Sublethal enteroviral infection exacerbates disease progression in an ALS mouse model

Background Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of the motor neuron system associated with both genetic and environmental risk factors. Infection with enteroviruses, including poliovirus and coxsackievirus, such as coxsackievirus B3 (CVB3), has been proposed as a possible causal/risk factor for ALS due to the evidence that enteroviruses can target motor neurons and establish a persistent infection in the central nervous system (CNS), and recent findings that enteroviral infection-induced molecular and pathological phenotypes closely resemble ALS. However, a causal relationship has not yet been affirmed. Methods Wild-type C57BL/6J and G85R mutant superoxide dismutase 1 (SOD1G85R) ALS mice were intracerebroventricularly infected with a sublethal dose of CVB3 or sham-infected. For a subset of mice, ribavirin (a broad-spectrum anti-RNA viral drug) was given subcutaneously during the acute or chronic stage of infection. Following viral infection, general activity and survival were monitored daily for up to week 60. Starting at week 20 post-infection (PI), motor functions were measured weekly. Mouse brains and/or spinal cords were harvested at day 10, week 20 and week 60 PI for histopathological evaluation of neurotoxicity, immunohistochemical staining of viral protein, neuroinflammatory/immune and ALS pathology markers, and NanoString and RT-qPCR analysis of inflammatory gene expression. Results We found that sublethal infection (mimicking chronic infection) of SOD1G85R ALS mice with CVB3 resulted in early onset and progressive motor dysfunction, and shortened lifespan, while similar viral infection in C57BL/6J, the background strain of SOD1G85R mice, did not significantly affect motor function and mortality as compared to mock infection within the timeframe of the current study (60 weeks PI). Furthermore, we showed that CVB3 infection led to a significant increase in proinflammatory gene expression and immune cell infiltration and induced ALS-related pathologies (i.e., TAR DNA-binding protein 43 (TDP-43) pathology and neuronal damage) in the CNS of both SOD1G85R and C57BL/6J mice. Finally, we discovered that early (day 1) but not late (day 15) administration of ribavirin could rescue ALS-like neuropathology and symptoms induced by CVB3 infection. Conclusions Our study identifies a new risk factor that contributes to early onset and accelerated progression of ALS and offers opportunities for the development of novel targeted therapies.

Cruzipain Sulfotopes-Specific Antibodies Generate Cardiac Tissue Abnormalities and Favor Trypanosoma cruzi Infection in the BALB/c Mice Model of Experimental Chagas Disease

Trypanosoma cruzi cruzipain (Cz) bears a C-terminal domain (C-T) that contains sulfated epitopes “sulfotopes” (GlcNAc6S) on its unique N-glycosylation site. The effects of in vivo exposure to GlcNAc6S on heart tissue ultrastructure, immune responses, and along the outcome of infection by T. cruzi, were evaluated in a murine experimental model, BALB/c, using three independent strategies. First, mice were pre-exposed to C-T by immunization. C-T-immunized mice (C-TIM) showed IgG2a/IgG1 <1, induced the production of cytokines from Th2, Th17, and Th1 profiles with respect to those of dC-TIM, which only induced IL-10 respect to the control mice. Surprisingly, after sublethal challenge, both C-TIM and dC-TIM showed significantly higher parasitemia and mortality than the control group. Second, mice exposed to BSA-GlcNAc6S as immunogen (BSA-GlcNAc6SIM) showed: severe ultrastructural cardiac alterations while BSA-GlcNAcIM conserved the regular tissue architecture with slight myofibril changes; a strong highly specific humoral-immune-response reproducing the IgG-isotype-profile obtained with C-TIM; and a significant memory-T-cell-response demonstrating sulfotope-immunodominance with respect to BSA-GlcNAcIM. After sublethal challenge, BSA-GlcNAc6SIM showed exacerbated parasitemias, despite elevated IFN-γ levels were registered. In both cases, the abrogation of ultrastructural alterations when using desulfated immunogens supported the direct involvement of sulfotopes and/or indirect effect through their specific antibodies, in the induction of tissue damage. Finally, a third strategy using a passive transference of sulfotope-specific antibodies (IgG-GlcNAc6S) showed the detrimental activity of IgG-GlcNAc6S on mice cardiac tissue, and mice treated with IgG-GlcNAc6S after a sublethal dose of T. cruzi, surprisingly reached higher parasitemias than control groups. These findings confirmed the indirect role of the sulfotopes, via their IgG-GlcNAc6S, both in the immunopathogenicity as well as favoring T. cruzi infection.

State of Immunological Reactivity of Rat’s Body after Exposure to Different Doses of γ-Radiation in a Long Period and their Offense of the 1st Generation

BACKGROUND: Recently, the problem of changes in immunological reactivity has become important with the growth of immunodeficiency states of a different nature. [4]. High radiosensitivity of the immune system, its stability in time and the irreversibility of some post-radiation changes can contribute to the development of long-term effects of radiation [2,3,6]. One of the tasks of modern medicine and biology is to study the effect of chronic or fractionated ionizing effects on the body's immune system both in the early and late periods of irradiation and their 1st generation descendants [1,2,3,11,12]. Therefore, it is necessary to study the long-term effects of sublethal and fractionated effects of g-radiation on the immunological reactivity of the organism, nonspecific phagocytic resistance and their 1st generation descendants. AIM: The aim of this study was to study the long-term effects of sublethal and fractionated effects of g-radiation on the immunological reactivity of the organism, nonspecific phagocytic resistance and their 1st generation descendants.. METHODS: 7 series of experiments were performed on 105 white outbred sexually mature rats. 1-series intact (n=15), 2nd series - (n = 15) irradiated with a sublethal dose of 6 Gr. (1 month), 3rd series - irradiated with a sublethal dose (3 months, n = 20), 4 - descendants 1 - generations after sublethal dose, 5 series - irradiated with a fractionated dose (1 month), 6 series - irradiated with a fractionated dose (3 months), 7 - descendants of the 1st generation after fractionated -irradiation. Each series used 15 animals. Irradiation of animals 2 - 3 - 4 series was carried out on the Russian radiotherapy device "Agat-RM" -rays 60Co, the dose of sublethal irradiation is 6 Gr. Irradiation of animals of 5-6-7 series was carried out on the Russian radiotherapy device "Agat-RM" with 60Co-rays with topometric and dosimetric preparation of experimental animals, which facilitates the administration of a fractionated dose of 2 Gr. to animals three times within 3 weeks. RESULTS: In the long-term period after fractionated g-irradiation in the T-system of immunity, the following changes occur: against the background of an increase in the total number of lymphocytes, there is a decrease in the pool of CD3 +, CD4 + lymphocytes, immunoregulatory index, normalization of the lymphokine-producing ability of lymphocytes and a decrease in the pool of CD8 + lymphocytes. In the long-term period after fractionated g-irradiation in the humoral link of immunity, an increase in the absolute amount of CD19 + by 3.5 times was noted, which significantly exceeded the indicators of both control and intact animals. The percentage of this pool of cells exceeded the data of intact ones by 1.7 times. In the studied time period, the antibody-producing ability in the spleen increased from 22 ± 1.3 to 45 ± 2.6, without reaching, however, the level of intact animals. At the same time, there was a significant decrease in the suppression index to 13% (P<0.001) and the CIC concentration by 14 times (P<0.001) in the blood serum. The indicators of F/n and NST-test were high by 1.43 and 2.46 times, respectively. So, in the long-term period after exposure to a fractionated dose of g-radiation, the nonspecific phagocytic resistance of the organism is increased. CONCLUSIONS: The tension in the humoral link of immunity is manifested by a decrease in the quantitative and qualitative indicators and an increase in the functional and metabolic activity of neutrophils.

Comparative Investigation of Five Cobras’ Venoms Analgesic Capacity

Every day, a lot of people suffer from different types and intensity pain. Pain is probably the most common symptomatic reason to seek medical consultation. Unfortunately, despite improved knowledge of underlying mechanisms and better treatments, many people who have any type of pain receive inadequate care and non-effective drugs. Although the pain transmission channels are intensively studied, and the drug market is constantly replenished with new analgesics, it is well known that existing medications for the treatment of pain are often associated with serious side effects and rapid development of tolerance (moderate efficiency, physical dependence, respiratory arrest, suffocation, cardiac arrest, etc.). Thus, there is a need for new, more effective remedies. For this reason, despite the presence of a large number of anti-pain drugs, research and development of more effective and safe means for anaesthesia continue. Natural resources, particularly venoms, are a perspective supplier of antinociceptive and anti-inflammatory medicines. Venoms are complex mixtures of bioactive substances with high selectivity for physiological processes, including modulation of different ion channels, receptors function, and metabolic pathways. Thus, venoms represent an extensive source of molecules for the development of therapeutic agents. The goal of this study was the comparison of antinociceptive effects of five different cobras' venom antinociceptive action of cobras’ venoms was carried out under the same experimental conditions, at the same doses, during acute and inflammatory pain in mice in “formalin test”. To avoid toxic effects, the sublethal dose of each cobra venom (approximately 1/10 LD 50 ) was selected. The behavioural study showed that all tested venoms had a slight sensitizing effect in the acute phase during the first 5 min. In the second, inflammatory phase (16*25min), all tested cobra’s venoms (3μg/0.1ml, intraperitoneal) showed significant antinociceptive action, particularly the Naja naja oxiana venom decreased pain sensitivity by 48.4%, the Naja naja pallida venom by 75.4%, the Naja naja nigricincta venom by 38.5%, the Naja naja kaouthia venom by 33.2%, and the Ophiophagus Hannah venom by 78.3%, (p<0.05). The analgesic capacity of Analgin and Diclofenac under the same conditions were 77.9% and 88.7%, respectively. Thus, the Naja n. pallida and Ophiophagus Hannah venoms have shown the most expressed antinociceptive action, and they have competitive effectiveness compared to classic analgesics. They may be chosen as the most effective from tested venoms for further development of pain relief remedies.

Extracellular release of mitochondrial DNA is triggered by cigarette smoke and is detected in COPD

Chronic obstructive pulmonary disease (COPD) is characterized by continuous and irreversible inflammation frequently caused by persistent exposure to toxic inhalants such as cigarette smoke (CS). CS may trigger mitochondrial DNA (mtDNA) extrusion into the cytosol, extracellular space, or foster its transfer by extracellular vesicles (EVs). The present study aimed to elucidate whether mtDNA is released upon CS exposure and in COPD. We measured cell-free mtDNA (cf-mtDNA) in the plasma of former smokers affected by COPD, in the serum of mice that developed CS-induced emphysema, and in the extracellular milieu of human bronchial epithelial cells exposed to cigarette smoke extract (CSE). Further, we characterized cells exposed to sublethal and lethal doses of CSE by measuring mitochondrial membrane potential and dynamics, superoxide production and oxidative stress, cell cycle progression, and cytokine expression. Patients with COPD and mice that developed emphysema showed increased levels of cf-mtDNA. In cell culture, exposure to a sublethal dose of CSE decreased mitochondrial membrane potential, increased superoxide production and oxidative damage, dysregulated mitochondrial dynamics, and triggered mtDNA release in extracellular vesicles. The release of mtDNA into the extracellular milieu occurred concomitantly with increased expression of DNase III, DNA-sensing receptors (cGAS, NLRP3), proinflammatory cytokines (IL-1B, IL-6, IL-8, IL-18, CXCL2), and markers of senescence (p16, p21). Exposure to a lethal dose of CSE preferentially induced mtDNA and nuclear DNA release in cell debris. Our findings demonstrate that CS-induced stress triggers mtDNA release and is associated with COPD, supporting cf-mtDNA as a novel signaling response to CS exposure.

Surface charge-dependent mitochondrial response to similar intracellular nanoparticle contents at sublethal dosages

Background Considering the inevitability for humans to be frequently exposed to nanoparticles (NPs), understanding the biosafety of NPs is important for rational usage. As an important part of the innate immune system, macrophages are widely distributed in vital tissues and are also a dominant cell type that engulfs particles. Mitochondria are one of the most sensitive organelles when macrophages are exposed to NPs. However, previous studies have mainly reported the mitochondrial response upon high-dose NP treatment. Herein, with gold nanoparticles (AuNPs) as a model, we investigated the mitochondrial alterations induced by NPs at a sublethal concentration. Results At a similar internal exposure dose, different AuNPs showed distinct degrees of effects on mitochondrial alterations, including reduced tubular mitochondria, damaged mitochondria, increased reactive oxygen species, and decreased adenosine triphosphate. Cluster analysis, two-way ANOVA, and multiple linear regression suggested that the surface properties of AuNPs were the dominant determinants of the mitochondrial response. Based on the correlation analysis, the mitochondrial response was increased with the change in zeta potential from negative to positive. The alterations in mitochondrial respiratory chain proteins indicated that complex V was an indicator of the mitochondrial response to low-dose NPs. Conclusion Our current study suggests potential hazards of modified AuNPs on mitochondria even under sublethal dose, indicates the possibility of surface modification in biocompatibility improvement, and provides a new way to better evaluation of nanomaterials biosafety.

Biochemical and histological alterations induced by nickel oxide nanoparticles in the ground beetle Blaps polychresta (Forskl, 1775) (Coleoptera: Tenebrionidae)

The present study evaluates the effect of nickel oxide nanoparticles on some biochemical parameters and midgut tissues in the ground beetle Blaps polychresta as an indicator organism for nanotoxicity. Serial doses of the NiO-NPs colloid (0.01, 0.02, 0.03, 0.04, 0.05, and 0.06 mg/g) were prepared for injecting into the adult beetles. Insect survival was reported daily for 30 days, and the sublethal dose of 0.02 mg/g NiO-NPs was selected for the tested parameters. After the treatment, nickel was detected in the midgut tissues by X-ray microanalysis. The treated group demonstrated a significant increase in aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities when compared to the untreated group. However, the treated group demonstrated a significant decrease in ascorbate peroxidase (APOX) activity when compared to the untreated group. Histological and ultrastructural changes in the midgut tissues of treated and untreated beetles were also observed. The current findings provide a precedent for describing the physiological and histological changes caused by NiO-NPs in the ground beetle B. polychresta.

A Rabbit Model for the Evaluation of Drugs for Treating the Chronic Phase of Botulism

Antitoxin, the only licensed drug therapy for botulism, neutralizes circulating botulinum neurotoxin (BoNT). However, antitoxin is no longer effective when a critical amount of BoNT has already entered its target nerve cells. The outcome is a chronic phase of botulism that is characterized by prolonged paralysis. In this stage, blocking toxin activity within cells by next-generation intraneuronal anti-botulinum drugs (INABDs) may shorten the chronic phase of the disease and accelerate recovery. However, there is a lack of adequate animal models that simulate the chronic phase of botulism for evaluating the efficacy of INABDs. Herein, we report the development of a rabbit model for the chronic phase of botulism, induced by intoxication with a sublethal dose of BoNT. Spirometry monitoring enabled us to detect deviations from normal respiration and to quantitatively define the time to symptom onset and disease duration. A 0.85 rabbit intramuscular median lethal dose of BoNT/A elicited the most consistent and prolonged disease duration (mean = 11.8 days, relative standard deviation = 27.9%) that still enabled spontaneous recovery. Post-exposure treatment with antitoxin at various time points significantly shortened the disease duration, providing a proof of concept that the new model is adequate for evaluating novel therapeutics for botulism.