Foamy virus as a gene transfer vector to the central nervous system.

Engineered foamy virus (FV) vectors have been lauded for their superior safety profiles and stable integration patterns compared to their gammaretroviral counterparts. The drawback has been the belief that FV incorporation is cell cycle-dependent, thereby limiting its utility in post-mitotic tissues such as the central nervous system. In this brief communication, we challenged this theory by examining FV in vivo. We injected equal titers of FV and lentivirus (LV) into the adult rat brain and found that at 1 week, FV transduced a significantly greater volume of bromodeoxyuridine (BrdU)-negative brain parenchyma than did LV. By 8 weeks, however, the volume of transduced tissue was greatly reduced--comparable to LV-and restricted to BrdU+. Taken together, these data implicate a role for FV in short-term gene delivery strategies to the CNS.

Influence of tumor-derived interleukin 1 on melanoma-endothelial cell interactions in vitro.

Human melanoma cell lines that express high constitutive levels of the metastasis-associated marker intercellular adhesion molecule 1 (ICAM-1) were found to secrete interleukin 1 (IL-1) in vitro. Experiments with neutralizing antibodies showed that this cytokine was responsible for their expression of ICAM-1 but not that of two other progression/metastasis markers, Muc-18 and Gp IIb/IIIa. The IL-1 present in melanoma-conditioned medium induced the expression of vascular cell adhesion molecule 1, endothelial-leukocyte adhesion molecule 1, and ICAM-1 on human umbilical vein endothelial cells (ECs) in culture and increased the rate at which melanoma cells and ECs adhered to each other. IL-1-producing melanoma lines adhered significantly more rapidly to ECs than did non-IL-1-producing lines, and this enhancement was reduced by prior incubation of the melanoma cells with neutralizing anti-IL-1 antibodies. Similarly, endothelial cells treated with conditioned medium from IL-1-producing melanoma lines adhered significantly more rapidly to melanoma cells than did ECs treated with medium from non-IL-1-producing melanoma lines, and this enhancement was abolished by addition of anti-IL-1 antibodies to EC cultures in conditioned medium. Blocking antibodies to endothelial vascular cell adhesion molecule 1, endothelial-leukocyte adhesion molecule 1, and ICAM-1 failed to inhibit melanoma-EC adhesion, but an antibody to tumor cell GpIIb/IIIa did block adhesion by up to 44%. The ability to secrete IL-1 could increase the metastatic potential of melanoma cells by stimulating tumor cell-EC adhesion.

Development and application of radioimmunoassays for interleukin-1 alpha and interleukin-1 beta.

Specific and sensitive radioimmunoassay (RIA) systems for human IL-1 alpha and IL-1 beta, using sheep polyclonal antisera, have been developed. The assays, which do not require prior extraction of IL-1, have been used to quantify IL-1 alpha and IL-1 beta in synovial fluid and plasma and to quantify intracellular and extracellular IL-1 alpha and IL-1 beta produced by human monocytes stimulated with endotoxin.

Cytokine-mediated inflammatory hyperalgesia limited by interleukin-10.

1. The effect of interleukin-10 (IL-10) upon the hyperalgesic activities in rats of bradykinin, tumor necrosis factor alpha (TNF alpha), interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), interleukin-8 (IL-8), prostaglandin E2 (PGE2) and carrageenin were investigated in a model of mechanical hyperalgesia. 2. Hyperalgesic responses to bradykinin (1 micrograms) were inhibited in a dose-dependent manner by prior treatment with IL-10 (1-100 ng). 3. Hyperalgesic responses to TNF alpha (2.5 pg), IL-1 beta (0.5 pg) and IL-6 (1.0 ng) but not to IL-8 (0.1 ng) and PGE2 (50 ng and 100 ng) were inhibited by prior treatment with IL-10 (10 ng). 4. Hyperalgesic responses to carrageenin (100 micrograms) were inhibited by IL-10 (10 ng) when this cytokine was injected before but not after the carrageenin. 5. A monoclonal antibody to mouse IL-10 potentiated the hyperalgesic responses to carrageenin (10 micrograms) and TNF alpha (0.025 pg) but not that to IL-8 (0.01 ng). 6. In in vitro experiments in human peripheral blood mononuclear cells (MNCs), IL-10 (0.25-4.0 ng ml-1) inhibited in a dose-dependent manner PGE2 production by MNCs stimulated with IL-1 beta (1-64 ng ml-1) or endotoxin (lipopolysaccharide, LPS, 1 iu = 143 pg ml-1) but evoked only small increases in IL-1ra production. 7. These data suggest that IL-10 limits the inflammatory hyperalgesia evoked by carrageenin and bradykinin by two mechanisms: inhibition of cytokine production and inhibition of IL-1 beta evoked PGE2 production. Our data suggest that the latter effect is not mediated via IL-10 induced IL-Ira and may result from suppression by IL-10 of prostaglandin H synthase-2 (COX-2).

Variability in LPS composition, antigenicity and reactogenicity of phase variants of Bordetella pertussis.

Comparison of lipopolysaccharides (LPS) from phase variants of different strains of Bordetella phase variants of different strains of Bordetella pertussis has shown a difference in their composition, antigenicity and reactogenicity. Phase I variants of B. pertussis, with the exception of strain 134, contain a preponderance of LPS I whereas the major component of LPS of phase IV variants is LPS II. Sera raised to LPSs of phase I strains, other than 134, cross-react with each other but not with phase IV LPSs; and similarly all sera raised to phase IV LPSs cross-react with each other and with LPS from 134 phase I. The LPSs of all phase I variants, including that of 134, are approximately ten-fold or more reactive in the limulus amoebocyte lysate assay (LAL) than phase IV LPSs. In the human mononuclear cell pyrogen assay phase IV LPSs also stimulated a lower response than phase I LPSs. The B. pertussis phase I LPSs are 10-times more reactive than Escherichia coli standard endotoxin in the LAL assay but 100-times less reactive than E. coli LPS in the monocyte test for pyrogen. The SDS-PAGE profiles of B. pertussis LPSs are quite different from those of B. parapertussis and B. bronchiseptica strains. B. pertussis LPSs produced a typical lipo-oligosaccharide (LOS) pattern. B. bronchiseptica LPS produced a similar pattern but was antigenically distinct from B. pertussis LPSs I and II. B. parapertussis in contrast produced a ladder pattern typical of smooth type LPS.

Assay of pyrogens by interleukin-6 release from monocytic cell lines.

A novel in-vitro system has been developed for the detection and quantification of pyrogen in pharmaceutical products. The measured variable was evoked secretion of the pyrogenic cytokine interleukin-6 from MONO MAC 6 monocytic cells incubated with the product. The interleukin-6 was detected using a specific and sensitive ELISA developed for this purpose. The test system detected pyrogenic contamination in 3 batches of therapeutic human serum albumin which had caused adverse reactions in recipients. The contamination was not detected in conventional tests: the rabbit pyrogen test and the limulus amoebocyte lysate test.

Gene therapy in clinical medicine.

Although the field of gene therapy has experienced significant setbacks and limited success, it is one of the most promising and active research fields in medicine. Interest in this therapeutic modality is based on the potential for treatment and cure of some of the most malignant and devastating diseases affecting humans. Over the next decade, the relevance of gene therapy to medical practices will increase and it will become important for physicians to understand the basic principles and strategies that underlie the therapeutic intervention. This report reviews the history, basic strategies, tools, and several current clinical paradigms for application.

Patterns of adjustment of discharged psychiatric patients as measured by mailed questionnaires.

Mailed questionnaires were administered to 34 former psychiatric patients of a provincial mental institution and their relatives in order to assess frequency of psychopathological and social behavior, and to estimate validity and return rate of the forms. The sample reported few symptoms of mental illness, but a relatively low degree of social activity and leisure activities. The return rate was very high (88.9%). The questionnaires did not correlate significantly with a measure of vocational adjustment.

Preparation of monoiodinated interleukin-1 beta and its use in a radioimmunoassay.

Polyclonal anti-interleukin-1 beta (IL-1 beta) sera raised in sheep and in rabbits bound to unlabelled IL-1 beta. IL-1 beta radioiodinated using chloramine-T or Bolton-Hunter procedures was bound by the anti-IL-1 beta sera but was not displaced by unlabelled IL-1 beta suggesting that IL-1 beta was damaged during iodination procedures, resulting in alterations to the immunological properties of the tracer. Iodination using the mild oxidant N-bromosuccinimide, followed by extensive tracer purification to remove the unlabelled IL-1 beta, produced a tracer with a specific activity of 89 microCi/micrograms (0.83 atoms iodine/molecule IL-1) which was fully displaced by unlabelled IL-1 beta, allowing the development of a sensitive and specific radioimmunoassay for IL-1 beta. The radioimmunoassay has been used to quantify intracellular IL-1 beta and IL-1 beta release by peripheral blood monocytes stimulated with endotoxin.

Syngeneic central nervous system transplantation of genetically transduced mature, adult astrocytes.

Advances in the development of highly infectious, replication-deficient recombinant retroviruses provide an efficient means of stable transfer of gene expression. Coupled with ex vivo transduction, surrogate cell populations can be readily implanted into the brain, thus serving as vehicles for delivering selected gene products into the central nervous system (CNS). Here we report that rat astrocytes can be routinely and safely isolated from brain tissue of a living donor by use of short-term gelatin sponge implants. The mature, nontransformed astrocytes were easily expanded, maintained in long-term tissue cultures and were efficiently transduced with an amphotropic retrovirus harboring a heterologous, fused transgene. In vitro retroviral infection rendered the nontransformed cells essentially 100% viable after exposure. The level of efficiency of infection (30-50% effective genome integration of provirus and expression of transgene in target cell populations) and minimal cell toxicity obviated the need to harvest large numbers of target cells. Cultured transduced astrocytes were resilient and exhibited select peptide expression for up to 1 year. Subsequently, transduced astrocytes were used in a series of experiments in which cells were transplanted intracerebrally in syngeneic animals. Post-implantation, astrocytes seeded locally and either insinuated into the surrounding parenchyma in situ or exhibited a variable degree of migration, depending on the anatomic source of astrocytes and the targeted brain implantation site. Transduced astrocytes remained viable in excess of 8 months post-transplantation and exhibited sustained transgenic peptide expression of green fluorescent protein/neomycin phosphotransferase in vivo. The sequential isolation and culture of nontransformed, mature, adult astrocytes and recombinant retrovirus-mediated transduction in vitro followed by brain reimplantation represents a safe and effective means for transferring genetic expression to the CNS. This study lays the foundation for exploring the utility of using a human autologous transplantation system as a potential gene delivery approach to treat neurological disorders. Prepared and utilized in this manner, autologous astrocytes may serve as a vehicle to deliver gene therapy to the CNS.

Fate of injected interleukin 1 in rats: sequestration and degradation in the kidney.

The tissue distribution and route of clearance of human recombinant interleukin 1 alpha (IL 1 alpha) injected intravenously in rats was studied. The plasma half-life was approximately 2.5 min, and this was increased after nephrectomy, the kidney being the major organ through which the IL 1 alpha was excreted. Two iodinated fragments of IL 1 alpha, of approximately 5 and 9 kDa, were excreted by the kidneys whereas only intact, 17-kDa IL 1 alpha was detected in plasma, suggesting that the protein was being degraded after uptake by the kidney. The results of in vivo experiments in which surface endopeptidase-24.11 was inhibited with phosphoramidon and in vitro experiments in which rat kidney homogenates were incubated with radiolabeled IL 1 alpha suggest that the cytokine was endocytosed and then hydrolysed by lysosomal proteinases.

Novel compound tetra-, dinucleotide microsatellite polymorphism in the tumor necrosis factor/lymphotoxin locus.

A polymorphic (TGCG)n, tetranucleotide repeat was discovered juxtaposed to the (GT)n dinucleotide repeat that comprises the tumor necrosis factor a microsatellite (TNF) located telomeric to the tumor necrosis factor/lymphotoxin gene cluster. The degree of complexity of this compound tetra-,dinucleotide microsatellite consists of 16 potential alleles of combined length ranging from 24 to 54 bp. The pattern of frequencies of individual alleles belonging to the compound TNFa microsatellite was established from 52 healthy volunteers and was found to be highly heterogeneous. The data diverges significantly from previously published statistics that recognized only a simple variable dinucleotide tandem repeat. The newly recognized compound tetra-, dinucleotide TNFa microsatellite polymorphism establishes a more accurate genetic basis to explore potential linkage with disease susceptibility genes located within this region of the class III major histocompatibility complex. In addition, variable tumor necrosis factor and lymphotoxin production may reflect the more complex polymorphic nature of this microsatellite region. Finally, compound microsatellites probably exist elsewhere, throughout the human genome. Recognition of their presence may have a considerable impact on the validity of past and future microsatellite-based genetic analyses.