Heterotopic ossification (HO), or bone formation in soft tissues, is often the result of traumatic injury. Much evidence has linked the release of BMPs (bone morphogenetic proteins) upon injury to this process. HO was once thought to be a rare occurrence, but recent statistics from the military suggest that as many as 60% of traumatic injuries, resulting from bomb blasts, have associated HO. In this study, we attempt to define the role of peripheral nerves in this process. Since BMP2 has been shown previously to induce release of the neuroinflammatory molecules, substance P (SP) and calcitonin gene related peptide (CGRP), from peripheral, sensory neurons, we examined this process in vivo. SP and CGRP are rapidly expressed upon delivery of BMP2 and remain elevated throughout bone formation. In animals lacking functional sensory neurons (TRPV1−/−), BMP2-mediated increases in SP and CGRP were suppressed as compared to the normal animals, and HO was dramatically inhibited in these deficient mice, suggesting that neuroinflammation plays a functional role. Mast cells, known to be recruited by SP and CGRP, were elevated after BMP2 induction. These mast cells were localized to the nerve structures and underwent degranulation. When degranulation was inhibited using cromolyn, HO was again reduced significantly. Immunohistochemical analysis revealed nerves expressing the stem cell markers nanog and Klf4, as well as the osteoblast marker osterix, after BMP2 induction, in mice treated with cromolyn. The data collectively suggest that BMP2 can act directly on sensory neurons to induce neurogenic inflammation, resulting in nerve remodeling and the migration/release of osteogenic and other stem cells from the nerve. Further, blocking this process significantly reduces HO, suggesting that the stem cell population contributes to bone formation.

The factors contributing to heterotopic ossification, the formation of bone in abnormal soft-tissue locations, are beginning to emerge, but little is known about microenvironmental conditions promoting this often devastating disease. Using a murine model in which endochondral bone formation is triggered in muscle by bone morphogenetic protein 2 (BMP2), we studied changes near the site of injection of BMP2-expressing cells. As early as 24 hours later, brown adipocytes began accumulating in the lesional area. These cells stained positively for pimonidazole and therefore generated hypoxic stress within the target tissue, a prerequisite for the differentiation of stem cells to chondrocytes and subsequent heterotopic bone formation. We propose that aberrant expression of BMPs in soft tissue stimulates production of brown adipocytes, which drive the early steps of heterotopic endochondral ossification by lowering oxygen tension in adjacent tissue, creating the correct environment for chondrogenesis. Results in misty gray lean mutant mice not producing brown fat suggest that white adipocytes convert into fat-oxidizing cells when brown adipocytes are unavailable, providing a compensatory mechanism for generation of a hypoxic microenvironment. Manipulation of the transcriptional control of adipocyte fate in local softtissue environments may offer a means to prevent or treat development of bone in extraskeletal sites. (Am

The Epstein-Barr virus (EBV)-encoded LMP1 protein is expressed in EBV-positive Hodgkin disease and is a potential target for cytotoxic T-lymphocyte (CTL) therapy. However, the LMP1-specific CTL frequency is low, and so far the generation of LMP1-specific CTLs has required T-cell cloning. The toxicity of LMP1 has prevented the use of dendritic cells (DCs) for CTL stimulation, and we reasoned that an inactive, nontoxic LMP1 mutant (⌬LMP1) could be expressed in DCs and would enable the activation and expansion of polyclonal LMP1-specific CTLs. Recombinant adenoviral vectors expressing LMP1 or ⌬LMP1 were tested for their ability to transduce DCs. LMP1 expression was toxic within 48 hours whereas high levels of ⌬LMP1 expression were achieved with minimal toxicity. ⌬LMP1-expressing DCs were able to reactivate and expand LMP1-specific CTLs from 3 healthy EBV-seropositive donors. LMP1-specific T cells were detected by interferon-␥ (IFN-␥) enzyme-linked immunospot assay (ELISPOT) assays using the HLA-A2-restricted LMP1 peptide, YLQQN-WWTL (YLQ). YLQ-specific T cells were undetectable (less than 0.001%) in donor peripheral blood mononuclear cells (PBMCs); however, after stimulation the frequency increased to 0.5% to 3.8%. Lysis of autologous target cells by CTLs was dependent on the level of LMP1 expression. In contrast, the frequency of YLQ-specific CTLs in EBV-specific CTLs reactivated and expanded using lymphoblastoid cell lines was low and no LMP1-specific cytotoxic activity was observed. IntroductionImmunotherapy with cytotoxic T cells (CTLs) is increasingly used to treat malignancies and viral infections. 1,2 For example, polyclonal Epstein-Barr virus (EBV)-specific CTLs have been used for the prevention and treatment of posttransplantation EBV-associated lymphoma (PTLD). 3 EBV-specific CTLs persisted long-term, reconstituted immunity against EBV, and produced antiviral and antilymphoma effects. We have also used EBV-specific CTLs to treat 13 patients with EBV-positive Hodgkin disease and, while the results have been promising, no patient with bulky disease has been cured. 1,4 One explanation for this failure is that current methods of EBV-specific CTL generation produce CTL lines that are dominated by clones reactive to EBV proteins not expressed in the malignant Reed-Sternberg cells (H-RS cells) of EBV-positive Hodgkin disease. 5 Only a limited number of EBV-derived antigens (EBNA1, BARF0, LMP1, and LMP2) are present in H-RS cells, 6,7 and the immunodominant response of CTL lines is against EBNAs 3A, B, and C, which are not expressed by the tumor cells. Of the EBV proteins expressed in H-RS cells, only LMP2 and LMP1 are potential targets for CD8 ϩ T cells, because EBNA 1 is mainly presented on major histocompatibility complex (MHC) class II molecules 8 and the level of BARF0 expression might be too low for CTL recognition. 9 Our group and others have recently reported the generation of LMP2-specific CTLs 10-12 ; however, for future clinical protocols it is desirable to generate CTLs against more than one tumor-as...

Recombinant adenoviral vectors have potential for the treatment of a variety of musculoskeletal defects and such gene therapy systems have been a recent research focus in orthopedic surgery. In studies reported here, two different adenovirus vectors have been compared for their ability to transduce human bone marrow mesenchymal stem cells (hBM-MSCs) and elicit bone formation in vivo. Vectors consisted either of standard adenovirus type 5 (Ad5) vector or a chimeric adenovirus type 5 vector that contains an adenovirus type 35 fiber (Ad5F35), which has been recently demonstrated to bestow a different cellular tropism, and a complete cDNA encoding human bone morphogenetic 2 (BMP2). Studies were also conducted to compare the transduction efficiency of these vectors using enhanced green fluorescent protein (GFP). hBM-MSCs transduced with Ad5F35 vectors had higher levels of transgene expression than those transduced with Ad5 vectors. The results also demonstrate that hBM-MSCs lack the coxsackie-adenovirus receptor (CAR), which is responsible for cellular adsorption of Ad5. Therefore, the data suggest that Ad5 virus adsorption to hBM-MSCs is inefficient. Ad5BMP2- or Ad5F35BMP2-transduced hBM-MSCs were also compared in an in vivo heterotopic bone formation assay. Mineralized bone was radiologically identified only in muscle that received the Ad5F35BMP2 transduced hBM-MSCs. In summary, Ad5F35BMP2 can efficiently transduce hBM-MSCs leading to enhanced bone formation in vivo.

Osteoblasts are continually recruited from stem cell pools to maintain bone. Although their immediate precursor is a plasticadherent mesenchymal stem cell able to generate tissues other than bone, increasing evidence suggests the existence of a more primitive cell that can differentiate to both hematopoietic and mesenchymal cells. We show here that the ''side population'' (SP) of marrow stem cells, defined by their ability to rapidly expel a DNA-binding dye and to regenerate the hematopoietic compartment, can differentiate to osteoblasts through a mesenchymal intermediate. When transplanted into lethally irradiated mice, single gene-marked murine SP cells reconstituted depleted osteoprogenitor pools, such that a large proportion of the osteogenic cells in the epiphysis of long bone carried the donor SP cell marker. These findings suggest that the developmental capacity of SP cells is not restricted to the hematopoietic lineages but extends to osteogenic differentiation. This property not only elucidates a previously unrecognized step in osteoblast development, but also has intriguing implications for the use of SP cells in clinical orthopedics and stem cell-based disorders of bone. Skeletal bone is unique among human tissues. It is continuously remodeled throughout life in a process that requires the recruitment and proliferation of stem cells with the capacity to differentiate to functional osteoblasts, which then deposit and mineralize extracellular bone matrix (1, 2). The identification of an osteogenic stem cell with competency for both self-renewal and robust differentiation to bone-forming osteoblasts has been elusive. Several studies have documented the ability of cells in whole bone marrow to form osteoblasts in vitro (3) and in vivo (4). Pittenger et al. (3) isolated mesenchymal stem cells (MSCs) that were able to differentiate to chondrocytes, adipocytes, and osteoblasts in culture, whereas the results of serial transplantation of single bone marrow-derived stem cells can be interpreted to suggest the existence of rare long-term repopulating cells that can regenerate not only the entire hematopoietic system, but also several different mesenchymal lineages (5, 6).Our efforts to identify an osteogenic stem cell have focused on a side population (SP) of bone marrow cells that display strong hematopoietic reconstituting activity, as measured by competitive repopulation assays (7,8). These so-called SP cells, which can be identified by their unique capacity to efflux fluorescent DNA-binding dye (7,8), also have a limited capacity to differentiate in vivo to skeletal myocytes (9) as well as vascular endothelial cells (10), suggesting multilineage potential. To test the candidacy of these adult stem cells as progenitors of the osteoblast lineage, we tracked the fate of gene-marked SP both in vitro and in vivo. Donor-derived mesenchymal progenitors differentiated to osteoblasts in clonogenic medium, and immunostaining of long-bone sections after transplantation of SP cells into lethally irradiated mice demonst...

Despite the potential of type 1 interferons (IFNs) for the treatment of cancer, clinical experience with IFN protein therapy of solid tumors has been disappointing. IFN-␤ has potent antiproliferative activity against most human tumor cells in vitro in addition to its known immunomodulatory activities. The antiproliferative effect, however, relies on IFN-␤ concentrations that cannot be achieved by parenteral protein administration because of rapid protein clearance and systemic toxicities. We demonstrate here that ex vivo IFN-␤ gene transduction by a replication-defective adenovirus in as few as 1% of implanted cells blocked tumor formation. Direct in vivo IFN-␤ gene delivery into established tumors generated high local concentrations of IFN-␤, inhibited tumor growth, and in many cases caused complete tumor regression. Because the mice were immune-deficient, it is likely that the anti-tumor effect was primarily through direct inhibition of tumor cell proliferation and survival. Based on these studies, we argue that local IFN-␤ gene therapy with replication-defective adenoviral vectors might be an effective treatment for some solid tumors.

The 3'-and 5'-terminal nucleotide sequences of the defective interfering (DI) RNAs present in a preparation of DI influenza virus were determined. It was found that all DI RNAs possessed identical terminal sequences for at least the first 13 nucleotides at the 5' end and at least the last 12 nucleotides at the 3' end. The sequence of the DI RNAs is (5')A-GU-A-G-A-A-AC-A-A-G-G-... -C-C-U-G-C-U-U-U-C-G-C-U-OH(3'). In addition, the same sequences were present at the 3' and 5' termini of the viral polymerase genes (P1, P2, and P3) from which these DI RNAs originate. These results indicate that DI RNAs of influenza virus are formed by an internal deletion of the genomic RNA.Defective interfering (DI) animal viruses are noninfectious viruses that interfere with the replication of standard viruses (1, 2). DI influenza virus is produced after repeated undiluted passage of the virus (3), and over 99% of the infectious virus population is replaced by DI virus (4). The DI influenza virus has small virus-specific RNA molecules not present in the standard virus (4-7). Recently it has been directly demonstrated that the ribonucleoprotein complexes of these new deleted forms of the viral RNA specifically cause interference (8).In a previous report (9) the sequence relationships among these DI-RNAs and their relationship to the standard eight viral genes were studied by oligonucleotide mapping. Because each clone of influenza virus produces a unique set of DI RNAs when passaged (4), several DI RNAs are present in each DI virus preparation. All the DI RNAs studied thus far (9) have been found to be forms of one of the viral polymerase genes (P1, P2, or P3) from which sequences have been deleted. Comparison of the oligonucleotide maps of DI RNAs and P genes revealed that the DI RNAs originating from the same polymerase gene were related in one of two ways. Either they contained completely overlapping regions or each contained both overlapping and nonoverlapping regions (9). This latter group of DI RNAs could not be formed from the progenitor RNA by a mechanism of common initiation and simple deletion from one end as reported for vesicular stomatis virus (10) and Sendai virus (11). We therefore proposed (9) internal deletion of the progenitor virion RNA as one possible model for the formation of influenza DI RNAs.We have now determined the sequences of the 3' and 5' ends of three DI RNAs present in a DI influenza virus preparation as well as the sequences of the 3' and 5' ends of the polymerase genes. The sequence analysis of the DI RNAs and their progenitor genes indicates a model for the formation of influenza DI RNAs quite different from that proposed for other negative-stranded viruses (1, 11). (Mr 1 X 106). The third, Li, is also a deleted form of the P3 gene (9). MATERIALS AND METHODSPreparation of 3'-and 5'-Labeled RNAs. Viral RNA (vRNA) was extracted from a suspension of purified virus (13). The RNA was labeled at its 3' end as described (14) in a reaction containing 50 mM N-2-hydroxyethylpiperazine-N'-2-ethanesu...

A combination AIDS vaccine approach consisting of priming with adenovirus-HIV-1MN gp160 recombinants followed by boosting with HIV-1SF2 gp120 was evaluated in chimpanzees. Long-lasting protection, requiring only three immunizations, was achieved against a low-dose challenge with the SF2 strain of HIV-1 and a subsequent high-dose SF2 challenge administered 1 year later without an intervening boost. Notably, neutralizing antibody responses against both clinical and laboratory isolates developed in three chimpanzees and persisted until the time of high-dose challenge. The possibility that cytotoxic T-lymphocytes contribute to low-dose protection of a chimpanzee lacking neutralizing antibodies is suggested. Our results validate the live vector priming/subunit booster approach and should stimulate interest in assessing this combination vaccine approach in humans.