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.

STAT5B restrains human B-cell differentiation to maintain humoral immune homeostasis.

BACKGROUND: Lymphocyte differentiation is regulated by coordinated actions of cytokines and signaling pathways. IL-21 activates STAT1, STAT3, and STAT5 and is fundamental for the differentiation of human B cells into memory cells and antibody-secreting cells. While STAT1 is largely nonessential and STAT3 is critical for this process, the role of STAT5 is unknown. OBJECTIVES: This study sought to delineate unique roles of STAT5 in activation and differentiation of human naive and memory B cells. METHODS: STAT activation was assessed by phospho-flow cytometry cell sorting. Differential gene expression was determined by RNA-sequencing and quantitative PCR. The requirement for STAT5B in B-cell and CD4+ T-cell differentiation was assessed using CRISPR-mediated STAT5B deletion from B-cell lines and investigating primary lymphocytes from individuals with germline STAT5B mutations. RESULTS: IL-21 activated STAT5 and strongly induced SOCS3 in human naive, but not memory, B cells. Deletion of STAT5B in B-cell lines diminished IL-21-mediated SOCS3 induction. PBMCs from STAT5B-null individuals contained expanded populations of immunoglobulin class-switched B cells, CD21loTbet+ B cells, and follicular T helper cells. IL-21 induced greater differentiation of STAT5B-deficient B cells into plasmablasts in vitro than B cells from healthy donors, correlating with higher expression levels of transcription factors promoting plasma cell formation. CONCLUSIONS: These findings reveal novel roles for STAT5B in regulating IL-21-induced human B-cell differentiation. This is achieved by inducing SOCS3 to attenuate IL-21 signaling, and BCL6 to repress class switching and plasma cell generation. Thus, STAT5B is critical for restraining IL-21-mediated B-cell differentiation. These findings provide insights into mechanisms underpinning B-cell responses during primary and subsequent antigen encounter and explain autoimmunity and dysfunctional humoral immunity in STAT5B deficiency.

Serum Citrulline and Ornithine: Potential Markers of Coeliac Disease Activity.

INTRODUCTION: To date, there is not generally accepted and universal indicator of activity, and functional integrity of the small intestine in patients with coeliac disease. The aim of our study was to investigate whether serum concentrations of the non-essential amino acids citrulline and ornithine might have this function. METHODS: We examined serum citrulline and ornithine concentrations in a subgroup of patients with proven coeliac disease and healthy controls (blood donors). RESULTS: A total of 94 patients with coeliac disease (29 men, mean age 53 ± 18 years; 65 women, mean age 44 ± 14 years) and 35 healthy controls (blood donors) in whom coeliac disease was serologically excluded (10 men, mean age 51 ± 14 years; 25 women, mean age 46 ± 12 years) were included in the study. Significantly lower concentrations of serum ornithine were found in patients with coeliac disease (mean 65 ± 3 µmol/L; median 63 µmol/L, IQR 34 µmol/L, p < 0.001). No statistically nor clinically significant differences were found in the citrulline concentrations between the study and control group. CONCLUSIONS: Serum ornithine (but not citrulline) may be useful for assessing the functional status of the small intestine in uncomplicated coeliac disease. Further studies involving more detailed analysis of dietary and metabolic changes in patients will be needed to reach definitive conclusions.

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.

Monitoring of Lymphocyte Populations During Treatment with Interferon-ß-1b to Predict Multiple Sclerosis Disability Progression.

The authors aim to understand how lymphocyte populations could predict the course of multiple sclerosis (MS) in people treated with interferon-ß (IFN-ß). Twenty-five male patients and 72 female patients were analyzed in the study. Peripheral blood samples were taken before and 5 years after the treatment with IFN-ß. Lymphocyte subsets were analyzed by flow cytometry. The authors compared lymphocyte parameters between confirmed sustained progression (CSP) and non-CSP groups by using Welch's one-way analysis of means or a chi-square test of independence. A penalized (lasso) logistic regression model was fitted to identify the combination of lymphocyte parameters for potential biomarkers. The combination of lymphocyte counts, relative CD3+/CD25+ cells, absolute CD8 T cells, absolute CD8+/CD38+ cells, absolute CD38+ cells, and relative CD5+/CD19+ cells was identified as potential biomarker for the IFN-ß treatment to monitor MS development in relation to CSP. The results suggest that other biomarkers aid in patient observation, predict a favorable outcome, and assist in the decision-making process for the early therapy escalation.

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.

Dominant-negative STAT5B mutations cause growth hormone insensitivity with short stature and mild immune dysregulation.

Growth hormone (GH) insensitivity syndrome (GHIS) is a rare clinical condition in which production of insulin-like growth factor 1 is blunted and, consequently, postnatal growth impaired. Autosomal-recessive mutations in signal transducer and activator of transcription (STAT5B), the key signal transducer for GH, cause severe GHIS with additional characteristics of immune and, often fatal, pulmonary complications. Here we report dominant-negative, inactivating STAT5B germline mutations in patients with growth failure, eczema, and elevated IgE but without severe immune and pulmonary problems. These STAT5B missense mutants are robustly tyrosine phosphorylated upon stimulation, but are unable to nuclear localize, or fail to bind canonical STAT5B DNA response elements. Importantly, each variant retains the ability to dimerize with wild-type STAT5B, disrupting the normal transcriptional functions of wild-type STAT5B. We conclude that these STAT5B variants exert dominant-negative effects through distinct pathomechanisms, manifesting in milder clinical GHIS with general sparing of the immune system.

Lymphocytes in the treatment with interferon beta-1 b.

BACKGROUND: Multiple sclerosis (MS) is a chronic inflammatory demyelinating and neurodegenerative disease affecting the central nervous system. One of the basic medications for the treatment of a clinically isolated syndrome (CIS) or relapsing-remitting MS is interferon beta (INFß). Although the exact mechanism of its effects is unknown, the medication has an anti-inflammatory and immunomodulatory effect. The goal of this study was to determine the characters which are affected in patients treated with INFß. METHODS: A total of 97 patients (25 males and 72 females) were included into the study. Patients were treated by INFß 1-b (subcutaneous injection, 250µg, each other day). Clinical evaluations were performed by an attending neurologist. Peripheral blood samples were obtained just prior to treatment and 5 years after INFß 1-b. Statistical analysis and processing of the obtained data were performed by using a comprehensive statistical software package MATLAB®. RESULTS: A significant decrease of the observed parameters after 5 years' of treatment (significant at the 1% significance level) was found in the absolute and relative CD69 count, absolute cytotoxic/suppressor T lymphocyte count, absolute total leukocyte count, absolute natural killer cells count. A significant decrease of the observed parameters after 5 years' of treatment (significant at the 5% significance level) was found in the absolute lymphocyte count, relative cytotoxic/suppressor T lymphocyte count, relative CD3+CD69+ count and absolute CD8+CD38+ count. CONCLUSION: The treatment with interferon beta reduces clinical exacerbations in multiple sclerosis (MS) through several known immunomodulatory mechanisms. However, the exact mechanism of effect of this medication is not known. This study presents some parameters that were affected by the long-term INFß 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 number of immunoregulatory T cells is increased in patients with psoriasis after Goeckerman therapy.

Regulatory T cells (Treg) are a specialized subpopulation of T cells that act to suppress inadequate immune response. Psoriasis is recognized as a T-cell driven immune-mediated systemic inflammatory disease with skin manifestation. Effective therapeutical approach to treat psoriasis is Goeckerman therapy (GT). The aim of this study was to compare the number of Treg in the peripheral blood of 27 psoriatic patients and 19 controls and to evaluate the influence of GT on Treg population in peripheral blood of patients with psoriasis. There was no significant difference in the relative number of Treg cells in the peripheral blood of healthy blood donors and patients with psoriasis before initiation of GT (P = 0.2668). In contrary, the relative number of Treg cells in peripheral blood of patients with psoriasis after GT was significantly higher than those found in healthy blood donors (P = 0.0019). Moreover, the relative number of Treg is significantly increased in psoriatic patients after Goeckerman therapy compared to the pre-treatment level (P = 0.0042). In conclusion, this significant increase in Treg count after GT is probably associated with amelioration of inflammation by GT, as disease activity expressed as PASI decreased in our patients by GT (P = 0.0001).

Telomere attrition occurs during ex vivo expansion of human dental pulp stem cells.

We provide a detailed characteristic of stem cells isolated and expanded from the human dental pulp. Dental pulp stem cells express mesenchymal cell markers STRO-1, vimentin, CD29, CD44, CD73, CD90, CD166, and stem cell markers Sox2, nestin, and nucleostemin. They are multipotent as shown by their osteogenic and chondrogenic potential. We measured relative telomere length in 11 dental pulp stem cell lines at different passages by quantitative real-time PCR. Despite their large proliferative capacity, stable viability, phenotype, and genotype over prolonged cultivation, human dental pulp stem cells suffer from progressive telomere shortening over time they replicate in vitro. Relative telomere length (T/S) was inversely correlated with cumulative doubling time. Our findings indicate that excessive ex vivo expansion of adult stem cells should be reduced at minimum to avoid detrimental effects on telomere maintenance and measurement of telomere length should become a standard when certificating the status and replicative age of stem cells prior therapeutic applications.

Regulatory T cells (TREG) and their roles in immune system with respect to immunopathological disorders.

Regulatory T cells (Tregs) are a specialized subpopulation of T cells that act to suppress immune response, thereby maintaining homeostasis and self-tolerance. It has been shown that Tregs are able to inhibit T cell proliferation and cytokine production and play a critical role in preventing autoimmunity. Different subsets with various functions of Treg cells exist. Tregs can be usually identified by flow cytometry. The most specific marker for these cells is FoxP3, which is localized intracellulary. Selected surface markers such as CD25high (high molecular density) and CD127low (low molecular density) could serve as surrogate markers to detect Tregs in a routine clinical practice. Dysregulation in Treg cell frequency or functions may lead to the development of autoimmune disease. Therapeutical Treg modulation is considered to be a promising therapeutical approach to treat some selected disorders, such as allergies, and to prevent allograft rejection.

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.

Licorice infusion: Chemical profile and effects on the activation and the cell cycle progression of human lymphocytes.

UNLABELLED: A licorice infusion (LI) and its major constituents were investigated for their capacity to stimulate the activation and the cell cycle progression of human lymphocytes, measured by the CD69 expression and DNA content, respectively. The chemical profile of LI was determined by high-performance liquid chromatography-diode array detection (HPLC-DAD). RESULTS: Two major components of LI were identified as liquiritin (1) and glycyrrhizin (2); total flavones and flavonols were shown as its minor constituents. The LI (100-800 mug/ml) stimulated the expression of CD69 on lymphocytes in a concentration-independent manner. Values of the activation index (AI) of total lymphocytes treated with LI (100-800 mug/ml) did not differ significantly among them (P < 0.05), but were 50% lower than the AI value exhibited by cells treated with phytohemagglutinin (PHA). The LI showed a similar effect on T cells, but on a lower scale. Compounds 1 and 2 (12-100 mug/ml) did not stimulate the CD69 expression on lymphocytes. The LI, 1 and 2 showed no meaningful effect on cell cycle progression of lymphocytes. The experimental data indicates that LI stimulates the activation of lymphocytes as a result of a proliferation-independent process. This finding suggests that LI could be considered as a potential specific immune stimulator.

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.