Florence Kanakoudi-Tsakalidou

Administration of recombinant human granulocyte-colony stimulating factor to septic neonates induces neutrophilia and enhances the neutrophil respiratory burst and β2 integrin expression Results of a randomized controlled trial

Abstract The objective of this study was to investigate the effect of treatment with recombinant human granulocyte-colony stimulating factor (rhG-CSF) on the neutrophil count and function of preterm neonates with documented sepsis. For this purpose 62 preterm neonates with proven sepsis and 19 healthy preterm ones were studied. Of the 62 patients, 27 septic neonates had an absolute neutrophil count (ANC) >5000/mm3 (group A) and were scheduled not to receive rhG-CSF and 35/62 had an ANC <5000/mm3 (n= 35) and were randomly assigned either to receive rhG-CSF (group B) or not to receive it (group C). rhG-CSF (10 μg/kg) was administered for 3 consecutive days (0, 1, 2). The ANC, plasma levels of G-CSF (ELISA), neutrophil respiratory burst activity (NRBA) and neutrophil expression of CD11a, CD11b and CD11c (flow cytometry) were measured in all septic neonates on days 0 (onset of sepsis), 1, 3 and 5 and in the healthy neonates once within the first 2 days of life. We found that on day 0, G-CSF levels of all groups of septic neonates were significantly higher than those of the healthy ones. The highest levels were observed in group A. NRBA was diminished only in groups B and C and the expression of CD11a and CD11c was reduced in all groups of septic neonates. Administration of rhG-CSF resulted in a rapid and significant increase in ANC, NRBA and CD11a, CD11b and CD11c expression that persisted throughout the follow up. Conclusion The administration of granulocyte colony stimulating factor to septic neonates significantly increases the absolute granulocyte count and enhances the neutrophil respiratory burst and β2 integrin expression.

Flow cytometric measurement of HLA-DR expression on circulating monocytes in healthy and sick neonates using monocyte negative selection

The aim of this study was to investigate the effect of prematurity, neonatal sepsis, respiratory distress syndrome (RDS) and perinatal asphyxia on monocyte HLA‐DR expression of neonates using a flow cytometric method based on monocyte negative selection. The subjects were one hundred and thirty‐one neonates (59 healthy, 44 septicaemic, 20 with RDS and eight with perinatal asphyxia) and 20 healthy adults. Monocyte HLA‐DR expression was measured using one‐colour HLA‐DR labelling in a gate for monocytes obtained using the combination of CD3‐CD19–PE/CD15–FITC MoAbs. In addition, the common dual staining method using MoAbs against two CD14 epitopes (TUK4, MO2) was evaluated. With the one‐colour HLA‐DR labelling higher purity and recovery values of monocytes were achieved than with the dual labelling method. Healthy neonates had significantly lower percentages of HLA‐DR+ monocytes than adults (69 ± 13% versus 91·5 ± 2·5%) and comparable mean fluorescence intensity (MFI) (119 ± 25 versus 131 ± 26). Values did not differ significantly between healthy term and preterm neonates. Preterm neonates with RDS had a significantly lower percentage of HLA‐DR+ monocytes than the healthy preterm neonates. In neonates with asphyxia both parameters were comparable to those of the healthy ones. Septicaemic neonates presented significantly lower values of both parameters than the healthy, RDS and asphyxiated neonates. Monocyte negative selection provides a reliable estimation of HLA‐DR expression on monocytes. Expression of monocyte HLA‐DR is lower in healthy neonates in comparison with adults and is further decreased in neonates with sepsis and RDS, but it is not influenced by prematurity and perinatal asphyxia.

Case Report. Hepatic abscesses due to Aspergillus terreus in an immunodeficient child

Summary. We report the first case of hepatitis due to Aspergillus terreus in a 13‐year‐old boy with common variable immunodeficiency that occurred while the patient was receiving secondary prophylaxis with fluconazole after an episode of pulmonary candidosis. The infection subsided after the addition of itraconazole to the combination of liposomal amphotericin B and granulocyte‐macrophage colony‐stimulating factor that he was receiving.

The significance of persistent newly developed autoantibodies in JIA patients under long-term anti-TNF treatment

OBJECTIVE To study the significance of persistent (12 months) new autoantibodies, in Juvenile Idiopathic Arthritis (JIA) patients treated with either Infliximab (INFL) or Etanercept (ET) for 2 years. PATIENTS-METHODS 26 children under INFL (n=12) or ET (n=14) were prospectively studied. A large panel of autoantibodies was tested using indirect immunofluorescence (ANA, anti-dsDNA, anti-ENA, SMA, LKM, AMA, PCA, anti-R1, ATA), ELISA (ANA, anti-ENA, anti-cardiolipin, ANCA), immunoblotting assay (anti-ENA: anti-Ro, anti-La, anti-Sm, anti-URNP, anti-Jo, anti-Scl70, anti-centromere, anti-ribosomal and anti-histone) and rate nephelometry (RF). RESULTS Apart from the positive patients for ANA (13/26) and RF (2/26) prior to anti-TNF treatment, 6/26 patients (23%) developed new autoantibodies (SMA, anti-R1, ATA) which persisted for 12-50 months. None developed antibodies to nuclear antigens. In only one case, ATA was associated with the development of Hashimotos thyroiditis. CONCLUSIONS These findings indicate that in JIA patients in contrast to adult RA patients, development of new autoantibodies to various nuclear antigens is rare. Other non relevant to rheumatic diseases autoantibodies, may appear and persist for >12 months, but very rarely they may be related to clinical entities, especially in the presence of a positive family history of autoimmunity.

IL-18 is correlated with type-2 immune response in children with steroid sensitive nephrotic syndrome

Children with steroid sensitive nephrotic syndrome (SSNS) is thought to have dysregulated type-1/type-2 cytokine network. Interleukin (IL)-18 is a cytokine, which may enhance both type-1 and type-2 responses, depending on the cytokines milieu. This prospective study aimed to assess type-1/type-2 cytokine synthesis and production profile in different stages of SSNS and define the potent involvement of IL-18. Twenty-three children with SSNS, aged 2.5-14 years, were studied; 23/23 both in active stage before treatment initiation and in remission still on steroids; 15/23 in remission off steroids as well. Data were compared with those obtained from 25 age-matched controls. The following parameters were assessed: Basic T cell populations, percentages of CD3+/CD69+/IFN-gamma+ and CD3+/CD69+/IL-4+ T cells as well as serum levels of IFN-gamma, IL-2, IL-4, IL-13 and IL-18. No difference in IL-2 levels was found between nephrotic children of all disease stages and controls (p>0.05). Percentage of CD3+/CD69+/IL-4+ T cells and serum levels of IL-4, IL-13 and IL-18 were significantly higher in the active stage of SSNS compared with the remission stages and controls (p<0.05). On the contrary, percentage of CD3+/CD69+/IFN-gamma+ T cells as well as serum IFN-gamma were significantly lower during active disease stage compared with remission stages and controls (p<0.05). In children with SSNS, of all disease stages, serum levels of IL-18 were significantly correlated with both IL-4 and IL-13 (r=0.628 and p<0.0001, r=0.71 and p<0.0001, respectively). It seems that a type-2 cytokine synthesis and production pattern prevails in children with active SSNS and IL-18 expression is significantly correlated with this type-2 immune response.

Antenatal Betamethasone Does Not Influence Lymphocyte Apoptosis in Preterm Neonates

Despite the widespread use of antenatal glucocorticosteroids (GCs), the possibility of adverse effects on the immune response in preterm neonates remains a major concern. GCs stimulate lymphocyte apoptosis, resulting in lymphopenia and functional disorders, which have been associated with sepsis-related death in critically ill neonates. We sought to assess the effect of antenatal betamethasone (BM) on lymphocyte apoptosis in preterm neonates. Fifty preterm neonates exposed to antenatal BM and 50 controls were studied prospectively. Lymphocyte apoptosis was assessed using the annexin-V/propidium iodide (PI) assay, analysis of cell cycle after staining with PI, and intracellular caspase-3 activity. The two groups did not differ significantly as regards absolute lymphocyte counts and the percentage of lymphocytes being annexin-V (+)/PI (-) (early apoptotic) or lymphocytes in the subG1 peak after staining with PI and those with intracellular caspase-3 activation. The lymphocyte number and apoptosis were not associated with the time elapsed between antenatal BM administration and delivery. A single course of antenatal BM does not influence apoptosis of neonatal lymphocytes. This is of significant importance with respect to the preservation of lymphocyte-associated immune response in preterm neonates.

Peripheral CD19+ B cells are increased in children with active steroid-sensitive nephrotic syndrome

Sir, Pathogenesis of steroid-sensitive nephrotic syndrome (SSNS) is thought to be related mainly to T-cell dysfunction [1]. However, the beneficial use of rituximab in cases of frequently relapsing SSNS provided evidence of B cell involvement [2–4]. Our aim was, thus, to investigate prospectively the levels of the circulating CD19+ B cells in children with a first episode of SSNS in sequential stages (presentation, remission on steroids and remission off steroids). Twenty-three children (M/F = 13/10, age = 2.5–14 years, median = 4.32 years) with a first episode of SSNS were studied; 23/23 both at presentation (before steroids initiation) and in remission on steroids (40 mg/m2 on alternate day); 15/23 were tested as well in remission off steroids for at least 6 months. Twenty-five age-matched children who had come to the outpatient haematology clinic in order to be tested for b-thalassaemia trait were found to be negative and served as healthy controls (controls 1). Considering that the presentation of SSNS may be associated with a recent infection, mainly a respiratory tract infection, twenty age-matched children with an upper respiratory tract infection acted as a second control group (controls 2). The percentages of CD3± T cells, as a pan T-cell marker, and the percentages of CD19+ and CD20± B cells were evaluated in all children. The above-mentioned parameters were determined in each sample by flow cytometry using the lysed whole blood method. The duochrome phycoerythrin-cyanin5 (PE-Cy5) conjugated CD3± monoclonal antibody (MoAb) and phycoerythrin (PE) conjugated CD19+ and CD20± MoAbs purchased from Beckman Coulter were used. The samples were analysed with a EPICS-XL flow cytometer. The results were expressed as percentages (%) of fluorescence-positive cells as well as actual numbers (cells/μL) of the circulating CD19+ and CD20+ B cells, based on the white blood cell count. Statistical analysis was performed using the Statistical Package for the Social Sciences for Windows (SPSS version 11.5). The paired t-test and independent-samples t-test were used to compare differences between study groups with and without paired data, respectively. Pearsons coefficient of correlation (r) was used to determine the correlations. A P ≤ 0.05 was considered to be statistically significant. In 5 of 23 children with a first episode of SSNS, there was a recent history of an upper respiratory tract infection. Remission was achieved in all children within 6–15 days after steroid initiation. During remission, all patients presented normoalbuminaemia and were free of proteinuria and albuminuria. Percentages of CD3± T cells were found to be within normal limits in all patients (at presentation of SSNS, in remission still on steroids and in remission off steroids) compared with the two control groups (P ≥ 0.05). As depicted in Figure ​Figure1,1, the circulating CD19+ B cells were significantly higher at presentation of SSNS (mean percentage = 18.13 ± 5.43, mean actual number = 695.34 ± 258.29) compared with remission on steroids (mean percentage = 13.57 ± 4.22 and P 0.05). Moreover, circulating CD19+ B cells were inversely correlated with disease activity (r = −0.465, P 0.05). Fig. 1 Percentage of CD19+ B cells in children with a first episode of SSNS in sequential stages (presentation, remission on steroids, remission off steroids) and in controls 1 and 2. The pathogenesis of SSNS has mainly been focused on T cells dysfunction. The success of rituximab, a chimeric anti-CD20 antibody, in the treatment of cases of frequently relapsing SSNS initiated interest in pathogenic pathways involving B cells [2–4]. There is an interaction between B cells and T cells, and B cells are involved in the presentation of antigens to T cells. However, the effect of B-cell depletion on T-cell function is unknown. Kemper et al. [5] suggested that in children with SSNS, there is a combined T- and B-cell activation. In agreement with our results, recent studies showed that CD19+ B cells may be elevated in relapsing nephrotic children [6,7]. Children with both steroid-sensitive and steroid-resistant nephrotic syndrome were included in this study, and there were no paired data of the circulating CD19+ B cells in remission off steroids. It is worth noting that in our study not all children with SSNS presented elevated CD19+ B cells. We intend to follow up this cohort of 23 nephrotic children in order to identify whether the long-term outcome of the nephrotic syndrome is associated with the up-regulation of CD19+ B cells in these patients taking into account steroid dependency and frequency of relapses. Our findings demonstrated an up-regulation of circulating CD19+ and CD20± B cells in children with a first episode of SSNS. This indicates that B cells may play a role in SSNS and that rituximab may be considered in certain cases. Conflict of interest statement. None declared.

Secondary osteonecrosis mimicking a femur bone tumor in an adolescent with juvenile idiopathic arthritis

To the Editor: Osteonecrosis (ON) of the femoral head has been described in patients with sickle cell disease [1], malignancies [2] and juvenile idiopathic arthritis (JIA) receiving prednisone (PDN) [3]. We present a 15-year-oldale with a systemic JIA onset, who developed secondary ON mimicking ‘‘skip’’ lesions of bone malignancy due to its unusual location in the lower part of her right thigh. Fourteen months following diagnosis, while in disease remission, she complained of a persistent femur pain. It was a continuous dull pain, with no history of trauma, aggravated by the routine physical activities, resolving with rest and responding to analgesics. Her previous regimen included methotrexate (MTX) 15 mg/m/week, cysclosporine (CSA) 3 mg/kg/day and PDN 2 mg/ kg for the first 2 months, following tapering that ended to 0.25 mg/ kg/day for the last 5 months. X-rays and MRI revealed lesions in the right femoral diaphysis and distal metaphysis suspicious of an osteosarcoma (Fig. 1). The double phase scan TI-201 did not show any radiotracer uptake and was felt to exclude malignancy. To make a definitive diagnosis, an open biopsy was performed. The specimen consisted of pieces of lamellar bone, woven bone, striated muscle and necrotic material. It was compatible either with a healing bone fracture and/or ON. She was conservatively managed by gradual withdrawal of PDN, restriction of weight bearing; she returned to her regular activities 2 months later. After a 5-year follow-up, her radiographs and MRI showed healing sclerotic lesions of the ON area with no bone marrow oedema or periosteal reaction or any soft tissue mass. ON of the femoral bone in patients with pediatric rheumatic diseases receiving long-term PDN is unusual [4,5]. We assume that PDN was the exclusive contributor to the development of ON, as data on the association of MTX or CSA with ON have not been reported. The femoral head is the most common and clinically significant site of ON, while the femoral shaft of JIA patients has not previously been reported. In adults using steroids or following radiation, insufficient fractures of the sacrum have been misdiagnosed as malignancies [6]. On the other hand, Fujimoto et al. [7] emphasized that a metastatic femur tumor can mimic spontaneous ON and have a misleading appearance on MRI imaging. We were faced with the same dilemma due to the unusual location of the ON lesions and the misleading imaging that was consistent with that of a bone tumor. Thallium scintigraphy was an important piece of data as it appeared inconsistent with malignancy [8]. However, a biopsy might be necessary to support such a diagnosis of ON, as occurred in our patient. This case highlights the importance of including ON in the differential diagnosis for bone pain of any patient receiving steroids.

The Greek version of the Juvenile Arthritis Multidimensional Assessment Report (JAMAR)

The Juvenile Arthritis Multidimensional Assessment Report (JAMAR) is a new parent/patient-reported outcome measure that enables a thorough assessment of the disease status in children with juvenile idiopathic arthritis (JIA). We report the results of the cross-cultural adaptation and validation of the parent and patient versions of the JAMAR in the Greek language. The reading comprehension of the questionnaire was tested in 10 JIA parents and patients. Each participating centre was asked to collect demographics, clinical data, and the JAMAR from 100 consecutive JIA patients or all consecutive patients seen in a 6-month period and to administer the JAMAR to 100 healthy children and their parents. The statistical validation phase explored descriptive statistics and the psychometric issues of the JAMAR: the three Likert assumptions, floor/ceiling effects, internal consistency, Cronbach’s alpha, interscale correlations, test–retest reliability, and construct validity (convergent and discriminant validity). The Greek JAMAR was fully cross-culturally adapted with two forward and three backward translations. A total of 272 JIA patients (5.9% systemic, 57.7% oligoarticular, 21.3% RF negative poly-arthritis, 15.1% other categories), and 100 healthy children were enrolled in all centres. The JAMAR components discriminated well-healthy subjects from JIA patients; notably, there was no significant difference between healthy subjects and their affected peers in psychosocial quality of life and school-related items. All JAMAR components revealed good psychometric performances. In conclusion, the Greek version of the JAMAR is a valid tool for the assessment of children with JIA and is suitable for use both in routine clinical practice and in clinical research.

B lymphocyte stimulator, interferon-α and HMGB 1 interrelation in childhood onset systemic lupus erythematosus: associations with disease activity and severity

The role of B lymphocyte stimulator (Blys) in Childhood onset Systemic Lupus Erythematosus (cSLE) has not been elucidated.