Biocompatibility of Hemoglobin Solutions. I. Reactions of Vascular Endothelial Cells to Pure and Impure Hemoglobins

Hemoglobin (Hb) solutions can cause vasoconstriction and activation of intravascular coagulation. Because the endothelium plays a major role in the regulation of vascular tone and hemostasis, a study was conducted of human umbilical vein endothelial cells (EC) incubated with various Hbs. Cell injury was evaluated by electron microscopy and the release of lactic dehydrogenase, H2O2, and procoagulant "tissue factor." Cell reaction was assessed by the measurement of 6-keto-prostaglandin F (PGF)1 alpha (metabolite of prostacyclin) and thromboxane B2 (metabolite of thromboxane A2). Incubation with unmodified bovine hemoglobin for 24 h caused no cell injury and a reaction characterized by 48.4 +/- 8.2% increase in 6-keto-PGF1 alpha production, accompanied by 40.2 +/- 9.4% reduction in thromboxane (Tx)B2 (compared with a control group of EC incubated with saline solution). Incubation with a nonpure Hb solution (Hb plus red blood cell membrane aminophospholipids; a-PLs) caused cell injury with significant release of tissue factor, plus a reaction characterized by 97.5 +/- 12.5% increase in TxB2 production accompanied by 25.3 +/- 3% reduction in 6-keto-PGF1 alpha. A second nonpure Hb [Hb plus bacterial environmental endotoxin (E)] caused cell injury, the release of tissue factor, and increased production of both prostaglandins, with greater release of TxB2 (197 +/- 17%) than of 6-keto-PGF1 alpha (112 +/- 8.3%). These data indicate that the endothelium reacts differently to pure and nonpure hemoglobins. The biocompatibility of Hb solutions, with regard to vasoconstriction and activation of intravascular coagulation, depends on the absence of stromal a-PLs and bacterial E.     

Effects of Pyridoxalated Hemoglobin Polyoxyethylene Conjugate and Stroma-Free Hemoglobin on Cardiac Function in Isolated Rat Heart

Effects of a newly introduced modified hemoglobin, pyridoxalated hemoglobin polyoxyethylene conjugate (PHP), and stroma-free hemoglobin (SFH) on cardiac rhythm and contractility were examined in isolated rat hearts. Hearts were perfused with HEPES-buffered Krebs solution containing 6% PHP, 6% SFH, or 6% hydroxyethylstarch (HES). Arrhythmia was not observed in PHP-perfused hearts, but marked sinus bradycardia occurred in SFH-perfused hearts. Ventricular contractility assessed by left ventricular pressure and pressure-volume relationship in both PHP and SFH-perfused hearts were well maintained during perfusion. There was no significant difference to the coronary flow resistance between hearts perfused with PHP and HES solutions, indicating that PHP has little vasoactive effect. The flow resistance in SFH-perfused hearts decreased significantly when compared with other groups. Results demonstrate that PHP solution can preserve cardiac function and have no toxic effects in isolated rat hearts.     

Lysophosphatidylcholine, a Component of Stromal Phospholipids, as a Candidate Vasoconstrictive Factor in Stroma-Free Hemoglobin

Abstract: Stroma-free hemoglobin (SFH) contains a trace amount of phospholipids, which has been implicated in the toxic reactions associated with SFH. We analyzed stromal phospholipids by high-performance liquid chromatography and found that SFH contained small quantities of lysophosphatidylcholine (LPC), which is known to be capable of producing a defect in endothe-lium-dependent arterial relaxation, in addition to major classes of constituent phospholipids in red cell membrane. LPC content was determined to be 1.65 nmol/ml (hemoglobin 8.1 g/dl). To evaluate the role of these stromal phospholipids in SFH-induced vasoconstriction, we next examined the effect of lipids on vascular tone in rabbit aortic strips. Preincubation with the crude lipid extract or the LPC purified from SFH significantly inhibited acetylcholine-induced relaxation in phenylephrine-precontracted tissues. The LPC-induced inhibition was reversed by incubation of the tissues in the absence of lipids, indicating the functional integrity of endothelium. From these results, we propose a possibility that LPC, a component of stromal phospholipids, is a candidate for vasoconstrictive factors present in SFH.     

Coronary Vascular Actions of Stroma-Free Hemoglobin Preparations

The left anterior descending coronary artery was cannulated in anesthetized, open-chest dogs for use as a coronary test bed to assess the coronary vascular actions of oxygenated, unmodified and pyridoxylated, partially cross-linked (polymerized), stroma-free hemoglobin solutions (SFHS). The actions of these SFHSs were assessed in this test bed, by comparison with perfusion with whole blood. Unmodified SFHS caused significant vasoconstriction, whereas pyridoxylated, partially polymerized SFHS did not do so. The existing coronary flow during perfusion with either SFHS preparation, under basal conditions, did not increase during intracoronary infusion of adenosine. Coronary flow-autoregulation was also altered during perfusion with either SFHS, because the "normal" reactive hyperemia response, observed in the control experiments, was not seen. These findings suggest that both unmodified and pyridoxylated cross-linked (70% polymers) hemoglobin preparations possess some vascular activity when tested in the canine coronary circulation.     

Effects of Pyridoxalated Hemoglobin Polyoxyethylene Conjugate and Stroma-free Hemoglobin on Renal Vascular Responsiveness to Vasoactive Substances in Isolated Perfused Rat Kidney

Abstract: The effects of pyridoxalated hemoglobin polyoxyethylene conjugate (PHP) and stroma-free hemoglobin (SFH) on vascular responsiveness to various vasoactive substances were examined in isolated perfused rat kidneys. The kidneys isolated from rats were perfused with 6% PHP, 6% SFH, and 6% hydroxyethylstarch (HES) solution at a constant flow rate. Vascular responsiveness to acetylcholine (ACh), nitroglycerin (NG), norepinephrine (NE), and angiotensin-II (ANG-II) was examined by measuring the perfusion pressure (PP). Effects of inhibition of endothelium-derived relaxing factor (EDRF) by N^G-monomethyl L-arginine (L-NMMA) on NE-induced and ANG-II-induced renal vascular responses were examined. ACh and NG induced a dose-dependent decrease in perfusion pressure (PP) in all groups. NE and ANG-II induced an increase in PP in all groups, but NE-induced and ANG-II-induced responses in the PHP-perfused and SFH-perfused groups were significantly larger than those in the HES-perfused group. L-NMMA did not alter vascular responsiveness to NE and ANG-II. These results indicate that PHP and SFH do not inhibit EDRF induced by ACh, but hemoglobin moiety per se does augment the vascular responsiveness to NE and ANG-II in the isolated perfused rat kidney.     

Effects of Pyridoxalated Hemoglobin Polyoxyethylene Conjugate and Stroma Free Hemoglobin on Pulmonary Vascular Responsiveness to Vasoactive Substances in Isolated Perfused Rat Lungs

We examined the effects of pyridoxalated hemoglobin polyoxyethylene conjugate (PHP) and stroma free hemoglobin (SFH) on vascular responsiveness to various vasoactive substances in isolated perfused rat lungs. The lungs isolated from rats were perfused with 6% PHP, 6% SFH, or 6% hydroxyethylstarch (HES) solution, and the effects of intrapulmonary arterial injection of norepinephrine (NE), angiotensin II (ANG-II), acetylcholine (ACh), and nitroglycerin (NG) were examined by measuring perfusion pressure. NE and ANG-II produced a dose-dependent increase in perfusion pressure in all groups. The NE response in the PHP- or SFH-perfused group was significantly larger than that in the HES-perfused one. ACh decreased perfusion pressure in both PHP- and HES-perfused groups but increased perfusion pressure in the SFH-perfused group. NG decreased perfusion pressure in all groups. Present results indicate that pulmonary arterial responses to endothelium-derived relaxing factor (EDRF) induced by ACh would not be affected in the presence of PHP.     

Human Osteoclast Formation from Blood Monocytes, Peritoneal Macrophages, and Bone Marrow Cells

Mononuclear precursors of the human osteoclast have been identified in both bone marrow and the circulation in man, but osteoclast membership of the mononuclear phagocyte system (MPS) and its precise cellular ontogeny remain controversial. We isolated human hematopoietic marrow cells, blood monocytes, and peritoneal macrophages and incubated each of these cell populations with UMR106 osteoblast-like cells on glass coverslips and dentine slices in both the presence and absence of 1,25 dihydroxyvitamin D₃ (1,25(OH)₂D₃), macrophage-colony stimulating factor (M-CSF), and dexamethasone. Cells isolated from peripheral blood and peritoneal dialysis fluid were positive only for monocyte/macrophage markers (CD11a, CD11b, CD14, and HLA-DR) and negative for osteoclast markers [tartrate-resistant acid phosphatase (TRAP), vitronectin reception (VNR), and calcitonin (CT) receptors and did not form resorption pits on dentine slices after 24 hours in culture. Similarly marrow cells did not form resorption pits on dentine slices after 24 hours in culture. However, after 14 days in co-culture with UMR106 cells, in the presence of 1,25(OH)₂D₃ and M-CSF, numerous TRAP, CT receptor, and VNR-positive multinucleated cells capable of extensive lacunar resorption were formed in co-cultures of all these preparations. The presence of 1,25 (OH)₂D₃, M-CSF, and UMR106 were absolute requirements for osteoclast differentiation. It is concluded that precursor cells capable of osteoclast differentiation are present in the marrow compartment, the monocyte fraction of peripheral blood, and in the macrophage compartment of extraskeletal tissues and that these cells are capable of differentiating into mature functional osteoclasts. These findings argue in favor of osteoclast membership of the human MPS. Received: 3 January 1997 / Accepted: 14 November 1997     

Biocompatibility Assessment of Peritoneal Dialysis Solutions With a New In Vitro Model of Preconditioned Human HL60 Cells

The purposes of this study were to test the human promyelocytic cell line HL60 for its usability as a new cell model for the immune barrier of the peritoneum, and to investigate the impact of different peritoneal dialysis (PD) solutions in the model. HL60 cells were stimulated by retinoic acid and recombinant human granulocyte and macrophage colony-stimulating factor to differentiate into neutrophilic granulocytes. Cells were incubated in different commercially available PD solutions. After a 4-h incubation, functional (chemiluminescence phagocytosis) and viability tests (Live-Dead, XTT) were performed. High glucose concentrations (>1.36%) and low pH values (<7.0) appeared to be detrimental for neutrophil functions and for neutrophil viability. There is a quantitative correlation between glucose concentration and the cytotoxicity of standard PD solutions (PD 1.36% glucose shows 42.6% higher chemiluminescence than PD 3.86% glucose [ P  < 0.05]). PD solution containing icodextrin shows 74.3% higher chemiluminescence than PD 3.86% glucose, and PD solution with amino acids shows 52.4% higher chemiluminescence than PD 3.86% glucose which is a sign for better biocompatibility in these tests ( P  < 0.05). The test system is useful for biocompatibility investigations of PD solutions and their effect on immune cells, for example, neutrophil granulocytes. It does not depend on donor variability and availability in comparison to models based on primary isolated leukocytes.     

Preparation and Evaluation of Hemoglobin-Polyethylene Glycol Conjugate (Pyridoxalated Polyethylene Glycol Hemoglobin) as an Oxygen-Carrying Resuscitation Fluid

Human hemoglobin was pyridoxalated and subsequently coupled with the activated ester of polyethylene glycol (PEG) to provide an oxygen-carrying resuscitation fluid. The PEG conjugate of pyridoxalated hemoglobin (pyridoxalated PEG hemoglobin) has a longer half-life in the circulation (12.8 h) than does hemoglobin (2.2 h) when the solutions are exchange-transfused by approximately 60%. The P50 of pyridoxalated PEG hemoglobin, which is the partial pressure of oxygen at which one-half of the absorbed oxygen is released, was 21.3 +/- 1.4 mm Hg (mean +/- SD, n = 5) (37 degrees C, pH = 7.4), which is close to that of human whole blood (25.9 +/- 0.6 mm Hg). The colloidal osmotic pressure and the viscosity were 36.5 +/- 2.4 mm Hg (n = 5) and 2.6 +/- 0.4 cp (n = 5), respectively. The results indicate that the pyridoxalated PEG hemoglobin solution has favorable properties for use as an oxygen-carrying resuscitation fluid.     

Ex Vivo Expansion of Human Tregs by Rabbit ATG Is Dependent on Intact STAT3‐Signaling in CD4+ T Cells and Requires the Presence of Monocytes

The addition of low, nondepleting doses of rabbit antithymocyte globulin (ATG) to human peripheral blood mononuclear cells has been shown to expand functional CD4⁺CD25⁺FoxP3⁺ regulatory T cells (Tregs) in vitro. This report is the first to elucidate the exact cellular mechanisms of ATG‐mediated Treg expansion. CD4⁺ T cells require monocytes, but not other antigen presenting cell subsets, to be present in coculture to expand Tregs. However, T cells do not require direct cell–cell contact with monocytes, suggesting the importance of soluble factors. Moreover, ATG initially “reprograms” CD4⁺ T cells, but not monocytes, and induces STAT3 and STAT5 signaling in CD4⁺ cells. These reprogrammed CD4⁺ T cells subsequently secrete GM‐CSF and IL‐10 only in case of intact STAT3 signaling, which in turn promote the generation of tolerogenic CD14⁺CD11c⁺ dendritic cells characterized by enhanced IL‐10 and decreased IL‐12 production. Treg expansion following ATG treatment is accompanied by enhanced gene expression of both GM‐CSF and Bcl‐2, but not TGF‐β, in peripheral blood mononuclear cells. These results demonstrate that ex vivo expansion of human Tregs by ATG is due to its ability to reprogram CD4⁺ T cells in a STAT3‐dependent but TGF‐β‐independent manner, leading to the generation of monocyte‐derived dendritic cells with a tolerogenic cytokine profile.     

大鼠面神经挤压缺血损伤对巨噬细胞募集的影响

目的 研究挤压缺血损伤对面神经修复过程中巨噬细胞募集的影响,为治疗外伤性面瘫提供理论依据。方法60只雄性SD大鼠,随机分为缺血组20只,单纯挤压组20只,对照组20只。缺血组给予左侧面神经挤压并切断面神经表面营养血管,同时在手术显微镜下剥离神经外膜(造成完全缺血);单纯挤压组仅给予左侧面神经挤压,保留表面营养血管;对照组为假手术组。于术后3 d、7 d、14 d检测大鼠行为学及电生理变化;每组5只大鼠取材,切片行组织学观察;使用巨噬细胞特异性抗体CD68进行免疫组化染色,进一步分析巨噬细胞募集情况。结果 术后3 d缺血组及单纯挤压组均出现面瘫表现;术后7 d时缺血组及单纯挤压组部分大鼠面瘫情况较术后3 d加重,部分大鼠已出现面瘫恢复迹象;术后14 d缺血组及单纯加压组大鼠面瘫表现较7 d时均有恢复,但缺血组大鼠恢复速度明显缓慢。电生理检测显示,随时间延长,缺血组及单纯挤压组大鼠口轮匝肌复合动作电位最大波幅和潜伏期逐渐恢复,但均未达正常水平。缺血组恢复幅度较单纯挤压组小。组织学观察显示,缺血组大鼠术后免疫组化结果表明巨噬细胞逐渐增多,7 d时达到高峰;单纯挤压组同缺血组类似,但巨噬细胞的数量明显多于缺血组;对照组无明显阳性表达。结论 面神经局部营养血管的挤压缺血损伤,可阻碍神经修复过程中巨噬细胞的募集,最终阻碍神经修复过程。     

The Comparative Pathology of Human and Mouse Mammary Glands

The mouse has emerged as a primary animal modelfor human breast cancer because the mammary glands ofthe two species are very similar in structure andfunction. In this regard the TDLU⁴ and LAhave similar morphology. The mouse, infected by MMTV,develops "spontaneous" tumors with specificbut limited tumor phenotypes. The advent of geneticmanipulation has created transgenic mice that develophyperplasias and tumors morphologically and cytochemicallycomparable to lesions in humans. Even experiencedpathologists have difficulty distinguishing betweenlesions from the two species, and the morphologicalsimilarities support the utility of the mouse model inunderstanding human breast cancer. In this essay wereview our experience with the histopathology of humanand mouse mammary disease by comparing the normal gland with hyperplastic, dysplastic and neoplasticlesions of traditional and transgenic origin.     

The Prevalence of Diabetes Mellitus and Routine Hemoglobin A1c Screening in Elective Total Joint Arthroplasty Patients

Background

Diabetes mellitus has been associated with significant perioperative complications in joint arthroplasty. In addition, many patients are unaware of their dysglycemic status, and the prevalence of undiagnosed dysglycemia in joint arthroplasty patients is unknown.

The influence of Patent Blue V on pulse oximetry and haemoximetry

Patent Blue V (PBV) is a blue dye solution which is used to visualize lymphatic vessels for surgical procedures. There has been some conflicting reports about the influence of this dye solution on pulse oximetry and haemoximetry, why we decided to: 1) produce a spectrum of PBV in plasma, 2) measure absorbances of full blood before and after addition of PBV and 3) to record the effect on the pulse oximeter and haemoximeter of an intradermal injection of PBV to a patient. Computer analysis of the blood gas measurements were carried out by the Oxygen Status Algorithm (OSA). The spectrum of PBV demonstrated a peak absorption at 640 nm and correspondingly the absorbances of the haemoximeter increased most significantly at 622 and 636 nm. These changes invalidated the determination of haemoglobin pigments, and the results should not be used. Computer analysis interpreted the measurements as a shift of the haemoglobin oxygen binding curve.     

The Relationship of Visfatin Levels to Inflammatory Cytokines and Left Ventricular Hypertrophy in Hemodialysis and Continuous Ambulatory Peritoneal Dialysis Patients

Visfatin was recently defined as an adipocytokine; however, the pathophysiological role of visfatin is not completely understood. A few studies suggest that visfatin may be a new proinflammatory adipocytokine. The aim of the present study was to compare serum visfatin levels between hemodialysis and continuous ambulatory peritoneal dialysis (CAPD) patients and evaluate the relationship between visfatin levels to IL-6, TNF-α, and left ventricular hypertrophy. Serum visfatin, IL-6, and TNF-α levels were measured by using the ELISA method, and echocardiographic evaluations were performed in 31 hemodialysis patients, 30 CAPD patients, and 21 healthy volunteers. Serum visfatin levels were higher in the CAPD group (265.27 ± 387.86 ng/mL) than hemodialysis (97.68 ± 244.96 ng/mL,) and control (41.33 ± 48.87 ng/mL) groups (p = 0.04, p = 0.01, respectively). No significant difference was observed between the hemodialysis and control groups. In univariate analysis, visfatin levels were positively correlated with IL-6 (r = 0.24, p = 0.03), TNF-α (r = 0.34, p = 0.002), and BMI (r = 0.26, p = 0.03) and negatively correlated with some left ventricular diastolic parameters [Em and Em/Am (r = ?0.305, p = 0.01), (r = ?0.251, p = 0.03), respectively]. No relationship was found between visfatin and left ventricular mass index. In the linear regression analysis, visfatin levels independently related with TNF-( (β = 0.369, p = 0.001) and IL-6 (β = 0.284, p = 0.015). This study has found significantly higher levels of serum visfatin in CAPD patients when compared to healthy individuals. Increased visfatin levels seem to associate with proinflammatory cytokines such as IL-6 or TNF-α. As for the effects of on left ventricular structure and functions, visfatin might have negative effects on left ventricular diastolic function parameters but have no effects on left ventricular mass index.     

The Relevance of Mouse Models to Understanding the Development and Progression of Human Breast Cancer

Mouse modeling of human breast cancer has developed tremendously over the past ten years. Human breast cancer is characterized by enormous biological diversity and, collectively, the new models have come much closer to encompassing this diversity. They have provided a deeper understanding of the fundamental events that mediate the initiation, development, and progression of breast cancer, and they offer new opportunities to develop and test strategies to treat and, perhaps, even prevent the disease. This chapter reviews the historical development of mouse models of breast cancer and highlights some of their major strengths, weaknesses, and contributions.     

The Role of Histamine and Serotonin in the Inflammatory Reaction in an Experimental Model of Open Wounds in the Rat

The role of histamine and serotonin in the inflammatory reaction in the granulation tissue of open wounds in the rat was studied. The model involved plastic chambers attached to the edges of two open circular full-thickness skin wounds. Five days post-wounding, agonists or antagonists were applied in one of the two chambers, the adjacent wound serving as control. Thereafter blood flow and albumin extravasation were measured. Application of histamine (100 µM) caused an increase in granulation tissue blood flow by 36%, but left albumin extravasation unaffected. Treatment with mepyramine (H₁ antagonist, 20 µM), cimetidine (H₂ antagonist, 20 µM) or methysergide (serotonin antagonist, 20 µM) did not influence the level of either blood flow or albumin extravasation. It is suggested that endogeneous histamine and serotonin play a minor role in the inflammatory process in the granulation tissue of this model of healing wounds.