Expansion in bioreactors of human progenitor populations from cord blood and mobilized peripheral blood

Umbilical cord blood (UCB) and mobilized peripheral blood (MPB) provide an alternate source to bone marrow for transplantation. Expansion in vitro of stem/progenitor cell populations from these sources may provide adult-sized grafts otherwise not attainable because of the limited cell numbers available in the case of UCB or because of numerous rounds of apheresis required for sufficient MPB cells. We asked whether continuous perfusion culture could be employed in ex vivo expansion to produce clinically relevant numbers of stem/progenitor cells from these sources. To evaluate MPB, 1-10 million leukocytes, from patients who had received either granulocyte colony-stimulating factor (G-CSF) or cyclophosphamide and granulocyte-macrophage colony-stimulating factor (GM-CSF), were inoculated into bioreactors, with or without irradiated, allogeneic stroma. The growth factor combination in the perfusion medium consisted of interleukin-3 (IL-3), stem cell factor (SCF), GM-CSF and erythropoietin (Epo). Under the best conditions tested, total cell numbers, granulocyte-macrophage colony-forming units (CFU-GM), and long-term culture-initiating cell (LTC-IC) populations were expanded by about 50-, 80-, and 20-fold, respectively, over 14 days. At low cell inocula (1 million), the presence of stroma enhanced the expansion of total cells and CFU-GM but not of LTC-IC. When SCF was not included in the medium, both total cells and CFU-GM expanded to a much lesser extent, but again the expansion of LTC-IC was not affected. At the higher cell inoculum (10 million), expansions of total cells and CFU-GM were equivalent with or without stroma. To evaluate UCB, cells were placed into bioreactors with or without irradiated, allogeneic stroma, and the bioreactors were perfused with medium containing the four standard growth factors. After 6-14 days, in several independent experiments, 20-24 million cells were harvested from bioreactors perfused with SCF-containing medium, irrespective of the presence or absence of preformed stroma. Similarly, in reactors perfused with SCF-containing medium (with or without stroma), an average 40- to 60-fold expansion of CFU-GM was obtained, yielding an average of 1.5-1.8 x 10(5) CFU-GM per reactor. Harvested cells were thus up to 40-fold enriched in CFU-GM in comparison to the inoculum. In the absence of SCF, cell expansions averaged 1.5- to 2-fold, and CFU-GM were expanded only 10- to 14-fold by day 14. As before, the presence of preformed stroma did not affect either cell or CFU-GM yields, provided the cell inoculum was at least 4.5 million cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Short-pulse, eye-safe Nd:YAG laser using cavity-dumping

A short-pulse 1.444-μm laser based on Nd:YAG technology has been demonstrated. The 1.444-μm is eye-safe. With the cavity-dump technique, a pulse of 50 m× and 14 ns was obtained. The beam quality was excellent with an M² of 1.6 by the use of a telescopic resonator. Silicon-window polarizers were used to suppress the 1.06-μm radiation but showed 1.444-μm absorption as well     

Propagation of granulocytic Ehrlichia spp. from human and equine sources in HL-60 cells induced to differentiate into functional granulocytes

Ehrlichia spp. from human and equine sources in the northeastern Unites States were detected by PCR, isolated, and propagated in the HL-60 promyelocytic leukemia cell line. Growth of Ehrlichia from both equine and human sources was enhanced by addition of retinoic acid, which causes granulocytic differentiation of the HL-60 cells. DNA sequencing of a portion of the 16S rDNA gene supported the hypothesis that the same pathogen was responsible for both equine and human granulocytic ehrlichiosis.     

A new actigraph for long-term registration of the duration andintensity of tremor and movement

Actigraphy, the long-term measurement of human movement with a small solid state recorder, is gaining acceptance as a useful method in many research fields. Currently available actigraphs assess or estimate the movement duration per time interval. However, the output gives no information on movement type or intensity, and cannot be used in subjects suffering from tremor. The present paper describes a new type of actigraph, that has been developed primarily for the long-term evaluation of motor symptoms in Parkinson patients. The device is the first to discriminate tremor from other movements and to assess both duration and intensity of the two types of movement. It is based on a Motorola 68HC805B6 microcontroller and contains: an accelerometer, programmable gain stages, programmable low- and highpass filters, a programmable level comparator, a peak detector, interface circuits, a real time clock, data storage, and control circuitry. The micro-controller performs a period amplitude sequence analysis (PASA) on the conditioned accelerometer signal, and stores four output variables (tremor duration, tremor amplitude, movement duration, and movement amplitude) at the end of programmable time intervals. The analysis of fluctuations in the motor symptoms of, e.g., Parkinson patients using this actigraph can be of great help in the pharmacological management of symptoms     

The Transmogrifier-2: a 1 million gate rapid-prototyping system

This paper describes the Transmogrifier-2 (TM-2), a second-generation multifield programmable gate array (FPGA) rapid-prototyping system. The largest version of the system will comprise 16 boards that each contain two Altera 10K50 FPGA's, four I-Cube interconnect chips, and up to 8 Mbytes of memory. The inter-FPGA routing architecture of the TM-2 uses a novel interconnect structure, a nonuniform partial crossbar, that provides a constant delay between any two FPGA's in the system. The TM-2 architecture is modular and scalable, meaning that systems of various sizes can be constructed from copies of the same board, while maintaining routability and the constant delay feature. Other features include a system-level programmable clock that allows single-cycle access to off-chip memory, and programmable clock waveforms with edge resolution of 10 ns. The first Transmogrifier-2 boards have been manufactured and are functional. They have recently been used successfully in some simple graphics acceleration applications     

HoxA is a transcriptional regulator for expression of the hup structural genes in free-living Bradyrhizobium japonicum

A chromosomally integrated Bradyrhizobium japonicum hoxA mutant is unable to oxidize hydrogen in free-living conditions. Derepressing conditions that induce hydrogenase activity in free-living, wild-type B. japonicum cells cannot induce expression of the hydrogenase structural genes in the hoxA mutant. The DNA-binding capacity of HoxA at the hup promoter region was studied by means of gel retardation. Both heterotrophically growing cells and cells induced to express hydrogenase activity contain a protein that specifically binds to the hup promoter region. Crude protein extracts isolated from a B. japonicum hoxA mutant do not contain this binding compound. The HoxA protein was overexpressed in E. coli and isolated in the form of a maltose-binding protein (MBP)-HoxA fusion. The MBP-HoxA hybrid protein specifically bound to a 50 bp region of the hupSL promoter known to be important for regulation of hupSL expression.     

High-resolution HLA typing for the DQB1 gene by sequence-based typing

BACKGROUND: Monocytic tissue factor (TF), initiating the extrinsic blood coagulation pathway, is often upregulated under septic or inflammatory conditions. The complex activating mechanism remains largely unclear and no effective strategy has been firmly established. In this study, we used a model monocytic cell line (human leukemic THP-1 promonocytes) to address (1) the nature of TF activation in response to bacterial endotoxin and (2) the application of anti-inflammatory cytokines in relieving monocytic hypercoagulation. RESULTS: TF in THP-1 cells was substantially activated by exposure to bacterial endotoxin (LPS; 5 micrograms/ml) for 6 h. Human recombinant IL-4 (500 ng/ml) and IL-10 (500 ng/ml) inhibited TF activation induced by LPS. To determine if these cytokines depressed LPS recognition resulting in such inhibition, we employed an anti-CD14 mAb (UCHM-1; Sigma Chemical) to address the role of CD14 in LPS transmembrane signaling. LPS-induced TF activation was depressed by 35% upon inclusion of the anti-CD14 mAb (1:10 dilution). This antibody alone mimicked TF activation which accounted for 35% of the LPS-induced TF activation, suggesting the activating role of CD14 ligation. In addition, the anti-CD14 mAb elicited the production of nitric oxide (NO) which was found to be independent of TF activation. NO production could serve as an independent index for monitoring LPS recognition. IL-4 depressed the anti-CD14 mAb-induced TF activation as well as NO elicitation, indicating the blockade of CD14 ligation. In contrast, IL-10 showed differential inhibitory activities. TF activation induced by either LPS or anti-CD14 mAb was inhibited by IL-10 which did not show any inhibition on NO elicitation under these conditions. In a separate approach, neither IL-4 nor IL-10 inhibited phorbol ester-induced NO elicitation. More direct evidence came from an epifluorescent demonstration showing that IL-4 blocked binding of FITC-conjugated LPS and anti-CD14 mAb to THP-1 cells. CONCLUSIONS: Taken together, the results suggest that LPS action in relation to TF activation consists of CD14-independent and -dependent signaling including CD14 ligation. We also showed that anti-inflammatory cytokines (IL-4 and -10) significantly depressed TF activation. IL-4 antagonized CD14-dependent LPS recognition leading to the depression in TF activation.