Francisco Gilabert

Jean-Noël Roux

Antonio Castellanos

Discrete simulations of

Keywords: cohesive granular material, rolling resistance, fractal, colloid aggregation, consolidation, plasticity index, strength PACS: 81.05.Rm, 81.20.Ev, 83.80.Kn, 81.40.Lm

come

Discrete simulation of model, loose cohesive powders: plastic consolidation, fractal microstructure and tensile strength

Francisco Gilabert * , Jean-Noël Roux † and Antonio Castellanos *

INTRODUCTION

Cohesive granular systems, such as some soil materials [START_REF] Mitchell | Fundamentals of soil behavior[END_REF] and powders [START_REF] Castellanos | [END_REF], can form very loose structures, which undergo considerable plastic compaction under growing stress intensity [START_REF] Mitchell | Fundamentals of soil behavior[END_REF]3] and can resist tension. The present communication investigates those phenomena by discrete element simulations in quasistatic conditions. Such a situation, with loose systems, has hardly been addressed by numerical means, as the recent literature rather explored dynamic compaction [START_REF] Wolf | Compaction of cohesive powders[END_REF][START_REF] Bartels | [END_REF], gravity deposition [6], steady flows [7], or denser materials [8,9]. We introduce a simple model material and report on its consolidation properties, in relation to its microstructure, thus presenting a brief account of studies published in two recent papers [10,11], to which a section with new results on the resistance to tension is added. The paper ends with a short discussion.

MATERIAL AND INTERACTION LAWS

Just like in Refs. [10,11], which might be consulted for more details on the model, we consider 2D assemblies of N disks, with diameters uniformly distributed in the interval [a/2, a]. The disks interact with their contacting or very close neighbors, with forces that combine normal and tangential elasticity (involving spring constants K N , K T ), Coulomb friction (with friction coefficient µ), some viscous damping (the values of which are irrelevant in quasistatic conditions), and some simplified form of van der Waals attraction. The latter consists in an attrac-tive force which vanishes beyond the value D 0 = a/1000 of the gap h separating neighbors, varies linearly with h to reach its maximum intensity F 0 for h = 0 and stays constant for h < 0, i.e., when the particles are in contact. We also introduce rolling resistance (RR) in some samples, hence a rolling friction parameter (a length) µ R such that the local rolling moment Γ at a contact satisfies |Γ| ≤ µ R F e N , with F e N the repulsive elastic part of normal force F N . RR is physically attributed to small asperities and modeled similarly to standard sliding friction [10], whence a rolling stiffness constant K R . For contact deflection -h 0 = F 0 /K N , while the static normal force -K N h+F 0 vanishes, the maximum tangential force is µF 0 and the maximum rolling moment is µ R F 0 .

Table 1 lists the values of micromechanical parameters used for most simulations, in dimensionless form.

µ aK N F 0 K T K N D 0 a K R K N a 2
µ R a 0.5 10 5 1 10 -3 10 -4 0 or 0.005

INITIAL CONFIGURATIONS

In an initial stage, a granular gas is generated with solid fraction Φ I and a Maxwell velocity distribution, corresponding to mean quadratic velocity V 0 for particles of diameter a and mass m. V 0 should be compared to the value V * = √ 2F 0 D 0 of the receding velocity in a contact that is necessary to overcome attraction. While the disks collide and stick to one another, in a process akin to isotropic ballistic aggregation, Φ is kept constant, until all particles form one unique cluster. Then, a nonvanishing pressure P is applied to the periodic rectangular simulation cell, the edge lengths of which vary according to an adapted Parrinello-Rahman scheme [10]. The strain rate ε is constrained by condition εT 0 < 0.05, involving characteristic time T 0 = am/F 0 . The dimensionless ratio P * = aP/F 0 conveniently expresses the competition of confining pressure with contact adhesion. This procedure is applied to 5 samples of N = 1400 disks, 4 with N = 5600 and 1 with N = 10976, with the parameters of Table 1, and Φ I = 0.36. The application of the first nonzero confining pressure, P * I = 0.01, is regarded as part of the assembling process. It entails a substantial density increase, to Φ = 0.524 ± 0.008 with RR and to Φ = 0.472 ± 0.008 without RR. One such configuration in Fig. 1. Coordination number z is about 3.1 without RR and 2.9 with RR in such states. It decreases to nearly 2 if a large value µ R /a = 0.5 is adopted.

PLASTIC CONSOLIDATION

The pressure is then stepwise increased, from cohesiondominated states at small P * to confinement-dominated ones at large P * . In each step, P * is multiplied by a constant factor 10 1/8 , and one waits for equilibrium to be satisfied with good accuracy: the net force -and moment -on each particle is lower than 10 -4 F 1 -resp. 10 -4 F 1 a -with F 1 = Max{F 0 , aP}, with a similar tolerance on controlled internal stresses. The stability of such equilibrium states can also be checked with stiffness matrcies [11]. Considerable structural changes are witnessed under growing P * , as the initial loose structure (Fig. 1) gradually collapses and loses its porosity. The compaction is stopped at P * = 13.3, above which no significant further compaction is observed. The sample structure and force distribution is then similar to that of a cohesionless system, as apparent in Fig. 2. The evolution of density with pressure during compaction is shown in Fig. 3. No size dependence is observed and the data of all samples are averaged over. Conforming to the literature, in which the void index e = -1 + 1/Φ is used as a state variable, such consolidation curves can be represented as 1/Φ versus ln P * . They are quite sensitive to a small RR, and can be modeled as follows, introducing three regimes for growing P * . In the first one, for P * of the order of P * I , the first applied nonvanishing pressure, the initial structure resists the pressure increase, and Φ stays constant. Then, in regime II, pressure increments cause irreversible compaction and a straight line is ap-FIGURE 3. Consolidation data and fit to Eq. ( 3), for systems with (red triangles) and without (black dots) a small RR. Dashed lines: fits to Eq. ( 1). Continuous lines: fits to Eq. ( 3) and (bottom) decompression from highest P * . proached as N → ∞. This is the classical form of consolidation curves, defining a plasticity index λ :

1 Φ = 1 Φ 0 -λ ln P * P * 0 . (1)
In Eq. ( 1), values Φ 0 and P * 0 correspond to the end of regime I, and depend on the initial assembling method. However, we could check [11] that various factors affecting Φ 0 and P * 0 , such as Φ I or V 0 /V * , become irrelevant once regime II is reached. The consolidation behavior expressed by Eq. 1 is thus intrinsic, independent on the initial state. Finally, a maximum solid fraction Φ max is approached at large P * , as a power law,

1 Φ = 1 Φ max + A (P * ) α . (2)
A possible complete formula interpolating between Eqs. 1 and 2 is (defining a crossover pressure P * 1 by setting Φ = Φ max in (1))

1 Φ = 1 Φ 0 -λ ln P * P * 0 1 -exp - P * 1 P * α 1/α , (3)
which is found (see Fig. 3) to fit the data appropriately. Eq. ( 3) requests A = λ (P * 1 ) α /(2α) in (2). Table 2 lists the values of fitting parameters. On reducing P * at any stage (as shown on Fig. 3 for P * = 13.3), Φ remains constant, apart from small, nearly elastic [11] changes. Subsequently, plastic compaction is resumed only if the highest value P * c applied in the past, the preconsolidation pressure, is exceeded.

DENSITY CORRELATIONS

Open, ramified structures such as the one of Fig. 1 are frequent in colloid aggregation processes, and exhibit a self-similar structure characterized by a fractal dimension d F . Ideal aggregation processes (e.g., diffusionlimited, reaction-limited or ballistic) have been studied a lot [12] and lead to different values of d F [13]. Since the density of a fractal object vanishes in the thermodynamic limit, arbitrarily large systems of finite solid fraction Φ only display a fractal structure below some correlation length or "blob size" ξ , which satisfies (in 2D) [12]

ξ ∝ Φ -1/(2-d F ) (4)
In the traditional models, aggregates are rigid, undeformable and unbreakable objects, whence a loopless structure and coordination number z = 2. We are dealing here with a more realistic version of ballistic aggregation, with a complete mechanical model for particle interactions. The result of the ideal aggregation process with rigid clusters is only approached for large RR with, then, z → 2 at small P * (or, also, in the limit of V 0 /V * → 0 under P * = 0 [10], as initial clusters are hardly disturbed as aggregation proceeds). d F is measured on computing the Fourier transform of the density autocorrelation function (or "scattering intensity"), I(k), which should be approximately constant for wavevector k below 2π/ξ and decrease as k -d F in the fractal range 2π/a k 2π/ξ . Fig. 4, in spite of large error bars at small k, shows that our results abide by such expectations. Correlations coincide for large k for different P * , indicating that consolidation essentially affects the large scale features of the microstructure. The slope of I(k) versus k on the logarithmic plot yields d F = 1.52 ± 0.04, which coincides with the fractal dimension (1.55±0.02) obtained in ideal ballistic aggregation [13], despite a different connectivity: z is well above 2, it increases with P * c from about 2.9 to 3.1. Correlation length ξ can be estimated from the abscissa of the intersection of the initial plateau with the line of slope -d F . ξ decreases as P * increases until all signatures of a fractal microstructure vanish (for P * c ∼ 1). Due to the limited fractal range and corrections to scaling, relation (4) cannot be accurately checked, although it correctly describes [10] the change of ξ between P * c = 0 (Φ = 0.36, ξ /a 9.5) and P * c = 0.01 (Φ = 0.515, ξ /a 5.0). Interestingly, the same data analysis in systems without RR yields quite a different value of d F , near 1.9: a small level of RR (µ R /a = 0.005) has a large effect on the fractal dimension.

TENSILE STRENGTH

Some values of tensile strength, as measured under isotropic stresses (i.e., a negative value P * T of P * ), are shown in Fig. 5, as a function of preconsolidation pressure P * c . P * T might be measured either with a stepwise loading (as used in compression), or on smoothly stretching the specimen until the inner tension reaches a maximum. Both procedures agree. Remarkably, P * T turns out to be very similar for small RR or without RR. This contrasts with the sensitivity of consolidation behavior -in which the system gets continuously broken and repaired -to a small RR level. P * T is reached for a very small strain level (< 10 -4 ), irrespective of P * c . The rupture can thus be regarded as brittle -although for low P * c samples may still withstand smaller tensions, as they get progressively torn apart in a strain-controlled test. Classically, the strength of a cohesive material is assessed with the so-called Rumpf formula [9], which amounts in the present case to replacing the average normal force F N by -F 0 in relation F N = 7πaP/(zΦ) (which is nearly exact [10] and directly stems from the classical expression of stresses as sums over contacts involving forces and branch vectors). The Rumpf formula was proposed decades ago [14], based on the assumption that a large population of contacts simultaneously break in tension. It ignores the specific disorder and microstructure of cohesive packings and overestimates the tensile strength by a large factor ( 3 for large P * , above 50 for P * = 0.01). On the other hand, a Rumpf-like formula can be tried [START_REF] Castellanos | [END_REF] at scale ξ , based on a description of the system as a packing of blobs joined by fragile bonds with tensile strength ∼ F 0 . This provides a rough estimate of P * T , up to some unknown numerical factor, and predicts P * T ∝ ξ -1 . The variations of P * T (Fig. 5) are compatible with such a relation, as it increases by a factor of about 6 while ξ decreases in the same proportion.

CONCLUSIONS

We have retrieved the classical consolidation behavior of cohesive granular assemblies and powders with a simple model. The study of the mechanical properties of loose cohesive granular assemblies can benefit from the concepts developed and applied in the fields of gels or colloidal aggregates (fractal dimension, blob size). Thus estimates for such quantities as tensile strength, on accounting for the characteristic blob size, are improved over simple, particle-level approaches. However, the scaling regime is not necessarily well developed, and the mechanical behavior depends on connectivity properties beyond the simplest fractal characteristics. Although consolidation regime II is observed to coincide with the self-similar scaling regime for ξ , a prediction of plasticity index -associated with complex rearrangement events -is still unavailable. Another clear conclusion is the importance of rolling friction in contacts (even a small µ R changes the geometric and mechanical proper- ties). Our observations call for both more detailed investigations of rupture and plasticity mechanisms, for varying RR levels, and more general classifications, in 2 and

FIGURE 1 .

 1 FIGURE 1. One configuration (N = 1400) with RR under P * = 0.01. Compressive forces are shown in red, tensile ones in green, line thickness encoding intensity. A few contacts (in blue) bear vanishing forces (h = h 0 ).

FIGURE 2 .

 2 FIGURE 2. Configuration of Fig 1, compacted to P * = 13.3.

FIGURE 4 .

 4 FIGURE 4. Scattering intensity per unit area versus wave vector k in systems with RR for 3 values of P * c .

FIGURE 5 .

 5 FIGURE 5. Isotropic tensile strength versus P * c in a sample with N = 1400.

TABLE 1 .

 1 Values of interaction parameters.

TABLE 2 .

 2 Values of parameters λ , Φ max and α used to fit the consolidation curve with parameters of Table1, and in a sample with larger RR, with Eq. (3). Corresponding P * 1 values are 0.271 ± 0.033, 0.900 ± 0.064 and 2.6 ± 0.4.

	µ R /a 0 0.005 0.0316 0.515 0.194 0.7745 1.08 ± 0.16 P * Φ 0 λ Φ max α 0 0.0237 0.469 0.349 0.7808 0.91 ± 0.10 0.5 0.0178 0.382 0.25 0.724 0.86 ± 0.24

dimensions, of the rich variety of structures appearing in loose cohesive systems (would mechanical properties change along with d F with diffusion-limited instead of ballistic aggregation ?)


Dr Rosa Zampino
email: rosa.zampino@unina2.it

Aldo Marrone

Antonietta Merola

Barbara Trani

Grazia Cirillo

Peter Karayiannis

Nicola ; Coppola

Rosario Zappalà

Riccardo Utili

Giuseppe ; Ruggiero

Aldo Merola

Antonietta Trani

Barbara Cirillo

Grazia ; Sun

Paediatrics Karayiannis

Sun

Antonietta Merola

Peter Karayiannis

Luigi E Adinolfi

LONG-TERM OUTCOME OF HEPATITIS B AND HEPATITIS C VIRUS CO-INFECTION AND SINGLE HBV INFECTION ACQUIRED IN YOUTH

Keywords: HBV-HCV co-infection, chronic hepatitis, long-term outcome HBV-HCV co-infection, chronic hepatitis, long-term outcome

published or not. The documents may come

F o r P e e r R e v i e w

3 Worldwide, hepatitis B (HBV) and hepatitis C (HCV) viruses are responsible for most of the cases of liver disease and hepatocellular carcinoma (HCC) [START_REF] Lavanchy | Hepatitis B virus epidemiology, disease burden, treatment, and current and emerging prevention and control measures[END_REF][START_REF] Lauer | Hepatitis C virus infection[END_REF].

Several factors have been suggested to influence the rate of progression of chronic hepatitis, such as co-infection with HBV, HCV or HIV, age at infection, gender, alcohol intake and viral genomic mutations. Retrospective cohort and cross-sectional studies have shown that co-infection with HBV and HCV is associated with a more severe liver disease, an increased risk of HCC [START_REF] Sagnelli | Virologic and clinical expression of reciprocal inhibitory effect of hepatitis B, C and delta viruses in patients with chronic hepatitis[END_REF]Mathrurin et al, 2000;[START_REF] Marcellin | Fibrosis and disease progression in hepatitis C[END_REF] and viral interference, with a dynamic dominance of one virus over the other [START_REF] Pontisso | Clinical and virological profiles in patients with multiple hepatitis virus infections[END_REF][START_REF] Utili | Dual and single Hepatitis B and C virus infections in childhood cancer survivors: long-term follow up and effect of interferon treatment[END_REF][START_REF] Liaw | Role of hepatitis C virus in dual and triple hepatitis virus infection[END_REF][START_REF] Raimondo | Longitudinal evaluation reveals a complex spectrum of virological profiles in hepatitis B virus/hepatitis C virus coinfected patients[END_REF].

In endemic areas, anti-HCV antibodies are detectable in about 7% of patients with chronic HBV infection; co-infection increases with age and is more common in patients older than 50 years [START_REF] Gaeta | Epidemiological and clinical burden of chronic hepatitis B virus/hepatitis C virus infection. A multicenter Italian study[END_REF].

The natural history of chronic HBV infection is characterised by the emergence of genomic mutations in the core-promoter and/or the pre-core regions. Such genomic mutations have been associated with the presence or absence of HBeAg, with a higher rate of progression of liver fibrosis, transaminase flares and fulminant liver failure [START_REF] Friedt | Mutations in the basic core promoter and the precore region of hepatitis B virus and their selection in children with fulminant and chronic hepatitis B[END_REF][START_REF] Lindh | Core promoter mutations and genotypes in relation to viral replication and liver damage in east Asian hepatitis B virus carriers[END_REF], Zampino et al, 2002]. In contrast, patients co-infected with HBV and HCV seem to have a lower rate of genomic mutations of HBV, and the appearance of these mutations does not seem to be involved in the reciprocal inhibition of viral replication [START_REF] Yeh | Absence of precore stop mutant in chronic dual (B and C) and triple (B, C, and D) hepatitis virus infection[END_REF][START_REF] Jardi | Role of hepatitis B, C and D viruses in dual and triple infection: influence of viral genotypes and hepatitis B precore and basal promoter mutations on viral replicative interference[END_REF].

The host immune response is another factor that may modulate viral replication and the progression of liver damage. It has been reported that HBV infection regulates TNF-α expression and production, both in vivo and in vitro, suggesting that cytokines may have a role in the development of liver disease [START_REF] Gonzales-Amaro | Induction of tumor necrosis factor alpha production by human hepatocytes in chronic viral hepatitis[END_REF]. In a transgenic mouse model it was

shown that the induction of TNF-α regulates negatively the gene expression of HBV [START_REF] Gilles | Tumor necrosis factor alpha negatively regulates hepatitis B virus gene expression in transgenic mice[END_REF]. In chronic HCV infection, a Th2 cytokine profile seems to be associated with a more severe outcome of the liver disease [START_REF] Gramenzi | Cytokine profile of peripheral blood mononuclear cells from patients with outcomes of hepatitis C virus infection[END_REF]. was scored according to [START_REF] Knodell | Formulation and application of a numerical scoring system for assessing histological activity in asymptomatic chronic active hepatitis[END_REF] and staging according to [START_REF] Scheuer | Classification of chronic viral hepatitis: a need for reassessment[END_REF].

At the end of the follow-up period a second liver biopsy was performed in 20 patients and fibrosis was scored according to [START_REF] Scheuer | Classification of chronic viral hepatitis: a need for reassessment[END_REF] and to METAVIR [START_REF] Bedossa | An algorithm for grading activity in chronic hepatitis C[END_REF]. The METAVIR fibrosis score was compared with that obtained by transient elastography (FibroScan®, Echosense, Paris, France), a validated non-invasive method to quantify hepatic fibrosis [START_REF] Sandrin | Transient elastography: a new noninvasive method for assessment of hepatic fibrosis[END_REF][START_REF] Castera | Non-invasive evaluation of liver fibrosis using transient elastography[END_REF]. The operator was an expert staff physician who had performed previously about 300 examinations in patients with chronic liver disease. Since a good correlation between the two methods was found, a liver biopsy was not performed for the other patients and the fibrosis score was assessed only by transient elastography. During the follow up, 41 of the 54 patients were treated with α-interferon (α-IFN), 5MU/m 2 t.i.w. for 12 months. The end points of treatment were HBV-DNA and/or HCV-RNA clearance. In HBeAg positive patients, HBeAg/anti-HBe seroconversion was also evaluated. Thirteen patients were not treated because of contraindications or because they refused treatment.

HBV genomic analysis, including core promoter, pre-core and core regions, and serum detection of Th1-Th2 cytokines were evaluated on serum samples taken simultaneously at the first liver biopsy.

The principles outlined in the Declaration of Helsinki were adhered to in carrying out this study and each patient's full consent was obtained.

Serological determinations

Serum markers for HBV, HCV, HDV and HIV infection were tested using commercially available immunoenzymatic assays (Abbott Laboratories, North Chicago, IL. U.S.A. and Ortho Diagnostic Systems, Raritan, NJ, U.S.A.).

Quantitative HBV DNA and HCV RNA

DNA was extracted with the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) and RNA by QIAamp Viral RNA equivalent (Qiagen, Hilden, Germany).

HBV DNA real-time PCR was performed in a Light-cycler 1.5 (Roche Diagnostics, Branchburg, NJ, USA). Forward and reverse primers, located in the core region of HBV, were 5 1 -TTTTTCACCTCTGCCTAATCATC-3 1 nt 8-30, and 5 1 -ACCCACCCAGGTAGCTAGAGTCAT-3 1 nt 306-289. Using a commercially available standard of HBV viral load (Accuran HBV DNA, BBI Diagnostics, West Bridgewater, MA, USA) the detection limit was 100 copies/mL.

HCV RNA real-time PCR was performed using the same Light-cycler 1.5. Forward and reverse primers, located in the 5-UTR region, were 5 1 -AGCGTCTAGCCATGGCGT-3 1 nt 74-91, and 5 1 -CAAGCACCCTATCAGGCAGT-3 1 nt 308-288. FRET hybridization probes were 5 1 - Using a commercial standard of HCV viral load (Accuran HCV RNA, BBI Diagnostics, West Bridgewater, MA, USA) the detection limit was 200 copies/mL.

HBV genomic analysis, HBV and HCV genotype

For the HBV molecular study, the extraction, amplification, sequencing and computer analysis of the sequences were performed as described previously [Zampino et al, 2002].

HBV and HCV genotypes were determined using Inno-Lipa HBVDR (Innogenetics N.V.;

Ghent, Belgium) and HCV genotype assay Lipa (Bayer, France), respectively, following the manufacturer's instructions.

Serum cytokines

IL-2, IL-4, IL-8, IL-10, IL-12, IL-13, γ-IFN and TNF-α were detected by multiple sandwich ELISA using Human Search Light TM Proteome Array for human Th1/Th2 cytokines (Pierce Biotechnology, Rockford, IL, USA), according to the manufacturer's instructions.

Statistical analysis

The comparisons between the groups were made by the Mann-Whitney U test and by Fisher's exact test. Multiple logistic regression analysis was used to evaluate the factors independently associated with the development of liver cirrhosis and HCC. The odds ratio (OR) with 95% confidence interval (CI) was determined. A p-value <0.05 was considered statistically significant.

Statistical analysis was performed using the SPSS (SPSS Inc.,Chicago, IL, USA) software for Windows (Version 12.0).

F

Results

Characteristics of patients

Table I shows the characteristics of the study population at first liver biopsy. The infection was acquired in 44% of patients by transfusion, in 32% by surgery, in 3% by intravenous drug use and in 21% by intra-family contact.

Serum HBV DNA and HCV RNA levels

Serum HBV DNA and HCV RNA levels were determined at least once a year. In the group with HBV and HCV co-infection, low and comparable HCV RNA serum levels were observed in HBeAg and anti-HBe positive patients (Table I). In the patients with HBV and HCV co-infection and patients with HBV infection only, the HBV DNA serum levels were higher in HBeAg positive than in anti-HBe positive patients (Table I). However, an analysis of the subgroups showed that the anti-HBe positive patients with HBV and HCV co-infection had significantly lower HBV DNA levels than those observed in the anti-HBe positive patients with HBV (Table I, p=0.001).

HBV genomic analysis: core promoter, pre-core and core regions

Table II shows the HBV mutations at the first liver biopsy.

A higher prevalence of HBV genomic mutations in the core promoter and pre-core regions was observed in patients infected with HBV than in patients co-infected with HBV and HCV.

In the co-infection group a significant difference between the anti-HBe positive versus HBeAg positive subgroup was observed for the G1896A/G1899A mutations (p=0.007 and p=0.01, respectively). In the pre-core region in anti-HBe positive patients, the presence of these mutations was associated with lower HBV DNA levels than in HBeAg positive patients, but the presence of these mutations had no impact on the HCV RNA levels.

Overall, the presence of mutations in the HBV genomic regions investigated, either single or multiple, did not correlate with the HAI or progression of fibrosis.

Serum cytokines

Table III shows the serum cytokine levels at the time of the first liver biopsy. There were no significant differences in the serum cytokine levels of the patients, except for the TNF-α and IL-8 levels, which were significantly higher in the anti-HBe positive subgroup of HBV patients. The levels of these cytokines were not correlated with the HBV DNA levels or histological damage of the liver or fibrosis scores.

Liver histology at the time of the first biopsy

The HAI and liver fibrosis scores, performed after a median period of about 12 years post infection, are shown in Table I. At the time of the first biopsy no patient showed liver cirrhosis.

Interferon treatment

Treatment with α-IFN was started after the first liver biopsy in 41 patients; it was well tolerated and no drop outs were observed.

At the end of the treatment none of the 18 co-infected patients cleared HBV DNA and/or HCV RNA (Figure 1).

In the HBV group, 5 (36%) HBeAg positive patients and none of the anti-HBe positive cleared HBV DNA (Figure 2).

Viral status during the post-treatment follow up

In the group with HBV and HCV co-infection, 8 HBeAg positive patients cleared HCV RNA and 1 both HBV DNA and HCV RNA; in the anti-HBe positive group only 1 patient cleared HCV RNA (p=0.06; Figure 1). During the follow up, 9 (50%) treated patients and 1 (11%) untreated patient with co-infection with HBV and HCV cleared HCV RNA (p=0.052). HCV RNA clearance was observed between 1 and 5 years of post-treatment follow up. There were no differences in the pre-treatment serum HCV RNA levels or HCV genotype between patients who cleared HCV RNA and those who did not. In patients who did not clear HBV and/or HCV viral load, the viremia levels decreased during the follow up (Table IV).

HBeAg seroconversion was observed in 7 (58%) of the co-infected patients and in 9 (64%)

with a single HBV infection.

Analysis of factors associated with the development of cirrhosis and HCC.

The factors associated with the development of liver cirrhosis and HCC were evaluated by uni-and multivariate analysis. Included in the analysis were the following variables: age at infection, duration of disease, gender, co-infection with HBV and HCV, HBeAg/anti-HBe-status, serum levels of HBV DNA and HCV RNA, genomic mutations of HBV, interferon treatment and alcohol intake (>20 g/day). At multiple logistic regression analysis, alcohol intake (OR = 9.5 ± 1.2; 95% CI = 6.6 -13.9; p< 0.0001) was an independent factor associated with the development of liver cirrhosis and HCC.

Long-term outcome: development of cirrhosis and HCC

The long-term outcome was evaluated from the date of infection until a stable (about 1 year)

HBV DNA or HCV RNA disappearance. Figure 3 shows the liver staging at the end of the follow up. None of the patients showed HBsAg/anti-HBs seroconversion.

Group with HBV and HCV co-infection:

After a median follow-up period of 23 years (range: 17-40 yrs), 2 patients (7.4%), one HBeAg positive and one anti-HBe positive, showed cirrhosis, 19 and 23 years post infection, respectively, and one anti-HBe positive patient developed HCC (3.4%) 36 years post infection.

These three patients were male, did not respond to previous α-IFN treatment and two of them had a significant alcohol intake (> 60 g/daily). The diagnosis of cirrhosis was histologically established; the patients were followed up for a further 6-8 years and showed no sign of liver de-compensation. After a median follow-up period of 21 years (range: 16-34 yrs), only one male developed cirrhosis.

Yearly rate of progression of fibrosis

Assuming a steady linear progression, the yearly rate of fibrosis was calculated by dividing the score of fibrosis by the duration of infection. Although in clinical practice and in the single patient the yearly rate of fibrosis may be questioned because progression of liver fibrosis may not be linear, it is used [START_REF] Poynard | Natural history of progression of liver fibrosis in patients with chronic hepatitis C. The OBSVIRC, METAVIR, CLINIVIR, and DOSVIRC groups[END_REF][START_REF] Adinolfi | Serum HCV RNA levels correlate with histological liver damage and concur with steatosis in progression of chronic hepatitis C[END_REF] to describe the overall long-term rate of progression of liver fibrosis in cohorts of chronic hepatitis patients. Among the co-infected patients, the mean annual progression rate was 0.07 ± 0.03, with no differences between the HBeAg positive and anti HBe positive groups.

In the patients infected with HBV only, the mean annual rates were 0.11±0.06 and 0.05±0.01 in the anti-HBe positive and HBeAg positive, respectively (p<0.004).

Discussion

This prospective study shows that co-infection with HBV and HCV acquired early in life has an overall low progression rate (0.07 per year) of liver fibrosis over a median follow up of 23 years and as long as 40 years. Advanced disease was observed in 11% of the cases and was associated with male gender and a long-term history of alcohol intake, which may have contributed to the development of the advanced liver disease [START_REF] Reuben | Alcohol and the liver[END_REF]. The progression of the liver disease in patients co-infected with HBV and HCV mirrors that of patients with HBV infection only. In the latter group the majority of cases had a relatively mild chronic liver disease, and cirrhosis was seen in one patient (3.7%). However, among the patients infected with HBV only, those who were anti-HBe positive had a significantly higher progression rate than the HBeAg positive.

The data of this study are in sharp contrast with those of retrospective cross-sectional studies conducted on adults, which reported a more severe liver disease with a higher prevalence of cirrhosis in patients co-infected with HBV and HCV [START_REF] Sagnelli | Virologic and clinical expression of reciprocal inhibitory effect of hepatitis B, C and delta viruses in patients with chronic hepatitis[END_REF][START_REF] Mathurin | Replication status and histological features of patients with triple (B, C, D) and dual (B, C) hepatic infection[END_REF].

The results of the cross-sectional studies may have been influenced by several factors. First, in those studies the precise moment of infection is unknown, so the follow up refers to the years of clinical observation rather than to the real duration of the disease, which may be much longer and account for the higher severity of the disease. This hypothesis is supported by the fact that the mean age of our patients was less than 40 years whereas the mean age of patients in the cross-sectional studies was higher. Second, cross-sectional studies [START_REF] Sagnelli | Virologic and clinical expression of reciprocal inhibitory effect of hepatitis B, C and delta viruses in patients with chronic hepatitis[END_REF][START_REF] Mathurin | Replication status and histological features of patients with triple (B, C, D) and dual (B, C) hepatic infection[END_REF] were conducted on patients hospitalised or referred to hospital, and therefore with more advanced liver disease. Third, we cannot exclude that the infection acquired in youth may have a more favourable long-term outcome. In this respect, it is known that children who acquire hepatitis viruses from their mother show an asymptomatic infection and mild chronic hepatitis in most cases. These patients often show the phenomenon of viral "tolerance" (particularly in HBV infection), with high viremia levels, low ALT levels and a slow progression rate of the disease [START_REF] Broderick | Hepatitis B in children[END_REF]; , 2005;[START_REF] Mast | Risk factors for perinatal transmission of hepatitis C virus (HCV) and natural history of HCV infection acquired in infancy[END_REF][START_REF] Bortolotti | Chronic hepatitis B in children after e antigen seroclearance: final report of a 29-year longitudinal study[END_REF][START_REF] Iorio | Long-term outcome in children with chronic hepatitis B: a 24-year observation period[END_REF]. Moreover, in patients who acquired HCV infection by transfusion, the risk of developing liver cirrhosis, after a median follow up of 18 years, was related to the age at infection, i.e., lower in patients who were 20 years old or less at the time of infection [START_REF] Minola | Age at infection affects the long-term outcome of transfusion-associated chronic hepatitis C[END_REF]]. To answer the question whether age at the acquisition of infection may modulate the progression of the disease a longer follow up of our patients is necessary, as well as prospective studies that include subjects who acquired their infection as adults.

HBV gene mutations seem to be associated with a more advanced liver disease and HCC, but no definitive conclusions can be drawn [START_REF] Brunetto | Wild type and e antigen-minus hepatitis B viruses and course of chronic hepatitis[END_REF][START_REF] Chu | Precore stop mutant in HBeAg positive patients with chronic hepatitis B: clinical characteristics and correlation with the course of HBeAg-to anti-HBe seroconversion[END_REF][START_REF] Liu | Role of hepatitis B viral load and basal core promoter mutation in hepatocellular carcinoma in hepatitis B carriers[END_REF]. In this study, HBV mutations were more frequent in anti-HBe positive patient,s regardless of HCV coinfection. None of the single or multiple mutations observed in the HBV genome was associated with increased liver inflammation or progression of fibrosis. De Metri et al. [2006] reported that mutation T1936C accelerates the progression of disease in patients co-infected with HBV and HCV.

In our co-infected patients this mutation was not found, and few mutations in the core region involving the well-defined B (aa. 74-81) and T (aa 12-21) cell epitopes were observed. Therefore, the absence of mutation T1936C, associated with the few core mutations found, supports the hypothesis that the antigen presentation and immune response are modified in these subjects, explaining the mild course of infection in these patients [START_REF] Jung | Immunology of hepatitis B infection[END_REF][START_REF] Barboza | Antigen-induced regulatory T cells in HBV chronically infected patients[END_REF].

However, in-vitro studies are necessary to clarify this point.

It has been reported that the progression of liver damage could be correlated with an imbalance in the Th1-Th2 cytokines in patients with HBV or HCV infection [START_REF] Gonzales-Amaro | Induction of tumor necrosis factor alpha production by human hepatocytes in chronic viral hepatitis[END_REF][START_REF] Gramenzi | Cytokine profile of peripheral blood mononuclear cells from patients with outcomes of hepatitis C virus infection[END_REF]. In this study there were no significant differences in the serum levels of Th1 and Th2 cytokines in the groups studied and, what is more pertinent, none of the alterations observed was associated with advanced liver disease.

A significant number of patients in this study were treated with a schedule of α-IFN that is considered certainly inadequate at present. However, in patients co-infected with HBV and HCV, α-IFN treatment did not produce any favourable response at the end of treatment, but during the post-treatment follow up, a higher number of patients cleared HCV RNA in the treated group than in the untreated group (50% vs 11%, respectively; p=0.052). Thus, the data seem to indicate that α-IFN treatment does not have a direct effect on HBV or HCV but it might boost the immune system in clearing HCV RNA, particularly in patients with low baseline levels of viremia, and in reducing HBV DNA levels. However, the clinical impact of HCV RNA clearance is doubtful, considering that over the observation period there was no difference in the progression of disease or viremia levels, regardless of a single or co-infection.

In patients with HBV infection only, α-IFN treatment seems to be more effective for the HBeAg positive patients, who cleared HBV DNA in 36% of cases, whereas no clearance of HBV DNA was observed in anti-HBe positive patients (Figure 2).

In conclusion, the results of this prospective long-term follow-up study (median 23 years)

show that patients with co-infection with HBV and HCV or HBV infection only, when acquired in youth, do not have spontaneous or α-IFN-induced seroconversion to anti-HBs. They also have a favourable clinical outcome, the majority of patients having a low rate of progression of the liver disease, no liver failure and a low emergence of HCC. Alcohol intake increased by 9.5 times the risk of developing "early" cirrhosis or HCC. Having acquired the infection in youth these patients have a long life-expectancy but a more severe status of liver disease is possible in the future. A longer follow up is necessary to evaluate further the progression of the disease and whether other factors, besides alcohol, may be associated with a delayed development of cirrhosis and HCC. Fibrosis* score at the end of followup period, median (range)

1 (1-4) 1 (1-4) 1 (0-2) 2 (1-4)

  Fig.1

  in the absence of prospective studies with a known date of infection and of other contributing factors over time, the real natural history remains obscure. In addition, no data are available on the natural history of co-infection with HBV and HCV acquired at a young age.Accordingly, the aim of this study was to assess the long-term clinical outcome in a cohort of patients with co-infection with HBV and HCV or HBV infection only, acquired in childhood or in adolescence, and to evaluate the factors associated with the possible progression of the hepatic disease, in particular, the impact of virological and host immunological parameters on the course of the disease. 54 consecutive Italian patients with active co-infection with HBV and HCV or active HBV infection acquired in youth and followed up prospectively at the Liver Unit of the Second University of Naples were evaluated for long-term liver disease outcome.Until 1990, before a serological test for HCV became available, the patients were referred as infected with HBV. All patients had a known date of HBV infection and had been on regular follow up since their infection or shortly thereafter. Sera were collected at least once a year and stored at -80°C. Stored sera were tested for the presence of HBV DNA and HCV RNA when these molecular tests became available. On the first serum samples, collected between 8 and 38 months (median 16 months) after infection with HBV, 27 patients had HBV DNA and HCV RNA and 27 patients were HBV DNA positive only. No patient was positive by serology for HDV and/or HIV at the first observation, nor became positive at any time. Patients were evaluated at least once a year with physical examination, measurement of liver function tests, α-fetoprotein, blood cell count, hepatic viral markers, liver ultrasound scan and other risk factors for liver disease, such as alcohol and drug use. All patients underwent at least one liver biopsy. The histological activity index (HAI)

  group 2 HBeAg positive patients and 2 anti-HBe positive cleared HBV DNA (Figure2). Clearance of HBV DNA throughout the observation period was higher in the HBeAg positive group (7 patients) than in the anti-HBe positive group (2 patients; p < 0.01).

Figure 1 .

 1 Figure 1. Effect of α-interferon treatment in patients co-infected with HBV and HCV.

Figure 2 .

 2 Figure 2. Effect of α-interferon treatment in patients infected with HBV only.

Figure 3 .

 3 Figure 3. Long-term outcome of co-infection with HBV and HCV and HBV infection alone

Table I . General features of patients at the first liver biopsy

 I

						22 23
	PATIENTS	HBV-HCV	HBV
		HBeAg +	Anti-HBe + HBeAg +	Anti-HBe +	P =
	Number of patients	15	12	14	13
	Gender M/F	8/7	9/3	8/6	11/2
	Median age at	6 (2-16)	10 (0.2-16)	14 (10-17)	9 (6-16)
	infection, yrs
	(range)
	F o r Median HBV DNA cps/ml x 10 5 (range) 387 (0.00007 -1400) F o r	0.17 (0.015-1.1)	330 (15-1000)	2 (0.056-890)	0.001* 0.001° 0.001^
	HBV genotype: A D Median HCV RNA cps/ml x 10 5 (range) HCV genotype:	P e e r 1 14 12 P 2 (0.0001-190) 11 (0.006-500) e e r	14 --	13 --
	1 2	11	R 8 2 R
	3 nd Median ALT x NV(range)	4 1.4 (1.2-9.3)	e v i e e 1.6(1.2-6.1) 2.0 (1.6-11)^ 3.2 (1.1-15.1)^ 0.002 § 2 0.002* v i e
	HAI score median (range)	2 (1-8)	3 (1-5)	w 3 (1-7) w	4 (1-10)
	Fibrosis score,
	median (range)	1 (1, all pts)	1 (1-3)	1 (0-1)	2 (1-2)
	nd= not detectable; HAI Histological Activity Index; * HBV-HCV group (anti-HBe positive) vs HBV group (anti-HBe
	positive); ° HBV group (HBeAg positive) vs HBV group (anti-HBe positive);^ HBV-HCVgroup (HBeAg positive) vs
	HBV-HCV group (anti-HBe positive); § HBV-HCVgroup (HBeAg positive) vs HBV group (HBeAg positive)

Table II . HBV genomic analysis at the first liver biopsy

 II HBV group (HBeAg positive) vs HBV group (anti-HBe positive); * HBV-HCV group (anti-HBe positive) vs HBV group (anti-HBe positive); ^ HBV-HCVgroup (HBeAg positive) vs HBV-HCV group (anti-HBe positive).

							24
	PATIENTS		HBV-HCV		HBV
		HBeAg +	Anti-HBe +	HBeAg +	Anti-HBe +	P =
	Number of	14		12		14	13
	patients
	Core promoter
	mutations:
	A1762T	2 (14%)		4(33%)		2(14%)	7(53%)	0.03°
	G1764A	2 (14%) F o r	4 (33%)		3 (21%)	9 (75%)	0.06* 0.01° 0.02^
	Pre-core mutations:		P
	G1896A G1899A	1 (7%) 1(7%)	7 (58%) e 6 (50%) e r	0 0	10 (76%) 7 (53%)	0.000003° 0.007^ 0.001°
	Core mutations: T12S E40D V73G/N A79I	0 0 12 (85%) 10 (71%)		2 (16%) 1 (8%) 1 (8%) 1 (8%)	R e 0 2 (14%) 8 (57%) v i e 7 (50%) w	8 (61%) 8 (61%) 9 (64%) 0	0.01^ 0.02* 0.007 * 0.003 *

°

Table III . Th1-Th2 cytokine profiles at the first liver biopsy

 III

	PATIENTS		HBV-HCV		HBV
		HBeAg +	Anti-HBe +	HBeAg +	Anti-HBe +	P=
	Number of patients	14	12	14	13
	Th1 cytokine values:
	pg/ml; median
	(range)
	IL 2	6.8(1-69.7) 4.35 (1.5-31.7) 11.4 (0.98-	13.5 (1-159)
				37.1)
	IL 12	3.75(2-34.6)	5.5 (4-609)	2 (0.2-6.7)	4.5 (1.5-37.8)
	γ-IFN	8.9 (3-65.9) 102 (0.5-1557) 8.64 (3.7-28-1) 7.6 (1-44.6)
	TNF-α	31.85 (7.5-	28 (1.5-833)	8.47 (2.6-53)	68.7 (1.5-	0.040*
		724)			3300)	0.016°
	Th2 cytokine values:
	pg/ml; median
	(range)
	IL 4	4 (2-12.8)	3.5 (2-7.5)	3.61 (2.1-15.3) 3.5 (2.5-53)
	IL 8	14.9 (6.7-66.1) 10 (2.5-299) 48.43 (1-155) 13.3 (4-46.3) 0.007*
						0.021°
	IL 10	3 (1-58.9)	3.45 (2-8.8)	2.7 (2.1-5.7)	4.5 (1-45.9)
	IL 13	233.3 (4-764) 101 (0.5-1577) 208 (75.5-701) 475 (24-4900)

Journal of Medical Virology

Acknowledgments

The Authors thank Dr. Adriana Boemio and Ms Geltrude Fiorillo for technical assistance and Ms Serafina Verde and Mr Vincenzo Vaccaro for nursing assistance.

Author disclosure: Authors have no financial or other conflict of interest to disclose.

Financial support: This study was supported by a grant from the Regione Campania, Italy.


Angélica De Vrankrijker
email: a.m.m.devrankrijker@umcutrecht.nl

Tom F W Wolfs

Oana Ciofu

Niels Høiby

Cornelis K Van Der Ent

Seier Steen

Helle Poulsen

Krogh Johansen

Angélica ; De Vrankrijker

Angélica M M De Vrankrijker

Steen S Poulsen

Helle Krogh Johansen

A M M De Vrankrijker

Respiratory Syncytial Virus infection facilitates acute colonization of Pseudomonas aeruginosa in mice

Keywords: viral-bacterial synergism, inflammation, pneumonia, cystic fibrosis

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

21

Pseudomonas aeruginosa occurs ubiquitously throughout the environment but rarely causes 22 disease in immunocompetent individuals, even though it is present in water and the 23 gastrointestinal or upper respiratory tract [START_REF] Sordelli | Replication rate of Pseudomonas aeruginosa in the 410 murine lung[END_REF]. In immunocompromised 24 individuals or patients ventilated mechanically P. aeruginosa causes opportunistic infections 25 like pneumonias, medical device-related infections and super infection of burn wounds 26 [START_REF] Sutterwala | Immune recognition of 414 Pseudomonas aeruginosa mediated by the IPAF/NLRC4 inflammasome[END_REF]. P. aeruginosa is the principal infectious pathogen in cystic fibrosis, 27 a hereditary disease characterized mainly by chronic infections of the respiratory tract. In 28 these patients up to 80% develop a chronic infection which is accompanied by significant 29 morbidity and eventually mortality [START_REF] Pressler | Early rise of anti-pseudomonas 398 antibodies and a mucoid phenotype of pseudomonas aeruginosa are risk factors for 399 development of chronic lung infection--a case control study[END_REF].

31

Factors resulting in a predisposition to infection with P. aeruginosa are poorly understood.

32

Decrease of innate or adaptive immunity in the immunocompromised host and decrease of 33 local defence mechanisms in patients with burn wounds or mechanical ventilation might play 34 a role. In patients with cystic fibrosis, dehydrated airway secretions and mucus plugging, 35 altered composition of airway surface liquid and impaired host defence against P. aeruginosa 36 are suggested [START_REF] Sadikot | Pathogen-host interactions in 404 Pseudomonas aeruginosa pneumonia[END_REF]. Additionally, ceramide accumulation has been reported 37 to facilitate P. aeruginosa infection in a cystic fibrosis mouse model [Teichgraber et al., 38 2008]. Furthermore, several bacterial factors of P. aeruginosa are thought to contribute to the 39 establishment and persistence of chronic infection, such as the ability to form biofilms, 40 conversion to a mucoid phenotype and the accumulation of multiple mutations over time, 41 including some which may promote persistence and chronic infection [START_REF] Smith | Genetic adaptation by 407 Pseudomonas aeruginosa to the airways of cystic fibrosis patients[END_REF].

83

After inoculation with RSV, the mice were housed in an isolated cabinet. All cages were 84 placed on a heat pad for several hours after infection (Scanbur, Karlslunde, Denmark). The 85 local animal care committee approved the procedures performed on the mice in this study.

86

Bacteria

87

A clinical P. aeruginosa isolate was obtained from a patient with cystic fibrosis who attended 88 the cystic fibrosis centre at the Rigshospitalet, Copenhagen, Denmark. It was the first isolate 89 obtained in this patient, and has been described [START_REF] Jelsbak | Molecular epidemiology and dynamics of Pseudomonas aeruginosa 374 populations in lungs of cystic fibrosis patients[END_REF]. Bacteria were 90 inoculated in Luria-Bertani (LB) medium and grown shaking overnight at 37°C. Serial 91 dilutions were made with sterile 0.9 % saline, which were plated on blue agar plates (a [START_REF] Hament | Direct binding of 360 respiratory syncytial virus to pneumococci: a phenomenon that enhances both pneumococcal 361 adherence to human epithelial cells and pneumococcal invasiveness in a murine model[END_REF]. Stock aliquots contained 99 1.26 x 10 7 plaque-forming units (PFU)/ml and were stored at -80°C. Nasal application of P. aeruginosa or RSV was described previously [START_REF] Allewelt | Acquisition of expression of the 346 Pseudomonas aeruginosa ExoU cytotoxin leads to increased bacterial virulence in a murine 347 model of acute pneumonia and systemic spread[END_REF] 112 [START_REF] Hament | Direct binding of 360 respiratory syncytial virus to pneumococci: a phenomenon that enhances both pneumococcal 361 adherence to human epithelial cells and pneumococcal invasiveness in a murine model[END_REF]. In order to investigate whether this would be a practical method of 113 administering a sufficient amount of inoculum to the lungs after nasal inoculation, a radio-114 labelled peptide ((125)I-TFF2) was used [START_REF] Poulsen | Injected TFF1 and TFF3 396 bind to TFF2-immunoreactive cells in the gastrointestinal tract in rats[END_REF]. Mice were anaesthetized as 115 described above. The mice were then held in an upright position and while pushing the 116 mandible to close the mouth, 50 µl of the peptide solution was placed over the nostrils. Mice 179 aeruginosa showed about 2000 times higher CFU counts of P. aeruginosa in the lung 180 homogenates when compared to mice infected only with P. aeruginosa (median 3.05 x 10 1 181 CFU/ml of lung homogenate for mice infected with bacteria only and 6.0 x 10 4 CFU/ml of 182 lung homogenate for co-infected mice (p<0.01) (Figure 1)).

242

pathogenic agents [START_REF] Hament | Respiratory viral infection predisposing for 367 bacterial disease: a concise review[END_REF]. Several mechanisms are suggested that could explain 243 an enhancing effect of viral infections like RSV on acute pulmonary infection with P.

244

aeruginosa. The first is that respiratory viral infections disrupt normal tissue architecture of 245 the respiratory epithelium and cause loss of cilia, which might allow bacteria to adhere more 246 easily [START_REF] Patel | Effect of respiratory syncytial virus on adherence, 390 colonization and immunity of non-typable Haemophilus influenzae: implications for otitis 391 media[END_REF]. This effect has been observed in a mouse model of influenza, where 247 P. aeruginosa was able to adhere only to the desquamating cells of influenza-infected 248 tracheas [START_REF] Ramphal | Adherence of 401 Pseudomonas aeruginosa to tracheal cells injured by influenza infection or by endotracheal 402 intubation[END_REF]. The second is that immunological changes caused by viruses 249 can lead to less efficient clearance of bacteria. In a chinchilla model, influenza infection has 250 been observed to reduce neutrophil functions including respiratory burst and bacterial killing 251 [START_REF] Abramson | Influenza A virus-induced polymorphonuclear leukocyte 343 dysfunction in the pathogenesis of experimental pneumococcal otitis media[END_REF]. In another study, clearance of S. pneumoniae after RSV infection in disease may occur as a consequence of the innate antiviral response [START_REF] Navarini | Increased susceptibility to bacterial 387 superinfection as a consequence of innate antiviral responses[END_REF].

269

The third mechanism is increased adherence of pathogenic bacteria as a result of virus-270 induced upregulation of cellular molecules that act as receptors for pathogenic bacteria.

271

Bacteria can bind to virus-induced glycoproteins expressed on the surface of infected cells, 272 e.g. glycoprotein G and F [START_REF] Hament | Respiratory viral infection predisposing for 367 bacterial disease: a concise review[END_REF]. Additionally, in vitro studies showed direct 273 binding between RSV and S. pneumoniae and RSV and P. aeruginosa, suggesting that RSV 274 acts as a coupling agent between these bacteria and respiratory cells [START_REF] Hament | Enhanced 364 adherence of Streptococcus pneumoniae to human epithelial cells infected with respiratory 365 syncytial virus[END_REF] 275 [START_REF] Avadhanula | Additionally, virus-induced loss of 295 ciliary function could have impaired the mucociliary clearance mechanism, leading to an 296 inability of virus-infected mice to successfully clear the bacteria. Furthermore, since the virus 297 and bacteria were added simultaneously, it is a possibility that direct binding between P. 298 aeruginosa and RSV, as observed in vitro[END_REF]. 281 of cytokine production. Histopathological evaluation of the lungs showed damage to the 282 airway epithelium but no alveolar tissue inflammation. This suggests that the inflammatory 283 response to RSV is not substantial 24 hours after inoculation. This is in accordance with 284 studies of primary RSV infections in Balb/c mice, where viral titres peak after 4-6 days, 285 around which time pathological changes are observed in lung tissue [START_REF] Graham | Primary respiratory syncytial virus infection 355 in mice[END_REF] simultaneously. Similarly, in a mouse model of pneumococci and influenza virus, the virus 305 increased mortality in pneumococcal infection in mice, and an additional effect in mortality 306 rates was observed when mice were inoculated simultaneously with virus and bacteria 318 aeruginosa, suggesting that the enhancing effect can take place during simultaneous infection.

319

For this purpose, prevention of respiratory viruses by immunization or prophylactic use of 320 antibiotics during the respiratory virus season could be possible treatment options, for 321 instance in patients with cystic fibrosis. For RSV no vaccine is available at present [Malfroot 322 et al., 2005], but the use of passive prophylaxis with palivizumab has been suggested to be of 323 possible benefit in reducing hospitalizations in a recent retrospective study in young CF 324 patients [START_REF] Giebels | Prophylaxis 352 against respiratory syncytial virus in young children with cystic fibrosis[END_REF]. Further prospective studies are needed to investigate the effect 325 of RSV infection prevention on reduction of bacterial infections.

  suggested that viruses can play a role in the initiation or facilitation of P.44aeruginosa respiratory infections in previously healthy subjects. In a 25 year retrospective 45 study in Danish patients with cystic fibrosis, the onset of initial colonization and chronic the initiation of an acute infection with P. aeruginosa in c mice aged 12-15 weeks were obtained from Taconic (Tornbjerg, Denmark).79After transportation the mice were allowed to recover for at least one week, before being used 80 in experiments. The mice were housed in cages of 5, in a clean experimental unit (specific 81 pathogen-free) with barrier provisions and systematic health monitoring, at the Panum 82 Institute, Copenhagen, Denmark. The mice received commercial food and water ad libitum.

  92 modified Conradi Drigalski's medium selective for Gram-negative rods; State Serum 93 Institute, Copenhagen, Denmark) for determination of colony-forming units (CFU) after 94 overnight incubation at 37°C. The dilution factor that yielded around 1 x 10 6 CFU/ml was 95 determined and used for diluting the overnight culture with sterile saline. was used in all experiments

  by intraperitoneal injection of a freshly prepared mixture of 102 ketamine (65 mg/kg), xylazine (13 mg/kg) (both Intervet, Skovlunde, Denmark) and sterile 103 isotonic saline. Mice were held in an upright position and while pushing their mandible to 104 close the mouth, 50 µl of RSV inoculum (activity 0.63 x 10 6 PFU) was applied drop wise to 105 the nostrils with a pipette, followed by 50 µl (∼0.5 x 10 5 CFU) of bacterial inoculum. Mice 106 were held upright until the inoculum was inhaled completely. A period of 60 minutes was 107 allowed between the viral inoculation and the bacterial challenge, to prevent the mice from 108 inhaling large amounts of fluid at once. Control mice received bacteria, RSV or sterile

  117were held in an upright position until the solution was inhaled completely. The mice were 118 then killed using pentobarbital and the lungs and stomach were removed. Radioactivity was 119 measured in the lungs, stomach and a control amount of 50 µl of the peptide solution using in respiratory physiology induced by infection Whole Body 123 Plethysmography (WBP) was used in unrestrained mice(Buxco, Troy, NY). Before each 124 measurement, each chamber was calibrated to ambient pressure and temperature by rapidly 125 introducing 1 ml of air into the chamber. Each mouse was placed inside a separate chamber 126 and was provided with fresh air through a bias flow inlet. Readings were taken for 5 minutes, 127 in which pressure differences between the main chamber (containing the mouse) and the 128 reference chamber were measured. Several respiratory variables including enhanced pause 129 (PenH; a non-dimensional measurement that correlates with airway resistance[Hamelmann et 130 al., 1997]) were calculated using software supplied by the manufacturer. Lung function was 131 measured at baseline in all mice (15 mice per treatment group), and again 24 hours after 132 inoculation, right before termination of the experiment. 133 Lung bacteriology evaluation 134 After completion of lung function measurements 24 hours after inoculation, mice were killed 135 using intraperitoneal pentobarbital (200mg/ml)/lidocaine (20 mg/ml) injection (10 mice per 136 group in each experiment). Lungs were removed and homogenized on ice at 13,500 rpm in 3 137 ml sterile saline, using a Heidolph diax 600 homogenizer (Struers, Denmark). The 138 homogenates were serially diluted in sterile saline. 100 µl of the homogenate and 100 µl of 139 every dilution were plated on blue agar plates and incubated overnight at 37°C for counting of 140 colony-forming units (CFU). Colonies were assessed by appearance and development of a 141 deep blue colour within 10 seconds when stained with oxydase reagens (State Serum Institute, 142 Copenhagen, Denmark) to detect P. aeruginosa (presence of the enzymecomplex cytochrome 143 c). 144 Bronchoalveolar lavage fluid studies and histopathology 145 In each group 5 mice were randomly selected to undergo bronco alveolar lavage after having 146 been killed as described above. The trachea was exposed and a venflon catheter (22G) was 147 trachea. Lavage was done by flushing the lungs once with 0.7 ml of sterile 148 0.9 % saline. Lavage fluid was immediately frozen on dry ice and later stored at -80°C. 149 Antibody bead kit assays (mouse 5-plex and 10-plex) were performed to measure cytokines 150 (Biosource, Camarillo, CA). Concentrations were measured of pro-inflammatory cytokines: 151 IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, GM-CSF (which promotes granulocyte and 152 monocyte production), IFN-γ, and TNF-α. Additionally 5 chemokines were included in the 153 assay: KC (an IL-8 homologue), MCP-1, MIG (monokine induced by IFN-γ), IP10 and MIP-154 1α. Measurements were done using a Luminex 100 IS (Luminex Corporation). STarStation 155 software (Applied Cytometry Systems, Sheffield, UK) was used for analysis and acquisition 156 of data. For histopathology studies, lungs were fixed in situ by intratracheal injection of 4% 157 buffered formaldehyde, removed and put into more fixative. After fixation the lungs were 158 embedded in paraffin wax and cut into 5µm sections, followed by staining with hematoxylin 159 and periodic-acid Schiff (PAS). The samples were evaluated blind by two pathologists. application was investigated by administration of 50µl of radio-labelled 168 peptide solution followed by excision of the lungs and stomach for radio-active particle 169 counting. The counts detected in the lungs and stomachs of the mice were compared to an 170 equivalent sample of the original peptide solution. The mean percentage of radioactive 171 particles detected in the lungs (relative to the control fluid) was 71% (range 61-76%). The 172 stomachs of the mice did not contain any detectable radio-active particles indicating that the 173 fluid was delivered to the lungs without also being swallowed. with RSV and P. aeruginosa simultaneously, or only with P. aeruginosa 177 (approximately 0.5 x 10 5 CFU/mouse). Lungs were harvested 24 hours after infection and 178 quantitative cultures of lung homogenates were performed. Mice exposed to both RSV and P.

  respiratory physiology changes, lung function measurements were performed 185 in unrestrained mice at baseline and 24 hours after inoculation. Measurements were also 186 performed in control mice inoculated with only saline or RSV. Baseline values of Penh 187 (enhanced pause, a measure of airway resistance), minute ventilation and breathing frequency 188 were similar in all groups of mice (Figure2). After 24 hours, co-infected mice showed a 189 when compared to the mice infected only with bacteria (332/min 190 vs. 381/min respectively, p<0.05). The breathing frequency in the RSV control mice was no 191 different from the saline control group (mean 494/min vs. 464/min respectively, p>0.05). A 192 similar effect was observed for minute ventilation. The co-infected mice had significantly 193 lower minute ventilation when compared to the group infected only with P. aeruginosa (65.6 194 and 85.6 ml respectively, p<0.05). RSV control mice showed no significant difference 195 compared to the saline group (103.6 ml and 95.7 ml respectively, p>0.05). Enhanced pause 196 (Penh) was elevated in both groups of mice infected with bacteria (1.74 for co-infected mice 197 and 1.37 for mice infected with P. aeruginosa, p>0.05) and was not different in the RSV 198 group compared to the saline group (0.63 and 0.57, p>0.05).199 Bronchoalveolar lavage fluid 200 Cytokine analysis of the lavage fluid was performed to evaluate the production of pro-201 inflammatory cytokines and chemokines in the lungs. IFN-γ was undetectable in all groups 202 and IL-2, IL-4 and IL-5 gave unreliable standard curves, and therefore these results were 203 omitted. For all cytokines, mice infected with RSV alone showed no significant production of 204 cytokines when compared to control mice that received saline. Production of KC (IL-8 205 homologue) was observed but this was minimal (mean 22.3 pg/ml (SD 19.32) for RSV 206 infected mice and 0.0 pg/ml (SD 0.0) for control mice, p=0.06). There was increased 207 production of the measured cytokines in mice infected with P. aeruginosa only and co-208 infected mice. However, comparison of these two groups of mice showed no significant 209 differences in cytokine production, except for a minimal difference in MIG (a T-cell 210 chemoattractant) production (54.8 pg/ml (SD 30.3) for P. aeruginosa infected and 22.1 pg/ml 211 (SD 8.5) for co-infected mice, p=0.03).212Histopathology 213 Histopathology slides were evaluated and scored for severity of inflammatory changes of lung 214 tissue and bronchial epithelial changes. Scoring was done on a 5-point scale, and the average results from the two observers (Table1). Examination of the lungs of mice 216 infected with P. aeruginosa and with P. aeruginosa and RSV showed a similar severity of 217 inflammation in the lung parenchyma. The inflammation was characterized by perivascular 218 and alveolar infiltration with inflammatory cells (Figure3 c and d). The lungs of control mice 219 receiving saline or RSV did not show substantial inflammatory changes in the lung 220 parenchyma. Epithelial damage was observed in all groups of mice to some degree, but was 221 most severe in the mice infected with RSV only. Bronchial epithelial cells of these mice 222 showed a high degree of vacuolization and sloughing of nonciliated epithelial cells (Clara 223 cells) and also damage to ciliated epithelial cells. This was more pronounced distally i.ein vitro experiments suggest that viral infections can have a facilitating role in 228 P. aeruginosa airway infection[START_REF] Avadhanula | Additionally, virus-induced loss of 295 ciliary function could have impaired the mucociliary clearance mechanism, leading to an 296 inability of virus-infected mice to successfully clear the bacteria. Furthermore, since the virus 297 and bacteria were added simultaneously, it is a possibility that direct binding between P. 298 aeruginosa and RSV, as observed in vitro[END_REF]. Clinical observations provide 229 circumstantial evidence for this enhancing effect in cystic fibrosis[Johansen and Hoiby, 230 1992;[START_REF] Petersen | Respiratory infections 393 in cystic fibrosis patients caused by virus, chlamydia and mycoplasma--possible synergism 394 with Pseudomonas aeruginosa[END_REF]. The aim of the experiments presented here was to study the in 231 vitro findings in an in vivo model. Lung bacterial load and associated lung function changes 232 were compared between mice infected with RSV and P. aeruginosa and mice infected only 233 with P. aeruginosa. This showed that co-infected mice had a higher bacterial load and more 234 severe lung function changes, suggesting that P. aeruginosa was better able to establish an 235 infection in the presence of an RSV infection. Evaluation of the associated immunological 236 response by histopathology and cytokine analysis showed inflammatory changes in both 237 groups of mice infected with bacteria, but no parenchymal inflammation in control mice 238 infected only with RSV. This suggests that in this early infection model the facilitating effect 239 of RSV occurred independent of an immunological response to the virus. 240 241 A facilitating role in bacterial infections by viruses has been reported widely for various

  reduced 1-7 days following RSV exposure[START_REF] Stark | Decreased bacterial clearance from the lungs 412 of mice following primary respiratory syncytial virus infection[END_REF]. This 253 effect of RSV on bacterial clearance was not specific to S. pneumoniae: similar decreases in 254 clearance were observed for Staphylococcus aureus. The effect of RSV infection on P. 255 aeruginosa (PA01) clearance after RSV infection was studied and in common with the 256 present observations significantly higher colony counts were found in mice with RSV 257 infection. These results were obtained after bacterial challenge 7 days following RSV 258 exposure as opposed to the present study in which the bacterial challenge was preformed 259 simultaneously with viral inoculation. In the S. pneumoniae experiments, analysis of 260 bronchoalveolar lavage fluid demonstrated a greater rise in macrophages and 261 polymorphonuclear neutrophils in RSV-infected mice with S. pneumoniae when compared to 262 controls. Neutrophil cellular myeloperoxidase levels were decreased in RSV infected mice 263 which is suggestive of an alteration in neutrophil function being the possible cause of 264 decreased bacterial clearance [Stark et al., 2006]. In another study, viral infection of mice 265 with Lymphocytic Choriomeningitis Virus (LCMV), through induction of type I IFN, led to 266 apoptosis of bone-marrow granulocytes and impaired granulocyte migration to sites of 267 bacterial infection, suggesting that increased susceptibility to bacterial superinfection in viral 268

  mechanisms responsible for the enhancing effect of RSV on P. aeruginosa airway 278 infection in the studies presented require further study. Virus-induced changes in the 279 immunologic response may have a role. However, control mice receiving only RSV did not in lung function and minimal upregulation (although not significant)

  of the present findings is that they provide some insight into the role 310 of respiratory viral infections in P. aeruginosa airway infections. The results confirm 311 observations from in vitro experiments. Furthermore, these data support circumstantial 312 clinical evidence for a facilitating role in colonization with P. aeruginosa as observed in 313 patients with cystic fibrosis. In contrast to other studies on virus-bacterial interaction, an early 314 infection model was used, in which the RSV infection did not last beyond 24 hours, showing 315 that the facilitating effect of RSV occurs within the initial phase of infection, without the 316 presence of a clinical effect of RSV or a substantial inflammatory response. This could have 317 implications for possible strategies to prevent the opportunistic colonization with P.

  present study shows that in a mouse model, acute airway colonization with 328 P. aeruginosa can be facilitated by viral infections like RSV: The presence of an RSV 329 infection led to increased lung bacterial loads, and more severe lung function changes. This 330 effect occurred during simultaneous infection with RSV, without the occurrence of a 331 significant immune response, suggesting that even without the virus being apparent clinically, 332 its facilitating effect can take place. This could be important for understanding and prevention 333 of opportunistic airway infection with P. aeruginosa. Gulbins E. 2008. Ceramide accumulation mediates inflammation, cell death and 419 infection susceptibility in cystic fibrosis. Nat Med 14:382-391.

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[START_REF] Mccullers | Lethal synergism between influenza virus and Streptococcus 380 pneumoniae: characterization of a mouse model and the role of platelet-activating factor 381 receptor[END_REF].

Tristram DA, Hicks W, Jr., Hard R. 1998. Respiratory syncytial virus and human bronchial epithelium.

Acknowledgements