Experimental upper bound and theoretical expectations for parity-violating neutron spin rotation in 4He.

Neutron spin rotation is expected from quark-quark weak interactions in the standard model, which induce weak interactions among nucleons that violate parity. We present the results from an experiment searching for the effect of parity violation via the spin rotation of polarized neutrons in a liquid 4He medium. The value for the neutron spin rotation angle per unit length in 4He, d ϕ / d z = [ + 2.1 ± 8.3 (stat.) - 0.2 + 2.9 (sys.) ] × 10 - 7 rad/m, is consistent with zero. The result agrees with the best current theoretical estimates of the size of nucleon-nucleon weak amplitudes from other experiments and with the expectations from recent theoretical approaches to weak nucleon-nucleon interactions. In this paper we review the theoretical status of parity violation in the n → + 4He system and discuss details of the data analysis leading to the quoted result. Analysis tools are presented that quantify systematic uncertainties in this measurement and that are expected to be essential for future measurements.

Transverse relaxation of cerebrospinal fluid depends on glucose concentration.

PURPOSE: To evaluate the biophysical processes that generate specific T2 values and their relationship to specific cerebrospinal fluid (CSF) content. MATERIALS AND METHODS: CSF T2s were measured ex vivo (14.1T) from isolated CSF collected from human, rat and non-human primate. CSF T2s were also measured in vivo at different field strength in human (3 and 7T) and rodent (1, 4.7, 9,4 and 11.7T) using different pulse sequences. Then, relaxivities of CSF constituents were measured, in vitro, to determine the major molecule responsible for shortening CSF T2 (2s) compared to saline T2 (3s). The impact of this major molecule on CSF T2 was then validated in rodent, in vivo, by the simultaneous measurement of the major molecule concentration and CSF T2. RESULTS: Ex vivo CSF T2 was about 2.0s at 14.1T for all species. In vivo human CSF T2 approached ex vivo values at 3T (2.0s) but was significantly shorter at 7T (0.9s). In vivo rodent CSF T2 decreased with increasing magnetic field and T2 values similar to the in vitro ones were reached at 1T (1.6s). Glucose had the largest contribution of shortening CSF T2in vitro. This result was validated in rodent in vivo, showing that an acute change in CSF glucose by infusion of glucose into the blood, can be monitored via changes in CSF T2 values. CONCLUSION: This study opens the possibility of monitoring glucose regulation of CSF at the resolution of MRI by quantitating T2.

A slow neutron polarimeter for the measurement of parity-odd neutron rotary power.

We present the design, description, calibration procedure, and an analysis of systematic effects for an apparatus designed to measure the rotation of the plane of polarization of a transversely polarized slow neutron beam as it passes through unpolarized matter. This device is the neutron optical equivalent of a crossed polarizer/analyzer pair familiar from light optics. This apparatus has been used to search for parity violation in the interaction of polarized slow neutrons in matter. Given the brightness of existing slow neutron sources, this apparatus is capable of measuring a neutron rotary power of dϕ/dz = 1 × 10(-7) rad/m.

Image-guided, direct convective delivery of glucocerebrosidase for neuronopathic Gaucher disease.

OBJECTIVE: To determine if convection-enhanced delivery (CED) of glucocerebrosidase could be used to treat targeted sites of disease progression in the brain and brainstem of a patient with neuronopathic Gaucher disease while monitoring enzyme distribution using MRI. METHODS: A CED paradigm in rodents (n = 8) and primates (n = 5) that employs co-infusion of a surrogate MRI tracer (gadolinium diethylenetriamine penta-acetic acid [Gd-DTPA]) with glucocerebrosidase to permit real-time monitoring of distribution was developed. The safety and feasibility of this delivery and monitoring paradigm were evaluated in a patient with type 2 Gaucher disease. RESULTS: Animal studies revealed that real-time, T1-weighted, MRI of Gd-DTPA accurately tracked enzyme distribution during CED. Targeted perfusion of clinically affected anatomic sites in a patient with neuronopathic Gaucher disease (frontal lobe and brainstem) with glucocerebrosidase was successfully performed. Real-time MRI revealed progressive and complete filling of the targeted region with enzyme and Gd-DTPA infusate. The patient tolerated the infusions without evidence of toxicity. CONCLUSIONS: Convection-enhanced delivery can be used to safely perfuse large regions of the brain and brainstem with therapeutic levels of glucocerebrosidase. Co-infused imaging surrogate tracers can be used to monitor and control the distribution of therapeutic agents in vivo. Patients with neuronopathic Gaucher disease and other intrinsic CNS disorders may benefit from a similar treatment paradigm.

AV.TK-mediated killing of subcutaneous tumors in situ results in effective immunization against established secondary intracranial tumor deposits.

Gene transfer vectors expressing herpes simplex thymidine kinase (HSVtk), in addition to direct killing of tumor cells, often have an associated local "bystander effect" mediated by metabolic coupling of tumor cells. A systemic antitumor effect mediated by the immune system, termed the distant bystander effect, has also been reported. We have observed the development of cytotoxic T-lymphocyte (CTL) populations and long-lasting antitumor immunity following treatment of subcutaneous tumors with an adenoviral vector expressing HSVtk (AV.TK) and ganciclovir (GCV) in rat glioma model. This vaccination effect seen with AV.TK/GCV treatment of subcutaneous tumor could even abrogate or retard growth of previously established secondary intracranial tumors.

Site-specific immune response to implanted gliomas.

OBJECT: Immunotherapy for glioblastoma has been uniformly ineffective. The immunological environment of the brain, with its low expression of major histocompatibility complex (MHC) molecules and limited access for inflammatory cells and humoral immune effectors due to the blood-brain barrier (BBB), may contribute to the failure of immunotherapy. The authors hypothesize that brain tumors are protected from immune surveillance by an intact BBB at early stages of development. To investigate the immunological characteristics of early tumor growth, the authors compared the host response to a glioma implanted into the brain and into subcutaneous tissue. METHODS: Samples of tumors growing in the brain or subcutaneously in rats were obtained for 7 consecutive days and were examined immunohistochemically for MHC Class I & II molecules, and for CD4 and CD8 lymphocyte markers. Additionally, B7-1 costimulatory molecule expression and lymphocyte-specific apoptosis were examined. CONCLUSIONS: On Days 3 and 4 after implantation, brain tumors displayed significantly lower MHC Class II expression and lymphocytic infiltration (p < 0.05). After Day 5, however, no differences were detected. The MHC Class II expressing cells within the brain tumors appeared to be infiltrating microglia. Minimal B7-1 expression combined with lymphocyte-specific apoptosis were detected in both brain and subcutaneous tumors. Low MHC Class II expression and low lymphocytic infiltration at early time points indicate the importance of the immunologically privileged status of the brain during early tumor growth. These characteristics disappeared at later time points, possibly because the increasing perturbation of the BBB alters the specific immunological environment of the brain. The lack of B7-1 expression combined with lymphocyte apoptosis indicates clonal anergy of glioma-infiltrating lymphocytes regardless of implantation site.

Neurocognitive sequelae of infants of diabetic mothers.

On the basis of animal models, it was hypothesized that infants of diabetic mothers (IDMs) would be at risk for suffering damage to the hippocampus primarily because of fetal iron deficiency, chronic hypoxia, and hypoglycemia. This, in turn, may result in impairments in recognition memory at a young age. To test this model, the memory of 6-month-old IDMs and control infants was evaluated with electrophysiological (event-related potential [ERP]) and behavioral (looking time) measures. At 12 months, the Bayley Scales of Infant Development was administered. Our ERP measures showed robust evidence consistent with memory deficits in the IDMs. In contrast, the looking time measures and the Bayley exam failed to distinguish between the groups. From these results it was concluded that the ERP, but not the behavioral, measures are able to detect, in an at-risk population, deficits in recognition memory that are thought to be mediated by damage to the hippocampus.

Production of reactive oxygen species after reperfusion in vitro and in vivo: protective effect of nitric oxide.

OBJECT: Thrombolytic treatments for ischemic stroke can restore circulation, but reperfusion injury, mediated by oxygen free radicals, can limit their utility. The authors hypothesized that, during reperfusion, nitric oxide (NO) provides cytoprotection against oxygen free radical species. METHODS: Levels of NO and oxygen free radicals were determined in both reoxygenation in vitro and reperfusion in vivo models using an NO electrochemical probe and high-performance liquid chromatography with the 2,3- and 2,5-dihydroxybenzoic acid trapping method, before and after addition of the NO donor diethanolamine nitric oxide (DEA/NO). Reoxygenation after anoxia produced a twofold increase in NO release by human fetal astrocytes and cerebral endothelial cells (p < 0.005). In both cell lines, there was also a two- to threefold increase in oxygen free radical production (p < 0.005). In human fetal astrocytes and cerebral endothelial cells given a single dose of DEA/NO, free radical production dropped fivefold compared with peak ischemic levels (p < 0.001). In a study in which a rat global cerebral ischemia model was used, NO production in a vehicle-treated group increased 48 +/- 16% above baseline levels after reperfusion. After intravenous DEA/NO infusion, NO reached 1.6 times the concentration of the postischemic peak in vehicle-treated animals. In vehicle-treated animals during reperfusion, free radical production increased 4.5-fold over basal levels (p < 0.01). After intravenous DEA/NO infusion, free radical production dropped nearly 10-fold compared with peak levels in vehicle-treated animals (p < 0.006). The infarct volume in the vehicle-treated animals was 111 +/- 16.9 mm3; after DEA/NO infusion it was 64.8 +/- 23.4 mm3 (p < 0.01). CONCLUSIONS: The beneficial effect of early restoration of cerebral circulation after cerebral ischemia is limited by reperfusion injury. These results indicate that NO release and oxygen free radical production increase during reperfusion, and suggest a possible early treatment of reperfusion injury using NO donors.

Vascular protection by chloroquine during brain tumor therapy with Tf-CRM107.

Tf-CRM107 is a conjugate of transferrin and a point mutant of diphtheria toxin that selectively kills cells expressing high levels of the transferrin receptor. Tf-CRM107 has been infused intratumorally into patients with malignant brain tumors. Although approximately half of the patients exhibit tumor responses, patients receiving higher doses of Tf-CRM107 may develop magnetic resonance image (MRI) evidence of toxicity indicative of small vessel thrombosis or petechial hemorrhage. Consistent with these clinical results we found that intracerebral injection of Tf-CRM107 into rats at total doses > or =0.025 microg causes brain damage detectable by MRI and histology. To widen the therapeutic window of Tf-CRM107, we explored ways to prevent this damage to the vasculature. We reasoned that the vasculature may be protected to a greater extent than tumor from Tf-CRM107 infused into brain parenchyma by i.v. injection of reagents with low blood-brain barrier permeability that block the toxicity of Tf-CRM107. Chloroquine, a well-characterized antimalarial drug, blocks the toxicity of diphtheria toxin and Tf-CRM107. Systemic administration of chloroquine blocked the toxicity of Tf-CRM107 infused intracerebrally in rats and changed the maximum tolerated dose of Tf-CRM107 from 0.2 to 0.3 microg. Moreover, chloroquine treatment completely blocked the brain damage detected by MRI caused by intracerebral infusion of 0.05 microg of Tf-CRM107. In nude mice bearing s.c. U251 gliomas, chloroquine treatment had little effect on the antitumor efficacy of Tf-CRM107. Thus, chloroquine treatment may be useful to reduce the toxicity of Tf-CRM107 for normal brain without inhibiting antitumor efficacy and increase the therapeutic window of Tf-CRM107 for brain tumor therapy.

Vascular endothelial growth factor (VEGF) modulates vascular permeability and inflammation in rat brain.

Vascular endothelial growth factor (VEGF) is an angiogenic growth factor that also induces vascular permeability and macrophage migration. VEGF expression is weak in normal adult brain, but is strongly upregulated in glioma cells and reactive astrocytes, suggesting that chronic overexpression of VEGF in the brain contributes to blood-brain barrier (BBB) breakdown. We examined the effects of chronic VEGF overexposure on the integrity of the BBB using the following approaches: 1) continuous intracerebral infusion of VEGF via miniosmotic pump; and 2) intracerebral injection of an adenoviral vector encoding the VEGF165 gene (AdCMV.VEGF). After 6 days both treatments produced approximately 10-fold breakdown of the BBB (as measured by transport of 14C-aminoisobutyric acid (AIB) from blood into brain) compared with the respective controls (albumin infusion or AdCMV.beta gal virus). BBB disruption in AdCMV.VEGF-treated brains was accompanied by a severe inflammatory response not observed in brains receiving AdCMV.beta gal or VEGF protein infusion, indicating that neither VEGF nor viral particles alone were responsible for the inflammatory response. However, injection of AdCMV.beta gal followed by VEGF infusion to the same site also elicited inflammation. Chronic overexposure of normal brain to VEGF also increased intercellular adhesion molecule-1 (ICAM-1) and major histocompatibility complex (MHC) class I and II expression. Although VEGF itself is not inflammatory, VEGF may modulate immune responses in the central nervous system (CNS) by opening the BBB, altering the immunoprivileged status of the brain, and allowing contact between normally sequestered CNS antigens and blood-borne immune mediators.

Vascular endothelial growth factor (vascular permeability factor) expression in injured rat brain.

We investigated the expression of vascular endothelial growth factor (VEGF)/vascular permeability factor (VPF) in stab and freeze brain injury models in rats. Immunohistochemical staining with anti-VEGF antibodies demonstrated an increase in VEGF-positive cells in and around both lesions. Morphologically, the injury-induced VEGF-positive cells resembled astrocytes. Double immunofluorescent staining for the astrocytic marker glial fibrillary acidic protein (GFAP) and VEGF demonstrated directly that VEGF-positive cells which appeared in response to these injuries were astrocytes. VEGF expression in astrocytes was maximal on days 3 and 4 after injury in terms of both cell number and affected area. The increase in VEGF-positive cells was more widespread in the freeze lesion than in the stab wound, and occurred in both the lesioned and nonlesioned hemispheres. VEGF-positive cells were still present 3 weeks after both injuries, but their numbers were reduced and their distribution became limited to the immediate vicinity of the lesions. These observations indicate that astrocytes react to injury by increasing VEGF expression, suggesting that VEGF might participate in the central nervous system response to injury.

Mechanism of dexamethasone suppression of brain tumor-associated vascular permeability in rats. Involvement of the glucocorticoid receptor and vascular permeability factor.

Brain tumor-associated cerebral edema arises because tumor capillaries lack normal blood-brain barrier function; vascular permeability factor (VPF, also known as vascular endothelial growth factor, VEGF) is a likely mediator of this phenomenon. Clinically, dexamethasone reduces brain tumor-associated vascular permeability through poorly understood mechanisms. Our goals were to determine if suppression of permeability by dexamethasone might involve inhibition of VPF action or expression, and if dexamethasone effects in this setting are mediated by the glucocorticoid receptor (GR). In two rat models of permeability (peripheral vascular permeability induced by intradermal injection of 9L glioma cell-conditioned medium or purified VPF, and intracerebral vascular permeability induced by implanted 9L glioma), dexamethasone suppressed permeability in a dose-dependent manner. Since 80% of the permeability-inducing activity in 9L-conditioned medium was removed by anti-VPF antibodies, we examined dexamethasone effects of VPF expression in 9L cells. Dexamethasone inhibited FCS- and PDGF-dependent induction of VPF expression. At all levels (intradermal, intracranial, and cell culture), dexamethasone effects were reversed by the GR antagonist mifepristone (RU486). Dexamethasone may decrease brain tumor-associated vascular permeability by two GR-dependent mechanisms: reduction of the response of the vasculature to tumor-derived permeability factors (including VPF), and reduction of VPF expression by tumor cells.

Toxicity studies and distribution dynamics of retroviral vectors following intrathecal administration of retroviral vector-producer cells.

The use of intrathecal, retroviral-mediated transfer of the herpes simplex thymidine kinase (HStk) gene and subsequent ganciclovir (GCV) administration has recently been shown to improve survival in a rat model of leptomeningeal carcinomatosis. Clinical application of this approach is attractive because access to the cerebrospinal fluid (CSF) space is relatively noninvasive and distribution of producer cells and vectors may be facilitated by circulation of CSF, overcoming distribution problems inherent in solid tumors. However, meningeal inflammation, transduction and injury to normal CNS tissue, proliferation of the xenogeneic producer cells in the subarachnoid space, immune-mediated injury, and development of hydrocephalus are possible complications of intraventricular or intrathecal administration of vector-producer cells. In addition, the dynamics of producer cell and vector distribution in the CSF are unknown. To address these issues, we evaluated the safety of this approach for gene delivery and assessed the dynamics of distribution of producer cells and retroviral vectors in rats and non-human primates. In rats, transduction of normal central nervous system (CNS) structures surrounding the subarachnoid space was evaluated after intrathecal and intraventricular injections of beta-galactosidase and HStk vector-producer cells, with and without GCV. In primates, beta-galactosidase and HStk vector-producer cells were injected intraventricularly and GCV was administered either intrathecally or intravenously. Toxicity was evaluated by neurologic examination, serial gadolinium-enhanced MRI scans of the brain, and blood and CSF profiles. A subgroup of monkeys received repeated intraventricular injection of vector-producer cells and intravenous GCV. The titer of retroviral-vector was measured in cisternal and lumbar CSF samples after repeated producer cell injection.(ABSTRACT TRUNCATED AT 250 WORDS)

In situ cyclopentenyl cytosine infusion for the treatment of experimental brain tumors.

Cyclopentenylcytosine (CPEC; NSC 375575) is a pyrimidine nucleoside analogue that has potent antitumor effects when tested in vitro and also when tested in experimental tumors outside the central nervous system. CPEC exerts its antiproliferative effect through inhibition of CTP synthetase and consequent depletion of CTP and dCTP pools required for cell replication. Due to its poor penetration of the bloodbrain barrier, CPEC has failed to demonstrate therapeutic efficacy in experimental brain tumors after systemic administration. We therefore examined the in vivo activation, distribution, and antitumor effect of CPEC after long-term regional infusion of the drug directly into experimental brain tumors in rats. HPLC analysis of CPEC incubated with homogenized human brain and brain tumor tissue showed minimal degradation of the drug over 24 h. Analysis of rat cerebral 9L gliosarcoma infused with tritium-labeled CPEC demonstrated intratumoral accumulation of the active metabolite CPEC-triphosphate and concomitant depletion of CTP to a much greater extent in tumor tissue than in the adjacent brain. Tumor tissue UTP also decreased, but no significant effects on other ribonucleoside triphosphates were detected. Only trace amounts (< 1%) of CPEC and its metabolites reached peripheral sites, including the liver and kidneys, after intratumoral infusion. Rats treated with continuous intratumoral infusion of CPEC for 4 weeks using s.c. implanted osmotic pumps survived significantly longer than control rats receiving intratumoral saline or i.p. CPEC (P < 0.0001). Long-term intratumoral infusion of CPEC was not associated with any detectable toxicity. Our results support the feasibility of using intratumoral administration of CPEC as a regional therapy for malignant brain tumors.

Adenovirally mediated gene transfer into experimental solid brain tumors and leptomeningeal cancer cells.

Among the appealing features of adenoviruses as vectors for transfer of genes into the central nervous system (CNS) are that they are not neurotoxic, they can accommodate the insertion of several large genes, they are not associated with the hazards of insertional mutagenesis, and they can be concentrated to a high-titer preparation. The authors evaluated the feasibility of using adenovirally mediated gene transfer into cultured human glioma cells and in rat models of solid brain tumors and meningeal cancer. Replication-deficient adenoviral vector particles carrying a nuclear-localizing lacZ gene were injected into established 9L cerebral gliomas in Fischer rats. In addition, the adenoviral vector was injected into the subarachnoid space, either simultaneously with intrathecal tumor inoculation or after establishing leptomeningeal cancer. The brains and spinal cords were removed at various intervals for histochemical evaluation for beta-galactosidase activity using X-Gal staining. Additional rats received a stereotactic intracerebral injection of the vector into normal brain. No clinical abnormalities were observed in the injected rats. Injection of the adenoviral vector into normal brain resulted in diffuse transduction of astrocytes, microglia, neurons, and endothelial cells at the injection site. Injection of a high-concentration vector preparation into cerebral gliomas resulted in effective tumor transduction. Intrathecal injection of the vector in rats with meningeal cancer resulted in transduction of the infiltrating tumor in the subarachnoid space when injections were given simultaneously with, or 7 days after, tumor inoculation. Transduction rates of both solid and leptomeningeal tumors correlated with the number of injected particles. These results suggest that adenoviral vectors can efficiently transduce solid brain tumors and that the vectors survive in the cerebrospinal fluid for a sufficient period of time to allow leptomeningeal tumor transduction. Adenoviral vector should be evaluated for its potential use in therapeutic gene transfer approaches in malignancies of the CNS.

The effect of thymidine kinase transduction and ganciclovir therapy on tumor vasculature and growth of 9L gliomas in rats.

Eradication of malignant brain tumors by in situ intratumoral, retrovirally mediated transfer of the herpes simplex virus thymidine kinase (HSVtk) gene, which sensitizes the tumor cells to ganciclovir, has recently been demonstrated in animal models. The observation that tumors studied in vitro and in animals can be completely eliminated despite only partial transduction of the tumor suggests a bystander mechanism that affects nontransduced tumor cells. Such a bystander effect is not completely understood and may represent a combination of several factors that lead to tumor eradication. Endothelial cells of the tumor blood vessels were shown to occasionally integrate the retroviral vector and thus become sensitized to ganciclovir. In the presence of vector-producer cells, which continuously release infectious viral particles, diffuse multifocal hemorrhages occurred during ganciclovir administration. When the tumor was composed of cells that had been transduced with the thymidine kinase gene before inoculation, no infectious viral particles were present within the tumor, no transduction of endothelial cells occurred, and no hemorrhages were observed during ganciclovir therapy. These observations suggest that tumor regression may be due, in part, to destruction of in vivo HSVtk-transduced endothelial cells after exposure to ganciclovir, resulting in tumor ischemia as one possible bystander mechanism. The authors investigated this hypothesis using the subcutaneous 9L gliosarcoma tumor model in Fischer rats. The tumors were evaluated with Doppler color-flow and ultrasound imaging during the various phases of the study. Twenty rats received intratumoral injections of HSVtk retroviral vector-producer cells (6 x 10(7) cells/ml) 21 days after bilateral flank tumor inoculation. Ten rats were subsequently treated with intraperitoneal ganciclovir (15 mg/kg/ml twice a day) for 14 days starting on Day 7 after producer cell injection; 10 control rats received intraperitoneal saline injections (1 ml twice a day) instead of ganciclovir. Ultrasound and flow images were obtained before cell injection, before and during ganciclovir or saline administration, and after cessation of treatment. The number, location, and ultrasonographic appearance of tumor vessels and the tumor volumes were recorded. The number of blood vessels in the tumors increased over time in both groups before treatment. Intratumoral cell injection without ganciclovir administration did not influence tumor growth or intratumoral vasculature. However, tumor vasculature decreased after initiation of ganciclovir therapy in the HSVtk-transduced tumors (p < 0.05). Early patchy or diffuse necrotic changes associated with ultrasonographic evidence of scattered intratumoral hemorrhage occurred in tumors treated with ganciclovir.(ABSTRACT TRUNCATED AT 400 WORDS)

Growth inhibition, tumor maturation, and extended survival in experimental brain tumors in rats treated with phenylacetate.

Phenylacetate is a naturally occurring plasma component that suppresses the growth of tumor cells and induces differentiation in vitro. To evaluate the in vivo potential and preventive and therapeutic antitumor efficacy of sodium phenylacetate against malignant brain tumors, Fischer 344 rats (n = 50) bearing cerebral 9L gliosarcomas received phenylacetate by continuous s.c. release starting on the day of tumor inoculation (n = 10) using s.c. osmotic minipumps (550 mg/kg/day for 28 days). Rats with established brain tumors (n = 12) received continuous s.c. phenylacetate supplemented with additional daily i.p. dose (300 mg/kg). Control rats (n = 25) were treated in a similar way with saline. Rats were sacrificed during treatment for electron microscopic studies of their tumors, in vivo proliferation assays, and measurement of phenylacetate levels in the serum and cerebrospinal fluid. Treatment with phenylacetate extended survival when started on the day of tumor inoculation (P < 0.01) or 7 days after inoculation (P < 0.03) without any associated adverse effects. In the latter group, phenylacetate levels in pooled serum and cerebrospinal fluid samples after 7 days of treatment were in the therapeutic range as determined in vitro (2.45 mM in serum and 3.1 mM in cerebrospinal fluid). Electron microscopy of treated tumors demonstrated marked hypertrophy and organization of the rough endoplasmic reticulum, indicating cell differentiation, in contrast to the scant and randomly distributed endoplasmic reticulum in tumors from untreated animals. In addition, in vitro studies demonstrated dose-dependent inhibition of the rate of tumor proliferation and restoration of anchorage dependency, a marker of phenotypic reversion. Phenylacetate, used at clinically achievable concentrations, prolongs survival of rats with malignant brain tumors through induction of tumor differentiation. Its role in the treatment of brain tumors and other cancers should be explored further.

Intrathecal gene therapy for malignant leptomeningeal neoplasia.

In meningeal carcinomatosis, retroviral vector-producer cells can be introduced into the thecal sac and circulate in the cerebrospinal fluid to reach malignant tumor cells in the leptomeninges, release vector particles, and selectively infect and transfer a gene of interest to these cells. Gene transfer experiments with the lacZ gene and in vitro retroviral titer measurements showed that retroviral vectors can survive in the cerebrospinal fluid, retain their infectivity, and successfully transduce tumor cells. To examine the potential of intrathecal gene therapy, we evaluated the antitumor efficacy of in situ transduction with the herpes simplex-thymidine kinase gene followed by ganciclovir therapy in a rat model of leptomeningeal neoplasia. Fischer rats were inoculated via a subarachnoid catheter implanted at the upper thoracic level, and thymidine kinase vector-producer cells were injected into the subarachnoid space the day of tumor inoculation. Seven days later, rats received ganciclovir for 14 days by daily i.p. injections (30 mg/kg/ml) or intrathecal injections (25 micrograms/kg or 600 micrograms/kg) for 14 days. To evaluate possible enhancement of tumor eradication by the ability of helper virus to package the vector in the cells and further extend gene transfer, additional rats received thymidine kinase vector-producer cells that had been previously coinfected with a replication-competent retrovirus (4070A). In all groups, control rats received i.p. or intrathecal saline injections. Ganciclovir administration i.p. resulted in significant prolongation of survival in rats given injections of thymidine kinase vector-producer cells. Injection of producer cells coinfected with the 4070A retrovirus did not improve antitumor efficacy. Intrathecal administration of ganciclovir (low and high doses) did not extend survival; histological examination of the spinal cords showed elimination of the infiltrative tumor in the leptomeninges, but residual tumor mass was present at the inoculation site, consistent with limited penetration of topical ganciclovir into the tumor. These results support the potential application of gene therapy using the thymidine kinase/ganciclovir approach for treatment of meningeal carcinomatosis.

In vivo transfer of the human interleukin-2 gene: negative tumoricidal results in experimental brain tumors.

The authors have recently shown the feasibility of eradicating brain tumors using in vivo retroviral-mediated transduction of tumors with the herpes simplex thymidine kinase (HStk) gene and ganciclovir therapy. However, thymidine kinase-transduced subcutaneous tumors in immunocompromised (athymic) mice were less responsive to this therapy than in immunocompetent animals, suggesting a role of the immune system in the process of tumor eradication. Broad suppression of humoral and cell-mediated immunity is found in patients with malignant gliomas. Interleukin-2 (IL-2) production and IL-2 receptor expression are decreased in gliomas patients. These findings and the proposed association between lymphocytic infiltration of brain tumors and survival suggest that immune response modifiers may be useful in treating glioma patients. To evaluate the role of local cytokine expression by tumor cells, alone or combined with HStk gene transfer and ganciclovir therapy, the authors investigated the efficacy of tumor (9L gliosarcoma) eradication in Fischer rats by in vitro and in vivo tumor transduction with the IL-2 gene alone or with a combined vector carrying both the HStk and IL-2 genes. Tumors injected with HStk vector-producer cells alone, with or without ganciclovir, and rats inoculated in the brain and subcutaneously with 9L cells that had previously been transduced in vitro served as controls. Murine vector-producer cells (3 x 10(6)/50 microliters) were injected into the brain tumors 7 days after tumor inoculation. Ganciclovir (15 mg/kg) was administered intraperitoneally twice daily for 10 days to animals that received HStk with or without IL-2 vector-producer cells, starting 5 days after producer-cell injection. The experiment was repeated with continuous daily treatment of all rats with oral dexamethasone (0.5 mg/kg). Rats were sacrificed 21 days after tumor inoculation, and the brains were removed for histological and immunohistochemical analysis for IL-2. Within each experimental group, tumors were found in a similar proportion in the dexamethasone-treated and untreated rats. Large brain tumors developed in all 10 rats that had been inoculated with 9L cells which had been pretransduced in vitro with the IL-2 gene, whereas only three of eight rats receiving subcutaneous inoculation of similar cells developed palpable tumors. No enhancement of tumor eradication was observed by adding the IL-2 gene in the HStk vector construct compared to the use of the vector with HStk alone. Lymphocytic infiltration was absent in all dexamethasone-treated rats but was observed in all treatment groups not receiving steroids.(ABSTRACT TRUNCATED AT 400 WORDS)

Toxicity of an antitumor ribonuclease to Purkinje neurons.

Purkinje cell toxicity is one of the characteristic features of the Gordon phenomenon, a syndrome manifested by ataxia, muscular rigidity, paralysis, and tremor that may lead to death (Gordon, 1933). Two members of the RNase superfamily found in humans, EDN (eosinophil-derived neurotoxin) and ECP (eosinophil cationic protein), cause the Gordon phenomenon when injected intraventricularly into guinea pigs or rabbits. We have found that another member of the RNase superfamily, an antitumor protein called onconase, isolated from Rana pipiens oocytes and early embryos, will also cause the Gordon phenomenon when injected into the cerebrospinal fluid of guinea pigs at a dose similar to that of EDN (LD50, 3-4 micrograms). Neurologic abnormalities of onconase-treated animals were indistinguishable from those of EDN-treated animals, and histology showed dramatic Purkinje cell loss in the brains of onconase-treated animals. The neurotoxic activity of onconase correlates with ribonuclease activity. Onconase modified by iodoacetic acid to eliminate 70% and 98% of the ribonuclease activity of the native enzyme displays a similar decrease in ability to cause the Gordon phenomenon. In contrast, the homologous bovine pancreatic RNase A injected intraventricularly at a dose 5000 times greater than the LD50 dose of EDN or onconase is not toxic and does not cause the Gordon phenomenon. A comparison of the RNase activities of EDN, onconase, and bovine pancreatic RNase A using three pancreatic RNA substrates demonstrates that onconase is orders of magnitude less active enzymatically than EDN and RNase A. Thus, another member of the RNase superfamily in addition to EDN and ECP can cause the Gordon phenomenon.(ABSTRACT TRUNCATED AT 250 WORDS)