Preparation, stability, and in vitro performance of vesicles made with diblock copolymers.

Vesicles made completely from diblock copolymers-polymersomes-can be stably prepared by a wide range of techniques common to liposomes. Processes such as film rehydration, sonication, and extrusion can generate many-micron giants as well as monodisperse, approximately 100 nm vesicles of PEO-PEE (polyethyleneoxide-polyethylethylene) or PEO-PBD (polyethyleneoxide-polybutadiene). These thick-walled vesicles of polymer can encapsulate macromolecules just as liposomes can but, unlike many pure liposome systems, these polymersomes exhibit no in-surface thermal transitions and a subpopulation even survive autoclaving. Suspension in blood plasma has no immediate ill-effect on vesicle stability, and neither adhesion nor stimulation of phagocytes are apparent when giant polymersomes are held in direct, protracted contact. Proliferating cells, in addition, are unaffected when cultured for an extended time with an excess of polymersomes. The effects are consistent with the steric stabilization that PEG-lipid can impart to liposomes, but the present single-component polymersomes are far more stable mechanically and are not limited by PEG-driven micellization. The results potentiate a broad new class of technologically useful, polymer-based vesicles.

Acetaminophen and other risk factors for excessive warfarin anticoagulation.

CONTEXT: Warfarin is highly effective in preventing thromboembolism, but increases the risk of hemorrhage, particularly at an international normalized ratio (INR) greater than 4.0. Identifying causes of excessive anticoagulation in clinical practice could help target patients at risk for elevated INRs. OBJECTIVE: To determine causes of INRs greater than 6.0 in a clinical practice setting. DESIGN: Case-control study. SETTING: Outpatient anticoagulant therapy unit. PATIENTS: Outpatients followed up prospectively from April 1995 to March 1996 who had been taking warfarin for more than 1 month, had a target INR of 2.0 to 3.0, and were able to be interviewed within 24 hours of their reported INR. Case patients had INRs greater than 6.0; controls were randomly selected from patients having INRs between 1.7 and 3.3. MAIN OUTCOME MEASURES: Factors associated with INRs greater than 6.0, including medication use, recent diet, illness, alcohol consumption, and actual warfarin use. RESULTS: A total of 93 cases and 196 controls were interviewed; they did not differ in age, indication for warfarin, length of therapy, warfarin dose, number of prescription medications, or previous INR or long-term INR variability. Acetaminophen ingestion was independently associated in a dose-dependent manner with having an INR greater than 6.0 (P for trend <.001). For the highest-dose category of acetaminophen intake, 9100 mg/wk or more, the odds of having an INR greater than 6.0 were increased 10-fold (95% confidence interval [CI], 2.6-37.9). Other factors independently associated with an INR greater than 6.0 were new medication known to potentiate warfarin (odds ratio [OR], 8.5; 95% CI, 2.9-24.7), advanced malignancy (OR, 16.4; 95% CI, 2.4-111.0), recent diarrheal illness (OR, 3.5; 95% CI,1.4-8.6), decreased oral intake (OR, 3.6; 95% CI, 1.3-9.7), and taking more warfarin than prescribed (OR, 8.1; 95% CI, 2.2-30.0). Higher vitamin K intake (OR, 0.7; 95% CI, 0.5-0.9) and habitual alcohol consumption of from 1 drink every other day to 2 drinks a day (OR, 0.2; 95% CI, 0.1-0.7) were associated with decreased risk. CONCLUSIONS: These data suggest that acetaminophen is an underrecognized cause of overanticoagulation in the outpatient setting. Several other clinically important risk factors were identified. Increased monitoring of INR values when such risk factors are present or modification of the risk factors themselves should reduce the frequency of dangerously high levels of anticoagulation.

IgM antibodies in renal transplantation.

A prospective final crossmatch with patient serum and donor lymphocytes using the complement-dependent cytotoxicity assay to identify any performed anti-donor antibody is required for kidney transplantation. The presence of pre-existing antibody may lead to hyperacute rejection of the transplanted kidney. Certain anti-donor antibodies have previously been shown to be ineffective in promoting hyperacute rejection, such as IgM autoantibodies and non-specific IgM lymphocytotoxic antibodies. In this report, we present evidence that IgM HLA alloantibody specific to the donor does not lead to hyperacute rejection and produces graft survival results equivalent to transplants with negative pre-transplant final crossmatches. Forty-eight (48) of 402 patients transplanted over and 8 yr period were transplanted across a positive final crossmatch due to IgM antibodies alone. Three patients exhibited IgM autoantibodies and 26 patients demonstrated non-specific IgM antibodies to lymphocytes. In 15 patients, following a detailed serum screening analysis, a significant correlation (r > 0.9, p < 0.001) was observed between HLA Class I antigens and the presence of corresponding IgM alloantibodies. Five of these patients were subsequently transplanted despite a positive final crossmatch that was clearly demonstrated to be the result of IgM alloantibody to donor HLA Class I specificities. All of these patients continue to have graft function. These results suggest that hyperacute rejection is not mediated by any type of IgM antibody to donor lymphocytes and that kidney transplantation when only IgM antibody is present against donor lymphocytes represents a reasonable opportunity for a safe transplant and successful long-term graft survival.

An analysis of the lowest effective intensity of prophylactic anticoagulation for patients with nonrheumatic atrial fibrillation.

BACKGROUND: To avert major hemorrhage, physicians need to know the lowest intensity of anticoagulation that is effective in preventing stroke in patients with atrial fibrillation. Since the low rate of stroke has made it difficult to perform prospective studies to resolve this issue, we conducted a case-control study. METHODS: We studied 74 consecutive patients with atrial fibrillation who were admitted to our hospital from 1989 through 1994 after having an ischemic stroke while taking warfarin. For each patient with stroke, three controls with nonrheumatic atrial fibrillation who were treated as outpatients were randomly selected from the 1994 registry of the anticoagulant-therapy unit (222 controls). We used the international normalized ratio (INR) to measure the intensity of anticoagulation. For the patients with stroke, we used INR at admission; for the controls, we selected the INR that was measured closest to the month and day of the matched case patient's hospital admission. RESULTS: The risk of stroke rose steeply at INRs below 2.0. At an INR of 1.7, the adjusted odds ratio for stroke, as compared with the risk at an INR of 2.0, was 2.0 (95 percent confidence interval, 1.6 to 2.4); at an INR of 1.5, it was 3.3 (95 percent confidence interval, 2.4 to 4.6); and at an INR of 1.3, it was 6.0 (95 percent confidence interval, 3.6 to 9.8). Other independent risk factors were previous stroke (odds ratio, 10.4; 95 percent confidence interval, 4.4 to 24.5), diabetes mellitus (odds ratio, 2.95; 95 percent confidence interval, 1.3 to 6.5), hypertension (odds ratio, 2.5; 95 percent confidence interval, 1.1 to 5.7), and current smoking (odds ratio, 5.7; 95 percent confidence interval, 1.4 to 24.0). CONCLUSIONS: Among patients with atrial fibrillation, anticoagulant prophylaxis is effective at INRs of 2.0 or greater. Since previous studies have indicated that the risk of hemorrhage rises rapidly at INRs greater than 4.0 to 5.0, tight control of anticoagulant therapy to maintain the INR between 2.0 and 3.0 is a better strategy than targeting lower, less effective levels of anticoagulation.

Effect of low-intensity warfarin anticoagulation on level of activity of the hemostatic system in patients with atrial fibrillation. BAATAF Investigators.

BACKGROUND AND PURPOSE: The Boston Area Anticoagulation Trial for Atrial Fibrillation (BAATAF) demonstrated that low-intensity warfarin anticoagulation can, with safety, sharply reduce the rate of stroke in patients with nonvalvular atrial fibrillation. The beneficial effect of warfarin was presumably related to a decrease in clot formation in the cardiac atria and subsequent embolization. METHODS: To assess the effect of warfarin therapy on in vivo clotting in patients in the BAATAF, we measured the plasma level of prothrombin activation fragment F1+2. One sample was obtained from 125 patients from the BAATAF; 62 were taking warfarin and 63 were not taking warfarin (control group). RESULTS: The warfarin group had a 71% lower mean F1+2 level than the control group (mean F1+2 of 1.57 nmol/L in the control group compared with a mean of 0.46 nmol/L in the warfarin group; P < .001). F1+2 levels were higher in older subjects but were consistently lower in the warfarin group at all ages. Fifty-two percent of patients in the control group were taking chronic aspirin therapy at the time their F1+2 level was measured. Control patients taking aspirin had F1+2 levels very similar to control patients not taking aspirin (mean of 1.52 nmol/L for control patients on aspirin compared with 1.64 nmol/L for control patients off aspirin; P > .1). CONCLUSIONS: We conclude that prothrombin activation was significantly suppressed in vivo by warfarin but not aspirin among patients in the BAATAF. These findings correlate with the marked reduction in ischemic stroke noted among patients in the warfarin treatment group observed in the BAATAF.

The effect of aspirin on the risk of stroke in patients with nonrheumatic atrial fibrillation: The BAATAF Study.

Recent randomized trials have consistently demonstrated the marked efficacy of warfarin in reducing the risk of stroke caused by nonrheumatic atrial fibrillation. These trials have provided conflicting evidence on the effect of aspirin. We report the aspirin analysis from the BAATAF study, a trial in which control patients could choose to take aspirin. There we two strokes in 446 person-years with warfarin (annual rate of 0.45%); eight strokes in 206 person-years with aspirin, most at 325 mg per day (annual rate of 3.9%); and five strokes in 271 person-years among patients taking neither aspirin nor warfarin (annual rate of 1.8%). Simultaneously controlling for the other significant determinants of stroke in the BAATAF study (age, mitral annular calcification, and clinical heart disease), the relative rates (95% confidence interval) of stroke were: (1) warfarin/aspirin = 0.135 (0.029 to 0.64); (2) aspirin/(no aspirin and no warfarin) = 1.95 (0.64 to 5.97); and (3) warfarin/(no aspirin and no warfarin) = 0.263 (0.051 to 1.36). Our "treatment received" analysis argues that warfarin is strikingly more effective than aspirin in preventing stroke in nonrheumatic atrial fibrillation.

The impact of long-term warfarin therapy on quality of life. Evidence from a randomized trial. Boston Area Anticoagulation Trial for Atrial Fibrillation Investigators.

To determine the effect of long-term warfarin sodium therapy on quality of life, we surveyed 333 patients participating in a randomized, controlled trial of warfarin for the prevention of stroke in nonrheumatic atrial fibrillation. No significant differences between warfarin-treated and control patients were found on well-validated measures of functional status, well-being, and health perceptions. For example, the summary score for health perceptions was 68.8 in the warfarin-treated vs 66.6 in the control group (scale of 0 to 100; 95% confidence intervals for the difference, -1.6 to 6.0). In contrast, patients taking warfarin who had a bleeding episode had a significant decrease in health perceptions (-11.9; 95% confidence interval, -4.1 to -19.6). Warfarin therapy is not usually associated with a significant decrease in perceived health, unless a bleeding episode has occurred. Negative effects of warfarin treatment on health perceptions may be balanced by confidence in its protective effects.

Nuclear structure and the control of DNA replication in the Xenopus embryo.

We have developed a cell-free system from frog eggs that efficiently initiates and completes a single round of semi-conservative replication. 70-100% of sperm chromatin and up to 40% of plasmid DNA molecules are completely replicated in vitro. Before DNA is replicated it is assembled into nuclei surrounded by a double unit membrane studded with nuclear pores. Flow cytometry shows that initiation events are co-ordinated within individual nuclei, although different nuclei can start to replicate at different times in the same extract. This demonstrates the importance of nuclear structure in the control of DNA replication in this system. Only a single round of semi-conservative replication occurs in the cell-free system. This mirrors the way that only one round of DNA replication occurs in each cell cycle in vivo. When replicated nuclei are transferred to fresh extract they are unable to undergo another round of replication. However, if the nuclear envelope is permeabilised before nuclei are transferred to fresh extract, the DNA becomes capable of undergoing a further round of semi-conservative replication. These results suggest a simple model for the control of DNA replication within the cell cycle, whereby an essential initiation factor can only gain access to DNA when the nuclear envelope breaks down during mitosis.

Steps in the assembly of replication-competent nuclei in a cell-free system from Xenopus eggs.

We have studied the pathway of nuclear assembly from demembranated sperm chromatin by fractionating a cell-free system from Xenopus eggs (Lohka, M. J., and Y. Masui. 1983. Science (Wash. DC). 220:719-721). Both the soluble fraction and a washed vesicular fraction are required for formation of normal nuclei that initiate replication in vitro. The soluble fraction alone decondenses chromatin and the vesicular fraction alone surrounds chromatin with membranes. Both fractions are required for formation of nuclear pore complexes. Recombining these two fractions recovers approximately 100% of the nuclear assembly and DNA replication activities. Restricting the proportion of the vesicular fraction slows acquisition of the nuclear membrane and allows observation of immature nuclear pores ("prepores"). These form as arrays around and within the chromatin mass before membranes form. Subsequently membrane vesicles bind to these prepores, linking them by a single membrane throughout the chromatin mass. At the periphery this single membrane is surrounded by an outer membrane. In mature nuclei all membranes are at the periphery, the two membranes are linked by pores, and no prepores are seen. Nuclear assembly and replication are inhibited by preincubating the chromatin with the vesicular fraction. However nuclear assembly is accelerated by preincubating the condensed chromatin with the soluble fraction. This also decreases the lag before DNA replication. Initiation of DNA replication is only observed after normal nuclei have fully reassembled, increasing the evidence that replication depends on nuclear structure. The pathway of nuclear assembly and its relationship to DNA replication are discussed.