A T-cell diagnostic test for cystic echinococcosis based on Antigen B peptides.

Cystic echinococcosis (CE) immunodiagnosis is still imperfect. We recently set-up a whole-blood test based on the interleukin (IL)-4 response to the native Antigen B (AgB) of Echinococcus granulosus. However, AgB is encoded by a multigene family coding for five putative subunits. Therefore, the aims of this study were to analyse the IL-4 response to peptides spanning the immunodominant regions of the five AgB subunits and to evaluate the accuracy of this assay for CE diagnosis. Peptides corresponding to each subunit were combined into five pools. A pool containing all peptides was also used (total pool). IL-4 evaluated by enzyme-linked immunosorbent assay was significantly higher in patients with CE compared to those without (NO-CE subjects) when whole-blood was stimulated with AgB1 and with the total pool. Moreover, IL-4 levels in response to the total pool were significantly increased in patients with active cysts. Receiver Operator Curve analysis identified a cut-off point of 0.59 pg/mL predicting active cysts diagnosis with 71% sensitivity and 82% specificity in serology-positive CE patients. These data, if confirmed in a larger cohort, offer the opportunity to develop new diagnostic tools for CE based on a standardized source of AgB as the peptides.

Analytical evaluation of QuantiFERON- Plus and QuantiFERON- Gold In-tube assays in subjects with or without tuberculosis.

The QuantiFERON-TB Gold Plus (QFT-Plus) represents the new QuantiFERON-TB Gold In-tube (QFT-GIT) to identify latent tuberculosis infection (LTBI). The main differences is the addition of a new tube containing shorter peptides stimulating CD8 T-cells. Aim of this study is to evaluate the accuracy of QFT-Plus compared with QFT-GIT in a cross sectional study of individuals with or without tuberculosis (TB). We enrolled 179 participants: 19 healthy donors, 58 LTBI, 33 cured TB and 69 active TB. QFT-Plus and QFT-GIT were performed. The two tests showed a substantial agreement. Moreover we found a similar sensitivity in active TB and same specificity in healthy donors. A higher proportion of the LTBI subjects responded to both TB1 and TB2 compared to those with active TB (97% vs 81%). Moreover, a selective response to TB2 was associated with active TB (9%) and with a severe TB disease, suggesting that TB2 stimulation induces a CD8 T-cell response in absence of a CD4-response. In conclusion, QFT-Plus and QFT-GIT assays showed a substantial agreement and similar accuracy for active TB detection. Interestingly, a higher proportion of the LTBI subjects responded concomitantly to TB1 and TB2 compared to those with active TB, whereas a selective TB2 response associated with active TB.

Blood and urine inducible protein 10 as potential markers of disease activity.

SETTING: Blood interferon-γ inducible protein 10 (IP-10) has been proposed as a biomarker of disease activity for both tuberculosis (TB) and human immunodeficiency virus (HIV) infection. Urine IP-10 has been detected in adults with active TB, and its level decreases after successful anti-tuberculosis treatment. OBJECTIVE: To evaluate blood and urine IP-10 as biomarker of disease activity. DESIGN: Patients with HIV-TB and active TB were enrolled. Individuals with HIV infection only and healthy donors were included as controls. Blood and urine IP-10 levels were measured using an enzyme-linked immunosorbent assay. RESULTS: Of 39 active TB patients enrolled, 24 were HIV-infected and 15 were HIV-uninfected. Of 87 control subjects without active TB, 54 were HIV-infected and 33 were HIV-uninfected. IP-10 analysis was performed in patients with concomitant blood and urine sample collection. Blood IP-10 was associated with active TB, regardless of HIV infection status; urine IP-10 levels were increased in active TB patients, although the difference was significant in HIV-infected individuals only. Finally, in HIV-infected patients, both blood and urine IP-10 levels were inversely correlated with CD4 T-cell counts. CONCLUSION: These findings suggest that IP-10 could be used as a biomarker for disease activity (inflammation).

Lack of Response to HBHA in HIV-Infected Patients with Latent Tuberculosis Infection.

Heparin-binding haemagglutinin (HBHA) has been proposed as an immunological biomarker for discriminating active tuberculosis (TB) from latent TB infection (LTBI) and to identify those at higher risk of progressing to active disease. Few data are available in immune-compromised patients, which are those with increased risk of TB reactivation. The aim of this stusy was to evaluate the immune response to HBHA in HIV-infected subjects with LTBI (HIV-LTBI) or active TB (HIV-TB) in comparison with the immune response to additional Mycobacterium tuberculosis (Mtb) or HIV and CMV antigens. The responses are evaluated in relation to TB status and in the LTBI subjects with the progression to active TB within 2 years. Forty-one HIV-infected antiretroviral-naïve subjects were prospectively enrolled: 18 were HIV-TB and 23 HIV-LTBI. Whole blood was in vitro stimulated overnight with several antigens and mitogen. Interferon-γ response in the harvested plasma was evaluated by ELISA. Despite that CD4 cell count was significantly different between HIV-LTBI and HIV-TB, no differences were observed in response to Mtb- or HIV-specific antigens. Differently, low responses to HBHA were observed in both HIV-LTBI and HIV-TB subjects. Importantly, none of the six HIV-LTBI responding to HBHA developed TB, while two of 17 non-HBHA responders developed active disease. HIV-TB-coinfected subjects, regardless of their TB status, showed low responses to HBHA despite maintaining detectable responses to other antigens; moreover, among the HIV-LTBI, the lack of HBHA responses indicated an increased risk to develop active TB. These results, although preliminary, suggest that a positive response to HBHA in HIV-LTBI correlates with Mtb containment.

Detection of interleukin-2 in addition to interferon-gamma discriminates active tuberculosis patients, latently infected individuals, and controls.

Effective control of tuberculosis (TB) includes discrimination of subjects with active TB from individuals with latent TB infection (LTBI). As distinct interferon (IFN)-gamma and interleukin (IL)-2 profiles of antigen-specific T-cells have been associated with different clinical stages and antigen loads in several viral and bacterial diseases, we analysed these cytokines in TB using a modified QuantiFERON-TB Gold In Tube test. Detection of IL-2 in addition to IFN-gamma distinguishes not only Mycobacterium tuberculosis-infected subjects from healthy controls, but also individuals with LTBI from active TB patients. This may help to improve diagnostic tests for TB.

Response to Rv2628 latency antigen associates with cured tuberculosis and remote infection.

Interferon-gamma release assays based on region of difference 1 antigens have improved diagnosis of latent tuberculosis infection (LTBI). However, these tests cannot discriminate between recently acquired infection (higher risk of progression to active tuberculosis) and remote LTBI. The objective of the present study was to evaluate the T-cell interferon-gamma responses to Mycobacterium tuberculosis DosR-regulon-encoded antigens (latency antigens) compared with QuantiFERON TB-Gold In-Tube (QFT-GIT) in subjects at different stages of tuberculosis. A total of 16 individuals with remote LTBI and 23 with recent infection were studied; 15 controls unexposed to M. tuberculosis and 50 patients with active tuberculosis and 45 with cured tuberculosis were also analysed. The results indicated that subjects with remote LTBI showed significantly higher whole-blood interferon-gamma responses to M. tuberculosis latency antigen Rv2628 than did individuals with recent infection, active tuberculosis and controls (p<0.003), whereas no significant differences between these groups were found for other latency antigens tested (Rv2626c, Rv2627c, Rv2031c and Rv2032). The proportion of responders to Rv2628 was five-fold higher among QFT-GIT-positive-individuals with remote infection than among those with recently acquired infection. These data suggest that responses to M. tuberculosis latency antigen Rv2628 may associate with immune-mediated protection against tuberculosis. In contact-tracing investigations, these preliminary data may differentiate recent (positive QFT-GIT results without responses to Rv2628) from remote infection (positive to both tests).

Characterization of regulatory T cells identified as CD4(+)CD25(high)CD39(+) in patients with active tuberculosis.

Forkhead box P3 (FoxP3) is a transcription factor whose expression characterizes regulatory T cells (T(reg)), but it is also present on activated T cells, thus hindering correct T(reg) identification. Using classical markers for T(reg) recognition, discordant results were found in terms of T(reg) expansion during active tuberculosis (TB) disease. Recently CD39 has been shown to be an accurate marker for T(reg) detection. The objectives of this study were: (i) to identify T(reg) expressing CD39 in patients with TB and to compare the results with those obtained by the standard phenotypic markers; (ii) to evaluate if T(reg) are expanded in vitro by exogenous interleukin (IL)-2 or by antigen-specific stimulation; and (iii) to characterize T(reg) function on the modulation of antigen-specific responses. We enrolled 13 patients with pulmonary TB and 12 healthy controls. T(reg) were evaluated by flow cytometry ex vivo and after antigen-specific in vitro stimulation using CD25, FoxP3, CD127 and CD39 markers. Results indicate that CD39(+) cells within the CD4(+)CD25(high) cells have T(reg) properties (absence of interferon-gamma production and transforming growth factor-beta1 release upon stimulation). Ex vivo analysis did not show significant differences between TB patients and controls of T(reg) by classical or novel markers. In contrast, a significantly higher percentage of T(reg) was found in TB patients after antigen-specific stimulation both in the presence or absence of IL-2. Depletion of CD39(+) T(reg) increased RD1-specific responses significantly. In conclusion, CD39 is an appropriate marker for T(reg) identification in TB. These results can be useful for future studies to monitor Mycobacterium tuberculosis-specific response during TB.

3,5,3'-Triiodothyronine deprivation affects phenotype and intracellular [Ca2+]i of human cardiomyocytes in culture.

OBJECTIVE: A decrease in plasma T3 concentration is a frequent finding in patients with heart failure. However, the role of this 'low T3 syndrome' on disease evolution has never been clarified. As phenotypic and functional cardiomyocyte impairments are alterations that correlate with the failing myocardium, we studied the long-term effects of T3 deprivation on human cardiomyocyte structure and calcium handling. METHODS: Atrial cardiomyocytes and myocardial tissue were cultured with or without 3 nM T3. Microscopical examination of structural features was followed by analysis of alpha-sarcomeric actinin and sarcoplasmic reticulum calcium ATP-ase (SERCA-2) content. Calcium handling was studied by [Ca2+](i) imaging. RESULTS: When stimulated with cyclopiazonic acid, a SERCA-2 inhibitor, T3-deprived cardiomyocytes showed significantly faster (P=0.03) and more transient (P=0.04) increases in [Ca(2+)](i) than T3-supplemented cells. Moreover, in the T3-free cultures a significantly lower number of cells (P=0.003) responded to caffeine, a typical activator of sarcoplasmic reticulum Ca(2+)-release channel. T3-deprived cardiomyocytes also presented altered morphology with larger dimensions than T3-supplemented cells (P < 0.0001). Additionally, in T3-deprived samples alpha-sarcomeric actinin and SERCA-2 protein levels were reduced to 65.6 +/- 3% (P < 0.0001) and 74.1 +/- 4% (P=0.005), respectively, when compared with the T3-supplemented group. CONCLUSIONS: Our data show that human cardiomyocyte calcium handling and phenotype are strongly influenced by T3 suggesting important implications of the 'low T3 syndrome' on the progression of heart failure.

Interferon-inducible protein 10, monokine induced by interferon gamma, and interferon-inducible T-cell alpha chemoattractant are produced by thymic epithelial cells and attract T-cell receptor (TCR) alphabeta+ CD8+ single-positive T cells, TCRgammadelta+ T cells, and natural killer-type cells in human thymus.

Strong reactivity for interferon-inducible protein 10 (IP-10), monokine induced by interferon gamma (Mig), and interferon-inducible T-cell alpha chemoattractant (I-TAC) was found in epithelial cells mainly localized to the medulla of postnatal human thymus. The CXC chemokine receptor common to the 3 chemokines (CXCR3) was also preferentially expressed in medullary areas of the same thymuses and appeared to be a property of 4 distinct populations: CD3+ T-cell receptor (TCR) alphabeta+ CD8+ single-positive (SP) T cells, TCRgammadelta+ T cells, natural killer (NK)-type cells, and a small subset of CD3+(low) CD4+ CD8+ TCRalphabeta+ double-positive (DP) T cells. IP-10, Mig, and I-TAC showed chemoattractant activity for TCRalphabeta+ CD8+ SP T cells, TCRgammadelta+ T cells, and NK-type cells, suggesting their role in the migration of different subsets of mature thymocytes during human thymus lymphopoiesis.

No variation in Hsp70 mRNA level during cardiac surgery in pediatric patients evaluated by semiquantitative RT-PCR.

We evaluated mRNA expression of the heat shock protein gene, Hsp70-1, by means of a semiquantitative RT-PCR in atrial tissue specimens from pediatric patients collected before and after cardiopulmonary bypass surgery for congenital heart diseases, to see whether surgical stress may affect the expression level of this mRNA. We studied thirty nine pediatric patients (aged 3 months to 15 years) undergoing surgical correction of congenital heart malformation. Twenty-one patients were affected by the tetralogy of Fallot, two by combined atrioventricular septal defects, six by ventricular septal defect, three by atrial septal defect, two by atrioventricular canal defect, two by pulmonary valve stenosis, one by mitral insufficiency, and one by double-outlet right ventricle. Our results showed no significant changes in the Hsp70-1 mRNA expression in atrial tissue of patients before and after cross-clamping (the mean relative expression after cross-clamping was 1.0+/-0.6 compared to the baseline value). Furthermore, no significant correlations were observed between cross-clamping time and temperature, cardiopulmonary bypass time and mRNA variation. Our study suggests that, during cardioplegia, myocardial tissue does not have an appropriate adaptive response to surgical stress.

Accessory mitral valve tissue causing left ventricular outflow tract obstruction: case reports and literature review.

Accessory mitral valve (AMV) tissue is a rare congenital malformation causing left ventricular outflow tract obstruction (LVOTO). We present three patients with AMV tissue undergoing surgery. A 60-year old man presented with an AMV leaflet, mild LVOTO and coronary artery disease and underwent accessory leaflet excision and coronary revascularization. A 24-year old man presented with an AMV leaflet, LVOTO and interatrial septal defect and underwent defect closure and accessory leaflet resection. An 8-month-old girl underwent interventricular septal closure and AMV leaflet resection but died on postoperative day 5 from progressive heart failure. Another 87 cases with AMV tissue were identified in the literature The anomaly was classified as: Type I (fixed: A = nodular, B = Membranous), and type II (mobile: A = pedunculated, B = leaflet like). Type IIB was further subdivided as rudimentary chordae and developed chordae. Patients with AMV tissue causing LVOTO may undergo mass removal with acceptable postoperative outcome. Prophylactic removal of AMV tissue should not be attempted in patients with no or mild LVOTO and no other associated heart defects. These patients should be followed and observed periodically by Doppler echocardiography to identify any progression in LVOTO.

Repair of hemitruncus with autologous arterial ring and valved bioconduit.

We present a technical variant to reconstruct the right outflow tract in truncus type A3 (single pulmonary artery branch originating from the ascending aorta with common arterial valve and ventricular septal defect) with interposition of a ring of autologous arterial tissue and a bioconduit between the right ventricular infundibulum and the pulmonary artery branches. The final result is shown by postoperative angiogram which demonstrates an anatomically adequate reconstruction of the right ventricular outflow tract.

Macrophage-derived chemokine and EBI1-ligand chemokine attract human thymocytes in different stage of development and are produced by distinct subsets of medullary epithelial cells: possible implications for negative selection.

The chemoattractant activity of macrophage-derived chemokine (MDC), EBI1-ligand chemokine (ELC), and secondary lymphoid tissue chemokine (SLC) on human thymocytes was analyzed. Both ELC and SLC caused the accumulation of CD4+CD8- or CD4-CD8+ CD45RA+ thymocytes showing high CD3 expression. By contrast, a remarkable proportion of MDC-responsive thymocytes were CD4+CD8+ cells exhibiting reduced levels of CD8 or CD4+CD8- cells showing CD3 and CD45R0, but not CD45RA. MDC-responsive thymocyte suspensions were enriched in cells expressing the MDC receptor, CCR4, selectively localized to the medulla, and in CD30+ cells, whereas ELC-responsive thymocytes never expressed CD30. Reactivity to both MDC and ELC was localized to cells of the medullary areas, but never in the cortex. Double immunostaining showed no reactivity for either MDC or ELC by T cells, macrophages, or mature dendritic cells, whereas many medullary epithelial cells were reactive to MDC or ELC. However, MDC reactivity was consistently localized to the outer wall of Hassal's corpuscles, whereas ELC reactivity was often found in cells surrounding medullary vessels, but not in Hassal's corpuscles. Moreover, while most MDC-producing cells also stained positive for CD30L, this molecule was never found on ELC-producing cells. We suggest therefore that CD30L-expressing MDC-producing medullary epithelial cells attract CCR4-expressing thymocytes, thus favoring the CD30/CD30L interaction, and therefore the apoptosis, of cells that are induced to express CD30 by autoantigen activation. By contrast, ELC production by CD30L-lacking medullary epithelial cells may induce the migration into periphery of mature thymocytes that have survived the process of negative selection.

High CD30 ligand expression by epithelial cells and Hassal's corpuscles in the medulla of human thymus.

CD30 is a member of tumor necrosis factor (TNF) receptor superfamily that is expressed by activated T cells in the presence of interleukin-4 (IL-4). Although CD30 can mediate a variety of signals, CD30-deficient mice have impaired negative selection of T cells, suggesting that at least in the context of murine thymus, CD30 is a cell death-mediating molecule. The ligand for CD30 (CD30L) is a membrane-associated glycoprotein related to TNF, which is known to be expressed mainly by activated T cells and other leukocytes. However, the nature of CD30L-expressing cells involved in the interaction with CD30+ thymocytes is unclear. We report here that in postnatal human thymus the great majority of CD30+ cells are double positive (CD4+CD8+), activated, IL-4 receptor-expressing T cells which selectively localize in the medullary areas. Moreover, many medullary epithelial cells and Hassal's corpuscles in the same thymus specimens showed unusually high expression of CD30L in comparison with other lymphoid or nonlymphoid tissues. These findings provide additional information on the nature and localization of CD30+ thymocytes and show that epithelial cells are the major holder of CD30L in the thymic medulla.

Surgical closure of muscular ventricular septal defects using double umbrella devices (intraoperative VSD device closure).

OBJECTIVES: Surgical closure of some muscular ventricular septal defects has been proven to be difficult. In order to simplify the surgical technique we have used intraoperatively Rashkind double umbrella devices to occlude muscular ventricular septal defects. METHODS: On the basis of haemodynamic and echocardiographic study five children aged 4, 6, 7, 14 and 41 months were considered suitable candidates for intraoperative closure of muscular ventricular septal defects (midmuscular in three cases, apical in two) by Rashkind devices. Three of them had previously undergone pulmonary artery banding at 10, 11 and 41 days, respectively. During hypothermic cardiopulmonary by pass a delivery system was introduced across the tricuspid valve into the right ventricle and then passed through the ventricular septal defect; the distal umbrella of a 17 mm device was opened in the left ventricular cavity; a traction was applied to the introducer and the proximal umbrella was opened on the right side straddling the interventricular septum; the device was then secured on the right side by few stitches. In one case because of the wide diameter of the ventricular septal defect two umbrellas were used. The surgical procedure was completed with debanding and/or closure of other defects close to the aortic or tricuspid valve. RESULTS: Immediate results, tested by epicardial or transesofageal echo, showed a minimal residual shunt in 4 patients and a moderate shunt in one. No early deaths occurred. A complete atrioventricular block developed in 1 patient who had an additional perimembranous defect closed with a prosthetic patch: a permanent pace maker was inserted 3 months after the operation. There was a late death for untractable right ventricular failure in 1 patient who had a large residual shunt erroneously considered moderate. In this patient, the size of the defect was underestimated both preoperatively then intraoperatively. The four survivors are doing well with no signs of hemodynamically significant residual shunts. CONCLUSIONS: The use of Rashkind umbrella devices for closing intraoperatively muscular defects can be helpful to standard surgical techniques when technical problems make patch closure difficult. Its use avoid the need of left ventriculotomy. Careful definition of the size of the defect is mandatory to select suitable candidates.

[Ectopia cordis and Cantrell's pentalogy: personal experience and considerations on the surgical treatment]. / Ectopia Cordis e Pentalogia di Cantrell: esperienza personale e considerazioni sul trattamento chirurgico.

Clefts of the sternum have always attracted attention whether for pathological and physiological features or for research of surgical correction. Two cases of sternal cleft, one with partial ectopia cordis, the other with Cantrell's pentalogy, are presented. Embryology, strategies and several surgical techniques are discussed on the grounds of personal experience. The pediatric surgeon can make a choice among a lot of surgical techniques, because the ectopia cordis and Cantrell's pentalogy are very uncommon and the surgical treatment has a difficult codification. The knowledge of several methods of surgical correction is necessary to reduce high mortality of ectopia cordis and Cantrell's pentalogy. Primary repair in the neonatal period is the best type of management for these rare conditions, because simple closure of the sternal defect during the first month of life avoids the more complex reconstruction necessary in older children.

Thyroid hormones homeostasis in pediatric patients during and after cardiopulmonary bypass.

The concentrations of thyroid hormones were measured in 14 pediatric patients before, during, and after cardiopulmonary bypass. The ages of the patients ranged between 18 months and 14 years. Patients were kept normothermic, or moderate or deep hypothermia was induced depending on the specific pathologic condition involved. A marked reduction in the levels of total triiodothyronine, total thyroxine, free triiodothyronine, and thyroid-stimulating hormone, and in the ratio of free triiodothyronine to free thyroxine was detected during the time frame of the study. The minimum levels of each hormone were reached between 12 and 48 hours after cardiopulmonary bypass, indicating that changes in thyroid function and in the conversion of thyroxine to triiodothyronine are triggered by cardiopulmonary bypass and represent specific phenomena, and that these changes are progressively exacerbated during the post-operative period. The thyroid-stimulating hormone level was markedly reduced versus its baseline values (24% +/- 0.13%), despite low levels of both total (40% +/- 18%) and free (39% +/- 20%) triiodothyronine: it returned to its preoperative level by the third postoperative day, but both the total (75% +/- 10%) and free (74% +/- 3%) triiodothyronine levels remained below their baseline values for 7 days postoperatively. Neither hemodilution nor hypothermia was responsible for the alteration observed. We conclude that pediatric patients undergoing cardiopulmonary bypass manifest changes in hormone metabolism similar to those seen in adult patients. These changes increase progressively during the postoperative period, and are still present 7 days postoperatively. The exact mechanism responsible for causing these changes is not thoroughly understood. Whether triiodothyronine replacement therapy is beneficial or deleterious remains controversial.