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Glycyrrhizin is known to exert antiviral and anti-inflammatory effects. Here, the effects of an approved parenteral glycyrrhizin preparation (Stronger Neo-Minophafen C) were investigated on highly pathogenic influenza A H5N1 virus replication, H5N1-induced apoptosis, and H5N1-induced pro-inflammatory responses in lung epithelial (A549) cells. Therapeutic glycyrrhizin concentrations substantially inhibited H5N1-induced expression of the pro-inflammatory molecules CXCL10, interleukin 6, CCL2, and CCL5 (effective glycyrrhizin concentrations 25 to 50 µg/ml) but interfered with H5N1 replication and H5N1-induced apoptosis to a lesser extent (effective glycyrrhizin concentrations 100 µg/ml or higher). Glycyrrhizin also diminished monocyte migration towards supernatants of H5N1-infected A549 cells. The mechanism by which glycyrrhizin interferes with H5N1 replication and H5N1-induced pro-inflammatory gene expression includes inhibition of H5N1-induced formation of reactive oxygen species and (in turn) reduced activation of NFκB, JNK, and p38, redox-sensitive signalling events known to be relevant for influenza A virus replication. Therefore, glycyrrhizin may complement the arsenal of potential drugs for the treatment of H5N1 disease.

H5N1 influenza vaccines, including live intranasal, appear to be relatively less immunogenic compared to seasonal analogs. The main influenza virus surface glycoprotein hemagglutinin (HA) of highly pathogenic avian influenza viruses (HPAIV) was shown to be more susceptible to acidic pH treatment than that of human or low pathogenic avian influenza viruses. The acidification machinery of the human nasal passageway in response to different irritation factors starts to release protons acidifying the mucosal surface (down to pH of 5.2). We hypothesized that the sensitivity of H5 HA to the acidic environment might be the reason for the low infectivity and immunogenicity of intranasal H5N1 vaccines for mammals. We demonstrate that original human influenza viruses infect primary human nasal epithelial cells at acidic pH (down to 5.4), whereas H5N1 HPAIVs lose infectivity at pH ≤ 5.6. The HA of A/Vietnam/1203/04 was modified by introducing the single substitution HA2 58K→I, decreasing the pH of the HA conformational change. The H5N1 reassortants containing the indicated mutation displayed an increased resistance to acidic pH and high temperature treatment compared to those lacking modification. The mutation ensured a higher viral uptake as shown by immunohistochemistry in the respiratory tract of mice and 25 times lower mouse infectious dose₅₀. Moreover, the reassortants keeping 58K→I mutation designed as a live attenuated vaccine candidate lacking an NS1 gene induced superior systemic and local antibody response after the intranasal immunization of mice. Our finding suggests that an efficient intranasal vaccination with a live attenuated H5N1 virus may require a certain level of pH and temperature stability of HA in order to achieve an optimal virus uptake by the nasal epithelial cells and induce a sufficient immune response. The pH of the activation of the H5 HA protein may play a substantial role in the infectivity of HPAIVs for mammals.

In April 2009, a new variant of influenza A virus, subtype H1N1v emerged in Mexico and spread all over the world producing the H1N1 pandemic in mankind after 1918-1920 and 1978/1979. Obviously there was no herd immunity against this new virus variant. Mainly young people, but less elderly were affected and presented severe and even lethal courses of disease. Since virus-specific antibodies are commonly regarded as markers of partial or complete immunoprotection, we performed antibody determinations in serum samples obtained from people before and after the pandemic has arrived in our region (Frankfurt/M., Germany). The assays were done by indirect immunofluorescence, by neutralization test, and by a haemagglutination inhibition test (HI), which was established in a practical modification for general and easy use. Among 145 individuals, of whom serum specimens had been drawn before the onset of pandemic, 19 revealed humoral immunity, i.e. titres of H1N1v neutralizing antibodies (at least 1:64). Eleven were older than 60 years, one belonged to the age group 40-59 years, three to the age group 20-39 years, and two to the age group 15-19 years. After the onset of pandemic in Frankfurt, serum specimens drawn from n = 225 randomly selected patients of our local university hospital were investigated for antibodies against H1N1v by HI, which is generally recommended for routine check of immunity. Twenty-eight individuals revealed the protecting antibody titre of at least 1:40. The age distribution had moved to mean age groups. The results fit to the incidence of influenza A/H1N1(09) disease, as confirmed by RT-PCR in patients admitted to our hospital, peaking in the younger age groups up to 30 years (second affected group: 30-40 years). While commonly used solid-phase antibody tests (like immunofluorescence) are not suitable to diagnose passed H1N1(09) infection and acquired immunity, this can be easily done by HI. Expecting the next waves of influenza A/H1N1v infections, HI testing may avoid vaccinations under special risk of severe or hidden adverse reactions.

Background. The nonstructural protein NS1 of influenza virus counteracts the interferon-mediated immune response of the host. By deleting the open reading frame of NS1, we have generated a novel type of influenza vaccine. We studied the safety and immunogenicity of an influenza strain lacking the NS1 gene (ΔNS1-H1N1) in healthy volunteers. Methods. Healthy seronegative adult volunteers were randomized to receive either a single intranasal dose of the ΔNS1-H1N1 A/New Caledonia vaccine at 1 of 5 dose levels (6.4, 6.7, 7.0, 7.4, and 7.7 log10 median tissue culture infective dose) (n= 36 recipients) or placebo (n= 12 recipients). Results. Intranasal vaccination with the replication-deficient ΔNS1-H1N1 vaccine was well tolerated. Rhinitis like symptoms and headache were the most common adverse events identified during the 28-day observation period. Adverse events were similarly distributed between the treatment and placebo groups. Vaccine-specific local and serum antibodies were induced in a dose-dependent manner. In the highest dose group, vaccine-specific antibodies were detected in 10 of 12 volunteers. Importantly, the vaccine also induced neutralizing antibodies against heterologous drift variants. Conclusions. We show that vaccination with an influenza virus strain lacking the viral interferon antagonist NS1 induces statistically significant levels of strain-specific and cross-neutralizing antibodies despite the highly attenuated replication-deficient phenotype. Further studies are warranted to determine whether these results translate into protection from influenza virus infection. Trial registration. ClinicalTrials.gov identifier: NCT00724997.

Objectives To investigate convergence of endoplasmic reticulum stress pathways and enhanced reactive oxygen species (ROS) production, due to intracellular retention of mutant tumour necrosis factor receptor 1 (TNFR1), as a disease mechanism in TNFR-associated periodic syndrome (TRAPS). Methods Peripheral blood mononuclear cells from patients with TRAPS (n=16) and healthy controls (HC) (n=22) were studied alongside HEK293T cells expressing wild type-TNFR1 or TRAPS-associated mutations. Unfolded protein response (UPR)-associated proteins (protein kinase-like endoplasmic reticulum kinase, PERK), phosphorylated-PERK (p-PERK), phosphorylated inositol-requiring enzyme 1α (p-IRE1α) and spliced X-box binding protein 1 (sXBP1)) were measured by flow cytometry. XBP1 splicing and UPR-associated transcript expression were assessed by reverse transcription PCR/quantitative real-time PCR. ROS levels were measured using CM-H2DCFDA and MitoSOX Red in patients' monocytes or HEK293T cells by flow cytometry. Results Mutant TNFR1-expressing HEK293T cells had increased TNFR1 expression associated with intracellular aggregation. TRAPS patients had increased sXBP1 transcripts (p<0.01) compared with HC. Raised p-PERK protein was seen, indicative of an UPR, but other UPR-associated transcripts were normal. Increased ROS levels were observed in TRAPS monocytes compared with HCs (p<0.02); these increased further upon IL-6 stimulation (p<0.01). Lipopolysaccharide-stimulated peripheral blood mononuclear cells of patients with TRAPS, but not HCs, demonstrated increased sXBP1 levels (p<0.01), which were reduced by antioxidant treatment (p<0.05). Conclusions Patients with TRAPS have evidence of increased sXBP1 and PERK expression but without other signs of classical UPR, and also with high ROS generation that may contribute to the pro-inflammatory state associated with TRAPS. The authors propose a non-traditional XBP1 pathway with enhanced sXBP1 as a novel disease-contributing mechanism in TRAPS.

Hypercytokinaemia is thought to contribute to highly pathogenic H5N1 influenza A virus disease. Glycyrrhizin is known to exert immunomodulatory and anti-inflammatory effects and therefore a candidate drug for the control of H5N1-induced pro-inflammatory gene expression. Here, the effects of an approved parenteral glycyrrhizin preparation were investigated on H5N1 virus replication, H5N1-induced pro-inflammatory responses, and H5N1-induced apoptosis in human monocyte-derived macrophages. Glycyrrhizin 100 μg/ml, a therapeutically achievable concentration, impaired H5N1-induced production of CXCL10, interleukin 6, and CCL5 and inhibited H5N1-induced apoptosis but did not interfere with H5N1 replication. Global inhibition of immune responses may result in the loss of control of virus replication by cytotoxic immune cells including natural killer cells and cytotoxic CD8⁺ T-lymphocytes. Notably, glycyrrhizin concentrations that inhibited H5N1-induced pro-inflammatory gene expression did not affect cytolytic activity of natural killer cells. Since H5N1-induced hypercytokinaemia is considered to play an important role within H5N1 pathogenesis, glycyrrhizin may complement the arsenal of potential drugs for the treatment of H5N1 disease.

Influenza A virus infection of macrophages and virus-induced pro-inflammatory gene expression are regarded to contribute to severity of influenza A virus-caused diseases. Although some data are available on cytokine production by influenza A virus-infected macrophages, systematic comparisons of the virus types are currently considered to be of high relevance in humans (pandemic H1N1/2009, seasonal H1N1, seasonal H3N2, highly pathogenic avian influenza H5N1) on pro-inflammatory potential, and relevant underlying cellular signalling events are missing. Here, we show that the infection of human monocyte-derived macrophages with pandemic H1N1/2009 (A/HH/01/2009), seasonal H1N1/1999 (A/New Caledonia/20/99), seasonal H3N2/2004 (A/California/7/2004) or highly pathogenic H5N1/2004 (A/Thailand/1(Kan-1)/04) results in similar infection rates. However, the investigated H1N1 strains caused delayed and decreased apoptosis in comparison with H3N2/2004 or H5N1/2004. Moreover, human macrophage infection with H3N2/2004 or H5N1/2004 but not with H1N1 viruses was associated with pronounced pro-inflammatory cytokine production and activation of relevant mitogen-activated protein kinase pathways as indicated by phosphorylation of p38, JNK and ERK 1/2. These findings are in line with clinical observations indicating enhanced disease severity in H3N2- or H5N1-infected patients compared to individuals infected with pandemic H1N1/2009 or seasonal H1N1.

Tumor resistance to lysis by resting natural killer (NK) cells may be overcome by priming of NK cells with cytokines or by binding of NK activating receptors to ligands expressed on target cells. In this study, major histocompatibility complex class I (MHC-I)-negative LNCaP and MHC-I-positive DU145 cells were infected with genetically modified influenza A virus lacking the non-structural gene 1 (∆NS1 IAV). The cells were used to investigate the influence of ∆NS1 IAV infection on NK cell lysis of tumor cells as well as to prime NK cells for lysis of LNCaP and DU145 cells. While LNCaP cells infected with ΔNS1 IAV showed enhanced lysis when compared with mock-infected cells (93% ± 1.47 vs. 52% ± 0.74), both mock-infected and ΔNS1 IAV-infected DU145 cells were resistant to NK cell lysis. Moreover, NK cells primed with ΔNS1 IAV-infected LNCaP/DU145 cells effectively lysed resistant DU145 and sensitive LNCaP cells to a greater extent than NK cells primed with mock-infected LNCaP/DU145 or non-primed NK cells. Also, NK cell priming with ΔNS1 IAV-infected tumor cells enhanced extracellular signal-regulated kinase phosphorylation and increased granule release in NK cells. The increased granule release was specifically mediated by NKp46, which eventually potentiated NK cells primed with ΔNS1 IAV-infected tumor cells to overcome the inhibitory effects posed by MHC-I expression on DU145 cells. These findings show that in addition to direct lytic activity of NK cells, ΔNS1 IAV may influence anti-tumoral responses by priming NK cells.

A coupled luminescent method (CLM) based on glyceraldehyde-3-phosphate dehydrogenase released from injured target cells was used to evaluate the cytotoxicity of antigen-specific HLA class I-restricted CTLs. In contrast to established methods, CLM does not require the pretreatment of target cells with radioactive or toxic labeling substances. CTLs from healthy HLA-A2 positive donors were stimulated by autologous dendritic cells (DCs) pulsed with HLA-A2 restricted HCMV-pp65 nonamer peptides. HLA-A2 positive T2 cells or autologous monocytes pulsed with HCMV-pp65 nonamer peptide served as target cells. Lysis was detected only in HCMV-pp65-pulsed target cells incubated with CTLs from seropositive donors stimulated by HCMV-pp65-pulsed DCs. After 3 days, stimulation 38% of T2 cells and 17% of monocytes were lysed at an effector to target ratio of 8:1. In conclusion, CLM represents a highly sensitive, fast, material-saving and non-toxic/non-radioactive method for the measurement of antigen-specific CTL cytotoxic activity.

Background Naturalistic developmental behavioural interventions (NDBI) have been shown to improve autism-specific symptoms in young children with Autism Spectrum Disorder (ASD). NDBI approaches, such as the ASD-specific Frankfurt Early Intervention Programme for ASD (A-FFIP), are based on ASD-specific developmental and learning aspects. A-FFIP is a low-intensity intervention which can easily be implemented in the local health care/social welfare system. The aim of the present study is to establish 1-year efficacy of the manualised early intervention programme A-FFIP in toddlers and preschool children with ASD. It is hypothesised that A-FFIP will result in improved ASD-specific symptoms compared to early intervention as usual (EIAU). Child- and family-specific secondary outcomes, as well as moderators and mediators of outcome, will be explored. Methods/design A prospective, multi-centre, parallel-group, randomised controlled, phase-III trial comparing A-FFIP versus EIAU. A total of 134 children (A-FFIP: 67, EIAU: 67) aged 24–66 months at baseline assessment meeting the criteria for ASD ( DSM-5 ) will be included. The primary outcome is the absolute change of the total score of the Brief Observation of Social Communication Change (BOSCC-AT) between baseline (T2) and 1-year follow-up (T6). The treatment effect will be tested, adjusted for relevant covariates applying a mixed model for repeated measures. Secondary outcomes are BOSCC social communication and repetitive-behaviour scores, single ASD symptoms, language, cognition, psychopathology, parental well-being and family quality of life. Predictors, moderators and mediating mechanisms will be explored. Discussion If efficacy of the manualised A-FFIP early intervention is established, the current study has the potential to change clinical practice strongly towards the implementation of a low-intensity, evidence-based, natural early intervention in ASD. Early intervention in ASD requires specialist training, which subsequently needs to be developed or included into current training curricula. Trial registration German Registry for Clinical Trials (Deutscher Register Klinischer Studien, DRKS); ID: 00016330. Retrospectively registered on 4 January 2019. URL: https://www.drks.de/drks_web/navigate.do?navigationId=trial.HTML&TRIAL_ID=DRKS00016330 .