OBJECTIVE: To analyze the preservation effect and related influencing factors of human peripheral blood mononuclear cells under serum-free condition at 4 ℃. METHODS: Human peripheral blood mononuclear cells were isolated by density gradient centrifugation, and stored at 4 ℃ under different cell concentrations, supplemented with human serum albumin, and glucose. The cell viability, total cell number, viable cell number and cell phenotype were detected during preservation of 72 h. RESULTS: With the prolongation of storage time, the number of human peripheral blood mononuclear cells gradually decreased(r=0.982). Compared with the cell concentration of (5-6)×10⁶ cells/ml, the cell number decreased more slowly when the cell storage concentration was (1-2)×10⁶ cells/ml; Adding human serum albumin and glucose can effectively improve the survival rate of human peripheral blood mononuclear cells, among which 2% human serum albumin has a better preservation effect; Compared with the blank control group, the analysis results of cell subsets showed that the downward trends of NK cells and T cells were significantly slowed after adding albumin and glucose. CONCLUSION The cell density of (1-2)×10⁶/ml and 2% human serum albumin are more suitable for the preservation of PBMC, and 5% glucose can improve the preservation effect of human peripheral blood mononuclear cells at 4 ℃.
Humans ; Leukocytes, Mononuclear

OBJECTIVETo establish a method for isolation, cryopreservation and recovery of the highly viable human peripheral blood monomuclear cells (PBMNCs) so as to achieve the long-term preservation of PBMNCs.

METHODSA total of 80-100 ml peripheral blood were collected from the healthy volumteers aged over 50 years old. The PBMNCs were isolated by the Ficoll density gradient technique and cryopreserved gradually by program control method in liquid nitrogen freezer of -196 °C. The serum-free medium and autoloqous plasma medium were test for preservation of PBMNCs. The cell viability was assessed at time point of 1, 2, 4, 8, 12 and 24 months after thawing. Finally, the proliferation ability, purity and cytotoxicity were compared between the autologous immune lymphocytes (AIL) induced from cryopreserved PBMNCs and AIL as control from fresh PBMNCs.

RESULTSAfter separating, the cell viability was 99.6%±0.4%, and the recovery rate of lymphocytes was 58.4%±6.52%. The cell recovery rate of lymphocyte was 89.7%±3.82% at 24 months. The quality assurance program was reliable within 2 years of running. The AIL cells induced with cryopreserved PBMNCs were not significantly different from those induced from fresh PBMNCs in terms of proliferative action, purity and cytotoxicity(CD3(+)CD8(+) ≥45%,CD3(+)CD56(+) NKT≥10%,CD4(+)CD25(+) NKT≤10%).

CONCLUSIONManual separation of lymphocytes in vitro can get enough high-quality PBMNCs. The long-term cryopreserved PBMNC still maintain their high viability. The reinfusion of the clinical autologous immune cells would be advantageous for early tumor immunotherapy. Human AIL induced from cryopreserved PBMNC maintain their anti-tumor ability. These findings have the important implications for the application of these cells to adoptive cellular therapy.

Cytokines ; metabolism ; Humans ; Leukocytes, Mononuclear ; immunology ; secretion ; Ubiquitin ; blood

OBJECTIVE: The aim of this study was to investigate the effect of saliva of patients with chronic periodontitis (CPD) on the differentiation, activation, and secretion of osteoclast-maturing mediators of macrophages. METHODS: A total of 40 saliva samples were collected from healthy donors (n=20) and severe periodontitis patients (n=20). Peripheral blood mononuclear cells (PBMCs) and THP-1 monocyte line cells were challenged with 15% saliva for 5 days. The phenotype, surface marker, and phagocytosis of macrophages were analyzed by flow cytometry and microscopy. Osteoclast-maturing mediators were assayed by using enzyme-linked immunosorbent assay (ELISA) kits. RESULTS: When PBMCs were treated with CPD saliva for 5 days, 61.25%±11.33% of cells were transformed into large granular cells; 86.78%±13.69% of large granular cells were identified as CD14⁺⁺CD16⁺ macrophages. When THP-1 cells were treated with CPD saliva, most cells attached to the bottom of cell culture plates, thereby exhibiting macrophage morphology and releasing additional osteoclast-maturing mediators. Furthermore, the phagocytosis of THP-1 cells considerably increased in the presence of CPD saliva (66.35%±9.67%) compared with medium control (33.33%±7.52%), or healthy saliva (40.71%±3.52%). CONCLUSIONS Saliva from patients with CPD can induce macrophage differentiation, activate phagocytose microorganisms, and secrete osteoclast-maturing mediators.
Cell Differentiation ; Humans ; Leukocytes, Mononuclear ; Macrophages ; Monocytes ; Periodontitis ; immunology ; Saliva

Johne's disease is a condition that refers to chronic granulomatous enteritis in ruminants. It is believed that survival and replication of Mycobacterium (M.) paratuberculosis in mononuclear phagocytes plays an important role in the pathogenesis of Johne's disease. However, it is not clear how M. paratuberculosis survives for long time periods in mononuclear phagocytes, nor is it clear which factors trigger multiplication of these bacilli and result in the development of Johne's disease. Investigating the intracellular fate of M. paratuberculosis is challenging because of its very slow growth (more than two months to form visible colonies on media). Existing animal models also have limitations. Despite those obstacles, there has been progress in understanding the intracellular survival tactics of M. paratuberculosis and the host response against them. In this review, we compare known aspects of the intracellular survival tactics of M. paratuberculosis with those of other mycobacterial species, and consider possible mycobactericidal mechanisms of mononuclear phagocytes.
Animals ; Leukocytes, Mononuclear/*microbiology ; Mycobacterium avium subsp. paratuberculosis/*physiology ; Phagocytes/*microbiology

OBJECTIVETo detect the mRNA expression levels of TERT and TIN2 in peripheral blood mononuclear cells of acquired aplastic anemia(AAA) patients, and to explore their correlation with pathogenesis of acquired aplastic anemia.

METHODSPeripheral blood mononuclear cells of 40 cases of AAA including 33 cases of non-severe aplastic anemia(NSAA), 7 cases of severe aplastic anemia (SAA) and 20 subjects as control group were collected to detect mRNA expression of TERT and TIN2 by using real-time quantitative polymerase chain reaction(RT-qPCR), the correlation of TERT and TIN2 mRNA expression levels with classification of peripheral blood cells were analyzed.

RESULTSThe expression levels of TERT and TIN2 mRNA in patients with AAA were lower significantly than those in control group (P<0.05), and the SAA (P<0.01). The expression levels of TERT and TIN2 mRNA in patients with SAA were all lower significantly than those in patients with NSAA (P<0.05). The expression levels of TIN2 mRNA in patients with NSAA were lower significantly than those in control group (P<0.01). There were no significant difference in the expression level of TERT mRNA between patients with NSAA and control group (P=0.082). There was significant correlation between the expression level of TERT mRNA and red blood cells count (r=0.437, P=0.029), and hemoglobin level (r=0.522, P=0.007). There was significant correlation between the expression levels of TIN2 mRNA and the lymphocyte percentage (r=-0.404, P=0.045).

CONCLUSIONThe expression level of TERT mRNA may be associated with the red blood cells and hemoglobin level. The expression level of TIN2 mRNA may be associated with the lymphocyte percentage.

This paper is aimed to establish and optimize proteomic research platform using isobaric tags for relative and absolute quantitation (iTRAQ) so as to facilitate further proteomic research of human peripheral blood mononuclear cells (hPBMC). We collected hPBMC, after protein extraction and trypsin digestion, we labeled the samples with iTRAQ reagents and then subjected to mass spectrometry. In triplicates, thirty precursors were randomly selected and detected; as a result, 26, 28 and 29 peptides were respectively tagged with ITRAQ reporter ions. The labeling efficiencies ranged between 86.7%-96.7%, with no significant difference among the groups (P>0.05). The coefficient of variance for the relative ratios of peptides from different proteins was ranged from 7.6% to 25.5% and there were no significant differences across the groups (P>0.05). The coefficient of variance for the relative ratios of different peptides from the same protein was varied from 9.3% to 19.1% and the differences across groups were not significant (P>0.05). The labeling of iTRAQ combined with tandem mass spectrometry in PBMC was successful with favourable reproducibility and accuracy, which could lay a foundation for further proteomic study of hPBMC in autoimmune disorders.
Humans ; Leukocytes, Mononuclear ; chemistry ; Proteins ; analysis ; chemistry ; Proteome ; Proteomics ; methods ; Staining and Labeling ; Tandem Mass Spectrometry ; methods

OBJECTIVETo evaluate the sensitivity of an interferon-gamma release assay T-SPOT. TB in the diagnosis of bacteriologically or histologically confirmed extrapulmonary tuberculosis.

METHODTotally 31 patients with bacteriologically or histologically confirmed extrapulmonary tuberculosis in Peking Union Medical College Hospital received T-SPOT. TB assay to detect early secreting antigen target 6 or culture filtrate protein 10 peptides-specific T cells in the peripheral blood mononuclear cells (PBMCs).

RESULTST-SPOT. TB assay showed positive results in 29 patients with extrapulmonary tuberculosis and the sensitivity was 93.5% (95% CI 84.8% - 100%). The median of spot forming cells (SFCs) in response to early secreting antigen target 6 peptides was 196/10(6) PBMCs (interquartile range, 72-532/10(6) PBMCs), the median of SFCs in response to culture filtrate protein 10 peptides was 276 SFCs/10(6) PBMCs (interquartile range, 72-568/10(6) PBMCs), and the median of the incorporate SFCs was 612/10(6) PBMCs (interquartile range, 192-1 152/10(6) PBMCs).

CONCLUSIONT-SPOT. TB is highly sensitive in diagnosing extrapulmonary tuberculosis.

OBJECTIVETo compare enzyme-linked immunospot assay (ELISPOT) and tuberculin skin test (TST) and explore their roles in the auxiliary diagnosis of initial pulmonary tuberculosis.

METHODSTotally 123 patients with initial pulmonary tuberculosis (tuberculosis group) and 102 patients with non-tuberculosis pulmonary disease (control group) were enrolled. The peripheral blood mononuclear cells of all participants were co-cultured with early secretiny antigen target-6/culture filtrate protein-10 fusion protein (ESAT-6/CFP-10), and spot forming cells (SFCs) were enumerated by ELISPOT (ESAT-6/CFP-10-ELISPOT). TST was also performed simultaneously.

RESULTSESAT-6/CFP-10-ELISPOT showed significantly higher numbers of SFCs after stimulation in tuberculosis group than in control group (P = 0.000). The sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, positive predictive value, and negative predictive value of ESAT-6/CFP-10-ELISPOT were 91.1% (111/123), 81.4% (82/102), 4.60, 0.12, 0.85, and 0.87 respectively, while the above values of TST were 65.6% (59/90), 45.1% (46/102), 1.31, 0.76, 0.51, and 0.60, respectively. The sensitivity and specificity of ESAT-6/CFP-10-ELISPOT were significantly higher than those of TST (all P = 0.000). The number of SFCs were not significantly different between smear-positive tuberculosis subgroup and smear-negative tuberculosis subgroup (P = 0.166). The sensitivities were 91.8% (67/73) and 88.0% (44/50) in these two subgroups, respectively, (P = 0.448).

CONCLUSIONSESAT-6/CFP-10-ELISPOT may be a more accurate approach for the auxiliary diagnosis of initial pulmonary tuberculosis; meanwhile, it offers certain diagnostic evidences for smear-negative tuberculosis. However, its specificity may be affected by latent tuberculosis infection. On the contrary, TST has poor value in the auxiliary diagnosis of initial pulmonary tuberculosis.

OBJECTIVETo investigate the expression of high mobility group box 1 (HMGB1) in gingival tissues of chronic periodontitis.

METHODSHuman peripheral blood mononuclear cells(PBMC) were stimulated with 1 microg x mL(-1) lipopolysaccharide (LPS) for 24 h or 48 h. Expression and release of HMGB1 were checked by immunofluorescence and enzyme-linked immunosorbent assay (ELISA), respectively. PBMC were stimulated with 100 ng x mL(-1) HMGB1 or 50 ng x mL(-1) tumor necrosis factor-alpha (TNF-alpha), the expressions of TNF-alpha and HMGB1 in the supernatant were studied by ELISA. Gingival tissues and gingival crevicular fluids (GCF) were collected from patients and healthy people. Expression of HMGB1 in gingival tissues and GCF was studied using immunofluorescence and ELISA, respectively.

RESULTSHMGB1 was translocated from nucleus to cytosol in PBMC after LPS stimulation for 24 h. The content of HMGB1 in the supernatant from stimulated cells was significantly higher than that from unstimulated cells after 48 h (P < 0.01). HMGB1 was released by PBMC in response to TNF-alpha stimulation, it also stimulated PBMC to release TNF-alpha (P < 0.01). Translocation of HMGB1 from nucleus to cytosol was also found in infiltrated cells in gingival tissues from patients, and HMGB1 in GCF from patients was significantly higher than that from healthy people P < 0.01).

CONCLUSIONThe results suggest that HMGB1 may play an important role in the pathological progress of chronic periodontitis.