Neurotensin inversely modulates maternal aggression.

Neurotensin (NT) is a versatile neuropeptide involved in analgesia, hypothermia, and schizophrenia. Although NT is released from and acts upon brain regions involved in social behaviors, it has not been linked to a social behavior. We previously selected mice for high maternal aggression (maternal defense), an important social behavior that protects offspring, and found significantly lower NT expression in the CNS of highly protective females. Our current study directly tested NT's role in maternal defense. Intracerebroventricular (i.c.v.) injections of NT significantly impaired defense in terms of time aggressive and number of attacks at all doses tested (0.05, 0.1, 1.0, and 3.0 microg). Other maternal behaviors, including pup retrieval, were unaltered following NT injections (0.05 microg) relative to vehicle, suggesting specificity of NT action on defense. Further, i.c.v. injections of the NT receptor 1 (NT1) antagonist, SR 48692 (30 microg), significantly elevated maternal aggression in terms of time aggressive and attack number. To understand where NT may regulate aggression, we examined Fos following injection of either 0.1 microg NT or vehicle. Thirteen of 26 brain regions examined exhibited significant Fos increases with NT, including regions expressing NT1 and previously implicated in maternal aggression, such as lateral septum, bed nucleus of stria terminalis, paraventricular nucleus, and central amygdala. Together, our results indicate that NT inversely regulates maternal aggression and provide the first direct evidence that lowering of NT signaling can be a mechanism for maternal aggression. To our knowledge, this is the first study to directly link NT to a social behavior.

17beta-estradiol regulates the secretion of TGF-beta by cultured human dermal fibroblasts.

Estrogen plays an important role in skin homeostasis, as demonstrated by the changes seen in the skin of post-menopausal women, changes reversed by HRT. Estrogen also has a role in wound healing, since estrogen deficiency as occurs post-menopausally and in ovariectomised animals, is associated with a reduced rate of wound healing. Estrogen appears to modulate all phases of wound healing with effects on inflammatory cells, epithelialization, angiogenesis, extracellular matrix deposition and tissue remodelling. This study was designed to investigate the effects of 17beta-estradiol on cultured human dermal fibroblasts using an in vitro wound-healing assay. The end points investigated were cell migration, proliferation, total collagen secretion and active TGF-beta1 secretion. 17beta-estradiol significantly increased the migration and proliferation of cultured dermal fibroblasts following mechanical wounding, although the secretion of total soluble collagen was not altered. An increase in TGF-beta1 was demonstrated by unwounded confluent dermal fibroblast monolayers in response to 17beta-estradiol, but paradoxically, a decrease in the secretion of TGF-beta1 was demonstrated in the mechanically wounded dermal fibroblasts. These results identify human dermal fibroblasts as estrogen target cells and provide further evidence for a role by which estrogen regulates this particular cell type as part of the wound-healing process. However, the paradoxical nature of the effect of estrogen on TGF-beta1 secretion following mechanical wounding suggests that the cellular mechanism of action is complex. A greater understanding of the cell-specific action of estrogen may help to develop therapies that will improve cutaneous wound healing in the future.

Altered gene expression in mice selected for high maternal aggression.

We previously applied selective breeding on outbred mice to increase maternal aggression (maternal defense). In this study, we compared gene expression within a continuous region of the central nervous system (CNS) involved in maternal aggression (hypothalamus and preoptic regions) between lactating selected (S) and nonselected control (C) mice (n= 6 per group). Using microarrays representing over 40,000 genes or expressed sequence tags, two statistical algorithms were used to identify significant differences in gene expression: robust multiarray and the probe logarithmic intensity error method. Approximately 200 genes were identified as significant using an intersection from both techniques. A subset of genes was examined for confirmation by real-time polymerase chain reaction (PCR). Significant decreases were found in S mice for neurotensin and neuropeptide Y receptor Y2 (both confirmed by PCR). Significant increases were found in S mice for neuronal nitric oxide synthase (confirmed by PCR), the K+ channel subunit, Kcna1 (confirmed by PCR), corticotrophin releasing factor binding protein (just above significance using PCR; P= 0.051) and GABA A receptor subunit 1A (not confirmed by PCR, but similar direction). S mice also exhibited significantly higher levels of the neurotransmitter receptor, adenosine A1 receptor and the transcription factors, c-Fos, and Egr-1. Interestingly, for 24 genes related to metabolism, all were significantly elevated in S mice, suggesting altered metabolism in these mice. Together, this study provides a list of candidate genes (some previously implicated in maternal aggression and some novel) that may play an important role in the production of this behavior.

Synthesis of adenoviral targeting molecules by intein-mediated protein ligation.

Adenoviral vectors infect cells through the binding of capsid proteins to cell-surface receptors. The ubiquitous expression of adenoviral receptors in human tissues represents an obstacle toward the development of systemically deliverable vectors for cancer therapy, since effective therapy may require delivery to specific sites. For these reasons, major efforts are directed toward the elimination of the native tropism combined with identification of ligands that bind to tumor-specific cell-surface proteins. Highthroughput technologies have identified potential targeting ligands, which need to be evaluated for their ability to retarget adenovirus to alternative receptors. Here, we present a strategy that permits the routine analysis of adenoviral targeting ligands. We use intein-mediated protein ligation as a means to produce functional biological molecules, that is, adenoviral targeting molecules that function as adapters between cellular receptors and the adenovirus fiber protein. We demonstrate the versatility of the present system by conjugating targeting ligands that differ in size and nature including an apolipoprotein E synthetic peptide, the basic fibroblast growth factor and folic acid. The resulting adenoviral targeting molecules mediate adenoviral gene delivery in cells that express the corresponding receptor.

Private specificities can dominate the humoral response to self-antigens in patients with cryptogenic fibrosing alveolitis.

BACKGROUND: The pathogenetic mechanisms that underlie the interstitial lung disease cryptogenic fibrosing alveolitis (CFA) may involve an immunological reaction to unidentified antigens in the lung, resulting in tissue damage. METHOD: In order to identify the range of target autoantigens, we used expression cloning, employing serum from an index patient as the probe against an expressed cDNA library that was derived from a tumour cell line. We screened over 5 x 105 recombinants and obtained sequence information on three antigens that had provoked strong responses with immunoglobulin heavy chain class switching, presumably as a consequence of T-cell recognition. RESULTS: All of the antigens were identifiable by comparison with sequence data from the US National Center for Biotechnology Information. Alanyl tRNA synthetase (ATS) was picked on six occasions; five of these incidences reflected independent recombination events, indicating that the library was not biased. Antibodies to ATS (anti-PL-12) represent the most common reactivity that defines the antisynthetase syndrome, which is typically expressed as polymyositis, dermatomyositis and interstitial lung disease (ILD). The index patient never showed symptoms other than those associated with alveolitis, even though sera obtained from him over a period of 2 years contained antibodies with the same specificity. Autoantibodies to ATS were never detected in serial bleeds from 11 other patients with CFA, and neither did we detect antibodies to the other two antigens identified from the serum of the index patient. CONCLUSION: The humoral response in patients with CFA can be dominated by autoantibodies with private specificities. This suggests that the antibodies are epiphenomenal and are a secondary feature of tissue damage induced by some other mechanism.

A genetically modified adenoviral vector exhibits enhanced gene transfer of human smooth muscle cells.

Adenoviral vector-based gene therapy is a promising approach for the treatment of restenosis postangioplasty. However, a high concentration of adenoviral vector can cause cellular activation, damage, and an enhanced immune response. One approach to solving this problem is to increase gene transfer efficiency by directing adenoviral vector entry via an alternate receptor system. We have constructed an adenoviral vector, Av9LacZ, that encodes the beta-galactosidase gene and contains a chimeric fiber protein that redirects viral vector binding to the Ad3 adenoviral receptor on the host cell. We examined the ability of Av9LacZ to transduce primary human smooth muscle cells (SMC) and found that it showed a 10- to 15-fold higher transduction efficiency when compared to the prototypic adenoviral vector currently used for preclinical and clinical studies. While both vectors were able to transduce rabbit, pig and monkey SMCs, the genetically modified vector transduced human SMC with much higher efficiency. SMC obtained from the aorta, coronary, renal, popliteal and pulmonary arteries were all efficiently transduced by Av9LacZ. Consistent with the data obtained from cultured cells, Av9LacZ also transduced fresh human arterial tissues considerably more efficiently than Av1LacZ. We conclude that the large discrepancy between transduction of animal and human cells by conventional vectors supports a cautious extrapolation of the results of in vivo animal studies to man. Furthermore, the genetically modified AV9 vector may deliver better efficacy and studies in large animal models with this vector could be more predictive of therapeutic efficacy in the treatment of human restenosis.

Enhanced gene transfer to rabbit jugular veins by an adenovirus containing a cyclic RGD motif in the HI loop of the fiber knob.

Gene therapy using recombinant adenoviral vectors represents a promising therapeutic tool to prevent vein graft stenosis, the main complication of coronary artery bypass grafting. However, the low transduction efficiency of vascular smooth muscle cells and endothelial cells (EC) is a potential limitation, presumably due to the low levels of functional adenovirus receptor (coxsackie:adenovirus receptor; CAR). Designing vectors specifically targeted to alpha(v) integrins is a strategy that might overcome the poor expression of CAR in vascular smooth muscle cells and EC. RGD, a receptor-binding motif that can interact with alpha(v) integrins, was inserted into the HI loop and at the C-terminus of the adenoviral fiber protein in two separate adenovirus vectors encoding a beta-galactosidase reporter gene. Av1nBgCRGD (C-terminus) and Av1nBgHIRGD (HI loop) were evaluated in EC in culture and in jugular vein organ culture. Transduction of primary rat and rabbit EC with Av1nBgHIRGD was significantly more efficient when compared to Av1nBgCRGD or Av1nBg. Transduction of mouse, rat and rabbit jugular veins in organ culture using Av1nBg showed that adenovirus-mediated gene expression was greatest in rabbit jugular veins compared to rat and mouse veins. Av1nBgHIRGD augmented gene expression approximately four-fold in rabbit jugular veins when compared to Av1nBg. Histochemical analysis showed that numerous EC but few smooth muscle cells were transduced at all vector concentrations. A substantial number of adventitial fibroblasts were transduced only at the highest vector concentrations of Av1nBgHIRGD. These findings demonstrate that integrin-targeted vectors allow for enhanced gene delivery to veins and strengthen the viability of adenoviral-mediated gene transfer of therapeutic transgenes to human veins prior to vein grafting.

Socioeconomic differentials in recurrent ischaemia and mortality after acute myocardial infarction.

OBJECTIVE: To examine the influence of socioeconomic deprivation on case fatality following acute myocardial infarction. DESIGN: Prospective cohort observational study. SETTING: General hospital. PATIENTS: 1417 white and south Asian patients admitted with acute myocardial infarction between January 1988 and December 1996, and classified by the Carstairs socioeconomic deprivation score of the enumeration district of residence. MAIN OUTCOME MEASURES: 30 day and one year survival. RESULTS: There was little variation across deprivation groups in age, sex, or smoking status, though a higher proportion of patients from more deprived enumeration districts were diabetic and of south Asian origin, and a higher proportion of them developed Q wave infarction and left ventricular failure. There was no appreciable variation in clinical treatment with deprivation. Patients from more deprived enumeration districts had a higher risk of recurrent ischaemic events (death, recurrent myocardial infarction, or unstable angina) over the first 30 days: event free survival (95% confidence interval (CI)) of the most deprived quartile was 0.79 (95% CI 0.74 to 0.83) compared with 0.85 (95% CI 0.80 to 0.88) in the least deprived quartile. The unadjusted hazard ratio corresponding to an increase from the 5th to 95th centile of the deprivation distribution was 1.54 (95% CI 1.02 to 2.32), and 1.59 (95% CI 1.03 to 2.44) after adjustment for age, sex, racial group, diabetes, acute treatment with thrombolysis and aspirin, and left ventricular failure. Survival from 30 days to one year, however, did not show a socioeconomic gradient (hazard ratio adjusted for the same variables was 1.07 (95% CI 0.68 to 1.70)). CONCLUSIONS: In patients hospitalised with acute myocardial infarction, there is a strong association between early recurrent ischaemic events and socioeconomic deprivation that is not accounted for by clinical presentation or treatment. This association appears to be attenuated over time.

Adenovirus type 5 viral particles pseudotyped with mutagenized fiber proteins show diminished infectivity of coxsackie B-adenovirus receptor-bearing cells.

A major limitation of adenovirus type 5 (Ad5)-based gene therapy, the inability to target therapeutic genes to selected cell types, is attributable to the natural tropism of the virus for the widely expressed coxsackievirus-adenovirus receptor (CAR) protein. Modifications of the Ad5 fiber knob domain have been shown to alter the tropism of the virus. We have developed a novel system to rapidly evaluate the function of modified fiber proteins in their most relevant context, the adenoviral capsid. This transient transfection/infection system combines transfection of cells with plasmids that express high levels of the modified fiber protein and infection with Ad5.beta gal.Delta F, an E1-, E3-, and fiber-deleted adenoviral vector encoding beta-galactosidase. We have used this system to test the adenoviral transduction efficiency mediated by a panel of fiber protein mutants that were proposed to influence CAR interaction. A series of amino acid modifications were incorporated via mutagenesis into the fiber expression plasmid, and the resulting fiber proteins were subsequently incorporated onto adenoviral particles. Mutations located in the fiber knob AB and CD loops demonstrated the greatest reduction in fiber-mediated gene transfer in HeLa cells. We also observed effects on transduction efficiency with mutations in the FG loop, indicating that the binding site may extend to the adjacent monomer in the fiber trimer and in the HI loop. These studies support the concept that modification of the fiber knob domain to diminish or ablate CAR interaction should result in a detargeted adenoviral vector that can be combined simultaneously with novel ligands for the development of a systemically administered, targeted adenoviral vector.

p53 autoantibodies in patients with malignant mesothelioma: stability through disease progression.

Malignant mesothelioma (MM) generally occurs as a pleural tumour, related to the inhalation of asbestos fibres. It is highly aggressive and largely unresponsive to treatment. The incidence of MM is particularly high in Western Australia because of the extensive blue asbestos mining operations that occurred in the north of the state until 1966. MM is unusual in that mutations in the tumour suppressor gene p53 are rarely observed, whilst over-expression of p53 protein is common. As the level of antibodies directed against p53 is thought to be of prognostic value in some cancers and as MM is known to be immunogenic, we studied a cohort of Western Australian patients to determine the prevalence of anti-p53 antibodies and their value as diagnostic markers or prognostic indicators. 6/88 (7%) of patients had high titres (>2 SD above the mean of controls) of anti-p53 antibodies. There was no correlation between antibody titre and survival. Although 3/38 (8%) of sera obtained from patients exposed to asbestos but prior to a diagnosis of MM contained antibodies, the same proportion of sera obtained from patients exposed to asbestos but who remained disease free also contained antibodies (2/40; 8%). Sera collected sequentially demonstrated a profound temporal stability in the titre of anti-p53 antibodies in patients with MM throughout the course of their illness. These results show that anti-p53 antibodies are observed only at a low frequency in the sera of MM patients and where they do occur, their elicitation is an early event that may be unrelated to antigen load. The occurrence of anti-p53 antibodies does not serve as either a useful prognostic or diagnostic indicator in MM.

Simian virus (SV) 40 like sequences in cell lines and tumour biopsies from Australian malignant mesotheliomas.

BACKGROUND: Simian virus (SV) 40 sequences have been found in some, but not all studies of mesotheliomas. This virus is known to cause tumours in rodents but its role in human oncogenesis remains controversial. AIMS: The aim of this study therefore was to determine whether SV40 is associated with the development of mesotheliomas in Australia. The absence of the virus or its gene products in tissue derived from mesotheliomas would detract from this possibility. METHODS: We used polymerase chain reaction from three pairs of primers to amplify different regions of the large T antigen from DNA from cell lines and cDNA from both cell lines and an independent set of tumour biopsies from patients with mesothelioma. RESULTS: We examined five human mesothelioma cell lines that were established in our laboratories. In addition, we examined several tumour biopsies from seven different patients. SV40 like sequences were present in all the cell lines and in at least one sample from each of the patients examined. CONCLUSIONS: The large T antigen of SV40 or an SV40 like virus is expressed in Australian mesotheliomas and therefore could be aetiologically-associated with tumourigenesis. Alternatively, these sequences could be expressed subsequent to the development of the disease.

The effects of processing and low dose irradiation on cortical bone grafts.

The authors studied the effects of standard processing and preprocessing low dose gamma irradiation (1.5 Mrad) on the strength and incorporation of syngeneic and allogeneic cortical bone grafts. Bilateral femoral middiaphyseal 8-mm segmental defects in 120 male Fisher rats were stabilized with internal fixation. Each defect received one of six types of grafts: fresh syngeneic, processed syngeneic, irradiated processed syngeneic, fresh allogeneic, processed allogeneic, and irradiated processed allogeneic grafts. Graft processing included soaking in 70% ethanol and deep freezing for preservation. Irradiation was performed by 60Co source immediately before processing. Grafts were evaluated by histologic analysis, histomorphometric analysis, and biomechanical testing at 4 and 6 months after surgery. Graft treatment, either processing or irradiation processing, did not affect consistently or significantly the incorporation of syngeneic or allogeneic grafts. Graft allogenicity was the major determinant of the revascularization and the histologic pattern of graft incorporation. Processed and irradiated processed allogeneic grafts gained compressive strength with time and were as strong as syngeneic grafts at 6 months. Biomechanical and histologic data from this study suggest that standard processing and preprocessing low dose irradiation do not compromise the natural course of allogeneic cortical bone graft incorporation.

Targetable gene delivery vectors.

Adenoviral vectors, which have targeting ligands for tumor cells on the capsid, no natural tropism, and carry a therapeutic payload should be constructed soon and tested in pre-clinical models. Nevertheless, there are still important considerations for the design and therapeutic use of targetable vectors. Perhaps the single greatest challenge in the future, as it was in the past, will be finding ligands that have a higher apparent affinity for tumor and/or tumor endothelial cells then normal cells. However, the advent of many rapidly advancing technologies and information including the sequencing of the human genome, in vivo and in vitro phage display, rapid analysis of gene and protein expression in any context, and new cellular targets such as angiogenic endothelial cells, may provide many opportunities for the discovery of novel and useful ligands. In addition, the interests in targeting vectors are rapidly growing with new journals and meetings solely devoted to this subject increasing annually. Within the next 5 years, we should have meaningful clinical data on targetable vectors to reassess our progress.

Serologic responses in patients with malignant mesothelioma: evidence for both public and private specificities.

Malignant mesothelioma (MM) is a pulmonary malignancy that appears to be immunogenic based on a large number of studies in both animals and humans. This notion is supported by our recent demonstration using Western blot analysis of immunoglobulin G antibodies reactive with a variety of autoantigens in many patients with MM. In view of the enormous potential of such antigens in early diagnosis, immunotherapy, and vaccination of at-risk individuals, it was essential to identify these antigens. We therefore applied the SEREX technique (serologic identification by recombinant expression cloning), using a serum pool from six patients as the probe against an expressed complementary DNA library derived from a cloned MM cell line. We screened over one million recombinants and obtained sequence information on eight antigens that had provoked immunoglobulin heavy chain class switching, presumably as a consequence of T-cell recognition. Six of these antigens were identifiable (U2AF[65], Siah binding protein, topoisomerase IIbeta, ZFM1, mIre1, and pendulin), and of the others, one was found as a single EST from a myotube library (Jemm-1); the other (Jemm-2) was not represented in any EST database even as a weak homolog. Consistent with our previous findings, each of the characterizable antigens would be expected to be associated with the cell nucleus. Each of the autoantibody specificities was uniquely associated with a single patient with the exception of antibodies to TOPIIbeta and U2AF(65). We found 13 of 14 (93%) patients with MM had antibodies to TOPIIbeta and two of 14 (14%) patients had antibodies to U2AF(65). The number of serum reactivities, taken as a measure of the complexity of the immune response, correlates with patient survival and with an index of systemic inflammation. These data suggest that a broader range of serologic reactivities reflects a more active host response to the presence of tumor.

Adenovirus vector pseudotyping in fiber-expressing cell lines: improved transduction of Epstein-Barr virus-transformed B cells.

While adenovirus (Ad) gene delivery vectors are useful in many gene therapy applications, their broad tropism means that they cannot be directed to a specific target cell. There are also a number of cell types involved in human disease which are not transducible with standard Ad vectors, such as Epstein-Barr virus (EBV)-transformed B lymphocytes. Adenovirus binds to host cells via the viral fiber protein, and Ad vectors have previously been retargeted by modifying the fiber gene on the viral chromosome. This requires that the modified fiber be able to bind to the cell in which the vector is grown, which prevents truly specific vector targeting. We previously reported a gene delivery system based on a fiber gene-deleted Ad type 5 (Ad5) vector (Ad5.betagal.DeltaF) and packaging cells that express the viral fiber protein. Expression of different fibers in packaging cells will allow Ad retargeting without modifying the viral chromosome. Importantly, fiber proteins which can no longer bind to the producer cells can also be used. Using this approach, we generated for the first time pseudotyped Ad5.betagal.DeltaF particles containing either the wild-type Ad5 fiber protein or a chimeric fiber with the receptor-binding knob domain of the Ad3 fiber. Particles equipped with the chimeric fiber bound to the Ad3 receptor rather than the coxsackievirus-adenovirus receptor protein used by Ad5. EBV-transformed B lymphocytes were infected efficiently by the Ad3-pseudotyped particles but poorly by virus containing the Ad5 fiber protein. The strategy described here represents a broadly applicable method for targeting gene delivery to specific cell types.

Adenovirus-mediated expresssion of the murine ecotropic receptor facilitates transduction of human hematopoietic cells with an ecotropic retroviral vector.

One factor limiting the ability to modify human repopulating hematopoietic cells genetically with retroviral vectors is the relatively low expression of the cognate viral receptor. We have tested sequential transduction of human hematopoietic cells with an adenoviral vector encoding the ecotropic retroviral receptor followed by transduction with an ecotropic retroviral vector. Adenoviral transduction of K562 erythroleukemia cells was highly efficiently with >95% of cells expressing the ecotropic receptor at a multiplicity of infection (MOI) of 103with a correspondingly high transduction with a retroviral vector. Ecotropic receptor expression in CD34+ cells following transduction with adenoviral vectors was increased by at least two-fold (from 20 to 48%) by replacing the RSV promoter with the CMV E1a promoter, resulting in a parallel increase in retroviral transduction efficiency. Replacing the head portion of the fiber protein in conventional adenoviral vectors (serotype 5) with the corresponding portion from an adenoviral 3 serotype resulted in ecotropic receptor expression in 60% of CD34+ cells at an MOI of 104 and a retroviral transduction of 60% of hematopoietic clonogenic progenitors. The sequential transduction strategy also resulted in efficient transduction of the primitive CD34+CD38- subset suggesting that it may hold promise for genetic modification of human hematopoietic stem cells.

The effect of regional gene therapy with bone morphogenetic protein-2-producing bone-marrow cells on the repair of segmental femoral defects in rats.

BACKGROUND: Recombinant human bone morphogenetic proteins (rhBMPs) can induce bone formation, but the inability to identify an ideal delivery system limits their clinical application. We used ex vivo adenoviral gene transfer to create BMP-2-producing bone-marrow cells, which allow delivery of the BMP-2 to a specific anatomical site. The autologous BMP-2-producing bone-marrow cells then were used to heal a critical-sized femoral segmental defect in syngeneic rats. METHODS: Femoral defects in five groups of rats were filled with 5 x 10(6) BMP-2-producing bone-marrow cells, created through adenoviral gene transfer (twenty-four femora, Group I); twenty micrograms of rhBMP-2 (sixteen femora, Group II); 5 x 10(6) beta-galactosidase-producing rat-bone-marrow cells, created through adenoviral gene transfer of the lacZ gene (twelve femora, Group III); 5 x 10(6) uninfected rat-bone-marrow cells (ten femora, Group IV); or guanidine hydrochloride-extracted demineralized bone matrix only (ten femora, Group V). Guanidine hydrochloride-extracted demineralized bone matrix served as a substrate in all experimental groups. Specimens that were removed two months postoperatively underwent histological and histomorphometric analysis as well as biomechanical testing. RESULTS: Twenty-two of the twenty-four defects in Group I (BMP-2-producing bone-marrow cells) and all sixteen defects in Group II (rhBMP-2) had healed radiographically at two months postoperatively compared with only one of the thirty-two defects in the three control groups (beta-galactosidase-producing rat-bone-marrow cells, uninfected rat-bone-marrow cells, and guanidine hydrochloride-extracted demineralized bone matrix alone). Histological analysis of the specimens revealed that defects that had received BMP-2-producing bone-marrow cells (Group I) were filled with coarse trabecular bone at two months postoperatively, whereas in those that had received rhBMP-2 (Group II) the bone was thin and lace-like. Defects that had been treated with bone-marrow cells producing beta-galactosidase (Group III), uninfected bone-marrow cells (Group IV), or guanidine hydrochloride-extracted demineralized bone matrix only (Group V) demonstrated little or no bone formation. Histomorphometric analysis revealed a significantly greater total area of bone formation in the defects treated with the BMP-2-producing bone-marrow cells than in those treated with the rhBMP-2 (p = 0.036). Biomechanical testing demonstrated no significant differences, with the numbers available, between the healed femora that had received BMP-2-producing bone-marrow cells and the untreated (control) femora with respect to ultimate torque to failure or energy to failure. CONCLUSIONS: This study demonstrated that BMP-2-producing bone-marrow cells created by means of adenoviral gene transfer produce sufficient protein to heal a segmental femoral defect. We also established the feasibility of ex vivo gene transfer with the use of biologically acute autologous short-term cultures of bone-marrow cells.

Enhanced neuroblastoma transduction for an improved antitumor vaccine.

BACKGROUND: A recent clinical trial of an antineuroblastoma vaccine used adenovirus serotype 5 (Ad5) vectors to transduce autologous tumor cells with the gene encoding IL-2. A method to improve transduction efficiency was sought to enable the use of lower viral titers, especially when in situ adenoviral-mediated tumor cell transduction is considered. MATERIALS AND METHODS: A chimeric adenoviral delivery vector was utilized in which the fiber head from adenovirus serotype 3 was incorporated into the backbone of Ad5. Since the fiber head protein is responsible for viral attachment to target cells, a different spectrum and range of infectivity might result. Both the chimeric (Av9LacZ4) and Ad5 (Av1LacZ4) vectors were constructed to carry a beta-galactosidase transgene. The relative transduction efficiency of these two vectors was then evaluated in five tumor-derived short-term neuroblastoma cultures and four established neuroblastoma cell lines. Enzyme activity was assessed using three different methods: in situ staining, flow cytometric analysis, and a quantitative assay. RESULTS: A significant improvement in transduction efficiency of the short-term neuroblastoma cultures with the new chimeric adenovector was demonstrated. A similar improvement in transduction efficiency was not observed in the established cell lines, suggesting that the cell surface receptor for the Ad 3 serotype had been lost in vitro. Increased transduction of tumor cells with N-myc amplification was also observed. CONCLUSIONS: The newly constructed chimeric adenoviral vector transduces short-term neuroblastoma cultures more efficiently than the standard Ad5 vector. This vector will permit the use of lower viral titers and may be useful in other adenoviral-based gene-therapy protocols. Increased transgene expression in N-myc-amplified cells offers possible selectivity for in situ gene delivery.

Photon-avalanche upconversion in thulium-doped lutetium aluminum garnet.

Strong blue fluorescence at 487 nm corresponding to the (1)G(4) --> (3)H(6) transition was generated from Tm(3+)-doped lutetium aluminum garnet on excitation with a 618-nm dye laser as a result of a photon-avalanche upconversion mechanism.