Modulation of secretion of human chorionic gonadotropin by biologic response modifiers on term placenta and choriocarcinoma cells

The effects of biologic response modifiers such as interferon-gamma, tumor necrosis factor alpha (TNF), and retinoic acid on the human chorionic gonadotropin (hCG) secretion of cultured choriocarcinoma cells (JAR) and term placenta have been studied. Although the proliferation of JAR cells was not inhibited by these agents, retinoic acid and TNF markedly increased both the intracellular levels as well as the secreted amounts of hCG. In the case of the term placenta, only retinoic acid increased the hCG secretion into the culture medium, whereas interferon-gamma and TNF both markedly reduced secretion. The cytostatic agent etoposide (VP-16) was able to augment the hCG secretion on the choriocarcinoma cells but did not alter its production on term placenta. The The data presented indicate different mechanisms of regulation of hCG secretion in the normal and malignant trophoblast.     

Modulation of cytokine secretion by pentacyclic triterpenes from olive pomace oil in human mononuclear cells

Olive pomace oil, also known as "orujo" olive oil, is a blend of refined-pomace oil and virgin olive oil, fit for human consumption. Maslinic acid, oleanolic acid, erythrodiol, and uvaol are pentacyclic triterpenes, found in the non-glyceride fraction of orujo oil, which have previously been reported to have anti-inflammatory properties. In the present work, we investigated the effect of these minor components on pro-inflammatory cytokine production by human peripheral blood mononuclear cells in six different samples. Uvaol, erythrodiol, and oleanolic acid significantly decreased IL-1beta and IL-6 production in a dose-dependent manner. All three compounds significantly reduced TNF-alpha production at 100microM; however, at 10microM, uvaol and oleanolic acid enhanced the generation of TNF-alpha. In contrast, maslinic acid did not significantly alter the concentration of those cytokines, with the exception of a slight inhibitory effect at 100microM. All four triterpenes inhibited production of I-309, at 50microM and 100microM. However, uvaol enhanced I-309 production at 10microM. The triterpenic dialcohols had a similar effect on MIG production. In conclusion, this study demonstrates that pentacyclic triterpenes in orujo oil exhibit pro- and anti-inflammatory properties depending on chemical structure and dose, and may be useful in modulating the immune response.     

Detection of hepatitis B virus DNA in mononuclear blood cells.

The Southern transfer hybridisation technique was used to test mononuclear blood cells for hepatitis B virus DNA. Viral DNA sequences were detected in mononuclear cells of 10 out of 16 patients with hepatitis B virus infection and in none of 21 normal controls. Blood contamination was excluded by the absence of hepatitis B virus DNA in the corresponding serum samples in all cases. Free monomeric hepatitis B virus DNA was found in three patients positive for hepatitis Be antigen (HBeAg) and one positive for anti-HBe, and integrated hepatitis B virus DNA was present in four patients positive for anti-HBe. In two other patients the small size of the samples did not allow a distinction between free and integrated viral DNA. The state of the virus in the mononuclear cells seemed to correlate with the HBeAg or anti-HBe state, as has been noted in the liver. These results indicate that hepatitis B virus may infect mononuclear blood cells, thereby expanding the tissue specificity of this agent beyond the liver, as has been reported for pancreatic, kidney, and skin tissue. They also suggest that hepatitis B virus infection of mononuclear cells might be related to immunological abnormalities observed in carriers of the virus.     

Compartmentalization of hepatitis C virus genotypes between plasma and peripheral blood mononuclear cells

Differences in hepatitis C virus (HCV) variants of the highly conserved 5' untranslated region (UTR) have been observed between plasma and peripheral blood mononuclear cells (PBMC). The prevalence and the mechanisms of this compartmentalization are unknown. Plasma and PBMC HCV variants were compared by single-strand conformation polymorphism (SSCP) and by cloning or by genotyping with a line probe assay (LiPA) in 116 chronically infected patients, including 44 liver transplant recipients. SSCP patterns differed between compartments in 43/109 analyzable patients (39%). Differences were significantly more frequent in patients with transplants (21/38 [55%] versus 22/71 [31%]; P < 0.01) and in those who acquired HCV through multiple transfusions before 1991 (15/20; 75%) or through drug injection (16/31; 52%) than in those infected through an unknown route (7/29; 24%) or through a single transfusion (5/29; 17%; P < 0.001). Cloning of the 5' UTR, LiPA analysis, and nonstructural region 5B sequencing revealed different genotypes in the two compartments from 10 patients (9%). In nine patients, the genotype detected in PBMC was not detected in plasma and was weak or undetectable in the liver in three cases. This genotypic compartmentalization persisted for years in three patients and after liver transplantation in two. The present study shows that a significant proportion of HCV-infected subjects harbor in their PBMC highly divergent variants which were likely acquired through superinfections.     

Compartmentalization of hepatitis C virus quasispecies in blood mononuclear cells of patients with mixed cryoglobulinemic syndrome

The aim of this study was to investigate the quasispecies heterogeneity of hepatitis C virus (HCV) in the plasma, cryoprecipitate, and peripheral lymphocytes of chronically infected HCV patients with mixed cryoglobulinemia (MC). We studied 360 clones from 10 HCV-positive patients with MC and 8 age-, gender- and HCV genotype-matched subjects with chronic HCV infection but without MC. A partial nucleotide sequence encompassing the E1/E2 region, including hypervariable region 1 (HVR1), was amplified and cloned from plasma, cryoprecipitates, and peripheral blood mononuclear cells (PBMC), and the genetic diversity and complexity and synonymous and nonsynonymous substitution rates were determined. Heterogeneous selection pressure at codon sites was evaluated. Compartmentalization was estimated by phylogenetic and phenetic (Mantel's test) approaches. The patients with MC had 3.3 times lower nonsynonymous substitution rates (1.7 versus 5.7 substitutions/100 sites). Among the subjects with HCV genotype 1, the MC patients had significantly less complexity than the controls, whereas the diversity and complexity were similar in the genotype 2 patients and controls. Site-specific selection analysis confirmed the low frequency of MC patients showing positive selection. There was a significant correlation between positive selection and the infecting HCV genotype. The quasispecies were less heterogeneous in PBMC than in plasma. Significant compartmentalization of HCV quasispecies was observed in the PBMC of four of nine subjects (three with MC) and seven of nine cryoprecipitates. In one subject with MC, we detected a 5-amino-acid insertion at codons 385 to 389 of HVR1. Our results suggest reduced quasispecies heterogeneity in MC patients that is related to a low selection pressure which is probably due to an impaired immune response, the HCV genotype, and/or the duration of the infection. The frequent HCV quasispecies compartmentalization in patients' PBMC suggests a possible pathogenetic significance.     

Anti-angiogenic effects of lycopene through immunomodualtion of cytokine secretion in human peripheral blood mononuclear cells

The carotenoid lycopene has been reported to possess anti-metastatic activity which may be associated with immunomodulation. However, the anti-angiogenic effects and mechanisms of action of lycopene have not been reported. In this study, we investigated the immunomodulatory effect on in vitro and ex vivo angiogenesis of lycopene. We found that the proliferation, migration and the matrigel tube formation of human umbilical vein endothelial cells (HUVECs) was remarkably inhibited by conditioned medium (CM) of human peripheral blood mononuclear cells (MNC-CM) stimulated with various dose (1-10 μmol/L) of lycopene (LP-MNC-CM). LP-MNC-CM treatment inhibited ex vivo angiogenesis, as revealed by chicken egg chorioallantoic membrane assay. We further examined the effects of lycopene stimulation on cytokine levels in MNC and showed that, as compared to the control, lycopene (10 μmol/L) significantly (P<.001) up-regulated interleukin (IL)-12 by 163% and interferon (IFN)-γ by 531%. Furthermore, pre-treatment of HUVECs with dexamethasone, an IL-12 inhibitor, blocked the anti-angiogenic effects of LP-MNC-CM in parallel with inhibition of IL-12 and IFN-γ induction in MNC. These results demonstrate that lycopene has a potent anti-angiogenic effect and that these effect may be associated with its up-regulation of IL-12 and IFN-γ.     

Piperine inhibits cytokine production by human peripheral blood mononuclear cells

Piperine, an amide isolated from Piper species (Piperaceae), has been reported to exhibit central nervous system depression, anti-pyretic and anti-inflammatory activity. Immunomodulatory and anti-tumor activity of piperine has been demonstrated in mouse carcinomas. However, there is little information available concerning the effect of piperine on humans. We evaluated the immunopharmacological activity of this compound in human immune cells. Human peripheral blood mononuclear cells (PBMCs) were exposed to piperine, and cell proliferation was determined by the MTS assay. Piperine significantly inhibited phytohemagglutinin-stimulated human PBMC proliferation after exposure for 72 h. This compound inhibited PBMC activity, with an IC(50) of 100.73 ± 11.16 μg/mL. Production of interleukin-2 (IL-2) and interferon-γ (IFN-γ) was measured using an ELISA assay and RT-PCR. Piperine inhibited IL-2 and IFN-γ production in the PBMCs. RT-PCR data indicated that IL-2 and IFN-γ mRNA expression in PBMCs is suppressed by piperine. This compound significantly inhibited the production of these two cytokines by activated PBMCs in a dose-dependent manner. In conclusion, piperine appears to have potential as an immunomodulatory agent for immune system suppression.     

Taurine chloramine modulates cytokine production by human peripheral blood mononuclear cells

Summary.  The effect of taurine (Tau) and taurine chloramine (Tau-Cl) on the production of TNF-α, IL-1β, and IL-6 by peripheral blood mononuclear cells of healthy volunteers was examined. Cells were stimulated with bacterial lipopolysaccharide (LPS) in the presence of either Tau or Tau-Cl. After 24 h culture the cytokine concentrations were measured in both culture supernatants (secreted) and cell lysates (cell-associated) using ELISA. In LPS-stimulated cells Tau-Cl inhibited both the secreted and cell-associated IL-1β and IL-6, while exerted dual effect on TNF-α production: raising it slightly at low and reducing at higher concentration. By contrast, Tau had no significant effect on the cytokine production. These results indicate that Tau-Cl modulates synthesis of pro-inflammatory cytokines, and therefore it may play a role in the initiation and propagation of immune response. Received November 29, 2001 Accepted January 18, 2002 Published online August 30, 2002 Acknowledgments This research was supported by grants from the State Committee for Scientific Research of Poland (No 4 P05B 01018) and the Institute of Rheumatology (No I/14). The Institute of Rheumatology is supported by a core grant from the State Committee for Scientific Research of Poland. Authors' address: Ewa Kontny, Ph.D., Department of Pathophysiology and Immunology, Institute of Rheumatology, Spartanska 1, 02-637 Warsaw, Poland, E-mail: zpatiir@warman.com.pl Abbreviations: Tau, taurine; Tau-Cl, taurine chloramine; LPS, lipopolysaccharide; TNF-α, tumor necrosis factor-α; IL-1β, interleukin 1β; IL-6, interleukin 6; PBMC, peripheral blood mononuclear cells     

IgE-dependent cytokine production by human peripheral blood mononuclear phagocytes.

These studies demonstrate the IgE-dependent production of IL-1 beta and TNF-alpha by circulating blood monocytes. IL-1 beta production was demonstrated biologically as the stimulation of proliferation of the cloned IL-1-dependent murine T cell line D10.G4.1 in the presence of a submitogenic concentration of PHA. In a representative experiment, 3H-thymidine uptake increased from 57826 cpm in the presence of supernatants obtained from unstimulated cells to 200774 cpm with supernatants from monocytes stimulated by IgE/alpha IgE immune complexes. By ELISA, IgE complexes increased IL-1 beta production from 0.54 +/- 0.06 ng (per 10(6) monocytes) to 2.60 +/- 0.62 ng (p less than 0.01; mean of eight experiments) and TNF-alpha production from 0.17 +/- 0.10 ng to 3.00 +/- 0.54 ng (p less than 0.01; mean of four experiments). No IL-1 alpha secretion was observed. RNA hybridization analysis demonstrated that IL-1 beta production represented de novo synthesis of the cytokine. Stimulated RNA production was observed after a minimal 1/2-h incubation and was maximal at 2 h. The IgE-dependent secretion of these pro-inflammatory cytokines by mononuclear phagocytic cells may contribute to the inflammation characteristic of allergic responses.     

Modulation of cytokine production by human mononuclear cells following impairment of Na,K-ATPase activity

Cytokines, including TNFα and IL-1β, are central to the chronic inflammatory process and tissue damage that characterises diseases such as rheumatoid arthritis. The mechanisms responsible for long-term generation of these molecules are poorly understood. We have previously demonstrated impaired activity of Na,K-ATPase, a key enzyme regulating intracellular cation levels, on rheumatoid mononuclear cells. Mimicking this `defect' on normal mononuclear cells with ouabain has been shown to induce TNFα and, in particular, IL-1β production, whereas IL-6 synthesis was suppressed. A similar pattern of cytokine generation was noted when mononuclear cells were treated with the sodium ionophore, monensin. Induction of cytokine production was related to up-regulation of the appropriate mRNA, although enhanced secretion of processed IL-1β was also observed. The mechanism underlying these cellular responses appears to involve sodium/calcium exchange across the cell membrane. Impaired Na,K-ATPase activity might promote pro-inflammatory cytokine secretion in patients with rheumatoid arthritis.     

Modulation of hepatitis B virus secretion by naturally occurring mutations in the S gene

Alteration in hepatitis B virus (HBV) secretion efficiency may have pathological consequences. Naturally occurring mutations that regulate virion secretion have not been defined. We recently identified HBV genomes displaying high (4B), substantially reduced (3.4), or negative (4C) virion secretion. In the present study, the underlying mutations were mapped. A T552C point mutation in the 4B genome was responsible for its enhanced virion secretion, whereas a G510A mutation in 3.4 and G660C in 4C impaired virus secretion. The three point mutations generate M133T, G119E, and R169P substitutions in the S domains of viral envelope proteins, respectively, without modifying the coding capacity of the overlapping polymerase gene. The mutated residues are predicted to lie in the luminal side of the endoplasmic reticulum (ER) or to be embedded in the ER membrane and thus are not involved in contact with core particles during envelopment. Of the two mutations inhibitory of virion secretion, G510A greatly reduced small envelope protein (hepatitis B surface antigen [HBsAg]) levels both inside cells and in culture medium, whereas G660C specifically abolished HBsAg secretion. Surprisingly, a T484G mutation in the 4B genome, generating an I110M substitution in the S domain, could also reduce HBsAg secretion and block virion secretion. However, its inhibitory effect was suppressed in the 4B genome by the T552C mutation, the enhancer of virion secretion. T552C can also override the inhibitory G510A mutation, but not the G660C mutation. These findings suggest a hierarchy in the regulation of virion secretion and a close link between defective virion secretion and impaired HBsAg formation or secretion.     

Beta-endorphin secretion by human peripheral blood mononuclear cells: regulation by glucocorticoids

Corticotropin-releasing factor (CRF), a 41-aminoacid neuropeptide, can induce lymphocytes to production of beta-endorphin (beta E). Furthermore, the neuropeptide Arginine-Vasopressin (AVP) can enhance CRF-induced production of beta E. We have demonstrated that CRF acts by stimulating monocytes to production of the cytokine interleukin-1 (IL-1). IL-1 can in its turn activate the lymphocytes to secretion of beta E. Here we demonstrate that the glucocorticoid analogue dexamethasone is capable of modulating CRF-induced beta E secretion by lymphocytes. It appeared that dexamethasone can inhibit secretion of lymphocyte-derived beta E. The mechanism by which dexamethasone exerts its inhibitory activity is by blocking CRF-induced production of IL-1, thereby preventing induction of beta E secretion by B cells. These results support the concept that peptide hormones and glucocorticoids are mediating a reciprocal modulation of neuroendocrine and immunological activities.     

Modulation of cytokine release from mononuclear cells by prostacyclin, IL-4 and IL-13

In a previous study, we reported that cicaprost, a stable prostacyclin analogue can inhibit the release of granulocyte-macrophage colony-stimulating factor (GM-CSF) from activated human peripheral mononuclear blood cells (PBMCs). Since interleukin (IL-4) and IL-13 have been shown to inhibit the release of cytokines from PBMCs we tested the hypothesis that prostacyclin in combination with IL-4 or IL-13 can act synergistically to modulate the release of IL-10, generally associated with anti-inflammatory properties, and the pro-inflammatory cytokine tumour necrosis factor alpha (TNF-alpha). For this purpose, PBMCs were isolated over Ficoll, stimulated with lipopolysaccharide (LPS) and incubated in the presence of cicaprost, IL-4 or IL-13. There was a significant reduction in TNF-alpha as well as IL-10 secretion from LPS-stimulated PBMCs following incubation with IL-4 or IL-13. In contrast, cicaprost reduced the secretion of TNF-alpha but led to a slight enhancement of IL-10 release from PBMCs. When LPS-activated PBMCs were incubated in the presence of cicaprost and IL-4 or IL-13 there was a selective, synergistic inhibition of the TNF-alpha release which was not observed for IL-10. Thus, our data suggest that prostacyclin can synergize with cytokines to selectively inhibit the release of pro-inflammatory cytokines from PBMCs.     

Sequence analysis of the hepatitis C virus genome recovered from serum, liver, and peripheral blood mononuclear cells of infected chimpanzees.

Of 13 different strains of hepatitis C virus (HCV) in the inoculum used, only 1 persisted in human lymphocyte cell lines infected in vitro (N. Nakajima, M. Hijikata, H. Yoshikura, and Y. K. Shimizu, J. Virol. 70:3325-3329, 1996). To determine whether that particular strain (designated H1-2) has a tropism for lymphocytes in vivo, we sequenced hypervariable region 1 (HVR1) of the genome of HCV recovered from the sera, livers, and peripheral blood mononuclear cells (PBMC) of chimpanzees infected with plasma H77, the same inoculum used for the in vitro studies. In the PBMC collected from two chimpanzees during the early phase of infection, H1-2 was detected as the only or predominant HVR1 sequence. H1-2 was also detected in PBMC obtained during persistent infection from a chimpanzee that had been treated with immunosuppressants. From the livers of these chimpanzees, two to six different strains were recovered but H1-2 was not detected. Thus, H1-2 appeared to have an affinity for lymphocytes not only in vitro but also in vivo. In samples collected from a chimpanzee after 6 years of infection, however, such tissue compartmentalization of the HCV genome was not observed; a single strain became predominant in the serum, liver, and PBMC. An HCV strain capable of replicating in both the liver and PBMC probably emerged during in vivo replication and persisted.     

Modulation of the immune response induced by gene electrotransfer of a hepatitis C virus DNA vaccine in nonhuman primates

Induction of multispecific, functional CD4+ and CD8+ T cells is the immunological hallmark of acute self-limiting hepatitis C virus (HCV) infection in humans. In the present study, we showed that gene electrotransfer (GET) of a novel candidate DNA vaccine encoding an optimized version of the nonstructural region of HCV (from NS3 to NS5B) induced substantially more potent, broad, and long-lasting CD4+ and CD8+ cellular immunity than naked DNA injection in mice and in rhesus macaques as measured by a combination of assays, including IFN-gamma ELISPOT, intracellular cytokine staining, and cytotoxic T cell assays. A protocol based on three injections of DNA with GET induced a substantially higher CD4+ T cell response than an adenovirus 6-based viral vector encoding the same Ag. To better evaluate the immunological potency and probability of success of this vaccine, we have immunized two chimpanzees and have compared vaccine-induced cell-mediated immunity to that measured in acute self-limiting infection in humans. GET of the candidate HCV vaccine led to vigorous, multispecific IFN-gamma+CD8+ and CD4+ T lymphocyte responses in chimpanzees, which were comparable to those measured in five individuals that cleared spontaneously HCV infection. These data support the hypothesis that T cell responses elicited by the present strategy could be beneficial in prophylactic vaccine approaches against HCV.     

Comparison of cytokine production in cultures of whole human blood and purified mononuclear cells.

In order to examine inter- and intra-individual variations in cytokine production, blood was collected from 48 healthy subjects on each of 4 occasions separated by 4 weeks. Whole blood (diluted 1:10) and mononuclear cell (MNC) cultures were stimulated for 24 h with either concanavalin A (Con A) or bacterial lipopolysaccharide (LPS) and the concentrations of IL-1alpha, IL-1beta, IL-2, TNF-alpha, IL-10 and IFN-gamma in the culture medium measured. There were highly significant inter-individual variations in the production of each of the cytokines measured. However, the level of the production of each cytokine appeared to be characteristic of an individual. There were significant correlations between production of each cytokine in whole blood and MNC cultures. It is concluded that there is significant inter-individual variation in cytokine production which is unaffected by time or by the stimulus used to elicit cytokine production, and that whole blood cultures can be used instead of MNC cultures to measure cytokine production.