Investigation into the Pathology of Idiopathic Systemic Amyloidosis in Four Captive Badgers (Meles meles).

Systemic idiopathic amyloidosis was described in four captive badgers (Meles meles). Two animals (B1 and B2) were not enrolled in any trial, while animals B3 and B4 took part in a vaccine efficacy study and had been challenged with Mycobacterium bovis. A full set of tissues was collected and processed routinely for histopathological, immunohistochemical and ultrastructural studies. Splenomegaly was found in three out of four animals. Histopathological evaluation revealed congophilic, permanganate-resistant systemic amyloid deposits in the tissues of all badgers. Animals B2 and B4 displayed a marked granulomatous response to amyloid within the spleen. Animals B1 and B2 also displayed clinicopathological findings suggestive of chronic kidney disease. Ultrastructural examination identified peculiar star-shaped arrays of amyloid. Immunohistochemical studies were unrewarding. Systemic amyloidosis should be considered among the differentials of wasting in captive badgers.

Escherichia coli O115 forms fewer attaching and effacing lesions in the ovine colon in the presence of E. coli O157:H7.

Escherichia coli O115 has been isolated from healthy sheep and was shown to be associated with attaching-effacing (AE) lesions in the large intestine. Following previous observations of interactions between E. coli O157 and O26, the aim of the present study was to assess what influence an O115 AE E. coli (AEEC) would have on E. coli O157 colonisation in vitro and in vivo. We report that E. coli O115- and O157-associated AE lesions were observed on HEp-2 cells and on the mucosa of ligated ovine spiral colon. In single strain inoculum, E. coli O115 associated intimately with HEp-2 cells and the spiral colon in greater numbers than E. coli O157:H7. However, in mixed inoculum studies, the number of E. coli O115 AE lesions was significantly reduced suggesting negative interference by E. coli O157. Use of the ligated colon model in the present work has allowed in vitro observations to be extended and confirmed whilst using a minimum of experimental animals. The findings support a hypothesis that some AEEC can inhibit adhesion of other AEEC in vivo. The mechanisms involved may prove to be of utility in the control of AE pathovars.

Pelioid hepatocellular carcinoma in an adult Eurasian badger (Meles meles).

A mass was identified within the left lateral lobe of the liver of a 10-year-old Eurasian badger (Meles meles). The mass was friable and multilobulated, with blood-filled spaces between the lobules. Microscopically, the lesion consisted of sheets and trabeculae of neoplastic hepatocytes often forming cystic spaces containing erythrocytes, fibrin and necrotic debris. The histological appearance was consistent with hepatocellular carcinoma (HCC). Immunohistochemically, the neoplastic cells expressed cytokeratin 18 but not von Willebrand factor. Multiple intranuclear (amphophilic or acidophilic) inclusion bodies were observed in hepatocytes at the junction between the tumour and normal hepatic tissue. HCCs have also been reported in other domestic and wild animals. As hepadnavirus infection has been associated with HCC in woodchucks, further histochemical and transmission electron microscopical studies were performed; however, these demonstrated that the inclusions consisted of lipid droplets and not viral particles. To our knowledge, this is the first report of a naturally occurring HCC in a Eurasian badger.

Influence of colostrum deprivation and concurrent Cryptosporidium parvum infection on the colonization and persistence of Escherichia coli O157 : H7 in young lambs.

Escherichia coli O157 : H7 and Cryptosporidium parvum infections of man have been associated with direct contact with small ruminants. Colostrum protects neonates against gastrointestinal pathogens, and orphan lambs, which are common on petting farms, may be deprived of this protection. In a recent study, it was demonstrated that high shedding of E. coli O157 : H7 by an 8-week-old goat kid was associated with coincidental C. parvum infection. Furthermore, both pathogens were co-located in the distal gastrointestinal tract. It was hypothesized that colostrum deprivation and pre-infection with C. parvum predisposed young ruminants to colonization and increased shedding of E. coli O157 : H7. To test this, 21 lambs 5 weeks of age were divided into four groups as follows: (A) colostrum-deprived and inoculated with E. coli O157 : H7, (B) colostrum-deprived and inoculated with C. parvum and then E. coli O157 : H7, (C) conventionally reared and inoculated with E. coli O157 : H7, (D) conventionally reared and inoculated with C. parvum and then E. coli O157 : H7. C. parvum was detected between 8 and 12 days post-inoculation in most of the infected lambs. At 24 h post-inoculation with E. coli O157 : H7, all lambs were shedding between 5 x 10(4) and 5 x 10(7) c.f.u. E. coli O157 : H7 per gram of faeces. E. coli O157 : H7 was shed in higher numbers in the groups pre-inoculated with C. parvum, whether conventionally reared or colostrum-deprived. Interestingly, for the colostrum-deprived lambs on day 3, a significant difference in shedding of E. coli O157 : H7 was observed (P = 0.038), with the lambs inoculated with E. coli alone yielding higher counts than those pre-inoculated with C. parvum. From day 15 onwards, shedding of E. coli O157 : H7 was highest from the colostrum-deprived C. parvum-infected lambs, then (in descending order of shedding) the colostrum-deprived lambs, the conventionally reared lambs infected with C. parvum, and the conventionally reared animals. In total, four animals were euthanized, two at 24 h and two at 96 h post inoculation with E. coli O157 : H7 (two conventionally reared and two colostrum-deprived). All animals euthanized were from groups pre-inoculated with C. parvum prior to challenge with E. coli O157 : H7. On examination of tissues, in three of the four animals examined, multifocal attaching and effacing lesions were observed in the caecum, colon, rectum and at the recto-anal junction, and were confirmed by immunohistochemistry to be associated with E. coli O157 : H7.

Colonization of 8-week-old conventionally reared goats by Escherichia coli O157 : H7 after oral inoculation.

Enterohaemorrhagic Escherichia coli O157 : H7 infections of man have been associated with consumption of unpasteurized goat's milk and direct contact with kid goats on petting farms, yet little is known about colonization of goats with this organism. To assess the contribution of flagella and intimin of E. coli O157 : H7 in colonization of the goat, 8-week-old conventionally reared goats were inoculated orally in separate experiments with 1x10(10) c.f.u. of a non-verotoxigenic strain of E. coli O157 : H7 (strain NCTC 12900 Nal(r)), an aflagellate derivative (DMB1) and an intimin-deficient derivative (DMB2). At 24 h after inoculation, the three E. coli O157 : H7 strains were shed at approximately 5x10(4) c.f.u. (g faeces)(-1) from all animals. Significantly fewer intimin-deficient bacteria were shed only on days 2 (P = 0.003) and 4 (P = 0.014), whereas from day 7 to 29 there were no differences. Tissues from three animals inoculated with wild-type E. coli O157 : H7 strain NCTC 12900 Nal(r) were sampled at 24, 48 and 96 h after inoculation and the organism was cultured from the large intestine of all three animals and from the duodenum and ileum of the animal examined at 96 h. Tissues were examined histologically but attaching-effacing (AE) lesions were not observed at any intestinal site of the animals examined at 24 or 48 h. However, the animal examined at 96 h, which had uniquely shed approximately 1x10(7) E. coli O157 : H7 (g faeces)(-1) for the preceding 3 days, showed a heavy, diffuse infection with cryptosporidia and abundant, multifocal AE lesions in the distal colon, rectum and at the recto-anal junction. These AE lesions were confirmed by immunohistochemistry to be associated with E. coli O157 : H7.

Functional characterization of the human neurokinin receptors NK1, NK2, and NK3 based on a cellular assay system.

The neurokinin receptor family is known to modulate phospholipase C activity. In order to find new compounds modulating the activity of these receptors we have developed a cellular screening system that measures the biological activity of receptors coupled to the IP3/DAG signal transduction pathway via the transcriptional activation of a reporter gene. For the establishment of neurokinin test cell lines the reporter cell line A20, stably transformed with the luciferase gene under the control of a promoter containing TPA response elements (TRE), which did not respond to neurokinin agonists, was used. Stable test cell lines were developed by transfecting the reporter cell line A20 with the genes for the human neurokinin receptors NK1, NK2 or NK3, respectively. In these cell lines, expression of luciferase was inducible by substance P, neurokinin A and neurokinin B, respectively. The order of potency of the three neurokinins substance P, neurokinin A and neurokinin B was consistent with published data and results from ligand binding studies performed with the NK1 and NK2 test cell lines. The agonistic effect of the neurokinins could be inhibited in a dose-dependent manner by simultaneous addition of neurokininspecific antagonists like the non-peptide antagonists CP-99,994 and SR 48968.

Establishment of a cellular assay system for G protein-linked receptors: coupling of human NK2 and 5-HT2 receptors to phospholipase C activates a luciferase reporter gene.

A functional cellular assay system was developed for the detection of substances modulating the activity of G protein-coupled receptors, linked to the phospholipase C second messenger system. The human adenocarcinoma cell line A549 was transformed with the Photinus pyralis luciferase gene under the control of the ICAM-1 gene 5'regulatory region and, subsequently, stably transfected with the human neurokinin 2 (NK2) receptor gene. The ICAM-1 promoter is known to be inducible via the phospholipase C signal transduction pathway. In this NK2 receptor test cell line, expression of luciferase was inducible by neurokinin A and other NK2-specific agonists. The order of potency of the three neurokinins substance P, neurokinin A and neuromedin K was consistent with published data and results from ligand binding studies performed with the same NK2 test cell line. The agonistic effect of neurokinin A could be inhibited in a dose-dependent manner by simultaneous addition of NK2-specific antagonists or protein kinase C-inhibitors. Similarly, a stable test cell line expressing the human serotonin 2 receptor was established. Agonist-induced luciferase expression in this cell line was abolished in the presence of 5-HT2-specific antagonists. These cellular assay systems can be employed for the identification of competitive, non-competitive and allosteric modulators of the NK2 and the 5-HT2 receptor, and they represent prototypes for analogous test cell lines for other phospholipase C-coupled receptors.

[Bowenoid actinic keratosis: therapy with intralesional injection of recombinant beta-interferon]. / Bowenoide aktinische Keratose: Therapie durch intraläsionale Injektion von rekombinantem beta-Interferon.

Biopsy-proven bowenoid actinic keratosis located in the pretibial region in each of two elderly women (61 and 81 years) were treated with intralesional injections with a new recombinant beta-interferon. The treatment took the form of intralesional injections three times a week over 3 consecutive weeks. The dose per single injection was 1.5 or 1.0 MU interferon, respectively. Clinical and histological examinations showed a complete response in both patients. Controls up to 18 months showed no relapse. There were no flu-like side-effects. Leukocyte counts decreased by 2700 and 500 cells per microliter during therapy.

A baculovirus dual expression vector derived from the Autographa californica nuclear polyhedrosis virus polyhedrin and p10 promoters: co-expression of two influenza virus genes in insect cells.

A baculovirus transfer vector, pAcUW3, was developed to facilitate the insertion of two influenza virus genes, those encoding the haemagglutinin (HA) and neuraminidase (NA) membrane glycoproteins, into the Autographa californica nuclear polyhedrosis virus genome in a single cotransfection experiment. The NA gene was inserted in place of the polyhedrin coding sequences under the control of the polyhedrin promoter, whereas the HA gene was placed under the control of a copy of the p10 promoter at a site upstream of and in opposite orientation to the polyhedrin promoter. After infection of Spodoptera frugiperda cells with the recombinant virus, AcUW3HANA, both HA and NA were expressed in the very late phase of infection and were shown to be functional in appropriate assays. Immunofluorescence assays demonstrated their localization at the surface of infected insect cells. The expression of both foreign genes in the recombinant virus was found to be stable for at least 12 passages in cell culture.

Successful treatment of primary progressive follicular mucinosis with interferons.

Follicular mucinosis is a primary idiopathic disease or a secondary, lymphoma-associated dermatosis. An effective standard therapy for the benign group is unknown. We describe a patient with primary benign disseminated progressive follicular mucinosis who was successfully treated with recombinant interferon alfa-2b and interferon-gamma. Interferons might act by down-regulation of activated inflammatory cells and/or by induction of enhanced elimination of extracellular mucin via increasing phagocytosis by macrophages.

[Association of arteriovenous and cavernous angioma of the head and neck area]. / Assoziation eines arteriovenösen und eines kavernösen Angioms im Kopf-Hals-Bereich.

A 20-year-old man complained of pulse-synchronous noise in the ear and recurrent bleedings in the nose and throat region. From birth he had had an extensive haemangioma, black-blue with dark-red parts. It had been diagnosed as a cavernous haemangioma, part of a Sturge-Weber syndrome. An angiogram was performed before intended dermatological treatment of the disfiguring venous angioma. It demonstrated the capillary venous angioma (slow-flow angioma) in the lateral triangle of the neck, extending up to the skull base. In addition there was an arteriovenous angioma (high flow angioma) in the region of the clivus, which was supplied bilaterally largely by the ascending pharyngeal artery. The arteriovenous angioma also had connections to the outflow area of the capillary venous angioma. These findings and absence of ocular changes excluded Sturge-Weber syndrome. Because of the risk of life-threatening bleedings, the arteriovenous malformation was superselectively embolized by multiple injections of nonresorbable polyvinyl-alcohol particles via a microcatheter. This brought about the collapse of the cutaneous angiomatous spaces. This case demonstrates that external appearance indicating a capillary venous angioma is not reliable. Before treatment of this malformation a neuroradiological diagnosis should be undertaken.

Construction of genetically engineered baculovirus insecticides containing the Bacillus thuringiensis subsp. kurstaki HD-73 delta endotoxin.

The delta-endotoxin gene from Bacillus thuringiensis subsp. kurstaki HD-73 was inserted into Autographa californica nuclear polyhedrosis virus (AcMNPV) using two transfer vector systems. In the first, the delta-endotoxin gene was placed under the control of the polyhedrin gene promoter in lieu of the polyhedrin coding sequences, thus deriving a polyhedrin-negative virus. In the second, it was inserted under the control of a copy of the AcMNPV p10 promoter positioned upstream of the polyhedrin gene to produce a polyhedrin-positive virus. Analysis of infected cell extracts showed that the delta-endotoxin was expressed in insect cells as 130K, 62K and 44K proteins, with peak syntheses at 18 h post-infection. Each of these products reacted with antisera specific for the complete protoxin and the cleaved, active form. When extracts from the cells infected with the polyhedrin-negative virus were fed to Trichoplusia ni larvae, feeding by the insects was inhibited and deaths occurred that were inconsistent with virus infection. This effect was also observed after the inoculum had been treated with detergents to inactivate virus particles prior to feeding to the larvae. These data indicate that the expression of the B. thuringiensis delta-endotoxin gene by a baculovirus in insect cells produces material with insecticidal activity. The biological activities of the two recombinant viruses were assessed in conventional bioassay tests by feeding virus particles or occlusion bodies to the insects. The polyhedrin-negative virus preparation appeared to be contaminated with endotoxin which inhibited feeding of the insects and prevented determination of the LD50 value. The polyhedrin-positive virus had an LD50 value about twofold higher than that of unmodified AcMNPV. The significance of these data for the genetic engineering of virus insecticides is discussed.

Analysis of very late gene expression by Autographa californica nuclear polyhedrosis virus and the further development of multiple expression vectors.

The consequences of locating the polyhedrin gene coding sequences and the p10 promoter at heterologous positions within the Autographa californica nuclear polyhedrosis virus (AcMNPV) genome were investigated. Positioning the polyhedrin or beta-galactosidase coding sequences under the control of the p10 gene promoter via the use of the new transfer vector, pAcUW1, resulted in viable recombinant viruses able to produce high levels of each non-fused gene product at the appropriate time. Polyhedra were also produced by the virus with the p10 promoter-polyhedrin hybrid gene and appeared normal in thin sections. Therefore the combination of polyhedrin promoter and coding sequences is evidently not essential for efficient expression of this protein. The p10 promoter can serve this function equally well. Viruses with the p10 promoter and beta-galactosidase coding sequences placed upstream from the polyhedrin gene in either orientation produced large amounts of beta-galactosidase protein in infected cells, thus demonstrating that the p10 promoter can function at an alternative position within the virus genome. A second transfer vector, pAcUW2B, was constructed, with a copy of the p10 gene promoter placed upstream and in opposition to the polyhedrin gene. This mediates the insertion of any foreign gene under the control of the p10 promoter while preserving normal p10 gene expression. The advantages of these constructs over the conventional vectors presently used to express foreign genes in insect cell systems and their utilization in the production of virus insecticides are discussed.

Recurrent papillomas of the nipple associated with human papillomavirus 41.

A case is described of a man with multiple papillomas on the nipple that resembled clinically and histologically condylomata acuminata. Human papillomavirus (HPV) type 41 DNA sequences were detected in the biopsy material. There was no evidence of any immunodeficiency.

Systemic therapy of advanced metastatic malignant melanoma with a combination of fibroblast interferon-beta and recombinant interferon-gamma.

Eighteen patients with advanced metastatic malignant melanoma (stage IVUICC 1987) were entered into a prospective trial with a combination of systemic fibroblast interferon-beta and recombinant interferon-gamma. Treatment was performed over a 6-week period with 3 x 5 x 10(6) U i.v. interferon-beta and 5 X 100 micrograms s.c. interferon-gamma every week. Under this therapy 16 patients showed a progressive disease, and 2 patients had a stable disease. The median time of survival was 7.5 months. Successive immunological examinations showed no significant immunomodulating effect after the 6 weeks of interferon treatment. We conclude that the combination of interferon-beta and -gamma is insufficient in the treatment of advanced metastatic malignant melanoma when administered by this dose, route and schedule.

[Immunomodulation by cryosurgery in malignant melanoma]. / Immunmodulation durch Kryochirurgie beim malignen Melanom.

Cryosurgery is a well-known, established method for the local destruction of tumor tissue by freezing. The assumption that, in addition to a physical and blood vascular phase, an immunological phase exists, has been discussed by many authors and tested using animal models. These results can only be transferred to humans in a limited sense. During the last year, we initiated a randomized study "Cryosurgery versus Conventional Surgery", whereby the peripheral blood and the normal skin from the areas surrounding the resection were compared. We were able to demonstrate in the peripheral blood of 8 cryosurgery patients a postoperative increase in the total and helper T-cells, HLA-DR-positive cells, and the ratio helper/suppressor T-cells in comparison to preoperative values. In the 8 patients treated with conventional surgery, these parameters decreased slightly or remained the same. The differences were highly significant (p = 0.001) to significant (p = 0.01). The results from the first 16 are patients studied presented and discussed here.

Defect of adenovirus type 12 replication in hamster cells: absence of transcription of viral virus-associated and L1 RNAs.

The nonpermissive interaction of hamster cells with human adenovirus type 12 (Ad12) is characterized by a total block of Ad12 DNA replication and late transcription, whereas most of the early functions of Ad12 DNA can be transcribed. Ad2 can replicate in hamster cells. The replication and late transcription defects of Ad12 DNA can be complemented to a certain extent by the E1B functions of Ad2 DNA. This complementation fails, however, to lead to the synthesis of the late Ad12 proteins and to the assembly of infectious virions. It will now be demonstrated that the Ad12 L1 (late genes of group 1) and virus-associated (VA) RNAs are not transcribed in hamster cells. Synthesis of these RNAs in productively infected human cells or Ad2-infected hamster cells is readily detectable by S1 nuclease protection experiments and Northern (RNA) blotting. Similarly, the Ad2-transformed hamster cell line BHK-Ad2E1 fails to complement L1 and VA RNA syntheses after superinfection with Ad12. However, Ad12 infection of the Ad5-transformed hamster cell line BHK297-C131 leads to the transcription of the Ad12 L1 and VA segments. This difference in complementation by the two transformed hamster cell lines might be accounted for by functions in the segment of Ad5 DNA extending between map units 30 and 40 and persisting in the Ad5-transformed hamster cells or by hamster host cell functions which might be operative in cell line BHK297-C131 but not in BHK-Ad2E1 or BHK-21 hamster cells.