Extracellular DNA from Serpulina hyodysenteriae consists of 6.5 kbp random fragments of chromosomal DNA.

Preparations of chromosomal DNA from a number of Serpulina hyodysenteriae strains have shown, using agarose gel electrophoresis, the presence of an additional band with a mobility similar to that of a 6.5 kbp linear DNA fragment. Analysis showed that this is not a plasmid but rather a form of extracellular DNA like that observed for Gram-negative bacteria. However, unlike the extracellular DNA from Gram-negative bacteria, which showed a similar band profile to that of the DNA from whole cells, that from S. hyodysenteriae consisted primarily of fragments of a fixed 6.5 kbp.

Distribution of the smpA gene from Serpulina hyodysenteriae among intestinal spirochaetes.

Forty intestinal spirochaete strains were investigated for nucleotide sequences related to the smpA locus from Serpulina hyodysenteriae by Southern hybridization of chromosomal DNA using the smpA locus from S. hyodysenteriae strain P18A as a probe and by PCR using primers internal to the smpA gene. The intensity of the hybridization signal at high stringency and positive PCR results suggested that 12 S. hyodysenteriae strains possessed a similar nucleotide sequence. PCR was negative for another 12 S. hyodysenteriae strains and the hybridization signal obtained from 11 of these was weak and one was negative. All S. hyodysenteriae strains hybridized under low stringency conditions. These results indicated that there is variation among the smpA loci of S. hyodysenteriae strains. Among seven strains of S. innocens, and the proposed species 'S. intermedius' and 'S. murdochii', hybridization was weak and no PCR products were obtained, suggesting that these species have sequences related to, but divergent from, the smpA sequences of strains of S. hyodysenteriae. Both gene probe hybridization and PCR analysis of nine strains of the proposed new genus 'Anguillina', including isolates from pigs and humans, gave negative results.

Expression of the SmpA outer membrane lipoprotein of Serpulina hyodysenteriae strain P18A in vivo.

An ELISA has been developed using a monoclonal antibody (F325 AC4) to the SmpA surface lipoprotein of Serpulina hyodysenteriae strain P18A when grown in vitro. The lower level of detection of the ELISA was approximately 5 x 10(6) spirochaetes/ml when spirochaetes were either resuspended in phosphate buffered saline or in pig faeces. When pigs were challenged with S. hyodysenteriae strain P18A the lipoprotein was detected in the faeces of pigs by ELISA when the numbers of spirochaetes excreted was greater than 10(6) per g of faeces. After onset of clinical signs in the pig, expression of SmpA was not detected by ELISA or by Western blotting using either monoclonal antibody F325 AC4 or polyclonal antiserum B50 against the SmpA antigen. However, when the in vivo grown spirochaetes were subsequently cultured in vitro expression of SmpA was detected by Western blotting. In the mouse model of swine dysentery S. hyodysenteriae spirochaetes obtained from mice with gross lesions also did not express SmpA. It was concluded that the apparent lack of expression may have been the result of environmental regulation of gene expression or antigenic variation and was not due to denaturation of the antigen in vivo.

Use of commercial enzyme kits and fatty acid production for the identification of Serpulina hyodysenteriae: a potential misdiagnosis.

The accuracy of identification of Serpulina hyodysenteriae strains grown in a complex medium was 90% when 2 commercial test kits were used. Unlike the other S. hyodysenteriae strains, S. hyodysenteriae strain P35/2 was unusual in being indole negative. The nonpathogenic intestinal spirochete PWS/A, which is from a different species, was indole positive and alpha-galactosidase negative. Identification of these spirochetes on the basis of these kits alone would have been incorrect. The analysis of volatile fatty acids by gas chromatography showed that the ratio of acetic to butyric acid was from 11:1 to 44:1 for S. hyodysenteriae strains, which distinguished them from the other spirochetes. The exception was PWS/A (acetic: butyric of 32:1), but this spirochete, unlike the S. hyodysenteriae spirochetes, also produced isobutyric acid. Short chain fatty acid (SCFA) analysis by high-performance liquid chromatography detected different SCFAs in addition to acetic and butyric acids. These additional SCFAs did not contribute to further differentiation of the porcine spirochetes.

Comparative study of the enzyme activities of Borrelia burgdorferi and other non-intestinal and intestinal spirochaetes.

Comparative analysis of the enzymatic profiles of 58 spirochaetal isolates clearly differentiated borrelias from leptospires, serpulinas and a treponeme. Strains of both Borrelia burgdorferi and Borrelia hermsii characteristically produced significant amounts of leucine arylamidase. This enzyme activity was not unique to borrelias but was also detected amongst pathogenic and non-pathogenic leptospira serovars. This fact, however, did not hamper a correct differentiation of borrelias from these spirochaetes, because leptospires possessed unique enzyme profiles. The API ZYM system could not differentiate the human strains of B. burgdorferi from those isolated from ticks, or from B. hermsii. Treponema phagedenis could be differentiated from all the other spirochaetes by the production of alpha-fucosidase. Our results confirm and extend previous studies indicating that human and animal intestinal spirochaetes have many common enzyme activities. All strains produced reactions of maximum intensity when tested for the presence of beta-galactosidase activity. However the avian strains lacked esterase (C4) which was present in human and swine intestinal spirochaetes. All strains of Serpulina hyodysenteriae, and Serpulina innocens as well as the human intestinal spirochaete strain HRM-14 showed alpha and beta glucosidase activity. Both enzyme activities were absent or insignificant in most other intestinal spirochaetes examined: 25 different human strains, non-pathogenic swine strain M1 and the avian strain 4742. However, swine strain LL3 and avian strain 1380 showed some beta-glucosidase activity.

A controlled trial of adjuvant tamoxifen, with or without prednisolone, in post-menopausal women with operable breast cancer.

A randomised clinical trial has been conducted to compare adjuvant tamoxifen, 20 mg daily, with tamoxifen and prednisolone, 7.5 mg daily, in post-menopausal women with operable breast cancer. There were 254 evaluable patients, of whom 128 were given tamoxifen alone and 126 received tamoxifen and prednisolone. After a median follow-up of 48 months there was no significant difference in relapse-free or overall survival of the two groups. Furthermore, with survival slightly favouring tamoxifen, confidence intervals on the hazard ratio established that a difference in favour of tamoxifen plus prednisolone of even 5% at 5 years was very unlikely (P < 0.02). Thus, despite the relatively small number of patients in this trial, the data clearly establish that prednisolone is not of value as an additional adjuvant agent.

Chemotactic response to mucin by Serpulina hyodysenteriae and other porcine spirochetes: potential role in intestinal colonization.

Chemotaxis of porcine spirochetes towards a variety of mucins was measured quantitatively by a capillary method. A chemotaxis buffer consisting of 0.01 M potassium phosphate buffer (pH 7.0) and 0.2 mM L-cysteine hydrochloride was necessary for chemotaxis of spirochetes. The optimum incubation time and incubation temperature were 1 h and 40 degrees C, respectively. The mucin concentration also affected the chemotaxis observed, and a concentration of 1% (wt/vol) was near the optimum. Virulent Serpulina hyodysenteriae strains were chemotactic towards 1% (wt/vol) hog gastric mucin and 1% (wt/vol) porcine colonic mucin but not towards 1% (wt/vol) bovine submaxillary mucin. Virulent S. hyodysenteriae strains were significantly more chemotactic than avirulent strains of S. hyodysenteriae (SA3 and VS1), Serpulina intermedius, and Serpulina innocens. Other spirochetes belonging to the proposed group of spirochetes Anguillina coli were also not chemotactic. Pathogenicity of S. hyodysenteriae strains that cause swine dysentery may, in part, be attributed to their attraction to porcine intestinal mucus.

Molecular cloning, expression, and DNA sequence analysis of the gene that encodes the 16-kilodalton outer membrane lipoprotein of Serpulina hyodysenteriae.

The gene (smpA) that encodes the 16-kDa outer membrane lipoprotein of Serpulina hyodysenteriae was cloned in Escherichia coli, and its primary structure was determined by nucleotide sequencing. The putative open reading frame encodes a prolipoprotein of 16.8 kDa which in its fully acylated and cleaved form is 15.1 kDa. Analysis of the N-terminal amino acid sequence derived from the DNA sequence revealed the presence of a signal sequence and a putative acylation and signal peptidase II cleavage site (Phe-Ala-Val-Ser-Cys). In E. coli, processing of the prolipoprotein was less efficient than that observed in S. hyodysenteriae, and globomycin, an inhibitor of signal peptidase II, inhibited cleavage of the lipoprotein expressed in E. coli but did not inhibit cleavage in S. hyodysenteriae.

Monoclonal antibodies to a 16-kDa antigen of Serpulina (Treponema) hyodysenteriae.

Monoclonal antibodies (MAbs) were produced to an outer-envelope preparation from Serpulina (Treponema) hyodysenteriae, the aetiological agent of swine dysentery. Three MAbs (isotype IgG1) were obtained. All three recognised a 16-kDa antigen that was common to most strains of S. hyodysenteriae of different serotypes but was absent from nonpathogenic, porcine intestinal spirochaetes. Immunofluorescence and immunogold labelling studies showed that the 16-kDa antigen was exposed on the surface of intact spirochaetes. The MAbs agglutinated freshly grown cultures of spirochaetes and inhibited growth of S. hyodysenteriae strains in vitro.

A 16-kilodalton lipoprotein of the outer membrane of Serpulina (Treponema) hyodysenteriae.

Serpulina (Treponema) hyodysenteriae P18A and VS1 were extracted by using the detergent Triton X-114 and separated into detergent and aqueous phases. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblot analysis confirmed that a membrane-associated 16-kDa antigen was hydrophobic, since it was found in the detergent phase. A 45-kDa antigen partitioned into the aqueous phase, suggesting that it was hydrophilic and may be of periplasmic origin. When spirochetes were grown in the presence of [3H]palmitic acid, a predominant 16-kDa antigen was labeled; from the results of immunoprecipitation experiments, this antigen appeared to be the same as that recognized by both polyclonal and monoclonal antisera to a previously described 16-kDa antigen. This antigen was proteinase K sensitive and was not a component of the lipopolysaccharide, which, although [3H]palmitate labeled, was resistant to proteinase K digestion. The most probable explanation is that the 16-kDa antigen is a membrane-associated, surface-exposed, immunodominant lipoprotein.

A cytotoxic haemolysin from Treponema hyodysenteriae--a probable virulence determinant in swine dysentery.

The haemolysin from a virulent strain of Treponema hyodysenteriae was extracted and injected into ligated loops of the ileum and colon of germ-free pigs. It caused severe epithelial damage, especially to the differentiated cells at the tips of the villi in the ileum and the cells in the intercrypt zones of the colon; goblet cells were less affected. The changes in the colon were similar to those seen in natural cases of swine dysentery. The ligated loop offers a means of investigating pathogenic mechanisms and the mode of action of the toxin. This study demonstrated that the haemolysin was a potent cytotoxin for pig enterocytes, and a probable virulence determinant in swine dysentery.

Fine-wire localization and biopsy of non-palpable breast lesions.

We have undertaken fine-wire localization and biopsy of 130 impalpable breast lesions identified by mammography and considered suspicious of malignancy. Histologically 22 of these lesions were invasive carcinomas and 24 were in situ carcinomas (35 per cent malignant). Twenty-nine per cent of the lesions were identified during the screening of asymptomatic women. In the remainder, the presenting symptoms bore no relation to the eventual histological diagnosis. Clusters of microcalcification were more often malignant than were abnormal soft-tissue masses. Malignancy in the absence of microcalcification was almost always invasive.

Antibodies to a common outer envelope antigen of Treponema hyodysenteriae with antibacterial activity.

Outer envelopes of Treponema hyodysenteriae strains P18A and VS1 were prepared and characterized by SDS-PAGE. In Western blot analysis of eleven strains of T. hyodysenteriae and two intestinal non-pathogenic spirochaetes, polyclonal antiserum raised to the outer envelopes of strain P18A contained antibodies primarily to two polypeptides. A 45 kDa polypeptide was present in only two strains of T. hyodysenteriae, P18A and MC52/80, whereas another antigen of 16 kDa was common to all eleven strains of T. hyodysenteriae but was not present in the two nonpathogens. Immunogold labelling of whole organisms suggested that the 16 kDa antigen was present on the surface of the spirochaetes. In in vitro tests the serum agglutinated and inhibited growth of only the T. hyodysenteriae strains, suggesting that antibodies to the 16 kDa antigen were responsible for these activities. Serum from a gnotobiotic pig infected with T. hyodysenteriae strain P18A had antibodies to the 16 kDa antigen alone and also possessed agglutinating and growth-inhibitory activities.

Analysis of the axial filaments of Treponema hyodysenteriae by SDS-PAGE and immunoblotting.

Purified axial filaments from eight serotypes of Treponema hyodysenteriae and two non-pathogenic intestinal spirochaetes were characterized by SDS-PAGE and Western blotting. Axial filaments of all ten strains had similar SDS-PAGE profiles; five major axial filament polypeptides were identified, with molecular masses of 43.8, 38, 34.8, 32.8 and 29.4 kDa. Hyperimmune gnotobiotic pig serum raised against purified axial filaments of strain P18A (serotype 4) cross-reacted with all other serotypes and with the non-pathogens, and convalescent serum taken from a pig with persistent swine dysentery also showed a strong response to the axial filament polypeptides. Hyperimmune gnotobiotic pig serum raised against axial filaments failed to agglutinate viable organisms and did not inhibit growth in vitro. Hence, the axial filaments of T. hyodysenteriae have been identified as major immunodominant antigens, although the role that antibodies to these antigens play in protection has yet to be established.

Extended role for needle biopsy in the management of carcinoma of the breast.

Treatment choice in primary breast cancer is wide and still controversial; it seems likely that the optimum treatment for individual patients could be dictated by biological indicators of tumour behaviour. If biopsy could provide prognostic information as well as detailed tissue diagnosis then definitive treatment, with or without adjuvant systemic therapy, could be planned from the outset. We studied 140 patients with a clinical diagnosis of primary breast cancer to determine how much information could be obtained from Tru-Cut needle biopsies performed at the first clinic visit. Ten patients were found to have benign disease. Of 130 carcinomas, 123 (95 per cent sensitivity) were diagnosed correctly from the needle biopsies, with seven false negative and no false positive results (100 per cent specificity). Precise histopathology was predicted in 121 (93 per cent). Grade was correctly assessed in 77 of 112 (69 per cent), but needle biopsy was not accurate for assessment of lymphatic invasion nor elastosis. Steroid hormone receptors were assayed in 59 needle biopsies, and the incidence of oestrogen receptor positivity (34, 58 per cent) was similar to the resected tumours (35, 59 per cent), but the incidence of progesterone receptor positivity (26, 44 per cent) was lower (33, 56 per cent, P less than 0.04). Immunostaining with monoclonal antibody human milk fat globule membrane was accurate in the needle biopsies. DNA analysis by flow cytometry was performed in 37 tumours and the concordance between needle biopsies and resected samples was high. Tru-Cut needle biopsy obviates open biopsy and gives reliable detailed information.

The expression of milk fat globule antigens within human mammary tumours: relationship to steroid hormone receptors and response to endocrine treatment.

The value of steroid hormone receptors for the management of advanced carcinoma of the breast is often limited by the lack of availability of fresh tissue. Differentiation antigens may be estimated on paraffin-embedded fixed material by immunostaining, and the aim of this study was to determine whether staining with the monoclonal antibody raised to human milk fat globule (HMFG-1) could replace receptor measurements. The indirect immunoperoxidase technique was used to stain formalin-fixed paraffin-embedded tumour samples from 168 patients. All received tamoxifen or ovarian ablation as first-line systemic therapy, and all were evaluable for response (UICC criteria). One hundred and sixty-seven had oestrogen (ER) and progesterone receptors (PR) estimated. HMFG-1 staining was assessed as the percentage of tumour cells stained, and by the site of stain. The proportion of cells stained was highly correlated with both ER (P less than 0.0001) and PR (P less than 0.0001) and with response. When greater than or equal to 30% cells stained, 53 of 69 (77%) responded; when 20-29% stained 10 of 19 (53%) responded, when 10-19% stained seven of 19 (37%) responded, and when less than or equal to 9% cells stained 16 of 61 (26%) responded (P less than 0.0001). The median survival of patients with tumours that stained greater than or equal to 30% cells was 36 months, and with no cells stained, 11 months (P less than 0.0001). ROC (receiver operator characteristic) curves found that the optimum threshold for sensitivity and specificity of response prediction was greater than or equal to 20% cells stained. Cox's multiple regression analysis of 42 variables indicated that PR was the most important predictor of survival (P less than 0.000001), but that after PR the percentage of cells stained with HMFG-1 was the most important (P less than 0.0001). We conclude that immunostaining for HMFG-1 gives similar information to receptor status, and has the advantage that fixed archival tissue may be used.

The definition of the 'no change' category in patients treated with endocrine therapy and chemotherapy for advanced carcinoma of the breast.

In the criteria used for assessment of response to treatment for advanced breast cancer the definition of no change (NC) is clear; however, there is no indication of the duration of stabilization required for patients to qualify for this category of response. We have made the assumption that NC is a worthwhile category of response if the overall time to progression (TTP) and survival of this group is not significantly different from patients with partial remissions (PR). Two hundred and sixty-three evaluable patients treated with endocrine therapy and 302 evaluable chemotherapy-treated patients were studied and the TTP and survival curves for PR and periods of NC from 1 to 6 months compared. For the endocrine-treated patients the TTP and survival curves for NC became non-significantly different from the PR curves after 4 and 5 months respectively. For chemotherapy-treated patients the TTP curves became non-significantly different from PR at 4 months and for survival the period was 3 months. In order to define NC as a useful category of response and to eliminate the possibility that NC taken for a shorter period could simply represent a slowly progressive tumour, we suggest that the minimum period of disease stabilization be taken as 5 months for both endocrine- and chemotherapy-treated patients.

Expression of differentiation antigens within human mammary tumours is related to response to endocrine therapy and survival.

Human mammary tumours which are histologically well differentiated are more likely to synthesize receptors for estrogen (ER) and progesterone (PR) and to respond to systemic endocrine therapy. The aim of this study was to explore the relationship between differentiation, receptors and endocrine responsiveness in more detail by relating the expression of putative differentiation antigens within tumours to ER, PR and response to treatment. Sections of the primary tumours of 160 patients with advanced evaluable breast cancer were immunostained with 2 monoclonal antibodies (MAbs) (HMFG1 and HMFG2) raised against putative differentiation antigens found on the membranes which surround the milk fat globule. Tumours were highly heterogeneous with respect to antigen expression. However, the number of cells which expressed the antigens was highly correlated with ER and PR concentrations and with response to endocrine therapy. In tumours where greater than or equal to 20% of cells expressed the antigen recognized by HMFG1, 73% responded to endocrine therapy; this was similar to the response predicted by ER (67%) and PR (73%). Expression of HMFG1 was correlated with survival from the start of endocrine therapy (p less than 0.0001) to the same degree as ER and PR. Patients with tumours which expressed ER, PR and HMFG1 had the highest response rate (87%) and survival (median 49 months); the response in tumours which expressed none of these phenotypes was 13% and the median survival of these patients was 9 months. These results suggest that cells which express differentiation antigens also express ER and PR. Differentiated cells within mammary tumours may therefore be the target cells for systemic hormone, and also the source of factors which control tumour growth.

Ultrastructural observations on the basal lamina in the normal human breast.

The ultrastructure of the basal lamina of histologically normal human breast tissue was determined in 19 women undergoing operations for removal of a fibroadenoma or reduction mammoplasty. The day of the menstrual cycle was determined by hormone assay and direct questioning. Previously documented ultrastructural appearances were confirmed: in addition, three morphological variants were found. In all tissue examined, there was reduplication of basal lamina in some areas, which has been described previously as a pathological feature. Also, there was complex branching of the basal lamina into the periductular connective tissue. Some projections contained cytoplasmic processes and, in almost all, hemidesmosomes were seen. The third variant consisted of loops of basal lamina thrown up in folds into the collagenous stromal cuff. Reduplication of basal lamina was detected in breast tissue removed at all stages of the menstrual cycle, looping was not and could not be related to any particular phase of the menstrual cycle. However, complex branching was seen predominantly in the periovulatory and early luteal phase. We conclude that these appearances are normal variants of basal lamina. The appearance of branching basal lamina in the luteal phase suggests that this may be produced in response to endocrine stimulation.

Fibroblasts from relatives of patients with hereditary breast cancer show fetal-like behaviour in vitro.

Fetal and normal adult skin fibroblasts show distinctive migratory behaviour when plated on three-dimensional collagen gels. Skin fibroblasts from 13 of 15 patients with hereditary breast cancer showed fetal-like behaviour compared with only 1 of 12 age-matched healthy controls (p less than 0.015; Wilcoxon signed-rank matched-pairs test). In addition, 10 of 15 first-degree relatives of patients with hereditary breast cancer showed a fetal-like fibroblast phenotype, compared with none of 7 surgical controls (p less than 0.009; chi 2 test). These results suggest that abnormalities expressed by skin fibroblasts may help identify people at increased risk of breast cancer developing.