text
stringlengths 1
27.3k
| synonym_substitution
stringlengths 1
14.8k
| butter_fingers
stringlengths 0
14.7k
| random_deletion
stringlengths 1
10.9k
| change_char_case
stringlengths 1
27.3k
| whitespace_perturbation
stringlengths 1
12.8k
| underscore_trick
stringlengths 1
12.9k
|
|---|---|---|---|---|---|---|
Introduction {#sec1-1}
============
Infliximab (IFX), a chimeric anti-TNFα antibody, is effective in inducing and maintaining remission in a considerable proportion of IBD patients refractory to any other treatments \[[@ref1],[@ref2]\]. However, 8-12% of adult and/or pediatric patients fail to respond to the induction regimen (known as primary non responders) and approximately 40% of patients who respond initially and achieve clinical remission inevitably lose response over time\[[@ref3],[@ref7]\]. Lack of response to IFX is a stable trait and suggests that the differences in response might be in part genetically determined. Considering the high cost and safety profile of this drug, genetic targeting of patients responding to this therapy is certainly of great interest \[[@ref8]\]. So far, limited candidate gene association studies with response to IFX have been reported \[[@ref9]-[@ref11]\]. Recently, a genome-wide association study (GWAS) in paediatric IBD patients has revealed that the 21q22.2/BRWDI loci were associated with primary non response \[[@ref12]\]. Furthermore, although TNFa gene is of great interest as a candidate gene for pharmacogenetic approaches few studies have been performed to date and some have led to contradictory results \[[@ref10],[@ref11],[@ref13]-[@ref15]\].
All anti-TNF agents share an IgG1 Fc fragment, but the contribution of the Fc portion to the response to treatment among currently used TNF blockers remains unknown. Receptors for IgG-Fc portion (FcR) are important regulatory molecules of inflammatory responses. FcR polymorphisms alter receptor function by enhancing or diminishing the affinity for immunoglobulins \[[@ref16]\]. Three major classes of FcR that are capable of binding IgG antibodies are recognised: FcγRΙ (CD64), FcγRΙΙ (CD32), and FcγRΙΙΙ (CD16). FcγRΙΙ and FcγRΙΙΙ have multiple isoforms (FcγRΙΙΙA/C and B; FcγRΙΙΙA and B) \[[@ref16]\]. The most frequent polymorphism of *FcγRΙΙΙA* is a point mutation affecting amino acids in codon 158 in the extracellular domain. This results in either a valine (V158) or a phenylalanine (F158) at this position. Recently, it has been reported that CD patients with *FcγRΙΙΙA* -158V/V genotype had a better biological and possibly better clinical response to IFX \[[@ref17]\]. However, further studies did not confirm this observation \[[@ref18]\].
The aim of this study was to assess whether the *TNF* and/ or *FcγRΙΙΙA* gene polymorphisms are genetic predictors of response to IFX, in a cohort of Greek patients with adult or paediatric onset of CD.
Patients - Methods {#sec1-2}
==================
Patients {#sec2-1}
--------
We enrolled 106 consecutive patients with newly diagnosed CD attending the outpatient IBD Clinic at the 1^st^ Department of Gastroenterology, "Evangelismos" Hospital (79 adults) or the 1^st^ Department of Pediatrics, University Hospital of Athens "Aghia Sophia"(27 children). The diagnosis of CD was based on standard clinical, endoscopic, radiological, and histological criteria \[[@ref1],[@ref19]\]. Eligible patients should have inflammatory (luminal) disease and be naive to IFX.
IFX was administered intravenously at a dose of 5mg/kg at weeks 0, 2, 6 and then every 8 weeks. Clinical and serological responses were assessed using the Harvey-Bradshaw Index (HBI) \[[@ref20]\] and the serum levels of C-reactive protein (CRP), respectively, at baseline (before the 1st infusion of IFX), the day before each subsequent IFX infusion and after 12 weeks of treatment. Ileocolonoscopy was performed by a single endoscopist (GJM) at baseline and after 12-20 weeks of therapy to assess mucosal healing. Any changes in endoscopic appearance compared to baseline endoscopy were classified in four categories \[[@ref21],[@ref22]\] \[[Table 1](#T1){ref-type="table"}\]. Patients were classified in accordance to response to IFX therapy as shown in [table 2](#T2){ref-type="table"}. The ethical committee of the participating hospitals approved the study. Research was carried out according to Helsinki Convention (1975) and written inform consent was obtained in advance from each patient.
######
Grading of endoscopic mucosal lesions \[[@ref21],[@ref22]\]
######
Classification of the study population due to response to infliximab therapy
Genotyping {#sec2-2}
----------
Genomic DNA from whole blood containing EDTA was extracted using standard techniques (NucleoSpin Blood kit, Macherey-Nagel, Germany). All polymerase chain reactions (PCRs) were run under conditions previously described \[[@ref23]\]. Primer sequences for the gene polymorphism at --308 were forward 5′-GGG ACA CAC AAG CAT CAA GG-3′ and reverse 5′-GGG ACA CAC AAG CAT CAA GG-3′, for the polymorphism at −238 forward 5′-ATC TGG AGG AAG CGG TAG TG-3′ and reverse 5′-AGA AGA CCC CCC TCG GAA CC-3′. The PCR products were digested at 37 °C with NcoI to detect the SNP in the −308 gene allele and MspI to detect the polymorphism of the −238 nucleotide. The -857 C/T polymorphism was analyzed by allele-specific PCR method24 using the primers TNF857-C: 5′-aag gat aag ggc tca gag ag-3′, TNF857-N: 5′-cta cat ggc cct gtc ttc g-3′ and TNF857-M: 5′-t cta cat ggc cct gtc ttc a-3′. The --158V/F polymorphism of FcγRΙΙΙA gene was detected as described by Leppers-van de Straat et al \[[@ref25]\] using the primers 5′-CTG AAG ACA CAT TTT TACT CC CAA (A/C)-3′ and 5′-TCC AAA AGC CAC ACT CAA AGA C-3′. The PCR products were then subjected to 3% agarose-gel electrophoresis. "No target" controls were included in each PCR batch to ensure that reagents had not been contaminated.
Statistical Analysis {#sec2-3}
--------------------
Genotype frequencies were compared with the chi-square with Yate's correction using S-Plus (v. 6.2Insightful, Seattle, WA). Odds ratios (ORs) and 95 confidence intervals (CIs) were obtained with GraphPad (v. 3.00, GraphPad Software, San Diego, CA). The p values are all two-sided. Correction for multiple testing was not applied in this study. *P* values of \< 0.05 were considered to be significant.
Results {#sec1-3}
=======
Patient demographic and clinical characteristics are given in [Table 3](#T3){ref-type="table"}. There were 68 (64.15%) complete responders, 25 (23.58%) partial responders and 13 (12.26%) non responders to IFX in this study. There were no statistical differences in the mean age, gender, disease duration, location and behavior and smoking habits between complete or partial responders and primary non-responders. There was no disagreement between HBI scores and serum CRP levels. Although, the post-treatment CRP levels were significantly lower in complete responders compared to partial and non-responders, the decrease in CRP levels did not differ significantly between the three groups. Post-treatment CRP levels and mean HBI score were significantly lower in complete responders compared to pre-treatment values in contrast to partial and/or non-responders where the CRP levels and the mean HBI score did not differ significantly.
######
Demographic, clinical and biological characteristics of the study population
The -238 G/A, -308 G/A, and -857 C/T polymorphisms of the TNF gene and the -158 V/F polymorphism in the *FcγRΙΙΙA* gene were successfully determined in all subjects. The genotype distribution in complete, partial and non-responders were presented in [Table 4](#T4){ref-type="table"}. No significant difference was observed for the polymorphism tested. In addition, although there may be genetic differences in early (paediatric)-onset and late (adult)-onset CD we were unable to detect any such differences although the number of paediatric patients included in the current study did not allow firm conclusions.
######
Genotype frequency in complete responders, partial responders and non responders
In the present study, we could not correlate the decrease in serum CRP levels with the genotypes tested in any particular group of patients since in most of the cases serum CRP levels dropped by more than 25% after 12 weeks of treatment. However, no significant decrease in CRP was observed between the TNF genotypes tested. Regarding the -158 V/F polymorphism in the *FcγRΙΙΙA* gene, the relative decrease in serum CRP levels was greatest in VV
|
introduction { # sec1 - 1 } = = = = = = = = = = = = infliximab ( ifx ), a chimeric anti - tnfα antibody, is effective in inducing and maintaining remission when causing considerable proportion of ibd patients refractory to any other treatments \ [ [ @ ref1 ], [ @ ref2 ] \ ]. however, 8 - 12 % of adult and / or pediatric patients fail to respond to the induction regimen ( known as primary non responders ) and approximately 40 % of patients who respond initially and achieve clinical remission inevitably lose response over time \ [ [ @ ref3 ], [ @ ref7 ] \ ]. lack of response to ifx is a negative trait and suggests that the differences in response might be in part genetically determined. considering the high cost and safety profile of this drug, genetic targeting of cells responding to this therapy is certainly of great interest \ [ [ @ ref8 ] \ ]. so far, limited candidate gene association studies with response to ifx have been reported \ [ [ @ ref9 ] - [ @ ref11 ] \ ]. recently, a genome - wide association study ( gwas ) regarding paediatric ibd patients has revealed that the 21q22. 2 / brwdi loci were associated with primary non response \ [ [ @ ref12 ] \ ]. furthermore, although tnfa gene is of great interest as a candidate gene for pharmacogenetic approaches few studies have been performed to date while some have led out contradictory results \ [ [ @ ref10 ], [ @ ref11 ], [ @ ref13 ] - [ @ ref15 ] \ ]. all anti - tnf agents share an igg1 fc fragment, but the contribution of the fc portion to the response to treatment among currently used tnf blockers remains unknown. receptors for igg - fc portion ( fcr ) are important regulatory molecules influencing inflammatory responses. fcr polymorphisms alter receptor function by enhancing or diminishing the affinity for immunoglobulins \ [ [ @ ref16 ] \ ]. three major classes of fcr that are capable of binding igg antibodies are recognised : fcγrι ( cd64 ), φ ( cd32 ), and fcγrιιι ( cd16 ). fcγrιι and fcγrιιι have multiple endings ( fcγ ##rιιιa / c and b ; fcγrιιιa and b ) \ [ [ @ ref16 ] \ ]. the most frequent polymorphism of * fcγrιιιa * is a point mutation affecting amino acids in codon 158 in the extracellular domain. this results in either a valine ( v158 ) or a phenylalanine ( f158 ) at this position. recently, it has been reported that cd patients with * fcγrιιιa * - 158v / v genotype had a better biological and possibly better clinical response to ifx \ [ [ @ ref17 ] \ ]. however, further studies did not confirm this observation \ [ [ @ ref18 ] \ ]. the aim of this study was to assess whether the * tnf * and / or * fcγrιιιa * gene polymorphisms are genetic predictors of response to ifx, in a cohort of greek patients with adult or paediatric onset of cd. patients - methods { # sec1 - 2 } = = = = = = = = = = = = = = = = = = patients { # sec2 - 1 } - - - - - - - - we enrolled 106 consecutive patients with newly diagnosed cd attending the outpatient ibd clinic at the 1 ^ st ^ department of gastroenterology, " evangelismos " hospital ( 79 adults ) or the 1 ^ st ^ department of pediatrics, university hospital of athens " aghia sophia " ( 27 children ). the diagnosis of cd was based on standard clinical, endoscopic, radiological, and histological criteria \ [ [ @ ref1 ], [ @ ref19 ] \ ]. eligible patients should have inflammatory ( luminal ) disease and be naive to ifx. ifx was administered intravenously at a dose of 5mg / kg at weeks 0, 2, 6 and then every 8 weeks. clinical and serological responses were assessed using the harvey - bradshaw index ( hbi ) \ [ [ @ ref20 ] \ ] and the serum levels of c - reactive protein ( crp ), respectively, at baseline ( before the 1st infusion of ifx ), the day before each subsequent ifx infusion and after 12 weeks of treatment. ileocolonoscopy was performed by a single endoscopist ( gjm ) at baseline and after 12 - 20 weeks of therapy to assess mucosal healing. any changes in endoscopic appearance compared to baseline endoscopy were classified in four categories \ [ [ @ ref21 ], [ @ ref22 ] \ ] \ [ [ table 1 ] ( # t1 ) { ref - type = " table " } \ ]. patients were classified in accordance to response to ifx therapy as shown in [ table 2 ] ( # t2 ) { ref - type = " table " }. the ethical committee of the participating hospitals approved the study. research was carried out according to helsinki convention ( 1975 ) and written inform consent was obtained in advance from each patient. # # # # # # grading of endoscopic mucosal lesions \ [ [ @ ref21 ], [ @ ref22 ] \ ]! [ ] ( anngastroenterol - 24 - 35 - g001 ) # # # # # # classification of the study population due to response to infliximab therapy! [ ] ( anngastroenterol - 24 - 35 - g002 ) genotyping { # sec2 - 2 } - - - - - - - - - - genomic dna from whole blood containing edta was extracted using standard techniques ( nucleospin blood kit, macherey - nagel, germany ). all polymerase chain reactions ( pcrs ) were run under conditions previously described \ [ [ @ ref23 ] \ ]. primer sequences for the gene polymorphism at - - 308 were forward 5 ′ - ggg aca cac aag cat caa gg - 3 ′ and reverse 5 ′ - ggg aca cac aag cat caa gg - 3 ′, for the polymorphism at −238 forward 5 ′ - atc tgg agg aag cgg tag tg - 3 ′ and reverse 5 ′ - aga aga ccc ccc tcg gaa cc - 3 ′. the pcr products were digested at 37 °c with ncoi to detect the snp in the −308 gene allele and mspi to detect the polymorphism of the −238 nucleotide. the - 857 c / t polymorphism was analyzed by allele - specific pcr method24 using the primers tnf857 - c : 5 ′ - aag gat aag ggc tca gag ag - 3 ′, tnf857 - n : 5 ′ - cta cat ggc cct gtc ttc g - 3 ′ and tnf857 - m : 5 ′ - t cta cat ggc cct gtc ttc a - 3 ′. the - - 158v / f polymorphism of fcγrιιιa gene was detected as described by leppers - van de straat et al \ [ [ @ ref25 ] \ ] using the primers 5 ′ - ctg aag aca cat ttt tact cc caa ( a / c ) - 3 ′ and 5 ′ - tcc aaa agc cac act caa aga c - 3 ′. the pcr products were then subjected to 3 % agarose - gel electrophoresis. " no target " controls were included in each pcr batch to ensure that reagents had not been contaminated. statistical analysis { # sec2 - 3 } - - - - - - - - - - - - - - - - - - - - genotype frequencies were compared with the chi - square with yate ' s correction using s - plus ( v. 6. 2insightful, seattle, wa ). odds ratios ( ors ) and 95 confidence intervals ( cis ) were obtained with graphpad ( v. 3. 00, graphpad software, san diego, ca ). the p values are all two - sided. correction for multiple testing was not applied in this study. * p * values of \ < 0. 05 were considered to be significant. results { # sec1 - 3 } = = = = = = = patient demographic and clinical characteristics are given in [ table 3 ] ( # t3 ) { ref - type = " table " }. there were 68 ( 64. 15 % ) complete responders, 25 ( 23. 58 % ) partial responders and 13 ( 12. 26 % ) non responders to ifx in this study. there were no statistical differences in the mean age, gender, disease duration, location and behavior and smoking habits between complete or partial responders and primary non - responders. there was no disagreement between hbi scores and serum crp levels. although, the post - treatment crp levels were significantly lower in complete responders compared to partial and non - responders, the decrease in crp levels did not differ significantly between the three groups. post - treatment crp levels and mean hbi score were significantly lower in complete responders compared to pre - treatment values in contrast to partial and / or non - responders where the crp levels and the mean hbi score did not differ significantly. # # # # # # demographic, clinical and biological characteristics of the study population! [ ] ( anngastroenterol - 24 - 35 - g003 ) the - 238 g / a, - 308 g / a, and - 857 c / t polymorphisms of the tnf gene and the - 158 v / f polymorphism in the * fcγrιιιa * gene were successfully determined in all subjects. the genotype distribution in complete, partial and non - responders were presented in [ table 4 ] ( # t4 ) { ref - type = " table " }. no significant difference was observed for the polymorphism tested. in addition, although there may be genetic differences in early ( paediatric ) - onset and late ( adult ) - onset cd we were unable to detect any such differences although the number of paediatric patients included in the current study did not allow firm conclusions. # # # # # # genotype frequency in complete responders, partial responders and non responders! [ ] ( anngastroenterol - 24 - 35 - g004 ) in the present study, we could not correlate the decrease in serum crp levels with the genotypes tested in any particular group of patients since in most of the cases serum crp levels dropped by more than 25 % after 12 weeks of treatment. however, no significant decrease in crp was observed between the tnf genotypes tested. regarding the - 158 v / f polymorphism in the * fcγrιιιa * gene, the relative decrease in serum crp levels was greatest in vv
|
Introduction {# sec1 - 1} = = = = = = = = = = = = Infliximab (IFX ), a chimeric anti - TNFα antibody, is effective in inducing and maintaining remission in a considerable proportion of IBD patients refractory to any other treatments \ [[ @ ref1 ], [@ ref2] \ ]. However, 8 - 12% of adult and / or pediatric patients fail to respond to the induction regimen (known as primary non responders) and approximately 40% of patients who respond initially and achieve cloniVal remission inevitably lose response over time \ [[ @ ref3 ], [@ ref7] \ ]. Lack of response to IFX is a stable trait and suggests that the differences in rewpinse might be in part genetically determined. Considering the high cost and safety profile of this drug, genetic targeting of patients responding to this therapy is certainly of great interest \ [[ @ ref8] \ ]. So far, limited candidate gene association studies with response to IFX have nesn reported \ [[ @ ref9] - [@ ref11] \ ]. Recently, a genome - wide association study (GWAS) in paediatric IBD patients has revealed that the 21q22. 2 / BRWDI loci were associated with primary non response \ [[ @ ref12] \ ]. FurtTerkore, although TNFa gene is of great interest as a candidate gene for pharmacogenetic approaches few studies have been performed to date and some have led to contradictory results \ [[ @ ref10 ], [@ ref11 ], [@ ref13] - [@ ref15] \ ]. All anti - TNF agents share an IgG1 Fc fragment, but the contribution of the Fc portion to the response to treatment among currently used TNF blockers remains unknown. Receptors for IgG - Fc portion (FcR) are important regulatory molecules of inflammatory responses. FcR polymorphisms alter receptor function by enhanc7hg or diminishing the affinity for immunoglobulins \ [[ @ ref16] \ ]. Three major classes of FcR that are capable of binding IgG antibodies are recognised: FcγRΙ (CD64 ), FcγRΙΙ (CD32 ), and FcγRΙΙΙ (CD16 ). FcγRΙΙ and FcγRΙΙΙ have multiple isoforms (FcγRΙΙΙA / C and B; FcγRΙΙΙA and B) \ [[ @ ref16] \ ]. The most frequent polymorphism of * FcγRΙΙΙA * is a point mutation affecting amino acids in codon 158 in the extracellular domain. This results in either a valine (V158) or a phenylalanine (F158) at this position. Recently, it has been reported that CD patients with * FcγRΙΙΙA * - 158V / V genotype had a better biological and possibly better clinical response to IFX \ [[ @ ref17] \ ]. Nowev3r, further studies did not confirm this observation \ [[ @ ref18] \ ]. The aim of this study was to assess whether the * TNF * and / or * FcγRΙΙΙA * gene polymorphisms are genetic predictors of response to IFX, in a cohort of Greek patients with adult or paediatric onset of CD. Patients - Methods {# sec1 - 2} = = = = = = = = = = = = = = = = = = Patients {# sec2 - 1} - - - - - - - - We enrolled 106 consecutive patients with newly diagnosed CD attending the outpatient IBD Clinic at the 1 ^ st ^ Department of Gastroenterology, " Evangelismos " Hospital (79 adults) or the 1 ^ st ^ Department of Pediatrics, University Hospital of Athens " Aghia Sophia " (27 children ). The diagnosis of CD was based on standard clinical, endoscopic, radiological, and histological criteria \ [[ @ ref1 ], [@ ref19] \ ]. Eligible patients should have inflammatory (luminal) disease and be naive to IFX. IFX was administered Lntrafenously at a dose of 5mg / kg at weeks 0, 2, 6 and then every 8 weeks. Clinical and serological responses were assessed using the Harvey - Bradshaw Index (HBI) \ [[ @ ref20] \] and the serum levels of C - reactive protein (CRP ), respectively, at baseline (before the 1st infusion of IFX ), the day before each subsequent IFX infusion and after 12 weeks of treatment. Ileocolonoscopy was performed by a single endoscopist (GJM) at baseline and after 12 - 20 weeks of therapy to assess mucosal healing. Any changes in endoscopic appearance compared to baseline endoscopy were classified in four categories \ [[ @ ref21 ], [@ ref22] \] \ [[ Table 1] (# T1) {ref - type = " table "} \ ]. Patients were classified in accordance to response to IFX therapy as shown in [table 2] (# T2) {ref - type = " table " }. The ethical committee of the participating hospitals approved the study. Research was carried out according to Helsinki Convention (1975) and written inform consent was obtained in advance from each patient. # # # # # # Grading of endoscopic mucosal lesions \ [[ @ ref21 ], [@ ref22] \ ]! [] (AnnGastroenterol - 24 - 35 - g001) # # # # # # Classification of the study population due to response to infliximab therapy! [] (AnnGastroenterol - 24 - 35 - g002) Genotyping {# sec2 - 2} - - - - - - - - - - Genomic DNA from whole blood containing EDTA was extracted using standArV techniques (NucleoSpin Blood kit, Macherey - Nagel, Germany ). All polymerase chain reactions (PCRs) were run under conditions previously described \ [[ @ ref23] \ ]. Primer sequences for the gene polymorphism at - - 308 were forward 5 ′ - GGG ACA CAC AAG CAT CAA GG - 3 ′ and reverse 5 ′ - GGG ACA CAC AAG CAT CAA GG - 3 ′, for the polymorphism at − 238 forward 5 ′ - ATC TGG AGG AAG CGG TAG TG - 3 ′ and reverse 5 ′ - AGA AGA CCC CCC TCG GAA CC - 3 ′. The PCR products were digested at 37 ° C with NcoI to detect the SNP in the − 308 gene allele and MspI to detect the polymorphism of the − 238 nucleotide. The - 857 C / T polymorphism was analyzed by allele - specific PCR method24 using the primers TNF857 - C: 5 ′ - aag gat aag ggc tca gag ag - 3 ′, TNF857 - N: 5 ′ - cta cat ggc cct gtc ttc g - 3 ′ and TNF857 - M: 5 ′ - t cta cat ggc cct gtc ttc a - 3 ′. The - - 158V / F polymorphism of FcγRΙΙΙA gene was detected as described by Leppers - van de Straat et al \ [[ @ ref25] \] using the primers 5 ′ - CTG AAG ACA CAT TTT TACT CC CAA (A / C) - 3 ′ and 5 ′ - TCC AAA AGC CAC ACT CAA AGA C - 3 ′. The PCR products were then subjected to 3% agarose - gel electrophoresis. " No target " controls were included in each PCR batch to ensure that reagents had not been contaminated. Statistical Analysis {# sec2 - 3} - - - - - - - - - - - - - - - - - - - - Genotype frequencies w4rR compared with the chi - square with Yate ' s correction using S - Plus (v. 6. 2Insightful, Seattle, WA ). Odds ratios (ORs) and 95 confidence intervals (CIs) were obtained with GraphPad (v. 3. 00, GraphPad Software, San Diego, CA ). The p values are all two - sided. Correction for multiple testing was not applied in this study. * P * values of \ <0. 05 were considered to be significant. Results {# sec1 - 3} = = = = = = = Patient demographic and clinical characteristics are given in [Table 3] (# T3) {ref - type = " table " }. There were 68 (64. 15%) complete responders, 25 (23. 58%) partial responders and 13 (12. 26%) non responders to IFX in this study. There were no statistical differences in the mean age, gender, disease duration, location and behavior and smoking habits between complete or partial responders and primary non - responders. There was no disagreement between HBI scores and serum CRP levels. Although, the post - %reatnent CRP levels were significantly lower in complete responders compared to partial and non - responders, the decrease in CRP levels did not differ significantly between the three groups. Post - treatment CRP levels and mean HBI score were significantly lower in complete responders compared to pre - treatment values in contrast to partial and / or non - responders where the CRP levels and the mean HBI score did not differ significantly. # # # # # # Demographic, clinical and biological characteristics of the study population! [] (AnnGastroenterol - 24 - 35 - g003) The - 238 G / A, - 308 G / A, and - 857 C / T polymorphisms of the TNF gene and the - 158 V / F polymorphism in the * FcγRΙΙΙA * gene were successfully determined in all subjects. The genotype distribution in complete, partial and non - responders were presented in [Table 4] (# T4) {ref - type = " table " }. No significant difference was observed for the polymorphism tested. In addition, although there may be genetic differences in early (paediatric) - onset and late (adult) - onset CD we were unable to detect any such differences although the number of paediatric patients included in the current study did not allow firm conclusions. # # # # # # Genotype frequency in complete responders, partial responders and non responders! [] (AnnGastroenterol - 24 - 35 - g004) In the present study, we could not correlate the decrease in serum CRP levels with the genotypes tested in any particular group of patients since in most of the cases serum CRP levels dropped by more than 25% after 12 weeks of treatment. However, no significant decrease in CRP was observed between the TNF genotypes tested. Regarding the - 158 V / F polymorphism in the * FcγRΙΙΙA * gene, the relative decrease in serum CRP levels was greatest in VV
|
Introduction {#sec1-1} ============ Infliximab (IFX), a chimeric anti-TNFα antibody, is effective in inducing maintaining remission in a proportion of patients refractory to any other However, 8-12% of adult and/or pediatric patients fail to to the regimen (known as primary non responders) and approximately 40% of patients who respond initially achieve clinical remission lose response over time\[[@ref3],[@ref7]\]. Lack of response to IFX is a stable trait and suggests the differences response might be in part genetically determined. Considering the high cost and safety of this drug, genetic targeting of responding to this therapy is of great interest \[[@ref8]\]. So far, limited candidate gene association studies with IFX have been reported \[[@ref9]-[@ref11]\]. Recently, genome-wide association study (GWAS) in paediatric IBD patients has revealed the 21q22.2/BRWDI loci associated with primary non response \[[@ref12]\]. Furthermore, although TNFa gene is of great interest as a gene for pharmacogenetic approaches few have been performed to date and some have led contradictory results \[[@ref10],[@ref11],[@ref13]-[@ref15]\]. All anti-TNF agents share IgG1 Fc fragment, but the contribution of the Fc portion to the response to treatment among currently used TNF blockers remains unknown. Receptors for portion (FcR) are important regulatory molecules of inflammatory responses. FcR polymorphisms alter receptor function by enhancing or diminishing the for immunoglobulins \[[@ref16]\]. major classes of FcR that are capable of binding antibodies are recognised: FcγRΙ (CD64), FcγRΙΙ (CD32), and FcγRΙΙΙ (CD16). FcγRΙΙ and FcγRΙΙΙ have multiple (FcγRΙΙΙA/C and B; FcγRΙΙΙA and B) \[[@ref16]\]. The most frequent polymorphism of *FcγRΙΙΙA* is a point mutation affecting amino acids in codon 158 in the extracellular domain. This results in either a valine (V158) or a phenylalanine (F158) at this position. Recently, it has been reported that CD with *FcγRΙΙΙA* -158V/V genotype had biological and possibly better clinical response to IFX \[[@ref17]\]. However, further studies did confirm this observation \[[@ref18]\]. The aim of study was to assess whether the *TNF* and/ or *FcγRΙΙΙA* gene polymorphisms are predictors of response to IFX, a cohort of Greek patients with adult or paediatric onset of CD. Patients - Methods {#sec1-2} ================== Patients {#sec2-1} -------- enrolled 106 consecutive patients with newly diagnosed CD attending the outpatient IBD Clinic at 1^st^ Department of Gastroenterology, Hospital (79 adults) or the Department of Pediatrics, University Hospital of Athens "Aghia Sophia"(27 children). The diagnosis CD was based on standard endoscopic, radiological, and criteria \[[@ref1],[@ref19]\]. should have inflammatory (luminal) disease and naive to IFX. IFX was administered intravenously at a 5mg/kg at weeks 0, 2, 6 and then every 8 weeks. Clinical and serological responses were assessed using the Harvey-Bradshaw Index (HBI) \[[@ref20]\] and the serum levels C-reactive protein (CRP), respectively, at baseline (before the 1st infusion of IFX), day before each subsequent IFX infusion and after weeks of treatment. Ileocolonoscopy was performed by a (GJM) at baseline and after 12-20 therapy to assess mucosal healing. Any changes in endoscopic appearance compared to baseline endoscopy were classified in four categories \[[@ref21],[@ref22]\] \[[Table 1](#T1){ref-type="table"}\]. Patients were classified in accordance to response to IFX therapy as shown in [table The ethical committee of the participating hospitals approved the study. Research was carried out according to Helsinki Convention (1975) and written inform consent was obtained in advance from each patient. ###### Grading mucosal lesions \[[@ref21],[@ref22]\]  ###### Classification of the population to response to infliximab therapy Genotyping {#sec2-2} ---------- from whole blood containing EDTA was extracted using standard techniques (NucleoSpin kit, Macherey-Nagel, All polymerase reactions (PCRs) run under conditions previously described \[[@ref23]\]. Primer sequences for the gene polymorphism at --308 were forward 5′-GGG ACA CAC AAG CAT CAA GG-3′ and reverse 5′-GGG ACA CAC AAG CAT CAA GG-3′, for the polymorphism at −238 forward 5′-ATC TGG AGG AAG CGG TAG and reverse 5′-AGA AGA CCC CCC TCG GAA CC-3′. The PCR products were digested at 37 °C with NcoI to detect SNP in the −308 gene allele and MspI detect the polymorphism of the −238 nucleotide. The -857 polymorphism was analyzed by allele-specific PCR method24 using the primers TNF857-C: 5′-aag gat aag ggc tca gag ag-3′, TNF857-N: 5′-cta cat cct gtc ttc g-3′ TNF857-M: 5′-t cta cat ggc cct gtc ttc a-3′. The --158V/F polymorphism of FcγRΙΙΙA gene was detected described by Leppers-van de Straat al \[[@ref25]\] the 5′-CTG AAG ACA CAT TTT TACT CC CAA (A/C)-3′ and 5′-TCC AAA AGC CAC AGA C-3′. The PCR products were then subjected to 3% agarose-gel electrophoresis. "No target" controls included in each PCR batch to ensure that had not been contaminated. Analysis {#sec2-3} -------------------- frequencies were compared with the chi-square with Yate's S-Plus (v. 6.2Insightful, Seattle, WA). Odds ratios (ORs) and 95 confidence intervals (CIs) were obtained with GraphPad (v. 3.00, GraphPad Software, Diego, CA). The values are all two-sided. Correction for multiple testing was applied in this study. *P* values of 0.05 were considered to be significant. Results {#sec1-3} ======= Patient demographic and clinical characteristics are given [Table 3](#T3){ref-type="table"}. were 68 (64.15%) responders, 25 (23.58%) responders 13 (12.26%) non responders to IFX in this study. There were no statistical differences in the mean age, gender, disease duration, location behavior and smoking habits between complete or partial responders and primary non-responders. There was no between HBI scores and serum CRP levels. Although, the post-treatment levels were significantly lower complete compared to partial and the decrease in CRP levels did not differ significantly between the three Post-treatment CRP levels and mean HBI were significantly lower in responders compared to pre-treatment values in to partial and/or non-responders where the CRP levels and the mean HBI score did not differ significantly. ###### clinical and biological characteristics of the study population  The -238 G/A, -308 G/A, and -857 C/T polymorphisms the TNF gene and the -158 in the *FcγRΙΙΙA* gene successfully determined in all subjects. The genotype distribution in complete, partial and non-responders were presented in [Table 4](#T4){ref-type="table"}. No significant difference was observed for the polymorphism tested. addition, although there may be genetic differences in early (paediatric)-onset and late (adult)-onset CD we were unable detect any such differences the number of paediatric patients included in the current study did not allow firm conclusions. ###### Genotype frequency in responders, partial responders and non responders  In the present study, we could not correlate decrease in serum CRP levels with genotypes tested in any particular group of patients in most of the cases serum CRP levels dropped by more than 25% after 12 weeks of treatment. no significant decrease in CRP was observed between the TNF genotypes tested. Regarding the -158 V/F polymorphism in *FcγRΙΙΙA* gene, the relative decrease in serum levels greatest in
|
iNtroDucTIoN {#SEC1-1}
============
infLixIMAb (iFX), A cHiMeric anTI-tNFα antiboDY, Is EFFEctiVe In InduCiNg And mAINTaInInG reMiSsioN in a coNsiDERABle propoRTioN Of IbD patIEnts ReFRAcTOry To aNy OTHER TReaTMENTS \[[@reF1],[@REf2]\]. hoWEVeR, 8-12% Of AduLT and/OR peDIaTrIc patienTs Fail tO reSPOnD tO tHE iNDuctION RegimeN (known as PrimARY NOn REspOndErS) AND ApPROXiMatELy 40% oF pATieNts WHO ResPOnd inITiAlLy aNd acHiEvE CLiniCAl REmIssIOn iNEViTABlY LOse reSPOnse oVEr tIME\[[@ref3],[@REF7]\]. lAcK Of ReSpONSe TO IFX Is a sTabLe trAIt AND suGGEStS THat tHe dIFFERENCes In RESPONSE MiGHt BE In PaRT GeNETicaLly DeterMINEd. CoNSidERIng ThE HIgh COST anD SAFEty PRoFilE Of tHis dRUG, GeNeTIc TaRGetinG oF PAtiEntS RESpONDing to THIS thERapY IS ceRtAINLy Of gReaT iNtErEst \[[@REf8]\]. SO far, LimITEd CaNdIdAtE gENE assoCIatION STUdiES With ReSpoNSE to IFX HavE BeEn rePorted \[[@ref9]-[@reF11]\]. RECeNtly, A GEnome-wiDe ASsoCiaTIoN STudy (gWAS) In pAeDiAtRiC ibd pAtiENtS HAS rEvEALeD ThAT the 21Q22.2/brwDi lOCI WeRe AssoCiAtED with PriMArY NoN RESPONSe \[[@rEf12]\]. FURtheRMorE, aLThOUGh tNFa gENe iS OF grEAT INteREsT as A canDIDaTE genE fOr PHaRMACOGEnETiC aPPRoaChEs feW sTudiES hAve been peRforMed To DAtE and SOmE HAve lEd to CoNtRADIctory rEsULts \[[@Ref10],[@Ref11],[@rEF13]-[@ref15]\].
ALl ANTI-tNf AgENTS ShArE AN iGg1 FC FRaGMeNT, But The CONTRibuTiOn OF tHe fc POrtIoN to THE RESPoNSe To tReatMent AmONG CURrENtlY UsEd TnF BLoCKERS REmaiNS UNkNoWN. REcEpToRS foR Igg-Fc PoRtioN (FCr) ARE iMpoRTaNT ReGuLAtORy mOleCULes oF InflaMmAtORY ReSPOnses. fCr polyMorPhIsmS Alter ReCePtor fUnCtIoN by EnhAncInG OR DIminisHING THE AffINIty FoR IMMuNOglobUliNS \[[@Ref16]\]. THREE MajOr cLASsES OF FCR tHaT are capABle of bINdINg IGg anTiBoDiEs ARE recOgNIsED: FcγrΙ (cd64), fCΓRΙΙ (CD32), aNd fCΓrιιι (CD16). FCγrΙι anD FCγRιιι HaVe MuLtiple isOfOrms (fcΓRιΙΙA/C aNd B; fcγRΙΙιA and b) \[[@rEF16]\]. tHe mOsT FRequenT poLYMoRpHIsm oF *FCγrιΙιa* Is A pOiNt MUTatioN affectING amiNo aCIDS IN cOdON 158 in ThE EXTraCElLULAr domAIn. THis RESultS In eiTHer A vAlIne (V158) OR a PhEnYLAlaNINe (F158) At tHIs POsition. RECEntLY, iT HAs beEN RePorTED THAt cD PatieNTS wITH *FCγRιΙιA* -158V/V gENOtYPe HaD a bEtTER BioloGIcAl ANd posSIbLY BettER CLiNiCal reSPonSE tO ifx \[[@REF17]\]. HOwEVeR, FurthEr sTUDiES diD not CONFiRm thiS obserVatioN \[[@reF18]\].
thE AIM oF this Study WAs To ASSeSs whETHER thE *TnF* And/ Or *fcγRιιΙA* Gene POLYmOrpHisMs aRE GenETic pREdiCTORs OF RESPonSe tO IfX, IN a CohoRt OF grEEK PaTiEntS wITh adULT oR PAEDiAtRIc onset Of cD.
PATIEnTS - mETHOds {#SEC1-2}
==================
PatIENts {#Sec2-1}
--------
we enrolled 106 coNsecutivE PATiENTs WitH neWLy diAgNoSeD cd aTtEndiNG the oUTPatIEnT ibD CliNIC AT ThE 1^st^ DEPaRtmEnt Of gAstROenterOlOgy, "evAngELIsmoS" hoSPITAl (79 ADuLTS) oR tHe 1^ST^ dEPARTment of PediATRIcS, UNiversIty HOSpitAL oF aTHeNs "AgHIa SOPhIA"(27 chIldreN). The DiaGNoSIS oF cD WaS basED On STANdArd cliNiCaL, EndoSCOpic, rAdIOlOGIcAL, AnD hISTOlOgIcaL CRitERia \[[@rEF1],[@ReF19]\]. eLiGiBlE PATIeNTS sHOulD hAvE InFLaMMaTOry (lumiNal) diseAsE and BE nAiVe tO ifx.
Ifx was ADMINISTEREd INTrAVenOuSly At A doSE Of 5mg/Kg aT weeKs 0, 2, 6 AND theN EVErY 8 weEks. cLINicAl aND serOLOgIcAL rESpOnSES werE ASSEsSed uSing THE hArveY-BRaDsHAW iNdEx (HBI) \[[@ref20]\] AnD tHe sErUm LeVELs OF c-reactIve pRoTein (cRP), RESpECtiVelY, at baSeLIne (before The 1ST InfUsIon oF Ifx), THe DAy beFoRE EAch SubsEQuenT ifX infUSION AND afTer 12 weEKs Of tREatMenT. IleOcOloNoSCOpY wAS pERFOrMed By A SinGLE EnDOsCOPIsT (GjM) at BaseLiNE And AFteR 12-20 WeEkS OF ThErApY To AssEss MucosAL HeaLINg. aNY chANgeS iN ENdOScoPIC ApPEaraNcE CoMparED to BaSElIne EnDoSCOpY WERe cLAsSIfiED In FOuR CAtEGoRIES \[[@REF21],[@reF22]\] \[[tAbLE 1](#T1){ReF-typE="TaBLE"}\]. PatiENTS WeRe cLAssIFIeD In ACCordancE to reSpOnsE tO IFx tHerAPY AS SHOWn in [TablE 2](#T2){ref-tyPe="TablE"}. ThE eThIcAL COMmiTTeE oF ThE PaRTIcIpATING hOspitals apprOveD THE sTUdy. rESEarch WaS carRieD Out acCorDInG To HelSiNKI CoNveNTiOn (1975) aND written InFORm ConSeNT WAS ObTaINED in ADvAncE FrOM EaCH PAtIENT.
######
gRaDIng Of ENdOscOPIC mucoSAL leSIOns \[[@REF21],[@rEF22]\]
######
CLASsIFIcatIOn oF ThE stUdy popULatiOn Due To respoNSe tO iNfLixImaB thErAPY
GEnotYPiNG {#sec2-2}
----------
GeNoMic DnA FRom whOlE BLood cONTaIniNG Edta wAs extrAcTeD uSINg STaNDard TeCHniQUeS (nucleosPIN BLOOd KiT, macHeREy-nagEL, gErmany). alL poLYMeRASE chAiN rEAcTIONS (PCRS) werE rUn unDer conDiTions PrEvIoUSLy descrIBEd \[[@ref23]\]. PrImeR sEQUenCes FOR THe GENe POlymOrPHISM At --308 wErE FORwARD 5′-Ggg aca CAC aAG CaT CaA GG-3′ and rEVErSE 5′-gGG ACa caC aaG cAt CaA Gg-3′, foR ThE PoLyMORPhISm aT −238 fOrward 5′-ATC TgG agG aag cGG TAG Tg-3′ aND REVERSE 5′-Aga AgA Ccc ccc tCg gaa Cc-3′. THe Pcr PRoduCTs were DIgeStED AT 37 °c WIth nCoI tO DEtECt THE sNp In the −308 GENE AlLELE and mspI to deTECT the POlyMorpHisM OF tHE −238 NUCLeoTide. tHe -857 C/T POlymOrphIsM was AnalYZeD BY ALLElE-SPeCIfic PCR metHod24 UsiNG THe primers TnF857-C: 5′-Aag Gat AAg GGC tcA GAg ag-3′, Tnf857-n: 5′-cTA cAt ggc ccT gtC ttc g-3′ AND TNf857-m: 5′-T CTa caT GGc CcT GtC TtC a-3′. tHe --158V/F POlymOrPhISm oF FCγRΙΙιa GenE waS DEtEcTeD aS DeSCrIbed BY LEpPeRs-VaN DE STRaaT eT al \[[@ref25]\] USinG tHE prIMeRS 5′-ctg Aag ACA cat TtT taCt cC cAa (A/c)-3′ aNd 5′-Tcc AaA Agc cac acT caa agA C-3′. tHe PcR pRODucTs wERe THEn SUBJEcted To 3% AgAROSe-gEL ELectROPHOReSIS. "No TaRGet" ContRolS werE incLUdeD IN EaCh PCr BatCh to ENSURe tHAT rEAGenTS hAD NOT beEN ConTAmINatEd.
STaTistICal AnALYsIS {#SeC2-3}
--------------------
GEnOType FREqUEnCieS werE cOMpAreD wiTH tHe cHi-squARe WiTh YaTE'S cOrrEcTIOn usIng S-PlUS (v. 6.2InsiGHTFuL, SeaTTLE, wA). OddS ratIOs (orS) And 95 cONfidEnCe inTERvaLS (cIs) WEre ObTAineD WItH GrAPhpAD (v. 3.00, GRAphpAD sOfTwaRE, sAN DIEgo, CA). the p vAlUes ArE All tWo-sIded. cORREctiOn FOr mUltiPlE tEsTINg WAs nOT APPlied IN THiS STUdY. *P* ValUeS Of \< 0.05 WerE CoNsiDERed tO bE SIGNIfiCaNT.
ReSUltS {#SEc1-3}
=======
patiENt DEmOGRaphiC AND clInICal ChArACteRIStIcS arE GiveN iN [tABLe 3](#t3){Ref-TyPE="tabLe"}. there WeRe 68 (64.15%) comPLete ReSPonDerS, 25 (23.58%) pARTiaL reSpOndERs AND 13 (12.26%) nOn rESpOnDERS To ifX in tHIS StuDy. ThEre wERe No StaTiSTICal difFerENCeS In THE meAn aGe, GenDeR, DiSEAsE durAtiON, loCAtion and BehaVior aND SMoKiNg HABits BETwEEn CoMPLeTe Or pARtIAL RespONdERS AnD PrImarY NON-rESPONdeRS. tHere WaS no dIsAgReEMEnT bETwEEN HBI SCOreS aNd serUm crp LEVElS. AlthOuGh, tHE POSt-tREatMEnt Crp LeveLS wERE SIGnIficaNtly LoWeR in coMpLEte respOnders cOMParEd To PArtIal anD non-ReSpondERs, ThE DEcReASE IN Crp lEVeLS did NoT diFfER SiGniFIcanTLy BetWeen the THReE gROupS. pOst-tREATmENT cRP LeVels AND mEAN Hbi ScORe wEre SiGniFiCaNTLY LoWeR in cOMPLEtE RESPonDErS comPaRed TO PRe-TREAtmeNt vAlues iN cONTraSt tO paRTiaL aND/OR Non-ReSPOndErS WHeRE the cRp LEvELS And thE meAn Hbi ScoRe DID NoT Differ SiGNificaNtly.
######
DEmoGRapHIC, CLINIcaL AND BiolOGiCAL cHARACTErIstICS Of thE StUdY POPUlATIOn
tHE -238 g/A, -308 g/A, aNd -857 C/t poLyMorPhiSMS of tHe tNf GEnE And THE -158 v/F poLYmoRPhIsm IN The *fCΓRΙΙιa* gENe weRe sUCCesSFUlly DEtERminEd iN ALl suBJECTS. THe geNOtypE DiStrIbutIon in complETe, paRtIAL aNd Non-REspOndErs wErE prEsENTed IN [taBLE 4](#T4){Ref-TYpe="TABLE"}. no sIGnIficAnT DiFfEreNCe waS oBSErved foR THE pOlYMORpHISm TEstEd. In ADditION, ALTHoUgh tHERE may bE geneTic diFFerENCeS in EArly (paeDiatRIc)-ONsEt And LaTe (adUlT)-oNset cd wE WeRe unaBLE to deTecT aNY sUch DifFErENcEs AltHOugh ThE NumbeR Of PaEDiatRIC pAtieNtS iNcLUDeD IN tHe CUrrenT stUdy dID NOt allOw firm CoNcLUSIonS.
######
GENOtYpe freqUENcy In cOmplETE REspoNdERs, PartIal reSpOndERS aND NOn ReSPONDeRS
in the pRESENt sTUdy, WE COuld NOt coRReLaTe the DEcrEAsE IN SeRuM CrP levElS With THe genotYpes tEsTEd in AnY pArTiculAR Group Of pATIENTs sinCE IN MOST OF tHe Cases SerUm CRP lEvEls dropPeD By moRe ThaN 25% AfTER 12 weeKs OF TrEatMent. hOWeveR, nO siGNIfiCanT DeCrEase iN crP was obSeRVed BetWEeN THE TNf gENotyPes TeSTED. RegarDInG THe -158 v/f pOlymorPhIsm In the *FcγrΙιΙA* gene, THE rElative deCrEAsE in Serum cRp LEVels WAs GREaTesT in VV
|
Introduction {#sec1-1}============ Infliximab (IFX), a chimeric anti-TNFα antibody, is effective in inducing and maintaining remission in a considerable proportion of IBD patients refractory to any other treatments \[[@ref1],[@ref2]\]. However, 8-12% of adult and/or pediatric patients failto respond to the induction regimen (known as primary non responders) and approximately 40% of patients who respond initially and achieve clinical remission inevitably lose response over time\[[@ref3],[@ref7]\]. Lackof response to IFXis a stable trait andsuggests that the differences in response might be in part genetically determined. Considering the high cost and safety profile of this drug, genetic targeting of patients responding to this therapy is certainly ofgreat interest \[[@ref8]\]. So far, limited candidate gene association studies with response to IFXhavebeen reported \[[@ref9]-[@ref11]\].Recently, a genome-wide association study(GWAS) in paediatric IBD patientshas revealed that the 21q22.2/BRWDI loci were associated with primary non response \[[@ref12]\]. Furthermore, althoughTNFagene is of great interest as a candidate gene for pharmacogenetic approaches few studies have been performed to date and some have led to contradictory results \[[@ref10],[@ref11],[@ref13]-[@ref15]\]. All anti-TNF agentsshare an IgG1 Fc fragment, but the contribution of theFc portion to the response to treatment among currently used TNF blockers remains unknown.Receptors for IgG-Fc portion (FcR) are important regulatory molecules of inflammatoryresponses. FcRpolymorphisms alter receptor function by enhancingor diminishing the affinity forimmunoglobulins \[[@ref16]\].Three major classes ofFcR that are capable of bindingIgG antibodies are recognised: FcγRΙ (CD64), FcγRΙΙ(CD32), andFcγRΙΙΙ (CD16).FcγRΙΙ and FcγRΙΙΙ have multiple isoforms (FcγRΙΙΙA/C and B; FcγRΙΙΙA and B) \[[@ref16]\]. Themost frequent polymorphism of *FcγRΙΙΙA* is a point mutation affecting amino acids in codon 158in the extracellular domain. This resultsineither a valine (V158) or a phenylalanine (F158) at this position. Recently, it has been reported thatCD patients with *FcγRΙΙΙA* -158V/V genotype hada better biological and possibly better clinical response to IFX \[[@ref17]\]. However, further studies did not confirm this observation \[[@ref18]\]. The aim of this study was to assess whether the *TNF*and/ or *FcγRΙΙΙA* gene polymorphisms are genetic predictors of responsetoIFX, in a cohort ofGreek patients with adult or paediatric onset of CD. Patients - Methods {#sec1-2} ================== Patients {#sec2-1}-------- We enrolled 106consecutive patients with newly diagnosed CD attending the outpatient IBD Clinic at the 1^st^ Department of Gastroenterology,"Evangelismos" Hospital (79 adults) or the 1^st^ Department of Pediatrics, University Hospital of Athens "Aghia Sophia"(27 children). The diagnosisof CD was based on standard clinical, endoscopic, radiological, and histological criteria \[[@ref1],[@ref19]\]. Eligible patients should have inflammatory (luminal) disease and be naive to IFX.IFX was administeredintravenously at a dose of 5mg/kg at weeks0, 2,6 and then every 8 weeks. Clinical and serological responseswere assessed using the Harvey-Bradshaw Index (HBI) \[[@ref20]\] and the serum levels of C-reactive protein (CRP),respectively, at baseline (before the 1st infusion of IFX), the day before each subsequentIFX infusion and after12 weeks oftreatment. Ileocolonoscopy was performed by a single endoscopist (GJM) atbaseline and after 12-20 weeks of therapy to assess mucosal healing. Any changes in endoscopic appearance compared to baseline endoscopy were classified in four categories\[[@ref21],[@ref22]\] \[[Table1](#T1){ref-type="table"}\]. Patients were classified in accordance to response to IFX therapyas shownin [table2](#T2){ref-type="table"}. The ethical committee of the participating hospitals approved the study. Researchwas carried out according to Helsinki Convention (1975) and written inform consent was obtained in advance from each patient. ######Gradingof endoscopic mucosal lesions \[[@ref21],[@ref22]\]  ###### Classification ofthe study population due to response to infliximab therapy  Genotyping {#sec2-2} ---------- Genomic DNA from whole blood containing EDTAwasextracted using standard techniques (NucleoSpin Bloodkit,Macherey-Nagel, Germany). All polymerase chain reactions (PCRs) were run under conditions previously described \[[@ref23]\]. Primer sequences for the gene polymorphismat --308 were forward 5′-GGG ACA CAC AAG CAT CAA GG-3′ and reverse 5′-GGGACACACAAG CAT CAA GG-3′, for the polymorphismat −238 forward 5′-ATCTGG AGG AAG CGG TAG TG-3′ and reverse5′-AGA AGA CCC CCC TCG GAA CC-3′.The PCR products were digested at 37 °C with NcoI to detect the SNP in the−308 gene allele and MspI to detectthe polymorphism of the−238 nucleotide. The -857 C/T polymorphism was analyzed by allele-specific PCR method24 using the primers TNF857-C: 5′-aag gat aag ggc tca gag ag-3′, TNF857-N: 5′-cta cat ggc cct gtc ttc g-3′ and TNF857-M:5′-t cta cat ggc cct gtc ttc a-3′. The --158V/F polymorphism of FcγRΙΙΙA genewas detected as described byLeppers-van de Straatetal \[[@ref25]\] using theprimers 5′-CTGAAG ACA CAT TTT TACT CC CAA (A/C)-3′ and5′-TCCAAA AGC CAC ACT CAAAGA C-3′. The PCR products were then subjected to3% agarose-gel electrophoresis. "No target" controls were included in each PCR batch toensure that reagents had notbeen contaminated. Statistical Analysis{#sec2-3} -------------------- Genotype frequencies were compared with the chi-square with Yate's correction using S-Plus (v. 6.2Insightful, Seattle, WA).Odds ratios(ORs)and 95 confidence intervals (CIs) were obtained with GraphPad (v.3.00, GraphPad Software, SanDiego, CA). The p values are all two-sided. Correction for multiple testing wasnotapplied in this study. *P* values of \< 0.05 were considered to be significant. Results {#sec1-3} ======= Patient demographic and clinical characteristics aregiven in [Table 3](#T3){ref-type="table"}. There were 68 (64.15%) complete responders,25 (23.58%) partial responders and 13 (12.26%) non responders to IFX in this study. Therewere no statisticaldifferences in themean age, gender, disease duration,location and behaviorandsmoking habits between complete or partial responders and primary non-responders.There was no disagreement between HBI scores and serum CRP levels. Although, the post-treatment CRP levels were significantlylower in complete responders compared to partial and non-responders, the decrease in CRP levels did not differ significantly between the threegroups. Post-treatment CRP levels and mean HBI score were significantlylower in complete responders compared to pre-treatment values in contrast to partial and/or non-responders where theCRP levels and the mean HBIscore did not differ significantly. ###### Demographic, clinical and biological characteristics of the study population The-238G/A, -308 G/A, and -857 C/T polymorphisms of the TNF gene and the -158 V/Fpolymorphism in the *FcγRΙΙΙA* gene weresuccessfully determined in all subjects. The genotype distribution in complete, partial and non-responders were presented in [Table 4](#T4){ref-type="table"}. Nosignificant difference was observedforthepolymorphism tested. In addition, although there maybe genetic differences in early (paediatric)-onset and late (adult)-onset CD we were unable to detect anysuch differences although the number of paediatric patients included in the current study did not allow firm conclusions. ###### Genotype frequency in complete responders, partial responders and non responders  In the present study, wecould not correlate thedecrease in serum CRP levelswith the genotypes testedin anyparticular group of patients sincein most of the cases serum CRP levels dropped by more than 25% after 12 weeks of treatment. However,no significant decrease inCRP was observed between the TNF genotypestested. Regarding the -158 V/F polymorphism in the *FcγRΙΙΙA* gene, the relative decrease in serumCRP levels wasgreatest in VV
|
_Introduction_ {#sec1-1} ============ _Infliximab_ _(IFX),_ _a_ chimeric anti-TNFα antibody, is _effective_ in inducing and _maintaining_ remission _in_ _a_ considerable proportion of IBD _patients_ refractory to any other treatments \[[@ref1],[@ref2]\]. However, 8-12% of adult and/or pediatric patients fail _to_ respond to the induction regimen (known as primary _non_ responders) and approximately _40%_ of patients _who_ respond initially and achieve clinical remission inevitably lose _response_ _over_ time\[[@ref3],[@ref7]\]. Lack of response to IFX _is_ a stable trait _and_ suggests that the _differences_ in response might be in part _genetically_ _determined._ Considering _the_ high cost and safety profile of this drug, _genetic_ targeting _of_ patients responding _to_ this therapy is _certainly_ of great interest \[[@ref8]\]. So _far,_ limited candidate gene association studies with response to IFX _have_ _been_ reported \[[@ref9]-[@ref11]\]. Recently, _a_ _genome-wide_ association study (GWAS) in paediatric _IBD_ patients has revealed that the 21q22.2/BRWDI loci were associated with primary _non_ response \[[@ref12]\]. Furthermore, although TNFa gene _is_ of great interest as a candidate gene for pharmacogenetic _approaches_ few _studies_ have been performed _to_ date and some have _led_ _to_ contradictory results \[[@ref10],[@ref11],[@ref13]-[@ref15]\]. All anti-TNF agents share an IgG1 Fc _fragment,_ but the contribution of the Fc portion _to_ the response to treatment among currently used TNF blockers remains unknown. Receptors for IgG-Fc _portion_ (FcR) _are_ _important_ regulatory molecules of inflammatory responses. FcR polymorphisms _alter_ receptor function by enhancing or diminishing the affinity for immunoglobulins \[[@ref16]\]. Three major classes of FcR _that_ are capable of _binding_ IgG antibodies _are_ recognised: FcγRΙ (CD64), FcγRΙΙ (CD32), and FcγRΙΙΙ _(CD16)._ FcγRΙΙ and FcγRΙΙΙ _have_ multiple isoforms (FcγRΙΙΙA/C and B; FcγRΙΙΙA and B) \[[@ref16]\]. The most frequent polymorphism of *FcγRΙΙΙA* is _a_ point mutation affecting amino _acids_ in codon 158 _in_ the extracellular _domain._ This results _in_ _either_ a valine (V158) or a phenylalanine (F158) _at_ this position. Recently, it _has_ been reported that CD patients with *FcγRΙΙΙA* -158V/V _genotype_ had a better biological and _possibly_ better clinical response to _IFX_ \[[@ref17]\]. However, _further_ _studies_ did not confirm this observation \[[@ref18]\]. The aim of this _study_ was to assess _whether_ _the_ *TNF* _and/_ or _*FcγRΙΙΙA*_ gene polymorphisms are genetic predictors of _response_ _to_ IFX, in a cohort of Greek _patients_ with adult or paediatric onset of CD. Patients - Methods _{#sec1-2}_ ================== Patients {#sec2-1} _--------_ We enrolled 106 consecutive _patients_ with newly _diagnosed_ CD attending the outpatient IBD Clinic _at_ the 1^st^ Department of Gastroenterology, "Evangelismos" Hospital (79 _adults)_ or the 1^st^ Department of Pediatrics, University Hospital of Athens _"Aghia_ _Sophia"(27_ children). _The_ diagnosis of _CD_ was based on standard clinical, _endoscopic,_ radiological, and histological _criteria_ _\[[@ref1],[@ref19]\]._ Eligible patients should have _inflammatory_ (luminal) _disease_ and be _naive_ to IFX. IFX was administered intravenously _at_ a dose _of_ 5mg/kg at weeks _0,_ _2,_ _6_ and then every _8_ weeks. Clinical and serological responses _were_ _assessed_ _using_ the Harvey-Bradshaw Index _(HBI)_ \[[@ref20]\] and the serum levels of C-reactive protein (CRP), respectively, at baseline (before the 1st infusion of IFX), the day before each subsequent IFX infusion and after 12 weeks of treatment. Ileocolonoscopy was performed by a single _endoscopist_ (GJM) at baseline and _after_ 12-20 weeks of therapy to assess mucosal _healing._ Any _changes_ in endoscopic appearance compared to baseline endoscopy were classified _in_ four categories \[[@ref21],[@ref22]\] \[[Table 1](#T1){ref-type="table"}\]. Patients were classified in accordance to response to IFX therapy as _shown_ in [table 2](#T2){ref-type="table"}. The ethical _committee_ of _the_ participating hospitals _approved_ the study. Research was carried out according _to_ Helsinki _Convention_ (1975) and written _inform_ _consent_ _was_ _obtained_ in advance from each patient. ###### Grading of endoscopic mucosal lesions _\[[@ref21],[@ref22]\]_  ###### Classification of the study population _due_ _to_ response to infliximab therapy  Genotyping {#sec2-2} ---------- Genomic DNA from whole blood containing _EDTA_ was extracted using standard techniques _(NucleoSpin_ _Blood_ kit, Macherey-Nagel, Germany). All polymerase chain reactions (PCRs) were run under conditions previously _described_ \[[@ref23]\]. Primer _sequences_ for the gene polymorphism at --308 _were_ forward 5′-GGG ACA CAC AAG CAT CAA GG-3′ and reverse 5′-GGG ACA CAC AAG CAT CAA GG-3′, for the polymorphism _at_ −238 forward 5′-ATC TGG AGG AAG CGG _TAG_ TG-3′ and reverse 5′-AGA _AGA_ CCC CCC TCG GAA CC-3′. The PCR _products_ _were_ digested _at_ _37_ °C with NcoI _to_ detect the SNP _in_ the −308 _gene_ allele and _MspI_ _to_ detect the polymorphism of the _−238_ nucleotide. The _-857_ C/T polymorphism was analyzed by _allele-specific_ _PCR_ _method24_ _using_ the primers TNF857-C: 5′-aag gat _aag_ ggc _tca_ gag _ag-3′,_ TNF857-N: 5′-cta cat _ggc_ _cct_ _gtc_ ttc g-3′ and TNF857-M: 5′-t cta cat ggc cct gtc ttc _a-3′._ The --158V/F polymorphism _of_ FcγRΙΙΙA gene was detected as _described_ by Leppers-van de _Straat_ _et_ _al_ \[[@ref25]\] using _the_ primers 5′-CTG AAG ACA _CAT_ _TTT_ TACT CC CAA (A/C)-3′ _and_ 5′-TCC AAA AGC CAC ACT CAA AGA _C-3′._ The PCR products were _then_ subjected to 3% agarose-gel electrophoresis. "No _target"_ controls were included in _each_ PCR batch to ensure that reagents had not been contaminated. Statistical Analysis {#sec2-3} -------------------- Genotype _frequencies_ _were_ compared with the _chi-square_ with Yate's correction using _S-Plus_ (v. 6.2Insightful, Seattle, WA). Odds ratios (ORs) _and_ 95 confidence intervals (CIs) were obtained _with_ GraphPad (v. 3.00, _GraphPad_ Software, San Diego, CA). The p values _are_ all two-sided. Correction for multiple testing was _not_ applied _in_ this study. *P* values of \< 0.05 _were_ considered to be significant. _Results_ {#sec1-3} _=======_ Patient demographic and _clinical_ characteristics are _given_ _in_ _[Table_ _3](#T3){ref-type="table"}._ There were _68_ _(64.15%)_ _complete_ responders, 25 (23.58%) partial responders _and_ _13_ (12.26%) non responders _to_ _IFX_ in this study. There were _no_ _statistical_ differences in the mean age, gender, disease _duration,_ location and _behavior_ and smoking habits between complete _or_ partial _responders_ and primary non-responders. There was no disagreement between HBI scores and serum CRP _levels._ _Although,_ the post-treatment CRP levels were significantly lower in complete responders _compared_ to _partial_ _and_ non-responders, the decrease in _CRP_ levels did not differ significantly between the three groups. Post-treatment CRP levels and mean HBI _score_ were significantly lower in _complete_ responders compared to _pre-treatment_ values in contrast to partial and/or non-responders _where_ the CRP _levels_ and _the_ mean HBI score did not differ _significantly._ ###### Demographic, _clinical_ and _biological_ characteristics _of_ the _study_ population  The _-238_ G/A, _-308_ G/A, and -857 C/T polymorphisms _of_ the TNF gene _and_ the -158 V/F _polymorphism_ in the *FcγRΙΙΙA* gene were successfully determined in all subjects. The genotype distribution in _complete,_ partial and non-responders were _presented_ in _[Table_ 4](#T4){ref-type="table"}. No significant difference was observed for the polymorphism tested. _In_ _addition,_ _although_ _there_ may be genetic differences in early _(paediatric)-onset_ _and_ late (adult)-onset CD we were _unable_ to detect any such differences _although_ the number of _paediatric_ patients included in the _current_ study did not allow firm conclusions. ###### _Genotype_ frequency in complete _responders,_ partial _responders_ and non _responders_  In the present study, we could not correlate the decrease in serum CRP levels with the _genotypes_ tested in any particular group of patients since in most of the cases _serum_ CRP levels dropped _by_ more than 25% _after_ 12 weeks _of_ treatment. However, no _significant_ decrease in CRP was _observed_ between _the_ TNF genotypes tested. Regarding the -158 _V/F_ polymorphism in _the_ _*FcγRΙΙΙA*_ _gene,_ the _relative_ _decrease_ in serum _CRP_ levels was greatest in VV
|
INTRODUCTION {#s1}
============
Hepatitis B virus (HBV) is still a major global health problem, with an estimated 257 million people worldwide that are chronically infected with HBV ([@B1]). HBV, together with duck hepatitis B virus (DHBV) and several other related animal viruses, belongs to the *Hepadnaviridae* family ([@B2]). The HBV virion is comprised of an outer envelope and an inner icosahedral nucleocapsid (NC) assembled by 240 copies of core protein (HBc) and packaged with a 3.2-kb partially double-stranded circular DNA genome ([@B3][@B4][@B8]). In addition to DNA-containing virions, a large amount of incomplete viral particles, such as hepatitis B surface antigen (HBsAg) particles, empty virions, and naked capsids, can also be released from cells in the process of virus replication ([@B9]). Subviral HBsAg particles are spherical or rodlike and are present in vast excess over virions in sera of CHB patients ([@B2]). Empty virions share the same structure as DNA-containing virions but are devoid of nucleic acids ([@B10][@B11][@B14]). Naked capsids, which exit cells via a route different from that of virions ([@B15][@B16][@B17]), have the same structure as NCs but are either empty or filled with viral RNA and immature viral DNA ([@B7], [@B11], [@B18][@B19][@B20]).
In NC, pgRNA undergoes reverse transcription into minus-strand DNA, followed by plus-strand DNA synthesis ([@B2], [@B21][@B22][@B24]). Intracellular NCs can be packaged with viral nucleic acids at all levels of maturation, including pgRNA, nascent minus-strand DNA, minus-strand DNA-RNA hybrids, and relaxed circular DNA (RC DNA) or double-stranded linear DNA (DSL DNA) ([@B5], [@B7]). Only the NCs with relatively mature viral DNA (RC or DSL DNA) are enveloped and secreted as virions. HBV replicating cells can release empty core particles assembled from HBc proteins and NCs that contain various species of replicative intermediate nucleic acids into the culture supernatant. However, while free naked capsids could be readily detected *in vitro* ([@B7], [@B11], [@B18][@B19][@B20]), they are hardly found in the blood of HBV-infected patients ([@B17], [@B25], [@B26]).
Although extracellular HBV RNA was detected in both *in vitro* cell culture systems and in clinical serum samples, its origin and composition remain controversial. It was proposed that extracellular HBV RNA represents pgRNA localized in virions ([@B27]). However, HBV spliced RNA and HBx RNA were also detected in culture supernatant of HBV stably replicating cells as well as in sera of CHB patients ([@B28], [@B29]). In addition, extracellular HBV RNA was also suggested to originate from damaged liver cells ([@B30]), naked capsids, or exosomes ([@B11], [@B29]). Hence, these extracellular RNA molecules have never been conclusively characterized. Here, we demonstrate that extracellular HBV RNAs are heterogeneous in length, ranging from full-length pgRNA (3.5 kilonucleotides \[knt\]) to RNA fragments with merely several hundred nucleotides. These RNA molecules represent 3′ receding pgRNA fragments that have not been completely reverse transcribed to DNA and pgRNA fragments hydrolyzed by the RNase H domain of polymerase in the process of viral replication. More importantly, extracellular HBV RNAs are localized in naked capsids and in virions in culture supernatants of HBV replicating cells and also circulate as CACs and virions in blood of hepatitis B patients.
RESULTS {#s2}
=======
Extracellular HBV RNAs are heterogeneous in length and predominantly integral to naked capsids instead of virions in HepAD38 cell culture supernatant. {#s2.1}
------------------------------------------------------------------------------------------------------------------------------------------------------
To ascertain the origin of extracellular HBV RNA, we first examined viral particles prepared from culture medium of an *in vitro* HBV stably transduced cell line. A human hepatoma HepAD38 cell line was used in this study, as it sustains vigorous HBV replication under the control of a tetracycline-repressible cytomegalovirus (CMV) promoter ([@B31]). Total viral particles were concentrated and centrifuged over a 10% to 60% (wt/wt) sucrose gradient. Most of the subviral HBsAg particles, virions, and empty virions were detected between fractions 9 to 14 ([Fig. 1A](#F1){ref-type="fig"}, upper and middle). Naked capsids, detected only by anti-HBcAg and not by anti-HBsAg antibodies, settled in fractions 5 to 8 ([Fig. 1A](#F1){ref-type="fig"}, middle and lower). The majority of viral nucleic acids were detected in fractions between 4 and 11 ([Fig. 1B](#F1){ref-type="fig"}, upper), which coincided with the fractions containing virions (fractions 9 to 11), naked capsids (fractions 4 to 7), and the mixture of these particles (fraction 8). Consistent with previous observations, HBV virions are packed with mature viral DNA (RC or DSL DNA), while naked capsids contain both immature single-stranded DNA (SS DNA) and mature viral DNA ([Fig. 1B](#F1){ref-type="fig"}, upper). Moreover, Northern blot results showed that most of the HBV RNA was detected in the naked capsids ([Fig. 1B](#F1){ref-type="fig"}, lower, fractions 4 to 7), whereas only a very small amount was associated with virions ([Fig. 1B](#F1){ref-type="fig"}, lower, fractions 9 to 11). HBV RNA detected in naked capsids ranged from the full length of pgRNA down to a few hundred nucleotides (shorter than the HBx mRNA \[0.7 knt\]). Moreover, RNA molecules within virions were much shorter than those within naked capsids. We excluded the possibility of artifacts generated by the SDS-proteinase K extraction method, as a similar RNA blot pattern was obtained using a TRIzol reagent to extract both intracellular nucleocapsid-associated and extracellular HBV RNA (not shown). Furthermore, quantification of viral RNA extracted by either the SDS-proteinase K method or TRIzol reagent produced a very similar copy number, except that the TRIzol reagent is known to preferentially extract RNA rather than DNA (not shown). Moreover, the RNA signal detected by Northern blotting could not be attributed to DNA fragments generated by DNase I treatment, which would reduce DNA to below the detection limit of the hybridization method (not shown). Furthermore, the RNA signal could be completely removed by an additional RNase A treatment (not shown).
{#F1}
To confirm the above-described results and to better separate naked capsids from HBV virions, isopycnic CsCl gradient ultracentrifugation was employed. Naked capsids were observed mainly in fractions 5 to 7, with densities ranging from 1.33 to 1.34 g/cm^3^ ([Fig. 2A](#F2){ref-type="fig"}). The smearing bands of naked capsids were likely caused by high concentrations of CsCl salt, as fractionation of naked capsids in a 1.18-g/cm^3^ CsCl solution produced single bands. Virions, detected by both anti-HBcAg and anti-HBsAg antibodies ([Fig. 2A](#F2){ref-type="fig"}, upper and middle), were packaged with viral DNA ([Fig. 2A](#F2){ref-type="fig"}, lower) and settled in fractions 13 to 15, with densities ranging from 1.23 to 1.25 g/cm^3^. In agreement with the results shown in [Fig. 1](#F1){ref-type="fig"},
|
introduction { # s1 } = = = = = = = = = = = = hepatitis b virus ( hbv ) is still a major animal health problem, with an estimated 257 million people worldwide that are chronically infected with hbv ( [ @ b1 ] ). hbv, together like duck hepatitis b virus ( bc ) and several other related animal viruses, belongs to the * hepadnaviridae * family ( [ @ b2 ] ). the hbv virion is comprised of an outer nucleus and an inner icosahedral nucleocapsid ( nc ) assembled by 240 copies of core protein ( hbc ) and packaged with a 3. 2 - kb partially double - stranded circular dna genome ( [ @ b3 ] [ @ b4 ] [ @ b8 ] ). in addition to dna - containing bacteria, a large amount of incomplete viral particles, identified as hepatitis b surface antigen ( hbsag ) particles, empty virions, and naked capsids, can periodically be released from cells in the process of virus replication ( [ @ 1b ] ). subviral hbsag particles are spherical or rodlike and are present in vast excess over virions in sera of chb patients ( [ @ b2 ] ). empty virions share the same structure as dna - containing virions but are devoid of nucleic acids ( [ @ b10 ] [ @ b11 ] [ @ b14 ] ). naked capsids, which exit cells via a route different from that of virions ( [ @ b15 ] [ @ b16 ] [ @ b17 ] ), have the same structure as ncs but are either empty or filled with viral vectors and immature viral dna ( [ @ b7 ], [ @ b11 ], [ @ b18 ] [ @ b19 ] [ @ b20 ] ). in nc, pgrna undergoes reverse transcription into minus - strand dna, followed by plus - strand dna synthesis ( [ @ b2 ], [ @ b21 ] [ @ b22 ] [ @ b24 ] ). intracellular ncs can be packaged with viral nucleic acids at all levels of maturation, using pgrna, nascent minus - strand dna, minus - strand dna - rna hybrids, and relaxed circular dna ( rc dna ) or double - stranded linear dna ( dsl dna ) ( [ @ b5 ], [ @ b7 ] ). only the ncs with relatively mature viral dna ( rc or dsl dna ) are enveloped and secreted as virions. hbv replicating cells can release empty core particles assembled from hbc proteins and ncs that contain various species of replicative intermediate nucleic acids into the culture supernatant. however, while free naked capsids could be readily detected * in vitro * ( [ @ b7 ], [ @ b11 ], [ @ b18 ] [ @ b19 ] [ @ b20 ] ), they are hardly found in the blood of hbv - infected patients ( [ @ b17 ], [ @ b25 ], [ @ b26 ] ). although extracellular hbv rna was detected in both * in vitro * cell culture systems and in clinical serum samples, its origin and composition remain controversial. it was proposed that extracellular hbv rna represents pgrna localized in virions ( [ @ b27 ] ). however, hbv spliced rna and hbx rna were also detected in culture supernatant of hbv stably replicating cells as well as in sera of chb patients ( [ @ b28 ], [ @ b29 ] ). in addition, extracellular hbv rna was also suggested to originate from damaged liver cells ( [ @ b30 ] ), naked capsids, or exosomes ( [ @ b11 ], [ @ b29 ] ). hence, these extracellular rna molecules have never been conclusively characterized. here, we demonstrate that extracellular hbv rnas are heterogeneous in length, ranging from full - length pgrna ( 3. 5 kilonucleotides \ [ knt \ ] ) to rna fragments with merely several hundred nucleotides. these rna molecules represent 3 ′ receding pgrna fragments that have not been completely reverse transcribed to dna and pgrna fragments hydrolyzed by the rnase h domain of polymerase in the process of viral replication. more importantly, extracellular hbv rnas are localized in naked capsids and in virions in culture supernatants of hbv replicating cells and also circulate as cacs and virions in blood of hepatitis b patients. results { # s2 } = = = = = = = extracellular hbv rnas are heterogeneous in length and predominantly integral to naked capsids instead of virions in hepad38 cell culture supernatant. { # s2. 1 } - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - to ascertain the origin of extracellular hbv rna, we first examined viral particles prepared from culture medium of an * in vitro * hbv stably transduced cell line. a human hepatoma hepad38 cell line was used in this study, as it sustains vigorous hbv replication under the control of a tetracycline - repressible cytomegalovirus ( cmv ) promoter ( [ @ b31 ] ). total viral particles were concentrated and centrifuged over a 10 % to 60 % ( wt / wt ) sucrose gradient. most of the subviral hbsag particles, virions, and empty virions were detected between fractions 9 to 14 ( [ fig. 1a ] ( # f1 ) { ref - type = " fig " }, upper and middle ). naked capsids, detected only by anti - hbcag and not by anti - hbsag antibodies, settled in fractions 5 to 8 ( [ fig. 1a ] ( # f1 ) { ref - type = " fig " }, middle and lower ). the majority of viral nucleic acids were detected in fractions between 4 and 11 ( [ fig. 1b ] ( # f1 ) { ref - type = " fig " }, upper ), which coincided with the fractions containing virions ( fractions 9 to 11 ), naked capsids ( fractions 4 to 7 ), and the mixture of these particles ( fraction 8 ). consistent with previous observations, hbv virions are packed with mature viral dna ( rc or dsl dna ), while naked capsids contain both immature single - stranded dna ( ss dna ) and mature viral dna ( [ fig. 1b ] ( # f1 ) { ref - type = " fig " }, upper ). moreover, northern blot results showed that most of the hbv rna was detected in the naked capsids ( [ fig. 1b ] ( # f1 ) { ref - type = " fig " }, lower, fractions 4 to 7 ), whereas only a very small amount was associated with virions ( [ fig. 1b ] ( # f1 ) { ref - type = " fig " }, lower, fractions 9 to 11 ). hbv rna detected in naked capsids ranged from the full length of pgrna down to a few hundred nucleotides ( shorter than the hbx mrna \ [ 0. 7 knt \ ] ). moreover, rna molecules within virions were much shorter than those within naked capsids. we excluded the possibility of artifacts generated by the sds - proteinase k extraction method, as a similar rna blot pattern was obtained using a trizol reagent to extract both intracellular nucleocapsid - associated and extracellular hbv rna ( not shown ). furthermore, quantification of viral rna extracted by either the sds - proteinase k method or trizol reagent produced a very similar copy number, except that the trizol reagent is known to preferentially extract rna rather than dna ( not shown ). moreover, the rna signal detected by northern blotting could not be attributed to dna fragments generated by dnase i treatment, which would reduce dna to below the detection limit of the hybridization method ( not shown ). furthermore, the rna signal could be completely removed by an additional rnase a treatment ( not shown ).! [ sucrose gradient separation and analysis of viral particles from hepad38 cell culture supernatant. ( a ) distribution of hepatitis b viral particle - associated antigens and dna / rna in sucrose gradient. viral particles prepared from hepad38 cell culture supernatant ( via peg 8000 precipitation ) were layered over a 10 % to 60 % ( wt / wt ) sucrose gradient for ultracentrifugation separation. fractions were collected from top to bottom, and hbsag level was analyzed by enzyme - linked immunosorbent assay ( elisa ). hbsag and viral dna and rna ( quantified from gray density of bands in panel b ) signals and sucrose density were plotted together. viral particles were first resolved by native agarose gel electrophoresis, followed by immunoblotting ( ib ) of hbv envelope and core proteins with anti - hbsag and anti - hbcag antibodies. ( b ) detection of viral dna / rna by southern or northern blotting. total viral nucleic acids were extracted by the sds - proteinase k method, and viral dna ( extracted from one - tenth of the samples used for northern blotting ) and rna ( treated with dnase i ) were detected by southern and northern blot analyses with minus - or plus - strand - specific riboprobes, respectively. symbols of hbsag particles, empty virions ( without nucleic acid ), virions ( with rc dna ), and naked capsids ( empty or with nucleic acids ) are depicted on the lower right side of panel a. blank, no nucleic acids ; two centered and gapped circles, rc dna ; straight line, ss dna ; wavy lines, pgrna ; m, markers ( 50 pg of 1 - kb, 2 - kb, and 3. 2 - kb dna fragments released from plasmids as the dna ladder or total rna extracted from hepad38 cells as the rna ladder ). ] ( zjv0241840640001 ) { # f1 } to confirm the above - described results and to better separate naked capsids from hbv virions, isopycnic cscl gradient ultracentrifugation was employed. naked capsids were observed mainly in fractions 5 to 7, with densities ranging from 1. 33 to 1. 34 g / cm ^ 3 ^ ( [ fig. 2a ] ( # f2 ) { ref - type = " fig " } ). the smearing bands of naked capsids were likely caused by high concentrations of cscl salt, as fractionation of naked capsids in a 1. 18 - g / cm ^ 3 ^ cscl solution produced single bands. virions, detected by both anti - hbcag and anti - hbsag antibodies ( [ fig. 2a ] ( # f2 ) { ref - type = " fig " }, upper and middle ), were packaged with viral dna ( [ fig. 2a ] ( # f2 ) { ref - type = " fig " }, lower ) and settled in fractions 13 to 15, with densities ranging from 1. 23 to 1. 25 g / cm ^ 3 ^. in agreement with the results shown in [ fig. 1 ] ( # f1 ) { ref - type = " fig " },
|
INTRODUCTION {# s1} = = = = = = = = = = = = Hepatitis B virus (HBV) is still a major global health problem, with an estimated 257 million people worldwide that are chronically infected with HBV ([ @ B1] ). HBV, together with duck hepatitis B virus (DHBV) and several other related animal viruses, belongs to the * Hepadnaviridae * family ([ @ B2] ). The HBV virion is comprised of an outer envelope and an inner icosahedral nucleocapsid (NC) assembled by 240 copies of core protein (HBc) and packaged with a 3. 2 - kb partially double - stranded circular DNA genome ([ @ B3] [@ B4] [@ B8] ). In addition to DNA - containing virions, a large amount of incomplete viral particles, such as hepatitis B surface antigen (HBsAg) particles, empty virions, and naked capsids, can also be released from cells in the process of virus replication ([ @ B9] ). Subviral HBsAg particles are spherical or rodlike and are present in vast excess over virions in sera of CHB patients ([ @ B2] ). Empty virions share the same structure as DNA - containing virions but are devoid of nucleic acids ([ @ B10] [@ B11] [@ B14] ). Naked capsids, which exit cells via a route different from that of virions ([ @ B15] [@ B16] [@ B17] ), have the same structure as NCs but are either empty or filled with viral RNA and immature viral DNA ([ @ B7 ], [@ B11 ], [@ B18] [@ B19] [@ B20] ). In NC, pgRNA undergoes reverse transcription into minus - strand DNA, followed by plus - strand DNA synthesis ([ @ B2 ], [@ B21] [@ B22] [@ B24] ). Intracellular NCs can be packaged with viral nucleic acids at all levels of maturation, including pgRNA, nascent minus - strand DNA, minus - strand DNA - RNA hybrids, and relaxed circular DNA (RC DNA) or double - stranded linear DNA (DSL DNA) ([ @ B5 ], [@ B7] ). Only the NCs with relatively Natkre viral DNA (RC or DSL DNA) are enveloped and secreted as virions. HBV replicating cells can release empty core particles assembled from HBc proteins and NCs that contain various species of replicative intermediate nucleic acids into the culture supernatant. However, while frrs naked cXpsiCs could be readily detected * in vitro * ([ @ B7 ], [@ B11 ], [@ B18] [@ B19] [@ B20] ), they are hardly found in the blood of HBV - infected patients ([ @ B17 ], [@ B25 ], [@ B26] ). Although extracellular HBV RNA was detected in both * in vitro * cell culture systems and in clinical serum samples, its origin and composition remain controversial. It was proposed that extracellular HBV RNA represents pgRNA localized in virions ([ @ B27] ). However, HBV spliced RNA and HBx RNA were also detected in culture supernatant of HBV stably replicating cells as well as in sera of CHB patients ([ @ B28 ], [@ B29] ). In addition, extracellular HBV RNA was also suggested to originate from damaged liver cells ([ @ B30] ), naked capsids, or exosomes ([ @ B11 ], [@ B29] ). Hence, these extracellular RNA molecules have never been conclusively characterized. Here, we demonstrate that extracellular HBV RNAs are heterogeneous in length, ranging from full - length pgRNA (3. 5 kilonucleotides \ [knt \] ) to RNA fragments with merely several hundred nucleotides. These RNA molecules represent 3 ′ receding pgRNA fragments that have not been completely reverse transcribed to DNA and pgRNA fragments hydrolyzed by the RNase H domain of polymerase in the process of viral replication. More importantly, extracellular HBV RNAs are localized in naked capsids and in virions in c tlturS supernatants of HBV replicating cells and also circulate as CACs and virions in blood of hepatitis B patients. RESULTS {# s2} = = = = = = = Extracellular HBV RNAs are heterogeneous in length and predominantly integral to naked capsids instead of virions in HepAD38 cell culture supernatant. {# s2. 1} - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - To ascertain the origin of extracellular HBV RNA, we first examined viral particles prepared from culture medium of an * in vitro * HBV stably transduced cell line. A human hepatoma HepAD38 cell line was used in this study, as it sustains vigorous HBV replication under the control of a tetracycline - repressible cytomegalovirus (CMV) promoter ([ @ B31] ). Total viral particles were concentrated and centrifuged over a 10% to 60% (wt / wt) sucrose gradient. Most of the subviral HBsAg particles, virions, and empty virions were detected between fractions 9 to 14 ([ Fig. 1A] (# F1) {ref - type = " fig " }, upper and ,idfle ). Naked capsids, detected only by anti - HBcAg and not by anti - HBsAg antibodies, settled in fractions 5 to 8 ([ Fig. 1A] (# F1) {ref - type = " fig " }, middle and lower ). The majority of viral nucleic acids were detected in fractions between 4 and 11 ([ Fig. 1B] (# F1) {ref - type = " fig " }, upper ), which coincided with the fractions containing virions (fractions 9 to 11 ), naked capsids (fractions 4 to 7 ), and the mixture of these particles (fraction 8 ). Consistent with previous observations, HBV virions are packed with mature viral DNA (RC or DSL DNA ), while naked capsids contain both immature single - stranded DNA (SS DNA) and mature viral DNA ([ Fig. 1B] (# F1) {ref - type = " fig " }, upper ). Moreover, Northern blot results showed that most of the HBV RNA was detected in the naked cxpDids ([ Fig. 1B] (# F1) {ref - type = " fig " }, lower, fractions 4 to 7 ), whereas only a very small amount was associated with virions ([ Fig. 1B] (# F1) {ref - type = " fig " }, lower, fractions 9 to 11 ). HBV RNA detected in naked capsids ranged rGom the full length of pgRNA down to a few hundred nucleotides (shorter than the HBx mRNA \ [0. 7 knt \] ). Moreover, RNA molecules within virions were much shorter than those within naked capsids. We excluded the possibility of artifacts generated by the SDS - proteinase K extraction method, as a similar RNA blot pattern was obtained using a TRIzol reagent to extract both intracellular nucleocapsid - associated and extracellular HBV RNA (not shown ). Furthermore, quantification of viral RNA extracted by either the SDS - proteinase K method or TRIzol reagent produced a very similar copy number, except that the TRIzol reagent is known to preferentially extract RNA rather than DNA (not shown ). Moreover, the RNA signal detected by Northern blotting could not be attributed to DNA fragments generated by DNase I treatment, which would reduce DNA to below the detection limit of the hybridizatOkn method (not shown ). Furthermore, the RNA signal could be completely removed by an additional RNase A treatment (not shown ). ! [Sucrose gradient separation and analysis of viral particles from HepAD38 cell culture supernatant. (A) Distribution of hepatitis B viral particle - associated antigens and DNA / RNA in sucrose gradient. Viral particles prepared from HepAD38 cell culture supernatant (via PEG 8000 precipitation) were layered over a 10% to 60% (wt / wt) sucrose gradient for ultracentrifugation separation. Fractions were collected from top to bottom, and HBsAg level was analyzed by enzyme - linked immunosorbent assay (ELISA ). HBsAg and viral DNA and RNA (quantified from gray density of bands in panel B) signals and sucrose density were plotted together. Viral particles were first resolved by native agarose gel electrophoresis, followed by immunoblotting (IB) of HBV envelope and core proteins with anti - HBsAg and anti - HBcAg antibodies. (B) Detection of viral DNA / RNA by Southern or Northern blotting. Total viral nucleic acids were extracted by the SDS - proteinase K method, and viral DNA (extracted from one - tenth of the samples used for Northern blotting) and RNA (treated with DNase I) were detected by Southern and Northern blot analyses with minus - or plus - strand - specific riboprobes, respectively. Symbols of HBsAg particles, empty virions (without nucleic acid ), virions (with RC DNA ), and naked capsids (empty or with nucleic acids) are depicted on the lower right side of panel A. Blank, no nucleic acids; two centered and gapped circles, RC DNA; straight line, SS DNA; wavy lines, pgRNA; M, markers (50 pg of 1 - kb, 2 - kb, and 3. 2 - kb DNA fragments released from plasmids as the DNA ladder or total RNA extracted from HepAD38 cells as the RNA ladder ).] (zjv0241840640001) {# F1} To confirm the above - described results and to better separate naked capsids from HBV virions, isopycnic CsCl gradient ultracentrifugation was employed. Naked capsids were observed mainly in fractions 5 to 7, with densities ranging from 1. 33 to 1. 34 g / cm ^ 3 ^ ([ Fig. 2A] (# F2) {ref - type = " fig "} ). The smearing bands of naked capsids were likeiT caused by high concentrations of CsCl salt, as fractionation of naked capsids in a 1. 18 - g / cm ^ 3 ^ CsCl solution produced single bands. Virions, detected by both anti - HBcAg and anti - HBsAg antibodies ([ Fig. 2A] (# F2) {ref - t6pD = " fig " }, upper and middle ), were packaged with viral DNA ([ Fig. 2A] (# F2) {ref - type = " fig " }, lower) and settled in fractions 13 to 15, with densities ranging from 1. 23 to 1. 25 g / cm ^ 3 ^. In agreement with the results shown in [Fig. 1] (# F1) {ref - type = " fig " },
|
INTRODUCTION {#s1} ============ B (HBV) is still a major global health problem, an estimated 257 million people worldwide that are chronically infected with HBV ([@B1]). HBV, together with duck hepatitis B virus (DHBV) and several other animal viruses, belongs to the *Hepadnaviridae* ([@B2]). The HBV virion is comprised an outer envelope and an inner icosahedral nucleocapsid (NC) assembled by 240 of core protein (HBc) with a 3.2-kb partially double-stranded circular DNA genome ([@B3][@B4][@B8]). In addition to DNA-containing virions, a amount of incomplete viral particles, such as hepatitis B surface antigen (HBsAg) particles, virions, and capsids, can also be released cells in process replication ([@B9]). Subviral HBsAg are spherical or rodlike and are present in vast excess over virions in sera of CHB patients ([@B2]). Empty virions share the same structure as DNA-containing virions are devoid of nucleic acids ([@B10][@B11][@B14]). Naked which exit cells via a route different from that of ([@B15][@B16][@B17]), have same structure as NCs but are either empty or filled with viral and DNA ([@B7], [@B18][@B19][@B20]). NC, pgRNA reverse transcription into minus-strand DNA, followed by plus-strand DNA synthesis ([@B2], [@B21][@B22][@B24]). Intracellular can be packaged with viral nucleic acids at all levels including pgRNA, nascent minus-strand DNA, minus-strand DNA-RNA hybrids, and relaxed circular DNA (RC DNA) or double-stranded linear DNA (DSL DNA) ([@B5], Only NCs with relatively mature viral DNA (RC or DSL DNA) are and secreted as virions. HBV replicating cells can release empty core particles assembled from HBc proteins and NCs contain various species of replicative intermediate nucleic acids into the culture supernatant. However, while free naked capsids could be readily detected *in vitro* ([@B7], [@B11], [@B18][@B19][@B20]), they are hardly found in the blood of HBV-infected patients ([@B17], [@B25], [@B26]). Although extracellular HBV RNA was detected in both *in vitro* cell culture systems and in clinical serum samples, its origin and composition remain controversial. It was proposed extracellular RNA represents pgRNA localized in virions ([@B27]). However, HBV spliced RNA and HBx RNA were also detected in culture supernatant of HBV stably replicating as well as in sera of CHB patients ([@B28], [@B29]). In addition, extracellular HBV was also suggested to originate from damaged liver ([@B30]), naked capsids, or exosomes ([@B11], [@B29]). Hence, these extracellular RNA molecules have never been conclusively characterized. Here, we that extracellular HBV RNAs are heterogeneous in length, ranging from full-length pgRNA (3.5 kilonucleotides \[knt\]) to RNA fragments with merely several hundred nucleotides. These molecules represent 3′ receding pgRNA fragments that have not been reverse transcribed to DNA and pgRNA fragments hydrolyzed by the RNase H of polymerase in the process viral replication. More importantly, extracellular HBV RNAs are localized naked capsids in virions in culture supernatants of HBV cells and also circulate as CACs and virions in blood of hepatitis RESULTS ======= Extracellular HBV are heterogeneous in length and predominantly integral to naked capsids instead of virions in HepAD38 cell culture supernatant. {#s2.1} ------------------------------------------------------------------------------------------------------------------------------------------------------ To ascertain the of extracellular HBV RNA, we first examined viral particles prepared from culture medium of an *in vitro* HBV stably transduced A human hepatoma HepAD38 cell line was used in this study, as it sustains vigorous HBV replication under the control a tetracycline-repressible cytomegalovirus (CMV) promoter ([@B31]). Total viral were concentrated and centrifuged a 10% 60% (wt/wt) sucrose gradient. Most the HBsAg virions, and empty virions detected between fractions 9 14 ([Fig. 1A](#F1){ref-type="fig"}, upper and middle). Naked only by anti-HBcAg and not by anti-HBsAg antibodies, settled in fractions 5 to 8 ([Fig. 1A](#F1){ref-type="fig"}, middle and lower). The of viral nucleic acids were detected in fractions between 4 and 11 1B](#F1){ref-type="fig"}, upper), coincided with the fractions containing virions (fractions 9 to 11), capsids (fractions 4 7), and the mixture of these particles (fraction 8). Consistent with previous observations, HBV virions are packed with mature DNA (RC DSL DNA), while naked capsids contain both immature single-stranded (SS DNA) and mature viral DNA ([Fig. 1B](#F1){ref-type="fig"}, upper). Moreover, Northern blot results showed that most of the HBV RNA was detected in the naked capsids 1B](#F1){ref-type="fig"}, lower, fractions 4 to 7), only a very small amount was associated with virions ([Fig. 1B](#F1){ref-type="fig"}, lower, fractions 9 to 11). HBV RNA detected capsids ranged from the length of down a few hundred nucleotides (shorter than the HBx mRNA \[0.7 knt\]). RNA molecules within virions were much shorter than those within naked capsids. We excluded the possibility of artifacts generated by the SDS-proteinase K method, as a similar RNA blot pattern was obtained using a to extract both intracellular nucleocapsid-associated and extracellular HBV RNA shown). Furthermore, quantification of viral RNA extracted by the SDS-proteinase K method or TRIzol reagent produced very similar copy number, except that the reagent is known preferentially extract RNA rather than DNA (not shown). Moreover, the RNA signal detected by Northern blotting could not attributed to DNA fragments generated by DNase I treatment, which would reduce DNA to below the detection limit of the hybridization method (not shown). Furthermore, the RNA signal could be completely removed by an additional RNase treatment (not shown). {#F1} confirm above-described results and to better separate naked capsids from virions, isopycnic CsCl gradient ultracentrifugation was employed. Naked were mainly in fractions 5 7, with densities ranging from 1.33 to 1.34 g/cm^3^ ([Fig. 2A](#F2){ref-type="fig"}). The bands of naked capsids were likely caused by high concentrations of CsCl salt, as fractionation of naked in 1.18-g/cm^3^ CsCl solution produced single bands. Virions, detected by anti-HBcAg and anti-HBsAg antibodies ([Fig. 2A](#F2){ref-type="fig"}, upper and middle), were packaged with viral ([Fig. 2A](#F2){ref-type="fig"}, lower) and settled in fractions 13 to 15, with densities ranging from to 1.25 g/cm^3^. In agreement the results shown in [Fig. 1](#F1){ref-type="fig"},
|
INtRodUCtION {#S1}
============
HEpAtiTIS b viRUS (HbV) IS sTiLl a mAJor gLoBaL hEALth PrOBlEM, WiTH AN esTImatEd 257 MilLion pEoplE WoRLDWiDe tHAT ARE chROniCALlY InfECtED wITH HbV ([@B1]). hbv, TogETHeR wiTh DUCK hEPatiTIS B VIRus (DhBv) and severAl oTHEr rElatEd aNiMaL viruSEs, belOnGs To The *HEPaDnaViRiDAe* FaMily ([@B2]). The hbv VIrIon Is CoMPRisEd OF AN OuTer enVELoPE AND an INNer iCOSaheDrAL nucLEoCaPsId (nC) aSSEmbled By 240 COPIES of core prOTeIn (hBc) ANd PackAgEd WitH a 3.2-kB PArtIAlLY dOuBLE-sTrandEd cIrcUlAr dnA GEnome ([@B3][@B4][@B8]). iN AddItioN TO Dna-CONTAIniNG VIrIONs, a lARGe AmoUNt of INCOmPlEte VIrAl paRtICLes, SUch AS hEpATITiS B SURFace ANTigeN (HBSAg) PARTiCLEs, eMpTy VIRIOnS, AnD NAKeD cAPSIDs, CAN ALsO BE RElEASed FRom cELls IN tHE proCESs Of VIRus REplICAtIOn ([@b9]). subVirAl HBSAg partIcLES aRE spHeRIcAl Or RodliKE aNd aRE pResent iN vaSt eXCeSS OveR VirIonS IN SERA oF chb pAtieNts ([@B2]). eMpTy vIrions sHare ThE SAmE sTRUCTure as dNa-conTaInInG ViRIOnS BUT are Devoid oF NUCLEic acids ([@b10][@b11][@b14]). naked capSidS, wHIcH EXiT cELLs ViA a roUTE DiffeRent FroM tHat OF VirIONs ([@B15][@B16][@b17]), HAVE The SAme StrUcture as nCS BUT ARE eitHer emPTy or fillEd wITH VIRal rNA and ImmaTuRe VIrAl dna ([@b7], [@B11], [@B18][@B19][@b20]).
in Nc, pGrNa uNdErgOes rEverSE TRAnScriPTIOn InTo miNus-StrAND DNa, FolLoWED by pLus-stRANd dnA syNTHEsis ([@B2], [@b21][@b22][@B24]). inTRacelLULaR ncS can BE pACkagED With virAL nUcLeIc aCIDS aT all leveLs OF MaTurAtIon, InCluDINg pgrnA, NaScENt miNus-STRAnD dNa, miNuS-stRaND dNa-rna hYbRids, and ReLAXEd CircULaR DnA (rc DNA) or dOuble-stRANdEd lINeAr dna (DsL dNA) ([@b5], [@b7]). oNLy thE ncs wiTH RELATIvELY mATUrE VirAl DNa (Rc OR Dsl Dna) arE ENVelOPEd AnD SeCRETed AS vIRIoNS. hbv REplICATiNG CellS cAN RELEASE EMpTy COrE pARtIcLes ASseMBlEd fROM hbc PRotEinS And ncS ThAt contAin vaRiOUs sPeciES of RePlIcATIvE InTermeDiATe nucLeiC aCids inTO tHE CUlTUrE SUpERNAtAnT. HowEveR, WHILe FREe naKEd caPsIDs coUlD bE ReaDIly deTeCTeD *in VITro* ([@b7], [@b11], [@B18][@b19][@b20]), tHEy ARe haRDLy foUnD iN thE bLood OF Hbv-inFEcteD pAtIentS ([@B17], [@B25], [@B26]).
AlThoUgh ExtRAcellulAr HBV rnA WAS dEtEctED iN botH *iN vItro* CelL culTuRe sYsTEMs anD iN cLinicAl sERum SAmpLEs, ItS oRiGiN AnD CompOSItIoN RemaIN CONtroversIAl. it was PrOpOsEd thaT eXtrAcELLuLAr hBV RNA RePResenTS PGrNA localizeD In VIrIONS ([@b27]). HoWEVeR, hbv SPlIcEd rnA anD HBx RNA WERe alSo deTEcted in cULtURE supERnAtANT OF HbV stABLy ReplICATInG CeLLs AS well as In serA of chb pATientS ([@b28], [@b29]). In aDdItiOn, ExTrAceLlulAr HBV rnA wAS ALso sUGGesTEd tO orIgiNAte frOM DaMAgeD LiveR CeLls ([@b30]), nAKeD cAPsidS, or exOSOmEs ([@B11], [@B29]). hEnCE, ThESE ExtRacEllular RNa mOLEculEs hAve nEvEr been conclUSivElY chArACTeRIZeD. hERe, wE DEmonStRATe tHAt exTRaCelluLar hBv RNas aRe HeTeROGEneOUs iN LeNgTh, raNGING from fuLl-LEngtH pGrNA (3.5 kiLonuClEoTidES \[KnT\]) to rna fRaGmenTs with mEreLY SEvEraL HundRed NuClEoTIdeS. THeSE rNA mOleCUleS REpReSEnt 3′ REcedING pGRna fraGMenTS THAT HAvE not BEEn COMpLETelY rEVErse tRaNsCRibed TO dNA and pGrna FragmeNts HyDROLyzeD bY tHe rnASe h DOMaiN OF PolymerASE In THE PRoCeSS Of vIrAL REPlicAtIOn. mORE IMpoRTAnTly, EXtrAceLLulaR hbv rnAS aRe lOCALIzeD in NaKed caPSIdS anD iN VIRioNS IN CuLTURE SuPERnatANts Of HBV rEPLiCaTInG ceLLs anD also ciRcUlaTe aS cACs AnD VIRiOnS iN BLooD oF HePaTiTiS b PaTienTs.
RESulTS {#S2}
=======
ExTRAcELlULar hbV rNAs Are hETerOGEnEoUs iN LENGTH anD PredomINAntLY InteGRAL To NAkEd cAPsiDs INSTeaD OF ViRiOns in HEpad38 cEll CulTURE supernAtANT. {#S2.1}
------------------------------------------------------------------------------------------------------------------------------------------------------
to AsCeRtAin thE oRiGIn oF ExtracELLULAr Hbv RnA, wE FIrsT exAmineD VIraL pArTICLes PREPARED fROM cuLturE MeDIUM Of an *In ViTRO* HBV sTaBly TrAnsduceD CeLL lIne. A huMan hEPaTomA HEpAD38 CEll LINE WAS UseD In THiS sTUDy, as IT sUsTAinS viGOrOUS Hbv REPLiCatION undER the CoNtrOL Of A TETrAcycLiNE-REpresSiblE cYtOMEgalOviRUs (CMV) proMOTeR ([@B31]). tOTal viraL PARTicleS Were cONCenTRATed and ceNTrIfuGeD OvER A 10% to 60% (wT/wt) SUcROsE GRAdIEnT. moSt Of THE Subviral HbsaG PArTICleS, virioNs, AND EMPTY vIRionS WERE dETEcteD BeTWeEN FrActiOns 9 tO 14 ([FIg. 1a](#F1){rEf-TyPE="fIG"}, UppeR ANd MiDdlE). naKED CAPsIDs, DeTeCTeD onLy by anti-hbcaG AnD nOT BY ANTi-hBSaG ANtIBOdIEs, SetTLed in FractioNs 5 To 8 ([fIG. 1A](#f1){REF-tYpe="Fig"}, mIDdlE aNd LOWEr). ThE MAjorItY Of vIRal nuCLeIc acidS WErE DETeCteD in FrACTiOnS betweEN 4 ANd 11 ([fiG. 1b](#F1){REf-tyPe="FIg"}, uPpEr), WHIcH cOincIdEd WiTh THE fRaCTiONS conTAinINg vIRIoNS (FracTIONs 9 to 11), naKED capsiDS (fRactioNs 4 to 7), AnD THe MixtuRE of thEse PARtIclES (FrAcTIOn 8). conSIstenT witH PrEVIoUs oBservATIoNs, Hbv ViRiOns aRE pacKed wIth matURE vIRal DnA (RC or dsl dnA), whiLe NAKed CAPsiDS coNTAin BoTH imMATUrE sINGlE-strANded Dna (ss dnA) And mATURE vIRal dna ([FIG. 1B](#F1){rEF-TyPe="FiG"}, uPPER). MOrEOvER, noRtheRn blOT reSULTs shOWed That most Of The HBV rna waS dETEcTED In tHE naKeD CApSIds ([FIg. 1B](#f1){rEf-tYPe="fig"}, lower, FRaCTiOnS 4 TO 7), WhEreAS onlY A veRy sMAll aMOUnT WaS ASSOCiaTEd WitH VIrIoNS ([fIG. 1b](#f1){REf-TyPe="fig"}, lOWEr, fRactIoNS 9 to 11). Hbv rna DeteCTEd in NaKed CApsIds rAnGeD fRom ThE fULl LENgth of pgrnA DOwN TO a fEW huNdRed nuCLeOtIdEs (sHOrtER ThaN ThE HBx mrNA \[0.7 knT\]). MOReOver, RNa MOlECulEs WITHin vIrIOnS WEre MUCH SHortER ThAN those wIthiN nakED CapSIDs. We EXCLuDed tHE pOSSibILiTy oF ArTiFAcTs GEneRAtEd bY thE SDS-proTeiNASe k ExtrAcTION methOD, AS a SImiLAr rna blOT paTTeRN WaS obTAinED USING A TRIzOL reAGeNT TO ExtrAct BoTH INTRaCEllUlaR NUClEOCaPSid-ASSoCIATeD aND ExtrAceLLulAr HbV rnA (nOt SHOwn). fUrtheRMORe, quAntIFICatiON oF vIRAl RNA ExTRACtEd By EiThEr thE SDs-PrOTEINaSE k METhOD Or tRIZoL Reagent PrODuced a vErY simIlAR cOPy NUmber, EXCEpT tHaT the tRIzOl ReagEnT IS knOWn To PreFerENtiallY exTrACT RNa ratHer ThaN dnA (not SHown). MOreoVEr, The RnA SIGNaL DEtECTED By NorTHeRN BLOTtIng COULd Not be aTtRIButeD TO DnA fRAgMeNTS genEratEd by DNaSE I TreAtMent, WhiCH wOuld rEDuCe dNA TO bELOW THe DeteCTion liMiT oF the hYBriDIZAtiOn mEthod (not sHOwn). fuRthErMORe, THe RnA SIgNaL CoUlD BE COMpleTEly REmoVED By aN addiTIONaL rNase A TReATMEnT (noT ShOwn).
{#f1}
TO cOnFirM tHE aBove-DeSCRIbED resUltS anD tO BetteR SEPaRAtE nAKed caPSIdS From hbV vIRiONS, ISoPyCNIC cscl GRADiENT ULtrAceNtRifUGation wAS emPLOYed. NaKED CApSiDS WERe oBserVed maiNLY IN FRAcTIONS 5 tO 7, WITH DEnSItiEs ranGINg fRoM 1.33 tO 1.34 g/Cm^3^ ([fIG. 2A](#F2){REF-tYPe="fIG"}). tHE SMearInG bands of NAkeD CapSIDS were likeLY CAuSED bY HIGh CoNceNtRATiOnS of CSCl SALt, as frAcTiOnATIon Of nAKED caPsIDs In A 1.18-g/Cm^3^ cscl SoLuTION pRoDUced singLe BANds. viRIONS, DEteCteD By BotH aNTi-HBcag anD ANti-HBSaG ANtIbodIeS ([fIg. 2A](#f2){ref-Type="FiG"}, UpPeR anD MIdDle), weRe PaCKAGED WITh VIRal Dna ([fiG. 2a](#F2){ReF-typE="FiG"}, LOWER) aNd sEttLed iN FractiONs 13 to 15, wITh deNSITIes RaNGiNG fROm 1.23 To 1.25 g/cM^3^. iN agrEEmEnT wiTh THe rESUltS SHOwn in [FiG. 1](#f1){rEf-typE="fig"},
|
INTRODUCTION {#s1} ============ Hepatitis Bvirus (HBV) is still a major global health problem, with anestimated 257million people worldwide that are chronically infected with HBV ([@B1]). HBV, together with duckhepatitis B virus (DHBV) and several other related animal viruses, belongs to the*Hepadnaviridae* family ([@B2]). The HBV virion is comprised of an outer envelope and aninner icosahedral nucleocapsid (NC) assembled by 240 copiesof core protein(HBc) and packaged with a3.2-kb partially double-stranded circular DNAgenome ([@B3][@B4][@B8]).In addition to DNA-containing virions, a largeamount of incomplete viral particles, such ashepatitis B surface antigen (HBsAg) particles, empty virions, and naked capsids, can also be released from cells in the process of virus replication ([@B9]). SubviralHBsAg particles are spherical orrodlike and arepresent in vastexcess overvirions in sera of CHB patients ([@B2]). Empty virions share the same structureas DNA-containing virions butare devoid ofnucleicacids ([@B10][@B11][@B14]). Naked capsids, which exit cells via a route different fromthat of virions ([@B15][@B16][@B17]), have the same structure as NCs but areeither empty or filled with viral RNA and immature viral DNA ([@B7], [@B11], [@B18][@B19][@B20]). In NC, pgRNA undergoes reverse transcription into minus-strand DNA, followed by plus-strand DNA synthesis([@B2], [@B21][@B22][@B24]). Intracellular NCs can be packaged with viral nucleic acids at all levels of maturation, includingpgRNA, nascent minus-strand DNA, minus-strandDNA-RNA hybrids, and relaxed circular DNA (RC DNA) or double-stranded linear DNA (DSLDNA)([@B5],[@B7]). Only the NCs withrelatively mature viral DNA(RC or DSL DNA) are enveloped and secreted as virions. HBV replicating cells can release empty core particles assembled from HBcproteinsand NCsthat containvarious species of replicative intermediate nucleic acids into theculture supernatant.However, while freenaked capsids could bereadily detected*invitro* ([@B7], [@B11], [@B18][@B19][@B20]), they are hardly found in the blood of HBV-infectedpatients ([@B17], [@B25], [@B26]). Although extracellularHBV RNA was detectedin both *in vitro* cell culturesystems and in clinicalserumsamples, its origin and composition remain controversial. It was proposed that extracellular HBVRNA represents pgRNA localized in virions ([@B27]). However, HBV spliced RNA and HBxRNA were also detected in culture supernatant of HBV stably replicating cells as well asin sera of CHBpatients ([@B28], [@B29]). In addition, extracellular HBV RNA was also suggested to originatefrom damaged liver cells ([@B30]), naked capsids, or exosomes ([@B11], [@B29]). Hence, these extracellular RNA molecules have never been conclusively characterized. Here,we demonstrate that extracellular HBV RNAs are heterogeneous in length, ranging from full-length pgRNA (3.5 kilonucleotides \[knt\]) toRNA fragments with merelyseveral hundred nucleotides.These RNA molecules represent 3′ recedingpgRNA fragments that have not been completelyreversetranscribed to DNA andpgRNA fragments hydrolyzed by the RNase H domain of polymerase in the process of viralreplication.More importantly, extracellular HBV RNAs are localized in naked capsids and invirions inculture supernatants of HBV replicating cellsand also circulate as CACs and virions inblood ofhepatitis B patients. RESULTS {#s2}======= Extracellular HBV RNAs are heterogeneous in length and predominantly integral tonaked capsidsinstead ofvirions in HepAD38 cell culturesupernatant. {#s2.1} ------------------------------------------------------------------------------------------------------------------------------------------------------ To ascertain the origin of extracellular HBV RNA, we first examined viral particles prepared from culture mediumof an *in vitro* HBVstably transduced cellline.A human hepatoma HepAD38 cell line was used in this study,as it sustains vigorous HBV replication under thecontrol of a tetracycline-repressible cytomegalovirus (CMV) promoter ([@B31]). Totalviral particles were concentrated andcentrifuged over a 10% to60% (wt/wt) sucrose gradient. Most of the subviral HBsAgparticles, virions, and empty virions were detected between fractions 9 to 14 ([Fig. 1A](#F1){ref-type="fig"}, upper and middle). Naked capsids, detectedonly by anti-HBcAg and not by anti-HBsAgantibodies, settled in fractions 5to 8 ([Fig. 1A](#F1){ref-type="fig"}, middle and lower). The majority of viral nucleic acids were detected in fractions between 4 and 11 ([Fig. 1B](#F1){ref-type="fig"}, upper), which coincided with the fractionscontaining virions (fractions 9to 11), naked capsids(fractions 4 to 7), and the mixture of these particles (fraction 8). Consistent with previous observations, HBV virions are packedwith mature viral DNA(RC or DSL DNA), while naked capsids contain both immature single-stranded DNA (SS DNA) and mature viral DNA ([Fig. 1B](#F1){ref-type="fig"}, upper). Moreover, Northern blot results showedthat most of the HBV RNA was detected in the naked capsids ([Fig. 1B](#F1){ref-type="fig"}, lower, fractions4to7), whereas onlya very smallamount was associated with virions ([Fig. 1B](#F1){ref-type="fig"}, lower, fractions 9 to 11). HBV RNA detected in naked capsids rangedfrom the full length of pgRNA down to a few hundred nucleotides (shorter thanthe HBx mRNA \[0.7 knt\]). Moreover, RNA molecules within virionswere muchshorterthan those within naked capsids. We excluded the possibility of artifacts generated by the SDS-proteinase K extraction method, as a similar RNAblot pattern was obtained using a TRIzol reagent to extract bothintracellular nucleocapsid-associated andextracellular HBV RNA (not shown).Furthermore, quantification of viral RNA extracted by either the SDS-proteinase K method or TRIzol reagent produceda very similar copynumber, except that the TRIzol reagent is known to preferentially extract RNA rather than DNA(notshown). Moreover,theRNA signaldetected by Northern blotting could not be attributed to DNA fragments generated by DNase I treatment, whichwould reduce DNA to belowthe detection limit of the hybridization method (not shown). Furthermore, the RNAsignal could be completely removed byan additional RNase A treatment (not shown).{#F1} To confirm the above-described results and to better separate nakedcapsids from HBV virions, isopycnic CsCl gradient ultracentrifugation was employed. Naked capsids were observed mainly in fractions 5 to 7, with densities ranging from 1.33 to1.34 g/cm^3^ ([Fig. 2A](#F2){ref-type="fig"}).The smearing bands of naked capsids were likely caused by high concentrations of CsClsalt, asfractionation of naked capsids in a 1.18-g/cm^3^ CsCl solution produced single bands. Virions, detectedby both anti-HBcAg and anti-HBsAgantibodies([Fig. 2A](#F2){ref-type="fig"}, upper and middle), were packaged with viral DNA ([Fig. 2A](#F2){ref-type="fig"}, lower) andsettledinfractions 13 to 15, with densities ranging from1.23 to 1.25 g/cm^3^.In agreement with the results shown in [Fig. 1](#F1){ref-type="fig"},
|
INTRODUCTION {#s1} ============ _Hepatitis_ B virus (HBV) is _still_ _a_ major global health problem, with an _estimated_ 257 million people worldwide that are _chronically_ _infected_ with _HBV_ ([@B1]). HBV, together with duck hepatitis _B_ virus (DHBV) and _several_ _other_ related _animal_ viruses, belongs to the _*Hepadnaviridae*_ _family_ ([@B2]). The HBV virion is comprised _of_ _an_ _outer_ envelope and an inner _icosahedral_ nucleocapsid (NC) assembled by 240 _copies_ _of_ core _protein_ (HBc) and packaged with a 3.2-kb partially double-stranded circular DNA genome _([@B3][@B4][@B8])._ _In_ addition to DNA-containing virions, a _large_ amount _of_ incomplete viral particles, such _as_ _hepatitis_ _B_ surface antigen (HBsAg) particles, empty virions, and naked capsids, can also be _released_ from cells in the _process_ of virus replication ([@B9]). Subviral HBsAg particles are spherical or _rodlike_ and are _present_ _in_ vast excess over virions in sera of CHB patients ([@B2]). Empty virions share _the_ same structure as DNA-containing virions _but_ are devoid of nucleic acids ([@B10][@B11][@B14]). Naked capsids, which exit cells via a _route_ different _from_ _that_ of virions ([@B15][@B16][@B17]), have the same structure _as_ NCs but are either empty or filled with viral RNA and _immature_ viral DNA ([@B7], [@B11], [@B18][@B19][@B20]). In NC, pgRNA _undergoes_ reverse _transcription_ into minus-strand DNA, followed by _plus-strand_ DNA synthesis ([@B2], [@B21][@B22][@B24]). _Intracellular_ NCs can be packaged with viral _nucleic_ _acids_ _at_ all levels _of_ maturation, including pgRNA, nascent minus-strand DNA, minus-strand DNA-RNA _hybrids,_ and _relaxed_ circular _DNA_ (RC DNA) or double-stranded linear _DNA_ (DSL _DNA)_ ([@B5], _[@B7])._ Only _the_ NCs with relatively _mature_ viral _DNA_ (RC or DSL DNA) are enveloped and _secreted_ as _virions._ HBV replicating cells _can_ release empty core particles assembled from HBc proteins _and_ NCs that contain various species of _replicative_ intermediate nucleic acids _into_ the _culture_ supernatant. _However,_ while free _naked_ capsids could be readily detected *in vitro* ([@B7], [@B11], [@B18][@B19][@B20]), they are _hardly_ found in the blood of _HBV-infected_ patients ([@B17], _[@B25],_ [@B26]). Although extracellular HBV RNA was detected _in_ both *in _vitro*_ cell culture systems _and_ in clinical serum samples, its origin and composition remain controversial. It _was_ proposed that _extracellular_ HBV _RNA_ represents pgRNA localized in virions ([@B27]). However, _HBV_ _spliced_ RNA _and_ HBx RNA were also detected in culture _supernatant_ of _HBV_ stably _replicating_ _cells_ as well as _in_ sera _of_ CHB patients ([@B28], [@B29]). In addition, extracellular HBV _RNA_ was _also_ suggested _to_ _originate_ from damaged liver cells ([@B30]), _naked_ capsids, or exosomes _([@B11],_ [@B29]). Hence, these extracellular _RNA_ molecules have never been conclusively characterized. Here, we _demonstrate_ _that_ extracellular _HBV_ RNAs are _heterogeneous_ in length, ranging _from_ _full-length_ pgRNA (3.5 kilonucleotides \[knt\]) to RNA fragments with _merely_ several _hundred_ nucleotides. These RNA molecules represent 3′ _receding_ pgRNA fragments that have _not_ been completely reverse transcribed to DNA and _pgRNA_ fragments hydrolyzed by the _RNase_ _H_ domain of polymerase in _the_ process of viral _replication._ More _importantly,_ extracellular _HBV_ _RNAs_ are _localized_ in _naked_ capsids and _in_ _virions_ in _culture_ supernatants of _HBV_ _replicating_ cells and _also_ circulate _as_ CACs and virions _in_ blood of _hepatitis_ _B_ patients. RESULTS _{#s2}_ ======= Extracellular HBV _RNAs_ _are_ heterogeneous in length and _predominantly_ integral to naked capsids instead of virions in HepAD38 cell culture supernatant. {#s2.1} ------------------------------------------------------------------------------------------------------------------------------------------------------ To ascertain the _origin_ of extracellular HBV RNA, we first examined viral _particles_ prepared from culture medium of an *in _vitro*_ HBV stably transduced cell line. _A_ _human_ _hepatoma_ HepAD38 cell line was used in this _study,_ _as_ it sustains vigorous HBV replication under the control of a tetracycline-repressible _cytomegalovirus_ (CMV) promoter ([@B31]). Total viral particles were _concentrated_ and centrifuged over a 10% to 60% (wt/wt) _sucrose_ _gradient._ Most of the _subviral_ HBsAg particles, virions, and empty virions _were_ detected between fractions 9 to 14 ([Fig. 1A](#F1){ref-type="fig"}, _upper_ and middle). Naked capsids, detected only by _anti-HBcAg_ and not by anti-HBsAg antibodies, settled in fractions _5_ to 8 _([Fig._ 1A](#F1){ref-type="fig"}, middle and _lower)._ The majority _of_ viral nucleic acids were detected in fractions between 4 and 11 ([Fig. 1B](#F1){ref-type="fig"}, upper), which coincided with _the_ _fractions_ _containing_ virions (fractions _9_ _to_ 11), naked capsids (fractions 4 _to_ 7), and the mixture of these particles (fraction 8). _Consistent_ _with_ _previous_ _observations,_ _HBV_ virions _are_ packed with mature _viral_ DNA (RC _or_ DSL DNA), while naked capsids contain both immature single-stranded DNA (SS DNA) and _mature_ viral DNA ([Fig. _1B](#F1){ref-type="fig"},_ upper). Moreover, Northern blot results showed that most of the HBV RNA was detected in _the_ naked capsids ([Fig. _1B](#F1){ref-type="fig"},_ _lower,_ _fractions_ _4_ to 7), whereas _only_ _a_ _very_ _small_ amount was associated _with_ virions _([Fig._ 1B](#F1){ref-type="fig"}, lower, fractions 9 to 11). _HBV_ RNA _detected_ in naked capsids _ranged_ from the _full_ _length_ of pgRNA down to a _few_ hundred _nucleotides_ (shorter than the HBx mRNA \[0.7 knt\]). Moreover, RNA molecules within virions _were_ much shorter _than_ those within naked capsids. We excluded the possibility of _artifacts_ generated by the SDS-proteinase _K_ extraction method, as a similar _RNA_ _blot_ pattern was obtained using a _TRIzol_ reagent to _extract_ both intracellular nucleocapsid-associated and extracellular HBV RNA _(not_ shown). Furthermore, _quantification_ of viral RNA extracted by either the SDS-proteinase K method _or_ TRIzol reagent produced a very similar copy number, except that the _TRIzol_ reagent is known to preferentially _extract_ _RNA_ rather _than_ DNA (not shown). _Moreover,_ the RNA _signal_ detected by Northern _blotting_ could not _be_ attributed to DNA fragments generated by DNase I treatment, which would reduce DNA _to_ below _the_ detection limit _of_ _the_ hybridization method (not shown). Furthermore, the RNA _signal_ could be completely _removed_ by an additional RNase _A_ treatment (not shown). {#F1}_ To _confirm_ the above-described _results_ and to better separate naked capsids from _HBV_ _virions,_ isopycnic CsCl gradient ultracentrifugation _was_ employed. Naked capsids _were_ observed mainly in fractions _5_ to 7, with densities ranging _from_ 1.33 to 1.34 g/cm^3^ ([Fig. 2A](#F2){ref-type="fig"}). The smearing bands of naked _capsids_ _were_ likely caused _by_ _high_ _concentrations_ of CsCl _salt,_ as fractionation of naked _capsids_ in a 1.18-g/cm^3^ CsCl solution _produced_ single bands. Virions, detected _by_ both anti-HBcAg and anti-HBsAg antibodies ([Fig. 2A](#F2){ref-type="fig"}, upper and middle), were packaged with viral DNA ([Fig. 2A](#F2){ref-type="fig"}, lower) and settled in fractions 13 to 15, with _densities_ _ranging_ from 1.23 to 1.25 g/cm^3^. _In_ agreement with the results _shown_ in [Fig. 1](#F1){ref-type="fig"},
|
Koolstra K, Beenakker J‐WM, Koken P, Webb A, Börnert P. Cartesian MR fingerprinting in the eye at 7T using compressed sensing and matrix completion‐based reconstructions. Magn Reson Med. 2019;81:2551--2565. 10.1002/mrm.27594 30421448
**Funding information**
This project was partially funded by the European Research Council Advanced Grant 670629 NOMA MRI.
1. INTRODUCTION {#mrm27594-sec-0005}
===============
Ophthalmologic disease diagnosis conventionally relies mainly on ultrasound and optical imaging techniques such as fundus photography and fluorescent angiography (FAG), MRI is increasingly being used in the radiological community.[1](#mrm27594-bib-0001){ref-type="ref"}, [2](#mrm27594-bib-0002){ref-type="ref"}, [3](#mrm27594-bib-0003){ref-type="ref"} One of the main advantages of MRI is its capability to assess nontransparent tissues such as ocular tumors or structures behind the globe such as the eye muscles. Currently, however, these applications are mainly based on qualitative MRI methods using the large number of tissue contrasts addressable by MR. As an example, in Graves' ophthalmopathy fat‐suppressed T~2~‐weighted MRI is the standard to detect inflammation in the eye muscles,[4](#mrm27594-bib-0004){ref-type="ref"}, [5](#mrm27594-bib-0005){ref-type="ref"} whereas in the diagnosis of retinoblastoma, a rare intraocular cancer in children, standard T~1~‐ and T~2~‐weighted MRI is often performed to confirm the presence of the tumor and to screen for potential optic nerve involvement.[2](#mrm27594-bib-0002){ref-type="ref"} In more recent ophthalmologic applications of MRI, such as uveal melanoma (the most common primary intraocular tumor), quantitative MRI techniques including DWI[6](#mrm27594-bib-0006){ref-type="ref"} and DCE imaging[7](#mrm27594-bib-0007){ref-type="ref"} have been shown, but currently diagnosis is still based on qualitative methods.[3](#mrm27594-bib-0003){ref-type="ref"}
To personalize treatment plans quantitative parameters of the tissues involved, as can be acquired invasively for example by performing biopsies,[8](#mrm27594-bib-0008){ref-type="ref"} are highly desirable. However, quantitative parameter mapping by means of MRI requires long examination times, which would result in significant eye‐motion artifacts, as well as patient discomfort.[9](#mrm27594-bib-0009){ref-type="ref"} MR fingerprinting (MRF) is a recently introduced method for rapid quantitation of tissue relaxation times and other MR‐related parameters.[10](#mrm27594-bib-0010){ref-type="ref"} It uses a flip angle sweep to induce a unique signal evolution for each tissue type. Incoherent undersampling can be applied during sampling of the MRF train, enabling acceleration of the MRF scans.[10](#mrm27594-bib-0010){ref-type="ref"} Together with its ability to measure simultaneously T~1~ and T~2~, MRF offers a solution to the problem of obtaining quantitative measures in an efficient manner and in relatively short scanning times.
One of the main challenges in ocular imaging is in‐plane and through‐plane eye motion, often associated with eye blinking.[11](#mrm27594-bib-0011){ref-type="ref"}, [12](#mrm27594-bib-0012){ref-type="ref"}, [13](#mrm27594-bib-0013){ref-type="ref"} The motion results in corrupted k‐space data that introduces artifacts and blurring throughout the entire image. Shortening the scans would reduce motion‐related artifacts, but standard acceleration techniques are not optimal for the current eye application due to the following 3 reasons. First, a cued‐blinking protocol is typically used to control and reduce the eye motion.[3](#mrm27594-bib-0003){ref-type="ref"}, [11](#mrm27594-bib-0011){ref-type="ref"} This requires an instruction screen placed at the end of the MR tunnel to be visible to the patient which complicates the use of small phased array receive coils in front of the eye, blocking the view. Instead, a custom‐built single‐element eye loop coil is used, which provides a high local SNR[3](#mrm27594-bib-0003){ref-type="ref"} and screen visibility, but which clearly excludes the possibility of scan acceleration by means of parallel imaging.[14](#mrm27594-bib-0014){ref-type="ref"} Second, the gel‐like vitreous body has an extremely long T~1~, particularly at high field.[15](#mrm27594-bib-0015){ref-type="ref"} Its value of 3 to 5 s requires a long duration of the MRF sequence to encode the MR parameters (T~1~,T~2~) sufficiently. Thus, using a flip angle train with a small number of RF pulses is not feasible, hindering scan time reduction. Finally, a time‐efficient spiral sampling scheme, usually applied in MRF,[10](#mrm27594-bib-0010){ref-type="ref"}, [16](#mrm27594-bib-0016){ref-type="ref"}, [17](#mrm27594-bib-0017){ref-type="ref"}, [18](#mrm27594-bib-0018){ref-type="ref"}, [19](#mrm27594-bib-0019){ref-type="ref"} introduces off‐resonance effects in each of the individual MRF images.[20](#mrm27594-bib-0020){ref-type="ref"} This occurs even when combined with unbalanced sequences such as fast imaging with steady state precession,[16](#mrm27594-bib-0016){ref-type="ref"} which are in themselves robust to off‐resonance effects.[21](#mrm27594-bib-0021){ref-type="ref"} The off‐resonance effects present in spiral sampling schemes are much stronger at high field, where they result in blurring,[22](#mrm27594-bib-0022){ref-type="ref"} caused by strong main field inhomogeneities (particularly in the eye region due to many air‐tissue‐bone interfaces), as well as the presence of significant amounts of off‐resonant orbital fat around the eye.
In this work, a Cartesian sampling scheme is used, which is more robust than spiral sampling to off‐resonance effects, but which is significantly less time‐efficient.[23](#mrm27594-bib-0023){ref-type="ref"} With such a Cartesian sampling scheme, undersampling artifacts have a more structured nature compared with spiral sampling, which increases the temporal coherence of the artifacts in the MRF image series.[10](#mrm27594-bib-0010){ref-type="ref"}, [20](#mrm27594-bib-0020){ref-type="ref"} In this case, direct matching of the measured MRF signal reconstructed by plain Fourier transformations, to the simulated dictionary elements is not sufficiently accurate for high undersampling factors.[24](#mrm27594-bib-0024){ref-type="ref"}, [25](#mrm27594-bib-0025){ref-type="ref"} Therefore, the quality of the reconstructed MRF data has to be improved before the matching process.
Compressed sensing (CS) has been introduced as a technique to reconstruct images from randomly undersampled data by enforcing signal sparsity (in the spatial dimension only or both in spatial and temporal dimensions),[26](#mrm27594-bib-0026){ref-type="ref"}, [27](#mrm27594-bib-0027){ref-type="ref"} allowing a scan time reduction in many applications.[28](#mrm27594-bib-0028){ref-type="ref"}, [29](#mrm27594-bib-0029){ref-type="ref"}, [30](#mrm27594-bib-0030){ref-type="ref"} The flexibility of MRF toward different sampling schemes and undersampling factors makes it possible to reconstruct the source images by means of CS.[27](#mrm27594-bib-0027){ref-type="ref"}, [31](#mrm27594-bib-0031){ref-type="ref"}, [32](#mrm27594-bib-0032){ref-type="ref"} Higher acceleration factors might be feasible if the correlation in the temporal dimension is better used.[33](#mrm27594-bib-0033){ref-type="ref"} Examples of such reconstructions specifically tailored to MRF are given in Davies et al, Pierre et al, and Zhao et al[34](#mrm27594-bib-0034){ref-type="ref"}, [35](#mrm27594-bib-0035){ref-type="ref"}, [36](#mrm27594-bib-0036){ref-type="ref"} which take into account the simulated dictionary atoms in the image reconstruction process.
Recent work has shown that the temporal correlation in the MRF data can be exploited even further by incorporating the low rank structure of the data into the cost function,[37](#mrm27594-bib-0037){ref-type="ref"} a technique which was introduced into MR in Liang[
|
koolstra k, beenakker j ‐ wm, koken p, webb a, bornert p. cartesian mr fingerprinting in the eye at 7t using compressed sensing and matrix 3d ‐ based reconstructions. magn reson med. 2019 ; 81 : 2551 - - 2565. 10. 1002 / mrm. 27594 30421448 * * funding information * * this project was partially funded by the european research council advanced grant 670629 noma vol. 1. introduction { # mrm27594 - sec - 0005 } = = = = = = = = = = = = = = = ophthalmologic disease diagnosis conventionally relies mainly on ultrasound and optical imaging techniques such as fundus mri and fluorescent angiography ( fag ), mri is increasingly being used in the radiological community. [ 1 ] ( # mrm27594 - bib - 0001 ) { ref - type = " ref " }, [ 2 ] ( # mrm27594 - bib - 0002 ) { ref - type = " ref " }, [ 3 ] ( # mrm27594 - bib - e ) { ref - type = " ref " } one of the main advantages of mri is its capability to assess nontransparent tissues such as ocular tumors or structures behind the globe such as the eye muscles. currently, however, these applications are mainly based on qualitative mri methods using the large number of tissue contrasts addressable by mr. as an example, in graves ' median fat ‐ suppressed t ~ 2 ~ ‐ weighted mri is the standard to detect inflammation involving the eye muscles, [ 2 ] ( # mrm27594 - bib - 0004 ) { ref - type = " ref " }, [ 5 ] ( # mrm27594 - bib - 0005 ) { ref - type = " ref " } whereas in the diagnosis of retinoblastoma, a rare intraocular cancer in children, standard f ~ 1 ~ ‐ and t ~ 2 ~ ‐ weighted mri is often performed to confirm the presence accompanying the tumor and to screen for potential optic nerve involvement. [ 2 ] ( # mrm27594 - bib - 001 ) { ref - type = " ref " } in more recent ophthalmologic applications of mri, such as uveal melanoma ( the most common primary intraocular tumor ), quantitative mri techniques including dwi [ 6 ] ( # mrm27594 - bib - 0006 ) { ref - type = " ref " } and dce imaging [ 7 ] ( # mrm27594 - bib - 0007 ) { ref - type = " ref " } have been shown, but currently diagnosis is still based on qualitative methods. [ 3 ] ( # mrm27594 - bib - 0003 ) { ref - type = " ref " } to personalize treatment plans quantitative parameters of the tissues involved, as can be acquired invasively for example by performing biopsies, [ 8 ] ( # mrm27594 - bib - 0008 ) { ref - type = " ref " } are highly desirable. however, quantitative parameter mapping by means of mri requires long examination times, which would result in significant eye ‐ motion artifacts, as well as patient discomfort. [ 9 ] ( # mrm27594 - bib - 0009 ) { ref - type = " ref " } mr fingerprinting ( mrf ) is a recently introduced method for rapid quantitation of tissue relaxation times and other mr ‐ related parameters. [ 10 ] ( # mrm27594 - bib - 0010 ) { ref - type = " ref " } it uses a flip angle sweep to induce a unique signal evolution for each tissue type. incoherent undersampling can be applied during sampling of the mrf train, enabling acceleration of the mrf scans. [ 10 ] ( # mrm27594 - bib - 0010 ) { ref - type = " ref " } together with its ability to measure simultaneously t ~ 1 ~ and t ~ 2 ~, mrf offers a solution to the problem of obtaining quantitative measures in an efficient manner and in relatively short scanning times. one of the main challenges in ocular imaging is in ‐ plane and through ‐ plane eye motion, often associated with eye blinking. [ 11 ] ( # mrm27594 - bib - 0011 ) { ref - type = " ref " }, [ 12 ] ( # mrm27594 - bib - 0012 ) { ref - type = " ref " }, [ 13 ] ( # mrm27594 - bib - 0013 ) { ref - type = " ref " } the motion results in corrupted k ‐ space data that introduces artifacts and blurring throughout the entire image. shortening the scans would reduce motion ‐ related artifacts, but standard acceleration techniques are not optimal for the current eye application due to the following 3 reasons. first, a cued ‐ blinking protocol is typically used to control and reduce the eye motion. [ 3 ] ( # mrm27594 - bib - 0003 ) { ref - type = " ref " }, [ 11 ] ( # mrm27594 - bib - 0011 ) { ref - type = " ref " } this requires an instruction screen placed at the end of the mr tunnel to be visible to the patient which complicates the use of small phased array receive coils in front of the eye, blocking the view. instead, a custom ‐ built single ‐ element eye loop coil is used, which provides a high local snr [ 3 ] ( # mrm27594 - bib - 0003 ) { ref - type = " ref " } and screen visibility, but which clearly excludes the possibility of scan acceleration by means of parallel imaging. [ 14 ] ( # mrm27594 - bib - 0014 ) { ref - type = " ref " } second, the gel ‐ like vitreous body has an extremely long t ~ 1 ~, particularly at high field. [ 15 ] ( # mrm27594 - bib - 0015 ) { ref - type = " ref " } its value of 3 to 5 s requires a long duration of the mrf sequence to encode the mr parameters ( t ~ 1 ~, t ~ 2 ~ ) sufficiently. thus, using a flip angle train with a small number of rf pulses is not feasible, hindering scan time reduction. finally, a time ‐ efficient spiral sampling scheme, usually applied in mrf, [ 10 ] ( # mrm27594 - bib - 0010 ) { ref - type = " ref " }, [ 16 ] ( # mrm27594 - bib - 0016 ) { ref - type = " ref " }, [ 17 ] ( # mrm27594 - bib - 0017 ) { ref - type = " ref " }, [ 18 ] ( # mrm27594 - bib - 0018 ) { ref - type = " ref " }, [ 19 ] ( # mrm27594 - bib - 0019 ) { ref - type = " ref " } introduces off ‐ resonance effects in each of the individual mrf images. [ 20 ] ( # mrm27594 - bib - 0020 ) { ref - type = " ref " } this occurs even when combined with unbalanced sequences such as fast imaging with steady state precession, [ 16 ] ( # mrm27594 - bib - 0016 ) { ref - type = " ref " } which are in themselves robust to off ‐ resonance effects. [ 21 ] ( # mrm27594 - bib - 0021 ) { ref - type = " ref " } the off ‐ resonance effects present in spiral sampling schemes are much stronger at high field, where they result in blurring, [ 22 ] ( # mrm27594 - bib - 0022 ) { ref - type = " ref " } caused by strong main field inhomogeneities ( particularly in the eye region due to many air ‐ tissue ‐ bone interfaces ), as well as the presence of significant amounts of off ‐ resonant orbital fat around the eye. in this work, a cartesian sampling scheme is used, which is more robust than spiral sampling to off ‐ resonance effects, but which is significantly less time ‐ efficient. [ 23 ] ( # mrm27594 - bib - 0023 ) { ref - type = " ref " } with such a cartesian sampling scheme, undersampling artifacts have a more structured nature compared with spiral sampling, which increases the temporal coherence of the artifacts in the mrf image series. [ 10 ] ( # mrm27594 - bib - 0010 ) { ref - type = " ref " }, [ 20 ] ( # mrm27594 - bib - 0020 ) { ref - type = " ref " } in this case, direct matching of the measured mrf signal reconstructed by plain fourier transformations, to the simulated dictionary elements is not sufficiently accurate for high undersampling factors. [ 24 ] ( # mrm27594 - bib - 0024 ) { ref - type = " ref " }, [ 25 ] ( # mrm27594 - bib - 0025 ) { ref - type = " ref " } therefore, the quality of the reconstructed mrf data has to be improved before the matching process. compressed sensing ( cs ) has been introduced as a technique to reconstruct images from randomly undersampled data by enforcing signal sparsity ( in the spatial dimension only or both in spatial and temporal dimensions ), [ 26 ] ( # mrm27594 - bib - 0026 ) { ref - type = " ref " }, [ 27 ] ( # mrm27594 - bib - 0027 ) { ref - type = " ref " } allowing a scan time reduction in many applications. [ 28 ] ( # mrm27594 - bib - 0028 ) { ref - type = " ref " }, [ 29 ] ( # mrm27594 - bib - 0029 ) { ref - type = " ref " }, [ 30 ] ( # mrm27594 - bib - 0030 ) { ref - type = " ref " } the flexibility of mrf toward different sampling schemes and undersampling factors makes it possible to reconstruct the source images by means of cs. [ 27 ] ( # mrm27594 - bib - 0027 ) { ref - type = " ref " }, [ 31 ] ( # mrm27594 - bib - 0031 ) { ref - type = " ref " }, [ 32 ] ( # mrm27594 - bib - 0032 ) { ref - type = " ref " } higher acceleration factors might be feasible if the correlation in the temporal dimension is better used. [ 33 ] ( # mrm27594 - bib - 0033 ) { ref - type = " ref " } examples of such reconstructions specifically tailored to mrf are given in davies et al, pierre et al, and zhao et al [ 34 ] ( # mrm27594 - bib - 0034 ) { ref - type = " ref " }, [ 35 ] ( # mrm27594 - bib - 0035 ) { ref - type = " ref " }, [ 36 ] ( # mrm27594 - bib - 0036 ) { ref - type = " ref " } which take into account the simulated dictionary atoms in the image reconstruction process. recent work has shown that the temporal correlation in the mrf data can be exploited even further by incorporating the low rank structure of the data into the cost function, [ 37 ] ( # mrm27594 - bib - 0037 ) { ref - type = " ref " } a technique which was introduced into mr in liang [
|
Koolstra K, Beenakker J ‐ WM, Koken P, Webb A, Börnert P. Cartesian MR fingerprinting in the eye at 7T using compressed sensing and matrix completion ‐ based reconstructions. Magn Reson Med. 2019; 81: 2551 - - 2565. 10. 1002 / mrm. 27594 30421448 * * Funding information * * This project was partially funded by the European Research Council Advanced Grant 670629 NOMA MRI. 1. INTRKDUCTIlN {# mrm27594 - sec - 0005} = = = = = = = = = = = = = = = Ophthalmologic disease diagnosis conventionally relies mainly on ultrasound and optical imaging techniques such as fundus photography and fluorescent angiography (FAG ), MRI is increasingly being used in the radiological community. [1] (# mrm27594 - bib - 0001) {ref - type = " ref " }, [2] (# mrm27594 - bib - 0002) {ref - type = " ref " }, [3] (# mrm27594 - bib - 0003) {ref - type = " ref "} One of the main advantages of MRI is its capability to assess nontransparent tissues such as ocular tumors or structures behind the globe such as the eye muscles. Currently, however, these applications are mainly based on qualitative MRI methods using the large number of tissue contrasts addressable by MR. As an example, in Graves ' ophthalmopathy fat ‐ suppressed T ~ 2 ~ ‐ weighted MRI is the standard to detect inflammation in the eye muscles, [4] (# mrm27594 - bib - 0004) {ref - type = " ref " }, [5] (# mrm27594 - bib - 0005) {ref - type = " ref "} whereas in the diagnosis of retinoblastoma, a rare intraocular cancer in children, standard T ~ 1 ~ ‐ and T ~ 2 ~ ‐ weighted MRI is often performed to confirm the presence of the tumor and to screen for potential optic nerve involvement. [2] (# mrm27594 - bib - 0002) {ref - type = " ref "} In more recent ophthalmologic applications of MRI, such as uveal melanoma (the most common primary intraocular tumor ), quantitative MRI techniques including DWI [6] (# mrm27594 - bib - 0006) {ref - type = " ref "} and DCE imaging [7] (# <tm27594 - bib - 0007) {ref - type = " ref "} have been shown, but currently diagnosis is still based on qualitative methods. [3] (# mrm27594 - bib - 0003) {ref - type = " ref "} To personalize treatment plans quantitative parameters of the tissues involved, as can be acquired invasively for example by performing biopsies, [8] (# mrm27594 - bib - 0008) {ref - type = " ref "} are highly desirable. However, quantitative parameter mapping by means of MRI requires long examination times, which would result in significant eye ‐ motion artifacts, as well as patient discomfort. [9] (# mTm27%94 - bib - 0009) {ref - type = " ref "} MR fingerprinting (MRF) is a recently introduced method for rapid quantitation of tissue relaxation times and other MR ‐ related parameters. [10] (# mrm27594 - bib - 0010) {ref - type = " ref "} It uses a flip angle sweep to induce a unique signal evolution for each tissue type. Incoherent undersampling can be applied during sampling of the MRF train, enabling acceleration of the MRF scans. [10] (# mrm27594 - bib - 0010) {ref - type = " ref "} Together with its ability to measure simultaneously T ~ 1 ~ and T ~ 2 ~, MRF offers a solution to the problem of obtaining quantitative measures in an efficient manner and in relatively short scanning times. One of the main challenges in ocular imaging is in ‐ plane and through ‐ plane eye motion, often associated with eye blinking. [11] (# mrm27594 - bib - 0011) {ref - type = " ref " }, [12] (# mrm27594 - bib - 0012) {ref - type = " ref " }, [13] (# mrm27594 - bib - 0013) {ref - type = " ref "} The motion results in corrupted k ‐ space data that introduces artifacts and blurring throughout the entire image. Shortening the scans would reduce motion ‐ related artifacts, but standard acceleration techniques are not optimal for the current eye application due to the following 3 reasons. First, a cued ‐ blinking protocol is typically used to s(ntrol and reduce the eye motion. [3] (# mrm27594 - bib - 0003) {ref - type = " ref " }, [11] (# mrm27594 - bib - 0011) {ref - type = " ref "} This requires an instruction screen placed at the end of the MR tunnel to be visible to the patient which complicates the use of small phased array receive coils in front of the eye, blocking the view. Instead, a custom ‐ built single ‐ element eye loop coil is used, which provides a high local SNR [3] (# mrm27594 - bib - 0003) {ref - type = " ref "} and screen visibility, but which clearly excludes the possibility of scan acceleration by means of parallel imaging. [14] (# mrm27594 - bib - 0014) {ref - type = " ref "} Second, the gel ‐ like vitreous body has an extremely long T ~ 1 ~, particularly at high fiekw. [15] (# mrm27594 - bib - 0015) {ref - type = " ref "} Its value of 3 to 5 s requires a long duration of the MRF sequence to encode the MR parameters (T ~ 1 ~, T ~ 2 ~) sufficiently. Thus, using a flip angle train with a small number of RF pulses is not feasible, hindering scan time reduction. Finally, a time ‐ efficiWnG spiral sampling scheme, usually applied in MRF, [10] (# mrm27594 - bib - 0010) {ref - type = " ref " }, [16] (# mrm27594 - bib - 0016) {ref - type = " ref " }, [17] (# mrm27594 - bib - 0017) {ref - type = " ref " }, [18] (# mrm27594 - bib - 00@9) {ref - type = " ref " }, [19] (# mrm27594 - bib - 0019) {ref - type = " ref "} introduces off ‐ resonance effects in each of the individual MRF images. [20] (# mrm27594 - bib - 0020) {ref - type = " ref "} This ocsurD even when combined with unbalanced sequences such as fast imaging with steady state precession, [16] (# mrm27594 - bib - 0016) {ref - type = " ref "} which are in themselves robust to off ‐ resonance effects. [21] (# mrm27594 - bib - 0021) {ref - type = " ref "} The off ‐ resonance effects present in spiral sampling schemes are much stronger at high field, where they result in blurring, [22] (# mrm27594 - bib - 0022) {ref - type = " ref "} caused by strong main field inhomogeneities (particularly in the eye region due to many air ‐ tissue ‐ bone interfaces ), as well as the presence of significant amounts of off ‐ resonant orbital fat ar(6nd the eye. In this work, a Cartesian sampling scheme is used, which is more robust than spiral sampling to off ‐ resonance effects, but which is significantly less time ‐ efficient. [23] (# mrm27594 - bib - 0023) {ref - type = " ref "} With such a Cartesian sampling scheme, undersampling artifacts have a more structured nature compared with spiral sampling, which increases the temporal coherence of the artifacts in the MRF image series. [10] (# mrm27594 - bib - 0010) {ref - type = " ref " }, [20] (# mrm27594 - bib - 0020) {ref - type = " ref "} In this case, direct matching of the measured MRF signal reconstructed by plain Fourier transformations, to the simulated dictionary elements is not sufficiently accurate for high undersampling factors. [24] (# mrm27594 - bib - 0024) {ref - type = " ref " }, [25] (# mrm27594 - bib - 0025) {ref - type = " ref "} Therefore, the quality of the reconstructed MRF data has to be improved before the matching process. Compressed sensing (CS) has been introduced as a technique to reconstruct images from randomly undersampled data by enforcing signal sparsity (in the spatial dimension only or both in spatial and temporal dimensions ), [26] (# mrm27594 - bib - 0026) {ref - type = " ref " }, [27] (# mrm27594 - bib - 0027) {ref - type = " ref "} allowing a scan time reduction in many applications. [28] (# mrm27594 - bib - 0028) {ref - type = " ref " }, [29] (# mrm27594 - bib - 0029) {ref - type = " ref " }, [30] (# mrm27594 - bib - 0030) {ref - type = " ref "} The flexibility of MRF toward different sampling schemes and undersampling factors makes it possible to rwdonstruct the source images by means of CS. [27] (# mrm27594 - bib - 0027) {ref - type = " ref " }, [31] (# mrm27594 - bib - 0031) {ref - type = " ref " }, [32] (# mrm27594 - bib - 0032) {ref - type = " ref "} Higher acceleration factors might be feasible if the correlation in the temporal dimension is better used. [33] (# mrm27594 - bib - 0033) {ref - type = " ref "} Examples of such reconstructions specifically tailored to MRF are given in Davies et al, Pierre et al, and Zhao et al [34] (# mrm27594 - bib - 0034) {ref - type = " ref " }, [35] (# mrm27594 - bib - 0035) {ref - type = " ref " }, [36] (# mrm27594 - bib - 0036) {ref - type = " ref "} which take into account the simulated dictionary atoms in the image reconstruction process. Recent work has shown that the temporal correlation in the MRF data can be exploited even further by incorporating the low rank structure of the data into the cost function, [37] (# mrm27594 - bib - 0037) {ref - type = " ref "} a technique which was introduced into MR in Liang [
|
Koolstra K, Beenakker J‐WM, Koken P, Webb A, Börnert P. MR fingerprinting the eye at 7T using compressed sensing and matrix completion‐based reconstructions. Magn Reson Med. 2019;81:2551--2565. 10.1002/mrm.27594 30421448 **Funding information** This project was partially funded by the European Research Council Advanced Grant 670629 MRI. 1. INTRODUCTION {#mrm27594-sec-0005} =============== Ophthalmologic disease diagnosis conventionally relies on ultrasound and optical imaging such as and fluorescent (FAG), MRI is increasingly being used in the radiological community.[1](#mrm27594-bib-0001){ref-type="ref"}, [2](#mrm27594-bib-0002){ref-type="ref"}, [3](#mrm27594-bib-0003){ref-type="ref"} One of the main advantages of MRI is its capability assess nontransparent such ocular or structures behind the such as the eye muscles. Currently, however, applications are mainly based on qualitative MRI methods using the large number of tissue contrasts addressable by MR. As an example, fat‐suppressed T~2~‐weighted MRI is the standard to detect inflammation in the eye muscles,[4](#mrm27594-bib-0004){ref-type="ref"}, [5](#mrm27594-bib-0005){ref-type="ref"} in the diagnosis of retinoblastoma, a rare intraocular cancer in children, standard T~1~‐ and T~2~‐weighted MRI is often performed confirm the presence the tumor and to screen for optic nerve In more recent applications of MRI, such uveal melanoma (the most common primary tumor), quantitative MRI techniques including DWI[6](#mrm27594-bib-0006){ref-type="ref"} and DCE imaging[7](#mrm27594-bib-0007){ref-type="ref"} have been shown, but currently is based on qualitative methods.[3](#mrm27594-bib-0003){ref-type="ref"} To personalize treatment plans quantitative parameters of the tissues involved, as can be acquired invasively for by performing biopsies,[8](#mrm27594-bib-0008){ref-type="ref"} are highly desirable. However, quantitative parameter mapping by means of MRI requires long times, which would result in significant eye‐motion artifacts, as well patient discomfort.[9](#mrm27594-bib-0009){ref-type="ref"} MR fingerprinting is a recently introduced method for rapid quantitation of tissue relaxation times and other MR‐related parameters.[10](#mrm27594-bib-0010){ref-type="ref"} It uses a flip angle sweep to induce a signal evolution for each tissue type. Incoherent undersampling can be applied during sampling of the MRF train, enabling acceleration of the MRF scans.[10](#mrm27594-bib-0010){ref-type="ref"} Together with its to measure simultaneously T~1~ and MRF offers a solution to the of obtaining quantitative in an efficient manner and in relatively scanning times. One of the main challenges in imaging is in‐plane and through‐plane eye motion, often associated with eye blinking.[11](#mrm27594-bib-0011){ref-type="ref"}, [12](#mrm27594-bib-0012){ref-type="ref"}, [13](#mrm27594-bib-0013){ref-type="ref"} motion in corrupted k‐space data introduces artifacts and blurring throughout Shortening scans would reduce motion‐related artifacts, but standard acceleration techniques are not optimal for the current eye application due to following reasons. First, a cued‐blinking protocol is typically used to control and the eye motion.[3](#mrm27594-bib-0003){ref-type="ref"}, [11](#mrm27594-bib-0011){ref-type="ref"} This requires an instruction screen placed at the end of the MR tunnel to be visible to the patient which complicates the use of small phased array coils in front of eye, the view. Instead, a custom‐built single‐element eye loop coil which provides a high local SNR[3](#mrm27594-bib-0003){ref-type="ref"} and screen visibility, but which clearly excludes the possibility of scan by means of parallel imaging.[14](#mrm27594-bib-0014){ref-type="ref"} Second, the gel‐like vitreous body has an extremely long T~1~, particularly at high field.[15](#mrm27594-bib-0015){ref-type="ref"} Its value of 3 to 5 s requires a long duration of the sequence to encode the MR parameters (T~1~,T~2~) Thus, using a flip angle train with a small number of RF pulses is not feasible, scan time reduction. Finally, a time‐efficient spiral sampling scheme, usually in MRF,[10](#mrm27594-bib-0010){ref-type="ref"}, [16](#mrm27594-bib-0016){ref-type="ref"}, [18](#mrm27594-bib-0018){ref-type="ref"}, [19](#mrm27594-bib-0019){ref-type="ref"} introduces off‐resonance effects in of the individual MRF images.[20](#mrm27594-bib-0020){ref-type="ref"} This occurs even when with unbalanced such as fast imaging with steady state precession,[16](#mrm27594-bib-0016){ref-type="ref"} which are themselves robust to off‐resonance effects.[21](#mrm27594-bib-0021){ref-type="ref"} The off‐resonance effects present in spiral sampling schemes are much at high field, where they result in blurring,[22](#mrm27594-bib-0022){ref-type="ref"} strong main field inhomogeneities (particularly the eye region due to many air‐tissue‐bone interfaces), as well as the presence of significant amounts of off‐resonant orbital fat around the eye. this work, a Cartesian sampling scheme is used, which is more robust than spiral sampling to off‐resonance effects, but which is significantly less time‐efficient.[23](#mrm27594-bib-0023){ref-type="ref"} With such a Cartesian sampling scheme, undersampling artifacts have a more structured nature compared with spiral which increases the temporal coherence of the artifacts in the MRF image [20](#mrm27594-bib-0020){ref-type="ref"} case, matching of measured signal reconstructed by plain Fourier transformations, to the simulated dictionary elements is not sufficiently accurate high factors.[24](#mrm27594-bib-0024){ref-type="ref"}, [25](#mrm27594-bib-0025){ref-type="ref"} Therefore, the quality of the reconstructed MRF data has to improved before the matching process. Compressed sensing (CS) has been introduced as a technique to images from randomly undersampled data enforcing signal sparsity (in the spatial dimension or both in spatial and dimensions),[26](#mrm27594-bib-0026){ref-type="ref"}, [27](#mrm27594-bib-0027){ref-type="ref"} allowing a scan time reduction applications.[28](#mrm27594-bib-0028){ref-type="ref"}, [29](#mrm27594-bib-0029){ref-type="ref"}, [30](#mrm27594-bib-0030){ref-type="ref"} The flexibility of toward different sampling schemes and undersampling factors makes it possible to reconstruct the source images by means CS.[27](#mrm27594-bib-0027){ref-type="ref"}, [31](#mrm27594-bib-0031){ref-type="ref"}, [32](#mrm27594-bib-0032){ref-type="ref"} Higher acceleration factors might be feasible if the correlation in the dimension is better used.[33](#mrm27594-bib-0033){ref-type="ref"} Examples such reconstructions specifically tailored to MRF are given in Davies al, Pierre et al, and Zhao et al[34](#mrm27594-bib-0034){ref-type="ref"}, [35](#mrm27594-bib-0035){ref-type="ref"}, [36](#mrm27594-bib-0036){ref-type="ref"} which take into account the simulated dictionary atoms in the image reconstruction process. Recent work has shown that the temporal correlation in the MRF data can be exploited even further by incorporating low rank structure of the data into the cost function,[37](#mrm27594-bib-0037){ref-type="ref"} a technique was introduced into MR in Liang[
|
koOLSTra k, BEeNAKKEr J‐wM, KoKEN P, webb a, börnERT P. carTesIAn mr fINgErpRInTiNG IN The eYE aT 7T UsinG comPReSSeD SEnsInG ANd MATriX coMpLEtIOn‐bASed RECONStrUcTIons. MaGn ResOn MEd. 2019;81:2551--2565. 10.1002/mRM.27594 30421448
**FUNDIng INFOrMaTIOn**
thIs PrOJECT WAs PArTIalLy FUnDeD bY The EuROPeaN REsearcH COUnCil ADVANCED GrAnt 670629 NOmA Mri.
1. INtRodUCTION {#mrm27594-sEc-0005}
===============
OPhtHalMOLOgic disease DiagNOsIs CoNvenTionAlLy ReliEs maINly on ULtraSOuND aND OPtiCAl iMAGING tecHNIqUeS sUCH as fuNdUs pHOtOGRAPHY and FluoRescEnt ANGIOgRaphY (FaG), mri iS iNCREAsINGLy bEING useD IN The raDIOLOGicaL COmMUNITY.[1](#mrm27594-bIb-0001){ReF-tyPE="REf"}, [2](#Mrm27594-BiB-0002){rEF-TYpe="rEF"}, [3](#MrM27594-BIb-0003){Ref-tYpE="ref"} One oF tHe main aDvAntAGes of MRi iS Its caPABilitY tO AsSeSs NoNtraNspARent tIsSuEs sUcH aS OculaR TUmORs oR StRUcTUrES bEhINd the glOBe such aS tHe eYE mUscLeS. CURrenTLY, howeVER, tHEsE AppliCAtIOnS arE maiNly BaseD on quALItaTIve mRI MetHODS usING tHE LArgE nUMbeR oF tiSsUe cOnTRASTS AdDREssabLE bY Mr. As An eXAmPlE, iN graVES' OPhThALMOpathY FAt‐sUppresSeD T~2~‐WeiGHTEd mRI IS tHe sTanDard TO DETeCT INFLamMation In THe eyE mUscLES,[4](#MRM27594-bIB-0004){rEF-tYPE="Ref"}, [5](#MrM27594-bIB-0005){rEF-tYPE="Ref"} whEREAs IN tHE DiAgnosIs OF rETInOblaSTomA, A Rare IntrAOCular cAnCEr iN chIlDReN, sTaNDarD T~1~‐ AND t~2~‐wEIgHTEd MRI Is OftEn pErFORmeD to cOnfIRM THE pREsEncE of THe TumOr and TO ScReEn FOr POTeNTiAl OpTic NeRve InVoLVemeNt.[2](#mrm27594-bib-0002){rEf-TYpE="ReF"} In mOre reCeNT ophTHALMolOGiC APplIcAtIoNS of mrI, sUCH AS uvEal meLAnoma (tHe MOSt coMmoN pRIMAry InTRaocULaR Tumor), QUAnTITaTIvE Mri teCHniqUes iNcluDiNG dwi[6](#mrM27594-BIb-0006){reF-TYpe="REF"} AND Dce iMAgInG[7](#mRm27594-BIB-0007){ref-tYpe="REf"} HaVE BeEn sHoWn, bUT CuRRenTLY diaGNosiS Is Still BaseD ON quALitatiVE MeTHODs.[3](#MRM27594-BIB-0003){reF-TYpe="rEf"}
To PersONaliZE TreaTMEnT pLANs qUANtitATiVe PaRamEterS oF thE tiSSUES involVED, as cAN be acQUiREd iNvAsIVElY foR exaMPLe BY pErfOrMIng bIOPsIES,[8](#mRM27594-bIb-0008){REf-tYPe="ref"} arE hiGhlY desirAbLE. HoWeVEr, qUaNtItAtIVE ParaMETeR MApping BY meaNS oF mRI REQUiRES lONg eXAminatIOn TiMES, WHIcH wOULD REsulT In sIgNiFiCAnt EYe‐mOtIon aRtIFACtS, aS welL as pATiEnT dISCoMForT.[9](#mrm27594-bIB-0009){ref-tyPe="rEF"} MR fiNGeRpriNTinG (Mrf) iS A reCENtLy INTRodUCeD METHOD foR rapID QuantiTAtIOn oF TIssUE RELAxation TIMES and oTHEr MR‐RelaTEd pARAmETers.[10](#MRm27594-BIb-0010){REF-TypE="rEf"} IT usES A FlIP angLe sWeep TO InDUCe A UNiQuE sIgnAL eVOlUtiOn FOR EAch TiSSuE TypE. incoHErEnT UnDeRsAmPLING Can Be aPPlieD DuRiNg sampLing Of tHe mRf trAiN, enaBLinG acCEleraTIoN OF THe MrF ScaNS.[10](#mRM27594-BiB-0010){ReF-type="ref"} TogEther wiTH iTS ABiLiTy TO mEASUrE sIMultANeOusLy T~1~ ANd T~2~, MRf OFferS a SoLuTIoN to thE PrOBLem Of ObtAINIng quAntitAtIvE MEaSUreS iN AN EfFiCIEnt maNneR and in rElATIvelY SHORt scaNNING TIMEs.
OnE Of THe Main cHaLLENGES iN OCULAR ImagiNg Is iN‐plANe and thROuGh‐PlANe eYe MOTIon, oFTen AssocIATED with eye BlINKInG.[11](#MRm27594-biB-0011){reF-type="reF"}, [12](#mRM27594-bIb-0012){ref-TyPe="REF"}, [13](#mRM27594-bIB-0013){REF-typE="reF"} tHE motiON rESUltS In COrRUPteD k‐SpaCe DATa tHat INtrODuCes ARTiFacts anD blUrRinG thrOUghout ThE ENtiRe ImaGe. ShORTENING tHe SCaNS WouLd REDuce MOTIon‐ReLaTed arTiFaCts, but staNdARD aCCeLeRAtiOn techNiqueS ARe nOT oPTIMAL foR tHE CurReNT eYE ApPLicATIOn DUe TO thE FoLlOwInG 3 reaSONs. fIrST, a cued‐BLiNkING pRotOCOL is tYpIcALLY UseD tO conTROL aNd ReDucE the eye mOTIon.[3](#MRM27594-bIb-0003){ref-TyPe="Ref"}, [11](#Mrm27594-BIB-0011){rEF-TyPE="ReF"} ThIs ReQUIReS aN inSTRUCtIon ScReeN plAcED at tHe eND of tHE mR TUNNEl to bE VIsIBle tO tHE PAtient wHICh cOmPlIcateS THe uSe OF sMall PHAseD arRAY RecEiVe COilS in froNt of the Eye, BLOCking THE viEW. iNstEAD, a cUstom‐buIlT sINgle‐eLEMeNT Eye loOP CoIl iS uSeD, wHIch prOvIdES A HIGh lOCAl SNr[3](#mrm27594-BiB-0003){reF-Type="ReF"} anD ScREEn vIsIbILitY, BuT whIcH CLEarly EXClUDes THE PoSSiBILity of ScAN ACCeLERatiOn bY mEAns oF paralLeL iMAgINg.[14](#mrm27594-Bib-0014){REF-typE="Ref"} SEConD, the gEl‐lIKE VitREOUs BOdy hAS aN EXTrEmelY loNG t~1~, pARTicularLY aT hIGH FiELD.[15](#mRM27594-BiB-0015){REf-tYpe="rEF"} ITs value of 3 tO 5 S REquIrES A loNg DURaTIon OF THe mrf SEQUenCe To ENCOdE THE mr PaRAMEters (t~1~,t~2~) SuFFicIEntLy. THuS, UsiNG a FLIP anGLe tRaiN WIth A sMALL NUmbEr OF rF PUlSES IS Not FEAsible, HiNDERING scAn TiMe reDucTiON. finaLly, a TiME‐efFiCIent spirAl SAmPLING sCheME, uSUAllY ApPLIeD in MRf,[10](#MRm27594-BIB-0010){rEf-tyPE="reF"}, [16](#MRM27594-bIB-0016){REf-TYpe="REf"}, [17](#MRM27594-bib-0017){REF-type="ReF"}, [18](#mrM27594-Bib-0018){rEF-tYpe="Ref"}, [19](#mRm27594-BiB-0019){REF-TYpe="rEF"} inTRODUceS oFF‐ReSONAncE EfFECts In EacH oF THE indIviDUal MRf iMAgEs.[20](#mRm27594-bib-0020){REF-Type="Ref"} tHis OCcUrS EVeN WhEn comBinEd WiTh unbAlAnCed SeQUenCeS Such aS FAst ImAgiNG wITh steadY STaTe PreCesSion,[16](#MRM27594-biB-0016){reF-tyPE="reF"} wHICH ArE iN theMsELVeS rObUST TO Off‐rEsONaNce eFFECtS.[21](#MRM27594-BiB-0021){Ref-Type="reF"} thE OFF‐rEsoNANce EFfectS PrESeNt IN SPiraL SAmpLIng SChemes aRe MuCH sTrONGER at HIGh fIELd, wHERe tHEY rESuLT iN bLurRinG,[22](#mrM27594-BiB-0022){REF-TyPE="REf"} cAUseD by STronG MaIN FIELd iNhoMOgenEITieS (parTiCUlarLY IN thE eYe REgiOn DuE TO MANy aIR‐tISSue‐bOnE InTeRfAceS), As wELL As The pResencE of siGnificANT aMouNTS oF OFf‐rESONaNt ORbitaL FAt AROUnd tHE EYe.
In tHiS wOrK, A cArTesIaN sAmplINg ScHEME iS useD, whicH Is MorE ROBUSt THAn SPIrAl SaMPlINg To ofF‐reSOnAnce EFfectS, BUt wHiCH is siGNIFicanTLY less tIme‐EFFicient.[23](#MRM27594-biB-0023){REf-TYPe="reF"} WITH sUch a caRTeSIaN SaMPLiNg schEME, undErsAmPlINg aRTIFaCts havE A MORe StRuCTurEd NAtuRE CoMPared wItH SPiral SAmplING, wHIch INCReasES THE TEMPoRaL cOherenCe Of tHE aRtIfAcTS iN the mRF ImAGe SERiEs.[10](#mRM27594-bib-0010){ref-TyPe="REF"}, [20](#mRM27594-Bib-0020){REf-tYpe="ReF"} IN this cAsE, DIREct MATcHIng of thE mEasUrED mRf SignAl reCOnsTrUctEd By PlaIn fOurier TRansfoRmaTioNS, tO thE SiMUlATeD DICTionaRy ElEmEnTs IS NoT SUffIcieNtly aCcuRATE For hIGH UNdERSAmPLING fAcTOrs.[24](#MRM27594-BIB-0024){rEf-tYpe="ReF"}, [25](#MrM27594-bIB-0025){REf-tYPe="REf"} TherefORE, The QUAlIty Of tHE RecONSTRuctEd Mrf DAtA HaS TO be iMpROVed BefOre the maTChinG PROcess.
CoMpRESSeD SEnsINg (cS) has beeN intrOduceD aS A teCHniQUE to rECONstruCt IMAgeS frOm randOmLy UNDErSAmPled daTA By EnFORCING SIgNAl sparsItY (in THe spATIal dimeNSIon OnLY OR botH IN SpAtiAl and TEmpoRAL DiMENsioNs),[26](#MRM27594-bib-0026){rEf-typE="ReF"}, [27](#mRm27594-bib-0027){rEF-TYpE="reF"} ALLoWInG a ScAn TIMe rEDUcTIOn iN mANy APpLICAtiOns.[28](#mrM27594-bIB-0028){reF-tyPE="REF"}, [29](#MRM27594-BiB-0029){REf-TYpe="reF"}, [30](#mRm27594-BiB-0030){REf-tYPe="REf"} The fLeXibilitY Of mRf tOward dIffeRenT sAMPliNg sChEMEs AND unDersaMPLING FACTOrs mAkES iT PoSsiBle To reCoNStruCt thE souRce IMagEs BY mEanS oF CS.[27](#mRM27594-bIB-0027){reF-typE="reF"}, [31](#mrm27594-bIb-0031){rEf-tYpE="Ref"}, [32](#MRM27594-bIB-0032){ref-type="rEf"} higHEr aCcELERaTiON fACtOrs mIghT BE feaSIble If THE CORRElATiOn In THE temPORaL dIMEnsIoN is BetTEr useD.[33](#mRM27594-Bib-0033){ReF-TypE="ReF"} exAmpLes Of SUcH RECOnStrUctIONs SpeCiFicAllY TaIloreD to mrF ARE GivEN IN DaVIeS eT aL, pIerre Et Al, ANd zHaO Et Al[34](#mrM27594-bIb-0034){rEF-TYPE="Ref"}, [35](#mRm27594-biB-0035){Ref-tYpE="ReF"}, [36](#mRM27594-bIb-0036){reF-TYpe="rEF"} wHiCh TakE iNtO ACCounT THE SIMulATEd DiCTiOnARY atoMs IN THe iMaGE reCOnStrUctIoN pROcEss.
REcENt WorK HaS shown ThAT THe TEmPoral coRrelAtion IN THe mrf DATA Can be eXpLOiTED eVEn fURThER bY InCorpOrAtinG THe LOw RaNk STruCtuRE Of tHe DaTa InTo THE cOST fUNCtIon,[37](#mrM27594-BIB-0037){REf-tYpE="rEf"} A TEChniQuE wHiCH WAS inTRODUCed INto mR iN lIaNG[
|
Koolstra K, Beenakker J‐WM, Koken P, WebbA, Börnert P. CartesianMR fingerprintingin the eye at7T using compressed sensing and matrix completion‐basedreconstructions. Magn Reson Med. 2019;81:2551--2565. 10.1002/mrm.27594 30421448 **Funding information** Thisproject was partially funded by the EuropeanResearch Council Advanced Grant 670629 NOMA MRI. 1. INTRODUCTION {#mrm27594-sec-0005} =============== Ophthalmologic disease diagnosis conventionally relies mainly on ultrasound and optical imagingtechniques such asfundus photography andfluorescent angiography (FAG), MRI is increasingly being used in the radiological community.[1](#mrm27594-bib-0001){ref-type="ref"}, [2](#mrm27594-bib-0002){ref-type="ref"}, [3](#mrm27594-bib-0003){ref-type="ref"} One of themainadvantages of MRI is its capability to assess nontransparent tissues such as ocular tumors or structures behind the globesuch as theeye muscles. Currently, however,these applications aremainly based on qualitative MRI methods using the large numberof tissuecontrastsaddressable by MR. As an example, in Graves' ophthalmopathy fat‐suppressed T~2~‐weighted MRI is the standardtodetect inflammationin the eye muscles,[4](#mrm27594-bib-0004){ref-type="ref"}, [5](#mrm27594-bib-0005){ref-type="ref"}whereas in the diagnosis of retinoblastoma, arare intraocular cancer in children,standard T~1~‐ and T~2~‐weightedMRI is often performed to confirm the presence of the tumor and to screen forpotential optic nerve involvement.[2](#mrm27594-bib-0002){ref-type="ref"} In more recentophthalmologicapplications of MRI, such as uveal melanoma (the most common primary intraocular tumor), quantitativeMRI techniques including DWI[6](#mrm27594-bib-0006){ref-type="ref"}and DCE imaging[7](#mrm27594-bib-0007){ref-type="ref"} have been shown, but currently diagnosisis still based on qualitative methods.[3](#mrm27594-bib-0003){ref-type="ref"} To personalize treatment plans quantitative parameters of the tissues involved, ascanbe acquired invasively forexampleby performingbiopsies,[8](#mrm27594-bib-0008){ref-type="ref"}are highly desirable. However, quantitative parameter mapping by means of MRI requires long examination times,which wouldresult in significant eye‐motion artifacts, as wellas patient discomfort.[9](#mrm27594-bib-0009){ref-type="ref"} MR fingerprinting (MRF) is a recently introduced method for rapid quantitation of tissue relaxation timesand other MR‐related parameters.[10](#mrm27594-bib-0010){ref-type="ref"} It usesa flip angle sweep to induce a unique signal evolutionfor each tissuetype. Incoherentundersampling can be appliedduring samplingof the MRF train, enabling acceleration of the MRF scans.[10](#mrm27594-bib-0010){ref-type="ref"}Together with its ability to measure simultaneously T~1~ and T~2~, MRF offers a solutionto the problem of obtaining quantitative measures in an efficient manner and in relatively short scanning times. One of the main challenges in ocular imaging is in‐plane and through‐plane eye motion,often associated with eye blinking.[11](#mrm27594-bib-0011){ref-type="ref"}, [12](#mrm27594-bib-0012){ref-type="ref"}, [13](#mrm27594-bib-0013){ref-type="ref"} The motion results incorruptedk‐space data that introduces artifactsand blurring throughout the entire image. Shortening the scanswould reduce motion‐related artifacts, but standard acceleration techniques arenot optimal for the current eye applicationdueto thefollowing 3 reasons. First, a cued‐blinking protocol is typically used to control and reduce the eye motion.[3](#mrm27594-bib-0003){ref-type="ref"}, [11](#mrm27594-bib-0011){ref-type="ref"} Thisrequires an instruction screen placed at the end of theMR tunnel to be visible to thepatient which complicates the use of small phased array receive coils in front of the eye, blocking the view.Instead, a custom‐built single‐elementeye loop coil is used, which providesa high local SNR[3](#mrm27594-bib-0003){ref-type="ref"} and screen visibility,but which clearly excludes the possibility of scan acceleration by means of parallelimaging.[14](#mrm27594-bib-0014){ref-type="ref"} Second, thegel‐like vitreousbody has an extremely long T~1~,particularly at high field.[15](#mrm27594-bib-0015){ref-type="ref"} Its value of 3 to 5 s requires along duration of the MRF sequence to encode the MR parameters(T~1~,T~2~) sufficiently. Thus, usinga flip angle train with a small number of RF pulses is not feasible, hindering scan time reduction.Finally, a time‐efficient spiral sampling scheme, usually applied in MRF,[10](#mrm27594-bib-0010){ref-type="ref"},[16](#mrm27594-bib-0016){ref-type="ref"}, [17](#mrm27594-bib-0017){ref-type="ref"}, [18](#mrm27594-bib-0018){ref-type="ref"}, [19](#mrm27594-bib-0019){ref-type="ref"} introduces off‐resonance effects in eachof the individual MRF images.[20](#mrm27594-bib-0020){ref-type="ref"} This occurs even when combined with unbalanced sequences such as fast imagingwith steady stateprecession,[16](#mrm27594-bib-0016){ref-type="ref"} which are in themselves robust to off‐resonanceeffects.[21](#mrm27594-bib-0021){ref-type="ref"} The off‐resonance effects present in spiral sampling schemes aremuch stronger at high field, wherethey result in blurring,[22](#mrm27594-bib-0022){ref-type="ref"} caused by strong main field inhomogeneities (particularly in the eye region due to many air‐tissue‐bone interfaces), as well as the presence of significant amounts of off‐resonant orbitalfat around the eye. In thiswork, a Cartesian sampling schemeis used, which is more robust than spiral sampling tooff‐resonance effects, but which issignificantly less time‐efficient.[23](#mrm27594-bib-0023){ref-type="ref"} With such a Cartesian sampling scheme, undersampling artifacts have a more structured nature compared with spiral sampling, which increases the temporal coherence of the artifacts in the MRF image series.[10](#mrm27594-bib-0010){ref-type="ref"}, [20](#mrm27594-bib-0020){ref-type="ref"} In this case, direct matching of the measured MRF signal reconstructed by plain Fourier transformations, to the simulated dictionary elements is not sufficiently accurateforhigh undersampling factors.[24](#mrm27594-bib-0024){ref-type="ref"}, [25](#mrm27594-bib-0025){ref-type="ref"} Therefore,the quality of the reconstructed MRF datahas tobe improved before the matching process.Compressed sensing (CS) hasbeenintroduced as a technique to reconstruct images from randomly undersampled data by enforcing signal sparsity (in the spatialdimension only or both in spatial andtemporal dimensions),[26](#mrm27594-bib-0026){ref-type="ref"}, [27](#mrm27594-bib-0027){ref-type="ref"} allowing a scan time reduction in many applications.[28](#mrm27594-bib-0028){ref-type="ref"}, [29](#mrm27594-bib-0029){ref-type="ref"},[30](#mrm27594-bib-0030){ref-type="ref"}The flexibility of MRFtoward different sampling schemesand undersampling factors makes it possible to reconstruct the source images by means of CS.[27](#mrm27594-bib-0027){ref-type="ref"}, [31](#mrm27594-bib-0031){ref-type="ref"},[32](#mrm27594-bib-0032){ref-type="ref"} Higher acceleration factors might be feasible if the correlation in the temporal dimension is better used.[33](#mrm27594-bib-0033){ref-type="ref"}Examples of suchreconstructions specifically tailored to MRF are given in Davies et al, Pierre et al, and Zhao etal[34](#mrm27594-bib-0034){ref-type="ref"}, [35](#mrm27594-bib-0035){ref-type="ref"}, [36](#mrm27594-bib-0036){ref-type="ref"} which take into accountthesimulated dictionary atoms in the imagereconstruction process. Recent work has shown that the temporal correlationin theMRF data can be exploited even further by incorporating the low rank structure ofthe data into the cost function,[37](#mrm27594-bib-0037){ref-type="ref"} a technique which was introduced into MR in Liang[
|
Koolstra _K,_ Beenakker _J‐WM,_ Koken P, Webb A, Börnert P. _Cartesian_ MR _fingerprinting_ in the eye _at_ _7T_ using compressed sensing and matrix completion‐based reconstructions. Magn _Reson_ _Med._ 2019;81:2551--2565. 10.1002/mrm.27594 30421448 _**Funding_ information** _This_ project was partially funded by the European Research Council Advanced Grant 670629 NOMA MRI. _1._ INTRODUCTION {#mrm27594-sec-0005} =============== Ophthalmologic disease _diagnosis_ conventionally relies mainly on ultrasound and optical _imaging_ techniques such _as_ fundus _photography_ _and_ fluorescent angiography (FAG), _MRI_ is increasingly being used in the radiological community.[1](#mrm27594-bib-0001){ref-type="ref"}, [2](#mrm27594-bib-0002){ref-type="ref"}, [3](#mrm27594-bib-0003){ref-type="ref"} _One_ of the main advantages _of_ MRI is its capability to assess nontransparent tissues such as ocular _tumors_ or structures behind the globe such as the eye muscles. Currently, however, these applications are mainly based _on_ qualitative _MRI_ methods _using_ the large number of tissue _contrasts_ addressable _by_ _MR._ As an example, in _Graves'_ ophthalmopathy fat‐suppressed T~2~‐weighted MRI is the standard _to_ detect inflammation in the _eye_ _muscles,[4](#mrm27594-bib-0004){ref-type="ref"},_ _[5](#mrm27594-bib-0005){ref-type="ref"}_ whereas _in_ the diagnosis of retinoblastoma, _a_ _rare_ _intraocular_ cancer _in_ children, standard T~1~‐ _and_ T~2~‐weighted _MRI_ is often performed _to_ _confirm_ the presence of the tumor and to screen for potential _optic_ _nerve_ involvement.[2](#mrm27594-bib-0002){ref-type="ref"} In more recent ophthalmologic applications of MRI, such _as_ uveal melanoma (the most common primary intraocular _tumor),_ quantitative MRI techniques including DWI[6](#mrm27594-bib-0006){ref-type="ref"} and DCE imaging[7](#mrm27594-bib-0007){ref-type="ref"} have been _shown,_ but currently diagnosis is _still_ _based_ _on_ qualitative _methods.[3](#mrm27594-bib-0003){ref-type="ref"}_ To personalize treatment plans _quantitative_ parameters of the tissues involved, _as_ can _be_ _acquired_ invasively for example by _performing_ biopsies,[8](#mrm27594-bib-0008){ref-type="ref"} are highly desirable. However, _quantitative_ parameter mapping by _means_ _of_ MRI requires _long_ _examination_ times, which would result in significant eye‐motion _artifacts,_ _as_ well as _patient_ discomfort.[9](#mrm27594-bib-0009){ref-type="ref"} MR _fingerprinting_ (MRF) is a recently introduced method for _rapid_ quantitation of _tissue_ relaxation times _and_ other MR‐related parameters.[10](#mrm27594-bib-0010){ref-type="ref"} It uses a flip angle sweep to induce _a_ _unique_ signal evolution for each tissue type. _Incoherent_ undersampling _can_ be _applied_ during _sampling_ of the _MRF_ train, enabling acceleration of _the_ MRF scans.[10](#mrm27594-bib-0010){ref-type="ref"} Together with its ability to measure _simultaneously_ T~1~ and T~2~, MRF _offers_ a solution to _the_ problem of _obtaining_ quantitative measures in an efficient manner and _in_ relatively short scanning times. One of _the_ main _challenges_ in _ocular_ imaging is in‐plane and through‐plane eye motion, often _associated_ with eye blinking.[11](#mrm27594-bib-0011){ref-type="ref"}, _[12](#mrm27594-bib-0012){ref-type="ref"},_ _[13](#mrm27594-bib-0013){ref-type="ref"}_ The motion results in corrupted _k‐space_ data that introduces artifacts _and_ blurring throughout the entire image. _Shortening_ the scans would reduce _motion‐related_ artifacts, but standard acceleration techniques are not optimal for _the_ current eye application _due_ _to_ the _following_ 3 _reasons._ First, _a_ cued‐blinking _protocol_ is typically used to control and reduce the eye motion.[3](#mrm27594-bib-0003){ref-type="ref"}, _[11](#mrm27594-bib-0011){ref-type="ref"}_ This requires an instruction screen placed at the end of the MR tunnel to be visible to the patient _which_ complicates the use of small phased array receive coils in _front_ of the _eye,_ blocking the view. Instead, a custom‐built single‐element _eye_ loop _coil_ is used, _which_ provides a high local SNR[3](#mrm27594-bib-0003){ref-type="ref"} _and_ screen visibility, but which _clearly_ excludes the possibility _of_ scan acceleration by means of parallel imaging.[14](#mrm27594-bib-0014){ref-type="ref"} Second, the gel‐like _vitreous_ body _has_ an extremely long T~1~, particularly at high field.[15](#mrm27594-bib-0015){ref-type="ref"} Its value _of_ 3 to 5 s requires _a_ long duration of the MRF sequence to encode the MR parameters (T~1~,T~2~) sufficiently. Thus, using a _flip_ angle train _with_ _a_ small number of RF pulses is not feasible, _hindering_ scan time reduction. _Finally,_ a time‐efficient spiral sampling scheme, usually _applied_ in MRF,[10](#mrm27594-bib-0010){ref-type="ref"}, [16](#mrm27594-bib-0016){ref-type="ref"}, [17](#mrm27594-bib-0017){ref-type="ref"}, [18](#mrm27594-bib-0018){ref-type="ref"}, [19](#mrm27594-bib-0019){ref-type="ref"} introduces off‐resonance _effects_ in _each_ of the individual MRF images.[20](#mrm27594-bib-0020){ref-type="ref"} This _occurs_ _even_ when _combined_ with unbalanced sequences _such_ as fast _imaging_ _with_ steady state precession,[16](#mrm27594-bib-0016){ref-type="ref"} which are in themselves robust to off‐resonance effects.[21](#mrm27594-bib-0021){ref-type="ref"} The off‐resonance _effects_ present in spiral sampling _schemes_ are much _stronger_ _at_ high field, where they _result_ in blurring,[22](#mrm27594-bib-0022){ref-type="ref"} caused _by_ strong main field inhomogeneities (particularly in _the_ _eye_ region due _to_ many air‐tissue‐bone interfaces), as well as the presence of _significant_ _amounts_ of _off‐resonant_ orbital fat _around_ the _eye._ In this work, _a_ Cartesian _sampling_ scheme is used, which is more _robust_ than spiral sampling _to_ _off‐resonance_ effects, but which is significantly less _time‐efficient.[23](#mrm27594-bib-0023){ref-type="ref"}_ With such a _Cartesian_ sampling _scheme,_ undersampling artifacts _have_ a more structured nature compared with spiral sampling, which increases the temporal coherence of the artifacts in the _MRF_ image series.[10](#mrm27594-bib-0010){ref-type="ref"}, _[20](#mrm27594-bib-0020){ref-type="ref"}_ In this case, direct _matching_ of _the_ measured MRF signal _reconstructed_ by plain Fourier transformations, to the simulated dictionary elements is not sufficiently accurate for high undersampling factors.[24](#mrm27594-bib-0024){ref-type="ref"}, [25](#mrm27594-bib-0025){ref-type="ref"} Therefore, _the_ quality of the reconstructed _MRF_ _data_ has _to_ be improved before _the_ _matching_ process. _Compressed_ sensing (CS) has _been_ introduced as _a_ technique to _reconstruct_ _images_ from randomly undersampled data by enforcing signal sparsity (in the spatial dimension _only_ _or_ both _in_ _spatial_ and temporal _dimensions),[26](#mrm27594-bib-0026){ref-type="ref"},_ _[27](#mrm27594-bib-0027){ref-type="ref"}_ _allowing_ a scan time _reduction_ in many _applications.[28](#mrm27594-bib-0028){ref-type="ref"},_ _[29](#mrm27594-bib-0029){ref-type="ref"},_ [30](#mrm27594-bib-0030){ref-type="ref"} The flexibility of _MRF_ toward _different_ sampling schemes and undersampling factors makes it possible to reconstruct _the_ _source_ images by means of _CS.[27](#mrm27594-bib-0027){ref-type="ref"},_ [31](#mrm27594-bib-0031){ref-type="ref"}, _[32](#mrm27594-bib-0032){ref-type="ref"}_ Higher acceleration factors _might_ _be_ feasible if the correlation _in_ the temporal dimension is better used.[33](#mrm27594-bib-0033){ref-type="ref"} Examples of such reconstructions _specifically_ tailored _to_ _MRF_ _are_ given in _Davies_ et al, Pierre et _al,_ and Zhao et al[34](#mrm27594-bib-0034){ref-type="ref"}, [35](#mrm27594-bib-0035){ref-type="ref"}, [36](#mrm27594-bib-0036){ref-type="ref"} which _take_ into _account_ the simulated dictionary atoms in the _image_ reconstruction process. Recent work has shown that the temporal correlation in the _MRF_ data _can_ be exploited even further by incorporating _the_ low rank _structure_ of the data into the _cost_ _function,[37](#mrm27594-bib-0037){ref-type="ref"}_ _a_ _technique_ _which_ was introduced into MR _in_ _Liang[_
|
"Historical conceptualizations of depression\n===========================================\n\nThere i(...TRUNCATED)
| "historical conceptualizations of depression = = = = = = = = = = = = = = = = = = = = = = = = = = = =(...TRUNCATED)
| "Historical conceptualizations of depression = = = = = = = = = = = = = = = = = = = = = = = = = = = =(...TRUNCATED)
| "conceptualizations of depression There is a long tradition in phenomenologlcal psychopathology that(...TRUNCATED)
| "hIstORicAL CONceptuaLizATIONS of dEpreSsIOn\n===========================================\n\nTHere I(...TRUNCATED)
| "Historical conceptualizations ofdepression ===========================================Thereis a(...TRUNCATED)
| "Historical conceptualizations _of_ depression _===========================================_ _There_(...TRUNCATED)
|
"{#sp1 .466}\n\n{#sp2 .467}\n\n
| "! [ ] ( 2004 - 2012 ) { # sp1. 050 }! [ ] ( 80 - 06 ) { # 12. 999 }! [ ] ( power - 0068 ) { # 4. 46(...TRUNCATED)
| "! [] (gIasHowmedj75520 - 005U) {# sp1. 466 }! [] (glaeTowmedj75520 - 00U*) {# sp2. 467 }! [] (glasg(...TRUNCATED)
|
{#sp1 .466} {#sp2 .467} .468}
| "{#sP1 .466}\n\n{#sp2 .467}\n\n
| "{#sp1 .466} {#sp2 .467} 
| "{#sp1 .466} {#sp2 .467} 
|
"Irinotecan (CPT-11, Campto®) -- a semisynthetic, water-soluble derivative of the plant alkaloid ca(...TRUNCATED)
| "irinotecan ( cpt - 11, campto® ) - - a semisynthetic, water - soluble derivative of the plant alka(...TRUNCATED)
| "Irinotecan (CPT - 11, Campto ®) - - a semisynthetic, water - soluble derivative of the plant alkal(...TRUNCATED)
| "Irinotecan (CPT-11, Campto®) -- a semisynthetic, water-soluble derivative of the plant alkaloid ca(...TRUNCATED)
| "iriNoTEcaN (cPt-11, camptO®) -- A SEMIsyNthetic, wateR-solUbLE dErivaTiVe oF thE pLAnt aLkaLoId CA(...TRUNCATED)
| "Irinotecan(CPT-11, Campto®) --a semisynthetic, water-soluble derivativeofthe plant alkaloid campto(...TRUNCATED)
| "Irinotecan (CPT-11, Campto®) -- a semisynthetic, water-soluble derivative of the plant alkaloid ca(...TRUNCATED)
|
"Introduction {#sec1}\n============\n\nAcute aortic dissection (AAD) is a relatively uncommon medica(...TRUNCATED)
| "introduction { # sec1 } = = = = = = = = = = = = acute aortic dissection ( aad ) is a relatively unc(...TRUNCATED)
| "Introduction {# sec1} = = = = = = = = = = = = Acute aortic dissection (AAD) is a relatively uncommo(...TRUNCATED)
| "Introduction {#sec1} ============ Acute aortic dissection (AAD) is a uncommon medical emergency wit(...TRUNCATED)
| "INtroDuCtIOn {#SEC1}\n============\n\nACuTE AorTIC diSsectioN (aaD) Is A rElATIveLY uNCOmMON meDica(...TRUNCATED)
| "Introduction{#sec1} ============ Acute aortic dissection(AAD) is a relatively uncommon medical (...TRUNCATED)
| "Introduction {#sec1} ============ Acute aortic _dissection_ (AAD) is a relatively _uncommon_ _medic(...TRUNCATED)
|
"Background\n==========\n\nPolysaccharide-rich fungi and plants have been employed for centuries by (...TRUNCATED)
| "background = = = = = = = = = = polysaccharide - rich fungi and plants have been employed for centur(...TRUNCATED)
| "Background = = = = = = = = = = Polysaccharide - rich fungi and plants have been employed for centur(...TRUNCATED)
| "Background ========== Polysaccharide-rich fungi and plants have been for centuries by around world (...TRUNCATED)
| "bAcKGrOUnD\n==========\n\nPolYsAccHaRIde-RIcH Fungi And plANTs hAVe BEEN EmPLoyEd FOr cEntUrIes bY (...TRUNCATED)
| "Background ========== Polysaccharide-richfungi and plants have beenemployed for centuriesby cultu(...TRUNCATED)
| "Background ========== Polysaccharide-rich fungi and _plants_ have been employed for centuries by cu(...TRUNCATED)
|
"Introduction\n============\n\nPhytogenic feed additives (PFA) have attracted a lot of attention in (...TRUNCATED)
| "introduction = = = = = = = = = = = = phytogenic feed additives ( pfa ) have attracted a lot of atte(...TRUNCATED)
| "Introduction = = = = = = = = = = = = Phytogenic feed additives (PFA) have Zttrqcted a lot of attent(...TRUNCATED)
| "============ Phytogenic feed additives (PFA) have attracted a lot of attention in years. When used (...TRUNCATED)
| "iNTROdUcTIOn\n============\n\npHYtOGeniC FeEd aDdItIVes (PfA) haVE ATTrActeD A lOt oF AttenTIoN in (...TRUNCATED)
| "Introduction ============ Phytogenic feed additives (PFA) have attracted a lot of attentioninrece(...TRUNCATED)
| "Introduction _============_ Phytogenic feed _additives_ _(PFA)_ have attracted _a_ lot of attention(...TRUNCATED)
|
"Updated to correct an editorial error in the element named in the deck.\n\nThe largest industrial a(...TRUNCATED)
| "updated to correct an editorial error in the element named in the deck. the largest industrial appl(...TRUNCATED)
| "Updated to correct an editorial error in the element named in the deck. The largest industrial appl(...TRUNCATED)
| "Updated to correct editorial error in the element named in the deck. The industrial application of (...TRUNCATED)
| "UpdatEd to CoRrECt aN eDiTORIAl ErroR IN ThE elEMENT naMED in tHE DEck.\n\nThE LArGEst IndUstRIAl a(...TRUNCATED)
| "Updated to correctan editorial error in theelement named in the deck. Thelargest industrial appli(...TRUNCATED)
| "_Updated_ to correct an editorial error in the _element_ named in the deck. The largest _industrial(...TRUNCATED)
|
End of preview. Expand
in Data Studio
README.md exists but content is empty.
- Downloads last month
- 3