Lactate transporters in the rat barrel cortex sustain whisker-dependent BOLD fMRI signal and behavioral performance.

Lactate is an efficient neuronal energy source, even in presence of glucose. However, the importance of lactate shuttling between astrocytes and neurons for brain activation and function remains to be established. For this purpose, metabolic and hemodynamic responses to sensory stimulation have been measured by functional magnetic resonance spectroscopy and blood oxygen level-dependent (BOLD) fMRI after down-regulation of either neuronal MCT2 or astroglial MCT4 in the rat barrel cortex. Results show that the lactate rise in the barrel cortex upon whisker stimulation is abolished when either transporter is down-regulated. Under the same paradigm, the BOLD response is prevented in all MCT2 down-regulated rats, while about half of the MCT4 down-regulated rats exhibited a loss of the BOLD response. Interestingly, MCT4 down-regulated animals showing no BOLD response were rescued by peripheral lactate infusion, while this treatment had no effect on MCT2 down-regulated rats. When animals were tested in a novel object recognition task, MCT2 down-regulated animals were impaired in the textured but not in the visual version of the task. For MCT4 down-regulated animals, while all animal succeeded in the visual task, half of them exhibited a deficit in the textured task, a similar segregation into two groups as observed for BOLD experiments. Our data demonstrate that lactate shuttling between astrocytes and neurons is essential to give rise to both neurometabolic and neurovascular couplings, which form the basis for the detection of brain activation by functional brain imaging techniques. Moreover, our results establish that this metabolic cooperation is required to sustain behavioral performance based on cortical activation.

Acidic tumor microenvironment abrogates the efficacy of mTORC1 inhibitors.

BACKGROUND: Blocking the mechanistic target of rapamycin complex-1 (mTORC1) with chemical inhibitors such as rapamycin has shown limited clinical efficacy in cancer. The tumor microenvironment is characterized by an acidic pH which interferes with cancer therapies. The consequences of acidity on the anti-cancer efficacy of mTORC1 inhibitors have not been characterized and are thus the focus of our study. METHODS: Cancer cell lines were treated with rapamycin in acidic or physiological conditions and cell proliferation was investigated. The effect of acidity on mTORC1 activity was determined by Western blot. The anticancer efficacy of rapamycin in combination with sodium bicarbonate to increase the intratumoral pH was tested in two different mouse models and compared to rapamycin treatment alone. Histological analysis was performed on tumor samples to evaluate proliferation, apoptosis and necrosis. RESULTS: Exposing cancer cells to acidic pH in vitro significantly reduced the anti-proliferative effect of rapamycin. At the molecular level, acidity significantly decreased mTORC1 activity, suggesting that cancer cell proliferation is independent of mTORC1 in acidic conditions. In contrast, the activation of mitogen-activated protein kinase (MAPK) or AKT were not affected by acidity, and blocking MAPK or AKT with a chemical inhibitor maintained an anti-proliferative effect at low pH. In tumor mouse models, the use of sodium bicarbonate increased mTORC1 activity in cancer cells and potentiated the anti-cancer efficacy of rapamycin. Combining sodium bicarbonate with rapamycin resulted in increased tumor necrosis, increased cancer cell apoptosis and decreased cancer cell proliferation as compared to single treatment. CONCLUSIONS: Taken together, these results emphasize the inefficacy of mTORC1 inhibitors in acidic conditions. They further highlight the potential of combining sodium bicarbonate with mTORC1 inhibitors to improve their anti-tumoral efficacy.

A New Ex Vivo Human Skin Burn Model.

Currently, most burn models for preclinical testing are on animals. For obvious ethical, anatomical, and physiological reasons, these models could be replaced with optimized ex vivo systems. The creation of a burn model on human skin using a pulsed dye laser could represent a relevant model for preclinical research. Six samples of excess human abdominal skin were obtained within one hour after surgery. Burn injuries were induced on small samples of cleaned skin using a pulsed dye laser on skin samples, at varying fluences, pulse numbers and illumination duration. In total, 70 burn injuries were performed on skin ex vivo before being histologically and dermato-pathologically analyzed. Irradiated burned skin samples were classified with a specified code representing burn degrees. Then, a selection of samples was inspected after 14 and 21 days to assess their capacity to heal spontaneously and re-epithelize. We determined the parameters of a pulsed dye laser inducing first, second, and third degree burns on human skin and with fixed parameters, especially superficial and deep second degree burns. After 21 days with the ex vivo model, neo-epidermis was formed. Our results showed that this simple, rapid, user-independent process creates reproducible and uniform burns of different, predictable degrees that are close to clinical reality. Human skin ex vivo models can be an alternative to and complete animal experimentation, particularly for preclinical large screening. This model could be used to foster the testing of new treatments on standardized degrees of burn injuries and thus improve therapeutic strategies.

Reactivation of AKT signaling following treatment of cancer cells with PI3K inhibitors attenuates their antitumor effects.

Targeting the phosphatidylinositol-3-kinase (PI3K) is a promising approach in cancer therapy. In particular, PI3K blockade leads to the inhibition of AKT, a major downstream effector responsible for the oncogenic activity of PI3K. However, we report here that small molecule inhibitors of PI3K only transiently block AKT signaling. Indeed, treatment of cancer cells with PI3K inhibitors results in a rapid inhibition of AKT phosphorylation and signaling which is followed by the reactivation of AKT signaling after 48 h as observed by Western blot. Reactivation of AKT signaling occurs despite effective inhibition of PI3K activity by PI3K inhibitors. In addition, wortmannin, a broad range PI3K inhibitor, did not block AKT reactivation suggesting that AKT signals independently of PI3K. In a therapeutical perspective, combining AKT and PI3K inhibitors exhibit stronger anti-proliferative and pro-apoptotic effects compared to AKT or PI3K inhibitors alone. Similarly, in a tumor xenograft mouse model, concomitant PI3K and AKT blockade results in stronger anti-cancer activity compared with either blockade alone. This study shows that PI3K inhibitors only transiently inhibit AKT which limits their antitumor activities. It also provides the proof of concept to combine PI3K inhibitors with AKT inhibitors in cancer therapy.

PAR2 promotes vaccine-induced protection against Helicobacter infection in mice.

BACKGROUND & AIMS: Protective immunization limits Helicobacter infection of mice by undetermined mechanisms. Protease-activated receptor 2 (PAR2) signaling is believed to regulate immune and inflammatory responses. We investigated the role of PAR2 in vaccine-induced immunity against Helicobacter infection. METHODS: Immune responses against Helicobacter infection were compared between vaccinated PAR2-/- and wild-type (WT) mice. Bacterial persistence, gastric pathology, and inflammatory and cellular responses were assessed using the rapid urease test (RUT), histologic analyses, quantitative polymerase chain reaction, and flow cytometry, respectively. RESULTS: Following vaccination, PAR2-/- mice did not have reductions in Helicobacter felis infection (RUT values were 0.01±0.01 for WT mice and 0.11±0.13 for PAR2-/- mice; P<.05). The vaccinated PAR2-/- mice had reduced inflammation-induced stomach tissue damage (tissue damage scores were 8.83±1.47 for WT mice and 4.86±1.35 for PAR2-/- mice; P<.002) and reduced T-helper (Th)17 responses, based on reduced urease-induced interleukin (IL)-17 secretion by stomach mononuclear cells (5182 ± 1265 pg/mL for WT mice and 350±436 pg/mL for PAR2-/- mice; P<.03) and reduced recruitment of CD4+ IL-17+ T cells into the gastric mucosa of PAR2-/- mice following bacterial challenge (3.7%±1.5% for WT mice and 2.6%±1.1% for PAR2-/- mice; P<.05). In vitro, H felis-stimulated dendritic cells (DCs) from WT mice induced greater secretion of IL-17 by ovalbumin-stimulated OT-II transgenic CD4+ T cells compared with DCs from PAR2-/- mice (4298±347 and 3230±779; P<.04), indicating that PAR2-/- DCs are impaired in priming of Th17 cells. Adoptive transfer of PAR2+/+ DCs into vaccinated PAR2-/- mice increased vaccine-induced protection (RUT values were 0.11±0.10 and 0.26±0.15 for injected and noninjected mice, respectively; P<.03). CONCLUSIONS: PAR2 activates DCs to mediate vaccine-induced protection against Helicobacter infection in mice.

Humoral Responses Against Variants of Concern by COVID-19 mRNA Vaccines in Immunocompromised Patients.

Importance: There are limited comparative data on the durability of neutralizing antibody (nAb) responses elicited by messenger RNA (mRNA) vaccines against the SARS-CoV-2 variants of concern (VOCs) in immunocompromised patients and healthy controls. Objective: To assess the humoral responses after vaccination with BNT162b2 (Pfizer-BioNTech) or mRNA-1273 (Moderna) vaccines. Design, Setting, and Participants: In this prospective, longitudinal monocentric comparative effectiveness study conducted at the Lausanne University Hospital, binding IgG anti-spike antibody and nAb levels were measured at 1 week, 1 month, 3 months, and 6 months after vaccination with mRNA-1273 (24.6% of participants) or BNT162b2 (75.3% of participants). Interventions: All participants received 2 doses of either mRNA-1273 or BNT162b2 vaccines 4 to 6 weeks apart. Main Outcomes and Measures: The primary outcome of the study was the persistence of nAb responses against the original, nonvariant SARS-CoV-2 (2019-nCoV) and different VOCs at 6 months after vaccination. Key secondary outcomes were associations of the type of mRNA vaccine, the underlying disease, and the treatment with the response to vaccination. Results: Among the 841 participants enrolled between January 14 and August 8, 2021, the patient population comprised 637 participants (mean [SD] age, 61.8 [13.7] years; 386 [60.6%] female), and the healthy control population comprised 204 participants (mean [SD] age, 45.9 [12.0] years; 144 [70.6%] female). There were 399 patients with solid cancers, 101 with hematologic cancers, 38 with solid organ transplants, 99 with autoimmune diseases, and 204 healthy controls. More than 15 000 nAb determinations were performed against the original, nonvariant 2019-nCoV and the Alpha, Beta, Gamma, and Delta variants. The proportions of nAbs and their titers decreased in all study groups at 6 months after vaccination, with the greatest decreases for the Beta and Delta variants. For Beta, the proportion decreased to a median (SE) of 39.2% (5.5%) in those with hematologic cancers, 44.8% (2.7%) in those with solid cancers, 23.1% (8.3%) in those with solid organ transplants, and 22.7% (4.8%) in those with autoimmune diseases compared with 52.1% (4.2%) in healthy controls. For Delta, the proportions decreased to 41.8% (5.6%) in participants with hematologic cancer, 51.9% (2.7%) in those with solid cancers, 26.9% (8.7%) in those with solid organ transplants, and 30.7% (5.3%) in those with autoimmune diseases compared with 56.9% (4.1%) healthy controls. Neutralizing antibody titers decreased 3.5- to 5-fold between month 1 and month 6, and the estimated duration of response was greater and more durable among those participants vaccinated with mRNA-1273. In participants with solid cancers, the estimated duration of nAbs against the Beta variant was 221 days with mRNA-1273 and 146 days with BNT162b2, and against the Delta variant, it was 226 days with mRNA-1273 and 161 with BNT162b2. The estimated duration of nAbs in participants with hematologic cancers was 113 and 127 days against Beta and Delta variants, respectively. Conclusions and Relevance: This comparative effectiveness study suggests that approximately half of patients with hematologic cancers and solid cancers, about 70% of patients with solid organ transplants or autoimmune diseases, and 40% of healthy controls have lost nAbs against the circulating VOCs at 6 months after vaccination. These findings may be helpful for developing the best boosting vaccination schedule especially in immunocompromised patients.

Interleukin-17 is a critical mediator of vaccine-induced reduction of Helicobacter infection in the mouse model.

BACKGROUND & AIMS: Despite the proven ability of immunization to reduce Helicobacter infection in mouse models, the precise mechanism of protection has remained elusive. This study explores the possibility that interleukin (IL)-17 plays a role in the reduction of Helicobacter infection following vaccination of wild-type animals or in spontaneous reduction of bacterial infection in IL-10-deficient mice. METHODS: In mice, reducing Helicobacter infection, the levels and source of IL-17 were determined and the role of IL-17 in reduction of Helicobacter infection was probed by neutralizing antibodies. RESULTS: Gastric IL-17 levels were strongly increased in mice mucosally immunized with urease plus cholera toxin and challenged with Helicobacter felis as compared with controls (654 +/- 455 and 34 +/- 84 relative units for IL-17 messenger RNA expression [P < .01] and 6.9 +/- 8.4 and 0.02 +/- 0.04 pg for IL-17 protein concentration [P < .01], respectively). Flow cytometry analysis showed that a peak of CD4(+)IL-17(+) T cells infiltrating the gastric mucosa occurred in immunized mice in contrast to control mice (4.7% +/- 0.3% and 1.4% +/- 0.3% [P < .01], respectively). Gastric mucosa-infiltrating CD4(+)IL-17(+) T cells were also observed in IL-10-deficient mice that spontaneously reduced H felis infection (4.3% +/- 2.3% and 2% +/- 0.6% [P < .01], for infected and noninfected IL-10-deficient mice, respectively). In wild-type immunized mice, intraperitoneal injection of anti-IL-17 antibodies significantly inhibited inflammation and the reduction of Helicobacter infection in comparison with control antibodies (1 of 12 mice vs 9 of 12 mice reduced Helicobacter infection [P < .01], respectively). CONCLUSIONS: IL-17 plays a critical role in the immunization-induced reduction of Helicobacter infection from the gastric mucosa.

Tau accumulation in astrocytes of the dentate gyrus induces neuronal dysfunction and memory deficits in Alzheimer's disease.

Alzheimer's disease (AD) is characterized by the accumulation of the tau protein in neurons, neurodegeneration and memory loss. However, the role of non-neuronal cells in this chain of events remains unclear. In the present study, we found accumulation of tau in hilar astrocytes of the dentate gyrus of individuals with AD. In mice, the overexpression of 3R tau specifically in hilar astrocytes of the dentate gyrus altered mitochondrial dynamics and function. In turn, these changes led to a reduction of adult neurogenesis, parvalbumin-expressing neurons, inhibitory synapses and hilar gamma oscillations, which were accompanied by impaired spatial memory performances. Together, these results indicate that the loss of tau homeostasis in hilar astrocytes of the dentate gyrus is sufficient to induce AD-like symptoms, through the impairment of the neuronal network. These results are important for our understanding of disease mechanisms and underline the crucial role of astrocytes in hippocampal function.

Development of Efficient AAV2/DJ-Based Viral Vectors to Selectively Downregulate the Expression of Neuronal or Astrocytic Target Proteins in the Rat Central Nervous System.

Viral vectors have become very popular to overexpress or downregulate proteins of interest in different cell types. They conveniently allow the precise targeting of well-defined tissue areas, which is particularly useful in complex organs like the brain. In theory, each vector should have its own cell specificity that can be obtained by using different strategies (e.g., using a cell-specific promoter). For the moment, there is few vectors that have been developed to alternatively target, using the same capsid, neurons and astrocytes in the central nervous system. There is even fewer examples of adeno-associated viral vectors able to efficiently transduce cells both in vitro and in vivo. The development of viral vectors allowing the cell-specific downregulation of a protein in cultured cells of the central nervous system as well as in vivo within a large brain area would be highly desirable to address several important questions in neurobiology. Here we report that the use of the AAV2/DJ viral vector associated to an hybrid CMV/chicken ß-actin promoter (CBA) or to a modified form of the glial fibrillary acidic protein promoter (G1B3) allows a specific transduction of neurons or astrocytes in more than half of the barrel field within the rat somatosensory cortex. Moreover, the use of the miR30E-shRNA technology led to an efficient downregulation of two proteins of interest related to metabolism both in vitro and in vivo. Our results demonstrate that it is possible to downregulate the expression of different protein isoforms in a cell-specific manner using a common serotype. It is proposed that such an approach could be extended to other cell types and used to target several proteins of interest within the same brain area.

Scalable Production of AAV Vectors in Orbitally Shaken HEK293 Cells.

Adeno-associated virus (AAV) vectors are currently among the most commonly applied for in vivo gene therapy approaches. The evaluation of vectors during clinical development requires the production of considerable amounts of highly pure and potent vectors. Here, we set up a scalable process for AAV production, using orbitally shaken bioreactors and a fully characterized suspension-adapted cell line, HEKExpress. We conducted a proof-of-concept production of AAV2/8 and AAV2/9 vectors using HEKExpress cells. Furthermore, we compared the production of AAV2/9 vectors using this suspension cell line to classical protocols based on adherent HEK293 cells to demonstrate bioequivalence in vitro and in vivo. Following upstream processing, we purified vectors via gradient centrifugation and immunoaffinity chromatography. The in vitro characterization revealed differences due to the purification method, as well as the transfection protocol and the corresponding HEK293 cell line. The purification method and cell line used also affected in vivo transduction efficiency after bilateral injection of AAV2/9 vectors expressing a GFP reporter fused with a nuclear localization signal (AAV2/9-CBA-nlsGFP) into the striatum of adult mice. These results show that AAV vectors deriving from suspension HEKExpress cells are bioequivalent and may exhibit higher potency than vectors produced with adherent HEK293 cells.

Therapeutic efficacy of regulable GDNF expression for Huntington's and Parkinson's disease by a high-induction, background-free "GeneSwitch" vector.

Gene therapy is currently an irreversible approach, without possibilities to fine-tune or halt the expression of a therapeutic gene product. Especially when expressing neurotrophic factors to treat neurodegenerative disorders, options to regulate transgene expression levels might be beneficial. We thus developed an advanced single-genome inducible AAV vector for expression of GDNF, under control of the approved small molecule drug mifepristone. In the rat brain, GDNF expression can be induced over a wide range up to three hundred-fold over endogenous background, and completely returns to baseline within 3-4 weeks. When applied with appropriate serotype and titre, the vector is absolutely free of any non-induced background expression. In the BACHD model of Huntington's disease we demonstrate that the vector can be kept in a continuous ON-state for extended periods of time. In a model of Parkinson's disease we demonstrate that repeated short-term expression of GDNF restores motor capabilities in 6-OHDA-lesioned rats. We also report on sex-dependent pharmacodynamics of mifepristone in the rodent brain. Taken together, we show that wide-range and high-level induction, background-free, fully reversible and therapeutically active GDNF expression can be achieved under tight pharmacological control by this novel AAV - "Gene Switch" vector.

The Self-Inactivating KamiCas9 System for the Editing of CNS Disease Genes.

Neurodegenerative disorders are a major public health problem because of the high frequency of these diseases. Genome editing with the CRISPR/Cas9 system is making it possible to modify the sequence of genes linked to these disorders. We designed the KamiCas9 self-inactivating editing system to achieve transient expression of the Cas9 protein and high editing efficiency. In the first application, the gene responsible for Huntington's disease (HD) was targeted in adult mouse neuronal and glial cells. Mutant huntingtin (HTT) was efficiently inactivated in mouse models of HD, leading to an improvement in key markers of the disease. Sequencing of potential off-targets with the constitutive Cas9 system in differentiated human iPSC revealed a very low incidence with only one site above background level. This off-target frequency was significantly reduced with the KamiCas9 system. These results demonstrate the potential of the self-inactivating CRISPR/Cas9 editing for applications in the context of neurodegenerative diseases.

Acidic pH reduces VEGF-mediated endothelial cell responses by downregulation of VEGFR-2; relevance for anti-angiogenic therapies.

Anti-angiogenic treatments targeting the vascular endothelial growth factor or its receptors have shown clinical benefits. However, impact on long-term survival remains limited. Solid tumors display an acidic microenvironment that profoundly influences their biology. Consequences of acidity on endothelial cells and anti-angiogenic therapies remain poorly characterized and hence are the focus of this study. We found that exposing endothelial cells to acidic extracellular pH resulted in reduced cell proliferation and migration. Also, whereas VEGF increased endothelial cell proliferation and survival at pH 7.4, it had no effect at pH 6.4. Furthermore, in acidic conditions, stimulation of endothelial cells with VEGF did not result in activation of downstream signaling pathways such as AKT. At a molecular level, acidity significantly decreased the expression of VEGFR-2 by endothelial cells. Consequently, anti-angiogenic therapies that target VEGFR-2 such as sunitinib and sorafenib failed to block endothelial cell proliferation in acidic conditions. In vivo, neutralizing tumor acidity with sodium bicarbonate increased the percentage of endothelial cells expressing VEGFR-2 in tumor xenografts. Furthermore, combining sodium bicarbonate with sunitinib provided stronger anti-cancer activity than either treatment alone. Histological analysis showed that sunitinib had a stronger anti-angiogenic effect when combined with sodium bicarbonate. Overall, our results show that endothelial cells prosper independently of VEGF in acidic conditions partly as a consequence of decreased VEGFR-2 expression. They further suggest that strategies aiming to raise intratumoral pH can improve the efficacy of anti-VEGF treatments.

Targeting carbonic anhydrase IX improves the anti-cancer efficacy of mTOR inhibitors.

The inhibition of the mechanistic target of rapamycin complex 1 (mTORC1) by chemical inhibitors, such as rapamycin, has demonstrated anti-cancer activity in preclinical and clinical trials. Their efficacy is, however, limited and tumors eventually relapse through resistance formation. In this study, using two different cancer mouse models, we identify tumor hypoxia as a novel mechanism of resistance of cancer cells against mTORC1 inhibitors. Indeed, we show that the activity of mTORC1 is mainly restricted to the non-hypoxic tumor compartment, as evidenced by a mutually exclusive staining pattern of the mTORC1 activity marker pS6 and the hypoxia marker pimonidazole. Consequently, whereas rapamycin reduces cancer cell proliferation in non-hypoxic regions, it has no effect in hypoxic areas, suggesting that cancer cells proliferate independently of mTORC1 under hypoxia. Targeting the hypoxic tumor compartment by knockdown of carbonic anhydrase IX (CAIX) using short hairpin RNA or by chemical inhibition of CAIX with acetazolamide potentiates the anti-cancer activity of rapamycin. Taken together, these data emphasize that hypoxia impairs the anti-cancer efficacy of rapalogs. Therapeutic strategies targeting the hypoxic tumor compartment, such as the inhibition of CAIX, potentiate the efficacy of rapamycin and warrant further clinical evaluation.

Manufacturing and characterization of a recombinant adeno-associated virus type 8 reference standard material.

Abstract Gene therapy approaches using recombinant adeno-associated virus serotype 2 (rAAV2) and serotype 8 (rAAV8) have achieved significant clinical benefits. The generation of rAAV Reference Standard Materials (RSM) is key to providing points of reference for particle titer, vector genome titer, and infectious titer for gene transfer vectors. Following the example of the rAAV2RSM, here we have generated and characterized a novel RSM based on rAAV serotype 8. The rAAV8RSM was produced using transient transfection, and the purification was based on density gradient ultracentrifugation. The rAAV8RSM was distributed for characterization along with standard assay protocols to 16 laboratories worldwide. Mean titers and 95% confidence intervals were determined for capsid particles (mean, 5.50×10(11) pt/ml; CI, 4.26×10(11) to 6.75×10(11) pt/ml), vector genomes (mean, 5.75×10(11) vg/ml; CI, 3.05×10(11) to 1.09×10(12) vg/ml), and infectious units (mean, 1.26×10(9) IU/ml; CI, 6.46×10(8) to 2.51×10(9) IU/ml). Notably, there was a significant degree of variation between institutions for each assay despite the relatively tight correlation of assay results within an institution. This outcome emphasizes the need to use RSMs to calibrate the titers of rAAV vectors in preclinical and clinical studies at a time when the field is maturing rapidly. The rAAV8RSM has been deposited at the American Type Culture Collection (VR-1816) and is available to the scientific community.

An adeno-associated virus-based intracellular sensor of pathological nuclear factor-κB activation for disease-inducible gene transfer.

Stimulation of resident cells by NF-κB activating cytokines is a central element of inflammatory and degenerative disorders of the central nervous system (CNS). This disease-mediated NF-κB activation could be used to drive transgene expression selectively in affected cells, using adeno-associated virus (AAV)-mediated gene transfer. We have constructed a series of AAV vectors expressing GFP under the control of different promoters including NF-κB -responsive elements. As an initial screen, the vectors were tested in vitro in HEK-293T cells treated with TNF-α. The best profile of GFP induction was obtained with a promoter containing two blocks of four NF-κB -responsive sequences from the human JCV neurotropic polyoma virus promoter, fused to a new tight minimal CMV promoter, optimally distant from each other. A therapeutical gene, glial cell line-derived neurotrophic factor (GDNF) cDNA under the control of serotype 1-encapsidated NF-κB -responsive AAV vector (AAV-NF) was protective in senescent cultures of mouse cortical neurons. AAV-NF was then evaluated in vivo in the kainic acid (KA)-induced status epilepticus rat model for temporal lobe epilepsy, a major neurological disorder with a central pathophysiological role for NF-κB activation. We demonstrate that AAV-NF, injected in the hippocampus, responded to disease induction by mediating GFP expression, preferentially in CA1 and CA3 neurons and astrocytes, specifically in regions where inflammatory markers were also induced. Altogether, these data demonstrate the feasibility to use disease-activated transcription factor-responsive elements in order to drive transgene expression specifically in affected cells in inflammatory CNS disorders using AAV-mediated gene transfer.

Rapid transgene expression in multiple precursor cell types of adult rat subventricular zone mediated by adeno-associated type 1 vectors.

The adult rat brain subventricular zone (SVZ) contains proliferative precursors that migrate to the olfactory bulb (OB) and differentiate into mature neurons. Recruitment of precursors constitutes a potential avenue for brain repair. We have investigated the kinetics and cellular specificity of transgene expression mediated by AAV2/1 vectors (i.e., adeno-associated virus type 2 pseudotyped with AAV1 capsid) in the SVZ. Self-complementary (sc) and single-stranded (ss) AAV2/1 vectors mediated efficient GFP expression, respectively, at 17 and 24 hr postinjection. Transgene expression was efficient in all the rapidly proliferating cells types, that is, Mash1(+) precursors (30% of the GFP(+) cells), Dlx2(+) neuronal progenitors (55%), Olig2(+) oligodendrocyte progenitors (35%), and doublecortin-positive (Dcx(+)) migrating cells (40%), but not in the slowly proliferating glial fibrillary acidic protein-positive (GFAP(+)) neural stem cell pool (5%). Because cell cycle arrest by wild-type and recombinant AAV has been described in primary cultures, we examined SVZ proliferative activity after vector injection. Indeed, cell proliferation was reduced immediately after vector injection but was normal after 1 month. In contrast, migration and differentiation of GFP(+) precursors were unaltered. Indeed, the proportion of Dcx(+) cells was similar in the injected and contralateral hemispheres. Furthermore, 1 month after vector injection into the SVZ, GFP(+) cells, found, as expected, in the OB granular cell layer, were mature GABAergic neurons. In conclusion, the rapid and efficient transgene expression in SVZ neural precursors mediated by scAAV2/1 vectors underlines their potential usefulness for brain repair via recruitment of immature cells. The observed transient precursor proliferation inhibition, not affecting their migration and differentiation, will likely not compromise this strategy.

Granulocyte-macrophage colony-stimulating factor elicits bone marrow-derived cells that promote efficient colonic mucosal healing.

BACKGROUND: Granulocyte-macrophage colony-stimulating factor (GM-CSF) therapy is effective in treating some Crohn's disease (CD) patients and protects mice from colitis induced by dextran sulfate sodium (DSS) administration. However, its mechanisms of action remain elusive. We hypothesized that GM-CSF affects intestinal mucosal repair. METHODS: DSS colitic mice were treated with daily pegylated GM-CSF or saline and clinical, histological, and inflammatory parameters were kinetically evaluated. Further, the role of bone marrow-derived cells in the impact of GM-CSF therapy on DSS colitis was addressed using cell transfers. RESULTS: GM-CSF therapy reduced clinical signs of colitis and the release of inflammatory mediators. GM-CSF therapy improved mucosal repair, with faster ulcer reepithelialization, accelerated hyperproliferative response of epithelial cells in ulcer-adjacent crypts, and lower colonoscopic ulceration scores in GM-CSF-administered mice relative to untreated mice. We observed that GM-CSF-induced promotion of mucosal repair is timely associated with a reduction in neutrophil numbers and increased accumulation of CD11b(+) monocytic cells in colon tissues. Importantly, transfer of splenic GM-CSF-induced CD11b(+) myeloid cells into DSS-exposed mice improved colitis, and lethally irradiated GM-CSF receptor-deficient mice reconstituted with wildtype bone marrow cells were protected from DSS-induced colitis upon GM-CSF therapy. Lastly, GM-CSF-induced CD11b(+) myeloid cells were shown to promote in vitro wound repair. CONCLUSIONS: Our study shows that GM-CSF-dependent stimulation of bone marrow-derived cells during DSS-induced colitis accelerates colonic tissue repair. These data provide a putative mechanism for the observed beneficial effects of GM-CSF therapy in Crohn's disease.