Hydroxyurea-induced augmentation of fetal hemoglobin production in patients with sickle cell anemia

Five patients with sickle cell anemia were treated with hydroxyurea (HU), in hopes of augmenting their production of fetal hemoglobin. Laboratory responses in two patients treated for more than 2 years were encouraging and there were suggestions of clinical improvement. Long- term HU therapy should be considered for severely affected adults with sickle cell anemia who are willing to accept what is probably a small risk of carcinogenesis. Preliminary chromosomal analysis and knowledge of the clastogenic properties of HU suggest that conception and pregnancy should be avoided. Pharmacokinetic studies will probably be necessary to adjust individual dosage schedules so that cytotoxicity is avoided. F cell responses can be seen in 2 to 3 weeks if the HU dose is optimal, but establishment of a large number of F cells in the circulation may take a month or longer.     

Pre-B acute lymphoblastic leukemia cells may induce T-cell anergy to alloantigen

Even if neoplastic cells express tumor associated antigens they still may fail to function as antigen presenting cells (APC) if they lack expression of one or more molecules critical for the induction of productive immunity. These cellular defects can be repaired by physiologic activation, transfection, or fusion of tumor cells with professional APC. Although such defects can be repaired, antitumor specific T cells may still fail to respond in vivo if they may have been tolerized. Here, human pre-B cell acute lymphoblastic leukemia (pre-B ALL) was used as a model to determine if primary human tumor cells can function as alloantigen presenting cells (alloAPC) or alternatively whether they induce anergy. In the present report, we show that pre-B cell ALL express alloantigen and adhesion molecules but uniformly lack B7-1 (CD80) and only a subset express B7-2 (CD86). Pre-B ALL cells are inefficient or ineffective alloAPC and those cases that lack expression of B7-1 and B7-2 also induce alloantigen specific T- cell unresponsiveness. Under these circumstances, T-cell unresponsiveness could be prevented by physiologic activation of tumor cells via CD40, cross-linking CD28, or signaling through the common gamma chain of the interleukin-2 receptor on T cells. Taken together, these results suggest that pre-B ALL may be incapable of inducing clinically significant T-cell-mediated antileukemia responses. This defect may be not only due to their inability to function as APC, but also due to their potential to induce tolerance. Attempts to induce clinically significant antitumor immune responses may then require not only mechanisms to repair the antigen presenting capacity of the tumor cells, but also reversal of tolerance.     

Quality control of multidrug resistance assays in adult acute leukemia: correlation between assays for P-glycoprotein expression and activity

We have compared multiple assays for the P-glycoprotein (Pgp/MDR1) phenotype in fresh and thawed adult acute leukemia to validate and quantitate measures for the expression and function of Pgp. The results are related to the Pgp-expressing KB8 and KB8-5 call lines. The most sensitive assay was the measurement of modulation of the rhodamine 123 (R123) fluorescence by 2 micromol/L PSC833, followed by the modulation of the probe calcein-AM. We also found a good intralaboratory and interlaboratory correlation between the values of the R123/PSC833 assay for fresh as well as thawed samples. In addition, the affects of PSC833 on 3H-daunorubicin (DNR) accumulation, DNR fluorescence, and 3H- vincristine accumulation were very similar. The correlation between the DNR/PSC833 and R123/PSC833 test was r = .86 (N = 51). The modulation of drug accumulation by 8 micromol/L verapamil was the some as the PSC833 effect for DNR (117%, N = 21), but was higher for vincristine in every single case (161% v 121%, N = 22; P< .001), indicating additional verapamil effects, not related to Pgp. The correlation of the staining of viable cells for Pgp with the monoclonal antibody MRK16 was r = .77 (N = 52) for the R123/PSC833 functional test and r = .84 (N = 50) for the DNR/PSC833 test. From these results it could be calculated that a maximal increase of the mean DNR accumulation of about 50% can be achieved by blocking Pgp pump activity with PSC833 in leukemic blast samples with the highest mean Pgp expression. Subpopulations of blast calls with higher Pgp activity are likely to be present. Their relevance has to be studied further. The methods outlined here allow the reliable, quantitative monitoring of the Pgp/MDR1 phenotype in leukemias in multicentered, clinical Pgp modulation studies.     

Task analysis of the preincision surgical period: an independent observer-based study of 1558 cases

Intense production pressure has focused on the preincision period (from patient-on-table to incision) as an important component of overall operating room efficiency. We conducted a prospective study in which trained independent observers measured the performance of anesthesiologists, surgeons, and nursing staff to determine anesthesia release time (ART, patient-on-table until release for surgical preparation) and surgical preparation time (SPT, start surgical preparation to incision) and the factors, including delays, that affect their duration. We enrolled 1558 patients undergoing elective surgery in a tertiary medical center. The mean ART was 21 +/- 16 min. Mean SPT was 22 +/- 13 min, and mean case length was 207 +/- 123 min. Significant variation was seen in both ART (range, 1-115 min) and SPT (range, 1-130 min). Multivariate regression analysis revealed ASA physical status, age, level of resident training, invasive monitoring, case length, and case number in the room were all positive predictors of ART duration (P < 0.05). In contrast, gender, body mass index, number of anesthesia personnel concurrently in the room, and number of rooms covered per anesthesia attending were not predictors for ART (P > 0.05). Delays affected both ART and SPT and were encountered in 24.5% of all procedures (surgery 66.8%, anesthesiology 21.7%, and logistical 11.5%). For operating room scheduling purposes, we conclude that assigning a constant fixed duration for anesthetic induction is inappropriate and will result in creating erroneous administrative expectations.     

Increased transvascular transport of WGA-peroxidase after chronic perinatal stress in the hippocampal microvasculature of the rat

Brain endothelial ultrastructural properties contribute to maintain proper blood-brain barrier (BBB) function. Several physiological and pathological conditions have been shown to alter BBB permeability to blood-borne molecules, acute and chronic stress among them. In the rat, early life stress increased transvascular transport of Evans blue, however, the route of tracer extravasation is not fully known; therefore the aim of the present experiment was to describe the ultrastructural changes in endothelial cells subsequent to chronic perinatal stress in order to ascertain the route for transvascular transport of an electrodense tracer. Pregnant Wistar rats and their litters were used. Four pregnant rats were subjected to forced swimming between gestational days 10 to 20. After delivery, half of the control litters underwent 180 min maternal separation from postnatal day 2 to 20. Controls were kept free of any stress manipulation. At sacrifice between postnatal days 1 to 30 subjects were given intracardially the lectin wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). WGA-HRP stained hippocampi were processed for ultrastructural analysis, transmission electron micrographs were obtained and endothelial ultrastructural parameters quantified using the ImageJ software. Both stress procedures accelerated gross microvessel development by decreasing capillary wall thickness and endothelial microvilli. However, early-life stress also neutralized endothelial glycocalyx, increased vesicle-mediated transport and tended to promote the formation of secondary lysosomes containing endocytosed WGA-HRP vesicles, all parameters of altered endothelial cell function. Tight junction development in both stress groups was similar to the control pups.