Negative selection by apoptosis enriches progenitors in naïve and expanded human umbilical cord blood grafts.

The influence of TNF-α and Fas-ligand (FasL) on viability and function was evaluated in fresh- and expanded-umbilical cord blood (UCB) cells. CD34(+) progenitors and T cells display outstanding survival, whereas ~30% and >50% B lymphocytes and myeloid cells undergo spontaneous apoptosis within 24 and 48 h, respectively. Although the impact of exposure to toxic doses of FasL and TNF-α was undetectable in measurements of apoptosis; removal of dead cells after 2 days of incubation with the ligands revealed a twofold increase in frequency of colony-forming cells (CFU). The sensitivity of progenitors to apoptosis was also unaffected by Fas cross-linking following TNF-induced upregulation of the receptor, increasing CFU frequency without impairing SCID repopulating cell (SRC) activity. Most significant enrichment in CD34(+) progenitors and corresponding increase in CFU frequency were observed when FasL was applied during the final week of ex vivo expansion under the influence of nicotinamide, without impairing SRC activity. These data emphasize differential sensitivities of UCB progenitors and lineage-positive cells to apoptotic signaling mediated by the Fas and TNF receptors, which might be useful in improving the efficiency of ex vivo expansion and improving UCB cell engraftment.

Transplantation of ex vivo expanded cord blood cells using the copper chelator tetraethylenepentamine: a phase I/II clinical trial.

The copper chelator tetraethylenepentamine (TEPA; StemEx) was shown to attenuate the differentiation of ex vivo cultured hematopoietic cells resulting in preferential expansion of early progenitors. A phase I/II trial was performed to test the feasibility and safety of transplantation of CD133+ cord blood (CB) hematopoietic progenitors cultured in media containing stem cell factor, FLT-3 ligand, interleukin-6, thrombopoietin and TEPA. Ten patients with advanced hematological malignancies were transplanted with a CB unit originally frozen in two fractions. The smaller fraction was cultured ex vivo for 21 days and transplanted 24 h after infusion of the larger unmanipulated fraction. All but two units contained <2 x 10(7) total nucleated cells (TNCs) per kilogram pre-expansion. All donor-recipient pairs were mismatched for one or two HLA loci. Nine patients were beyond first remission; median age and weight were 21 years and 68.5 kg. The average TNCs fold expansion was 219 (range, 2-620). Mean increase of CD34+ cell count was 6 (over the CD34+ cell content in the entire unit). Despite the low TNCs per kilogram infused (median=1.8 x 10(7)/kg), nine patients engrafted. Median time to neutrophil and platelet engraftment was 30 (range, 16-46) and 48 (range, 35-105) days. There were no cases of grades 3-4 acute graft-versus-host disease (GVHD) and 100-day survival was 90%. This strategy is feasible.

Pre-clinical development of cord blood-derived progenitor cell graft expanded ex vivo with cytokines and the polyamine copper chelator tetraethylenepentamine.

BACKGROUND: We have previously demonstrated that the copper chelator tetraethylenepentamine (TEPA) enables preferential expansion of early hematopoietic progenitor cells (CD34+CD38-, CD34+CD38-Lin-) in human umbilical cord blood (CB)-derived CD34+ cell cultures. This study extends our previous findings that copper chelation can modulate the balance between self-renewal and differentiation of hematopoietic progenitor cells. METHODS: In the present study we established a clinically applicative protocol for large-scale ex vivo expansion of CB-derived progenitors. Briefly, CD133+ cells, purified from CB using Miltenyi Biotec's (Bergisch Gladbach, Germany) CliniMACS separation device and the anti-CD133 reagent, were cultured for 3 weeks in a clinical-grade closed culture bag system, using the chelator-based technology in combination with early-acting cytokines (SCF, thrombopoietin, IL-6 and FLT-3 ligand). This protocol was evaluated using frozen units derived from accredited cord blood banks. RESULTS: Following 3 weeks of expansion under large-scale culture conditions that were suitable for clinical manufacturing, the median output value of CD34+ cells increase by 89-fold, CD34+CD38- increase by 30-fold and CFU cells (CFUc) by 172-fold over the input value. Transplantation into sublethally irradiated non-obese diabetic (NOD/SCID) mice indicated that the engraftment potential of the ex vivo expanded CD133+ cells was significantly superior to that of unexpanded cells: 60+/-5.5% vs. 21+/-3.5% CD45+ cells, P=0.001, and 11+/-1.8% vs. 4+/-0.68% CD45+CD34+ cells, P=0.012, n=32, respectively. DISCUSSION: Based on these large-scale experiments, the chelator-based ex vivo expansion technology is currently being tested in a phase 1 clinical trial in patients undergoing CB transplantation for hematological malignancies.

Immune response to a diphtheria and tetanus toxoid administration in a three-dose diphtheria tetanus whole-cell pertussis/enhanced inactivated poliovirus vaccination schedule: a 7-year follow up.

The immune response to diphtheria and tetanus toxoid components of a combined diphtheria tetanus whole-cell pertussis/enhanced inactivated poliovirus (DTwP/eIPV) vaccine, administered in a three-dose schedule to infants at 2, 3 1/2 and 10 months of age and followed by a booster at the age of 8 years, was compared with the immune profile of a group of children at the same ages given the customary DTwP vaccine schedule at 2, 4, 6, and 12 months of age and a booster at the age of 8. Diphtheria- and tetanus-antitoxin titers were measured in parallel enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA). After the reinforcing dose given at 10 months of age, diphtheria antitoxin concentrations of > or = 0.01 IU/ml were found in 100% of infants in the study group, 91.7% of whom reached a titer of > or = 0.1 IU/ml and a geometric mean titer (GMT) of 0.40 and 0.93 IU/ml in ELISA and RIA, respectively. At 3 and 6 years of age, diphtheria antitoxin values of > or = 0.01 IU/ml were detected in 100 and 94% of children with GMT of 0.043 and 0.024 IU/ml, respectively. Seropositivity and GMT values indicative of protection were measured by both ELISA and RIA after the booster at the age of 8 years. Similar results were found in the control group, although the GMT tended to be higher. A good correlation between results obtained by ELISA vs. RIA was evident throughout. Priming at 2 and 3 1/2 months with diphtheria and tetanus antitoxin, as a component of a DTwP program, and reinforcing 6 months later induced an immune response indicative of protection against the diseases, which persisted up to the age of the booster recommended at school entry.

Hepatitis A virus seropositivity among hospital and community healthcare workers in Israel-the role of occupation, demography and socioeconomic background.

Hospital and community-clinic workers were tested for hepatitis A virus antibodies (HAV)-IgG to identify variables associated with presence of (HAV-IgG) and to determine whether sociodemographic background may explain all differences in HAV seropositivty among healthcare workers. Logistic regression analysis was used to identify variable associated with HAV-immunity. Multivariate logistic regression analysis revealed that HAV-seroprevalence correlated significantly (P<0.01) with age, siblings, residence in rural areas and origin. Nurse aides had an increased risk for HAV seropositivity (OR=5.04; 95% CI: 1.49-17.08) whereas physicians had a lower risk (OR=0.54: 95% CI: 0.30-0.98). Age and socioeconomic background were independently correlated with HAV immunity but did not explain all difference in HAV-seroprevalence. The higher susceptibility and elevated incidence of hepatitis A amongst physicians, prioritize primary prevention in this group.

Cost-benefit analysis of active vaccination campaigns against hepatitis A among daycare centre personnel in Israel.

OBJECTIVE: To evaluate, in economic terms, active vaccination campaigns against hepatitis A in comparison with the use of nonspecific immune globulin for the prevention of the disease among daycare centre employees in Israel. SETTING: Hypothetical analysis of the costs and benefits related to vaccination campaigns of workers currently employed in daycare centres in Israel. METHODS: A cost-benefit analysis was performed, comparing mass and selective active vaccination strategies for the daycare centre working force. Direct and indirect costs of diagnosis, treatment and immunisation as well as productivity loss were considered. A Markov-based model was developed using data from previous epidemiological studies and literature. RESULTS: The benefit-to-cost ratios of selective and mass active vaccination strategies were 1.50 [net present value (NPV) $US606 396] and 0.04 (NPV-$US2.36 million), respectively (2000 values). CONCLUSION: Under these study assumptions, the practice of administering hepatitis A active vaccine to serologically proven non-immune daycare centre workers has a cost-benefit justification, and should be widely considered in countries with a similar hepatitis A epidemiology to that in this study.

Influenza surveillance during winter 1997-1998 in Israel.

BACKGROUND: Each winter influenza activity is a major cause of morbidity and mortality both in Israel and worldwide. OBJECTIVES: To identify the influenza viruses active in Israel during the winter season and to assess the extent of influenza morbidity. METHODS: Information was collected on a population of 18,684 individuals enrolled in two community clinics in central Israel. It included the total number of visits for acute respiratory infection--including influenza and influenza-like illness (ARI/flu-like)--during a 20 week surveillance period (23 November 1997 to 27 March 1998) and the percent of influenza virus isolates in nasopharyngeal specimens from a sample of patients with ARI/flu-like collected on a weekly basis during the same period. RESULTS: A total of 5,947 visits for ARI/flu-like were recorded among 18,684 enrolled patients in two community clinics (18.1%). The progressive increase in the number of visits for ARI/flu-like reached a peak on week 2/98 with 597 visits and a rate of 31.95 visits per 1,000 population. After this, a decrease to the initial values was evident by week 12/98. Most affected patients were in the age groups 5-14 and 65 years and over, with a rate of 733.5 and 605.3 visits per 1,000 population, respectively. Influenza virus was isolated from 92 of the 426 nasopharyngeal specimens (21.6%). The most commonly detected strain was A/Sydney/5/97 (H3N2) like (77.2%). The peak rate of isolates was recorded at the beginning of January (01/98). CONCLUSIONS: A/Sydney/5/97 (H3N2) like-strain was the dominant influenza virus. Its presence did not prevent the simultaneous activity of influenza A/H1N1 virus. The dynamic of the clinical disease as expressed by the weekly visit rate for ARI/flu-like was similar to the temporal pattern of the virological findings. The extent of morbidity suggests moderate epidemic activity.

The effect of human myelomonocytic leukemic cell line (M20) derived IL-1 inhibitor on human erythroid cell development.

The effect of an inhibitor of IL-1, purified from a human myelomonocytic cell line (M20) on the development of human erythroid cell development was studied. The inhibitor, is a protein of 52 kD molecular weight that is distinct immunologically and functionally from other reported IL-1 inhibitors. The experiments were performed in a two-phase culture system that allows separation of the erythroid cell development into an erythropoietin (EPO)-independent phase, where early erythroid-committed BFUe proliferate and differentiate into the more mature progenitors, CFUe, and EPO-dependent phase, where CFUe further proliferate and mature into hemoglobin-containing orthochromatic normoblasts. The results indicated that in both developmental stages the M20-derived inhibitor reversibly blocked cell proliferation without interfering with cell differentiation.

Effect of M20 interleukin-1 inhibitor on normal and leukemic human myeloid progenitors.

This study aimed to assess the effect of the M20 interleukin-1 (IL-1) inhibitor on normal and leukemic hematopoietic cells. The M20-derived IL-1 inhibitor was found to inhibit the growth of various hematopoietic cells. The in vitro proliferation of myeloid cell lines in serum-containing medium or proliferation of these cells induced by IL-1 in serum-free medium (measured by 3H-TdR) were inhibited by the M20 IL-1 inhibitor. In addition, growth of normal progenitors and fresh leukemic cells stimulated by granulocyte-macrophage colony-stimulating factor (GM-CSF) (as measured by colony and liquid systems) was also inhibited by this factor. After the removal of the IL-1 inhibitor at the peak of growth inhibition, leukemic and normal progenitor cells retain their ability to grow and develop into GM-CSF colonies. These results show that the growth inhibition phenomena were reversible and did not result from a cytotoxic effect. Our data suggest that the M20-derived IL-1 inhibitor might function as a true negative growth regulator of normal and leukemic hematopoietic cells.

Isolation and characterization of HL-60 cell variants with different potentials for spontaneous differentiation.

Although HL-60 cells, an in vitro established cell line derived from a patient with acute promyelocytic leukemia, are blocked at the promyelocytic stage of myeloid differentiation, certain chemicals can induce the cells to undergo terminal differentiation into either granulocytes or macrophages. Moreover, a small fraction of the cell population undergoes differentiation spontaneously without the addition of any inducing agent. In this paper it is demonstrated that this cell line is heterogeneous with respect to the ability of the cells to differentiate spontaneously: some clones (SD+) have a higher tendency to do so than others (SD-). In semi-solid medium, SD+ cells developed diffused colonies containing mature monocytes and macrophages, whereas SD- cells developed compact colonies of promyelocytes. Based on these morphological differences the various clones were isolated and analysed. Although only a small fraction of the population actually became differentiated at any particular time, practically all the cells in the SD+ clones had the potential to differentiate spontaneously. The clones also differ in their response to differentiation inducers; whereas some agents induced complete differentiation in both types of clones, others (e.g. actinomycin C and cytosine arabinoside) induced only SD+ clones, suggesting that differentiation induced by the latter agents is related to the ability of the cells to differentiate spontaneously. Thus the potential of leukemic cells to undergo spontaneous differentiation may be an important factor when considering differentiation-inducing therapy for leukemic patients.

[Immediate breast reconstruction with tissue expander].

The technique of immediate breast reconstruction, using the tissue expander and replacing it with a permanent silicone prosthesis, has been applied with increasing frequency during the past several years. We chose this technique for immediate breast reconstruction because of its simplicity, rapidity when combined with mastectomy, and good esthetic results. We have performed 29 immediate breast reconstructions in the past 1.5 years in patients ranging from 22 to 72 years (mean 47). The insertion of the tissue expander added only 20-30 minutes of operating time for the mastectomy, and did not prolong hospitalization. Before we began to replace the expander with a permanent silicone prosthesis there were complications in 4 patients (14%). These included development of a seroma in 3, 1 of which became infected 40 days after insertion of the tissue expander, and capsular contraction in a patient who had previously had radiotherapy to her breast. The case with the infection was the only failure and the expander had to be removed. Immediate reconstruction of the breast is easily performed and can be of tremendous psychological benefit to mastectomy patients. Chemotherapy or radiotherapy are not contraindications to this type of reconstruction.

Self-renewal and commitment to differentiation of human leukemic promyelocytic cells (HL-60).

More than 80% of cells from a human promyelocytic leukemic cell line (HL-60) possess the capacity for self-renewal as evidenced by their ability to form large primary colonies in semisolid medium and the presence within these colonies of cells capable of subsequent colony formation. Colony development is independent of the normal regulator--the myeloid colony stimulating factor. The observed autostimulation suggests the production of specific growth promoters by the cells. Differentiation either to mature granulocytes or macrophages, induced by various agents, was associated with reduced cloning potential. Nevertheless, colonies containing differentiated cells could be developed either by cloning cells in the presence of suboptimal concentrations of inducer or by adding inducers over colonies developed in its absence. Upon differentiation, there was a morphological change from compact to diffused colony morphology due to cell mobility in the semisolid medium. Even at suboptimal concentrations of inducer more than 95% of the colonies became diffused, indicating clonal homogeneity of the population with respect to differentiation capacity. The loss of self-renewal was found to be one of the early properties which changed following the initiation of differentiation. The loss preceded not only the overt expression of maturation-specific functions but also cellular commitment to terminal differentiation; shorter contact with the inducer was required to cause loss of self-renewal than to induce an irreversible transition to differentiation. This resulted in cells that lost their self-renewal potential without being able to complete their program of differentiation.

Modulation of the maturation of human leukemic promyelocytes (HL-60) to granulocytes or macrophages.

The relationship between the effects of two types of inducers on the maturation of a line of human promyelocytic cells (HL-60) was studied. The dual potentiality of these promyelocytes was demonstrated by the ability of isolated colonies to differentiate into granulocytes, following induction by dimethylsulphoxide (DMSO) or express properties specific to macrophages following exposure to 12-O-tetradecanoylphorbol-13-acetate (TPA). The differentiation process involved an irreversible step which occurred 48 h after exposure to DMSO, and a few h after exposure to TPA. This implies that the presence of the inducer in the culture medium is no more required for completion of the differentiation programme. Removal of the inducer prior to this step resulted in reversal of all the inducer-mediated effects. During the period of non-commitment the specific pathway of maturation was still undetermined; cells in which differentiation was initiated by exposure to DMSO were able to develop into macrophages after substitution of DMSO by TPA. Moreover, pre-exposure to DMSO and other inducers of granulocyte differentiation resulted in higher sensitivity to TPA, as indicated by the cell response to low TPA concentrations and more rapid expression of macrophage specific properties. These results suggest that the early stages in HL-60 differentiation are probably common to the granulocyte and the macrophage pathways.