Genotoxicity evaluation of drinking water sources in human peripheral blood lymphocytes using the comet assay

The potential harm of organic pollutants in drinking water to human health is widely focused on in the wodd; more and more pollutants with genotoxic substances are released into the aquatic environment. Water source samples were collected from 7 different localities of Nanjing City. The potential genotoxicity of organic extracts from drinking water sources were investigated by means of the comet assay in human peripheral lymphocytes. The results showed that all the organic extracts from all the water source samples could induce DNA damages of human peripheral blood lymphocytes at different levels. A significant difference （P 〈 0.01） was observed when compared with the solvent control, The DNA damage increased with the increase of the dosage of the original water source. Significant differences of DNA damage were observed in different drinking water sources, as shown by the multiple comparisons analysis at the dosage of 100x; the degree of DNA damage treated by Hushu waterworks （at town level） was the most serious, the arbitrary units （AU） was 141.62±6.96, however, that of Shangyuanmen waterworks （at city level） was only 109.64±2.97. The analysis also revealed that the genotoxicity of town＇s water sources was higher than that of the city. The results demonstrated that the comet assay can be successfully applied to the genotoxicity monitoring programs of drinking water sources.

Early Changes of Peripheral Blood Lymphocyte Subpopulations in Patients with Occupational 2,4-dinitrophenol Poisoning

2,4-dinitrophenol （DNP）, an organic compound which frequently used in industry, is considered to have high toxicity. This study aimed to investigate the early changes of lymphocyte subpopulations in patients with occupational 2,4-DNP poisoning. Totally 9 patients with acute occupational 2,4-DNP poisoning and 30 healthy volunteers as control were enrolled. The patients received immediately comprehensive supportive treatments, including large-dose glucocorticoid and repeated hemoperfusion （HP）. The ratio of CD4＋/CD8＋ T cells were significantly higher in patients upon admission compared to healthy controls （P 〈 0.01）; however, counts of total lymphocytes, CD3＋, CD3＋CD4＋, CD3＋CD8＋, B （CD19＋）, and natural killer （NK） cells （CD16＋CD56＋） were significantly reduced （all P 〈 0.001）. The NK cell count was negatively correlated with initial plasma 2,4-DNP concentration （r = -0.750, P = 0.026）. Thus, acute occupational 2,4-DNP poisoning was accompanied by immediate complex immune cell reactions, especially NK cells might play important role in severe 2,4-DNP poisoning.

DNA Damage Response in Resting and Proliferating Peripheral Blood Lymphocytes Treated by Camptothecin or X-ray

DNA damage response （DDR） in different cell cycle status of human peripheral blood lymphocytes （PBLs） and the role of H2AX in DDR were investigated. The PBLs were stimulated into cell cycle with phytohemagglutinin （PHA）. The apoptotic ratio and the phosphorylation H2AX （S139） were flow cytometrically measured in resting and proliferating PBLs after treatment with camptothecin （CPT） or X-ray. The expressions of γH2AX, Bcl-2, caspase-3 and caspase-9 were detected by Western blotting. DDR in 293T cells was detected after H2AX was silenced by RNAi method. Our results showed that DNA double strand breaks （DSBs） were both induced in quiescent and proliferating PBLs after CPT or X-ray treatment. The phosphorylation of H2AX and apoptosis were more sensitive in proliferating PBLs compared with quiescent lymphocytes （P0.05）. The expression levels of anti-apoptotic proteins Bcl-2 were reduced and cleaved caspase-3 and caspase-9 were increased. No significant changes were observed in CPT-induced apoptosis in 293T cells between H2AX knocking down group and controls. It was concluded that proliferating PBLs were more vulnerable to DNA damage compared to non-stimulated lymphocytes and had higher apoptosis rates. γH2AX may only serve as a marker of DNA damage but exert no effect on apoptosis regulation.

Metallothionein 1 Isoform Gene Expression Induced by Cadmium in Human Peripheral Blood Lymphocytes

Objective To study the gene expression of metallothionein 1 （MT-1） isoforms in human peripheral blood lymphocytes （HPBLs）. Methods The expression of mRNA representing the seven active MT-I genes was determined in HPBLs by quantitative RT-PCR before and after exposure to cadmium. Results Basal expressions of MT-IX, and MT-1A in HPBLs were similar to expression of housekeeping gene. In contrast, the basal gene expressions of MT- 1 H, IF, 1E, and 1G were a little transcripts in human HPBLs. No signal was detected for MT-1B. There was a sex difference （P〈0.05）. in basal gene expression of MT-1E. The levels of gene expression of MT-1A, 1E, IF, 1G, 1H, and 1X increased, but the level of MT-1B did not increase after exposure to cadmium. Conclusions Gene expressions of MT-1 G, MT-1 H, MT-1 F, and MT-1X in HPBLs can be used as a potential biomarker of cadmium exposure.

EXPRESSION OF IL-2R ON PERIPHERAL BLOOD LYMPHOCYTES OF PATIENTS WITH COLORECTAL CANCER AND ITS CLINICAL SIGNIFICANCE

Objective: To study expression of membrane receptors of interleukin-2 (CD25) on the peripheral blood lymphocytes (PBL) of patients with colorectal cancer and its clinical significance. Methods: CD25 percentages (CD25%) in PBL of 105 colorectal cancer patients before operation and 100 normal individuals were examined by flow cytometer, and the results were clinically and pathologically analyzed. Results: The mean of CD25% in PBL of the normal individuals was 17.24±5.33, it was significantly lower (P<0.01) than that of the colon cancer patients (21.29±7.95) or rectal cancer patients (21.62±6.11). In contrast to the normal individuals, the means of CD25% in PBL in ulcer type (20.53±6.50) or protruded type (21.56±6.16) colorectal cancer patients were notably elevated (P<0.01). The significant difference (P<0.01) of means of CD25% in PBL was observed between the normal individuals and patients with less than 4 cm mass (22.10±5.43) or 4cm–8cm mass (20.90±6.96). The significant difference (P<0.05) of means of CD25% in PBL was also observed between the normal individuals and patients with greater than 8 cm mass (21.56±5.41). The mean of CD25% in PBL in patients with well differentiation colorectal cancer was 22.20±5.50, it was significantly higher than that in normal individuals (P<0.05). The means of CD25% in PBL in patients with middle or poor differentiation colorectal cancer were 21.30±6.89 and 22.15±5.71 respectively, they were obviously higher than that in normal individuals (P<0.01). The significant difference (P<0.01) of means of CD25% in PBL was present between the colorectal cancer patients without metastatic lymph nodes (22.06±6.90) and normal individuals. The significant difference (P<0.05) of means of CD25% in PBL was present between the colorectal cancer patients with metastatic lymph nodes (20.73±6.40) and normal individuals. The means of CD25% in PBL in colorectal cancer patients in various clinic stages were significantly higher than that in the health subjects (P<0.01). The significant difference (P<0.01) of means of CD25% in PBL was present between the patients whose ages were equal to or less than 60 (21.00±5.76) and normal individuals in the same age group. The significant difference (P<0.05) of means of CD25% in PBL was also present between the patients whose ages were greater than 60 (22.54±7.75) and normal individuals in the same age group. The significant difference (P<0.01) of means of CD25% in PBL was present between the male patients (22.55±7.05) and normal men. The significant difference (P<0.05) of means of CD25% in PBL was also present between the female patients (20.09±5.48) and normal women. Conclusion: The mean of CD25% in PBL of colorectal cancer patients was significantly higher than that in health subjects. Abnormally elevated CD25% were correlative with site of tumor growth, macropathology type of tumor, the degree of tumor differentiation, clinical stage and patient’s age and sex. It may be helpful to detect CD25% in PBL of colorectal cancer patients before operation for diagnosis, immune treatment and judging prognosis.

Potassium channel in peripheral blood lymphocytes of turbot (Scophthalmus maximus)

In order to provide pertinent evidence of ion channel with immune response in the fish, whole cell patch-clamp technique was employed for potassium ion channel study in turbot （Scophthalmus maximus）. Lymphocytes were isolated by Percoll density gradient centrifugation from peripheral blood samples, and electrophysiological characters of the channel were analyzed. In the recorded cells, activated voltage of the channels was -42.5±3.7 mV and the average peak current was 313.12±28.2 pA. The channel was identified as voltage dependent, the current was outward and it could be inhibited by 10 mmol/dma TEA or 5 mmol/dm^3 4-AP, a specific potassium channel inhibitor, identifying the existence of potassium channel in peripheral lymphocytes of the turbot.

Study on Peripheral Blood Lymphocytes ChromosomeAbnormality of People Exposed to Cadmium in Environment

Chromosome aberration (CA) and micmnucleus (MN) tests were appied to investigate Peripheral blood lymphocytes in 56 people environmentally exposed to cadwhum (Cd) for a period up to 30years, and in 10 unexposed People as controls. As indicator of body-load of Cd, urineq Cd (UCd)concentrations were measured simultaneously. The People in polluted villages were divided into four groups according to vallous levels of UCd concentrations: ~ 2 .5, 2 .5 ~, 5 .0 ~, 10.0 ~ (μg/l).There was significant difference in MN rates between the exposed and control groups (3 .47, 5 .06,8.06, 12 .75‰ for the exposed groups respectively, and 3. 10‰ for the controls), and significant correlation between MN rates and UCd was observed. Although no markesd difference in CA rates was noted between UCd 5 .0 ~ and 10 .0 ~ groups, there was significant difference in CA rates between the exposed and control groups (3. 07,5. 21, 7. 21, 8. 50% for exposed groups respectively, and2 .33% for the controls) and significant correlation between CA rates and UCd. CA was presented mainly in the form of chromatid and chromosome gaps and breaks. Together with our another study 'An Investigation on Human Health Effects by Envimnmental Cadmium Pollution', the results suggest that Cd may injure human chromosomes and that the damage appears to be concentrated on cytogenetic material and may happen earlier than renal disfunction

Isolation of Sturgeon Peripheral Blood Lymphocytes and the Optimal Proliferation Response Conditions

In this study, sterlet （ Acipenser ruthenus ） was chosen as the model species of sturgeon, different solutions were used to isolate the sturgeon peripheral blood lymphocytes and study their optimal proliferate response condition. Phytohemagglutinin （PHA）, concanavalin A （ConA）and lipopolysaccharide （LPS） were used as lymphocyte proliferation mitogen, respectively. According to L 25 （5^6） five-factor five-level orthogonal experimental design, the conditions for sturgeons proliferation response of peripheral blood lymphocytes were optimized using enhanced cell counting Kit-8 （enhanced CCK-8 or WST-8）. Five factors were selected to explore the optimal response conditions, including culture time, culture temperature, cell concentration, fetal bovine serum （FBS） concentration, and mitogen concentration. The results showed that 70% Percoll （1.092 g/ml） used as the sturgeon lymphocyte separation solution had the best separating effect. The optimal proliferation conditions were as follows： 3.625×10 6 initial cells, 20 μg/ml PHA or 50 μg/ml ConA or 10 μg/ml LPS as mitogen, 10%-20% FBS, the temperature at 20-25 ℃, and the culture time of 2 d.

Retinoic acid regulated insulin-like growth factor gene expression in cord blood lymphocytes

Objective To explore the immune promotion mechanism of retinoic acid (RA).Methods Reverse Transcription-Polymerase chain reaction (RT-PCR) was used to detect the gene expression of insulin like growth factor Ⅰ (IGF-Ⅰ) and IGF receptors (IGF-IR and IGF-IIR) in vitro, and their regulation by RA in human cord blood lymphocyte (CBL). Results Significant upregulation of IGF-I, IGF-IR and IGF-IIR mRNA was found at 6-24 hours in CBL incubated with physiologic concentrations of RA as compared to those without RA. Conclusion The enhancement of IGF expression may be an important pathway for vitamin A to promote immune cellular functions.

Investigation of the cytotoxicity,antioxidative and immune-modulatory effects of Ligusticum porteri(Osha) root extract on human peripheral blood lymphocytes

OBJECTIVE： Ligusticum ported is a traditional Native American herb. The roots of L. ported are traditionally used in the treatment of many diseases, however, its cytotoxicity, antioxidative and immune- modulatory effects need to be investigated. In this study, we evaluated the effects of the root extract at different doses on human peripheral blood lymphocytes （PBLs）. METHODS： The lymphocytes were incubated with different concentrations of the root extracts （0, 50, 100, 200, and 400 μg/mL） and harvested every 6 h for 2 d （P〈0.05）. The protective effect of the herb against oxidative damage was determined by inducing oxidative stress with the administration of 50 μmol/L of hydrogen peroxide （H202）. RESULTS： Treatments with L. ported at 200 and 400 pg/mL increased the viability of PBLs. The deleterious effect of H2O2 was ameliorated by 400μg/mL L. ported treatment. Addition of 400 μg/mL L. ported reduced lipid peroxidation in stressed PBLs by 94% （P〈0.05）. Treatment with 400 μg/mL of L. ported resulted in a 26.4% increase of reduced glutathione levels. Activities of superoxide dismutase and catalase increased by 17.5% and 55.2% respectively, when stressed PBLs were treated with 400 μg/mL L. ported for 2 d （P〈0.05）. Treatment with 400 μg/mL L. ported increased interferon-γand interleukin-2 expressions in H2O2-challenged PBLs （P〈0.05）, however, the root extract did not cause a significant difference in interleukin-10 levels compared to the control （P〉0.05）. CONCLUSION： The findings suggest that L involving protective effects against oxidative ported might be a potential immune-modulating agent damage.

Tim-3 mRNA expression in peripheral blood lymphocytes from asthmatic patients

The study aimed to detect the expression of the Th1-specific cell surface protein T cell Ig and mucin domain-containing molecule-3(Tim-3)mRNA peripheral blood lymphocytes isolated from asthmaticin patients and to examine the correlation among Tim-3 mRNA,interleukin-4(IL-4),interferon-γ(IFN-γ)level,and FEV1/FVC(force expiratory volume in one second/forced vital capacity)to explore the role of Tim-3 in the development and progression of asthmatic inflammation.Tim-3 mRNA expression was detected by reverse transcription polymerase chain reaction(RT-PCR).The IL-4 and IFN-γlevels were determined by using enzyme-linked immunosorbent assay(ELISA).The correlation among Tim-3 mRNA,IL-4,IFN-γlevel,and pulmonary ventilatory capacity was analyzed.The expres-sion of Tim-3 mRNA in patients with acute asthma exacerbation was 0.39�0.06,significantly higher than that in patients at the remission stage and controls(0.18�0.05 and 0.07�0.03,P<0.05).The level of IL-4 in patients with acute asthma exacerbation was 68.42�10.54,significantly higher than that in the patients at the remission stage and controls(41.83�9.37 and 32.75�8.16,P<0.05).The level of IL-4 in patients in remission was significantly higher than that in controls(P<0.05).The level of IFN-γin patients with acute asthma exacerbation was 65.74�7.85,significantly lower than that in patients in remission and the control group(120.84�11.62 and 139.65�13.47,P<0.05).The level of IFN-γin patients in asthma remission was significantly lower than that in controls(P<0.05).Tim-3 mRNA expression was positively correlated with the level of IL-4(r=0.68,P<0.05)and negatively with the level of IFN-γand pulmonary ventilatory capacity(r=–0.85,r=–0.76,both P<0.01).The increased expression of Tim-3 mRNA in peripheral blood lymphocytes might be involved in the development and progression of asthmatic inflammation.

Chromosome aberration and micronucleus frequency in peripheral blood lymphocytes in radiation workers

Objective To investigate chromosome aberration and micronucleus frequency in peripheral blood lymphocytes in workers engaged in radiation for a long time,to reduce occupational hazard caused by ionizing radiation and to further strengthen health surveillance.Methods A total of 366 members of medical staff engaged in radiation work who underwent physical examinations in

Correlation Study Between T Lymphocyte Subsets and Rheumatoid Arthritis

Objective:To study the effect of Helicobacter pylori infection on rheumatoid arthritis and T-lymphocyte subpopulations in patients with rheumatoid arthritis and to provide a new method for the treatment of rheumatoid arthritis by removing Helicobacter pylori from patients.Methods:60 patients with rheumatoid arthritis admitted to the hospital from May 2022 to May 2023 were selected for the study,and all patients underwent a 13-carbon urea breath test to detect gastric H.pylori and the test results showed that 20 cases were negative and 40 cases were positive.The 40 positive patients were divided into the treatment group(n=20)and non-treatment group(n=20)by random number table method and the treatment group was given anti-Helicobacter pylori treatment,and the non-treatment group was given maintenance rheumatoid basic treatment,comparing the anti-cyclic citrulline peptide(CCP),DS28 score,peripheral blood T-lymphocyte subsets(CD4^(+)T-lymphocytes,CD8^(+)T-lymphocytes,CD4^(+)/CD8^(+)ratio)before and after the treatment of patients by 13-carbon urea respiration test(pylori-negative group,20 patients)and those who were positive for the treatment of H pylori(pylori-positive group,40 patients).Besides,the correlation of peripheral blood T-lymphocyte subsets and disease activity between treatment and non-treatment groups in the pylori-positive group was identified together with the correlation of DS28 scores,TNF-αlevels,sedimentation and immunoglobulin,lymphocyte subsets in the pylori-positive treatment group and positive non-treatment group as well as the level of globulin,lymphocyte subsets,and peripheral blood lymphocytes before and after treatment.Results:Before treatment,CCP,DS28 score,CD8^(+)T lymphocyte level of the pylori-negative group were lower than that of the positive group,and CD4^(+)T lymphocyte and CD4^(+)/CD8^(+)ratio were higher than that of the positive group(P<0.05);after treatment,the indexes of the pylori-positive group improved,and there was no significant difference in the comparison of the indexes with those of the pylori-negative group(P>0.05);the positive treatment group had a DS28(3.19±1.02)points,positive non-treatment group DS28(5.36±1.85)points,non-treatment group DS28 score and CD4^(+)T lymphocytes,CD4^(+)/CD8^(+)negative correlation with CD8^(+)T lymphocytes showed a positive correlation(P<0.05);before the treatment,pylori-positive treatment group and non-treatment group DS28 scores,TNF-αlevels,peripheral blood T lymphocyte subpopulation levels were not significantly different(P>0.05);after treatment,DS28 score,TNF-αlevel,CD8^(+)T of the treatment group were lower than those of the non-treatment group,and CD4^(+)T lymphocytes and CD4^(+)/CD8^(+)ratio were higher than those of the non-treatment group(P<0.05).Conclusion:H.pylori affects the level of T lymphocyte subsets in patients with rheumatoid arthritis,and there is a certain correlation between the two.Removal of H.pylori can improve the level of T lymphocyte subsets,which is important for the treatment of patients with rheumatoid arthritis.

Cytogenetic Effects of Pulsing Electromagnetic Field on Domestic Pig Lymphocytes in Vitro

The effects of pulsing electromagnetic fields(PEMFs)on cells are very important subjects in the field of bioelectromagnetics.In this experiment,the cytogenetic effects of PEMF on domestic pig lymphocytes were tested in vitro.Pig lymphocytes in RPMI 1640 medium were exposed to PEMFs of 100 kHz and 200 kHz for 12,24 and 48 hours.Chromosomal aberrations(aneuploidy,breaks,gaps,et al)were significantly increased in exposed cultures,and of these aberrations,56%chromosomal or chromatid breaks and 42%gaps induced by PEMFs were the points of pig chromosomal fragile sites.The baseline frequency of sister chromatid exchange(SCE)increased after exposing lymphocytes continuously to PEMFs of 100 kHz and 200 kHz for 48 hours.These results suggested that the exposure to PEMFs might induce a type of DNA lesion and chromosomal aberrations.

EFFECT OF ELECTROACUPUNCTURE ON RED BLOOD CELL IMMUNE AND T-CELL SUBGROUP IN THE RAT

In the present study, the effect of electroacupuncture (EA) on immune system was observed in the rat by using micro- whole blood direct immunofluorescence staining assay to detect changes of the peripheral blood T lymphocyte subgroup and employing red blood cell (RBC) C 3b receptor- yeast rosette test and red blood cell-IC rosette test to analyze erythrocytic immune function. Results showed that after EA of “Zusanli" (ST 36), CD+ 4, RBC-C 3bRR and RBC-ICR in the peripheral blood of the normal rats increased significantly while CD+ 8 had no any considerable changes and a positive correlation between CD+ 4 and RBC-C 3bRR was found. In immunosuppression model rats, the values of CD+ 4 and RBC-C 3bRR were obviously lower than those of the normal control group while CD+ 8 had no any striking changes; but after EA treatment, there were no evident differences between EA group and normal control group in the above-mentioned indexes. There were also no any significant differences between non-acupoint group and normal control group in those indexes. Results suggest that EA of “Zusanli" (ST 36) can raise T cell immune function and RBC adhesion function in both normal rats and immunosuppression model rats, both of which present a positive correlation.

Human papilloma virus 16 E6 oncoprotein associated with p53 inactivation in colorectal cancer

AIM:To investigate the association between human papilloma virus(HPV) infection and colorectal cancer.METHODS:Sixty-nine patients with pathologically confirmed primary colorectal cancer including 6 stage Ⅰ,24 stage Ⅱ,21 stage Ⅲ,and 18 stage Ⅳ patients were enrolled in this study to investigate whether HPV 16 could be involved in colorectal tumorigenesis.Nested-polymerase chain reaction(nested-PCR) was used to detect HPV16 DNA in colorectal tumor tissues and further confirmed by in situ hybridization(ISH).In addition,immunohistochemistry analysis was performed to examine the E6 oncoprotein in colorectal tumors.To verify whether E6 could inactivate the p53 transcriptional function,the levels of p21 and Mdm2 mRNA expression were evaluated by real-time reverse transcription(RT)-PCR.RESULTS:Of the 69 colorectal tumors,HPV16 DNA was detected in 11(16%) by nested-PCR,and HPV16 DNA was present in 8 of the 11(73%) tumors which was confirmed by ISH.The presence of HPV16 DNA in colorectal tumors was not associated with patients' clinical parameters including age,gender,smoking status,tumor site;however,HPV16 infection was more common in stage Ⅰ patients than in late-stages patients(Ⅱ,Ⅲ and Ⅳ).We next asked whether HPV16 infection could be linked with colorectal cancer development.Immunohistochemical data indicated that 8 of the 11 HPV16 DNA-positive tumors had E6 oncoprotein expression.Moreover,we also observed that the adjacent normal tissues including endothelial cells,lymphocytes,fibroblasts,and gland cells in E6-positive tumors had E6 oncoprotein expression.In addition,3 of the 4(75%) E6-positive tumors carrying p53 wild-type had negative immunostaining,but one tumor had less p53 immunostaining.We further examined whether E6-positive and/or p53 mutated tumors reduce p53 transcriptional activity.Real-time RT-PCR analysis indicated that p21 and mdm2 mRNA expression levels in E6/p53-wildtype tumors were significantly lower than in their adjacent normal tissues;as expected,E6-positive/p53-mutated tumors had lower p21 and mdm2 mRNA expression levels compared with their adjacent normal tissues.These results clearly indicate that the E6 oncoprotein expressed in p53 wildtype tumors may reduce p21 and mdm2 expression via p53 inactivation.CONCLUSION:These results suggest that HPV16 infection may be involved in a subset of colorectal cancer,and we suggest that the transmission of HPV to the colon and rectum might occur through peripheral blood lymphocytes.

A machine learning model to predict efficacy of neoadjuvant therapy in breast cancer based on dynamic changes in systemic immunity

Objective:Neoadjuvant therapy(NAT)has been widely implemented as an essential treatment to improve therapeutic efficacy in patients with locally-advanced cancer to reduce tumor burden and prolong survival,particularly for human epidermal growth receptor 2-positive and triple-negative breast cancer.The role of peripheral immune components in predicting therapeutic responses has received limited attention.Herein we determined the relationship between dynamic changes in peripheral immune indices and therapeutic responses during NAT administration.Methods:Peripheral immune index data were collected from 134 patients before and after NAT.Logistic regression and machine learning algorithms were applied to the feature selection and model construction processes,respectively.Results:Peripheral immune status with a greater number of CD3^(+)T cells before and after NAT,and a greater number of CD8^(+)T cells,fewer CD4^(+)T cells,and fewer NK cells after NAT was significantly related to a pathological complete response(P<0.05).The post-NAT NK cell-to-pre-NAT NK cell ratio was negatively correlated with the response to NAT(HR=0.13,P=0.008).Based on the results of logistic regression,14 reliable features(P<0.05)were selected to construct the machine learning model.The random forest model exhibited the best power to predict efficacy of NAT among 10 machine learning model approaches(AUC=0.733).Conclusions:Statistically significant relationships between several specific immune indices and the efficacy of NAT were revealed.A random forest model based on dynamic changes in peripheral immune indices showed robust performance in predicting NAT efficacy.

The approaches in detecting cell cycle specificity of Fas-mediated apoptosis in leukemia cell lines and activated PBLs in vitro

Objective： To establish a system in detecting the cell cycle specificity induced by recombinant human Fas ligand in vitro, so as to provide a reliable platform for further exploring the mechanism of cell cycle control and regulation in Fas-mediated apoptosis. Methods： The target cells-leukaemia cell lines and activated peripheral blood lymphocytes stimulated by phytohemagglutinin were incubated with recombinant human Fas ligand for 6 to 36 h, apoptosis was detected by sub-G1, common annexin-Ⅴ/PI and modified annexin Ⅴ and propidium iodide （API） methods and analysed by flow cytometry. Results： The modified API method demonstrated that Fas-mediated apoptosis was cell cycle specific and initiated at G1 phase. The common annexinⅤ/PI method showed the most appropriate condition for the detection of typical cell cycle-specific apoptosis. The sub-G1 method could only illuminate late apoptosis and DNA histogram. Conclusion： Fas-mediated apoptosis was cell cycle-specific and initiated at G 1 phase. Based on the modified API and common AnnexinⅤ/PI methods, the establishment of stable and typical cell cycle-specific model in Fas-mediated apoptosis in vitro was feasible.

Pharmacokinetics of H002, a novel S1PR_1 modulator, and its metabolites in rat blood using liquid chromatography–tandem mass spectrometry

A rapid and sensitive liquid chromatography–tandem mass spectrometry(LC–MS/MS) method was developed and validated for the simultaneous determination of H002 and its phosphorylated metabolite, H002-P and hydroxylated metabolite H002-M, in rat blood. H001, an analogue of H002, was used as the internal standard.Blood samples were prepared by simple protein precipitation. The analytes and internal standard were separated on a Zorbax SB-C18 column with a gradient mobile phase consisting of methanol and water containing 0.1% formic acid at a flow rate of 0.2 mL /min with an operating temperature of 20 1C. The detection was performed on a triple quadrupole tandem mass spectrometer with positive electrospray ionization in multiple-reaction monitoring mode.Linear detection responses were obtained from 0.2–100 ng/mL for H002 and H002-M, while 0.5–100 ng/mL for H002-P. The intra- and inter-day precision(RSD%) was within 11.76%, with the accuracy(RE%) ranging from –9.84% to 9.12%. The analytes were shown to be stable during sample storage, preparation and analytic procedures.The method was applied to determine the pharmacokinetics of H002 in rats, and a preliminary study showed that the pharmacokinetics of H002 correlated with its biological effect on peripheral blood lymphocytes.

An improved anti-leukemic effect achieved with donor progenitor cell infusion for relapse patients after allogeneic bone marrow transplantation

Objective To observe the antileukemic effect in relapse patients by infusion of donor immunocompetent cells with or without granulocyte colony-stimulating factor (G-CSF) mobilization.Methods Twenty patients with leukemia in relapse after allogeneic bone marrow transplantation (allo-BMT) were treated with chemotherapy followed by donor-derived lymphocytes (DDL) without G-CSF mobilization (Group A, n=11), or donor peripheral blood progenitor cells (PBPCs) with G-CSF mobilization (Group B, n=9).Results Five patients in Group A were in hematologic relapse. After DDL infusion, 3 of 5 patients had a temporary complete remission (CR) and relapsed after 3, 7 and 10 months, respectively. One achieved partial remission and died of interstitial pneumonia; and the other one showed no response. Another 6 patients in Group A were in cytogenetic relapse or central nerve system (CNS) leukemia, and all achieved CR and remained in disease free survival (DFS) for 10 to 98 months after DDL infusion. All 9 patients in group B were in hematologic relapse. Three patients with chronic myeloid leukemia (CML) had cytogenetic and molecular remission for 16, 35 and 51 months, respectively after PBPC infusion; and 5 patients with acute lymphoid leukemia (ALL) had CR and were still in CR for 10 to 18 months except 1 patient relapsed soon. And the other one with AML showed no response to the therapy.Conclusion Donor immunocompetent cells infusion is an effective therapy for relapsed leukemia after allo-BMT, especially for the patients with early (molecular and cytogenetic) or CNS relapse. Infusion of donor PBPC mobilized by G-CSF seems to have more potentiated graft-versus-leukemia (GVL) effect than DDL infusion.