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.

Cell Survival ; Cryopreservation ; Humans ; Immunocompetence ; Leukocytes, Mononuclear

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

OBJECTIVEThe aim of this study was to determine the expression of genes of the oligodendrocyte lineage-myelin basic protein (Golli-MBP) in peripheral blood mononuclear cell (PBMC) in oral lichen planus (OLP) and to further understand the pathogenesis of OLP.

METHODSPBMC was obtained by density gradient centrifugation, and the expression of Golli-MBP in PBMC was investigated in erythematous/erosive OLP (20 cases), reticular OLP (16 cases) and normal controls (19 cases) using reverse transcription-polymerase chain reaction (RT-PCR) and Western blot methods.

RESULTSRT-PCR results showed that Golli-MBP mRNA was overexpressed in erythematous/erosive and reticular OLP as compared with normal control group (P < 0.01). Western blot assay indicated that erythematous/erosive and reticular OLP patients had a higher expression level of Golli-MBP protein in PBMC than normal controls (P < 0.01). However, there were no significant differences between erythematous/erosive and reticular groups in Golli-MBP mRNA and protein expression (P > 0.05).

CONCLUSIONThe data accumulated here strongly indicate that Golli-MBP was involved in the pathogenesis of OLP.

Humans ; Leukocytes, Mononuclear ; Lichen Planus, Oral ; Mouth Mucosa ; Myelin Basic Protein ; Oligodendroglia ; RNA, Messenger

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 study myeloid-derived suppressor cells (MDSC) levels in peripheral blood of infants with recurrent wheezing, and the role of MDSC in the development of recurrent wheezing.

METHODSThirty-one infants with recurrent wheezing at wheezing attacks were randomly enrolled in the study. Twenty-seven infants with bronchopneumonia and 27 preoperative infants (hernia or renal calculus), without infectious or neoplastic diseases, were selected as controls. The proportion of MDSC in peripheral blood mononuclear cells (PBMC) was measured by flow cytometry.

RESULTSThe proportion of MDSC in PBMC in infants with wheezing was significantly higher than in those with bronchopneumonia and preoperative infants (P<0.05).

CONCLUSIONSMDSC levels increase in infants with recurrent wheezing, suggesting that MDSC may play a crucial role in the development of this disorder.

Child, Preschool ; Female ; Humans ; Infant ; Leukocytes, Mononuclear ; immunology ; Male ; Myeloid Cells ; immunology ; Recurrence ; Respiratory Sounds ; immunology

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.

Anemia, Aplastic ; Gene Expression Regulation ; Humans ; Leukocytes, Mononuclear ; RNA, Messenger ; Telomerase

OBJECTIVETo investigate the correlation of immunologic thrombocytopenia(ITP) pathogenesis with the abnormal expression of IL-21, and to explore the association of high-dose dexamethasone (HD-DEX) treatment with the IL-21 expression.

METHODS26 newly diagnosed ITP patients and 24 healthy controls were enrolled in this study. The mononuclear cells and serum were obtain from density gradient centrifugation in the newly diagnosed ITP patients before HD-DXM treatment, and the samples of healthy controls were also used for assays. The protein and mRNA expression of IL-21 on peripheral blood mononuclear cells(MNC) were determined by flow cytometry and real-time reverse-transcription polymerase chain reaction. Plasma levels of IL-21, IFN-γ and IL-4 were determined by enzyme-linked immunoabsorbent assay (ELISA).

RESULTSIL-21 expression on mononuclear cells was significantly higher in ITP patients (13.07%) than that in normal controls (8.2%), the ratio of IL-21/GAPDH mRNA expression on MNC was significantly higher in ITP patients (9.524±0.97) than that in normal controls (3.701±0.60, P<0.01). After HD-DXM therapy, the ratio of IL-21/GAPDH mRNA decreased significantly (5.87±1.21) as compared with the level before treatment. Significantly high levels of serum IL-21, IFN-γ and lower IL-4 were found in ITP patients, as compared with healthy controls. Serum IL-21 and IFN-γ levels in ITP patients decreased significantly after HD-DXM administration (P<0.01), while post-treatment levels of IL-4 were increased significantly, compared with the levels before treatment (P<0.01).

CONCLUSIONTherapeutic effect of DXM on ITP associates with down-regalation of IL-21 expression. The increased expression of IL-21 involves in the pathogenesis of ITP.

Dexamethasone ; Flow Cytometry ; Humans ; Interleukin-4 ; Interleukins ; Leukocytes, Mononuclear ; Purpura, Thrombocytopenic, Idiopathic ; RNA, Messenger