CD45 fraction bone marrow cells as potential delivery vehicles for genetically corrected dystrophin loci.

Targeted correction of mutations in muscle can be delivered by direct i.m. injection of corrective DNA to the dystrophic muscle or by autologous injection of cells that have been genetically corrected after isolation from the individual with the dystrophic muscle. The successful application of chimeraplasty and short fragment homologous replacement to correct the exon 23 nonsense mdx transition at the mouse dys locus has opened up the possibility that with further development, targeted gene correction may have some future application for the treatment of muscular dystrophies. In vitro, application of targeted gene correction at the mdx dys locus results in better correction efficiencies than when applied directly to dystrophic muscle. This suggests that at least for the time being, a strategy involving ex vivo correction may be advantageous over a direct approach for delivery of gene correction to dystrophic muscle. This, particularly in view of recent developments indicating that bone-marrow-derived cells are able to systemically remodel dystrophic muscle, whilst penetration of DNA introduced to muscle is limited to individually injected muscles. Application of targeted gene correction to Duchenne dystrophy needs to account for the fact that about 65% of Duchenne muscular dystrophy cases involve large frame-shift deletion of gene sequence at the dys locus. Traditionally, whilst targeted gene correction is able to restore point mutations entirely, it remains to be seen as to whether a strategy for the 'correction' of frame shift deletions may be engineered successfully. This communication discusses the possibility of applying targeted gene correction to dystrophic muscle in Duchenne dystrophy.

Periwound dopaminergic sprouting is dependent on numbers of wound macrophages.

Injury to many regions of the central nervous system, including the striatum, results in a periwound or 'abortive' sprouting response. In order to directly evaluate whether macrophages play an important role in stimulating periwound sprouting, osteopetrotic (op/op) mice, which when young are deficient in a variety of macrophage subtypes, were given striatal wounds and the degree of dopaminergic sprouting subsequently assessed. Two weeks postinjury, significantly fewer wound macrophages were present in the striata of op/op mice compared with controls (144 +/- 30.1 in op/op mice vs. 416.6 +/- 82.3 in controls, P < 0.005, analysis performed on a section transecting the middle of the wound). Dopamine transporter immunohistochemistry revealed a marked decrease in the intensity of periwound sprouting in the op/op group of animals. Quantification of this effect using [H3]-mazindol autoradiography confirmed that periwound sprouting was reduced significantly in the op/op mice compared with controls (71.4 +/- 21.7 fmol/mg protein in op/op mice vs. 210.7 +/- 27.1 fmol/mg protein in controls, P < 0.0005). In the two groups of animals the magnitude of the sprouting response in individuals was closely correlated with the number of wound macrophages (R = 0.83, R2 = 0.69). Our findings provide strong support for the crucial involvement of macrophages in inducing dopaminergic sprouting after striatal injury.

Gene targeting of Desrt, a novel ARID class DNA-binding protein, causes growth retardation and abnormal development of reproductive organs.

We have cloned and characterized a novel murine DNA-binding protein Desrt, with a motif characteristic of the ARID (A-T rich interaction domain) family of transcription factors. The Desrt gene encodes an 83-kD protein that is shown to bind DNA and is widely expressed in adult tissues. To examine the in vivo function of Desrt, we have generated mice with a targeted mutation in the ARID domain of Desrt. Homozygous mutants have reduced viability, pronounced growth retardation, and a high incidence of abnormalities of the female and male reproductive organs including cryptorchidism. This may thus serve as a model to dissect the mechanisms involved in the development of the reproductive tract including testicular descent. Gene-targeted mice also display a reduction in the thickness of the zona reticularis of the adrenal gland and transient aberrations of the T and B cell compartments of primary lymphoid organs. These data show that this novel DNA-binding protein, Desrt, has a nonredundant function during growth and in the development of the reproductive system.

Defective stem cell factor expression in c-myb null fetal liver stroma.

High levels of c-Myb are observed in immature precursor myeloid and lymphoid cells, while downregulation of c-myb accompanies terminal differentiation to a mature phenotype. This has established c-Myb as a crucial transcription factor for hematopoiesis. Further evidence for this is the embryonic death of the c-myb homozygous mutant mouse at ED15 due to defective fetal liver erythropoiesis. Cells from fetal liver of wild-type and c-myb-/- embryos were examined in detail for their hematopoietic potential and the capacity of the stroma to support wild-type hematopoiesis. The c-myb-/- fetal liver was shown to harbor sevenfold fewer spleen focus-forming cells and a similarly lower number of cells with long-term repopulating capacity (high proliferative potential cells). However, shorter term repopulating cells were not substantially reduced. c-myb-/- stromal cells were unable to support the proliferation of wild-type bone marrow lineage-negative cells. This was found to be partly due to a decrease in stem cell factor (SCF) expression while partial rescue of the stromal cell cultures was achieved through the addition of exogenous SCF. DNA binding studies for two sites within the SCF promoter demonstrated an in vitro interaction between the SCF promoter and c-Myb and transient transfection studies demonstrated that c-Myb could substantially transactivate the SCF promoter in HEK293 cells. These data explain why the c-myb-/- embryos are so impaired in their ability to establish hematopoiesis.

c-Myb is critical for murine colon development.

The mammalian colon develops from a simple tube of undifferentiated cells into a complex, highly ordered organ, with a continuously self-renewing epithelial layer. We have previously described c-Myb expression in the epithelia of murine and human colon crypts and documented increased expression in colorectal adenocarcinoma cells. To investigate the role of c-Myb in colonic epithelium development, we have used embryos with a disrupted c-myb gene. Prior to the in utero death of these embryos at E15, we excised colon tissue and transplanted it under the kidney capsule of recipient mice to allow further development and cyto-differentiation. Compared to the colons of wildtype and heterozygous littermates, the c-myb homozygous knockout colon is highly irregular with a disordered epithelium and abnormal crypts. In addition, the expression of Bcl-2, a known target of c-Myb, is reduced and apoptosis is increased, indicating a critical requirement for c-Myb in normal colon development.

Cathepsin K knockout mice develop osteopetrosis due to a deficit in matrix degradation but not demineralization.

Cathepsin K is a cysteine protease expressed predominantly in osteoclasts. Activated cathepsin K cleaves key bone matrix proteins and is believed to play an important role in degrading the organic phase of bone during bone resorption. Mutations in the human cathepsin K gene have been demonstrated to be associated with a rare skeletal dysplasia, pycnodysostosis. The degree of functional activity of the mutated forms of cathepsin K in these individuals has not been elucidated, but is predicted to be low or absent. To study the role of cathepsin K in bone resorption, we have generated mice deficient in the cathepsin K gene. Histologic and radiographic analysis of the mice revealed osteopetrosis of the long bones and vertebrae, and abnormal joint morphology. X-ray microcomputerized tomography images allowed quantitation of the increase in bone volume, trabecular thickness, and trabecular number in both the primary spongiosa and the metaphysis of the proximal tibiae. Not all bones were similarly affected. Chondrocyte differentiation was normal. The mice also had abnormalities in hematopoietic compartments, particularly decreased bone marrow cellularity and splenomegaly. The heterozygous animals appeared normal. Close histologic examination of bone histology revealed fully differentiated osteoclasts apposed to small regions of demineralized bone. This strongly suggests that cathepsin K-deficient osteoclasts are capable of demineralizing the extracellular matrix but are unable to adequately remove the demineralized bone. This is entirely consistent with the proposed function of cathepsin K as a matrix-degrading proteinase in bone resorption.

Fluoro-Gold: An alternative viability stain for multicolor flow cytometric analysis.

BACKGROUND: The viability stains propidium iodide (PI) and 7-amino-actinomycin D (7-AAD) are excited at 488 nm, as are the commonly used antibody conjugates fluorescein isothiocyanate (FITC), phycoerythrin (PE), and cyanine 5 dye covalently coupled to R-phycoerythrin (RPE-Cy5). When excited by a single laser, spectral overlap in the emission of PI and 7-AAD with RPE-Cy5 precludes the use of these viability stains for three-color immunophenotyping, particularly when evaluating low levels of marker expression in viable target cells. The ultraviolet excitable dye hydroxystilbamidine methanesulfonate (Fluoro-Gold, or FG) binds to DNA at the A-T-rich regions of the minor groove in permeabilized or dead cells. We assessed the suitability of this dye as a viability stain. METHODS: The ability of FG to detect nonviable cells in fresh and cryopreserved human apheresed peripheral blood cells was compared with that of PI and 7-AAD. The stability of FG staining and the effects of dye and cell concentration on the discrimination of nonviable cells was determined by measuring changes in the median fluorescence of viable and nonviable cells. RESULTS: FG labeling at dye concentrations of 2-8 microM is stable for at least 3 h over a wide range of cell concentrations (4 x 10(5) to 4 x 10(7) cells/ml). Costaining studies and linear regression analysis show that cell viability as determined by FG is strongly correlated with estimates using PI (r = 0.9636) and 7-AAD (r = 0.9879). CONCLUSIONS: FG is a reliable, alternative viability stain that can be used in conjunction with fluorochromes including FITC, PE, and RPE-Cy5 for multicolor analysis using dual-laser instruments.

Lymphohematopoietic stem cell engraftment.

Traditional dogma has stated that space needs to be opened by cytoxic myeloablative therapy in order for marrow stem cells to engraft. Recent work in murine transplant models, however, indicates that engraftment is determined by the ratio of donor to host stem cells, i.e., stem cell competition. One hundred centigray whole body irradiation is stem cell toxic and nonmyelotoxic, thus allowing for higher donor chimerism in a murine syngeneic transplant setting. This nontoxic stem cell transplantation can be applied to allogeneic transplant with the addition of a tolerizing step; in this case presensitization with donor spleen cells and administration of CD40 ligand antibody to block costimulation. The stem cells that engraft in the nonmyeloablated are in G0, but are rapidly induced (by 12 hours) to enter the S phase after in vivo engraftment. Exposure of murine marrow to cytokines (IL-3, IL-6, IL-11 and steel factor) expands progenitor clones, induces stem cells into cell cycle, and causes a fluctuating engraftment phenotype tied to phase of cell cycle. These data indicate that the concepts of stem cell competition and fluctuation of stem cell phenotype with cell cycle transit should underlie any new stem cell engraftment strategy.

Ultrastructure of primitive hematopoietic stem cells isolated using probes of functional status.

The most primitive hematopoietic stem cells capable of longterm reconstitution of the entire hematopoietic system following transplantation are characterized by their ability to exclude both Rhodamine 123 and Hoechst 33342 dyes (Rh/Ho(dull)), and are an appropriate target population for the determination of stem cell ultrastructure. We have used a fluorescence-activated cell sorter to enrich to near purity these rare, highly quiescent cells. Analysis of the in vitro growth characteristics of Rh/Ho(dull) cells demonstrated an obligatory requirement for multiple growth factors, with 62% of the sorted population having the capacity to form colonies in the presence of CSF-1 + IL-1alpha + IL-3 + SCF. The Rh/Ho(dull) cells were small, with profiles having a mean diameter of 4.6 microm. Ultrastructural examination showed numerous ribosomes and several mitochondria in the thin rim of cytoplasm surrounding the nucleus, with other cytoplasmic organelles revealed in serial sections. The cells were generally homogeneous in appearance apart from the nucleus, which had an irregular shape with a single deep indentation. The heterochromatin around the margin was distinctly more pronounced in about 50% of nuclei. The findings provide a basis for studying the structural changes that occur with progressive differentiation of early hematopoietic cells.

Peripheral blood CD34+ cell count reliably predicts autograft yield.

A reliable measure to predict peripheral blood progenitor cell (PBPC) autograft CD34+ cell content is required to optimize the timing of PBPC collection. We prospectively examined the peripheral blood (PB) CD34+ cell count in 59 consecutive patients with various malignancies and analyzed the correlation between the PB CD34+ cell count and various parameters in the PBPC autograft. Two hundred and thirty-five collections were performed with a median of 4.0 collections per patient (range, 2-10). The median PB CD34+ cell count at the time of collection was 39 x 10(6)/1 (range, 0.0-285.6). The PBPC autograft parameters measured were the CD34+ cell, colony-forming unit granulocyte-macrophage (CFU-GM) and mononuclear cell (MNC) content. There was a strong linear correlation between PB CD34+ cells/l and autograft CD34+ cells/kg (r = 0.8477). The correlation with CFU-GM/kg (r = 0.5512) was weaker. There was no correlation between autograft CD34+ cells/kg and PB WBC (r= 0.0684), PB MNC (r = 0.1518) or PB platelet count (r = 0.2010). At our institution we aim to obtain a minimum of 0.5 x 10(6) CD34+ cells/kg with each day of collection. We demonstrate that such a collection can be reliably obtained if the PB CD34+ cell count exceeds 5.0 x 10(6)/l.

Macrophage lineage cells in inflammation: characterization by colony-stimulating factor-1 (CSF-1) receptor (c-Fms), ER-MP58, and ER-MP20 (Ly-6C) expression.

Macrophage populations resident in tissues and at sites of inflammation are heterogeneous and with local proliferation sometimes evident. Using the convenient murine peritoneal cavity as an inflammation model, the appearance of macrophage lineage cells was followed with time in both thioglycollate- and sodium periodate-induced exudates. The cells were characterized by their proliferative response in vitro in response to colony-stimulating factor-1 (CSF-1) (or macrophage colony-stimulating factor [M-CSF]), particularly by their ability to form colonies in agar, in combination with flow cytometry (surface marker expression and forward and side scatter characteristics). We propose that c-Fms (CSF-1 receptor), unlike other markers, is a uniformly expressed and specific marker suitable for the detection of macrophage-lineage cells in tissues, both in the steady state and after the initiation of an inflammatory reaction. It was shown that the bone marrow myeloid precursor markers, ER-MP58 and ER-MP20 (Ly-6C), but not ER-MP12 (PECAM-1), are expressed by a high proportion of macrophage-lineage cells in the inflamed peritoneum. The macrophage colony-forming cells (M-CFCs) in a 16-hour thioglycollate-induced exudate were phenotyped as c-Fms+ERMP12-20+58+, properties consistent with their being more mature than bone marrow M-CFCs. It is proposed that ER-MP58, as well as ER-MP20, may be a useful marker for distinguishing inflammatory macrophage-lineage cells from the majority of those residing normally in tissues.

Tissue hyperplasia and enhanced T-cell signalling via ZAP-70 in c-Cbl-deficient mice.

The c-Cbl protein is tyrosine phosphorylated and forms complexes with a wide range of signalling partners in response to various growth factors. How c-Cbl interacts with proteins, such as Grb2, phosphatidylinositol 3-kinase, and phosphorylated receptors, is well understood, but its role in these complexes is unclear. Recently, the Caenorhabditis elegans Cbl homolog, Sli-1, was shown to act as a negative regulator of epidermal growth factor receptor signalling. This finding forced a reassessment of the role of Cbl proteins and highlighted the desirability of testing genetically whether c-Cbl acts as a negative regulator of mammalian signalling. Here we investigate the role of c-Cbl in development and homeostasis in mice by targeted disruption of the c-Cbl locus. c-Cbl-deficient mice were viable, fertile, and outwardly normal in appearance. Bone development and remodelling also appeared normal in c-Cbl mutants, despite a previously reported requirement for c-Cbl in osteoclast function. However, consistent with a high level of expression of c-Cbl in the hemopoietic compartment, c-Cbl-deficient mice displayed marked changes in their hemopoietic profiles, including altered T-cell receptor expression, lymphoid hyperplasia, and primary splenic extramedullary hemopoiesis. The mammary fat pads of mutant female mice also showed increased ductal density and branching compared to those of their wild-type littermates, indicating an unanticipated role for c-Cbl in regulating mammary growth. Collectively, the hyperplastic histological changes seen in c-Cbl mutant mice are indicative of a normal role for c-Cbl in negatively regulating signalling events that control cell growth. Consistent with this view, we observed greatly increased intracellular protein tyrosine phosphorylation in thymocytes following CD3epsilon cross-linking. In particular, phosphorylation of ZAP-70 kinase in thymocytes was uncoupled from a requirement for CD4-mediated Lck activation. This study provides the first biochemical characterization of any organism that is deficient in a member of this unique protein family. Our findings demonstrate critical roles for c-Cbl in hemopoiesis and in controlling cellular proliferation and signalling by the Syk/ZAP-70 family of protein kinases.

Quiescence, cycling, and turnover in the primitive hematopoietic stem cell compartment.

Continuous oral bromodeoxyuridine (BrdU) administration was used for the non-invasive measurement of the in vivo cell cycling characteristics of hematopoietic stem and progenitor cell populations of increasing maturity, isolated on the basis of their relative levels of Rhodamine 123 (Rh) and Hoechst 33342 (Ho) fluorescence. The results showed that whereas primitive hematopoietic stem cells (PHSCs) are hierarchically ordered on the basis of quiescence, the most primitive of these, characterized by their Rh/Ho(dull) phenotype and their capacity for long-term hematopoietic reconstitution, are not dormant, but cycle slowly in normal steady-state bone marrow (BM). Cell cycle analysis showed that 30 +/- 7% of Rh/Ho(dull) PHSCs had cycled and incorporated BrdU following continuous administration over 1 week, whereas 60 +/- 14% and 89 +/- 3% of these cells were BrdU positive at 4 and 12 weeks, respectively. Linear regression analysis of these data showed that Rh/Ho(dull) PHSCs cycle with an average turnover time of 4.3 weeks (30 days), and a t1/2 (time to 50% cycled) of 2.75 weeks (19 days).

Expression of mdr1 and mrp in the normal B-cell homologue of B-cell chronic lymphocytic leukaemia.

B-cell chronic lymphocytic leukaemia (CLL) cells commonly express the multidrug resistance phenotype. The aim of this study was to establish whether the normal homologue in B-cell ontogeny of B-CLL also expressed the multidrug resistance (mdr) phenotype. Human tonsillar lymphocytes were sorted to yield two B-cell subsets based on the expression of CD19, CD5 and CD10. The normal homologue was represented by a population of B cells that was CD19 positive, CD10 negative and weakly expressed CD5. Based upon functional analysis and the detection of mdr1 mRNA by semi-quantitative PCR, these cells expressed the mdr phenotype. In contrast, functional multidrug resistance could not be demonstrated in CD19-positive CD10-positive cells with strong expression of CD5, nor could mdr1 mRNA be found in these cells. MRP was variably expressed in both B-cell subsets with no discernable differences in the pattern of expression. We conclude that normal B cells with a phenotype resembling that of B-CLL cells express the multidrug resistance phenotype.

Mutation in Sos1 dominantly enhances a weak allele of the EGFR, demonstrating a requirement for Sos1 in EGFR signaling and development.

We have investigated the role of the mammalian Son of sevenless 1 (Sos1) protein in growth factor signaling in vivo by generating mice and cell lines that lacked the Sos1 protein. Homozygous null embryos were smaller than normal, died mid-gestation with cardiovascular and yolk sac defects, and their fibroblasts showed reduced mitogen-activated protein kinase activation in response to epidermal growth factor (EGF). An intercross of mice mutant for Sos1 and the EGF receptor (EGFR) demonstrated that a heterozygous mutation in Sos1 dominantly enhanced the phenotype of a weak allele of the EGFR allele (wa-2). These animals had distinctive eye defects that closely resembled those seen in mice that were null for the EGFR or its ligand, TGF alpha. Our findings provide the first demonstration of a functional requirement for Sos1 in growth factor signaling in vivo. They also show that the genetic test of enhancement of weak receptor allele by heterozygous mutation in one component represents a powerful tool for analyzing the ras pathway in mammals.

A null mutation in the gene encoding a type I interferon receptor component eliminates antiproliferative and antiviral responses to interferons alpha and beta and alters macrophage responses.

To examine the in vivo role(s) of type I interferons (IFNs) and to determine the role of a component of the type I IFN receptor (IFNAR1) in mediating responses to these IFNs, we generated mice with a null mutation (-/-) in the IFNAR1 gene. Despite compelling evidence for modulation of cell proliferation and differentiation by type I IFNs, there were no gross signs of abnormal fetal development or morphological changes in adult IFNAR1-/- mice. However, abnormalities of hemopoietic cells were detected in IFNAR1 -/- mice. Elevated levels of myeloid lineage cells were detected in peripheral blood and bone marrow by staining with Mac-1 and Gr-1 antibodies. Furthermore, bone marrow macrophages from IFNAR1 -/- mice showed abnormal responses to colony-stimulating factor 1 and lipopolysaccharide. IFNAR1 -/- mice were highly susceptible to viral infection: viral titers were undetected 24 hr after infection of IFNAR1 +/+ mice but were extremely high in organs of IFNAR1 -/- mice, demonstrating that the type I IFN system is a major acute antiviral defence. In cell lines derived from IFNAR1 -/- mice, there was no signaling in response to IFN-alpha or -beta as measured by induction of 2'-5' oligoadenylate synthetase, antiviral, or antiproliferative responses. Importantly, these studies demonstrate that type I IFNs function in the development and responses of myeloid lineage cells, particularly macrophages, and that the IFNAR1 receptor component is essential for antiproliferative and antiviral responses to IFN-alpha and -beta.

Granulocyte-macrophage colony-stimulating factor is not responsible for the correction of hematopoietic deficiencies in the maturing op/op mouse.

Osteopetrotic (op/op) mice are characterized by an autosomal recessive inactivating mutation resulting in the absence of biologically active colony-stimulating factor-1 (CSF-1). Consequently, young op/op mice have a severe deficiency of macrophages and osteoclasts resulting in excessive bone formation, occlusion of the marrow cavity, and reduced marrow hematopoietic activity. Recently, we showed that the osteopetrosis and hematopoietic deficiencies evident in young op/op mice are not permanent but are progressively corrected with age. There are increases in osteoclast activity; bone resorption; femoral marrow space; and marrow hematopoietic activity, cellularity, and macrophage content. In the present study we show that CSF-1-/- granulocyte-macrophage colony-stimulating factor (GM-CSF)(-/-)-deficient mice also undergo the same pattern of hematopoietic correction as the op/op mouse. Also, like the op/op mouse, the peritoneal cellularity and macrophage content of CSF-1/GM-CSF-deficient mice remains severely reduced. Our data show that the "knockout" of GM-CSF does not change the op/op phenotype, and that GM-CSF is not essential for the correction of the hematopoietic deficiencies in the op/op mouse. Importantly, the data also show that neither GM-CSF nor CSF-1 is an absolute requirement for the commitment of primitive hematopoietic stem cells to the macrophage lineage or for the differentiation of at least some classes of macrophages. This finding suggests that an alternate regulatory factor can be involved in macrophage and osteoclast commitment, differentiation, and function in vivo.