Lipopolysaccharide effect on vascular endothelial factor and matrix metalloproteinases in leukemic cell lines in vitro

Angiogenesis, the process of new vessels generation, plays a critical role in tumor invasion and metastasis. Vascular Endothelial Growth Factor [VEGF], as a cytokine, and Matrix Metalloproteinases [MMPs], has been the important factors that involved in angiogenesis. Lipopolysaccharide [LPS] has an essential effect on angiogenesis. In this study the effect of LPS on VEGF production and MMP-2/MMP-9 activity in two leukemic cell lines has been assessed in vitro. Human leukemic U937 and THP1 cells were cultured in complete RPMI medium. Then the cells at the exponential growth phase were incubated with different concentrations of LPS [0 - 4 micro g/mL] for 48 hours. Then the level of VEGF production and MMP-2/MMP-9 activity in cell culture supernatants were evaluated with the ELISA standard kits and gelatin zymography respectively. U937 cells have produced a large amount of VEGF without any stimulus and LPS has not shown any substantial effect on VEGF production by these cells. However THP1 cells have produced a small amount of VEGF without stimulation and LPS significantly has increased VEGF production in these cells dose-dependently. Moreover LPS significantly has augmented the MMP-2/MMP-9 activity in the both leukemic cell lines in a dose-dependent manner. Our results have shown that LPS might be a potential inducer/enhancer of VEGF production and MMP-2/MMP-9 activity [angiogenic factors] in leukemia. Moreover the LPS effect on angiogenesis might be in part, due to its stimulatory effects on VEGF and MMPs. Overall LPS-stimulated leukemic cells might be good models for study and planning the useful therapeutic approaches for angiogenesis- dependent diseases

Suppression of gelatinase activity in human peripheral blood mononuclear cells by verapamil

Gelatinases are a large group of proteolytic enzymes that belong to the matrix metalloproteinases [MMPs]. MMPs are a broad family of peptidases, which proteolyse the extracellular matrix and have an important role in inflammation. Verapamil is a calcium channel blocker extensively used in the treatment of numerous cardiovascular diseases such as arrhythmia and hypertension. The anti-tumor and anti-inflammatory effects of verapamil have also been shown. In this study, the effect of verapamil on gelatinase activity in human peripheral blood mononuclear cells [PBMCs] has been assessed in vitro. In this experimental study, PBMCs from healthy adult volunteers were isolated by ficoll-hypaque-gradient centrifugation. The cells were then cultured in complete RPMI-1640 medium and after that incubated with different concentrations of verapamil [0-200 Mirco M] in the presence or absence of phytoheamagglutinin [PHA] [10 Mirco g/ml] for 48 hours. The gelatinase A [MMP-2]/gelatinase B [MMP-9] activity in cell-conditioned media was then evaluated by gelatin zymography. Statistical comparisons between groups were made by analysis of variance [ANOVA]. Verapamil significantly decreased the MMP-2/MMP-9 activity in human PBMCs after 48 hours incubation time compared with untreated control cells. The association was dose-dependent. In this study verapamil exhibited a dose-dependent inhibitory effect on gelatinase A and gelatinase B activity in human PBMCs. It seems that the anti-inflammatory properties of verapamil may be in part due to its inhibitory effects on gelatinase activity. Regarding the beneficial effects of MMPs- inhibitors in the treatment of some cardiovascular diseases, the positive effect of verapamil on such diseases may be in part due to its anti-MMP activity. Verapamil with its inhibitory effects on gelatinases activity may be a useful MMP-inhibitor. Given the beneficial effect of MMP-inhibitors in some cancerous, inflammatory and autoimmune disorders, it seems likely that verapamil could also be used to treat these diseases

Production and characterization of mouse monoclonal antibodies recognizing human Pan-IgG specific conformational or linear epitopes

Pan-IgG specific monoclonal antibodies [MAbs] are essential tools for assessment of humoral immunity, immune deficiency and gammopathy. In this study, four hybridoma clones producing MAbs with different specificities for human IgG isotypes were established. Splenocytes from Balb/c mice immunized with Fc fractions of human IgG were fused with SP2/0 myeloma cells. Hybridoma cells were selected in HAT selective medium and cloned by limiting dilution assay. Antibody-secreting cells were screened by enzyme-linked immunosorbent assay [ELISA] and the specificity of secreted MAbs was further analyzed using a panel of purified myeloma IgG proteins by ELISA and immunoblotting. Cross-reactivity to immunoglobulins [Igs] of other species was studied by indirect ELISA using serum samples collected from 9 animals. Immunoblotting studies revealed recognition of either linear or conformational epitopes by these MAbs. The most abundant cross-reactivity [100%] was observed with monkey Igs, while no cross-reactivity was detected with rabbit, guinea pig, sheep, goat, cat and hen sera. Two of the MAbs crossreacted with either dog or horse sera. The affinity constant of two MAbs was measured by ELISA and found to be 0.74×10[8] M[-1] and 0.96×10[7] M[-1]. Our results indicate that these pan-IgG specific MAbs recognize restricted linear or conformational epitopes located on all human IgG subclasses with no cross-reactivity to IgG from most species studied. These MAbs are potentially useful tools for quantification of total or antigen-specific IgG levels

effects of isosorbide dinitrate on in vitro proliferation of WEHI-164 cells and peripheral blood mononuclear cells

Isosorbide dinitrate has been broadly used in the treatment of various ischemic heart diseases. Isosorbide is a nitric oxide donor which increases blood flow to tumors through vasodilatation and consequently accelerates the access of chemo-drugs to them. Furthermore, this drug has inhibitory effects on angiogenesis, tumor growth and metastasis in vivo. Moreover, its antinflammatory effects have also been reported. In the present study we evaluated the effects of isosorbide on the proliferative activity of fibrosarcoma WEHI-164 cell line and peripheral blood mononuclear cells [PBMCs]. WEHI-164 fibrosarcoma cells and human PBMCs were cultured in complete Roswell Park Memorial Institute [RPMI] 1640 medium with 10% fetal bovine serum and 2x104 cells/mL for WEHI-164 and 2x105 cells/mL for PBMCs. The cells were then incubated at the exponential growth phase with different concentrations of isosorbide [4xl0[-6]-1.6xl0[-3] M] for 24, 48 and 72 hours. Subsequently, isosorbide effects on proliferation of the cells were evaluated by trypan blue dye exclusion [TB] test and MTT assay. Statistical comparisons between groups were made by analysis of variance. The proliferative activity of WEHI-164 fibrosarcoma cells and human PBMCs treated with different concentrations of isosorbide, did not show any significant difference with untreated control cells. The results of this study showed that isosorbide neither had any significant effects on the proliferative activity of fibrosarcoma WEHI-164 cells nor on human PBMCs. Our findings suggest that anti-tumoral effects of isosorbide reported by other investigators may be mediated through non-cytotoxic mechanisms

Profiles of MMP-2 expression in Jurkat, Molt-4 and U937 cells

Leukemia is a malignant proliferative disorder of the hematopoietic cells. The important role of angiogenesis in leukemia has been reported by several studies. Matrix metalloproteinases [MMPs] are a large group of endopeptidases which degredate the extracellular matrix and play an important role in angiogenesis. The present study was conducted to evaluate the patterns of MMP-2 activity in three leukemic cell lines. Human leukemic monocyte [U937] and T cells [Molt-4 and Jurkat] were cultured in complete RPMI-1640 medium. The cells were then seeded at a density of 106 cells/ml and were incubated with different concentrations of phorbol myristate acetate [PMA] [1-25 ng/ml] or phytoheamagglutinin [PHA] [2-10 micro g/ml] for 24 hours. The MMP-2 activity in cell-conditioned media was then evaluated by gelatin zymography. Statistical comparisons between groups were made by analysis of variance [ANOVA]. PHA/PMA significantly and dose-dependently increased MMP-2 activity in U937 cells after 24 hours of incubation compared with untreated control cells. Moreover, PHA/PMA significantly induced MMP-2 activity in Molt-4 and Jurkat cells after 24 hours of incubation in a dosedependent manner compared with untreated control cells. We conclude that human leukemic Jurkat, U937 and Molt-4 cells could potentially display MMP-2 activity with different degrees. Thus, these cell lines could provide an appropriate system to study the mechanisms regulating MMPs production in leukemia patients

Production and characterization of mouse monoclonal antibodies recognizing multiple subclasses of human IgG

Different IgG subclass profiles are produced in response to different antigenic stimuli in a variety of diseases. IgG subclass levels may reflect disease severity. Quantification of IgG subclasses depends on the availability if specific Monoclonal antibodies [MAbs]. In the present study seven hybridoma clones producing MAbs reactive with multiple subclasses of human IgG were established. Splenocytes form Balb/c mice immunized with Fc fractions of human IgG1 or IgG2 myeloma proteins were fused with mouse myeloma cells. Fused cells were selected and cloned by limiting dilution assay. Antibody secreting cells were screened by Enzyme-linked immunosorbent assay [ELISA] and the specificity of secreted MAbs was further analyzed, using a panel of purified human myeloma paraproteins of different IgG subclasses by ELISA and immunoblotting. Cross-reactivity to immunoglobulins [Igs] of other species was studied by indirect ELISA using serum samples collected from 9 animals. The MAbs were found to react with triple IgG subclasses, including IgG1,2,4 [n=4] and IgG1,2,3 [n=3]. Immunoblotting studies revealed recognition of linear [n=4] or conformational [n=3] epitopes by these MAbs. The most abundant cross-reactivity [71.4%] was observed with monkey Ig while no cross-reactivity was detected with hen and cat sera. The MAbs mostly displayed a restricted pattern of cross-reactivity and one of them did not bind to any of the animal sera tested. The affinity constant of 3 MABs was measured by ELISA. Based on the data obtained from this study, mouse MAbs reactive with multiple human IgG subclasses are directed to a variety of immunogenic epitopes, mostly shared with IgG of other species. Thes MAbs are valuable tools for purification of non-reactive IgG subclasses through negative affinity chromatography. These Mabs could also provide an opportunity for epitope mapping of the Fc region of IgG, as well as serological phylogenetic studies

Generation and characterization of mouse hybridomas secreting monoclonal antibodies specific for human lgG3

Mammalians express several subclasses of the IgG molecule. In human being there are four homologous IgG subclasses, each of which is structurally unique and has different functions. Quantification of IgG subclasses is fundamental to clinical assessment and diagnosis of many diseases as such assessments depends on the availability of subclassspecific antibodies [Abs], particularly monoclonal antibodies [MAbs]. In the present study, we produced and characterized two murine MAbs specific for human lgG3 molecule. These MAbs were obtained by the fusion of myeloma cells with splenocytes from Balb/c mice immunized with heavy chain of a human IgG3 myeloma protein. Fused cells were selected in, hypoxanthine, aminopterine and thymidine [HAT] medium and cloned by limiting dilution assay. Ab-secreting cells were screened by enzyme-linked immunosorbent assay [ELISA] and the specificity of secreted MAbs was further analyzed, using a panel of purified myeloma proteins by ELISA and immunoblotting. Two stable hybridomas designated 1F18G7 and 1F18A11 were obtained secreting MAbs specific for Fc fragment of human IgG3. None of these MAbs showed cross-reactivity with other immunoglobulin isotypes derived from human and nine other animals, except 1F18A11 which displayed a weak cross-reactivity with only dog serum. Immunoblotting results indicate that these MAbs react with linear epitope [s] located in the heavy chain of human IgG3 molecules. The affinity constant of 1F18G7 and 1F18A11 MAbs was found to be 0.81x10[9] Mol[-1] and 0.7x10[9] Mol[-1], respectively, as measured by ELISA. These two MAbs with relatively high affinity can be useful tools for quantification of IgG3 subclass levels in human serum

Effect of propranolol on angiogenic factors in human hematopoietic cell lines in vitro

Beta-adrenergic blocking agents have been broadly used for treatment of many cardiovascular diseases such as arterial hypertension and ischemic heart failure. Anti-tumoral, anti-inflammatory and antiangiogenesis effects of propranolol [a non-selective beta-adrenergic blocker] have been shown. Angiogenesis [replenish of the pre-existing vascular networks] plays a critical role in some pathological conditions such as tumor expansion and metastasis. In this study, we investigated the effects of propranolol on vascular endothelial growth factor [VEGF] production and matrix metalloproteinase-2 [MMP-2] activity [two important angiogenic factors] in human leukemic cell lines in vitro. Two human leukemic T [Molt-4 and Jurkat] and one monocyte [U937] cell lines were used in this study. The cells were cultured in complete RPMI medium and then incubated with different concentrations of propranolol [0.3-30 micro M] in the presence or absence of phorbol myristate acetate [PMA, 25 ng/ml] for 48 hours. The level of VEGF secreted in the cell culture supernatants was measured with enzyme-linked immunosorbent assay kits [R and D systems] and MMP-2 activity in cell-conditioned media was evaluated by gelatin zymography. Propranolol significantly decreased VEGF production and also MMP-2 activity in PMA-activated human leukemic cell lines Molt-4, Jurkat and U937 at 30 micro M concentration of the drug compared to untreated control cells [P<0.05]. Propranolol might be a useful anti-angiogenic agent in hematopoietic malignancies. Thus, propranolol along with its chronic long-term usage in cardiac problems may have potential implication in treatment of leukemia

Production and characterization of murine monoclonal antibodies recognizing conformational and linear epitopes localized on human IgA2 molecules

There are two subclasses of human IgA [IgA1 and IgA2] that differ in antigenic properties and in chemical composition. The constant domains of alpha1 and alpha2 heavy chains have >95% sequence homology though major structural differences exist in the hinge region. Quantitation of IgA subclass levels depends on the availability of monoclonal antibodies [MAbs] specific for conserved conformational or linear epitopes restricted to each subclass. To produce, select and characterize monoclonal antibodies specific for human IgA2. Splenocytes from BALB/C mice immunized with a human IgA2 myeloma protein were fused with SP2/0 myeloma cells. Fused cells were grown in hypoxanthine, aminopterine and thymidine [HAT] selective medium and cloned by limiting dilution assay. Antibody [Ab] secreting cells were screened by enzyme-linked immunosorbent assay [ELISA] and the specificity of secreted MAbs was further analyzed, using a panel of purified myeloma proteins and some animal sera by ELISA and immunoblotting. The affinity constant [K[aff]] was also determined by ELISA. Four murine hybridoma clones designated 2F20G5, 2F20B5, 3F20E3 and 6F20H11 were obtained that secreted MAbs specific for the human IgA2. 2F20G5 and 6F20H11 MAbs react with linear epitope[s] while 2F20B5 and 3F20E3 react with conformational epitope[s] located to human IgA2 subclass. 2F20G5 MAb displays a weak cross-reactivity with monkey and rabbit sera and a strong cross-reactivity with cat and dog sera while the other three MAbs showed no cross-reactivity with the animal sera tested. These MAbs, especially 6F20H11 with high affinity constant [6.03 x10[9] M[-1]] are useful tools for quantitation of human IgA2 subclass levels in various diseases. Cross-reactivity of 2F20G5 MAb with some animal sera suggests phylogenic conservation of the epitope recognized by this MAb