Sanna Siitonen

Autoimmunity, hypogammaglobulinemia, lymphoproliferation, and mycobacterial disease in patients with activating mutations in STAT3

The signal transducer and activator of transcription (STAT) family of transcription factors orchestrate hematopoietic cell differentiation. Recently, mutations in STAT1, STAT5B, and STAT3 have been linked to development of immunodysregulation polyendocrinopathy enteropathy X-linked-like syndrome. Here, we immunologically characterized 3 patients with de novo activating mutations in the DNA binding or dimerization domains of STAT3 (p.K392R, p.M394T, and p.K658N, respectively). The patients displayed multiorgan autoimmunity, lymphoproliferation, and delayed-onset mycobacterial disease. Immunologically, we noted hypogammaglobulinemia with terminal B-cell maturation arrest, dendritic cell deficiency, peripheral eosinopenia, increased double-negative (CD4(-)CD8(-)) T cells, and decreased natural killer, T helper 17, and regulatory T-cell numbers. Notably, the patient harboring the K392R mutation developed T-cell large granular lymphocytic leukemia at age 14 years. Our results broaden the spectrum of phenotypes caused by activating STAT3 mutations, highlight the role of STAT3 in the development and differentiation of multiple immune cell lineages, and strengthen the link between the STAT family of transcription factors and autoimmunity.

Onset of mechanical ventilation is associated with rapid activation of circulating phagocytes in preterm infants.

OBJECTIVE. In preterm infants with respiratory distress syndrome (RDS), circulating neutrophils are activated. Kinetics and effects of surfactant therapy on this activation are unknown. Therefore, we studied activation of circulating neutrophils and monocytes in newborn preterm infants with and without RDS. PATIENTS AND METHODS. Preterm infants with RDS who were mechanically ventilated and received surfactant (“ventilated infants”: n = 38; mean gestational age ± SD: 28.3 ± 2.2 weeks; mean birth weight ± SD: 1086 ± 353 g) and preterm infants who received nasal continuous positive airway pressure (n = 8) or no ventilatory support (n = 17) (“control infants”: mean gestational age ± SD: 32.1 ± 1.2 weeks; mean birth weight ± SD: 1787 ± 457 g) were recruited. Blood samples were taken from ventilated infants at birth, before surfactant treatment, at 1 and 2 hours after surfactant, and at 12 to 24 hours of age. Blood samples were taken from control infants at birth, at 2 to 6 hours, and at 12 to 24 hours of age. Phagocyte CD11b expression was analyzed by flow cytometry. RESULTS. In ventilated infants, phagocyte CD11b expression increased from birth to the first postnatal samples. It increased further by 12 to 24 hours of age. Control infants with or without nasal continuous positive airway pressure showed no significant increase after birth. At 12 to 24 hours of age, phagocyte CD11b expression was higher in ventilated infants than in control infants. In ventilated infants, neutrophil CD11b expression at 1 and 2 hours after surfactant correlated positively with gestational age. CONCLUSIONS. In preterm infants with RDS, significant activation of circulating phagocytes occurs within 1 to 3 hours of the onset of mechanical ventilation, independent of surfactant administration, which indicates that mechanical ventilation may be the inducer of this systemic inflammatory response.

Increased CD11b-Density on Circulating Phagocytes as an Early Sign of Late-Onset Sepsis in Extremely Low-Birth-Weight Infants

Late-onset hospital-acquired sepsis is common in extremely low birth-weight (<1000g) (ELBW) infants. The diagnosis is difficult since, at early stages of sepsis, routine laboratory tests are neither specific nor sensitive. In term infants with sepsis neutrophil surface expression of CD11b/CD18, a β2-integrin, is significantly increased. Here we studied whether increased CD11b/CD18 density on blood neutrophils and monocytes serves as an early sepsis marker in ELBW infants. Blood samples were obtained from 30 ELBW infants on a daily basis for 3–4 postnatal weeks, and neutrophil and monocyte CD11b/CD18 expression was determined by flow-cytometry. Patients were assigned one of 3 groups: 1) an infected group, comprised of infants who had blood culture-positive sepsis and/or necrotizing enterocolitis, 2) a non-infected group, and 3) a potentially infected group, comprised of infants in whom infection was suspected but could not be confirmed microbiologically. One patient had blood culture contamination and was excluded from the analysis. In the infected group, CD11b expression gradually increased during the three days preceding sampling for blood culture. At the day of sampling, median expression of CD11b in neutrophils and monocytes was higher in the infected group than in the control group. For neutrophils the sensitivity and specificity were 1.00 and 0.56, respectively, and for monocytes, 0.86 and 0.94, respectively. From these data, we conclude that determination of CD11b/CD18 density on neutrophils and monocytes may improve diagnosis of late-onset sepsis in ELBW infants.

Monocyte CD14 and soluble CD14 in predicting mortality of patients with severe community acquired infection.

Monocyte membrane CD14 (mCD14) and soluble CD14 (sCD14) both associate with poor outcome in sepsis. Because the value of combined use of the markers is unknown we measured both in patients with severe community acquired infections. The study comprised 142 acutely ill patients with community acquired pneumonia and/or blood culture-positive sepsis. Expression of mCD14 was measured, on admission to hospital, by whole blood flow cytometry and sCD14 by ELISA. There was no significant correlation between mCD14 and sCD14. Patients in the lowest tertile of mCD14 were 9.79 times (95% CI 1.31– >50, p =0.006) more likely to die than patients in the middle/highest tertiles. Survival rates in the highest and middle/lowest tertiles of sCD14 levels were comparable. After stratification by sCD14, patients in the lowest tertile of mCD14 were 14.4 times (95% CI 1.90–39.44) more likely to die than patients in the middle/highest tertiles. A significant positive correlation was detected between C-reactive protein and sCD14 levels, providing evidence that sCD14 may serve as an acute phase reactant. In conclusion, low monocyte mCD14 level, unlike the concurrent sCD14 level, predicts 28-d mortality in patients with community acquired infections.

T cell regeneration in pediatric allogeneic stem cell transplantation

Delayed and/or insufficient T cell recovery post hematopoietic stem cell transplantation (HSCT) leads to an increased risk of morbidity and mortality. We evaluated thymic function and its association with T cell regeneration post HSCT and identified factors involved in the process among pediatric stem cell transplant recipients. T cell regeneration in 66 pediatric patients was prospectively followed by naive T cell phenotyping, measuring of T cell receptor excision circles (TRECs) and expression of Foxp3 by regulatory T cells for the first 18 months post HSCT. TRECs were lower pre-HSCT in children with a malignant than non-malignant primary disease or immunosuppressed controls (P=0.001). Naive T lymphocyte reconstitution and thymic recovery were slow in the recipients of allogeneic stem cell grafts post HSCT. Infections caused by herpesviruses had a prognostic impact on mortality. Children with low TRECs had a high mortality (P=0.05) and low TRECs were also associated with extensive chronic graft-versus-host disease from 6 months onwards. Low amount of Foxp3 pre-HSCT was associated with an increased mortality post HSCT (P=0.03). Our study indicates an association between impaired T cell regeneration and thymic dysfunction and the clinical post transplant complications in pediatric allogeneic stem cell transplantation.

Low TNF-induced NF-κB and p38 phosphorylation levels in leucocytes in tumour necrosis factor receptor-associated periodic syndrome

OBJECTIVE TNF receptor-associated periodic syndrome (TRAPS) is a systemic autoinflammatory disorder caused by mutations in the type 1 TNF receptor (TNFRSF1A) gene. Because the pathomechanism of TRAPS may involve aberrant TNF-mediated intracellular signalling, we examined phosphorylation levels of nuclear factor kappaB (NF-kappaB) and p38 in response to TNF in 10 patients with three different TNFRSF1A mutations (C73R, C88Y and F112I). METHODS Phosphorylation levels of NF-kappaB p65 and p38 were determined in fresh leucocytes stimulated with TNF (0-100 ng/ml) for 2.5-20 min and permeabilized for phospho-specific antibodies in a whole blood flow cytometry assay. As control agonists, we used bacterial lipopolysaccharide (LPS) and IFN-gamma, the latter mediating phosphorylation of the signal transducer and activator of transcription 1. Areas under curve values for dose-response and time course of NF-kappaB and p38 phosphorylation were calculated for the comparison of patients and reference subjects. RESULTS NF-kappaB and p38 phosphorylation levels of monocytes, lymphocytes and neutrophils stimulated with TNF were significantly lower in TRAPS patients than in reference subjects. Phosphorylation levels induced by LPS, or by IFN-gamma, in patient and reference samples were comparable, indicating that the defect was confined to TNF-mediated signalling. CONCLUSIONS In the three families studied, TRAPS was associated with low TNF-mediated signalling in leucocytes. This deficiency of the innate immune system may result in the activation of as yet unidentified compensatory regulatory mechanisms yielding the hyperinflammatory phenotype of TRAPS.

Quality control of flow cytometry data analysis for evaluation of minimal residual disease in bone marrow from acute leukemia patients during treatment.

Low levels of leukemia cells in the bone marrow, minimal residual disease (MRD), are considered to be a powerful indicator of treatment response in acute lymphatic leukemia (ALL). A Nordic quality assurance program, aimed on standardization of the flow cytometry MRD analysis, has been established before implementation of MRD at cutoff level 10−3 as one of stratifying parameters in next Nordic Society of Pediatric Hematology and Oncology (NOPHO) treatment program for ALL. In 4 quality control (QC) rounds 15 laboratories determined the MRD levels in 48 follow-up samples from 12 ALL patients treated according to NOPHO 2000. Analysis procedures were standardized. For each QC round a compact disc containing data in list-mode files was sent out and results were submitted to a central laboratory. At cutoff level 10−3, which will be applied for clinical decisions, laboratories obtained a high concordance (91.6%). If cutoff level 10−4 was applied, the concordance would be lower (85.3%). The continuing standardization resulted in better concordance in QC3 and QC4 compared with QC1 and QC2. The concordance was higher in precursor B as compared with T-cell ALL. We conclude that after standardization, flow cytometry MRD detection can be reliably applied in international, multicenter treatment protocols.

Activated Protein C Reduces Graft Neutrophil Activation in Clinical Renal Transplantation

We studied the role of endogenous activated protein C (APC), the major physiological anti‐coagulant with concomitant anti‐inflammatory properties, on ischemia/reperfusion (I/R) in 45 patients participating in a larger trial comparing three immunosuppressive protocols in cadaveric renal transplantation: perioperative anti‐thymocyte globulin (ATG, Fresenius AG, Bad Homburg, Germany), perioperative basiliximab and conventional triple therapy. Blood samples for assessing plasma APC, protein C, and lactoferrin concentrations, neutrophil CD11b and L‐selectin expressions and blood leukocyte differential counts were obtained preoperatively and before reperfusion from central venous cannula, complemented with simultaneous samples from iliac artery and graft vein for calculation of transrenal differences (Δ) of study parameters at 1 and 5 min after reperfusion. Unlike basiliximab or conventional therapy groups, ATG infusion induced a substantial increase in plasma APC concentration (119 [88–144]% before infusion vs. 232 [85–1246]% after infusion, p < 0.001), resulting in renal graft sequestration of APC at 1 min after reperfusion (Δ=−72 [−567 to 12]%, p < 0.001). Graft APC consumption was associated with transrenal reduction of neutrophil activation markers (L‐selectin r= 0.7, p = 0.01; lactoferrin r=−0.6, p = 0.02; CD11b r=−0.8, p = 0.001), and with both warm (r= 0.6, p = 0.01) and cold ischemia time (r= 0.6, p = 0.02) and donor age (r= 0.6, p = 0.01). These findings suggest that APC has an anti‐inflammatory role in I/R injury in clinical renal transplantation.

Activation of T Cells in Preterm Infants with Respiratory Distress Syndrome

Background: Preterm infants with respiratory distress syndrome (RDS) present with systemic inflammation. The role of lymphocytes in RDS is less studied. Activation of lymphocytes could mediate chronic inflammation and development of bronchopulmonary dysplasia (BPD). Objective: To evaluate whether T cells are activated in preterm infants with RDS and whether T cell activation is associated with the development of BPD. Methods: Thirty-four infants with RDS [mean gestational age 27.1 (SD 2.0) weeks, birth weight 900 (216) g] were compared with 21 infants without RDS [32.6 (1.4) weeks, 1,697 (406) g]. From blood samples taken on postnatal days 1, 3, and 7, CD4 and CD8 cell counts and their expressions of co-stimulatory molecule CD54 and adhesion molecule CD62L were determined by flow cytometry. In activated cells, expression of CD54 is increased and CD62L is decreased. Results:As compared with infants without RDS, infants with RDS had less CD4 and CD8 cells on day 3 (both p = 0.02). On day 1 and day 3, RDS was associated with increased CD54 expression on CD4 cells (p = 0.001; p = 0.03) and decreased CD62L expression on CD8 cells (both p = 0.02). Infants with RDS who developed BPD (n = 18) had higher CD54 expression on CD4 cells on day 3 (p = 0.01) and on CD8 cells on day 1 and day 3 (p = 0.01; p = 0.04) as compared with infants without BPD (n = 16). Conclusions: In preterm infants, RDS is associated with a lower T cell count and a higher proportion of activated cells. Increased proportion of activated T cells predicts the development of BPD. Systemic T cell activation could mediate inflammation and development of BPD.

Comparative analysis of minimal residual disease detection by multiparameter flow cytometry and enhanced ASO RQ-PCR in multiple myeloma

Multiparameter flow cytometry (MFC) and allele-specific oligonucleotide real-time quantitative PCR (ASO RQ-PCR) are the two most sensitive methods to detect minimal residual disease (MRD) in multiple myeloma (MM). We compared these methods in 129 paired post-therapy samples from 22 unselected, consecutive MM patients in complete/near complete remission. Appropriate immunophenotypic and ASO RQ-PCR-MRD targets could be detected and MRD analyses constructed for all patients. The high PCR coverage could be achieved by gradual widening of the primer sets used for clonality detection. In addition, for 13 (55%) of the patients, reverse orientation of the ASO primer and individual design of the TaqMan probe improved the sensitivity and specificity of ASO RQ-PCR analysis. A significant nonlinear correlation prevailed between MFC-MRD and PCR-MRD when both were positive. Discordance between the methods was found in 32 (35%) paired samples, which were negative by MFC-MRD, but positive by ASO RQ-PCR. The findings suggest that with the described technique, ASO RQ-PCR can be constructed for all patients with MM. ASO RQ-PCR is slightly more sensitive in MRD detection than 6−10-color flow cytometry. Owing to technical demands ASO RQ-PCR could be reserved for patients in immunophenotypic remission, especially in efficacy comparisons between different drugs and treatment modalities.