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Platelet-derived growth factor (PDGF) has been implicated in the processes regulating gliogenesis in the CNS. Conflicting in vivo data in rodents have variously implicated either glia or neurons as being the primary source of PDGF. We have used in situ hybridization and immunocytochemical analysis to study the in vivo expression and cellular localization of PDGF-A, sis/PDGF-B, together with the two PDGF receptors alpha and beta, in developing human forebrain. In this study we demonstrate the strong expression of mRNA and protein of both PDGF chains, A and B, and their receptors, alpha and beta, in human embryonic glial cells. The neurons, in contrast to glial cells, expressed lower levels of PDGF and PDGF-receptor mRNAs and protein. Identification of the cell types expressing the PDGF and PDGF-receptor mRNAs was achieved by counterstaining with antibodies specific for glial cells (GFAP) and neurons (NF). The predominant glial-specific expression of both PDGF-A and PDGF-B, together with the coexpression of their receptors alpha and beta, suggests an important role for the PDGF isoforms in the development of human embryonic glial cells and neurons in vivo.

Canine bone marrow stromal cells (BMSCs), transduced ex vivo with retroviral vectors, expressed and secreted biologically active human and canine coagulation factor IX (hFIX and cFIX) in vitro, and on autologous reinfusion expressed hFIX into the circulation of normal (nonhemophiliac) dogs. Human FIX, when expressed in vitro by BMSCs of two dogs at 1.22 and 1.39 microg/10(6) cells/24 hr in medium supplemented with vitamin K, respectively, exhibited 28.1 and 27.3% normal biological activity as determined on the basis of a one-stage clotting assay. BMSCs of two additional dogs expressed 1.54 and 4.81 microg of cFIX/10(6) cells/24 hr in vitamin K-supplemented medium and the expressed cFIX possessed 58.4 and 32.9% normal activity, respectively. Between 2.33 and 3.35 x 10(8) transduced BMSCs, expressing 1.22 and 2.61 microg of hFIX/10(6) cells/24 hr or 3.24 and 7.82 microg of cFIX/10(6) cells/24 hr were reintroduced into the four donor dogs by intravenous infusion. Human FIX was detected in plasma for 7 or 12 days after BMSC reinfusion, with peak levels of 85.8 and 233.0 ng/ml observed at 2 days. Canine anti-hFIX antibodies, which were detected as early as 2-4 days after reinfusion of BMSCs expressing hFIX, may have masked potentially longer duration expression in vivo. Peak plasma levels of hFIX represented 2.1 and 5.8% normal human hFIX levels. When adjusted for percent normal one-stage clotting activity determined in vitro, these levels represented 0.6 and 1.6% normal human hFIX activity levels. Thus, we have demonstrated that retroviral vector-modified BMSCs can deliver human therapeutic levels of hFIX to the circulation of dogs.

Meningiomas are primarily benign brain tumors thought to arise through multistep tumorigenesis, involving both the activation of oncogenes and the loss of tumor suppressor genes. The recently isolated neurofibromatosis 2 (NF2) tumor suppressor gene has been found to be mutated in a large proportion of meningiomas. Almost all cases of familial meningioma occur in association with NF2. Familial meningioma in isolation from NF2 (sporadic) is exceedingly rare, with only 14 reports since 1959. The authors report the existence of a family lacking any stigmata of NF2, in which two members had spinal meningiomas. Tumor specimens were subjected to immunocytochemical analysis for the NF2 protein product Merlin, which has been implicated in the tumorigenesis of meningioma. Merlin immunoreactivity was present in both tumor specimens, implying that the NF2 tumor suppressor gene was not deleted in these tumors. This supports the hypothesis that a second tumor suppressor gene locus, other than NF2, acts in the formation of familial sporadic meningioma. The results are discussed in the context of putative oncogenic mechanisms of familial meningiomas.

Canine bone marrow stromal cells were expanded to numbers in excess of 10(9) cells from the initial 10-20 ml of marrow aspirates and transfected to express high levels of human growth hormone (hGH) in vitro. Ex vivo-modified marrow stromal cells were used in a gene therapy model system for the systemic delivery of transgene products in dogs. Adherent bone marrow stromal cell cultures, established and expanded from iliac crest marrow aspirates from each of 8 dogs, were transfected with a hGH gene plasmid expression vector and shown to express from 0.54-3.84 micrograms/10(6) cells per 24 hr hGH in vitro. The transfected plasmid vector does not possess a eukaryotic origin of replication nor does it possess sequences required for efficient integration into the host cell genome. As such, expression was expected to be transient. Transfected cells were autologously reintroduced into each dog by either infusion into a foreleg vein or directly into iliac crest marrow. In two cases, the stromal cells were cryopreserved following transfection, and subsequently thawed and infused. In one case, the expanded stromal cells were first cryopreserved, and then thawed, recultured, transfected, and infused. Reintroduced cell numbers ranged from 2.2 x 10(7) to 2.6 x 10(9), with total hGH expression capacities ranging from 62 to 1,400 micrograms/24 hr. Plasma of each of the dogs contained detectable hGH for a mean of 3.1 days (SD +/- 0.8 day) ranging from 2 to 5 days following reinfusion of cells. Peak plasma levels ranged from 0.10 to 1.76 ng/ml. Similar hGH expression values, based upon total expression capacity of the cells infused and dog body weight, were obtained for all dogs. Vector-modified stromal cells were detectable, by polymerase chain reaction (PCR) analysis, in the peripheral circulation following reinfusion in all 4 dogs analyzed. In 3 of the dogs, modified stromal cells were detected for 8.5-15 weeks. In addition, modified stromal cells were detected in iliac crest marrow of 2 dogs for 9 and 13 weeks, respectively, following reinfusion. In another experiment, cultured bone marrow stromal cells were transfected with a human factor IX (hFIX) plasmid vector. Modified cells (5.57 x 10(8)), with a total hFIX expression capacity of 281 micrograms/24 hr, were reinfused, resulting in detectable hFIX in plasma continuously for 9 days with a peak level of 8 ng/ml on day 1. These results demonstrate that the ex vivo bone marrow stromal cell system is a potentially powerful method by which to deliver secreted transgene product to the systemic circulation of large animals.

We report that acute injury induces the expression of selective growth factor and growth factor receptors in the epithelial cells of the wounded tissue. In situ hybridization analysis of skin biopsy specimens obtained after cutaneous injury in swine demonstrated the induction of the expression of transforming growth factor-alpha, its receptor, epidermal growth factor-R, acidic fibroblast growth factor, and basic fibroblast growth factor messenger RNAs in the skin epithelial cells of the wounded tissue. There was no significant expression in the epithelial cells of control, uninjured tissues. The expression levels were maximal during the period of active tissue repair (1 to 5 days after injury) and were totally suppressed upon the healing of the wounded tissues. In contrast, insulinlike growth factor-I, (IGF-I), IGF-I receptor, and IGF-II receptor messenger RNAs were expressed in the epithelial cells of both the control, uninjured tissues and in tissue specimens obtained after injury. There was no significant expression of IGF-II messenger RNA in the epithelial cells before or after injury. It seems that injury induces the coordinated expression of selective growth factor and growth factor receptor genes whose products contribute to the regulation of the complex processes involved in tissue repair and remodeling.

Meningiomas are common brain tumors that show a predilection for females and become more aggressive during pregnancy and menses. The existence of gender-specific hormone receptors in meningiomas has long been a matter of controversy; the recent cloning of androgen, estrogen, and progesterone receptors has facilitated their direct evaluation. The authors have demonstrated the expression of androgen and progesterone receptor messenger ribonucleic acid and protein product in nine primary human meningiomas by Northern blot analysis. Cellular localization was achieved by in situ hybridization analysis. Estrogen receptor expression was not detected. Normal adult meninges were shown to express very low levels of both androgen and progesterone receptors.

Glioblastomas are malignant brain tumors that are attended by an immunosuppressed state. The authors have studied the expression of transforming growth factor-beta 2, which is known to have potent immunosuppressive and angiogenic properties. Transforming growth factor-beta 2 messenger ribonucleic acid and its protein product are both found to be greatly overexpressed in these tumors and are absent from normal brain tissue. The overexpression of this growth factor may contribute to the escape of neoplastic astrocytes from immune surveillance and, furthermore, to the immunosuppressed state that is characteristic of many of these patients.

These studies demonstrate the expression of IGF-1, IGF-11, and their respective receptor mRNAs in primary human astrocytomas and meningiomas. In situ hybridization and immunocytochemistry have localized a strong expression of both IGF-1 and IGF-11 mRNAs and of their protein products in the tumor cells of astrocytomas and meningiomas. The expression of IGF-1 and IGF-11 mRNAs in the tumor cells was accompanied by the co-expression of their respective type-1 and type-11 IGF receptor mRNAs. Control, non-malignant human brain expressed IGF-1 mRNA and IGF-1 and IGF-11 receptor mRNAs. There was no significant expression of IGF-11 MRNA in the control brain specimens. Control pachymeninges (dura mater) expressed low levels of IGF-1 mRNA and IGF-1 receptor mRNA. There was no significant expression of IGF-11 and IGF-11 receptor mRNAs in pachymeninges. The co-expression of IGFs and their receptors in brain tumors may contribute in their development and maintenance. The strong inappropriate expression of IGF-11 mRNA and its protein product in the tumor cells of astrocytomas and meningiomas, but not in normal brain specimens, may serve as molecular markers for the early detection of these tumors.

A common feature of human melanoma is infiltration by monocytes at early stages of tumorigenesis. This infiltration may be highly significant since macrophages have the capacity to alter the behavior of tumor cells. The authors previously demonstrated that the predominant monocyte chemoattractant produced by tumor cells in vitro was monocyte chemotactic protein-1 (MCP-1). The authors identify the expression of MCP-1 in pathologic specimens of both primary and metastatic human melanoma but not in normal skin. The finding that MCP-1 is produced by malignant melanoma suggests that specific genes are expressed in tumor cells that can induce the recruitment of monocytes in vivo.

Astrocytomas are highly malignant brain tumors and are among the most neovascularized solid tumors. We have investigated the expression of the angiogenic growth factors acidic fibroblast growth factor and transforming growth factor-alpha, together with its receptor epidermal growth factor receptor, in 30 primary astrocytomas. Both acidic fibroblast growth factor and transforming growth factor-alpha, together with epidermal growth factor receptor, are found to be greatly overexpressed in these tumors when compared with normal brain. This overexpression of angiogenic growth factors may underlie the intense neovascularization characteristic of astrocytomas.

The present studies investigated the expression of the platelet-derived growth factor (PDGF) and PDGF-receptor genes in human meningiomas. Northern blot analysis demonstrated that all meningiomas examined expressed both the c-sis/PDGF-2 proto-oncogene and the PDGF-receptor gene. In situ hybridization localized the c-sis mRNA and the PDGF-receptor mRNA in the tumor cells of the meningioma tissues. Control pachymeninges derived from adult individuals, without meningiomas, expressed only PDGF-receptor mRNA but not the c-sis mRNA. Immunocytochemistry studies detected both the c-sis and the PDGF-receptor protein products in meningioma tissues but only the PDGF-receptor protein products in control pachymeninges. These findings indicate the presence of an autocrine mechanism in human meningiomas based on the co-expression of the c-sis/PDGF-2 proto-oncogene and PDGF-receptor gene and their protein products. This co-expression of a potent mitogen and its receptor may contribute to the growth and maintenance of human meningiomas.

Several HTLV-I-infected T-cell lines express the two genes encoding platelet-derived growth factor (PDGF). Therefore, we examined the question of a possible self-stimulatory mechanism of proliferation involving PDGF in these cells. Using a nucleic acid probe and an antibody specific for the PDGF-B receptor (PDGFR-B), we examined four established human T-cell lines infected with HTLV-I, as well as several noninfected T-cell lines for expression of the PDGF-B receptor. Previous reports indicate that lymphocytes do not display receptors for PDGF; our results show that two noninfected T-cell lines (HUT-78, CCRF-HSB-2) expressed the canonical 5.5-kb PDGFR mRNA, and two HTLV-I-infected T-cell lines (C10/MJ, MT-2) expressed a novel PDGFR mRNA of 4.8 kb. Concomitantly, the cell lines expressing PDGFR mRNA also synthesize PDGFR proteins immunoprecipitated by the antibody to PDGFR-B. Differences were observed in the molecular weight of PDGFR molecules immunoprecipitated from uninfected and HTLV-infected T-cells. Immunofluorescence studies demonstrated that the PDGFR-B proteins are localized primarily on the cell external membrane. The results suggest that the HTLV-I-infected T-cells acquire an autostimulatory mechanism of cell proliferation that involves PDGF.