Investigation of the transforming growth factor-beta 1 signalling pathway as a possible link between hyperphosphataemia and renal fibrosis in feline chronic kidney disease.

Chronic kidney disease (CKD) is common in geriatric cats, and is characterised in the majority of cases by tubulointerstitial inflammation and fibrosis. Hyperphosphataemia is a frequent complication of CKD and is independently associated with severity of renal fibrosis and disease progression. Transforming growth factor-beta 1 (TGF-ß1) signalling is thought to be a convergent pathway which mediates the progression of renal fibrosis in CKD. The aims of this study were to explore the interaction between increased extracellular phosphate and the TGF-ß1 signalling pathway by investigating: (a) the effect of a commercially available, phosphate-restricted, diet on urinary TGF-ß1 excretion in cats with CKD; and (b) the role of increased extracellular phosphate in regulating proliferation, apoptosis, and expression of genes related to TGF-ß1 signalling and extracellular matrix (ECM) production in feline proximal tubular epithelial cells (FPTEC) and cortical fibroblasts from cats with azotaemic CKD (CKD-FCF). The dietary intervention study revealed no effect of dietary phosphate restriction on urinary active TGF-ß1 excretion after 4-8 weeks (P=0.98), despite significantly decreasing serum phosphate (P<0.001). There was no effect of increased growth media phosphate concentration (from 0.95mM to 2mM and 3.5mM) on proliferation (P=0.99) and apoptotic activity in FPTEC (P=0.22), or expression of genes related to ECM production and the TGF-ß1 signalling pathway in FPTEC and CKD-FCF (P>0.05). These findings suggest the beneficial effects of dietary phosphate restriction on progression of feline CKD may not occur through modulation of renal TGF-ß1 production, and do not support a direct pro-fibrotic effect of increased extracellular phosphate on feline renal cells.

Characterisation of Crandell-Rees Feline Kidney (CRFK) cells as mesenchymal in phenotype.

The Crandell-Rees Feline Kidney Cell (CRFK) is an immortalised cell line derived from the feline kidney that is utilised for the growth of certain vaccinal viruses. Confusion exists as to whether CRFK are epithelial or mesenchymal in phenotype. The aim of this study was to characterise CRFK cells via immunofluorescence, enzyme cytochemistry, western blotting, RT-qPCR for S100A4 and comparison to primary feline proximal tubular epithelial cells (FPTEC) and feline cortical fibroblasts (FCF). CRFK cells were of fusiform morphology and appeared similar to FCF. CRFK expressed the mesenchymal intermediate filament (IF) protein vimentin together with two cell adhesion molecules associated with feline fibroblasts (CD29 and CD44), and lacked expression of the epithelial IF cytokeratin, myogenic IF desmin and endothelial marker von Willebrand factor (vWF). In addition, CRFK did not demonstrate brush border enzyme activity typical of FPTEC. S100A4 gene expression, implicated in both neoplastic transformation and epithelial to mesenchymal transition, was highly upregulated in CRFK in comparison to the primary feline renal cells. CRFK appear phenotypically similar to fibroblasts, rather than tubular epithelial cells, and may have undergone neoplastic transformation or epithelial-to-mesenchymal transition after extensive passaging. This finding may have potential implications for future research utilising this cell line.

Correction to: mouse mammary tumour virus (MMTV) and human breast cancer with neuroendocrine differentiation.

[This corrects the article DOI: 10.1186/s13027-017-0135-8.].

Human papilloma virus is associated with breast cancer.

BACKGROUND: There is increasing evidence that high-risk human papilloma virus (HPV) is involved in cancers in addition to cervical cancer. For example, it is generally accepted that HPV has a role in a significant proportion of head and neck tumours, and it has long been hypothesised that hormone dependent oncogenic viruses, such as HPV may have causal roles in some human breast cancers. A number of reports have identified HPV DNA in breast tissue and breast cancer specimens, but these rely on standard polymerase chain reaction (PCR), which is criticised for its propensity for contamination. METHODS: We have used two different technologies, in situ and standard PCR (with sequencing), and histology based on light microscopy. RESULTS: We unambiguously demonstrate the presence of high-risk HPV in the cells of breast cancer specimens and breast cancer cell lines. In addition, we also show that the oncogenic characteristics of HPV associated breast cancer are very similar to HPV-associated cervical cancer. Specifically, that putative koilocytes are present in some HPV associated breast cancers. INTERPRETATION: The above observations indicate a likely causal role for high-risk HPV in human breast cancer and offer the possibility of primary prevention of some breast cancers by vaccination against HPV.

Koilocytes indicate a role for human papilloma virus in breast cancer.

BACKGROUND: High-risk human papilloma viruses (HPVs) are candidates as causal viruses in breast cancer. The scientific challenge is to determine whether HPVs are causal and not merely passengers or parasites. Studies of HPV-related koilocytes in breast cancer offer an opportunity to address this crucial issue. Koilocytes are epithelial cells characterised by perinuclear haloes surrounding condensed nuclei and are commonly present in cervical intraepithelial neoplasia. Koilocytosis is accepted as pathognomonic (characteristic of a particular disease) of HPV infection. The aim of this investigation is to determine whether putative koilocytes in normal and malignant breast tissues are because of HPV infection. METHODS: Archival formalin-fixed normal and malignant breast specimens were investigated by histology, in situ PCR with confirmation of the findings by standard PCR and sequencing of the products, plus immunohistochemistry to identify HPV E6 oncoproteins. RESULTS: human papilloma virus-associated koilocytes were present in normal breast skin and lobules and in the breast skin and cancer tissue of patients with ductal carcinoma in situ (DCIS) and invasive ductal carcinomas (IDCs). INTERPRETATION: As koilocytes are known to be the precursors of some HPV-associated cervical cancer, it follows that HPVs may be causally associated with breast cancer.

Presence of mouse mammary tumour-like virus gene sequences may be associated with morphology of specific human breast cancer.

BACKGROUND: Mouse mammary tumour virus (MMTV) has a proven role in breast carcinogenesis in wild mice and genetically susceptible in-bred mice. MMTV-like env gene sequences, which indicate the presence of a replication-competent MMTV-like virus, have been identified in some human breast cancers, but rarely in normal breast tissues. However, no evidence for a causal role of an MMTV-like virus in human breast cancer has emerged, although there are precedents for associations between specific histological characteristics of human cancers and the presence of oncogenic viruses. AIM: To investigate the possibility of an association between breast cancer and MMTV-like viruses. METHODS: Histological characteristics of invasive ductal human breast cancer specimens were compared with archival MMTV-associated mammary tumours from C3H experimental mice. The presence of MMTV-like env DNA sequences in the human breast cancer specimens was determined by polymerase chain reaction and confirmed by Southern hybridisation. RESULTS: MMTV-like env gene sequences were identified in 22 of 59 (37.3%) human breast cancer specimens. Seventeen of 43 (39.5%) invasive ductal carcinoma breast cancer specimens and 4 of 16 (25%) ductal carcinoma in situ specimens had some histological characteristics, which were similar to MMTV-associated mouse mammary tumours. However, these similarities were not associated with the presence or absence of MMTV-like gene sequences in the human breast tumour specimens. A significant (p = 0.05) correlation was found between the grade of the human breast cancer and similarity to the mouse mammary tumours. The lower the grade, the greater the similarity. CONCLUSION: Some human breast cancer specimens, in which MMTV-like env DNA sequences have been identified, were shown to have histological characteristics (morphology) similar to MMTV-associated mouse mammary tumours. These observations are compatible with, but not conclusive of, an association between the presence of MMTV-like env DNA sequences and some human breast cancers.

Identification of human papillomavirus DNA gene sequences in human breast cancer.

Human papilloma viruses (HPVs) are accepted as being carcinogenic in human cervical and anogenital cancers. The suspicion that HPVs may also have a role in human breast cancer is based on the identification of HPVs in human breast tumours and the immortalisation of normal human breast cells by HPV types 16 and 18. For this investigation, DNA that had been previously extracted and fresh frozen at -70 degrees C from 50 unselected invasive ductal breast cancer specimens were screened by polymerase chain reaction (PCR) for HPV type 16, 18 and 33 gene sequences. We show that HPV 18 gene sequences are present in DNA extracted from breast tumours in Australian women. Overall, 24 (48%) of the 50 samples were HPV positive. Overall no correlations with tumour grade, patient survival, steroid receptor status, ERB-2, p53 expression and mutation were observed. Human papilloma viruses may have a role in human breast cancer. We speculate that HPVs may be transmitted by hand from the female perineum to the breast.

Hormone replacement therapy use dramatically increases breast oestrogen receptor expression in obese postmenopausal women.

BACKGROUND: It is known that use of hormone replacement therapy (HRT) by postmenopausal women increases the risk of breast cancer. METHOD: In this study, oestrogen receptor (ER)-alpha expression is examined using standard immunoperoxidase technique. RESULTS: Normal breast samples of 11 Australian postmenopausal women have been included in the ER-alpha study; the result showed a strong correlation (r(2) = 0.80) between ER-alpha expression in normal breast epithelial cells and body mass index (BMI) in normal women who currently use HRT. CONCLUSION: This finding confirms that the possibility of increased risk of breast cancer associated with increased ER-alpha expression in normal breast epithelial cells, in turn associated with high BMI and the use of HRT.

From Bittner to Barr: a viral, diet and hormone breast cancer aetiology hypothesis.

It is hypothesized that the human homologue of the mouse mammary tumour virus (HHMMTV) and other viruses, such as human papillomavirus (HPV) and Epstein-Barr virus (EBV), act as cofactors with diet, oestrogens and other hormones in the initiation and promotion of some types of breast cancer in genetically susceptible women. It is further hypothesized that diet influences the risk of breast cancer, through its influence on oestrogen metabolism and that of other hormones, in combination with genetic and infectious agents.

Low oestrogen receptor alpha expression in normal breast tissue underlies low breast cancer incidence in Japan.

Among white Australians without breast cancer, the median of the percentage of oestrogen receptor alpha positive cells was 12% for women younger than 50 years and 17% for those 50 years or older; among Japanese women who had no breast cancer and are generally at low risk for this disease, the corresponding values were both significantly lower and around 9%.

The link between socioeconomic status and breast cancer--a possible explanation.

The aim of this ecological study was to explore possible associations between childhood nutrition and breast cancer. The study compared heights and weights of children of different socioeconomic status and subsequent breast cancer risk in Queensland, Australia. In 1950 12-year-old girls from families of high socioeconomic status were 3 cm taller and 6.6 kg heavier than girls of the same age of lower socioeconomic status. Approximately 35 years later, age standardized mortality rates for breast cancer among all Queensland women were approximately 10% higher for women of high compared with low socioeconomic status. Taking into account the limitations of ecological studies and risk factors other than nutrition, these results are compatible with the hypothesis that there is an association between childhood nutritional experiences and subsequent risk of breast cancer.

Capillary gas chromatography of trihexyphenidyl, procyclidine and cycrimine in biological fluids.

A sensitive (50-100 pg/ml) method is described for the analysis of the anticholinergic drugs cycrimine, procyclidine and trihexyphenidyl by capillary gas chromatography with flame thermionic detection. Since these anticholinergic drugs are frequently administered in combination with antipsychotic medication for the treatment of mental illness, the potential interference by antipsychotic drugs in this assay was examined. No interference was observed from a series of antipsychotic drugs in the quantitation of cycrimine, procyclidine or trihexyphenidyl. The use of this technique to study trihexyphenidyl pharmacokinetics in man is described.

Effect of calcium channel modulators on isolated endothelial cells.

Indirect evidence, using organic calcium channel modulators suggests that calcium channels exist in endothelial cells. Using freshly prepared and cultured bovine aortic endothelial cells, we have studied the effect of calcium channel modulators on Fura-2 fluorescence and have examined the binding of the dihydropyridine, (+)[3H]PN200-110. In both isolated primary and cultured cells, external calcium (0.5-2 mM) and bradykinin (10(-8) M) increased the intracellular calcium concentration. In cultured cells, the increase in calcium was not significantly attenuated by preincubation with nitrendipine (10(-8) M) or d-cis-diltiazem (10(-6) M). The calcium agonists (-)Bay k8644 and (+)202-791 had no effect on intracellular calcium concentration, but other agonists including ATP (10(-4) M) and thrombin (1.5 micrograms/ml) significantly increased the calcium concentration. Competition binding studies with (+)[3H]PN200-110 indicated specific binding of this ligand with a KD of 57 nM and a Bmax of 2.1 pmol/10(6) cells. While these data do not provide convincing evidence for the existence of calcium channels in cultured or fresh bovine aortic endothelial cells, explanations may yet reconcile our observations with the presence of calcium channels in these cells.

Pilot study: effective health and personal development. An experiment in school education.

This paper describes an experimental school-based health education and personal development programme conducted jointly by the Health Commission and Department of Education in northern Sydney, New South Wales. We evaluated the programme after it had been taught for five school terms, by adopting a control/experimental group design based on multiple choice questionnaires. We recorded large and significant gains in the health status of the secondary students from the experimental schools compared with the control students. Notable successes were in the areas of cigarette smoking (reductions as high as 13%), alcohol consumption (reductions of up to 12%) and exercise (increase of 17.5%). The long-term effectiveness of such programmes may depend on support from the medical profession.

Early progress report of practical experience with lifestyle programmes in a suburban population.

Lifestyle of individuals and families is now postulated to be one of the major determinants of morbidity and mortality in the Australian community. Experimental (lifestyle) programmes using techniques of physical screening of volunteers, group and individual counselling, and mass-media health promotion have been tried and evaluated during the past five years. The results have shown that it is possible to encourage and achieve behavioural and attitudinal changes in the short term for a range of lifestyle problems. Behaviour in relation to cigarette smoking and alcohol consumption has proved very difficult to alter. Physical screening was found to be much more expensive and no more effective than much simpler education and group-involvement programmes. The programmes have been reorganized to take account of these results, with new emphases being given to parents and children and the role of the doctor, as well as various adult programmes. The aims of these programmes are to prevent the establishment of harmful lifestyles as well as to change the behaviour of those who have already established poor lifestyles.