Multimodal Biomarkers That Predict the Presence of Gleason Pattern 4: Potential Impact for Active Surveillance.

PURPOSE: Latent grade group ≥2 prostate cancer can impact the performance of active surveillance protocols. To date, molecular biomarkers for active surveillance have relied solely on RNA or protein. We trained and independently validated multimodal (mRNA abundance, DNA methylation, and/or DNA copy number) biomarkers that more accurately separate grade group 1 from grade group ≥2 cancers. MATERIALS AND METHODS: Low- and intermediate-risk prostate cancer patients were assigned to training (n=333) and validation (n=202) cohorts. We profiled the abundance of 342 mRNAs, 100 DNA copy number alteration loci, and 14 hypermethylation sites at 2 locations per tumor. Using the training cohort with cross-validation, we evaluated methods for training classifiers of pathological grade group ≥2 in centrally reviewed radical prostatectomies. We trained 2 distinct classifiers, PRONTO-e and PRONTO-m, and validated them in an independent radical prostatectomy cohort. RESULTS: PRONTO-e comprises 353 mRNA and copy number alteration features. PRONTO-m includes 94 clinical, mRNAs, copy number alterations, and methylation features at 14 and 12 loci, respectively. In independent validation, PRONTO-e and PRONTO-m predicted grade group ≥2 with respective true-positive rates of 0.81 and 0.76, and false-positive rates of 0.43 and 0.26. Both classifiers were resistant to sampling error and identified more upgrading cases than a well-validated presurgical risk calculator, CAPRA (Cancer of the Prostate Risk Assessment; P < .001). CONCLUSIONS: Two grade group classifiers with superior accuracy were developed by incorporating RNA and DNA features and validated in an independent cohort. Upon further validation in biopsy samples, classifiers with these performance characteristics could refine selection of men for active surveillance, extending their treatment-free survival and intervals between surveillance.

A prospective study of type 2 diabetes, metformin use, and risk of breast cancer.

BACKGROUND: Type 2 diabetes (T2D) has been associated with increased breast cancer risk, but commonly prescribed antidiabetic medications such as metformin may reduce risk. Few studies have investigated T2D and medications together in relation to breast cancer. PATIENTS AND METHODS: Data came from 44 541 Sister Study participants aged 35 to 74 years at enrollment (2003-2009) who satisfied eligibility criteria, followed through 15 September 2017. Information on time-varying, self-reported, physician-diagnosed, prevalent and incident T2D, use of antidiabetic medications, and covariates was obtained from baseline and follow-up questionnaires. Incident breast cancers were confirmed with medical records. Hazard ratios (HRs) and 95% confidence intervals (CIs) were estimated. RESULTS: During follow-up (median, 8.6 years), 2678 breast cancers were diagnosed at least 1 year after enrollment. There were 3227 women (7.2%) with prevalent and 2389 (5.3%) with incident T2D, among whom 61% (n = 3386) were ever treated with metformin. There was no overall association between T2D and breast cancer risk (HR 0.99; 95% CI, 0.87-1.13). However, T2D was associated with increased risk of triple-negative breast cancer (HR 1.40; 95% CI, 0.90-2.16). Compared with not having T2D, T2D with metformin use was not associated with overall breast cancer risk (HR 0.98; 95% CI, 0.83-1.15), but it was associated with decreased risk of estrogen receptor (ER)-positive breast cancer (HR 0.86; 95% CI 0.70-1.05) and increased risk of ER-negative (HR 1.25; 95% CI, 0.84-1.88) and triple-negative breast cancer (HR 1.74; 95% CI, 1.06-2.83). The inverse association with ER-positive cancer was stronger for longer duration (≥10 year) metformin use (HR 0.62; 95% CI, 0.38-1.01; P for trend = 0.09). Results were supported by sensitivity analyses. CONCLUSION: Our findings suggest that associations between T2D and breast cancer may differ by hormone receptor status and that associations between T2D and ER-positive breast cancer may be reduced by long-term metformin use.

Impact of an integrated model of care on potentially preventable hospitalizations for people with Type 2 diabetes mellitus.

AIMS: To evaluate the impact of an integrated model of care for patients with complex Type 2 diabetes mellitus on potentially preventable hospitalizations. METHODS: A prospective controlled trial was conducted comparing a multidisciplinary, community-based, integrated primary-secondary care diabetes service with usual care at a hospital diabetes outpatient clinic. Study and hospital admissions data were linked for the period from 12 months before to 24 months after commencement of the trial. The primary outcome was the number of potentially preventable hospitalizations with diabetes-related principal diagnoses. Length of stay once hospitalized was also reported. RESULTS: Of 327 adult participants, 206 were hospitalized and accounted for 667 admissions during the study period. Compared with the usual care group, patients in the integrated model of care group were nearly half as likely to be hospitalized for a potentially preventable diabetes-related principal diagnosis in the 24 months after study commencement (incidence rate ratio 0.53, 95% CI 0.29, 0.96; P = 0.04). The magnitude of the result remained similar after adjusting for age, sex, education and baseline HbA1c concentration (incidence rate ratio 0.54, 95% CI 0.29, 1.01; P = 0.05).When hospitalized, patients in the integrated care group had a similar length of stay compared with those in the usual care group (median difference -2 days, 95% CI -6.5, 2.3; P = 0.33). CONCLUSIONS: Patients receiving the integrated model of care had a reduction in the number of hospitalizations when the principal diagnosis for admission was a diabetes-related complication. Integrated models of care for people with complex diabetes can reduce hospitalizations and help attempts to curtail increasing demand on finite health services.

Model of care for the management of complex Type 2 diabetes managed in the community by primary care physicians with specialist support: an open controlled trial.

AIMS: To evaluate patient outcomes for a novel integrated primary/specialist model of community care for complex Type 2 diabetes mellitus management compared with outcomes for usual care at a tertiary hospital for diabetes outpatients. METHODS: This was a prospective open controlled trial performed in a primary and tertiary care setting in Australia. A total of 330 patients with Type 2 diabetes aged >18 years were allocated to an intervention (n=185) or usual care group (n=145). The intervention arm was a community-based model of care led by a general practitioner with advanced skills and an endocrinologist partnership. Usual care was provided via the hospital diabetes outpatient department. The primary end point was HbA(1c) concentration at 12 months. Secondary end points included serum lipids and blood pressure. RESULTS: The mean change in HbA1c concentration in the intervention group was -9 mmol/mol (-0.8%) at 12 months and in the usual care group it was -2 mmol/mol (-0.2%) (95% CI -5,1). The percentage of patients in the intervention group achieving the HbA(1c) target of ≤53 mmol/mol (7%) increased from 21 to 42% (P<0.001); for the usual care group there was a 1% increase to 39% of patients attaining this target (P=0.99). Patients in the intervention group experienced significant improvements in blood pressure and total cholesterol compared with those in the usual care group. The percentage of patients achieving clinical targets was greater in the intervention group for the combined target of HbA(1c) concentration, blood pressure and LDL cholesterol. CONCLUSIONS: A community-based, integrated model of complex diabetes care, delivered by general practitioners with advanced skills, produced clinical and process benefits compared with a tertiary diabetes outpatient clinic.

Transforming growth factor beta signalling and matrix metalloproteinases in the mucosa overlying Crohn's disease strictures.

BACKGROUND AND AIMS: In addition to its crucial role in dampening tissue-damaging immune responses in the gut, transforming growth factor beta (TGFbeta) is a potent profibrogenic agent inducing collagen synthesis and regulating the balance between matrix-degrading matrix metalloproteinases (MMPs) and their inhibitors (TIMPs). TGFbeta signalling was investigated by analysis of Smad proteins and MMPs/TIMPs in the mucosa overlying strictures in patients with Crohn's disease (CD). METHODS: Specimens were collected from macroscopically normal mucosa overlying strictured and non-strictured gut of patients with fibrostenosing CD. Isolated myofibroblasts were cultured with anti-TGFbeta blocking antibody or TGF beta 1. TGFbeta transcripts were analysed by quantitative reverse transcription-PCR (RT-PCR). Smad proteins and MMPs were determined by immunoblotting. MMP-12 activity was measured by a real-time MMP-12 activity assay. An in vitro wound-healing scratch assay was used to assess myofibroblast migration. RESULTS: TGFbeta transcripts, phosphorylated Smad2-Smad3 (pSmad2-3) and TIMP-1 proteins were higher in mucosa overlying strictures than in mucosa overlying non-strictured areas. In contrast, mucosa overlying strictured gut had lower expression of Smad7, MMP-12 and MMP-3. Myofibroblasts from mucosa overlying strictured gut showed higher TGFbeta transcripts, a greater pSmad2-3 response to TGFbeta, increased TIMP-1, lower Smad7, increased collagen production and reduced migration ability compared with myofibroblasts from mucosa overlying non-strictured gut. TGFbeta blockade increased myofibroblast MMP-12 production and migration, more obviously in myofibroblasts isolated from mucosa overlying non-strictured compared with strictured gut. CONCLUSIONS: Changes in TGF-beta signalling and MMP production were identified in the mucosa overlying strictures in CD which may give a window into the process of fibrosis.

Is there life after plaque rupture?

Little is known of the relationship between plaque rupture and adaptive geometric remodelling, especially in the context of unstable atherosclerosis. We have assessed remodelling in the proximal brachiocephalic arteries of fat-fed apoE (apolipoprotein E)-knockout mice. The rate of vessel expansive remodelling is similar in vessels with plaques and without plaques, suggesting that the presence of plaque is not necessary for remodelling to occur. In vessels with plaques, the degree of expansive remodelling was strongly associated with the stability of the plaque. Vessels with stable plaques (i.e. with neither buried fibrous caps nor acute plaque ruptures) showed no expansion, whereas those with evidence of plaque rupture expanded at a significant rate. Vessels with stable plaques suffered significant loss of lumen over time, but those with unstable plaques maintained lumen area over time. Pravastatin treatment of male apoE-knockout mice caused a 5-fold increase in fibrous cap thickness and, although it did not influence overall rates of vessel remodelling, it significantly increased both the amount of vessel expansion and the period of time between plaque ruptures, suggesting that it increases the ability of the plaque to resist the rupturing force caused by vessel expansion. These results suggest that vessel expansion in brachiocephalic arteries of fat-fed apoE-knockout mouse does not require the presence of plaque. When a plaque is present, the outward remodelling force is exerted across its cap: vessels with smaller outward remodelling forces cannot overcome the strength of the cap, and the plaque remains stable. When the remodelling force is greater than the strength of the cap, the plaque ruptures. Thus plaque rupture can be viewed as a consequence of vessel remodelling. Interventions that strengthen the plaque, such as pravastatin therapy, do not alter remodelling parameters but instead allow for more outward remodelling before a rupture is caused.

Effects of growth hormone on leucine absorption, intestinal morphology, and ultrastructure of the goldish intestine.

The mechanisms whereby exogenous growth hormone modulates intestinal structure and function in fish were investigated. Goldfish (Carassius auratus) were fed commercial flake diet sprayed with recombinant carp growth hormone (cGH) daily for 1 month. Control animals received food sprayed with the vehicle. After 1 month of daily feedings, body mass and length were determined, and animals were sacrificed to study intestinal characteristics. Sections of foregut were removed after determination of total gut length for measurement of leucine uptake, histology, and epithelial ultrastructure. Oral administration of cGH for 1 month resulted in a 40% increase in body mass and an 8% increase in body length above controls. Gut length was 43% greater and the gut length to body length ratio was 32% greater as a result of the cGH treatment. Feeding with cGH also resulted in a significant increase in leucine uptake and increased gut mucosal thickness. Analysis of transmission electron micrographs revealed significant increases in the microvillous height and density and epithelial surface area. The findings indicate that growth hormone added to feed may increase growth in fish, in part by significantly increasing gut length, mucosal thickness, and epithelial brush border surface area, leading to enhanced epithelial absorption.

Immune-dependent thrombocytopaenia in mice infected with Schistosoma mansoni.

As has been shown previously, immunologically intact mice with patent Schistosoma mansoni infections had a significantly lower mean platelet number than intact uninfected mice (P<0.0001). However, platelet numbers in T-cell deprived mice with patent infections were not significantly different from those in uninfected T-cell deprived mice. Also, platelet counts in both the infected and uninfected T-cell deprived groups were not significantly different from those in intact uninfected mice. The S. mansoni-induced thrombocytopaenia in mice is thus seemingly immune dependent. Immunologically intact mice with chronic 12-week-old S. mansoni infections had IgG antibodies that were reactive in an ELISA-type assay with whole fixed platelets of both mouse and human origin. In Western immunoblots the IgG antibodies from chronically-infected mice reacted in particular against mouse and human platelet antigens of 90, 37 and 30 kDa. Antisera raised from 2 rabbits, immunized respectively with mouse and human platelet antigens, cross-reacted with antigens of the larval, adult worm and egg stages of S. mansoni. These results support the hypothesis that an anti-platelet antibody response may be the cause of the thrombocytopaenia observed in mice with patent schistosome infections.

Identification of a fibrinolytic enzyme in Schistosoma mansoni eggs and modulated blood fibrinogen metabolism in S. mansoni-infected mice.

Aqueous extracts of Schistosoma mansoni eggs have been shown to have fibrinolytic activity inhibitable by a serine protease inhibitor. Fibrinolytic activity was not present in extracts of either adult worms or cercariae. A 27 kDa enzyme that was proteolytically active on fibrinogen in zymography and that degraded fibrinogen in a pattern similar to that of plasmin, is presumed to be responsible for the schistosome egg fibrinolytic activity. Anti-human fibrinogen antisera were shown to have antibodies that cross-reacted with mouse fibrinogen in Western immunoblots. Electroblotted sera from S. mansoni-infected and control uninfected mice displayed different antigenic profiles when probed with the cross-reactive anti-human fibrinogen antibodies, suggesting an alteration in mouse host fibrinogen metabolism as a result of the parasitic infection. We discuss the possibility that modulation of fibrinogen metabolism is a factor in a recently discovered anti-atherogenic effect exerted by schistosomes.

An anti-atherogenic effect of Schistosoma mansoni infections in mice associated with a parasite-induced lowering of blood total cholesterol.

In affluent societies the prevalences of so-called 'Western' diseases such as atherosclerosis, allergies and autoimmune disorders appear to have increased, while many diseases caused by communicable infections are now relatively less common. To test whether there may be a causal relationship we examined the effects of Schistosoma mansoni infections in mice that develop cardiovascular pathology as a result of a genetic deficiency in apolipoprotein E (apoE-/-). The development of atherosclerotic lesions in the aortic arch and brachiocephalic artery of the apoE-/- mice was reduced by approximately 50% in mice with the parasitic infection, when comparison was made with uninfected control mice fed the same diet. Observations on S. mansoni-infected conventional laboratory mice indicate that patent schistosome infections could be counteracting the effects of an atherogenic diet by modulating host lipid metabolism and inducing a reduction in blood total cholesterol concentrations.

Urologic laparoscopy.

Urologic laparoscopy initially was confined to a diagnostic role or the treatment of benign conditions. Many of these initial procedures, however, have been abandoned because they offered no significant benefit over open surgery. The treatment of urologic malignancies, however, recently has emerged as the most common indication for laparoscopic urologic surgery. Maturing data for laparoscopic radical nephrectomy, nephroureterectomy and partial nephrectomy demonstrate equivalent oncologic results. Newer applications such as laparoscopic radical prostatectomy are evolving. Despite these data demonstrating many advantages as compared with open techniques, urologists have been slow to include laparoscopy in their practice. This reluctance has been seen in other disciplines and is no doubt, at least in part, caused by the steep learning curve. Laparoscopic management of urologic malignancies is complicated and difficult to learn. In a multi-institutional review of laparoscopic complications, 71% of the complications occurred in the first 20 cases. The risk of complications and operative time significantly declines with experience, however. Higashihara et al reported a decline in laparoscopic operative time to levels comparable to open radical nephrectomy. The learning curve seems to be approximately 30 to 40 cases. The scope and practice of urology does not provide a common procedure of relatively low complexity such as the general surgical cholecystectomy or gynecologic tubal ligation to facilitate the adoption of laparoscopy by urologists. Nevertheless, evolving techniques and equipment coupled with the incorporation of laparoscopic training in residency and fellowship programs will help secure laparoscopy a prominent place in the treatment of urologic malignancy.

Modulation of smooth muscle proliferation in rat carotid artery by platelet-derived mediators and fibroblast growth factor-2.

Endothelial denuding injury to the rat carotid artery stimulates smooth muscle cell proliferation in the tunica media. Fibroblast growth factor-2 (FGF2) is responsible for a significant portion of this proliferation but other factors may contribute, particularly those released from adherent platelets. We therefore tested the effects of a range of platelet-derived factors. After filament injury, which minimises FGF2 release, the proliferation rate in thrombocytopaenic rats was decreased by 74% (P < 0.02). After balloon injury, antibody neutralisation of platelet-derived growth factor (PDGF) caused a 27% decrease in proliferation (P < 0.05), while inhibition of histamine H(1) receptors caused a 53% increase (P < 0.05). When filament injury was performed 1 h after FGF2 injection, the proliferation rate increased from 2.3+/-0.7 to 32.8+/-2.7% (P < 0.001), while filament injury alone caused a proliferation rate of only 18.3+/-2.9% (P < 0.01 versus filament plus FGF2). These data suggest that platelet-derived factors interact with FGF2 that is adsorbed to the vessel wall in the control of smooth muscle cell proliferation, and that the net effect of platelets is to stimulate smooth muscle cell proliferation. PDGF, but no other platelet agonist tested, contributes to that stimulation.

Plasmin-mediated fibroblast growth factor-2 mobilisation supports smooth muscle cell proliferation in human saphenous vein.

The focus of this study was identification of the contribution of the plasminogen activator-plasmin system to smooth muscle cell proliferation and migration in human saphenous vein. Segments of human saphenous vein were grown in organ culture for up to 14 days. Smooth muscle cell proliferation and migration were measured by incubating vein segments in bromodeoxyuridine, and smooth muscle cell death was detected by in situ end-labelling. Tissue-type (tPA) and urokinase-type (uPA) plasminogen activator enzymic activities were detectable in cultured saphenous vein segments, and were concentrated in focal zones. Inhibition of plasmin activity with alpha-N-acetyl-L-lysine methyl ester (NALME) or of uPA activity with a neutralising antibody caused significant decreases in smooth muscle cell proliferation in the media and the intima, but no significant changes in smooth muscle cell migration. Intimal thickness was also significantly decreased. Incubation with plasminogen or plasmin caused fibroblast growth factor-2 (FGF2) to be released into the medium. Addition of FGF2 to segments cultured with NALME reversed the inhibition of smooth muscle cell proliferation, and blocking the activity of FGF2 with a neutralising antibody caused a significant decrease in medial smooth muscle cell proliferation. These data suggest that plasmin mobilises FGF2 bound to the extracellular matrix of human saphenous vein, so that it can support smooth muscle cell proliferation and intimal thickening.

Three dimensional configuration of the secretory pathway and segregation of secretion granules in the yeast Saccharomyces cerevisiae.

The structural elements of the secretory pathway in the budding yeast Saccharomyces cerevisiae were analyzed by 3D stereo-electron microscopy using relatively thick sections in which membranes were selectively impregnated. In a wild-type strain, tubular networks of various sizes and staining properties were distributed throughout the cytoplasm. As a rule, wide-meshed, lightly stained polygonal networks were connected to more or less fenestrated sheets of endoplasmic reticulum (ER). Some of these networks were continuous with more intensely stained networks and narrower meshes that displayed at their intersections nodular dilations that progressively increased in size and staining properties to reach those of secretion granules. Such networks presumably corresponded to Golgi elements. Indeed, stacked cisternae typical of the mammalian Golgi apparatus are rarely found in wild-type cells. However, if it is assumed that the Golgi apparatus plays a key role in the segregation and maturation of secretion granules, then tubular networks with nodular dilations should be equivalent to parts of this organelle. In correlation with the increase in size and density of the nodules there was a decrease in diameter and staining intensity of the interconnecting tubules. These results parallel observations on the formation of secretory granules in mammalian cells and suggest that the segregation of secretory material is concomitant with the progressive perforation and tubulization of previously unperforated sheets. When the sec21-3 thermosensitive mutant was examined at the nonpermissive temperature (37 degrees C), the secretory pathway was blocked at exit from the ER, which started to accumulate as clusters of narrow, anastomosed, unperforated ribbon-like elements. When the block was released by shifting down to permissive temperature (24 degrees C), tubular networks of various sizes and caliber, presumably Golgi in nature, formed as soon as 5 minutes after release of the block. At later time intervals, granules of various sizes and densities appeared to be released by rupture of these tubular networks or even to form at the edges of ER fenestrae. These observations support a dynamic maturation process in which the formation of secretion granules occurs by means of an oriented series of membrane transformations starting at the ER and culminating with the liberation of secretion granules from Golgi networks.

The ARF exchange factors Gea1p and Gea2p regulate Golgi structure and function in yeast.

The Sec7 domain guanine nucleotide exchange factors (GEFs) for the GTPase ARF are highly conserved regulators of membrane dynamics. Their precise molecular roles in different trafficking steps within the cell have not been elucidated. We present a functional analysis of two members of this family, Gea1p and Gea2p, in the yeast Saccharomyces cerevisiae. Gea1p and Gea2p can functionally replace each other, but at least one is necessary for viability. Temperature sensitive gea mutants were generated and found to have defects in ER-Golgi and intra-Golgi transport. Similar to mutants in COPI subunits in yeast, gea mutants had a cargo-selective secretion defect, in that some proteins continued to be secreted whereas others were blocked in the ER or early Golgi. Like yeast arf mutants, the rate of transport of those proteins that continued to be secreted was slowed. In addition, the structure of Golgi elements was severely perturbed in gea mutants. We conclude that Gea1p and Gea2p play an important role in the structure and functioning of the Golgi apparatus in yeast.

Physical and transcriptional map of the hereditary inclusion body myopathy locus on chromosome 9p12-p13.

Hereditary inclusion body myopathy (HIBM) is a group of neuromuscular disorders characterised by adult-onset, slowly progressive distal and proximal muscle weakness and typical muscle pathology. Previously, we have mapped the gene responsible for a recessive form of HIBM to chromosome 9p1 and narrowed the interval to one single YAC clone of 1 Mb in size. As a further step towards the identification of the HIBM gene, we have constructed a detailed physical and transcriptional map of this region. A high resolution BAC contig that includes the HIBM critical region, flanked by marker 327GT4 and D9S1859, was constructed. This contig allowed the precise localisation of 25 genes and ESTs to the proximal region of chromosome 9. The expression pattern of those mapped genes and ESTs was established by Northern blot analysis. In the process of refining the HIBM interval, 13 new polymorphic markers were identified, of which 11 are CA-repeats, and two are single nucleotide polymorphisms. Certainly, this map provides an important integration of physical and transcriptional information corresponding to chromosome 9p12-p13, which is expected to facilitate the cloning and identification not only of the HIBM gene, but also other disease genes which map to this region.

Controlling small guanine-nucleotide-exchange factor function through cytoplasmic RNA intramers.

ADP-ribosylation factor (ARF) GTPases and their regulatory proteins have been implicated in the control of diverse biological functions. Two main classes of positive regulatory elements for ARF have been discovered so far: the large Sec7/Gea and the small cytohesin/ARNO families, respectively. These proteins harbor guanine-nucleotide-exchange factor (GEF) activity exerted by the common Sec7 domain. The availability of a specific inhibitor, the fungal metabolite brefeldin A, has enabled documentation of the involvement of the large GEFs in vesicle transport. However, because of the lack of such tools, the biological roles of the small GEFs have remained controversial. Here, we have selected a series of RNA aptamers that specifically recognize the Sec7 domain of cytohesin 1. Some aptamers inhibit guanine-nucleotide exchange on ARF1, thereby preventing ARF activation in vitro. Among them, aptamer M69 exhibited unexpected specificity for the small GEFs, because it does not interact with or inhibit the GEF activity of the related Gea2-Sec7 domain, a member of the class of large GEFs. The inhibitory effect demonstrated in vitro clearly is observed as well in vivo, based on the finding that M69 produces similar results as a dominant-negative, GEF-deficient mutant of cytohesin 1: when expressed in the cytoplasm of T-cells, M69 reduces stimulated adhesion to intercellular adhesion molecule-1 and results in a dramatic reorganization of F-actin distribution. These highly specific cellular effects suggest that the ARF-GEF activity of cytohesin 1 plays an important role in cytoskeletal remodeling events of lymphoid cells.