A novel method for human hematopoietic stem/progenitor cell isolation from umbilical cord blood based on immunoaffinity aqueous two-phase partitioning

A novel cell separation process based on immunoaffinity aqueous two phase systems is presented to isolate and purify CD34⁺ stem/progenitor cells directly from the whole umbilical cord blood (UCB). A system, composed of polyethylene glycol and dextran, was evaluated for the selective recovery of CD34⁺ cells from UCB. A monoclonal antibody against the CD34 surface antigen was used for the direct partitioning of CD34⁺ cells in UCB to the PEG-rich phase. The initial population of CD34⁺ cells (0.2% of the initial sample) was enriched to values up to 42% in a single partitioning step, while the majority of contaminant cells were partitioned to the dextran-rich phase (1.37 × 10⁻² < K_P < 2.76 × 10⁻²). This novel selection method allowed a recovery yield of 95% of CD34⁺ cells with a purification factor of 245 and is expected to pave a new way to purify hematopoietic stem/progenitor cells for use in a variety of clinical settings.     

Differential expression of CD14, CD36 and the LDL receptor on human monocyte-derived macrophages

 Macrophages are key players in many aspects of human physiology and disease. It has been hypothesized that in a given microenvironment monocytes differentiate into specific subpopulations with distinct functions. In order to study the role of macrophage heterogeneity in atherogenesis, we established a novel isolation and culture technique for human monocyte-derived macrophages. The present technique does not select for monocyte subpopulations prior to the onset of differentiation. Monocytes were cultured for 2 weeks in the presence of autologous lymphocytes before being plated quantitatively. They differentiated into mature macrophages in terms of morphology, lipid composition, and biological activity. Based on phagocytic activity as well as on the expression of CD14, CD36, and the low-density lipoprotein (LDL) receptor, we have identified macrophage subpopulations that may play distinct roles in atherogenesis. While virtually all adherence-purified monocytes expressed CD14, CD36, and the LDL-R, we characterized three subpopulations of macrophages based on the expression of these antigens: CD36⁺CD14^–LDL-R^– (58±12%), CD36⁺CD14⁺LDL-R⁺(18±5%), the remaining cells being CD36^–CD14^–LDL-R^–. The first two subsets decreased in size during further differentiation (51±12% and 8±3%, respectively). Our culture technique may also serve as a good model for studying the implications of macrophage heterogeneity in diseases other than atherosclerosis. Accepted: 13 February 1998     

Optimization of ex vivo hematopoietic stem cell expansion in intermittent dynamic cultures

For the ex vivo expansion of CD34⁺ cells, culture conditions were optimized using cytokine cocktails and media change methods. In addition, static, orbital-shake, and stirred cultures were compared. After cultivation, total cell expansion, immunophenotypes, clonogenic ability, and metabolite concentration in media were analyzed. Optimized media change methods enhanced the number of total nucleated cells (TNCs) by 600-fold (from 10⁴ to 6 × 10⁶ cells) in static cultures. Furthermore, intermittent orbital-shake cultures gave the highest fold increase of TNCs and CD34⁺/CD38⁻ cells. These results imply that proliferation of CD34⁺ cells in intermittent shake cultures was more efficient than that in static cultures under optimized culture conditions.     

Higher efficacy of Etoposide + Cytarabine Plus Pegfilgrastim in poorly mobilizing Multiple Myeloma and lymphoma Patients

Background aimsAn optimal strategy for mobilizing hematopoietic stem cells in poorly mobilizing patients with multiple myeloma (MM) and lymphoma has not yet been determined.MethodsWe retrospectively analyzed the efficacy and safety of etoposide combined with cytarabine (etoposide 75 mg/m², daily d1∼2; Ara-C 300 mg/m², every 12 h d1∼2), plus pegfilgrastim (6 mg d6) in 32 patients with MM or lymphoma, among whom 53.1% were defined as “proven poor mobilizers.”ResultsThis approach resulted in adequate mobilization (≥2.0 × 10⁶ CD34⁺ cells/kg) in 93.8% of patients and optimal mobilization (≥5.0 × 10⁶ CD34⁺ cells/kg) in 71.9% of patients. A total of 100% of patients with MM reached at least 5 × 10⁶ CD34⁺ cells/kg collected, the amount required for double autologous stem cell transplant. In total, 88.2% of patients with lymphoma reached at least 2 × 10⁶ CD34⁺ cells/kg collected, the amount required for a single autologous stem cell transplant. This was achieved with a single leukapheresis in 78.1% of cases. A median peak number of 42.0/μL circulating CD34⁺ cells and a median number of blood CD34⁺ cells counts in 6.7 × 10⁶/L were collected among 30 successful mobilizers. Approximately 6.3% of patients required plerixafor rescue, which was successful. Nine (28.1%) of the 32 patients suffered grade 2∼3 infections, and 50% required platelet transfusions.ConclusionsWe conclude that chemo-mobilization with etoposide, Ara-C and pegfilgrastim in poorly mobilizing patients with MM or lymphoma is very effective and has acceptable toxicity.     

Expansion of CD34+ cells from human umbilical cord blood by FL and/or TPO gene transfected human marrow stromal cell lines

To elucidate the effect of gene transfected marrow stromal cell on expansion of human cord blood CD34⁺ cells, a culture system was established in which FL and TPO genes were transfected into human stromal cell line HFCL. To establish gene transfected stromal cells co-culture system, cord blood CD34⁺ cells were purified by using a magnetic beads sorting system. The number of all cells and the number of CD34⁺ cells and CFC (CFU-GM and BFU-E) were counted in different culture systems. The results showed that in all 8 culture systems, SCF+IL-3+HFT manifested the most potent combination, with the number of total nucleated cells increasing by (893.3 ±52.1)-fold, total progenitor cells (CFC) by (74.5 ±5.2)-fold and CD34⁺ cells by 15.7-fold. Maximal expansions of CFC and CD34⁺ cells were observed at the end of the second week of culture. Within 14 days of culture, (78.1 ± 5.5)-fold and (57.0 ± 19.7)-fold increases in CFU-GM and BFU-E were obtained. Moreover, generation of LTC-IC from amplified CD34⁺ cells within 28 days was found only in two combinations, i.e. SCF+IL-3+FL+TPO and SCF+IL-3+HFT, and there was no significant difference between these two groups statistically. These results suggest that human umbilical cord blood CD34⁺ cells can be extensively expandedex vivo by using gene transfected stromal cells along with cytokines.     

Murine mesenchymal stem cell isolated and expanded in low and high density culture system: surface antigen expression and osteogenic culture mineralization

Marrow culture from mice has been reported to be overgrown by non-mesenchymal cells. In almost all protocols for isolation of murine mesenchymal stem cells (MSCs), high density culture systems have been employed. Since MSCs are colonogenic cells, the initiating cell seeding density may have significant impact on their cultures. This subject was explored in this study. For this purpose, the bone marrow cells from NMRI mice were plated at 2.5 × 10⁶ cells/cm² and upon confluency were reseeded as either low density (50 cells/cm²) or high density (8 × 10⁴ cells/cm²) cultures. The cells were expanded through an additional subculture and the passage 2 cells as a product of two culture systems were statistically compared with respect to their surface antigen profiles and osteogenic culture mineralization. While low density culture grew with multiple colony formation, there were no distinct colonies in high density cultures. In contrast to high density cultures, passage 2 cells from low density system possessed typical homogenous fibroblastic morphology. Some cells from high density system but not the low density cultures expressed hematopoietic and endothelial cell markers including CD135, CD34, CD31, and Vcam surface antigens. Furthermore, osteogenic cultures from low density system displayed significantly more mineralization than those from high density system. Taken together, it seems that low density culture system resulted in more purified MSC culture than its counterpart as high density culture system.     

Depletion of T-cell receptor alpha/beta and CD19 positive cells from apheresis products with the CliniMACS device

Background aimsThe CliniMACS device (Miltenyi Biotec, Bergisch Gladbach, Germany) was used for depletion of T-cell receptor alpha/beta positive (TCRαβ⁺) and CD19 positive (CD19⁺) cells from apheresis products.MethodsInvestigators performed 102 separations. Apheresis products with a median 5.8 (minimum to maximum, 1.2–10.4) × 10¹⁰ mononuclear cells were used with a median 358 (92–1432) × 10⁶ CD34⁺ cells. There were 24.8% (6.1–45.7%) median TCRαβ⁺ cells and 4.4% (1.2–11.7%) median B cells in the apheresis product.ResultsAfter depletion, a median 0.00097% (0.00025–0.0048%) of TCRαβ⁺ cells could be detected, and B cells, as determined as CD20⁺ cells, were reduced to 0.0072% (0.0008–0.072%). TCRαβ⁺ cells were depleted by log 4.7 (3.8–5.5), and B cells were depleted by log 4.1 (3.0–4.7). Recovery of mononuclear cells was 55% (33–77%), and recovery of CD34⁺ cells was 73% (43–98%). Recovery of CD56⁺/3^? natural killer cells was 80% (35–142%), recovery of TCR gamma/delta positive (TCRγδ⁺) T cells was 83% (39–173%) and recovery of CD14⁺ cells was 79% (22–141%). Viability of cells was 98% (93–99%) after separation (all values median).ConclusionsProfound depletion of TCRαβ⁺ T cells can be achieved with the CliniMACS system. Recovery of CD34⁺ stem cells is in the same range than after CD34⁺ enrichment and CD3/CD19 depletion. Transplantation with >4 × 10⁶ CD34⁺ cells/kg can be performed for every patient with 1–5 × 10⁴ TCRαβ⁺ cells/kg and about 5–10 × 10⁶ TCRγδ⁺ cells/kg with two rounds of apheresis.     

Metallothionein in human immunomagnetically selected CD34+ haematopoietic progenitor cells

Human umbilical CD34⁺ immature haematopoietic cells were rapidly and efficiently obtained from light density MNC (mononuclear cells) by MACS (magnetic cell sorting). An ex vivo expanded population of CD34⁺ was cultured in serum‐free medium supplemented with cytokines FL (flt3 ligand), SCF (stem cell factor) and TPO (thrombopoietin) in order to obtain a sufficient number of CD34⁺ cells. CD34⁺ cells expanded from cord blood for 7 days were demonstrated to increase in the absolute number of CD34⁺ cells by 5.12±2.47‐fold (mean±S.D., n=3). Flow cytometric analysis demonstrated that the percentage of CD34 antigen expression after expansion of the culture was 97.81±1.07%, whereas it was 69.39±10.37% in none‐expanded CD34⁺ cells (mean±S.D., n=3), thus defining a system that allowed extensive amplification accompanied by no maturation. MTs (metallothioneins), low molecular weight, cysteine‐rich metal‐binding proteins, exhibit various functions, including metal detoxification and homoeostasis. We here examined the expression pattern of functional members of the MT gene family in immature CD34⁺ cells and compared it with more mature CD34^? cells in order to strengthen the proposed function of MT in differentiation. Cells were cultured in RPMI 1640 medium, with or without different zinc supplements for 24 h. Relative quantitative expression of MT isogenes in the mature CD34^? cells was higher than in the immature CD34⁺ cells. IHC (immunohistochemical staining) revealed an increased MT protein biosynthesis in CD34^? cells, greater than in CD34⁺ cells. Therefore, the role of MT in differentiation of human haematopoietic progenitor cells from human cord blood is reported for the first time.     

Effective expansion of umbilical cord blood hematopoietic stem/progenitor cells by regulation of microencapsulated osteoblasts under hypoxic condition

The expansion of hematopoietic stem/progenitor cells (HSPCs) from umbilical cord blood (UCB) with the support of microencapsulated osteoblasts under hypoxia environment was investigated. The expansion of HSPCs was evaluated through the total number of UCB mononuclear cells (MNCs) produced, their repopulating potential with the colony-forming unit assay (CFU-Cs) and CD34⁺ phenotypic analysis with flow cytometry. At the end of 7 days of culture, the UCB-MNCs, CFU-Cs and CD34⁺ cells had achieved 18.7 ± 1.6, 11.6 ± 0.9 and 23.4 ± 2-fold expansions, respectively, in the test groups. These were significantly different from those in control groups. Microencapsulated osteoblasts under hypoxia conditions had therefore a significant effect on the expansion potential of HSPCs in vitro.     

Evaluation of natural killer cell expansion and activation in vivo with daily subcutaneous low-dose interleukin-2 plus periodic intermediate-dose pulsing

 Natural killer (NK) cells may be expanded in vivo with a prolonged course of daily subcutaneous interleukin-2 (IL-2). However, cellular activation requires higher concentrations of IL-2 than are achieved with low-dose therapy. The objective of the current trial was to determine the toxicity and immunological effects of periodic subcutaneous intermediate-dose IL-2 pulses in patients receiving daily low-dose therapy. A group of 19 patients were treated with daily subcutaneous low-dose IL-2 at 1.25×10⁶ International Units (1.25 MIU) m^–2 day^–1. After 4–6 weeks, patients received escalating 3-day intermediate-dose IL-2 pulses administered as single daily subcutaneous injections, repeated at 2-week intervals. The maximum tolerated pulse dose was 15 MIU m^–2 day^–1, with transient hypotension, fatigue, and nausea/vomiting dose-limiting. Subcutaneous IL-2 resulted in in vivo expansion of CD56⁺ NK cells (796±210%) and CD56^bright natural killer (NK) cells (3247±1382%). Expanded NK cells coexpressed CD16, and showed lymphokine-activated killer activity and antibody-dependent cellular cytotoxicity in vitro. Intermediate-dose pulsing resulted in serum IL-2 concentrations above 100 pM. Cellular activation was suggested by rapid margination of NK cells following pulsing, coincident with peak IL-2 levels, with return to baseline by 24 h. In addition, interferon γ production in response to lipopolysaccharide was augmented. Subcutaneous daily low-dose IL-2 with intermediate-dose pulsing is a well-tolerated outpatient regimen that results in in vivo expansion and potential activation of NK cells, with possible application in the treatment of malignancy and immunodeficiency. Received: 31 December 1997 / Accepted: 20 April 1998     

Mobilization and engraftment of peripheral blood stem cells in healthy related donors >55 years old

Background aimsThe increasing scarcity of young related donors has led to the use of older donors for related allogeneic hematopoietic stem cell transplantation (HSCT). This study analyzed the influence of age on the results of mobilization of peripheral blood stem cells (PBSCs) in healthy donors as well as on the engraftment and outcome of HSCT.MethodsA retrospective analysis from a single center was performed comparing the results of PBSC mobilization from related healthy donors according to their age.ResultsThe study included 133 consecutive related donors. The median age was 50 years (range, 4–77 years); 70 (53%) donors were males, and 44 (33%) were >55 years old. All donors were mobilized with granulocyte colony-stimulating factor for 5 days. The peak CD34⁺ cell count in peripheral blood was higher in younger than in older donors (median, 90.5 CD34⁺ cells/μL [range, 18–240 CD34⁺ cells/μL] versus 72 CD34⁺ cells/μL [range, 20–172.5 CD34⁺ cells/μL], P = 0.008). The volume processed was lower in younger than in older donors (16,131 mL [range, 4424–36,906 mL] versus 18,653 mL [range, 10,003–26,261 mL], P = 0.002) with similar CD34⁺ cells collected (579.3 × 10⁶ cells [range, 135.14 × 10⁶–1557.24 × 10⁶ cells] versus 513.69 × 10⁶ cells [range, 149.81 × 10⁶–1290 × 10⁶ cells], P = 0.844). There were no differences in time to recovery of neutrophils and platelets or in the incidences of acute and chronic graft-versus-host disease, overall survival, non-relapse mortality and relapse incidence.ConclusionsDonors >55 years old mobilized fewer CD34⁺ cells and required a greater volume to collect a similar number of CD34⁺ cells. The outcome of HSCT was not influenced by donor age. Donor age should not be a limitation for related allogeneic HSCT.     

CD4 + CD25<Superscript>high</Superscript> Regulatory T Cell Numbers and FOXP3 mRNA Expression in Patients with Advanced Esophageal Cancer Before and After Chemotherapy

We evaluated the changes in CD4 + CD25^high regulatory T (Treg) cells and FOXP3 mRNA expression in patients with advanced esophageal cancer as well as its clinical significance. For this purpose, the frequencies of peripheral blood Treg cells in 68 patients with advanced esophageal cancer and 40 healthy controls were determined by flow cytometry, and FOXP3 mRNA expression in Treg cells of 40 patients was determined by RT–PCR. The data show that Treg cell numbers were significantly higher (P < 0.01) in esophageal cancer patients (1.82 ± 0.54% of CD4 + T cells) as compared with healthy controls (1.52 ± 0.70% of CD4⁺ T cells). Treg cell numbers in the patients were significantly higher (P < 0.05) before chemotherapy (1.82 ± 0.54% of CD4 + T cells) than after chemotherapy (1.66 ± 0.58% of CD4 + T cells). Expression of the FOXP3 mRNA in the patients was significantly lower (P < 0.05) after chemotherapy (0.266 ± 0.028% of CD4 + T cells) than before chemotherapy (0.318 ± 0.027% of CD4 + T cells). It was, therefore, concluded that Treg cell numbers as well as FOXP3 mRNA expression in advanced esophageal cancer patients were significantly decreased after chemotherapy. Notably, FOXP3 gene may thus be involved in regulating the numbers and function of Treg cells in advanced esophageal cancer patients receiving chemotherapy.     

Zoledronate-activated Vγ9γδ T cell-based immunotherapy is feasible and restores the impairment of γδ T cells in patients with solid tumors

Gamma/delta (γδ) T cells play a role in innate immunity and exhibit cytotoxicity toward a large range of tumor types. Recent studies have shown that aminobisphosphonates may be applied to a culture in which a large number of γδ T cells are proliferated ex vivo. We carried out a clinical study of 25 patients with various solid tumors to determine further the safety, immunologic effect and feasibility of zoledronate-activated Vγ9γδ T cell-based immunotherapy. No severe toxicity was observed. In the cells used for the first treatment, the total cell number, frequency and number of CD3⁺ Vγ9⁺ γδ T cells were 409 ± 284 × 10⁷ cells, 56 ± 33% and 255 ± 242 × 10⁷ cells, respectively. Aminobisphosphonate therapy or chemotherapy resulted in the suppression of CD3⁺ Vγ9⁺ γδ T-cell proliferation. The numbers of CD3⁺ T cells, CD3⁺ Vγ9⁺ γδ T cells and CD27^? CD45RA^? Vγ9⁺ subsets in peripheral blood were significantly lower in patients than in healthy subjects (P <y 0.05). From such an impaired immunologic condition, the numbers and frequencies of CD3⁺ Vγ9⁺ γδ T cells and CD27^? CD45RA^? subsets significantly increased in patients treated with this immunotherapy. Zoledronate-activated Vγ9γδ T cell-based immunotherapy that restores the number of Vγ9γδ T cells in cancer patients may provide another mode of adoptive immunotherapy.     

XIAP antisense oligonucleotide (AEG35156) achieves target knockdown and induces apoptosis preferentially in CD34<Superscript>+</Superscript>38<Superscript>−</Superscript> cells in a phase 1/2 study of patients with relapsed/refractory AML

XIAP, a potent caspase inhibitor, is highly expressed in acute myeloid leukemia (AML) cells and contributes to chemoresistance. A multi-center phase 1/2 trial of XIAP antisense oligonucleotide AEG35156 in combination with idarubicin/cytarabine was conducted in 56 patients with relapsed/refractory AML. Herein we report the pharmacodynamic studies of the patients enrolled at M. D. Anderson Cancer Center. A total of 13 patients were enrolled in our institution: five in phase 1 (12–350 mg/m² AEG35156) and eight in phase 2 (350 mg/m² AEG35156) of the protocol. AEG35156 was administered on 3 consecutive days and then weekly up to a maximum of 35 days. Blood samples were collected from patients on days 1 through 5 and on day 28–35 post-chemotherapy for detection of XIAP levels and apoptosis. AEG35156 treatment led to dose-dependent decreases of XIAP mRNA levels (42–100% reduction in phase 2 patients). XIAP protein levels were reduced in all five samples measured. Apoptosis induction was detected in 1/4 phase 1 and 4/5 phase 2 patients. Importantly, apoptosis was most pronounced in CD34 ⁺ 38 ⁻ AML stem cells and all phase 2 patients showing apoptosis induction in CD34 ⁺ 38 ⁻ cells achieved response. We conclude that at 350 mg/m², AEG35156 is effective in knocking down XIAP in circulating blasts accompanied by the preferential induction of apoptosis in CD34 ⁺ 38 ⁻ AML stem cells.     

<Emphasis Type="Italic">Ex vivo</Emphasis> expansion of hematopoietic cells from CD34<Superscript>+</Superscript> cord blood cells in various culture conditions

This study compared the cell expansion and colony-forming ability of human cord blood stem cells cultured ex vivo with 2 types of cytokine combinations, and 2 types of media characterized by the presence or absence of fetal bovine serum (FBS) in 2 or 3 dimensional (2D or 3D) culture environments. Purified CD34⁺ cells derived from different donors were cultured in Iscove’s Modified Dulbecco’s Medium (IMDM) and Ultraculture serum-free medium (SFM) containing the cytokine cocktail-I (coc-I) (EPO, GM-CSF, SCF, and IL-3) or cytokine cocktail-II (coc-II) (TPO, G-CSF, SCF, IL-6, and Flt3/Flk-2 ligand) with or without FBS. Generally, higher CFU-GM values were observed in the IMDM compared to the SFM. In the coc-I conditions, the ‘IMDM + coc-I’ and ‘IMDM + coc-I + FBS’ conditions gave the greatest cell (1,667 ± 274 and 1,600 ± 140-fold, respectively) and colony-forming units (CFU) expansions (BFU-E: 21 ± 3, 36 ± 5; CFU-GM: 95 ± 19, 81 ± 17; and CFU-GEMM: 2 ± 1, 3 ± 1-fold, respectively) in 26 day culture, respectively. In the coc-II conditions, the ‘SFM + coc-II’ condition gave the greatest cell expansion (2,143 ± 134-fold), but the ‘IMDM + coc-II’ condition gave the best CFU-GM expansion (924 ± 110-fold) in 26 day culture. In conclusion, ‘IMDM + coc-I’ and ‘IMDM + coc-II’ were the most accessible conditions for CFU expansion for all culture cases. The 2D stationary culture had affirmative effect on CFU expansion compared to the 3D culture using semisolid Methocult™. These results are believed to be significant in the ex vivo expansion of hematopoietic stem cells.     

Initial CD34+ cell-enrichment of cord blood determines hematopoietic stem/progenitor cell yield upon ex vivo expansion

Since umbilical cord blood (UCB), contains a limited hematopoietic stem/progenitor cells (HSC) number, successful expansion protocols are needed to overcome the hurdles associated with inadequate numbers of HSC collected for transplantation. UCB cultures were performed using a human stromal‐based serum‐free culture system to evaluate the effect of different initial CD34⁺ cell enrichments (Low: 24 ± 1.8%, Medium: 46 ± 2.6%, and High: 91 ± 1.5%) on the culture dynamics and outcome of HSC expansion. By combining PKH tracking dye with CD34⁺ and CD34⁺CD90⁺ expression, we have identified early activation of CD34 expression on CD34^? cells in Low and Medium conditions, prior to cell division (35 ± 4.7% and 55 ± 4.1% CD34⁺ cells at day 1, respectively), affecting proliferation/cell cycle status and ultimately determining CD34⁺/CD34⁺CD90⁺ cell yield (High: 14 ± 1.0/3.5 ± 1.4‐fold; Medium:22 ± 2.0/3.4 ± 1,0‐fold; Low:31 ± 3.0/4.4 ± 1.5‐fold) after a 7‐day expansion. Considering the potential benefits of using expanded UCB HSC in transplantation, here we quantified in single UCB units, the impact of using one/two immunomagnetic sorting cycles (corresponding to Medium and High initial progenitor content), and the average CD34⁺ cell recovery for each strategy, on overall CD34⁺ cell expansion. The higher cell recovery upon one sorting cycle lead to higher CD34⁺ cell numbers after 7 days of expansion (30 ± 2.0 vs. 13 ± 1.0 × 10⁶ cells). In particular, a high (>90%) initial progenitor content was not mandatory to successfully expand HSC, since cell populations with moderate levels of enrichment readily increased CD34 expression ex‐vivo, generating higher stem/progenitor cell yields. Overall, our findings stress the importance of establishing a balance between the cell proliferative potential and cell recovery upon purification, towards the efficient and cost‐effective expansion of HSC for cellular therapy. J. Cell. Biochem. 112: 1822–1831, 2011. © 2011 Wiley‐Liss, Inc.     

Multidimensional flow-cytometric analysis of dendritic cells in peripheral blood of normal donors and cancer patients

 We studied the potential of multidimensional flow cytometry to evaluate the frequency and maturation/activation status of dendritic cells in minimally manipulated peripheral blood mononuclear cell preparations (i.e., only separated on Ficoll-Hypaque) of normal donors and cancer patients. A rare subset of HLA-DR⁺ leukocytes (less than 1% mononuclear cells) was detected in blood of normal donors that displayed all the features of dendritic cells: these cells had high forward-light-scatter characteristics and coexpressed CD4, CD86 and CD54 surface antigens, but lacked the lineage-associated surface markers of T cells, B cells, monocytes, granulocytes or NK i.e. they were CD3^–, CD19^–, CD20^–, CD14^–, CD11b^–, CD16^–, CD56^–). These physical and phenotypic properties were virtually identical to those of immunomagnetically sorted leukocytes characterized as dendritic-cells on the basis of morphology, phenotype and high stimulatory activity in allogeneic mixed-lymphocyte cultures. Using this flow-cytometric approach we observed that the frequency of dendritic cell-like cells in peripheral blood mononuclear cell specimens of cancer patients receiving chemotherapy alone or those recovering from stem cell transplantation was significantly lower than that of normal individuals (mean ± SE: 0.36 ± 0.05%, 0.14 ± 0.06%, and 0.75 ± 0.04% respectively). Multidimensional flow-cytometric analysis of dendritic cells might represent an important new tool for assessing immunocompetence, and for monitoring the effects of therapeutic regimens on the immune system. Received: 20 June 1997 / Accepted: 14 August 1997     

Preparation of cross-linked carboxymethyl chitosan for repairing sciatic nerve injury in rats

A successful nerve regeneration process was achieved with nerve repair tubes made up of 1-ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) cross-linked carboxymethyl chitosan (CM-chitosan) with improved biodegradability. Chitosan has a very slow degradation rate, while the EDC cross-linked CM-chitosan tubes degraded to 30% of original weight during 8 weeks of incubation in lysozyme solution. In vitro cell culture indicated that the CM-chitosan films presented no cytotoxicity to Schwann cells. From in vivo studies using a 10 mm rat sciatic nerve defect model investigated by histomorphometry analysis, the average diameter of the fibers and the average thickness of myelin sheath in the CM-chitosan tubes were 3.7 ± 0.33 and 0.33 ± 0.04 μm, respectively, which demonstrated equivalence to nerve autografts (the current “gold” standard); furthermore, the average fiber density in the CM-chitosan tubes was 20.5 × 10³/mm², which was similar to that of autografts (21 × 10³/mm²) and significantly higher than that of common chitosan tubes (15.3 × 10³/mm²).     

The influence of differentiation-inducing agents on plasma membrane surface characteristics of THP-1 cells

Undifferentiated THP-1 cells from Cell Culture Collection of the Institute of Cytology, RAS (St. Petersburg), are characterized by weak expression of Toll-like receptor-4 (TLR4) on the cell surface (up to 2%) and by almost undetectable expression of CD14 and CD11b receptors. Differentiation agent phorbol-12-myristate-13-acetate independently of its concentration (2 × 10⁻⁷ M or 10⁻⁸ M) and incubation time (24 or 48 h) did not initiate CD11b surface expression and did not change the parameter S_app (0.605 ± 0.005 at 37°C) reflecting the cell membrane viscosity. Differentiation of THP-1 cells induced by another differentiation agent, 1α,25-dihydroxyvitamin D₃, caused expression of CD14 (up to 70–80%) and CD11b (up to 15–20%) receptors, again without changes in plasma membrane viscosity. The rate constants of the reduction of 5- and 16-doxyl-stearic acids by THP-1 cells were in the range of 6–8 × 10⁻³ s⁻¹ at 37°C. During cell differentiation significant changes in cell electrophoretic mobility (EM, μm s⁻¹ V⁻¹ cm) were observed. Mean value of EM for undifferentiated THP-1 cells was −1.332 ± 0.011, whereas for phorbol-12-myristate-13-acetate- and 1α,25-dihydroxyvitamin D₃-treated cells it was −1.432 ± 0.030 and −1.212 ± 0.016, respectively.     

Effect of Dietary Selenium and Cancer Cell Xenograft on Peripheral T and B Lymphocytes in Adult Nude Mice

Selenium (Se) is known to regulate tumorigenesis and immunity at the nutritional and supranutritional levels. Because the immune system provides critical defenses against cancer and the athymic, immune-deficient NU/J nude mice are known to gradually develop CD8⁺ and CD4⁺ T cells, we investigated whether B and T cell maturation could be modulated by dietary Se and by tumorigenesis in nude mice. Fifteen homozygous nude mice were fed a Se-deficient, Torula yeast basal diet alone (Se−) or supplemented with 0.15 (Se+) or 1.0 (Se++) mg Se/kg (as Na₂SeO₄) for 6 months, followed by a 7-week time course of PC-3 prostate cancer cell xenograft (2 × 10⁶ cells/site, 2 sites/mouse). Here, we show that peripheral B cell levels decreased in nude mice fed the Se −  or Se++ diet and the CD4⁺ T cell levels increased in mice fed the Se++ diet. During the PC-3 cell tumorigenesis, dietary Se status did not affect peripheral CD4⁺ or CD8⁺ T cells in nude mice whereas mice fed with the Se++ diet appeared to exhibit greater peripheral CD25⁺CD4⁺ T cells on day 9. Dietary Se status did not affect spleen weight in nude mice 7 weeks after the xenograft. Spleen weight was associated with frequency of peripheral CD4⁺, but not CD8⁺ T cells. Taken together, dietary Se at the nutritional and supranutritional levels regulates peripheral B and T cells in adult nude mice before and after xenograft with PC-3 prostate cancer cells.