Biomedical subjects

J H Pringle

Publications and source records attributed to J H Pringle.

At least 19 recordsLinked to original sources

PubMed2006

Trimegestone differentially modulates the expression of matrix metalloproteinases in the endometrial stromal cell.

Matrix metalloproteinases (MMP) are considered to be of critical importance in the initiation of menstruation where MMP protein levels are reciprocally modulated by the actions of the gonadal steroid hormones, estradiol (E(2)) and progesterone (P4), with P4 being considered the principal suppressor of endometrial MMP expression. Trimegestone (T) is a novel progestagen that tightly controls menstruation timing and duration through mechanisms that might involve MMP suppression. Endometrial stromal cells treated with 10(-6) M E(2), P4 or T in the presence and absence of 10(-6)M RU486 showed that both T and P4 suppressed the expression of MMP-1 and MMP-3 transcripts and secreted protein, whereas MMP-9 was not produced in culture. The suppressive effect of T or P4 on MMP-1 and MMP-3 transcript levels was enhanced in the presence of E(2) and attenuated in the presence of RU486, although MMP-1 proteins were unaffected by the presence of RU486, which alone acted as a partial progesterone agonist in these cultures. Immunohistochemistry with MMP-1, MMP-3 and MMP-9-specific antibodies performed on endometrial biopsies obtained from non-treated, LH-dated, normally cycling women and endometrial biopsies obtained from postmenopausal women treated with T-based HRT showed that immunoreactive MMP-1 and MMP-3 was higher in the menstrual phase, whilst MMP-9 expression was higher in the late luteal phase (P = 0.03) and T significantly inhibited the presence of MMP-9(+) cells. These data suggest that T acts in a similar manner to P4, but causes subtle differences in expression patterns of MMPs that may explain the different clinical effect that this progestagen has on endometrial behaviour compared to P4.

Adult↗

PubMed2005

Patterns of expression of Bax, Bcl-2 and Bcl-x(L) in the implantation site in rat during pregnancy.

The uterine endometrium responds to blastocyst implantation with extensive proliferation and differentiation of stromal cells into decidual cells, forming the antimesometrial and mesometrial decidua. These undergo regression by apoptosis but as this process occurs at different time periods suggest that there is spatially dependent temporal control of apoptosis in these specific regions. To elucidate the role of the mitochondrion-dependent signalling pathway in tissue regression, we investigated the spatial and temporal pattern of expression of the Bcl-2 family members in uterine tissues of the implantation site, from the post-implantation period to parturition. Furthermore, the activities of the initiator caspases-8 and -9, and of the executioner caspase-3 were determined. Overall Bax and Bcl-2 were expressed from day 8 till day 19, whilst Bcl-x(L) was extinguished by day 16. In the antimesometrial and in the mesometrial decidua both Bcl-2 and Bax declined from days 10 to 12. In the latter Bcl-2 immunoreactivity decreased till the end of pregnancy, whilst for Bax a constant level remained thereafter. The pattern of variation of enzymatic activities throughout pregnancy for all the enzymes was similar, increasing from days 10 to 14 and decreasing towards the end of pregnancy. The increased levels of active caspase-9 correlated with Bax/Bcl-2 and Bcl-x(L) expression suggesting that the apoptotic mitochondrion-dependent pathway is involved in decidual regression during pregnancy progression.

Animals↗

PubMed2005

Vitamin D receptor gene polymorphisms, particularly the novel A-1012G promoter polymorphism, are associated with vitamin D3 responsiveness and non-familial susceptibility in psoriasis.

Psoriasis is a genetically determined disease characterized by hyperproliferation and disordered maturation of the epidermis. Th1 lymphocytes are implicated in its pathogenesis. The vitamin D receptor (VDR) is a candidate modifying gene, having immunosuppressive effects and being involved in anti-proliferative and pro-differentiation pathways in keratinocytes. There is suggestive evidence that the A allele of the A-1012G polymorphism is associated with down-regulation of the Th1 response, via GATA-3. The F and T alleles of Fok1 and Taq1 have been associated with increased VDR activity. The present study aimed to test the hypothesis that the A allele of A-1012G is protective for occurrence and severity of psoriasis and enhances therapeutic response to vitamin D analogues and that these effects would be additive to those of Fok1 and Taq1. The study group comprised 206 psoriasis patients who had received topical calcipotriol treatment and 80 controls. There was no significant linkage disequilibrium between any pair of the three polymorphic sites (P=0.3-0.8). The A, F and T alleles were positively associated with calcipotriol response: AA genotype (compared to AG/GG), odds ratio (OR)=2.18 (P=0.04); TT, OR=1.97 (P=0.03); AAFF genotype combination, OR=4.11 (P=0.03); AATT, OR=5.64 (P=0.005); and FFTT, OR=3.22 (P=0.01). Comparing patients without, to patients with, a family history of psoriasis, the A allele was under represented (P=0.01) and the AAFF genotype combination even more so (compared to residual genotypes) (OR=0.24; P=0.005). AAFF was also under-represented in patients without a family history compared to controls (OR=0.31; P=0.04). There were no associations of family history with Fok1 and Taq1. There were no associations of severity of psoriasis with any polymorphism. In conclusion, the A-1012G, Fok1 and Taq1 VDR polymorphisms were associated with response to calcipotriol. A-1012G and Fok1 were associated with susceptibility to non-familial psoriasis.

Adolescent↗

PubMed2004

A novel polymorphism in the 1A promoter region of the vitamin D receptor is associated with altered susceptibilty and prognosis in malignant melanoma.

The association of Taq 1 and Fok 1 restriction fragment length polymorphisms of the vitamin D receptor with occurrence and outcome of malignant melanoma (MM), as predicted by tumour (Breslow) thickness, has been reported previously. We now report a novel adenine-guanine substitution -1012 bp relative to the exon 1a transcription start site (A-1012G), found following screening by single-stranded conformational polymorphism of this promoter region. There was a total of 191 MM cases, which were stratified according to conventional Breslow thickness groups, cases being randomly selected from each group to form a distribution corresponding to the known distribution of Breslow thickness in our area, and this population (n=176) was compared to 80 controls. The A allele was over-represented in MM patients and, with GG as reference, odds ratio (OR) for AG was 2.5, 95% confidence interval (CI) 1.1-5.7, (P=0.03) and AA 3.3, CI 1.4-8.1, (P=0.007). The outcome was known in 171 of 191 patients and the A allele was related to the development of metastasis, the Kaplan-Meier estimates of the probability of metastasis at 5 years being: GG 0%; AG 9%, CI 4-16%; AA 21%, CI 12-36%; (P=0.008), and to thicker Breslow thickness groups (P=0.04). The effect on metastasis was independent of tumour thickness and A-1012G may have predictive potential, additional to Breslow thickness. Neither the Fok 1 nor Taq 1 variants (f and t) were significantly related to the development of metastasis, although there was a strong relationship of fftt with the thickest Breslow thickness group (P=0.005). There was an interaction between the A-1012G and Fok 1 polymorphisms (P=0.025) and the Fok 1 variant enhanced the effect of the A allele of the A-1012G polymorphism on metastasis, the probability of metastasis for AAff at 5 years follow-up being 57%, CI 24-92%.

DNA Mutational Analysis↗

PubMed2004

Patterns of uterine cellular proliferation and apoptosis in the implantation site of the rat during pregnancy.

During gestation, the balance between cell proliferation and death is crucial for successful embryo implantation and maintenance of pregnancy. The uterine endometrium responds to blastocyst implantation with extensive proliferation and differentiation of stromal cells into decidual cells, forming the antimesometrial and mesometrial decidua, which regress by apoptosis. In the latter region it is also observed the growth of metrial gland. To elucidate the events underlying this tissue remodelling we investigated the spatial and temporal pattern of expression of the proliferating cell nuclear antigen (PCNA) and localized the apoptotic cells, by the TUNEL assay and by the expression of active caspase-3. We found that PCNA is expressed at high levels during decidualization until day 12 of gestation declining thereafter abruptly. On the contrary, the appearance of apoptotic cells was detected, by the TUNEL and active caspase-3 expression, in the mesometrial decidua on day 12, increasing from days 14 to 16 in the decidua and metrial gland. In the antimesometrial decidua apoptosis was observed from early to day 12 of pregnancy. However, on day 13 only cell debris and neutrophils were observed, indicating also the presence of necrosis. These results suggest that decidual cells undergo, in distinct regions and at different stages of pregnancy, cell death by apoptosis and secondary necrosis.

Animals↗

PubMed2003

Primary breast myoepithelial cells exert an invasion-suppressor effect on breast cancer cells via paracrine down-regulation of MMP expression in fibroblasts and tumour cells.

In normal breast and ductal carcinoma in situ, myoepithelial cells form an incomplete layer separating the epithelial compartment from the stromal environment. Transition to invasive disease is marked by penetration of the myoepithelial-basement membrane (BM) interface. One mechanism involved in tumour invasion is breakdown of extracellular matrices by matrix metalloproteinases (MMPs). It was hypothesized that myoepithelial cells may modulate tumour invasion by controlling MMP gene expression, both in tumour cells and in peri-ductal fibroblasts. To investigate this, myoepithelial cells from normal breast were purified and characterized and their effect on tumour cell invasive potential was assessed. The effect on MMP gene expression of breast cancer cells cultured alone or in combination with primary normal breast fibroblasts was also analysed using RT-PCR with ELISA quantitation, with zymographic analysis to measure enzyme activity. Normal breast myoepithelial cells significantly reduced invasion by the breast cancer cell lines MCF-7, T47D, MDA-MB 231, and MDA-MB 468 when they were cultured alone or in the presence of a fibroblast population. Reduced invasion was associated with changes in MMP gene expression. In those tumour cells expressing MMP, there was a significant down-regulation of MMP-2 (MDA-MB 468, p<0.001), MMP-9 (MDA-MB 231, p=0.05; MDA-MB 468, p<0.001), and MT1-MMP (p<0.001 for both MDA-MB 231 and MDA-MB 468). Myoepithelial cells also caused a significant decrease in MMP gene expression in co-cultured fibroblasts. Furthermore, this was associated with reduced gelatinolytic activity as identified by zymography. This study demonstrates for the first time that primary myoepithelial cells from normal breast reduce breast cancer cell invasion and that this is mediated via modulation of both tumour cell and fibroblast function. This emphasizes the importance of the myoepithelial cell in controlling the breast microenvironment and focuses on the potential significance of the loss of this population with disease progression.

Antineoplastic Agents↗

PubMed2003

There is more than one kind of myofibroblast: analysis of CD34 expression in benign, in situ, and invasive breast lesions.

AIMS: Smooth muscle actin (SMA) positive myofibroblasts have been implicated in tumour invasion; however, acquisition of SMA is not limited to peritumorous fibroblasts and other changes in fibroblasts may be more specifically related to the malignant environment. CD34 is a sialomucin expressed by normal breast fibroblasts but lost in invasive carcinomas. The aim of this study was to establish the relation between CD34 and SMA expression in breast fibroblasts and to analyse whether loss of CD34 is specific for invasive disease. METHODS: Immunohistochemistry for CD34 and SMA was performed on 135 cases including 10 normal, 10 fibroadenomas, 40 infiltrating ductal carcinomas, 55 cases of ductal carcinoma in situ (DCIS), and 20 radial scar/complex sclerosing lesions. The relation between staining pattern and histopathological features was recorded as positive, negative, or reduced. RESULTS: Fibroblasts around all normal duct-lobule units and those showing epithelial hyperplasia were CD34 positive and mainly SMA negative. In fibroadenomas, fibroblasts retained CD34 but acquired SMA expression. In contrast, fibroblasts around invasive carcinoma were CD34 positive and SMA negative. In DCIS, loss of CD34 was significantly more frequent in high grade tumours than in low or intermediate grade ones (p < 0.001). The acquisition of SMA was seen more frequently than the loss of CD34, particularly in non-high grade DCIS. In all radial scars, fibroblasts were SMA positive but CD34 negative, and a similar pattern was seen in stromal cells in areas of fibrosis following core biopsy. CONCLUSIONS: These results show that SMA positive myofibroblasts exhibit variable expression of CD34, indicating that these markers are not coordinately controlled. Loss of CD34 is strongly related to the malignant phenotype, in both invasive and preinvasive disease, but is not entirely specific because radial scar fibroblasts and fibroblasts in reactive fibrosis exhibit a similar phenotype. The functional relevance of altered CD34 expression is unclear but the very focal changes implicate local signalling mechanisms probably of epithelial origin.

Actins↗

PubMed2002

Th1/Th2 cytokine expression and its relationship with tumor growth in B cell non-Hodgkin's lymphoma (NHL).

AT helper 1 (Th1) immune response is considered more effective than T helper 2 (Th2) for anti-tumor immunity, but either response could potentially stimulate tumor cell growth in lymphomas. Moreover, both IL-4 and IL-2/IL-12 are used in experimental treatment models for non-Hodgkin's lymphoma (NHL) despite their differing ability to elicit Th2 or Th1 responses, respectively. Here, we investigate which T helper cytokines (Th1 or Th2) predominate in B cell NHL tissue and determine whether cytokine expression correlates with tumor cell growth, cell death, and survival in a series of 44 NHL patients. Overall, we observed both Th1 and Th2 cytokine expression at the mRNA level, detecting high levels of IFN-gamma, IL6 and IL-10 expression in the majority of tumors. Transcripts for the IL-12 subunits p35 (38 of 38) and p 40 (23 of 38) were frequently detected in NHL tissue, and high p40 levels were common in patients with a good prognosis. Furthermore, high IL-4 levels correlated with greater survival duration (P < 0.0024) but nor overall survival. Cytokine expression of IL-2, IFNgamma and IL-4 was significantly reduced in the high grade tumor group. Interestingly, there was a strong correlation between high IL-4 levels and reduced levels of apoptosis (P < 0.006) or proliferation (P < 0.0001), which has also been reported in leukemic models. This has important implications for the success of IL-4 as a treatment for low and high grade tumors.

Adolescent↗

PubMed2001

Specific inhibition of estrogen receptor alpha function by antisense oligodeoxyribonucleotides.

We have tested the effect of a range of antisense oligodeoxyribonucleotides (ODN) directed against the human estrogen receptor alpha (ERalpha) on ERalpha protein expression and function. Antisense ERalpha ODN transfected into the ERalpha-positive human breast carcinoma cell line MCF7-K2 showed variable responses dependent on the oligo used. The most active antisense ODN (oligo 7) decreased the levels of ERa protein by 61% as measured by Western blot analysis. Exogenous 17beta-estradiol (17beta-E2), but not 17alpha-E2, augmented this effect, with a threshold effect at 10(-8) M 17beta-E2. The inhibitory effect of antisense ERa oligo 7 was confirmed by measurement of functional ERalpha protein. 3H-17beta-E2 binding to MCF7 cell extracts was inhibited to approximately 40% of control values in the presence of oligo 7. Antisense-transfected MCF7-K2 cell cultures produced a further 30% binding reduction in the presence of exogenous 17beta-E2. An inhibitory effect on 17beta-E2-dependent cell function was confirmed by the demonstration that ERalpha oligo 7-transfected MCF7-K2 cells failed to exhibit 17beta-E2-stimulated cell proliferation. Exogenous 17beta-E2 enhanced the inhibitory effect of the antisense ODN by increasing ODN transfection efficiency but without ERalpha catabolism via the proteosomal pathway, suggesting an effect of 17beta-E2 on the plasma membrane and the existence of different ERalpha degradation pathways in the MCF7-K2 cell subclone. As 17beta-E2 had no effect on ERalpha protein degradation, we conclude that the observed reduction of ERalpha protein levels is due solely to the presence of the antisense ERalpha ODN. Antisense ERalpha ODN molecules, therefore, may form the basis of effective therapies against ERalpha-dependent malignancies.

Adenocarcinoma↗

PubMed2001

Regional and cellular localization of osteonectin/SPARC expression in connective tissue and cytotrophoblastic layers of human fetal membranes at term.

Fetal membranes overlying the cervix in patients prior to and during labour, and within the rupture tear after spontaneous delivery at term, exhibit altered morphology. In this study we report that in comparison to mid-zone fetal membranes biopsies, these regions are characterized by increased expression of the matricellular protein osteonectin or SPARC (Secreted Protein Acidic and Rich in Cysteine). In the reticular layer, the percentage of vimentin positive mesenchymal cells immunoreactive for osteonectin increased in these regions from 3-4% to 25-33% and represented a fraction of the alpha-smooth muscle actin positive myofibroblasts elevated in the same regions. In the fibroblastic layer, the percentage of osteonectin positive cells increased from 1-5% to 8-13%; however, these did not exhibit the same relationship to the alpha-smooth muscle actin positive myofibroblasts in this layer. In the cytotrophoblastic layer the percentage of cytotrophoblastic cells immunoreactive for osteonectin increased from 1% to 6-12%. Elevation of in-situ detectable mRNA was also observed in the same cellular populations in this region. The incidence of cells positive for osteonectin mRNA or protein in the reticular layer correlated with morphological changes. Osteonectin has been implicated in the regulation of extracellular matrix turnover, and its pattern of expression suggests a role in the regional connective tissue and cytotrophoblastic changes proposed to be involved in the cleavage and rupture of fetal membranes.

Connective Tissue↗

PubMed2000

Inactive matrix metalloproteinase 2 is a normal constituent of human glomerular basement membrane. An immuno-electron microscopic study.

Remodelling of the extracellular matrix requires tight control not only of matrix synthesis, but also of matrix degradation. Control of matrix degradation is achieved mainly through the matrix metalloproteinase (MMP) enzymes. In the glomerulus, MMP-2 and MMP-9 are believed to be particularly important, as they have activity against type IV collagen. This study has demonstrated by immuno-electron microscopy that most of the immunoreactivity for MMP-2 in the normal glomerulus is located within the glomerular basement membranes and mesangial matrix. mRNA for MMP-2 is also detectable in normal glomeruli, but the other main gelatinase, MMP-9, could not be localized by immuno-electron microscopy. In the normal glomerulus, it seemed likely that MMP-2 is present in an inactive form. To confirm this, in situ zymography was carried out using frozen sections of normal kidney. Baseline activity of normal kidney was relatively weak, but this was dramatically increased by chemical activation of metalloproteinases. The results imply that MMP-2, in an inactive form, is a normal constituent of the extracellular matrix and glomerular basement membranes. Activation would presumably render the matrix 'self-degrading'; membrane-bound MMPs (MT-MMPs) seem particularly likely to be involved in leukocyte penetration of basement membranes in inflammation.

Basement Membrane↗

PubMed2000

Differential effects of cyclosporin and tacrolimus on the expression of fibrosis-associated genes in isolated glomeruli from renal transplants.

BACKGROUND: Chronic allograft nephropathy is characterized by an excessive accumulation of extracellular matrix proteins leading to glomerular and interstitial fibrosis. The aim of this study was to determine the effects of two different immunosuppressive agents (cyclosporin and tacrolimus) on the expression of the genes controlling extracellular matrix deposition in renal transplant glomeruli. METHODS: Fifty-one renal transplant recipients were randomized to receive immunosuppression with either microemulsion cyclosporin or tacrolimus. Isolated glomeruli were plucked from protocol transplant biopsies performed 1 week, 3 months and 6 months after transplantation. Expression of the genes for collagen IValpha2, collagen III, matrix metalloproteinase 2, tissue inhibitor of metalloproteinases (TIMP) 1 and TIMP-2, tenascin and transforming growth factor (TGF) beta1 was studied by quantitative reverse transcriptase-polymerase chain reaction. RESULTS: The expression of messenger RNA (mRNA) for collagen III and TIMP-1 was significantly higher in patients receiving cyclosporin therapy than in those having tacrolimus (P < 0.01); this finding was accounted for by differences in the biopsy material at 1 week. A significant difference in collagen III, TIMP-1 and TIMP-2 mRNA expression was also detected between patients depending on the source of renal donor (cadaveric or living). There were no significant differences in the level of glomerular TGF-beta1. CONCLUSION: The data provide new in vivo evidence that tacrolimus may exert a less fibrogenic influence on transplant glomeruli than cyclosporin.

Adult↗

PubMed1999

Immunohistochemical localization of caspase-3 correlates with clinical outcome in B-cell diffuse large-cell lymphoma.

Although B-cell diffuse large-cell lymphoma (DLCL) can respond to chemotherapy and radiotherapy, a large number of patients are still resistant to treatment. Caspase-3 is an enzyme crucial to the apoptotic process and may be important in the clinical outcome of these patients. The pattern of caspase-3 expression was studied in 54 cases of DLCL using immunohistochemistry and quantitative reverse-transcription PCR. Tumor cells displayed both a diffuse cytosolic and a punctate cytosolic staining for caspase-3. Kaplan-Meier survival curves indicated that tumor cells with a diffuse cytosolic expression of caspase-3 correlated with a poor prognosis (P > 0.0004). In addition, a punctate cellular localization was associated with complete response to treatment (P = 0.011). Cases with a small percentage of lymphoma cells expressing caspase-3 also tended to show poor survival (P > 0.09). Levels of caspase-3 mRNA were not significant (P > 0.17), although a weak trend was observed similar to the immunohistochemical analysis. The pattern of expression of caspase-3 was also assessed with respect to terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) positivity in both reactive lymph nodes and B-cell DLCL cases. Our results suggest that TUNEL-positive cells are not caspase-3-positive and that there is no correlation between DLCL cases with a high degree of DNA fragmentation and caspase-3 immunostaining. Furthermore, a survival curve indicated that a high TUNEL positivity was associated with a poor survival probability (P < 0.02) and a poor response to treatment (P = 0.04). These results confirm the dynamic nature of caspase-3 expression in DLCL and suggests that the pattern of expression of the enzyme has prognostic significance.

Adolescent↗

PubMed1999

Expression of mRNA encoding insulin-like growth factors I and II by uterine tissues and placenta during pregnancy in the rat.

The uterus and the placenta synthesize insulin-like growth factors (IGFs) and insulin-like binding proteins (IGFBPs). These growth factors are implicated in processes of proliferation and differentiation that occur in the uterus. To determine the patterns of expression of IGFs during rat pregnancy we used in situ hybridization with digoxigenin labeled probes on uterus from day 7 to day 16 of pregnancy. In early gestation days (7-8) both IGF mRNAs showed similar tissue distribution with relative abundance in the stroma and circular muscle layer. On days 11 and 12 expression for IGF-I mRNA was found in the mesometrial decidua and metrial gland and in the ectoplacental cone while clear expression of IGF-II mRNA could only be found in the latter. On days 13 and 14, expression for IGF-I mRNA could be detected in the mesometrial decidua and metrial gland but no expression was observed for IGF-II mRNA. A gradient of IGF-I mRNA expression could be observed in the placenta on day 16, with the trophoblastic cells of the basal zone expressing the signal with stronger intensity than in the labyrinthine zone. For IGF-II mRNA the highest expression was associated with the labyrinthine zone. Endovascular trophoblast was positive for both mRNAs. The spatial and temporal patterns of expression suggests a role for IGFs in the process of decidualization as well as in the establishment, growth and differentiation of the various trophoblast cells of the placenta.

Animals↗

PubMed1999

Characterisation of Kir2.0 proteins in the rat cerebellum and hippocampus by polyclonal antibodies.

Rabbit polyclonal antibodies were raised to rat Kir2.0 (Kir2.1, Kir2.2 and Kir2.3) inwardly rectifying potassium ion channel proteins. The antibody specificities were confirmed by immunoprecipitation of [35S]-methionine-labelled in vitro translated channel proteins and western blotting. Immunohistochemistry revealed a different patterns of expression of Kir2.0 subfamily proteins in the rat hind-brain (cerebellum and medulla) and fore-brain (hippocampus). Notably, only Kir2.2 protein was detected in the cerebellum and medulla, Kir2.1, Kir2.2 and Kir2.3 proteins were expressed in the hippocampus and immunostaining was not limited to neuronal cell types. Anti-Kir2.1 (fore-brain only) and anti-Kir2.2 (fore- and hind-brain) antibodies showed positive staining in macroglia, endothelia, ependyma and vascular smooth muscle cells. In contrast, anti-Kir2.3 (fore-brain only) immunostaining was limited to neurons, macroglia and vascular smooth muscle. These results indicate that specific regions within the rat fore- and hind-brain have differential distributions of inwardly rectifying potassium ion channel proteins.

Amino Acid Sequence↗

PubMed1999

Proteinuria induces tubular cell turnover: A potential mechanism for tubular atrophy.

BACKGROUND: Proteinuria and tubular atrophy have both been closely linked with progressive renal failure. We hypothesized that apoptosis may be induced by tubular cell exposure to heavy proteinuria, potentially leading to tubular atrophy. Apoptosis was studied in a rat model of "pure" proteinuria, which does not induce renal impairment, namely protein-overload proteinuria. METHODS: Adult female Lewis rats underwent intraperitoneal injection of 2 g of bovine serum albumin (BSA, N = 16) or sham saline injections (controls, N = 8) daily for seven days. Apoptosis was assessed at day 7 in tissue sections using in situ end labeling (ISEL) and electron microscopy. ISEL-positive nuclei (apoptotic particles) were counted in blinded fashion using image analysis with NIH Image. Cell proliferation was assessed by detection of mRNA for histone by in situ hybridization, followed by counting of positive cells using NIH Image. RESULTS: Animals injected with saline showed very low levels of apoptosis on image analysis. BSA-injected rats had heavy proteinuria and showed both cortical and medullary apoptosis on ISEL. This was predominantly seen in the tubules and, to a lesser extent, in the interstitial compartment. Overall, the animals injected with BSA showed a significant 30-fold increase in the number of cortical apoptotic particles. Electron microscopy of tubular cells in a BSA-injected animal showed a progression of ultrastructural changes consistent with tubular cell apoptosis. The BSA-injected animals also displayed a significant increase in proximal tubular cell proliferation. This increased proliferation was less marked than the degree of apoptosis. CONCLUSION: Protein-overload proteinuria in rats induces tubular cell apoptosis. This effect is only partially balanced by proliferation and potentially provides a direct mechanism whereby heavy proteinuria can induce tubular atrophy and progressive renal failure.

Animals↗

PubMed1999

Alternatively spliced tenascin-C mRNA isoforms in human fetal membranes.

Tenascin-C is an extracellular matrix glycoprotein whose monomers include eight consecutive fibronectin type III-like repeats, encoded by exons 10-16, and which are subject to alternative splicing. Transcripts containing these exons are expressed during tissue wounding and active tissue remodelling. Human fetal membranes have been proposed to undergo active tissue remodelling as part of the mechanisms leading to their rupture and immunoreactive tenascin-C has been detected in this tissue. Employing reverse transcription-polymerase chain reaction (RT-PCR) and exon-specific primers, products corresponding to multiple splicing events in the alternatively spliced region have now been identified. The overall splicing pattern would indicate that the major transcripts correspond to complete exclusion of the alternatively spliced region; inclusion of only exon 16; and inclusion of exons 10-14 and 16, including or excluding exon 12. The sole site in tenascin-C susceptible to cleavage by matrix metalloproteinases (MMP)-2 and MMP-3 is found within the exon 12 encoded repeat, therefore translation of isoforms which include or exclude exon 12 may produce 'large' tenascins mediating functions ascribed to this form but susceptible or resistant to these MMPs. The demonstration of expression of 'large' tenascin mRNA isoforms supports the concept that fetal membranes at term are a site of active tissue remodelling.

Alternative Splicing↗

PubMed1999

Glomerular expression of nephrin is decreased in acquired human nephrotic syndrome.

BACKGROUND: Nephrin recently has been identified as a putative adhesion molecule, expressed in the glomerulus, in which mutations cause congenital nephrotic syndrome of Finnish type. We sought to determine whether expression of nephrin is altered in human glomeruli in patients with acquired nephrotic syndrome. METHODS: We performed PCR amplification of nephrin cDNA, using cDNA previously prepared from single human glomeruli plucked fresh from the surface of human renal biopsies. We had available four cases of nephrotic syndrome (one membranous, three minimal change) and six normal controls. PCR product quantitation was by gel densitometry, confirmed by enzyme-linked immunosorbent assay using a specific oligonucleotide probe. Results were corrected for reaction efficiency and glomerular cellularity by expression as a ratio to levels of the 'housekeeping gene' glyceraldehyde phosphate dehydrogenase. RESULTS: Glomerular levels ofnephrin mRNA are significantly decreased in cases of minimal change nephrotic syndrome. An apparent reduction was also seen in the single case of membranous nephropathy which was available for study. CONCLUSIONS: Abnormalities of nephrin expression appear to be associated with acquired as well as congenital causes of human nephrotic syndrome.

Adult↗