Search

In the SARS-CoV-2 virus pandemic, the questions about specific activity of this virus in induction and/or inhibition of the innate and adaptive immune response are still open. Clinical observations of the severe and critical course of infection showed the hyperinflammation and cytokine storm. In organs and tissues that are a target for viral entry the lymphocytes and monocytes are dominant cells in tissue infiltration. There are different ways and different mechanisms leading to immune response disorders. In peripheral blood in the severe and fatal course of disease lymphopenia is frequent as a poor prognosis factor. Reduced numbers of lymphocytes, mainly T cells and NK cells, are noted in the majority of these patients. The NK cells belonging to the innate immunity system are dedicated to the antiviral response due to production of interferon (IFN) and direct lysis of virus infected cells. In SARS-CoV-2 infection NK cells' activity against this pathogen is impaired based on inhibition of IFN production and functional exhaustion. The restoration of NK cell number and function might support elimination of the SARS-CoV-2 virus, increasing chances of recovery. The use of activated NK cells in SARS-CoV-2 therapy is under clinical trials.

Background The phenotypic heterogeneity of circulating tumor cells (CTC) in peripheral blood and disseminated tumor cells (DTC) in bone marrow is an important constraint for clinical decision making. Here, we investigated the implications of two different subpopulations of these cells in gastric cancer (GC). Methods GC patients (n = 228) who underwent elective gastric resections were prospectively examined for CTC/DTC. The cells obtained from peripheral blood and bone marrow aspirates were sorted by flow cytometry and CD45− cells expressing cytokeratins (8, 18, and 19) and CD44 were identified by immunofluorescent double staining. Results Ninety-three (41%) patients had cytokeratin-positive tumor cells in either blood or bone marrow, while cells expressing CD44 were found in 22 (10%) cases. CK+CD44+ cells were significantly more common among patients with distant metastases (50 vs 19%, P = 0.001), while no such correlations were demonstrated for CK+CD44− cells. Detection of CK+CD44+ cells, but not CK+CD44−, was associated with significantly shortened survival. Moreover, the Cox proportional hazards model identified CK+CD44+ cells as a negative prognostic factor with an odds ratio of 2.38 (95% CI 1.28–4.41, P = 0.006). Conclusion CD44+ phenotype of cytokeratin-positive cells in blood and bone marrow is an independent prognostic factor in patients with gastric cancer. Electronic supplementary material The online version of this article (10.1007/s10120-018-0858-2) contains supplementary material, which is available to authorized users.

The extracellular matrix (ECM) is a dynamic environment involved in the regulation of haematopoiesis. A crucial role of this structure is the promotion of proliferation, maturation, and differentiation of haematopoietic stem cells (HSC), and adhesion and migration of HSC in bone marrow. In the present study the effect of ECM proteins (fibronectin, collagens, laminin, thrombospondin, and vitronectin) on proliferation and apoptosis of acute lymphoblastic leukaemia cells isolated from acute lymphoblastic leukaemia (ALL) patients (iiiiiin vitro/i/i/i/i/i) was assessed. The leukaemia cells were obtained as interphase on Ficoll/Isopaque (Pancoll human, PAN-Biotech) density gradient and, after washing, counted in a chamber. Subsequently, cells were used for culture and apoptosis assay. Presence of fibronectin, collagen type IV, and laminin was associated with inhibition of lymphoblastic leukaemia cell proliferation. Analysis of the culture of lymphoblastic leukaemia cells in the presence of ECM showed fibronectin as the most active protein.

Common variable immunodeficiency (CVID) comprise different kinds of primary hypogammaglobulinemias of mostly unknown etiology. The onset of the disease and its clinical symptoms may appear as well in children as in adults. Apart from susceptibility to bacterial, viral and fungal infections, other clinical signs (autoimmunization, non-infectious lung and gastrointestinal diseases, neoplasm) are frequently observed. About 58% of patients with CVID develops granulomatous and lymphocytic interstitial lung disease (GLILD) associated with early mortality.

Common variable immunodeficiency (CVID) is a primary immunodeficiency of humoral immunity with heterogeneous clinical features. Diagnosis of CVID is based on hypogammaglobulinaemia, low production of specific antibodies, and disorders of cellular immunity. The standard therapy includes replacement of specific antibodies with human immunoglobulin, prophylaxis, and symptomatic therapy of infections. High prevalence of autoimmunity is characteristic for CVID, most commonly: thrombocytopaenia and neutropaenia, celiac disease, and systemic autoimmune diseases. The study included seven children diagnosed with CVID and treated with immunoglobulin substitution from 2 to 12 years. Thrombocytopenia was diagnosed prior to CVID in four children, developed during immunoglobulin substitution in three children. In one boy with CVID and thrombocytopaenia, haemolytic anaemia occurred, so a diagnosis of Evans syndrome was established. Therapy of thrombocytopaenia previous to CVID included steroids and/or immunoglobulins in high dose, and azathioprine. In children with CVID on regular immunoglobulin substitution, episodes of acute thrombocytopaenia were associated with infections and were treated with high doses of immunoglobulins and steroids. In two patients only chronic thrombocytopaenia was noted. Splenectomy was necessary in one patient because of severe course of thrombocytopaenia. The results of the study indicated a supportive role of regular immunoglobulin substitution in patients with CVID and chronic thrombocytopaenia. However, regular substitution of immunoglobulins in CVID patients did not prevent the occurrence of autoimmune thrombocytopaenia episodes or exacerbations of chronic form. In episodes of acute thrombocytopaenia or exacerbations of chronic thrombocytopaenia, infusions of immunoglobulins in high dose are effective, despite previous regular substitution in the replacing dose.

Introduction In this study we describe a patient with gross deletion containing the BTK and TIMM8A genes. Mutations in these genes are responsible for X-linked agammaglobulinemia and Mohr-Tranebjaerg syndrome, respectively. X linked agammaglobulinemia is a rare primary immunodeficiency characterized by low levels of B lymphocytes and recurrent microbial infections, whereas, Mohr-Tranebjaerg syndrome is a progressive neurodegenerative disorder with early onset of sensorineural deafness. Material and methods For neuroimaging, the magnetic resonance imaging and magnetic resonance spectroscopy of the brain were performed. Microarray analysis was performed to establish the extent of deletion. Results The first clinical symptoms observed in our patient at the age of 6 months were connected with primary humoral immunodeficiency, whereas clinical signs of MTS emerged in the third year of live. Interestingly, the loss of speech ability was not accompanied by hearing failure. Neuroimaging of the brain suggested leukodystrophy. Molecular tests revealed contiguous X-chromosome deletion syndrome encompassing BTK (from exons 6 through 19) and TIMM8A genes. The loss of the patient’s DNA fragment was accurately localized from 100 601 727 to 100 617 576 bp on chromosome’s loci Xq22.1. Conclusions We diagnosed XLA-MTS in the first Polish patient on the basis of particular molecular methods. We detected neurodegenerative changes in MRI and MR spectroscopy in this patient. Our results provide further insight into this rare syndrome.

We described mutation of STAT3 gene, typical for autosomal dominant hiperimmunoglobulin E syndrome (HIES), in a child with recurrent skin abscesses and respiratory tract infections without skeletal and connective tissue anomalies.

We have studied the effect of intravenous immunoglobulins (IVIG) on monocyte subpopulations and cytokine production in patients with CVID. The absolute number of CD14(+)CD16(++) monocytes decreased on average 2.5-fold 4h after IVIG and after 20h returned to the baseline. The cytokine level in the supernatants of peripheral blood mononuclear cells (PBMC) after ex vivo LPS stimulation demonstrated the >2-fold decrease in TNF production 4h after IVIG. The TNF expression, which is higher in the CD14(+)CD16(++) monocytes, was decreased in these cells by IVIG in 4/7 CVID cases. In vitro exposure of the healthy individuals' monocytes to the IVIG preparation resulted in reduced TNF production, which was overcome by blockade of the FcγRIIB in the CD14(+)CD16(++) CD32B(high) monocytes. Our data suggest that reduction in the number of CD14(+)CD16(++) monocytes and the blockade of their cytokine production via triggering CD32B can contribute to the anti-inflammatory action of IVIG.

Chronic granulomatous disease (CGD) is phagocytic cell metabolic disorder resulting in recurrent infections and granuloma formation. This paper reports the favourable outcome of allogeneic transplantation in six high-risk CGD patients. The following donors were used: HLA-matched, related (two) and unrelated (three), and HLA-mismatched, unrelated (one). One patient was transplanted twice using the same sibling donor because of graft rejection at 6 months after reduced-intensity conditioning transplant (fludarabine and melphalan). Myeloablative conditioning regimen consisted of busulphan and cyclophosphamide. Stem cell source was unmanipulated bone marrow containing: 5.2 (2.6-6.5) × 10(8) nucleated cells, 3.8 (2.0-8.0) × 10(6) CD34+ cells and 45 (27-64) × 10(6) CD3+ cells per kilogramme. Graft-versus-host disease prophylaxis consisted of cyclosporine A and, for unrelated donors, short course of methotrexate and anti-T-lymphocyte globulin. Mean neutrophile and platelet engraftments were observed at day 22 (20-23) and day 20 (16-29), respectively. Pre-existing infections and inflammatory granulomas resolved. With the follow-up of 4-35 months (mean, 20 months), all patients are alive and well with full donor chimerism and normalized superoxide production.

Recent studies in breast cancer suggest that monitoring the isolated tumour cells (ITC) may be used as a surrogate marker to evaluate the efficacy of systemic chemotherapy. In the present study, we have investigated the effects of preoperative chemotherapy on ITC in the blood and bone marrow of patients with potentially resectable gastric cancer. After sorting out the CD45-positive cells, the presence of ITC defined as cytokeratin-positive cells was examined before and after preoperative chemotherapy. The patients received two courses of preoperative chemotherapy with cisplatin (100 mg m(-2), day 1) and 5-fluorouracil (1000 mg m(-2), days 1-5), administered every 28 days. Fourteen of 32 (44%) patients initially diagnosed with ITC in blood and/or bone marrow were found to be negative (responders) after preoperative chemotherapy (P0.01). The incidence of ITC in bone marrow was also significantly (P0.01) reduced from 97 (31 of 32) to 53% (17 of 32). The difference between patients positive for ITC in the blood before (n=7, 22%) and after (n=5, 16%) chemotherapy was statistically insignificant. The overall 3-year survival rates were 32 and 49% in the responders and non-responders, respectively (P=0.683). These data indicate that preoperative chemotherapy can reduce the incidence of ITC in patients with gastric cancer.

Vascular endothelial growth factor (VEGF) induces proliferation of endothelial cells, stimulates angiogenesis and increases vascular permeability. Epidermal growth factor (EGF) induces proliferation of epidermal cells and stimulates epidermal migration. Increased VEGF and EGF expression have been associated with poor clinical outcome in many malignancies. Several recent reports have shown overexpression of VEGF and EGF in papillary thyroid cancer (PTC). The study aimed to determine the intensity of expression of VEGF and EGF in patients with PTC and to find any correlation between the intensity of the expression and staging of the disease at the moment of surgery The study comprised a group of 48 consecutive patients with PTC who underwent radical surgery. The group consisted of 11, 25, eight and four patients at pT1N0M0, pT2N0M0, pT3N1M0 and pT4N1M0 stages, respectively. The control group was composed of 20 healthy bone marrow transplant donors, age-, gender- and BMI-matched with PTC patients. The immuno-chemiluminescence enzyme linked immunoassay (ELISA) method was used to determine the expression and level of VEGF and EGF in serum samples. Patients’ characteristics, serum levels of VEGF and EGF, final histology and pTNM were analysed. The mean serum level of VEGF and EGF was remarkably higher in PTC patients than in controls. A negative correlation between staging in pTNM classification and the mean serum VEGF level (r=−0.5168; P

Background: Cancers characterized by microsatellite instability may be biologically different from their counterparts with stable microsatellite sequences. Circulating cancers cell present in blood prior to surgery may constitute an adverse prognostic finding. Aim: To correlate these two phenomena with morphological features and survival in advanced gastric cancer. Methods: We examined 76 cases of resected sporadic, advanced gastric cancer by means of routine morphology and a panel of microsatellite markers. Sixty-six cases were screened for presence of cancer cells circulating in blood prior to the surgery using combined morphological and immunocytochemical approach. Results: Twenty-one (27.6%) cases demonstrated microsatellite instability in at least one locus. Among them 11 (14.5%) showed microsatellite instability in more than 30% (4/12) examined loci, and they were therefore designated as replication error positive (RER+). Circulating cancer cells were detected in 2/19 microsatellite instability and in 11/47 remaining cases (difference not significant). The survival of the microsatellite instability cases was significantly better. The presence of circulating cancer cells did not correlate with survival. Conclusion: It is possible that the microsatellite instability status, but not circulating cancer cells, constitutes a prognostic predictive factor in advanced gastric carcinoma. Confirmation of this hypothesis requires continuation of patient follow-up.

Common variable immunodeficiency (CVID) is a primary immunodeficiency of humoral immunity with heterogeneous clinical features. Diagnosis of CVID is based on hypogammaglobulinaemia, low production of specific antibodies, and disorders of cellular immunity. The standard therapy includes replacement of specific antibodies with human immunoglobulin, prophylaxis, and symptomatic therapy of infections. High prevalence of autoimmunity is characteristic for CVID, most commonly: thrombocytopaenia and neutropaenia, celiac disease, and systemic autoimmune diseases. The study included seven children diagnosed with CVID and treated with immunoglobulin substitution from 2 to 12 years. Thrombocytopenia was diagnosed prior to CVID in four children, developed during immunoglobulin substitution in three children. In one boy with CVID and thrombocytopaenia, haemolytic anaemia occurred, so a diagnosis of Evans syndrome was established. Therapy of thrombocytopaenia previous to CVID included steroids and/or immunoglobulins in high dose, and azathioprine. In children with CVID on regular immunoglobulin substitution, episodes of acute thrombocytopaenia were associated with infections and were treated with high doses of immunoglobulins and steroids. In two patients only chronic thrombocytopaenia was noted. Splenectomy was necessary in one patient because of severe course of thrombocytopaenia. The results of the study indicated a supportive role of regular immunoglobulin substitution in patients with CVID and chronic thrombocytopaenia. However, regular substitution of immunoglobulins in CVID patients did not prevent the occurrence of autoimmune thrombocytopaenia episodes or exacerbations of chronic form. In episodes of acute thrombocytopaenia or exacerbations of chronic thrombocytopaenia, infusions of immunoglobulins in high dose are effective, despite previous regular substitution in the replacing dose.

Seven boys with diagnosis of X-linked agammaglobulinemia on regular substitution of immunoglobulins were included into study. The patients showed episodes of infections but the clinical course was mild with good response to antibiotics. All patients developed, with time, the chronic sinusitis with proliferation of mucous membrane, two patients showed bronchiectases. The number of T lymphocytes, ratio of CD4:CD8 subpopulations, response to stimulation and NK number were assayed with flow cytometry and cell culture. Results showed CD4:CD8 ratio within normal value in majority of patients, reverse ratio in 2 boys, increased number of activated T cells (CD3/HLA-DR) in one of them. The number of NK cells was different from lack of these cells to high number. Response of T cells to stimulation (mitogens and CD3) were normal in majority of assays. There were no associations between clinical course and observed changes in T or NK cell populations. Further studies on number and function of NK cells are needed.

bcl-2 expression is associated with the expression of the multidrug resistance molecule (p-gp) and the resistance of leukaemia cells to the induction of apoptosis. The activity of p-gp is the main mechanism of resistance of leukaemia cells to chemotherapy. This study assessed the induction of apoptosis of acute lymphoblastic leukaemia (ALL) and non-Hodgkin's lymphoma (NHL) blastic cells following in vitro treatment with dexamethasone (DXM), vincristine (VCR), and tumour necrosis factor (TNF) in relation to the expression of bcl-2 and p-gp. Common ALL (cALL; n = 24 patients), common ALL with co-expression of myeloid antigens (cALL + My; n = 9), ALL-T (n = 9), and NHL [n = 6 (T type, n = 2; B type, n = 4)] were included. The expression of bcl-2 and p-gp and apoptosis were assayed by flow cytometry. Spontaneous apoptosis was low (5%) in cALL and ALL-T and higher (8%) in NHL and cALL + My. A high frequency of bcl-2 expression was noted in cALL and cALL + My. A high frequency of p-gp expression was observed in cALL + My, ALL-T, and NHL. There was a reverse association between bcl-2 expression and spontaneous apoptosis. DXM-induced apoptosis was observed in 52.63%, TNF-induced in 42.85%, VCR-induced in 36.36%, and GM-CSF-induced in 33.3% of leukaemia and lymphoma cases. DXM and GM-CSF-driven apoptosis was reversibly associated with bcl-2-expression (bcl-2-dependent mechanism). VCR and TNF-driven apoptosis was not associated with bcl-2 expression, suggesting a different, bcl-2-independent, mechanism(s) of its induction. The in vitro induction of apoptosis was not associated with expression of p-gp.

— The photodynamic effect of Victoria blue BO (VB-BO) and photoirradiation on peripheral blood mononuclear cells was studied. The cells were preincubated with VB-BO followed by photoirradiation and overnight culture. The highest percentage of dead cells (propidium iodide assay in flow cytometry) was seen in the monocyte population. The lymphocytes showed a lower sensitivity to VB-BO photodynamic action than the monocytes (12%vs 80% of Pi-positive cells). The effect of VB-BO and phototreatment on lymphocyte function was studied using a mitogen-induced proliferation assay. A decrease of mitogen response was observed. The VB-BO and photoirradiation were also used on leukemic cells. The leukemic cells from acute myeloid leukemia and B precursors leukemia were sensitive to VB-BO photodynamic action. The high VB-BO sensitivity of monocytes and leukemic cells (myeloid and lymphoid B derived) suggests possible application of VB-BO for selective depletion of monocytes or sensitive leukemic cells.

The cytokines play an important role in the cascade of the pathological events leading to septic shock. The TNF alpha produced by monocytes/macrophages upon stimulation with bacterial fragments may contribute to induction of this cytokine cascade. Moreover, the antibiotics used for antimicrobial therapy may cause the increase of TNF alpha production due to massive bacterial killing and exposure of monocytes/macrophages to bacterial cell constituents. To investigate the effect of Vancomycin on TNF alpha production, an in vitro model of LPS-stimulated monocytes was used. The level of TNF alpha protein or TNF biological activity were tested in the culture supernatants of monocytes with LPS. Vancomycin down-regulated, in dose-dependent manner, the TNF alpha production. Vancomycin also inhibited TNF alpha-mRNA accumulation in LPS-stimulated monocytes, as assessed by fluorescence in situ hybridization (FISH) in cell suspension. The down-regulation of TNF alpha production in LPS-stimulated monocytes may indicate that inhibition of this cytokine release is one of the important therapeutic effects of Vancomycin in sepsis.

Background V(D)J recombination takes place during lymphocyte development to generate a large repertoire of T- and B-cell receptors. Mutations in recombination-activating gene 1 (RAG1) and RAG2 result in loss or reduction of V(D)J recombination. It is known that different mutations in RAG genes vary in residual recombinase activity and give rise to a broad spectrum of clinical phenotypes. Objective We sought to study the immunologic mechanisms causing the clinical spectrum of RAG deficiency. Methods We included 22 patients with similar RAG1 mutations (c.519delT or c.368_369delAA) resulting in N-terminal truncated RAG1 protein with residual recombination activity but presenting with different clinical phenotypes. We studied precursor B-cell development, immunoglobulin and T-cell receptor repertoire formation, receptor editing, and B- and T-cell numbers. Results Clinically, patients were divided into 3 main categories: T − B − severe combined immunodeficiency, Omenn syndrome, and combined immunodeficiency. All patients showed a block in the precursor B-cell development, low B- and T-cell numbers, normal immunoglobulin gene use, limited B- and T-cell repertoires, and slightly impaired receptor editing. Conclusion This study demonstrates that similar RAG mutations can result in similar immunobiological effects but different clinical phenotypes, indicating that the level of residual recombinase activity is not the only determinant for clinical outcome. We postulate a model in which the type and moment of antigenic pressure affect the clinical phenotypes of these patients.

The control of expression of MHC class II molecules on antigen-presenting cells is important for the induction of immunity, while aberrant expression of these molecules plays a role in the immunopathology of autoimmune diseases. This study explored the role of tumor necrosis factor alpha (TNF) in controlling the level of HLA class II mRNA in human monocytes. Exposure of monocytes to exogenous recombinant TNF (rTNF) selectively up-regulated DR alpha-mRNA but not DP or DQ alpha-mRNA. Inhibitors of TNF synthesis, pentoxifylline (PTX) and thalidomide, inhibited TNF mRNA accumulation in LPS-activated monocytes and down-regulated DR mRNA but not DP or DQ mRNA. The inhibitory effect of anti-TNF monoclonal antibody (MAb) indicated that endogenously generated TNF acted extracellularly. Anti-p75 TNF-R2 receptor and to a lesser extent anti-p55 TNF-R1 MAbs inhibited TNF-mediated up-regulation of DR mRNA and TNF mRNA. Taken together, this implies that endogenously generated TNF plays a role in controlling isotype-specific MHC class II gene expression in human monocytes/macrophages. These results may have some implications for anti-tumor response and autoimmunity.

© 2012 Pituch-Noworolska and Zwonarz, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Celiac and Inflammatory Bowel Diseases in Children with Primary Humoral Immunodeficiency

In the population of children and adolescents, epilepsy affects approximately 1% of cases, nonepileptic seizures are seen in approximately 3%, and endocrine disorders are several times more common. For this reason, coincidence of endocrine disorders and epilepsy and psychoneurologic disorders is frequent. Much less common are structural abnormalities (tumors, developmental abnormalities), and especially non-structural CNS abnormalities, resulting in coincidence of both disorders. There are no reports available in the literature that would address the problem.1) Assessment of the frequency of coincidental epilepsy and endocrine disorders in patients without structural CSN abnormalities treated as outpatients and inpatients of Department of Endocrinology University Children's Hospital of Krakow. 2) Presentation of diagnostic and therapeutic difficulties in these patients, and 3) An attempt at defining the common etiology of both disorders.On the basis of ICD code patients with coincidance of endocrine disorders, epilepsy and psychoneurologic disorders were selected from several thousands of children treated between 2000 and 2009 in Pediatric Endocrinology Department. The neurologic disorders were diagnosed and treated in Chair and Department of Children's and Adolescents Neurology or in another pediatric neurology center.Various forms of epilepsy (symptomatic or idiopathic) and other psychoneurological disorders (disorders of behavior and emotions, obsession-compulsion syndromes, stereotypias, aggression, autoaggression, or hypothalamic obesity) coincident with one or more endocrine disorders, such as growth disorders, disorders of pubertal development, obesity, thyroid diseases, adrenal diseases, hyperprolactinemia, hypoparathyroidism and ion metabolism disorders were diagnosed in 49 patients. The group included: i) children after cranial irradiation and chemotherapy due to medulloblastoma (3 patients), oligodenroglioma (1 patient), ependymoma (1 patient), optic chiasm glioma (2 patients), suprasellar germinal tumor (1 patient), ii) children with Hashimoto encephalopathy (2 patients), iii) children with Prader-Willi syndrome (20 patients), with Klinefelter syndrome (10 patients), with Albright syndrome (9 patients). Of the 49 patients, a group of 6 children representative for individual disorders was selected. In those patients, the etiology of both endocrine disorders, epilepsy and neuropsychiatric disorders was suspected to be common, and the diagnosis was usually delayed.1. Cranial irradiation and chemotherapy, encephalopathy associated with Hashimoto disease and some of the syndromes with the chromosomal and genetic background are the causes of non-structural CNS abnormalities and coincidence of endocrinopathies, epilepsy and psychoneurologic disorders. 2. MR/CT CNS imaging should be performed in any case of central neurological disorders, disorders of behavior, epilepsy or seizures, but also in patients with delayed psycho-motor development, delayed or accelerated growth and pubertal development. All of the above-mentioned manifestations may be symptoms of structural CNS abnormalities and their early treatment determines the child's future. 3. Excluding structural CNS abnormalities allows for forming suspicions associated with diseases resulting in non-structural disorders of the CNS function, predisposing to coincidence of endocrine and neurological disorders. 4. In the diagnosis of Hashimoto's encephalopathy, a decisive factor is exclusion of structural, infectious, traumatic and metabolic causes, intoxications, epilepsy and presence of neuropsychiatric symptoms in patients with high level of against TPO antibodies. In cases of steroids resistance, a good therapeutic effect may be achieved by plasmapheresis, Rituximab therapy and progestagene inhibition of the menstrual cycle.

The primary immunodeficiency, mainly humoral immunity, secondary immunodeficiency and autoimmune diseases are the indications for immunoglobulins substitution. The prolonged substitution in primary immunodeficiency includes regular intravenous infusion of immunoglobulins in 0.4-0.6 g/kg of body weight every 21-28 days. The purpose of such substitution is decrease of frequency and diminishes the clinical course of infections. The high-dose use of immunoglobulins (1-2 g/kg body weight) is preferred in autoimmune diseases based on suppressive and anti-inflammatory activity of immunoglobulins. The subcutaneous administration of immunoglobulins is an alternative to intravenous way, but the singular dose (0.1 g/kg body weight) is too low for suppressive and anti-inflammatory activity of immunoglobulins, so this substitution is indicated in primary immunodeficiency only. The adverse events of immunoglobulins differentiate because of time of occurrence and clinical character. The rapid symptoms occurred just after beginning of infusion and often present the clinical features of anaphylactoid reaction. During the infusion the occurring adverse symptoms are mild and the life-threatening situations are very rare. The next periods of typical adverse reaction are 24-48 hrs after infusion, 72 hrs and later. The mechanisms leading to adverse reaction to immunoglobulins are based on presence of IgG dimmers, stimulating high production of pro-inflammatory cytokines by immunocompetent cells. High level of cytokines is associated with high fever, chills, flu-like symptoms, feeling malaise and sick. The reaction of anti-IgA antibodies present in patient serum with IgA in immunoglobulins preparation is responsible for moderate and severe adverse clinical symptoms. The late adverse events present the symptoms of aseptic meningo-encephalitis. In case of adverse events the stopping of infusion, additions saline/ glucose infusion, anti-histaminic drugs of I and II generation and steroids are used. Severe adverse events are indication for withdraw the immunoglobulins intravenous infusions. In these patients the subcutaneous way of immunoglobulins substitution is an alternative with hope that low singular dose and slow uptake may be better tolerated.

The loss of the CD16a, Fc receptor for IgG type III, (FcgammaRIIIa) B73.1/Leu11c binding epitope, detected by the monoclonal antibody (mAb) used in routine enumeration of NK cells or monocytes, has been observed in children with recurrent viral infections. It has also been linked with the change of leucine (L) to histidine (H) or arginine (R) at amino acid position 48 (FcgammaRIIIa-48L/R/H) in the CD16a receptor. The reactivity of the anti-CD16a clone B73.1/Leu11c mAb with monocytes and NK cells was examined in patients with primary immunodeficiencies (n=167), gastrointestinal malignancies (n=91) and healthy subjects (n=88). Cells of only 12 children, 11 with diagnosed primary immunodeficiency and one with recurrent bacterial infections were not reactive with B73.1/Leu11c mAb. In contrast to previous findings, no linkage between the loss of B73.1/Leu11c binding epitope and herpes virus infections was observed. Furthermore, the sequence analysis of the FcgammaRIIIa gene performed in these 12 patients and 11 healthy subjects revealed that all of them had FcgammaRIIIa-48L/L genotype. Thus, the loss of B73.1/Leu11c binding epitope was not associated with the FcgammaRIIIa-48 polymorphism. The commonly described FcgammaRIIIa-158 polymorphism was determined to be 158V/V in 11 patients and 5 healthy subjects. Moreover, no linkage between FcgammaRIIIa-48L/L and -158F/F genotypes was observed. It is suggested that the loss of the B73.1/Leu11c binding epitope is connected with primary immunodeficiency disorders, but not associated with the FcgammaRIIIa-48 polymorphism.

The loss of the CD16a, Fc receptor for IgG type III, (FcgammaRIIIa) B73.1/Leu11c binding epitope, detected by the monoclonal antibody (mAb) used in routine enumeration of NK cells or monocytes, has been observed in children with recurrent viral infections. It has also been linked with the change of leucine (L) to histidine (H) or arginine (R) at amino acid position 48 (FcgammaRIIIa-48L/R/H) in the CD16a receptor. The reactivity of the anti-CD16a clone B73.1/Leu11c mAb with monocytes and NK cells was examined in patients with primary immunodeficiencies (n=167), gastrointestinal malignancies (n=91) and healthy subjects (n=88). Cells of only 12 children, 11 with diagnosed primary immunodeficiency and one with recurrent bacterial infections were not reactive with B73.1/Leu11c mAb. In contrast to previous findings, no linkage between the loss of B73.1/Leu11c binding epitope and herpes virus infections was observed. Furthermore, the sequence analysis of the FcgammaRIIIa gene performed in these 12 patients and 11 healthy subjects revealed that all of them had FcgammaRIIIa-48L/L genotype. Thus, the loss of B73.1/Leu11c binding epitope was not associated with the FcgammaRIIIa-48 polymorphism. The commonly described FcgammaRIIIa-158 polymorphism was determined to be 158V/V in 11 patients and 5 healthy subjects. Moreover, no linkage between FcgammaRIIIa-48L/L and -158F/F genotypes was observed. It is suggested that the loss of the B73.1/Leu11c binding epitope is connected with primary immunodeficiency disorders, but not associated with the FcgammaRIIIa-48 polymorphism.

The basic mechanisms of specific immune response in children to pathogens with characteristics of cells involved in the process were described. The characteristics of commonly used stimulators (thymus-derived peptides, bacterial lysates and ribosoms, glukans) and the mechanisms of action, target cells were shown. In general remarks the limitations of immunostimulation in children with recurrent infections with or without known immunodeficiency were stressed.

The activity of multidrug resistance (MDR) proteins in tumour cells is associated with an increased resistance to therapy and in consequence with a decreased effectiveness of chemotherapy. The majority of MDR molecules belong to a family of ABC (ATP binding cassette) transporters. Neuroblastoma (NBL) and rhabdomyosarcoma (RMS) are common solid tumours of childhood. The response to therapy is better in NBL, worse in RMS, but still unsatisfactory despite surgery and aggressive chemotherapy. The immunohistochemical staining for p-gp (p-glycoprotein), MRP1 (multidrug resistance associated protein 1), BCRP (breast cancer resistance protein) and LRP (lung resistance protein) expression was performed in primary tumour sections of NBL (10 cases) and RMS (10 cases). A different pattern of MDR expression in NBL and RMS were noted. In NBL, MRP1 was expressed in all studied tumours, p-gp, BCRP only in 3 out of 10 tumours, LRP, in 4 cases. The combination of more than one protein was noted in the majority of NBL tumours. In RMS, the expression of 3 or 4 MDR proteins was noted in 9 cases. The high expression of an MDR protein profile in RMS suggests various mechanisms acting simultaneously, which might explain chemotherapy resistance and a low percentage of long-time survival in this tumour.

Introduction: Warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome is a rare immunodeficiency disorder with an autosomal-dominant pattern of inheritance and low fatality rate but significant lifelong morbidity. Materials and Methods: A 27-year-old mother of two children has been suffering from severe neutropenia and recurrent infections with the diagnosis of sporadic WHIM syndrome established by sequencing the CXCR4 gene and the finding of a heterozygous 1000 C→T nonsense mutation in the second CXCR4 exon. The first child was an apparently healthy boy delivered at full term. Umbilical cord blood cells were obtained for genetic analysis. Peripheral blood cells were also analyzed at 8 months of life. Both analyses revealed the same mutation as that of his mother. The child was in a good condition, manifesting neutropenia without infections until 11 months of life. He subsequently developed pneumonia requiring a more aggressive treatment. After that, the regular substitution of immunoglobulins (IVIGs) and G-CSF has been preventing serious infections. Six months ago the second boy was delivered who also demonstrated neutropenia without severe infections. Genetic studies using cord blood and also peripheral blood cells in the fourth month showed an identical mutation of the CXCR4 gene as in his mother. Moreover, the mother and her first son demonstrated monocytopenia. Results: The results indicate that genetic defects connected with WHIM syndrome may influence not only the granulocyte, but also the monocytic lineage. Moreover, a perinatal diagnosis of WHIM syndrome made by sequencing the CXCR4 gene should be performed in cases where either parent is known to be affected with this disease. Conclusions: This would facilitate an earlier detection of the deficiency in children, thereby allowing a more comprehensive follow-up and administration of appropriate therapy.

Hepatitis B is a serious epidemiological problem in uremic patients treated with renal replacement therapy. A high proportion of hemodialyzed patients do not respond to the standard method of intramuscular (i.m.) hepatitis B vaccination. Low-dose intradermal (i.d.) inoculations and supplementary i.m. injections have been reported to improve the responsiveness in formerly non responding uremic patients. We applied a inoculation schedule of 10 microg Engerix B i.d. in 49 pts and i.m. (control group) in 13 pts once a week during 12 consecutive weeks in order to compare the effectiveness of the various ways of immunization in maintenance dialyzed patients not responding to standard vaccination. Serum anti-HBs antibody level, as well as biochemical and immunological parameters were examined. Already one month after initiation of the cycle, 57.1% of patients in the i.d. group responded by achieving the minimum protective anti-HBs antibody level (10 IU/I.); while 14.3% reached full adequate anti-HBs antibody level (100 IU/I.). After the full therapy period, anti-HBs antibody level100 IU/I. was achieved in 42.9% of the patients, while a total of 81.7% of patients reached the anti-HBs antibody level10 IU/I. In 18.4% of patients no response was observed. Surprisingly similar results were achieved in the i.m. group. Twelve months after termination of the inoculation cycle we noted decrease of anti-HBs antibody level; the values100 IU/ I. was observed only in 18.4% of the study group, while 87.8% reached a titre10 IU/I. We found a relationship between the effectiveness of immunization and RBC count, total serum protein and albumin levels and GGTP activity. Mitogen stimulation indexes in both groups were 4-5 times lower in comparison to reference values in the general population. In the study group that did not respond to vaccination, mitogen stimulation indexes were 2 times lower as compared to the group characterized as having a good response. In conclusion, the route of injection seems to be less important than the frequency and number of doses of the vaccine. Anemia and malnutrition may be responsible for the worse response to vaccination against hepatitis B virus.

Immunophenotype of isolated (disseminated or circulating) tumour cells (ITC) in the blood, bone marrow and lymph nodes were studied in patients with gastric cancer. Coexpression of metalloproteinases inducer (EMMPRIN), chemokine receptors (CCR6, CXCR4) and adhesion molecules (Ep-CAM, CD44) was determined on cytokeratin positive (CK+) cells in CD45- cell population sorted out from the blood and/or bone marrow. Eight cytospin samples of blood and 69 samples of bone marrow containing CK+ cells from patients with gastric cancer were included into study. Expression of EMMPRIN and CCR6 were noted in a half of CK+ samples (of blood/bone marrow) whereas the expression of CXCR4 and Ep-CAM was much lower. Analysis of paired data of these determinants expression on CK+ cells showed no association between them. Expression of EMMPRIN, Ep-CAM, CCR6, CCR7, CXCR1, and CXCR4 on ITC in lymph nodes was determined by flow cytometry. In 18 lymph nodes (out of 36 assayed) CK+ cells were found. The expression of CCR6 and Ep-CAM on CK+ cells was observed in almost all studied lymph nodes, CXCR1--in half of them. The expression of EMMPRIN and CCR7 cells was lower. These results suggest that ITC of gastric cancer express variably several molecules that may be involved in metastasis formation.

The authors evaluated the prevalence of TPO Ab and thyroid disorders in 219 children and adolescents (119/54% girls) with type 1 diabetes from southeast Poland aged 3.2-22.3 years (mean age-13.7 +/- 3.9 years). Their age upon diagnosis ranged from 1.6 to 17.2 years (mean age--8.1 +/- 3.6 years), while diabetes duration was between 1 and 18.7 years (mean, 6.4 +/- 3.7 years). In addition to clinical assessment of all patients, determinations were made of serum TPO Ab, FT4 and TSH; thyroid ultrasound was performed in each patient with abnormal thyroid morphology and/or positive TPO Ab titer. Positive TPO Ab titer was demonstrated in 76 (34.7%) patients with type 1 diabetes; in this group 49 showed no other overt thyroid pathological symptoms. Hashimoto's disease was detected in 26 children, Graves's disease in 1 girl. Twenty children (9.1%) with negative TPO Ab titter were shown to have euthyrotic goiter. Thus, thyroid abnormalities were demonstrated in 43.8% of the patients and were seen twice as often in girls than in boys (+ n = 69n = 27). Thyroid dysfunction was detected in 11 (5.05%) patients. These 11 patients with thyroid dysfunction constituted 14.5% of the entire group of children with both type 1 diabetes and positive TPO Ab titer (n = 76). Ten patients were hypothyroid (including 8 with previously undiagnosed disease) and 1 girls had hyperthyroidism. The present results indicate that in each child with type 1 diabetes--apart from diabetes control--thyreologic assessment should be done, and the frequency and type of examinations should depend on the comprehensive preliminary evaluation.

The immunophenotyping of leukaemia and non-Hodgkin's lymphoma cells is based on staining the cells with monoclonal antibodies against surface and cytoplasmic determinants followed with flow cytometry analysis. The problems of immuno-phenotyping are associated with technical difficulties, changes in expression of determinants and the rare types of leukaemia and haematological disorders typical for newborns and infants. The lack of blast cells within cell suspension obtained for test may be the result of bone marrow disorder (aplastic anaemia, preleukaemic cytopenia) or technical pitfall. The changed expression of determinants on blastic cells observed as weak expression or overexpression or atypical combination of determinants requires a careful interpretation. In the diagnosis of rare types of acute leukaemia (e.g. erythroleukaemia, megakaryoblastic leukaemia, mixed lineage or undifferentiated leukaemia) the additional monoclonal antibodies beyond routine set are needed. A special concern is necessary in diagnosis of newborns and infants leukaemia or bone marrow disorders like myelodisplastic syndrome particularly in children with other systemic diseases e.g. congenital immunological deficiencies, Down's syndrome. The problems of immunophenotyping in non-Hodgkin's lymphoma are frequently associated with obtaining a representative material e.g. surgical tumour biopsy, lymph node. In some case the differential diagnosis including small round cell tumours and anaplastic type of lymphoma is necessary what requires an additional set of monoclonal antibodies. Despite of modern technology, morphology, immunophenotyping and histopathology remain the standard of complex diagnosis of lymphoproliferative diseases and haematopoietic disorders in children.

In the present study 13 lectins of plant, fungal and animal origin, characterised by different glycan-binding specificities were used to examine the structure and distribution of specific glycans in the tissues of human placenta. Histochemical analysis was focused on villi (villous syncytiotrophoblast and stroma), cytotrophoblast of the basal plate and amniotic epithelium. It was found that glycoconjugates containing mannose and N-acetyl glucosamine were widely expressed while external fucosyl residues were absent on all studied structures of placenta. Lectins GNA (revealing non-reducing terminal mannosyl residues) and LPA (revealing sialic acid) bound selectively to the villus structures. The presence of sialic acid residues was observed in the superficial plasmalemma of syncytiotrophoblast and on the surface of the foetal capillary endothelium. Lectins LCA and PSA showed specific affinity to the plasmalemma of the cytotrophoblast and to the amniotic epithelium basement membrane. N-acetyl lactosamine-specific fungal lectin PSL selectively labelled amniotic epithelial and cytotrophoblast cell membranes and superficial plasmalemma of the syncytiotrophoblast.

Proliferating Reed-Sternberg and Hodgkin’s cells within the lymph node in Hodgkin’s disease (HD) affect the surrounding cells, which results in alterations of the proportion between T and B lymphocytes and the activation of these cells. The purpose of this study was to assay the changes within lymphocyte populations in lymph nodes in different types of HD. The lymph nodes of 10 children with nonmalignant, reactive changes, and 31 children with HD of the following types: mixed cellularity (MC; 11 cases), nodular sclerosis (NS; 17 cases) and lymphocyte depletion (LD; 3 cases) were included in the study. The percentage of T lymphocytes (CD3+), B lymphocytes (CD22+), T cell (CD4+ and CD8+) and B cell subsets (kappa+ and lambda+ light chain) were assayed with flow cytometry. The ratio of T:B lymphocytes in reactive lymph nodes and in NS type of HD was similar. An increase of the T cell percentage (resembling an increase of the CD4+ subset) and a decrease of B cells resulted in a high ratio of T:B and was noted in the MC and LD types of HD. These HD types were associated with marked changes in lymphocytes within the lymph node, which suggested a different effect of proliferating Reed-Sternberg and Hodgkin cells in HD on the surrounding cells.

The trans-differentiation hypothesis of adult tissue-specific stem cells has been recently questioned because of insufficient proof that the so-called plasticity experiments were performed on pure populations of tissue-specific stem cells. It was shown recently, for example, that the formation of haematopoietic colonies by muscle cells depended on the presence of haematopoietic stem/progenitor cells residing within the muscle tissue and hence was not related to the plasticity of the muscle stem cells. The explanation for the presence in, or homing into, muscles of haematopoietic stem cells is, however, not clear. In our study, we hypothesised that muscle tissues secrete stromal-derived factor (SDF)- 1, an alpha-chemokine for haematopoietic stem cells (HSC), which could attract HSC circulating in peripheral blood into muscle tissue. We found, using RT-PCR and immunocytochemistry, that SDF-1 was expressed in human heart and skeletal muscles. Moreover, muscle satellite cells, which are pivotal for regeneration of muscle, highly expressed on their surface CXCR4, a G-protein-coupled receptor that binds SDF-1. To determine whether the CXCR4 receptor is functional on muscle satellite/progenitor cells, we stimulated murine satellite cells (the C2C12 cell line) with SDF-1 and demonstrated the phosphorylation of p42/44 MAPK and AKT serine-threonine kinase in these cells. Moreover, we showed that SDF-1 gradient chemoattracts these cells. We postulate that the CXCR4-positive muscle satellite and CXCR4-positive HSC circulating in the peripheral blood compete for occupancy of SDF-1-positive stem cell niches that are present in bone marrow and muscle tissues. Thus, we suggest that competition for common niches by various circulating CXCR4-positive stem cells and their ability to home to the SDF-1-positive niches in various organs, is a better explanation than stem cell plasticity of why (i) haematopoietic colonies can be cultured from muscles and (ii) early muscle progenitors could be cultured from bone marrow.

We report here a mother and her two daughters suffering from systemic lupus erythematosus, accompanied by the presence of anti-Ro (SSA-52kD and 60kD) and anti-La (SSB) antibodies, and peripheral lymphadenopathy as the first manifestation of the disease. The expression of DR3 and B8 in all three affected family members suggests familiar predisposition to SLE. The differential diagnosis and clinical course are discussed.

The objective of the authors was to evaluate the prevalence of TPO Ab and thyroid diseases in children with newly diagnosed type 1 diabetes. The examination included 153 patients (85/55.6% girls) from southeast Poland aged 11 months to do 17.4 years (mean age 9.5 +/- 3.9 years). Apart from clinical assessment, all children had determinations made of serum TPO Ab, FT4 and TSH, while thyroid ultra sound was performed in each patients with abnormal thyroid morphology and/or positive TPO Ab titter. Positive TPO Ab was detected in 45 patients (29.4%). In this group 26 had isolated serum TPO Ab elevation, 18 had Hashimoto's disease, 1 Graves's disease. Another 12 children (7.8%) were demonstrated to have euthyroid goiter. Thyroid abnormalities were thus seen in 37.2% children with newly diagnosed type 1 diabetes. No association was demonstrated between the prevalence of thyroid abnormalities and sex. Children with subclinical stage of autoimmune thyreoiditis were significantly younger in comparison to patients with Hashimoto's disease (8.9 +/- 4.2 vs. 12.0 +/- 3.1 years) and had significantly lower serum TPO Ab and TSH levels (314.2 +/- 232.4 vs. 2076.8 +/- 1300.8 U/ml, 1.7 +/- 0.82 vs. 4.1 +/- 2.9 ulU/ml, respectively). Thyroid dysfunction was detected in 7 (4.6%) children with newly diagnosed type 1 diabetes. In comparison to the entire group with positive serum TPO Ab titer in these 7 children the percentage of patients with thyroid dysfunction was significantly higher (15.5%). Six patients were hypothyroid and 1 had hyperthyreosis. The present results justify the need for comprehensive screening for thyroid disorders in all children with newly diagnosed type 1 diabetes.

The atypical immunophenotype (expression of determinant from the another cell lines than line of origin) of acute leukaemia blast cells are noted in a part of cases. The characteristics and classification of atypical immunophenotypes are not unified and the clinical significance is not yet fully described. The purpose of the study was: precise description of atypical immunophenotypes and analysis of their frequency in different types of acute leukaemia, analysis of association between expression of atypical immunophenotypes and the level of initial leukocytosis, percentage of blast cells in peripheral blood, expression of CD34, analysis of frequency of multidrug resistance molecule (MDR) expression and association between MDR and immunophenotypes of leukaemia cells, analysis of association between atypical immunophenotypes and proliferation, secretion of cytokines (IL-6, TNF) and spontaneous apoptosis of leukaemia cells, analysis of association between atypical immunophenotypes and sensitivity to induction therapy. The bone marrow samples used for routine diagnosis were the basic source of leukaemia cells for the study. The morphological examination and the immunophenotypes of leukaemia cells were done for classification of leukaemia. The immunophenotype and the expression of MDR determination was performed with flow cytometry after staining the cells with monoclonal antibodies (directly labelled) for CD determinants and MDR. The spontaneous proliferation of leukaemia cells was studied with 3H-Thymidine uptake after 3-days culture in vitro. The type of proliferation (autocrine, paracrine) was defined based on comparison of shorter (3-days) and longer (6-days) culture of leukaemia cells. The percentage of apoptotic leukaemia cells was analysed with flow cytometry after staining of leukaemia cells with propidium iodide in subdiploidal region of DNA profile. The secretion of cytokines (IL-6 and TNF) was determined by ELISA technique in supernatants of leukaemia cells cultured for 24 hr in vitro. The biological activity of TNF was determined in the bioassay using L929 mouse cells line. The effect of induction therapy was estimated base on time of cytoreduction in peripheral blood and time of reaching the haematological remission in bone marrow. The study included 230 children with acute leukaemia: lymphoblastic (ALL)--189 children (ALL-proB--19, common ALL--139, ALL-B--5 and ALL-T--26) and myeloid (AML)--34 children. Moreover, into the study 2 cases of acute undifferentiated leukaemia (AUL) and 3--acute mixed lineage leukaemia (AMLL) and 2--biphenotypic leukaemia were included. The all studies of leukaemia cells had been done before the therapy was installed. Basing on the assay of immunophenotypes the following forms of atypical immunophenotypes were distinguished: immunophenotype incomplete, hyperexpression of determinants, asynchronic immunophenotype, coexpression of determinants from the other line than origin of leukaemia cells, balanced expression of determinants from two cells lines (biphenotypic leukaemia) and three cells lines (mixed lineage leukaemia). The atypical immunophenotypes were observed in: 21.1% ALL-proB cases, 34.5% common ALL cases, 42.3% ALL-T and 58.8% AML. The most common form of atypical immunophenotypes was coexpression of determinants from the other cell line. There were no associations between atypical immunophenotypes and the level of initial leukocytosis and percentage of blast cells in peripheral blood. The expression of CD34, recognised as the one of markers of poorer prognosis, was analysed regarding the leukaemia type and immunophenotype of leukaemia cells. The lowest frequency of CD34 expression was noted in ALL-T (28.5%), the highest one in common ALL (62.3%). The significant association between frequency of CD34 and atypical immunophenotypes was observed in AML and ALL-T. Moreover, in common ALL the expression of CD34 was significantly higher when myeloid determinants were present on common ALL cells (common ALL + My) in comparison to coexpression of lymphoid determinants (common ALL + Ly). The frequency of MDR expression (cases with more than 10% of MDR positive cells) was in range between 16.6% in ALL-proB and 78.9% in AML. The mean percentage of cells expressing MDR was low in ALL-proB (10.7%) and high in ALL-T (39.6%). In ALL the atypical immunophenotype was associated with expression of MDR whereas in AML this association did not appear. The common ALL + My leukaemia cells showed higher ability to proliferation in vitro compare with common ALL without atypical immunophenotype. The opposite results were observed in AML. AML leukaemia cells with coexpression of lymphoid determinants (AML + Ly) showed lower proliferation in vitro than AML without atypical immunophenotype. The autocrine type of proliferation was observed frequently in AML (35.3% of cases) than in ALL (14.2%). This type of spontaneous proliferation was observed only when the leukaemia cells without changes in immunophenotype had been cultured. The low level in common ALL and high in AML of spontaneous release of IL-6 and TNF were noted. AML leukaemia cells without changes in immunophenotype released significantly higher amount of these cytokines than AML cells with atypical immunophenotypes (AML + Ly). The above observations suggested that coexpression of myeloid determinants in ALL and lymphoid determinants in AML were leading to changes of some biological properties of these cells. The ALL + My leukaemia cells behaved similarly to myeloid leukaemia cells, while AML + Ly cells showed features of lymphoid leukaemia cells. The common ALL and AML leukaemia cells with atypical immunophenotype showed higher percentage of apoptotic cells (16.1% and 16.9% respectively) comparing to common ALL and AML without changes in immunophenotype (9.0% and 9.2% respectively). The weak negative association of MDR expression and apoptosis suggested the indirect inhibiting influence of MDR on ability of cells to undergo into the apoptosis process. In common ALL and AML with typical immunophenotype of leukaemia cells and ALL-T the level of apoptosis was associated positively with the spontaneous proliferation, whereas this relation was negative in AML with atypical immunophenotype. There were no differences of the time of cytoreduction of leukaemia cells in peripheral blood in B cell origin ALL and AML with or without changes in immunophenotype of blastic cells. In ALL-T + My the time of cytoreduction was significantly longer. However, the expression of CD10 in ALL-T had no effect on cytoreduction time. The expression of MDR in ALL-T with typical immunophenotype was independent marker associated with elongation of cytoreduction time. The time of reaching the complete haematological remission was analysed in 186 children with ALL (ALL-proB--18 children, common ALL--137 children, ALL-T--26) and only 19 children with AML. The longest period of time for reaching the remission was observed in AML, shortest--in ALL-T. In common ALL and ALL-T the expression of myeloid determinants was associated with significant elongation of time of reaching the remission. In the majority of AML cases with coexpression lymphoid determinants, the complete remission was reached. The time needed for the reaching of remission was similar in AML with or without coexpression of lymphoid determinants. The results of this study suggest that coexpression of determinants from the other cell line modify the biological properties of leukaemia cells into the cells from the line of origin of these additional determinants. In ALL the combined expression of MDR and atypical immunophenotype of leukaemia cells were associated with poorer response to induction therapy. In AML the combined expression of CD34 and atypical immunophenotype were associated with response to induction therapy by reaching the complete remission, but without any influence on the time of reaching this remission. The results of analysis of cytoreduction time and time of reaching the remission improved the usefulness of these parameters for the estimation of response to the induction therapy. The clinical importance of these observations consist in characterisation of leukaemia cells potentially resistant to the induction therapy what may suggest the modification and individualization of the induction therapy.

Changes in the expression of integrins and cadherins might contribute to the progression, invasion and metastasis of transitional cell cancer of the bladder and of melanomas. The expression of alpha5 (P0.001), alpha2 and beta1 (P0.05 - P0.001) integrin subunits in melanoma cells from noncutaneous metastatic sites (WM9, A375) were significantly increased as compared to cutaneous primary tumor (WM35) and metastatic (WM239) cell lines. These differences might be ascribed to the invasive character of melanoma cells and their metastasis to the noncutaneous locations. The significantly heterogeneous expression of beta1 integrin subunit in two malignant bladder cancer cell lines (T24 and Hu456) and nonsignificant differences in the expression of alpha2, alpha3, and alpha5 subunits between malignant and non-malignant human bladder cell lines do not allow an unanimous conclusion on the role of these intergrin subunits in the progression of transitional cancer of bladder. The adhesion molecule, expressed in all studied melanoma and bladder cell lines, that reacted with anti-Pan cadherin monoclonal antibodies was identified as N-cadherin except in the HCV29 non-malignant ureter cell line. However, neither this nor any other bladder or melanoma cell line expressed E-cadherin. The obtained results imply that the replacement of E-cadherin by N-cadherin accompanied by a simultaneous increase in expression of alpha2, alpha3 and alpha5 integrin subunits clearly indicates an increase of invasiveness of melanoma and, to a lesser extent, of transitional cell cancer of bladder. High expression of N-cadherin and alpha5 integrin subunit seems to be associated with the most invasive melanoma phenotype.

Cancers characterized by microsatellite instability may be biologically different from their counterparts with stable microsatellite sequences. Circulating cancers cell present in blood prior to surgery may constitute an adverse prognostic finding.To correlate these two phenomena with morphological features and survival in advanced gastric cancer.We examined 76 cases of resected sporadic, advanced gastric cancer by means of routine morphology and a panel of microsatellite markers. Sixty-six cases were screened for presence of cancer cells circulating in blood prior to the surgery using combined morphological and immunocytochemical approach.Twenty-one (27.6%) cases demonstrated microsatellite instability in at least one locus. Among them 11 (14.5%) showed microsatellite instability in more than 30% (4/12) examined loci, and they were therefore designated as replication error positive (RER+). Circulating cancer cells were detected in 2/19 microsatellite instability and in 11/47 remaining cases (difference not significant). The survival of the microsatellite instability cases was significantly better. The presence of circulating cancer cells did not correlate with survival.It is possible that the microsatellite instability status, but not circulating cancer cells, constitutes a prognostic predictive factor in advanced gastric carcinoma. Confirmation of this hypothesis requires continuation of patient follow-up.

CD20 determinant expressed on B precursors is associated with regulation of proliferation, apoptosis and maturation of these cells. The acute lymphoblastic leukemia "common" type (cALL) based on expression of CD20 is subdivided in type I and II. However, the clinical significance of CD20 expression on cALL and significance of cALL type I and II discernment are not fully elucidated. The association of CD20 expression with the expression of multidrug resistance molecule (MDR), CD34, atypical immunophenotypes of leukemia cells and response to induction therapy were determined in the group of 147 patients with acute lymphoblastic leukemia (ALL) B progenitor type (ALL-proB -14 patients) and common type (cALL-133 patients). The expression of CD20 on leukemia cells was studied routinely at diagnosis before the therapy. This expression was noted on leukemia cells of 6 ALL-proB patients (42.8%) and 66 cALL patients (49.6%). The expression of CD20 showed no association with the expression of CD34, CD22 and MDR. The reverse association was observed between CD20 expression and the presence of co-expression of myeloid (CD13, CD33, CD65, CD15) and T lymphoid determinants (CD2, CD5, CD7) on leukemia cells. The effect of induction therapy analyzed as time of blast cells cytoreduction in peripheral blood and time of reaching the complete remission showed the slower clearance of peripheral blood from blast cells associated with expression of CD20. There was no association of CD20 expression with the time of reaching the hematological remission. The above results suggested a "protective" role of CD20 against co-expression of other determinants (myeloid and lymphoid) and no association with the results of induction therapy.

The aim of the study was an analysis of renal transplantation results in the Krakow Transplant Center during 1992-2000. The analysis concerned 94 cadaveric transplant recipients. The study group included 31 females aged 23 to 61 years (mean 40.4 years) and 63 males aged 16 to 60 years (mean 41.8 years). The time of pre-transplant renal replacement therapy ranged from 4 to 120 months (mean 32 months). The mean time of total ischaemia was 22 hours 20 minutes. The majority of the recipients had three identical antigens out of six typed. Most of the recipients were treated with three immunosuppressive drugs including: Cyclosporine A, Azathioprine and steroids. Immediately after kidney transplantation 25.6% of the patients had urine output and did not require dialysis. Acute renal failure (ARF) of the graft was observed in 73.2% recipients. The average number of hemodialysis sessions in patients presenting ARF was 10. Acute rejection was diagnosed in 41.5% of the patients. The most frequent complications were: CMV (cytomegalovirus) infection, UTI (urinary tract infection) and policytemia. In the study group 1-year survival rate of the patients was 97.8% and 1-year graft survival was 93.61%. The 5-year survival rates both in the patients and the grafts were very satisfactory (96.96% and 87.7% respectively).

The classification of acute leukemias based on the phenotype of leukemic cells was shown. The technical problems and difficulties in interpretation were discussed including atypical phenotypes, biphenotypic and mixed lineage forms of leukemias.

Tumor progression is associated with the clonal expansion of surviving cell variants. These results in cancer cell heterogeneity and selection of cells with a high malignant potential reflected also by the ability to metastasize. In seeding and implantation of cancer cells at the distant site cell adhesion molecules play a crucial role. Of particular interest is CD44 adhesion molecule, which possibly is involved in tumor metastasis development. Forty cases of an advanced gastric cancer were studied. Paraffin block were collected from the files. In addition to routine tumor typing, grading (Lauren and Goseki classifications) and staging, CD44 (standard, v5 and v6) was studied by immunohistochemistry and RT-PCR. CD44 immunoreactivity was found in 36 of the 40 studied cases. A significant overexpression of CD44v5 was noticed in gastric cancer. This was especially seen in Goseki's grades I and III (72.7% of cases) and was less common in Goseki's grades II and IV (44.4% of cases). CD44v6 was less commonly expressed. In some cases CD44 heterogeneity of neoplastic intravascular emboli was noticed and in some other cases stronger expression of CD44 was present in deeper parts of cancer infiltrate. Immunohistochemical expression was mostly in concert with CD44 gene expression as shown by RT-PCR results. Some discrepancies are discussed. These findings are interesting in view of better prognosis and different route of dissemination of Goseki's grades I + III compared with Goseki's grades II + IV of the gastric cancer. We have shown an overexpression of CD44v5 in an advanced gastric cancer, especially in Goseki's grades I and III. This could reflect a different malignant potential and a different route of dissemination of gastric well differentiated adenocarcinomas.

The occurrence of circulating tumour cells in the blood of 51 patients with gastric cancer (stages I-IV) was studied using flow cytometry, cell sorting, immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR). The lysed whole blood samples were stained with monoclonal antibodies against common leukocyte antigen (CD45), epithelial membrane antigen (EMA), tumour associated glycoprotein 72kD (TAG72), CD44 variants (v5 and v6) and analysed by flow cytometry within ungated or CD45-gated populations. The frequency of detection of TAG72+, CD44v5+ and v6+ cells within CD45- gate was considerably increased in comparison to the ungated population. Furthermore, the presence of tumour cells was directly demonstrated by immunostaining for cytokeratin 18 of sorted CD45- population. The presence of CD44v5+, v6- cells and CD44v-mRNA in the blood was compared to their expression in the primary tumour. The occurrence of circulating CD44v+ cells was associated with their presence in the primary tumour and CD44v-mRNA in the blood. The method described may provide a sensitive tumour marker-independent tool for detection of circulating tumour cells in cancer patients.

The tumour necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6) mRNA accumulation and the release of the cytokines TNF-alpha and IL-6 was determined in leukemic cells isolated from bone marrow biopsy from patients with acute lymphoblastic leukemia: ALL-common type (cALL), 11 patients; ALL-T type, nine patients. The non-leukemic bone marrow cells (BMMC) and peripheral blood mononuclear cells (PBMC) from healthy donors were used as a control. The mRNA was assessed by fluorescent in situ hybridization in cell suspension and analyzed with flow cytometry. The accumulation of cytokine mRNA was higher in cALL cells as compared to ALL-T and PBMC (control) and was comparable to cytokines mRNA accumulation in BMMC. The production of IL-6 by leukemic cells from both types of leukemia was significantly lower than in BMMC. The bioactive TNF was not detected in either of the leukemia groups studied. TNF-alpha protein was produced by ALL-T cells and BMMC but not by cALL type of leukemic cells. The synthesis of IL-6 was significantly enhanced by TNF-alpha in BMMC and ALL-T while the presence of TNF-alpha had no effect on IL-6 synthesis in the culture of cALL leukemic cells. It was concluded that despite IL-6 and TNF-alpha mRNA contents, leukemic cells representing early stage of B-cell development (CD10+) showed disregulation of production of these cytokines.

The parasitic infections (Toxocara can is, Trichinella spiralis) are characterized by general and local symptoms including fever, muscle pain and swelling. The question was asked whether the muscle changes that occur due to larva migrans give rise to the autoimmune response. The presence of antistriational antibodies (aStrAbs) was determined in the following groups of patients: group 1--66 patients with toxocariasis and the presence of anti-Toxocara antibodies, group 2--22 patients suspected of Toxocara canis infection without anti-Toxocara antibodies, group 3--20 patients with active trichinellosis confirmed by anti-Trichinella antibodies. As control 25 healthy persons (group 4) were studied. The aStrAb were tested by the indirect fluorescence using unfixed cryostat sections of human striated muscle. The following results were obtained: group 1--42 sera positive, group 2--5 sera were weakly positive, group 3--all sera showed the presence of aStrAbs, group 4--no aStrAbs. The presence of aStrAbs in patients with parasitic infections may suggest the occurrence of anti-muscle autoimmune response.