Peripheral T-lymphocyte subpopulations in different clinical stages of chronic HBV infection correlate with HBV load

AIM： To characterize the peripheral T-cell subpopulation profiles and their correlation with hepatitis B virus （HBV） replication in different dinical stages of chronic HBV infection. METHODS： A total of 422 patients with chronic HBV infection were enrolled in this study. The patients were divided into three stages： immune-tolerant stage, immune active stage, and immune-inactive carrier stage. Composition of peripheral T-cell subpopulations was determined by flow cytometry. HBV markers were detected by enzyme-linked immunosorbent assay. Serum HBV DNA load was assessed by quantitative real-time poiymerase chain reaction.RESULTS： CD8^＋ T-cells were significantly higher in patients at the immune-tolerant stage than in patients at the immune-active and -inactive carrier stages （36.87 ± 7.58 vs 34.37 ± 9.07, 36.87 ± 7.58 vs 28.09 ± 5.64, P 〈 0.001）. The peripheral blood in patients at the immune-tolerant and immune active stages contained more CD8^＋ T-cells than CD4^＋ T-cells （36.87 ± 7.58 vs 30.23 ± 6.35, 34.37 ± 9.07 vs 30.92 ± 7.40, P 〈 0.01）, whereas the peripheral blood in patients at the immune- inactive carrier stage and in normal controls contained less CD8^＋ T-cells than CD4^＋ T-cells （28.09 ± 5.64 vs 36.85 ±6.06, 24.02 ± 4.35 vs 38.94 ± 3.39, P 〈 0.01）. ANOVA linear trend test showed that CD8^＋ T-cells were significantly increased in patients with a high viral load （39.41 ± 7.36, 33.83 ± 7.50, 31.81 ± 5.95 and 26.89 ± 5.71, P 〈 0.001）, while CD4^＋ T-cells were significantly increased in patients with a low HBV DNA load （37.45 ± 6.24, 33.33 ± 5.61, 31.58 ± 6.99 and 27.56 ± 5.49, P 〈 0.001）. Nultiple regression analysis displayed that log copies of HBV DNA still maintained its highly significant coefficients for T-cell subpopulations, and was the strongest predictors for variations in CD3^＋, CD4^＋ and CD8^＋ cells and CD4^＋/CD8^＋ ratio after adjustment for age at HBV-infection, maternal HBV-infection status, presence of hepatitis B e antigen and HBV mutation.CONCLUSION： Differences in peripheral T-cell subpopulation profiles can be found in different clinical stages of chronic HBV infection. T-cell impairment is significantly associated with HBV load.

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.

Correlation of Quantitative Changes in Bone Marrow Lymphocyte Subpopulations with Some Rhabdomyosarcoma Prognosis Factors in Children

Rationale: The known prognosis factors for rhabdomyosarcoma (RMS) in children do not always explain the unsatisfactory outcome of treatment. Changes in the subpopulation composition of Bone Marrow (BM) effector cells during the development of RMS may indicate new directions for the search for prognostic factors and points for the impact of targeted therapy. Purpose: To identify correlations between quantitative changes in the levels of subpopulations of T, B and NK-lymphocytes of BM and known risk factors for RMS in children. Objects: The study included 31 patients. The main group included 16 patients with RMS, average age—6.8 ± 1.0 years, while children 1 - 10 years old—13 (81.3%), over 10 years old—3 (18.8%) people, girls and boys were 8 people each. The embryonic variant of RMS was established in 10 (62.5%) cases, the alveolar variant—in 4 (25%) cases, in two patients (12.5%), the histological variant was not established. In 12 (75%) patients, an unfavorable localization of the RMS (parameningeal, extremities, prostate, bladder) was revealed, in 4 patients (25%), the localization of the tumor was regarded as favorable. Patients with T2b—13 (81.2%) and T2a—2 (12.5%) stages prevailed. Regional and distant metastases were detected in 10 (52.6%) patients. The comparison group included 15 children in whom the presence of malignant neoplasia was excluded, the average age was 8.4 ± 1.5 years, 11 boys (73.7%) and 4 girls (26.3%). Methods: All patients underwent morphological (myelogram counting) and immunological (quantitative analysis of lymphocytic subpopulations) bone marrow studies. Immunophenotyping in all patients was carried out by direct immunofluorescence using a triple fluorescent label. Results: Significant differences in the levels of subpopulations of BM T-lymphocytes were found when comparing the values of the main group, distributed by localization and histological variant, with the data obtained in the control group of patients. For example, the percentage of CD3+ T cells with the co-stimulatory molecule CD28+ was significantly higher in patients with parameningeal RMS (p = 0.010). Conclusion: Each clinical group of patients has its own individual immunological characteristics. The results obtained by us can be considered indicative and regarded as starting points for further study of the peculiarities of the subpopulation composition of BM in patients with RMS.

Study on the blood-borne virus co-infection and T lymphocyte subset among intravenous drug users

AIM： To investigate the features of various blood- borne virus infections and co-infection in intravenous drug users （IDUs）, and to examine the correlation of T lymphocyte subsets with virus co-infection. METHODS： Four hundred and six IDUs without any clinical manifestation of hepatitis and 102 healthy persons were enrolled in this study. HBV-DNA and HCV-RNA were detected by fluorescence quantitative PCR. HBsAg, HBeAg, anti-HBc, anti-HCV, HDV-Ag, anti-HGV, anti-HIV, and HCMV-IgM were assayed by enzyme-linked immunosorbent assay （ELISA） and immunochromatographic tests. The levels of Th1 and Th2 cytokines were measured by ELISA and radioactive immune assay （RIA）. The T lymphocyte subpopulation was detected by using fluorescence immunoassay. The similar indices taken from the healthy persons served as controls. RESULTS： The viral infection rate among IDUs was 36.45% for HBV, 69.7% for HCV, 47.3% for HIV, 2.22% for HDV, 1.97% for HGV, and 3.45% for HCMV. The co- infection rate of blood-borne virus was detected in 255 of 406 （62.81%） IDUs. More than 80% （161/192） of subjects infected with HIV were co-infected with the other viruses, such as HBV, HCV. In contrast, among the controls, the infection rate was 17.65% for HBV and 0% for the other viruses. Our investigation showed that there was a profound decrease in the proportion of CD4/CD8 and the percentage of CD3 and CD4, but not in the percentage of CD8. The levels of PHA-induced cytokines （IFN-γ and IL-4） and serum IL-2 were obviously decreased in IDUs. On the other hand, the level of serum IL-4 was increased. The level of IFN-γ and the percentage of CD4 were continuously decreased when the IDUs were infected with HIV or HIV co-infection. IDUs with HIV and HBV co-infection was 15.1% （29/192）. Of those 29 IDU with HIV and HBV co-infection, 51.72% （15/29） and 37.93% （11/29） were HBV-DNA-positive and HBeAg-positive, respectively. But, among IDUs without HIV infection, only 1.68% （2/119） of cases were HBV- DNA-positive.CONCLUSION： HCV, HBV and HIV infections are common in this population of IDU, leading to a high incidence of impaired Thl cytokine levels and CD4 lymphocyte. IDUs with HIV and HBV/HCV co-infection have lower expression of Th1 cytokine with enhancement of the Th2 response. HIV may be causing HBV replication by decreasing Thl function.

Increase of CD4^+CD25^+ T cells in Smad3^(-/-) mice

AIM： To investigate the changes of lymphocyte subpopulations, especially CD4^＋CD25^ T regulatory cells in Smad3^-/- mice. METHODS： Hematological changes and changes of lymphocyte subpopulations were detected in Smad3＂/- mice using cell counter and flow cytometry, respectively, and compared to their littermate controls. RESULTS： The numbers of neutrophils and lymphocytes in peripheral blood were significantly increased in Smad3^-/- mice compared to littermate controls. CD19^＋ expressing cells in blood and spleen, and CD8^＋ T cells in thymus were all markedly decreased in Smad3^-/- mice. More important, Smad3^-/- mice had an increased population of CD4^＋CD25^＋ T cells in peripheral lymphoid tissues, including thymus, spleen, and lymph nodes. CONCLUSION： These observations suggest that the changes of lymphocyte subpopulations might play a role in susceptibility to inflammation of Smad3^-/- mice.

Clinical Study of Gushen Tablet(固肾片)in Reducing Children's Nephrotic Syndrome Relapse

Objective:To explore the effect of Gushen tablet (固肾片,GST) in reducing the relapse of children's nephrotic syndrome and the possible mechanism of drugs used. Methods: Fifty children with primary nephrotic syndrome who had been induced and alleviated with regular glucocorticoid (GC) were randomly divided into two groups: the GST group used GST and standard middle-long term course of GC, and the control group adopted standard middle-long term course of GC and immunoinhibitory or immuno-modulatory agents for treatment. The 0.5,1 and 2 years after the treatment the relapse episodes, time for urinary protein negative conversion after relapse, the episodes of patient's infection and relapse after infection were evaluated. Before and after treatment the plasma cortisol and T lymphocyte subpopulation were determined. Results: The relapse rate of GST group: the rates after 0. 5, 1, 2 years were 20.0%, 30. 0% and 40. 9%, and the frequent relapse rate were 0, 6. 7% and 9. 2% respectively, which were lower than those of control group (60. 0%, 70. 0%, 69. 2% and 25. 0%, 15. 0%, 15. 4% respectively) ; in the GST group no relapse occurred within 0. 5 year, the relapse rate after 1 and 2 years reduced by 40. 0% and 28. 3%, compared with those of the control group (all P<0. 05) ; during the observation period, the mean infection/every child patient was 1. 86 episodes in GST group, after infection the nephrotic relapse rate was 28.3%, which was lower than that of the control group (2. 25 episodes, 71.1%, P<0. 05) > the relapse per patient in GST group was 0. 8 episodes, time for urinary protein negative conversion was 12. 00± 8. 98 days, lower than those of control group (1. 6 episodes, 20. 75±11. 95 days, P<0. 05) ; 3 months after GST treatment the plasma cortisol level normalized, and the CD4/CD8 ratio elevated (P<0. 05). Conclusion:GST could possibly reduce the relapse of children nephrosis, and the frequent relapse and relapse episodes, and the time for post-relaptic urinary protein negative conversion shortened, the plasma cortisol elevated, and the adjustment of cellular immunity disturbance promoted.

Decrease of Peripheral Blood CD8+/CD28- Suppressor T Cell Followed by Dentritic Cells Immunomodulation among Metastatic Breast Cancer Patients

Objective： To explore the effects of dentritic cells on the peripheral blood lymphocyte subpopulations of metastatic breast cancer patients treated with chemotherapy. Methods： The current study involved 44 metastatic breast cancer patients treated with docetaxel-based chemotherapy. Among them, 25 cases were treated with dendritic cells derived from CD34＋ hematopoietic stem cells enriched autologous peripheral mononuclear cells after chemotherapy, and 19 cases received chemotherapy alone. Peripheral blood samples were collected from each patient before and after treatment, and lymphocyte subpopulations including CD3＋, CD3＋/CD4＋, CD3＋/CD8＋, CD3-/CD16＋56＋, CD3＋/CD16＋56＋, CD4＋/CD25＋, CD8＋/CD28-, CD8＋/CD28＋, CD4/CD8, DC1, DC2 and DC1/DC2 were analysed by a 3-color flow cytometric analysis. Results： The two treatment groups were well matched with regard to demographic and baseline disease characteristics. Comparing the changes of lymphocyte subpopulations between the two groups, it showed that the difference of the change of CD8＋/CD28-lymphocyte had statistic significance. The percentage of CD8＋/CD28-lymphocyte was lower in the chemotherapy＋DC group, but higher in the chemotherapy-alone group. Conclusion： As CD8＋/CD28-lymphocyte represent a kind of suppressive T lymphocyte, we conclude that dentritic cell therapy can relieve immunosuppression to some extent.

Effect of panel reactive antibodies on T cell immunity reinstatement following renal transplantation

BACKGROUND Chronic kidney disease is associated with immunological disorders,presented as phenotypic alterations of T lymphocytes.These changes are expected to be restored after a successful renal transplantation;however,additional parameters may contribute to this process.AIM To evaluate the impact of positive panel reactive antibodies(PRAs)on the restoration of T cell phenotype,after renal transplantation.METHODS CD4CD28null,CD8CD28null,natural killer cells(NKs),and regulatory T cells(Tregs)were estimated by flow cytometry at T0,T3,and T6 which were the time of transplantation,and 3-and 6-mo follow-up,respectively.Changes were estimated regarding the presence or absence of PRAs.RESULTS Patients were classified in two groups:PRA(-)(n=43)and PRA(+)(n=28)groups.Lymphocyte and their subtypes were similar between the two groups at T0,whereas their percentage was increased at T3 in PRA(-)compared to PRA(+)[23(10.9-47.9)vs 16.4(7.5-36.8)μ/L,respectively;P=0.03].Lymphocyte changes in PRA(-)patients included a significant increase in CD4 cells(P<0.0001),CD8 cells(P<0.0001),and Tregs(P<0.0001),and a reduction of NKs(P<0.0001).PRA(+)patients showed an increase in CD4(P=0.008)and CD8(P=0.0001),and a reduction in NKs(P=0.07).CD4CD28null and CD8CD28null cells,although initially reduced in both groups,were stabilized thereafter.CONCLUSION Our study described important differences in the immune response between PRA(+)and PRA(-)patients with changes in lymphocytes and lymphocyte subpopulations.PRA(+)patients seemed to have a worse immune profile after 6 mo follow-up,regardless of renal function.

Sex Hormones Affect Aging Process by Influencing Lipid Profiles, Cellular Immunological Function and Lipid Peroxides and Oxidation System

Objectives To investigate the correlation between sex hormones(SH) and aging. Methods Through epidemiological investigation in our country, the levels of SH were measured by radioimmunoassy; lipid profile, glucose and apolipoprotein by automatic biochemic analytical instrument; T cell subsets by flow cytometer; and MDA, SOD were evaluated by the thiobarbituric acid (TBA) test and the nitrite method modified by Oyanagui respectively using spectrophotometry. Results In men, the serum levels of follicle stimulating hormone ( FSH)、luteinizing hormone(LH) increased significantly with aging, but serum prolactin(PRL) and progesterone(P) levels remained unchanged in all life; Both testosterone (T) and free testosterone (FT) all decreased greatly with aging, but 17β - estradiol( 17β - E_2) was reverse ; E_2 was negatively correlated with T and E_2/T increased with aging. The level of serum total cholesterol (TC) increased with aging, but triglycerides (TG) remain unchanged; compared with young group, high - density lipoprotein cholesterol ( HDL - C) ; HDL - C/TC of other groups decreased significantly, but low - density lipoprotein cholesterol ( LDL - C ) changed inversely; HDL- C/LDL- C reduced slightly with aging and showed no difference between groups. Apolipoprotein A1 (apoA1) and apoB all enhanced greatly with aging; meanwhile the ratio of apoA1/apoB decreased. The concentration of serum glucose (GLU) was unchanged in all life. To compare with those in the young group, CD3 + , CD4 + in other groups reduced greatly, CD4 + remained unchanged. Meanwhile, CD8 + increased significantly with aging. Compared with the young group, serum malondialdehyde(MDA) value of the old ones increased obviously, but the activity of superoxide dismutase(SOD) was reverse. By partial correlation analysis (controlling BMI, FSH, LH and PRL), TC、 LDL-C、apoA1、apoB、CD8 + 、MDA of men all presented a positive correlation with E_2/T respectively, their correlation coefficients (γ) were 0. 262、0. 136、 0. 532、0. 379、0. 394、0. 234 (P < 0. 001 ) ; HDL - C、 HDL- C/TC、HDL- C/LDL- C、CD3 + 、CD4 +/CD8 + 、SOD showed a negatively correlation with E_2/T respectively, γequaled - 0.563、- 0.332、- 0.654、- 0.1530、-0.4140、-0.236(P<0.001). In women, the serum concentrations of FSH、LH increased significantly after menopause; PRL increased little with aging; compared with young group, E_2 and P in postmenopausal groups reduced obviously, E_2/P revealed significant reduce with aging. T enhanced significantly after menopause, but nor did FT. E_2, P and the ratio of E_2/P were negatively correlated with age respectively by bivariate correlation analysis, and a positive relation between T and age. After 70 years old, the level of TC increased obviously, and so did that of TG after menopause; HDL decreased with aging, but LDL increased after 70, with the result that the ratios of HDL- C/TC and HDL- C/LDL- C all reduced with aging; apoA1 decreased gently after 70, but apoB increased signifi- cantly after menopause; correspondingly, the ratio of apoA1/apoB declined obviously. The concentration of GLU increased with aging. CD3 + and CD4 + didn't change until 60, but reduced after 60. Compared with the young groups, CD8 + remained unchanged, CD4 +/CD8 + reduced greatly with aging, CD4 + and CD8 + presented a negatively correlation with age respectively. The value of MDA in serum of women increased notably after 70 years old, but SOD activity already decreased significantly from 60. By partial correlation analysis (controlling BMI, FSH, LH and PRL), HDL-C、CD4 +、CD4 +/CD8 + showed a certain correlation with E_2/P respectively; γ were 0. 245、 0.157、0. 154 ( P <0.05 ) ; TG、 LDL、 apoB、 apoA1/ apoB、SOD presented a negatively correlation with E_2/P respectively, γ were 0. 452、 0. 236、 0.321、 0. 135、 0.156、0.154、0.426 ( P < 0.05 ). Conclusions The Disequilibrium of SH had correlations with lipid profile, cellular immunological function and lipid peroxides and oxidation system; these suggested SH took an important role in the process of aging.

Effects of Tripterygii Totorum in Treating Insulin DependentDiabetes Mellitus Patients with lslet Transplantation

The authors determined both the numbers of T Iymphocyte subpopulations in peripheralblood and the concentration of Cpeptide in serum before experimentation at 15 days, 1 month, 6 months,and 1 year atter islet transplantation in tripterygii totorum (T Ⅱ ) group in which 30 insulin dependent dia-betes mellitus (IDDM) patients were given T Ⅱ and the control group in which 24 IDDM patients did notuse any immunosuppressive agents, and 20 healthy individuals were used as the healthy group. Results:Before transplantation, the numbers of T lymphocyte subpopulations such as CD2,CD4 in the two groupswere lower than those in the healthy group (P<0. 01) the ratio of CD4/CD8 was between 1. 2 and 2. 0.Atter transplantation the numbers of CD2, CD4 in the two groups were elevated, particulary in T Ⅱ groupthe numbers of CD2, CD4 were close to those in the healthy group (P>0. 05) . The ratio of CD4/CD8 in thecontrol group was more than 2. 0. The average dose ot insulin reduced by 84. 2% in 19 patients of the T Ⅱgroup, 2 cases stopped insulin, the peak value of C-peptide was 3. 24±1. 2 ng/ml. In the control groupthe average dose of insulin and the peak value of C-peptide was similar to that in the T Ⅱ group in the first6 months atter transplantation (P>0. 05) , then the dose ot insulin was gradually increased and the con-centration of C-peptide began decreasing. One year after transplantation both the dose of insulin and theconcentration of C-peptide were close to those before transplantation. The study suggests that T Ⅱ haveimmunosuppressive effects in islet transplantation and prolong the survival time of gratts in IDDM patients.

Manipulation of the immune system by non-small cell lung cancer and possible therapeutic interference

Pulmonary carcinomas have developed mechanisms by which they escape the attack of immune cells.Immune checkpoint molecules programmed death 1-programmed death ligand 1(PD1-PDL1)and the cytotoxic T-lymphocyte antigen 4 system have gained attention.The expression of PDL1 by tumor cells causes immune tolerance,and further influences the microenvironment via orchestration by cytokines.Therapy with PDL1 antibodies could restore the cytotoxicity of T-lymphocytes towards tumor cells.Many patients will respond to this treatment.However,resistance mechanisms will counteract this therapy.New investigations have identified additional immune checkpoint inhibitors such as lymphocyte activation gene 3 and T cell immunoglobulin and mucin-domain containing-3.Tumor cells also induce tolerance by manipulating cells of the innate immune system.Macrophages are polarized to tumor-friendly M2,neutrophils into N2 types,and dendritic cells and myeloid suppressor cells are switched to assist tumor cells.Regulatory T cells enter the tumor microenvironment and signal tolerance to cytotoxic cells,inhibiting the influx of NK cells.Soluble mediators either released by tumor cells or cells of the tumor stroma induce immune tolerance,examples including tryptophan and indolamine dioxygenases,arginine and adenosine.Treatment options to counteract these molecules are currently being tested.The tumor stroma has been classified as immune-inflamed,immune-excluded,and immune-desert types.The latter might be switched to an inflamed type by induction of tertiary lymph follicles.Dendritic cells and macrophages normally phagocytose tumor antigens,but inhibitors of phagocytosis can block this.Interference with these molecules is another option for re-establishing the cytotoxic action of the immune system against tumor cells.In this review we will discuss these aspects with a special emphasis on non-small cell lung cancer.