Estrogen receptor-beta mRNA is associated with adverse outcome in patients with breast cancer.

BACKGROUND/AIMS: Estrogen receptor (ER) is the prototype therapy predictive marker in oncology. The ER is now known to exist in two main forms with similar overall structure: ER-alpha and ER-beta. Both forms may be expressed in breast cancer. The aim of this study was to examine breast cancer outcome in relation to expression of ER-beta. METHODS: In this investigation, we measured the expression of ER-alpha protein and ER-beta mRNA in 121 extracts of invasive breast cancer. Association of expression with clinical outcome was examined using Kaplan-Meier and Cox regression analyses. RESULTS: While ER-alpha expression was associated with good patient outcome [hazard ratio (HR) for death from breast cancer 0.37; 95% confidence interval (CI) 0.17-0.84; p = 0.017], ER-beta predicted poor outcome (HR for death from breast cancer 2.49; 95% CI 1.10-5.63; p = 0.028). CONCLUSION: Based on these findings, we conclude that ER-beta may have a different biological role from that of ER-alpha in breast cancer.

Characterisation of monoclonal antibodies for detection of Mobiluncus spp. in genital specimens.

Monoclonal antibodies were raised to the anaerobic curved rods, Mobiluncus curtisii subsp. curtisii NCTC 11656, M. curtisii subsp. holmesii NCTC 11657 and M. mulieris NCTC 11658. Three antibodies reacted with the two subspecies of M. curtisii and, when used in combination against clinical isolates, showed 100% sensitivity and specificity in immunofluorescence studies. Immunoblotting showed that two of these antibodies reacted with an epitope on a protein which had an electrophoretic mobility corresponding to a Mr of 75 Kda in the absence of a reducing agent and 82 Kda in its presence in both type strains and in clinical isolates. The third antibody reacted with an epitope in type strains which had a mobility corresponding to 75 Kda and was unaffected by a reducing agent. However, in clinical isolates the epitope was present on a protein of 75 Kda or 71 Kda, or on both. A fourth antibody showed reactivity with M. mulieris NCTC 11658 alone, but only 6 (24%) of 25 clinical isolates gave positive results by immunofluorescence. The epitope is believed to be present on a protein of greater than 90 Kda. All four antibodies were shown by immunogold staining to be directed against epitopes exposed on the cell surface.

Flow cytometric analysis of within-strain variation in polysaccharide expression by Bacteroides fragilis by use of murine monoclonal antibodies.

The reactivity of four different monoclonal antibodies (MAbs) with populations of Bacteroides fragilis NCTC 9343, enriched by density gradient centrifugation for a large capsule, small capsule and electron-dense layer (EDL) only visible by electronmicroscopy, was examined. The MAbs reacted strongly with polysaccharides present in both the large capsule- and EDL-enriched populations but not in the small capsule-enriched populations. The pattern of labelling was determined by immunoblotting, immunofluorescence and immuno-electronmicroscopy, and flow cytometry. The MAbs labelled cell membrane-associated epitopes in the large capsule- and EDL-enriched populations and cell-free material in the EDL population. By immunoblotting, ladders of repeating polysaccharide subunits were evident in the EDL population but not in the large capsule population. The proportion of cells labelled within each population was determined by flow cytometry. The reactivity of another MAb with the small capsule population was confirmed by flow cytometry. A qualitative indication of epitope expression was obtained by examination of the flow cytometric profiles. Differential expression of the same saccharide epitope was observed both between and within structurally distinct B. fragilis populations. The MAbs were species-specific and cross-reacted with several recent clinical isolates. These polysaccharides may be relevant to the virulence of B. fragilis.

Intraoperative margin assessment and re-excision rate in breast conserving surgery.

AIM: The aim of this study was to assess the efficacy of intraoperative margin assessment in obtaining clear margins in conserving surgery for breast cancer. METHODS: Two hundred and twenty patients undergoing wide local excision (WLE) for core biopsy proven primary invasive breast cancer, during a 30 months period, were included in the study. Following surgical excision the breast specimen was orientated with sutures, inked using India ink and coloured pigments and incised to identify the tumour, maintaining orientation. The distance to the individual radial margins were estimated macroscopically by the pathologist and conveyed intraoperatively to the surgeon. A macroscopic tumour-margin distance of less than 10 mm was considered compromised and the margin(s) in question was then excised if feasible. RESULTS: Eighty-one patients (37%) were judged to have compromised margins following intraoperative macroscopic evaluation and had at least one margin re-excised. Sixteen of the 81 patients (20%) in this subgroup had compromised margins on microscopy and required a second operation. One hundred and thirty-nine patients (63%) were deemed to have clear margins intraoperatively, subsequently confirmed on microscopic examination in 135 patients (97%). Intraoperative macroscopic assessment of margin status was associated with 9.1% of patients requiring a second operation. In the absence of intraoperative assessment of margin status a further 47 patients (21.4%) would have required a second operation. CONCLUSION: Intraoperative macroscopic margin assessment is an effective technique in reducing the number of second operative procedures in patients undergoing conserving surgery for primary invasive breast cancer.

An Otofuke-like virus associated with diarrhoea. Case report and electronmicroscopic study.

Virus particles similar to Otofuke virus have been found, together with rotaviruses and astroviruses, by electronmicroscopy in faeces from an infant with diarrhoea in Northern Ireland. Previously Otofuke virus has been found only in Japan whence it may have been carried to this country by a businessman.

Novel tubular inclusions in the bone marrow in multiple sclerosis: an ultrastructural study of early autopsy material.

Samples of bone marrow taken at early autopsy from patients who died with multiple sclerosis and from control cases, were examined ultrastructurally with the aim of detecting any infectious agents which might be present. No recognizable virus or mycoplasma was detected. However, rare bizarre cellular inclusions were found in 2 cases. The inclusions which are unlike anything previously described in MS consisted of fine (ca 17 nm) sinuous tubules occasionally showing dilated discoid ends. They occurred together with fragmentary electron opaque material in large membrane bound vacuoles in unidentified cells. Despite superficial resemblance to some viral nucleocapsids it is considered more likely that they have been formed as a result of degenerative phagocytic or autolytic activity. The specificity or otherwise of these inclusions to MS remains to be demonstrated.

Morphogenesis of Bittner virus.

The morphogenesis of Bittner virus (mouse mammary tumor virus) was studied in sectioned mammary tumor cells. Internal components of the virus (type A particles) were seen being assembled in virus factories close to the nucleus and were also seen forming at the plasma membrane. The particles in virus factories became enveloped by budding through the membrane of cytoplasmic vacuoles which were derived from dilated endoplasmic reticulum. Complete virus particles were liberated from these vacuoles by cell lysis. Particles budding at the plasma membrane were released into intercellular spaces. Maturation of enveloped virus occurred after release, but mature internal components were rarely seen in the cytoplasm before envelopment. Direct cell-to-cell transfer of virus by pinocytosis of budding particles by an adjacent cell was observed, and unusual forms of budding virus which participated in this process are illustrated and described. There was evidence that some virus particles contained cytoplasmic constituents, including ribosomes. Certain features of the structure of internal components are discussed in relation to a recently proposed model for the internal component of the mouse leukemia virus. Intracisternal virus-like particles were occasionally seen in tumor cells, but there was no evidence that these structures were developmentally related to Bittner virus.

Some characteristics of salt-dependent haemagglutinating measles viruses.

Several strains of measles virus which did not agglutinate monkey erythrocytes in phosphate-buffered saline did so in buffer containing 0-8 M-ammonium sulphate. Haemadsorption to cells infected with these viruses was also salt-dependent. In a series of tests salt-dependent agglutinin was shown to be a stable structural component of the infectious virion. The relevance of these findings is discussed in the light of previous reports that many measles virus preparations do not agglutinate erythrocytes.

A comparison of the haemagglutinating and enzymic activities of Bacteroides fragilis whole cells and outer membrane vesicles.

The haemagglutinating and enzymic activities of the obligately anaerobic pathogenic bacterium Bacteroides fragilis were examined. Outer membrane vesicles are released from the surface of B. fragilis. They can be detected by electron microscopy in ultrathin sections and bacterial suspensions after negative staining. Electron microscopy and immunogold labelling with a MAb specific for surface polysaccharide of B. fragilis confirmed that the vesicles carried outer membrane associated epitopes. The haemagglutinating activity of whole cells from populations of B. fragilis strains NCTC9343, BE3 and LS66 enriched by Percoll density gradient centrifugation for a large capsule (LC), electron dense layer (EDL); non-capsulate by light microscopy) and outer membrane vesicles (OMV) which had been purified by centrifugation from EDL-enriched populations were compared using human and horse erythrocytes. The enzymic activity of OMV, LC- and EDL-enriched populations, as detected by the API ZYM kit, was compared for strains NCTC 9343 and BE3. Purified OMV from the strains examined exhibited both haemagglutinating and enzymatic activity. Haemagglutination by the EDL-enriched population was sensitive to treatment with sodium periodate. The LC-enriched population haemagglutinated only after ultrasonic removal of the capsule. This indicates that the LC masks a haemagglutinin. The results suggest a potential role for OMV in the virulence of B. fragilis.

Characterization of Mapuera virus: structure, proteins and nucleotide sequence of the gene encoding the nucleocapsid protein.

The molecular biology of Mapuera virus was studied at both the protein and nucleic acid levels. Seven virus-encoded proteins were detected in infected Vero cells. The sizes and characteristics of each of the proteins determined from various radiolabelling experiments allowed preliminary identification of the proteins as the large (L; 190 kDa), haemagglutinin neuraminidase (HN; 74 kDa), nucleocapsid (N; 66 kDa), fusion (F0; 63 kDa), phosphoprotein (P; 49 kDa), matrix (M; 43 kDa) and non-structural (V; 35 kDa) proteins. Western blot analysis showed that the HN, N and P proteins were major antigens recognized in the mouse. A cDNA library of total virus-infected cellular mRNA was created and screening of the library resulted in the detection of cDNA sequences representing the N mRNA transcript of Mapuera virus. The N mRNA sequence determined from the clones was 1731 nt in length and contained an ORF that encoded 537 amino acids, the complete 3' untranslated region and part of the 5' non-coding region. The calculated M(r) of the N protein was 59 kDa, which is close to the 66 kDa protein observed by SDS-PAGE.

Large, virus-like sinuous tubules in the endoplasmic reticulum of human neurons: report from a case of encephalopathy and brief critical review.

We report a case of a rapidly progressive, fatal non-inflammatory demyelinating disease, distinct from multiple sclerosis and lysosomal disorders, in a patient with progressive dementia. Electron microscopy of stereotactic brain biopsy samples revealed the presence in neurons of sinuous, double-walled cylindrical membranous structures within the cisterns of the endoplasmic reticulum. These structures were 75-80 nm in overall diameter, up to 1.5 mm long and had a 40- to 45-nm diameter core. The possibility that they might be viruses of the Filoviridae or Paramyxoviridae families was considered, but the inclusions differed in key morphological aspects from members of both virus families and there were no supporting clinical or pathological data. Neither was it possible to assign the structures to any other known virus family on the basis of their morphology. Such inclusions have been the subject of only three published reports over the past 20 years. Evidence suggests that they may be confined to human central nervous system neurons, but occur in unrelated disorders (Alzheimer's disease, amyotrophic lateral sclerosis, meningoencephalitis, low-pressure hydrocephalus, demyelination). The possibility that they are formed in certain neurons by an abnormal internal budding process, as a response to a variety of pathological insults, is considered most likely, although an infectious origin (such as an unrecognised virus with variable clinical effect) cannot be ruled out.

Carrier cultures of simian foamy virus.

The production of cultures of HEp-2 and BHK-21 cells persistently infected with a type 1 simian foamy virus is described. After infection, HEp-2 cells showed no structural changes, whereas BHK-21 cells lost their normal spindle shape and showed mitochondrial damage, and some cells contained many lysosomes. Thin sections also showed that a few BHK-21 cells contained virus particles in low concentration, and infectious virus could be isolated from both the cells and the supernatant fluid. No virus was seen in thin sections of HEp-2 cells, although infectious virus in low titer could be recovered intermittently from lysed cells. Both carrier cultures were immune to challenge with homologous virus and antigen could be detected in over 90% of the cells even after growth for 9 weeks in the presence of virus-neutralizing serum. The distribution of antigen in carrier cultures of both cell types is described and compared with that seen in cytocidal infections.

Immunoelectron microscopic studies on haemagglutinin and haemolysin of measles virus in infected HEp2 cells.

The antigenic determinants of the haemagglutinin and haemolysin antigens of measles virus were located at the surface of HEp2 cells infected with measles virus and on measles virions released from these cells, using immunoelectron microscopy. Antisera specific for haemagglutinin or haemolysin antigen and peroxidase-conjugated antiglobulin were used. Treatment of the infected cells with trypsin removed the virus spikes and prevented binding by the anti-haemagglutinin serum, while the reaction with anti-haemolysin serum was unaltered. This suggests that the antigenic determinants for measles haemagglutinin reside on the spike, while the antigenic determinants for haemolysin reside on, or are close to, the virus membrane.

Carotid artery thrombosis, encephalitis, myelitis and optic neuritis associated with rubella virus infections.

The clinical, virological and pathological findings in 5 patients with neurological complications associated with rubella virus infection are described. The neurological illnesses began four to ten days after the rubella illnesses. The patients were all males aged between 6 and 17 years and were diagnosed during one non-epidemic year in a population of 1-5 million people. All the patients had rubella specific IgM in their sera. Two patients had no rash. In one of the patients who died, left internal carotid artery thrombosis and cerebral infarction were found at post-mortem. Rubella virus antigen and particles resembling rubella virus were found in the brain together with IgG and IgM in the same areas. This patient also had extensive liver necrosis. The other patient had a severe meningomyelitis and radiculitis and he recovered completely after two years. His serum rubella antibody rose significantly and was shown to leak into CSF during the acute stage of his illness. Three patients had a rash. Two of these patients had encephalitis: one recovered completely and the other had residual disability. The third patient had bilateral optic neuritis from which he recovered completely. Rubella specific IgM was, however, present in his serum for the abnormally long time of twenty-eight weeks indicating possible persistence of rubella virus.