Harry M. Charlton

Adenovirus gene transfer causes inflammation in the brain.

We report that injecting an E1-deleted, non-replicating, human adenovirus type 5 vector into the brain leads to an inflammatory response. Much of this inflammation is induced directly by the virion particles themselves rather than through the expression of new proteins from the vector. The severity of inflammation was found to depend on the strain of inbred rat used: PVG rats have less inflammation than AO rats in response to a vector injection. Twelve hours after injection of adenovirus vectors into the striatum of AO rats, leukocytes were seen marginating to the walls of nearby blood vessels. By two days there was a large increase in major histocompatibility complex class I expression and a heavy infiltration of leukocytes, mainly macrophages and T cells. Retrograde transport of adenovirus to neurons of the substantia nigra was associated with a delayed and less intense inflammation at this distant site. Although AO and PVG rats showed comparable responses in the striatum up to six days, at later times PVG rats had less intense inflammation. In spite of the inflammatory response, vector-driven expression of the marker protein beta-galactosidase and an adenovirus early protein was seen for at least two months following the injection, although expression declined with time. The observation that adenovirus gene transfer leads to an inflammatory response in the brain must be taken into account when planning and interpreting experiments with these vectors. Furthermore, we conclude that using an appropriate strain of rat can diminish some aspects of the inflammation.

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.

The immune response to intracerebral neural grafts.

Neural transplantation offers a potential therapeutic approach to a variety of neurological disorders, most notably those of a degenerative nature. However, the degree of immunological privilege (i.e. isolation from an immune response) in the brain, which is not absolute, may be a significant impediment to the survival of histoincompatible grafts. The nature of this privilege, together with the specific immune events leading to neural graft rejection, are discussed. As a consequence of this immune-mediated rejection, immunosuppression in some form might be necessary to guarantee long-term graft survival. Various strategies are being explored to suppress the immune response to neural grafts, not only for future use in clinical therapies, but also to bring intracerebral allo- and xenotransplantation to the attention of the general neurobiologist.

Indefinite survival of neural xenografts induced with anti-CD4 monoclonal antibodies.

Xenografts of neural tissue are usually rapidly rejected when transplanted into the central nervous system of adult recipient animals. This study has examined the cell mediated immune response to both concordant (between closely related species) and discordant (between distantly related species) neural xenografts in the mouse, and has investigated the role of the CD4+ and CD8+ T lymphocyte subsets in this process using monoclonal antibodies specific for the CD4 and CD8 cell surface glycoproteins. We have established that: (1) in this model system concordant neural xenograft rejection occurs within 15-30 days; however, xenograft survival can be dramatically prolonged with CD4+, but not CD8+, T lymphocyte depletion; (2) the administration of two successive courses of a high dose of anti-CD4 monoclonal antibody treatment results in indefinite concordant neural xenograft survival; (3) the mechanism by which the high dose anti-CD4 monoclonal antibody therapy appears to function involves the depletion of intrathymic CD4+ cells; (4) anti-CD4 monoclonal antibody treatment enhances discordant neural xenograft survival, to beyond 60 days in many cases. These results demonstrate that CD4+ T lymphocytes are of central importance in the immune response to both concordant and discordant neural xenoantigens. Thus the use of anti-CD4 monoclonal antibody therapy is an effective strategy to prolong significantly the survival of xenogeneic neural transplants. Furthermore this treatment caused no obvious deleterious side-effects. These findings have implications for future cross-species studies in experimental neurobiology and, possibly, in clinical neural transplantation.

Spermatogenesis and Sertoli cell activity in mice lacking Sertoli cell receptors for follicle stimulating hormone and androgen

Spermatogenesis in the adult male depends on the action of FSH and androgen. Ablation of either hormone has deleterious effects on Sertoli cell function and the progression of germ cells through spermatogenesis. In this study we generated mice lacking both FSH receptors (FSHRKO) and androgen receptors on the Sertoli cell (SCARKO) to examine how FSH and androgen combine to regulate Sertoli cell function and spermatogenesis. Sertoli cell number in FSHRKO-SCARKO mice was reduced by about 50% but was not significantly different from FSHRKO mice. In contrast, total germ cell number in FSHRKO-SCARKO mice was reduced to 2% of control mice (and 20% of SCARKO mice) due to a failure to progress beyond early meiosis. Measurement of Sertoli cell-specific transcript levels showed that about a third were independent of hormonal action on the Sertoli cell, whereas others were predominantly androgen dependent or showed redundant control by FSH and androgen. Results show that FSH and androgen act through redundant, additive, and synergistic regulation of spermatogenesis and Sertoli cell activity. In addition, the Sertoli cell retains a significant capacity for activity, which is independent of direct hormonal regulation.

Implantation of normal fetal preoptic area into hypogonadal mutant mice: Temporal relationships of the growth of gonadotropin-releasing hormone neurons and the development of the pituitary/testicular axis

Central nervous system tissue which included the preoptic area (an area rich in gonadotropin-releasing hormone neurons) was taken from normal 17-day fetal mice and transplanted into the infundibular recess of the third ventricle of the hypothalamus of 90-day male mutant hypogonadal mouse hosts that are unable to synthesize the neurohormone, gonadotropin-releasing hormone. The growth and development of gonadotropin-releasing hormone neurons and fibers in the donor and host tissue as well as recovery of the pituitary-testicular axis were followed from 10 to 120 days post-implantation. Testicular growth was evident in 94% of the hypogonadal animals within 30 days post-implantation, continued for 90 days but showed no further increase during the remainder of the experiment. Increases in seminal vesicle weight, an index of testosterone secretion, were measurable at 30 days and continued through to the end of the experiment. Pituitary concentrations of gonadotropins were doubled at 30 days over that seen in the control mutant mouse and were maintained thereafter at normal or supranormal concentrations. In contrast plasma levels of gonadotropins, although above baseline at 30 days, never reached normal circulating levels. Nevertheless, it appeared that the concentration of luteinizing hormone achieved was sufficient to initiate and maintain testicular growth and testosterone secretion for the entire duration of the experiment. Immunocytochemical analysis of brain tissue was used to determine the presence and numbers of gonadotropin-releasing hormone neurons in the transplant and the distribution of their fibers in the donor and host tissue. The numbers of immunoreactive gonadotropin-releasing hormone neurons present at the time of sacrifice ranged from 3 to 140. Fiber outgrowth from the donor cells into the host was noted as early as 10 days post-implantation and the density of outgrowth continued to increase over the course of the experiment. Positive fibers tended to accumulate over the tuberoinfundibular sulci as they do in normal animals. In those instances where the transplant was placed a long distance from the median eminence, the gonadotropin-releasing hormone axons grew on the internal surface of the third ventricle until they reached these specific exit zones. These studies indicate that in the mutant hypogonadal mouse, central nervous system transplants from normal fetal mice can maintain the function of the pituitary-gonadal axis for periods of up to 120 days post-implantation. Outgrowth of the neurosecretory fibers begins very soon after implantation and the axons tend to follow pathways seen in normal tissue.(ABSTRACT TRUNCATED AT 400 WORDS)

Cytokines and peripheral tolerance to alloantigen

The induction of peripheral tolerance to alloantigen is accompanied in many cases by a decrease in the production of cytokines such as IL-2 and IFN gamma, yet a sustained production of cytokines such as IL-10 and IL-4. Whether or not this altered pattern of cytokine production in tolerant animals is causally related to the induction and/or maintenance of the tolerant state has yet to be fully determined, although experiments blocking selectively the action of IL-2 with CD25 antibodies suggest that manipulation of cytokine production may at least be a route to tolerance. Alternative methods for directly influencing the cytokine balance are sought and recent experiments on the CD28/CTLA-4-B7 interaction suggest a possible approach.

Chapter 12 Angiogenesis and the blood-brain barrier in solid and dissociated cell grafts within the CNS

Available evidence suggests that blood vessels indigenous to solid CNS and peripheral tissues grafted to the brain are sustained and maintain the morphological and permeability characteristics they manifest in normal life. Furthermore, these vessels of graft origin anastomose (albeit not rapidly) with vessels of the surrounding host tissue predominantly at the host-graft interface and less so, or not at all, within the graft itself. For these reasons, blood-brain and brain-blood barriers, evident in the late fetal and neonatal CNS, can be expected to exist within CNS grafts placed intracerebrally or extracerebrally, providing the graft remains viable. Peripheral neural and non-neural tissues not possessing cellular barriers to circulating macromolecules do not acquire such barriers subsequent to their transplantation within the CNS. The absence of a blood-brain barrier in the adrenal gland grafted intracerebrally may be relevant for the treatment of Parkinsons disease with blood-borne therapeutics. Compared to solid tissue grafts, cell suspension grafts have the potential of becoming vascularized rapidly. That cell suspensions of neurons and of glia are supplied with BBB vessels of host origin and that the permeability characteristics of host BBB vessels are altered by a tumor cell suspension reaffirm the belief that the type of transplanted cell/tissue indeed determines the permeability characteristics of the blood vessels supplying it. The suspected immunologic privilege of the CNS is not absolute. Eventual host rejection of allografts placed within the third ventricle may be a dual consequence of the absence of a BBB at the level of the host median eminence and involvement of the minor histocompatibility complex.

Allografts of CNS tissue possess a blood-brain barrier: I. Grafts of medial preoptic area in hypogonadal mice

This study represents the first part of a three-part investigation of blood vessels supplying CNS tissue transplanted within the brains of adult mammalian hosts. The results emphasize that blood vessels in solid CNS grafts contribute a blood-brain barrier to that of the host. Neurosecretory cells in basal forebrain grafts placed intraventricularly on the dorsal surface of the host median eminence, a neurosecretory site containing fenestrated blood vessels, do not stimulate similar blood vessels to inhabit the transplanted tissue. Solid grafts of the medial preoptic area containing neurons that synthesize and secrete gonadotropic hormone-releasing hormone (GnRH) were obtained from AKR mice and placed into the third cerebral ventricle of hypogonadal (HPG) mice genetically incapable of synthesizing GnRH. GnRH neurons in the allografts were confirmed immunohistochemically. Blood vessels supplying the host median eminence and the allograft at 10 days to 3 months post-transplantation were analyzed with peroxidase cytochemistry applied in three ways: to HPG mice injected systemically with native horseradish peroxidase; to HPG mice infused into the aorta with peroxidase subsequent to perfusion fixation; and to HPG mice brains fixed by immersion and incubated for endogenous peroxidase activity in red cells retained within blood vessels. The median eminence of the HPG mouse was innervated by GnRH neurons residing within the graft, and blood vessels traversing the median eminence-allograft interface were seen rarely. The allografts contained no fenestrated endothelia, and no extravasations of blood-borne HRP were related directly to leaky blood vessels supplying the grafted tissue. Endothelial cells throughout the CNS grafts were similar morphologically to blood-brain barrier endothelia; they were nonfenestrated, exhibited interendothelial tight junctional complexes and an endomembrane system of organelles, and they endocytosed blood-borne HRP that eventually was sequestered within dense body lysosomes. The results support the belief that blood vessels supplying CNS tissue transplanted to a host brain manifest endothelial characteristics identical to those of the tissue in normal life and to those of the host CNS.

A γ34.5 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.