Highly efficient targeted transduction of undifferentiated human hematopoietic cells by adenoviral vectors displaying fiber knobs of subgroup B.

Human hematopoietic stem cells (HSCs) are poorly transduced by vectors based on adenovirus serotype 5 (Ad5). This is primarily due to the paucity of the coxsackievirus-Ad receptor on these cells. In an attempt to change the tropism of Ad5, we constructed a series of chimeric E1-deleted Ad5 vectors in which the shaft and knob of the capsid fibers were exchanged with those of other Ad serotypes. In all these vectors, the Ad E1 region was replaced by an expression cassette containing the cytomegalovirus immediate-early promoter and the gene for enhanced green fluorescent protein (GFP). Experiments performed in vitro showed an efficient transduction of umbilical cord blood (UCB) monocytes, granulocytes, and their precursors as well as the undifferentiated CD34(+) CD33(-) CD38(-) CD71(-) cells by Ad5 vectors carrying Ad subgroup B-specific fiber chimeras (Ad5FBs). In the latter subpopulation, which comprises less than 1% of the CD34(+) cells and is highly enriched with cells repopulating immunodeficient mice, more than 90% of the cells were GFP(+). Transduction by Ad5FBs of the less primitive fraction within UCB CD34(+) cells was significant lower. Actually, the transduction frequency and GFP level declined gradually with increased expression of the CD33, CD38, and CD71 antigens. Flow cytometric analysis of transduced UCB CD34(+) cells that were cultured for 5 days on an allogeneic human bone marrow stroma layer showed maintenance of the phenotypically defined HSCs at levels similar to those of control cultures. The latter finding indicates that neither the transduction procedure nor the high levels of GFP were toxic for these cells.

Intrinsic potential of phenotypically defined human hemopoietic stem cells to self-renew in short-term in vitro cultures.

In search for culture conditions that will facilitate hemopoietic stem cell (HSC) replication while preserving their primitive properties, we have made use of a multi-parameter FACS assay to define HSCs on basis of their phenotypic characteristics, i.e., CD34++CD33,38,71(-). Bone marrow and umbilical cord blood samples of CD34(+) cells from 31 donors were loaded with the membrane dye PKH26 and each exposed to various culture conditions for 6 days. The cells that retained the primitive CD34(++)CD33,38,71(-) phenotype were analysed for the number of cell replications they underwent, by measuring loss of PKH26 fluorescence after 6 days. A most striking observation was the large inter-sample variation in the proliferative response of cells that retained the CD34(++)CD33,38,71(-) phenotype. In general, samples could be characterised as either good- or poorly-replicating, according to the proliferation property of their CD34(++)CD33,38,71(-) subset. In comparison to this 'intrinsic' potential, the effects of the applied growth stimuli on CD34(++)CD33,38,71(-) cell replication were negligible. In contrast, the overall recovery of the CD34(++)CD33,38,71(-) cells was clearly dependent on the culture stimuli. Of the various conditions tested, serum-free cultures with pre-established stroma maintained the cells with this primitive phenotype most effectively. In cultures supplemented with various combinations of recombinant HGFs, HSC differentiation prevailed. These findings with phenotypically defined HSCs should assist in the design of systems for expansion and ex vivo gene therapy of early hemopoietic cells.

Frequency analysis of multidrug resistance-1 gene transfer into human primitive hematopoietic progenitor cells using the cobblestone area-forming cell assay and detection of vector-mediated P-glycoprotein expression by rhodamine-123.

Transfer of the multidrug resistance-1 (MDR1) gene into hematopoietic progenitor cells may reduce myelotoxicity of MDR1-related cytotoxic agents and therefore allow dose intensification. Mobilized peripheral blood progenitor cells (PBPC) can be obtained in ample quantity and are a suitable target cell population. CD34-selected PBPC samples (n = 6) were transduced with cell-free supernatant (SNT) of a cell line producing recombinant retrovirus containing the human MDR1 gene. Limiting-dilution long-term cultures were employed that allow continuous monitoring of stroma-adherent cobblestone areas (CA) and comparison of their frequency in a 5-log range over time. MDR1 provirus integration in CA-containing wells followed single-hit kinetics. According to Poisson statistics, proviral DNA was contained in 22% of unselected cobblestone area-forming cells (CAFC) at week 6, which represent primitive hematopoietic precursors. In comparison, 1.0 +/- 0.44% (mean +/- SEM) of week-6 CAFC were expressing P-glycoprotein at sufficient levels to convey vincristine resistance, suggesting low expression of the retroviral vector or splicing of the vector-drived mRNA in hematopoietic progenitor cells. Next we analyzed lineage-committed progenitors. The proviral DNA was detectable in 20-66% of colony-forming units granulocyte-macrophage (CFU-GM) while corresponding percentages (25-52%) of CD34+ PBPC were in the S/G2M phase of the cell cycle at the end of the transduction period. The proportion of vincristine-resistant CFU-GM was similar to the CAFC data and no significant differences were found between various MDR1-SNT transduction schedules whereas MDR1 co-cultivation, which served as a positive control, yielded significantly higher proportions of resistant colonies (5.3 +/- 1.4%, IL-3, 96 hr, p < or = 0.05). Assessment of rhodamine-123 (Rh-123) efflux in the myelo-monocytic progeny of MDR1-transduced cells mirrored the colony assay results in the SNT and co-cultivation groups. Less culture effort was required in the Rh-123 assay and functional characterization of the transferred P-glycoprotein was possible using cyclosporin A. Further development toward an effective MDR1 gene therapy should be facilitated by the CAFC assay, which allows estimation of the retroviral gene transfer frequency into primitive hematopoietic cells, and by the Rh-123 assay, which permits tractable side-by-side assessments of numerous MDR1 transduction protocols or different MDR1-SNT lots.

Cell cycle state, response to hemopoietic growth factors and retroviral vector-mediated transduction of human hemopoietic stem cells.

A multi-parameter fluorometric analysis was applied to study in vitro proliferation and expansion of a candidate hemopoietic stem cell population, ie CD34brightLin cells. In primary hemopoietic cell samples the CD34brightLin population comprised on average, 1% of CD34+ cells from bone marrow (BM) or umbilical cord blood and 0.1% of mobilized peripheral blood CD34+ cells. The fraction of CD34brightLin cells engaged in the S+G2/M phases of the cell cycle was largest in regenerating BM (10.6% versus 1-2% in the other sources). Maintenance of CD34+ BM cells in hemopoietic growth factor supplemented liquid cultures resulted in a decrease of the CD34brightLin population in most samples. Cell cycle analysis showed the constant existence of proliferating CD34+ cells during the culture period. The fraction of cycling CD34brightLin cells was, however, very small and only occasionally exceeded input values. At all tested time-points during culture, BM CD34+ cells could be transduced by a single incubation with a recombinant retrovirus supernatant. CD34brightLin cells, however, were much more refractory to retroviral vector-mediated transduction. This could be explained only in part by quiescence of CD34brightLin cells. Hence, factors other than cell proliferation clearly influence the early stages of retroviral transduction of human hemopoietic stem cells.

Occurrence of a soluble nonspecific suppressor factor in the serum early after birth.

A nonspecific suppressor factor has been identified in serum of newborn rats and calves. This factor, designated SUF-s, was shown to interfere--across species barriers--with lymphocyte responses in vitro and in vivo. SUF-s interferes in vitro with T- and B-cell proliferation induced by different mitogens and IL-2. Our findings indicate that the activity of SUF-s in vitro, which is of a reversible nature, is directed at an early event in the cascade of T-cell activation. SUF-s does not affect intrinsically regulated proliferation, such as that of tumor cells or established cell lines. In vivo, SUF-s prevents graft-vs-host disease induced by transplantation of allogeneic bone marrow cells in lethally irradiated mice. Using of affinity chromatography, hydrophobic interaction chromatography, and gel filtration, a 15,000-fold purification of the suppressive factor was attained. The moiety engaged in suppression was identified as a 20- to 40-kDa protein. The biological activity is destroyed at temperatures above 70 degrees C, by proteolytic enzyme digestion and under alkaline conditions but was resistant to acidic and reducing conditions. Judged by its biological activity and some of its physical properties, SUF-s is most likely distinct from other described suppressor factors or known cytokines with suppressor activity, such as IL-4, IL-10, interferon-gamma, transforming growth factor-beta or alpha-fetoprotein.

Dietary fish oil supplementation alters LTB4:LTB5 ratios but does not affect the expression of acute graft versus host disease in mice.

One of the mechanisms by which corticosteroids may modify acute graft vs host disease (GvHD) is via inhibition of arachidonic acid (AA) metabolism. Leukotriene B4 (LTB4) is a product of that pathway which may take part in the pathogenesis of GvHD through the stimulation of T-lymphopoiesis and T-lymphocyte activation. LTB4 is a metabolite of AA (20:4n-6). Alternate dietary sources of polyunsaturated fatty acids (PUFA), specifically eicosapenteinoic acid (20:5n-3) (EPA) and docosahexaenoic acid (22:6n-3) (DHA), shift the LTs formed with a decrease in LTB4 an increase in LTB5. LTB5 is a less potent agonist than LTB4 and this results in a theoretical decrease of LTB4 mediated events. Supplementation of in vitro bone marrow cultures with EPA or DHA had no detrimental effect on myeloid colony formation. Dietary EPA/DHA supplementation in mice with induced GvHD appeared to be safe and well tolerated. The LTB4:LTB5 ratio shifted from 7.65 +/- 1.75 in control-fed animals to 1.03 +/- 0.18. Fish-oil-supplementation did not compromise engraftment or stem cell content. Alone, this therapy was unable to modify GvHD.

Remission induction of adjuvant arthritis in rats by total body irradiation and autologous bone marrow transplantation.

Following the demonstration that adjuvant arthritis in rats can be cured with total body irradiation (TBI) and allogeneic or syngeneic bone marrow, the efficacy of autologous bone marrow was investigated in the experiments reported here. Bone marrow from arthritic rats, harvested at the same time that the recipients were irradiated, and real autologous bone marrow were found to be similarly effective as bone marrow grafts from naive syngeneic donors. Sublethal TBI with lower doses was less effective, but the highest tolerated doses of 8 Gy approached the effect of 9 Gy and bone marrow rescue. In contrast, partial body irradiation of either the affected limbs, or of the whole body except the limbs, resulted in only partial and temporary regression of the arthritis.

Antiarthritic activity of the newly developed neutrophil oxidative burst antagonist apocynin.

The plant-phenol 4-hydroxy-3-methoxyacetophenone (trivial name apocynin) is a strong inhibitor of neutrophil superoxide anion (O2-) release in vitro. In vitro the inhibitory effect of apocynin is restricted to cells with the capacity to release peroxidase and reactive oxygen species (ROS). Peroxidase deficient cells are insensitive to apocynin. In the present study the antiinflammatory activity of apocynin was tested in collagen-induced arthritis in rats. Collagen-immunized rats were treated with different doses of apocynin in the drinking water starting at the onset of joint-swelling and terminating 14 days later, at the time when joint swelling in the control group was maximal. Apocynin-treated animals had a normal plasma level of collagen-specific antibodies, but showed a significant reduction of the joint swelling. Also the plasma IL-6 level in apocynin-treated animals was substantially lower than in control animals. No flare-up of joint swelling after termination of the treatment was observed in the apocynin-treated groups.

Regression of adjuvant-induced arthritis in rats following bone marrow transplantation.

Total body irradiation followed by bone marrow transplantation was found to be an effective treatment for adjuvant arthritis induced in rats. This treatment is most effective when applied shortly after the clinical manifestation of arthritis--i.e., 4-7 weeks after administration of Mycobacterium tuberculosis. Transplantation of bone marrow at a later stage results in a limited recovery, in that the inflammatory reaction regresses but the newly formed excessive bone is not eliminated. Local irradiation of the affected joints had no effect on the disease. It could also be excluded that the recovery of arthritis following marrow transplantation is due to lack of available antigen. Transplantation of syngeneic bone marrow is as effective as that of allogeneic bone marrow from a rat strain that is not susceptible to induction of adjuvant arthritis. The beneficial effect of this treatment cannot be ascribed to the immunosuppressive effect of total body irradiation, since treatment with the highly immunosuppressive drug Cyclosporin A resulted in a regression of the joint swelling but relapse occurred shortly after discontinuation of the treatment.

Soluble factors secreted by naturally occurring suppressor cells that interfere with in vivo graft-vs.-host disease and with T cell responsiveness in vitro.

A potent immunosuppressive factor (SUF) is found in the supernatant of short-term cultures of unstimulated thymocytes or spleen cells of neonatal mice and rats and in culture medium of hybridoma cell lines established by fusing neonatal mouse spleen cells with T lymphoma cells (the BW 5147 line). In vitro incubation of spleen cells with SUF suppresses the acute in vivo graft-vs.-host disease, normally induced by allogeneic spleen cells in lethally irradiated mice. Incubation of bone marrow cells with SUF does not affect the hemopoietic stem cells. The addition of SUF to mixed lymphocyte reaction cultures strongly suppresses lymphocyte proliferation. The non-species-restricted inhibition of cell proliferation induced by SUF is shown not to be due to toxicity or nonspecific interference with DNA synthesis. Molecular size fractionation of crude SUF revealed two active moieties: a large moiety of molecular mass greater than 100 kDa and a small moiety of less than 3 kDa. The high kDa moiety mediates T cell unresponsiveness both in vivo and in vitro. In vitro studies revealed that this moiety primarily affects an early event in the proliferative response to alloantigen and mitogen, that prevents interleukin 2 (IL 2) receptor expression and, consequently, blastogenesis and DNA duplication. It does not affect, however, the synthesis of IL 2. The suppressive activity of the low kDa moiety can be demonstrated only in in vitro systems. Pre-treatment of donor lymphocytes with this fraction cannot prevent graft-vs.-host disease mortality. The inhibition of cell proliferation induced by this fraction in vitro is most likely due to interference with the utilization of IL 2, as suggested by its suppressive effect on the proliferation of CTLL-2 cells (an IL 2-dependent cell line).

Prevention of GvHD by a lymphokine.

T lymphocytes which are capable of preventing GvHD when mixed with GvHD inducing cells before grafting and of inhibiting MLR, have been demonstrated in spleen and thymus of neonatal mice and rats. The supernatant of short term cultures of neonatal thymus and spleen contains a factor termed SUF, that produces the same suppressions. SUF has been produced by a number of hybridoma cell lines, that were obtained by fusion of a T lymphoma cell line with neonatal spleen cells. SUF does not reduce CFU-S upon incubation with mouse bone marrow. Suppression by SUF in MLR is not mediated via a cytotoxic mechanism, neither does SUF inhibit proliferation of normal and malignant lymphocytes. Preliminary data suggest that SUF interferes with the action of IL-2 on T effector lymphocytes.

Idiopathic paraproteinaemia. IV. The role of genetic factors in the development of monoclonal B cell proliferative disorders--a study in the ageing C57BL/KaLwRij and CBA/BrARij mouse radiation chimeras.

Mouse radiation chimeras, employing strains with a low (CBA/BrARij) and a high (C57BL/KaLwRij) frequency of idiopathic paraproteinaemia (IP), were used in a study on genetic influences in the development of IP, a benign B cell monoclonal proliferative disorder. Taking advantage of the different Igh1 allotypic markers between the two strains, the development of IP with increasing age was investigated by agar electrophoresis, immunoelectrophoresis and immunofixation. Four of 18 CBA recipients transplanted with C57BL bone marrow cells were shown to develop IP of the IgG2a isotype and the Igh1b (donor) allotype during their life. In contrast, none of the 23 C57BL recipients of CBA bone marrow developed an IgG2a paraprotein of the Igh1a allotype. However, in three of these 23 chimeras, an IgG2a and Igh1b (recipient) allotype paraprotein appeared with age; two of these mice proved to be reversals at 12 months and one at 15 months of age. The frequencies of homogeneous immunoglobulins of the donor type in the chimeras corresponded roughly to those of normal mice of the donor strain. Histopathological examination excluded a malignant origin of these monoclonal proliferations. These findings support the view that intrinsic cellular genetic factors are of major importance in the development of IP, a benign B cell neoplasia.

Characterization of a subpopulation in neonatal thymus which suppress the graft-vs.-host reaction.

Thymus cells from neonatal and infant mice were found to have a high capacity to prevent mortality from acute graft-vs.-host disease as compared with spleen cells from stable radiation chimeras. This suppressive capacity of thymocytes decreases with age after birth as was demonstrated by semi-quantitative cell titrations. This suppressor activity is restricted to syngeneity of the graft-vs.-host disease-including cells. The thymic suppressor cells are Thy-1+ and Lyt-1+ and IgG- and IgM-. They do not agglutinate with peanut agglutinin and have a high electrophoretic mobility. In vitro irradiation experiments showed that the suppressor cells are radiation sensitive. These results are compared with the available information on cells suppressing delayed-type hypersensitivity reactions and those suppressing B cell responses.