Decrease in interferon-γ production by peripheral blood mononuclear cells in patients with uterine cervical cancer

The interferon- (IFN-) productivity of peripheral blood mononuclear cells (PBMCs) was examined in 30 patients with uterine cervical cancer. The patients under 50 years of age had decreased IFN- production compared with the age-matched controls. The IFN- productivity in the patients over 50 years of age was decreased as well as in the age-matched controls. The proportion of monocytes in PBMCs did not correlate with the IFN- productivity. The prostaglandin E₂ (PGE₂) productivity of PBMCs increased with the progress of cancer. PGE₂ inhibited the IFN- production by PBMCs, and the sensitivity of PBMCs to PGE₂ was increased in the patients and controls over 60 years of age. The addition of indomethacin resulted in an increase in IFN- production by PBMCs. These results suggest that the increased production of PGE₂ and/or increased sensitivity to PGE₂ are responsible for the decreased IFN- production in patients with cervical cancer.     

Paeoniflorin regulates the function of human peripheral blood mononuclear cells stimulated by rhIL-1β by up-regulating Treg expression

Abstract

The aim of the present study was to investigate the effect of paeoniflorin (Pae) on recombinant human interleukin-1β (rhIL-1β)-stimulated human peripheral blood mononuclear cells (PBMCs) in vitro. PBMCs were collected by Ficoll density gradient centrifugation and were co-cultured with rhIL-1β for different time periods. The proliferation response was determined by a cell counting kit-8 (CCK-8) assay. The production of IL-17 and IL-10 was measured by enzyme-linked immunosorbent assay (ELISA). The percentage of CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Treg) was detected by flow cytometry analysis. These results indicated that rhIL-1β stimulation induced the proliferation of PBMCs in a concentration- and time-dependent manner; it also increased the level of IL-17 and decreased the level of IL-10 in a concentration-dependent manner. The flow cytometry analysis demonstrated that the stimulation of rhIL-1β significantly downregulated the percentage of CD4⁺CD25⁺Foxp3⁺ Treg in CD4⁺ T cells. However, administration of Pae significantly suppressed the proliferation response of rhIL-1β-induced PBMCs and regulated the secretion function of IL-17 and IL-10. Additional experiments demonstrated that Pae treatment significantly reduced rhIL-1β-induced decreases in PBMCs CD4⁺CD25⁺Foxp3⁺ subpopulation numbers. These results suggest that the anti-inflammatory action of Pae is attributable to its regulation of IL-17/IL-10 secretion and Treg expression.     

Coculture with mesenchymal stem cells facilitates the proliferation of hematopoietic stem cells under different coculture modes北大核心

BACKGROUND: Although a large number of related studies have been carried out, there is still a lack of practical methods to amplify hematopoietic stem cells (HSCs) in vitro. Mesenchymal stem cells (MSCs) secrete a variety of cytokines that promote the HSCs proliferation and inhibit their differentiation. These cytokines play an important role in maintaining the hematopoietic microenvironment and regulating HSCs function. OBJECTIVE: To investigate the effect of bone marrow MSCs on the proliferation of HSCs in vitro under different coculture modes. METHODS: Mesenchymal stem cells from the bone marrow of C57BL/6 mice were cultured in vitro using the whole bone marrow adherent culture. CD117+ cells (HSCs) were sorted from passage 3 cells by using miniMACS magnetic beads sorting. Then, CD117+ cells were co-cultured with MSCs under different coculture models, including single culture of HSCs (control group), Transwell coculture (upper chamber, HSCs; lower chamber, MSCs) and two-dimensional contact coculture (coculturing HSCs and MSCs in 24-well platts). The morphology of HSCs was observed under phase contrast microscope and fluorescence microscope, and the number of active cells of HSCs was counted at 1,3,5, and 7 days after coculture. RESULTS AND CONCLUSION: During the coculture of 1-7 days, the number of HSCs in the two groups was increased with culture time (P < 0.05). After 3 days of coculture, HSCs in each group was grown into the logarithmic growth phase, and morphological changes in some HSCs were detected at 5 days of coculture. Ait 7 days of coculture, the viabilities of HSCs in different culture models were ranked as follows: single culture model < Transwell coculture model < two-dimensional contact coculture model (P < 0.05). These findings suggest that MSCs can effectively promote the proliferation of HSCs in vitro, and the promotion effect is increased under contact coculture conditions. © 2018, Journal of Clinical Rehabilitative Tissue Engineering Research. All rights reserved.     

How Glycosylphosphatidylinositol-phospholipase D acts in homing of hematopoietic stem/progenitor cells?

Homing of hematopoietic stem/progenitor cells (HS/PC) to the bone marrow (BM) microenvironment is the first and essential step in HS/PC engraftment and initiation of the marrow reconstitution during clinical hematopoietic stem cell transplantation (HSCT). How to improve the homing efficiencies and make full use of HS/PC resources, especially umbilical cord blood (UCB), are of great importance in clinical practice. However, the cellular and molecular mechanisms that govern this process are poorly understood. Glycosylphosphatidylinositol-phospholipase D (GPI-PLD) is an enzyme which can regulate the expression of Glycosylphosphatidylinositol (GPI)-anchored proteins and modulate their correspondent functions by releasing GPI-anchored proteins from cell membrane. Recent studies suggested that the mechanisms of the malignancy and prognosis of certain tumors were correlated with GPI-PLD. HS/PC homing was similar to tumor invasion and metastasis in some process. Here we proposed the hypothesis that GPI-PLD might also has played a role in the homing of HS/PC by modulating the adhesion and migration of these cells. If GPI-PLD did participate in HS/PC homing, maybe the mechanisms of homing can herefrom be partly elucidated, which would benefit transplantation in clinical practice.     

Aberrant cytokine production by peripheral blood mononuclear cells in recurrent pregnancy loss?

BACKGROUND: Successful pregnancy may depend on a Th2-type cytokine response, whilst, conversely, a poor pregnancy outcome may be associated with an increase in Th1 cytokines and a concomitant decrease in Th2 cytokines. This prospective study was designed to elucidate whether a failure of the cytokine shift pre-dated miscarriage and was therefore likely to be an aetiological factor in recurrent pregnancy loss (RPL). METHODS: Cytokine production by stimulated peripheral blood mononuclear cells from 46 pregnant women who had previously suffered idiopathic RPL during early pregnancy was compared with 25 gestationally age-matched pregnant controls and 11 non-pregnant women. RESULTS: Production of IFN-gamma was lower in pregnant than in non-pregnant women and even lower in RPL pregnant women (P = 0.0191). IL-10 was increased in pregnant women compared with non-pregnant controls, and further increased in RPL patients (P = 0.026). IL-4 was also increased in women with RPL (P = 0.0001). No differences in IFN-gamma, IL-10 or IL-4 secretion were observed in RPL patients who subsequently miscarried compared with those who successfully completed the pregnancy. RPL women with a successful reproductive outcome had similar concentrations of TNF-alpha to pregnant women, RPL women who subsequently miscarried had significantly lower levels than either pregnant women (P = 0.02) or non-pregnant controls (P = 0.0004). CONCLUSIONS: Contrary to our hypothesis, the cytokine shift, which appears to characterize normal pregnancy, was accentuated rather than diminished in RPL pregnant women.     

Disproportion of helper T cell subsets in peripheral blood of patients with primary Sjögren's syndrome

We studied the proportions of Th1 and Th2 cells in peripheral blood of 15 patients with primary Sj?gren's syndrome (p-SS), by using a procedure to enumerate the cells synthesizing cytokines such as INF-gamma or IL-4 in cytoplasm of CD4+ lymphocytes. The frequency of Th1 (INF-gamma containing) cells in p-SS patients was significantly reduced as compared to normal control (20.57+/-7.48% vs 28.78+/-11.56%, p < 0.05), while that of Th2 (IL-4 containing) cells was not different from normal control (3.33+/-0.98% vs 2.85+/-1.88%). The ratio of Th1 to Th2 cells in p-SS patients was significantly decreased as compared to normal control (6.60+/-3.15 vs 11.55+/-6.72, p < 0.05). There was no difference in frequency of Th1 or of Th2 cells between 8 patients given small amounts of prednisolone (PSL) and 7 patients not given PSL (21.44+/-9.39% vs 19.57+/-5.05%, 3.12+/-0.80% vs 3.56+/-1.17%). The percentage of Th1 cells was not different between 7 patients with glandular symptoms (G) and 8 patients with extraglandular symptoms (EG) (18.61+/-9.63% vs 22.27+/-5.02%). Although the frequency of Th2 cells was higher in EG-patients than that in G-patients (3.84+/-0.78% vs 2.74+/-0.86%) with tendency of elevated IgG level in sera, the ratio of Th1 to Th2 cells was not different among them (6.26+/-2.84 vs 6.99+/-3.64). These results suggest that the reduced ratio of Th1 to Th2 cells is essential and is related to the dysfunction of cellular immunity in p-SS.     

Ultrastructural findings of cutaneous nerves in patients with Hunter’s syndrome following hematopoietic stem cell transplant

Hematopoietic stem cell transplant (HSCT) has been performed on patients with Hunters syndrome. If applied, evaluation of recovery in various organs is needed for long-term follow-up. However, it remains unclear whether HSCT is effective against the neurological involvement in Hunters syndrome, and morphological evaluation of recovery is inconsistent. We observed the degree of cutaneous nerve involvement in patients with Hunters syndrome ultrastructurally before and after HSCT. Electron microscopic studies revealed that membrane-bound clear vacuoles were still observed in the cytoplasm of endoneurial fibroblasts and Schwann cells 2 months and 2 years, respectively, after HSCT. On the other hand, only a few vacuoles were present in dermal fibroblasts at 2 months after HSCT, and these disappeared within 2 years. These results suggest that the persistence of clear vacuoles in endoneurial fibroblasts and Schwann cells indicates a disturbed internal condition in the endoneurium 2 years after HSCT. Skin biopsies can be used in patients with Hunters syndrome to study peripheral nerves for long-term follow-up to evaluate morphological efficacy.     

GSK-3β inhibition promotes engraftment of ex vivo-expanded hematopoietic stem cells and modulates gene expression

Glycogen synthase kinase-3β (GSK-3β) has been identified as an important regulator of stem cell function acting through activation of the wingless (Wnt) pathway. Here, we report that treatment with an inhibitor of GSK-3β, 6-bromoindirubin 3'-oxime (BIO) delayed cell cycle progression by increasing cell cycle time. BIO treatment resulted in the accumulation of late dividing cells enriched with primitive progenitor cells retaining the ability for sustained proliferation. In vivo analysis using a Non-obese diabetic/severe combined immunodeficient (NOD/SCID) transplantation model has demonstrated that pretreatment with BIO promotes engraftment of ex vivo-expanded hematopoietic stem cells. BIO enhanced the engraftment of myeloid, lymphoid and primitive stem cell compartments. Limiting dilution analysis of SCID repopulating cells (SRC) revealed that BIO treatment increased human chimerism without increasing SRC frequency. Clonogenic analysis of human cells derived from the bone marrow of transplant recipient mice demonstrated that a higher level of human chimerism and cellularity was related to increased regeneration per SRC unit. Gene expression analysis showed that treatment with BIO did not modulate the expression of canonical Wnt target genes upregulated during cytokine-induced cell proliferation. BIO increased the expression of several genes regulating Notch and Tie2 signaling downregulated during ex vivo expansion, suggesting a role in improving stem cell engraftment. In addition, treatment with BIO upregulated CDK inhibitor p57 and downregulated cyclin D1, providing a possible mechanism for the delay seen in cell cycle progression. We conclude that transient, pharmacologic inhibition of GSK-3β provides a novel approach to improve engraftment of expanded HSC after stem cell transplantation.     

Detection of Hantavirus gene from peripheral blood of patients with HFRS in Heilongjiang province and the epidemiological significance

The aim of this study is to further understand the genotype of Hantavirus(HV) from peripheral blood of patients with hemorrhagic fever with renal syndrome (HFRS) and the epidemiological significance of this disease in Heilongjiang province in recent years. Thirty-one serum samples of clinically diagnosed patients with HFRS were examined by RT-PCR to decide the genetic subtype. On the basis of infection season, the serum samples were divided into two groups: winter (Nov, 2003—Feb, 2004) , spring and summer (April, 2004—Sep, 2004). Further analysis was performed in combination with clinical symptoms. It was found that among the total 31 samples, 22 were sero-positive. Among 14 serum samples in winter, 8 were sero-positive, of which 5 cases were of typeⅠ(Hantaan virus, HTNV) and 3 of typeⅡ(Seoul virus, SEOV). Among 17 samples in spring and summer, 14 were sero-positive, of which 5 cases were of typeⅠand 9 of typeⅡ. So it concludes that both of the two types of Hantavinis exist in Heilongjiang. The typeⅠis the main pathogen of HFRS in winter, and typeⅡis the main in spring and summer.     

Significantly depressed percentage of CD27+ (memory) B cells among peripheral blood B cells in patients with primary Sjögren's syndrome

CD27 has been found to be expressed on somatically mutated B cells and is thus a positive marker for memory B cells in peripheral blood (PB). Since abnormal immunogloblin (Ig) production is characteristic of the autoimmune diseases primary Sjögren's syndrome (pSS) and rheumatoid arthritis (RA), we have analyzed in detail the CD27 expression on PB B cell from these patient groups. Staining of PB B cells with monoclonal antibodies (MoAb) specific for CD19 and CD27 revealed a significantly depressed percentage of CD27+ PB B cells in patients with pSS (14.8 ± 1.6%) compared to both healthy donors (31.3 ± 4.7%, P = 0.005) and patients with RA (40.8 ± 4.1%, P = 0.0001). In addition, the percentages of both the IgD+CD27+ and the IgD‐CD27+ B‐cell subpopulations were significantly lower in pSS patients compared to RA patients and healthy donors. However, the relative proportion of IgD‐ and IgD+ cells among the CD27+B cells were almost the same for the three groups. Our data suggest a disturbance in the differentiation of peripheral B cells and possibly a bias towards plasma cell differentiation, resulting in a depressed percentage of CD27+ memory PB B cells in pSS. These results are potentially of pathological significance and of diagnostic value.     

Modulation of the function of dendritic cells in adolescents with chronic HBV infection by IFN-λ1

The exact immunology pathogenesis of hepatitis B virus (HBV) infection remains unclear currently. The dendritic cells (DCs) dysfunction is evident in adolescents with chronic HBV infection in the immune tolerant phase. DCs, as the most efficient professional antigen-presenting cells (APCs), possess the strongest antigen presenting the effect in the body and can stimulate the initial T cell activation and proliferation, depending on their stage of maturation. The recently classified type III interferon group, interferon-λ1 (IL-29), interferon-λ2 (IL-28A), and interferon-λ3 (IL-28B) displays immunomodulatory and antiviral activity. In the current study, we describe a way to stimulate the DCs maturation. As a result, IFN-λ1 combined with recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF) and recombinant human interleukin-4 (rhIL-4) can induce the DCs maturation and promote the costimulatory molecules such as CD80, CD83, CD86 and human leucocyte antigen DR (HLA-DR) expression in the immune tolerance and the clearance phases. This study demonstrates that the DCs function is remarkably impaired both in the immune tolerant phase and the immune clearance phase in adolescents with chronic HBV infection compared with healthy youth control. At the same time, this study has developed a theoretical basis for the application of IFN-λ1 breaking immune tolerance and improving the body’s immune system to clear HBV.     

Altered synthesis of interferon-γ and expression of interferon-γ receptor by peripheral blood mononuclear cells from patients with IgA nephropathy and non-IgA proliferative glomerulonephritis

Previously we reported disease-specific interaction between interferon- (IFN-) and interleukin-4 (IL-4) in patients with IgA nephropathy (IgAN), suggesting the existence of unusual T cell behavior in this disease. In the present study, we investigated characteristic synthesis of interferon- (IFN-) and expression of IFN- receptor (IFN-R) in the peripheral blood mononuclear cells (PBMC) from patients with IgAN and other chronic proliferative glomerulonephritis (PGN). Heparinized peripheral blood samples were obtained from 38 patients with chronic mesangial proliferative glomerulonephritis (CGN; including 24 with IgA nephropathy) and 20 healthy controls. PBMC were isolated by gradient centrifugation and fragments were cultured in Iscove's modified Dulbecco's medium (IMDM) supplemented with 10% fetal calf serum (FCS) for 72 hr. IFN- concentrations in supernatants were evaluated by the enzyme-linked immunosorbent assay (ELISA). Other parts of PBMC pellets were reacted with anti-human IFN-R monoclonal antibody and FITC-labeled anti-mouse second antibody for analysis of IFN-R expression on these cells by FACScan. The remaining PBMC were fractionated into CD4⁺ T cells, CD8⁺ T cells, B cells, NK, cells and macrophages using the MACS cell sorting system. The isolated cells were evaluated for IFN- or IFN-R mRNA expression by the semiquantitative RT-PCR method.In vitro IFN- synthesis was enhanced in patients with CGN, and NK cells were revealed to be responsible for such enhancement. On the other hand, the expression of IFN-R on macrophages was suppressed in CGN patients. These results suggest that impairment of regulation of the IFN- system might be involved in the development of CGN.     

Phenotypic diversity of peripheral blood plasma cells in primary Sjögren's syndrome

Production of autoantibodies is one of the main features of primary Sjögren’s syndrome (pSS). Long‐lived plasma cells (PC) can produce autoantibodies for prolonged period of times without being affected by immunosuppressive therapies. As of today, little is known about the long‐lived PC subset and their contribution to autoimmunity. We have characterized the phenotypic and migratory properties of peripheral blood PC isolated from pSS patients (grouped by focus score, FS) and compared them to PC from rheumatoid arthritis (RA) patients and normal non‐autoimmune subjects. We observed two populations of PC in all study groups, CD19+ PC and CD19? PC. Interestingly, the CD19? PC subset was most prominent in autoimmune patients (pSS and RA) compared to normal controls. Further investigation of the PC phenotype revealed that a high percentage of both CD19+ and CD19? PC isolated from pSS and RA patients did not express the CD27 marker, which is normally highly expressed on all types of PC. Differences in the expression of markers such as IgM, IgG, CD95 and CXCR3 in the group with high FS compared to FS = 1, underscore the heterogeneity of pSS patient group and demonstrate that phenotypic pattern of circulating PC associates with the severity of inflammation in the salivary glands of these patients. Our migration experiments show that addition of CXCL12 to PC in vitro, do not alter the migration potential of PC in any group tested. However, we observed an overall higher spontaneous migration of PC from pSS compared to both RA and normal controls.     

Immunomodulatory responses of peripheral blood mononuclear cells from multiple sclerosis patients upon in vitro incubation with the flavonoid luteolin: additive effects of IFN-β

The study is aimed to determine the role of luteolin (3',4',5,7-tetrahydroxyflavone), alone and in combination with human interferon-beta (IFN-β), in modulating the immune response(s) of peripheral blood mononuclear cells (PBMCs) isolated from multiple sclerosis (MS) patients. PBMC proliferation in the presence or absence of these drugs was determined and the production of pro-inflammatory cytokines (IL-1β, TNF-α), and the ratio of cell migration mediator MMP-9, and its inhibitor, TIMP-1 was assessed in the culture supernatants. Luteolin reduced, in a dose-dependent manner, the proliferation of PBMCs, and modulated the levels of IL-1β and TNF-α released by PBMCs in the culture supernatants. Luteolin reduced the MMP-9/TIMP-1 ratio via lowering MMP-9 production. In the majority of cases, luteolin, when combined with IFN-β, had additive effects in modulating cell proliferation, IL-1β, TNF-α, MMP-9 and TIMP-1.     

Multiple mechanisms for HSV-1 induction of interferon α production by peripheral blood mononuclear cells

Summary.  UV-inactivated, infectious, and other forms of herpes simplex virus 1 (HSV-1) induced interferon (IFN) production by different routes in myeloid origin mononuclear cells (MOMC) (consisting predominantly of monocytes). GM-CSF activated the MOMC (G-MOMC) to produce greater amounts of interferon while differentiation to DC, by the addition of granulocyte macrophage colony stimulating factor (GM-CSF) and calcium ionophore (GA-MOMC), reduced the levels of interferon production upon challenge with some HSV strains. UV-inactivated virus induced more interferon than infectious virus. L-fucose, an antagonist of the mannose receptor, inhibited the induction of IFN-α by UV-inactivated virus and gB⁻ virus (defective in penetration) in MOMC and GA-MOMC but not G-MOMC. L-fucose had little effect on interferon induction by infectious HSV-1. The insensitivity of the G-MOMC to fucose inhibition distinguishes these interferon producing cells from the pDC2 cells previously described as natural interferon producing cells. The mannose receptor appears to be involved in the response to non-infectious forms of HSV but infectious virus appears to use a different pathway. These studies suggest that non-infectious virions and HSV infected cell debris effectively stimulate monocytes and pre-dendritic cells to produce IFN-α to initiate host protection against HSV infection. Received April 1, 2002; accepted August 17, 2002