Vassiliki Tzimouli

Impact of prematurity, stress and sepsis on the neutrophil respiratory burst activity of neonates

The aim of this study was to evaluate the impact of prematurity, sepsis and stress on the neutrophil respiratory burst activity (NRBA) of neonates. For this purpose 122 healthy neonates (89 term and 33 preterm), 33 preterm stressed neonates, 59 septic neonates (12 term and 47 preterm) and 26 healthy adults were studied. The NRBA was assessed after in vitro stimulation by PMA using a whole blood flow cytometric microassay with dihydrorhodamine 123 (DHR 123). It was found that the percentage of responding neutrophils in term neonates was comparable to that found in adults (medians 83.5 and 89.8%, respectively), whereas it was significantly lower in the healthy preterm neonates (median 70.6%, p < 0.05). The NRBA was further depressed in the stressed (median = 63%) and septic neonates, both term and preterm (medians 60.5 and 54.3%, respectively). No correlation with the levels of G-CSF, TNF-alpha and IL-1 beta, which were found to be higher in the stressed and septic neonates, was observed. These findings indicate that prematurity, sepsis and stress significantly depress the respiratory burst activity of neonatal neutrophils.

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.

Renal dysfunction in patients with beta-thalassemia major receiving iron chelation therapy either with deferoxamine and deferiprone or with deferasirox.

There are limited studies on renal involvement in β-thalassemia, mainly involving patients on deferoxamine, reporting both glomerular and tubular dysfunction. The aim of the present study was to investigate renal involvement in young thalassemia patients, using both conventional and early markers of renal dysfunction, and to correlate findings to iron chelation therapy. Forty-two patients aged 4–23 years were studied and, for analysis purposes, were divided into two groups based on chelation therapy (group A receiving deferasirox and group B receiving deferoxamine and deferiprone combination therapy). In addition to conventional renal biochemistries, creatinine clearance, estimated glomerular filtration rate, serum cystatin C (Cys C), fractional excretion of sodium, tubular phosphorus reabsorption and urine calcium, protein, β2-microglobulin (β2-MG) and glucose levels were measured. A considerable number of patients demonstrated impaired renal function with elevated Cys C levels (36%), glomerular dysfunction with proteinuria (24%) and tubulopathy with hypercalciuria (35.5%) and elevated excretion of β2-MG (33.5%). Renal involvement seems to be present even in young patients with β-thalassemia, therefore, routine use of early markers of renal dysfunction is recommended. Further studies are needed in order to investigate the role of new chelators in tubular function parameters.

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.

Simultaneous changes in serum HMGB1 and IFN-α levels and in LAIR-1 expression on plasmatoid dendritic cells of patients with juvenile SLE.. New therapeutic options?

We investigated the simultaneous changes in serum levels of HMGB1 and IFN-α as well as in LAIR-1 expression on plasmatoid dendritic cells (pDCs) of juvenile systemic lupus erythematosus (jSLE) patients in order to explore their involvement in the disease pathogenesis and their correlation with disease activity and other characteristics. In total, 62 blood samples were studied from 26 jSLE patients (18 girls), aged 8–16 years. Twenty healthy subjects (16 girls) of comparable age were included as healthy controls (HCs). Concentrations of serum HMGB1 and IFN-α were assessed by ELISA and LAIR-1 expression on pDCs by five-color flow cytometry. The disease activity index was assessed by SLEDAI and ECLAM scores. It was found that mean serum levels both of HMGB1 and IFN-α were significantly increased in jSLE patients compared to HCs and in jSLE patients with active disease with or without active nephritis compared to those with inactive disease. Mean serum levels of HMGB1 were positively correlated with levels of IFN-α and both were positively correlated with the SLEDAI and ECLAM scores. The expression of LAIR -1 on pDCs of jSLE patients was significantly lower than that of HCs. In conclusion, our findings indicate that serum HMGB1 not only represents a potential marker of disease activity but together with the lack of LAIR-1 inhibitory function may contribute to the sustained inflammatory action of IFN-α in jSLE. In this regard, blocking the action of HMGB1 and its receptors or enhancing the expression/inhibitory function of LAIR-1 on pDCs should be included in future immune interventions for controlling jSLE.

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.

Novel biomarkers for the assessment of paediatric systemic lupus erythematosus nephritis

The discovery of serum biomarkers specific for paediatric lupus nephritis (pLN) will facilitate the non‐invasive diagnosis, follow‐up and more appropriate use of treatment. The aim of this study was to explore the role of serum high‐mobility group box 1 (HMGB1) protein, antibodies against nucleosomes (anti‐NCS), complement factor C1q (anti‐C1q) and glomerular basement membrane (anti‐GBM) in pLN. Serum samples of 42 patients with paediatric systemic lupus erythematosus (pSLE) (22 with pLN and 20 without renal involvement), 15 patients with other autoimmune nephritis (AN) and 26 healthy controls (HCs) were examined using enzyme‐linked immunosorbent assay (ELISA). The activity of both pSLE and pLN was assessed by the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) tool. The levels of all four biomarkers were significantly higher in pLN compared to AN and to HCs. The anti‐NCS, anti‐GBM and HMGB1 serum levels were significantly higher in pLN than in pSLE without renal involvement. The anti‐C1q and the HMGB1 serum levels were correlated positively with pSLE activity. The HMGB1 serum levels were also correlated positively with pLN activity. These findings suggest that serum anti‐NCS, anti‐GBM and HMGB1 may serve as biomarkers specific for the presence of nephritis in pSLE. HMGB1 emerged as a useful biomarker for the assessment of pLN and pSLE activity, whereas anti‐C1q only of pSLE activity.

IMMUNE AND NEURAL STATUS OF THALASSEMIC PATIENTS RECEIVING DEFERIPRONE OR COMBINED DEFERIPRONE AND DEFEROXAMINE CHELATION TREATMENT

Deferiprone (L1), has previously been reported to be associated with immunological abnormalities in iron loaded thalassemia patients. However, other factors may also have similar effects such as the level of iron overload, chronic immuno-stimulation due to transfusions, splenectomy and deferoxamine (DFO). During chelation therapy with DFO, several complications have been reported, which were due to pharmacological activity and high dose toxicity with regard to both acoustic and visual effects, as well as peripheral nerve disorders that were measured by nerve conduction velocities. The immune and neural status of 44 β-thalassemic patients, aged 10–30 years (mean 19.4 ± 4.9), receiving L1 as a monotherapy (n = 21), or in combination with DFO (n = 23), has been followed for 2 years by monitoring the level of immunoglobulins (IgG, IgM, IgA), the level of T and B lymphocytes (CD4/CD8), the auto antibodies: anti nuclear (ANA), anti-double-stranded (anti-ds DNA), anti reticulin (anti-R1), anti-extra nuclear (anti-ENA), anti histone (anti-AHA), anti liver-kidney-muscle (anti-LKM), anti-smooth muscle (anti-SMA), anti-thyroid (anti-ATA), anti-mitochondrial (anti-AMA) antibodies and the C-reactive protein. The percentage of patients with disorders of the immune and nervous system concerned very few cases. None of our patients with pathological findings in their immunological or neurophysiological examinations presented any signs or symptoms of involvement of the immune or nervous system. Further advantages have been identified for the oral use of L1 and its combination with DFO, including synergistic efficacy and lower dosing with limited toxicity.

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.

The portrait of Familial Mediterranean Fever in N. Greek pediatric patients: a 30-year experience

Familial Mediterranean Fever (FMF), is the second commonest autoinflammatory disease in pediatric Greek patients (pts) after PFAPA. So far, long-term follow-up case series in Greek FMF pts have not been emerged.