Cases of cat-associated human plague in the Western US, 1977-1998.

Exposure to cats infected with Yersinia pestis is a recently recognized risk for human plague in the US. Twenty-three cases of cat-associated human plague (5 of which were fatal) occurred in 8 western states from 1977 through 1998, which represent 7.7% of the total 297 cases reported in that period. Bites, scratches, or other contact with infectious materials while handling infected cats resulted in 17 cases of bubonic plague, 1 case of primary septicemic plague, and 5 cases of primary pneumonic plague. The 5 fatal cases were associated with misdiagnosis or delays in seeking treatment, which resulted in overwhelming infection and various manifestations of the systemic inflammatory response syndrome. Unlike infections acquired by flea bites, the occurrence of cat-associated human plague did not increase significantly during summer months. Plague epizootics in rodents also were observed less frequently at exposure sites for cases of cat-associated human plague than at exposure sites for other cases. The risk of cat-associated human plague is likely to increase as residential development continues in areas where plague foci exist in the western US. Enhanced awareness is needed for prompt diagnosis and treatment.

Effects of atmospheric pollution on a Lichenophagous lepidopteran.

Terrestrial epiphytic algae were used to absorb atmospheric pollutants and this material was then fed to larvae of a bagmoth to assess the toxicological effects of the pollutants. Samples of algae were amended with pesticides to simulate agricultural spray drift and with vehicle exhaust gases to simulate road traffic emissions. Larval mortality, immobilization, and feeding were used as the toxicological endpoints. Feeding was the most sensitive of these, but clear dose-response relationships were observed with all three endpoints. The effects of atmospheric pollution on an impacted environment were then investigated using these organisms. Algal samples were collected along a transect that included an area of arable farming, a motorway, and a woodland. Feeding was reduced when the larvae were fed algae from the areas proximal to the arable land and to the motorway. It is suggested that pesticide spray drift and vehicle exhaust emissions may have been the causal agents for these effects.

Use of a beta1 integrin-deficient human T cell to identify beta1 integrin cytoplasmic domain sequences critical for integrin function.

T cell activation rapidly and transiently regulates the functional activity of integrin receptors. Stimulation of CD3/T cell receptor, CD2 or CD28, as well as activation with phorbol esters, can induce within minutes an increase in beta1 integrin-mediated adhesion of T cells to fibronectin. In this study, we have produced and utilized a mutant of the Jurkat T cell line, designated A1, that lacks protein and mRNA expression of the beta1 integrin subunit but retains normal levels of CD2, CD3, and CD28 on the cell surface. Activation-dependent adhesion of A1 cells to fibronectin could be restored upon transfection of a wild-type human beta1 integrin cDNA. Adhesion induced by phorbol 12-myristate 13-acetate-, CD3-, CD2-, and CD28 stimulation did not occur if the carboxy-terminal five amino acids of the beta1 tail were truncated or if either of two well-conserved NPXY motifs were deleted. Scanning alanine substitutions of the carboxy-terminal five amino acids demonstrated a critical role for the tyrosine residue at position 795. The carboxy-terminal truncation and the NPXY deletions also reduced adhesion induced by direct stimulation of the beta1 integrin with the activating beta1 integrin-specific mAb TS2/16, although the effects were not as dramatic as observed with the other integrin-activating signals. These results demonstrate a vital role for the amino-terminal NPXY motif and the carboxy-terminal end of the beta1 integrin cytoplasmic domain in activation-dependent regulation of integrin-mediated adhesion in T cells. Furthermore, the A1 cell line represents a valuable new cellular reagent for the analysis of beta1 integrin structure and function in human T cells.

Structural requirements for beta1 integrin-mediated tyrosine phosphorylation in human T cells.

The beta1 integrin adhesion receptors activate signal transduction pathways that induce tyrosine phosphorylation of a variety of substrates. Increased tyrosine phosphorylation is mediated by the beta1 subunit cytoplasmic domain, which consists of 46 amino acids and contains no intrinsic kinase activity. In the H9 T cell line, beta1 integrin engagement leads to the increased tyrosine phosphorylation of three 105 to 115-kDa substrates that are distinct from focal adhesion kinase (FAK): HEF1 (human enhancer of filamentation 1), a protein with structural homology to p130Cas, and two novel substrates, pp105 and pp115. DNA-mediated gene transfer was used to explore the role of the beta1 cytoplasmic domain in integrin-mediated tyrosine phosphorylation of HEF1, pp105, and pp115 in human T cells. Using a chimeric receptor composed of the cytoplasmic domain of the beta1 integrin subunit and the extracellular and transmembrane domains of the CD2 Ag, we demonstrate that the beta1 cytoplasmic domain is necessary and sufficient for inducing tyrosine phosphorylation of each of these three substrates in H9 T cells. Analysis of a series of beta1 cytoplasmic domain truncations reveals that a truncation of only five amino acids from the carboxyl-terminal end of the beta1 cytoplasmic domain abrogates the ability of the CD2/beta1 chimera to activate tyrosine phosphorylation of HEF1, pp105, or pp115. Thus, the carboxyl-terminal five amino acids, Lys-Tyr-Glu-Gly-Lys (KYEGK), of the beta1 integrin cytoplasmic domain are critical for the coordinate tyrosine phosphorylation of three non-FAK substrates in human T cells.

Baroreflex frequency-response characteristics to aortic depressor and carotid sinus nerve stimulation in rats.

Dynamic cardiovascular regulation depends on baroreflexes and the processing of sensory information. We evaluated the influence of choice of anesthetic on the frequency-response characteristics of the baroreflex of rats by electrical stimulation of two major baroreceptor-containing nerves, the carotid sinus (CSN) and aortic depressor nerves (ADN). The ADN contains baroreceptors alone, and the CSN has both chemoreceptors and baroreceptors. Most studies were performed under pentobarbital sodium (PB; 65 mg/kg) anesthesia. We compared this to a combination of alpha-chloralose (80 mg/kg) and urethan (800 mg/kg) (CU). Stimulus trains were fixed at 60-s periods (0.1-ms shocks, supramaximal intensities, 1-200 Hz) and delivered in steady and burst patterns. Unilateral steady-frequency ADN stimulation in PB-anesthetized rats evoked reflex decreases in mean arterial pressure and heart rate that increased with frequencies between 1 and approximately 10 Hz before reaching a maximum. From 10 to 200 Hz, PB ADN reflex responses were sustained at these maximal levels. Cutting the opposite ADN or both CSNs did not alter ADN baroreflex relationships. Heart rate and mean arterial pressure depressor responses evoked by CSN stimulation in PB-anesthetized rats were smaller compared with ADN stimulation and were biphasic, with small pressor responses at 1 Hz. Maximal CSN depressor responses in PB-anesthetized rats occurred at approximately 20 Hz and were sustained at 20-200 Hz. Baroreflex responses for ADN stimulation in CU-anesthetized rats were similar to those in PB-anesthetized rats. In contrast, in CU-anesthetized rats, maximal CSN responses occurred at 20 Hz but declined at 50-200 Hz. Constant- and burst-stimulation responses were equivalent. The results suggest that rat aortic baroreflex responses are sustained even at very high input frequencies (> 100 Hz). The sustained high-frequency baroreflex responses seem to present a paradox in understanding central integration because other studies show substantial depression of sensory transmission at the first synapse in the nucleus tractus solitarius at frequencies as low as 10 Hz.

Heterogeneous functional expression of calcium channels at sensory and synaptic regions in nodose neurons.

1. In the present study we have taken advantage of the unique anatomy of visceral sensory neurons that enabled us to isolate and examine the role of calcium channel subtypes at the soma, central synaptic terminals, and peripheral sensory endings. 2. N-type calcium channels dominated somatic currents (60%), with lesser (16% and 12%) contributions from P- and L-type channels, respectively, in patch-clamped dispersed nodose neurons using toxins selective for each calcium channel subtype. 3. These toxins also blocked the release of neurotransmitters from these visceral synaptic terminals in a brain stem slice. Similar to the profile at the soma, N-type calcium channels were most responsible for neurotransmission at this central glutamatergic synapse (57%), with P- and L-type channels making small contributions (12% and 11%, respectively). 4. In contrast to the soma and central synapses, these calcium channel toxins failed to affect the sensory transduction at aortic baroreceptor endings. 5. Therefore calcium channel subtypes have dramatically heterogenous distributions in sensory neurons that presumably subserve the specialized functions that occur at different cellular regions.

Tyrosine kinase activity associated with the CD7 antigen: correlation with regulation of T cell integrin function.

Rapid up-regulation of the functional activity of integrin adhesion receptors is a hallmark of T cell activation. Monoclonal antibody engagement of the CD7 antigen on human T cells results in an increase in beta 1 and beta 2 integrin-mediated adhesion within minutes. This suggests that CD7 is capable of transducing intracellular signals, and is consistent with other indirect studies implicating CD7 as a signaling receptor on T cells. In this report, we have explored the intracellular mechanism by which CD7 modulates integrin functional activity. First, CD7-mediated up-regulation of T cell adhesion was found to be unique when compared to phorbol ester stimulation and CD3/T cell receptor cross-linking, based on differences in the kinetics of activation-dependent integrin-mediated adhesion and lack of increase in CD2 functional activity. Second, up-regulation of integrin activity mediated by CD7 cross-linking was completely inhibited by the tyrosine kinase inhibitor herbimycin A. Third, antiphosphotyrosine immunoblotting demonstrated that antibody engagement of CD7 results in a rapid but transient increase in tyrosine phosphorylation in human T cells. Finally, CD7 immunoprecipitates contain in vitro kinase activity, as demonstrated by phosphorylation of a predominant band of 80 kDa and multiple other bands. Phosphoamino acid analysis of the 80-kDa substrate revealed phosphorylation on tyrosine as well as serine and threonine residues. Together, our results suggest that CD7 is associated with tyrosine kinase activity and that this tyrosine kinase activity correlates with the ability of CD7 to regulate T cell integrin functional activity.

Contribution of potassium channels to the discharge properties of rat aortic baroreceptor sensory endings.

The expression of several types of membrane potassium channel at the cell body and central synaptic terminal of the rat aortic arch baroreceptor has been reported by others. It is not known if any of the same channels function at the peripheral sensory terminal of these afferent nerves. Our study examined the effect of three potassium channel blocking agents on the pressure-evoked discharge of such baroreceptors. Thirty-one single unit, regularly discharging baroreceptors were studied using an in vitro aortic arch-aortic nerve preparation. Discharge thresholds and suprathreshold pressure sensitivities were derived from responses of receptors to slowly rising ramps of pressure applied to the aortic arch. Vessel diameter was recorded along with receptor discharge to assess any drug-induced changes in vascular smooth muscle. The blocking agents tested have a range of specificities for classes of potassium channels: tetraethylammonium (TEA), 4-aminopyridine (4-AP) and charybdotoxin. TEA depressed the pressure sensitivity of all baroreceptors tested (n = 3) in a dose-dependent manner. Baroreceptor responses to 4-AP were complex (n = 22) and varied widely across individuals. Three were unaffected by 5 mM 4-AP. Most baroreceptors were generally depressed by 4-AP. Some of the 4-AP effects appeared to be related to actions at vascular smooth muscle. None of the baroreceptors tested (n = 6) was affected by charybdotoxin. The results of selective potassium channel blockade are generally consistent with what would be expected from a sustained depolarization of baroreceptor endings such as has been reported with raising extracellular potassium and probably includes effects of inactivation of other voltage-dependent channels.(ABSTRACT TRUNCATED AT 250 WORDS)

Establishing the incidence of myelodysplastic syndrome.

We studied the incidence of myelodysplastic syndrome (MDS) in a population served by one district general hospital over a 10-year period (1981-90). Cases were sought through investigation of individuals presenting with suggestive peripheral blood findings. 279 new cases were diagnosed in a population rising from 203,000 to 226,000 over the study period. The observed incidence rate did not change over the study period. The crude incidence rate was 12.6/100,000/year. Age-specific incidence rates per 100,000 per year were 0.5 for age < 50, 5.3 for age 50-59, 15 for age 60-69, 49 for age 70-79, and 89 for age 80+. A cohort study of one large general practice revealed several previously unknown and asymptomatic cases, indicating that the observed figures for the population overall may still underestimate the real incidence. We conclude that MDS is more common than reported in previous studies and that incidence continues to rise into very old age. Difficulties in case ascertainment hamper reliable estimates of true incidence.

Characterization of CTLA-4 structure and expression on human T cells.

CTLA-4 is an adhesion receptor expressed on activated T cells. The amino acid sequence of CTLA-4 is related to CD28, and although the function of CTLA-4 remains unknown, it shares several features with CD28, including a common counter-receptor, B7, that is present on Ag-presenting cells. In a recent study we found that CD28 and CTLA-4 were coexpressed at the mRNA level on activated T cells but that only CD28 was expressed on resting T cells. Here we show that within the T cell population, CTLA-4 expression is restricted to the subset of T cells that also express cell surface CD28. CTLA-4 mRNA expression can be induced on quiescent T cells via phorbol ester-mediated activation of protein kinase C but not with calcium ionophore treatment alone. Phorbol ester-induced expression of CTLA-4 mRNA could be enhanced with calcium ionophore treatment, and treatment of cells in this manner resulted in a reciprocal decrease in expression of CD28 mRNA. Ligation of CD28 with monoclonal antibody also resulted in the specific and rapid induction of CTLA-4 mRNA. To study the expression of CTLA-4 at the protein level, a rabbit antiserum against a recombinant protein derived from CTLA-4 cDNA was generated. When activated T cells were labeled with [35S]methionine, the rabbit antiserum precipitated a 41- to 43-kDa protein from whole cell lysates. Similar results were found when detergent-soluble lysates from 125I surface-labeled resting and activated T cells were analyzed by SDS-PAGE. Surprisingly, under the conditions tested, CTLA-4 migrated primarily as a monomer at the cell surface, and could not be shown to exist as a disulfide-bonded homodimer or as a heterodimer consisting of CTLA-4 and CD28. These results suggest that B7 can bind to T cells via distinct receptor complexes consisting of either CD28 or CTLA-4, and that these complexes may potentially mediate distinct biologic functions. Further, the present results suggest that noncovalent interactions might mediate association of CTLA-4 and/or CD28 at the cell surface.

Regulatory mechanisms underlying T cell integrin receptor function.

Adhesion molecules allow lymphocytes to interact with and respond to the extracellular environment. Since these interactions must be essentially transient in nature, the function of lymphocyte adhesion molecules must be precisely regulated. Studies of integrin receptors vividly illustrate the various mechanisms by which the function of these adhesion molecules can be regulated. These include: (1) activation-dependent changes in functional activity; (2) changes in levels of expression due to differentiation events; (3) cell-specific differences in integrin binding; and (4) differential binding to distinct ligands by the same integrin. These mechanisms provide highly precise and specific modes of regulating lymphocyte interactions with a wide variety of potential counter-receptors and ligands.

Functional analysis of the SH2 and SH3 domains of the lck tyrosine protein kinase.

p56lck is a lymphoid cell-specific member of the src family of cytoplasmic tyrosine kinases. In helper and cytotoxic T cells it is physically associated with the CD4 and CD8 surface antigens and appears to play a role in signal transduction during T-cell activation. p56lck contains both an SH3 and an SH2 Src homology domain. Such domains have been suggested to play a role in the regulation of the activity or function of both receptor and non-receptor tyrosine protein kinases. Deletion of either or both domains in p56lck was found here to activate the protein and to lead to increased phosphorylation of the autophosphorylation site, Tyr-394, in vivo. These findings are consistent with the hypothesis that these domains participate in repression of the kinase activity of p56lck. None of the deleted forms was capable of transformation of fibroblasts. Deletion of the SH3 domain of a constitutively activated form of p56lck, p56lckF505, did not diminish the transforming activity of this protein. This suggests that this domain is dispensable for the transformation of fibroblasts by p56lck. In contrast, deletion of the SH2 domain abolished the transforming potential of activated p56lckF505. However, interpretation of this effect is made somewhat difficult because the mutation also lowered the steady-state abundance of the protein.

The effect of residence and social class on dental caries experience in 15-16-year-old children living in three towns (natural fluoride, adjusted fluoride and low fluoride) in the north east of England.

Caries experience in 1374 children aged 15-16 years from three towns in the north east of England with varying concentrations of fluoride in drinking water, was determined. The mean DMFT values for 15-year-old continuous residents was 1.7 in Hartlepool (natural F 1.0-1.3 ppm), 2.5 in Newcastle (F adjusted to 1.0 ppm) and 3.3 in Middlesbrough (F = 0.2 ppm). Forty per cent of Hartlepool 15-year-olds were caries free, compared with 30% in Newcastle and 24% in Middlesbrough. Caries prevalence for both Hartlepool and Newcastle 'continuous residents' was lower than for non-continuous residents, whereas in Middlesbrough, the low fluoride area, non-continuous residents had a lower DMF value than those who had lived in Middlesbrough all their lives. There was a slight trend in both Newcastle and Middlesbrough for DMFT values to increase from social class I to social class V, but no discernable trend was observed in Hartlepool. The results for Hartlepool 15-year-olds were very similar to those reported by Weaver in 1949.

Effects of diet forage-to-concentrate ratio and intake on energy metabolism in growing beef heifers: net nutrient metabolism by visceral tissues.

Effects of diet forage-to-concentrate ratio and intake on metabolism of nutrients by portal-drained viscera (PDV), liver and kidneys were measured in seven growing beef heifers. Isonitrogenous pelleted diets containing approximately 75% alfalfa or 75% concentrate were fed every 2 h at two metabolizable energy intakes. Greater intake increased net PDV removal of glucose and urea nitrogen and net PDV release of NH3N, alpha-amino nitrogen (AAN), alanine and lactate. Lower net PDV release of NH3N, liver removal of NH3N and AAN and liver release of urea nitrogen accompanied lower N intake and digestion in heifers fed the 75% concentrate vs. the 75% alfalfa diet. Lower net PDV glucose removal resulted in greater total splanchnic glucose release when heifers were fed the 75% concentrate vs. the 75% alfalfa diet. In addition, net PDV, liver and total splanchnic release of beta-hydroxybutyrate (BOHB) were lower when heifers were fed the 75% concentrate vs. the 75% alfalfa diet. Greater tissue energy retention in heifers fed the 75% concentrate vs. the 75% alfalfa diet at equal metabolizable energy intake accompanied differences in net PDV metabolism of glucose, NH3N and BOHB and liver metabolism of nitrogenous compounds, BOHB and lactate.

Effects of diet forage-to-concentrate ratio and intake on energy metabolism in growing beef heifers: whole body energy and nitrogen balance and visceral heat production.

Effects of diet forage-to-concentrate ratio and intake on balances of energy and nitrogen and portal-drained viscera (PDV), liver and kidney blood flow and O2 consumption were measured in seven growing beef heifers. Isonitrogenous pelleted diets containing approximately 75% alfalfa or 75% concentrate were fed daily as 12 equal meals every 2 h at two isoenergetic metabolizable energy intakes. A split-plot design was used, with 4 wk for adaptation to diet followed by 3-wk intake periods within 6-wk diet periods. Heifers consumed and digested less dry matter, energy and nitrogen when fed the 75% concentrate vs. 75% alfalfa diet at equal metabolizable energy. Heifers fed the 75% concentrate diet produced less heat energy and retained more tissue energy than when fed the 75% alfalfa diet. Blood flow for PDV, liver and kidneys increased with intake and was greater when heifers were fed the 75% alfalfa vs. 75% concentrate diet. Increased PDV and liver O2 uptake accounted for 44 and 72% of heat increment for the 75% concentrate and 75% alfalfa diets, respectively. Greater PDV uptake of O2 accounted for 72% of the decrease in tissue energy of heifers fed the 75% alfalfa vs. 75% concentrate diet at equal metabolizable energy.

Suggestive hypnotherapy for nocturnal bruxism: a pilot study.

Although one can find many case reports of hypnotherapy for bruxism, there is a paucity of scientific research on the subject. This study describes the use of suggestive hypnotherapy and looks at its effectiveness in treating bruxism. Eight subjects who reported bruxism with symptoms such as muscle pain and complaints of bruxing noise from sleep partners were accepted into the study. An objective baseline of the bruxing was established using a portable electromyogram (EMG) detector attached over the masseter muscle during sleep. Hypnotherapy was then employed. Both self-reports and posttreatment EMG recordings were used to evaluate the hypnotherapy. Long-term effects were evaluated by self-reports only. The bruxers showed a significant decrease in EMG activity; they also experienced less facial pain and their partners reported less bruxing noise immediately following treatment and after 4 to 36 months.

Net absorption of macrominerals by portal-drained viscera of lactating Holstein Cows and beef steers.

Net absorption of macrominerals by portal-drained viscera of cattle was measured in two experiments. In Experiment 1, net portal-drained visceral and liver flux of Na, K, Ca, P, and Mg were measured in four lactating Holstein cows fed a 60:40 corn silage:concentrate diet ad libitum and milked at 12-h intervals. Twelve measurements of net flux (venous-arterial concentration difference x blood plasma flow) were obtained hourly at 4 and 8 wk postpartum. Intake of DM and milk yield averaged 15.6 and 32.2 kg/d, respectively. Excluding Na, net absorption of these macrominerals was measurable across portal-drained viscera. Flux of Na was negative at wk 4 postpartum (net removal from blood) and positive at wk 8 (net absorption into blood). Excluding Mg, differences in venous-arterial concentrations for macrominerals across the liver were not different from zero. In Experiment 2, net portal-drained visceral flux of Na, K, Ca, P, and Mg was partitioned into stomach and poststomach sections in two Hereford steers. Poststomach tissues accounted for essentially all such absorption of K, Ca, and P and, on a net basis, removed Na and Mg. Stomach tissues absorbed Mg and small amounts of K, Ca, and P. Net stomach flux of Na was positive, denoting net absorption, but highly variable. Net portal-drained visceral absorption of K increased with intake in both experiments. Net portal-drained visceral absorption of P was greater than total intake in both experiments, reflecting recycling of P via saliva and other digestive secretions.

Food intake regulation by diet-induced thermogenesis.

Diet-induced thermogenesis represents energy wasted in the assimilation of food. Since this source of energy loss is variable, it has long been linked to obesity in people who assimilate food at high efficiency, the saved energy converting to adipose tissue. In this report we provide evidence to dispel that notion and, instead, show that the variability in DIT may be linked to the amount of food one consumes during the course of a meal. We call upon the well known temperature sensitive neurons in the hypothalamus acting in concert with the ventromedial nuclei to present a new version of an old 'thermostatic hypothesis' of food intake regulation.

Changes in the relative abundance of type I and type II lck mRNA transcripts suggest differential promoter usage during T-cell development.

The lck gene, which encodes the lymphoid cell-specific tyrosine protein kinase p56lck, is expressed from two widely separated promoters. The proximal promoter gives rise to a type I lck transcript, and the distal promoter gives rise to a type II transcript. We found that the ratio of the two transcripts changed during T-cell maturation. Type I lck mRNA was twofold more abundant than the type II transcript in early fetal thymocytes. In the adult, the type I and type II lck mRNAs were present in approximately equal amounts in immature thymocytes expressing the heat-stable antigen. In contrast, there was five- to ninefold more type II lck than type I lck mRNA in more mature thymocytes that did not express the heat-stable antigen and in splenic T cells. This change in relative transcript abundance probably reflects activation of the distal promoter and inactivation of the proximal promoter during T-cell maturation in the thymus. It is possible that the two promoters are regulated by different trans-acting factors whose expression is regulated during T-cell maturation.

Mechanisms of microbial movement in subsurface materials.

The biological factors important in the penetration of Escherichia coli through anaerobic, nutrient-saturated, Ottawa sand-packed cores were studied under static conditions. In cores saturated with galactose-peptone medium, motile strains of E. coli penetrated four times faster than mutants defective only in flagellar synthesis. Motile, nonchemotactic mutants penetrated the cores faster than did the chemotactic parental strain. This, plus the fact that a chemotactic galactose mutant penetrated cores saturated with peptone medium at the same rate with or without a galactose gradient, indicates that chemotaxis may not be required for bacterial penetration through unconsolidated porous media. The effect of gas production on bacterial penetration was studied by using motile and nonmotile E. coli strains together with their respective isogenic non-gas-producing mutants. No differences were observed between the penetration rates of the two motile strains through cores saturated with peptone medium with or without galactose. However, penetration of both nonmotile strains was detected only with galactose. The nonmotile, gas-producing strain penetrated cores saturated with galactose-peptone medium five to six times faster than did the nonmotile, non-gas-producing mutant, which indicates that gas production is an important mechanism for the movement of nonmotile bacteria through unconsolidated porous media. For motile strains, the penetration rate decreased with increasing galactose concentrations in the core and with decreasing inoculum sizes. Also, motile strains with the faster growth rates had faster penetration rates. These results imply that, for motile bacteria, the penetration rate is regulated by the in situ bacterial growth rate.(ABSTRACT TRUNCATED AT 250 WORDS)