Treatment of experimental autoimmune encephalomyelitis by feeding myelin basic protein conjugated to cholera toxin B subunit.

Oral administration of autoantigens can prevent and partially suppress autoimmune diseases in a number of experimental models, Depending on the dose of antigen fed, this approach appears to involve distinct yet reversible and short-lasting mechanisms (anergy/deletion and suppression) and usually requires repeated feeding of large (suppression) to massive (anergy/deletion) amounts of autoantigens to be effective. Most importantly, this approach is relatively less effective in animals already systemically sensitized to the fed antigen, such as in animals already harboring autoreactive T cells and, thus, presumably also in humans suffering from an autoimmune disorder. We have previously shown that feeding a single dose of minute amounts of antigens conjugated to cholera toxin B subunit (CTB) can effectively suppress delayed-type hypersensitivity reactions in systemically immune animals. We now report that feeding small amounts of myelin basic protein (MBP) conjugated to CTB either before or after disease induction protected rats from experimental autoimmune encephalomyelitis. Such treatment was as effective in suppressing interleukin 2 production and proliferative responses of lymph node cells to MBP as treatment involving repeated feeding with much larger (50- to 100-fold) doses of free MBP. Different from the latter treatment, which led to decreased production of interferon-gamma in lymph nodes, low-dose oral CTB-MBP treatment was associated with increased interferon-gamma production. Most importantly, low-dose oral CTB-MBP treatment greatly reduced the level of leukocyte infiltration into spinal cord tissue compared with treatment with repeated feeding of large doses of MBP. These results suggest that the protection from experimental autoimmune encephalomyelitis achieved by feeding CTB-conjugated myelin autoantigen involves immunomodulating mechanisms that are distinct from those implicated by conventional protocols of oral tolerance induction.     

Cholera toxin B subunit: an efficient transmucosal carrier-delivery system for induction of peripheral immunological tolerance.

Oral administration of antigens, including allergens and autoantigens, may be an efficient way to prevent diseases associated with untoward immune responses to self- and non-self-antigens. However, this approach has met with limitations because it usually requires repeated administrations of large doses of antigen and is less efficient in an already immune host, and the effect is of short duration. We report that a single oral administration of minute amounts of particulate or soluble antigen coupled to the B subunit of cholera toxin (CTB) can markedly suppress systemic immune responses in naive and in systemically immune animals. Both early (2-4 hr) and late (24-48 hr) delayed type-hypersensitivity reactivities were strongly suppressed after feeding a single dose of CTB-conjugated antigen. Serum antibody responses were also decreased, although moderately, after oral administration of CTB-conjugated antigen. This strategy of tolerance induction, based on oral administration of small amounts of antigens conjugated to a mucosa-binding molecule, may find broad applications for preventing or abrogating untoward immune responses.     

Mechanisms and applications of oral tolerance

The term oral tolerance (OT) describes the antigen-specific suppression of immune responses following the feeding of the antigen. While some common features with other forms of induction of systemic tolerance have been disclosed, OT can be distinguished by certain immunologic characteristics. Thus, work in experimental animal models revealed the importance of intestinal antigen processing, especially antigen processing in the Peyer's patches, in inducing OT. It has become clear that suppressive T cell cytokines derived from mucosal sites play a major role in mediating OT. The variation of the dose of fed antigen and the modulation of the cytokine milieu both have influences on the underlying immunologic mechanisms active suppression, clonal anergy and clonal deletion following oral antigen uptake. In several animal models of autoimmunity the disease activity can be suppressed by feeding oral autoantigen. Based on these results, recent clinical studies have begun to explore OT as a means to treat autoimmune disorders such as multiple sclerosis, rheumatoid arthritis and diabetes.     

Timing of introduction of gluten and celiac disease risk

Breast milk is the natural nutrition for infants, but in the second half of the first year of life, complementary feeding is needed. Many complementary foods contain gluten, but gluten exposure is associated with the risk of developing celiac disease (CD). CD is a disease with considerable morbidity and mortality. Although CD is associated with certain genetic features, carrying the human leukocyte antigen haplotypes DQ2 or DQ8 (a prerequisite for CD development) cannot fully explain who will or who will not develop CD. Potential risk factors for CD include perinatal events and infant feeding practice. With the exception that children who are breastfed at and beyond gluten introduction into the diet probably may be at a lower risk of developing CD, and that heavy gluten load early in life may increase the risk of future CD, data on the impact of infant feeding are inconsistent.     

Isolation and characterization of salivary antigens from the female tick, Dermacentor andersoni

The salivary glands of ixodid ticks are complex organs which are known to contain the antigens responsible for tick resistance in animals. We have identified a large number of proteins from salivary gland extracts (SGE), at least some of which are immunologically recognized by tick resistant animals and which are therefore presumed to be secreted salivary components. During the 6 to 10 day feeding process, a number of these antigens alter in concentration according to individual kinetics, and some of these changes correlate with the kinetics of skin test reactivity of SGE obtained at different times throughout the feeding period. By use of immunoaffinity chromatography we have isolated large quantities of many of the salivary antigens (SGA) contained in SGE, and found that they contain several esterase activities. SGA stimulates both immediate and delayed skin reactions in tick resistant guinea-pigs, and these reactions are about 200-fold more intense, per unit protein, than those elicited by SGE. The skin reactions to SGA are basophil-mediated and have many features in common with the cutaneous basophil hypersensitivity reactions of tick resistant animals. The demonstrated antigenic complexity of the glands may have profound implications for attempts to develop anti-tick vaccines, as it may eventually be found that candidate vaccines will have to incorporate more than one tick antigen in order to be effective.     

Oral tolerance in myelin basic protein T-cell receptor transgenic mice: suppression of autoimmune encephalomyelitis and dose-dependent induction of regulatory cells.

Orally administered antigens induce a state of immunologic hyporesponsiveness termed oral tolerance. Different mechanisms are involved in mediating oral tolerance depending on the dose fed. Low doses of antigen generate cytokine-secreting regulatory cells, whereas high doses induce anergy or deletion. We used mice transgenic for a T-cell receptor (TCR) derived from an encephalitogenic T-cell clone specific for the acetylated N-terminal peptide of myelin basic protein (MBP) Ac-1-11 plus I-Au to test whether a regulatory T cell could be generated from the same precursor cell as that of an encephalitogenic Th1 cell and whether the induction was dose dependent. The MBP TCR transgenic mice primarily have T cells of a precursor phenotype that produce interleukin 2 (IL-2) with little interferon gamma (IFN-gamma), IL-4, or transforming growth factor beta (TGF-beta). We fed transgenic animals a low-dose (1 mg x 5) or high-dose (25 mg x 1) regimen of mouse MBP and without further immunization spleen cells were tested for cytokine production. Low-dose feeding induced prominent secretion of IL-4, IL-10, and TGF-beta, whereas minimal secretion of these cytokines was observed with high-dose feeding. Little or no change was seen in proliferation or IL-2/IFN-gamma secretion in fed animals irrespective of the dose. To demonstrate in vivo functional activity of the cytokine-secreting cells generated by oral antigen, spleen cells from low-dose-fed animals were adoptively transferred into naive (PLJ x SJL)F1 mice that were then immunized for the development of experimental autoimmune encephalomyelitis (EAE). Marked suppression of EAE was observed when T cells were transferred from MBP-fed transgenic animals but not from animals that were not fed. In contrast to oral tolerization, s.c. immunization of transgenic animals with MBP in complete Freund's adjuvant induced IFN-gamma-secreting Th1 cells in vitro and experimental encephalomyelitis in vivo. Despite the large number of cells reactive to MBP in the transgenic animals, EAE was also suppressed by low-dose feeding of MBP prior to immunization. These results demonstrate that MBP-specific T cells can differentiate in vivo into encephalitogenic or regulatory T cells depending upon the context by which they are exposed to antigen.     

Transport of large breakdown products of dietary protein through the gut wall.

Ferritin or tritium labelled immunoglobulin G may, by electron microscopy, be demonstrated entering, within, and leaving the epithelial cells. Quantitative studies using various proteins labelled with radioiodine show that large amounts of protein bound radioactivity may be demonstrated in the tissues after feeding the labelled protein to adult rats by stomach tube. The molecular size of this material as determined by sugar gradient ultracentrifugation of tissue extracts ranges when IgG is fed from 50,000-20,000 Daltons. The material retains its ability to react as antigen with antisera specific to the original molecule: precipitation reactions may be obtained in gels and quantitative studies show that cnosiderable amounts of the protein-bound radioactivity are still specifically precipitable. Such studies have been carried out with alpha-gliadin as well as bovine IgG. At 100 days old rats may absorb as much as 40% of a dose of bovine IgG in the form of these large molecular breakdown products.     

Isolation and characterization of salivary antigens from Hyalomma anatolicum anatolicum

This study demonstrates the involvement of a large number of salivary proteins in the acquisition of resistance to Hyalomma anatolicum anatolicum. Using immunoblotting, sera from hypersensitized rabbits were shown to react with nine proteins in the saliva and 17 in salivary gland extracts (SGE) from 96 h fed female ticks. The salivary antigens had molecular weights in the range of 14 400 to 130 000. All the antigens identified in the saliva and 12 of the SGE antigens were glycoprotein in nature and a majority of them appeared to be common to different stages of feeding. In addition antigen I (molecular weight 130 000) showed acid phosphatase and antigen III (molecular weight 96 000) showed both non-specific esterase and aminopeptidase activity. Three high molecular weight proteins isolated from saliva (antigen I, antigen II--molecular weight 103 000 and antigen III), gave immediate hypersensitivity reactions in intradermal inoculation into rabbits which had previously been exposed to ticks. Antigens II and III also elicited a strong delayed hypersensitivity reaction. These results may help to explain the nature of the immune mechanisms which effect resistance against H. a. anatolicum.     

Oral tolerance: lessons on treatment of food allergy

Oral tolerance is the active non-response by the immune system to an antigen administered through the oral route. It is postulated that food hypersensitivity results from a breakdown in oral tolerance induction, and the importance of oral tolerance in food hypersensitivity can be traced back to classic experiments from 1911 in which guinea pigs were protected from anaphylaxis by prior feeding of antigen. Host and antigenic factors play a role in determining the pathways and mechanisms to which a fed antigen can gain tolerance. Recent studies have demonstrated the potential of using oral tolerance to treat food allergies, and additional studies are necessary to further our understanding of mechanisms of oral tolerance induction.     

Direct evidence for cytotoxicity associated with expression of hepatitis delta virus antigen.

It has been postulated that hepatocyte injury resulting from infection with hepatitis D virus may be caused by a direct virus cytotoxicity in contrast to immune-mediated injury associated with hepatitis B virus. We have transfected HeLa and HepG2 continuous cell lines with a recombinant plasmid containing the hepatitis D antigen gene under the inducible control of the human metallothionein promoter. The addition of zinc to the cell culture medium then led to the expression of hepatitis D antigen associated with, in the short term, a significant reduction in the rate of RNA but not DNA synthesis and, in the longer term, cell death. The necrotic cells had pyknotic nuclei and shrunken eosinophilic cytoplasm; these necrotic cells resembled the apoptotic bodies seen in hepatitis D virus-related hepatitis. The level of hepatitis D antigen in individual cells that produced these changes was similar to the level of hepatitis D antigen in hepatocytes from a chimpanzee with acute hepatitis D virus infection. We conclude that the expression of hepatitis D antigen resulted in significant cytotoxic changes in these cells, providing strong support for the view that hepatitis D antigen may be specifically cytotoxic to infected hepatocytes in vivo.     

口服耐受与炎症性肠病

口服耐受是机体对口服抗原产生的特异性无应答或低应答状态.低剂量口服抗原主要通过调节性T细胞分泌抑制性细胞因子介导免疫抑制,高剂量口服抗原主要引起T细胞清除或T细胞失能.多次低剂量口服结肠炎提取蛋白或正常结肠提取蛋白均能缓解实验性结肠炎.目前,多次低剂量口服自体结肠提取蛋白已初步应用于临床试验,并证明是安全的治疗方案.充分发挥口服耐受对炎症性肠病的临床疗效仍需深入研究.     

Ethanol feeding of micropigs alters methionine metabolism and increases hepatocellular apoptosis and proliferation

Chronic alcoholism is associated with increased cancer risk that may be related to ethanol-induced alterations in methionine and deoxynucleotide metabolism. These metabolic relationships were studied in micropigs fed diets for 12 months that contained 40% ethanol or cornstarch control with adequate folate. Ethanol feeding altered methionine metabolism without changing mean terminal liver folate levels. After initial equilibration to diet, ethanol feeding significantly increased monthly serum homocysteine levels while reducing serum methionine levels over the time course of the experiment. After 12 months, hepatic methionine synthase activity and the ratio of S-adenosylmethionine (SAM) to S-adenosylhomocysteine (SAH) were significantly reduced in ethanol-fed animals, whereas the ratio of liver deoxyuridine triphosphate (dUTP) to deoxythymidine triphosphate (dTTP) was increased and correlated inversely with methionine synthase activity. These findings were associated with increased frequency of hepatocytes with apoptotic bodies and positivity for proliferating cell nuclear antigen (PCNA) in livers from ethanol-fed minipigs. These studies suggest that chronic ethanol feeding perturbs methionine metabolism by impairment of methionine synthase activity, resulting in deoxynucleoside triphosphate (dNTP) imbalance, increased apoptosis, and regenerative proliferation. These biochemical alterations may provide a promoting environment for carcinogenesis during long-term ethanol exposure. (Hepatology 1996 Mar;23(3):497-505)     

Effect of chronic antigen exposure on growth and intestinal histamine content of sensitized rats.

The effects of chronic antigen feeding on systemically sensitized rats were investigated. Findings include a reduction of water and antigen intake in egg albumin (EA), sensitized rats receiving EA in their drinking water for an 8 day period, compared to that of sensitized rats fed bovine serum albumin and of naive rats. Feeding EA to sensitized animals also induced a decrease in daily weight gain. This decline did not seem to be a consequence of a decreased food intake, but might rather reflect a decreased water consumption and an alteration of nutrient absorption in the gut. Indeed, sensitized rats fed EA exhibited a significant increase in jejunal and ileal histamine content compared to control rats, which may indicate the development of an inflammatory reaction in the small intestinal mucosa. Intestinal troubles experienced because of this inflammatory reaction might explain the reduction of antigen and water intake observed in sensitized rats.     

Prevalence of ankylosing spondylitis and its association with HL-A 27.

The close relationship between ankylosing spondylitis and the presence of the transplantation antigen HL-A 27 is well recognized. An attractive explanation for this has been that the gene coding for this antigen exists in marked linkage disequilibrium with an abnormal immune response gene. We have suggested, in contrast, that HL-A 27 may have a more direct importance because of a defective epistatic interaction between the gene coding for this antigen and a distinct disease susceptibility gene for AS, which may therefore be situated some distance from the major histocompatibility complex.     

An association of hepatitis B (Australia) antigen with platelets.

Hepatitis B surface antigen (HBsAg, Australia antigen) was detected by radioimmunoassay in freeze-thaw lysates of washed platelets from HBsAg carriers. Incubation of platelets from an HBsAg-negative person with medium containing HBsAg resulted in the platelets becoming positive for HBsAg. We suggest that platelets may phagocytize HBsAg-coated particles, and this may be an important transport mechanism for the hepatitis B agent.     

Dissemination, replication, and trans-stadial persistence of Dugbe virus (Nairovirus, Bunyaviridae) in the tick vector Amblyomma variegatum

The dissemination and replication of Dugbe (DUG) virus and its tissue tropisms in the tick vector Amblyomma variegatum were examined by immunohistochemical analysis using specific antibody, in situ hybridization with a viral-complementary riboprobe, and infectivity assays of dissected tissues. Dugbe virus was localized in both unfed and feeding adults inoculated as nymphs or orally infected by capillary feeding, and in nymphs infected by capillary feeding. In non-feeding ticks, the main sites of DUG virus replication were the epidermis, hemocytes associated with loose connective tissue, and a small number of phagocytic digestive cells in the gut lumen. Virus infectivity in the hemolymph was associated entirely with hemocytes. Dugbe viral antigen or infectivity was not detected in the salivary glands until after the start of feeding. Viral titers in the salivary glands of feeding ticks were about ten-fold higher than in gut, ovary, or loose connective tissue. The level of infection decreased during molting and increased during feeding. Viral particles and pathologic effects were not detected in infected ticks. The primary site of trans-stadial persistence of DUG virus is the hemocytes. Tick hemocytes and other motile cells may be important in the transmission of persistent virus infection from one cell or organ to another by diapedesis.     

Detection of hepatitis B virus DNA in hepatocellular carcinoma: analysis by hybridization with subgenomic DNA fragments

We have previously reported an analysis of DNA extracted from 31 primary liver tumors where, in 25 cases, we found chromosomal integration of hepatitis B virus DNA sequences. We describe here an investigation of the extent of the viral genome at each integration site in 15 of the hepatitis B virus DNA-positive tumors using subgenomic fragments of the viral genome as probes. Probes were roughly equivalent to the pre-S region, the surface antigen gene, the region containing the enhancer, the x gene and the core antigen gene. We found the core antigen gene to be that most underrepresented in the tumors and speculate that, since cells which express core antigen in the infected liver may be targeted for lysis by the immune system, modifications of the integrated viral DNA which prevent core antigen expression may be selected. Conversely, the region of the genome present in the greatest number of integrations was the surface antigen gene and, because it is known that the major surface antigen promoter is active in the integrated state, we find promoter insertion an attractive hypothesis to explain oncogenesis by hepatitis B virus.     

Immunization against rabies with plant-derived antigen

We previously demonstrated that recombinant plant virus particles containing a chimeric peptide representing two rabies virus epitopes stimulate virus neutralizing antibody synthesis in immunized mice. We show here that mice immunized intraperitoneally or orally (by gastric intubation or by feeding on virus-infected spinach leaves) with engineered plant virus particles containing rabies antigen mount a local and systemic immune response. After the third dose of antigen, given intraperitoneally, 40% of the mice were protected against challenge infection with a lethal dose of rabies virus. Oral administration of the antigen stimulated serum IgG and IgA synthesis and ameliorated the clinical signs caused by intranasal infection with an attenuated rabies virus strain.     

Detection of hepatitis C core antigen in the antibody negative 'window' phase of hepatitis C infection

BACKGROUND AND OBJECTIVES: Despite improvements in assays for anti-HCV, there remains a significant delay before the appearance of antibodies following infection, during which, circulating viral RNA is present. We have evaluated a prototype assay for the serological detection of hepatitis C virus (HCV) core antigen with specimens derived from the early phase of HCV infection. MATERIALS AND METHODS: Serial specimens from 24 individuals undergoing HCV seroconversion were tested for the presence of anti-HCV, HCV RNA and HCV core antigen. RESULTS: HCV antigen was detected at the same time as HCV RNA in 83% (20/24) cases. The mean time to the first detection of HCV antigen was approximately 1 day later than HCV RNA. Overall, 87% of HCV-RNA-positive specimens contained detectable HCV core antigen. CONCLUSION: These results indicate that HCV core antigen can be identified by routine serological ELISA in specimens from the early antibody-negative phase of HCV infection. A test for HCV core antigen may be a useful test for identifying window phase blood donations from antibody negative donors infected with HCV.