Innate immunity and inflammation – two facets of the same anti‐infectious reaction

Innate immunity is the host's first line of defence against infection. In this review, we present the innate immune response implicated in three examples of pulmonary infection of viral, fungal and bacterial origin. We show that this defence against infection can be a double‐edged sword. Thus, the same cells, molecules and mechanisms involved in this protective process can also be involved in deleterious inflammation. A delicate balance between immunity and inflammation is therefore required, making it possible to fight pathogens effectively while limiting inflammation that might be damaging to the host.     

Epstein–Barr virus antibodies mark systemic lupus erythematosus and scleroderma patients negative for anti‐DNA

Systemic lupus erythematosus (SLE) is an autoimmune disease that can attack many different body organs; the triggering event is unknown. SLE has been associated with more than 100 different autoantibody reactivities – anti‐dsDNA is prominent. Nevertheless, autoantibodies to dsDNA occur in only two‐thirds of SLE patients. We previously reported the use of an antigen microarray to characterize SLE serology. We now report the results of an expanded study of serology in SLE patients and scleroderma (SSc) patients compared with healthy controls. The analysis validated and extended previous findings: two‐thirds of SLE patients reacted to a large spectrum of self‐molecules that overlapped with their reactivity to dsDNA; moreover, some SLE patients manifested a deficiency of natural IgM autoantibodies. Most significant was the finding that many SLE patients who were negative for autoantibodies to dsDNA manifested abnormal antibody responses to Epstein–Barr virus (EBV): these subjects made IgG antibodies to EBV antigens to which healthy subjects did not respond or they failed to make antibodies to EBV antigens to which healthy subjects did respond. This observation suggests that SLE may be associated with a defective immune response to EBV. The SSc patients shared many of these serological abnormalities with SLE patients, but differed from them in increased IgG autoantibodies to topoisomerase and centromere B; 84% of SLE patients and 58% of SSc patients could be detected by their abnormal antibodies to EBV. Hence an aberrant immune response to a ubiquitous viral infection such as EBV might set the stage for an autoimmune disease.     

Characterization of ELISA detection of broad‐spectrum anti‐Epstein–Barr virus antibodies associated with nasopharyngeal carcinoma

Epstein–Barr virus (EBV) infection is associated with undifferentiated nasopharyngeal carcinomas (NPC). A distinct seroreactivity pattern to EBV is predictive of subsequent risk of sporadic and familial nasopharyngeal carcinomas. There are currently no accepted screening tools for guiding the clinical management of individuals at high‐risk for nasopharyngeal carcinomas, particularly unaffected relatives from nasopharyngeal carcinoma multiplex families. Therefore, the reproducibility of a panel of largely synthetic peptide‐based anti‐EBV antibody ELISAs was evaluated and their ability to distinguish nasopharyngeal carcinoma cases from controls was explored. IgG and IgA antibodies against 6 different EBV antigens (10 assays, total) were tested on sera from 97 individuals representing the full spectrum of anti‐EBV seroprevalence (i.e., healthy individuals with no known EBV seroreactivity, healthy individuals with known EBV seroreactivity, and nasopharyngeal carcinoma cases). Each specimen was tested in triplicate to assess within‐batch and across‐batch variation, and the triplicate testing was repeated on two separate days. Reproducibility was assessed by the coefficients of variation (CVs) and intraclass correlation coefficients (ICCs). All markers were detectable in 17% or more of samples. For all but one marker, the overall, within‐batch, and across‐batch CVs were below 15%, and the ICCs were above 70% for all but three markers. Sensitivity of these markers to detect prevalent nasopharyngeal carcinomas ranged from 22% to 100%, and among unaffected controls, most distinguished those with and without known seropositivity. In conclusion, a large number of EBV markers can be measured reliably in serum samples using peptide‐based anti‐EBV ELISAs. J. Med. Virol. 85:524–529, 2013. Puiblished 2012. This is a US government work, and, as such, is in the public domain of The United States of America.     

Characterization of the anti‐HBV activity of HLP1–23, a human lactoferrin‐derived peptide

Lactoferrin (Lf) was shown to exhibit its antiviral activity at an early phase of viral infection and a mechanism whereby the protein interacts with host cell surface molecules has been suggested. In this study, human Lf (HLf) and seven HLf‐derived synthetic peptides (HLP) corresponding to the N‐terminal domain of the native protein (1–47 amino acids sequence) were assayed for their capacity to prevent hepatitis B virus (HBV) infection and replication using the HepaRG and HepG2.2.2.15 cell lines. Of the series tested, four peptides showed 40–75% inhibition of HBV infection in HepaRG cells, HLP_1–23, containing the GRRRR cationic cluster, being the most potent. Interestingly, this cluster is one of the two glycosaminoglycan binding sites of the native HLf involved in its antiviral activity; however, the mechanism of the HLP_1–23 action was different from that of the full‐length protein, the peptide inhibiting HBV infection when pre‐incubated with the virus, while no effect was observed on the target cells. It is suggested that the cationic cluster is sufficient for the peptide to interact stably with negatively charged residues on the virion envelope, while the absence of the second glycosaminoglycan binding site prevents its efficient attachment to the cells. In conclusion, this peptide may constitute a non‐toxic approach for potential clinical applications in inhibiting HBV entry by neutralizing the viral particles. J. Med. Virol. 85:780–788, 2013. © 2013 Wiley Periodicals, Inc.     

Long‐term anti‐endotoxin/E. coli efficacy in mice transfected with AAV2/1‐muBPI25‐muFcγ1

Bactericidal/permeability increasing (BPI) is an antibiotic protein which kills Gram‐negative bacteria and neutralizes endotoxin. We have previously developed a recombinant adeno‐associated virus which contains human BPI amino acid residues 1–199 and Fc fragment of human IgG1 gene (AAV‐hBPI‐Fc) and shown that the recombinant virus can protect mice from lethal endotoxemia. However, whether AAV‐hBPI‐Fc can be used in vivo for the long term remains unclear. To address this, we established an adeno‐associated virus‐containing mouse BPI and Fc fragment genes (muBPI‐Fc) and compared antigenicity of these recombinant proteins in murine models. Immunohistochemistry showed the expression of both fusion proteins at injected sites. ELISA and Western blotting showed that the muBPI‐Fc protein was detected in serum up to 8 weeks after injection, without generation of autoantibodies against muBPI‐Fc. In contrast, expressed hBPI‐Fc protein was only detected on the 2nd week, whereas the autoantibody against hBPI‐Fc protein occurred in serum from the 4th week to the end of study. muBPI‐Fc also reduced production of proinflammatory cytokines and protected mice from endotoxemia and bacteremia. Our data showed that AAV‐muBPI‐Fc has potential long‐term efficacy as an anti‐endotoxin and has anti‐bacterial activity in mice, suggesting the potential clinical application of AAV‐hBPI‐Fc, such as in endotoxin shock.     

The antimalarial drug amodiaquine possesses anti‐ZIKA virus activities

Zika virus (ZIKV) outbreak has emerged as a global health threat, particularly in tropical areas, over the past few years. No antiviral therapy or vaccine is available at present. For these reasons, repurposing clinically approved drugs against ZIKV infection may provide rapid and cost‐effective global health benefits. Here, we explored this strategy and screened eight FDA‐approved drugs for antiviral activity against ZIKV using a cell‐based assay. Our results show that the antimalarial drug amodiaquine has anti‐ZIKV activity with EC₅₀ at low micromolar concentrations in cell culture. We further characterized amodiaquine antiviral activity against ZIKV and found that it targets early events of the viral replication cycle. Altogether, our results suggest that amodiaquine may be efficacious for the treatment of ZIKV infection.

     

Human dominant‐negative class II transactivator transgenic pigs – effect on the human anti‐pig T‐cell immune response and immune status

Swine leucocyte antigen (SLA) class II molecules on porcine (p) cells play a crucial role in xenotransplantation as activators of recipient human CD4⁺ T cells. A human dominant‐negative mutant class II transactivator (CIITA‐DN) transgene under a CAG promoter with an endothelium‐specific Tie2 enhancer was constructed. CIITA‐DN transgenic pigs were produced by nuclear transfer/embryo transfer. CIITA‐DN pig cells were evaluated for expression of SLA class II with/without activation, and the human CD4⁺ T‐cell response to cells from CIITA‐DN and wild‐type (WT) pigs was compared. Lymphocyte subset numbers and T‐cell function in CIITA‐DN pigs were compared with those in WT pigs. The expression of SLA class II on antigen‐presenting cells from CIITA‐DN pigs was significantly reduced (40–50% reduction compared with WT; P < 0·01), and was completely suppressed on aortic endothelial cells (AECs) even after activation (100% suppression; P < 0·01). The human CD4⁺ T‐cell response to CIITA‐DN pAECs was significantly weaker than to WT pAECs (60–80% suppression; P < 0·01). Although there was a significantly lower frequency of CD4⁺ cells in the PBMCs from CIITA‐DN (20%) than from WT (30%) pigs (P < 0·01), T‐cell proliferation was similar, suggesting no significant immunological compromise. Organs and cells from CIITA‐DN pigs should be partially protected from the human cellular immune response.     

Adenosine induces prolonged anti‐β‐adrenergic effects in guinea‐pig papillary muscle

A sustained anti‐β‐adrenergic effect of adenosine has been reported. This study was initiated to investigate this topic and especially elucidate the role of protein kinase C (PKC). Contractile force amplitude and action potential duration at 90% repolarization (APD₉₀) were measured in guinea‐pig papillary muscles before and after 5 min challenge with 5 nm isoproterenol. Protocols contained 30 min exposure to the test agents adenosine 33 μm (ado), adenosine + PKC‐inhibitor bisindolylmaleimide 20 nM (ado + BIM), PKC‐activator 1,2‐dioctanoyl‐sn‐glycerol 10 μm (DOG) and α‐agonist phenylephrine 5 μm (phe). Isoproterenol was given at the end of test exposure and after 15 min washout. Results are mean ± SEM of percentage‐change, P ≤ 0.05 considered significant and labelled *. The first isoproterenol challenge significantly increased contractile force (27 ± 7%*) in the control group. Responses in the test groups were 2 ± 4 (ado), 1 ± 5 (ado + BIM), 14 ± 4* (DOG), 0 ± 2% (phe). After washout of adenosine, DOG and phenylephrine, isoproterenol induced 3 ± 8 (ado), 23 ± 5* (ado + BIM), 13 ± 5* (DOG), 15 ± 7% (phe) increase in test groups compared with 22 ± 5%* increase in contractile force in the control group. After 45 min washout of adenosine the inotropic response was still significantly reduced compared with control (29 ± 4 vs. 79 ± 8%*). Isoproterenol stimulation shortened APD₉₀ in controls at both time points (5 ± 1%* and 4 ± 1%*), with no significant shortening in test groups. Adenosine induces sustained anti‐β‐adrenergic effects on contractile force as well as APD₉₀. A role for PKC in signal transduction is supported with respect to contractile force.     

The prevalence of “anti‐HBc alone” and HBV DNA detection among anti‐HBc alone in Korea

The “anti‐HBc alone” is a frequent serological finding in clinical laboratories, making it difficult to determine whether the HBV infection has resolved. The objectives of this study were to investigate the prevalence of anti‐HBc alone and HBV DNA detection (occult HBV infection) among anti‐HBc alone, and to describe the demographic and clinical characteristics of anti‐HBc alone. A total of 17,677 sera referred from the Health Promotion Center (HPC group, 4,014 sera) as well as all the hospital clinical departments (Patient group, 13,663 sera) were tested for HBs Ag, anti‐HBc, and anti‐HBs. HBV DNA test using real‐time PCR was performed on 230 anti‐HBc alone. The prevalence of anti‐HBc alone was 8.9%, significantly higher in the Patient group than in the HPC group. The prevalence of anti‐HBc was higher in men than women and was increased with age. Very low levels of HBV DNA were found in only 4 (1.7%) out of 230 subjects with anti‐HBc alone. They were patients with conditions unrelated to chronic liver disease. Considering the high prevalence of anti‐HBc alone, the frequency of occult HBV infection among anti‐HBc alone was unexpectedly low. In addition, HBV viral load was low in these patients. Further studies are required to determine the clinical significance and infectivity of anti‐HBc alone, in conjunction with very low levels of HBV DNA and to standardize the detection methodology for both anti‐HBc alone and HBV DNA. J. Med. Virol. 82:1508–1514, 2010. © 2010 Wiley‐Liss, Inc.     

An in‐house‐anti‐hepatitis A virus (HAV)‐specific immunoglobulin M capture enzyme‐linked immunosorbent assay: Evaluation and application to an HAV outbreak

An anti‐hepatitis A virus (HAV)‐specific immunoglobulin M capture enzyme‐linked immunosorbent assay (anti‐HAV IgM ELISA) kit was re‐designed for laboratory use and compared with a commercial anti‐HAV IgM detection system using 58 serum samples collected from patients, vaccines, and healthy individuals. Because concordance between the two systems was high (r = 0.93, P < 0.05), 19 sets of serum and fecal samples obtained from individuals exposed to an HAV outbreak were also examined. Serum levels of anti‐HAV IgM were determined using the in‐house ELISA kit and the HAV genome in fecal samples was detected using the polymerase chain reaction (PCR). Among the 19 sets of sample, 14 were positive for both anti‐HAV IgM and the HAV genome. All of those whose serum samples were anti‐HAV IgM negative were also negative for the HAV genome in fecal samples. The results of the in‐house IgM ELISA were consistent with those of the HAV genome detected by PCR and with the commercial IgM ELISA. The in‐house anti‐HAV IgM ELISA kit was therefore proven suitable for laboratory use and applicable to epidemiological studies of HAV infection. J. Med. Virol. 81:1513–1516, 2009. © 2009 Wiley‐Liss, Inc.     

Cover Picture: Involvement of nonclassical MHC class Ib molecules in heat shock protein‐mediated anti‐tumor responses – Eur. J. Immunol. 6/2007

The image of the jumping frog was kindly provided by Jacques Robert and created by his son by Adrien. Anti‐tumor responses, both in vitro and in vivo, are studied using Xenopus laevis by Robert and colleagues (pp. 1494–1501); non‐classical MHC class Ib molecules are shown, by RNA interference, to be important for the gp96‐induced CTL response against class Ia‐negative tumors in Xenopus. The conservation of the class Ia‐unrestricted response from Xenopus to mammals indicates the importance of this anti‐tumor immune mechanism.     

Dual‐Functional PEI–Poly(γ‐Cholesterol‐l‐Glutamate) Copolymer for Drug/Gene Co‐delivery

Novel dual‐functional PEI‐poly(γ‐cholesterol‐l ‐glutamate) (PEI‐PCHLG) copolymers are developed for the first time. A series of PEI‐PCHLG (PEI‐1, PEI‐2, PEI‐3, and PEI‐4) with various PEI percentages and molecular weights are successfully synthesized, among which the poor organic solvent solubility of PEI‐1 precludes its further application. The other three copolymers can spontaneously self‐assemble into micelles; the critical micelle concentration (CMC) values are 0.66, 1.3, and 0.95 μmol L^?1, respectively. PEI‐2 and PEI‐4, with lower CMC, are worth being further developed as promising drug carriers because of their resistance to dilution in circulation after systemic administration. However, PEI‐4 can form smaller‐sized micelles than PEI‐2 and has similar in vitro cytotoxicity to PEI. Thus, PEI‐4 is further investigated. PEI‐4 micelles can not only incorporate docetaxel (DTX) with high encapsulation efficiency (91.0%) and drug loading (4.3%), but also load pDNA efficiently at a ratio of 8:1 (w/w). DTX‐loaded PEI‐4 micelles (DTX‐PEI‐4) can also carry genes with the same gene‐binding capacity as PEI‐4 micelles. The above three micelles (DTX‐PEI‐4, pDNA‐PEI‐4, and pDNA/DTX‐PEI‐4) are sub‐micrometer‐sized and spherical. The results indicate that PEI‐4 containing 28.9% PEI, one of the PEI‐PCHLG copoly­mers, is a potential carrier for gene delivery, drug delivery, or even drug/gene co‐delivery.