Tissue culture surface characteristics influence the expansion of human bone marrow cells

Human cell therapy applications in tissue engineering, such as the ex vivo production of hematopoietic cells for transplantation, have recently entered the clinic. Although considerable effort has been focused on the development of biological processes to generate therapeutic cells, little has been published on the design and manufacture of devices for implementation of these processes in a robust and reproducible fashion at a clinical scale. In this study, the effect of tissue culture surface chemistry and texture was assessed in human bone marrow (BM) mononuclear cell (MNC) and CD34-enriched cell cultures. Growth and differentiation was assessed by total, progenitor (CFU-GM), stromal (CFU-F), and primitive (LTC-IC) cell output. Tissue culture treated (TCT) plastic significantly increased MNC culture output as compared with non-TCT plastic, whereas CD34-enriched cell cultures gave lower output (than MNC cultures) that was unaffected by TCT plastic. Interestingly, the level of MNC culture output was significantly different on four commercial TCT surfaces, with the best performing surface giving output that was 1.6- to 2.8-fold greater than the worst one. The surface giving the highest output was the best at supporting development of a distinct morphological feature in the adherent layer (i.e. cobblestone area) indicative of primitive cells, and X-ray photoelectron spectroscopy (XPS) was used to characterize this surface. For custom injection molding of culture devices, the use of three different resins resulted in MNC culture output that was equivalent to commercial cultureware controls, whereas CD34-enriched cell cultures were highly sensitive to resins containing additives. When the texture of molded parts was roughened by sandblasting of the tool, MNC culture output was significantly reduced and higher spikes of IL-6 and G-CSF production were observed, presumably due to macrophage activation. In conclusion, the manufacture of BM MNC culture devices for clinical applications was optimized by consideration of plastic resin, surface treatment, and texture of the culture substratum. Although CD34-enriched cells were insensitive to surface treatment, they were considerably more sensitive to biocompatibility issues related to resin selection. The development of robust systems for BM MNC expansion will enable clinical trials designed to test the safety and efficacy of cells produced in this novel tissue engineering application.     

Swelling detection for volume regulation in the primitive eukaryote Giardia intestinalis: a common feature of volume detection in present-day eukaryotes

Interleukin (IL)-12, a natural killer (NK) cell stimulatory factor, is a heterodimeric cytokine that is known to be a potent activator of non-major histocompatibility complex-restricted cytotoxicity by peripheral blood-derived NK cells. NK cells (CD3-CD16+/CD56+) represent approximately 15% of human umbilical cord blood mononuclear cells (HUCB MNCs) and are known to be highly sensitive to activation by IL-2. In the present study, we monitored the effect of IL-12 on the cytotoxic activity, proliferation, and phenotypic expression of HUCB-derived resting and IL-2-activated cytotoxic cells and compared these parameters with those of bone marrow (BM)-derived cells. Lymphocytes were separated from HUCB by 3% gelatin sedimentation and incubated with IL-12 and/or IL-2 for 18 hours. At effector:target ratios of 40:1 and 20:1, IL-12 (50 U/mL) significantly increased both resting and IL-2-activated NK cell-mediated cytotoxicity in a standard 51Cr-release assay against both NK-sensitive (K562) and NK-resistant (Colo-205) cell lines. In addition, resting and IL-2-activated cytotoxic cells derived from HUCB exhibited superior cytolytic ability compared with BM-derived cells. This increase was observed in resting cells as well as in those that were preincubated with IL-12. Moreover, HUCB-derived cells were found to be more sensitive to IL-12 activation than cytotoxic cells from BM. To evaluate the involvement of accessory cells, NK cells were purified from HUCB using immunomagnetic beads, and these cells were found to have a lower response to treatment with IL-12 than unpurified populations. HUCB MNCs exhibited a nonsignificant increase in proliferation after IL-12 treatment and were better able to respond to IL-12 activation than BM MNCs. Following an 18-hour incubation, IL-12 was able to cause upregulation of CD25 and CD69 activation antigens, whereas no significant change in expression of CD16 and CD56 NK cell surface antigens, CD3 on T cells, or IL-12 receptor was observed. Similarly, IL-12 did not affect NK cell:target cell conjugation as assessed by fluorescence-activated cell sorting. Our results indicate that HUCB-derived NK-mediated cytotoxic capabilities can be increased by IL-12, a finding that may have clinical relevance.     

Prostaglandins suppress an outward potassium current in embryonic rat sensory neurons

One of the challenges in understanding ciliary and flagellar motility is determining the mechanisms that locally regulate dynein-driven microtubule sliding. Our recent studies demonstrated that microtubule sliding, in Chlamydomonas flagella, is regulated by phosphorylation. However, the regulatory proteins remain unknown. Here we identify the 138-kD intermediate chain of inner arm dynein I1 as the critical phosphoprotein required for regulation of motility. This conclusion is founded on the results of three different experimental approaches. First, genetic analysis and functional assays revealed that regulation of microtubule sliding, by phosphorylation, requires inner arm dynein I1. Second, in vitro phosphorylation indicated the 138-kD intermediate chain of I1 is the only phosphorylated subunit. Third, in vitro reconstitution demonstrated that phosphorylation and dephosphorylation of the 138-kD intermediate chain inhibits and restores wild-type microtubule sliding, respectively. We conclude that change in phosphorylation of the 138-kD intermediate chain of I1 regulates dynein-driven microtubule sliding. Moreover, based on these and other data, we predict that regulation of I1 activity is involved in modulation of flagellar waveform.     

Structural effects of the binding of GTP to the wild-type and oncogenic forms of the ras-gene-encoded p21 proteins

Molecular dynamics calculations have been performed to determine the average structures of ras-gene-encoded p21 proteins bound to GTP, i.e., the normal (wild-type) protein and two oncogenic forms of this protein, the Val 12- and Leu 61-p21 proteins. We find that the average structures for all of these proteins exhibit low coordinate fluctuations (which are highest for the normal protein), indicating convergence to specific structures. From previous dynamics calculations of the average structures of these proteins bound to GDP, major regional differences were found among these proteins [Monaco et al. (1995), J. Protein Chem., in press]. We now find that the average structures of the oncogenic proteins are more similar to one another when the proteins are bound to GTP than when they are bound to GDP [Monaco et al. (1995), J. Protein Chem., in press]. However, they still differ in structure at specific amino acid residues rather than in whole regions, in contradistinction to the results found for the p21-GDP complexes. Two exceptions are the regions 25-32, in an alpha-helical region, and 97-110. The two oncogenic (Val 12- and Leu 61-) proteins have similar structures which differ significantly in the region of residues 97-110. This region has recently been identified as being critical in the interaction of p21 with kinase target proteins. The differences in structure between the oncogenic proteins suggest the existence of more than one oncogenic form of the p21 protein that can activate different signaling pathways.     

Sleep and nitric oxide: effects of 7-nitro indazole, inhibitor of brain nitric oxide synthase

The in vitro amplification method for heterologous gene expression in mammalian cells is based on the stable transfection of cells with long, linear DNA molecules having several copies of complete expression units, coding for the gene of interest, linked to one terminal unit, coding for the selectable marker. DNA concatenamers containing additional expression units can also be prepared: we exploited this feature by co-polymerizing expression units coding for granulocyte colony-stimulating factor (G-CSF) with cassettes for dihydrofolate reductase (DHFR) and for neomycin (Nm) resistance, as selectable markers. We were thus able to obtain high level production of G-CSF in chinese hamster ovary (CHO) dhfr- cells by combining in vitro amplification to just one step of in vivo amplification. This approach required a considerably shorter time than the classical, stepwise amplification by methotrexate.     

Discrepancies in normative data between Lunar and Hologic DXA systems

Many studies have shown the high correlation between Lunar and Hologic DXA bone mineral density (BMD) measurements despite differences in absolute calibration. However, in clinical practice, raw BMD values (in g/cm2) are not normally used for assessing skeletal status and fracture risk. Instead, the BMD values are expressed in terms of the number of standard deviations above or below the young normal value (commonly referred to as the T-score). If the normative populations of the various systems are consistent, the standard deviation scores should also be consistent. For this reason, the World Health Organization (WHO) recently established diagnostic criteria for osteoporosis based on T-scores and not BMD. However, few studies have compared the instruments in terms of their standard deviation scores. In this study, we used linear regression to compare T-scores in 83 women at L1-4 and 120 women at the femoral neck obtained on a Lunar DPX and a Hologic QDR-1000/W system. patient BMD and T-score measurements were highly correlated between the two systems (r > 0.95). No clinically significant difference in L1-4 T-scores was seen (less than 0.1 SD). However, linear regression analysis confirmed a systematic difference of 0.9 SD between the femoral neck T-scores. This discrepancy is caused by: (1) differences in the normal populations, and (2) differences in statistical models used to determine the young normal mean and standard deviation. In an attempt to correct the discrepancy, the female young normal mean and standard deviation were recalculated for the femoral neck using published epidemiological data from NHANES and existing DXA cross-calibration equations. The Hologic young normal value (mean +/- SD) was redefined as 0.85 +/- 0.11 g/cm2, while the Lunar value was redefined as 1.00 +/- 0.11 g/cm2. When the femoral neck T-scores for the study population were recalculated on the basis of these new values, the results were equivalent between manufactures, effectively eliminating the discrepancy. However, the revised values should be confirmed by additional measurements in young normal adults.     

Role of nitric oxide-related mechanisms in renal function in ageing rats

BACKGROUND: The impaired renal function and vasodilatation that accompany age need to be re-addressed based upon the new knowledge concerning vascular nitric oxide (NO)-dependent systems. The present study examined the effects of age on the NO-related renal response. METHODS: The study was performed in euvolaemic, conscious Wistar rats, aged 5 and 18 months. Renal function and haemodynamic measurements with fluorescent microspheres were employed to assess differences between groups. RESULTS: A first set of experiments showed that ageing rats had a reduced natriuretic and diuretic response to acetylcholine, whereas the response to sodium nitroprusside was preserved. In the same regard, a reduction of the renal functional effects of L-arginine (L-Arg) and L-glycine (L-Gly) was found in the older rats. In the ageing rats, these responses were accompanied by an enhanced effect of the L-Arg competitive analogue, NwNLA, which provoked a marked reduction of renal function. This effect of NwNLA was blocked by the simultaneous administration of a small dose of L-Arg in the ageing but not in the young rats. Systemic haemodynamic studies revealed that in ageing rats, NwNLA reduced renal blood flow and increased renal vascular resistances in a significantly higher proportion than in younger animals. However, flow to other organs, namely, brain, spleen or liver, was affected in a similar manner in both young and old rats. Ultrastructural alterations were found in endothelial cells, which might constitute the anatomical basis for the observed functional derangements. CONCLUSIONS: The present experiments reveal that ageing is accompanied by significant differences in NO-related responses in the kidney which do not appear to affect blood flow to other organs. The response to L-Arg and L-Arg competitive analogues supports the existence of a marked dependency on NO-related mechanisms in the ageing rats, but not of a decreased baseline activity of the NO-dependent pathways.     

Extracorporeal membrane oxygenator support for cardiopulmonary failure. Experience in 28 cases

We have used extracorporeal membrane oxygenation (ECMO) for 28 patients (14 children and 14 adults) over a 5 year period. Nine patients improved on ECMO and 5 were long-term survivors. ECMO was used for pulmonary insufficiency in 24 patients. Initially, only moribund patients were treated, but recently the combination of open lung biopsy and pulmonary insufficiency index (PII) has been used to select patients. The best results have been obtained in newborn cases and the adult capillary leak syndromes; the major problem has been progression to fibrosis despite ECMO support. ECMO was used for cardiac failure in 4 patients. Children with postoperative cardiac failure did the best; profound shock was not reversed with venoarterial bypass. ECMO support is lifesaving in selected cases of pulmonary insufficiency. Initial trials in cardiac failure and the infant age group in this series suggest that ECMO will have an even greater role in those applications.