Laboratory infection of primates with Ascaris suum to provide a model of allergic bronchoconstriction

Wild-caught non-human primates are naturally sensitive to Ascaris antigen and provide a useful model for studying atopic asthma. The present study was carried out to determine the effect of experimentally infecting home-bred macaques with the nematode Ascaris suum and hence provide an alternative for the naturally occurring model. Following oral infection with the parasite the animals developed a blood eosinophilia and specific antibodies to purified Ascaris antigen. These antibodies appeared to be of the IgE class as they could be detected by a radiometric assay using a radiolabelled antibody to human IgE. However, on further investigation, using the passive cutaneous anaphylaxis test, two classes of antibody were found, a heat labile (56 degrees C) and a heat stable antibody. Lung lavage cells taken from monkeys infected with Ascaris suum were shown to include cells morphologically characteristic of mast cells and released histamine when challenged in vitro with Ascaris antigen. Hence this model of immediate hypersensitivity provides a simple alternative to the less accessible natural model.     

Identification of a 33-kilodalton immunodominant antigen of Trypanosoma congolense as a cysteine protease.

A 33-kDa protein of Trypanosoma congolense is a major antigen in infected cattle and the production of antibody to this antigen appeared to correlate with enhanced resistance to trypanosomiasis [4]. Immunoelectron microscopy using a monoclonal antibody (mAb 4C5) raised against the 33-kDa antigen showed a lysosomal localisation, similar to that of a previously described 32-kDa cysteine protease of T. congolense. Both mAb 4C5 and anti-33 kDa antibody from infected cattle bound on Western blots to the cysteine protease that had been purified by affinity chromatography on cystatin-Sepharose. Sepharose-coupled mAb 4C5 was used to affinity purify the antigen from bloodstream forms of T. congolense. On sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), the affinity-purified antigen had a molecular mass of 33 kDa under non-reducing conditions, and 40 kDa under reducing conditions. Anti-33-kDa antibody from infected cattle bound to both non-reduced and reduced affinity-purified antigen on Western blots. Serum from a rabbit immunised with the biochemically purified enzyme also bound the affinity-purified antigen. The affinity-purified antigen displayed proteolytic activity in fibrinogen-containing SDS-PAGE and against Azocoll. It hydrolysed benzyloxycarbonyl-Phe-Arg-7-amino-methyl coumarin (Z-Phe-Arg-NHMec) with a Km similar to that of the biochemically purified enzyme. Proteolytic and peptidolytic activities of the antigen were inhibited by the inhibitors of cysteine proteases, cystatin and trans-epoxysuccinyl-L-leucyl-amido (4-guanidino)butane (E-64). On two-dimensional gel electrophoresis, the antigen displayed similar characteristics to those of the biochemically purified enzyme. We conclude that the 33-kDa antigen of T. congolense and the cysteine protease are the same molecule.     

Adeno-associated virus vector gene transfer and sarcolemmal expression of a 144 kDa micro-dystrophin effectively restores the dystrophin-associated protein complex and inhibits myofibre degeneration in nude/mdx mice

Duchenne muscular dystrophy is a severe life-threatening X-linked recessive disorder, caused by mutations in the dystrophin gene, for which currently there is no effective treatment. Because of the large size of the dystrophin cDNA (14 kb) this precluded it from being used in early adenovirus- or retrovirus-based gene therapy vectors. However, some therapeutic success has been achieved in mdx mice using adenovirus- and retrovirus-mediated transfer of a 6.3 kb recombinant mini-dystrophin cDNA. Despite this, problems with immunogenicity and inefficient transduction of mature myofibres make these vectors less than ideal for gene transfer to skeletal muscle. Adeno-associated viral (AAV) vectors overcome many of the problems associated with other vector systems. However, AAV vectors can only accommodate <5 kb of foreign DNA. For this reason we have produced a micro-dystrophin cDNA gene construct that is <3.8 kb. This construct, driven by a CMV promoter, was introduced into the skeletal muscle of 12-day-old nude/mdx mice using an AAV vector, resulting in specific sarcolemmal expression of micro-dystrophin in >50% of myofibres up to 20 weeks of age, and effective restoration of the dystrophin-associated protein (DAP) complex components. Additionally, evaluation of central nucleation indicated a significant inhibition of degenerative dystrophic muscle pathology. We have therefore shown that the current micro-dystrophin gene delivered in vivo using an AAV vector is not only capable of restoring sarcolemmal DAP complexes, but can also ameliorate dystrophic pathology at the cellular level.     

Adherence and surface properties ofTritrichomonas mobilensis,an intestinal parasite of the squirrel monkey

Adherence properties of the potentially enteropathogenicTritrichomonas mobilensis were studied in vitro. Axenically cultivated trichomonads readily attached to isolated intestinal epithelial cells and mucus of the squirrel monkey. The kinetics and nature ofT. mobilensis cytadherence were microscopically evaluated in cell-suspension assay using Chinese hamster ovary (CHO) cells and in microplate hemagglutination assay with human erythrocytes. Adherence of the parasites to target cells was concentration- and time-dependent; it was inhibited by sialic acid (N-acetylneuraminic orN-glycolylneuraminic acid) and sialyllactose. Neither trypsinization of the flagellates nor their exposure to low temperature (4° C) affected their cytadherence capacities. The data indicate the presence of adhesin(s) with lectin properties onT. mobilensis. Aggulutination of live protozoa by animal and plant lectins with various carbohydrate-binding specificities as well as the occurrence of an electron-dense cell coat on plasma membrane suggest marked glycosylation of the parasite surface.     

Laminin and fibronectin in adenoid cystic carcinoma.

The distribution of fibronectin and laminin was examined by immunohistochemistry in 11 adenoid cystic breast carcinomas, six adenoid cystic carcinomas of mouth and salivary gland, and six cribriform ductal breast carcinomas. Both proteins were present lining cystic lumina and around tumour islands in all the adenoid cystic breast carcinomas and in five of six salivary gland tumours. Abundant laminin and fibronectin were dispersed among adenoid cystic tumour cells arranged in sheets. One adenoid cystic carcinoma from buccal mucosa showed a transition from a cribriform tumour positive for both fibronectin and laminin to a cribriform tumour negative for fibronectin and laminin to undifferentiated carcinoma. Fibronectin and laminin seemed to disappear simultaneously from tumour cell surfaces. Another adenoid cystic carcinoma from buccal mucosa was negative for fibronectin and laminin from the time of initial biopsy. This was the only tumour that gave rise to disseminated metastases, resulting in the death of the patient within two years of surgery. In cribriform invasive ductal breast carcinomas the linings of cystic lumina were always negative for fibronectin and laminin. Varying quantities were present at the tumour boundaries. We suggest that staining for fibronectin and laminin may be a valuable aid to the diagnosis of adenoid cystic carcinomas and that the absence of these proteins may have important prognostic implications.     

Lack of lysosomal fusion with phagosomes containing Ehrlichia risticii in P388D1 cells: abrogation of inhibition with oxytetracycline.

Fusion of lysosomes with phagosomes containing Ehrlichia risticii, an obligate intracellular parasite, was evaluated in P388D1 murine macrophagelike cells. Lysosomes in cells ranging in infectivity from 30 to 70% were labeled cytochemically with acid phosphatase or via endocytosis of thorium dioxide or cationized ferritin to document phagosome-lysosome (P-L) fusion in untreated cells and cells treated with oxytetracycline. Regardless of the marker used, P-L fusion was generally not observed in E. risticii-containing vacuoles in untreated cells, while significantly greater P-L fusion with ehrlichia-containing vacuoles was observed after oxytetracycline treatment. When latex beads were introduced into uninfected cell cultures, P-L fusion was observed with vacuoles containing latex. Fusion of lysosomes with latex-containing vacuoles in cells was significantly greater than fusion of lysosomes with ehrlichia-containing vacuoles in the same infected cells. These findings indicate that E. risticii is able to inhibit P-L fusion, whereas oxytetracycline deprives organisms of this ability.     

The ATAC ('Arimidex', Tamoxifen, Alone or in Combination) adjuvant breast cancer trial: baseline endometrial sub-protocol data on the effectiveness of transvaginal ultrasonography and diagnostic hysteroscopy

BACKGROUND: The 'Arimidex', Tamoxifen, Alone or in Combination (ATAC) trial is a randomized, double-blind trial comparing anastrozole ('Arimidex'), alone or in combination with tamoxifen, relative to tamoxifen alone as 5 year adjuvant treatment for post-menopausal women with early breast cancer. Since tamoxifen is associated with endometrial pathology, the ATAC endometrial sub-protocol was initiated to establish the background prevalence of intrauterine pathology, and to assess prospectively the incidence and nature of intrauterine changes following endocrine therapy. Another aim was to provide data from which advice could be generated on the best endometrium screening method for patients receiving tamoxifen. METHODS: Patients underwent endometrial assessments at entry to the sub-protocol. The baseline investigations comprised transvaginal ultrasound scanning (TVUS), a hysteroscopy and an endometrial biopsy. RESULTS: A total of 285 gynaecologically asymptomatic women from 31 centres in 10 countries entered the endometrial sub-protocol. The mean uterine volume was 47.7 cm3. The median endometrial thickness overall was 3 mm. Twenty-four histologically confirmed, pathological changes were observed. Twenty-three pathologies were confirmed by TVUS, and 21 were identified by hysteroscopy and confirmed by histopathology. Women with or without intrauterine pathology had median endometrial thickness of 5 and 3 mm respectively. CONCLUSIONS: The presence of pathology was associated with increased endometrial thickness. The relative sensitivity and specificity of hysteroscopy and endometrial thickness for the diagnosis of endometrial pathology was comparable to other studies. If screening of the endometrium prior to treatment is appropriate, this study supports the use of an endometrial thickness of 3 mm, as assessed by TVUS, as a threshold for needing further investigation. This study demonstrates that if the endometrial thickness is >3 mm, hysteroscopy and biopsy is the optimal method of detecting intrauterine pathology in women with breast cancer who are about to commence endocrine treatment.     

Stable micro-dystrophin gene transfer using an integrating adeno-retroviral hybrid vector ameliorates the dystrophic pathology in mdx mouse muscle

The ability to transfer the dystrophin gene stably to the skeletal muscle of DMD patients is a major confounding issue in establishing an effective gene therapy for this disease. To overcome this problem, we have examined the ability of muscle fibres from mdx mice to act as in situ factories of retroviral vector production. Tibialis anterior (TA) muscles from 4-week-old mdx mice were injected with an adenoviral vector expressing LacZ within a retroviral expression cassette (AdLZIN). Retroviral vector production was induced by the inclusion of two additional adenoviral vectors expressing retroviral gag-pol (AdGagPol) and 10A1 env genes (Ad10A1). Upon introduction of infected muscles into cell culture, colonies of beta-galactosidase-expressing myotubes formed only in cultures where the muscle was injected with AdLZIN, AdGagPol and Ad10A1, but not from muscle injected with AdLZIN only. Muscles from mdx/nude mice producing retroviral vector displayed a 4.6-fold increase in beta-galactosidase-positive myofibres after 1 month, compared with contralateral muscle in the same animal injected with AdLZIN and AdGagPol only. By constructing a hybrid adeno-retroviral vector expressing a truncated micro-dystrophin construct (AdmicroDyIN), we were able to partially correct the mdx dystrophic phenotype. AdmicroDyIN-mediated expression of micro-dystrophin in mdx TA muscle restored the formation of the dystrophin-associated glycoprotein complex and significantly reduced the level of muscle degeneration over uninjected controls. By stimulating in situ production of retroviral vector expressing micro-dystrophin, we achieved 92%+/-6% transduction of myofibres in the TA muscle by 4 weeks. Strikingly, by 3 months post injection, micro-dystrophin was still expressed to high levels in nearly all the myofibres of the TA muscle. By comparison, there was a pronounced drop in the levels of micro-dystrophin expressed by muscles injected with AdmicroDyIN only. Finally, using a novel PCR approach, we detected reverse-transcribed, integrated proviral sequences in TA muscle genomic DNA by 4 weeks post injection, the levels of which were found to increase after 3 months.