Release of prostacyclin,endothelium-derived relaxing factor and endothelin by freshly harvested cells attached to microcarrier beads

Summary Cultured endothelial cells have been used in the past as a source of endothelium-derived relaxing factor (EDRF) and of prostacyclin (PGI₂). Although cell cultures are essential for observation of prolonged exposure to media or when there is delayed response, they are time consuming and sterile conditions are essential. In the present study, we report that endothelial cells, freshly harvested from bovine aortas, readily attached themselves to cytodex-3 microcarrier beads and released an endothelium-derived relaxing factor (EDRF), prostacyclin (PGI₂) and increased the amount of cyclic GMP in vascular smooth muscle. Attachment to microcarrier beads was essential since it increased the surface area and the number of attached cells and permited collection of cell free filtrates because of the formation of dense networks of cells and beads. As a result superfusion of cells and beads on the filter did not dislodge bound cells which remain on the filter. Conditioned filtrates from freshly harvested endothelial cells attached to microcarrier beads caused marked relaxation of endothelium-deprived bovine pulmonary artery strips. The degree of relaxation depended on the number of cells; maximal relaxation occurred with 50 million cells at ED₅₀ of 14 million. High values of cyclic GMP were found in vascular smooth muscle exposed to conditioned filtrate. The calcium ionophore A23187 further increased the amount of cyclic GMP. Large amounts of PGI₂ were released by freshly harvested endothelial cells particularly after stimulation with the calcium ionophore. In contrast, endothelin production by freshly harvested cells attached to microcarrier beads was barely detectable after 30 min incubation and was beyond the limit of detection by bioassay procedures. Freshly harvested endothelial cells attached to microcarrier beads appear to be a useful adjunct to tissue cultures under specific experimental conditions.Abbreviations EDRF Endothelium-Derived Relaxing Factor - PGI₂ Prostacyclin - K-H Krebs-Henseleit solution - cyclic GMP cyclic Guanosine Monophosphate - fmoles femtomoles - IB Ibuprofen     

The use of microcarrier beads in the production of endothelium-derived relaxing factor by freshly harvested endothelial cells.

This study is concerned with the use of freshly harvested bovine endothelial cells attached to microcarrier beads in the production of the endothelium-derived relaxing factor (EDRF). The results are compared to production of EDRF by endothelial cells grown in tissue cultures. We found that freshly harvested cells attach themselves to microcarrier beads within minutes. This results in large surface/area volume ratio and permits superfusion of cells suspension on a filter (pore size of 25-30 microns), resulting in cell free filtrate. When superfusing an endothelium-deprived pulmonary artery strip, the effluent causes relaxation; the response depends on the number of superfused endothelial cells. The number of viable freshly harvested cells attached to microcarrier beads in 5 ml Krebs-Henseleit solution is small (30%), as compared to almost 100% for cultured cells. Despite this difference, percent relaxation induced for the same number of viable cells is identical for both groups. Scanning electromicrographs confirm anchorage of endothelial cells to microcarrier beads. While cultured cells cover the entire surface and are individually attached, freshly harvested cells are anchored as cell aggregates leaving some of the surface free. Attachment of freshly harvested endothelial cells to microcarrier beads offers an alternative for the study of the role of endothelial cells in the production of vasoactive substances.     

Arachidonic acid and freshly isolated human bone marrow mononuclear cells

Arachidonic acid (AA), a fatty acid found in the human bone marrow plasma, is the precursor of eicosanoids that modulate bone marrow haematopoiesis. To further our understanding of the role of AA in the bone marrow physiology, we have assessed its incorporation in human bone marrow mononuclear cells. Gas chromatography analysis indicates the presence of AA in their fatty acid composition. In bone marrow mononuclear cells, [3H]-AA is incorporated into triglycerides and is later delivered into phospholipids, a result not observed with blood mononuclear cells. Prelabelling-chase experiments indicate a trafficking of labelled AA from phosphatidylcholine to phosphatidylethanolamine. Stimulation of prelabelled bone marrow mononuclear cells with granulocyte-macrophage colony-stimulating factor (GM-CSF) results in the release of a part of the incorporated labelled AA. Finally, exogenous AA (up to 1 microM) has no significant effect on cell growth. In conclusion, human bone marrow mononuclear cells participate to the control of marrow AA concentrations by incorporating AA into phospholipids and triglycerides. In turn, bone marrow mononuclear cells can release AA in response to the potent haematopoietic growth factor GM-CSF.     

Cytokine production by peripheral blood mononuclear cells in recurrent miscarriage

It has been postulated that a proportion of recurrent miscarriage (RM) might be due to immune causes. The objective was to determine whether cytokine expression in peripheral blood mononuclear cell is altered in patients with a history of RM. We compared the levels of IL-2, IL-4, IL-10, IL-13, TGFbeta1 and IFNgamma in the supernatant of Phytohemagglutinin stimulated mononuclear cells in 21 women with RM at the time of 3rd or higher abortion (group I), 32 women who were at least 3 months past their 3rd or higher abortion (group II) and 32 pregnant women with no history of abortion (group III). Gestational age was matched between groups I and III. Group I had higher level of IL-2 than group III (P=0.001). Group II showed higher level of IL-2 (P=0.001) and IFNgamma (P=0.015) than group III. The production of IL-10 by mononuclear cells of group III was higher than both group I (P=0.002) and group II (P=0.001). There was no difference in the levels of IL-2, IL-10 and IFNgamma between groups I and II. Also, the levels of IL-4, IL-13, and TGFbeta1 were similar among the groups. The data indicate an elevation of Th1 cytokines in women with RM as compared to normal pregnant women, and IL-10 is an important cytokine in the maintenance of pregnancy.     

Mycotoxigenic potential of fungi isolated from freshly harvested Argentinean blueberries

Alternaria alternata, A. tenuissima, Fusarium graminearum, F. semitectum, F. verticillioides, Aspergillus flavus, and Aspergillus section Nigri strains obtained from blueberries during the 2009 and 2010 harvest season from Entre Ríos, Argentina were analyzed to determine their mycotoxigenic potential. Taxonomy status at the specific level was determined both on morphological and molecular grounds. Alternariol (AOH), alternariol monomethyl ether (AME), aflatoxins (AFs), zearalenone (ZEA), fumonisins (FBs), and ochratoxin A (OTA) were analyzed by HPLC and the trichotecenes deoxynivalenol (DON), nivalenol (NIV), HT-2 toxin (HT-2), T-2 toxin (T-2), fusarenone X (FUS-X), 3-acetyl-deoxynivalenol (3-AcDON), and 15-acetyl-deoxynivalenol (15-AcDON) by GC. Twenty-five out of forty two strains were able to produce some of the mycotoxins analyzed. Fifteen strains of Aspergillus section Nigri were capable of producing Fumonisin B₁ (FB₁); two of them also produced Fumonisin B₂ (FB₂) and one Fumonisin B₃ (FB₃). One of the F. graminearum isolated produced ZEA, HT-2, and T-2 and the other one was capable of producing ZEA and DON. Two A. alternata isolates produced AOH and AME. Four A. tenuissima were capable of producing AOH and three of them produced AME as well. One Aspergillu flavus strain produced aflatoxin B₁ (AFB₁), aflatoxin B₂ (AFB₂), and aflatoxin G₁ (AFG₁). To our knowledge, this is the first report showing mycotoxigenic capacity of fungal species isolated from blueberries that include other fungi than Alternaria spp.     

Occurrence and toxigenicity of shape Fusarium moniliforme from freshly harvested maize ears with special references to fumonisin production in Egypt

Using the seed-plate technique, 18 different isolates of Fusarium moniliforme were isolated on pentachloronitrobenzene (PCNB) agar medium from 18 samples of a local variety of corn collected from locations in Minia Governorate. The isolates of F moniliforme were screened for their ability to produce fumonisins on polished rice grains using the solid state fermentation technique. Based on thin layer chromatographic (TLC) analyses using silica gel plates, 14 of the 18 isolates tested produced FB₁ and FB₂ with R _f (0.17) and (0.24), respectively. Concentration of FB₁ was estimated using high performance liquid chromatography (HPLC). Production of FB₁ by the 14 isolates of F. moniliforme tested ranged from 69 to 4495 ppm indicating that mouldy corn may represent a health hazard to consumers. This revised version was published online in August 2006 with corrections to the Cover Date.     

Mycoflora of freshly harvested flint corn from Northwestern Provinces in Argentina

A mycological survey was carried out for the first time, on red flint corn samples from the northwestern Andinian region of Argentina in the 1999 and 2000 harvest seasons. Species of the genus Fusarium were the most prevalent component of the flint corn mycoflora present in all provinces.F. verticillioides was the predominant Fusarium isolated in the 1999 harvest season in the the region, and was found at higher incidence level than those observed on commercial semident corn hybrids harvested in the main corn production area in Argentina (Pampean region). During the 2000 harvest season, Fusarium graminearum was most commonly isolated species in Salta province.This revised version was published online in October 2005 with corrections to the Cover Date.     

Mycoflora of freshly harvested flint corn from Northwestern Provinces in Argentina

A mycological survey was carried out for the first time, on red flint corn samples from the northwestern Andinian region of Argentina in the 1999 and 2000 harvest seasons. Species of the genus Fusarium were the most prevalent component of the flint corn mycoflora present in all provinces. F. verticillioides was the predominant Fusarium isolated in the 1999 harvest season in the the region, and was found at higher incidence level than those observed on commercial semident corn hybrids harvested in the main corn production area in Argentina (Pampean region). During the 2000 harvest season, Fusarium graminearum was most commonly isolated species in Salta province.     

Cytokine regulation of interleukin 6 production by human endothelial cells

The influence of recombinant (r) human tumor necrosis factor alpha (rTNF-alpha), r human interleukin 1 beta (rIL-1 beta), and r human interferon gamma (rIFN-gamma) on the production of interleukin 6 (IL-6) by human endothelial cells (HEC) was investigated. The addition of 1-100 U/ml of either rTNF-alpha or rIL-1 beta to cultures of HEC monolayers caused a dose-related increase in IL-6 production as detected after 24 hr of incubation. In contrast to rIL-1 beta and rTNF-alpha, the use of up to 1000 U/ml of rIFN-gamma caused only a moderate increase in IL-6 production. However, significantly greater quantities of IL-6 were produced by HEC monolayers subjected to 1000 U/ml of rIFN-gamma in combination with 1-100 U/ml of rTNF-alpha. Furthermore, the addition of graded concentrations of human transforming growth factor beta (TGF-beta) to cultures resulted in a dose-related inhibition of rIL-1 beta- and rTNF-alpha-induced IL-6 production by HEC. The results demonstrate that rIL-1 beta and rTNF-alpha share the ability to stimulate HEC for production of IL-6 and indicate that TGF-beta may act as an immunosuppressive agent, at least partially, through its ability to inhibit the action of TNF-alpha and IL-1 on endothelial cells.     

Stimulation of production of prostaglandin E in gingival cells exposed to products of human blood mononuclear cells.

1. Supernatant media from cultures of unstimulated human peripheral blood mononuclear cells contained one or more factors that increased by several hundred-fold the production of prostaglandin E by fibroblast-like cells derived from both inflamed and normal human gingival tissue. 2. This stimulation occurred in a dose-dependent manner and was completely inhibited by 14 microM-indomethacin. 3. Responsiveness to the factor declined as the age of the cell culture increased. 4. An increase in prostaglandin E production was first observed after a 2h exposure to the mononuclear cell factor(s) and could be prevented by cycloheximide. 5. Brief exposure (0.5 and 1.0 h) to mononuclear cell factor did not increase prostaglandin E production by the cells in a subsequent 72 h incubation in the absence of mononuclear cell factor. 6. Addition of arachidonate (10 microM and 15 microM) further enhanced stimulation of prostaglandin E production in response to mononuclear cell factor. 7. The stimulatory activity was resistant to digestion by trypsin, but was heat-labile, so that only 17% remained after treatment at 56 degrees C for 30 min.     

Effects of PDE4 inhibitors on lipopolysaccharide-induced priming of superoxide anion production from human mononuclear cells.

AIMS: Phosphodiesterase 4 (PDE4) inhibitors have been described as potent anti-inflammatory compounds, involving an increase in intracellular levels of cyclic 3'',5''-adenosine monophosphate (AMP). The aim of this study was to compare the effects of selective PDE4 inhibitors, rolipram and RP 73-401 with the cell permeable analogue of cyclic AMP, dibutyryl-cyclic AMP (db-cAMP) and the anti-inflammatory cytokine interleukin-10 (IL-10) on superoxide anion production from peripheral blood mononuclear cells preincubated with lipopolysaccharide (LPS). MAJOR FINDINGS: We report that, after incubation of the cells with LPS, a large increase in superoxide anion production was observed. Rolipram or RP 73-401 (10(-8) to 10(-5) M) induced significant reductions of fMLP-induced superoxide anion production in cells incubated with or without LPS. The db-cAMP (10(-5) to 10(-3) M) also elicited dose-dependent inhibitions of the fMLP-induced superoxide anion production. In contrast, IL-10 (1 or 10 ng/ml) did not elicit a reduction in fMLP-induced superoxide anion production in both conditions. PRINCIPAL CONCLUSION: These results suggest that the inhibitory activity of PDE4 inhibitors on fMLP-induced production of superoxide anion production is mediated by db-cAMP rather than IL-10.     

Cryopreservation-induced enhancement of interleukin-2 production in human peripheral blood mononuclear cells.

To better understand the effects of cryopreservation on various immunocompetent cell functions, we have examined the interleukin-2 (IL-2)-producing activities of frozen mononuclear cells (MNCs) from healthy subjects. The mechanisms responsible for the observed effects were also analyzed. Both the unfractionated and monocyte-depleted, frozen MNCs produced significantly larger quantities of IL-2 than fresh cells. Similar to freezing, L-leucine methyl ester (Leu-OMe) treatment (to eliminate IL-1 and prostaglandin E-2 (PGE-2)-secreting cells) also increased the IL-2-producing activities of fresh cells, but freezing no longer enhanced the production of IL-2 by Leu-OMe-treated cells, suggesting that (1) both the freezing process and Leu-OMe treatment have similar effects on IL-2 production, (2) the increased IL-2 secretion by frozen MNCs is independent of IL-1, and (3) inactivation of PGE-2-secreting cells during the freezing procedure is responsible for increased IL-2 secretion. Elimination of CD8+ T cells (putative suppressor cells) from MNCs has also resulted in the production of increased amounts of IL-2 by fresh cells, and again, freezing did not further enhance the IL-2-secreting activities of MNCs, that are devoid of CD8+ T cells. This confirms that the increased IL-2 production is due to the inactivation of immuno-down-regulatory cells. The results provide further evidence that the lack of active, suppressor T cells, monocytes, and increased IL-1 and -2 production may be responsible for the previously reported enhanced immunoglobulin-producing abilities of cryopreserved cells from healthy subjects and from patients with lung cancer.     

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.     

The effect of somatostatin on the production of human interferons by mononuclear cells.

Somatostatin (SMS) is a tetradecapeptide which can inhibit the secretion of a number of peptides produced by the endocrine or nervous systems. SMS 201-995 (octreotide) is a somatostatin analogue with very potent somatostatin activities. We have been investigating the effects of both SMS and octreotide on the production of human interferon (IFN). We obtained human peripheral blood mononuclear cells from normal donors and induced them to produce IFN in the presence or absence of a number of peptides possessing somatostatin activities. SMS and octreotide were shown to inhibit the secretion of INF-gamma but not IFN-alpha. Concentrations of 10(-6) M were shown to decrease yields when Concanavalin A or phytohemagglutin were used as the inducer. Higher concentrations had a progressively greater effect. No effects were observed on IFN-gamma production if interleukin 2, ionomycin, or various natural antigens were used to induce the cells. The 28-amino acid form of somatostatin had some effects on gamma IFN yields but the first 14-amino acid fragment of this peptide moiety did not. No effect of any of these compounds was observed on IFN bioactivity. These studies indicate SMS may have some regulatory action on the secretion of immunomodulators in vitro but the concentrations required are well above those encountered under physiologic circumstances, suggesting SMS may not play an important regulatory role governing such secretion in vivo.     

A histamine-releasing factor from activated human mononuclear cells

Human mononuclear cells activated by streptokinase-streptodornase have been shown to elaborate a factor capable of releasing histamine from human basophils. We have developed reproducible methods for its production in large quantities by using cells obtained from leukapheresis packs, by detection utilizing donor basophils known to release well with anti IgE, and by quantitation of histamine by the radioenzyme method. Human histamine-releasing factor (HRF) gave a single peak upon gel filtration with an estimated m.w. of 32,000; SDS gel electrophoresis revealed a single major band as seen at m.w. 30,000. HRF can be resolved into at least two forms separable by ion-exchange chromatography on QAE Sephadex, and two peaks of activity were obtained by chromatofocusing or isoelectric focusing in gels at pH 6.9 and between 7.4 and 8.3. This factor represents an important potential link between cellular immunity and immediate hypersensitivity.     

Substance P enhances interferon-gamma production by human peripheral blood mononuclear cells

Substance P (SP), a neuropeptide widely distributed in the organism, has been shown to stimulate lymphocyte proliferation and immunoglobulin synthesis. However, the effect of SP on specific lymphokines is unknown. Therefore we investigated the influence of SP on mitogen-induced interferon-gamma (IFN-gamma) production in vitro. Peripheral blood mononuclear cells (PBMC) of healthy donors were isolated by density gradient centrifugation and cultured in supplemented RPMI 1640 medium with phytohemagglutinin (PHA) or pokeweed mitogen (PWM), 0.125 and 0.25 mg/liter each, and varying concentrations of SP (10(-12) to 10(-6) M). After 24 and 48 h, IFN-gamma was measured in the supernatant using radioimmunoassay. Results were expressed as percent change of controls. SP alone had no relevant IFN-gamma inducing properties. It enhanced the IFN-gamma production of PWM-stimulated cells significantly up to 18%. The maximal effect was observed at 10(-8) M. PHA-stimulated cells also increased their IFN-gamma production after addition of SP. However, due to great interindividual variations this effect did not attain statistical significance. Stimulation of IFN-gamma production by SP might be of physiological importance, since the effect was seen at concentrations comparable to those found in the body. Our data lend further support to the immunoregulatory functions of SP.     

Opioid peptides modulate production of interferon gamma by human mononuclear cells

The opioid neuropeptides have previously been shown to bind to and affect leukocyte function including lymphocyte proliferation, NK-cell activity, mononuclear cell chemotaxis, immunoglobulin synthesis, and lymphokine production. The effect of the opioid peptides beta-endorphin and Met-enkephalin on interferon gamma (IFN) production by concanavalin A-stimulated human mononuclear cells was examined. Both beta-endorphin and Met-enkephalin enhanced IFN production by the majority of donor mononuclear cells tested and did so at concentrations between 10(-14) and 10(-10) M. When 10(-12) M beta-endorphin or Met-enkephalin were included in concanavalin A-stimulated mononuclear cell cultures, IFN concentrations were significantly enhanced to 205 +/- 45 and 252 +/- 67% of control, respectively. Although the majority of cell preparations tested exhibited an enhanced production of IFN in response to these opioid peptides, some did not. When beta-endorphin or Met-enkephalin were utilized at 10(-11) M, 10 of 15 and 7 of 11 responded with IFN production greater than 20% above the control (untreated) level. There was not an absolute correlation between an enhanced response to beta-endorphin and Met-enkephalin, suggesting the presence of multiple receptor types on these cells for opioids. The opioid receptor antagonist, naloxone, did not significantly prevent the opiate effect. When 10(-8) M naloxone was included in cultures containing 10(-12) M beta-endorphin or Met-enkephalin no significant inhibition of the effect of either opioid on IFN production was observed.