[Evaluation of selected parameters of specific immunity in children with osteosarcoma at various stages of antitumour treatment].

UNLABELLED: 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. THE AIM OF STUDY: was evaluation by flow cytometry selected elements of specific cellular immunity in children with osteosarcoma at various stages of antitumour treatment. MATERIALS AND METHODS: 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). RESULTS: 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). CONCLUSIONS: 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 evaluation of selected parameters of calcium and phosphorus metabolism in children with cow's milk allergy]. / Ocena wybranych parametrów gospodarki wapniowo-fosforanowej u dzieci z alergia na bialka mleka krowiego.

UNLABELLED: THE AIM of this study was to evaluate selected parameters of calcium and phosphorus metabolism in children with CMA treated with the following milk substitute formulas: lactose-containing extensively hydrolyzed wheat protein formula, lactose-free extensively hydrolyzed casein protein formula, as well as soy-based formula. MATERIAL AND METHODS: The study involved 66 children with CMA aged 2-5 years treated with milk-free diet for at least one year. Group I included 31 children fed with a lactose-containing formula, group II - 35 children treated with lactose-free formula. In all children the mean energy intake and nutritional value of daily food rations were assessed. Serum concentrations of calcium (Ca), phosphorus (P), sodium (Na) and magnesium (Mg) were determined using standard methods. Serum values of 25 hydroxyvitamin D (25-OH D) and parathormone (PTH) were assessed by chemiluminescence, whereas concentrations of biochemical markers of bone formation-bone alkaline phosphatase (BALP), osteocalcin (OC) and bone resorption marker-collagen type I crosslinked C-telopeptide (CTX) were determined by immunoenzymatic methods (ELISA), using specific monoclonal antibodies. RESULTS: There were no significant differences in the mean dietary supply of calcium, phosphorus, magnesium, sodium, total protein and vitamin C in children from both groups. In the diets of children from group II, the mean content of lactose (0.5±1.0 vs 10.0±6.8 g/d) and 25-OH vitamin D (4.1±2.3 vs 8.5±4.0 ug/d) were significantly lower and dietary fibre content (14.7±3.9 vs 10.4±3.9 g/d) was higher. Calcium and vitamin D dietary supply was lower with respect to nutritional recommendations in all the studied children, whereas the dietary deficiency of vitamin D was higher in children from group II. The mean serum concentrations of evaluated biochemical parameters did not reveal any differences in children from the study groups and were in the normal ranges. There were also no differences in the mean serum concentration of 25-OH vitamin D, ALP, BALP, CTX and PTH in patients from both groups. The mean concentration of OC was significantly higher in group II (71±26.6 ng/ml) than in children from group I (61.1±23.4 ng/ml) <0.01. Positive correlation was found between OC and CTX in both study groups. CONCLUSIONS: 1. In children with CMA basic blood laboratory tests may have limited importance in the evaluation of calcium and phosphorus metabolism. 2. Our results suggest that the disturbances in the balance between bone formation and bone resorption processes may occur in children with CMA treated with lactose-free formulas. 3. In order to assure optimal conditions for achieving adequate bone mass by children with CMA, it is necessary to provide them with regular medical and nutritional care.

[Evaluation of selected parameters of cellular immunity in children with osteosarcoma at diagnosis]. / Ocena stanu immunologicznego dzieci I mlodzinezy z miesakiem kosciopochodnym w momencie rozpoznania choroby.

UNLABELLED: 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 Taä 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. MATERIALS AND METHODS: 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. RESULTS: 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, p<0.001) CD4+(598.0 vs 1071.0 kom/l; p<0.001) and their cytotoxic subpopulation CD8+ (386.0 vs. 866.0 cells/µL; p<0.001), activated T lymphocytes CD3+/HLA-DR+(39.0 vs. 81.0 cells/µL; p<0.025), B lymphocytes CD19+(205.0 vs. 381.0 cells/µL; p<0.025) and NK cells (161.0 vs. 339.0 cells/µL; p<0.005). 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. CONCLUSIONS: 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.

[Evaluation of selected cytokines in children and adolescents with osteosarcoma at diagnosis - preliminary report]. / Ocena stezenia wybranych cytokin u dzieci i mlodziezy z miesakiem kosciopochodnym w momencie rozpoznania choroby - doniesienie wstepne.

BACKGROUND: 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. MATERIALS AND METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.

[Contribution of indicators of sensitized T lymphocytes in the diagnosis of IgE independent and IgE dependent cow's allergy in children]. / Udzial wskazników uczulenia limfocytów T w diagnostyce alergii IgE niezaleznej i IgE zaleznej na bialka mleka krowiego u dzieci.

BACKGROUND: 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. AIM: To investigate, the usefulness of flow cytometry for estimation of specific sensitization of subpopulation of lymphocytes on food allergens in the allergy diagnosis. PATIENTS AND METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.

[Immunological and clinical study on therapeutic efficacy of inosine pranobex]. / Immunologiczne i kliniczne badania nad przydatnoscia terapeutyczna inozyny pranobeks.

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).

[Similarity of immunological mechanisms responsible for immunotolerance in healthy subjects and mechanisms induced by specific immunotherapy]. / Podobienstwo procesów immunologicznych warunkujacych immunotolerancje w stanie zdrowia i procesów wyzwalanycnh przez swoista immunoterapie.

The immonomodulatory mechanisms responsible for allergens tolerance in healthy subjects were described on the basis of the newest medical literature, including own papers and experience. It was detected that the same immunological mechanisms are built by specific immunotherapy. IL-10 and TGF-beta cooperate in the regulatory T-cell response to aeroallergens, food allergens and allergens of bee and yellow jacket venoms in healthy subjects as well as during specific immunotherapy making the tolerance to these allergens. The actual progress in improvement of specific immunotherapy methods is described.

[Immunological confirmation of allergy in children with hypoimmunoglobulinemia]. / Badania nad immunologicznym potwierdzeniem alergii u dzieci z hipoimmunoglobulinemia.

UNLABELLED: The recognition of allergy in children with hypoimmunoglobulinemia is very difficult because they have very low level or even lack of specific IgE antibodies. Our research hypothesis was the possibility of detecting the specific sensitization of lymphocytes T to food allergens in children with hypoimmunoglobulinemia and with clinical symptoms of allergy. MATERIAL: 32 children actually treated in our Immunology Outpatients Department due to immunodeficiency connected with hypoimmunoglobulinemia and with clinical suspicion of allergy. METHODS: IgE concentration in serum and IgE specific antibodies to food and pollens were examined by FEIA CAP-SYSTEM. Lymphocyte specific response to allergens (cow's milk, gluten) was examined by classical blast transformation test. Immunological profile of children was also evaluated. RESULTS: In the group of 32 children we recognised: 20 children with predominantly antibody deficiencies (2 - agammaglobulinemia; 8 - CVID; 5 - selective immunoglobulins deficiency; 5 - hypoimmunoglobulinemia of infants) and 12 children who had combined immunodeficiency with hypoimmunoglobulinemia ( 3 - AT; 8 - Nijmegen S; 1 - Di George). The children had atopic dermatitis and clinical symptoms of allergy in respiratory and digestive tracts. Concentration of IgE in serum of children was very low, even < 2KU/L, Pediatric Phadiatop was negative, f2 and f79 were absent. Using lymphocytes transformation test (culture stimulated with cow's milk and gluten antigens) we detected sensitivity to cow's milk in 24 children and additionally in 4 of them sensitivity to gluten. CONCLUSIONS: 1) Laboratory difficulties in allergy diagnosis in children with hypoimmunoglobulinemia by evaluation of specific IgE concentration in serum may by caused by defect in biosynthesis of mentioned antibodies. 2) In children with hypoimmunoglobulinemia the lymphocyte blastic transformation test with specific allergens in cultures has a diagnostic value indicating lymphocytes specific response to allergens.

[Detection of the T-lymphocyte-specific sensitivity in the diagnosis of food allergies]. / Badania nad wykrywaniem swoistego uczulenia limfocytów T w diagnostyce nadwrazliwosci na pokarmy.

BACKGROUND: 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. METHODS/DATA BASE: 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). RESULTS/CONCLUSIONS: The significance of such investigation is underlined especially for young children during early diagnosis of food allergy and monitoring of the treatment.

[CD69 and PCNA expression of T-lymphocytes from children with cow's milk allergy (CMA)]. / Ekspresja CD69 i PCNA limfocytów dzieci z alergia na bialka mleka krowiego (cow's milk allergy CMA).

BACKGROUND: 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. AIM: The study on usefulness of lymphocytes CD69 and PCNA detection for diagnosis of CMA in children and for monitoring of the treatment. PATIENTS AND METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.