TXM peptides inhibit SARS-CoV-2 infection, syncytia formation, and lower inflammatory consequences.

After three years of the SARS-CoV-2 pandemic, the search and availability of relatively low-cost benchtop therapeutics for people not at high risk for a severe disease are still ongoing. Although vaccines and new SARS-CoV-2 variants reduce the death toll, the long COVID-19 along with neurologic symptoms can develop and persist even after a mild initial infection. Reinfections, which further increase the risk of sequelae in multiple organ systems as well as the risk of death, continue to require caution. The spike protein of SARS-CoV-2 is an important target for both vaccines and therapeutics. The presence of disulfide bonds in the receptor binding domain (RBD) of the spike protein is essential for its binding to the human ACE2 receptor and cell entry. Here, we demonstrate that thiol-reducing peptides based on the active site of oxidoreductase thioredoxin 1, called thioredoxin mimetic (TXM) peptides, can prevent syncytia formation, SARS-CoV-2 entry into cells, and infection in a mouse model. We also show that TXM peptides inhibit the redox-sensitive HIV pseudotyped viral cell entry. These results support disulfide targeting as a common therapeutic strategy for treating infections caused by viruses using redox-sensitive fusion. Furthermore, TXM peptides exert anti-inflammatory properties by lowering the activation of NF-κB and IRF signaling pathways, mitogen-activated protein kinases (MAPKs) and lipopolysaccharide (LPS)-induced cytokines in mice. The antioxidant and anti-inflammatory effects of the TXM peptides, which also cross the blood-brain barrier, in combination with prevention of viral infections, may provide a beneficial clinical strategy to lower viral infections and mitigate severe consequences of COVID-19.

Rehoming and Other Refinements and Replacement in Procedures Using Golden Hamsters in SARS-CoV-2 Vaccine Research.

Effective vaccines are needed to fight the COVID-19 pandemic. Forty golden hamsters were inoculated with two promising vaccine candidates and eighteen animals were used in pilot trials with viral challenge. ELISA assays were performed to determine endpoint serum titres for specific antibodies and virus neutralisation tests were used to evaluate the efficacy of antibodies. All tests with serum from vaccinated hamsters were negative even after booster vaccinations and changes in vaccination protocol. We concluded that antibodies did not have sufficient neutralising properties. Refinements were observed at all steps, and the in vitro method (virus neutralisation test) presented a replacement measure and ultimately lead to a reduction in the total number of animals used in the project. The institutional animal welfare officer and institutional designated veterinarian approved the reuse or rehoming of the surplus animals. Simple socialization procedures were performed and ultimately 19 animals were rehomed, and feedback was collected. Recently, FELASA published recommendations for rehoming of animals used for scientific and educational purposes, with species-specific guidelines, including mice, rats, and rabbits. Based on our positive experience and feedback from adopters, we concluded that the rehoming of rodents, including hamsters, is not only possible, but highly recommended.

Potential revival of cholinesterase inhibitors as drugs in veterinary medicine.

The cholinergic system is involved in the regulation of all organ systems and has acetylcholine (ACh) as almost its only neurotransmitter. Any substance is called cholinergic if it can alter the action of acetylcholine. Cholinesterases (ChEs) are enzymes that enable the hydrolysis of acetylcholine and in this way ensure homeostasis in cholinergic synapses. Cholinesterase inhibitors (ChEi) are a group of indirect-acting cholinergic agonists that influence the activity of the cholinergic system. Several compounds that can inhibit cholinesterases are of importance to veterinary medicine from pharmacological and toxicological perspective. The frequency of their use in veterinary medicine has fluctuated over the years and is now reduced to a minimum. They are mainly used in agriculture as pesticides, and some are rarely used as parasiticides for companion animals and livestock. In recent years, interest in the use of new cholinesterase inhibitors has increased since canine cognitive dysfunction (CCD) became a recognized and extensively studied disease. Similar to Alzheimer's disease (AD) in humans, CCD can be treated with cholinesterase inhibitors that cross the blood-brain barrier. In this review, the mammalian cholinergic system and the drugs that interact with cholinesterases are introduced. Cholinesterase inhibitors that can be used for the treatment of CCD are described in detail.

Treatment of canine cognitive dysfunction with novel butyrylcholinesterase inhibitor.

Canine cognitive dysfunction (CCD) is common in aged dogs and has many similarities with Alzheimer's disease. Unfortunately, like Alzheimer's disease, CCD cannot be cured. In the present study, we treated dogs with CCD with our newly developed and characterized butyrylcholinesterase inhibitor (BChEi). Seventeen dogs were randomized into two groups (treated with BChEi and untreated) and followed for 6 months at regular check-ups. The dogs' cognitive status was determined by a Canine Dementia Scale (CADES) questionnaire and two cognitive tests. In dogs with moderate cognitive impairment, treatment caused significant improvement in the clinical rating of cognitive abilities and the performance-based tests of cognitive functioning when compared to the untreated group (p < 0.001). Dogs treated with BChEi showed markedly improved cognitive function with enhanced quality of life. No side effects were observed in the treated dogs with moderate cognitive impairment. According to the results of this preliminary study, there is an indication that novel BChEi may be a promising drug for the treatment of CCD in dogs and may be an interesting candidate for the treatment of Alzheimer's disease in humans. However, further clinical studies are needed to confirm this.

Nitrosative Stress in the Frontal Cortex From Dogs With Canine Cognitive Dysfunction.

Canine cognitive dysfunction (CCD) is an age-related disorder similar to human Alzheimer's disease (AD) that occurs in elderly dogs. Nitrosative stress has been implicated as one of the causes leading to neurodegenerative diseases, particularly AD. Its involvement in the development of CCD has not been studied so far. In the present study, immunohistochemical staining detected all three isoforms of nitric oxide synthases (nNOS, eNOS, and iNOS) and 3-nitrotyrosine (3-NT) in brains from CCD-affected dogs and non-demented control dogs in all layers of the canine frontal cortex. In CCD-affected and non-demented brains, nNOS was highly expressed in pyramidal-like neurons in the upper cortical layers. nNOS has also been observed in astrocytes in the CCD frontal cortex. The nNOS immunohistochemical staining was statistically significantly elevated in dogs with CCD in comparison to non-demented dogs. Blood vessel wall cells were positive for eNOS, which was also expressed in astrocytes and neurons. Intense 3-NT immunoreactivity was observed in the upper cortical layers, where amyloid-beta deposits spread in the last stage of CCD. Brain cells in the same area were highly immunoreactive for iNOS. This infers that neuroinflammation and nitrosative stress might exacerbate the neurodegenerative process in CCD-affected brains, ultimately leading to cognitive impairment.

Intraneural and perineural inflammatory changes in piglets after injection of ultrasound gel, endotoxin, 0.9% NaCl, or needle insertion without injection.

BACKGROUND: Ultrasound gel nerve inflammation has been reported. We evaluated the extent and nature of inflammation after gel injection with endotoxin (positive), saline, or dry needle puncture (negative) controls after peripheral blocks in piglets. METHODS: Selected nerves of 12 piglets were localized by landmarks and nerve stimulator. Forty-eight hours after injection, specimens were examined for immunohistochemical cell differentiation/quantification and cytokine expression by using quantitative polymerase chain reaction. RESULTS: Both gel and endotoxin injections resulted in a significantly higher density of inflammatory cells (lymphocytes/granulocytes) as compared with needle insertions and/or saline injections (both P < 0.001). Cytokines were not detected in any of the specimens. CONCLUSIONS: Perineural gel injections cause significant inflammation. The lack of cytokines suggests injectate-related changes rather than mechanical trauma.

Inflammatory response after injection of aqueous gel into subarachnoid space in piglets.

BACKGROUND: Ultrasound-guided neuraxial anesthesia requires the application of ultrasound gel between the transducer and the skin to avoid signal drop-off. As the needle is inserted, the gel may be introduced intrathecally. The purpose of this study was to examine the evidence of an inflammatory response in the subarachnoid space after intrathecal gel introduction. METHODS: Twelve piglets were sedated with azaperone 0.5 mg/kg intramuscularly and anesthetized via face mask (2%-4% isoflurane in 50% air-oxygen mixture). After collection of cerebrospinal fluid by lumbar puncture with a 22-gauge needle (baseline), 0.2 mL of ultrasound gel mixed with 1 mL of saline was injected intrathecally into 9 piglets (gel group). In 3 piglets (control group), 1 mL of saline was administered. Behavioral and neurologic assessments were recorded on a 4-grade scale. Following the preinjection and postinjection cerebrospinal fluid collection, the piglets were killed, and samples of spinal cord with meninges were excised. Five cross sections (1 mm apart) were processed using immunohistochemistry. RESULTS: After anesthesia, all piglets displayed short-lived mild (grade 1) motor and behavioral deficits. Mean ± SD protein concentrations in the gel and baseline samples were 14.1 ± 3.0 and 1.3 ± 0.5 g/L, respectively (P = 0.001). No differences were found in protein concentration between baseline (1.8 ± 0.7 g/L) and control samples (2.8 ± 0.8 g/L) (P = 0.4). In the gel group, numerous immunopositive cells were found in the pia, arachnoid, and inner layer of dura. CONCLUSION: Subarachnoid injection of ultrasound gel in piglets results in an inflammatory response within neuraxial space.

Enzyme- and immunohistochemical aspects of skeletal muscle fibers in brown bear (Ursus arctos).

To further elucidate the pattern of MHC isoform expression in skeletal muscles of large mammals, in this study the skeletal muscles of brown bear, one of the largest mammalian predators with an extraordinary locomotor capacity, were analyzed. Fiber types in longissimus dorsi, triceps brachii caput longum, and rectus femoris muscles were determined according to the myofibrillar ATPase (mATPase) histochemistry and MHC isoform expression, revealed by a set of antibodies specific to MHC isoforms. The oxidative (SDH) and glycolytic enzyme (alpha-GPDH) capacity of fibers was demonstrated as well. By mATPase histochemistry five fiber types, i.e., I, IIC, IIA, IIAX, IIX were distinguished. Analyzing the MHC isoform expression, we assume that MHC-I, -IIa, and -IIx are expressed in the muscles of adolescent bears. MHC-I isoform was expressed in Type-I fibers and coexpressed with presumably -IIa isoform, in Type-IIC fibers. Surprisingly, two antibodies specific to rat MHC-IIa stained those fast fibers, that were histochemically and immunohistochemically classified as Type IIX. This assumption was additionally confirmed by complete absence of fiber staining with antibody specific to rat MHC-IIb and all fast fiber staining with antibody that according to our experience recognizes MHC-IIa and -IIx of rat. Furthermore, quite high-oxidative capacity of all fast fiber types and their weak glycolytic capacity also imply for MHC-IIa and -IIx isoform expression in fast fibers of bear. However, in adult, full-grown animal, only MHC-I and MHC-IIa isoforms were expressed. The expression of only two fast isoforms in bear, like in many other large mammals (humans, cat, dog, goat, cattle, and horse) obviously meets the weight-bearing and locomotor demands of these mammals.

Myosin heavy chain isoform transitions in canine skeletal muscles during postnatal growth.

To gain a better understanding of the normal characteristics of developing canine muscles, myosin heavy chain (MHC) isoform expression was analysed in the axial and limb skeletal muscles of 18 young dogs whose ages ranged from the late prenatal stage to 6 months. We compared the results of immunohistochemistry using ten monoclonal antibodies, specific to different MHC isoforms, and enzyme-histochemical reactions, which demonstrate the activity of myofibrillar ATPase, succinate dehydrogenase (SDH) and alpha-glycerophosphate dehydrogenase (alpha-GPDH). In the skeletal muscles of fetuses and neonatal dogs the developmental isoforms MHC-emb and MHC-neo were prevalent. In all muscles the primary fibres, located centrally in each muscle fascicle, strongly expressed the slow isoform MHC-I. The adult fast isoform MHC-IIa was first noted in some of the secondary fibres on fetal day 55. During the first 10 days after birth, the expression of MHC-emb declined, as did that of MHC-neo during the second and third weeks. Correspondingly, the expression of MHC-IIa, and later, of MHC-I increased in the secondary fibres. Between the sixth week and second month the expression of MHC-IIx became prominent. The slow rhomboideus muscle exhibited an early expression of the slow isoform in the secondary fibres. Our results indicate that the timing of muscle maturation depends on its activity immediately following birth. The fastest developing muscle was the diaphragm, followed by the fast muscles. A pronounced changeover from developmental to adult isoforms was noted at 4-6 weeks of age, which coincides with the increased physical activity of puppies.