Embryonic exposure to aflatoxin-B1: mutagenicity and influence on development and immunity

Chick embryos were mutagenized in ovo in order to study developmentally related alterations in immune functions in survivors of this prenatal toxicant insult. In this experimental system, a single exposure of 6-day chick embryos to 0.1 microgram aflatoxin-B1 (AF-B1) in 10 microliters of acetone was employed, and the control embryos received 10 microliters of solvent alone. This dosage of AF-B1 administered to 6-day embryos was found to increase the incidence of sister chromatid exchanges in blood cells approximately fivefold above the baseline observed in solvent controls. A second sham control, where no solvent was administered, was included in some experiments. The cell cycle times in blood increased slightly during the initial exposure to AF-B1. However, a majority of the AF-B1 and acetone exposed embryos survived and hatched without incident. Losses occurred mainly in the latter part of embryogenesis. After hatching, no significant differences were observed in body weight between different treatment groups up to 26 weeks of age and no change in primary humoral immunity was detected. In contrast, two parameters of cell-mediated immunity, graft vs host (GvH), and cutaneous basophil hypersensitivity (CBH) reactions were both depressed as a result of exposure to AF-B1. The AF-B1 treatment group was significantly reduced in the GvH reaction compared with sham-treated controls. In the CBH assay, AF-B1-exposed chicks showed reduced immunity compared with acetone controls. These results suggest that long-term selective immune depression can occur following embryonic exposure to AF-B1.     

Early-onset inflammatory responses in vivo to adenoviral vectors in the presence or absence of lipopolysaccharide-induced inflammation.

Adenoviral vectors (Ad) have potential for use in pulmonary gene transfer for treating cystic fibrosis (CF). However, Ad may induce inflammation even in the absence of gene expression. Endotoxin from gram-negative bacteria in the airways of CF patients may also induce inflammation, and may further inhibit vector delivery and gene transfer. We used a mouse model to study the time course of Ad-induced lung inflammation and to assess additivity with lipopolysaccharide (LPS)-induced inflammatory responses. C3H/HeJ endotoxin-resistant (RES) mice hyporesponsive to inflammatory stimuli and normoresponsive C3HeB/FeJ endotoxin-sensitive (SEN) mice were studied to characterize inflammatory responses that follow intratracheal instillation of inactivated Ad, with or without simultaneous inhalation exposure to LPS. Instillation of 10(10) Ad particles dramatically increased bronchoalveolar lavage fluid (BALF) concentrations of tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 at 3 to 6 h and induced profound neutrophilia, maximal at 12 to 24 h. SEN mice had tenfold greater responses than did RES mice at 6, 12, and 24 h. Mice exposed to Ad alone, LPS alone, or Ad + LPS had significant inflammation at the 3-h time point as demonstrated by BALF neutrophils, TNF-alpha, and IL-6. With all three treatments, SEN mice had a five- to 300-fold greater response than did RES mice. Importantly, Ad + LPS yielded no greater inflammatory response than LPS without Ad. These data demonstrate that replication-deficient Ad induce early inflammation and LPS-induced inflammation is not augmented by concurrent treatment with Ad.     

Heterologous immunity in the absence of variant-specific antibodies after exposure to subpatent infection with blood-stage malaria

We examined immunity induced by subpatent blood-stage malaria (undetectable by microscopy) using the rodent malaria parasite, Plasmodium chabaudi chabaudi, postulating that limited infection may allow expansion of antigen-specific T cells that are normally deleted by apoptosis. After three infections drug cured at 48 h, mice were protected against high-dose challenge with homologous or heterologous parasites (different strain or variant). Immunity differed from that generated by three untreated, patent infections. Subpatently infected mice lacked immunoglobulin G (IgG) to variant surface antigens, despite producing similar titers of total malaria-specific IgG to those produced by patently infected mice, including antibodies specific for merozoite surface antigens conserved between heterologous strains. Antigen-specific proliferation of splenocytes harvested prechallenge was significantly higher in subpatently infected mice than in patently infected or naive mice. In subpatently infected mice, lymphoproliferation was similar in response to homologous and heterologous parasites, suggesting that antigenic targets of cell-mediated immunity were conserved. A Th1 cytokine response was evident during challenge. Apoptosis of CD4+ and CD8+ splenic lymphocytes occurred during patent but not subpatent infection, suggesting a reason for the relative prominence of cell-mediated immunity after subpatent infection. In conclusion, subpatent infection with blood stage malaria parasites induced protective immunity, which differed from that induced by patent infection and targeted conserved antigens. These findings suggest that alternative vaccine strategies based on delivery of multiple parasite antigens at low dose may induce effective immunity targeting conserved determinants.     

Mucosal penetration of antigen in the presence or absence of serum-derived antibody: An in vitro study of rabbit oral and intestinal mucosa

Rabbit mucous membranes were mounted in diffusion chambers within 30 min after excision. The epithelial side was exposed to cell culture medium containing `cold' and ¹²⁵I-labelled human albumin. The basal side was exposed to normal rabbit serum (control chambers) or to rabbit anti-serum against human albumin. The chambers were incubated in a humidified atmosphere of CO₂ in air for 2–3 h at 37°. The radioactive material recovered on the basal side of the sublingual control membranes sedimented virtually like native albumin on ultracentrifugation. The amount of radioactive material recovered after penetration through anti-serum-exposed sublingual mucosa was reduced by 50–80% and showed a very heterogeneous sedimentation pattern including aggregates, presumably immune complexes, as well as a considerable amount of degradation products.

In a second series of experiments the concurrent penetration of human albumin and transferrin through the sublingual mucosa of rabbits immunized parenterally with albumin was compared with that occurring through control membranes. With reference to immunochemical quantification, scintillation counting was found to overestimate the penetration of intact albumin considerably, and jeopardized evaluation of the influence of serum-derived antibody. Radial immunodiffusion showed that in controls the basal antigen concentration, expressed in percentage of the oral (30 mg/ml for both molecules), was after 2 h 0.0032±0.0023 for albumin and 0.0016±0013 for transferrin. Penetration of immunoreactive albumin through mucosa from immunized animals was clearly reduced (0.0007±0.0003), whereas there was a significant tendency toward increased penetration of transferrin (0.0035±0.0023). These results suggest that antibody within the mucosa retards the penetration of intact homologous antigen, while immune reactions may enhance the penetration of unrelated macromolecules.

In similar experiments with colon mucosa penetration was 50–100 times increased, but the membranes did not discriminate between albumin and transferrin and there was no effect of immunization. Histological and immunohistochemical studies of the latter membranes indicated marked defects in cell viability after 2 h in vitro.

     

Transfer of maternal specific cell-mediated immunity to the fetus.

The extent of specific cell-mediated immunity was measured in 67 consecutive newborns and their mothers. The stimulation index of blast transformation of the infants' lymphocytes in the presence of purified protein derivative, Candida extract and streptokinase was greater than 2.0 in 54%, 18% and 23% respectively. This was seen only in infants whose mothers' index was also greater than 2.0 to the same antigen. Leucocyte inhibition factor generated from lymphocytes of four babies in the presence of purified protein derivative inhibited migration of indicator cells over 50%; their stimulation index with purified protein derivative was greater than 2.0. Newborns have cell mediated immunity to the same antigens as their mothers, and this wanes during the first few months of life.     

Mucosal immunogenicity of a recombinant Salmonella typhimurium-cloned heterologous antigen in the absence or presence of coexpressed cholera toxin A2 and B subunits.

An avirulent Salmonella typhimurium vaccine strain expressing a streptococcal protein adhesin and a similar clone which produces the same streptococcal antigen linked to the cholera toxin (CT) A2 and B subunits (CTA2/B) were compared for the ability to induce antibody responses to the expressed heterologous antigen after oral or intranasal immunization of mice. Expression of cloned immunogens in these systems is temperature regulated, being optimal at 37 degrees C, and the two clones under comparison were shown to produce similar levels of the streptococcal antigen. Both clones were found to stimulate high levels of serum immunoglobulin G (IgG) and mucosal IgA antibodies to the cloned immunogen. A consistent trend was observed toward higher mucosal IgA but lower serum IgG responses in the case of the S. typhimurium vector that coexpressed CTA2/B, a potential mucosal adjuvant, regardless of the route of administration. Also noteworthy was the capacity of these antigen delivery systems to induce anamnestic systemic and secretory responses to the cloned immunogen 15 weeks after the primary immunization, despite preexisting immunity to the Salmonella vectors. These antibody responses were sustained for at least 7 months following the booster immunization, at which time the secretory IgA antibody levels were significantly higher in mice given the Salmonella clone that coexpressed CTA2/B. Although the serum IgG response against the Salmonella vector was characterized by a high IgG2a/IgG1 ratio (indicative of the T helper type 1 [Th1]/Th2 profile), a mixed IgG1 and IgG2a pattern was observed for the carried heterologous antigen, which displayed a dominant IgG1 response when administered as a purified immunogen. Our findings indicate that the recombinant streptococcal antigen and CTA2/B are strong immunogens when expressed by the antigen delivery system used in this study and suggest that CTA2/B may have an additional immunoenhancing activity in the mucosal compartment besides its ability to target antigen uptake into the mucosal inductive sites. CTA2/B may thus be useful as an S. typhimurium-cloned adjuvant for coexpressed protein antigens.     

Alkaline-treated poly(epsilon-caprolactone) films: degradation in the presence or absence of fibroblasts

In the first stage, we observed the study of the degradation behavior of alkaline-treated poly(epsilon-caprolactone) (PCL) in two biologically-related media: phosphate buffered saline (PBS) and Dulbecco's modified Eagle's medium (DMEM) for 18 months, finding a much accelerated degradation in the last one. As expected, the degradation in the presence of cells is much pronounced even considering that the study is limited to 6 months. The characterization of the degraded substrates by chemiluminescence (CL) allows to explain the modifications of the substrate and their relations with transitory oxidative stress phenomena described in the fibroblasts seeded onto the PCL membranes.     

Neutralization or absence of the interleukin-23 pathway does not compromise immunity to mycobacterial infection

Interleukin-23 (IL-23), a member of the IL-12 family, is a heterodimeric cytokine that is composed of the p40 subunit of IL-12 plus a unique p19 subunit. IL-23 is critical for autoimmune inflammation, in part due to its stimulation of the proinflammatory cytokine IL-17A. It is less clear, however, if IL-23 is required during the immune response to pathogens. We examined the role of IL-23 during Mycobacterium bovis BCG infection. We found that IL-23 reduces the bacterial burden and promotes granuloma formation when IL-12 is absent. However, IL-23 does not contribute substantially to host resistance when IL-12 is present, as the ability to control bacterial growth and form granulomata is not affected in IL-23p19-deficient mice and mice treated with a specific anti-IL-23p19 antibody. IL-23p19-deficient mice are also able to mount an effective memory response to secondary infection with BCG. While IL-23p19-deficient mice do not produce IL-17A, this cytokine is not necessary for effective control of infection, and antibody blocking of IL-17A in both wild-type and IL-12-deficient mice also has little effect on the bacterial burden. These data suggest that IL-23 by itself does not play an essential role in the protective immune response to BCG infection; however, the presence of IL-23 can partially compensate for the absence of IL-12. Furthermore, neutralization of IL-23 or IL-17A does not increase susceptibility to mycobacterial BCG infection.     

The accuracy of Masson's trichrome stain in predicting the presence or absence of glomerular immune complexes

The accuracy of Masson's trichrome stain to predict the presence of immune complexes was determined in 63 renal biopsies. When immunofluorescence was defined as the reference method, the histologic method correctly predicted the presence or absence of deposits in 70% of biopsy specimens, while electron microscopy was accurate in 79% of specimens. This difference was not statistically significant. The commonest error in our assessment of the Masson's trichrome-stained specimens was the erroneous interpretation of specimens showing minimal change nephropathy or ischemic glomerulopathy by immunofluorescence and electron microscopy. This resulted in a falsely positive diagnosis of one or another of the glomerulonephritides in 13% of cases. Thus, the routine study of renal biopsies with Masson's trichrome stain is clearly useful and should be applied with caution, but it does not replace electron-microscopic and immunofluorescence studies.     

The influence of low doses 131I-induced maternal hypothyroidism on the development of rat embryos.

The aim of our research was to create and verify a model for studying the effects of a low dose of 131I and 131I-induced maternal hypothyroidism on the development of the embryo's thyroid gland and brain. The given dose (150 microCi) corresponds to the absorbed dose of 0.5 Gy. This dose is similar to that dose received by large numbers of the population of the C.I.S. regions polluted by radioactive isotopes of iodine as a result of the Chernobyl accident in 1985. Thirty-five female Wistar rats and their 168 newborn pups were used for observation. The females were divided into a control group and four experimental groups (each distinguished by the time of 131I injection: group I - no less than 12 days before mating; groups II, III and IV - on 5th, 10th and 16th days of gestation, respectively). In all the experimental female groups the incorporate dose of 131I led to hypothyroidism accompanied by a 43% reduction in the thyroxin level and by a nearly 8-fold increase in the TSH level. However, the influence of maternal hypothyroidism on the development of the thyroid gland and brain of embryos depends on the time when 131I took effect. There is a reduction in the weight of the newborns' brain and thyroid gland, total body mass. The hormonal status of the newborns' thyroid gland also changes. The proposed model will allow us to study many aspects of induced changes in the brain and thyroid gland of the embryos which develop under conditions of maternal hypothyroidism resulting from a low dose of 131I, administered at the critical times of development.     

The influence of dietary protein antigen on Th1/Th2 balance and cellular immunity.

Dietary administration of ovalbumin (OVA) antigen (Ag) into OVA-specific T cell receptor alphabeta-transgenic (TCR-Tg) mice resulted in the induction of activated CD4+ Th cells expressing CD69 early activation Ag. However, the number of CD4+ Th cells rather decreased by dietary administration of OVA antigen. The production of Th1-cytokines such as IFN-gamma and IL-2 markedly reduced in spleen of OVA-fed mice compared to mice fed with normal diet. In sharp contrast, the production of Th2-cytokine, IL-4 greatly increased in spleen of OVA-fed mice though the number of CD4+ T cells decreased to less than 10% of control mouse spleen. The decrease of IFN-gamma production and the increase of IL-4 production by CD4+ T cells was demonstrated at a single cell level by intracellular cytokine staining analysis. Moreover, such a polarized cytokine production pattern was also demonstrated using highly purified CD4+ T cells obtained from mice fed with OVA. In addition to the decrease of Th1-cytokine production, TCR-Tg mice fed with OVA-containing diet showed greatly reduced in vivo generation of NK cells, LAK cells and CTL. These results suggested that dietary protein antigen caused the polarization of Th1/Th2 balance into Th2-dominant immunity and inhibited cellular immunity.     

The demonstration of cell-associated immunity to viruses. In vitro lymphocyte responsiveness to Varicella-zoster antigen.

The demonstration of in vitro lymphocyte responsiveness to common pediatric viruses has previously been fraught with many technical and conceptual problems. Based upon our prior experience in demonstrating cell-associated immunity to mumps, rubella and measles viruses we illustrate our methodology and conceptual framework by documenting in vitro lymphocyte responsiveness to the Varicella-zoster virus, another ubiquitous virus of childhood. We discuss our approach to the problems of reactivity, specificity and reliability in the use of membrane-associated viral antigens.     

The effects of prenatal exposure to predictable or unpredictable stress on early development in the rat

Pregnant rats were exposed to different schedules of noise and light stress, and the development of their offspring was studied during the first 2 weeks of life. Motor development was measured by different tests: Righting reflex (Days 2-4); cliff avoidance (Days 4-10); turning on an inclined plane (Days 5-10); and swimming behavior (Days 6-10). Development of motivation-involved behavior was measured with a home-seeking test (Days 6-16). Other developmental landmarks such as acoustic response (Days 12-14) and eye-opening (Days 14-17) were also recorded. Thrice-weekly random stress resulted in a delay in the development of all behaviors studied. Daily stress exposure throughout pregnancy resulted in smaller litters with heavier pups, but otherwise normal behavioral and physical development. Rats exposed to daily stress during the last week of pregnancy only produced litters that did not differ in size and body weight, but that displayed accelerated development of all parameters (except for eye-opening) from Day 6 onwards. It is concluded that the unpredictable nature of prenatal stress is responsible for delays in behavior of offspring.     

Regulation of experimental autoimmune orchitis by the presence or absence of testicular antigens during immunological development in SCID mice reconstituted with fetal liver cells.

Severe combined immunodeficient (SCID) mice were immunologically reconstituted by the transfer of fetal liver cells (FLC) of BALB/c mice (SCID-FLC mice). In peripheral blood (PB) of SCID-FLC mice, B and T cells started to appear 2 and 5 weeks, respectively, after the transfer of FLC, and had attained normal levels by 7 weeks. Orchidectomy and transplantation of testis under the kidney capsule were conducted at various stages of immunological maturation, and the induction of experimental autoimmune orchitis (EAO) was performed after immunological maturation in SCID-FLC mice. The experimental system was used to establish that the presence of testicular antigens in the early stage of immunological development influences the induction of EAO; grade of EAO was reduced in the presence of the antigens, and enhanced in their absence. In other words, the existence of self tissue antigens in the early stage of immunological development was essential for proper establishment of tolerance to the self tissue. These findings suggested that the SCID-FLC mouse is a suitable model with which to analyse the interaction between self antigens and cells of the developing immune system, which is otherwise observed only in the fetal or perinatal stage in experimental animals.     

Immune responses of mice to orally administered asialo GM1-specific rabbit IgG in the presence or absence of cholera toxin.

Cholera toxin (CT) has been shown to be a most potent mucosal immunogen and an adjuvant to orally administered unrelated antigens. We investigated the effect of the oral administration of substances with the ability to bind to intestinal epithelial cells on the immune responses against themselves in the presence or absence of CT. Mice were fed non-specific rabbit IgG (RGG) or rabbit IgG (a-GA1) specific to asialo GM1 glycolipid, a major component of the apical membrane of mouse small intestinal epithelial cells, with or without CT. Oral administration of a-GA1 evoked stronger antibody responses than that of RGG in both the serum and intestinal fluid in the presence of CT. However, when the antigens were administered singly without CT, no significant antibody response was detected. In this case, oral administration of RGG induced severe suppression of the systemic antibody response to a subsequent intraperitoneal injection of RGG. In contrast, a-GA1 could not induce oral tolerance. Together these findings suggest that substances with the ability to bind to intestinal epithelial cells are strong immunogens in the presence of CT and weak tolerogens in the absence of CT.