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.

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.

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.

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.

Seroepidemiological and clinical survey of feline immunodeficiency virus infection in northern Italy.

Four hundred and thirty-nine feline serum samples from cats with different living conditions in the north of Italy were tested for antibodies to feline immunodeficiency virus (FIV) and for antigen of Feline Leukemia Virus by enzyme-linked immunosorbent assay. A Western blot technique was also used on the positive sera in order to confirm the presence of specific antibodies to FIV. The Western blot enabled the detection of a false positive serum. The prevalence of FIV infection in this population was 12.5% and among the seropositive cats a greater proportion was male (74.5%) than female (25.5%). A correlation between the clinical status and the evolution of the pathology is described together with a score based on the severity of the stomatitis in infected cats. The Western blot patterns of positive samples were then compared with the stage of the pathology. Statistical analysis on the distribution of FIV in stray cats, cats with garden and courtyard access and strictly house-confined cats showed a highly significant risk of the infection in the first group.

Monoclonal antibodies recognized bovine leukocyte antigens used in immuno-scanning electron microscopy.

Antibovine leukocyte monoclonal antibodies were tested in order to use them for scanning electron microscopy in backscattered electron imaging mode. The tests revealed that numerous antibovine leukocyte monoclonal antibodies still recognize lightly glutaraldehyde prefixed antigens and can be used to identify various blood cell types. The results of these tests are presented and discussed. Clearcut differences in the surface morphology exist among bovine peripheral blood normal lymphocytes. Expression of IgM and IgG molecules was observed first of all on the surface of B lymphocyte microvilli.

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.

Aphidicolin inhibits in vitro the activity of pseudorabies virus (PRV) DNA polymerase and in vivo the viral proliferation.

We have purified and characterized Pseudorabies virus (PRV) DNA polymerase from infected TK- mouse cells. PRV DNA polymerase has a 3'- > 5' exonuclease activity; it is stimulated by ionic strength, requires magnesium for optimal activity and it is more sensitive to aphidicolin than eukaryotic and HSV-1 replicative DNA polymerases. Aphidicolin inhibits in vitro PRV DNA polymerase competitively with respect to dCTP with a Ki of 0.06 microM and completely blocks viral growth in vivo at 4.4 microM. The high sensitivity to aphidicolin of animal herpesvirus DNA polymerases might allow a topical use of this drug in the treatment of animal herpesvirus keratitis and stomatitis.

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.

Expression and quantification of IgG and IgM molecules on the surface of lymphocytes of cattle infected with bovine leukaemia virus.

Immunogold-labelled antibodies were used with scanning electron microscopy (SEM) and fluorescent-labelled antibodies were used with flow cytometry (FACS) to evaluate the expression and quantity of IgG and IgM molecules on the surface of the lymphocytes of cattle infected with bovine leukaemia virus (BLV). The BLV-infected animals were divided serologically and haematologically into groups with (BLV+PL+) and without (BLV+PL-) persistent lymphocytosis (PL). The percentage of IgM-bearing cells was significantly higher in the BLV+PL+ group than in the BLV-PL- and BLV+PL- groups by FACS. There was a significantly higher percentage of IgG-bearing cells in the BLV+PL+ group than in the BLV-PL- and BLV+PL- groups by SEM, but no differences were found by FACS. A significantly higher intensity of IgM expression was observed in the BLV+PL+ group by SEM. A higher intensity of IgG expression in some animals was detected only by SEM. An increase in the number of larger IgM cells were observed in the BLV+PL- and BLV+PL+ groups by SEM. The SEM analysis was more sensitive than FACS in this experiment.

Use of polymerase chain reaction to diagnose bovine leukemia virus infection in calves at birth.

A specific polymerase chain reaction (PCR) assay was devised, allowing detection of 1 bovine leukemia virus (BLV)-infected cell in 10(4) bovine lymphocytes. The efficacy of field application of the developed method was verified by evaluating the rate of viral transmission to calves from infected cows, whether they have persistent lymphocytosis. With this objective, 43 calves were simultaneously tested at birth and at 6 months of age for viral antibodies in serum and for proviral DNA in lymphocytes. At birth, 36 calves were BLV-negative and 3 were BLV-positive by results of serologic and DNA-based assays. Conversely, results for 4 calves had lack of correlation between the diagnostic methods. In particular, 2 calves were DNA-positive and antibody-negative for BLV and 2 other calves had the opposite test results. At 6 months of age, when the immunologic pattern more closely reflects the status of calves' immune response, independent of maternal antibodies, all calves DNA-negative for BLV at birth (n = 38), were consistently PCR- and antibody-negative for BLV. On the contrary, the cattle DNA-positive for BLV at birth (n = 5), whether seropositive or not, were PCR- and antibody-positive for BLV, at the time of the second screening. Thus, these results indicate reliability of the PCR to diagnose perinatal BLV infection. Furthermore, the observation that all calves found to be infected at birth were born to BLV-positive cows with persistent lymphocytosis, indicates that the persistent lymphocytosis status of the cow may represent a factor associated with BLV infection in utero.

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.

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.