Overexpression of C-terminally but not N-terminally truncated Myb induces fibrosarcomas: a novel nonhematopoietic target cell for the myb oncogene.

The myb oncogene encodes a DNA-binding transcriptional transactivator which can become a hematopoietic cell-transforming protein following the deletion of amino acid sequences from either its amino or carboxyl terminus. Although a number of hematopoietic tumors express terminally deleted variants of Myb, the involvement of truncated Myb in nonhematopoietic tumors has not been adequately investigated. To assess the full spectrum of Myb's oncogenic capability, a replication-competent retroviral vector (RCAMV) was used to express a full-length protein (C-Myb), an amino-terminally truncated protein (VCC- or delta N-Myb), a carboxyl-terminally truncated protein (T-Myb), or a doubly truncated protein (VCT-Myb) in vivo. These viruses were injected intravenously into 10-day chicken embryos, and the infected chicks were monitored for tumors. Approximately 4 to 8 weeks after hatching, the majority (30 of 39 [77%]) of animals infected with the T-Myb retrovirus (without 214 carboxyl-terminal residues) developed nodular muscle tumors which could be identified by both morphologic and immunohistochemical criteria as fibrosarcomas. Identically appearing tumors could also be found in the kidney of some T-Myb-infected animals. The T-Myb-induced fibrosarcomas expressed the appropriately sized T-Myb protein, contained an unaltered proviral T-myb gene, and showed clonal proviral integration sites. In comparison, no sarcomas were observed in any of the animals infected with the amino-terminally truncated (VCC- and delta N-Myb) or doubly truncated (VCT-Myb) viruses. A loss of carboxyl-terminal but not amino-terminal sequences can thus convert Myb into a potent in vivo transforming protein for nonhematopoietic mesenchymal cells. In comparison, a truncation of either or both ends of the protein can activate Myb into a hematopoietic cell-transforming protein.

Bin1 mediates apoptosis by c-Myc in transformed primary cells.

The Bin1 gene encodes a c-Myc-interacting adapter protein with tumor suppressor and cell death properties. In this study, we offer evidence that Bin1 participates in a mechanism through which c-Myc activates programmed cell death in transformed primary chick or rat cells. Antisense or dominant inhibitory Bin1 genes did not affect the ability of c-Myc to drive proliferation or transformation, but they did reduce the susceptibility of cells to c-Myc-induced apoptosis. Protein-protein interaction was implicated, suggesting that Bin1 mediates a death or death sensitization signal from c-Myc. Our findings offer direct support for the "dual signal" model of Myc apoptotic function, based on interactions with a binding protein. Loss of Bin1 in human tumors may promote malignant progression in part by helping to stanch the death penalty associated with c-Myc activation.

Retroviruses that express different ras mutants cause different types of tumors in chickens.

We have used replication-competent retroviral vectors to express avian and murine ras genes in cultured chick embryo fibroblasts (CEF) and in chickens. Since the viral vectors are identical, it is possible to compare the oncogenic potential of the ras genes directly. The normal (12 gly) form of chicken c-Ha-ras is not oncogenic in vivo, nor does high-level expression transform CEF. Expression of murine v-ras or modified forms of chicken c-Ha-ras with either lysine or glutamine at position 12 transforms CEF and causes tumors in birds. However, the oncogenic potential of the transforming ras genes is different; the viruses that express the genes with lysine and glutamine at position 12 cause a distinct spectrum of tumors.

Induction of B cell lymphomas by overexpression of a Myb oncogene truncated at either terminus.

The c-myb oncogene encodes a nuclear transcriptional transactivator that is often terminally truncated in hematopoietic tumors. To directly assess the tumorigenic activity of full length and terminally-truncated variants of c-myb, we have overexpressed several structurally-altered forms of myb within an avian retroviral vector and have shown that overexpression of truncated (but not full length) myb transforms both myeloid cells in vitro and mesenchymal cells in vivo. In vivo infection with these truncated myb viruses is now shown to induce metastatic B cell lymphomas in a significant minority of animals. Evaluation of the lymphomas revealed two distinct mechanisms of myb-induced tumorigenesis. In most of the lymphomas, proviral DNA inserted into the endogenous chicken c-myb gene and promoted the expression of a 5'-truncated myb transcript encoding an amino terminal truncated protein. In comparison, some animals infected with a virus encoding a carboxyl (C) terminal truncated myb (T-myb) developed non-insertional B cell lymphomas that directly expressed the provirally-encoded T-myb gene. The lymphomagenic T-myb protein lacks 214 C terminal amino acids including all of the myb transcription inhibition domain. This novel lymphomagenic activity for a C terminal truncated myb suggests that a loss of regulatory sequences at either end of c-myb is sufficient to create a B cell-specific transforming gene.

Blocked B cell differentiation and emigration support the early growth of Myc-induced lymphomas.

Avian leukosis virus induces lymphoma in chickens after proviral integration within the c-Myc gene, and subsequent expansion of Myc-overexpressing lymphocytes within transformed bursal follicles. The clonal expansion of these follicles allowed us to examine how Myc influences cell differentiation, growth, and apoptosis in lymphoid progenitors soon after the onset of Myc overexpression. Immunohistochemical analysis of developmental markers established that Myc overexpression consistently blocks lymphocyte differentiation at a late embryonic stage. Myc-transformed follicles also grow much more rapidly than normal follicles. This rapid growth is not mediated by suppression of apoptosis, as normal and Myc-transformed follicles showed similar rates of cell death by TUNEL immunohistochemical analysis of cells undergoing DNA degradation. Measurements of DNA synthesis and mitotic index showed modest effects of Myc to increase lymphocyte proliferation, as normal lymphocytes already divide rapidly. The major mechanism mediating rapid growth of transformed follicles instead involved failure of myc-overexpressing lymphocytes to emigrate from transformed follicles, while normal lymphocytes actively emigrate after hatching, as measured by BrdU pulse-chase labeling and immunohistochemical measurements. This failure to undergo the normal program of differentiation and subsequent bursal retention of lymphocytes accounts for most of the growth of transformed follicles, while Myc-induced proliferation makes a smaller contribution.

The B-L (Ia-like) antigens of the chicken. Lymphocyte plasma membrane distribution and tissue localization.

Specific antisera reacting with B-L (Ia-like) antigens were prepared by reciprocal immunization of animals from the congeneic lines CB and CC. The resulting antisera were tested either in direct or indirect immunofluorescence tests and stained 10-16% of peripheral blood cells (PBL). Of the B-L+ cells, 90% were B cells and 8% were T cells. After in vitro stimulation of PBL with ConA, 58% were B-L+ and 91% of these were T cells. B and T cells were defined by means of rabbit antisera raised against bursa and thymus cells made specific by absorption with the relevant tissues. Antigens determined by anti-B-L antisera, rabbit anti-bursa (ABS) and rabbit anti-thymus (ATS) sera showed an independent distribution on the membrane of PBL. The tissue distribution of B-L+ cells, defined by means of allo-antisera and monoclonal antibodies, was studied by direct and indirect immunofluorescence on sections of skin, liver, kidney and brain. In all organs, in addition to B cells and a small number of, presumably activated, T cells, macrophages and dendritic cells were positive. Notably, glia cells in the brain were also shown to express B-L antigen.

Acute cardiovascular changes of partial left ventriculectomy without mitral valve repair.

We assessed the acute cardiovascular changes of partial left ventriculectomy (PLV) in a patient with idiopathic dilated cardiomyopathy (IDCM) without mitral regurgitation. Acutely, PLV reduced left ventricular (LV) end-diastolic dimension and volume while increasing LV ejection fraction and cardiac output due to increased HR and SV. Substantial increases in LV filling pressure, possibly due to high LV end-systolic and diastolic elastances, were of concern clinically and the mechanism(s) of change remain unclear. However, one year follow-up showed remarkable improvements in NYHA and VO2 max while maintaining reduced LV volume, increased LVEF, and trivial MR.

Improvement in specificity of ultrasonography for diagnosis of breast tumors by means of artificial intelligence.

A set of ultrasonograms of lesions from 200 patients between the ages of 14 and 93 years who underwent mammography followed by ultrasonographic examination and excisional biopsy has been studied with computer vision techniques to improve the ultrasonographic specificity of the diagnosis. Selected features representing the texture of the lesion were calculated and then classified by an artificial neural network. This network was biased toward correctly classifying all the malignant cases at the expense of some misclassification of the benign cases. The network diagnosed the malignant cases with 100% sensitivity and 40% specificity (compared with 0% specificity for the radiologists diagnosing the same set of cases in the breast imaging setting), and tests performed with a leave-one-out technique indicate that the network will generalize well to new cases. This suggests that methods based on neural network classification of texture features show promise for potentially decreasing the number of unnecessary biopsies by a significant amount in patients with sonographically identifiable lesions.

Neural network pattern recognition analysis of graft flow characteristics improves intra-operative anastomotic error detection in minimally invasive CABG.

OBJECTIVE: The intra-operative assessment of the quality of anastomosis in minimally invasive coronary artery bypass surgery (CABG) is critical. Recent investigations demonstrated that flow probes used intra-operatively to assess anastomotic errors may give the surgeon a false sense of confidence as only severely stenotic anastomoses (>90%) could be reliably detected. We developed a neural network system using graft flow data and assessed its potential to improve anastomotic error detection. METHODS: Mammary to LAD grafts (n = 46) were constructed in mongrel dogs off-pump. Continuous beat-to-beat graft flow was recorded using transit-time flow probes. Various degrees of anastomotic stenoses (0-100%) were created by an additional suture. The degree of anastomotic stenosis was confirmed by postoperative angiography. A learning vector quantization neural network was created using heart rate, mean aortic pressure, mean systolic, maximum systolic, minimum systolic, mean diastolic, maximum diastolic, minimum diastolic, and mean graft flows. In addition, a spectral analysis of the flow waveforms was performed and the magnitude and phase of the first five harmonics were used to further develop the neural network. RESULTS: The neural network pattern recognition system was 94% accurate in detecting any stenosis >50%. To validate the model, a testing set was used with 20% of the data values, and the accuracy remained at 100% above chance alone. CONCLUSION: Pattern recognition of transit-time flow probe tracings using neural network systems can detect anastomotic errors significantly better than the surgeon's visual assessment, thereby improving the clinical outcome of minimally invasive CABG.

Spectral analysis of graft flow for anastomotic error detection in off-pump CABG.

OBJECTIVE: Flow probes have been introduced as a non-invasive means of anastomotic quality assessment in off-pump coronary artery bypass graft (CABG). Flow waveform morphology cannot reliably be assessed visually unless severe anastomotic stenosis is present ( > 90%). We applied spectral analysis techniques to determine whether the frequency content of graft flow can improve the surgeon's ability to detect anastomotic errors. METHODS: Forty-six mammary to left anterior descending artery (LAD) anastomoses were created in mongrel dogs during off-pump CABG surgery. Graft flow was measured using transit-time flow probes with the LAD closed, and the mammary graft patent and with varying degrees of stenosis. The degree of anastomotic stenosis was created by an artificial stitch and verified by random postoperative angiography. Spectral analysis of the graft flow waveforms was performed. Differences in the magnitude and phase components of the graft flow for the first five harmonics were determined for the varying anastomosis test conditions. Differences were determined using analysis of variance and least square means techniques. RESULTS: The magnitude of the fundamental (zeroth) harmonic was statistically different in the internal mammary artery (IMA) with 0-25% stenosis compared to IMA with 50-75% stenosis (P < 0.01 ). Further, the magnitude of the first, second, and fourth harmonics were statistically different in IMA with 0-25% compared to IMA with 75% (P < 0.01). The phase of the first harmonic was statistically different in IMA with 25% stenosis than IMA with 50% stenosis (P < 0.01 ). No differences in interaction between the LAD and IMA for all ranges of stenosis were detected (P > 0.50). CONCLUSION: Spectral analysis of graft flow waveforms may be beneficial in detecting lesser degrees of anastomotic stenosis (i.e. < 90%) compared to traditional visual assessment of mean graft flow and/or graft flow waveform morphology.

A technique for sequential segmental neuromuscular stimulation with closed loop feedback control.

In dynamic myoplasty, dysfunctional muscle is assisted or replaced with skeletal muscle from a donor site. Electrical stimulation is commonly used to train and animate the skeletal muscle to perform its new task. Due to simultaneous tetanic contractions of the entire myoplasty, muscles are deprived of perfusion and fatigue rapidly, causing long-term problems such as excessive scarring and muscle ischemia. Sequential stimulation contracts part of the muscle while other parts rest, thus significantly improving blood perfusion. However, the muscle still fatigues. In this article, we report a test of the feasibility of using closed-loop control to economize the contractions of the sequentially stimulated myoplasty. A simple stimulation algorithm was developed and tested on a sequentially stimulated neo-sphincter designed from a canine gracilis muscle. Pressure generated in the lumen of the myoplasty neo-sphincter was used as feedback to regulate the stimulation signal via three control parameters, thereby optimizing the performance of the myoplasty. Additionally, we investigated and compared the efficiency of amplitude and frequency modulation techniques. Closed-loop feedback enabled us to maintain target pressures within 10% deviation using amplitude modulation and optimized control parameters (correction frequency = 4 Hz, correction threshold = 4%, and transition time = 0.3 s). The large-scale stimulation/feedback setup was unfit for chronic experimentation, but can be used as a blueprint for a small-scale version to unveil the theoretical benefits of closed-loop control in chronic experimentation.

Cardiovascular responses to propofol and etomidate in long-term instrumented rhesus monkeys (Macaca mulatta).

BACKGROUND AND PURPOSE: Cardiac and arterial responses to prescribed doses of propofol and etomidate in rhesus monkeys were compared. METHODS: Intravenously administered induction doses of propofol (2 mg/kg of body weight) or etomidate (1 mg/kg) followed by continuous intravenous infusions of propofol (200 microg/kg/min) or etomidate (100 microg/kg/min) were administered. Left ventricular and right atrial access catheters were implanted for long-term use, along with a transit-time flow probe on the ascending aorta, and pericardial electrocardiogram leads. A dual sensor 3-F micromanometer was used to measure left ventricular pressure and aortic pressure, and an active redirectional transit-time probe measured aortic flow. Noordergraaf's four-element model was used to estimate total peripheral resistance and systemic arterial compliance. RESULTS: Significant (P < 0.01) decreases in mean arterial pressure, heart rate, and myocardial contractility were accompanied by an increase in systemic arterial compliance associated with propofol and etomidate. Only minimal changes in left ventricular diastolic pressure, cardiac output, stroke volume, and total peripheral resistance were found for both drugs. The changes associated with propofol are comparable to results in human beings, whereas the changes associated with etomidate did not agree with results of published human studies. CONCLUSION: The significant cardiovascular alterations associated with both agents were attributed to reductions in heart rate, although the possibility exists that negative inotropic effects may have had a role.

Electric analog model of the aortic valve for calculation of continuous beat-to-beat aortic flow using a pressure gradient.

The objective was to develop a technique for calculating continuous, beat-to-beat aortic flow (AoF) using only left ventricular pressure (LVP) and aortic pressure (AoP). An electric analog model of the aortic valve was developed that includes resistance (R), inertance (L), and compliance (C) parameters, and resulting second order differential equations were derived. Aortic flow, AoP, and LVP recorded in eight subjects during a 5 day period and during lower body negative pressure (LBNP) were used to validate the model. Resistance, L, and C were estimated using a least-squares fit to the measured AoF on day 0 and during 0 mm Hg LBNP. For days 1-4, AoF was calculated using measured values of AoP and LVP and the R, L, and C values from day 0. Similarly, for LBNP, AoF was calculated using measured values of AoP and LVP, and the R, L, and C values from 0 mm Hg LBNP. The calculated and measured AoF were compared. Differences in cardiac output between the calculated and measured flows were less than 13.1+/-17% across days and under minor altered physiologic conditions (LBNP). Waveform morphology for the calculated AoF also agreed well with the measured AoF. Spectral analysis showed differences in magnitude and phase between measured and calculated aortic flow for the first five harmonics across days, less than 20+/-6% and 25+/-14 degrees, respectively. Preliminary evaluation indicates that our model works well for calculating flow through a biologic valve using LVP and AoP. We speculate that it may perform better for a mechanical valve, and if so it may be possible to develop an instrumented mechanical valve capable of continuous LVP, AOP, and AoF measurements.

Can a linear electrical analog model of a mechanical valve predict flow by using a pressure gradient?

The objective was to determine whether a previously developed technique for biological aortic valves could predict flow through a mechanical valve. An electrical analog model of the aortic valve that includes compliance, resistance, and inertance parameters, and corresponding second order differential equations was used to predict flow given a pressure gradient, as previously reported. Simulated pressures and flow were recorded by using a pulse duplicator system. The heart rate was varied from 60 to 180 bpm, and the stroke volume was varied from 22 to 67 cc. Resistance, inertance, and compliance parameters of the governing differential equation were estimated by using a least-squares fit to the measured flow at 120 bpm and 50 cc stroke volume. By using these parameter estimates, flow was calculated for other heart rates and stroke volumes. To achieve a better flow prediction, a nonlinear filter (third order polynomial range calibration equation) was applied to the output of the linear model (flow). The mean error, full-scale error, and spectral error in magnitude and phase between measured and predicted flow were compared. Error in mean flow ranged from 3% at medium flow rates to 90% at low flow rates. The maximum and minimum full scale errors were 12% and 5%, respectively. Error in the harmonics of measured and calculated flow ranged from 0% to 55%. Larger errors were usually present at the higher harmonics. The agreement between measured and calculated flow was better at normal and high flows but rather poor at low flows. The nonlinear filter (range calibration equation) was unable to account for the discrepancies between the measured and calculated flow over all flow ranges. It seems that this linear model and nonlinear filter have limited application, and an alternate nonlinear approach may produce better results.

Development of a flow feedback pulse duplicator system with rhesus monkey arterial input impedance characteristics.

An in vitro pulsatile pump flow system that is capable of producing physiologic pressures and flows in a mock circulatory system tuned to reproduce the first nine harmonics of the input impedance of a rhesus monkey was developed and tested. The system was created as a research tool for evaluating cardiovascular function and for the design, testing, and evaluation of electrical-mechanical cardiovascular models and chronically implanted sensors. The system possesses a computerized user interface for controlling a linear displacement pulsatile pump in a controlled flow loop format to emulate in vivo cardiovascular characteristics. Evaluation of the pump system consisted of comparing its aortic pressure and flow profiles with in vivo rhesus hemodynamic waveforms in the time and frequency domains. Comparison of aortic pressure and flow data between the pump system and in vivo data showed good agreement in the time and frequency domains, however, the pump system produced a larger pulse pressure. The pump system can be used for comparing cardiovascular parameters with predicted cardiovascular model values and for evaluating such items as vascular grafts, heart valves, biomaterials, and sensors. This article describes the development and evaluation of this feedback controlled cardiovascular dynamics simulation modeling system.

In-line pressure-flow module for in vitro modelling of haemodynamics and biosensor validation.

An in-line pressure-flow module for in vitro modelling of haemodynamics and biosensor validation has been developed. Studies show that good accuracy can be achieved in the measurement of pressure and of flow, in steady and pulstile flow systems. The model can be used for development, testing and evaluation of cardiovascular-mechanical-electrical anlogue models, cardiovascular prosthetics (i.e. valves, vascular grafts) and pressure and flow biosensors.

Evaluation of dual-tip micromanometers during 21-day implantation in goats.

Investigative research efforts using a cardiovascular model required the determination of central circulatory haemodynamic and arterial system parameters for the evaluation of cardiovascular performance. These calculations required continuous beat-to-beat measurement of pressure within the four chambers of the heart and great vessels. Sensitivity and offset drift, longevity, and sources of error for eight 3F dual-tipped micromanometers were determined during 21 days of implantation in goats. Subjects were instrumented with pairs of chronically implanted fluid-filled access catheters in the left and right ventricles, through which dual-tipped (test) micromanometers were chronically inserted and single-tip (standard) micromanometers were acutely inserted. Acutely inserted sensors were calibrated daily and measured pressures were compared in vivo to the chronically inserted sensors. Comparison of the pre- and post-gain calibration of the chronically inserted sensors showed a mean sensitivity drift of 1.0 +/- 0.4% (99% confidence, n = 9 sensors) and mean offset drift of 5.0 +/- 1.5 mmHg (99% confidence, n = 9 sensors). Potential sources of error for these drifts were identified, and included measurement system inaccuracies, temperature drift, hydrostatic column gradients, and dynamic pressure changes. Based upon these findings, we determined that these micromanometers may be chronically inserted in high-pressure chambers for up to 17 days with an acceptable error, but should be limited to acute (hours) insertions in low-pressure applications.

Markers of B lymphocyte differentiation in the chicken.

A study was made of the ontogeny and tissue distribution of seven antigen systems associated with B lymphocyte development. A panel of seven monoclonal antibodies (MAbs) directed against avian bursal and peripheral B lymphocytes was developed. The spectra of cellular reactivity and size of respective antigens indicate that these MAbs appear to react with determinants of chicken B lymphocytes that had not been previously described. Immunofluorescence analysis of cell surface antigens using this panel indicated that dynamic changes in antigen expression are associated with early ontogeny and maturation of the B cell lineage. The yolk sac contained subpopulations of hematopoietic cells reactive with three MAbs (CB10, CLA3 and Hy86b5) which possibly mark the pre-bursal stem cell population. At or near the time of surface IgM expression in the embryonic bursa, B cells expressed antigens detected by the CB7, CB8, CB9 and CB11. In the juvenile chicken, CB7 reacted with immature bursal lymphocytes, while CB8, CB9 and CB10 reacted with bursal lymphocytes and a subpopulation of peripheral B lymphocytes. The antigens defined by Hy86b5 and CB11 were expressed on mature B lymphocytes but on only a subpopulation of bursal lymphocytes. The MAb CLA3 detected an antigen expressed on all leukocytes but not erythroid cells. Expression of some of these antigens by cells of the myelomonocytic lineages suggests a possible functional or ontogenetic relationship between the B lymphocyte and myeloid lineages.

Effect of aflatoxin on the humoral and cell-mediated immune systems of the chicken.

Chickens fed 2.5 microgram of aflatoxin/g of diet from 2 to 4 weeks of age or from hatching to 4 weeks were deficient in cell-mediated immunity, as measured at 4 weeks of age by the graft-versus-host reaction. Delayed-type hypersensitive skin reactions to tuberculin were also reduced in chickens given dietary aflatoxin from hatching to 7 weeks of age. Humoral immunity, as measured by the ability of 4-week-old chicks to produce natural agglutinins to rabbit red blood cells, was not significantly altered by dietary aflatoxin. A significant decrease in concentrations of serum immunoglobulins (Ig) IgG and IgA, but no IgM, however, did occur in chicks given dietary aflatoxin from hatching to 4 weeks or between 2 and 4 weeks of age. Aflatoxin consumption from 0 to 2 weeks of age produced no marked effect on either cell-mediated or humoral immunity in 4-week-old chicks.

Effect of infectious bursal disease virus on the immunological responsiveness of the chicken.

The immunological response of chickens infected at 28 days of age with infectious bursal disease virus (IBDV) was examined. Cellular immunity was studied by the graft-versus-host reaction and delayed type hypersensitivity skin reactions to tuberculin. Antibody responses to sheep red blood cells (S-RBC's) were tested by a direct hemagglutination test. Circulating levels of immunoglobulins (Ig's) IgM, IgG and IgA were monitored by the radial immunodiffusion test. The results of the graft-versus-host reaction and delayed type hypersensitivity reactions were similar for both the infected and control chicks. IBDV significantly reduced the primary antibody response to S-RBC's, but had no marked effect on the secondary antibody response. Chicks infected with IBDV had normal levels of serum IgM and IgG, but significantly lower levels of IgA when compared to uninfected control birds.