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Human apolipoprotein E is the major apolipoprotein expressed in the brain and exists as three isoforms, designated E2, E3, and E4. Although evidence suggests that apolipoprotein E plays an important role in modifying systemic and brain inflammatory responses, there is little data investigating apoE isoform-specific effects in vivo. In this study, we compared the inflammatory responses of targeted-replacement mice expressing the human APOE3 and APOE4 genes after intravenous administration of lipopolysaccharide. Animals expressing the E4 allele had significantly greater systemic and brain elevations of the pro-inflammatory cytokines TNFα and IL-6 as compared with their APOE3 counterparts, suggesting an isoform-specific effect of the immunomodulatory properties of apoE. Furthermore, intravenous administration of a small apoE-mimetic peptide derived from the receptor-binding region of the apoE holoprotein (apoE-(133–149)) similarly suppressed both systemic and brain inflammatory responses in mice after lipopolysaccharide administration. These results suggest that apoE plays an isoform-specific role in mediating the systemic and brain inflammatory responses. Moreover, because exogenous administration of this apoE mimetic peptide is effective at suppressing both systemic and brain inflammation, it may represent a novel therapeutic strategy for diseases characterized by systemic or central nervous system inflammation, such as septic shock, multiple sclerosis, and traumatic brain injury. Human apolipoprotein E is the major apolipoprotein expressed in the brain and exists as three isoforms, designated E2, E3, and E4. Although evidence suggests that apolipoprotein E plays an important role in modifying systemic and brain inflammatory responses, there is little data investigating apoE isoform-specific effects in vivo. In this study, we compared the inflammatory responses of targeted-replacement mice expressing the human APOE3 and APOE4 genes after intravenous administration of lipopolysaccharide. Animals expressing the E4 allele had significantly greater systemic and brain elevations of the pro-inflammatory cytokines TNFα and IL-6 as compared with their APOE3 counterparts, suggesting an isoform-specific effect of the immunomodulatory properties of apoE. Furthermore, intravenous administration of a small apoE-mimetic peptide derived from the receptor-binding region of the apoE holoprotein (apoE-(133–149)) similarly suppressed both systemic and brain inflammatory responses in mice after lipopolysaccharide administration. These results suggest that apoE plays an isoform-specific role in mediating the systemic and brain inflammatory responses. Moreover, because exogenous administration of this apoE mimetic peptide is effective at suppressing both systemic and brain inflammation, it may represent a novel therapeutic strategy for diseases characterized by systemic or central nervous system inflammation, such as septic shock, multiple sclerosis, and traumatic brain injury. Apolipoprotein E (apoE protein; APOE gene) 1The abbreviations used are: apoEapolipoprotein E proteinAPOEapolipoprotein E geneCNScentral nervous systemTRtargeted replacementTNFαtumor necrosis factor-αIL-6interleukin-6PBSphosphate-buffered salineELISAenzyme-linked immunosorbent assay. is a 34-kDa protein originally studied for its role in cholesterol metabolism. There are three common human isoforms of apoE, designated E2, E3, and E4, encoded for by distinct alleles on human chromosome 19. The isoforms differ by single amino acid interchanges at residues 112 and 158: E3(Cys112-Arg158), E4 (Arg112-Arg158), and E2(Cys112-Cys158) (1.Weisgraber K.H. Adv. Protein Chem. 1994; 45: 249-302Crossref PubMed Google Scholar). The allele frequency of APOE4 is ∼14% and has been associated with the early onset of Alzheimer's disease and poor prognosis in neuroinflammatory disorders such as multiple sclerosis (2.Strittmatter W.J. Roses A.D. Annu. Rev. Neurosci. 1996; 19: 53-77Crossref PubMed Scopus (392) Google Scholar, 3.Schmidt S. Barcellos L.F. DeSombre K. Rimmler J.B. Lincoln R.R. Bucher P. Saunders A.M. Lai E. Martin E.R. Vance J.M. Oksenberg J.R. Hauser S.L. Pericak-Vance M.A. Haines J.L. Am. J. Hum. Genet. 2002; 70: 708-717Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). The presence of the APOE4 allele is also associated with poor prognosis following traumatic brain injury (4.Sorbi S. Nacmias B. Piacentini S. Repice A. Latorraca S. Forleo P. Amaducci L. Nat. Med. 1995; 1: 852Crossref PubMed Scopus (149) Google Scholar, 5.Friedman G. Froom P. Sazbon L. Grinblatt I. Shochina M. Tsenter J. Babaey S. Yehuda B. Groswasser Z. Neurology. 1999; 52: 244-248Crossref PubMed Google Scholar) and with an increase in the systemic inflammatory response in patients following cardiopulmonary bypass (6.Grocott H.P. Newman M.F. El-Moalem H. Bainbridge D. Butler A. Laskowitz D.T. J. Thorac. Cardiovasc. Surg. 2001; 122: 622-623Abstract Full Text PDF PubMed Scopus (64) Google Scholar). apolipoprotein E protein apolipoprotein E gene central nervous system targeted replacement tumor necrosis factor-α interleukin-6 phosphate-buffered saline enzyme-linked immunosorbent assay. ApoE is the major apolipoprotein produced in the central nervous system (CNS). Independently of its role in cholesterol metabolism, apoE modulates innate and acquired immune responses in vitro and in vivo (7.Laskowitz D.T. Lee D.M. Schmechel D. Staats H.F. J. Lipid Res. 2000; 41: 613-620Abstract Full Text Full Text PDF PubMed Google Scholar). ApoE-deficient animals have impaired immunity after bacterial challenge with Listeria monocytogenes (8.Roselaar S.E. Daugherty A. J. Lipid Res. 1998; 39: 740-743Abstract Full Text Full Text PDF Google Scholar). They also have increased susceptibility to endotoxemia after intravenous lipopolysaccharide (LPS) administration and inoculation with Klebsiella pneumonia (9.de Bont N. Netea M.G. Demacker P.N. Verschueren I. Kullberg B.J. van Dijk K.W. van der Meer J.W. Stalenhoef A.F. J. Lipid Res. 1999; 40: 680-685Abstract Full Text Full Text PDF PubMed Google Scholar). A recent report confirmed the observation that apoE-deficient animals had an increased systemic inflammatory response and higher mortality following LPS injection, and that the administration of exogenous apoE improved mortality by down-regulating the inflammatory cascade (10.Van Oosten M. Rensen P.C. Van Amersfoort E.S. Van Eck M. Van Dam A.M. Breve J.J. Vogel T. Panet A. Van Berkel T.J. Kuiper J. J. Biol. Chem. 2001; 276: 8820-8824Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). The immunomodulatory properties of apoE may be of particular relevance in the CNS, where the absence of endogenous apoE has been shown to exacerbate cerebral edema and functional deficit after traumatic brain injury (11.Lynch J.R. Pineda J.A. Morgan D. Zhang L. Warner D.S. Benveniste H. Laskowitz D.T. Ann. Neurol. 2002; 51: 113-117Crossref PubMed Scopus (98) Google Scholar, 12.Chen Y. Lomnitski L. Michaelson D.M. Shohami E. Neuroscience. 1997; 80: 1255-1262Crossref PubMed Scopus (192) Google Scholar). The mechanisms by which apoE exerts these immunomodulatory effects remain incompletely understood. Recent evidence suggests that, in addition to its role in lipid transport, apoE is capable of binding high affinity receptors and initiating a calcium-dependent signaling response in immunocompetent cells (13.Misra U.K. Adlakha C.L. Gawdi G. McMillian M.K. Pizzo S.V. Laskowitz D.T. J. Leukocyte Biol. 2001; 70: 677-683PubMed Google Scholar). Specifically, the lipoprotein receptor-related protein/α-2 macroglobulin receptor is capable of initiating signal transduction and modulating immune responses (14.Misra U.K. Chu C.T. Gawdi G. Pizzo S.V. J. Biol. Chem. 1994; 269: 18303-18306Abstract Full Text PDF PubMed Google Scholar). Although not extensively studied, isoform-specific differences in immune regulation may also play a particularly important role in mediating the CNS response to injury. For example, apoE4 is less effective than apoE3 or apoE2 at suppressing the activation of microglia in cell culture paradigms of brain inflammation (15.Laskowitz D.T. Thekdi A.D. Thekdi S.D. Han S.K. Myers J.K. Pizzo S.V. Bennett E.R. Exp. Neurology. 2001; 167: 74-85Crossref PubMed Scopus (183) Google Scholar, 16.Barger S.W. Harmon A.D. Nature. 1997; 388: 878-881Crossref PubMed Scopus (554) Google Scholar). In humans, isoform-specific differences are suggested by autopsy reports demonstrating increased numbers of scattered microglia and microglial activation in Alzheimer's disease patients carrying the APOE4 allele (17.Egensperger R. Kosel S. von Eitzen U. Graeber M.B. Brain Path. 1998; 8: 439-447Crossref PubMed Scopus (130) Google Scholar). Although there is increasing evidence that apoE plays a biologically relevant role in modulating immune responses, the mechanism(s) by which this occurs remain unclear. It has been postulated that the protective effect of lipoproteins in endotoxemia is mediated by the binding and redirection of LPS from Kupffer cells to parenchymal liver cells, where endotoxin is inactivated and secreted into bile (18.Harris H.W. Grunfeld C. Feingold K.R. Read T.E. Kane J.P. Jones A.L. Eichbaum E.B. Bland G.F. Rapp J.H. J. Clin. Invest. 1993; 91: 1028-1034Crossref PubMed Scopus (183) Google Scholar, 19.Read T.E. Harris H.W. Grunfeld C. Feingold K.R. Calhoun M.C. Kane J.P. Rapp J.H. Infect. Immun. 1993; 61: 3496-3502Crossref PubMed Google Scholar, 20.Harris H.W. Rockey D.C. Chau P. Hepatology. 1998; 27: 1341-1348Crossref PubMed Scopus (32) Google Scholar). An alternative hypothesis is that, independent of lipid binding, apoE down-regulates activation of immune cells by binding to high affinity receptors and initiating a signaling cascade (13.Misra U.K. Adlakha C.L. Gawdi G. McMillian M.K. Pizzo S.V. Laskowitz D.T. J. Leukocyte Biol. 2001; 70: 677-683PubMed Google Scholar). To investigate the mechanism by which apoE exerts its immunomodulatory effects, we looked at the ability of an apoE mimetic peptide to suppress the systemic and CNS inflammatory responses in vivo. This peptide is derived from the receptor-binding region of apoE and is composed of amino acid residues 133–149 (15.Laskowitz D.T. Thekdi A.D. Thekdi S.D. Han S.K. Myers J.K. Pizzo S.V. Bennett E.R. Exp. Neurology. 2001; 167: 74-85Crossref PubMed Scopus (183) Google Scholar). We have shown previously that apoE-(133–149) retains the bioactivity of the intact apoE holoprotein in its ability to suppress brain macrophage activation and initiate a macrophage signaling cascade in cultured cells (13.Misra U.K. Adlakha C.L. Gawdi G. McMillian M.K. Pizzo S.V. Laskowitz D.T. J. Leukocyte Biol. 2001; 70: 677-683PubMed Google Scholar). We utilized an LPS model of induced inflammation in targeted replacement (TR) mice expressing the human E3 and E4 genes to establish whether systemic and brain inflammatory responses were influenced by apoE in an isoform-specific manner. These animals have the human apoE3 or E4 genes at the correct murine genetic locus and do not express murine apoE (20.Harris H.W. Rockey D.C. Chau P. Hepatology. 1998; 27: 1341-1348Crossref PubMed Scopus (32) Google Scholar). The inflammatory response was monitored by measuring the temporal secretion and expression profiles of two pro-inflammatory cytokines, TNFα and IL-6, in the central nervous system and peripheral circulation. In this series of experiments, wild-type C57BL6/J mice were treated with a single intravenous injection of the apoE-mimetic peptide after LPS administration with TNFα and IL-6 levels were determined. TNFα and IL-6 were selected to monitor the systemic and CNS immune response because they are both released from immune cells in the periphery during inflammation, are expressed in many CNS cells including microglia, neurons, and astrocytes. In addition, TNFα and IL-6 play an important role in neuronal cell death and survival during injury and pathological conditions (22.Sternberg E.M.J. Clin. Invest. 1997; 100: 2641-2647Crossref Scopus (300) Google Scholar). Mice—APOE TR model was created by gene targeting of E14TG2a embryonic stem cells from 129P2/OlaHsd mice with the human apoE3 or human apoE4 construct in combination with flanking sequences from the 129 mouse. The targeted embryonic stem cells were injected into C57BL6/J blastocysts, and the resulting chimeras were bred to C57BL6/J mice. Then, they underwent eight generations of back-crossing to C57BL6/J. The colony was maintained by homozygous matings. Genotype was confirmed prior to each experiment. In addition, age-matched male C57BL6/J mice were used in the peptide experiments. Peptide Synthesis—Peptides were synthesized from the Peptide Synthesis Laboratory at the University of North Carolina (Chapel Hill, NC) to a purity of 95% and were reconstituted in sterile isotonic PBS. For each peptide, the amino terminus was acetylated, and the carboxyl terminus was blocked with an amide moiety. The 17-amino acid peptide was derived from apoE residues 133–149 (the receptor-binding region): Ac-LRVRLASHLRKLRKRLL-amide. Control injections utilized the identical vehicle of PBS. To rule out the possibility of nonspecific peptide effects, controls included a scrambled peptide of identical size and amino acid composition. LPS and Peptide Injections—This study was approved by the Duke University Animal and Care Use Committee. 14–16-week-old male C57-BL6 and matched APOE3-TR and APOE4-TR mice were injected with LPS (40 μg/kg in 100 μl of PBS) via tail vein and then immediately with vehicle (100 μl of isotonic sterile PBS) or peptide (apoE-(133–149)) (6.0 mg/kg, prepared in isotonic saline). Serum samples were obtained in LPS + vehicle and LPS + peptide groups (n = 20 animals/group) at the following timepoints: baseline and at 1, 3, and 24 h after injection. Mice were anesthetized with isoflurane anesthesia and ventilated with 21% oxygen. Blood was collected by transcardiac puncture and allowed to clot for 30 min. Serum samples were centrifuged at 16,000 × g for 5 min, quick-frozen by immersing in liquid nitrogen, and stored at –80 °C. To remove systemic from the cerebral mice were with 20 of via transcardiac were in liquid nitrogen, and stored at –80 °C. of levels in murine and brain were by for murine IL-6 and TNFα following the were and quick-frozen by immersing in liquid The were into a in a liquid were by in and and by a and a were maintained on the the was by at 5 for at × g to remove The was and into and stored at of brain was by to the gene expression be by gene following the This and to relevant the of in It a in which two were used in a single was used to the of and a was used to an endogenous In addition to for the gene and a of are included to of The were on and a and For each the signal obtained for the was by the signal obtained for the a for the in each differences in and protein were by data was and a for of apoE on whether apoE systemic inflammatory responses in an isoform-specific APOE3-TR and APOE4-TR animals were injected with LPS via the tail Serum levels for TNFα and IL-6 were by at to h after injection. of LPS in a of TNFα protein levels in both groups of h after injection, TNFα was significantly higher in APOE4-TR animals compared with APOE3-TR animals there was TNFα protein in the groups of animals a of IL-6 after injection of LPS IL-6 at h in both groups was significantly higher in the APOE4-TR animals to the APOE3-TR mice. The observation that animals the human APOE3 gene had levels for two of inflammation suggests that apoE3 is a protein in a murine model of inflammation compared with We whether endogenous apoE similarly the brain inflammatory response in an isoform-specific manner. brain TNFα h after the peripheral LPS injection and was significantly higher in APOE4-TR animals compared with APOE3-TR TNFα was baseline at there were differences at this Brain IL-6 was also at h after injection, and at APOE4 animals had significantly higher levels at and differences were at h 24 both TNFα and IL-6 were the of of this assay. these results suggest an isoform-specific effect of apoE on modulating systemic and CNS inflammatory responses, with endogenous E4 less effective than we whether a peptide derived from the receptor-binding region of apoE, inflammatory responses in the as the intact apoE. with the apoE-mimetic peptide significantly TNFα levels at and IL-6 levels at and h in wild-type C57BL6/J mice There was TNFα or IL-6 protein at 24 h after injection in The effects of the peptide were in the where the single intravenous injection of peptide significantly TNFα and IL-6 expression h after LPS injection A and The protein levels in the brain the and both TNFα and IL-6 protein were after peptide injection apoE-mimetic peptide significantly levels in the brain after LPS inflammatory protein levels in brain were h after LPS injection (40 at and there was a in brain TNFα protein after LPS injection in animals treated with apoE-(133–149) peptide compared with controls at there was a in brain IL-6 protein after LPS injection in animals treated with apoE-(133–149) peptide compared with controls of the data eight animals and is as To rule out the possibility that the of inflammatory cytokines in the CNS and systemic were a nonspecific peptide we these the scrambled apoE-(133–149) with We differences in IL-6 the scrambled peptide and vehicle in IL-6 at h or h we in TNFα the scrambled and vehicle groups at h and at There was in brain IL-6 at h after injection in vehicle and scrambled peptide treated Brain TNFα levels were also not significantly in scrambled peptide and vehicle These results that the scrambled apoE peptide immunomodulatory bioactivity and suggest for the apoE-(133–149) peptide derived from the apoE receptor-binding ApoE has been to in vitro immunomodulatory effects by a of G. N. J.A. J. Biol. Chem. Full Text PDF PubMed Google Scholar, M.G. J. Google Scholar, J. Google Scholar). The study that apoE is a biologically relevant immunomodulatory protein that exerts isoform-specific In mice expressing the human APOE4 gene had higher levels of the pro-inflammatory cytokines TNFα and IL-6 in after LPS injection. Although the of APOE on immune has not been these results are with a report demonstrating that patients with the APOE4 allele had a systemic inflammatory response after cardiopulmonary bypass (6.Grocott H.P. Newman M.F. El-Moalem H. Bainbridge D. Butler A. Laskowitz D.T. J. Thorac. Cardiovasc. Surg. 2001; 122: 622-623Abstract Full Text PDF PubMed Scopus (64) Google Scholar). In addition, apoE to after injury in an isoform-specific D. M. Saunders A.M. Schmechel K. 2002; PubMed Scopus Google Scholar, E. Laskowitz D.T. Biol. Med. 2002; PubMed Scopus Google Scholar). We also that the APOE4-TR mice have a CNS inflammatory than APOE3-TR mice following peripheral injection of animals were to remove systemic from the cerebral the increase in inflammatory that we a brain inflammatory These results are with a of apoE in modulating activation and the CNS response to injury. In cell culture experiments, apoE down-regulates activation and the of inflammatory This effect is apoE4 is less effective than apoE3 (15.Laskowitz D.T. Thekdi A.D. Thekdi S.D. Han S.K. Myers J.K. Pizzo S.V. Bennett E.R. Exp. Neurology. 2001; 167: 74-85Crossref PubMed Scopus (183) Google Scholar, 16.Barger S.W. Harmon A.D. Nature. 1997; 388: 878-881Crossref PubMed Scopus (554) Google Scholar). The possibility that apoE4 is associated with a of is also with prior data demonstrating that apoE-deficient mice have a systemic and CNS inflammatory response to a of Bont N. Netea M.G. Demacker P.N. Kullberg B.J. van der Meer J.W. Stalenhoef A.F. J. Clin. Invest. 2000; PubMed Scopus Google Scholar, J.R. Morgan D. J. Laskowitz D.T. J. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). It is that the of the apoE4 at suppressing the brain inflammatory response may play an important role in diseases associated with and In the presence of the APOE4 allele has been associated with poor in multiple sclerosis, and an increased susceptibility of Alzheimer's disease (2.Strittmatter W.J. Roses A.D. Annu. Rev. Neurosci. 1996; 19: 53-77Crossref PubMed Scopus (392) Google Scholar, 3.Schmidt S. Barcellos L.F. DeSombre K. Rimmler J.B. Lincoln R.R. Bucher P. Saunders A.M. Lai E. Martin E.R. Vance J.M. Oksenberg J.R. Hauser S.L. Pericak-Vance M.A. Haines J.L. Am. J. Hum. Genet. 2002; 70: 708-717Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, S. Nacmias B. Piacentini S. Repice A. Latorraca S. Forleo P. Amaducci L. Nat. Med. 1995; 1: 852Crossref PubMed Scopus (149) Google Scholar, 5.Friedman G. Froom P. Sazbon L. Grinblatt I. Shochina M. Tsenter J. Babaey S. Yehuda B. Groswasser Z. Neurology. 1999; 52: 244-248Crossref PubMed Google Scholar). The mechanism by which apoE is to from is by binding and LPS from macrophage to with (18.Harris H.W. Grunfeld C. Feingold K.R. Read T.E. Kane J.P. Jones A.L. Eichbaum E.B. Bland G.F. Rapp J.H. J. Clin. Invest. 1993; 91: 1028-1034Crossref PubMed Scopus (183) Google Scholar, 19.Read T.E. Harris H.W. Grunfeld C. Feingold K.R. Calhoun M.C. Kane J.P. Rapp J.H. Infect. Immun. 1993; 61: 3496-3502Crossref PubMed Google Scholar, 20.Harris H.W. Rockey D.C. Chau P. Hepatology. 1998; 27: 1341-1348Crossref PubMed Scopus (32) Google Scholar). apoE down-regulates brain macrophage activation after to a of including and acid (15.Laskowitz D.T. Thekdi A.D. Thekdi S.D. Han S.K. Myers J.K. Pizzo S.V. Bennett E.R. Exp. Neurology. 2001; 167: 74-85Crossref PubMed Scopus (183) Google Scholar, 16.Barger S.W. Harmon A.D. Nature. 1997; 388: 878-881Crossref PubMed Scopus (554) Google Scholar). This suggests that apoE may also immunomodulatory effects that are independent of LPS the that apoE a signaling cascade in mechanism of from LPS is that apoE modulates inflammatory responses by receptor In apoE a of lipoprotein receptor-related which have immunomodulatory properties and are on macrophage and microglia (13.Misra U.K. Adlakha C.L. Gawdi G. McMillian M.K. Pizzo S.V. Laskowitz D.T. J. Leukocyte Biol. 2001; 70: 677-683PubMed Google Scholar, U.K. Chu C.T. Gawdi G. Pizzo S.V. J. Biol. Chem. 1994; 269: 18303-18306Abstract Full Text PDF PubMed Google Scholar). It has been that both apoE and the apoE mimetic peptide used in the study for the high affinity receptor on and initiate a signaling cascade associated with increased of and of This signaling cascade is by with protein and and it is mediated by a protein (13.Misra U.K. Adlakha C.L. Gawdi G. McMillian M.K. Pizzo S.V. Laskowitz D.T. J. Leukocyte Biol. 2001; 70: 677-683PubMed Google Scholar). To this we created a series of small derived from the receptor-binding region of apoE. of these such as a of the of the receptor-binding region in the with the intact apoE protein for macrophage receptor binding, and the ability of the intact protein to microglial activation (15.Laskowitz D.T. Thekdi A.D. Thekdi S.D. Han S.K. Myers J.K. Pizzo S.V. Bennett E.R. Exp. Neurology. 2001; 167: 74-85Crossref PubMed Scopus (183) Google Scholar). It is that the apoE peptide used in this study not residues 112 or which are associated with the common human In there is in the protein the apoE isoforms, the on 112 and E3 to (1.Weisgraber K.H. Adv. Protein Chem. 1994; 45: 249-302Crossref PubMed Google Scholar). Although this region is distinct from the receptor-binding it is that the induced by these amino acid the receptor-binding region and For example, the of a for an at significantly the ability of apoE2 to the lipoprotein this the receptor-binding region (1.Weisgraber K.H. Adv. Protein Chem. 1994; 45: 249-302Crossref PubMed Google Scholar, K.H. J. Biol. Chem. Full Text PDF PubMed Google Scholar). results suggest that endogenous apoE plays an important role in modifying both systemic and brain inflammatory responses. These effects to be with the apoE4 less effective than apoE3 at down-regulating inflammatory cytokines in the brain and peripheral circulation. Moreover, in this study we that an apoE-mimetic peptide suppressed the of the inflammatory cytokines TNFα and IL-6 after LPS injection. These results suggest that the apoE-mimetic peptide retains the bioactivity of the the intact apoE which of amino acid residues and has into the CNS small apoE-mimetic be to This possibility has for the of novel therapeutic targeting diseases characterized by systemic or CNS inflammation, such as septic shock, multiple sclerosis, and traumatic brain injury.
Lynch et al. (Wed,) studied this question.