Effects of thermal processing on the allergenicity, structure, and critical epitope amino acids of crab tropomyosin.

Food processing can change the structure and immunoreactivity of purified allergens, but the effect of food processing on the immunoreactivity of the processed and purified allergen is still poorly understood. In this study, tropomyosin (TM) was obtained from Scylla paramamosain and purified after different treatments. A basophil activation test was employed to detect the allergenicity of allergens. The protein structure was detected by mass spectrometry, circular dichroism spectroscopy and surface hydrophobicity. Critical amino acids were identified by Dot blot. Heating obviously affects the biochemical characteristics of TM. The allergenicity of TM was decreased in high temperature-pressure-processed crabs, due to alteration in the protein structure (e.g. denaturation). Seven critical amino acids, namely, R21, E103, E104, E115, A116, E122 and E156, related to the maintenance of the IgE-binding activity of TM were identified. This research of thermal processing helps to accurately reduce or eliminate the immunoreactivity of crabs by food processing.

Analysis of the mechanism of unresponsiveness produced by haptens painted on skin exposed to low dose ultraviolet radiation.

Acute, low dose ultraviolet B radiation of murine body wall skin followed by local application of DNFB produces a state of antigen-specific unresponsiveness. This state is maintained at least in part by an Lyt-1+ T cell that effects unresponsiveness by impairing the induction phase of contact hypersensitivity. The absence of suppressor cells capable of acting at the effector stage of immunity suggests that tolerogenic signals derived from the skin establish suppressor networks that are incomplete and perhaps different from networks that are induced by systemic administration of tolerogens. It is proposed that ultraviolet radiation produces its effects by impairing the antigen-presenting potential of resident Langerhans cells in whose absence hapten-derivatized keratinocytes (or their products) are able to deliver a tolerogenic signal.

How microwave treatment of gluten affects its toxicity for celiac patients? A study on the effect of microwaves on the structure, conformation, functionality and immunogenicity of gluten.

The microwave heating of wheat kernels, flour, and gluten, has attracted attention lately because it has been claimed to abolish gluten toxicity for celiac patients. Nevertheless, contradictory results have been reported regarding the effect on gluten celiac-immunotoxicity. In order to better understand the effect of the microwave treatment on gluten structure, conformation, functionality and celiac-immunotoxicity, a central composite design with two factors, power level, and treatment time, was used to investigate a possible quadratic and interaction effects between both factors. Extractable gliadins content was affected by the power and time in a linear and quadratic fashion; extractable glutenins were not affected. Gluten secondary structure was affected by the microwave treatment and related to the polymer's disaggregation phenomenon observed. In fact, the microwave treatment increased the amount of potentially toxic epitopes released after peptic and tryptic digestion, showing inefficiency as a treatment to detoxify the gluten for celiac disease patients.

Specificity of antigens on UV radiation-induced antigenic tumor cell variants measured in vitro and in vivo.

In vitro exposure of nonimmunogenic murine tumor cells to UV radiation (UVR) generates highly antigenic variants that are immunologically rejected by normal, syngeneic mice. The purpose of this study was to determine whether these antigenic variants cross-react immunologically with the parental tumor and whether the UVR-associated antigen unique to UVR-induced tumors is also present on the variants. Antigenic (regressor) variants and nonimmunogenic (progressor) clones derived from UV-irradiated cultures of the C3H K1735 melanoma and SF19 spontaneous fibrosarcoma cell lines were used to address these questions. In an in vivo immunization and challenge assay, the antigenic variants did not induce cross-protection among themselves, but each induced immunity against the immunizing variant, the parent tumor cells, and nonimmunogenic clones derived from UV-irradiated parent cultures. Therefore, the variants can be used to induce in mice a protective immunity that prevents the growth of the parent tumor and nonimmunogenic clones, but not other antigenic variants. In contrast, immunization with cells of the parental tumor or the nonimmunogenic clones induced no protective immunity against challenge with any of the cell lines. Utilizing the K1735 melanoma-derived cell lines in vitro, T-helper (Th) cells isolated from tumor-immunized mice were tested for cross-reactivity by their ability to collaborate with trinitrophenyl-primed B-cells in the presence of trinitrophenyl-conjugated tumor cells. Also, the cross-reactivity of cytotoxic T-lymphocytes from tumor-immunized mice was assessed by a 4-h 51Cr-release assay. Antigenic variants induced cytotoxic T-lymphocytes and Th activity that was higher than that induced by the parent tumor and nonimmunogenic clones from the UVR-exposed parent tumor and cross-reacted with the parental tumor cells and nonimmunogenic clones, but not with other antigenic variants. Furthermore, upon transplantation, the UVR-induced antigenic variants grew in UV-irradiated and immunosuppressed mice, but not in untreated mice indicating that the variants expressed the determinant recognized by suppressor T-cells present in UV-irradiated mice. These results demonstrate that highly antigenic cells generated by the in vitro exposure of two different murine tumors to UV radiation express a determinant shared with the parental tumor cells and nonimmunogenic clones, a unique variant-specific determinant and the suppressor cell-defined determinant present on UVR-induced tumors. Based on these results, two models are proposed to explain the make-up of the antigenic determinants present on the UVR-induced antigenic variants.

Radiation inactivation of human intestinal mucin: determination of the size of the functional antigenic unit.

Previously we have shown that the major antigenic determinant of human intestinal mucin is associated with its glycopeptide monomers and not the 118 kDa 'link' component. In the present study, the size and nature of the functional unit containing the antigenic determinant has been assessed by radiation inactivation and immunological assays. Increasing doses of radiation led to a monoexponential decay in antigenic reactivity due to a progressive loss of antigenic determinants. From three independent mucin preparations, a value of 78500 +/- 7000 was determined for the Mr of the functional antigenic unit. Prolonged pronase digestion of native mucin released large degraded glycopeptide monomers containing all the mucin carbohydrate, and low molecular weight peptides. The antigenicity of the glycopeptides decreased with digestion but could not be recovered in the peptide fractions, suggesting that determinants were released and destroyed by the enzyme. Treatment of native mucin with trifluoromethanesulphonic acid caused a major loss of carbohydrate (approx. 70%), but the protein component was unchanged in amino acid profile and remained antigenic. Subsequent thiol reduction, however, abolished the antigenicity of the deglycosylated mucin. We conclude that antigenicity is associated with a non-glycosylated segment of the peptide backbone of the glycopeptides and that a large functional unit of Mr 78500 which is stabilized by disulphide bonds is important for full antigenic activity.

Crystal structure of the 64M-2 antibody Fab fragment in complex with a DNA dT(6-4)T photoproduct formed by ultraviolet radiation.

DNA photoproducts with (6-4) pyrimidine-pyrimidone adducts formed by ultraviolet radiation are implicated in mutagenesis and cancer, particularly skin cancer. The crystal structure of the Fab fragment of the murine 64M-2 antibody specific to DNA T(6-4)T photoproducts is determined as a complex with dT(6-4)T, a (6-4) pyrimidine-pyrimidone photodimer of dTpT, at 2.4 A resolution to a crystallographic R-factor of 0.199 and an R(free) value of 0.279. The 64M-2 Fab molecule is in an extended arrangement with an elbow angle of 174 degrees, and its five complementarity-determining regions, except L2, are involved in the ligand binding. The bound dT(6-4)T ligand adopting a ring structure with (6-4) linked 5' thymine-3' pyrimidone bases is fully accommodated in an antigen-binding pocket of about 15 Ax10 A. The 5'-thymine and 3'-pyrimidone bases are in half-chair and planar conformations, respectively, and are nearly perpendicular to each other. The 5'-thymine base is hydrogen-bonded to Arg95H and Ser96H, and is in van der Waals contact with Tyr100iH. The 3'-pyrimidone base is hydrogen-bonded to His35H, and is in contact with Trp33H. Three water molecules are located at the interface between the bases and the Fab residues. Hydrogen bonds involving these water molecules also contribute to Fab recognition of the dT(6-4)T bases. The sugar-phosphate backbone connecting the bases is surrounded by residues His27dL, Tyr32L, Ser92L, Trp33H, and Ser58H, but is not hydrogen-bonded to these residues.

Immunogenic and antigenic epitopes of immunoglobulin--XX. Denaturation of human IgG3 by free radicals.

It has previously been demonstrated that exposure of polyclonal IgG to free radicals results in denaturation evidenced by aggregation, auto-fluorescence and destruction of cysteine, proline and aromatic amino acids. In the present study we have used a panel of monoclonal antibodies (McAb) to epitopes expressed on the IgG3 heavy chain to detect changes in antigenicity. When IgG3 was exposed to u.v. irradiation, as a source of free radicals, subclass specific epitopes were rapidly lost whilst other epitopes were unaffected. Prolonged exposure resulted in further denaturation and a progressive loss of expression of further epitopes. The IgG3 subclass specific McAb are specific to epitopes localized to the hinge region of IgG3. Thus, this exposed cysteine and proline rich region is shown to be particularly vulnerable to free radical attack; however, prolonged exposure results in structural alterations throughout the heavy chain.

Action spectrum and mechanisms of UV radiation-induced injury in lupus erythematosus.

Photosensitivity associated with lupus erythematosus (LE) is well established. The photobiologic basis for this abnormal response to ultraviolet radiation, however, has not been determined. This paper summarizes the criteria for elucidating possible photobiologic mechanisms and reviews the literature relevant to the mechanism of photosensitivity in LE. In patients with LE, photosensitivity to wavelengths shorter than 320 nm has been demonstrated; wavelengths longer than 320 nm have not been adequately evaluated. DNA is a possible chromophore for photosensitivity below 320 nm. UV irradiation of skin produces thymine photodimers in DNA. UV-irradiated DNA is more antigenic than native DNA and the antigenicity of UV-irradiated DNA has been proposed, but not proven, to be involved in the development of clinical lesions. UV irradiation of mice previously injected with anti-UV-DNA antibodies produces Ig deposition and complement fixation that appears to be similar to the changes seen in lupus lesions. Antibodies to UV-irradiated DNA occur in the serum of LE patients although a correlation between antibody titers and photosensitivity was not observed. Defective repair of UV-induced DNA damage does not appear to be a mechanism for the photosensitivity in LE. Other mechanisms must also be considered. The chromophore for photosensitivity induced by wavelengths longer than 320 nm has not been investigated in vivo. In vitro studies indicate that 360-400 nm radiation activates a photosensitizing compound in the lymphocytes and serum of LE patients and causes chromosomal aberrations and cell death. The mechanism appears to involve superoxide anion. Further research is required to establish the action spectrum for long wavelength photosensitivity in vivo and to elucidate the mechanisms for the photosensitivity at all wavelengths.

Ionizing radiation modulates the exposure of the HUIV26 cryptic epitope within collagen type IV during angiogenesis.

PURPOSE: The majority of the research on the biologic effects of ionizing radiation has focused on the impact of radiation on cells in terms of gene expression, DNA damage, and cytotoxicity. In comparison, little information is available concerning the direct effects of radiation on the extracellular microenvironment, specifically the extracellular matrix and its main component, collagen. We have developed a series of monoclonal antibodies that bind to cryptic epitopes of collagen Type IV that are differentially exposed during matrix remodeling and are key mediators of angiogenesis. We have hypothesized that ionizing radiation might affect the process of angiogenesis through a direct effect on the extracellular matrix and specifically on collagen Type IV. METHODS AND MATERIALS: Angiogenesis was induced in a chick chorioallantoic membrane (CAM) model; 24 h later, a single-dose treatment with ionizing radiation (0.5, 5, and 20 cGy) was administered. Angiogenesis was assessed, and the exposure of two cryptic regulatory epitopes within collagen Type IV (HUI77 and HUIV26) was studied in vitro by solid-phase ELISA and in vivo by immunofluorescence staining. RESULTS: A dose-dependent reduction of angiogenesis with maximum inhibition (85%-90%) occurring at 20 cGy was demonstrated in the CAM model. Exposure of the cryptic HUIV26 site, an angiogenesis control element, was inhibited both in vitro and in vivo by the same radiation dose, whereas little if any change was observed for the HUI77 cryptic epitope. CONCLUSIONS: A dose-dependent alteration of the functional exposure of the HUIV26 cryptic epitope is induced by radiation in vitro and in the CAM model in vivo. This radiation-induced change in protein structure and function may contribute to the inhibitory effects of ionizing radiation on new blood vessel growth and warrants further studies in other models.

Suppression of the elicitation of the immune response to alloantigen by ultraviolet radiation.

Previous studies have established that exposure of mice to ultraviolet radiation followed by injection of alloantigen can suppress the induction of delayed hypersensitivity and the rejection of allografts in an antigen-specific manner. In the clinical situation, however, UV irradiation several days prior to transplantation may prove impractical due to the difficulty in predicting when a donor organ will be available. Thus, the purpose of this study was to determine if exposure to UV radiation can suppress the elicitation of the immune response in mice sensitized with alloantigen. The data demonstrate that exposure of mice to UV radiation 1, 3, or 5 days after the injection of alloantigen can significantly suppress the delayed hypersensitivity response to that alloantigen. Present in the spleens of these mice are suppressor T lymphocytes. These suppressor cells are specific for the antigen originally used to sensitize the mice, in that they do not suppress the response to an irrelevant alloantigen. In addition, spleen cells from mice sensitized with alloantigen and exposed to UV radiation 1, 3, or 5 days later are unable to proliferate in response to the alloantigen in a mixed lymphocyte response. These cells do respond to irrelevant third-party cells, demonstrating again the specificity of the suppression. These data demonstrate that exposure of mice in vivo to UV radiation can inhibit the elicitation of the immune response to alloantigen. Since the immunosuppression is specific for the sensitizing antigen, these data suggest that this may provide a novel method of suppressing the immune response to tissue allografts.

In vivo studies of the maintenance of peripheral transplant tolerance after cyclosporine. Radiosensitive antigen-specific suppressor cells mediate lasting graft protection against primed effector cells.

Cellular mechanisms responsible for maintenance of peripheral transplant tolerance in a rodent model were evaluated. Donor-specific tolerance was established in ACI rats given a vascularized heterotopic cardiac allograft followed by a 10-day course of cyclosporine. Tolerance was associated with a reduction in donor-specific cytotoxic T lymphocyte precursors and the presence within the spleen of cells capable of transferring suppression in adoptive transfer assays. Experiments using thymectomized animals revealed that the establishment and maintenance of tolerance occurred peripherally, independently of the thymus. Adoptive transfer experiments demonstrated that ongoing graft tolerance was mediated by suppressor cells that were antigen-restricted, radiosensitive, and capable of preventing allograft rejection by naive as well as sensitized cells in vivo. Studies designed to disrupt tolerance demonstrated a remarkable durability of graft protection once established, and give insight into the identity and mechanism of action of suppressor cells generated in this model.

Stress-induced cell surface expression and antigenic alteration of the Ro/SSA autoantigen.

Recent studies have shown that Ro/SSA autoantigen is heterogeneous. There are two isoform families; the 60 kD forms (Ro60) and the 52 kD forms (Ro52). Recently we have found that autoantibodies to the Ro/SSA proteins are conformation dependent. Anti-Ro60 antibodies are mainly directed to the native protein and conversely anti-Ro52 antibodies are directed only to the denatured protein. It has been known that UV irradiation to cultured keratinocytes induces cell surface expression of Ro/SSA and this phenomenon has been thought to be related with photosensitivity in patients with anti-Ro/SSA antibodies. We studied the quantitative and qualitative changes of the Ro/SSA protein induced by stress, such as with heat shock and UV irradiation, and found that only Ro52 could be expressed on the cell surface of human peripheral lymphocytes by either heat shock or UV irradiation. Moreover, flow cytometric analysis revealed that HS-treated and UV-treated lymphocytes could be stained with patient sera, and by using a technique which combined immunoprecipitation and Western immunoblotting, it has been confirmed that Ro52 expressed on the cell surface can be recognized by anti-Ro/SSA antibodies in native form while cytoplasmic Ro52 cannot be recognized. These data suggest that Ro52 can be antigenic in vivo when expressed on the cell surface and may explain the mechanism of direct tissue damage by anti-Ro/SSA antibodies.

Piroxicam has at least two epitopes for contact photoallergy.

We have demonstrated previously in guinea pigs that the induction of photocontact sensitivity to piroxicam (PXM) also induces a state of cross-reactive contact hypersensitivity to two compounds having structurally related elements, thimerosal (TMS) and thiosalicylate (TOS). The present study was conducted to determine whether oral administration of TOS would desensitize guinea pigs previously photosensitized with PXM. At the same time, the spectrum of reactivities against these compounds and against tenoxicam (TXM) which resembles only piroxicam was assessed by appropriate sensitizing and eliciting protocols. As expected, animals photosensitized to PXM developed reactivities against all four compounds, PXM and TXM (photosensitivity) and TMS and TOS (contact sensitivity). By contrast, photosensitization with TXM induced cross-reactivity only against PXM. Moreover, the induction of contact sensitivity against TMS or TOS induced photosensitive cross-reactivity to PXM, but not to TXM. Finally, the oral administration of TOS produced a transient desensitization only for TMS and TOS. These results suggest that photosensitization with PXM induces two distinct reactivities. The first reactivity cross-reacts with TMS and TOS and is suppressible with orally administered TOS. The second cross-reacts only with TXM and is not suppressible with oral TOS. We conclude that PXM acquires at least two distinct immunogenic epitopes when exposed to UVA irradiation.

Microwave antigen retrieval of beta-amyloid precursor protein immunoreactivity.

Formalin fixation reduces beta-amyloid precursor protein (beta APP) immunoreactivity which restricts its study in archival tissue. Formic acid pretreatment has previously been used in an attempt to overcome this problem, but makes the sections very friable. In the present study, a microwave antigen retrieval method has been compared with formic acid pretreatment for retrieving beta APP immunoreactivity in formalin-fixed, paraffin-embedded human brain tissue. Microwave treatment resulted in superior retrieval of beta APP antigenicity in dystrophic neurites in Alzheimer's disease and in injured axons after head injury, using antibodies to three different epitopes. Unlike formic acid, microwave treatment causes minimal adverse effects on the strength and slide adhesion of the sections.

Regulation of mu binding sites after chronic administration of antibodies directed against specific anti-opiate peptides.

There is some indication that anti-opiate peptides (AOP) modulate opioid receptor systems by altering mu-receptor density. To further characterize this phenomenon, we investigated the effects of continuous infusion of anti-AOP IgG on mu binding sites in the brains of rats. Specifically, male Sprague-Dawley rats received intracerebroventricular (i.c.v.) infusions for 13 days of either control (rabbit) IgG or test IgGs: anti-dynorphin A IgG, anti-dynorphin A1-8 IgG, anti-alpha-MSH IgG, or the monoclonal anti-NPFF IgG. Administration of anti-NPFF IgG or the anti-dynorphin1-8 IgG significantly increased mu labeling by 40-70% in several brain regions at the caudate level. Contrary to these findings, anti-alpha-MSH IgG decreased (19-32%) [125I]-DAMGO labeling in several thalamic nuclei. The results suggest that the density of mu-opioid receptors is regulated in part by anti-opiate peptides in the extracellular fluid of the brain.

Red light-induced appearance of phosphotyrosine-like epitopes on nuclear proteins from pea (Pisum sativum L.) plumules.

As assayed by western blot analysis, red light induces the appearance of epitopes recognized by anti-phosphotyrosine antibodies in several pea nuclear proteins. The immunostaining is blocked by preadsorbing the antibodies with phosphotyrosine but not by preadsorbing them with phosphoserine or phosphothreonine. This light response is observed whether the red light irradiation is given to pea plumules or nuclei isolated from the plumules. The red-light-induced response seen in plumules is reversible by a subsequent far-red-light irradiation, indicating that the likely photoreceptor for this response may be phytochrome. By immunoblot analysis pea phytochrome A, but not phytochrome B, can be detected in proteins extracted from pea nuclear chromatin-matrix preparations. Phytochrome A and the protein bands immunostained by anti-phosphotyrosine antibodies can be solubilized from unirradiated pea chromatin by 0.3 M NaCl, but irradiating this preparation with red light does not induce the appearance of phosphotyrosine-like epitopes in any nuclear proteins. These results suggest that the association of phytochrome with purified pea nuclei is such that its conversion to the far-red light-absorbing form can induce a post-translational epitope change in nuclear proteins in vivo.

Porphyrin-induced photodynamic cross-linking of hepatic heme-binding proteins.

Three types of hepatic proteins, a heme-binding Z protein, a mixture of the glutathione S-transferases and a cytochrome P450 isozyme, were shown to be susceptible to photodynamic cross-linking and loss in antigenicity by naturally occurring porphyrins. At 50 microM, uroporphyrin caused the most and protoporphyrin the least photodecomposition. Hemopexin, a specific serum heme carrier, was photodecomposed but no cross-linking was detected. Heme and scavengers of singlet oxygen partially prevented protein photodecomposition.

Further characterisation of a polyclonal antiserum for DNA photoproducts: the use of different labelled antigens to control its specificity.

Synthetic polynucleotides irradiated with far (254 nm) or near (320 nm) UV-light were used to characterise 3 different radioimmunoassay systems. Antiserum raised against DNA irradiated with a high dose of far-UV-light was found to have at least 2 antibody populations. A competitive assay in which the labelled antigen was irradiated at 254 nm was found to be specific for Pyr(6-4)Pyo adducts, the antibody-binding sites being sensitive to a secondary photolytic dose of 320-nm light. When the labelled antigen was irradiated with 320-nm light the assay was specific for cyclobutane dimers. This assay had the same specificity as one consisting of labelled DNA irradiated with 254-nm light and an antiserum raised against DNA irradiated at 320 nm in the presence of acetophenone. These assay systems were used to demonstrate the dose-dependence of the induction and photolytic degradation of Pyr(6-4)Pyo adducts by a near-UV-light source.

Effect of radiotherapy on the levels of secretory immunoglobulin A against indigenous and virulent streptococci.

It is well known that the frequency of upper respiratory infection is clinically increased after radiotherapy of the head and neck region. This study found higher antibacterial secretory immunoglobulin A (S-IgA) activity against three indigenous streptococci (Streptococcus mitis, S. salivarius, and S. sanguis I) and S. pneumoniae in patients who had undergone radiation therapy of the head and neck region than in control subjects. This showed no relation to the extent of the radiation field. Compared with before radiotherapy, the S-IgA titer against S. pneumoniae and its ratio to the activities against the indigenous streptococci were significantly higher in patients with fully irradiated major salivary glands. These results indicated that the radiotherapy promoted the antigen-specific S-IgA production of virulent streptococci in most patients with head and neck cancer, even more than 6 months after radiotherapy. The resulting altered balance in the S-IgA system of normal indigenous streptococci may also impair the ability to maintain the stable bacterial interference between normal indigenous and virulent streptococci in the oropharyngeal cavity.

[Principles of selective inactivation of the virus genome. IV. The effect of UV-irradiation of phage MS2 on its binding with anti-MS2-immunoglobulins]. / Printsipy selektivnoi inaktivatsii virusnogo genoma. IV. Vliianie UF-oblucheniia faga MS2 na ego sviazyvanie s anti-MS2-immunoglobulinami.

Ultraviolet (254 nm) irradiation of the bacteriophage MS2 results in the decrease of the number of antigenic determinants exposed on the virion surface. The cross-section of the decrease, as measured by the number of anti-MS2 IgG molecules bound per virion, is 10(-16) mm2 per photon. The decrease of the phage-antibody binding proceeds after irradiation with a rate constant of about 5 x 10(-3) min-1. Since the antigenic determinants of the phage MS2 coat protein does not contain photoreactive amino acid residues, the irradiation-induced decrease of the phage antibody binding is determined, most probably, by the shielding of the antigenic determinants. Such shielding could be caused by rearrangement of coat protein molecules and/or of the capsid induced by photomodification of non-antigenic fragments of coat protein and/or of intraphage RNA.