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Human herpesvirus-6 dysregulates monocyte-mediated anticryptococcal defences

¹ Department of Hygiene, Microbiology and Biostatistics, University of Modena and Reggio Emilia, Via Campi 287, I-41100 Modena, Italy

² Department of Experimental and Diagnostic Medicine, University of Ferrara, Ferrara, Italy

Correspondence
Claudio Cermelli
c.cermelli{at}unimo.it

Received 3 January 2006
Accepted 17 February 2006

Abbreviations: Cn, Cryptococcus neoformans; CNS, central nervous system; CPE, cytopathic effect; HHV-6, human herpesvirus-6; IFA, immunofluorescence assay; IHC, immunohistochemistry.

	INTRODUCTION

TOP INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Clinical cases of double infections by fungi and viruses are increasing, especially in immunocompromised hosts (Marti Cabane & Alvarez Rubio, 2004; Weinberg et al., 2005; Fonseca et al., 2003; Kobayashi et al., 2003; Karino et al., 1998). To date, the biomolecular events that characterize the outcome of polymicrobic diseases remain poorly investigated and little is known of the mutual interactions occurring between pathogens, and their concomitant synergistic or antagonistic effects.

Because of the role of macrophages as the first-line defence against micro-organisms, the mechanisms involved in phagocytosis and microbial killing have been thoroughly investigated (Langermans et al., 1994; Nathan 1987; Vàquez-Torres & Balish, 1997). In addition, evidence exists of the variety of strategies through which micro-organisms can elude macrophage-mediated host defences (Rathman et al., 1996; Russell 1994; Tardieux et al., 1992); the relevance of such event(s) to pathogen persistence and eventually to disease outcome remains a matter of debate.

Cryptococcus neoformans (Cn) is a basidiomycetous yeast causing asymptomatic pulmonary infections in immunocompetent individuals, while in immunodeficient hosts, especially AIDS patients, it frequently disseminates from the lungs, causing severe and often recurrent meningoencephalitis.

Human herpesvirus-6 (HHV-6) is the aetiologic agent of exanthema subitum, also known as roseola infantum, a benign febrile infantile disease with high fever and cutaneous rash (Salahuddin et al., 1986; Yamanishi et al., 1988). Virus primary infection is usually asymptomatic; rarely, it may evolve as encephalitis, hepatitis, mononucleosis-like syndrome or fatal haemocytophagic syndrome (Ward, 2005). As with the other members of the Herpesviridae family, after primary infection, HHV-6 persists latently in the infected host. Peripheral blood lymphocytes and monocyte/macrophages, together with central nervous system (CNS) cells, support viral latency. Virus reactivation may occur and cause disease, especially in immunocompromised hosts. HHV-6 is highly neurotropic, and virus reinfections and/or reactivations are often associated with pathologies of the CNS, such as encephalitis, meningitis, radiculitis and demyelinating diseases (De Bolle et al., 2005). Moreover, by infecting immune cells, HHV-6 can directly cause immune dysfunctions, with a dramatic alteration of the secretion pattern that, in turn, can induce an imbalance of the immune response (Flamand et al., 1995; Arena et al., 1999, 2002; Milne et al., 2000; Yoshikawa et al., 2002; Caruso et al., 2003).

Multiple reasons support the rationale for investigating HHV-6 and Cn. First of all, both pathogens are highly neurotropic and represent important causes of CNS disease (Dewhurst, 2004). Second, both pathogens have monocytes, among others, as target cells, and both have the capability to affect macrophage functional parameters. In particular, Cn has been found as an intracellular pathogen within macrophages (Levitz, 2001). Moreover, it must be considered that both pathogens can interfere with the host defence pathways. Different mechanisms allow Cn to avoid macrophage-mediated defences, significantly impairing Cn ingestion and/or enhancing intramacrophage survival and replication of the pathogen (Tucker & Casadevall, 2002; Levitz, 2001). In addition, Cn can alter the cytokine secretory response of macrophages (Blasi et al., 1995, 2001a; Kawakami et al., 2000; Goldman et al., 2001; Mucci et al., 2003; Uicker et al., 2005).

The clinical evidence of simultaneous infections by fungi and HHV-6 (Vuorinen et al., 2004), and the finding that HHV-6 viraemia is significantly associated with invasive fungal infections in liver transplant recipients (Rogers et al., 2000), raise the question of whether and to what extent the wide-spectrum immunomodulating activity of HHV-6 may affect the outcome of fungal disease. By an in vitro model, we evaluated the consequences of HHV-6 infection on macrophages as antifungal defence elements. The effects of HHV-6 infection were measured as the capability to alter macrophage-mediated effector and secretory functions, namely phagocytosis and killing of Cn, and secretion of selected cytokines.

	METHODS

TOP INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Cn. We used a clinical isolate of Cn var. neoformans (serotype D) (laboratory name: 1526), obtained from the cerebrospinal fluid of a patient with meningitis (Blasi et al., 2001b) and cultured on Sabouraud plates at room temperature.

HHV-6. The prototype strain U1102 of HHV-6 variant A (Downing et al., 1987) was used for this study. The viral stock was obtained by infecting JJHAN cells with HHV-6; when more than 50 % of cells showed the typical cytopathic effect (CPE), cells were concentrated to 10⁷ ml^–1 and then frozen and thawed three times. The cell lysate was then centrifuged at low speed (800 r.p.m.) for 5 min to remove cell debris and stored in small aliquots at –20 °C (cell-free inocula).

Cell lines. The human acute monocytic leukaemia cell line THP-1 and the human T cell lymphoblast line JJHAN were maintained with RPMI medium with 10 % fetal bovine serum (FBS), 2 mM L-glutamine, 100 U penicillin ml^–1 and 100 µg streptomycin ml^–1 (complete medium), at 37 °C and 5 % CO₂. Both cell lines were split twice a week in fresh medium.

Protocols for THP-1 cell infection by HHV-6. We used two protocols to infect THP-1 cells with HHV-6. Protocol I was as follows. THP-1 cells were infected with cell-free HHV-6 inocula, obtained as described above, at an m.o.i. of 0.01 tissue culture infecting dose (TCID₅₀) per cell. After 2 h incubation at 37 °C, cells were centrifuged, washed with PBS and resuspended in complete medium to a final concentration of 10⁶ cells ml^–1. Mock infection was carried out using a cell lysate from uninfected JJHAN cells that was processed in the same way as infected lysates. Protocol II was as follows. To expose THP-1 cells to HHV-6, cells were co-cultured with uninfected JJHAN cells (THP1/JJHAN^mock) or with HHV-6-infected JJHAN cells (THP1/JJHAN^HHV-6) at a ratio of 1 : 1 and at a final cell concentration of 10⁶ cells ml^–1, and seeded in wells of a 24-well cell-culture plate. At different time-points, co-cultures were employed in the assays described below. Co-cultures were also carried out in transwell plates in which the two cell populations were cultured in separate compartments; in this case, 5x10⁵ THP-1 cells were seeded in the lower chamber and 5x10⁵ JJHAN cells (either HHV-6- or mock-infected) were seeded in the upper chamber.

Immunofluorescence assay (IFA). The percentage of HHV-6 infected cells was evaluated by IFA according to a protocol reported previously (Cermelli et al., 1992), using a HHV-6 hyperimmune human serum.

Molecular analysis. Total RNA was extracted with EUROzol (Euroclone), as recommended by the manufacturer. DNA contamination was eliminated by three cycles of digestion with 40 U RNase-free DNase at room temperature for 30 min. The complete absence of DNA contaminants was checked by PCR amplification of 200 ng total RNA without previous retrotranscription. Retrotranscription was carried out on 2 µg total purified RNA, using dNTP, random hexamer primers and 20 U of murine leukaemia virus reverse transcriptase. The presence of virus transcripts was analysed by nested RT-PCR of regions corresponding to the U42 (immediate-early) and U22 (late) genes of HHV-6, as previously described (Caruso et al., 2002).

Phenotypical analysis. Phenotypical analysis of THP1/JJHAN co-cultures was carried out by means of haematoxylin/eosin and May–Grunwald/Giemsa staining according to a standard protocol, and by immunohistochemistry (IHC) microscopy using anti-CD3 mAb clone PS1. The secondary antibody was biotinylated anti-mouse IgG. After incubation with biotin-streptavidin conjugated peroxidase, diaminobenzidine was used to visualize the reaction.

Cytokine quantification. THP1/JJHAN^mock, THP1/JJHAN^HHV-6 and THP1/JJHAN^HHV-6/Cn co-cultures were carried out for 1 and 2 days; then, cell-free supernatants were tested for IL-12 (Euroclone human IL-12) and IFN- content (Human IFN- ELISA kit, R&D Systems) according to the manufacturers' instructions.

Phagocytic activity. The phagocytosis assay was performed as previously described (Blasi et al., 1990). THP-1 cells alone (10⁶ cells ml^–1) or co-cultured with JJHAN at a ratio of 1 : 1 (THP1/JJHAN^mock and THP1/JJHAN^HHV-6) were infected with Cn (10⁷ cells ml^–1; effector : target=1 : 10) in 24-well plates and then incubated for 2 h. The excess of micro-organisms was removed by centrifugation of the cell suspension on a Ficoll cushion at 300 g for 10 min. The cells at the interface were recovered and washed. Fungal uptake was directly evaluated in May–Grunwald/Giemsa-stained cytospin preparations. A minimum of 200 macrophages were scored, and any cells containing one or more yeast were counted as phagocytic. The phagocytosis index was calculated as the total number of phagocytosed yeasts divided by the total number of phagocytic cells.

Anticryptococcal activity. Anticryptococcal activity was evaluated by means of the colony forming unit inhibition assay. THP1/JJHAN^mock and THP1/JJHAN^HHV-6 co-cultures (10⁵ cells ml^–1) were infected with Cn (10⁶ cells ml^–1; effector : target=1 : 10) in 96-well microplates. After 2, 3, 4 and 6 h incubation at 37 °C, Triton X-100 was added at 0.2 % final concentration. Serial dilutions from each well were seeded on Sabouraud agar plates in triplicate. After 48–72 h incubation at room temperature, c.f.u. were counted. Control cultures consisted of Cn without effector cells. The results, derived from the arithmetical means of the c.f.u. values, were expressed as percentage anticryptococcal activity, according to the formula:

Statistical analysis. Each experiment was performed three times and each sample was in duplicate. The results were analysed by the Mann–Whitney test. The data reported in figures and tables are the means of values from three different experiments (the SD was mostly less than 10 %).

	RESULTS

TOP INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Susceptibility of THP-1 cells to HHV-6 infection

Viral infection of THP-1 cells was carried out employing a lysate of HHV-6-infected JJHAN cells, according to Protocol I (Methods). Microscope observation of the cultures up to 10 days post-infection did not show evidence of any CPE. Moreover, IFA performed 1, 2 and 6 days after infection showed that approximately 1 % of THP-1 cells were positive for virus antigen expression (Table 1). In parallel, RT-PCR analysis was carried out on RNAs extracted from HHV-6-infected THP-1 cells; as shown in Fig. 1, single-round PCR did not give a positive signal, while nested PCR showed amplification bands for U42 (transcribed during the immediate-early phase of infection) and U22 (transcribed in the late phase of infection). The absence of amplification bands in RNA samples without previous retrotranscription confirmed that the bands corresponded to viral transcripts and not to contamination from residual DNA. Since the detection limit of our single-round PCR was 10³ copies (Caruso et al., 2002), we were able to assume that less than 1 % of cells had one copy of the transcript (Table 1, Fig. 1), thus confirming the IFA results.

View larger version (46K): [in this window] [in a new window]   Fig. 1. Molecular analysis of THP-1 cells infected with cell-free HHV-6 inocula. THP-1 cells were infected with cell-free HHV-6. Total RNA was extracted from HHV-6-infected THP-1 cells, and RT-PCR amplification was performed with reactions specific for HHV-6 U42 (immediate-early gene), HHV-6 U22 (late gene) and cellular ß-actin. Lanes 1 and 2 show the results of single-step PCR after retrotranscription of THP-1 cells harvested, respectively, 24 and 48 h post-infection. Lanes 3 and 4 show the results of nested PCR of THP-1 cells 24 and 48 h post-infection, respectively. Lanes 5 and 6 show, as control for the absence of contamination from residual DNA, the results of nested amplification of RNA extracted from THP-1 cells, without previous retrotranscription. ß-Actin RNA was amplified by single-step PCR: lanes 1 and 2, PCR results of TPH-1 cells, 24 and 48 h post-infection; lanes 3 and 4, negative controls (RNA without retrotranscription). The expected size of amplimers is shown to the right of the figure.

Phenotypical analysis of THP1/JJHAN co-cultures

Since direct exposure of THP-1 cells to HHV-6 inocula did not result in a productive infection, we set up a model of co-culture between mock- or HHV-6-infected JJHAN and THP-1 cells at a 1 : 1 cell ratio (THP1/JJHAN^mock and THP1/JJHAN^HHV-6, respectively). To verify whether the initial 1 : 1 monocyte : lymphocyte ratio was maintained over time, May–Grunwald/Giemsa, haematoxylin/eosin and IHC staining of the co-cultures was performed after 1 and 2 days of co-culture. A comparable percentage of lymphocytes and monocytes was observed under both HHV-6-infected and uninfected conditions (Table 2): in both THP1/JJHAN co-culture groups about 50 % of cells were consistently CD3 positive. No differences were observed at later times of co-culture (data not shown). On the other hand, both HHV-6-infected and uninfected JJHAN cells remained 100 % CD3 positive. Interestingly, THP1/JJHAN^HHV-6 showed a decrease in the percentage of mitotic figures (from 20 to 15 %), the appearance of apoptotic cells (from 0 to 9 %), and an increase in the percentage of giant cells (from 2 to 6 %) compared with THP1/JJHAN^mock (Table 2).

THP1/JJHAN co-culture: effects on IL-12 and IFN- production

View larger version (16K): [in this window] [in a new window]   Fig. 2. IL-12 production by THP1/JJHAN co-cultures. THP-1 cells were co-cultured with mock- and HHV-6-infected JJHAN for 1 and 2 days; IL-12 was then measured in supernatants by ELISA, as reported in Methods. P<0.01 between each type of co-culture at each time-point. Grey bars, THP1/JJHANmock; white bars, THP1/JJHANHHV-6; black bars, THP1/JJHANHHV-6/Cn.

View larger version (11K): [in this window] [in a new window]   Fig. 3. Phagocytic activity of THP1/JJHAN co-cultures. THP-1 cells were co-cultured with mock- and HHV-6-infected JJHAN for 1 and 2 days, and then infected with Cn. After 2 h incubation, phagocytosis was assessed by May–Grunwald/Giemsa staining, as reported in Methods. P<0.01 between the two types of co-culture at each time-point. Grey bars, THP1/JJHANmock; white bars, THP1/JJHANHHV-6.

View larger version (10K): [in this window] [in a new window]   Fig. 4. Increase in phagocytic activity of THP1/JJHANHHV-6 is independent of direct cell-to-cell contact. THP-1 cells were co-cultured with mock- and HHV-6-infected JJHAN for 1 and 2 days in transwell plates and then infected with Cn. After 2 h incubation, phagocytosis was assessed by May–Grunwald/Giemsa staining, as reported in Methods. P=0.04 at day 1; P=0.01 at day 2. Grey bars, THP1/JJHANmock; white bars, THP1/JJHANHHV-6.

View larger version (12K): [in this window] [in a new window]   Fig. 5. Kinetics of anticryptococcal activity of THP1/JJHAN. THP-1 cells were co-cultured with mock- and HHV-6-infected JJHAN overnight and then infected with Cn. After 2, 3, 4 and 6 h incubation, anticryptococcal activity was assessed by the colony forming unit inhibition assay, as reported in Methods. *P<0.01. Grey bars, THP1/JJHANmock; white bars, THP1/JJHANHHV-6.

	DISCUSSION

TOP INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Initially, we addressed the susceptibility of THP-1 cells to infection by HHV-6 by employing cell-free virus inocula (Methods, protocol I). Although several conditions were employed, THP-1 infection by HHV-6 was not efficient, as documented by the absence of CPE and by the IFA results (about 1 % positive cells). Moreover, RT-PCR analysis confirmed that only a minority of cells expressed early and late HHV-6 mRNAs. The amplification of both immediate-early and late viral mRNAs indicated that productive infection of THP-1 cells took place; yet, the transcription occurred with only low efficiency, as shown by the presence of bands using nested PCR alone. Furthermore, HHV-6 infection of THP-1 cell cultures did not affect their phagocytic activity. Using a similar approach, Li et al. (1997) have reported a dysregulation of monocyte secretory functions by HHV-6, showing induction of IL-10 and IL-12 in HHV-6-infected THP-1 cells. Nevertheless, they do not provide direct evidence of the efficiency of the viral infection of THP-1 cells.

With the aim of enhancing the efficacy of HHV-6 infection, another approach (protocol II) was chosen. In particular, THP-1 cells were exposed to HHV-6-producing lymphocytes. This experimental approach may be particularly relevant, since it closely reproduces the situation that potentially occurs in patients, in whom infected CD4+ T cells are the main source of HHV-6 (Lusso et al., 1988). Moreover, while this paper was in preparation, Meeuwsen et al. (2005) published a study of the effects of HHV-6A on human astrocytes. In that experimental system also, the co-culture model was employed, since the authors could not obtain infection of astrocytes using cell-free HHV-6 inocula. In our study, THP-1 cells were co-cultured with JJHAN cells that had been infected with HHV-6 5 or 6 days earlier. Under these conditions, the viral CPE on JJHAN cells was at its peak (usually about 50–60 % of cells were IFA positive for HHV-6 antigens). As negative controls, THP-1 cells were co-cultured with mock-infected JJHAN under the same experimental conditions. The co-cultures were subsequently exposed to Cn for different times; then, phagocytosis, cytokine secretory response and anticryptococcal activity were assayed. Here, we show that THP1/JJHAN^HHV-6 displayed increased phagocytic activity in comparison with uninfected THP1/JJHAN^mock co-cultures; this increase occurred regardless of the duration of the co-culture, and did not require direct contact between lymphocytes and monocytes, as established by experiments in transwell plates. It is worth noting that the enhanced percentage of phagocytic activity cannot be attributed to an alteration of the monocyte : lymphocyte ratio. As shown in Table 2, phenotypic analysis of the co-cultures disclosed that the initial 1 : 1 ratio was consistently maintained after 24 and 48 h, in both THP1/JJHAN^mock and THP1/JJHAN^HHV-6 co-cultures. Nevertheless, a reduced number of mitotic figures and an increased percentage of apoptotic cells were observed in THP1/JJHAN^HHV-6 with respect to THP1/JJHAN^mock, although these differences were not statistically significant. These data were consistent with the findings that HHV-6 induces an arrest of the cell cycle and apoptosis (Inoue et al., 1997; Oster et al., 2005).

Since the observed upregulation of phagocytosis was independent of direct contact between lymphocytes and monocytes, it is likely that released soluble factor(s) account for the observed phenomenon. With this in mind, we tested the THP1/JJHAN supernatants for IFN- and IL-12. We chose these cytokines for different reasons. IFN- is an important antiviral effector, produced by virus-infected cells, and it has also been reported to reduce the adhesiveness, intracellular multiplication and invasiveness of Shigella flexneri in HEp-2 cells preinfected with coxsackie B1 virus (Modalsli et al., 1992). IL-12 is crucial in the development of an effective immune response, favouring protective Th1-dependent resistance (Trinchieri, 1997). Moreover, we (Blasi et al., 2001a) and other authors (Kawakami et al., 1999) have shown that Cn can inhibit IL-12 production, whereas, as said above, HHV-6 can induce its production. In this work, we found that THP1/JJHAN^HHV-6 produced higher amounts of IL-12 and IFN- with respect to the control cultures in a time-dependent manner. The presence of Cn further increased IL-12 production by HHV-6-infected co-cultures. Although we cannot rule out some involvement of HHV-6-infected JJHAN cells, we favour the conclusion that, in our system, THP-1 cells are the main IL-12 producers, in accordance with an earlier report describing the IL-12 response of THP-1 cells exposed to cell-free HHV-6 inocula (Li et al., 1997). The observed production of IL-12, a powerful IFN- inducer, together with that of IFN-, also detected in supernatants from THP1/JJHAN^HHV-6, may in turn contribute to the increased phagocytosis of Cn by macrophages. In this respect, Siren et al. (2005) have recently shown that IFN- upregulates toll-like receptor (TLR) expression in human monocyte-derived macrophages as well as the expression of adapter molecules and kinases involved in TLR signalling, known to be critically involved in pathogen–host cell recognition events (Roeder et al., 2004; Netea et al., 2004).

Smith et al. (2003, 2005) have shown that HHV-6 inhibits IL-12 production by dendritic cells and macrophages, suggesting an immunosuppressive effect of the viral infection. The discrepancy between these data and those of the present study can be explained by the use of different cells (primary macrophages and dendritic cells versus a monocytic cell line). As suggested by other authors (Inoue et al., 1997), HHV-6 may have different effects in different cell types. Also, we cannot rule out differences in the experimental model employed. Smith et al. (2005) directly infected the immune cells, whereas we used infected lymphocytes as carriers of HHV-6 to macrophages. In our model, cytokine response by macrophages may be influenced not only by HHV-6 per se but also by the secretory response of HHV-6-infected T cells. In this regard, Mayne et al. (2001) have demonstrated a major modification in the pattern of cytokine response by lymphocytes, with an increase in pro-inflammatory cytokines and a down-regulation of anti-inflammatory cytokines in HHV-6-infected JJHAN cells. In particular, an increase of IL-18 was observed. IL-18, as well as IL-12, is a known IFN- inducer; moreover, Li et al. (1997) have proved that HHV-6-induced IFN- enhances IL-12 production by monocytes. Consequently, these two cytokines could have a synergistic effect, leading to the increase in phagocytic activity observed in our experimental model. Although Kawakami et al. (1999) have demonstrated a down-regulation of IL-12 by monocytes exposed to Cn, we observed the opposite outcome. First of all, different monocytic cell lines were used: in our case the human cell line THP-1 without any stimulation, while in the work of Kawakami and co-workers the murine cell line J774 was also stimulated with LPS and IFN-. Therefore, the two models are very different. More importantly, in our model, IL-12 induction by HHV-6 could have overcome the inhibiting effect of Cn, confirming the results reported by Li et al. (1997) of IL-12 upregulation by HHV-6 in monocytes. Moreover, the simultaneous presence of two different cell populations must be considered. Retini et al. (1999) have demonstrated that Cn-exposed monocytes increase their IL-12 production in the presence of lymphocytes.

Constitutively, THP-1 cells did not exhibit efficient killing of Cn. This activity was also influenced by the fungal strain employed [a more virulent Cn was killed even less efficiently (Blasi et al., 2001b)], and it was not significantly affected by THP-1 pre-treatment with phorbol ester (data not shown). Here we show that, when assessed for anticryptococcal activity, the co-cultures were unable to counteract fungal infection at least at early time-points. Furthermore, when assessed after 6 h, Cn exposed to THP1/JJHAN co-cultures grew more rapidly with respect to control yeast cells, giving rise to the observed negative values. Interestingly, such a phenomenon was more pronounced in THP1/JJHAN^HHV-6. While the molecular events mediating such phenomena remain to be elucidated, this study provides initial evidence of the dual role of HHV-6 on macrophage-mediated anticryptococcal defences. The net result of HHV-6 presence may be interpreted as a pro-Cn effect, since the fungus is better ingested, but it is also promoted in its survival and replication. Both conditions certainly favour dissemination of the pathogen and progression of the disease.

In conclusion, bearing in mind the limitations peculiar to any in vitro study, our co-culture cell model points to the possibility that virus infection may indeed affect the course of fungal disease. In particular, our model using lymphocytes and monocytes/macrophages could mirror the interactions between the two pathogens in the peripheral nervous system. A possible scenario suggested by these results is that in vivo HHV-6 reactivation may represent an additional risk factor for disseminated cryptococcosis from the periphery to the CNS. We are presently setting up an in vitro model of co-culture using cells of the CNS to verify that our results are confirmed in this system too.

	ACKNOWLEDGEMENTS

 
This work was partially supported by a grant to E. B. from Ministero dell'Istruzione, dell'Università e della Ricerca PRIN2003, n. 2003062784. We thank Professor Giuseppe Barbolini (Department of Laboratory Medicine, University of Modena and Reggio Emilia) for morphological analysis of the co-cultures used in this work.

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