Platelet size for distinguishing between inherited thrombocytopenias and immune thrombocytopenia: a multicentric, real life study.

The most frequent forms of inherited thrombocytopenia (IT) are characterized by platelet size abnormalities and it has been suggested that this parameter is useful for their differentiation from immune thrombocytopenia (ITP). Recently, a monocentric study identified cut-off values for mean platelet volume (MPV) and mean platelet diameter (MPD) with good diagnostic accuracy in this respect. To validate these cut-off values in a different and larger case series of patients, we enrolled 130 subjects with ITP and 113 with IT in six different centres. The platelet count and MPV was each measured by the instrument routinely used in each institution. In some centres, platelet count was also measured by optical microscopy. MPD was evaluated centrally by image analysis of peripheral blood films. The previously identified cut-off value for MPV had 91% specificity in distinguishing ITP from inherited macrothrombocytopenias (mono and biallelic Bernard-Soulier, MYH9-related disease), while its sensitivity was greatly variable depending on the instrument used. With an appropriate instrument, specificity was 83%. The diagnostic accuracy of MPD was lower than that obtained with MPV. We concluded that MPV is a useful parameter for differentiating ITP from IT provided that it is measured by appropriate cell counters.

Diagnostic performance of triggering receptor expressed on myeloid cells-1 and CD64 index as markers of sepsis in preterm newborns.

OBJECTIVE: CD64 index and triggering receptor expressed on myeloid cells-1 are biomarkers on neutrophil polymorphonuclear cells with crucial role in sepsis. The study aim is to assess diagnostic performance, individually and combined, of CD64 index and triggering receptor expressed on myeloid cells-1 (surface marker/soluble form), in late-onset sepsis of preterm infants. DESIGN: Observational study. SETTING: Neonatal ICU. PATIENTS: Sixteen septic and 16 control preterm infants, gestational age younger than 32 weeks and/or birth weigh less than 1500 g. MEASUREMENT AND MAIN RESULTS: Seventy preterm infants, free of sepsis were enrolled into the study. CD64 index and triggering receptor expressed on myeloid cells-1 were measured once between day 5 and 15 of life (T0) and once between day 16 and 25 (T1). At T1, 16 infants were assigned to septic group because of reported signs of sepsis and positive blood culture. From the remaining 54 infants, 16 of them who always remained free of sepsis had a blood sample at T1 and constituted the control group (n = 16). Comparing T1 vs T0, triggering receptor expressed on myeloid cells-1 polymorphonuclear cells percentage was significantly lower (p = 0.002) in septic group but not in control group; soluble triggering receptor expressed on myeloid cells-1 concentration did not show significant differences in both groups; CD64 index significantly increased (p = 0.0004) in septic group, while no difference was found in control group. Comparing septic with control group at T0, no differences were found in any markers. At T1, triggering receptor expressed on myeloid cells-1 polymorphonuclear cells percentage was significantly lower (p = 0.003) and CD64 index was higher (p = 0.00019) in septic infants. Triggering receptor expressed on myeloid cells-1 polymorphonuclear cells receiver operating characteristic curve indicated cutoff 62.12%, sensitivity 56.2%, specificity 93.5%, and area under the curve 0.8. CD64 index receiver operating characteristic curve indicated cutoff 2.85, sensitivity 87.5%, specificity 100%, and area under the curve 0.95. Combination of the two indexes was not useful in increasing individual diagnostic power. CONCLUSIONS: Despite limited sample size, CD64 index demonstrated to be a promising biomarker, with high specificity, to diagnose late-onset sepsis. Further investigations are needed to substantiate these findings. Triggering receptor expressed on myeloid cells-1 showed less valuable diagnostic role.

Induction and survival of binucleated Purkinje neurons by selective damage and aging.

Fusion of bone marrow-derived cells with adult Purkinje cells in the cerebellum gives rise to binucleated Purkinje cells. Whether fusion can be modulated by epigenetic factors and whether fused neurons are stable has remained unclear. Here, we show that in mice and rats, partial ablation of Purkinje cells and local microglial activation in the absence of structural damage to the cerebellum increase the rate of fusion. Moreover, mouse Purkinje cells once fused with bone marrow-derived cells are viable for at least 7 months. We also show that cerebellar irradiation is unnecessary for the generation of binucleated Purkinje cells after bone marrow grafting. Moreover, binucleated Purkinje cells can be found in aged mice that did not receive any treatment, suggesting that fusion events occasionally occur throughout the whole lifespan of healthy, unmanipulated individuals. However, in aged chimeric mice that, after bone marrow transplant, have the majority of their nucleated blood cells fluorescent, the number of binucleated fluorescent Purkinje cells is two orders of magnitude less than the total number of binucleated Purkinje cells. This suggests that, in the majority of heterokaryons, either the incoming nucleus is quickly inactivated or fusion is not the only way to generate a binucleated Purkinje cell.

Early and accurate diagnosis of congenital toxoplasmosis.

OBJECTIVE: Early diagnosis of congenital toxoplasma infection is difficult to establish using serological methods. We explored specific T cell immunity to Toxoplasma gondii antigens to identify more accurate diagnostic tests for an early diagnosis of toxoplasma infection in newborns at risk for congenital toxoplasmosis. STUDY DESIGN: T lymphocyte proliferation, interferon (IFN)-gamma production and lymphocyte activation antigens expression were evaluated in 23 infected and 65 uninfected neonates at different times, in the first year of life. RESULTS: The immunologic tests accurately discriminated when tested 90 days of age, respectively and were significantly lower in uninfected than in infected infants: activation antigen CD25, P < 0.001 and P < 0.00001; activation antigen histocompatibility leukocyte antigen (HLA)-DR, P < 0.01 and P < 0.00001; T cell proliferation, P < 0.0001 and P < 0.00001; IFN-gamma production, P < 0.001 and P < 0.00001. Evaluation of the specific T cell response allowed identification at 3 months of age or younger, 2 of 23 infected neonates, who had negative serologic tests. Moreover specific T lymphocyte activity increased with age even in neonates undergoing therapy, suggesting that medical treatment does not affect lymphocyte response. CONCLUSIONS: Evaluation of T cell immunity is important for an early and accurate diagnosis of congenital toxoplasmosis.

Regulatory CD4+CD25+ T cells in the peripheral blood of lung transplant recipients: correlation with transplant outcome.

BACKGROUND: The subset of CD4+CD25+ regulatory T cells, recently identified in humans, may play a central role in the regulation of immune tolerance to graft survival. METHODS: This study assesses the frequency and functional profile of CD4+CD25+CD69- cells in the peripheral blood of lung transplant recipients (>3 years from transplantation), 10 of whom were in a stable clinical condition and 11 of whom demonstrated chronic rejection (bronchiolitis obliterans syndrome). We also studied a group of seven healthy subjects. RESULTS: The frequency of CD4+ T cells expressing CD25 (CD4+CD25+) and the highest levels (CD25) were lower in patients with bronchiolitis obliterans syndrome compared with healthy subjects and subjects in a stable clinical condition (P < or = 0.01). Purified CD4+CD25+ cells exhibited a regulatory functional profile in vitro: they were hyporesponsive, suppressed the proliferation of CD4+CD25- cells, and produced interleukin-10. CONCLUSION: These results provide in vivo evidence that peripheral CD4+CD25+ T cells may represent an important regulatory subset in lung transplantation.

The red cell distribution width (RDW): value and role in preterm, IUGR (intrauterine growth restricted), full-term infants.

OBJECTIVE: To measure the red cell distribution width (RDW) ranges at birth and to evaluate potential association with typical neonatal diseases: patent of the ductus arteriousus (PDA), bronchopulmonary dysplasia (BPD), and late-onset sepsis (LOS) mortality. METHODS: Forty-six full-term, 41 preterm, and 35 intrauterine growth restricted (IUGR) infants participated in this retrospective, observational study. RDW was measured before 3 days of life (T0) in all infants, and at first month of life (T1) in preterm/IURG patients. RESULTS: RDW% mean (standard deviation) at T0 was: 15.65 (1.18) in full-term newborns; 17.7 (2.06) in preterm; 17.45 (1.81) in IUGR. A negative correlation (r = -0.51; P < 0.001) between RDW and gestational age was found. RDW at T1 was: 17.25 (2.19) in the preterm group; 17.37 (2.56) in IUGR group. Fourteen preterm infants reported: 12 PDA, 5 LOS, 4 BPD, and 3 died; 10 IUGR infants had: 4 PDA, 6 LOS, 3 BPD, and 1 died. RDW of IUGR infants suffering from those pathologies was not statistically different compared with unaffected infants, while preterm newborns with pathologies reported higher RDW: PDA vs. PDA absent: P = 0.008 at T0; P < 0.002 at T1. BPD vs. BPD absent: P < 0.005 at T1. LOS vs. LOS absent: P < 0.005 at T0. RDW in preterm/IUGR population was associated with early mortality, T0: dead 21.2 (2.7) vs. alive 16.7 (1.7), P < 0.0001. CONCLUSION: RDW and gestational age at birth were negatively correlated. High RDW resulted to be an indication of risk for critical newborns. This parameter can be inexpensively and routinely verified and further studies are required to confirm its prognostic role in neonatal pathologies.

Correlation between cord blood, perinatal BNP values and echocardiographic parameters in healthy Italian newborns.

We evaluated the correlation between brain natriuretic peptide (BNP) in umbilical cord blood after normal pregnancy, in blood samples of twenty-nine Italian healthy newborns and paired echocardiographic parameters. Plasma BNP was evaluated in UCB (T0) and in blood on day 3 (T1), 30 (T2) of life. Echocardiographic parameters were recorded at T1 and T2. Median of BNP concentrations in cord blood was 8.6 pg/ml. Median BNP concentrations on T1 was 59.2 pg/ml, on T2 was 8.7 pg/ml. Significantly higher BNP concentrations were reported on T1 than T0 and T2 (p<0.0001), while no significant difference resulted between T0 and T2. Plasma BNP at T2 was significantly correlated with mVTI (p=0.006), E wave (p=0.004), LA (p=0.047), LVPW (p=0.004), M (p=0.025). No correlation was found with SF% and E/A. Our results confirm that in healthy and term neonates the cord blood BNP concentrations are low. On T1 BNP values are high with wide ranges because of physiological adjustment to postnatal circulation. When echocardiographic parameters are in normal ranges, BNP concentrations return to low levels on day 30. In healthy newborns left ventricular filling, LA size and M seem to influence BNP levels rather than left ventricular systolic and diastolic function.

Mature antigen-experienced T helper cells synthesize and secrete the B cell chemoattractant CXCL13 in the inflammatory environment of the rheumatoid joint.

OBJECTIVE: Synovial B cells play a critical role in rheumatoid arthritis (RA), being involved in autoantibody synthesis, T cell activation, and cytokine production. CXCL13 is a B cell chemoattractant that is instrumental in synovial B cell organization; the regulatory determinants of CXCL13 in inflammation are poorly characterized. This study was undertaken to investigate the functional involvement of synovial T cells in the ectopic expression of CXCL13 in RA. METHODS: CXCL13 production and regulation were addressed using immunohistochemistry, in situ hybridization, quantitative polymerase chain reaction, multicolor flow cytometry, and enzyme-linked immunosorbent assay, by in situ-ex vivo analysis and in vitro functional assays with rheumatoid synovial tissue and primary cells. RESULTS: CXCL13 messenger RNA and protein expression and spontaneous CXCL13 secretion were detected in RA synovial fluid T cells but were not detected (or were detected only occasionally) in peripheral blood T cells. Analysis of tissue expression confirmed cytoplasm localization of CXCL13 in T lymphocytes infiltrating B cell follicles and small perivascular aggregates. Multicolor characterizations in synovial fluid demonstrated CXCL13 expression in antigen-experienced T helper cells, frequently characterized by terminal differentiation and the lack of the follicular helper T cell markers CXCR5 and BCL6 protein. In vitro functional assays revealed the enhancing effect of T cell receptor-CD28 engagement on CXCL13 production and secretion in primary cells. CONCLUSION: Our findings define a new functional property of synovial T cells, demonstrating their active involvement in the local production of B cell chemoattractants, and support a direct contribution of the adaptive immune system and antigen-dependent signals in the mechanisms of B cell localization in RA.

Immune system development in infants born to mothers with autoimmune disease, exposed in utero to immunosuppressive agents.

Exposure to immunosuppressant agents during gestation can affect the fetal immune system. The aim of this study was to evaluate the immune function of infants whose mothers were administered immunosuppressants during pregnancy for the treatment of autoimmune disorders. Circulating lymphocyte subsets and in vitro immunoglobulin production were assessed at birth, and months 1, 6, and 12 of life in 19 infants exposed in utero to glucocorticoid alone or in combination with azathioprine, cyclosporine A, or hydroxychloroquine. The results were compared with those obtained in 15 age-matched infants from mothers with autoimmune diseases not exposed to immunosuppressants. No differences in terms of absolute lymphocyte count, percentage of B and T lymphocytes, and immunoglobulin production were observed. No immune system dysfunction was found in the two studied groups, suggesting a lack of interference between the immunosuppressive treatment and the immune systems of the offspring.

Differential recognition of heat-shock protein dnaJ-derived epitopes by effector and Treg cells leads to modulation of inflammation in juvenile idiopathic arthritis.

OBJECTIVE: To identify epitopes on Escherichia coli heat-shock protein (HSP) dnaJ or on homologous human HSP dnaJ involved in the induction/modulation of autoimmune inflammation in patients with oligoarticular juvenile idiopathic arthritis (JIA). METHODS: We used a proliferation assay and cytokine production to evaluate the immune responses of synovial fluid mononuclear cells (SFMCs) to pan-HLA-DR binder peptides derived from either homologous or nonhomologous regions on bacterial and human HSP dnaJ. Cytofluorometric analysis was performed in order to phenotype and sort Treg cells. Sorted cells were then analyzed for the expression of the forkhead box P3 (FoxP3) transcription factor, and their regulatory capacity was tested in coculture assays. RESULTS: T cell responses to E coli HSP dnaJ-derived peptides were eminently proinflammatory. Conversely, peptides derived from human HSP dnaJ induced interleukin-10 (IL-10) production from SFMCs of patients with oligoarticular JIA. A positive correlation was found between disease with a better prognosis (persistent oligoarticular JIA) and recognition of 3 human HSP dnaJ-derived peptides. The recognition of the human peptide H134-148 also induced a significantly greater amount of IL-10 in patients with persistent oligoarticular JIA than in those with extended oligoarticular JIA (P = 0.0012). Incubation of SFMCs from patients with persistent oligoarticular JIA with this human epitope increased the percentage of Treg cells and FoxP3 expression. It also induced the recovery of suppressor activity by Treg cells. CONCLUSION: This is the first description of a self-regulating immune modulator circuit active during autoimmune inflammation through recognition of HSP epitopes with different functional properties. These epitopes induce T cells with regulatory function. Such induction correlates with disease severity and prognosis.

Age-related changes in intracellular TH1/TH2 cytokine production, immunoproliferative T lymphocyte response and natural killer cell activity in newborns, children and adults.

To evaluate the development of the neonatal immune system, we measured T lymphocyte response to Con A, intracellular IL-2, IL-4, IFN-gamma and IL-10 production, and natural killer cell (NKC) activity in 12 very preterm, 12 preterm and 20 term neonates, 10 children and 10 adults. Immunoproliferation to Con A was significantly lower in cord blood than in children or adults. The percentage of CD4+ lymphocytes was significantly higher in newborns while CD8+ cells were higher at older ages, with a resulting gradual decline of the CD4+/CD8+ ratio. The percentage of IL-2-producing CD4+ and CD8+ cells was higher in all newborn groups than in children and adults, while the percentage of IL-4-producing cells was higher for CD8+ and lower for CD4+ cells in cord blood than in children and adults. Neonates had substantially lower percentages of CD4+ and CD8+ IFN-gamma-producing cells. A significant negative correlation was observed between gestational age and IFN-gamma-CD4+-, IL-2-CD8+-, and IL-10- CD4+-producing cells. In addition, a positive correlation was found between gestational age and IL-10-CD8+-producing cells. Percentages of CD4+/CD45RA+ cells were higher and CD4+/CD45RO+ percentages were lower in newborns than in children and adults. NKC activity in infants was significantly correlated with gestational age and significantly impaired compared to children and adults. On the whole, these results suggest a gradual development of immunity during gestation and show significant immaturity of cellular immune response at birth. The reduction of NKC activity, the lower proliferative response of T cells, the reduced cytotoxic response and a dysregulated cytokine production may contribute to the neonatal increased risk of infection and to the low incidence of graft-versus-host disease after cord blood transplantation.

Freshly dissociated fetal neural stem/progenitor cells do not turn into blood.

Earlier studies suggested that stem cells from one somatic tissue may generate differentiated elements of another, embryologically unrelated, tissue after an exchange in their positions through transplantation. Two reports indicated that murine and human neural stem cells of clonogenic origin after in vitro expansion in growth factor-supplemented media, may sustain hematopoiesis when injected into sublethally irradiated mice. Here we investigated if freshly dissociated fetal neural cells (fNC) share the reported hemopoietic potential of in vitro expanded neural cells. In order to minimize the risk of hemopoietic contamination, donor cells were taken from mouse E10.5 developing brains, before completion of blood vessel ingrowth into the brain; 10(6) fNC derived directly from fetal brains of transgenic mouse expressing an enhanced version of the green fluorescent protein were injected into the tail vein or directly into the bone marrow of sublethally irradiated (6 Gy) C57B16 mice. After transplantation, the presence of donor-derived cells was assessed at different survival times by FACS analysis, PCR, and clonogenic stem cell assays on peripheral blood and bone marrow. While bone marrow-derived cells were detected from 2 weeks onward after grafting, none of the mice grafted with neural embryonic cells demonstrated any sign of transdifferentiation into hemopoietic cells up to 16 months after transplantation. Our data indicate that ability to transdifferentiate from neural into the hematopoietic phenotype, if present, is acquired only after in vitro expansion of neural stem/progenitor cells and it is not present in vivo.

Human amniotic fluid cells are able to produce IL-6 and IL-8.

PROBLEM: In order to investigate the role of amniotic fluid cells (AFC) in the establishment of feto-maternal immune relationship, we evaluated their phenotype and capacity to produce cytokines. METHODS OF STUDY: CK 7-8, human leukocyte antigen (HLA)-I, HLA-DR, HLA-G, CD1d, CD34, CD45, CD14 surface antigens expression and the intracellular production of IL-4, IL-6, IL-8, IL-10, IL-12, interferon-gamma and tumor necrosis factor-1 were studied in cultured AFC and in eight samples immediately after amniocentesis using flow cytometry. IL-6 and IL-8 were detected by ELISA in all amniotic fluids and in all culture supernatants. Moreover, IL-6 and IL-8 mRNA were tested in nine samples. RESULTS: Cultured AFC express HLA-I, HLA-G and CK 7-8 and are able to produce IL-6 and IL-8, confirmed by presence of their mRNA. We quantified IL-6 and IL-8 levels in all amniotic fluids and in all supernatants. CONCLUSION: Surface antigen expression of AFC are not specific of immune cells, but AFC are able to produce cytokines and we can postulate that they may participate in mechanisms involved in normal as well as pathological pregnancy.