Blood Mononuclear Cells Of Children Research Articles

Expression of monocyte chemotactic protein-1 in peripheral blood mononuclear cells of children with Kawasaki disease and its relation to coronary artery impairment

Kawasaki disease (KD) is a febrile illness of childhood. The etiology of KD remains unknown. Multiple theories exist, including an infectious etiology and an immunological abnormality. Cardiac involvement ranges from myocarditis and pericarditis in the acute stage to the development of coronary artery aneurysms later in the course. The present study aimed to explore the effect of monocyte chemotactic protein-1 (MCP-1) in Kawasaki disease and its relationship with damage to the coronary arteries during the development of KD. Plasma MCP-1 concentrations were measured by enzyme-linked immunosorbent assay (ELISA), and MCP-1 mRNA expression in peripheral blood mononuclear cells (PBMC) was measured by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) in comparison of three groups: 56 patients with KD, 60 age-matched patients with non-infectious diseases, and 66 age-matched febrile patients with various diseases. Plasma MCP-1 concentration and MCP-1 mRNA expression in PBMC of patients with active KD [(409.55 +/- 97.42) pg/ml] and (1.97 +/- 0.77) were higher than those of control group. Plasma MCP-1 levels and MCP-1 mRNA expression of inactive KD group [(301.64 +/- 71.55) pg/ml] and (1.31 +/- 0.39) were significantly higher than those of non-infectious diseases patients. There was a marked increase in patients with inactive KD than those of non-infective patients, but there were no significant differences between inactive KD and febrile patients. Plasma MCP-1 levels and MCP-1 mRNA expression were markedly increased in KD patients with coronary artery lesions than those in patients without coronary artery lesions. Plasma MCP-1 concentration and MCP-1 mRNA expression in PBMC were significantly increased in patients with KD, and they were higher in KD with coronary artery lesions. It indicates that MCP-1 may be a useful parameter for monitoring disease activity in patients with KD.

RESEARCH OF MANNOSE-BINDING LECTIN AND INTERLEUKINS 10, 12, AND 18 IN CHILDREN WITH HENOCH-SCHÖNLEIN PURPURA

INTRODUCTION: Henoch-Schönlein Purpura (HSP) is a common vasculitis in children, and the unbalance of T-helper 1/T-helper 2 plays an important part in its pathogenesis. Mannose-binding lectin (MBL) is an important component of innate immunity and related with a lot of diseases with immunologic derangement. However, we do not know the relationship between MBL and HSP. OBJECTIVE: We aimed to explore the serum level and gene polymorphisms of MBL and the levels of interleukin 10 (IL-10), IL-12, and IL-18 in supernatant of peripheral blood mononuclear cells of children with HSP and HSP nephritis (HSPN) and of healthy children. METHODS: The concentrations of MBL and IL-10, IL-12, and IL-18 were measured by enzyme-linked immunosorbent assay: MBL gene polymorphisms were analyzed by polymerase chain reaction-restriction fragment length polymorphism and polymerase chain reaction with sequence-specific primers. RESULTS: The serum MBL levels in the 23 children with HSP were not significantly different from 27 children with HSPN (P = .95) or 18 normal children. The levels of IL-18 in the supernatant of peripheral blood mononuclear cells in the 3 groups were not significantly different from each other (P = .47, .15, and .14). The levels of IL-10 in children with HSP and HSPN were not different from each other (P = .70), but both were significantly different from those in the normal children (P = .04 and .01). The levels of IL-12 in children with HSP were different from those in the children with HSPN (P = .04). The MBL promoter genotype and the frequency of alleles were not different between the children with HSP and HSPN or between those 2 groups compared with the normal group. CONCLUSIONS: IL-12 probably plays an important role in the renal involvement in HSP. The position of MBL in the pathogenesis of HSP and HSPN remains to be confirmed.

Nuclear factor-κB activation in peripheral blood mononuclear cells in children with sepsis

To determine the activation of nuclear factor-[kappa]B in peripheral blood CD14+ monocyte/macrophages and CD3+, CD4+, and CD8+ T cells in children with sepsis. Observational study. University hospital. Twenty-six children with sepsis (nine females and 17 males, aged between 10 days and 15 yrs; median, 4.3 yrs) on admission to our hospital between August 1999 and November 2005. None. The percentages of peripheral blood CD14+ monocyte/macrophages and CD3+, CD4+, and CD8+ T cells exhibiting nuclear factor-[kappa]B activity were determined by flow cytometry. In addition, relationships among the degree to which nuclear factor-[kappa]B was activated, serum levels of cytokines (interferon-[gamma], tumor necrosis factor-[alpha], interleukin-2, interleukin-4, interleukin-6, and interleukin-10), and clinical variables were analyzed. The percentage of cells exhibiting nuclear factor-[kappa]B activity was increased among CD14+, CD3+, CD4+, and CD8+ cells in the sepsis group and was significantly higher among CD14+, CD3+, and CD4+ cells of the patients with severe sepsis (n = 9) than those of patients with nonsevere sepsis (n = 17). The percentage of cells exhibiting nuclear factor-[kappa]B activity was significantly higher among CD14+ cells than CD3+ cells in the patients with severe sepsis. In addition, this percentage was significantly higher among CD14+ cells than CD3+, CD4+, and CD8+ cells in septic patients who had positive blood cultures (n = 16). Serum interleukin-6 levels were correlated with the percentages of CD14+, CD3+, CD4+, and CD8+ cells exhibiting nuclear factor-[kappa]B activity, and serum IL-10 levels were correlated with the percentages of CD14+, CD3+, and CD4+ cells exhibiting nuclear factor-[kappa]B activity. Nuclear factor-[kappa]B in peripheral blood mononuclear cells was activated in children with sepsis and was related the severity of sepsis.

Nuclear Factor κB Activity is Increased in Peripheral Blood Mononuclear Cells of Children Affected by Atopic and Non-Atopic Eczema

Atopic and non-atopic eczema is an inflammatory cutaneous disease which is common in childhood and is associated with a dysregulation of the immune system. Many genes encoding immune receptors, cytokines, chemokines, chemokine receptors, and adhesion molecules involved in the development of the disease are under the control of transcription factors belonging to the nuclear factor (NF)-kappaB family. To investigate the role of NF-kappaB in the development of eczema, 20 children, affected by relapsing chronic eczema, were enrolled in this study. Eleven of the 20 children showed IgE immunoreactivity and had a positive prick test. The DNA binding activity of NF-kappaB in nuclear extracts of the patients' peripheral blood mononuclear cells (PBMC) was examined by electrophoretic mobility shift assay. We found that basal NF-kappaB-DNA binding activity in PBMC was significantly higher in the eczema patient group in comparison with the same parameter in the healthy age-matched control group. Moreover, we observed a significant correlation between NF-kappaB-DNA binding activity and patients clinical score (SCORAD). Based on these observations we speculate that NF-kappaB can play an important role in the immunopathogenesis of eczema and therefore could be considered as a potential therapeutic target.

No Evidence of Persisting Measles Virus in Peripheral Blood Mononuclear Cells From Children With Autism Spectrum Disorder

Despite epidemiologic evidence to the contrary, claims of an association between measles-mumps-rubella vaccination and the development of autism have persisted. Such claims are based primarily on the identification of measles virus nucleic acids in tissues and body fluids by polymerase chain reaction. We sought to determine whether measles virus nucleic acids persist in children with autism spectrum disorder compared with control children. Peripheral blood mononuclear cells were isolated from 54 children with autism spectrum disorder and 34 developmentally normal children, and up to 4 real-time polymerase chain reaction assays and 2 nested polymerase chain reaction assays were performed. These assays targeted the nucleoprotein, fusion, and hemagglutinin genes of measles virus using previously published primer pairs with detection by SYBR green I. Our own real-time assay targeted the fusion gene using novel primers and an internal fluorescent probe. Positive reactions were evaluated rigorously, and amplicons were sequenced. Finally, anti-measles antibody titers were measured by enzyme immunoassay. The real-time assays based on previously published primers gave rise to a large number of positive reactions in both autism spectrum disorder and control samples. Almost all of the positive reactions in these assays were eliminated by evaluation of melting curves and amplicon band size. The amplicons for the remaining positive reactions were cloned and sequenced. No sample from either autism spectrum disorder or control groups was found to contain nucleic acids from any measles virus gene. In the nested polymerase chain reaction and in-house assays, none of the samples yielded positive results. Furthermore, there was no difference in anti-measles antibody titers between the autism and control groups. There is no evidence of measles virus persistence in the peripheral blood mononuclear cells of children with autism spectrum disorder.

Expression of Toll-like receptors and their association with cytokine responses in peripheral blood mononuclear cells of children with acute rotavirus diarrhoea

To understand virus and host interactions and host responses to rotavirus infection in children, we analysed by real-time polymerase chain reaction (PCR) the expression of mRNA for five Toll-like receptors (TLRs) (TLR2, TLR3, TLR4, TLR7 and TLR8) and four T helper (Th)1 and Th2 cytokines [interleukin (IL)-2, IL-12, interferon (IFN)-gamma and IL-4) in peripheral blood mononuclear cells (PBMC) of children with acute rotavirus diarrhoea. We observed significantly higher expression of genes encoding TLR2, TLR3, TLR4, TLR7 and TLR8 in PBMC of 41% (31/75) patients within 3 days of illness onset than those in healthy children. After 3 days of illness onset, only TLR3 and TLR8 mRNA expressions were still significantly (P<0.05) increased in 59% (44/75) children with diarrhoea. We also observed significantly (P<0.05) elevated expression of IL-12p40 and IFN-gamma in PBMC of patients during the entire period of illness and the first 3 days of illness, respectively. We further demonstrated a weak but significant association between elevated levels of gene expression of four TLRs (TLR2, TLR3, TLR4 and TLR8) and IFN-gamma. Our results suggest that multiple TLRs may modulate the immune response in the acute phase of rotavirus infection and play a role in the activation of IFN-gamma.

Different lymphocyte markers and cytokine expression in peripheral blood mononuclear cells in children with acute atopic dermatitis*

Different lymphocyte markers and cytokine expression in peripheral blood mononuclear cells in children with acute atopic dermatitis*

Different lymphocyte markers and cytokine expression in peripheral blood mononuclear cells in children with acute atopic dermatitis

BackgroundThe pathogenesis of atopic dermatitis (AD) is still not completely understood. AD is characterized by the presence of clinical symptoms of both IgE antibody-mediated immediate hypersensitivity and specific T lymphocyte-mediated delayed hypersensitivity ObjectiveTo evaluate the immunological mechanisms involved in children with acute AD lesions Material and methodsTen children with acute AD lesions and 10 non-atopic controls were studied. Total IgE was measured by immunoassay. T cell marker expression (CD3, CD4, CD8, cutaneous lymphocyte-associated antigen [CLA]) and cytokine production (interferon [IFN]-μ, interleukin [IL]-13) were analyzed in peripheral blood mononuclear cells by flow cytometry ResultsIn children with AD the percentage of CD3+ cells (p = 0.015) increased while that of CD8+ cells (p = 0.023) decreased, with no differences in CLA expression. We found increased IL-13 production in CD3+ cells (p = 0.01) and CD3+CD4+ (p = 0.001) cells with no difference in IFN-μ. Total IgE was significantly higher in patients with AD (p = 0.01). Comparison of IL-13 production in CD4+ cells categorized by total IgE level showed that IL-13 production was significantly increased in subjects with a higher IgE level ConclusionPeripheral blood from children with AD showed an increase in IgE levels and a Th2 pattern. There was a correlation between IL-13 production and total IgE levels

Cytokine gene expression and protein production in peripheral blood mononuclear cells of children with acute dengue virus infections.

Plasma leakage in dengue hemorrhagic fever (DHF) is associated with elevated plasma levels of cytokines. To define further the contribution of immune activation to DHF and the source of cytokines, we analyzed the production of cytokines in peripheral blood mononuclear cells (PBMC) obtained from children with dengue, using RT-PCR and immunostaining. Tumor necrosis factor-alpha (TNF-alpha) and TNF-beta expression was detected in all samples by PCR and in < 50% of samples by immunostaining. Interferon-gamma (IFN-gamma) expression was detected in < 50% of samples by either method. Interleukin-2 (IL-2) and IL-4 expression was detected in a few samples by immunostaining but was not detectable by PCR. We found greater expression of TNF-alpha and IL-4 in DHF than in dengue fever or other (non-dengue) febrile illnesses. These results support the model of immunopathogenesis of DHF. However, low levels of cytokine expression in PBMC suggest that cellular activation in tissues may contribute to high serum cytokine levels in DHF.

Different drug sensitivity profiles of acute myeloid and lymphoblastic leukemia and normal peripheral blood mononuclear cells in children with and without Down syndrome

Children with Down syndrome (DS) have an increased risk for leukemia. The prognosis for DS acute myeloid leukemia (AML) is better than for non-DS AML, but the clinical outcome of DS acute lymphoblastic leukemia (ALL) is equal to that of non-DS ALL. Differences in prognosis may reflect differences in cellular drug resistance. In vitro drug resistance profiles were successfully investigated on leukemic cells from 13 patients with DS AML and 9 patients with DS ALL and were compared with reference data from 151 non-DS AML and 430 non-DS B-cell precursor (BCP) ALL. DS AML cells were significantly more sensitive to cytarabine (median, 12-fold), the anthracyclines (2-7-fold), mitoxantrone (9-fold), amsacrine (16-fold), etoposide (20-fold), 6-thioguanine (3-fold), busulfan (5-fold), vincristine (23-fold), and prednisolone (more than 1.1-fold), than non-DS AML cells. Compared with DS ALL, DS AML cells were significantly more sensitive to cytarabine only (21-fold). After short-term exposure to methotrexate, DS AML cells were 21-fold more resistant than non-DS AML cells, but no difference was observed after continuous exposure. DS ALL cells and non-DS BCP-ALL cells were equally sensitive to all drugs, including methotrexate. Normal peripheral blood mononuclear cells from DS and non-DS children without leukemia showed highly resistant drug profiles. It was concluded that the better prognosis of DS AML might, at least partially, be explained by a specific, relatively sensitive drug-resistance profile, reflecting the unique biology of this disease. (Blood. 2002;99:245-251)

Vincristine-induced apoptosis in vivo in peripheral blood mononuclear cells of children with acute lymphoblastic leukaemia (ALL)

We conducted a study to demonstrate vincristine-induced apoptosis in vivo in peripheral blood mononuclear cells of children with newly diagnosed acute lymphoblastic leukaemia (ALL). In five children, apoptosis was detected by terminal deoxynucleotide transferase-mediated dUTP-digoxigenin nick-end labelling (TUNEL) assay in blood samples collected during an up-front window study of vincristine monotherapy. In one patient, we found a striking increase of apoptotic cells from 2% (SEM 0.6) before to 40.7% (SEM 2.9) 3 h after vincristine injection. In the other patients, the maximum increase ranged from 0.8% to 4.3%. Wilcoxon matched pairs analysis of all patients confirmed that vincristine induces apoptosis in vivo in peripheral blood mononuclear cells in childhood ALL (P = 0.043).

Vincristine-induced apoptosis in vivo in peripheral blood mononuclear cells of children with acute lymphoblastic leukaemia (ALL)

Vincristine-induced apoptosis in vivo in peripheral blood mononuclear cells of children with acute lymphoblastic leukaemia (ALL)

Detection of human herpesvirus 8 DNA sequences in peripheral blood mononuclear cells of children

Detection of human herpesvirus 8 DNA sequences in peripheral blood mononuclear cells of children

Detection of human herpesvirus 8 DNA sequences in peripheral blood mononuclear cells of children

Human herpesvirus 8 (HHV-8) DNA sequences were examined in peripheral blood mononuclear cell (PBMC) DNA samples of 56 children, 15 healthy adults, and 10 renal transplant patients by the polymerase chain reaction (PCR). The PCR amplification was carried out using the published KS330(233) primer pairs to amplify HHV-8 DNA sequences. The PCR-amplified products were confirmed by Southern blot hybridization with radiolabeled 233 bp HHV-8 DNA fragment, which was cloned and sequenced from the PCR-amplified product of Kaposi's sarcoma tissue. Six PCR-amplified product of four children and two renal transplant patients were cloned and sequenced. HHV-8 DNA sequences were detected in 36 of 56 (64%) normal children, in 12 of 15 (80%) healthy adults, and in all 10 renal transplant patients by Southern blot hybridization of PCR-amplified products. Six PCR-amplified products were confirmed by sequencing. These results suggest that HHV-8 infection is prevalent in the Japanese population with infection occurring in early childhood.

Detection of human herpesvirus 8 DNA sequences in peripheral blood mononuclear cells of children

Detection of human herpesvirus 8 DNA sequences in peripheral blood mononuclear cells of children

In utero exposure to Onchocerca volvulus: relationship to subsequent infection intensity and cellular immune responsiveness.

Afro-Ecuadorian individuals from an area where Onchocerca volvulus is hyperendemic have been monitored for infection over the past 16 years. To determine whether in utero exposure to O. volvulus biases a child's subsequent immune responses, children (9 to 16 years old) for whom the mother's infection status was known were chosen for study. Children of infected mothers (n = 19) had significantly higher levels of skin microfilariae than children of uninfected mothers (n = 13; P = 0.021). While the serum levels of O. volvulus-specific immunoglobulin G (IgG), IgG subclasses, and IgE showed no significant differences between the two groups of children, peripheral blood mononuclear cells of children of infected mothers produced higher levels of Th2-type cytokines to several parasite antigens and lower levels of Th1-type cytokines to nonparasite antigens than those of children of uninfected mothers. Thus, in utero exposure to O. volvulus has a long-term effect on the child's subsequent cellular immune response that may render the child more susceptible to O. volvulus infection postnatally.

Increase of tumour necrosis factor α synthesis and gene expression in peripheral blood mononuclear cells of children with idiopathic nephrotic syndrome

The pathogenesis of idiopathic nephrotic syndrome (minimal change nephropathy and its variants) is not completely understood. In recent years it has been speculated that a cytokine released by circulating blood mononuclear cells could alter the permeability of the glomerular capillary wall. In this study we have explored the potential participation of tumour necrosis factor alpha (TNF alpha), a cytokine mainly produced by monocytes, in 25 children with idiopathic nephrotic syndrome. TNF alpha was determined by cytotoxicity bioassay in the L-929 cell line and by double antibody/RIA. Patients in activity had higher serum TNF alpha levels and TNF alpha production by monocytes than patients in remission and controls. TNF alpha mRNA expression in blood mononuclear cells was analysed by Northern blot. The TNF alpha mRNA levels in patients in activity were increased compared to controls and to patients in remission. No significant differences in IL-1 beta and IL-6 synthesis were found between patients and controls. Our results suggest that TNF alpha, but not other cytokines such as IL-1 beta and IL-6, could play a role in the pathogenesis of the proteinuria in idiopathic nephrotic syndrome.

Clinical value of measuring the interferon-induced enzyme 2'-5'-oligoadenylate synthetase in children

2'-5'-oligoadenylate synthetase, an interferon-induced enzyme and a sensitive indicator of the presence of interferon, was measured in peripheral blood mononuclear cells in children with inflammatory disorders of known and unknown origin in order to assess the value of such a test in patient management. Differences in median 2'-5'-oligoadenylate synthetase values in groups of children with viral or bacterial diseases or healthy children were observed. Considerable overlap of the 2'-5'-oligoadenylate synthetase range in the three groups was observed. All children with acute viral infections had increased levels early in their disease and a low value clearly argued against an acute viral infection. However, as more than one-third of children with bacterial diseases displayed elevated 2'-5'-oligoadenylate synthetase concentrations, distinction between a viral and bacterial cause of disease by measuring this enzyme was difficult. Estimation of 2'-5'-oligoadenylate synthetase concentrations in patients with inflammatory diseases of unknown origin, including juvenile rheumatoid arthritis, systemic lupus erythematosus, idiopathic uveitis and glomerulonephritis did not contribute to establishing a diagnosis or measuring disease activity.

Low constitutive activity of aryl hydrocarbon hydroxylase in phytohaemoagglutinin-stimulated peripheral blood mononuclear cells of children after treatment for acute lymphoblastic leukemia

In six children in the first remission after treatment of acute lymphoblastic leukemia (ALL) the constitutive activity of aryl hydrocarbon (benzo[ a] pyrene) hydroxylase (AHH) (0.14 ± 0.04 U, i.e. pmol of 3-OH-benzo[ a] pyrene × 10 −6 cells × min −1) in phytohaemoagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC) was significantly lower ( P 0.05) than the constitutive activities in the parents of the children (0.25 ± 0.09 U) and in the control adult group (0.26 ± 0.09 U). In methylcholanthrene (MC)-induced activities of AHH no significant differences were observed between the above groups.

893 MORPHOLOGY, DEVELOPMENT AND DISTRIBUTION OF HUMAN NK CELLS IDENTIFIED BY A MONOCLONAL ANTIBODY (HNK-1)

A monoclonal IgM antibody (HNK-1) has been shown to react with a subset of human lymphocytes that can function as natural killer (NK) and antibody dependent killer (K) cells. HNK-1 antigen is present on 60% of Tγ cells (Fcγ receptor+, E-rosette+) and 56% of “null” cells (FcγR+ER−sIg-) but not on sIg-bearing B cells and monocytes. In the present studies we have used immunofluorescent and cell sorter techniques to examine the development, distribution, morphology and function of cells expressing HNK-1. Fewer than 1% of cells in thymus and newborn blood samples were HNK-1+. The number of circulating HNK-1+ cells increased as a function of age: <5% of blood mononuclear cells in children under 15, 5-35% in adults 15 to 40 and 25-45% in adults >40 years of age. HNK-1+ cells comprised a mean of 12% of spleen cells but <1% of lymph node cells. The level of NK and K cell functions was closely related with the numbers of HNK-1+ cells in all tissues examined. Morphologically, HNK-1+ cells were defined as a homogeneous population of lymphocytes with abundant cytoplasm containing azurophilic granules. A significant proportion of HNK-1+ cells coexpressed antigens previously defined by monoclonal antibodies on T cells and monocytes. Our studies define a morphologically distinct subpopulation of lymphocytes of unknown origin with NK and K activities, the HNK-1+ cells were shown to expand as a function of age and exhibit selective migration. NIH CA 16673 and CA 27197.