Pathogenic cellulase assay of pine wilt disease and immunological localization

The pine wilt disease caused by Bursaphelenchus xylophilus (BX), also known as the pine wood nematode (PWN), is the most devastating disease of pine trees. In this work, a high molecular weight B. xylophilus cellulase antigen (BXCa) was purified from total homogenates of nematodes. BXCa was found to be able to hydrolyze carboxymethyl cellulose (CMC) efficiently (155.65 U/mg) and to have an approximate molecular mass of 58.9 kDa. We harvested anti-BXCa antibodies and performed immunocytochemical assays, which revealed the localization of cellulase pools in the esophageal gland cells of the PWN. It was also discovered that cellulase was secreted from the stylet and was used to hydrolyze cellulose to facilitate the PWN entering host cells. These results are consistent with other plant parasitical nematodes. Interestingly, strong fluorescence signals from cellulase staining were observed in tracheid cells in naturally infected pine wood, in addition to ray cells and the resin canal zone. These results strongly suggest that the cellulase released by the PWN is one of the pathogenic substances of pine wilt disease and is responsible for the development of the early symptoms of the disease.     

Ultrastructural localization of factor VIII-related antigen in cultured human brain microvessel endothelial cells.

Immunogold staining of primary cultures of human brain microvessel endothelial cells demonstrated the presence of Factor VIII-related antigen within cytoplasmic vesicles in close association with the rough endoplasmic reticulum and Golgi apparatus. Immunoperoxidase staining, at the light microscopic level, revealed a similar granular, perinuclear staining. The morphology and location of these vesicular profiles indicate that they are part of the trans-Golgi region where terminal processing and short-term storage of Factor VIII-related antigen takes place. Weibel-Palade bodies, specific storage organelles for von Willebrand factor in large vessel endothelium, were not observed in cerebral microvessel endothelium. The release of Factor VIII-related antigen from the cytoplasmic vesicles was influenced by some of the factors known to stimulate or inhibit the regulated pathway of secretion from Weibel-Palade bodies. Thus, stimulation of endothelial cells with calcium ionophore A23187 resulted in almost complete loss of staining, while addition of EGTA to the culture medium led to slight increase of intracellular pools of Factor VIII-related antigen. Pre-incubation of monolayers with interferon-gamma was associated with significant increase in the number of labeled vesicles, suggesting an additional role of this cytokine in the localized immune reaction within the central nervous system.     

Pathogenic Cellulase Assay of Pine Wilt Disease and Immunological Localization

The pine wilt disease caused by Bursaphelenchus xylophilus (BX), also known as the pine wood nematode (PWN), is the most devastating disease of pine trees. In this work, a high molecular weight B. xylophilus cellulase antigen (BXCa) was purified from total homogenates of nematodes. BXCa was found to be able to hydrolyze carboxymethyl cellulose (CMC) efficiently (155.65 U/mg) and to have an approximate molecular mass of 58.9 kDa. We harvested anti-BXCa antibodies and performed immunocytochemical assays, which revealed the localization of cellulase pools in the esophageal gland cells of the PWN. It was also discovered that cellulase was secreted from the stylet and was used to hydrolyze cellulose to facilitate the PWN entering host cells. These results are consistent with other plant parasitical nematodes. Interestingly, strong fluorescence signals from cellulase staining were observed in tracheid cells in naturally infected pine wood, in addition to ray cells and the resin canal zone. These results strongly suggest that the cellulase released by the PWN is one of the pathogenic substances of pine wilt disease and is responsible for the development of the early symptoms of the disease.     

Immunocytochemical differentiation between adipokinetic hormone (AKH)-like peptides in neurons and glandular cells in the corpus cardiacum of Locusta migratoria and Periplaneta americana with C-terminal and N-terminal specific antisera to AKH

Summary An immunocytochemical method was used to differentiate between immunoreactive substances in glandular cells in the corpora cardiaca (CC) and in certain cerebral neurons in 2 insect species, Locusta migratoria migratorioides and Periplaneta americana. The staining properties of antisera raised to different parts of the decapeptide adipokinetic hormone (AKH) were compared and their specificity was determined by preabsorption with AKH and related peptides. Antibodies raised to the N-terminal part of AKH (serum 433) and the central and C-terminal part (serum 241) were found to have different staining properties.In the CC of the locust both antisera show a strong immunoreactivity with glandular cells, we therefore suggest that at least one of the compounds revealed is AKH. Some of the glandular cells in the locust and large numbers of glandular cells in the CC of the cockroach are revealed by the N-terminal specific antiserum. On the other hand, neurons in the central nervous system are revealed only by the C-terminal specific antiserum. The possible identity of the various substances revealed by these two antisera is discussed.     

Sensitivity and efficiency of four immunohistochemical methods as defined by staining of artificial sections

Direct (DIF) and indirect (IIF) immunofluorescence, indirect immunoperoxidase conjugate (IPC) and unlabelled antibody peroxidase antiperoxidase (PAP) staining was performed on sections of artificial substrate containing different concentrations of human immunoglobulin (Ig)A or IgG. Detection sensitivity, in terms of the lowest amount of discernible antigen, was evaluated by direct microscopy and by microphotometry. Staining efficiency (signal-to-noise ratio) was evaluated by microphotometry. Only minor differences in antigen detection sensitivity were found when IPC and PAP were compared with DIF and IIF under appropriate conditions. The sensitivity of DIF was only marginally improved by raised conjugate concentration and prolonged incubation time. Microphotometry of DIF on ethanol-fixed IgA substrate revealed that the staining intensity increased proportionally with the antigen concentration whereas on formaldehyde-fixed substrate a progressive masking of the antigen was indicated which, however, could be overcome by applying raised conjugate concentration and prolonged incubation time. Such antigenic self masking was of relatively little importance to IPC and PAP staining, probably because of the inherent amplification in these methods. An additional masking effect due to extraneous protein was revealed by DIF when ethanol-fixed sections had been soaked in bovine serum albumin and postfixed with formaldehyde; unmasking was achieved by proteolytic treatment of the sections.     

Sensitivity and efficiency of four immunohistochemical methods as defined by staining of artificial sections

Summary Direct (DIF) and indirect (IIF) immunofluorescence, indirect immunoperoxidase conjugate (IPC) and unlabelled antibody peroxidase antiperoxidase (PAP) staining was performed on sections of artificial substrate containing different concentrations of human immunoglobulin (Ig)A or IgG. Detection sensitivity, in terms of the lowest amount of discernible antigen, was evaluated by direct microscopy and by microphotometry. Staining efficiency (signal-to-noise ratio) was evaluated by microphotometry. Only minor differences in antigen detection sensitivity were found when IPC and PAP were compared with DIF and IIF under appropriate conditions. The sensitivity of DIF was only marignally improved by raised conjugate concentration and prolonged incubation time. Microphotometry of DIF on ethanol-fixed IgA substrate revealed that the staining intensity increased proportionally with the antigen concentration whereas on formaldehyde-fixed substrate a progressive masking of the antigen was indicated which, however, could be overcome by applying raised conjugate concentration and prolonged incubation time. Such antigenic self masking was of relatively little importance to IPC and PAP staining, probably because of the inherent amplification in these methods. An additional masking effect due to extraneous protein was revealed by DIF when ethanol-fixed sections had been soaked in bovine serum albumin and postfixed with formaldehyde; unmasking was achieved by proteolytic treatment of the sections.This work was supported by the Norwegian Cancer Society, the Norwegian Research Council for Science and the Humanities, and Anders Jahres Foundation     

Relation between ultrastructural presence of CEA using peroxidase-antiperoxidase (PAP) method and tissue and serum levels of CEA in patients with breast cancer.

Ultrastructural studies were performed to detect the presence of carcinoembryonic antigen (CEA) in breast cancer by peroxidase-antiperoxidase (PAP) method. The evidenced presence of CEA was compared with the serum and tissue concentrations. A correlation between the presence of CEA at the ultrastructural level and tissue concentration was observed but not with serum levels. These studies revealed positive immunocytochemical staining for CEA when antigen concentration was 700 ng/g tissue and the reaction was strongly positive when the concentration was greater than or equal to 2000 ng/g tissue.     

Detection of Proliferating Cell Nuclear Antigen in Tissues of Three Small Fish Species

An immunohistochemical assay for proliferating cell nuclear antigen (PCNA) identifies cells in all active phases of the cell cycle. In this study, PCNA methodology, which was developed primarily for mammalian tissues, was adapted to three small fish species, medaka (Ory-zias latipes), guppy (Poecilia reticulata), and western mosquitofish (Gambusia affiinis) that are used in carcinogenesis bioassays and environmental sentinel studies. Our study showed that PCNA can be identified in routinely processed, paraffin embedded specimens of these fishes. Optimum staining conditions were dependent on fixative, primary antibody, antigen retrieval processing. and protein blocking reagent. Best results were achieved using 10% neutral buffered formalin as the fixative, clone PC10 as the primary antibody, and a combination of powdered milk and bovine serum albumin as a protein block. Except for medaka specimens, antigen retrieval was not required for specimens preserved in 10% neutral buffered formalin. but was required for the other fixatives tested. In whole fish specimens, PCNA marked cells in normally proliferating tissues such as testis. ovary, primary filament epithelium of the gill, hematopoietic tissues, thymus, retina and alimentary tract. The study demonstrated the successful application of mammalian-based PCNA technology to these aquatic species. Further applications of the assay will aid in understanding the role of cell proliferation in normal, diseased, and toxicant-affected tissues of aquatic animals.     

蝗虫消化道形态结构研究的一种新方法

采用扫描仪和扫描电镜相结合的方法对中华稻蝗Oxyachinensis(Thunberg)消化道内部的超微结构进行定位研究。结果表明直接将蝗虫消化道的内壁翻出(不染色)铺展于扫描仪的玻璃板上进行扫描,就能得到蝗虫消化道在自然状态下的完整图像,将扫描仪扫描后的样品用于扫描电子显微镜的样品制备,并对照上述研究结果,即可定位蝗虫消化道在扫描电子显微镜下的超微结构。为蝗虫消化道的形态学研究提供一个简便有效的方法,同时也为蝗虫消化道超微结构的定位研究提供新的手段。     

Correlation between Production of Inflammatory Substances and Generation of Effector T Cells Mediating Delayed-Type Hypersensitivity in Mice

In vitro exposure to human serum albumin (HSA) of splenic lymphocytes from mice sensitized for delayed-type hypersensitivity (DTH) against HSA resulted in the release of substances that could induce a footpad inflammatory reaction with a maximum 6 hr after injection into normal mice. The substances were fractionated mainly in a molecular weight range of 30,000 to 70,000 daltons on Sephadex G-200. The ability of sensitized lymphocytes to produce the substances was dependent on T cells, was antigen specific, and correlated well with the ability of the lymphocytes to mediate DTH reactions. Moreover, the substances were produced efficiently by the DTH effector cell population generated in the in vitro culture system and also by the effector cell-enriched fractions on discontinuous bovine serum albumin gradients. These results suggest that the substances are produced by DTH-effector cells.     

Propagation of human parvovirus B19 in primary culture of erythroid lineage cells derived from fetal liver.

Erythroid lineage cells derived from fetal liver were demonstrated to be target cells for human parvovirus B19 infection. B19 virus antigen-positive serum was inoculated into primary cultures containing erythroid lineage cells enriched from fetal liver. The B19 virus antigen was detected on about 5% of cells in the culture by immunofluorescence staining, and the stained cells were identified as erythroid lineage cells by double staining with anti-B19 virus-positive serum and anti-erythroid lineage monoclonal antibody. The immunofluorescence staining study also revealed that the B19 virus antigen localized in the nucleus and the periphery of cytoplasm. We also detected B19 virus DNA, which was generated by replication in the infected cells, not only in the cells but also in the culture supernatants, in which the amount of B19 DNA increased depending on the period of culture, indicating that the cells infected with B19 virus produced B19 virus and released it into the medium. The ability of B19 virus released into the medium to infect fetal erythroid lineage cells was demonstrated quantitatively. Because of the absence of any cytopathic effect of B19 virus during culture periods of at least 15 days, this culture system should be useful in the study of B19 virus replication and in vitro generation of B19 virus. In addition, the present study may contribute to a better understanding of the pathogenesis of hydrops fetalis, which is probably associated with B19 virus infection during pregnancy.     

Expression of carbohydrate antigens in the goat uterus during early pregnancy and on steroid-treated polarized uterine epithelial cells in vitro

Our objectives were to determine whether specific fucosylated carbohydrate antigens, associated with uterine receptivity in rodents, are expressed in pregnant caprine uterine tissues and polarized uterine luminal epithelial (ULE) cells in culture. Immunofluorescence microscopy on frozen endometrium revealed that expression of the H-type 1 antigen, confined to epithelial cells, was regulated during early pregnancy. Staining was high on Day 5 and low on Days 11 and 13. Strong, uniform apical staining was characteristic of ULE cells between Days 15 and 19 but declined markedly by Day 25. Immunofluorescence analysis of the apical surface of polarized ULE cells cultured in steroid-free medium revealed weak and diffuse staining for the H-type 1 antigen, while progesterone (P(4)) treatment resulted in the formation of aggregates of punctate staining along the apical surface. Domain-specific biotinylation of polarized ULE cells, coupled with streptavidin precipitation and Western blotting, revealed that six apical surface proteins (31, 33, 42, 55, 60, and 70 kDa) carry the H-type 1 antigen. Therefore, H-type 1 antigen expression is up-regulated in vivo during the periimplantation period, stimulated by P(4) on polarized ULE cells in culture, and may be a useful marker for uterine receptivity in this species.     

Immunological study of lung development in the mouse embryo. Appearance of a lung-specific antigen, localized in the great alveolar cell.

After preparation of an antiserum specifically recognizing the antigenic determinants of adult mouse lung tissue, the existence of a lung-specific antigen could be demonstrated by immunodiffusion studies in agar. This specific anti-adult lung serum was used in immunofluorescence studies to localize the corresponding antigen in adult mouse lung tissue. Comparison of the results of immunofluorescent staining with those of histochemical enzyme staining showed that the lung-specific antigen is localized in the great alveolar cell.Subsequently, the same antiserum was used in immunodiffusion studies to follow the development of the lung-specific antigen in the mouse embryo. The results indicate that the lung-specific antigen first appears in embryos with a weight of between 760 and 900 mg, which corresponds with a developmental age of between 16.76 and 17.23 days.     

Studies on Complement-Fixation Reaction in Equine Infectious Anemia

The substances responsible for inhibiting complement-fixation (CF) reaction of the late-stage serum of an equine infectious anemia (EIA)-infected horse were investigated. It was found that the IgG and IgG(T) classes in the late-stage serum were responsible for the CF inhibition. IgA could not be detected in partially purified IgG(T) by an immunodiffusion test using rabbit anti-human IgA serum. Other serum components could not be demonstrated in purified IgG by immunoelectrophoresis using rabbit anti-horse serum. The IgG class simultaneously showed CF and CF-inhibiting (CFI) activities, whereas the IgG(T) class showed only CFI activity. The IgG(T) class could exert CFI activity only when it had been reacted with the EIA antigen before addition of the reference CF serum and complement. In contrast, the IgG class converted the CF-active reference serum into a non-CF-reactive one irrespective of whether it was simultaneously reacted with the EIA antigen and the reference CF serum, whether it was added to the reaction mixture of the EIA antigen and the reference CF serum, or whether it was sensitized with the EIA antigen before addition of the reference CF serum. Inhibitory activities of the IgG and IgG(T) classes seemed to be different from each other in their reaction pattern as far as tested under our experimental conditions. Their CFI activities seemed to be specific for EIA, being negative in CFI activity in reaction with other antigens.     

Studies on complement-fixation reaction in equine infectious anemia. II. Identification of complement-fixing inhibitors.

The substances responsible for inhibiting complement-fixation (CF) reaction of the late-stage serum of an equine infectious anemia (EIA)-infected horse were investigated. It was found that the IgG and IgG(T) classes in the late-stage serum were responsible for the CF inhibition. IgA could not be detected in partially purified IgG(T) by an immunodiffusion test using rabbit anti-human IgA serum. Other serum components could not be demonstrated in purified IgG by immunoelectrophoresis using rabbit anti-horse serum. The IgG class simultaneously showed CF and CF-inhibiting (CFI) activities, whereas the IgG(T) class showed only CFI activity. The IgG(T) class could exert CFI activity only when it had been reacted with the EIA antigen before addition of the reference CF serum and complement. In contrast, the IgG class converted the CF-active reference serum into a non-CF-reactive one irrespective of whether it was simultaneously reacted with the EIA antigen and the reference CF serum, whether it was added to the reaction mixture of the EIA antigen and the reference CF serum, or whether it was sensitized with the EIA antigen before addition of the reference CF serum. Inhibitory activities of the IgG and IgG(T) classes seemed to be different from each other in their reaction pattern as far as tested under our experimental conditions. Their CFI activities seemed to be specific for EIA, being negative in CFI activity in reaction with other antigens.     

Cuterebra buccata: immune response in myiasis of domestic rabbits

Naturally infected rabbits (Oryctolagus) were used to define further the nature of the immune response in myiasis due to Cuterebra buccata. Third instar larvae were dissected into four fractions; (1) alimentary tract with attached organs, (2) hemolymph, (3) fat body with tracheae, and (4) cuticle with attached muscles. The antigens provoking immune phenomena in naturally infected rabbits were found to reside in the alimentary tract and hemolymph fractions only. All rabbits which were skin tested were found to exhibit delayed hypersensitivity and to have serum precipitins with specificity against these antigens. Passive cutaneous anaphylaxis activity was demonstrated only in sera from rabbits also exhibiting reaginic and/or Arthus-type skin hypersensitivities. With the use of immunoelectrophoresis four separate antigens were demonstrated in alimentary tract fractions. Larval dissections revealed the alimentary tract to be filled with cellular elements of rabbit whole blood. The immunologic findings are discussed in relation to this newly recognized feeding pattern and it is proposed that sensitization of the host occurs as a consequence of exogenous larval secretions injected at the time of feeding.     

Histochemical reactivity of soybean agglutinin with blood group antigens and their precursor substances in acinar cells of human pancreas

In human pancreas, soybean agglutinin (SBA) conjugated to horseradish peroxidase reacted with the acinar cells secreting blood group A and/or H antigen, but not with those secreting only B antigen. For detailed histochemical characterization of SBA staining, the effects of treatment with unlabeled lectins and of digestion of certain enzymes on SBA staining were investigated in formalin-fixed, paraffin-embedded pancreatic tissue from individuals of different blood groups. Pre-incubation of sections with unlabeled Dolichos biflorus agglutinin to block A antigen eliminated subsequent SBA staining in the cells secreting A antigen, although failing to induce any effects in those secreting H antigen. In contrast, pre-incubation with unlabeled Ulex europaeus agglutinin-I (UEA-I) to block H antigen abolished SBA staining in cells secreting H antigen but not in those secreting A antigen. Treatment with galactose oxidase yielded the same results as those with unlabeled UEA-I, i.e., SBA reactivity was significantly diminished in cells secreting H antigen but not in those secreting A antigen. Digestion with beta-galactosidase resulted in a slight decrease of SBA staining in the cells secreting H antigen. Accompanying the decrease of SBA staining, reactivity with Griffonia simplicifolia agglutinin-II (GSA-II) appeared for the first time in the enzyme-susceptible, SBA-reactive cells secreting H antigen. Pre-treatment with galactose oxidase abolished this effect of beta-galactosidase. The GSA-II reactivity disclosed by treatment with galactosidase was completely eliminated by digestion with beta-N-acetylhexosaminidase, indicating that GSA-II staining after digestion with galactosidase is due to exposed penultimate beta-N-acetyl-D-glucosamine residues. These results demonstrate that at least two substances react with SBA in acinar cells of human pancreas, one being terminal beta-N-acetyl-D-galactosamine residues of A antigen, and the other being terminal beta-D-galactose-(1----3 or 1----4)-beta-N-acetyl-D-glucosamine dimers in the precursor of blood group H antigen. Such dimers may exist in close proximity to L-fucose residues of H antigen, since unlabeled UEA-I blocked SBA staining.     

An unpleasant souvenir: Endoscopic finding of Trichuris trichiura (Nematoda: Trichuridae)

Whipworms are responsible for up to 500 million cases of trichuriasis worldwide, with higher endemicity in tropical and sub-tropical countries. In non-endemic countries, trichuriasis can be accidentally diagnosed upon colonoscopy, often in the presence of negative microscopy. Here, we describe an incidental diagnosis of trichuriasis in an HIV patient residing in a non-endemic area (i.e., Turin, Italy), six months after his return from Antigua. The species-level diagnosis was made thanks to PCR-based molecular identification of Trichuris sp. following optical microscopy detection. Overall, this case highlights the importance of improving parasitic diseases diagnosis through cutting-edge clinical and laboratory diagnostic tools alongside advanced training of specialists in the area of parasitology.