Hp-Hb Complex Research Articles

Hypoxic pulmonary hypertension and the role of extra-cellular hemoglobin in perivascular macrophage accumulation

Hemolytic anemia associated pulmonary hypertension (PH) typically occurs in chronic diseases such as sickle cell anemia and thalassemia. The contributions of chronic and intermittent hypoxia caused by the anemic state and the role of free hemoglobin (Hb) and iron have been debated in the literature. The lifetime cycle of hypoxia in hemolytic anemia is an important contributor to the worsening of pulmonary vascular remodeling and increased pulmonary vascular disease. However, the role of low, but continuous plasma Hb in combination with hypoxia has not been well studied. As a result we initially designed a rat model to evaluate the specific effects of hypoxia and continuous levels of Hb on the progression of PH in a chronic setting. In this model we observed that Hb delivered by an implanted refillable infusion pump had an additive effect on hypoxia driven PH, even at low Hb plasma concentrations (10-20 µM heme). Our observations were consistent with increased adventitia macrophage accumulation, oxidation and inflammation that consistently lead to more pronounced metrics of enhanced pulmonary vascular remodeling. As a result we suggested this model would be useful to evaluate potential treatments for PH, specifically PH associated with hemolysis. Effective treatments targeted directly at Hb induced PH associated with hemolytic anemia do not presently exist. Sickle cell anemia patients have undetectable levels of the Hb binding protein, haptoglobin (Hp). Therefore we focused our efforts on the study of repeated dose Hp therapy to increase the circulatory Hp pool and maintain plasma Hb in a non-reactive intravascular compartmentalized Hb-Hp complex. Here we review results from several studies utilizing this model, discuss the role of macrophages in PH disease progression, dissect the contributions of hypoxia and Hb in our model and suggest treatment strategies tested to reduce Hbs contribution of progressive complications of PH.

SERS-based quantitative detection of ovarian cancer prognostic factor haptoglobin.

Surface-enhanced Raman spectroscopy (SERS) is increasingly being used for biosensing because of its high sensitivity and low detection limit, which are made possible by the unique Raman ‘fingerprint’ spectra from the biomolecules. Here we propose a novel SERS method for the fast, sensitive, and reliable quantitative analysis of haptoglobin (Hp), an acute phase plasma glycoprotein that is widely gaining application as a prognostic ovarian cancer biomarker. We exploited the peroxidase activity of the hemoglobin–haptoglobin (Hb–Hp) complex formed by the selective and specific binding of Hp to free Hb to catalyze the reaction of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate and hydrogen peroxide to result in the final product of strongly SERS-active TMB2+. We observed a linear increase in the SERS signal of TMB2+ with increasing concentrations of Hb–Hp complex from 50 nM to 34 μM. Based on this concentration-dependent SERS spectrum, we quantified Hp in clinical samples. We observed that our inference about the prognosis of the disease coincided with the histology data and that our method was much more sensitive than the enzyme-linked immunosorbent assay method.

Structural basis for trypanosomal haem acquisition and susceptibility to the host innate immune system.

Sleeping sickness is caused by trypanosome parasites, which infect humans and livestock in Sub-Saharan Africa. Haem is an important growth factor for the parasites and is acquired from the host by receptor-mediated uptake of haptoglobin (Hp)-haemoglobin (Hb) complexes. The parasite Hp-Hb receptor (HpHbR) is also a target for a specialized innate immune defence executed by trypanosome-killing lipoprotein particles containing an Hp-related protein in complex with Hb. Here we report the structure of the multimeric complex between human Hp-Hb and Trypanosoma brucei brucei HpHbR. Two receptors forming kinked three-helical rods with small head regions bind to Hp and the β-subunits of Hb (βHb), with one receptor at each end of the dimeric Hp-Hb complex. The Hb β-subunit haem group directly associates with the receptors, which allows for sensing of haem-containing Hp-Hb. The HpHbR-binding region of Hp is conserved in Hp-related protein, indicating an identical recognition of Hp-Hb and trypanolytic particles by HpHbR in human plasma.

Utilization of host iron sources by Corynebacterium diphtheriae: multiple hemoglobin-binding proteins are essential for the use of iron from the hemoglobin-haptoglobin complex.

The use of hemin iron by Corynebacterium diphtheriae requires the DtxR- and iron-regulated ABC hemin transporter HmuTUV and the secreted Hb-binding protein HtaA. We recently described two surface anchored proteins, ChtA and ChtC, which also bind hemin and Hb. ChtA and ChtC share structural similarities to HtaA; however, a function for ChtA and ChtC was not determined. In this study, we identified additional host iron sources that are utilized by C. diphtheriae. We show that several C. diphtheriae strains use the hemoglobin-haptoglobin (Hb-Hp) complex as an iron source. We report that an htaA deletion mutant of C. diphtheriae strain 1737 is unable to use the Hb-Hp complex as an iron source, and we further demonstrate that a chtA-chtC double mutant is also unable to use Hb-Hp iron. Single-deletion mutants of chtA or chtC use Hb-Hp iron in a manner similar to that of the wild type. These findings suggest that both HtaA and either ChtA or ChtC are essential for the use of Hb-Hp iron. Enzyme-linked immunosorbent assay (ELISA) studies show that HtaA binds the Hb-Hp complex, and the substitution of a conserved tyrosine (Y361) for alanine in HtaA results in significantly reduced binding. C. diphtheriae was also able to use human serum albumin (HSA) and myoglobin (Mb) but not hemopexin as iron sources. These studies identify a biological function for the ChtA and ChtC proteins and demonstrate that the use of the Hb-Hp complex as an iron source by C. diphtheriae requires multiple iron-regulated surface components.

Can gas replace protein function? CO abrogates the oxidative toxicity of myoglobin.

Outside their cellular environments, hemoglobin (Hb) and myoglobin (Mb) are known to wreak oxidative damage. Using haptoglobin (Hp) and hemopexin (Hx) the body defends itself against cell-free Hb, yet mechanisms of protection against oxidative harm from Mb are unclear. Mb may be implicated in oxidative damage both within the myocyte and in circulation following rhabdomyolysis. Data from the literature correlate rhabdomyolysis with the induction of Heme Oxygenase-1 (HO-1), suggesting that either the enzyme or its reaction products are involved in oxidative protection. We hypothesized that carbon monoxide (CO), a product, might attenuate Mb damage, especially since CO is a specific ligand for heme iron. Low density lipoprotein (LDL) was chosen as a substrate in circulation and myosin (My) as a myocyte component. Using oxidation targets, LDL and My, the study compared the antioxidant potential of CO in Mb-mediated oxidation with the antioxidant potential of Hp in Hb-mediated oxidation. The main cause of LDL oxidation by Hb was found to be hemin which readily transfers from Hb to LDL. Hp prevented heme transfer by sequestering hemin within the Hp-Hb complex. Hemin barely transferred from Mb to LDL, and oxidation appeared to stem from heme iron redox in the intact Mb. My underwent oxidative crosslinking by Mb both in air and under N2. These reactions were fully arrested by CO. The data are interpreted to suit several circumstances, some physiological, such as high muscle activity, and some pathological, such as rhabdomyolysis, ischemia/reperfusion and skeletal muscle disuse atrophy. It appear that CO from HO-1 attenuates damage by temporarily binding to deoxy-Mb, until free oxygen exchanges with CO to restore the equilibrium.

Haptoglobin Genotype-dependent Differences in Macrophage Lysosomal Oxidative Injury

The major function of the Haptoglobin (Hp) protein is to control trafficking of extracorpuscular hemoglobin (Hb) thru the macrophage CD163 receptor with degradation of the Hb in the lysosome. There is a common copy number polymorphism in the Hp gene (Hp 2 allele) that has been associated with a severalfold increased incidence of atherothrombosis in multiple longitudinal studies. Increased plaque oxidation and apoptotic markers have been observed in Hp 2-2 atherosclerotic plaques, but the mechanism responsible for this finding has not been determined. We proposed that the increased oxidative injury in Hp 2-2 plaques is due to an impaired processing of Hp 2-2-Hb complexes within macrophage lysosomes, thereby resulting in redox active iron accumulation, lysosomal membrane oxidative injury, and macrophage apoptosis. We sought to test this hypothesis in vitro using purified Hp-Hb complex and cells genetically manipulated to express CD163. CD163-mediated endocytosis and lysosomal degradation of Hp-Hb were decreased for Hp 2-2-Hb complexes. Confocal microscopy using lysotropic pH indicator dyes demonstrated that uptake of Hp 2-2-Hb complexes disrupted the lysosomal pH gradient. Cellular fractionation studies of lysosomes isolated from macrophages incubated with Hp 2-2-Hb complexes demonstrated increased lysosomal membrane oxidation and a loss of lysosomal membrane integrity leading to lysosomal enzyme leakage into the cytoplasm. Additionally, markers of apoptosis, DNA fragmentation, and active caspase 3 were increased in macrophages that had endocytosed Hp 2-2-Hb complexes. These data provide novel mechanistic insights into how the Hp genotype regulates lysosomal oxidative stress within macrophages after receptor-mediated endocytosis of Hb.

Abstract 42: Haptoglobin Genotype as a Marker of Vascular Intraplaque Hemorrhage

Background: Intraplaque hemorrhage (IPH), a component of late stage atherosclerosis, is a critical factor in plaque destabilization leading to stroke and myocardial infarction. Magnetic resonance imaging (MRI) is able to identify IPH and allows for identification of high risk patients. Currently however, no routinely available tests are conducted to identify individuals at risk of IPH. IPH is a rich source of free hemoglobin (Hb), a potent oxidant. The plasma protein haptoglobin (Hp) binds Hb forming a Hb-Hp complex, that can be engulfed by tissue macrophages.The reduction in oxidative stress decreases vascular inflammation. In humans, the Hp gene has three different genotypes: Hp1-1, Hp2-2 and Hp1-2. The Hb-Hp2-2 complex has a lower binding affinity to macrophages, resulting in higher oxidative burden. Numerous studies have demonstrated a higher risk of cardiovascular events in Hp2-2 individuals. We therefore hypothesized that patients of a Hp2-2 genotype have a greater risk of MR-IPH. Methods: From 2010 to 2014, patients with non-surgical carotid artery disease (30-95% stenosis) underwent 3T carotid MRI that included a MR-IPH sequence. A radiologist with 25 years of expertise in MR-IPH imaging determined the presence of MR-IPH using widely accepted methods. Patients’ charts were blindly reviewed for demographics and medical history. Patients were genotyped for Hp1/2 using an established PCR genotyping method. Statistical association analysis was performed using logistic regression. Results and Conclusions: Out of the 80 recruited patients (mean age, 72.8 years; range 52-100), those with Hp2-2 genotype vs. Hp1-1/Hp1-2 had a prevalence of MR-IPH of 65% compared to 41%(p=0.09). Non-diabetics (HbA1c <6.5%) with Hp2-2 genotype had an increased risk of IPH compared to non-diabetics with Hp1-1/Hp 1-2 genotype (OR=3, 95% CI 1.04-8.73, p=0.03). Males were also at an increased risk of IPH (OR=4.05, 95% CI 1.53-10.70, p=0.004) and the majority (71%) of males with IPH were of the Hp2-2 genotype. In conclusion, non-diabetic Hp2-2 male patients with history of vascular disease are the population with the highest risk of developing IPH and they can be identified using simple haptoglobin genotype testing.

Molecular Modeling of the Human Hemoglobin-Haptoglobin Complex Sheds Light on the Protective Mechanisms of Haptoglobin

Hemoglobin (Hb) plays a critical role in human physiological function by transporting O2. Hb is safe and inert within the confinement of the red blood cell but becomes reactive and toxic upon hemolysis. Haptoglobin (Hp) is an acute-phase serum protein that scavenges Hb and the resulting Hb-Hp complex is subjected to CD163-mediated endocytosis by macrophages. The interaction between Hb and Hp is extraordinarily strong and largely irreversible. As the structural details of the human Hb-Hp complex are not yet available, this study reports for the first time on insights of the binding modalities and molecular details of the human Hb-Hp interaction by means of protein-protein docking. Furthermore, residues that are pertinent for complex formation were identified by computational alanine scanning mutagenesis. Results revealed that the surface of the binding interface of Hb-Hp is not flat and protrudes into each binding partner. It was also observed that the secondary structures at the Hb-Hp interface are oriented as coils and α-helices. When dissecting the interface in more detail, it is obvious that several tyrosine residues of Hb, particularly β145Tyr, α42Tyr and α140Tyr, are buried in the complex and protected from further oxidative reactions. Such finding opens up new avenues for the design of Hp mimics which may be used as alternative clinical Hb scavengers.

Redox Reactions of Hemoglobin: Mechanisms of Toxicity and Control

In the last several years, significant work has been done studying hemoglobin (Hb) oxidative reactions and clearance mechanisms using both in vitro and in vivo model systems. One active research area involves the study of molecular chaperones and other proteins that are thought to mitigate the toxicity of acellular Hb. For example, the plasma protein haptoglobin (Hp) and the pre-erythroid protein alpha-hemoglobin-stabilizing protein (AHSP) bind to acellular Hb and alpha-subunits of Hb, respectively, to reduce these adverse effects. Moreover, there has been significant work studying hemopexin and alpha-1 microglobulin, both of which are thought to be involved with hemin degradation. These studies have coincided with the timely publication of the first crystal structure of the Hb-Hp complex. In constructing this Forum, we have invited a number of researchers in the area of Hb and myoglobin (Mb) redox biochemistry, as well as those who have contributed fundamentally to our knowledge of Hp function. Our goal has been to update this critically important research area, because we believe that it will ultimately impact the practice of transfusion medicine in a number of important ways.

Haptoglobin gene polymorphisms in peri-implantitis and chronic periodontitis

The haptoglobin-hemoglobin (Hp-Hb) complex plays a significant role in regulating immune responses and acts as a bacteriostatic agent. Haptoglobin polymorphisms result in different Hb binding affinities. This study sought to assess whether Hp2-2 could be a genetic determinant for increasing the risk of peri-implantitis and chronic periodontitis. Of the Iranian subjects referred to the Department of Periodontics, Shahid Beheshti University, Tehran, 203 were entered into the study, including 117 patients and 86 periodontally healthy individuals. Polymerase chain reaction (PCR) was performed for genotyping. Data were analyzed by Kruskal-Wallis test using the SPSS statistics software package. The prevalence of Hp2-2, 2-1, and 1-1 did not differ significantly between patients and healthy subjects (P>0.05). The highest frequencies of Hp1-1, 2-1, and 2-2 genotypes were seen in the control (7%), peri-implantitis (51%) and periodontitis (64%) groups, respectively. Haptoglobin polymorphisms may not play a role in development of peri-implantitis or chronic periodontitis among Iranians but we strongly suggest researchers to evaluate this polymorphism in further studies on larger sample sizes, different populations, and other types of periodontitis.

Haptoglobin polymorphism affects nitric oxide bioavailability in preeclampsia

Studies showed elevated cell-free hemoglobin (Hb) in preeclampsia (PE), and Hb reacts with nitric oxide (NO), decreasing its bioavailability. Haptoglobin (Hp) is a polymorphic protein (Hp1-1, Hp2-1 and Hp2-2) that binds Hb to form a complex that is removed from circulation, thus preventing Hb-driven oxidative stress and NO scavenging. Hp protein products differ in biochemical and biophysical properties, which reflects on the Hb-Hp complex clearance rate. We hypothesized that Hp phenotypes modulate NO bioavailability by influencing NO consumption in PE. We studied 92 PE subjects and 105 normal pregnant women (NP). Hp genotypes were determined using real-time PCR. To assess NO bioavailability, we measured plasma nitrite using an ozone-based chemiluminescence assay. Plasma Hb and Hp were assessed with commercial immunoassays. A NO consumption assay was used to measure NO consumption. We found no differences in Hp genotype frequencies between PE and NP groups. Hp genotypes had no effects on plasma heme levels, NO consumption and plasma nitrite in NP. However, in PE, Hp2-1 and Hp2-2 were associated with higher plasma heme levels (48 and 55% higher, respectively; P<0.05), increased NO consumption (42 and 44% more, respectively; P<0.05) and lower plasma nitrite (39% less for Hp2-2; P<0.05) compared with Hp1-1. These findings indicate that although Hp genotype does not affect the risk of PE, Hp1-1 genotype may exert a protective role in PE by reducing NO scavenging, whereas Hp2-1 and Hp2-2 further may aggravate PE by reducing NO bioavailability.

Trapping of Human Hemoglobin by Haptoglobin: Molecular Mechanisms and Clinical Applications

Haptoglobin (Hp) is an abundant plasma protein controlling the fate of hemoglobin (Hb) released from red blood cells after intravascular hemolysis. The complex formed between Hp and Hb is extraordinary strong, and once formed, this protein-protein association can be considered irreversible. A model of the Hp-Hb complex has been generated and the first steps toward understanding the mechanism behind the shielding effects of Hp have been taken. The clinical potential of the complex for modulating inflammatory reactions and for functioning as an Hb-based oxygen carrier have been described. The three-dimensional structure of the Hp-Hb complex is unknown. Moreover, Hp is not a homogeneous protein. There are two common alleles at the Hp genetic locus denoted Hp1 and Hp2, which when analyzed on the protein levels result in differences between their physiological behavior, particularly in their shielding against Hb-driven oxidative stress. Additional cysteine residues on the α-subunit allow Hp2 to form a variety of native multimers, which influence the biophysical and biological properties of Hp. The multimeric conformations, in turn, also modulate the glycosylation patterns of Hp by steric hindrance. A detailed analysis of the influence of Hp glycosylation will be instrumental to generate a deeper understanding of its biological function. Several pathological conditions also modify the glycan compositions allowing Hp to be potentially used as a marker protein for these disorders.

Plasma Clearance of Hemoglobin and Haptoglobin in Mice and Effect of CD163 Gene Targeting Disruption

In humans, plasma haptoglobin (Hp) and the macrophage receptor CD163 promote a fast scavenging of hemoglobin (Hb). In the present study, we have compared the mouse and human CD163-mediated binding and uptake of Hb and HpHb complex in vitro and characterized the CD163-mediated plasma clearance of Hb in CD163 gene knockout mice and controls. Contrary to human Hp, mouse Hp did not promote high-affinity binding to CD163. This difference between mouse and man was evident both by analysis of the binding of purified proteins and by ligand uptake studies in CD163-transfected cells. Plasma clearance studies in mice showed a fast clearance (half-life few minutes) of fluorescently labeled mouse Hb with the highest uptake in the kidney and liver. HPLC analysis of serum showed that the clearance curve exhibited a two-phase decay with a faster clearance of Hb than plasma-formed HpHb. In CD163-deficient mice, the overall clearance of Hb was slightly slower and followed a one-phase decay. In conclusion, mouse Hp does not promote high-affinity binding of mouse Hb to CD163, and noncomplexed mouse Hb has a higher CD163 affinity than human Hb has. Moreover, CD163-mediated uptake in mice seems to only account for a part of the Hb clearance. The new data further underscore the fact that the Hp system in man seems to have a broader and more sophisticated role. This has major implications in the translation of data on Hb metabolism from mouse to man.

Lesional Accumulation of CD163+ Macrophages/microglia in Rat Traumatic Brain Injury

A robust neuroinflammation, contributing to the development of secondary injury, is a common histopathological feature of traumatic brain injury (TBI). Characterization of leukocytic subpopulations contributing to the early infiltration of the damaged tissue might aid in further understanding of lesion development. Reactive macrophages/microglia can exert protective or damaging effects in TBI. CD163 is considered a marker of M2 (alternatively activated) macrophages. Therefore we investigated the accumulation of CD163+ macrophages/microglia in the brain of TBI rats. TBI was induced in rats using an open skull weight-drop contusion model and the accumulation of CD163+ cells was analyzed by immunohistochemistry. In normal rat brains, CD163 was expressed by meningeal, choroid plexus and perivascular macrophages. Significant parenchymal CD163+ cell accumulation was observed two days post TBI and continuously increased in the investigated survival time. The accumulated CD163+ cells were mainly distributed to the lesional areas and exhibited macrophage phenotypes with amoeboid morphologic characteristics but not activated microglial phenotypes with hypertrophic morphology and thick processes. Double-labeling experiments showed that most CD163+ cells co-expressed heme oxygenase-1 (HO-1). In addition, in vitro incubating of macrophage RAW264.7 cells or primary peritoneal macrophages with hemoglobin– haptoglobin (Hb-Hp) complex suppressed LPS-induced inflammatory macrophages phenotype and induced CD163 and HO-1 upregulation, indicating that CD163+ macrophages/microglia in TBI might have anti-inflammatory effects. And further study is necessary to identify functions of these cells in TBI.

Slow Histidine H/D Exchange Protocol for Thermodynamic Analysis of Protein Folding and Stability Using Mass Spectrometry

Described here is a mass spectrometry-based protocol to study the thermodynamic stability of proteins and protein-ligand complexes using the chemical denaturant dependence of the slow H/D exchange reaction of the imidazole C(2) proton in histidine side chains. The protocol is developed using several model protein systems including: ribonuclease (Rnase) A, myoglobin, bovine carbonic anhydrase (BCA) II, hemoglobin (Hb), and the hemoglobin-haptoglobin (Hb-Hp) protein complex. Folding free energies consistent with those previously determined by other more conventional techniques were obtained for the two-state folding proteins, Rnase A and myoglobin. The protocol successfully detected a previously observed partially unfolded intermediate stabilized in the BCA II folding/unfolding reaction, and it could be used to generate a K(d) value of 0.24 nM for the Hb-Hp complex. The compatibility of the protocol with conventional mass spectrometry-based proteomic sample preparation and analysis methods was also demonstrated in an experiment in which the protocol was used to detect the binding of zinc to superoxide dismutase in the yeast cell lysate sample. The yeast cell sample analyses also helped define the scope of the technique, which requires the presence of globally protected histidine residues in a protein's three-dimensional structure for successful application.

Erythrocytes, leukocytes and platelets as a source of oxidative stress in chronic vascular diseases: Detoxifying mechanisms and potential therapeutic options

Oxidative stress is involved in the chronic pathological vascular remodelling of both abdominal aortic aneurysm and occlusive atherosclerosis. Red blood cells (RBCs), leukocytes and platelets present in both, aneurysmal intraluminal thrombus and intraplaque haemorraghes, could be involved in the redox imbalance inside diseased arterial tissues. RBCs haemolysis may release the pro-oxidant haemoglobin (Hb), which transfers heme to tissue and low-density lipoproteins. Heme-iron potentiates molecular, cell and tissue toxicity mediated by leukocytes and other sources of reactive oxygen species (ROS). Polymorphonuclear neutrophils release myeloperoxidase and, along with activated platelets, produce superoxide mediated by NADPH oxidase, causing oxidative damage. In response to this pro-oxidant milieu, several antioxidant molecules of plasma or cell origin can prevent ROS production. Free Hb binds to haptoglobin (Hp) and once Hp-Hb complex is endocytosed by CD163, liberated heme is converted into less toxic compounds by heme oxygenase-1. Iron homeostasis is mainly regulated by transferrin, which transports ferric ions to other cells. Transferrin-bound iron is internalised via endocytosis mediated by transferrin receptor. Once inside the cell, iron is mainly stored by ferritin. Other non hemo-iron related antioxidant enzymes (e.g. superoxide dismutase, catalase, thioredoxin and peroxiredoxin) are also involved in redox modulation in vascular remodelling. Oxidative stress is a main determinant of chronic pathological remodelling of the arterial wall, partially linked to the presence of RBCs, leukocytes, platelets and oxidised fibrin within tissue and to the imbalance between pro-/anti-oxidant molecules. Understanding the complex mechanisms underlying redox imbalance could help to define novel potential targets to decrease atherothrombotic risk.

Patients With Colorectal Cancer Are Characterized by Increased Concentration of Fecal Hb-Hp Complex, Myeloperoxidase, and Secretory IgA

The aim of our study was to evaluate the use of fecal hemoglobin-haptoglobin (Hb-Hp) complex, myeloperoxidase (MPO), and secretory IgA as potential diagnostic markers for the screening of colorectal cancer. Our study consisted of 190 participants (150 patients and 40 healthy individuals) aged 40 to 70 years who underwent complete colonoscopy. From each participant, a stool sample was collected 1 day before colonoscopy. The patients were instructed to collect another sample after the fourteenth postoperative day. No special diet was recommended. In the control group, all fecal markers were within normal values in most participants. Colorectal tumors were accompanied by a highly significant increase in all markers. The median value of Hb-Hp complex was 23.22±10.02 (P<0.0001) whereas the median values of MPO and sIgA were 14936.43±9580.83 (P<0.0001) and 6503.38±2794.87 (P<0.0001), respectively. The sensitivity and specificity of Hb-Hp complex, MPO, and sIgA are 95.3% and 87.5%, 96% and 87%, and 90.8% and 85%, respectively. The difference of preoperative and postoperative values of the patients was statistically significant for all markers. After surgical intervention, the value of Hb-Hp complex was normalized in 133 of the 150 patients (88.7%). The postoperative values of MPO were within normal limits in 131 patients (87.3%), whereas those of secretory IgA were within normal limits in 103 of the 150 patients (68.5%). Fecal Hb-Hp complex, MPO, and sIgA can effectively be used as screening tools for the early diagnosis of colorectal cancer, and for the postoperative follow-up of patients.

Liposomes surface conjugated with human hemoglobin target delivery to macrophages

Current strategies to deliver therapeutic molecules to specific cell and tissue types rely on conjugation of antibodies and other targeting ligands directly to the therapeutic molecule itself or its carrier. This work describes a novel strategy to deliver therapeutic molecules into macrophages that takes advantage of the native hemoglobin (Hb) scavenging activity of plasma haptoglobin (Hp) and the subsequent uptake of the Hb-Hp complex into macrophages via CD163 receptor-mediated endocytosis. The drug delivery system described in this work consists of Hb decorated liposomes that can encapsulate any therapeutic molecule of interest, in this case the model fluorescent dye calcein was used in this study. The results of this study clearly demonstrate that this delivery system is specific towards macrophages and demonstrates the feasibility of using this approach in targeted drug delivery.

Heterotropic Effect of β-lactam Antibiotics on Antioxidant Property of Haptoglobin (2-2)-Hemoglobin Complex.

Haptoglobin (Hp) is a mammalian serum glycoprotein showing a genetic polymorphism with three types, 1-1, 2-2 and 1-2. Hp appears to conserve the recycling of heme-iron by forming an essentially irreversible but non-covalent complex with hemoglobin which is released into the plasma by erythrocyte lysis. As an important consequence, Haptoglobin-Hemoglobin complex (Hp-Hb) shows considerable antioxidant property. In this study, antioxidant activity of Hp (2-2)-Hb complex on hydrogen peroxide has been studied and analyzed in the absence and presence of two beta-lactam antibiotics in-vitro. For this purpose, non-Michaelis behavior of peroxidase activity of Hp (2-2)-Hb complex was analyzed using Eadie-Hofstee, Clearance and Hill plots, in the absence and presence of pharmaceutical dose of ampicillin and coamoxiclav. The results have shown that peroxidase activity of Hp (2-2)-Hb complex is modulated via homotropic effect of hydrogen peroxide as an allostric substrate. On the other hand antioxidant property of Hp (2-2)-Hb complex increased via heterotropic effect of both antibiotics on the peroxidase activity of the complex. Both drugs also have mild effect on quality of homotropic property of the peroxidase activity of Hp (2-2)-Hb complex. Therefore, it can be concluded from our study that both beta-lactam antibiotics can increase peroxidase activity of Hp (2-2)-Hb complex via heterotropic effect. Thus, the two antibiotics (especially ampicillin) may help those individuals with Hp (2-2) phenotype to improve the Hp-Hb complex efficiency of removing hydrogen peroxide from serum under oxidative stress. This can be important in the individuals with phenotype Hp 2-2 who have less antioxidant activity relative to other phenotypes and are susceptible to cardiovascular disorders, as has been reported by other researchers.

Cellular and Molecular Remodeling of the Endocytic Pathway during Differentiation of Trypanosoma brucei Bloodstream Forms

During the course of mammalian infection, African trypanosomes undergo extensive cellular differentiation, as actively dividing long slender (SL) forms progressively transform into intermediate (I) forms and finally quiescent G(1)/G(0)-locked short stumpy (ST) forms. ST forms maintain adaptations compatible with their survival in the mammalian bloodstream, such as high endocytic activity, but they already show preadaptations to the insect midgut conditions. The nutritional requirements of ST forms must differ from those of SL forms because the ST forms stop multiplying. We report that the uptake of several ligands was reduced in ST forms compared with that in SL forms. In particular, the haptoglobin-hemoglobin (Hp-Hb) complex was no longer taken up due to dramatic downregulation of its cognate receptor, TbHpHbR. As this receptor also allows uptake of trypanolytic particles from human serum, ST forms were resistant to trypanolysis by human serum lipoproteins. These observations allowed both flow cytometry analysis of SL-to-ST differentiation and the generation of homogeneous ST populations after positive selection upon exposure to trypanolytic particles. In addition, we observed that in ST forms the lysosome relocates anterior to the nucleus. Altogether, we identified novel morphological and molecular features that characterize SL-to-ST differentiation.