OBJECTIVETo study whether human placenta contains hematopoietic stem/progenitor cells (HSPCs), and analyze phenotypes of lymphocyte subpopulations in the placenta.

METHODSNucleated cells from fresh human placenta were analyzed for phenotypes of HSPCs and lymphocyte subpopulations by flow cytometry (FCM). And CD(34)(+) cells were sorted from human placenta nucleated cells by FCM or MiniMACS.

RESULTS(1) CD(34)(+) cells, CD(34)(+)/CD(38)(+) cells, and CD(34)(+)/CD(38)(-) cells from a human placenta were 8.8, 4.6 and 11.9 times higher than those from umbilical cord blood (UCB), respectively. (2) The yields and purity of CD(34)(+) cells isolated from human placenta by FCM sorting system were (63.05 +/- 10.14)% and (86.39 +/- 11.27)%, respectively. (3) Lymphocytes, T cells (CD(3)(+)/CD(2)(+)), B cells (CD(19)(+)), Th cells (CD(3)(+), CD(4)(+)), and Th/Ts ratio in the placenta tissue were apparently lower than those in the UCB, while the CD(8)(+)/CD(28)(-) T suppressor cells were higher in the placenta than in the UCB.

CONCLUSIONSHuman placenta is rich in HSPCs, and has important hematopoietic function in ontogeny. It is probable that human placenta would be graft resource for HSPCs transplantation. CD(8)(+)/CD(28)(-) T suppressor cells might play an important role in feto-maternal immunologic tolerance.

Cells, Cultured ; Female ; Hematopoietic Stem Cells ; cytology ; immunology ; Humans ; Lymphocyte Count ; Lymphocyte Subsets ; cytology ; immunology ; Male ; Placenta ; cytology ; immunology ; Pregnancy

OBJECTIVETo investigate the frequency distribution features of innate-like lymphocytes (iNKT cells, γΔT cells and B1 cells) in peripheral blood of normal adults.

METHODSThe flow cytometry with 6 fluorescence staining was used to detect the percentages of iNKT lymphocytes, γΔT lymphocytes, B1 lymphocytes and adaptive T lymphocyte, B2 lymphocytes in peripheral blood lymphocytes of 50 normal adults. The difference and correlation between these lymphocyte subsets were analyzed by statistical software.

RESULTSThe percentage of iNKT cells in peripheral blood of 50 normal adults was 0.18% (0.01%-2.01%), the percentage of γΔT cells was 4.90% (1.45%-20.14%), the percentage of B1 lymphocytes was 1.62% (0.20%-3.77%), the percentage of adaptive T cells was 63.52% (33.20%-83.22%), the percentage of B2 cells was 6.64% (3.07%-13.80%). B1 and B2 were two subsets of B lymphocyte, the percentage of B2 in B lymphocyte was 81.43% (57.90%-94.12%) and more than that of B1 lymphocyte; the percentage of B1 lymphocytes was 17.28% (5.28%-41.13%). In T lymphocyte group the percentage of iNKT cell was 0.32% (0.01%-3.6%), the percentages of γΔT cells and adaptive T cells were 7.55% (3.04%-27.66%) and 91.98% (72.22%-96.86%) respectively. Spearman correlation analysis was used to analyze the correlation between the percentages of several lymphocyte subsets. There was a positive correlation between iNK T cells and γΔT cells, γΔT cells and adaptive T cells, B1 cells and B2 cells (r=0.39, P=0.0056; r=0.6028, P<0.0001; r=0.4791, P=0.0004). It was also found that the percentage of iNKT cells in female peripheral blood lymphocytes was 0.29% (0.06%-2.01%), and significantly higher than that in male peripheral blood lymphocytes 0.12% (0.01%-1.37%) (P<0.05).

CONCLUSIONThe percentages of γΔT cells, B1 cells and iNKT cells in peripheral blood lymphocytes of normal adults are significantly lower than that of adaptive lymphocytes, and their contents in peripheral blood decrease in turn. There are no sex differences in the percentages of these lymphocyte subsets except iNKT cells.

Adult ; B-Lymphocytes ; cytology ; Female ; Flow Cytometry ; Humans ; Male ; Natural Killer T-Cells ; cytology ; T-Lymphocyte Subsets ; cytology

OBJECTIVETo investigate the value of lymphocyte count in assessing cellular immune function in patients with community-acquired pneumonia.

METHODSNinety-three patients with community-acquired pneumonia (including 53 non-severe and 40 severe cases) and 52 healthy adults were examined for routine blood test and T lymphocyte count. Blood lymphocyte counts and absolute T lymphocyte counts were compared among the 3 groups and their correlation was analyzed.

RESULTSCompared with the healthy control subjects, patients with community-acquired pneumonia showed significantly lower blood lymphocyte counts and CD3(+), CD4(+), and CD8(+) levels (P<0.05). CD3(+), CD4(+), and CD8(+) levels were positively correlated with blood lymphocyte counts. With blood lymphocyte count as the independent variable (L), and the regression equations for CD3(+), CD4(+), and CD8(+) levels were CD3(+)=485.45L+313.48 (F=59.68, P<0.01), CD4(+)=192.57L+290.11 (F=24.62, P<0.01), and CD8(+)=275.14L+18.04 (F=23.46, P<0.01) in the control group; CD3(+)=564.15L+25.04 (F=96.56, P<0.01), CD4(+)=381.91L-37.45 (F=68.60, P<0.01), and CD8(+)=165.61L+61.83 (F=55.47, P<0.01) in non-severe pneumonia group; and CD3(+)=565.44L+49.09 (F=31.87, P<0.01), CD4(+)=332.34L-17.37 (F=43.64, P<0.01), and CD8(+)=223.46L+54.39 (F=13.90, P<0.01) in severe pneumonia group.

CONCLUSIONPatients with community-acquired pneumonia have decreased cellular immune function. Absolute T lymphocyte count can be estimated by blood lymphocyte count to save the cost of laboratory tests.

Adult ; Case-Control Studies ; Community-Acquired Infections ; immunology ; Humans ; Lymphocyte Count ; Pneumonia ; immunology ; T-Lymphocyte Subsets ; cytology

Lymphocyte subset analysis is widely used in clinical laboratories, and more than two levels of daily QC materials are required for reliable results. Commercially available, expensive QC materials have short shelf lives and may not be suitable in resource-poor settings. We compared different methods for preparing homemade QC material, including fixation with 1%, 2%, or 4% paraformaldehyde (PFA); freezing with 10% dimethylsulfoxide (DMSO), 0.1% bovine serum albumin-phosphate buffered saline, or after ethanolic dehydration; and using cryopreservation temperatures of -20℃, -80℃, or -196℃. We found an optimal experimental condition, which is 'fixation with 4% PFA, freezing with 10% DMSO, and storage at 80℃'. To evaluate long-term stability of QC materials prepared in this optimal condition, two levels of QC materials (QM1 and QM2) were thawed after 30, 33, 35, 37, 60, 62, 64, and 67 days of cryopreservation. Lymphocyte subset was analyzed with BD Multitest IMK kit (BD Biosciences, USA). QM1 and QM2 were stable after 1-2 months of cryopreservation (CV <3% for CD3, CD4, and CD8 and 5-7% for CD16/56 and CD19). We propose this method as an alternative cost-effective protocol for preparing homemade internal QC materials for lymphocyte subset analysis in resource-poor settings.
Cryopreservation ; Cryoprotective Agents/chemistry ; *Flow Cytometry/standards ; Lymphocyte Subsets/*cytology ; Quality Control ; Reagent Kits, Diagnostic ; Time Factors

OBJECTIVETo measure the quantities and apoptosis-related protein levels of B lymphocyte in the patients with immunorelated pancytopenia (IRP) and explore the action of B lymphocyte in the pathogenic mechanism of IRP.

METHODSQuantities of whole B lymphocytes and CD5+ B lymphocytes as well as the expressions of Fas and Bcl-2 in B lymphocytes in 35 patients with untreated IRP, 15 IRP patients in complete remission (CR), and 10 normal controls were assayed by flow cytometry.

RESULTSThe percentages of B lymphocyte and CD5+ B lymphocyte were significantly higher in untreated IRP patients than in CR IRP patients and normal controls (P < 0.05), and there was no significant difference between the latter two groups (P > 0.05). There was no significant difference of Fas expression in B lymphocyte among three groups (P > 0.05). The expression of Bcl-2 in B lymphocyte was significantly higher in untreated patients than in CR patients or normal controls (P < 0.01), and significantly higher in CR patients than in normal controls (P < 0.01). The apoptosis-related index was significantly lower in untreated patients than in CR patients or normal controls (P < 0.05), and significantly lower in CR patients than in normal controls (P < 0.05). The percentage of B lymphocyte was positively correlated with post-treated response time (r = 0.53, P < 0.01).

CONCLUSIONThe production of auto-antibodies in IRP patients probably has some relationship with the abnormal quantities of B lymphocyte and its subpopulations as well as with the inhibition of B lymphocyte apoptosis.

Adolescent ; Adult ; B-Lymphocytes ; cytology ; immunology ; Case-Control Studies ; Child ; Female ; Humans ; Lymphocyte Subsets ; Male ; Middle Aged ; Pancytopenia ; immunology ; pathology

OBJECTIVESTo observe the changes of serum interleukins (IL), T-lymphocyte subsets, and white blood cell (WBC) count in patients with severe acute respiratory syndrome (SARS), and to investigate the relationship between injured immune function, immune response and disturbed immune adjustment in SARS patients.

METHODSThe levels of serum IL-2, IL-10, IL-12 and T-lymphocyte subset counts were measured in 35 clinically diagnosed SARS patients by using enzyme linked immunosorbant assay (ELISA). The relationship between the measured results and WBC count was further analyzed.

RESULTSThe level of serum IL was increased to a great extent in the 35 SARS patients, and the levels of serum IL-2, IL-10 and IL-12 were 242.53 (92.69) pg/ml, 77.43 (63.37) pg/ml and 65.94 (43.21) pg/ml, respectively. The level of serum IL-2 increased markedly (P < 0.01). The peripheral blood CD(3)(+), CD(4)(+) and CD(8)(+) counts were lower than normal in 23 patients (67.7%), 26 patients (74.3%) and 15 patients (42.9%), respectively. The peripheral blood WBC counts were lower than 4.0 x 10(9)/L in 10 patients, and their CD(3)(+), CD(4)(+) and CD(8)(+) counts were 583.90 (315.58) x 10(6)/L, 272.00 (94.13) x 10(6)/L and 209.00 (72.21) x 10(6)/L, respectively. The peripheral blood WBC counts were (4.0 - 10.0) x 10(9)/L in 20 patients, and their CD(3)(+), CD(4)(+) and CD(8)(+) counts were 700.00 (502.96) x 10(6)/L, 347.00 (247.58) x 10(6)/L and 322.05 (228.47) x 10(6)/L, respectively. The peripheral blood WBC counts were higher than 10.0 x 10(9)/L in 5 patients, and their CD(3)(+), CD(4)(+) and CD(8)(+) counts were 1466.00 (630.86) x 10(6)/L, 783.00 (311.14) x 10(6)/L and 640.00 (294.40) x 10(6)/L, respectively. The decreased CD(3)(+), CD(4)(+) and CD(8)(+) counts were consistent with the decreased WBC counts. The level of IL in SARS patients was significantly higher than that in patients with chronic hepatitis B (P < 0.01).

CONCLUSIONSThe level of serum IL is closely related to cell immunity in SARS patients. The level of serum IL is increased evidently while CD(3)(+), CD(4)(+) and CD(8)(+) counts decrease. Both serum IL and CD are associated with injury of immune function, and thus they could be regarded as a monitoring index for judging the condition of SARS patients and prescribing immune therapy.

Adult ; Female ; Humans ; Interleukins ; blood ; Leukocyte Count ; Male ; Middle Aged ; Severe Acute Respiratory Syndrome ; immunology ; T-Lymphocyte Subsets ; cytology

The study was aimed to investigate the changes of T-cell subgroups in the peripheral blood (PB) of patients with aplastic anemia (AA) and the relationships between these changes and the pathogenesis of AA and the immunosuppressive therapeutic effects in AA, in order to provide a basis for selecting rational therapy of AA patients. T-cell subtype and the ratio of CD4+/CD8+ cell in the PB of 88 AA patients which had been diagnosed clearly and given conventional therapy or conventional therapy combined with immunotherapy were analyzed by tri-colour fluorescence-labeled monoclonal antibody and using multiparameter flow cytometry. The patients with AA were divided into normal type of ratio, inverted type of ratio, hypernormal type of ratio according to the ratio of CD4+/CD8+ cell in normal group, and then the relations of these subtype with patients' conditions and therapeutic effects were investigated. The results showed that the percentage of normal type of ratio in all patients was 39.8%, the percentage of inverted type of ratio in all patients was 44.3%, The percentage of hypernormal type of ratio in all patients was 15.9%. In the conventional therapy alone, there was no significant difference on therapeutic effects among these three immunological subtypes. In combined immunotherapy, total therapeutic efficacy of AA patients with inverted type of ratio and AA patients with immunologic abnormality (inverted type + hypernormal type) was 84.2% and 82.6% respectively, which were more than that in conventional therapy (45.5% and 42.8%) (p < 0.05). Total therapeutic efficacy in these patients was better than that in AA patients with normal type. It is concluded that significant abnormal ratios of CD4+/CD8+ exist in the majority of AA patients, abnormal ratios of CD4+/CD8+ both may be showed as increase or decrease, immunologic abnormality may play a role in pathogenesis of the patients with AA. The detection of PB T-cell subtype in patients with aplastic anemia contributes to evaluation of patients' condition and choice of rational treatment prescription, and enhancement of diagnostic level and therapeutic efficacy significantly, which is an important indicator for therapeutic strategy also.
Adult ; Anemia, Aplastic ; immunology ; CD4-CD8 Ratio ; Female ; Humans ; Male ; Middle Aged ; T-Lymphocyte Subsets ; cytology ; immunology ; Young Adult

The aim of this study was to investigate the effect of human bone marrow mesenchymal stem cells on human T-cell proliferation resulted from stimulation with PHA and possible immunomodulating mechanism. T cells were positively selected by CD3(+) magnetic beads, and were then co-cultured with irradiated MSCs overnight before the addition of PHA. T-cell proliferation was measured by BrdU assay and the degree of apoptosis was assessed by flow cytometry with Annexin V/PI. T cells co-cultured with or without MSCs were treated with PHA for 72 hours, then harvested. They were labeled with anti-CD4, anti-CD8, anti-CD25 antibodies and analyzed by flow cytometry. The results showed that MSCs inhibited T-cell proliferation, but did not induce T cell apoptosis. There were no significant changes in the ratio of CD4(+) and CD8(+) T cells of MSC-treated group, as compared with the control group. After stimulation with PHA, there was an increase in CD4(+) T cells and decrease of CD4(+)CD25(+) cells in MSC co-cultured group. It is concluded that the MSCs inhibit T-cell proliferation after stimulation with PHA, and show more inhibitive effects on CD8(+) and CD4(+) T cells, but CD25(+) regulatory T cells may not be involved in this process.
Bone Marrow Cells ; cytology ; CD4-CD8 Ratio ; Cell Proliferation ; Cells, Cultured ; Coculture Techniques ; Humans ; Mesenchymal Stromal Cells ; cytology ; physiology ; T-Lymphocyte Subsets ; cytology

OBJECTIVETo explore the mechanism of electroacupuncture of "Shuanggu Yitong" prescription on postponing aging.

METHODSForty 3-month SD rats, male only, 30 rats were made sub-acute aging model by D-galactose s.c. injection continuously for 42 d, and rest of the rats, 10, were divided into a normal control group. After the modeling, the sub-acute aging model rats were randomly into a Shuanggu Yitong group [electroacupuncture at "Guanyuan" (CV 4) and "Zusanli" (ST 36), hand needle at "Baihui" (GV 20)], an acupuncture control group [electroacupuncture at "Weizhong" (BL 40) and "Shuifen" (CV 9), hand needle at "Yintang" (GV 29)] and an aging model group, ten in each one. The treatment was given once in a day, six of which made a course. The rats in the normal control group and aging model group were not received any treatment. After the treatment for three weeks, the rats were put to death and their spleen index, thymus index and the T lymphocytes subgroups (CD8(+) T/T cell and CD8(+) CD28(-) T/CD8(+) T cell) were tested.

RESULTSThe spleen index (1.74 +/- 0.059) and thymus index (0.64 +/- 0.039) in the aging model group was obviously lower than those in the normal control group (1.93 +/- 0.061), (0.81 +/- 0.053) respectively (both P < 0.05); the CD8(+) CD28(-) T/CD8(+) T cell percentages (26.28 +/- 4.69)% and CD8(+) T/T cell percentages (43.33 +/- 2.84)% in the aging model group were both significantly higher than those (15.08 +/- 5.58)% (P < 0.01), (34.70 +/- 4.24)% (P < 0.01) in the normal control group. Compared with the aging model group, the spleen index (1.91 +/- 0.081) and thymus index (0.79 +/- 0.080) in the Shuanggu Yitong group were significantly higher (both P < 0.05), but obviously decreased with the percentage of CD8(+) CD28(-) T/CD8(+) T cell (18.07 +/- 1.73) (P < 0.01); the percentage of CD8(+) CD28(-) T/CD8(+) T cell (18.07 +/- 1.73)% in the acupuncture control group was also lower than the aging model group (P < 0.05), but more obvious reduce for the Shuanggu Yitong group (P < 0.05).

CONCLUSIONThe treatment of Shuanggu Yitong prescription could regulate the proportions of the T lymphocyte subset, and slow down the immunosenescence of subacute aging model rats induced by D-galactose.

Acupuncture Points ; Aging ; physiology ; Animals ; Electroacupuncture ; Flow Cytometry ; Humans ; Male ; Rats ; Rats, Sprague-Dawley ; Spleen ; cytology ; T-Lymphocyte Subsets ; cytology ; Thymus Gland ; cytology

OBJECTIVETo verify the clinical efficacy on chronic fatigue syndrome of qi deficiency syndrome treated with acupuncture at selective time and explore the effect mechanism.

METHODSEighty patients were randomized into a selective-time-acupuncture group and an acupuncture group, 40 cases in each one. Qihai (CV 6), Guanyuan (CV 4), Hegu (LI 4), Taichong (LR 3), Sanyinjiao (SP 6) and Zusanli (ST 36) were selected in the two groups. In the selective-time-acupuncture group, acupuncture was used at 9:00am to 11:00am. In the acupuncture group, acupuncture was used at any time except in the range from 9:00am to 11:00am. No any manipulation was applied after the arrival of needling sensation. The treatment was given once every day, 10 day treatment made one session and two sessions of treatment were required. The fatigue scale was adopted to evaluate the efficacy before and after treatment in the patients of the two groups. The ratios among CD3+, CD4+ and CD8+ T cells in the peripheral blood were detected before ad b a after treatment.

RESULTSIn the acupuncture group, the total score of fatigue and the score of physical fatigue were reduced after treatment as compared with those before treatment (all P<0.05). In the selective-time -acupuncture group, the total score of fatigue, the s core of physical fatigue and the score of mental fatigue after treatment were reduced obviously as compared with those hefore treatment (all P<0. 01). The improvements in the scores of the selective-time-acupuncture group were superior to the acupuncture group (all P<0. 05). The ratio of CD3+ and CD8+ T cells was increased obviously after treatment in the two groups (all P<0. 05) and the ratio of CD4+ and CD8+ T cells was reduced obviously in the selective-time-acupuncture group (P<0. 05), which was better than that in the acupuncture group (all P<0.05). The total effective rate was 95.0% (38/40) in the selective-time-acupuncture group, which was better than 80.0% (32/40) in the acupuncture group (P<0.05).

CONCLUSIONThe acupuncture therapy at selective time is effective in the treatment of chronic fatigue syndrome of qi deficiency syndrome, which is especially better at relieving mental fatigue. The effect of this therapy is achieved probably by improving the immune function via the regulation of the ratios among CD3+, CD4+ and CD8+ T cells.

Acupuncture Points ; Acupuncture Therapy ; Adult ; Fatigue Syndrome, Chronic ; immunology ; therapy ; Female ; Humans ; Lymphocyte Count ; Male ; Middle Aged ; Qi ; T-Lymphocyte Subsets ; cytology ; immunology ; Treatment Outcome ; Young Adult