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Basic science
Original article
Association of Trichomonas vaginalis with its symbiont Mycoplasma hominis synergistically upregulates the in vitro proinflammatory response of human monocytes
  1. Pier Luigi Fiori,
  2. Nicia Diaz,
  3. Anna Rita Cocco,
  4. Paola Rappelli,
  5. Daniele Dessì
  1. Dipartimento di Scienze Biomediche, Università degli Studi di Sassari, Sassari, Italy
  1. Correspondence to Dr Daniele Dessì, Dipartimento di Scienze Biomediche, Viale San Pietro 43B, Sassari 07100, Italy; danieled{at}uniss.it

Abstract

Objectives Trichomonas vaginalis is the causative agent of trichomoniasis, one of the most common sexually transmitted diseases worldwide. In recent years we have described the symbiotic relationship between T vaginalis and Mycoplasma hominis. How this biological association might affect the pathogenicity of one or both the microorganisms is still unknown. Since local inflammation is thought to play a central role in T vaginalis infection, we investigated the in vitro response of human macrophages to naturally mycoplasma-free T vaginalis, as compared to a mycoplasma-infected trichomonad isolate.

Methods THP-1 cells were stimulated with two isogenic T vaginalis isolates, one naturally mycoplasma-free and one stably associated with M hominis, and secreted cytokines measured by ELISA. Nuclear factor κB (NFκB) involvement in THP-1 response to T vaginalis and M hominis was evaluated by means of a reporter system based on detection of alkaline phosphatase activity.

Results We found that the presence of M hominis upregulates the expression of a panel of proinflammatory cytokines in a synergistic fashion. We also found that the upregulation of the proinflammatory response by THP-1 cells involves the transcription factor NFκB.

Conclusions These findings suggest that the presence of M hominis in T vaginalis isolates might play a key role in inflammation during trichomoniasis, thus affecting the severity of the disease. The synergistic upregulation of the macrophage proinflammatory response might also affect some important clinical conditions associated with T vaginalis infection, such as the increased risk of acquiring cervical cancer or HIV, which are thought to be affected by the inflammatory milieu during trichomoniasis.

  • TRICHOMONAS
  • MYCOPLASMA
  • INFLAMMATION

https://doi.org/10.1136/sextrans-2012-051006

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    Introduction

    Trichomonas vaginalis is the no. 1 non-viral sexually transmitted pathogen worldwide, with 170 million new cases each year.1 The clinical presentation of trichomoniasis in women may vary from asymptomatic to severe vaginitis,2 while the infection is mainly asymptomatic in men. Trichomoniasis is associated with a number of pregnancy and postpartum complications, such as preterm birth, premature membrane rupture and low birth weight.3

    The symbiotic relationship between T vaginalis and Mycoplasma hominis is the first described involving two obligated human parasites. Both microorganisms are independently capable of causing disease in the same anatomical area, the human urogenital tract. Despite its potential clinical impact, several fundamental aspects of the symbiosis between T vaginalis and M hominis still need to be characterised. Initial studies from our research group led to the demonstration that T vaginalis is able to pass the mycoplasmal infection to mycoplasma-free trichomonad isolates and human cervical cells in vitro.4 In addition, we also showed that M hominis resides and replicates within trichomonad cells.5 Interestingly, T vaginalis and M hominis share a common biochemical trait: they both possess the arginine dihydrolase pathway (ADH), a major energetic pathway for M hominis.6 In anaerobic conditions, T vaginalis can exploit the ADH pathway to obtain up to the 10% of its energy requirements.7 Trichomonas cultures stably infected with M hominis showed an increased ADH pathway activity as compared to the parental mycoplasma-free isolate.8 This suggests one of the possible mechanisms that might be important for the establishment of the association between the two microorganisms. On the one hand, M hominis could gain access to molecules that is not able to synthesise, such as putrescine. On the other hand, the metabolic interaction between T vaginalis and M hominis might be important in the evasion from the host innate defences. An increased arginine scavenging from the vaginal environment could reflect a lower arginine availability for nitric oxide synthesis by the phagocyte system of the host, with benefits to both microorganisms.

    Symptomatic and asymptomatic T vaginalis infections are both usually accompanied by a heavy leukocyte infiltration, as well as by high levels of secreted proinflammatory cytokines in vivo and in vitro.9 ,10 Thus, it appears that the innate immune response plays a crucial role in T vaginalis pathogenesis. Importantly, T vaginalis infection has been associated with an increased risk of invasive cervical cancer,11 prostate cancer,12 and of HIV acquisition and shedding.13 Several hypothetical mechanisms by which T vaginalis might directly contribute to neoplastic transformation and to HIV transmission have been proposed:14 the local inflammatory response to Trichomonas attracts at the site of infection CD4+ T lymphocytes, macrophages and dendritic cells, which are highly receptive to HIV infection. Moreover, the epithelial damage caused on the one hand by the direct effect of trichomonad cytotoxins and on the other hand by the inflammation, could disrupt the physical barrier to HIV entry. As regard to the increased risk of cervical and prostate cancer linked to T vaginalis infection, a speculative model involving chronic inflammation as a key factor leading to neoplastic transformation has been proposed.15

    Despite the important biological and clinical implications of this unique association, many aspects still need to be studied. In particular, given the importance of inflammatory phenomena in T vaginalis immunopathogenesis, we hypothesised a possible influence of the mycoplasmal symbiont over the host innate immune response. In this work, we describe the stimulation of the human monocytic cell line THP-1 with two isogenic T vaginalis isolates (one naturally mycoplasma-free and one experimentally infected with M hominis). We showed that the presence of M hominis upregulates the proinflammatory response of macrophages to T vaginalis. These data suggest that the symbiotic relationship with M hominis may contribute to Trichomonas immunopathogenesis.

    Methods

    Organisms and cultivation

    T vaginalis isolates G3 (reference strain, naturally mycoplasma-free) and MOZ-2 (naturally M hominis-infected) were cultivated with 1:16 daily passages in Diamond's TYM (trypticase, yeast extract, maltose) medium supplemented with 10% fetal bovine serum. M hominis cells were isolated from T vaginalis TvMOZ-2 as previously described,4 maintained in BEA medium (2.2% heart infusion broth; 15% horse serum; 1.9% yeast extract; 40 IU/ml benzylpenicillin; 0.23% l-arginine; 0.0023% phenol red, pH 7.2) and named by the originating trichomonad host (MhMOZ-2). T vaginalis G3 was stably infected with M hominis in order to obtain an isogenic mycoplasma-infected trichomonad strain (G3/MOZ-2).8 Briefly, 1 ml of an overnight culture of M hominis MhMOZ-2 (corresponding to approximately 109 colour-changing units (CCUs)) was added daily to a 10-ml mid-log phase culture of the M hominis-free T vaginalis isolate G3 for 5 days. Parasites were subsequently cultivated for 10 days with 1:16 daily passages. Stable M hominis infection was then assessed by PCR with specific primers16 and by isolating M hominis in liquid and solid BEA medium.

    Cell cultivation and stimulation

    The human monocytic cell line THP-1 was grown in RPMI 1640 medium supplemented with 10% fetal bovine serum. THP-1 cells were incubated at a concentration of 5×105 cells/ml in the presence of 50 ng/ml phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich, St Louis, Missouri, USA) for 24 h to induce differentiation to a macrophage-like phenotype (dTHP-1).17 Medium was replaced and cells allowed to rest for 48 h, and then maintained with two daily medium changes for further 5 days prior to infections. dTHP-1 cells were then stimulated with T vaginalis G3 and G3/MOZ-2 at different multiplicities of infection (MOI), ranging from 1:5 to 1:50 (T vaginalis/dTHP-1 ratio). Macrophages were also infected with M hominis MhMOZ-2 alone or in combination with mycoplasma-free T vaginalis G3. Bacterial inocula (expressed in CCUs) were assessed to match the number of M hominis CCUs of the T vaginalis G3/MOZ-2 samples.

    T vaginalis G3 cell lysates were prepared by sonication in phosphate-buffered saline (PBS), and then used to stimulate dTHP-1 cultures at MOIs equivalent to the live infections. Negative and positive controls of macrophage stimulation were PBS as a mock infection and Escherichia coli O55:B5 lipopolysaccharide (LPS; Sigma Aldrich) at a final concentration of 1 μg/ml, respectively. Cell supernatants were collected 8 h post infection for subsequent quantification of secreted proinflammatory cytokines. All conditions were tested in duplicate.

    Titration of M hominis inocula

    To determine the amount of bacteria used to stimulate THP-1 cells, M hominis (T vaginalis-associated and in pure culture) was titrated in a 96-well plate. Aliquots of T vaginalis G3/MOZ2 and M hominis MhMOZ-2 were added to BEA broth and then serially diluted at a 1:5 ratio in the same medium. Plates were incubated at 37°C for 4 days and monitored daily for broth colour change, which indicates bacterial growth. The endpoint (CCU/ml) was expressed as the reciprocal of the last dilution that showed a stable colour change.

    Quantification of secreted cytokines

    Tumour necrosis factor (TNF)α, IL-1β, IL-6, IL-8, IL-10, IL-12 and IL-23 in the supernatants of treated and control dTHP-1 cells were quantified by sandwich ELISA using matched monoclonal antibody pairs (R&D Systems, Minneapolis, Minnesota, USA, for TNFα, IL-1β, IL-6, IL-8 and IL-10; Ebiosciences, San Diego, California, USA, for IL-12 and IL-23), following the manufacturer's instructions. dTHP-1 supernatants were tested undiluted (IL-1β, IL-6, IL-10, IL-12 and IL-23), diluted 1:10 (TNFα) or 1:100 (IL-8). All cytokines assayed were determined from the same supernatant samples. Each sample was tested in duplicate. Colourimetric reactions were read at 405 nm with a VersaMax instrument and raw data elaborated with the SoftMaxPro software (Molecular Devices, Sunnyvale, California, USA).

    Nuclear factor κB (NFκB) reporter assay

    THP-1 Blue (Invivogen, San Diego, California, USA) are THP-1 cells that have been stably transfected with a reporter construct expressing secreted embryonic alkaline phosphatase (SEAP) under the control of a NFκB-inducible promoter. THP-1 Blue were maintained in complete RPMI 1640 with 200 μg/ml Zeocin, seeded at a density of 105 cells/ml and differentiated to a macrophage-like phenotype (dTHP-1 Blue) as described above. dTHP-1 Blue cells were then stimulated with isogenic T vaginalis G3 and G3/MOZ-2 at different MOIs (1:20, 1:50 and 1:100), and with M hominis MOZ-2 alone. After 24 h of incubation at 37°C, cell-free supernatants from treated and control THP-1 Blue cells were collected and NFκB activation was measured by spectrophotometric analysis of SEAP activity on Quanti-Blue alkaline phosphatase substrate (Invivogen). Negative and positive controls were the same as used for dTHP-1 stimulation.

    Statistical analysis

    We performed statistical analysis using the unpaired Student t test (Microsft Excel; Microsoft, Redmond, Washington, USA). A p value <0.05 was considered significant.

    Results

    T vaginalis and M hominis induce a qualitatively and quantitatively different proinflammatory response in human macrophages

    THP-1 cells were differentiated to a macrophage-like phenotype by treatment with PMA (dTHP-1), and were subsequently infected with either M hominis MhMOZ-2 or the naturally mycoplasma-free T vaginalis isolate G3, by using different MOIs, ranging from 1:5 to 1:50 trichomonads per dTHP-1 cell. M hominis MhMOZ-2 was shown to be a more potent inducer of proinflammatory cytokines (IL-1B, IL-8, IL-23, TNFα) than T vaginalis G3 (figure 1). Neither G3 nor MOZ-2 was able to elicit the production of IL-10, IL-12 or IL-6, while IL-23 and TNFα secretion was induced by MhMOZ-2 only. In all cases, we observed a marked dose-dependent cytokine production by dTHP-1 after stimulation. E coli LPS and PBS were used as positive and negative controls of stimulation, respectively, throughout the experiment. dTHP-1 cells were also challenged with T vaginalis G3 cell lysate, by using MOIs equivalent to those of live infections. Surprisingly, the levels of proinflammatory cytokines (IL-1B, IL-8, TNFa) secreted in response to G3 cell lysates were higher than those observed when macrophages were stimulated with live trichomonad cells. In particular, G3 lysate induced TNFα secretion, while live G3 trichomonads did not (figure 1).

    Figure 1
    Figure 1

    Trichomonas vaginalis and Mycoplasma hominis induce a qualitatively and quantitatively different proinflammatory response in human macrophages. G3, naturally mycoplasma-free T vaginalis; MOZ-2, M hominis; G3 lysates, T vaginalis cell lysate. Values on the x-axis represent multiplicities of infections (trichomonad cells/dTHP-1 ratio for G3, colour-changing units (CCUs) for MOZ-2). Values on the y-axis represent pg/ml of secreted cytokines. Values for interleukin (IL)-1β are shown in log scale. The graphs shown are representative of three independent experiments. Bars represent mean values of duplicate experimental conditions+SEM, in one of three independent experiments. MOI, multiplicities of infection; TNF, tumour necrosis factor.

    Symbiotically-associated M hominis synergistically upregulates the secretion of proinflammatory cytokines in THP-1 cells in response to T vaginalis infection

    We measured the concentration of a panel of proinflammatory cytokines in the supernatants of dTHP-1 cells after stimulation with T vaginalis G3, M hominis MhMOZ-2 or isogenic mycoplasma-associated T vaginalis G3/MOZ-2, using the same MOIs described above. G3/MOZ-2 was a more potent inducer of IL-1B, IL-8, IL-23 and TNFα than either T vaginalis or M hominis alone. As shown in figure 2, the upregulation of the proinflammatory response was synergistic: in all cases, the concentration of cytokines detected in the supernatants of cells stimulated with G3/MOZ-2 were greater than the sum of concentrations induced by individual microorganisms (twofold increase in TNFα and IL-23; fourfold increase in IL-1β). We also compared the cytokine profile induced by G3/MOZ-2 with that obtained after simultaneous costimulation of dTHP-1 cells with G3 and MhMOZ-2 in combination. Symbiotically-associated T vaginalis and M hominis (G3/MOZ-2) induced a greater cytokine production than simultaneous coinfection with G3 and MOZ-2 (figure 2).

    Figure 2
    Figure 2

    Symbiotically-associated Mycoplasma hominis synergistically upregulates the secretion of proinflammatory cytokines in THP-1 cells in response to Trichomonas vaginalis infection. dTHP-1 cells were exposed to M hominis (MOZ-2), mycoplasma-free T vaginalis (G3) (separately or simultaneously) and its isogenic counterpart stably associated with M hominis (G3/MOZ-2). The data shown refer to a stimulation carried with a multiplicity of infection (MOI) of 1:5 (T vaginalis:THP-1 ratio) and 107 colour-changing units (CCUs) (M hominis MOZ-2). A titration experiment was conducted in parallel, and allowed us to assess that the stimulation with G3/MOZ-2 with a MOI of 1:5 carried 107 M hominis CCUs stably associated with T vaginalis. No interleukin (IL)-6, IL-10 or IL-12 secretion could be detected in the supernatants of dTHP-1 cells stimulated with M hominis-infected T vaginalis G3/MOZ-2. * p<0.001, when concentration of secreted cytokine induced by G3/MOZ-2 are compared to the sum of concentrations induced by G3 and MOZ-2 or by the two in combination. The graph shown is representative of three independent experiments. Bars represent mean values of duplicate experimental conditions+SEM, in one of three independent experiments. A comparable pattern of THP-1 activation and relative proportions could also be observed at different MOIs (1:10, 1:20, 1:50) (data not shown). TNF, tumour necrosis factor.

    Activation of the transcription factor NFκB

    THP-1 Blue cells are equipped with a reporter system that allows quantifying NFκB activation after stimulation by detecting SEAP activity in cell supernatants. We stimulated PMA-differentiated cells (dTHP-1 Blue) with M hominis MhMOZ-2 and isogenic T vaginalis isolates G3 and G3/MOZ-2, by using MOIs ranging from 1:20 to 1:100 (T vaginalis/THP-1 ratio). The pattern of activation, expressed as NFκB-driven SEAP reporter activity, was dose dependent and was consistent with the synergistic production of proinflammatory cytokines induced by G3/MOZ-2, as compared to either G3 or MhMOZ-2 single stimulations (figure 3).

    Figure 3
    Figure 3

    Activation of the transcription factor nuclear factor κB (NFκB). The reporter cell line THP-1 Blue was stimulated with different conditions, incubated 24 h and supernatants collected and assayed for secreted embryonic alkaline phosphatase (SEAP) activity with a colorimetric reaction. G3, naturally mycoplasma-free Trichomonas vaginalis; MOZ-2, Mycoplasma hominis; G3/MOZ-2, symbiotically-associated T vaginalis G3 and M hominis MOZ-2. The x-axis shows multiplicities of infections (MOIs) (T vaginalis/dTHP-1 ratios; M hominis colour-changing units (CCUs)). The y-axis shows SEAP activity, absorbance at 620 nm. Cytokine secretion after stimulation with G3, MOZ-2, G3/MOZ-2 is significantly different from mock control at all MOIs used throughout the experiment (unpaired Student t test; p<0.01).

    Discussion

    In recent years our group has showed that T vaginalis establishes a symbiotic relationship with another sexually transmitted pathogen, the bacterium M hominis.18 This is the first case of a biological association between two obligated parasites: neither of them is able to live outside the human host. We showed that T vaginalis is able to transmit in vitro the bacterial infection to naturally mycoplasma-free trichomonad isolates and to human cervical epithelial cells.4 Furthermore, we demonstrated how M hominis is able to localise, replicate and persist within protozoan cells.5 Altogether, this data suggest how T vaginalis may act as a ‘Trojan horse’ for M hominis infection, protecting the bacterium from the harsh vaginal environmental conditions and from antibiotic treatments.19

    While some light was shed on several aspects of the symbiosis, its influence over the host–parasite interaction still needs to be studied. The current model of T vaginalis pathogenesis involves three fundamental aspects: (a) adhesion of T vaginalis to the vaginal epithelium and interaction with the vaginal environment, (b) production of cytotoxins that cause the host cell lysis and (c) the host inflammatory response.20–22 While the adhesion of T vaginalis to the host epithelium and the subsequent cytopathic effect are intensively investigated and many aspects have been elucidated, the contribution of T vaginalis-induced inflammation to the pathology remains elusive. The data present in the literature are somehow contradictory and sparse, since proinflammatory and anti-inflammatory responses to T vaginalis have both been described.3 Some authors described anti-inflammatory and proapoptotic responses of murine macrophages and human neutrophils,23 ,24 while others reported the production of the proinflammatory chemokine IL-8 by neutrophils and primary monocytes after challenge with T vaginalis. We hypothesise that one possible explanation for this variability could be related to the symbiotic relationship with M hominis: indeed, the T vaginalis isolates used in these studies did not undergo preliminary tests for the presence of M hominis.

    In this work, we tested the possibility of an influence of the symbiotic association between T vaginalis and M hominis over the interaction in vitro of both microorganisms with the human monocytic cell line THP-1. To address this question, we set up an experimental model consisting of two isogenic T vaginalis isolates. T vaginalis G3 was experimentally infected with M hominis MhMOZ-28 and then used to stimulate the human monocytic cell line THP-1. The pattern of secreted proinflammatory cytokines was compared to that obtained using the parental mycoplasma-free G3 isolate as a stimulus.

    We first characterised the macrophage response to single infections. dTHP-1 macrophages responded in a qualitatively and quantitatively different fashion to T vaginalis and M hominis, with the latter shown to be a more potent inducer of inflammatory mediators such as IL-8, TNFα, IL-1β, IL-23. Interestingly, T vaginalis cell lysates induced levels of cytokines higher than those induced by live trichomonad cells. This may be an indication that cell lysis causes the exposition to the macrophage surface receptors of intracellular protozoan molecules that have proinflammatory activity. Alternatively, we could speculate that live T vaginalis is able to evade the recognition of the innate immunity receptors and to possibly downregulate the inflammatory response. dTHP-1 cells thus appear to have a certain degree of plasticity in their response to these two microorganisms, similarly to human dendritic cells.25 A previous report showed that T vaginalis cells and membrane preparations are able to induce the production of IL-8 by primary human monocytes.9 This is in contrast with our findings, since we could not detect IL-8 in dTHP-1 supernatants after stimulation with G3 mycoplasma-free T vaginalis. One explanation could be that the authors of the previous report did not consider the possible presence of M hominis in the trichomonad isolates used in their study.

    We then compared the responses of dTHP-1 to the isogenic G3 and G3/MOZ-2 T vaginalis isolates. The stable association between T vaginalis and M hominis synergistically upregulates the secretion of proinflammatory cytokines by dTHP-1 cells to either T vaginalis or M hominis single infection. This finding is suggestive of a possible role of the symbiosis in the high degree of variability of signs and symptoms observed in T vaginalis infections. Possibly, the stable association with M hominis may also exacerbate the inflammatory condition in vivo. We also showed that the stable association between Trichomonas and M hominis is essential for the synergistic increase in cytokine secretion. In fact, G3/MOZ-2 led to higher concentrations of secreted cytokines than simultaneous costimulation with G3 and MhMOZ-2 in combination.

    We also tested the possible involvement of the transcription factor NFkB in the synergistic upregulation of the macrophage response. THP-1 cells expressing a reporter system showed that NFkB activation is also synergistically upregulated by the symbiotic association between T vaginalis and M hominis, consistent with the upregulation observed for cytokine secretion. This suggests that this phenomenon involves signalling pathways leading to the activation of NFkB.

    The ability of symbiotically-associated T vaginalis and M hominis to upregulate the proinflammatory response of human macrophages has important potential implications. The synergistic increase in the secretion of inflammatory mediators such as IL-1β, TNFα and IL-8 could also happen in vivo, thus affecting the severity of the disease. Interestingly IL-23, which polarises the adaptive immunity to a T helper (Th)17 type of response, is produced only when M hominis is present. The Th17 response is involved in host defence to infections by playing a key role in inflammatory processes at the mucosal surfaces,26 and T vaginalis appears to evade this defence by not inducing IL-23 secretion by macrophages. The modulation of the inflammatory response to one or both microorganisms is certainly more complex, and involves a series of interactions among the different components of the unique immunological niche represented by the vaginal mucosa.27 From our data it appears that macrophages are able, at least in vitro, to respond in a peculiar way to T vaginalis when symbiotically associated to M hominis by modulating the pattern of secreted proinflammatory cytokines.

    T vaginalis hosts an endosymbiont other than M hominis, a dsRNA virus highly prevalent in Trichomonas isolates (T vaginalis virus (TVV)).28 Recently Fichorova and colleagues29 elegantly showed how TVV is sensed by vaginal epithelial cells with mechanisms independent from those involved in T vaginalis recognition. Furthermore, the authors demonstrated that TVV amplifies the inflammatory response to T vaginalis. This is further evidence supporting the idea that T vaginalis infection involves a complex series of interactions among multiple actors on the host and the pathogen sides. Further studies will be aimed at elucidating the mechanisms underpinning this phenomenon. In particular the host receptors involved in the recognition of the two symbionts, and the signalling pathways leading to the synergistic upregulation of the inflammatory host response should be explored, as well as the possibility of an influence of the symbiosis on other aspects of Trichomonas pathogenesis, such as adhesion to host cells and subsequent cytolysis.

    The synergistic upregulation of the proinflammatory response might impact on Trichomonas pathogenesis, especially on those aspects for which the T vaginalis-induced inflammation is thought to have a role: an increased risk of cervical and prostate cancer and HIV acquisition. Altogether, our data reinforce the idea that when diagnosing T vaginalis infections in men and women, the likely presence of M hominis30 ,31 must be taken into account for subsequent treatment indications.

    Key messages

    • Trichomonas vaginalis and Mycoplasma hominis establish a unique symbiosis, with growing evidences supporting the idea of an influence of this relationship over the pathobiology of both microorganisms.

    • Symbiotically-associated T vaginalis and M hominis elicit a synergistic effect over the proinflammatory response of human macrophages in vitro.

    • The synergistic upregulation of inflammatory cytokines may have important implications on several risks associated with trichomoniasis (HIV, cancer), which are thought to be linked to chronic inflammatory phenomena.

    Acknowledgments

    The authors are grateful to Giuliana Solinas (University of Sassari, Italy) for her advice in statistical analysis.

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    Footnotes

    • Contributors PLF conceived the research, designed the experimental outline and helped to draft the manuscript. ND performed experiments and helped to draft the manuscript. ARC was involved in data analysis and interpretation. PR critically revised the manuscript and made important scientific contributions. DD conceived the research, designed the experimental outline, performed experiments, analysed data and was the primary writer of the manuscript. All authors were involved in the finalisation of the manuscript and gave their approval for the final version.

    • Funding This work was supported by Fondazione Banco di Sardegna grant number FBSDESS2010.

    • Competing interests The authors declare that they have no competing financial interests.

    • Provenance and peer review Not commissioned; externally peer reviewed.