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approximately , one - third of patients with symptomatic vte manifests pe , whereas two - thirds manifest dvt alone . both dvt and pe can be clinically silent ( asymptomatic ) and hence not suspected . if undiagnosed , asymptomatic vte can lead to chronic venous disease or recurrent vte and long - term debilitating sequelae such as postthrombotic syndrome and chronic thromboembolic pulmonary hypertension . vte is not only disabling but also prolongs hospital stay and increases the cost of treatment . along with myocardial infarction and arrhythmia ( due to electrolyte imbalance ) , pe is one of the commonest causes of sudden unexplained deaths in hospitalized patients . it is estimated that 20 million cases of lower extremity dvt occur in the usa alone . the prevailing notion that the incidence of vte in asians is less than that in the western population has been disproved by recent studies . the incidence of postoperative dvt in indian patients undergoing major lower limb surgery is as high ( 43.2% and 60% patients in the groups with and without prophylaxis , respectively ) as seen in the western world . given the growing burden of vte in india and lack of substantial indian data on characteristics of vte patients , use of diagnostics tools , prophylaxis , treatment options , and clinical outcomes in vte , there was a need to systematically collect such data . data on patient characteristics , clinical outcomes , predictors of mortality in acute dvt , management strategies and temporal trends in vte . the intent was to collect and provide data that would reflect actual day - to - day clinical practice , rather than results of highly controlled clinical trials with restricted study populations and imposed experimental intervention . consecutive medical records of inpatients and outpatients between january 2006 and december 2010 , meeting eligibility criteria ( confirmed diagnosis of acute or acute - on - chronic dvt by doppler ultrasound scan and/or pe by chest computed tomography scan , pulmonary angiography or v / q scan ) were identified and collected from the general medical records and/or radiology departments at each of the three participating hospitals . hospital data were used to obtain the total number of patients who were annually registered at the hospital from 2006 to 2010 . data were processed and analyzed using sas ( version 9.1 , statistical analysis system ) . for the purpose of analysis , acute - on - chronic descriptive statistics were used to present patient characteristics , management strategies , and clinical outcomes of patients . annual incidence rates ( 95% ci ) of vte per 100,000 hospital registrations over a period of 5 years were reported for each site . fisher 's exact test was used to determine differences in the incidence of acute dvt ( pe ) over the years 20062010 . armitage trend test was used to examine the direction ( positive or negative ) of the trend . as primary analyses the remaining 41% ( 393/949 ) medical records were not included because they did not satisfy the inclusion criteria . data from seven patients were excluded as there was no radiologically confirmed diagnosis of pe . a total of 64% ( 352/549 ) patients had acute dvt without pe , 23% ( 124/549 ) had acute dvt with pe , and 13% ( 73/549 ) had pe . eighty - seven percent ( 476/549 ) of patients had acute dvt ( pe ) , and 36% ( 197/549 ) had pe ( acute dvt ) [ figure 1 ] . overall distribution of venous thromboembolism patients ( n = 549 ) a total of 21% ( 115/549 ) of patients visited the hospitals directly without being referred by a physician . venous thromboembolism patients referred from different medical specialties ( n=434 ) the demographic characteristics of the vte patients are mentioned in table 2 . demographic characteristics of venous thromboembolism patients ( n=549 ) a total of 182 patients had evidence of one risk factor , 126 had evidence of two risk factors , 70 had evidence of three risk factors and 31 had four or more risk factors recorded . patients undergoing orthopedic surgery constituted 22% ( 33/152 ) of all surgical patients [ table 3 ] . risk factors for venous thromboembolism based on a review of the available records , 157 patients had a single co - morbidity , 81 had two co - morbidities , 23 had three co - morbidities , and 16 had four or more co - morbidities . ( myocardial infarction , heart failure , chronic obstructive pulmonary disease , ventilator dependency , sepsis , or pneumonia ) [ table 4 ] . co - morbidities in venous thromboembolism patients of the 476 patients with dvt , 2% ( 9 ) had upper extremity dvt , 97% ( 462 ) had lower extremity dvt and the site of dvt was not known in 5 patients . a total of 31% ( 143/462 ) patients had dvt in the right limb , 54% ( 249/462 ) in the left limb and 9% ( 41/462 ) in both limbs ( site not known in 29 patients ) . of the 462 patients with lower extremity dvt , 61% had proximal dvt , 13% had distal dvt , and 7% had proximal and distal dvt . a total of 39% ( 215/549 ) patients were diagnosed with vte during their hospital stay , 54% ( 296/549 ) were admitted to hospital with a diagnosis of vte , and 7% ( 38/549 ) were diagnosed and continued to be managed in the outpatient department [ figure 2 ] . place of detection of venous thromboembolism ( n = 549 ) duration of hospitalization after diagnosis of venous thromboembolism a smaller proportion of patients ( 15% ; 81/549 ) was diagnosed with vte during the postoperative period . figure 3 shows the proportion of patients with vte at different time points during the postoperative period . of those diagnosed beyond 6 weeks , 21% ( 3/14 ) had orthopedic surgery ( hip fracture surgery ) . diagnosis of venous thromboembolism during the postoperative period ( n = 81 ) the most common ( 73% ) symptom was swelling of the limb among patients with vte [ table 6 ] . symptoms in venous thromboembolism patients in merely 4% of all the patients , dvt was also confirmed by venography . pe was confirmed by pulmonary angiography in 27% of all the patients [ table 7 ] . . heparin ( low molecular weight heparin [ lmwh]/unfractionated heparin [ ufh ] ) alone , a combination of heparin ( lmwh / ufh ) and oral anticoagulant ( warfarin ) , and fondaparinux sodium alone were recommended to 82% ( 420/515 ) , 13% ( 66/515 ) , and 2% ( 12/515 ) patients , respectively as initial anticoagulation . five percent ( 25/515 ) of patients were recommended lmwh alone , and 76% ( 393/515 ) were recommended either warfarin or acenocoumarol alone for long - term anticoagulation . the median duration of initial anticoagulation was 5 days while that of long - term anticoagulation was 180 days ( 6 months ) . anticoagulants were needed to be stopped because of bleeding in only 2% ( 9/515 ) patients . clinical outcomes in patients diagnosed with venous thromboembolism during hospital stay clinical outcomes in patients admitted to hospital with a diagnosis of venous thromboembolism the annual incidence of acute dvt ( pe ) increased from 2006 to 2010 at all the three sites [ figure 4 ] . however , a formal site - wise statistical analysis could not be performed to analyse trends in the incidence rates in acute dvt ( pe ) and pe alone as there were zero observations in some instances . incidence of acute deep vein thrombosis ( with or without pulmonary embolism ) over a 5 years period ( 20062010 ) at three sites demographic characteristics of venous thromboembolism patients ( n=549 ) a total of 182 patients had evidence of one risk factor , 126 had evidence of two risk factors , 70 had evidence of three risk factors and 31 had four or more risk factors recorded . patients undergoing orthopedic surgery constituted 22% ( 33/152 ) of all surgical patients [ table 3 ] . risk factors for venous thromboembolism based on a review of the available records , 157 patients had a single co - morbidity , 81 had two co - morbidities , 23 had three co - morbidities , and 16 had four or more co - morbidities . ( myocardial infarction , heart failure , chronic obstructive pulmonary disease , ventilator dependency , sepsis , or pneumonia ) [ table 4 ] . 2% ( 9 ) had upper extremity dvt , 97% ( 462 ) had lower extremity dvt and the site of dvt was not known in 5 patients . a total of 31% ( 143/462 ) patients had dvt in the right limb , 54% ( 249/462 ) in the left limb and 9% ( 41/462 ) in both limbs ( site not known in 29 patients ) . of the 462 patients with lower extremity dvt , 61% had proximal dvt , 13% had distal dvt , and 7% had proximal and distal dvt . a total of 39% ( 215/549 ) patients were diagnosed with vte during their hospital stay , 54% ( 296/549 ) were admitted to hospital with a diagnosis of vte , and 7% ( 38/549 ) were diagnosed and continued to be managed in the outpatient department [ figure 2 ] . place of detection of venous thromboembolism ( n = 549 ) duration of hospitalization after diagnosis of venous thromboembolism a smaller proportion of patients ( 15% ; 81/549 ) was diagnosed with vte during the postoperative period . figure 3 shows the proportion of patients with vte at different time points during the postoperative period . of those diagnosed beyond 6 weeks diagnosis of venous thromboembolism during the postoperative period ( n = 81 ) the most common ( 73% ) symptom was swelling of the limb among patients with vte [ table 6 ] . a total of 182 patients had evidence of one risk factor , 126 had evidence of two risk factors , 70 had evidence of three risk factors and 31 had four or more risk factors recorded . patients undergoing orthopedic surgery constituted 22% ( 33/152 ) of all surgical patients [ table 3 ] . based on a review of the available records , 157 patients had a single co - morbidity , 81 had two co - morbidities , 23 had three co - morbidities , and 16 had four or more co - morbidities . ( myocardial infarction , heart failure , chronic obstructive pulmonary disease , ventilator dependency , sepsis , or pneumonia ) [ table 4 ] . of the 476 patients with dvt , 2% ( 9 ) had upper extremity dvt , 97% ( 462 ) had lower extremity dvt and the site of dvt was not known in 5 patients . a total of 31% ( 143/462 ) patients had dvt in the right limb , 54% ( 249/462 ) in the left limb and 9% ( 41/462 ) in both limbs ( site not known in 29 patients ) . of the 462 patients with lower extremity dvt , 61% had proximal dvt , 13% had distal dvt , and 7% had proximal and distal dvt . a total of 39% ( 215/549 ) patients were diagnosed with vte during their hospital stay , 54% ( 296/549 ) were admitted to hospital with a diagnosis of vte , and 7% ( 38/549 ) were diagnosed and continued to be managed in the outpatient department [ figure 2 ] . place of detection of venous thromboembolism ( n = 549 ) duration of hospitalization after diagnosis of venous thromboembolism a smaller proportion of patients ( 15% ; 81/549 ) was diagnosed with vte during the postoperative period . figure 3 shows the proportion of patients with vte at different time points during the postoperative period . of those diagnosed beyond 6 weeks diagnosis of venous thromboembolism during the postoperative period ( n = 81 ) the most common ( 73% ) symptom was swelling of the limb among patients with vte [ table 6 ] pe was confirmed by pulmonary angiography in 27% of all the patients [ table 7 ] . heparin ( low molecular weight heparin [ lmwh]/unfractionated heparin [ ufh ] ) alone , a combination of heparin ( lmwh / ufh ) and oral anticoagulant ( warfarin ) , and fondaparinux sodium alone were recommended to 82% ( 420/515 ) , 13% ( 66/515 ) , and 2% ( 12/515 ) patients , respectively as initial anticoagulation . five percent ( 25/515 ) of patients were recommended lmwh alone , and 76% ( 393/515 ) were recommended either warfarin or acenocoumarol alone for long - term anticoagulation . the median duration of initial anticoagulation was 5 days while that of long - term anticoagulation was 180 days ( 6 months ) . anticoagulants were needed to be stopped because of bleeding in only 2% ( 9/515 ) patients . clinical outcomes in patients diagnosed with venous thromboembolism during hospital stay clinical outcomes in patients admitted to hospital with a diagnosis of venous thromboembolism the annual incidence of acute dvt ( pe ) increased from 2006 to 2010 at all the three sites [ figure 4 ] . however , a formal site - wise statistical analysis could not be performed to analyse trends in the incidence rates in acute dvt ( pe ) and pe alone as there were zero observations in some instances . incidence of acute deep vein thrombosis ( with or without pulmonary embolism ) over a 5 years period ( 20062010 ) at three sites pe was confirmed by pulmonary angiography in 27% of all the patients [ table 7 ] . heparin ( low molecular weight heparin [ lmwh]/unfractionated heparin [ ufh ] ) alone , a combination of heparin ( lmwh / ufh ) and oral anticoagulant ( warfarin ) , and fondaparinux sodium alone were recommended to 82% ( 420/515 ) , 13% ( 66/515 ) , and 2% ( 12/515 ) patients , respectively as initial anticoagulation . five percent ( 25/515 ) of patients were recommended lmwh alone , and 76% ( 393/515 ) were recommended either warfarin or acenocoumarol alone for long - term anticoagulation . the median duration of initial anticoagulation was 5 days while that of long - term anticoagulation was 180 days ( 6 months ) . anticoagulants were needed to be stopped because of bleeding in only 2% ( 9/515 ) patients . clinical outcomes in patients diagnosed with venous thromboembolism during hospital stay clinical outcomes in patients admitted to hospital with a diagnosis of venous thromboembolism the annual incidence of acute dvt ( pe ) increased from 2006 to 2010 at all the three sites [ figure 4 ] . however , a formal site - wise statistical analysis could not be performed to analyse trends in the incidence rates in acute dvt ( pe ) and pe alone as there were zero observations in some instances . incidence of acute deep vein thrombosis ( with or without pulmonary embolism ) over a 5 years period ( 20062010 ) at three sites to our knowledge , this is the first multicenter , retrospective registry in india involving patients with vte that reflect real - world clinical practice . in contrast with the western data in which vte is predominantly a disease of older age , 44% patients in our study were between 40 and 59 years of age while 34% were below 40 years , particularly those with pe . in a study from north india , men constituted 70% of our registry , more than those reported from vellore registry ( 48% ) , but similar to those reported in the endorse ( epidemiologic international day for the evaluation of patients at risk for vte in the acute hospital care setting ) study ( 69% ) . one of the reasons for this could be significantly high levels of homocysteine ( thrombophilia marker ) in males as compared to females as reported in an indian study . fewer indian women use oral contraceptives and postmenopausal hormone replacement therapy , which are known to be risk factors for thrombosis . this is supported by the fact that only 1% of women in this registry reported the use of oral contraceptives , and none reported use of hormonal replacement therapy . a total of 28% of the overall referrals were from cardiologists . the majority ( 82% ) of the referrals were from medical rather than surgical ( 15% ) specialties as against a referral rate of 93% from surgeons at vellore . our finding complements that from the endorse study in which 55% of the medical patients at risk of vte had cardiovascular disease . majority ( 53% ) of patients in our study had co - morbid cardiovascular disease including diabetes mellitus ; it is possible that these patients visited a cardiologist for their cardiovascular ailment ( s ) and were then referred by the cardiologist to vascular disease specialist ( investigator ) . most ( 89% ) of these patients had swelling of the ( lower ) limb . it is possible that these patients may not have felt the need to visit a specialist for a symptom like swelling of limb , instead visited their family physician . it is very encouraging to know that family physicians suspected dvt in these situations and referred the patient to a specialist . patients with a history of vte are about 8 times more likely to develop a new episode during a subsequent high - risk period compared with patients without a history of dvt or pe . prior history of dvt was the most ( 34% ) common risk factor in patients who had only dvt , whereas past history of pe , trauma , and immobilization for more than 3 days were the most common risk factors in patients who had only pe . our results ( major lower limb surgery as a risk factor in 3% patients ) appear to be consistent with those reported in the endorse study , which reported dvt in 4.4% patients undergoing major lower limb surgery . other studies from india have reported a dvt incidence rate ranging from 8% to 20% in major lower limb surgery . however , in our study , only 7% of patients had malignancy as a predisposing factor . among the malignancies , genitourinary cancer had the highest incidence ( 45% ) . hypertension ( 25% ) was the most common co - morbidity followed by diabetes mellitus ( 19% ) in this patient population . in addition , obesity ( 11% ) was a common risk factor in dvt complicated by pe . our findings support an asian ( korean ) study that demonstrated prevalence of the metabolic syndrome in 48% patients with vte . co - morbid neurological disease ( other than stroke ) and ventilator dependency were also commonly found in patients with dvt ( 10% ) and pe ( 11% ) respectively . both these conditions immobilize patients for prolonged periods of time , predisposing them to vte . venography and pulmonary angiography are the gold standard for diagnosis of dvt and pe respectively . in our study , venography was used in just 4% patients and pulmonary angiography in less than one - third of the patients . perhaps the relatively high cost of these tests and limited availability of such procedures may be the limiting factors . overall , most ( 93% ) patients were managed as inpatients ( 39% diagnosed with vte during hospital stay and 54% admitted to hospital with a diagnosis of vte ) . a mean duration of hospitalization of 79 days after diagnosis of vte is supported by published data . in selected low - risk patients , outpatient treatment of dvt and pe may be considered . this approach was observed in a small proportion ( 7% ) of patients who were managed on an outpatient basis , nearly all ( 97% ) of whom had only dvt . the reported prevalence of postsurgical vte in our study ( 15% ) was half of that ( 30% ) reported in vellore registry . this could be explained by higher referral rate from surgeons at vellore compared to that of our sites . most ( 40% ; 32/81 ) dvt cases were diagnosed between 2 and 6 postoperative weeks , but pe in most cases ( 70% ; 7/10 ) was diagnosed during the first postoperative week . we notice that acute dvt complicated by pe was less ( 6% ; 7/124 ) frequently diagnosed during the postoperative period as against 18% ( 64/352 ) and 14% ( 10/73 ) of acute dvt alone and pe alone , respectively . the use and duration of anticoagulants in our registry appears to be consistent with the american college of chest physicians treatment guidelines , which recommend at least 5 days of initial anticoagulation with parenteral anticoagulation ( lmwh , fondaparinux , intravenous ufh , or subcutaneous ufh ) and at least 3 months of long - term anticoagulation treatment with vitamin k antagonist . bleeding is the most serious complication of anticoagulation treatment and is a major concern for clinicians particularly as the patient 's age advances . in this registry , anticoagulant treatment was needed to be stopped because of bleeding in only 2% of the study population . the prospective reite registry has reported a rate of 3% for major / fatal bleeds . thus , the fear of bleeding complications , which decreases the use of anticoagulant treatment , appears to be minimal . dvt complicated by pe ( 60% ) and pe alone ( 75% ) were more frequently shifted to intensive care unit than those who had dvt alone ( 25% ) . similar to published data in which hospital readmission rate for vte was 5% for primary and 14% for secondary diagnosis , we report a hospital readmission rate of 6% ; however we do not know the cause for readmission . the death rate was 7% among those diagnosed with vte during hospital stay as against a rate of 1% among those who were hospitalized with a diagnosis of vte . over 90% of patients treated on an outpatient basis obtained symptomatic relief with treatment . in our study , the hospital discharge rate ( 97% ) was more than triple and death rate was a quarter of that reported by pandey et al . ( hospital discharge rate 31% and death rate 16% ) at a university hospital in delhi . our data show a significant increase in acute dvt ( pe ) from 2006 to 2010 . this can be explained by the increased awareness of vte in india as well as the advent of better diagnostic modalities , such as duplex ultrasonography becoming more readily available and accepted . although there was no significant change in the number of pe cases from 2006 to 2010 , the burden of pe is almost double ( 13% of all vte ) of 7% , rate reported at christian medical college , vellore during a 10-year period from 1996 to 2005 . our finding is consistent with a study from north india that reported a 16% incidence of pe in adult medical autopsies . this study has the expected limitations of any retrospective review including the availability of complete records for all patients , although a robust review of the data on medical charts was conducted . controlling for bias and confounders is difficult as there is no randomization and no blinding . follow - up data of patients after hospital discharge were not available . in cases of death , further , the clinic charts reviewed in this study included a mix of those from vascular surgery and hematology departments , limiting the generalizability of the study results . despite these limitations , this study provides large amount of useful information in a short span of time on patient characteristics , clinical outcomes , management strategies , and temporal trends in vte , based on real world data that reflect actual day - to - day clinical practice over a period of 5 years across three sites in india . we believe that this information will serve as a guide in the optimal implementation of vte prophylaxis and treatment , to improve patient outcomes and to decrease the occurrence of vte in india . real world data reflecting actual day - to - day clinical practice in vte over a period of 5 years across three sites in india showed that vte is not uncommon in indian patients and that acute dvt was responsible for the substantial burden of vte . we believe that this information will serve as a guide in the optimal implementation of vte prophylaxis and treatment , to improve patient outcomes and to decrease the occurrence of vte in india . liesel c. dsilva is and dr . sadhna j. joglekar was full - time employee of glaxosmithkline pharmaceuticals limited .

background and aim : there is lack of substantial indian data on venous thromboembolism ( vte ) . the aim of this study was to provide real - world information on patient characteristics , management strategies , clinical outcomes , and temporal trends in vte.subjects and methods : multicentre retrospective registry involving 549 medical records of patients with confirmed diagnosis of vte ( deep vein thrombosis [ dvt ] confirmed by doppler ultrasonography ; pulmonary embolism [ pe ] by computed tomography , pulmonary angiography and/or v / q scan ) from 2006 to 2010 at three indian tertiary care hospitals.results:acute dvt without pe , acute dvt with pe , and pe alone were reported in 64% ( 352/549 ) , 23% ( 124/549 ) , and 13% ( 73/549 ) patients , respectively . mean age was 47 ( 16 ) years , and 70% were males . h / o dvt ( 34% ) , surgery including orthopedic surgery ( 28% ) , trauma ( 16% ) , and immobilization > 3 days ( 14% ) were the most common risk factors for vte . hypertension ( 25% ) , diabetes ( 19% ) , and neurological disease ( other than stroke ) ( 8% ) were the most common co - morbidities . most ( 94% ) were treated with heparin alone ( 82% ) or fondaparinux ( 2% ) for initial anticoagulation ; low molecular weight heparin alone ( 5% ) or warfarin / acenocoumarol ( 76% ) for long - term anticoagulation . anticoagulant treatment was stopped because of bleeding in 2% ( 9/515 ) patients . mortality was 7% among patients diagnosed with vte during hospital stay versus 1% in those hospitalized with diagnosed vte . the annual incidence of dvt ( pe ) increased from 2006 to 2010.conclusion:acute dvt alone was responsible for the substantial burden of vte in indian patients . bleeding was not the limiting factor for anticoagulant treatment in most patients .

Introduction Subjects and Methods Results Demographics and characteristics of venous thromboembolism patients Risk factors for venous thromboembolism patients Co-morbidities in venous thromboembolism patients Clinical presentation of venous thromboembolism Management strategies for venous thromboembolism patients Diagnostic tools for venous thromboembolism Anticoagulant treatment in venous thromboembolism Annual incidence of acute deep venous thrombosis including the trend over a period of 5 years Discussion Conclusion None Financial support and sponsorship Conflicts of interest

given the growing burden of vte in india and lack of substantial indian data on characteristics of vte patients , use of diagnostics tools , prophylaxis , treatment options , and clinical outcomes in vte , there was a need to systematically collect such data . data on patient characteristics , clinical outcomes , predictors of mortality in acute dvt , management strategies and temporal trends in vte . consecutive medical records of inpatients and outpatients between january 2006 and december 2010 , meeting eligibility criteria ( confirmed diagnosis of acute or acute - on - chronic dvt by doppler ultrasound scan and/or pe by chest computed tomography scan , pulmonary angiography or v / q scan ) were identified and collected from the general medical records and/or radiology departments at each of the three participating hospitals . for the purpose of analysis , acute - on - chronic descriptive statistics were used to present patient characteristics , management strategies , and clinical outcomes of patients . a total of 64% ( 352/549 ) patients had acute dvt without pe , 23% ( 124/549 ) had acute dvt with pe , and 13% ( 73/549 ) had pe . heparin ( low molecular weight heparin [ lmwh]/unfractionated heparin [ ufh ] ) alone , a combination of heparin ( lmwh / ufh ) and oral anticoagulant ( warfarin ) , and fondaparinux sodium alone were recommended to 82% ( 420/515 ) , 13% ( 66/515 ) , and 2% ( 12/515 ) patients , respectively as initial anticoagulation . clinical outcomes in patients diagnosed with venous thromboembolism during hospital stay clinical outcomes in patients admitted to hospital with a diagnosis of venous thromboembolism the annual incidence of acute dvt ( pe ) increased from 2006 to 2010 at all the three sites [ figure 4 ] . of those diagnosed beyond 6 weeks diagnosis of venous thromboembolism during the postoperative period ( n = 81 ) the most common ( 73% ) symptom was swelling of the limb among patients with vte [ table 6 ] pe was confirmed by pulmonary angiography in 27% of all the patients [ table 7 ] . heparin ( low molecular weight heparin [ lmwh]/unfractionated heparin [ ufh ] ) alone , a combination of heparin ( lmwh / ufh ) and oral anticoagulant ( warfarin ) , and fondaparinux sodium alone were recommended to 82% ( 420/515 ) , 13% ( 66/515 ) , and 2% ( 12/515 ) patients , respectively as initial anticoagulation . clinical outcomes in patients diagnosed with venous thromboembolism during hospital stay clinical outcomes in patients admitted to hospital with a diagnosis of venous thromboembolism the annual incidence of acute dvt ( pe ) increased from 2006 to 2010 at all the three sites [ figure 4 ] . heparin ( low molecular weight heparin [ lmwh]/unfractionated heparin [ ufh ] ) alone , a combination of heparin ( lmwh / ufh ) and oral anticoagulant ( warfarin ) , and fondaparinux sodium alone were recommended to 82% ( 420/515 ) , 13% ( 66/515 ) , and 2% ( 12/515 ) patients , respectively as initial anticoagulation . clinical outcomes in patients diagnosed with venous thromboembolism during hospital stay clinical outcomes in patients admitted to hospital with a diagnosis of venous thromboembolism the annual incidence of acute dvt ( pe ) increased from 2006 to 2010 at all the three sites [ figure 4 ] . incidence of acute deep vein thrombosis ( with or without pulmonary embolism ) over a 5 years period ( 20062010 ) at three sites to our knowledge , this is the first multicenter , retrospective registry in india involving patients with vte that reflect real - world clinical practice . prior history of dvt was the most ( 34% ) common risk factor in patients who had only dvt , whereas past history of pe , trauma , and immobilization for more than 3 days were the most common risk factors in patients who had only pe . despite these limitations , this study provides large amount of useful information in a short span of time on patient characteristics , clinical outcomes , management strategies , and temporal trends in vte , based on real world data that reflect actual day - to - day clinical practice over a period of 5 years across three sites in india . real world data reflecting actual day - to - day clinical practice in vte over a period of 5 years across three sites in india showed that vte is not uncommon in indian patients and that acute dvt was responsible for the substantial burden of vte .

the need for magnetic resonance imaging ( mri ) in patients with an implanted pacemaker or implantable cardioverter - defibrillator ( icd ) is a growing clinical issue . it is estimated that as many as 75% of active cardiac device recipients will become indicated for mri . currently , the vast majority of such devices are contraindicated for use with an mri . in european heart rhythm association survey , published recently for non - mri - certified icds ( 0.51.5 t field strength ) , the totally subcutaneous icd ( s - icd ) system , an implantable defibrillator with no leads that touch the heart , has recently been demonstrated to be a safe and effective defibrillator option for patients at risk for sudden cardiac death . it provides shock therapy and post - shock pacing therapy , but no long - term bradycardia pacing . although it has been shown as an alternative to the standard transvenous icd , its compatibility with mri remains unclear . various types of clinical mri systems currently use a superconductive magnet that creates a static magnetic field strength , typically 1.5 or 3 t. the use of mri with most pacemakers and icds is considered a contraindication due to potential hazards , including heating of the electrode that resides in or on the heart , damage to myocardium , elevation of pacing thresholds , unintended induction of ventricular tachycardia ( vt ) or ventricular fibrillation ( vf ) , pacing inhibition , permanent device malfunction , and distortion of the mri scan . recently , mr - conditional. mr - conditional indicates a lack of known hazards in a specified mri environment with specified conditions of use . due to the variety of mri scanners and scanning protocols , it is not practical to test even a single device under all conditions . hence , mr - conditional labelling dictates that the device is safe for use under certain scanning conditions , as well as how the cardiac device should be programmed before an exposure to the magnetic field in a mri scanner . the literature , although limited , provides some guidance for imaging patients with implanted pacemakers or icds that do not have mr - conditional labelling . this single - centre prospective non - controlled study describes the first use of mri in patients with an implanted s - icd . patients with implanted s - icd systems ( boston scientific sqrx model 1010 and q - trak model 3010 ) were enrolled for mri testing over a period of 18 months . the s - icd system implanted in this patient cohort was composed of a can implanted in a left mid - lateral pocket and a para - sternal subcutaneous electrode . the s - icd is currently not certified for use with an mri ; therefore , the ethics committee of homolka hospital , prague , czech republic approved our clinical study . patients with newly implanted s - icd systems ( < 6 weeks ) were excluded , and none of the patients had any intravascular leads . the patients were randomized for either a cardiac , brain , cervical , or lumbar spinal scan . one of the subjects underwent an additional knee examination , due to reported chronic pain . a total of 15 patients were enrolled into this study ( 12 males and three females , aged 2283 years , mean 53 years . subjects in our cohort ( table 1 ) underwent a total of 22 mri scans between 6 june 2012 and 24 december 2013 . in total , five brain scans , three cardiac scans , 12 lumbar scans , one knee , and one cervical spine scan were conducted ( table 2 ) . however , in one patient a minor disc protrusion was found , in other mri revealed stenosis of intervertebral foramen which was causing radicular pain of the nerve root l4 and based on this examination the patient was referred to ct - navigated periradicular therapy . table 1summary of patient anatomical data and scan locations , along with noted clinical eventsidagesexbmidgef , % indication for s - icdheating0164f20.5hcmp / vfs85secondary preventionnone0283m30.0post - mi / smvts post - catheter ablation/35secondary prevention ( post - transvenous icd extraction)none0331m25.3arvc / d / smvts68secondary preventionin - tolerable re - scanned0458m23.6post - mi / post - cabg30primary preventionnone0577m25.5post - mi30primary preventionnone0663m27.0post - mi30primary preventionnone0768m23.7post - mi / vfs / vts60secondary prevention post - transvenous icd extraction / svc occlusiontolerable0822m29.4brugada sy / vfs68secondary preventionin - tolerable re - scanned0959m27.1dcmp / vfs / post - mitral valve surgery/60secondary prev./post - transvenous icd extractionnone1041f24.6arvc / d70primary preventionnone1123f21.5lqts / vf60secondary preventionnone1266m36.9post - mi / vf / post - cabg50secondary prevention / post - repeat transvenous icd extractiontolerable1348m22.9dcmp(non - compaction)/vfs35secondary preventionnone1470m29systolic dysfunction of lv35primary preventionnone1526m33brugada sy65primary preventionnonehcmp , hypertrophic cardiomyopathy ; smvt , sustained monomorphic ventricular tachycardia ; mi , myocardial infarction ; arvc , arrhythmogenic right ventricular cardiomyopathy ; cabg , coronary artery by - pass graft ; lqts , long qt syndrom . table 2parmeters of s - icd and patient sensation during individual mri scansscan # idbody partheating sensationsshock zone ( b.p.m.)condit . shock zone ( b.p.m.)bat % episode num.101brainnone2302101001202brainnone240220861303l spinein - tolerable240220831403brainnone240220831504brainnone220190691605l spinenone220210541706l spinenone240220681807l spinetolerable240220582908l spinein - tolerablenananana1008brainnonenananana1108l spinenone2302108411209heartnone2402208911310l spinenone2301807911410heartnonenananana1511heartnone2301909711612l spinetolerable2001709721712l spinenone2001709421813c spinenone23019010041913l spinenone23019010042014l spinenone2301908612115kneenone25021010012215l spinenone2502101001s - icd parameters acquired prior- and post - mri were without any change , therefore only one value is presented.indices : na , not available ; l spine , lumbar spine ; c spine , cervical spine . summary of patient anatomical data and scan locations , along with noted clinical events hcmp , hypertrophic cardiomyopathy ; smvt , sustained monomorphic ventricular tachycardia ; mi , myocardial infarction ; arvc , arrhythmogenic right ventricular cardiomyopathy ; cabg , coronary artery by - pass graft ; lqts , long qt syndrom . parmeters of s - icd and patient sensation during individual mri scans s - icd parameters acquired prior- and post - mri were without any change , therefore only one value is presented . indices : na , not available ; l spine , lumbar spine ; c spine , cervical spine . studies were performed using a siemens avanto 1.5 t mri scanner ( vb17 software , quantum gradient coils ) . all scans were run in normal operating mode , which is limited to 2 w / kg whole body averaged specific absorption rate ( sar ) . clinically relevant mri sequences were used for evaluation ( see table 3 ) . table 3types of pulse sequences typically used for imaging of respective anatomical areasscan locationscan sequencesflairdwiflashfsehastesestirtruefispbrainxxxxheartxxxxcervical spinexxkneexxxxlumbar spinexxflair , fluid attenuated inversion recovery ; dwi , diffusion weighted imaging ; flash , fast low angle shot ; fse , fast spin echo ; haste , half acquisition single - shot turbo spin echo ; se , spin echo ; stir , short tau inversion recovery ; truefisp , true fast imaging with steady - state precession.fse sequence caused heating in subjects with a thermistor probe during lumbar spine examination ( see the text for details ) . types of pulse sequences typically used for imaging of respective anatomical areas flair , fluid attenuated inversion recovery ; dwi , diffusion weighted imaging ; flash , fast low angle shot ; fse , fast spin echo ; haste , half acquisition single - shot turbo spin echo ; se , spin echo ; stir , short tau inversion recovery ; truefisp , true fast imaging with steady - state precession . fse sequence caused heating in subjects with a thermistor probe during lumbar spine examination ( see the text for details ) . patients were asked to report immediately any pain , torqueing movement , or heating sensation in the area of the pocket or the electrode by pressing an emergency bulb . furthermore , all patients were questioned immediately following the mri procedure to ascertain any discomfort in the vicinity of the can or electrode . pulse oximetry and standard lead electrocardiogram ( ecg ) if discomfort occurred , the patient was asked if the scan could be repeated at a later time using a revised scan sequence or the subject was again randomized for another anatomical area . since none of the components of the s - icd system are on or in the heart , heating near or around however , heating near the electrode or can with the s - icd system may still cause serious patient discomfort . therefore , along with education of subjects , each patient was instrumented by taping an oesophageal temperature probe ( beta - therm model g22k7mcd8 ) on the skin over the mid - lateral implant site to record any temperature excursions that might be correlated to patient symptoms of heating / discomfort near the pocket . to minimize the risk of inappropriate therapy , the s - icd system was programmed to therapy each s - icd system was evaluated prior to and immediately after the scan to verify proper functioning , including interrogation , sensing , and battery voltage . after the completion of the mri , long - term regular clinical follow - up and checking of the device were performed . patients with implanted s - icd systems ( boston scientific sqrx model 1010 and q - trak model 3010 ) were enrolled for mri testing over a period of 18 months . the s - icd system implanted in this patient cohort was composed of a can implanted in a left mid - lateral pocket and a para - sternal subcutaneous electrode . the s - icd is currently not certified for use with an mri ; therefore , the ethics committee of homolka hospital , prague , czech republic approved our clinical study . patients with newly implanted s - icd systems ( < 6 weeks ) were excluded , and none of the patients had any intravascular leads . the patients were randomized for either a cardiac , brain , cervical , or lumbar spinal scan . one of the subjects underwent an additional knee examination , due to reported chronic pain . a total of 15 patients were enrolled into this study ( 12 males and three females , aged 2283 years , mean 53 years . subjects in our cohort ( table 1 ) underwent a total of 22 mri scans between 6 june 2012 and 24 december 2013 . in total , five brain scans , three cardiac scans , 12 lumbar scans , one knee , and one cervical spine scan were conducted ( table 2 ) . however , in one patient a minor disc protrusion was found , in other mri revealed stenosis of intervertebral foramen which was causing radicular pain of the nerve root l4 and based on this examination the patient was referred to ct - navigated periradicular therapy . table 1summary of patient anatomical data and scan locations , along with noted clinical eventsidagesexbmidgef , % indication for s - icdheating0164f20.5hcmp / vfs85secondary preventionnone0283m30.0post - mi / smvts post - catheter ablation/35secondary prevention ( post - transvenous icd extraction)none0331m25.3arvc / d / smvts68secondary preventionin - tolerable re - scanned0458m23.6post - mi / post - cabg30primary preventionnone0577m25.5post - mi30primary preventionnone0663m27.0post - mi30primary preventionnone0768m23.7post - mi / vfs / vts60secondary prevention post - transvenous icd extraction / svc occlusiontolerable0822m29.4brugada sy / vfs68secondary preventionin - tolerable re - scanned0959m27.1dcmp / vfs / post - mitral valve surgery/60secondary prev./post - transvenous icd extractionnone1041f24.6arvc / d70primary preventionnone1123f21.5lqts / vf60secondary preventionnone1266m36.9post - mi / vf / post - cabg50secondary prevention / post - repeat transvenous icd extractiontolerable1348m22.9dcmp(non - compaction)/vfs35secondary preventionnone1470m29systolic dysfunction of lv35primary preventionnone1526m33brugada sy65primary preventionnonehcmp , hypertrophic cardiomyopathy ; smvt , sustained monomorphic ventricular tachycardia ; mi , myocardial infarction ; arvc , arrhythmogenic right ventricular cardiomyopathy ; cabg , coronary artery by - pass graft ; lqts , long qt syndrom . table 2parmeters of s - icd and patient sensation during individual mri scansscan # idbody partheating sensationsshock zone ( b.p.m.)condit . shock zone ( b.p.m.)bat % episode num.101brainnone2302101001202brainnone240220861303l spinein - tolerable240220831403brainnone240220831504brainnone220190691605l spinenone220210541706l spinenone240220681807l spinetolerable240220582908l spinein - tolerablenananana1008brainnonenananana1108l spinenone2302108411209heartnone2402208911310l spinenone2301807911410heartnonenananana1511heartnone2301909711612l spinetolerable2001709721712l spinenone2001709421813c spinenone23019010041913l spinenone23019010042014l spinenone2301908612115kneenone25021010012215l spinenone2502101001s - icd parameters acquired prior- and post - mri were without any change , therefore only one value is presented.indices : na , not available ; l spine , lumbar spine ; c spine , cervical spine . summary of patient anatomical data and scan locations , along with noted clinical events hcmp , hypertrophic cardiomyopathy ; smvt , sustained monomorphic ventricular tachycardia ; mi , myocardial infarction ; arvc , arrhythmogenic right ventricular cardiomyopathy ; cabg , coronary artery by - pass graft ; lqts , long qt syndrom . parmeters of s - icd and patient sensation during individual mri scans s - icd parameters acquired prior- and post - mri were without any change , therefore only one value is presented . indices : na , not available ; l spine , lumbar spine ; c spine , cervical spine . studies were performed using a siemens avanto 1.5 t mri scanner ( vb17 software , quantum gradient coils ) . all scans were run in normal operating mode , which is limited to 2 w / kg whole body averaged specific absorption rate ( sar ) . clinically relevant mri sequences were used for evaluation ( see table 3 ) . table 3types of pulse sequences typically used for imaging of respective anatomical areasscan locationscan sequencesflairdwiflashfsehastesestirtruefispbrainxxxxheartxxxxcervical spinexxkneexxxxlumbar spinexxflair , fluid attenuated inversion recovery ; dwi , diffusion weighted imaging ; flash , fast low angle shot ; fse , fast spin echo ; haste , half acquisition single - shot turbo spin echo ; se , spin echo ; stir , short tau inversion recovery ; truefisp , true fast imaging with steady - state precession.fse sequence caused heating in subjects with a thermistor probe during lumbar spine examination ( see the text for details ) . types of pulse sequences typically used for imaging of respective anatomical areas flair , fluid attenuated inversion recovery ; dwi , diffusion weighted imaging ; flash , fast low angle shot ; fse , fast spin echo ; haste , half acquisition single - shot turbo spin echo ; se , spin echo ; stir , short tau inversion recovery ; truefisp , true fast imaging with steady - state precession . fse sequence caused heating in subjects with a thermistor probe during lumbar spine examination ( see the text for details ) . patients were asked to report immediately any pain , torqueing movement , or heating sensation in the area of the pocket or the electrode by pressing an emergency bulb . furthermore , all patients were questioned immediately following the mri procedure to ascertain any discomfort in the vicinity of the can or electrode . pulse oximetry and standard lead electrocardiogram ( ecg ) if discomfort occurred , the patient was asked if the scan could be repeated at a later time using a revised scan sequence or the subject was again randomized for another anatomical area . since none of the components of the s - icd system are on or in the heart , heating near or around the electrode can not harm the myocardium . however , heating near the electrode or can with the s - icd system may still cause serious patient discomfort . therefore , along with education of subjects , each patient was instrumented by taping an oesophageal temperature probe ( beta - therm model g22k7mcd8 ) on the skin over the mid - lateral implant site to record any temperature excursions that might be correlated to patient symptoms of heating / discomfort near the pocket . to minimize the risk of inappropriate therapy , the s - icd system was programmed to therapy each s - icd system was evaluated prior to and immediately after the scan to verify proper functioning , including interrogation , sensing , and battery voltage . after the completion of the mri , the s - icd system was reprogrammed to original settings . long - term regular clinical follow - up and checking of the device were performed . no anomalies were noted via pulse oximetry or ecg during the scans for any of the patients . eleven of 15 patients reported no sensation or pain from heating of the can , two of 15 patients reported feeling some heating , and two patients reported intolerable heating ( see table 2 ) . in patients with intolerable heating , the scan was halted within seconds and changed to a scan of the brain , which proceeded without incident . patient reports of heating in the vicinity of the can occurred only during lumbar scans with a thermistor probe ; no such reports occurred during scans of the brain , cardiac area , cervical spine , or without the probe . in two cases where heating in the vicinity of the can was reported by the patient , the scan sequence was altered to reduce the intensity of radiofrequency ( rf ) field exposure by reducing the turbo factor ( e.g. from 21 to 7 ) , increasing the repetition time ( e.g. to > 4000 ms ) , and reducing the flip angle ( e.g. from 170 to 120 ) . the target values were chosen arbitrarily to maintain image contrast ( flip angle ) and keep scan time at reasonable limits ( turbo factor and repetition time ) . less heating was noted by patients after these modifications to the scan parameters were made . 03 ) was observed to have a skin lesion , appearing to be a circular rash or ulcer on the surface of the skin over the can , approximately 35 mm in diameter . the cause of this skin anomaly is not known ; it was later noted to have fully healed at a follow - up 10 days after the scan . to ascertain the effect of heating due to the instrumented thermistor catheter , the two patients who experienced the heating ( examinations 9 and 16 , see table 2 ) were rescanned several weeks later without the thermistor catheter in place ( examinations 11 and 17 ) . first , modified sequence ( with even lower amount of energy deposited in the tissue ) was used , which caused no heating . as no sensation was reported by the subjects , they were asked to report even a minimal discomfort , and the lumbar scans were performed using the same settings that resulted in heating with the thermistor catheter in place in the first imaging session . the results of the rescans revealed that no heating was felt by the patients when the thermistor catheter was absent . there were no noted changes to battery voltage , ability to detect the qrs signal or stored diagnostic data . pacing thresholds can not be assessed by the s - icd system , so this was not evaluated . none of the patients reported any pulling or twisting of the can or pain from heating of the s - icd electrode . for scans of the brain , lumbar spine , knee , and cervical spine , no effect from image artefact was noted in the anatomical area of interest . however , for scans of the cardiac area , image artefact was noted to interfere with the ability to see parts of the left ventricle , though the right ventricle of the heart was unaffected and could be imaged usefully . this was due to the can and not the electrode ( see figure 1 ) , modifications to the protocol for the lumbar spine resulted in a lower signal - to - noise ratio ; however , the images remain in diagnostic quality ( see figure 2 ) . figure 1kinetic study in four - chamber view : the systolic ( a and c ) and diastolic ( b and d ) images of cine sequences , four - chamber view . the steady - state free precession ( ssfp ) sequence ( a and b ) shows more artefacts . in ssfp kinetic study , an inflow of dark blood from the left pulmonary veins was seen . it could be caused by s - icd but also by metallic ring in mitral annulus . the spoiled gradient echo ( gre ) sequence ( c and d ) is better , but an artefact at the lateral wall is obvious . figure 2lumbar spine imaging with icd : low sar t2 fse sequence ( upper image ) compared with normal t2 fse in the same subject ( lower image , for the scanning parameters see the discussion section ) . kinetic study in four - chamber view : the systolic ( a and c ) and diastolic ( b and d ) images of cine sequences , four - chamber view . the steady - state free precession ( ssfp ) sequence ( a and b ) shows more artefacts . in ssfp kinetic study , an inflow of dark blood from the left pulmonary veins was seen . it could be caused by s - icd but also by metallic ring in mitral annulus . the spoiled gradient echo ( gre ) sequence ( c and d ) is better , but an artefact at the lateral wall is obvious . lumbar spine imaging with icd : low sar t2 fse sequence ( upper image ) compared with normal t2 fse in the same subject ( lower image , for the scanning parameters see the discussion section ) . there were no noted changes to battery voltage , ability to detect the qrs signal or stored diagnostic data . pacing thresholds can not be assessed by the s - icd system , so this was not evaluated . none of the patients reported any pulling or twisting of the can or pain from heating of the s - icd electrode . for scans of the brain , lumbar spine , knee , and cervical spine , no effect from image artefact was noted in the anatomical area of interest . however , for scans of the cardiac area , image artefact was noted to interfere with the ability to see parts of the left ventricle , though the right ventricle of the heart was unaffected and could be imaged usefully . this was due to the can and not the electrode ( see figure 1 ) , modifications to the protocol for the lumbar spine resulted in a lower signal - to - noise ratio ; however , the images remain in diagnostic quality ( see figure 2 ) . figure 1kinetic study in four - chamber view : the systolic ( a and c ) and diastolic ( b and d ) images of cine sequences , four - chamber view . the steady - state free precession ( ssfp ) sequence ( a and b ) shows more artefacts . in ssfp kinetic study , an inflow of dark blood from the left pulmonary veins was seen . it could be caused by s - icd but also by metallic ring in mitral annulus . the spoiled gradient echo ( gre ) sequence ( c and d ) is better , but an artefact at the lateral wall is obvious . figure 2lumbar spine imaging with icd : low sar t2 fse sequence ( upper image ) compared with normal t2 fse in the same subject ( lower image , for the scanning parameters see the discussion section ) . kinetic study in four - chamber view : the systolic ( a and c ) and diastolic ( b and d ) images of cine sequences , four - chamber view . the steady - state free precession ( ssfp ) sequence ( a and b ) shows more artefacts . in ssfp kinetic study , an inflow of dark blood from the left pulmonary veins was seen . it could be caused by s - icd but also by metallic ring in mitral annulus . the spoiled gradient echo ( gre ) sequence ( c and d ) is better , but an artefact at the lateral wall is obvious . lumbar spine imaging with icd : low sar t2 fse sequence ( upper image ) compared with normal t2 fse in the same subject ( lower image , there are several reports in the current literature about mr - conditional pacemakers from several companies , but very limited reports about mr - conditional icds . biotronik announced in late 2011 release of their first mr - compatible icd device and defibrillator leads pro mri , but in the conditions of use excluded scanning of the torso and focused more on the extremities examination . in european heart rhythm association survey , 60% of centres did not implant any mri - certified icds , 34.3% implanted < 10 icd devices , and only 5.6% implanted 10 and more icds ; one - fifth of responders stated that mri - certified icds should be implanted in all patients but lack of reimbursement was indicated as a possible obstacle to implant more mri - certified pacemakers / icds by 47.1% of responding centres . none of the components of the s - icd system are on or in the heart . the s - icd depends less upon being in direct contact with the myocardium to function and instead uses far - field sensing and stimulation to provide the shock and post - shock pacing therapy . as a consequence , unlike transvenous systems heating near or around the electrode can not harm the myocardium , which could present with possible safety consequences such as an elevation in pacing thresholds or scarring of the myocardium , but it may still cause serious patient discomfort . because the s - icd is larger than modern transvenous icd 's , there may be more potential for the can to experience heating due to the magnetic gradient or rf field . we report results from what we believe is the first experience of mri scanning in patients with an implanted s - icd and in various anatomical areas . overall , mri was performed safely in all patients , which is in contrast to the current literature with mri imaging in patients with electrical - active devices which are not mri - conditional . in our study , the primary clinically significant event attributable to the mri scan was the occurrence of heating in the area of the pocket in the four patients that underwent lumbar scans . it was not known if this was due to the s - icd can itself or an artefact of the thermistor catheter used to measure skin temperature over the pocket . this required a revision of our protocol , which was to re - scan two of the patients who complained of heating . re - scanning of these patients without the thermistor probe resulted in no complaints of heating , so it is assumed that the thermistor catheter itself heated during the lumbar scans and caused the discomfort . as further evidence , all the heating complaints occurred during rf - intensive scan sequences ( namely fast spin echo ) with the temperature probe located axially near the centre of the bore , where rf fields are the highest . the thermistor catheter is constructed of insulated conductive cables connected to electrodes and should couple to the rf fields efficiently , causing heating at the electrodes and pain or damage on the surface of the skin where the probe was placed over the s - icd can . if the heating was due to the s - icd can itself , it would more likely occur during gradient - intensive scan sequences ( which can generate eddy currents on can surfaces and internal components ) and at locations in the bore where there are high gradient fields , such as near the bore edges . however , when the patient was scanned with gradient - intensive scan sequences ( e.g. flair dwi ) and with the s - icd system in high gradient field locations in the bore ( e.g. such as during a brain scan ) , patients did not detect any heating or discomfort . in addition , the subcutaneous lead , which was not instrumented with a thermistor catheter , never resulted in any heating sensation noted by the patient , even when exactly the same sequence that resulted in heating in the first session was used . the use of mri - compatible temperature monitors such as fibre optic temperature probes would have provided better confirmation of possible skin temperature elevation and would not have been affected by the rf fields . for cardiac imaging , the main problem to solve is metallic artefact , especially on the gradient - echo sequences . like in research performed by nazarian et al . , several scan protocols were used to see if any yielded different effects or reduced the qualitative extent of artefact . gradient mode was changed from normal to whisper , resulting in slower ramping of the field and therefore diminishing the changes of the magnetic field in time . artefacts when present were limited to blurring of the left ventricle during cardiac scans and most yielded clinically useful information . standard interrogation of the s - icd revealed no adverse effects upon the functioning of the system . while no adverse effects upon the post - scan s - icd device function were noted , not all possible scanning protocols were tested . it should be noted that , four of the s - icd 's were exposed to repeat mri scans without adverse effects to device function . in addition , because the s - icd does not provide long - term bradycardia pacing , it is assumed that pacemaker - dependent patients would not be implanted with this system . the inhibition of the pacemaker function during the scanning sequence and possible pacing threshold changes are a unique concern in patients implanted with transvenous icds . this study included only 15 patients and 22 scans done on the same 1.5 t mri scanner . thus , even these preliminary results should only be applied to 1.5 t mri scanners ( similarly as reported in the present literature for other implantable devices ) . device functionality was tested immediately after the scan but not for long - term effects . in addition , not all device functions were tested although the s - icd system does have a beeper / interrogation warning if battery levels or memory irregularities occur . however , patients were scheduled for regular check - up and no defect of the device was observed in following 725 months ( mean observation time 18 months ) . delayed enhancement mri for determining cardiac scarring was also not tested . also , there are other anatomical areas that were not evaluated , such as shoulder and knees . this study included only 15 patients and 22 scans done on the same 1.5 t mri scanner . thus , even these preliminary results should only be applied to 1.5 t mri scanners ( similarly as reported in the present literature for other implantable devices ) . device functionality was tested immediately after the scan but not for long - term effects . in addition , not all device functions were tested although the s - icd system does have a beeper / interrogation warning if battery levels or memory irregularities occur . however , patients were scheduled for regular check - up and no defect of the device was observed in following 725 months ( mean observation time 18 months ) . delayed enhancement mri for determining cardiac scarring was also not tested . also , there are other anatomical areas that were not evaluated , such as shoulder and knees . while more data are required to support a claim of mri - conditional , this study is the study to demonstrate the feasibility of exposing s - icd patients to mri using the scanning and monitor protocol described , with some precautionary measures including : ( i ) programming the device therapy off ; ( ii ) limiting the sar to 2.0 w / kg ; ( iii ) continuous monitoring of the patients pulse oximetry and ecg by qualified personnel and especially for any feelings of heating ; ( iv ) evaluate device function post scan ; ( v ) availability of full resuscitation facilities at the mri site . given the variables of different mri scanners , the decision to perform mri on patients with an implanted s - icd system should be balanced against the potential risks . in our study , the only heating was very likely introduced by not fully mri - compatible thermometer probe ; subjects rescanned without the probe did not report any abnormalities during the scan of any body area listed ( brain , cervical and lumbar spine , heart , and knee ) . this study was supported by iga mz r nt12094/2011 , research project charles university in prague , prvouk p34 and unce 204010/2012 . funding to pay the open access publication charges for this article was provided by iga mz r nt12094/2011 .

aimsour aim was to evaluate the potential for safely imaging patients with a new type of implantable cardioverter - defibrillator called the subcutaneous implantable cardioverter - defibrillator ( s - icd ) in a 1.5 t magnetic resonance imaging ( mri ) scanner . with the increasing number of patients with cardiac implantable devices who are indicated for mri , there is a growing need for establishing mri compatibility of cardiac implantable devices.methods and resultspatients with implanted s - icd systems underwent one or more types of anatomical mri scans . the s - icd was programmed off and patients were monitored throughout the imaging procedure . device function was evaluated pre- and post - scan . patients were asked to report immediately any pain , torqueing movement , or heating sensation in the area of the pocket or electrode . fifteen patients underwent a total of 22 examinations at 1.5 t. scans included brain , spine , knee , and heart . two patients were re - scanned due to complaints of heating over the can during lumbar scans , which was caused by a thermistor probe placed on the skin to measure skin temperature . all the remaining scans occurred without incident . no evidence of device malfunction was observed.conclusionthis study is the first to domonstrate the feasibility of exposing s - icd patients to mri using the scanning and monitoring protocol described . more data are required to support s - icd as a mri conditional device .

Introduction Methods Patient selection Magnetic resonance imaging Device assessment Results Subcutaneous implantable cardioverter-defibrillator status Image artefacts Discussion Limitations Conclusions Funding

the need for magnetic resonance imaging ( mri ) in patients with an implanted pacemaker or implantable cardioverter - defibrillator ( icd ) is a growing clinical issue . various types of clinical mri systems currently use a superconductive magnet that creates a static magnetic field strength , typically 1.5 or 3 t. the use of mri with most pacemakers and icds is considered a contraindication due to potential hazards , including heating of the electrode that resides in or on the heart , damage to myocardium , elevation of pacing thresholds , unintended induction of ventricular tachycardia ( vt ) or ventricular fibrillation ( vf ) , pacing inhibition , permanent device malfunction , and distortion of the mri scan . patients with implanted s - icd systems ( boston scientific sqrx model 1010 and q - trak model 3010 ) were enrolled for mri testing over a period of 18 months . patients with newly implanted s - icd systems ( < 6 weeks ) were excluded , and none of the patients had any intravascular leads . patients were asked to report immediately any pain , torqueing movement , or heating sensation in the area of the pocket or the electrode by pressing an emergency bulb . since none of the components of the s - icd system are on or in the heart , heating near or around however , heating near the electrode or can with the s - icd system may still cause serious patient discomfort . to minimize the risk of inappropriate therapy , the s - icd system was programmed to therapy each s - icd system was evaluated prior to and immediately after the scan to verify proper functioning , including interrogation , sensing , and battery voltage . patients with implanted s - icd systems ( boston scientific sqrx model 1010 and q - trak model 3010 ) were enrolled for mri testing over a period of 18 months . patients with newly implanted s - icd systems ( < 6 weeks ) were excluded , and none of the patients had any intravascular leads . patients were asked to report immediately any pain , torqueing movement , or heating sensation in the area of the pocket or the electrode by pressing an emergency bulb . to minimize the risk of inappropriate therapy , the s - icd system was programmed to therapy each s - icd system was evaluated prior to and immediately after the scan to verify proper functioning , including interrogation , sensing , and battery voltage . patient reports of heating in the vicinity of the can occurred only during lumbar scans with a thermistor probe ; no such reports occurred during scans of the brain , cardiac area , cervical spine , or without the probe . as no sensation was reported by the subjects , they were asked to report even a minimal discomfort , and the lumbar scans were performed using the same settings that resulted in heating with the thermistor catheter in place in the first imaging session . for scans of the brain , lumbar spine , knee , and cervical spine , no effect from image artefact was noted in the anatomical area of interest . for scans of the brain , lumbar spine , knee , and cervical spine , no effect from image artefact was noted in the anatomical area of interest . because the s - icd is larger than modern transvenous icd 's , there may be more potential for the can to experience heating due to the magnetic gradient or rf field . in our study , the primary clinically significant event attributable to the mri scan was the occurrence of heating in the area of the pocket in the four patients that underwent lumbar scans . it was not known if this was due to the s - icd can itself or an artefact of the thermistor catheter used to measure skin temperature over the pocket . this required a revision of our protocol , which was to re - scan two of the patients who complained of heating . the thermistor catheter is constructed of insulated conductive cables connected to electrodes and should couple to the rf fields efficiently , causing heating at the electrodes and pain or damage on the surface of the skin where the probe was placed over the s - icd can . in addition , the subcutaneous lead , which was not instrumented with a thermistor catheter , never resulted in any heating sensation noted by the patient , even when exactly the same sequence that resulted in heating in the first session was used . it should be noted that , four of the s - icd 's were exposed to repeat mri scans without adverse effects to device function . while more data are required to support a claim of mri - conditional , this study is the study to demonstrate the feasibility of exposing s - icd patients to mri using the scanning and monitor protocol described , with some precautionary measures including : ( i ) programming the device therapy off ; ( ii ) limiting the sar to 2.0 w / kg ; ( iii ) continuous monitoring of the patients pulse oximetry and ecg by qualified personnel and especially for any feelings of heating ; ( iv ) evaluate device function post scan ; ( v ) availability of full resuscitation facilities at the mri site .

most adults with autoimmune diabetes non - insulin - requiring at diagnosis become so within 36 years ( 1 , 2 ) . the optimal treatment for this second largest group of patients with diabetes is still unknown ( 37 ) . adult patients with autoimmune diabetes usually have larger remaining -cell mass at diagnosis and many develop -cell destruction more slowly . latent autoimmune diabetes in adults ( lada ) is therefore a suitable group for evaluating new therapies in autoimmune diabetes and may also serve as a model for intervention in classical type 1 diabetes ( 3 , 4 , 69 ) . the incidence of autoimmune diabetes is about equal in almost all age groups ( 10 , 11 ) . abrupt onset , often with ketoacidosis , is most frequent during childhood , a more modest onset is more frequent in adolescents and younger adults , and among adults and elders a slowly progressive onset , termed lada , is frequent ( 3 , 4 , 11 , 12 ) . classical type 1 diabetes and lada patients often have normal c - peptide levels at diagnosis , but further progressive decline occurs after onset , and insulin dependency occurs almost inevitably ( 3 , 4 , 8 , 9 , 13 ) . most trials in early type 1 diabetes have been performed in children , whose remaining -cell mass is limited , and short - term evaluation of intervention may be difficult also due to not infrequently occurring remission periods ( 14 , 15 ) . no therapy has yet been demonstrated to promote long - term insulin independency ( 3 , 5 , 7 , 16 ) . rodent studies have demonstrated potential positive effects of insulin treatment ( 17 , 18 ) . a pilot study of small doses of insulin versus sulphonylurea ( su ) to ten ica - positive patients with slowly progressive -cell failure favoured insulin for the preservation of c - peptide ( 19 ) . c - peptide is the outcome measure of choice of -cell function in trials of autoimmune diabetes ( 20 ) . even modest preservation of -cell function has been demonstrated to have positive effects on the frequency of hypoglycaemic events , and on the prevalence of retinopathy ( 21 ) . to investigate the effect of early insulin treatment in lada patients , for 3 years , on residual -cell function and metabolic control , compared with a group initially treated with diet and/or oral hypoglycaemic agents ( oha ) . adults , aged 30 years , diagnosed with diabetes in lund and kronoberg counties in southern sweden , non - insulin - requiring at diagnosis and positive to at least one of gadabs and/or icas were eligible for participation . two thirds had to be excluded due to mental conditions or severe physical illness , but also unwillingness to risk the early start of insulin injections . the majority of the patients were randomised into two groups , in blocks of eight , by pre - prepared closed envelopes kept at the two hospital policlinics . however complete strict randomisation was not possible , as some patients refused randomisation to possible insulin treatment before it was unavoidable . there were 20 patients in the intervention group ( i ) , treated with insulin from baseline , starting with 26 units intermediate - acting insulin at night ; and 17 patients in the control group ( c ) who received regular treatment with dietoha , mostly metformin , and some su ( 5/17 ; 30% ) . for both the groups , goals for glucose levels were in accordance with general guidelines ( fasting plasma glucose ( fpg ) 4.57 , preprandial pg 57 and postprandial decisions to increase treatment in doses , number of doses and addition of ohas or insulin were at the discretion of the treating physician . if two doses / day of intermediate - acting insulin or mix - insulin were not satisfactory , doses of direct or rapid - acting insulin before meals were added , resulting in 14 doses / day . glucagon - stimulation tests were performed at baseline and after 12 , 24 and 36 months during annual policlinic visits at the two research clinics . after an overnight fast , c - peptide was determined before and 6 min after i.v . c - peptide was analysed by commercial ria ( md315 , euro - diagnostica ab , malm , sweden ) , total variation ( sum of intra - and inter - assay variations ) 7% , reference range 0.251.0 c - peptide level , to reflect a preserved normal -cell function , was arbitrarily set at 0.5 nmol / l . icas were analysed with immunofluorescence assay , with detection limit 9 jdf - u , sensitivity 100% and specificity 88% . gadabs were analysed with radioimmuno - precipitation with lower reference limit at an index of 0.08 , corresponding to 21 who - u / ml , sensitivity 70% and specificity 100% . all analyses were standardised according to the diabetes antibody standardization program ( 25 , 26 ) . metabolic control was assessed by hbalc ( mono - s ) , and values were converted to dcct standard ( 27 ) . regarding the metabolic syndrome , complete information was available regarding only body mass index ( bmi ) and prevalence of hypertension , defined as blood pressure > 140/80 mmhg at the baseline visit , or taking antihypertensive medication . all subjects provided informed consent . analyses were carried out according to intention - to - treat . for comparisons between the groups , student 's t - test , or fisher 's exact test and the mann c - peptide and hbalc at baseline and after 12 , 24 , and 36 months were analysed with repeated measures anova using time as covariate ; for three - group analyses , anova with bonferroni correction was performed where appropriate . risk factors and relations were analysed in several models with simple , multiple , linear and logistic regressions ( forward stepwise , wald ) . spss software , version 17.0 ( chicago , il , usa ) , was used . analyses were carried out according to intention - to - treat . for comparisons between the groups , student 's t - test , or fisher 's exact test and the mann c - peptide and hbalc at baseline and after 12 , 24 , and 36 months were analysed with repeated measures anova using time as covariate ; for three - group analyses , anova with bonferroni correction was performed where appropriate . risk factors and relations were analysed in several models with simple , multiple , linear and logistic regressions ( forward stepwise , wald ) . spss software , version 17.0 ( chicago , il , usa ) , was used . for the baseline characteristics of the groups , see table 1 . there were no significant gender differences in the whole study , except that in the control group there were only men with hypertension ( p=0.03 ) . most subjects were overweight since both the i and c groups had mean bmi27 kg / m . median duration of diabetes at inclusion in the study was 5.0 ( quartiles 3.09.0 ) months . in both the groups , 90% of the patients , 18/20 in i and 15/17 in c , completed 36 months of follow - up . the conventionally treated ( c ) , 30% ( 5/17 ) started insulin treatment due to clinical necessity within 6612182430 months . for the levels of glucagon - stimulated c - peptide , see table 2 and fig . c - peptide levels were unchanged for four patients , increased by mean 0.73 ( 0.5 ) nmol / l for six and declined in all others after 36 months . mean glucagon - stimulated c - peptide decreased significantly in both the groups during the 36 months ( p<0.0001 ) . there was a significant time trend for the decrease in c - peptide of 0.17 nmol / l per year ( p=0.03 ) over 36 months without any significant difference between the groups . in repeated measures , anova with time as covariate , that analyses the changes in c - peptide over time with the levels at baseline taken into account , no differences could be found regarding mean - stimulated c - peptide at any time point , although with the mann whitney u test the difference in c - peptide at baseline ( cp0 ) was significant , p=0.03 . there were large variations in c - peptide levels between different individuals , at all time points , within both the groups ( p<0.0001 ) , explaining all the variation between them ( fig . cp0 explained 43% of level of c - peptide at 36 months ( cp36 ) , r 0.43 ( p<0.0001 ) . furthermore , age was the only other factor that had a weak and non - significant influence on cp36 , explaining about 5% of cp36 , r 0.049 ( p=0.2 ) . gender , baseline or end - of - study values of bmi , titres of gadab or ica , hbalc or diabetes duration before the study start did not influence c - peptide at end - of - study . it was 86% ( 13/15 ) in c , and 61% ( 11/18 ) in i ( p=0.13 ) . the odds ratio ( or ) for having a cp36 0.5 nmol / l was 2.4 for every increase in cp0 with 0.10 nmol / l ( p=0.02 ) , and 1.06 for each increase in baseline age by 1 year ( p=0.03 ) . if corrected for cp0 and age , the two factors found to have any influence on cp36 , the insulin treated had a non - significant or of 2.5 ( 0.04184 ) of having a cp36 of 0.5 the results of levels of fasting c - peptide ( fcp ) , when comparing the two treatment groups ( data not shown ) , were in accordance with the described results of stimulated c - peptide . among the controls , the level of hbalc had increased significantly at 36 months from 7.0 ( 1.3)% to 7.5 ( 1.5)% ( p=0.006 ) ( fig . , there was no significant difference between hbalc at baseline , 7.3 ( 1.3)% , and after 36 months , 7.2 ( 0.7)% ( p=0.6 ) ( fig . 2 ) . for the levels of hbalc at baseline and during follow - up , see table 2 and fig . the differences between the groups in absolute levels of hbalc were not significant either at baseline or after 12 , 24 or 36 months . the levels of hbalc were not influenced by age , gender , bmi , antibody prevalences or titres or c - peptide levels . at baseline , prevalences of gadab were 94% , ica 67% , 72% had both antibodies , 22% gadab only and 6% only ica , with no significant differences between the two treatment groups . of the 32 patients whose ia-2a status was known , 7 ( 22% ) were positive , all of them were also positive to gadab and all but one was positive to ica , meaning that 75% of all were positive to at least two antibodies . at baseline , mean indexes of gadab were 0.78 ( 0.39 ) in i , 0.78 ( 0.48 ) in c ( ns ) and mean ica titres were 29.4 ( 40 ) in i , 72.4 ( 119 ) jdf - u in c ( ns ) . at baseline , there were no significant differences in the prevalences of any of the three antibodies between the two treatment groups or between genders , different ages , duration of diabetes before study start or levels of bmi or hbalc . neither were there any significant differences between the groups in titres of gadab or ica after 12 , 24 or 36 months . the titres of gadab or ica were not related to patient age , gender , bmi , diabetes duration , treatment or hbalc at baseline or during the follow - up . c - peptide at baseline or 36 months was not influenced by baseline titre of gadab , ica or ia-2a . no episodes of major hypoglycaemia were reported for any of the patients , and only a few minor ones . mean weight at baseline was 77.4 ( 14.5 , range 57.8110 ) kg in group i , and 83.0 ( 17.8 , range 50.8117 ) kg in group c ( ns ) ; at end - of - study 79.3 ( 12.4 , 57.7101 ) kg in i , 82.3 ( 14.8 , range 50.4115 ) kg in c ( ns ) . mean weight change during the study was 2.5 ( 4.8 , range 8.8 to + 9.3 ) kg in i ; 1.0 ( 10.5 , range 27.3 to + 16.4 ) kg in c ( ns ) . for the three groups , those treated with insulin from baseline , those never treated with insulin and those who were originally treated with dietoha , but had to start insulin treatment during the study , the influences of age , bmi , hbalc , diabetes duration before study start or antibody titres were analysed , with no significant findings except for the influence of cp0 on cp36 . the tests for all the relevant parameters were also carried out with the 37 patients divided into groups of ever- versus never - insulin treated ( during the study ) , again with no significant results ( p=0.120.87 ) , apart from the significant influence of cp0 on cp36 ( p<0.0001 ) . for the levels of glucagon - stimulated c - peptide , see table 2 and fig . c - peptide levels were unchanged for four patients , increased by mean 0.73 ( 0.5 ) nmol / l for six and declined in all others after 36 months . mean glucagon - stimulated c - peptide decreased significantly in both the groups during the 36 months ( p<0.0001 ) . there was a significant time trend for the decrease in c - peptide of 0.17 nmol / l per year ( p=0.03 ) over 36 months without any significant difference between the groups . in repeated measures , anova with time as covariate , that analyses the changes in c - peptide over time with the levels at baseline taken into account , no differences could be found regarding mean - stimulated c - peptide at any time point , although with the mann whitney u test the difference in c - peptide at baseline ( cp0 ) was significant , p=0.03 . there were large variations in c - peptide levels between different individuals , at all time points , within both the groups ( p<0.0001 ) , explaining all the variation between them ( fig . cp0 explained 43% of level of c - peptide at 36 months ( cp36 ) , r 0.43 ( p<0.0001 ) . furthermore , age was the only other factor that had a weak and non - significant influence on cp36 , explaining about 5% of cp36 , r 0.049 ( p=0.2 ) . gender , baseline or end - of - study values of bmi , titres of gadab or ica , hbalc or diabetes duration before the study start did not influence c - peptide at end - of - study . it was 86% ( 13/15 ) in c , and 61% ( 11/18 ) in i ( p=0.13 ) . the odds ratio ( or ) for having a cp36 0.5 nmol / l was 2.4 for every increase in cp0 with 0.10 nmol / l ( p=0.02 ) , and 1.06 for each increase in baseline age by 1 year ( p=0.03 ) . if corrected for cp0 and age , the two factors found to have any influence on cp36 , the insulin treated had a non - significant or of 2.5 ( 0.04184 ) of having a cp36 of 0.5 the results of levels of fasting c - peptide ( fcp ) , when comparing the two treatment groups ( data not shown ) , were in accordance with the described results of stimulated c - peptide . among the controls , the level of hbalc had increased significantly at 36 months from 7.0 ( 1.3)% to 7.5 ( 1.5)% ( p=0.006 ) ( fig . , there was no significant difference between hbalc at baseline , 7.3 ( 1.3)% , and after 36 months , 7.2 ( 0.7)% ( p=0.6 ) ( fig . 2 ) . for the levels of hbalc at baseline and during follow - up , see table 2 and fig . 2 . the differences between the groups in absolute levels of hbalc were not significant either at baseline or after 12 , 24 or 36 months . the levels of hbalc were not influenced by age , gender , bmi , antibody prevalences or titres or c - peptide levels . at baseline , prevalences of gadab were 94% , ica 67% , 72% had both antibodies , 22% gadab only and 6% only ica , with no significant differences between the two treatment groups . of the 32 patients whose ia-2a status was known , 7 ( 22% ) were positive , all of them were also positive to gadab and all but one was positive to ica , meaning that 75% of all were positive to at least two antibodies . at baseline , mean indexes of gadab were 0.78 ( 0.39 ) in i , 0.78 ( 0.48 ) in c ( ns ) and mean ica titres were 29.4 ( 40 ) in i , 72.4 ( 119 ) jdf - u in c ( ns ) . at baseline , there were no significant differences in the prevalences of any of the three antibodies between the two treatment groups or between genders , different ages , duration of diabetes before study start or levels of bmi or hbalc . neither were there any significant differences between the groups in titres of gadab or ica after 12 , 24 or 36 months . the titres of gadab or ica were not related to patient age , gender , bmi , diabetes duration , treatment or hbalc at baseline or during the follow - up . c - peptide at baseline or 36 months was not influenced by baseline titre of gadab , ica or ia-2a . no episodes of major hypoglycaemia were reported for any of the patients , and only a few minor ones . mean weight at baseline was 77.4 ( 14.5 , range 57.8110 ) kg in group i , and 83.0 ( 17.8 , range 50.8117 ) kg in group c ( ns ) ; at end - of - study 79.3 ( 12.4 , 57.7101 ) kg in i , 82.3 ( 14.8 , range 50.4115 ) kg in c ( ns ) . mean weight change during the study was 2.5 ( 4.8 , range 8.8 to + 9.3 ) kg in i ; 1.0 ( 10.5 , range 27.3 to + 16.4 ) kg in c ( ns ) . for the three groups , those treated with insulin from baseline , those never treated with insulin and those who were originally treated with dietoha , but had to start insulin treatment during the study , the influences of age , bmi , hbalc , diabetes duration before study start or antibody titres were analysed , with no significant findings except for the influence of cp0 on cp36 . the tests for all the relevant parameters were also carried out with the 37 patients divided into groups of ever- versus never - insulin treated ( during the study ) , again with no significant results ( p=0.120.87 ) , apart from the significant influence of cp0 on cp36 ( p<0.0001 ) . few prospective intervention studies have been conducted in lada patients and there is still no general agreement on the best treatment aimed to preserve -cell function ( 57 , 16 ) . there has not been any general consensus definition of lada , which complicates comparisons and pooling of results . the most common denominators are adult age , positivity to at least one pancreatic autoantibody , and non - insulin dependency at diagnosis ( 4 ) . age , bmi , duration of diabetes and of insulin independency , which of the antibodies that were analysed , gadab titres and the expression of the essential outcome variable c - peptide vary ( 2 , 4 , 5 , 2830 ) . a cochrane review also noted the heterogeneity between studies , and the conclusion about early insulin treatment was uncertain ( 5 ) . our study included patients aged 30 years , non - insulin dependent at the times of diagnosis and inclusion and positive to at least one pancreatic autoantibody , for 75% two antibodies , thereby fulfilling the main criteria for lada ( 4 ) . the results of the study indicated that none of the baseline parameters , except initial c - peptide level , significantly influenced the outcome , eliminating the importance of several criteria in comparisons with other studies . in other studies , patients aged > 65 years have often been excluded , but lada exists also in these older age groups ( 11 ) . the decline in residual -cell function was progressive for the majority of our lada patients , as is usual in autoimmune diabetes ( 4 , 8 , 13 , 16 ) . we observed great variation in the rates and magnitudes of -cell loss between patients and between different time periods during the study , with no consistent patterns . mechanisms such as more step - wise losses due to , for instance , partial remissions might explain this ( 15 ) . the decline in c - peptide was irrespective of age , gender , bmi , antibody titres , hbalc or treatment modality . the lack of influence of bmi , age , diabetes duration and baseline hbalc on disease progression was also seen in a non - interventional observation study of lada that followed 13 ab - positive patients by stimulated c - peptide for 2 years ( 16 ) . similar to ukpds , we found no association between gadab levels and disease progress ( 31 ) , in contrast to observational studies that described this ( 30 , 32 ) . we could not , in a number of regression analyses , define any other factor besides cp0 that significantly influenced the level of cp36 . the significance of initial c - peptide level was also demonstrated in a large swedish study of new - onset 1534 year olds and in the tokyo intervention study ( 28 , 33 ) . the length of our study may explain that some patients with initially higher levels of c - peptide , overrepresented in the control group , by 36 months had not yet lost enough -cell function to be clinically insulin dependent . some antibody - positive patients were described to take up to 12 years to become insulin dependent , but practically all eventually did ( 34 ) . a significant beneficial effect of early insulin treatment on the preservation of -cell function could not be demonstrated , but level of hbalc after 36 months was better preserved in the insulin treated , in keeping with observations by chaillous et al . incidentally , the shape of the curve of the development of hbalc levels over time for the conventionally treated group in our study had a likeness to that observed in the ukpds ( 35 ) . our study , as most prospective intervention studies of lada , was not large . the tokyo study , with 60 patients , found a preference for insulin treatment versus su , possibly due to the differences in treatment , but longer duration of diabetes , up to 5 years without insulin before inclusion , rendering a selection of patients with better endogenous insulin production from the start , would have excluded those who progressed earlier to insulin dependence , so the trial population differed from ours ( 28 ) . baseline level of c - peptide was an important independent predictor of the ability to preserve a sufficient amount of c - peptide over time , just as in our study . many reports end with a general recommendation of insulin treatment in lada , but the evidence has not been compelling , as concluded by the 2007 cochrane review , which scrutinised seven insulin intervention studies in lada , two insulin versus su , the rest insulin alone versus different combinations of insulin+oha ( 5 ) . ukpds recorded hbalc , weight and treatment , randomised to insulin versus su , and found that 60% of the su treated were insulin dependent after 2 years ( 36 ) . one conclusion was that su might promote insulin dependency and apart from not recommending su the cochrane review found no preference for any special type of treatment for the lada group ( 5 ) . in our study after 3 years , 65% of the conventionally treated patients were not yet treated with insulin . in contrast to both ukpds and the tokyo study , only 30% of our control patients were treated with su , which could be of importance for -cell function . in another study , of 54 patients in four groups , the insulin treated , all with low fcp , 0.3 nmol , received either insulin alone or combined with rosiglitazone ( rgz ) , and the oha treated , all with fcp > 0.3 to our knowledge , this study is the first prospective controlled european intervention study of treatment in lada . differences in defining the lada population regarding age , diabetes duration before start of intervention , antibody prevalences and gadab titres , bmi and , not least , baseline levels of c - peptide , if available , have contributed to difficulties in interpreting and comparing the results of the few existing lada intervention studies ( 4 , 5 , 28 , 29 , 37 ) . in this study , none of these factors , except baseline level of c - peptide , significantly affected the outcome . the similar findings also seen in trials of classical type 1 diabetes and of prevention of autoimmune diabetes in high - risk individuals substantiate the observation of the influence of initial level of c - peptide on outcome level ( 38 , 39 ) . we also saw a non - significant or favouring insulin treatment for preservation of -cell function , indicating the possibility that a larger study population and/or a longer period of follow - up might demonstrate significant preference for early insulin treatment also regarding -cell function . this study indicated that early insulin treatment in lada patients lead to better preservation of level of metabolic control , and that it was safe and well tolerated . the decline in c - peptide was progressive irrespective of age , gender , bmi , hbalc levels and antibody titres . only baseline level of c - peptide significantly influenced c - peptide level after 3 years . m landin - olsson has received a part - time professorship sponsored by novo - nordisk scandinavia . the other authors have no dualities of interest in relation to this article . the work was financed by the healthcare regions of skane and kronoberg , southern sweden ; lund university funding of clinical research ( alf ) ; and the swedish council of medical research .

objectivesthe optimal treatment of latent autoimmune diabetes in adults ( lada ) is not established . we explored whether early insulin treatment , which has shown beneficial effects in rodents and in human pilot studies , would result in better preservation of -cell function or metabolic control , compared with conventional treatment.subjects and methodsglucagon - stimulated c - peptide and hbalc were evaluated at baseline and after 12 , 24 and 36 months in 37 patients recently diagnosed with diabetes , aged 30 years , non - insulin - requiring and gadab and/or ica positive . twenty patients received early insulin and 17 received conventional treatment ( dietoral hypoglycaemic agents ( oha ) , metformin , some and/or sulfonylurea ) and insulin when necessary.resultslevel of metabolic control , hbalc , was preserved in the early insulin treated , while it significantly deteriorated in the conventionally treated . there was no significant difference between the groups in c - peptide after 12 , 24 or 36 months , or in the decline of c - peptide . only baseline c - peptide predicted a c - peptide of 0.5 nmol / l at 36 months . gender , body mass index , antibody titres or hbalc did not influence the levels of c - peptide or hbalc at baseline or end - of - study , or the decline in c - peptide . among the dietoha - treated , 5/17 ( 30% ) developed insulin dependency during the follow - up . no major hypoglycaemic events occurred.conclusionsearly insulin treatment in lada leads to better preservation of metabolic control and was safe . superior preservation of c - peptide could not be significantly demonstrated . only baseline level of c - peptide significantly influenced c - peptide level after 3 years . further studies exploring the best treatment in lada are warranted .

Introduction Objective Subjects and methods Statistical analyses Results -Cell function Metabolic control Autoimmunity Adverse events Three-group analysis and ever- versus never-insulin treated Discussion Further considerations Conclusions Declaration of interest Funding

to investigate the effect of early insulin treatment in lada patients , for 3 years , on residual -cell function and metabolic control , compared with a group initially treated with diet and/or oral hypoglycaemic agents ( oha ) . adults , aged 30 years , diagnosed with diabetes in lund and kronoberg counties in southern sweden , non - insulin - requiring at diagnosis and positive to at least one of gadabs and/or icas were eligible for participation . glucagon - stimulation tests were performed at baseline and after 12 , 24 and 36 months during annual policlinic visits at the two research clinics . c - peptide was analysed by commercial ria ( md315 , euro - diagnostica ab , malm , sweden ) , total variation ( sum of intra - and inter - assay variations ) 7% , reference range 0.251.0 c - peptide level , to reflect a preserved normal -cell function , was arbitrarily set at 0.5 nmol / l . for comparisons between the groups , student 's t - test , or fisher 's exact test and the mann c - peptide and hbalc at baseline and after 12 , 24 , and 36 months were analysed with repeated measures anova using time as covariate ; for three - group analyses , anova with bonferroni correction was performed where appropriate . for comparisons between the groups , student 's t - test , or fisher 's exact test and the mann c - peptide and hbalc at baseline and after 12 , 24 , and 36 months were analysed with repeated measures anova using time as covariate ; for three - group analyses , anova with bonferroni correction was performed where appropriate . there was a significant time trend for the decrease in c - peptide of 0.17 nmol / l per year ( p=0.03 ) over 36 months without any significant difference between the groups . gender , baseline or end - of - study values of bmi , titres of gadab or ica , hbalc or diabetes duration before the study start did not influence c - peptide at end - of - study . if corrected for cp0 and age , the two factors found to have any influence on cp36 , the insulin treated had a non - significant or of 2.5 ( 0.04184 ) of having a cp36 of 0.5 the results of levels of fasting c - peptide ( fcp ) , when comparing the two treatment groups ( data not shown ) , were in accordance with the described results of stimulated c - peptide . , there was no significant difference between hbalc at baseline , 7.3 ( 1.3)% , and after 36 months , 7.2 ( 0.7)% ( p=0.6 ) ( fig . the differences between the groups in absolute levels of hbalc were not significant either at baseline or after 12 , 24 or 36 months . neither were there any significant differences between the groups in titres of gadab or ica after 12 , 24 or 36 months . there was a significant time trend for the decrease in c - peptide of 0.17 nmol / l per year ( p=0.03 ) over 36 months without any significant difference between the groups . gender , baseline or end - of - study values of bmi , titres of gadab or ica , hbalc or diabetes duration before the study start did not influence c - peptide at end - of - study . if corrected for cp0 and age , the two factors found to have any influence on cp36 , the insulin treated had a non - significant or of 2.5 ( 0.04184 ) of having a cp36 of 0.5 the results of levels of fasting c - peptide ( fcp ) , when comparing the two treatment groups ( data not shown ) , were in accordance with the described results of stimulated c - peptide . , there was no significant difference between hbalc at baseline , 7.3 ( 1.3)% , and after 36 months , 7.2 ( 0.7)% ( p=0.6 ) ( fig . the differences between the groups in absolute levels of hbalc were not significant either at baseline or after 12 , 24 or 36 months . neither were there any significant differences between the groups in titres of gadab or ica after 12 , 24 or 36 months . the titres of gadab or ica were not related to patient age , gender , bmi , diabetes duration , treatment or hbalc at baseline or during the follow - up . the decline in c - peptide was irrespective of age , gender , bmi , antibody titres , hbalc or treatment modality . a significant beneficial effect of early insulin treatment on the preservation of -cell function could not be demonstrated , but level of hbalc after 36 months was better preserved in the insulin treated , in keeping with observations by chaillous et al . this study indicated that early insulin treatment in lada patients lead to better preservation of level of metabolic control , and that it was safe and well tolerated . only baseline level of c - peptide significantly influenced c - peptide level after 3 years .

following a clinically predictable progression pattern , advanced prostate cancer ( pca ) metastasizes to distant organs , with a striking predisposition for bone . despite a decreasing overall mortality rate for pca patients , the development of effective treatments has been hindered , in part , by the lack of cell lines and/or xenograft models that accurately recapitulate the complex metastatic microenvironment . without appropriate models to reflect the disease , mechanistic studies to accurately elucidate the players involved in pca progression in bone have been difficult to implement , impeding the development of clinically effective therapeutics targeted to bone metastases . some of the more commonly used pca cell lines ( e.g. , pc-3 and du-145 ) offer convenience to investigators because they adhere well to tissue culture plastic and therefore are amenable to high throughput screening of drug libraries . however , these same cell lines , when inoculated in bone , generate a largely osteolytic response , in contrast to the predominantly osteoblastic nature of the native human disease . to more accurately model the disease , we have employed the lncap cell line progression series ( lncap , c4 , c4 - 2 , and c4 - 2b ) , with c4 - 2b cells forming osseous lesions in bone . however , we and other investigators continue to search for pca cell models with greater fidelity to the disease that will foster the translation of preclinical findings into the clinic , particularly to satisfy the need to identify new treatments that will eradicate pca metastases growing in bone . to address the need for the highest - possible fidelity in pca cell sources , patient - derived xenograft ( pdx ) models have been established for the preclinical investigation of various aspects of pca biology including angiogenesis , identification of castrate - resistant stem - like cells , and effect of anti - androgen therapies . pdx models are generated when tumor tissue from the patient is surgically resected and engrafted directly into immunocompromised mice . tumors are subsequently maintained solely in vivo via mouse - to - mouse passage , requiring both careful monitoring of the tumor burden and labor intensive animal transfers . through serial passaging in mice and the absence of any in vitro manipulation , pdx tumors remain biologically stable , preserving much of the molecular , genetic , and histological features as well as heterogeneity of the original tumor . however , given the high costs of animal maintenance , lengthy latency period following engraftment , variable engraftment rates , and rare access to patient tissue specimens , pdx in vivo models are generally not yet widely employed in cancer research . most notably , given the poor viability exhibited when grown in vitro under standard tissue culture conditions , it is extremely challenging to culture pdx bone metastatic pca cells for any brief ex vivo manipulation needed to conduct controlled mechanistic studies in vivo . mirroring the in vitro behavior of pdx prostate tumors , pca cell lines belonging to the lncap series also adhere poorly to two - dimensional ( 2d ) tissue culture surfaces , precluding them from use in standard drug screening platforms . to circumvent this problem , our laboratory recently demonstrated the feasibility of using three - dimensional ( 3d ) hyaluronan ( ha)-based hydrogel systems to support the growth and viability of these pca cell lines for mechanistic studies and drug testing . while the commonly employed human tumor spheroid model is an alternative to culturing these poorly adherent cells , hydrogel encapsulation provides the means to fully recapitulate the tumor microenvironment with precise , tunable control over architectural and mechanical cues and/or critical cell specifically , as a ubiquitous component of the bone marrow where bone metastatic pca cells reside , ha plays an active role in regulating several biological processes , including tumorigenesis , strongly justifying its use as an extracellular matrix analogue for culturing bone metastatic tumor cells in vitro . on the basis of the suitability of ha - based hydrogel systems for the culture of pca cell lines , we hypothesized that these systems would similarly support the viability of pdx pca cells in vitro . hence , in the present study , we developed a novel protocol to encapsulate pdx pca cells within 3d ha - based hydrogels and examined tumor cell morphology , viability , proliferative capacity and phenotype . we also tested the potential of this 3d pdx pca model as a diagnostic platform for evaluating rapid patient - specific drug response , a significant advance toward achieving a more personalized therapeutic regimen . phenol red - free dulbecco s modified eagle medium / nutrient mixture f-12 ( dmem / f-12 ) , t - medium , penicillin / streptomycin , trypsin - edta , l - glutamine , live / dead viability / cytotoxicity kit , and quant - it picogreen dsdna assay kit were obtained from life technologies ( grand island , ny ) . fetal bovine serum ( fbs ) phosphate buffered saline ( pbs ) was obtained from lonza ( walkersville , md ) . thiol - modified ha ( ha - sh , glycosil , average mw = 240 kda , degree of thiolation = 1 mol / mg ha - sh ) and poly(ethylene glycol)-diacrylate ( peg - da , extralink , average mw = 3350 da ) were obtained from biotime inc . this ha - sh / peg - da hydrogel system previously has been characterized extensively . dmso , l - cysteine , and papain papaya latex were obtained from sigma - aldrich ( st . louis , mo ) . primary antibodies used were human - specific anti - nuclei antibody from millipore ( mab1281 , billerica , ma ) , anti - androgen receptor from santa cruz biotechnology ( sc-816 , dallas , tx ) , anti - cleaved caspase-3 from cell signaling technology ( nb11089717 , danver , ma ) , and anti - ki-67 from novus biologicals ( # 9664s , littleton , co ) . sylgard 184 poly(dimethylsiloxane ) ( pdms ) elastomer kit was from dow corning ( midland , mi ) . mda pca 183 and mda pca 118b cells were routinely maintained as subcutaneous tumors in cb-17 scid mice ( charles river ) . all experiments for the propagation of pdx tumors in mice were conducted under iacuc approval from the university of texas md anderson cancer center . the mda pca 183 xenograft was derived from androgen - dependent prostate carcinoma , whereas the mda pca 118b was derived from androgen receptor - negative castrate - resistant prostate carcinoma . for this study , mda pca 183 and mda pca 118b pdxs of passage 12 and 8 respectively , were used . on the day of harvest , tumor specimens were removed immediately , rinsed six times with pbs , minced with a scalpel blade , and digested with accumax enzymatic solution for 15 min at 37 c . the enzyme solution was inactivated with fbs , the resultant tumor slurry was filtered through a 70 m cell strainer , and the filtrate was centrifuged . the supernatant was removed , the resulting cell pellet was resuspended , and a cell count was performed . custom - made pdms molds were made by mixing sylgard 184 elastomer and cross - linker 10:1 ( v / v ) according to manufacturer s instructions . the liquid silicone solution was centrifuged at 4 c/3000 rpm/5 min to remove bubbles and poured into a square aluminum frame with a 1.5 mm - thick spacer . an x-660 automated co2 laser cutter ( universal laser systems , scottsdale , az ) was used at 2.5% speed and 60% power to cut the slabs into 24 60 mm rectangles with multiple 6 mm diameter cylindrical cavities . the resultant molds were steam - autoclaved before each use and were sealed onto sterile glass slides before being employed as molds for hydrogel fabrication . each cylindrical cavity could hold approximately 50 l of hydrogel . prior to pdx tumor culture , ha - sh and peg - da were solubilized at 10 and 20 mg / ml , respectively , in degassed water per the manufacturer s instructions . the solutions were mixed at 4:1 ( v / v ) ratio , 35 l of the combined solution was placed in each cavity of the silicone molds , and the mixture was allowed to cross - link for 1 h at 37 c . the bottom layer of acellular hydrogel served as a cushion layer to prevent cell clusters from settling out of the cell hydrogel construct during culture . following tumor harvest , were plated on 6-well plates at densities determined to yield hydrogel constructs with 150,000 and 300,000 encapsulated mda pca 183 and mda pca 118b cells respectively , per construct . mda pca 183 and mda pca 118b cells were cultured in dmem / f-12 supplemented with 10% and 30% ( v / v ) fbs , respectively , in the presence of 100 u / ml of penicillin and 100 g / ml of streptomycin . tumor cells then were incubated at 37 c with 5% co2 for 2 days , after which tumor cell aggregates that formed in suspension from all wells were collected into sterile 15 ml tubes and gently centrifuged . supernatant was removed from each tube , and the remaining cell pellets were resuspended in complete dmem / f-12 medium , combined , and split into microcentrifuge tubes and centrifuged again . notably , as these cells form clusters in suspension , we could not perform cell counts immediately prior to encapsulation . instead , cell counting for the preparation of hydrogel constructs was performed at the step of 2d culture in 6-well plates ( immediately after the tumors were dissociated ) . the resulting cell pellets then were resuspended in solutions of ha - sh and peg - da ( 4:1 v / v ) as described above , and 25 l of the cell - hydrogel suspension was immediately pipetted into each mold cavity , over the 35 l cushion layer of cross - linked ha - sh / peg - da . seeded hydrogels were returned to the incubator for 45 min , then immersed in cell culture medium and incubated overnight . the following day , each mold cavity was scored with a 26-gauge needle , cell - hydrogel constructs were transferred into 24-well plates and submerged in culture medium . the c4 - 2b bone metastatic pca cell line was maintained in t - medium containing 5% fbs ( v / v ) and 2 mm l - glutamine in the presence of 100 u / ml of penicillin and 100 g / ml of streptomycin . for the docetaxel drug study , 50,000 cells were encapsulated within ha - sh / peg - da as described above for the pdx tumors . a lower cell density was used for the cell line because high - density culture was associated with poor cell viability ( data not shown ) . cell - hydrogel constructs ( n = 3 ) were maintained for 2 days before treatment with docetaxel for 3 days . docetaxel was diluted in dimethyl sulfoxide ( dmso ) such that the final concentration of dmso was 1% ( v / v ) in complete medium across all drug concentrations . morphology of the cells encapsulated within the hydrogel was monitored by differential interference contrast microscopy at days 1 , 3 , 5 , and 7 postencapsulation using a nikon eclipse te300 inverted microscope and nis elements software ( nikon instruments , melville , ny ) . fluorescently labeled samples were imaged using a nikon a1-rsi confocal microscope and images processed using the nikon nis - elements ar software ( nikon instruments , melville , ny ) . cell viability was assessed using the live / dead viability / cytotoxicity kit as per the manufacturer s instructions . briefly , cell - hydrogel constructs at the designated time - points were incubated in 2 m calcein - am and 4 m ethidium homodimer-1 in pbs for 30 min at 37 c before confocal imaging . cell - hydrogel constructs ( n = 3 or 4 ) were collected into individual microcentrifuge tubes at the designated time - points , flash - frozen using liquid nitrogen , and stored at 80 c . frozen samples then were lyophilized overnight and digested in pbe buffer ( 0.10 m na2hpo4 and 0.010 m na2edta in demineralized water at ph 6.5 ) containing 125 g / ml papain in the presence of 14.5 mm l - cysteine at 65 c overnight . the digested samples then were sonicated using a probe sonicator , and the liquid supernatant was assayed using the quant - it picogreen dsdna quantification assay as per the manufacturer s instructions . excitation and emission wavelengths of 485 and 528 nm , respectively , were used to measure the fluorescence ( flx800 fluorescence microplate reader ; biotek instruments ) . cell - hydrogel constructs were washed with pbs and fixed with 4% ( v / v ) paraformaldehyde for 10 min at room temperature . after fixation , constructs were washed with pbs and stored at 4 c until staining . constructs were immersed in 0.2% ( v / v ) triton x-100 for 5 min at room temperature to permeabilize cells , then blocked with 500 l of 3% ( w / v ) bsa and 0.2% triton x-100 in pbs at 4 c overnight . all antibodies were diluted at 1:200 in 3% bsa and 0.2% triton - x-100 in pbs . antibody staining was performed using 200 l of the mixed solution to each sample , which were placed on a rocking platform shaker at 4 c overnight . samples were washed with pbs before adding fluorophore - labeled secondary antibodies directed against the appropriate host . secondary antibodies were diluted 1:500 in 3% bsa and 0.2% triton - x-100 in pbs , and 200 l of that solution was added to each sample . dapi ( 5 g / ml ) was added to each sample at room temperature for 5 min . when phalloidin was used , it was diluted 1:20 in pbs , and 100 l of that mixture was added to each sample for 15 min . in initial experiments , following tumor digestion , we encapsulated the entire pdx cell population directly into hydrogels . when we did so , a large number of dead cells was transferred to the hydrogels as observed after 1 day in 3d culture ( data not shown ) . these dead cells likely were generated during the tumor harvest and digestion , and also contain mouse - derived cells that die immediately within the hydrogels . as the presence of high numbers of dead cells would complicate any biochemical assays that were envisioned , we sought to develop an alternative procedure designed to encapsulate cells with high viability ( figure 1a ) . ( a ) mda pca 183 and mda pca 118b pdx tumors ( indicated by red arrows ) that were grown subcutaneously in scid mice were harvested , then mechanically and enzymatically digested . the tumor slurry was subsequently plated onto 6-well plates and cultured for 2 days during which the majority of mouse - derived cells ( red ) attached . the resulting tumor aggregates that formed ( blue ) then were encapsulated within ha - sh / peg - da hydrogels and cultured for up to 14 days . ( b ) dissociated tumor cells plated on tissue culture plastic for 2 days formed multicellular clusters in suspension with other cell types adhering to the plastic surface . ( c ) mixture of dapi - stained ( blue ) cells within tumoroids and adherent cells after 2 days in culture . scale bars = 100 m . in optimizing the culture conditions for the pdx pca cells , we observed that most of the mda pca 183 and mda pca 118b pca cells that had undergone mechanical and enzymatic digestion and been plated onto 6-well plates formed multicellular aggregates in suspension after 2 days in culture , presumably reflecting their characteristic poor adherence onto tissue culture plastic ( figure 1b ) . additionally , during this period , we noted that a population of cells in the tumor slurry ( shown in red in figure 1a ) adhered to the tissue culture plastic surface . leveraging this phenomenon , we collected the aggregates in suspension ( leaving behind the adherent cells ) and found that the process of gentle centrifugation resulted in the removal of dead cells . given that the mda pca 183 and mda pca 118b tumors were grown as subcutaneous tumors in mice , they carry along with them a subpopulation of mouse - derived cells in addition to the human pca cells . indeed , we found that among the poorly adherent pca cells that formed multicellular aggregates in suspension after 2 days on tissue culture plastic , the cells that had adhered stained positive for vimentin ( figure 1c ) . henceforth , we employed this pre - encapsulation 2d culture method , which not only removes dead cells , but also enriches the pdx tumor population via the depletion of mesenchymal cells . a summary of the process used to form 3d pdx tumoroids within the ha - sh / peg - da hydrogel is illustrated in figure 1a . in the first week after encapsulation , mda pca 183 cells were maintained as large tumoroids with most having diameters between 60100 m . mda pca 118b cells were maintained as smaller tumoroids , typically smaller than 60 m in diameter ( figure 2a and supplementary figure 1 ) . cells in tumoroids were in close contact in multicellular clusters , observed via staining with phalloidin for f - actin ( figure 2b ) . to demonstrate that human pca cells comprise the 3d pdx tumoroids , we stained the encapsulated cells for epithelial cell adhesion molecule ( epcam ) , an established marker for epithelial cells . as expected , both the mda pca 183 and mda pca 118b 3d pdx tumoroids stained positive for epcam ( figure 2c ) , indicating that the tumoroids form from self - sorting epithelial cells despite the presence of mesenchymal cells in the original xenograft tumors . additionally , we also confirmed this by probing for the human - specific anti - nuclei antibody and found that the majority of cells within the tumoroids stained positive ( data not shown ) , indicating that the 3d pdx tumoroids form from largely human cells . next , we asked if the pca cells in tumoroids retained their androgen receptor activation status . probing specifically for the androgen receptor , we found that while the androgen receptor was mainly localized in the nucleus of cells in the mda pca 183 tumoroids , nuclear localization of the receptor was not observed in the mda pca 118b cells as would be expected in vivo . generation of pdx tumoroids encapsulated within 3d ha - sh / peg - da hydrogels . ( a ) mda pca 183 and mda pca 118b cells remained as multicellular tumoroids post - encapsulation , over 1 week in culture . cells were stained with dapi ( blue ) or phalloidin ( green ) ; a merged image is shown on the right panel . ( c ) hydrogel - encapsulated mda pca 183 and mda pca 118b cells were stained with dapi ( blue ) , and with antibodies against epcam ( green ) and the androgen receptor ( red ) . while the mda pca 183 cells exhibited nuclear localization of the androgen receptor ( indicated by yellow arrows ) , the mda pca 118b cells stained negative for nuclear localization of the androgen receptor . encapsulation of the nonadherent tumoroids into ha - sh / peg - da hydrogels maintained cell cell contacts for at least 2 weeks in culture . given that these pdx pca tumors exhibit poor viability in vitro on 2d , we next investigated whether the encapsulated pdx tumoroids remained viable within the hydrogels over time using the live / dead viability / cytotoxicity assay . as shown in figure 3 , cells in both the mda pca 183 and mda pca 118b tumoroids were predominantly viable at days 1 , 5 , and 14 . remarkably , pdx cells had the highest survival when they had formed large clusters with other cells . dead or dying cells in each hydrogel were observed to be one of two types : either single cells that had not aggregated with other cells to form multicellular clusters or cells on the periphery of each cluster ( particularly prominent in figure 3b , day 14 ) . beyond viability , to establish if the 3d pdx tumoroids demonstrate tumor growth characteristics necessary for the model to serve as a drug - testing platform , particularly for drug candidates that target actively dividing cells , we monitored their growth over 1 week in culture , using dna content as a surrogate measure of cellularity . notably , the overall cellularity of the mda pca 118b constructs was lower than that of the mda pca 183 because the mda pca 118b cells formed smaller clusters , therefore the retrieval of cells for hydrogel encapsulation was less efficient than from the mda pca 183 cultures . this is apparent from the finding that even though a higher theoretical seeding density ( 150,000 and 300,000 cells per construct for mda pca 183 and 118b , respectively ) was employed for the mda pca 118b constructs , the average initial dna content of the mda pca 118b constructs still was lower than that of the mda pca 183 constructs ( day 1 , figure 4a ) . in analyzing the cellularity of the 3d pdx constructs over time , while dna content remained constant with no significant difference over time for the mda pca 183 tumoroids , there was a significant decrease in cellularity for the mda pca 118b from day 1 to 5 , after which an increase in cellularity was observed ( figure 4a ) . probing the 3d pdx tumoroids for ki-67 and cleaved caspase-3 , markers of proliferation and apoptosis , respectively , we found that differences in the proportion of proliferative and apoptotic cells at day 5 in culture was not apparent for either mda pca 183 or 118b ( figure 4b , c ) . panels in ( a ) and ( b ) show mda pca 183 and 118b after 1 , 5 , and 14 days in culture , respectively . cells were stained with calcein - am ( green , left panel ) or ethidium homodimer-1 ( red , middle panel ) . ( a ) average cellularity of the 3d mda pca 183 and 118b constructs over 1 week in culture . upper panel of ( b ) and ( c ) shows the mda pca 183 and mda 118b tumoroids , respectively , stained with dapi ( left panel , blue ) and antibodies against ki-67 ( middle panel , red ) . lower panel of ( b ) and ( c ) shows the mda pca 183 and mda pca 118b tumoroids , respectively , stained with dapi ( left panel , blue ) and antibodies against cleaved caspase-3 ( middle panel , red ) the right panel shows a merged image where green indicates f - actin . having demonstrated that the encapsulated 3d pdx tumoroids are ( 1 ) made up of viable pca cells , ( 2 ) maintain in vivo - like androgen receptor distribution , and ( 3 ) are proliferative in the hydrogels , we next determined the suitability of this in vitro platform for drug testing of primary pca cells . given the inherent differences in origin between the mda pca 118b and mda pca 183 pdx models , we hypothesized that they would demonstrate a differential sensitivity to chemotherapeutic drugs . to test this hypothesis , we exposed the 3d mda pca 183 and mda pca 118b tumoroids to docetaxel , currently part of the first line regimen to treat patients with castrate - resistant metastatic pca . surprisingly , we found that not only was there no overall difference in docetaxel sensitivity between the 3d mda pca 183 and mda pca 118b tumoroids , no significant reduction in cell number was detected within the range of docetaxel concentrations tested for either 3d tumoroid systems ( figure 5a ) . to confirm these findings , we performed the live / dead viability / cytotoxicity assay and found that cells were largely viable even at the highest docetaxel concentration ( figure 5b ) . additionally , there were no apparent differences in the proportion of apoptotic cells as indicated by cleaved caspase-3 staining at the various docetaxel concentrations tested ( supplementary figure 2 ) . to demonstrate that the lack of cell - kill was cell source - dependent , we similarly exposed hydrogel - encapsulated cells from a bone metastatic prostate cancer cell line , c4 - 2b , to docetaxel . interestingly , a significant decrease in cellularity was already apparent between 1 and 10 nm ( figure 5a , b ) . this finding was corroborated by an observed increase in the proportion of ethidium homodimer-1-stained cells with increasing concentration of docetaxel ( figure 5b ) . notably , given that viable c4 - 2b cells were present at 10,000 nm as observed by calcein - am staining , it is likely that the decrease in the proportion of calcein - am - stained c4 - 2b cells due to drug cytotoxicity could not be clearly discerned when compared to controls . we also showed that the pdx cells were readily killed with 1 m sodium azide ; thus , it was likely not an issue of drug accessing the pca cells ( data not shown ) . ( a ) average cellularity of 3d mda pca 183 and 118b constructs after exposure to increasing docetaxel concentrations , compared to the c4 - 2b cell line , also hydrogel - encapsulated . c4 - 2b cells exhibited significantly lower resistance to docetaxel as compared to both pdxs ( p < 0.05 ) . ( b ) viability of cells after exposure to 0 , 10 , or 10,000 nm docetaxel , assessed by calcein - am staining ( green , left panels ) and ethidium homodimer-1 ( red , middle panels ) . in this study , we assessed a 3d ha - based hydrogel system for the culture of primary pdx bone metastatic pca tumors , well - known to exhibit poor viability when cultured on tissue culture plastic in vitro . given the ubiquity of ha in the bone marrow extracellular matrix and our previous studies that demonstrated the feasibility of using 3d ha - based hydrogels to culture poorly adherent bone metastatic pca cell lines , we hypothesized that these biologically active hydrogels would serve as excellent matrices to support the long - term culture of primary pdx pca tumor tissue in vitro . the inability of bone metastatic pca cells , whether derived from cell lines or primary pdx tumor tissue , to grow in 2d culture is an established phenomenon that indicates the lack of critical components from the bone microenvironment upon which these highly adapted cells depend . in optimizing the culture conditions for the 3d pdx pca cells , we found that the cancer cells failed to adhere to tissue culture plastic , as was expected . instead , these cells aggregated to form multicellular aggregates in suspension , where the formation of cell cell contact is a likely mechanism employed by the pca cells to adapt to the loss of native cell - matrix interactions . indeed , as proposed by shen and kramer , cancer cells are capable of undergoing synoikis , a term that was used to describe the avoidance of apoptosis by relying on intercellular adhesions in the absence of cell matrix interactions . focusing specifically on squamous cell carcinoma cells , the authors demonstrated that e - cadherin - mediated cell cell contact resulted in the generation of compensatory survival signaling via the epidermal growth factor receptor . interestingly , a similar observation was made by a landmark report by kondo et al . cell interactions were necessary for the survival of primary patient - derived colorectal cancer cells in suspension culture . in optimizing the culture method for the primary pdx pca cells , we leveraged the ability of the cancer cells to spontaneously aggregate to form multicellular clusters in suspension as a means to reduce the presence of contaminant cells such as dead cells , including those that were originally from the tumor tissue itself ( in regions of necrosis ) and those that were generated during the process of tumor digestion . we found that the application of gentle centrifugation to retrieve the pca aggregates in suspension after 2 days on tissue culture plastic was an effective method to separate the pca cells from dead cells in suspension prior to encapsulation within the hydrogel . given that the pdx pca cells were maintained in mice prior to harvest , the cancer cells were also likely contaminated with mouse - derived cells ( such as blood cells , endothelial cells , or fibroblasts ) , which consequently introduces a source of heterogeneity and complicates downstream biochemical studies . furthermore , considering the importance of the tumor microenvironment in influencing tumor behavior , the presence of mouse - derived stroma may confound studies evaluating species - specific stroma - targeting drug candidates , hence making it a major hindrance in current pdx tumor models . indeed , while the pca cells formed aggregates in suspension , a population of cells was consistently observed to adhere to the tissue culture plastic surface . for example , in mda pca 183 cultures , we found that the adherent cells expressed vimentin and originated from mouse tissue ( figure 1c ) . while further characterization is necessary to determine the actual yield of the cancer cells , these results , in conjunction with the observation that epcam , an epithelial cell marker , was near - ubiquitously expressed in all of the hydrogel - encapsulated pdx clusters ( figure 2c ) , highly suggest that the pre - encapsulation 2d culture serves as a desirable selective method to generate viable and enriched pdx pca cells in 3d . in this study , we optimized the culture of pdx pca cells in vitro , which to our knowledge is unprecedented . while robust growth was limited by a balance between proliferation and apoptosis , viability of the hydrogel - encapsulated pca cells was notably well maintained even up to 14 days in culture ( figure 3 ) . ten to 20% of cells within the 3d pdx tumoroids stained positive for ki-67 at day 5 in culture ( figure 4b , c ) , indicating that a subset of cells was still progressing through the cell cycle . comparing the proportion of proliferative cells within the 3d mda pca 118b pdx tumoroids to their in vivo counterparts , we found that our estimate of ki-67-positive cells is well within the range of that in vivo . we are currently modifying the ha - based hydrogel to study the effects of ecm moieties and other cell types on the growth behavior of the pdx pca tumoroids in 3d culture . with the shift in focus from traditional chemotherapy to biologically targeted approaches based on a mechanistic understanding of pca biology , preservation of salient features of the original tumor is a critical criterion in evaluating the success of this culture system as a drug - testing platform . to this end the androgen receptor is an important modulator for prostate growth and currently the primary therapeutic target in pca . additionally , this nuclear receptor regulates psa expression , which is used clinically as a biochemical marker to track the progression of pca and response to therapy . predominantly cytoplasmic in the absence of ligands , ligand binding to the androgen receptor results in conformational changes , nuclear translocation of the ligand - bound receptor , and transcriptional activity . mirroring the in vivo tumor , detection of nuclear androgen receptor in the 3d mda pca 183 tumoroids indicates that the ha - sh / peg - da hydrogel system is capable of maintaining critical androgen receptor signaling in these androgen - dependent cells . similarly , nuclear androgen receptor was not observed in the 3d mda pca 118b tumoroids as would be expected in in vivo , indicating that these 3d pdx models may have utility as drug - testing platforms evaluating androgen receptor - targeting agents currently under intense clinical investigation . drug discovery and screening has historically relied on cancer cell lines , which have generated a wealth of knowledge in cancer biology . however , the dichotomy in drug efficacy between preclinical and clinical testing is increasingly apparent , primarily attributed to the genetic and epigenetic changes that accrue when cells acclimatize to the in vitro culture environment in extended periods of cell culture . indeed , gillet et al . evaluated the multidrug resistance transcriptome of six cancer types and found no correlation between clinical samples and established cell lines , underscoring the need for new in vitro cancer models and primary tumor models . hypothesizing that inherent differences exist between traditional pca cell lines and pdx pca tumors , we evaluated the response of the 3d pdx pca tumoroids ( mda pca 183 and 118b ) to docetaxel and compared their response to the c4 - 2b bone metastatic prostate cancer cell line . docetaxel is a microtubule stabilizer and is currently part of the first - line standard treatment for metastatic castrate - resistant pca . we found that despite the differences between the mda pca 183 and 118b cells in patient origin , their overall drug sensitivity to docetaxel was similar . given that both pdx - derived cultures showed a low number of proliferating cells and that docetaxel targets actively dividing cells , it thus was unsurprising that both demonstrated similar resistance to the drug in 3d culture . however , interestingly , the 3d pdx pca cells exhibited a significantly higher resistance to the drug as compared to the c4 - 2b cells . it is unlikely that the increased resistance is due to impaired diffusion of the drug into the hydrogel since it demonstrated clear efficacy against the hydrogel - encapsulated c4 - 2b cells . while results from this drug study are promising , studies are ongoing to establish clinical relevance and understand the mechanisms underlying this difference . with the increasing awareness that irreversible biological changes occur when conventional cell lines are established in vitro , beyond prostate cancer , there is general shift toward the use of pdx tumor models for cancer research . however , their utility depends on the ability to manipulate the tumor cells ex vivo prior to implantation . given the alterations in biological properties associated with 2d culture , a few groups have taken steps to optimize methods to reliably grow primary tumor cells in 3d . one such example is in colorectal cancer , where it has been shown that primary colorectal cancer cells can be cultured as 3d spheroids or colospheres in vitro , enabling the manipulation of the cancer cells before engraftment in vivo for controlled studies . in our current study , we demonstrate for the first time that primary bone metastatic pca cells can similarly be cultured in vitro with the use of a 3d ha - based hydrogel system . lastly , this 3d pdx model may ultimately be adapted to a rapid and high throughput platform for assessing drug efficacy , rational drug combinations , and development of predictive biomarkers for novel targeted therapies , while reducing the need for low throughput animal hosts . as an example , we showed here that 3d pdx pca cells exhibited an increased resistance to docetaxel as compared to a standard cell line commonly used in pca research . ultimately , it is envisioned that the use of 3d pdx models may greatly accelerate the advancement of novel drug candidates together with predictive biomarkers that enable patient selection when translated into early phase clinical trials . on the basis of previous success culturing bone metastatic pca cell lines using 3d ha - based hydrogels , we demonstrate for the first time that never in 2d pdx pca tumors can be cultured in vitro and maintained for at least 2 weeks . the resulting hydrogel - encapsulated 3d pdx tumoroids retained viability , proliferative capacity , and the androgen receptor expression , indicating that this novel 3d pdx model may serve as a valuable platform for drug development . while it has yet to be tested , this system promises the possibility of culturing tumor tissue directly from the patient for rapid drug screening , thereby eliminating the middle mouse and its associated problems , a major leap toward personalized medicine .

the lack of effective therapies for bone metastatic prostate cancer ( pca ) underscores the need for accurate models of the disease to enable the discovery of new therapeutic targets and to test drug sensitivities of individual tumors . to this end , the patient - derived xenograft ( pdx ) pca model using immunocompromised mice was established to model the disease with greater fidelity than is possible with currently employed cell lines grown on tissue culture plastic . however , poorly adherent pdx tumor cells exhibit low viability in standard culture , making it difficult to manipulate these cells for subsequent controlled mechanistic studies . to overcome this challenge , we encapsulated pdx tumor cells within a three - dimensional hyaluronan - based hydrogel and demonstrated that the hydrogel maintains pdx cell viability with continued native androgen receptor expression . furthermore , a differential sensitivity to docetaxel , a chemotherapeutic drug , was observed as compared to a traditional pca cell line . these findings underscore the potential impact of this novel 3d pdx pca model as a diagnostic platform for rapid drug evaluation and ultimately push personalized medicine toward clinical reality .

Introduction Materials and Methods Results Discussion Conclusions

however , we and other investigators continue to search for pca cell models with greater fidelity to the disease that will foster the translation of preclinical findings into the clinic , particularly to satisfy the need to identify new treatments that will eradicate pca metastases growing in bone . to address the need for the highest - possible fidelity in pca cell sources , patient - derived xenograft ( pdx ) models have been established for the preclinical investigation of various aspects of pca biology including angiogenesis , identification of castrate - resistant stem - like cells , and effect of anti - androgen therapies . on the basis of the suitability of ha - based hydrogel systems for the culture of pca cell lines , we hypothesized that these systems would similarly support the viability of pdx pca cells in vitro . we also tested the potential of this 3d pdx pca model as a diagnostic platform for evaluating rapid patient - specific drug response , a significant advance toward achieving a more personalized therapeutic regimen . in optimizing the culture conditions for the pdx pca cells , we observed that most of the mda pca 183 and mda pca 118b pca cells that had undergone mechanical and enzymatic digestion and been plated onto 6-well plates formed multicellular aggregates in suspension after 2 days in culture , presumably reflecting their characteristic poor adherence onto tissue culture plastic ( figure 1b ) . indeed , we found that among the poorly adherent pca cells that formed multicellular aggregates in suspension after 2 days on tissue culture plastic , the cells that had adhered stained positive for vimentin ( figure 1c ) . having demonstrated that the encapsulated 3d pdx tumoroids are ( 1 ) made up of viable pca cells , ( 2 ) maintain in vivo - like androgen receptor distribution , and ( 3 ) are proliferative in the hydrogels , we next determined the suitability of this in vitro platform for drug testing of primary pca cells . to demonstrate that the lack of cell - kill was cell source - dependent , we similarly exposed hydrogel - encapsulated cells from a bone metastatic prostate cancer cell line , c4 - 2b , to docetaxel . in this study , we assessed a 3d ha - based hydrogel system for the culture of primary pdx bone metastatic pca tumors , well - known to exhibit poor viability when cultured on tissue culture plastic in vitro . given the ubiquity of ha in the bone marrow extracellular matrix and our previous studies that demonstrated the feasibility of using 3d ha - based hydrogels to culture poorly adherent bone metastatic pca cell lines , we hypothesized that these biologically active hydrogels would serve as excellent matrices to support the long - term culture of primary pdx pca tumor tissue in vitro . in optimizing the culture conditions for the 3d pdx pca cells , we found that the cancer cells failed to adhere to tissue culture plastic , as was expected . while further characterization is necessary to determine the actual yield of the cancer cells , these results , in conjunction with the observation that epcam , an epithelial cell marker , was near - ubiquitously expressed in all of the hydrogel - encapsulated pdx clusters ( figure 2c ) , highly suggest that the pre - encapsulation 2d culture serves as a desirable selective method to generate viable and enriched pdx pca cells in 3d . hypothesizing that inherent differences exist between traditional pca cell lines and pdx pca tumors , we evaluated the response of the 3d pdx pca tumoroids ( mda pca 183 and 118b ) to docetaxel and compared their response to the c4 - 2b bone metastatic prostate cancer cell line . however , interestingly , the 3d pdx pca cells exhibited a significantly higher resistance to the drug as compared to the c4 - 2b cells . as an example , we showed here that 3d pdx pca cells exhibited an increased resistance to docetaxel as compared to a standard cell line commonly used in pca research . on the basis of previous success culturing bone metastatic pca cell lines using 3d ha - based hydrogels , we demonstrate for the first time that never in 2d pdx pca tumors can be cultured in vitro and maintained for at least 2 weeks . the resulting hydrogel - encapsulated 3d pdx tumoroids retained viability , proliferative capacity , and the androgen receptor expression , indicating that this novel 3d pdx model may serve as a valuable platform for drug development .

organisms often employ more than one mechanism to accomplish a task . for instance , animals typically navigate with multiple input channels. the classic example is homing by the rock dove columba livia , for which magnetic fields , the sun , landmarks and geophysical processes have all been shown to be used ( wiltschko and wiltschko 2003 ) . the range of behaviours or mechanisms organisms employ may be puzzling . at times , an apparent simplicity is observed . either have chemical defences to ward off herbivores or have symbiotic relationships with protective ants ( rehr et al . 1973 ) . hosts parasitised by eurasian cuckoos cuculus canorus famously reject cuckoo eggs , but never reject cuckoo chicks ( davies 2000 ) . this simplicity may be the result of evolutionary lag but , more interestingly , may also be caused by one strategy making another maladaptive by reducing predator abundance ( planqu et al . different mechanisms may complement one another , and true redundancy is often hard to show ( able and bingman 1987 ) . indeed , the existence of a suite of mechanisms against a broad ensemble of predators is readily understandable . another striking example of a system in which different mechanisms augment and complement one another , but now at a collective level , is house hunting in social insects . this has become one of the model systems to study distributed decision making in animals . when the nest is destroyed , the colony has to decide collectively where to settle next during a time of crisis ( franks et al . individual ants or bees have been shown to combine sophisticated assessments of potential nest sites ( seeley 1977 ; seeley and morse 1978 ; mallon and franks 2000 ; franks et al . 2003b ) with various recruitment mechanisms to collate information , and thus make collective decisions ( mallon et al . 2001 ; pratt et al . 2002 ; pratt 2005 ; seeley and visscher 2003 , 2004 ; visscher 2007 ) . when the old nest is destroyed , a fraction of ants goes out scouting to find a new home . upon finding a nest , the nest is assessed ( mallon and franks 2000 ) and ants start recruiting other ants to it with a latency that is inversely proportional to the perceived nest quality ( mallon et al . 2001 ) , using a process called forward tandem running ( mglich et al . 1974 ) . during a forward tandem run , a knowledgeable ant teams up with a nave ant . the leader slowly progresses towards the new nest , each time waiting for the follower to catch up , thereby teaching her the way ( franks and richardson 2006 ) . through this slow recruitment process , once a nest population reaches a certain quorum threshold , the recruiters switch from slow tandem running to much faster social carrying , and transport the remaining passive ants and brood to the new nests ( pratt et al . this description has been the basis of several models ( pratt et al . 2002 ; pratt et al . . however , a behaviour commonly employed by these ants is usually not included ( but see pratt et al . recruiter ants are not only engaged in social carrying , but also regularly perform tandem runs from the new back to the old nest . these so - called reverse tandem runs ( mglich 1978 ) are often more common than forward tandem runs ( mallon et al . 2002 ) , but their function is much less well understood . to maximise fitness , the colony should emigrate as quickly as possible to avoid predation and other hazards . therefore , during house hunting , a fast build up of recruiters is essential . why then do ants mix fast carrying with slow reverse tandem running , when they already have forward tandem running at their disposal ? in particular , through the use of mathematical models , we explore under what circumstances reverse tandem running can have a positive influence on emigration speed . we present two mathematical models to investigate the possible role of reverse tandem runs in ant colony emigrations . reverse tandem running does not contribute to the decision - making process of which new nest to choose ( pratt et al . 2002 ; franks et al . only a small fraction of the ants in a colony are actively involved during emigrations ( pratt et al . 2002 ; langridge 2006 ) . in this paper , we thus divide the colony s n ants into fractions fn of active ants a , and ( 1 f)n passive ants p. this assumption to divide ants into active and passive is a crucial one , without which the models collapse . the rates at which active ants leave the nest and become scouts , and scouts recruiters , are given by and k , respectively . forward tandem running occurs at a rate until the quorum q is met , after which recruiters carry passive ants and brood at a rate . to incorporate reverse tandem runs , we need to model which ants follow these tandem runs . available data from nest - choice experiments ( mallon et al . 2001 ) suggest two possibilities : either the reverse tandem runs are followed by ants that have not found the new nest yet , or by ants that have been carried to the new nest . these two options are not necessarily mutually exclusive : the carried ants could have been scouts . model 1 assumes that reverse tandem runs are followed by uncommitted scouts in the arena , and model 2 assumes they are followed by passive ants that were carried to the new nest . 1diagrams of the two different models for which reverse tandem runs are hypothesised to increase speed . the parameters are explained in table 1 diagrams of the two different models for which reverse tandem runs are hypothesised to increase speed . the parameters are explained in table 1 to capture in detail the influence of reverse tandem runs on emigration dynamics , we need to consider the following points : both tandem running and social carrying involve a pair of ants from two different classes . hence , recruitment can only occur if ants of both participating classes are available;once the quorum has been met , recruiters can not carry and perform reverse tandem runs simultaneously ( we also assume recruiters are not involved in other activities than these two ) . both tandem running and social carrying involve a pair of ants from two different classes . hence , recruitment can only occur if ants of both participating classes are available ; once the quorum has been met , recruiters can not carry and perform reverse tandem runs simultaneously ( we also assume recruiters are not involved in other activities than these two ) . we assume that ants of both classes are well mixed in the part of the arena ( or nest ) where they meet . with populations of ants of size x and y meeting , the number of ants that on average meet is then proportional to xy/(x + y ) . importantly , the smallest class limits the interaction rate , as is to be expected . we thus also have to specify how much post - quorum time recruiters spend on carrying or reverse tandem running ( they are assumed not to spend any time on other behaviours ) . before the quorum is met , the rate at which active ants at the old nest , a , become recruiters , r , through tandem running is given by ra/(a + r ) . now let f be the fraction of post - quorum time spent on reverse tandem runs , and the remainder 1 f spent on social carrying . then the mean number of scouts becoming recruiters through reverse tandem runs is 1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f\lambda rs/(s+r).$$\end{document}for carried ants becoming recruiters through reverse tandem running , we have by analogy 2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f\lambda rc/(c+r).$$\end{document}similarly , the mean number of passive ants p that become carried ants is given by ( 1 f ) rp/(p + r ) . recruiter ants should not perform reverse tandem runs when there are no scouts or carried ants left . therefore , we replace f by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\bar f$\end{document } in eqs . 1 and 2 , where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\bar f = \min\{s f , f\}$\end{document } and min { cf , f } , respectively . the min operation is for computational reasons only and ensures that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\bar f$\end{document } decreases continuously but rapidly to zero as s or c decreases , respectively . we drop the bar on \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\bar f$\end{document } in the rest of the paper . forward tandem running only occurs before the quorum is met and carrying and reverse tandem running only after . these are modelled with functions l , c and r , respectively , as follows . \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { l{\left ( { \lambda , r , q , a } \right ) } = } { \left\ { { \begin{array}{*{20}l } { { \lambda \frac{{ra}}{{r + a } } } \hfill } & { { { \text{if}}\,r < q , } \hfill } \\ { 0 \hfill } & { { { \text{otherwise } } , } \hfill } \\ \end{array } } \right . } \\ { c{\left ( { \phi , r , q , p } \right ) } = } { \left\ { { \begin{array}{*{20}l } { { \phi \frac{{rp}}{{r + p } } } \hfill } & { { { \text{if}}\,r \geqslant q , } \hfill } \\ { 0 \hfill } & { { { \text{otherwise } } , } \hfill } \\ \end{array } } \right . } \\ \end{array}$$\end{document}and , setting b to a for model 1 , and to p for model 2 , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$r(\lambda , r , q , b ) = \left\ { \begin{array}{ll } \lambda \frac{rb}{r+b}&\quad\text{if } r \ge q,\\ 0 & \quad\text{otherwise}. \end{array } \right.$$\end{document}to aid the reader , we state the full equations for both models . the equations for model 1 are given by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\left\ { \begin{array}{ll } \dot a & = -\mu a - l(\lambda , r , q , a),\\ \dot s & = \mu a - ks - f r(\lambda , r , q , s)\\ \dot r & = ks + l(\lambda , r , q , a ) + f r(\lambda , r , q , s)\\ \dot p & = -(1-f ) c(\phi , r , q , p),\\ \dot c & = ( 1-f ) c(\phi , r , q , p ) , \end{array } \right.$$\end{document}with initial conditions ( a , s , r , p , c)(0 ) = ( fn 2 , ,,(1 f)n,0 ) . model 2 is specified by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\left\ { \begin{array}{ll } \dot a & = -\mu a -l(\lambda , r , q , a),\\ \dot s & = \mu a - ks,\\ \dot r & = ks + l(\lambda , r , q , a ) + f r(\lambda , r , q , c),\\ \dot p & = -(1-f ) c(\phi , r , q , p),\\ \dot c & = ( 1-f ) c(\phi , r , q , p ) - f r(\lambda , r , q , c ) , \end{array } \right.$$\end{document}with the same initial conditions as model 1 . models 1 and 2 only differ in the placement of the term fr(,r , q , c ) . in both models , the in the initial conditions is necessary to avoid singularities in the denominators of the interaction functions l , r and c. we have used = 0.01 throughout . the main hypothesis we tested on both these models was : reverse tandem runs speed up the emigration if recruiter numbers failed to increase early in the emigration ; this occurs through a combination of the new nest being hard to find and forward tandem running being prohibited . we tested this hypothesis by finding the fastest emigration strategy for given parameter settings and determining whether reverse tandem runs formed part of this optimal strategy . we first fixed the scouting parameter , , the fraction of active ants at the old nest , f , and k , the rate at which scouts become recruiters . then quorum size q and fraction of post - quorum reverse tandem running time f were varied to optimise emigration speed . emigrations were termed completed when all passive ants and active ants at the old nest had disappeared . in particular , we set the threshold for emigration completeness at p + a = 0.01 . optimal strategies were found using the nelder mead simplex method ( nelder and mead 1965 ) . parameters and f were varied on an equidistant 2020 grid spanning [ 0.01,0.2 ] [0.05,0.5 ] . the ranges of these parameters are inspired by experimental estimates in ( pratt et al . table 1values or ranges , where applicable , for the parameters used in models 1 and 2 depicted in fig . 1parametersdescriptionvalue / rangencolony size250ffraction of active ants[0.05,0.5]qquorum thresholdn.a.ffraction of post - quorum reverse tandem running timen.a.rate at which active ants at old nest become scouts ( ant min)[0.01,0.2]rate at which ants following tandem runs become recruiters ( ant min)0.1rate at which passive ants are carried to new nest ( ant min)0.2krate at which scouts independently become recruiters ( ant min){0.0001,0.001}parameter choices for , and n were taken from the ranges in pratt et al . values or ranges , where applicable , for the parameters used in models 1 and 2 depicted in fig . 1 parameter choices for , and n were taken from the ranges in pratt et al . for both models , the optimal emigration strategy included reverse tandem runs for a wide range of parameters , together with low quorum thresholds ( fig . 2 ) . fixing k whilst varying and f , the optimal strategy often contained more reverse than forward tandem runs for a large part of the parameter range ( fig . 2 ) . the fraction of time spent on reverse tandem running f and the quorum threshold q were negatively correlated . when either the fraction of active ants f decreased or the scouting parameter increased , fraction f increased , and the quorum q decreased . choosing a higher recruitment latency by decreasing k gave more reverse tandem running and lower quorum thresholds ( fig . 2 ) . 2optimal fractions of post - quorum time spent on reverse tandem runs ( top figures ) , and optimal quorum thresholds ( bottom figures ) for models 1 ( left two columns ) and 2 ( right two columns ) for varying fractions of active ants f and scouting probabilities and for two values of recruitment latencies k. other parameter values used are given in table 1 . reverse tandem runs are part of the optimal emigration strategy when scouting probability is high or when fraction of active ants is low . lowering k enhances the use of reverse tandem running optimal fractions of post - quorum time spent on reverse tandem runs ( top figures ) , and optimal quorum thresholds ( bottom figures ) for models 1 ( left two columns ) and 2 ( right two columns ) for varying fractions of active ants f and scouting probabilities and for two values of recruitment latencies k. other parameter values used are given in table 1 . reverse tandem runs are part of the optimal emigration strategy when scouting probability is high or when fraction of active ants is low . lowering k enhances the use of reverse tandem running note that , although models 1 and 2 broadly give similar predictions , they differ in the amount of post - quorum time spent on reverse tandem runs . in model 1 , this reaches a full 100% in model 1 , but never so in model 2 . overall , the models predict that reverse tandem running should be used more than forward tandem running , and the quorum threshold lowered , if the recruitment latency increases by decreasing k ( scouting ants wait longer before starting their first recruitment act ) , in combination with either a decreasing fraction of active ants f , or an increasing scouting parameter . for all but very large f , the optimal quorum threshold corresponded to the time when all active ants have left the old nest to go scouting . in the absence of multiple new nests , the decision when to switch from forward tandem running to social carrying is thus best made at the old nest . recruiters should thus apply the following rule : continue forward tandem running until there are no ants left to perform them with and then switch to social carrying ; if few forward tandem runs have been performed ( by the recruiters ) , combine carrying with reverse tandem runs ; otherwise , do not . the numbers of forward tandem runs , reverse tandem runs , the numbers of carried ants in the new nest and total emigration time were computed for each of the optimal strategies of models 1 and 2 ( fig . first , for a large parameter range , there are more reverse tandem runs than forward tandem runs . this is broadly consistent with the experimental data from nest choice experiments in mallon et al . numbers there range between 3 and 17 forward tandems , and between 9 and 25 reverse tandems , and reverse tandems were always performed more often than forward tandems . second , as a validation of our optimisation method , note that the optimal emigration time varies smoothly under parameter changes , as is to be expected for this type of model . third , for model 1 , despite a clear drop in the post - quorum time spent on reverse tandem runs with increasing f ( see fig . 2 , top left ) , the number of reverse tandem runs in fact varies smoothly . fourth , the number of carried ants that remained in the nest at the end of the emigration is clearly different between models 1 and 2 . in model 1 , this number is just ( 1 f)n , the number of passive ants in the colony . in model 2 , however , over half of the colony may end up being recruiters by drawing recruits from the carried class using reverse tandem runs . 3numbers of forward and reverse tandem runs , number of carried ants and emigration time , computed for each of the optimal strategies for models 1 ( top row ) and 2 ( bottom row ) , illustrated in fig . 2 . here we have only illustrated k = 0.001 numbers of forward and reverse tandem runs , number of carried ants and emigration time , computed for each of the optimal strategies for models 1 ( top row ) and 2 ( bottom row ) , illustrated in fig . to maximise their fitness , ants should try to achieve the fastest emigrations to minimise vulnerability ( franks et al . therefore , the active ants either have to become scouts , discover a new site and then become recruiters or wait at the old nest until a recruiter leads them to the new nest . both of these processes may be hampered : when all the active ants go out scouting , recruiter numbers slowly increase if the new nest is hard to find or if those few can not find any active ant back at the old nest to tandem run with . in terms of the models , this could occur if scouts slowly become recruiters ( low value for k ) , in combination with either a small class of active ants at the old nest ( f is small ) or all active ants having gone scouting ( high value for scouting rate ) . under either or both of these circumstances , the model predicts that ants should not waste time trying to recruit by forward tandem runs but should do the next best thing and use a low quorum threshold to quickly switch to carrying . the recruiters should then invest a fraction of their time to recruit scouts or carried ants using reverse tandem runs , thus boosting the recruiter population and speeding up the emigration . conversely , the model also predicts that reverse tandem runs should not be used if either the new nest is easy to find ( recruiter numbers then build quickly anyway ) , or when there are many ants to follow a forward tandem run . ants have been shown to leave their intact old nest if the new nest is sufficiently better , but have lower standards when their nest is destroyed ( dornhaus et al . reverse tandem runs were mainly observed when the old nest was destroyed , combined with few forward tandem runs . the model offers a simple explanation for this : the greater panic might have caused fewer scouts to remain at the old nest , thereby obstructing early recruitment . whilst investigating speed accuracy trade - offs , franks et al . , this difference in reverse tandem running activity between mild and harsher conditions was found to be non - significant ( franks et al . although evidence for which ants follow reverse tandem runs is scarce , both models give the same qualitative predictions . note , however , that the dynamics of the different ant classes during a simulated emigration in model 2 poorly match observed experimental dynamics ( see , e.g. , planqu et al . the final number of ants active in an emigration is much greater than the original number of active ants . these recruiter numbers far exceed observed numbers of active ants ( available in table 3 in pratt et al . indeed , simulated emigrations often end with half the colony being recruiters and less than half carried into the new nest ( fig . 3 , bottom row , third from left ) . 4examples of temporal dynamics for models 1 and 2 . at = 0.05 , f = 0.1447 , we have taken parameters optimal for models 1 and 2 , respectively . notice that , in model 1 , the number of recruiters rises to about 35 , but in model 2 , there are no less than 100 recruiters at the end of the emigration , indicating that recruitment from the carried class ( model 2 ) may give rise to very unrealistic emigration dynamics examples of temporal dynamics for models 1 and 2 . at = 0.05 , f = 0.1447 , we have taken parameters optimal for models 1 and 2 , respectively . notice that , in model 1 , the number of recruiters rises to about 35 , but in model 2 , there are no less than 100 recruiters at the end of the emigration , indicating that recruitment from the carried class ( model 2 ) may give rise to very unrealistic emigration dynamics figure 2 ( top left ) shows that recruiters in model 1 should use a sequential strategy if f is small ( and is large ) : when the quorum is met , they first spend all of their time on reverse tandem runs until all scouts have become recruiters , and then switch to carrying . in contrast , the total number of recruiters is bounded by fn , the number of active ants in the colony . as f decreases , the remaining recruiters take longer to carry all the passive ants . hence , the time costs for not having the recruiters increases and the time to recruit the remaining scouts decreases ( as there are fewer scouts too ) . hence , in this situation , recruiters should devote their post - quorum time , first , all on reverse tandem running before starting carrying . one does not have to make many hands if the work was light to start with . note that this behaviour for f 0 is different for model 2 . in this paper top right ) , as there is no end to building recruiter numbers but by completing the entire emigration . this argument also explains another difference between these models : the number of reverse tandem runs during an emigration . in model 1 , there is a clear maximum for intermediate f , whereas in model 2 the number of reverse tandems strictly decreases with f ( fig . 3 , second from left , top and bottom ) . contrary to the models in this paper , two previous models of house hunting by t. albipennis ants ( pratt et al . the predictions of the current models proved to be strongly dependent on the assumption of non - linearity of these terms . the corresponding linear models predicted that reverse tandem running should not be used for practically any parameter choices in f and or k. another ingredient in this model shared with the previous ( pratt et al . 2006 ) , models of this collective decision - making system is the division between active and passive ants . all we know at present is that a limited fraction of ants is actively engaged during an emigration . we have thus also explored models in which this division was absent , using both linear and non - linear interaction terms such as those in models 1 and 2 presented in this paper . in none of these models the division between active and passive ants is thus a crucial ingredient for reverse tandem running to have a positive impact on emigration speed , which should be experimentally validated . several hypotheses on the potential role of reverse tandem running have been put forward ( pratt et al . 2002 ) . first , the ants might have a home nest , which changes during the emigration , thereby reversing the direction of any recruitment events from home to another nest ( pratt et al . if true , this would predict a change of direction when about half of the colony had been displaced . moreover , this hypothesis does not offer a suggestion why tandem running often occurs early in the emigration . in other words , it might explain the direction , but not the occurrence itself . second , it has been suggested that reverse tandem running may re - allocate recruitment ( pratt et al . reverse tandem runs were nearly always observed between the best nest and the old nest . the models in this paper do not incorporate choice between nest sites , but we conjecture that early flexible commitment ( planqu et al . 2006 ) will be more efficient in redirecting ants to better nests than late recruitment . other experimental results also corroborate that reverse tandem running does not influence the decision - making process ( franks et al . the first models in which reverse tandem runs have been explicitly incorporated to analyse their role have yielded clear predictions : under a range of conditions , we expect a negative correlation between levels of early and late recruitment . this finding lends itself well to simple experiments , and we aim to present those in the near future . the build up of recruiter numbers serves two purposes : to decide on a nest and to increase the number of ants actively involved in transport . this in itself is a side - effect of the distributed nature of this system . this suggests that such additional backup behaviours could be a common feature of decentralised collective decision - making systems . although evidence for which ants follow reverse tandem runs is scarce , both models give the same qualitative predictions . note , however , that the dynamics of the different ant classes during a simulated emigration in model 2 poorly match observed experimental dynamics ( see , e.g. , planqu et al . the final number of ants active in an emigration is much greater than the original number of active ants . these recruiter numbers far exceed observed numbers of active ants ( available in table 3 in pratt et al . indeed , simulated emigrations often end with half the colony being recruiters and less than half carried into the new nest ( fig . 3 , bottom row , third from left ) . 4examples of temporal dynamics for models 1 and 2 . at = 0.05 , f = 0.1447 , we have taken parameters optimal for models 1 and 2 , respectively . notice that , in model 1 , the number of recruiters rises to about 35 , but in model 2 , there are no less than 100 recruiters at the end of the emigration , indicating that recruitment from the carried class ( model 2 ) may give rise to very unrealistic emigration dynamics examples of temporal dynamics for models 1 and 2 . at = 0.05 , f = 0.1447 , we have taken parameters optimal for models 1 and 2 , respectively . notice that , in model 1 , the number of recruiters rises to about 35 , but in model 2 , there are no less than 100 recruiters at the end of the emigration , indicating that recruitment from the carried class ( model 2 ) may give rise to very unrealistic emigration dynamics figure 2 ( top left ) shows that recruiters in model 1 should use a sequential strategy if f is small ( and is large ) : when the quorum is met , they first spend all of their time on reverse tandem runs until all scouts have become recruiters , and then switch to carrying . the total number of recruiters is bounded by fn , the number of active ants in the colony . as f decreases , the remaining recruiters take longer to carry all the passive ants . hence , the time costs for not having the recruiters increases and the time to recruit the remaining scouts decreases ( as there are fewer scouts too ) . hence , in this situation , recruiters should devote their post - quorum time , first , all on reverse tandem running before starting carrying . one does not have to make many hands if the work was light to start with . note that this behaviour for f 0 is different for model 2 . in this paper top right ) , as there is no end to building recruiter numbers but by completing the entire emigration . this argument also explains another difference between these models : the number of reverse tandem runs during an emigration . in model 1 , there is a clear maximum for intermediate f , whereas in model 2 the number of reverse tandems strictly decreases with f ( fig . 3 , second from left , top and bottom ) . contrary to the models in this paper , two previous models of house hunting by t. albipennis ants ( pratt et al . the predictions of the current models proved to be strongly dependent on the assumption of non - linearity of these terms . the corresponding linear models predicted that reverse tandem running should not be used for practically any parameter choices in f and or k. another ingredient in this model shared with the previous ( pratt et al . 2006 ) , models of this collective decision - making system is the division between active and passive ants . all we know at present is that a limited fraction of ants is actively engaged during an emigration . we have thus also explored models in which this division was absent , using both linear and non - linear interaction terms such as those in models 1 and 2 presented in this paper . in none of these models the division between active and passive ants is thus a crucial ingredient for reverse tandem running to have a positive impact on emigration speed , which should be experimentally validated . several hypotheses on the potential role of reverse tandem running have been put forward ( pratt et al . 2002 ) . first , the ants might have a home nest , which changes during the emigration , thereby reversing the direction of any recruitment events from home to another nest ( pratt et al . if true , this would predict a change of direction when about half of the colony had been displaced . moreover , this hypothesis does not offer a suggestion why tandem running often occurs early in the emigration . in other words , it might explain the direction , but not the occurrence itself . second , it has been suggested that reverse tandem running may re - allocate recruitment ( pratt et al . reverse tandem runs were nearly always observed between the best nest and the old nest . the models in this paper do not incorporate choice between nest sites , but we conjecture that early flexible commitment ( planqu et al . 2006 ) will be more efficient in redirecting ants to better nests than late recruitment . other experimental results also corroborate that reverse tandem running does not influence the decision - making process ( franks et al . the first models in which reverse tandem runs have been explicitly incorporated to analyse their role have yielded clear predictions : under a range of conditions , we expect a negative correlation between levels of early and late recruitment . this finding lends itself well to simple experiments , and we aim to present those in the near future . the build up of recruiter numbers serves two purposes : to decide on a nest and to increase the number of ants actively involved in transport . the decision - making process and the implementation of this decision are thus conflated . this in itself is a side - effect of the distributed nature of this system . this suggests that such additional backup behaviours could be a common feature of decentralised collective decision - making systems .

to perform tasks , organisms often use multiple procedures . explaining the breadth of such behavioural repertoires is not always straightforward . during house hunting , colonies of temnothorax albipennis ants use a range of behaviours to organise their emigrations . in particular , the ants use tandem running to recruit nave ants to potential nest sites . initially , they use forward tandem runs ( ftrs ) in which one leader takes a single follower along the route from the old nest to the new one . later , they use reverse tandem runs ( rtrs ) in the opposite direction . tandem runs are used to teach active ants the route between the nests , so that they can be involved quickly in nest evaluation and subsequent recruitment . when a quorum of decision - makers at the new nest is reached , they switch to carrying nestmates . this is three times faster than tandem running . as a rule , having more ftrs early should thus mean faster emigrations , thereby reducing the colony s vulnerability . so why do ants use rtrs , which are both slow and late ? it would seem quicker and simpler for the ants to use more ftrs ( and higher quorums ) to have enough knowledgeable ants to do all the carrying . in this study , we present the first testable theoretical explanation for the role of rtrs . we set out to find the theoretically fastest emigration strategy for a set of emigration conditions . we conclude that rtrs can have a positive effect on emigration speed if ftrs are limited . in these cases , low quorums together with lots of reverse tandem running give the fastest emigration .

Introduction Materials and methods Results Discussion Critique on model 2 Reverse tandem activity Nonlinearities in the models and divisions between active and passive ants Hypothesised explanations

in particular , through the use of mathematical models , we explore under what circumstances reverse tandem running can have a positive influence on emigration speed . model 1 assumes that reverse tandem runs are followed by uncommitted scouts in the arena , and model 2 assumes they are followed by passive ants that were carried to the new nest . the parameters are explained in table 1 to capture in detail the influence of reverse tandem runs on emigration dynamics , we need to consider the following points : both tandem running and social carrying involve a pair of ants from two different classes . the main hypothesis we tested on both these models was : reverse tandem runs speed up the emigration if recruiter numbers failed to increase early in the emigration ; this occurs through a combination of the new nest being hard to find and forward tandem running being prohibited . 1parametersdescriptionvalue / rangencolony size250ffraction of active ants[0.05,0.5]qquorum thresholdn.a.ffraction of post - quorum reverse tandem running timen.a.rate at which active ants at old nest become scouts ( ant min)[0.01,0.2]rate at which ants following tandem runs become recruiters ( ant min)0.1rate at which passive ants are carried to new nest ( ant min)0.2krate at which scouts independently become recruiters ( ant min){0.0001,0.001}parameter choices for , and n were taken from the ranges in pratt et al . for both models , the optimal emigration strategy included reverse tandem runs for a wide range of parameters , together with low quorum thresholds ( fig . in the absence of multiple new nests , the decision when to switch from forward tandem running to social carrying is thus best made at the old nest . recruiters should thus apply the following rule : continue forward tandem running until there are no ants left to perform them with and then switch to social carrying ; if few forward tandem runs have been performed ( by the recruiters ) , combine carrying with reverse tandem runs ; otherwise , do not . the numbers of forward tandem runs , reverse tandem runs , the numbers of carried ants in the new nest and total emigration time were computed for each of the optimal strategies of models 1 and 2 ( fig . therefore , the active ants either have to become scouts , discover a new site and then become recruiters or wait at the old nest until a recruiter leads them to the new nest . both of these processes may be hampered : when all the active ants go out scouting , recruiter numbers slowly increase if the new nest is hard to find or if those few can not find any active ant back at the old nest to tandem run with . under either or both of these circumstances , the model predicts that ants should not waste time trying to recruit by forward tandem runs but should do the next best thing and use a low quorum threshold to quickly switch to carrying . conversely , the model also predicts that reverse tandem runs should not be used if either the new nest is easy to find ( recruiter numbers then build quickly anyway ) , or when there are many ants to follow a forward tandem run . notice that , in model 1 , the number of recruiters rises to about 35 , but in model 2 , there are no less than 100 recruiters at the end of the emigration , indicating that recruitment from the carried class ( model 2 ) may give rise to very unrealistic emigration dynamics figure 2 ( top left ) shows that recruiters in model 1 should use a sequential strategy if f is small ( and is large ) : when the quorum is met , they first spend all of their time on reverse tandem runs until all scouts have become recruiters , and then switch to carrying . in none of these models the division between active and passive ants is thus a crucial ingredient for reverse tandem running to have a positive impact on emigration speed , which should be experimentally validated . the first models in which reverse tandem runs have been explicitly incorporated to analyse their role have yielded clear predictions : under a range of conditions , we expect a negative correlation between levels of early and late recruitment . notice that , in model 1 , the number of recruiters rises to about 35 , but in model 2 , there are no less than 100 recruiters at the end of the emigration , indicating that recruitment from the carried class ( model 2 ) may give rise to very unrealistic emigration dynamics figure 2 ( top left ) shows that recruiters in model 1 should use a sequential strategy if f is small ( and is large ) : when the quorum is met , they first spend all of their time on reverse tandem runs until all scouts have become recruiters , and then switch to carrying . in none of these models the division between active and passive ants is thus a crucial ingredient for reverse tandem running to have a positive impact on emigration speed , which should be experimentally validated . the first models in which reverse tandem runs have been explicitly incorporated to analyse their role have yielded clear predictions : under a range of conditions , we expect a negative correlation between levels of early and late recruitment .

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" background and objectives : the aim of this study was to compare the in vitro fertilization ( ivf (...TRUNCATED)

"INTRODUCTION\nMATERIALS AND METHODS\nSubjects\nLaparoscopic Endometrioma Resection Surgery\nControl(...TRUNCATED)

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1. Introduction 2. Patients and Methods 3. Results 4. Discussion 5. Recommendation

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"Introduction\nMaterials and methods\nPopulation and data collection\nDefinitions and classification(...TRUNCATED)

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1. Introduction 2. Methods 3. Results 4. Discussion

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