Comparative analysis of IFN-gamma B7.1 and antisense TGF-beta gene transfer on the tumorigenicity of a poorly immunogenic metastatic mammary carcinoma.

Cancer progression is attributed in part to immune evasion strategies that include lack of co-stimulation, down-regulation of cell surface MHC molecules, and secretion of immunosuppressive factors, such as transforming growth factor-beta (TGF-beta). Gene therapy has been employed to counter these mechanisms of immune evasion by transference of B7.1, IFN-gamma or antisense TGF-beta genes into tumor cells, resulting in cell surface expression of B7.1, upregulation of MHC class I and class II molecules, or elimination of tumor-derived TGF-beta, respectively. Although each of these transgenes has been shown to alter tumorigenicity in murine models, a direct comparison of their efficacy has not been performed. In this study, we have employed a very aggressive, poorly immunogenic and highly metastatic mammary model, 4T1, to compare the efficacy of B7.1, IFN-gamma and antisense TGF-beta gene transfer in stimulating an anti-tumor response. We demonstrate that both IFN-gamma and antisense TGF-beta gene expression significantly reduced the tumorigenicity of these cells compared to mock transduced cells, with IFN-gamma having a greater effect. In contrast, B7.1 gene transfer did not affect the tumorigenicity of 4T1 cells. The anti-tumor response directed against antisense TGF-beta-expressing 4T1 tumors was mediated by CD4+ and CD8+ T cells. However, CD8+ T cells, and not CD4+ T cells, appeared to mediate the anti-tumor response against IFN-gamma-expressing tumors. Treatment of tumor-bearing animals with IFN-gamma or antisense TGF-beta gene-modified tumor cell vaccines reduced the number of clonogenic metastases to the lungs and liver compared to treatment with mock-transduced cells. Finally, in a residual disease model in which the primary tumor was excised and mice were vaccinated with irradiated tumor cells, treatment of mice with vaccinations consisting of 4T1 cells expressing both antisense TGF-beta and IFN-gamma genes was the most effective in prolonging survival.

Induction of melanoma-associated antigen systemic immunity upon intratumoral delivery of interferon-gamma retroviral vector in melanoma patients.

A total of 17 patients with metastatic melanoma were treated with intratumoral interferon-gamma (IFN-gamma) retroviral vector in a phase I clinical trial. A cycle of treatment consisted of five daily injections every 2 weeks. Patients were divided into two treatment arms that involved a single course (one cycle) of treatment (group I; n = 9) and multiple cycles (six cycles) of treatment (group II; n = 8). Patients received intratumoral injections of IFN-gamma (10(7) plaque-forming units/mL administered at 0.3, 0.5, and 1.0 mL per cohort of patients). All patients receiving multiple injections either maintained stable disease (n = 5) or achieved a partial or complete response (n = 3) of the injected lesion, whereas in patients receiving a single cycle of treatment, only one of nine patients had a response. Patients were assessed for immunoglobulin G antibody (Ab) responses to the melanoma-associated antigens (MAA) tyrosinase, gp100, TRP-2, and MAGE-A1 by affinity enzyme-linked immunosorbent assay. Anti-MAGE-A1 and tyrosinase Ab were significantly elevated from baseline (day 0) to week 16 during treatment (P = .005; P = .002, respectively) in patients who received multiple injections. Patients undergoing treatment who had a clinical response (stable disease or better) also had significantly more elevated Ab responses to a greater number of MAA (P = .0004). The induction of systemic Ab responses to multiple MAA also correlated with systemic clinical responses. These studies suggest that multiple anti-MAA Ab responses are associated with clinical responses to IFN-gamma retroviral treatment and may be used as surrogate response markers.

The use and development of retroviral vectors to deliver cytokine genes for cancer therapy.

In this review, we describe technical advancements of retroviral vectors to address issues of safety, titer, and clinical scale manufacturing to produce high-quality retroviral vector preparations that have made direct intratumoral administration of cytokine encoding recombinant vectors a feasible cancer therapy in the clinic. We also review possible further advances in retroviral vector design, which may prove important in expanding these clinical applications.

Ablation of tumor cells in vivo by direct injection of HSV-thymidine kinase retroviral vector and ganciclovir therapy.

The introduction of therapeutic genes into proliferating tumor cells in vivo by direct intralesional injection of retroviral vectors can provide an effective and valuable approach for the treatment of a variety of solid tumor types. Efficient transduction of tumor cells in situ by direct injection was demonstrated using a retroviral vector containing the beta-galactosidase (beta-gal) gene. Ablation therapy in vivo was demonstrated using a retroviral vector containing the Herpes simplex virus thymidine kinase gene (HSV-TK) to deliver the TK gene into the murine colorectal tumor cell line CT26. Ablation of CT26 tumor cells in situ was achieved by directly injecting high-titer HSV-TK retroviral vector preparations into the site of tumor cell inoculation followed by intraperitoneal (i.p.) delivery of ganciclovir (GCV). This gene therapy strategy demonstrated a markedly lower rate of tumor progression, with several complete regressions, compared to animals in control groups. We also demonstrated that resistance to subsequent challenges with unmodified CT26 cells and an enhanced cellular immune response is associated with tumor regression in immunocompetent animals. Our results demonstrate the feasibility of direct in situ administration of HSV-TK retroviral vectors for the treatment of cancer and suggest that a cellular immune response may be elicited by this therapy.

Generation of a systemic antitumor response with regional intratumoral injections of interferon gamma retroviral vector.

The generation of a lasting systemic immune response is a primary goal for cancer immunotherapy. Here we examine the ability of high-titer IFN-gamma retroviral vector injected into an accessible tumor to generate significant antitumor responses at a distal untreated site. CT26 or B16F10 murine tumors were inoculated subcutaneously to form solid tumors in BALB/c or C57BL/6 mice. Seven to 10 days postinoculation, high-titer IFN-gamma retroviral vector was directly injected into the subcutaneous tumor nodule, and optimal dose and course of therapy were determined. As a model for disseminated disease, mice were inoculated intravenously with CT26 cells to form pulmonary lesions, at the same time as the subcutaneous injections. Regression of subcutaneous tumor correlated with a systemic response at the distal lung metastases in the IFN-gamma-treated group (p < 0.0005). Splenocytes from mice with completely regressed tumors had a twofold increase in percent specific cytotoxicity in a standard CTL assay as compared with nonresponding mice. CD8+ T cells were shown to be essential for the regional and systemic antitumor response, as determined by in vivo cell depletion experiments. These data demonstrate that IFN-gamma retroviral vector gene therapy delivered intralesionally can generate significant inhibition of pulmonary tumor formation distal to the treatment site. The data from these preclinical studies suggest the potential clinical value of retroviral vector-mediated cytokine gene therapy for systemic cancer.

Establishment of parameters for optimal transduction efficiency and antitumor effects with purified high-titer HSV-TK retroviral vector in established solid tumors.

Suicide gene therapy using the herpes simplex thymidine kinase gene and ganciclovir is an attractive strategy for solid tumors. Early animal studies involved intratumoral injection of retroviral producer cells or unprocessed supernatant to generate an antitumor effect. Xenotransplantation of producer cells proved effective in several models, but the crude supernatants from the same cells were of insufficient titer to produce antitumor effects. We have developed new non-murine producer lines that yield replication-defective retroviral vectors encoding thymidine kinase at high titer which are then further purified and processed, resulting in pharmaceutical grade retroviral vectors with titers of up to 10(8) cfu/ml. Purified, high-titer retroviral preparations were injected directly into solid tumors in two syngeneic mouse tumor models. Significant antitumor responses and some cures were observed following systemic ganciclovir therapy. Assays using monoclonal antibodies to measure thymidine kinase protein expression at the single cell level in vitro and in vivo were developed so that therapeutic transgene expression could be quantified. Intralesional delivery resulted in transduction of over 20% of tumor cells in a protocol designed to maximize transduction on the basis of separate analyses of route, dosage, and schedule of vector administration. A consensus strategy evolved in which the combined effects of increased titer and a longer duration of retroviral vector administration interact to maximize transduction efficiency. These results indicate that purified high-titer retroviral vectors have the potential to transfer effective quantities of therapeutic genes into solid tumors in human subjects and highlight some pharmacologic factors that could be valuable in the design of clinical gene therapy protocols.

Identification of a promoter element that regulates tissue-specific expression of the human CD80 (B7.1) gene.

We isolated the promoter region of the gene encoding human CD80 to examine for elements responsible for the regulated expression of this important costimulatory molecule. Using CAT reporter constructs containing a heterologous general enhancer, we demonstrate that the CD80 promoter is active in CD80-expressing Raji cells, but has no significant activity in Jurkat cells that are CD80 negative. Transcriptional activity in Raji increases as the promoter is truncated from nucleotide position -906 to -84. However, truncation of this promoter to -41 significantly decreases its activity. Within this region is one stretch of DNA that is protected in DNase I footprint analysis and that shows some sequence similarity to the NF-kappaB element. Site-specific mutation of the 5' purine-rich portion of this element (B7-RE, or B7 regulatory element) abrogates expression. Nuclear extracts prepared from Raji, or from leukemic cells induced to express CD80, form a distinct complex(es) with B7-RE in electromobility shift assays. Moreover, a consensus NF-kappaB oligonucleotide can compete with B7-RE for nuclear extract binding. However, no super-shifted bands are observed when extracts are preincubated with Abs to p50, p65, or other Rel proteins. Moreover, we find that recombinant p49 (RelB), p50, p65 (RelA), or p49/p65 heterodimers do not bind B7-RE in vitro. These data indicate that B7-RE may help govern expression of genes independent of a tissue-specific enhancer and that this element is bound by nuclear factor(s) other than those that commonly bind NF-kappaB.

MR findings in methanol intoxication.

We report the MR and CT findings with pathologic correlation in a case of severe methanol intoxication. There was bilateral hemorrhagic necrosis of the putamen and caudate nuclei and, in addition, extensive subcortical necrosis and symmetric bilateral necrosis of the pontine tegmentum and optic nerves, which may indicate poor prognosis.

Magnetic resonance features in carbon monoxide poisoning.

The authors describe unusual magnetic resonance findings due to carbon monoxide poisoning in a 34-year-old woman. With T2-weighted imaging, increased signal intensity was observed bilaterally in the putamen and the caudate nucleus. Lesions of high signal intensity in the globus pallidus, which have been previously reported, were also observed.

Pseudodelta sign on computed tomography: an indication of bilateral interhemispheric hemorrhage.

The "pseudodelta sign" is introduced as an indicator of interhemispheric hemorrhage. This sign, appearing in computed tomography (CT) scans, consists of hyperattenuated extra-axial blood surrounding blood flowing in the superior sagittal sinus, which shows lower attenuation. To determine the prevalence of interhemispheric hemorrhage and the pseudodelta sign in patients with post-traumatic extra-axial hemorrhage, the authors reviewed the charts and CT scans of 198 such patients. The patients had been admitted to a tertiary-care hospital between Jan. 1, 1986, and Dec. 1, 1990. Interhemispheric hemorrhage was visible in 18 cases; the pseudodelta sign was seen in 14 of these. In 8 of the 14 cases the interhemispheric hemorrhage consisted of bilateral subdural hematoma, and in 3 it consisted of unilateral subdural hematoma. In the remaining three cases the appearance of the interhemispheric blood was nonspecific, and the hemorrhage may have been subdural or subarachnoid. The pseudodelta sign may be a good indicator of interhemispheric hemorrhage in either the subdural or subarachnoid space.

Cystic intracranial schwannoma.

The development of cystic components in intracranial schwannoma is not rare but has not been emphasized in any previous series or reviews. Cystic areas usually develop from coalescence of mucinous or microcystic areas in Antoni B tissue of the schwannoma. Hemorrhagic degeneration or necrosis due to the characteristic vascular abnormalities of these lesions may cause the formation of tiny cysts but not large ones. The formation of an associated arachnoid cyst and, rarely, the formation of cysts in glandular or pseudoglandular elements have also been reported. Awareness of the potential for partially or largely cystic schwannoma of the acoustic or trigeminal nerve is important for both the differential diagnosis and surgical planning. Surgically proven large cysts (occupying more than 50% of tumour volume) were detected preoperatively by computed tomography (CT) in 7 of the 35 cases of acoustic nerve schwannoma and both cases of trigeminal nerve schwannoma managed surgically at the authors' institution between 1980 and 1990. In a review of the literature the authors found descriptions of low-attenuation regions in CT scans for an average of 13% of acoustic and 29% of trigeminal nerve sheath tumours. Magnetic resonance imaging, ideally performed after intravenous administration of contrast material, also plays an important role in the detection and delineation of these tumour cysts.

Multiple sclerosis: the impact of MR imaging.

MR imaging has had a significant impact on the understanding of multiple sclerosis. The procedure now plays an important role in initial diagnostic workup, replacing some other radiologic and paraclinical tests and often confirming clinically suggested locations of lesions. It also has contributed greatly to the understanding of the natural history of this disease, allowing objective assessment of disease load, detection of asymptomatic lesions, and differentiation between acute and chronic lesions. MR imaging is highly sensitive to inflammation and demyelination caused by multiple sclerosis, and although there is a long differential diagnosis for some of the MR findings, increasing experience has defined a number of relatively specific criteria for multiple sclerosis. Recent advances may allow faster imaging and highly objective lesion quantification, which will aid in therapeutic trials.

The erythroid-specific protein cGATA-1 mediates distal enhancer activity through a specialized beta-globin TATA box.

The erythroid-specific protein cGATA-1 regulates the chick beta-globin gene through GATA sequences present at the canonical TATA location in the promoter as well as the distal 3' enhancer. We have analyzed beta-globin transcription in transfected erythroid cells and in erythroid extracts to determine whether cGATA-1 binding at -30 regulates promoter or enhancer activity. The interaction of both cGATA-1 and TFIID at different times with the -30 GATA site is required for efficient beta-globin expression in vivo, and the GATA enhancer site can functionally replace the TATA element in the beta-globin promoter. TFIID initiates transcription in vitro by complexing with adaptor proteins and displacing cGATA-1 from the -30 GATA site. Mutations that abolish TFIID binding to the -30 GATA box inactivate the promoter, whereas elimination of cGATA-1 binding to this site selectively diminishes enhancer-dependent transcription. We propose that interaction of cGATA-1 with the distal 3' enhancer and the specialized TATA box confers erythroid specificity to the initiation complex by mediating promoter-enhancer communication. Thus, one mechanism of action for tissue-specific proteins that recognizes noncanonical TATA motifs is to enable TFIID to be regulated by distal control elements. In this way, the initiation complex can be responsive to specific regulators that may not recognize a canonical TFIID-TATA structure.

Calcified herniations of the thoracic disk: role of magnetic resonance imaging and computed tomography in surgical planning.

Herniations of thoracic disks are uncommon, and their surgical management can be challenging. Magnetic resonance imaging (MRI) is rapidly becoming the method of choice for assessing degenerative disease in thoracic disks. However, calcification may be difficult to detect with MRI and plain films alone. The authors report a case in which MRI and myelography underestimated the true extent of disk calcification, the detection of which would have altered the initial surgical approach.

Erythroid-specific activation and derepression of the chick beta-globin promoter in vitro.

Transcriptionally active extracts were prepared from chick red cells isolated at different stages of development. The template activity of cloned beta-globin genes is highest in extracts from definitive red cells, where the endogenous gene is normally expressed, and lowest in extracts from primitive red cells or nonerythroid tissues. This system has been used to identify regulatory elements and to assign functions to the proteins that bind within the beta-globin promoter. Regulation of expression is achieved, in part, by factors whose composition changes during red cell development. Two proteins, PAL and CON, bind at adjacent sites but have opposite effects on transcription in vitro. Levels of PAL, a potent repressor, are highest in mature red cells while those of CON, an activator, are highest in actively transcribing red cells. The effect of PAL can be overcome by blocking its binding site with a protein having a similar recognition sequence but a dissimilar function.

Preferential enhancement by 2-mercaptoethanol of IL-2 responsiveness of T blast cells from old over young mice is associated with potentiated protein kinase C translocation.

Certain thiol compounds have been shown to enhance the T cell-dependent immune response of mice in vivo and the proliferation of T cells in vitro. The magnitude of augmentation is often greater in old than young mice. We hypothesized that the metabolic process that is preferentially up-regulated by thiol compounds in T cells from old mice may reflect a rate-limiting process which contributes to immunosenescence in aging mice. Because IL-2 dependent T cell proliferation in vitro is positively correlated with the strength of T cell-dependent immune response in vivo, we investigated the effects of 2-ME on (a) IL-2 synthesis in vitro, (b) the IL-2-IL-2R binding interaction, and (c) the translocation of PKC from the cytosol to the membrane in Con A-activated splenic T cells from young and old C57BL/6 and C57BL/s mice. The results demonstrated that 2ME does not preferentially enhance the synthesis or secretion of IL-2. Neither the binding affinity of IL-2 to the IL-2R nor the number of receptors on activated T cell blasts differed between young and old mice. At the post-receptor binding level, the magnitude of the translocation of PKC from the cytosol to the membrane was significantly greater in the T blast cells from old than young mice. The preferential enhancement of IL-2-dependent proliferation of T cells from old mice by 2ME is therefore associated with a potentiated translocation of PKC. This would suggest that the metabolic event involved in the translocation of PKC in T cells is vulnerable to aging.

Increase in Tac mRNA expression in natural killer-like cell line in response to interleukin-1 and interleukin-2 shown at the population and cellular level by in situ hybridization.

Our previous results have shown that the increased expression of the Tac component of the interleukin-2 (IL-2) receptor at the surface of a natural killer-like cell line (YTN10) in response to interleukin-1 (IL-1) is associated with an eightfold increase in Tac mRNA production. Tac expression was detected by flow cytometry and anti-Tac binding studies, and mRNA synthesis was assessed by Northern and dot blots. These findings have now been extended utilizing recombinant IL-1 and autoradiographic techniques in experiments using both the parent YT and YTN10 cells. In situ hybridization experiments have shown that the increase in mRNA expression is manifested both in the percentage of cells expressing it and in the level of expression per cell. Consistent results were obtained in both sublines. In addition, exposure of both sublines to recombinant IL-2 also induced increased Tac mRNA synthesis, while inducing only a marginal increase in Tac expression at the membrane level.

In situ hybridization analysis of the age-associated decline in IL-2 mRNA expressing murine T cells.

Only a small decrease in the number of L3T4- cells was observed in the Con A-stimulated splenocyte cultures of old mice as compared to young, which cannot account for the threefold decrease in IL-2 production. Northern and dot blot analysis of RNA from splenocytes containing equivalent numbers of L3T4+ cells from young and old mice showed that cells from old mice express less IL-2 mRNA after mitogenic stimulation than cells from young mice. Direct analysis by in situ hybridization of stimulated splenocytes from young and old mice then showed approximately a threefold decrease in the percentage of IL-2 mRNA expressing cells in the spleens of old mice as compared to young (8.7 +/- 4.1% old; 28.7 +/- 11.7% young). The average level of expression of IL-2 mRNA was not significantly different between cells from young and old mice; however, there were approximately 40% fewer cells expressing an intermediate to high amount of IL-2 mRNA in old mice as compared to young (26.3% vs 41.8%). These data suggest that the decrease in IL-2 production with age is associated primarily with a decrease in the frequency of IL-2 mRNA-expressing cells in old mice, especially in those cells expressing intermediate to high levels of IL-2 mRNA.