A Case of Acute Myeloid Leukemia with Novel Translocation t(6;11)(p22.2;q23) and Concurrent Insertion ins(11;9)(q23;p21.3p21.3).

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.

Impairment of Immune Function in Children with Familial Hemophagocytic Lymphohistiocytosis.

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.

Pro- and antiangiogenic markers in patients with pulmonary complications of systemic scleroderma.

Systemic sclerosis (SSc) is an autoimmune disorder characterized by skin and internal organs fibrosis and concomitant vascular abnormalities. Although SSc is considered mainly fibrosing disease, underlying vascular pathology plays a fundamental role in its pathogenesis. We have focused on positive and negative serum markers of angiogenesis and fibrosis (pigment epithelium-derived factor [PEDF], vascular endothelial growth factor [VEGF], and soluble VEGF receptor [sVEGFR]), in progressive SSc patients at baseline and after follow-up in relation to cardiopulmonary complications (systemic hypertension [HT], pulmonary arterial hypertension [PAH] and pulmonary fibrosis [PF]). VEGF and PEDF but not sVEGFR were reciprocally regulated in SSc progression. Moreover, VEGF/PEDF ratio significantly increased during follow up suggesting that it might be used as a biomarker of disease progression. No correlation between the studied markers and cardiopulmonary complications was observed. In conclusion, VEGF and PEDF level, and the VEGF/PEDF ratio are significantly changed in the course of SSc progression and these markers can be used to assess SSc activity.

Immunological Characteristics of Children with Hashimoto's Autoimmune Thyroiditis.

The main cause of autoimmune thyroiditis of Hashimoto's type (HT) is a pathological immune response to thyroid antigens. The aim of the study was to present clinical characteristics and immune profile of children with HT. Ninety five children were examined: 45 with HT (age: 8-18 years) and 50 healthy age-matched controls. The peripheral blood mononuclear cells' (PBMC) phenotype was evaluated using a Beckman Coulter flow cytometer with the following monoclonal antibodies: CD4-FITC, CD28-PC5, CD152-PE and CD8-FITC, CD28-PC5, CD152-PE. The thyroid stimulating hormone, thyroid hormones, and antibodies against thyroid peroxidase (TPO) and thyroglobulin (TG) were evaluated by a microparticle enzyme immunoassay. We found that goiter was present in 53% of children, the thyroid had an increased density in palpation in 98%, and hypothyroidism was diagnosed in 11% of HT patients. The number of CD152+ was lower in HT than in healthy children (p<0.05). CD4+ and CD8+ PBMC subsets did not differ between the groups at baseline. After stimulation with phytohemagglutinine (PHA), CD4+ cells decreased in healthy controls and remained constant in HT children. Anti-TPO and anti-TG antibodies were higher in children with a lower percentage of CD152+. No other markers correlated with the immunological profile of PBMC. The percentages of CD4+ and CD152+ negatively correlated with the anti-TG concentration. We conclude that children with HT have a different PBMC profile than healthy children and show a different pattern of response to stimulation.

Antiendothelial cells antibodies in patients with systemic sclerosis in relation to pulmonary hypertension and lung fibrosis.

Although scleroderma is generally considered a fibrosing disease, it is now recognized that the underlying vascular pathology is playing a fundamental role in its pathogenesis. The present study was aimed at testing the prevalence of anti-endothelial cell antibodies (AECA) in systemic scleroderma (SSc) patients with and without pulmonary hypertension (PH) and in relation to the presence of pulmonary fibrosis. Fifty four SSc patients (50 females and 4 male, mean age 55.7 ± 16.3 years) were prospectively screened. All patients underwent transthoracic echocardiography with the estimation of pulmonary artery pressure (PAP) and tricuspid regurgitant peak gradient (TRPG). All patients suspected to have pulmonary hypertension were referred for right heart catheterization. Restrictive lung disease was confirmed by HRCT. A healthy control group included (n = 27; 7 men and 20 women, mean age 49.8 ± 12.1 years). The study of AECA was performed using the indirect immunofluorescence method on commercially available human umbilical vein endothelial cells. The HRCT scans in patients with suspected interstitial lung disease revealed signs of lung fibrosis in 15 (out of the 36 examined patients). TRPG at rest of 31 mmHg was demonstrated in 14 (21%) patients. During cardiac catheterization, arterial PH was found in two patients. Resting venous PH was found in one patient and an excessive post capillary PAP elevation at rest was demonstrated in 11 patients. At the baseline, 14/54 patients (26%) were positive for AECA. In the control group, the frequency of the antibodies was 3/27 (11%). No statistical correlation between antibody titter and the presentation of the disease existed. AECA were highly prevalent in a subgroup of patients suffering from interstitial pulmonary fibrosis. Out of the 15 patients suffering from lung fibrosis, 7 were AECA positive. The presence of AECA correlated very well with antinuclear antibodies (ANA), but was not related to the profile of ANA. Our findings support evidence that endothelial cell damage is involved in SSc, as there was increased prevalence of circulating AECA of the IgG isotype in SSc patients. AECA may also be related to the complications of SSc, like pulmonary fibrosis.

Leptin receptor in childhood acute leukemias.

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.

Flow cytometry in detection of abnormalities of natural killer cell.

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.

Decreased CD4+CD152+ T cell subset and its correlation with the level of antithyroid antibodies in children with chronic autoimmune thyroiditis.

OBJECTIVE: Cytotoxic T lymphocyte antigen-4 (CTLA-4) is one of the basic antigens involved in immune responses regulation associated with autoimmune thyroid diseases. The aim of the study was to evaluate whether the surface expression of CTLA-4(CD152) on T cells is correlated with laboratory autoimmune markers in children with Hashimoto's disease. MATERIAL AND METHODS: Blood samples were obtained from 45 children with Hashimoto's thyroiditis of the mean age 14.8 ±2.35 years, and from 55 healthy age-matched children, free of allergic, immune and hematological disorders, and with a normal thyroid function. The anti-thyroid antibodies were measured with Microparticle Enzyme Immunoassay (AxSYM Anti-Tg, AxSYM Anti-TPO). The T cell phenotype was evaluated flow cytometery, with the use of monoclonal antibodies combination: CD4- FITC/ CD28 -PC5/ CD152 -PE and CD8 -FITC/ CD28 -PC5/ CD152 -PE. - RESULTS: The percentage of T cells with CD152 expression was significantly decreased in children with Hashimoto's thyroiditis compared with healthy controls (P<0.001). A significant negative correlation was found between the level of anti-thyroglobulin antibodies and the percentage of CD4+CD152+ T cells (r = -0.34; P<0.05). Anti-thyroperoxidase antibodies did not correlate with CD152 expression. CONCLUSIONS: In children with Hashimoto's thyroiditis, the number of CD4+CD152+ T cells is decreased and negatively correlates with the level of anti-thyroglobulin antibodies.

Proinflammatory cytokines Il-6 and TNF-α and the development of inflammation in obese subjects.

BACKGROUND: The development of obesity and related disorders, e.g., type II diabetes (T2D), hypertension, and metabolic disturbances is strongly related to increased levels in proinflammatory cytokines (IL-1, IL-6, and TNF-α). Both IL-6 and TNF-α are secreted by adipocytes and their concentration correlates with the percentage and distribution of fat tissue in the body. Both cytokines are the main factors responsible for the induction of acute phase proteins production (e.g., CRP) and to inflammatory state. OBJECTIVE: To compare of TNF-α and IL-6 concentrations in serum from obese subjects with those in subjects with normal BMI and to analyze the relation between TNF-α, IL-6, BMI and the inflammatory state as measured by the level of CRP. MATERIAL AND METHODS: The study included 80 obese subject (54 males and 26 females) BMI >25 kg/m⊃2. A control group consisted of 53 healthy subjects (24 males and 29 females) with BMI <25 kg/m⊃2. To determine the blood plasma concentration of IL-6 and TNF, commercial ELISA assay kits were used. RESULTS: The concentration of IL-6 was lower in the control compared with the obese patients, but a significance difference concerned only female subjects (P = 0.001). TNF-α concentration was significantly higher in all obese subjects (P<0.001). A higher level of this cytokine was also found in patients with obesity suffering from T2DM. A positive correlation was present between IL-6 and TNF-α concentrations. Only did the IL-6 level correlate with the concentration of CRP in serum. CONCLUSIONS: The study confirmed that increased inflammatory cytokines lead to the persistence of inflammation in obese subjects. However, some other factors, such as gender, may contribute to the development of obesity-related inflammatory states.

Influence of interleukin-6 and G174C polymorphism in IL-6 gene on obesity and energy balance.

Obesity is a multifactor disease with a very complicated etiology. Genetic factors play an important role in the development of primary obesity. They may be responsible for up to 40% of causes leading to obesity. There are a great number of genes affecting food intake and energy expenditure. Serious consequences accompanying obesity, e.g., type 2 diabetes and lipid abnormalities may be caused by increased level of proinflammatory cytokines, such as IL-1, IL-6, and TNF. It is possible that polymorphisms located in cytokine genes affect the level of protein expression. It is known that IL-6 plays a role in lipid metabolism and energy expenditure. The polymorphism found in point 174 (G174C) of a promoter region of IL-6 gene affects the level of interleukin-6 expression and, consequently, may lead to obesity and correlated conditions.

Expression of CD152 (CTLA-4) in children with autoimmune thyroiditis and +49 A/G polymorphism of exon 1 of the CTLA-4 gene.

CTLA-4 gene is one of the strongest locus of genetic susceptibility to autoimmune thyroid diseases. The aim of the present study was to investigate surface expression of CTLA-4 on peripheral T cells in homozygotes AA and GG at position +49 of CTLA-4 gene in children with Hashimoto's thyroiditis and in healthy controls. Blood samples were obtained from 100 children: 45 with Hashimoto's thyroiditis and 55 controls. CTLA-4 exon 1 polymorphism was defined by SSCP and RFLP with BbvI enzyme. T cells were analyzed with three color flow cytometry by Coulter EPICS XL. We found that CTLA-4 expression was significantly lower in the thyroiditis patients than in controls, but CTLA-4 expression in homozygotes GG and AA was comparable. We therefore conclude that decreased expression of CTLA-4 on T cells in children with Hashimoto's thyroiditis is not dependent on polymorphic changes at position +49 of CTLA-4 gene.

Influence of proinflammatory cytokine gene polymorphism on childhood obesity.

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.

Increased serum fractalkine in systemic sclerosis. Down-regulation by prostaglandin E1.

OBJECTIVES: To evaluate serum levels of fractalkine (FKN), a mediator of leukocyte transmigration, C-reactive protein (CRP) and expression of integrins CD11a and CD49d on peripheral blood lymphocytes in systemic sclerosis (SSc) and to investigate whether they are modulated by intravenous prostaglandin E1 (PGE1). METHODS: Serum levels of fractalkine and C-reactive protein and expression of CD11a and CD49d on peripheral blood lymphocytes were assessed in 50 SSc patients and in 18 healthy controls. In 25 SSc patients studied parameters were evaluated also after 3 consecutive daily PGE1 infusions (20 microg-40 microg-60 microg) and after 4 weeks. RESULTS: In SSc fractalkine basal level was significantly higher than in controls (9.04+/-1.79 ng/ml vs. 1.17+/-0.1 ng/ml; p<0.0001) and decreased significantly after PGE1 (5.16+/-1.27 ng/ml, p<0.05). After four weeks fractalkine level was still significantly lower than baseline 7.70+/-2.19 ng/ml (p<0.05). Basal percentage of CD11a (+) nor CD49d (+) lymphocytes in SSc (82.38+/-1.60%, 70.74+/-1.68%, respectively) did not differ from controls (85.73+/-2.04%, 75.62+/-2.48%; respectively, p>0.05). PGE1 treatment resulted in decrease of both CD11a (+) (67.72+/-3.34%, p<0.0001) and CD49d (+) lymphocytes (65.32+/-1.62%, p<0.0001). After 4 weeks the percentage of CD11a (+) and CD49d (+) lymphocytes remained significantly lower than at baseline (77.80+/-2.47% and 65.32+/-1.62%, respectively, both p<0.001). In SSc CRP basal level was significantly higher than in controls (4.70+/-2.01 mg/dl vs. 1.40+/-1.79 mg/dl, p<0.005) and reduced significantly after PGE1 (3.39+/-2.06 mg/dl, p<0.05). After 4 weeks, CRP level (4.38+/-2.19 ng/ml) was significantly lower than baseline (p<0.05). CONCLUSION: Fractalkine may play an important role in the pathogenesis of vascular dysfunction in systemic sclerosis. Prostaglandin E1 down-regulates serum fractalkine level, as well as CD11a and CD49d expression on peripheral blood lymphocytes, which suggests additional mechanisms in which this vasodilatatory agent exerts its efficacy in systemic sclerosis.

Altered expression of T lymphocyte surface markers in children with chronic autoimmune thyroiditis.

CTLA-4 and CD28+ are regulators of T cell activation. The CTLA-4 gene is associated with variety of autoimmune diseases. The aim of the study was to evaluate changes in basic T cell subpopulations, and the expression of CD152+ and CD28+ before and after T cell stimulation in children with autoimmune thyroiditis (AT), as compared with control subjects. Blood samples were obtained from 35 AT children and 25 healthy children. CD markers were evaluated by flow cytometry at baseline, after the culture with phytohemagglutinin and without stimulation. At baseline, CD152+ expression was lower in patients than in controls (P<10(-6)). After stimulation, there were an increase in CD152+ T cells and decreases in CD28+ and CD4+ cells in controls (P<0.01). In AT children, CD152+ T cells remained stable. CD4+CD152+ T cells correlated inversely with antithyroglobulin antibodies. We conclude that alterations in lymphocyte markers are associated with AT. Stimulation leads to differing changes in T-lymphocyte subsets in both examined children populations.

Frequency of distribution of inflammatory cytokines IL-1, IL-6 and TNF-alpha gene polymorphism in patients with obstructive sleep apnea.

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.

Frequency of distribution of leptin receptor gene polymorphism in obstructive sleep apnea patients.

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.

The influence of low-power helium-neon laser irradiation on function of selected peripheral blood cells.

The effects of low-level laser light irradiation are debatable and the mechanisms of its action are still unclear. This study was conducted to test the effects of low-level laser irradiation on human blood cells: erythrocytes, granulocytes, and lymphocytes. Whole blood obtained by phlebotomy was irradiated at 632.8 nm by using energy fluences 0.6 J/cm2. An analysis of blood gases revealed an increase in PO2 and SaO2 (P<0.001) in irradiated blood. No shifts in PCO2 and pH were recorded. Spontaneous synthesis of DNA in T and B blood lymphocytes decreased significantly after laser irradiation (P<0.02 and P<0.04, respectively). Phytohemagglutinin (PHA)-induced proliferation of T cells and SAC proliferation of B cells, expressed as a stimulation index, were statistically higher in the samples of irradiated than in non-irradiated blood (P<0.01). Chemiluminescence of fMLP-stimulated granulocytes from irradiated blood increased in comparison with non-irradiated samples (P<0.001). No changes of spontaneous and stimulated chemiluminescence kinetics in irradiated samples were observed. These results reveal the influence of photodynamic reactions on the ability of blood to transport oxygen and on immunomodulatory effects on leukocytes.

The Gln223Arg polymorphism of the leptin receptor gene and peripheral blood/bone marrow leptin level in leukemic children.

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.

Degradation of cellulose by nitrogen-fixing strain of Bacillus polymyxa.

During an intensive screening programme, several strains of cellulolytic bacteria were isolated. One nitrogenase-positive strain able to degrade filter paper, Avicel cellulose, carboxymethyl cellulose and cellobiose was selected for further study. On the basis of biochemical characteristics and Mol % of G+C content, the selected strain was identified as Bacillus polymyxa. The highest production of the enzymes degrading filter paper (FP-ase) and carboxymethyl cellulose (CMCase) by B. polymyxa was observed in Park's medium suplemented with Avicel cellulose. The investigated strain of bacteria produced cellulosome-like structures as was shown by transmission electron microscopy.