Mesenteric Lymph Node Cell Cultures Research Articles

Effect of high pressure-assisted crosslinking of ovalbumin and egg white by transglutaminase on their potential allergenicity

Abstract Transglutaminase (TG) modifies proteins by amine incorporation, crosslinking and deamination. It is used as an important tool in food processing. The aim of this work was to crosslink ovalbumin (OVA) and egg white (EW) with TG, either under high pressure (HP) or under atmospheric pressure on previously HP-treated proteins, to assess the allergenic potential of polymerized allergens. The susceptibility of OVA and EW to TG-mediated crosslinking was enhanced by HP (400 MPa, 40 °C, 30 min). Simultaneous or sequential HP and TG treatments led to the formation of high-molecular weight polymers, but left a substantial amount of monomeric proteins. Results of digestibility experiments, binding to immunoglobulin E (IgE) from egg allergic patients and stimulation of spleen and mesenteric lymph node cell cultures of EW-sensitized BALB/c mice showed that resistance to proteolysis, IgE-binding and immunostimulatory properties of the HP and TG-treated proteins were not substantially different from those of the native ones. Industrial Relevance The use of transglutaminase as a crosslinking agent has been suggested for many industrial applications, as in the case of meat, fish, milk or soy proteins, which are good substrates for this enzyme. This work widens the possible use of transglutaminase, for modifying less suitable substrates for crosslinking, as egg proteins, through the use of high pressure before or during crosslinking. In our study, HP-assisted TG-induced OVA and EW crosslinking enhanced gastroduodenal digestion but did not reduce the allergenicity of egg proteins.

Perinatal maternal administration of Lactobacillus paracasei NCC 2461 prevents allergic inflammation in a mouse model of birch pollen allergy.

BackgroundThe hygiene hypothesis implies that microbial agents including probiotic bacteria may modulate foetal/neonatal immune programming and hence offer effective strategies for primary allergy prevention; however their mechanisms of action are poorly understood. We investigated whether oral administration of Lactobacillus paracasei NCC 2461 to mothers during gestation/lactation can protect against airway inflammation in offspring in a mouse model of birch pollen allergy, and examined the immune mechanisms involved.MethodsBALB/c mice were treated daily with L. paracasei in drinking water or drinking water alone in the last week of gestation and during lactation. Their offspring were sensitized with recombinant Bet v 1, followed by aerosol challenge with birch pollen extract.ResultsMaternal exposure to L. paracasei prevented the development of airway inflammation in offspring, as demonstrated by attenuation of eosinophil influx in the lungs; reduction of IL-5 levels in bronchoalveolar lavage, and in lung and mediastinal lymph node cell cultures; and reduced peribronchial inflammatory infiltrate and mucus hypersecretion. While allergen-specific IgE and IgG antibody levels remained unchanged by the treatment, IL-4 and IL-5 production in spleen cell cultures were significantly reduced upon allergen stimulation in offspring of L. paracasei treated mice. Offspring of L. paracasei supplemented mothers had significantly reduced Bet v 1-specific as well as Concanavalin A-induced responses in spleen and mesenteric lymph node cell cultures, suggesting the modulation of both antigen-specific and mitogen-induced immune responses in offspring. These effects were associated with increased Foxp3 mRNA expression in the lungs and increased TGF-beta in serum.ConclusionOur data show that in a mouse model of birch pollen allergy, perinatal administration of L. paracasei NCC 2461 to pregnant/lactating mothers protects against the development of airway inflammation in offspring by activating regulatory pathways, likely through TLR2/4 signalling.

Neonatal colonization of mice with Lactobacillus plantarum producing the aeroallergen Bet v 1 biases towards Th1 and T-regulatory responses upon systemic sensitization

The use of recombinant lactic acid bacteria (LAB) as vehicles for mucosal delivery of recombinant allergens is an attractive concept for antigen-defined allergy prevention/treatment. Interventions with LAB are of increasing interest early in life when immune programming is initiated. Here, we investigated the effect of neonatal colonization with a recombinant LAB producing the major birch pollen allergen Bet v 1 in a murine model of type I allergy. We constructed a recombinant Lactobacillus (L.) plantarum NCIMB8826 strain constitutively producing Bet v 1 to be used for natural mother-to-offspring mono-colonization of germ-free BALB/c mice. Allergen-specific immunomodulatory effects of the colonization on humoral and cellular immune responses were investigated prior and after sensitization to Bet v 1. Mono-colonization with the Bet v 1 producing L. plantarum induced a Th1-biased immune response at the cellular level, evident in IFN-γ production of splenocytes upon stimulation with Bet v 1. After sensitization with Bet v 1 these mice displayed suppressed IL-4 and IL-5 production in spleen and mesenteric lymph node cell cultures as well as decreased allergen-specific antibody responses (IgG1, IgG2a, and IgE) in sera. This suppression was associated with a significant up-regulation of the regulatory marker Foxp3 at the mRNA level in the spleen cells. Intervention at birth with a live recombinant L. plantarum producing a clinically relevant allergen reduces experimental allergy and might therefore become an effective strategy for early intervention against the onset of allergic diseases.

VIP balances innate and adaptive immune responses induced by specific stimulation of TLR2 and TLR4

Crohn's disease (CD) is a chronic intestinal inflammatory pathology, which develops as a result of innate immune signals, such as the activation of Toll-like receptors (TLRs), and adaptive immune signals, including Th1 cytokine release. We have recently demonstrated in TNBS-induced colitis, a murine model of CD, that VIP plays a homeostatic role, by reducing TNBS-induced TLR2 and TLR4 expression to control levels. The purpose of this paper is to elucidate for the first time, the physiological relevance of VIP specific control of innate and adaptive immune responses through TLR2 and TLR4 ligands. In addition, we investigated the effect of VIP on regulatory activity of T regulatory (Treg) cells in the TNBS-colitis model. First, we found that VIP downregulated the inflammatory response elicited in mesenteric lymph node cell cultures by treatment with the TLR2 ligand Pam3Cys, or the TLR4 ligand lipopolysaccharide (LPS), reducing the production of the chemokine CXCL1. Also, treatment with VIP impaired the induction of Th1 responses by decreasing p70 interleukin (IL)-12 and interferon gamma (IFN-gamma) levels after TLR2/TLR4 stimulation in culture. Besides, VIP treatment restored in vivo the numbers of TLR2 and TLR4 positive CD4+CD25+ T lymphocytes, augmented by TNBS administration, and increased the expression of molecules involved in regulatory T cell function, such as Foxp3 and TGF-beta. In conclusion, the ability of VIP to down-regulate uncontrolled inflammation by targeting TLR-mediated responses and regulatory T cell activity could be used as a new alternative therapy for intestinal inflammatory/autoimmune disorders.

Immunostimulatory potential of smokeless tobacco extract in in vitro cultures of murine lymphoid tissues.

Despite numerous studies on the general toxicologic effects of smokeless tobacco (ST) little immunotoxicologic information is available. As a first step in assessing the potential activity of ST on the immune system, the effects of an aqueous extract of ST was studied in in vitro cultures of mouse lymphoid cells. There was a significant increase in the proliferation of spleen cells cultured with different concentrations of ST extract. The polyclonal IgM antibody responses as determined by protein A plaque assay were also elevated in ST stimulated spleen cell cultures. Similar immunostimulatory results were seen in the mesenteric lymph node cell cultures also. ST extract was able to stimulate the spleen cells of the immune defective CBA/N mice. The mitogenic ability of ST extract may not be due to lipopolysaccharide (LPS) contamination as determined by its response in the LPS resistant C3H/HeJ mice spleen cells. ST extract was mitogenic not only to B cells but also to T cells. However the magnitude of response was less in T cells than in B cells. The proliferation of T cells was not accompanied by secretion of IL-2 or expression of IL-2 receptors on T cells. However there was an increase of IL-1 activity in spleen cells cultured with ST extract. Finally, activation of B or T lymphocytes by ST did not result in the elevation of intracellular calcium levels. Since ST is consumed orally, the chronic immunostimulation by ST in oral mucosal lymphoid tissues may be associated with the increased incidence of gingivitis, leukoplakia and oral cancer seen in human ST users.

Effect of ricin on the proliferation of rat spleen and mesenteric lymph node cells.

The effect of the castor bean toxin, ricin, on rat spleen and mesenteric lymph node cell cultures was tested. Rat lymphocytes were cultured for 2 days in the presence of optimal doses of the mitogens phytohaemagglutinin, conconavalin A, and lipopolysaccharide. At concentrations of 10(-3) to 10(-5) mg/ml ricin, spontaneous and mitogen-driven proliferation were inhibited. At lower concentrations, spleen but not mesenteric lymph node cells showed enhanced proliferation. Studies are continuing to identify subpopulations of lymphocytes with increased or decreased sensitivity to ricin and their role in the regulation of IgE.

Analysis of suppressor factor in delayed immune responses of a bat, [formula omitted][formula omitted

Bat spleen and mesenteric lymph node cell cultures treated with varying doses of LPS showed significant blastogenic and DNA synthetic responses between 72–96h. Peak responses were not different when the cell culture medium was supplemented either with autologous serum or heterologous serum indicating the absence of any significant suppressor factor in autologous bat serum. In contrast, blastogenesis and DNA synthesis peaks appeared early, at 48h, in lymphocytes depleted of suppressor T cells by pretreatment with cyclophosphamide. Direct antibody producing cells against SRBC were studied in normal spleen and mesenteric lymph node cell cultures. The peak PFC response was lacking even at 120h while CY pretreated bat lymphocytes, showed peak PFC responses at 96h. Thus the delayed immune response in bats seems to be a function of suppressor T cells but serum suppressor factor(s) possibly exerts no significant effect. The function of suppressor cells in bats in relation to their role as carriers of several dreaded bacteria and viruses is discussed.

Giardia muris-induced depression of the primary immune response in spleen and mesenteric lymph node cell cultures to sheep red blood cells.

Primary in vitro plaque-forming cell (PFC) responses to sheep red blood cells (SRBC) were examined for spleen and mesenteric lymph node (MLN) cell populations from susceptible (A/J) and resistant (B10.A) mice during the infection with Giardia muris. Spleen and MLN cells isolated from mice during the acute phase of the infection were less responsive to SRBC in vitro than those from uninfected mice. Depressed anti-SRBC PFC response was detected earlier and was more pronounced in MLN cell cultures when compared to the response of spleen cell cultures. Spleen and MLN cells from donors infected with G. muris for 15 days had the capacity of depressing PFC response to SRBC of cells isolated from uninfected mice. This suppressor activity was localized in the plastic-adherent fraction of spleen cell populations isolated from A/J and B10.A mice. Since G. muris is a gastro-intestinal infection of mice, lower capacity of the MLN cells to respond to an antigenic stimulation in vitro may explain, in part, the proliferation of the trophozoites during the acute phase of the infection.

In vitro development of a primary antibody response with lymph node cells

Utilizing a variety of lymphoid tissues from three common laboratory species, comparative studies were performed to investigate the competence of the dissociated cells to respond to a heterologous erythrocyte with the development of specific plaque-forming cells. Dissociated spleen cells harvested from BDF1 mice consistently developed specific plaque-forming cells (PFC) to sheep red blood cells (SRBC), while hamster spleen cells inconsistently developed specific antibody-forming cells to SRBC. Under identical conditions, guinea pig spleen cells did not develop significant numbers of PFC to SRBC. However, lymph node cell cultures of all three species tested yielded specific PFC. In the mouse and hamster lymph node cell cultures, the yield of PFC per culture or per 10 6 recovered viable cells was always greater than the yield from companion spleen cell cultures. Guinea pig mesenteric lymph node cell cultures developed the major PFC response to SRBC, while both mesenteric and peripheral lymph node cell cultures from hamsters were equivalent in their response to SRBC. The data demonstrate that it is possible to develop a primary antibody response to SRBC in vitro utilizing normal endogenous hamster or guinea pig lymphoid cells, if lymph nodes are the source of cells.

IgE-B Cell-Generating Factor from Lymph Node Cells of Rats Infected with Nippostrongylus Brasiliensis

Abstract Culture supernatants of mesenteric lymph node cells obtained from Nippostrongylus brasiliensis (Nb)-infected rats have the ability to increase the proportion of IgE-bearing cells in normal bone marrow cell cultures. In order to determine the cell source of the active substance, mesenteric lymph node cells and thoracic duct cells from Nb-infected rats were fractionated by various methods, and culture supernatants of each fraction were examined for the ability to generate IgE-bearing cells in normal bone marrow cell cultures. It was found that culture supernatants of the B cell-enriched fraction were substantially more active than those of the unfractionated cells or T cell-enriched fraction. Residual activity in the culture supernatant of the T cell-enriched fraction was eliminated by prior removal of contaminating immunoglobulin (Ig)-bearing cells in the fraction. The mesenteric lymph node cells depleted of IgE-bearing cells by rosette technique failed to produce IgE-B cell-generating factor. The results indicated that a major source of the soluble factor is IgE-bearing B cells. This conclusion was supported by the fact that treatment of mesenteric lymph node cells with antibodies specific for IgE accelerated the formation of IgE-B cell-generating factor in culture fluids. Formation of IgE-B cell-generating factor in culture fluids. Formation of IgE-B cell-generating factor by the mesenteric lymph node cells was reduced by pretreatment of the cells with pactamycin but not with mitomycin C, indicating that DNA synthesis is not required for the formation of the factor. The appearance of IgE-B cell-generating activity in the culture fluids of mesenteric lymph node cell cultures was inhibited in the presence of dibutyryl cyclic AMP or aminophilline but not by cyclic GMP.