Measurement of mast cell cytokine release by multiplex assay.

Mast cells are highly responsive cells that are capable of secreting a variety of inflammatory mediators, including histamine, heparin, serine proteases, leukotrienes, prostaglandins, and thromboxanes. Studies from several laboratories have demonstrated that mast cells have the capacity to produce a variety of cytokines in response to various stimuli. Characterization of the cytokine profiles in mast cells has routinely been determined by the performance of individual enzyme-linked immunosorbent assays. This process is expensive, time-consuming, and requires a great deal of material to characterize multiple cytokines. In this chapter, we describe a multiplex cytokine assay to detect 17 cytokines simultaneously in 50 microL of culture supernatant derived from stimulated human cord blood-derived mast cells.

Proapoptotic activated T-cells in the blood of children with Down's syndrome: relationship with dietary antigens and intestinal alterations.

Immune defects, thyroid abnormalities, infections and coeliac disease are often associated with Down's syndrome (DS). However, the basis of the immune defects is still unclear in DS. In the present study, we show that peripheral CD4 T-cells were decreased in children with DS, while mean values of cytotoxic CD8 T-cells were comparable with those from healthy children. Circulating activated (CD3/HLA-DR positive) T-cells were increased and a large proportion of purified T-cells from DS were also positive for APO-I/FAS (CD95) antigen. To further explore the functional status of circulating activated T-cells, enriched CD3 lymphocytes were cultured for 3 h and were tested for positivity to annexin-V (ANX-V) and propidium iodide. T-cells with the early apoptotic phenotype were increased in cell cultures from DS children. Plasma levels of inteleukin-6 (IL-6) were higher in DS children than in healthy children. The incidence of coeliac disease was also increased in this group of children. Most DS children showed increased levels of circulating IgG or IgA specific for gliadin, and their plasma IL-6 levels correlated with those of antigliadin IgG. The number of CD4 circulating cells was very low in DS children with coeliac disease, was low in those with serum antigliadin antibodies and was normal in DS without antigliadin antibodies. An overload of dietary antigens and impaired nutrient absorption secondary to altered functioning of the gastrointestinal mucosa might interfere with normal immune responses by inducing programmed cell death in CD4 T-cells.

Nanomolar level of resveratrol (trans-3,5,4'-trihydroxystilbene) is required, but is not sufficient, to inhibit the growth of human monocytoid tumor cells through an apoptotic-like mechanism.

The chemopreventive activity of resveratrol, a stilbene found in grapes and wine, was evaluated in a human monocytic leukemia cell line at the same concentration (100 nM to 1 microM) as that found in the blood-stream after moderate wine intake. As early as at 4 h after intake, resveratrol exhibited antiproliferative and cytotoxic activity. At the same time, some apoptotic-like phenomena were detected such as cell membrane perturbation (phosphatidylserine-annexin V binding), apolipoprotein (APO)-1/FAS (CD95) expression and mitochondrial (delta psi) depolarization. The anticancer drug camptothecin, used as a positive control, did not significantly increase APO-1/FAS (CD95) levels, while only a modest increase in APO-1/FAS-CD95 ligand (CD95-L) was detected. At 12 h, however, resveratrol at concentrations of 100 nM and 1 microM did not exhibit the same antiproliferative activity and increased cell proliferation was correlated to a significant increase in FAS-L expression. We conclude that treatment with low doses of resveratrol, such as those found after moderate wine intake, is not sufficient to stop human leukemia cell line proliferation and that cell resistance, marked by high FAS-L (CD95-L) expression, could be mediated by low (delta psi) mitochondria-released antiapoptotic factors such as BCL-2. It is also suggested that the synergistic action of other wine components with resveratrol might, at least partially, explain its chemopreventive activity.

Cannabinoids inhibit nitric oxide production in bone marrow derived feline macrophages.

Feline immunodeficiency virus (FIV) infection causes a widespread natural immunodeficiency syndrome in cats that is considered a suitable animal model for studying human immunodeficiency virus (HIV) infection and pathogenesis. Short term cultures of bone marrow derived feline macrophages stimulated with recombinant feline interferon-gamma (r-IFN-gamma) and lipopolysaccharide (LPS) were shown to produce nitric oxide. Feline macrophages were shown to express cannabinoid receptors, and nitric oxide production decreased after in vitro exposure to synthetic cannabinoid CP-55940. Both cannabinoid receptors, CB1 and CB2, were involved in this process, since the inhibition was reversed by selective cannabinoid antagonists for both of these receptors.

Value of the dorsal cutaneous guinea pig model in selecting topical antiviral formulations for the treatment of recurrent herpes simplex type 1 disease.

Recurrent herpes simplex labialis represents a disease still difficult to treat, despite the availability of many established antiviral drugs used in clinical research since 30 years ago. Although differences between the human disease and that obtained in experimental animal suggest caution in predicting an effective clinical response from the experimental results, some of the animal models seem to be useful in optimising the topical formulation of single antiviral drugs. In the present work the dorsal cutaneous guinea pig model was used to compare 5 different topical antiviral formulations with clinical promise (active molecule: 5% w/w micronized aciclovir, CAS 59277-89-3), using both roll-on and lipstick application systems. The aim being to evaluate which vehicle (water, oil, low melting and high melting fatty base) and application system (roll-on, lipstick) enhances the skin penetration and the antiviral activity of the drug, after an experimental intradermal infection with Herpes simplex virus type 1 (HSV-1). As reference, a commercial formulation (5% aciclovir ointment) was used. The cumulative results of this study showed that the formulation A, containing 5% aciclovir in an aqueous base in a roll-on application system, has the better antiviral efficacy in reducing the severity of cutaneous lesions and the viral titer; among the lipsticks preparations, the formulation D, containing 5% aciclovir in a low melting fatty base, demonstrates a very strong antiviral activity, though slightly less than formulation A. This experimental work confirms the validity of the dorsal cutaneous guinea pig model as a rapid and efficient method to compare the antiviral efficacy of new formulations, with clinical promise, to optimise the topical formulation of the active antiviral drugs.

Decreased expression of L-selectin (CD62L) on lymphocytes in enzootic bovine leukaemia.

Expression of L-selectin was determined by single- and two-colour immunofluorescence on granulocytes, peripheral blood mononuclear cells (PBMC) and blasts of bovine origin by means of a monoclonal antibody IVA94 which recognizes bovine L-selectin (CD62L). Cells were separated from peripheral blood of healthy cattle and colleagues infected with bovine leukaemia virus (BLV). BLV-infected animals comprised lymphocytotic and non-lymphocytotic cows. L-selectin was expressed on 90-98% of granulocytes in all tested animals. The percentage of PBMC expressing L-selectin was lower in cattle with persistent lymphocytosis than in non-lymphocytotic or BLV-free cattle, and inversely correlated with lymphocyte counts. The ratio of B lymphocytes stained for L-selectin was significantly decreased from 60.2 +/- 1.9% in BLV-free cattle to 43.8 +/- 3.6 and 22.5 +/- 5.7% in non-lymphocytotic and lymphocytotic cattle, respectively. B-lymphocytes stained for L-selectin exhibited about 50% reduction in L-selectin expression in BLV-infected cattle compared with BLV-free cattle, as judged by the mean fluorescence intensity (MFI). The percentage of L-selectin-positive PBMC not bearing surface immunoglobulin M (predominantly T lymphocytes) was comparable in BLV-free and BLV-infected cattle. However, L-selectin expression on T lymphocytes was reduced (about 50%) in BLV-infected cattle, as judged by the MFI. We suppose that BLV infection results in a decreased L-selectin expression on lymphocytes, and accordingly, it may contribute to deregulation of the host immune system.

Resveratrol, a natural stilbene in grapes and wine, enhances intraphagocytosis in human promonocytes: a co-factor in antiinflammatory and anticancer chemopreventive activity.

Trans-resveratrol, a natural stilbene present in wine and grapes, has been studied mainly for its antiinflammatory and anticancer activities. In this study the activity of resveratrol on proliferative immunological parameters (differentiation, apoptosis, phagocytosis and intracellular killing) was studied using a U937 human promonocytic cell line in comparison with another polyphenol, quercetin. After incubation of the pathogen, Candida albicans, intracellular killing by macrophage-like cells was decreased by quercetin and resveratrol 10 microM but was enhanced by resveratrol 1 microM after 20 h of treatment. Phagocytosis rate, expressed as phagocytosis frequency, (i.e., percentage number of phagocytosing cells/total cells) at 20 h was highest with resveratrol 10 microM and was higher with quercetin 10 microM than with resveratrol 1 microM. The phagocytosis index exhibited the same trend. While both polyphenols demonstrated cytostatic activity on U937 growth, a prointraphagocytic effect for resveratrol 10 microM-treated cells at 10 min, resveratrol 1 microM-treated cells at 20 h and resveratrol 10 microM-treated cells at 48 h was observed. Morphological examination with optic microscopy demonstrated both apoptotic and differentiating cells, even after 10 min treatment. Resveratrol-induced apoptosis (following 4 h treatment) was confirmed by flow cytometry at concentrations as low as 1 microM and 100 nM in the assay for detection of membrane phosphatidylserine. Resveratrol- or quercetin-treated, but unstimulated cells, did not produce tumor necrosis factor-alpha protein. As phosphatidylserine externalization triggers specific recognition by monocytes and macrophages, removal of intact apoptotic cells is important a) in cell population selection and differentiation for antiblastic therapy, and b) in preventing the release of toxic inflammatory substances such as reactive oxygen substances and proteolytic enzymes by dying cells. This observation suggests that wine polyphenols, at the same concentrations as those found in plasma after moderate wine consumption, are important cofactors in antiinfective, antiinflammatory and anticancer nonspecific immune reactions.

Expression of bovine leukemia virus ENV glycoprotein in insect cells by recombinant baculovirus.

The gp51-p30 glycoprotein constituting BLV envelope was expressed in Sf-21 insect cells by means of recombinant baculoviruses. Post-infection cell lysates were analyzed, in order to define the immunologic reactivity of recombinant products. Oligosaccharide chains, containing N-acetylglucosamine, mannose, galactose and sialic acid were found on recombinant gp51-p30. In order to investigate the timing of transcription and translation of the glycoprotein, kinetic assays were carried out on cell lysates and directly in situ on Sf-21 cells during the course of baculovirus infection. The use of different solubilizing reagents was also evaluated in order to rescue recombinant glycoprotein from its subcellular location.

Immune function alterations in mice tolerant to delta9-tetrahydrocannabinol: functional and biochemical parameters.

We studied the effect of acute (1 h) or chronic exposure (7 and 14 days) to delta9-tetrahydrocannabinol (delta9-THC) on immune parameters in male Swiss mice. One hour after a dose of 10 mg/kg s.c., the splenocyte proliferative response to ConA and NK activity were not inhibited, but there was a significant decrease in the production of IL-2. After 7 days of treatment, when mice were tolerant to delta9-THC-induced analgesia, these functional parameters were strongly inhibited and there was a persistent reduction in IL-2 and IFNgamma. With 14 days exposure to the drug, splenocyte proliferation was significantly reduced only with 5 microg/ml ConA, and NK activity was still significantly depressed (about 37%). IL-2 had returned to the control value, whereas IFNgamma was still 40% down. Flow cytometry analysis of spleen cell composition indicated no changes after the acute and 7 day treatments, but at 14 days there was a 20% decrease in the number of T lymphocytes, mirrored by a 26% increase of B lymphocytes. In conclusion, in vivo exposure to psychoactive doses of delta9-THC has profound effects on immune function. This implies some important questions in relation to the liberalization of marijuana and its therapeutic uses.

Association of BoLA class II haplotypes with subclinical progression of bovine leukaemia virus infection in Holstein-Friesian cattle.

The influence of bovine lymphocyte antigen (BoLA) complex polymorphism on subclinical progression of bovine leukaemia virus (BLV) infection was investigated in 41 Holstein-Friesian cows from two herds in Italy. All cows were seropositive for BLV and 22 had persistent lymphocytosis (PL). BoLA-A specificities were defined by serology, and class II haplotypes were defined based on restriction fragment length polymorphism (RFLP) and polymerase chain reaction (PCR)-RFLP analysis of DQ and DR genes. Chi-square analysis revealed a significant and absolute association of haplotype DQA*3A-DQB*3A-DRB2*2A-DRB3.2*11 with resistance to PL (P chi 2 = 0.028, relative risk (RR) = 0.061). Consistent with this observation, multiple regression analysis revealed that animals carrying this haplotype had lower lymphocyte counts (P = 0.0057). By contrast, haplotype DQA*12-DQB*12-DRB2*3A-DRB3.2*8 was associated with susceptibility to PL (P chi 2 = 0.043, RR = 9.625) and increased lymphocyte counts (P = 0.0537). These results confirm the association of haplotype DQA*3A-DQB*3A-DRB2*2A-DRB3.2*11 with resistance to PL, and substantiate earlier findings of haplotype DQA*12-DQB*12-DRB2*3A-DRB3.2*8 as a risk factor for subclinical progression to PL in BLV-infected Holstein-Friesian cattle.

Reactivity of monoclonal antibodies of the B cell panel on PBM from BLV-infected and lymphocytotic cows.

The monoclonal antibodies included in the B cell panel of the Third Workshop on Ruminant Leukocyte Antigens were tested by flow cytometry for their reactivity with peripheral blood mononuclear cells (PBM) from normal, BLV (bovine leukemia virus)-infected but non-lymphocytotic and lymphocytotic cows. Three MoAbs probably detected pan-B cell antigens. They detected an increase in the B/T cell ratio in peripheral blood from lymphocytotic cattle. However, no changes were observed in the surface phenotypes of B cells from infected animals with MoAbs of the workshop panel.

Surface analysis of IgM-expressing bovine lymphocytes in BLV-infected cattle by immuno-scanning electron microscopy.

Surface of IgM-expressing lymphocytes infected by bovine leukemia virus and with/without persistent lymphocytosis (BLV+PL-, BLV+PL+) in cattle is heterogenous. Three distinct morphological structures of B lymphocytes were found in these groups by immuno-scanning electron microscopy (ISEM). B lymphocytes with generally elongated and pleomorphic microvilli and sometimes short ruffles were observed in infected, and as well as in non-infected animals. A second morphological type endowed with a relatively small number of elongated villi-like veils, and a third morphological type characterized by a relatively small number of short microvilli or blunt stub-like microvilli covering the smooth surface were seen, mainly in BLV+PL+ and seldom in BLV+PL- Cells of the second type were more intensively labeled, than lymphocytes of the third type.