ALBERT

Description: The article addresses old ‘west-east discourses’ and how they continue to develop in the high north, and, not least, in the Norwegian petroleum debate. Adopting a discourse analytical perspective the author shows how environmental safety is used as an argument in favour of Norway producing oil in the Barents Sea at the earliest possible moment. This is only feasible if a connection is made in the public mind between Russia and the environment. These views, it is argued, stem from ideas about Russia that gained currency after the demise of the Soviet Union. While they perhaps have less to do with Russia's petroleum industry and environmental performance today, they nevertheless have a strong impact on how challenges in the high north and Arctic region are perceived. And, perhaps even more importantly, they define freedom of action and available options. In this paper all references to government departments refer to those of Norway.

Description: A study of the morphology of the vortical skeleton behind a flapping NACA0030 wing with a finite aspect ratio of 3, is undertaken. The motivation for this work originates with the proposal that thrust can be efficiently produced by flapping aerofoils. The test condition corresponds to a Strouhal number of 0.35, Reynolds number, based on aerofoil chord, of 600 and an amplitude of flapping, equal to the chord length of the wing. This test condition corresponds to the optimal thrust-producing case in infinite-span flapping wings. This study investigates the effect of wing three-dimensionality on the structure of the wake-flow. This is accomplished here, by quantitatively describing the spatio-temporal variations in the velocity, vorticity and Reynolds stresses for the finite-span-wing case.Preliminary flow visualizations suggest that the presence of wingtip vortices for the three-dimensional-wing case, create a different vortical structure to the two-dimensional-wing case. In the case of a two-dimensional-wing, the flow is characterized by the interaction of leading- and trailing-edge vorticity, resulting in the formation of a clear reverse Kármán vortex street at the selected test condition. In the case of a three-dimensional-wing, the flow exhibits a high degree of complexity and three-dimensionality, particularly in the midspan region. Using phase-averaged particle image velocimetry measurements of the forced oscillatory flow, a quantitative analysis in the plane of symmetry of the flapping aerofoil was undertaken. Using a triple decomposition of the measured velocities, the morphological characteristics of the spanwise vorticity is found to be phase correlated with the aerofoil kinematics. Reynolds stresses in the direction of oscillation are the dominant dissipative mechanism. The mean velocity profiles resemble ajet, indicative of thrust production. Pairs of strong counter-rotating vortices from the leading- and trailing-edge of the aerofoil are shed into the flow at each half-cycle. The large-scale structure of the flow is characterized by constructive merging of spanwise vorticity. The midspan region is populated by cross-sections of interconnected vortex rings.

Description: The effectiveness of international environmental cooperation is a theoretical and methodological challenge that has captured the interest of scholars and students of international relations in the recent years. This article is an evaluation of international environmental cooperation in northwest Russia by applying an approach developed in the Institutional Dimensions of Global Environmental Change (IDGEC) research programme of the International Human Dimension Programme (IHDP). The IDGEC approach emphasises the importance of complex diagnostics of institutional environmental arrangements in terms of performance. The approach relates to the effects of international environmental cooperation in terms of sustainability, efficiency, fairness, and robustness. The article focuses on Russian and Nordic experiences of international environmental cooperation, and the assessment of performance has been done by the participants themselves. According to the results of interviews and questionnaires, the cooperation is considered institutionally effective and robust in general. However, views about the environmental effects vary. Some problems exist that are related to sustainability and fairness, but the most critical issue is the lack of domestic environmental capacity in Russia.

Description: In this study, we performed simulations of turbulent flow over rectangular ribs transversely mounted on one side of a plane in a channel, with the other side being smooth. The separation between ribs is large enough to avoid forming stable vortices in the spacing, which exhibitsk-type, or sand-grain roughness. The Reynolds numberReτof our representative direct numerical simulation case is 460 based on the smooth-wall friction velocity and the channel half-width. The roughness heighthis estimated as 110 wall units based on the rough-wall friction velocity. The velocity profile and kinetic energy budget verify the presence of an equilibrium, logarithmic layer aty≳2h. In the roughness sublayer, however, a significant turbulent energy flux was observed. A high-energy region is formed by the irregular motions just above the roughness. Visualizations of vortical streaks, disrupted in all three directions in the roughness sublayer, indicate that the three-dimensional flow structure of sand-grain roughness is replicated by the two-dimensional roughness, and that this vortical structure is responsible for the high energy production. The difference in turbulence structure between smooth- and rough-wall layers can also be seen in other flow properties, such as anisotropy and turbulence length scales.

Description: A model for simulating the process of growth, collapse and rebound of a cavitation bubble travelling along the flow through a convergent–divergent nozzle producing a cavitating water jet is established. The model is based on the Rayleigh–Plesset bubble dynamics equation using as inputs ambient pressure and velocity profiles calculated with the aid of computational fluid dynamics (CFD) flow modelling. A variable time-step technique is applied to solve the highly nonlinear second-order differential equation. This technique successfully solves the Rayleigh–Plesset equation for wide ranges of pressure variation and bubble original size and saves considerable computing time. Inputs for this model are the pressure and velocity data from CFD calculation. To simulate accurately the process of bubble growth, collapse and rebound, a heat transfer model, which includes the effects of conduction plus radiation, is developed to describe the thermodynamics of the incondensable gas inside the bubble. This heat transfer model matches previously published experimental data well. Assuming that single bubble behaviour also applies to bubble clouds, the calculated distance from the nozzle exit travelled by the bubble to the point where the bubble size becomes invisible is taken to be equal to the bubble cloud length observed. The predictions are compared with experiments carried out in a cavitation cell and show good agreement for different nozzles operating at different pressure conditions.

Description: An experimental study, investigating the mean flow and turbulence in the wind drift layer formed beneath short wind waves was conducted. The degree to which these flows resemble the flows that occur in boundary layers adjacent to solid walls (i.e. wall-layers) was examined. Simultaneous DPIV (digital particle image velocimetry) and infrared imagery were used to investigate these near-surface flows at a fetch of 5.5 m and wind speeds from 4.5 to 11 m s−1. These conditions produced short steep waves with dominant wavelengths from 6 cm to 18 cm. The mean velocity profiles in the wind drift layer were found to be logarithmic and the flow was hydrodynamically smooth at all wind speeds. The rate of dissipation of turbulent kinetic energy was determined to be significantly greater in magnitude than would occur in a comparable wall-layer. Microscale breaking waves were detected using the DPIV data and the characteristics of breaking and non-breaking waves were compared. The percentage of microscale breaking waves increased abruptly from 11% to 80% as the wind speed increased from 4.5 to 7.4 m s−and then gradually increased to 90% as the wind speed increased to 11 m s−. At a depth of 1 mm, the rate of dissipation was 1.7 to 3.2 times greater beneath microscale breaking waves compared to non-breaking waves. In the crest–trough region beneath microscale breaking waves, 40% to 50% of the dissipation was associated with wave breaking. These results demonstrated that the enhanced near-surface turbulence in the wind drift layer was the result of microscale wave breaking. It was determined that the rate of dissipation of turbulent kinetic energy due to wave breaking is a function of depth, friction velocity, wave height and phase speed as proposed by Terrayet al. (1996). Vertical profiles of the rate of dissipation showed that beneath microscale breaking waves there were two distinct layers. Immediately beneath the surface, the dissipation decayed as ζ−0.7and below this in the second layer it decayed as ζ−2. The enhanced turbulence associated with microscale wave breaking was found to extend to a depth of approximately one significant wave height. The only similarity between the flows in these wind drift layers and wall-layers is that in both cases the mean velocity profiles are logarithmic. The fact that microscale breaking waves were responsible for 40%–50% of the near-surface turbulence supports the premise that microscale breaking waves play a significant role in enhancing the transfer of gas and heat across the air–sea interface.

Description: Results are presented from experimental investigations into the motion of a heavy ellipsoid in a horizontal rotating cylinder, which has been completely filled with highly viscous fluid. The motion can be conveniently classified using the ratio between the maximum radius of curvature of the ellipsoid κmaxand the radius of the drumRd. If κmax

Description: Electric conduction from an electrolyte solution into a charge selective solid, such as ion exchange membrane or electrode, becomes unstable when the electrolyte concentration near the interface approaches zero owing to diffusion limitation. The sequence of events leading to instability is as follows: upon the decrease of the interface concentration, the electric double layer at the interface transforms from its common quasi-equilibrium structure to a different, non-equilibrium one. The key feature of this new structure is an extended space charge added to the usual one of the quasi-equilibrium electric double layer. The non-equilibrium electro-osmotic slip related to this extended space charge renders the quiescent conductance unstable. A unified asymptotic picture of the electric double-layer undercurrent, encompassing all regimes from quasi-equilibrium to the extreme non-equilibrium one, is developed and employed for derivation of a universal electro-osmotic slip formula. This formula is used for a linear stability study of quiescent electric conduction, yielding the precise parameter range of instability, compared with that in the full electroconvective formulation. The physical mechanism of instability is traced both kinematically, in terms of non-equilibrium electro-osmotic slip, and dynamically, in terms of forces acting in the electric double layer.

Description: The water-shipping problem is modelled in a two-dimensional framework and studied experimentally and numerically for the case of a fixed barge-shaped structure. The analysis represents the second step of the research discussed in Grecoet al. (J. Fluid Mech., vol. 525, 2005, p. 309). The numerical investigation is performed by using both a boundary element method and a domain-decomposition strategy. The model tests highlight the occurrence of dam-breaking-type water on deck, (a) with and (b) without an initial plunging phase, and (c) an unusual type of water shipping connected with blunt water–deck impacts here called a hammer-fist type event never documented before. Cases (a) and (c) are connected with the most severe events and the related features and green-water loads are discussed in detail. A parametric analysis of water-on-deck phenomena has also been carried out in terms of the local incoming waves and bow flow features. We classify such phenomena in a systematic way to provide a basis for further investigations of water-on-deck events. The severity of (a)-type water-on-deck events is analysed in terms of initial cavity area and water-front velocity along the deck. The former increases as the square power of the modified incoming-wave (front-crest) steepness while the latter scales with its square-root. The two-dimensional investigation gives useful quantitative information in terms of water-front velocity for comparison with three-dimensional water-on-deck experiments on fixed bow models interacting with wave packets.

Description: The irrotational flow past two slender bodies of revolution at angles of yaw, translating in parallel paths in very close proximity, is analysed by extending the classical slender body theory. The flow far away from the two bodies is shown to be a direct problem, which is represented in terms of two line sources along their longitudinal axes, at the strengths of the variation rates of their cross-section areas. The inner flow near the two bodies is reduced to the plane flow problem of the expanding (contracting) and lateral translations of two parallel circular cylinders with different radii, which is then solved analytically using conformal mapping. Consequently, an analytical flow solution has been obtained for two arbitrary slender bodies of revolution at angles of yaw translating in close proximity. The lateral forces and yaw moments acting on the two bodies are obtained in terms of integrals along the body lengths. A comparison is made among the present model for two slender bodies in close proximity, Tuck & Newman's (1974) model for two slender bodies far apart, and VSAERO (AMI)–commercial software based on potential flow theory and the boundary element method (BEM). The attraction force of the present model agrees well with the BEM result, when the clearance,h0, is within 20% of the body length, whereas the attraction force of Tuck & Newman is much smaller than the BEM result whenh0is within 30% of the body length, but approaches the latter whenh0is about half the body length. Numerical simulations are performed for the three typical manoeuvres of two bodies: (i) a body passing a stationary body, (ii) two bodies in a meeting manoeuvre (translating in opposite directions), and (iii) two bodies in a passing manoeuvre (translating in the same direction). The analysis reveals the orders of the lateral forces and yaw moments, as well as their variation trends in terms of the manoeuvre type, velocities, sizes, angles of yaw of the two bodies, and their proximity, etc. These irrotational dynamic features are expected to provide a basic understanding of this problem and will be beneficial to further numerical and experimental studies involving additional physical effects.

Description: The flow associated with a synthetic jet transitioning to turbulence in an otherwise quiescent external flow is examined using time-accurate three-dimensional numerical simulations. The incompressible Navier–Stokes solver uses a second-order accurate scheme for spatial discretization and a second-order semi-implicit fractional step method for time integration. The simulations are designed to model the experiments of C. S. Yaoet al. (Proc. NASA LaRC Workshop, 2004) which have examined, in detail, the external evolution of a transitional synthetic jet in quiescent flow. Although the jet Reynolds and Stokes numbers in the simulations match with the experiment, a number of simplifications have been made in the synthetic jet actuator model adopted in the current simulations. These include a simpler representation of the cavity and slot geometry and diaphragm placement. Despite this, a reasonably good match with the experiments is obtained in the core of the jet and this indicates that for these jets, matching of these key non-dimensional parameters is sufficient to capture the critical features of the external jet flow. The computed results are analysed further to gain insight into the dynamics of the external as well as internal flow. The results indicate that near the jet exit plane, the flow field is dominated by the formation of counter-rotating spanwise vortex pairs that break down owing to the rapid growth of spanwise instabilities and transition to turbulence a short distance from the slot. Detailed analyses of the unsteady characteristics of the flow inside the jet cavity and slot provide insights that to date have not been available from experiments.

Description: A general proof that more energy flows upscale than downscale in two-dimensional turbulence and barotropic quasi-geostrophic (QG) turbulence is given. A proof is also given that in surface QG turbulence, the reverse is true. Though some of these results are known in restricted cases, the proofs given here are pedagogically simpler, require fewer assumptions and apply to both forced and unforced cases.

Description: The local linear stability of forced, stationary long waves produced by topography or potential vorticity (PV) sources is examined using a quasi-geostrophic barotropic model. A multiple scale analysis yields coupled equations for the background stationary wave and low-frequency (LF) disturbance field. Forcing structures for which the LF dynamics are Hamiltonian are shown to yield conservation laws that provide necessary conditions for instability and a constraint on the LF structures that can develop. Explicit knowledge of the forcings that produce the stationary waves is shown to be crucial to predicting a unique LF field. Various topographies or external PV sources can be chosen to produce stationary waves that differ by asymptotically small amounts, yet the LF instabilities that develop can have fundamentally different structures and growth rates. If the stationary wave field is forced solely by topography, LF oscillatory modes always emerge. In contrast, if the stationary wave field is forced solely by PV, two LF structures are possible: oscillatory modes or non-oscillatory envelope modes. The development of the envelope modes within the context of a linear LF theory is novel.An analysis of the complex WKB branch points, which yields an analytical expres-sion for the leading-order eigenfrequency, shows that the streamwise distribution of absolute instability and convective growth is central to understanding and predicting the types of LF structures that develop on the forced stationary wave. The location of the absolute instability region with respect to the stationary wave determines whether oscillatory modes or envelope modes develop. In the absence of absolute instability, eastward propagating wavetrains generated in the far field can amplify via local convective growth in the stationary wave region. If the stationary wave region is streamwise symmetric (asymmetric), the local convective growth results in a local change in wave energy that is transient (permanent).

Description: This paper presents combined theoretical and experimental studies of the two-dimensional piston-like steady-state motions of a fluid in a moonpool formed by two rectangular hulls (e.g. a dual pontoon or catamaran). Vertical harmonic excitation of the partly submerged structure in calm water is assumed. A high-precision analytically oriented linear-potential-flow method, which captures the singular behaviour of the velocity potential at the corner points of the rectangular structure, is developed. The linear steady-state results are compared with new experimental data and show generally satisfactory agreement. The influence of vortex shedding has been evaluated by using the local discrete-vortex method of Graham (1980). It was shown to be small. Thus, the discrepancy between the theory and experiment may be related to the free-surface nonlinearity.

Description: This paper presents a study of the global response of a fluid to impulsive and localized forcing; it has been motivated by the recent laboratory experiments on the locomotion of water-walking insects reported in Hu, Chan & Bush (Nature, vol. 424, 2003, p. 663). These insects create both waves and vortices by their rapid leg strokes and it has been a matter of some debate whether either form of motion predominates in the momentum budget. The main result of this paper is to argue that generically both waves and vortices are significant, and that in linear theory they take up the horizontal momentum with share 1/3 and 2/3, respectively.This generic result, which depends only on the impulsive and localized nature of the forcing, is established using the classical linear impulse theory, with adaptations to weakly compressible flows and flows with a free surface. Additional general comments on experimental techniques for momentum measurement and on the wave emission are given and then the theory is applied in detail to water-walking insects.Owing to its generality, this kind of result and the methods used to derive it should be applicable to a wider range of wave–vortex problems in the biolocomotion of water-walking animals and elsewhere.

Description: The Regenerative Enclosed Life Support Module Simulator (REMS) was designed to simulate the conditions aboard the International Space Station (ISS). This unique terrestrial, encapsulated environment for humans and their associated organisms allowed investigations into the microbial communities within an enclosed habitat system, primarily with respect to diversity, phylogeny and the possible impact on human health. To assess time- and/or condition-dependent changes in microbial diversity within REMS, a total of 27 air samples were collected during three consecutive months. The microbial burden and diversity were elucidated using culture-dependent and culture-independent molecular methods. The results indicate that during controlled conditions the total microbial burden detected by culture-dependent techniques (below a detectable level to 102 cells m−3of air) and intracellular ATP assay was significantly low (102–103 cells m−3of air), but increased during the uncontrolled post-operation phase (∼104 cells m−3of air). Only Gram-positive and α-proteobacteria grew under tested culture conditions, with a predominant occurrence ofMethylobacterium radiotolerans, andSphingomonas yanoikuyae. Direct DNA extraction and 16S rDNA sequencing methodology revealed a broader diversity of microbes present in the REMS air (51 species). Unlike culture-dependent analysis, both Gram-positive and proteobacteria were equally represented, while members of a few proteobaterial groups dominated (Rhodopseudomonas,Sphingomonas,Acidovorax,Ralstonia,Acinetobacter,Pseudomonas, andPsychrobacter). Although the presence of several opportunistic pathogens warrants further investigation, the results demonstrated that routine maintenance such as controlling the humidity, crew’s daily cleaning, and air filtration were effective in reducing the microbial burden in the REMS.

Description: Experimentation and theory are used to study the long-term dynamics of a two-dimensional density current flowing into a two-layer stratified basin. When the initial Richardson number,$hbox{it Ri}_{ ho}^{hbox{scriptsizeit in}}$, characterizing the ratio of the background stratification to the buoyancy flux of the density current, is less than the critical value of$hbox{it Ri}_{ ho}^{*} ,{=}, 21-27$, it is found that the density current penetrates the stratified interface. This result is ostensibly independent of slope for angles between 30° and 90°. If the current does not initially penetrate the interface, then it slowly increases the density of the top layer until the interfacial density difference is reduced sufficiently to drive penetration. The time scale for this to occur,$t_{p} ,{=}, (hbox{it Ri}^{hbox{scriptsizeit in}}_{ ho} - hbox{it Ri}_{ ho}^{*}) L/B^{1/3}$, is explicitly a function of the buoyancy fluxBand the length of the basinL. The initial Richardson number,$hbox{it Ri}^{hbox{scriptsizeit in}}_{ ho}$, is a function of depth, the initial reduced gravity of the interface and a weak function of slope angle. In the absence of initial penetration for very steep slopes of 75° and 90°, we observe that penetrative convection at the interface leads to significant local entrainment. In consequence, the top layer thickens and the interfacial entrainment rate increases as the fifth power of the interfacial Froude number. In contrast, such a process is not observed at comparable interfacial Froude numbers on lower slopes of 30°, 45° and 60°, thereby demonstrating the important role of impact angle on penetrative convection. We attribute the increased interfacial entrainment by the steep density currents as the result of the transition from an undular bore to a turbulent hydraulic jump at the point where the density current intrudes. We discuss the applicability of the observed circulation to the stability of the Arctic halocline where we find$0.56,{lesssim}, t_{p} ,{lesssim},1.2$years for a range of contemporary oceanographic conditions.

Description: Direct numerical simulations (DNS) of stenotic flows under conditions of steady inlet flow were discussed in Part 1 of this study. DNS of pulsatile flow through the 75% stenosed tube (by area) employed for the computations in Part 1 is examined here. Analogous to the steady flow results, DNS predicts a laminar post-stenotic flow field in the case of pulsatile flow through the axisymmetric stenosis model, in contrast to previous experiments, in which intermittent disturbed flow regions and turbulent breakdown were observed in the downstream region. The introduction of a stenosis eccentricity, that was 5% of the main vessel diameter at the throat, resulted in periodic, localized transition to turbulence. Analysis in this study indicates that the early and mid-acceleration phases of the time period cycle were relatively stable, with no turbulent activity in the post-stenotic region. However, towards the end of acceleration, the starting vortex, formed earlier as the fluid accelerated through the stenosis at the beginning of acceleration, started to break up into elongated streamwise structures. These streamwise vortices broke down at peak flow, forming a turbulent spot in the post-stenotic region. In the early part of deceleration there was intense turbulent activity within this spot. Past the mid-deceleration phase, through to minimum flow, the inlet flow lost its momentum and the flow field began to relaminarize. The start of acceleration in the following cycle saw a recurrence of the entire process of a starting structure undergoing turbulent breakdown and subsequent relaminarization of the post-stenotic flow field. Peak wall shear stress (WSS) levels occurred at the stenosis throat, with the rest of the vessel experiencing much lower levels. Turbulent breakdown at peak flow resulted in a sharp amplification of instantaneous WSS magnitudes across the region corresponding to the turbulent spot, accompanied by large axial and circumferential fluctuations, even while ensemble-averaged axial shear stresses remained mostly low and negative. WSS levels dropped rapidly after the mid-deceleration phase, when the relaminarization process took over, and were almost identical to laminar, axisymmetric shear levels through most of the acceleration phase.

Description: We investigate experimentally the force generated by the unsteady vortex formation of low-aspect-ratio normal flat plates with one end free. The objective of this study is to determine the role of the free end, or tip, vortex. Understanding this simple case provides insight into flapping-wing propulsion, which involves the unsteady motion of low-aspect-ratio appendages. As a simple model of a propulsive half-stroke, we consider a rectangular normal flat plate undergoing a translating start-up motion in a towing tank. Digital particle image velocimetry is used to measure multiple perpendicular sections of the flow velocity and vorticity, in order to correlate vortex circulation with the measured plate force. The three-dimensional wake structure is captured using flow visualization. We show that the tip vortex produces a significant maximum in the plate force. Suppressing its formation results in a force minimum. Comparing plates of aspect ratio six and two, the flow is similar in terms of absolute distance from the tip, but evolves faster for aspect ratio two. The plate drag coefficient increases with decreasing aspect ratio.

Description: Using the Boussinesq approximation, a set of depth-integrated wave equations for long-wave propagation over a mud bed is derived. The wave motions above the mud bed are assumed to be irrotational and the mud bed is modelled as a highly viscous fluid. The pressure and velocity are required to be continuous across the water–mud interface. The resulting governing equations are differential–integral equations in terms of the depth-integrated horizontal velocity and the free-surface displacement. The effects of the mud bed appear in the continuity equation in the form of a time integral of weighted divergence of the depth-averaged velocity. Damping rates for periodic waves and solitary waves are calculated. For the solitary wave case, the velocity profiles in the water column and the mud bed at different phases are discussed. The effects of the viscous boundary layer above the mud–water interface are also examined.

Description: During extended deployment at an ocean observatory off the coast of New Jersey, a bottom-mounted five-beam acoustic Doppler current profiler measured large-scale velocity structures that we interpret as Langmuir circulations filling the entire water column. These circulations are the large-eddy structures of wind-wave-driven turbulent flows that occur episodically when a shallow water column experiences prolonged strong wind forcing. Many observational characteristics agree with former descriptions of Langmuir circulations in deep water. The three-dimensional velocity field reveals quasi-organized structures consisting of pairs of surface-intensified counter-rotating vortices, aligned approximately downwind. Maximum downward velocities are stronger than upward velocities, and the downwelling region of each cell, defined as a pair of vortices, is narrower than the upwelling region. Maximum downward vertical velocity occurs at or above mid-depth, and scales approximately with wind speed. The estimated crosswind scale of cells is roughly 3–6 times their vertical scale, set under these conditions by water depth. The long axis of the cells appears to lie at an angle ∼10°–20° to the right of the wind. A major difference from deep-water observations is strong near-bottom intensification of the downwind ‘jets’ found typically centred over downwelling regions. Accessible observational features such as cell morphology and profiles of mean velocities, turbulent velocity variances, and shear stress components are compared with the results of associated large-eddy simulations (reported in Part 2) of shallow water flows driven by surface stress and the Craik–Leibovich vortex forcing generally used to represent generation of Langmuir cells. A particularly sensitive diagnostic for identification of Langmuir circulations as the energy-containing eddies of the turbulent flow is the depth trajectory of invariants of the turbulent stress tensor, plotted in the Lumley ‘triangle’ corresponding to realizable turbulent flows. When Langmuir structures are present in the observations, the Lumley map is distinctly different from that of surface-stress-driven Couette flow, again in agreement with the large-eddy simulations (LES). Unlike the LES, observed velocity fields contain two distinct and significant scales of variability, documented by wavelet analysis of observational records of vertical velocity. Variability with periods of many minutes is that expected from Langmuir cells drifting past the instrument at the slowly time-varying crosswind velocity. Shorter period variability, of the order of 1–2 min, has roughly the observed periodicity of surface wave groups, suggesting a connection with the wave groups themselves and/or the wave breaking associated with them in high wind conditions.

Description: Lagrangian auto- and cross-correlation functions of the rate of strains2, enstrophy ω2, their respective production terms −sijsjkskiand ωiωjsij, and material derivatives, Ds2/Dtand Dω2/Dtare estimated using experimental results obtained through three-dimensional particle tracking velocimetry (three-dimensional-PTV) in homogeneous turbulence atReλ=50. The autocorrelation functions are used to estimate the Lagrangian time scales of different quantities, while the cross-correlation functions are used to clarify some aspects of the interaction mechanisms between vorticity ω and the rate of strain tensorsij, that are responsible for the statistically stationary, in the Eulerian sense, levels of enstrophy and rate of strain in homogeneous turbulent flow. Results show that at the Reynolds number of the experiment these quantities exhibit different time scales, varying from the relatively long time scale of ω2to the relatively shorter time scales ofs2, ωiωjsijand −sijsjkski. Cross-correlation functions suggest that the dynamics of enstrophy and strain, in this flow, is driven by a set of different-time-scale processes that depend on the local magnitudes ofs2and ω2. In particular, there are indications that, in a statistical sense, (i) strain production anticipates enstrophy production in low-strain–low-enstrophy regions (ii) strain production and enstrophy production display high correlation in high-strain–high-enstrophy regions, (iii) vorticity dampening in high-enstrophy regions is associated with weak correlations between −sijsjkskiands2and between −sijsjkskiand Ds2/Dt, in addition to a marked anti-correlation between ωiωjsijand Ds2/Dt. Vorticity dampening in high-enstrophy regions is thus related to the decay ofs2and its production term, −sijsjkski.

Description: Transverse jets arise in many applications, including propulsion, effluent dispersion, oil field flows, and V/STOL aerodynamics. This study seeks a fundamental, mechanistic understanding of the structure and evolution of vorticity in the transverse jet. We develop a high-resolution three-dimensional vortex simulation of the transverse jet at large Reynolds number and consider jet-to-crossflow velocity ratiosrranging from 5 to 10. A new formulation of vorticity-flux boundary conditions accounts for the interaction of channel wall vorticity with the jet flow immediately around the orifice. We demonstrate that the nascent jet shear layer contains not only azimuthal vorticity generated in the jet pipe, but wall-normal and azimuthal perturbations resulting from the jet–crossflow interaction. This formulation also yields analytical expressions for vortex lines in the near field as a function ofr.Transformation of the cylindrical shear layer emanating from the orifice begins with axial elongation of its lee side to form sections of counter-rotating vorticity aligned with the jet trajectory. Periodic roll-up of the shear layer accompanies this deformation, creating complementary vortex arcs on the lee and windward sides of the jet. Counter-rotating vorticity then drives lee-side roll-ups in the windward direction, along the normal to the jet trajectory. Azimuthal vortex arcs of alternating sign thus approach each other on the windward boundary of the jet. Accordingly, initially planar material rings on the shear layer fold completely and assume an interlocking structure that persists for several diameters above the jet exit. Though the near field of the jet is dominated by deformation and periodic roll-up of the shear layer, the resulting counter-rotating vorticity is a pronounced feature of the mean field; in turn, the mean counter-rotation exerts a substantial influence on the deformation of the shear layer. Following the pronounced bending of the trajectory into the crossflow, we observe a sudden breakdown of near-field vortical structures into a dense distribution of smaller scales. Spatial filtering of this region reveals the persistence of counter-rotating streamwise vorticity initiated in the near field.

Description: The decay of a passive scalar in a sinusoidal shear flow translating in the cross-stream direction at a constant speeduis studied in the limit of small diffusivity κ. The decay rate, obtained by solving an eigenvalue problem, is found to tend to a non-zero constant as κ→0 whenuis of order κ1/2. This result, establishing that fast decay is possible in shear flows, is fragile however: because of the existence of pseudomodes, the addition of a small noise leads to decay rates that decrease to zero with κ as κ2/5.

Description: Direct numerical simulation is used to study a round turbulent jet in a laminar crossflow. The ratio of bulk jet velocity to free-stream crossflow velocity is 5.7 and the Reynolds number based on the bulk jet velocity and the jet exit diameter is 5000. The mean velocity and turbulent intensities from the simulations are compared to data from the experiments by Su & Mungal (2004) and good agreement is observed. Additional quantities, not available from experiments, are presented. Turbulent kinetic energy budgets are computed for this flow. Examination of the budgets shows that the near field is far from a state of turbulent equilibrium – especially along the jet edges. Also – in the near field – peak kinetic energy production is observed close to the leading edge, while peak dissipation is observed toward the trailing edge of the jet. The results are used to comment upon the difficulty involved in predicting this flow using RANS computations. There exist regions in this flow where the pressure transport term, neglected by some models and poorly modelled by others, is significant. And past the jet exit, the flow is not close to established canonical flows on which most models appear to be based.

Description: The theory of Wagner from 1932 for the normal symmetric impact of a two-dimensional body of small deadrise angle on a half-space of ideal and incompressible liquid is extended to derive the second-order corrections for the locations of the higher-pressure jet-root regions and for the upward force on the impactor using a systematic matched-asymptotic analysis. The second-order predictions for the upward force on an entering wedge and parabola are compared with numerical and experimental data, respectively, and it is concluded that a significant improvement in the predictive capability of Wagner's theory is afforded by proceeding to second order.

Description: SUMMARYThe purpose of the current work was to study phosphorus (P) metabolism in growing sheep supplemented with different levels of dicalcium phosphate using an extant mathematical model. Twelve male non-castrated Santa Inês sheep, weighing 23 (±2·2) kg, received a basal diet unsupplemented or supplemented with dicalcium phosphate to provide 1·5, 3·0, 4·5 g of P/animal per day (treatments T1 to T4, respectively). After 3 weeks adaptation, 7·4 MBq of32P was injected into the jugular vein of each animal. Samples of blood, faeces and urine were collected every day during a 7-day period and thereafter the animals were sacrificed and samples from liver, kidney, heart, muscle and bone were collected for specific activity and inorganic P determinations. The flows between gut and plasma were similar for each treatment except for T1, which showed the lowest values for both flows (P

Description: SUMMARYMedial claws from the right hind feet were obtained post mortem from four 19–20-month-old beef heifers and from four 28-month-old first-calving dairy heifers 3–4 days postpartum. X-ray diffraction (XRD) studies were undertaken on samples of soft and hard (cornified) integumental tissues of dorsal wall, sole and heel (bulb) for varying sites and planes of exposure. The measurements were interpreted as defining diffraction patterns and intermolecular spacings of cytoskeletal and extracellular fibrous structural proteins. The orientation of these proteins was examined in relation to physical characteristics and function including bearing of body weight by these tissues.Physical measurements taken included impression hardness which showed typically greater values for wall than sole and variable differences between horn of dairy and beef origin and husbandry systems. Claws from dairy heifers had significantly smaller values for toe (dorsal wall) angle, claw height and heel height and thickness of solear horn and heel soft tissue. Although few were studied, the results reflected typical husbandry origins and indicated the susceptibility to the lesion formation well recognized in postpartum dairy cattle.Typical XRD patterns for horn samples showed defined arcs of reflectance on the equatorial axis consistent with findings for the presence of α-helices in fibrils reported to occur in other hard-keratin-containing integumental tissues. However, reflectance on the meridional axis also reported for these other tissues was not detected. A similar defined pattern was obtained for less than 0·10 of samples of internal soft pre-cornified epidermal and attached dermal tissue although the values for intermolecular ‘d’ spacing for these were consistent with those reported for type I collagen. Diffuse reflection patterns were thus evident for the majority of samples of soft tissue epidermis and dermis and also for adipose tissue of the digital cushion.The formation of defined arcs of reflectance allowed the determination of fibril alignment in wall and solear horn. For the orientated samples of dorsal wall horn tissue, the outer layer showed a longitudinal angle of orientation essentially maintained proximal to distal. This pattern was maintained throughout the depth of horn at the proximal site. In contrast, layers in mid-wall and towards the distal edge showed a greater circumferential (horizontal) orientation in sections collected anterior to posterior towards the inner corial, including laminar, tissues. The orientation of fibrils in inner wall horn appears to relate to the direction of load-bearing forces in connecting horn to the distal phalanx. Horizontal alignment of fibrils was observed in the sole. In presenting the long axis of cells to the ground surface this orientation may facilitate erosive forces and contribute to the thinning of cornified sole horn under adverse underfoot conditions.

Description: The flapping coupling between two filaments is studied theoretically and experimentally in this paper. A temporal linear instability analysis is carried out based on a simplified hydrodynamic model. The dispersion relationship between the eigen-frequency ω and wavenumberkis expressed by a quartic equation. Two special cases of flapping coupling, i.e. two identical filaments having the same length and two filaments having different lengths, are studied in detail. In the case of two identical filaments, the theoretical analysis predicts four coupling modes, i.e. the stretched-straight mode, the antisymmetrical in-phase mode, the symmetrical out-of-phase mode and the indefinite mode. The theory also predicts the existence of an eigenfrequency jump during transition between the in-phase and out-of-phase modes, which has been observed in previous experiments and numerical simulations. In the case of two filaments having different lengths, four modes similar to those in the former case are identified theoretically. The distribution of coupling modes for both the cases is shown in two planes. One is a dimensionless plane ofSvs.U, whereSis the density ratio of solid filament to fluid andU2is the ratio of fluid kinetic energy to solid elastic potential energy. The other is a dimensional plane of the half-distance (h) between two filaments vs. the filament length (L). Relevant experiments are carried out in a soap-film tunnel and the stable and unstable modes are observed. Theory and experiment are compared in detail. It should be noted that the model used in our analysis is a very simplified one that can provide intuitional analytical results of the coupling modes as well as their qualitative distributions. The factors neglected in our model, such as vortex shedding, viscous and nonlinear effects, do not allow the model to predict results precisely consistent with the experiments. Moreover, the Strouhal numbers of the flapping filaments are found to be generally around a fixed value in the experiments for both cases, implying that the filaments try to maintain a lower potential energy state.

Description: Numerical simulations on a β-plane are used to further understand the formation of zonal flows from small-scale fluctuations. The dynamics of ‘reduced models’ are computed by restricting the nonlinear term to include a subset of triad interactions in Fourier space. Reduced models of near-resonant triads are considered, as well as the complement set of non-resonant triads. At moderately small values of the Rhines number, near-resonant triad interactions are shown to be responsible for the generation of large-scale zonal flows from small-scale random forcing. Without large-scale drag, both the full system and the reduced model of near resonances produce asymmetry between eastward and westward jets, in favour of stronger westward jets. When large-scale drag is included, the long-time asymmetry is reversed in the full system, with eastward jets that are thinner and stronger than westward jets. Then the reduced model of near resonances exhibits a weaker asymmetry, but there are nevertheless more eastward jets stronger than a threshold value.

Description: Free surface potential flows past disturbances in a channel are considered. Three different types of disturbance are studied: (i) a submerged obstacle on the bottom of a channel; (ii) a pressure distribution on the free surface; and (iii) an obstruction in the free surface (e.g. a sluice gate or a flat plate). Surface tension is neglected, but gravity is included in the dynamic boundary condition. Fully nonlinear solutions are computed by boundary integral equation methods. In addition, weakly nonlinear solutions are derived. New solutions are found when several disturbances are present simultaneously. They are discovered through the weakly nonlinear analysis and confirmed by numerical computations for the fully nonlinear problem.

Description: A numerical description of heterogeneous propellant combustion enables us to examine the spatial and temporal fluctuations in the flow field arising from the heterogeneity. Particular focus is placed on the fluctuations in a zone intermediate between the combustion field (where reaction is important) and the chamber flow domain, for these define boundary conditions for simulations of the turbulent chamber flow. The statistics of the temperature field and the normal velocity field are described, and characteristic length scales and time scales are identified. The length scales are small compared to any relevant length scale of the chamber flow, and so the boundary conditions for this flow at any mesh point are statistically independent of those at any other mesh point. But the temporal correlations at a fixed point are significant, and affect the nature of the chamber flow in a variety of ways. We describe the fluctuations in the head-end pressure that arise because of them, and contrast these results with those calculated using a white-noise assumption.

Description: We consider the flapping stability and response of a thin two-dimensional flag of high extensional rigidity and low bending rigidity. The three relevant non-dimensional parameters governing the problem are the structure-to-fluid mass ratio, μ = ρsh/(ρfL); the Reynolds number,Rey=VL/ν; and the non-dimensional bending rigidity,KB=EI/(ρfV2L3). The soft cloth of a flag is represented by very low bending rigidity and the subsequent dominance of flow-induced tension as the main structural restoring force. We first perform linear analysis to help understand the relevant mechanisms of the problem and guide the computational investigation. To study the nonlinear stability and response, we develop a fluid–structure direct simulation (FSDS) capability, coupling a direct numerical simulation of the Navier–Stokes equations to a solver for thin-membrane dynamics of arbitrarily large motion. With the flow grid fitted to the structural boundary, external forcing to the structure is calculated from the boundary fluid dynamics. Using a systematic series of FSDS runs, we pursue a detailed analysis of the response as a function of mass ratio for the case of very low bending rigidity (KB= 10−4) and relatively high Reynolds number (Rey= 103). We discover three distinct regimes of response as a function of mass ratio μ: (I) a small μ regime of fixed-point stability; (II) an intermediate μ regime of period-one limit-cycle flapping with amplitude increasing with increasing μ; and (III) a large μ regime of chaotic flapping. Parametric stability dependencies predicted by the linear analysis are confirmed by the nonlinear FSDS, and hysteresis in stability is explained with a nonlinear softening spring model. The chaotic flapping response shows up as a breaking of the limit cycle by inclusion of the 3/2 superharmonic. This occurs as the increased flapping amplitude yields a flapping Strouhal number (St= 2Af/V) in the neighbourhood of the natural vortex wake Strouhal number,St≃ 0.2. The limit-cycle von Kármán vortex wake transitions in chaos to a wake with clusters of higher intensity vortices. For the largest mass ratios, strong vortex pairs are distributed away from the wake centreline during intermittent violent snapping events, characterized by rapid changes in tension and dynamic buckling.

Description: The governing equations for the two-point correlations of the turbulent fluctuating velocity in the temporally evolving wake were analysed to determine whether they could have equilibrium similarity solutions. It was found that these equations could have such solutions for a finite-Reynolds-number wake, where the two-point velocity correlations could be written as a product of a time-dependent scale and a function dependent only on similarity variables. It is therefore possible to collapse the two-point measures of all the scales of motions in the temporally evolving wake using a single set of similarity variables. As in an earlier single-point analysis, it was found that the governing equations for the equilibrium similarity solutions could not be reduced to a form that was independent of a growth-rate dependent parameter. Thus, there is not a single ‘universal’ solution that describes the state of the large-scale structures, so that the large-scale structures in the far field may depend on how the flow is generated.The predictions of the similarity analysis were compared to the data from two direct numerical simulations of the temporally evolving wakes examined previously. It was found that the two-point velocity spectra of these temporally evolving wakes collapsed reasonably well over the entire range of scales when they were scaled in the manner deduced from the equilibrium similarity analysis. Thus, actual flows do seem to evolve in a manner consistent with the equilibrium similarity solutions.

Description: This paper examines the consistency of the exact scaling laws for isotropic magnetohydrodynamic (MHD) turbulence in numerical simulations with large magnetic Prandtl numbersPmand withPm= 1. The exact laws are used to elucidate the structure of the magnetic and velocity fields. Despite the linear scaling of certain third-order correlation functions, the situation is not analogous to the case of Kolmogorov turbulence. The magnetic field is adequately described by a model of a stripy (folded) field with direction reversals at the resistive scale. At currently available resolutions, the cascade of kinetic energy is short-circuited by the direct exchange of energy between the forcing-scale motions and the stripy magnetic fields. This non-local interaction is the defining feature of isotropic MHD turbulence.

Description: SUMMARYThe development of the genetic control of nematode resistance in growing lambs is of biological interest, as well as being important in terms of designing practical strategies to breed for increased nematode resistance. The current paper demonstrates the use of random regression techniques for quantifying the development of the heritability of faecal egg count (Fec), the indicator of nematode resistance, in growing lambs and predicted inter-age genetic and phenotypic correlations for Fec. Fec data from 732 lambs, collected at 4-week intervals fromc. 8–24 weeks of age, were analysed using random regression techniques. Random effects fitted in the model included genetic, individual animal environmental, litter and residual random effects. Output (co)variance components were interpolated to weekly time points. Individual variance components showed complex patterns of change over time; however, the estimated heritability increased smoothly with age, from 0·10 to 0·38, and showed more stable time trends than were obtained from univariate analyses of Fec at individual time points. Inter-age correlations decreased as the time interval between measurements increased. Genetic correlations were always positive, with 0·6 of all possible inter-age correlations being greater than 0·80. Phenotypic correlations were lower, and decreased more quickly as the time interval between measurements increased. The results presented confirm biological understanding of the development of immunity to nematode infections in growing lambs. Additionally, they provide a tool to determine optimal sampling ages when assessing lambs' relative resistance to nematode infections.

Description: SUMMARYSince many soils used for growing potatoes in the UK are likely to be close to their plastic limit for cultivation during early spring, there exists the potential for soil compaction to occur during planting which will restrict root penetration. A series of experiments showed that soil compaction delayed emergence, reduced rate of leaf appearance and ground cover expansion, shortened canopy cover duration and restricted light interception, which combined to reduce tuber yield. Rooting density and maximum depth of rooting were reduced, particularly where compaction was shallow. In some soils, irrigation helped alleviate some of the effects of compaction but in others it exacerbated their severity. Using a cone penetrometer, relationships between rate of root penetration and soil resistance (Ω) were established from a number of experiments and replicated blocks in commercial fields and an overall relationship of the formy=16·3–4·08Ω mm/day was produced. Root penetration rates ofc. 20 mm/day were measured in the intensively-cultivated ridge zone but growth rates were halved at a Ω of 1·5 MPa. A survey of 602 commercial fields showed that two thirds of fields had Ωs ⩾3 MPa (where root growth rates would be

Description: The hypothesis of the explosion of a number of planets and moons of our Solar System during its 4.6-billion-year history is in excellent accord with all known observational constraints, even without adjustable parameters orad hochelper hypotheses. Many of its boldest predictions have been fulfilled. In most instances, these predictions were judged highly unlikely by the current standard models. Moreover, in several cases, the entire exploded planet model was at risk of being falsified if the predictions failed. The successful predictions include: (1) satellites of asteroids; (2) satellites of comets; (3) salt water in meteorites; (4) ‘roll marks’ leading to boulders on asteroids; (5) the time and peak rate of the 1999 Leonid meteor storm; (6) explosion signatures for asteroids; (7) the strongly spiked energy parameter for new comets; (8) the distribution of black material on slowly rotating airless bodies; (9) splitting velocities of comets; (10) the asteroid-like nature ofDeep Impacttarget Comet Tempel 1; and (11) the presence of high-formation-temperature minerals in theStardustcomet dust sample return. In physics and astronomy, hypotheses are either falsified if their predictions fail, or proved to be of value if they succeed. By all existing evidence, the exploded planet hypothesis has proved far more useful than the half-dozen or so hypotheses it would replace. Among the many important corollaries are these. (a) Perhaps as many as six former planets of our Solar System have exploded over its 4.6-billion-year history. (b) In particular, Mars is not an original planet, but a former moon of an exploded planet. (c) As a major player in Solar System evolution, the exploded planet scenario must be considered as a likely propagation vehicle for the spread of biogenic organisms. We conclude with a brief mention of three possible planetary explosion mechanisms.

Description: The degree and nature of association between trace metals (Cu, Pb, Zn, Ni, Ag, & Cd) and cyanobacterial mats, phytoplankton and sediments has been assessed in the Lake Vanda region of the Wright Valley, Victoria Land. Trace metal:Fe ratios and SEM imaging confirmed that apparent trace metal enrichment in cyanobacterial mats, relative to the sediment beneath, was due to incorporation of fine (sub-micron) sediment particles in the muciligenous matrix of the mat. In suspended particulate material (SPM) filtered from the oxic water of Lake Vanda and the Onyx River, the degree of trace metal binding to the SPM did not appear to correlate with phytoplankton content. Instead a positive correlation was observed between Fe and trace metal content. The SPM at the top of the lake water column, where only the finest sediment remains suspended, had the highest trace metal concentrations. It is concluded that the trace metal content of cyanobacterial mats and phytoplankton samples is primarily due to incorporation of fine sediment particles of high surface area which therefore enhance trace metal adsorption capacity. This reinforces the existing hypothesis that trace metal solubility in this environment is primarily controlled by abiotic processes.

Description: The statistical properties of fully developed planar turbulent Couette–Poiseuille flow result from the simultaneous imposition of a mean wall shear force together with a mean pressure force. Despite the fact that pure Poiseuille flow and pure Couette flow are the two extremes of Couette–Poiseuille flow, the statistical properties of the latter have proved resistant to scaling approaches that coherently extend traditional wall flow theory. For this reason, Couette–Poiseuille flow constitutes an interesting test case by which to explore the efficacy of alternative theoretical approaches, along with their physical/mathematical ramifications. Within this context, the present effort extends the recently developed scaling framework of Weiet al. (2005a) and associated multiscaling ideas of Fifeet al. (2005a,b) to fully developed planar turbulent Couette–Poiseuille flow. Like Poiseuille flow, and contrary to the structure hypothesized by the traditional inner/outer/overlap-based framework, with increasing distance from the wall, the present flow is shown in some cases to undergo abalance breakingandbalance exchangeprocess as the mean dynamics transition from a layer characterized by a balance between the Reynolds stress gradient and viscous stress gradient, to a layer characterized by a balance between the Reynolds stress gradient (more precisely, the sum of Reynolds and viscous stress gradients) and mean pressure gradient. Multiscale analyses of the mean momentum equation are used to predict (in order of magnitude) the wall-normal positions of the maxima of the Reynolds shear stress, as well as to provide an explicit mesoscaling for the profiles near those positions. The analysis reveals a close relationship between the mean flow structure of Couette–Poiseuille flow and two internal scale hierarchies admitted by the mean flow equations. The averaged profiles of interest have, at essentially each point in the channel, a characteristic length that increases as a well-defined ‘outer region’ is approached from either the bottom or the top of the channel. The continuous deformation of this scaling structure as the relevant parameter varies from the Poiseuille case to the Couette case is studied and clarified.

Description: In this paper, we use numerical simulation and laboratory experimental observation to show that fire whirls can be generated spontaneously through the interaction between a central flame and surrounding organized or randomly distributed flames. The momentum of the air stream entrained by the main flame decreases as it crosses a surrounding flame, so that the main flame rotates if surrounding flames are arranged in such a way as to block the passage of the air stream directed towards the centre of the main flame and to favour flows in a particular circumferential direction. An analysis is performed to study the role of the rotation speed in the flame height. It is found that the flame height initially decreases to a minimum owing to the inflow boundary layer wind reducing the initial vertical velocity of gas for low rotation speed and to entrainment enhancement reducing the rising time, and then it increases owing to the pressure reduction at the centre of the rotating vortex and entrainment suppression extending the rising time.

Description: We investigate aspects of hovering insect flight by finding the optimal wing kinematics which minimize power consumption while still providing enough lift to maintain a time-averaged constant altitude over one flapping period. In particular, we study the flight of three insects whose masses vary by approximately three orders of magnitude: fruitfly (Drosophila melanogaster), bumblebee (Bombus terrestris), and hawkmoth (Manduca sexta). Here, we model an insect wing as a rigid body with three rotational degrees of freedom. The aerodynamic forces are modelled via a quasi-steady model of a thin plate interacting with the surrounding fluid. The advantage of this model, as opposed to the more computationally costly method of direct numerical simulation via computational fluid dynamics, is that it allows us to perform optimization procedures and detailed sensitivity analyses which require many cost function evaluations. The optimal solutions are found via a hybrid optimization algorithm combining aspects of a genetic algorithm and a gradient-based optimizer. We find that the results of this optimization yield kinematics which are qualitatively and quantitatively similar to previously observed data. We also perform sensitivity analyses on parameters of the optimal kinematics to gain insight into the values of the observed optima. Additionally, we find that all of the optimal kinematics found here maintain the same leading edge throughout the stroke, as is the case for nearly all insect wing motions. We show that this type of stroke takes advantage of a passive wing rotation in which aerodynamic forces help to reverse the wing pitch, similar to the turning of a free-falling leaf.

Description: Direct numerical simulations (DNS) of steady and pulsatile flow through 75% (by area reduction) stenosed tubes have been performed, with the motivation of understanding the biofluid dynamics of actual stenosed arteries. The spectral-element method, providing geometric flexibility and high-order spectral accuracy, was employed for the simulations. The steady flow results are examined here while the pulsatile flow analysis is dealt with in Part 2 of this study. At inlet Reynolds numbers of 500 and 1000, DNS predict a laminar flow field downstream of an axisymmetric stenosis and comparison to previous experiments show good agreement in the immediate post-stenotic region. The introduction of a geometric perturbation within the current model, in the form of a stenosis eccentricity that was 5% of the main vessel diameter at the throat, resulted in breaking of the symmetry of the post-stenotic flow field by causing the jet to deflect towards the side of the eccentricity and, at a high enough Reynolds number of 1000, jet breakdown occurred in the downstream region. The flow transitioned to turbulence about five diameters away from the stenosis, with velocity spectra taking on a broadband nature, acquiring a -5/3 slope that is typical of turbulent flows. Transition was accomplished by the breaking up of streamwise, hairpin vortices into a localized turbulent spot, reminiscent of the turbulent puff observed in pipe flow transition, within which r.m.s. velocity and turbulent energy levels were highest. Turbulent fluctuations and energy levels rapidly decayed beyond this region and flow relaminarized. The acceleration of the fluid through the stenosis resulted in wall shear stress (WSS) magnitudes that exceeded upstream levels by more than a factor of 30 but low WSS levels accompanied the flow separation zones that formed immediately downstream of the stenosis. Transition to turbulence in the case of the eccentric stenosis was found to be manifested as large temporal and spatial gradients of shear stress, with significant axial and circumferential variations in instantaneous WSS.

Description: Panspermia, the dissemination of life through space, would require resistance to the conditions found in space, including UV light. All known life forms depend on DNA to store information. In an effort to understand the liabilities of DNA to UV light and modes of DNA protection in terrestrial life forms, we established UV–VUV (125–340 nm) absorption spectra for dry DNA and its polymerized components and mononucleotides, as well as for a selection of potential UV screens ubiquitous in all organisms, including proteins, selected amino acids and amines (polyamines and tyramine). Montmorillonite clay was included as a potential abiotic UV screen. Among the potential screens tested, adenosine triphosphate (ATP) appeared to be particularly attractive, because its UV absorption spectrum was similar to that of DNA. We suggest that the use of ATP in UV protection could have pre-dated its current role in energy transfer. Spectroscopy also showed that UV absorption varied according to nucleotide content, suggesting that base pair usage could be a factor in adaptation to given UV environments and the availability of UV screens.

Description: About 5400 cal yr BP, a large landslide formed a 〉 400-m-tall dam in the upper Marsyandi River, central Nepal. The resulting lacustrine and deltaic deposits stretched 〉 7 km upstream, reaching a thickness of 120 m.14C dating of 7 wood fragments reveals that the aggradation and subsequent incision occurred remarkably quickly (∼ 500 yr). Reconstructed volumes of lacustrine (∼ 0.16 km3) and deltaic (∼ 0.09 km3) deposits indicate a bedload-to-suspended load ratio of 1:2, considerably higher than the ≤ 1:10 that is commonly assumed. At the downstream end of the landslide dam, the river incised a new channel through ≥ 70 m of Greater Himalayan gneiss, requiring a minimum bedrock incision rate of 13 mm/yr over last 5400 yr. The majority of incision presumably occurred over a fraction of this time, suggesting much higher rates. The high bedload ratio from such an energetic mountain river is a particularly significant addition to our knowledge of sediment flux in orogenic environments.

Description: Cases of acute leukemia showing both terminal deoxynucleotidyl transferase (TdT) and myeloperoxidase (MPO) activities are usually classified as acute myelogenous leukemia (AML). Although some reports have implied overlap of TdT and MPO based on population percentages, direct evidence for simultaneous expression of TdT and MPO by a leukemic blast is lacking. By use of a simple new technique developed in our laboratory for identifying TdT and MPO in individual cells by light microscopy, we examined three cases of acute leukemia with both TdT and MPO positivity and found that the incidence of cells positive for both TdT and MPO was 0%, 1%, and 23%. Cytogenetic analysis showed a single leukemic clone in all patients, providing additional evidence that these leukemias arose from a single cell capable of expressing both MPO and TdT. These findings have implications for understanding the relation between MPO and TdT expression in leukemia.

Description: Biosynthesis and molecular structure of major histocompatibility complex (MHC) class II antigens of DR2/DR7 hairy cells were analyzed by two-dimensional polyacrylamide-gel electrophoresis (2D-PAGE). Two anti- human Ia monoclonal antibodies (mAb) were used to immunoprecipitate DR and DR-linked DC/DS molecules. Monoclonal antibody VI 15 C recognizes DR (I-E-like) molecules and CA 2.06 precipitates DR and DR-linked DC/DS (I-A-like) molecules in DR7 allotypes. Studies were performed on a pure population of hairy cells before and after culture with phorbol ester: 12-O-tetradecanoyl phorbol-13-acetate (TPA), 5 azacytidine (5 Aza), sodium butyrate (NA-BU), and phytohemagglutinin (PHA-P). Before any treatment, hairy cells expressed and synthesized DR antigens: DR alpha and beta subunits appeared both qualitatively and quantitatively normal by 2D-PAGE profile. In contrast, the hairy cells failed to express and synthesized any DC/DS molecule. The lack of DC/DS molecular expression was restored after culture in presence of TPA, sodium butyrate, and 5 azacytidine, but not after PHA-P treatment. Differential molecular expression of MHC class II antigens in leukemic cells provides a model to define further discrete stages of hemopoietic differentiation and study the role of these molecules in the cellular interactions occurring during differentiation.

Description: The activity of delta-aminolevulinic acid (ALA) dehydratase, an enzyme involved in heme biosynthesis, has been shown to increase in Friend virus-transformed murine erythroleukemia (MEL) cells during erythroid differentiation. In this study, the nature of the increase in ALA dehydratase activity in MEL cells was examined using a monospecific antibody directed to the enzyme. A sevenfold increase in ALA dehydratase activity was observed after cells had been treated with 1.5% Me2SO for 5 days. Ouchterlony double immunodiffusion analysis showed that lysates from untreated and Me2SO-treated MEL cells formed a single precipitin line with rabbit IgG directed to the normal mouse liver ALA dehydratase. A single arc of identity was also observed with the lysates from normal mouse erythrocytes, spleen, liver, and lysates from both uninduced and induced MEL cells. Rocket immunoelectrophoresis demonstrated that lysates from both uninduced and induced cells formed rockets with the IgG and that the peak height of the rocket was proportional to the ALA dehydratase activity applied. The slope of linear plots of rocket peak heights v ALA dehydratase activity was identical for lysates from uninduced and Me2SO-induced cells. Succinylacetone, a potent inhibitor of ALA dehydratase, was shown to markedly inhibit the activity of the enzyme, but did not interfere with the synthesis of ALA dehydratase induced by Me2SO treatment. Me2SO- induced increases in ALA dehydratase activity and the enzyme protein were both blocked by the simultaneous treatment of cells with 5-bromo- 2′-deoxyuridine (BrdU). BrdU-mediated repression of ALA dehydratase was partially overcome by treating the cells with thymidine. These data demonstrate that increased ALA dehydratase activity in MEL cells undergoing erythroid differentiation after Me2SO treatment is due to de novo synthesis of the same enzyme protein present in uninduced MEL cells as well as in normal erythrocytes. This represents the first direct demonstration of an increase in a heme biosynthetic pathway enzyme protein in erythroid cells undergoing differentiation.

Description: Hebbel and colleagues have proposed that increased adherence of sickle red cells to vascular endothelium may initiate vasoocclusive events in sickle cell disease. We have developed a micropipette technique to obtain direct, quantitative measure of the adherence of individual red cells to vascular endothelial cells. Using this technique, we found that the vast majority of sickle cells suspended in autologous plasma were strongly adherent to endothelial cells, whereas only a small fraction of normal cells were weakly adherent. Influence of plasma factors on adherence was determined by measuring adherence of sickle cells suspended in normal plasma and normal cells suspended in sickle plasma. Although over 90% of sickle cells adhered to endothelial cells in autologous plasma, the percentage of adherent cells decreased dramatically to less than 20% when the same sickle cells were suspended in normal plasma. In contrast, adhesion of normal red cells suspended in sickle plasma was only modestly increased compared to adhesion in autologous normal plasma. Our results provide direct evidence for markedly enhanced adherence of sickle cells to endothelial cells. In addition, they suggest that both cell membrane changes and plasma factors contribute to this interaction. The requirement for sickle plasma further implies that temporal changes in plasma factors may play an important role in determining the onset of vasoocclusive crisis.

Description: The effect of splenectomy on the response to random donor platelet transfusion in 15 multitransfused thrombocytopenic patients is presented. Eight patients responded poorly, with low corrected platelet count increments at 1 and 24 hours posttransfusion. These eight patients were clinically alloimmunized and had lymphocytotoxic antibody ( LCTAb ) in their sera. They responded well to closely HLA-matched transfusions. In contrast, seven splenectomized patients responded well to random donor platelets. Five of these patients had no LCTAb and no other evidence of immunization. Two patients who responded well to random donor platelets had “weak” LCTAb , and one responded to platelets presplenectomy in the presence of this antibody. Splenectomy does not improve the response to random donor platelets in alloimmunized recipients.

Description: Prenatal diagnosis of hematologic diseases can now be performed with fetal blood, fetal amniotic fluid cell DNA, and fetal chorionic villi DNA. Some hemoglobinopathies can be detected by all three methods, and the choice will depend on the available obstetric and laboratory techniques, as well as the time of presentation of the pregnancy. Hopefully, further development of molecular probes and techniques will soon expand these options to all of the globin disorders. Detection of coagulation disorders in utero currently requires samples of pure fetal blood. Gene cloning is accomplished for some (factor IX and antithrombin III) and is underway for others (factor VIII), and further investigation is necessary to determine whether deficiencies in these gene products are due to gene deletion or to mutant genes linked to polymorphic restriction enzyme sites of diagnostic use. Thus, molecular biology may be applied to prenatal diagnosis of the clotting problems, but this has not yet been accomplished. Disorders affecting the number and/or function of erythrocytes, leukocytes, and platelets can be diagnosed by analysis of fetal blood. Blood samples will continue to be required until more is known about the molecular biology of hematopoiesis. Syndromes that can be diagnosed by chromosome studies should be revealed in cultures of amniotic fluid cells, fetal blood lymphocytes, and chorionic villi cells. Cultured cells can be examined for karyotypes, Y-chromatin, spontaneous or induced chromosome breakage, DNA repair, SCEs, and translocations. The techniques for culturing amniotic cells and fetal blood white cells are established, and those for growing cells from chorionic villi are improving rapidly. Direct preparations of cells from villi only may suffice for some of the above analyses. The study of hematologic disease in utero has thus come full circle, from the use of amniotic cells to determine the sex in X-linked disorders, to fetal blood sampling for the analysis of gene products, then back to amniocentesis for DNA, and now earlier in gestation to chorionic villi. All of this has occurred in less than ten years, and it is anticipated that developments in the next ten years will be equally dramatic. The future should bring all prenatal testing into the first trimester, use molecular probes, and provide for both early diagnosis and early treatment of genetic hematologic disease.

Description: Methotrexate has been used as the mainstay therapy to prevent or ameliorate graft-versus-host disease (GVHD) in allogeneic bone marrow transplantation. We began a nonrandomized study in which methotrexate was not given routinely. Fifty-five patients underwent transplant for acute leukemia (44 patients), aplastic anemia (6 patients), and other malignancies (5 patients). Methotrexate was given to 34 patients (MTX +) and was withheld in 21 patients (MTX -). Median (range) age of patients was 12 (0.8–43) years in the MTX + group, and 16 (3–45) years in the MTX- group. Mean days (+/- SEM) to engraftment (neutrophils greater than 500/microL, and platelets greater than 20,000/microL untransfused) occurred earlier in the MTX- patients (19.6 +/- 1.4 v 24.9 +/- 1.8 days for granulocytes, and 19.3 +/- 1.5 v 27.4 +/- 2.8 days for platelets, P less than .05). There were no statistically significant differences between the patient groups for the incidence or severity of GVHD (10/34 in the MTX + group had grade O-l GVHD compared to 9/21 in the MTX- group). The interstitial pneumonitis occurred at a significantly increased rate in patients who received methotrexate (15/34) compared to those patients who did not (3/21) (P = .02). However, there was also a significant relationship between the interstitial pneumonitis and the preparative regimen: if the preparative regimen contained 1,000 rad single fraction total body irradiation, 8/14 patients were affected compared to 5/22 patients affected when 1,200 rad fractionated total body irradiation was used (P = .03). Because methotrexate significantly retards hematopoietic reconstitution, randomized trials for GVHD prevention are recommended.

Description: Acute graft-versus-host disease (GVHD), a major complication of allogeneic bone marrow transplantation (BMT), is probably mediated by T lymphocytes present in the marrow graft. In this study, the repopulation of the peripheral blood with T4+ and T8+ T cells was investigated during the period preceding the occurrence of acute GVHD. Twenty-four allogeneic and 11 autologous BMT recipients were monitored from day 4 post-BMT onward by the use of monoclonal antibodies, indirect immunofluorescence, and flow cytometry. The recipients of allogeneic transplants received methotrexate as GVHD prophylaxis. Similar recovery patterns for T4+ and T8+ T cells were found following autologous and allogeneic BMT. However, lymphoid repopulation occurred at a clearly faster rate after autologous BMT. T4+ T cells were the first to reappear in the peripheral blood, followed by T8+ T cells 4–7 days later. The T8+ T cell reconstitution occurred at an even faster rate in patients who were to develop grade II-IV GVHD, as compared with those with grade O-I GVHD, thus leading to an earlier decrease in the T4/T8 ratio. Of 10 patients with a T4/T8 ratio less than 2.5 at day 19, 9 developed grade II-IV GVHD and 1 showed no GVHD. Of 14 patients with a ratio greater than 2.5 at that time, only 2 developed grade II-IV and 12 grade O-I GVHD (p less than 0.001). In the 11 patients developing grade II-IV GVHD, the T4/T8 ratio decreased to values less than 2.5 before the first clinical symptoms of GVHD in 9; it coincided in one and occurred later in another patient. Thus, early monitoring of the T4/T8 ratio can distinguish patients at risk of developing grade II-IV GVHD.

Description: We have studied the role of factor VIII-von Willebrand factor (FVIII- vWF) in both platelet adherence to subendothelium and ristocetin- induced platelet aggregation using monoclonal antibodies to human FVIII- vWF. Twenty-five monoclonal antibodies were obtained, two of which were directed to the factor VIII moiety of FVIII-vWF; one of these two completely inhibited the procoagulant activity (FVIII:C). The remaining 23 monoclonal antibodies were directed to the von Willebrand factor moiety of FVIII-vWF. The ability of the latter monoclonal antibodies to inhibit platelet adherence to arterial subendothelium was investigated with a perfusion model. According to the number of platelets adhering to the subendothelium, three groups of monoclonal antibodies could be discerned: (A) antibodies not affecting platelet adherence; (B) antibodies that inhibited platelet adherence to the level as observed when von Willebrand's disease plasma was tested; and (C) antibodies that completely inhibited both platelet adherence to subendothelium and ristocetin-induced platelet aggregation. The two antibodies present in group C competed for the same or closely related epitope(s) present on FVIII-vWF. These results demonstrate that a domain is present on the FVIII-vWF molecule that is associated both with ristocetin-induced aggregation and with the ability of FVIII-vWF to support platelet adherence to the subendothelium. Based on these observations, it is concluded that ristocetin-induced binding of FVIII-vWF to platelets reflects, at least in part, a physiologic mechanism regulating the function of FVIII-vWF in primary hemostasis.

Description: We have analyzed a cloned beta O-thalassemia (beta O-thal) gene from a patient doubly heterozygous for hemoglobin Lepore and beta O- thalassemia. Studies of 3H-uridine incorporation into beta-globin mRNA in this patient's erythroblasts suggested an intranuclear defect in both beta and Lepore (delta beta) mRNA synthesis, as did S1 nuclease analysis of nuclear RNA. However, the nucleotide sequence of the beta O- thal gene revealed only a single base change in codon 39 (CAG----UAG), which created a premature translation termination codon. The 5′ flanking sequence, including transcription promotor boxes and the mRNA initiation (CAP) site, were normal. The unexpected effect of this mutation on intranuclear beta-mRNA synthesis in vivo was studied by insertion of the cloned gene into a plasmid expression vector and transfection into tissue culture (COS-1) cells. beta-Globin mRNA produced by the transfected cells was assessed by S1 nuclease analysis. The beta O-39 thalassemia gene generated five- to tenfold less beta- mRNA than a normal beta-gene in both nuclear and cytoplasmic RNA, simulating the results observed in vivo. Moreover, the small amount of beta O-39 mRNA produced was as stable as normal beta-mRNA during an actinomycin D chase, ruling out rapid cytoplasmic turnover as a cause of the reduced accumulation. Cotransfection of the beta O-39 thalassemia gene with a mutant tyrosine suppressor tRNA gene resulted in restoration of the beta O-39 mRNA accumulation to near-normal levels. On the basis of these results, we suggest that the low levels of beta-mRNA known to exist in the common form of beta O-thalassemia, beta O-39 thalassemia, result from a lesion in transcription, or early posttranscriptional processes; the defect appears to be corrected by restoration of proper translational potential to the mutant mRNA, at least in a gene transfer-expression system in tissue-culture cells.

Description: Two new cell surface antigens specific for the erythroid lineage were defined with cytotoxic IgM monoclonal antibodies (McAb) (EP-1; EP-2) that were produced using BFU-E-derived colonies as immunogens. These two antigens are expressed on in vivo and in vitro derived adult and fetal erythroblasts, but not on erythrocytes. They are not detectable on resting lymphocytes, concanavalin-A (Con-A) activated lymphoblasts, granulocytes, and monocytes or granulocytic cells or macrophages present in peripheral blood or harvested from CFU-GM cultures. Cell line and tissue distributions distinguish McAb EP-1 and EP-2 from all previously described monoclonal antibodies. McAb EP-1 (for erythropoietic antigen-1) inhibits the formation of BFU-E and CFU-E, but not CFU-GM, colonies in complement-dependent cytotoxicity assays. By cell sorting analysis, about 90% of erythroid progenitors (CFU-E, BFU-E) were recovered in the antigen-positive fraction. Seven percent of the cells in this fraction were progenitors (versus 0.1% in the negative fraction). The expression of EP-1 antigen is greatly enhanced in K562 cells, using inducers of hemoglobin synthesis. McAb EP-2 fails to inhibit BFU-E and CFU-E colony formation in complement-dependent cytotoxicity assays. EP-2 antigen is predominantly expressed on in vitro derived immature erythroblasts, and it is weakly expressed on mature erythroblasts. The findings with McAb EP-1 provide evidence that erythroid progenitors (BFU-E and CFU-E) express determinants that fail to be expressed on other progenitor cells and hence appear to be unique to the erythroid lineage. McAb EP-1 and EP-2 are potentially useful for studies of erythroid differentiation and progenitor cell isolation.

Description: This study was undertaken to examine the interaction of platelet size and age in determining in vitro platelet function. Baboon megakaryocytes were labeled in vivo by the injection of 75Se- methionine. Blood was collected when the label was predominantly associated with younger platelets (day 2) and with older platelets (day 9). Size-dependent platelet subpopulations were prepared on both days by counterflow centrifugation. The reactivity of each platelet subpopulation was determined on both days by measuring thrombin-induced aggregation. Platelets were fixed after partial aggregation had occurred by the addition of EDTA/formalin. After removal of the aggregated platelets by differential centrifugation, the supernatant medium was assayed for remaining platelets and 75Se radioactivity. Comparing day 2 and day 9, no significant difference was seen in the rate of aggregation of a given subpopulation. However, aggregation was more rapid in the larger platelet fractions than in the smaller ones on both days. A greater percentage of the 75Se radioactivity appeared in the platelet aggregates on day 2 than on day 9. This effect was independent of platelet size, as it occurred to a similar extent in the unfractionated platelets and in each of the size-dependent platelet subpopulations. The data indicate that young platelets are more active than older platelets. This study demonstrates that size and age are both determinants of platelet function, but by independent mechanisms.

Description: We have measured the fully carboxylated (native) prothrombin antigen and the undercarboxylated (abnormal) prothrombin antigen in patients treated with sodium warfarin using specific immunoassays to evaluate a new approach for monitoring oral anticoagulant therapy. Plasma and serum samples (391) were assayed for the prothrombin time, native prothrombin antigen, and abnormal prothrombin antigen. The results were correlated with the presence of bleeding or thromboembolic complications at the time of phlebotomy. The native prothrombin antigen correlated with the occurrence of complications in 95% of samples. Of 13 samples from patients with bleeding complications, 13/13 (100%) had a native prothrombin of 12 micrograms/mL or lower. Of seven samples from patients with thromboembolic complications, 6/7 (86%) had a native prothrombin of 24 micrograms/mL or greater. By comparison, a prothrombin time index of 1.5 to 2.5, 1.5 to 2.2, 1.5 to 2.0, or 1.3 to 1.8 identified 6/20 (30%), 9/20 (45%), 11/20 (55%), or 12/20 (60%) patients at risk, respectively. Although the prothrombin time index did correlate with the presence of bleeding complications, the native prothrombin antigen correlated closely with the presence of bleeding and thromboembolic complications. According to these results, the native prothrombin antigen, maintained in a range of 12 to 24 micrograms/mL by regular adjustment of the warfarin dosage, may be associated with a reduced risk of complications due to excessive or insufficient warfarin therapy. On the basis of these preliminary data, we recommend that the native prothrombin antigen be considered to monitor warfarin therapy.

Description: High proliferative potential macrophage progenitor cells (HPP-CFC) in 5- fluorouracil (FU) treated and normal mouse bone marrow (BM) have been shown to be less sensitive to inhibition of proliferation by prostaglandins of the E series (PGE) than low proliferative potential macrophage progenitor cells (LPP-CFC) in normal BM in agar cultures. The growth of large colonies (diameter greater than 0.5 mm) derived from HPP-CFC in FU BM, which require a combination of macrophage colony- stimulating factor (CSF-1) plus a new growth factor called synergistic activity (SA), are inhibited by 50% in the presence of 5.5 X 10(-6) M PGE1. On the other hand, LPP-CFC in normal BM, which form smaller colonies (diameter less than or equal to 0.5 mm) in the presence of CSF- 1 alone, require only 5 X 10(-8) M PGE1 for the same level of inhibition. Addition of appropriate concentrations of PGE1 to the agar culture assay should improve detection of HPP-CFC by inhibiting the proliferation of LPP-CFC. These observations suggest that the apparent negative feedback control of macrophage production by PGE operates largely on the LPP-CFC, which respond to CSF-1 alone, and is probably not involved in the regulation of the more primitive HPP-CFC.

Description: We used both radiolabeled and fluorescein-labeled antiglobulins in assays to detect antibodies against platelets in multiply transfused patients to determine the value of these tests in predicting the outcome of platelet transfusion in such patients. In 15 allosensitized patients, we studied 68 single-donor platelet transfusions, 43 (63%) of which had a poor outcome, defined as a corrected count increment (CCI), less than 10,000. The results obtained with either test were significantly correlated with the CCI following transfusion (p less than 0.001), but the assay using the radiolabeled antiglobulin had slightly better sensitivity, specificity, and predictive value. When the assays were used in combination, there was again significant correlation with the CCI of the transfusion, p less than 0.001. When both assays predicted failure of the transfusions, 31/31 (100%) such transfusions resulted in a CCI of less than 10,000, and when both assays predicted success of the transfusions, 14/15 (93%) such transfusions resulted in a CCI of greater than 10,000. Both assays are useful in predicting the outcome of the platelet transfusions; when the assay results were concordant, almost total predictive accuracy was obtained.

Description: A 42-yr-old woman with systemic lupus erythematosus without bleeding diathesis developed a prolonged activated partial thromboplastin time that was not corrected by normal plasma. An inhibitor that acted rapidly and inactivated 0.5 U/ml plasma thromboplastin antecedent (PTA, factor XI) at a 1:200 plasma dilution was demonstrated. In addition to a low titer of PTA (less than 0.01 U/ml), plasma assayed at 20-fold dilution also showed low titers of Hageman (factor XII, 0.02 U/ml), Fletcher (plasma prekallikrein, 0.02 U/ml), and Fitzgerald (high molecular weight kininogen, less than 0.01 U/ml) factors. The titer of these factors, except PTA, returned to normal upon further plasma dilution or upon removal of the inhibitor by protein A adsorption. Thus, the inhibitor appeared to interfere with these clotting factor assays, possibly by inactivating PTA in the substrate plasmas in the test system. Its specificity was further confirmed. The inhibitor did not interfere with surface-induced proteolytic cleavage of Hageman factor. Surface-induced generation of plasma kallikrein activity (amidolysis of H-D-pro-phe-arg-pNa and cold-promoted factor VII activity enhancement) requires only Hageman, Fletcher, and Fitzgerald factors and was normal. Reactions requiring all 4 contact phase factors, including PTA, such as surface-induced generation of plasmin activity (amidolysis of H-D-val-leu-lys-pNa) and activated Christmas factor (factor IXa) activity, were defective. Furthermore, the inhibitor bound to agarose-protein A inactivated and removed PTA selectively from normal plasma. The inhibitor was an IgG-lambda autoantibody that precipitated PTA. The inactivated activated PTA (factor XIa) without the requirement for an additional cofactor. Furthermore, it inhibited surface-induced activation of PTA by interfering with its proteolytic cleavage upon glass surface exposure and with its binding onto the reactive surfaces.

Description: Because clinical disorders of spontaneous iron overload have no experimental counterpart, we studied iron distribution (atomic absorption analysis) and intestinal absorption (59Fe) in mice with hereditary alpha-thalassemia. Mice heterozygous for a radiation-induced alpha-Hb gene deletion exhibit a mild hemolytic anemia, like the human condition, with microcytosis, reticulocytosis, splenomegaly, and chemical evidence of defective alpha-chain synthesis. Quantitative iron determination showed that total iron content in spleen, liver, and kidney, but not heart or lung, of adult alpha-thalassemic mice was greater (P less than .05) than that in unaffected littermates. Iron concentration was also increased in liver (P less than .001), spleen (P = .025), kidney (P = .058), and heart (P = .010); in general, the greater the iron concentration in liver, the greater that in spleen (r = .39, P = .009), kidney (r = .70, P less than .001), and heart (r = .46, P less than .001). In mice examined 8 months postoperatively, splenectomy, as compared to sham operation, significantly raised iron content in extrasplenic tissues, but did not affect total body iron. At 10–11 weeks of age, but no longer at 12–14 weeks, thalassemic mice showed higher rates of iron absorption than age-matched controls. Thus, alpha-thalassemic mice display an early occurring iron absorption defect, leading to a modest, sustained, nonprogressive iron overload, and thereby represent a valuable model for exploring disorders of iron homeostasis.

Description: The role of divalent cations in platelet adherence to deendothelialized human arteries in flowing blood was investigated in an annular perfusion chamber. Spreading of platelets on the subendothelium was impaired below 30 microM of free Ca2+ ions (Ca2+). When Ca2+ was replaced by Mg2+, adherence was unchanged in perfusates without exogenous factor VIII-von Willebrand factor (FVIII-vWF), but the ability of FVIII-vWF to support platelet adherence was lost. Binding of FVIII-vWF to the vessel wall was independent of divalent cations, but bound FVIII-vWF was only able to mediate adherence after exposure to Ca2+. Pretreatment of FVIII-vWF with the calcium chelator EGTA (10 mM) resulted in loss of the ability to facilitate platelet adherence, while the ristocetin cofactor activity remained intact. Full restoration of the ability to mediate platelet adherence could only be obtained by prolonged dialysis against Ca2+ in the millimolar range. These data indicate that divalent cations have at least two separate roles to play in supporting platelet adherence: (1) platelet spreading on the subendothelium requires Ca2+ or Mg2+; (2) FVIII-vWF should be exposed to Ca2+ to obtain its optimal biologic activity in supporting platelet adherence.

Description: Peripheral blood granulocytes from patients with chronic myelogenous leukemia (CML) were studied for accessibility of membrane sialic acid and galactose residues to sodium borohydride-3H radiolabeling after oxidation with sodium metaperiodate (PI/B3H4) or with galactose oxidase (GO/B3H4). Granulocytes from untreated patients with chronic myelogenous leukemia showed increased radiolabeling with PI/B3H4, and decreased labeling with GO/B3H4 when compared to normal granulocytes. Granulocytes from leukemic patients receiving chemotherapy showed normal labeling patterns. Gel electrophoresis of membrane extracts showed that the changes in PI/B3H4 and GO/B3H4 reactivity of CML cells were distributed over all membrane protein bands. Our data suggest that CML granulocyte membrane proteins are aberrantly sialylated, with decreased accessibility of galactose residues, and that these changes may be reversed by clinical drug treatment.

Description: We present a colony assay system that allows in situ identification of human basophil/mast cell (basophil) colonies. In methylcellulose culture, in the presence of phytohemagglutinin-leukocyte conditioned media (PHA-LCM), human peripheral blood and bone marrow cells form colonies that can be distinguished by their unique morphological characteristics. Pure basophil colonies are diffuse, small colonies containing small, round, highly refractile cells. These characteristics of the constituent cells led us to the observation that a significant number of basophils are found in combination with eosinophils. The mixed eosinophil/basophil colonies have the distinctive elements of pure eosinophil and pure basophil colonies. Usually, these are diffuse colonies with compact clusters of slightly larger, darker-appearing cells. We also found colonies that contained basophils and neutrophils/monocytes, but this type could not be consistently identified by in situ morphology. Cytochemical analysis confirmed the metachromatic nature of the granules in the basophils. The presence of IgE receptors on the cells was documented by indirect immunofluorescent staining after passive sensitization with purified human IgE. Peripheral blood cells from six healthy volunteers formed 5.7 +/- 1.0 (mean +/- SEM) pure colonies in 2 X 10(5) cells. Cultures of bone marrow cells from patients with various types of anemia had 9.0 +/- 1.5 colonies in 10(5) cells. This is the first description of a colony assay system for in situ identification of a pure population of basophilic granulocytes.

Description: Morphological, immunologic, and functional properties of peripheral blood cells from two patients with chronic proliferations of granular lymphocytes are described. Cells from both patients showed a heterogeneous pattern from both a morphological and immunologic standpoint, indicating a polyclonal, rather than a monoclonal, expansion of these cells. In fact, both large and small-to-medium-sized granular lymphocytes were observed, and different percentages of positivity were found in the analysis with a large panel of monoclonal antibodies. Serologic and histologic features support the hypothesis that this lymphocytosis could be secondary to bacterial or viral infections rather than a primary event, suggesting that these patients may have chronic reactive immunoregulatory disorders.

Description: This report describes the experience of the Southeastern Cancer Study Group (SECSG) with a transport medium used for immunologic phenotyping of non-Hodgkin’s lymphomas. In a 2-mo pilot study, portions of 53 specimens of non-Hodgkin's lymphoma from four member institutions of the SECSG and affiliated community hospitals were sent by regular mail to a central laboratory. Immunologic phenotyping was carried out using a frozen section immunoperoxidase technique. In 48 of the cases, a clear-cut immunologic phenotype was obtained. Thirty-four tumors were of B cell origin and 7 had T cell markers. Six of the remaining lymphomas had neither B nor T cell markers, and the seventh had both. In 12 cases, phenotyping was also carried out at the originating institution using conventional cell suspension techniques; agreement between the two methods was excellent. The immunologic results were correlated with histopathologic diagnosis standardized using the Working Formulation for non-Hodgkin’s lymphomas. It was found that the low grade tumors were all B cell, but that the intermediate grade tumors were very heterogeneous immunologically. About one-fourth of the diffuse, intermediate grade or miscellaneous tumors had T cell markers. Our results indicate that immunologic phenotyping may be performed satisfactorily on transported material, making multiinstitution studies on the prognostic significance of immunologic phenotype in non- Hodgkin’s lymphomas feasible.

Description: In the baboon (Papio species), the two nonallelic gamma-genes produce gamma-chains that differ at a minimum at residue 75, where isoleucine (I gamma-chain) or valine (V gamma) may be present. This situation obtains in baboons that are sometimes designated as Papio anubis, Papio hamadryas, and Papio papio. However, in Papio cynocephalus, although the I gamma-chains are identical with those in the above mentioned types, the V gamma-chains have the substitutions ala----gly at residue 9 and ala----val at residue 23. The V gamma-chains of P. cynocephalus are called V gamma C to distinguish them from the V gamma A-chains of P. anubis, etc. A single cynocephalus animal has been found to have only normal I gamma-chains and I gamma C-chains (that is, glycine in residue 9, valine in 23, and isoleucine in 75). When HbF is produced in response to stress with 5-azacytidine, P. anubis baboons respond with greater production than do P. cynocephalus, and hybrids fall between. Minimal data on P. hamadryas and P. papio suggest an even lower response than P. cynocephalus. As HbF increases under stress, the ratio of I gamma to V gamma-chains changes from the value in the adult or juvenile baboon toward the ratio in the newborn baboon. However, it does not attain the newborn value. The V gamma A and V gamma C-genes respond differently to stress. In hybrids, the production of V gamma A- chains exceeds that of V gamma C-chains. A controlling factor in cis apparently is present and may be responsible for the species-related extent of total HbF production. It may be concluded that the more primitive the cell in the erythroid maturation series that has been subjected to 5-azacytidine, the more active is the I gamma-gene.

Description: In the baboon (Papio species), the two nonallelic gamma-genes produce gamma-chains that differ at a minimum at residue 75, where isoleucine (I gamma-chain) or valine (V gamma) may be present. This situation obtains in baboons that are sometimes designated as Papio anubis, Papio hamadryas, and Papio papio. However, in Papio cynocephalus, although the I gamma-chains are identical with those in the above mentioned types, the V gamma-chains have the substitutions ala----gly at residue 9 and ala----val at residue 23. The V gamma-chains of P. cynocephalus are called V gamma C to distinguish them from the V gamma A-chains of P. anubis, etc. A single cynocephalus animal has been found to have only normal I gamma-chains and I gamma C-chains (that is, glycine in residue 9, valine in 23, and isoleucine in 75). When HbF is produced in response to stress with 5-azacytidine, P. anubis baboons respond with greater production than do P. cynocephalus, and hybrids fall between. Minimal data on P. hamadryas and P. papio suggest an even lower response than P. cynocephalus. As HbF increases under stress, the ratio of I gamma to V gamma-chains changes from the value in the adult or juvenile baboon toward the ratio in the newborn baboon. However, it does not attain the newborn value. The V gamma A and V gamma C-genes respond differently to stress. In hybrids, the production of V gamma A- chains exceeds that of V gamma C-chains. A controlling factor in cis apparently is present and may be responsible for the species-related extent of total HbF production. It may be concluded that the more primitive the cell in the erythroid maturation series that has been subjected to 5-azacytidine, the more active is the I gamma-gene.

Description: Fibrin prepared from 15 pathologic thrombi was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to determine the extent and pattern of fibrinolysis that occurs in vivo. Two groups of patients could be distinguished on the basis of the polypeptide chain composition of fibrin in their thrombi. Those patients who presented with acute vascular obstruction, either arterial or venous, showed a minimal degree of fibrin degradation, with a dominance of intact, undegraded crosslinked gamma-gamma dimers. On the other hand, patients with long-standing symptoms associated with chronic aortic aneurysms had thrombi containing extensively degraded fibrin. Thrombi in large aortic aneurysms were dissected into concentric layers that showed different degrees of fibrinolysis. The luminal surface consisted of fresh, red thrombus and contained undegraded crosslinked fibrin similar to that found in patients with acute occlusive disease. Deeper layers of the thrombus showed gamma-gamma chain degradation throughout, indicating that this portion was undergoing active thrombolysis. The findings demonstrate that the variability in the pathophysiologic balance between coagulation and fibrinolysis is reflected in vivo by the polypeptide chain composition of crosslinked fibrin in thrombi. The results support the hypothesis of a dynamic equilibrium between clotting and lysis, but indicate that the balance between these two processes may be distinctly different in separate areas of a single clot.

Description: The fucose-binding lectin from Lotus tetragonolobus ( FBL -L) has been previously shown to bind specifically to normal cells of the myeloid and monocytic lineages. The purpose of this study was to explore the utility of fluoresceinated FBL -L as a leukemia differentiation marker in conjunction with a panel of other frequently used surface markers (Fc receptor, HLA-DR, OKM1, and antimonocyte antibody). FBL -L reacted with leukemic cells in 8/9 cases of clinically recognized acute myeloid leukemia, including myeloid blast crisis of chronic granulocytic leukemia, 3/3 cases of chronic phase chronic myelogenous leukemia, and in 2/7 cases of clinically undifferentiated acute leukemia. Correlations were noted between reactivity with FBL -L, and DR and Fc receptor expression. Among continuous cell lines, FBL -L bound with high intensity to a majority of HL-60 and U937 cells. The less well differentiated myeloblast cell lines, KG-1, KG1a , and HL-60 blast II, exhibited less FBL -L binding than HL-60 and U937. A moderate proportion of K562 cells exhibited low level binding of FBL -L. Several lymphoblastic cell lines exhibited a pattern of low intensity binding that was distinguishable from the high intensity binding pattern of the myeloblastic lines. FBL -L reactivity of U937 was enhanced by induction of differentiation with leukocyte conditioned medium, but not dimethylsulfoxide. Such treatments induced contrasting patterns of change of HL-60 and U937 when labeled with OKM1, alpha-Mono, and HLA- DR. These studies demonstrate the application of FBL -L to analysis and quantitation of myelomonocytic leukemic differentiation.

Description: To investigate the cellular events that accompany erythroid hyperplasia, we studied several effects of erythropoietin (Epo) on marrow CFU-E in sickle cell anemia (SCA). We measured CFU-E number, CFU- E growth as a function both of Epo exposure time and of Epo concentration, and suppression of Epo-induced CFU-E formation by anti- Epo antiserum. With 0.5 U Epo/ml, the number of CFU-E was elevated in SCA (1,087 +/- 520) compared to normal (430 +/- 130). CFU-E were formed even when Epo was immediately neutralized by a 1/150 dilution of anti- Epo. After 40 hr of Epo exposure, only 2% of total CFU-E were expressed in normal marrow, whereas 12%-40% of CFU-E were expressed in SCA. Inhibition of CFU-E growth required at least 1/50 dilution of anti-Epo in SCA and a 1/300 dilution in normal marrow. In contrast to normal, a small number (5%-20%) of CFU-E were expressed in the absence of added Epo in SCA, and this pool required a 1/150 dilution of anti-Epo for inhibition. The Epo dose-response curve in SCA revealed a peak in colony formation around 0.1 U Epo/ml and 0.5 U Epo/ml, whereas only one peak at 0.5 U Epo/ml was seen in normals. These data strongly suggest that, in response to the demands of chronic erythroid hyperplasia in SCA, a pool of CFU-E is present characterized by increased in vitro sensitivity to Epo.

Description: Thirteen patients with acute leukemias that were difficult to classify by the use of cytochemical staining and terminal deoxyribonucleotidyl transferase (TdT) activity are reported. The phenotype of the leukemic cells was characterized by the presence of mature or early monocyte lineage antigens and intense Ia antigen expression detected by monoclonal antibodies, terminal deoxytransferase activity, and cytochemical features, including both Sudan black B and periodic acid- Schiff activity. The mean age of this group of patients was 60 years. Five patients had leukemia occurring after chemotherapy or radiotherapy of a prior malignant disease, and two patients had a refractory anemia prior to development of acute leukemia. These patients had a low response rate to chemotherapy. This series of leukemia appears to form a distinct nosologic entity, representing a leukemic transformation among early cells of the monocyte lineage, resulting in a predominant neoplastic cell that is less mature than either the French-American- British M4 acute myelomonocytic leukemia or M5 acute monoblastic leukemia. The presence of terminal deoxytransferase activity was interpreted as indicating the primitive state of the cells in the differentiation sequence, rather than as implying any significance with respect to lineage.

Description: Levels of platelet-associated IgG (PA-IgG) were studied in 72 patients with Hodgkin–s (HD) and non-Hodgkin–s lymphoma (NHL). Thirty-nine percent of patients with HD and 20% of patients with NHL had elevated PA-IgG levels. There was a positive correlation between disease activity and the presence of PA-IgG in HD and NHL. In patients with HD, PA-IgG strongly correlated with extent of disease and may serve as a marker of disease activity. PA-IgG may have facilitated platelet destruction in 5 of 11 thrombocytopenic patients with HD and increased PA-IgG and in 2 patients with HD and increased PA-IgG who developed severe thrombocytopenia when treated with chemotherapy.

Description: Sedimentation analysis of factor VIII complex was performed in the analytical ultracentrifuge using partition cells. This method allowed for the calculation of three different sedimentation coefficients from each run: one based on ristocetin agglutination activity for von Willebrand protein, SWF; one based on coagulant activity for factor VIIIC, SVIIIC; and one based on the schlieren or adsorption data for protein concentration, Sconc. In most cases, there was no agreement between the three values calculated from the same run, indicating a heterogeneous system. The calculated functional sedimentation coefficients give values that require the molecules to be highly asymmetric to be consistent with a glycoprotein of high molecular weight, which is in agreement with results observed in electron microscope studies. The dissociation of VIIIC into a smaller form can be demonstrated by this method. Determination of the three sedimentation coefficients in a series of fractions from gel filtration indicates a uniform size for the VIIIC activity but not for the WF activity. These observations are in agreement with the concept of a copolymer between WF and VIIIC and also with the concept of separate polymers for the two activities.

Description: The malignant monoclonal population in hairy cell leukemia (HCL) has been variously ascribed to be of myeloid, B, or even T cell origin. Recent data have been interpreted as suggesting that hairy cells (HC) may concomitantly or serially express both B and T surface determinants, a phenomenon which, if verified, would be unique among the lymphoproliferative malignancies. Data described here, however, demonstrate that (1) at least the majority of HCL are phenotypically of B cell derivation, and (2) the initial B cell phenotype is retained and solely expressed on cultured as well as phytohemagglutinin (PHA) activated monoclonal malignant HC.

Description: To study the influence of a biologic environment on cultured human leukemia cells, KG-1, KG-1a, and HL-60 cells were inoculated subcutaneously into newborn nude mice. The cells developed myelosarcomas at the site of inoculation and in lungs and kidneys. KG-1 and HL-60 myelosarcomas were successfully passaged through adult nude mice, whereas KG-1a tumors proliferated only after transplantation into newborn hosts. The human nature of the cells forming myelosarcomas in mice was assessed by chromosomal analyses and detection of cross- reactivity with an antibody to the human leukemia cell line K562. We undertook electron microscopic and cytochemical examinations of the cells proliferating in vitro and in the mice. The granules of KG-1 cells in vivo did not react for acid phosphatase, as observed in vitro, and the HL-60 cells proliferating in mice lost the perinuclear myeloperoxidase (MPO) demonstrated in cultured cells. Although the influence of an in vivo selection of cell subpopulations cannot be ruled out, the enzymatic changes are compatible with induced cell differentiation. Conclusive evidence of differentiation in vivo was observed in the KG-1a cell subline. The undifferentiated KG-1a blasts developed cytoplasmic granules and synthesized MPO during proliferation in vivo. These observations indicate that human leukemia cells from established cell lines proliferate in nude mice and may acquire new differentiated properties in response to the in vivo environment.

Description: The Southeastern Cancer Study Group conducted a post-remission induction randomized trial in adult acute myelogenous leukemia to assess the efficacy of alternate drug therapy during consolidation and of immunotherapy during maintenance. Of 508 evaluable patients entered into the study, 335 (66%) achieved a complete remission treated with a 7-day infusion of cytosine arabinoside at a dose of 100 mg/sq m/day and 3 days of daunorubicin at a dose of 45 mg/sq m/day. Those in remission were randomized to receive 3 courses of 1 of 3 consolidation regimens: (A) a continuous infusion of 5-azacytidine, 150 mg/sq m/day for 5 days; (B) 5-azacytidine plus beta-deoxythioguanosine, 300 mg/sq m/day for 5 days; or (C) cytosine arabinoside, 100 mg/sq m/day intravenously, and thioguanine, 100 mg/sq m orally every 12 hr, plus daunorubicin, 10 mg/sq m every 24 hr daily for 5 days. There was no difference in relapse rate among the 3 arms. Those completing consolidation and remaining in remission were randomized to 1 of 3 maintenance regimens: (D) chemotherapy, 5-day infusion of cytosine arabinoside and 2 days of daunorubicin (same doses as induction) given every 13 wk for 1 yr; (E) BCG given twice weekly for 1 mo and then monthly for 1 yr; or (F) the combination of regimens D and E. The median duration of remission was significantly better on regimen D (17.4 versus 9.4 and 9.5 mo), and median survival was 29 mo compared to 21 mo for the other regimens. Those given different drugs during consolidation than used for induction (regimens A and B) and subsequent chemotherapy for maintenance (regimen D) had the longest remission durations and survival. Immunotherapy was not as good as intensive chemotherapy for maintenance.

Description: Samples of leukemic cell DNA from 14 children with acute nonlymphocytic leukemia (ANLL) and 4 human myeloid leukemia cell lines were analyzed for rearrangement in the heavy chain region of the immunoglobulin gene. The diagnosis of ANLL was confirmed in all patients by morphological, cytochemical, and immunologic studies. By restriction endonuclease digestion and hybridization with cloned heavy chain immunoglobulin gene probes for the constant (Cmu) and joining (JH) regions, the DNA of 2 patients and 1 cell line (ML-1) was found to contain rearrangements. The DNA from the remaining 12 patients and 3 cell lines was not rearranged (germline configuration). Both patients with apparent immunoglobulin gene rearrangement achieved complete remission on therapy for ANLL. Immunoglobulin gene rearrangement in phenotypically defined ANLL suggests (1) that such changes may not be limited to lymphoid leukemia of B cell lineage, or (2) that, in some patients, the leukemic transforming event may involve stem cells capable of both B cell and myeloid differentiation.

Description: Patients with multiple myeloma (MM) are at an increased risk for infections with bacteria that require opsonization with complement. Because Streptococcus pneumoniae is the most frequently encountered pathogen in these patients, we investigated the ability of serum from patients with MM to mediate the binding of C3b, the major opsonin of the complement system, to S. pneumoniae. S. pneumoniae types 3, 14, and 25 were chosen for study, since S. pneumoniae type 3 activates primarily the classical complement pathway (CCP), type 25 primarily the alternative complement pathway (ACP), and type 14 both pathways. S. pneumoniae were treated with normal serum or serum from 17 patients with MM, and the bound C3b was quantified with fluorescein-conjugated anti-C3 in a spectrophotofluorometric assay. Despite normal or elevated serum concentrations of C3, total hemolytic complement, and C-reactive protein in all of the MM sera, factor B in 16/17 such sera, and C4 in 14/17 MM sera studied, all 17 sera demonstrated a defect in C3b binding to type 3 (32.7% +/- 6% of normal). In addition, serum from 15/17 patients bound decreased amounts of C3b to types 14 (39.6% +/- 8%) and 25 (52.2% +/- 8%). Mixing normal serum with MM serum restored MM C3b binding activity to all three S. pneumoniae types, suggesting that the defect was related to a deficiency rather than an inhibitor of C3 activation. Although MM patients are unable to produce specific antibodies to bacterial antigens, the addition of anti-S. pneumoniae antibodies to MM serum did not enhance C3b binding to any of the S. pneumoniae types. However, when S. pneumoniae were opsonized in a mixture of MM serum and C3-depleted normal serum, C3b binding was restored to all three S. pneumoniae types, demonstrating that MM C3 functions normally in the presence of other normal serum factors. In the present studies, the MM C3b binding defect appeared to correlate with the incidence of S. pneumoniae infections. Serum from patients with a history of an S. pneumoniae infection bound significantly less C3 (20.5% +/- 4%) than those study patients without a history of an S. pneumoniae infection (55.8% +/- 8%) (p less than 0.0025). Thus, MM serum has a defect in the activation of C3, and this may contribute to the increased susceptibility of MM patients to S. pneumoniae infections.

Description: The effects of hemin on the conversion of pyruvate kinase (PK) isozymes from the M2-type to the L-type in K562 cells were investigated. Immunofluorescence, ion exchange chromatography, and electrophoretic studies showed that the untreated K562 cells contained only the M2-type PK, while eight to 20 days after induction with hemin, concomitant with hemoglobin F synthesis, L-type PK levels increased while M2-type PK levels decreased. Electrophoretic study revealed three hybrid isozymes of the L-type and M2-type PK. We conclude that the conversion of PK isozymes from the M2-type to the L-type in erythroid precursor cells occurs in the early stage of maturation.

Description: In this study, the effects of 5-aza-2′-deoxycytidine on differentiation of human leukemic cells in primary suspension culture are reported for the first time. Morphological and functional differentiation was induced in cells from two acute monoblastic leukemias and two of three acute myeloid leukemias following repeated exposures to 1 mumol/L 5-aza- 2′-deoxycytidine. The observation that nontoxic concentrations of the drug are able to induce the in vitro differentiation of both monoblastic and myeloblastic leukemic cells into mature elements may encourage the exploitation of the differentiating properties of 5-aza- 2′-deoxycytidine in chemotherapy protocols for acute non-lymphoblastic leukemias.

Description: We describe four patients with impaired platelet aggregation and 14C- serotonin secretion during stimulation with adenosine diphosphate (ADP), epinephrine, collagen, and platelet-activating factor. The response to arachidonic acid was normal in all patients with regard to aggregation and in three of the four with regard to 14C-serotonin secretion. The total platelet adenosine triphosphate (ATP) and ADP content and the ATP to ADP ratio was normal in all patients, thereby excluding storage pool deficiency as the cause of the secretion defect. Studies with 3H-arachidonic acid-labeled platelets revealed that the thrombin-induced liberation of arachidonic acid from membrane-bound phospholipids was impaired in these patients. Further, platelet thromboxane B2 production, measured using a radioimmunoassay, was diminished during stimulation with ADP and thrombin, but was normal with arachidonic acid, indicating that the oxygenation of arachidonic acid was normal and that the diminished thromboxane production was due to a defect in the liberation of arachidonic acid. Release of arachidonic acid is mediated by phospholipases that are Ca++ dependent. To examine whether these patients may have a defect in making intracellular Ca++ available, another Ca++-dependent process, myosin light chain phosphorylation, was studied during thrombin stimulation. Platelets from three of the patients were found to behave the same as normal ones, suggesting that the deficiency in phospholipase activity may not be due to impaired Ca++ mobilization. Our studies demonstrate a novel group of patients with platelet secretion defects associated with impaired liberation of arachidonic acid from phospholipids. These patients exemplify a congenital defect, other than deficiencies of cyclooxygenase and thromboxane synthetase, by which thromboxane production may be impaired in platelets.

Description: Restriction endonuclease mapping defined a partial deletion of about 1.35 kb in the beta-globin gene of a black American patient with hemoglobin S-beta zero-thalassemia and in his uncle with a beta zero- thalassemia trait. The 5′ endpoint of the deletion is about 600 bases upstream from the cap site, and the 3′ endpoint lies within about 500 bases from the 5 splice junction of the second intervening sequence. The deletion is different from that of a previously reported Indian beta zero-thalassemia allele, where 0.6 kb is deleted at the 3′ end of the beta-globin gene.

Description: Hereditary persistence of fetal hemoglobin (HPFH) is a genetically heterogeneous and clinically benign condition characterized by persistent expression of fetal hemoglobin (Hb F) into adulthood. In the G gamma beta + type, no major deletions in the globin gene cluster occur; adult heterozygotes produce approximately 20% Hb F, which results from overproduction of G gamma chains, with no apparent increase in production from the adjacent A gamma gene. We have recently described a point mutation 202 base pairs 5′ to the cap site of the G gamma gene in an individual with G gamma beta + HPFH. This mutation abolishes a normal ApaI restriction endonuclease site, and thus can be detected by blotting of genomic DNA. We present here further data on the ApaI mutation: (1) It occurs in six of seven families with G gamma beta + HPFH. (2) In three families, detailed haplotype analysis using 11 polymorphic restriction sites in the beta globin cluster has been done. The two that carry the missing ApaI site are identical but the third, which has a normal ApaI pattern, differs from the other two in at least two sites, one of which is a new polymorphic Nco I site between the delta and beta globin genes. This suggests the possibility of a different HPFH mutation in the third family. (3) The haplotype of the G gamma beta + HPFH chromosome carrying the ApaI mutation is different from that of 108 beta A chromosomes of black individuals that have been tested. (4) The G gamma ApaI site is normal in 61 beta A and 109 beta S alleles from non-HPFH black individuals, including 22 who share the same haplotype for the intragenic G gamma, A gamma HindIII polymorphisms. These data add support to the possibility that the -202 mutation is actually causative of the G gamma beta + HPFH phenotype.

Description: Potential limitations of prenatal diagnosis of hemophilia B, as compared to hemophilia A, include (1) occurrence of far more frequent defects with abnormal circulating antigen, (2) lower levels of factor IX in fetal plasma at 16 to 20 weeks gestation, and (3) the presence of factor IX antigen in amniotic fluid. In addition, proteolysis could occur, especially with amniotic fluid contamination of fetal plasma. A sensitive polyclonal immunoradiometric assay for factor IX antigen was used to characterize the range of levels in amniotic fluids and fetal plasma samples. To assess for altered forms, factor IX species were compared to those of a homologous clotting factor, prothrombin. Fourteen postmortem abortus blood samples from fetuses of 14 to 23 weeks gestation had factor IX antigen levels that averaged 5.1 U/dL and ranged from 1.7 to 15 U/dL. Amniotic fluid factor IX antigen averaged 2.9 U/dL, with a range from 1.4 to 8.5 U/dL in 19 separate amniocentesis samples. Thus, in a male fetus at risk of hemophilia B and with a low circulating level of gene product, mixture of fetal plasma with amniotic fluid could severely limit prenatal diagnosis, assuming that the amniotic fluid factor IX is of maternal origin. Despite rapid processing of amniotic fluid samples, the prothrombin was extensively cleaved, suggesting that it had been activated in vivo. On gel electrophoresis of amniotic fluid samples, however, factor IX was only minimally cleaved. In the postmortem fetal blood specimens, prothrombin was partially cleaved. On crossed-immunoelectrophoresis, fetal plasma prothrombin showed decreased migration in calcium, compared to EDTA, indicative of mature gamma-glutamyl carboxylation. The latter presumably resulted from fetal hepatic synthesis.

Description: Colony-forming cells in ten cases of acute myeloid leukemia (AML) were studied with six cytotoxic monoclonal antibodies that react with antigens expressed at discrete stages of differentiation of normal and leukemic hematopoietic cells. The reactivity of the whole leukemic population was measured by indirect immunofluorescence, and the reactivity of the colony-forming cells was established by complement- mediated cytotoxicity and by fluorescence activated cell sorting. Comparison of the immunofluorescent reactivity with cytotoxicity and cell sorting showed that colony-forming cells were found within a fraction of the leukemic subpopulations that expresses these antigens. This finding implies that immunofluorescence reactivity of the total leukemic population does not necessarily predict the phenotype of the clonogenic cells. When the surface phenotype of the clonogenic leukemic cells was compared to that previously established for normal marrow hemopoietic clonogenic cells, several patterns were seen: (1) in four of ten cases, the clonogenic cells expressed a phenotype like that of relatively mature normal granulocyte-macrophage colony-forming cells (late CFU-GM) or, (2) in two cases, a phenotype similar to the less mature colony-forming cells (early CFU-GM or CFU-GEMM), and (3) in four cases, a composite phenotype of early and late CFU-GM. Thus, the level of impairment of differentiation in AML may vary from case to case. In those cases phenotypically similar to the late CFU-GM, it may be possible to separate leukemic clonogenic cells from less mature normal clonogenic cells using monoclonal antibodies selectively cytotoxic for the late CFU-GM.

Description: We evaluated 37 patients with moderate or severe hemophilia A and six patients with severe factor IX deficiency for clinical or laboratory evidence of immune abnormalities. Patients were assigned to one of four groups according to the type of clotting factor replacement. Twenty patients had received only cryoprecipitate during the two years preceding the evaluation (group I); 11 additional patients were treated predominantly with cryoprecipitate but had also received up to nine bottles of factor VIII concentrate (group II); six patients received factor VIII concentrate (group III); six patients received factor IX concentrate (group IV). There was no clinical or laboratory evidence of immunodeficiency among the 43 patients. The mean absolute number of Th cells was normal in all patient groups, but the mean absolute number of Ts cells was increased compared with controls, both in patients treated with cryoprecipitate and in patients treated with factor VIII or factor IX concentrate. There was no correlation between the Th/Ts ratio and patient age, alanine aminotransferase level, hepatitis serology, in vitro lymphocyte function, or amount of clotting factor administered. Our observations demonstrate that the volunteer or commercial origin of clotting factor replacement cannot fully explain the alterations in lymphocyte subset distribution previously described in patients with hemophilia A.

Description: Human monocytes generate the procoagulant tissue factor (MTF) following exposure to a variety of immune stimuli in vitro. The generation of MTF is modified by T cells, lymphokines, and immunoregulatory lipoproteins, and recent studies have shown that MTF can be activated in an immune- specific manner following exposure to antigen. We have examined the role of serum factors in the regulation of MTF generation. Low concentrations (less than 1%) of heat-inactivated normal human serum greatly enhanced MTF generation in cultures of normal peripheral blood mononuclear cells. The stimulatory effect was observed in cultures of both unstimulated cells and cells exposed to bacterial lipopolysaccharide. Stimulation was not observed at high serum concentrations (greater than 10%) and could not be explained by endotoxin contamination or activation of the assay system. Stimulatory activity was present in plasma and BaSO4-adsorbed plasma as well as autologous and allogeneic serum, was not abolished by removal of serum lipoproteins, and did not require the presence of T cells for its expression. Sera from 28 different normal volunteers were screened for stimulatory activity and demonstrated a wide variation in potency. These results suggest that a potent factor is present in sera that enhances the expression of MTF activity in vitro. This factor is distinct from previously described lipoprotein regulators and may play a role in the initiation of coagulation in both normal hemostasis and pathologic states.

Description: Non-Hodgkin's lymphoma (NHL) is a very rare complication of acute lymphoblastic leukemia (ALL). We present the pathologic, clinical, immunologic, and ultrastructural features of the third reported example of NHL following successfully treated ALL. This white girl developed ALL with predominantly L1 cells at 3.5 yr of age. The lymphoblasts were terminal deoxynucleotidyl transferase (TdT) positive and were non-B, non-T cells. She achieved a complete remission with standard induction therapy and has remained in continuous complete remission. Four and one- third years after the onset of ALL, she developed multifocal, pleomorphic large cell lymphoma of the small bowel, which resulted in episodes of intussusception and obstruction. These pleomorphic and frequently multinucleated lymphoma cells lacked TdT, common ALL antigen, and all tested markers of B cell, T cell, and histiocyte differentiation. Following three small bowel resections, systemic multiagent chemotherapy, and abdominal irradiation, she is currently free of disease.

Description: Leukemia in the newborn is an infrequent disease that has not been well defined using modern laboratory techniques. We describe two infants, one at birth and one at four weeks, with acute lymphoblastic leukemia. The blasts from each patient were studied in great detail, using a battery of cytochemical and immunologic procedures in addition to ultrastructural studies. Immunologic cell marker studies, not previously reported in congenital leukemia, showed the lymphoblasts from each infant to be of the pre-B cell phenotype. Each infant relapsed, one after a 17-week clinical remission and the other after a 44-week remission. The former has died while the latter is in a second remission. The subtype of pre-B cell acute lymphoblastic leukemia (ALL) which in childhood appears to confer an unfavorable prognosis, may have the same significance in neonatal ALL.

Description: The proliferation and differentiation of granulocyte and monocyte progenitor cells (CFU-C) in vitro is dependent on the presence of a group of closely related glycoproteins termed colony-stimulating factors (CSF). In order to investigate the interaction of these factors with CFU-C, we purified CFU-C from the peripheral blood of chronic myeloid leukemia patients with an immune rosette technique using specific monoclonal antibodies (mean 74-fold enrichment, 45% cloning efficiency). Colony formation by purified CFU-C demonstrated an absolute dependence on an exogenous source of CSF. Liquid culture of small aliquots of enriched CFU-C with CSF-containing medium resulted in a rapid, time- and concentration-dependent induction of DNA synthesis as measured by 3H-thymidine incorporation. This specific CSF induction of DNA synthesis by enriched CFU-C was used to develop a microassay system for CSF activity. CSF activity could be reproducibly quantitated in 24–48 hr. The proliferating cells in this assay system were shown to be myeloid progenitor cells by examining the morphology of their progeny and by determining the surface antigen phenotype of the responding cells (Ia+, T3-, B1-, Mo1-). This microassay provides a quantitative assessment of CSF activity that may be useful in the purification of human CSF and in the generation of monoclonal antibodies to CFU-C surface structures.