Walking and Running of Children with Decreased Femoral Torsion.

Understanding the implications of decreased femoral torsion on gait and running in children and adolescents might help orthopedic surgeons optimize treatment decisions. To date, there is limited evidence regarding the kinematic gait deviations between children with decreased femoral torsion and typically developing children, as well as the implications of the same on the adaptation of walking to running. A three-dimensional gait analysis study was undertaken to compare gait deviations during running and walking among patients with decreased femoral torsion (n = 15) and typically developing children (n = 11). Linear mixed models were utilized to establish comparisons within and between the two groups and investigate the relationship between clinical examination, spatial parameters, and the difference in hip rotation between running and walking. Patients exhibited increased external hip rotation during walking in comparison to controls, accompanied by higher peaks for the same as well as for knee valgus and external foot progression angle. A similar kinematic gait pattern was observed during running, with significant differences noted in peak knee valgus. In terms of variations from running to walking, patients internally rotated their initially externally rotated hip by 4°, whereas controls maintained the same internal hip rotation. Patients and controls displayed comparable kinematic gait deviations during running compared to walking. The passive hip range of motion, torsions, and velocity did not notably influence the variation in mean hip rotation from running to walking. This study underlines the potential of 3D gait kinematics to elucidate the functional implications of decreased FT and, hence, may contribute to clinical decision making.

Antibody verification of HLA class I and class II eplets by human monoclonal HLA antibodies.

In solid organ transplantation, formation of de novo donor-specific HLA antibodies is induced by mismatched eplets on donor HLA molecules. While several studies have shown a strong correlation between the number of eplet mismatches and inferior outcomes, not every eplet mismatch is immunogenic. Eplets are theoretically defined entities, necessitating formal proof that they can be recognised and bound by antibodies. This antibody verification is pivotal to ensure that clinically relevant eplets are considered in studies on molecular matching. Recombinant human HLA-specific monoclonal antibodies (mAbs) were generated from HLA-reactive B cell clones isolated from HLA immunised individuals using recombinant HLA molecules. Subsequently, the reactivity patterns of the mAbs obtained from single antigen bead assay were analysed using HLA-EMMA software to identify single or configurations of solvent accessible amino acids uniquely present on the reactive HLA alleles and were mapped to eplets. Two HLA class I and seven HLA class II-specific human mAbs were generated from four individuals. Extensive mAb reactivity analysis, led to antibody verification of three HLA-DR-specific eplets, and conversion of five eplets (one HLA-A, one HLA-B, two HLA-DR, and one HLA-DP), from provisionally verified to truly antibody-verified. Finally, one HLA-DQ-specific eplet was upgraded from level A2 to level A1 verification evidence. The generation of recombinant human HLA-specific mAbs with different specificities contributes significantly to the antibody verification of eplets and therefore is instrumental for implementation of eplet matching in the clinical setting.

HLA-DQ-Specific Recombinant Human Monoclonal Antibodies Allow for In-Depth Analysis of HLA-DQ Epitopes.

HLA-DQ donor-specific antibodies (DSA) are the most prevalent type of DSA after renal transplantation and have been associated with eplet mismatches between donor and recipient HLA. Eplets are theoretically defined configurations of surface exposed amino acids on HLA molecules that require verification to confirm that they can be recognized by alloantibodies and are therefore clinically relevant. In this study, we isolated HLA-DQ specific memory B cells from immunized individuals by using biotinylated HLA-DQ monomers to generate 15 recombinant human HLA-DQ specific monoclonal antibodies (mAb) with six distinct specificities. Single antigen bead reactivity patterns were analyzed with HLA-EMMA to identify amino acids that were uniquely shared by the reactive HLA alleles to define functional epitopes which were mapped to known eplets. The HLA-DQB1*03:01-specific mAb LB_DQB0301_A and the HLA-DQB1*03-specific mAb LB_DQB0303_C supported the antibody-verification of eplets 45EV and 55PP respectively, while mAbs LB_DQB0402_A and LB_DQB0602_B verified eplet 55R on HLA-DQB1*04/05/06. For three mAbs, multiple uniquely shared amino acid configurations were identified, warranting further studies to define the inducing functional epitope and corresponding eplet. Our unique set of HLA-DQ specific mAbs will be further expanded and will facilitate the in-depth analysis of HLA-DQ epitopes, which is relevant for further studies of HLA-DQ alloantibody pathogenicity in transplantation.

Generation and reactivity analysis of human recombinant monoclonal antibodies directed against epitopes on HLA-DR.

In kidney transplantation, eplet mismatches between donor and recipient have been associated with de novo donor-specific antibody development. Eplets are theoretically defined configurations of polymorphic amino acids and require experimental verification to establish whether they can be bound by alloantibodies. Human HLA-specific monoclonal antibodies (mAbs) have been instrumental for this purpose but are largely lacking for HLA class II. In this study, we isolated single HLA-DR-specific memory B cells from peripheral blood of immunized individuals (n = 3) using HLA class II tetramers to generate recombinant human HLA-DR antigen-reactive mAbs (n = 5). Comparison of the amino acid composition of the reactive HLA alleles in relation to the antibody reactivity patterns led to identification of 3 configurations, 70Q 73A, 31F 32Y 37Y, and 14K 25Q recognized, respectively, by HLA-DRB1*01:01, HLA-DRB1*04:01, and HLA-DRB1*07:01 antigen-reactive mAbs. The first 2 correspond to eplets 70QA and 31FYY and can now be considered antibody verified. The latter indicates that eplet 25Q needs to be redefined before being considered as antibody verified. Generation and reactivity analysis of human HLA-DR mAbs allowed for identification of amino acid configurations corresponding to known eplets, whereas the other patterns may be used to redefine eplets with similar, but not identical predicted amino acid composition.

Recombinant human monoclonal HLA antibodies of different IgG subclasses recognising the same epitope: Excellent tools to study differential effects of donor-specific antibodies.

In the field of transplantation, the humoural immune response against mismatched HLA antigens of the donor is associated with inferior graft survival, but not in every patient. Donor-specific HLA antibodies (DSA) of different immunoglobulin G (IgG) subclasses may have differential effects on the transplanted organ. Recombinant technology allows for the generation of IgG subclasses of a human monoclonal antibody (mAb), while retaining its epitope specificity. In order to enable studies on the biological function of IgG subclass HLA antibodies, we used recombinant technology to generate recombinant human HLA mAbs from established heterohybridomas. We generated all four IgG subclasses of a human HLA class I and class II mAb and showed that the different subclasses had a comparable affinity, normal human Fc glycosylation, and retained HLA epitope specificity. For both mAbs, the IgG1 and IgG3 isotypes were capable of binding complement component 3d (C3d) and efficient in complement-dependent cell lysis against their specific targets, while the IgG2 and IgG4 subclasses were not able to induce cytotoxicity. Considering the fact that the antibody-binding site and properties remained unaffected, these IgG subclass HLA mAbs are excellent tools to study the function of individual IgG subclass HLA class I and class II-specific antibodies in a controlled fashion.

The avidity of cross-reactive virus-specific T cells for their viral and allogeneic epitopes is variable and depends on epitope expression.

Virus-specific T cells can recognize allogeneic HLA (allo-HLA) through cross-reactivity of their T-cell receptor (TCR). In a transplantation setting, such allo-HLA cross-reactivity may contribute to harmful immune responses towards the allograft, provided that the cross-reactive T cells get sufficiently activated upon recognition of the allo-HLA. An important determinant of T-cell activation is TCR avidity, which to date, has remained largely unexplored for allo-HLA-cross-reactive virus-specific T cells. For this purpose, cold target inhibition assays were performed using allo-HLA-cross-reactive virus-specific memory CD8+ T-cell clones as responders, and syngeneic cells loaded with viral peptide and allogeneic cells as hot (radioactively-labeled) and cold (non-radioactively-labeled) targets. CD8 dependency of the T-cell responses was assessed using interferon γ (IFNγ) enzyme-linked immunosorbent assay (ELISA) in the presence and absence of CD8-blocking antibodies. At high viral-peptide loading concentrations, T-cell clones consistently demonstrated lower avidity for allogeneic versus viral epitopes, but at suboptimal concentrations the opposite was observed. In line, anti-viral reactivity was CD8 independent at high, but not at suboptimal viral-peptide-loading concentrations. The avidity of allo-HLA-cross-reactive virus-specific memory CD8+ T cells is therefore highly dependent on epitope expression, and as a consequence, can be both higher and lower for allogeneic versus viral targets under different (patho)physiological conditions.

Pregnancy complications in singleton pregnancies with isolated fetal heart defects.

INTRODUCTION: As the prenatal detection rates of congenital heart defects (CHDs) increase, obstetricians are more frequently faced with pregnancies complicated by a fetal CHD. Congenital anomalies in general are associated with preterm birth and fetal demise. The aim of this study was to gain insight into the prevalence of preterm birth and fetal demise in singleton pregnancies with fetuses with isolated CHDs. MATERIAL AND METHODS: A geographical cohort study was performed in a large region in the Netherlands. Fetuses and infants from singleton pregnancies diagnosed with severe isolated CHD, born between 1 January 2002 and 1 January 2012, were included. All cases in the CHD cohort were assessed for preterm birth or fetal demise. The proportions of preterm birth and fetal demise were compared with those in a control group and odds ratios were calculated. RESULTS: The proportion of preterm births in the CHD cohort (n = 1013) was 9.1% (95% CI 7.3-10.9) compared with 5.6% (95% CI 5.4-5.8) in the control group, with an odds ratio of 1.7 (95% CI 1.4-2.1). The preterm birth started spontaneously in 49.5% and 38.4% were induced. In 15 cases fetal demise occurred (1.5%; 95% CI 0.8-2.2), compared with 0.7% (95% CI 0.6-0.8) in the control group, odds ratio 2.0 (95% CI 1.2-3.4). CONCLUSIONS: Higher rates of preterm birth and fetal demise occur in fetuses with isolated CHD compared with the general population. Prenatal specialists should be vigilant for signs of heart failure, premature closure of the foramen ovale or fetal distress in fetuses with isolated CHDs.

Chromosomal abnormalities and copy number variations in fetal left-sided congenital heart defects.

OBJECTIVES: To demonstrate the spectrum of copy number variants (CNVs) in fetuses with isolated left-sided congenital heart defects (CHDs), and analyse genetic content. METHODS: Between 2003 and 2012, 200 fetuses were identified with left-sided CHD. Exclusion criteria were chromosomal rearrangements, 22q11.2 microdeletion and/or extra-cardiac malformations (n = 64). We included cases with additional minor anomalies (n = 39), such as single umbilical artery. In 54 of 136 eligible cases, stored material was available for array analysis. CNVs were categorized as either (likely) benign, (likely) pathogenic or of unknown significance. RESULTS: In 18 of the 54 isolated left-sided CHDs we found 28 rare CNVs (prevalence 33%, average 1.6 CNV per person, size 10.6 kb-2.2 Mb). Our interpretation yielded clinically significant CNVs in two of 54 cases (4%) and variants of unknown significance in three other cases (6%). CONCLUSIONS: In left-sided CHDs that appear isolated, with normal chromosome analysis and 22q11.2 FISH analysis, array analysis detects clinically significant CNVs. When counselling parents of a fetus with a left-sided CHD it must be taken into consideration that aside from the cardiac characteristics, the presence of extra-cardiac malformations and chromosomal abnormalities influence the treatment plan and prognosis.

Characterization and quantification of dynamic eccentric regurgitation of the left atrioventricular valve after atrioventricular septal defect correction with 4D Flow cardiovascular magnetic resonance and retrospective valve tracking.

BACKGROUND: To characterize and directly quantify regurgitant jets of left atrioventricular valve (LAVV) in patients with corrected atrioventricular septal defect (AVSD) by four-dimensional (4D)Flow Cardiovascular Magnetic Resonance (CMR), streamline visualization and retrospective valve tracking. METHODS: Medical ethical committee approval and informed consent from all patients or their parents were obtained. In 32 corrected AVSD patients (age 26 ± 12 years), echocardiography and whole-heart 4DFlow CMR were performed. Using streamline visualization on 2- and 4-chamber views, the angle between regurgitation and annulus was followed throughout systole. On through-plane velocity-encoded images reformatted perpendicular to the regurgitation jet the cross-sectional jet circularity index was assessed and regurgitant volume and fraction were calculated. Correlation and agreement between different techniques was performed with Pearson's r and Spearman's rho correlation and Bland-Altman analysis. RESULTS: In 8 patients, multiple regurgitant jets over the LAVV were identified. Median variation in regurgitant jet angle within patients was 36°(IQR 18-64°) on the 2-chamber and 30°(IQR 20-40°) on the 4-chamber. Regurgitant jets had a circularity index of 0.61 ± 0.16. Quantification of the regurgitation volume was feasible with 4DFlow CMR with excellent correlation between LAVV effective forward flow and aortic flow (r = 0.97, p < 0.001) for internal validation and moderate correlation with planimetry derived regurgitant volume (r = 0.65, p < 0.001) and echocardiographic grading (rho = 0.51, p = 0.003). CONCLUSIONS: 4DFlow CMR with streamline visualization revealed multiple, dynamic and eccentric regurgitant jets with non-circular cross-sectional shape in patients after AVSD correction. 4DFlow with retrospective valve tracking allows direct and accurate quantification of the regurgitation of these complex jets.

Is there any impact of HLA-DPB1 disparity in 10/10 HLA-matched unrelated hematopoietic SCT? Results of a French multicentric retrospective study.

We retrospectively analyzed the impact of HLA-DPB1 mismatches in a large cohort of 1342 French patients who underwent 10/10 HLA-matched unrelated HSCT. A significant impact of HLA-DPB1 allelic mismatches (2 vs 0) was observed in severe acute GVHD (aGVHDIII-IV) (risk ratio (RR)=1.73, confidence interval (CI) 95% 1.09-2.73, P=0.019) without impact on OS, TRM, relapse and chronic GVHD (cGVHD). According to the T-cell epitope 3 (TCE3)/TCE4 HLA-DPB1 disparity algorithm, 37.6% and 58.4% pairs had nonpermissive HLA-DPB1, respectively. TCE3 and TCE4 disparities had no statistical impact on OS, TRM, relapse, aGVHD and cGVHD. When TCE3/TCE4 disparities were analyzed in the graft-vs-host or host-vs-graft (HVG) direction, only a significant impact of TCE4 nonpermissive disparities in the HVG direction was observed on relapse (RR=1.34, CI 95% 1.00-1.80, P=0.048). In conclusion, this French retrospective study shows an adverse prognosis of HLA-DPB1 mismatches (2 vs 0) on severe aGVHD and of nonpermissive TCE4 HVG disparities on relapse after HLA-matched 10/10 unrelated HSCT.

HLA phenotypes of candidates for HSCT: comparing transplanted versus non-transplanted candidates, resulting in the predictive estimation of the probability to find a 10/10 HLA matched donor.

In order to study the impact of human leucocyte antigen (HLA) polymorphism distribution in identifying a matched haematopoietic stem cells unrelated donor (UD), we performed a multi-centric retrospective analysis with the aim of comparing the HLA-A, HLA-B, HLA-C, HLA-DRB1 and HLA-DQB1 phenotypes of 2126 patients (772 patients for whom a donor search failed to identify a matched UD, and 1354 patients who received a 10/10 allele level matched UD). Our results showed that rare HLA-C is often responsible for difficulty in identifying a donor. This locus may add a degree of complexity to a supposed 'frequent' HLA-A HLA-B and HLA-DRB1 phenotype, turning this phenotype into a less frequent one. For example, 32.5% of the phenotypes in the non-transplanted patients could not be explained by any of the pairs of known HLA-A, HLA-B, HLA-C and HLA-DRB1 haplotypes while this percentage dropped to less than 2% if combinations of only HLA-A, HLA-B and HLA-DRB1 haplotypes were considered. Such situations can be anticipated by computing an index, based on HLA haplotype frequency, the average registry sample size (ARS). ARS is defined as the inverse of the phenotype frequency computed using all corresponding pairs of haplotype frequencies. ARS confirmed that the most significant difference between transplanted and non-transplanted patients was correlated with the introduction of the locus HLA-C in the analysis (median: 8.3e + 4 vs 3.1e + 6, P < 0.0001). The higher the ARS the lower the likelihood of finding a 10/10 match UD reflecting the rareness of the patient's HLA. The area under receiver operator characteristics (AUROC) values of the ARS computation for HLA-A, HLA-B and HLA-DRB1 was 0.82 (0.80; 0.84) at a low-resolution level (two digits). Overall, our study promotes the use of haplotype frequency-based computations to develop computer-assisted donor search.

[A report of the SFGM-TC on the interactions between national transplant coordination and local coordinators in allogeneic stem cell transplantation activity]. / Interactions entre coordination nationale de greffe et coordinateurs locaux.

In the attempt to harmonize clinical practices between different French transplantation centers, the French Society of Bone Marrow Transplantation and Cell Therapy (SFGM-TC) set up the third annual series of workshops which brought together practitioners from all member centers and took place in October 2012 in Lille. The main aim of this session was to describe the relations between the national transplant coordination office of the French registry and local stem cell transplantation coordinators throughout France.

Volumetric Computed Tomography Angiography in the Evaluation of Mediastinal Fluid Collections following Congenital Cardiac Surgery.

We present 3 patients with 4 causes of mediastinal fluid collection after congenital cardiac surgery in this extended case report. Volumetric computed tomography played an essential role in diagnosing causes and extent, relevant to subsequent management. Recent advances in volumetric computed tomography allow fast and accurate imaging of cardiovascular and extravascular structures in children with acceptable radiation dose, providing a powerful imaging tool for the evaluation of complications after congenital cardiac surgery.

Mild residual pulmonary stenosis in tetralogy of fallot reduces risk of pulmonary valve replacement.

BACKGROUND: Current surgical strategies that aim at preventing pulmonary regurgitation in patients with corrected tetralogy of Fallot (cToF) may result in a certain grade of residual pulmonary stenosis (PS). The clinical implications of a postoperative residual PS in cToF patients remain unclear. Pulmonary valve replacement (PVR) is frequently needed during follow-up of cToF patients. The aim of the current study was to determine the role of residual PS in the need for PVR during follow-up in cToF patients. METHODS: cToF patients were included if clinical follow-up after primary surgical correction had taken place for a minimum of 5 years. Patient characteristics, surgical factors, and postoperative factors were reviewed, with a special focus on the transpulmonic systolic gradient. Cox proportional hazards regression analysis was performed to identify predictors of PVR. RESULTS: Of 171 cToF patients, 71 (41.5%) underwent PVR after 24.2 years (interquartile range, 16.8-31.6 years). Year of birth, older age at corrective operation, and patch use significantly predicted PVR during follow-up. By contrast, a mild residual PS in cToF patients (peak systolic gradient, 15-30 mm Hg) independently reduced the risk of PVR, as compared with patients without PS (hazard ratio, 0.47; p=0.02) and with moderate-to-severe PS (hazard ratio, 0.35; p=0.01). CONCLUSIONS: In addition to the known risks factors for PVR, a postoperative mild residual PS reduces the risk of PVR during follow-up of cToF patients. This finding provides clinical evidence for a conservative PS relief during correction of ToF.

Disturbed myocardial connexin 43 and N-cadherin expressions in hypoplastic left heart syndrome and borderline left ventricle.

OBJECTIVES: Borderline left ventricle is the left ventricular morphology at the favorable end of the hypoplastic left heart syndrome. In contrast to the severe end, it is suitable for biventricular repair. Wondering whether it is possible to identify cases suitable for biventricular repair from a developmental viewpoint, we investigated the myocardial histology of borderline and severely hypoplastic left ventricles. METHODS: Postmortem specimens of neonatal, unoperated human hearts with severe hypoplastic left heart syndrome and borderline left ventricle were compared with normal specimens and hearts from patients with transposition of the great arteries. After tissue sampling of the lateral walls of both ventricles, immunohistochemical and immunofluorescence stainings against cardiac troponin I, N-cadherin, and connexin 43, important for proper cardiac differentiation, were done. RESULTS: All severely hypoplastic left hearts (7/7) and most borderline left ventricle hearts (4/6) showed reduced sarcomeric expressions of troponin I in left and right ventricles. N-cadherin and connexin 43 expressions were reduced in intercalated disks. The remaining borderline left ventricle hearts (2/6) were histologically closer to control hearts. CONCLUSIONS: Four of 6 borderline left ventricle hearts showed myocardial histopathology similar to the severely hypoplastic left hearts. The remainder were similar to normal hearts. Our results and knowledge regarding the role of epicardial-derived cells in myocardial differentiation lead us to postulate that an abnormal epicardial-myocardial interaction could explain the observed histopathology. Defining the histopathologic severity with preoperative myocardial biopsy samples of hearts with borderline left ventricle might provide a diagnostic tool for preoperative decision making.

[Genetic diversity and bone marrow transplantation]. / Diversité génétique et greffe de moelle osseuse.

The genetic origin of the patients, for whom a bone marrow transplantation has been proposed, is a key determinant in the possibility of identifying or not a compatible unrelated donor, and consequently in the possibility of performing the bone marrow transplantation. The required strict HLA compatibility, in the context of a bone marrow transplantation, increases the difficulty. A patient has one chance over four to have a compatible donor within his brothers and sisters, if any. This chance becomes one over a million, as an average, in the context of unrelated donor search. Taking into consideration the genetic history of the populations, their evolution and the large actual HLA diversity, the probability of finding an unrelated donor for a defined patient varies according to the frequency and the combination of the patient's HLA antigens, genetic markers inherited not only from his parents, but also from his ancestries. In the unrelated context, the HLA compatible donor most probably shares the same genetic history than the patient, and consequently belongs to the same population group. The study of the genetic of populations explains the difficulties in finding an unrelated compatible donor in the migrant populations, particularly those originated from Africa and from the middle east, due to their HLA specificities and to the small number of donors sharing the same origins registered on a volunteer bone marrow donors' file worldwide.

Long-term results of reoperation for left atrioventricular valve regurgitation after correction of atrioventricular septal defects.

BACKGROUND: Long-term results of reoperation for left atrioventricular valve regurgitation (LAVVR) after previous correction of atrioventricular septal defect (AVSD) are scarce. We evaluated long-term outcome of reoperation for LAVVR and identified risk factors for reoperation. METHODS: Between December 1976 and July 2006, 45 of 312 patients with correction of different AVSDs underwent reoperation for LAVVR. The cohort of 267 patients who did not need reoperation for LAVVR allowed for the identification of risk factors for reoperation and evaluation of overall survival after primary AVSD repair in a competing risk scenario. Clinical data were obtained by retrospective review. RESULTS: The left atrioventricular valve (LAVV) was repaired in 31 patients (68.9%) and replaced in 14 (31.1%). There were 3 in-hospital deaths (6.7%) and 2 late deaths (4.4%). Estimated overall survival was 88.1% at 15 years after the reoperation, and estimated incidence of death after reoperation in the total patient cohort was 2% at 15 years after the primary AVSD repair. Overall survival was significantly higher after LAVV repair than after replacement (p=0.010). Ten patients with LAVV repair required a second reoperation for LAVVR. At follow-up, survivors were in New York Heart Association functional class I (n=36) or II (n=4). Independent risk factors for first reoperation for LAVVR were associated cardiovascular anomalies (p<0.001), LAVV dysplasia (p<0.001), and nonclosure of the cleft (p=0.027). CONCLUSIONS: After previous correction of AVSD, LAVVR can usually be corrected by valve repair. A very dysplastic valve may necessitate replacement. Overall survival is higher after repair than after replacement. In general, overall survival of patients reoperated on for LAVVR is favorable. The overall mortality rate after primary repair of AVSD is explained only for a small part by mortality after reoperation for LAVVR.

Tissue specificity of cross-reactive allogeneic responses by EBV EBNA3A-specific memory T cells.

BACKGROUND: The crossreactivity of Epstein-Barr virus (EBV Epstein-Barr virus nuclear antigen 3A [EBNA3A])-specific CD8 T cells against allogeneic human leukocyte antigen (HLA)-B*44:02 has been shown to be dependent on presentation of self-peptide EEYLQAFTY by the target antigen. In this study, we report that allogeneic HLA-B*44:02 proximal tubular epithelial cells (PTECs) and human umbilical vein endothelial cells (HUVECs) are poor targets for EBV EBNA3A-specific T cells. METHODS: The EEY peptide was exogenously loaded onto HLA-B*44:02 and HLA-B*44:03-expressing PTECs and HUVECs. EEY-peptide-loaded, and unloaded, PTECs and HUVECs were then incubated with serial dilutions of our EBNA3A T-cell clone, in a cytotoxicity assay. RESULTS: Although HLA-B*44:02-expressing PTECs were specifically lysed in proportion to the effector/target ratio by the EBNA3A T-cell clone, without peptide loading, lysis was greatly increased by exogenous EEY peptide loading (15% vs. 75%; P<0.0001). HLA-B*44:02-expressing HUVECs were only lysed when loaded with exogenous EEY peptide (0% vs. 64%; P<0.0001). Lack of HLA expression and lack of ABCD3 gene expression were excluded as a cause for these results. PTECs and HUVECs were specifically targeted by another alloreactive T-cell clone without exogenous peptide loading, suggesting that the lack of recognition of HLA-B*44:02 epithelial and endothelial cells by the EBV EBNA3A T-cell clone was due to lack of EEYLQAFTY peptide presentation. CONCLUSIONS: Tissue-specific (peptide dependent) alloreactivity may have important implications for transplantation monitoring and rejection.

The clinical spectrum of complete FBN1 allele deletions.

The most common mutations found in FBN1 are missense mutations (56%), mainly substituting or creating a cysteine in a cbEGF domain. Other mutations are frameshift, splice and nonsense mutations. There are only a few reports of patients with marfanoid features and a molecularly proven complete deletion of a FBN1 allele. We describe the clinical features of 10 patients with a complete FBN1 gene deletion. Seven patients fulfilled the Ghent criteria for Marfan syndrome (MFS). The other three patients were examined at a young age and did not (yet) present the full clinical picture of MFS yet. Ectopia lentis was present in at least two patients. Aortic root dilatation was present in 6 of the 10 patients. In three patients, the aortic root diameter was on the 95th percentile and in one patient, the diameter of the aortic root was normal, the cross-section, however, had a cloverleaf appearance. Two patients underwent aortic root surgery at a relatively young age (27 and 34 years). Mitral valve prolapse was present in 4 of the 10 patients, and billowing of the mitral valve in 1. All patients had facial and skeletal features of MFS. Two patients with a large deletion extending beyond the FBN1 gene had an extended phenotype. We conclude that complete loss of one FBN1 allele does not predict a mild phenotype, and these findings support the hypothesis that true haploinsufficiency can lead to the classical phenotype of Marfan syndrome.