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.

A kinetic analysis of D-xylose transport in Rhodotorula glutinis.

The kinetics of D-xylose transport were studied in Rhodotorula glutinis. Analysis of the saturation isotherm revealed the presence of at least two carriers for D-xylose in the Rhodotorula plasma membrane. These two carriers exhibited Km values differing by more than an order of magnitude. The low-Km carrier was repressed in rapidly growing cells and derepressed by starvation of the cells. Several hexoses were observed to inhibit D-xylose transport. In the studies reported here, the inhibitions produced by D-galactose and 2-deoxy-D-glucose were examined in some detail in order to define the interactions of these sugars with the D-xylose carriers. 2-Deoxy-D-glucose competitively inhibited both of the D-xylose carriers. In contrast, only the low-Km carrier was competitively inhibited by D-galactose.

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.

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.

Autologous bone marrow transplantation in acute lymphoblastic leukemia--preclinical immunologic studies.

Immunologic aspects of autologous bone marrow transplantation (ABMT), immunodiagnosis, patient monitoring, and the purging of bone marrow have been studied in individual patients. It was demonstrated that the most sensitive method for detecting lymphoid cells which show the phenotypes of ALLs of B or T lineage was double immunofluorescence staining for nuclear terminal transferase (TdT) and B or T lineage antigens. With the help of these sensitive tests in the presence of rabbit complement (C'), MAbs CD10 (RFAL3 of IgM class), CD19 (SB4 of IgM class), and their cocktail were capable of eliminating greater than 3 log blast cells of B lineage ALL in 84%, 75.5%, and 90% of cases, respectively. The same reagents lysed 26.8%, 0%, and 45% of blasts in the presence of human C'. CD7 (RFT2, IgG2) eliminated greater than 3 log T-ALL blast cells in 73% of cases. The proliferative fractions of leukemic blasts were also TdT+ and sensitive to lysis with MAb and C'. On the basis of these observations MABs were selected for purging in 36 patients undergoing ABMT in first remission (10 patients considered to be at a high risk of relapse), second and third remissions (23 and 2 patients), and without entering into remission (1 patient). The efficacy of eliminating the MAb-reactive cells from the bone marrow inoculum was also documented in five patients. By the use of sensitive immunologic assay (TdT/cytoplasmic CD3 double staining) in patients with T-ALL, no residual leukemia (less than 10(-4] could be detected at the time of transplantation. Following an observation period of 5-34 months, 24 of the 36 patients are alive and well with no procedure-related mortality.

Prevention of graft-versus-host disease by ex vivo T cell depletion: reduction in graft failure with augmented total body irradiation.

Graft-versus-host disease prevention was attempted in 35 consecutive patients with hematological malignancy who received bone marrow from an HLA match sibling donor who was depleted of T cells ex vivo. Five of the first 8 patients who received cyclophosphamide 60 mg/kg on 2 consecutive days followed by fractionated total body irradiation (TBI) (6 x 2 Gy) had graft failure. The subsequent 27 patients had received an extra fraction of TBI (7 x 2 Gy), and only one failed to have stable engraftment. There were no differences in nucleated cell dose, granulocyte-macrophage colony-forming units, or T cell numbers given to the two groups. Neutrophil but not platelet regeneration of those patients who successfully grafted was slower than in a group of historical controls receiving unmanipulated marrow. Significant graft-versus-host disease was prevented with no increase in relapse rate. We suggest that engraftment can be reliably achieved by augmenting the TBI conditioning in recipients of T cell-depleted matched allogeneic bone marrow.

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 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.

Exceptions to Hick's law: explorations with a response duration measure.

Five experiments used a new response-duration measure in explorations of the conditions necessary for confirmation of Hick's law. Hick's law states that reaction time increases logarithmically with number of choices. Exceptions to the law, venerable as it is, have been reported. They have always included the following conditions: a verbal response; a familiar stimulus with a single dominant name; and a large number of practice trials. These conditions have carried a heavy explanatory burden in accounting for the anamolous results. The present studies use none of these conditions and yet manage to replicate the anamolous result of a very shallow slope across set size, a slope less than one-tenth the usual value. This was accomplished by using a novel task in which the initial component of the response is the same for all stimuli (depression of a single response key) but the termination of the response is different (different durations for each stimulus). Using this task, a slope in the neighborhood of 15 ms per bit of stimulus uncertainty is found, as compared with the usual value of about 150 ms. A number of possible explanations are examined. Among the most important are the possibilities that response overlap is the critical factor (i.e., duration errors overlap); possible stimuli are simply ignored when more than one is involved; and the duration decision is made after the reaction-time interval rather than during it. All three possibilities, as well as some others, are found to be inconsistent with the various experimental outcomes. Instead, a new theory of choice reaction time is presented, which emphasizes the nature of the S-R code that is assumed to represent various reaction-time tasks. This theory leads to a new "law" that is put forward as a replacement for Hick's law. It is RT = a + b(1 - N-1).

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.

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.

Giant adrenal myelolipoma: case report and review of the literature.

Adrenal myelolipomas are rare tumors that consist of mature fat and bone-marrow elements. The majority that have been reported are small, asymptomatic lesions incidentally observed at the time of autopsy. In recent years, larger, symptomatic myelolipomas have been successfully resected. We studied the case of a giant adrenal myelolipoma in a 70-year-old woman. She was obese, hypertensive, and had abdominal pain, findings frequently associated with these lesions. Unusual features included formation of prominent bony spicules, a 52-year history of an abdominal mass, and massive size (5,500 g). To our knowledge, it is the largest myelolipoma yet reported.

Economic effects of clinical chicken anemia agent infection on profitable broiler production.

An outbreak of anemia dermatitis syndrome caused by chicken anemia agent (CAA) occurred in 15 broiler flocks. An average of 29% of chickens in these flocks were derived from a common breeder flock. The breeder flock had no antibody to CAA at 20 weeks of age but had seroconverted by 31 weeks. Diseased broiler flocks were derived from eggs laid by the breeder flock between 25 and 30 weeks of age. CAA infection in the breeder flock was subclinical, with no apparent effects on mortality or performance. A strategic program of therapeutic and/or prophylactic antibiotic therapy was begun in affected broiler flocks as soon as the disease was diagnosed. Nevertheless, when the cost of therapy was taken into account, affected broiler flocks had a net income 17.3% to 19.6% lower than normal flocks. Average bird weights were 3.3% to 3.5% lower in affected flocks than in unaffected flocks, and affected flocks had a significantly greater proportion of lighter birds. Average mortality in affected flocks was 2.0% to 2.3% higher than in normal flocks, with peak mortality occurring in the third week of life. There was no apparent effect on feed-conversion ratio.

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.

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.

Factors influencing haematological recovery in 53 patients with acute myeloid leukaemia in first remission after autologous bone marrow transplantation.

The kinetics of haematological recovery were retrospectively analysed in 53 patients with acute myeloid leukaemia in first remission after myeloablative chemoradiotherapy followed by autologous bone marrow transplantation. The median time to achieve a neutrophil count of 1 x 10(9)/l was 46 d (22-196 d) and median time to achieve unsupported platelet counts of 20 x 10(9)/l and 50 x 10(9)/l was 70 d (24-310 d) and 126 d (29-497 d) respectively. Multivariate analysis revealed two factors that were significantly associated with delayed neutrophil and platelet recovery: (1) use of high dose fractionated TBI and mononuclear cell cryopreservation, and (2) low platelet count at the time of bone marrow harvest. There was no correlation with: number of courses of chemotherapy, remission to ABMT interval, CMV status, indices of autograft quality or the development of elevated platelet associated immunoglobulin. Delayed haematological recovery did not predict for relapse or death. Delayed platelet recovery did, however, present significant problems with increased blood and platelet requirements and lengthening of hospital stay.

Endometriosis of the male urinary system: a case report.

Tissue histologically indistinguishable from endometrium was removed from the bladder of a 73-year-old man. The lesional tissue involved the right ureterovesical junction, producing hydronephrosis on the right side. The patient had an adenocarcinoma of the prostate and had been on estrogen therapy for 5 years before diagnosis of endometriosis. Two previous reports of endometriosis in male subjects, who were also on estrogen therapy, are reviewed briefly.

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.