Functional consequences of sphingomyelinase-induced changes in erythrocyte membrane structure.

Inflammation enhances the secretion of sphingomyelinases (SMases). SMases catalyze the hydrolysis of sphingomyelin into phosphocholine and ceramide. In erythrocytes, ceramide formation leads to exposure of the removal signal phosphatidylserine (PS), creating a potential link between SMase activity and anemia of inflammation. Therefore, we studied the effects of SMase on various pathophysiologically relevant parameters of erythrocyte homeostasis. Time-lapse confocal microscopy revealed a SMase-induced transition from the discoid to a spherical shape, followed by PS exposure, and finally loss of cytoplasmic content. Also, SMase treatment resulted in ceramide-associated alterations in membrane-cytoskeleton interactions and membrane organization, including microdomain formation. Furthermore, we observed increases in membrane fragility, vesiculation and invagination, and large protein clusters. These changes were associated with enhanced erythrocyte retention in a spleen-mimicking model. Erythrocyte storage under blood bank conditions and during physiological aging increased the sensitivity to SMase. A low SMase activity already induced morphological and structural changes, demonstrating the potential of SMase to disturb erythrocyte homeostasis. Our analyses provide a comprehensive picture in which ceramide-induced changes in membrane microdomain organization disrupt the membrane-cytoskeleton interaction and membrane integrity, leading to vesiculation, reduced deformability, and finally loss of erythrocyte content. Understanding these processes is highly relevant for understanding anemia during chronic inflammation, especially in critically ill patients receiving blood transfusions.

Microplate spectrophotometry for high-throughput screening of cytotoxic molecules.

OBJECTIVES: High-throughput chemical and biochemical technologies are now exploited by modern pharmacology and toxicology to synthesize a multitude of new molecules with bioactive potential, or to isolate them from living matter. Testing molecules in cell systems on a large scale, however, is a rate-limiting step in drug discovery or in toxicity assessment. In this study, we developed a low-cost high-throughput method for first-level screening of cytotoxic molecules. MATERIALS AND METHODS: We used microplate spectrophotometry to measure growth kinetics of human tumour cells that grow in suspension (Molt3) or adherent to the plastic surface of culture wells (HeLa) in standard RPMI medium. Cells were treated with colchicin, idarubicin or paclitaxel under various treatment schedules. The effects were quantified and compared with those measured by optical microscopy using the trypan blue dye exclusion method to reveal dead cells. RESULTS: Proliferation kinetics of tumour cells can be quantified by measuring variations in optical densities of cell samples at 410 and 560 nm wavelengths. For cells that grow in suspension, one single reading at 730 nm may be sufficient to reconstruct growth curves that parallel those obtained by direct cell counting. Effects of the cytotoxic treatments could also be quantified and results compared very favourably with those obtained using standard techniques. CONCLUSIONS: Microplate spectrophotometry is a robust and sensitive method to monitor growth of animal cell populations both in the absence and in the presence of cytotoxic drugs. This method implements existing technologies and can be fully automated.

A new electromechanical trainer for sensorimotor rehabilitation of paralysed fingers: a case series in chronic and acute stroke patients.

BACKGROUND: The functional outcome after stroke is improved by more intensive or sustained therapy. When the affected hand has no functional movement, therapy is mainly passive movements. A novel device for repeating controlled passive movements of paralysed fingers has been developed, which will allow therapists to concentrate on more complicated tasks. A powered cam shaft moves the four fingers in a physiological range of movement. METHODS: After refining the training protocol in 2 chronic patients, 8 sub-acute stroke patients were randomised to receive additional therapy with the Finger Trainer for 20 min every work day for four weeks, or the same duration of bimanual group therapy, in addition to their usual rehabilitation. RESULTS: In the chronic patients, there was a sustained reduction in finger and wrist spasticity, but there was no improvement in active movements. In the subacute patients, mean distal Fugl-Meyer score (0-30) increased in the control group from 1.25 to 2.75 (ns) and 0.75 to 6.75 in the treatment group (p < .05). Median Modified Ashworth score increased 0/5 to 2/5 in the control group, but not in the treatment group, 0 to 0. Only one patient, in the treatment group, regained function of the affected hand. No side effects occurred. CONCLUSION: Treatment with the Finger Trainer was well tolerated in sub-acute & chronic stroke patients, whose abnormal muscle tone improved. In sub-acute stroke patients, the Finger Trainer group showed small improvements in active movement and avoided the increase in tone seen in the control group. This series was too small to demonstrate any effect on functional outcome however.

A quantitative study of growth variability of tumour cell clones in vitro.

OBJECTIVES: In this study, we quantify growth variability of tumour cell clones from a human leukaemia cell line. MATERIALS AND METHODS: We have used microplate spectrophotometry to measure growth kinetics of hundreds of individual cell clones from the Molt3 cell line. Growth rate of each clonal population has been estimated by fitting experimental data with the logistic equation. RESULTS: Growth rates were observed to vary between different clones. Up to six clones with growth rates above or below mean growth rate of the parent population were further cloned and growth rates of their offspring were measured. Distribution of growth rates of the subclones did not significantly differ from that of the parent population, thus suggesting that growth variability has an epigenetic origin. To explain observed distributions of clonal growth rates, we have developed a probabilistic model, assuming that fluctuation in the number of mitochondria through successive cell cycles is the leading cause of growth variability. For fitting purposes, we have estimated experimentally by flow cytometry the average maximum number of mitochondria in Molt3 cells. The model fits nicely observed distributions in growth rates; however, cells in which mitochondria were rendered non-functional (rho(0) cells) showed only 30% reduction in clonal growth variability with respect to normal cells. CONCLUSIONS: A tumour cell population is a dynamic ensemble of clones with highly variable growth rates. At least part of this variability is due to fluctuations in the initial number of mitochondria in daughter cells.

Children of heroin addicts.

Fourteen Black male, opiate addicts, their wives, and their children were studied intensively using psychiatric interviews and psychological tests. Their 32 children were compared to 37 pediatric clinic children. The children raised in a home where father is an opiate addict function cognitively less well than their father, and the teenagers show earlier and stronger antisocial trends than pediatric clinic peers. On the other hand, there is a surprising absence of other psychopathology that one might expect, taking into consideration the deviant environment from which they come.

Who are the "committed"?

A record review of patients who were committed by the court during the course of a hospitalization at an acute urban facility was carried out. Court-committed patients represented 4% of total patients admitted during a 2-year period. Black patients and patients over the age of 70 were more likely to reach the stage of a court hearing and be committed. Schizophrenia was the most frequent diagnosis, being present in well over one half of court-committed patients. Approximately one third of the patients had a hospital stay exceeding 3 months, and transfer to a long term inpatient program occurred significantly more often among court-committed patients as compared to the rest of the hospitalized population. The majority of court-committed patients were eventually returned to the community; about one fifth were placed in intermediate facilities such as boarding or nursing homes. When legal status of previous and subsequent hospitalizations of this sample of court-committed patients was examined, a clear predominance of uncomplicated voluntary hospitalization became apparent.