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We describe the case of a boy with acute myeloid leukemia with translocation t(6;11)(p22.2;q23) and insertion ins(11;9)(q23;p21.3p21.3). Translocation t(6;11)(p22.2;q23) involving the short arm of chromosome 6 has not been previously described. The LDI-PCR showed the presence of KMT2A-MLLT3 fusion and identified the BTN3A1 (butyrophilin subfamily 3 member A1) gene on 6p22.2 as the other KMT2A translocation partner. The BTN3A1 gene has never been described in the context of acute leukemia. Although this fusion is out of frame, as the antisense strand of BTN3A1 is fused to the sense strand of KMT2A, the loss of heterozygosity of the BTN3A1 gene might contribute to the malignancy of leukemic cells.

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Hemophagocytic lymphohistiocytosis (HLH) is a severe systemic syndrome associated with hyperactivation of macrophages and impaired regulation of the immune system. Two forms of HLH are currently recognized: genetically determined or familial (FHLH), and secondarily developed in the course of primary diseases, like autoimmune disorders, rheumatoid disorders, cancers, or infections. In the Polish population, FHLH is rather rare. The aim of the present study was to assess the immune function in a group of children with clinical symptoms suggesting FHLH. Forty five children with suspected HLH of the median age of 4 years and 15 healthy children, taken as a control group, were enrolled into the study. All presented results were obtained with the use of flow cytometry. In the HLH group, there were only three cases identified with the UNC13D gene mutation responsible for the FHLH3 phenotype. Another four children, without known mutation, were classified as FHLH because of frequent recurrence of the disease. In all cases of FHLH, cell cytotoxicity was impaired compared with healthy children (p = 0.003). Perforin expression in FHLH was normal or higher than that observed in controls (p = 0.09). In case of patients with mutation in the Munc13 protein, degranulation was lower than that in healthy children (<5 %). The findings of this study demonstrate that children with known mutations responsible for the FHLH development are immunocompromised. However, it requires further elucidation whether the presence of currently unknown mutations could lead to a similar phenotype.

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Ob-R receptor is encoded by db gene and belongs to class I cytokine receptors family. Its expression was observed in hematopoietic CD34+ stem cells, erythropoietic, myeloid and lymphoblastic lineages cell lines and in human leukemic blast cells in lymphomas, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and chronic myeloid leukemia (CML). The studies on human bone marrow cells show that JAK/STAT pathway plays a substantial role in signal transduction in young bone marrow cells. The aim of the study was to examine the relationship between leptin receptor expression and the proliferation of neoplastic hematopoietic cells in bone marrow. The study was performed in a total of 57 children of both sexes aged 3 months to 16 years. A group of 46 patients with acute leukemia involved 25 children with ALLB, 11 children with ALLT and 10 children with ANNL. The control group consisted of 11 non-obese children with non-malignant hematological disturbances. The tests were performed on bone marrow samples. The assessments of membrane expression of Ob-R and the antigens determining the phenotype of bone marrow cells were performed using a flow cytometry method. In acute lymphoblastic leukemia, a significant decrease of Ob-R expression on leukemic blasts was observed in comparison with respective populations of normal bone marrow cells. Also in progenitor cells populations a significant decrease of CD34+Ob-R+w ALLT and ALLB was observed in comparison with the cells from normal bone marrow. No statistically significant differences in the percentage of Ob-R+ cells in ANNL bone marrow and in control bone marrow were observed.

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The population of natural killer (NK) cells is very heterogeneous and plays a role in the immune system. Several NK cells subpopulations are recognized, differing in phenotype, cytokine release and cytotoxic ability. Different expression of biologically relevant molecules on the surface of NK cells may indicate their multiple functions. The activity of NK cells has mainly to do with their cytotoxic nature. A complete analysis of NK cells function requires application of many tests because a defect may be present at different stages of the cytotoxic process, from signal transduction through lysosome degranulation to target cells destruction. Flow cytometry is actually one of the best methods for the identification of NK cells and tracking their defects.

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Leptin or obesity receptor (Ob-R) is a member of class I cytokine receptor family. Ob-R, expressed in six isoforms, is the product of alternative RNA splicing of db gene. According to its structural differences, the receptor's isoforms are divided into three classes: long, short, and secretory isoforms. A long, fully active isoform of Ob-Rb is expressed mainly in the hypothalamus, where it takes part in energy homeostasis and in the regulation of secretory organs' activity. Ob-Rb is also present on all types of immune cells, involved in innate and adaptive immunity. Short leptin isoforms (Ob-Ra, Ob-Rc, Ob-Rd, and Ob-Re) that contain box 1 motif are able to bind JAK kinases (Janus kinases) as well as to activate some other signal transduction cascades. A soluble isoform (Ob-Re) can regulate serum leptin concentration and serve as a carrier protein delivering the hormone to its membrane receptors and is able to transduce the signal into the cell. JAK/STAT pathway plays the major role in leptin signal transduction through membrane receptors. Among all Ob-R isoforms, only full-length isoform (Ob-Rb) is able to fully transduce activation signal into the cell.

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INTRODUCTION: TNF--α is one of the most important factors in the development and course of inflammation. It is suggested that polymorphism located in the 5'regulatory region of the TNF-α gene at position 308 (guanine [G]→adenine[A]) may increase the expression of this cytokine in fat tissue and influence the fat mass and insulin resistance. OBJECTIVE: To investigate whether the G-308A polymorphism of the TNF-α gene may influence obesity, insulin resistance, fasting plasma lipids, serum leptin levels, and the incidence of metabolic syndrome. MATERIAL AND METHODS: The obese group included 124 children with simple obesity (72 girls and 52 boys) aged 10-18 (mean age 15 years) with SDS of BMI ≥2.0. A control group consisted of 56 healthy non-obese children (36 girls and 20 boys) aged 11-18 (mean age 14 years) with SDS of BMI <1.0. Polymorphism identification was performed in total genomic DNA, using PCR-RFLP method. RESULTS: Carriers of A (AG+AA) allele among the obese children were significantly more frequent than in the control group (OR = 2.29, 95% CI 1.2-4.4, χ⊃2 = 6.24, P<0.05). Carriers of A alleles showed a higher concentrations of fasting glucose (81.3 ±10.5 vs. 77.4 ±10.3 mg/dl; P<0.05), but lower values of fasting insulin (15.1 ±7.3 vs. 19.0 ±9.5 µIU/ml; P<0.05), lower values of HOMA index (3.0 ±1.5 vs. 3.7 ±2.0; P <0.05). In the group of boys, carriers of A alleles showed a tendency for lower concentrations of HDL (43.8 ±12.6 vs. 48.3 ±11.8 mg/dl; P<0.05). Blood pressure and leptin level did not differ between the obese children with gene polymorphism and those of wild homozygous. The incidence of the full metabolic syndrome (MetS) in the children, according to the IDF definition, was 33%. The presence of the MetS in children with wild homozygous GG and carriers of A allele of TNF-α polymorphism gene did not show statistical differences (OR = 1.38; 95% CI 0.6-3.1, χ⊃2 = 0.58). CONCLUSIONS: 1/ Polymorphism G-308A of the TNF-α gene is more common in children with obesity; and 2/ Polymorphism G-308A of the TNF-α gene does not seem to be associated with the grade of obesity, insulin resistance, lipid profile, leptin levels, and the incidence of metabolic syndrome in obese children.

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The prevalence of obesity in children and adolescents has been increasing worldwide. As in adults, childhood obesity is closely related to hypertension, dyslipidemia, type 2 diabetes, and insulin resistance (IR) syndrome. Moreover, obese children have been found to be at increased risk of becoming obese adults. Obese children and adolescents tend to develop serious medical and psychosocial complications and also are at greater risk morbidity and mortality in adulthood. The molecular basis of the pathogenesis of obesity-linked disorders has not been fully elucidated. Adipose tissue serves not only as an energy storage organ, but also as an endocrine organ. It releases many factors with autocrine, paracrine and endocrine functions. Adipokines such as leptin, resistin, tumor necrosis factor-α, interleukin-6, adipsin, visfatin, and adiponectin are biologically active molecules produced by adipose tissue. They play a role in energy homeostasis, and in glucose and lipid metabolism. Adiponectin level, unlike that of other adipocytokines, is decreased in obesity and increased after weight reduction. Adiponectin has been associated with both central obesity and increased visceral adipose tissue and it has anti-inflammatory, anti-atherogenic, and potent insulin-sensitizing (anti-diabetic) effects.

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BACKGROUND: Obesity development is a complex process which can be influenced by genetic predisposition modified by environmental factors. Nowadays, the problem of overweight and obesity, including related complications, occurs in increasingly younger children. Thus, there is a need for new genetic markers of increased risk of excessive body mass. OBJECTIVE: The aim of the present study was to examine the relation between polymorphisms located in promoter regions of IL-1beta, IL-6, and TNF-alpha genes and obesity development in children. Fifty obese and 55 normal weighing children were enrolled into the study. Genetic examination was performed using PCR-RFLP technique. RESULTS: We found a relation between G174C polymorphism in IL-6 gene and G308A in TNF-alpha gene with the occurrence of obesity. Allele A in G308A was more frequent in the obese group than in the control one (P=0.04). The presence of allele C in promoter region of IL-6 gene was more frequent in obese children and connected with a statistically significant increase in the sum of 10 skin fold thickness measurements (P=0.03). CONCLUSIONS: The polymorphism C3954T in IL-1beta gene showed no such relation. The examined polymorphisms of proinflammatory cytokines play a role in the regulation of body mass through their influence on metabolism and energetic homeostasis.

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The goal of the study was to evaluate the process of Ca(2+)-mediated transduction of signals into neutrophils from patients with type I diabetes and its modification by insulin. The study was performed with the use of isolated peripheral blood neutrophils from 20 diabetic patients and 30 healthy volunteers. Isolated granulocytes were stimulated separately by fMLP or insulin, or by both substances added to the medium in combinations: fMLP + insulin (after 20 min) or insulin + fMLP (after 20 min). fMLP evoked fast intracellular increase of free Ca(2+) concentration in neutrophils compared with the resting state (P<0.001). Similarly, the peak of fluorescence, as measured by Fluo 3 to Fura Red ratio, was significantly higher in neutrophils stimulated by insulin. Insulin did not cause any changes in intracellular Ca(2+) level when it was added to the previously fMLP-stimulated cells. Prestimulation with insulin significantly decreased fMLP-induced intracellular free Ca(2+) concentration, expressed as Fluo3/Fura Red ratio compared with fMLP alone (1.77 +/- 0.6 vs. 2.63 +/- 0.8, P<0.001). No relation between initial intracellular Ca(2+) in the resting state and the response to insulin was found. Nor was the response to fMLP alone related to intracellular Ca(2+) before stimulation. A strong correlation was observed between initial intracellular Ca(2+) after incubation with insulin and the response to fMLP (r=0.90, P<0.0001). In diabetic granulocytes, the intracellular Ca(2+) was significantly lower than in those from healthy donors in unstimulated cells (P<0.001), after fMLP stimulation (P<0.0001), in medium enriched by insulin (P<0.05), and after fMLP stimulation in insulin rich medium (P<0.001). Only in fMLP prestimulated samples, the emission of light did not differ after stimulation with insulin in granulocytes from both diabetic and healthy subjects. In conclusion, patients with type I diabetes have decreased levels of cytosolic Ca(2+) after insulin and fMLP stimulation in polymorphonuclear granulocytes. This abnormality is probably primarily responsible for the impaired neutrophilic function seen in these patients.

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OBJECTIVE: The aim of the study was to investigate whether the G-174C polymorphism of the IL-6 gene is related to obesity and the incidence of the metabolic syndrome (MetS) according to IDF definition in children. MATERIALS AND METHODS: The examined group included 124 obese children with BMI > or = 2 SDS, and the control group consisted of 56 non-obese children with BMI <1.0 SDS. Polymorphism identification was performed in total genomic DNA using PCR-RFLP method. RESULTS: In the obese children, carriers of C allele in homozygotic and heterozygotic genotypes were more frequent than in the control group. The carriers of C alleles presented with lower thickness of subcutaneous tissue and higher concentrations of HDL-C than the wild type. The incidence of MetS was 33% of the group of obese children. Analysis of the presence of MetS factors showed that there is more frequent MetS in the group with the wild homozygous genotype type. CONCLUSION: Polymorphism 174G>C in the IL-6 gene does not seem to be associated with obesity and with the incidence of MetS in children.

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The goal of the study was to evaluate the process of neutrophil activation via Ca(2+)-mediated transduction signal and its modification by insulin. The study was performed with the use of isolated peripheral blood neutrophils obtained from 20 healthy volunteers. Isolated granulocytes were stimulated by fMLP or insulin alone, or by both substances added to the medium in combinations: fMLP + insulin (after 20 min) or insulin + fMLP (after 20 min). To explore the mechanism of intracellular Ca(2+) changes, receptor signal transduction pathway was blocked by tyrosine kinase inhibitors: tyrphostin 25 and genistein. fMLP evoked fast intracellular increase of free Ca(2+) concentration in neutrophils, compared with the resting state (P< 0.001). Insulin did not cause any changes in intracellular Ca(2+) when was added to the previously fMLP stimulated cells. Prestimulation with insulin significantly decreased fMLP-induced intracellular free Ca(2+) concentration compared with fMLP alone (P<0.01). A strong correlation was observed between initial intracellular Ca(2+) concentration after incubation with insulin and the response to fMLP (P<0.0001). The tyrphostin 25 did not influence the Ca(2+) concentration in control granulocytes, but inhibited the fMLP-induced intracellular Ca(2+) increase when added before fMLP (P<0.05). In a Ca(2+)-free medium, a strong relationship between intracellular Ca(2+) and the response to fMLP after incubation with tyrphostin was found (P<0.001) The genistein did not influence the intracellular Ca(2+) in non-stimulated cells. However, it inhibited the fMLP-induced Ca(2+) increase when added before fMLP (P<0.05). The genistein added to the suspension of cells after fMLP stimulation did not influence intracellular Ca(2+) level. A positive correlation was found between the initial intracellular Ca(2+) and the response to fMLP of genistein preincubated cells. This effect was seen in both Ca(2+)-rich, and Ca(2+)-free medium We conclude that insulin is a potent immunomodulator and its signaling pathways are mediated by Ca(2+) concentration changes. The process of intracellular Ca(2+) changes following insulin signaling is, at least partly, tyrosine kinase-related. Derangements in the concentration of intracellular Ca(2+) may represent a link between the mechanisms of insulin resistance in diabetes.

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Obesity is one of the most commonly identified factors for the obstructive sleep apnea syndrome (OSAS). Adipose tissue is the source of many cytokines, among them there are IL-6, IL-1, and TNF-alpha. The level of inflammatory cytokines increases in people with OSAS and obesity. The aim of this study was to evaluate the distribution of genotypes in inflammatory cytokine genes in people with obesity-related OSAS. The examined group consisted of 102 person with obesity related-OSAS and 77 normal weight person without OSAS. Genotyping of DNA sequence variation was carried out by restriction enzyme (IL-1: Taq I, IL-6: Lwe I, TNF-alpha: Nco I) analysis of PCR amplified DNA. The study revealed a significant correlation between polymorphism located in the promoter region of inflammatory cytokine genes and obesity-related OSAS.

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Leptin is an adipocyte-derived hormone regulating energy homeostasis and body weight. Leptin concentration is increased in patients with the obstructive sleep apnea syndrome (OSAS). Leptin receptor (LEPR) is a single transmembrane protein belonging to the superfamily of cytokine receptors related by a structure to the hemopoietin receptor family. The aim of the present study was to evaluate the frequency of distribution of leptin receptor gene polymorphism GLN223ARG in OSAS patients compared with healthy controls. The examined group included 179 subjects: 102 OSAS patients (74 men and 28 women) and 77 non-apneic controls (39 men and 38 women). Genomic DNA was isolated with the use of a column method and genotyping of DNA sequence variation was carried out by restriction enzyme analysis of PCR-amplified DNA. The results revealed a significant correlation between the polymorphism of LEPR and OSAS. Carriers of Arg allele in homozygotic genotype Arg/Arg and heterozygotic genotype Gln/Arg were more often obese and developed OSAS than the group of carriers of homozygotic Gln/Gln genotype. This tendency was observed in the whole examined population and in the group of obese women. We also found the highest levels of total cholesterol, LDL, HDL, and triglycerides in the group of homozygotic Arg/Arg genotype carriers, lower in heterozygotic Gln/Arg genotype carriers, and the lowest in the group of persons carring homozygotic Gln/Gln genotype. The presence of Arg allel seems linked to a higher risk of obesity and higher lipid levels in OSAS patients. OSAS may have a strong genetic basis due to the effects from a variety of genes including those for leptin receptor.

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Increased susceptibility to infections can be a consequence of altered function of immune cells including neutrophils. The goal of the study was to evaluate the process of neutrophil activation via Ca(2+)-mediated signal. The study was performed on isolated peripheral blood neutrophils obtained from 41 children with recurrent infections (21 girls, 20 boys) 3-10 years old with more than five episodes of respiratory tract infection (RI) per year and from a control group of 30 healthy children age and sex matched, free from allergic, immune and hematological disorders. Neutrophils were activated by bacterial peptide fMLP, opsonized zymosan (OZ), and phorbol myristat acetate (PMA). The kinetics of the intracellular Ca(2+) concentration i[Ca(2+) ] was assessed by flow cytometry (Coulter Epics XL) with the use of Fluo3 and Fura Red fluorescent dyes. Data were collected in histograms displaying Fluo3 fluorescence vs. time and Fura Red fluorescence vs. time and the mean channels of fluorescence intensity were used for calculations. fMLP and OZ-induced Ca(2+) mobilization lasted shorter in the RI group (P<0.05). The peak influx of free Ca(2+) and i[Ca(2+) ] in the resting state after stimulation with fMLP were lower in the patients (P<0.05). In the RI group stimulation with OZ was delayed compared with that in the control group (P<0.01). In response to PMA, i[Ca(2+) ] decreased faster. The kinetic slopes of i[Ca(2+) ] in both groups examined differed statistically at all points measured. A decrease in i[Ca(2+) ] after PMA stimulation was greater (P<0.01) and lasted longer in the RI group. We conclude that increased sensitivity to infections in RI children may be related to the disturbance in neutrophil activation that is mediated by changes in i[Ca(2+) ] with the subsequent production of free oxygen radicals. Such a disturbance may be inheritable or secondary to infection and antibiotic therapy.

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Leptin is an adipocyte-derived hormone regulating energy homeostasis and body weight. Leptin also plays a role in hematopoiesis, cell cycle regulation, and in oncogenesis. The leptin receptor is a single transmembrane protein belonging to the superfamily of cytokine receptors, structurally related to the hemopoietin receptor family. The aim of the study was to evaluate bone marrow and peripheral blood leptin level and frequency of distribution of leptin receptor gene polymorphism Gln223Arg in children with acute leukemia. The examined group included 92 children with acute leukemia (83 ALL and 9 AML) and 39 non-leukemic control children. Leptin level was measured by ELISA method at the day of leukemia diagnosis. Genomic DNA was isolated with the use of a column method and the genotyping of DNA sequence variation was carried out by the restriction enzyme analysis of PCR - amplified DNA. The samples were then electrophoresed on 2.5% agarose gel. Leptin level in leukemic children was lower than in healthy children. Bone marrow leptin level was significantly lower than that in the blood in leukemic children with ALL-T and AML. An analysis of frequency distribution of the Gln233Arg polymorphism in the leptin receptor gene in leukemic children showed lack of differences between the patients and controls. There was no difference in the genotype frequencies between the leukemic AML and ALL groups either. The results indicate a possible relation between the leptin level and leukemia development in children. The effectory effect of the hormone seems not related to Gln223Arg polymorphism of its receptor.

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