Haptoglobin concentrations in free-range and temporarily captive juvenile steller sea lions

Haptoglobin (Hp) is an acute-phase protein synthesized in the liver that circulates at elevated concentrations in response to tissue damage caused by inflammation, infection, and trauma. As part of a larger study, sera Hp concentrations were measured in temporarily captive (n = 21) and free-range (n = 38) western stock juvenile Steller sea lions (Eumetopias jubatus) sampled from 2003 to 2006. Baseline Hp concentration at time of capture was 133.3 +/- 17.4 mg/dl. Temporarily captive animals exhibited a 3.2-fold increase in Hp concentrations during the first 4 wk of captivity, followed by a return to entry levels by week 5. Haptoglobin levels were not influenced by age, season, or parasite load. There was a significant positive correlation between Hp concentrations and white blood cell count (P < 0.001) and globulin levels (P < 0.001) and a negative correlation to red blood cell count and hematocrit (P < 0.001 for both). There was no correlation between Hp levels and platelet count (P = 0.095) or hemoglobin (P = 0.457). Routine blubber biopsies collected under gas anesthesia did not produce a measurable Hp response. One animal with a large abscess had an Hp spike of 1,006.0 mg/dl that returned to entry levels after treatment. In conclusion, serum Hp levels correlate to the stable clinical health status observed during captivity, with moderate Hp response during capture and initial acclimation to captivity and acute response to inflammation and infection.     

Haptoglobin and lipoperoxides in rabbit plasma under turpentine- and endotoxin-induced inflammation

Inflammation was induced in rabbits by injection of turpentine or/and endotoxin. Haptoglobin (Hp) and lipoperoxide (as malondialdehyde - MDA) concentrations in rabbit plasma, were measured. In some experiments indomethacin was administered, as a specific control of anti-inflammatory action. In the course of the reagents-induced inflammation there existed positive correlation between changes in Hp and MDA levels. Indomethacin was found to eliminate this effect. Under the model system of experiments, Hp as presumed endogenous inhibitor of prostaglandin synthesis, even in concentrations in rabbits' blood 15-20 times higher than normal, did not exert any protective action against inflammation.     

Elevated plasma concentrations of haptoglobin in European brown bears during hibernation

Haptoglobin (Hp), a hemoglobin-binding protein, is known as an acute phase protein and increases during the acute phase of inflammation in most mammals. We reported previously in brown bears that the mean Hp concentrations were higher in blood samples obtained in winter than those in spring. To examine a possible relation of the seasonal variations of Hp to hibernation, in the present study, we measured the plasma concentrations of Hp as well as some other acute phase proteins (alpha(2)-macroglobulin, alpha(1)-antitrypsin, C-reactive protein) in 6 European brown bears (Ursus arctos), from which blood samples were obtained at 5-6 different months of year including February, the time of hibernation. The Hp concentrations showed clear seasonal variations, being highest in February. The alpha(2)-macroglobulin concentrations also showed a similar but much smaller rise in February, but those of alpha(1)-antitrypsin and C-reactive protein did not show any seasonal variations. Our results suggest that the seasonal variation of plasma Hp concentration in brown bears is associated with a hibernation-specific mechanism more than that of acute phase response.     

Acute suppression of albumin synthesis in systemic inflammatory disease: an individually graded response of rat hepatocytes.

The effects of an inflammatory insult on albumin of the rat liver were investigated at the cellular level and were correlated with serum albumin concentration. After SC injection of turpentine, the livers were perfused and fixed in vivo; serial liver sections were stained using a streptavidin-ABC-immunoperoxidase technique with an antibody to rat albumin. Albumin and total protein were measured at intervals after turpentine injection in whole livers and in serum. Fibrinogen was determined in plasma only. Twenty-four hours after turpentine injection serum albumin had dropped by 25% and was at 50% of its initial value at Day 3. Serum fibrinogen increased 2.4-fold within 24 hr and decreased thereafter. Liver homogenates showed no significant changes in albumin concentration. Immunohistochemically, all hepatocytes stained positive for albumin in normal animals. During inflammation, the immunostainable albumin content vanished entirely in a majority of all hepatocytes while remaining unchanged in other cells, thus producing a strikingly patchy staining pattern. No signs of resumption of albumin accumulation in depleted hepatocytes were seen after 8 days, despite a clear trend towards normalization of serum albumin concentration. These results suggest that individual hepatocytes differ widely in their response to agents that suppress albumin synthesis in an acute-phase reaction.     

Modulation of the acute phase response by altered expression of the IL-1 type 1 receptor or IL-1ra

A complete understanding of the role for endogenously produced interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-alpha), and IL-1 receptor antagonist (IL-1ra) in the acute phase response to inflammation remains unknown. In the present studies, knockout mice lacking either a functional IL-1 type I receptor (IL-1RI(-/-)), a TNF type I receptor (TNFR-I(-/-)), or both IL-1 type I and TNF type I receptors (IL-1RI(-/-)/TNFR-I(-/-)) received a turpentine abscess. Additional mice deficient in IL-1ra protein (IL-1ra(-/-)) or overexpressing IL-1ra protein (IL-1ra(tg)) were similarly treated. After a turpentine abscess, IL-1 receptor knockout mice exhibited an attenuated inflammatory response compared with wild-type or animals lacking a functional TNFR-I. Mice overexpressing IL-1ra also had an attenuated hepatic acute phase protein response, whereas IL-1ra knockout mice had a significantly greater hepatic acute phase response. We conclude that the inflammatory response to a turpentine abscess is the result of a balance between IL-1ra expression and IL-1 binding to its type I receptor. Endogenously produced IL-1ra plays a central role in mitigating the magnitude of the IL-1-mediated inflammatory response and, ultimately, the outcome to a turpentine abscess.     

A genome-wide association study identifies rs2000999 as a strong genetic determinant of circulating haptoglobin levels

Haptoglobin is an acute phase inflammatory marker. Its main function is to bind hemoglobin released from erythrocytes to aid its elimination, and thereby haptoglobin prevents the generation of reactive oxygen species in the blood. Haptoglobin levels have been repeatedly associated with a variety of inflammation-linked infectious and non-infectious diseases, including malaria, tuberculosis, human immunodeficiency virus, hepatitis C, diabetes, carotid atherosclerosis, and acute myocardial infarction. However, a comprehensive genetic assessment of the inter-individual variability of circulating haptoglobin levels has not been conducted so far.We used a genome-wide association study initially conducted in 631 French children followed by a replication in three additional European sample sets and we identified a common single nucleotide polymorphism (SNP), rs2000999 located in the Haptoglobin gene (HP) as a strong genetic predictor of circulating Haptoglobin levels (P(overall) = 8.1 × 10(-59)), explaining 45.4% of its genetic variability (11.8% of Hp global variance). The functional relevance of rs2000999 was further demonstrated by its specific association with HP mRNA levels (β = 0.23 ± 0.08, P = 0.007). Finally, SNP rs2000999 was associated with decreased total and low-density lipoprotein cholesterol in 8,789 European children (P(total cholesterol) = 0.002 and P(LDL) = 0.0008).Given the central position of haptoglobin in many inflammation-related metabolic pathways, the relevance of rs2000999 genotyping when evaluating haptoglobin concentration should be further investigated in order to improve its diagnostic/therapeutic and/or prevention impact.     

The Haptoglobin β chain as a supportive biomarker for human lung cancers

Haptoglobin (Hp) is produced as an acute phase reactant during inflammation, infection, malignant diseases, and several cancers. In proteomics analysis using human blood samples, the Hp peptide levels were about 3-fold higher in lung cancer patients versus normal individuals. This study is aimed at analyzing the elevation of which chain of Hp is closely related to lung cancers and can be a serum biomarker for lung cancers. In Western blot (WB) analysis, we found that the Hp β chain can be a better diagnostic biomarker for lung cancers. In the result of the Hp β chain ELISA developed by us, the concentrations of the Hp β chain in the sera increased about 4-fold in 190 lung adenocarcinoma patients versus 190 healthy controls (8.0 ± 3.8 μg ml(-1)vs. 1.9 ± 1.2 μg ml(-1)). ELISA data showed that the serum levels of the Hp β chain in breast cancer (1.5 ± 0.5 μg ml(-1)) and hepatocellular carcinoma (HCC) (1.4 ± 1.0 μg ml(-1)) patients remained similar to those of healthy controls. Compared to lung adenocarcinoma, the Hp β chain levels in the plasma of patients with other respiratory diseases such as tuberculosis (TBC), idiopathic pulmonary fibrosis (IPF) and bronchial asthma (BA) were closer to those of healthy controls. Our data suggest that an increase of the Hp β chain can be a potential serum biomarker for lung cancers.     

Acute phase protein haptoglobin in blood plasma samples of harbour seals (Phoca vitulina) of the Wadden Sea and of the isle Helgoland

Haptoglobin (Hp) which is synthesized in response to infection, inflammation, trauma or toxicological damage is known as a major acute phase protein in numerous species. Quantification of the circulating concentration of this protein can provide an objective measure of the health status, but there is a lack of investigations on harbour seals. We investigated the Hp concentration in samples of 123 seals (Phoca vitulina) from the German and Danish Wadden Sea to study physiological ranges of Hp levels. Hp levels between 2002, the end of the phocine distemper virus epidemic (PDV), and 2007 were considered, and Hp concentrations between animals of different sex, ages as well as living areas were compared. Furthermore, as a case study, six animals from the open sea isle Helgoland were investigated in 2006. Influences on the health status of the seal population e.g. the PDV epidemic were reflected by increased Hp levels in North Sea seals in 2002. The results of the Wadden Sea seals showed no significant age-, sex-, or geographical area-related differences. Interestingly, for the seals of the open sea isle Helgoland higher Hp values were measured compared to the Wadden Sea seals. The present study demonstrates that Hp can be used as a diagnostic tool to monitor the health status of harbour seals.     

Proximal tubule haptoglobin gene activation is an integral component of the acute kidney injury "stress response"

Haptoglobin (Hp) synthesis occurs almost exclusively in liver, and it is rapidly upregulated in response to stress. Because many of the pathways that initiate hepatic Hp synthesis are also operative during acute kidney injury (AKI), we tested whether AKI activates the renal cortical Hp gene. CD-1 mice were subjected to six diverse AKI models: ischemia-reperfusion, glycerol injection, cisplatin nephrotoxicity, myoglobinuria, endotoxemia, and bilateral ureteral obstruction. Renal cortical Hp gene induction was determined either 4-72 h or 1-3 wk later by measuring Hp mRNA and protein levels. Relative renal vs. hepatic Hp gene induction during endotoxemia was also assessed. Each form of AKI induced striking and sustained Hp mRNA increases, leading to ～10- to 100-fold renal Hp protein elevations (ELISA; Western blot). Immunohistochemistry, and isolated proximal tubule assessments, indicated that the proximal tubule was the dominant (if not only) site of the renal Hp increases. Corresponding urinary and plasma Hp elevations were surrogate markers of this response. Endotoxemia evoked 25-fold greater Hp mRNA increases in kidney vs. liver, indicating marked renal Hp gene reactivity. Clinical relevance of these findings was suggested by observations that urine samples from 16 patients with established AKI had statistically higher (～12×) urinary Hp levels than urine samples from either normal subjects or from 15 patients with chronic kidney disease. These AKI-associated urinary Hp increases mirrored those seen for urinary neutrophil gelatinase-associated lipoprotein, a well accepted AKI biomarker gene. In summary, these studies provide the first evidence that AKI evokes rapid, marked, and sustained induction of the proximal tubule Hp gene. Hp's known antioxidant, as well as its protean pro- and anti-inflammatory, actions imply potentially diverse effects on the evolution of acute tubular injury.     

Study of biochemical behavior of some exported and nonexported hepatic proteins during an acute inflammatory reaction in the rat

Haptoglobin, albumin, glucose-6-phosphatase, p-nitrophenol uridine diphosphate (UDP)-glucuronosyltransferase and cytochrome P-450 were measured in liver microsomes from normal rats and from rats undergoing an acute inflammatory reaction (AIR) induced either by subcutaneous administration of turpentine or by intrapleural injection of calcium pyrophosphate. 24 h after the beginning of the AIR induced by subcutaneous administration of turpentine, haptoglobin and albumin, two exported proteins, had risen to a peak (+313%), and dropped considerably (-52%) whereas nonexported protein levels did not change except for cytochrome P-450, which diminished (-38%). In the same way, intrapleural injection of calcium pyrophosphate was followed after 24 h by significant but smaller variations in haptoglobin (+60%) and cytochrome P-450 (-20%) concentrations. Albumin levels, glucose-6-phosphatase and p-nitrophenol UDP-glucuronosyltransferase activities were unchanged in this experimental model. The drop in cytochrome P-450 under all these conditions and also the diminution of albumin in the first model suggest that all the proteins produced by liver cells might not be synthesized in equal amounts. The decrease in cytochrome P-450 could interfere in hepatic drug metabolism during an AIR.     

Haptoglobin gene subtypes in three Brazilian population groups of different ethnicities

Haptoglobin is a plasma hemoglobin-binding protein that limits iron loss during normal erythrocyte turnover and hemolysis, thereby preventing oxidative damage mediated by iron excess in the circulation. Haptoglobin polymorphism in humans, characterized by the Hp(*1) and Hp (*2) alleles, results in distinct phenotypes known as Hp1-1, Hp2-1 and Hp2-2, whose frequencies vary according to the ethnic origin of the population. The Hp(*1) allele has two subtypes, Hp (*1F) and Hp (*1S) , that also vary in their frequencies among populations worldwide. In this work, we examined the distribution frequencies of haptoglobin subtypes in three Brazilian population groups of different ethnicities. The haptoglobin genotypes of Kayabi Amerindians (n = 56), Kalunga Afro-descendants (n = 70) and an urban population (n = 132) were determined by allele-specific PCR. The Hp(*1F) allele frequency was highest in Kalunga (29.3%) and lowest in Kayabi (2.6%). The Hp(*1F)/Hp(*1S) allele frequency ratios were 0.6, 1.0 and 0.26 for the Kayabi, Kalunga and urban populations, respectively. This variation was attributable largely to the Hp(*1F) allele. However, despite the large variation in Hp(*1F) frequencies, results of F (ST) (0.0291) indicated slight genetic differentiation among subpopulations of the general Brazilian population studied here. This is the first Brazilian report of variations in the Hp(*1F) and Hp(*1S) frequencies among non-Amerindian Brazilians.     

Variation in inflammation as a correlate of range expansion in Kenyan house sparrows

Many introduced animals harbor fewer parasites than native ones. This “enemy release” can select for individuals that bias resources away from parasite resistance traits, including immune functions, and towards traits that enhance success in new areas. One vertebrate example that supports this hypothesis involves house sparrows (Passer domesticus) and Eurasian tree sparrows (Passer montanus) introduced to St. Louis, MO, USA, over 150 years ago. Since ca. 1850, house sparrows have colonized most of North America whereas tree sparrows have expanded little from the area of introduction. The more successful house sparrows now exhibit weaker inflammatory responses than the less successful tree sparrows, which supports the possibility that diminished investments in immune defense may have been conducive to the initial colonization by the more successful species. The goal of the present study was to determine whether damped inflammation generally facilitates invasion by comparing inflammatory markers between house sparrows invading Kenya and a native congener. House sparrows arrived in Mombasa, Kenya, about 50 years ago whereas rufous sparrows (Passer ruficinctus) are native but ecologically similar. We predicted that if inflammation mediated invasion success, Kenyan house sparrows would mount weaker inflammatory responses than the native species. Complete Freund’s adjuvant (CFA), a strong inflammatory stimulus, increased body mass in house sparrows, a result unprecedented in any other vertebrate. Haptoglobin (Hp), a multi-functional acute phase protein, was elevated by CFA in both species but rufous sparrows maintained more Hp than house sparrows irrespective of treatment. Lysozyme, a broadly effective antimicrobial enzyme, was reduced by CFA in both species, but not differentially so. Corticosterone was unaffected by CFA in either species, but elevated in both relative to free-living individuals.     

Plasma Haptoglobin Concentrations Are Elevated in Patients with Coronary Artery Disease

Inflammation underlies the development and progression of coronary artery plaques. Haptoglobin (Hp) is an acute phase protein, the synthesis of which is increased during inflammation. The aim of this study was to investigate plasma Hp concentrations and phenotype in patients with coronary artery disease (CAD). We recruited 359 patients with fixed luminal stenosis ≥50% in at least one coronary artery (CAD group) and 83 patients with luminal stenosis ≤40%, normal ejection fraction, and normal regional wall motion (control group). Plasma Hp concentrations were measured using a phenotype-specific enzyme-linked immunosorbent assay. Hp phenotype was determined by native polyacrylamide gel electrophoresis. Plasma lipid concentrations were measured. Plasma Hp concentrations were significantly higher in the CAD compared with the control group (262.4±144.2 vs 176.0±86.7 ng/mL, P<0.001); however, there was no between group difference in the distribution of Hp phenotype (1-1 = 7.5% vs 7.2%; 2-1 = 40.4% vs 42.2%; 2-2 = 52.1% vs 50.6%). Stepwise multivariate logistic regression revealed that high Hp concentrations (odds ratio [OR] = 5.865), male sex (OR = 3.689), hypertension (OR = 2.632), diabetes mellitus (OR = 3.300), and low-density lipoprotein concentrations (OR = 1.480) were independently associated with CAD (all P<0.05). Hp phenotype was not associated with CAD. Plasma Hp concentrations were significantly correlated with the severity of luminal stenosis (r = 0.236, P<0.001). Our findings suggest that plasma Hp concentrations may be elevated in patients with CAD. There does not appear to be any relationship between Hp phenotype and CAD.     

Turpentine-induced inflammation reduces the hepatic expression of the multiple drug resistance gene, the plasma cholesterol concentration and the development of atherosclerosis in apolipoprotein E deficient mice

We aimed to investigate the effect of turpentine-induced inflammation in an atherosclerosis-prone murine model. We have induced a chronic aseptic inflammation in apolipoprotein E-deficient mice, with or without a dietary supplement of aspirin (n = 10, each), by the injection of a mixture (1:1) of turpentine and olive oil in the hind limb twice weekly for a period of 12 weeks. Control animals were injected with olive oil alone (n = 10). The control mice did show any alteration neither in plasma nor at the site of injection. Turpentine-treated mice showed a significant increase in plasma TNF-alpha and SAA concentrations which indicated a systemic inflammatory response that was not substantially affected by aspirin. Also, turpentine injections significantly reduced the plasma cholesterol concentration, probably decreasing intestinal cholesterol re-absorption, and attenuated the size of atherosclerotic lesion. Both effects were minimally influenced by aspirin. The burden of atherosclerosis correlated with plasma lipid levels but not with plasma inflammatory markers. Finally, there was a concomitant decrease in the expression of the hepatic mdr1b gene that correlated with the decrease in plasma cholesterol concentration. Therefore, we conclude that mdr1 is an additional factor to consider in the complexity of alterations in cholesterol metabolism that occur in this model.     

Acute phase response in the horse: plasma protein changes associated with adjuvant induced inflammation

The induction of an acute phase response in four horses by adjuvant administration was used to examine the effect on the levels of plasma proteins. Blood parameters (packed cell volume, total plasma protein, red blood cell count, haemoglobin concentration) were monitored to follow the progress of the acute phase response in parallel with the examination of plasma proteins. Plasma protein levels were determined by densitometry from the electrophoretic patterns of three different gel systems. Haptoglobin and alpha 1 B glycoprotein were shown to be positive acute phase reactants whereas albumin was a negative acute phase reactant. Plasma esterase and proteins of the Pi system did not change following the experimental inflammation.     

Individual Variation in Levels of Haptoglobin-Related Protein in Children from Gabon

Background

Haptoglobin related protein (Hpr) is a key component of trypanosome lytic factors (TLF), a subset of high-density lipoproteins (HDL) that form the first line of human defence against African trypanosomes. Hpr, like haptoglobin (Hp) can bind to hemoglobin (Hb) and it is the Hpr-Hb complexes which bind to these parasites allowing uptake of TLF. This unique form of innate immunity is primate-specific. To date, there have been no population studies of plasma levels of Hpr, particularly in relation to hemolysis and a high prevalence of ahaptoglobinemia as found in malaria endemic areas.

Methods and Principal Findings

We developed a specific enzyme-linked immunosorbent assay to measure levels of plasma Hpr in Gabonese children sampled during a period of seasonal malaria transmission when acute phase responses (APR), malaria infection and associated hemolysis were prevalent. Median Hpr concentration was 0.28 mg/ml (range 0.03–1.1). This was 5-fold higher than that found in Caucasian children (0.049 mg/ml, range 0.002–0.26) with no evidence of an APR. A general linear model was used to investigate associations between Hpr levels, host polymorphisms, parasitological factors and the acute phase proteins, Hp, C-reactive protein (CRP) and albumin. Levels of Hpr were associated with Hp genotype, decreased with age and were higher in females. Hpr concentration was strongly correlated with that of Hp, but not CRP.

Conclusions/Significance

Individual variation in Hpr levels was related to Hp level, Hp genotype, demographics, malaria status and the APR. The strong correlations between plasma levels of Hp and Hpr suggest that they are regulated by similar mechanisms. These population-based observations indicate that a more dynamic view of the relative roles of Hpr and Hpr-Hb complexes needs to be considered in understanding innate immunity to African trypanosomes and possibly other pathogens including the newly discovered Plasmodium spp of humans and primates.     

Partial characterization of alpha 1-acid glycoprotein isolated from mouse serum

AGP was purified from mouse serum by perchloric acid treatment and CM-Sepharose chromatography. Induction of inflammation with turpentine resulted in a 10-fold increase in the serum level of mouse AGP, indicating mouse AGP is an acute phase reactant. Biochemical characterization of mouse AGP indicated similarity with human and rat AGP.     

Haptoglobin in lung defence

Haptoglobin (Hp) has been known to be associated with the host defence response to infection and inflammation. The biological functions of Hp can be related to its ability to bind haemoglobin or to modulate immune response. Hp is expressed at a high level in lung cells, yet its protective role(s) in the lung is not known. Using transgenic mice overexpressing Hp, we demonstrated that Hp can reduce blood-induced lung injury. Hp-mediated haemoglobin catabolism in lung cells appears to be linked to iron mobilization, and may be an efficient mechanism to reduce oxidative damage associated with haemolysis.