Oseltamivir Prophylaxis Reduces Inflammation and Facilitates Establishment of Cross-Strain Protective T Cell Memory to Influenza Viruses

CD8⁺ T cells directed against conserved viral regions elicit broad immunity against distinct influenza viruses, promote rapid virus elimination and enhanced host recovery. The influenza neuraminidase inhibitor, oseltamivir, is prescribed for therapy and prophylaxis, although it remains unclear how the drug impacts disease severity and establishment of effector and memory CD8⁺ T cell immunity. We dissected the effects of oseltamivir on viral replication, inflammation, acute CD8⁺ T cell responses and the establishment of immunological CD8⁺ T cell memory. In mice, ferrets and humans, the effect of osteltamivir on viral titre was relatively modest. However, prophylactic oseltamivir treatment in mice markedly reduced morbidity, innate responses, inflammation and, ultimately, the magnitude of effector CD8⁺ T cell responses. Importantly, functional memory CD8⁺ T cells established during the drug-reduced effector phase were capable of mounting robust recall responses. Moreover, influenza-specific memory CD4⁺ T cells could be also recalled after the secondary challenge, while the antibody levels were unaffected. This provides evidence that long-term memory T cells can be generated during an oseltamivir-interrupted infection. The anti-inflammatory effect of oseltamivir was verified in H1N1-infected patients. Thus, in the case of an unpredicted influenza pandemic, while prophylactic oseltamivir treatment can reduce disease severity, the capacity to generate memory CD8⁺ T cells specific for the newly emerged virus is uncompromised. This could prove especially important for any new influenza pandemic which often occurs in separate waves.     

Effects of Clinacanthus siamensis leaf extract on influenza virus infection

Ethanolic extracts of 20 medicinal plants were screened for influenza virus NA inhibition and in vitro antiviral activities using MDCK cells in an MTT assay. The vaccine proteins of influenza virus A/New Caledonia/20/99 (H1N1), mouse-adapted influenza virus A/Guizhou/54/89 (A/G)(H3N2) and mouse-adapted influenza virus B/Ibaraki/2/85 (B/I) were used in the NA inhibition assay, and mouse-adapted influenza viruses A/PR/8/34 (H1N1), A/G and B/I were used in the in vitro antiviral assay. The results of the in vitro antiviral assay indicated that the A/G virus was the most susceptible and an extract of the leaf of CS possessed the highest in vitro anti-A/G virus activity (41.98%). Therefore, the A/G virus and the CS extract were selected for studying in vivo anti-influenza virus activity. BALB/c mice were treated with CS extract (100 mg/kg per day, 5 times) orally from 4 hr before to 4 days after infection. CS extract elicited significant production of anti-influenza virus IgG₁ antibody in BAW and increased mouse weight compared to oseltamivir (0.1 mg/kg per day) on day 19 or water on days 17–19 of infection. Moreover, CS extract produced a higher anti-influenza virus IgA antibody level in BAW compared to oseltamivir, and a tendency towards an increase in anti-influenza virus IgA compared to water was shown. The results suggest that CS extract has a protective effect against influenza virus infection.     

Induction and Role of Indoleamine 2,3 Dioxygenase in Mouse Models of Influenza A Virus Infection

Influenza infection stimulates protective host immune responses but paradoxically enhances lung indoleamine 2,3 dioxygenase (IDO) activity, an enzyme that suppresses helper/effector T cells and activates Foxp3-lineage regulatory CD4 T cells (Tregs). Influenza A/PR/8/34 (PR8) infection stimulated rapid elevation of IDO activity in lungs and lung-draining mediastinal lymph nodes (msLNs). Mice lacking intact IDO1 genes (IDO1-KO mice) exhibited significantly lower morbidity after sub-lethal PR8 infection, and genetic or pharmacologic IDO ablation led to much faster recovery after virus clearance. More robust influenza-specific effector CD8 T cell responses manifested in lungs of PR8-infected IDO1-KO mice, though virus clearance rates were unaffected by IDO ablation. Similar outcomes manifested in mice infected with a less virulent influenza A strain (X31). IDO induction in X31-infected lungs was dependent on IFN type II (IFNγ) signaling and was restricted to non-hematopoietic cells, while redundant IFN type 1 or type II signaling induced IDO exclusively in hematopoietic cells from msLNs. Memory T cells generated in X31-primed IDO1-KO mice protected mice from subsequent challenge with lethal doses of PR8 (100×LD₅₀). However recall T cell responses were less robust in lung interstitial tissues, and classic dominance of TCR Vβ8.3 chain usage amongst memory CD8⁺ T cells specific for influenza nucleoprotein (NP₃₆₆) did not manifest in IDO1-KO mice. Thus, influenza induced IDO activity in lungs enhanced morbidity, slowed recovery, restrained effector T cell responses in lungs and shaped memory T cell repertoire generation, but did not attenuate virus clearance during primary influenza A infection.     

2009流行性感冒患者血清细胞因子的变化特点

本文旨在通过比较30例2009甲型H1N1流行性感冒(简称流感)患者经奥司他韦治疗前、后血清中细胞因子的变化特点,探讨其可能的发病机制.30例患者分为奥司他韦治疗前组和治疗后组,另选取健康志愿者20例为对照组.采用酶联免疫吸附试验,检测患者血清白细胞介素10(IL-10)、IL-2、IL-8及γ干扰素(IFN-γ)水平...     

DBA/2 mouse as an animal model for anti-influenza drug efficacy evaluation

Influenza viruses are seasonally recurring human pathogens. Vaccines and antiviral drugs are available for influenza. However, the viruses, which often change themselves via antigenic drift and shift, demand constant efforts to update vaccine antigens every year and develop new agents with broad-spectrum antiviral efficacy. An animal model is critical for such efforts. While most human influenza viruses are unable to kill BALB/c mice, some strains have been shown to kill DBA/2 mice without prior adaptation. Therefore, in this study, we explored the feasibility of employing DBA/2 mice as a model in the development of anti-influenza drugs. Unlike the BALB/c strain, DBA/2 mice were highly susceptible and could be killed with a relatively low titer (50% DBA/2 lethal dose = 10^2.83 plaque-forming units) of the A/Korea/01/2009 virus (2009 pandemic H1N1 virus). When treated with a neuraminidase inhibitor, oseltamivir phosphate, infected DBA/2 mice survived until 14 days post-infection. The reduced morbidity of the infected DBA/2 mice was also consistent with the oseltamivir treatment. Taking these data into consideration, we propose that the DBA/2 mouse is an excellent animal model to evaluate antiviral efficacy against influenza infection and can be further utilized for combination therapies or bioactivity models of existing and newly developed anti-influenza drugs.     

Influenza Virus Targets Class I MHC-Educated NK Cells for Immunoevasion

The immune response to influenza virus infection comprises both innate and adaptive defenses. NK cells play an early role in the destruction of tumors and virally-infected cells. NK cells express a variety of inhibitory receptors, including those of the Ly49 family, which are functional homologs of human killer-cell immunoglobulin-like receptors (KIR). Like human KIR, Ly49 receptors inhibit NK cell-mediated lysis by binding to major histocompatibility complex class I (MHC-I) molecules that are expressed on normal cells. During NK cell maturation, the interaction of NK cell inhibitory Ly49 receptors with their MHC-I ligands results in two types of NK cells: licensed (“functional”), or unlicensed (“hypofunctional”). Despite being completely dysfunctional with regard to rejecting MHC-I-deficient cells, unlicensed NK cells represent up to half of the mature NK cell pool in rodents and humans, suggesting an alternative role for these cells in host defense. Here, we demonstrate that after influenza infection, MHC-I expression on lung epithelial cells is upregulated, and mice bearing unlicensed NK cells (Ly49-deficient NKC^KD and MHC-I-deficient B2m^-/- mice) survive the infection better than WT mice. Importantly, transgenic expression of an inhibitory self-MHC-I-specific Ly49 receptor in NKC^KD mice restores WT influenza susceptibility, confirming a direct role for Ly49. Conversely, F(ab’)₂-mediated blockade of self-MHC-I-specific Ly49 inhibitory receptors protects WT mice from influenza virus infection. Mechanistically, perforin-deficient NKC^KD mice succumb to influenza infection rapidly, indicating that direct cytotoxicity is necessary for unlicensed NK cell-mediated protection. Our findings demonstrate that Ly49:MHC-I interactions play a critical role in influenza virus pathogenesis. We suggest a similar role may be conserved in human KIR, and their blockade may be protective in humans.     

甲型流感病毒合并肺部感染与非病毒性肺部感染的表现及治疗分析

目的:通过与非病毒性肺部感染对比,分析甲型流感病毒合并肺部感染患者的常见临床实验检查及治疗效果,为经验性治疗甲型流感病毒提供一定依据。方法:选择2017年11月至2018年4月期间在我科接受治疗的已确诊存在甲型流病毒合并肺部感染(A组)及非病毒性肺感染(B组)各25例,对于确诊的甲型流感患者给予奥司他韦抗病毒治疗一周外,两组均正常给予常规抗感染治疗,对比两组患者的炎症指标白细胞计数、中性粒细胞比例、C-反应蛋白及治疗天数、住院天数、症状消失时间、不良反应发生率。结果:两组患者的CT检查表现无统计学差异(P0.05),A组患者的白细胞计数水平明显低于B组,差异有统计学意义(P0.05),而两组患者的中性粒细胞比例及C反应蛋白水平差异无统计学意义(P0.05)。两组患者的治疗天数及住院天数差异均无统计学意义(P0.05)。两组患者在体温恢复正常时间、咳嗽消失时间、肺部湿罗音消失时间、气促消失时间等比较差异无统计学意义(P0.05)。A组不良反应发生率为16.00%(4/25),B组不良反应发生率为12.00%(3/25),组间差异无统计学意义(P0.05)。结论:甲型病毒性肺部感染常表现为血常规未见异常,常伴发不同程度炎症反应,除常规抗感染治疗外,口服抗病毒药物奥司他韦可促进疾病转归。     

A GFP Expressing Influenza A Virus to Report In Vivo Tropism and Protection by a Matrix Protein 2 Ectodomain-Specific Monoclonal Antibody

The severity of influenza-related illness is mediated by many factors, including in vivo cell tropism, timing and magnitude of the immune response, and presence of pre-existing immunity. A direct way to study cell tropism and virus spread in vivo is with an influenza virus expressing a reporter gene. However, reporter gene-expressing influenza viruses are often attenuated in vivo and may be genetically unstable. Here, we describe the generation of an influenza A virus expressing GFP from a tri-cistronic NS segment. To reduce the size of this engineered gene segment, we used a truncated NS1 protein of 73 amino acids combined with a heterologous dimerization domain to increase protein stability. GFP and nuclear export protein coding information were fused in frame with the truncated NS1 open reading frame and separated from each other by 2A self-processing sites. The resulting PR8-NS1(1–73)GFP virus was successfully rescued and replicated as efficiently as the parental PR8 virus in vitro and was slightly attenuated in vivo. Flow cytometry-based monitoring of cells isolated from PR8-NS1(1–73)GFP virus infected BALB/c mice revealed that GFP expression peaked on day two in all cell types tested. In particular respiratory epithelial cells and myeloid cells known to be involved in antigen presentation, including dendritic cells (CD11c⁺) and inflammatory monocytes (CD11b⁺ GR1⁺), became GFP positive following infection. Prophylactic treatment with anti-M2e monoclonal antibody or oseltamivir reduced GFP expression in all cell types studied, demonstrating the usefulness of this reporter virus to analyze the efficacy of antiviral treatments in vivo. Finally, deep sequencing analysis, serial in vitro passages and ex vivo analysis of PR8-NS1(1–73)GFP virus, indicate that this virus is genetically and phenotypically stable.     

Comparison of ribavirin and oseltamivir in reducing mortality and lung injury in mice infected with mouse adapted A/California/04/2009 (H1N1)

AimTo compare the efficacy of ribavirin and oseltamivir in reducing mortality, lung injury and cytokine response profile in pandemic influenza H1N1 (2009) infection.Main methodsWe assessed survival, weight loss, lung viral load (by RT-PCR), lung injury (by protein content in bronchoalveolar lavage), and inflammation (cell counts, differentials and cytokines in the bronchoalveolar lavage) in BALB/c mice after infection with mouse-adapted pandemic influenza strain A/California/04/2009.Key findingsOur results indicate that ribavirin (80 mg kg^? 1) and oseltamivir (50 mg kg^? 1) are equally effective in improving survival (100% vs. 0% in water treated controls), while ribavirin proved to be more effective in significantly preventing weight loss. Both drugs diminished the injury of the alveolar-capillary barrier by decreasing the protein detected in the BAL to baseline levels, and they were also equally effective in reduction lung viral loads by 100-fold. Administration of either drug did not decrease the amount of inflammatory infiltrate in the lung, but ribavirin significantly reduced the percentage comprised of lymphocytes. This study shows that these antivirals differentially regulate inflammatory cytokines and chemokines with ribavirin significantly reducing most of the cytokines/chemokines measured. Ribavirin treatment leads to a Th1 cytokine response while oseltamivir leads to a Th2 cytokine response with significant increase in the levels of the anti-inflammatory cytokine IL-10.SignificanceThis study reveals new mechanistic insights in the way that ribavirin and oseltamivir exert their antiviral activity and supports the theory that ribavirin could potentially serve as an efficacious therapeutic alternative for oseltamivir resistant pandemic H1N1 strains.     

Triple Combination of Amantadine,Ribavirin, and Oseltamivir Is Highly Active and Synergistic against Drug Resistant Influenza Virus Strains In Vitro

The rapid emergence and subsequent spread of the novel 2009 Influenza A/H1N1 virus (2009 H1N1) has prompted the World Health Organization to declare the first pandemic of the 21^st century, highlighting the threat of influenza to public health and healthcare systems. Widespread resistance to both classes of influenza antivirals (adamantanes and neuraminidase inhibitors) occurs in both pandemic and seasonal viruses, rendering these drugs to be of marginal utility in the treatment modality. Worldwide, virtually all 2009 H1N1 and seasonal H3N2 strains are resistant to the adamantanes (rimantadine and amantadine), and the majority of seasonal H1N1 strains are resistant to oseltamivir, the most widely prescribed neuraminidase inhibitor (NAI). To address the need for more effective therapy, we evaluated the in vitro activity of a triple combination antiviral drug (TCAD) regimen composed of drugs with different mechanisms of action against drug-resistant seasonal and 2009 H1N1 influenza viruses. Amantadine, ribavirin, and oseltamivir, alone and in combination, were tested against amantadine- and oseltamivir-resistant influenza A viruses using an in vitro infection model in MDCK cells. Our data show that the triple combination was highly synergistic against drug-resistant viruses, and the synergy of the triple combination was significantly greater than the synergy of any double combination tested (P<0.05), including the combination of two NAIs. Surprisingly, amantadine and oseltamivir contributed to the antiviral activity of the TCAD regimen against amantadine- and oseltamivir-resistant viruses, respectively, at concentrations where they had no activity as single agents, and at concentrations that were clinically achievable. Our data demonstrate that the TCAD regimen composed of amantadine, ribavirin, and oseltamivir is highly synergistic against resistant viruses, including 2009 H1N1. The TCAD regimen overcomes baseline drug resistance to both classes of approved influenza antivirals, and thus may represent a highly active antiviral therapy for seasonal and pandemic influenza.     

小儿豉翘清热颗粒联合奥司他韦对急性上呼吸道感染患儿血清炎性因子和外周血T淋巴细胞亚群的影响

摘要 目的：探讨小儿豉翘清热颗粒联合奥司他韦对急性上呼吸道感染患儿血清炎性因子和外周血T淋巴细胞亚群的影响。方法：选择2019年9月至2022年1月期间合肥市第二人民医院收治的急性上呼吸道感染患儿180例。按照双色球法将患儿分为对照组和研究组,在常规治疗的基础上,对照组（给予磷酸奥司他韦颗粒治疗）和研究组（给予小儿豉翘清热颗粒联合磷酸奥司他韦颗粒治疗）,两组各为90例。对比两组疗效、临床症状缓解情况、T淋巴细胞亚群指标、炎性因子水平和不良反应发生情况。结果：研究组的临床总有效率为95.56%,高于对照组的83.33%（P<0.05）。研究组的咳嗽、发热、咽痛、鼻塞流涕等症状缓解时间均短于对照组（P<0.05）。治疗5 d后,研究组CD3⁺、CD4⁺、CD4⁺/CD8⁺高于对照组,CD8⁺低于对照组（P<0.05）,研究组血清降钙素原（PCT）、白介素6（IL-6）、血清淀粉样蛋白A（SAA）、超敏C反应蛋白（hs-CRP）低于对照组（P<0.05）。两组不良反应发生率组间对比无明显差异（P>0.05）。结论：奥司他韦联合小儿豉翘清热颗粒治疗急性上呼吸道感染,可提高患儿的免疫力,降低血清PCT、IL-6、SAA、hs-CRP水平,有助于临床症状改善。     

Neuraminidase inhibitor-resistant recombinant A/Vietnam/1203/04 (H5N1) influenza viruses retain their replication efficiency and pathogenicity in vitro and in vivo

Effective antiviral drugs are essential for early control of an influenza pandemic. It is therefore crucial to evaluate the possible threat posed by neuraminidase (NA) inhibitor-resistant influenza viruses with pandemic potential. Four NA mutations (E119G, H274Y, R292K, and N294S) that have been reported to confer resistance to NA inhibitors were each introduced into recombinant A/Vietnam/1203/04 (VN1203) H5N1 influenza virus. For comparison, the same mutations were introduced into recombinant A/Puerto Rico/8/34 (PR8) H1N1 influenza virus. The E119G and R292K mutations significantly compromised viral growth in vitro, but the H274Y and N294S mutations were stably maintained in VN1203 and PR8 viruses. In both backgrounds, the H274Y and N294S mutations conferred resistance to oseltamivir carboxylate (50% inhibitory concentration [IC(50)] increases, >250-fold and >20-fold, respectively), and the N294S mutation reduced susceptibility to zanamivir (IC(50) increase, >3.0-fold). Although the H274Y and N294S mutations did not compromise the replication efficiency of VN1203 or PR8 viruses in vitro, these mutations slightly reduced the lethality of PR8 virus in mice. However, the VN1203 virus carrying either the H274Y or N294S mutation exhibited lethality similar to that of the wild-type VN1203 virus. The different enzyme kinetic parameters (V(max) and K(m)) of avian-like VN1203 NA and human-like PR8 NA suggest that resistance-associated NA mutations can cause different levels of functional loss in NA glycoproteins of the same subtype. Our results suggest that NA inhibitor-resistant H5N1 variants may retain the high pathogenicity of the wild-type virus in mammalian species. Patients receiving NA inhibitors for H5N1 influenza virus infection should be closely monitored for the emergence of resistant variants.     

Inhibition of Influenza A Virus Infection In Vitro by Peptides Designed In Silico

Influenza A viruses are enveloped, segmented negative single-stranded RNA viruses, capable of causing severe human respiratory infections. Currently, only two types of drugs are used to treat influenza A infections, the M2 H⁺ ion channel blockers (amantadine and rimantadine) and the neuraminidase inhibitors (NAI) (oseltamivir and zanamivir). Moreover, the emergence of drug-resistant influenza A virus strains has emphasized the need to develop new antiviral agents to complement or replace the existing drugs. Influenza A virus has on the surface a glycoprotein named hemagglutinin (HA) which due to its important role in the initial stage of infection: receptor binding and fusion activities of viral and endosomal membranes, is a potential target for new antiviral drugs. In this work we designed nine peptides using several bioinformatics tools. These peptides were derived from the HA1 and HA2 subunits of influenza A HA with the aim to inhibit influenza A virus infection. The peptides were synthetized and their antiviral activity was tested in vitro against several influenza A viral strains: Puerto Rico/916/34 (H1N1), (H1N1)pdm09, swine (H1N1) and avian (H5N2). We found these peptides were able to inhibit the influenza A viral strains tested, without showing any cytotoxic effect. By docking studies we found evidence that all the peptides were capable to bind to the viral HA, principally to important regions on the viral HA stalk, thus could prevent the HA conformational changes required to carry out its membranes fusion activity.     

Type I interferon signaling regulates Ly6C(hi) monocytes and neutrophils during acute viral pneumonia in mice

Type I interferon (IFN-I) plays a critical role in the homeostasis of hematopoietic stem cells and influences neutrophil influx to the site of inflammation. IFN-I receptor knockout (Ifnar1 ^−/−) mice develop significant defects in the infiltration of Ly6C^hi monocytes in the lung after influenza infection (A/PR/8/34, H1N1). Ly6C^hi monocytes of wild-type (WT) mice are the main producers of MCP-1 while the alternatively generated Ly6C^int monocytes of Ifnar1 ^−/− mice mainly produce KC for neutrophil influx. As a consequence, Ifnar1 ^−/− mice recruit more neutrophils after influenza infection than do WT mice. Treatment of IFNAR1 blocking antibody on the WT bone marrow (BM) cells in vitro failed to differentiate into Ly6C^hi monocytes. By using BM chimeric mice (WT BM into Ifnar1 ^−/− and vice versa), we confirmed that IFN-I signaling in hematopoietic cells is required for the generation of Ly6C^hi monocytes. Of note, WT BM reconstituted Ifnar1 ^−/− chimeric mice with increased numbers of Ly6C^hi monocytes survived longer than influenza-infected Ifnar1 ^−/− mice. In contrast, WT mice that received Ifnar1 ^−/− BM cells with alternative Ly6C^int monocytes and increased numbers of neutrophils exhibited higher mortality rates than WT mice given WT BM cells. Collectively, these data suggest that IFN-I contributes to resistance of influenza infection by control of monocytes and neutrophils in the lung.     

Prevention and treatment of influenza with hyperimmune bovine colostrum antibody

Background

Despite the availability of specific vaccines and antiviral drugs, influenza continues to impose a heavy toll on human health worldwide. Passive transfer of specific antibody (Ab) may provide a useful means of preventing or treating disease in unvaccinated individuals or those failing to adequately seroconvert, especially now that resistance to antiviral drugs is on the rise. However, preparation of appropriate Ab in large scale, quickly and on a yearly basis is viewed as a significant logistical hurdle for this approach to control seasonal influenza.

Methodology/Principal Findings

In this study, bovine colostrum, which contains approximately 500 g of IgG per milking per animal, has been investigated as a source of polyclonal antibody for delivery to the respiratory tract. IgG and F(ab'')2 were purified from the hyperimmune colostrum of cows vaccinated with influenza A/Puerto Rico/8/34 (PR8) vaccine and were shown to have high hemagglutination-inhibitory and virus-neutralizing titers. In BALB/c mice, a single administration of either IgG or F(ab'')2 could prevent the establishment of infection with a sublethal dose of PR8 virus when given as early as 7 days prior to exposure to virus. Pre-treated mice also survived an otherwise lethal dose of virus, the IgG- but not the F(ab'')2-treated mice showing no weight loss. Successful reduction of established infection with this highly virulent virus was also observed with a single treatment 24 hr after virus exposure.

Conclusions/Significance

These data suggest that a novel and commercially-scalable technique for preparing Ab from hyperimmune bovine colostrum could allow production of a valuable substitute for antiviral drugs to control influenza with the advantage of eliminating the need for daily administration.     

Persistent Salmonellosis Causes Pancreatitis in a Murine Model of Infection

Pancreatitis, a known risk factor for the development of pancreatic ductal adenocarcinoma, is a serious, widespread medical condition usually caused by alcohol abuse or gallstone-mediated ductal obstruction. However, many cases of pancreatitis are of an unknown etiology. Pancreatitis has been linked to bacterial infection, but causality has yet to be established. Here, we found that persistent infection of mice with the bacterial pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) was sufficient to induce pancreatitis reminiscent of the human disease. Specifically, we found that pancreatitis induced by persistent S. Typhimurium infection was characterized by a loss of pancreatic acinar cells, acinar-to-ductal metaplasia, fibrosis and accumulation of inflammatory cells, including CD11b⁺ F4/80⁺, CD11b⁺ Ly6C^int Ly6G⁺ and CD11b⁺ Ly6C^hi Ly6G⁻ cells. Furthermore, we found that S. Typhimurium colonized and persisted in the pancreas, associated with pancreatic acinar cells in vivo, and could invade cultured pancreatic acinar cells in vitro. Thus, persistent infection of mice with S. Typhimurium may serve as a useful model for the study of pancreatitis as it relates to bacterial infection. Increased knowledge of how pathogenic bacteria can cause pancreatitis will provide a more integrated picture of the etiology of the disease and could lead to the development of new therapeutic approaches for treatment and prevention of pancreatitis and pancreatic ductal adenocarcinoma.     

Ly6C+Ly6G− Myeloid‐derived suppressor cells play a critical role in the resolution of acute inflammation and the subsequent tissue repair process after spinal cord injury

Acute inflammation is a prominent feature of central nervous system (CNS) insult and is detrimental to the CNS tissue. Although this reaction spontaneously diminishes within a short period of time, the mechanism underlying this inflammatory resolution remains largely unknown. In this study, we demonstrated that an initial infiltration of Ly6C⁺Ly6G^? immature monocyte fraction exhibited the same characteristics as myeloid‐derived suppressor cells (MDSCs), and played a critical role in the resolution of acute inflammation and in the subsequent tissue repair by using mice spinal cord injury (SCI) model. Complete depletion of Ly6C⁺Ly6G^? fraction prior to injury by anti‐Gr‐1 antibody (clone: RB6‐8C5) treatment significantly exacerbated tissue edema, vessel permeability, and hemorrhage, causing impaired neurological outcomes. Functional recovery was barely impaired when infiltration was allowed for the initial 24 h after injury, suggesting that MDSC infiltration at an early phase is critical to improve the neurological outcome. Moreover, intraspinal transplantation of ex vivo‐generated MDSCs at sites of SCI significantly reduced inflammation and promoted tissue regeneration, resulting in better functional recovery. Our findings reveal the crucial role of an Ly6C⁺Ly6G^? fraction as MDSCs in regulating inflammation and tissue repair after SCI, and also suggests an MDSC‐based strategy that can be applied to acute inflammatory diseases.     

Comparison of anti-influenza virus activity and pharmacokinetics of oseltamivir free base and oseltamivir phosphate

Influenza viruses are major human respiratory pathogens that cause high morbidity and mortality worldwide. Currently, prophylactic vaccines and therapeutic antiviral agents are used to prevent and control influenza virus infection. Oseltamivir free base (OSV-FB), a modified generic antiviral drug of Tamiflu (oseltamivir phosphate, OSV-P), was launched in the Republic of Korea last year. Here, we examine the bioequivalence of these two compounds by assessing their antiviral efficacy in infected cells and in a mouse model. It was observed that both antivirals showed comparable efficacy against 11 different influenza A and B viruses in vitro. Moreover, in mice infected with influenza A virus (mouse-adapted A/Puerto Rico/8/34), they showed a dose-dependent therapeutic activity and alleviated infection-mediated reductions in body weight, leading to significantly better survival. There was histopathological disappearance of virus-induced inflammatory cell infiltration of the lung after oral treatment with either antiviral agent (at 10 mg/kg). Pharmacokinetic analysis also exhibited similar plasma concentrations of the active drug, oseltamivir carboxylate, metabolised from both OSV-B and OSV-P. This is the first report showing bioequivalence of OSV-FB to its phosphate salt form in the mouse system. The free base drug has some beneficial points including simple drug formulation process and reduced risk of undesirable cation-phosphate precipitation within solution. The long term stability of OSV-FB requires further monitoring when it is provided as a national stock in readiness for an influenza pandemic.     

Detection of the Antiviral Drug Oseltamivir in Aquatic Environments

Oseltamivir (Tamiflu®) is the most important antiviral drug available and a cornerstone in the defence against a future influenza pandemic. Recent publications have shown that the active metabolite, oseltamivir carboxylate (OC), is not degraded in sewage treatment plants and is also persistent in aquatic environments. This implies that OC will be present in aquatic environments in areas where oseltamivir is prescribed to patients for therapeutic use. The country where oseltamivir is used most is Japan, where it is used to treat seasonal flu. We measured the levels of OC in water samples from the Yodo River system in the Kyoto and Osaka prefectures, Japan, taken before and during the flu-season 2007/8. No OC was detected before the flu-season but 2–58 ng L⁻¹ was detected in the samples taken during the flu season. This study shows, for the first time, that low levels of oseltamivir can be found in the aquatic environment. Therefore the natural reservoir of influenza virus, dabbling ducks, is exposed to oseltamivir, which could promote the evolution of viral resistance.