Peripheral benzodiazepines enhance the respiratory burst of macrophage-like P388D1 cells stimulated by arachidonic acid

P388D1, a murine cell with macrophage properties, responds to exogenous arachidonic acid with superoxide anion production. This oxidative burst is enhanced by peripheral and mixed type benzodiazepines and this stimulation is specifically reversed by the peripheral antagonist PK 11195. In contrast, PK 11195 is unable to antagonize a stimulation caused by a non-benzodiazepine ligand such as the chemotactic peptide fMet-Leu-Phe. The optimal concentrations were close to 10 nM and corresponded to the affinities of the compounds for the peripheral benzodiazepine receptor detected on these cells. Compared to other tissues where peripheral benzodiazepines acted only at micromolar concentrations, the macrophage with its functional receptor appears as a privileged site of action for these molecules.     

Infection enhancement of influenza A NWS virus in primary murine macrophages by anti-hemagglutinin monoclonal antibody.

Antibody-dependent enhancement (ADE) of influenza A NWS virus infection was investigated in primary murine macrophages (M phi) using anti-hemagglutinin(HA) monoclonal antibody (mAB). Contrary to previous reports of abortive influenza virus infection in primary M phi, this study demonstrated that the NWS virus replicated productively in both resident peritoneal M phi and thioglycolate-elicited peritoneal M phi providing cleavage of the HA was achieved by trypsin; 5 micrograms/ml of trypsin was the optimum concentration for the induction of infectivity. Under multiple-cycle growth conditions in the presence of mAB at various concentrations in trypsin-containing media, ADE was demonstrated in both M phi in the presence of subneutralizing concentrations of mAB. Flow cytometric analysis showed that the mechanism of virus entry into M phi could be through HA to specific virus receptors, or HA plus antibody to Fc receptors. These results indicate that ADE of the NWS virus infection actually occurs on Fc receptor-bearing primary murine M phi depending on the concentration of antibody in the presence of the appropriate protease for cleavage of viral HA.     

H1N1亚型流感病毒在A549和BEAS-2B细胞中复制情况的初步研究

目的 探讨不同宿主来源的H1N1亚型流感病毒在A549和BEAS-2B细胞的复制情况.方法 用分离自人、禽、猪三种宿主的7株H1N1甲型流感病毒分别接种A549和BEAS-2B细胞,分析病毒感染细胞后不同时段的特点;应用受体类型不同的红细胞进行微量血凝试验,检测流感病毒的受体结合特性;同时检测了A549和BEAS-2B细胞表面的受体分布情况.结果 三种宿主来源的H1N1亚型流感病毒感染A549细胞,24 h后CPE十分明显,36 h病毒滴度达到最高值;而感染BEAS-2B细胞后,从24 h-120 h CPE都不是很明显,且所有病毒的病毒滴度都很低.对6株H1N1流感病毒的受体结合特性进行了筛查,发现部分测试病毒具有SA a-2,6Gal受体结合特异性.而A549和BEAS-2B细胞表面均含有SA a-2,3Gal及SA a-2,6Gal受体,且A549细胞表面糖受体含量明显高于BEAS-2B细胞.结论 不同宿主来源的H1N1亚型流感病毒对A549细胞都易感并能有效增殖复制,而对具有相似受体特性、上皮组织来源的BEAS-2B细胞不易感,提示支持流感病毒有效感染、复制存在宿主内的调节机制.     

Monoclonal antibodies isolated from human B cells neutralize a broad range of H1 subtype influenza A viruses including swine-origin Influenza virus (S-OIV)

The new H1N1 swine-origin influenza virus (S-OIV) strain is a global health problem. The elucidation of the virus-host relationship is crucial for the control of the new infection. Two human monoclonal antibody Fab fragments (HMab) neutralizing the novel H1N1 influenza strain at very low concentrations were cloned before the emergence of S-OIV from a patient who had a broad-range H1N1 serum neutralizing activity. The two HMabs neutralized all tested H1N1 strains, including S-OIV and a swine strain with IC₅₀ ranging from 2 to 7 μg/ml. Data demonstrate that infection with previously circulating H1N1 strains can elicit antibodies neutralizing S-OIV. Finally, the human genes coding for the neutralizing HMabs could be used for generating full human monoclonal IgGs that can be safely administered being potentially useful in the prophylaxis and the treatment of this human infection.     

Treatment of the macrophage-like P388D.1 cells with bacterial lipopolysaccharide and interferon-gamma causes long-term alterations in calcium metabolism.

The intracellular calcium concentrations ([Ca2+]i) of P338D.1 macrophage-like cells, activated with interferon-gamma (IFN-gamma) and/or bacterial lipopolysaccharide (LPS) were determined using fura-2/AM and ratiometric imaging techniques. Treatment of macrophages with IFN-gamma and LPS resulted in significant downward shift in [Ca2+]i, 8, 16 and 24 h but not at 1 and 4 h after treatment. The decrease in [Ca2+]i also occurred when macrophages were treated with LPS only, but not after exposure of the cells to recombinant IFN-gamma, indicating that LPS was an essential signal in the observed changes in [Ca2+]i of activated macrophages. The IFN-gamma and/or LPS alteration in the [Ca2+]i, paralleled the in vitro nitric oxide production of the activated macrophages, 8, 16 and 24 h after treatment. The decrease in the [Ca2+]i may be caused by vigorous buffering and storing of Ca2+ by macrophages to below the normal resting quantities, following the reported transient increase in Ca2+ during the priming stage of macrophage activation. Thus, the downward shift in [Ca2+]I may play a physiological role in the activation processes of macrophages for antimicrobial responses.     

Antigenic and genetic variation in the hemagglutinins of H1N1 and H3N2 human influenza a viruses in the Shanghai area from 2005 to 2008

Continued rapid evolution of the influenza A virus is responsible for annual epidemics and occasional pandemics in the Shanghai area. In the present study, the representative strains of A/H1N1 and A/H3N2 influenza viruses isolated in the Shanghai area from 2005 to 2008 were antigenically and genetically characterized. The antigenic cartography method was carried out to visualize the hemagglutination-inhibition data. Antigenic differences were detected between circulating A/H1N1 strains isolated from 2005 to 2006 and the epidemic A/H1N1 strains isolated in 2008, which were found to be associated with the amino acid substitution K140E in HA1. The present vaccine strain A/Brisbane/59/2007 is considered to be capable of providing sufficient immunity against most of the circulating A/H1N1 viruses isolated in 2008 from the Shanghai population. The study showed that there were significant antigenic differences between the epidemic A/H3N2 strains isolated in 2007 and 2008, suggesting that antigenic drift had occurred in the A/H3N2 strains isolated in 2008. The P194L mutation was thought to be responsible for the antigenic evolution of influenza A/H3N2 viruses isolated from Shanghai in 2008. Evidence of antigenic drift suggests that the influenza A/H3N2 vaccine component needs to be updated.     

Receptor specificity of subtype H1 influenza A viruses isolated from swine and humans in the United States

The evolution of classical swine influenza viruses receptor specificity preceding the emergence of the 2009 H1N1 pandemic virus was analyzed in glycan microarrays. Classical swine influenza viruses from the α, β, and γ antigenic clusters isolated between 1945 and 2009 revealed a binding profile very similar to that of 2009 pandemic H1N1 viruses, with selectivity for α2-6-linked sialosides and very limited binding to α2-3 sialosides. Despite considerable genetic divergence, the ‘human-like’ H1N1 viruses circulating in swine retained strong binding preference for α2-6 sialylated glycans. Interspecies transmission of H1N1 influenza viruses from swine to humans or from humans to swine has not driven selection of viruses with distinct novel receptor binding specificities. Classical swine and human seasonal H1N1 influenza viruses have conserved specificity for similar α2-6-sialoside receptors in spite of long term circulation in separate hosts, suggesting that humans and swine impose analogous selection pressures on the evolution of receptor binding function.     

Generation and characterisation of monoclonal antibodies against influenza virus A, subtype H5N1

The emergence of highly pathogenic avian influenza A virus (HPAIV) subtype H5N1 in 1997 has since resulted in large outbreaks in poultry and in transmission from poultry to humans, mostly in southeast Asia, but also in several European countries. Effective diagnosis and control measures are essential for the management of HPAIV infections. To develop a rapid diagnostic test, a panel of murine monoclonal antibodies (mAbs) against influenza virus A subtype H5 was generated. Eleven mAbs were produced and characterised according to their reactivity by indirect and sandwich ELISA and western blotting against different H5 subtypes representing past and viruses currently circulating. Ten out of 11 mAbs reacted strongly with the haemagglutinin (HA) protein of H5 viruses, whereas one mAb reacted with the M1 protein. Targeted HA protein epitopes seemed to be conformational. One hybridoma clone binds to a linear epitope of the M1 protein. One specific mAb reacts with HPAIV H5 in the immunofluorescence test, and two antibodies neutralised H5 viruses. On the basis of the results, the set of seven mAbs is appropriate for developing diagnostic tests. With the generated mAbs, a sandwich ELISA was developed recognising all H5N1 strains tested but no other influenza viruses. With this ELISA, as little as 0.005 HA units or 0.1 ng/ml H5N1 was detected, surpassing other ELISA tests. The novel reagents have the potential to improve significantly available rapid antigen detection systems.     

Antigenic determinant study of the hemagglutinin in the composition of influenza viruses A subtype H1N1 by an indirect solid-phase radioimmunological analytical method

A comparative study of antigenic determinants of influenza A virus, subtype H1N1, hemagglutinins isolated in 1947-1953 and in 1977-1979 was carried out. The competitive capacity of 8 virus strains for antibody of 4 hyperimmune antisera to A/Fm/1/47, A/Moscow/Pan/52, A/USSR/090/77, and A/Brazil/11/78 viruses was studied by solid-phase radioimmunoassay (SPRIA) in two modifications: in the homologous and heterologous systems. The analysis of competition in the heterologous system permits one to study isolated subpopulations of antibody homologous to viruses fixed on the solid phase. The combination of both modifications allows investigations of a wider spectrum of antibodies than those possible in the homologous system alone. The results of the study showed the antigenic determinants of hemagglutinin exposed on the surface of particles of different influenza A virus strains to differ in their capacity to stimulate antibody synthesis. The analysis of the time course of changes in the antigenic properties of hemagglutinin regions of similar structure in various viral strains may facilitate prognosing of evolution of virus strains of the same subtype.     

Divergent evolution of hemagglutinin and neuraminidase genes in recent influenza A:H3N2 viruses isolated in Canada

Limited information is available concerning the molecular drift of the influenza neuraminidase (NA) genes. We report on the genetic variability of the NA gene from 31 influenza A:H3N2 viruses isolated in the Province of Québec (Canada) during the last three flu seasons (1997-2000). Amino acid substitutions within the NA protein were observed at rates of 1.01% and 0.45% between strains of the 1997-1998 and 1998-1999 seasons and between those of the 1998-1999 and 1999-2000 seasons, respectively. In most strains (28/31), amino acid changes occurred within at least one of four codons (197, 339, 370, and 401) previously implicated as antigenic sites. The 8 functional and 10 framework residues that compose the catalytic site of the NA enzyme were completely conserved over the study period. All isolates contained the seven conserved asparagine-linked glycosylation sites found in the NA of the progenitor A/Hong Kong/8/68 strain. In addition, most strains (30/31) had an eighth potential glycosylation site at position 329, whereas a ninth one was found at position 93 in 16 strains. The NA of all strains in this study was related to that of the vaccine strain A/Beijing/353/89, whereas the HA genes of these strains were related to the A/Beijing/32/92 vaccine strain. Thus, it appears that recent influenza strains continue to evolve from a reassortant produced during the cocirculation of the two above variants. Interestingly, some strains whose HA genes were closely related showed significant differences in their NA genes and conversely. This study confirms the importance of analyzing both the NA and the HA genes to assess the evolution of recent influenza epidemic strains.     

Emergent 2009 influenza A(H1N1) viruses containing HA D222N mutation associated with severe clinical outcomes in the Americas

Background

During the 2010-2011 influenza season, a small sub-group of 2009 influenza A(H1N1) viruses (hereafter referred to as 2009 A(H1N1)) emerged that was associated with more severe clinical outcomes in Ecuador and North America. Genetically, the haemagglutinin (HA) of this sub-clade was distinct from HAs found in viruses associated with severe outbreaks in 2010 from the United Kingdom and from other global specimens isolated earlier in the season.

Objective

We report the emergence of a novel 2009 A(H1N1) variant possessing a re-emergent HA D222N mutation obtained from patients with severe respiratory illnesses and phylogenetically characterise these D222N mutants with other severe disease-causing variants clustering within a common emerging sub-clade.

Case reports

In early 2011, three cases of 2009 A(H1N1) infection, two from Quito, Ecuador, and one from Washington, DC, USA, were complicated by severe pneumonia requiring mechanical ventilation, resulting in one fatality. These cases were selected due to the reported nature of the acute respiratory distress (ARD) that were captured in Department of Defence (DoD)-sponsored global influenza surveillance nets.

Results

Genetically, the 2009 A(H1N1) strains isolated from two of the three severe cases carried a prominent amino acid change at position 222 (D222N) within the primary HA receptor binding site. Furthermore, these cases represent an emerging sub-clade of viruses defined by amino acid changes within HA: N31D, S162N, A186T and V272I. Phylogenetically, these viruses share a high degree of homology with strains associated with recent fatal cases in Chihuahua, Mexico.

Discussion

Previously, enhanced virulence associated with the change, D222G, has been clinically linked to severe morbidity and mortality. Initial observations of the prevalence of a novel sub-clade of strains in the Americas suggest that viruses with a re-emergent D222N mutation may too correlate with severe clinical manifestations. These findings warrant heightened vigilance for emerging sub-clades of 2009 A(H1N1) and presumptive clinical implications.     

Possible involvement of adenosine 3':5'-cyclic monophosphate and extracellular calcium ions in histamine stimulation of interleukin-1 release from macrophage-like P388D1 cells.

Culture of macrophage-like P388D1 cells led to a spontaneous increase in release of interleukin-1 (IL-1). Addition of histamine enhanced the process as a function of its dose; histamine was effective at a concentration of as low as 10(-6)M and maximally at a dose of 10(-3)M. The effect of histamine was partially blocked by an H1-antagonist, diphenhydramine, at 10(-6)M - 10(-4)M. Ranitidine, an H2-antagonist, had no appreciable effect on the histamine-stimulated IL-1 release, even at a dose of 10(-5)M. At 10(-4)M it attenuated the histamine effect. Combination of the H1- and H2- antagonists resulted in a larger magnitude of attenuation of the histamine effect than that caused by either the H1-antagonist or the H2-antagonist alone. An H1-agonist, 2-pyridylethylamine, markedly augmented IL-1 release, reaching a maximum at 10(-4)M. Dimaprit, an H2-agonist, also stimulated IL-1 release, but the effect was far less than that of the H1 agonist. Dibutyryl adenosine 3':5'cyclic monophosphate (DBc-AMP) caused a marked rise in IL-1 production by the cells. The effects of DBc-AMP were synergistic to the effects of histamine at all doses examined. Histamine significantly augmented the uptake of 45Ca2+ by the cell as a function of time and dose of the amine. Addition of Co2+ attenuated the histamine effect at doses between 10(-5)M and 10(-4)M. These results suggest that the histamine-stimulated IL-1 release from P388D1 cells is dependent on both H1- and H2-receptors and involves both influx of calcium ions to the cells and altered intracellular concentration of adenosine 3':5'cyclic monophosphate.     

Transmission of influenza A/H5N1 viruses in mammals

Highly pathogenic avian H5N1 influenza A viruses occasionally infect humans and cause severe respiratory disease and fatalities. Currently, these viruses are not efficiently transmitted from person to person, although limited human-to-human transmission may have occurred. Nevertheless, further adaptation of avian H5N1 influenza A viruses to humans and/or reassortment with human influenza A viruses may result in aerosol transmissible viruses with pandemic potential. Although the full range of factors that modulate the transmission and replication of influenza A viruses in humans are not yet known, we are beginning to understand some of the molecular changes that may allow H5N1 influenza A viruses to transmit via aerosols or respiratory droplets among mammals. A better understanding of the biological basis and genetic determinants that confer transmissibility to H5N1 influenza A viruses in mammals is important to enhance our pandemic preparedness.     

Specific and nonspecific indicators of activation of influenza viruses before an epidemic caused by influenza viruses subtype A/H3N2 and type B in Czechoslovakia in 1986

The authors submit an aetiological and epidemiological analysis of the influenza epidemic which occurred in the CSR between the 4th and 14th week of 1986 and was caused by the influenza virus subtype A/H3N2/ and type B. The epidemic affected a total of 27.1% of the population, in the age group of 0-5 years 63.7%, in the age group 6-14 years 52.7% and in the age group above 15 years 17.1%. In the course of the epidemic 77,458 cases of pneumonia and bronchitis were reported and 1,412 deaths with the diagnosis influenza, bronchitis, pneumonia and chronic affection of the lungs. The authors analyze also specific indicators of the activation of influenza viruses and reach the conclusion that serological evidence of the circulation of influenza viruses in the population was detected already in the third quarter of 1985, the first isolations were made six weeks before the influenza epidemic. Activation of the influenza viruses is indicated already during the pre-epidemic period by some non-specific indicators which include the rising number of patients with acute respiratory affections in surgeries and the rising number of children absent from nurseries and nursery schools on account of these diseases. The most sensitive non-specific indicator is the rising number of patients with respiratory diseases in surgeries of the First aid medical service.     

Infection of lung epithelial cells with pandemic 2009 A(H1N1) influenza viruses reveals isolate-specific differences in infectivity and host cellular responses

To better understand the early virus-host interactions of the pandemic 2009 A(H1N1) viruses in humans, we examined early host responses following infection of human epithelial cell cultures with three 2009 A(H1N1) viruses (A/California/08/2009, A/Mexico/4108/2009, and A/Texas/15/2009), or a seasonal H1N1 vaccine strain (A/Solomon Islands/3/2006). We report here that infection with pandemic A/California/08/2009 and A/Mexico/4108/2009 viruses resulted in differences in virus infectivity compared to either pandemic A/Texas/15/2009 or the seasonal H1N1 vaccine strain. In addition, IFN-β levels were decreased in cell cultures infected with either the A/California/08/2009 or the A/Mexico/4108/2009 virus. Furthermore, infection with A/California/08/2009 and A/Mexico/4108/2009 viruses resulted in lower expression of four key proinflammatory markers (IL-6, RANTES, IP-10, and MIP-1β) compared with infection with either A/Texas/15/2009 or A/Solomon Islands/3/2006. Taken together, our results demonstrate that 2009 A(H1N1) viruses isolated during the Spring wave induced varying degrees of early host antiviral and inflammatory responses in human respiratory epithelial cells, highlighting the strain-specific nature of these responses, which play a role in clinical disease.     

Characterization of influenza A/Fujian/411/2002(H3N2)-like viruses isolated in Portugal between 2003 and 2005

In Portugal, influenza surveillance is achieved through the National Influenza Surveillance Programme (NISP), in close collaboration with other European and global surveillance networks. The NISP integrates epidemiological, clinical and virological data based on the information collected by a Network of Sentinel Medical Practitioners and by a network of Emergency Units of Hospitals and Health Care Centres. In this study, genetic and antigenic characterization of influenza A viruses of the A/Fujian/411/2002 lineage, isolated during the 2003/2004 and 2004/2005 influenza winter seasons, in the context of the NISP, are described. Antigenic analysis of A/Fujian/411/2002-like viruses, first detected and isolated during the 2003/2004 winter season, revealed a close similarity with the reference strains A/Kumamoto/102/2002 and A/Wyoming/3/2003. Genetic analysis confirmed this similarity and revealed two different phylogenetic branches. The 2004/2005 influenza A(H3) isolates formed, both antigenic and genetically, a more homogeneous group and were closely related to A/Oslo/807/2004 and A/California/7/2004. During this season, the characterization of the influenza viral strains has shown continuous evolution to variants close related to A/Oslo/807/2004. The majority of amino acid substitutions detected in the haemagglutinin occurred at antigenic sites. This study reflects the contribution of individual countries for the surveillance and knowledge of the molecular epidemiology of the infection, essential for a concerted action towards the global monitoring of the disease. It also reflects the importance of constant monitoring of genetic and antigenic characteristics of circulating influenza strains, which will certainly be a major contribution to the formulation of influenza vaccines.     

Infection of P388D1 macrophages and respiratory epithelial cells by Histoplasma capsulatum: selection of avirulent variants and their potential role in persistent histoplasmosis.

We evaluated P388D1 macrophagelike cells as model host cells for studying the intracellular survival and strain-specific virulence of Histoplasma capsulatum. Previously characterized strains which were virulent for mice destroyed monolayers of these cells within a few days. In contrast, related avirulent "smooth" variants failed to do so even after 20 days, although they persisted within P388D1 cells for at least 7 days. On the basis of this observation, we developed a quantitative radiolabel assay to use as an initial screen for virulence. Another cell type lining the respiratory tract was then examined as a potential host for H. capsulatum. Hamster trachea epithelial (HTE) cells readily internalized a variety of strains lacking alpha-(1,3)-glucan in their cell walls; however, the tracheal cells were only rarely infected by organisms possessing this polysaccharide. We subsequently inoculated HTE cells with alpha-(1,3)-glucan-positive strains and enriched for the few yeasts infecting these cells. The progeny resembled smooth variants in terms of colony morphology, the absence of alpha-(1,3)-glucan in their cell walls, and their inability to kill macrophages. Did the HTE cells select for these variant yeasts from the parent inoculum or instigate a change from the parental phenotype? Following a 3-h uptake period, only 2% of the ingested yeasts lacked alpha-(1,3)-glucan. One day later, nearly half of the intracellular organisms lacked this polymer. This rapid conversion of a large proportion of the inoculum suggests some type of environmentally triggered change, perhaps analogous to phase variation seen in many other pathogens. Infection of epithelial cells or some other nonprofessional phagocyte during natural histoplasmosis might give rise to similar variants, thus establishing a reservoir of organisms capable of causing chronic or latent infections.     

2009甲型H1N1流感大流行期间北京儿童的流感监测

目的 了解2009年甲型H1N1流感大流行期间北京地区儿童中流感流行的情况.方法 采用WHO推荐的实时荧光定量RT-PCR和国家流感中心推荐的分型方法,对2009年甲型H1N1流感大流行期间因流感样症状来首都儿科研究所附属儿童医院就诊患儿的咽拭子标本进行流感病毒核酸检测.结果 2009年6月1日至2010年2月28日期间共检测了4363份咽拭子标本,其中623例为甲型H1N1阳性,阳性率为14.3%,657例为其他甲型流感病毒阳性(15.1%),所有甲型流感病毒的总阳性率为29.3%.623例中有23例为危重症病例(占阳性患者的3.7%),其中5例死亡.618例信息完整的甲型H1N1病例中,患儿年龄为14天～16岁,性别比例为男比女为1.3:1.1～3岁儿童占25.2%,3～6岁学龄前儿童和6～12岁学龄儿童所占比例相近,各约占30%.在监测期间,仅呈现了一个甲型H1N1的流行波.2009年11月达到最高峰,随后减弱,2010年2月快速下降至2.7%.对监测期间每周20～30份临床标本同时进行季节性流感的监测显示,季节性H3N2、甲型H1N1和乙型流感交替流行.呼吸道合胞病毒(RSV)在甲型H1N1流行趋势减缓后逐渐流行成为流行优势株.结论 2009年6月至2010年2月北京地区儿童中出现甲型H1N1的流行,主要累及学龄前和学龄儿童.季节性流感和RSV与甲型H1N1交替流行.