Systemic Immune Bias Delineates Malignant Astrocytoma Survival Cohorts.

Patients with grade III anaplastic astrocytomas (AA) separate into survival cohorts based on the presence or absence of mutations in isocitrate dehydrogenase (IDH). Progression to glioblastoma (GBM), morphologically distinguishable by elevated microvascular proliferation, necrosis, and cell division in tumor tissues, is considerably more rapid in IDH wild-type tumors such that their diagnosis as AA is relatively rare. More often initially presenting as GBM, these contain higher numbers of tumor-associated macrophages (TAMs) than most AA, and GBM patients also have higher levels of circulating M2 monocytes. TAM and M2 monocytes share functional properties inhibitory for antitumor immunity. Yet, although there is a wealth of data implicating TAM in tumor-immune evasion, there has been limited analysis of the impact of the circulating M2 monocytes. In the current study, immune parameters in sera, circulating cells, and tumor tissues from patients with primary gliomas morphologically diagnosed as AA were assessed. Profound differences in serum cytokines, glioma extracellular vesicle cross-reactive Abs, and gene expression by circulating cells identified two distinct patient cohorts. Evidence of type 2-immune bias was most often seen in patients with IDH wild-type AA, whereas a type 1 bias was common in patients with tumors expressing the IDH1R132H mutation. Nevertheless, a patient's immune profile was better correlated with the extent of tumor vascular enhancement on magnetic resonance imaging than IDH mutational status. Regardless of IDH genotype, AA progression appears to be associated with a switch in systemic immune bias from type 1 to type 2 and the loss of tumor vasculature integrity.

Dual Role of Toll-Like Receptor 7 in the Pathogenesis of Rabies Virus in a Mouse Model.

Rabies, caused by rabies virus (RABV), is a fatal encephalitis in humans and other mammals, which continues to present a public health threat in most parts of the world. Our previous study demonstrated that Toll-like receptor 7 (TLR7) is essential in the induction of anti-RABV antibodies via the facilitation of germinal center formation. In the present study, we investigated the role of TLR7 in the pathogenicity of RABV in a mouse model. Using isolated plasmacytoid dendritic cells (pDCs), we demonstrated that TLR7 is an innate recognition receptor for RABV. When RABV invaded from the periphery, TLR7 detected viral single-stranded RNA and triggered immune responses that limited the virus's entry into the central nervous system (CNS). When RABV had invaded the CNS, its detection by TLR7 led to the production of cytokines and chemokines and an increase the permeability of the blood-brain barrier. Consequently, peripheral immune cells, including pDCs, macrophages, neutrophils, and B cells infiltrated the CNS. While this immune response, triggered by TLR7, helped to clear viruses, it also increased neuroinflammation and caused immunopathology in the mouse brain. Our results demonstrate that TLR7 is an innate recognition receptor for RABV, which restricts RABV invasion into the CNS in the early stage of viral infection but also contributes to immunopathology by inducing neuroinflammation.IMPORTANCE Developing targeted treatment for RABV requires understanding the innate immune response to the virus because early virus clearance is essential for preventing the fatality when the infection has progressed to the CNS. Previous studies have revealed that TLR7 is involved in the immune response to RABV. Here, we establish that TLR7 recognizes RABV and facilitates the production of some interferon-stimulated genes. We also demonstrated that when RABV invades into the CNS, TLR7 enhances the production of inflammatory cytokines which contribute to immunopathology in the mouse brain. Taken together, our findings suggest that treatments for RABV must consider the balance between the beneficial and harmful effects of TLR7-triggered immune responses.

Type 1 Immune Mechanisms Driven by the Response to Infection with Attenuated Rabies Virus Result in Changes in the Immune Bias of the Tumor Microenvironment and Necrosis of Mouse GL261 Brain Tumors.

Immunotherapeutic strategies for malignant glioma have to overcome the immunomodulatory activities of M2 monocytes that appear in the circulation and as tumor-associated macrophages (TAMs). M2 cell products contribute to the growth-promoting attributes of the tumor microenvironment (TME) and bias immunity toward type 2, away from the type 1 mechanisms with antitumor properties. To drive type 1 immunity in CNS tissues, we infected GL261 tumor-bearing mice with attenuated rabies virus (RABV). These neurotropic viruses spread to CNS tissues trans-axonally, where they induce a strong type 1 immune response that involves Th1, CD8, and B cell entry across the blood-brain barrier and virus clearance in the absence of overt sequelae. Intranasal infection with attenuated RABV prolonged the survival of mice bearing established GL261 brain tumors. Despite the failure of virus spread to the tumor, infection resulted in significantly enhanced tumor necrosis, extensive CD4 T cell accumulation, and high levels of the proinflammatory factors IFN-γ, TNF-α, and inducible NO synthase in the TME merely 4 d postinfection, before significant virus spread or the appearance of RABV-specific immune mechanisms in CNS tissues. Although the majority of infiltrating CD4 cells appeared functionally inactive, the proinflammatory changes in the TME later resulted in the loss of accumulating M2 and increased M1 TAMs. Mice deficient in the Th1 transcription factor T-bet did not gain any survival advantage from RABV infection, exhibiting only limited tumor necrosis and no change in TME cytokines or TAM phenotype and highlighting the importance of type 1 mechanisms in this process.

T-bet Is Required for the Rapid Clearance of Attenuated Rabies Virus from Central Nervous System Tissue.

Much of our understanding of CNS immunity has been gained from models involving pathological inflammation. Attenuated rabies viruses (RABV) are unique tools to study CNS immunity in the absence of conventional inflammatory mechanisms, as they spread from the site of inoculation to the CNS transaxonally, thereby bypassing the blood-brain barrier (BBB), and are cleared without neutrophil or monocyte infiltration. To better understand the role of CD4 T cell subsets in the clearance of the virus from CNS tissues, we examined the development of antiviral immunity in wild-type (WT) and T-bet knockout mice (T-bet(-/-)), which lack Th1 cells. Early control of RABV replication in the CNS tissues of WT mice is associated with the production of IFN-γ, with antiviral effects likely mediated through the enhanced expression of type I IFNs. Of interest, IFN-α and -γ are overexpressed in the infected T-bet(-/-) by comparison with WT CNS tissues, and the initial control of RABV infection is similar. Ultimately, attenuated RABV are cleared from the CNS tissues of WT mice by Ab locally produced by the activities of infiltrating T and B cells. Although T and B cell infiltration into the CNS of infected T-bet(-/-) mice is comparable, their activities are not, the consequence being delayed, low-level Ab production and prolonged RABV replication. More importantly, neither T-bet(-/-) mice immunized with an attenuated virus, nor WT mice with Th2 RABV-specific immunity induced by immunization with inactivated virus, are protected in the long term against challenge with a pathogenic RABV.

Expression of interferon gamma by a recombinant rabies virus strongly attenuates the pathogenicity of the virus via induction of type I interferon.

UNLABELLED: Previous animal model experiments have shown a correlation between interferon gamma (IFN-γ) expression and both survival from infection with attenuated rabies virus (RABV) and reduction of neurological sequelae. Therefore, we hypothesized that rapid production of murine IFN-γ by the rabies virus itself would induce a more robust antiviral response than would occur naturally in mice. To test this hypothesis, we used reverse engineering to clone the mouse IFN-γ gene into a pathogenic rabies virus backbone, SPBN, to produce the recombinant rabies virus designated SPBNγ. Morbidity and mortality were monitored in mice infected intranasally with SPBNγ or SPBN(-) control virus to determine the degree of attenuation caused by the expression of IFN-γ. Incorporation of IFN-γ into the rabies virus genome highly attenuated the virus. SPBNγ has a 50% lethal dose (LD50) more than 100-fold greater than SPBN(-). In vitro and in vivo mouse experiments show that SPBNγ infection enhances the production of type I interferons. Furthermore, knockout mice lacking the ability to signal through the type I interferon receptor (IFNAR(-/-)) cannot control the SPBNγ infection and rapidly die. These data suggest that IFN-γ production has antiviral effects in rabies, largely due to the induction of type I interferons. IMPORTANCE: Survival from rabies is dependent upon the early control of virus replication and spread. Once the virus reaches the central nervous system (CNS), this becomes highly problematic. Studies of CNS immunity to RABV have shown that control of replication begins at the onset of T cell entry and IFN-γ production in the CNS prior to the appearance of virus-neutralizing antibodies. Moreover, antibody-deficient mice are able to control but not clear attenuated RABV from the CNS. We find here that IFN-γ triggers the early production of type I interferons with the expected antiviral effects. We also show that engineering a lethal rabies virus to express IFN-γ directly in the infected tissue reduces rabies virus replication and spread, limiting its pathogenicity in normal and immunocompromised mice. Therefore, vector delivery of IFN-γ to the brain may have the potential to treat individuals who would otherwise succumb to infection with rabies virus.

Glioblastoma exosomes and IGF-1R/AS-ODN are immunogenic stimuli in a translational research immunotherapy paradigm.

Glioblastomas are primary intracranial tumors for which there is no cure. Patients receiving standard of care, chemotherapy and irradiation, survive approximately 15 months prompting studies of alternative therapies including vaccination. In a pilot study, a vaccine consisting of Lucite diffusion chambers containing irradiated autologous tumor cells pre-treated with an antisense oligodeoxynucleotide (AS-ODN) directed against the insulin-like growth factor type 1 receptor was found to elicit positive clinical responses in 8/12 patients when implanted in the rectus sheath for 24 h. Our preliminary observations supported an immune response, and we have since reopened a second Phase 1 trial to assess this possibility among other exploratory objectives. The current study makes use of a murine glioma model and samples from glioblastoma patients in this second Phase 1 trial to investigate this novel therapeutic intervention more thoroughly. Implantation of the chamber-based vaccine protected mice from tumor challenge, and we posit this occurred through the release of immunostimulatory AS-ODN and antigen-bearing exosomes. Exosomes secreted by glioblastoma cultures are immunogenic, eliciting and binding antibodies present in the sera of immunized mice. Similarly, exosomes released by human glioblastoma cells bear antigens recognized by the sera of 6/12 patients with recurrent glioblastomas. These results suggest that the release of AS-ODN together with selective release of exosomes from glioblastoma cells implanted in chambers may drive the therapeutic effect seen in the pilot vaccine trial.

Enhancement of glioma-specific immunity in mice by "NOBEL", an insulin-like growth factor 1 receptor antisense oligodeoxynucleotide.

Autologous glioblastoma multiforme tumor cells treated with an antisense oligodeoxynucleotide (AS-ODN) targeting insulin-like growth factor receptor-1 (IGF-1R) are the basis of a vaccine with therapeutic effects on tumor recurrence in a pilot clinical trial. As a preface to continued clinical investigation of this vaccination strategy, we have studied the contribution of an optimized IGF-1R AS-ODN, designated "NOBEL", to the induction of immunity to mouse GL261 glioma cells. The impact of NOBEL on mechanisms contributing to the development of GL261 immunity was first examined in the periphery. GL261 cells are naturally immunogenic when implanted into the flanks of congenic C57BL/6 mice, immunizing rather than forming tumors in around 50 % of these animals but causing tumors in the majority of mice lacking T and B lymphocytes. Overnight treatment with NOBEL in vitro reduces IGF-1R expression by GL261 cells but has minimal effect on cell viability and does not reduce the capacity of the cells to form tumors upon implantation. In contrast, tumors are extremely rare when GL261 cells are mixed with NOBEL at inoculation into the flanks of C57BL/6, and the recipient mice become immune to subcutaneous and intracranial challenge with untreated GL261. Adaptive immune mechanisms contribute to this effect, as immunocompromised mice fail to either fully control tumor formation or develop immunity following flank administration of the GL261/NOBEL mix. NOBEL's structure has known immunostimulatory motifs that likely contribute to the immunogenicity of the mix, but its specificity for IGF-1R mRNA is also important as a similarly structured sense molecule is not effective.

Cannabinoid receptor type-2 stimulation, blockade, and deletion alter the vascular inflammatory responses to traumatic brain injury.

BACKGROUND: Immunomodulatory therapies have been identified as interventions for secondary injury after traumatic brain injury (TBI). The cannabinoid receptor type-2 (CB2R) is proposed to play an important, endogenous role in regulating inflammation. The effects of CB2R stimulation, blockade, and deletion on the neurovascular inflammatory responses to TBI were assessed. METHODS: Wild-type C57BL/6 or CB2R knockout mice were randomly assigned to controlled cortical impact (CCI) injury or to craniotomy control groups. The effects of treatment with synthetic, selective CB2R agonists (0-1966 and JWH-133), a selective CB2R antagonist, or vehicle solution administered to CCI groups were assessed at 1-day after injury. Changes in TNF-α, intracellular adhesion molecule (ICAM-1), inducible nitric oxide synthase (iNOS), macrophage/microglial ionized calcium-binding adaptor molecule, and blood-brain-barrier (BBB) permeability were assessed using ELISA, quantitative RT-PCR, immunohistochemistry, and fluorometric analysis of sodium fluorescein uptake. CB2R knockouts and wild-type mice with CCI injury were treated with a CB2R agonist or vehicle treatment. RESULTS: TNF-α mRNA increased at 6 hours and 1 to 3 days after CCI; a CB2R antagonist and genetic knockout of the CB2R exacerbated TNF-α mRNA expression. Treatment with a CB2R agonist attenuated TNF-α protein levels indicating post-transcriptional mechanisms. Intracellular adhesion molecule (ICAM-1) mRNA was increased at 6 hours, and at 1 to 2 days after CCI, reduced in mice treated with a CB2R agonist, and increased in CB2R knockout mice with CCI. Sodium fluorescein uptake was increased in CB2R knockouts after CCI, with and without a CB2R agonist. iNOS mRNA expression peaked early (6 hours) and remained increased from 1 to 3 days after injury. Treatment with a CB2R agonist attenuated increases in iNOS mRNA expression, while genetic deletion of the CB2R resulted in substantial increases in iNOS expression. Double label immunohistochemistry confirmed that iNOS was expressed by macrophage/microglia in the injured cortex. CONCLUSION: Findings demonstrate that the endogenous cannabinoid system and CB2R play an important role in regulating inflammation and neurovascular responses in the traumatically injured brain. CB2R stimulation with two agonists (0-1966 and JWH-133) dampened post-traumatic inflammation, while blockade or deletion of the CB2R worsened inflammation. Findings support previous evidence that modulating the CB2R alters infiltrating macrophages and activated resident microglia. Further investigation into the role of the CB2R on specific immune cell populations in the injured brain is warranted.

Intramuscular inoculation of mice with the live-attenuated recombinant rabies virus TriGAS results in a transient infection of the draining lymph nodes and a robust, long-lasting protective immune response against rabies.

A single intramuscular application of the live but not UV-inactivated recombinant rabies virus (RABV) variant TriGAS in mice induces the robust and sustained production of RABV-neutralizing antibodies that correlate with long-term protection against challenge with an otherwise lethal dose of the wild-type RABV. To obtain insight into the mechanism by which live TriGAS induces long-lasting protective immunity, quantitative PCR (qPCR) analysis of muscle tissue, draining lymph nodes, spleen, spinal cord, and brain at different times after TriGAS inoculation revealed the presence of significant copy numbers of RABV-specific RNA in muscle, lymph node, and to a lesser extent, spleen for several days postinfection. Notably, no significant amounts of RABV RNA were detected in brain or spinal cord at any time after TriGAS inoculation. Differential qPCR analysis revealed that the RABV-specific RNA detected in muscle is predominantly genomic RNA, whereas RABV RNA detected in draining lymph nodes is predominantly mRNA. Comparison of genomic RNA and mRNA obtained from isolated lymph node cells showed the highest mRNA-to-genomic-RNA ratios in B cells and dendritic cells (DCs), suggesting that these cells represent the major cell population that is infected in the lymph node. Since RABV RNA declined to undetectable levels by 14 days postinoculation of TriGAS, we speculate that a transient infection of DCs with TriGAS may be highly immunostimulatory through mechanisms that enhance antigen presentation. Our results support the superior efficacy and safety of TriGAS and advocate for its utility as a vaccine.

BP1003 Decreases STAT3 Expression and Its Pro-Tumorigenic Functions in Solid Tumors and the Tumor Microenvironment.

Overexpression and aberrant activation of signal transducer and activator of transcription 3 (STAT3) contribute to tumorigenesis, drug resistance, and tumor-immune evasion, making it a potential cancer therapeutic target. BP1003 is a neutral liposome incorporated with a nuclease-resistant P-ethoxy antisense oligodeoxynucleotide (ASO) targeting the STAT3 mRNA. Its unique design enhances BP1003 stability, cellular uptake, and target affinity. BP1003 efficiently reduces STAT3 expression and enhances the sensitivity of breast cancer cells (HER2+, triple negative) and ovarian cancer cells (late stage, invasive ovarian cancer) to paclitaxel and 5-fluorouracil (5-FU) in both 2D and 3D cell cultures. Similarly, ex vivo and in vivo patient-derived models of pancreatic ductal adenocarcinoma (PDAC) show reduced tissue viability and tumor volume with BP1003 and gemcitabine combination treatments. In addition to directly affecting tumor cells, BP1003 can modulate the tumor microenvironment. Unlike M1 differentiation, monocyte differentiation into anti-inflammatory M2 macrophages is suppressed by BP1003, indicating its potential contribution to immunotherapy. The broad anti-tumor effect of BP1003 in numerous preclinical solid tumor models, such as breast, ovarian, and pancreatic cancer models shown in this work, makes it a promising cancer therapeutic.

Neuroendocrine gene subsets are uniquely dysregulated in prostate adenocarcinoma.

Prostate cancer has heterogeneous growth patterns, and its prognosis is the poorest when it progresses to a neuroendocrine phenotype. Using bioinformatic analysis, we evaluated RNA expression of neuroendocrine genes in a panel of five different cancer types: prostate adenocarcinoma, breast cancer, kidney chromophobe, kidney renal clear cell carcinoma and kidney renal papillary cell carcinoma. Our results show that specific neuroendocrine genes are significantly dysregulated in these tumors, suggesting that they play an active role in cancer progression. Among others, synaptophysin (SYP), a conventional neuroendocrine marker, is upregulated in prostate adenocarcinoma (PRAD) and breast cancer (BRCA). Our analysis shows that SYP is enriched in small extracellular vesicles (sEVs) derived from plasma of PRAD patients, but it is absent in sEVs derived from plasma of healthy donors. Similarly, classical sEV markers are enriched in sEVs derived from plasma of prostate cancer patients, but weakly detectable in sEVs derived from plasma of healthy donors. Overall, our results pave the way to explore new strategies to diagnose these diseases based on the neuroendocrine gene expression in patient tumors or plasma sEVs.

Postexposure treatment with the live-attenuated rabies virus (RV) vaccine TriGAS triggers the clearance of wild-type RV from the Central Nervous System (CNS) through the rapid induction of genes relevant to adaptive immunity in CNS tissues.

Postexposure treatment (PET) of wild-type rabies virus (RV)-infected mice with a live-attenuated triple-glycoprotein RV variant (TriGAS) promotes survival but does not prevent the pathogenic RV from invading and replicating in the brain. Successful PET is associated with the induction of a robust virus-neutralizing antibody response and clearance of the wild-type RV from brain tissues. Comparison of the transcriptomes of normal mouse brain with those of wild-type-RV-infected mice that had received either mock or TriGAS PET treatment revealed that many of the host genes activated in the mock-treated mice represent type I interferon (IFN) response genes. This indicates that RV infection induces an early type I IFN response that is unable to control the infection. In contrast, most of the activated genes in the brain of the RV-infected, TriGAS-treated mouse play a role in adaptive immunity, including the regulation of T cell activation, T cell differentiation, and the regulation of lymphocyte and mononuclear cell proliferation. These findings were confirmed by quantitative PCR (qPCR) array studies, which showed that 3 genes in particular, encoding chemokine ligand 3 (Ccl3), natural killer cell activator 2 (interleukin 12B [IL-12B]), and granzyme A (GzmA), were activated earlier and to a greater extent in the brains of RV-infected mice treated with TriGAS than in the brains of mock-treated mice. The activation of these genes, known to play key roles in the regulation of lymphocyte and mononuclear cell proliferation, is likely an important part of the mechanism by which TriGAS mediates its PET activity.

Autoantibodies Drive Heart Damage Caused by Concomitant Radiation and PD-1 Blockade.

Concurrent PD-1 blockade and thoracic radiotherapy is being investigated in clinical trials for locally advanced, non-small cell lung cancer and small cell lung cancer, despite a potential overlapping risk of cardiotoxicity. Our prior studies demonstrate that cardiotoxicity from concurrent cardiac irradiation and anti-PD-1 administration in a mouse model is CD8+ T-cell dependent. The objective of this study was to determine whether humoral immunity contributed to the observed cardiac tissue damage, as measured by creatine kinase MB and cardiac troponin 1 release and decline in cardiac function. In the current study, we demonstrate the presence of cardiac autoantibodies, which were essential for the occurrence of cardiotoxicity from the combined therapy. Mice subjected to cardiac irradiation, while being treated with anti-PD-1, developed high levels of antibodies that reacted with cardiac tissues in vivo and cardiac antigens in vitro. Moreover, mice deficient in B cells were protected against cardiotoxicity, whereas the transfer of autoantibody-containing sera from mice that had received combined treatment reproduced the same pathologic phenotype in mice exposed to cardiac irradiation but was not observed in normal recipients. The cardiotoxic effect of the sera, which associated with CD8+ T-cell accumulation in cardiac tissue, was limited by IgG depletion. In conclusion, concurrent cardiac irradiation and PD-1 blockade leads to production of cardiac autoantibodies, likely due to antigen exposure within the irradiated cardiac tissues, which play a key role in the resulting cardiotoxicity.

Targeting the αVß3/NgR2 pathway in neuroendocrine prostate cancer.

Highly aggressive, metastatic, neuroendocrine prostate cancer, which typically develops from prostate cancer cells acquiring resistance to androgen deprivation therapy, is associated with limited treatment options and hence poor prognosis. We have previously demonstrated that the αVß3 integrin is over-expressed in neuroendocrine prostate cancer. We now show that LM609, a monoclonal antibody that specifically targets the human αVß3 integrin, hinders the growth of neuroendocrine prostate cancer patient-derived xenografts in vivo. Our group has recently identified a novel αVß3 integrin binding partner, NgR2, responsible for regulating the expression of neuroendocrine markers and for inducing neuroendocrine differentiation in prostate cancer cells. Through in vitro functional assays, we here demonstrate that NgR2 is crucial in promoting cell adhesion to αVß3 ligands. Moreover, we describe for the first time co-fractionation of αVß3 integrin and NgR2 in small extracellular vesicles derived from metastatic prostate cancer patients' plasma. These prostate cancer patient-derived small extracellular vesicles have a functional impact on human monocytes, increasing their adhesion to fibronectin. The monocytes incubated with small extracellular vesicles do not show an associated change in conventional polarization marker expression and appear to be in an early stage that may be defined as "adhesion competent". Overall, these findings allow us to better understand integrin-directed signaling and cell-cell communication during cancer progression. Furthermore, our results pave the way for new diagnostic and therapeutic perspectives for patients affected by neuroendocrine prostate cancer.

Temporal changes in innate immune signals in a rat model of alcohol withdrawal in emotional and cardiorespiratory homeostatic nuclei.

BACKGROUND: Chronic alcohol use changes the brain's inflammatory state. However, there is little work examining the progression of the cytokine response during alcohol withdrawal, a period of profound autonomic and emotional upset. This study examines the inflammatory response in the central nucleus of the amygdala (CeA) and dorsal vagal complex (DVC), brain regions neuroanatomically associated with affective and cardiorespiratory regulation in an in vivo rat model of withdrawal following a single chronic exposure. METHODS: For qRT-PCR studies, we measured the expression of TNF-α, NOS-2, Ccl2 (MCP-1), MHC II invariant chain CD74, and the TNF receptor Tnfrsf1a in CeA and DVC samples from adult male rats exposed to a liquid alcohol diet for thirty-five days and in similarly treated animals at four hours and forty-eight hours following alcohol withdrawal. ANOVA was used to identify statistically significant treatment effects. Immunohistochemistry (IHC) and confocal microscopy were performed in a second set of animals during chronic alcohol exposure and subsequent 48-hour withdrawal. RESULTS: Following a chronic alcohol exposure, withdrawal resulted in a statistically significant increase in the expression of mRNAs specific for innate immune markers Ccl2, TNF-α, NOS-2, Tnfrsf1a, and CD74. This response was present in both the CeA and DVC and most prominent at 48 hours. Confocal IHC of samples taken 48 hours into withdrawal demonstrate the presence of TNF-α staining surrounding cells expressing the neural marker NeuN and endothelial cells colabeled with ICAM-1 (CD54) and RECA-1, markers associated with an inflammatory response. Again, findings were consistent in both brain regions. CONCLUSIONS: This study demonstrates the rapid induction of Ccl2, TNF-α, NOS-2, Tnfrsf1a and CD74 expression during alcohol withdrawal in both the CeA and DVC. IHC dual labeling showed an increase in TNF-α surrounding neurons and ICAM-1 on vascular endothelial cells 48 hours into withdrawal, confirming the inflammatory response at the protein level. These findings suggest that an abrupt cessation of alcohol intake leads to an acute central nervous system (CNS) inflammatory response in these regions that regulate autonomic and emotional state.

Effective preexposure and postexposure prophylaxis of rabies with a highly attenuated recombinant rabies virus.

Rabies remains an important public health problem with more than 95% of all human rabies cases caused by exposure to rabid dogs in areas where effective, inexpensive vaccines are unavailable. Because of their ability to induce strong innate and adaptive immune responses capable of clearing the infection from the CNS after a single immunization, live-attenuated rabies virus (RV) vaccines could be particularly useful not only for the global eradication of canine rabies but also for late-stage rabies postexposure prophylaxis of humans. To overcome concerns regarding the safety of live-attenuated RV vaccines, we developed the highly attenuated triple RV G variant, SPBAANGAS-GAS-GAS. In contrast to most attenuated recombinant RVs generated thus far, SPBAANGAS-GAS-GAS is completely nonpathogenic after intracranial infection of mice that are either developmentally immunocompromised (e.g., 5-day-old mice) or have inherited deficits in immune function (e.g., antibody production or type I IFN signaling), as well as normal adult animals. In addition, SPBAANGAS-GAS-GAS induces immune mechanisms capable of containing a CNS infection with pathogenic RV, thereby preventing lethal rabies encephalopathy. The lack of pathogenicity together with excellent immunogenicity and the capacity to deliver immune effectors to CNS tissues makes SPBAANGAS-GAS-GAS a promising vaccine candidate for both the preexposure and postexposure prophylaxis of rabies.

Blood-brain barrier changes and cell invasion differ between therapeutic immune clearance of neurotrophic virus and CNS autoimmunity.

CNS tissues are protected from circulating cells and factors by the blood-brain barrier (BBB), a specialization of the neurovasculature. Outcomes of the loss of BBB integrity and cell infiltration into CNS tissues can differ vastly. For example, elevated BBB permeability is closely associated with the development of neurological disease in experimental allergic encephalomyelitis (EAE) but not during clearance of the attenuated rabies virus CVS-F3 from the CNS tissues. To probe whether differences in the nature of BBB permeability changes may contribute to the pathogenesis of acute neuroinflammatory disease, we compared the characteristics of BBB permeability changes in mice with EAE and in mice clearing CVS-F3. BBB permeability changes are largely restricted to the cerebellum and spinal cord in both models but differ in the extent of leakage of markers of different size and in the nature of cell accumulation in the CNS tissues. The accumulation in the CNS tissues of CD4 T cells expressing mRNAs specific for IFN-gamma and IL-17 is common to both, but iNOS-positive cells invade into the CNS parenchyma only in EAE. Mice that have been immunized with myelin basic protein (MBP) and infected exhibit the features of EAE. Treatment with the peroxynitrite-dependent radical scavenger urate inhibits the invasion of iNOS-positive cells into the CNS tissues and the development of clinical signs of EAE without preventing the loss of BBB integrity in immunized/infected animals. These findings indicate that BBB permeability changes can occur in the absence of neuropathology provided that cell invasion is restricted.

Glioblastoma Contains Topologically Distinct Proliferative and Metabolically Defined Subpopulations of Nestin- and Glut1-Expressing Cells.

The difficulty in treatment of glioblastoma is a consequence of its natural infiltrative growth and the existence of a population of therapy-resistant glioma cells that contribute to growth and recurrence. To identify cells more likely to have these properties, we examined the expression in tumor specimens of several protein markers important for glioma progression including the intermediate filament protein, Nestin (NES), a glucose transporter (Glut1/SLC2A1), the glial lineage marker, glial fibrillary acidic protein, and the proliferative indicator, Ki-67. We also examined the expression of von Willebrand factor, a marker for endothelial cells as well as the macrophage/myeloid markers CD163 and CD15. Using a multicolor immunofluorescence and hematoxylin and eosin staining approach with archival formalin-fixed, paraffin embedded tissue from primary, recurrent, and autopsy IDH1 wildtype specimens combined with high-resolution tissue image analysis, we have identified highly proliferative NES(+)/Glut1(-) cells that are preferentially perivascular. In contrast, Glut1(+)/NES(-) cells are distant from blood vessels, show low proliferation, and are preferentially located at the borders of pseudopalisading necrosis. We hypothesize that Glut1(+)/NES(-) cells would be naturally resistant to conventional chemotherapy and radiation due to their low proliferative capacity and may act as a reservoir for tumor recurrence.

Phase Ib Clinical Trial of IGV-001 for Patients with Newly Diagnosed Glioblastoma.

PURPOSE: Despite standard of care (SOC) established by Stupp, glioblastoma remains a uniformly poor prognosis. We evaluated IGV-001, which combines autologous glioblastoma tumor cells and an antisense oligonucleotide against IGF type 1 receptor (IMV-001), in newly diagnosed glioblastoma. PATIENTS AND METHODS: This open-label protocol was approved by the Institutional Review Board at Thomas Jefferson University. Tumor cells collected during resection were treated ex vivo with IMV-001, encapsulated in biodiffusion chambers with additional IMV-001, irradiated, then implanted in abdominal acceptor sites. Patients were randomized to four exposure levels, and SOC was initiated 4-6 weeks later. On the basis of clinical improvements, randomization was halted after patient 23, and subsequent patients received only the highest exposure. Safety and tumor progression were primary and secondary objectives, respectively. Time-to-event outcomes were compared with the SOC arms of published studies. RESULTS: Thirty-three patients were enrolled, and median follow-up was 3.1 years. Six patients had adverse events (grade ≤3) possibly related to IGV-001. Median progression-free survival (PFS) was 9.8 months in the intent-to-treat population (vs. SOC, 6.5 months; P = 0.0003). In IGV-001-treated patients who met Stupp-eligible criteria, PFS was 11.6 months overall (n = 22; P = 0.001) and 17.1 months at the highest exposure (n = 10; P = 0.0025). The greatest overall survival was observed in Stupp-eligible patients receiving the highest exposure (median, 38.2 months; P = 0.044). Stupp-eligible patients with methylated O6-methylguanine-DNA methyltransferase promoter (n = 10) demonstrated median PFS of 38.4 months (P = 0.0008). Evidence of immune activation was noted. CONCLUSIONS: IGV-001 was well tolerated, PFS compared favorably with SOC, and evidence suggested an immune-mediated mechanism (ClinicalTrials.gov: NCT02507583).

Brain tissue-resident immune memory cells are required for long-term protection against CNS infection with rabies virus.

Immune memory cells residing in previously infected, nonlymphoid tissues play a role in immune surveillance. In the event that circulating antibodies fail to prevent virus spread to the tissues in a secondary infection, these memory cells provide an essential defense against tissue reinfection. CNS tissues are isolated from circulating immune cells and antibodies by the blood-brain barrier, making the presence of tissue-resident immune memory cells particularly needed to combat recurrent infection by neurotropic viruses. Wild-type and laboratory-engineered rabies viruses are neurotropic, differ in pathogenicity, and have varying effects on BBB functions. These viruses have proven invaluable tools in demonstrating the importance of tissue-resident immune memory cells in the reinfection of CNS tissues. Only Type 1 immune memory is effective at therapeutically clearing a secondary infection with wild-type rabies viruses from the CNS and does so despite the maintenance of blood-brain barrier integrity.