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.

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.

Working hypothesis to redefine endothelial progenitor cells.

Since 1997, postnatal vasculogenesis has been purported to be an important mechanism for neoangiogenesis via bone marrow (BM)-derived circulating endothelial progenitor cells (EPCs). Based on this paradigm, EPCs have been extensively studied as biomarkers to assess severity of cardiovascular disease and as a cell-based therapy for several human cardiovascular disorders. In the majority of studies to date, EPCs were identified and enumerated by two primary methodologies; EPCs were obtained and quantified following in vitro cell culture, or EPCs were identified and enumerated by flow cytometry. Both methods have proven controversial. This review will attempt to outline the definition of EPCs from some of the most widely cited published reports in an effort to provide a framework for understanding subsequent studies in this rapidly evolving field. We will focus this review on studies that used cell culture techniques to define EPCs.

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.

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.

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.

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.

Juvenile-onset bulbospinal muscular atrophy with deafness: Vialetta-van Laere syndrome or Madras-type motor neuron disease?

A girl with rapid-onset, bulbospinal muscular atrophy and deafness is described. The patient's mother and brother showed EMG features consistent with subclinical involvement. T is bulbospinal form of spinal muscular atrophy associated with deafness described by Vialetto and van Laere closely resembles the Madras type of motor neuron disease, also associated with deafness, described by Jagganathan and colleagues.

Brain-stem auditory evoked responses in diagnosis of central pontine myelinolysis.

Central pontine myelinolysis (CPM) developed in association with acute adrenocortical insufficiency during correction of severe hyponatraemia in a 58-year-old woman. Repeated CT scanning and NMR imaging were normal from the onset of the illness. Electroencephalography and brain-stem auditory evoked responses showed abnormalities consistent with a brain-stem lesion, which resolved as the patient made a gradual but incomplete recovery. Our observations illustrate the value of electrophysiological monitoring in CPM and support the proposed association between this condition and the rapid correction of an electrolyte imbalance.

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.

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.

Endothelial colony-forming cells and mesenchymal stem cells from ECMO circuits of term infants.

OBJECTIVE: Endothelial progenitor cells (EPCs) have been examined in numerous adult diseases and have been suggested as a cellular-based therapy. However, there are no reports describing EPCs being isolated from newborn peripheral blood. STUDY DESIGN: Endothelial colony-forming cells (ECFCs), a subtype of EPCs, were isolated from blood collected from 12 neonatal extracorporeal membrane oxygenation (ECMO) circuits. RESULT: ECFCs were isolated in all samples. We unexpectedly isolated a distinctly different colony of mesenchymal stem cells (MSCs) in seven samples. Both cell types expressed the expected endothelial or mesenchymal cell surface antigens. CONCLUSION: To our knowledge, this is the first report of ECFCs and MSCs isolated from peripheral blood of critically ill term newborns. Both cells types may be mobilized in response to critical illness or to the ECMO circuit. Further studies evaluating the role of stem cells in various newborn conditions are warranted.

Accuracy of clinical signs, SEP, and EEG in predicting outcome of hypoxic coma: a meta-analysis.

OBJECTIVE: Accurate prediction of neurologic outcome after hypoxic coma is important. Previous systematic reviews have not used summary statistics to summarize and formally compare the accuracy of different prognostic tests. We therefore used summary receiver operating characteristic curve (SROC) and cluster regression methods to compare motor and pupillary responses with sensory evoked potential (SEP) and EEG in predicting outcome after hypoxic coma. METHODS: We searched PubMed, MEDLINE, and Embase (1966-2007) for reports in English, German, and French and identified 25 suitable studies. An SROC was constructed for each marker (SEP, EEG, M1 and M < or = 3), and the area under the curve (AUC), a measure of diagnostic accuracy, was determined. For comparison, we calculated the differences between the AUC for each test and M1 reference standard. RESULTS: The AUC for absent SEP was larger than those for M1, M < or = 3, absent pupillary response, and EEG when the examinations were performed within the first 24 hours. The difference between the AUC for SEP (AUC 0.891) and that for M1 (AUC 0.786) was small (0.105, 95% confidence interval 0.023-0.187), only reaching significance on day 1 after coma onset. The use of M < or = 3 improved the diagnostic accuracy of motor signs. CONCLUSIONS: This study demonstrated that sensory evoked potential (SEP) is marginally better than M1 at predicting outcome after hypoxic coma. However, the superiority of SEP diminishes after day 1 and when M < or = 3 is used. The findings therefore caution against the tendency to generalize that SEP is a better marker than clinical signs.

Human papillomavirus causes an angiogenic switch in keratinocytes which is sufficient to alter endothelial cell behavior.

One of the requirements for tumor growth is the ability to recruit a blood supply, a process known as angiogenesis. Angiogenesis begins early in the progression of cervical disease from mild to severe dysplasia and on to invasive cancer. We have previously reported that expression of human papillomavirus type 16 E6 and E7 (HPV16 E6E7) proteins in primary foreskin keratinocytes (HFKs) decreases expression of two inhibitors and increases expression of two angiogenic inducers [Toussaint-Smith, E., Donner, D.B., Roman, A., 2004. Expression of human papillomavirus type 16 E6 and E7 oncoproteins in primary foreskin keratinocytes is sufficient to alter the expression of angiogenic factors. Oncogene 23, 2988-2995]. Here we report that HPV-induced early changes in the keratinocyte phenotype are sufficient to alter endothelial cell behavior both in vitro and in vivo. Conditioned media from HPV16 E6E7 expressing HFKs as well as from human cervical keratinocytes containing the intact HPV16 were able to stimulate proliferation and migration of human microvascular endothelial cells. In addition, introduction of the conditioned media into immunocompetent mice using a Matrigel plug model resulted in a clear angiogenic response. These novel data support the hypothesis that HPV proteins contribute not only to the uncontrolled keratinocyte growth seen following HPV infection but also to the angiogenic response needed for tumor formation.

Central motor conduction is abnormal in motor neuron disease.

Conduction in the central motor pathways of the brain and spinal cord was studied in 12 patients with motor neuron disease. Six healthy volunteers served as controls. Transcutaneous electrical stimulation of the cortex, cervical cord, thoracic cord and conus medullaris was used to determine motor latencies to the biceps brachii, thenar eminence and tibialis anterior muscles. Prominent, and often asymmetrical, slowing of central motor conduction was demonstrated in seven of the 12 patients; these findings were most marked in the spinal cord and in most cases correlated with clinical features of corticospinal involvement. In general it was more difficult to excite motor pathways in the central nervous system in the patients with motor neuron disease than in control subjects. Evidence of subclinical involvement of central motor pathways was found in five patients. The central lesion in motor neuron disease may thus contribute more significantly to the clinical deficit than has been realised, since the clinical signs of the upper motor neuron lesion are often masked by the more obvious lower motor neuron features.

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.

Effect of nitrous oxide on visual, auditory and somatosensory evoked potentials.

The effects of 10%, 30% and 50% nitrous oxide on visual, auditory and somatosensory evoked potentials were studied in seven healthy volunteers. The evoked potentials were averaged from the electroencephalogram following repeated peripheral sensory stimulation of the appropriate modality. Latencies and amplitudes of the resulting potentials were measured and compared with control values. In five subjects, increasing concentrations of nitrous oxide were associated with a graded reduction in amplitude of the visual (P less than 0.02) and somatosensory (P less than 0.02) evoked potentials. The latency of the first major negative potential of the visual evoked potential was significantly increased (P less than 0.02). Latencies of brainstem auditory evoked potentials did not alter. In the other two subjects the amplitudes of the visual and somatosensory evoked potentials showed graded increase with decreasing concentrations of nitrous oxide, confirming that the changes are dose related. As nitrous oxide is used almost universally during anaesthesia, these changes must be taken into account when assessing variations observed during operation in anaesthetic-related evoked potential studies.

Mirror movements studied in a patient with Klippel-Feil syndrome.

1. Electromyographic (EMG) recordings have been made from upper limb muscles in a patient with well-defined congenital mirror movements occurring in association with Klippel-Feil syndrome and the results compared to those obtained in normal control subjects. 2. In the patient, liminal percutaneous electrical or magnetic brain stimulation applied over either hemisphere elicited bilateral and symmetrical short-latency muscle responses in relaxed intrinsic hand muscles. In the normal subjects unilateral brain stimulation only elicited contralateral muscle responses. 3. F response and H reflex studies for the patient's ulnar-supplied intrinsic hand muscles were normal. No crossed responses were recorded in the homologous muscles of the contralateral hand. 4. Scalp-recorded somatosensory-evoked responses following ulnar or median nerve stimulation were of normal latency and distribution in the patient. 5. In the patient, cross-correlation analysis of on-going single and multiunit needle EMGs recorded between muscles of left and right hands revealed a central peak in the cross-correlogram. No cross-correlogram peaks were found between left- and right-hand muscles in normal subjects. The magnitude and time course of the central peaks in the cross-correlograms constructed between the firing of motor units on opposite sides of the body in the patient were similar to those found in cross-correlograms constructed between the firing of motor units from muscles on the same side of the body in the patient and in normal subjects. 6. The magnitude of cross-correlogram peaks detected within a muscle and those detected between left and right homologous muscles showed a gradient in which the largest peaks were found in the intrinsic hand and forearm extensor muscles. The smallest peaks were observed in the forearm flexor muscles. No peaks were detected between left and right biceps brachii muscles. In intrinsic hand muscles, the size of the cross-correlogram peak detected between the EMGs of homologous muscle pairs was greater than that found for non-homologous muscle pairs. 7. Cutaneous reflex responses were recorded from first dorsal interosseous muscle following unilateral electrical stimulation of the digital nerves of the index finger. In the patient, this produced an early excitatory (E1) response on the stimulated side. Later excitatory (E2 and E3) responses, of approximately equal size and latency, were distributed bilaterally. In the normal subjects, reflex responses were confined to the stimulated side.(ABSTRACT TRUNCATED AT 400 WORDS)

On the localization of the stretch reflex of intrinsic hand muscles in a patient with mirror movements.

1. The patient studied showed the typical mirror movements of the Klippel-Feil syndrome. Earlier intensive electrophysiological analysis suggests that many of her corticospinal axons branch abnormally to supply motoneurones on both sides of the spinal cord. Thus, in her, a long-latency reflex utilizing the motor cortex should manifest itself bilaterally. 2. EMG recordings were made simultaneously from the first dorsal interosseous (FDI) muscles of both hands while they were being voluntarily activated by the subject. The FDI of one hand was then briefly stretched by forcibly adducting the index finger. Similar but more limited studies were made using flexor pollicis brevis. 3. The reflex response of the stretched muscle consisted of a typical mixture of early (M1) and late (M2) components, with mean latencies of 32 and 49 ms respectively. 4. Unlike normal controls, the contralateral muscle responded on stretch of the ipsilateral muscle. However, its response consisted solely of a long-latency reflex. This was comparable in size, latency and waveform to the ipsilateral late component. (Mean size, 84% of the ipsilateral M2 response; mean latency 46 ms, or 3 ms less than the ipsilateral response due to the absence of M1). 5. The short-latency response did not spread to the homologous contralateral muscle even when it was large ipsilaterally. The long-latency response elicited from FDI did not spread to the abductor digiti minimi muscle of either hand. 6. Reducing the duration of the stretch reduced the duration of the crossed response by an equivalent amount. Unloading the ipsilateral muscle produced a delayed reduction of EMG activity contralaterally. Thus her long-latency pathway can act tonically as well as phasically. 7. These findings strongly support the hypothesis that delayed components of the human stretch reflex are relayed via the motor cortex and the corticospinal tract.

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.