Target Site of Prepulse Inhibition of the Trigeminal Blink Reflex in Humans.

Despite the clinical significance of prepulse inhibition (PPI), the mechanisms are not well understood. Herein, we present our investigation of PPI in the R1 component of electrically induced blink reflexes. The effect of a prepulse was explored with varying prepulse test intervals (PTIs) of 20-600 ms in 4 females and 12 males. Prepulse-test combinations included the following: stimulation of the supraorbital nerve (SON)-SON [Experiment (Exp) 1], sound-sound (Exp 2), the axon of the facial nerve-SON (Exp 3), sound-SON (Exp 4), and SON-SON with a long trial-trial interval (Exp 5). Results showed that (1) leading weak SON stimulation reduced SON-induced ipsilateral R1 with a maximum effect at a PTI of 140 ms, (2) the sound-sound paradigm resulted in a U-shaped inhibition time course of the auditory startle reflex (ASR) peaking at 140 ms PTI, (3) facial nerve stimulation showed only a weak effect on R1, (4) a weak sound prepulse facilitated R1 but strongly inhibited SON-induced late blink reflexes (LateRs) with a similar U-shaped curve, and (5) LateR in Exp 5 was almost completely absent at PTIs >80 ms. These results indicate that the principal sensory nucleus is responsible for R1 PPI. Inhibition of ASR or LateR occurs at a point in the startle reflex circuit where auditory and somatosensory signals converge. Although the two inhibitions are different in location, their similar time courses suggest similar neural mechanisms. As R1 has a simple circuit and is stable, R1 PPI helps to clarify PPI mechanisms.SIGNIFICANCE STATEMENT Prepulse inhibition (PPI) is a phenomenon in which the startle response induced by a startle stimulus is suppressed by a preceding nonstartle stimulus. This study demonstrated that the R1 component of the trigeminal blink reflex shows clear PPI despite R1 generation within a circuit consisting of the trigeminal and facial nuclei, without startle reflex circuit involvement. Thus, PPI is not specific to the startle reflex. In addition, PPI of R1, the auditory startle reflex, and the trigeminal late blink reflex showed similar time courses in response to the prepulse test interval, suggesting similar mechanisms regardless of inhibition site. R1 PPI, in conjunction with other paradigms with different prepulse-test combinations, would increase understanding of the underlying mechanisms.

Immunization with inactivated whole virus particle influenza virus vaccines improves the humoral response landscape in cynomolgus macaques.

Although antibody-inducing split virus vaccines (SV) are currently the most effective way to combat seasonal influenza, their efficacy can be modest, especially in immunologically-naïve individuals. We investigated immune responses towards inactivated whole influenza virus particle vaccine (WPV) formulations, predicated to be more immunogenic, in a non-human primate model, as an important step towards clinical testing in humans. Comprehensive analyses were used to capture 46 immune parameters to profile how WPV-induced responses differed to those elicited by antigenically-similar SV formulations. Naïve cynomolgus macaques vaccinated with either monovalent or quadrivalent WPV consistently induced stronger antibody responses and hemagglutination inhibition (HI) antibody titres against vaccine-matched viruses compared to SV formulations, while acute reactogenic effects were similar. Responses in WPV-primed animals were further increased by boosting with the same formulation, conversely to modest responses after priming and boosting with SV. 28-parameter multiplex bead array defined key antibody features and showed that while both WPV and SV induced elevated IgG responses against A/H1N1 nucleoprotein, only WPV increased IgG responses against A/H1N1 hemagglutinin (HA) and HA-Stem, and higher IgA responses to A/H1N1-HA after each vaccine dose. Antibodies to A/H1N1-HA and HA-Stem that could engage FcγR2a and FcγR3a were also present at higher levels after one dose of WPV compared to SV and remained elevated after the second dose. Furthermore, WPV-enhanced antibody responses were associated with higher frequencies of HA-specific B-cells and IFN-γ-producing CD4+ T-cell responses. Our data additionally demonstrate stronger boosting of HI titres by WPV following prior infection and support WPV administered as a priming dose irrespective of the follow up vaccine for the second dose. Our findings thus show that compared to SV vaccination, WPV-induced humoral responses are significantly increased in scope and magnitude, advocating WPV vaccination regimens for priming immunologically-naïve individuals and also in the event of a pandemic outbreak.

Evaluation of mechanomyogram efficacy as a tool for assessing paired-pulse inhibition of blink reflex early R1 component.

INTRODUCTION/AIMS: Paired-pulse stimulation provides clinically useful information regarding sensory inhibition. When supraorbital nerve stimulation is repeated within a short interval, the response to the second stimulation is reduced to varying degrees. This magnitude of change in stimulation response can be monitored by electromyogram (EMG) or by mechanomyogram (MMG) as in this report. MMG has some advantages such as being less time consuming and lacking stimulus artifact. We compared the use of MMG and EMG to validate MMG as an effective method of assessing blink reflex paired-pulse inhibition. METHODS: Eight volunteers participated. Participants received electrical stimulation to the supraorbital nerve of each side. A paired-pulse paradigm was employed, varying the conditioning-test interval between 5 and 800 ms. The R1 component of the induced blink reflex was simultaneously recorded by EMG using a pair of electrodes placed on the lower eyelid and by MMG using an accelerometer placed between the electrodes. RESULTS: The correlation coefficient of the R1 amplitude between MMG and EMG of the grand-averaged waveforms was 0.99. The average participant r value was .91 (range .76-.99). Similar analyses were performed for the amplitude variation of the second response relative to the first response. Results correlated well, yielding r values of .97 and .86 for the grand-averaged waveform and the average for each subject. DISCUSSION: The present results demonstrate that MMG could be an alternative to EMG in assessing paired-pulse inhibition of the electrical blink reflex R1 component.

Inactivation of SARS-CoV-2 variants by nitrogen-doped titanium dioxide loaded with metals as visible-light photocatalysts.

PURPOSE: We examined the inactivation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by a nitrogen-doped titanium dioxide (N-TiO2) visible-light photocatalyst that was activated via light irradiation in the natural environment and was safe for human use as a coating material. METHODS: The photocatalytic activity of glass slides coated with three types of N-TiO2 without metal or loaded with copper or silver and copper was investigated by measuring acetaldehyde degradation. The titer levels of infectious SARS-CoV-2 were measured using cell culture after exposing photocatalytically active coated glass slides to visible light for up to 60 min. RESULTS: N-TiO2 photoirradiation inactivated the SARS-CoV-2 Wuhan strain and this effect was enhanced by copper loading and further by the addition of silver. Hence, visible-light irradiation using silver and copper-loaded N-TiO2 inactivated the Delta, Omicron, and Wuhan strains. CONCLUSION: N-TiO2 could be used to inactivate SARS-CoV-2 variants, including emerging variants, in the environment.

Efficacy of a Cap-Dependent Endonuclease Inhibitor and Neuraminidase Inhibitors against H7N9 Highly Pathogenic Avian Influenza Virus Causing Severe Viral Pneumonia in Cynomolgus Macaques.

H7N9 highly pathogenic avian influenza virus (HPAIV) infection in a human was first reported in 2017. A/duck/Japan/AQ-HE29-22/2017 (H7N9) (Dk/HE29-22), found in imported duck meat at an airport in Japan, possesses a hemagglutinin with a multibasic cleavage site, indicating high pathogenicity in chickens, as in the case of other H7 HPAIVs. In the present study, we examined the pathogenicity of Dk/HE29-22 and the effectiveness of a cap-dependent endonuclease inhibitor (baloxavir) and neuraminidase inhibitors (oseltamivir and zanamivir) against infection with this strain in a macaque model (n = 3 for each group). All of the macaques infected with Dk/HE29-22 showed severe signs of disease and pneumonia even after the virus had disappeared from lung samples. Virus titers in macaques treated with baloxavir were significantly lower than those in the other treated groups. After infection, levels of interferon alpha and beta (IFN-α and IFN-ß) in the blood of macaques in the baloxavir group were the highest among the groups, whereas levels of tumor necrosis factor alpha (TNF-α) and interleukin 13 (IL-13) were slightly increased in the untreated group. In addition, immune checkpoint proteins, including programmed death 1 (PD-1) and T cell immunoreceptor with Ig and ITIM domains (TIGIT), were expressed at high levels in the untreated group, especially in one macaque that showed severe signs of disease, indicating that negative feedback responses against vigorous inflammation may contribute to disease progression. In the group treated with baloxavir, the percentages of PD-1-, CTLA-4-, and TIGIT-positive T lymphocytes were lower than those in the untreated group, indicating that reduction in virus titers may prevent expression of immune checkpoint molecules from downregulation of T cell responses.

Immune profiling of influenza-specific B- and T-cell responses in macaques using flow cytometry-based assays.

Influenza remains a significant global public health burden, despite substantial annual vaccination efforts against circulating virus strains. As a result, novel vaccine approaches are needed to generate long-lasting and universal broadly cross-reactive immunity against distinct influenza virus strains and subtypes. Several new vaccine candidates are currently under development and/or in clinical trials. The successful development of new vaccines requires testing in animal models, other than mice, which capture the complexity of the human immune system. Importantly, following vaccination or challenge, the assessment of adaptive immunity at the antigen-specific level is particularly informative. In this study, using peripheral blood mononuclear cells (PBMCs) from cynomolgus macaques, we describe detection methods and in-depth analyses of influenza virus-specific B cells by recombinant hemagglutinin probes and flow cytometry, as well as the detection of influenza virus-specific CD8+ and CD4+ T cells by stimulation with live influenza A virus and intracellular cytokine staining. We highlight the potential of these assays to be used with PBMCs from other macaque species, including rhesus macaques, pigtail macaques and African green monkeys. We also demonstrate the use of a human cytometric bead array kit in detecting inflammatory cytokines and chemokines from cynomolgus macaques to assess cytokine/chemokine milieu. Overall, the detection of influenza virus-specific B and T cells, together with inflammatory responses, as described in our study, provides useful insights for evaluating novel influenza vaccines. Our data deciphering immune responses toward influenza viruses can be also adapted to understanding immunity to other infections or vaccination approaches in macaque models.

Efficacy of Neuraminidase Inhibitors against H5N6 Highly Pathogenic Avian Influenza Virus in a Nonhuman Primate Model.

Attention has been paid to H5N6 highly pathogenic avian influenza virus (HPAIV) because of its heavy burden on the poultry industry and human mortality. Since an influenza A virus carrying N6 neuraminidase (NA) has never spread in humans, the potential for H5N6 HPAIV to cause disease in humans and the efficacy of antiviral drugs against the virus need to be urgently assessed. We used nonhuman primates to elucidate the pathogenesis of H5N6 HPAIV as well as to determine the efficacy of antiviral drugs against the virus. H5N6 HPAIV infection led to high fever in cynomolgus macaques. The lung injury caused by the virus was severe, with diffuse alveolar damage and neutrophil infiltration. In addition, an increase in interferon alpha (IFN-α) showed an inverse correlation with virus titers during the infection process. Oseltamivir was effective for reducing H5N6 HPAIV propagation, and continuous treatment with peramivir reduced virus propagation and the severity of symptoms in the early stage. This study also showed pathologically severe lung injury states in cynomolgus macaques infected with H5N6 HPAIV, even in those that received early antiviral drug treatments, indicating the need for close monitoring and further studies on virus pathogenicity and new antiviral therapies.

The Natural History of Spontaneously Occurred Endometriosis in Cynomolgus Monkeys by Monthly Follow-Up Laparoscopy for Two Years.

Endometriosis, a disease in which endometrial tissue proliferates outside the uterus, is a progressive disease that affects women in reproductive age. It causes abdominal pain and infertility that severely affects the quality of life in young women. The mechanism of the onset and development of endometriosis has not been fully elucidated because of the complex mechanism involved in the disease. Nonhuman primates have been used to study the pathogenesis of spontaneous endometriosis because of their gynecological and anatomical similarities to humans. To reveal the natural history of endometriosis in cynomolgus monkeys, we selected 11 female cynomolgus monkeys with spontaneous endometriosis and performed monthly laparoscopies, mapping endometriotic lesions and adhesions up to two years. At the initial laparoscopy, endometriotic lesions were exclusively found in the vesicouterine pouch in 45.4% (5/11) of the monkeys and spread to the Douglas' pouch over time. Appearance of small de novo lesions and disappearance of some of the small lesions were observed in 100% (11/11) and 18.2% (2/11) of the monkeys, respectively. Endometriosis developed in all monkeys, and the speed of progression varied greatly among individuals that could be attributed to the degree or frequency of retrograde menstruation and genetic factors; these findings support the similarities between humans and monkeys, thus verifying the value of this nonhuman primate model. Finding reliable quantification markers and unravelling the underlying factors in correlation with the spatiotemporal development of the disease using a nonhuman primate model would be useful for the better management of endometriosis in humans.

Histological analysis of fundic gland polyps secondary to PPI therapy.

AIMS: The aim of this study was to clarify the histopathological features of fundic gland polyps (FGPs) in patients treated with proton pump inhibitors (PPIs) and to investigate the mechanism of enlargement of FGPs after PPI treatment. METHODS AND RESULTS: A total of 196 biopsy specimens of FGPs, which consisted of 87 FGPs in patients treated with PPIs (PPI group) and 109 FGPs in patients treated without PPIs (non-PPI group) were compared histologically using haematoxylin and eosin staining, Ki67 immunohistochemistry and multiplex immunohistochemical stain with Ki67, MUC5AC and MUC6. The significant histological features of FGPs in the PPI group were: larger size of dilated fundic gland cysts, larger number of foveolar and mixture type fundic gland cysts, foveolar cell hyperplasia, parietal cell protrusion, mononuclear cell infiltration and a higher percentage of Ki67-positive cells in the deeper layers of the glands. Multiplex immunohistochemical stain showed that Ki67-positive cells were also positive for MUC5AC, and the Ki67-positive rate was significantly higher in MUC5AC-positive cells of the PPI group than of the non-PPI group. Gene mutations of ß-catenin were found in only 9.7% of FGPs in the PPI group. CONCLUSIONS: Enlargement of fundic gland cysts due to foveolar cell proliferation and parietal cell protrusion might promote the enlargement of FGPs in patients treated with PPIs. ß-catenin gene mutations might not be associated with these histological changes of FGPs after PPI treatment.

Emergence of Oseltamivir-Resistant H7N9 Influenza Viruses in Immunosuppressed Cynomolgus Macaques.

Antiviral compounds (eg, the neuraminidase inhibitor oseltamivir) are invaluable for the treatment of individuals infected with influenza A viruses of the H7N9 subtype (A[H7N9]), which have infected and killed hundreds of persons. However, oseltamivir treatment often leads to the emergence of resistant viruses in immunocompromised individuals. To better understand the emergence and properties of oseltamivir-resistant A(H7N9) viruses in immunosuppressed individuals, we infected immunosuppressed cynomolgus macaques with an A(H7N9) virus and treated them with oseltamivir. Disease severity and mortality were higher in immunosuppressed than in immunocompetent animals. Oseltamivir treatment at 2 different doses reduced A(H7N9) viral titers in infected animals, but even high-dose oseltamivir did not block viral replication sufficiently to suppress the emergence of resistant variants. Some resistant variants were not appreciably attenuated in cultured cells, but an oseltamivir-resistant A(H7N9) virus did not transmit among ferrets. These findings are useful for the control of A(H7N9) virus infections in clinical settings.

Protective efficacy of passive immunization with monoclonal antibodies in animal models of H5N1 highly pathogenic avian influenza virus infection.

Highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype often cause severe pneumonia and multiple organ failure in humans, with reported case fatality rates of more than 60%. To develop a clinical antibody therapy, we generated a human-mouse chimeric monoclonal antibody (MAb) ch61 that showed strong neutralizing activity against H5N1 HPAI viruses isolated from humans and evaluated its protective potential in mouse and nonhuman primate models of H5N1 HPAI virus infections. Passive immunization with MAb ch61 one day before or after challenge with a lethal dose of the virus completely protected mice, and partial protection was achieved when mice were treated 3 days after the challenge. In a cynomolgus macaque model, reduced viral loads and partial protection against lethal infection were observed in macaques treated with MAb ch61 intravenously one and three days after challenge. Protective effects were also noted in macaques under immunosuppression. Though mutant viruses escaping from neutralization by MAb ch61 were recovered from macaques treated with this MAb alone, combined treatment with MAb ch61 and peramivir reduced the emergence of escape mutants. Our results indicate that antibody therapy might be beneficial in reducing viral loads and delaying disease progression during H5N1 HPAI virus infection in clinical cases and combined treatment with other antiviral compounds should improve the protective effects of antibody therapy against H5N1 HPAI virus infection.

Translational research on influenza virus infection using a nonhuman primate model.

Influenza virus infection is a seasonal infectious disease for humans, whereas it is also a zoonosis that is originally transmitted from animals to humans. Therefore, several animal models are used in research on influenza virus infection. We have used a nonhuman primate (NHP) model to extrapolate pathogenicity of various influenza viruses and efficacy of vaccines and antiviral drugs against the influenza viruses in humans. NHPs have genes, anatomical structure, and immune responses similar to those of humans as compared to other animal models. Using an NHP model, we revealed that the pandemic 2009 influenza A virus caused viral pneumonia as reported in human patients. Thus, it is thought that NHP models can be used to predict replication of emerging viruses in humans. We also examined the pathogenicity of highly pathogenic avian influenza viruses and evaluated a new therapeutic antibody in macaques under an immunocompromised condition. NHP models have provided promising results in research on other infectious diseases including Ebola virus and human/simian immunodeficiency virus infections. Thus, NHPs are important in biomedical research for determining the pathogenesis and for development of treatments, especially when clinical trials are difficult. We summarize the characteristics and advantages of research using NHP models in this review.

Vaccination against H9N2 avian influenza virus reduces bronchus-associated lymphoid tissue formation in cynomolgus macaques after intranasal virus challenge infection.

H9N2 avian influenza virus causes sporadic human infection. Since humans do not possess acquired immunity specific to this virus, we examined the pathogenicity of an H9N2 virus isolated from a human and then analyzed protective effects of a vaccine in cynomolgus macaques. After intranasal challenge with A/Hong Kong/1073/1999 (H9N2) (HK1073) isolated from a human patient, viruses were isolated from nasal and tracheal swabs in unvaccinated macaques with mild fever and body weight loss. A formalin-inactivated H9N2 whole particle vaccine derived from our virus library was subcutaneously inoculated to macaques. Vaccination induced viral antigen-specific IgG and neutralization activity in sera. After intranasal challenge with H9N2, the virus was detected only the day after inoculation in the vaccinated macaques. Without vaccination, many bronchus-associated lymphoid tissues (BALTs) were formed in the lungs after infection, whereas the numbers of BALTs were smaller and the cytokine responses were weaker in the vaccinated macaques than those in the unvaccinated macaques. These findings indicate that the H9N2 avian influenza virus HK1073 is pathogenic in primates but seems to cause milder symptoms than does H7N9 influenza virus as found in our previous studies and that a formalin-inactivated H9N2 whole particle vaccine induces protective immunity against H9N2 virus.

Cancer-associated fibroblast-targeted strategy enhances antitumor immune responses in dendritic cell-based vaccine.

Given the close interaction between tumor cells and stromal cells in the tumor microenvironment (TME), TME-targeted strategies would be promising for developing integrated cancer immunotherapy. Cancer-associated fibroblasts (CAFs) are the dominant stromal component, playing critical roles in generation of the pro-tumorigenic TME. We focused on the immunosuppressive trait of CAFs, and systematically explored the alteration of tumor-associated immune responses by CAF-targeted therapy. C57BL/6 mice s.c. bearing syngeneic E.G7 lymphoma, LLC1 Lewis lung cancer, or B16F1 melanoma were treated with an anti-fibrotic agent, tranilast, to inhibit CAF function. The infiltration of immune suppressor cell types, including regulatory T cells and myeloid-derived suppressor cells, in the TME was effectively decreased through reduction of stromal cell-derived factor-1, prostaglandin E2 , and transforming growth factor-ß. In tumor-draining lymph nodes, these immune suppressor cell types were significantly decreased, leading to activation of tumor-associated antigen-specific CD8(+) T cells. In addition, CAF-targeted therapy synergistically enhanced multiple types of systemic antitumor immune responses such as the cytotoxic CD8(+) T cell response, natural killer activity, and antitumor humoral immunity in combination with dendritic cell-based vaccines; however, the suppressive effect on tumor growth was not observed in tumor-bearing SCID mice. These data indicate that systemic antitumor immune responses by various immunologic cell types are required to bring out the efficacy of CAF-targeted therapy, and these effects are enhanced when combined with effector-stimulatory immunotherapy such as dendritic cell-based vaccines. Our mouse model provides a novel rationale with TME-targeted strategy for the development of cell-based cancer immunotherapy.

Emergence of H7N9 Influenza A Virus Resistant to Neuraminidase Inhibitors in Nonhuman Primates.

The number of patients infected with H7N9 influenza virus has been increasing since 2013. We examined the efficacy of neuraminidase (NA) inhibitors and the efficacy of a vaccine against an H7N9 influenza virus, A/Anhui/1/2013 (H7N9), isolated from a patient in a cynomolgus macaque model. NA inhibitors (oseltamivir and peramivir) barely reduced the total virus amount because of the emergence of resistant variants with R289K or I219T in NA [residues 289 and 219 in N9 of A/Anhui/1/2013 (H7N9) correspond to 292 and 222 in N2, respectively] in three of the six treated macaques, whereas subcutaneous immunization of an inactivated vaccine derived from A/duck/Mongolia/119/2008 (H7N9) prevented propagation of A/Anhui/1/2013 (H7N9) in all vaccinated macaques. The percentage of macaques in which variant H7N9 viruses with low sensitivity to the NA inhibitors were detected was much higher than that of macaques in which variant H5N1 highly pathogenic influenza virus was detected after treatment with one of the NA inhibitors in our previous study. The virus with R289K in NA was reported in samples from human patients, whereas that with I219T in NA was identified for the first time in this study using macaques, though no variant H7N9 virus was reported in previous studies using mice. Therefore, the macaque model enables prediction of the frequency of emerging H7N9 virus resistant to NA inhibitors in vivo. Since H7N9 strains resistant to NA inhibitors might easily emerge compared to other influenza viruses, monitoring of the emergence of variants is required during treatment of H7N9 influenza virus infection with NA inhibitors.

Discovery of novel MHC-class I alleles and haplotypes in Filipino cynomolgus macaques (Macaca fascicularis) by pyrosequencing and Sanger sequencing: Mafa-class I polymorphism.

Although the low polymorphism of the major histocompatibility complex (MHC) transplantation genes in the Filipino cynomolgus macaque (Macaca fascicularis) is expected to have important implications in the selection and breeding of animals for medical research, detailed polymorphism information is still lacking for many of the duplicated class I genes. To better elucidate the degree and types of MHC polymorphisms and haplotypes in the Filipino macaque population, we genotyped 127 unrelated animals by the Sanger sequencing method and high-resolution pyrosequencing and identified 112 different alleles, 28 at cynomolgus macaque MHC (Mafa)-A, 54 at Mafa-B, 12 at Mafa-I, 11 at Mafa-E, and seven at Mafa-F alleles, of which 56 were newly described. Of them, the newly discovered Mafa-A8*01:01 lineage allele had low nucleotide similarities (<86%) with primate MHC class I genes, and it was also conserved in the Vietnamese and Indonesian populations. In addition, haplotype estimations revealed 17 Mafa-A, 23 Mafa-B, and 12 Mafa-E haplotypes integrated with 84 Mafa-class I haplotypes and Mafa-F alleles. Of these, the two Mafa-class I haplotypes, F/A/E/B-Hp1 and F/A/E/B-Hp2, had the highest haplotype frequencies at 10.6 and 10.2%, respectively. This suggests that large scale genetic screening of the Filipino macaque population would identify these and other high-frequency Mafa-class I haplotypes that could be used as MHC control animals for the benefit of biomedical research.

Disease severity is associated with differential gene expression at the early and late phases of infection in nonhuman primates infected with different H5N1 highly pathogenic avian influenza viruses.

UNLABELLED: Occasional transmission of highly pathogenic avian H5N1 influenza viruses to humans causes severe pneumonia with high mortality. To better understand the mechanisms via which H5N1 viruses induce severe disease in humans, we infected cynomolgus macaques with six different H5N1 strains isolated from human patients and compared their pathogenicity and the global host responses to the virus infection. Although all H5N1 viruses replicated in the respiratory tract, there was substantial heterogeneity in their replicative ability and in the disease severity induced, which ranged from asymptomatic to fatal. A comparison of global gene expression between severe and mild disease cases indicated that interferon-induced upregulation of genes related to innate immunity, apoptosis, and antigen processing/presentation in the early phase of infection was limited in severe disease cases, although interferon expression was upregulated in both severe and mild cases. Furthermore, coexpression analysis of microarray data, which reveals the dynamics of host responses during the infection, demonstrated that the limited expression of these genes early in infection led to a failure to suppress virus replication and to the hyperinduction of genes related to immunity, inflammation, coagulation, and homeostasis in the late phase of infection, resulting in a more severe disease. Our data suggest that the attenuated interferon-induced activation of innate immunity, apoptosis, and antigen presentation in the early phase of H5N1 virus infection leads to subsequent severe disease outcome. IMPORTANCE: Highly pathogenic avian H5N1 influenza viruses sometimes transmit to humans and cause severe pneumonia with ca. 60% lethality. The continued circulation of these viruses poses a pandemic threat; however, their pathogenesis in mammals is not fully understood. We, therefore, investigated the pathogenicity of six H5N1 viruses and compared the host responses of cynomolgus macaques to the virus infection. We identified differences in the viral replicative ability of and in disease severity caused by these H5N1 viruses. A comparison of global host responses between severe and mild disease cases identified the limited upregulation of interferon-stimulated genes early in infection in severe cases. The dynamics of the host responses indicated that the limited response early in infection failed to suppress virus replication and led to hyperinduction of pathological condition-related genes late in infection. These findings provide insight into the pathogenesis of H5N1 viruses in mammals.

In vitro and in vivo characterization of new swine-origin H1N1 influenza viruses.

Influenza A viruses cause recurrent outbreaks at local or global scale with potentially severe consequences for human health and the global economy. Recently, a new strain of influenza A virus was detected that causes disease in and transmits among humans, probably owing to little or no pre-existing immunity to the new strain. On 11 June 2009 the World Health Organization declared that the infections caused by the new strain had reached pandemic proportion. Characterized as an influenza A virus of the H1N1 subtype, the genomic segments of the new strain were most closely related to swine viruses. Most human infections with swine-origin H1N1 influenza viruses (S-OIVs) seem to be mild; however, a substantial number of hospitalized individuals do not have underlying health issues, attesting to the pathogenic potential of S-OIVs. To achieve a better assessment of the risk posed by the new virus, we characterized one of the first US S-OIV isolates, A/California/04/09 (H1N1; hereafter referred to as CA04), as well as several other S-OIV isolates, in vitro and in vivo. In mice and ferrets, CA04 and other S-OIV isolates tested replicate more efficiently than a currently circulating human H1N1 virus. In addition, CA04 replicates efficiently in non-human primates, causes more severe pathological lesions in the lungs of infected mice, ferrets and non-human primates than a currently circulating human H1N1 virus, and transmits among ferrets. In specific-pathogen-free miniature pigs, CA04 replicates without clinical symptoms. The assessment of human sera from different age groups suggests that infection with human H1N1 viruses antigenically closely related to viruses circulating in 1918 confers neutralizing antibody activity to CA04. Finally, we show that CA04 is sensitive to approved and experimental antiviral drugs, suggesting that these compounds could function as a first line of defence against the recently declared S-OIV pandemic.

Efficacy of repeated intravenous administration of peramivir against highly pathogenic avian influenza A (H5N1) virus in cynomolgus macaques.

Highly pathogenic avian influenza A (H5N1) viruses cause severe and often fatal disease in humans. We evaluated the efficacy of repeated intravenous dosing of the neuraminidase inhibitor peramivir against highly pathogenic avian influenza virus A/Vietnam/UT3040/2004 (H5N1) infection in cynomolgus macaques. Repeated dosing of peramivir (30 mg/kg/day once a day for 5 days) starting immediately after infection significantly reduced viral titers in the upper respiratory tract, body weight loss, and cytokine production and resulted in a significant body temperature reduction in infected macaques compared with that of macaques administered a vehicle (P < 0.05). Repeated administration of peramivir starting at 24 h after infection also resulted in a reduction in viral titers and a reduction in the period of virus detection in the upper respiratory tract, although the body temperature change was not statistically significant. The macaque model used in the present study demonstrated that inhibition of viral replication at an early time point after infection by repeated intravenous treatment with peramivir is critical for reduction of the production of cytokines, i.e., interleukin-6 (IL-6), tumor necrosis factor α, gamma interferon, monocyte chemotactic protein 1, and IL-12p40, resulting in amelioration of symptoms caused by highly pathogenic avian influenza virus infection.

Short-latency prepulse inhibition of the trigeminal blink reflex.

Prepulse inhibition (PPI) is a well-established phenomenon wherein a weak sensory stimulus attenuates the startle reflex triggered by a subsequent strong stimulus. Within the circuit, variations in target responses observed for PPI paradigms represent prepulse-induced excitability changes. However, little is known about the mechanism of PPI. Here, we focused on short-latency PPI of the trigeminal blink reflex R1 signal with an oligosynaptic reflex arc through the principal sensory trigeminal nucleus and the facial nucleus. As the facial nucleus is facilitatory to any input, R1 PPI is the phenomenon in the former nucleus. Considering that GABAergic modulation may be involved in PPI, this study investigated whether the PPI mechanism includes GABA-A equivalent inhibition, which peaks at approximately 30 ms in humans. In 12 healthy volunteers, the reflex was elicited by electrical stimulation of the supraorbital nerve, and recorded at the ipsilateral lower eyelid by accelerometer. Stimulus intensity was 1.5 times the R1 threshold for test stimulus and 0.9 times for the prepulse. The prepulse-test interval (PTI) was 5-150 ms. Results showed significant inhibition at 40-and 80-150-ms PTIs but not at 20-, 30-, 50-, 60-, and 70-ms PTIs, yielding two distinct inhibitions of different time scales. This corresponds well to the early and late components of inhibitory post synaptic potentials by GABA-A and GABA-B receptor activation. Thus, the data support the contribution of inhibitory post synaptic potentials elicited by the prepulse to the observed PPI. As inhibitory function-related diseases may impair the different inhibition components to varying degrees, methods deconvoluting each inhibitory component contribution are of clinical importance.