Intranasal Immunotherapy with M2 Macrophage Secretome Ameliorates Language Impairments and Autistic-like Behavior in Children.

Background/Objectives: The intranasal delivery of various neurotropic substances is considered a new attractive therapeutic approach for treating neuropathologies associated with neuroinflammation and altered regeneration. Specific language impairment (SLI) that arises as a result of damage to the cortical speech zones during the developmental period is one of the most common problems in preschool children, and it is characterized by persistent difficulties in the acquisition, understanding, and use of language. This study's objective is to evaluate the efficacy and safety of intranasal immunotherapy using the M2 macrophage secretome as a rich source of immunoregulatory and neurotrophic factors for the treatment of severe language impairment in children. Methods: Seventy-one children (54 boys and 17 girls, aged 3 to 13 years) were recruited to participate in a clinical trial (NCT04689282) in two medical centers. The children were examined before, 1 month after, and 6 months after the start of therapy. In the vast majority of children (55/71), language impairment was associated with autistic-like symptoms and attention deficit hyperactivity disorder (ADHD). Results: Daily intranasal inhalations of M2 macrophage-conditioned medium (for 30 days) were well tolerated and led to a decrease in the severity of language impairments, autistic-like behavior, and ADHD symptoms. The clinical effect appeared within a month after the first procedure and persisted or intensified during a 6-month follow-up. Two-thirds of the children showed a clear clinical improvement, while the rest had less pronounced improvement. Conclusions: Thus, the use of the M2 macrophage secretome and its intranasal delivery is safe, well tolerated, and clinically effective in children with severe language impairments.

Doxorubicin aqueous systems at low concentrations: Interconnection between self-organization, fluorescent and physicochemical properties, and action on hydrobionts.

Doxorubicin (Dox) is a highly effective cytostatic antibiotic that exhibits activity against a wide range of malignant neoplasms and is often used as the basis of various anti-tumor compositions. However, the use of Dox in therapeutic doses is associated with high systemic toxicity, which makes it urgent to find ways to reduce therapeutic concentrations, which is necessary primarily to minimize the side effects on the patient's body, as well as to reduce the harmful effects on aquatic ecosystems, commonly polluted by toxic pharmaceuticals. Studying the self-organization, physicochemical and spectral patterns, and their relation to bioeffects of Dox solutions in the range of low concentrations can reveal useful insights into the unknown effects of Dox as a cytostatic and potential pollutant of ecosystems. The self-organization in solutions and on substrates, physicochemical and spectral properties, and action of Dox solutions on hydrobionts were studied in the range of calculated concentrations from 1·10-20 to 1·10-4 M by methods of dynamic and electrophoretic light scattering (DLS and ELS), scanning electron microscopy (SEM), scanning probe microscopy (SPM), fluorescence spectroscopy, UV absorption spectroscopy, conductometry, tensiometry, pH-metry. Certified techniques for monitoring the toxicity of natural water and wastewater were used to establish the interconnection between these phenomena. It was shown that aqueous solutions of Dox are dispersed systems which rearrange their dispersed phase measuring hundreds of nm in size (nanoassociates) at dilution, followed by concerted changes in nanoassociates' parameters (size and ζ-potential) and properties of systems, as well as their bioassay results. SPM and SEM results confirm and complement the DLS and ELS data indicating the existence of nanoassociates in dilute Dox solutions.

L-Tryptophan Aqueous Systems at Low Concentrations: Interconnection between Self-Organization, Fluorescent and Physicochemical Properties, and Action on Hydrobionts.

As shown by fluorescence monitoring of dissolved organic matter, amino acid L-Trp can be present in natural water. The consequences of the presence of L-Trp at low concentrations in surface water systems are not yet established for hydrobionts. Studying the physicochemical patterns, as well as their relationships to the bioeffects of L-Trp solutions in the low concentration range, can provide new and important information regarding the unknown effects of L-Trp. The self-organization, physicochemical properties, fluorescence, UV absorption, and action of L-Trp solutions on Paramecium caudatum infusoria, Chlorella vulgaris algae were studied in the calculated concentrations range of 1 × 10-20-1 × 10-2 mol/L. The relationship between these phenomena was established using the certified procedures for monitoring the toxicity of natural water and wastewater. It was shown for the first time that aqueous solutions of L-Trp are dispersed systems in which the dispersed phase (nanoassociates) undergoes a rearrangement with dilution, accompanied by coherent changes in the nanoassociates' parameters and the properties of systems. The non-monotonic concentration dependence of fluorescence intensity (λex at 225 nm, λem at 340 nm) is in good agreement with the data on the nanoassociates' parameters, as well as with both the physicochemical properties of the systems and their bioassay results.

The role of Sp1 in the detection and elimination of cells with persistent DNA strand breaks.

Maintenance of genome stability suppresses cancer and other human diseases and is critical for organism survival. Inevitably, during a life span, multiple DNA lesions can arise due to the inherent instability of DNA molecules or due to endogenous or exogenous DNA damaging factors. To avoid malignant transformation of cells with damaged DNA, multiple mechanisms have evolved to repair DNA or to detect and eradicate cells accumulating unrepaired DNA damage. In this review, we discuss recent findings on the role of Sp1 (specificity factor 1) in the detection and elimination of cells accumulating persistent DNA strand breaks. We also discuss how this mechanism may contribute to the maintenance of physiological populations of healthy cells in an organism, thus preventing cancer formation, and the possible application of these findings in cancer therapy.

Sp1-independent downregulation of NHEJ in response to BER deficiency.

Base excision repair (BER) is the major repair pathway that removes DNA single strand breaks (SSBs) arising spontaneously due to the inherent instability of DNA. Unrepaired SSBs promote cell-cycle delay, which facilitates DNA repair prior to replication. On the other hand, in response to persistent DNA strand breaks, ATM-dependent degradation of transcription factor Sp1 leads to downregulation of BER genes expression, further accumulation of SSBs and renders cells susceptible to elimination via apoptosis. In contrast, many cancer cells are not able to block replication and to downregulate the expression of Sp1 in response to DNA damage. However, knockdown of BER in cancer cells leads to the accumulation of DNA double strand breaks (DSBs), suggesting deficiency in non-homologous end joining (NHEJ) repair of DSBs. Here we investigated whether DNA repair deficiency caused by knockdown of the XRCC1 gene expression in proliferating cells results in downregulation of NHEJ genes expression. We find that knockdown of the XRCC1 gene expression does not cause degradation of Sp1, but leads to downregulation of Lig4/XRCC4 and Ku70/80 at the transcription and protein levels. We thus propose the existence of Sp1-independent backup mechanism that in response to BER deficiency downregulates NHEJ in proliferating cells.

2.3THz radiation: Absence of genotoxicity/mutagenicity in Escherichia coli and Salmonella typhimurium.

The mutagenicity and genotoxicity in bacteria of 2.3THz radiation (THz) produced by a free-electron laser (NovoFEL) were evaluated; exposures were 5, 10, or 15min at average power 1.4W/cm(2). Two Ames mutagenicity test strains of Salmonella typhimurium, TA98 and TA102, were used. For the genotoxicity test, we measured SOS induction in Escherichia coli PQ37. No significant differences were found between exposed and control cells, indicating that THz radiation is neither mutagenic nor genotoxic under these conditions. Nevertheless, a small increase in total cell number of S. typhimurium after 15min exposure, and an increase in ß-galactosidase and alkaline phosphatase activities in E.coli PQ37, were observed, indicating some effect of THz radiation on cell metabolism. We also examined the combined effect of 4-NQO (8µM; positive control) and THz exposure (5min) on genotoxicity in E.coli PQ37. Unexpectedly, THz radiation decreased 4-NQO genotoxicity.

Renin-Angiotensin-Aldosterone Signaling Inhibitors-Losartan, Enalapril, and Cardosten-Prevent Infarction-induced Heart Failure Development in Rats.

CONTEXT: The activation of the renin-angiotensin-aldosterone system (RAAS) plays an important role in the pathophysiology of congestive heart failure, which is the reason that angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin 2 receptor blockers (ARBs) have become established therapies for heart failure. However, it is still not known whether preventive treatment with losartan or enalapril can reduce symptoms of infarction-induced heart failure. Ultra-low dose (ULD) drug therapy is thought to exert specific activity, with a lower chance of side effects. OBJECTIVES • The research team had hypothesized that preventive treatment with inhibitors of RAAS signaling-losartan, enalapril, and a preparation of a ULD antibody (ie, cardosten), which target the angiotensin type 1 (AT1) receptor-might alleviate pathological hypertrophy and/or functional decline in infarction-induced heart failure. METHODS: The research team treated male Wistar rats orally for 30 d with 20 mg/kg of losartan, 10 mg/kg enalapril, 5 or 7.5 mL/kg of cardosten, or a control solution, started 1 d prior to permanent coronary occlusion. A sham-operated group functioned as a second control group. SETTINGS: The study was conducted at the Department of Biochemistry of the Faculty of Medicine at the University of Szeged in Szeged, Hungary, in cooperation with the Pharmahungary Group, also in Szeged, Hungary, and with OOO "NPF" Materia Medica Holding Ltd in Moscow, Russia. OUTCOME MEASURES: To determine cardiac functional parameters in vivo, the research team inserted a catheter into the left ventricle of the rats and measured the parameters of ventricular pressure, and cardiac output was determined by thermodilution. Morphological parameters were measured after heart isolation in transverse sections by a digital caliper. RESULTS: A total of 30 d after permanent coronary ligation, both losartan and enalapril, significantly decreased mean arterial blood pressure (MABP), attenuated the development of the left-ventricular anterior-wall and septum hypertrophy, and reduced scar thickness compared with the vehicle control group. The deterioration of cardiac output and the increase in total peripheral resistance (TPR) due to coronary ligation were significantly inhibited by both losartan and enalapril. The effects of cardosten were comparable with those of losartan and enalapril on cardiac morphology, left ventricular function, and TPR; however, it did not influence MABP. Moreover, in contrast to losartan and enalapril, cardosten did not decrease the rate of survival. CONCLUSIONS: The study was the first to have demonstrated that preventive treatment with losartan, enalapril, or cardosten can attenuate pathological hypertrophy in infarction-induced heart failure in rats.

Adjunctive use of interferon γ inducer for treatment of patients with schizophrenia.

OBJECTIVE: The present paper is devoted to evaluation of clinical and immunomodulatory effect of ultra-high dilutions of antibodies to human interferon γ, included in the complex therapy of patients with schizophrenia. Materials and methods The study was carried out at the Mental Health Research Institute, Tomsk, Russian Federation. This double-blind, placebo-controlled randomised in parallel-group study enrolled 40 patients. As a part of complex therapy, patients from the main group (n=20) received anaferon, a drug containing ultra-high dilutions of affinity-purified antibodies to human interferon γ as the active pharmaceutical ingredient; patients from the comparative group (n=20) received placebo. Duration of the therapy was 30±5 days. Assessment of severity of symptoms and changes in them were made using clinical scales: Positive and Negative Syndrome Scale, Clinical Global Impression, Abnormal Involuntary Movements Scale. Spontaneous and phytohemagglutinin-induced production of interferon γ by immunocompetent cells in supernatants of 48 h whole blood culture of patients was measured by enzyme-linked immunosorbent assay (ELISA) method. RESULTS: The reduction of interferon-producing potential by immunocompetent cells in comparison with reference normal value was shown in total group of patients (n=40) before combined therapy. During the treatment, increase of spontaneous interferon γ production and favourable changes in psychopathological symptoms as compared with placebo were shown in subjects receiving anaferon. It was found that favourable changes in clinical symptoms assessed using clinical scales with a high degree of confidence correlated with high level of spontaneous interferon γ production. CONCLUSION: Anaferon as a part of complex therapy of patients with schizophrenia contributes to enhancement of its efficacy acting via mechanism of psychoimmunomodulation.

Activity of ultra-low doses of antibodies to gamma-interferon against lethal influenza A(H1N1)2009 virus infection in mice.

BACKGROUND: The influenza A virus is a highly infective agent that causes acute pulmonary diseases. In serious cases, it causes pneumonia which is particularly fatal in patients with cardiopulmonary diseases, obesity, young children and elderly people. The present study shows a protective effect of ultra-low doses of purified antibodies to gamma-interferon (Anaferon for children®, AC®) against lethal influenza virus infection caused by pandemic influenza virus A(H1N1) in mice. METHODS: Balb/c mice were infected with mouse-adapted pandemic influenza virus A/California/07/09 (H1N1)v. Mortality, weight loss, infectious titer of the virus in lungs and lung morphology were monitored in the groups of AC®-, oseltamivir- and placebo-treated animals. RESULTS: The protective action of AC® was demonstrated by prolongation of life of the infected animals, reduction of infectious titer of the virus in the lung tissue, normalization of weight dynamics in the course of disease, decrease in mortality of treated animals compared to a placebo control and normalization of lung tissue structure. The protective activity of AC® was similar to that of the reference compound oseltamivir. Combination of AC® with oseltamivir resulted in a higher protective effect comparing to oseltamivir alone. CONCLUSION: Based on the results obtained, AC® should be considered as an important part of anti-influenza prophylaxis and therapy, in particular in severe cases of the disease.

Stability and reactivity of free radicals: a physicochemical perspective with biological implications.

Several factors control the reactivity of radicals and can provide the strategies to convert highly reactive species into more persistent species that are easier to detect in an experiment. A reaction can only proceed if sufficient mobility and thermodynamic driving force are provided and the reaction is allowed by steric considerations and by electronic states of the reagents and products. A violation of at least one of these conditions can make the radical relatively stable. In certain cases, these factors occur naturally, in other situations, they can be purposefully manipulated to reduce the reactivity of highly reactive radicals, prolonging their lifetime and increasing their concentration. The discussed examples cover a vast range of lifetimes, from 10(-9) seconds to 10(9) years, at concentration levels down to 10(3) radicals per sample (10(-18) M), and stress that stability and reactivity are not independent notions and are the two sides of the same coin.

Kinetic magnetic-field effect involving the small biologically relevant inorganic radicals NO and O2(·-).

Oxidation of dihydrorhodamine 123 (DHR) to rhodamine 123 (RH) by oxoperoxonitrite (ONOO(-)), formed through recombination of NO and O(2)(·-) radicals resulting from thermal decomposition of 3-morpholinosydnonimine (SIN-1) in buffered aerated aqueous solution at pH 7.6, represents a kinetic model system of the reactivity of NO and O(2)(·-) in biochemical systems. A magnetic-field effect (MFE) on the yield of RH detected in this system is explored in the full range of fields between 0 and 18 T. It is found to increase in a nearly linear fashion up to a value of 5.5±1.6 % at 18 T and 23 °C (3.1±0.7 % at 40 °C). A theoretical framework to analyze the MFE in terms of the magnetic-field-enhanced recombination rate constant k(rec) of NO and O(2)(·-) due to magnetic mixing of T(0) and S spin states of the radical pair by the Δg mechanism is developed, including estimation of magnetic properties (g tensor and spin relaxation times) of NO and O(2)(·-) in aqueous solution, and calculation of the MFE on k(rec) using the theoretical formalism of Gorelik at al. The factor with which the MFE on k(rec) is translated to the MFE on the yield of ONOO(-) and RH is derived for various kinetic scenarios representing possible sink channels for NO and O(2)(·-). With reasonable assumptions for the values of some unknown kinetic parameters, the theoretical predictions account well for the observed MFE.

Sildenafil and a compound stimulating endothelial NO synthase modify sexual incentive motivation and copulatory behavior in male Wistar and Fisher 344 rats.

INTRODUCTION: Earlier studies have shown that sildenafil may modify some aspects of male rat sexual behavior and sexual incentive motivation. Stimulation of endothelial nitric oxide synthase (eNOS) has also been reported to affect sexual motivation in old rats. AIM: To determine the effects of sildenafil and a compound stimulating eNOS on copulatory behavior and sexual incentive motivation in young adult Fisher 344 and Wistar male rats. METHODS: The rats were selected for a low intromission ratio, and then treated with Impaza (stimulator of eNOS), sildenafil, or Impaza + sildenafil for 28 days. Tests for copulatory behavior and sexual incentive motivation were performed before the beginning of treatment and at days 7, 14, and 28 of treatment. MAIN OUTCOME MEASURES: Standard parameters of copulatory behavior and sexual incentive motivation. Measurements of penis length at mount, intromission, and ejaculation. RESULTS: The Fisher 344 rats displayed a higher level of sexual incentive motivation than the Wistar rats, while the copulatory behavior was similar in both strains. Impaza and sildenafil enhanced the sexual incentive motivation after 28 days of treatment in the Wistar rats, but failed to do so in the Fisher 344 rats. The copulatory behavior was unaffected in the Wistar strain, while the Fisher 344 males had an enhanced intromission ratio after treatment with Impaza and sildenafil for 28 days. CONCLUSIONS: The nitric oxide-guanylyl cyclase pathway seems to be of importance for sexual incentive motivation in animals with a modest baseline level. The different drug effects in the Wistar and Fisher 344 rats can be attributed to baseline differences. The importance of eNOS for sexual functions should not be overlooked.

Antibodies to S100 proteins have anxiolytic-like activity at ultra-low doses in the adult rat.

S100 proteins are small calcium-binding proteins interacting with numerous intra- and extra cellular targets involved in diverse physiological functions. In particular, S100 proteins may be involved in the regulation of anxiety-related behaviour. In the present study, the effects of affinity-purified antibodies to S100 proteins administered orally at ultra-low doses were evaluated in pre-clinical tests for anxiolytic-like activity in the adult rat. In the Vogel conflict test in the rat, antibodies to S100 proteins increased punished drinking (anti-conflict effect) at 5 and 7.5 mL kg(-1), but not at 2.5 or 10 mL kg(-1). Antibodies to S100 proteins increased the percentage of entries into the open arms of an elevated plus-maze at 10 mL kg(-1), but not at lower doses. Taken together, these results indicate the presence of anxiolytic-like activity for antibodies to S100 proteins over the dose range 5-10 mL kg(-1) in the adult rat.

19F NMR measurements of NO production in hypertensive ISIAH and OXYS rats.

Recently we demonstrated the principal possibility of application of 19F NMR spin-trapping technique for in vivo *NO detection [Free Radic. Biol. Med. 36 (2004) 248]. In the present study, we employed this method to elucidate the significance of *NO availability in animal models of hypertension. In vivo *NO-induced conversion of the hydroxylamine of the fluorinated nitronyl nitroxide (HNN) to the hydroxylamine of the iminonitroxide (HIN) in hypertensive ISIAH and OXYS rat strains and normotensive Wistar rat strain was measured. Significantly lower HIN/HNN ratios were measured in the blood of the hypertensive rats. The NMR data were found to positively correlate with the levels of nitrite/nitrate evaluated by Griess method and negatively correlate with the blood pressure. In comparison with other traditionally used methods 19F NMR spectroscopy allows in vivo evaluation of *NO production and provides the basis for in vivo *NO imaging.