Genomic instability in long-term culture of human adipose-derived mesenchymal stromal cells.

Mesenchymal stromal/stem cells stem (MSC) have been widely studied due to their great potential for application in tissue engineering and regenerative and translational medicine. In MSC-based therapy for human diseases, cell proliferation is required to obtain a large and adequate number of cells to ensure therapeutic efficacy. During in vitro culture, cells are under an artificial environment and manipulative stress that can affect genetic stability. Several regulatory agencies have established guidelines to ensure greater safety in cell-based regenerative and translational medicine, but there is no specific definition about the maximum number of passages that ensure the lowest possible risk in MSC-based regenerative medicine. In this context, the aim of this study was to analyze DNA damage and chromosome alterations in adipose-derived mesenchymal stromal cells (ADMSC) until the eleventh passage and to provide additional subsidies to regulatory agencies related to number of passages in these cells. Thus, two methods in genetic toxicology were adopted: comet assay and micronucleus test. The comet assay results showed an increase in DNA damage from the fifth passage onwards. The micronucleus test showed a statistically significant increase of micronucleus from the seventh passage onwards, indicating a possible mutagenic effect associated with the increase in the number of passages. Based on these results, it is important to emphasize the need to assess genetic toxicology and inclusion of new guidelines by regulatory agencies to guarantee the safety of MSC-based therapies for human diseases.

Genomic instability in long-term culture of human adipose-derived mesenchymal stromal cells

Mesenchymal stromal/stem cells stem (MSC) have been widely studied due to their great potential for application in tissue engineering and regenerative and translational medicine. In MSC-based therapy for human diseases, cell proliferation is required to obtain a large and adequate number of cells to ensure therapeutic efficacy. During in vitro culture, cells are under an artificial environment and manipulative stress that can affect genetic stability. Several regulatory agencies have established guidelines to ensure greater safety in cell-based regenerative and translational medicine, but there is no specific definition about the maximum number of passages that ensure the lowest possible risk in MSC-based regenerative medicine. In this context, the aim of this study was to analyze DNA damage and chromosome alterations in adipose-derived mesenchymal stromal cells (ADMSC) until the eleventh passage and to provide additional subsidies to regulatory agencies related to number of passages in these cells. Thus, two methods in genetic toxicology were adopted: comet assay and micronucleus test. The comet assay results showed an increase in DNA damage from the fifth passage onwards. The micronucleus test showed a statistically significant increase of micronucleus from the seventh passage onwards, indicating a possible mutagenic effect associated with the increase in the number of passages. Based on these results, it is important to emphasize the need to assess genetic toxicology and inclusion of new guidelines by regulatory agencies to guarantee the safety of MSC-based therapies for human diseases.

Geochemistry of silicate-rich rocks can curtail spreading of carbon dioxide in subsurface aquifers.

Pools of carbon dioxide are found in natural geological accumulations and in engineered storage in saline aquifers. It has been thought that once this CO2 dissolves in the formation water, making it denser, convection streams will transport it efficiently to depth, but this may not be so. Here, we assess theoretically and experimentally the impact of natural chemical reactions between the dissolved CO2 and the rock formation on the convection streams in the subsurface. We show that, while in carbonate rocks the streaming of dissolved carbon dioxide persists, the chemical interactions in silicate-rich rocks may curb this transport drastically and even inhibit it altogether. These results challenge our view of carbon sequestration and dissolution rates in the subsurface, suggesting that pooled carbon dioxide may remain in the shallower regions of the formation for hundreds to thousands of years. The deeper regions of the reservoir can remain virtually carbon free.

The behaviour of Sal'nikov's reaction, P --> A --> B, in a spherical batch reactor with the diffusion of heat and matter.

The behaviour of a simple chemical reaction, occurring with the release of heat in a closed batch reactor, is considered for the situation when matter and heat are transported only by diffusive processes; thus, the reacting fluid has negligible velocity, so that heat transfer is by thermal conduction. The reaction is Sal'nikov's, which consists of two, consecutive first-order steps, producing a product B, from a precursor P, via an active intermediate A, in P --> A --> B. The first of these steps is assumed to be thermoneutral, with zero activation energy, whilst the second is exothermic, with an appreciable activation energy. These features make Sal'nikov's reaction the simplest to display thermokinetic oscillations that characterise many, more complex schemes, e.g. cool flames in hydrocarbon combustion. This study involves identifying the regions of parameter space, in which these oscillations in the temperature and the concentration of the intermediate A occur, by means of numerical simulation. These regions are compared with previous analytical stability analyses in one-dimensional systems. It was found that oscillations occur over a much larger range of conditions in the case considered here, i.e. a reactor with spherical symmetry, than in the simple 1-D case, previously studied by Gray and Scott (P. Gray and S. K. Scott, Chemical Oscillations and Instabilities, Clarendon Press, Oxford, 1990, pp. 264-291). In addition, approximate analytical solutions for the temperature and concentration of A are presented for two limiting cases of non-oscillatory behaviour. These analytical solutions have been verified by comparison with full numerical solutions of the governing equations.

Assessment and significance of 24-h energy intake patterns among young and aged non-affluent southern US women.

Energy intake patterns that may impact health status among non-affluent southern U.S. women from small urban communities have not been evaluated extensively. Usual intake estimates are confounded by factors such as validity of intake methods and socioeconomic status. Typical 24-h energy intakes were reported by Caucasian (CA, n=149) and African-American (AA, n=110) women; at 43% of this sub-population, AA women are appropriately and proportionately represented. Daily energy intake was examined for these non-pregnant females, 24 to 93 y of age, to define typical energy, carbohydrate, protein, and fat intake. Study groups were: 24-29 y, 30-39 y, 40-49 y, 50-59 y, 60-69 y, 70-79 y, and 80-93 y. Statistical comparisons of nutrient variables by age were made by least squares means between groups. Body mass index (BMI) calculations accounted for differences in height and relative body mass. Both races reported similar energy intakes and significant (P<0.05) decreases with age were noted. Energy intakes were 15-40% below recommended levels, similar to reported values; senior lunch programs ameliorated declines among some women >60 y. More daily calories (52-62%) were provided by carbohydrates, followed by fat (26-35%) and protein (14-17%) findings in close agreement with health recommendations. Time-of-day intake patterns suggest women >59 y consume larger noon meals. BMI for AA women was greater (P<0.05) than that of CA women between 30-59 y. At 24-29 y, AA women had lowest BMI values; BMI decreases occurred in CA women after 80 y. These factors may impact the health of non-affluent southern AA and CA women, particularly the elderly who may require guidance for diet planning and intake intervention programs.

Reliability and validity of cardiovascular and vasomotor autonomic function tests.

OBJECTIVE: To determine the reliability and validity of autonomic function tests (AFTs) as clinical tools for diagnosing diabetic autonomic dysfunction. RESEARCH DESIGN AND METHODS: Twenty-one healthy control subjects and 21 insulin-dependent diabetes mellitus (IDDM) patients (11 with no symptomatology and 10 with symptomatic diabetic autonomic neuropathy [DAN]) were matched for age, and administered three standard cardiovascular tests and two new vasomotor tests of autonomic function. Each of the cardiovascular tests (change in heart rate [delta bpm], Valsalva ratio [VR], change in systolic blood pressure [delta sBP]) and vasomotor tests (total pulse amplitude [TPA] and percent vasoconstriction [%VC]) were repeated within 1 week. Infrared photoplethysmography measured sympathetic-mediated vasomotor function. Reliability was determined by intraclass correlation coefficients. Validity was determined by analysis of variance procedures to test for differences between known groups and by computing sensitivity, specificity, and positive and negative predictive values. RESULTS: All AFTs were reliable, with %VC having highest reproducibility (r = 0.90). AFT scores were not different from time 1 to time 2. After controlling for age, two cardiovascular tests had significantly different values for control subjects and asymptomatic diabetic patients. AFTs, except delta sBP, were significantly different between symptomatic diabetic patients and asymptomatic diabetic patients after controlling for age and duration of disease simultaneously. Sensitivity, specificity, and predictive values for %VC were comparable to the values for delta bpm and VR. TPA indexes were lower but clinically acceptable. CONCLUSIONS: AFTs were found to be reliable and valid tests for detecting DAN. TPA and %VC are important because they measure an aspect of sympathetic function not assessed by standard cardiovascular AFTs, and they do not depend on the patient's cooperation or ability to exert effort.

Chronobiology of exercise: the influence of scheduling upon glycemic responses of control and of subjects with diabetes mellitus.

Exercise programs contribute significantly to: a) improve cardiovascular fitness; b) lower blood pressure in hypertension; and c) facilitate glycemic control in subjects with Diabetes Mellitus (DM). The mechanisms involved in promoting the above responses remain undetermined. Circadian phases at which exercise is applied could play a major role in optimizing the individual's physiological responses to exercise. In a pilot study, controls and a subject with Insulin Dependent Diabetes Mellitus (IDDM) were exercised at 0800 and 2000 hours: pedaling on an exercycle for 30 minutes at rates that maintained tachycardia at 70% of maximum reserve heart rates (Karvomen et al., 1957). All subjects were on "standard" rest-activity schedules (rest 2300-2400 to 0600-0700) and were allowed a single 360 ml Sustacal meal either at 2400 hours or at 1200 hours; i.e., 8 hours preceding the exercise program. Blood samples were obtained for determinations of glucose (finger tip) just before exercise (0 time) and at 10, 20, 30, 40 and 60 minutes of exercise/post exercise periods. The results suggest that non-diabetics experience a rapid decrease in blood glucose levels which does not recover during the 60 minute sampling time when exercise was in the morning. However, when these individuals exercised during the evening, glucose levels did not fall as much on a percentage basis and full recovery was obtained within 40-60 minutes. In the case of the diabetic patient, after morning exercise, glucose levels did not decline and after 40 minutes had risen above resting glucose levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Prevention of arteriolar rarefaction in the spontaneously hypertensive rat by exposure to simulated high altitude.

Microvascular alterations associated with reversal of hypertension by exposure to simulated high altitude were investigated in the gracilis muscle of the spontaneously hypertensive rat (SHR). Male, 4-week-old SHR were either placed in a hypobaric chamber (SHR-HA) at 430 mmHg or maintained at ambient barometric pressure (SHR-SL) for 8-10 or 12-14 weeks. Measurements of microvascular diameter and density were made by closed-circuit television microscopy. Mean arterial blood pressure was significantly lower in the SHR-HA groups. Functional arteriolar rarefaction occurred in SHR-SL at 8-10 weeks while structural arteriolar rarefaction occurred after 12-14 weeks. In contrast, neither functional nor structural rarefaction of arterioles was observed in the SHR-HA groups. Although functional rarefaction of capillaries did occur in SHR-HA, it was less than that in SHR-SL. Total capillary density, however, was similar in SHR-SL and SHR-HA after 12-14 weeks. Vasoconstriction was decreased in SHR-HA and vasodilated arteriolar diameters were greater than those of SHR-SL. These results show that simulated high altitude lowered blood pressure in SHR and resulted in a microcirculatory bed resembling that seen in a normotensive animal.

Depressed growth of Morris hepatomas in altitude- and heat-stressed but not in cold-stressed buffalo rats.

Female inbred BUF rats bearing Morris hepatomas 5123C, 5123D, 7795, and 7800 bilaterally in the femoral musculature were exposed for 3 weeks to ether 4,500-m simulated altitude or sea level or to an ambient temperature of either 7, 23, or 33 degrees C. Rats were given inoculations 12 days before these exposures. Tumor size, body weight, food consumption, and body temperatures were measured weekly in these treated rats and in normal rats. At time of killing, tumor mass, DNA synthesis (by [3H]thymidine incorporation), and respiration (by conversion of [1,4-14C]succinic acid to 14CO2) were measured in each of the 4 hepatoma lines, in the livers of normal and host rats, and in regenerated livers 10 days post 70% hepatectomy. Growths of all 4 tumors and regenerated livers were significantly impaired in rats stressed by exposure to altitude and heat but not to cold. Neither DNA synthesis nor respiration was altered in the hepatomas and livers by any environmental stress. The environmentally stressed rats gained weight at a slower rate and consumed less food than did their controls, but no differences were found in these variables for tumor-bearing and non-tumor-bearing rats. However, whereas the ratio of body weight gain to food consumed was reduced under the three stressful environments, that of tumor weight gain to food consumed was not altered by any environment. Host survivorship was not influenced by any of these effects.

Control of cell division in the cornea of rats. III. Mitogenic effect of isoproterenol and theophylline.

The effects of isoproterenol, theophylline and the combination of the two drugs were studied on the circadian mitotic rhythm in rat corneal epithelium. The drugs induced profound alterations in the normal circadian mitotic pattern. The alterations included an initial suppression of cell division, followed by two distinct rises above control; the first immediately following suppression, the second 12-16 hr following drug administration. The second peak of cell division occurred at the circadian trough in controls. The simultaneous administration of isoproterenol and theophylline greatly magnified the effects seen with either drug alone.

Control of cell division in the cornea of rats II. Age-dependent effects of dexamethasone upon circadian mitotic activity.

Data are presented here which verify that immature rats exhibit no circadian fluctuation of mitoses in the corneal epithelium prior to the third week of life. These data indicate that dexamethasone administration induces age-dependent alterations in the circadian mitotic pattern of the corneal epithelium. Nine day old animals still revealed no rhythmic component in circadian mitotic activity after dexamethasone treatment. Eleven-, 12- and 13-day old animals treated with dexamethasone exhibited circadian cycles which looked qualitatively like the circadian rhythm of the 15-day old animal. Fifteen-day old animals responded with a synchrony of mitotic activity at 0700 hr and a generalized decrease in mitotic activity. The possible dependence of these responses upon existing circadian mitotic activity and maturation of the central nervous system and/or the glucocorticoid target tissue is discussed.