In a search for efficient treatment for amyotrophic lateral sclerosis: Old drugs for new approaches.

Recent progress in understanding the pathological changes in the nervous system and in certain other body systems (e.g., immune system) that lead to the development and progression of amyotrophic lateral sclerosis (ALS) revealed a number of molecular and cellular processes that can potentially be used as therapeutic targets. Many of these processes are compromised not only in ALS but also in other diseases and a repertoire of drugs able to restore, at least partially, their functionality has been developed. In this review, we briefly describe current approaches to the repurposing of such "old" drugs for treatment of patients with ALS.

The peptide semax affects the expression of genes related to the immune and vascular systems in rat brain focal ischemia: genome-wide transcriptional analysis.

BACKGROUND: The nootropic neuroprotective peptide Semax (Met-Glu-His-Phe-Pro-Gly-Pro) has proved efficient in the therapy of brain stroke; however, the molecular mechanisms underlying its action remain obscure. Our genome-wide study was designed to investigate the response of the transcriptome of ischemized rat brain cortex tissues to the action of Semax in vivo. RESULTS: The gene-expression alteration caused by the action of the peptide Semax was compared with the gene expression of the "ischemia" group animals at 3 and 24 h after permanent middle cerebral artery occlusion (pMCAO). The peptide predominantly enhanced the expression of genes related to the immune system. Three hours after pMCAO, Semax influenced the expression of some genes that affect the activity of immune cells, and, 24 h after pMCAO, the action of Semax on the immune response increased considerably. The genes implicated in this response represented over 50% of the total number of genes that exhibited Semax-induced altered expression. Among the immune-response genes, the expression of which was modulated by Semax, genes that encode immunoglobulins and chemokines formed the most notable groups. In response to Semax administration, 24 genes related to the vascular system exhibited altered expression 3 h after pMCAO, whereas 12 genes were changed 24 h after pMCAO. These genes are associated with such processes as the development and migration of endothelial tissue, the migration of smooth muscle cells, hematopoiesis, and vasculogenesis. CONCLUSIONS: Semax affects several biological processes involved in the function of various systems. The immune response is the process most markedly affected by the drug. Semax altered the expression of genes that modulate the amount and mobility of immune cells and enhanced the expression of genes that encode chemokines and immunoglobulins. In conditions of rat brain focal ischemia, Semax influenced the expression of genes that promote the formation and functioning of the vascular system.The immunomodulating effect of the peptide discovered in our research and its impact on the vascular system during ischemia are likely to be the key mechanisms underlying the neuroprotective effects of the peptide.

Immunogenicity and In Vivo Protective Effects of Recombinant Nucleocapsid-Based SARS-CoV-2 Vaccine Convacell®.

The vast majority of SARS-CoV-2 vaccines which are licensed or under development focus on the spike (S) protein and its receptor binding domain (RBD). However, the S protein shows considerable sequence variations among variants of concern. The aim of this study was to develop and characterize a SARS-CoV-2 vaccine targeting the highly conserved nucleocapsid (N) protein. Recombinant N protein was expressed in Escherichia coli, purified to homogeneity by chromatography and characterized by SDS-PAGE, immunoblotting, mass spectrometry, dynamic light scattering and differential scanning calorimetry. The vaccine, formulated as a squalane-based emulsion, was used to immunize Balb/c mice and NOD SCID gamma (NSG) mice engrafted with human PBMCs, rabbits and marmoset monkeys. Safety and immunogenicity of the vaccine was assessed via ELISA, cytokine titer assays and CFSE dilution assays. The protective effect of the vaccine was studied in SARS-CoV-2-infected Syrian hamsters. Immunization induced sustainable N-specific IgG responses and an N-specific mixed Th1/Th2 cytokine response. In marmoset monkeys, an N-specific CD4+/CD8+ T cell response was observed. Vaccinated Syrian hamsters showed reduced lung histopathology, lower virus proliferation, lower lung weight relative to the body, and faster body weight recovery. Convacell® thus is shown to be effective and may augment the existing armamentarium of vaccines against COVID-19.

Genomic epidemiology of SARS-CoV-2 in Russia reveals recurring cross-border transmission throughout 2020.

In 2020, SARS-CoV-2 has spread rapidly across the globe, with most nations failing to prevent or substantially delay its introduction. While many countries have imposed some limitations on trans-border passenger traffic, the effect of these measures on the global spread of COVID-19 strains remains unclear. Here, we report an analysis of 3206 whole-genome sequences of SARS-CoV-2 samples from 78 regions of Russia covering the period before the spread of variants of concern (between March and November 2020). We describe recurring imports of multiple COVID-19 strains into Russia throughout this period, giving rise to 457 uniquely Russian transmission lineages, as well as repeated cross-border transmissions of local circulating variants out of Russia. While the phylogenetically inferred rate of cross-border transmissions was somewhat reduced during the period of the most stringent border closure, it still remained high, with multiple inferred imports that each led to detectable spread within the country. These results indicate that partial border closure has had little effect on trans-border transmission of variants, which helps explain the rapid global spread of newly arising SARS-CoV-2 variants throughout the pandemic.

Targeted depletion of TRBV9+ T cells as immunotherapy in a patient with ankylosing spondylitis.

Autoimmunity is intrinsically driven by memory T and B cell clones inappropriately targeted at self-antigens. Selective depletion or suppression of self-reactive T cells remains a holy grail of autoimmune therapy, but disease-associated T cell receptors (TCRs) and cognate antigenic epitopes remained elusive. A TRBV9-containing CD8+ TCR motif was recently associated with the pathogenesis of ankylosing spondylitis, psoriatic arthritis and acute anterior uveitis, and cognate HLA-B*27-presented epitopes were identified. Following successful testing in nonhuman primate models, here we report human TRBV9+ T cell elimination in ankylosing spondylitis. The patient achieved remission within 3 months and ceased anti-TNF therapy after 5 years of continuous use. Complete remission has now persisted for 4 years, with three doses of anti-TRBV9 administered per year. We also observed a profound improvement in spinal mobility metrics and the Bath Ankylosing Spondylitis Metrology Index (BASMI). This represents a possibly curative therapy of an autoimmune disease via selective depletion of a TRBV-defined group of T cells. The anti-TRBV9 therapy could potentially be applicable to other HLA-B*27-associated spondyloarthropathies. Such targeted elimination of the underlying cause of the disease without systemic immunosuppression could offer a new generation of safe and efficient therapies for autoimmunity.

Single-Cell Gene Expression Analysis Revealed Immune Cell Signatures of Delta COVID-19.

The coronavirus disease 2019 (COVID-19) is accompanied by a cytokine storm with the release of many proinflammatory factors and development of respiratory syndrome. Several SARS-CoV-2 lineages have been identified, and the Delta variant (B.1.617), linked with high mortality risk, has become dominant in many countries. Understanding the immune responses associated with COVID-19 lineages may therefore aid the development of therapeutic and diagnostic strategies. Multiple single-cell gene expression studies revealed innate and adaptive immunological factors and pathways correlated with COVID-19 severity. Additional investigations covering host-pathogen response characteristics for infection caused by different lineages are required. Here, we performed single-cell transcriptome profiling of blood mononuclear cells from the individuals with different severity of the COVID-19 and virus lineages to uncover variant specific molecular factors associated with immunity. We identified significant changes in lymphoid and myeloid cells. Our study highlights that an abundant population of monocytes with specific gene expression signatures accompanies Delta lineage of SARS-CoV-2 and contributes to COVID-19 pathogenesis inferring immune components for targeted therapy.

Inhaled [D-Ala2]-Dynorphin 1-6 Prevents Hyperacetylation and Release of High Mobility Group Box 1 in a Mouse Model of Acute Lung Injury.

COVID-19 is a respiratory infection caused by the SARS-CoV-2 virus that can rapidly escalate to life-threatening pneumonia and acute respiratory distress syndrome (ARDS). Recently, extracellular high mobility group box 1 (HMGB1) has been identified as an essential component of cytokine storms that occur with COVID-19; HMGB1 levels correlate significantly with disease severity. Thus, the modulation of HMGB1 release may be vital for treating COVID-19. HMGB1 is a ubiquitous nuclear DNA-binding protein whose biological function depends on posttranslational modifications, its redox state, and its cellular localization. The acetylation of HMGB1 is a prerequisite for its translocation from the nucleus to the cytoplasm and then to the extracellular milieu. When released, HMGB1 acts as a proinflammatory cytokine that binds primarily to toll-like receptor 4 (TLR4) and RAGE, thereby stimulating immune cells, endothelial cells, and airway epithelial cells to produce cytokines, chemokines, and other inflammatory mediators. In this study, we demonstrate that inhaled [D-Ala2]-dynorphin 1-6 (leytragin), a peptide agonist of δ-opioid receptors, significantly inhibits HMGB1 secretion in mice with lipopolysaccharide- (LPS-) induced acute lung injury. The mechanism of action involves preventing HMGB1's hyperacetylation at critical lysine residues within nuclear localization sites, as well as promoting the expression of sirtuin 1 (SIRT1), an enzyme known to deacetylate HMGB1. Leytragin's effects are mediated by opioid receptors, since naloxone, an antagonist of opioid receptors, abrogates the leytragin effect on SIRT1 expression. Overall, our results identify leytragin as a promising therapeutic agent for the treatment of pulmonary inflammation associated with HMGB1 release. In a broader context, we demonstrate that the opioidergic system in the lungs may represent a promising target for the treatment of inflammatory lung diseases.

Semax and Pro-Gly-Pro activate the transcription of neurotrophins and their receptor genes after cerebral ischemia.

Consisting of a fragment of ACTH(4-7) and C-terminal PGP tripeptide, the polypeptide Semax is successfully used for acute stroke therapy. Previous experiments showed rapid induction of Bdnf, Ngf, and TrkB expression in intact rat hippocampus following Semax treatment. To investigate the mRNA expression of neurotrophins and their receptors after treatment with either Semax or PGP, the rat brains were analyzed at three time points following a permanent middle cerebral artery occlusion (pMCAO). We have shown for the first time that both Semax and PGP activate the transcription of neurotrophins and their receptors in the cortex of rats subjected to pMCAO. The profiles of transcription alteration under PGP and Semax treatment were partially overlapped. Semax enhanced the transcription of Bdnf, TrkC, and TrkA 3 h after occlusion, Nt-3 and Ngf 24 h after occlusion, and Ngf 72 h after occlusion. PGP enhanced the transcription of Bdnf and TrkC 3 h after pMCAO and Ngf, TrkB, TrkC, and TrkA 24 h after pMCAO. The analysis of the transcription alterations under PGP and Semax treatment in the cortex of rats without surgery, sham-operated rats and rats subjected to pMCAO revealed that Semax selectively affected the transcription of neurotrophins and their receptors in the ischemic rat cortex, whereas the influence of PGP was mainly unspecific.

Expression of sphingomyelin synthase 1 gene in rat brain focal ischemia.

Metabolites of the sphingomyelin cycle are reported to play an important role in neuronal death after ischemia. To elucidate the involvement of the key enzyme of this cycle, sphingomyelin synthase (SMS), in the mechanism underlying cerebral ischemia, we, for the first time, investigated changes in the mRNA expression of the SMS1 gene in rats after focal cerebral ischemia. According to our histological analysis, the damaged area is localized only in the ipsilateral cortex. In the ischemic cortex, the level of SMS1 transcripts was decreased at 3 and 24 h after occlusion, and at 72 h it had returned to the control level. A reduced level of SMS1 mRNA expression in the subcortex of rats with occlusion and sham-operated animals also was appeared during the first 24 h after surgery. This could be attributed to the effect of surgical stress. Seventy-two hours after occlusion, SMS1 mRNA expression in subcortex of ischemic rats was still at a decreased level; this may be considered to be a somewhat distant extended effect. Our results show the early response of the SMS1 gene that can be induced by both ischemia and stress. The results also suggest that inhibition of SMS1 mRNA expression may contribute to ceramide accumulation in a damaged cortex.

C9ORF72 hexanucleotide repeat expansion in ALS patients from the Central European Russia population.

Cohorts of amyotrophic lateral sclerosis (ALS) patients and control individuals of Caucasian origin from the Central European Russia (Moscow city and region) were analyzed for the presence of hexanucleotide repeat GGGGCC expansion within the first intron of the C9ORF72 gene. The presence of a large (>40) repeat expansion was found in 15% of familial ALS cases (3 of 20 unrelated familial cases) and 2.5% of sporadic ALS cases (6 of 238) but in none of control cases. These results suggest that the frequency of C9ORF72 hexanucleotide repeats expansions in the Central European Russian ALS patients is significantly lower than in Western European or Northern American ALS patients of Caucasian origin but higher than in Asian ALS patients.

Effect of semax and its C-terminal fragment Pro-Gly-Pro on the expression of VEGF family genes and their receptors in experimental focal ischemia of the rat brain.

The synthetic peptide Semax (Met-Glu-His-Phe-Pro-Gly-Pro) is used successfully in acute stroke therapy. In spite of numerous studies on the subject, many aspects of the neuroprotective effects of the peptide remain unknown. We studied the action of Semax and its C-terminal tripeptide Pro-Gly-Pro on the expression of the VEGF gene family (Vegf-a, Vegf-b, Vegf-c, Vegf-d, and Plgf) and their receptors (Vegfr-1, Vegfr-2, and Vegfr-3) in the frontoparietal cortex region of the rat brain at 3, 24, and 72 h after permanent left middle cerebral artery occlusion (pMCAO). The relative mRNA level of the genes studied was assessed using real-time reverse transcription-PCR. The Vegf-b and Vegf-d genes were most affected by the peptides, which resulted in their most noticeable activation at 3 h after pMCAO. The level of Vegf-d transcripts decreased considerably, whereas the mRNA level of the Vegf-b gene was significantly increased after 72 h of treatment with each of the peptides. In addition, the effects of the peptides on the expression of the Vegf-b and Vegf-d genes were the opposite of the action of ischemia. It is suggested that the identified effects of the peptides diminish the effects of ischemia, thus participating in the positive therapeutic effect of Semax on ischemic stroke.

The effect of Semax and its C-end peptide PGP on the morphology and proliferative activity of rat brain cells during experimental ischemia: a pilot study.

The neuropeptide preparation Semax (Met-Glu-His-Phe-Pro-Gly-Pro) has been employed successfully in clinical practice for treating patients with severe brain blood circulation disorders. In spite of numerous studies, many aspects of the therapeutic effects of this preparation remain unknown. In this context, the effects of Semax and its C-end tripeptide PGP on the functional morphology of nervous tissue cells were studied in the normal rat brain and in a model of incomplete global rat brain ischemia. In control animals, both peptides activated the capillary network and caused similar morphological changes to neurons and the neuropil regions. We show here for the first time at the histological level that Semax and PGP increased proliferation of the neuroglia, blood vessel endothelium, and progenitor cells in the subventricular zone. In these experimental conditions, only Semax abated the manifestation of ischemic damage to the nervous tissue. This was probably attributable to a decrease in vascular stasis symptoms as well as the trophic effect of the peptide.

Recent advances in rehabilitation of stroke survivors.

This report discusses the newest approaches to rehabilitation of post-stroke patients. Recent studies have clinical implications for the treatment of stroke at all stages, and chronic aphasia.

Long-Term Study of Interferon System State in Patients with Multiple Sclerosis Received the Individual Immune Therapy with Human Recombinant IFN-alpha.

Three patients with an evident multiple sclerosis (MS) were observed in a dynamics of long-term immune therapy based on administration of rIFN-alpha preparation. Production of IFN-alpha and IFN-gamma by induced peripheral blood leukocytes (PBL) of the patients was tested simultaneously with detection of a response of PBL to in vitro IFN-alpha and IFN-beta priming action every 10th day of observation. It has been shown that positive clinical effect of rIFN-alpha therapy was associated with a partial restoration of initially decreased IFN-alpha and IFN-gamma production by PBL, and paralleled by increasing the cell IFN-alpha sensitivity. On the other hand, decrease of PBL response to in vitro IFN-beta action was related to augmentation of IFN-alpha and IFN-gamma production in the secondary progressive MS. The effect of rIFN-alpha administration on blood cell sensitivity to in vitro IFN-beta treatment was manifested by augmentation of PBL response in patient with the remitting MS. The data obtained allowed to suggest a possibility to combine a usage of rIFN-beta and rIFN-alpha in pathogenetical therapy of the remitting MS.

Dynamics of Interferon System State Parameters during the Immune Therapy of Multiple Sclerosis: Individual Analysis.

Three patients with obvious multiple sclerosis (MS) were observed in a dynamics of immune therapy based on administration of rIFN-beta preparation. The ability to produce IFN-alpha and IFN-gamma by stimulated peripheral blood leukocytes (PBL) of the patients was tested simultaneously with detection of PBL response to the in vitro IFN-beta priming action on the every 10th day of observation. It has been shown that positive clinical effect of recombinant IFN-beta therapy was associated with a partial restoration of initially decreased IFN-beta and IFN-beta production by PBL, and paralleled by reducing the cell IFN-beta sensitivity. On the other hand, increase of PBL response to the in vitro IFN-beta action was related with diminishing IFN-alpha and IFN-gamma production, and did precede the following MS exacerbation. Mechanisms of IFN-beta therapeutical action in patients with MS in a context of changing relations between the parameters of IFN system functioning are discussed. Possibility is underlined to use the IFN system state parameters, combined with the cell IFN sensitivity determination, for prognosis of disease progression.

Possibility for Combination of Human rIFN-alpha and rIFN-beta in Complex Immune Therapy of Multiple Sclerosis.

Six patients with an evident multiple sclerosis (MS) were observed in dynamics of long term immune therapy based on administration of rIFN preparations. One half of them received the therapy with rIFN-alpha. Three other patients received rIFN-beta administration that was interrupted, when sensitivity of blood cells to in vitro IFN-beta action was decreased; the trial was continued by rIFN-alpha courses. Production of IFN-alpha and IFN-gamma by stimulated peripheral blood leukocytes (PBL) of the patients was tested simultaneously with detection of in vitro PBL response to IFN-alpha, IFN-beta and IFN-gamma priming action every 10th day of observation. It has been shown that positive clinical effect of rIFN-alpha therapy following rIFN-beta administration was comparable or even surpassed neurological effects of rIFN-alpha and rIFN-beta applied alone. Similarly rIFN-alpha and rIFN-beta therapy caused a partial restoration of initially decreased IFN-alpha and IFN-gamma production by PBL. It has been also accompanied by the dissociation appearance between IFN-alpha and IFN-gamma production by PBL, when in accordance with physiological IFN system state restoration the IFN-alpha production was augmented while IFN-gamma synthesis was reduced. Such a dissociation was paralleled by decrease in PBL response to IFN-alpha treatment, by increase in blood cell sensitivity to IFN-beta action, and by keeping IFN-gamma response at lower level than during rIFN-alpha or rIFN-beta caused dissociation. The data obtained allowed to confirm the possibility to combine rIFN-beta pathogenetical therapy with rIFN-alpha administration in patients with MS when sensitivity of blood cells to IFN-beta action displayed a tendency to a fall.