Key points are not available for this paper at this time.
Lymphotoxin ॆ receptor (LTॆR)-induced activation of NF-κB in mouse embryo fibroblasts was mediated by the classical pathway and by an alternative or second pathway. The classical pathway involved the IκB kinase (IKK)ॆ- and IKKγ-dependent degradation of IκBα and resulted in the rapid but transient activation of primarily RelA-containing NF-κB dimers. The alternative or second pathway proceeded via NF-κB-inducing kinase (NIK)-, IKKα-, and protein synthesis-dependent processing of the inhibitory NF-κB2 p100 precursor protein to the p52 form and resulted in a delayed but sustained activation of primarily RelB-containing NF-κB dimers. This second pathway was independent of the classical IKK complex, which is governed by its central IKKγ regulatory subunit. The sequential engagement of two distinct pathways, coupled with the negative feedback inhibition of RelA complexes by NF-κB-induced resynthesis of IκBα, resulted in a pronounced temporal change in the nature of the NF-κB activity during the course of stimulation. Initially dominant RelA complexes were replaced with time by RelB complexes. Therefore, the alternative activation path mediated by processing of p100 was necessary for sustained NF-κB activity in mouse embryo fibroblasts in response to LTॆR stimulation. Based on the phenotype of mice deficient in various components of the LTॆR-induced activation of p100 processing, we conclude that this pathway is critically involved in the function of stromal cells during the generation of secondary lymphoid organ microarchitectures. Lymphotoxin ॆ receptor (LTॆR)-induced activation of NF-κB in mouse embryo fibroblasts was mediated by the classical pathway and by an alternative or second pathway. The classical pathway involved the IκB kinase (IKK)ॆ- and IKKγ-dependent degradation of IκBα and resulted in the rapid but transient activation of primarily RelA-containing NF-κB dimers. The alternative or second pathway proceeded via NF-κB-inducing kinase (NIK)-, IKKα-, and protein synthesis-dependent processing of the inhibitory NF-κB2 p100 precursor protein to the p52 form and resulted in a delayed but sustained activation of primarily RelB-containing NF-κB dimers. This second pathway was independent of the classical IKK complex, which is governed by its central IKKγ regulatory subunit. The sequential engagement of two distinct pathways, coupled with the negative feedback inhibition of RelA complexes by NF-κB-induced resynthesis of IκBα, resulted in a pronounced temporal change in the nature of the NF-κB activity during the course of stimulation. Initially dominant RelA complexes were replaced with time by RelB complexes. Therefore, the alternative activation path mediated by processing of p100 was necessary for sustained NF-κB activity in mouse embryo fibroblasts in response to LTॆR stimulation. Based on the phenotype of mice deficient in various components of the LTॆR-induced activation of p100 processing, we conclude that this pathway is critically involved in the function of stromal cells during the generation of secondary lymphoid organ microarchitectures. nuclear factor-κB inhibitor of κB IκB kinase complex NF-κB-inducing kinase lymphotoxin lymphotoxin ॆ receptor alymphoplasia tumor necrosis factor mouse embryo fibroblast electrophoretic mobility shift assay B cell-activating factor belonging to the TNF family NF-κB1 transcription factors are critical mediators in the fight of the host against invading pathogens (reviewed in Refs. 1Ghosh S. Karin M. Cell. 2002; 109: S81-S96Google Scholar, 2Li Q. Verma I.M. Nat. Rev. Immunol. 2002; 2: 725-734Google Scholar, 3Caamaño J. Hunter C.A. Clin. Microbiol. Rev. 2002; 15: 414-429Google Scholar, 4Karin M. Ben-Neriah Y. Annu. Rev. Immunol. 2000; 18: 621-663Google Scholar, 5Brown K. Claudio E. Siebenlist U. Targeted Therapies in Rheumatology. Martin Dunitz Ltd., London, UK2002: 381-401Google Scholar). These factors are integral parts of the innate machinery that translates initial detection of foreign pathogens, for example by epithelial cells, into activation of these cells, including production of chemokines and cytokines to in turn attract and activate professional immune cells. The innate system further involves NF-κB factors to produce antipathogenic effectors as well as chemokines and cytokines to mediate evolving cell-cell communications needed to coordinate responses. Depending on the exact nature of the initial innate response, NF-κB factors then help to develop the appropriate adaptive responses by lymphocytes. In the final phase of the immune response, NF-κB factors have important roles during the expansion and differentiation of lymphocytes involved in the adaptive response. Beyond the innate and adaptive antipathogenic responses, NF-κB factors are also essential during development and maintenance of lymphoid organ structures (6Franzoso G. Carlson L. Poljak L. Shores E.W. Epstein S. Leonardi A. Grinberg A. Tran T. Scharton-Kersten T. Anver M. Love P. Brown K. Siebenlist U. J. Exp. Med. 1998; 187: 147-159Google Scholar, 7Caamaño J.H. Rizzo C.A. Durham S.K. Barton D.S. Raventos-Suarez C. Snapper C.M. Bravo R. J. Exp. Med. 1998; 187: 185-196Google Scholar), and they make important contributions during the development of hematopoietic cells, including B cells and osteoclasts (8Franzoso G. Carlson L. Xing L. Poljak L. Shores E.W. Brown K.D. Leonardi A. Tran T. Boyce B.F. Siebenlist U. Genes Dev. (1997). 1997; 11: 3482-3496Google Scholar, 9Gugasyan R. Grumont R. Grossmann M. Nakamura Y. Pohl T. Nesic D. Gerondakis S. Immunol. Rev. 2000; 176: 134-140Google Scholar). To carry out its diverse physiologic roles, NF-κB factors not only help to induce expression of various factors and effectors, but depending on the cellular context, they also transcriptionally induce proteins that function to protect cells from apoptosis and that help to stimulate proliferation (1Ghosh S. Karin M. Cell. 2002; 109: S81-S96Google Scholar, 2Li Q. Verma I.M. Nat. Rev. Immunol. 2002; 2: 725-734Google Scholar, 5Brown K. Claudio E. Siebenlist U. Targeted Therapies in Rheumatology. Martin Dunitz Ltd., London, UK2002: 381-401Google Scholar, 10Karin M. Lin A. Nat. Immunol. 2002; 3: 221-227Google Scholar). NF-κB is a collective term for a family of dimeric complexes comprised of combinations of five polypeptides, RelA, c-Rel, RelB, p50/NF-κB1, and p52/NF-κB2. p50 and p52 are the N-terminal parts of the longer p105/NF-κB1 and p100/NF-κB2 proteins, respectively, and they are generated by proteolytic processing (1Ghosh S. Karin M. Cell. 2002; 109: S81-S96Google Scholar, 2Li Q. Verma I.M. Nat. Rev. Immunol. 2002; 2: 725-734Google Scholar,4Karin M. Ben-Neriah Y. Annu. Rev. Immunol. 2000; 18: 621-663Google Scholar, 5Brown K. Claudio E. Siebenlist U. Targeted Therapies in Rheumatology. Martin Dunitz Ltd., London, UK2002: 381-401Google Scholar). High levels of p50 are produced constitutively by a cotranslational mechanism. In contrast, usually only small amounts of p52 exist in cells, but higher amounts may be induced by select signals. To activate NF-κB, appropriate environmental signals must bring about the release of NF-κB dimers from their bound cytoplasmic inhibitors, in particular from the prototypical inhibitor IκBα and its close relatives, IκBॆ and IκBκ (1Ghosh S. Karin M. Cell. 2002; 109: S81-S96Google Scholar, 2Li Q. Verma I.M. Nat. Rev. Immunol. 2002; 2: 725-734Google Scholar, 4Karin M. Ben-Neriah Y. Annu. Rev. Immunol. 2000; 18: 621-663Google Scholar, 5Brown K. Claudio E. Siebenlist U. Targeted Therapies in Rheumatology. Martin Dunitz Ltd., London, UK2002: 381-401Google Scholar). NF-κB factors are in addition subject to various direct and indirect mechanisms that modulate their ability to stimulate transcription, dependent also on promoter context (1Ghosh S. Karin M. Cell. 2002; 109: S81-S96Google Scholar, 2Li Q. Verma I.M. Nat. Rev. Immunol. 2002; 2: 725-734Google Scholar, 5Brown K. Claudio E. Siebenlist U. Targeted Therapies in Rheumatology. Martin Dunitz Ltd., London, UK2002: 381-401Google Scholar), but the release from the inhibitors is a first and necessary step in the activation process. Most of the NF-κB activation signals, and in particular inflammatory cytokines, such as TNFα and IL-1, induce the phosphorylation of the IκBs followed by the rapid ubiquitin- and proteasome-mediated degradation of the inhibitors, thus freeing NF-κB dimers to migrate to the nucleus to initiate gene transcription (1Ghosh S. Karin M. Cell. 2002; 109: S81-S96Google Scholar, 2Li Q. Verma I.M. Nat. Rev. Immunol. 2002; 2: 725-734Google Scholar, 4Karin M. Ben-Neriah Y. Annu. Rev. Immunol. 2000; 18: 621-663Google Scholar, 5Brown K. Claudio E. Siebenlist U. Targeted Therapies in Rheumatology. Martin Dunitz Ltd., London, UK2002: 381-401Google Scholar). IκBs are phosphorylated on two conserved serines by the IκB kinase (IKK) complex. IKKs consist of the catalytic subunits, IKKα and IKKॆκ, and the regulatory subunit IKKγ (also known as Nemo). Most signals have been shown to activate NF-κB by the classical, IKK-dependent pathway and, in particular, to be dependent on the IKKॆ catalytic and IKKγ/Nemo regulatory subunit to bring about the degradation of small IκB inhibitors (1Ghosh S. Karin M. Cell. 2002; 109: S81-S96Google Scholar, 2Li Q. Verma I.M. Nat. Rev. Immunol. 2002; 2: 725-734Google Scholar, 5Brown K. Claudio E. Siebenlist U. Targeted Therapies in Rheumatology. Martin Dunitz Ltd., London, UK2002: 381-401Google Scholar, 11Smahi A. Courtois G. Rabia S.H. Doffinger R. Bodemer C. Munnich A. Casanova J.L. Israel A. Hum. Mol. Genet. 2002; 11: 2371-2375Google Scholar). In addition to the small IκBs, the long forms of the NF-κB1 and NF-κB2 proteins, p105 and p100, can also act as cytoplasmic inhibitors of bound Rel proteins due to the of inhibitory in their (1Ghosh S. Karin M. Cell. 2002; 109: S81-S96Google Scholar, 2Li Q. Verma I.M. Nat. Rev. Immunol. 2002; 2: 725-734Google Scholar, 4Karin M. Ben-Neriah Y. Annu. Rev. Immunol. 2000; 18: 621-663Google Scholar, 5Brown K. Claudio E. Siebenlist U. Targeted Therapies in Rheumatology. Martin Dunitz Ltd., London, UK2002: 381-401Google Scholar). p105 may be in response to signals in a to that of small IκBs, including phosphorylation of two serines in a small phosphorylation D. C. Mol. Cell. Scholar). a second or alternative path been to NF-κB activity via induced processing of p100 inhibitor G. E.W. Mol. Cell. Scholar, U. Y. G. G. G. Y. Y. A. Karin M. Scholar). physiologic signals for this pathway were not processing was mediated by the NF-κB-inducing kinase and In the we that physiologic via the lymphotoxin ॆ receptor in stromal cells induced the degradation of IκBα via the classical and induced processing of p100 via an alternative pathway. p100 processing was shown to be dependent on and IKKα but independent of IKKॆ and Therefore, the p100 processing pathway was independent of the IKK complex, not of the IKKॆ kinase subunit. also that transient activation of the classical pathway the transient activation of the delayed and protein synthesis-dependent p100 processing to the delayed and sustained of and complexes. also an and for p100 processing complexes. and mouse fibroblasts were by Q. and M. and were by M. and K. and were by E. To fibroblasts from and were and were and parts were and to for The cells were and in were in with and were into To stimulation. cells were in and to were with were for and were by R. and C. R. S. Lin Y. M. Mol. Cell. 1998; 18: Scholar). p100 was from a expression Brown K. J. S. Bravo R. K. Siebenlist U. Mol. Cell. and into nuclear cells were to stimulation. was nuclear and cytoplasmic were as 11: Scholar). fibroblasts were in and in of with and a on was to a final of and the was for The were and the as cytoplasmic The were in of with and and for an on of this were in from the was as a were in and were with of were The was out for in a of nuclear were with for on to the of were to The of the was in including by the that of factors was dependent on and by the that RelA not in cells, RelB cytoplasmic and nuclear were generated from These were to and with or as a negative that been to a the were in and the were to The LTॆR were by J. and the to proteins were the were RelA, and p100 (also were against the the N-terminal and the N-terminal TNFα was from and factor were from was The inhibitor was by J. To protein cells were with for to stimulation. mice are in their they and they are in primarily due to the stromal (6Franzoso G. Carlson L. Poljak L. Shores E.W. Epstein S. Leonardi A. Grinberg A. Tran T. Scharton-Kersten T. Anver M. Love P. Brown K. Siebenlist U. J. Exp. Med. 1998; 187: 147-159Google Scholar, 7Caamaño J.H. Rizzo C.A. Durham S.K. Barton D.S. Raventos-Suarez C. Snapper C.M. Bravo R. J. Exp. Med. 1998; 187: 185-196Google Scholar, L. Carlson L. K. Siebenlist U. J. Immunol. Scholar, S. M. M. G. S. K. 2002; Scholar). These in secondary lymphoid which also of in turn to immune responses in these have been mice M. K. T. R. A. M. S. M. J. Immunol. Scholar, T. T. K. D. A. K. S. M. J. Immunol. 2000; and in mice lymphotoxin ॆ receptor or its and M. K. T. R. A. M. S. M. J. Immunol. Scholar, A. K. A. K. 1998; Scholar, Annu. Rev. Immunol. Scholar, S. J. T. U. K. J. Exp. Med. 2002; Scholar, K. J. G. M. L. J. Immunol. 2002; Scholar), of the TNF mice are in R. K. K. K. M. K. T. T. Nat. Genet. Scholar). of the form of processing of the p100 protein of NF-κB2 to but its form not G. E.W. Mol. Cell. Scholar). mice of the p52 protein of NF-κB2 mice levels of p100 T. T. K. D. A. K. S. M. J. Immunol. 2000; Scholar). processing in B cells on IKKα U. Y. G. G. G. Y. Y. A. Karin M. Scholar), and thus B cells p52 protein U. Y. G. G. G. Y. Y. A. Karin M. Scholar). which their be shown that these mice are deficient in A. T. K. D. K. S. M. J. Exp. Med. Scholar). Based on these we that critical of the receptor on stromal cells on via and NF-κB2 and thus may processing of To for this we to an against the with for the expression of the NF-κB2 proteins p100 and as well as the NF-κB1 proteins p105 and the inhibitor of NF-κB RelA, RelB, and time during an with also for expression of these proteins and of with The a in p100 and a in and by of via the such in p100 and p52 levels were with TNFα stimulation. TNFα the degradation of IκBα and the degradation of IκBॆ by of IκBα levels were by of stimulation. LTॆR induced only a degradation of IκBα, which a delayed with that induced by The amounts of IκBα to of via the LTॆR and were levels by Most this was due to in response to NF-κB, in the of degradation of this inhibitor to in the amounts of the proteins in with the of RelB, amounts were and amounts were of with In to the amounts of the p50 form of NF-κB1 not LTॆR stimulation. These that receptor in processing of p100 to p52 and a degradation of the IκBα inhibitory engagement of two to activate and the are of the In addition to the also be shown to induce processing of p100 to p52 in factor not the delayed of processing, we the mechanisms protein processing of p100 was to the protein inhibitor and this was cells were with the to the receptor not processing of p100 the or of a which the of processing stimulation. we the LTॆR-induced processing by of mice or in various generated which carry a in to the LTॆR to induce processing of p100 to p52 in Therefore, was for processing of p100, with the ability of to induce processing in cells and the LTॆR-induced NF-κB activity in and A. T. K. D. K. S. M. J. Exp. Med. Scholar, L. L. Scholar). was shown to on IKKα to induce processing in B cells, and with LTॆR also to induce processing in from mice the for these cells but the of p100 to p52 not from mice IKKॆ or IKKγ (also known as were for LTॆR-induced processing of p100, as were deficient in NF-κB1 and mice are in Q. Q. D. Verma I.M. Genes Dev. Scholar, Q. D. Verma I.M. Scholar, M. Courtois G. K. Israel A. K. M. Mol. Cell. 2000; The regulatory subunit IKKγ and the two catalytic IKKα and IKKॆ the classical IKK complex. Therefore, LTॆR-induced processing the IKKα was independent of the classical, IKKγ IKK complex that degradation of the small IκB inhibitors in response to signals. with the amounts of p100 to be in and but were in processing in response to LTॆR in a of p100 in the cells longer of the is shown in and LTॆR-induced activation of NF-κB via the classical IKK to IκB degradation path may be for p100 deficient in IKKγ may have levels of p100 the classical activation pathway was in these cells, activity may have in the due to the of also an to further for the of the classical activation in p100 levels in To for the activity of this we a inhibition of degradation of IκBα of amounts of also the amounts of p100 of via the due to of p100, processing to p52 was Therefore, expression of p100 on and induced activation of the classical, pathway for NF-κB, but processing of p100 not and only on the IKKα subunit. that the to p100 in and in response to was not to of IKKα or of these and with p100 with or the with of IKKα and of in induced processing of p100 to IKKॆ not IKKα and were to induce processing in IKKγ and This that the classical pathway was for processing and that the of to processing be by or This also IKKα of which was by the that of IKKα in resulted in processing, of not Therefore, we that processing of p100 to p52 as induced by LTॆR on and proceeded via and then IKKα but was independent of classical IKKγ and NF-κB classical IKK activity help p100 we NF-κB activation in to the of κB activity primarily of p50 and and as in with to the various NF-κB amounts of were and this was in in which activity to the for the transcription factor was not LTॆR for resulted in amounts of activity primarily of and, to a RelA of the activity of dimers activity and activity be as well RelB and p52 In p50 activity to have activity was in these this was to in lymphoid cells not The with complexes with an of complexes. must that the are not and not with the to which a NF-κB been from cytoplasmic dimers κB with and dimers in particular have not been for the various in of and may in addition be in the is that these the amounts of complexes in This was by which of p52 and RelB into by of LTॆR and the of RelA in was of RelB as two with the to In RelB that RelB was with p52 in the nucleus p52 is by an the an activation of which activation of RelB dimers to that the and transient degradation of IκBs LTॆR be for the and transient in the the processing of p100 be for the in activity and also in To this we the activation of NF-κB with in the of and IKKγ for resulted in activation of and, to an to was in and RelB to with activation of RelA-containing dimers was and not of not also These that the classical activation pathway via was not for activation of or but was for activation of To that of IKKγ of p52 and RelB into we also shown in RelB and p52 of these cells and to during the course of to was with cells RelA to be into as for these cells, which also as a for the the of the alternative pathway in the activation of κB activity in response to LTॆR stimulation. and to activate or of and in was of in not and was and RelA these that activation of IKKॆ and IKKγ but not IKKα or the activation of and IKKα and but not IKKॆ or also that activation of to be in the of IKKα or activation of and to be in the of IKKॆ and IKKγ not The that the alternative pathway of activation via and IKKα and, by processing of p100 were for the activation of not only but also for the activation of dimers from processing of was dimers be To mechanisms we LTॆR-induced activation in these cells the p100 inhibitor to In these was in the of and this activity was further with via the LTॆR RelB The activity that was not the act of processing of p100 but the of the p100 inhibitor that to RelB is to with p100 J. 2002; Scholar), and in the of p100, RelB is to with that LTॆR of may have to a further levels of activity as a of of RelB and NF-κB1 induced via the classical NF-κB activation In of the that RelB complexes were in the of p100, we that TNFα activity to levels in of which p100, not in such activation of RelB complexes RelB in and are The RelB activity long term of with TNFα was due to the amounts of RelB protein induced via the classical NF-κB activation of which may have by p100 These the that the or of p100 was the to dimers were or these that and IKKα were for activation of they to processing of p100 and thus of the and inhibitory for The of also that LTॆR-induced activation of RelB activity in was dependent on protein with the of p100 processing on protein RelB activation in the of the protein inhibitor were with the LTॆR In the of p100 RelB proteins are to be with p105 and is that or induced of the inhibitory p105 have to a further release and thus activation of RelB as p105 not be replaced in this LTॆR-induced activation of RelA complexes was in the of in and in due to the of inhibitors in the of The that of mouse embryo fibroblasts resulted in the engagement of two pathways, to activation of distinct NF-κB complexes. that the initial or classical activation was mediated by and IKKγ-dependent degradation of IκBα and of primarily independent of and further that the second or alternative activation was mediated by and processing of the inhibitory p100 protein of independent of IKKॆ and This pathway to activation of primarily and dimers. These were from of and in or or LTॆR-induced activation was and with an only and transient of IκBα induced by the classical pathway. time of amounts of IκBα to levels to RelA complexes. In contrast, activity of RelB complexes and to the NF-κB of the LTॆR on two to activate NF-κB complexes. The change in the of NF-κB complexes is to in a change in the with time during the course of via The the LTॆR as a physiologic of the second pathway of The the of this pathway long term κB activity in response to LTॆR was dependent on p100 of gene to via the LTॆR but not the L. L. Scholar). the LTॆR-induced κB activity was not in the is by the classical activation path as shown was that LTॆR-induced in have been in the of that is also to in response to LTॆR that the of processing of p100 in and thus the of sustained activation of RelB complexes in particular also the of gene The alternative activation path and p100 processing not only which be generated from but also of this complex was a of of p100 via processing, the and inhibitory for RelB, thus RelB to with NF-κB1 proteins, including the constitutively generated The of p100 in was to to activation of was not in and this was further in the via the and in particular, with This was the of induced of RelB by the classical activation which was with TNFα and in have resulted from degradation of p105 D. C. Mol. Cell. Scholar), the only known inhibitor of RelB in the of p100 IκBs are not to be physiologic inhibitors of RelB, they have to inhibition in the of Based on these we conclude that the processing of p100 to activation of complexes the of and signals in the process. The of p100 processing to protein inhibitors is that induced or of or proteins was a is with the long in p100 processing, in or of LTॆR a in p52 was of p100 be processing is to or induced of be cells may only small amounts of that the amounts of be to induce processing, on the that is in the of signals induced or of proteins be in which the alternative pathway was to a sustained activation of RelA in which the classical pathway was to have RelB complexes these to be they the that the two may in cells. IKKα be and thus may not have been to the second pathway. The a of the and complexes with time of stimulation. of of the components of the classical activation pathway of to the amounts of p100/NF-κB2 in In addition to RelB, c-Rel, and NF-κB1 are also known of via the classical may be needed to NF-κB including NF-κB2 and RelB, to of the alternative activation pathway. signals may the activation to and to the course of depending on we that of the B cell-activating factor by on B cells processing of the NF-κB2 protein p100 to p52 E. Brown K. S. Siebenlist U. Nat. Immunol. 2002; 3: Scholar). the and of processing in B cells not be as as was with p100 processing in B cells be shown to on and protein to be independent of IKKγ and to to activation of RelB complexes. In in a of E. M. E. Y. C. Karin M. 2002; to with LTॆR is to activate the classical pathway as well as p100 processing E. M. E. Y. C. Karin M. 2002; and not to stimulate the classical activation pathway in B cells, p100 of this second or alternative activation path by in B cells was shown to to of these cells. the roles of activation are not they of stromal cells in lymphoid Based on in mice deficient in LTॆR and is that processing of p100 in stromal cells to the stromal cells and lymphoid cells and to secondary lymphoid of p100 processing in stromal cells the B E. M. E. Y. C. Karin M. 2002; Scholar), which is in with that induced expression of this is in stromal cells L. Carlson L. K. Siebenlist U. J. Immunol. and that mice deficient in LTॆR or NF-κB2 are in of B (6Franzoso G. Carlson L. Poljak L. Shores E.W. Epstein S. Leonardi A. Grinberg A. Tran T. Scharton-Kersten T. Anver M. Love P. Brown K. Siebenlist U. J. Exp. Med. 1998; 187: 147-159Google Scholar, 7Caamaño J.H. Rizzo C.A. Durham S.K. Barton D.S. Raventos-Suarez C. Snapper C.M. Bravo R. J. Exp. Med. 1998; 187: 185-196Google Scholar, L. Carlson L. K. Siebenlist U. J. Immunol. Scholar, S. M. M. G. S. K. 2002; Scholar, M. K. T. R. A. M. S. M. J. Immunol. Scholar, T. T. K. D. A. K. S. M. J. Immunol. 2000; Scholar, A. K. A. K. 1998; Scholar, Annu. Rev. Immunol. Scholar, S. J. T. U. K. J. Exp. Med. 2002; Scholar, K. J. G. M. L. J. Immunol. 2002; Scholar). the of by Q. M. M. K. R. C. E. and J. are to of the for E. Claudio and K. and to A. S. for
Müller et al. (Fri,) studied this question.