To further understand the delayed inducing effects of simvastatin, we added simvastatin at different time-points after the initiation of TCR stimulation with TGF-β or added simvastatin at culture initiation and then blocked its action at different time-points by the addition of mevalonate. All cultures were analysed for the expression of Foxp3+ cells after 72 hr of stimulation (Fig. 4b). The maximal inducing

effects of simvastatin could be observed even when it was added as late as 24 hr after the initiation of the cultures, but its synergistic activity was completely abolished when it was added after 48 hr (Fig. 4b). Similarly, the neutralization of the effects AZD6244 mouse of simvastatin with mevalonate was only observed when mevalonate was added during the first 24–32 hr of the culture.

This study suggested that simvastatin mediated its activity between 24 and 48 hr after T-cell activation. We confirmed this result by adding simvastatin at 24 hr and neutralizing its effects with mevalonate at 48 hr (Fig. 4c). The magnitude of the enhancement of the induction of Foxp3-expressing cells was similar in cells pulse-exposed to simvastatin only between 24 and 48 hr after activation to that in cells that had been exposed for the entire 72-hr culture period. To address whether synergistic action of simvastatin on TGF-β-mediated induction is controlled at the transcriptional level, we assayed the Foxp3 messenger RNA (mRNA) levels in cells treated with TGF-β alone or with the combination of TGF-β and simvastatin (Fig. 5a). Up-regulation of Foxp3 mRNA was observed after 24 hr of culture in the TGFβ only treated group compared to cells selleck inhibitor cultured with vehicle alone and no enhancement of Foxp3 mRNA was seen in cultures with simvastatin. In contrast, marked enhancement of Foxp3 mRNA levels were seen after 48 and 72 hr in cultures containing both TGF-β and simvastatin, whereas levels of Foxp3 mRNA in cultures with TGF-β alone were slightly diminished. This result together with the results of the time–course study strongly suggest that

the effects of simvastatin are not related to enhancement of the initial Selleckchem Paclitaxel signals induced by TGF-β and raise the possibility that simvastatin might regulate epigenetic control of Foxp3 transcription. Recent studies6,15 have identified two or three CpG islands within the promoter and enhancer regions of the Foxp3 gene that regulate the induction of Foxp3 transcription and the stabilization of Foxp3 expression. We focused on one site in the Foxp3 promoter that contains six CpGs within a 173-base-pair sequence of the mouse Foxp3 promoter that are located close to the proximal transcription start site. To verify if this candidate site is specific for Foxp3 promoter activity, Foxp3− and Foxp3+ CD4+ cells were isolated from Foxp3gfp male mice, and methylation profiles of both were analysed by bisulphite-modified sequence reading.

Cells were left in culture for 4 days in 5% CO2 at 40 °C. At day 4, EDTA (20 mm) was added to all wells to a final concentration of 2 mm EDTA. The plate was left for 10 min in the CO2 incubator at 40 °C to detach cells from the well. Finally, each sample was mixed by carefully pipetting up and down before transferring

it to FACS tubes. Flow cytometry.  Staining was carried out in tubes by adding 110 μl cell suspension to 90 μl of FACS buffer (0.2% BSA, 0.2% sodium azide, 0.05% normal horse serum in PBS) containing CD4-RPE (Clone CT4) and CD8α-APC (Clone CT8 or Clone 3-298) or CD8α-RPE (Clone EP72 or Clone 3-298). All antibodies were purchased from SouthernBiotech (Birmingham, AL, AZD2014 datasheet USA). In addition, propidium iodide (Fluka BioChemica, Buchs, Switzerland) was added to exclude dead cells. Cells were incubated at 4 °C for 15 min and then washed once with 2 ml FACS buffer by centrifugation at 295 g for 5 min. All flow cytometry analyses were

performed on a BD FACSCanto™ (BD Biosciences, San Jose, CA, USA) equipped with a 488-nm blue laser and a 633-nm red laser. Using the FACSDiva software, we aimed at collecting a minimum of 10,000 live cells from each sample. Titration of all antibodies was performed prior to the experiment in order to determine the optimal staining concentrations, Ku-0059436 supplier and the multicolour panel was carefully evaluated using fluorescence minus one (FMO) controls [15]. Statistical analysis.  Antigen-specific stimulations were run in triplicates with the exception of the experiment where the effect of anticoagulant and type of serum were tested, as this experiment was run in duplicates. For all optimization steps Acetophenone and for experiment 1, the mean percentage of proliferated cells ± SE was calculated and shown. The CFSE proliferation data for experiment 2 were tested using standard anovaF-tests on a 5% significance level, with dose and breeding line as the classification variables. Normally, blood is stabilized with heparin, and FBS is used as an additive to growth medium

in cellular stimulation assays. We wanted to test EDTA as a substituent for heparin as anticoagulant in the blood samples and autologous serum from an NDV-vaccinated chicken (CIS) as a substituent for FBS in the cell culture medium used for our recall proliferation assay assessed for both CD4+ and CD8α+ (Fig. 1A) T cells. The strategy for gating on CD4+ and CD8α+ T cells was debris exclusion on the Forward Scatter (FSC) – Side Scatter (SSC) dot plot followed by exclusion of dead cells by PI staining. Out of the live cells, CD4 cells were gated positive at the PE axis and CD8α cells were gated positive at the APC axis in a PE-APC dot plot (Fig. 1A). Finally, the CD4+ and CD8α+ T cells were shown in a dot plot with CFSE on the x-axis, and the percentage of proliferated CD4+ and CD8α+ T cells were measured. Fig. 1B shows the results from one representative sample.

Recently, p.N352S mutation in TARDBP was first identified in

a German family by Kühnlein et al [5] (Table 2). Their case showed fine motor skill impairments of the right hand selleck chemical as the first sign at the age of 40 years. In this pedigree, the patient’s aunt with onset in the distal upper extremity and a distant female relative with onset in the right distal upper extremity were also affected by the motor neurone disease. Kamada et al. [1] reported the same mutation in one of 30 Japanese patients with SOD1-negative FALS (Table 2). Their case exhibited weakness of the right hand as the first sign at the age of 55 years. Although the clinical features have not been described, five families with FALS with p.N352S mutation in TARDBP, including 15 cases diagnosed with FALS and three cases diagnosed with sporadic

ALS, have been reported [11]. p.N352S mutation in TARDBP have been reported in two cases with motor neurone disease who were clinically diagnosed with progressive muscular learn more atrophy [10] (Table 2) whose onset sites and ages were cervical at 68 years and lumbosacral at 61 years, respectively. Our case exhibited upper extremity impairment at onset similar to the previously reported cases of FALS with p.N352S mutation in TARDBP. Furthermore, all reported cases with p.N352S mutation in TARDBP, including our case, showed LMN signs with no detectable UMN and no cognitive impairment (Table 2). Their duration of illness was at least 4 years. Among the clinical features, the major symptoms of this FALS mutation type seemed to be as follows: (i) a tendency for onset in the upper extremities;

(ii) presence of LMN signs and no detectable UMN sign; (iii) no cognitive impairments; and (iv) a relatively long prognosis. The clinicopathological features of autopsy-confirmed FALS cases with several TARDBP mutations have been described (Table 2) [6–9]. Their sites of onset were variable, and most had both UMN and LMN signs during the disease course. Cognitive impairment was not observed in all cases, which was similar to those with p.N352S mutation in TARDBP. Thus, although the clinical features of several types of TDP-43-mutated FALS 4-Aminobutyrate aminotransferase seem to vary, none of them was affected by cognitive impairment (Table 2). As described in the Table 2, the previously reported cases of autopsy-confirmed FALS with TARDBP mutations [6–9] exhibited several common neuropathological features, including (i) degeneration of both the UMN and LMN systems; (ii) presence of Bunina bodies; and (iii) widespread TDP-43-immunopositive NCIs and GCIs. Similar to the previously reported FALS cases with TARDBP mutations, our present case showed LMN system degeneration with Bunina bodies, suggesting the possible presence of TARDBP mutations in several sporadic ALS cases.

4B). Available data indicate that the induction of efficient antiviral CD8+ cytotoxic T lymphocyte (CTL) response for viral clearance depends on the early CD4+ T cell priming to HBV infection [1]. However, the mechanisms by which CD4 T help cells required to control HBV infection has yet to be elucidated. In this study, we

investigated HBcAg-specific IL-21 producing CD4+ T cell responses in patients with HBV infection. We found a significantly higher frequency of HBcAg-specific IL-21+ CD4+ T cells in AHB patients than that in patients with chronic HBV infection, suggesting a role for IL-21 production of HBcAg-specific CD4+ T cells in inducing an effective immune response for viral clearance in patients with HBV infection. Because all of the patients with AHB enrolled in this study completely cleared the virus in the end, 3-MA mouse we have not yet been able to demonstrate a role for IL-21 in converting a self-limited HBV infection to chronic infection. In CHB patients, however, the frequency of HBcAg-specific IL-21+ CD4 T cells did not change significantly between IA patients and IHC individuals. This is different from recent findings where HBV-specific CD4+ T cells producing IL-21 were significantly higher in IHC versus HBeAg-positive IA CHB patients [16]. The cause of this difference may be

related to patients’ selection. Although IL-21 is induced only in the presence of large amounts of Ag [15], it is well known that there are lower circulating HBV-specific www.selleckchem.com/products/OSI-906.html CD4+ T cells or CD8+ T cells in IA CHB patients with too high levels of serum HBV DNA (especially more than 108 copies/ml), compared with relative low HBV DNA levels. This means that too high viral loads or viral antigen may sharply suppress HBV-specific CD4+ T cell response in CHB patients. The study

by Ma et al. [16] was focused on CHB patients with median 8.5 log10 copies/ml levels of serum HBV DNA. However, the HBV DNA levels of IA CHB patients Farnesyltransferase were moderate (6.1 log10 copies/ml) in our study. So, circulating HBV-specific CD4+ T cells producing IL-21 in our study may be relative high. This may explain the discrepancy of findings between the two studies. Interestingly, we found a significantly negative correlation between HBV DNA levels and IL-21-producing CD4+ T cell response to HBcAg in IA CHB patients. The immune state between IHC and IA stage in patients with CHB is different. There is a kind of balance between antiviral response and low HBV replication in IHC CHB patients. However,it is fluctuant between antiviral response and HBV replication in IA CHB patients. HBV replication would be suppressed if the antiviral response was strong. Studies in murine models with human hepatitis B have shown that IL-21-producing CD4+ T cells are necessary for HBV antigen clearance [20]. Recently, Li et al.

78 Similarly, other purified TLR agonists and inflammatory cytokines that induce the maturation of dendritic cells and augment expression of cell surface molecules that promote T-cell stimulation (e.g. CD80, CD86 and MHC) have also been reported to override Treg-cell suppression through IL-6-independent pathways.79–81 Even in the absence of APCs, cell-intrinsic stimulation through defined TLRs can also trigger shifts in Treg-cell suppression. For example, purified TLR2 agonists stimulate reductions in suppressive potency for mouse Treg cells, and TLR8 agonists trigger similar reductions in potency for human Treg cells.82–84

On the other hand, microbial ligands can also augment Treg-suppressive potency. Mouse CD25+ Treg cells selectively express TLR4,

and lipopolysaccharide stimulation augments their suppressive potency;85 whereas flagellin stimulation via Stem Cells antagonist TLR5 augments the suppressive potency of human Treg cells.86 Taken together, these in vitro studies illustrate the enormous potential whereby microbes and the response to infection can influence immune activation through shifts in Treg-cell suppression. The cumulative impacts whereby pathogens that express multiple TLR ligands and the ensuing immune response on shifts in Treg-suppressive potency have also been characterized for green fluorescent protein-positive (GFP+) cells recovered from Foxp3GFP reporter mice directly ex vivo following infection.87 For example, at Barasertib manufacturer relatively early time-points during persistent Salmonella infection, when the activation of effector T cells is blunted and the pathogen burden is progressively increasing, the suppressive potency for GFP+ Treg cells is augmented.59 Conversely, at later infection time-points when effector T cells are highly activated and progressive reductions in pathogen burden occur, the suppressive potency for Foxp3+ cells is reduced. Together Rolziracetam with the waning impacts of Foxp3+ cell ablation with infection progression, these results illustrate how shifts

in Treg-cell suppression can dictate the tempo of persistent infection.59 Similarly, following acute Listeria infection, reductions in suppressive potency are found for GFP+ Treg cells that immediately precede the expansion of pathogen-specific effector T cells.88 The expansion of circulating Treg cells with increased suppressive potency is associated with increased parasite burdens for patients with severe malaria infection.26 However, no significant changes in suppressive potency were found for Foxp3+ Treg cells isolated directly ex vivo after Plasmodium berghei infection in mice.31 Nevertheless, these findings illustrate how infection-induced shifts in Foxp3+ Treg-cell suppressive potency may play important and increasingly appreciated roles in infection outcomes.

It is likely that if a place is found for Helicobacter spp. within IBD pathogenesis, other organisms

with similar traits may be equally able to fulfill the same role. Gradel et al. (2009) demonstrated recently that infection with NVP-BGJ398 datasheet either Campylobacter or Salmonella predisposed to subsequent IBD development. We recently discussed the methodology utilized to identify the Campylobacter within this study, suggesting that further investigation may be warranted to define whether all Campylobacter attribute this risk or whether there are specific candidates (Hansen et al., 2010). Further exploration of the role that infectious triggers play in IBD in association with the host genetic factors involved may lead us to a better understanding of IBD, which may in turn take us far from the convenient, but imprecise labels of CD and UC. This may subsequently improve the accuracy of IBD research in much the same way that detailed genotyping and phenotyping of cancer variants has led to increased scientific accuracy of treatment studies and, as a result, the efficacy of cancer therapies. The other benefit of such understanding would, of course, be click here new therapeutic targets for IBD including perhaps immunization against

potential pathogenic triggers, targeted antibiotic therapies and probiotics designed to compete for the same ecological niche

as the pathogenic organism in question. We have recently come through a genetic revolution in our understanding of IBD. Perhaps the next revolution will be in understanding the colonic bacteria of IBD and both the route from ‘normal’ microbiota to dysbiosis, Rolziracetam and the microbial factors that foster disease chronicity. Organisms from the genus Helicobacter may well be involved in both areas. The authors wish to acknowledge funding from the Broad Foundation, USA, and the Chief Scientist Office, Scotland. R.H. is funded by a fellowship from the Chief Scientist Office in Scotland. We declare no conflicts of interest with the data included in this manuscript. [Correction added 8 November after online publication: Acknowledgements section has been added]. “
“Mature lymphocyte immigration into the thymus has been documented in mouse, rat, and pig models, and highly increases when cells acquire an activated phenotype. Entrance of peripheral B and T cells into the thymus has been described in healthy and pathological situations. However, it has not been proposed that leukocyte recirculation to the thymus could be a common feature occurring during the early phase of a Th1 inflammatory/infectious process when a large number of peripheral cells acquire an activated phenotype and the cellularity of the thymus is seriously compromised.

Vetrano (Rozzano) who had studied colonic sections from patients suffering from inflammatory bowel disease. S. Vetrano had also generated PC–/– transgenic mice, which were found to develop spontaneous intestinal inflammation and severe colitis, along with decreased expression of JAM-A, claudin-3 and alterations

in ZO-1 expression. A joint session held in collaboration with the Italian Society for Rheumatology hosted different talks. The effects TNF-α-blocking agents on monocytes and T cells in rheumatoid arthritis (U. Wagner, Leipzig), the role of biological drugs in the treatment of ANCA-associated systemic vasculitis (R. A. Sinico, Milan), as well as novel pathways and possible new targets in SLE (F. R. Spinelli, Rome), were all discussed. In the afternoon, talks were given by M. Cassatella (Verona) who reviewed the ability of neutrophils to undertake bidirectional STI571 in vitro cross talks with different cell types, including DCs, NK, iNKT and also unpolarized T cells. T. Laskay (Lübeck) showed that intracellular pathogens can globally diminish the expression of IFN-γ-regulated genes. The development of the thymus during evolution and, in particular, its phylogenetic

pendant in jawless vertebrates (agnathans) was discussed by T. Boehm (Freiburg), while L. Screpanti (Rome) presented data on the relative contributions and interconnectivity of Notch3, the pre-TCR and NF-kB in the development of T cells. A. Diefenbach (Freiburg) reported that dietary AhR ligands dynamically regulated postnatal development of lymphoid follicles by controlling the pool size of LTI-like ILCs. Concerning tumor

immunology, Ruxolitinib datasheet T. Blankenstein (Berlin) reported an aberrant rather than protective T-cell response, resulting in tolerance at the premalignant stage, while P. Yu (Marburg) showed that TLR-3, -7 and -9 can protect against murine T-cell lymphomas caused by endogenous retroviruses. The role of protein kinase CK2 as a pro-survival molecule that protects multiple myeloma cells from bortezomib, the first therapeutic proteasome inhibitor, was discussed by F. Cinetto (Padova). In the late afternoon, after viewing and discussing more than 300 posters and attending Selleck Osimertinib several workshops, members participated in the Assembly of their respective Society, both meetings being characterized by a very relaxed atmosphere (p<0.000001 versus several other assemblies that we have attended). The new SIICA board with Prof. V. Barnaba (Rome) as the new President was elected during the SIICA Assembly. Finally, one of the most exciting moments of the meeting arrived. If you put together any number of randomly selected Italians and Germans aged 4 years or older, you can be sure that after a couple of nanoseconds a challenging discussion starts about who are, or who were, the best football (or soccer, for readers in the new world) players, trainers, or national teams.

In this report, 14 heterozygous mutations in the FI gene (CFI, complement factor I), previously identified by different groups 4, 7, 8, 31, 32, have been studied to determine their effects on protein expression, secretion and function. To date, only the locations

of these CFI mutations and the clinical descriptions of patient symptoms have been reported. At the molecular level, the functional effects of only three of the currently analyzed 14 mutations have been investigated previously using eukaryotic expression system; one of these three was not secreted and therefore not amenable to functional analysis 9. It is important to understand how the complement system is regulated in these patients, especially with a view to developing therapeutic options. We found that the presence of pre-mature stop codons affected mainly protein secretion, whereas the amino acid see more substitutions affected either the secretion or the function of the FI protein. Thus, mutations in CFI lead to impaired regulation of the complement alternative pathway because of either impaired secretion or impaired function of FI, in turn predisposing patients to aHUS. In this study, we have investigated the functional effects of 14 CFI mutations identified in aHUS patients 4, 7, 8, 31, 32. These mutations are present in different domains: the FIMAC, CD5, LDLr1, region of unknown

homology and SP domain (Fig. 1A). Of the mutations, 11 are point mutations, eight

resulting in amino acid changes, and another three generate pre-mature stop codons. Another two of the mutations are MLN0128 cost deletions, (del C or del Selleckchem Erastin CACTT) and the final mutation was due to the insertion of an AT dinucleotide. These last three mutations also generated pre-mature stop codons (Fig. 1A, Table 1). Transient transfections were performed to determine how the mutations affect the expression and secretion of FI. Human embryonic kidney (HEK) 293 cells were transfected with different FI constructs and the FI concentrations in the cell lysates and supernatants were analyzed by ELISA. The C25F, P32A, M120V, H165R, R299W, W468x and D501N mutants were all expressed as efficiently as the WT FI, but the remaining seven mutants were expressed at significantly decreased levels (Fig. 1B). Only three of the mutants (P32A, H165R and D501N) were secreted at similar levels as WT. The mutants M120V, A222G and R299W were secreted, but at significantly lower levels compared with WT FI (Fig. 1C). The remaining eight mutants (C25F, W127x, N133S, L289x, R456x, W468x, T520x and W528x) were not secreted (Fig. 1C). The ratio of FI concentrations between the supernatant and cell lysate for each mutant shows that the P32A, H165R and R299W mutants were secreted as efficiently as WT FI from the HEK 293 cells (Fig. 1D). The remaining mutants were secreted less efficiently than WT FI.

To date, this has only been achieved with attenuated N. caninum isolates used as live vaccines (10,11). However, application of a live vaccine poses a series of logistic and economical problems, which render inactivated and/or subunit vaccines much more attractive, provided a reasonable degree of protection against infection and disease can be achieved. Several research groups have reported promising results using recombinant antigens for vaccination studies, but others have reported failures or even anti-protective effects (3,9). This shows that the antigen repertoire of N. caninum contains both protective and immunomodulatory or

even immunosuppressive molecules, and these need to be defined and investigated. In addition, the route of antigen delivery and VX-770 concentration the type of adjuvant employed also need further investigation, mTOR inhibitor considering that they can also alter the efficacy of a given vaccine candidate (41,43,44). Infection studies in cattle do not represent a cost-effective system to work with, and only

a limited number of research groups have taken up the enormous task to work with cattle directly (9,12). Accordingly, murine models have been extensively used for proof-of-concept studies on how an immune response against a vaccine could limit parasite dissemination and pathology. The currently used experimental murine models include (i) cerebral infection models with challenge infections of nonpregnant mice leading to cerebral disease and death, (ii) foetal infection models where mice are challenged during pregnancy and (iii) transplacental transmission of N. caninum tachyzoites leading to stillbirth, abortion or birth of infected offspring (9,49). In

the present study, we employed the acute disease model of cerebral infection in nonpregnant animals. For the vaccine, we employed an innovative approach by analysing the relative efficacy of recNcPDI vaccine antigen associated with nanogel vaccine delivery formulations. RecNcPDI has been previously shown to be ineffective when applied i.p. emulsified in SAPs, but highly effective and mediating protection against cerebral infection and disease when applied i.n. in the presence of cholera toxin (19). The purpose of the current work was to use chitosan-based nanogels, combined with different adjuvants (saponin and CT), as carriers for the E. coli Succinyl-CoA expressed recNcPDI antigen. Thereby, the aims were to investigate whether this nanogel association would influence the immunogenic and efficacy characteristics of the vaccine antigen upon i.p. and i.n. vaccination. SDS–PAGE and immunoblotting showed that recNcPDI was efficiently associated with both types of nanogels employed – alginate-coated chitosan nanogels and mannosylated, alginate-coated nanogels. The vaccine antigen was well associated with the nanogels, in terms of no nanogel-free material being detected. It also retained its antigenic reactivity with a polyclonal anti-recNcPDI antiserum.

All Rickettsia genomes available were compared to discover specific sequences to design new sets of primers and probes. The specificity was verified in silico and against a panel of 30 rickettsial species. Sensitivity was determined using 10-fold serial dilutions. Finally, primers and probes that were both specific and sensitive were routinely used for the diagnosis of rickettsial infections from

clinical specimens. We retained sets of primers and probes to detect spotted fever group Rickettsia, typhus group Rickettsia,Rickettsia conorii,Rickettsia slovaca,Rickettsia africae and Rickettsia australis; 643 clinical samples were screened for the presence of Rickettsia DNA. Overall, 45 positive samples were detected, including 15 Rickettsia africae, nine R. conorii, five Rickettsia sibirica mongolitimonae, see more four R. slovaca, two R. australis, four Rickettsia massiliae, Nutlin-3 research buy one Rickettsia honei, one Rickettsia typhi and eight Rickettsia sp. Positive samples were detected mainly from cutaneous biopsies and swabs (31/45). Widespread use of real-time PCR is inexpensive and reduces delay in the diagnosis of rickettsial infections. These real-time PCR assays could be implemented easily in laboratories that have molecular facilities and may be added to existing molecular tools as a point-of-care strategy. Members of the genus

Rickettsia may be classified into spotted fever group (SFG) Rickettsia, typhus group (TG) Rickettsia, Rickettsia bellii group and Rickettsia canadensis group (Merhej & Raoult, 2011). Rickettsiae can be transmitted to humans by blood-sucking arthropods and are associated with specific diseases termed rickettsioses. For example, Rickettsia conorii is associated with Mediterranean spotted fever (MSF) (Parola et al., 2005), Rickettsia

africae with African tick-bite fever (ATBF) (Jensenius et al., 2003), Rickettsia sibirica ssp. Sorafenib molecular weight mongolitimonae with lymphangitis-associated rickettsiosis (LAR) (Fournier et al., 2005), Rickettsia slovaca with ‘scalp eschar and neck lymphadenopathy after tick bite’ (SENLAT) (Angelakis et al., 2010), Rickettsia australis with Queensland tick typhus (QTT) (Parola et al., 2005), Rickettsia typhi with murine typhus (Civen & Ngo, 2008) and Rickettsia honei with Flinders Island spotted fever (FISF) (Parola et al., 2005). When a rickettsiosis is clinically suspected, biological diagnosis can be obtained using serology, cell culture and/or molecular tools (Parola et al., 2005); among the molecular tools, real-time quantitative PCR (qPCR) is rapid and sensitive (Stenos et al., 2005; Henry et al., 2007; Kidd et al., 2008). Genomic approaches have recently increased our knowledge of Rickettsia sp., and massive amounts of genomic data have become available (Ogata et al., 2001; Fournier et al., 2007; Merhej & Raoult, 2011).