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Biomedical subjects

L Wakefield

Publications and source records attributed to L Wakefield.

17 recordsLinked to original sources
PubMed2006

Patellar resurfacing in total knee replacement: a ten-year randomised prospective trial.

A series of 100 consecutive osteoarthritic patients was randomised to undergo total knee replacement using a Miller-Galante II prosthesis, with or without a cemented polyethylene patellar component. Knee function was evaluated using the American Knee Society score, Western Ontario and McMaster University Osteoarthritis index, specific patellofemoral-related questions and radiographic evaluation until the fourth post-operative year, then via questionnaire until ten years post-operatively. A ten-point difference in the American Knee Society score between the two groups was considered a significant change in knee performance, with alpha and beta levels of 0.05. The mean age of the patients in the resurfaced group was 71 years (53 to 88) and in the non-resurfaced group was 73 years (54 to 86). After ten years 22 patients had died, seven were suffering from dementia, three declined further participation and ten were lost to follow-up. Two patients in the non-resurfaced group subsequently had their patellae resurfaced. In the resurfaced group one patient had an arthroscopic lateral release. There was no significant difference between the two treatment groups: both had a similar deterioration of scores with time, and no further patellofemoral complications were observed in either group. We are unable to recommend routine patellar resurfacing in osteoarthritic patients undergoing total knee replacement on the basis of our findings.

Aged↗
PubMed2001

Translational regulation of renal proximal tubular epithelial cell transforming growth factor-beta1 generation by insulin.

We have previously demonstrated that the proximal tubular cell may contribute to the pathogenesis of renal interstitial fibrosis in diabetes. Transforming growth factor (TGF)-beta1 is one of a group of pro-fibrotic cytokines and growth factors, which have been associated with the development of interstitial fibrosis. The aim of the current study was to examine the effect of insulin on the generation of TGF-beta1 by proximal tubular cells. HK-2 cells were grown to confluence in the absence of insulin, and serum deprived for 48 hours before all experimental manipulations. Addition of insulin (5 microg/ml) to the culture medium led to a time-dependent increase in TGF-beta1 concentration in the cell culture supernatant, and increased incorporation of radiolabeled amino acids into TGF-beta1 suggestive of de novo TGF-beta1 protein synthesis. Addition of insulin did not alter TGF-beta1 mRNA expression as assessed by reverse transcriptase-polymerase chain reaction or Northern analysis. Insulin-induced increase in TGF-beta1 concentration was not abrogated by actinomycin D, however, stimulation by insulin, in the presence of cycloheximide led to a dose-dependent decrease in TGF-beta1 production. Addition of insulin had no effect on TGF-beta1 mRNA stability as assessed by actinomycin D chase, but led to increased binding of a cytoplasmic protein to a putative stem loop structure in the 5'-UTR of TGF-beta1 mRNA, previously implicated in the posttranscriptional control of TGF-beta1 synthesis. To address the functional significance of insulin-induced alteration in TGF-beta1 synthesis, we examined its effect on matrix turnover. Insulin stimulated type IV collagen gene expression and an increase in the concentrations of the type IV collagen laid down in the extracellular matrix. This increase in type IV collagen was abrogated when cells were stimulated by insulin in the presence of an anti-TGF-beta1-blocking antibody. In conclusion the data demonstrate that insulin may directly alter the production of TGF-beta1 by renal proximal tubular cells by a posttranscriptional mechanism, and that this may have implications for the increase in extracellular matrix that accompanies diabetic nephropathy.

5' Untranslated Regions↗
PubMed1999

Tamoxifen and fenretinide in women with metastatic breast cancer.

BACKGROUND: Tamoxifen and fenretinide combination therapy has been shown to be an active treatment regimen in metastatic breast cancer patients. This pilot study sought to determine whether the addition of fenretinide to tamoxifen would be associated with antitumor activity in metastatic breast cancer patients who had been previously treated with tamoxifen or who had hormone receptor negative disease. The effect of this therapy on circulating plasma transforming growth factor-beta (TGF-beta) levels and serum lipids was also examined. PATIENTS AND METHODS: Thirty-one patients were treated with tamoxifen (20 mg p.o. daily), and fenretinide (400 mg p.o. daily with a 3-day drug holiday each month). Plasma TGF-beta testing was performed using isoform specific sandwich ELISA. RESULTS: Twenty four of the 31 patients were evaluable for an antitumor response including 14 estrogen receptor (ER) positive patients who had failed prior tamoxifen therapy, seven ER-negative patients, and three hormone therapy naive ER-positive patients. There were no objective antitumor responses; three patients had stable disease for 8, 8, and 24 months. Five patients (16%) discontinued therapy for toxicity (one for grade 3 skin rash and four for abnormal dark adaptation). There was a statistically significant decrease in total cholesterol (median change per patient of -13.5 mg/dl; p = 0.049, a 6.5% decrease), and an increase in HDL levels (median change per patient of +18 mg/dl, p = 0.0001, a 35% increase) with tamoxifen and fenretinide therapy. TGF-beta1 plasma levels were normal in 26 of 28 patients, and no changes in these levels post-treatment were demonstrated. CONCLUSIONS: Tamoxifen and fenretinide therapy is not an active combination in ER negative metastatic breast cancer or in patients whose disease has progressed on tamoxifen. This combination had a beneficial effect on total serum cholesterol and HDL levels with no associated rise in serum triglyceride levels. The 400 mg dose of fenretinide was associated with symptomatic nyctalopia in one-third of patients making it an unsuitable dose for use in breast cancer prevention studies.

Adult↗
PubMed1998

Fine-needle aspiration cytology of malignant retroperitoneal paraganglioma.

The cytologic findings in a fine-needle aspiration sample are described from a large retroperitoneal mass in a 56-yr-old male. The aspiration was performed under CT guidance using a 22-gauge needle, maintaining negative pressure. For cytologic study, on-site smears were prepared and stained by the Papanicolaou method. Additionally, cytospin, filter preparations, cell blocks from the aspirate, histology, and electron microscopy of the tumor tissue were performed. Also, immunodiagnostic staining for neuron-specific enolase (NSE), chromogranin, S-100 protein, vimentin, HMB45, cytokeratin, and Grimelius preparation was performed on cytologic and histologic material. The cytologic material was characterized by cords, suggestive acinar structures, and small follicle-like clusters of cells, while cell blocks from the aspirate showed oval or spindle-shaped nuclei with a somewhat fasciculated appearance. However, sections from the tumor tissue showed an organoid "zellballen" pattern. The nuclei were round, oval, spindly, and epithelioid, with moderate to scanty cytoplasm, smooth nuclear membranes, fine, evenly dispersed chromatin, slight hyperchromasia, and mild pleomorphism. No intranuclear vacuoles were seen. Positive immunohistochemical staining for NSE, chromogranin, and Grimelius preparation was noted in the tumor cells, while staining for vimentin, HMB45, and cytokeratin was negative. Electron microscopy of the tumor tissue revealed the presence of variable numbers of round, membrane-bound, electron-dense neurosecretory granules. The cytohistologic and ultrastructural findings are presented, as well as the results of immunodiagnostic staining which helped in the diagnosis of retroperitoneal paraganglioma, an infrequently reported tumor.

Biopsy, Needle↗
PubMed1996

Modulation of B16 melanoma growth and metastasis by anti-transforming growth factor beta antibody and interleukin-2.

Earlier evidence suggests that transforming growth factor beta (TGF beta) plays a significant role in tumor progression and metastasis. The most likely mechanism of the action of TGF beta is induction of immunosuppression in the host, allowing for unchecked tumor growth and metastasis. We attempted to test that hypothesis and to compare antitumor effects of anti-TGF beta antibody alone and in combination with interleukin-2 (IL-2). Six- to 8-week-old female C57B1-6 mice were induced with murine B16 melanoma by tail vein injection. Therapy was started 48 h after tumor injections. Monoclonal anti-TGF beta antibody (2G7) was administered intraperitoneally (i.p.) at 500 micrograms every other day, and IL-2 at 10,000 U i.p. twice daily, for 21 days. A threefold decrease in the number of lesions in the anti-TGF beta/IL-2 treatment group compared with the control group was observed, a highly significant statistical difference (p = 0.002). No statistically significant differences were seen between the control group and other studied groups (IL-2 alone, anti-TGF beta alone). Analysis of TGF beta levels in plasma by the TGF beta-1 Quantikine assay indicated normal levels in the control and IL-2 groups, and significantly diminished levels in the two groups that received TGF beta antibody. However, acid-ethanol extraction of plasma (to reverse antibody binding before assay) showed normal plasma TGF beta levels in all groups, suggesting that the antibody may alter the availability of TGF beta in vivo. Microscopic analysis of metastases revealed a decrease in the average size of lesions in the groups treated with IL-2. Thus, combination therapy using anti-TGF beta antibody and IL-2 may be a novel, less toxic approach to tumor immunotherapy.

Animals↗
PubMed1995

Hepatic expression of mature transforming growth factor beta 1 in transgenic mice results in multiple tissue lesions.

Aberrant expression of transforming growth factor beta 1 (TGF-beta 1) has been implicated in a number of disease processes, particularly those involving fibrotic and inflammatory lesions. To determine the in vivo effects of overexpression of TGF-beta 1 on the function and structure of hepatic as well as extrahepatic tissues, transgenic mice were generated containing a fusion gene (Alb/TGF-beta 1) consisting of modified porcine TGF-beta 1 cDNA under the control of the regulatory elements of the mouse albumin gene. Five transgenic lines were developed, all of which expressed the Alb/TGF-beta 1 transgene selectively in hepatocytes. The transgenic line 25 expressing the highest level of the transgene in the liver also had high (> 10-fold over control) plasma levels of TGF-beta 1. Hepatic fibrosis and apoptotic death of hepatocytes developed in all the transgenic lines but was more pronounced in line 25. The fibrotic process was characterized by deposition of collagen around individual hepatocytes and within the space of Disse in a radiating linear pattern. Several extrahepatic lesions developed in line 25, including glomerulonephritis and renal failure, arteritis and myocarditis, as well as atrophic changes in pancreas and testis. The results from this transgenic model strongly support the proposed etiological role for TGF-beta 1 in a variety of fibrotic and inflammatory disorders. The transgenic model may also provide an appropriate paradigm for testing therapeutic interventions aimed at neutralizing the detrimental effects of this important cytokine.

Animals↗
PubMed1995

Release and activation of platelet latent TGF-beta in blood clots during dissolution with plasmin.

Transforming growth factor beta 1 (TGF-beta 1) is a platelet-derived cytokine involved in both normal wound healing and scarring. We show that human platelets contain two pools of latent TGF-beta 1, which constitute more than 95% of the total TGF-beta assayed in whole platelets. During clotting, one pool, the large latent TGF-beta complex consisting of latent TGF-beta binding protein (LTBP), the latency-associated peptide (LAP) and the 25-kD mature TGF-beta 1 dimer is released into the serum. A second pool, which contains LAP but not LTBP, is retained in the clot, but can be released by RGD peptide. When the clot is dissolved by plasmin this bound TGF-beta 1 is gradually activated and released. If similar mechanisms operate in vivo, the clot will act as a slow-release capsule of TGF-beta 1 activity during wound healing.

Blood Platelets↗
PubMed1991

Latent forms of transforming growth factor-beta (TGF beta) derived from bone cultures: identification of a naturally occurring 100-kDa complex with similarity to recombinant latent TGF beta.

Transforming growth factor-beta (TGF beta) is produced by most tissues, including bone, as a complex that is biologically inert. Release of TGF beta homodimer from this latent complex is necessary for TGF beta to exert effects on target cells. Thus, the nature of the latent complex and the mechanisms responsible for TGF beta release are the key to understanding TGF beta actions. We have found that murine calvarial bone cultures secrete multiple latent forms of TGF beta. Using analytical chromatography and Western blot analysis, we have compared bone latent TGF beta with the previously characterized latent complex present in platelets and with simian TGF beta precursor, which is stably expressed in a latent form by Chinese hamster ovarian (CHO) cells. A major component of the bone material appears to be a latent complex of 100 kDa, consisting of mature TGF beta (25-kDa homodimer). Like the recombinant TGF beta precursor, it elutes from a Mono-Q fast pressure liquid chromatography anion exchange column at 0.2 M NaCl and shows a very similar banding pattern on Western blots. Thus, this bone complex closely resembles recombinant TGF beta precursor expressed in a latent form by CHO cells and differs from the naturally occurring platelet complex, which has an additional 135-kDa binding protein that is bound through disulfide bonds to the precursor proregion. Western blot analysis also indicates that, like CHO cells, which express recombinant TGF beta precursor, but unlike other cell types, the bone cultures secrete detectable amounts of uncleaved TGF beta precursor. The bone calvarial culture is the first example of a naturally occurring system that expresses the 100-kDa latent TGF beta complex.

Animals↗
PubMed1990

Regulation of transforming growth factor-beta subtypes by members of the steroid hormone superfamily.

Transforming growth factor-beta s (TGF-beta s) are potent regulators of cell growth and differentiation. Expression of the closely related TGF-beta subtypes in vivo is differentially regulated both temporally and spatially. Members of the steroid hormone superfamily may play an important role in this gene- and tissue-specific regulation. We have shown that anti-estrogens induce the production of TGF-beta 1 in mammary carcinoma cells and fetal fibroblasts, whereas retinoic acid specifically induces TGF-beta 2 in primary epidermal keratinocytes. The induction of TGF-beta 2 by retinoids is accompanied by an increase in TGF-beta 2 mRNAs, but little change in transcription rates, suggesting an effect of retinoids on message stability or processing. In contrast, TGF-beta 1 mRNA levels are unchanged by anti-estrogen treatment, suggesting these compounds may regulate the translatability of the TGF-beta 1 message or some post-translational processing event. We have identified a stable stem-loop structure in the 5' untranslated region (UTR) of the TGF-beta 1 mRNA that inhibits translation of a heterologous reporter gene, and we are investigating the possibility that anti-estrogens may regulate the activity of this element, and hence the translatability of the TGF-beta 1 message. A significant fraction (25-90%) of the TGF-beta induced by retinoids and anti-estrogens is in the biologically active rather than the latent form. We have shown that active TGF-beta has a much shorter in vivo half-life than latent TGF-beta, suggesting that the TGF-beta induced by retinoids and steroids may act locally at the site of production. Since many tumor cells retain sensitivity to the growth inhibitory effects of active TGF-beta, the use of members of the steroid hormone superfamily for inducing this potent growth inhibitor locally at the tumor site may have therapeutic potential.

Animals↗
PubMed1989

RNA-binding properties of influenza A virus matrix protein M1.

The matrix protein (M1) of influenza A virus, which has a critical role in viral assembly and can inhibit the viral transcriptase complex, has the ability to bind RNA. The RNA-binding property of M1 is specific for single-stranded RNA, like that of influenza nucleoprotein (NP) and shows similar sensitivity to pH and to salt concentration. M1:RNA complexes are stable, once formed, to competition from excess single-stranded RNA. The possible location of the RNA-binding regions in the M1 protein is discussed.

Animals↗
PubMed1983

Cytoplasmic regulation of 5S RNA genes in nuclear-transplant embryos.

In the normal development of Xenopus laevis, genes for oocyte-type and somatic-type 5S RNAs are both expressed in late blastulae. Estimates of rates of synthesis indicate that the oocyte-type genes (5Sooc) undergo at least a 100-fold reduction in transcriptional activity between the end of oogenesis and the late blastula stage, and at least a further 20-fold reduction during gastrulation. When neurula nuclei, with inactive 5Sooc genes, were transplanted to enucleated eggs, the resulting nuclear-transplant embryos showed a reactivation of 5Sooc genes in blastulae and a subsequent inactivation after this stage. This effect is not explicable by a differential stability of the two types of 5S RNA. We conclude that egg or early embryo cytoplasm must contain components which can continuously regulate 5S gene expression, and that the mechanism by which 5Sooc genes are developmentally inactivated does not persist through mitosis in early embryos. These results have been obtained by a new procedure in which oocyte- and somatic-type 5S RNAs are separated in a 4 M urea-15% acrylamide gel.

Animals↗
PubMed1983

The activation of RNA synthesis by somatic nuclei injected into amphibian oocytes.

Previous work has shown that nuclei of cultured cells or erythrocytes are transcriptionally activated when injected into the germinal vesicle of Xenopus oocytes. We now find that the total amount of stable RNA synthesized by an oocyte with injected nuclei is about twice that of an uninjected oocyte (approximately 15 ng/day). At least half of the RNA transcribed by the injected nuclei is low-molecular-weight RNA of discrete sizes, and is transcribed by RNA polymerase III. Part of this is attributable to a new class of gene (referred to as OAX) which makes a transcript about 180 nucleotides long, and which is activated by at least 50-fold in somatic nuclei injected into oocytes. OAX RNA is not a degradation or processing variant of ribosomal or 5 S RNA, and is not coded by mitochondrial DNA. It enters oocyte cytoplasm, but turns over with a half-life of less than 3 hr, as judged by the culture of enucleate oocyte cytoplasms for several days during which 4 and 5 S RNAs are stable. OAX RNA synthesis is not detected by labelling cultured cells or their nuclei. The results emphasize the selectivity with which components of an oocyte germinal vesicle activate genes in injected somatic nuclei.

Animals↗