SEPSIS AND ICU (J RUSSELL, SECTION EDITOR)

Noncanonical Nuclear Factor Kappa B (NF-κB) Signalingand Potential for Therapeutics in Sepsis

Simone Thair & James A. Russell

Published online: 22 August 2013# Springer Science+Business Media New York 2013

Abstract NF-κB signaling plays a central role in the patho-physiology of severe sepsis and septic shock. Despite tremen-dous and missed efforts, novel therapeutics for severe sepsisand septic shock are still needed. Many drugs have beendesigned to target the canonical NF-κB signaling pathwaywith limited success, potentially due to the nonspecificity ofthe drugs for other kinases and the interaction of canonicalsignaling with other pathways. Here, we review the canonicaland noncanonical signaling pathways of NF-κB, the cross talkand negative regulation of the two pathways, and the potentialfor therapeutics arising from the noncanonical NF-κB path-way in relation to the pathophysiology of septic shock.

Keywords Sepsis . Septic shock . Therapeutics .

Noncanonical NF-κB signaling . Personalizedmedicine

Introduction

The documented incidence of sepsis worldwide is 1.8 millioneach year, but there is speculation that the value is confoundedby a low diagnostic rate and difficulties tracking sepsis incertain countries. The surviving sepsis campaign has proposedthat a more realistic rate would be 3 per 1,000, bringing thetrue number of cases per year world wide up to 18 million.Combined with a mortality rate of approximately 30 %, sepsisbecomes a leading cause of death worldwide [1]. Tumornecrosis factor alpha (TNFα) is one of the most extensivelystudied cytokines in relation to the development of sepsis,

severe sepsis, and septic shock. TNFα stimulates the nuclearfactor kappa B (NF-κB) canonical pathway, whereas thenoncanonical pathway responds to TNFα superfamily ligands[2]. Here, we review the canonical and noncanonical signalingpathways of NF-κB, the cross talk and negative regulation ofthe two pathways, and the potential for therapeutics arisingfrom the noncanonical pathway in relation to the pathophys-iology of septic shock.

The Pathophysiology of Septic Shock

TNFα Superfamily Induced Nuclear Factor Kappa BSignaling in Septic Shock

TNFα is centrally involved in the pathophysiology of septicshock [3]. The existence of a TNFα superfamily was firstrealized as the true nature of lymphotoxin (LT) and TNFαwereelucidated. Using hundreds of liters of culture medium from alymphotoxic cell line, human LTwas the first cytotoxic factor tobe isolated and characterized from a B-lymphoblastoid cell line[4, 5]. Since the actions of LTand TNFαwere determined to besimilar, experiments done using the neutralization of LT withspecific antibodies resulted in the isolation of a second cytotoxicfactor, TNFα [6]. Shortly after the cloning and characterizationof TNFα [6], experiments in baboons immunized with anti-TNFα demonstrated protection from hypotension and lethalrenal and respiratory failure due to bacteraemic shock,suggesting TNFα as a major root of sepsis and anti-TNFα apotential therapy [7, 8]. Further characterization of the action ofthese two ligands indicated a common receptor [9] and, hence,the beginnings of a TNFα super family, which today encom-passes 19 ligands acting on 29 receptors (as reviewed in [2]).These ligands and their receptors activate various signalingpathways, including NF-κB [2]. Depending on the TNFα su-perfamily ligand present, this can activate NF-κB via the ca-nonical or noncanonical pathways [2].

S. Thair (*) : J. A. RussellUniversity of British Columbia, Heart Lung Innovation ResearchCentre, Critical Care Research Laboratories, St. Paul’s Hospital,#166-1081 Burrard St., Vancouver, BC V6Z 1Y6, Canadae-mail: Simone.thair@hli.ubc.ca

J. A. Russelle-mail: Jim.russell@hli.ubc.ca

Curr Infect Dis Rep (2013) 15:364–371DOI 10.1007/s11908-013-0362-0

Canonical Signaling

Canonical NF-κB signaling can be induced by many ligandsbinding to receptors, including, but not limited to, cytokine-induced signaling (e.g., IFNγ), toll-like receptors (TLRs), or theTNFα superfamily receptors [10]. All of these events are im-portant in the pathophysiology of septic shock; here, we focuson the events following TNFα-induced NF-κB signaling.

Following binding of TNFα, tumor necrosis factor receptorassociated factor (TRAF2) is recruited to the tumor necrosisfactor receptor (TNFR1) through its interaction with tumornecrosis factor receptor type 1 associated death domain(TRADD) [11]. Interestingly, experiments demonstrated thatTRAF2-deficient cells have relatively intact TNF signaling toNF-κB [12]. Simultaneously, evidence of an interaction ofTRAF5 with TNFR1 signaling complex was uncovered, yetTRAF5 knockouts could still activate NF-κB [13]. It was thenshown that TRAF2/5 double knockout cells, however, wereunable to activate IκB kinase (IKK) [12–14]. Therefore, thecurrent consensus is that TRAF2 and TRAF5 are both re-quired for NF-κB activation by TNFR1.

TRAF2 and 5 form a complex with TRADD and receptorinteracting protein (RIP-1), at which point TRAF2 is releasedand RIP-1 recruits mitogen-activated kinase kinase (MEKK-3)and transforming growth factor β activating kinase 1 (TAK1)[15]. This leads to the phosphorylation of the IKKs (IKKα andIKKβ) that reside in a ~900-kDa multicomponent complextermed the signalsome [16–18]. The most common IKKα/βheterodimeric complex also includes NF-κB essential modula-tor (NEMO)/IKKγ/ inhibitor of kappa light polypeptide geneenhancer in B-cells, kinase gamma (IKKAP1), and a scaffold-ing protein called inhibitor of kappa light polypeptide geneenhancer in B-cells, kinase complex-associated protein(IKAP) [19]. IKBα, which serves to sequester NF-κB homo-and heterodimers in the cytoplasm by binding the Rel homol-ogy domain to interfere with its nuclear localization sequence(NLS) [20–22], is phosphorylated, ubiquitinated, and sent forproteosomal degradation, thus releasing the dimers for translo-cation to the nucleus [23]. The signalsome is where many of thedifferent stimuli-induced paths merge, and the steps betweenthis axis point and the nucleus that follows are considered thecanonical pathway [2].

Overall, the NF-κB family consists of five key molecules:NF-κB1 (p105/ p50), NF-κB2 (p100/p52), RelA (p65), RelB,and c-Rel [10]. p105 and p100 are processed via theproteosome to produce p50 and p52, respectively [10]. Varioushomo- or heterodimer combinations of the five molecules allowfor differential transcription depending on the signal induction[10]. NF-κB dimers are retained in the cytoplasm by IκBs, theaforementioned degradation of IκBs reveals the NLS, and thedimers can translocate to the nucleus. Phosphorylated NF-κBdimers bind to κB DNA elements and induce transcription oftarget genes.

Noncanonical Signaling

Until approximately 1997, the dogma of NF-κB signaling wasthat all signals converged at the IKKs and IκBα and proceededthrough to the nucleus from there. At this time, Malinin et al.discovered a novel TRAF2 interacting protein that appeared tobe a potent stimulator of NF-κB signaling in response to TNFand IL-1, called MAP3K14 or NF-κB inducing kinase (NIK)[24]. Concurrently, it was shown that NIK was capable ofactivating IKKα by phosphorylation at Ser 176 [25]. Scrutinyof this novel NIK protein in relation to the canonical pathwayand its molecular mechanism revealed several key aspects:There appeared to be negative regulation [26]; NIK was adocking molecule for p100, and this docking led to p100activation and proteasomal processing [27, 28]. Further studieselucidated the mechanism of regulation whereby NIK is consti-tutively transcribed, translated, and degraded, through interac-tion with TRAF3 [29, 30]. Simultaneously, Senftleben et al.found IKKα preferentially phosphorylates NF-κB2 (p100), amechanism requiring upstream kinases—specifically, NIK—suggesting that NIK is not involved in IKBα degradation butthat a second evolutionarily conserved pathway had been dis-covered [31]. Furthermore, it was shown that NIK −/− miceexhibited normal canonical NF-κB activity upon stimulationwith TNF and IL-1, with no changes in NIK transcription.Rather, LTβ stimulation increased NIK transcription, furthersupporting the evidence of a novel separate pathway based onregulated NF-κB processing, rather than IκB degradation [32].Taken together, the unique characteristics of this noncanonicalpathway include stimulation of receptors with TNFα superfam-ily ligands such as CD40L, LT/ LTβ, and BAFF [33]. Degra-dation of TRAF3 stabilizes NIK protein, which recruits IKKαhomodimers to NIK, while simultaneously acting as a dockingmolecule for p100 so that IKKα can phosphorylate p100 [27].Subsequently, p100:RelB-associated heterodimers areprocessed to p52:RelB heterodimers for translocation to thenucleus [10, 27]. This is in contrast to canonical signalingtranslocation of RelA, c-Rel, and/or p50 dimers [10].

Cross Talk Between Canonical and Noncanonical Signaling

The discovery of two distinct and separate pathways furtherexplains more of the incredibly diverse and elegant signalingorchestrated by NF-κB. As the mechanism of the noncanonicalpathway was understood, sophisticated studies to further under-stand the mechanism of the signalsome led to the discovery of adirectional activation of the heterodimeric IKKα/β complex,proposed as the pivot point for cross talk between the twopathways [34]. Recent evidence suggests that the IKK–NIKaxis may be a pivot point for control of both noncanonical andcanonical signaling [33–35].

Zarnegar et al. demonstrated that while TRAF3 inhibits NIKand noncanonical signaling, TRAF3 also inhibits canonical

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signaling [35]. Furthermore, in TRAF3-deficient mice, canon-ical signaling is dysregulated in concert with an accumulationof NIK, suggesting that TRAF3 controls levels of NIK thatfacilitate cross talk between the two pathways [35]. Inspectionof the LTβR-associated signaling complexes showed thatoverexpression of TRAF3 increased TRAF3 accumulation withLTβR complexes and decreased IκBα phosphorylation, henceinhibiting canonical signaling [36]. Conversely, knockdown ofTRAF3 increased the presence of noncanonical pathway mem-bers such as p100, RelB, and NIK, as well as increased pro-cessing of p100 to p52 [36]. Upon examination of timing ofexperimental procedures, it is interesting to note that manystudies that separated the two pathways and excluded NIK fromcanonical signaling focused on early events (from minutes toless than 2 h) [31, 32]. However, many studies that collecteddata at much later time points (several hours to days) havefound that NIK plays a pivotal role in canonical signaling[24, 34, 35].

Negative Regulation of NF-κB

NF-κB initiates the signaling for the inflammatory response,whether proinflammatory or antiinflammatory. It is critical forthe human host to terminate these responses when no longerneeded, and abberations of these termination processes are theroot of many conditions, including sepsis and septic shock [37].Termination is accomplished via a wide array of evolutionarilyconserved members, including decoy receptors, ubiquitinases,de-ubiquitinases, transcriptional regulators, splice variants,microRNAs, and direct binding inhibitors [38]. The negativeregulation of toll-like receptor (TLR) induced NF-κB signalingsuggests a complex system of tightly control processes normal-ly [39]. One of the most famous examples of TNFα-inducedNF-κB signaling is A20 (TNFAIP3). Initially discovered as aTNF inducible gene in human umbilical vein endothelial cells[40], A20 plays an essential role in regulating the NF-κB-driveninflammatory response [41]. A20 expression is up-regulated inresponse to NF-κB signaling [42]. A20 is both a ubiquitinizingand de-ubiquitinizing protein, its induction creating a negativefeedback loop to inhibit further signaling [43].

There is less known about the negative regulation of thenoncanonical pathway, since the focus has largely been in theshifts in the signaling that occur as the pools of noncanonicaland canonical NF-κB homo- and heterodimers change overtime depending on the stimulus, as described above. It has beenshown that lupus patients have abnormal CD40-induced NF-κB signaling [44]; however, instead of observing constitutivetranslocation of p52/RelB in response to excess CD40 and itsligand CD154, Zhang et al. found that there was increasedIKBα degradation and increased canonical signaling via p50,RelA, and c-Rel, but not RelB and p52 [44]. Again, thissuggests that a shift from noncanonical signaling to canonicalover time acts as the “inhibition” of the noncanonical signaling

and it is yet to be determined whether there are direct inhibitorsspecific to noncanonical signaling.

Potential Therapeutics from the Alternative/NoncanonicalNuclear Factor Kappa B (NF-κB) Signaling Pathway

The Potential Role of NIK in Immunosuppression

To suppress or not to suppress, that is the question! Despiteyears of research, the optimal balance between pro- andantiinflammatory signals needed for the resolution of severeinfections has yet to be pinpointed. There is mounting evi-dence to support a shift in dogma from a biphase responsewhereby initial early response proinflammatory signals(termed the systemic inflammatory response syndrome) arefollowed temporally by antiinflammatory signals (the com-pensatory antiinflammatory response syndrome) to a model inwhich both events happen simultaneously and the balance (orlack thereof) between the two is key for resolution [45••].Furthermore, it is becoming apparent that assessing theimmunostatus of a patient to determine whether treatmentshould proceed to control the hyperinflammatory responseor stimulate the immunosupressed is a viable and promisingavenue as we progress toward personalizedmedicine [46••]. Arecent review by LaRosa and Opal revealed that microarraysof blunt trauma and burn victims have a common responsetranscriptional response similar to that of LPS challenge;hence, advancements in therapeutics for sepsis may be appliedin numerous settings [47].

NIK acts as a pivot point in the control of NF-κB signaling,both noncanonical and canonical, and may demonstrate agood target for potential therapeutics, especially in relationto treatment of immunosuppression. There is a growing bodyof research supporting the evidence of a critical balance be-tween T helper 17 (Th17) and regulatory T cells (Tregs), suchthat unbalanced T helper/T reg may contribute to autoimmu-nity and inflammation [48, 49]. Interestingly, in sepsis andseptic shock, quantification of T cell populations revealed anexcessive amount of Tregs, which can contribute to lympho-cyte anergy and immunoparalysis and has been associatedwith a poor outcome [50–52, 53•]. The balance between Tregsand Th17 cells demonstrated in autoimmunity and inflamma-tion prompted a study of the Th17 population in patients withsevere sepsis, which found that survivors had restoration ofTh17 differentiation and counts [54•]. Jin et al. demonstratedthat suppression of MAP3K14 (NIK) resulted in the inabilityof T cells to differentiate into Th17 cells. This reduction in theTh17 population was associated with resistance to experimen-tal automimmune encephalitis [49]. Interestingly, Murray andcolleagues demonstrated that NIK-deficient mouse T cellstransferred into MHC class II mismatched recipients failedto cause graft versus host disease. Furthermore, when NIK

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was overexpressed in mouse T cells, fatal autoimmunity en-sued, characterized by hyperactive effector T cells and a lackof suppressive Tregs [55•]. This suggests that less NIK wouldresult in a smaller Th17 population, potentially increasing theamount of Tregs.This could be beneficial for resistance toautoimmunity, but in sepsis may be detrimental, as highlight-ed by increased Treg populations in patients with poor out-come [50–52]. We showed an association of rs7222094 (Callele, minor) in NIK (MAP3K14) with increased organ dys-function and mortality in septic shock patients in two inde-pendent septic shock cohorts [56•]. Furthermore, nonsuvivorshad decreased plasma levels of CXCL10, a cytokine depen-dent on NIK and known for its recruitment of T cells andactivated natural killer cells to the site of infection [35, 57].Patients who carried the major T allele had a higher survivaland more CXCL10 [56•]. We did not measure NIK directly; itcould be inferred that reduced CXCL10 is a product of re-duced NIK activity. Since NIK is also required for Th17proliferation, it is possible that less NIK (marked by lessCXCL10, found in the patients homozygous for the C allele)would also result in fewer Th17 cells, leading to in an increaseof Tregs. This shift in the T cell population could contribute tothe poor outcomes we observed.

Noncanonical Signaling and Glucocorticoids

A recent randomized double-blind, placebo controlled trial ofcorticosteroid therapy of septic shock (CORTICUS) found thatlow-dose hydrocortisone did not alter 28-day mortality, regard-less of the patients’ adrenal responsiveness to corticosteroids.However, the duration of time until the reversal of shock wasshorter in those treated with hydrocortisone, but the proportionsof patients whose shock was reversed was not significantlydifferent between the hydrocortisone and placebo groups [58].Therefore, it would appear that if the patients’ condition isgoing to improve, hydrocortisone may accelerate this process;however, the treatment itself may not alter the outcome forthose whose condition will not reverse. Previous studies hadshown that hydrocortisone was beneficial [59–61]; hence, theseresults are somewhat but not entirely unexpected [62–64].Several key differences between their study and the study byAnnane et al. [59] may explain the different results, such asdissimilar patient populations, different time windows for in-clusion criteria, an increased incidence of superinfection, andnew septic episodes.

Importantly, CORTICUS may have been underpowered,since the study was stopped after recruitment of 500 patientsinstead of the intended 800. Corticosteroids inhibit inflamma-tory pathways emphasized by the increased proinflammatorymediators and hemodynamic deterioration after abrupt cessa-tion of treatment [65]. Glucocorticoids (GCs) bind to the GCreceptor (GR) [66, 67]. The ligand bound receptor complextranslocates into the cell nucleus, where it binds to GC

response elements in the promoter region of the target genes,a process referred to as transactivation, which up-regulatesexpression of target genes [66]. Transrepression can alsooccur whereby the ligand bound receptor complex interactswith specific transcription factors (such as AP-1 and NF-κB)and prevents the transcription of targeted genes [66]. Thefindings of the CORTICUS trial in light of other conflictingdata suggest that further molecular studies regarding the spe-cific effect of GCs on key inflammatory pathways known tobe involved in sepsis are needed, as well as extremely uniforminclusion and dosage criteria across trials.

An elegant series of experiments have been publishedspecifically related to the action of GCs on receptors thatfacilitate the communication between Tregs and plasmacytoidDCs (pDCs) [68, 69]. Members of the TNFα superfamilyligands can also act as receptors, not only inducing signalingin the cell that possesses the receptor (such as Tcells), but alsocausing “reverse signaling” in the cell possessing the liganditself (pDCs) [70]. The GC inducible TNF receptor (GITR)can be found on Treg cells [71], while its natural ligand,GITRL, belongs to the TNF superfamily and is present on avariety of cells, including mature and immature dendritic cells(DCs) [72]. GC treatment in vivo increased the amount bothof GITRTcells and of GITRL expressed by pDCs, which thenresulted in an increase in indoleamine 2,3-dioxygenase (IDO),a molecule originally discovered for its role in tryptophancatabolism [68]. GC acts on the GITR–GITRL co-receptorsystem to induce IDO expression via noncanonical NF-κBsignaling, and this leads to IDO-mediated Treg generation[68]. Interestingly, a recent study in asthmatic patients foundthat GC treatment (while immunosuppressive andantiinflammatory) promotes the differentiation of naive Tcellstoward a Treg phenotype in a FOXP3-dependent manner [73].

We have provided examples of noncanonical signaling con-trolling differentiation of Th17s or Tregs, and this process maybe influenced byGCs.Most of this work has arisen from studiesof autoimmunity or allergic diseases. Further studies to deter-mine the status of these mechanisms under acute inflammatoryconditions could provide a platform to support personalizedmedicine for GC use. One could postulate a scenario in whichhigh levels of Tregs and proinflammatory cytokines contributeto poor outcome in sepsis and septic shock, and so a patientdiagnosed with low Tregs and high inflammatory cytokinescould benefit from GC treatment. Conversely, a patient with aprofile of increased Tregs and high antiinflammatory cytokinesmay be at risk for immunosuppresion upon treatment with GCs.

Noncanonical Signaling and GM-CSF Treatment

IRAK-M was discovered as an inhibitor of NF-κB signaling,and hence, in order to further define the mechanism, Su et al.[74] found that IRAK-M selectively inhibits the noncanonicalsignaling pathway, rather than the canonical. Specifically, in

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IRAK-M-deficient mice, granulocyte macrophage colonystimulating factor (GM-CSF) expression is increased; howev-er, upon knockdown of the noncanonical NF-κB subunitRelB, this effect is ablated. Hence, the noncanonical signalingpathway plays a role in the production of GM-CSF.

GM-CSF is a growth factor with immunostimulatory effectssuch as improving survival, proliferation, differentiation, phago-cytosis, and bacterial killing of neutrophils and monocytes/macrophages [75]. GM-CSF also promotes migration and ad-hesion of neutrophils [75]. Importantly, GM-CSF increasesHLA-DR expression on monocytes in patients with severesepsis [76, 77]. That, combined with evidence from recent pilottrials observing monocyte deactivation reversal in vivo byapplication of GM-CSF [49], resulted in a randomized con-trolled trial of GM-CSF treatment for septic shock [78•].

Immunosuppression or immunoparalysis has been previous-ly defined in patients with monocytic HLA-DR expression ofless than 30 % for two consecutive days and is characterized byan impaired ability to mount a further response [79]. Reports ofmonocyte inactivation for more than 2 days was associated with58 % mortality, which then rose to 88 % if the inactivation waspresent for more than 5 days [79, 80]. Initially, inadequateIFN-γ production was implicated in the down-regulation ofHLA-DR expression and IFN-γ stimulation of monocytesin vitro. A small trial of inhaled IFN-γ showed promisingresults [79–82].

The trial of GM-CSF enrolled septic shock patients and usedbiomarker assessment of monocytic HLA-DR expression todetermine which patients were candidates for treatment withGM-CSF [78•]. Of those treated, HLA-DR levels normalized in19/19 patients, accompanied by less time on mechanical venti-lation, an improved APACHE II score, and a trend toward ashorter hospital stay, with no reported side effects [78•]. Thishighlights the fact that proper functioning of noncanonicalsignaling is important for GM-CSF levels. Another potentialscenario is one in which less NIK (patients homozygous for Callele of rs7222094) results in less GM-CSF, lower expressionof HLA-DR, and without treatment, a worse outcome.

NIK Targeted Therapies for Multiple Myeloma and Potentialfor Treatment of Sepsis

Dysregulation of NIK due to mutations that inactivate or deleteTRAF2, TRAF3, cIAP1, or cIAP2 as well as duplications ortranslocations of NIK itself have been identified as responsiblefor a large portion of multiple myeloma (MM) cases [83, 84].These aberrations result in hyperactivity of the noncanonicalsignaling pathway, as well as canonical signaling, most likelydue to the accumulation of NIK over time, similar to themechanism described above in the lupus study [44]. Hence,inhibitors of NIK and/or the noncanonical signaling pathwayare being developed. A virtual screen of small molecule NIKinhibitors revealed 4H-isoquinoline-1.3-dione and 2,7-

naphthydrine-1,3,6,8-tetrone [85•]. Two patents were filed for6-azaindole aminopyrimidine and derivatives of alkynyl alco-hols [86, 87]. If any of these drugs were to succeed in devel-opment for treatment of MM, it would present an interestinghypothesis to test in sepsis. In light of the complex effects of thenoncanonical signaling pathway on sepsis, especially in termsof hyperinflammation or immunosuppresion, personalizedmedicine to determine the status of a patient would beimperative.

New Pharmaceutical Targets for Noncanonical Signaling

Many attempts have been made to inhibit NF-κB signaling,largely targeted at the canonical pathway (as reviewed in[88]). In light of the evidence of cross talk between the twopathways and the increasing list of effects, muchmore specificinhibitors need to be developed. Furthermore, the specificaction of these two pathways is cell specific, which results intheir specialized differentiation and signaling [89]. Inhibitingthe phosphorylation of IkBα prevents DC maturation, andimmature DCs are tolerogenic and induce Treg differentiation,a situation that would be beneficial in autoimmune diseases.Hence, ex vivo DCs were treated with BAY 11–7082, aninhibitor of IkBα, and then the cells were injected into theknee joints of arthritic mice; it was found that inflammationand erosion were suppressed [90]. While this strategy sup-presses autoimmunity in specific tissues (rather than treatingsystemic acute inflammation), it does highlight the ability touse “retrained” cells to assist resolution of inflammation intissues that are unable to reinstate homeostasis themselves.

Conclusion

Both the noncanonical and canonical NF-κB pathways playkey roles in both the development and resolution of severesepsis and septic shock. The need for further understanding ofthe exact roles of the two pathways in order to utilize them isimperative and highlights the need for personalized medicinein the heterogeneous population of patients with sepsis. Thedetermination of immune status [46••] and genotyping for keypolymorphisms that may influence immune status [56•,91–96] will allow for more informed decisions.

Compliance with Ethics Guidelines

Conflict of Interest Simone Thair has received a graduate studentstipend from CIHR/NSA and has been a consultant for LKLConsulting/Infinity Cohort Systems.

James Russell declares no conflicts of interest.

Human and Animal Rights and Informed Consent This article doesnot contain any studies with human or animal subjects performed by anyof the authors.

368 Curr Infect Dis Rep (2013) 15:364–371

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