Apoptosis In Lymphocytes Of Patients Research Articles

Features of apoptosis and blebbing of the lymphocyte plasma membrane in bronchial asthma

Given a persistent global trend towards an increase in the number of patients with bronchial asthma (BA) over the past decades, researchers are facing challenges related to a comprehensive study of the pathogenesis of BA. Numerous studies have shown that BA is associated with long-term persistence of leukocytes (lymphocytes, macrophages, and eosinophils) in the bronchial tissues. However, the causes of this phenomenon remain understudied. The article provides an overview of modern research on the mechanisms of disorders of lymphocyte apoptosis in patients with BA. Our study considers the main mechanisms of molecular regulation of lymphocyte apoptosis, including transcription factors, the Fas/FasL system, and bcl-2/bcl-XL factors. We present the data on the role of reduced lymphocyte apoptosis in the formation of a severe BA phenotype. Taking into account high prevalence of obesity among patients with BA, we analyzed a few existing articles on the apoptosis of immunocompetent cells in obesity. In addition, the article highlights the key mechanisms of development of lymphocyte plasma membrane blebbing (PMB) with formation of microvesicles, as well as their influence on the course of pathological processes in BA. The authors believe that further in-depth study of apoptosis, lymphocyte necrosis, and plasma membrane blebbing can help improve the principles of diagnosis and treatment of BA.

P035 NUDT15 variance increases DNA-incorporated thiopurine metabolites and lymphocyte apoptosis in patients with inflammatory bowel disease

Abstract Background A novel thiopurine metabolizing enzyme, nucleotide diphosphate-linked moiety X-type motif 15 (NUDT15) was associated with drug-induced leukopenia in patients with non-synonymous genetic polymorphisms. Thiopurine-induced leukopenia in Japanese patients with genetic variance in NUDT15 (c.415C>T) appears to be independent of the 6-thioguanine nucleotide concentration in red blood cells. However, detailed molecular mechanism how NUDT15 variance causes thiopurine-induced leukopenia remains unclear and NUDT15-associated subcellular thiopurine metabolism has not been investigated in patients with inflammatory bowel diseases (IBD). Methods DNA-incorporated deoxythioguanosine (dTG) was measured in the peripheral blood mononuclear cells (PBMCs) of Japanese patients with IBD under thiopurine treatment. Association of a single-nucleotide polymorphism for NUDT15 (c.415C>T) with dTG in PBMCs (dTGPBMC) was examined. Peripheral blood T lymphocytes were cultured in vitro with 6-thioguanine (6-TG) to examine the Impact of NUDT15 genotypes on incorporation into DNA, cell proliferation and apoptosis. Results NUDT15 variants had significantly higher dTGPBMC per thiopurine dosage than non-variants (homozygous variants (TT) vs. heterozygous variants (CT) vs. non-variants (CC), 4418.0 vs. 663.0 vs. 295.3 dTG mol/106 moles dA per mg/kg/day of 6-MP (Figure A)). dTGPBMC and peripheral lymphocyte counts showed a negative correlation (r = −0.30, p = 0.015) (Figure B). Peripheral blood lymphocytes from patients with NUDT15 variance showed a higher DNA-incorporated dTG associated with increased apoptosis (increase of Annexin V+ PI+ CD4+ lymphocytes; TT vs. CT vs. CC, 158.5 % vs. 80.1 % vs. 57.9 % (p = 0.0427)) (Figure C) and decreased proliferation (decrease of proliferative CD4+ lymphocytes, TT vs. CT vs. CC, 49.0 % vs. 25.0 % vs. 19.1 % (p = 0.0098)) (Figure D) when cultured with 6-thioguanine in vitro. Conclusion DNA-incorporated dTG affected by NUDT15 genotypes induces T lymphocyte apoptosis in patients with IBD.

Evaluation of lymphocyte apoptosis in patients with oral cancer.

Objectives To evaluate apoptotic levels of peripheral blood mononuclear cells (PBMCs) and apoptotic regulatory proteins (Bax and Bcl-2) in lymphocyte subsets of oral cancer (OC) patients and healthy controls (HC).Methodology The percentage of apoptotic cells and lymphocyte counts were measured in the first cohort using PBMCs obtained from 23 OC patients and 6 HC. In the second cohort, (OC, 33; HC, 13), the mean fluorescence intensity (MFI) of Bax and Bcl-2 in CD19+ B, CD4+ T, CD8+ T, and CD16+56+ natural killer (NK) cells was determined via flow cytometry.Results The percentage of apoptotic cells was higher in the PBMCs of OC patients than in HC patients, particularly in patients with stage IV cancer (p<0.05). However, lymphocyte counts were significantly lower in stage IV patients (p<0.05). NK CD19+ B and CD16+56+ cell counts were significantly lower in OC patients compared with HC patients (p<0.001 and p<0.01, respectively), but CD4+ T cells were interestingly significantly higher in OC patients (p<0.001). While Bax MFI was slightly higher, Bcl-2 MFI was significantly lower for all four lymphocyte subsets in OC samples, particularly in stage IV patients, when compared with HC. Consequently, Bax/Bcl-2 ratios showed an upward trend from HC to OC patients, particularly those in stage IV. We found similar trends in Bax and Bcl-2 MFI for tumor stage, tumor size, and lymph node involvement.Conclusions The increased lymphocyte apoptosis in stage IV OC patients may be related to higher Bax levels and lower Bcl-2 levels. The Bax/Bcl-2 ratio in lymphocytes may be useful to determine the prognosis of OC patients, and could be considered a mean for supportive treatment in the future.

Effects of Levofloxacin on Blood Lymphocyte Apoptosis in Patients with Pulmonary Tuberculosis: an In Vitro Study.

The effects of a fluroquinolone levofloxacin on apoptosis of peripheral blood lymphocytes from patients with infiltrative pulmonary tuberculosis were studied in vitro. It was found that levofloxacin stimulated apoptotic cell death in tuberculosis. Addition of levofloxacin to cell suspension from patients with drug-susceptible form of tuberculosis led to an increase in the number of CD95+ and AnnV+ lymphocytes. In patients with drug-resistant form of tuberculosis, only the number of apoptotic lymphocytes, but not the count of CD95+ cells increased under these conditions.

强直性脊柱炎患者单个核细胞TLR2、TLR4与淋巴细胞凋亡的研究

强直性脊柱炎患者单个核细胞TLR2、TLR4与淋巴细胞凋亡的研究

Activation of caspases in lymphocytes of patients with borderline personality disorders

Activities of caspases involved in cell death have been investigated in lymphocytes of patients with borderline personality disorders (BPD). In the group of patients with organic mental disorders (OMD) caspase activity decreased in a depressive disorder and increased in an anxiety disorder, thus suggesting activation of apoptosis in lymphocytes of patients with the anxiety disorder. Caspase activities differed in lymphocytes of OMD patients with the depressive disorder and a recurrent depressive disorder. In BPD patients with OMD lymphocyte caspase activity was higher thus indicating activation of apoptosis in these cells. Results can be used for specification of mechanisms of pathogenesis of mental disorders and search for new markers of these diseases.

Nuclease Activity Associated with Secreting Granules by Lymphocytes in Patients with Bronchial Asthma

Background: We know, through recent studies, the existence of some morpho-biochemical peculiarities in the process of type 1 programmed death of patients’ lymphocytes suffering from bronchial asthma, but little convincing data exist on the activity of enzymes involved in this physiological process. Therefore, the aim of our research was to study the enzymatic activity of secreting granules of patients’ lymphocytes with bronchial asthma, according to the degree of severity. Method: The study was based on the role of granular extracts in the process of programmed death isolated lymphocytes from peripheral blood of relatively healthy individuals and asthmatic patients with different severity. The immunological characteristics of lymphocytes was done with the radial immune-diffusion method and ELISA test but the method of agarose gel electrophoresis help us to detect the catalytic activity of protein extracts of secreting granules of lymphocytes. Results: The results obtained showed that lymphocytes from asthmatic patients with severe severity are characterized by a decrease in cytotoxic T lymphocytes content balanced by an increase in T-Helper lymphocytes. We also noticed the enzymatic activity at all the groups studied but this activity was relatively high in asthmatics with severe severity. Furthermore, the study of the cationic dependence has allowed to establish an increase in enzymatic activity in all the groups studied after incubation of DNA in a medium containing Ca2+ with a pH of 7.5 unlike ions Mn2+ which seem to reduce the enzymatic activity. The expression of enzymatic activity in the presence of zinc allows us to suggest the presence of DNase acid in granules, which activity is not necessarily associated with divalent metal ions. Conclusion: Based on the above results, one might conclude that the secreting granules have a high enzymatic activity but with a strong cationic dependence. This not only allows a better understanding of the morphological changes observed during the course of apoptosis in lymphocytes of patients but also brings more to the knowledge of the enzymatic influence in the process of type 1 programmed death.

The Particularities of Protein fraction in the Apoptosis of Lymphocytes of Patients with Asthma

The emergence of various diseases specifically severe diseases such as bronchial asthma is associated with apoptosis of lymphocytes. One of the major biochemical features of apoptosis is chromosome DNA fragmentation implemented by apoptotic nucleases. The inactivation of these apoptotic nucleases produces undigested DNA and is linked to a number of autoimmune disorders. Instead of this we have studied the enzymatic activities of the cytoplasmic and nucleic proteins of lymphocytes from healthy donors and patients with bronchial asthma. The study of enzymatic activities of the nuclease of lymphocytes was assessed by flow cytometry, spectrofluorimetry and electrophoresis method in agarose gel. In the peripheral blood cells of healthy donors undergoing apoptosis, we found a DNAse activated by Ca2+ and Mg2+ ions. Lymphocytes of patients with bronchial asthma contain DNAses, the activity of which depends on the seriousness of the desease. In patients cells, the activity of the Mn2+-dependent DNAse increases, whereas the activity of the Ca2+, Mg(2+)-dependent DNase decreases. Taking into consideration the role of the Ca2+, Mg(2+)-dependent DNAse in apoptosis, we can propose that there is a link between the reduction of the rate of apoptosis of lymphocytes in patients with bronchial asthma and the dysfunction of the induction of "apoptotical" Ca2+, Mg(2+)-dependent nuclease. According to the results obtained, we can assume that why apoptosis of lymphocytes resist in patients with bronchial asthma is a reduction in concentration of endocellular calcium and an increase of manganese ions content, which results in the triggering of activation mechanism for Mn(2+)-dependent endonuclease activity. This leads to the change of DNA fragmentation nature in lymphocytes and as a consequence, to disorders in process of apoptotic bodies' formation, thus, hindering apoptosis of lymphocytes in patients with bronchial asthma.

Apoptosis in peripheral blood lymphocytes of patients with LRRK2-associated Parkinson's disease

Apoptosis in peripheral blood lymphocytes of patients with LRRK2-associated Parkinson's disease

Estimation of lymphocyte apoptosis in patients with chronic, non healing diabetic foot ulcer

Background: Lymphocytes play an important role in wound healing and the removal of circulating T lymphocytes inhibits the healing cascade. Decreased stimulation of survival factors and increased levels of dead signals may lead to the malfunction of many cells, including lymphocytes. Aims & Objective: To explore lymphocytes involvement in wound healing. Material and Methods: Study participants were divided into three groups: group A, group B and group C (30 participants each). Annexin-V-FITC+CD-3-PE kit were used for the lymphocyte apoptosis estimation in diabetic foot patients by florescent activated cell sorter (FACS). Results: We find out significantly higher total T cell apoptosis in type 2 diabetes mellitus patients having chronic, non healing diabetic foot ulcer as compared with healthy individuals. CD-3 + Annexin-V-FITC positive lymphocytes were statistically significant in group C (P<0.01) and group B (p<0.001) when compared from group A. Conclusion: This study suggests the importance of T-lymphocytes in wound healing. From the present study we can suggest diabetic patients to maintain their immune system for the normal wound healing. Key-Words: Diabetic Foot; Apoptosis; Lymphocyte

Apoptosis of peripheral blood lymphocytes in patients with LRRK2-associated Parkinson’s disease

Mutations in the Leucine Reach Repeat Kinase 2 (LRRK2) gene are the most frequent cause of familial Parkinson's disease (PD). Although the precise physiological and pathological role of LRRK2 is unclear, a direct link between mutant LRRK2 and apoptosis has been suggested. Using flow cytometric analysis (PI+Annexin V(FITC)) we showed increased spontaneous apoptosis of peripheral blood lymphocytes in patients with LRRK.2-associated PD compared to controls after 24 (P < 0.016) and 48 (P < 0.031 ) h of incubation (5 % CO2, 37 degrees C). We found the increased FAS mRNA level in peripheral blood lymphocytes of patients with LRRK2-associated PD compared to controls (P < 0.05) and to sporadic PD (sPD) (P < 0.002). Significant difference in FAS expression between patients with LRRK2-associated PD and controls remained after three years and was detected after 1 and 24 h during lymphocyte incubation (P < 0.03 and 0.05, respectively). Increased spontaneous lymphocytes apoptosis along to increased FAS expression in patients with LRRK2-associated PD suggest that LRRK2 mutations may lead to the activation of extrinsic apoptotic way.

Immunosuppression following surgical and traumatic injury

Severe sepsis and organ failure are still the major causes of postoperative morbidity and mortality after major hepatobiliary pancreatic surgery. Despite recent progress in understanding the immune conditions of abdominal sepsis, the postoperative incidence of septic complications after major visceral surgery remains high. This review focuses on the clinical and immunological parameters that determine the risk of the development and lethal outcome of postoperative septic complication following major surgery and trauma. A review of the literature indicates that surgical and traumatic injury profoundly affects the innate and adaptive immune responses, and that a marked suppression in cell-mediated immunity following an excessive inflammatory response appears to be responsible for the increased susceptibility to subsequent sepsis. The innate and adaptive immune responses are initiated and modulated by pathogen-associated molecular-pattern molecules and by damage-associated molecular-pattern molecules through the pattern-recognition receptors. Suppression of cell-mediated immunity may be caused by multifaceted cytokine/inhibitor profiles in the circulation and other compartments of the host, excessive activation and dysregulated recruitment of polymorphonuclear neutrophils, induction of alternatively activated or regulatory macrophages that have anti-inflammatory properties, a shift in the T-helper (Th)1/Th2 balance toward Th2, appearance of regulatory T cells, which are potent suppressors of the innate and adaptive immune system, and lymphocyte apoptosis in patients with sepsis. Recent basic and clinical studies have elucidated the functional effects of surgical and traumatic injury on the immune system. The research studies of interest may in future aid in the selection of appropriate therapeutic protocols.

Effects of low dose of hydrocortisone on circulating thymus-dependent lymphocyte apoptosis in patients with septic shock

Objective To investigate the effects of low-dose of hydrocortisone on circulating thymus-dependent lymphocyte (T lymphocyte) apoptosis in patients with septic shock. Method fifty-seven patients with septic shock admitted into ICU from January 2006 to January 2009 were prospectively randomized (random number) to treatment group and control group. Another 20 healthy volunteers and 18 patients with sepsis alone were included as external control groups.The patients of treatment group and control group were treated with low-dose of hydrocortisone and placebo,respectively. Samples of peripheral blood were taken from healthy volunteers and patients 0 hr,24 hrs,48 hrs,72 hrs and 168 hrs after onset of the disease to determine the circulating T lymphocyte apoptosis by using theassays of Annexin V and flow cytometry. Least significant difference t -test was used for multiple comparisons. Results The percentage of Annexin V-positive CD4+ T lymphocytes in the primary stage was (11.01 +4.52)% in septic shock patients, (4.41 + 1.45)% in healthy volunteers, and (7.87 + 3. 82)% in patients with sepsis alone. And in the initial setting, the percentage of Annexin V-positive CD4+ T lymphocytes in the septic shock patients was higher than that in healthy volunteers ( P 0.05 between three groups). The percentages of Annexin V-positive CD4+ T lymphocytes in control group after 24 his, 48 hrs and 72 hrs were(13.51+6.85)%, (19.39 + 6.63)% and (15.33+ 6.21)%, respectively. And the percentages of Annexin V-positive CD4+ T lymphocytes in treatment patients after 24 hrs, 48 hrs and 72 hrs were (17.4 + 7.21)%, (22.61 + 5.64)%, and (25.73 + 6.91)%, respectively. The percentage of Annexin V-positive CD4+ T lymphocytes in septic shock patients was higher than that in control groups ( P 0.05 between two groups). In the meantime, the percentages of Annexin V-positive CDS'1' T lymphocytes in control group and in treatment group were (12.72+ 19.69)% and (13.88 + 13.28)%, respectively (P >0.05). Conclusions Low-dose of hydrocortisone could induce CD4+ T lymphocyte apoptosis and has no effects on CD8+ T lymphocyte apoptosis when it is used to treat septic shock. Key words: Septic shock; Lymphocyte; Apoptosis; Hydrocortisone

P.1.107 Influence of pharmacotherapy on fas-receptorsand spontaneous apoptosis of lymphocytes in patients with depressive disorders

P.1.107 Influence of pharmacotherapy on fas-receptorsand spontaneous apoptosis of lymphocytes in patients with depressive disorders

In vitro spontaneous and UVB-induced lymphocyte apoptosis are not specific to SLE.

We studied in vitro spontaneous and ultraviolet light (UV)-induced lymphocyte apoptosis in patients with systemic lupus erythematosus (SLE, n = 11), cutaneous lupus erythematosus (CLE, n = 8), and other collagen diseases (n = 6), as well as normal individuals (n = 6). Apoptosis was confirmed by the presence of a 180 bp DNA ladder on gel electrophoresis. UVB-induced apoptosis was observed in 4 of 11 patients with SLE (36.3%), 3 of 8 patients with CLE (37.5%) and 2 of 6 patients (33.3%) with other collagen diseases. There was no clinical correlation between clinical photosensitivity and UV-induced apoptosis. Similarly, spontaneous apoptosis was also found in lymphocytes from patients with diseases other than SLE. No apoptosis was found in normal subjects with or without UVB irradiation (25 mJ/cm2). These data suggest that UV-induced lymphocyte apoptosis may not be specific to SLE but may be common in collagen diseases.

Induction of Apoptosis by Chlorodeoxyadenosine and Dexamethasone in B-Chronic Lymphocytic Leukemia

Apoptosis, programmed cell death, occurs in a variety of cellular systems and in response to many different stimuli. In the present study we examined the ability of dexamethasone (Dex) and chlorodeoxyadenosine (2-CdA) to induce apoptosis in lymphocytes of patients with B-chronic lymphocytic leukemia (B-CLL). Lymphocytes of 29 untreated patients and 9 healthy controls were isolated and incubated for 24 hours in the presence or absence of either Dex (2 m¨M) (n = 15) or 2-CdA (3 m¨M) (n = 14). Following incubation the cells were harvested and their DNA extracted and analysed for internucleosomal DNA cleavage by UV illumination after electrophoresis on agarose slab gel containing ethidium bromide. In the Dex group, 10 patients showed dexamethasone independent spontaneous apoptosis appearing 24 hours after the start of incubation. These were the only instances of dexamethasone-enhanced apoptosis. Five patients showed no spontaneous or dexamethasone induced apoptosis. Of the 2-CdA group, 5 showed spontaneous apoptosis enhanced by 2-CdA. No spontaneous apoptosis was observed in the cells from 9 other patients, however, 2-CdA induced apoptosis in 8 cases in this group. This study shows that monitoring of apoptosis in CLL may provide important information regarding susceptibility of the cells to drug induced apoptosis.

IL-2 rescues in vitro lymphocyte apoptosis in patients with HIV infection: correlation with its ability to block culture-induced down-modulation of Bcl-2.

IL-2 administration in vivo has been shown to increase CD4+ T cell counts in HIV+ patients. We have previously reported that PBMC from HIV-infected patients undergo marked spontaneous apoptosis in vitro. In this study, we examined the effect of IL-2 added in vitro upon culture-induced apoptosis in PBMC from 80 HIV-infected patients by flow cytometry. IL-2 at concentrations of > or = 10 U/ml significantly reduced spontaneous apoptosis in CD3+ T lymphocytes in patients but not in healthy volunteers. Interestingly, we observed that Bcl-2 expression in patient lymphocytes decreased rapidly upon in vitro culture while that in cells of healthy volunteers was relatively unaffected. The most significant decrease in Bcl-2 expression was noted in the apoptotic cell population. The IL-2-mediated reduction in lymphocyte apoptosis was found to be associated with the blocking of this culture-induced down-modulation of Bcl-2 expression. IL-2 did not induce significant expansion of lymphocytes during the culture period nor did it affect Fas Ag expression in patient cells, which were already expressing Fas maximally. These findings strongly suggest that IL-2 mediates its apoptosis-blocking effects via suppressing down-modulation of Bcl-2. Our findings also provide an experimental basis for the ongoing therapies utilizing this cytokine for slowing HIV disease progression.

APOPTOSIS AS AN INDICATOR FOR STEROID SENSITIVITY OF LYMPHOCYTES IN B-CHRONIC LYMPHOCYTIC-LEUKEMIA

Apoptosis, programmed cell death, occurs in a variety of cellular systems and in response to many different stimuli. One group of apoptosis inducers are glucocorticosteroids which are also found in the battery of cytotoxic drugs used to treat CLL. In the present study we have examined the potency of the glucocorticosteroid-dexamethasone to induce apoptosis in lymphocytes of patients with B-CLL. Lymphocytes of 15 nontreated patients and 5 controls were isolated and incubated for 24 h in the presence or absence of dexamethasone (2 mu M) Following incubation the cells were harvested and their DNA extracted. The extracted DNA samples were analysed for internucleosomal DNA cleavage by UV illumination after electrophoresis on agarose slab gel containing ethidium bromide. Five patients showed neither spontaneous nor dexamethasone induced apoptosis. Whereas, 10 patients, showed a dexamethasone-non-dependent spontaneous apoptosis which appeared 24 h after the start of incubation. The cells of these patients were the only ones to respond to dexamethasone showing an enhanced apoptosis effect. This study shows that apoptosis monitoring in CLL may provide important information regarding susceptibility of the cells to steroid induced apoptosis.