Are we assuming too much with our statistical assumptions? Lessons learned from the ALTTO trial.

BACKGROUND: Design, conduct, and analysis of randomized clinical trials (RCTs) with time to event end points rely on a variety of assumptions regarding event rates (hazard rates), proportionality of treatment effects (proportional hazards), and differences in intensity and type of events over time and between subgroups. DESIGN AND METHODS: In this article, we use the experience of the recently reported Adjuvant Lapatinib and/or Trastuzumab Treatment Optimization (ALTTO) RCT, which enrolled 8381 patients with human epidermal growth factor 2-positive early breast cancer between June 2007 and July 2011, to highlight how routinely applied statistical assumptions can impact RCT result reporting. RESULTS AND CONCLUSIONS: We conclude that (i) futility stopping rules are important to protect patient safety, but stopping early for efficacy can be misleading as short-term results may not imply long-term efficacy, (ii) biologically important differences between subgroups may drive clinically different treatment effects and should be taken into account, e.g. by pre-specifying primary subgroup analyses and restricting end points to events which are known to be affected by the targeted therapies, (iii) the usual focus on the Cox model may be misleading if we do not carefully consider non-proportionality of the hazards. The results of the accelerated failure time model illustrate that giving more weight to later events (as in the log rank test) can affect conclusions, (iv) the assumption that accruing additional events will always ensure gain in power needs to be challenged. Changes in hazard rates and hazard ratios over time should be considered, and (v) required family-wise control of type 1 error ≤ 5% in clinical trials with multiple experimental arms discourages investigations designed to answer more than one question. TRIAL REGISTRATION: clinicaltrials.gov Identifier NCT00490139.

Characterisation of the HLA-DRB1*07:01 biomarker for lapatinib-induced liver toxicity during treatment of early-stage breast cancer patients with lapatinib in combination with trastuzumab and/or taxanes.

HLA-DRB1*07:01 allele carriage was characterised as a risk biomarker for lapatinib-induced liver injury in a large global study evaluating lapatinib, alone and in combination with trastuzumab and taxanes, as adjuvant therapy for advanced breast cancer (adjuvant lapatinib and/or trastuzumab treatment optimisation). HLA-DRB1*07:01 carriage was associated with serum alanine aminotransferase (ALT) elevations in lapatinib-treated patients (odds ratio 6.5, P=3 × 10-26, n=4482) and the risk and severity of ALT elevation for lapatinib-treated patients was higher in homozygous than heterozygous HLA-DRB1*07:01 genotype carriers. A higher ALT case incidence plus weaker HLA association observed during concurrent administration of lapatinib and taxane suggested a subset of liver injury in this combination group that was HLA-DRB1*07:01 independent. Furthermore, the incidence of ALT elevation demonstrated an expected correlation with geographic HLA-DRB1*07:01 carriage frequency. Robust ALT elevation risk estimates for HLA-DRB1*07:01 may support causality discrimination and safety risk management during the use of lapatinib combination therapy for the treatment of metastatic breast cancer.

GABA(A) receptors containing (alpha)5 subunits in the CA1 and CA3 hippocampal fields regulate ethanol-motivated behaviors: an extended ethanol reward circuitry.

GABA receptors within the mesolimbic circuitry have been proposed to play a role in regulating alcohol-seeking behaviors in the alcohol-preferring (P) rat. However, the precise GABA(A) receptor subunit(s) mediating the reinforcing properties of EtOH remains unknown. We examined the capacity of intrahippocampal infusions of an alpha5 subunit-selective ( approximately 75-fold) benzodiazepine (BDZ) inverse agonist [i.e., RY 023 (RY) (tert-butyl 8-(trimethylsilyl) acetylene-5,6-dihydro-5-methyl-6-oxo-4H-imidazo [1,5a] [1,4] benzodiazepine-3-carboxylate)] to alter lever pressing maintained by concurrent presentation of EtOH (10% v/v) and a saccharin solution (0.05% w/v). Bilateral (1.5-20 microgram) and unilateral (0.01-40 microgram) RY dose-dependently reduced EtOH-maintained responding, with saccharin-maintained responding being reduced only with the highest doses (e.g., 20 and 40 microgram). The competitive BDZ antagonist ZK 93426 (ZK) (7 microgram) reversed the RY-induced suppression on EtOH-maintained responding, confirming that the effect was mediated via the BDZ site on the GABA(A) receptor complex. Intrahippocampal modulation of the EtOH-maintained responding was site-specific; no antagonism by RY after intra-accumbens [nucleus accumbens (NACC)] and intraventral tegmental [ventral tegmental area (VTA)] infusions was observed. Because the VTA and NACC contain very high densities of alpha1 and alpha2 subunits, respectively, we determined whether RY exhibited a "negative" or "neutral" pharmacological profile at recombinant alpha1beta3gamma2, alpha2beta3gamma2, and alpha5beta3gamma2 receptors expressed in Xenopus oocytes. RY produced "classic" inverse agonism at all alpha receptor subtypes; thus, a neutral efficacy was not sufficient to explain the failure of RY to alter EtOH responding in the NACC or VTA. The results provide the first demonstration that the alpha5-containing GABA(A) receptors in the hippocampus play an important role in regulating EtOH-seeking behaviors.

Synthesis of the isoleucyl- and valyl-tRNA synthetases and the isoleucine-valine biosynthetic enzymes in a threonine deaminase regulatory mutant of Escherichia coli K-12.

A mutation in the structural gene for threonine deaminase, ilvA538 , results in lower than normal levels of the isoleucyl, valyl- and leucyl-tRNA synthetases. Moreover, this regulatory mutation decreases the level of expression of the ilv biosynthetic operons and renders their expression non-responsive to limitations of the branched-chain amino acids. In this paper, we present in vitro evidence for the inhibition of isoleucyl- and valyl-tRNA synthetase activity by threonine deaminase and 2-ketobutyrate, the product of the threonine deaminase reaction, through the formation of a high molecular weight complex of the three molecules. Based on these results, we propose a model to explain the regulation of the isoleucyl- and valyt -tRNA synthetases in which transient inhibition of the synthetase enzyme activities by threonine deaminase and 2-ketobutyrate increases the expression of ileS and valS , the structural genes for isoleucyl- and valyt -tRNA synthetase, respectively. Further, the results suggest that the hyperattenuated expression of the ilv biosynthetic operons is due to an increased rate of complex formation of valyl and isoleucyl-tRNA synthetases and the altered form of threonine deaminase of the ilvA538 mutant strain.

Bleeding risk, physical functioning and non-use of anticoagulation among patients with stroke and atrial fibrillation.

BACKGROUND: Atrial fibrillation (AF) is common among people with stroke. Anticoagulation medications can be used to manage the deleterious impact of AF after stroke, however, may not be prescribed due to concerns about post-stroke falls and decreased functioning. Thus, the purpose of this study was to identify, among people with stroke and AF, predictors of anticoagulation prescription at hospital discharge. METHODS: This is a secondary analysis of a retrospective cohort study of data retrieved via medical records, including National Institutes of Health Stroke Scale score, Functional Independence Measure (FIM) motor score (motor or physical function), ambulation on second day of hospitalization, Morse Falls Scale (fall risk) and HAS-BLED score (Hypertension; Abnormal renal and liver function; Stroke; Bleeding; Labile INRs; Elderly >65; and Drugs or alcohol). Data analyses included bivariate comparisons between people with and without anticoagulation at discharge. Logistic-regression modeling was used to assess predictors of discharge anticoagulation. RESULTS: There were 334 subjects included in the analyses, whose average age was 75 years old. Anticoagulation was prescribed at discharge for 235 (70%) of patients. In the adjusted regression analyses, only the FIM motor score (adjusted OR = 1.015, 95% CI 1.001-1.028) and the HAS-BLED score (adjusted OR = 0.36, 95% CI 0.22-0.58) were significantly associated with anticoagulation prescription at discharge. CONCLUSION: It appears that in this sample, post-stroke anticoagulation decisions appear to be made based on clinical factors associated with bleed risk and motor deficits or physical functioning. However, opportunities may exist for improving clinician documentation of specific reasoning for non-anticoagulation prescription.

Validating the Questionnaire for Verifying Stroke-Free Status (QVSFS) by neurological history and examination.

BACKGROUND AND PURPOSE: The Questionnaire for Verifying Stroke-Free Status (QVSFS) is an 8-item structured interview designed to identify stroke-free individuals. Previously, the QVSFS was validated with medical record review in a cohort with a low prevalence (7.1%) of stroke or transient ischemic attack (TIA). The objective of this study was to evaluate the validity of the QVSFS by comparing it with stroke status as determined by neurological history and examination in a population with a higher prevalence of stroke. METHODS: A research assistant administered the QVSFS to outpatients from Veterans Administration stroke and general medicine clinics. Subjects were defined as QVSFS negative if responses to all 8 questions were negative. Questions requiring rephrasing or clarification were noted. Neurologists, blinded to QVSFS scores, interviewed and examined all subjects to determine stroke-free status, defined as no history or examination findings of previous stroke and/or TIA. RESULTS: One hundred fifty-five subjects were examined; mean age was 70 years; 98.1% were male. Seventy-eight subjects were determined to be stroke free by the neurologist. The negative predictive value of the QVSFS was 0.96, with positive predictive value of 0.71. No question required rephrasing or clarification >5 times. Twenty-two subjects (14.2%) required rephrasing or clarification of at least 1 question. CONCLUSIONS: The QVSFS can effectively identify stroke-free individuals with a high degree of accuracy, even in a population with a large proportion of patients with prior stroke or TIA. Accuracy for identifying subjects with stroke and/or TIA is lower, but the QVSFS may still be useful as a screening tool in that regard.

Protocol violations in community-based rTPA stroke treatment are associated with symptomatic intracerebral hemorrhage.

BACKGROUND: Recombinant tissue plasminogen activator (rTPA) is an established treatment for acute ischemic stroke. The rate and type of protocol violations in rTPA use and their effect on patient outcomes in this setting are not well understood. OBJECTIVE: The objective of this study was to examine associations between protocol violations and outcomes in community-based rTPA use. METHODS: We reviewed medical records of stroke patients treated with rTPA in 10 acute-care hospitals in Indianapolis from July 1996 to February 1998 and assessed complications and outcome. Retrospective National Institute of Health Stroke Scale (on admission and discharge), Canadian Neurological Scale, and length of hospital stay were calculated. Appropriate use of rTPA was determined by the National Institute of Neurological Disorders and Stroke (NINDS) protocol. RESULTS: Fifty patients (mean age, 66 years; 76% white; 56% men) were treated by general neurologists (70%), stroke neurologists (24%), or emergency physicians (6%). Mean times to hospital arrival, brain CT, and start of rTPA infusion were 44, 86, and 141 minutes, respectively. In-hospital mortality rate was 10% (4 intracerebral hemorrhage [ICH], 1 cardiogenic shock). Complications were more frequent among patients with protocol violations (n=8) compared with those without all hemorrhages (75% versus 10%, P:<0.001), symptomatic ICH (38% versus 5%, P:<0.02), and ICH attributable to rTPA, occurring within 36 hours (38% versus 2.4%, P:<0.01), respectively. CONCLUSIONS: NINDS protocol violations are relatively common and are associated with symptomatic cerebral and systemic hemorrhages. When the NINDS protocol is strictly followed, hemorrhage rates in community-based rTPA use are similar to those in the NINDS trial.

The transient syndrome of headache with neurologic deficits and CSF lymphocytosis.

We describe seven patients who experienced a transient syndrome consisting of headache with neurologic deficits and CSF lymphocytic pleocytosis. Thirty-three similar patients have been reported previously. All had from one to more than 20 episodes of a severe migrainous headache accompanied by a temporary neurologic deficit and lymphocytes in the CSF. Some patients had a preceding viral syndrome, increased intracranial pressure, elevated CSF protein, or focal EEG abnormalities. All stopped having episodes within 3 months. We present diagnostic criteria for this benign syndrome. Its cause remains unknown.

Measuring quality of life in a way that is meaningful to stroke patients.

OBJECTIVE: To identify predictors of poststroke quality of life. BACKGROUND: Health-related quality of life (HRQOL) measures assess the impact of disease on the physical, emotional, and social aspects of patients' lives. Although HRQOL measures are used increasingly, factors associated with HRQOL poststroke and the ability of stroke-specific versus generic HRQOL measures to predict patient-reported HRQOL are not well known. METHODS: A total of 71 patients were evaluated 1 month postischemic stroke with a new stroke-specific HRQOL measure-the SS-QOL-and the SF-36, a generic HRQOL measure. Stroke severity, impairments, and functional limitations were also measured. Demographic variables and outcome measure scores were compared between patients rating their overall HRQOL the same as pre-stroke versus those with overall HRQOL worse than prestroke. Independent predictors of overall HRQOL were identified using multivariable modeling. RESULTS: Variables associated with better overall HRQOL were higher (better) SS-QOL and Barthel Index scores, and lower (better) NIH Stroke Scale and Beck Depression Inventory scores. Independent predictors of good overall HRQOL were the SS-QOL score (odds ratio [OR], 2.97; 95% CI, 1.3, 7.1; p = 0.01) and NIH Stroke Scale score (OR, 0.69; 95% CI, 0.47, 0.99; p = 0.05). Demographic factors and SF-36 scores were not associated with overall HRQOL ratings. CONCLUSIONS: Stroke-specific quality of life score and patient impairments predict patient-reported overall health-related quality of life (HRQOL) poststroke. SF-36 scores were not associated with overall HRQOL ratings. Disease-specific HRQOL measures are more sensitive to meaningful changes in poststroke HRQOL and may thus aid in identifying specific aspects of poststroke function that clinicians and "trialists" can target to improve patients' HRQOL after stroke.

Subtypes of ischemic stroke in children and young adults.

Specific strategies for primary and secondary stroke prevention in children and young adults can only be recommended once the causes of stroke in these age groups are well described. ICD-9 codes were used to identify children aged 1 to 18 years with acute ischemic stroke. Young adults aged > 18 to 45 years were identified from the Indiana University and Northwestern University Young Adults Stroke Registries. Validated criteria were used to subtype ischemic stroke as atherothrombotic (AT), cardioembolic (CE), small-vessel (SV), other determined cause, or unknown cause. Ninety-two children and 116 young adults were identified. Stroke subtypes in children/young adults (percentages) were as follows: AT 0/16 (p < 0.001), CE 15/14 (p = 1.0), SV 0/3 (p = 0.26), other 49/44 (p = 0.40), and unknown 36/23 (p = 0.04). Children had more prothrombotic causes (25% versus 14%, p = 0.03), and young adults had more dissections (3% versus 15%, p = 0.005). Children aged 15 to 18 years had causes of ischemic stroke more similar to those in young adults. The cause of ischemic stroke is less often identified in children than it is in young adults. Children have more prothrombotic causes of stroke, and adults have more atherothrombotic causes and dissections. Lacunar strokes are rare in both children and young adults. The age of 15 years should be used to separate childhood from young-adult ischemic stroke.

Effects of admission hyperglycemia on mortality and costs in acute ischemic stroke.

BACKGROUND: Hyperglycemia at the time of acute ischemic stroke has been linked to worse outcome in both human and animal studies. OBJECTIVE: To describe the prevalence and severity of hyperglycemia on hospital admission among acute ischemic stroke patients, to examine the independent relationship of admission hyperglycemia to all-cause mortality, and to document the inpatient management of hyperglycemia. METHODS: Patients hospitalized with acute ischemic stroke at one hospital from July 1993 to June 1998 (n = 656) were identified. Demographic data, diagnoses, and blood glucose (BG) values were retrieved from the electronic medical record system. Admission stroke severity, fingerstick BG results, and new diabetes diagnoses were obtained by chart review. Hyperglycemia was defined as admitting random serum BG > or = 130 mg/dL. Hazard ratios (HR) for 30-day, 1-year, and 6-year mortality were calculated using multivariable Cox regression models. RESULTS: Hyperglycemia at admission to hospital was present in 40% of patients with acute stroke. Patients with hyperglycemia were more often women and more likely to have prior diagnoses of diabetes and heart failure. Almost all of these patients remained hyperglycemic during their hospital stay (mean BG = 206 mg/dL), and 43% received no inpatient hypoglycemic drugs. Hyperglycemic patients had longer hospital stay (7 vs 6 days, p = 0.015) and higher inpatient hospital charges ($6,611 vs $5,262, p < 0.001). Hyperglycemia independently increased the risk for death at 30 days (HR 1.87, p < or = 0.01), 1 year (HR 1.75, p < or = 0.01), and 6 years after stroke (HR 1.41, p

Acute blood glucose level and outcome from ischemic stroke. Trial of ORG 10172 in Acute Stroke Treatment (TOAST) Investigators.

OBJECTIVE: To study the relation between acute blood glucose level and outcome from ischemic stroke. BACKGROUND: Hyperglycemia may augment acute ischemic brain injury and increase the risk of hemorrhagic transformation of the infarct. METHODS: The authors analyzed the relation between admission blood glucose level (within 24 hours from ischemic stroke onset) and clinical outcome in 1,259 patients enrolled in the Trial of ORG 10172 in Acute Stroke Treatment (TOAST)-a placebo-controlled, randomized, double-blind trial to test the efficacy of a low-molecular weight heparinoid in acute ischemic stroke. Very favorable outcome was defined as a Glasgow Outcome Scale score of 1 and a modified Barthel index of 19 or 20. Neurologic improvement at 3 months was defined as a decrease by > or =4 points on the NIH Stroke Scale compared with baseline or a final score of 0. Hemorrhagic transformation of infarct was assessed within 10 days after onset of stroke with repeat cerebral CT. Stroke subtype as lacunar or nonlacunar (atherothromboembolic, cardioembolic, and other or undetermined etiology) was classified by one investigator after completion of stroke evaluation according to study protocol. RESULTS: In all strokes combined (p = 0.03) and in nonlacunar strokes (p = 0.02), higher admission blood glucose levels were associated with worse outcome at 3 months according to multivariate logistic regression analysis adjusted for stroke severity, diabetes mellitus, and other vascular risks. In lacunar strokes, the relationship between acute blood glucose level and outcome was related to treatment. In the placebo group, higher admission blood glucose levels were associated with better outcome at 3 months. However, in the active drug group, as the glucose level increased from 50 to 150 mg/dL, the probability of a very favorable outcome decreased sharply and remained relatively unchanged as the glucose level increased further (p = 0.002, for overall effect of glucose on outcome). Acute blood glucose level was not associated with symptomatic hemorrhagic transformation of infarcts or with neurologic improvement at 3 months. CONCLUSIONS: During acute ischemic stroke hyperglycemia may worsen the clinical outcome in nonlacunar stroke, but not in lacunar stroke, and is not associated with an increased risk of hemorrhagic transformation of the infarct.

Perineural spread of cutaneous squamous and basal cell carcinoma: CT and MR detection and its impact on patient management and prognosis.

PURPOSE: To analyze the pretreatment imaging findings and outcome of patients with perineural spread of squamous or basal cell carcinoma of the face and scalp treated with radiotherapy, to determine whether CT (computed tomography) or MR (magnetic resonance) imaging can be effectively used to identify patients who would benefit from aggressive treatment, and to characterize the imaging features associated with cure. METHODS: Thirty-five patients had perineural spread of squamous and basal cell carcinoma along the divisions of the trigeminal and/or facial nerves based on clinical findings and/or histopathological proof. Perineural extension seen on imaging was divided into three zones of involvement. The volume of perineural disease was graded semiquanitatively. All patients received radiotherapy with curative intent. RESULTS: Eighteen of the 35 patients had imaging evidence of perineural spread of tumor, and the remaining 17 were imaging negative for perineural spread. The absolute 5-year survival of the imaging positive group was 50% compared with 86% in the imaging-negative group (p = 0.048). CONCLUSIONS: Imaging can be used to identify patients with advanced perineural spread who warrant aggressive radiotherapy. Imaging evidence of perineural invasion worsens prognosis; however, low-volume and peripheral perineural disease is radiocurable. Greater perineural tumor volume with more central disease was associated with an unfavorable outcome.

Characterization of the conjunctival vasopermeability response to leukotrienes and their involvement in immediate hypersensitivity.

The microvascular permeability response of the guinea pig conjunctiva to sulfidopeptide leukotrienes (LTs) was quantified as extravasation of radiolabeled bovine serum albumin. The LTs were potent inducers of increased microvascular permeability, with relative potencies LTE4 greater than or equal to LTD4 greater than LTC4. The response to LTs was unaffected by indomethacin or a pyrilamine/cimetidine combination, but the LT antagonists FPL 55712 and SKF 102922 significantly inhibited the response to LTC4, LTD4 and LTE4. In guinea pigs actively sensitized to ovalbumin, topical ocular administration of ovalbumin markedly increased conjunctival microvascular permeability; this response was reduced by approximately 50% following histaminergic blockade by pyrilamine/cimetidine. FPL 55712 and SKF 102922 and the 5-lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) had no effect on the response to antigen when used alone. However, each agent significantly reduced the non-histaminergic component of the response when given in conjunction with pyrilamine/cimetidine. Thus, it appears that the immediate hypersensitivity response in guinea pig conjunctiva has a possible non-histaminergic component which is at least partly mediated by LTs.

Studies on the ocular pharmacology of prostaglandin D2.

Prostaglandin D2 (PGD2) exerts a variety of biologic actions in the eye; these include ocular hypotension and inflammatory effects on the conjunctiva. The profile of activity of PGD2 in ocular tissues was compared to that of BW 245C, a selective agonist for the PGD2-sensitive (DP) receptor, and to that of the biologically active metabolites of PGD2, 9 alpha,11 beta-prostaglandin F2 (9 alpha,11 beta-PGF2) and prostaglandin J2 (PGJ2). PGD2 produced a dose-dependent decrease in intraocular pressure and in the conjunctiva it caused increased conjunctival microvascular permeability, eosinophil infiltration and goblet cell depletion. Although BW 245C was equipotent to PGD2 as an ocular hypotensive agent, it did not cause pathological effects in the conjunctiva. Thus, the ocular hypotensive effect of PGD2 may be separated from inflammatory effects on the conjunctiva by employing a selective DP-receptor agonist such as BW 245C. 9 alpha,11 beta-PGF2 was a weak ocular hypotensive and did not cause conjunctival inflammation. PGJ2 produced no significant effect on intraocular pressure. PGJ2 did not elicit a microvascular permeability response in the conjunctiva, but was inflammatory in other respects and caused eosinophil infiltration and goblet cell depletion similar to PGD2. Thus, both the ocular hypotensive actions and the conjunctival pathology of PGD2 may be replicated individually by employing PGD2 analogues and metabolites.

Prostaglandin F2 alpha effects on intraocular pressure negatively correlate with FP-receptor stimulation.

According to the current working classification for prostanoid receptors, the prostaglandin F2 alpha-sensitive receptor (FP-receptor) may be identified by comparing the rank order of activity of prostaglandin F2 alpha (PGF2 alpha) and its analogues. In order to further understand the pharmacology of PGF2 alpha-induced ocular hypotension, the intraocular pressure response to PGF2 alpha and selected analogues was compared with their rank order of activity in typical FP-receptor preparations such as contraction of the cat iris sphincter and affinity for corporal luteal membrane binding sites. The rank order of potency for decreasing intraocular pressure was as follows: PGF2 alpha greater than PGF1 alpha greater than 16-phenoxytetranor PGF2 alpha greater than 17-phenyltrinor PGF2 alpha = fluprostenol (inactive). For cat iris sphincter contraction, the rank order of potency appears to be fluprostenol = 17-phenyltrinor PGF2 alpha greater than 16-phenoxytetranor PGF2 alpha = PGF2 alpha greater than PGF1 alpha. The rank order of potency for PGF2 alpha analogues in decreasing intraocular pressure appears to negatively correlate with the rank order for cat iris sphincter contraction and literature values for corporal luteal membrane binding. It is concluded that the ocular hypotensive effect of PGF2 alpha is not mediated by the FP-receptor.

Non-isotopic method for quantitation of platelets and erythrocytes in experimental thrombi.

Experimental animal models of thrombosis have been established in several species to examine factors responsible for thrombotic disorders in man. One technical facet of all thrombosis models is the need to quantitate cell deposition on thrombogenic surfaces, and this is routinely accomplished with radioisotopic labeling of specific components. Data reported here demonstrate that formalin-fixed thrombi can be hydrolyzed with chymotrypsin allowing recovery and quantitation of platelets and erythrocytes incorporated within the clot. Recovery of platelets from in vitro generated, model thrombi averaged 99 +/- 10% (mean +/- 1 SD; n = 7; range 88-116%) of calculated content; recovery of erythrocytes was 94.1 +/- 1.1% (n = 6) as measured by recovery of cellular hemoglobin after chymotrypsin hydrolysis of clots. Chymotrypsin was also shown to release platelets and erythrocytes from string-bound thrombi generated in vivo with an arterio-venous shunt model in beagle dogs. Platelet recovery from these string clots after chymotrypsin hydrolysis was independently verified with a quantitative Western blot assay of platelet antigens. These data demonstrate that experimental thrombi can be hydrolyzed with chymotrypsin, thereby not only eliminating the need for radioisotopes, but also permitting flow cytometric analysis of cells comprising the thrombus.

Evidence for human thromboxane receptor heterogeneity using a novel series of 9,11-cyclic carbonate derivatives of prostaglandin F2 alpha.

1. The pharmacological activity of a novel series of 9,11-cyclic carbonate derivatives of prostaglandin F2 alpha (PGF2 alpha) was investigated in various isolated smooth muscle preparations possessing different prostanoid receptor subtypes as well as in human platelets. Since subdivision of thromboxane (TP-) receptors into vascular/smooth muscle and platelet subtypes is a controversial subject, our studies included a human smooth muscle preparation (myometrium) in addition to the widely used rat aorta and human platelets as TP-receptor preparations. 2. Two members of that series, AGN191976 and AGN192093 were found to be highly potent and selective thromboxane-mimetics. AGN191976 and AGN192093 contracted isolated tissues of the rat thoracic aorta with EC50 values of 0.32 +/- 0.08 and 1.30 +/- 0.53 nM, respectively. Both agonists were at least 10 times more potent than the benchmark TP-agonist, U-46619, in this preparation, whilst being at least 500 times less potent at other prostanoid receptors (DP, EP1, EP3, FP, IP) in vitro. 3. In human myometrial strips from pregnant and non-pregnant donors, both AGN191976 and AGN192093 were potent contractile agonists. The rank order of potency in myometrium of AGN191976 > AGN192093 > U-46619 correlated well with that in the rat aorta. In human platelet-rich plasma (PRP), however, AGN191976 had potent proaggregatory activity (EC50 = 16.3 +/- 1.4 nM), which is a TP-receptor-mediated event, whereas AGN192093 was a much weaker agonist (EC50 = 37.9 +/- 2.0 microM). AGN192093 did not behave as an antagonist in the platelets, since it did not antagonize platelet aggregation induced by ADP, arachidonic acid, U-46619 or AGN191976. In human washed platelets, the activity profile of AGN191976 (EC50 = 4.15 +/- 0.52 nM) and AGN192093 (no aggregation up to 10 microM) was similar to that obtained in PRP. 4. The involvement of TP-receptors was verified with the potent TP-antagonist, SQ29548. SQ29548 (0.1 microM in myometrium; 1 microM in aorta; 1 microM and 10 microM in platelets) antagonized responses to U-46619, AGN191976 and AGN192093 as expected. 5. In conclusion, AGN191976 and AGN192093, both 9,11-cyclic carbonate derivatives of PGF2 alpha, were found to be highly potent and selective thromboxane-mimetics in rat vascular and human myometrial smooth muscle. However, only AGN 191976 was a potent agonist at TP-receptors in human platelets. The differential activity of AGN192093 on TP-receptor-mediated events in platelets and smooth muscle provides further evidence for a subdivision of TP-receptors. AGN192093 appears to be a useful tool for the pharmacological distinction of TP-receptor subtypes.

Clinical problem-solving in geriatric medicine: obstacles to rehabilitation.

This case illustrates how multifactorial medical, neurological and psychiatric conditions can synergistically effect the functional status of elderly individuals. This case also illustrates how iatrogenically induced illness, most commonly in the form of drug therapy, can significantly affect patients' conditions and their progress in rehabilitative therapies. This case also illustrates how the atypical presentation of disease in the elderly, as well as the medical ramifications of rehabilitative therapies, need to be strongly considered during trials at functional rehabilitation. More importantly, however, this case demonstrates how even a community-based geriatrician can be instrumental in recognizing the need of more appropriate evaluations in elderly patients with functional disabilities and how even in the setting of a multidisciplinary team there can still be a consulting relationship, with each person seeing the patient individually and communicating with other members of the team as necessary. Although each team member may have a specific area of expertise, there is still a very important role for the geriatrician to play in making sure that all the different components of the rehabilitation and the medical care proceed in a way that will provide the most favorable outcome for the patient.

Platelet-activating factor causes goblet cell depletion in the conjunctiva.

Platelet-activating factor (PAF) (1-O-hexadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine) produced dose-dependent depletion of the goblet cell population associated with the conjunctival epithelium. Reductions in goblet cell numbers did not correspond to leukocyte infiltration and were consistent with a direct effect of PAF. In contrast, LTD4 and LTE4 did not affect the goblet cell population although they caused massive eosinophil infiltration into the conjunctival epithelium. Histamine also produced conjunctival goblet cell depletion, but this appeared secondary to eosinophil degranulation and resultant epithelial desquamation. In addition to goblet cell expulsion, PAF produced an increase in conjunctival microvascular permeability over an identical dose-range. PAF-induced leukocyte emigration was small or absent and comprised a neutrophil infiltrate which exhibited no clear dose-dependent relationship. Lyso-PAF produced effects only at the highest dose employed where pathological changes and a distinct increase in conjunctival microvascular permeability were evident. Lyso-PAF- and PAF-induced increases in conjunctival microvascular permeability were virtually abolished by the PAF antagonist CV-6209. The pronounced inhibitory activity of CV-6209 suggests that high doses of lyso-PAF may either weakly stimulate conjunctival PAF receptors or that there may be sufficient conversion of lyso-PAF to biologically active levels of PAF.