Mycobacterium avium Subsp. paratuberculosis Induces Specific IgE Production in Japanese People with Allergies.

Background. The prevalence of allergies is steadily increasing worldwide; however, the pathogenesis is still unclear. We hypothesized that Mycobacterium avium subsp. paratuberculosis (MAP) may contribute to allergy development. This organism can be present in dairy foods, it can elicit an immunomodulatory switch from a Th1 to a Th2 response, and it has been speculated that it is linked to several human autoimmune diseases. To determine the contribution, sera from 99 individuals with various atopic disorders and 45 healthy nonallergic controls were assessed for total IgE levels and successively for MAP-specific IgE by ELISA. Results. The mean total serum IgE level in allergic patients was 256 ± 235 IU/mL, and in the healthy controls it was 62 ± 44 IU/mL (AUC = 0.88; p < 0.0001). Among the patient groups, 50 of the 99 subjects had increased IgE total level ≥ 150 IU/mL, while 49 subjects had IgE ≤ 150 IU/mL (mean level: 407 ± 256 IU/mL versus 106 ± 16 IU/mL; p < 0.0001). Additionally, 6 out of 50 subjects (12%) with IgE ≥ 150 IU/mL and none (0%) with IgE ≤ 150 IU/mL were positive for specific MAP IgE (AUC = 0.63; p = 0.03). Conclusion. The present study revealed that MAP has the ability to induce specific IgE and might contribute to the induction of allergic inflammation in genetically predisposed individuals.

Role of TNF-alpha in muscularis inflammation and motility disorder in a TNBS-induced colitis model: clues from TNF-alpha-deficient mice.

Macroscopic and histological analysis revealed that the colonic inflammation induced by 2,4,6-trinitrobenzenesulphonic acid (TNBS) was of lower grade in tumour necrosis factor-alpha (TNF-alpha)(-/-) mice than in wild-type mice. Myeloperoxidase activity, an indicator of neutrophilic infiltration, was also low in both the mucosal and smooth muscle layer of the TNF-alpha(-/-) mouse colon. After the induction of inflammation with TNBS, the levels of proinflammatory cytokines, such as TNF-alpha, interleukin-1beta and interleukin-6, were elevated both in the inflamed mucosa and muscle layers in the wild-type mice; however, the productions of these cytokines were greatly reduced in the TNF-alpha(-/-) mouse colon. The contractions of isolated colonic smooth muscle strips induced by several stimulatory agents were significantly decreased after treatment with TNBS in wild-type mice; however, these contractions were scarcely affected in TNF-alpha(-/-) mice. Finally, using the organ culture method, we found that TNF-alpha directly (independent of mucosal inflammation) disturbs the smooth muscle function. These results suggest that TNF-alpha plays an essential role not only in mucosal inflammation but also in muscularis inflammation in the colon of mice with TNBS-induced colitis, and that TNF-alpha directly induces motor dysfunctions by acting on the smooth muscle.

Myxoid leiomyosarcoma of the gizzard in a broiler chicken.

A leiomyosarcoma was found in the gizzard of a 57-day-old female broiler chicken weighing 1.8 kg. Grossly, the tumor mass, 13.0 x 8.5 x 10.0 cm, enveloped the gizzard and had a gelatinous appearance due to the rich production of mucin. Miliary metastatic tumors were noted in the liver. Histopathologically, there was marked production of mucus throughout the tumor tissue, and densely or loosely arranged long spindle-shaped leiomyosarcoma cells proliferated. The tumor cells had a low rate of mitosis, showed slight cellular atypia, and, immunohistochemically, were positive for actin, alpha-smooth muscle actin, and desmin. Electron microscopically, various amounts of microfibrils with focal densities, dense patches, and basal plates were observed.

Salivary gland extract of Rhipicephalus appendiculatus ticks inhibits in vitro transcription and secretion of cytokines and production of nitric oxide by LPS-stimulated JA-4 cells.

There is increasing evidence that compounds in tick saliva and salivary gland extract (SGE) have a suppressive effect on host immunity and that tick-borne pathogens exploit this situation to their benefit thus causing diseases. We have demonstrated that SGE derived from Rhipicephalus appendiculatus ticks has a suppressive effect on a macrophage like cell line, JA-4, in terms of secretion as well as mRNA transcription of three cytokines. Percent suppression of cytokine secretion by JA-4 cells cultured in the presence of lipopolysaccharide (LPS) and SGE in comparison to JA-4 cells cultured in the presence of LPS alone was 67.8, 89.1 and 82.0% for IL-1alpha, TNF-alpha and IL-10, respectively (P<0.05). A similar pattern of results was demonstrated in terms of mRNA transcription where SGE-induced suppression was 36.9% for IL-1alpha, 25.0% for TNF-alpha and 31.5% for IL-10 (P<0.05). In addition, we have demonstrated that SGE partially inhibited nitric oxide production by JA-4 activated with LPS. The results of the present study suggest that tick salivary gland compounds may exert their effect in vivo by blocking the functions of macrophages in the transcription of cytokines and production of nitric oxide. This SGE-induced immunomodulation may comprise a major gateway in the facilitation of tick feeding and transmission of pathogens in hosts.

Expression of Kit, the receptor for stem cell factor, in bovine peripheral blood.

The expression of Kit, the receptor for stem cell factor (SCF), on bovine peripheral blood cells (PBCs) was examined by using monoclonal antibodies against the bovine Kit protein. Flow cytometric analysis showed that approximately 1.5% of PBCs expressed Kit. In cytospin preparations, the morphology of most Kit+ PBCs was similar to that of large lymphocytes. Subsets of Kit+ PBCs coexpressed CD3, IgM, and/or CD11b but not CD14 or G1. SCF did not induce the proliferation of Kit+ PBCs in vitro. These results indicate that Kit is expressed on subsets of lymphocytes in bovine peripheral blood, but the ligand of Kit, SCF, does not directly induce the proliferation of this cell population.

Dissecting aortic aneurysm in a horse.

A case of dissecting aortic aneurysm in a 4-year-old male thoroughbred horse is reported. The horse had a history of inflammation in the right thigh and a fever 2 weeks before sudden death. At necropsy, aortic aneurysms were observed from the aortic valve to the aortic arch, spreading over a distance of 40 cm. An irregular rupture of the intima of the ascending aorta was located in the cardiac side of a ramification to the tunica branchiocephalicus communis. An intramural haematoma, apparent on the cut surface and in the pericardium, had caused cardiac tamponade and sudden death.

Synthesis and characterization of bactericidal oligopeptides designed on the basis of an insect anti-bacterial peptide.

Defensin from a beetle, Allomyrina dichotoma, is known to have anti-bacterial activity against Gram-positive bacteria. This peptide, which comprises 43 amino acid residues, was effective against methicillin-resistant Staphylococcus aureus. We identified the active site of beetle defensin by measuring anti-bacterial activity against S. aureus of 64 overlapping 12-mer peptides with either a free carboxylate or a free amide group at their C-termini. An LCAAHCLAIGRR-NH2 (19L-30R-NH2) fragment showed the greatest activity of the synthetic oligopeptides. The 19L-30R-NH2 fragment was effective against both Gram-positive and Gram-negative bacteria. CD spectra showed that the 19L-30R-NH2 fragment formed an alpha-helical structure in the lipidic environment. The anti-bacterial effect of the 19L-30R-NH2 fragment was due to its interaction with bacterial membranes, judging from the leakage of liposome-entrapped glucose. Its anti-bacterial activity was increased when certain amino acid residues were replaced. Truncated peptides having had some amino acids removed from the N-terminus of the 19L-30R-NH2 fragment (8-10-mer peptides) still had strong anti-bacterial activity. Deleting some amino acids from the C-terminal region of the fragment dramatically reduced activity, indicating that the C-terminal region of the 19L-30R-NH2 fragment, i.e. RR-NH2, is important for exerting anti-bacterial activity. The AHCLAIGRR-NH2 (22A-30R-NH2) fragment and its analogues exhibited about 3-fold and 9-12-fold higher activity against S. aureus than did the 19L-30R-NH2 fragment, and these analogues were effective against methicillin-resistant S. aureus and Pseudomonas aeruginosa isolated from patients. These oligopeptides showed no haemolytic activity and did not inhibit the growth of murine fibroblast cells.

Exacerbated spongiform lesions in the cerebral cortex in Japanese sheep, in an outbreak of scrapie during 1984-1987.

The present study dealt with the pathology of natural scrapie in Japanese Suffolk sheep in a certain selected area. Vacuolations in the cytoplasm of neurons were conspicuous. They were particularly evident in many areas of the medulla and pons, extending into and through pedunculus cerebri and thalamus to the septal area and olfactory tubercle. Proliferation of astrocytes was also easily observed with glial fibrillary acidic protein staining. Neural vacuolations in the cerebral cortex were observed in 73% of the cases. Abnormal prion protein deposits were seen in all cases observed by hydrolytic autoclaving, and subsequent peroxidase and anti-peroxidase immunostaining. Abnormal prion protein staining was the most conspicuous in the polymorphic layers of the hippocampus.

Cloning and sequencing of cDNA encoding bovine macrophage colony-stimulating factor (bM-CSF) and expression of recombinant bM-CSF using baculovirus.

The cDNAs encoding bovine macrophage colony-stimulating factors alpha and beta (M-CSF alpha and M-CSF beta) were cloned and recombinant bovine M-CSF alpha (rbM-CSF beta) in its dimeric form was expressed by using a recombinant baculovirus/insect cell system. The predicted amino acid sequence of rbM-CSF alpha and rbM-CSF beta shared 83.3 and 75.9% (alpha), 75.3 and 65.9% (beta) similarity with the sequence for human and murine M-CSFs, respectively. The biological activity of rbM-CSF beta was confirmed by the colony-forming assay using mouse bone marrow cells. SDS-PAGE under a reducing condition showed that the molecular weight of rbM-CSF beta was approximately 34 kDa. On the other hand, Western blot analysis under a non-reducing condition revealed that this rbM-CSF beta was secreted in dimeric form into the cell supernatant.

Expression of biologically active recombinant porcine GM-CSF by baculovirus gene expression system.

The full length porcine granulocyte/macrophage colony stimulating factor (GM-CSF) cDNA, including secretion signal peptide coding region was recloned into baculovirus transfer vector pAcYM1. The vector was then transfected with Autographica californica nuclear polyhedrosis virus (AcNPV) DNA into SF21AE cells and the recombinant virus AcPGM was recovered. Recombinant porcine GM-CSF (rpGM-CSF) was obtained from the serum-free culture medium of Tn5 cells infected with the AcPGM virus, and was shown to be a glycosylated 21 kDa protein as confirmed by tunicamycin treatment and [3H]-glucosamine uptake. The biological activities of rpGM-CSF in AcPGM-infected cell culture supernatants were demonstrated by porcine bone marrow cell proliferation and haematopoietic cell colony formation assays. The use of rpGM-CSF enabled us to culture porcine monocytes/macrophage and dendritic-like cells, derived from either porcine bone marrow or peripheral blood, for up to 4 months.

The effect of oligosaccharides on the production of tumor necrosis factor-alpha by macrophage-like cell line J774/JA-4.

Stimulation and modulation of tumor necrosis factor-alpha (TNF-alpha) production during treatment of the murine macrophage-like cell line J774/JA-4 with 25 oligosaccharides were studied. Direct stimulation of TNF-alpha production by oligosaccharides was measured with a cytotoxic assay using the L929 cell line. Twelve samples showed a significantly higher production (P < or = 0.01) of TNF-alpha than the controls. Modulation of TNF-alpha production by treatment with oligosaccharides, followed by stimulation with lipopolysaccharide (LPS) from E. coli, was examined using the L929 bioassay system. In three samples TNF-alpha production increased significantly, but in four samples, production was reduced significantly (P < or = 0.01). No samples showed modulation of growth or viability of L929 cells within the first 26 hr. The present results are useful in the application of these oligosaccharides which is potentially applicable in medical and food technology.

Failure of germinal center formation and impairment of response to endotoxin in tumor necrosis factor alpha-deficient mice.

To study the pathophysiologic roles of TNF alpha, we produced TNF alpha gene-disrupted mice by gene targeting. TNF alpha-deficient mice develop normally without any alteration in the lymphocyte populations. However, in these mice, the germinal center formation in the peripheral lymphoid organs failed in response to the T cell-dependent antigens. TNF alpha-deficient mice are resistant to lethal doses of endotoxin and D-galactosamine without hepatocyte apoptosis, yet demonstrate thymus apoptosis. Our results indicated an important role for TNF alpha in germinal center formation and in the sepsis-induced hepatocyte apoptosis that precedes liver failure.

Immunoreactivity of specific epitopes of PrPSc is enhanced by pretreatment in a hydrated autoclave.

An abnormal protein (PrPSc) accumulates in animals affected with scrapie. Immunoblotting procedures have been used widely to detect PrPSc. Blotted membranes were subjected to pretreatment in a hydrated autoclave, and the subsequent immunoreactivity of PrPSc was examined. The immunoreactivity of PrPSc to antisera against the synthetic peptides of the mouse PrP amino acid sequences 199 to 208 and 213 to 226 was enhanced by the pretreatment. However, the reactivity to antisera of peptide sequences 100 to 115 and 165 to 174 was not affected. The antibody-binding ability of the specific epitopes which are located close to the C-terminal end of PrP27-30 the proteinase-resistant portion of PrPSc, was enhanced by pretreatment in a hydrated autoclave. This pretreatment increased the sensitivity of PrPSc, and it would be useful for diagnosis of scrapie.

Granulomatous lesions caused by Pseudomonas aeruginosa in the ostrich (Struthio camelus).

Granulomatous lesions were observed in imported ostriches aged 3 months. Clinically, the birds showed lassitude, incoordination, and inappetence. At necropsy, yellowish white nodules often accompanied by a pseudodiphtheritic membrane were found in the oral, pharyngeal, tracheal and air sac mucosae, the lungs, oesophageal serosa, and abdominal peritoneum. Histopathological examination revealed purulent granulomatous lesions containing central bacterial colonies with an outer shell and club formation. The bacteria were small Gram-negative bacilli, which showed positive immunohistochemical staining for Pseudomonas aeruginosa. The bacterial colonies were positive for chicken IgM. Clubs around the colonies were negative for P. aeruginosa and chicken IgM. Such findings have not previously been reported in the ostrich.

Endogenous tumor necrosis factor (TNF) production and modification of pathological lesions in experimental Escherichia coli endotoxemia of piglets.

We examined the kinetics of tumor necrosis factor (TNF) production induced by Escherichia coli lipopolysaccharide (LPS) in relation to LPS tolerance and endotoxemic lesions of piglets. The plasma of piglets demonstrated cytotoxicity to TNF-sensitive L929 cells between 0.5 and 4 h after inoculation with 200 micrograms kg-1 of LPS. This cytotoxicity was neutralized by anti-bovine TNF serum. These piglets had disseminated intravascular coagulation (DIC) and meningoencephalitis. However, if piglets were first treated with three doses of 40 micrograms kg-1 of LPS, both TNF production and the occurrence of DIC were inhibited when 200 micrograms kg-1 of LPS was inoculated into these piglets. Repetitive inoculation with increasing doses of LPS induced fibrinoid vasculitis, meningoencephalitis and pneumonitis, while hemorrhage was minimal. A very low amount of TNF activity was detected from most of the samples of a piglet after repeated LPS inoculation. These results suggested that severity of the hemorrhagic and thrombotic lesions might relate to the amount of endogenous TNF activity, and that LPS tolerance might relate to inhibition of TNF production.

Preparation of anti-canine serum amyloid A (SAA) serum and purification of SAA from canine high-density lipoprotein.

Antiserum to canine serum amyloid A (SAA) was prepared in rabbits by immunization with crude SAA which was prepared from high-density lipoprotein 3 (HDL3) obtained from canine acute-phase serum. The antiserum was absorbed for contaminating antibodies by affinity chromatography using Sepharose 4B coupled with normal canine serum proteins. The rabbit anti-canine SAA serum reacted with a protein and formed a single precipitin line at the position of the alpha 1-region of the immunoelectrophoresis of canine acute-phase serum but did not react with the normal canine serum on immunoelectrophoresis. The antibody to canine SAA was also confirmed by Western blotting analysis. Canine SAA was purified as a low molecular weight protein component from crude SAA by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) after gel filtration chromatography. Purified canine SAA had a molecular weight of 15,000 as estimated by SDS-PAGE. This SAA level was found by enzyme-linked immunosorbent assay (ELISA) to increase 1 day after inoculation with Bordetella bronchiseptica to 9.0-20.1 times the preinoculation value.

Isolation of a cDNA clone for a cytokinin-repressed gene in excised cucumber cotyledons.

Rapid changes in gene expression were studied during incubation of cucumber (Cucumis sativus L.) cotyledons with cytokinins in darkness. Complementary-DNA clones for mRNAs whose levels decreased within 4 h of treatment with N6-benzyladenine (BA) were isolated by differential hybridization. One of them (CR9) was sequenced. It is 588 bp long, and would encode a protein consisting of 137 amino-acid residues and having a molecular mass of 15 kDa. The sequence shows a high homology with a light-induced gene from rice. Northern blot analysis of the CR9 transcript showed the level of the mRNA (0.7 kb) to decrease tenfold within 4 h of BA treatment, i.e. well before BA-induced cotyledon expansion was observed. The repression became greater with increasing concentration of BA (10(-8)-10(-5) M). The expression of the CR9 gene was repressed specifically by cytokinins (BA, isopentenyladenine and t-zeatin), but not by adenine or 2,4-dichlorophenoxyacetic acid (auxin). The results are discussed in relation to the primary action of cytokinin.

Immunohistochemical distribution of S-100 alpha-positive cells in bovine mycobacterial and non-mycobacterial granulomas.

By means of immunohistochemistry, the distribution of the alpha-subunit (S-100 alpha) and the beta-subunit (S-100 beta) of S-100 protein was studied in bovine granulomas caused by Actinomyces bovis, Actinobacillus lignieresi, Actinomyces (Corynebacterium) pyogenes, Pseudomonas aeruginosa, Staphylococcus aureus, Mycobacterium bovis and Mycobacterium paratuberculosis. S-100 alpha-positive epithelioid cells or dendritic cells were scattered among the predominantly S-100 alpha-negative cells of the mononuclear phagocyte system (MPS). S-100 beta was not found in the MPS cells of granulomas but was observed in the endothelial cells of blood vessels. A positive reaction to S-100 was also seen in normal cells in the lymphoid and mammary tissues. Mycobacterial granulomas contained more S-100 alpha-positive cells than did non-mycobacterial ones.

Induction of acute phase protein by recombinant human interleukin-6 (IL-6) in calves.

Interleukin-6 (IL-6) is a major inducer of acute phase proteins in human and murine species. However, the effects of IL-6 have not yet been investigated in cattle. Following continuous infusion of recombinant human IL-6, serum concentrations of bovine haptoglobin and fibrinogen increased in a manner similar to those in cattle with acute phase reaction. In contrast, C-reactive protein and alpha 1-acid glycoprotein, as well as the other hematologic parameters, did not change significantly. Intravenous administration of recombinant human IL-6 resulted in only a mild and transient increase of bovine haptoglobin. These results suggest that the regulation of acute phase protein production in cattle is similar, but not identical, to that observed in human and murine species.

Phenotyping of lymphocyte subsets in the vascular and epithelial lesions of a cow with malignant catarrhal fever.

Surface marker analysis of lymphoid cells infiltrating the vascular and epithelial lesions of a cow with malignant catarrhal fever (MCF) was conducted by immunohistochemistry using ten monoclonal antibodies. The majority of lymphoid cells in these lesions had BoCD8, BoCD6 or BoCD2, but they rarely possessed N-cell (BoCD5+/BoCD4-/BoCD8-, non-T non-B) markers. Similar reactivity was seen in lymphoid cells of perivascular infiltrates in the liver, heart and brain, and in T-dependent areas of lymph nodes. These results suggest involvement of cytotoxic T-lymphocytes in the pathogenesis of MCF.