Using hemoglobin vesicles to treat operative hemorrhagic shock after pneu- monectomy in dog models: an experimental study.

Hemoglobin vesicles (HbVs), considered as red blood cell substitutes, are liposomes encapsulating purified hemoglobin, with a phospholipid bilayer membrane (diameter: 250 nm; P50, 28 Torr). In this study, we aimed to investigate HbV function during hemorrhagic shock in lung resection and analyze the details of oxygen delivery. Left pneumonectomy was performed in dogs under mechanical ventilation, followed by rapid exsanguination of approximately 30% of the total circulating blood volume, which led to shock, reducing the mean arterial pressure (MAP) by approximately 60% of baseline. Subsequently, either 5% human serum albumin (HSA) or HbVs suspended in 5% HSA were infused for resuscitation. The MAP only recovered to 75% of baseline after HSA administration, but fully recovered (100%) after HbV administration, with significant differences between the groups (P < 0.005). Oxygen delivery was restored in the HbV group and was significantly higher than that in the HSA group (P < 0.0001). The infusion of HbVs dispersed in a 5% HSA solution compensated for the rapid loss of approximately 30% of the total circulating blood volume in a dog pneumonectomy model, even with impaired lung function. Thus, HbVs can be used for resuscitation from hemorrhagic shock during thoracic surgery.

Poly(2-ethyl-2-oxazoline)-Conjugated Hemoglobins as a Red Blood Cell Substitute.

Hemoglobin wrapped covalently with poly(2-ethyl-2-oxazoline)s (POx-Hb) is characterized physicochemically and physiologically as an artificial O2 carrier for use as a red blood cell (RBC) substitute. The POx-Hb is generated by linkage of porcine Hb surface-lysines to a sulfhydryl terminus of the POx derivative, with the average binding number of the polymers ascertained as 6. The POx-Hb shows moderately higher colloid osmotic activity and O2 affinity than the naked Hb. Human adult HbA conjugated with POx also possesses equivalent features and O2 binding properties. The POx-Hb solution exhibits good hemocompatibility, with no influence on the functions of platelets, granulocytes, and monocytes. Its circulation half-life in rats is 14 times longer than that of naked Hb. Hemorrhagic shock in rats is relieved sufficiently by infusion of the POx-Hb solution, as revealed by improvements of circulatory parameters. Serum biochemistry tests and histopathological observations indicate no acute toxicity or abnormality in the related organs. All results indicate that POx-Hb represents an attractive alternative for RBCs and a useful O2 therapeutic reagent in transfusion medicine.

Core-Shell Structured Hemoglobin Nanoparticles as Artificial O2 Carriers.

This paper describes the synthesis and O2 binding properties of core-shell structured hemoglobin (Hb) nanoparticles (NPs), artificial O2 carriers of five types, as designed for use as red blood cell (RBC) substitutes. Human adult Hbs were polymerized using α-succinimidyl-ω-maleimide and dithiothreitol in spheroidal shapes to create parent particles. Subsequent covalent wrapping of the sphere with human serum albumin (HSA) yielded 100 nm-diameter Hb nanoparticles (HbNPs). The HbNP showed higher O2 affinity than that of RBC, but NPs prepared under a N2 atmosphere exhibited low O2 affinity. Entirely synthetic particles comprising recombinant human adult Hb and recombinant HSA were also fabricated. Using a recombinant Hb (rHb) variant in which Leu-ß28 of the heme pocket had been replaced with Phe, we found somewhat low O2 affinity of rHb(ßL28F)NP. Particles made of stroma-free Hb (SFHb) containing natural antioxidant enzyme catalase (SFHbNP) formed a very stable O2 complex, even in aqueous H2O2 solution. The SFHbNP showed good blood compatibility and did not affect the blood cell component functionality. The circulation half-life of SFHbNP in rats was considerably longer than that of naked Hb. All results indicate these Hb-based NPs as useful alternative materials for RBC and as a useful O2 therapeutic reagent in diverse medical scenarios.

Early reperfusion with hemoglobin vesicles into tracheal subepithelial capillaries in a mouse tracheal transplant model.

Hemoglobin vesicles (HbVs), liposomes containing concentrated hemoglobin extracted from outdated human red blood cells (RBC), are artificial oxygen carriers with a small particle size. To evaluate the reperfusion of capillaries with HbVs in a tracheal transplant model and compare it with that of RBC. Isogenic mice were used as donors and recipients in a parallel trachea transplant model. Both ends of the donor trachea were anastomosed end-laterally to the recipient trachea to form in parallel. After transplantation, 0.3 mL of HbV solution (Hb concentration, 10 g/dL) was administered via the tail vein. The recipients were euthanized 1, 4, 6, and 8 h after surgery (n = 5 in each group). The tracheas were harvested, and tracheal subepithelial capillaries (SEC) reperfusion was histologically evaluated. A significant number of particles defined as HbV by electron microscopy were observed in the SEC of the grafted tracheas 4 h after the transplant surgery and HbV administration when no RBC were found in the SECs. The number increased 6 and 8 h later. Our findings suggest that HbVs, which are smaller than RBC, can reperfuse the capillaries of grafts earlier than RBCs after transplantation and contribute to the oxygenation of transplanted tissues.

Hemoglobin-albumin clusters as an artificial O2 carrier: Physicochemical properties and resuscitation from hemorrhagic shock in rats.

A bovine hemoglobin (HbBv) or human adult hemoglobin (HbA) wrapped covalently by human serum albumins (HSAs), hemoglobin-albumin clusters (HbBv-HSA3 and HbA-HSA3 ), are artificial O2 carriers used as a red blood cell substitute. This article describes the physicochemical properties of the HbBv-HSA3 and HbA-HSA3 solutions, and their abilities to restore the systemic condition after resuscitation from hemorrhagic shock in anesthetized rats. The HbBv-HSA3 and HbA-HSA3 , which have high colloid osmotic activity, showed equivalent solution characteristics and O2 binding parameters. Shock was induced by 50% blood withdrawal. Rats exhibited hypotension and significant metabolic acidosis. After 15 min, the rats were administered shed autologous blood (SAB), HbBv-HSA3 , HbA-HSA3 , or Ringer's lactate (RL) solution. Survival rates, circulation parameters, hematological parameters, and blood gas parameters were monitored during the hemorrhagic shock and for 6 h after administration. All rats in the SAB, HbBv-HSA3 , and HbA-HSA3 groups survived for 6 h. The HbBv-HSA3 and HbA-HSA3 groups restored mean arterial pressure after the resuscitation. No remarkable difference was observed in the time courses of blood gas parameters in any resuscitated group except for the RL group. Serum biochemical tests showed increases in aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the HbBv-HSA3 and HbA-HSA3 groups compared to the SAB group. Therefore, we observed other rats awakened after resuscitation with HbA-HSA3 for 7 days. The blood cell count, AST, and ALT recovered to the baseline values by 7 days. All the results implied that HbBv-HSA3 and HbA-HSA3 clusters provide restoration from hemorrhagic shock as an alternative material for SAB transfusion.

Lung adenocarcinoma in a patient with a cis EGFR L858R-K860I doublet mutation identified using NGS-based profiling test: Negative diagnosis on initial companion test and successful treatment with osimertinib.

Tyrosine kinase inhibitors are used as first-line treatment for non-small cell lung cancer (NSCLC) patients harboring driver mutations in EGFR, ALK, ROS1, and BRAF. Currently, standard molecular testing approaches help identify single genes for such targetable driver mutations in NSCLC; however, next-generation sequencing (NGS)-based genetic profiling provides a more comprehensive approach and is hence strongly recommended. This case study aimed to highlight the benefits of NGS-based tests for the diagnosis of complex EGFR L858R mutations. A patient was diagnosed with stage IVB NSCLC using a government-approved in vitro diagnostic test and was noted to have a high programmed death-ligand 1 tumor proportion score. This patient was treated with pembrolizumab monotherapy followed by cisplatin and pemetrexed owing to the lack of actionable driver gene mutations, including EGFR mutations. After treatment failure, a sample harvested from the same transbronchial lung biopsy specimen (formalin-fixed and paraffin-embedded) used for the initial EGFR test was subjected to NGS-based broad genetic profiling. The NGS-based test identified an EGFR L858R-K860I cis doublet mutation; however, neither of these mutations was identified upon initial molecular testing. The patient was then successfully treated with a third-generation EGFR-tyrosine kinase inhibitor, osimertinib. In this study, we delved deeper into the realm of L858R and K860I mutations in NSCLC and discuss the potential causes underlying our initial negative diagnosis. Furthermore, this study highlighted the additional benefits of replacing typical molecular tests with NGS-based broad profiling approaches. KEY POINTS: SIGNIFICANT FINDINGS OF THE STUDY: The EGFR L858R-K860I cis doublet mutation was not detected by a PCR-based EGFR test. A next generation sequencing (NGS)-based test was able to identify the L858R-K860I cis doublet mutation. WHAT THIS STUDY ADDS: Osimertinib was effective in an NSCLC patient with EGFR L858R and K860I mutations.

Immediate effects of systemic administration of normal and high O2-affinity haemoglobin vesicles as a transfusion alternative in a rat pneumonectomy model.

BACKGROUND: Haemoglobin vesicles (HbVs) are red blood cell (RBC) substitutes with a phospholipid bilayer membrane and a polyethylene modified surface (diameter=250 nm; P50=28 Torr). They can be preserved for years and can be used in patients of all blood types without the risk of infection. Their oxygen affinity can be modified by changing the allosteric effectors. METHODS: Left pneumonectomy was performed under mechanical ventilation on rats, followed by rapid exsanguination of ~30% of the total circulating blood volume. Rat RBCs shed in 5% human serum albumin (HSA) solution (rat RBC), HbV with high oxygen affinity in 5% albumin solution (low-P50 HbV, P50=9 Torr), normal HbV suspended in 5% albumin (HbV, P50=28 Torr) or 5% HSA was infused for resuscitation. Haemodynamics and oxygenation were evaluated. RESULTS: Systemic arterial blood pressure significantly decreased after exsanguination and increased after each infusion. In the HbV, low-P50 HbV and rat RBC groups, all rats were liberated from mechanical ventilation and blood pressure was stabilised, whereas 50% of the rats in the HSA group died within 1 hour after weaning from mechanical ventilation. The PaO2 in arterial blood for 1 hour after liberation from mechanical ventilation in the rat RBC, HbV and low-P50 HbV groups was 59.4±12.5, 58.3±10.1 and 70.5±14.5 mm Hg, respectively. The PaO2 in the low-P50 HbV group was significantly higher than those in the rat RBC and HbV groups (p=0.05 for both). Serum lactate elevations due to hypoxic damage were minimised by HbV, low-P50 HbV as well as rat RBCs. CONCLUSIONS: The oxygen-carrying ability of HbV was comparable to that of rat RBCs, even under impaired lung function after pneumonectomy. HbVs with high oxygen affinity may have more beneficial effects on oxygenation in pulmonary resection.

Epithelial-mesenchymal Transition (EMT) is Correlated with Patient's Prognosis of Lung Squamous Cell Carcinoma.

Epithelial-mesenchymal transition (EMT) is an important step leading to invasion and migration of various cancer cells, and are characterized by decreased E-cadherin as an epithelial marker, and increased vimentin as a mesenchymal marker. The present study focused on the clinicopathological significance of E-cadherin and vimentin expression in lung squamous cell carcinoma (SqCC). Immunohistochemically, E-cadherin expression patterns were classified into two types: preserved or reduced; and vimentin expression patterns were also divided into two types: positive or negative. The univariate analyses showed six factors associated with increased mortality: tumor size (P = 0.031), lymph node metastasis (P < 0.001), lymphatic invasion (P < 0.001), histological differentiation (P = 0.036), E-cadherin reduced expression (P < 0.001), and vimentin positive expression (P = 0.004). Multivariate analysis demonstrated that E-cadherin reduced expression (P < 0.001), vimentin positive expression (P = 0.028), lymph node metastasis (P < 0.001), and age (P = 0.020) were independent predictors of patient mortality. There may be some correlation between E-cadherin and vimentin expression (P = 0.017), but the correlation coefficient was 0.235. The complete EMT and the incomplete EMT type were associated with a poor prognosis (p < 0.001 and p=0.036, respectively). The overall survival rate after curative resection was significantly lower in patients with the complete EMT type (reduced E-cadherin / positive vimentin). In conclusion, both E-cadherin and vimentin are independent predictors of mortality, and the EMT phenotype is a significant indicator of poor prognosis in lung SqCC.