The in vitro effects of artificial and natural sweeteners on the immune system using whole blood culture assays.

This article investigates the effects of commercially available artificial (aspartame, saccharin, sucralose) and natural sweeteners (brown sugar, white sugar, molasses) on the immune system. Human whole blood cultures were incubated with various sweeteners and stimulated in vitro with either phytohemagglutinin or endotoxin. Harvested supernatants were screened for cytotoxicity and cytokine release. Results showed that none of the artificial or natural sweeteners proved to be cytotoxic, indicating that no cell death was induced in vitro. The natural sweetener, sugar cane molasses (10 ug/mL), enhanced levels of the inflammatory biomarker IL-6 while all artificial sweeteners (10 ug/mL) revealed a suppressive effect on IL-6 secretion (P < 0.001). Exposure of blood cells to sucralose-containing sweeteners under stimulatory conditions reduced levels of the biomarker of humoral immunity, Interleukin-10 (P < 0.001). The cumulative suppression of Interleukin-6 and Interleukin-10 levels induced by sucralose may contribute to the inability in mounting an effective humoral response when posed with an exogenous threat.

Development of a bio-assay for estrogens using estrogen receptor alpha gene expression by MCF7 cells as biomarker.

Estrogenic endocrine disruptors (EDCs) have been identified in soil, food, air, and water, and may produce adverse health effects in both humans and wildlife. Various in vitro assays, including the E-screen that measures estrogen dependent proliferation of the MCF-7 human breast cancer cell line, have been developed and implemented to screen for environmental estrogenic EDCs. This study describes a new amendment to the well known E-screen. A direct ELISA to quantify ERalpha protein levels on MCF-7 cells cultured in a high through put 96-well format were validated as a biomarker for estrogenicity. The ELISA shows that there is an inverse correlation between ERalpha levels and 17beta-estradiol (E2) concentration (R(2) = 1). The detection range of the assay is between 1 and 1000 nM for E2. Results obtained with the ERalpha ELISA showed a good inverse correlation with total cellular LDH levels that is conventionally used to quantify MCF-7 cell proliferation. This ELISA was employed to assess environmental water extracts for estrogenicity.

The development and validation of a quantitative ELISA for in vivo and in vitro synthesized vitellogenin from mossambicus tilapia (Oreochromis mossambicus).

The induction of vitellogenin (VTG), an egg yolk precursor found in most oviparious vertebrates, has been well established as a valuable biomarker for assessing estrogenic endocrine disruption. Oreochromis mossambicus is a tilapiine species indigenous to Southern Africa and, therefore, represents a potential African bio-indicator for the development of in vitro and in vivo screens for estrogenicity using VTG as biomarker. However, few ELISAs have been developed to quantify tilapia VTG protein levels. In the present study, commercially available VTG antiserum that shows cross reactivity with tilapia VTG was used to set up and validate a quantitative competition ELISA for tilapia VTG. This ELISA has a broad detection range between 80 ng/ml-5.4 microg/ml VTG and is able to detect both in vivo and in vitro synthesized VTG. This ELISA is highly repeatable with intra- and inter-assay variations less than 3.4% at the lowest detection limit.

Molasses as a possible cause of an "endocrine disruptive syndrome" in calves.

During the mid 1990s a potentially serious, chronic syndrome was reported in well-managed beef and dairy herds from unrelated parts of South Africa. Farmers reported that it manifested as various combinations of decreased production, decreased weaning masses, apparent immune breakdown in previously immunocompetent animals, increased reproductive disorders, various mineral imbalances in non-deficient areas and goitre, noticeable as enlarged thyroid glands. The farmers associated this syndrome with certain batches of sugar cane molasses and molasses-based products. The syndrome was reminiscent of an "endocrine disruptive syndrome". The objective of this study was to evaluate the suspected endocrine disruptive effect of molasses included in cattle feed. Using existing in vitro assays, four batches of molasses syrup were screened for possible inclusion in a calf feeding trial. Two batches were selected for the trial. Thirty-two, 4- to 6-week-old, weaned Holstein bull calves were included in the single phase, three treatment, parallel design experiment. In two of the groups of calves, two different batches of molasses were included in their rations respectively. The control group was fed a ration to which no molasses was added, but which was balanced for energy and mineral content. The mass gain of the calves was recorded over the 6-month study period. The calves were clinically examined every week and clinical pathology parameters, immune responses and endocrine effects were regularly evaluated. Even though endocrine disrupting effects were detected with the in vitro screening assays, these could not be reproduced in the calves in the experiment. The two batches of molasses utilized in the calf feeding trial did not induce major differences in any of the parameters measured, with the exception of a lower mass gain in one of the molasses-fed groups (Group 1), which tended towards significance. The results of the study indicate that the two batches of molasses had no endocrine disruptive or immunosuppressive effects in calves.

The use of IL-6 induction as a human biomarker for inflammatory agents in water.

A rapid whole blood culture (WBC) assay system was developed to monitor the inflammatory potential of water samples collected in the Manguang Municipal area, South Africa. Several systems associated with the Modder River catchment area were investigated in this preliminary study to find out whether there were potential problems in this catchment area. Three focus areas were investigated: (a) the impact of the Klein Modder River, which passes through a densely populated residential area, on the inflammatory activity of the Modder River; (b) the efficiency of the treatment plants in eliminating inflammatory activity; and (c) the efficiency of chlorination of treated water in eliminating inflammatory activity that was not eliminated by the treatment system. Water contaminated with inflammatory substances induced the pro-inflammatory hormone interleukin 6 (IL-6). All water samples collected from the Modder River induced IL-6 secretion, and the quantity of IL-6 secreted was dependent on the concentration and origin of the sample. The lowest IL-6 inducing activity for river water was obtained for samples collected near the origin of the river (Rustfontein). Samples at subsequent points downstream from confluence with the Klein Modder River showed a major increase in IL-6 inducing activity. The two treatment plants along the Modder River also received water of different inflammatory activity. Both treatment plants eliminated some of the inflammatory activity during processing, however inflammatory residues remained after processing which might have been potentially harmful for infants and people with suppressed immune systems. Chlorination eliminated most of the inflammatory activity, indicating that chlorination of water was a necessary process in ensuring water of low inflammatory content.

The effects of anti-androgenic and estrogenic disrupting contaminants on breeding gland (nuptial pad) morphology, plasma testosterone levels, and plasma vitellogenin levels in male Xenopus laevis (African clawed frog).

Nuptial pads are areas, digits, and forearm characterized by epidermal keratin hooks and dermal breeding glands opening on the epidermis surface in male frogs. The presence of the epidermal hooks and secretory activity of the breeding glands are androgen-dependent and considered functioning during mating. Chemical pollutants released into the aquatic environment by humans are suspected of disrupting the normal hormonal pathways and functioning. Among the best known of these endocrine-disrupting contaminants (EDCs) are compounds that mimic the steroid 17beta-estradiol. However, recently specific anti-androgenic activity by certain EDCs, including DDE and several fungicides, have been shown in mammalian studies. The African clawed frog, Xenopus laevis, has been proposed as an ideal species to be used as a model system for screening of endocrine disruptors. In this study we employed a newly developed ELISA for the measurement of plasma vitellogenin (VTG), normally produced in the liver of females under the influence of estrogens but also in males when exposed to exogenous estrogens. Furthermore, we investigated the potential of breeding (nuptial) gland activity to be employed as a biomarker system for screening for anti-androgenic activity by certain EDCs. Our results indicate that the pharmaceutic anti-androgen flutamide did significantly (p < 0.05) effect the androgen-dependent breeding (nuptial) glands and plasma testosterone concentrations in male X. laevis. Our results further confirm that the dicarboximide fungicide vinclozolin mimics the anti-androgenic action of flutamide. Vinclozolin, however, did not significantly effect the plasma testosterone concentration. Results also confirm the ability of 17beta-estradiol to stimulate production and release of VTG in the liver of male X. laevis, although we could not confirm the in vivo induction of VTG by estrogenic mimics octylphenol and nonylphenol. Although more dose-response research is needed, this preliminary study confirms the potential of VTG production and breeding gland activity as biomarkers to screen chemicals and environmental samples for estrogenic and anti-androgenic activity using X. laevis males.

The development of an ELISA for chicken apolipoprotein II quantitation.

Polyclonal antibodies raised against chicken apoll was characterised for its use in Western blotting and ELISA detection systems of apoll in chicken plasma. The antibody has a high avidity and specificity for apolipoprotein II (apoII). Western blots show that the antibody reacts with a single band at 15 kDa. The antibody was used for setting up both direct and indirect ELISA assays for apoll. The indirect ELISA has a broader detection range (10-1,600U/mL) than the direct ELISA (10-100U/mL). It was found that both ELISA systems discriminate very well between vitellogenic (laying hen) and non-vitellogenic (rooster) plasma. The in- direct ELISA, due to its broad detection range, can potentially be used for monitoring female reproductive cycles, accidental and environmental exposure of males to estrogen, and for apoII secretion by cultured hepatocytes and hepatomas.

Validation of an ex vivo Xenopus liver slice bioassay for environmental estrogens and estrogen mimics.

A sensitive ex vivo bioassay for detecting environmental estrogens and estrogen mimics was developed using Xenopus liver slice culture. Vitellogenin synthesis by these cultures was used as biomarker for estrogenic activity of environmental water samples. Sensitization of the assay for estrogens and mimics was accomplished by employing tissue from animals after in vivo preexposure to estrogen. Effects of various tissue culture factors were investigated in order to obtain optimum conditions for the bioassay. It was found that endogenous vitellogenin and/or estrogen could be "washed out" of cultures and that not only uncontaminated males but also estrogen-pretreated males and females can successfully be used as bioindicators. Estrogenicity was detected, using the ex vivo Xenopus liver slice culture assay, in sewage effluent, lake water, and dam water. This article presents an optimized protocol for effective estrogen detection in environmental water samples.

IL-6 secretion by ex vivo whole blood cultures upon allergen stimulation.

Allergen induced IL-6 synthesis by whole blood cultures was compared with skin prick allergen test results for the same group of individuals. Whole blood cultures from both allergic and non-allergic individuals secrete IL-6 at high allergen concentrations. When whole blood cultures from controls were incubated with serial dilutions of allergens it was found that IL-6 induction was abolished at lower allergen dilutions (allergen threshold concentration or ATC). When whole blood cultures from patients with allergic rhinitis were stimulated with ATC it was found that some allergens induced IL-6 secretion. The allergens inducing IL-6 and the level of IL-6 secreted were dependent on the patient. The induction of IL-6 secretion by the cultures at ATC correlated very significantly with the patient's skin prick test results (r = 0.71 1; p = 0.0003).

Inflammatory activity as an indicator of water quality: the use of human whole blood cultures.

A rapid whole blood culture (WBC) assay system was developed to monitor the inflammatory potential of water samples collected in the Western Cape, South Africa. Water contaminated with inflammatory substances induced the pro-inflammatory hormone interleukin 6 (IL-6). All water samples collected from the Eerste River, Stellenbosch, induced IL-6 secretion, and the quantity of IL-6 secreted is dependent on the concentration and origin of the sample. The lowest IL-6 inducing activity for river water was obtained for samples collected near the origin of the river. Samples at subsequent points downstream showed an increase in IL-6 inducing activity. Drinking water samples collected from selected towns in the Western Cape showed that there were major differences between the inflammatory potential of the water. Of the 15 samples assayed, 7 had low inflammatory activity, 4 had an intermediate inflammatory activity and 4 had high inflammatory activity. The water sources that have a high inflammatory activity may pose a health risk to consumers.

The effects of B-sitosterol (BSS) and B-sitosterol glucoside (BSSG) mixture on selected immune parameters of marathon runners: inhibition of post marathon immune suppression and inflammation.

A pilot study was undertaken to investigate the effects of the intake of capsules containing the plant sterols and sterolins (BSS:BSSG mixture) on selected immune parameters of volunteers participating in an ultra-marathon in Cape Town, South Africa. Those runners having received active capsules (n=9) showed less neutrophilia, lymphopenia and leukocytosis when compared to their counterparts having received placebo capsules (n=8): the placebo treated individuals showed significant increases in their total white blood cell numbers as well as in their neutrophils (p=0.03 and 0.03 respectively). Furthermore, statistically significant increases within lymphocyte subsets were observed in the runners having received the active capsules: CD3+ cells increased (p=0.02) as did CD4+ cells (p=0.03). In parallel, the BSS:BSSG capsules decreased the plasma level of IL6 in the runners using the active capsules (p=0.08) and significantly decreased the cortisol: DHEAs ratio (p=0.03), suggesting that these volunteers had less of an inflammatory response and were less immune suppressed during the post-marathon recovery period. These findings justify further investigations into the use of the phytosterols to prevent the subtle immunosuppression associated with excessive physical stress.

The detection of pyrogens in sera from patients with symptoms of sepsis using an ex vivo whole blood culture assay.

The aim of this study was to investigate whether the ex vivo whole blood culture (WBC) assay system can be used to detect pyrogens in blood from patients with symptoms of sepsis. Blood samples from 35 patients with symptoms of sepsis were assayed for bacterial contamination using the radiometric blood culture assay. Serum from the same patients were screened for IL-6, C-reactive protein (CRP) and pyrogens using the whole blood culture assay. Serum samples from 26 patients tested positive for pyrogens. Of the 26 patients with pyrogenic serum, 15 had elevated serum IL-6 levels and 19 had elevated CRP levels. Only two of the samples had positive blood cultures as detected by the routine radiometric assay. Both of these patients had high serum CRP and pyrogen levels, while only one of them had an elevated serum IL-6 level. These results show that the WBC is very sensitive in detecting pyrogens in serum of patients. This technique can be a useful tool to quantitate pyrogens in sera from patients with symptoms of sepsis and to determine whether their clinical symptoms are caused by pyretic substances in their circulatory system.

Differentiation between endotoxin and non-endotoxin pyrogens in human albumin solutions using an ex vivo whole blood culture assay.

Purified E.coli endotoxin, Gram negative bacteria and Gram positive bacteria induce IL-6 secretion by whole blood cultures (WBC's). Polymyxin B at concentrations greater than 2 U/ml completely inhibits IL-6 secretion caused by 10 EU/ml of endotoxin. Polymyxin B has no effect on IL-6 secretion by WBC's in the absence of endotoxin. The inhibition of endotoxin induced IL-6 secretion is Polymyxin B concentration dependent at concentrations less than 1 U/ml. IL-6 induction caused by E.coli is only partially inactivated by 8 U/ml Polymyxin B. Polymyxin B has no effect on IL-6 secretion caused by B.subtilis. Two pyrogenic batches of human serum albumin (HSA), as tested by the rabbit assay for pyrogens, were also investigated. Polymyxin B at 4 U/ml inhibits less than 40 % of IL-6 secretion caused by these pyrogenic HSA batches. All the endotoxin activity in HSA samples spiked with purified endotoxin is inhibited by Polymyxin B indicating that HSA does not protect endotoxin against Polymyxin B inhibition. These results indicate that the pyrogenicity of these HSA batches are caused by Polymyxin B inhibitable and non-inhibitable fractions. This study shows that pyrogenic substances other than endotoxin can contaminate batches of pharmaceutical products and that results obtained using the Limulus Amoebocyte Lysate (LAL) assay does not necessarily indicate the pyrogenic status of pharmaceutical products. The WBC assay for pyrogens, having a broader sensitivity range than the LAL assay, is a better indicator of the pyrogenic status of pharmaceutical products.

The detection of pyrogens in blood products using an ex vivo whole blood culture assay.

Induction of interleukin-6 (IL-6) secretion by whole blood cultures (WBC) was used as an in vitro assay system for pyrogen-induced inflammatory reactions. The assay system was very sensitive to Eschericia coli (E coli) endotoxin (< 10 pg/ml). The potential pyrogenic effects of human serum albumin (HSA), Fibronectin (Fn) and stabilised human serum (SHS) solutions were analyzed using this system. None of the products assayed had an effect on the sensitivity of the WBC assay. Spike recovery studies with isolated endotoxin, gram positive and gram negative bacteria showed that none of the products had an effect on the spike recovery of these pyrogenic substances. Good correlations were found between the WBC assay and the rabbit assay for pyrogens for all the production batches tested. When these samples were analysed by the limulus amoebocyte lysate (LAL) assay, the LAL test gave anomalous results for 1 out of the 22 production batches tested. This batch gave a false negative result on the LAL assay and might be indicative of the inability of the LAL assay to detect pyrogens other than endotoxin.

The use of peroxidase anti-peroxidase (PAP) complexes in the detection of plant viruses by ELISA.

An ELISA test system for detection of plant viruses in field samples is described, based on the unlabelled antibody method using a peroxidase-antiperoxidase (PAP) complex. Novel features of the system include the use of acid-treated naked bacteria as combined carrier-adjuvants for the production of rabbit antiviral antibodies, and the use of acid-treated chicken antibodies (IgY) for antigen trapping in the ELISA. Systems for detection of potato virus Y (PVY), potato leafroll virus (PLRV), grapevine fanleaf virus (GFV) and grapevine virus A (GVA) were developed and compared with conventional direct double antibody sandwich (DAS) systems in tests with both purified virus and field samples. The PAP systems offer improved sensitivity, no background problems in the outer rows of the microtitre plates and are much easier to visualize with the naked eye if no plate reader is available.