Biobanks have a primary responsibility to collect tissues that are a true reflection of their local population and thereby promote translational research, which is applicable to the community. The Infectious Diseases BioBank (IDB) at King's College London is located in the southeast of the city, an area that is ethnically diverse. Transplantation programs have frequently reported a low rate of donation among some ethnic minorities. To determine whether patients who volunteered peripheral venous blood samples to the IDB were representative of the local community, we compared local government demographic data to characteristics of patients who have donated to the IDB. There was a good match between these statistics, indicating that the IDB's volunteer population of human immunodeficiency virus patients was similar to local demographics.
We have studied the functional characteristics and clinical importance of the natural killer (NK) cytotoxicity of peripheral blood mononuclear cells (PBMNC) from patients with ulcerative colitis. Normal NK activity was observed in PBMNC from patients with inactive disease, but a pronounced decrease was found in those with active disease. Clinical change from active to inactive disease was associated with enhancement of the depressed NK activity. The impairment of NK cytotoxicity found in patients with active disese could not be ascribed to a deficient number of NK cells as the amounts of HNK-1+, CD16+ (Leu 11), and CD11b (OKM1) cells in PBMNC were within normal ranges. This defective cytotoxic PBMNC activity was normalised by short term (18 hour) incubation with recombinant interleukin 2 (rIL-2). Moreover, long term (5 day) incubation of these effector cells with rIL-2 induced strong cytotoxic activity against NK resistant and NK sensitive target cells in patients with active and inactive disease. We also found that both precursors and effectors of cytotoxic activity promoted by short term and long term incubation with rIL-2 of PBMNC from the patients showed the phenotype of NK cells (CD16+, CD3-). Taken together, these results show that active ulcerative colitis is associated with a defective function of NK cells that is found to be normal in the inactive stage of the disease. The possible pathogenic and therapeutic implications of these findings are discussed.
The activation and proliferation of peripheral blood mononuclear cells (PBMNCs) are complex processes involving several surface molecules, cell secretion and response to cytokines. This paper investigates the immunomodulatory effect of prophylactic treatment with interferon alpha 2b (IFN-alpha 2b) upon the blastogenic response of PBMNCs from patients with superficial transitional cell carcinoma (STCC) of the bladder to mitogenic signals that interact with surface molecules [phytohaemagglutinin, PHA and anti-CD3 monoclonal antibodies, (MAbs)]. PBMNCs from the patients were studied prior to the transurethral resection (TUR) of the tumour, during the second month of prophylactic intravesical instillation of IFN-alpha 2b and 3 and 6 months after finishing the instillation treatment. The [3H]thymidine uptake of PBMNCs from 17 patients with STCC of the bladder after 5 days of PHA and anti-CD3 MAb stimulus was found to be significantly lower than that of healthy controls (P < 0.05). The addition of interleukin 2 (IL-2) to the culture medium did not correct this defective proliferative response to PHA and the anti-CD3 MAb. There were no significant differences between IL-2 production in PBMNCs from STCC patients after stimulation with PHA and in PBMNCs from healthy controls (P > 0.05). Patients without evidence of recurrence showed a significantly enhanced proliferative response in PBMNC to PHA and anti-CD3 MAb after intravesical prophylactic treatment with interferon-alpha 2b in the follow-up examinations 3 and 6 months after treatment (P < 0.01). However, three patients had evidence of tumour recurrence, and they showed no enhancement of the PBMNC proliferative response to these mitogens in the same examinations. In conclusion, the prophylactic intracavitary treatment of STCC with IFN-alpha 2b may induce a systemic immunomodulatory effect which is associated to the clinical evolution of the disease.
Peripheral blood mononuclear cells (PBMNC) from patients with type 2 diabetes (DM2) have generated higher levels of reactive oxygen species (ROS) that were higher than those in cells from healthy individuals. In the presence of a cAMP-elevating agent, ROS production was significantly activated in PBMNC from DM2 patients but it was inhibited in cells from healthy subjects. Higher levels of IL-6 has been detected in the supernatant of PBMNC cultures from DM2 patients in comparison with healthy controls. When cells were cultured in the presence of a cAMP-elevating agent, the level of IL-6 decreased has by 46% in the supernatant of PBMNC from DM2 patients but it remained unaltered in controls. No correlations between ROS and IL-6 levels in PBMNC from DM2 patients or controls have been observed. Secretions of IL-4 or IFN by PBMNC from patients or controls have not been affected by the elevation of cAMP. cAMP elevating agents have activated the production of harmful reactive oxidant down modulated IL-6 secretion by these cells from DM2 patients, suggesting an alteration in the metabolic response possibly due to hyperglicemia. The results suggest that cAMP may play an important role in the pathogenesis of diabetes.
ROS; IL-6; cAMP; diabetes; peripheral blood mononuclear cells
Basal, lipopolysaccharide (LPS) and silica-stimulated prostaglandin (PG) production were compared between peripheral blood mononuclear cells (PBMNC) from UC patients and healthy subjects (HS). Basal and LPS-stimulated PBMNC PGI2, but not PGE2, production was greater in UC. LPS stimulated both PGE2 and PGI2 by PBMNC from HS and UC patients. Silica stimulated production of both PGs by cells from HS but only PGE2 by cells from UC patients. The differences in responses to silica and LPS may result from differences in activation of NFkappaB or, alternatively, prior sensitisation to one of these agents. That PBMNC PGE2 production is not increased in UC, as it is in Crohn's disease, suggests that there are differences in PBMNC behaviour between these two diseases.
To measure the virus load in patients with symptomatic Epstein-Barr virus (EBV) infections, we used a real-time PCR assay to quantify the amount of EBV DNA in blood. The real-time PCR assay could detect from 2 to over 107 copies of EBV DNA with a wide linear range. We estimated the virus load in peripheral blood mononuclear cells (PBMNC) from patients with symptomatic EBV infections. The mean EBV-DNA copy number in the PBMNC was 103.7 copies/μg of DNA in patients with EBV-related lymphoproliferative disorders, 104.1 copies/μg of DNA in patients with chronic active EBV infections, and 102.2 copies/μg of DNA in patients with infectious mononucleosis. These numbers were significantly larger than those in either posttransplant patients or immunocompetent control patients without EBV-related diseases. In a patient with infectious mononucleosis, the virus load decreased as the symptoms resolved. The copy number of EBV DNA in PBMNC from symptomatic EBV infections was correlated with the EBV-positive cell number determined by the in situ hybridization assay (r = 0.842; P < 0.0001). These results indicate that the real-time PCR assay is useful for diagnosing symptomatic EBV infection and for monitoring the virus load.
Since the identification of xenotropic murine leukemia virus–related virus (XMRV) in prostate cancer patients in 2006 and in chronic fatigue syndrome patients in 2009, conflicting findings have been reported regarding its etiologic role in human diseases and prevalence in general populations. In this study, we screened both plasma and peripheral blood mononuclear cells (PBMNCs) collected in Africa from blood donors and human immunodeficiency virus Type 1 (HIV-1)-infected individuals to gain evidence of XMRV infection in this geographic region.
STUDY DESIGN AND METHODS
A total of 199 plasma samples, 19 PBMNC samples, and 50 culture supernatants from PBMNCs of blood donors from Cameroon found to be infected with HIV-1 and HIV-1 patients from Uganda were screened for XMRV infection using a sensitive nested polymerase chain reaction (PCR) or reverse transcription (RT)-PCR assay.
Using highly sensitive nested PCR or RT-PCR and real-time PCR assays capable of detecting at least 10 copies of XMRV plasmid DNA per reaction, none of the 268 samples tested were found to be XMRV DNA or RNA positive.
Our results failed to demonstrate the presence of XMRV infection in African blood donors or individuals infected with HIV-1. More studies are needed to understand the prevalence, epidemiology, and geographic distribution of XMRV infection worldwide.
OBJECTIVE—To use a panel of monoclonal antibodies (Mab) which recognise HLA class II alleles associated with rheumatoid arthritis for fluorescence activated cell sorter (FACS) analysis of peripheral blood mononuclear cells (PBMNC) from patients with early and established rheumatoid arthritis and to compare these results against DNA oligotyping of HLA class II molecules in the same patients.
METHODS—27 patients (18 from an early arthritis clinic, nine with established rheumatoid arthritis) were studied using both techniques. PBMNC were stained with Mab which recognise the shared epitope, the HLA-DRB1*04 molecule and its *0401, *0404 subtypes in the presence of bound peptide. Mab stained cells were analysed by FACS. Genomic DNA was prepared from PBMNC and used for DNA oligotyping and sequencing by standard methods.
RESULTS—FACS analysis of Mab stained PBMNC gave identical results to those obtained by DNA oligotyping in 26/27 patients. The antibodies identified the shared epitope in 14/14 cases and the presence of an HLA-DRB1*04 molecule in 12/12 cases. HLA-DRB1*0404 was identified in 4/4 cases. HLA-DRB1*0401 was identified in 5/6 cases. One patient oligotyped as HLA-DRB1*0401, but consistently failed to react with the *0401 Mab. DNA sequencing of the second exon of the HLA-DRB1*0401 allele in this patient confirmed a normal HLA-DRB1*0401 genotype.
CONCLUSIONS—FACS analysis of PBMNC stained with Mab recognising the shared epitope and rheumatoid arthritis associated HLA susceptibility molecules provides a rapid, reliable, and more accessible alternative to DNA oligotyping. The apparent discordance between phenotypic and genetic analysis of HLA-DRB1*0401 in one patient, may reflect variability in HLA-DRB1*0401 gene expression or in class II peptide presentation.
A subpopulation of mononuclear cells (PBMNC) that expresses Fc receptors with specificity for the C gamma 2 region of IgG may be detected by rosette formation with calf erythrocytes coated with the Facb fragment of rabbit IgG. These Facb-R+ cells are found in increased numbers in the peripheral blood of patients with rheumatoid arthritis (RA). Studies have been carried out to identify the functional properties of these cells in healthy and rheumatoid subjects. Facb-R+ cells were shown to lack both natural killer and antibody-dependent cytotoxic activity. Depletion of Facb-R+ cells from both healthy and rheumatoid PBMNC resulted in a marked suppression of pokeweed mitogen (PWM) stimulated IgG synthesis but had no effect on T cell proliferation induced by phytohaemagglutinin, concanavalin A, or PWM. The addition of Facb fragments to PBMNC cultures also caused inhibition of PWM-driven IgG production. In this assay rheumatoid PBMNC were significantly less sensitive to Facb-mediated suppression than healthy control cells. Our results suggest that Facb-R+ cells are involved in the antibody-mediated feedback regulation of immunoglobulin synthesis and that this mechanism is impaired in patients with RA.
The functional profile of natural killer (NK) cells has been reported to be lower in auto-immune haemolytic anaemia (AIHA). In this study, we report a comparative analysis of peripheral blood mononuclear cells (PBMNCs) and the in vitro expansion of NK cells in a patient with AIHA and cancer, with that of other cancer patients without AIHA. PBMNCs and in vitro NK-cell expansion of a 64-year old female patient with ovarian cancer and AIHA was compared with that of four other patients with cancer without AIHA who underwent autologous immune enhancement therapy (AIET). The NK cells were cultured using autologous plasma without feeder layers. The quantities of PBMNCs, NK cells and CD3−CD56+ cells were compared. The average quantity of PBMNCs per ml in Cases I to V were 10.71, 39.2, 49.26, 65.16 and 49.33×104, respectively, and the average maximum count of NK cells was 3.9, 1730.03, 1824.16, 1058.61 and 761×106, respectively. The average percentage of CD3−CD56+ cells in Cases I to V following in vitro expansion was 1.2, 65.7, 28.63, 65.9 and 40%, respectively. In the present study, probably the first in the literature, the in vitro expansion of NK cells was found to be significantly lower in the AIHA patient. Previously, only a lower NK-cell functional profile was reported. Further studies are required to establish the association between AIHA and NK-cell profile and in vitro expansion, and to find common antibodies between red blood cells (RBCs) and NK cells.
auto-immune hemolytic anemia; natural killer cells; CD3+CD56− cells; red blood cell and natural killer cell antibodies; autologous immune enhancement therapy
We examined the effect of a probiotic strain, Lactobacillus casei strain Shirota, on cytokine production and natural killer (NK) cell activity in human peripheral blood mononuclear cells (PBMNC). The cellular mechanisms of immunoregulation by L. casei strain Shirota were also investigated. L. casei strain Shirota stimulated PBMNC to secrete interleukin-12 (IL-12), gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), and IL-10. However, depletion of monocytes from PBMNC eliminated the induction of these cytokines. L. casei strain Shirota was phagocytosed by monocytes and directly stimulated them to secrete IL-12, TNF-α, and IL-10. IFN-γ production was diminished by the addition of anti-IL-12 antibody to the PBMNC cultures. Purified T cells, but not NK cells, produced IFN-γ effectively when stimulated with L. casei strain Shirota in the presence of monocytes, indicating that monocytes triggered by L. casei strain Shirota help T cells to produce IFN-γ through secreting IL-12. In addition, NK cell activity and CD69 expression on NK cells increased after cultivation of PBMNC with L. casei strain Shirota. When monocytes were depleted from PBMNC, L. casei strain Shirota did not enhance NK cell activity. These results demonstrate that monocytes play critical roles in the induction of cytokines and following the augmentation of NK cell activity during the stimulation of human PBMNC with L. casei strain Shirota.
Dendritic cells (DCs) generated ex vivo from peripheral blood monocytes or mobilized CD34+ cells and intended for clinical immunotherapy are typically characterized by morphologic, phenotypic, and functional assays. Assay results are highly dependent on conditions used to prepare the cells, so there is no standard assay battery for clinical DC products. This study evaluated gene expression profiling for characterization of immature DCs prepared from monocytes that had been elutriated from normal donor peripheral blood mononuclear cells (PBMNCs) immediately after collection or after storage at 4°C for 48 hours.
RNA was isolated from fresh and 48-hour-stored PBMNCs, elutriated monocytes, elutriated lymphocytes and immature DCs from 5 healthy subjects and was analyzed using a cDNA gene expression microarray with 17,500 genes.
Unsupervised hierarchical clustering separated the 40 products into 4 groups: one with all 10 immature DCs, one with all 10 elutriated lymphocytes, one with 7 PBMNCs, and one with 10 elutriated monocytes and 3 PBMNCs. However, within each of the 4 groups, fresh and stored products, or products derived from fresh or stored products, clustered together. Comparison of genes differentially expressed by fresh vs stored products (paired t-tests, p<0.005) found 273 genes that differed between fresh and stored PBMCs, 429 between lymphocytes elutriated from fresh vs stored PBMNCs, 711 between monocytes elutriated from fresh vs stored PBMNCs, and 3 between immature DCs prepared from monocytes elutriated from fresh vs stored PBMCs.
This study demonstrates the potential utility of gene expression profiling for characterization of cell therapy products.
Immunomagnetic enrichment followed by RT-PCR (immunobead RT-PCR) is an efficient methodology to identify disseminated carcinoma cells in the blood and bone marrow. The RT-PCR assays must be both specific for the tumor cells and sufficiently sensitive to enable detection of single tumor cells. We have developed a method to test RT-PCR assays for any cancer. This has been investigated using a panel of RT-PCR markers suitable for the detection of breast cancer cells.
In the assay, a single cell line-derived tumor cell is added to 100 peripheral blood mononuclear cells (PBMNCs) after which mRNA is isolated and reverse transcribed for RT-PCR analysis. PBMNCs without added tumor cells are used as specificity controls. The previously studied markers epidermal growth factor receptor (EGFR), mammaglobin 1 (MGB1), epithelial cell adhesion molecule (EpCAM/TACSTD1), mucin 1 (MUC1), carcinoembryonic antigen (CEA) were tested. Two new epithelial-specific markers ELF3 and EphB4 were also tested.
MUC1 was unsuitable as strong amplification was detected in 100 cell PBMNC controls. Expression of ELF3, EphB4, EpCAM, EGFR, CEA and MGB1 was found to be both specific for the tumor cell, as demonstrated by the absence of a signal in most 100 cell PBMNC controls, and sensitive enough to detect a single tumor cell in 100 PBMNCs using a single round of RT-PCR.
ELF3, EphB4, EpCAM, EGFR, CEA and MGB1 are appropriate RT-PCR markers for use in a marker panel to detect disseminated breast cancer cells after immunomagnetic enrichment.
Systemic lupus erythematosus (SLE) is associated with premature and accelerated atherosclerosis. Circulating progenitor cells (CPCs) are circulating bone-marrow derived cells that play an important role in the repair of vascular damage that underlies the development of atherosclerosis. The objective of this study was to determine the number and functionality of CPCs in patients with SLE. The study included 44 female SLE patients in an inactive stage of disease and 35 age-matched female controls. CPC numbers in the circulation were determined by FACS with monoclonals against CD14, CD34 and CD133. Peripheral blood-derived mononuclear cell (PBMNC) fractions were cultured in angiogenic medium. The endothelial-like phenotype was confirmed and the colony forming unit (CFU) capacity, migratory capacity and the potential to form clusters on Matrigel were determined. Expression of apoptosis inhibiting caspase 8L was analyzed in PBMNCs and CPCs by gene transcript and protein expression assays. The number of CD34–CD133 double-positive cells (P < 0.001) as well as the CFU capacity (P = 0.048) was reduced in SLE patients. Migratory activity on tumor necrosis factor-α tended to be reduced in patient CPCs (P = 0.08). Migration on vascular endothelial growth factor showed no significant differences, nor were differences observed in the potential to form clusters on Matrigel. The expression of caspase 8L was reduced at the transcriptional level (P = 0.049) and strongly increased at the protein level after culture (P = 0.003). We conclude that CPC numbers are reduced in SLE patients and functionality is partly impaired. We suggest these findings reflect increased susceptibility to apoptosis of CPCs from SLE patients.
In the event of chronic diabetes or burn wounds, accomplishing skin regeneration is a major concern. Autologous skin grafting is the most effective remedy, but the tissue harvest may create more nonhealing wounds. Currently available skin substitutes have a limited clinical outcome because of immune reactions arising from the xenobiotic scaffold or allogenous cells. Autologous stem cells that can be collected without an additional injury may be a viable option for skin-tissue engineering. Presence of a low number of keratinocyte progenitor cells (KPCs) within the peripheral blood mononuclear cell (PBMNC) population has been indicated. Identification, isolation, expansion, and differentiation of KPCs is necessary before they are considered for skin regeneration, which is the focus of this study.
Culture of isolated human PBMNCs on a cell-specific matrix was carried out to induce differentiation of KPCs. Flow cytometry and reverse transcriptase polymerase chain reaction were done for epithelial stem cell marker p63 and lineage markers cytokeratin 5 and cytokeratin 14, to track differentiation. Proliferation was confirmed by quantifying the proliferating cell nuclear antigen-expressing cells. Immunostaining with epithelial cell markers, involucrin and filaggrin, was carried out to establish terminal differentiation. Microscopic analysis confirmed growth and survival of KPCs on the dermal fibroblast monolayer and on a transplantable fibrin sheet.
We demonstrated that KPCs are p63+ and CD34-. The specifically designed composition of the extracellular matrix was found to support selective adhesion, proliferation, and differentiation of p63+ KPCs. The PBMNC culture for 12 days under controlled conditions resulted in a homogenous population that expressed cytokeratins, and >90% of the cells were found to proliferate. Subculture for 5 days resulted in expression of filaggrin and involucrin, suggesting terminal differentiation. Transfer of matrix-selected KPCs to a dermal fibroblast monolayer or fibrin supported cell proliferation and showed typical hexagonal morphology of keratinocytes within 15 days.
Circulating KPCs were identified with p63, which differentiated into keratinocytes with expression of the cytokeratins, involucrin and filaggrin. Components of the specifically designed matrix favored KPC attachment, directed differentiation, and may turn out to be a potential vehicle for cell transplantation.
Keratinocyte progenitor cells; p63; Cytokeratins; Peripheral blood mononuclear cells; Epithelial cells; Fibrin; Matrix-directed differentiation; Skin-tissue engineering
About one third of patients with epilepsy become refractory to therapy despite receiving adequate medical treatment, possibly from multidrug resistance. P-glycoprotein, encoded by multidrug resistance protein-1 (MDR1) gene, at the blood brain barrier is considered as a major factor mediating drug efflux and contributing to resistance. Given that peripheral blood mononuclear cells (PBMNCs) express MDR1, we investigated a MDR1 status of PBMNCs in various subsets of epilepsy patients and demonstrated their association with clinical characteristics.
Clinical and MDR1 data were collected from 140 patients with epilepsy, 30 healthy volunteers, and 20 control patients taking anti-epileptic drugs. PBMNCs were isolated, and basal MDR1 levels and MDR1 conformational change levels were measured by flow cytometry. MDR1 profiles were analyzed according to various clinical parameters, including seizure frequency and number of medications used in epilepsy patients. Epilepsy patients had a higher basal MDR1 level than non-epilepsy groups (p<0.01). Among epilepsy patients, there is a tendency for higher seizure frequency group to have higher basal MDR1 level (p = 0.059). The MDR1 conformational change level was significantly higher in the high-medication-use group than the low-use group (p = 0.028). Basal MDR1 (OR = 1.16 [95% CI: 1.060–1.268]) and conformational change level (OR = 1.11 [95% CI: 1.02–1.20]) were independent predictors for seizure frequency and number of medications, respectively.
The MDR1 profile of PBMNCs is associated with seizure frequency and medication conditions in patients with epilepsy.
RNA quality is believed to decrease with ischaemia time, and therefore open radical prostatectomy has been advantageous in allowing the retrieval of the prostate immediately after its devascularization. In contrast, robotic-assisted laparoscopic radical prostatectomies (RALP) require the completion of several operative steps before the devascularized prostate can be extirpated, casting doubt on the validity of this technique as a source for obtaining prostatic tissue. We seek to establish the integrity of our biobanking process by measuring the RNA quality of specimens derived from robotic-assisted laparoscopic radical prostatectomy.
We describe our biobanking process and report the RNA quality of prostate specimens using advanced electrophoretic techniques (RNA Integrity Numbers, RIN). Using multivariate regression analysis we consider the impact of various clinicopathological correlates on RNA integrity.
Our biobanking process has been used to acquire 1709 prostates, and allows us to retain approximately 40% of the prostate specimen, without compromising the histopathological evaluation of patients. We collected 186 samples from 142 biobanked prostates, and demonstrated a mean RIN of 7.25 (standard deviation 1.64) in 139 non-stromal samples, 73% of which had a RIN ≥ 7. Multivariate regression analysis revealed cell type - stromal/epithelial and benign/malignant - and prostate volume to be significant predictors of RIN, with unstandardized coefficients of 0.867(p = 0.001), 1.738(p < 0.001) and -0.690(p = 0.009) respectively. A mean warm ischaemia time of 120 min (standard deviation 30 min) was recorded, but multivariate regression analysis did not demonstrate a relationship with RIN within the timeframe of the RALP procedure.
We demonstrate the robustness of our protocol - representing the concerted efforts of dedicated urology and pathology departments - in generating RNA of sufficient concentration and quality, without compromising the histopathological evaluation and diagnosis of patients. The ischaemia time associated with our prostatectomy technique using a robotic platform does not negatively impact on biobanking for RNA studies.
biobanking; prostate collection; ischaemia time; robotic-assisted radical prostatectomy; RNA quality; RIN
Tissue quality control measures are routinely performed in brain banks with the assessment of brain pH being the most common measure. In some brain banks the assessment of the RNA integrity number is also performed, although this requires access to specialised equipment and is more expensive. The aim of this study is to determine if there is a correlation between the visual assessment of cerebellar granule cell integrity and brain pH or RIN. One hundred and five consecutive cases from the NSW Tissue Resource Centre, Sydney, Australia were accessed. The cerebrum was hemisected and one hemisphere sliced parasagittally at approximately 1–2 cm intervals and frozen. The other hemisphere was fixed in 15% buffered formalin for 2–3 weeks. The contralateral cerebellar hemisphere was preserved in the same manner as the cerebral hemisphere. Samples of fixed tissue were embedded in paraffin, 7 μm sections cut and stained routinely with hematoxylin and eosin. The granular cell layer (GCL) was assessed microscopically to determine the degree of autolytic degradation. Degradation was graded as nil, mild, moderate or severe. Brain tissue pH and RIN were measured using standardised protocols. This study showed that both brain pH and RIN significantly correlated with the severity of the degradation of the cerebellar granule cell layer. This additional screening tool can be performed during routine histological review of the cerebellar tissue to assess the suitability for further investigation of tissue quality.
Postmortem; Brain pH; RIN
Increasing evidence shows that some cells from peripheral blood fibroblast-like mononuclear cells have the capacity to differentiate into mesenchymal lineages. However, the insufficiency of these cells in the circulation challenges the cell isolation and subsequently limits the clinical application of these cells. In the present study, the peripheral blood mononuclear cells (pbMNCs) were isolated from wound animals and treated with the supernatant of bone marrow mesenchymal stromal cells (bmMSCs). Results showed these pbMNCs were fibroblast-like, had stromal morphology, were negative for CD34 and CD45, but positive for Vimentin and Collagen I, and had the multipotency to differentiate into adipocytes and osteoblasts. We named these induced peripheral blood-derived mesenchymal stromal cells (ipbMSCs). Skin grafts in combination with ipbMSCs and collagen I were applied for wound healing, and results revealed ipbMSC exhibited similar potency and effectiveness in the promotion of wound healing to the bmMSCs. Hereafter, we speculate that the mixture of growth factors and chemokines secreted by bmMSCs may play an important roles in the induction of the proliferation and mesenchymal differentiation of mononuclear cells. Our results are clinically relevant because it provide a new method for the acquisition of MSCs which can be used as a candidate for the wound repair.
Bone marrow mesenchymal stromal cells; Peripheral blood mesenchymal stromal cells; Cell differentiation; Wound healing
Mounting evidence suggests that opiate addiction and stress are associated with impaired cell-mediated immunity. We tested the hypothesis that morphine and the endogenous opioid beta-endorphin (beta-END), a pituitary peptide released in increased concentrations during stress, can suppress the production of the key macrophage-activating lymphokine interferon-gamma (IFN-gamma) by cultured human peripheral blood mononuclear cells (PBMNC). Using a radioimmunoassay to measure IFN-gamma, we found that exposure of PBMNC to biologically relevant concentrations of both opioids significantly inhibited IFN-gamma generation by cells stimulated with concanavalin A and varicella zoster virus. Studies of the mechanism of suppression revealed (a) a classical opioid receptor is involved (suppression was antagonized by naloxone and was specific for the NH2 terminus of beta-END), (b) monocytes are the primary target cell for opioids (monocyte-depleted lymphocyte preparations showed little suppression), and (c) reactive oxygen intermediates (ROI) and prostaglandin E2 are important mediators (scavengers of ROI and indomethacin eliminated the suppression). Based on these findings we suggest that opioid-triggered release of inhibitory monocyte metabolites may play a role in the immunodeficiency associated with narcotic addiction and stress.
AIM: To determine the anti-inflammatory activity of probiotic Bifidobacteria in Bifidobacteria-fermented milk (BFM) which is effective against active ulcerative colitis (UC) and exacerbations of UC, and to explore the immunoregulatory mechanisms.
METHODS: Peripheral blood mononuclear cells (PBMNC) from UC patients or HT-29 cells were co-cultured with heat-killed probiotic bacteria or culture supernatant of Bifidobacterium breve strain Yakult (BbrY) or Bifidobacterium bifidum strain Yakult (BbiY) to estimate the amount of IL-10 or IL-8 secreted.
RESULTS: Both strains of probiotic Bifidobacteria contained in the BFM induced IL-10 production in PBMNC from UC patients, though BbrY was more effective than BbiY. Conditioned medium (CM) and DNA of both strains inhibited IL-8 secretion in HT-29 cells stimulated with TNF-α, whereas no such effect was observed with heat-killed bacteria. The inhibitory effect of CM derived from BbiY was greater than that of CM derived from BbrY. DNAs of the two strains had a comparable inhibitory activity against the secretion of IL-8. CM of BbiY induced a repression of IL-8 gene expression with a higher expression of IκB-ζ mRNA 4 h after culture of HT-29 cells compared to that in the absence of CM.
CONCLUSION: Probiotic Bifidobacterium strains in BFM enhance IL-10 production in PBMNC and inhibit IL-8 secretion in intestinal epithelial cells, suggesting that BFM has anti-inflammatory effects against ulcerative colitis.
Bifidobacterium; Ulcerative colitis; Anti-inflammatory; Interleukin-10; Interleukin-8
We tested the hypothesis that altered Toll-like receptor (TLR) signaling may be involved in early stages of type 1 diabetes (T1D). To do so, we analyzed TLR-induced interleukin (IL)-1β and IL-6 responses in freshly isolated peripheral blood mononuclear cells (PBMNCs) from seropositive compared with seronegative subjects. Similar frequencies of myeloid dendritic cells (mDCs), plasmacytoid DCs (pDCs), and monocytes were observed in seropositive and seronegative subjects. Subjects with autoantibodies had increased proportions of monocytes expressing IL-1β ex vivo. Activating PBMNCs with TLR3, TLR4, or TLR7/8 agonists in vitro led to increased percentages of IL-1β–expressing monocytes and mDCs from seropositive versus seronegative subjects. TLR ligation also resulted in a diminished IL-6 response in seropositive individuals as lower frequencies of IL-6–expressing monocytes and mDCs were induced. The dysregulated TLR-induced IL-1β and IL-6 pathways were more readily detectable in children aged <11 years and from 11 to <21 years, respectively, and did not involve altered HbA1c or the presence of one or more autoantibodies. Finally, subjects with autoantibodies had lower amounts of serum chemokine (C-X-C motif) ligand 10 compared with autoantibody-negative subjects. Our data may imply that alterations in innate immune pathways are detectable in genetically susceptible individuals and could be linked with the early course of T1D.
Vitamin E (VE) has been added to ultrahigh-molecular-weight polyethylene (UHMWPE) acetabular cups and tibial trays primarily to reduce oxidative damage to the polymer. The aim of this study was to investigate the relative wear rates of UHMWPE-containing VE compared with virgin UHMWPE. The ability of VE to reduce the amount of inflammatory cytokines produced from stimulated peripheral blood mononuclear cells (PBMNCs) was also investigated. Stimulation was achieved by exposure of PBMNCs to either lipoplysaccharide (LPS) or VE-containing UHMWPE (VE-UHMWPE). In the present study, results showed that the wear rates of UHMWPE with or without VE were not significantly different. Particles generated by UHMWPE with and without VE were not significantly different in size distribution. The production of osteolytic mediators, tumor necrosis factor-alpha, interleukin 1β (IL-β), IL-6, and IL-8 were significantly reduced in (PBMNCs) stimulated with either LPS + VE compared with LPS or VE-UHMWPE particles compared to virgin UHMWPE particles. This trend was also observed when VE was added as a liquid to UHMWPE wear particle-stimulated PBMNCs. The exact mechanism of how VE affects the release of inflammatory mediators from particle-stimulated macrophages is not yet understood. It is likely to involve the anti-inflammatory and/or antioxidant effects of VE.
vitamin E; UHMWPE; oxidative damage; oxidative stress; osteolytic mediators; novel bearing materials
The success of basic molecular research using biospecimens strongly depends on the quality of the specimen. In this study, we evaluated the effects of delayed freezing time on the stability of DNA and RNA in fresh frozen tissue from patients with colorectal cancer.
Tissues were frozen at 10, 30, 60, and 90 minutes after extirpation of colorectal cancer in 20 cases. Absorbance ratio of 260 to 280 nm (A260/A280) and agarose gel electrophoresis were evaluated. In addition, the RNA integrity number (RIN) was assayed for the analysis of the RNA integrity.
Regardless of delayed freezing time, all DNA and RNA samples revealed A260/A280 ratios of more than 1.9, and all DNA samples showed a discrete, high-molecular-weight band on agarose gel electrophoresis. The RINs were 7.53 ± 2.04, 6.70 ± 1.88, 6.47 ± 2.58, and 4.22 ± 2.34 at 10, 30, 60, and 90 minutes, respectively. Though the concentration of RNA was not affected by delayed freezing, the RNA integrity was decreased with increasing delayed freezing time.
According to the RIN results, we recommend that the collection of colorectal cancer tissue should be done within 10 minutes for studies requiring RNA of high quality and within 30 minutes for usual RNA studies.
Colorectal neoplasms; Tissue banks; DNA; RNA; Quality control
Banked tissue is essential to the study of neurological disease but using postmortem tissue introduces a number of possible confounds. Foremost amongst these are factors relating to variation in postmortem interval (PMI). Currently there are conflicting reports on how PMI affects overall RNA integrity, and very few reports of how gene expression is affected by PMI. We analyzed total RNA extracted from frozen cerebellar cortex from 79 deceased human subjects enrolled in the Banner Sun Health Research Institute Brain and Body Donation Program. The PMI, which ranged from 1.5 to 45 hours, correlated with overall RNA quality measures including RNA Integrity Number (RIN) (r = − 0.34, p = 0.002) and RNA quantitative yield (r = − 0.25, p = 0.02). Additionally, we determined the expression of 89 genes using a PCR-based gene expression array (RT2 ProfilerTM PCR Array: Human Alzheimer’s Disease; SABiosciencesTM, Frederick, MD). A greater proportion of genes had decreased rather than increased expression with increasing PMI (65/89 vs 20/89; p < 0.0001). Of these, transcripts from the genes ADAM9, LPL, PRKCG, and SERPINA3 had significantly decreased expression with increasing PMI (p < 0.01). No individual gene transcripts had significantly increased expression with increasing PMI. In conclusion, it is apparent that RNA degrades progressively with increasing PMI and that measurement of gene expression in brain tissue with longer PMI may give artificially low values. For tissue derived from autopsy, a short PMI optimizes its utility for molecular research.
RIN; RNA; postmortem; brain; gene expression; Alzheimer’s disease; neurological disease; research; methods