The direct impact of pegvisomant on osteoblast functions and bone development.

PURPOSE: Acromegaly is a chronic disease characterized by growth hormone (GH) hypersecretion, usually caused by a pituitary adenoma, resulting in elevated circulating levels of insulin-like growth factor type I (IGF-I). Pegvisomant (PEG), the GH-receptor (GHR) antagonist, is used in treating acromegaly to normalize IGF-I hypersecretion. Exposure to increased levels of GH and IGF-I can cause profound alterations in bone structure that are not completely reverted by treatment of GH hypersecretion. Indeed, there is evidence that drugs used for the treatment of acromegaly might induce direct effects on skeletal health regardless of biochemical control of acromegaly. METHODS: We investigated, for the first time, the effect of PEG on cell proliferation, differentiation, and mineralization in the osteoblast cell lines MC3T3-E1 and hFOB 1.19 and its potential impact on bone development in zebrafish larvae. RESULTS: We observed that PEG did not affect osteoblast proliferation, apoptosis, alkaline phosphatase (ALP) activity, and mineralization. After PEG treatment, the analysis of genes related to osteoblast differentiation showed no difference in Alp, Runx2, or Opg mRNA levels in MC3T3-E1 cells. GH significantly decreased cell apoptosis (- 30 ± 11%, p < 0.001) and increased STAT3 phosphorylation; these effects were suppressed by the addition of PEG in MC3T3-E1 cells. GH and PEG did not affect Igf-I, Igfbp2, and Igfbp4 mRNA levels in MC3T3-E1 cells. Finally, PEG did not affect bone development in zebrafish larvae at 5 days post-fertilization. CONCLUSION: This study provides a first evidence of the impact of PEG on osteoblast functions both in vitro and in vivo. These findings may have clinically relevant implications for the management of skeletal health in subjects with acromegaly.

Conservative management of posterior tracheal wall injury by endoscopic stent placement in children: Preliminary data of three cases.

The management of tracheal wall lacerations is debated. Current treatments are mainly derived by the experience on adults and include conservative or surgical treatments depending on the clinical condition of the patient. We report our preliminary data with removable tracheal stents in 3 children with tracheal tears and respiratory failure. If performed in specialized centers with appropriate endoscopic and clinical follow-up, airway stents can be considered a valid and safe conservative treatment for tracheal tears and an alternative to intubation or tracheostomy. Further studies are needed to compare different therapeutic options and better define the management and duration of stent treatment.

The role of antral resection in sleeve gastrectomy. An observational comparative study.

OBJECTIVE: The role of antral resection (AR) in laparoscopic sleeve gastrectomy (LSG) is still a greatly debated topic in the literature. The aim of this study was to evaluate the results and complications of AR in LSG. PATIENTS AND METHODS: In this observational comparative study, 101 patients who underwent LSG were divided into two groups based on the extent of antral resection: 1 cm from the pylorus (1-DP group), or 6 cm from the pylorus (6-DP group). The %EWL (%Excess weight loss), resolution of T2D (Type 2 Diabetes Mellitus) and GLP-1 were investigated 48 hours before surgery and 3, 6 and 12 months after LSG. Postoperative complications in the first 30 days after surgery were also compared between the two groups using the Clavien-Dindo (CD) score. RESULTS: A significant difference in %EWL was observed at 3 and 6 months in favor of the 1-DP group (38.9% and 57.8%, respectively) compared to the 6-DP group (31.4% and 49.7%, respectively). No difference in T2D resolution was observed between two groups during the follow-up period, with similar changes in GLP-1. Statistically significant differences were found between 1-DP and 6-DP group for the reintervention rate (CD III, 7.7% and 1.9%, respectively; p = 0.02) and life-threatening complications requiring intensive care unit management (CD IV, 3.8% and 0%, respectively, p = 0.03). CONCLUSIONS: In LSG, sparing the antrum is associated with a significant reduction in the rate of postoperative complications, but the metabolic and weight results are comparable to those for antrum resection.

Water quality and human health: A simple monitoring model of toxic cyanobacteria growth in highly variable Mediterranean hot dry environments.

Due to population growth, urbanization and economic development, demand for freshwater in urban areas is increasing throughout Europe. At the same time, climate change, eutrophication and pollution are affecting the availability of water supplies. Sicily, a big island in southern Italy, suffers from an increasing drought and consequently water shortage. In the last decades, in Sicilian freshwater reservoirs several Microcystis aeruginosa and more recently Planktothrix rubescens blooms were reported. The aims of the study were: (1) identify and quantify the occurring species of cyanobacteria (CB), (2) identify which parameters, among those investigated in the waters, could favor their growth, (3) set up a model to identify reservoirs that need continuous monitoring due to the presences, current or prospected, of cyanobacterial blooms and of microcystins, relevant for environmental and, consequentially, for human health. Fifteen artificial reservoirs among the large set of Sicilian artificial water bodies were selected and examined for physicochemical and microbiological characterization. Additional parameters were assessed, including the presence, identification and count of the cyanobacterial occurring species, the measurement of microcystins (MCs) levels and the search for the genes responsible for the toxins production. Principal Component Analysis (PCA) was used to relate environmental condition to cyanobacterial growth. Water quality was poor for very few parameters, suggesting common anthropic pressures, and PCA highlighted clusters of reservoirs vulnerable to hydrological conditions, related to semi-arid Mediterranean climate and to the use of the reservoir. In summer, bloom was detected in only one reservoir and different species was highlighted among the Cyanobacteria community. The only toxins detected were microcystins, although always well below the WHO reference value for drinking waters (1.0 µg/L). However, molecular analysis could not show the presence of potential cyanotoxins producers since a few numbers of cells among total could be sufficient to produce these low MCs levels but not enough high to be proved by the traditional molecular method applied. A simple environmental risk-based model, which accounts for the high variability of both cyanobacteria growth and cyanotoxins producing, is proposed as a cost-effective tool to evaluate the need for monitoring activities in reservoirs aimed to guarantee supplying waters safety.

Metal removal processes in a pilot hybrid constructed wetland for the treatment of semi-synthetic stormwater.

This study investigates the reliability of a pilot hybrid constructed wetland (H-CW), located in Eastern Sicily (Italy). To address the uncertainty associated with implementing representative monitoring during highly variable storm events, unique to Mediterranean conditions, a recipe for semi-synthetic stormwater was used to evaluate the removal efficiency of the system. This was characterised by metals (Cd, Cr, Fe, Pb, Cu, Zn) and relative concentrations typically found in urban stormwater runoff (SR). Approximately one month of intensive monitoring activities were carried out and quality analyses were conducted on three matrices comprising the pilot H-CW: water, biomass (Canna indica, Typha latifolia), and volcanic gravel substrate. Metal retention in early clogging matter (SS) was also examined. The results showed a significantly high H-CW efficiency for the removal of all metals (70-98%) already at the horizontal flow unit outflow, confirming its strategic role. A metal mass balance analysis was also conducted to describe the retention capacity and influence of each system component on the overall efficiency (ranging from 87.8% for Cr to 99.2% for Pb). Metal removal was mostly related to sediment and substrate processes, while plants exhibited root bioaccumulation and phytostabilisation capacity even with a limited impact on overall system retention. The pilot H-CW exhibits characteristics suitable for the treatment of metal-enriched stormwater runoff and validates the useful application of decentralised natural systems for water resource management.

Impact of the environment on the health: From theory to practice.

The Erice 56 Charter titled "Impact of the environment on the health: from theory to practice" was unanimously approved at the end of the 56th course of the "International School of Epidemiology and Preventive Medicine G. D'Alessandro" held from 3rd to November 7, 2019 in Erice - Sicily (Italy) and promoted by the Study Group of "Environment and Health" of the Italian Society of Hygiene, Preventive Medicine and Public Health. The course, that included lectures, open discussions and guided working groups, was aimed to provide a general training on epidemiological and toxicological aspects of the environmental health impact, to be used by public health professionals for risk assessment, without forgetting the risk communications. At the end of the course 12 key points were agreed among teachers and students: they underlined the need of specific training and research, in the perspective of "One Health" and "Global Health", also facing emerging scientific and methodological issues and focusing on communication towards stakeholders. This Discussion highlight the need to improve knowledge of Health and Environment topic in all sectors of health and environmental prevention and management.

Contraception during Coronavirus-Covid 19 pandemia. Recommendations of the Board of the Italian Society of Contraception.

Purpose: The Italian Society of Contraception identified as one of its priorities the need to give recommendations on management of contraception during Coronavirus-Covid 19 pandemiaMaterials and methods: A concise communication was produced which summarises in an easy-to-read format suitable for clinicians the management of the different contraceptives mostly used. Information how to manage contraception in different conditions is presented.Results: Women may, in general, continue to use either intrauterine and or hormonal contraceptives. The use of condom should be added to any hormonal contraceptive, when the contraceptive efficacy is reduced or when women stop the contraceptive method.Conclusion: At the present time, during the Coronavirus-Covid 19 pandemia, no data contraindicate the use of intrauterine or hormonal contraceptives. Conversely the use of an appropriate contraception is advocate to prevent unintended pregnancies.

Cilostazol reduces dry eye symptoms and improve walking distance in patients with peripheral artery disease.

AIM OF THE STUDY: Cilostazol is a phosphodiesterase III inhibitor that has anti-inflammatory and immunomodulatory effects and can act with beneficial effect in Dry Eye Syndrome (DES). This clinical trial evaluates the effects of cilostazol on the tear film. MATERIALS AND METHODS: Following the run-in period, subjects were randomly into two groups: 40 subjects treated with cilostazol and 40 no-treated subjects. The Walking Impairment Questionnaire (WIQ) has been administered to all patients. RESULT: The data obtained from comparison of the two study groups A and B were, respectively, the following: Schirmer I: 10.2±0.2 Vs 13.8±0.4 (p< 0.001); Schirmer II: 3.8±0.1 Vs 4.6±0.2 (p<0.001); Break-up time (BUT) 4.2±0.3 Vs 6.5±0.2 (p<0.001) with disappearing of symptoms. The WIQ showed a significant difference in the walking distance (p<0.05) and calf pain severity (p<0.005) of treated patients. In comparison with the placebo group, treated patients showed an improvement (p<0.03) in calf pain severity. CONCLUSION: The administration of cilostazol was effective, in reducing DES and improve walking distance questionnaire.

Reply for comment on "Exposure to microplastics (<10 µm) associated to plastic bottles mineral water consumption: The first quantitative study by Zuccarello et al. [Water Research 157 (2019) 365-371]".

Microplastics (MPs) are a threat to both the environment and human health. In the absence of an official method that would allow the analysis of all MPs with diameters less than 10 µm, it was not possible to estimate the exact exposure to these contaminants, yet. In the last years, few studies are available in literature with the first data concerning the concentrations of MPs on bottle mineral waters. Zuccarello et al. (2019) carried out an exposure assessment study where a high value of EDI (Estimated Daily Intake) has been reported. This rebuttal letter aims to reply to the comments of Oßmann et al. (2018) on the article of Zuccarello et al. (2019) about the new analytical method and both the use and the interpretation of Principal Component Analysis.

Vascular endothelial growth factor expression as a biomarker of prognosis in patients with chondrosarcoma, Ewing's sarcoma and osteosarcoma. Current concepts.

With the advent of the molecularly targeted therapies, identifying molecular therapeutic targets and molecolar marker is increasingly important, especially in neoplastic diseases. Several studies show VEGF is involved in neo-angiogenesis in many solid cancers, as breast, lung, renal, gastric carcinomas, through promoting endothelial cell growth and migration. Conversely the relationship between VEFG and tumours of the musculoskeletal system is yet unclear, in particular the role of VEGF has not yet been completely understood in these tumours. Chondrosarcoma, Ewing's Sarcoma and Osteosarcoma are the tumours of the musculoskeletal system in which the activity of VEGF has been closely studied. The present study aims to give an overview focused on the relationship between VEGF and these three cancers.

The combined use of platelet rich plasma and hyaluronic acid: prospective results for the treatment of knee osteoarthritis.

Osteoarthritis represents an important social economic burden with a high incidence worldwide. Conservative management of knee OA consists in several therapeutic options: pharmacologic therapy such as analgesics, non-steroid and steroid anti-inflammatory drugs, physical therapy, and injective therapy with hyaluronic acid (HA) and platelet-rich plasma injections (PRP). The aim of our study is to evaluate the effect of combined autologous PRP and HHA (Hybrid Hyaluronic Acid) viscosupplementation on clinical outcomes of patients with knee OA, by assessing the subjects before and after injective treatment. The study was conducted on 60 patients with an age between 40 and 70 years old affected by unilateral symptomatic knee osteoarthritis (stage II and III of Kellgren-Lawrence scale) nonresponsive to pharmacologic and rehab treatment. We divided the patients in two groups, and we treated the group A with injection of HHA and group B with HHA+PRP. Each patient received 3 injections at an interval of 1 week for 3 consecutive weeks. The patients were evaluated by the Knee Injury and Osteroartrhitis Outcome Score (KOOS) and Visual Analog Scale (VAS) at 3, 6 and 12 months after treatment. Statistical comparison between groups showed a significantly better result for the group B concerning the KOOS value, at 3 months and at 6 months. This difference, although clinically relevant, lost the statistical significance at 12 months. The VAS trend differently showed a significant difference at 3 and 12 months, while at 6 months the superiority of group B did not achieve statistical significance. Few studies investigated the effects of HA+PRP combined treatment for knee OA. Numerous studies demonstrated the efficacy of HA injection therapy in knee OA for a clinical point of view, reducing the pain and improving the quality of life. PRP preparations also improved functional outcome scores compared to hyaluronic acid and placebo in patients affected by knee OA. Based on our results we can conclude that the combined PRP and HHA treatment is not only a safe and efficacious procedure which can provide functional benefit but is also significantly better than HHA injective therapy alone, as demonstrated by the comparison within our cohort.

Hip megaprosthesis in oncological surgery: open questions.

Prosthetic replacement with modular implants has become the most common reconstructive tech¬nique of bone loss of the lower limb after tumour resection. The use of the megaprosthesis in bone metastasis, silver-coated megaprosthesis and the use of Trevira tube are not uniform and represent an "open question" about the use of megaprosthesis. The following paper aims to review the current literature in this topic.

Sport activity as risk factor for early knee osteoarthritis.

There is wide discussion about the association between sport activity and musculoskeletal disorders, as sports-related joint loading increases the risk of osteoarthritis (OA). The present article reviews the current available literature on the connections between participation in several sports and athletic activities and prevalence of knee OA, especially focusing on early knee OA. The study was based on an electronic search through web databases including Medline, Cochrane and Google Scholar. Articles were retrieved and evaluated, and case series, retrospective studies, case-control studies, prospective cohort studies and randomized controlled trials were considered for inclusion. The main data were extracted and summarized in tables and text. Athletic individuals do show an increased prevalence of knee OA, especially for professional athletes when compared to general population or non-professional athletes. Furthermore, several features related to sport activity were associated to increased risk of early knee OA, such as knee ligamentous injury, concussion, high-impact sports and different team roles. Methodology and results of the included studies are barely comparable, thus preventing the authors to carry out an accurate and systematic comparison of the results of the included studies. Only low level evidence studies are available, and better designed studies, with radiological and functional evaluation of the knee based on internationally validated measures, should be planned. Also, follow-up of patients during and after their life-period of sport involvement should be considered.

Exposure to microplastics (<10 µm) associated to plastic bottles mineral water consumption: The first quantitative study.

The uncontrolled introduction into the environment of plastic polymers have caused the dispersion of plastic fragments, known as Microplastics (MPs), that represent an important topic for public health. This study was the first to investigate the cause of the release of MPs in mineral waters and to estimate the concentration of MPs smaller than 10 µm both in number of particles and in mass unit. This study was carried out using a patent method regarding the extraction and analysis of MPs in more kind of matrix. Therefore, aims of this study were a) to assess the number of MPs with diameters of between 0.5 and 10 µm in mineral waters contained in plastic bottles, b) to evaluate if the physical-chemical properties of mineral waters and bottle quality could influence the release of MPs and, finally, c) to estimate the human daily exposure to MPs due to mineral water consumption. The Mps were found in every sample. The main concentration of MPs was 656.8 µg/L ± 632.9 or 5.42E+07 p/L ± 1.95E+07. The main diameter of detected MPs was 2.44 µm ± 0.66 (where p/L, where p was the number of MPs). The Estimated Daily Intake (EDI) for adults and children were 1,531,524 p/kg/body-weight/day corresponding to 40.1 µg/kg/body-weight/day and 3,350,208 p/kg/body-weight/day corresponding to 87.8 µg/kg/body-weight/day, respectively. The number of MPs contamination in bottled mineral waters was strongly correlated to the pH of waters and to plastic density of bottle. Otherwise, micrograms of MPs per liter and the MPs diameters were strongly affected by plastic thickness. The most mineral water brand contaminated by MPs was the one whose bottles were made from poor quality plastic. In absence of reference values, it was no possible carried out a risk assessment for MPs exposure. It is fundamental to establish the reference method of analysis to monitoring every source of human intake.

Gene-ecology of durum wheat HMW glutenin reflects their diffusion from the center of origin.

The production of many food items processed from wheat grain relies on the use of high gluten strength flours. As a result, about 80% of the allelic variability in the genes encoding the glutenin proteins has been lost in the shift from landraces to modern cultivars. Here, the allelic variability in the genes encoding the high molecular weight glutenin subunits (HMW-GSs) has been characterized in 152 durum wheat lines developed from a set of landraces. The allelic composition at the two Glu-1 loci (Glu-A1 and -B1) was obtained at both the protein and the DNA level. The former locus was represented by three alleles, of which the null allele Glu-A1c was the most common. The Glu-B1 locus was more variable, with fifteen alleles represented, of which Glu-B1b (HMW-GSs 7 + 8), -B1d (6 + 8) and -B1e (20 + 20) were the most frequently occurring. The composition of HMW-GSs has been used to make inferences regarding the diffusion and diversification of durum wheat. The relationships of these allelic frequencies with their geographical distribution within the Mediterranean basin is discussed in terms of gene-ecology.

Etiology of chronic skin lesions in subjects with peripheral arterial disease.

INTRODUCTION: Skin lesions can be defined as lesions that result in loss of tissues and their joints, and often this cutaneous skin process is a primary or secondary consequence of the structural changes in the skin itself. Subjects with peripheral arteripathies that develop chronic skin lesions in the lower extremities of the Western world are constantly increasing. We conducted a study on the etiologic incidence of chronic skin lesions in peripheral arterial disease CSLpa subjects in the lower limbs compared to subjects with chronic skin lesions CSL (controls). MATERIALS AND METHODS: 30 subjects with peripheral atheropathies PA (22 F - 8 M mean age 74,5 ± 4,9) and with chronic skin lesions (CSLpa) in the lower limbs "A" group were admitted to our study according to a randomized and compared to 30 no peripheral atheropathies subjects (19 F-11 M, mean age 81,5 ± 7,3 - controls) group B with chronic skin lesions (CSL). These two groups "A" and "B" have been studied and compared on the basis of infectious etiology responsible for the infectious skin process. RESULTS: In the subjects of the "A" group we found a 12 positive assay of 40.0% of the examinations, while in the group "B" we achieved a total cultured positivity of 9 cases corresponding to 30.0% of the examinations . For the number of bacterial species identified for "A" group we obtained 3 mono microbial and 6 poly microbial bacteriological tests and for group "B" we observed 7 mono microbial and 2 poly microbial tests. All bacteriological isolates showed "in vitro" sensitivity to satisfactory ciprofloxacin with MICs range of 0.78-1.56mg/L. The data observed after 4 weeks after the amniotic membrane (MA) in the two study groups A and B were respectively the following: and for group A 50% scarring, 46.6% partial resolution and in one case worsening for the B-healing group in 63.3%, the partial resolution in the remaining 36.6. CONCLUSIONS: The data from this study show a different etiology between subjects with CSLpa than subjects with CSL. This phenomenon confirms an alteration of the skin microbioma of subjects with peripheral arteriopathy and chronic skin lesions with modification of the opportunistic role of some species of cutaneous bacterial flora.

Live surgery: highly educational or harmful?

PURPOSE: Live surgery (LS) is considered a useful teaching opportunity. The benefits must be balanced with patient safety concerns. To evaluate the rate of complications of a series of urologic LS performed by experts during the Congress Challenge in Laparoscopy and Robotics (CILR). METHODS: We present a large, multi-institution, multi-surgeon database that derives from 12 CILR events, from 2004 to 2015 with a total of 224 cases. Radical prostatectomy (RP) was the most common procedure and a selection of complex cases was noted. The primary measure was postoperative complications and use of a Postoperative Morbidity Index (PMI) to allow quantitative weighing of postoperative complications. RESULTS: From 12 events, the number of cases increased from 11 in 2004 to 27 in 2015 and a total of 27 surgeons. Of 224 cases (164 laparoscopic and 60 robotic), there were 26 (11.6%) complications: 5 grade I, 5 grade II, 3 grade IIIa, 12 grade IIIb and 1 grade V, the latter from laparoscopic cystectomy. Analysis of PMI was 23 times higher from cystectomy compared to RP. CONCLUSIONS: In the setting of live surgery, the overall rate of complications is low considering the complexity of surgeries. The PMI is not higher in more complex procedures, whereas RP seems very safe.

Can an adjustable-loop length suspensory fixation device reduce femoral tunnel enlargement in anterior cruciate ligament reconstruction? A prospective computer tomography study.

BACKGROUND: The aim of this study was to compare bone femoral tunnel enlargement in patients who underwent anterior cruciate ligament (ACL) transtibial reconstruction using an adjustable-loop length suspensory fixation device and a fixed-loop length suspensory fixation device. METHODS: All patients treated for ACL reconstruction with an ipsilateral hamstring between March 2013 and March 2014 were evaluated. Subjects were assigned to Group A (TightRope™ (TR) femoral fixation) or Group B (EndoButton® (EB) femoral fixation). All patients were evaluated with the Lachman test, pivot-shift test, 2000 International Knee Documentation Committee (IKDC) knee examination and KT1000 arthrometer. The subjective evaluation was performed using the 2000 IKDC Subjective Knee score, the Lysholm knee score, and the Tegner activity scale. CT examination was performed to evaluate femoral tunnel enlargement at four different levels. All patients were assessed at a 12month follow-up visit. Power analysis was performed a priori in accordance with the femoral tunnel enlargement values from the CT scans. Differences with P-values of ≤0.05 were considered to be statistically significant. RESULTS: The groups were homogenous at baseline with regard to age, gender, BMI, dominance and disease duration. At the final follow-up, no statistically significant differences (P>0.05) were found according to subjective and objective clinical outcome measures. According to the femoral tunnel enlargement, no statistically significant difference was found between the two groups (P>0.05). CONCLUSION: In transtibial ACL reconstruction, the use of a fixed or adjustable-loop length device products, on the femoral side, led to similar clinical and radiological results.

Hsa-miR-30d, secreted by the human endometrium, is taken up by the pre-implantation embryo and might modify its transcriptome.

During embryo implantation, the blastocyst interacts with and regulates the endometrium, and endometrial fluid secreted by the endometrial epithelium nurtures the embryo. Here, we propose that maternal microRNAs (miRNAs) might act as transcriptomic modifier of the pre-implantation embryo. Microarray profiling revealed that six of 27 specific, maternal miRNAs were differentially expressed in the human endometrial epithelium during the window of implantation--a brief phase of endometrial receptivity to the blastocyst--and were released into the endometrial fluid. Further investigation revealed that hsa-miR-30d, the expression levels of which were most significantly upregulated, was secreted as an exosome-associated molecule. Exosome-associated and free hsa-miR-30d was internalized by mouse embryos via the trophectoderm, resulting in an indirect overexpression of genes encoding for certain molecules involved in the murine embryonic adhesion phenomenon--Itgb3, Itga7 and Cdh5. Indeed, this finding was supported by evidence in vitro: treating murine embryos with miR-30d resulted in a notable increase in embryo adhesion. Our results suggest a model in which maternal endometrial miRNAs act as transcriptomic modifiers of the pre-implantation embryo.

Robot-assisted radical prostatectomy: recent advances.

Robot assisted radical prostatectomy (RARP) represents the leading application of robotic surgery in the urologic field and it has become the main treatment option for localized prostate cancer (PCa) in the USA In the present review we summarized and critically analyzed the literature of the past five years about this widely used robotic procedure. RARP has continuously evolved in terms of technical modifications and procedural steps. Long-term data are now available, suggesting comparable oncological outcomes to those of open and laparoscopic radical prostatectomy. Good functional outcomes have also been demonstrated. Technological innovations and the introduction of more advanced robotic platforms featuring novel arm-integrated equipment, together with a mature clinical experience with the robotic approach, are likely to lead towards optimal outcomes. Despite the expanding clinical implementation of RARP in the management of prostate cancer, some issues related to this procedure remain matter of debate, such as costs, comparative outcomes versus other approaches, and its role in high risk disease.