Characterisation of feline renal cortical fibroblast cultures and their transcriptional response to transforming growth factor ß1.

BACKGROUND: Chronic kidney disease (CKD) is common in geriatric cats, and the most prevalent pathology is chronic tubulointerstitial inflammation and fibrosis. The cell type predominantly responsible for the production of extra-cellular matrix in renal fibrosis is the myofibroblast, and fibroblast to myofibroblast differentiation is probably a crucial event. The cytokine TGF-ß1 is reportedly the most important regulator of myofibroblastic differentiation in other species. The aim of this study was to isolate and characterise renal fibroblasts from cadaverous kidney tissue of cats with and without CKD, and to investigate the transcriptional response to TGF-ß1. RESULTS: Cortical fibroblast cultures were successfully established from the kidney tissue of cats with normal kidney function (FCF) and cats with chronic kidney disease (CKD-FCF). Both cell types expressed the mesenchymal markers vimentin, CD44 and CD29, and were negative for the epithelial marker cytokeratin, mesangial cell marker desmin and endothelial cell marker vWF. Only CKD-FCF expressed VCAM-1, a cell marker associated with inflammation. Incubation with TGF-ß1 (0-10 ng/ml) induced a concentration dependent change in cell morphology, and upregulation of myofibroblast marker gene α-SMA expression alongside collagen 1α1, fibronectin, TGF-ß1 and CTGF mRNA. These changes were blocked by the TGF-ß1 receptor 1 antagonist SB431542 (5 µM). CONCLUSIONS: FCF and CKD-FCF can be cultured via a simple method and represent a model for the investigation of the progression of fibrosis in feline CKD. The findings of this study suggest TGF-ß1 may be involved in fibroblast-myofibroblast transition in feline CKD, as in other species.

Urinary extracellular vesicles as a source of protein-based biomarkers in feline chronic kidney disease and hypertension.

OBJECTIVES: To validate a methodology for isolating feline urinary extracellular vesicles and characterise the urinary extracellular vesicle population and proteome in cats with normal renal function and cats with normotensive or hypertensive chronic kidney disease. METHODS: Feline urinary extracellular vesicles were isolated using three different methods (precipitation alone, precipitation followed by size exclusion chromatography and ultrafiltration followed by size exclusion chromatography, which were compared via transmission electron microscopy and nanoparticle tracking analysis. Cats with normal renal function (n=9), normotensive chronic kidney disease (n=10) and hypertensive chronic kidney disease (n=9) were identified and urinary extracellular vesicles isolated from patient urine samples via ultrafiltration followed by size exclusion chromatography. Extracellular vesicle size and concentration were determined using nanoparticle tracking analysis, and subsequently underwent proteomic analysis using liquid chromatography with tandem mass spectrometry to identify differences in protein expression between categories. RESULTS: Urinary extracellular vesicle preparations contained particles of the expected size and morphology, and those obtained by ultrafiltration + size exclusion chromatography had a significantly higher purity (highest particle: protein ratio). The urinary extracellular vesicle proteomes contained extracellular vesicle markers and proteins originating from all nephron segments. Urinary extracellular vesicle concentration and size were unaffected by renal disease or hypertension. There were no differentially expressed proteins detected when comparing urinary extracellular vesicles derived from cats in the healthy category with the combined chronic kidney disease category, but five differentially expressed proteins were identified between the normotensive chronic kidney disease and hypertensive chronic kidney disease categories. CLINICAL SIGNIFICANCE: Feline urinary extracellular vesicles can be successfully isolated from stored urine samples. Differentially expressed urinary extracellular vesicle proteins were discovered in cats with hypertensive chronic kidney disease, and warrant further investigation into their utility as biomarkers or therapeutic targets.

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.

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.

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.

Sex differences in the effects of unilateral brain damage on intelligence.

A sexual dimorphism in the functional asymmetry of the damaged human brain is reflected in a test-specific laterality effect in male but not in female patients. This sex difference explains some contradictions concerning the effects of unilateral brain damage on intelligence in studies in which the influence of sex was overlooked.

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.].

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.

Urinary active transforming growth factor ß in feline chronic kidney disease.

The cytokine transforming growth factor beta 1 (TGF-ß1) has been widely implicated in the development and progression of renal fibrosis in chronic kidney disease (CKD) in humans and in experimental models. The aims of this study were to assess the association between urinary active TGF-ß1 and (a) development of CKD in a cross-sectional study, (b) deterioration of renal function over 1 year in a longitudinal study, and (c) renal histopathological parameters in cats. A human active TGF-ß1 ELISA was validated for use in feline urine. Cross-sectional analysis revealed no significant difference in urinary active TGF-ß1:creatinine ratio (aTGF-ß1:UCr) between groups with differing renal function. Longitudinally, non-azotaemic cats that developed CKD demonstrated a significant (P = 0.028) increase in aTGF-ß1:UCr approximately 6 months before the development of azotaemia, which remained elevated (P = 0.046) at diagnosis (approximately 12 months prior, 8.4 pg/mg; approximately 6 months prior, 22.2 pg/mg; at CKD diagnosis, 24.6 pg/mg). In the histopathology study, aTGF-ß1:UCr was significantly higher in cats with moderate (P = 0.02) and diffuse (P = 0.005) renal fibrosis than in cats without fibrosis. Cats with moderate renal inflammation had significantly higher urinary active aTGF-ß1 concentrations than cats with mild (P = 0.035) or no inflammatory change (P = 0.004). The parameter aTGF-ß1:UCr was independently associated with Log urine protein:creatinine ratio in a multivariable analysis of clinicopathological parameters and interstitial fibrosis score in a multivariable analysis of histopathological features. These results suggest that urinary aTGF-ß1 reflects the severity of renal pathology. Increases in urinary aTGF-ß1 followed longitudinally in individual cats may indicate the development of CKD.

Functional correlates of reduced central conduction velocity in diabetic subjects.

In a previous publication, we presented evidence of slowed conduction speed in the central nervous systems of insulin-dependent diabetic subjects, manifest in a delay in the latency of the brainstem auditory-evoked response (BAER). In this article, we present the results of a multivariate study conducted on a larger sample of 50 insulin-dependent, adult diabetic subjects. The purpose of the study was to determine some of the functional correlates of the BAER delay; each patient received an assessment of the BAER, the late auditory-evoked potential (EP), the conduction velocities of the sural, median, and common peroneal nerves, and intellectual and emotional function, in addition to neurologic and audiologic examinations. A nondiabetic control group was matched with the diabetic group as to age and sex. The results indicated a delay in the latency of wave V, and in interpeak latencies I-III and I-V, of the BAER. The most reliable effect was on interpeak latency I-V; this suggested that the locus of the delay was in the central auditory projections, rather than in the acoustic nerve. In addition, BAER waves I, II, III, IV, and V were reduced in amplitude, as was the N1 component of the late auditory EP; the most reliable reduction in amplitude was in wave V. The effect was similar in magnitude for males and females, relative to their counterparts in the control group. The slowed BAER response appeared early in the disease and was not related to the duration of insulin treatment. It was correlated with a chronic loss of energy and the presence of sexual dysfunction.(ABSTRACT TRUNCATED AT 250 WORDS)

Osteopenia, pathological fractures, and increased urinary calcium excretion in schizophrenic patients with polydipsia.

Ten male chronic schizophrenic patients with polydipsia and 10 nonpolydipsic controls, matched for gender, diagnosis, duration of illness, age, and race, were studied by dual- and single-photon absorptiometry to estimate bone density of the lumbar spine and radius and by 24-hr urine collections to estimate urinary electrolyte excretion. Bone density was normal in the control group, but was abnormally low in the polydipsic group, which had a markedly increased incidence of fractures. Electrolyte excretion was normal in the control group and in the polydipsic group when water intake was restricted to normal amounts; increased urinary sodium and calcium excretion occurred in proportion to polydipsia. As polydipsia is associated with a number of physiological changes, the cause of the osteopenia is unclear; we suggest that a negative calcium balance caused by increased urinary calcium excretion induced by extracellular space expansion may play an important role in the causation of the skeletal changes.

Thyroid function and response to 48-hour sleep deprivation in treatment-resistant depressed patients.

BACKGROUND: Clinical depression is associated with abnormalities of the hypothalamic-pituitary-thyroid axis. Changes in thyroid function during sleep deprivation may be related to its antidepressant effects. METHODS: Levels of thyroid-stimulating hormone, tri-iodothyronine, tri-iodothyronine uptake, thyroxine, and free thyroxine were measured before, during, and after a 48-hour sleep deprivation in nine treatment-resistant depressed patients. Clinical state was assessed every 4 hours. A retrospective study of 26 similar patients was added for cross-validation. RESULTS: Significant increases in thyroid-stimulating hormone and tri-iodothyronine during sleep deprivation were not correlated with clinical improvement. Sleep deprivation responders had lower tri-iodothyronine uptake levels than nonresponders in both the prospective (p <.02) and the retrospective (p <.03) samples. CONCLUSIONS: The lower tri-iodothyronine uptake values in responders may identify a subgroup of depressed patients who respond to sleep deprivation by virtue of some abnormality of the hypothalamic-pituitary-thyroid axis that is temporarily corrected by sleep deprivation.

The fragile X syndrome I: familial variation in the proportion of lymphocytes with the fragile site in males.

A review of 61 males with the fragile X positive form of the Martin Bell syndrome from 30 families seen in the past 4 years suggests that the number of lymphocytes with the fragile site on the X chromosome (fra(X) ) in retarded males tends to be characteristic for the individual and similar to that found in other retarded males in the same family. The number of lymphocytes with fra(X) was not correlated with height, weight, occipitofrontal circumference, ear length or mean testicular volume in adults nor with age over the whole series.

Elderly applicants to long-term care institutions. I. Their characteristics, health problems and state of mind.

As part of a study of long-term institutional care of the elderly, this report presents a comparison of the characteristics, health problems, and state of mind of 193 elderly applicants for such care (Group A) with those of 141 elderly persons living independently in the community (Group I). Group A members were older, living with a spouse less often, and had low incomes. They showed much more cerebrovascular disease, incontinence, recent loss of independence in the activities of daily living, dementia, recent hospitalization, loneliness, and depression. They had had much less recent involvement in social and recreational activities, although most had not been socially isolated. They had received more extensive help from relatives and friends, and it seemed unlikely that additional help from these sources would keep many more of these elderly persons out of institutions. Community agencies and services had been used by a relatively low proportion of Group A, and hardly at all by Group I.

Elderly applicants to long-term care institutions. II. The application process; placement and care needs.

As part of a study of long-term institutional care of the elderly, this report presents salient data concerning 193 elderly applicants. Physicians play an important role in decisions to apply for such care. More than half of the applicants and about 80% of their family members approved of the proposed move. The number applying to homes for the aged vs. other institutions seemed much greater than appropriate; many of these applicants appeared more elibible for placement in foster homes and specialized facilities for the demented, or for remaining in their own homes. About a fifth of the applicants could have continued living independently if they had received a reasonable amount of community assistance. Action implications include: 1) expansion and more informed use of community services, 2) more programs of supervised foster homes for the elderly, 3) construction of specialized institutions for the demented, and 4) a greater effort by institutions to satisfy the desire of many new residents to be involved there in useful tasks.

Handedness, sex and intelligence.

On the basis of data from a large and representative population (N = 1880), we have now had the opportunity to examine the relationships that have been claimed to exist between handedness, sex, and the patterning of intellectual abilities, as these are reflected in performance on the Wechsler Adult Intelligence Scale--Revised (Wechsler, 1981). Such relationships had previously been studied using rather small samples of men and women. Our analyses show a reliable, if negligible, effect of sex on these test results, but no effect of handedness.

Sex differences in the cognitive effects of unilateral brain damage: comparison of stroke patients and normal control subjects.

Our original paper on the influence of the sex of the patients on the cognitive effects of unilateral brain damage mainly described the differences found between the various lesion groups (left/right, male/female) and said little about comparisons between these groups and our control subjects. Such comparisons are examined here; they confirm that the major sex differences after such brain damage appear in tasks intended to involve nonverbal processing. This evidence supports the hypothesis that women, to a greater extent than men, may employ verbal, left hemisphere processing to solve ostensibly nonverbal problems.

Sex differences in the cognitive effects of unilateral brain damage.

One hundred subjects (50 men, 50 women), of whom 80 had suffered a unilateral cerebrovascular accident (40 left, 40 right), were tested on the WAIS. In the case of left hemisphere damage the male patients showed lower Verbal than Performance Scale IQ scores; for the right brain damaged men Performance Scale scores were Lower than their scores on the Verbal Scale. Women with unilateral brain damage showed no such reliable discrepancies between their Verbal and Performance Scale scores. This difference in the patterning of WAIS IQs in male and female stroke patients persisted even after the scores of those few patients with any significant degree of expressive aphasia had been excluded from consideration.

Reading disability subtypes and the test of memory and learning.

For Study 1, 24 readers with dysphonetic dyslexia and 21 with dyseidetic dyslexia, classified by Boder criteria, were compared to 90 control group participants (45 matched for age and 45 for reading level) on the Composite Memory Index (CMI) score from the Test of Memory and Learning (TOMAL). CMI scores were significantly lower for children with dyslexia (p <.0001). Plotting average subtest score profiles for all reader groups revealed auditory sequential memory impairments for both types of readers with dyslexia, and multiple strengths for good readers. Dysphonetic and dyseidetic dyslexia profiles were nearly identical. For Study 2, average linkage cluster analysis was performed using principal components derived from subtests of the TOMAL. Homogeneous clusters of normal readers and children with reading disabilities emerged. Results indicated that qualitatively distinct subtypes of readers with dyslexia exist.