Human Peripheral Blood Mononuclear Cells Cells Research Articles

Monoclonal enolase-1 blocking antibody ameliorates pulmonary inflammation and fibrosis

BackgroundIdiopathic pulmonary fibrosis (IPF) is a chronic fatal disease with limited therapeutic options. The infiltration of monocytes and fibroblasts into the injured lungs is implicated in IPF. Enolase-1 (ENO1) is a cytosolic glycolytic enzyme which could translocate onto the cell surface and act as a plasminogen receptor to facilitate cell migration via plasmin activation. Our proprietary ENO1 antibody, HL217, was screened for its specific binding to ENO1 and significant inhibition of cell migration and plasmin activation (patent: US9382331B2).MethodsIn this study, effects of HL217 were evaluated in vivo and in vitro for treating lung fibrosis.ResultsElevated ENO1 expression was found in fibrotic lungs in human and in bleomycin-treated mice. In the mouse model, HL217 reduced bleomycin-induced lung fibrosis, inflammation, body weight loss, lung weight gain, TGF-β upregulation in bronchial alveolar lavage fluid (BALF), and collagen deposition in lung. Moreover, HL217 reduced the migration of peripheral blood mononuclear cells (PBMC) and the recruitment of myeloid cells into the lungs. In vitro, HL217 significantly reduced cell-associated plasmin activation and cytokines secretion from primary human PBMC and endothelial cells. In primary human lung fibroblasts, HL217 also reduced cell migration and collagen secretion.ConclusionsThese findings suggest multi-faceted roles of cell surface ENO1 and a potential therapeutic approach for pulmonary fibrosis.

Human peripheral blood mononuclear cells as an ex vivo model to study the host parasite interaction in Toxoplasma gondii

Toxoplasma gondii is a parasite that can invade any cell in the human body. Here, we implemented and described an ex vivo model with human peripheral blood mononuclear cells (PBMCs) without using culture supplements/antibiotics and without cryopreserved cells (EXMOWS) to study the interactions between T. gondii and human cells. To establish the EXMOWS, three independent tests were carried out. Firstly, blood samples from 5 individuals were included to assess the viability and adherence of PBMCs in plate culture. In a second trial, blood samples from three seropositive and two seronegative individuals for T. gondii were used to evaluate human PBMCs cells: parasites, multiplicity of infection (MOI) 1:1, 1:3 and 1:5 at different times post infection (1 h, 6 h and 24 h). The possible immunomodulatory effect of the infection for this EXMOWS were evaluated in a third trial where HFF cells were infected with T. gondii and co-cultured with PBMCs obtained from anti-Toxoplasma IgG positive and IgG negative individuals. One hour was enough time for T. gondii infection of human PBMCs and 2 h was the minimum incubation time to guarantee adherence before carrying out any infection assay. A minimum of 1:3 MOI was necessary to guarantee efficient infection in human PBMCs with T. gondii RH-GFP. All protocols, including PBMCs isolation and stimulation, should be conducted the same day. This EXMOWS can be adapted to study the early stages of interaction with other microorganisms of human interest, without need of using cryopreservation and supplements/antibiotics.

In vitro regulation of IL-6 and TGF-ß by mesenchymal stem cells in systemic lupus erythematosus patients.

Aim To analyse the ability of mesenchymal stem cells (MSCs) to regulate interleukin 6 (IL-6) and transforming growth factor (TGF-β) expression in vitro under co-culture conditions in human systemic lupus erythematosus (SLE). Method This study used a post-test group design that used peripheral blood mononuclear cells (PBMCs) from SLE patients at Kariadi Hospital, Semarang, Indonesia, and MSCs from a human umbilical cord. The cells were divided into two groups. The control group of PBMCs was treated with a standard medium, and the treatment group was co-cultured with the MSCs at a 1:40 ratio. Following 24 h incubation, the levels of IL-6 and TGF-β released in the culture medium were measured using a specific ELISA assay. Results This study showed a significant decrease in IL-6 level (p<0.05) and a significant increase in TGF-β level (p<0.001) following 24 h of co-culture incubation of human SLE PBMCs cells and MSCs. Conclusion The PBMCs-to-MSCs ratio of 1:40 can regulate the IL-6 and TGF-β levels in human SLE PBMCs.

Anticancer and Immunomodulatory Activities of a Novel Water-Soluble Derivative of Ellipticine.

Cancer still remains a major public health concern around the world and the search for new potential antitumor molecules is essential for fighting the disease. This study evaluated the anticancer and immunomodulatory potential of the newly synthetized ellipticine derivate: sodium bromo-5,11-dimethyl-6H-pyrido[4,3-b]carbazole-7-sulfonate (Br-Ell-SO3Na). It was prepared by the chlorosulfonation of 9-bromoellipticine. The ellipticine-7-sulfonic acid itself is not soluble, but its saponification with sodium hydroxide afforded a water-soluble sodium salt. The cytotoxicity of Br-Ell-SO3Na was tested against cancerous (K562 cell line) and non-cancerous cells (Vero cell line and human peripheral blood mononuclear cells (PBMC)) using a Methylthiazoletetrazolium (MTT) assay. Cell cycle arrest was assessed by flow cytometry and the immunomodulatory activity was analyzed through an enzyme-linked immunosorbent assay (ELISA). The results showed that the Br-Ell-SO3Na molecule has specific anticancer activity (IC50 = 35 µM) against the K562 cell line, once no cytotoxicity effect was verified against non-cancerous cells. Cell cycle analysis demonstrated that K562 cells treated with Br-Ell-SO3Na were arrested in the phase S. Moreover, the production of IL-6 increased and the expression of IL-8 was inhibited in the human PBMC treated with Br-Ell-SO3Na. The results demonstrated that Br-Ell-SO3Na is a promising anticancer molecule attested by its noteworthy activity against the K562 tumor cell line and immunomodulatory activity in human PBMC cells.

Design and synthesis of novel nitrogen mustard-evodiamine hybrids with selective antiproliferative activity

A series of novel nitrogen mustard-evodiamine hybrids were synthesized and evaluated for their antitproliferative properties. The antiproliferative activities of 10a–d, 11a–d, and 12a–d against four different kinds of human cancer cell lines (PC-3, HepG2, THP-1 and HL-60) and human normal peripheral blood mononuclear cells (PBMC) were determined. The results showed that all the target hybrid compounds exhibited antiproliferative activities against tested human tumor cell lines to some extent and no antiproliferative activities (>200 μM) against human normal PBMC cells. The antiproliferative selectivity between tumorous and normal cells was very useful for further antitumor drug development. Among the target compounds, 12c showed the strongest cytotoxicity against two tumor cell lines (THP-1 and HL-60) with IC50 values of 4.05 μM and 0.50 μM, respectively, and selected for further mechanism study in HL-60 cells. The results showed that 12c could induce HL-60 cells apoptosis and arrest at G2 phase at low sub-micromolar concentrations via mitochondria-related pathways.

566. Selective and Stable Transduction of Human CD4+ T Cells In Vivo Upon Systemic Administration of CD4-Targeted Lentiviral Vectors

Playing a central role in both innate and adaptive immunity, CD4+ T cells are the key target for genetic modifications in basic research and immunotherapy. Specific and stable delivery of therapeutic genes into these cells is therefore highly desirable. Here, we describe novel lentiviral vectors (CD4-LV) that have been rendered selective for human or simian CD4+ cells by surface engineering. This novel CD4-LV was highly specific and effective in genetic modification of human CD4+ T cells both in vitro and in vivo. When applied to peripheral blood mononuclear cells (PBMC), CD4-LV transduced CD4+ but not CD4− cells. Notably, also unstimulated T cells were stably genetically modified. Upon systemic or intrasplenic administration into mice reconstituted with human PBMC or hematopoietic stem cells, reporter gene expression was predominantly detected in lymphoid organs. Evaluation of GFP expression in organ-derived cells and blood by flow cytometry demonstrated exclusive gene transfer into CD4+ human lymphocytes. In bone marrow and spleen memory T cells were preferentially hit. Towards potential therapeutic applications, we show that CD4-LV can deliver genes protecting T cells from infection with HIV-1 or encoding chimeric antigen receptors thereby inducing tumor cell killing. This study demonstrates for the first time highly selective and stable gene delivery into human CD4+ T lymphocytes upon in vivo administration of lentiviral vectors, opening an avenue towards novel strategies in immunotherapy.

Probiotic attributes ofLactobacillusstrains isolated from food and of human origin

Lactobacilli isolated from various sources were identified on the basis of 16S-23S rRNA gene intergenic region amplification and subsequent sequencing of the smaller intergenic region. An in vitro analysis of probiotic properties including binding, ability to tolerate different concentrations of bile, survival in acidic buffer and antimicrobial activity of four different isolates and two standard strains (Lactobacillus plantarum American Type Culture Collection (ATCC) 8014 and L. rhamnosus GG (LGG)) was carried out. The ability of each isolate to stimulate Caco-2 cells, human peripheral blood mononuclear cells (PBMC) and THP-1 cells resulting in immunomodulation of these cells was analysed. Isolates L. rhamnosus CS25 and L. delbrueckii M and standard strain ATCC 8014 showed broad antimicrobial activity, and isolates CS25 (percentage of survival 6.9 % at pH 2.5, 5.1 % at pH 2.0) and L. plantarum CS23 (5.7 % at pH 2.5, 4.9 % at pH 2.0) have shown good tolerance to acidic pH. Isolate CS23 showed a good survival (14 %) after 2 h incubation in de Man, Rogosa and Sharpe (MRS) medium containing 3 % bile salts. Isolates CS23, CS25 and L. fermentum ASt1 could stimulate Caco-2 cells, human PBMC and THP-1 cells for a strong and varied immunomodulatory response in these cells. Though LGG showed poor antimicrobial activity as well as bile and acid tolerance, it was found to be the best binding strain tested. Child faecal isolate CS23 from the present study showed high binding ability (seventeen bacteria/Caco-2), high tolerance to acidic pH and bile salts and significant immunomodulation; therefore it is a good potential probiotic candidate.

New pterocarpanquinones: Synthesis, antineoplasic activity on cultured human malignant cell lines and TNF-α modulation in human PBMC cells

A new pterocarpanquinone (5a) was synthesized through a palladium catalyzed oxyarylation reaction and was transformed, through electrophilic substitution reaction, into derivatives 5b–d. These compounds showed to be active against human leukemic cell lines and human lung cancer cell lines. Even multidrug resistant cells were sensitive to 5a, which presented low toxicity toward peripheral blood mononuclear cells (PBMC) cells and decreased the production of TNF-α by these cells. In the laboratory these pterocarpanquinones were reduced by sodium dithionite in the presence of thiophenol at physiological pH, as NAD(P)H quinone oxidoredutase-1 (NQO1) catalyzed two-electron reduction, and the resulting hydroquinone undergo structural rearrangements, leading to the formation of Michael acceptors, which were intercepted as adducts of thiophenol. These results suggest that these compounds could be activated by bioreduction.

Immune cell activation and subsequent epithelial dysfunction by Staphylococcus enterotoxin B is attenuated by the green tea polyphenol (−)-epigallocatechin gallate

Bacterial superantigens (SAg) are potent T cell activators and when delivered systemically elicit a self-limiting enteropathy in mice. Also, SAg-stimulated human peripheral blood mononuclear cells (PBMC) increase enteric epithelial cell monolayer permeability in vitro. Epigallocatechin gallate (EGCG), the major polyphenol component of green tea ( Camilla sinesis) leaf, has been presented as an anti-inflammatory agent. We tested the hypothesis that EGCG (10–100 μM) would block PBMC activation by the SAg, Staphylococcus aureus enterotoxin B (SEB, 1 μg/ml), thus preventing disruption of the epithelial barrier. Pretreatment or co-treatment of human PBMC or murine lymphnode cells with EGCG significantly reduced SEB-induced proliferation and IL-2, IFNγ, and TNFα production. ConA-induced proliferation was also inhibited by EGCG (50 μM) co-treatment. These effects of EGCG were not due to induction of immune cell apoptosis, and were independent of EGCGs anti-oxidant activity, and inhibition of NF-κB or AP-1 activation. Moreover, addition of exogenous IL-2 (20 ng/ml) to the cultures could not overcome the immunosuppressive effect of EGCG. Culture supernatant from PBMC stimulated in the presence of EGCG failed to increase the permeability of T84 epithelial cell monolayers: a finding consistent with the reduced IFNγ and TNFα production by SAg + EGCG treated PBMC. These data promote EGCG as a suppressor of T cell activation, and given the prominent role that bacteria and T cells play in inflammatory disease we suggest that EGCG could be a useful addition to current treatments for enteric immune disorders and T cell driven immunopathologies.

Conversion of normal rats into SCID-like animals by means of bone marrow transplantation from SCID donors allows engraftment of human peripheral blood mononuclear cells.

We have recently shown that lethally irradiated normal strains of mice, radioprotected with SCID bone marrow, can be engrafted with human peripheral blood mononuclear cells (PBMC). We now demonstrate that lethally irradiated Lewis rats can also be radioprotected with a transplant of SCID bone marrow cells, administered 1 day after total body irradiation. Split chimerism was found in PBMC, 30 days after transplantation, with predominance of SCID donor-type cells. The average percentages of CD4 and CD8 T cells, of mouse or rat origin, were < 1%. This chimerism status could be maintained for over 3 months. When human PBMC (300-1000 x 10(6) cells) were transplanted intraperitoneally 1 day after the administration of SCID bone marrow, prompt engraftment of human CD4 and human CD8 T cells, as well as human CD20 B cells, was found in the peritoneum and in internal organ (such as liver, lung, spleen, thymus, and lymph nodes). T cell activation was high: about 50% of the cells expressed HLA-DR and almost all expressed CD45RO. High titers of human Ig (> 1 mg/ml) were initially found after 2 weeks; these levels were similar to those found in the irradiated mouse model and in the SCID model. Likewise, marked human anti-tetanus response, predominantly of the IgG type, was recorded 2 weeks after the immunization, reaching maximal levels at 4 weeks. The triple-chimeric SCID-like rats, which accept as much as 1000 x 10(6) human PBMC, can potentially be used to elicit both antibody responses and T cell responses against specific antigens, with the advantages of a larger animal.

Inhibitory effect of azelastine, a potent antiallergic agent, on release of tumor necrosis factor‐α from activated human peripheral blood mononuclear cells and U937 cells

It is generally accepted that tumor necrosis factor-alpha (TNF-alpha) is a multifunctional cytokine which is involved in the regulation of inflammation as well as immunity. In the present study, we investigated whether azelastine, a potent antiallergic agent, affects release of TNF-alpha from peripheral blood mononuclear cells (PBMC) and U937 cell line in vitro. When human PBMC and U937 cells were stimulated by phytohemagglutinin (PHA) and 12-0-tetradecanoyl-phorbol-13-acetate (TPA), respectively, the cells released significant amounts of TNF-alpha as determined by TNF-alpha-specific enzyme immunoassay. TNF-alpha levels in the culture supernatant of PHA-stimulated human PBMC and TPA-activated U937 cells decreased in a dose-dependent manner when these cells were cultured in the presence of azelastine. This inhibitory effect of azelastine was obtained at concentrations where the drug produced no toxicity. Moreover, azelastine also inhibited release of TNF-alpha from U937 cells which were already activated by TPA. These results suggest that the inhibitory effect of azelastine on TNF-alpha release plays an important role in its antiallergic action in addition to inhibition and/or antagonism of histamine and leukotrienes, which has been previously reported.

Monoclonal Antibodies to Human Osteosarcoma-Associated Antigen(s)&lt;xref ref-type="fn" rid="FN2"&gt;2&lt;/xref&gt;&lt;xref ref-type="fn" rid="FN3"&gt;3&lt;/xref&gt;

Monoclonal antibodies (MoAbs) against human osteosarcoma cells were obtained by the production and cloning of hybrids resulting from the fusion of mouse myeloma cells P3 X 63Ag8.653 with spleen cells from partially purified, osteosarcoma-associated antigen (OSAA)-immunized BALB/c mice. OSAAs were isolated from the spent culture medium of a human osteosarcoma cell line (TE-85). Five hybrid clones were established and designated as OSA1, OSA2, OSA3, OSA4, and OSA5. OSA1 and OSA2 had similar activity. All 5 MoAbs reacted strongly with most osteosarcoma cell lines and with all osteosarcoma tissues tested but not with 10 tumor cell lines and 2 tumor tissues from other cancers. OSA3, OSA4, and OSA5 cross-reacted with a fibrosarcoma cell line, a colon cell line, and fibrosarcoma, respectively, as well as with a melanoma cell line. None of the MoAbs were reactive with activated normal human peripheral blood mononuclear cells (PBMC). Immunoprecipitation of membrane protein isolated from LM cells and TE-85 cells with the MoAbs OSA1 and OSA2 conjugated with Staphylococcus aureus yielded a molecule with molecular weight of approximately 92,000. No detectable membrane protein was precipitated when 125I-labeled membrane protein from pooled activated human PBMC and tumor cells of other histologic types were used in the immunoprecipitation.