Studies of Factors V and VIII:C in an animal model of disseminated intravascular coagulation.

An experimental animal model of disseminated intravascular coagulation (DIC) induced by the co-infusion of coagulant-active phospholipid and activated Factor X (Factor Xa) is described. The infusion of Factor Xa at a dose of 6.6 X 10(-12) mol/kg with phosphatidylcholine/phosphatidylserine (PCPS) lipid vesicles at a dose of 4.0 X 10(-8) mol/kg was associated with significant falls in the levels of fibrinogen and Factors V and VIII, and a bleeding diathesis developed. Assays of Factors V and VIII were performed by a one-stage prothrombin time and activated partial thrombin time system, respectively. In additional experiments, the effect of the same dose combination of Factor Xa/PCPS on Factor V kinetics was studied by preinfusing 125I-labeled Factor V. After Factor Xa/PCPS infusion, Factors VIII and V were reduced at 2 min by 90 and 50% of the preinfusion levels, respectively, and at 1 h by 80 and 75%, respectively. During the same period, there was little change in the total circulating radioactivity. Autoradiography indicated small but detectable levels of circulating proteolytic products of Factor V that comigrated with peptides obtained by the incubation of Factor V with Factor Xa and activated protein C. The majority of radioactivity remained associated with the intact single-chain precursor Factor V. These observations suggested maintenance of the precursor pool after the onset of DIC. This was confirmed by performing two-stage assays of Factors V and VIII, whereby each was completely converted to the active cofactor, i.e., Va and VIII:Ca, by preincubation of the test sample with thrombin before assaying in a one-stage system as before. The Factor V levels assayed by the two-stage procedure did not change appreciably over 1 h. The Factor VIII levels fell but corrected within 1 h at a time when the level measured by a one-stage assay remained depressed. These results indicate that in the dog, infusion of Factor Xa/PCPS induces changes characteristic of DIC, and this is associated with the appearance of Factor V peptides characteristic of the expression of Factor Xa and activated protein C-like activities. The differences noted between the one-stage and two-stage assays suggest that the one-stage assay is measuring the activated fraction of each cofactor and not the total level of the available precursor for each activated species. The results suggest a close correlation between the activated fraction of both cofactors and the hemostatic abnormality that occurs in DIC.     

Generation of an HLA-restricted cytotoxic T cell line reactive against cultured tumor cells from a patient infected with human T cell leukemia/lymphoma virus

Lymphocytes from a patient who had an unusually long survival after therapy for a human T cell leukemia/lymphoma virus (HTLV)-associated T cell lymphoma were stimulated in vitro with an autologous tumor cell line, and the generation of cytotoxic T lymphocytes (CTL) was studied. CTL generated were directed against autologous (HTLV-associated tumor cells. These propagated CTL were OKT3+, OKT4-, and OKT8+. The cytotoxic activity required target tumor cells that were infected with HTLV and also expressed histocompatibility antigens in common with the patient, suggesting a major histocompatibility complex-restricted associative recognition of target antigens expressed on the tumor cell membrane.     

A comparison of bolus versus continuous cardiac output in an experimental model of heart failure

OBJECTIVE: The majority of studies examining cardiac output measurement have been done in physiologically stable models with low thermal background noise. Research comparing continuous cardiac output (CCO) with bolus thermodilution cardiac output (COTD) measures in human and animal models have reported high correlations, negligible bias, but large limits of agreement. The purpose of this project was to compare CCO with COTD measures in an experimental model of heart failure where the cardiac output values were low and the range was narrow. DESIGN: A one-group experimental design with preintervention control measures and repeated CCO and COTD measures across nine time points. SETTING: Cardiovascular research laboratory. SUBJECTS: Thirty dogs. MEASURES AND MAIN RESULTS: Univariate and multivariate versions of repeated-measures analysis were used to assess the influences of temperature, weight, and stage of the experimental protocol on CCO, COTD, and the differences between them. The two measures CCO and COTD were assessed for agreement by using methods proposed by Bland and Altman. Two hundred and fifty pairs of measurements were obtained during sinus rhythm. The range for COTD measures was 0.5-4.67 L/min and for CCO measures 1.0-5.40 L/min. Of the 250 cardiac outputs estimated by the continuous method, 73.4% of the values were within +/-15% of that estimated by the repetitive, single thermodilution method. The mean bias for the entire protocol was 0.01 (SD = 0.51) with a range of 4.33 L/min. CONCLUSION: Agreement between the two measures may be the function of biological variability, responses to anesthesia, and technique. Bland and Altman evaluation demonstrated low bias and precision and similar levels of agreement when compared with previous studies in an experimental model where the cardiac output was low and the range was narrow.     

Prolonged gastric emptying half-time and gastric hypomotility after drug overdose

A prospective study was undertaken to determine if gastric motility and emptying are altered by the ingestion of overdoses of tricyclic antidepressants, acetaminophen, opioid-acetaminophen mixtures, carbamazepine or phenytoin. Gastric scintigraphy was used to measure gastric emptying half-time and assess gastric motility in 104 patients at initial presentation and again at follow-up (n = 85). Patients were imaged for 5 hours after being given 20 MBq of 99mTc tin colloid to drink. Drug serum levels were measured on all patients at initial presentation and at follow-up. We observed markedly prolonged gastric emptying half-times and severe hypomotility at initial presentation compared with follow-up in the vast majority of patients, except for a small group of patients with phenytoin poisoning. Twelve patients had gastric emptying half-times of over 300 minutes, a further 14 had half-times of over 200 minutes and 21 others had half-times of over 120 minutes. Poisoning is associated with hypomotility and a marked delay in gastric emptying that could influence the clinical course and patient management. These abnormalities may not be due to a direct effect of the ingested drug and factors such as stress may play a role.     

The use of phones by elders with disabilities: problems, interventions, costs

Recognizing the important role the telephone plays in the life of frail elders, this study sought to gain a better understanding of the problems these elders encounter in using their phones to meet their needs. Starting with a sample of 354 frail elders, it was determined that 35, or just under 10%, were having some difficulty in the use of their phones. Twenty-two of these 35 subjects were randomly selected for an assessment of their impairments and phone setups in their homes. Interventions were provided to 19 of the 22 subjects, with two subjects refusing an intervention. At a 6-week follow-up call, all subjects were satisfied with the new phone or phone-related equipment. At a 6-month follow-up, 95% of subjects expressed satisfaction with the intervention. Phone usage increased by 50% for subjects provided a phone intervention. Average cost of equipment was $70.45; cost of personnel time was significantly higher. Recommendations are made for addressing the phone-related problems of frail elders.     

Use of the patient at risk scores in the emergency department: a preliminary study

Objectives: To assess the feasibility of using a modified patient at risk (PAR) scoring system in the emergency department.

Methods: Modified PAR scores were calculated for 30 triage category 1 and 2 patients on arrival in the department and at 15 minutes after the initial resuscitation was underway using observation datasheets designed for this project.

Results: Three patients (10%) were admitted to the intensive care unit (ICU). Twenty six patients (88%) were admitted to a general ward and one was discharged. Fifty per cent of the patients had a PAR score of 3 or more on arrival. Eighty seven per cent of the general ward patients had a PAR score that was stable or improved during the first 15 minutes of resuscitation. The other three ward patients, whose scores deteriorated, were assessed in the emergency department by the appropriate teams and deemed appropriate for ward beds. Two of the three ICU patients deteriorated in the resuscitation room. The third ICU patient maintained a PAR score of 2 but had such overwhelming injuries that ICU was the only option.

Discussion: Serial PAR scores are far more informative than isolated scores. This study suggests that serial PAR scores could be used in three ways in the emergency department. Firstly, in the assessment of individual patients and their response to initial resuscitation. Secondly, as a guide to the need further specialist assessment before leaving the emergency department. Finally, as an audit tool to measure the effectiveness of initial treatments in large numbers of patients during the initial resuscitation.

     

Introduction of H-2Dd determinants into the H-2Ld antigen by site-directed mutagenesis

We used site-directed mutagenesis to localize serologically defined (s) and CTL (c)-defined alloantigenic determinants to discrete amino acid sequences of a murine MHC class I antigen. Based on the prediction that amino acid position 63-73 of the H-2Dd antigen forms s-allodeterminants, the H-2Ld gene was mutated in a sequential fashion to replace codons for amino acid positions 63, 65, 66, 70, and 73 with those of the H-2Dd amino acids. Epitopes of the mutant antigens expressed in L-cells were examined by the binding of a series of mAbs specific for the H-2Dd antigen. The mutant antigen M66 had substitutions at residues 63, 65, and 66, and resulted in the acquisition of a number of H-2Dd-specific s-epitopes. Mutant M70 had an additional substitution at residue 70, which led to the gain of multiple additional H-2Dd s-epitopes. Together, more than half of all the relevant H-2Dd s-epitopes were mapped into amino acid position 63-70 of the H-2Dd molecule, which was expressed in the mutant H-2Ld gene. The final mutation at residue 73 (M73) caused no new epitope gains, rather, a few Dd s-epitopes acquired by the preceding mutations were lost. All of the H-2Ld-specific s-determinants were retained in the mutant molecules, as were H-2Dd s-determinants specific for the alpha-2 or alpha-3 domains. Changes of these residues affected c-determinants defined by CTL. Anti-H-2Dd CTL cultures and an anti-H-2Dd CTL clone recognized the mutant H-2Ld molecules, M66 and M70. Some CTL clones generated against the Q10d molecule, which has an identical sequence to H-2Dd between residues 61 and 73, failed to recognize native H-2Dd or Ld but did crossreact with mutant Ld. While bulk-cultured anti-H-2Ld CTL cultures reacted strongly against M73, bulk-cultured H-2Ld restricted anti-vesicular stomatitis virus CTL did not. Finally, at the clonal level two of three anti-H-2Ld CTL clones lost reactivity with some or all of these mutant molecules. From these results we conclude that a stretch of amino acids from position 63 to 70 of the alpha-1 domain controls major s- and c-antigenic sites on the H-2Dd antigen and c-sites on H-2Ld antigen.     

Sonographic changes of the pancreas in chronic renal failure

Several abnormalities regarding pancreatic morphology and function have been reported in patients with chronic renal failure (CRF) with an incidence as high as 72%. In a prospective study we investigated 96 outpatients from our chronic ambulatory hemodialysis program by abdominal ultrasound. Of the patients with CRF, 20.6% were found to have morphologic alterations of the pancreas compared to 4.7% of controls. Although pathologic sonograms of the pancreas correlated with biliary disease, hyperparathyroidism and years of hemodialysis, the most obvious etiologic factor appeared to be the duration of CRF. Possible pathogenetic mechanisms are discussed and screening abdominal ultrasound examinations in patients with long-standing CRF are recommended.     

The high-affinity HSP90-CHIP complex recognizes and selectively degrades phosphorylated tau client proteins

A primary pathologic component of Alzheimer's disease (AD) is the formation of neurofibrillary tangles composed of hyperphosphorylated tau (p-tau). Expediting the removal of these p-tau species may be a relevant therapeutic strategy. Here we report that inhibition of Hsp90 led to decreases in p-tau levels independent of heat shock factor 1 (HSF1) activation. A critical mediator of this mechanism was carboxy terminus of Hsp70-interacting protein (CHIP), a tau ubiquitin ligase. Cochaperones were also involved in Hsp90-mediated removal of p-tau, while those of the mature Hsp90 refolding complex prevented this effect. This is the first demonstration to our knowledge that blockade of the refolding pathway promotes p-tau turnover through degradation. We also show that peripheral administration of a novel Hsp90 inhibitor promoted selective decreases in p-tau species in a mouse model of tauopathy, further suggesting a central role for the Hsp90 complex in the pathogenesis of tauopathies. When taken in the context of known high-affinity Hsp90 complexes in affected regions of the AD brain, these data implicate a central role for Hsp90 in the development of AD and other tauopathies and may provide a rationale for the development of novel Hsp90-based therapeutic strategies.     

Developing research criteria to define medical necessity in emergency medical services

“The Neely Conference: Developing Research Criteria to Define Medical Necessity in EMS” convened emergency medical services (EMS) physicians, researchers, administrators, providers, and federal agency representatives to begin the development of a set of uniform triage criteria and outcome measures that could be used to study and evaluate medical necessity among EMS patients. These standardized criteria might be used in research studies examining EMS dispatch and response (e.g., dispatch triage protocols, alternative response configurations), and EMS treatment and transport (e.g., field triage protocols, alternative care destinations). The conference process included review and analysis of the literature, expert judgment, and consensus building. There was general agreement on the following: 1. Any dispatch triage or field triage system that is developed must be designed to offer patients alternatives to EMS, not to refuse care to patients. 2. It is theoretically possible to develop a set of clinical criteria for need. Some groups of patients will clearly need a traditional EMS response and other groups will not, but this has yet to be defined. 3. In addition to clinical criteria, certain social and other nonclinical criteria such as pain or potential abuse may be used to justify a response. 4. Communication barriers, patient age, special needs, and other conditions complicate patient assessment but should not exclude patients from consideration for alternate triage or transport. 5. These research questions are important, and standard sets of outcome measures are needed so that different studies and innovative programs can be compared.