PubMed Health⌕ Search

Biomedical subjects

H M Charlton

Publications and source records attributed to H M Charlton.

At least 19 recordsLinked to original sources
PubMed2004

Local adenoviral expression of Fas ligand upregulates pro-inflammatory immune responses in the CNS.

The central nervous system (CNS) is a site of relative immunological privilege; despite this it can be a target of the immune system under certain conditions. For example, adenoviral vectors elicit an immune response strong enough to result in antigen elimination, in immunologically primed animals. Fas ligand (FasL) contributes to the immune privilege of certain tissues by inducing apoptosis in activated T cells. We therefore investigated whether local overexpression of FasL could downregulate the immune response to adenovirus in the brain. Adenoviral vectors expressing FasL (AdFasL) and the reporter gene beta-galactosidase (Adbetagal) were co-injected into the striatum of naïve or immunologically primed mice. A co-injection of an adenovirus lacking a transgene (Ad0) and Adbetagal acted as a control. At 2 weeks after inoculation, reporter protein expression was significantly reduced with the AdFasL:Adbetagal combination compared with the Ad0:Adbetagal controls. This was accompanied by a strong inflammatory cell infiltrate, local demyelination and upregulation of pro-inflammatory cytokine gene expression. These experiments demonstrate that FasL overexpression elicits a pro-inflammatory response in the CNS rather than immunosuppression. This was characterized by chronic inflammation and accelerated loss of transgene expression. Induction of such an unexpected pro-inflammatory response caused by introducing FasL may be a peculiarity of the relative immunoprivilege of the unique environment of the brain.

Adenoviridae↗
PubMed2003

Differentiation of adult-type Leydig cells occurs in gonadotrophin-deficient mice.

During mammalian testis development distinct generations of fetal and adult Leydig cells arise. Luteinising hormone (LH) is required for normal adult Leydig cell function and for the establishment of normal adult Leydig cell number but its role in the process of adult Leydig cell differentiation has remained uncertain. In this study we have examined adult Leydig cell differentiation in gonadotrophin-releasing hormone (GnRH)-null mice which are deficient in circulating gonadotrophins. Adult Leydig cell differentiation was assessed by measuring expression of mRNA species encoding four specific markers of adult Leydig cell differentiation in the mouse. Each of these markers (3beta-hydroxysteroid dehydrogenase type VI (3betaHSD VI), 17beta-hydroxysteroid dehydrogenase type III (17betaHSD III), prostaglandin D (PGD)-synthetase and oestrogen sulphotransferase (EST)) is expressed only in the adult Leydig cell lineage in the normal adult animal. Real-time PCR studies showed that all four markers are expressed in adult GnRH-null mice. Localisation of 3betaHSD VI and PGD-synthetase expression by in situ hybridisation confirmed that these genes are expressed in the interstitial tissue of the GnRH-null mouse. Treatment of animals with human chorionic gonadotrophin increased expression of 3betaHSD VI and 17betaHSD III within 12 hours further indicating that differentiated, but unstimulated cells already exist in the GnRH-null mouse. Thus, while previous studies have shown that LH is required for adult Leydig cell proliferation and activity, results from the present study show that adult Leydig cell differentiation will take place in animals deficient in LH.

17-Hydroxysteroid Dehydrogenases↗
PubMed2003

Age-related uterine and ovarian hypertrophy in FSH receptor knockout and FSHbeta subunit knockout mice.

Female mice in which the gene encoding the follicle-stimulating hormone FSH receptor (FSHR) knockout (KO) or its ligand (FSHbetaKO) have been disrupted were infertile. Ovaries of these mice were significantly smaller than those of heterozygous littermates but significantly larger than those of hypogonadal mice of the same age. Uterine masses in all three mutants were <6 mg, significantly reduced compared with heterozygous mice. At 1 year of age uterine mass had increased to >12 mg in 63% of FSHRKO females and 88% of FSHbetaKO females. Despite the increase in uterine size there was no evidence of contractility: uteri were flaccid and unresponsive to electrical or pharmacological stimulation. In most females in which uterine growth had occurred there was evidence of ovarian growth with hypertrophy of the interstitial tissue, occurrence of ovarian cysts and epithelial and tubular inclusions. There was no evidence of uterine or ovarian hypertrophy in hypogonadal (hpg) mice at any age or in 1 year old females in which the FSH mutations were bred onto the hpg background. There was an inverse correlation of plasma LH concentrations and uterine mass in 1 year old mutant females with uterine hypertrophy. Ovariectomy of both FSHRKO and FSHbetaKO females with large uteri resulted in decreased uterine mass and increased plasma concentration of LH. The number of mice with ovarian pathology, reminiscent of the serous ovarian adenocarcinomas found in humans, was significantly greater in the FSHbetaKO mice, indicating that the presence of an intact FSH receptor on ovarian cells of FSHbetaKO females may allow constitutive basal stimulation of the ovary, which is absent in mice lacking FSH receptors.

Aging↗
PubMed2002

Increasing local levels of IGF-I mRNA expression using adenoviral vectors does not alter oligodendrocyte remyelination in the CNS of aged rats.

IGF-I, a growth factor that contributes to developmental myelination, shows increased levels of expression within experimental models of remyelination. The pattern of IGF-I mRNA expression changes with the rate of remyelination, with peak levels of expression occurring earlier during rapid remyelination in young adult rats compared to the slower remyelination in old adult rats. In this study we have attempted to accelerate remyelination in old adult rats by using an IGF-expressing adenoviral vector (IGF-I-Ad) to bring forward the timing of peak level of IGF-I expression. Following injection of IGF-I-Ad into focal areas of lysolecithin-induced demyelination in the spinal white matter of old adult rats we created levels of IGF-I mRNA expression at 10 days that were considerably higher than those normally occurring at this time and more similar to those in young animals. However, despite the elevated levels of IGF-I mRNA expression there was no significant change in the extent of oligodendrocyte remyelination compared to saline controls or animals injected with an adenoviral vector expressing LacZ (NT-LacZ-Ad). There was a small increase in Schwann cell remyelination in IGF-I-Ad- and NT-LacZ-Ad-injected animals compared to saline controls. These results indicate that changing the levels of IGF-I directly within demyelinating lesions undergoing remyelination is not sufficient to alter remyelination and that the proremyelinating effects of systemically delivered IGF-I are unlikely to be due to direct effects on the oligodendrocyte lineage.

Adenoviridae↗
PubMed2000

Humoral immune responses to adenovirus vectors in the brain.

We have investigated the humoral immune response to E1-deleted adenovirus vectors encoding the lacZ gene introduced into the brains of mice. Injection of these non-replicating vectors into the brain induced systemic antibody production to adenovirus vectors in dose dependent manner. Apparent antibody production to beta-galactosidase, the product of the lacZ gene, was detected later than anti-adenovirus antibody. Neutralizing antibody was not detected. This study demonstrates that E1-deleted adenovirus vectors injected into the brain trigger humoral immune responses to the adenovirus and its gene products, but they are not sufficient to block the infection of cells by adenovirus upon repeat injection.

Adenoviridae↗
PubMed2000

The effect of a null mutation in the follicle-stimulating hormone receptor gene on mouse reproduction.

To investigate further brain-pituitary-gonadal interrelationships we have generated mice in which the gene encoding the FSH receptor has been disrupted. Female FSH receptor knockout (FSHRKO) mice were infertile. The ovaries were significantly reduced in size, with follicular development arrested at the preantral stage, but there was evidence of stromal hypertrophy. The vagina was imperforate, and the uterus was atrophic. There was no response to administration of PMSG. Inhibins A and B were undetectable in both the serum and gonads. Compared with those in control animals, serum concentrations of FSH and LH were significantly elevated in mutant females. The pituitary content of FSH, but not LH, was also significantly elevated. Estrogen administration in FSHRKO female mice suppressed serum LH levels to those seen in control mice, whereas FSH levels were reduced by only 50%. Male FSHRKO mice were fertile, although testis weight was significantly reduced. However, testicular inhibin A and B concentrations did not differ from those in normal littermates. Serum levels of FSH and LH were elevated in the null mutant male mice, whereas no differences were found in the pituitary content of these hormones. In conclusion, ovarian follicular development cannot progress beyond the preantral stage without FSH. In the absence of mature follicles ovarian estrogen remains low, and consequently accessory sex tissue growth and negative feedback regulation of gonadotropin secretion are severely compromised. In the male, however, inability to respond to FSH does not impair fertility, although testicular weight is reduced, and feedback regulation of pituitary gonadotropins and intratesticular paracrine interactions may be disturbed.

Animals↗
PubMed1999

Cytokine gene expression following neural grafts.

A reverse-transcription polymerase chain reaction study of molecular events following solid neural tissue xenogeneic and syngeneic grafts into the mouse caudate nucleus was made between 1 day and 30 days post-grafting. Constitutive expression of interleukin (IL)-1 and transforming growth factor (TGF)-beta1 mRNAs was detected. The sham operation produced minimal cytokine upregulation, indicating that surgical trauma caused minimal damage. A similar picture was seen with syngeneic grafts, which showed good graft survival. Xenografts, however, were rejected within 30 days and cytokine mRNAs for IL-2, IL-2R, IL-4, IL-10 and interferon (IFN)-gamma were detected from 7 days post-graft, correlating with histological identification of a leukocytic infiltrate. This method provides a quick, comparative screen for detecting cytokine mRNA profiles in neural grafts and may assist the diagnosis of early rejection phenomena. As such, it is a potential analytical tool for strategies aimed at depleting/blocking the activity of different cell types and/or cytokines in future neural transplant therapy.

Animals↗
PubMed1999

Variation in the immune response to adenoviral vectors in the brain: influence of mouse strain, environmental conditions and priming.

E1-deleted adenoviral vectors expressing the bacterial beta-galactosidase gene were inoculated into the brain of unprimed and primed C3H.He or C57BL/6J mice housed under either conventional or specific-pathogen-free (SPF) conditions. The kinetics of immune responses to both the vector and the transgene were investigated. In mice previously sensitized to adenovirus, the leukocyte infiltrate in the brain was dominated by CD8+ T cells, whereas in unprimed mice CD4+ T cells were present at higher levels. As expected, antibody titres to both adenovirus and beta-galactosidase were higher in primed mice than in unprimed mice after intracranial inoculation. C3H.He mice consistently made higher antibody responses than C57BL/6J mice. Although adenoviral vectors induced an inflammatory response under all conditions, mice housed in SPF facilities exhibited less inflammation than conventional mice and transgene expression persisted for longer. Irrespective of whether the mice had been deliberately primed to adenovirus, antibody titres were consistently lower in SPF mice compared with conventional mice. This study clearly demonstrates that environmental conditions, as well as previous priming to adenovirus, will affect both the quality and duration of the immune response triggered by gene delivery to the brain.

Adenoviridae↗
PubMed1999

A model for long-term transgene expression in spinal cord regeneration studies.

In order to determine the suitability of first generation adenoviral vectors for gene delivery into spinal cord white matter, four different titres of beta-galactosidase-expressing adenovirus were injected into spinal cord white matter of adult rats. At titres > or =106 p.f.u., transgene expression was extensive but severe tissue damage was observed in the form of axon degeneration, demyelination and astrocyte loss. When < or = 105 p.f.u. were injected, only low levels of axon degeneration and demyelination were observed. beta-Galactosidase activity was detectable at 72 days and did not diminish significantly with time. The immune response in the spinal cord to 105 p.f.u. over 72 days was minimal, and indistinguishable from that to injection of buffer. A prominent immune response was observed when 107 p.f.u. was injected into the spinal cord of PVG rats, and when 105 or 107 p.f.u. was injected into AO rats. These results indicate that the immune response in PVG rats to betagal-expressing adenovirus is both strain and titre dependent. First generation adenoviral vectors, therefore, induce moderate and long-lived transgene expression with minimal tissue damage and immune response when an appropriate titre is injected into the low responder PVG rat strain, providing a suitable model for assessing the effect of gene delivery in models of spinal cord injury.

Adenoviridae↗
PubMed1998

A gamma34.5 mutant of herpes simplex 1 causes severe inflammation in the brain.

A number of viral vectors are currently being evaluated as potential gene therapy vectors for gene delivery to the brain. As well as evaluating their ability to express a transgene for extended periods of time it is also essential to examine any cytotoxic immune response to such vectors as this may not only limit transgene expression but also cause irreparable harm. This work describes the effect of inoculating a gamma34.5 mutant of herpes simplex type 1 (1716lacZ) into the brain of different strains of rats and mice. Animals were monitored for weight loss and signs of illness, and their brains were evaluated for inflammation, beta-galactosidase expression and recoverable infectious virus. We report that there is (i) a powerful immune response consisting of an early non-specific phase and a later presumably T-cell-mediated phase; (ii) significant weight loss in some animals strains accompanied by severe signs of clinical illness and (iii) transient reporter gene expression in all animal strains examined. To be useful for gene therapy we suggest this virus requires further modification, it should be tested in several animal strains and the dose of virus used may be critical in order to limit damage.

Animals↗
PubMed1998

Potential and limitations of a gamma 34.5 mutant of herpes simplex 1 as a gene therapy vector in the CNS.

Direct injection of viral vectors into the central nervous system has become a valuable technique for exploring the function of neurological systems and is a potential therapy for neural disease. To this end a number of herpes simplex virus (HSV)-derived vectors are currently being developed for the introduction of foreign DNA into the brain. In this study a non-neurovirulent HSV 17+ mutant, variant 1716, deleted in the gamma 34.5 gene and expressing the marker gene lacZ under the control of the latency-associated transcripts promoter was injected stereotactically into the central nervous system of two strains of rat (AO and PVG). We show (1) that transgene expression was low at the site of injection, in the striatum, at all times studied (12 h to 30 days after injection); (2) dramatically more transgene expression was observed at distant sites which contain neurons projecting directly to the site of injection, with maximal expression at these sites being at 1-2 days; (3) immunostaining with a polyclonal anti-HSV antibody and with an antibody which detects a 65 kDa HSV DNA binding protein (the product of the UL42 gene of the virus) demonstrated that viral gene products could be detected at the injection site as early as 12 h and up to 1 week after injection. Moreover these could also be detected at several secondary sites not all of which have direct connections with the injection site. These findings suggest that gamma 34.5 negative vectors have potential for gene transfer but may require further attenuation to limit viral antigen expression before they can be used successfully for gene therapy in the brain.

Animals↗
PubMed1998

Fetal development of Leydig cell activity in the mouse is independent of pituitary gonadotroph function.

During fetal development the testes secrete anti-Mullerian hormone and testosterone to induce formation of the male phenotype. Adult Leydig cells secrete testosterone under the control of LH, but the role of the fetal pituitary in regulating fetal Leydig cell function is unclear. To study the early relationship between pituitary and Leydig cell function, we have examined the development of fetal pituitary LH levels and Leydig cell function in normal mice and in hypogonadal (hpg) mice that lack GnRH and, thus, circulating gonadotropins. In normal and hpg mice, pituitary LH content was barely detectable until embryonic day 17 (E17), when levels began to increase significantly in both groups. Pituitary levels of LH in hpg mice were, however, only about 10% of normal at all ages. Full-length LH receptor transcripts were first detectable in fetal testes on E16 in both normal and hpg mice. In normal mice, levels of testicular messenger RNA (mRNA) encoding cytochrome P450 side-chain cleavage and 17alpha-hydroxylase increased from E13 to reach a peak around birth. In hpg mice, levels of mRNA encoding these enzymes were normal until around birth, at which time there was a significant decline. Levels of testicular mRNA encoding 3beta-hydroxysteroid dehydrogenase type I were similar in normal and hpg mice and showed little change during development. Intratesticular testosterone reached a peak on E18 in normal animals before declining again after birth. In hpg mice, intratesticular testosterone levels were normal throughout fetal development and on the day of birth, but were barely detectable by postnatal day 5. Results show 1) that fetal Leydig cell function in the mouse is normal in the absence of endogenous circulating gonadotropins; 2) that Leydig cells become dependent on gonadotropins shortly after birth; and 3) that pituitary LH synthesis can start in the absence of GnRH but is dependent on LH for a normal level of synthesis and secretion.

Animals↗
PubMed1998

Relaxin-like factor expression as a marker of differentiation in the mouse testis and ovary.

Expression of the relaxin-like factor (RLF) was studied at the messenger RNA (mRNA) and protein levels in the testes and ovaries of the mouse, as well as through testicular development and differentiation in the mouse testis. In situ hybridization or RT-PCR, and immunohistochemistry using a polyclonal antibody raised against a recombinant protein, provided mutually confirmatory results for a high expression of RLF in the Leydig cells of the adult testis and at a much lower level of expression in the luteal cells of the ovary through the cycle, pregnancy, and in lactation. Analysis of protein and mRNA expression, through postnatal testicular development, indicated moderate RLF expression also in the fetal population of Leydig cells, even in the hpg mutant mouse, lacking an active pituitary-gonadal axis. Prepubertal Leydig cells, however, exhibit only very low-level RLF gene expression, this phenotype persisting in the adult hpg mouse. In summary, fetal Leydig cells express RLF in an LH/human CG-independent fashion, whereas LH/human CG is essential to induce RLF expression in the adult-type Leydig cell. In cultured adult Leydig cells or in the mouse tumor MA-10 cell line, RLF mRNA is expressed in a constitutive fashion. RLF thus seems to be a useful marker of Leydig cell differentiation status.

Animals↗
PubMed1997

Immune responses to adenoviral vectors during gene transfer in the brain.

We have investigated the immune response to E1-deleted adenovirus vectors encoding the lacZ gene introduced into the brains of adult mice. Injection of these nonreplicating vectors caused a marked inflammatory response in the brain as assessed by immunocytochemistry and flow cytometry of leukocytes. Infiltrating leukocytes were detectable within 2 days of injection and reached a maximum by 9 days. Thereafter, the number of infiltrating cells decreased, but a small number persisted in the brain until day 60. Between 2 and 4 days after injection, the percentage of CD8+ cells detectable increased whereas the percentage of CD4+ cells present in the infiltrating population did not significantly increase until day 6, peaking on day 15. Activated CD25+ T cells were detectable between days 6 and 15. beta-Galactosidase (beta-Gal), the product of the lacZ gene encoded by the vector, was also detected, both at the injection site in the striatum and also in the substantia nigra. Expression peaked between 4 and 6 days but a small number of beta-Gal+ cells was still seen at 60 days after injection. This study demonstrates that a quantitative analysis of the immune responses caused by a nonreplicating adenovirus vector is possible in the brain. E1-deleted adenoviral vectors trigger a strong inflammatory response in the brain, but this immune response is not sufficient to eliminate completely expression of genes encoded by the adenoviral construct.

Adenoviridae↗
PubMed1996

Immunological instability of persistent adenovirus vectors in the brain: peripheral exposure to vector leads to renewed inflammation, reduced gene expression, and demyelination.

Nonreplicating adenovirus vectors are being developed as vehicles for the delivery of therapeutic genes in vivo. Whereas in many organs an antiviral T cell response eliminates the vector and damages local tissue, when adenovirus vectors are injected into the brain the subsequent immune attack can be ineffective, allowing the vector to persist. In the present study, E1-deleted human adenovirus vectors were injected into the caudate nucleus of rats. Two months later, expression of protein from the vector was still evident and little inflammation was seen. A subcutaneous injection of adenovirus vector at this time, however, led within 2 weeks to severe mononuclear inflammation and microglial activation in the caudate. This caused local demyelination and a decrease in detectable protein expression from the vector. Interestingly, intense microglial activation and numerous lymphocytes and monocytes were also seen in brain areas containing neurons capable of retrogradely transporting the adenovirus vector from the caudate. Control experiments established that this inflammation in distant brain areas was not a nonspecific consequence of degeneration. These experiments demonstrate that although adenovirus vectors can persist in the brain without causing chronic inflammation, they remain the potential target of a damaging cell-mediated immune response brought about by a subsequent peripheral exposure to vector. The finding of lymphocytes in brain areas that project to the caudate further shows that viral antigens that are retrogradely transported by neurons can also be the target of a T cell attack.

Adenoviridae↗
PubMed1996

Contributions of donor and host blood vessels in CNS allografts.

The contributions of blood vessels in various transplantation paradigms of solid CNS tissue or cell suspension allografts placed into adult host brains were investigated immunohistochemically using the PVG-RT1C and PVG-RT1U inbred rat strains and a panel of highly specific monoclonal antibodies. The monoclonal antibodies included OX-27 and U9F4 against major histocompatibility complex (MHC) class I antigens of the PVG-RT1C and PVG-RT1U rats, respectively; OX-26 against the rat transferrin receptor located on blood-brain barrier (BBB) endothelia; and OX-7 against rat neuronal Thy 1.1 for evaluating graft survival. Our study is the first to address the immunogenicity of blood vessels in surviving CNS allografts. Solid fetal or neonatal PVG-RT1C cortex was grafted into the third or lateral cerebral ventricle or caudate/putamen of PVG-RT1U adult hosts for 30 days to 7 months. All allografts expressed demonstrable Thy 1.1 immunoreactivity with OX-7 antibody and appeared well-vascularized with blood vessels that immunostained with the OX-26 antibody against the transferrin receptor. For the most part, the allografts were supplied sparsely with donor (PVG-RT1C) MHC class I-positive (OX-27) blood vessels clustered in pockets. Donor MHC class I-positive vessels entered the host brain only from allografts in the third ventricle; these vessels were restricted to the host median eminence and no longer immunostained with OX-26 for the transferrin receptor (normally the median eminence is supplied with non-BBB vessels that do not possess the transferrin receptor and do not stain with OX-26). In host brains harboring a third ventricle allograft, host MHC class I-positive vessels immunostained with the U9F4 antibody were evident throughout the host CNS, including the median eminence, and throughout the allografts excluding sites inhabited by donor PVG-RT1C vessels. Cell suspension neural allografts (donor PVG-RT1C) placed within the brain parenchyma of PVG-RT1U hosts revealed no significant differences in vascular contributions between donor and host when compared to results obtained from solid CNS allografts. A unique immunohistochemical approach of introducing ascites fluid OX-27 as the primary antibody intravenously to the PVG-RT1U host demonstrated that in donor PVG-RT1C posterior pituitary allografts, donor and not host vessels predominate and are restricted to the graft. Finally, blood vessels isolated from adult PVG-RT1C brains were mixed with solid fetal PVG-RT1U cortical tissue and grafted into the brain parenchyma of adult PVG-RT1U hosts. Immunostaining with OX-27 antibody against MHC class I of the PVG-RT1C rat strain disclosed that the PVG-RT1C blood vessels survived and were confined to the PVG-RT1U syngeneic graft. The results suggest that blood vessels supplying CNS allografts placed within the host brain are predominantly of host origin; surviving donor vessels are restricted to the allograft with rare exceptions, which may be dictated by the type of neural allograft and the host CNS site receiving the allograft. The survival of isolated allogeneic CNS blood vessels grafted into the host brain suggests that such blood vessels can present an endothelial genotype and phenotype different from those of host vessels indigenous to the CNS site receiving the allogeneic vessel graft. This finding may have implications in the circumvention of the blood-brain fluid barriers for the CNS delivery of blood-borne therapeutics.

Age Factors↗
PubMed1996

Immune responses to adenovirus vectors in the nervous system.

Non-replicating adenovirus vectors are being developed as vehicles for gene transfer into cells of the nervous system. An important requirement for successful gene transfer is the absence of deleterious cytotoxic or inflammatory side effects of the delivery system. Despite offering relatively stable reporter gene expression, currently available adenovirus vectors also elicit immune responses in the brain, both at the site of vector delivery and at synaptically linked distant sites. However, although an anti-viral T-lymphocyte response eliminates the vector and damages local tissue in many peripheral organs, the immune response to adenovirus in the brain is less effective and enables the vector to persist. Nevertheless, in this persistent state the adenovirus vector remains a potential target for a destructive immune response that can also cause local demyelination. The development of strategies to minimize this damaging immune response, through either vector modification or immunomodulation, will be crucial for the future success of genetic therapies in the brain.

Adenoviridae↗