TLR dependent XBP-1 activation induces an autocrine loop in rheumatoid arthritis synoviocytes.

X-box binding protein 1 (XBP1) is a central regulator of the endoplasmic reticulum (ER) stress response. It is induced via activation of the IRE1 stress sensor as part of the unfolded protein response (UPR) and has been implicated in several diseases processes. XBP1 can also be activated in direct response to Toll-like receptor (TLR) ligation independently of the UPR but the pathogenic significance of this mode of XBP1 activation is not well understood. Here we show that TLR-dependent XBP1 activation is operative in the synovial fibroblasts (SF) of patients with active rheumatoid arthritis (RA). We investigated the expression of ER stress response genes in patients with active RA and also in patients in remission. The active (spliced) form of (s)XBP1 was significantly overexpressed in the active RA group compared to healthy controls and patients in remission. Paradoxically, expression of nine other ER stress response genes was reduced in active RA compared to patients in remission, suggestive of a UPR-independent process. However, sXBP1 was induced in SF by TLR4 and TLR2 stimulation, resulting in sXBP1-dependent interleukin-6 and tumour necrosis factor (TNF) production. We also show that TNF itself induces sXBP1 in SF, thus generating a potential feedback loop for sustained SF activation. These data confirm the first link between TLR-dependent XBP1 activation and human inflammatory disease. sXBP1 appears to play a central role in this process by providing a convergence point for two different stimuli to maintain activation of SF.

Involvement of X-box binding protein 1 and reactive oxygen species pathways in the pathogenesis of tumour necrosis factor receptor-associated periodic syndrome.

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-H(2)DCFDA 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.

Multiplexing immunoassays for cytokine detection in the serum of patients with rheumatoid arthritis: lack of sensitivity and interference by rheumatoid factor.

OBJECTIVES: Accurately measuring cytokines in clinical material remains an important challenge in the development of biomarkers. Enzyme-linked immunoabsorbent assays (ELISAs) are considered 'gold standard'; however, their use is limited by the relatively large sample volume required for multiple analyte testing. Several alternatives (including membrane or bead-ELISA) have been developed particularly to enable multiplexing. Concerns were raised regarding their use in rheumatology due to interference by heterophilic antibodies, notably rheumatoid factor (RF). In this report, we compared several multiplex assays using serum from rheumatoid arthritis (RA) patients with respect to the presence of residual RF following attempted removal employing commonly used procedures. METHODS: Healthy control and RF-positive/negative RA sera were used to compare 4 multiplex assays with ELISA: bead-based 'Luminex' immunoassay, cytometric bead assays (CBAs), membrane-based and Mosaic™ ELISAs. Sera were tested following Ig blockade (mixed species serum) or removal (using PEG6000 or sepharose-L). RESULTS: Ig removal was only partially efficient and residual RF was detected in most sera. RF had no impact on cytokine measurement by ELISA. In single and multiplex Luminex, cytokine levels associated with false positive results correlated directly with RF titres. Following Ig-blockade/removal, these relationship remained suggesting false positivity was still associated with the presence of residual RF. Conversely, detection of cytokines in multiplex membrane-based or Mosaic- ELISA were not affected by the presence of RF; however, levels of cytokines readily detected by ELISA were often below the detection threshold of these assays. CBA assays were also low on sensitivity but unaffected by RF. CONCLUSIONS: False positivity, due to the presence of heterophilic antibodies, mainly affected Luminex assays. Other assays however remained limited in their sensitivity. Multiplexing of cytokine measurement remains a challenge, particularly in rheumatological pathologies, until assays of adequate sensitivity are developed. ELISA remains the gold standard.

Comparison of pro-inflammatory cytokine expression and cellular signal transduction in human macrophages infected with different influenza A viruses.

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.

A novel type of influenza vaccine: safety and immunogenicity of replication-deficient influenza virus created by deletion of the interferon antagonist NS1.

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 (DeltaNS1-H1N1) in healthy volunteers. METHODS. Healthy seronegative adult volunteers were randomized to receive either a single intranasal dose of the DeltaNS1-H1N1 A/New Caledonia vaccine at 1 of 5 dose levels (6.4, 6.7, 7.0, 7.4, and 7.7 log(10) median tissue culture infective dose) (n = 36 recipients) or placebo (n = 12 recipients). RESULTS. Intranasal vaccination with the replication-deficient DeltaNS1-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 .

Determination of serum antibodies against swine-origin influenza A virus H1N1/09 by immunofluorescence, haemagglutination inhibition, and by neutralization tests: how is the prevalence rate of protecting antibodies in humans?

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.

Glycyrrhizin inhibits highly pathogenic H5N1 influenza A virus-induced pro-inflammatory cytokine and chemokine expression in human macrophages.

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.

Tumor cells infected with oncolytic influenza A virus prime natural killer cells for lysis of resistant tumor cells.

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 DeltaNS1 IAV showed enhanced lysis when compared with mock-infected cells (93% +/- 1.47 vs. 52% +/- 0.74), both mock-infected and DeltaNS1 IAV-infected DU145 cells were resistant to NK cell lysis. Moreover, NK cells primed with DeltaNS1 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 DeltaNS1 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 DeltaNS1 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, DeltaNS1 IAV may influence anti-tumoral responses by priming NK cells.

Measurement of cytotoxic T lymphocyte activity of human cytomegalovirus seropositive individuals by a highly sensitive coupled luminescent method.

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.

Anti-TNF treatment in rheumatoid arthritis.

Rheumatoid arthritis (RA), the most common autoimmune disease, is characterized by persistent synovitis and systemic inflammation. Genetic predisposition as well as autoantibodies and environmental factors, such as smoking, are associated with an increased risk of RA. Traditionally RA has been treated with disease modifying anti-rheumatic drugs (DMARDs) but in the last 15 years or so the introduction of biological response modifiers has revolutionized the treatment of RA. Among these anti-tumor necrosis factor (TNF) agents were the first to be successfully used in treating RA. The goal in treating RA is to induce remission or very low disease activity; remission is now accepted as the ultimate therapeutic goal by adoption of a "treat to target" strategy to achieve tight disease control. Therefore early diagnosis, as well as immediate intervention, are of the utmost importance. This review of the role of TNF in RA pathogenesis describes the mechanisms of action of currently used anti-TNF agents and the adverse events and safety of these drugs. Guidance on the use of anti-TNFs during pregnancy and prior to surgical procedures is also discussed. The intense efforts currently being made to identify biomarkers of response to anti-TNF therapy and recent progress in defining genetic predictors of response using genome- wide association studies (GWAS) are covered. However, so far, none of these studies have been translated into clinical application. The development of biosimilars or follow-on biologicals is also discussed and the first reported study of a biosimilar, involving a multicenter study of an etanercept biosimilar, Etanar, is described.

Glycyrrhizin exerts antioxidative effects in H5N1 influenza A virus-infected cells and inhibits virus replication and pro-inflammatory gene expression.

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.

Single HA2 mutation increases the infectivity and immunogenicity of a live attenuated H5N1 intranasal influenza vaccine candidate lacking NS1.

BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSION/SIGNIFICANCE: 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.

Gevokizumab, an anti-IL-1ß mAb for the potential treatment of type 1 and 2 diabetes, rheumatoid arthritis and cardiovascular disease.

The inflammatory cytokine IL-1ß has an essential role in the innate immune response. High levels of IL-1ß have been implicated in the development of many diseases, including type 1 and 2 diabetes (T1D and T2D), rheumatoid arthritis (RA) and cardiovascular disease. XOMA is developing gevokizumab (XOMA-052), an IgG2 humanized mAb against human IL-1ß, for the potential treatment of these diseases. Gevokizumab has a high affinity for IL-1ß and a long t1/2, which would allow for once-monthly dosing and offer a considerable advantage for patients over agents requiring more frequent dosing. Data from preclinical studies and clinical trials suggest that gevokizumab is a potentially effective and well-tolerated treatment for the indicated diseases. At the time of publication, phase II clinical trials were ongoing in patients with T1D, T2D and RA, with the T2D trials assessing key cardiovascular markers. Following promising data from a recent pilot trial, XOMA was also planning a phase I/II trial of gevokizumab for the potential treatment of uveitis in patients with the vasculitic inflammatory disorder Behçet's disease and the autoinflammatory conditions familial cold autoinflammatory syndrome and Muckle-Wells syndrome.

N-acetyl-L-cysteine (NAC) inhibits virus replication and expression of pro-inflammatory molecules in A549 cells infected with highly pathogenic H5N1 influenza A virus.

The antioxidant N-acetyl-L-cysteine (NAC) had been shown to inhibit replication of seasonal human influenza A viruses. Here, the effects of NAC on virus replication, virus-induced pro-inflammatory responses and virus-induced apoptosis were investigated in H5N1-infected lung epithelial (A549) cells. NAC at concentrations ranging from 5 to 15 mM reduced H5N1-induced cytopathogenic effects (CPEs), virus-induced apoptosis and infectious viral yields 24 h post-infection. NAC also decreased the production of pro-inflammatory molecules (CXCL8, CXCL10, CCL5 and interleukin-6 (IL-6)) in H5N1-infected A549 cells and reduced monocyte migration towards supernatants of H5N1-infected A549 cells. The antiviral and anti-inflammatory mechanisms of NAC included inhibition of activation of oxidant sensitive pathways including transcription factor NF-kappaB and mitogen activated protein kinase p38. Pharmacological inhibitors of NF-kappaB (BAY 11-7085) or p38 (SB203580) exerted similar effects like those determined for NAC in H5N1-infected cells. The combination of BAY 11-7085 and SB203580 resulted in increased inhibitory effects on virus replication and production of pro-inflammatory molecules relative to either single treatment. NAC inhibits H5N1 replication and H5N1-induced production of pro-inflammatory molecules. Therefore, antioxidants like NAC represent a potential additional treatment option that could be considered in the case of an influenza A virus pandemic.

Anti-cancer effects of artesunate in a panel of chemoresistant neuroblastoma cell lines.

Artemisinin derivatives are well-tolerated anti-malaria drugs that also exert anti-cancer activity. Here, we investigated artemisinin and its derivatives dihydroartemisinin and artesunate in a panel of chemosensitive and chemoresistant human neuroblastoma cells as well as in primary neuroblastoma cultures. Only dihydroartemisinin and artesunate affected neuroblastoma cell viability with artesunate being more active. Artesunate-induced apoptosis and reactive oxygen species in neuroblastoma cells. Of 16 cell lines and two primary cultures, only UKF-NB-3(r)CDDP(1000) showed low sensitivity to artesunate. Characteristic gene expression signatures based on a previous analysis of artesunate resistance in the NCI60 cell line panel clearly separated UKF-NB-3(r)CDDP(1000) from the other cell lines. l-Buthionine-S,R-sulfoximine, an inhibitor of GCL (glutamate-cysteine ligase), resensitised in part UKF-NB-3(r)CDDP(1000) cells to artesunate. This finding together with bioinformatic analysis of expression of genes involved in glutathione metabolism showed that this pathway is involved in artesunate resistance. These data indicate that neuroblastoma represents an artesunate-sensitive cancer entity and that artesunate is also effective in chemoresistant neuroblastoma cells.

Infection of human retinal pigment epithelial cells with influenza A viruses.

PURPOSE: Ocular involvement in influenza A virus diseases is common but usually limited to mild conjunctivitis. Rarely, inflammation of the choriocapillaris may result in atrophia of the retinal pigment epithelium (RPE). Primary human retinal pigment epithelial (RPE) cells were infected with seasonal (H1N1 A/New Caledonia/20/99, H3N2 A/California/7/2004) or highly pathogenic avian H5N1 (A/Thailand/1(Kan-1)/04, A/Vietnam/1203/04, A/Vietnam/1194/04) influenza strains. METHODS: Influenza A virus replication was studied by investigation of cytopathogenic effects, immune staining for influenza A virus nucleoprotein, determination of virus titers, and electron microscopy. Apoptosis induction was examined by immune staining for activated caspase 3 and cleaved PARP. Proinflammatory gene expression was investigated by quantitative PCR. RESULTS: H5N1 but not seasonal influenza strains replicated to high titers (>10(8) TCID(50)/mL; 50% tissue culture infectious dose/milliliter) in RPE cells. H5N1 infection resulted in RPE cell apoptosis that was abolished by the antiviral drug ribavirin. Pretreatment with type I interferons (interferon-alpha and -beta) or the type II interferon, (interferon-gamma), inhibited H5N1 replication. Moreover, H5N1 infection induced expression of proinflammatory genes (tumor necrosis factor-alpha, CXCL8, CXCL10, CXCL11, and interleukin-6), which was inhibited by ribavirin in a concentration-dependent manner. CONCLUSIONS: A novel cell type derived from the central nervous system was permissive to H5N1 influenza virus replication. This findings supports those suggesting H5N1 influenza strains to own a greater potential to spread to nonrespiratory tissues than seasonal human influenza viruses. Moreover, the data warrant the further study of the role of influenza A virus replication in retinal diseases associated with influenza A virus infections.

NK sensitivity of neuroblastoma cells determined by a highly sensitive coupled luminescent method.

The measurement of natural killer (NK) cells toxicity against tumor or virus-infected cells especially in cases with small blood samples requires highly sensitive methods. Here, a coupled luminescent method (CLM) based on glyceraldehyde-3-phosphate dehydrogenase release from injured target cells was used to evaluate the cytotoxicity of interleukin-2 activated NK cells against neuroblastoma cell lines. In contrast to most other methods, CLM does not require the pretreatment of target cells with labeling substances which could be toxic or radioactive. The effective killing of tumor cells was achieved by low effector/target ratios ranging from 0.5:1 to 4:1. CLM provides highly sensitive, safe, and fast procedure for measurement of NK cell activity with small blood samples such as those obtained from pediatric patients.