Functional signaling test identifies HER2 negative breast cancer patients who may benefit from c-Met and pan-HER combination therapy.

BACKGROUND: Research is revealing the complex coordination between cell signaling systems as they adapt to genetic and epigenetic changes. Tools to uncover these highly complex functional linkages will play an important role in advancing more efficacious disease treatments. Current tumor cell signal transduction research is identifying coordination between receptor types, receptor families, and transduction pathways to maintain tumor cell viability despite challenging tumor microenvironment conditions. METHODS: In this report, coactivated abnormal levels of signaling activity for c-Met and HER family receptors in live tumor cells were measured by a new clinical test to identify a subpopulation of breast cancer patients that could be responsive to combined targeted therapies. The CELsignia Multi-Pathway Signaling Function (CELsignia) Test uses an impedance biosensor to quantify an individual patient's ex vivo live tumor cell signaling response in real-time to specific HER family and c-Met co-stimulation and targeted therapies. RESULTS: The test identified breast tumors with hyperactive HER1, HER2, HER3/4, and c-Met coordinated signaling that express otherwise normal amounts of these receptors. The supporting data of the pre-clinical verification of this test included analyses of 79 breast cancer patients' cell response to HER and c-Met agonists. The signaling results were confirmed using clinically approved matching targeted drugs, and combinations of targeted drugs in addition to correlative mouse xenograft tumor response to HER and c-Met targeted therapies. CONCLUSIONS: The results of this study demonstrated the potential benefit of a functional test for identifying a subpopulation of breast cancer patients with coordinated abnormal HER and c-Met signaling for a clinical trial testing combination targeted therapy. Video Abstract.

Cu-BTC metal-organic framework as a biocompatible nanoporous carrier for chlorhexidine antibacterial agent.

Antibacterial materials are an essential part of modern life and many efforts have been made to find a new and effective type of them. In this study, chlorhexidine (CHX) was loaded on Cu-BTC metal-organic framework (MOF), that both of them are known to have antibacterial properties. The antibacterial properties of Cu-BTC, CHX and CHX@Cu-BTC were investigated against Gram-positive and Gram-negative bacteria. Agar well-diffusion method and MIC test showed that CHX@Cu-BTC has high antibacterial activity. Characterization methods, such as FT-IR, XRD, N2 adsorption-desorption isotherm, TGA, SEM, EDX, TEM and zeta potential, were employed to characterize their structures. Cu-BTC MOF nanoparticles were synthesized and used as nanoporous carriers for chlorhexidine. The loading was about 10%, which was absorbed into the pores. Antibacterial activity was investigated against Gram-negative and Gram-positive bacteria by Agar well diffusion method and MIC (minimal inhibitory concentration) assay. The CHX@Cu-BTC had synergistic antibacterial activity of Cu-BTC and chlorhexidine.

Feasibility of two-dimensional speckle-tracking echocardiography of aortic valve in patients with calcific aortic valve disease.

Calcific aortic valve (AV) disease is associated with increased stiffness and reduced motion of AV leaflets, has a progressive course, and can develop into aortic stenosis (AS). Our aim was to evaluate whether two-dimensional speckle-tracking echocardiography (STE) may be used for the assessment of AV stiffness. We applied STE to AV leaflets in patients with various degrees of degenerative changes of AV and measured strain as an absolute value of the radial deformation of AV leaflets. Deformation of AV expressed as averaged AV strain was greatest in patients with a normal AV (23.4 ± 6.4%), compared to those with aortic sclerosis (12.9 ± 3.2%), moderate-to-severe AS (11.9 ± 4%), and severe AS (10.9 ± 3.5%) (p < 0.01). A non-linear relationship and moderate correlation of AV strain with transvalvular hemodynamic parameters was observed. In patients with mild-to-moderate AS, the strain of AV leaflets also correlated negatively with AV calcification (r = -0.59, p = 0.008). Good inter-observer agreement was obtained for averaged AV strain with a coefficient of variation of 0.15 and an interclass correlation coefficient of 0.94 (p < 0.0001). In this study we demonstrated that deformation of AV leaflets as assessed by STE might be a potential method for a non-invasive evaluation of AV biomechanical properties and of the progression of calcific aortic disease. Further development of the two-dimensional speckle tracking technique specifically for valve structures is needed to enable a better quantification of leaflet deformation.

Validity of visual assessment of aortic valve morphology in patients with aortic stenosis using two-dimensional echocardiography.

The diagnostic value of a visual assessment of aortic valve (AV) morphology for grading aortic stenosis (AS) remains unclear. A visual score (VS) for assessing the AV was developed and its reliability with respect to Doppler measurements and the calcium score (ctCS) derived by multislice computed tomography was evaluated. 99 Patients with AS of various severity and 38 patients without AS were included in the analysis. Echocardiographic studies were evaluated using the new VS which includes echogenicity, thickening, localization of lesions and leaflet mobility, with a total score ranging from 0 to 11. The association of VS with ctCS and the severity of AS was analyzed. There was a significant correlation of VS with AV hemodynamic parameters and with ctCS. The cut-off value for the detection of AS of any grade was a VS of 6 (sensitivity 95%, specificity 85% for women; sensitivity 85%, specificity 88% for men). A VS of 9 for women and of 10 for men was able to predict severe AS with a high specificity (96% in women and 94% in men, AUC 0.8 and 0.86, respectively). The same cut-off values were identified for the detection of ctCS of ≥ 1600 AU and ≥ 3000 AU with a specificity of 77% and 82% (AUC 0.69 and 0.81, respectively). Assessment of aortic valve morphology can serve as an additional diagnostic tool for the detection of AS and an estimation of its severity.

Global work index correlates with established prognostic parameters of heart failure.

AIM: Identification of patients with heart failure and a poor prognosis is paramount to ensure timely and adequate treatment. We investigated the relationship between the new measures of noninvasive pressure-strain analysis, such as the global work index (GWI), and established prognostic parameters of echocardiography, cardiopulmonary exercise test (CPX), and N-terminal pro-B-type natriuretic peptide (NT-pro-BNP). METHODS AND RESULTS: We retrospectively analyzed data of 51 patients with heart failure. Echocardiography and CPX were performed, and NT-pro-BNP was determined. Patients with a GWI < 500 mm Hg% had a mean LVEDV of 286.1 ± 100.8 mL, an LVEF of 21.3 ± 5.7%, and a stroke volume (SV) of 45.9 ± 11.6 mL, and patients with a GWI > 1000 mm Hg% had an LVEDV of 147.9 ± 39.6 mL, an LVEF of 42.6 ± 4.8%, and a SV of 70.9 ± 14.3 mL. The GWI also showed a significant correlation with peak oxygen consumption (peak VO2 ) (r = .521; P < .001) and with NT-pro-BNP (r = .635; P < .001). Patients with a GWI of <500 mm Hg% had a significantly higher NT-pro-BNP (median 2415 pg/mL [IQR 1071, 5933]) and a lower peak VO2 (9.5 mL/min/kg ± 2.6) compared to patients with a GWI of >1000 mm Hg% (NT-pro-BNP median 253 pg/mL [IQR 150, 549]; peak VO2 15.6 ± 4.2 mL/min/kg). CONCLUSION: GWI correlates with known prognostic markers of heart failure. A GWI of <500 mm Hg% was a predictor of severely impaired ejection fraction, very low exercise capacity, and strongly elevated NT-pro-BNP, indicating a poor prognosis.

New HER2-negative breast cancer subtype responsive to anti-HER2 therapy identified.

PURPOSE: HER2 signaling functional activity may be important to measure in addition to HER2 protein quantification when identifying patients eligible for HER2 therapies. A HER2 Signaling Function (CELx HSF) Test for HER2-negative patients uses patient's live tumor cells on a biosensor to identify patients with abnormally high HER2-related signaling (HSFs+) likely to respond to anti-HER2 therapies. METHODS: The CELx HSF test was employed to: (1) characterize the sensitivity and specificity of the test to detect abnormal levels of HER2 signaling; (2) evaluate the inhibitory effectiveness of five different anti-HER2 therapies; (3) assess the correlation between CELx HSF test detection of abnormal HER2 signaling and response to HER2 therapy using xenograft models; and (4) confirm the prevalence of abnormal HER2 signaling amongst HER2-negative breast cancer patients (HER2-/HSFs+). RESULTS: HER2-/HSFs+ breast cancer patient samples were identified and showed sensitivity to five approved anti-HER2 therapies. Xenograft studies using both HER2+ and HER2- cell lines confirmed that CELx HER2 signaling status better predicts HER2 inhibitor efficacy than HER2 receptor status. In a study of 114 HER2-negative breast tumor patient samples, 27 (23.7%; 95% CI = 17-32%) had abnormal HER2 signaling (HSFs+). A ROC curve constructed with this dataset projects the CELx HSF Test would have greater than 90% sensitivity and specificity to detect the HER2-/HSFs+ patient population. CONCLUSIONS: The CELx HSF test is a well-characterized functional biomarker assay capable of identifying dynamic HER2-driven signaling dysfunction in tumor cells from HER2-negative breast cancer patients. This test has demonstrated efficacy of various HER2 targeted therapies in live tumor cells from the HSFs+ population and correlated the test result to HER2 drug response in mouse xenograft studies. The proportion of HER2-negative breast cancer patients found to have abnormal HER2 signaling in a 114 patient sample study, 20-25%, is significant. A clinical trial to evaluate the efficacy of anti-HER2 therapies in this patient population is warranted.

Impact of preoperative atrial fibrillation on thromboembolic events and pump thrombosis in long-term left ventricular assist device therapy.

OBJECTIVES: Pump thrombosis (PT) and thromboembolic events (TEs) remain major adverse events in left ventricular assist device (LVAD) therapy at an annual rate of 6-8% supported with the HeartWare HVAD and HeartMate II. PT and TEs are multifactorial events. Understanding the predisposing risk factors for PT and TE is paramount to define preventive strategies. Preoperative atrial fibrillation (AF) is considered a significant and potentially modifiable risk factor. This study investigates whether LVAD patients with AF exhibit a higher rate of PT and TE than those in sinus rhythm (SR). METHODS: We evaluated medical records of consecutive patients who underwent implantation of the HeartMate II (n = 195; 25.4%) and HeartWare HVAD (n = 574; 74.6%) at our institution between 2006 and 2015. Only visually confirmed PT was included in the study. TE was defined as any peripheral embolism or cerebral embolism according to the INTERMACS definitions. RESULTS: SR was documented preoperatively in 211 patients (SR group) and AF in 558 patients (AF group). The median duration of support was 0.78 years in the AF group and 1.03 years in the SR group. The mean age was 60.27 years in the AF group and 52.04 years in the SR group. In the AF group, 83.0% of the patients were male, compared to 77.3% in the SR group. The cumulative incidence of PT in the SR group was 2.5% [95% confidence interval (CI) 0.3-4.7%] after 1 year and 5.7% (95% CI 2.2-9.2%) after 2 years, and in the AF group 4.9% (95% CI 3.0-6.7%) and 7.8% (95% CI 5.4-10.2%), respectively (P = 0.129). TEs were recorded in the SR group in 4.4% (95% CI 1.6-7.2%) after 1 year and in 6.3% (95% CI 2.8-9.8%) after 2 years, and occurred after a median support time of 214 days (range 120-768). In the AF group, the cumulative incidence was 8.4% (95% CI 6.0-10.7%) and 10.7% (95% CI 8.0-13.4%), respectively, after a median support time of 116 days (range 37-375), P-value = 0.163. In the multivariate analysis, event-free survival was not influenced by the preoperative SR [hazard ratio (HR) 0.86, 95% CI 0.68-1.1; P = 0.19]. CONCLUSIONS: Our study showed that the preoperative rhythm has no impact on survival, PT and TEs despite different preoperative risk factor profiles in the AF and SR group. Thus, the effect of a maze procedure, catheter ablation or left atrial appendage closure on PT and TE's for AF patients during LVAD implantation is questionable. However, to obtain a definitive answer, a prospective study would be of value.

The role of fibroblast - Cardiomyocyte interaction for atrial dysfunction in HFpEF and hypertensive heart disease.

AIMS: Atrial contractile dysfunction is associated with increased mortality in heart failure (HF). We have shown previously that a metabolic syndrome-based model of HFpEF and a model of hypertensive heart disease (HHD) have impaired left atrial (LA) function in vivo (rat). In this study we postulate, that left atrial cardiomyocyte (CM) and cardiac fibroblast (CF) paracrine interaction related to the inositol 1,4,5-trisphosphate signalling cascade is pivotal for the manifestation of atrial mechanical dysfunction in HF and that quantitative atrial remodeling is highly disease-dependent. METHODS AND RESULTS: Differential remodeling was observed in HHD and HFpEF as indicated by an increase of atrial size in vivo (HFpEF), unchanged fibrosis (HHD and HFpEF) and a decrease of CM size (HHD). Baseline contractile performance of rat CM in vitro was enhanced in HFpEF. Upon treatment with conditioned medium from their respective stretched CF (CM-SF), CM (at 21 weeks) of WT showed increased Ca2+ transient (CaT) amplitudes related to the paracrine activity of the inotrope endothelin (ET-1) and inositol 1,4,5-trisphosphate induced Ca2+ release. Concentration of ET-1 was increased in CM-SF and atrial tissue from WT as compared to HHD and HFpEF. In HHD, CM-SF had no relevant effect on CaT kinetics. However, in HFpEF, CM-SF increased diastolic Ca2+ and slowed Ca2+ removal, potentially contributing to an in-vivo decompensation. During disease progression (i.e. at 27 weeks), HFpEF displayed dysfunctional excitation-contraction-coupling (ECC) due to lower sarcoplasmic-reticulum Ca2+ content unrelated to CF-CM interaction or ET-1, but associated with enhanced nuclear [Ca2+]. In human patients, tissue ET-1 was not related to the presence of arterial hypertension or obesity. CONCLUSIONS: Atrial remodeling is a complex entity that is highly disease and stage dependent. The activity of fibrosis related to paracrine interaction (e.g. ET-1) might contribute to in vitro and in vivo atrial dysfunction. However, during later stages of disease, ECC is impaired unrelated to CF.

Mode-of-action of the PROPELLA concept in fulminant myocarditis.

AIMS: Haemodynamic load induces cardiac remodelling via mechano-transduction pathways, which can further trigger inflammatory responses. We hypothesized that particularly in an inflammatory disorder such as myocarditis, a therapeutic strategy is required which, in addition to providing adequate circulatory support, unloads the left ventricle, decreases cardiac wall stress, and mitigates inflammatory responses. METHODS AND RESULTS: Axial flow pumps such as the Impella systems comply with these requirements. Here, we report a potential mode-of-action of prolonged Impella support (PROPELLA concept) in fulminant myocarditis, including a decrease in cardiac immune cell presence, and integrin α1, α5, α6, α10 and ß6 expression during unloading. CONCLUSION: PROPELLA may provide benefits beyond its primary function of mechanical circulatory support in the form of additional disease-altering effects, which may contribute to enhanced myocardial recovery/remission in patients with chronic fulminant myocarditis.

Outcomes from a recovery protocol for patients with continuous-flow left ventricular assist devices.

BACKGROUND: In this retrospective analysis we evaluated a standardized echocardiographic assessment and an invasive technique for patient selection for successful continuous-flow left ventricular assist device (CF-LVAD) explantation. METHODS: Inclusion criteria for LVAD recovery assessment were: clinically stable condition; LVAD support for >6 months; physical activity; normal echocardiography findings; and no more than mild valvular disease and aortic valve opening. In a second step, echocardiography was performed under CF-LVAD reduction and stop conditions (PStopE). In the third step, patients who presented with stable parameters underwent right heart catheterization under CF-LVAD stoppage and occlusion of the outflow graft with a balloon catheter. Criteria for explantation were normal pulmonary artery pressure and pulmonary capillary wedge pressure <16 mmHg. RESULTS: Thirty-three of 424 patients entered the second step of evaluation and 20 entered the third step. Fourteen presented positive results and the pump was successfully explanted. The PCWP at baseline was 8.5 (2.8) mmHg in the explantation group and 10.6 (2.8) mmHg in the non-explantation group (p = 0.105). It increased to 10.9 (3.0) mmHg vs 20.8 (4.9) mmHg under outflow graft occlusion. The wedge pressure was significantly higher in the non-explantation group (p < 0.001). Median duration of follow-up after explantation was 9.74 (interquartile range 4.3 to 20.60) months, with survival of 93%. CONCLUSIONS: The protocol presented is feasible and safe. The criteria applied provide good patient selection for sustained mid-term myocardial recovery after LVAD explantation.

Development of a test that measures real-time HER2 signaling function in live breast cancer cell lines and primary cells.

BACKGROUND: Approximately 18-20% of all human breast cancers have overexpressed human epidermal growth factor receptor 2 (HER2). Standard clinical practice is to treat only overexpressed HER2 (HER2+) cancers with targeted anti-HER2 therapies. However, recent analyses of clinical trial data have found evidence that HER2-targeted therapies may benefit a sub-group of breast cancer patients with non-overexpressed HER2. This suggests that measurement of other biological factors associated with HER2 cancer, such as HER2 signaling pathway activity, should be considered as an alternative means of identifying patients eligible for HER2 therapies. METHODS: A new biosensor-based test (CELxTM HSF) that measures HER2 signaling activity in live cells is demonstrated using a set of 19 human HER2+ and HER2- breast cancer reference cell lines and primary cell samples derived from two fresh patient tumor specimens. Pathway signaling is elucidated by use of highly specific agonists and antagonists. The test method relies upon well-established phenotypic, adhesion-related, impedance changes detected by the biosensor. RESULTS: The analytical sensitivity and analyte specificity of this method was demonstrated using ligands with high affinity and specificity for HER1 and HER3. The HER2-driven signaling quantified ranged 50-fold between the lowest and highest cell lines. The HER2+ cell lines were almost equally divided into high and low signaling test result groups, suggesting that little correlation exists between HER2 protein expression and HER2 signaling level. Unexpectedly, the highest HER2-driven signaling level recorded was with a HER2- cell line. CONCLUSIONS: Measurement of HER2 signaling activity in the tumor cells of breast cancer patients is a feasible approach to explore as a biomarker to identify HER2-driven cancers not currently diagnosable with genomic techniques. The wide range of HER2-driven signaling levels measured suggests it may be possible to make a distinction between normal and abnormal levels of activity. Analytical validation studies and clinical trials treating HER2- patients with abnormal HER2-driven signaling would be required to evaluate the analytical and clinical validity of using this functional biomarker as a diagnostic test to select patients for treatment with HER2 targeted therapy. In clinical practice, this method would require patient specimens be delivered to and tested in a central lab.

A functional signal profiling test for identifying a subset of HER2-negative breast cancers with abnormally amplified HER2 signaling activity.

The results of clinical trials evaluating the efficacy of HER2 inhibitors in patients with breast cancer indicate that the correlation between HER2 receptor levels and patient outcomes is as low as 50%. The relatively weak correlation between HER2 status and response to HER2-targeting drugs suggests that measurement of HER2 signaling activity, rather than absolute HER2 levels, may more accurately diagnose HER2-driven breast cancer. A new diagnostic test, the CELx HER2 Signaling Profile (CELx HSP) test, is demonstrated to measure real-time HER2 signaling function in live primary cells. In the present study, epithelial cells extracted fresh from breast cancer patient tumors classified as HER2 negative (HER2-, n = 34 of which 33 were estrogen receptor positive) and healthy subjects (n = 16) were evaluated along with reference breast cancer cell lines (n = 19). Live cell response to specific HER2 agonists (NRG1b and EGF) and antagonist (pertuzumab) was measured. Of the HER2- breast tumor cell samples tested, 7 of 34 patients (20.5%; 95% CI = 10%-37%) had HER2 signaling activity that was characterized as abnormally high. Amongst the tumor samples there was no correlation between HER2 protein status (by cell cytometry) and HER2 signaling activity (hyperactive or normal) (Regression analysis P = 0.144, R2 = 0.068). One conclusion is that measurement of HER2 signaling activity can identify a subset of breast cancers with normal HER2 receptor levels with abnormally high levels of HER2 signaling. This result constitutes a new subtype of breast cancer that should be considered for treatment with HER2 pathway inhibitors.

Design changes in continuous-flow left ventricular assist devices and life-threatening pump malfunctions.

OBJECTIVES: The implantable continuous-flow left ventricular assist devices (LVADs) HeartMate II (HM II) and HeartWare HVAD (HW) underwent design modifications. The impact of these changes on life-threatening pump malfunctions was evaluated. METHODS: We retrospectively analysed pump malfunctions due to thrombosis or cable damage in patients supported with primarily implanted HM II (n = 191) and HW (n = 347), separated into patients supported with the old and new pump designs. In 2010, the cable strain relief of the HM II device was improved (132 patients with old and 79 with new) and sealed grafts were introduced (68 patients with sealed inflow connector and outflow graft and 125 without). In 2011, titanium sintering of the inflow cannula of HW pumps was introduced (137 patients with a non-sintered and 210 with a sintered inflow cannula). RESULTS: The median support time was 1.12 (0-6.1) years for all HM II and 0.59 (0-4.2) years for all HW patients. The cumulative rate of events per patient-year (EPPY) was 0.11 in HM II patients, compared with 0.09 EPPY in HW patients (P = 0.32). After introduction of the new cable design, incidence of cable damage in HM II patients dropped from 0.06 to 0 EPPY (P = 0.03), whereas pump thrombosis increased from 0.02 to 0.14 EPPY (P < 0.001) after the sealed graft was introduced. Pump thrombosis occurred in 4% of patients supported with HW with a sintered inflow cannula vs 15% with a non-sintered pump; the incidence changed from 0.10 to 0.07 EPPY in sintered pumps (P = 0.45). Kaplan-Meier analysis showed no differences over a period of 2.5 years for events when the HM II cohort with sealed graft and new cable design (n = 68) was compared with the HW group with a sintered cannula (P = 0.14). CONCLUSIONS: The modified cable strain relief of the HM II pump and the sintering of the inflow cannula of the HW pump demonstrated a significant reduction in the incidence of life-threatening pump-related complications, whereas the sealed inflow connector and outflow graft seem to be associated with a higher incidence of pump thrombosis. However, the overall incidence of pump-related complications after the latest design changes was similar for both pumps over a 2.5-year period.

Acoustic spectral analysis for determining pump thrombosis in rotary blood pumps.

Implantation of rotary blood pumps is an established treatment for end-stage heart failure. Anticoagulation is routinely used for reducing pump thrombosis, but this severe complication still occurs in 5-8% of patients. In 105 patients, the sound produced by the left ventricular assist device HeartWare HVAD was prospectively and sequentially recorded. The acoustic spectra calculated by runtime fast Fourier transformation were investigated. Results were compared with respect to patients without thrombotic complications (97 patients) and those with signs of pump thrombosis (8 patients). All eight pumps exchanged were opened and investigated. Thrombus formation was confirmed in all eight exchanged pumps. The existence of a sound peak with the threefold frequency of the pump speed correlated with the presence of thrombi inside the pump (p < 0.001). An increase in the sound amplitude of the rotary frequency of approximately 75% compared to previous measurements also denotes pump thrombosis. Analysis of the acoustic spectrum of the HVAD pump can be used as a reliable method to detect pump thrombosis. The appearance of third harmonics indicates the presence of thrombotic particles independently of previous baseline measurements. The goal of this study is to define and validate such a method.

Factors affecting transplant outcomes in diabetic nude mice receiving human, porcine, and nonhuman primate islets: analysis of 335 transplantations.

BACKGROUND: In the absence of a reliable islet potency assay, nude mice (NM) transplantation is the criterion standard to assess islet quality for clinical transplantation. There are factors other than islet quality that affect the transplant outcome. METHODS: Here, we analyzed the transplant outcomes in 335 NM receiving islets from human (n=103), porcine (n=205), and nonhuman primate (NHP; n=27) donors. The islets (750, 1000, and 2000 islet equivalents [IEQ]) were transplanted under the kidney capsule of streptozotocin-induced diabetic NM. RESULTS: The proportion of mice that achieved normoglycemia was significantly higher in the group implanted with 2000 IEQ of human, porcine, or NHP islets (75% normoglycemic) versus groups that were implanted with 750 IEQ (7% normoglycemic) and 1000 IEQ (30% normoglycemic). In this study, we observed that the purity of porcine islet preparations (P≤0.001), islet pellet size in porcine preparations (P≤ 0.01), and mice recipient body weight for human islet preparations (P=0.013) were independently associated with successful transplant outcome. NHP islets of 1000 IEQ were sufficient to achieve normoglycemic condition (83%). An islet mass of 2000 IEQ, high islet purity, increased recipient body weight, and high islet pellet volume increased the likelihood of successful reversal of diabetes in transplanted mice. Also, higher insulin secretory status of islets at basal stimulus was associated with a reduced mouse cure rate. The cumulative incidence of graft failure was significantly greater in human islets (56.12%) compared with porcine islets (35.57%; P≤0.001). CONCLUSION: Factors affecting NM bioassay were identified (islet mass, islet purity, pellet size, in vitro insulin secretory capability, and mouse recipient body weight) and should be considered when evaluating islet function.

A new enzyme mixture to increase the yield and transplant rate of autologous and allogeneic human islet products.

BACKGROUND: The optimal enzyme blend that maximizes human islet yield for transplantation remains to be determined. In this study, we evaluated eight different enzyme combinations (ECs) in an attempt to improve islet yield. The ECs consisted of purified, intact or truncated class 1 (C1) and class 2 (C2) collagenases from Clostridium histolyticum (Ch), and neutral protease (NP) from Bacillus thermoproteolyticus rokko (thermolysin) or Ch (ChNP). METHODS: We report the results of 249 human islet isolations, including 99 deceased donors (research n=57, clinical n=42) and 150 chronic pancreatitis pancreases. We prepared a new enzyme mixture (NEM) composed of intact C1 and C2 collagenases and ChNP in place of thermolysin. The NEM was first tested in split pancreas (n=5) experiments and then used for islet autologous (n=21) and allogeneic transplantation (n=10). Islet isolation outcomes from eight different ECs were statistically compared using multivariate analysis. RESULTS: The NEM consistently achieved higher islet yields from pancreatitis (P<0.003) and deceased donor pancreases (P<0.001) than other standard ECs. Using the NEM, islet products met release criteria for transplantation from 8 of 10 consecutive pancreases, averaging 6510 ± 2150 islet equivalent number/gram (IEQ/g) pancreas and 694,681 ± 147,356 total IEQ/transplantation. In autologous isolation, the NEM yielded more than 200,000 IEQ from 19 of 21 pancreases (averaging 422,893 ± 181,329 total IEQ and 5979 ± 1469 IEQ/kg recipient body weight) regardless of the severity of fibrosis. CONCLUSIONS: A NEM composed of ChNP with CIzyme high intact C1 collagenase recovers higher islet yield from deceased and pancreatitis pancreases while retaining islet quality and function.

Insulin degradation by acinar cell proteases creates a dysfunctional environment for human islets before/after transplantation: benefits of α-1 antitrypsin treatment.

BACKGROUND: Pancreatic acinar cells are commonly cotransplanted along with islets during auto- and allotransplantations. The aims of this study were to identify how acinar cell proteases cause human islet cell loss before and after transplantation of impure islet preparations and to prevent islet loss and improve function with supplementation of α-1 antitrypsin (A1AT). METHODS: Acinar cell protease activity, insulin levels, and percent islet loss were measured after culture of pure and impure clinical islet preparations. The effect of proteases on ultrastructure of islets and ß-cell insulin granules were examined by transmission electron microscopy. The number of insulin granules and insulin-labeled immunogold particles were counted. The in vivo effect of proteases on islet function was studied by transplanting acinar cells adjacent to islet grafts in diabetic mice. The effects of A1AT culture supplementation on protease activity, insulin levels, and islet function were assessed in pure and impure islets. RESULTS: Islet loss after culture was significantly higher in impure relative to pure preparations (30% vs. 14%, P<0.04). Lower islet purity was associated with increased protease activity and decreased insulin levels in culture supernatants. Reduced ß-cell insulin granules and insulin degradation by proteases were confirmed by transmission electron microscopy. Transplantations in mice showed delayed islet graft function when acinar cells were transplanted adjacent to the islets under the kidney capsule. Supplementation of A1AT to impure islet cultures maintained islet cell mass, restored insulin levels, and preserved islet functional integrity. CONCLUSION: Culture of impure human islet fractions in the presence of A1AT prevents insulin degradation and improves islet recovery.

Prediction of pancreatic tissue densities by an analytical test gradient system before purification maximizes human islet recovery for islet autotransplantation/allotransplantation.

BACKGROUND: Using standard density gradient (SDG) ranges for human islet purification frequently results in islet loss and transplantation of lower islet mass. Measuring the densities of islet and acinar tissue beforehand to customize the gradient range for the actual COBE 2991 cell processor (COBE) purification is likely to maximize the recovery of islets. We developed an analytical test gradient system (ATGS) for predicting pancreatic tissue densities before COBE purification to minimize islet loss during purification. METHODS: Human islets were isolated from deceased donor (n=30) and chronic pancreatitis pancreata (n=30). Pancreatic tissue densities were measured before purification by the ATGS, and the density gradient range for islet purification in a COBE was customized based on density profiles determined by the ATGS. The efficiency of custom density gradients (CDGs) to recover high islet yield was compared with predefined SDGs. RESULTS: Pancreatic tissue densities from autografts were significantly higher than in allograft preparations. In allograft purifications, a higher proportion of islets were recovered using ATGS-guided CDGs (85.9%±18.0%) compared with the SDG method (69.2%±27.0%; P=0.048). Acinar contamination at 60%, 70%, and 80% cumulative islet yield for allografts was significantly lower in the CDG group. In autograft purifications, more islets were recovered with CDGs (81.9%±28.0%) than SDGs (55.8%±22.8%; P=0.03). CDGs effectively reduced islet loss by minimizing islet sedimentation in the COBE bag. CONCLUSIONS: Using ATGS-guided CDGs maximizes the islet recovery for successful transplantations by reducing acinar contamination in allograft preparations and by reducing sedimentation of islets in the COBE bag in autograft preparations.