The role of stem cells in limb regeneration.

Limb regeneration is a complex yet fascinating process observed to some extent in many animal species, though seen in its entirety in urodele amphibians. Accomplished by formation of a morphologically uniform intermediate, the blastema, scientists have long attempted to define the cellular constituents that enable regrowth of a functional appendage. Today, we know that the blastema consists of a variety of multipotent progenitor cells originating from a variety of tissues, and which contribute to limb tissue regeneration in a lineage-restricted manner. By continuing to dissect the role of stem cells in limb regeneration, we can hope to one day modulate the human response to limb amputation and facilitate regrowth of a working replacement.

Apnea-hypopnea indices and snoring in children diagnosed with ADHD: a matched case-control study.

OBJECTIVES: To measure apnea-hypopnea indices and snoring in children diagnosed with attention-deficit hyperactivity disorder (ADHD) in a case-control design. Additionally, the study design allowed us to investigate whether or not methylphenidate had any effect on breathing variables. METHODS: Twenty-eight children (22 boys) aged 6-12 years meeting diagnostic criteria for Diagnostic and Statistical Manual of Mental Disorders (DSM)-IV ADHD were studied together with matched controls. Two nights of polysomnography (PSG) were conducted that included recordings of snoring waveforms. A randomly assigned 48-h on-off medication protocol was used for ADHD children. Control children's recordings were matched for PSG night, but medication was not used. A low apnea-hypopnea index (AHI) threshold of >1 event per hour was used to define sleep-disordered breathing (SDB) because of a clinical relevance in ADHD. RESULTS: Categorical analyses for paired binary data showed no significant differences between control and ADHD children for presence of an AHI >1 or snoring. Variables were extracted from a significantly shorter total sleep time (67 min) on the medication night in children with ADHD. Eight (28%) control and 11 (40%) ADHD children snored >60 dB some time during the night. Methylphenidate had no effect on central apneas, AHI, desaturation events, or any snoring data. CONCLUSIONS: Our PSG findings show no strong link between ADHD and SDB although our findings could be limited by a small sample size. Findings from PSG studies in the literature argue both for and against an association between ADHD and SDB. Our results suggest medication is not a factor in the debate.

The sleep of children with attention deficit hyperactivity disorder on and off methylphenidate: a matched case-control study.

In the present study, we assessed the effects of regular use of methylphenidate medication in children diagnosed with attention deficit hyperactivity disorder (ADHD) on sleep timing, duration and sleep architecture. Twenty-seven children aged 6-12 years meeting diagnostic criteria for Diagnostic and Statistical Manual version IV ADHD and 27 control children matched for age (+/-3 months) and gender. Two nights of standard polysomnographic (PSG) recordings were conducted. ADHD children were allocated randomly to an on- or 48 h off-methylphenidate protocol for first or second recordings. Control children's recordings were matched for night, but no medication was used. Mixed modelling was employed in the analyses so that the full data set was used to determine the degree of medication effects. Methylphenidate in ADHD children prolonged sleep onset by an average of 29 min [confidence interval (CI) 11.6, 46.7], reduced sleep efficiency by 6.5% (CI 2.6, 10.3) and shortened sleep by 1.2 h (CI 0.65, 1.9). Arousal indices were preserved. Relative amounts of stages 1, 2 and slow wave sleep were unchanged by medication. Rapid eye movement sleep was reduced (-2.4%) on the medication night, an effect that became non-significant when control data were incorporated in the analyses. PSG data from ADHD children off-medication were similar to control data. Our findings suggest that methylphenidate reduces sleep quantity but does not alter sleep architecture in children diagnosed with ADHD. An adequate amount of sleep is integral to good daytime functioning, thus the sleep side effects of methylphenidate may affect adversely the daytime symptoms the drug is targeted to control.

Changes in behavior and attentional capacity after adenotonsillectomy.

The objective of this study was to quantify behavioral and attention capacity changes in children aged 4-11 y before and 3 mo after adenotonsillectomy (A/T). Overnight cardiorespiratory recordings were performed in 61 "behaviorally normal" children 1 wk before A/T. Tests of sustained attention using visual and auditory continuous performance tests (CPT) were completed by children 1 wk before and 3 mo after A/T. Behavioral Assessment Scales for Children (BASC) and a sleep questionnaire were completed by the parent/s at these same times. Results from overnight cardiorespiratory recordings showed that the children had mild sleep-related breathing disorders (SRBD) preoperatively with a mean apnea/hypopnea index of 3.0/h and a movement awakening index of 2.5/h. The majority had parent-perceived sleep and breathing difficulties that significantly improved post-A/T. BASC T scores for externalizing and internalizing behaviors improved post-A/T, e.g., behavioral symptom index mean pre-A/T was 56.2 (95% confidence interval, 52.8-59.6) compared with 50.9 (48.5-53.5) post-A/T. Some measures indicative of impulsivity and attentiveness obtained from the visual CPT before surgery, improved post-A/T, but no change was observed in any auditory CPT measures. Our data confirm improvements in subjective measures of sleep problems in children treated for SRBD and strengthen the notion of treating the disorder, not only related to the obvious clinical condition but also to the underlying sleep problems and adverse effects on daytime behavior and attention.

Comparison of two fluorochromes for flow cytometric assay of cellular glutathione content in human malignant melanoma.

Because the resistance of melanoma to cytotoxic therapy may be due to high intracellular glutathione (GSH), we wished to monitor GSH in melanoma cells during treatment. This required an assay suitable for very small samples. So we chose analytical flow cytometry. We calibrated the flow cytometric assays against biochemically determined GSH content in a range of cultured human melanoma cell lines, then applied the assays to fine-needle tumour biopsies. Mercury orange was the first fluorochrome suitable for use in a flow cytometer with a 488 nm light source, but many technical factors influenced the results. Chloromethyl fluorescein diacetate (CMFDA) allowed assay conditions less dependent on details of cell handling. Correlations between biochemical GSH content and fluorescence intensities in cell lines were good for mercury orange and CMFDA. CMFDA is the preferred fluorochrome for estimating cellular GSH content in biopsies from human melanomas. Tumours metastatic to or beyond regional lymph nodes had significantly more cells with high GSH than primary tumours or regional recurrences.

Glutathione content of human malignant melanoma cell lines: correlation of flow cytometric and biochemical assays, and application to human tumour aspirates.

We measured the glutathione content of a panel of human malignant melanoma cell lines by flow cytometry after staining with mercury orange, using visible light (488 nm) excitation, and compared the values obtained with those measured biochemically using a modified Tietze assay. Glutathione levels were depleted by culturing cells with various concentrations of buthionine sulphoximine to provide a suitable spread of glutathione concentrations. The two assays showed good correlation (r = 0.93). We found a number of technical factors to be critically important. In particular, the conditions of staining, cell number, and method of mixing media, cells and stain were responsible for wide variations in fluorescence intensity. We applied the flow cytometric technique to cell suspensions obtained by fine needle aspiration biopsy of human malignant melanoma deposits, and observed a proportion of cells with high glutathione levels in many samples. The results confirm the feasibility of measuring glutathione content by visible light flow cytometry, and raise the possibility of monitoring glutathione content as an indicator of drug resistance in clinical therapy of human malignant melanoma.

Test-retest reliability of isokinetic knee extension and flexion torque measurements in persons with spastic hemiparesis.

The purpose of this study was to evaluate and compare the test-retest reliability of isokinetic torque measurements in the involved and uninvolved knee musculature of 20 subjects with spastic hemiparesis. An isokinetic dynamometer was used to measure maximal voluntary knee extension and flexion at 60 degrees and 120 degrees/s. Peak torque (PT) and average peak torque (APT) data were collected from five repetitions on two separate occasions. Average peak torque was defined as the mean of the PT values obtained during each of the five repetitions. Spasticity was measured in the involved knee musculature prior to isokinetic testing using the Ashworth Scale. Pearson Product-Moment Correlation Coefficients and intraclass correlation coefficients (ICCs) were high (greater than or equal to .90) for both knees for PT and APT at both angular velocities. No clinically meaningful differences were found between the Pearson correlation coefficients and the ICCs of the involved versus the uninvolved knee for any testing conditions. We concluded that isokinetic evaluation of torque, as measured by PT and APT in subjects with spastic hemiparesis, can yield reliable results in both extremities.

Flow cytometric measurement of glutathione content of human cancer biopsies.

Rice et al. (1986) have described a flow cytometric method where the non-fluorescent probe monochlorobimane (mBCl) forms a fluorescent adduct with cellular glutathione (GSH) under the action of glutathione-S-transferase. We show here that for EMT6 carcinosarcoma cells there is a close correlation between mean cell fluorescence, expressed as a ratio to that of fluorescence calibration beads, and biochemically determined GSH content over the range 0.2-2.0 fmol cell-1. Single cell suspensions from 14 human cancers were prepared by 23-gauge needle aspiration or mechanical disaggregation of surgical specimens, stained using mBCl and examined by flow cytometry. There was a wide range in individual cell fluorescence, which in contrast to EMT6 cells was not strongly correlated with Coulter volume. By comparing tumour cell fluorescence to that of calibration beads, and assuming that the relationship with GSH content for EMT6 holds for other cells, a mean GSH content of 0.95 fmol cell-1 was derived for nine carcinomas, and 0.21 fmol cell-1 for five non-Hodgkin's lymphomas. Although this semi-quantitation needs further validation, the method used here is rapid, gives an indication of heterogeneity of tumour cell GSH content, and can be applied to fine needle biopsy samples. It therefore shows promise as a means for studying prospectively the relationship of GSH content to clinical drug and radiation sensitivity, and for monitoring the effects of agents such as buthionine sulphoximine which are intended to improve treatment results through tumour cell GSH depletion.

Adenosine A1 receptor activation mediates suppression of (-) bicuculline methiodide-induced seizures in rat prepiriform cortex.

The protective effects of a series of stable adenosine analogs against generalized seizures initiated by focal injection of bicuculline methiodide into the rat prepiriform cortex (PPC) were studied by microinjection of these compounds into this brain area. The adenosine agonists, 5'-N-(ethyl)carboxamido-adenosine (NECA), cyclohexyladenosine, cyclopentyadenosine, 2-chloroadenosine and R- and S-phenylisopropyladenosine (R- and S-PIA), protected animals against seizures in a dose-dependent, and extremely potent manner. NECA, the most potent compound evaluated, completely prevented seizures at doses greater than or equal to 6.8 pmol. In contrast, heroic doses of the A2 selective ligand, 2-phenylaminoadenosine, afforded no protection against seizures. The rank order of potency of these compounds in suppressing seizures is as follows: NECA greater than cyclohexyladenosine greater than cyclopentyladenosine greater than or equal to R-PIA greater than 2-chloroadenosine greater than S-PIA much greater than 2-phenylaminoadenosine. These data suggest that the antiseizure activity of these compounds in the PPC results from activation of A1 adenosine receptors. Quantitative autoradiographic analysis of the distribution of tritiated adenosine agonists 30 min after microinjection in the PPC reveals that [3H]NECA diffuses to a significantly greater extent than R-[3H]PIA, which may contribute to the relatively greater potency of the former compound in suppressing bicuculline methiodide-induced seizures. These results suggest that adenosine A1 receptors may participate in the normal inhibitory regulation of the PPC, a forebrain area which may play a significant role in the pathobiology of epilepsy.

Vinblastine sensitivity of leukaemic lymphoblasts modulated by serum lipid.

The high-resolution proton magnetic resonance spectrum of leukaemic lymphoblasts is characteristic of neutral lipid in an isotropic environment. When such lymphoblasts are selected for resistance to the anticancer drug vinblastine, the intensity of this spectrum increases with increasing drug resistance. A reversal of this trend can be achieved by growing cells in delipidated serum, whereby lipid spectrum and drug resistance are diminished. However, both can be restored by subsequent regrowth in normal medium. Thus, although detectable genetic changes accompany the development of vinblastine resistance, the expression of these changes can be modulated by environmental lipid.

Effect of gallium on DNA synthesis by human T-cell lymphoblasts.

We have studied the antiproliferative effects of gallium nitrate in cultured CCRF-CEM lymphoblasts. The 50% inhibitory dose for these cells was 120 microM, and after 24 h at a cytostatic concentration (480 microM) S-phase arrest was observed by DNA flow cytometry. Deoxyribonucleoside triphosphate pools were all reduced (dATP, dGTP, and dCTP by 50%, dTTP by 25%), suggesting inhibition of ribonucleotide reductase. Administration of tracer amounts (0.5 microM) of either [3H]uridine or [3H]deoxyuridine confirmed that DNA synthesis had been inhibited to 20% of control rates by gallium. Further, the flow of the ribonucleoside into the dTTP pool and DNA was selectively reduced compared to that of the deoxyribonucleoside. Gallium decreased the specific activity of dTTP labeled from uridine by 50%, whereas the specific activity of dTTP labeled from deoxyuridine was increased 2.5-fold. Thus counts in DNA derived from [3H]uridine were decreased by more than 80%, while counts in DNA derived from [3H]deoxyuridine were virtually unaltered. Uridine incorporation into RNA was not affected. Gallium did not significantly alter the capacity of permeabilized naive cells to incorporate [3H]dTTP into DNA, while 24-h gallium pretreatment (which increased the percentage of S-phase cells) produced a modest increase in [3H]dTTP incorporation, indicating that any effect of gallium on DNA polymerase alpha is minor. Gallium treatment did not induce or inhibit the repair of DNA single strand breaks. These data demonstrate that gallium inhibits replicative DNA synthesis, with the major specific enzyme target probably being ribonucleotide reductase.

Analytical DNA flow cytometric analysis of deoxyadenosine toxicity in cultured human leukemic lymphoblasts.

Human T leukemic lymphocytes are highly susceptible to growth inhibition by dAdo. We have investigated this phenomenon using analytical DNA flow cytometry. By using (a) bromodeoxyuridine quenching of Hoechst 33342 fluorescence and (b) the drug ICRF-159, a selective G2 - M-blocking agent, we show that dAdo causes a G1 block in cultured T leukemic cells and that cells in the S phase exposed to dAdo are able to complete that S phase, pass through G2 + M, and return to the G1 phase. Centrifugal elutriation was used to enrich cells for various phases of the cell cycles. dAdo caused elevation of the dATP pool to a similar extent in G1, S, and G2 - M-enriched cell fractions, but did not cause a fall in the dCTP pool. These findings indicate that dAdo induces a G1 block via elevation of dATP pools, apparently independently of inhibition of ribonucleotide reductase.

G1-phase arrest of cultured human leukemic T-cells induced by deoxyadenosine.

Cultured human T-cell leukemia lymphocytes have enhanced sensitivity to growth inhibition by deoxyadenosine. We have used flow cytometry to investigate the mechanism of deoxyadenosine toxicity in cultured T-leukemic cells. Comparative studies on deoxyadenosine-resistant Epstein-Barr virus-transformed B-lymphocyte cell lines were also performed. After exposure of T-cells to low concentrations of deoxyadenosine (3 microM), in the presence of an adenosine deaminase inhibitor (erythro-9-[3-(2-hydroxynonyl)]adenosine), accumulation of cells of cells with a G1 DNA content was demonstrated. In contrast, B-cell lines showed a similar degree of growth inhibition after exposure to 200 to 400 microM deoxyadenosine but were blocked in S phase. The T-cell G1 block was associated with a rise in the deoxyadenosine triphosphate pool, and both these phenomena were prevented by the addition of deoxycytidine. The biochemical mechanism of this G1 block induced by deoxyadenosine in T-cells is not understood.

Ecto-adenosine triphosphatase deficiency in cultured human T and null leukemic lymphocytes. A biochemical basis for thymidine sensitivity.

Cultured leukemic T and null lymphocytes are highly sensitive to growth inhibition by thymidine, as well as the other deoxynucleosides, deoxyguanosine and deoxyadenosine. By contrast, Epstein-Barr virus-transformed B lymphocytes are relatively resistant to deoxynucleosides. Growth inhibition is associated with the development of high deoxyribotriphosphate pools after exposure to the respective deoxynucleotides. We show that malignant T and null lymphocytes are deficient in ecto-ATPase activity. We show this cell surface enzyme to be of broad specificity, capable of degrading both ribotriphosphates and deoxyribotriphosphates. High levels of this ecto-enzyme are found in deoxynucleoside-resistant, Epstein-Barr virus-transformed B lymphocytes. Ecto-ATPase deficiency may represent a mechanism for increased sensitivity to deoxynucleoside growth inhibition.

Sensitivity of leukemic human null lymphocytes to deoxynucleosides.

The growth of cultured leukemic T-lymphocytes is readily inhibited by deoxynucleosides, particularly thymidine, deoxyguanosine, and deoxyadenosine. By contrast, Epstein-Barr virus-transformed B-lymphocytes are relatively resistant to deoxynucleosides. Growth inhibition correlates with the development of high deoxyribonucleoside triphosphate pools following exposure to deoxynucleosides. Leukemic T-lymphocytes are deficient in ecto-5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) activity, and it has been postulated that deficiency of this enzyme decreases the capacity of these cells to degrade deoxyribonucleotides, rendering them sensitive to deoxynucleosides. We find that the sensitivity of cultured null-type leukemic lymphocytes to growth inhibition of deoxynucleosides is similar to that of T-cells. However, the null cells contain normal levels of ecto-5'-nucleotidase. We infer that ecto-5'-nucleotidase deficiency does not have a central role in determining the deoxynucleoside sensitivity of leukemic lymphocytes.