Methanol-induced optic neuropathy: a still-present problem

Methanol-induced optic neuropathy (Me-ION) is a serious condition that may result in long-term or irreversible visual impairment or even blindness secondary to damage and loss of function of the optic nerve and retina. Me-ION shows a tendency to occur as mass poisonings around the world with a clear predilection for poor societies in developing countries. The main mechanism underlying the molecular basis of Me-ION is the inhibition of the mitochondrial oxidative phosphorylation process through the binding of the toxic metabolite of methanol—formic acid—with the key enzyme of this process—cytochrome c oxidase. However, other mechanisms, including damage to the eye tissues by oxidative stress causing the intensification of the oxidative peroxidation process with the formation of cytotoxic compounds, as well as an increase in the synthesis of pro-inflammatory cytokines and influence on the expression of key proteins responsible for maintaining cell homeostasis, also play an important role in the pathogenesis of Me-ION. Histopathological changes in the eye tissues are mainly manifested as the degeneration of axons and glial cells of the optic nerve, often with accompanying damage of the retina that may involve all its layers. Despite the development of therapeutic approaches, persistent visual sequelae are seen in 30–40% of survivors. Thus, Me-ION continues to be an important problem for healthcare systems worldwide.

Systemic high-dose dexamethasone treatment may modulate the efficacy of intratumoral viral oncolytic immunotherapy in glioblastoma models

BackgroundIntratumoral viral oncolytic immunotherapy is a promising new approach for the treatment of a variety of solid cancers. CAN-2409 is a replication-deficient adenovirus that delivers herpes simplex virus thymidine kinase to cancer cells, resulting in local conversion of ganciclovir or valacyclovir into a toxic metabolite. This leads to highly immunogenic cell death, followed by a local immune response against a variety of cancer neoantigens and, next, a systemic immune response against the injected tumor and uninjected distant metastases. CAN-2409 treatment has shown promising results in clinical studies in glioblastoma (GBM). Patients with GBM are usually given the corticosteroid dexamethasone to manage edema. Previous work has suggested that concurrent dexamethasone therapy may have a negative effect in patients treated with immune checkpoint inhibitors in patients with GBM. However, the effects of dexamethasone on the efficacy of CAN-2409 treatment have not been explored.MethodsIn vitro experiments included cell viability and neurosphere T-cell killing assays. Effects of dexamethasone on CAN-2409 in vivo were examined using a syngeneic murine GBM model; survival was assessed according to Kaplan-Meier; analyses of tumor-infiltrating lymphocytes were performed with mass cytometry (CyTOF - cytometry by time-of-flight). Data were analyzed using a general linear model, with one-way analysis of variance followed by Dunnett’s multiple comparison test, Kruskal-Wallis test, Dunn’s multiple comparison test or statistical significance analysis of microarrays.ResultsIn a mouse model of GBM, we found that high doses of dexamethasone combined with CAN-2409 led to significantly reduced median survival (29.0 days) compared with CAN-2409 treatment alone (39.5 days). CyTOF analyses of tumor-infiltrating immune cells demonstrated potent immune stimulation induced by CAN-2409 treatment. These effects were diminished when high-dose dexamethasone was used. Functional immune cell characterization suggested increased immune cell exhaustion and tumor promoting profiles after dexamethasone treatment.ConclusionOur data suggest that concurrent high-dose dexamethasone treatment may impair the efficacy of oncolytic viral immunotherapy of GBM, supporting the notion that dexamethasone use should be balanced between symptom control and impact on the therapeutic outcome.

A novel enzyme synthesized by Acinetobacter sp. SM04 is responsible for zearalenone biodegradation

Zearalenone (ZEA), a non-steroidal estrogenic mycotoxin produced by multiple Fusarium species, contaminates cereals and threatens the health of both humans and animals by inducing hepatotoxicity, immunotoxicity, and genotoxicity. A new alkali tolerant enzyme named Ase, capable of degrading ZEA without H2O2, was derived from Acinetobacter sp. SM04 in this study. The Ase gene shares 97% sequence identity with hypothetical proteins from Acinetobacter pittii strain WCHAP 100004 and YMC 2010/8/T346 and Acinetobacter calcoaceticus PHEA-2, respectively. Based on the Acinetobacter genus database, the gene encoding Ase was cloned and extracellularly expressed in E. coli BL21. After degrading 88.4% of ZEA (20 μg/mL), it was confirmed through MCF-7 cell proliferation assays that Ase can transform ZEA into a non-estrogenic toxic metabolite. Recombinant Ase (molecular weight: 28 kDa), produced by E. coli BL21/pET32a(+)-His-Ase, was identified as an oxygen-utilizing and cytochrome-related enzyme with optimal activity at 60 °C and pH 9.0.

Safety Evaluation of Weissella cibaria JW15 by Phenotypic and Genotypic Property Analysis

Weissella cibaria is one of the bacteria in charge of the initial fermentation of kimchi and has beneficial effects such as immune-modulating, antagonistic, and antioxidant activities. In our study, we aimed to estimate the safety of W. cibaria JW15 for the use of probiotics according to international standards based on phenotypic (antibiotic resistance, hemolysis, and toxic metabolite production) and genotypic analysis (virulence genes including antibiotic resistance genes). The results of the safety assessment on W. cibaria JW15 were as follows; (1) antibiotic resistance genes (ARGs) (kanamycin and vancomycin etc.) were intrinsic characteristics; (2) There were no acquired virulence genes including Cytolysin (cylA), aggregation substance (asa1), Hyaluronidase (hyl), and Gelatinase (gelE); (3) this strain also lacked β-hemolysis and the production of toxic metabolites (D-lactate and bile salt deconjugation). Consequently, W. cibaria JW15 is expected to be applied as a functional food ingredient in the food market.

Krabbe Disease: Prospects of Finding a Cure Using AAV Gene Therapy

Krabbe Disease (KD) is an autosomal metabolic disorder that affects both the central and peripheral nervous systems. It is caused by a functional deficiency of the lysosomal enzyme, galactocerebrosidase (GALC), resulting in an accumulation of the toxic metabolite, psychosine. Psychosine accumulation affects many different cellular pathways, leading to severe demyelination. Although there is currently no effective therapy for Krabbe disease, recent gene therapy-based approaches in animal models have indicated a promising outlook for clinical treatment. This review highlights recent findings in the pathogenesis of Krabbe disease, and evaluates AAV-based gene therapy as a promising strategy for treating this devastating pediatric disease.

CTIM-13. PHASE 1 CLINICAL TRIAL OF ONCOLYTIC VIRAL IMMUNOTHERAPY WITH CAN-2409 + VALACYCLOVIR IN COMBINATION WITH NIVOLUMAB AND STANDARD OF CARE (SOC) IN NEWLY DIAGNOSED HIGH-GRADE GLIOMA (HGG)

BACKGROUND CAN-2409 is a replication-deficient adenovirus that delivers HSV thymidine kinase to cancer cells, resulting in local conversion of orally administered valacyclovir into a toxic metabolite. Previously, a phase 1b/2 clinical trial of CAN-2409 combined with standard-of-care (SOC) demonstrated safety and improved survival in HGG patients. Addition of CAN-2409 to nivolumab has the potential to activate locally recruited lymphocytes and teach them to recognize tumor neoantigens, changing the ‘cold’ immunosuppressive tumor microenvironment, and synergizing with the activity mediated by immune checkpoint inhibitors. This notion is supported by preclinical experiments showing that the combination of CAN-2409 with anti-PD1 therapy was more effective than either treatment alone. METHODS This ongoing phase 1 clinical trial evaluates safety and initial efficacy of CAN-2409 combined with nivolumab and SOC in newly diagnosed HGG. CAN-2409 is injected during neurosurgery into the tumor bed, followed by 14-days of valacyclovir. Radiation starts within 8 (+/-4) days of surgery. Temozolomide is administered to MGMT-methylation positive patients only. Nivolumab is given every 2 weeks, up to 52-weeks. Deep immune profiling studies are ongoing and initial results will be available shortly. RESULTS From February 2019 to March 2021, 41 patients were enrolled and 35 were evaluable for safety from the combination of CAN-2409, nivolumab and SOC: 24 male and 11 female; 34 glioblastoma, 1 diffuse astrocytoma; 33 IDH-wildtype, 2 IDH-mutant; 15 MGMT-methylated, 20 unmethylated. Median age was 62-years (range 28-79), median KPS 90 (range 80-100). With 13 months median follow-up, no unexpected serious adverse events were observed, and 23 patients are still alive. The most frequent possibly related adverse events (>10%) were nausea, fatigue, fever, headache, and increased ALT. CONCLUSIONS The combination of CAN-2409 + nivolumab + SOC was well tolerated. Clinical follow-up and extensive biomarker analyses will provide a better understanding of the therapeutic potential of this approach.

55 Baseline glycolytic tumor burden predicts response and survival in NSCLC and melanoma patients treated with immune checkpoint inhibitors

BackgroundTumor metabolic remodeling is considered one of the hallmarks of cancers and has been implicated in immune evasion. Highly glycolytic tumors can lead to immune suppression by both nutrient competition and toxic metabolite accumulation. Immune checkpoint inhibitors (ICIs) are essential in treating melanoma and non-small cell lung cancer (NSCLC) patients, but novel predictive biomarkers can significantly contribute to clinical practice. Herein we analyzed the predictive and prognostic value of baseline metabolic tumor volumes in NSCLC and melanoma patients treated with ICIs.MethodsThis retrospective, single-center study includes patients with metastatic NSCLC or melanoma and a baseline 18F-FDG-PET/CT (PET) performed before ICI. PET studied parameters were SUV (maximum standardized uptake value), wMTV (whole metabolic tumor value), and wTLG (whole total lesion glycolysis). Best response rates were analyzed through RECIST. High or low glycolytic tumor burden (GTB) patients were defined according to SUV, wMTV, and wTLG cut-offs that best discriminate progressive disease. Overall survival (OS) and progression-free survival (PFS) were estimated using the Kaplan-Meier method, and curves were compared with log-rank.ResultsAmong 151 patients included, 107 had NSCLC and 44 melanoma. Regarding NSCLC patients, there was a significant correlation between GTB and responses among all PET parameters (figure 1). Noteworthily, only one patient with a low wTLG (<146) showed progressive disease after ICIs (figure 2C). In regard to the potential in predicting response to ICIs, the area under the curve (AUC) obtained for each glycolytic parameter was higher than that for PD-L1 expression (figure 2D-H). Furthermore, when patients were categorized according to ICI exposure (1st or ≥ 2nd line), AUC for glycolytic parameters was higher in ≥ 2nd line versus 1st line (figure 3). Lastly, a low GTB was associated with improved PFS (figure 4) and OS (figure 5).Regarding melanoma patients, wMTV and wTLG were significantly associated with response (figure 6), and a low GTB was significantly associated with improved PFS and OS (figure 7).Abstract 55 Figure 1Abstract 55 Figure 2Abstract 55 Figure 3Abstract 55 Figure 4Abstract 55 Figure 5Abstract 55 Figure 6Abstract 55 Figure 7ConclusionsGTB accessed through a baseline PET is associated with survival and response to ICIs in NSCLC and melanoma patients, suggesting that the tumor metabolism might impact immune responses and be a promising predictive tool.Ethics ApprovalThe study obtained ethics approval by Sírio-Libanês Hospital institutional review board. Study participants were not required to give an informed consent before taking part.

Oxidative Transformations of 3,4-Dihydroxyphenylacetaldehyde Generate Potential Reactive Intermediates as Causative Agents for Its Neurotoxicity

Neurogenerative diseases, such as Parkinson’s disease, are associated, not only with the selective loss of dopamine (DA), but also with the accumulation of reactive catechol-aldehyde, 3,4-dihydroxyphenylacetaldehyde (DOPAL), which is formed as the immediate oxidation product of cytoplasmic DA by monoamine oxidase. DOPAL is well known to exhibit toxic effects on neuronal cells. Both catecholic and aldehyde groups seem to be associated with the neurotoxicity of DOPAL. However, the exact cause of toxicity caused by this compound remains unknown. Since the reactivity of DOPAL could be attributed to its immediate oxidation product, DOPAL-quinone, we examined the potential reactions of this toxic metabolite. The oxidation of DOPAL by mushroom tyrosinase at pH 5.3 produced conventional DOPAL-quinone, but oxidation at pH 7.4 produced the tautomeric quinone-methide, which gave rise to 3,4-dihydroxyphenylglycolaldehyde and 3,4-dihydroxybenzaldehyde as products through a series of reactions. When the oxidation reaction was performed in the presence of ascorbic acid, two additional products were detected, which were tentatively identified as the cyclized products, 5,6-dihydroxybenzofuran and 3,5,6-trihydroxybenzofuran. Physiological concentrations of Cu(II) ions could also cause the oxidation of DOPAL to DOPAL-quinone. DOPAL-quinone exhibited reactivity towards the cysteine residues of serum albumin. DOPAL-oligomer, the oxidation product of DOPAL, exhibited pro-oxidant activity oxidizing GSH to GSSG and producing hydrogen peroxide. These results indicate that DOPAL-quinone generates several toxic compounds that could augment the neurotoxicity of DOPAL.

Clostridioides difficile infection increases circulating p-cresol levels and dysregulates brain dopamine metabolism: linking gut-brain axis to autism and other neurologic disorders?

Gastrointestinal illnesses are one of the most common comorbidities reported in patients with neurodevelopmental diseases, including autism spectrum disorders (ASD). Gut dysbiosis, overgrowth of C. difficile in the gut, and gut microbiota-associated alterations in central neurotransmission have been implicated in ASD, where the dopaminergic axis plays an important role in the disease pathogenesis. Human C. difficile strains produce a significant amount of the toxic metabolite p-cresol, an inhibitor of dopamine beta-hydroxylase (DBH), which catalyzes the conversion of dopamine (DA) to norepinephrine (NE). p-cresol is known to precipitate and exacerbate autistic behavior in rodents by increasing DA levels and altering DA receptor sensitivity in brain regions relevant to ASD. Therefore, we hypothesized that C. difficile infection dysregulates dopaminergic metabolism in the brain by increasing p-cresol levels in the gut and circulation and by inhibiting DBH, ultimately leading to elevated DA in the brain. For testing this hypothesis, we induced antibiotic-associated C. difficile in mice and determined the gut and serum p-cresol levels, serum DBH activity, and dopamine and its metabolite levels in different brain regions relevant to ASD. The results showed that C. difficile infection causes significant alterations in the dopaminergic axis in mice (p < 0.05). In addition, significantly increased circulating p-cresol levels and reduced DBH activity was observed in C. difficile infected animals (p < 0.05). Therefore, the results from this study suggest a potential link between C. difficile infection and alterations in the dopaminergic axis implicated in the precipitation and aggravation of ASD.