Normal Human Peripheral Blood Mononuclear Cells Research Articles

The Anti-Tumor and Immunomodulatory Effects of PLGA-Based Docetaxel Nanoparticles in Lung Cancer: The Potential Involvement of Necroptotic Cell Death through Reactive Oxygen Species and Calcium Build-Up.

Taxanes, microtubule stabilizing agents, are extensively used in the treatment of non-small cell lung cancer (NSCLC). However, their clinical effectiveness remains restricted owing to significant adverse effects and drug resistance. Nanotechnology may guide chemotherapeutic drugs directly and selectively to malignant cells, improving their therapeutic efficiency. In the present study, we synthesized polylactic-co-glycolic acid (PLGA) based nanoparticles encapsulating docetaxel and evaluated their efficacy in non-small cell lung carcinoma (A549) cells and primary immune cells derived from humans. Docetaxel-PLGA nanoparticles (PLGA-Dtx) were synthesized and characterized using distinct methods. Moreover, the cytotoxicity of free docetaxel (Dtx) and Dtx-conjugated nanoparticles (PLGA-Dtx) was studied in A549 cells and peripheral blood mononuclear cells derived from humans. Furthermore, annexin V-FITC/PI staining was used to assess the mode of cell death. Additionally, human peripheral blood mononuclear cells (PBMCs) were used for assessing the associated immune response and cytokine profile following PLGA-Dtx treatment. Spherical PLGA-Dtx nanoparticles with a 150 ± 10 nm diameter and 70% encapsulation efficiency (EE) were synthesized. The MTT assay showed that the IC50 of PLGA-Dtx nanoparticles was significantly lower than free docetaxel in A549 cells. Cytotoxicity data also revealed the selective nature of PLGA-Dtx with no significant effects in normal human bronchial epithelial cells (BEAS-2B) and PBMCs derived from healthy donors. Interestingly, PLGA-Dtx exerted an improved effect and tempted both apoptosis and necroptosis, as evidenced by annexin V and propidium iodide-positive cells. Further, PLGA-Dtx-exposed A549 cells showed increased Cas-3, Cas-9, RIP-1, and RIP-3, indicating apoptosis and necroptosis. An increased pro-inflammatory response manifested from the enhancement of IFN-γ and TNF-α in PLGA-Dtx-exposed PBMCs, posed by the occurrence of necroptosis and the immune stimulatory effect of PLGA-Dtx. In conclusion, PLGA-Dtx has a selective anticancer potential and better immunostimulatory potential. Therefore, PLGA-Dtx may be useful for the treatment of non-small cell lung carcinoma.

Toll-like Interleukin -1 Receptor Regulator (TILRR) Protein, a Major Modulator of Inflammation, is Expressed in Normal Human and Macaque Tissues and PBMCs.

PurposeTILRR is a modulator of genes in the NF-κB inflammation pathway. It regulates inflammation-responsive genes, the secretion of inflammatory mediators, and the migration of immune cells. Because inflammation drives the pathogenesis of many infectious and inflammatory diseases, it is important to know the expression of TILRR protein in tissues and cells. This study examined TILRR protein expression in healthy adult human and macaques’ tissues and PBMCs (peripheral blood mononuclear cells).Methods and ResultsTissues (trachea, lungs, stomach, small intestine [ileum], cecum, colon, rectum, vagina, cervix, uterus, and penis) and PBMCs from humans and macaques were lysed in RIPA (radioimmunoprecipitation assay) lysis buffer. The TILRR protein was examined by fluorescent Western blot analysis. The relative fluorescence units (rfu) of TILRR protein expression were quantified by Image Studio software (LI-COR). The results showed that adult healthy female (n=1) rectal and cervicovaginal tissues expressed a higher level of TILRR protein than the other tissues (trachea, lungs, stomach, small intestine [ileum], cecum, colon, uterus, and penis) examined. Like humans, the lungs, colon, and rectal tissues of healthy adult female cynomolgus monkeys (Macaca fascicularis) (n=2) expressed the TILRR protein. In addition, PBMCs of healthy adult women (n=4), adult female cynomolgus monkeys (Macaca fascicularis) (n=4), and adult male and female rhesus monkeys (Macaca mulatta) (n=4) showed a similar expression level of TILRR protein (p= 0.2858). TILRR protein was not detected in most of the human cell lines examined, except in Jurkat cells.ConclusionOur study for the first time showed that TILRR protein is expressed in healthy adult human and monkey tissues and PBMCs. The TILRR protein in these tissues and PBMCs may play a role in the inflammatory response of these tissues and cells in response to infectious pathogens.

Synthesis, biological activity, structure activity relationship study and liposomal formulation development of some arylsulfonyl pyroglutamic acid derivatives

In this study, we have synthesized some 4-biphenylsulphonyl-L(+)-pyroglutamic acid and pyroglutamines as we have already reported the corresponding glutamic acids and glutamines. The synthesized compounds were characterized and screened against human cancer cell lines (K562, Jurkat E6.1, U937 and MDA-MB-231) as well as normal human peripheral blood mononuclear cells (PBMCs) for their possible cytotoxic activity by MTT assay. All these synthesized compounds are cytotoxic towards leukemia cell lines (K562, Jurkat E6.1 and U937) and inactive against breast cancer cell line (MDA-MB-231) as well as normal human PBMCs. Two most active compounds in cytotoxicity study (compound 7 and compound 10) were further investigated by flow cytometric in-vitro apoptosis assay against Jurkat E6.1 and K562 cell lines. Compound 7 and compound 10 induced apoptosis significantly in both Jurkat E6.1 and K562 cell lines (p < 0.05) in a time dependent fashion. Mitochondrial membrane potential detection study as well as DNA deformation assay also confirmed apoptosis mediated cellular cytotoxicity. Compound 7 and compound 10 were encapsulated into liposomes (7a and 10a). Drug loaded liposomes, 7a and 10a are more than 8 fold cytotoxic in comparison with compound 7 and compound 10 against Jurkat E6.1 and K562 cell lines. In conclusion, compound 7 and compound 10 may be considered as potential lead molecules for the development of target specific anti-leukemic agents with minimal untoward events to normal human cells.

Cross-mixing study of a poisonous Cestrum species, Cestrum diurnum in herbal raw material by chemical fingerprinting using LC-ESI-QTOF-MS/MS

Poisonous plants are widely distributed and may have risk of phytotoxicity upon mixing with medicinal plants. Several species of Cestrum genus are poisonous and linked with many serious health issues. In the present study, cross-mixing of a toxic plant, Cestrum diurnum with morphologically resembling medicinal plant, Adhatoda vasica was studied using chemical fingerprinting approach. LC-ESI-MS/MS tool was used to develop the chemical fingerprints of three toxic species of Cestrum, including, C. diurnum, C. nocturnum and C. parqui. Total forty-three compounds were identified using high-resolution LC-ESI-MS/MS data comparison. Chemometric analyses were done to compare the distribution of identified compounds present in these Cestrum species. One of the identified compounds, nornicotine (a toxic compound) was also quantified using LC-IT-MS/MS. Adulteration study was conducted by mixing toxic C. diurnum in A. vasica with various ratios (w/w) and five differentiable compounds were identified to detect the adulteration. The method was able to detect up to the limit of 5% mixing of toxic C. diurnum. Moreover, cytotoxicity of the methanolic extracts of these three species were also studied on normal human PBMC (peripheral blood mononuclear cells) and all found to be toxic, while the C. nocturnum showed the highest level of toxicity with the IC50 12.5 μg/mL.

Sulforaphane enhances apoptosis induced by Lactobacillus pentosus strain S-PT84 via the TNFα pathway in human colon cancer cells.

Sulforaphane and Lactobacilli induce apoptosis in several cancer cells. Sulforaphane, a dietary isothiocyanate, is an attractive agent due to its potent anticancer effects. Sulforaphane suppresses the proliferation of various cancer cells in vitro and in vivo. The present study investigated the effect of sulforaphane and a co-culture with Lactobacillus-treated peripheral blood mononuclear cells (PBMCs) in human colon cancer cells. The combination markedly induced apoptosis in human colon cancer HCT116 and SW480 cells. A pan-caspase inhibitor markedly inhibited apoptosis, and a tumor necrosis factor (TNF) receptor/Fc chimera partially inhibited apoptosis in both cells. The amount of TNFα secretion in the culture supernatant was significantly increased by co-culture with Lactobacillus-treated normal human PBMCs. On the other hand, the expression of cellular inhibitor of apoptosis-2 (cIAP-2), an anti-apoptotic protein, was increased by co-culture with Lactobacillus-treated PBMCs in colon cancer cells, but sulforaphane treatment significantly suppressed the induction of cIAP-2. The present results revealed that sulforaphane enhances apoptosis in human colon cancer cells under co-culture with Lactobacillus-treated PBMCs via the TNFα signaling pathway.

Modulation of P-glycoprotein by Stemona alkaloids in human multidrug resistance leukemic cells and structural relationships

BackgroundMultidrug resistance (MDR) is a major reason for the failure of chemotherapy in the treatment of cancer patients. P-gp over-expression in MDR cancer cells is a multifactorial phenomenon with biochemical resistance mechanisms. Stemofoline (STF), isolated from Stemona bukillii, has been reported to be an MDR reversing compound. PurposeThis study investigated whether other Stemona alkaloids that had been purified from Stemonaceae plants exerted MDR modulation activity. MethodsMTT assay was performed to determine the MDR reversing property of the alkaloids. Modulation of P-gp function by these compounds was investigated using cell cycle analysis and P-gp fluorescent substrate accumulation assays. P-gp expression was determined by Western blot analysis. We preliminarily examined the safety of these compounds in normal human fibroblasts and human peripheral blood mononuclear cells (PBMCs) using the MTT assay, and in red blood cells (human and rat) through in vitro hemolysis assays. ResultsThree of the eight alkaloids tested, isostemofoline (ISTF), 11Z -didehydrostemofoline (11Z-DSTF) and 11E-didehydrostemofoline (11E-DSTF), enhanced the chemotherapeutic sensitivity of MDR leukemic K562/Adr cells, which overexpressed P-gp. The P-gp functional studies showed that these three alkaloids increased the accumulation of P-gp substrates, calcein-AM (C-AM) and rhodamine123 (Rho 123) in K562/Adr cells, while this effect was not seen in drug sensitive parental K562 cells. Whereas, the alkaloids did not alter P-gp expression as was determined by Western blotting analysis. ConclusionThe alkaloids reversed MDR via the inhibition of P-gp function. For pharmaceutical safety testing, the alkaloids were found to be not toxic to normal human fibroblasts and PBMCs. Moreover, the effective compounds did not induce hemolysis in either human or rat erythrocytes. These compounds may be introduced as potential candidate molecules for treating cancers exhibiting P-gp-mediated MDR.

Effects of the Commercial Flame Retardant Mixture DE-71 on Cytokine Production by Human Immune Cells.

IntroductionAlthough production of polybrominated diphenyl ethers (PBDEs) is now banned, release from existing products will continue for many years. The PBDEs are assumed to be neurotoxic and toxic to endocrine organs at low concentrations. Their effect on the immune system has not been investigated thoroughly. We aimed to investigate the influence of DE-71 on cytokine production by peripheral blood mononuclear cells (PBMCs) stimulated with Escherichia Coli lipopolysaccharide (LPS) or phytohaemagglutinin-L (PHA-L).Material and MethodsPBMCs isolated from healthy donors were pre-incubated with DE-71 at various concentrations and subsequently incubated with the monocyte stimulator LPS, or the T-cell activator PHA-L. Interferon (IFN)-γ, interleukin (IL)-1β, IL-2, IL-4, IL-6, IL-8, IL-10, tumor necrosis factor (TNF)-α, IL-17A, and IL-17F were quantified in the supernatants by Luminex kits.ResultsAt non-cytotoxic concentrations (0.01–10 μg/mL), DE-71 significantly enhanced secretion of IL-1β, IL-6, CXCL8, IL-10, and TNF-α (p<0.001–0.019; n = 6) from LPS-stimulated PBMCs. IFN-γ, TNF-α, IL-17A, and IL-17F (p = <0.001–0.043; n = 6) secretion were enhanced from PHA-L-stimulated PBMCs as well. Secretion of IL-1β, IL-2, IL-10, IL-8 and IL-6 was not significantly affected by DE-71.ConclusionsWe demonstrate an enhancing effect of DE-71 on cytokine production by normal human PBMCs stimulated with LPS or PHA-L ex vivo.

Regulatory CD4+ T lymphocytes after allogeneic transfusion in orthopaedic surgical patients.

Regulatory CD4+ T lymphocytes after allogeneic transfusion in orthopaedic surgical patients.

Effect of citral on the cytotoxicity of doxorubicin in human B-lymphoma cells

Context: Doxorubicin is a chemotherapy agent used in non-Hodgkin's lymphoma but side effects limit its use. Citral is a mixture of neral and geranial found in essential oils of lemon grass.Objectives: We evaluated the activity of citral, doxorubicin, and combination on cytotoxicity, apoptosis, and anti-proliferative effects using human lymphoma Ramos cells.Materials and methods: Cells were treated with doxorubicin alone or in combination with citral (10, 20, and 40 μM). Cytotoxic and apoptosis studies were done after 24 and 18 h incubations, respectively. Cytotoxic effects of citral on normal human peripheral blood mononuclear cells (PBMCs) were also investigated for its safety. Changes in the expression of BCL-2 family genes were analyzed by quantitative RT-PCR.Results: Citral had cytotoxicity on cells with an IC50 value of 77.19 ± 4.95 µM. Citral at concentrations of 10, 20, and 40 µM additively increased the cytotoxic and apoptotic effects of doxorubicin, leading to decreased IC50 (µM) of the drug from 2.50 ± 0.01 to 2.16 ± 0.03, 1.90 ± 0.04, and 1.23 ± 0.04, respectively. Enhanced cytotoxicity was not observed in normal human PBMCs. Citral (40 µM) in combination with doxorubicin (1.5 µM) increased the expression of pro-apoptotic protein BAK but significantly decreased the expression of anti-apoptotic protein BCL-XL to 5.26-fold compared with doxorubicin-treated cells. It did not change the anti-proliferative activity of drug.Discussion and conclusion: Citral potentiated cytotoxicity of doxorubicin by increasing apoptotic effects. We conclude that citral may have beneficial effects in patients with B cell lymphoma treated with chemotherapy.

Ciprofloxacin as a potential radio-sensitizer to tumor cells and a radio-protectant for normal cells: differential effects on γ-H2AX formation, p53 phosphorylation, Bcl-2 production, and cell death.

Ionizing radiation increases cell mortality in a dose-dependent manner. Increases in DNA double strand breaks, γ-H2AX, p53 phophorylation, and protein levels of p53 and Bax also occur. We investigated the ability of ciprofloxacin (CIP), a widely prescribed antibiotic, to inhibit DNA damage induced by ionizing radiation. Human tumor TK6, NH32 (p53 (-/-) of TK6) cells, and human normal peripheral blood mononuclear cells (PBMCs) were exposed to 2-8 Gy (60)Co-γ-photon radiation. γ-H2AX (an indicator of DNA strand breaks), phosphorylated p53 (responsible for cell-cycle arrest), Bcl-2 (an apoptotic protein, and cell death were measured. Ionizing irradiation increased γ-H2AX amounts in TK6 cells (p53(+/+)) within 1 h in a radiation dose-dependent manner. CIP pretreatment and posttreatment effectively inhibited the increase in γ-H2AX. CIP pretreatment reduced Bcl-2 production but promoted p53 phosphorylation, caspase-3 activation and cell death. In NH32 cells, CIP failed to significantly inhibit the radiation-induced γ-H2AX increase, suggesting that CIP inhibition involves in p53-dependent mechanisms. In normal healthy human PBMCs, CIP failed to block the radiation-induced γ-H2AX increase but effectively increased Bcl-2 production, but blocked the phospho-p53 increase and subsequent cell death. CIP increased Gadd45α, and enhanced p21 protein 24 h postirradiation. Results suggest that CIP exerts its effect in TK6 cells by promoting p53 phosphorylation and inhibiting Bcl-2 production and in PBMCs by inhibiting p53 phosphorylation and increasing Bcl-2 production. Our data are the first to support the view that CIP may be effective to protect normal tissue cells from radiation injury, while enhancing cancer cell death in radiation therapy.

Tetraspanin CD9 Inhibits ADAM17-Mediated LR11 Shedding In Leukemia Cells

IntroductionLR11, which is also known as SorLA or SORL1, is a type-I membrane protein from which a large extracellular part, soluble LR11 (sLR11), is released by proteolytic shedding. We have demonstrated that serum sLR11 levels are significantly elevated in patients with acute leukemia and that sLR11 levels are associated with the peripheral blast percentage (Sakai et al. Clin Chim Acta. 2012). In addition, we found that high sLR11 levels have a significant negative prognostic impact on progression-free survival in patients with follicular lymphoma (FL; Kawaguchi et al. Br J Haematol. 2013). In this FL analysis, the immunohistological intensity of LR11 in lymph nodes of FL patients did not show a significant association with serum sLR11 levels. Therefore, a shedding mechanism is presumed to play a key role in the functions of LR11. A disintegrin and metalloproteinase 17 [ADAM17, also known as tumor necrosis factor (TNF)-α converting enzyme (TACE)] has been identified as the enzyme that cleaves TNF-α from its transmembrane precursor form, and it has been found to cleave the ectodomains of other cell surface proteins. LR11 is also shed by ADAM17. Tetraspanin CD9 has been recently shown to inhibit the shedding activity of ADAM17. Therefore, we investigated the role of CD9 in sLR11 release. Materials and methodsFirst, we examined the gene expression levels of LR11 by quantitative real-time PCR and the cell surface expression of LR11 and CD9 by flow cytometric analysis in normal human peripheral-blood mononuclear cells (PBMCs). Second, we investigated the cellular expression and the released levels of LR11 and cellular CD9 in THP-1 monocytes, phorbol 12-myristate-13-acetate (PMA)-induced THP-1 macrophages (PMA/THP-1), and the B lymphoblastoid cell line CCRF-SB by immunoblot analysis. Furthermore, to assess the relationship between LR11 and CD9 in PMA/THP-1, we performed double immunofluorescence staining of these molecules followed by confocal microscopy analysis. Third, we examined the effects of ectopic neoexpression of CD9, anti-CD9 mAbs, and CD9 silencing. THP-1 cells stably interfered with CD9 were generated by the transfection of the shRNA expression vector that is specific for CD9. ResultsThe gene expression levels of LR11 were significantly higher in CD3+ T cells than in CD14+ monocytes, and there was no difference between monocytes and CD19+ B cells. However, LR11 was only expressed on the cell surface of monocytes in normal PBMCs. CD9 was broadly expressed on the cell surface of every PBMCs. Because it has been reported that CD9 associates with ADAM-17 on the cell surface, we hypothesized that CD9 associates with the shedding of LR11 on the cell surface. To verify this hypothesis, we chose the monocytic cell line THP-1 because monocytes have both LR11 and CD9 on their surface. In addition, because the gene expression levels of LR11 were significantly higher in human monocyte-derived macrophages than that in monocytes, we investigated the levels in PMA/THP-1. LR11 was not expressed or released in undifferentiated THP-1 cells, but it was expressed or released in differentiated PMA/THP-1. CD9 was poorly expressed in THP-1 cells, and the CD9 expression levels were increased in PMA/THP-1. The confocal microscopy analysis showed colocalization of LR11 and CD9 proteins in PMA/THP-1. We next investigated the effects of ectopic neoexpression of CD9 in CD9-negative cells, neutralizing anti-CD9 mAbs (clone ALB-6) in CD9-positive cells, and CD9 silencing in THP-1 on the shedding of LR11. When CCRF-SB, LR11-positive and CD9-negative cells were transiently overexpressed with CD9, the amount of sLR11 released from CD9-overexpressing cells was decreased compared with that from control cells. The amount of sLR11 released from LR11-transfected THP-1 cells that were cultured with ALB-6 was significantly increased compared with that cultured with an isotypic control antibody. The PMA-stimulated release of sLR11 was significantly increased in CD9 shRNA-interfered THP-1 cells compared with cells that were transduced with the control shRNA. The increase in CD9 shRNA-interfered THP-1 was nullified by treatment with the metalloproteinase inhibitor GM6001 (Figure 1). ConclusionThese results suggested that the tetraspanin CD9 inhibits ADAM17-mediated LR11 shedding in leukemia cells and that it may have a role in controlling the function of LR11.▪ Disclosures:No relevant conflicts of interest to declare.

The Polymerization Agent, 2-Methyl-4′-(methylthio)-2-morpholinopropiophenone Induces Caspases-3/7 in Human Blood Mononuclear Cells &lt;i&gt;in Vitro&lt;/i&gt;

Our previous studies detected the presence of a photoinitiator 2-methyl-4'-(methylthio)-2-morpholinopropiophenone (MTMP) in an intravenous (i.v.) injection bag solution. Importantly, MTMP has demonstrated cytotoxicity for normal human peripheral blood (PB) mononuclear cells (MNC). Cell death pathways have two well-known modes, apoptosis and necrosis. But it has not been clear whether MTMP induced apoptosis or necrosis in normal human PB MNC. In the present in vitro study, we examined normal human PB MNC for the frequencies of apoptosis and necrosis and changes upon exposure to MTMP. We observed time-dependent changes in MNC viability with MTMP. We also assessed the activity of caspases-3/7. The results demonstrated that MTMP induced apoptosis in normal human PB MNC after 24 h. In addition, MTMP induced caspases-3/7 in a time-dependent manner. In conclusion, we suggest that MTMP induces apoptosis in a caspase-dependent pathway in vitro.

Abstract 4625: IMGN529, a CD37-directed antibody-drug conjugate for the treatment of B-cell malignancies, has a favorable in vitro safety profile

Abstract The B-cell surface antigen CD37 is an attractive target for antibody-drug conjugate (ADC) mediated therapies, as it is widely expressed on malignant B cells in NHL and CLL. In normal human tissues, high CD37 expression is mainly restricted to B cells, with substantially lower expression seen in T cells, NK cells, monocytes and granulocytes. The CD37-targeting K7153A antibody was selected from a panel of antibodies for its potent pro-apoptotic, CDC and ADCC activity against B-cell lymphoma cell lines in vitro. The K7153A antibody was conjugated to the maytansinoid DM1 via the SMCC linker to yield the IMGN529 conjugate, which retained the intrinsic functions of the antibody and demonstrated enhanced in vitro and in vivo activity through targeted delivery of its payload. To complement preclinical in vivo toxicity studies with IMGN529 in animal models, we conducted extensive in vitro safety studies, with the aim of identifying any potential negative effects on normal human blood cells. Depletion of B cells, T cells and monocytes from peripheral blood cells by K7153A, IMGN529, the anti-CD20 antibody rituximab or the anti-CD52 antibody alemtuzumab was determined by flow cytometry. Proliferation of peripheral blood mononuclear cells (PBMCs) in response to K7153A or IMGN529 was evaluated by CFDA staining. Cytokine release of TNF-α, IFN-γ, IL-2, IL-4, IL-6 and IL-10 by normal human PBMCs was measured by ELISpot and cytometric bead array following exposure to compounds in both soluble and immobilized form. K7153A and IMGN529 specifically depleted B cells in a dose-dependent manner, but had no effect on T cells or monocytes, consistent with the much lower CD37 expression levels in these cells. K7153A and IMGN529 were more potent than rituximab and more specific than alemtuzumab, which depleted T cells and monocytes in addition to B cells. Incubation with IMGN529 did not increase the percentage of proliferating cells, while the anti-CD3 control antibody was able to strongly stimulate T cell proliferation. Treatment with K7153A or IMGN529 did not specifically induce cytokine release as compared to non-binding antibody controls, while treatment with the anti-CD3 control antibody resulted in significant cytokine release for all donors. Rituximab treatment caused release of low levels of IFN-γ and TNF in some donors. In contrast, treatment with alemtuzumab resulted in release of IFN-γ, TNF-α and IL-6 for most donors consistent with its potential for cytokine release in patients. In conclusion, the CD37-directed IMGN529 maytansinoid conjugate demonstrated potent in vitro and in vivo efficacy combined with a favorable in vitro safety profile with respect to cytokine release. Our analysis confirmed that the primary target cells of IMGN529 are B cells. Together, these data support the clinical development of this ADC for the treatment of B-cell malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4625. doi:1538-7445.AM2012-4625

Walterinnesia aegyptia venom combined with silica nanoparticles enhances the functioning of normal lymphocytes through PI3K/AKT, NFkappaB and ERK signaling

BackgroundThe toxicity of snake venom varies over time in some species. The venom of newborn and small juvenile snakes appears to be more potent than adults of the same species, and a bite from a snake that has not fed recently, such as one that has just emerged from hibernation, is more dangerous than one that has recently fed due to the larger volume of venom injected. Therefore, the potency of a snake's venom is typically determined using the LD50 or IC50 tests. In the present study, we evaluated the anti-tumor potential of snake venom from Walterinnesia aegyptia (WEV) on the human breast carcinoma cell line MDA-MB-231, as well as its effect on the normal mice peripheral blood mononuclear cells (PBMCs).ResultsThis venom was used alone (WEV) or in combination with silica nanoparticles (WEV+NP). The IC50 values of WEV alone and WEV+NP in the MDA-MB-231 cells were determined to be 50 ng/ml and 20 ng/ml, respectively. Interestingly, at these concentrations, the venom did not affect the viability of normal human PBMCs. To investigate the in vivo effects of this venom further, three groups of mice were used (15 mice in each group): Group I was the control, Group II was subcutaneously injected with WEV, and Group III was injected with WEV+NP. Using flow cytometry and western blot analysis, we found that the blood lymphocytes of WEV-injected mice exhibited a significant increase in actin polymerization and cytoskeletal rearrangement in response to CXCL12 through the activation of AKT, NF-κB and ERK. These lymphocytes also showed a significant increase in their proliferative capacity in response to mitogen stimulation compared with those isolated from the control mice (P < 0.05). More importantly, in the WEV+NP-treated mice, the biological functions of normal lymphocytes were significantly (P < 0.05) enhanced in comparison with those of WEV-treated mice.ConclusionOur data reveal the unique biological effects of WEV, and we demonstrated that its combination with nanoparticles strongly enhanced these biological effects.

Role of semaphorin 7a signaling in transforming growth factor β1–induced lung fibrosis and scleroderma‐related interstitial lung disease

Semaphorin 7a regulates transforming growth factor β1 (TGFβ1)-induced fibrosis. This study was undertaken to test the hypothesis that semaphorin 7a exerts its profibrotic effects in part by promoting the tissue accumulation of CD45+ fibrocytes. A murine model of pulmonary fibrosis in which an inducible, bioactive form of the human TGFβ1 gene is overexpressed in the lung was used. Fibrosis and fibrocytes were evaluated in TGFβ1-transgenic mice in which the semaphorin 7a locus had been disrupted. The effect of replacement or deletion of semaphorin 7a on bone marrow-derived cells was ascertained using bone marrow transplantation. The role of the semaphorin 7a receptor β1 integrin was assessed using neutralizing antibodies. The applicability of these findings to TGFβ1-driven fibrosis in humans was examined in patients with scleroderma-related interstitial lung disease (ILD). The appearance of fibrocytes in the lungs of TGFβ1-transgenic mice required semaphorin 7a. Replacement of semaphorin 7a on bone marrow-derived cells restored lung fibrosis and fibrocytes. Immunoneutralization of β1 integrin reduced pulmonary fibrocytes and fibrosis. Peripheral blood mononuclear cells (PBMCs) from patients with scleroderma-related ILD showed increased levels of messenger RNA for semaphorin 7a and its receptors, with semaphorin 7a located on collagen-producing fibrocytes and CD19+ lymphocytes. Peripheral blood fibrocyte outgrowth was enhanced in these patients. Stimulation of normal human PBMCs with recombinant semaphorin 7a enhanced fibrocyte differentiation; these effects were attenuated by β1 integrin neutralization. Our findings indicate that interventions that reduce semaphorin 7a expression or prevent the semaphorin 7a-β1 integrin interaction may ameliorate TGFβ1-driven or fibrocyte-associated autoimmune fibroses.

Cytotoxic effects of crude kaffir lime (Citrus hystrix, DC.) leaf fractional extracts on leukemic cell lines

Crude extracts from the leaves of kaffir lime (Citrus hystrix, DC.) were extracted using hexane, ethanol, ethyl acetate, butanol and methanol. The fractions were investigated in vitro for their potential cytotoxic activity on HL60, K562, Molt4, U937 cell lines, and normalhuman peripheral blood mononuclear cells (PBMCs) using the MTT assay. The in vitrocytotoxicity bioassays on 4 leukemic cell lines showed that the ethyl acetate fraction exhibited the highest cytotoxicity, with IC50 values of 19.0±0.6, 35.3±1.4, 21.8±0.4, and 19.8±1.0 µg/ml, in response to HL60, K562, Molt4, and U937 cell lines, respectively. These were higher than those of fractions from hexane, ethanol, and butanol. The methanol fraction had no cytotoxic activity (IC50 value > 100 µg/ml). None of the fractions had cytotoxic effects on PBMCs. The active compound in the ethyl acetate fraction should be investigated for possible use in chemotherapy. Key words: Kaffir lime, Citrus hystrix, DC., cytotoxicity, leukemic cell line.

The Ruthenium Complex cis-(Dichloro)Tetraammineruthenium(III) Chloride Presents Immune Stimulatory Activity on Human Peripheral Blood Mononuclear Cells

Ruthenium compounds in general are well suited for medicinal applications. They have been investigated as immunosuppressants, nitric oxide scavengers, antimicrobial agents, and antimalarials. The aim of this study is to evaluate the immunomodulatory activity of cis-(dichloro)tetraammineruthenium(III) chloride (cis-[RuCl(2)(NH(3))(4)]Cl) on human peripheral blood mononuclear cells (PBMC). The cytotoxic studies performed here revealed that the ruthenium(III) complex presents a cytotoxic activity towards normal human PBMC, only at very high concentration. Results also showed that cis-[RuCl(2)(NH(3))(4)]Cl presents a dual role on PBMC stimulating proliferation and interleukin-2 (IL-2) production at low concentration and inducing cytotoxicity, inability to proliferate, and inhibiting IL-2 production at high concentration. The noncytotoxic activity of cis-[RuCl(2)(NH(3))(4)]Cl at low concentration towards PBMC, which correlates with the small number of annexin V positive cells and also the absence of DNA fragmentation, suggest that this compound does not induce apoptosis on PBMC. For the first time, we show that, at low concentration (10-100 microg L(-1)), the cis-[RuCl(2)(NH(3))(4)]Cl compound induces peripheral blood lymphocytes proliferation and also stimulates them to IL-2 production. These results open a new potential applicability of ruthenium(III) complexes as a possible immune regulatory compound acting as immune suppressor at high concentration and as immune stimulator at low concentration.

131 The gp41-derived immunosuppressive (isu) peptide of HIV-1 modulates cytokine release and gene expression in human immune cells

The transmembrane envelope protein gp41 of HIV-1 and a synthetic peptide corresponding to a highly conserved domain of gp41, the so-called immunosuppressive (isu) domain, have been shown to inhibit mitogen-triggered stimulation and to modulate cytokine production of human immune cells. The expression of cytokines such as IL-10, IL-6, IL-8, RANTES, MCP-1, MCP-2, TNF-alpha, MIP-1alpha, MIP-1beta, MIP-3 increased upon exposure to gp41 or the corresponding isu-peptide. In contrast, the expression of IL-2 and MIG (CXCL-9) decreased. The modulation of cytokine expression induced by the isu-peptide is similar to the changes in cytokine expression in HIV-1 infected individuals. To analyse changes in gene expression in peripheral blood mononuclear cells (PBMCs) from healthy donors when incubated with the isu-peptide, a microarray analysis (AB1700, 29098 human genes) was performed. The microarray analysis confirmed the cytokine data and showed elevated expression of more than 400 genes, among them some of the above mentioned cytokines, MMP-1 (matrix metallopeptidase 1), TREM-1 (triggering receptor expressed on myeloid cells 1) and others. By real time PCR studies overexpression of these genes was confirmed and kinetics of the changes in gene expression were analysed. The changes in gene expression were also shown at the protein level using ELISA and Western blot analysis. In addition, preliminary data showed an elevated expression of FoxP3 and an increase in the number of CD4+CD25+FoxP3+ regulatory T cells. These data indicate that gp41 and the corresponding isu-peptide of HIV-1 modulate cytokine release and gene expression in normal human PBMCs.

Rabbit ATG but Not Horse ATG Promotes Expansion of Functional CD4+CD25highFoxP3 Regulatory T Cells In Vitro.

CD4+CD25+ regulatory T cells (Treg) are believed to play important roles in suppressing immune responses and maintaining tolerance. Treg have the ability to prevent the development of autoimmune diseases, graft rejection and graft versus host disease (GVHD) in mice and perhaps also in humans. Immunosuppressive drugs such as rabbit ATG (rATG), horse ATG (hATG) and cyclosporine A (CsA) are widely used in conditioning for transplantation and for the treatment of autoimmune diseases and GVHD, but their effects on Treg remain to be fully elucidated. Lopez et al. ( J Am Soc Nephrol . 2006; 17:2844–2853.) first reported that in vitro culture of human peripheral blood mononuclear cells (PBMC) with rATG resulted in expansion of CD4+CD25+ T cells. In the current study, we show that in vitro culture of normal human PBMC with low dose rATG (10 μg/ml) resulted in marked expansion of CD4+CD25high T cells (rATG 8.00±0.95% versus untreated 0.99±0.11%, n=10, p<0.0001) and CD4+CD25high FoxP3 T cells (rATG 2.24±0.11% versus untreated 0.90±0.10%, n=10, p<0.0001). rATG exposure converted CD4+CD25− T cells into CD4+CD25+ T cells, which proliferated better in comparison to CD4+CD25− T cells. In immunoblots of protein extracted from PBMC treated with rATG, there was increased expression of FoxP3 and nuclear factor of activated T cells (NFAT1) in CD4+CD25− and CD4+CD25+ T cells; rATG-induced NFAT1 expression correlated with FoxP3 expression. Expanded Treg suppressed autologous T-cell proliferation after T-cell receptor (TCR) stimulation by 64% when cultured with autologous PBMC at a 1:1 ratio, consistent with functional activity. Culture supernatants of PBMC treated with rATG showed increased levels of IL-10, compared with supernatants of PBMC treated with hATG or CsA, but no differences in INF-γ, IL-2, and IL-4. Unexpectedly, hATG did not expand but rather decreased Treg [For CD4+CD25high T cells (n=10): hATG 0.67±0.10% versus untreated 0.99±0.11%, p=0.0386; For CD4+CD25high FoxP3 T cells (n=10): hATG 0.62±0.08% versus untreated 0.90±0.10%, p=0.0435]. Furthermore, rATG and hATG showed differences in binding to lymphocytes, they contained different amounts of CD3 and TCRαβ antibodies, and they induced different activation states (expression of glucocorticoid-induced tumor necrosis factor receptor, cytotoxic T lymphocyte-associated antigen-4, and CD62L) for CD4+ T cells. In vitro, Treg expansion mediated by rATG occurred at submitogenic concentrations (< 50 μg/ml) rather than at lymphocyte depletion levels (50–100 μg/ml). Our findings suggest that rATG expanded Treg by converting CD4+CD25− T cells into CD4+CD25+ T cells, probably through a mechanism of transcription regulation, and enhanced NFAT1 expression, in turn conferring on CD4+CD25− T cells FoxP3 expression and regulatory activity. The therapeutic effects of rATG in the treatment of autoimmune diseases and GVHD may occur due to not only lymphocyte depletion but also enhanced Treg cell number and function. Our observation may provide a useful method for expansion of Treg in cellular treatment in transplantation and autoimmune diseases.

Anti-apoptotic activity of caffeic acid, ellagic acid and ferulic acid in normal human peripheral blood mononuclear cells: A Bcl-2 independent mechanism

Polyphenols have been shown to induce apoptosis in a variety of tumor cells including leukemia both in vitro and in vivo. However, their action on normal human peripheral blood mononuclear cells (PBMCs) during oxidative stress remains to be explored. In this study, we have evaluated the anti-apoptotic and radical scavenging activities of dietary phenolics, namely caffeic acid (CA), ellagic acid (EA) and ferulic acid (FA). H 2O 2-induced apoptosis in normal human PBMCs was assayed by phosphotidylserine externalization, nucleosomal damage and DNA fragmentation. Incubation of PBMCs with 5 mM H 2O 2 led to increased Annexin-V binding to externalized phosphatidyl serine (PS), an event of pre-apoptotic stage of the cell. Peripheral blood mononuclear cells pretreated with phenolics could resist H 2O 2-induced apoptotic damage. Caffeic acid (60 and 120 μM) and EA (100 and 200 μM) caused no change in externalization of PS, whereas FA (100 and 200 μM) increased externalization of PS in PBMCs treated with H 2O 2. The effects of phenolics were abolished to a large extent by culturing the PBMCs for 24 h after washing the phenolics from the medium. Inhibitory activities of these phenolics on lipid peroxidation were in the order of EA < CA < FA. DPPH-scavenging activities of EA, CA and FA were found to be 31.2 ± 1.36, 50 ± 1.86 and 73.0 ± 1.58 μM respectively. Although, the phenolics significantly inhibited DNA damage and lipid peroxidation, they could not alter the Bcl-2 expression in PBMCs. In conclusion, the anti-apoptotic effect of EA, CA and FA in PBMCs seems to be through the Bcl-2 independent mechanism.