Phase 1 Trial of Vaccination with Autologous Tumor Cells and Antisense Directed Against the Insulin Growth Factor Type 1 Receptor (IGF-1R AS ODN) in Patients with Recurrent Glioblastoma

Background: Extending a previous Phase I study, we report the results of a second Phase I autologous tumor cell vaccination trial for patients with recurrent glioblastomas (IND 14379-101, NCT01550523). Methods: Following surgery, subjects were treated by 24 hour implantation in the rectus sheath of ten biodiffusion chambers containing irradiated autologous tumor cells and IGF-1R AS ODN with the objective of stimulating tumor immunity. Patients were monitored for safety, clinical and radiographic as well as immune responses. Results: There were no Grade 3 toxicities related to protocol treatment and overall median survival from initial diagnosis was 91.4 weeks. Two protocol survival cohorts with median survivals of 48.2 and 10 weeks were identified and predicted by our pre-treatment assessments of immune function, corroborated by post-vaccination pro-inflammatory cytokine profiles. Longer survival subjects had imaging findings including transient elevations in cerebral blood volume (rCBV) and sustained elevations of apparent diffusion coefficient (ADC) interpreted as transient hyperemia and cell loss. Conclusions: The vaccine paradigm was well-tolerated with a favorable median survival. Our data support this as a novel treatment paradigm that promotes anti-tumor immunity

Cerebrospinal Fluid Cytokine and Chemokine Patterns in Central Nervous System Infections, Hemorrhage and Neoplasms

Cytokines and chemokines are soluble proteins that act as regulators of cellular functions throughout the body. Cytokines and chemokines released in the setting of various CNS disorders appear in the CSF compartment where determination of their levels can provide insight into pathogenic processes such as neuroinflammation. We utilized the Millipore HCYTOMAG 60K assay/kit/system to perform multiplex profiling of 42 different cytokines/chemokines in the CSF of patients with a variety of distinct CNS disease processes, including infection, hemorrhage and neoplasia. CNS infections included viral (Chronic Parechovirus type 3 (HPeV3), Enterovirus (EV) 68, Adenovirus, JC virus, West Nile virus), bacterial (Mycobacterium tuberculosis, Borrelia burgdorferi, Propionibacterium acnes, Staphylococcus epidermidis, Streptococcus sp.), fungal (Cryptococcus neoformans) and single celled parasite (Toxoplasma gondii). CSF specimens negative for infectious organisms in noninflammatory conditions were selected as controls. Additional non-infectious samples tested were obtained from patients with subarachnoid hemorrhage (SAH) and following surgery for glioblastoma. The glioblastoma samples were noteworthy in having negligible elevations in the cytokines/chemokines tested. CSF from patients with SAH was elevated in only MCP-1/CCL2. Distinct patterns of cytokine/chemokine expression were detected for each infectious patient population. Picornavirus infections HPeV3 and EV68 were associated with increased levels of the monocyte chemoattractant protein MCP-1/CCL2 when compared to non-infectious, non-inflammatory samples. In contrast to chronic HPeV3 infection, EV68 encephalitis was associated with increased CSF levels of additional cytokines; CCLX1, IL-4 and IL-7. Adenovirus infection was associated with markedly higher levels of fractalkine in CSF when compared to any of the other non-inflammatory, infectious, hemorrhage or tumor cases. CSF from a Mycobacterium tuberculosis infection demonstrated increased levels of a greater variety of cytokines/chemokines than any of the other groups tested. Patterns of cytokine/chemokine expression in the CNS reveal characteristics of the host innate response that provide insight into the disease process and potential targets for therapeutic intervention

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

Pharmacologic or Genetic Targeting of Glutamine Synthetase Skews Macrophages toward an M1-like Phenotype and Inhibits Tumor Metastasis.

Glutamine-synthetase (GS), the glutamine-synthesizing enzyme from glutamate, controls important events, including the release of inflammatory mediators, mammalian target of rapamycin (mTOR) activation, and autophagy. However, its role in macrophages remains elusive. We report that pharmacologic inhibition of GS skews M2-polarized macrophages toward the M1-like phenotype, characterized by reduced intracellular glutamine and increased succinate with enhanced glucose flux through glycolysis, which could be partly related to HIF1α activation. As a result of these metabolic changes and HIF1α accumulation, GS-inhibited macrophages display an increased capacity to induce T cell recruitment, reduced T cell suppressive potential, and an impaired ability to foster endothelial cell branching or cancer cell motility. Genetic deletion of macrophagic GS in tumor-bearing mice promotes tumor vessel pruning, vascular normalization, accumulation of cytotoxic T cells, and metastasis inhibition. These data identify GS activity as mediator of the proangiogenic, immunosuppressive, and pro-metastatic function of M2-like macrophages and highlight the possibility of targeting this enzyme in the treatment of cancer metastasis

The Production of Antibody by Invading B Cells Is Required for the Clearance of Rabies Virus from the Central Nervous System

Every year over 50,000 people die from rabies worldwide, primarily due to the poor availability of rabies vaccine in developing countries. However, even when vaccines are available, human deaths from rabies occur if exposure to the causative virus is not recognized and vaccination is not sought in time. This is because rabies virus immunity induced by the natural infection or current vaccines is generally not effective at removing disease-causing rabies virus from brain tissues. Our studies provide insight into why this is the case and how vaccination can be changed so that the immune response can clear the virus from brain tissues. We show that the type of immune response induced by a live-attenuated rabies virus vaccine may be the key. In animal models, live-attenuated rabies virus vaccines are effective at delivering the immune cells capable of clearing the virus into CNS tissues and promote recovery from a rabies virus infection that has spread to the brain while conventional vaccines based on killed rabies virus do not. The production of rabies-specific antibody by B cells that invade the CNS tissues is important for complete elimination of the virus. We hypothesize that similar mechanisms may promote rabies virus clearance from individuals who are diagnosed after the virus has reached, but not extensively spread, through the CNS

Aleuria Aurantia Lectin (AAL)-Reactive Immunoglobulin G Rapidly Appears in Sera of Animals following Antigen Exposure.

We have discovered an Aleuria Aurantia Lectin (AAL)-reactive immunoglobulin G (IgG) that naturally occurs in the circulation of rabbits and mice, following immune responses induced by various foreign antigens. AAL can specifically bind to fucose moieties on glycoproteins. However, most serum IgGs are poorly bound by AAL unless they are denatured or treated with glycosidase. In this study, using an immunogen-independent AAL-antibody microarray assay that we developed, we detected AAL-reactive IgG in the sera of all animals that had been immunized 1-2 weeks previously with various immunogens with and without adjuvants and developed immunogen-specific responses. All of these animals subsequently developed immunogen-specific immune responses. The kinetics of the production of AAL-reactive IgG in mice and rabbits were distinct from those of the immunogen-specific IgGs elicited in the same animals: they rose and fell within one to two weeks, and peaked between four to seven days after exposure, while immunogen-specific IgGs continued to rise during the same period. Mass spectrometric profiling of the Fc glycoforms of purified AAL-reactive IgGs indicates that these are mainly comprised of IgGs with core-fucosylated and either mono-or non-galactosylated Fc N-glycan structures. Our results suggest that AAL-reactive IgG could be a previously unrecognized IgG subset that is selectively produced at the onset of a humoral response

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\u27s 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

A Genome-Wide Association Study for Host Resistance to Ostreid Herpesvirus in Pacific Oysters (Crassostrea gigas)

Ostreid herpesvirus (OsHV) can cause mass mortality events in Pacific oyster aquaculture. While various factors impact on the severity of outbreaks, it is clear that genetic resistance of the host is an important determinant of mortality levels. This raises the possibility of selective breeding strategies to improve the genetic resistance of farmed oyster stocks, thereby contributing to disease control. Traditional selective breeding can be augmented by use of genetic markers, either via marker-assisted or genomic selection. The aim of the current study was to investigate the genetic architecture of resistance to OsHV in Pacific oyster, to identify genomic regions containing putative resistance genes, and to inform the use of genomics to enhance efforts to breed for resistance. To achieve this, a population of approximate to 1,000 juvenile oysters were experimentally challenged with a virulent form of OsHV, with samples taken from mortalities and survivors for genotyping and qPCR measurement of viral load. The samples were genotyped using a recently-developed SNP array, and the genotype data were used to reconstruct the pedigree. Using these pedigree and genotype data, the first high density linkage map was constructed for Pacific oyster, containing 20,353 SNPs mapped to the ten pairs of chromosomes. Genetic parameters for resistance to OsHV were estimated, indicating a significant but low heritability for the binary trait of survival and also for viral load measures (h2 0.12 - 0.25). A genome-wide association study highlighted a region of linkage group 6 containing a significant QTL affecting host resistance. These results are an important step toward identification of genes underlying resistance to OsHV in oyster, and a step toward applying genomic data to enhance selective breeding for disease resistance in oyster aquaculture.Peer reviewe