Dysregulated expression of the major telomerase components in leukaemic stem cells.

Telomere loss is rapid during the progression of chronic myeloid leukaemia (CML) and correlates with prognosis. We therefore sought to measure expression of the major telomerase components (hTR and hTERT) in CD34+ cells from CML patients and normal controls, to determine if their altered expression may contribute to telomere attrition in vivo. High-purity (median 94.1%) BCR-ABL+ CD34+ cells from CML (n=16) and non-CML (n=14) patients were used. CML samples had a small increase in telomerase activity (TA) compared to normal samples (approximately 1.5-fold, P=0.004), which was inversely correlated with the percentage of G0 cells (P=0.02) suggesting TA may not be elevated on a cell-to-cell basis in CML. Consistent with this, hTERT mRNA expression was not significantly elevated; however, altered mRNA splicing appeared to play a significant role in determining overall full length, functional hTERT levels. Interestingly, Q-RT-PCR for hTR demonstrated a mean five-fold reduction in levels in the chronic phase (CP) CML samples (P=0.002), raising the possibility that telomere homeostasis is disrupted in CML. In summary, the molecular events regulating telomerase gene expression and telomere maintenance during the CP of CML may influence the disease progression observed in these patients.

CML leukapheresis products can be enriched for CD34+ cells and simultaneously depleted of CD15+ cells using a simple Ab cocktail.

BACKGROUND: CML progenitor-cell studies would be greatly facilitated if samples could be repeatedly accessed from a source of well-characterized cells. The present study was designed to develop a simple, inexpensive Ab cocktail that would provide subpopulations of cells enriched for CD34+ cells and simultaneously depleted of CD15+ mature myeloid cells. METHODS: Cells from leukapheresis products from CML patients at diagnosis were incubated with each of two Ab cocktails. The standard cocktail (debulking, DB), containing 11 Abs, is recommended for obtaining a highly enriched population of CD34+ cells. The efficacy of an alternative, simpler cocktail (CML custom, CC), containing only four Abs was tested. The recoveries of CD34+ cells, CD15+ cells, colony-forming unit granulocyte-macrophage, and LTCIC were monitored. The samples were then cryopreserved, thawed, and the recoveries remeasured. RESULTS: The purity of CD34+ cells was significantly superior using the DB cocktail than with the CC cocktail. Conversely, using the CC cocktail, the yield of CD34+ cells was significantly higher compared to the DB cocktail. These results were maintained even when the amount of Ab was reduced 10-fold. Both Ab cocktails consistently removed > 99% of the CD15+ cells. Consistent with the CD34+ cell-enrichment data, higher colony-forming cell (CFC) frequencies were obtained with the DB cocktail, although superior yields of CFC were obtained with the CC cocktail. After cryopreservation and thawing the yield of CD34+ cells remained high, and a further reduction in the number of CD15+ cells was obtained. DISCUSSION: A method is described that allows the rapid and efficient debulking of large CML samples. This strategy will provide a source of well-characterized CML stem/progenitor cells that can be repeatedly accessed.

Adenovirus type 7 induces interleukin-8 production via activation of extracellular regulated kinase 1/2.

Infection with adenovirus serotype 7 (Ad7) frequently causes lower respiratory pneumonia and is associated with severe lung inflammation and neutrophil infiltration. Earlier studies indicated release of proinflammatory cytokines, specifically interleukin-8 (IL-8), by pulmonary epithelial cells following infection by Ad7. However, the mechanism of IL-8 induction by Ad7 is unclear. We have explored the role of the Ras/Raf/MEK/Erk pathway in the Ad7-associated induction of IL-8 using a model system of A549 epithelial cells. We found that Ad7 infection induced a rapid activation of epithelial cell-derived Erk. The MEK-specific inhibitors PD98059 and U0126 blocked Erk activation and release of IL-8 following infection with Ad7. Treatment with PD98059 is cytostatic and not cytotoxic, as treated cells regain the ability to phosphorylate Erk and secrete IL-8 after removal of the drug. The expression of a mutated form of Ras in A549 epithelial cells blocked the induction of IL-8 promoter activity, and MEK inhibitor blocked induction of IL-8 mRNA. These results suggest that the Ras/Raf/MEK/Erk pathway is necessary for the Ad7 induction of IL-8 and that induction occurs at the level of transcription. Further, the kinetics of Erk activation and IL-8 induction suggest that an early viral event, such as receptor binding, may be responsible for the observed inflammatory response.

Up-regulation of a gene homologous to the human tumor necrosis factor receptor associated factor 6 gene in the obstructed rabbit bladder determined by differential display polymerase chain reaction.

PURPOSE: We identified differentially expressed genes in the rabbit bladder after partial outlet obstruction. MATERIALS AND METHODS: Differential display polymerase chain reaction (PCR) was performed on smooth muscle tissue from normal, 2 and 6-week obstructed rabbit bladders. Semiquantitative reverse transcriptase PCR, Western and RNA blot analysis were done to confirm messenger RNA and protein up-regulation. RESULTS: A signal transducing protein human tumor necrosis factor receptor associated factor 6 (TRAF6)-like protein was identified on differential display PCR. TRAF6-like protein was up-regulated in rabbit bladders after 2 weeks of partial outlet obstruction. Reverse transcriptase PCR demonstrated TRAF6-like protein in bladder muscle tissue and semiquantitative analysis confirmed up-regulation in 2-week obstructed tissue. These findings were confirmed by RNA and Western blot analysis. CONCLUSIONS: TRAF6-like protein is up-regulated during the early phase of bladder outlet obstruction in rabbits. To our knowledge involvement of this gene in bladder outlet obstruction has not been described previously. TRAF6 may have a role in the regulation of molecular changes during the early bladder response to outlet obstruction, such as the up-regulation of growth factors and proto-oncogenes. Further understanding of this signaling pathway and its role in bladder outlet obstruction may open new avenues for treating detrusor dysfunction.

Primitive quiescent leukemic cells from patients with chronic myeloid leukemia spontaneously initiate factor-independent growth in vitro in association with up-regulation of expression of interleukin-3.

It was previously shown that patients with chronic myeloid leukemia (CML) have a rare but consistently detectable population of quiescent (G0) leukemic (Philadelphia chromosome-positive and BCR-ABL-positive [BCR-ABL+]) CD34+ cells. In the study described here, most such cells expressed a primitive phenotype (CD38-, CD45RA-, CD71-, and HLA-DR(lo)) and cultures of these cells containing growth factors produced ultimately larger, but initially more slowly growing clones than do cultures of initially cycling CD34+ leukemic cells. Initially quiescent leukemic cells expressing BCR-ABL proliferated in single-cell cultures in the absence of added growth factors, thereby demonstrating their ability to spontaneously exit G0 and enter a continuously cycling state. Interestingly, on isolation, few of these quiescent BCR-ABL+ cells contained either interleukin-3 (IL-3) or granulocyte colony-stimulating factor (G-CSF) transcripts, whereas both were present in most cycling BCR-ABL+ CD34+ cells. However, after 4 days of culture in the absence of added growth factors and in association with their entry into the cell cycle (as indicated by up-regulation of Ki-67 and cdc25 transcripts), IL-3 transcripts became detectable. These findings show that entry of leukemic (BCR-ABL-expressing) progenitors into a quiescent (G0) state in vivo is highest among the most primitive leukemic cell populations, associated with a down-regulation of IL-3 and G-CSF gene expression, and spontaneously reversible in association with up-regulation of IL-3 expression. These results highlight the potential physiologic relevance of quiescent CML progenitors, even in treated patients, in whom these cells would be predicted to have a proliferative advantage over their quiescent normal counterparts when cytokine concentrations are low.

Appropriateness of bed usage for inpatients admitted as emergencies to internal medicine services.

OBJECTIVE: To establish the appropriateness of bed usage for acute care within the medical directorates of two district general hospitals using a validated assessment tool, the Emergency Admission Review (EAR). This tool assesses the appropriateness of day of care against strict criteria and allows classification of care as either acute or non-acute. DESIGN: Prospectively, 200 medical emergency admissions, 100 in each of the hospitals, were selected. Following identification patients were assessed every two days during the first fortnight of admission or until discharge. Those patients staying longer than two weeks were then assessed weekly until conclusion of the audit period or discharge whichever was reached first. SETTING: The medical directorates of two District General Hospitals within one acute NHS trust. SUBJECTS: All patients admitted as medical emergencies, who were 14 years or older and had a length of stay of 24 hours or more. RESULTS: A total of 787 acute in-patient bed days were analysed in Hospital A of which 363 (46%) were deemed inappropriate for acute care. In Hospital B 810 bed days were analysed and 44% (363) were deemed inappropriate. In Hospital A the most common reason for bed-days not meeting the acute care criteria was short-term waiting, accounting for 60% (217 days) of the total bed days deemed non-acute. In Hospital B the most common reason for patients receiving non-acute care was that they were having active rehabilitation. This accounted for 29% (105 days) of the total number of non-acute care days. In Hospital B three patients accounted for 28% of the total occupied bed days. CONCLUSIONS: The use of the EAR is a systematic and objective approach to the assessment of appropriateness of acute care. It applies strict criteria to determine the reason for a patient's continued hospital stay. From the results it is clear that a significant proportion of medical emergency admissions in both Hospital A and B remain in hospital for care that is deemed non-acute and therefore in theory could be performed in another setting. This information has significant potential in identifying the opportunities for streamlining services within hospitals to reduce short-term delays and also to inform the development of intermediate care services both within and outwith the acute hospital setting.

The number of CD34+ cells mobilized into the peripheral blood can predict the quality of subsequent collections.

PBPC were mobilized using a variety of chemotherapy regimens plus G-CSF in a group of 126 consecutive patients. Data are presented that show a close correlation between the number of CD34+ cells mobilized into the peripheral blood (PB) and the number of CD34+ cells subsequently collected by leukapheresis (R = 0.904). On the basis of this correlation, a regression formula was calculated that could give an estimate of the total number of CD34+ cells likely to be collected by leukapheresis from a given number of CD34+ cells per microliter PB. An easy-to-read table has been compiled to show how this type of analysis can be applied to predict the likely dose of CD34+ cells that will be obtained by leukapheresis over a wide range of patient weights.

Selection of CD34+ cells from cryopreserved PBPC can be significantly improved by the addition of recombinant human DNase (Pulmozyme).

BACKGROUND: It has been reported previously that PBPC can be recovered from cryopreservation and can be efficiently CD34-selected, to provide a product of high purity (> 80% CD34) with good yield (> 50% recovery). METHODS: In this study, we have investigated the effects of thawing and CD34-selecting cryopreserved PBPC in the presence of recombinant human deoxyribonuclease (rhDNase; Pulmozyme) and magnesium chloride (MgCl2 injection). RESULTS: The addition of Pulmozyme and MgCl2 significantly improves the yield of CD34+ cells, compared with the standard procedure (65.2% and 39.7%, respectively). Following CD34 selection, significantly greater recovery of CFC in the selected fraction can be obtained from Pulmozyme-treated cells, compared with standard cells. The use of recombinant human Pulmozyme and i.v. grade MgCl2 should facilitate the application of this procedure to the clinical setting. CD34+ cells selected from cryopreserved PBPC, can in turn be cryopreserved for a second time. When thawed, these cells still retained good viability (> 80%). DISCUSSION: Cells originally processed in the presence of Pulmozyme gave significantly superior yields of CD34+ cells and CFC compared with standard cells. The functional ability of these CD34+ cells was demonstrated further in an ex vivo expansion culture system with extensive proliferation of cells and CFC. In addition, the presence of significant numbers of primitive hemopoietic cells could be readily demonstrated in a cobblestone-area forming assay.

An update on the epidemiology of contact dermatitis in commercial broilers.

A computerized management and disease information retrieval system for commercial broiler production was established in 1985. The database presently contains information on over 950 flocks (15 million birds) per annum. The system has enabled the effectiveness of strategies which have been implemented to control contact dermatitis to be monitored. Between 1986 and 1994, the prevalence of breast burn lesions has been practically eradicated (0.2 to 0.00%) and the prevalence of hock burn lesions has been dramatically reduced from 21 to 7%. This reduction has been mainly due to the use of better litter management techniques and improvements in house design, particularly changing the type of drinker system. Production factors during 1993 and 1994 which were found to be consistently related to increased prevalence of hock burn lesions were male only flocks, inferior food conversion ratio, failure to meet target income and increased mortality rates.

CD34+ cells can be selected efficiently from cryopreserved peripheral blood progenitor cells and can retain their proliferative potential.

Hematopoietic stem and progenitor cells express the CD34 antigen. Techniques have been developed that enable purified populations of CD34+ cells to be selected from hematopoietic tissues. These selected CD34+ cells have several potential applications, including CD34 selection to obtain a tumor purging effect in autologous transplantation studies and using CD34+ cells as the starting cells for ex vivo expansion studies and as a vehicle for gene transduction protocols. We have investigated the feasibility of using cryopreserved peripheral blood progenitor cells (PBPC) for CD34 selection. Cells could be recovered from cryopreservation with good yields and high viability. After CD34 selection, the final product was, on average, 84% pure, with a recovery of 54%. These cells retained extensive proliferative potential, as demonstrated by ex vivo expansion culture. We believe that cryopreserved PBPC could be thawed, and CD34+ cells could be selected and used for transplantation following high-dose chemotherapy.

CD34 positive PBPC expanded ex vivo may not provide durable engraftment following myeloablative chemoradiotherapy regimens.

We have previously demonstrated that CD34+ cells, selected from peripheral blood progenitor cells (PBPC), can be expanded in ex vivo culture and can be infused in tandem with unmanipulated PBPC with little or no toxicity. In this study, four patients (two non-Hodgkin's lymphoma (NHL), two multiple myeloma (MM)) received myeloablative conditioning prior to stem cell rescue using ex vivo expanded cells alone. The two patients with NHL received cyclophosphamide and total body irradiation (CY/TBI) and the two patients with MM, busulphan and melphalan (Bu/M). One case received an inadequate CFU-GM dose, despite expansion, and in one case the expanded cells were contaminated. No definitive conclusions may therefore be drawn concerning engraftment in these two cases. However, the other two cases received high doses of committed progenitors. Following infusion of the expanded material, all four patients failed to show sustained neutrophil engraftment and none showed evidence of platelet engraftment. Back-up, unmanipulated PBPC were therefore infused on days 14, 34, 32 and 28 and subsequently all four cases achieved satisfactory engraftment of both neutrophils and platelets. In conclusion, we feel that, CD34+ cells, expanded ex vivo using the conditions described in this report, may not provide durable engraftment following fully myeloablative conditioning.

CD34 Selection and EX Vivo Expansion of Haemopoietic Progenitor Cells: A Review of Laboratory Methodology.

The CD34 antigen is expressed on haemopoietic stem and progenitor cells. A number of strategies have been developed which allow the selection and purification of CD34(+) cells from bone marrow, peripheral blood, and umbilical cord blood. Transplantation studies have amply demonstrated that rapid and durable engraftment can be achieved following reinfusion of selected CD34(+) cells. More recently, techniques have become available which can produce extensive proliferation of haemopoietic progenitor cells in ex vivo culture systems. The most popular method involves a simple liquid suspension culture system supplemented with a range of cytokines. The degree of expansion and, indeed, the types of cells produced can be significantly influenced by culture conditions like the choice of cytokines, duration of culture, starting cell concentration, and type of culture vessel. Despite many laboratory investigations, there have been few clinical trials using ex vivo expanded cells. Although it has been shown that infusion of ex vivo cultured cells is well tolerated with no associated toxicity, there is no evidence to date that these culture systems sustain sufficient numbers of haemopoietic long-term repopulating cells to secure durable engraftment following myeloablative therapy. Clearly, the major goal is to define culture conditions which will produce true stem cell expansion.

Peripheral blood progenitor cell mobilisation in patients with multiple myeloma following oral idarubicin and dexamethasone (Z-Dex) induction therapy.

Difficulties associated with current intensive induction regimens for multiple myeloma and uncertainty as to how to achieve optimal peripheral blood progenitor cell mobilisation (PBPC) prompted this study of an oral induction regimen, Z-Dex (oral idarubicin and dexamethasone) followed by PBPC mobilisation using four different regimens. Thirty-patients received Z-Dex (median age 56 years, range 46-66 years) including 24 patients with previously untreated disease. The overall response rate was 75% with a CR rate of 16.7% and PR rate of 75.7% in patients with previously untreated disease. We compared four mobilisation regimens: low-dose (LD) cyclophosphamide, high-dose (HD) cyclophosphamide, cis-platin/VP16 and cis-platin, Ara-C and dexamethasone (DHAP). Failure to mobilise optimal numbers of PBPCs (>1.0 x 10(6) CD34+ cells/kg and >20 x 10(4) CFU-GM/kg) was seen in two patients who received LD cyclophosphamide, in two patients who received HD cyclophosphamide and three patients who received cis-platin/VP16. No patient failed to mobilise adequate numbers of PBPCs following DHAP. In previously untreated patients, DHAP mobilised significantly more PBPC than LD cyclophosphamide (P=0.02), HD cyclophosphamide (P=0.0015) and cis-platin/VP16 (P=0.021). This study demonstrates the efficacy of Z-Dex in inducing tumour responses in patients with multiple myeloma without limiting PBPC mobilisation in subsequent dose-intensive schedules. Furthermore, we also demonstrate that DHAP is superior to cyclophosphamide (low- and high-dose) and cis-platin/VP16 in mobilising PBPCs and demonstrated a degree of tumour control.

Ex vivo expansion of haemopoietic progenitor cells.

Over the last few years, techniques have become available that allow the extensive proliferation of haemopoietic progenitor cells in ex vivo culture systems. The most commonly used method involves a simple liquid suspension culture system supplemented with a range of cytokines. Alternatively, more complex systems have been devised in which the formation of a stromal layer is required. Large increases in total cell numbers and committed progenitor cells can be readily obtained and, with some techniques, significant expansion of primitive haemopoietic cells has been demonstrated. Although these strategies have several potential applications, few clinical studies have been performed. It has been shown that infusion of ex vivo cultured cells is well tolerated with no associated toxicity. However, it is still unclear whether these culture systems sustain sufficient numbers of long-term repopulating cells to secure durable engraftment following myeloablative therapy. In gene therapy studies, ex vivo expansion of stem cells should improve the efficiency of gene transduction to enable the production of genetically modified cells that are capable of expressing the gene of interest for extended periods of time.

CD34-positive cells isolated from cryopreserved peripheral-blood progenitor cells can be expanded ex vivo and used for transplantation with little or no toxicity.

PURPOSE: The objectives of this phase I study were to assess the feasibility of using cryopreserved peripheral-blood progenitor cells (PBPC) for large-scale CD34 selection and subsequent expansion, and the safety of their use for reinfusion following chemoradiotherapy. PATIENTS AND METHODS: For 10 patients with nonmyeloid malignancy, an aliquot from a PBPC harvest was recovered from liquid nitrogen, and CD34 selected using the Isolex system (Baxter Healthcare, Newbury, United Kingdom) and expanded for 8 days ex vivo in a medium free of animal proteins but supplemented with autologous serum, stemcell factor (SCF), interleukin-1 beta (IL-1 beta), IL-3, IL-6, and erythropoietin. RESULTS: The mean increase for cell number was 21-fold, for colony-forming units-granulocyte/macrophage (CFU-GM) 139-fold, and for burst-forming units-erythroid (BFU-E) 114-fold. The expanded cells were reinfused in tandem with unmanipulated material (> or = 25 x 10(4) CFU-GM/kg). The patients did not experience any adverse effects immediately on cell infusion or within 48 hours. The 10 index patients were compared with 10 historical controls for parameters of myelosuppressive morbidity. In this small study, there were no differences in either neutrophil or platelet recovery between the patients who received expanded cells and historical controls. CONCLUSION: These data demonstrate that CD34 cells can successfully be selected from cryopreserved material, expanded ex vivo on a large scale, and safely reinfused following myeloablative conditioning regimens.

The CD34 antigen: potential clinical advantages of CD34 selection.

The availability of monoclonal antibodies directed towards the haemopoietic cell surface antigen CD34 has facilitated accurate measurement, by flow cytometry, of CD34 positive cell frequencies in bone marrow and peripheral blood. In addition, a range of CD34 selection techniques, to purify peripheral blood progenitor cells or bone marrow prior to transplantation, have been developed. CD34 positive stem and progenitor cells may be selected with final purities in excess of 90%. Such pure populations of CD34 positive stem cells may be useful in several clinical areas, including tumour cell purging and T-cell depletion, and as a basis for gene therapy and stem cell expansion.

Injuries suffered by dogs from riding in the back of open pickup trucks: a retrospective review of seventy cases.

Case records of 70 dogs injured while riding in the back of open pickup trucks during the period January 1, 1982, to May 1, 1993, were reviewed. Most dogs were young (mean age 2.4 y) and of medium to large size (average weight 22.6 kg). Sixty-five dogs (93%) were injured during the months of April through October. Forty-nine dogs (70%) had single injuries and 21 dogs (30%) sustained multiple injuries. Fractures were the most frequent injury incurred, with fractures of the femur the most common. Surgical repair was recommended in all but 2 cases.

Filgrastim fails to improve haemopoietic reconstitution following myeloablative chemotherapy and peripheral blood stem cell rescue.

The morbidity of high-dose chemotherapy has been considerably reduced by the use of autologous peripheral blood progenitor cell reinfusion. Most studies have used myeloid colony-stimulating factors after stem cell reinfusion, making it difficult to determine the relative contribution of each of these variables to the early recovery of blood cells. The financial implications of colony-stimulating factor use are an area of concern as dose intensification in chemosensitive malignancies is increasingly employed. We have studied 19 consecutive patients receiving high-dose chemotherapy with and without filgrastim (Amgen, granulocyte colony-stimulating factor, G-CSF) after stem cell infusion to examine its effect on the kinetics of blood cell recovery, the complications of myelosuppression and the associated costs. Analysis of the two treatment groups reveals that administration of filgrastim 10 micrograms kg-1 day-1 following stem cell reinfusion does not further accelerate haemopoietic recovery, fails to reduce the incidence of neutropenic fever or antibiotic usage and significantly increases the cost of the procedure. The results of this study do not support the routine use of filgrastim after high-dose chemotherapy and peripheral blood stem cell reinfusion.

CD34+ positive haemopoietic cells: biology and clinical applications.

CD34+ is a heavily glycosylated surface antigen which is preferentially expressed on haemopoietic stem/progenitor cells. No definitive function has been attributed to CD34+, but it appears to play a role in cell to cell adhesion and may be involved in signal transduction to regulate the expression of other haemopoiesis-associated genes. A number of monoclonal antibodies to CD34+ have been raised and these have allowed the identification and characterization of a whole range of haemopoietic progenitor cells. CD34+ is expressed most strongly on the most primitive cells and is progressively lost as cells differentiate. The restricted expression of CD34+ to haemopoietic stem/progenitor cells has been exploited for transplantation studies. Several techniques have been developed to select cells expressing CD34+ from haemopoietic tissues. Successful sustained engraftment can be achieved using such positively selected cells. Alternatively, CD34+ cells may be expanded in vitro by incubation with synergistic cytokine combinations before being re-infused. An exciting new development has been the use of purified populations of CD34+ cells as the targets for gene marking and gene therapy protocols.