Vitiligo non-responding lesions to narrow band UVB have intriguing cellular and molecular abnormalities that may prevent epidermal repigmentation.

We have discovered that human vitiligo patients treated with narrow-band UVB (NBUVB) demonstrated localized resistance to repigmentation in skin sites characterized by distinct cellular and molecular pathways. Using immunostaining studies, discovery-stage RNA-Seq analysis, and confirmatory in situ hybridization, we analyzed paired biopsies collected from vitiligo lesions that did not repigment after 6 months of NBUVB treatment (non-responding) and compared them with repigmented (responding) lesions from the same patient. Non-responding lesions exhibited acanthotic epidermis, had low number of total, proliferative, and differentiated melanocyte (MC) populations, and increased number of senescent keratinocytes (KCs) and of cytotoxic CD8+ T cells as compared with responding lesions. The abnormal response in the non-responding lesions was driven by a dysregulated cAMP pathway and of upstream activator PDE4B, and of WNT/ß-catenin repigmentation pathway. Vitiligo-responding lesions expressed high levels of WNT10B ligand, a molecule that may prevent epidermal senescence induced by NBUVB, and that in cultured melanoblasts prevented the pro-melanogenic effect of α-MSH. Understanding the pathways that govern lack of NBUVB-induced vitiligo repigmentation has a great promise in guiding the development of new therapeutic strategies for vitiligo.

An ex vivo comparison of partial thromboplastin time and activated clotting time for heparin anticoagulation in an ovine model.

BACKGROUND: Sheep are a primary model of mechanical circulatory support (MCS) with heparin anticoagulation therapy frequently being monitored by activated clotting time (ACT) due to ease and cost. In patients undergoing long-term heparin therapy, other anticoagulation monitoring strategies, such as activated partial thromboplastin time (aPTT), have proven to be more reliable indicators for the adequacy of anticoagulation, frequently determined by heparin concentration. As there is a paucity of similar studies in sheep, we sought to investigate the correlation between heparin concentration and ACT and aPTT using whole sheep blood in an ex vivo model. METHODS: Fresh whole blood was serially drawn from an adult female Dorset-hybrid sheep and aliquots were placed into tubes containing heparin saline solutions with concentrations ranging from 0 to 7.81 U heparin per mL of whole blood. ACT and aPTT values were measured on each of the samples. The experiment was performed four times with the same animal. A simple linear regression was performed to determine correlation, and subgroup analysis was performed on low versus high heparin concentrations typically seen in human patients on long-term MCS, such as extracorporeal membrane oxygenation (ECMO), versus cardiopulmonary bypass, respectively. RESULTS: aPTT measurements versus the heparin concentration had an R2  = 0.7295. ACT measurements versus the heparin concentration had a R2  = 0.4628. aPTT measurements versus the ACT measurements had a R2  = 0.2974. The strength of the correlation between aPTT and heparin concentration increased at low heparin concentrations (R2  = 0.8392). CONCLUSION: aPTT had a more reliable correlation to heparin concentration and thus anticoagulation level than ACT. This was particularly true at lower heparin concentrations, similar to ranges seen for patients on ECMO. The correlation between aPTT and ACT values was poor. Further in vivo studies should be performed to confirm our results.

Neutrophil dysfunction due to continuous mechanical shear exposure in mechanically assisted circulation in vitro.

OBJECTIVE: Leukocytes play an important role in the body's immune system. The aim of this study was to assess alterations in neutrophil phenotype and function in pump-assisted circulation in vitro. METHODS: Human blood was circulated for four hours in three circulatory flow loops with a CentriMag blood pump operated at a flow of 4.5 L/min at three rotational speeds (2100, 2800, and 4000 rpm), against three pressure heads (75, 150, and 350 mm Hg), respectively. Blood samples were collected hourly for analyses of neutrophil activation state (Mac-1, CD62L, CD162), neutrophil reactive oxygen species (ROS) production, apoptosis, and neutrophil phagocytosis. RESULTS: Activated neutrophils indicated by both Mac-1 expression and decreased surface expression of CD62L and CD162 receptors increased with time in three loops. The highest level of neutrophil activation was observed in the loop with the highest rotational speed. Platelet-neutrophil aggregates (PNAs) progressively increased in two loops with lower rotational speeds. PNAs peaked at one hour after circulation and decreased subsequently in the loop with the highest rotational speed. Neutrophil ROS production dramatically increased at one hour after circulation and decreased subsequently in all three loops with similar levels and trends. Apoptotic neutrophils increased with time in all three loops. Neutrophil phagocytosis capacity in three loops initially elevated at one hour after circulation and decreased subsequently. Apoptosis and altered phagocytosis were dependent on rotational speed. CONCLUSIONS: Our study revealed that the pump-assisted circulation induced neutrophil activation, apoptosis, and functional impairment. The alterations were strongly associated with pump operating condition and duration.

Flow characteristics and hemolytic performance of the new Breethe centrifugal blood pump in comparison with the CentriMag and Rotaflow pumps.

Blood pumps have been increasingly used in mechanically assisted circulation for ventricular assistance and extracorporeal membrane oxygenation support or during cardiopulmonary bypass for cardiac surgery. However, there have always been common complications such as thrombosis, hemolysis, bleeding, and infection associated with current blood pumps in patients. The development of more biocompatible blood pumps still prevails during the past decades. As one of those newly developed pumps, the Breethe pump is a novel extracorporeal centrifugal blood pump with a hybrid magnetic and mechanical bearing with attempt to reduce device-induced blood trauma. To characterize the hydrodynamic and hemolytic performances of this novel pump and demonstrate its superior biocompatibility, we use a combined computational and experimental approach to compare the Breethe pump with the CentriMag and Rotaflow pumps in terms of flow features and hemolysis under an operating condition relevant to ECMO support (flow: 5 L/min, pressure head: ~350 mmHg). The computational results showed that the Breethe pump has a smaller area-averaged wall shear stress (WSS), a smaller volume with a scalar shear stress (SSS) level greater than 100 Pa and a lower device-generated hemolysis index compared to the CentriMag and Rotaflow pumps. The comparison of the calculated residence times among the three pumps indicated that the Breethe pump might have better washout. The experimental data from the in vitro hemolysis testing demonstrated that the Breethe pump has the lowest normalized hemolysis index (NIH) than the CentriMag and Rotaflow pumps. It can be concluded based on both the computational and experimental data that the Breethe pump is a viable pump for clinical use and it has better biocompatibility compared to the clinically accepted pumps.

Evaluation of an autoregulatory ECMO system for total respiratory support in an acute ovine model.

Extracorporeal membrane oxygenation (ECMO) has become a mainstay of therapy for patients suffering from severe respiratory failure. Ambulatory ECMO systems aim to provide long-term out-of-hospital respiratory support. As a patient's activity level changes, the required level of ECMO support varies with oxygen consumption and metabolic fluctuations. To compensate for such changes, an autoregulatory ECMO system (AR-ECMO) has been developed and its performance was evaluated as a proof of concept in an acute ovine model. The AR-ECMO system consists of a regular ECMO circuit and an electromechanical control system. A custom fuzzy logic control algorithm was implemented to adjust the blood flow and sweep gas flow of the ECMO circuit to meet the varying respiratory demand by utilizing two noninvasive sensors for venous oxyhemoglobin saturation and the oxygenator exhaust gas CO2 concentration. Disturbance responses of the AR-ECMO to induced acute respiratory distress were assessed for six hours in four juvenile sheep cannulated with a veno-pulmonary artery ECMO configuration, including acute ventilator shutoff, ventilator step change (off-on-off), and forced desaturation. All sheep survived for the study duration. The AR-ECMO system was able to respond and maintain stable hemodynamics and physiological blood gas contents (SpO2  = 96.3 % ± 4.29, pH 7.44 ± 0.09, pCO2  = 38.9 ± 9.9 mm Hg, and pO2 =237.9 ± 123.6 mm Hg) during simulated respiratory distress. Acceptable correlation between oxygenator exhaust gas CO2 and oxygenator outlet pCO2 were observed (R2  = 0.84). In summary, the AR-ECMO system successfully maintained physiologic control of peripheral oxygenation and carbon dioxide over the study period, utilizing only measurements taken directly from the ECMO circuit. The range of system response necessitates an adaptable system in the setting of variable metabolic demands. The ability of this system to respond to significant disturbances in ventilator support is encouraging. Future work to evaluate our AR-ECMO system in long-term, awake animal studies is necessary for further refinement.

Evaluation of in vitro hemolysis and platelet activation of a newly developed maglev LVAD and two clinically used LVADs with human blood.

In vitro hemolysis testing remains one of the most important performance measures to judge the hemocompatibility of a left ventricular assist device (LVAD). Clinically relevant operating conditions and appropriate testing blood are essential to infer in vitro data for potential clinical use. This in vitro study was carried out to evaluate and compare the hemolytic performance of a newly developed magnetically levitated (maglev) LVAD (CH-VAD) with two clinically used LVADs (HVAD and HeartMate II (HMII)) using fresh human blood. A small volume (~300 mL) in vitro circulating flow loop was constructed with a LVAD generated flow of 4.5 L/min at the nominal or reported clinical operating speed for each LVAD. The blood was circulated in the loop for 4 hours with samples drawn at baseline and hourly. Plasma-free hemoglobin (PFH) concentrations in the hourly blood samples were determined with spectrophotometry. Normalized index of hemolysis (NIH) was calculated to compare the hemolytic performance of the CH-VAD and the two reference LVADs. Platelet activation was measured with flow cytometry. The experimental test for each device was repeated at least 7 times. The data from this study showed that all the three LVADs generated very low hemolysis (NIH <0.01 g/100 L). The CH-VAD was found to have a significantly lower NIH value (0.00135 ± 0.00032 g/100 L) compared to the HVAD (0.00525 ± 0.00183 g/100 L) and the HMII (0.00583 ± 0.00182 g/100 L). No statistically significant difference in device-generated hemolysis was found between the HVAD and the HMII. The level of platelet activation induced by the CH-VAD is significantly lower than those by the HVAD and the HMII. The data suggest that the shear-induced hemolysis and platelet activation of the CH-VAD are acceptable relative to the two LVADs currently in clinical use.