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Spontaneous abortion occurs in 8–20% of recognized pregnancies and usually takes place in the first trimester (7–11 weeks). There are many causes of pregnancy loss, but the most important (about 75%) is the presence of chromosomal aberrations. We present the results of oligonucleotide array application in a cohort of 62 miscarriage cases. The inclusion criteria for the study were the loss after 8th week of pregnancy and the appearance of recurrent miscarriages. DNA was extracted from trophoblast or fetal skin fibroblasts. In the 62 tested materials from recurrent miscarriages, the detection rate was 56.5% (35/62). The most commonly found were aneuploidies (65%) (chromosomal trisomy 14, 16, 18, 21, and 22), Turner syndrome, and triploidy (17.1%). Other chromosomal abnormalities included pathogenic and likely pathogenic structural aberrations: 1) pathogenic: deletion 7p22.3p12.3 and duplication 9p24.3p13.2 inherited from the normal father, deletion 3q13.31q22.2 and deletion 3q22.3q23 of unknown inheritance and duplication of 17p12 inherited from father with foot malformation; 2) likely pathogenic variants: deletion 17p13.1 inherited from normal mother, deletion 5q14.3 of unknown inheritance and de novo deletion 1q21.1q21.2. Among these aberrations, six CNVs (copy number variants) were responsible for the miscarriage: deletion 7p22.3p12.3 and duplication 9p24.3p13.2, deletion 3q13.31q22.2 and deletion 3q22.3q23, and deletion 17p13.1 and deletion 1q21.1q21.2. Other two findings were classified as incidental findings (deletion 5q14.3 and 17p12 duplication). Our research shows that 17% of the aberrations (6/35 abnormal results) that cannot be identified by the routine kariotype analysis are structural aberrations containing genes important for fetal development, the mutations of which may cause spontaneous abortion.

An important aspect of the chemopreventive activity of isothiocyanates (ITC) is their ability to induce cell growth inhibition and apoptosis. In this study, the effect of two sulforaphane analogues, 2-oxoheksyl isothiocyanate and alyssin, on lymphoblastoid cells, derived from people carrying four different germ-line mutations in BRCA1 gene, was tested and compared to the effect on wild type cells. The mutations studied were: C61G; 3819del5; 4153delA, and 5382INSC. Changes in cell viability and density after 2-oxoheksyl isothiocyanate and alyssin treatment were evaluated, as well as cell cycle progression, mitochondrial membrane potential changes, and phosphatidylserine externalization. Both isothiocyanates were shown to reduce cell viability and density in all cell lines tested, as well as the change in cell cycle phase's distribution. The response of cells to two ITC tested was various, as well as mutation type-modulated. We found that change of cellular maintenance by chemopreventive agents can be modulated by single allele BRCA1 mutation. Drug Dev. Res. 65:84–92, 2005. © 2005 Wiley-Liss, Inc.

Typical cells of specific immunity are lymphocytes. T cells may specifically recognize antigens using T Cell Receptors (TCRs). Antigen presentation by specialized cells is a necessary element of specific immunity. It leads to initiating an immune response. After antigen dependent activation T cells transform to memory and effector lymphocytes. Cells engaged directly in destruction of the tumour are CD8+cytotoxic T lymphocytes, CD4+ lymphocytes, Tγδ lymphocytes, NK, NKT cells and indirectly B lymphocytes. One of the main methods of osteosarcoma treatment in children is chemotherapy. The goal is to destroy the cancer cells by putting them in a state of apoptosis. All of the medications used as chemotherapy carry the risk of short-term and long-term problems including leukopenia, immune disorders such as immunodeficiency.was evaluation by flow cytometry selected elements of specific cellular immunity in children with osteosarcoma at various stages of antitumour treatment.The study was performed on the group of 44 children with osteosarcoma, aged from 6 to 20 years (average 14.9 years; median 15.0 years). T and B lymphocytes and subpopulations: CD4+, CD8+, CD3+?HLA-DR+, CD3+γδ; NK, NKT cells were analyzed in peripheral blood with use of flow cytometry method with monoclonal antibodies. Examinations were performed before the therapy - in diagnostic period (examination I), after the neoadjuvant chemotherapy (examination II), 10-14 days after the surgery (examination III), 5 months after the surgery (after adjuvant chemotherapy, examination IV).The number of T and B lymphocytes was decreasing after each stage of cytostatic therapy, with the biggest differences for CD19+ cells (medians: I examination - 205.0; II exam. - 62.0; IV exam. - 24.0 cells/mL); in single cases the number of cells decreased even under 10/mL (norm 200-500 cells/mL).1. In children and youth with osteosarcoma antineoplastic treatment contributes to the suppression of the immune system, decreasing definitely the number and percentage of B lymphocytes, T helper lymphocytes and NK cells. 2. Decreased number of CD3+, CD4+, CD19+ and NK lymphocytes during chemotherapy may contribute to the progression of neoplastic disease in the future, after treatment. 3. Evaluation of immunologic status in patients with osteosarcoma may be helpful in monitoring of antineoplastic therapy effectiveness, may prevent the formation of unfavourable clinical changes and may be the basis for correction of the cytostatic agents' administration.

The causes of osteosarcoma (OS) and effector mechanisms of the immune response against OS and other neoplastic diseases remain unknown. According to current knowledge, the major role is attributed to cytotoxic T lymphocytes, NK, NKT and Tăä lymphocytes, which are engaged directly in the destruction of the tumour cells. Helper T lymphocytes (CD4+) and indirectly B lymphocytes are of special importance. There is sparse data on the state and efficiency of the immune system in children with neoplastic disease, with bone tumours in particular. THE AIM OF THE STUDY was the evaluation of selected elements of cellular immunity in children with osteosarcoma at the time of diagnosis.The study was performed on a group of 44 children with osteosarcoma, aged from 6 to 20 years (median 15.0 years). The control group consisted of 22 children of the same age (median 14.5 years) without the diagnosis of neoplastic disease and active inflammatory state. T lymphocytes with their subpopulations, activated T lymphocytes (CD3+/HLA-DR+), B lymphocytes, NK and NKT cells were analyzed in peripheral blood using the flow cytometry method. Examinations were performed before the therapy - in the diagnostic period.A lower number of peripheral blood lymphocyte population in children with osteosarcoma at diagnosis, compared to the control group was observed. The differences concerned T lymphocytes CD3+(1609.0 vs 3038.0 kom/μl, p0.001) CD4+(598.0 vs 1071.0 kom/l; p0.001) and their cytotoxic subpopulation CD8+ (386.0 vs. 866.0 cells/μL; p0.001), activated T lymphocytes CD3+/HLA-DR+(39.0 vs. 81.0 cells/μL; p0.025), B lymphocytes CD19+(205.0 vs. 381.0 cells/μL; p0.025) and NK cells (161.0 vs. 339.0 cells/μL; p0.005). The number and percentage of peripheral blood lymphocytes in children and youth with osteosarcoma at diagnosis is over 50% lower compared to the patients without neoplastic disease.1. The general analysis of peripheral blood without differentiating lymphocyte subpopulations is insufficient to determine disturbances which are forming in the immune system of patients developing the neoplastic disease. 2. The course of the neoplastic disease (osteosarcoma) in patients of developmental age is very diverse, and associated with individual biological variation. 3. The evaluation of the immunologic status in patients with osteosarcoma may have prognostic meaning for the further course of the disease, may prevent the formation of unfavourable clinical changes, and be the basis for correcting the administration of cytostatic agents.

Osteosarcoma (OS) is the most frequent primary malignant bone tumour, mainly affecting children in the first and second decade of life. Causes of the disease are still unknown and reaction of the immune system on its development is very individual. Particular emphasis must be placed on the role of cytokines in the immunoregulatory and coordinating function and tumour cell disruption. Knowledge about cytokines concentration in serum, regarding mechanisms of oncogenesis, may have prognostic significance for the further course of OS in children. The aim of study was evaluation of IL-2, IL-4, IL-8, IFN-gamma, and TNF-a concentrations in children with osteosarcoma at diagnosis.The study was performed on the group of44 children with osteosarcoma, aged from 6 to 20 years (average 14.9 years; median 15.0 years). 22 children ofthesame age (median 14.5years) without neoplastic disease and active inflammatory state formed the control group. Investigations were performed before the therapy. The inclusion criteria were: diagnosis of primary osteosarcoma, upper or lower limbs tumour localization, patients who were not treated by chemo- or radiotherapy before biopsy patients' age at diagnosis was 6-20 years. Concentrations of selected cytokines were analyzed in peripheral blood with using ELISA method with 99.8% sensitivity and 99,5% specificity.In children with osteosarcoma, at diagnosis the following concentration of peripheral blood cytokines (medians) was observed: IL-2 10.7 pg/ml (min-max: 0.0-894.0); IFN-gamma 1,3 pg/ml (min-max: 0.2-147); TNF-alpha 28.3 pg/ml (min-max: 0.0-188.8); IL-4 2.0 pg/ml (min-max: 0.0-32.0); IL-8 13.5 pg/ml (min-max: 0.0-2154.0). A large scatter among individual children results was found. Analysis of cytokines concentration showed significant statistical differences between patients with OS and the control group in case of IL-4 (p=0.005) and IL-8 (p=0.01).Results of studies obtained at diagnosis did not give a specific answer about the prognosis and further course of OS disease in patients in the developmental age. Big differences in cytokines concentration in children and youth with OS might be associated with individual biological variation and individual reaction to the development of neoplastic disease and further studies in this direction are needed - before the start of cytostatic therapy and in therapy monitoring.

The purpose of the study was to compare concentrations of inflammatory and Th1/Th2 cytokines in serum obtained from women with preeclampsia or severe pregnancy hypertension versus normotensive controls.The study group consisted of 34 pregnant women with hypertension over 140/90mmHg and proteinuria over 0.3 g/day or severe pregnancy hypertension. 16 healthy pregnant women comprised the control group. The concentration of IL-2, IL-4, IL-6, IL-10, TNFalpha and IFNgamma was measured with Cytometric Bead Array Human Th1/Th2 Cytokine Kit II (Becton Dickinson). U-Mann Whitney test was used for the comparison of the results.We found statistically significantly increased concentrations of IFNgamma: 8.4 +/- 5.3 pg/ml vs. 4.2 +/- 3.2 pg/ml (p = 0.02), TNFalpha: 1.5 +/- 0.7 pg/ml vs. 0.7 +/- 0.3 pg/ml (p = 0.04) and IL-2: 1.3-0.6 pg/ml vs. 0.6 +/- 0.4 pg/ml (p = 0.01) in the studied group. The level of IL-6 35.5 +/- 21.0 pg/ml vs. 19.8 +/- 12.3 pg/ml was also increased but the difference did not reach statistical significance. Concentrations of IL-4 and IL- 10 were similar in both groups.Increased concentrations of Th1 cytokines (IFNgamma, IL-2) in the serum of women with preeclampsia suggests an exaggerated cytotoxic activity of blood in this pathology accompanied by an increase in the levels of inflammatory cytokines TNFalpha and IL-6.

Food allergy is an abnormal response of the immunological system, especially of mucosal immunological system on antigens supplied per os. There are very complicated and still unexplained immunological mechanisms, which lead to hypersensitivity reaction. Most often food hypersensitivity is identified as the effect of atopy, which is connected with humoral response (specific IgE antibody). On the other hand cell immunological response are less investigated, however they can be very important, especially as a significant factor to initiate pathological allergic processes.To investigate, the usefulness of flow cytometry for estimation of specific sensitization of subpopulation of lymphocytes on food allergens in the allergy diagnosis.The investigations were performed on 60 children from 6 months to 5 years old: 20 children with CM A IgE dependent, 20 with CM A IgE independent and 20 healthy children. IgE total, sIgE, IgG, IgA, IgM, basic immunological panel, CD 23, CD25, CD26, CD30, CD69, PCNA were measured.We noticed decrease of expression of CD4+CD30+ between I and II examination (p=0.029), between I and III (p=0.009); decrease of expression of CD8+CD26+ between I and III test (p=0.038); decrease of expression of CD19+CD23+ between I and II examination (p=0.012) in I type of hypersensitivity. We observed a decrease of expression of CD4+CD25+ between I and III examine (p=0.026) and decrease of expression of CD4+ CD26+ between I and III examination (p=0.036) in IV type of hypersensitivity. Expression of CD69 was decreased after diet in IgE dependent allergy. Values of expression of PCNA are similar in I and IV type of hypersensitivity in children with CM A. Decrease of expression of PCNA in II examination was observed in both cases. Reintroduced allergen caused increase of expression of PCNA in both types of allergy (p=0.048 and p=0.041).Our recent research confirms changes of the expression of T lymphocytes activation markers. It is connected with in vivo stimulation to allergen or with allergen elimination. The study of expression of activation markers using flow cytometry in food allergy in children can be helpful in observation of the dynamic progress process, but it cannot be used as a single diagnosis test.

Many studies in vitro and in vivo have shown immunomodulating and antiviral activities of inosine pranobex. The object of this research was to examine the potential beneficial effects of inosine pranobex (Groprinosin) on immune system in children with cellular immunodeficiency as a prophylaxis of recurrent infections, mainly of viral origin. 50 mg/kg b.w/day of inosine pranobex in divided doses was given to the group of 30 children aged 3-15 years for 10 days in 3 following months. Clinical and immunological investigations were done before and after the treatment. Statistically significant rise of CD3T lymphocytes number (p = 0.02) and in this CD4T lymphocytes number (p = 0.02) as well as statistically significant improvement of their function (p = 0.005) evaluated with blastic transformation method were found. These laboratory findings were parallel to clinical benefits. Control study was performed in the group of children completed by randomization and treated in the same way with garlic (Alliofil).

The important aspect of sulforaphane (SFN) chemopreventive activity is its ability to induce cell growth inhibition and apoptosis. In this study, the effect of SFN on lymphoblastoid cells derived from people carrying four different germ-line mutations in BRCA1 gene was tested and compared to the effect of SFN on wild-type cells. The mutations studied were C61G, 3819del5, 4153delA and 5382INSC. Changes in cell viability and density after SFN treatment were evaluated, as well as cell cycle progression, changes in mitochondrial membrane potential, and phosphatidylserine externalization. SFN was shown to reduce cell viability and density in all cell lines tested. Cell cycle block in S-phase and the occurence of simultaneous apoptosis were observed. The concentration of SFN needed to elicit a comparable effect on each cell line was varied. We found that the effect of SFN on cells carrying different inherited mutations depended on mutation type.

As the frequency of allergic disease and especially the food allergy in children is still increasing - we undertook studies to determine the specific sensitivity of lymphocytes T in order to improve the diagnostics and monitoring of treatment. The usefulness of evaluation CD69 and PCNA expression of T cells from children with cow's milk allergy (CMA) in these procedures was examined.The study on usefulness of lymphocytes CD69 and PCNA detection for diagnosis of CMA in children and for monitoring of the treatment.The subjects in this study included 26 children from 1.5 to 15 years old with CMA who were under clinical care of our Immunological Department. Patients were examined (medical history of the child, physical examination, specific food provocation) and laboratory investigations were made before therapy and after 6 months of elimination diet. Allergen-specific IgE antibody was determined. Children's T cell cultures were stimulated by beta-lactoglobulin, alpha-casein, whole cow's milk, phytohaemoglutinin PHA and without stimulation. Expression of T cells CD69 and PCNA from patients with CMA and 10 healthy children were analysed by use of flow cytometry. 10 children with CMA after 6 months of elimination diet and next 2 weeks with cow's milk provocation were examined also for CD69 and PCNA expression in lymphocytes from peripheral blood.After 6 months of elimination diet it seems that the expression of CD69 receptor has tendency to fall, these differences, however, are not statistically significant. But after next 2 weeks with cow's milk diet the expression of CD69 was increased with statistically significance (p=0.04). T cells PCNA expression of children with CMA was lower than in healthy children in PHA stimulated cultures. Our study indicates that PCNA expression of T cells from patients with CMA increased even more after 6 months of elimination diet in all cultures but there was statistically significant differences only after stimulation with cow's milk and PHA (p=0.05 and p=0.04 - respectively). Subsequently through 2 weeks children were provoked with cow's milk and after that PCNA expression in vivo lowered (no statistically significant differences).CD69 expression of T lymphocytes was significantly higher after specific stimulation of children with cow's milk allergy (p=0.04). The evaluation of CD69 expression of children with CMA may be also useful in monitoring of treatment. Estimation of PCNA expression suggests the efficiency of T cells connected with DNA synthesis related to the stage of allergy disease and demands further investigations.

The possibility of detection of lymphocytes T specific sensitivity after contact with the allergen (mainly food allergen) even in early age of children has been reported in this paper.This is the literature review of basic applied methods such as: lymphocyte blastic transformation in allergen stimulated culture, macrophage migration inhibition test and flow cytometric analysis of in vivo and in vitro lymphocyte specific activation by using activation markers (CD69, PCNA - proliferating cell nuclear antigen and others).The significance of such investigation is underlined especially for young children during early diagnosis of food allergy and monitoring of the treatment.

Results Lower number of lymphocytes population in children with osteosarcoma compared to the control group was observed. The differences concerned lymphocytes CD3+ (1609,0 vs 3038,0 kom/μl, p