorocarbon combination activates complement, an inert dense liquid (mineral oil or silicon oil) substituted for the perfluorocarbons produced comparable complement activation both in vitro and in vivo. These observations suggest that creation of a larger pluronic micelle around a core of perfluorocarbons or any inert dense substance, causes formation of a specific surface configuration, resulting in activation of the complement cascade.

Animals↗

Changes in motor unit synchronization following central nervous lesions in man.

1. Single motor unit spike trains have been recorded during voluntary isometric contraction of the affected intrinsic hand muscles of patients with unilateral central nervous lesions. These have been compared with similar recordings made from the patients' unaffected hand muscles and with recordings made from the hand muscles of healthy subjects. 2. Cross-correlation analysis was performed between the times of occurrence of the motor unit spike trains. The time course of central cross-correlogram peaks constructed for normal subjects and stroke patients was used to infer properties of the underlying common EPSPs and the impulse-generating properties of the motoneurones. The results of this analysis were compared between the two groups. In addition, the size and time course of cross-correlogram peaks obtained from the patients were related both to the patients' clinical state and to their hand and fine finger function. 3. Central nervous lesions were found to result in either a narrowing or broadening of the time course of motor unit synchronization. These changes were attributed either to an increase in the size of common EPSPs with respect to synaptic noise, or to the effects of presynaptic synchronization of motoneurone inputs. 4. Longitudinal studies of motor unit discharges in the year following the stroke demonstrated, in some patients, differences in the level of motor unit synchronization. These paralleled improvements in the patients' fine motor control. Pooled data from patients with varying deficits of fine motor control confirmed that loss or reduction of motor unit synchronization was associated with a corresponding slowing in the performance of rapidly alternating finger movements. 5. The results of the present study suggest that the branched common presynaptic inputs that generate motor unit synchronization are either of corticospinal tract origin or are intimately dependent on its function. Differences in the strength and time course of motor unit synchronization are demonstrated that may reflect the altered behaviour of presynaptic inputs to motoneurones following central nervous damage in man.

Adult↗

Suicide in the elderly: staying in control.

Suicide remains one of the major causes of death among the fastest growing segment of the US population--the elderly aged 65 and over. Individuals 65 and over comprised 12.4% of the population in 1988, but accounted for 20.9% of all reported suicides (McIntosh, 1992). The purpose of this qualitative study was to explore the meaning of suicide to the elderly and how suicide becomes an alternative for them. Results indicate that elderly subjects gave considerable thought to the end of their lives--including planning for death. For them, suicide was frequently viewed as a way of maintaining control over the dying process. In this study, the elderly described their views on who makes the decision about when death will occur, when suicide is acceptable, and how they would like others to respond to their suicidal ideation. Risk factors and causes of suicide in the elderly emerged from the data.

Aged↗

Adverse effect of verapamil in myasthenia gravis.

Verapamil, a class IV anti-arrhythmic drug that blocks voltage-dependent calcium channels in cardiac and smooth muscle, also has effects on presynaptic and postsynaptic voltage-dependent calcium channels at the neuromuscular junction. In a postoperative patient with pre-existent myasthenia gravis, oral verapamil caused a marked exacerbation in myasthenic weakness.

Contraindications↗

Cyclosporin A therapy in paraprotein-associated neuropathy.

A case of IgG paraprotein-associated demyelinating neuropathy complicated by respiratory failure, which was unresponsive to standard immunosuppressive drug therapy, is reported. Cyclosporin A therapy resulted in a marked clinical recovery with objective improvement in nerve conduction and vital capacity. The beneficial response suggests that cell-mediated immunity is an important pathogenetic mechanism, and that cyclosporin A may be useful in the treatment of other refractory cases of paraprotein-associated neuropathy.

Adult↗

Role of perfluorochemical emulsions in the treatment of myocardial reperfusion injury.

Perfluorochemicals are substances with small particle size, low viscosity, and high oxygen-carrying capacity. The role of one perfluorochemical preparation. Fluosol, an emulsion of two perfluorocarbons, a detergent Pluronic F-68 (poloxamer 188), and phospholipids on myocardial reperfusion injury was investigated in a closed-chest canine model of regional ischemia. Intracoronary and intravenous infusions of Fluosol in the perireperfusion period significantly reduced infarct size and improved ventricular function in animals that were examined for up to 2 weeks after reperfusion. Fluosol preserved endothelial structure and endothelium-dependent relaxation of large and small vessels. Fluosol reduced neutrophil plugging of capillaries and attenuated neutrophil infiltration into the reperfused bed. Ex vivo studies of neutrophil function demonstrated apparent suppression of chemotaxis and lysozyme degranulation in cells from animals that were treated with Fluosol. However, treatment of cells in vitro manifested enhanced superoxide anion production within 5 minutes of incubation even with low concentrations of Fluosol. This effect was found to be almost entirely attributable to the detergent, Pluronic F-68. The stimulation of neutrophils by Fluosol was found to result directly from phagocytosis and indirectly from activation of the complement cascade. These findings suggest that perfluorochemicals may provide a novel form of therapy to enhance myocardial salvage after successful reperfusion. The mechanism appears to be due to stimulation and subsequent "deactivation" of neutrophils peripherally, which thereby reduces their cytotoxic potential in the reperfused myocardium. The role of the oxygen-carrying ability of the perfluorocarbons in the reduction of reperfusion injury remains to be determined. In a pilot study in human beings, Fluosol that was used as adjunctive therapy with angioplasty has also been shown to improve regional ventricular function. Clinical trials with perfluorochemical emulsions appear warranted to determine the role of reperfusion injury in limiting myocardial salvage in patients who are undergoing pharmacologic or mechanical reperfusion.

Animals↗

Pharmacologic perturbation of neutrophils by Fluosol results in a sustained reduction in infarct size in the canine model of reperfusion.

Previous studies have demonstrated that intravenous administration of large doses of Fluosol, a perfluorochemical preparation, reduced infarct size 24 h after reperfusion, an effect that was associated with reduced neutrophil infiltration. The effect of a clinically tolerable dose of Fluosol on infarct size after a prolonged period of reperfusion and its mechanism of action on neutrophils remain unknown. Twenty-one anesthesized closed chest dogs were subjected to 90 min of proximal left anterior descending coronary artery occlusion and 72 h of reperfusion. An additional five dogs that did not undergo regional myocardial ischemia were utilized to explore the mechanism of action of Fluosol on neutrophil function. In the infarct study, animals were randomized to receive either intravenous Fluosol (n = 10) or an equivalent volume of Ringer's lactate solution (control; n = 11) at 15 ml/kg body weight during the last 30 min of occlusion and for the 1st 30 min of reperfusion. Fluosol significantly reduced infarct size when expressed as percent area at risk 72 h after reperfusion (13.7 +/- 2.7% vs. 38.3 +/- 4.5%, respectively, p less than 0.001). This reduction was associated with significant improvement in regional wall motion (18.4 +/- 2.3% vs. 5.5 +/- 2%, p less than 0.001). Endocardial blood flow in the ischemic bed was significantly higher 3 h after reperfusion in Fluosol-treated dogs (0.63 +/- 0.08 vs. 0.34 +/- 0.07 ml/min per g, p = 0.01). Reduced capillary plugging by neutrophils with relative preservation of endothelial cell structure was observed in Fluosol-treated animals. Infusion of Fluosol produced a marked transient decrease in peripheral neutrophil and platelet counts in both ischemic and nonischemic dogs and was associated with a significant reduction in total hemolytic complement levels. Studies of neutrophil function ex vivo revealed a reduction in chemotaxis and lysozyme degranulation after infusion of Fluosol. In vitro experiments showed that Fluosol produced a rapid and sustained activation of neutrophils determined by superoxide anion production. These data demonstrate that low dose intravenous Fluosol produces a sustained reduction in infarct size in the canine model. The beneficial effect may be in part due to the suppression of various neutrophil functions in the reperfused myocardium subsequent to peripheral activation by Fluosol. Such interventions may offer a novel therapy to enhance myocardial salvage by sequestration of circulating neutrophils during the critical early reperfusion period.

Animals↗

Phagocytic activation of human neutrophils by the detergent component of fluosol.

Fluosol (Alpha Therapeutic Corporation, Los Angeles, CA) an emulsion of perfluorocarbons with a high oxygen-carrying capacity, was approved as an adjunct to alleviate myocardial ischemia during coronary angioplasty. This drug also significantly enhances myocardial salvage presumably related to an action on the neutrophil. The mechanism by which fluosol and its individual components, including the detergent Pluronic F-68, affected neutrophil function was examined. During the incubation of neutrophils with fluosol, a rapid stimulation of superoxide anion production and degranulation which progressively increased over a 30-minute period was detected. Neutrophils incubated with only Pluronic F-68 produced similar amounts of superoxide anion. Cytochalasin B, an inhibitor of phagocytosis, significantly inhibited this superoxide anion generation. As shown previously, neutrophils incubated with fluosol for 30 minutes and then subsequently stimulated manifested a reduction in lysozyme release as compared with untreated cells. Results of an electron microscopic examination confirmed the cellular uptake of the fluosol within phagocytic vacuoles. Neutrophil viability determined by trypan blue was unaffected after fluosol treatment. These observations show that the fluosol emulsion, primarily through micelles formed by the detergent Pluronic F-68, activates human neutrophils by serving as a phagocytic stimulus, which produces a cell refractory to subsequent stimulation.

Blood Substitutes↗

Abdominal wall weakness due to thoracic syringomyelia.

We describe a patient who presented with unilateral abdominal herniation due to weakness of abdominal musculature, associated with extensive syringomyelia. Electromyographic evidence of focal denervation and reinnervation was present in the weak muscles consistent with anterior horn cell loss. We compare these unusual clinical features with the more typical presentations of syringomyelia and with other neurologic causes of abdominal weakness.

Abdominal Muscles↗

Plasticity of central motor pathways in children with hemiplegic cerebral palsy.

To obtain neurophysiologic evidence for a reorganization of central motor pathways in children who had suffered a cerebral lesion at birth, we performed cross-correlation analyses of multiunit EMG recordings obtained from children with hemiplegic cerebral palsy and marked mirror movements. We found that the motoneuron pools of homologous left and right hand muscles received common synaptic input from abnormally branched presynaptic axons. The results of electromagnetic brain stimulation, cutaneomuscular, and tendon reflex testing suggested that these common inputs are provided by abnormally branched corticospinal tract fibers whose origin is the undamaged motor cortex.

Cerebral Palsy↗

Variability of electrocardiographic precordial lead placement: a method to improve accuracy and reliability.

Variability in precordial lead placement is a recognized source of electrocardiographic inaccuracy and lack of reproducibility. In an attempt to reduce error, we evaluated a new device to facilitate and guide precordial lead placement. This study involved three phases: (1) comparison of device-guided electrocardiogram with ECGs obtained by deliberate misplacement of precordial leads on the same patient; (2) electrocardiograms obtained by using the precordial lead device versus those obtained by standard technician methods; (3) reproducibility of precordial electrocardiographic leads between two technicians using the device to guide lead placement. Deliberate misplacement of precordial leads by 2 cm resulted in significant electrocardiographic interpretation changes in all patients. Comparing electrocardiograms obtained after device-guided precordial placement with those obtained after technician placement resulted in variations in 60% of patients including changes in R-wave amplitude, ST segments, Q waves, and transition zone. Significant Q-wave appearance/disappearance and/or significant ST-segment elevation/depression occurred in 19% of patients in Phase II. Sixteen percent of electrocardiograms showed significant changes when analyzed by an experienced electrocardiographer and 10% when interpreted by computer. Variable lead placements and resulting electrocardiographic alterations were not seen by either of two technicians when the device was used. This study confirms the widespread variability in precordial electrocardiograms secondary to lead misplacement. The use of a device to assist in the placement of precordial leads ensures accuracy and reproducibility of electrocardiography. Improved precision and quality control in this laboratory test have important implications in health care and its costs.

Electrocardiography↗