Dermatophagoides farinae induces conjunctival epithelial cell damage to promote neutrophil migration and neutrophil extracellular traps formation / 中国血吸虫病防治杂志

OBJECTIVE@#To investigate the mechanisms underlying allergic conjunctivitis caused by conjunctival epithelial cell damage, neutrophil migration and neutrophil extracellular traps (NETs) formation induced by crude extracts of Dermatophagoides farinae mite (CDM).@*METHODS@#Human conjunctival epithelial cells were stimulated with 500, 1 000, 2 000, 4 000 ng/mL, and the expression levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ) and IL-8 were detected using quantitative real-time PCR (qPCR) assay and enzyme-linked immunosorbent assay (ELISA). The culture supernatant of human conjunctival epithelial cells was collected and co-cultured with neutrophils. Neutrophil migration was measured using Transwell migration assay, and the expression of NETs markers myeloperoxidase (MPO) and citrullinated histone H3 (CitH3) was quantified using immunofluorescence staining. Neutrophils were stimulated with phorbol 12-myristate 13-acetate (PMA), and then NETs were collected for treatment of human conjunctival epithelial cells. Cell apoptosis was detected using flow cytometry, and the levels of IL-6, TNF-α, IFN-γ and IL-8 were measured in the cell culture supernatant using ELISA.@*RESULTS@#Treatment with CDM at concentrations of 2 000 ng/mL and 4 000 ng/mL up-regulated IL-6, TNF-α, IFN-γ and IL-8 expression in human conjunctival epithelial cells. Following treatment with CDM at concentrations of 2 000 ng/mL and 4 000 ng/mL, the culture supernatant of human conjunctival epithelial cells promoted neutrophil migration and induced increases in the staining intensity of MPO and CitH3. In addition, increased NETs triggered the apoptosis of human conjunctival epithelial cells and IL-6, TNF-α, IFN-γ and IL-8 secretion in the culture supernatant of human conjunctival epithelial cells.@*CONCLUSIONS@#CDM induces human conjunctival epithelial cell damages, thereby promoting neutrophil migration and NETs formation, while the release of NETs further aggravates human conjunctival epithelial cell damages.

Modulatory effects of Azadirachta indica leaf extract on cutaneous and hepatic biochemical status during promotion phase of DMBA/TPA-induced skin tumorigenesis in mice.

The modulation in biochemical status of skin and hepatic tissue at the time point of commencement of promotion stage of skin carcinogenesis in mice and its intervention with aqueous Azadirachta indica leaf extract (AAILE) were investigated. 7,12-Dimethylbenz(a)anthracene (DMBA, 500 nmol/100 ul of acetone) was applied topically for 2 weeks (twice weekly), followed by phorbol-12-myristate-13-acetate (TPA, 1.7 nmol/100 ul) twice weekly for 6 weeks on the depilated skin of mice and AAILE was administered orally at a dose level of 300 mg/kg body wt thrice a week for 10 weeks. DMBA/TPA treatment upregulated the phase I enzymes in skin and hepatic tissue, as revealed by the increased cytochrome P450 (CYP) and cytochrome b5 (cyt b5) levels and aryl hydrocarbon hydroxylase (AHH) activity when compared to the control group and differentially modulated the activities of phase II enzymes like glutathione-s-transferase (GST), DT-diaphorase (DTD) and uridine diphosphate glucuronosyltransferase (UDP-GT). AAILE treatment decreased the DMBA/TPA-induced increase in cutaneous CYP level and enhanced the DTD and UDP-GT activities when compared with DMBA/TPA group. In the hepatic tissue of AAILE + DMBA/TPA group, an increase in UDP-GT activity was observed when compared to DMBA/TPA group. DMBA/TPA treatment did not alter the skin lipid peroxidation (LPO) level when compared to control group, however, in the animals that received AAILE treatment along with DMBA/TPA, a significant increase in LPO was observed when compared to control group. This was associated with a decrease in cutaneous reduced glutathione (GSH) level of AAILE + DMBA/TPA group. Enhanced LPO level was observed in the hepatic tissue of DMBA/TPA and AAILE + DMBA/TPA groups when compared to control group. However, no alteration was observed in their hepatic GSH levels. The micronuclei score in hepatic tissue did not exhibit significant inter-group differences. The results of the present study suggest that apart from skin, liver may be affected during DMBA/TPA-induced skin tumorigenesis. AAILE treatment has the ability to modulate these changes potentially influencing the process of tumor formation. These findings seem to be important to carcinogenesis and its intervention with anti-cancer agents.

Effect of the monocyte locomotion inhibitory factor (MLIF) produced by E. histolityca on cytokines and chemokine receptors in T CD4+ lymphocytes

Entamoeba histolytica produces Monocyte Locomotion Inhibitory Factor (MLIF), which may contribute to the delayed inflammation observed in amoebic hepatic abscesses. Leukocytes are affected through the modulation of cytokine expression and/or production. We evaluated the effects of MLIF on the activation and production of intracellular cytokines in human CD4+ T lymphocytes by flow cytometry. Cells were stimulated for 24 h with PMA, MLIF, or PMA+MLIF. Cellular activation was measured using anti-CD69. Th1/Th2 production was studied by the expression of intracellular cytokines and cytokine/chemokine receptors. MLIF increased CD69 and induced the over-expression of the IL-l©¬, IFN-¥ã, IL-2, IL-4, and IL-10 intracellular cytokines; PMA+MLIF inhibited Th1 cytokine (IFN-¥ã) and increased Th2 cytokines (IL-4 and IL-10). The co-expression of the cytokine and chemokine receptors IFN-¥ã/CCR5 and IL-1©¬/CCR5 was inhibited by PMA+MLIF and Th2 co-expression was increased. MLIF effects varied depending on the conditions. MLIF alone activated the Th1 and Th2 cytokines and cytokine/receptor expression; however, PMA+MLIF increased the expression of Th2 but inhibited it in Th1.

Cyclooxygenase-2 Expression Is Related to the Epithelial-to-Mesenchymal Transition in Human Colon Cancers

PURPOSE: Down-regulation of E-cadherin is a hallmark of the epithelial-to-mesenchymal transition (EMT). EMT progression in cancer cells is associated with the loss of certain epithelial markers and the acquisition of a mesenchymal phenotype, as well as migratory activities. Cyclooxygenase-2 (COX-2) expression is associated with tumor invasion and metastasis in colon cancer. This study investigated the relationship between E-cadherin and COX-2 in colon cancer cells and human colon tumors. MATERIALS AND METHODS: Colon cancer cell lines and immunohistochemistry were used. RESULTS: E-cadherin expression was inversely related to the expressions of COX-2 and Snail in colon cancer cells. Ectopic expression of COX-2 or Snail reduced E-cadherin and induced a scattered, flattened phenotype with few intercellular contacts in colon cancer cells. Treatment of cancer cells with phorbol 12-myristate 13-acetate increased the expressions of COX-2 and Snail, decreased 15-hydroxyprostaglandin dehydrogenase expression, and increased the cells' motility. In addition, exposure to prostaglandin E2 increased Snail expression and cell motility, and decreased E-cadherin expression. Membranous E-cadherin expression was lower in adenomas and cancers than in the adjacent, non-neoplastic epithelium. In contrast, the expressions of Snail and COX-2 were higher in cancers than in normal tissues and adenomas. The expressions of COX-2 and Snail increased in areas with abnormal E-cadherin expression. Moreover, COX-2 expression was related to higher tumor stages and was significantly higher in nodal metastatic lesions than primary cancers. CONCLUSION: This study suggests that COX-2 may have a role in tumor metastasis via EMT.

Biological evaluation of novel 6-Arylbenzimidazo [1.2-c] quinazoline derivatives as inhibitors of LPS-induced TNF- alpha secretion

This study describes the effect of novel 6-Arylbenzimidazo [1,2-c] quinazoline derivatives as tumor necrosis factor alpha (TNF-á) production inhibitors. The newly synthesized compounds were tested for their in vitro ability to inhibit the lipolysaccharide (LPS) induced TNF-á secretion in the human promyelocytic cell line HL-60. The compound 6-Phenyl-benzimidazo [1,2-c] quinazoline, coded as Gl, resulted as the most potent inhibitor and with no significant cytotoxic activity. Thus, 6-Arylbenzimidazo [1,2-c] quinazoline derivatives may have a potential as anti-inflammatory agents.

Effect of sphingosine and phorbol-12-myristate-13-acetate on the growth and dimethylsulfoxide-induced differentiation in the insect trypanosomatid Herpetomonas samuelpessoai

We investigated the effect of two modulators of protein kinase C, sphingosine and phorbol-12-myristate-13-acetate (PMA), on the growth and dimethylsulfoxide (DMSO)-induced differentiation in Herpetomonas samuelpessoai. Sphingosine did not stimulate the transformation of undifferentiated-promastigotes in differentiated-paramastigotes. PMA alone or in association with DMSO increased the number of paramastigotes in comparison to control cells. DMSO inhibited the parasite growth (35 percent) and several unusual morphological features resembling aberrant cell division were observed. Sphingosine did not significantly reduce the growth in contrast to PMA. Collectively, our results demonstrated that the reduction of the proliferation translates in an increase of the differentiation rate in the insect trypanosomatid H. samuelpessoai.

PMA-induced up-regulation of MMP-9 is regulated by a PKCalpha-NF-kappaB cascade in human lung epithelial cells

Expression of matrix metalloproteinase-9 (MMP-9) is associated with airway remodeling and tissue injury in asthma. However, little is known about how MMP-9 is up-regulated in airway epithelial cells. In this study, we show that phorbol myristate acetate (PMA) induces MMP-9 expression via a protein kinase Calpha(PKCalpha)-dependent signaling cascade in BEAS-2B human lung epithelial cells. Pretreatment with either GF109203X, a general PKC inhibitor, or Go6976, a PKCalpha/beta isozyme inhibitor, inhibited PMA-induced activation of the MMP-9 promoter, as did transient transfection with PKCalpha antisense oligonuclotides. PMA activated NF-kappaB by phosphorylating IkappaB in these cells and this was also inhibited by GF109203X and Go6976, suggesting that PKCalpha acts as an upstream regulator of NF-kappaB in PMA-induced MMP-9 induction. Our results indicate that a "PKCalpha-NF-kappaB"-dependent cascade is involved in the signaling leading to PMA-induced MMP-9 expression in the lung epithelium.

Cytokine measurement in lymphocyte culture supernatant of inactive lepromatous leprosy patients.

The aim of the present study was to determine the effects of stimulation of sonicated Mycobacterium leprae (MLS) extract and phorbol myristate acetate (PMA) on the pattern of cytokine production in peripheral blood mononuclear cells (PBMC) and to find out whether there is any difference between stimulation of MLS extract and PMA. Blood samples were collected and PBMC isolated from 43 inactive lepromatous leprosy patients. After culture for 24 hours, lymphocytes were stimulated with MLS extract and PMA. In the culture supernatant, IL-2, 4, 6, 8, TNF-alpha and TGF-beta levels were measured by using ELISA. M. leprae stimulated group IL-6, IL-8, TNF-alpha, TGF-beta levels were found significantly higher than PMA stimulated group (P < 0.05). However, there was no difference between the two groups for IL-4. Only IL-2 levels were higher in PMA stimulated group than M. leprae stimulated group. Sonicated M. leprae extract have a strong effect on cytokine levels in vitro. Our results suggest that antigens with varying specificities favour the production of distinct cytokine patterns following in vitro restimulation.

Phospholipase D is activated and phosphorylated by casein kinase-II in human U87 astroglioma cells

Elevated expression of protein casein kinase II (CKII) stimulated basal phospholipase D (PLD) activity as well as PMA-induced PLD activation in human U87 astroglioma cells. Moreover, CKII-selective inhibitor, emodin and apigenin suppressed PMA-induced PLD activation in a dose-dependent manner as well as basal PLD activity, suggesting the involvement of CKII in the activation of both PLD1 and PLD2. CKII was associated with PLD1 and PLD2 in co-transfection experiments. Furthermore, CKII induced serine/threonine phosphorylation of PLD2 in vivo, and the multiple regions of PLD2 were phosphorylated by CKII in vitro kinase assay using glutathione S-transferase-PLD2 fusion protein fragments. Elevated expression of CKII or PLD increased cell proliferation but pretreatment of cells with 1-butanol suppressed CKII-induced cell proliferation. These results suggest that CKII is involved in proliferation of U87 cells at least in part, through stimulation of PLD activity.

Tyrosine phosphatase and cytochrome P450 activity are critical in regulating store-operated calcium channels in human fibroblasts

Diverse signaling pathways have been proposed to regulate store-operated calcium entry (SOCE) in a wide variety of cell types. However, it still needs to be determined if all of these known pathways operate in a single cell type. In this study, we examined involvement of various signaling molecules in SOCE using human fibroblast cells (HSWP). Bradykinin (BK)-stimulated Ca2+ entry, previously shown to be via SOCE, is enhanced by the addition of vanadate, an inhibitor of tyrosine phosphatases. Furthermore, SOCE is regulated by cytochrome P-450, as demonstrated by the fact that the products of cytochrome P-450 activity (14,15 EET) stimulated SOCE while econazole, an inhibitor of cytochrome P450, suppressed BK-stimulated Ca2+ entry. In contrast, Ca2+ entry was unaffected by the guanylate cyclase inhibitor LY83583, or the membrane permeant cyclic GMP analog 8-bromo-cyclic GMP (8-Br-cGMP). Neither nitric oxide donors nor phorbol esters affected BK-stimulated Ca2+ entry. SOCE in HSWP cells is primarily regulated by tyrosine phosphorylation and the cytochrome P-450 pathway, but not by cyclic GMP, nitric oxide, or protein kinase C. Thus, multiple pathways do operate in a single cell type leading to the activation of Ca2+ entry and some of these signaling pathways are more prominently involved in regulating calcium entry in different cell types.

Mechanism of CD11b down-regulation from phorbol myristate acetate stimulated polymorphonuclear neutrophils

To investigate the mechanism of CD11b down-regulation in phorbol myristate acetate [PMA] stimulated polymorphonuclear leukocytes [PMN]. Purified PMN were stimulated with PMA in the presence, or absence, of various enzyme inhibitors. Following stimulation, PMN CD11b expression was examined by flow cytometry and Western-blotting. The entire work was carried out at Liverpool University between the period of 1998 and 2001. Stimulation of PMN with PMA induced the down-regulation of CD11b by a mechanism involving a combination of an oxidant and a serine protease; most likely the primary granule derived elastase. The present study shows for the first time the cooperation of both oxidants and enzymes in the down-regulation of PMN adhesion receptors

Downstream components of RhoA required for signal pathway of superoxide formation during phagocytosis of serum opsonized zymosans in macrophages

Rac1 and Rac2 are essential for the control of oxidative burst catalyzed by NADPH oxidase. It was also documented that Rho is associated with the superoxide burst reaction during phagocytosis of serum- (SOZ) and IgG-opsonized zymosan particles (IOZ). In this study, we attempted to reveal the signal pathway components in the superoxide formation regulated by Rho GTPase. Tat-C3 blocked superoxide production, suggesting that RhoA is essentially involved in superoxide formation during phagocytosis of SOZ. Conversely SOZ activated both RhoA and Rac1/2. Inhibition of RhoA-activated kinase (ROCK), an important downstream effector of RhoA, by Y27632 and myosin light chain kinase (MLCK) by ML-7 abrogated superoxide production by SOZ. Extracellular signaling-regulated kinase (ERK)1/2 and p38 mitogen-activated protein kinase (MAPK) were activated during phagocytosis of SOZ, and Tat-C3 and SB203580 reduced ERK1/2 and p38 MAPK activation, suggesting that RhoA and p38 MAPK may be upstream regulators of ERK1/2. Inhibition of ERK1/2, p38 MAPK, phosphatidyl inositol 3-kinase did not block translocation of RhoA to membranes, suggesting that RhoA is upstream to these kinases. Inhibition of RhoA by Tat-C3 blocked phosphorylation of p47 PHOX. Taken together, RhoA, ROCK, p38MAPK, ERK1/2, and p47 PHOX may be subsequently activated, leading to activation of NADPH oxidase to produce superoxide.

Curcumin-induced inhibition of cellular reactive oxygen species generation: novel therapeutic implications.

There is evidence for increased levels of circulating reactive oxygen species (ROS) in diabetics, as indirectly inferred by the findings of increased lipid peroxidation and decreased antioxidant status. Direct measurements of intracellular generation of ROS using fluorescent dyes also demonstrate an association of oxidative stress with diabetes. Although phenolic compounds attenuate oxidative stress-related tissue damage, there are concerns over toxicity of synthetic phenolic antioxidants and this has considerably stimulated interest in investigating the role of natural phenolics in medicinal applications. Curcumin (the primary active principle in turmeric, Curcuma longa Linn.) has been claimed to represent a potential antioxidant and antiinflammatory agent with phytonutrient and bioprotective properties. However there are lack of molecular studies to demonstrate its cellular action and potential molecular targets. In this study the antioxidant effect of curcumin as a function of changes in cellular ROS generation was tested. Our results clearly demonstrate that curcumin abolished both phorbol-12 myristate-13 acetate (PMA) and thapsigargin-induced ROS generation in cells from control and diabetic subjects. The pattern of these ROS inhibitory effects as a function of dose-dependency suggests that curcumin mechanistically interferes with protein kinase C (PKC) and calcium regulation. Simultaneous measurements of ROS and Ca2+ influx suggest that a rise in cytosolic Ca2+ may be a trigger for increased ROS generation. We suggest that the antioxidant and antiangeogenic actions of curcumin, as a mechanism of inhibition of Ca2+ entry and PKC activity, should be further exploited to develop suitable and novel drugs for the treatment of diabetic retinopathy and other diabetic complications.

Formation of reactive oxygen species in rat epithelial cells upon stimulation with fly ash.

Fly ash was used as a model for ambient particulate matter which is under suspicion to cause adverse pulmonary health effects. The fly ash was pre-sized and contained only particles < 20 microm including an ultrafine fraction (< 100 nm) that contributed 31% to the particle number. In our study, we investigated the influence of fly ash on the promotion of early inflammatory reactions like the formation of reactive oxygen species (ROS) in rat lung epithelial cells (RLE-6TN). Furthermore, we determined the formation of nitric oxide (NO). The cells show a clear dose-response relationship concerning the formation of ROS with regard to the mass of particles applied. Lipopolysaccharide (LPS) added as a co-stimulus did not increase the formation of ROS induced by fly ash. Furthermore, in LPS (0.1 microg/ml) and tumour necrosis factor-alpha (TNF-alpha; 1 ng/ml) pre-treated cells no increase in reactive oxygen species comparable to fly ash alone is observable. In presence of the metal chelator, desferrioxamine (DFO), ROS formation can be significantly reduced. Neither fly ash nor LPS induced a significant NO release in RLE-6TN cells.

Neutrophil superoxide-anion generating capacity in chronic smoking: effect of long-term alpha-tocopherol therapy.

We investigated whether long-term alpha-tocopherol therapy in chronic smoking affects superoxide generating capacity of neutrophils ex vivo. To this purpose, we randomly assigned 128 male chronic smokers (37 +/- 21 pack years of smoking) to treatment with placebo (n = 64) or alpha-tocopherol (400 IU dL-a-tocopherol daily, n = 64). After two years of therapy, we measured phorbol 12-myristate 13-acetate-induced superoxide production of isolated neutrophils and of diluted whole blood by monitoring reduction of ferricytochrome c and luminol-enhanced peroxidase-catalyzed chemiluminescence. Plasma lipids and lipoproteins were not different between the two treatment groups. As expected, concentrations of alpha-tocopherol in plasma and in low-density lipoproteins were markedly elevated in the supplemented group compared to the placebo group (+ 120%, P < 0.0001 and + 83%, P < 0.0001, respectively). Consequently, resistance to in vitro oxidation of low-density lipoproteins (reflected by lag time of conjugated diene formation) was higher in the supplemented group than in the placebo group (+ 22%, P < 0.0001). Superoxide generating capacity of neutrophils and superoxide production in diluted whole blood did not differ between alpha-tocopherol and placebo group. It is concluded that in chronic smoking long-term supranormal alpha-tocopherol intake does not reduce neutrophil superoxide-anion generating capacity, despite large increases in the concentrations of alpha-tocopherol in plasma and in low-density lipoproteins.

Modulation of interleukin-8 receptor expression by lipopolysaccharide (LPS) and phorbol myristate acetate (PMA) in human peripheral monocytes--a preliminary study.

Interleukin-8, a monocyte derived neutrophil chemotactic agent is known to play as a key mediator in the pathogenesis of a large number of neutrophil driven inflammatory diseases. Since the cytokine activates the target cells through a cell surface receptor, study of the regulation of IL-8 receptor expression in monocytes is very important. We found that two very known modulators, lipopolysaccharide (LPS) in presence of homologous serum and Phorbol myristate acetate (PMA) resulted in induction of IL-8 receptor by 100-120% and 75-125% respectively within 1 h in monocytes. Based on the inhibitory effect of cycloheximide, actinomycin-D we may suggest that PMA and LPS could upregulate IL-8 receptor in monocytes through denovo protein synthesis. Prior incubation of polymixin B and anti-CD-14 antibody to the monocytes and subsequent stimulation of the cells with ser.act.LPS resulted in > 90% inhibition of IL-8 binding. Scatchard analysis showed that estimated receptor number in control cell was 7,500 per cell and it increased to 15,500 per cell in ser.act.LPS stimulated cell. The receptor number in PMA stimulated cells was 13,000 per cell. Chemical cross-linking of the IL-8 receptor with 125I labelled IL-8 in the ser.act.LPS and PMA stimulated cells-indicated that the signals at 59 kD were considerably increased with respect to control. A correlation between LPS and ser.act.LPS induced upregulation of IL-8 receptor expression has been shown. The study with bacterial product and co-carcinogenic agent thus provides information about the differential expression of IL-8 receptor for sustained IL-8 mediated biological response.

Cell-type specific regulation of thrombospondin-1 expression and its promoter activity by regulatory agents

Thrombospondin-1 (TSP-1), a multifunctional protein that is able to function as a negative regulator of solid tumor progression and angiogenesis, is normally present at a very low level but rapidly elevated in pathological tissues. To understand the cellular regulation of TSP-1 expression, the mode of it's expression in Hep3B, SK-HEP-1, and porcine aortic endothelial (PAE) cells was examined in the presence of all-trans retinoic acid (ATRA), interleukin-6 (IL-6), interferon-gamma (IFN-gamma), or phorbol 12-myristate 13-acetate (PMA). ATRA or IL-6 induced a dose-dependent increase of TSP-1 protein and mRNA levels in PAE cells, while they negatively regulated TSP-1 expression in the Hep3B and SK-HEP-1 cells. In contrast, PMA showed just the opposite effects on the TSP-1 expression in the same cells. IFN-gamma had little effect on TSP-1 level in Hep3B and PAE cells. The TSP-1 expression in SK-HEP-1 cells by these agents showed a close resemblance to that of liver cells rather than that of the endothelial cell line. Possible TSP-1 promoter-mediated responses by ATRA, IL-6, IFN-gamma, or PMA in Hep3B and PAE cells examined with luciferase activity of TSP-LUC reporter plasmid showed that levels of TSP-1 promoter activity were lower than that of the expressed TSP-1 protein and mRNA levels. Transfection of c-Jun and/or RARalpha expression vectors into Hep3B and PAE cells resulted in the enhanced TSP-1 promoter activity as well as the increments of of its protein and mRNA level. These results suggest that regulatory agents-induced TSP-1 expression may be attributed to mRNA stability and/or translational activation in concert with transcriptional activation and TSP-1 expression may be independently controlled via each signal pathway stimulated by PMA or ATRA.

Protein kinase C modulates telomerase activity in human cervical cancer cells

Telomerase, a ribonucleoprotein reverse transcriptase that extends telomeres of eukaryotic chromosomes is repressed in normal somatic cells but is activated during development and neoplasia. The regulation mechanism of telomerase activity in cancer cells is not clearly known. In this report, a possible affect of PKC on telomerase activity was examined using HeLa and CUMC-6 cervical cancer cell lines. Exposure of cells to PKC inhibitor, bisindolylmaleimide I and Go6976, and high levels of PKC activator, 12-O-tetradecanoyl phorbol 13-acetate (TPA) resulted in the inhibition of PKC activity in both cells. Telomerase activities were also inhibited by bisindolyl-maleimide I and Go6976, respectively, in a time-dependent manner. As PKC activity changes in TPA-treated cervical cancer cells, telomerase activities were increased at low dose of TPA and decreased at high dose. The expression levels of human telomerase subunits, human telomerase RNA (hTR) were not influenced by PKC modulating drugs. In contrast, the expression of full-length human telomerase reverse transcriptase (hTERT) was decreased after exposure to bisindolylmaleimide I and Go6976 in a time-dependent manner. hTERT expression was not affected by low dose of TPA. In contrast, high dose of TPA inhibited hTERT expression level. But the expression patterns of beta-deletion transcript of hTERT after 72 h of treatment with PKC inhibitors or high dose of TPA exposure were not discernable as compared with those of full-length hTERT transcripts to PKC modulating drugs. These results suggest that PKC-modulating drugs altered telomerase activities by affecting full-length hTERT expression profile in human cervical cancers.

Varying Effects of Intravenous Immunoglobulin on Mononuclear Cell Proliferation In Vitro

Intravenous immunoglobulin (IVIG) is being increasingly used to treat numerous immune-mediated diseases. However, there is a paucity of knowledge on the specific mode of action of IVIG in vivo. In this study, the in vitro effects of IVIG on peripheral blood mononuclear cell (PBMC) proliferation using phytohemagglutinin (PHA), anti-CD3 monoclonal antibody (MAb), phorbol myristate acetate (PMA), or purified protein derivatives (PPD) have been analyzed. The PBMCs were obtained from more than 10 individual donors. In all cases, IVIG almost completely inhibited PBMC proliferation at concentration above 20 mg/mL except when used in conjunction with PMA. PHA-induced proliferation of PBMCs at concentrations ranging from 1 to 15 mg/mL did not show significant differences. Anti-CD3 MAb-induced proliferation showed dose-dependent inhibition at concentrations ranging from 1 to 10 mg/mL. Interestingly, PMA-induced proliferation of PBMCs showed a dose-dependent increase at the same concentration range. PPD-induced proliferation of PBMC at concentrations ranging from 1 to 10 mg/mL did not show any statistically significant differences. These results suggest that high dose IVIG may be necessary to immune modulation in vivo and IVIG has various effects on PBMCs proliferation in limited concentration in vitro.

Differential regulation of vitamin D receptor expression in distinct leukemic cell lines upon phorbol ester-induced growth arrest

A close correlation between vitamin D receptor (VDR) abundance and cell proliferation rate has been shown in NIH-3T3 fibroblasts, MCF-7 breast cancer and in HL-60 myeloblastic cells. We have now determined if this association occurs in other leukemic cell lines, U937 and K562, and if VDR content is related to c-myc expression, which is also linked to cell growth state. Upon phorbol myristate acetate (PMA) treatment, cells from the three lineages (HL-60, U937 and K562) differentiated and expressed specific surface antigens. All cell lines analyzed were growth inhibited by PMA and the doubling time was increased, mainly due to an increased fraction of cells in the G0/G1 phase, as determined by flow cytometry measurements of incorporated bromodeoxyuridine and cell DNA content. C-myc mRNA expression was down-regulated and closely correlated to cell growth arrest. However, VDR expression in leukemic cell lines, as determined by immunofluorescence and Northern blot assays, was not consistently changed upon inhibition of cell proliferation since VDR levels were down-regulated only in HL-60 cells. Our data suggest that VDR expression cannot be explained simply as a reflection of the leukemic cell growth state.