Variability in pulmonary function following rapid altitude ascent to the Amundsen-Scott South Pole station.

The impact of acute altitude exposure on pulmonary function is variable. A large inter-individual variability in the changes in forced expiratory flows (FEFs) is reported with acute exposure to altitude, which is suggested to represent an interaction between several factors influencing bronchial tone such as changes in gas density, catecholamine stimulation, and mild interstitial edema. This study examined the association between FEF variability, acute mountain sickness (AMS) and various blood markers affecting bronchial tone (endothelin-1, vascular endothelial growth factor (VEGF), catecholamines, angiotensin II) in 102 individuals rapidly transported to the South Pole (2835 m). The mean FEF between 25 and 75% (FEF(25-75)) and blood markers were recorded at sea level and after the second night at altitude. AMS was assessed using Lake Louise questionnaires. FEF(25-75) increased by an average of 12% with changes ranging from -26 to +59% from sea level to altitude. On the second day, AMS incidence was 36% and was higher in individuals with increases in FEF(25-75) (41 vs. 22%, P = 0.05). Ascent to altitude induced an increase in endothelin-1 levels, with greater levels observed in individuals with decreased FEF(25-75). Epinephrine levels increased with ascent to altitude and the response was six times larger in individuals with decreased FEF(25-75). Greater levels of endothelin-1 in individuals with decreased FEF(25-75) suggest a response consistent with pulmonary hypertension and/or mild interstitial edema, while epinephrine may be upregulated in these individuals to clear lung fluid through stimulation of ß(2)-adrenergic receptors.

Alterations in central hemodynamic in patients with COPD after acute high intensity exercise.

The present study investigated the relationship between central hemodynamics and lung function and the response to an acute bout of exercise in COPD. METHODS: Based on the severity of COPD, moderate group (MOD, n = 12) and more mild group (MLD, n = 12) underwent central hemodynamic assessments pre- and post-peak exercise. RESULTS: In the entire cohort (n = 24), central diastolic blood pressure (cDBP) was associated with pulmonary function. Post-exercise, cDBP remained elevated (p < 0.01), however, peripheral diastolic blood pressure (pDBP) was reduced (p = 0.02). Prior to exercise, the MOD showed higher cDBP and heart rate (HR) than the MLD (p = 0.02 and p = 0.01, respectively), but no difference in central aortic/arterial stiffness (p > 0.05). These findings remained similar post-exercise. CONCLUSION: Central diastolic blood pressure is linked to pulmonary function in COPD and it is elevated after exercise-induced reductions in pDBP. Central diastolic blood pressure is higher in the MOD than the MLD, however, there was no difference in central aortic/arterial stiffness between groups.

Generation of stabilized microbubbles in seawater.

Bubbles of less than 1 micrometer and as large as 13.5 micrometers in diameter, stabilized by an apparent compression of substances sorbed onto their surfaces, were examined to determine their physical and temporal stability. Their ease of formation is related to the qualities of the water in which they are formed. Their presence in the water column must now be considered when interpreting acoustic data gathered to determine marine bubble populations.

A cytoskeletal mechanism for Ca2+ channel metabolic dependence and inactivation by intracellular Ca2+.

Many different types of voltage-dependent Ca2+ channels inactivate when intracellular ATP declines or intracellular Ca2+ rises. An inside-out, patch-clamp technique was applied to the Ca2+ channels of Lymnaea neurons to determine the mechanism(s) underlying these two phenomena. Although no evidence was found for a phosphorylation mechanism, agents that act on the cytoskeleton were found to alter Ca2+ channel activity. The cytoskeletal disrupters colchicine and cytochalasin B were found to speed Ca2+ channel decline in ATP, whereas the cytoskeletal stabilizers taxol and phalloidin were found to prolong Ca2+ channel activity without ATP. In addition, cytoskeletal stabilizers reduced Ca(2+)-dependent channel inactivation, suggesting that both channel metabolic dependence and Ca(2+)-dependent inactivation result from a cytoskeletal interaction.

Primary structure and function of an A kinase anchoring protein associated with calcium channels.

Rapid, voltage-dependent potentiation of skeletal muscle L-type calcium channels requires phosphorylation by cAMP-dependent protein kinase (PKA) anchored via an A kinase anchoring protein (AKAP). Here we report the isolation, primary sequence determination, and functional characterization of AKAP15, a lipid-anchored protein of 81 amino acid residues with a single amphipathic helix that binds PKA. AKAP15 colocalizes with L-type calcium channels in transverse tubules and is associated with L-type calcium channels in transfected cells. A peptide fragment of AKAP15 encompassing the RII-binding domain blocks voltage-dependent potentiation. These results indicate that AKAP15 targets PKA to the calcium channel and plays a critical role in voltage-dependent potentiation and regulation of skeletal muscle contraction. The expression of AKAP15 in the brain and heart suggests that it may mediate rapid PKA regulation of L-type calcium channels in neurons and cardiac myocytes.

Lentivirus-mediated platelet-derived factor VIII gene therapy in murine haemophilia A.

BACKGROUND: Previous studies from our laboratory have demonstrated that lineage-targeted synthesis of factor VIII (FVIII) under the direction of the platelet-specific integrin alphaIIb gene promoter (2bF8) can correct the murine haemophilia A phenotype even in the presence of high titer inhibitory antibodies in a transgenic mouse model. OBJECTIVE: In this study, we assessed the efficacy of using a genetic therapy approach to correct haemophilia A in FVIII-deficient (FVIII(null)) mice by transplantation of bone marrow (BM) transduced with a lentivirus (LV)-based gene transfer cassette encoding 2bF8. RESULTS: Functional FVIII activity (FVIII:C) was detected in platelet lysates from treated mice and the levels were similar to 2bF8 heterozygous transgenic mice. Mice transplanted with 2bF8 LV-transduced BM survived tail clipping and we did not detected inhibitory or non-inhibitory FVIII antibodies over the period of this study (11 months). Furthermore, BM transferred from the primary transplant recipients into FVIII(null) secondary recipients demonstrated sustained platelet-FVIII expression leading to correction of the haemophilia A phenotype showing that gene transfer occurred within long-term repopulating haematopoietic stem cells. CONCLUSIONS: These results demonstrate that ectopic expression of FVIII in platelets by lentivirus-mediated bone marrow transduction/transplantation may be a promising strategy for gene therapy of haemophilia A in humans.

Increased expression of the cardiac L-type calcium channel in estrogen receptor-deficient mice.

Steroid hormones control the expression of many cellular regulators, and a role for estrogen in cardiovascular function and disease has been well documented. To address whether the activity of the L-type Ca2+ channel, a critical element in cardiac excitability and contractility, is altered by estrogen and its nuclear receptor, we examined cardiac myocytes from male mice in which the estrogen receptor gene had been disrupted (ERKO mice). Binding of dihydropyridine Ca2+ channel antagonist isradipine (PN200-110) was increased 45.6% in cardiac membranes from the ERKO mice compared to controls, suggesting that a lack of estrogen receptors in the heart increased the number of Ca2+ channels. Whole-cell patch clamp of acutely dissociated adult cardiac ventricular myocytes indicated that Ca2+ channel current was increased by 49% and action potential duration was increased by 75%. Examination of electrocardiogram parameters in ERKO mice showed a 70% increase in the QT interval without significant changes in PQ or QRS intervals. These results show that the membrane density of the cardiac L-type Ca2+ channel is regulated by the estrogen receptor and suggest that decreased estrogen may lead to an increase in the number of cardiac L-type Ca2+ channels, abnormalities in cardiac excitability, and increased risk of arrhythmia and cardiovascular disease.

Effect of Army basic training in sickle-cell trait.

In order to determine if sickle-cell trait (SCT) represents an inherent adverse effect on response to training, we objectively evaluated exercise performance in 22 healthy, black men with SCT (hemoglobin AS) and 15 controls (hemoglobin AA) before and after seven weeks of army basic training at an altitude of 1270 m. An incremental exercise test to exhaustion on a cycle ergometer was used. Before basic training, peak exercise measurements did not reveal significant differences between groups other than a slightly lower, albeit significant, value for oxygen uptake (VO2) per kilogram (42 +/- 1 vs 45 +/- 1.4 mL/min per kilogram) for the SCT group. Both groups experienced modest overall cardiovascular improvement reflected in both peak and submaximal exercise responses. No statistically significant difference was observed between the SCT and the control groups at the end of basic training for any of the measured variables at peak exercise, including power (258 +/- 6 vs 266 +/- 9W), VO2 (3.24 +/- .06 vs 3.36 +/- .16 L/min), VO2 per kilogram (46 +/- 0.7 vs 46 +/- 1.2), minute ventilation (138 +/- 4 vs 147 +/- 8 L/min), heart rate (185 +/- 2 vs 184 +/- 3 beats per minute), oxygen pulse (17.6 +/- .3 vs 18.4 +/- 1 mL/min per beat), as well as anaerobic threshold (1.81 +/- .04 vs 1.80 +/- .06 L/min), respectively. No medical problems directly attributed to SCT were reported; it remains uncertain, however, whether a seizure experienced by one of the other SCT basic trainees after a two-mile run was SCT related. The results of this study would, therefore, suggest that for the majority of individuals who possess SCT, the response to the moderate training regimen provided by army basic training is not impaired.

Sleep disordered breathing and acute mountain sickness in workers rapidly transported to the South Pole (2835 m).

BACKGROUND: Sleep disordered breathing may be a risk factor for high altitude illness. Past Antarctic sleep studies suggest that rapid transport from sea level (SL) to the Amundsen Scott South Pole Station (SP, 2835 m) increases risk of Acute Mountain Sickness (AMS). We analyzed sleep studies in 38 healthy polar workers to explore the association between sleep disordered breathing and AMS after rapid transport to the South Pole. METHODS: Subjects completed a baseline questionnaire, performed basic physiology tests, and were evaluated for AMS and medication use using an extended Lake Louise Questionnaire (LLQ) during their first week at the South Pole. Participants were included in this study if they took no medications and underwent polysomnography on their first nights at Sea Level and the South Pole using the Vivometrics LifeShirt(®). Within group changes were assessed with Wilcoxon signed rank tests and between group differences were assessed with Kruskal-Wallis rank sum tests. RESULTS: Overall, 21/38 subjects met criteria for AMS at some time on or prior to the third morning at the South Pole. Subjective poor sleep quality was reported by both AMS (65%) and no AMS (41%) groups. The Apnea Hypopnea Index (AHI) increased significantly in both the AMS and no AMS groups, but the difference in the increase between the two groups was not statistically significant. Increased AHI was not associated with increased AMS symptoms. Previous altitude illness (p=0.06) and residence at low altitudes (p = 0.02) were risk factors for AMS. CONCLUSION: AMS was not significantly associated with sleep architecture changes or increased AHI. However, AHI sharply increased at South Pole (19/38 participants) primarily due to central apneas. Those developing AMS were more likely to have experienced previous problems at altitude and reported living at lowland altitudes within the 3 months prior to rapid transport to the South Pole than those without AMS.

Synaptic connectivity of local circuit neurons in laminae III and IV of hamster spinal cord.

The present study was undertaken to examine the morphological bases of local synaptic interactions between dorsal horn interneurons. Seven interneurons responding to innocuous mechanical stimuli were intracellularly recorded in lamina III/IV of an isolated preparation of hamster spinal cord with partially intact innervation from an excised patch of hairy skin. Axonal arborizations were stained with horseradish peroxidase (HRP) and examined with an electron microscope. Five cells had extensive synaptic terminations (375-1,785 boutons/axon) with localized distributions (rostrocaudal distance, 425-1,251 microns) overlapping the dendritic trees. Two cells gave rise to deep stem axons that bifurcated into rostrocaudal daughter branches with collaterals ventral to the parent cell bodies (79-661 boutons/axon). Axons of local interneurons were thinly myelinated and formed terminal and en passant enlargements (mean [+/- S.D.] diameter = 0.88 +/- 0.24 microns, n = 157) containing clear, round vesicles 20-60 nm in diameter. Collateral branches of deep axon cells produced round, vesicle-containing boutons comparable in diameter (0.93 +/- 0.22 microns, n = 31) to local axon cells. Both types of interneurons formed asymmetric synaptic contacts with dendritic profiles, but not with cell bodies or axon terminals. Postsynaptic profiles contained sparse ribosomes and had a mean diameter of 1.0 +/- 0.5 microns (n = 49), significantly smaller than a population of identified proximal dendrites (2.3 +/- 0.9 microns, n = 47). HRP-labeled boutons were rarely (5/45 or 11%) in synaptic contact with more than one profile. We conclude that lamina III/IV interneurons make axodendritic synapses predominantly with distal dendrites. Thus, terminations of deep dorsal horn interneurons appear to have a postsynaptic distribution overlapping with axodendritic contacts formed by several functional classes of cutaneous sensory fibers signaling innocuous mechanical stimuli. Such overlap suggests that local spinal networks selectively and strongly influence afferent signals at initial stages of somatosensory integration.

Induction of megakaryocytes to synthesize and store a releasable pool of human factor VIII.

von Willebrand factor (VWF) is a complex plasma glycoprotein that modulates platelet adhesion at the site of a vascular injury, and it also serves as a carrier protein for factor (F)VIII. As megakaryocytes are the only hematopoietic lineage to naturally synthesize and store VWF within alpha-granules, this study was performed to determine if expression of a FVIII transgene in megakaryocytes could lead to trafficking and storage of FVIII with VWF in platelet alpha-granules. Isolex selected CD34+ cells from human G-CSF mobilized peripheral blood cells (PBC) and murine bone marrow were transduced with a retrovirus encoding the B-domain deleted form of human FVIII (BDD-FVIII). Cells were then induced with cytokines to form a population of multiple lineages including megakaryocytes. Chromogenic analysis of culture supernatant from FVIII-transduced human cells demonstrated synthesis of functional FVIII. Treatment of cells with agonists of platelet activation (ADP, epinephrine, and thrombin receptor-activating peptide) resulted in the release of VWF antigen and active FVIII into the supernatant from transduced cells. Immunofluorescence analysis of cultured human and murine megakaryocytes revealed a punctate pattern of staining for FVIII that was consistent with staining for VWF. Electron microscopy of transduced megakaryocytes using immunogold-conjugated antibodies colocalized FVIII and VWF within the alpha-granules. FVIII retained its association with VWF in human platelets isolated from the peripheral blood of NOD/SCID mice at 2-6 weeks post-transplant of transduced human PBC. These results suggest feasibility for the development of a locally inducible secretory pool of FVIII in platelets of patients with hemophilia A.

Cardiopulmonary and gas exchange responses to acute strenuous exercise at 1,270 meters in sickle cell trait.

The impact of strenuous exercise and environmental hypoxia on sickle cell trait (SCT) remains controversial. To determine if these factors induce cardiopulmonary and gas exchange abnormalities in SCT, healthy, young black male volunteers, 25 with SCT (HbAS) and 16 control subjects (HbAA), were evaluated during incremental and steady-state exercise tests using a cycle ergometer at 1,270 meters and 24 degrees C. Peak incremental exercise values for power (242 +/- 7 versus 253 +/- 10 watts), oxygen consumption (3.08 +/- 0.1 versus 3.26 +/- 0.14 liters/minute), heart rate (188 +/- 2 versus 189 +/- 3 beats/minute), minute ventilation (129 +/- 4.6 versus 144 +/- 7.7 liters/minute), oxygen pulse (16.4 +/- 0.5 versus 17.3 +/- 0.8 ml/beat), and respiratory exchange ratio (1.31 +/- 0.01 versus 1.33 +/- 0.02) revealed no significant differences (p less than 0.05) between the SCT and control groups, respectively. Peak incremental exercise values for arterial oxygen tension (82 +/- 1.7 versus 82 +/- 2.2 mm Hg), arterial carbon dioxide tension (32 +/- 0.7 versus 31 +/- 0.9 mm Hg), and alveolar-arterial oxygen pressure differences (19 +/- 1.4 versus 21 +/- 1.9 mm Hg) were similar for the SCT and control groups, respectively. Steady-state exercise results corroborate incremental exercise findings. It is concluded that cardiopulmonary and gas exchange responses to a brief period of strenuous exercise performed at low altitude at 24 degrees C in a well-characterized SCT sample of recruits were within normal limits and comparable to those of a carefully selected control sample.

Effect of moderate inspiratory hypoxia on exercise performance in sickle cell trait.

In previous work (Weisman IM, Zeballos RJ, Johnson BD: Cardiopulmonary and gas exchange responses to acute strenuous exercise at 1,270 meters in sickle cell trait. Am J Med 1988; 84: 377-383), no significant differences in cardiopulmonary and gas exchange responses to acute, strenuous exercise were observed between volunteers with sickle cell trait (SCT) and control subjects at an altitude of 1,270 meters. The current study was designed to evaluate the effect of a greater hypoxic stimulus on the response of healthy, black male basic recruits, 11 with SCT (HbAS) and 11 control subjects, to acute strenuous exercise. Simulated 2,300-meter and simulated sea-level conditions were achieved by adjustment of the fraction of inspired oxygen (simulated condition of 2,300 meters equal to 18 percent; simulated sea-level condition equal to 24 percent) at the same barometric pressure (656 mm Hg). For each simulated condition, the subjects performed an incremental exercise test to exhaustion on a cycle ergometer. One steady-state exercise test with radial arterial access for arterial blood gases was performed under each condition on Day 2. Peak incremental exercise values for oxygen consumption (2.9 versus 2.81 liters/minute), heart rate (189 versus 187 beats/minute), oxygen pulse (15.4 versus 15.1 ml/beat), and anaerobic threshold (1.59 versus 1.62 liters/minute), at the simulated 2,300-meter height revealed no significant differences between men with SCT and control subjects, respectively. A 5 to 9 percent decrement in exercise performance at the simulated 2,300-meter level compared with exercise performance at the simulated sea-level condition was noted for both groups. Steady-state exercise values for arterial oxygen tension (64 versus 65 mm Hg), arterial oxygen saturation (90 versus 90 percent), alveolar-arterial oxygen pressure difference (22 versus 21 mm Hg), and physiologic dead space to tidal volume ratio (12 versus 11) at the simulated condition of 2,300 meters were similar for the SCT and control groups, respectively. It is concluded that in a moderate hypoxic environment, the cardiopulmonary and gas exchange responses of persons with SCT during brief episodes of exhaustive exercise were comparable to those of control subjects.

Potential antiarthritic agents. 2. Benzoylacetonitriles and beta-aminocinnamonitriles.

Benzoylacetonitrile and beta-aminocinnamonitrile are shown to possess potent antiinflammatory activity in the rat adjuvant arthritis model. In a series of phenyl-substituted analogues, only o-, m-, and p-fluorobenzoylacetonitrile and m- and p-fluoro-beta-aminocinnamonitrile retained activity. Additionally, beta-amino-2- and beta-amino-3-thiopheneacrylonitrile and beta-oxo-2- and beta-oxo-3-thiophenepropionitrile exhibited similar activity. These agents are not believed to be acting via prostaglandin synthetase inhibition. The metabolic profile of benzoylacetonitrile is also described.

Preparation of substituted N-phenyl-4-aryl-2-pyrimidinamines as mediator release inhibitors.

The role of immunologically released mediators, such as histamine, leukotrienes, and platelet-activating factor, is well-established for asthma and other allergic disorders. Developing therapeutic agents which would block mediator release from mast cells and other relevant cell types would provide a rational approach to asthma therapy. Using human basophil as a screen, a series of 4-aryl-2-(phenylamino)pyrimidines was found which inhibited mediator release. These compounds were prepared by condensing acetyl heterocycles with dimethylformamide dimethyl acetal to form enaminones which are cyclized with aryl guanidines to give pyrimidines. After examining a large number of analogs, N-[3-(1H-imidazol-1-yl)phenyl]-4-(2-pyridinyl)-2- pyrimidinamine (1-27) was chosen for toxicological evaluation.

Analogues of platelet activating factor. 7. Bis-aryl amide and bis-aryl urea receptor antagonists of PAF.

A series of bis-aryl amide (13-57 and 66-81) and bis-aryl urea (58 and 85) antagonists of platelet-activating factor (PAF) was prepared that contain, separating the two aromatic rings, linear amide linkages of the form -(CH2)nCONH- (n = 0-2), -OCH2CONH-, and -(CH2)nNHCO- (n = 0-1), branched amide linkages of the form -(CH2)nN(COR)- (n = 1-3, R = CH3 or n-C3H7), and -N(COCH3)CH2-, and urea linkages of the form -NHCONH- and -CH2N(CONHCH3)-. These compounds were examined for their ability to inhibit PAF-induced platelet aggregation of rabbit platelets. These in vitro data were compared to similar data obtained for a number of known PAF antagonists. The compounds were evaluated in vivo, in the mouse, for their ability to prevent death induced by a lethal challenge of PAF. The relationships between the biological activity and the nature, lipophilicity, and position of substituents of the aromatic rings were studied. Best activity was observed for compounds having linkages of the type -CH2CONH-, -CH2N(COR)-, and -CH2NHCO-. Many of these compounds inhibit PAF-induced platelet aggregation with IC50's under 1 microM.

Imidazo[1,5-d][1,2,4]triazines as potential antiasthma agents.

By using inhibition of histamine release from antigen-challenged, sensitized human basophils as a means of identifying a potentially prophylactic drug for the treatment of asthma, a series of substituted imidazo[1,5-d][1,2,4]triazines were found, which were active. These compounds were prepared by treating imidazolecarboxaldehydes with excess Grignard agent and then oxidizing the resulting alcohols to ketones with Jones reagent. Pyrolysis of a mixture of ketone and methyl carbazate at 200 degrees C in diphenyl ether produced the desired imidazo[1,5-d][1,2,4]triazines. Those compounds with the greatest basophil activity were tested for in vivo activity in the mouse passive cutaneous anaphylaxis (PCA) and the guinea pig passive anaphylaxis tests. The best compounds, 1-ethyl-8-methyl-6-propylimidazo[1,5-d][1,2,4]triazin-4(3H)- one (4-17) and 1,8-dimethyl-6-propylimidazo[1,5-d][1,2,4]triazin-4-(3H)-one (4-16) were chosen for further study.

4-Anilino-6,7-dialkoxyquinoline-3-carbonitrile inhibitors of epidermal growth factor receptor kinase and their bioisosteric relationship to the 4-anilino-6,7-dialkoxyquinazoline inhibitors.

The synthesis and SAR of a series of 4-anilino-6, 7-dialkoxyquinoline-3-carbonitrile inhibitors of epidermal growth factor receptor (EGF-R) kinase are described. Condensation of 3, 4-dialkoxyanilines with ethyl (ethoxymethylene)cyanoacetate followed by thermal cyclization gave, regiospecifically, 6,7-dialkoxy-4-oxo-1, 4-dihydroquinoline-3-carbonitriles. Chlorination (POCl(3)) followed by the reaction with substituted anilines furnished the 4-anilino-6, 7-dialkoxyquinoline-3-carbonitrile inhibitors of EGF-R kinase. An alternate synthesis of these compounds starts with a methyl 3, 4-dialkoxybenzoate. Nitration followed by reduction (Fe, NH(4)Cl, MeOH-H(2)O) gave a methyl 2-amino-4,5-dialkoxybenzoate. Amidine formation using DMF-acetal followed by cyclization using LiCH(2)CN furnished a 6,7-dialkoxy-4-oxo-1,4-dihydroquinoline-3-carbonitrile, which was transformed as before. Compounds containing acid, ester, amide, carbinol, and aldehyde groups at the 3-position of the quinoline ring were also prepared for comparison, as were several 1-anilino-6,7-dimethoxyisoquinoline-4-carbonitriles. The compounds were evaluated for their ability to inhibit the autophosphorylation of the catalytic domain of EGF-R. The SAR of these inhibitors with respect to the nature of the 6,7-alkoxy groups, the aniline substituents, and the substituent at the 3-position was studied. The compounds were further evaluated for their ability to inhibit the growth of cell lines that overexpress EGF-R or HER-2. It was found that 4-anilinoquinoline-3-carbonitriles are effective inhibitors of EGF-R kinase with activity comparable to the 4-anilinoquinazoline-based inhibitors. A new homology model of EGF-R kinase was constructed based on the X-ray structures of Hck and FGF receptor-1 kinase. The model suggests that with the quinazoline-based inhibitors, the N3 atom is hydrogen-bonded to a water molecule which, in turn, interacts with Thr 830. It is proposed that the quinoline-3-carbonitriles bind in a similar manner where the water molecule is displaced by the cyano group which interacts with the same Thr residue.

6-Substituted-4-(3-bromophenylamino)quinazolines as putative irreversible inhibitors of the epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor (HER-2) tyrosine kinases with enhanced antitumor activity.

A series of new 6-substituted-4-(3-bromophenylamino)quinazoline derivatives that may function as irreversible inhibitors of epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor (HER-2) tyrosine kinases have been prepared. These inhibitors have, at the C-6 position, butynamide, crotonamide, and methacrylamide Michael acceptors bearing water-solublilizing substituents. These compounds were prepared by acylation of 6-amino-4-(3-bromophenylamino)quinazoline with unsaturated acid chlorides or mixed anhydrides. We show that attaching a basic functional group onto the Michael acceptor results in greater reactivity, due to intramolecular catalysis of the Michael addition and/or an inductive effect of the protonated basic group. This, along with improved water solubility, results in compounds with enhanced biological properties. We present molecular modeling and experimental evidence that these inhibitors interact covalently with the target enzymes. One compound, 16a, was shown to have excellent oral activity in a human epidermoid carcinoma (A431) xenograft model in nude mice.

A decrease in graft-vs.-host disease without loss of graft-vs.-leukemia reactivity after MHC-matched bone marrow transplantation by selective depletion of donor NK cells in vivo.

It is thought that natural killer cells may play a role in graft-vs.-host reactions after allogeneic bone marrow transplantation, but the use of NK cell-specific reagents has been limited. In this report, an NK allele-specific monoclonal antibody, anti-NK 1.1, was used to study the impact of in vivo donor NK cell depletion on GVH disease, graft-vs.-leukemia (GVL) reactivity and donor T cell chimerism after allogeneic murine BMT. AKR/J (H-2k) recipient mice were preconditioned with suboptimal irradiation (9 Gy = LD50) and transplanted with major histocompatibility complex-matched B10.BR (H-2k) BM cells with or without added spleen cells as a source of T cells. The addition of increasing numbers of spleen cells to the BM inoculum produced GVHD of varying intensities. The beneficial effect of NK depletion on GVHD was dependent on the intensity of the GVH reaction. Donor NK cell depletion had no effect on the survival of mice with severe GVHD after MHC-matched BMT (B10.BR into AKR) or after MHC-mismatched BMT (B10.BR into DBA/2; H-2k into H-2d). However, donor NK depletion increased survival of AKR hosts given sufficient B10.BR splenic T cells to induce mild-to-moderate GVHD. Ex vivo depletion of donor CD8+ T cells also reduced GVH-associated mortality, but the use of both CD8 and NK depletion offered no improvement over either alone, suggesting an interaction between CD8+ and NK 1.1+ cells. In contrast to CD8 depletion, donor NK depletion did not compromise the rapid and complete establishment of donor T cell chimerism nor the ability of chimeras to mount an effective GVL reaction. Thus, elimination of donor NK cells provides an alternate strategy for reducing GVHD without loss of GVL reactivity following MHC-matched allogeneic BMT.