Long-term antibody response and protective effect induced by attenuated scorpion toxins: Involvement of memory plasma cells.

In Algeria, Androctonus australis hector scorpion envenomation remains a major problem of public health because of non-efficient therapy. The development of safe vaccine against scorpion venom could be one key strategy for the envenomation prevention. The irradiation of venom by γ-rays develops suitable immunogens which produced effective antivenom and safe vaccine. In this study, we investigated the ability of the irradiated toxic fraction (γ-FtoxG50) to induce long-term memory humoral response in immunized animals (mice and rabbits), by involving the long-lived plasma cells to prevent efficiently the lethality of scorpion envenomation. For this purpose, an appropriate immunization schedule was established in mice and rabbits using three (3) similar doses of γ-FtoxG50 associated with Alum adjuvant. Obtained results indicate that the long-term immunogenicity of γ-FtoxG50 is able to induce the long-term memory humoral response with a high level of specific antibodies. The long-term persistence of antibody levels could depend on bone marrow memory plasma cells. These cells produce continuously antibodies without antigen stimulus. Furthermore, an enhanced memory response was obtained post-repeated envenomation with toxic native venom that leads to improved protection of animals. Together, pre-existing protective antibodies and the activation of memory B-cells could induce a rapid neutralization of scorpion toxins and long-term protection against scorpion envenomation.

Immunohistochemical evaluation of the effects of paraffin section storage on biomarker stability.

Environmental stresses can alter immunoreactivity of biomarkers in stored tissue sections. The effect of temperature and lighting on 49 cellular or microbial antigens was evaluated in 4 serial paraffin sections, cut 12 months, 10 months, 8 months, 5 months, 3 months, 1 month, 3 days, and 1 day before immunohistochemistry. Slides were stored at room temperature (RT) in the dark, at 4°C in the dark, at RT under fluorescent light, or at RT with windowpane exposure to sunlight. Immunohistochemistry was performed simultaneously in an automated immunostainer. Immunoreactivity was compared with that in the corresponding 1-day-old section and scored as 4 (<10% reduction), 3 (10%-25% reduction), 2 (26%-60% reduction), 1(>60% reduction), or 0 (no reactivity). Any loss of immunoreactivity was proportional to the tissue section age and was least in sections stored in the dark. Immunoreactivity was only completely lost in light-exposed sections and as early as 1 month for CD45. Other markers with complete loss of immunoreactivity were bovine viral diarrhea virus, CD18 (only with fluorescent light), CD31, CD68, canine parvovirus, chromogranins, and thyroid transcription factor-1. Markers with complete loss after light exposure also had reduced immunoreactivity when stored in the dark, as early as day 3. Eight markers (Bartonella spp, CD11d, high molecular weight cytokeratins, feline coronavirus, GATA-4, insulin, p63, progesterone receptor) had minimal decrease in immunoreactivity, regardless of treatment. In conclusion, light-induced antigen decay (tissue section aging) is antigen dependent and could explain unexpectedly weak or negative immunohistochemical reactions in stored paraffin sections.

Expression of the progenitor marker NG2/CSPG4 predicts poor survival and resistance to ionising radiation in glioblastoma.

Glioblastoma (GBM) is a highly aggressive brain tumour, where patients respond poorly to radiotherapy and exhibit dismal survival outcomes. The mechanisms of radioresistance are not completely understood. However, cancer cells with an immature stem-like phenotype are hypothesised to play a role in radioresistance. Since the progenitor marker neuron-glial-2 (NG2) has been shown to regulate several aspects of GBM progression in experimental systems, we hypothesised that its expression would influence the survival of GBM patients. Quantification of NG2 expression in 74 GBM biopsies from newly diagnosed and untreated patients revealed that 50% express high NG2 levels on tumour cells and associated vessels, being associated with significantly shorter survival. This effect was independent of age at diagnosis, treatment received and hypermethylation of the O(6)-methylguanine methyltransferase (MGMT) DNA repair gene promoter. NG2 was frequently co-expressed with nestin and vimentin but rarely with CD133 and the NG2 positive tumour cells harboured genetic aberrations typical for GBM. 2D proteomics of 11 randomly selected biopsies revealed upregulation of an antioxidant, peroxiredoxin-1 (PRDX-1), in the shortest surviving patients. Expression of PRDX-1 was associated with significantly reduced products of oxidative stress. Furthermore, NG2 expressing GBM cells showed resistance to ionising radiation (IR), rapidly recognised DNA damage and effectuated cell cycle checkpoint signalling. PRDX-1 knockdown transiently slowed tumour growth rates and sensitised them to IR in vivo. Our data establish NG2 as an important prognostic factor for GBM patient survival, by mediating resistance to radiotherapy through induction of ROS scavenging enzymes and preferential DNA damage signalling.

Pathogenic mechanisms of polymorphic light eruption.

Polymorphic light eruption (PLE) is a photodermatosis (i.e. "sun allergy") with a high prevalence, particularly among young women in temperate climates. It is characterized through itchy skin lesions of variable morphology, occurring in spring or early summer on sun exposed body sites. As yet the exact etiology and pathogenesis of PLE are unknown although a resistance to ultraviolet (UV)-radiation-induced immunosuppression (i.e. a physiologic phenomenon in normal healthy subjects) and a subsequent delayed type hypersensitivity (DTH) response to a UV-modified skin antigen (i.e. neo-antigen) has been suggested as a key factor in the disease. This article reviews the cellular and molecular disturbances associated with and most likely playing a role in pathogenesis of the disease.

Cambios en la actividad biológica de un antígeno debido a la temperatura generada durante la electroforesis de poliacrilamida / Changes in the biological activity of an antigen due to the temperature generated during the polyacrylamide electrophoresis

Se evaluó el efecto que origina la temperatura sobre la actividad biológica de un antígeno durante la electroforesis en geles no reductores de poliacrilamida, porque en ocasiones la técnica precede a la electroelusión, método empleado en la purificación de determinadas proteínas. Se utilizó el antígeno específico prostático para el ensayo, este se purificó a partir del semen de pacientes voluntarios, precipitado con sulfato de amonio 70 por ciento. Las corridas (3 para cada temperatura), se llevaron a cabo a 4 y 25 °C, y se realizaron a 20 mA. Para cada temperatura ensayada, los fragmentos provenientes de los geles cortados a la talla esperada para el antígeno específico prostático (33 kDa), se electroeluyeron a 4 °C. Mediante la comparación de las concentraciones de proteínas empleando la técnica de Lowry y el método radioinmunoenzimático, se cuantificó la cantidad de proteína biológicamente activa posterior a la electroforesis. Se utilizó un antígeno específico prostático comercial como control. Los resultados indican que el antígeno a 4 °C, conserva 80 por ciento de su actividad biológica, lo que no sucede a 25 °C donde más de 50 por ciento de la proteína es biológicamente inactiva

Cambios en la actividad biológica de un antígeno debido a la temperatura generada durante la electroforesis de poliacrilamida / Changes in the biological activity of an antigen due to the temperature generated during the polyacrylamide electrophoresis

Se evaluó el efecto que origina la temperatura sobre la actividad biológica de un antígeno durante la electroforesis en geles no reductores de poliacrilamida, porque en ocasiones la técnica precede a la electroelusión, método empleado en la purificación de determinadas proteínas. Se utilizó el antígeno específico prostático para el ensayo, este se purificó a partir del semen de pacientes voluntarios, precipitado con sulfato de amonio 70 por ciento. Las corridas (3 para cada temperatura), se llevaron a cabo a 4 y 25 °C, y se realizaron a 20 mA. Para cada temperatura ensayada, los fragmentos provenientes de los geles cortados a la talla esperada para el antígeno específico prostático (33 kDa), se electroeluyeron a 4 °C. Mediante la comparación de las concentraciones de proteínas empleando la técnica de Lowry y el método radioinmunoenzimático, se cuantificó la cantidad de proteína biológicamente activa posterior a la electroforesis. Se utilizó un antígeno específico prostático comercial como control. Los resultados indican que el antígeno a 4 °C, conserva 80 por ciento de su actividad biológica, lo que no sucede a 25 °C donde más de 50 por ciento de la proteína es biológicamente inactiva(AU)

Detection of intracellular antigens by flow cytometry: comparison of two chemical methods and microwave heating.

Detection of intracellular antigens by flow cytometry requires effective fixation and permeabilization of the cell membrane. This study compares three fixation/permeabilization techniques: two commercial chemical reagents, the ORTHOPermeaFix (OPF) and the FIX&PERM Cell Permeabilization Kit (F&P), and a novel method based on microwave heating (MWH). They have been applied to the detection of two nuclear (p53 and rb/p105) and two cytoplasmic (bcl-2 and mdr-1/gp-170) antigens, using positive- and negative-control cell lines and peripheral blood mononuclear cells. Western blotting was performed as a control of protein expression. For the four antigens assessed, cellular morphology, discrimination between intact cells and debris, percentage of positive cells, and mean fluorescence intensity were examined. For this last parameter, the assessment of the MWH technique was performed using SD and a graphical approach inspired by the concepts described by Bland and Altman (Lancet 1986;346: 1085-7) as well as Petersen et al. (Clin Chem 1997;43: 2039-46). The statistical analysis shows that MWH is comparable to the commercial methods and that its reproducibility is also equivalent to OPF and F&P. As assessed for some of the most clinically relevant intracytoplasmic and intranuclear antigens, the MWH method appears to be a valuable and inexpensive alternative. It is worth noting that, unlike commercial reagents, MWH altered surface antigens. Interestingly, this feature, which would prevent cell selection on the basis of combined membrane and intracellular epitopes, is associated with a decrease of nonspecific background fluorescence.

Immunoreactivity of normal rabbit serum with epinephrine (E) cells of the rat adrenal medulla after microwave antigen retrieval.

Normal rabbit serum (NRS) produces intense staining of epinephrine (E) cells in microwave-heated sections of rat and mouse adrenal gland. This staining is not eliminated by liver adsorption, complement inactivation, high salt buffer, Triton X-100 or dilution in normal goat serum and bovine serum albumin (BSA), suggesting that it may result from specific antigen-antibody interactions. Western blots of adrenal medullary protein probed with NRS reveal several bands. The major band does not correspond to rat chromogranin A, which is a major constituent of E-cell secretory granules. The findings suggest that NRS may contain autoantibodies against a secreted rabbit E-cell protein with a homologous counterpart in rats and mice, and that this protein may be immunologically unmasked in situ by microwave heating. This phenomenon is a potential source of error in immunohistochemical studies of the adrenal medulla, and has potential biological significance in neuroimmunology.

Evaluation of immunohistochemical staining of human duodenal endocrine cells after microwave antigen retrieval.

The effect of microwave antigen retrieval on the immunostaining of human duodenal endocrine cells in formaldehyde-fixed, paraffin-embedded material was investigated. The sections were immunostained by the avidin-biotin complex (ABC) and immunogold-silver autometallography (IGSS) methods with and without prior microwave treatment. Dilutions of up to 1:30,000 of the following antisera/antibodies were used: anti-chromogranin A, anti-chromogranin AB, anti-secretin, anti-gastrin, anti-gastric inhibitory polypeptide, anti-somatostatin and anti-serotonin. The detection threshold for all the antibodies was lower after antigen retrieval, and the primary antibody could be used in higher dilutions. The dilutions varied for different antibodies and were between two and ten times the optimal dilution without antigen retrieval. At extremely high dilutions of, or without, the primary antibody, non-specific staining of some lymphocytes and the mucus of some goblet cells was observed when the avidin method was applied, but not with the immunogold technique. This phenomenon was not observed when optimal dilution or a lower dilution was used. This seems to have been caused by the binding of the avidin-biotin complex to epitopes in these structures unmasked by microwave treatment when competition with specific binding sites was absent.

Modulation of arrestin release in the light-driven regeneration of Rh1 Drosophila rhodopsin.

We report studies of the in vitro regeneration of Rh1 Drosophila rhodopsin using immunochemical and spectroscopic probes for the release of arrestin (49 kDa). Upon illumination of metarhodopsin-containing membrane suspensions isolated from homogenized Drosophila heads, arrestin was released into the aqueous medium. In contrast, no release of arrestin was observed upon illumination of metarhodopsin in lipid/detergent micellar extracts. The spectroscopic changes associated with the transition from metarhodopsin to rhodopsin were, however, similar in membrane suspensions and in micellar extracts. The light-driven release of arrestin was restored in reconstituted liposomes formed by dialysis of detergent from the micellar extracts. We conclude that micellar solubilization of membranes decouples the light-driven release of arrestin from rhodopsin structural changes which are responsible for altering the lambda max of the chromophore. The finding that arrestin release from rhodopsin can be modulated by changes in the local membrane environment provides an opportunity to further characterize the nature of rhodopsin conformational changes during regeneration.

Antigen retrieval by microwave oven heating for immunohistochemical analysis of bone marrow trephine biopsies.

Immunohistochemical analysis of bone marrow trephine (BMT) biopsies with monoclonal antibodies for the analysis of hemopoietic disorders has been hindered by the fixation and decalcification regimens which mask or destroy tissue antigens. This study evaluated the effect of microwave oven treatment on the quality of immunostaining of fixed decalcified trephine biopsies. The aim was to establish whether this method of pre-treatment would enable additional antigens to be detected. Fifty-eight monoclonal and 4 polyclonal antibodies to hemopoietic antigens were assessed to compare no tissue pre-treatment, proteolytic (trypsin) enzyme digestion and microwave oven heating. The microwave heating of the sections was performed by placing them in a boiling solution of 0.01M tri-sodium citrate for a total of 10 mins. Following microwave heating 14 antibodies that previously showed no reactivity in BMT biopsies gave positive staining and 9 antibodies previously known to detect antigens in the absence of pre-treatment gave enhanced staining. Other antibodies showed no staining improvement with microwave heating and some failed to give a positive reaction by any of the pre-treatment methods. Antigen retrieval utilizing microwave oven heating can expose antigenic sites for antibody binding in bone marrow trephine sections. However not all antigens are retrieved and there is variation between epitopes on the one molecule and their ability to be exposed by microwave heating. Utilizing antigen retrieval methods, the range of antibodies applicable to BMT sections is greatly expanded enabling the immunophenotypic analysis of the majority of hemopoietic disorders.

In vitro effects of ultraviolet B radiation on human Langerhans cell antigen-presenting function.

The effects of ultraviolet B radiation (UBV) on the immune function of human epidermal Langerhans cells (LC) were studied by using the mixed epidermal cell-lymphocyte reaction (MELR). Exposure of both enriched LC suspensions (eLC, 8-20% LC) and purified LC suspensions (pLC, 70-90% LC) to increasing doses of UVB radiation (25 to 200 J/m2) decreased the proliferative T cell response in a very similar dose-dependent way, suggesting that keratinocytes did not play a major role in the UVB-induced inhibition of MELR. Supernatants from irradiated cultured eLC or pLC failed to inhibit T cell proliferation induced by untreated pLC. Furthermore, addition of irradiated eLC to untreated pLC did not affec the allogeneic T cell response. Taken together, these results provide evidence that in vitro UVB-induced immunosuppression was not mediated by inhibitory soluble factors that could affect either LC allostimulatory property or T cell proliferative response. UVB irradiation of human LC inhibited the capacity of these cells to induce CD4+ as well as CD8+ T cell proliferation. UVB-irradiated LC also induced a decreased T cell response to recall antigen or mitogen. Moreover, addition of exogeneous cytokines such as IL-1 beta, IL-1 alpha, or IL-2 did not reverse the defective function of UVB-irradiated LC in MELR. The inhibitory effect of UVB radiation on human LC was not related to a decreased HLA-DR expression. Because cultured LC appeared to be less sensitive than freshly isolated LC to UVB-induced suppressive effects, the deleterious effects of UVB radiation on human LC allostimulatory properties may be associated with an impaired development of LC accessory function.

Reduction of antigenic protein levels in latex gloves after gamma irradiation.

Gamma irradiation is currently the method most commonly used to sterilize surgical gloves. In this study, the effect of gamma irradiation on antigenic proteins in latex gloves was examined. Protein extraction and quantitation were carried out using latex gloves before and after sterilization. Antigenic protein levels were determined by an ELISA assay specific for latex proteins (LEAP). LEAP analysis revealed a significant decrease after gamma-irradiation sterilization. This observation may partially explain the lower levels of extractable antigenic proteins found in sterile surgical gloves compared with nonsterile examination gloves. However, gamma irradiation was less effective than autoclave sterilization in reducing protein levels.

Direct covalent attachment of small peptide antigens to enzyme-linked immunosorbent assay plates using radiation and carbodiimide activation.

Direct adsorption of small peptide antigens to unaltered, commercially available polystyrene surfaces may be too weak to permit suitable assay by ELISA. We therefore developed a simple method for the covalent attachment of small, potentially single epitope antigens to polystyrene surfaces. Chemical activation of polystyrene plates with carbodiimide considerably improves the total and covalent attachment of radioactive octapeptides. The covalent attachment was demonstrated by washing with hot detergent. A 3.5 Mrad gamma-irradiation of plates also increases total binding, particularly in combination with chemical activation. The covalent attachment presumably occurs through formation and chemical activation of carboxylate functions on the polystyrene surface which form amide bonds with peptides. ELISA test was performed with CGRP and successive smaller CGRP fragments. Covalent attachment of C-terminal peptide fragments as detection antigens allows optimal recognition and sensitivity even for hexapeptides, while decapeptide antigens were already poorly recognized using a conventional antigen plating technique. Repetitive detergent washes and/or prolonged storage of plates with covalently bound antigens did not reduce their ELISA sensitivity. The method with storage and reutilization capacities that we present here will be useful for the development of preplated antibody screening test.

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.

Microwave antigen retrieval in immunocytochemistry: a study of 80 antibodies.

AIMS: To evaluate the effect of microwave irradiation on the staining quality of a range of commonly used primary antibodies in archival, formalin fixed, paraffin wax embedded material, with emphasis on antibodies that have previously worked successfully only on frozen tissue. METHODS: Immunocytochemistry (streptavidin-biotin complex technique) was performed on histological sections of a range of normal and pathological tissues, after varying treatment with microwave irradiation. The staining quality of each antibody was compared with that achieved without prior treatment of the sections or after enzyme predigestion. RESULTS: Microwave irradiation permitted successful immunostaining with 20 antibodies that stained only frozen tissues before. The staining characteristics of 21 antibodies that were already known to stain formalin fixed, paraffin wax embedded material were improved. Another 39 antibodies did not show enhanced staining with microwave irradiation. The method preserves tissue morphology and produces more consistent staining than that achieved by enzyme predigestion with many antibodies. Microwave irradiation may also allow some primary antibodies to be used at higher working dilutions. The citrate buffer used in this study avoids the necessity of exposure to heavy metal salts. CONCLUSIONS: Microwave antigen retrieval represents an important technical advance within immunocytochemistry that will greatly increase the range of antibodies which can be used to study formalin fixed, paraffin wax embedded tissues.

Phosphorylated rhodopsin and heparin induce similar conformational changes in arrestin.

Photoactivated rhodopsin is quenched upon its phosphorylation in the reaction catalyzed by rhodopsin kinase and the subsequent binding of a regulatory protein, arrestin. We have found that heparin and other polyanions compete with photoactivated, phosphorylated rhodopsin to bind arrestin (48-kDa protein, S-antigen). This is shown (a) by the suppression of stabilized metarhodopsin II; (b) by changes in the digestion of arrestin in the presence of heparin; and (c) by the restoration of arrestin-quenched phosphodiesterase activity. When bound to arrestin, heparin also mimics phosphorylated rhodopsin by similarly exposing arrestin to limited proteolysis. We conclude that heparin and rhodopsin have similar means of binding to arrestin, and we propose a cationic region of arrestin (beginning with Lys163 of the bovine sequence) as the interaction site. In agreement with previous kinetic data we interpret the results in terms of a binding conformation of arrestin which is stabilized by rhodopsin or heparin and is open to proteolytic attack.

Arrestin from nucleated red blood cells binds to bovine rhodopsin in a light-dependent manner.

Using a panel of monoclonal antibodies, it has previously been demonstrated that the cytosol of nucleated red cells (trout and turkey) contains a protein similar to arrestin, a soluble protein found so far only in the photosensitive cells and which, by binding to photoexcited rhodopsin, inhibits the phototransduction process. The role of this arrestin-like protein in non-photosensitive cells is questionable. In this report we present evidence that partially purified red blood cell arrestin (RBC arrestin) behaves functionally like bovine retinal arrestin: it binds to phosphorylated bovine rhodopsin only when this receptor has been photoactivated. Thus RBC arrestin and bovine retinal arrestin are closely related both structurally and functionally. By analogy with the function of retinal arrestin, it is proposed that RBC arrestin is involved in desensitization of membrane transport proteins and/or adrenergic receptors.

The effect of short-wavelength ultraviolet light on antigens, lection receptors and the ultraestructure of Crithidia fasciculata

Crithidia fasciculata is an important trypanosomatid parasite commonly affecting insects and is used extensively as a model for the study of the biochemistry, ultrastructure and organization of the kDNA network of trypanosomatids. The present study describes the evolution of UV-induced morphological changes detectable by transmission electron microscopy in Crithidia fasciculata. Although only rare and minor changes in Kinetoplast DNA were demonstrable 7 h after UV irradiation, alterations of this orgtanelle were present in almost al flagellates observed 24 h and 48 h after irradiation. Other cell structures were apparently undamaged. Ultrastructural changes in kDNA did not correspond to changes in antigenicity of protein bands in western blotting against serum from Chagas' disease patients or in the presence of 3 different lectin receptors on the surface of the parasite