Investigation of effect of particle size and rumen fluid addition on specific methane yields of high lignocellulose grass silage.

This work examines the digestion of advanced growth stage grass silage. Two variables were investigated: particle size (greater than 3 cm and less than 1cm) and rumen fluid addition. Batch studies indicated particle size and rumen fluid addition had little effect on specific methane yields (SMYs). In continuous digestion of 3 cm silage the SMY was 342 and 343 L CH4 kg(-1)VS, respectively, with and without rumen fluid addition. However, digester operation was significantly affected through silage floating on the liquor surface and its entanglement in the mixing system. Digestion of 1cm silage with no rumen fluid addition struggled; volatile fatty acid concentrations rose and SMYs dropped. The best case was 1cm silage with rumen fluid addition, offering higher SMYs of 371 L CH4 kg(-1)VS and stable operation throughout. Thus, physical and biological treatments benefited continuous digestion of high fibre grass silage.

Pegfilgrastim compared with filgrastim for cytokine-alone mobilization of autologous haematopoietic stem and progenitor cells.

Haematopoietic stem and progenitor cells (HSPC) mobilization, using cytokine-alone, is a well-tolerated regimen with predictable mobilization kinetics. Single-dose pegfilgrastim mobilizes HSPC efficiently; however, there is surprisingly little comparative data on its use without chemotherapy for HSPC mobilization. Pegfilgrastim-alone and filgrastim-alone mobilization regimens were compared in 52 patients with haematological malignancy. Pegfilgrastim 12 mg (n=20) or 6 mg (n=2) was administered Day 1 (D1) in 22 patients (lymphoma n=17; myeloma n=5). Thirty historical controls (lymphoma n=18; myeloma n=12) received filgrastim 10 mcg/kg daily from D1. Peripheral blood (PB) CD34(+) counts reached threshold (5 × 10(6)/L) and apheresis commenced on D4(4-5) and D4(4-6). Median PB CD34(+) cell count on D1 of apheresis was similar (26.0 × 10(6)/L (2.5-125.0 × 10(6)/L) and 16.2 × 10(6)/L (2.6-50.7 × 10(6)/L); P=0.06), for pegfilgrastim and filgrastim groups, respectively. Target yield (2 × 10(6) per kg CD34(+) cells) was collected in 20/22 (91%) pegfilgrastim patients and 24/30 (80%) in the filgrastim group (P=0.44), in a similar median number of aphereses (3(1-4) versus 3(2-6), respectively; P=0.85). A higher proportion of pegfilgrastim patients tended to yield 4 × 10(6) per kg CD34(+) cells; 16/22 (73%) versus 14/30 (47%) filgrastim patients (P=0.09). One pegfilgrastim patient developed hyperleukocytosis that resolved without incident. Pegfilgrastim-alone is a simple, well-tolerated, and attractive option for outpatient-based HSPC mobilization with similar mobilization kinetics and efficacy to regular filgrastim.

Human dendritic cells pulsed with specific lipopeptides stimulate autologous antigen-specific T cells without the addition of exogenous maturation factors.

SUMMARY: Serum-free culture conditions to generate immature human monocyte-derived DC (Mo-DC) were optimized, and the parameters that influence their maturation after exposure to lipopeptides containing CD4(+) and CD8(+) T-cell epitopes were examined. The lipopeptides contained a single CD4(+) helper T-cell epitopes, one of a number of human leucocyte antigen (HLA)-A2-restricted cytotoxic T-cell epitope and the lipid Pam2Cys. To ensure complete maturation of the Mo-DC, we examined (i) the optimal lipopeptide concentration, (ii) the optimal Mo-DC density and (iii) the appropriate period of exposure of the Mo-DC to the lipopeptides. The results showed that a high dose of lipopeptide (30 microm) was no more efficient at upregulating maturation markers on Mo-DC than a low dose (6 microm). There was an inverse relationship between Mo-DC concentration and the mean fluorescence intensity of maturation markers. In addition, at the higher cell concentrations, the chemotactic capacity of the Mo-DC towards a cognate ligand, CCL21, was reduced. Thus, high cell concentrations during lipopeptide exposure were detrimental to Mo-DC maturation and function. The duration of exposure of Mo-DC to the lipopeptides had little effect on phenotype, although Mo-DC exposed to lipopeptides for 48 rather than 4 h showed an increased ability to stimulate autologous peripheral blood mononuclear cells to release interferon-gamma in the absence of exogenous maturation factors. These findings reveal conditions for generating mature antigen-loaded DC suitable for targeted immunotherapy.

In vivo tracking for cell therapies.

The success of a particular cellular therapy regime requires the therapeutic agent to migrate expeditiously to the intended target in sufficient numbers and to provoke a desirable response. There are many variables associated with the production, administration and host that need to be investigated to maximize the resulting therapeutic benefit. The large number of factors which may contribute to, or detract from, treatment efficacy can make therapy optimization an arduous procedure. Direct visualization of in vivo migration patterns using nuclear medicine techniques greatly assists the appraisal of the multitude of variables. Conventional radionuclide cell labeling is a proven, simple and sensitive technique which can provide whole body biodistribution information. Labeling with a PET isotope offers greater sensitivity, much improved 3-dimensional resolution and quantification. In general, current efforts are increasingly concentrating on this technology. Imaging studies can supply definitive evidence of successful targeting and allow quantification of the degree of migration to a particular site. Incorporating tracking studies into clinical trials of cell-based therapy at the earliest stage can provide proof of mechanism of the therapy and permit evaluation of the many contributory variables, even on a patient-by-patient basis.

Regulation of cellular therapies: the Australian perspective.

The first step in the process of regulating cell-based products in Australia was taken in 1991, when the code of good manufacturing practice (cGMP) for 'Blood and Blood Components' was instituted. Paradoxically, it focused on the regulation of plasma fractionation, the non-cellular component of blood. Subsequently, Australia's regulatory body for medicinals, the Therapeutic Goods Administration (TGA), has clearly stated that all cell-based therapies utilizing components of blood and/or tissues will be regulated. The final landscape for the regulation of cellular therapies has yet to be defined, but is likely to be clarified within the next 12 months. The current cGMP for 'Blood and Tissues' is the regulatory document for all aspects of cell processing, including standard blood components (cellular and plasma), cord blood and allogeneic cells for storage. Currently, there are some exemptions to government regulation, and the most important of these is autologous hemopoietic stem cells (HSC). Indeed, no licensing is required for processing of HSC at the moment, although most centers subject themselves to a self-imposed auditing system through the National Association of Testing Authorities, Australia. However, it is anticipated that within 12 months this and the other exemptions within the Act will be removed. The TGA will become the formal regulator of all cell-based therapies, and laboratories will be required to apply for cGMP auditing and licensing. It is likely that the Foundation for the Accreditation of Cellular Therapy (FACT) guidelines or others of a similar nature, will form the basis of one of the regulatory standards for HSC processing. Of particular note is the inclusion of apheresis as an integral component of cGMP licensing.

Expression of mdr1 and mrp in the normal B-cell homologue of B-cell chronic lymphocytic leukaemia.

B-cell chronic lymphocytic leukaemia (CLL) cells commonly express the multidrug resistance phenotype. The aim of this study was to establish whether the normal homologue in B-cell ontogeny of B-CLL also expressed the multidrug resistance (mdr) phenotype. Human tonsillar lymphocytes were sorted to yield two B-cell subsets based on the expression of CD19, CD5 and CD10. The normal homologue was represented by a population of B cells that was CD19 positive, CD10 negative and weakly expressed CD5. Based upon functional analysis and the detection of mdr1 mRNA by semi-quantitative PCR, these cells expressed the mdr phenotype. In contrast, functional multidrug resistance could not be demonstrated in CD19-positive CD10-positive cells with strong expression of CD5, nor could mdr1 mRNA be found in these cells. MRP was variably expressed in both B-cell subsets with no discernable differences in the pattern of expression. We conclude that normal B cells with a phenotype resembling that of B-CLL cells express the multidrug resistance phenotype.

Altered methylation of the human MDR1 promoter is associated with acquired multidrug resistance.

One of the most important forms of drug resistance in acute myeloid leukemia is the multidrug resistance (MDR) phenotype, which is characterized by the expression of the MDR1 gene product, P-glycoprotein. Although a number of factors affect MDR1 gene expression, the genetic events that "switch on" the human MDR1 gene in tumor cells that were previously P-glycoprotein negative have remained elusive. Here, we report evidence that the methylation status of the human MDR1 promoter may serve as a basis for this "switch." Based on Southern analysis using methylation-sensitive and methylation-insensitive restriction enzymes, a tight correlation was found between MDR phenotype and demethylation of the 5' region of the MDR1 gene in a human T cell leukemia cell line. Similar results were obtained from the analysis of P-glycoprotein-positive and P-glycoprotein-negative samples of chronic lymphocytic leukemia. Treatment of the cell lines with the demethylating agent 5'-azadeoxycytidine altered the methylation pattern of the MDR1 promoter in P-glycoprotein-negative cells to resemble that of P-glycoprotein-positive cells and activated the promoter such that MDR1 mRNA was now detectable. Treatment also resulted in an increased resistance to epirubicin and decreased daunomycin accumulation, both of which were reversible by verapamil, a characteristic of the classical MDR phenotype in cells expressing P-glycoprotein. These results suggest that the MDR phenotype may be acquired as a result of changes in methylation of the MDR1 promoter.

Modulation of the function of human MDR1 P-glycoprotein by the antimalarial drug mefloquine.

MDR1 P-glycoprotein in membranes of human tumor cells of the CEM/VBL100 line was selectively labelled using photoreactive analogs of verapamil, N-(p-azido-3-[125I]salicyl)amino-verapamil ([125I]ASA-V) and prazosin, 2-[4-(4-azido-3-[125I]iodobenzoyl)piperazin-1-yl]4 -amino-6,7-dimethoxyyquinazoline ([125I]ASA-P). Mefloquine, a quinolinemethanol antimalarial drug, was shown to inhibit the labelling of P-glycoprotein with an efficiency similar to that for verapamil, a known chemosensitizer. By contrast, chloroquine competed poorly for the binding site on P-glycoprotein. Mefloquine also inhibited the functional activity of P-glycoprotein. It decreased the rates of extrusion of [3H]vinblastine and the fluorescent dyes, fluo-3 acetomethoxy ester and rhodamine 123, from drug-resistant cells and decreased the level of resistance of these cells to vinblastine. The ability of mefloquine to inhibit P-glycoprotein function may be involved in the neurotoxic side-effects occasionally associated with the use of mefloquine as an antimalarial drug.

Cyclosporin A and PSC 833 prevent up-regulation of MDR1 expression by anthracyclines in a human multidrug-resistant cell line.

We have previously demonstrated that within 24 h of exposure of the CEM/A7R cell line to epirubicin (EPI), MDR1 gene expression is induced. The aim of the current study was to investigate the role of cyclosporin A (CyA) and PSC 833, two biochemical modulators of the classical multidrug-resistant phenotype, in this model. CEM/A7R cells were exposed to EPI in the presence or absence of various concentrations of CyA or PSC 833. MDR1 expression was assessed using Northern blot analysis and quantitated using a phosphorimager. P-glycoprotein (P-gp) expression was analyzed by the determination of MRK16 binding using flow cytometry. P-gp function was measured in an assay of [3H]daunomycin accumulation. The coincubation of CyA or PSC 833 with EPI prevented the increase in MDR1 gene expression induced by EPI alone. This effect of the two modulators was dose dependent. Neither modulator alone had any significant effect on the expression of MDR1. In these experiments, changes in MDR1 expression correlated with changes in P-gp levels (based on MRK16 binding) and P-gp function. Thus, both PSC 833 and CyA appear to prevent the induction of MDR1 gene expression caused by the short-term exposure of CEM/A7R cells to EPI.

Inheritance of chromosome 7 is associated with a drug-resistant phenotype in somatic cell hybrids.

A major form of drug resistance in tumour cells known as classical multidrug resistance (MDR) is associated with the overexpression of the mdr1 gene product, the membrane protein P-glycoprotein (P-gp), which acts as an energy-dependent drug efflux pump. In this study the inheritance of P-gp expression was examined using hybrids formed after somatic cell fusion between a drug-sensitive human T-cell leukaemia cell line, CEM/CCRF, and a drug-resistant derivative, CEM/A7, which is characterized by a clonal chromosomal duplication dup(7)(q11.23q31.2). Fourteen hybrids, chosen at random, were analysed by reverse transcriptase-polymerase chain reaction (RT-PCR) and by binding studies involving the monoclonal antibody MRK16, which recognises an external P-gp epitope. Only two hybrids were positive for both MRK16 antibody labelling and mdr1 mRNA. Partial karyotypic analysis of all hybrids revealed that only the MRK16-positive hybrids contained the duplication in chromosome 7 seen in the CEM/A7 parental MDR line. Therefore, P-gp overexpression in the MRK16-positive hybrids may be linked to the inheritance of chromosome 7 from CEM/A7 and possibly associated with the chromosome 7 abnormality.

Rapid up-regulation of mdr1 expression by anthracyclines in a classical multidrug-resistant cell line.

Studies were carried out in a variant human multidrug-resistant (MDR) cell line CEM/A7R, which expresses very low levels of mdr1 mRNA and P-glycoprotein (P-gp). The induction of mdr1 RNA expression by three anthracyclines, (doxorubicin, daunorubicin, epirubicin), VP-16 and two vinca alkaloids (vincristine, vinblastine) was semiquantitatively assessed by scanning Northern blots on a phosphorimager. The relative level of mdr1 expression was expressed as ratio of mdr1 to the internal RNA (actin). A significant increase (P < 0.02) in expression of mdr1 was noted within 4 hrs of exposure to 1.5 micrograms ml-1 daunorubicin or epirubicin. Neither vinblastine nor vincristine had any effect on mdr1 levels after an 8 h exposure. With increasing concentrations of daunorubicin or epirubicin in a fixed 24 h time period, mdr1 expression increased, although a biphasic response was seen. Based on MRK 16 binding, an increase in P-gp levels was seen in the CEM/A7R line after a 24 h exposure to 1 microgram ml-1 daunorubicin or epirubicin. The rapid increase in mdr1 expression after a short period of exposure to doxorubicin, daunorubicin or epirubicin suggests that induction of mdr1 expression may have an important role in the development of drug-resistant tumours.

Cellular and karyotypic characterization of two doxorubicin resistant cell lines isolated from the same parental human leukemia cell line.

Separate mechanisms underlying the multidrug resistant (MDR) phenotype were identified in 2 independent approaches to select tumour cells resistant to low concentrations of doxorubicin (Dox) from the sensitive T cell leukemia cell line CCRF-CEM. The CEM/A7 cell line was selected at an initial concentration of 0.005 microgram/ml of Dox and maintained at 0.07 microgram/ml. In contrast, the CEM/A5 line was selected using an initial concentration of 0.01 microgram/ml and maintained in Dox at a concentration of 0.05 microgram/ml. P-glycoprotein expression was demonstrated in the CEM/A7 line but not the CEM/A5 line. Amplification of the mdrI gene was not observed in the CEM/A7 cell line. Both cell lines showed cross-resistance to a number of structurally unrelated cytotoxic drugs including anthracyclines and etoposide (VP-16), although only the CEM/A7 line was cross resistant to Vinca alkaloids. Immunoblots of total cell lysates of the CEM/A5 line have revealed almost undetectable levels of topoisomerase II alpha and beta in this line. Cytogenetic analyses of both lines revealed numerous karyotypic abnormalities which were present in the parental cell line as well as both resistant cell lines. The CEM/A7 line also demonstrated a duplication of part of the long arm of chromosome 7 which included the region containing the mdrI gene, a finding not seen in the parental or CEM/A5 line. CEM/A5, however, demonstrated an abnormality of chromosome 7, outside the region of the mdrI gene, and it also contained a deletion of the short arm of chromosome 2. Abnormalities in this latter region of genome have been associated with non-P-glycoprotein-mediated MDR.

P-glycoprotein expression in classical multi-drug resistant leukaemia cells does not correlate with enhanced chloride channel activity.

1. P-glycoprotein (Pgp) is an ATP-dependent drug efflux pump responsible for classical multi-drug resistance (MDR). 2. Pgp is part of a supergene family of membrane transport proteins that includes the cystic fibrosis gene product. 3. Transfection of cells with the MDR1 gene has been previously shown to generate volume-regulated chloride channel activity in association with Pgp expression. 4. We have used whole-cell patch clamping to examine the drug-sensitive T lymphoblastic cell line CEM-CCRF and its classical MDR derivative CEM/VLB100. The results suggest that expression of Pgp is not associated with increased chloride channel activity in this multi-drug resistant cell line. 5. We were unable to confirm previously reported results in MDR1 transfected cell lines that suggested that Pgp was associated with the presence of volume-regulated chloride channels.

Clinical application of a rapid, functional assay for multidrug resistance based on accumulation of the fluorescent dye, fluo-3.

A rapid and simple functional assay for P-glycoprotein (Pgp) using flow cytometry to measure the accumulation of the flurophore fluo-3 has been applied to samples from patients with B-cell chronic lymphocytic leukaemia (B-CLL). Peripheral blood lymphocytes from 37 patients with B-CLL were studied for Pgp. Pgp expression, using MRK-16, a monoclonal antibody recognising an external surface epitope of Pgp, was detected in 92% of patients with B-CLL. The functional assays for Pgp expression were positive in 78 and 59% of patients using the fluo-3 and doxorubicin (dox) assays, respectively. When compared with the MRK-16 assay, the fluo-3 assay had a sensitivity of 82% compared to a sensitivity of 56% for the dox assay (P = 0.004). The specificity of the fluo-3 and dox assays could not be evaluated because of the low number of MRK-16 negative CLL cells.

Differential effects of estrogen, tamoxifen and the pure antiestrogen ICI 182,780 in human drug-resistant leukemia cell lines.

ICI 182,780, a potent, new steroidal antiestrogen without apparent agonist activity, appears to be a potent modulator of the classic multidrug resistance (MDR) phenotype in the CEM/A7, CEM/VLB100 and K562/VIN100 MDR cell lines. This reagent had no effect on the respective parental CCRF-CEM and K562 cell lines. The use of 1.25 microM ICI 182,780 resulted in a 6- to 7-fold decrease in doxorubicin resistance in the CEM/A7 and CEM/VLB100 cell lines. A dose-response effect was observed at ICI 182,780 concentrations of up to 5 microM. As compared with tamoxifen (TAM), ICI 182,780 was 2 and 4 times more effective in the K562/VIN100 and CEM/A7 cell lines, respectively. ICI 182,780 at 0.625 microM increased [3H]-daunomycin uptake (P < 0.0001) as effectively as 5 microM TAM in the resistant CEM/A7 line. Drug-efflux studies showed that 5 microM ICI 182,780 significantly decreased drug efflux as compared with 5 microM TAM (P < 0.0001). Estradiol (EST) at 10 microM increased doxorubicin resistance by 1.2-1.3 times in the CEM/A7 and CEM/VLB100 cell lines and significantly decreased drug accumulation (P = 0.002) and retention (P < 0.001) in the CEM/A7 cell line. However, the addition of 10 microM EST to 1-2 microM ICI 182,780 did not inhibit the ability of ICI 182,780 to modulate doxorubicin resistance in the two resistant cell lines. Using reverse-phase high-performance liquid chromatography (HPLC) to measure lipophilicity, we found no apparent association between the ability of ICI 182,780, TAM or EST to modulate resistance and their relative hydrophobicity.

The Rancho mounting technique for the Ilizarov method. A preliminary report.

To improve implant tolerance and muscle function associated with circular external fixation, the authors substituted divergent titanium pins for the tensioned steel wires used to mount the Ilizarov apparatus on a limb. The first ten patients treated with half-pins were compared to the last ten patients managed with tensioned wires. While the conditions were not exactly comparable, the half-pin group showed improvement over the wire group in categories including time in fixation, implant-site sepsis, range of joint motion, pain medication requirements, and ambulatory capacity. Half-pin mountings require special techniques for a successful application.

Management of segmental defects by the Ilizarov intercalary bone transport method.

Seventeen patients with segmental skeletal defects were managed with the Ilizarov intercalary bone transport method, whereby an osseous defect is eliminated by elongating one fragment. On average, the regenerate new bone length measured 5.14 cm, corresponding to the creation of new osseous tissue equaling 13.7% of the bone's original length (range, 4.2%-35%). The average time in fixation was 9.6 months, including 4.8 months to transport the bone fragment throughout the limb. Numerous complications were encountered, most commonly wire-site sepsis and fixator instability. No serous nerve or vessel complications occurred. All but one patient eventually healed, although six patients required bone grafts, five at the target site and one at the level of the regenerate. Most of the difficulties encountered were due to a lack of technical knowledge with the method.

Update on combination oral contraceptives.

Oral contraceptives in current use appear to be safe and effective. Studies found that early contraceptive pills, which had a high estrogen content, were associated with a number of serious side effects and long-term sequelae. Studies of the newer low-dose preparations demonstrate a much lower incidence of adverse effects. The beneficial effects of oral contraceptives in disease prevention are now widely recognized. The diversity of available formulations allows the physician to tailor therapy to the individual patient, maintaining contraceptive efficacy while minimizing adverse effects and long-term risks.