Cell Squeeze: driving more effective CD8 T-cell activation through cytosolic antigen delivery.

Cell Squeeze is a novel technology that relies on temporarily disrupting the cell membrane to deliver cargo directly into the cytosol. This approach is applicable to a broad range of cell types (peripheral blood mononuclear cells, red blood cells, hematopoietic stem cells, etc.) and cargos (peptides, proteins, small molecules, nucleic acids, and gene-editing complexes) while minimally disrupting normal cell function. By enabling direct cytosolic delivery, one can use this technology to dramatically enhance major histocompatibility complex (MHC) class I presentation of antigens (Ags) for CD8+ T-cell activation-a longstanding challenge for the therapeutic cancer vaccine field that has generally relied on cross-presentation of endocytosed Ags. In addition, by coupling improved MHC class I presentation with coexpression of additional stimulatory factors or systemic immune modulators, one can further enhance the potential impact of an antitumor CD8 response. Pursuing a more direct cellular engineering strategy, which is independent of viral transduction, genetic manipulation, and expansion steps, enables <24 h manufacturing of autologous cell therapies. Through generation of more sophisticated, multifunctional, cell-based vaccines, clinical testing of this technology will elucidate its potential for impact across multiple tumor types.

Neuronal expression of the transcription factor Gli1 using the Talpha1 alpha-tubulin promoter is neuroprotective in an experimental model of Parkinson's disease.

Nigrostriatal neurons degenerate during Parkinson's disease. Experimentally, neurotoxins such as 6-hydroxydopamine (6-OHDA) in rodents, and MPTP in mice and non-human primates, are used to model the disease-induced degeneration of midbrain dopaminergic neurons. Glial-cell-derived neurotrophic factor (GDNF) is a very powerful neuroprotector of dopaminergic neurons in all species examined. However, recent reports have indicated the possibility that GDNF may, in the long term and if expressed in an unregulated manner, exert untoward effects on midbrain dopaminergic neuronal structure and function. Although GDNF remains a powerful neurotrophin, the search for alternative therapies based on alternative and complementary mechanisms of action to GDNF is warranted. Recently, recombinant adenovirus-derived vectors encoding the differentiation factor Sonic Hedgehog (Shh) and its downstream transcriptional activator (Gli1) were shown to protect dopaminergic neurons in the substantia nigra pars compacta from 6-OHDA-induced neurotoxicity in rats in vivo. A pancellular human CMV (hCMV) promoter was used to drive the expression of both Shh and Gli1. Since Gli1 is a transcription factor and therefore exerts its actions intracellularly, we decided to test whether expression of Gli1 within neurons would be effective for neuroprotection. We demonstrate that neuronal-specific expression of Gli1 using the neuron-specific Talpha1 alpha-tubulin (Talpha1) promoter was neuroprotective, and its efficiency was comparable to the pancellular strong viral hCMV promoter. These results suggest that expression of the transcription factor Gli1 solely within neurons is neuroprotective for dopaminergic neurons in vivo and, furthermore, that neuronal-specific promoters are effective within the context of adenovirus-mediated gene therapy-induced neuroprotection of dopaminergic midbrain neurons. Since cell-type specific promoters are known to be weaker than the viral hCMV promoter, our data demonstrate that neuronal-specific expression of transcription factors is an effective, specific, and sufficient targeted approach for neurological gene therapy applications, potentially minimizing side effects due to unrestricted promiscuous gene expression within target tissues.

Differentiation and transcription factor gene therapy in experimental parkinson's disease: sonic hedgehog and Gli-1, but not Nurr-1, protect nigrostriatal cell bodies from 6-OHDA-induced neurodegeneration.

We tested the activity of the dopaminergic neuron differentiation factor sonic hedgehog, its downstream transcription factor target Gli-1, and an orphan nuclear receptor, Nurr-1, necessary for the induction of the dopaminergic phenotype of nigrostriatal neurons, in an in vivo model of nigrostriatal neurodegeneration. Our preliminary experiments demonstrated that all three constructs expressed the proper molecules and that these had the predicted biological activities in vitro. We expressed the N-terminal of sonic hedgehog (ShhN) and the Gli-1 and Nurr-1 entire coding regions from highly purified, and quality controlled, replication-defective adenoviral vectors injected into the brains of rats and used the dopaminergic growth factor GDNF as a positive control. The neurotoxin 6-hydroxydopamine was used to lesion the nigrostriatal dopaminergic innervation; RAd-ShhN and RAd-Gli-1 protected dopaminergic neuronal cell bodies in the substantia nigra, but not axonal terminals in the striatum, from 6-OHDA-induced cell death, while RAd-Nurr-1 was ineffective in protecting either cell bodies or axons. RAd-GDNF was able to protect both the dopaminergic cell bodies and the striatal axon terminals. Our results establish for the first time, to the best of our knowledge, that gene transfer of ShhN and one of its target transcription factors can selectively protect dopaminergic nigrostriatal neuronal cell bodies from a specific neurotoxic insult. Selective protection of nigrostriatal dopaminergic cell bodies by the differentiation factor ShhN and the transcription factor Gli-1 was achieved in a neurotoxic model that eliminates more than 70% of the nigral neurons under consideration. Differentiation and transcription factors can thus be used for the treatment of neurodegeneration by gene therapy.

Gene therapy strategies for intracranial tumours: glioma and pituitary adenomas.

Intracranial tumours such as brain gliomas and pituitary adenomas pose a challenging area of research for the development of gene therapy strategies, both from the point of view of the severity of the diseases, to the physiological implication of gene delivery into the central nervous system and pituitary gland. On the one hand, brain gliomas are very malignant tumours, with a life expectancy of six months to a year at the most after the time of diagnosis, in spite of advances in treatment modalities which involve chemotherapy, surgery and radiotherapy. Gene therapy for these tumours is therefore a very attractive therapeutic modality which due to the severity of the disease is already in clinical trials. On the other hand, pituitary tumours are usually benign, and in most cases, treatment is successful. Nevertheless, there are some instances, especially with the macroadenomas and some invasive tumours in which treatment fails. Gene therapy strategies for these adenomas therefore needs to progress substantially in terms of safety, adverse side effects and physiological impact on the normal pituitary gland before clinical implementation. In this paper, we will review gene delivery systems both viral and non-viral and several therapeutic strategies which could be implemented for the treatment of these diseases. These include cytotoxic approaches both conditional and direct, immune-stimulatory strategies, anti-angiogenic strategies and approaches which harness pro-apoptotic and tumour suppressor gene targets. We will also review the models which are currently available in which these gene therapy strategies can be tested experimentally. This new therapeutic modality holds enormous promise, but we still need substantial improvements both from the delivery, efficacy and safety stand points before it can become a clinical reality.

Fas ligand-transfected myoblasts and islet cell transplantation.

BACKGROUND: Expression of Fas ligand (FasL, CD95L) within the local environment of an allograft may protect from rejection by inducing apoptosis of infiltrating T cells. However, there is mounting evidence that ectopic expression of FasL stimulates an inflammatory response and targets the FasL-expressing tissue for destruction. Given the potential therapeutic applicability of FasL-based immune protection, we sought to determine whether ectopic FasL expression was detrimental and to analyze the inflammatory response induced by ectopic FasL expression in the absence of any confounding allo-immune responses. METHODS AND RESULTS: Two myoblast cell lines expressing different levels of functional FasL were produced. Co-implantation of FasL-expressing myoblasts with syngeneic islets allowed examination of the inflammatory response induced by ectopic FasL expression. In contrast to the suggested benefits of localized FasL expression, islets co-implanted with FasL-expressing myoblasts were destroyed in a vigorous inflammatory response predominated by neutrophils. Interestingly, FasL expression also had a marked anti-tumor effect. CONCLUSIONS: Unless FasL-dependent neutrophil-mediated inflammation can be prevented, it is unlikely that this strategy will be useful for preventing allograft rejection.

Neuropeptide tyrosine-like immunoreactivity (NPY-LI) in ganglion neurons in the adrenal gland of the flat snake (Waglerophis merremii)

In previous morphological and histochemical studies on the adrenal gland of the flat snake, no data demonstrating the existence of ganglion neurons has been reported. The aim of this paper was therefore to establish the presence of ganglion neurons in the adrenal gland of the flat snake Waglerophis merremii and, further to study their chemical phenotype using immunohistochemistry. Our results showed the presence of cells which were immunolabelled with the neuronal marker neurofilament 10 and were thus identified as large ganglion neurons. These cells were localized in the dorsal ribbon of the gland, suggesting a noradrenergic phenotype, exhibited long processes with a longitudinal direction and co-expressed neuropeptide tyrosine- (NPY) and tyrosine hydroxylase-like immunoreactivities (-LI).In addition, NPY-immunoreactive (-IR) fibers were recognized with a wide distribution throughout the gland whereas vasoactive intestinal polypeptide (VIP)-IR fibers were only observed between clusters of cortical and adrenergic chromaffin cells. No cells containing VIP-LI were detected within the gland. Based on their histochemical phenotype, ganglion cells containing NPY and TH could correspond to ganglion neurons type I of the rat. The possible absence of type II ganglion neurons in the adrenal gland of the snake is discussed

Neuropeptide tyrosine-like immunoreactivity (NPY-LI) in ganglion neurons in the adrenal gland of the flat snake (Waglerophis merremii)

In previous morphological and histochemical studies on the adrenal gland of the flat snake, no data demonstrating the existence of ganglion neurons has been reported. The aim of this paper was therefore to establish the presence of ganglion neurons in the adrenal gland of the flat snake Waglerophis merremii and, further to study their chemical phenotype using immunohistochemistry. Our results showed the presence of cells which were immunolabelled with the neuronal marker neurofilament 10 and were thus identified as large ganglion neurons. These cells were localized in the dorsal ribbon of the gland, suggesting a noradrenergic phenotype, exhibited long processes with a longitudinal direction and co-expressed neuropeptide tyrosine- (NPY) and tyrosine hydroxylase-like immunoreactivities (-LI).In addition, NPY-immunoreactive (-IR) fibers were recognized with a wide distribution throughout the gland whereas vasoactive intestinal polypeptide (VIP)-IR fibers were only observed between clusters of cortical and adrenergic chromaffin cells. No cells containing VIP-LI were detected within the gland. Based on their histochemical phenotype, ganglion cells containing NPY and TH could correspond to ganglion neurons type I of the rat. The possible absence of type II ganglion neurons in the adrenal gland of the snake is discussed

Neuropeptide tyrosine-like immunoreactivity (NPY-LI) in ganglion neurons in the adrenal gland of the flat snake (Waglerophis merremii).

In previous morphological and histochemical studies on the adrenal gland of the flat snake, no data demonstrating the existence of ganglion neurons has been reported. The aim of this paper was therefore to establish the presence of ganglion neurons in the adrenal gland of the flat snake Waglerophis merremii and, further to study their chemical phenotype using immunohistochemistry. Our results showed the presence of cells which were immunolabelled with the neuronal marker neurofilament 10 and were thus identified as large ganglion neurons. These cells were localized in the dorsal ribbon of the gland, suggesting a noradrenergic phenotype, exhibited long processes with a longitudinal direction and co-expressed neuropeptide tyrosine- (NPY) and tyrosine hydroxylase-like immunoreactivities (-LI). In addition, NPY-immunoreactive (-IR) fibers were recognized with a wide distribution throughout the gland whereas vasoactive intestinal polypeptide (VIP)-IR fibers were only observed between clusters of cortical and adrenergic chromaffin cells. No cells containing VIP-LI were detected within the gland. Based on their histochemical phenotype, ganglion cells containing NPY and TH could correspond to ganglion neurons type I of the rat. The possible absence of type II ganglion neurons in the adrenal gland of the snake is discussed.

Galanin and cholecystokinin in cultured magnocellular neurons isolated from adult rat supraoptic nuclei: a correlative light and scanning electron microscopical study.

Cultured magnocellular neurons, isolated from adult rat supraoptic nuclei, were characterized by immunocytochemistry, using the avidin-biotin-peroxidase complex and antisera to vasopressin, oxytocin, galanin and cholecystokinin. Light microscope examination of the immunostained cultures revealed the presence of vasopressin- and oxytocin-like immunoreactivity, as well as neurons containing either galanin- or cholecystokinin-like immunoreactivity. In contrast, no significant galanin- or cholecystokinin-like immunoreactivity could be observed in freshly dispersed cells. Correlative scanning electron microscopical observations in the secondary electron imaging mode revealed that the stained neurons appeared significantly brighter than the unstained structures. Complementary observations with toad brain sections (preoptic area), immunostained for galanin, led to the same result. Considering previous results, it is suggested that the presence of galanin- and cholecystokinin-like immunoreactivity in the cultured neurons and its virtual absence in freshly dispersed cells is indicating a participation of these peptides in the regenerative processes taking place during culture. It is further concluded that the avidin-biotin-peroxidase method is suitable for correlative light and scanning electron microscopical studies of smooth surfaces and cultured cells.

Dispersion and culture of magnocellular neurons from the supraoptic nucleus of the adult rat.

A simple and efficient procedure for the dispersion and culture of magnocellular neurons of the adult rat hypothalamus was developed. The enzymatically and mechanically dispersed cells were highly viable and showed neurite outgrowth after 3 days of culture. The neurons could be maintained for more than 4 weeks without any sign of deterioration. Light-optic immunocytochemistry of the cultured cells revealed the presence of many oxytocin- and vasopressin-immunoreactive neurons. The results indicate that the method may be suitable for many experimental purposes.

Galanin-like immunoreactive expression in the central nervous system of the toad.

The distribution of galanin (GAL)-like immunoreactivity (-LI) was studied in the CNS of the toad (Bufo arenarum Hensel). Tissue sections were incubated with antibodies directed toward rat or porcine GAL and processed either for the avidin-biotin complex, or for the indirect immunofluorescence techniques. In the telencephalon GAL-immunoreactive (-IR) perikarya were observed in the ventral part of the striatum and in the septal accumbens nuclei. Immunopositive neurons were also observed in the medial amigdala with some intermingled cells between the fibers of the anterior commissure. Numerous GAL-IR perikarya were present along the rostrocaudal medial preoptic nucleus. Occasionally lightly immunoreactive cells were detected in the magnocellular region. The most numerous accumulation of GAL-IR cells was present in the ventral hypothalamus around the infundibular region, in the posterior tubercle and in the nucleus of the paraventricular organ. Immunostained cells were also present in the pretectal gray, solitary nucleus, gracil nucleus and in the spinal cord in the intermediate gray and in large motoneurons of the ventral horn. The widespread distribution found of GAL-LI suggests that GAL in the toad, as well as in mammalian species, may serve a variety of functions with a preponderant role in neuroendocrine processes. A role for GAL as a trophic factor in the brain of the toad is also suggested.

NPY- and CGRP-like immunoreactive nerve fibers in the testis and mesorchium of the toad (Bufo arenarum).

The presence and distribution of peptidergic nerve fibers were studied in the testis and mesorchium of the toad by means of immunohistochemistry. Cryostat sections of the testis and whole-mount preparations of mesorchia were immunostained with antisera to calcitonin gene-related peptide (CGRP) and neuropeptide tyrosine (NPY). After leaving the mesorchium CGRP-immunoreactive (IR) fibers were seen predominantly running in between the seminiferous tubules. In addition, a small population of CGRP-IR nerve fibers formed thin plexuses around blood vessels. Conversely, NPY-like immunoreactivity predominated in nerve fibers that formed dense plexuses around vessels both in the mesorchium and testis. Additionally, some single NPY-IR nerve fibers could be seen in both structures studied. The functional significance of these peptidergic systems in the testis of the toad remains to be analyzed.

Galanin-like immunoreactive expression in the central nervous system of the toad

The distribution of galanin (GAL)-like immunoreactivity (-LI) was studied in the CNS of the toad (Bufo arenarum Hensel). Tissue sections were incubated with antibodies directed toward rat or porcine GAL and processed either for the avidin-biotin complex, or for the indirect immunofluorescence techniques. In the telencephalon GAL-immunoreactive (-IR) perikarya were observed in the ventral part of the striatum and in the septal accumbens nuclei. Immunopositive neurons were also observed in the medial amigdala with some intermingled cells between the fibers of the anterior commissure. Numerous GAL-IR perikarya were present along the rostrocaudal medial preoptic nucleus. Occasionally lightly immunoreactive cells were detected in the magnocellular region. The most numerous accumulation of GAL-IR cells was present in the ventral hypothalamus around the infundibular region, in the posterior tubercle and in the nucleus of the paraventricular organ. Immunostained cells were also present in the pretectal gray, solitary nucleus, gracil nucleus and in the spinal cord in the intermediate gray and in large motoneurons of the ventral horn. The widespread distribution found of GAL-LI suggests that GAL in the toad, as well as in mammalian species, may serve a variety of functions with a preponderant role in neuroendocrine processes. A role for GAL as a trophic factor in the brain of the toad is also suggested.(AU)

Galanin-like immunoreactive expression in the central nervous system of the toad

The distribution of galanin (GAL)-like immunoreactivity (-LI) was studied in the CNS of the toad (Bufo arenarum Hensel). Tissue sections were incubated with antibodies directed toward rat or porcine GAL and processed either for the avidin-biotin complex, or for the indirect immunofluorescence techniques. In the telencephalon GAL-immunoreactive (-IR) perikarya were observed in the ventral part of the striatum and in the septal accumbens nuclei. Immunopositive neurons were also observed in the medial amigdala with some intermingled cells between the fibers of the anterior commissure. Numerous GAL-IR perikarya were present along the rostrocaudal medial preoptic nucleus. Occasionally lightly immunoreactive cells were detected in the magnocellular region. The most numerous accumulation of GAL-IR cells was present in the ventral hypothalamus around the infundibular region, in the posterior tubercle and in the nucleus of the paraventricular organ. Immunostained cells were also present in the pretectal gray, solitary nucleus, gracil nucleus and in the spinal cord in the intermediate gray and in large motoneurons of the ventral horn. The widespread distribution found of GAL-LI suggests that GAL in the toad, as well as in mammalian species, may serve a variety of functions with a preponderant role in neuroendocrine processes. A role for GAL as a trophic factor in the brain of the toad is also suggested.

Immunohistochemical localization of neurotensin in a subpopulation of noradrenergic chromaffin cells of the adrenal gland of the flat snake (Waglerophis merremii).

The distribution of neurotensin (NT)-like immunoreactivity (LI) in the adrenal gland of the snake Waglerophis merremii has been examined immunohistochemically. Double staining, combining NT with tyrosine hydroxylase (TH) or calcitonin gene-related peptide (CGRP) antibodies and TH with CGRP antibodies, was also carried out. Results were analyzed by conventional and by confocal fluorescence microscopy. Immunostaining revealed a subpopulation of chromaffin cells containing NT-LI within the dorsal noradrenergic ribbon. In addition, there were some NT-immunoreactive (IR) fibers in this region. NT immunoreactivity was not present within adrenergic chromaffin cells or in cortical tissue. Double staining revealed CGRP-IR fibers innervating most of the chromaffin adrenergic cells. Within the dorsal noradrenergic ribbon, two groups of chromaffin TH-IR cells were present, one receiving a dense CGRP-IR innervation and another without contact with CGRP-IR terminals. The latter chromaffin cells displayed NT-LI. These results show, for the first time, the presence of a neuropeptide in chromaffin noradrenergic cells of a reptilian adrenal gland and open up the possibility that other peptides may also be present in these cells.

Galanin-like immunoreactive expression in the central nervous system of the toad.

The distribution of galanin (GAL)-like immunoreactivity (-LI) was studied in the CNS of the toad (Bufo arenarum Hensel). Tissue sections were incubated with antibodies directed toward rat or porcine GAL and processed either for the avidin-biotin complex, or for the indirect immunofluorescence techniques. In the telencephalon GAL-immunoreactive (-IR) perikarya were observed in the ventral part of the striatum and in the septal accumbens nuclei. Immunopositive neurons were also observed in the medial amigdala with some intermingled cells between the fibers of the anterior commissure. Numerous GAL-IR perikarya were present along the rostrocaudal medial preoptic nucleus. Occasionally lightly immunoreactive cells were detected in the magnocellular region. The most numerous accumulation of GAL-IR cells was present in the ventral hypothalamus around the infundibular region, in the posterior tubercle and in the nucleus of the paraventricular organ. Immunostained cells were also present in the pretectal gray, solitary nucleus, gracil nucleus and in the spinal cord in the intermediate gray and in large motoneurons of the ventral horn. The widespread distribution found of GAL-LI suggests that GAL in the toad, as well as in mammalian species, may serve a variety of functions with a preponderant role in neuroendocrine processes. A role for GAL as a trophic factor in the brain of the toad is also suggested.

Histochemical study of chromaffin cells and nerve fibers in the adrenal gland of the flat snake (Waglerophis merremii).

The distributions of tyrosine hydroxylase (TH), protein gene product (PGP) 9.5, calcitonin gene-related peptide (CGRP), and peptide histidine isoleucine (PHI) have been examined immunohistochemically in the adrenal gland of the snake Waglerophis merremii. The morphology of chromaffin cells and the presence of ganglionic neurons in the gland revealed by means of the glutaraldehyde-silver technique and electron microscopy are also described. Two distinct types of TH-immunoreactive (-IR) cells are present in the dorsal noradrenergic ribbon: small chromaffin cells and a larger type identified as ganglionic neurons. Small, mostly round or fusiform cells often displayed long processes. Ganglionic cells, arranged in patches, had long processes entering the cortex of the gland. Chromaffin adrenergic cells, forming small groups of 4-7 cells, were scattered within the interrenal tissue and had a wide variety of shapes with processes that appeared to contact other chromaffin cells. Bundles of PGP 9.5-IR fibers occurred in the subcapsular zone of the adrenal gland with fibers entering the cortex and dorsal noradrenergic ribbon of the gland. Thick and thin TH-IR fibers were seen. Thick TH-IR fibers were nonvaricose and appeared to originate mainly in ganglionic neurons. Thin TH-IR fibers with small varicosities were numerous in the interrenal tissue and were frequently seen between clusters of adrenergic cells in close apposition to cortical cells and vessels. CGRP-IR fibers were present throughout the entire adrenal gland, whereas PHI-IR fibers had a preferential distribution in the interrenal tissue. Both CGRP- and PHI-IR fibers were closely associated with vessels and cortical cells.