A comprehensive assessment of lymphocyte subsets, their prognostic significance, and changes after first-line therapy administration in patients with chronic lymphocytic leukemia.

BACKGROUND: In chronic lymphocytic leukemia (CLL), changes in the peripheral blood lymphocyte subsets play an important role in disease progression and infectious complications. The impact of chemoimmunotherapy (CIT) on these changes has not been extensively studied METHODS: We used multi-color flow cytometry, to prospectively measure absolute and relative numbers of CD4+ and CD8+ T-cells and their subsets in 45 patients with indolent untreated CLL, 86 patients indicated for first-line treatment, and 34 healthy controls. In 55 patients, we analyzed the impact of CIT RESULTS: CLL patients had a significant increase in most cell populations in comparison to controls. Progression of CLL was characterized by significantly elevated counts with the exception of a lower percentage of naïve T-cells. After treatment, the percentage of naïve T-cells further decreased at the expense of effector memory T-cells (TEM). In patients with indolent CLL, higher percentages of naïve CD4+ (p = 0.0026) and naïve CD8+ (p = 0.023) T-cells were associated with a longer time to first treatment (TTFT). The elevation of CD4+ central memory T-cells (TCM) (p = 0.27) and TEM (p = 0.003) counts and a higher percentage of CD4+ TEM (p = 0.0047), were linked with shorter TTFT. In treated patients, increased regulatory T-cells count was associated with shorter time to next treatment (TTNT) (p = 0.042), while higher CD4+ TCM count with shorter TTNT (p = 0.035) and shorter overall survival (p = 0.041). CONCLUSION: Our results indicate that naïve cell depletion and CD4+ TCM and TEM increases are detrimental to CLL patients' prognosis.

CD4+/CD45RO+: A Potential Biomarker of the Clinical Response to Glatiramer Acetate.

Background: Glatiramer acetate (GA) is an effective treatment for the earliest stages of multiple sclerosis (MS)-clinically isolated syndrome (CIS) or clinically definite MS (CDMS). Objective: This study aims to determine the differences in the lymphocyte population (at baseline and the course of five years) between confirmed sustained progression (CSP) and non-CSP groups and to identify potential biomarkers among these parameters that can predict a positive response to the treatment. Methods: Twelve male and 60 female patients were included in the study. Peripheral blood samples were collected before and five years after treatment with GA. The authors compared lymphocyte parameters between the CSP and non-CSP groups by statistical analyses. Univariate and penalized logistic regression models were fitted to identify the best lymphocyte parameters at baseline and their combination for potential biomarkers. Subsequently, the ROC analysis was used to identify cut-offs for selected parameters. Results: The parameter CD4+/CD45RO+ was identified as the best single potential biomarker, demonstrating the ability to identify patients with CSP. Moreover, a combination of four lymphocyte parameters at baseline, relative lymphocyte counts, CD3+/CD69+, CD4+/CD45RO+, and CD4+/CD45RA+ab, was identified as a potential composite biomarker. This combination explains 23% of the variability in CSP, which is better than the best univariate parameter when compared to CD4+/CD45RO+ at baseline. Conclusions: The results suggest that other biomarkers can help monitor the conditions of patients and predict a favourable outcome.

Lymphocyte populations and their change during five-year glatiramer acetate treatment.

BACKGROUND: The goal of this study was to determine the characteristics that are affected in patients treated with glatiramer acetate (GA). METHODS: A total of 113 patients were included in this study. Patients were treated with glatiramer acetate (subcutaneous injection, 20 mg, each day). Peripheral blood samples were obtained just prior to treatment as well as 5 years after GA treatment. All the calculations were performed with the statistical system R (r-project.org). RESULTS: After 5 years of treatment, a significant decrease was found in the absolute and relative CD3+/CD69+ counts, the absolute and relative CD69 counts, the relative CD8+/CD38+ count and the relative CD38 count. A significant increase was found in the absolute and relative CD5+/CD45RA+ counts and the absolute CD5+/CD45RO+ count after 5 years of treatment. CONCLUSION: This study presents some parameters that were affected by long-term GA treatment.

Azorella compacta Infusion Activates Human Immune Cells and Scavenges Free Radicals In vitro.

BACKGROUND: Azorella compacta is traditionally used in the form of tea (infusion), in the Andean region of South America, to treat various chronic diseases. However, the health-promoting properties of this herbal tea have not yet been extensively explored. MATERIALS AND METHODS: The free radical scavenging activity of A. compacta infusion (ACI) was evaluated by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical and superoxide anion radical assays. The activation of immune cells by ACI, as determined by cell surface cluster of differentiation 69 expression, was measured by flow cytometry. The qualitative polyphenolic composition of ACI was investigated by HPLC/PDA/ESI-MS, (High-performance liquid chromatography coupled with photodiode array detection and electrospray ionization - mass spectrometry) and the total content of polyphenols was estimated by spectrophotometric methods. RESULTS: Eight polyphenols including chlorogenic acid, 6,8-di-C-hexosyl apigenin, isoorientin, orientin, dicaffeoylquinic acid, biochanin A-O-glucoside, biochanin A-O-(malonyl)-glucoside, and licoisoflavone A were tentatively identified in ACI. The total contents of phenols, flavonoids, and tannins in lyophilized ACI were 5.40 mg/100 mg ACI, 1.79 mg/100 mg ACI, and 1.76 mg/100 mg ACI, respectively. ACI, within the range of 25-400 µg/mL, scavenged DPPH and O2.- by 15-90% and 20-88%, respectively. The human natural killer (NK) cells were substantially activated by ACI, whereas T cells and granulocytes were slightly stimulated. CONCLUSION: Overall, the results demonstrate the free radical scavenging and immune-stimulating properties of ACI, and support, at least in part, its potential utilization as a functional herbal tea. for preventing chronic diseases and as a nonspecific immune stimulator during human immunosenescence. SUMMARY: The total contents of phenols, flavonoids, and tannins in Azorella compacta infusion (ACI) were 5.40 mg/100 mg ACI, 1.79 mg/100 mg ACI, and 1.76 mg/100 mg ACI, respectively.Eight polyphenols including chlorogenic acid, 6,8-di-C-hexosyl apigenin, isoorientin, orientin, dicaffeoylquinic acid, biochanin A-O-glucoside, biochanin A-O-(malonyl)-glucoside, and licoisoflavone A were tentatively identified in ACI by HPLC/PDA/ESI-MS.ACI, within the range of 25-400 µg/ml, scavenged 1,1-diphenyl-2-picrylhydrazyl (DPPH) and O2. by 15-90% and 20-88%, respectively.The human natural killer (NK) cells were substantially activated by ACI, whereas T cells and granulocytes were slightly stimulated. Abbreviations used: ESI: electrospray ionization, HPLC: high performance liquid chromatography, PDA: photodiode array detector, MS: mass spectrometry, MS/MS: tandem mass spectrometry, MW: molecular weight, m/z: mass-to-charge ratio, FITC: fluorescent isothiocyanate, PE: phycoerythrin.

Comparative study of various subpopulations of cytotoxic cells in blood and ascites from patients with ovarian carcinoma.

AIM OF THE STUDY: A number of observations have indicated that the immune system plays a significant role in patients with epithelial ovarian cancer (EOC). In cases of EOC, the prognostic significance of tumour infiltrating lymphocytes has not been clearly explained yet. The aim is to determine the phenotype and activation molecules of cytotoxic T cell and NK cell subpopulations and to compare their representation in malignant ascites and peripheral blood in patients with ovarian cancer. MATERIAL AND METHODS: Cytotoxic cells taken from blood samples of the cubital vein and malignant ascites were obtained from 53 patients with EOC. Their surface and activation characteristics were determined by means of a flow cytometer. Immunophenotype multiparametric analysis of peripheral blood lymphocytes (PBLs) and tumour infiltrating lymphocytes (TILs) was carried out. RESULTS: CD3(+) T lymphocytes were the main population of TILs (75.9%) and PBLs (70.9%). The number of activating T cells was significantly higher in TILs: CD3(+)/69(+) 6.7% vs. 0.8% (p < 0.001). The representation of (CD3(-)/16(+)56(+)) NK cells in TILs was significantly higher: 11.0% vs. 5.6% (p = 0.041); likewise CD56(bright) and CD-56(bright) from CD56(+) cells were higher in TILs (both p < 0.001). The activation receptor NKG2D was present in 45.1% of TILs vs. 32.3% of PBLs (p = 0.034), but we did not find a significant difference in the numbers of CD56(+)/NKG2D(+) in TILs and PBLs. CONCLUSIONS: These results prove that the characteristics and intensity of anti-tumour responses are different in compared compartments (ascites/PBLs). The knowledge of phenotype and functions of effector cells is the basic precondition for understanding the anti-tumour immune response.

[Secondary humoral immunodeficiency in patiens with systemic lupus erythematosus]. / Sekundární humorální imunodeficience u nemocných se systémovým lupus erythematodes.

INTRODUCTION: Systemic lupus erythematosus (SLE) is a chronic autoimmune multisystem disease. The aim of our study was to clarify the frequency of decreased serum immunoglobulin levels in SLE patients. There were evaluated 799 results of serum immunoglobulin levels gained from 157 patients fulfilling revised ACR criteria in the retrospective study. RESULTS: The immunoglobulin levels under the normal range were found in 29/157 (18.5 %) patients. The most frequent was isolated reduction of IgG 12/157 (7.6 %), two persons fulfilled criteria for selective IgA deficiency, and one case possible diagnosis of common variable immunodeficiency (CVID). Additionally we report two cases of SLE patients complicated by severe hypogammaglobulinaemia and infectious complications with necessity of long-term immunoglobulin substitution therapy. The diagnosis of CVID is highly probable in the first case. The second case presents sever drug-induced hypogammaglobulinaemia. This female with lymphoma history and multiorgan impairment due to acute SLE was treated with rituximab after convention therapy failure. CONCLUSION: Humoral immunodeficiency may occur in SLE patients. The monitoring of serum immunoglobulin levels could be a routine in these patients. The CVID diagnosis is possible in patients suffering from recurrent sinopulmonary infections, especially in combination with absence of lupus activity. Rituximab therapy could cause long-term suppression of B lymphocytes with secondary humoral deficiency requiring immunoglobulin substitution therapy.

Cytomegalovirus disease in patients with common variable immunodeficiency: three case reports.

Common variable immunodeficiency (CVID) is the most frequent clinically relevant primary immunodeficiency and shows enormous heterogeneity in clinical presentation. Despite clinical immunodeficiency, opportunistic infections are not a typical manifestation of CVID. A retrospective study of 32 patients followed up for 335 patient-years was performed to determine the frequency of cytomegalovirus (CMV) disease. Symptomatic CMV infection was documented in 3 CVID patients. Patients No. 1 and 2 suffered from CMV pneumonia, with complications due to atypical mycobacteriosis in patient No. 1. Patient No. 3 suffered from CMV enteritis. A history of cancer and chronic hepatitis C infection (patient No. 1), immunosuppressive therapy for interstitial lung disease (patient No. 2) and serious enteropathy complicated with malnutrition (patient No. 3) may have contributed to the complications despite only mild abnormalities in T-cell subpopulations. The direct detection of CMV in bronchoalveolar lavage, stool or tissue samples was the most beneficial diagnostic laboratory method, whereas the detection of CMV DNA in blood did not produce positive results. Adequate treatment of CMV disease led to significant clinical improvement in all 3 patients. The frequency of CMV disease appears to be higher than previously described. In our experience, the probability of opportunistic infections in CVID patients increases with secondary comorbidities and their management.

The effect of chronic iron losses on liver regeneration in male and female rats.

BACKGROUND: We studied the effect of iron deficiency on liver regeneration and innate immunity - respiratory burst of PMN. METHODS: Wistar rats, males (M) and females (F) had sham withdrawals or males (M-w) and females (F-w) had nine blood withdrawals every week. All rats were sacrificed in 10(th) week after 67% hepatectomy (PH) after (3)H-thymidin application. We determined erythrocyte and leukocyte count, respiratory burst (RB), serum prohepcidin, estradiol, iron, iron binding capacity (TIBC) and liver iron stores. RESULTS: Liver DNA synthesis in M-w and F-w increased versus M and F (p=0.05). Serum prohepcidin after PH decreased in M, F (p=0.001) and F-w (p=0.05), but not in M-w. Blood withdrawals increased spontaneous RB (p<0.05), stimulated RB at females (p<0.01). Stimulated RB was lower in M-w then in M (p<0.01). Serum iron was lower in males than in females, but higher in rats with withdrawals than in rats without withdrawals. TIBC decreased after PH in M, F, F-w groups (p<0.001), less at M-w (p<0.05). Liver iron stores decreased in M, less in F. CONCLUSIONS: Both genders with blood withdrawals had early beginning of liver regeneration after PH. The preconditioning (withdrawals) leads to increase in iron turnover and stores following best reactivity of PMN, rapid decrease in serum prohepcidin, and early initiation of liver regeneration, mainly in females. We assume, the females have higher iron turnover, liver iron stores more easily mobilized for blood losses, because next gravidity physio logically begin immediately after birth. Simply transfer of experimental results to human medicine is difficult.

Ionizing radiation sensitizes leukemic MOLT-4 cells to TRAIL-induced apoptosis.

One of perspective approaches in treatment of hematological malignancies is activation of death receptors for TRAIL. However, leukemia cells studied to date have shown variable susceptibility to TRAIL. Our study demonstrates that cells of acute T-lymphoblastic leukemia MOLT-4 are resistant to TRAIL and that ionizing radiation in the therapeutically achievable dose of 1 Gy sensitizes TRAIL-resistant cells MOLT-4 to the TRAIL-induced apoptosis by increase in death receptors for TRAIL DR5. When TRAIL is applied after the irradiation in the time of increased DR5 positivity more efficient cell killing is achieved.

Changes in phosphorylation of histone H2A.X and p53 in response of peripheral blood lymphocytes to gamma irradiation.

The main aim of this study was to compare the reaction of quiescent and proliferating, i.e. phytohemagglutinin (PHA)-stimulated, human peripheral blood mononuclear cells (PBMCs) to gamma-radiation, and analyse changes of proteins related to repair of DNA damage and apoptosis, such as gammaH2A.X, p53, p53 phosphorylation at serines-15 and -392, and p21 and their dose dependence. Freshly isolated PBMCs in peripheral blood are predominantly quiescent, in G(0) phase, and with very low amounts of proteins p53 and p21. Using confocal microscopy we detected dose dependent (0.5-5 Gy) induction of foci containing gammaH2A.X (1 h after gamma-ray exposure), which are formed around radiation-induced double strand breaks of DNA. Apoptosis was detected from 24 h after irradiation by the dose of 4 Gy onwards by Annexin V binding and lamin B cleavage. Seventy two hours after irradiation 70% of CD3(+) lymphocytes were A(+). Neither increase in p53 nor its phosphorylation on serine-392 after irradiation was detected in these cells. However, massive increase in p21 (cyclin-dependent kinase inhibitor 1A) was detected after irradiation, which can be responsible for late occurrence of apoptosis in these quiescent cells. PHA-stimulation itself (72 h) caused an increase in early apoptosis (A(+)PI(-)) in comparison to non-stimulated PBMCs (38% A(+) resp. 13.4%). After PHA-stimulation also the amount of gammaH2A.X, p53, and p21 increased, but no phosphorylation of p53 on serine-392 or -15 was detected. Reaction to gamma-radiation was different in PHA-stimulated lymphocytes: the p53 pathway was activated and p53 was phosphorylated on serines-15 and -392 4 h after irradiation by the dose of 4 Gy. Phosphorylation of p53 at serine-15 increased in a dose-dependent manner in the studied dose range 0.2-7.5 Gy. Also the amount of p21 increased after irradiation. Seventy two hours after irradiation of PHA-stimulated CD3(+) T lymphocytes by the dose of 4 Gy 65% of cells were A(+).

Is defect in phosphorylation of Nbs1 responsible for high radiosensitivity of T-lymphocyte leukemia cells MOLT-4?

Mutations in NBS1 gene are related to higher occurrence of malignancies. In this work we studied response of T-lymphocyte leukemia cells MOLT-4 to ionizing radiation. We detected IRIF (ionizing radiation forming foci) containing histone gammaH2A.X, protein 53BP1, and Nbs1, which were formed around double-strand breaks of DNA. We found dose-dependent increase in foci number (colocalization of gammaH2A.X and 53BP1) and gammaH2A.X amount (integral optical density) 1h after irradiation. After the dose of 1.5 Gy the number of foci decreases with time, but 72 h after irradiation 9% of live cells still contained big foci around unrepaired DNA damage. Western blot method revealed massive phosphorylation of H2A.X during apoptosis induction, 6-24 h after irradiation by the doses 1.5 and 3 Gy. Cells with apoptotic morphology showed strong phosphorylation of H2A.X, but it was not accompanied by 53BP1. 1h after irradiation by the lethal doses 5 and 10 Gy we detected by Western blot a decrease in repair proteins Mre11, Rad50, and Nbs1. While phosphorylation of H2A.X 1h after irradiation was detected by both confocal microscopy and Western blot, phosphorylation of Nbs1 on serine 343 was not detectable in MOLT-4 cells. Despite functional ATM and p53 the phosphorylation of Nbs1 on serine 343 was impaired in these cells, and might be responsible for high radiosensitivity of MOLT-4 cells.

Monitoring of serum levels of angiogenin, ENA-78 and GRO chemokines in patients with renal cell carcinoma (RCC) in the course of the treatment.

Tumour progression requires the presence of a rich vascular supply. A number of cytokines, chemokines and proteases participate in the process of tumour angiogenesis. We evaluated serum levels of angiogenin, panGRO (Growth Related Oncogene) (CXCL 1,2,3) and ENA-78 (Epithelial Neutrophil Activating) (CXCL5) in the serum of 32 patients with RCC (renal cell carcinoma) and 14 healthy blood donors by means of a protein array analysis. The patients were divided into three groups according to their disease stages (I+II, III, IV). We discovered significant differences between the blood donors and patients with RCC both in pre-operative and post-operative angiogenin, panGRO and ENA-78 levels. The increase in angiogenic factors lasted in patients even without metastases 2 months after surgery. We found no correlation between the levels of angiogenin and stages I+II, III and IV RCC. Patients with advanced carcinoma (stage III) had pre-operatively higher serum levels of ENA-78 than patients with stages I+II (p = 0,009) and IV (p< 0.001). Eight weeks after surgery the patients with stages I+II had significantly higher levels of panGRO than patients with stage IV.

Immunologic changes in TNF-alpha, sE-selectin, sP-selectin, sICAM-1, and IL-8 in pediatric patients treated for psoriasis with the Goeckerman regimen.

Psoriasis is a chronic inflammatory skin disease which is often manifested during childhood. The present study investigated changes in the serum levels of proinflammatory cytokines and soluble forms of adhesion molecules in children with psoriasis. The observed patient group of 26 children was treated with the Goeckerman regimen. This therapy combines dermal application of crude coal tar with ultraviolet radiation. The Psoriasis Area Severity Index decreased significantly after treatment by with the Goeckerman regimen (p < 0.001). Serum levels of the proinflammatory cytokine TNF-alpha and adhesion molecules sICAM-1, sP-selectin and sE-selectin decreased after the Goeckerman regimen. The TNF-alpha and sICAM-1 decreased significantly (p < 0.05). Our findings support the complex role of these immune parameters in the immunopathogenesis of psoriasis in children. The serum level of IL-8 increased after the Goeckerman regimen. This fact indicates that the chemokine pathway of IL-8 activity could be modulated by this treatment, most likely by polycyclic aromatic hydrocarbons.

Gamma-radiation-induced ATM-dependent signalling in human T-lymphocyte leukemic cells, MOLT-4.

ATM kinase (ATM) is essential for activation of cell cycle check points and DNA repair in response to ionizing radiation (IR). In this work we studied the molecular mechanisms regulating DNA repair and cell death in human T-lymphocyte leukemic cells, MOLT-4. Apoptosis was evaluated by flow-cytometric detection of annexin V. Early apoptotic cells were determined as sub-G1 cells and late apoptotic cells were determined as APO2.7-positive ones. Proteins involved in ATM signalling pathway were analysed by Western-blotting. We observed a rapid (0.5 h) phosphorylation of ATM declining after 6 h after irradiation by all the doses studied (1.5, 3.0, and 7.5 Gy). Checkpoint kinase-2 (Chk-2) was also phosphorylated after 0.5 h but its phosphorylated form persisted 4, 2, and 1 h after the doses of 1.5, 3.0, and 7.5 Gy, respectively. The amount of p53 protein and its form phosphorylated on Ser-392 increased 1 h after irradiation (1-10 Gy). The lethal dose of 7.5 Gy caused an immediate induction and phosphorylation of p53 after 0.5 h post-irradiation. At the time of phosphorylation of p53, we found simultaneous phosphorylation of the oncoprotein Mdm2 on Ser-166. Neither ATM nor its downstream targets showed a dose-dependent response after 1 h when irradiated by the doses of 1-10 Gy. MOLT-4 cells were very sensitive to the effect of IR. Even low doses, such as 1.5 Gy, induced apoptosis 16 h after irradiation (evaluated according to the cleavage of nuclear lamin B to a 48-kDa fragment). IR-induced molecular signalling after exposure to all the tested doses was triggered by rapid phosphorylation of ATM and Chk-2. Subsequent induction of p53 protein and its phosphorylation was accompanied by concomitant phosphorylation of its negative regulator, oncoprotein Mdm2, and followed by induction of apoptosis.

Proteomic analysis of MOLT-4 cells treated by valproic acid.

The effect of valproic acid (VA) on protein expression in human T-lymphocytic leukemia cells MOLT-4 was studied. VA is an inhibitor of histonedeacetylases and has a potential use as antitumor agent in leukemia treatment. The authors in this work prove that 4 h long incubation with 2 mmol/l VA causes phosphorylation of histone H2A.X and its colocalization with 53BP1 in nuclear foci. Their co-localization is typical for DSB signaling machinery. These foci were detected in cells after 4 h exposure without increase of Annexin V positive apoptotic cells. Slight increase in apoptosis (Annexin V positivity) after 24 h is accompanied by more intensive increase in phosphorylation of H2A.X and also by formation of nuclear foci containing gammaH2A.X and 53BP1. Treatment of cells with 2 mmol/l VA resulted in induction of apoptosis affecting about 30% of cells after incubation for 72 h. The changes in protein expression were examined after cell incubation with 2 mmol/l VA for 4 h. Proteins were separated by two-dimensional electrophoresis and quantified using image evaluation system. Those exhibiting significant VA-induced abundance alterations were identified by mass spectrometry. Changes in expression of 22 proteins were detected, of which 15 proteins were down-regulated. Proteomic analysis resulted in successful identification of three proteins involving alfa-tubulin 3, tubulin-specific chaperone and heterogeneous nuclear ribonucloprotein F. Expression of seven proteins was up-regulated, including heterogeneous nuclear ribonucloprotein A/B. Identified proteins are related to microtubular system and hnRNP family. Suppression of microtubular proteins and changes of balance among hnRNPs can contribute to proliferation arrest and apoptosis induction.

Phenotype analysis of tumour-infiltrating lymphocytes and lymphocytes in peripheral blood in patients with renal carcinoma.

INTRODUCTION: When checking tumour growth, a number of observations indicate that the immune system plays a significant role in patients with renal cell carcinoma (RCC). Infiltration by lymphocytes (tumour infiltrating lymphocytes, TILs) is more prevalent in RCC than any other tumours. T lymphocytes are the dominant population of TIL cells. Views concerning the role of T lymphocytic subpopulations, B lymphocytes and NK cells in an anti-tumour response are not established. AIM: The aim is to determine the phenotype and activation of T and B lymphocytic subpopulations and NK cells and to compare their representation in tumour stroma and peripheral blood lymphocytes (PBL) in patients with RCC. MATERIAL AND METHODS: Samples of peripheral blood taken from the cubital and renal veins and tumour stroma cells were obtained from 44 patients in the course of their surgeries carried out due to primary RCC. TILs were isolated from mechanically disintegrated tumour tissue. Immunophenotype multiparametric analysis of PBL and TILs was carried out. Their surface and activation characteristics were determined by means of flow cytometer. RESULTS: CD3+ T lymphocytes (69.7%) were the main population of TILs. The number of CD3+/CD8+ T lymphocytes was significantly higher in TILs, 42.6% (p < 0.01), while CD4+ T lymphocytes were the majority population in peripheral blood, 41.35% (p < 0.001). The representation of CD3+/69+ T lymphocytes was significantly higher in TILs, 32.9%, compared to PBL (p < 0.001). On the contrary, the numbers of CD3+/CD25+, CD8+/57+ and CD4+/RA+ (naive CD4+ T lymphocytes) were higher in PBL (p < 0.001). The differences in representation of (CD3-/16+56+) NK cells and CD3+/DR+ T cells in TILs and PBL were not significant. CONCLUSION: The above-mentioned results prove that the characteristics and intensity of anti-tumour responses are different in compared compartments (tumour/PBL). CD3+/CD8+ T lymphocytes are the dominant lymphocytic population of TILs. The knowledge of the phenotype and functions of effector cells, which are responsible for anti-tumour response, are the basic precondition for understanding the anti-tumour immune response and the cause of its failure.

Surface activation leukocyte markers and humoral factors in cord blood of newborns at risk of early infection.

Due to immaturity of both specific and non-specific immune mechanisms, neonates are at risk of serious infections. The risk group definition is vague, clinical signs are non-specific and common laboratory markers are not as useful as in later ages, especially due to delayed reactions. In an attempt to find early, sensitive and specific markers, we assessed a defined set of surface leukocyte markers and humoral factors in cord blood. Several differences were noted--children in the risk group had a higher proportion of CD19+/23+, CD16+/64+, CD45RO cells and higher levels of IL-6. We find it promising that already at birth there are notable signs of reaction to infection and that a follow-up of a set of infectious markers could be useful to identify the children in need of antibiotic treatment and for diminishing unnecessary treatment.

Effect of valproic acid, a histone deacetylase inhibitor, on cell death and molecular changes caused by low-dose irradiation.

Valproic acid (VA), a histone deacetylase inhibitor (HDACI), in vitro induces differentiation of promyelocyte leukemia cell (HL-60) and proliferation arrest and apoptosis of various leukemia cell lines. In MOLT-4 cells (human T lymphocyte leukemia) the cell cycle arrest is caused by 2 mM VA, while 4 mM VA induces mainly apoptosis. In our work we studied effect of VA on molecular mechanisms responsible for cell cycle arrest (2 mM VA) or apoptosis induction (4 mM VA). The aim of our article was to evaluate a cotreatment by low (cytostatic) concentrations of VA with ionizing radiation and an effect of this combination on apoptosis induction in tumor cells MOLT-4. We prove that 24-h long incubation with VA causes acetylation of histones H3 and H4 in concentration-dependent manners. During first hours after the beginning of cultivation with VA in both studied concentrations (2 and 4 mM) an increase of p53 and its phosphorylation on serine 392 is detected, as well as a phosphorylation of Mdm2 on serine 166. After 8 and 24 h after the beginning of cultivation with 2 mM VA we detect p21, which is not observed after exposure to 4 mM VA. Cleavage of lamin B to 46 kDa fragment as an indicator of apoptosis was apparent after 24-h long incubation with 4 mM VA. In this article we prove radiosensitizing effect of VA. After 3-days long cultivation of cells with 2 mM VA the D(0) value decreased from 0.7 to 0.2 Gy. Also the EC70 value fell from 0.97 to 0.38 mM when the cells were irradiated with a dose of 1 Gy before the continual cultivation with VA. Continual cultivation of MOLT-4 cells irradiated by the dose of 1 Gy with VA caused during 14 days after irradiation significant increase of apoptotic cells in comparison to the cells exposed to only one factor. As a conclusion it can be postulated that continual exposure of MOLT-4 cells to VA increases apoptosis and decreases colony-forming capacity of the cells irradiated with small dose of radiation.

The importance of abrogation of G2-phase arrest in combined effect of TRAIL and ionizing radiation.

BACKGROUND: In this work we studied the relationship between the enhanced expression of DR5 receptor and the effect of combination of TRAIL and ionizing radiation on cell cycle arrest and apoptosis induction in human leukemia cell line HL-60. MATERIAL AND METHODS: DR5, APO2.7 and cell cycle were analyzed by flow cytometry. Proteins Bid and Mcl-1 were analyzed by Western-blotting. For clonogenic survival, colony assay on methylcellulose was used. RESULTS: Ionizing radiation caused significantly enhanced positivity of DR5 receptors 24 h after irradiation with high doses (6 and 8 Gy). An increase of DR5 receptor positivity after a dose of 2 Gy was not statistically significant and application of TRAIL 48 h after irradiation did not increase the apoptosis induction. However, a decrease of radiation-induced G(2) phase arrest and an increase of apoptosis were observed when TRAIL was applied 16 h before irradiation with the dose of 2 Gy. Incubation with 6 microg/l TRAIL for 16 h reduced D(0) value from 2.9 Gy to 1.5 Gy. The induction of apoptosis by TRAIL was accompanied by Bid cleavage and a decrease of antiapoptotic Mcl-1 16 h after incubation with TRAIL. CONCLUSION: TRAIL in concentration of 6 microg/l applied 16 h before irradiation by the dose of 1.5 Gy caused the death of 63% of clonogenic tumor cells, similarly as the dose of 2.9 Gy alone, which is in good correlation with the enhanced apoptosis induction.

Transplantation of bone marrow derived progenitor cells in acute myocardial infarction. The first results.

The intracoronary administration of autologous bone marrow cells (BMCs) has been shown to improve the left ventricle function in the course of acute myocardial infarction. Therefore we have started a clinical trial using transplantation of BMCs in the acute phase of myocardial infarction. The aim of our study is to assess the feasibility and safety of this procedure, and effect on the left ventricle function of these patients. We describe the first experience in two patients with acute myocardial infarction reperfused using direct stenting. The aspiration of bone marrow from the sternum provided sufficient amount of the cells for transplantation. No serious ischemia and no changes in coronary artery patency were detected after intracoronary infusion. The left ventricle ejection fraction was increasing throughout the time of three-month follow-up. No other complications (ventricular arrhythmias, reinfarction, thrombus formation) were detected.