Exploring the immune-inflammatory mechanism of Maxing Shigan Decoction in treating influenza virus A-induced pneumonia based on an integrated strategy of single-cell transcriptomics and systems biology.

BACKGROUND: Influenza is an acute respiratory infection caused by influenza virus. Maxing Shigan Decoction (MXSGD) is a commonly used traditional Chinese medicine prescription for the prevention and treatment of influenza. However, its mechanism remains unclear. METHOD: The mice model of influenza A virus pneumonia was established by nasal inoculation. After 3 days of intervention, the lung index was calculated, and the pathological changes of lung tissue were detected by HE staining. Firstly, transcriptomics technology was used to analyze the differential genes and important pathways in mouse lung tissue regulated by MXSGD. Then, real-time fluorescent quantitative PCR (RT-PCR) was used to verify the changes in mRNA expression in lung tissues. Finally, intestinal microbiome and intestinal metabolomics were performed to explore the effect of MXSGD on gut microbiota. RESULTS: The lung inflammatory cell infiltration in the MXSGD group was significantly reduced (p < 0.05). The results of bioinformatics analysis for transcriptomics results show that these genes are mainly involved in inflammatory factors and inflammation-related signal pathways mediated inflammation biological modules, etc. Intestinal microbiome showed that the intestinal flora Actinobacteriota level and Desulfobacterota level increased in MXSGD group, while Planctomycetota in MXSGD group decreased. Metabolites were mainly involved in primary bile acid biosynthesis, thiamine metabolism, etc. This suggests that MXSGD has a microbial-gut-lung axis regulation effect on mice with influenza A virus pneumonia. CONCLUSION: MXSGD may play an anti-inflammatory and immunoregulatory role by regulating intestinal microbiome and intestinal metabolic small molecules, and ultimately play a role in the treatment of influenza A virus pneumonia.

Antiviral activity of Epimedium koreanum Nakai water extract against influenza viruses.

Epimedium koreanum Nakai (EKN) is a popular plant in Korean and Chinese medicine for treating a variety of ailments. The aqueous extract of EKN has a significant inhibitory impact on influenza A virus (IAV) infection by directly blocking viral attachment and having a virucidal effect, according to this study. Using fluorescent microscopy and fluorescence-activated cell sorting (FACS) with a green fluorescent protein (GFP)-tagged Influenza A/PR/8/34 virus, we examined the effect of EKN on viral infection. By viral infection, EKN strongly suppresses GFP expression, and at a dosage of 100 µg/mL, EKN decreased GFP expression by up to 90% of the untreated infected control. Immunofluorescence and Western blot analyses against influenza viral proteins revealed that EKN decreased influenza viral protein expression in a dose-dependent manner. EKN inhibited the H1N1 influenza virus's hemagglutinin (HA) and neuraminidase (NA), preventing viral attachment to cells. Furthermore, EKN had a virucidal impact and inhibited the cytopathic effects of H1N1, H3N2 and influenza B virus infection. Finally, our findings show that EKN has the potential to be developed as a natural viral inhibitor against influenza virus infection.

Carvacrol inhibits the excessive immune response induced by influenza virus A via suppressing viral replication and TLR/RLR pattern recognition.

ETHNOPHARMACOLOGICAL RELEVANCE: Carvacrol, a monoterpene phenol from Mosla chinensis Maxim, which is a commonly Chinese herbal medicine. The most important pharmacology of it is dispelling exogenous evils by increasing perspiration. And it is the gentleman medicine in the Chinese herbal compound prescription of Xin-Jia-Xiang-Ru-Yin, mainly for the treatment of summer colds with dampness including influenza virus A infection. AIM OF THE STUDY: Our preliminary study verified that the Xin-Jia-Xiang-Ru-Yin could inhibit acute lung injury of mice with influenza virus A infection. And there have been some reports implicating the high antimicrobial activity of carvacrol for a wide range of product preservation, but little research including the effects of it on viral infection. The aim of this study was to reveal the antiviral effects of carvacrol, the main constituent in Mosla chinensis Maxim. MATERIALS AND METHODS: Initially, C57BL/6 mice were grouped and intranasally administered FM1 virus to construct viral infection models. After treatment with ribavirin and carvacrol for 5 days, all mice were euthanized, and specimens were immediately obtained. Histology, flow cytometry and Meso Scale Discovery (MSD) analysis were used to analyze pathological changes in lung tissue, the expression levels of cytokines and the differentiation and proportion of CD4+ T cells subsets, while Western blot and qRT-PCR were used to detect the expression of related proteins and mRNA. RESULTS: Carvacrol attenuated lung tissue damage, the proportions of Th1, Th2, Th17 and Treg in CD4+ T cells and the relative proportions of Th1/Th2 and Th17/Treg cells. Carvacrol inhibited the expression of inflammation-associated cytokines including IFN-γ, IL-2, IL-4, IL-5, IL-12 and TNF-ɑ, IL-1, IL-10, IL-6. Decreased levels of TLR7, MyD88, IRAK4, TRAK6, NF-κB, RIG-I, IPS-I and IRF mRNA in carvacrol-treated mice were observed comparing to the mice in VC group. Further, the total expression of RIG-I, MyD88 and NF-κB proteins had increased significantly in the VC group but reduced obviously in the group treated with ribavirin or carvacrol. CONCLUSIONS: These results indicate that carvacrol is a potential alternative treatment for the excessive immune response induced by influenza virus A infection, the cold-fighting effect of Mosla chinensis Maxim may depend on the anti-virus of carvacrol.

Theacrine and strictinin, two major ingredients for the anti-influenza activity of Yunnan Kucha tea.

ETHNOPHARMACOLOGICAL RELEVANCE: Kucha tea plant (Camellia assamica var. kucha Chang et Wang) is regarded as a mutant variety of wild Pu'er tea plant found in few mountain areas of Yunnan, China. Its fresh young leaves and shoots are picked by the indigenous aborigines in these local areas to prepare an herbal tea for the treatment of common cold empirically. MATERIALS AND METHODS: Two extra compounds of relative abundance were detected in Kucha tea in comparison with Pu'er tea, and their chemical structures were identified as chlorogenic acid and theacrine. These two compounds as well as two major compounds, strictinin and caffeine, in Kucha tea were evaluated for their cytotoxicity and inhibitory effects on human influenza virus A/Puerto Rico/8/34 by analyzing viral protein expression and progeny production. RESULTS: No or low cytotoxicity was detected for the four Kucha compounds when their concentrations were below 100 µM. Expression of viral NS1 protein was significantly inhibited by chlorogenic acid, theacrine or strictinin, but not caffeine at a concentration of 100 µM. The relative inhibitory potency was detected as chlorogenic acid < theacrine < strictinin, and both theacrine and strictinin displayed significant inhibition at a concentration of 50 µM. According to a plaque assay, viral progeny production was significantly reduced by theacrine or strictinin, but not by chlorogenic acid or caffeine under the same concentration of 100 µM. CONCLUSION: It is suggested that theacrine and strictinin are two major ingredients responsible for the anti-influenza activity of Yunnan Kucha tea traditionally used for the treatment of common cold.

Isofraxidin ameliorated influenza viral inflammation in rodents via inhibiting platelet aggregation.

Platelets have been proved to exacerbate influenza infection and its complications. Inhibition of platelet activation may be a feasible method for preventing severe infection and secondary acute lung injury (ALI). Isofraxidin (IFD) is a natural coumarin isolated from the plants Sarcandra glabra and Siberian ginseng, and exerts anticancer, antioxidant and antiinflammatory effects. In the present study, we examined the therapeutic effects of IFD in ADP- or arachidonic acid (AA)-induced platelet aggregation model and in influenza A virus (IAV)-induced ALI mouse model. The results showed that IFD significantly inhibited platelet aggregation induced by ADP and AA in vitro in a concentration-dependent manner as well as the release of soluble P-selectin and platelet factor 4. Moreover, IFD significantly relieved IAV-induced lung inflammation, reduced the expressions of platelet activation biomarkers (P-selectin and CD61), decreased the serum levels of TNF-α, IL-1ß, IL-6 and MIP-2, suppressed peripheral platelet aggregation and prolonged the survival time of infected mice. The western blotting results also demonstrated that IFD reduced the phosphorylation levels of PI3K, AKT and p38 in the activated platelets stimulated by ADP and IAV infection. But IFD did not have any effects on IAV replication. It indicated that IFD ameliorated IAV-induced severe lung damage and lethal infection by suppressing platelet aggregation via regulating PI3K/AKT and MAPK pathways.

Does Vitamin D Deficiency Affect the Immunogenic Responses to Influenza Vaccination? A Systematic Review and Meta-Analysis.

Influenza virus infection is a major global public health problem, and the efficacy of influenza vaccination is not satisfactory. Vitamin D is involved in many immune-mediated inflammatory processes. The impact of vitamin D levels on the immunogenic response to influenza vaccination is not clear. We performed a comprehensive literature search and systematic review of studies that investigated vitamin D and influenza vaccination. Data pertaining to study population, vaccine components, vitamin D levels, and immunogenic response were analyzed. Nine studies, with a combined study population of 2367 patients, were included in the systematic review. Four studies were included in the meta-analysis to investigate the influence of vitamin D deficiency (VDD) on the seroprotection (SP) rates and seroconversion (SC) rates following influenza vaccination. We found no significant association between vitamin D level and the immunogenic response to influenza vaccination. However, strain-specific differences may exist. We observed lower SP rates of influenza A virus subtype H3N2 (A/H3N2) and B strain in VDD patients than patients with normal vitamin D levels (A/H3N2: 71.8% vs. 80.1%, odds ratio (OR): 0.63, 95% confidence interval (CI): 0.43-0.91, p = 0.01; B strain: 69.6% vs. 76.4%, OR: 0.68, 95% CI: 0.5-0.93, p = 0.01). However, the SP rates of A/H1N1 and SC rates of all three strains were not significantly different in VDD and control groups. In conclusion, no association was observed between VDD and immunogenic response to influenza vaccination.

Antibody and cell-mediated immune response to whole virion and split virion influenza vaccine in patients with inflammatory bowel disease on maintenance immunosuppressive and biological therapy.

OBJECTIVE: Influenza vaccination is recommended for inflammatory bowel disease (IBD) patients on immunosuppressive therapy. The objective was to evaluate the antibody and cell-mediated immune response to the split and whole virion influenza vaccine in patients with IBD treated with anti-TNF-α and immunosuppressive therapy. PATIENTS AND METHODS: One hundred and fifty-six immunocompromised IBD patients were vaccinated. Fifty-three patients (control group) refused vaccination. Split virion vaccine and whole virion vaccine were used. Serum samples were obtained for pre- and postimmunization antibody titers to influenza vaccine (A/California/7/2009 [H1N1], A/Victoria/361/2011 [H3N2], B/Wisconsin/1/2010-like B/Hubei-Wujiagang/158/2009). Cell-mediated response was evaluated using an interferon (INF)-γ, interleukine (IL)-2 and tumor necrosis factor (TNF)-α ELISA. RESULTS: Postimmunization titers of both influenza subtypes increased significantly after the administration of split virion vaccines compared to the controls and to those who received whole virion vaccine. The antibody titers of Influenza B also increased significantly in patients immunized with split vaccine and treated with anti-TNF-α therapy. After influenza vaccination, the level of serum IL-2 significantly decreased. No serious side effects developed occurred after influenza vaccination, and the influenza-like symptoms did not differ significantly between vaccinated versus control patients. The relapse of the disease was observed in only 10% of the patients and was more common in vaccinated than in control subjects. CONCLUSION: Split virion vaccines seem to be more effective than whole virion vaccines. Measuring the antibody responses is worthwhile in patients treated with immunosuppressants to determine the efficacy of influenza vaccination.

[A research on the influence of two herbal concoctions on Toll-like receptor signal pathways of influenza virus induced pneumonia in mice].

OBJECTIVE: To investigate the effect of Shufeng Xuanfei and Jiebiao Qingli concoctions on Toll-like receptor (TLR) signal pathway of pneumonia infected with influenza virus in mice. METHODS: The pneumonia model was reproduced by nasal dropping of influenza virus A in mice. The mice were randomly divided into nine groups: normal group (C), model group (M), tamiflu group (D), Shufeng Xuanfei low-dose (SL), medium-dose (SM) and high-dose (SH) groups, Jiebiao Qingli low-dose (JL), medium-dose (JM) and high-dose (JH) groups, each n=12. Two hours after model-reproduction, the mice in C group and M group received distilled water by gavage. The mice in D group received 2.5 g×mL(-1)×d(-1) oseltamivir phosphate. Shufeng Xuanfei formula in doses of 3.76, 1.88, 0.94 g×kg(-1)×d(-1) were respectively administered to SH, SM and SL groups by gavage, Jiebiao Qingli formula in doses of 4.37, 2.18, 1.09 g×kg(-1)×d(-1) was given to JH, JM and JL groups by gavage, respectively. Each group was in equal dose of 0.2 mL daily over a 4-day period. Total RNA was extracted in each group. Then gene chips were used to screen these RNA samples. Some genes that were involved in TLR signal pathways were selected. These candidate genes were verified by real-time reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: TLR7, MYD88, CCL5, IFNB1, IL6, IL12a, NFKBIA and IKBKB were up-regulated in model group compared with control group. Compared with model group, down-regulated genes in medium-dose, low-dose Shufeng Xuanfei formula and medium-dose Jiebiao Qingli formula included TLR3, TLR7, MYD88, CCL5, IFNB1, IL6, IL12a, NFKBIA and IKBKB (log2 signal intensity of SM/M in medium-dose Shufeng Xuanfei formula group were -1.24, -2.02, -1.36, -1.95, -0.63, -1.33, -3.50, -1.33, -1.33, log2 signal intensity of SL/M in low-dose Shufeng Xuanfei group were -1.07, -2.43, -2.63, -2.30, -5.09, -3.19, -3.53, -1.95, -1.95, log2 signal intensity of JM/M in medium-dose Jiebiao Qingli formula group were -1.78, -0.55, -1.35, -1.47, -1.65, -2.03, -3.02, -1.57, -1.57, respectively). The results suggested that the effect of Shufeng Xuanfei formula was better than that of Jiebiao Qingli formula. By RT-PCR, compared with model group, low-dose, medium-dose and high-dose groups of Shufeng Xuanfei formula, medium-dose and high-dose groups of Jiebiao Qingli formula, and tamiflu group, significant decrease in TLR7, nuclear factor-ΚB (NF-ΚB), myeloid differential protein-88 (MyD88) mRNA expression were found. Medium-dose and low-dose Shufeng Xuanfei formula group (TLR7 mRNA: 3.6±0.3, 3.5±1.2 vs. 7.4±1.6, NF-ΚB mRNA: 1.1±0.2, 1.0±0.2 vs. 2.2±0.4; MyD88 mRNA: 1.4±0.4, 1.0±0.3 vs. 3.4±0.9, all P<0.01) and medium-dose Jiebiao Qingli formula group (TLR7 mRNA: 4.9±0.3 vs. 7.4±1.6, NF-ΚB mRNA: 1.3±0.7 vs. 2.2±0.4, MyD88 mRNA: 1.6±0.8 vs. 3.4±0.9, P<0.05 or P<0.01) were shown statistically significant decreases compared with the model group. CONCLUSIONS: Medium-dose and low-dose Shufeng Xuanfei formula and medium-dose Jiebiao Qingli formula can inhibit the inflammatory reaction induced by influenza virus by down-regulating the NF-ΚB through TLR signal pathways dependent on MyD88. The regulation of Shufeng Xuanfei formula in TLR signal pathways was superior to that of Jiebiao Qingli formula.

[Establishment of drug screening assay and pharmacodynamic evaluation method targeting influenza RNA polymerase].

Influenza virus RNA-dependent RNA polymerase (RdRP) is essential for replication and expression of influenza virus genome. Viral genomic sequences encoding RdRP are highly conservative, thus making it a potential anti-influenza drug target. A cell-based influenza RdRP inhibitor screening assay was established by a luciferase reporter system to analyze the activity of RdRP. Specificity study and statistic analysis showed that the screening assay is sensitive and reproducible.

Utility of Maoto in an influenza season where reduced effectiveness of oseltamivir was observed - a clinical, non-randomized study in children.

BACKGROUND: In an influenza season where reduced effectiveness of oseltamivir was observed, we investigated the effectiveness of Maoto for influenza infection in children. METHODS: Patients diagnosed with influenza by rapid diagnostic kit underwent treatment in one of the following groups: Maoto-treated group (group 1 (M)); oseltamivir-treated group (group 2 (O)); Maoto+oseltamivir-treated group (group 3 (M+O)); zanamivir-treated group (group 4 (Z)); and Maoto+zanamivir-treated group (group 5 (M+Z)). RESULTS: In influenza A patients who completed the study (n = 150), the mean duration of fever after administration (DFA) was significantly shorter in group 3 (M+O) (31.1 h, p < 0.01) and in group 4 (Z) (35.2 h, p < 0.05), as compared to group 2 (O) (56.0 h). Among these, in patients aged ≤5 years (n = 54), DFA was significantly shorter in group 1 (M) (33.2 h, p < 0.05) and in group 3 (M+O) (34.6 h, p < 0.05), as compared to group 2 (O) (61.4 h). In influenza B patients who completed the study (n = 70), no significant differences in DFA were observed among the groups. CONCLUSION: Maoto may be useful, particularly in cases of influenza with low sensitivity to oseltamivir and in patients aged ≤5 years for whom the use of zanamivir is difficult.

In vitro inhibition of influenza virus infection by a crude extract from Isatis indigotica root resulting in the prevention of viral attachment.

Isatis indigotica root (IIR) has been widely used as a Chinese medicinal herb to treat regular seasonal influenza over the long history of traditional Chinese medicinal practice. However, its inhibitory activities against influenza virus infections along with the associated mechanisms have not been investigated comprehensively. In this study, the chemical nature, mode of action and in vitro anti-influenza activities of a crude extract (G2) of IIR were characterized. The extract was found to inhibit different subtypes of human or avian influenza viruses at various magnitudes of activity (IC50 0.39­4.3 mg/ml) in vitro, including A/PR/8/34 (H1N1), A/FM/1/47 (H1N1), A/Aichi/2/68 (H3N2), seasonal influenza (A/Guangzhou/GIRD/02/09 H1N1, B/Guangzhou/GIRD/08/09), novel swine-originating influenza (A/Guangzhou/GIRD/07/09, H1N1), A/Duck/Guangdong/09 (H6N2), A/Duck/Guangdong/94 (H7N3) and A/Chicken/Guangdong/96 (H9N2), while G2 was inactive against respiratory syncytial virus (RSV), adenovirus 3 (ADV3), parainfluenza virus 3 (PIV3) and enterovirus 71 (EV71). An apparent virus titer reduction was detected when the influenza viruses were pretreated with G2, and it was also shown that G2 exhibited inhibitory effects on influenza virus hemagglutination. In addition, G2 played a role in the early stages of infection, which did not easily result in the emergence of virus drug resistance. Thus, G2 may affect the attachment of influenza virus by interfering with the viral particles, thereby preventing the binding of influenza virus to the host cell surface.

Oral administration of patchouli alcohol isolated from Pogostemonis Herba augments protection against influenza viral infection in mice.

Seasonal influenza A infection results in considerable morbidity and mortality. The limited efficacy of available therapeutic strategies stresses the need for development and study of new molecules against influenza virus (IFV). Patchouli alcohol (PA), the major chemical constituent of Pogostemonis Herba, was previously found to strongly inhibit influenza H1N1 replication in vitro. In the present study, the in vivo anti-IFV effect of PA was investigated. In a mouse model infected with lethal levels of FM1, oral administration of PA (20 mg/kg to 80 mg/kg) for 7 d post IFV infection significantly increased the survival rate and survival time. For IFV infection at nonlethal levels, the quantity of IFV in the lungs 5 d after infection was significantly reduced after PA (20 mg/kg to 80 mg/kg) administration. Anti-IFV IgA, IgM, and IgG titers in serum on day 6 were significantly higher in the PA-treated group than the IFV-control group. Anti-IFV immune response augmentation was further confirmed by the elevated production of CD3+, CD4+, and CD8+ T cell levels in blood. Furthermore, the levels of inflammatory cytokines, including TNF-alpha, IL-10 and IFN-gamma in serum of mice, were regulated. Lung inflammation was reduced significantly after PA administration, and the effect may be mediated, at least in part, by regulating the lung levels of inflammatory cytokines. Thus, oral administration of PA appears to be able to augment protection against IFV infection in mice via enhancement of host immune responses, and attenuation of systemic and pulmonary inflammatory responses.

Establishment of drug screening assay and pharmacodynamic evaluation method targeting influenza RNA polymerase / 药学学报

Influenza virus RNA-dependent RNA polymerase (RdRP) is essential for replication and expression of influenza virus genome. Viral genomic sequences encoding RdRP are highly conservative, thus making it a potential anti-influenza drug target. A cell-based influenza RdRP inhibitor screening assay was established by a luciferase reporter system to analyze the activity of RdRP. Specificity study and statistic analysis showed that the screening assay is sensitive and reproducible.

Avaliação do potencial genotóxico e mutagênico de um nosódio vivo obtido com o vírus Influenza A / Evaluation of the genotoxic and mutagenic potentials of a living nosode compounded with Influenza A virus

Introdução: O vírus da gripe tem sido responsável por doenças respiratórias contagiosas com altas taxas de mortalidade [1]. Algumas drogas tem sido usadas para tratar a gripe humana. No entanto, esses medicamentos causam muitos efeitos colaterais e induzem o aparecimento de cepas virais resistentes [2]. O impacto causado pelo vírus influenza tem motivado o desenvolvimento de novas abordagens para a prevenção e controle da influenza [3]. Portanto, um novo medicamento homeopático foi desenvolvido utilizando, como ponto de partida, o vírus influenza infecciosa [4]. Este pertence a um grupo chamado nosódios vivos [5]. No entanto, seus potenciais mutagênicos e genotóxicos, especialmente quando usado em diluições baixas, ainda não foram avaliados e é importante porque este bioterápico é preparado a partir de microorganismos vivos. Diferentes métodos podem ser usados para detectar efeitos mutagênicos e genotóxicos. Objetivos: Este estudo visa avaliar o potencial genotóxico e mutagênico do nosódio vivo do vírus influenza A, em diferentes potências homeopáticas.Metodologia: 1 ml de suspensão viral purificada foi diluída em 9 ml de água destilada estéril. Esta amostra foi submetida a 100 sucussões mecânicas (aproximadamente 3 Hz), usando o aparato Autic ® brasileira, originando a primeira diluição, chamada decimal (1x). 1 ml desta solução foi diluída em 9 ml de solvente e foi submetido a 100 sucussões, gerando o bioterápico 2x. Este procedimento foi repetido sucessivamente, de acordo com a Farmacopéia Homeopática Brasileira, para obter o bioterápico 30x [6]. Pela mesma técnica, a água foi preparada até a potência 30x, para ser utilizada como controle. Todas as amostras foram preparadas sob condições estéreis e assépticas, utilizando-se fluxo laminar, classe II, e foram armazenados em geladeira (8ºC). As amostras 1x, 6x, 12x, 18x 24x, 30x e 30x e água (controle do veículo) foram analisadas por: Induteste, avalia a capacidade dos agentes físicos ou químicos de promover a indução lisogênico como um reflexo dos danos nas moléculas de DNA em bactérias lisogênicas, e o teste de Ames, que utiliza linhagens indicadoras de Salmonella typhimurium, sensíveis a substâncias que podem induzir diferentes tipos de mutação. Resultados: Os resultados obtidos no Induteste não mostraram diminuição da fração de sobrevivência das bactérias utilizadas, e nenhum aumento na formação de indução lisogênica, em quaisquer potências testadas. O mesmo perfil foi obtido após o teste de Ames, com resultados semelhantes ao controle negativo. Conclusão: Conclui-se que nosódio vivo obtido com vírus Influenza A não é capaz de induzir danos no DNA de células procarióticas. Este resultado nos permite concluir que pacientes que usam este medicamento não tem efeitos colaterais relacionados com a mutagênese e genotoxicidade.(AU)

Background: The influenza virus has been responsible for contagious respiratory diseases with high mortality rates [1]. Some drugs have been used to treat human influenza. However, these drugs cause many common side effects and induce the appearance of resistant viral strains [2]. The impact caused by the influenza virus has motivated the development of new approaches for the prevention and control of influenza [3]. Therefore, a new homeopathic medicine was developed using, as a starting point, the infectious influenza virus [4]. This belongs to a group called living nosodes [5]. However, its mutagenic and genotoxic potentials, especially when used in low dilutions, has not yet been evaluated and it is important because this biotherapic is prepared from living microorganisms. Different methods can be used to detect mutagenic and genotoxicic effects. Aims: This study aims to evaluate the genotoxic and mutagenic potentials of influenza A living nosode at different homeopathic potencies. Methodology: 1 ml of purified viral suspension was diluted in 9 ml of sterile distilled water. This sample was submitted to 100 mechanical succussions (approximately 3 Hz), using Autic® Brazilian machine, originating the first dilution, named decimal (1x). 1 ml of this solution was diluted in 9 ml of solvent and was submitted to 100 sucussions, generating biotherapic 2x. This procedure was successively repeated, according to Brazilian Homeopathic Pharmacopoeia, to obtain the biotherapic 30x [6]. By the same technique, water vehicle was prepared until 30x potency to be used as control. All samples were prepared in sterile and under aseptic conditions, using laminar flow cabinet, class II, and were stored in the refrigerator (8ºC). The samples 1x, 6x, 12x, 18x, 24x and 30x and water 30x (vehicle control) were analysed by: the Inductest, which assesses the ability of physical or chemical agents to promote lysogenic induction as a reflection of damage in DNA molecules in lysogenic bacteria, and the Ames test, which uses indicator strains of Salmonella typhimurium, sensitive to substances that can induce different types of mutation. Results: The Inductest showed no decrease in the survival fraction of the bacteria used, and no increase in the formation of lysogenic induction, in any tested potency. The same profile was obtained after the Ames test, with similar results to negative control. Conclusion: We can conclude that this living nosode compounded with Influenza A virus is not able to induce DNA damage in prokaryotic cells. This result permits us to conclude that patients who use this medicine have no side effects related to mutagenesis and genotoxicity.(AU)

Respostas celulares e bioquímicas induzidas por bioterápicos preparados a partir de influenza A (H3N2) íntegro e influenza A (H3N2) inativado nas potências 12x e 30x na linhagem MDCK / Cellular and biochemical responses induced by Biotherapics prepared from intact influenza A (H3N2) and inactivated influenza A (H3N2) virus at 12x and 30x in the MDCK cells

Biotherapics are homeopathic remedies prepared from organic products that are chemically undefined and can be used for treatment of diseases like influenza. There are several classes of biotherapics and, among these, there are some called "living biotherapics" or "Roberto Costa?s Biotherapics". This study aimed to compare the cellular and biochemical effects of biotherapics prepared from intact influenza virus diluted in water and the one obtained from the same viral sample inactivated by ethanol 70% (v / v), both in the potencies of 12x and 30x. Transmission electron microscopy (TEM) analyses were performed on both preparations to assess the integrity of viral particles, which showed that ethanol 70% (v/v) induced a complete denaturation of viral particles. In contrast, the integrity of virus particles was preserved when water was used as the biotherapic solvent. Cellular and biochemical alterations induced by the preparations on MDCK cells were analyzed and compared with those induced by respective controls (water 30x-treated and untreated cells). Cellular viability analyzed by MTT method showed statistically significant differences (p <0.05) in MDCK cells treated with intact biotherapic for 5 (3 stimuli) and 30 (18 stimuli) days in comparison with untreated control. TEM analysis did not show significant cellular changes when the different experimental groups were compared. The enzymatic activity of phosphofructokinase 1 (PFK), an important enzyme in the glycolytic pathway, presented a statistically significant increase (p <0.05) after 30 days of treatment when compared to control groups. The results obtained suggest that inactivation of viral sample with ethanol 70% induces lysis and disruption of viral particles. In addition, preliminary results indicated that treatment with intact biotherapic seems to induce higher variations on MDCK cells responses when compared to inactivated-biotherapic-treated cells. Further analyses are ongoing, including scanning electron microscopy and quantification of the number of mitosis, in order to elucidate the mechanisms involved with biochemical and cellular responses induced by theses biotherapics.(AU)

[Influence of four parts of forming of Yiqi Qingwen Jidu Heji (YQQWJDHJ) to lung inflammatory cytokines of model rats infected with influenza virus FM1].

OBJECTIVE: To observe the influence of the 4 parts of forming of Yiqi Qingwen Jidu Heji(YQQWJDHJ) to lung inflammatory cytokines of the model rats infected with influenza virus dynamically, and to discuss the mechanism of 4 parts of forming to anti-influenza immune injury and restoration. METHOD: At the different stages of infection with the model rats infected by FM1 influenza, expression in lung of TNF-alpha, IL-6, IL-1, IL-10 and IFN-gamma was detected after the intervention of 4 parts of forming using ELISA method. RESULT: The expression of TNF-alpha, IL-6, IL-1 and IFN-gamma of model rats infected by FM1 were higher than the control group, the expression of IL-10 did not change. The expression of TNF-alpha was significantly reduced in 3 to 5 days after infection. By the method of relieving superficies with acrid-cold, clearing away heat and poison and replenishing Qi, the lung expression of IFN-gamma was significantly increased in the stage after infection. The method of relieving superficies with acrid-warm significantly reduced lung expression of IL-6 after infection in 1 to 3 days and on the 7th day, decreased the expression of IL-1 in 3 to 7 days, increased IFN-gamma expression on the 3rd day and the 7th day, and significantly increased the expression of IL-10 on the 1st day and in 5 to 7 days. The method of relieving superficies with acrid-cold reduced the expresssion of IL-6 after infection, and significantly increased the expression of IL-10. It could increase the expression of IL-1 after infection on the 3rd day, but reduced IL-1 expression after infection 7 days. The method of clearing away heat and poison reduced lung IL- 6 expression after infection in 3 to 7 days significantly, decreased the expression of IL-1 in 5 to 7 days, also increased the lung expression of IL-10 in 1 to 5 days significantly. The method of replenishing Qi significantly reduced the expression of IL-6 after infection on the 1st day and in 5 to 7 days, decreased the expression of IL-1 in 3 to 7 days, also significantly increased the lung IL-10 on the 5th day after infection. CONCLUSION: The method of clearing away heat and poison and replenishing Qi could be against the lung immune inflammatory damage and repair damage. The method of relieving superficies with acrid-warm demonstrated some immunity against lung injury on the 3rd day after infection and the method of relieving superficies with acrid-cold demonstrated some immunity against lung injury on the 5th days after infection.

Influence of four parts of forming of Yiqi Qingwen Jidu Heji (YQQWJDHJ) to lung inflammatory cytokines of model rats infected with influenza virus FM1 / 中国中药杂志

<p><b>OBJECTIVE</b>To observe the influence of the 4 parts of forming of Yiqi Qingwen Jidu Heji(YQQWJDHJ) to lung inflammatory cytokines of the model rats infected with influenza virus dynamically, and to discuss the mechanism of 4 parts of forming to anti-influenza immune injury and restoration.</p><p><b>METHOD</b>At the different stages of infection with the model rats infected by FM1 influenza, expression in lung of TNF-alpha, IL-6, IL-1, IL-10 and IFN-gamma was detected after the intervention of 4 parts of forming using ELISA method.</p><p><b>RESULT</b>The expression of TNF-alpha, IL-6, IL-1 and IFN-gamma of model rats infected by FM1 were higher than the control group, the expression of IL-10 did not change. The expression of TNF-alpha was significantly reduced in 3 to 5 days after infection. By the method of relieving superficies with acrid-cold, clearing away heat and poison and replenishing Qi, the lung expression of IFN-gamma was significantly increased in the stage after infection. The method of relieving superficies with acrid-warm significantly reduced lung expression of IL-6 after infection in 1 to 3 days and on the 7th day, decreased the expression of IL-1 in 3 to 7 days, increased IFN-gamma expression on the 3rd day and the 7th day, and significantly increased the expression of IL-10 on the 1st day and in 5 to 7 days. The method of relieving superficies with acrid-cold reduced the expresssion of IL-6 after infection, and significantly increased the expression of IL-10. It could increase the expression of IL-1 after infection on the 3rd day, but reduced IL-1 expression after infection 7 days. The method of clearing away heat and poison reduced lung IL- 6 expression after infection in 3 to 7 days significantly, decreased the expression of IL-1 in 5 to 7 days, also increased the lung expression of IL-10 in 1 to 5 days significantly. The method of replenishing Qi significantly reduced the expression of IL-6 after infection on the 1st day and in 5 to 7 days, decreased the expression of IL-1 in 3 to 7 days, also significantly increased the lung IL-10 on the 5th day after infection.</p><p><b>CONCLUSION</b>The method of clearing away heat and poison and replenishing Qi could be against the lung immune inflammatory damage and repair damage. The method of relieving superficies with acrid-warm demonstrated some immunity against lung injury on the 3rd day after infection and the method of relieving superficies with acrid-cold demonstrated some immunity against lung injury on the 5th days after infection.</p>

[Effects of Yinqiao detoxifcation oral liquid on NK cell viability and serum concentration of IFN-gamma in SCID mouse infected by influenza virus FM1].

OBJECTIVE: To explore the dynamic effects of Yinqiao detoxifcation oral liquid (YQD) on NK cell lysis in spleen and serum concentration of IFN-gamma in SCID mouse infected by influenza A virus FM1. METHOD: The mice were divided into six different groups randomly. Except normal control, other mice were intranasally instilled with 15 TCID of virus. Three dosage groups of YQD (5.0, 10.0, 20.0 g x kg(-1)) were respectively fed with YQD. Positive control group was administrated orally with 0.07 g x kg(-1) of ribavirin. Normal control and model group were fed with physiological saline. After 1, 3, 5 and 7 days' infection, spleens and serum were collected. Then the NK cell lysis was detected by LDH release kit and the concentration of IFN-gamma was examined by ELISA assay. RESULT: Contents of IFN-gamma reached to peak value on the 3th day and until the 5th day. Later, the level of IFN-gamma returned to normal level. The variation tendency of activity of NK cell in spleen was according with that of IFN-gamma. But it reached the maximum value until the 5th day. The activity of NK cell lysis in three groups of YQD was well above that i n model group. In addition, therapeutic action of both 10.0 g x kg(-1) and 20.0 g x kg(-1) of YQD treatment groups was better than that of 5.0 g x kg(-1). CONCLUSION: The data showed that serum concentration of IFN-gamma and NK cell lysis were improved by YQD at different time, which was demonstrated YQD could perform well in immune system.

Effects of Yinqiao detoxifcation oral liquid on NK cell viability and serum concentration of IFN-gamma in SCID mouse infected by influenza virus FM1 / 中国中药杂志

<p><b>OBJECTIVE</b>To explore the dynamic effects of Yinqiao detoxifcation oral liquid (YQD) on NK cell lysis in spleen and serum concentration of IFN-gamma in SCID mouse infected by influenza A virus FM1.</p><p><b>METHOD</b>The mice were divided into six different groups randomly. Except normal control, other mice were intranasally instilled with 15 TCID of virus. Three dosage groups of YQD (5.0, 10.0, 20.0 g x kg(-1)) were respectively fed with YQD. Positive control group was administrated orally with 0.07 g x kg(-1) of ribavirin. Normal control and model group were fed with physiological saline. After 1, 3, 5 and 7 days' infection, spleens and serum were collected. Then the NK cell lysis was detected by LDH release kit and the concentration of IFN-gamma was examined by ELISA assay.</p><p><b>RESULT</b>Contents of IFN-gamma reached to peak value on the 3th day and until the 5th day. Later, the level of IFN-gamma returned to normal level. The variation tendency of activity of NK cell in spleen was according with that of IFN-gamma. But it reached the maximum value until the 5th day. The activity of NK cell lysis in three groups of YQD was well above that i n model group. In addition, therapeutic action of both 10.0 g x kg(-1) and 20.0 g x kg(-1) of YQD treatment groups was better than that of 5.0 g x kg(-1).</p><p><b>CONCLUSION</b>The data showed that serum concentration of IFN-gamma and NK cell lysis were improved by YQD at different time, which was demonstrated YQD could perform well in immune system.</p>

Poly-silicon nanowire field-effect transistor for ultrasensitive and label-free detection of pathogenic avian influenza DNA.

Enhanced surveillance of influenza requires rapid, robust, and inexpensive analytical techniques capable of providing a detailed analysis of influenza virus strains. Functionalized poly-crystalline silicon nanowire field-effect transistor (poly-SiNW FET) was demonstrated to achieve specific and ultrasensitive (at fM level) detection of high pathogenic strain virus (H5 and H7) DNA of avian influenza (AI) which is an important infectious disease and has an immediate need for surveillance. The poly-SiNW FET was prepared by a simple and low-cost method that is compatible with current commercial semiconductor process without expensive E-beam lithography tools for large-scale production. Specific electric changes were observed for AI virus DNA sensing when nanowire surface of poly-SiNW FET was modified with complementary captured DNA probe and target DNA (H5) at fM to pM range could be distinguished. With its excellent electric properties and potential for mass commercial production, poly-SiNW FET can be developed to become a portable biosensor for field use and point-of-care diagnoses.