Single-Molecule Analysis of Genome Uncoating from Individual Human Rhinovirus Particles, and Modulation by Antiviral Drugs.

Infection of humans by many viruses is typically initiated by the internalization of a single virion in each of a few susceptible cells. Thus, the outcome of the infection process may depend on stochastic single-molecule events. A crucial process for viral infection, and thus a target for developing antiviral drugs, is the uncoating of the viral genome. Here a force spectroscopy procedure using an atomic force microscope is implemented to study uncoating for individual human rhinovirus particles. Application of an increasing mechanical force on a virion led to a high force-induced structural transition that facilitated extrusion of the viral RNA molecule without loss of capsid integrity. Application of force to virions that h ad previously extruded the RNA, or to RNA-free capsids, led to a lower force-induced event associated with capsid disruption. The kinetic parameters are determined for each reaction. The high-force event is a stochastic process governed by a moderate free energy barrier (≈20 kcal mol-1 ), which results in a heterogeneous population of structurally weakened virions in which different fractions of the RNA molecule are externalized. The effects of antiviral compounds or capsid mutation on the kinetics of this reaction reveal a correlation between the reaction rate and virus infectivity.

Electrostatic Screening, Acidic pH and Macromolecular Crowding Increase the Self-Assembly Efficiency of the Minute Virus of Mice Capsid In Vitro.

The hollow protein capsids from a number of different viruses are being considered for multiple biomedical or nanotechnological applications. In order to improve the applied potential of a given viral capsid as a nanocarrier or nanocontainer, specific conditions must be found for achieving its faithful and efficient assembly in vitro. The small size, adequate physical properties and specialized biological functions of the capsids of parvoviruses such as the minute virus of mice (MVM) make them excellent choices as nanocarriers and nanocontainers. In this study we analyzed the effects of protein concentration, macromolecular crowding, temperature, pH, ionic strength, or a combination of some of those variables on the fidelity and efficiency of self-assembly of the MVM capsid in vitro. The results revealed that the in vitro reassembly of the MVM capsid is an efficient and faithful process. Under some conditions, up to ~40% of the starting virus capsids were reassembled in vitro as free, non aggregated, correctly assembled particles. These results open up the possibility of encapsidating different compounds in VP2-only capsids of MVM during its reassembly in vitro, and encourage the use of virus-like particles of MVM as nanocontainers.

Molecular Determinants of Human Rhinovirus Infection, Assembly, and Conformational Stability at Capsid Protein Interfaces.

Human rhinovirus (HRV), one of the most frequent human pathogens, is the major causative agent of common colds. HRVs also cause or exacerbate severe respiratory diseases, such as asthma or chronic obstructive pulmonary disease. Despite the biomedical and socioeconomic importance of this virus, no anti-HRV vaccines or drugs are available yet. Protein-protein interfaces in virus capsids have increasingly been recognized as promising virus-specific targets for the development of antiviral drugs. However, the specific structural elements and residues responsible for the biological functions of these extended capsid regions are largely unknown. In this study, we performed a thorough mutational analysis to determine which particular residues along the capsid interpentamer interfaces are relevant to HRV infection as well as the stage(s) in the viral cycle in which they are involved. The effect on the virion infectivity of the individual mutation to alanine of 32 interfacial residues that, together, removed most of the interpentamer interactions was analyzed. Then, a representative sample that included many of those 32 single mutants were tested for capsid and virion assembly as well as virion conformational stability. The results indicate that most of the interfacial residues, and the interactions they establish, are biologically relevant, largely because of their important roles in virion assembly and/or stability. The HRV interpentamer interface is revealed as an atypical protein-protein interface, in which infectivity-determining residues are distributed at a high density along the entire interface. Implications for a better understanding of the relationship between the molecular structure and function of HRV and the development of novel capsid interface-binding anti-HRV agents are discussed. IMPORTANCE The rising concern about the serious medical and socioeconomic consequences of respiratory infections by HRV has elicited a renewed interest in the development of anti-HRV drugs. The conversion into effective drugs of compounds identified via screening, as well as antiviral drug design, rely on the acquisition of fundamental knowledge about the targeted viral elements and their roles during specific steps of the infectious cycle. The results of this study provide a detailed view on structure-function relationships in a viral capsid protein-protein interface, a promising specific target for antiviral intervention. The high density and scattering of the interfacial residues found to be involved in HRV assembly and/or stability support the possibility that any compound designed to bind any particular site at the interface will inhibit infection by interfering with virion morphogenesis or stabilization of the functional virion conformation.

Optimization and validation of a chiral CE-LIF method for quantitation of aspartate, glutamate and serine in murine osteocytic and osteoblastic cells.

Asp, Glu, and D-Ser are chiral amino acids and neurotransmitters binding to the N-methyl-D-aspartate receptor (NMDA) and they participate in glutamate signalization. D-amino acids are increasingly being recognized as important signaling molecules and variations in their levels are considered a marker of different pathologies, however, there is still a lack of knowledge about the role of most of D-amino acids in living organisms such as bone cells. A method for determination of concentrations of L/D-Asp, L/D-Glu and L/D-Ser in two types of bone cell lines: murine osteocytes (MLOY4) and osteoblasts (MC3T3-E1) is presented. It is based on capillary electrophoresis coupled to laser-induced fluorescence detection in normal polarity with 4-fluoro-7-nitro-2,1,3-benzoxadiazole as derivatizing agent suitable for an Argon ion laser source. The electrolyte consists of 137.5 mM borate buffer and 12.5 mM ß-cyclodextrins as chiral selectors and the separation lasts 25 min. The method was optimized and validated for specificity, sensitivity, linearity, accuracy, and precision in murine osteocytes and osteoblasts. LLOQ was 0.25 µmol L-1 for the three D-amino acids and linearity was confirmed with r > 0.995 for all D-and L-amino acids. Accuracy ranged between 81.9% and 111.7% and intra-day precision ranged between 1.8% and 10.9%. Concentrations of D- and L- Asp, Glu, and Ser are given and statistical differences between osteocytes and osteoblasts were found. The highest differences corresponded to L- and D-Glu. This method could play a fundamental role in the study of therapeutic targets in the treatment of bone diseases.

Pancreatic human islets and insulin-producing cells derived from embryonic stem cells are rapidly identified by a newly developed Dithizone.

We developed an optimized Dipheylthiocarbazone or Dithizone (DTZ) with improved physical and chemical properties to characterize human islets and insulin-producing cells differentiated from embryonic stem cells. Application of the newly formulated iDTZ (i stands for islet) over a range of temperatures, time intervals and cell and tissue types found it to be robust for identifying these cells. Through high transition zinc binding, the iDTZ compound concentrated in insulin-producing cells and proved effective at delineating zinc levels in vitro.

Ovarian Hormone-Dependent Effects of Dietary Lipids on APP/PS1 Mouse Brain.

The formation of senile plaques through amyloid-ß peptide (Aß) aggregation is a hallmark of Alzheimer's disease (AD). Irrespective of its actual role in the synaptic alterations and cognitive impairment associated with AD, different therapeutic approaches have been proposed to reduce plaque formation. In rodents, daily intake of omega-3 (n-3) long-chain polyunsaturated fatty acids (LC-PUFAs) is required for neural development, and there is experimental and epidemiological evidence that their inclusion in the diet has positive effects on several neurodegenerative diseases. Similarly, estradiol appears to reduce senile plaque formation in primary mouse cell cultures, human cortical neurons and mouse AD models, and it prevents Aß toxicity in neural cell lines. We previously showed that differences in dietary n-6/n-3 LC-PUFAs ratios modify the lipid composition in the cerebral cortex of female mice and the levels of amyloid precursor protein (APP) in the brain. These effects depended in part on the presence of circulating estradiol. Here we explored whether this potentially synergistic action between diet and ovarian hormones may influence the progression of amyloidosis in an AD mouse model. Our results show that a diet with high n-3 LC-PUFA content, especially DHA (22:6n-3), reduces the hippocampal accumulation of Aß1 - 4 0, but not amyloid Aß1 - 42 in female APPswe/PS1 E9A mice, an effect that was counteracted by the loss of the ovaries and that depended on circulating estradiol. In addition, this interaction between dietary lipids and ovarian function also affects the composition of the brain lipidome as well as the expression of certain neuronal signaling and synaptic proteins. These findings provide new insights into how ovarian hormones and dietary composition affect the brain lipidome and amyloid burden. Furthermore, they strongly suggest that when designing dietary or pharmacological strategies to combat human neurodegenerative diseases, hormonal and metabolic status should be specifically taken into consideration as it may affect the therapeutic response.

Ovarian Function Modulates the Effects of Long-Chain Polyunsaturated Fatty Acids on the Mouse Cerebral Cortex.

Different dietary ratios of n-6/n-3 long-chain polyunsaturated fatty acids (LC-PUFAs) may alter brain lipid profile, neural activity, and brain cognitive function. To determine whether ovarian hormones influence the effect of diet on the brain, ovariectomized and sham-operated mice continuously treated with placebo or estradiol were fed for 3 months with diets containing low or high n-6/n-3 LC-PUFA ratios. The fatty acid (FA) profile and expression of key neuronal proteins were analyzed in the cerebral cortex, with intact female mice on standard diet serving as internal controls of brain lipidome composition. Diets containing different concentrations of LC-PUFAs greatly modified total FAs, sphingolipids, and gangliosides in the cerebral cortex. Some of these changes were dependent on ovarian hormones, as they were not detected in ovariectomized animals, and in the case of complex lipids, the effect of ovariectomy was partially or totally reversed by continuous administration of estradiol. However, even though differential dietary LC-PUFA content modified the expression of neuronal proteins such as synapsin and its phosphorylation level, PSD-95, amyloid precursor protein (APP), or glial proteins such as glial fibrillary acidic protein (GFAP), an effect also dependent on the presence of the ovary, chronic estradiol treatment was unable to revert the dietary effects on brain cortex synaptic proteins. These results suggest that, in addition to stable estradiol levels, other ovarian hormones such as progesterone and/or cyclic ovarian secretory activity could play a physiological role in the modulation of dietary LC-PUFAs on the cerebral cortex, which may have clinical implications for post-menopausal women on diets enriched with different proportions of n-3 and n-6 LC-PUFAs.

Evaluation of collagenase gold plus BP protease in isolating islets from human pancreata.

Selection of enzymes for optimal pancreas digestion is essential for successful human islet isolations. The aim of this study was to evaluate the efficacy and outcome of using Collagenase Gold plus BP protease (VitaCyte) (n = 8) by comparing it to two commercially available enzymes, Liberase MTF C/T (Roche) (n = 48) and Collagenase NB1/NP (Serva) (n = 15). The isolation outcomes were assessed by islet counting, viability, glucose-stimulated oxygen consumption rate (OCR), and successful graft-rate following transplantation in diabetic NOD scid mice. The pancreas donor characteristics were not significantly different between the tested enzyme groups regarding their BMI, pancreas weight, cold ischemia time (CIT) and HbA1c. The results show that digested tissue volume was not statistically significant between the VitaCyte enzyme (34.25 ± 5.4 mL) and the Roche enzyme (55.25 ± 3.42 mL, p = 0.073), however, this was significant with Serva enzyme (64.07 ± 7.95 mL, p = 0.020). Interestingly, the islet yields were not statistically different between all enzyme groups. Moreover, when islets were transplanted into NOD scid mice, the reversal rate of diabetes for the VitaCyte enzyme group was similar to all enzyme groups. In conclusion, the effectiveness of Collagenase Gold plus BP protease is comparable to the MTF C/T and the Collagenase NB1/NP enzymes; the low cost could facilitate the use of more pancreata for islet isolations.

Correlation of Blood Pressure Variability as Measured By Clinic, Self-measurement at Home, and Ambulatory Blood Pressure Monitoring.

BACKGROUND: Blood pressure variability (BPV) has been postulated as a potential predictor of cardiovascular outcomes. No agreement exists as to which measurement method is best for BPV estimation. We attempt to assess the correlation between BPV obtained at the doctor's office, self-measurement at home (SMBP) and ambulatory BP monitoring (ABPM). METHODS: Eight weekly clinic BP measurements, 2 SMBP series, and 1 24-hour ABPM recording were carried out in a sample of treated hypertensive patients. BPV was calculated using the SD, the "coefficient of variation" and the "average real variability." Determinants of short-, mid-, and long-term BPV (within each measurement method) were also calculated. The different BPV determinants were correlated "intramethod" and "intermethod" by linear regression test. RESULTS: For the 104 patients (66.5 ± 7.7 years, 58.7% males), the ABPM BPV (SD, systolic/diastolic: 14.5 ± 3.1/9.8 ± 2.5 mm Hg) was higher than the SMBP (12.2 ± 9.8/7.4 ± 5.8 mm Hg; P < 0.001) and clinic BPV (10 ± 8.9/5.9 ± 4.9 mm Hg; P = 0.001). The main BPV correlation between methods was weak, with a maximum R2 = 0.17 (P < 0.001) between clinic and SMBP systolic BPV. The "intramethod" correlation of BPV yielded a maximum R2 = 0.21 (P < 0.001) between morning diastolic SMBP intershift/intermeans variability. The "intermethod" correlation of short-, mid-, and long-term BPV determinants was weak (maximum R2 = 0.22, P < 0.001, between clinic intraday variability/SMBP morning intershift variability). CONCLUSIONS: The "intramethod" and "intermethod" correlation between BPV determinants was weak or nonexistent, even when comparing determinants reflecting the same type of temporal BPV. Our data suggest that BPV reflects a heterogeneous phenomenon that strongly depends on the estimation method and the time period evaluated.

Influence of renal function on anticoagulation control in patients with non-valvular atrial fibrillation taking vitamin K antagonists.

BACKGROUND AND PURPOSE: Chronic kidney disease (CKD) has been related to poor anticoagulation control and an increased risk of bleeding. This study aims to evaluate the association between impaired renal function (eGFR <60 mL/min/1.73 m2 ) and anticoagulation control in patients with non-valvular atrial fibrillation (AF) on vitamin K antagonists (VKA) therapy. We also assessed whether the predictive value of the SAMe-TT2 R2 score prevailed for subgroups both with and without CKD. METHODS: This is an ancillary analysis of 1381 patients from the PAULA study, which was a cross-sectional, retrospective and nationwide multicenter study. RESULTS: A total of 370 patients had eGFR <60 mL/min/1.73 m2 . Anticoagulation control levels progressively worsened across each stage of CKD. Multiple linear regression analysis showed CKD as an independent predictor of time in therapeutic range (TTR). In the subgroup of patients with preserved renal function, female sex, diet affecting INR, polypharmacy and amiodarone were associated with poorer TTR. The SAMe-TT2 R2 score had a significant but modest predictive value for TTR<65% (AUC, area under the curve 0.558, P = .002). In the subgroup of patients with CKD, the SAMe-TT2 R2 (>2 points) showed no significant predictive capacity for TTR (AUC 0.528, P = .354). The average TTR was similar for both sexes (P = .255), but with a higher percentage of males subjects with TTR ≥65% (P = .013). CONCLUSION: Chronic kidney disease is associated with poor anticoagulation control in patients with non-valvular AF taking VKA. The SAMe-TT2 R2 score was not predictive of poor TTR in the subgroup with CKD, although a modest predictive value for poor TTR was found in those without CKD.

Blood pressure control in anticoagulated patients with hypertension and atrial fibrillation.

OBJECTIVE: To determine the clinical profile and blood pressure (BP) control rates of anticoagulated patients with hypertension and atrial fibrillation (AF). METHODS: The PAULA study was a multicenter cross-sectional/retrospective observational study conducted throughout Spain. The study included patients with nonvalvular AF who were receiving vitamin K antagonist therapy during the past year and were attended at primary care setting. Adequate BP control was defined according to 2013 ESC/ESH guidelines. RESULTS: A total of 1,222 hypertensive patients were included (mean age 77.9 ± 8.3 years; 51.2% women; CHADS2 2.5 ± 1.1; CHA2DS2-VASc 4.2 ± 1.4; HAS-BLED 1.6 ± 0.9). 33.5% of patients had diabetes, 24.9% heart failure and 14.0% prior stroke/transient ischemic attack. Mean BP was 131.4 ± 14.5/74.9 ± 9.8 mm Hg. With regard to antihypertensive treatment, most of patients were on combined therapy (67.9%). The commonest prescribed antihypertensive drugs were diuretics (64.4%), followed by angiotensin receptor blockers (30.1%), and beta blockers (29.4%). 75.2% of hypertensive patients achieved BP control targets; 86.6% of patients ≥80 years and 67.6% of diabetics. CONCLUSIONS: More than 75% of hypertensive patients with AF achieved BP goals, and this rate was higher in elderly. More than 2 thirds of patients were on combined therapy. BP control appears to be better in AF patients than in general hypertensive population.

Effectiveness of dry needling for chronic nonspecific neck pain: a randomized, single-blinded, clinical trial.

Chronic neck pain attributed to a myofascial pain syndrome is characterized by the presence of muscle contractures referred to as myofascial trigger points. In this randomized, parallel-group, blinded, controlled clinical trial, we examined the effectiveness of deep dry needling (DDN) of myofascial trigger points in people with chronic nonspecific neck pain. The study was conducted at a public Primary Health Care Centre in Madrid, Spain, from January 2010 to December 2014. A total of 130 participants with nonspecific neck pain presenting with active myofascial trigger points in their cervical muscles were included. These participants were randomly allocated to receive: DDN plus stretching (n = 65) or stretching only (control group [n = 65]). Four sessions of treatment were applied over 2 weeks with a 6-month follow-up after treatment. Pain intensity, mechanical hyperalgesia, neck active range of motion, neck muscle strength, and perceived neck disability were measured at baseline, after 2 sessions of intervention, after the intervention period, and 15, 30, 90, and 180 days after the intervention. Significant and clinically relevant differences were found in favour of dry needling in all the outcomes (all P < 0.001) at both short and long follow-ups. Deep dry needling and passive stretching is more effective than passive stretching alone in people with nonspecific neck pain. The results support the use of DDN in the management of myofascial pain syndrome in people with chronic nonspecific neck pain.

Involvement of a proapoptotic gene (BBC3) in islet injury mediated by cold preservation and rewarming.

Long-term pancreatic cold ischemia contributes to decreased islet number and viability after isolation and culture, leading to poor islet transplantation outcome in patients with type 1 diabetes. In this study, we examined mechanisms of pancreatic cold preservation and rewarming-induced injury by interrogating the proapoptotic gene BBC3/Bbc3, also known as Puma (p53 upregulated modulator of apoptosis), using three experimental models: 1) bioluminescence imaging of isolated luciferase-transgenic ("Firefly") Lewis rat islets, 2) cold preservation of en bloc-harvested pancreata from Bbc3-knockout (KO) mice, and 3) cold preservation and rewarming of human pancreata and isolated islets. Cold preservation-mediated islet injury occurred during rewarming in "Firefly" islets. Silencing Bbc3 by transfecting Bbc3 siRNA into islets in vitro prior to cold preservation improved postpreservation mitochondrial viability. Cold preservation resulted in decreased postisolation islet yield in both wild-type and Bbc3 KO pancreata. However, after culture, the islet viability was significantly higher in Bbc3-KO islets, suggesting that different mechanisms are involved in islet damage/loss during isolation and culture. Furthermore, Bbc3-KO islets from cold-preserved pancreata showed reduced HMGB1 (high-mobility group box 1 protein) expression and decreased levels of 4-hydroxynonenal (4-HNE) protein adducts, which was indicative of reduced oxidative stress. During human islet isolation, BBC3 protein was upregulated in digested tissue from cold-preserved pancreata. Hypoxia in cold preservation increased BBC3 mRNA and protein in isolated human islets after rewarming in culture and reduced islet viability. These results demonstrated the involvement of BBC3/Bbc3 in cold preservation/rewarming-mediated islet injury, possibly through modulating HMGB1- and oxidative stress-mediated injury to islets.

Evaluation of SAMe-TT2R2 score and other clinical factors influencing the quality of anticoagulation therapy in non-valvular atrial fibrillation: a nationwide study in Spain.

OBJECTIVE: To assess the major clinical factors affecting the quality of anticoagulation and evaluate the predictive value of the SAMe-TT2R2 score to identify patients who will achieve a high average time in therapeutic range (T.T.R.) with vitamin K antagonist (V.K.A.) treatment. RESEARCH DESIGN AND METHODS: This observational, cross-sectional, retrospective and nationwide multicenter study included 1524 patients from the primary care setting with non-valvular atrial fibrillation receiving V.K.A. (≥12 months). We performed a bivariate analysis to identify factors individually associated with the T.T.R. and a multiple regression analysis to identify the independent predictive factors. For the validation of the SAMe-TT2R2 score, the receiver operating characteristic (R.O.C.) curve was calculated and the Hosmer-Lemeshow test was used to test calibration. RESULTS: A total of 94.8% of patients received acenocumarol (4.8% warfarin). A progressive decrease in mean T.T.R. was found when the SAMe-TT2R2 score increased from 0 points (72.1 ± 17.1%) to 4 points (64.1 ± 23.2%), p < 0.001. Other risk scores (CHADS2 and CHA2DS2-VASc, HAS-BLED) were also associated with the mean T.T.R. We found a significant association between low T.T.R. and the following clinical factors: female sex, three or more comorbidities, amiodarone treatment, dietary habits, bleeding history and the intake of ≥7 tablets per day besides V.K.A. (p < 0.01). Regarding SAMe-TT2R2 score validation, the R.O.C. curve showed significant capability, although not high, of discriminating good anticoagulation control (T.T.R. ≥65%) with an area under the curve of 0.562 (95% C.I. 0.533-0.592, p < 0.001) which increased, remaining modest, to 0.594 (95% C.I. 0.564-0.624, p < 0.001) when the factors not included in SAMe-TT2R2 score were added. CONCLUSION: In this cohort, the SAMe-TT2R2 score had a significant, although modest, ability to assess the likelihood of good international normalized ration (I.N.R.) control, and its predictive value might slightly improve by adding other simple clinical factors. Further research is needed to refine the predictive scales.

Prophylactically Decontaminating Human Islet Product for Safe Clinical Application: Effective and Potent Method.

BACKGROUND: Transplanting pancreatic islets into recipients must be safe and effective to treat Type 1 diabetes. Islet quality and quantity are important, however, the final product must also be free from microbial contamination and low endotoxin levels. METHODS: This study explored a method to eliminate contamination in manufacturing islets for transplantation. A simple (single antibiotic, n=164) and refined (triple antimicrobial agents, n=279) pancreas decontaminating methods were used to test their effects on reducing the contamination rates in the islet final product. A total of 443 pancreata were processed for islet isolations. Three samples for microbial tests (Gram stain, aerobic, and anaerobic culture) were taken at pre-process (pancreas preservation), post-isolation, and post-culture. Endotoxin levels were measured only for islets considered for transplantation. RESULTS: Out of 443 pancreata used for islet isolation, 79 (17.8%) showed signs of contamination in pre-process samples; 10 (2.3%) were contaminated in both pre-process and in the final product (post-isolation and post-culture) samples. Contamination rates in which pre-process and final product samples were positive for contamination was significantly lower using the refined method (refined vs. simple method: 5% vs. 20.5%, p=0.045). Identical microbial species were present in both pre-process and in the final product. CONCLUSIONS: This study demonstrated that the refined method reduces the rate of contamination of the islet final product and is safe for clinical application. Moreover, it may be used as a standard method during human islet manufacturing facilitating the application of a biological license agreement from United States Food and Drug Administration.

The Choice of Enzyme for Human Pancreas Digestion is a Critical Factor for Increasing the Success of Islet Isolation.

BACKGROUND: We evaluated three commercially available enzymes for pancreatic digestion by comparing key parameters during the islet isolation process, as well as islet quality post-isolation. METHODS: Retrospectively compared and analyzed islet isolations from pancreata using three different enzyme groups: Liberase HI (n=63), Collagenase NB1/Neutral Protease (NP) (n=43), and Liberase Mammalian Tissue Free Collagenase/Thermolysin (MTF C/T) (n=115). A standardized islet isolation and purification method was used. Islet quality assessment was carried out using islet count, viability, in vitro glucose-stimulated insulin secretion (GSIS), glucose-stimulated oxygen consumption rate (ΔOCR), and in vivo transplantation model in mice. RESULTS: Donor characteristics were not significantly different among the three enzyme groups used in terms of age, sex, hospital stay duration, cause of death, body mass index (BMI), hemoglobin A1c (HbA1c), cold ischemia time (CIT), and pancreas weight. Digestion efficacy (percentage of digested tissue by weight) was significantly higher in the Liberase MTF C/T group (73.5 ± 1.5 %) when compared to the Liberase HI group (63.6 ± 2.3 %) (p<0.001) and the Collagenase NB1/NP group (61.7 ± 2.9%) (p<0.001). The stimulation index for GSIS was significantly higher in the Liberase MTF C/T group (5.3 ± 0.5) as compared to the Liberase HI (2.9 ± 0.2) (p<0.0001) and the Collagenase NB1/NP (3.6 ± 2.9) (p=0.012) groups. Furthermore, the Liberase MTF C/T enzymes showed the highest success rate of transplantation in diabetic NOD Scid mice (65%), which was significantly higher than the Liberase HI (42%, p=0.001) and the Collagenase NB1/NP enzymes (41%, p<0.001). CONCLUSIONS: Liberase MTF C/T is superior to Liberase HI and Collagenase NB1/NP in terms of digestion efficacy and glucose-stimulated insulin secretion in vitro. Moreover, Liberase MTF C/T had a significantly higher success rate of transplantation in diabetic NOD Scid mice compared to Liberase HI and Collagenase NB1/NP enzymes.

Human Pancreatic Islets Isolated From Donors With Elevated HbA1c Levels: Islet Yield and Graft Efficacy.

UNLABELLED: The aim of this study was to investigate the effects of elevated donor HbA1c levels (type 2 diabetes, T2D) on the islet yield and functionality postisolation. In this retrospective analysis, donors for islet isolations were classified into two groups: T2D group (HbA1c ≥ 6.5%, n = 18) and normal group (HbA1c < 6.5%, n = 308). Optimum pancreas digestion time (switch time) was significantly higher in the T2D group compared to the normal group (13.7 ± 1.2 vs. 11.7 ± 0.1 min, respectively, p = 0.005). Islet yields were significantly lower in the T2D group compared to the control (T2D vs. control): islet equivalent (IEQ)/g (prepurification 2,318 ± 195 vs. 3,713 ± 114, p = 0.003; postpurification 1,735 ± 175 vs. 2,663 ± 89, p = 0.013) and islet particle number (IPN)/g (prepurification, 2,519 ± 336 vs. 4,433 ± 143, p = 0.001; postpurification, 1,760 ± 229 vs. 2,715 ± 85, p = 0.007). Islets from T2D pancreata had significantly lower viability (T2D vs. CONTROL: 91.9 ± 1.6 vs. 94.4 ± 0.3%, p = 0.004) and decreased oxygen consumption rate (ΔOCR) (T2D vs. CONTROL: 0.09 ± 0.01 and 0.21 ± 0.03 nmol O2 100 islets(-1) min(-1), p = 0.049). The islets isolated from T2D donor pancreata reversed diabetes in NOD-SCID mice in 9% (2/22) compared to islets from control donor pancreata, which reversed diabetes in 67% (175/260, p < 0.001). In conclusion, this study demonstrates that elevated HbA1c (≥ 6.5%) is associated with impairment of islet function and lower islet yield; however, these islets could not be suitable for clinical applications.

The effects of digestion enzymes on islet viability and cellular composition.

The choice of enzyme blend is critical for successful islet isolation. Islet yield, viability, integrity, and function are important factors that influence the outcome of islet transplantation. Liberase HI has been used as a standard enzyme for pancreas digestion and has successfully produced islets that reversed diabetes. However, the replacement of Liberase HI with collagenase NB1 has significantly influenced the process outcome, both in quality and quantity of the isolated islets. The assessment of islet cells by Flow Cytometry (FC) has been reported to be useful for evaluating islet quality. The aim of this study was to assess the isolation outcomes and islet quality when comparing human islet cell processed with Liberase HI and NB1. A total of 66 islet isolations, 46 processed using Liberase HI and 20 using Serva NB1, were retrospectively analyzed. Islet yield, function in vitro, islet cell viability by FC, as well as isolation-related factors were compared. There was no significant difference in donor characteristics such as age and height; however, body mass index (BMI) in the Liberase HI group was significantly higher. There was also no significant difference in prepurification, postisolation, or postculture IEQ or percent recovery between the two groups. Flow data showed Liberase HI preparations had a significantly higher percent of live cells (DAPI(-)) and NG(+)/TMRE(+) when compared to NB1. Stimulation Indices (SI) for Liberase HI (n = 45) showed 3.17 and NB1 (n = 18) 2.71 (p = NS). The results of Annexin V/DAPI staining for live, apoptotic, and necrotic cells were 50.7 ± 2.24%, 14.4 ± 1.02%, and 27.8 ± 1.92% for Liberase HI versus 48.1 ± 1.93%, 12.3 ± 0.92%, and 33.9 ± 2.28% for NB1. Islets isolated using Liberase HI showed higher viable ß cells by NG/TMRE staining and decreased necrosis by Annexin V/DAPI staining. FC assessment may be useful for determining the choice of digestion enzyme to maximize viable islets.

Quantitative assessment of ß-cell apoptosis and cell composition of isolated, undisrupted human islets by laser scanning cytometry.

BACKGROUND: Assays for assessing human islet cell quality, which provide results before transplantation, would be beneficial to improve the outcomes for islet transplantation therapy. Parameters such as percent ß-cell apoptosis and cell composition are found to vary markedly between different islet preparations and may serve as markers of islet quality. We have developed fluorescence-based assays using laser scanning cytometry for assessing ß-cell apoptosis and islet cell composition on serial sections of intact isolated islets. METHODS: Isolated human islets were fixed in formalin and embedded in paraffin. Serial sections were immunostained for the pancreatic hormones and acinar and ductal cell markers. DNA fragmentation was used to label apoptotic cells. Stained cells were quantified using an iCys laser scanning cytometer. RESULTS: Islet preparations from 102 human pancreatic islet isolations were analyzed. For the whole set of islet preparations, we found a mean islet cell composition of 54.5%±1.2% insulin-positive, 33.9%±1.2% glucagon, 12.1%±0.7% somatostatin, and 1.5%±0.2% pancreatic polypeptide-positive cells. The apoptotic ß cells were 2.85%±0.4% with a range of 0.27% to 18.3%. The percentage of apoptotic ß cells correlated well (P<0.0001, n=59) with results obtained in vivo by transplantation of the corresponding islets in diabetic NODscid mice. CONCLUSIONS: The analysis of whole, nondissociated islets for cell composition and ß-cell apoptosis using laser scanning cytometry gives reliable and reproducible results and could be performed both before islet transplantation and on preserved cell blocks at any time in future. Thus, they can be a powerful tool for islet quality assessment.