Human monocytes expressing a CEA-specific chimeric CD64 receptor specifically target CEA-expressing tumour cells in vitro and in vivo.

Antibody-dependent cellular cytotoxicity (ADCC) is one means by which macrophages (as well as natural killer cells and granulocytes) elicit a cytotoxic response. This is achieved via interaction of the Fc-gamma-receptor (CD64) with the Fc portion of antibody bound to target cells. We have created a chimeric CD64 molecule that incorporates a single chain Fv molecule, targeted against human carcinoembryonic antigen (CEA), fused to the membrane spanning and cytosolic domains of human CD64. Following adenoviral transfer to primary human monocytes, this chimeric CD64 receptor induced antigen-specific cytokine secretion during culture on immobilised CEA protein or on CEA-expressing tumour cells. Moreover, CEA targeted, but not control, monocytes effectively retarded CEA-positive tumour cell growth in vitro. Importantly, targeted monocyte cultures significantly reduced in vivo tumour growth rates in xenograft studies resulting in improved survival rates over that of control monocyte cultures. These data suggest that genetically directing monocytes against tumour antigens may be a useful means of achieving an immunotherapeutic response.

Molecular therapy in a model neuroendocrine disease: developing clinical gene therapy for pituitary tumours.

The main objectives of pituitary tumour treatment are to restore normal function of the pituitary gland and prevent tumour recurrences. In spite of the success of current therapies in the treatment of relatively small tumours, new therapeutic alternatives need to be explored for large invasive tumours, tumour recurrences postsurgery, and when intolerance to drug treatment develops. Gene therapy, which uses nucleic acids as drugs, is a very attractive alternative to classic therapeutic modalities. With the development of efficient gene delivery vectors, which allow widespread distribution and long-term transgene expression with limited side effects, the clinical implementation of gene therapy for the treatment of pituitary tumours will become a reality within the next five to ten years.

Long-term transgene expression within the anterior pituitary gland in situ: impact on circulating hormone levels, cellular and antibody-mediated immune responses.

Adenoviral vectors have been identified as useful tools for gene transfer to the pituitary gland with the aim of providing therapeutic treatments for pituitary diseases. Although successful adenovirus-mediated gene transfer to the pituitary has been shown, the duration of transgene expression, local immune responses and consequences on circulating pituitary hormone levels have not been investigated. These are critical not only for the successful implementation of these gene transfer techniques both for physiological and/or therapeutic applications but also for assessing the safety of these approaches. We have therefore assessed duration and levels of transgene expression 3 days, 14 days, 1, 2, and 3 months after delivery of adenoviruses expressing herpes simplex virus type 1 thymidine kinase (HSV1-TK), under the control of the major immediate early human cytomegalovirus (RAd-hCMV/TK) or human PRL (RAd-hPrl/TK) promoters, to the anterior pituitary (AP) gland in situ. The presence of vector genome and cellular immune infiltrates within the AP gland were also studied along with the levels of circulating anti-adenovirus neutralizing antibodies and AP hormones in sera. Ubiquitous or cell-type specific expression of HSV1-TK within the AP gland was seen from RAd-hCMV/TK and RAd-hPrl/TK respectively at all time points, although a reduction in expression was seen over time. PCR amplification of HSV1-TK specific sequences showed the persistence of adenoviral genomes for up to 3 months. Analysis of the AP showed the presence of a virus-induced inflammation that peaked around day 14 and was resolved between 2-3 months. ED1-positive macrophages, CD8-positive T-cells and CD161-positive NK cells were identified up to 1 month after virus administration. A virus-induced humoral immune response was also present as anti-adenovirus neutralizing antibodies were detected from 14 days after virus administration. Levels of circulating pituitary hormones were unaffected by virus administration with the exception of the stress hormone ACTH which was increased at 3 days but normalized by 14 days. In conclusion, our data indicates that adenovirus-mediated delivery to the AP gland in situ may be a useful tool for the treatment of pituitary diseases as no major cytotoxicity or disruption of AP hormonal functions are seen. Despite of this, further developments to this approach still need to be made to combat the reduced transgene expression seen over time and the induction of virus-induced immune responses.

Gene transfer into neural cells in vitro using adenoviral vectors.

Adenoviruses (Ads) have become a very attractive and versatile vector system for delivering genes into brain cells in vitro and in vivo. One of the main attractions of Ads is that they can mediate gene transfer into post-mitotic cells, i.e. neurons. Ads are easy to grow and manipulate, stable, and their biology is very well understood. This unit is designed to help newcomers into the field, to design, prepare and grow replication-defective recombinant adenovirus vectors with the aim of transferring genes into neurons and glial cells in primary culture. It provides step-by-step methods describing the preparation of brain cell cultures, their infection using recombinant adenovirus vectors and also the assessment of transgene expression using a variety of techniques including fluorescence immunocytochemistry and fluorescence activated cell-sorting (FACS) analysis. The methods described will be useful to scientists wishing to enter the adenovirus field to construct adenovirus vectors to be used for gene transfer into neural cells.

Transcriptional targeting to anterior pituitary lactotrophic cells using recombinant adenovirus vectors in vitro and in vivo in normal and estrogen/sulpiride-induced hyperplastic anterior pituitaries.

The use of pituitary cell type-specific promoters is a powerful molecular tool to achieve pituitary cell type-specific transcriptional targeting of transgenes encoded by viral vectors. It has recently been proposed that transcriptional targeting of therapeutic genes could be harnessed as a gene therapy strategy for the treatment of pituitary disease. We describe the successful use of the human PRL promoter (hPrl) encoded within recombinant adenovirus vectors to target transgene expression of Herpes Simplex Virus Type 1-Thymidine Kinase (HSV1-TK) or beta-galactosidase to lactotrophic cells in vitro and in vivo. Functionally, the restriction of expression of HSV1-TK to lactotrophic tumor cells, using the hPrl promoter, resulted in the cell type-specific induction of apoptosis in the lactotrophic GH3 tumor cell line, in the presence of ganciclovir (GCV). In the corticotrophic AtT20 cell line, we detected neither HSV1-TK expression, nor apoptosis in the presence of GCV. The hPrl promoter encoded within a recombinant adenoviral vector also restricted transgene expression to lactotrophic cells in primary anterior pituitary (AP) cultures, and importantly, within the anterior pituitary gland in vivo. When the HSV1-TK driven by hPrl promoter was used in an in vivo model ofestrogen/sulpiride lactotroph induced hyperplasia within the AP in situ, the treatment was not effective in either reducing the weight of the gland, the number of lactotrophic cells within the transduced area in vivo, or the circulating PRL levels. This is in contrast to the human cytomegalovirus promoter (hCMV) driving expression of HSV1-TK in the same experimental paradigm, which was effective in reducing pituitary weight and circulating PRL levels. Our results have important implications in the design of gene therapy strategies for pituitary tumors. We demonstrate that both the choice of the in vivo animal model, i.e. adenoma in the AP gland in situ, and the particular gene therapy strategy chosen, i.e. use of strong ubiquitous promoters vs. weaker but cell type-specific promoters, determine the experimental therapeutic outcome.

Adenovirus-mediated gene transfer to human lens epithelial cells in organ culture.

PURPOSE: To assess the feasibility of using recombinant adenovirus vectors to transduce the human lens epithelial cells (LECs) involved in posterior capsule opacification (PCO). SETTING: Department of Ophthalmology and Molecular Medicine Unit, University of Manchester, Manchester, United Kingdom. METHODS: Seventeen human lens capsules were maintained in organ culture to allow LECs to proliferate onto the posterior capsule. Partly covered and completely covered capsules were infected with a recombinant adenovirus vector RAd35, encoding for the marker gene beta-galactosidase at plaque-forming units per milliliter (pfu/mL) ranging from 10(7) to 10(10) for up to 48 hours. Assessment of infection and transduction of the marker gene were achieved by calculating the percentage of cells exhibiting X-gal staining both macroscopically and microscopically. RESULTS: Staining appeared to be dependent on virus dose, with most intense staining at doses of 10(8) and 10(9) pfu/mL with decreased staining at higher and lower viral doses. Microscopic assessment demonstrated that all cells expressed beta-galactosidase when infected with 10(9) pfu, 84% at 10(8) pfu, and 45% at 10(7) pfu. At 10(10) pfu, some cytotoxicity was observed. CONCLUSIONS: These results indicate that recombinant adenoviruses can be used to transfer genes to the LECs involved in PCO. The transfer of cytotoxic genes after cataract surgery may be considered a preventive measure for PCO.

Central nervous system toxicity of two adenoviral vectors encoding variants of the herpes simplex virus type 1 thymidine kinase: reduced cytotoxicity of a truncated HSV1-TK.

Herpes simplex virus type 1-thymidine kinase (HSV1-TK) in combination with ganciclovir is an efficient and widely used strategy in brain tumour gene therapy. Recently, we have shown effective inhibition of glioma growth in a syngeneic rat model using recombinant adenoviruses expressing the full-length HSV1-TK and an N-terminus truncated variant, HSV1-DeltaTK in the presence of ganciclovir. We also showed active chronic brain inflammation in the long-term survivors (3 months) treated with HSV1-TK plus GCV. Furthermore, our results indicated loss of myelinated fibres, oedema and indices of ongoing axonal degeneration. In this study, we assessed the cytotoxicity of both HSV1-TK variants in the presence or absence of ganciclovir, in primary cultures of neurones and glia, and in the rat brain in vivo. Our results indicate that, at viral doses where tumour cells are sensitive to the enzyme/prodrug system, (1) there is no major cytotoxicity for either neurones or glial cells grown in primary cultures, (2) on its own the full-length HSV1-TK is more cytotoxic than its truncated version HSV1-DeltaTK for a population of non-neuronal and non-glial cells within neocortical primary cultures, and (3) in vivo, when delivered into the striatum, RAds encoding HSV1-TK are more cytotoxic than RAds encoding HSV1-DeltaTK, after administration of ganciclovir. The effectiveness of HSV1-DeltaTK in preventing brain tumour growth in vivo, combined with its reduced cytotoxicity, both in vivo and in primary cultures of CNS cells, could represent an advantage for treatment of brain tumours using gene therapy.

Adenovirus-mediated herpes simplex virus type-1 thymidine kinase gene therapy suppresses oestrogen-induced pituitary prolactinomas.

We tested the hypothesis that gene transfer using recombinant adenovirus vectors (RAds) expressing herpes simplex virus type 1 thymidine kinase (HSV1-TK) might offer an alternative therapeutic approach for the treatment of pituitary prolactinomas that do not respond to classical treatment strategies. HSV1-TK converts the prodrug ganciclovir (GCV) to GCV monophosphate, which is in turn further phosphorylated by cellular kinases to GCV triphosphate, which is toxic to proliferating cells. One attractive feature of this system is the bystander effect, whereby untransduced cells are also killed. Our results show that RAd/HSV1-TK in the presence of GCV is nontoxic for the normal anterior pituitary (AP) gland in vitro, but causes cell death in the pituitary tumor cell lines GH3, a PRL/GH-secreting cell line, and AtT20, a corticotrophic cell line. We have used sulpiride- and oestrogen-induced lactotroph hyperplasia within the rat AP gland as an in vivo animal model. Intrapituitary infection of rats bearing oestrogen-induced lactotroph hyperplasia, with RAd/ HSV1-TK and subsequent treatment with GCV, decreases plasma PRL levels and reduces the mass of the pituitary gland. More so, there were no deleterious effects on circulating levels of other AP hormones, suggesting that the treatment was nontoxic to the AP gland in situ. In summary, our results show that suicide gene therapy using the HSV1-TK transgene could be further developed as a useful treatment to complement current therapies for prolactinomas.

Chronic brain inflammation and persistent herpes simplex virus 1 thymidine kinase expression in survivors of syngeneic glioma treated by adenovirus-mediated gene therapy: implications for clinical trials.

The long-term consequences of adenovirus-mediated conditional cytotoxic gene therapy for gliomas remain uncharacterized. We report here detection of active brain inflammation 3 months after successful inhibition of syngeneic glioma growth. The inflammatory infiltrate consisted of activated macrophages/microglia and astrocytes, and T lymphocytes positive for leucosyalin, CD3 and CD8, and included secondary demyelination. We detected strong widespread herpes simplex virus 1 thymidine kinase immunoreactivity and vector genomes throughout large areas of the brain. Thus, patient evaluation and the design of clinical trials in ongoing and future gene therapy for brain glioblastoma must address not only tumor-killing efficiency, but also long-term active brain inflammation, loss of myelin fibers and persistent transgene expression.

Neuronal and glial cell type-specific promoters within adenovirus recombinants restrict the expression of the apoptosis-inducing molecule Fas ligand to predetermined brain cell types, and abolish peripheral liver toxicity.

Gene therapy using Fas ligand (FasL) for treatment of tumours and protection of transplant rejection is hampered because of the systemic toxicity of FasL. In the present study, recombinant replication-defective adenovirus vectors (RAds) encoding FasL under the control of either the neuronal-specific neuronal-specific enolase (NSE) promoter or the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter have been constructed. The cell type-specific expression of FasL in both neurons and glial cells in primary cultures, and in neuronal and glial cell lines is demonstrated. Furthermore, transgene expression driven by the neuronal and glial promoter was not detected in fibroblastic or epithelial cell lines. Expression of FasL driven by a major immediate early human cytomegalovirus promoter (MIEhCMV) was, however, achieved in all cells tested. As a final test of the stringency of transgene-specific expression, the RAds were injected directly into the bloodstream of mice. The RAds encoding FasL under the control of the non-cell type-specific MIEhCMV promoter induced acute generalized liver haemorrhage with hepatocyte apoptosis, while the RAds containing the NSE or GFAP promoter sequences were completely non-toxic. This demonstrates the specificity of transgene expression, enhanced safety during systemic administration, and tightly regulated control of transgene expression of highly cytotoxic gene products, encoded within transcriptionally targeted RAds.

Interleukin-1 mediates a rapid inflammatory response after injection of adenoviral vectors into the brain.

Adenovirus-mediated gene transfer into the brain is associated with significant inflammation and activation of anti-vector and anti-transgene immune responses that curtail the gene delivery of adenoviruses and therapeutic efficacy. Elucidating the molecular mediators of inflammatory and immune responses to adenoviruses injected into the brain should allow us to inhibit their inflammatory actions, thereby reducing vector clearance and enhance adenoviral-mediated gene transfer into the CNS. Cytokines are primary mediators of the immune response and are released during inflammation. Here we report for the first time that injection of replication-deficient adenovirus vectors into the cerebral ventricles of rats causes a rapid increase in body temperature. This fever response precedes any vector-encoded transgene expression and occurs with vectors encoding no transgene, as well as with vectors encoding a therapeutic transgene i.e., HSV1-thymidine kinase. No fever is detected after infection of the striatum, an important brain target in studies on neurodegeneration. After infection of the brain ventricles, CSF levels of immunoreactive tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta increase significantly (up to 300-fold). In the hypothalamus, the locus of thermoregulation in the brain, only IL-1beta and IL-6 are significantly elevated. A neutralizing TNF-alpha antibody has no effect on adenovirus-induced fever. However, pretreatment with either the IL-1 receptor antagonist or the cyclooxygenase inhibitor flurbiprofen completely abolishes adenovirus-induced fever, suggesting that IL-1 and prostaglandins are direct mediators of this response. These results are the first to demonstrate that IL-1, but not TNF-alpha, is the main mediator of a very early inflammatory response to adenovirus in the brain.

Adenoviruses encoding HPRT correct biochemical abnormalities of HPRT-deficient cells and allow their survival in negative selection medium.

The Lesch-Nyhan syndrome is an X-linked disorder caused by a virtually complete absence of the key enzyme of purine recycling, hypoxanthine-guanine phosphoribosyltransferase (HPRT). It is characterized by uric acid overproduction and severe neurological dysfunction. No treatment is yet available for the latter symptoms. A possible long-term solution is gene therapy, and recombinant adenoviruses have been proposed as vectors for gene transfer into postmitotic neuronal cells. We have constructed an adenoviral vector expressing the human HPRT cDNA under the transcriptional control of a short human cytomegalovirus major immediate early promoter (RAd-HPRT). Here we show that infection of human 1306, HPRT-negative cells with RAd-HPRT, expressed high enough levels of HPRT enzyme activity, as to reverse their abnormal biochemical phenotype, thus enhancing hypoxanthine incorporation and restoring purine recycling, increasing GTP levels, decreasing adenine incorporation, and allowing cell survival in HAT medium in which only cells expressing high levels of HPRT can survive. Infection of murine STO cells, increased hypoxanthine incorporation and restored purine recycling, thus allowing cell survival in HAT medium, and reduced de novo purine synthesis. Although both cells were able to survive in HAT medium post infection with RAd-HPRT, some of the biochemical consequences differed. In summary, even though adenoviral vectors do not integrate into the genome of target HPRT-deficient human or murine cells, RAd-HPRT mediated enzyme replacement corrects abnormal purine metabolism, increases intracellular GTP levels, and allows cells to survive in a negative selection medium.

Gene transfer into enteric neurons of the rat small intestine in organ culture using a replication defective recombinant herpes simplex virus type 1 (HSV1) vector, but not recombinant adenovirus vectors.

We have designed a system in which to test gene transfer into gut neurons consisting of an organ culture of neonatal rat small intestine. The tissue was exposed to herpes simplex- and adenovirus-derived vectors: (1) a temperature-sensitive herpes simplex virus-1 (HSV1) vector (tsK-beta gal) containing the lacZ gene encoding beta-galactosidase (beta-gal), under the transcriptional control of the HSV1 immediate-early 3 (IE3) promoter; (2) RAd35, an E1-/E3- replication-deficient adenovirus expressing lacZ under the control of a truncated HCMV major IE promoter; and (3) RAd122, an E1-/E3- replication-deficient adenovirus expressing the lacZ under the control of the RSV LTR. Forty-eight hours after the vector was added to the organ culture, we detected beta-gal using immunohistochemistry or X-gal histochemistry in tissue sections examined by light microscopy. We encountered a distinctive staining of cells arranged in two concentric circles corresponding in location to the myenteric and submucosal plexuses. Cells in these areas were of similar size and morphology to neonatal enteric neurons, as visualized by NADPH-diaphorase histochemistry and immunocytochemical staining with antibodies to the neuronally expressed proteins PGP 9.5, or neurofilaments. Double labelling with antibodies recognizing neurofilaments and beta-galactosidase revealed that most cells infected by tsK were neurons, while the RAd35 and 122 vectors only infected non-neuronal cells. We thus demonstrate that both HSV1- and adenovirus-derived vectors can be used to transfer genes to the gut in vitro, but they transduce different populations of target cells.

Relationship between GAP-43 expression in the dentate gyrus and synaptic reorganization of hippocampal mossy fibres in rats treated with kainic acid.

Kainic acid-induced seizures, in adult rats produce neurodegeneration in the hippocampus followed by sprouting of the mossy fibres in the inner molecular layer of the dentate gyrus and changes in GAP-43 expression in the granule cells. In the present study we observed that 4 days after kainic acid injection a dense plexus of silver-impregnated degenerating terminals detected by Gallyas's method and a decrease of GAP-43 immunostaining was observed in the inner molecular layer of the dentate gyrus indicating deafferentiation of this region. This was associated with the formation of an intense GAP-43 immunostained band in the supragranular layer. MK-801, a non-competitive inhibitor of the NMDA receptor, which partially inhibited the behavioural seizures induced by KA, also protected from the inner molecular layer deafferentation and markedly reduced the expression of GAP-43 mRNA in the granule cells and the intense GAP-43 immunostained band in the supragranular layer, suggesting a relationship among these events. Two months after kainic acid injection the intense supragranular GAP-43 positive band was no longer evident but the whole inner molecular layer appeared more labelled in association with the formation of the collateral sprouting of the mossy fibres in the inner molecular layer as detected by Timm's staining. These effects were also markedly reduced by the pretreatment with MK-801. Taken together, these experiments indicate for the first time a direct relationship between the increase of GAP-43 immunostaining in the inner molecular layer of the dentate gyrus and the collateral sprouting of mossy fibres in this district in response to kainic acid induced seizures. This further supports the hypothesis that the early induction of GAP-43 in granule cells may be one of the molecular mechanisms required for the synaptic reorganization of the mossy fibres.

Heparinless cardiopulmonary bypass with ancrod.

A case is reported of a 22-year-old man with heparin-induced thrombocytopenia and thrombosis syndrome and a right atrial foreign body (Greenfield filter). Heparinless cardiopulmonary bypass for removal of the foreign body was conducted by pretreatment with ancrod, a rapid-acting antifibrinolytic of pit viper venom origin. Treatment protocol and a literature review are included in this article.

Technique for repair of left ventricular free wall rupture.

A case of postinfarction left ventricular free wall rupture is reported. The technique used to repair the rupture is described, along with a modification of the technique.