A mutation in X-linked inhibitor of apoptosis (G466X) leads to memory inflation of Epstein-Barr virus-specific T cells.

Mutations in the X-linked inhibitor of apoptosis (XIAP) gene have been associated with XLP-like disease, including recurrent Epstein-Barr virus (EBV)-related haemophagocytic lymphohystiocytosis (HLH), but the immunopathogenic bases of EBV-related disease in XIAP deficiency is unknown. We present the first analysis of EBV-specific T cell responses in functional XIAP deficiency. In a family of patients with a novel mutation in XIAP (G466X) leading to a late-truncated protein and varying clinical features, we identified gradual hypogammaglobulinaemia and large expansions of T cell subsets, including a prominent CD4(+) CD8(+) population. Extensive ex-vivo analyses showed that the expanded T cell subsets were dominated by EBV-specific cells with conserved cytotoxic, proliferative and interferon (IFN)-γ secretion capacity. The EBV load in blood fluctuated and was occasionally very high, indicating that the XIAP(G466X) mutation could impact upon EBV latency. XIAP deficiency may unravel a new immunopathogenic mechanism in EBV-associated disease.

Long-term efficacy of bosentan in treatment of pulmonary arterial hypertension in children.

The aim of the present study was to evaluate a 5-yr experience of bosentan in children with pulmonary arterial hypertension (PAH). A retrospective, observational study was made of children in the UK Pulmonary Hypertension Service for Children (Great Ormond Street Hospital for Children, London, UK) who were given bosentan as monotherapy or in combination, from February 2002 to May 2008 and followed up for ≥ 6 months. Detailed studies were made of 101 children with idiopathic PAH (IPAH) (n = 42) and PAH associated with congenital heart disease (n = 59). Before treatment, World Health Organization (WHO) functional class, 6-min walk distance (6MWD), height, weight and haemodynamic data were determined. Evaluations were analysed after 6 months and annually to a maximum of 5 yrs. Median duration of treatment was 31.5 months. Initial improvement in WHO functional class and 6MWD was maintained for up to 3 yrs. Height and weight increased but the z-scores did not improve. After 3 yrs, bosentan was continued as monotherapy in only 21% of children with IPAH, but in 69% of repaired cases and 56% of those with Eisenmenger syndrome. The Kaplan-Meier survival estimates for the 101 patients were 96, 89, 83 and 60% at 1, 2, 3 and 5 yrs, respectively. A treatment regime that includes bosentan is safe and appears to be effective in slowing disease progression in children with PAH.

Childhood idiopathic pulmonary arterial hypertension: a national cohort study.

OBJECTIVE: To clarify the clinical characteristics and epidemiology of idiopathic pulmonary arterial hypertension (IPAH) in childhood, a rare condition with a bad prognosis, poorly documented in children. Also, to describe the long-term outcome. DESIGN: A retrospective study of 7 years' experience. SETTING: UK Service for Pulmonary Hypertension in Children based at a tertiary referral centre. PATIENTS: 64 children. INTERVENTIONS: Patients were initially treated with prostanoids (n=15), bosentan (n=23), sildenafil (n=9), combination therapy (n=11) or calcium channel antagonists (n=6). MAIN OUTCOME MEASURES: WHO functional class, distance walked in 6 minutes, escalation of therapy, survival, transplant-free survival. RESULTS: Incidence of IPAH was 0.48 cases per million children per year and the prevalence was 2.1 cases per million. 31% presented with syncope. Oedema was rare. During the first year of follow-up WHO functional class and 6-minute walk distance improved significantly. Survival at 1, 3 and 5 years was 89%, 84% and 75%, respectively; while transplant-free survival was 89% 76% and 57%, respectively. Factors predicting worse survival were WHO functional class (HR 2.4, p=0.04) and poor height and weight z-score (p<0.05 for both) at presentation. CONCLUSIONS: We showed, for the first time, that the incidence of IPAH is lower in children than adults and that the clinical features can be different. Most children present with clinical evidence of advanced disease and clinical status at presentation is predictive of outcome. This 7-year experience confirms the significant improvement in survival over historical controls.

Prenatal diagnosis of primary pulmonary hypoplasia in fraternal twins.

Primary pulmonary hypoplasia is a rare, usually lethal, condition presenting only after birth without other congenital abnormalities. We describe the first case of fraternal twins diagnosed prenatally with primary pulmonary hypoplasia. Both had diffuse hypoplasia of the pulmonary arteries initially identified by fetal echocardiography and confirmed at autopsy following termination. These cases permit examination of the histopathology of this disease in the fetal stage of development.

Treatment and survival in children with pulmonary arterial hypertension: the UK Pulmonary Hypertension Service for Children 2001-2006.

OBJECTIVE: A retrospective study of the UK Pulmonary Hypertension Service for Children for the first 5-year period of its existence. DESIGN AND PATIENTS: Records of 216 children with idiopathic pulmonary arterial hypertension (IPAH) and associated pulmonary arterial hypertension (APAH) were reviewed. Kaplan-Meier survival curves were constructed for different diagnostic groups and for different therapies. RESULTS: At cardiac catheterisation only 7.4% of those with IPAH and 6% of those with APAH responded positively to vasodilator testing and so were treated with nifedipine. Others needing treatment were given continuous intravenous epoprostenol, bosentan or sildenafil singly or in combination. For IPAH survival rates were 85.6%, 79.9% and 71.9% at 1, 3 and 5 years, respectively, compared with a survival time of less than a year in historical untreated controls. A combination of intravenous epoprostenol with either bosentan or sildenafil, or both, appeared to achieve the best outcome. Six children underwent lung transplantation. In APAH survival rates were 92.3%, 83.8% and 56.9% at 1, 3 and 5 years, respectively, postoperative congenital heart disease with severe pulmonary hypertension having the worst outcome. CONCLUSION: New pulmonary hypertension-specific medicines have improved survival in children as in adults. Outcome in this series compares favourably with international outcome data.

Prevalence and associations of psychological distress in young adults with Type 1 diabetes.

AIMS: To determine the prevalence of psychological distress in young adults with Type 1 diabetes and to explore associated factors. METHODS: Ninety-two participants with Type 1 diabetes (46 male, 46 female) attending a young adult clinic completed two psychological self-report assessments; the Centre for Epidemiological Studies-Depression Scale (CES-D) and Adult Self-Report Scale (ASR). The mean age was 21.6 +/- 2.8 years (sd) and mean duration of diabetes was 9.3 +/- 5.4 years. A questionnaire identified the method of insulin delivery, the frequency of blood glucose monitoring and hypoglycaemia requiring third-party assistance. HbA(1c) was measured. RESULTS: Of the participants, 35.2% reported depressive symptoms (CES-D > or = 16), 23.1% indicating severe depressive symptoms (CES-D > or = 24), and 32.2, 40.4 and 35.5% of participants reported significant distress (ASR > or = 60) on the ASR total problem scales, ASR internalizing and ASR externalizing scores, respectively. Mean HbA(1c) levels were higher in participants with depressive symptoms compared with those with normal scores (CES-D > or = 16, HbA(1c)= 9.4% vs. CES-D < 16, HbA(1c)= 8.4%, P = 0.01). Factors associated with psychological distress included use of continuous subcutaneous insulin infusion (CSII) (P = 0.02) and increased frequency of hypoglycaemic episodes (P = 0.03). CSII users had higher CES-D (21.3 vs. 11.9, P = 0.001) and ASR-Total (59.7 vs. 53.0, P = 0.02) scores than non-CSII users. CONCLUSIONS: Approximately one-third of young adults with Type 1 diabetes experience psychological distress, which is associated with poorer glycaemic control. Psychological distress was related to frequency of hypoglycaemic episodes and method of insulin administration, with significantly greater distress being observed in those using CSII. These findings support inclusion of a psychologist in the diabetes team.

The 6-minute walk test: normal values for children of 4-11 years of age.

OBJECTIVE: The 6-minute walk test (6MWT) is an established measure of exercise capacity in adults and children with chronic cardiac or respiratory disease. Despite its widespread use, there are no normal values for healthy children under 12 years of age. We aimed to provide normal values for children between 4 and 11 years. METHODS: Healthy children were recruited prospectively from two UK primary schools and also children visiting Great Ormond Street Hospital. 328 children (54% male) aged 4 to 11 years were included in the study. Main outcome measures were the distance walked in 6 minutes, and oxygen saturation and heart rate during the 6 minutes and during a 3-minute recovery period. RESULTS: Mean oxygen saturation at baseline and during the 6MWT was 97-99%. Heart rate increased from 102+/-19 bpm at baseline to a maximum of 136+/-12 bpm. Overall, the mean distance walked in 6 minutes was 470+/-59 m. Distance walked correlated with age (r = 0.64, p<0.0001), weight (r = 0.51, p<0.0001) and height (r = 0.65, p<0.0001) with no significant difference between boys and girls. The distance walked increased significantly year on year from 4 to 7 years (4 years 383+/-41 m; 5 years 420+/-39 m, 6 years 463+/-40 m; 7 years 488+/-35 m; p<0.05 between each); further modest increases were observed beyond 7 years of age. CONCLUSIONS: Performing a 6MWT is feasible and practical in young children. This study provides data on normal children against which the performance of sick children and the response to therapeutic intervention can be judged.

IL-7R alpha versus CCR7 and CD45 as markers of virus-specific CD8+ T cell differentiation: contrasting pictures in blood and tonsillar lymphoid tissue.

In humans, circulating CD8(+) memory T cells to a nonpersistent virus (influenza) lie within CCR7(+)CD45RA(-) central memory, whereas memory to Epstein-Barr virus (EBV) latent, EBV lytic, and cytomegalovirus (CMV) antigens are progressively larger in size and are more biased toward CCR7(-)CD45RA(-) effector memory and CCR7(-)CD45RA(+) terminally differentiated compartments. We found that these populations are also distinguished by progressively lower expression of the interleukin-7 receptor (IL-7R alpha) and by lower IL-7 responsiveness; indeed, percentage IL-7R alpha -positive values showed a tight inverse correlation with population size. However, these relationships among size, differentiation phenotype, and IL-7R alpha status in blood did not hold in tonsillar tissue. In tonsil tissue, although EBV reactivities outnumbered their CMV and influenza counterparts, the distinct CCR7/CD45 isoform signatures of the different virus-specific populations were retained. Moreover, all detectable reactivities showed high levels of IL-7R alpha expression. As a discriminator between different virus-specific populations, IL-7R alpha therefore appears to be more susceptible to tissue location than the classical CCR7/CD45 markers.

The T cell response to persistent herpes virus infections in common variable immunodeficiency.

We show that at least half of patients with common variable immunodeficiency (CVID) have circulating CD8(+) T cells specific for epitopes derived from cytomegalovirus (CMV) and/or the Epstein-Barr virus (EBV). Compared to healthy age-matched subjects, more CD8(+) T cells in CVID patients were committed to CMV. Despite previous reports of defects in antigen presentation and cellular immunity in CVID, specific CD4(+) and CD8(+) T cells produced interferon (IFN)-gamma after stimulation with CMV peptides, and peripheral blood mononuclear cells secreted perforin in response to these antigens. In CVID patients we found an association between a high percentage of circulating CD8(+) CD57(+) T cells containing perforin, CMV infection and a low CD4/CD8 ratio, suggesting that CMV may have a major role in the T cell abnormalities described previously in this disease. We also show preliminary evidence that CMV contributes to the previously unexplained severe enteropathy that occurs in about 5% of patients.

Response to bosentan in children with pulmonary hypertension.

OBJECTIVE: To describe an early experience of treating 40 children with the dual endothelin receptor antagonist bosentan, which is known to be safe and effective in adults with pulmonary hypertension (PH). DESIGN: In this retrospective, observational study the UK Service for Pulmonary Hypertension for children treated 40 children with bosentan, 20 with idiopathic pulmonary arterial hypertension (IPAH) (mean age 8.03 years, range 1.2-17) and 20 with PH associated with other conditions (congenital heart disease, parenchymal lung or connective tissue disease, or HIV). Their mean age was 8.3 years (range 0.6-16 years). PATIENTS: 39 patients were in World Health Organization (WHO) class III and IV, and all had shown recent deterioration. In IPAH the mean pulmonary vascular resistance (PVR) was 21.7 units.m2 (range 5.6-42.8). In secondary PH the mean PVR was 18 units.m2 (range 4.9-49). No child had a positive response to vasodilator testing with nitric oxide. INTERVENTIONS: Bosentan was given as first line treatment to 25. Nine were given intravenous epoprostenol. Children were treated for a mean of 12.7 months (range 2-24 months). MAIN OUTCOME MEASURES: Response to treatment was judged by WHO functional class, six minute walk test, weight, ECG and echocardiographic findings, and need to add additional treatment. RESULTS: Bosentan was well tolerated. In the IPAH group 19 (95%) stabilised with bosentan treatment but 12 (60%) patients needed combined treatment with epoprostenol. In secondary PH, WHO class, six minute walk test, and weight gain improved significantly. CONCLUSION: Bosentan helped stabilise children with IPAH but intravenous epoprostenol was also needed by 60%. Children with secondary PH improved.

Low oxygen exposure does not cause pulmonary injury in the newborn rat.

BACKGROUND: Two recent studies have suggested that low levels of supplemental inspired oxygen may cause lung injury in preterm infants. AIMS: To assess lung injury of newborn rats exposed to 14 days of low-level variation of oxygen. STUDY DESIGN: Four groups were compared with 12 animals per group and 4 lung sections per animal. These were, a control group raised in room air and three groups raised in levels of inspired oxygen fluctuating around the following mean values: group Lo (mean FiO(2) 0.179), group N (mean FiO(2) 0.213), and group Hi (mean FiO(2) 0.247). The degree of oxygen variability was identical for each group. Lungs were inflated at 20 cm H(2)O, fixed and stained with H and E and Millers Elastin. SUBJECTS: Sprague Dawley albino newborn rats. OUTCOME MEASURES: Random alveolar areas were studied and analysed using imaging software to assess total amount of tissue and elastin, number of secondary septa, and mean linear intercept. RESULTS: There were no significant differences between the three experimental oxygen groups and the control group in terms of lung/body weight ratio and the measured markers of lung development. CONCLUSION: We conclude that low-level oxygen supplementation during early lung development does not affect alveolar development in the newborn rat.

Role of atrial septostomy in the treatment of children with pulmonary arterial hypertension.

OBJECTIVES: To assess in retrospect the safety and effectiveness of atrial septostomy in children with severe pulmonary arterial hypertension without an intracardiac communication. METHODS: 20 patients were reviewed retrospectively, 19 with idiopathic pulmonary arterial hypertension. The mean age at septostomy was 8.4 years (range 3 months to 17 years). Graded balloon septostomy alone was carried out in eight patients, a blade septostomy was done in two, a blade septostomy plus graded balloon septostomy was done in three, and a fenestrated device was inserted in seven. RESULTS: There were no fatalities. Four children suffered complications during the procedure. None had further syncope and all improved symptomatically with a significant (p < 0.01) decrease in World Health Organization functional class (mean shift -0.6) and a significant improvement in the semiquantitative echocardiographic assessment of right ventricular function (p < 0.03). The mean oxygen saturation decreased by 7.8 percentage points. The atrial communication closed in two children, necessitating a repeat procedure. After a mean follow up of 2.1 years (range one month to 6.7 years), 18 of 20 children are still alive. CONCLUSION: Atrial septostomy improved symptoms and quality of life in a group of children deteriorating with severe pulmonary arterial hypertension. This procedure is to be recommended for severely symptomatic children, before they become critically ill. Fenestrated devices may help ensure indefinite patency of the atrial communication.

HLA-A11-restricted epitope polymorphism among Epstein-Barr virus strains in the highly HLA-A11-positive Chinese population: incidence and immunogenicity of variant epitope sequences.

An individual's CD8(+)-cytotoxic-T-lymphocyte (CTL) response to Epstein-Barr virus (EBV) latent cycle antigens focuses on a small number of immunodominant epitopes often presented by just one of the available HLA class I alleles; for example, HLA-A11-positive Caucasians frequently respond to two immunodominant HLA A11 epitopes, IVTDFSVIK (IVT) and AVFDRKSDAK (AVF), within the nuclear antigen EBNA3B. Here, we reexamine the spectrum of EBV strains present in the highly HLA-A11-positive Chinese population for sequence changes in these epitopes relative to the Caucasian type 1 prototype strain B95.8. The IVT epitope was altered in 61 of 64 Chinese type 1 viruses, with four different sequence variants being observed, and the AVF epitope was altered in 46 cases with six different sequence variants; by contrast, all 10 Chinese type 2 viruses retained the prototype 2 epitope sequences. All but one of the type 1 epitope variants were poorly recognized by IVT- or AVF-specific CTLs in pulse-chase assays of peptide-mediated target cell lysis. More importantly, we screened HLA-A11-positive Chinese donors carrying viruses with known epitope mutations for evidence of epitope-specific CTL memory by enzyme-linked immunospot assays: none of the type 1 variants tested, nor the type 2 prototype, appeared to be immunogenic in vivo. The data remain consistent with the possibility that, during virus-host coevolution, pressure from the host CTL-mediated immune response has given A11 epitope-loss viruses a selective advantage.

Birth upregulates nitric oxide synthase activity in the porcine lung.

Developmental changes in the lung occur at birth, allowing for the transition from placental to air breathing. Here we have measured nitric oxide synthase (NOS) activity in the porcine lung pre and post partum. NOS activity, which was predominantly calcium dependent, was low in full term fetal tissue compared to that present in lungs from the newborn (5 minutes post partum), 1, 3, 6 and 14 day old animals. No increase in activity was seen when fetal pigs were allowed to breathe for 5 minutes. Specific activity remained low in fetal tissue following partial purification. By contrast, levels of NOS III protein in tissue extracts and in pulmonary arterial endothelial cells, demonstrated by immunohistochemistry, were similar in tissue from the fetal and newborn animals. Thus NOS activity is significantly lower in fetal when compared to postnatal lung tissue despite comparable amounts of NOS III protein being expressed, and birth is followed by an abrupt increase in enzyme activity in animals born at term which correlates with an increase in protein expression.

EBV-specific CD8+ T cell memory: relationships between epitope specificity, cell phenotype, and immediate effector function.

EBV infection in humans induces CD8+ T cell memory to viral epitopes derived from both lytic and latent cycle Ags. We have analyzed the relationship between the phenotype and function of the memory pool of T cells specific for these Ags. Lytic epitope-specific populations were heterogeneous in terms of CD45RO/RA and CD28 expression, whereas latent epitope-specific populations were uniformly CD45RO+ and CD28+, consistent with the higher antigenic challenge from lytic epitopes driving some memory cells toward a CD45RA+, CD28- phenotype. However, both types of memory population showed immediate epitope-specific cytotoxicity and type 1 cytokine production in ex vivo assays. Cytotoxic function was not associated with preactivated T cells, as EBV-specific populations were negative for activation markers such as CD69 or CD38, nor could cytotoxic function be ascribed to CD27- or CD56+ subsets, as such cells were not detected in EBV-specific memory. Furthermore, cytotoxicity was not limited to CD45RA+ and/or CD28- fractions, but also was observed in CD45RO+, CD28+ populations in lytic and latent epitope-specific memory. Cytokine (IFN-gamma, TNF-alpha) responses, measured by intracytoplasmic staining after peptide stimulation, also were detectable in CD45RO+ and RA+ subsets as well as CD28+ and CD28- subsets. Of other markers that were heterogeneous in both lytic and latent epitope populations, CCR7 gave the best discrimination of functionality; thus, CCR7+ cells consistently failed to give an IFN-gamma or TNF-alpha response, whereas many CCR7- cells were responsive. Our data are consistent with effector functions having a broad distribution among phenotypically distinct subsets of "effector memory" cells that have lost the CCR7 marker.

Memory T cells constitute a subset of the human CD8+CD45RA+ pool with distinct phenotypic and migratory characteristics.

Using HLA class I-viral epitope tetramers to monitor herpes virus-specific CD8(+) T cell responses in humans, we have shown that a significant fraction of responding cells revert from a CD45RO(+) to a CD45RA(+) state after priming. All tetramer-binding CD45RA(+) cells, regardless of epitope specificity, expressed a phenotype LFA-1(high)CCR7(low) that was stable for at least 10 years in infectious mononucleosis patients and indefinitely in asymptomatic carriers. CD8(+)CD45RA(+)LFA-1(high) cells were not present in cord blood but in adults account for up to 50% of CD8(+)CD45RA(+) cells. These CD45RA(+)LFA-1(high) cells have significantly shorter telomeres than CD45RA(+)LFA-1(low) cells, suggesting that the latter represent a naive population, while the former are memory cells. CD45RA(+) memory cells are a stable population of noncycling cells, but on stimulation they are potent producers of IFN-gamma, while naive CD8(+) cells produce only IL-2. The chemokine receptor profile and migratory potential of CD45RA(+) memory cells is very similar to CD45RO(+) cells but different to naive CD8 cells. In accord with this, CD45RA(+) memory cells were significantly underrepresented in lymph nodes, but account for virtually all CD8(+)CD45RA(+) T cells in peripheral tissues of the same individuals.

Frequent recognition of BCRF1, a late lytic cycle protein of Epstein-Barr virus, in the HLA-B*2705 context: evidence for a TAP-independent processing.

Using a transient COS transfection assay, allowing a rapid estimation of the dominant CD8(+) T cell responses against a large number of HLA/viral protein combinations within polyclonal cell lines, we searched for HLA-B*2705-restricted CD8 T cell responses to Epstein-Barr virus (EBV) within T cell samples enriched for EBV-reactive cells. Among the 18 EBV proteins tested, only 2, the latent protein EBNA3A and the late lytic protein BCRF1 (viral IL-10), appeared dominant in the B27 context, as they triggered significant TNF and cytolytic responses in some donors. We provide evidence that the B27/BCRF1 epitope (RRLVVTLQC) is located in the signal sequence and that it can be presented in a TAP-independent manner. Using B27/BCRF1 monomeric complexes coated on immunomagnetic beads, we sorted out BCRF1-specific CD8 T cells from 8 of 15 HLA-B27(+) donors. This is, to our knowledge, the first demonstration of a recognition of BCRF1, suggesting that some immune control against EBV exists even during the late stage of the lytic cycle. This result also strengthens the unusual ability of HLA-B*2705 to present peptide in a TAP-independent manner.

The flow cytometric analysis of telomere length in antigen-specific CD8+ T cells during acute Epstein-Barr virus infection.

Acute infectious mononucleosis (AIM) induced by Epstein-Barr virus (EBV) infection is characterized by extensive expansion of antigen-specific CD8+ T cells. One potential consequence of this considerable proliferative activity is telomere shortening, which predisposes the EBV-specific cells to replicative senescence. To investigate this, a method was developed that enables the simultaneous identification of EBV specificity of the CD8+ T cells, using major histocompatibility complex (MHC) class I/peptide complexes, together with telomere length, which is determined by fluorescence in situ hybridization. Despite the considerable expansion, CD8+ EBV-specific T cells in patients with AIM maintain their telomere length relative to CD8+ T cells in normal individuals and relative to CD4+ T cells within the patients themselves and this is associated with the induction of the enzyme telomerase. In 4 patients who were studied up to 12 months after resolution of AIM, telomere lengths of EBV-specific CD8+ T cells were unchanged in 3 but shortened in one individual, who was studied only 5 months after initial onset of infection. Substantial telomere shortening in EBV-specific CD8+ T cells was observed in 3 patients who were studied between 15 months and 14 years after recovery from AIM. Thus, although telomerase activation may preserve the replicative potential of EBV-specific cells in AIM and after initial stages of disease resolution, the capacity of these cells to up-regulate this enzyme after restimulation by the persisting virus may dictate the extent of telomere maintenance in the memory CD8+ T-cell pool over time.