Pedro R. Lowenstein

Nicotinic acetylcholine binding sites in Alzheimer's disease

In Alzheimers disease (AD), there is a loss of presynaptic cholinergic markers in the cerebral cortex, but the nature of cholinergic receptor changes is unclear. In this study, [3H]acetylcholine and [3H]nicotine were used to label nicotinic cholinergic binding sites in cerebral cortical tissues obtained at autopsy from patients with AD and from matched controls. A consistent and severe loss of nicotinic receptors was found in AD.

Chronic brain inflammation and persistent herpes simplex virus 1 thymidine kinase expression in survivors of syngeneic glioma treated by adenovirus-mediated gene therapy: Implications for clinical trials

The long-term consequences of adenovirus-mediated conditional cytotoxic gene therapy for gliomas remain uncharacterized. We report here detection of active brain inflammation 3 months after successful inhibition of syngeneic glioma growth. The inflammatory infiltrate consisted of activated macrophages/microglia and astrocytes, and T lymphocytes positive for leucosyalin, CD3 and CD8, and included secondary demyelination. We detected strong widespread herpes simplex virus 1 thymidine kinase immunoreactivity and vector genomes throughout large areas of the brain. Thus, patient evaluation and the design of clinical trials in ongoing and future gene therapy for brain glioblastoma must address not only tumor-killing efficiency, but also long-term active brain inflammation, loss of myelin fibers and persistent transgene expression.

Consensus guidelines for the detection of immunogenic cell death

Apoptotic cells have long been considered as intrinsically tolerogenic or unable to elicit immune responses specific for dead cell-associated antigens. However, multiple stimuli can trigger a functionally peculiar type of apoptotic demise that does not go unnoticed by the adaptive arm of the immune system, which we named “immunogenic cell death” (ICD). ICD is preceded or accompanied by the emission of a series of immunostimulatory damage-associated molecular patterns (DAMPs) in a precise spatiotemporal configuration. Several anticancer agents that have been successfully employed in the clinic for decades, including various chemotherapeutics and radiotherapy, can elicit ICD. Moreover, defects in the components that underlie the capacity of the immune system to perceive cell death as immunogenic negatively influence disease outcome among cancer patients treated with ICD inducers. Thus, ICD has profound clinical and therapeutic implications. Unfortunately, the gold-standard approach to detect ICD relies on vaccination experiments involving immunocompetent murine models and syngeneic cancer cells, an approach that is incompatible with large screening campaigns. Here, we outline strategies conceived to detect surrogate markers of ICD in vitro and to screen large chemical libraries for putative ICD inducers, based on a high-content, high-throughput platform that we recently developed. Such a platform allows for the detection of multiple DAMPs, like cell surface-exposed calreticulin, extracellular ATP and high mobility group box 1 (HMGB1), and/or the processes that underlie their emission, such as endoplasmic reticulum stress, autophagy and necrotic plasma membrane permeabilization. We surmise that this technology will facilitate the development of next-generation anticancer regimens, which kill malignant cells and simultaneously convert them into a cancer-specific therapeutic vaccine.

HMGB1 Mediates Endogenous TLR2 Activation and Brain Tumor Regression

Background Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor that carries a 5-y survival rate of 5%. Attempts at eliciting a clinically relevant anti-GBM immune response in brain tumor patients have met with limited success, which is due to brain immune privilege, tumor immune evasion, and a paucity of dendritic cells (DCs) within the central nervous system. Herein we uncovered a novel pathway for the activation of an effective anti-GBM immune response mediated by high-mobility-group box 1 (HMGB1), an alarmin protein released from dying tumor cells, which acts as an endogenous ligand for Toll-like receptor 2 (TLR2) signaling on bone marrow-derived GBM-infiltrating DCs. Methods and Findings Using a combined immunotherapy/conditional cytotoxic approach that utilizes adenoviral vectors (Ad) expressing Fms-like tyrosine kinase 3 ligand (Flt3L) and thymidine kinase (TK) delivered into the tumor mass, we demonstrated that CD4+ and CD8+ T cells were required for tumor regression and immunological memory. Increased numbers of bone marrow-derived, tumor-infiltrating myeloid DCs (mDCs) were observed in response to the therapy. Infiltration of mDCs into the GBM, clonal expansion of antitumor T cells, and induction of an effective anti-GBM immune response were TLR2 dependent. We then proceeded to identify the endogenous ligand responsible for TLR2 signaling on tumor-infiltrating mDCs. We demonstrated that HMGB1 was released from dying tumor cells, in response to Ad-TK (+ gancyclovir [GCV]) treatment. Increased levels of HMGB1 were also detected in the serum of tumor-bearing Ad-Flt3L/Ad-TK (+GCV)-treated mice. Specific activation of TLR2 signaling was induced by supernatants from Ad-TK (+GCV)-treated GBM cells; this activation was blocked by glycyrrhizin (a specific HMGB1 inhibitor) or with antibodies to HMGB1. HMGB1 was also released from melanoma, small cell lung carcinoma, and glioma cells treated with radiation or temozolomide. Administration of either glycyrrhizin or anti-HMGB1 immunoglobulins to tumor-bearing Ad-Flt3L and Ad-TK treated mice, abolished therapeutic efficacy, highlighting the critical role played by HMGB1-mediated TLR2 signaling to elicit tumor regression. Therapeutic efficacy of Ad-Flt3L and Ad-TK (+GCV) treatment was demonstrated in a second glioma model and in an intracranial melanoma model with concomitant increases in the levels of circulating HMGB1. Conclusions Our data provide evidence for the molecular and cellular mechanisms that support the rationale for the clinical implementation of antibrain cancer immunotherapies in combination with tumor killing approaches in order to elicit effective antitumor immune responses, and thus, will impact clinical neuro-oncology practice.

Platelets mediate cytotoxic T lymphocyte–induced liver damage

We found that platelet depletion reduces intrahepatic accumulation of virus-specific cytotoxic T lymphocytes (CTLs) and organ damage in mouse models of acute viral hepatitis. Transfusion of normal but not activation-blocked platelets in platelet-depleted mice restored accumulation of CTLs and severity of disease. In contrast, anticoagulant treatment that prevented intrahepatic fibrin deposition without reducing platelet counts did not avert liver injury. Thus, activated platelets contribute to CTL-mediated liver immunopathology independently of procoagulant function.

Response of Foot-and-Mouth Disease Virus to Increased Mutagenesis: Influence of Viral Load and Fitness in Loss of Infectivity

ABSTRACT Passage of foot-and-mouth disease virus (FMDV) in cell culture in the presence of the mutagenic base analog 5-fluorouracil or 5-azacytidine resulted in decreases of infectivity and occasional extinction of the virus. Low viral loads and low viral fitness enhanced the frequency of extinction events; this finding was shown with a number of closely related FMDV clones and populations differing by up to 106-fold in relative fitness in infections involving either single or multiple passages in the absence or presence of the chemical mutagens. The mutagenic treatments resulted in increases of 2- to 6.4-fold in mutation frequency and up to 3-fold in mutant spectrum complexity. The largest increase observed corresponded to the 3D (polymerase)-coding region, which is highly conserved in nonmutagenized FMDV populations. As a result, nucleotide sequence heterogeneity for the 3D-coding region became very similar to that for the variable VP1-coding region in FMDVs multiply passaged in the presence of chemical mutagens. The results suggest that strategies to combine reductions of viral load and viral fitness could be effectively associated with extinction mutagenesis as a potential new antiviral strategy.

Neuronal and glial cell type-specific promoters within adenovirus recombinants restrict the expression of the apoptosis-inducing molecule Fas ligand to predetermined brain cell types, and abolish peripheral liver toxicity.

Gene therapy using Fas ligand (FasL) for treatment of tumours and protection of transplant rejection is hampered because of the systemic toxicity of FasL. In the present study, recombinant replication-defective adenovirus vectors (RAds) encoding FasL under the control of either the neuronal-specific neuronal-specific enolase (NSE) promoter or the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter have been constructed. The cell type-specific expression of FasL in both neurons and glial cells in primary cultures, and in neuronal and glial cell lines is demonstrated. Furthermore, transgene expression driven by the neuronal and glial promoter was not detected in fibroblastic or epithelial cell lines. Expression of FasL driven by a major immediate early human cytomegalovirus promoter (MIEhCMV) was, however, achieved in all cells tested. As a final test of the stringency of transgene-specific expression, the RAds were injected directly into the bloodstream of mice. The RAds encoding FasL under the control of the non-cell type-specific MIEhCMV promoter induced acute generalized liver haemorrhage with hepatocyte apoptosis, while the RAds containing the NSE or GFAP promoter sequences were completely non-toxic. This demonstrates the specificity of transgene expression, enhanced safety during systemic administration, and tightly regulated control of transgene expression of highly cytotoxic gene products, encoded within transcriptionally targeted RAds.

Efficient FLPe recombinase enables scalable production of helper-dependent adenoviral vectors with negligible helper-virus contamination.

Helper-dependent (HD), high-capacity adenoviruses are one of the most efficient and safe gene therapy vectors, capable of mediating long-term expression. Currently, the most widely used system for HD vector production avoids significant contamination with helper virus by using producer cells stably expressing a nuclear-targeted Cre recombinase and an engineered first-generation helper virus with parallel loxP sites flanking its packaging signal. The system requires a final, density-based separation of HD and residual helper viruses by ultracentrifugation to reduce contaminating helper virus to low levels. This separation step hinders large-scale production of clinical-grade HD virus. By using a very efficient recombinase, in vitro–evolved FLPe (ref. 14), to excise the helper virus packaging signal in the producer cells, we have developed a scalable HD vector production method. FLP has previously been shown to mediate maximum levels of excision close to 100% compared to 80% for Cre (ref. 15). Utilizing a common HD plasmid backbone, the FLPe-based system reproducibly yielded HD virus with the same low levels of helper virus contamination before any density-based separation by ultracentrifugation. This should allow large-scale production of HD vectors using column chromatography–based virus purification.

Molecular indetermination in the transition to error catastrophe: Systematic elimination of lymphocytic choriomeningitis virus through mutagenesis does not correlate linearly with large increases in mutant spectrum complexity

Studies with several RNA viruses have shown that enhanced mutagenesis resulted in decreases of infectivity or virus extinction, as predicted from virus entry into error catastrophe. Here we report that lymphocytic choriomeningitis virus, the prototype arenavirus, is extremely susceptible to extinction mutagenesis by the base analog 5-fluorouracil. Virus elimination was preceded by increases in complexity of the mutant spectra of treated populations. However, careful molecular comparison of the mutant spectra of several genomic segments suggests that the largest increases in mutation frequency do not predict virus extinction. Highly mutated viral genomes have escaped detection presumably because lymphocytic choriomeningitis virus replicates at or near the error threshold, and genomes in the transition toward error catastrophe may have an extremely short half-life and escape detection with state-of-the-art cloning and sequencing technologies.

Efficient Virus Extinction by Combinations of a Mutagen and Antiviral Inhibitors

ABSTRACT The effect of combinations of the mutagenic base analog 5-fluorouracil (FU) and the antiviral inhibitors guanidine hydrochloride (G) and heparin (H) on the infectivity of foot-and-mouth disease virus (FMDV) in cell culture has been investigated. Related FMDV clones differing up to 106-fold in relative fitness in BHK-21 cells have been compared. Systematic extinction of intermediate fitness virus was attained with a combination of FU and G but not with the mutagen or the inhibitor alone. Systematic extinction of high-fitness FMDV required the combination of FU, G, and H. FMDV showing high relative fitness in BHK-21 cells but decreased replicative ability in CHO cells behaved as a low-fitness virus with regard to extinction mutagenesis in CHO cells. This confirms that relative fitness, rather than a specific genomic sequence, determines the FMDV response to enhanced mutagenesis. Mutant spectrum analysis of several genomic regions from a preextinction population showed a statistically significant increase in the number of mutations compared with virus passaged in parallel in the absence of FU and inhibitors. Also, in a preextinction population the types of mutations that can be attributed to the mutagenic action of FU were significantly more frequent than other mutation types. The results suggest that combinations of mutagenic agents and antiviral inhibitors can effectively drive high-fitness virus into extinction.