Identification of Annexin A1 interacting proteins in chronic myeloid leukemia KCL22 cells.

In the present study, we used a functional proteomic approach to identify Annexin A1 (Anxa1) interacting proteins in the Philadelphia-positive KCL22 cell line. We focused on Anxa1 because it is one of the major proteins upregulated in imatinib-sensitive KCL22S cells versus imatinib-resistant KCL22R. Our proteomic strategy revealed 21 interactors. Bioinformatic analysis showed that most of these proteins are involved in cell death processes. Among the proteins identified, we studied the interaction of Anxa1 with two phosphatases, Shp1 and Shp2, which were recently identified as biomarkers of imatinib sensitivity in patients affected by chronic myeloid leukemia. Our data open new perspectives in the search for annexin-mediated signaling pathways and may shed light on mechanisms of resistance to imatinib that are unrelated to Bcr-Abl activity. All mass spectrometry data have been deposited in the ProteomeXchange with identifier PXD000030.

SHP-1 expression accounts for resistance to imatinib treatment in Philadelphia chromosome-positive cells derived from patients with chronic myeloid leukemia.

We prove that the SH2-containing tyrosine phosphatase 1 (SHP-1) plays a prominent role as resistance determinant of imatinib (IMA) treatment response in chronic myelogenous leukemia cell lines (sensitive/KCL22-S and resistant/KCL22-R). Indeed, SHP-1 expression is significantly lower in resistant than in sensitive cell line, in which coimmunoprecipitation analysis shows the interaction between SHP-1 and a second tyrosine phosphatase SHP-2, a positive regulator of RAS/MAPK pathway. In KCL22-R SHP-1 ectopic expression restores both SHP-1/SHP-2 interaction and IMA responsiveness; it also decreases SHP-2 activity after IMA treatment. Consistently, SHP-2 knocking-down in KCL22-R reduces either STAT3 activation or cell viability after IMA exposure. Therefore, our data suggest that SHP-1 plays an important role in BCR-ABL-independent IMA resistance modulating the activation signals that SHP-2 receives from both BCR/ABL and membrane receptor tyrosine kinases. The role of SHP-1 as a determinant of IMA sensitivity has been further confirmed in 60 consecutive untreated patients with chronic myelogenous leukemia, whose SHP-1 mRNA levels were significantly lower in case of IMA treatment failure (P < .0001). In conclusion, we suggest that SHP-1 could be a new biologic indicator at baseline of IMA sensitivity in patients with chronic myelogenous leukemia.

Preferential nuclear accumulation of JAK2V617F in CD34+ but not in granulocytic, megakaryocytic, or erythroid cells of patients with Philadelphia-negative myeloproliferative neoplasia.

Recently, Dawson et al identified a previously unrecognized nuclear role of JAK2 in the phosphorylation of histone H3 in hematopoietic cell lines. We searched nuclear JAK2 in total bone marrow (BM) cells and in 4 sorted BM cell populations (CD34(+), CD15(+), CD41(+), and CD71(+)) of 10 myeloproliferative neoplasia (MPN) patients with JAK2V617F mutation and 5 patients with wild-type JAK2 MPN. Confocal immunofluorescent images and Western blot analyses of nuclear and cytoplasmic fractions found nuclear JAK2 in CD34(+) cells of 10 of 10 JAK2-mutated patients but not in patients with wild-type JAK2. JAK2 was predominantly in the cytoplasmic fraction of differentiated granulocytic, megakaryocytic, or erythroid cells obtained from all patients. JAK2V617F up-regulates LMO2 in K562 and in JAK2V617F-positive CD34(+) cells. The selective JAK2 inhibitor AG490 normalizes the LMO2 levels in V617F-positive K562 and restores the cyto-plasmic localization of JAK2.

Gaining insights into the Bcr-Abl activity-independent mechanisms of resistance to imatinib mesylate in KCL22 cells: a comparative proteomic approach.

Imatinib mesylate is a potent inhibitor of Bcr-Abl tyrosine kinase, an oncoprotein that plays a key role in the development of chronic myeloid leukemia. Consequently, imatinib is used as front-line therapy for this disease. A major concern in imatinib treatment is the emergence of resistance to the drug. Here we used the imatinib-resistant KCL22R and imatinib-sensitive KCL22S cells in which none of the known resistance mechanisms has been detected and hence novel Bcr-Abl activity-independent mechanisms could be envisaged. We characterized proteins that were differentially expressed between the KCL22R and KCL22S cells. Using two-dimensional differential gel electrophoresis coupled with mass spectrometry and Western blot analysis we identified 51 differentially expressed proteins: 27 were over-expressed and 24 were under-expressed in KCL22R versus KCL22S cells. Several of these proteins are likely to be involved in such survival mechanisms as modulation of redox balance and activation of anti-apoptotic pathways mediated by NF-kappaB and Ras-MAPK signaling. The data reported may be useful for further studies on mechanisms of imatinib resistance and for the screening of biomarkers to develop new combinatorial therapeutic approaches.

Monitoring Wound Healing With Contactless Measurements and Augmented Reality.

OBJECTIVE: This work presents a device for non-invasive wound parameters assessment, designed to overcome the drawbacks of traditional methods, which are mostly rough, inaccurate, and painful for the patient. The device estimates the morphological parameters of the wound and provides augmented reality (AR) visual feedback on the wound healing status by projecting the wound border acquired during the last examination, thus improving doctor-patient communication. METHODS: An accurate 3D model of the wound is created by stereophotogrammetry and refined through self-organizing maps. The 3D model is used to estimate physical parameters for wound healing assessment and integrates AR functionalities based on a miniaturized projector. The physical parameter estimation functionalities are evaluated in terms of precision, accuracy, inter-operator variability, and repeatability, whereas AR wound border projection is evaluated in terms of accuracy on the same phantom. RESULTS: The accuracy and precision of the device are respectively 2% and 1.2% for linear parameters, and 1.7% and 1.3% for area and volume. The AR projection shows an error distance <1 mm. No statistical difference was found between the measurements of different operators. CONCLUSION: The device has proven to be an objective and non-operator-dependent tool for assessing the morphological parameters of the wound. Comparison with non-contact devices shows improved accuracy, offering reliable and rigorous measurements. Clinical Impact: Chronic wounds represent a significant health problem with high recurrence rates due to the ageing of the population and diseases such as diabetes and obesity. The device presented in this work provides an easy-to-use non-invasive tool to obtain useful information for treatment.

Dissolution of radiolucent renal stones by oral alkalinization with potassium citrate/potassium bicarbonate.

AIM: Uric acid stone disease is dependent on three pathogenetic factors: acid urine pH, low urine volume, and hyperuricosuria. However, the most important factor for uric acid stone formation is persistently acidic urine that represents a prerequisite for uric acid stone formation and growth. Urinary alkalization with alkali administration has been advocated for dissolution of stones on the basis of estabilished clinical experience. The aim of this study was to evaluate the clinical efficacy of therapy with potassium citrate/potassium bicarbonate for dissolution of radiolucent stones. PATIENTS AND METHODS: A total of 8 patients with radiolucent stones (< or = 15 mm) in functioning kidneys were enrolled (4 M, 4 F; mean age 66 +/- 2 years) Ultrasonography (or computed tomography scan) was done to confirm stone presence and burden and plain X-ray to exclude calcified stones. At basal a blood sample was drawn for glucose, creatinine and uric acid measurement and a 24 hour urine sample was collected for evaluation of daily uric acid excretion. Urine cultures were also performed in order rule out urinary tract infection. All patients at presentation and weekly during the study period filled out urinary pH and volume diaries. Each study day three samples of urine were collected for pH and volume measurement (morning from 8 AM to 2 PM; afternoon from 2 PM to 8 PM, and night from 8 PM to 8 AM). Two study periods were considered: during the first 6 week period a daily water intake of 1500 ml was suggested whereas in the following 6 week period the same water intake plus potassium citrate 40 mEq and potassium bicarbonate 20 mEq (divided in two doses). Potassium alkali were chosen in order to reduce the risk of calcium precipitation because of their calcium-lowering effect. The effects of treatment on stone dissolution was evaluated by ultrasonography after each study period (6 weeks and 12 weeks). RESULTS: During the first period of treatment stone burden remained unchanged in all patients. On the contrary after 6 weeks of potassium citrate/bicarbonate treatment, complete stone dissolution was found in three of the patients. In the other five cases a partial dissolution was observed and in two of them complete dissolution of the stone was achieved after prolongation of the treatment for 4 and 6 month respectively. Mean urinary volumes were unchanged during all the two study periods. Mean urinary pH was significantly higher during the potassium citrate/bicarbonate treatment period in comparison to the first study period (morning 6.60 +/- 1.06 vs 5.53 +/- 0.51, p = 0.030; afternoon 6.53 +/- 0.70 vs 5.63 +/- 0.41, p = 0.007; night 6.57 +/- 0.51 vs 5.98 +/- 0.80, p = 0.092). Tolerance of the drug was good, and no serious effects were observed sufficient to interrupt treatment. None of the patients required subsequent interventions for stone treatment. CONCLUSION: Urinary alkalization with potassium citrate/bicarbonate is a well tolerated and highly effective treatment, resulting in dissolution of nonobstructing uric acid stones.

Prospective assessment of the efficacy of the EAU guidelines for the prevention of nosocomial acquired infections after genitourinary surgery in a district hospital.

OBJECTIVES: To evaluate incidence and risk factors for nosocomial acquired infections in an urology ward after the application of the European Association of Urology (EAU) guidelines for surgical prophylaxis after genitourinary surgery in a district hospital. METHODS: A three-month survey was performed according with the definitions and methods of the National Nosocomial Infections Surveillance System. The study was conducted at the Urology Unit of a district hospital of about 800 care beds. The unit has 24 beds with approximately 1000 patients admitted per year and 750 surgical procedures performed each year. During the study period antimicrobial prophylaxis was administered according to 2006 EAU Guidelines. The following events by CDC criteria were considered: site specific infection (SSI), symptomatic urinary tract infection (SUTI), other infection of urinary tract (OUTI) and blood stream infection (BSI). RESULTS: SUTI incidence density was 31/1000 patients-days and 34/1000 urinary catheter days. SSI and BSI incidence density were respectively 44/1000 and 25/1000 patients day. A total of 177 patients (146 M, 32 F) underwent surgical procedures (17 renal surgeries, 12 retropubic radical prostatectomies, 11 prostatectomies for benign prostatic hyperplasia, 9 cystectomies (with ileal neobladder or external urinary diversion respectively in 3 and 6), 19 male genital surgeries, 21 ureterorenoscopies (with/without lithotripsy), 10 ureteral catheterizations or stenting, 6 nephrostomies, 17 TURP, 50 TURB and 5 other procedures). Perioperative antibiotic prophylaxis was administered as a single dose of cefazolin 2 gr i.v. (or as an association of gentamicin and ampicillin) in 92 patients (51%). Nine patients with positive urine culture were treated with antibiotics prior to treatment (5%) and 40 patients were treated postoperatively (22%). After surgery UTI was diagnosed in 6 patients, SSI in 3 and BSI in 11. Risk factors for infection were indwelling catheter in 22, previous history of UTI in 15, long pre operative hospital stay in 2, diabetes in 24 patients. CONCLUSIONS: Antimicrobial prophylaxis according to EAU guidelines together with an active surveillance seems to be adequate to prevent symptomatic/febrile genito-urinary infections as well as serious wound infections in the majority of patients. A further effort should be made in order to identify sub-populations of patients for which the actual prophylactic regimen proved to be less efficacious.

Italian guidelines for the prevention and management of dental trauma in children.

Dental trauma is a frequent occurrence in children and adolescent and a correct diagnosis and treatment are essential for a favourable long-term prognosis. The present Guidelines aim to formulate evidence-based recommendations to assist dentists, paediatricians, surgeons, teachers, school and sport staff, parents in the prevention and first aid of dental trauma in children and to provide a careful assessment of the medico-legal implications, reviewing the first draft of the guidelines published in 2012. A multidisciplinary panel on the behalf of the Italian Ministry of Health and in collaboration with the WHO Collaborating Centre for Epidemiology and Community Dentistry of Milan, developed this document. The following four queries were postulated: 1) Which kind of precautions the health personnel, parents, sports and educational personnel must activate in order to prevent the dental trauma damage? 2) How an orofacial trauma in paediatric patients should be managed either in the Emergency Care Unit and/or in private dental office? 3) What criteria should be adopted by a dentist private practitioner to fill in a certificate in cases of dental and/or tempomandibular joint trauma occurring in children and adolescents? 4) What are the elements that should lead clinicians to suspect a non-accidental dental trauma? A systematic review and analysis of the scientific literature published in English, Italian and French from 2007 to 2017 regarding dental trauma in children and adolescents aged 0-18 years was performed, and about 100 papers were analysed and included. The following four domains were analysed and discussed: Dental Trauma Prevention Strategies and Health Education, First aid in orofacial and dental trauma, Certificate of the dental trauma, Oral and dental signs of child abuse and neglect. Twenty-eight recommendations were draw up and codified by the panel according to the Methodological handbook, produced by the Istituto Superiore di Sanità, in order to guide physicians in the prevention and first aid of dental trauma in children and adolescents. In addition, a careful assessment of the medico-legal implications is reported in this document.

Epidemiology of renal colic in a district general hospital.

BACKGROUND/AIMS: To assess the incidence of renal colic and the results of emergency management. METHODS: During a 12 month period data of patients with symptoms of renal colic were collected. RESULTS: A total of 495 visits were registered. The M/F was 2.19. Mean age was higher in males (45.5+/-13.0 vs 42.5+/-15.5 years, P=0.025). Three patients were hospitalised for immediate urinary diversion due to anuria or sepsis. Fifty-three patients recovered without performing any pharmacological treatment. Analgesic treatment (mainly NSAID) was offered to 439 patients. After a 6 hour period 36 patients were admitted to the hospital owing to persistent pain. Pain was reduced in 403 patients (91.8%) who were offered outpatient renal ultrasound within 48 hours. Twenty-five patients (6.2%) required deferred hospitalisation. Follow up with renal ultrasound was obtained in 213. CONCLUSION: Renal colics accounted for 0.9% of ambulatory care visits to our emergency departments with an annual rate of 0.158 visits per 100 in the general population. NSAIDs were efficacious in the management of colic. Diagnostic work up was able to demonstrate the presence of a stone in 56% of the subjects presenting with renal colic whereas alternative diagnoses were demonstrated in 12%.

Augmented Reality to Improve Surgical Simulation. Lessons Learned Towards the Design of a Hybrid Laparoscopic Simulator for Cholecystectomy.

Hybrid surgical simulators based on Augmented Reality (AR) solutions benefit from the advantages of both the box trainers and the Virtual Reality simulators. This paper reports on the results of a long development stage of a hybrid simulator for laparoscopic cholecystectomy that integrates real and the virtual components. We first outline the specifications of the AR simulator and then we explain the strategy adopted for implementing it based on a careful selection of its simulated anatomical components, and characterized by a real-time tracking of both a target anatomy and of the laparoscope. The former is tracked by means of an electromagnetic field generator, while the latter requires an additional camera for video tracking. The new system was evaluated in terms of AR visualization accuracy, realism and hardware robustness. Obtained results show that the accuracy of AR visualization is adequate for training purposes. The qualitative evaluation confirms the robustness and the realism of the simulator. The AR simulator satisfies all the initial specifications in terms of anatomical appearance, modularity, reusability, minimization of spare parts cost, and ability to record surgical errors and to track in real-time the Calot's triangle and the laparoscope. The proposed system could be an effective training tool for learning the task of identification and isolation of Calot's triangle in laparoscopic cholecystectomy. Moreover, the presented strategy could be applied to simulate other surgical procedures involving the task of identification and isolation of generic tubular structures, such as blood vessels, biliary tree and nerves, which are not directly visible.

A new head-mounted display-based augmented reality system in neurosurgical oncology: a study on phantom.

PURPOSE: Benefits of minimally invasive neurosurgery mandate the development of ergonomic paradigms for neuronavigation. Augmented Reality (AR) systems can overcome the shortcomings of commercial neuronavigators. The aim of this work is to apply a novel AR system, based on a head-mounted stereoscopic video see-through display, as an aid in complex neurological lesion targeting. Effectiveness was investigated on a newly designed patient-specific head mannequin featuring an anatomically realistic brain phantom with embedded synthetically created tumors and eloquent areas. MATERIALS AND METHODS: A two-phase evaluation process was adopted in a simulated small tumor resection adjacent to Broca's area. Phase I involved nine subjects without neurosurgical training in performing spatial judgment tasks. In Phase II, three surgeons were involved in assessing the effectiveness of the AR-neuronavigator in performing brain tumor targeting on a patient-specific head phantom. RESULTS: Phase I revealed the ability of the AR scene to evoke depth perception under different visualization modalities. Phase II confirmed the potentialities of the AR-neuronavigator in aiding the determination of the optimal surgical access to the surgical target. CONCLUSIONS: The AR-neuronavigator is intuitive, easy-to-use, and provides three-dimensional augmented information in a perceptually-correct way. The system proved to be effective in guiding skin incision, craniotomy, and lesion targeting. The preliminary results encourage a structured study to prove clinical effectiveness. Moreover, our testing platform might be used to facilitate training in brain tumour resection procedures.

Conservative treatment of high grade superficial bladder tumours.

AIM OF THE STUDY: To review our previous experience in the treatment of high grade superficial bladder tumour. MATERIAL AND METHODS: Data from 71 consecutive patients (12 women and 59 men) with high grade (G3) superficial (Ta-T1) bladder tumour were considered. After endoscopic treatment 31 patients received intravesical immunotherapy with Bacillus Calmette Guerin (BCG), 14 patients were treated with a combined scheme of intravesical chemotherapy and immunotherapy and respectively 3 patients and one patient with intravesical chemotherapy with mitomycin (MMC) or epirubicin (EPI) alone. A group of 16 patients received no further treatment after TUR and other 6 patients was treated with systemic chemotherapy or radiotherapy. Patients were followed up with urine cytology and cystoscopy every three months, renal sonography every six months and intravenous pyelography every 2 years or in the case of positive cytology associated with negative cystoscopy. RESULTS: Out of 16 patients treated with TUR alone 11 recurred (69%) BCG was initiated in 31 patients and 12 out of them suffered recurrences (42%), whereas 7 out of 14 patients who had combined epirubicin/BCG therapy developed recurrences (50%). Out of 19 patients who recurred after treatment with BCG alone or combined with epirubicin, five underwent immediate cystectomy and other five delayed cystectomy after a second course of intravesical therapy. In the latter group higher pathological stages were observed. We were not able to identify any factor predictive of recurrence after BCG treatment: sex (M 16/40 vs F 3/5 p = 0.35), stage (Ta 1/6 vs T1 18/39 p = 0.11), associated carcinoma in situ (CIS) (No CIS 1/8 vs CIS 18/37 p = 0.11), multifocal (single 13/31 vs multiple 6/14 p = 1.00), age (66.8 +/- 7.8 vs 62.9 +/- 11.9 years p = 0.19) and number of previous recurrences (0.42 +/- 0.90 vs 1.31 +/- 2.16 p = 0.06). Under chemotherapy (MMC or EPI) all 4 treated patients suffered recurrences (100%). Out of all recurrent patients after conservative treatment of an high grade superficial bladder tumours, seven presented tumours with a low grade histology (downgrading). In all but one of these patients an high grade tumour recurred within 30 months. CONCLUSIONS: The adoption of a correct strategy of intravesical metaphylaxis with BCG allows to spare cistectomy in up to 60% of the patients with high grade superficial bladder carcinoma and the optimisation of treatment protocols will lead to even more effective results. However this tumour still demands the highest level of attention in the follow up and in the case of recurrence primary conservative treatment with BCG should be promptly converted to radical surgical treatment considering that the development of less invasive surgical procedures has reduced the impact on the patient's quality of life

Selective strong synergism of Ruxolitinib and second generation tyrosine kinase inhibitors to overcome bone marrow stroma related drug resistance in chronic myelogenous leukemia.

The IC50 of TKIs is significantly increased when BCR-ABL+ K562 cell line is cultured in stroma conditioned media produced by BM mesenchymal cells. In particular, while the Imatinib IC50 in the stromal co-cultures was well above the in vivo through levels of the drug, the IC50s of second generation TKIs were still below their through levels. Moreover, we provide a formal comparison of the synergy between first and second generation TKIs with the JAK inhibitor Ruxolitinib to overcome BM stroma related TKI resistance. Taken together, our data provide a rationale for the therapeutic combination of TKIs and Ruxolitinib with the aim to eradicate primary BCR-ABL+ cells homed in BM niches.