An optimization study on unprogrammed cryopreservation of human CD34+ bone marrow cells and their subsequent storage in an -80 degrees C mechanical freezer

Forty-one patients for whose direct care at least one machine was used and 33 registered nurses from the same five non-critical care units as the patients and from one related unit, participated in a semistructured tape-recorded interview to identify the core categories of the human-machine interface in clinical nursing practice and the relationships between them. Constant comparative analysis was used to organize and process the data. Patients perceived the machines as neutral because of their view of health care and because nurses were the interface between them and the machines. Nurses perceived the machines as either positive or negative, depending on their effect on the nurses' professional competence and the extent to which they worked directly with them.     

Comparison of the different strategies for cryopreserving and storage of the bone marrow CD34+ cells. Possibility of unprogrammed rate freezing and storage at -80 degrees C mechanical freezer

We have compared the efficiency of the different strategies allowing for a long term storage of a human CD34+ bone marrow cells. Accordingly, the aliquots of CD34+ cells isolated from bone marrow were frozen using: controlled rate freezing equipment, or freezer unprogrammed in a -80 degrees C mechanical freezer. After freezing, CD34+ cells were subsequently stored for one month in a liquid nitrogen tank at -196 degrees C or in mechanical freezer at -80 degrees C. We have found that both the viability and the recovery of clonogeneic progenitors of CD34+ cells samples stored at different temperature were similar. Therefore, regarding the costs and simplicity, we recommend the unprogrammed freezing and storage of human CD34+ cells at -80 degrees C in a mechanical freezer as a convenient, inexpensive, and reliable method for storing marrow for transplantation. This data also indirectly indicate that the aliquots of the CD34+ cells can be shipped frozen on dry ice (-80 degrees C), and that these cells will maintain viability under this conditions. Furthermore, in this study we have confirmed validity of our earlier observation that human CFU-Meg progenitors are more sensitive to cryopreservation.     

A cryopreservation method of human peripheral blood mononuclear cells for efficient production of dendritic cells

We studied whether inducers of cell differentiation alone could have cytotoxic effect on the promonocytic U937 and Mono Mac 6 cells in vitro. The cells were incubated with standard differentiating doses of interferon (IFN)-gamma, dibutyryl cAMP (Bt2cAMP) or the phorbol ester phorbol-12-myristate-13-acetate (PMA), with or without lipopolysaccharide (LPS), and both protein synthesis and viability were examined. In both U937 and Mono Mac 6 cells the incorporation of [3H]leucine was significantly reduced after PMA plus LPS stimulation, but not after IFN-gamma stimulation, when compared with controls. For U937 cells there was also reduced incorporation after Bt2cAMP stimulation. Trypan blue exclusion experiments and the number of cells remaining in the cultures indicated that Bt2cAMP-, PMA- and/or LPS-stimulated, but not IFN-gamma-stimulated, cells were less viable than unstimulated U937 or Mono Mac 6 cells. The results suggest that Bt2cAMP, PMA and LPS, but not IFN-gamma, are cytotoxic towards promonocytic cancer cell lines in vitro.     

Measurement of intrahepatic shunted blood flow by ethanol-1-14C method as compared with D-galactose-1-14C method

The interaction of aflatoxin B1 with a number of biologically important substances has been investigated by spectrophotometric and fluorimetric techniques. The ultra-violet absorption of tryptophan, adenosine, RNA and DNA were altered by the addition of aflatoxin B1. Some amino-acids, purine and pyrimidine derivatives, bovine-serum albumin and DNA quenched the fluorescence of aflatoxin B1. Fluorescence polarization data suggested that the interaction of aflatoxin B1 with bovine-serum albumin was stronger than that with DNA.     

Allogenic transplantation of hematopoietic stem cells in the treatment of children with high-risk acute leukemia

BACKGROUND: Most children with acute lymphoblastic leukemia (ALL) and increasing number of children with acute myelogenous leukemia (AML) are currently cured with conventional chemotherapy. Despite of this success there is a subset of patients with high-risk features at diagnosis who are predisposed to a very high risk of relapse. Relapse of AML and early bone marrow relapse of ALL can not be cured by conventional chemotherapy. Allogeneic hematopoietic stem cell transplantation (HSCT) is therapeutic option in these children with very high-risk acute leukemia. METHODS AND RESULTS: Between XI/1989-XII/1996 33 children with acute leukemia (ALL: 22, AML: 11) underwent an allogeneic HSCT from HLA identical related donors (HLA-identical sibling: 30, twin: 1, other HLA-identical relative: 2) at the 2nd Dept. of Pediatrics, University Hospital Motol. Median age of our group was 9 years (1.5-19 y.), boys (n = 23) clearly dominated over the girls (n = 10). The resource of stem cells was bone marrow in 31 children, bone marrow and peripheral blood progenitor cells (PBPC) and PBPC in one child respectively. Myeloablative conditioning regimen varied, consisting of total body irradiation and chemotherapy in 21 children and chemotherapy in 12 children. HSCT was performed in first complete remission of acute leukemia in 9 children (AML: 7, ALL: 2), in second remission in 14 children (AML: 2, ALL: 12), in third remission in 4 children (ALL: 4). Six children underwent HSCT in first partial remission (n = 1) and in second (n = 4) or third (n = 1) chemoresistant relapse. Seven (21%) children died due to post-transplant complications. Nine (28%) children suffered from clinically significant acute graft-versus-host reaction (GVH) and 15% (4/27) children who survived 100 days post-transplant suffered from chronic GVH disease. Relapse of leukemia was diagnosed in 39% (12/31) children. Fourteen (42%) children are alive and well in continuous remission with median follow-up 42 months. CONCLUSIONS: Allogeneic HSCT can cure children with very high-risk acute leukemia in the situations where conventional chemotherapy fails. Relapse of leukemia and GVH reaction are most important causes of post-transplant morbidity and mortality.     

Efficiency of high-dose FEC chemotherapy for the mobilization of hematopoietic stem cells into peripheral blood

The best regimen for mobilizing hematopoietic stem cells (HSC) into peripheral blood is not yet defined. The efficiency of FEC chemotherapy in the treatment of breast cancer is well established and the effects of FEC on HSC mobilization have been characterized. We tested the feasibility and the toxicity of a high-dose FEC regimen which may improve the mobilizing capacity of conventional FEC. Twenty patients with poor prognosis breast cancer received high-dose FEC and filgrastim 5 micrograms/kg. Three leukaphereses were performed on each patient for 3 consecutive days. Total numbers of CFU-GM and CD34+ cells were assessed, and a retrospective analysis was made. The numbers of CFU-GM/kg and CD34+ cells/kg collected (mean +/- standard error) were respectively 12.2 x 10(5) (+/- 2.4) and 14.6 x 10(6) (+/- 2.5). Extra-hematologic toxicity was negligible. Hematologic recovery after CTCb high-dose chemotherapy and HSC infusion was rapid. High-dose FEC is efficient for collecting HSC in peripheral blood. Extra-hematologic toxicity is weak and hematologic recovery after autograft is normal. Increased dosage of epirubicin and cyclophosphamide could allow a single leukapheresis collection of sufficient HSC from peripheral blood.     

Effect of treatment with 13-cis-retinoic acid and ara-C on the hematopoietic stem cells of patients with myelodysplastic syndromes

PURPOSE: To assess the "in vivo" effect of 13-cis-retinoic acid and low dose Ara-C in MDS as well as to establish "in vitro" advantage of retinoid dose-related growth pattern on bone marrow cultures as defined by culture timing and CFU-GM proliferative response. PATIENTS AND METHODS: We evaluated 28 patients diagnosed of MDS according to FAB classification, of whom 4 cases had RA, 8 cases SRA, 14 cases RAEB and 2 cases RAEB-T. Patients who had RA and SRA were treated with oral 13-cis-retinoic acid at doses of 20-40 mg daily for 4 months and those cases with RAEB and RAEB-T had subcutaneous Ara-C at doses of 3 mg/m2 twice a day for 21 days. The "in vivo" and "in vitro" effect of retinoic acid on the haemopoietic differentiation was evaluated by the growth CFU-GM in semisolid cell culture methods. RESULTS: Increasing in vitro concentrations of 13-cis retinoic acid did not enhance the growth of myelodysplastic progenitors. Nevertheless, our study did not find any beneficial therapeutic effect of retinoic compounds in MDS patients. In this study, low-dose Ara-C (3 mg/m2) showed similar effects when compared with higher doses reported by others. Furthermore, in terms of CFU-GM proliferation the concentration of colonies before and after treatment were fairly similar in all but two patients. CONCLUSIONS: The results drawn from our study demonstrated that there is no beneficial advantage of 13-cis-retinoic acid as a differentiation inducing agent on myelodysplastic patients. In contrast, lower doses of Ara-C showed similar effects on haemopoiesis of MDS patients than standard doses of 10-20 mg/m2 but with less side effects.     

Induction of c-kit molecules on human CD34+/c-kit < low cells: evidence for CD34+/c-kit < low cells as primitive hematopoietic stem cells

c-kit, a receptor for stem cell factor, has been widely accepted as a distinctive marker for hematopoietic stem cells. However, the level of c-kit expression on pluripotent hematopoietic stem cells is still controversial in mice and humans. We purified CD34+/c-kit < low cells (phenotypically c-kit-negative but only detectable at the message level) from human cord blood and examined their maturational steps in relation to the expression of c-kit molecules. When the CD34+/c-kit < low cells were cultured with cytokines (flt 3 ligand, interleukin 6 and interleukin 7) plus immobilized anti-CD34 monoclonal antibody (to crosslink CD34 molecules), c-kit molecules were clearly induced within 24 h. The c-kit expression gradually increased until day 8. When CD34+/c-kit(low) or CD34+/c-kit+ cells that had been induced from CD34+/c-kit < low cells were resorted and recultured using a methylcellulose culture system, they showed the same colony-forming ability as the freshly isolated CD34+/c-kit(low) or CD34+/c-kit+ cells, respectively. Furthermore, CD34+/c-kit < low cells have a similar hematopoietic potential to CD34+/c-kit(low) cells in assays for long-term culture initiating cell and colony-forming unit culture generated from long-term cultures. These findings suggest that CD34+/c-kit < low cells mature into CD34+/c-kit(low) and CD34+/c-kit+ cells, and acquire the reactivity to various humoral hematopoietic stimuli. Moreover, CD34+/c-kit < low cells showed a low level of rhodamine 123 retention, suggesting that CD34+/c-kit < low cells have multidrug resistance. Therefore, the CD34+/c-kit < low cells without colony-forming unit-granulocyte-erythroid-macrophage-megakaryocyte activity are also a pluripotent hematopoietic stem cell population, and the expression of c-kit on c-kit < low cells is the first maturational step of hematopoiesis.     

A simplified method for the preparation of succinimidyl carbonate polyethylene glycol for coupling to proteins

An improved method for the activation of polyethylene glycol with commercially available succinimidyl carbonate is described. The activated polyethylene glycol was coupled to proteins in high yield.     

Presence of methylthioadenosine phosphorylase (MTAP) in hematopoietic stem/progenitor cells: its therapeutic implication for MTAP (-) malignancies

Methylthioadenosine phosphorylase (MTAP) is important for the salvage of adenine and methionine. Recently, we found frequent deletion of MTAP in T-cell acute lymphoblastic leukemia (T-ALL) patients both at diagnosis and at relapse (A. Batova et al., Blood, 88: 3083-3090, 1996). In addition, MTAP deficiency has been reported in other cancers. Thus, MTAP deficiency in cancer may offer opportunities for developing selective therapy, which would spare normal cells. It is therefore important to document the presence of MTAP activity in hematopoietic stem/progenitor cells. Our approach was to investigate whether hematopoietic stem/progenitor cells can be rescued from the cytotoxicity of an AMP synthesis inhibitor, L-alanosine, by 5'-deoxyadenosine, a process that requires MTAP. Erythroid burst-forming unit, granulocyte/monocyte colony-forming unit, or granulocyte/erythrocyte/macrophage/megakaryocyte colony-forming unit progenitors and the primitive high proliferative potential colony-forming cells in the purified CD34(+) cells were cultured in horse serum-containing medium, and their colony growth was found to be suppressed by incubation with 5 microM or greater concentrations of L-alanosine. However, in the presence of 5-10 microM of 5'-deoxyadenosine, colony formation of hematopoietic stem/primitive progenitors was restored. On the other hand, 5'-deoxy-5'-methylthioadenosine, the endogenous substrate of MTAP, was toxic to hematopoietic stem/progenitors (ID50 < 1 microM), presumably due to inhibition of methylation reactions or polyamine synthesis. We also compared the effects of L-alanosine and 5'-deoxyadenosine on MTAP (+) and MTAP (-) T-ALL cell lines. Treatment of MTAP (+) Molt 4 and MTAP (-) CEM cell lines with L-alanosine in the presence of 5'-deoxyadenosine resulted in killing of MTAP (-), but not MTAP (+) cells. Therefore, our findings demonstrate the presence of MTAP in human hematopoietic stem/progenitor cells and support the possibility of targeting MTAP in the design of an enzyme-selective therapy for T-ALL and other MTAP-deficient malignancies.     

A simplified method for growth of human microvascular endothelial cells results in decreased senescence and continued responsiveness to cytokines and growth factors

With recent health care policy changes and the implementation of direct access in many states, physical therapists must be able to identify pathology that is beyond their scope of practice. The five case reports presented in this series required the differential diagnosis of hip vs. lumbar spine pathology. All of the cases required a referral from the physical therapist to either the patient's physician or a specialist because of abnormal screening test results. Each referral resulted in a new diagnosis of pathology that was beyond the scope of physical therapy. Cyriax's concepts of capsular and noncapsular patterns of joint restriction and the "Sign of the Buttock" proved useful in differentiating between hip and lumbar spine pathology in each patient. Our clinical experience indicates that utilizing the presence/absence of a capsular pattern and a "Sign of the Buttock" to screen out hip pathology in patients may be effective; however, further research is needed to support these claims.     

A kinetic nephelometric method for the assay of serum C3 and C4

A two-point, fixed interval kinetic, nephelometric method for the immunoassay of serum C3 and serum C4 using a laser-modified, centrifugal fast analyzer is described. The procedure shows a precision of 2.45 percent (coefficient of variation, C.V.) for C3 at a mean concentration of 151 mg per dl and 3.65 percent (C.V.) for C4 at a mean concentration of 26.6 mg per dl. The kinetic method was found to give a satisfactory correlation with a continuous flow immunonephelometric procedure.     

Conjugation of the monoclonal antibody 17-1A with the nitroacridine compound C921 with the poly-L-lysine as an intermediate agent

Transferrin and transferrin receptors play an important role in the transport of iron into the brain. To determine whether gallium enters the brain by the same mechanism, uptakes of 67Ga and 59Fe have been compared under controlled conditions. Rates of gallium penetration into brain (K(in)) were four times slower than those for 59Fe. K(in) for 67Ga when infused with citrate were 0.88 +/- 0.24 and 0.94 +/- 0.39 x 10(-3) ml g-1h-1 for cerebral hemisphere and cerebellum, respectively. When infused as the transferrin complex, 67Ga uptake into the brain was not different from that when infused with citrate. The presence of the anti-transferrin receptor antibody OX-26 significantly reduced uptake of 59Fe by 60% and 64% into cerebral hemisphere and cerebellum, respectively. By contrast, pretreatment of rats with OX-26 enhanced the uptake of 67Ga into brain, particularly when infused with citrate; mean increases in uptake of 67Ga were 120% and 144% for cerebral hemisphere and cerebellum, respectively. Purified 67Ga-transferrin was also taken up into both brain regions examined in the presence of OX-26. These results indicate that the transport of non-transferrin bound gallium is an important mechanism for gallium uptake into brain.     

Comparison between a lyse-and-then-wash method and a lyse-non-wash technique for the enumeration of CD34+ hematopoietic progenitor cells

The flow cytometric enumeration of CD34+ hemopoietic precursor cells (HPC) present in samples used for transplantation of HPC has proven to be the most powerful single parameter for prediction of engraftment. At present, several different methodological approaches are used for the flow cytometric enumeration of CD34+ HPC. In the present study we have compared two of these methods as regards enumeration of CD34+ HPC and their CD34+/CD19- and CD34+/CD19+ subsets: a lyse-non-wash procedure based on the use of a recently commercialized red cell lysing solution (Quicklysis, Cytognos, Salamanca, Spain) and a lyse-and-then-wash method in which the Becton Dickinson (San Jose, CA) FACS Lysing Solution was used. For that purpose a total of 52 samples corresponding to 20 G-CSF mobilized peripheral blood (PB) samples and 21 PB-derived leucapheresis products from patients undergoing autologous PB stem cell harvest, together with 11 bone marrow (BM) samples from healthy volunteers were analyzed. Our results show that for each of the three types of samples analyzed the use of the lyse-and-then-wash method is associated with significantly lower numbers of both total CD34+ HPC (P < or = 0.003) and its major CD34+/CD19- subset (P < or = 0.01) while no significant changes are detected in the number of CD34+/CD19+ HPC in BM samples (P > 0.05). The use of an internal standard (reference beads) added just prior to data acquisition, showed that the differences between both methods are due to a selective loss of CD34+ HPC and its major CD34+/CD19- subset in BM (P=0.002 and P=0.003), PB (P < 0.0001 and P < 0.0001) and PB-derived leucapheresis products (P < 0.0001 and P=0.0001). Finally, addition of a centrifugation and washing step to a group of 11 leucapheresis samples lysed with Quicklysis showed that they did not significantly affect the overall number of total CD34+, CD34+/CD19- and CD34+/CD19+ HPC obtained. In line with these findings elimination of centrifugation and washing steps when FACS Lysing Solution was used to lyse mature red cells almost corrected for the selective loss of CD34+ HPC. In spite of these differences a significant degree of correlation (r > 0.83 in all cases) was found between both methods regarding the total number of CD34+, CD34+/CD19- and CD34+/CD19+ HPC present in the BM, PB and PB-derived leucapheresis samples analyzed in this study.