Isolation of immunomodulatory triterpene acids from a standardized rose hip powder (Rosa canina L.).

A previously published systematic review and a metaanalysis have concluded that the consumption of standardized rose hip powder (Rosa canina L.) can reduce pain in osteoarthritis patients. Synovial inflammation has been suggested to play an important role in the pathogenesis of osteoarthritis and mainly to involve infiltration of the synovial membrane by macrophages. Therefore, the immunomodulatory effect of standardized rose hip powder of Rosa canina L. was investigated and active principles isolated using the Mono Mac 6 cell line as a model for human macrophages. Treatment of Mono Mac 6 cells with the residue of a crude dichloromethane extract of rose hip powder significantly and concentration dependently inhibited the lipopolysaccharide induced interleukin-6 release. Through bioassay-guided fractionation the immunomodulatory effect of the dichloromethane extract was correlated to a mixture of three triterpene acids; oleanolic acid, betulinic acid and ursolic acid (IC(50) 21 ± 6 µm). Further studies revealed that only oleanolic acid and ursolic acid, but not betulinic acid, could inhibit the lipopolysaccharide induced interleukin-6 release from Mono Mac 6 cells when tested separately. Combination of either oleanolic acid or ursolic acid with betulinic acid enhanced the immunomodulatory effect of the two triterpene acids.

Assessment of the total inflammatory potential of bioaerosols by using a granulocyte assay.

Occupational health symptoms related to bioaerosol exposure have been observed in a variety of working environments. Bioaerosols contain microorganisms and microbial components. The aim of this study was to estimate the total inflammatory potential (TIP) of bioaerosols using an in vitro assay based on granulocyte-like cells. A total of 129 bioaerosol samples were collected in the breathing zone of workers during their daily working routine at 22 biofuel plants. The samples were analyzed by traditional assays for dust, endotoxin, fungal spores, (1-->3)-beta-d-glucan, total number of bacteria, the enzyme N-acetyl-beta-d-glucosaminidase (NAGase; primarily originating from fungi), Aspergillus fumigatus, and mesophilic and thermophilic actinomycetes; the samples were also assayed for TIP. In a multilinear regression four factors were significant for the TIP values obtained: endotoxin (P < 0.0001), fungal spores (P < 0.0001), (1-->3)-beta-d-glucan (P = 0.0005), and mesophilic actinomycetes (P = 0.0063). Using this model to estimate TIP values on the basis of microbial composition, the correlation to the measured values was r = 0.91. When TIP values obtained in the granulocyte assay were related to the primary working area, we found that bioaerosol samples from personnel working in straw storage facilities showed high TIP values ( approximately 50 times the TIP of unstimulated controls). In contrast, bioaerosol samples from personnel with work functions in offices or laboratories showed low TIP values ( approximately 5 times the TIP of the unstimulated control). This indicates, as expected, that these areas were less contaminated. In conclusion, the granulocyte assay reacts to multiple contaminants in the environmental samples and can be used to obtain a measurement of TIP. Therefore, potential occupational health effects related to inflammation of the airways in a working environment can be estimated using this assay.

Contamination versus preservation of cosmetics: a review on legislation, usage, infections, and contact allergy.

Cosmetics with high water content are at a risk of being contaminated by micro-organisms that can alter the composition of the product or pose a health risk to the consumer. Pathogenic micro-organisms such as Staphylococcus aureus and Pseudomonas aeruginosa are frequently found in contaminated cosmetics. In order to avoid contamination of cosmetics, the manufacturers add preservatives to their products. In the EU and the USA, cosmetics are under legislation and all preservatives must be safety evaluated by committees. There are several different preservatives available but the cosmetic market is dominated by a few preservatives: parabens, formaldehyde, formaldehyde releasers, and methylchloroisothiazolinone/methylisothiazolinone. Allergy to preservatives is one of the main reasons for contact eczema caused by cosmetics. Concentration of the same preservative in similar products varies greatly, and this may indicate that some cosmetic products are over preserved. As development and elicitation of contact allergy is dose dependent, over preservation of cosmetics potentially leads to increased incidences of contact allergy. Very few studies have investigated the antimicrobial efficiency of preservatives in cosmetics, but the results indicate that efficient preservation is obtainable with concentrations well below the maximum allowed.

Cryopreservation of differentiated HL-60 cells for pyrogen testing.

All-trans retinoic-acid (ATRA) differentiated HL-60 cells can be used to detect pyrogens such as bacteria, bacterial components, yeasts and fungi. Differentiated HL-60 cells obtain neutrophil like characteristics and if stimulated the differentiated HL-60 cells produce reactive oxygen species in a dose dependent manner. Culturing and differentiation of cell lines are time consuming activities and require suitable facilities; cryopreservation of pre-differentiated cells could provide the basis for an easily distributable pyrogen testing kit. Cryopreservation of granulocytes has proven to be very complicated and neutrophils are especially difficult to cryopreserve, most likely due to their large degree of granulation. Here we present evidence that HL-60 cells can be differentiated with ATRA and subsequently cryopreserved. Upon thawing the cells retain their ROS producing capabilities and reactivity towards pyrogens. Further, the cells retain their ability to react dose dependently towards lipopolysaccharide (LPS), lipoteichoic acid (LTA) and zymosan. At pathophysiologically relevant concentrations of LPS, LTA and zymosan the cells retain full reactivity for at least two months when stored in liquid nitrogen. In conclusion, ATRA differentiated HL-60 cells are cryopreservable and retain reactivity upon thawing. It is therefore possible to produce an in-vitro in-house pyrogen test kit for medicines and related products.

Effect of moist heat sterilisation on the pyrogenicity of cell wall components from Staphylococcus aureus.

As opposed to endotoxins very little is known about the heat resistance of Gram positive pyrogens. The aim of this study is to examine the pyrogenic activity of the cell wall components lipoteichoic acid and peptidoglycan from Staphylococcus aureus after moist heat sterilisation. The pyrogenic activity is determined as the ability of the substances to induce interleukin-6 secretion in Mono Mac 6 cells. The standard terminal moist heat sterilisation procedures (121 degrees C for 15min and 134 degrees C for 3min) are not able to inactivate the pyrogenic activity of S. aureus lipoteichoic acid and peptidoglycan. However after longer treatment times the pyrogenic activity of lipoteichoic acid is removed at both 121 degrees C and 134 degrees C. In contrast the activity of peptidoglycan is not removed after 160min at neither 121 degrees C nor 134 degrees C where only a 2-log reduction is obtained. In conclusion the terminal moist heat sterilisation procedures described by the European Pharmacopoeia are not able to inactivate the interleukin-6 inducing activity of S. aureus lipoteichoic acid and peptidoglycan.

Utilization of the human cell line HL-60 for chemiluminescence based detection of microorganisms and related substances.

In this paper we describe a new pyrogen assay using the human leukemia cell line HL-60. The cell line is differentiated using all-trans retinoic acid (ATRA) to generate a cell population that resembles mature granulocytes. The differentiated HL-60 cell is capable of generating reactive oxygen species (ROS) when challenged with pyrogenic substances. In a luminol enhanced chemilumimetric assay the responsiveness of differentiated HL-60 cells is tested towards Salmonella typhimurium, Bacillus subtilis, Saccharomyces cerevisiae, Candida albicans, lipopolysaccharide (LPS) and lipoteichoic acid (LTA). The results show a poor sensitivity to S. typhimurium but displays good sensitivity towards B. subtilis, LTA and LPS. Furthermore, the sensitivity towards the yeasts C. albicans and S. cerevisiae is considerably better than obtained in other in vitro cell systems. Overall these results indicate that the HL-60 cell assay possibly could be evolved to a supplementary assay for the known pyrogenic detection assays. Furthermore, the utilization of the assay for pyrogenic examination of recombinant drugs derived from yeast expression systems would be relevant to examine.

Regulation of interleukin-6 secretion in murine pituicytes.

Pituicytes, the astrocytic glial cells of the neural lobe, are known to secrete interleukin-6 and nitric oxide upon stimulation with various inflammatory mediators, i.e. interleukin-1beta. Nitric oxide is described to modulate the secretion of interleukin-6 in various cell types. The aim of the present study was to investigate the effect of nitric oxide on interleukin-1beta induced interleukin-6 secretion. Furthermore the effect of interferon-gamma on interleukin-6 and nitric oxide release was investigated. Cultures of pituicytes were prepared of neural lobes from male mice. The effect of interleukin-1beta and interferon-gamma on interleukin-6 and nitric oxide secretion was investigated in pituicytes cultured for 14 days. The secretion of interleukin-6 and nitric oxide was determined after 24 h of stimulation. Pituicytes secrete interleukin-6 upon stimulation with interleukin-1beta dose dependently but did not induce any detectable nitric oxide release. Co-stimulation with interferon-gamma and interleukin-1beta induced a significant nitric oxide release. In addition interferon-gamma inhibits interleukin-1beta induced interleukin-6 secretion dose dependently. The observed effect of interferon-gamma on interleukin-6 secretion was not affected by the specific inducible nitric oxide synthase inhibitor 1400W (N-(3-[aminomethyl]benzyl)acetamidine). Furthermore interferon-gamma dose dependently inhibits unstimulated interleukin-6 secretion. Use of the nitric oxide releaser DETA/NO (2,2'-(hydroxynitrosohydrazono)bis-ethanimine) demonstrated that nitric oxide does not inhibit interleukin-1beta induced interleukin-6 secretion. These results demonstrated that nitric oxide has no influence on interleukin-1beta induced interleukin-6 secretion in cultured pituicytes. However the results are showing that interferon-gamma has an inhibitory effect on interleukin-6 secretion.

Dry and moist heat sterilisation cannot inactivate pyrogenicity of Gram positive microorganisms.

In the monocytic cell line Mono Mac 6 pyrogens induce interleukin-6 secretion dose dependently. The aim of this study is to examine the interleukin-6 inducing capacity of Gram positive Staphylococcus aureus and Bacillus subtilis endospores after moist and dry heat sterilisation. Moist heat sterilisation of B. subtilis endospores for 15 min at 121 degrees C and 134 degrees C can only reduce the interleukin-6 inducing capacity to 57% and 63%, respectively, compared to untreated. Moist heat sterilisation of S. aureus for 60 min at 121 degrees C and 134 degrees C does not reduce the interleukin-6 inducing capacity of S. aureus. On the contrary moist heat sterilisation at 134 degrees C for 10, 20 and 40 min significantly increases the interleukin-6 inducing capacity compared to untreated S. aureus. This is confirmed in the rabbit pyrogen test. Dry heat sterilisation of B. subtilis endospores at 220 degrees C for 45 min reduces the interleukin-6 inducing capacity to 2% compared to untreated endospores. Dry heat treatment of S. aureus at 220 degrees C for 30 min only reduces the activity to 55%. However, after 250 degrees C for 30 min or 220 degrees C for 6h there is no detectable activity of S. aureus. In conclusion, neither the interleukin-6 inducing activity nor the pyrogenicity of S. aureus and endospores of B. subtilis can be inactivated by the heat sterilisation procedures described by the European Pharmacopoeia.

Lipidated α-peptide/ß-peptoid hybrids with potent anti-inflammatory activity.

In this study, we investigated, optimized, and characterized a novel subclass of host defense peptide (HDP) mimics based on α-peptide/ß-peptoid hybrid oligomers with an alternating cationic/hydrophobic design with respect to their ability to modulate the pro-inflammatory response by human primary leukocytes upon exposure to bacterial components. Structure-activity studies revealed that certain lipidated α-peptide/ß-peptoid hybrid oligomers possess anti-inflammatory activities in the submicromolar range with low cytotoxicity, and that the anti-inflammatory activity of the HDP mimics is dependent on the length and position of the lipid element(s). The resulting lead compound, Pam-(Lys-ßNSpe)6-NH2, blocks LPS-induced cytokine secretion with a potency comparable to that of polymyxin B. The mode of action of this HDP mimic appears not to involve direct LPS interaction since it, in contrast to polymyxin B, displayed only minor activity in the Limulus amebocyte lysate assay. Flow cytometry data showed specific interaction of a fluorophore-labeled lipidated α-peptide/ß-peptoid hybrid with monocytes and granulocytes indicating a cellular target expressed by these leukocyte subsets.

Baclofen influences lipopolysaccharide-mediated interleukin-6 release from murine pituicytes.

Pituicytes, the glial cells of the neurohypophysis, secrete interleukin-6 upon stimulation with various inflammatory mediators, i.e. lipopolysaccharide. Previous studies have identified several receptors on pituicytes. This study investigates the effect of GABA(B) receptor activation on interleukin-6 release from pituicytes. Cultured murine pituicytes were stimulated for 24 h with lipopolysaccharide (0.5 ng/ml) to give a significant interleukin-6 release compared to control. The interleukin-6 release was significantly potentiated by the GABA(B) receptor agonist (R)-4-amino-3-(4-chlorophenyl) butanoic acid (R-baclofen; 10, 100 or 500 microM). However, R-baclofen itself (10, 100 or 500 microM) did not stimulate the interleukin-6 secretion. Furthermore, the potent GABA(B) receptor antagonists 3-[[(3,4-Dichlorophenyl)methyl]amino]propyl]diethoxymethyl) phosphinic acid (CGP52432; 30 or 300 microM) and (RS)-3-Amino-2-(4-chlorophenyl)-2-hydroxypropyl-sulphonic acid (2-OH-saclofen; 10 or 100 microM) did not remove the effect of R-baclofen (100 microM). Gamma-amino butyric acid (GABA; 30 or 300 microM) did not alter the lipopolysaccharide-mediated interleukin-6 response. After 30 min, intracellular cyclic AMP (cAMP) was higher in cells stimulated with lipopolysaccharide compared to control, and R-baclofen significantly inhibited this increase in cAMP. Nevertheless, neither lipopolysaccharide nor R-baclofen had any effect on intracellular cAMP after 24 h of stimulation. The results suggest that the effect of R-baclofen on lipopolysaccharide-stimulated interleukin-6 secretion is independent of GABA(B) receptors.

Endospores of B subtilis are pyrogenic and activate Mono Mac 6 cells: importance of the CD14 receptor.

The monocytic cell line Mono Mac 6 is sensitive to pyrogens and interleukin-6 secretion is induced after exposure to pyrogens. The aim of this study is to examine the pyrogenic activity and the interleukin-6-inducing capacity of the Gram-positive B. subtilis bacteria, endospores and isolated cell wall components. Furthermore the involvement of CD14 in activation of interleukin-6 release is investigated. All test substances are pyrogenic in the rabbit pyrogen test. The test substance is incubated with monocytic cells (Mono Mac 6) for 24 h and the secreted interleukin-6 is determined in a sandwich immunoassay. B. subtilis bacteria and endospores induce interleukin-6 in a dose-dependent manner. Endospores are less potent than bacteria. Lipoteichoic acid (LTA) isolated from B. subtilis induces interleukin-6 in a dose-dependent manner, whereas muramyl dipeptide (MDP) is unable to induce interleukin-6. Lipopolysaccharides (LPS) dose-dependently induce interleukin-6 release, but the curve differs from that of LTA both in shape and offset. The interleukin-6 secretion induced by LPS, LTA and B. subtilis bacteria can be blocked by 73-85% by an antibody directed against CD14, whereas the antibody only blocks 25% of B. subtilis endospores-induced interleukin-6 release. The results might indicate that B. subtilis endospores use an additional pathway to CD14 to activate mononuclear cells.

Considerations regarding use of solvents in in vitro cell based assays.

Cell culture systems are widely used for the investigation of in vitro immunomodulatory effects of medicines and natural products. Since many pharmacological relevant compounds are water-insoluble, solvents are frequently used in cell based assays. Although many reports describe the cellular effects of solvents at high concentrations, only a few relate the effects of solvents used at low concentrations. In this report we investigate the interference of three commonly used solvents: Dimethyl sulfoxide (DMSO), ethanol and ß-cyclodextrin with five different cell culture systems. The effects of the solvents are investigated in relation to the cellular production of interleukin (IL)-6 or reactive oxygen species (ROS) after lipopolysaccharide (LPS) stimulation. We show that DMSO above 1 % reduces readout parameters in all cell types but more interestingly the 0.25 and 0.5 % solutions induce inhibitory effects in some cell types and stimulatory effects in others. We also found that LPS induced ROS production was more affected than the IL-6 production in the presence of ethanol. Finally we showed that ß-cyclodextrin at the investigated concentrations did not have any effect on the LPS induced IL-6 production and only minor effects on the ROS production. We conclude that the effects induced by solvents even at low concentrations are highly relevant for the interpretation of immunomodulatory effects evaluated in cell assays. Furthermore, these results show the importance of keeping solvent concentrations constant in serial dilution of any compound investigated in cell based assays.

Immunomodulatory effects of testosterone evaluated in all-trans retinoic acid differentiated HL-60 cells, granulocytes, and monocytes.

The sex hormones are known to affect innate immunity in humans. In this study we evaluated the immunomodulatory effects of testosterone in a model system comprising of all-trans retinoic acid differentiated HL-60 cells, and confirmed the results in human granulocytes and monocytes. Results showed that testosterone at pharmacological doses reduced the production of interleukin-8 and reactive oxygen species from differentiated HL-60 cells in a concentration dependent manner without affecting phagocytosis. The cells were stimulated with zymosan, lipopolysaccharide, or Bacillus subtilis. At the highest concentration of testosterone (120 µM), interleukin-8 secretion was reduced 42-80%, and production of reactive oxygen species was reduced 32-46%. Flutamide, an antagonist of the classical intracellular androgen receptor, was unable to antagonize the immunosuppressive effect of testosterone. We further demonstrated that the suppressive effect of testosterone has a short onset time. Our results suggest that testosterone affects the fast operating membrane bound androgen receptor or a rapid acting enzyme system. Testosterone, at pharmacological doses, was also shown to suppress generation of reactive oxygen species and interleukin-8 in human granulocytes and monocytes, respectively, to a similar extent as observed in differentiated HL-60 cells.