From the Cover: Distinctive properties of identical twins' TCR repertoires revealed by high-throughput sequencing

Adaptive immunity in humans is provided by hypervariable Ig-like molecules on the surface of B and T cells. The final set of these molecules in each organism is formed under the influence of two forces: individual genetic traits and the environment, which includes the diverse spectra of alien and self-antigens. Here we assess the impact of individual genetic factors on the formation of the adaptive immunity by analyzing the T-cell receptor (TCR) repertoires of three pairs of monozygous twins by next-generation sequencing. Surprisingly, we found that an overlap between the TCR repertoires of monozygous twins is similar to an overlap between the TCR repertoires of nonrelated individuals. However, the number of identical complementary determining region 3 sequences in two individuals is significantly increased for twin pairs in the fraction of highly abundant TCR molecules, which is enriched by the antigen-experienced T cells. We found that the initial recruitment of particular TCR V genes for recombination and subsequent selection in the thymus is strictly determined by individual genetic factors. J genes of TCRs are selected randomly for recombination; however, the subsequent selection in the thymus gives preference to some α but not β J segments. These findings provide a deeper insight into the mechanism of TCR repertoire generation.Adaptive immunity is provided by B and T cells bearing B-cell receptors (BCRs) and Ig-like T-cell receptors (TCRs), respectively. These hypervariable molecules are the key part of the adaptive immune system as they can potentially recognize any alien agent and drive specific immune responses. The α/β TCRs recognize short peptides in the complex with major histocompatibility complex (MHC) molecules and play the key role in the targeted immune response. The total diversity of TCR molecules in an individual human organism is initially formed via genomic recombination with subsequent positive and negative selection at several stages of maturation and activation. The maximal theoretical diversity of TCRβ chain’s amino acid sequences in humans is estimated between 5 × 10¹¹ (1) and 10¹⁴ (2), whereas the maximal number of α/β pairs reaches 10¹⁸ (3). This huge number of variants is probably never achieved: the whole TCRβ chain repertoire size in a single human organism is estimated at 1–5 × 10⁶ (1, 4–6), although this is only a lower bound estimate. Two driving forces shape the final face of individual TCR repertoire: the individual genetics and the complexity of environmental factors. The genes coding for proteins involved in VDJ recombination, antigen processing and presentation, and products of genes participating in the immune response signaling belong to the first type of the repertoire-forming factors. The spectrum of the organism’s self-peptides presented in the thymus also depends on the individual’s set of the MHC molecules. Moreover, this spectrum of peptides is determined by the amino acid sequences of the organism’s proteins, which thus can also be considered a genetic factor. Furthermore, TCRs arising to the same alien antigenic peptides are known to be MHC restricted (7). The environmental factors include the whole range of pathogens met by the individual including disease-causing bacteria and viruses, as well as vaccines, symbionts, etc. The genetic component can potentially have a major impact on the initial recombination and selection in the thymus forming the naïve TCR repertoire, whereas the subsequent interference with antigens provides the selective expansion of some TCRs and forms the final repertoire structure. However, the particular impact of genetic factors on TCR repertoire structure and diversity is unknown.All genes of monozygous (MZ) twins are identical (including those responsible for the TCR repertoire formation), and therefore, MZ twins are widely used in the studies where the genetic impact is evaluated. Several studies of TCR repertoires were performed mainly focusing on diseases concordant and discordant MZ twins and using complementarity determining region 3 (CDR3) spectra-typing and/or low depth sequencing (8–11). Some of these studies reported the common use of particular V genes and common clonotypes. In recent years, the high-throughput sequencing technologies paved the way to whole-repertoire studies of individual TCRs that led to new findings in the field of adaptive immunity (1, 5, 6, 12–22). In this study, for the first time to the best of our knowledge, we obtain and compare the α and β chain TCR repertoires of three pairs of MZ twins using next-generation sequencing (NGS).     

Duplex study of the carotid and femoral arteries of patients with systemic lupus erythematosus: a controlled study

OBJECTIVE: To identify atherosclerosis in the common carotid (CCA) and common femoral arteries (CFA) of patients with systemic lupus erythematosus (SLE) and matched controls. METHODS: Fifty-one consecutive patients with SLE were enrolled in the study. Controls were matched by age, sex, ethnicity, and atherosclerosis risk factors. All patients and controls underwent ultrasonic biopsy (U-B) of the CFA and CCA, a noninvasive screening technique that detects early atherosclerotic plaques and changes. The U-B features were classified and scored as follows: class A: normal (score 0); class B: interface disruption (score 2); class C: intima-media granulation (score 4); class D: plaque without hemodynamic disturbance (score 6); class E: stenotic plaque (score 8); and class F: plaque with symptoms (score 10). Total score was calculated. Classes A and B indicate an intact media; classes D to F point to a significant medial involvement; class C signifies a borderline lesion with a potential for regression to normal or progression to a plaque. RESULTS: Mean ages were 40.5 years for SLE patients and 41 years for controls (p = 0.6). Ninety-six percent of the patients and controls were women. The mean disease duration of SLE was 8.65 years. Frequencies of risk factors among the SLE patients compared to controls were hypertension (30% vs 24%), smoking (23% vs 24%), and dyslipidemia (17.7% vs 17%). No patient had diabetes mellitus or family history of cardiovascular disease. A 3.17-fold increased rate of atherosclerotic plaques was detected in the SLE patients compared with controls (95% CI 1.08-10.9). Twenty-eight percent of SLE patients had at least a single class D-F lesion in one of the 4 vessels tested, compared with 10% in the control group (p = 0.02). In addition, the mean total U-B score of the SLE patients was significantly higher than that of the controls (5.65 vs 3.14; p = 0.02). Univariate analyses showed that the development of plaques in SLE was associated with a history of ischemic heart disease, hypertension, cardiovascular accident, and anemia. Multivariate analysis found plaques to be strongly associated with age, particularly in those older than 50 (OR 2.66, p = 0.000). CONCLUSION: Patients with SLE have a high rate of atherosclerotic changes compared to controls. The development of atherosclerosis is strongly associated with age.     

Perioperative hemodynamic and geometric changes of the left ventricle during cardiomyoplasty in goats with dilated left ventricle

OBJECTIVE: Clinical data have suggested the occurrence of temporary short-term deterioration of the heart following cardiomyoplasty. The purpose of this study was to monitor the short-term hemodynamic effects of cardiomyoplasty in a goat model of a dilated left ventricle, using conductance catheters (ie, pressure-volume loops) and cardiac output measurements. METHODS: Eight female goats underwent acute cardiomyoplasty 8 to 12 weeks after left ventricular (LV) dilatation was induced by a carotid jugular arteriovenous shunt. The cardiomyoplasty procedure was monitored using a Swan-Ganz catheter for cardiac output measurements and a 12-electrode (dual-field) conductance catheter to LV pressure-volume loops. RESULTS: After wrapping the heart with the latissimus dorsi muscle, there was a significant reduction in both cardiac output and LV end-diastolic volume (LVEDV) at 10 min. Partial recovery was observed 45 min later. CONCLUSION: A decrease in both cardiac output and LVEDV was observed following myocardial wrapping. This may explain some of the perioperative and postoperative morbidity and mortality observed following cardiomyoplasty.     

Living with a child with familial Mediterranean fever: does it affect the quality of life of the parents?

OBJECTIVE: The aim of the present study was to assess the quality of life (QOL) and the psychological status of parents of children with familial Mediterranean fever (FMF). METHODS: The QOL, anxiety and depression of the parents of 35 children with FMF were evaluated and compared to the parents of 23 healthy children. RESULTS: Mothers of FMF children had lower QOL scores than mothers of healthy children: 5.5 +/- 1.1 versus 6.0 +/- 0.6 (p = 0.048). They also expressed higher levels of anxiety and depression. Within each group, mothers were more anxious and depressed than fathers. Parents with several FMF children were not significantly different from parents with only one FMF child. CONCLUSION: The QOL and psychological well being of parents with FMF children were found to be slightly impaired, especially that of the mothers.     

Serum levels of IL-7 in bone marrow transplant recipients: relationship to clinical characteristics and lymphocyte count

IL-7 is produced by stromal cells and is the major lympho- and thymopoietic cytokine. IL-7 induces proliferation and differentiation of immature thymocytes, and protects thymocytes from apoptosis by induction of bcl-2 expression. The regulation of IL-7 production is poorly characterized, although down-regulation by transforming growth factor-beta (TGF-beta) has been described. We measured the serum levels of IL-7 before and after bone marrow transplant (BMT) in 32 children undergoing BMT for genetic diseases (severe combined immune deficiency (SCID) and thalassemia), aplastic anemia, and acute lymphoblastic and non-lymphoblastic leukemia (ALL and ANLL). Prior to BMT, the highest IL-7 levels were observed in patients with SCID and ALL, i.e. those patients with genetic or acquired lymphopenia. Patients with thalassemia and ANLL had normal levels of IL-7. Over the 8 weeks following BMT, the IL-7 levels of patients with SCID and ALL fell as the absolute lymphocyte count (ALC) increased. No detectable change in IL-7 levels was observed in the patients with thalassemia and ANLL. Levels of IL-7 were highest in the young infants with SCID compared to the age-matched controls. Together, the data demonstrate that serum levels of IL-7 in lymphopenic patients are inversely related to patient age and the absolute lymphocyte count (ALC). The inverse relationship to ALC suggests that there is either direct regulation of stromal production or more likely, binding of secreted IL-7 to lymphocytes expressing IL-7 receptors.     

High-dose therapy with hematopoietic stem cell rescue in patients with poor prognosis Ewing family tumors

The purpose of the study was to evaluate the feasibility and safety of two cycles of high-dose chemotherapy (HDT) followed by autologous hematopoietic SCT (HSCT) in patients with poor prognosis Ewing family of tumors (EFT). Twenty patients with primary metastatic bulky disease or recurrent EFT were enrolled to a treatment protocol with two cycles of HDT and HSCT. Patients tolerated well the first (n=20) and second (n=13) cycles, with limited and predictable toxicities. Only one (5%) TRM occurred during the second cycle. Myeloid engraftment occurred at the median of 11 days after both cycles. At 3 years, the overall and EFS were 45% (confidence interval; CI 0.22, 0.69) and 47% (CI 0.25, 0.70), respectively, for the entire group and 58% (CI 0.30, 0.86) for patients who completed two cycles. Dose intensification with two cycles of HDT and HSCT is feasible and safe, with low and acceptable treatment-related morbidity and mortality. Adding a second course of therapy does not impair engraftment. However, only 65% of the patients were able to proceed to the second cycle. Further studies are required to define the optimal mode of delivery of HDT and HSCT in treatment of advanced EFT.Bone Marrow Transplantation (2008) 42, 311-318; doi:10.1038/bmt.2008.169; published online 30 June 2008.     

Duplex study of the carotid and femoral arteries of patients with rheumatoid arthritis: a controlled study

BACKGROUND: "Ultrasonic biopsy" (U-B) is a noninvasive screening technique to detect early atherosclerotic plaques and arterial wall changes. AIM: To identify atherosclerosis (AS) in the common carotid artery (CCA) and common femoral artery (CFA) of patients with rheumatoid arthritis (RA) and their matched controls. METHODS: Fifty-seven consecutive RA patients were enrolled in the study. Controls were matched by age, sex, ethnicity, and AS risk factors. All patients and controls underwent U-B study of the CCA and CFA. The U-B features were classified and scored as follows: Class A, normal (score 0); Class B, interface disruption (score 2); class C, intima-media (I-M) granulation (score 4); Class D, plaque without hemodynamic disturbance (score 6); Class E, stenotic plaque (score 8); and Class F, plaque with symptoms (score 10). Total score per patient was calculated. Classes A-B indicate an intact media or minimal interphase changes; classes D-F point to a significant medial involvement. Class C signifies a borderline lesion, with a potential for regression to normal, being unchanged, or progression to a plaque. RESULTS: Mean ages were 52.1 years for RA and 51.4 years for controls (P = 0.81). Eighty-six percent of the patients and 85% of controls were women. The mean disease duration of RA was 12.8 years. Frequencies of risk factors among the RA patients compared with controls were hypertension (28% versus 32%), smoking (37% versus 29%), dyslipidemia (23% versus 25%), diabetes mellitus (DM) (14% versus 14%), and family history of cardiovascular disease (CVD) (4% versus 7%). Forty-five percent of the RA patients had at least a single Classes D-F lesion (plaque) in 1 of the 4 vessels tested, compared with 40% in the control group (P = 0.19). The mean total U-B scores of the RA patients and controls were not significantly different (8.87 versus 9.49, P = 0.7). Univariate analyses have shown that the development of plaques in RA patients was associated with age >50 years, disease duration, hypertension, dyslipidemia, and smoking. Multivariate analysis found plaques to be strongly associated with age above 50 years and dyslipidemia. CONCLUSION: In unselected RA patients, besides classic AS risk factors, older age and longstanding disease may help predict the development of a severe morphological expression of AS.     

Inaccuracies inherent in patient-specific dual-energy X-ray absorptiometry bone mineral density measurements: comprehensive phantom-based evaluation.

An extensive series of dual-energy X-ray absorptiometry (DXA) scans and dual polyenergetic X-ray simulation studies of 150 different phantom arrays were carried out to evaluate quantitatively the extent of systematic inaccuracies inherent in DXA in vivo bone mineral density (BMD). These measurements are particularly relevant to lumbar vertebral and proximal femoral sites. The phantoms were specially fabricated near perfect absorptiometric representations of bone material, red marrow (RM) and yellow marrow (YM), and extraosseous mixtures of fat (F) and lean muscle that spanned the full range of soft tissue anthropometrics encountered clinically. In each case, the DXA-measured BMD values obtained using Hologic, Lunar, and Norland densitometers were found to be virtually the same and to be in excellent agreement with the corresponding quantitative simulation study BMD results. Comparisons of the known phantom BMD values and DXA-measured BMD in each case allowed the BMD inaccuracies to be evaluated. These present findings show that these ubiquitous inaccuracies in DXA BMD methodology are of in vivo soft tissue anthropometric genesis. It is found that patient-specific DXA-measured in vivo BMD inaccuracies as high as 20% or more can be readily anticipated clinically, particularly in cases of osteopenic, osteoporotic, and elderly patients. As these inaccuracies exceed considerably DXA precision errors, they may compromise patient-specific evaluations of fracture risk and, in prospective studies, mask or exaggerate clinically significant true changes in BMD. It is concluded that the magnitudes and variability of inherent inaccuracies in DXA-measured in vivo BMD underscore the need for prudence and circumspection in interpretations and assessments of DXA-based clinical studies.     

Imaging and quantification of myocardial perfusion using real-time three-dimensional echocardiography.

OBJECTIVES: We tested the feasibility of real-time three-dimensional echocardiographic (RT3DE) perfusion imaging and developed and validated an algorithm for volumetric analysis of myocardial contrast inflow. The study included three protocols wherein perfusion was measured: 1) in an ex-vivo model of controlled global coronary flow, 2) in an in-vivo model during regional perfusion variations, and 3) in humans during pharmacologically induced hyperemia. BACKGROUND: The RT3DE technology offers an opportunity for myocardial perfusion imaging without multi-slice reconstruction and repeated contrast maneuvers. METHODS: Electrocardiographically triggered harmonic RT3DE datasets were acquired (Philips 7500) while infusion of Definity was initiated and reached a steady state. Protocol 1 was performed in nine isolated rabbit hearts and included three coronary flow levels. In protocol 2, changes in regional perfusion caused by partial left anterior descending artery occlusion were measured in five pigs. In protocol 3, adenosine-induced changes in perfusion were measured in eight normal volunteers. Myocardial video-intensity (MVI) was measured over time in three-dimensional (3D) slices to calculate peak contrast inflow rate (PCIR). In pigs, PCIR was measured on a regional basis and validated against microspheres. RESULTS: The RT3DE imaging allowed selection of slices for perfusion analysis in rabbit hearts, pigs, and humans. Administration of contrast resulted in clearly visible and quantifiable changes in MVI. In rabbits, The PCIR progressively decreased with coronary flow (p < 0.0001). In pigs, coronary occlusion caused a 59 +/- 26% decrease in PCIR exclusively in the left anterior descending artery territory (p < 0.05) in agreement with microspheres. In humans, adenosine increased PCIR to 198 +/- 57% of baseline (p < 0.05). CONCLUSIONS: Contrast-enhanced RT3DE imaging provides the basis for volumetric imaging and quantification of myocardial perfusion.