Chronic hepatitis B: clinical issues and current treatment

Infection by hepatitis B virus (HBV) can be resolved after an acute episode, or leads to persistence and chronic hepatitis B (CHB). Chronic hepatitis B virus infection is a serious clinical problem and a major cause of severe liver-related morbidity and premature mortality. Indeed, patients with CHB have an increased risk of developing decompensated liver disease, cirrhosis and hepatocellular carcinoma (HCC) [START_REF] Dienstag | Hepatitis B virus infection[END_REF]. The primary treatment goal is the suppression of HBV replication, ideally followed by a seroconversion (anti-HBe, and then anti-HBs), and the prevention of active disease in the long-term [START_REF] Zoulim | Hepatitis B: reflections on the current approach to antiviral therapy[END_REF][START_REF] Zoulim | Management and prevention of drug resistance in chronic hepatitis B[END_REF]. In this respect, the treatment of CHB with either interferons or nucleos(t)ides analogs (NAs), including lamivudine (LMV), adefovir dipivoxil (ADV), entecavir (ETV), telbivudine (LdT) and tenofovir (TDF), has resulted in a significant reduction in patient morbidity and mortality. Yet the efficacy of treatments for CHB can be affected by a number of factors, including the development of adverse side effects, poor patient compliance, previous treatment with suboptimal regimes and/or inadequate drug exposure, individual genetic variation, or infection with drug-resistant virus. As therapy with interferons ("naked" or pegylated) alone remains quite inefficient, the clinical used of nucleos(t)ide analogs has played a major part in the substantial advances in CHB treatment that have occurred over the past decade. CHB requires long-term therapy, and resistance to therapy is a frequent consequence of treatment duration. The emergence of drug resistance during long-term therapy with NAs is almost inevitable, due to the high adaptability of viruses and the quasispecies nature of HBV, and represents a clinical challenge [START_REF] Zoulim | Hepatitis B: reflections on the current approach to antiviral therapy[END_REF][START_REF] Zoulim | Management and prevention of drug resistance in chronic hepatitis B[END_REF].

For clinicians, the close monitoring and management of resistance has become a key issue in clinical practice. For HBV virologists, the understanding of the mechanism of emergence of specific mutant strains in the viral quasispecies during treatment is also an important issue. If a particular viral strain can emerge in the quasispecies within a particular environment, it is likely because its fitness has become superior to other strains. The present review will focus on viral fitness as well as infectivity, and in particular on technical means that are available to study this viral fitness in vitro or in animal models.

Treatment failure and HBV resistance

HBV is a DNA virus that replicates its genome via an RNA intermediate, the pregenomic RNA (pgRNA), that comes from the transcription of cccDNA, i.e. covalently-closed-circular-DNA, the nuclear form of HBV genome and main template for viral transcription. The pgRNA is reversetranscribed by covalently-linked-HBV-polymerase after incorporation in the nucleocapsid [START_REF] Seeger | Fields Virology[END_REF]. This step of the viral life cycle is currently the target of NA-based therapy. Long-term therapies with NAs, which are theoretically necessary to get a chance to clear cccDNA from cells, are confronted with the emergence of drugs resistant strains in the viral quasispecies. HBV mutants are spontaneously produced by the low fidelity HBV polymerase, and a drug pressure may select for viral species that exhibit the best replication capacity in this new treatment environment. Mutations conferring resistance to NAs are located in the viral polymerase gene. The rapidity of selection of drug resistant mutants depends on their replication capacity and fitness, their level of resistance, and free liver space available for infection by these mutants [START_REF] Litwin | The competing roles of virus replication and hepatocyte death rates in the emergence of drug-resistant mutants: theoretical considerations[END_REF][START_REF] Zhang | Low dynamic state of viral competition in a chronic avian hepadnavirus infection[END_REF]. This may explain, at least in part, the differences in the rate of resistance for the different drugs that are clinically available.

Different mechanisms are involved in drug-resistance under antiviral therapy [START_REF] Zoulim | Management and prevention of drug resistance in chronic hepatitis B[END_REF]. First, a complex mixture of genetically distinct variants develops under selective pressure. A pre-existing or newly acquired mutation conferring a selective advantage to a variant will give rise to virions, which are fitter and can spread more rapidly in the liver. This mutant may accumulate and become the dominant (or at least a well represented) species in the infected liver, under the pressure of the antiviral drug. The kinetics of replacement of wild type virus in liver cells by a dominant mutant are generally slow. As resistant mutants mainly infect uninfected cells, the efficient spreading of the dominant mutant depends on its intrinsic fitness and the availability of free liver space for its propagation and replication [START_REF] Litwin | The competing roles of virus replication and hepatocyte death rates in the emergence of drug-resistant mutants: theoretical considerations[END_REF][START_REF] Zhang | Low dynamic state of viral competition in a chronic avian hepadnavirus infection[END_REF]. During antiviral therapy, several months may be needed for the immune system to clear hepatocytes infected with wild type virus and to generate new cells that are susceptible to infection by viral drug-resistant mutants. On the other hand, the specific infectivity of drug resistant mutants may have a major impact on the rapidity of selection of these strains during therapy. Indeed, some mutations in the viral polymerase gene may result in nucleotide changes in overlapping surface genes, which in turn may lead to reduced viral fitness, due to impaired assembly, secretion or infectivity [START_REF] Villet | In vitro characterization of viral fitness of therapy-resistant hepatitis B variants[END_REF][START_REF] Warner | The antiviral drug selected hepatitis B virus rtA181T/sW172* mutant has a dominant negative secretion defect and alters the typical profile of viral rebound[END_REF]. The level of resistance to a drug conferred by a given mutation may have profound implication on the fitness of the mutant. This may explain the difference in drug resistance rates observed with the different antivirals.

Definition of viral fitness and rationale for studying it

In a simple way, viral fitness can be defined as the sum of parameters that quantify the adaptation of a viral strain to a given environment. In vitro, it can be seen as the growth properties of a given strain as compared to a reference or wild type viral strain in a defined environment (i.e., in tissue culture or in vivo in the presence of drugs or an intact immune system). The fitness of a given viral strain is of great importance to understand its emergence or elimination in a particular microenvironment. Of course in the case of in vivo infection other features including the genetic makeup of host and immune system are also important to explain the emergence of a particular strain, but this will not be further developed here as this review focuses on virus related parameters.

The therapeutic pressure represents also an important environmental parameter to explain the emergence of mutant strains in patients. In general, NA-resistant strains do not replicate as well as wild type counterparts, because mutations in the polymerase gene tend to affect the enzymatic activity of the protein. But this pattern of replication can be reversed in the presence of the drug(s) that selected the mutant in the first place [START_REF] Villet | In vitro characterization of viral fitness of therapy-resistant hepatitis B variants[END_REF][START_REF] Brunelle | Susceptibility to antivirals of a human HBV strain with mutations conferring resistance to both lamivudine and adefovir[END_REF][START_REF] Durantel | A new strategy for studying in vitro the drug susceptibility of clinical isolates of human hepatitis B virus[END_REF].

The virologic parameters of HBV fitness are the capacity of a strain to synthesize its genome, to produce infectious particles and (re)-infect cells (i.e. enter and deliver the genome to nucleus). The synthesis of the genome itself includes the ability of the strain to produce pgRNA (i.e. transcription via precore promoter, some mutations may alter the level of transcription mediated by host RNA polymerase II) that is subsequently packaged into the nucleocapsid before being used for reverse transcription. The study of viral fitness is a complex matter as it relies on the models used to perform analysis. Cell culture systems or animal models capable of full replication and propagation of the virus are necessary to properly measure the fitness of a viral strain. For HBV such models do no exist. Therefore to get insight into HBV fitness, several more or less artificial assays that will be hereafter named replication, assembly and infectivity assays have been developed.

Studying viral fitness in vitro: critical review of assays

Replication assays. Several in vitro assays have been developed to determine the replication capacity of clinically relevant HBV strains, including wild-type and drug-resistant strains. These assays are also critical to determine the role of a given mutation profile in drug resistance, as well as to determine the cross-resistance profile of those mutants. Several approaches and assays, also termed phenotypic assays as they describe the phenotype (i.e. replication capacity + drug resistance profile) of a given strain in vitro, have been described in literature. These assays are mainly based on the transfection of full-length HBV PCR-amplicons (i.e. vector-free) or plasmids containing 1.1 to 2 HBV genome units into hepatoma cell lines (e.g. Huh7 or HepG2).

The vector-free method relies on an original and efficient PCR amplification of full-length HBV genomes isolated from patients [START_REF] Gunther | A novel method for efficient amplification of whole hepatitis B virus genomes permits rapid functional analysis and reveals deletion mutants in immunosuppressed patients[END_REF]. The linear amplicon is transfected into cells and serves as a ccc-like DNA template for the initiation of the intracellular HBV replication after re-circularization by host machinery. This step of circularization is rate limiting and explains the rather low absolute level of replication obtained with this approach; however the relative replication capacity of two different strains can be analyzed by this approach. Another advantage of this approach comes from the fact that the expression of pgRNA is driven by the HBV promoter itself, which is interesting if precore or core promoter mutants are analyzed.

Alternative approaches are based on vectors, that can be plasmid or alternatively recombinant adenovirus or baculovirus, carrying 1.1 to 3 HBV genome units [START_REF] Wet | Hepatitis B virus replication in human HepG2 cells mediated by hepatitis B virus recombinant baculovirus[END_REF][START_REF] Durantel | Resistance of human hepatitis B virus to reverse transcriptase inhibitors: from genotypic to phenotypic testing[END_REF][START_REF] Lucifora | Initiation of hepatitis B virus genome replication and production of infectious virus following delivery in HepG2 cells by novel recombinant baculovirus vector[END_REF][START_REF] Ren | Hepatitis B virus (HBV) virion and covalently closed circular DNA formation in primary tupaia hepatocytes and human hepatoma cell lines upon HBV genome transduction with replication-defective adenovirus vectors[END_REF]. These vectors contain the HBV genetic information necessary and sufficient to initiate an HBV replication cycle after transfection or transduction into cells. The synthesis of HBV pgRNA can be driven either by the HBV promoter (i.e. 1.3 to 2 genome unit) or a strong mammalian promoter (i.e. 1.1 genome unit).

Until recently, the analysis of the replication capacity of naturally occurring or drug induced HBV mutants relied either on PCR-mediated transfer of HBV genome cassettes or on site directed mutagenesis of a well established replication-competent laboratory strain [START_REF] Allen | Identification and characterization of mutations in hepatitis B virus resistant to Lamivudine[END_REF][START_REF] Seigneres | Evolution of Hepatitis B Virus Polymerase Gene Sequence during Famciclovir Therapy for Chronic Hepatitis B[END_REF]. Despite its obvious utility to quickly characterize new mutations in vitro, the methods based on the exchange of a cassette or site directed mutagenesis do not take into account the HBV genome variability existing in other parts of the genome.

Several methods based on a standardized cloning strategy of the entire HBV genome isolated from patient into plasmid vector have been recently described [START_REF] Durantel | A new strategy for studying in vitro the drug susceptibility of clinical isolates of human hepatitis B virus[END_REF][START_REF] Yang | In vitro antiviral susceptibility of full-length 10 clinical hepatitis B virus isolates cloned with a novel expression vector[END_REF] and have proven useful to study replication capacity and drug susceptibility, as well as cross-susceptibility of clinically relevant HBV strains [START_REF] Durantel | A new strategy for studying in vitro the drug susceptibility of clinical isolates of human hepatitis B virus[END_REF][START_REF] Yang | In vitro antiviral susceptibility of full-length 10 clinical hepatitis B virus isolates cloned with a novel expression vector[END_REF][START_REF] Lacombe | A novel hepatitis B virus mutation with resistance to adefovir but not to tenofovir in an HIV-hepatitis B virus-co-infected patient[END_REF][START_REF] Villeneuve | Selection of a hepatitis B virus strain resistant to adefovir in a liver transplantation patient[END_REF][START_REF] Villet | Stepwise process for the development of entecavir resistance in a chronic hepatitis B virus infected patient[END_REF][START_REF] Villet | Impact of hepatitis B virus rtA181V/T mutants on hepatitis B treatment failure[END_REF][START_REF] Villet | Selection of a multiple drugresistant hepatitis B virus strain in a liver-transplanted patient[END_REF]. These cloning techniques enable the assembly of molecular clones, containing either 1.1 or 1.3 HBV genome unit, which allows the study of viral replication upon transfection of one clone or a mixture of clones into eukaryotic cells. By using a vector in which the expression of HBV pgRNA is driven by heterologous mammalian promoters (e.g. CMV or actin promoters), the replication level detected post-transfection, as measured by the neo-synthesis of encapsidated relaxed-circular DNA (rcDNA), is artificially elevated. However the replication capacity of two given HBV strains can be determined in a relative fashion as it measures the intrinsic ability of HBV polymerase to transform pgRNA into rcDNA [START_REF] Durantel | A new strategy for studying in vitro the drug susceptibility of clinical isolates of human hepatitis B virus[END_REF][START_REF] Yang | In vitro antiviral susceptibility of full-length 10 clinical hepatitis B virus isolates cloned with a novel expression vector[END_REF].

Vector-free and vector-based replication assays are complementary approaches to determine the phenotype (replication capacity + drug susceptibility) of clinical HBV strains. The vector-free method is interesting as the whole viral population is represented in transfected amplicons, if we hypothesize that there is no bias during amplification. Vector based assays present the advantage of strong relative level of DNA synthesis, which render the approach more universal, but the disadvantage of a cloning step. Altogether, it may be interesting to use both approaches in order to generate complementary information. According to the approach used several methods can be utilized to detect HBV replication, including Southern blotting (if the replication level is high, i.e.

when vector based assays are used) or qPCR. The first method is work intensive but gives qualitatively relevant results (i.e. visualization of all DNA forms of HBV genome), whereas the second is very sensitive but might be confronted to false positive (i.e. detection of infra-length DNA sequences). Moreover HBV replication may be monitored by quantifying intracellular or secreted DNA. If qPCR can be used in both cases, Southern blotting is more adapted for the detection of intracellular DNA due to the rather low sensibility of the methodology.

Beside the molecular approach used, the choice of the cell line for the transfection is also important as differences in term of replication capacity and drug susceptibility have been observed [START_REF] Brunelle | Susceptibility to antivirals of a human HBV strain with mutations conferring resistance to both lamivudine and adefovir[END_REF][START_REF] Seigneres | Inhibitory activity of dioxolane purine analogs on wild-type and lamivudine-resistant mutants of hepadnaviruses[END_REF].

Altogether an important effort to standardize assays intra and inter laboratory remains to be made in order to move from current data that are mainly valid in a relative context (i.e. comparison with the same approach in the same laboratory) to fully comparable data.

It is worth noting that another approach to characterize viral drug resistance in tissue culture is the use of cell lines permanently expressing the mutants, to allow a more reproducible measure of drug susceptibility [START_REF] Fu | Characterization of novel human hepatoma cell lines with stable hepatitis B virus secretion for evaluating new compounds against lamivudine-and penciclovirresistant virus [In Process Citation][END_REF][START_REF] Qi | In vitro susceptibility of adefovirassociated hepatitis B virus polymerase mutations to other antiviral agents[END_REF][START_REF] Sun | Stable HepG2-and Huh7-based human hepatoma cell lines for efficient regulated expression of infectious hepatitis B virus[END_REF][START_REF] Yang | Cross-resistance testing of next-generation nucleoside and nucleotide analogues against lamivudine-resistant HBV[END_REF]. These cell lines are extremely useful to assess the antiviral activity of approved drugs and those in development on the main resistant mutants. However, one of the problems of these permanent cell lines, is the need to design and produce new cell lines when new resistant mutants are identified.

Virion production, secretion capacity and infectivity -Assembly and infectivity assays.

Beside the ability of a strain to replicate its genome in the absence or presence of drugs, the next important step to ensure a strain a better propagation is the ability to produce particles. Some mutations occurring in the polymerase gene have a consequence in overlapping envelope genes, in particular in the S gene. For instance, the rtM204V mutation is associated with the mutation sI195M in the surface antigen, whereas the rtM204I change is associated with three possible changes: sW196S, sW196L, or a termination codon. The mutation selected by adefovir, lamivudine or telbivudine at rtA181T typically results in a stop mutation in the envelope gene (sW172stop), and the ADV-resistance mutation at rtA181V results in a concomitant change sL173F. Mutations that result in a stop codon mutation in the envelope gene are present in association with a low percentage of wild type HBV in order to ensure rescue of viral packaging and release [START_REF] Warner | The antiviral drug selected hepatitis B virus rtA181T/sW172* mutant has a dominant negative secretion defect and alters the typical profile of viral rebound[END_REF].

Beside their impact on viral assembly, mutations may also impact the antigenicity and therefore the ability of a strain to be neutralised by circulating or administrated antibodies [START_REF] Pawlotsky | The concept of hepatitis B virus mutant escape[END_REF][START_REF] Sloan | Antiviral resistance mutations potentiate hepatitis B virus immune evasion through disruption of its surface antigen a determinant[END_REF]. This may also have an impact on viral propagation and therefore represent a component of viral fitness. Several studies have examined the altered antigenicity of common antiviral drug selected mutations that also alter HBsAg. In the study by Torresi et al., the mutations rtV173L+rtL180M+rtM204V that resulted in the mutations sE164D+ sI195M in HBsAg were found to reduce antigen -antibody binding [START_REF] Torresi | Reduced antigenicity of the hepatitis B virus HBsAg protein arising as a consequence of sequence changes in the overlapping polymerase gene that are selected by lamivudine therapy[END_REF]. Although the reduction was not as great as for the classical vaccine-escape mutant at sG145R, it was greatly reduced compared with the wild-type virus. These results were recently confirmed and extended by Sloan and colleagues using cell derived HBVs [START_REF] Sloan | Antiviral resistance mutations potentiate hepatitis B virus immune evasion through disruption of its surface antigen a determinant[END_REF].

To study the impact of a given mutation on HBV assembly there are two main methodologies. The first is based on hepatomas cells replicating HBV after stable or transient transfection. Stably transformed cell lines are useful to study the assembly and secretion of HBV strains, but are workintensive to generate. Moreover, it can be quite difficult to compare two different cell lines as the sites and numbers of integration into the genome may vary from one cell line to another. By contrast, the transient transfection of cells with plasmids carrying 1.1 to 2 HBV genome units is more flexible, although, due to transfection efficiency, a lower number of cells produce HBV particles compared to stably transformed cell lines. The assembly and secretion of HBV particles is measured by quantification of secreted HBV DNA by qPCR (or Southern blot) and/or envelope proteins by Elisa or Western blot. Those measurements are not precise, as they do not distinguish between enveloped and non-envelopped nucleocapsids for qPCR analysis, and between subviral particles and Dane particles for Elisa or Western blot analysis. The amount of Dane particle produced can be more precisely quantified after immunoprecipitation with an anti-PreS1 antibody followed by qPCR [START_REF] Villet | In vitro characterization of viral fitness of therapy-resistant hepatitis B variants[END_REF]. A more laborious approach is to purify particles by ultracentrifugation in density gradients, and to analyse the production of each particles (Dane, subviral particles and non-enveloped nucleocapsids). Electron microscopy can be used to some extend to visualise and distinguish HBV particles, but is not a quantitative approach.

Other approaches to study the impact of mutation in envelope genes on viral assembly are based on the utilisation of naturally pseudotyped hepatitis delta virus (HDV) particles. Indeed, HDV utilizes the envelope proteins of HBV for propagation. When introduced into permissive cells, the HDV RNA genome replicates and associates with multiple copies of the HDV-encoded proteins to assemble a ribonucleoprotein (RNP) complex. The mechanism necessary to export the RNP from the cell is provided by the HBV envelope proteins, which have the capacity to assemble lipoprotein vesicles that bud into the lumen of a pre-Golgi compartment before being secreted [START_REF] Sureau | The role of the HBV envelope proteins in the HDV replication cycle[END_REF]. Hence the co-tranfection of HDV genome (contained as trimers in a plasmid) and plasmid either expressing HBV pgRNA (i.e. like the ones used for replication assays; see above) or coding HBV envelope proteins leads to the production of either a mixture of HBV/HDV or pure HDV particles. These models have been successfully used to determine domains and residues of S protein important for HBV/HDV assembly [START_REF] Blanchet | Analysis of the cytosolic domains of the hepatitis B virus envelope proteins for their function in viral particle assembly and infectivity[END_REF][START_REF] Jaoude | Role of the antigenic loop of the hepatitis B virus envelope proteins in infectivity of hepatitis delta virus[END_REF]. They can be used as surrogate model to study the impact of naturally or drug-selected mutations on HBV assembly. In this case assembly and secretion of HBV/HDV or HDV particles is measured by quantification of secreted HDV RNA by qRT-PCR (or Northern blot) and/or envelope proteins by Elisa or Western blot.

The infectivity of HBV particles represents another important component of viral fitness to be analyzed. To perform infectivity assays, HBV particles have to be produced. All the approaches to produced in vitro HBV particles have been already described above and include transientlytransfected or stably-transformed cells [START_REF] Villet | In vitro characterization of viral fitness of therapy-resistant hepatitis B variants[END_REF], as well as HDV based models [START_REF] Abou-Jaoude | Entry of hepatitis delta virus requires the conserved cysteine residues of the hepatitis B virus envelope protein antigenic loop and is blocked by inhibitors of thiol-disulfide exchange[END_REF][START_REF] Salisse | A function essential to viral entry underlies the hepatitis B virus "a" determinant[END_REF]. To perform comparative infectivity analysis, the critical issue is to standardize the inoculum to be used.

Standardization based only on the dosing of HBV (or HDV) genome is not ideal as non-enveloped nucleocapsid can also be produced together with infectious Dane (or HDV) particles. Therefore a double standardization based on genome and protein dosing is necessary. An interesting method is to dose immunoprecipitated (with anti preS1 antibody) HBV (or HDV) genome. Infectivity assays are performed with cells that can be infected in vitro. Currently there are three main models available: primary human hepatocytes (PHH) [START_REF] Gripon | Hepatitis B virus infection of adult human hepatocytes cultured in the presence of dimethyl sulfoxide[END_REF], primary tupaia hepatocytes [START_REF] Walter | Hepatitis B virus infection of tupaia hepatocytes in vitro and in vivo[END_REF], or HepaRG cells [START_REF] Gripon | Infection of a human hepatoma cell line by hepatitis B virus[END_REF], the last being the easiest to use because it is an established cell line. After inoculation with HBV or HDV particles, the intensity of infection is measured by either Northern blot or qRT-PCR to detect HBV or HDV RNAs. There are differences between HBV and HDV based models.

With HDV, there is a disconnection between entry and replication, as the latter occurs irrespective of the HBV mutations that are assayed for their impact on entry. With HBV, the measurement of infectivity includes the ability to enter the cells and to initiate HBV replication, and both aspects are linked. One main problem with these infectivity models is that there is no propagation of infection in PHH and HepaRG cells. Thus no competition experiments have been reported with this experimental setting, although it is one of the main objectives when it comes to compare viral fitness of two strains.

Studying viral fitness in vivo: from human to animals models. Longitudinal studies in patients

with CHB treated with antivirals are per se very interesting as they provide useful information on viral fitness. Hence, the emergence of a particular HBV mutant strain in patients treated with NAs indicates that this strain is fitter than others in this particular microenvironment. Villet et al. managed to obtain in vitro data on the viral fitness of a multi-resistant strain that emerged after treatment with lamivudine, adefovir, and anti-HBV immunoglobulins [START_REF] Villet | In vitro characterization of viral fitness of therapy-resistant hepatitis B variants[END_REF]. The in vitro data confirmed that the finally selected strain was the fittest one amongst the four strains that were yet present after few weeks of treatment in terms of replication capacity, assembly efficiency and specific infectivity. This was the first demonstration of a good correlation between clinical and laboratory findings. The main problem with longitudinal studies on patient cases is that they are retrospective. It would be interesting to have a small animal model to perform prospective studies.

One particularly important point when the viral fitness of a given drug resistant strain is concerned is to determine whether this strain can be transmitted. The first report of transmission of LMV resistant HBV was from Thibault et al. [START_REF] Thibault | Primary infection with a lamivudineresistant hepatitis B virus[END_REF]. In this case report, the transmission of LMV-resistant HBV (rtL180M+rtM204V) was associated with an incubation period of 2-3 months and resulted in a typical acute hepatitis. Obtaining evidence for the transmission of HBV mutants in humans remains difficult and opportunistic. Animal models are necessary to get further information on the infectivity of HBV mutant strains. Using the chimpanzee infection model, Kamili et al. [START_REF] Kamili | Efficacy of hepatitis B vaccine against antiviral drug-resistant hepatitis B virus mutants in the chimpanzee model[END_REF] tested the efficacy of the immunity induced by a commercial hepatitis B vaccine against challenge with a tissue culture-derived, clonal HBV polymerase mutant that contained a combination of three polymerase mutations (rtV173L, rtL180M, rtM204V), two of which resulted in changes to the overlapping viral envelope of the HBsAg (sE164D, sI195M). Evidence of HBV replication was observed in the vaccinated chimpanzees after challenge with the mutant, as well as after rechallenge with serum-derived wild-type HBV, despite robust humoral and cellular anti-HBV immune responses after hepatitis B vaccination.

Although useful, the chimpanzee model remains difficult to handle. Amongst other animals that could be used for studying the infectivity of HBV mutant strains, liver-humanized mouse represents one of the most convenient models [START_REF] Kremsdorf | New animal models for hepatitis C viral infection and pathogenesis studies[END_REF][START_REF] Meuleman | The human liver-uPA-SCID mouse: a model for the evaluation of antiviral compounds against HBV and HCV[END_REF][START_REF] Sugiyama | Direct cytopathic effects of particular hepatitis B virus genotypes in severe combined immunodeficiency transgenic with urokinase-type plasminogen activator mouse with human hepatocytes[END_REF] SCID/Alb-uPA mice can be efficiently infected with in vitro produced HBV particles [START_REF] Petersen | Prevention of hepatitis B virus infection in vivo by entry inhibitors derived from the large envelope protein[END_REF], and can be therefore used to study the infectivity of HBV mutants. Moreover HBV infected SCID/Alb-uPA mice can be treated with LMV and ADV, whereas infection with LMV-resistant strain was not sensitive to LMV treatment [START_REF] Dandri | Chronic infection with hepatitis B viruses and antiviral drug evaluation in uPA mice after liver repopulation with tupaia hepatocytes[END_REF][START_REF] Tsuge | Infection of human hepatocyte chimeric mouse with genetically engineered hepatitis B virus[END_REF]. Altogether this model could be potentially used to study viral fitness of HBV strains in mono or competitive challenges in the presence or absence of drugs. However, it is worth noting that quantitative and qualitative species-dependent differences in the enzymology of nucleos(t)ide metabolism in mouse tissues might limit the usefulness of this model since the hepatocyte's environment is influenced by other cells and tissues, that are of mouse origin in this model.

Concluding remarks

The study of HBV viral fitness in vitro is limited as there is currently no really relevant and unique cell culture model to perform such studies. In the absence of such a model, several assays have been developed to get insight into various parameters that define viral fitness, including the replication capacity, assembly efficiency, and specific infectivity of a given HBV strain. These assays have proven useful to get correlation between in vitro and in vivo data, and explain the emergence of a given strain in vivo by a better viral fitness of this one, as compared to other wild-type and mutants strains. Besides in vitro testing, animal models could be used to get further information about the fitness and the infectivity of HBV mutant strains. So far, using chimpanzees, it has been possible to demonstrate the transmissibility of HBV drug resistant strains, and using HBV vaccinated chimpanzees it has been possible to demonstrate the transmissibility of HBV vaccine escape mutants therefore pointing out the potential risk of spreading of such strains. Mice with humanized liver may also represent an interesting model to get prospective data on the fitness of clinically relevant and laboratory engineered HBV strains.


Dorit M W Zander

Dr Matthias H F Klinger
email: klinger@anat.uni-luebeck.de

The blood platelets contribution to innate host defense -what they have learned from their big brothers

Keywords: blood platelets, innate immunity, blood plasma, thrombin, reactive oxygen species blood platelets, thrombin, reactive oxygen species, plasma, innate immunity

Bactericidal effects of blood platelets have been known for more than 120 years, but the underlying mechanisms are largely obscure. Keeping in mind structural and functional analogies of platelets to neutrophiles, three different mechanisms are thinkable: Engulfment of pathogens, release of microbicidal proteins, and production of reactive oxygen species (ROS). Here, we focus on the release of ROS and a possible contribution of blood plasma and thrombin to the bactericidal effects.

Killing of bacteria was evaluated by DNA fluorescence labeling and electron microscopy.

Release of ROS by platelets was measured photometrically by cytochrome C and phenolred/peroxidase assays and was further evaluated by topological methods.

We found that (1) platelets produce 1500 times less O 2 -and 4000 times less H 2 O 2 compared to neutrophils, (2) ROS do not effect the killing rates, (3) no local enrichment of ROS was detectable. On the other hand, thrombin and plasma proteins with a MW >100 kDa are essential for bactericidal effects.

We suggest that platelets contribute to the innate host defence by providing a catalytical surface for synthesis of thrombin. In the presence of a heat-instable plasma protein, thrombin may generate a strong bactericidal complex, which is only effective in close vicinity to the platelet membrane.

Introduction

Platelets are primarily known for their crucial role in haemostatic processes but they do have far more functions. They play a role in inflammation by quickly reacting to various specific signals and releasing inflammatory mediators [START_REF] Klinger | Role of blood platelets in infection and inflammation[END_REF]. They modulate vascular reactions, release mediators for chemotaxis and stimulation of leukocytes, and participate in wound repair by release of PDGF, Angiopoietin or EGF [START_REF] Klinger | Role of blood platelets in infection and inflammation[END_REF]. In this way, platelets chip into all stages of inflammation.

Inflammation is often the consequence of invading pathogens, and it seems to be logical that platelets participate in antimicrobial host defense as they are the first cellular elements in the blood that accumulate at any site of a vascular lesion [START_REF] Yeaman | Antimicrobial host defense[END_REF][START_REF] Clawson | Platelets in bactericidal infections[END_REF][START_REF] Nachman | The platelet as an inflammatory cell[END_REF]. Various bacteria bind to platelets [START_REF] Simonet | Human platelet aggregation by Yersinia pseudotuberculosis is mediated by invasin[END_REF][START_REF] Alugupalli | Platelet activation by a relapsing fever spirochaete results in enhanced bacterium-platelet interaction via integrin alphaIIbbeta3 activation[END_REF][START_REF] Herzberg | Platelets and bacterial infections[END_REF] and are embedded and killed in platelet aggregates [8;9]. The binding of bacteria can be a direct one via receptors or can be indirect via a bridging molecule like fibrinogen [START_REF] Clawson | Platelet interaction with bacteria. VI. contrasting the role of fibrinogen and fibronectin[END_REF].

Although a bactericidal function of platelets has been described for more than 120 years now, the underlying mechanisms are not clear [START_REF] Fodor J | Die Fähigkeit des Blutes Bakterien zu vernichten[END_REF]. Analogous to neutrophils, there are 3 potential killing mechanisms that may be proposed to operate in platelets: 1. platelets store and process bactericidal peptides, 2. they have a phagocytic capacity, and 3. they synthesize reactive oxygen species (ROS). As summarized below, mechanisms 1 and 2 have been under intensive investigation for longer times, but the possible role of ROS in killing bacteria by platelets has not been studied so far and stimulated the present study.

In 1972 Nachman and Weksler detected bactericidal peptides in platelet granules [START_REF] Nachman | The platelet as an inflammatory cell[END_REF]. They were identified and described in detail in rabbits by Yeaman et al. [START_REF] Yeaman | Purification and in vitro activities of rabbit platelet microbicidal proteins[END_REF] and later on in humans by Krijgsveld et al. [START_REF] Krijgsveld | Thrombocidins, microbicidal proteins from human blood platelets, are C-terminal deletion products of CXC chemokines[END_REF] and Tang et al. [START_REF] Tang | Antimicrobial peptides from human platelets[END_REF]. But Tang et al. reported about conflicting data on bactericidal activities of purified peptides, possibly due to inhibitory effects of sodium phosphate [START_REF] Tang | Antimicrobial peptides from human platelets[END_REF], which is also used in our experiments. Moreover, Krijgsveld et al. [START_REF] Krijgsveld | Thrombocidins, microbicidal proteins from human blood platelets, are C-terminal deletion products of CXC chemokines[END_REF] needed 100-fold higher platelet concentrations to extract thrombocidins in bactericidal amounts than we used in our killing model. Therefore we asked if additional mechanisms exist that are responsible for the observed strong killing effects and one of them could be phagocytosis.

Platelets show three main phagocytic features that are typical for leucocytes [4;15]: they interact with particulate material, they contain lysosomes in the cytoplasm and they store inflammatory mediators. But true phagocytosis of bacteria, which means that the vacuoles have no permanent contact to the extracellular space, is not established in platelets yet.

Platelets enclose bacteria trapped within an aggregate or act as "covercytes" by covering the bacterial surface [START_REF] White | Platelets are covercytes, not phagocytes: uptake of bacteria involves channels of the open canalicular system[END_REF]. They also may take up bacteria into the open canalicular system [3;16]. White [START_REF] White | Platelets are covercytes, not phagocytes: uptake of bacteria involves channels of the open canalicular system[END_REF] concluded that bacteria-platelet interaction occurs spontaneously after prolonged incubation times or prestimulation of platelets. Since enclosing by platelets does not seem to alter bacteria morphologically, it is questionable if this mechanism of ingestion is bactericidal.

And finally, platelets could have the possibility to kill bacteria by synthesis of ROS [START_REF] Krotz | Reactive oxygen species: players in the platelet game[END_REF][START_REF] Maresca | Generation of hydrogen peroxide in resting and activated platelets[END_REF][START_REF] Krotz | NAD(P)H oxidase-dependent platelet superoxide anion release increases platelet recruitment[END_REF][START_REF] Wachowicz | Generation of reactive oxygen species in blood platelets[END_REF][START_REF] Leo | Platelet activation by superoxide anion and hydroxyl radicals intrinsically generated by platelets that had undergone anoxia and then reoxygenated[END_REF][START_REF] Caccese | Superoxide anion and hydroxyl radical release by collagen-induced platelet aggregation--role of arachidonic acid metabolism[END_REF][START_REF] Pignatelli | Hydrogen peroxide is involved in collagen-induced platelet activation[END_REF][START_REF] Sanner | Effects of glucocorticoids on generation of reactive oxygen species in platelets[END_REF],

which act as potent killing agents with particularly high efficacy in leukocytes [START_REF] Babior | Biological defense mechanisms. The production by leukocytes of superoxide, a potential bactericidal agent[END_REF]. In this way, neutrophils and macrophages increase oxygen consumption during phagocytosis in what has been termed respiratory burst [START_REF] Jandl | Termination of the respiratory burst in human neutrophils[END_REF]. It was observed that ROS production in neutrophils and osteoclasts may be locally restricted to areas of "ruffled membranes" [27;28]. Since we found these ruffled membranes also in platelets, especially at platelet-bacteria binding sites [START_REF] Klinger | Participation of human blood platelets in antimicrobial host defense[END_REF], we assumed that platelets could kill bacteria by the local release of ROS. Keeping in mind the analogies to neutrophils, we always investigated the ROS release by platelets and neutrophils simultaneously, taking the latter "big brothers" as the positive control.

Thus, we asked if ROS production in platelets is high enough to kill bacteria in general and if not so, if locally restricted ROS release might explain the observed killing effects. Since the amount of ROS released by platelets was not sufficient alone to explain the bactericidal effects, we further investigated the role of plasma proteins and the effect of thrombin in the observed killing model.

We tested the killing potential of gel-filtered platelets (plasma-free) in buffer and determined the killing rates after adding increasing plasma portions, furthermore after addition of heatinactivated plasma and after the addition of high (> 100 kDa) and low (<100 kDa) molecular weight plasma protein fractions.

Thrombin performs its platelet stimulation in humans via the protease activating receptors (PAR) 1 and 4 on the platelet surface [29;30] but also cleaves plasma proteins to generate fibrin [START_REF] Blomback | Structure of N-terminal fragments of fibrinogen and specificity of thrombin[END_REF].

Because the addition of thrombin was crucial to get bactericidal effects, we dissected the action of thrombin by selecting the stimulation of the protease activating receptors (PAR) 1 and 4 on the platelet surface. We activated PAR 1 with the synthetic thrombin receptor agonist SFLLRN [START_REF] Dohlman | Model systems for the study of seven-transmembrane-segment receptors[END_REF] and the PAR 4 with GYPGKF [START_REF] Hollenberg | International Union of Pharmacology. XXVIII. Proteinase-activated receptors[END_REF], and determined the killing rates.

Furthermore, we mimicked the proteolytical function of thrombin by the addition of trypsin, we inhibited thrombin activity by hirudin, and compared all killing rates. MgCl 2 and 5 mM glucose [START_REF] Krotz | NAD(P)H oxidase-dependent platelet superoxide anion release increases platelet recruitment[END_REF]. The number of contaminating leukocytes was < 0.1 %.

F

Neutrophils were separated from other cells in the BC using a Histopaque1077 ® gradient centrifugation (Sigma, Schnelldorf, Germany) for 30 min at 400 x g followed by a subsequent ammonium chloride haemolysis [START_REF] Cabanis | A rapid density gradient technique for separating polymorphonuclear granulocytes[END_REF]. Neutrophils were resuspended in RPMI 1640 ® medium (Sigma) and were counted in a Neubauer chamber.

For Killing assays, PC were prepared by plateletpheresis (Amicus ® cell separator, Baxter).

Platelet counts were adjusted to 6x10 8 /ml. PC were stored for a maximum of 3 days at 22°C under constant agitation for better gas exchange and prevention of clumping.

Gel-filtered platelets were obtained by spinning down PC for 12 min at 1200 x g onto a 50 % albumin cushion [START_REF] Timmons | Isolation of Platelets[END_REF]. Platelets were collected and resuspended in HEPES buffer containing 0.35 % BSA, 137 mM NaCl, 2.7 mM KCl, 3 mM Na 2 HPO 4 , 1 mM MgCl 2 , 5.5 mM glucose and 3.5 mM HEPES and then applied to a Sepharose 2B column (Sigma).

Bacteria suspensions

Bacillus subtilis (B. subtilis), Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus)

were grown on tryptone-soya agar plates (Oxoid, Wesel, Germany) at 37°C for 16 h. For killing experiments, bacteria were collected and suspended in 0.9 % sodium chloride to10 9 bacteria/ml.

Plasma preparation and fractionation

Human plasma was obtained by centrifugation of PC for 90 sec at 14 000 x g. To prepare heat-inactivated plasma, the obtained plasma were incubated for 20 min at 56°C. To fractionate the plasma proteins, the plasma were centrifuged through a Microcon ® filter (Millipore) YM-10 for 30 min at 14 000 g x and YM-100 for 12 min at 14 000 x g. We got 4 plasma protein fractions: < 10 kDa, > 10 kDa, < 100 kDa and > 100 kDa.

Serum preparation

Whole blood without any anticoagulants was allowed to clot until retraction of the fibrin clot.

After 30 min at 20 °C, the samples were centrifuged for 10 min at 3000 x g and the supernatant was collected.

Platelet stimulation and inhibition

To achieve maximum killing rates, platelets were stimulated with thrombin (Sigma) at a concentration of 2 U/ml for 10 min at 37°C. Inhibition of thrombin was performed by hirudin (Sigma) at a concentration of 5 antithrombin units/ml.

To stimulate the NAD(P)H-oxidase, we used phorbol myristate acetate (PMA, Sigma) at concentrations up to 1 µM. To block the NAD(P)H-oxidase, we used the unspecific NAD(P)H-oxidase inhibitor Diphenylene iodonium chloride (DPI; Sigma, 50-500 nM). Cells were preincubated with DPI for 30 min before any stimulation, and DPI was also present in the incubation medium.

For selective stimulation of the thrombin receptors, we stimulated PAR 1 with SFLLRN (SF, Sigma, 75 µM) and PAR 4 with GYPGKF (GYP, Sigma, 750 µM). To mimic a proteolytical action of thrombin, we used trypsin (Sigma, 64 000 U/ml).

Superoxide assay

The superoxide (O 2 -) production was measured by the reduction of cytochrom C as described by Krötz et al. [START_REF] Krotz | NAD(P)H oxidase-dependent platelet superoxide anion release increases platelet recruitment[END_REF]. Platelets were stimulated with PMA and thrombin or inhibited by DPI.

After incubation times of 15 min (neutrophils) or 30 min (platelets) at 37°C, the absorbance of the supernatants was measured with the spectrometer (Spectronic 20 Genesis) at 550 nm. The O 2 -dependent part of cytochrome C reduction was calculated from the absorbance difference between samples with or without addition of superoxide dismutase (SOD, 380 U/ml). The tests were performed in triplicates. The data were translated into nanomole (nM) of O 2 -using the extinction coefficient of E 550nM = 29.5 mM -1 x cm -1 .

Hydrogen

Topological detection of H 2 O 2 by fluorescence microscopy

Cells were dropped on prewarmed slides to adhere. After washing with phosphate buffered saline (PBS), 10 µM of the fluorescence dye Oxyburst Green H 2 HFF ® (Molecular Probes, Eugene, Oregon) was added. Cells were stimulated by 0.5 µM of PMA (Sigma), 160 mg/l horseradish-peroxidase were added, and samples were incubated for 30 min. Samples were washed with PBS and were imaged using the confocal laser scanning microscope (LSM 510

Meta, Zeiss, Jena, Germany). The microscope was operated at 488 nm excitation, and a 510-580 nm emission filter was used.

Topological detection of H 2 O 2 by electron microscopy

Cerium chloride reacts with H 2 O 2 to form ceriumperhydroxide, which precipitates in the form of electron dense crystals at the side of H 2 O 2 synthesis [START_REF] Briggs | Localization of NADH oxidase on the surface of human polymorphonuclear leukocytes by a new cytochemical method[END_REF]. Neutrophils or platelets were incubated on Thermanox ® Plastic Coverslips (Nunc, Wiesbaden, Germany ) for 30 min in the presence of cerium chloride (CeCl 3 ,10 mM, Sigma), with or without 1 µM PMA and B.

subtilis. Cells were diluted in ringer lactate solution (Fresenius, Bad Homburg, Germany)

with addition of 5 mM glucose according to Telek et al. [START_REF] Telek | Cerium-based histochemical demonstration of oxidative stress in taurocholate-induced acute pancreatitis in rats. A confocal laser scanning microscopic study[END_REF]. Samples were fixed with 2.5 % glutaraldehyde for 2 h and were washed in 0.1 M cacodylate puffer, pH 7.2. Postfixation was performed with 1 % osmiumtetroxide (OsO 4 ). Samples were dehydrated in ethanol and embedded in araldite (Fluka, Buchs, Switzerland). Ultrathin sections were stained with uranyl acetate and lead citrate and were viewed with a Philips EM 400.

Killing assays

For quantitative detection of dead bacteria, we used DNA double labelling with the fluorescence dye LIVE/DEAD ® BacLight ™ (Molecular Probes, Eugene, USA). The rate of The validity of the LIVE/DEAD ® staining method was proven by the conventional colony counting method: mixed bacteria suspensions with 0, 25, 50, 75 and 100 % of dead bacteria were checked both by manual counting following LIVE/DEAD ® staining and by counting of the colony forming units (CFU) after plating bacteria on agar and incubation for 24 h at 37°C.

In agreement with the LIVE/DEAD ® data sheet, we found an excellent correlation between growing on agar plates and LIVE/DEAD ® staining.

To appraise the direct hurtful effect of H 2 O 2 on B. subtilis we developed an automated method. Killing of bacteria was achieved by adding increasing concentrations of H 2 O 2 .

96-well microtiter plates were loaded with 50 µl of bacteria suspension and H 2 O 2 in concentration between 50 µM to 50 mM. After 10 min incubation time, bacteria were labelled with 5 µM LIVE/DEAD® and killing was determined by measuring the emitted fluorescence signal with the LSM 510 Meta.

Three images per well were recorded using the multitrack mode. Numbers of green and red pixels between intensities 50-255 were counted automatically. We calculated the killing rates by dividing the number of red pixels by the sum of red and green pixels. Double coloured pixels (colocalised) were excluded.

Statistics

All data are expressed as mean ± standard deviation (SD to 260 ± 15 nM (Fig. 2).

Topological fluorescence analysis of H 2 O 2 -production by platelets

Since the level of ROS in the supernatant is not able to reflect a possible local enrichment of ROS, we tried to identify ROS topologically by fluorescence microscopy. PMA-stimulated neutrophils showed very bright fluorescent dots, likely phagolysosomes (Fig. 3A). If platelets were stimulated with PMA, we found a very weak fluorescence signal surrounding some aggregated platelets (Fig. 3B). At bacteria-platelet-binding sites, no particular H 2 O 2 signal was detectable.

No colocalization of H 2 O 2 -production and killing of bacteria

Since fluorescence detection was unable to demonstrate any locally restricted ROS-synthesis in platelets, we further tried to detect H 2 O 2 -release by ceriumperhydroxide precipitation as visualized by electron microscopy.

As reported by Repine et al. [START_REF] Repine | The influence of phorbol myristate acetate on oxygen consumption by polymorphonuclear leukocytes[END_REF], in PMA-stimulated neutrophils the external surface of the plasma membrane was coated with a thick layer of electron dense material representing this precipitation. To assure about the validity of the H 2 O 2 detection by the CeCl 3 method, we added catalase to the activated neutrophils. Catalase abolishes the released H 2 O 2 . Following addition of catalase, PMA-activated neutrophils showed no ceriumperhydroxide crystals on the plasma membrane. Unstimulated neutrophils displayed ceriumperhydroxide crystals only at a few sites.

In coincubation experiments of PMA-stimulated neutrophils with B. subtilis, most bacteria were phagocytosed. Within vacuoles of some neutrophils, ceriumperhydroxide crystals are seen, which indicates the local synthesis of H 2 O 2 (Fig. 4 A-D).

In contrast to neutrophils, platelets never showed any ceriumperhydroxide crystals at the plasma membrane, regardless of any stimulation. Although a lot of viable and dead B. subtilis were enclosed in platelet aggregates or in platelets themselves, no electron dense material was detectable at binding sites of B. subtilis, suggesting that no H 2 O 2 was produced at these sites (Fig. 4 E-H).

Promoting platelet ROS-generation does not lead to higher killing rates B. subtilis is highly susceptible to thrombin-stimulated platelets with killing rates to nearly 100 % within 10 min. We compared these killing rates with those achieved following PMAstimulation of platelets. PMA does not yield any significant killing effect within 10 min

Inhibition of O 2 -does influence killing rates partially

To determine if the killing is ROS dependent, we also inhibited the superoxide generating that would be needed for effective killing of this bacteria.

Killing rates are dependent on addition of plasma

Blood plasma contains many adhesive proteins, which could enhance the binding of bacteria to platelets and thereby change the rate of killing. To determine the role of plasma components in the bactericidal mechanism of platelets, we used gel-filtered platelets with addition of increasing plasma portions.

Thrombin-stimulated platelets in buffer killed only 22 ± 22 % of the added bacteria, which was significantly less than the maximum control in plasma (p<0.001, Wilcoxon, n=3). But 13 already a proportion of 20 % plasma completely regains the bactericidal effect (100 %). If the plasma was heat-inactivated, no killing was observed (8 ± 3 % versus baseline 11.1 ± 2.8 %).

Plasma but not Serum is bactericidal after addition of thrombin

Because plasma seemed to be crucial for yielding high killing rates of B. subtilis, we tested if plasma can kill these bacteria without the addition of platelets.

Plasma without platelets showed no significant killing (19 ± 4 %). The addition of thrombin to plasma resulted in a high killing rate of 84 ± 17 %, which was still significantly below the maximum control shown in fig. 5 (p=0.0011, Wilcoxon). The inhibition of thrombin activity by hirudin completely aborted bactericidal effects, also the incubation of B. subtilis with thrombin alone, without addition of plasma, did not show any significant killing (17 ± 8 %).

To exclude complement or antibody effects in the killing assay, we also tested the bactericidal effects of serum. But in the presence of serum there was no killing observed with (18 ± 7 %)

or without the addition of thrombin (19 ± 6 %).

Plasma protein fraction > 100 kDa mediates the high killing rates

To determine the plasma component, which cause the high killing rates, we fractionated the plasma proteins with two filters in the MW ranges > 10 kDa, < 10 kDa, > 100 kDa and < 100 kDa and tested the killing rates after addition of these fractions to gel-filtered platelets.

It was determined, that the fraction > 100 kDa was responsible for a killing as high as 91 ± 8 %. The effects of the fractions > 100 kDa and > 10 kDa were not significantly different from killing rates in normal plasma (max-control), which reached 95 ± 5 % (p= 0.34, Wilcoxon). In contrast, the fractions < 100 kDa showed a decreased killing of 33 ± 8 % and the fraction < 10 kDa reached only 19 ± 8 % (Fig. 6).

Thrombin interaction with platelet receptors is not responsible for killing B. subtilis

In our experiments thrombin was always crucial for killing of B. subtilis. Therefore we studied which effect of thrombin was responsible for the observed killing. Thrombin activates human platelets via the protease activating receptors (PAR) 1 and 4. We dissected these effects by using the synthetic thrombin receptor agonists SFLLRN (SF) for activating the platelet PAR 1 and GYPGKF (GYP) for activating PAR

Discussion

While the critical role of platelets in haemostasis, thrombosis and inflammation is clearly identified, their contribution to the innate host defense has received much less attention though the bactericidal properties of blood and platelets have been described for the first time as early as one century ago [START_REF] Fodor J | Die Fähigkeit des Blutes Bakterien zu vernichten[END_REF].

In analogy to leukocytes, 3 basic bactericidal mechanisms are thinkable to exist in platelets:

1. storage and processing of bactericidal proteins, 2. phagocytosis and 3. synthesis of reactive oxygen species (ROS).

Storage and processing of bactericidal proteins

In 1960 Hirsch et al. [START_REF] Hirsch | Comparative bactericidal activities of blood serum and plasma serum[END_REF] demonstrated bactericidal activities of rabbit serum that are not present prior to clotting. He noticed that plasma regains its bactericidal effect after addition of platelets but not after addition of leukocytes or erythrocytes. Investigations on that subject were intensified in the 1970`s. Donaldson et al. [START_REF] Donaldson | beta-Lysin of platelet origin[END_REF][START_REF] Roberts | Release of beta-lysin from platelets caused by antigen-antibody complexes, purified enzymes, and platelet-aggregating substances[END_REF][START_REF] Tew | Release of beta-lysin from platelets by thrombin and by a factor produced in heparinized blood[END_REF] isolated β-lysin as one bactericidal protein, which is stored in rabbit platelets at high concentrations but also occurs to a lower extend in human platelets. Since then several authors focussed on isolating the supposed bactericidal proteins derived from platelets. Nachman et al. [4;44] and the group leaded by Yeaman [START_REF] Yeaman | Purification and in vitro activities of rabbit platelet microbicidal proteins[END_REF][START_REF] Yeaman | Partial characterization and staphylocidal activity of thrombin-induced platelet microbicidal protein[END_REF][START_REF] Yeaman | Platelet microbicidal protein enhances antibiotic-induced killing of and postantibiotic effect in Staphylococcus aureus[END_REF][START_REF] Yeaman | Staphylococcus aureus susceptibility to thrombin-induced platelet microbicidal protein is independent of platelet adherence and aggregation in vitro[END_REF] isolated and characterized platelet microbicidal proteins in rabbits. Later on Krijgsveld et al. described analogous bactericidal proteins in human platelets that are released upon thrombin stimulation [START_REF] Krijgsveld | Thrombocidins, microbicidal proteins from human blood platelets, are C-terminal deletion products of CXC chemokines[END_REF]. Surprisingly, these proteins differ from not bactericidal proteins by a proteolytical truncation of only 2 amino acids. The possibility exist, that thrombin or other extracellular proteases cleave a precursor protein, which contributes to the generation of bactericidal proteins [START_REF] Yeaman | Antimicrobial host defense[END_REF]. According to these findings, Tang et al. [START_REF] Tang | Antimicrobial peptides from human platelets[END_REF] could identify more bactericidal proteins in human platelets, but two of them are only present in stimulated platelets.

In our model of killing of B. subtilis, however, several lines of evidence militate against a main role of bactericidal proteins. Platelet releasates, in our hands, only exert moderate killing effects [START_REF] Klinger | Participation of human blood platelets in antimicrobial host defense[END_REF], and we observed a massive killing of bacteria with thrombin-stimulated platelets at about 100fold lower platelet concentrations in comparison to Krijgsveld et al. [START_REF] Krijgsveld | Thrombocidins, microbicidal proteins from human blood platelets, are C-terminal deletion products of CXC chemokines[END_REF].

Moreover, in earlier investigations we could show, that even completely degranulated platelets have bactericidal capacity [START_REF] Klinger | Participation of human blood platelets in antimicrobial host defense[END_REF]. A possible explanation for these conflicting data on bactericidal activity of platelet releasates is presented by Tang et al. [START_REF] Tang | Antimicrobial peptides from human platelets[END_REF], who supposed an inhibiting effect of sodium phosphate which is used in most buffer systems.

Phagocytosis

Lewis et al. [START_REF] Lewis | Phagocytosis in human platelets: localization of acid phosphatase-positive phagosomes following latex uptake[END_REF] demonstrated for the first time that platelets really phagocytose small particles (<0.1 µm), which means that lysosomes fuse with the phagosome and no luminal continuity exists to the outer space. Latex particles were endocytosed through the OCS and reached the cytoplasm in small vesicles. But this way of ingestion is doubtful for bacteria with its much larger size (2 µm). Resuming the works of decades, Clawson found no evidences for a real phagocytosis of bacteria [START_REF] Clawson | Platelets in bactericidal infections[END_REF]. He observed that stimulated platelets recruit other platelets and the growing platelet aggregate surrounds initially adhered bacteria. There they are well separated from the surrounding media and usually appear within interspaces. But really engulfed bacteria are rarely to be seen in platelets [START_REF] Clawson | Proceedings: Products of platelet aggregation and release as a chemotactic factor for human leucocytes[END_REF]. Although granule secretion at plateletbacteria binding sites was observed, no killing of bacteria was described [START_REF] Clawson | Platelets in bactericidal infections[END_REF]. That is contrary to our investigations. Youssefian et al. [START_REF] Youssefian | Host defense role of platelets: engulfment of HIV and Staphylococcus aureus occurs in a specific subcellular compartment and is enhanced by platelet activation[END_REF] postulated an engulfment of Staphylococcus aureus in platelets, which is enhanced after stimulation. But he did not point out that killing occurs after engulfing. From White's updated point of view, bacteria are too big to be actively and completely enclosed within platelets. Therefore platelets may just cover bacteria as "covercytes" to separate this foreign material from the blood stream in vivo [START_REF] White | Platelets are covercytes, not phagocytes: uptake of bacteria involves channels of the open canalicular system[END_REF].

Synthesis of reactive oxygen species

After phagocytosis by neutrophils, ROS play an important role in the definitive killing of pathogens. Several clues exist that also platelet-derived ROS could be engaged in host defense: ROS-dependent cytocidal effects were described in the platelet-mediated killing of helminths [START_REF] Joseph | IgE-dependent platelet cytotoxicity against helminths[END_REF], ruffled platelet membranes were observed at bacteria binding sites [START_REF] Klinger | Participation of human blood platelets in antimicrobial host defense[END_REF], and a decreased killing of bacteria was observed to occur in the presence of antioxidants (own unpublished data). Thus we tried to establish the role of ROS in killing B. subtilis by platelets. First hints that platelet-derived ROS have cytocidal effects was given by Joseph et al. in IgEmediated killing of helminths [52;55]. This hypothesis is aided by our findings of ruffled membranes at platelet-bacteria binding sites which suggested similarities to ROS generating neutrophils [START_REF] Robinson | The NADPH oxidase complex of phagocytic leukocytes: a biochemical and cytochemical view[END_REF] and osteoclasts [START_REF] Key | Superoxide and bone resorption[END_REF]. In these cells, ruffled membranes are generated by actin polymerization and become sites of assembly of the NAD(P)H-oxidase complex. In this way, neutrophils and osteoclasts produce locally restricted large quantities of ROS to kill bacteria or to remodel the bone matrix. In addition to ruffled membranes, we observed a decreased killing rate by addition of the antioxidant vitamin E (α-tocopherol) in earlier investigations in our laboratory (unpublished data).

Based on these reports and observations, we assumed a possible role for platelet-derived ROS in our bactericidal model. We focussed in our experiments on detecting the release of O 2 -and H 2 O 2 quantitatively as well as topologically, and we studied the influence of stimulation and inhibition of ROS-synthesis on the resulting killing rates.

B. subtilis is highly susceptible to the killing abilities of thrombin-stimulated platelets. It is a

Gram-and catalase-positive bacterium, which has 3 catalases [START_REF] Loewen | Multiple catalases in Bacillus subtilis[END_REF], and it should be well protected against the cytocidal effects of H 2 O 2 . In our experiments we detected that one platelet does produce about 1500 times less superoxide and 4000 times less H 2 O 2 compared to one neutrophil. Although the concentration of platelets in native blood is 30 times higher and under our experimental conditions even 100-500fold higher than the physiological The detection of very low levels of ROS with subsequent high standard deviations and the disappointing weak reaction to stimulators led us to the assumption, that aggregated platelets can not release all generated ROS into the supernatant. In this way, freshly synthesized ROS inside a platelet aggregate will be converted before it will be detected in the supernatant. That might explain the low ROS detection when strong aggregating agents like thrombin were used.

Another critical factor in the ROS assay is that the isolation process prestimulates platelets in a donor-dependent fashion, so that it is difficult to further increase ROS production with Platelets might have found their own way, to participate in host defense.

Role of Thrombin and Plasma Proteins

In all experiments it was remarkable that the presence of plasma as well as a stimulation by thrombin was essential to kill B. subtilis. Gel-filtered and thrombin-stimulated platelets showed no killing effects until at least a portion of 20 % plasma was added. In contrast, heatinactivated plasma, a plasma protein fraction < 100 kDa, serum or thrombin alone offered no killing potential. Therefore complement or antibody action in that killing can be excluded, because these components are also present in the serum.

Furthermore, the action of thrombin was mimicked by the synthetic thrombin receptor agonists SFLLRN or GYPGKF, which stimulated the platelets without having any proteolytic effects [32;33]. Although the platelets were activated and degranulated, no remarkable killing of B. subtils occured.

In conclusion, the observed bactericidal effects do not seem to be mainly due to an active contribution of platelets, like bactericidal peptides, the production of ROS or phagocytosis.

The present work rather suggests that platelets contribute to the innate host defense by providing a catalytical surface for synthesis of thrombin. Thrombin was always added in our in-vitro experiments, but can be generated in vivo only in close contact to the platelet surface.

Inhibition of thrombin by hirudin completely abolishes all bactericidal activity of plasma in our experiments. In the presence of a heat-instable plasma protein with a MW > 100 kDa, the Compared to controls, thrombocytopenic rabbits had higher densities of streptococci per valve [START_REF] Sullam | Effect of thrombocytopenia on the early course of streptococcal endocarditis[END_REF].

In several clinical studies it was described, that thrombocytopenia in man is also accompanied by higher morbidity and mortality. An increased incidence and severity of lobar pneumonia in elderly individuals with thrombocytopenia was described [START_REF] Feldman | Comparison of bacteraemic community-acquired lobar pneumonia due to Streptococcus pneumoniae and Klebsiella pneumoniae in an intensive care unit[END_REF], thrombocytopenia was found to be a significant predictor of mortality in pneumococcal meningitis [START_REF] Kirkpatrick | A review of the clinical presentation, laboratory features, antimicrobial therapy and outcome of 77 episodes of pneumococcal meningitis occurring in children and adults[END_REF], and in liver transplant recipients thrombocytopenia is an independent and significant predictor associated with increased infection and related morbidity and mortality [START_REF] Chang | Thrombocytopenia in liver transplant recipients: predictors, impact on fungal infections, and role of endogenous thrombopoietin[END_REF].

However, the less pathogenic bacteria species B. subtilis and E. coli were steadily killed in our experiments, whereas S. aureus could not be killed. Thus, the effect of platelet-mediated antibacterial defense mechanisms seems to be limited to less pathogenic species, or vice versa, the ability to escape from the platelet-mediated defense makes bacteria much more pathogenic.

F

  The blood platelets contribution to innate host defensewhat they have learned from their big brothers

  calculated by dividing the number of red stained bacteria by the sum of red and green stained bacteria and was expressed as % killing.For killing assays, platelets were mixed with B. subtilis in a ratio of 1:1. After addition of stimulators or inhibitors (thrombin, PMA, DPI), samples were incubated on glass slides for 10 up to 45 min. Cells were labelled with LIVE/DEAD® and were imaged using the fluorescence microscope (Axiophot ® , Zeiss, Oberkochen, Germany) at 630 x magnifications for every sample. Three optical fields containing a continuous layer of platelets were chosen. 300 bacteria were counted with respect to their labels. Baseline percentages of dead bacteria were determined by LIVE/DEAD ® labelling of B. subtilis after suspension in sodium chloride without any additives. Maximum controls were performed by 10 min coincubation of LIVE/DEAD ® -labelled B. subtilis and thrombin-stimulated platelets (2 U/ml).

12 ( 13 ± 6 %

 12136 versus baseline 8 ± 3 %, p>0.05, Wilcoxon). After 20, 30 and 45 min incubation time, the PMA-stimulated platelets only reached a moderate killing up to 50 ± 20 %, which was significantly higher than the control but did not reach the killing efficacy of stimulation with thrombin (Fig.5, p<0.001, Wilcoxon, n=5).

2 -) 2 -)

 22 The term ROS contains several molecular species with different properties. The key role has superoxide anion (O as the first molecule in the complete reaction chain. Being unstable, it is converted to peroxynitrite or by superoxide dismutase (SOD) to hydrogen peroxide (H 2 O 2 ). H 2 O 2 itself is strongly bactericidal and can be further converted into other bactericidal oxygen species. In 1977, Marcus et al.[53] found superoxide (O production and in 1982 Finazzi-Agro et al. [54] could detect hydrogen peroxide (H 2 O 2 ) release from platelets.

  stimulators. Low amounts and prestimulated, aggregated platelets hamper the exact quantification of ROS release. Another delicate point is that platelet concentrates are not totally free of neutrophils. To assure about the cellular source of ROS (neutrophils or platelets), we used a direct local detection of ROS in addition to the cytochrom C or phenolred/peroxidase assays. Both electron and fluorescence microscopy revealed that no locally restricted ROS synthesis could be found at platelet-bacteria binding sites. In contrast neutrophils show a strong ROS production as visualized with both microscopical methods, whereas in platelets nearly no detection of H 2 O 2 is possible. Finally we investigated and compared the killing effects of PMA-and thrombin-stimulated platelets to determine the role of ROS in killing B. subtilis. Stimulation by PMA yields only a moderate killing rate after prolonged time compared to platelet stimulation by thrombin, which kill with high efficiency within a few minutes. Furthermore we preincubated platelets with DPI, an unspecific inhibitor of NAD(P)H-oxidase in neutrophils. DPI preincubated platelets do change their thrombin-dependent killing of bacteria partially. Hence neither PMA did increase the killing of B. subtilis nor did DPI inhibit the enormous thrombin-mediated killing clearly. So until now, neither bactericidal peptides nor phagocytosis or ROS release can explain the mechanism of platelet-mediated bactericidal action, and we have to conclude that the platelets obviously have learned not enough from their big brothers, the granulocytes. But what establishes their bactericidal activity, which is clearly observed both in vitro and in vivo?

  to generate a bactericidal compound, which is responsible for the observed killing.Distinct evidence from animal and clinical studies exist, that platelets are effectively engaged in innate host defense. Several in vitro and animal studies have shown that the susceptibility of bacteria to platelet-mediated defense mechanisms influences the induction and progression of endovascular infections[START_REF] Sullam | Effect of thrombocytopenia on the early course of streptococcal endocarditis[END_REF][START_REF] Bayer | In vitro resistance to thrombin-induced platelet microbicidal protein among clinical bacteremic isolates of Staphylococcus aureus correlates with an endovascular infectious source[END_REF][START_REF] Mercier | Influence of platelets and platelet microbicidal protein susceptibility on the fate of Staphylococcus aureus in an in vitro model of infective endocarditis[END_REF][START_REF] Kupferwasser | In vitro susceptibility to thrombin-induced platelet microbicidal protein is associated with reduced disease progression and complication rates in experimental Staphylococcus aureus endocarditis: microbiological, histopathologic, and echocardiographic analyses[END_REF]. For example, Sullam et al. tested the effects of an induced thrombocytopenia in rabbits on heart valve endocarditis after infection with Streptococcus sanguis.

Fig. 1 Release

 1 Fig.1

Fig. 2 Release

 2 Fig.2

Fig. 3 Topological 26 Fig. 4 AFig. 5 Killing 27 Fig. 6 Killing

 32645276 Fig.3

peroxide assay

	The rate of hydrogen peroxide (H 2 O 2 ) production was detected by the peroxidase/phenolred
	assay as described by Cabanis et al. [34]. Cells were incubated for 30 min (neutrophils) or
	45 min (platelets) at 37°C in the presence of 17 U/ml horseradish-peroxidase (Sigma) and
	0.2 mg/ml phenolred in buffer. After addition of NaOH the absorbance in the supernatant was
	measured at 610 nm. The tests were performed in triplicate. Concentrations of H 2 O 2 were
	calculated by means of a standard curve ranging till 0.6 µmol/l. To additionally detect
	released O 2	-, we applied superoxide dismutase (SOD), which catalyzes the conversion of O 2	-
	into H 2 O 2 .
		F o r
		P
		e
		e r
		R
		e
		v i e
		w

Low H 2 O 2 -formation in platelets in contrast to neutrophils

  Thrombin-stimulated platelets in plasma are capable to kill B. subtilis and E. coli up to 97 %, but were not able to kill S. aureus. All following killing tests were performed with the most susceptible bacteria, B. subtilis, which also yielded the brightest fluorescence labeling.

	Coincubation with SOD further enhances the maximum H 2 O 2 production from 219 ± 22 nM
	O 2	--production in platelets is very low compared to neutrophils
	To see if platelets release ROS in bactericidal amounts, we first investigated the O 2	-release
	photometrically. Platelets produce a small and hardly detectable amount of superoxide.
	F o r Resting platelets show a basal production of 0.2 ± 0.27 µM O 2 Stimulation with 1 µM PMA led to a 2.4-fold increase of O 2 -release on average (0.47 ± 0.46 -/min/7x10 8 platelets (n=9). µM O 2 -). Thrombin (2 U/ml) led to an even lower increase of O 2 -production up to 1.5-fold, again in only some preparations (0.33 ± 0.14 µM O 2 -, n=5). F o r
				P P
				e e
				e r e r
	more O 2	R -after PMA stimulation compared to one platelet. By addition of DPI, the maximum R
	O 2	-release was reduced from 1.58 ± 0.17 µM O 2	e /min/10 6 neutrophils to 0.66 ± 0.08 µM -e
	v i e (p<0,001, Wilcoxon, n=5). Thrombin had no effect on neutrophils (Fig.1). v i e
	O 2	w -is unstable and has a short half live time in the millisecond range. It is converted into w
	H 2 O 2 , which is detectable with the phenolred/peroxidase assay. Resting platelets produce
	80 ± 50 nM H 2 O 2 /min/10 9 platelets (n=5). Following stimulation with 1 µM PMA, platelets
	release significantly more H 2 O 2 (115 ± 30 nM H 2 O 2 , p= 0.03, Wilcoxon). Addition of DPI
	holds the H 2 O 2 basal release to 64 ± 41 nM. SOD converts generated O 2	-into H 2 O 2 . Thus the
	addition of SOD in this assay can detect indirectly the O 2	-release, too. In the presence of
	SOD, PMA stimulation fails to further enhance the H 2 O 2 production (Fig. 2) These findings
	correspond to the low detection of O 2
				11

). Statistical significance (p<0.05) was determined by using the Wilcoxon rank sum test performed by JMP® software (SAS Institute) and was characterized at the figures by an asterisk (*). 10 Results Killing rates of B. subtilis, E. coli and S. aureus diverge In general, no stimulators were able to induce platelet O 2 -formation significantly, since platelets of some donors did not respond. If platelet stimulation with PMA was performed in the presence of the unspecific NAD(P)H-oxidase inhibitor DPI, we observed a tendency of decreasing O 2 -production from 0.47 ± 0.46 µM to 0.34 ± 0.51 µM O 2 -.

In contrast, neutrophils strongly respond to PMA. One neutrophil produced about 1500-fold -in the cytochrome C assay (Fig.

1

).

In contrast to platelets, neutrophils produce about 4000-fold more H 2 O 2 on a single cell basis. 1 µM PMA increases the H 2 O 2 production from 134 ± 36 nM H 2 O 2 /min/10 5 neutrophils up to 219 ± 22 nM (p<0.001, Wilcoxon). DPI reduces the basal production to 25 ± 15 nM.

  concentration of neutrophils, the detected ROS release does not reach bactericidal dimensions for B. subtilis at all. Consistent with your observation that 50 mM H 2 O 2 kills 25 % of B. subtilis within 10 min, Murphy et al. demonstrated that 10 mM H 2 O 2 is enough to kill almost all B. subtilis by a longer incubation time of 1 h [56].

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Acknowledgements

This work was supported by the Deutsche Forschungsgemeinschaft (GRK 288).

We want to thank Dr. P. Schlenke and Dr. S. Görg from the Institute of Immunology and Transfusion Medicine at the University of Lübeck for providing us with platelet concentrates and buffy coats. The excellent technical assistance by Tine Asmus, Kerstin Fibelkorn and Jutta Endler is gratefully acknowledged.

The authors have declared no conflict of interest.

Wiley-VCH Biotechnology Journal