Presence Of Haptoglobin Research Articles

Treatment of Marmoset Intracerebral Hemorrhage with Humanized Anti-HMGB1 mAb.

Intracerebral hemorrhage (ICH) is recognized as a severe clinical problem lacking effective treatment. High mobility group box-1 (HMGB1) exhibits inflammatory cytokine-like activity once released into the extracellular space from the nuclei. We previously demonstrated that intravenous injection of rat anti-HMGB1 monoclonal antibody (mAb) remarkably ameliorated brain injury in a rat ICH model. Therefore, we developed a humanized anti-HMGB1 mAb (OKY001) for clinical use. The present study examined whether and how the humanized anti-HMGB1 mAb ameliorates ICH injury in common marmosets. The results show that administration of humanized anti-HMGB1 mAb inhibited HMGB1 release from the brain into plasma, in association with a decrease of 4-hydroxynonenal (4-HNE) accumulation and a decrease in cerebral iron deposition. In addition, humanized anti-HMGB1 mAb treatment resulted in a reduction in brain injury volume at 12 d after ICH induction. Our in vitro experiment showed that recombinant HMGB1 inhibited hemoglobin uptake by macrophages through CD163 in the presence of haptoglobin, suggesting that the release of excess HMGB1 from the brain may induce a delay in hemoglobin scavenging, thereby allowing the toxic effects of hemoglobin, heme, and Fe2+ to persist. Finally, humanized anti-HMGB1 mAb reduced body weight loss and improved behavioral performance after ICH. Taken together, these results suggest that intravenous injection of humanized anti-HMGB1 mAb has potential as a novel therapeutic strategy for ICH.

Evidence of haptoglobin in the porcine female genital tract during oestrous cycle and its effect on in vitro embryo production

Recent evidence supports involvement of the acute phase protein haptoglobin in numerous events during mammalian reproduction. The present study represents an in-depth investigation of haptoglobin expression and secretion in the porcine oviduct and uterus, and assesses its effect on porcine in vitro embryo production. A systematic study was made of sows in different oestrous stages: late follicular, early luteal and late luteal stages. Relative haptoglobin mRNA abundance was quantified by RT-qPCR. In addition, expression of the protein was analysed by immunohistochemistry and the results were complemented by Western-blot and proteomic analyses of the oviductal and uterine fluids. In vitro porcine fertilization and embryo culture were carried out in the presence of haptoglobin. The results indicate that haptoglobin mRNA expression in the porcine oviduct and uterus is most abundant during the late luteal stage of the oestrous cycle. By means of Western blot and proteomic analyses haptoglobin presence was demonstrated in the oviduct epithelium and in the oviductal and uterine fluids in different stages of the oestrous cycle. The addition of haptoglobin during gamete co-incubation had no effect on sperm penetration, monospermy or efficiency rates; however, compared with the control group, blastocyst development was significantly improved when haptoglobin was present (haptoglobin: 64.50% vs. control: 37.83%; p < 0.05). In conclusion, the presence of haptoglobin in the oviduct and uterus of sows at different stages of the oestrous cycle suggests that it plays an important role in the reproduction process. The addition of haptoglobin during in vitro embryo production improved the blastocyst rates.

Nano-LC–MS/MS for the identification of proteins trapped in sorbent cartridges used for coupled plasma filtration-adsorption treatments of healthy pigs

A dedicated proteomic approach based on nano-Liquid Chromatography coupled with tandem mass spectrometry in ion trap is proposed for the analysis of proteins trapped in sorbent resin cartridges used to remove inflammatory mediators from blood by coupled plasma filtration adsorption (CPFA). The final purpose of the proposed proteomic approach was to obtain a reference map of plasma proteins trapped in CPFA sorbents used for the extracorporeal blood purification of healthy pigs, with the potential impact to design new bio-filters able to control the inflammatory imbalance under pathological conditions, such as severe sepsis. The five main steps of the proteomics analysis, (i) protein extraction from resin cartridges, (ii) two-dimensional gel electrophoresis (2D-PAGE) for protein separation and profiling, (iii) in-gel proteolytic digestion, (iv) tandem mass analysis of peptides resulting from enzymatic cleavage and (v) bioinformatics, for protein identification and post-processing validation of MS/MS data sets, have been carefully evaluated. Prior to electrophoresis, the efficiency of different extraction solutions and procedures to recovery plasma proteins trapped into the sorbents were tested. Then, a rapid one-step procedure for protein extraction was optimized. Protein bands corresponding to the main plasma proteins, namely porcine serum albumin, serotransferrin and immunoglobulins, were identified. In addition, the presence of haptoglobin, hemopexin, α-1 acid glycoprotein and fetuin-A, that are known as acute-phase reaction proteins, was observed, suggesting that CPFA resins led to a non-specifically protein depletion from plasma, rather than targeting specific molecules.

Structure and biological activity of a conformational constrained apolipoprotein A-I-derived helical peptide targeting the protein haptoglobin

The development of novel pharmacological treatments for atherosclerosis is an active research field in medicinal chemistry. It has been shown that the acute phase protein haptoglobin (Hpt) plays a role in modulating the reverse cholesterol transport binding to the HDL-major protein Apolipoprotein A-I, and it also plays a role in impairing the activity of the lecithin–cholesterol acyltransferase (LCAT). We reported that the peptide P2a in vitro and in vivo restores the LCAT activity in the presence of Hpt. Now, we have designed and characterized a conformational constrained P2a analogue, ApoAib, with the intention of improving Hpt binding and metabolic stability. Using non-proteogenic aminoisobutyric acid residues, we have obtained a well folded α-helical peptide with high proteolytic stability in serum. It binds to Hpt, impairs haptoglobin binding to HDL, and restores LCAT activity in the presence of haptoglobin. Furthermore, an interaction analysis using NMR revealed the peptide binding site involved in haptoglobin molecular recognition. In conclusion, ApoAib represents a promising candidate to improve reverse cholesterol transport for application in cardiovascular diseases.

PH‐Dependent structural ensembles of haptoglobin suppress β2‐microglobulin amyloidosis: role in lysosomal localization and extracellular protein quality control (752.7)

Several diseases involve extracellular deposition of misfolded proteins, which is associated with inflammation and local acidosis. We have investigated the chaperone‐like activity of haptoglobin towards the amyloid aggregation of β2‐microglobulin (β2m) and of α‐synuclein and its role in clearing the amyloid deposits from the extracellular space. At neutral pH, haptoglobin prevents the formation of β2m amyloid fibrils but not that of α‐synuclein. Under conditions of local acidosis, pH 5.5‐6.5, it prevents β2m fibril formation with relatively decreased efficiency. We have investigated the structural and stability aspects of haptoglobin as a function of pH. Between pH 5.5‐8.0, it exists in a continuum of conformational states with subtle perturbation in tertiary structure, moderately exposed hydrophobic surfaces and distinct latent heat of unfolding. Below pH 5.0 it exists in a molten‐globule‐like conformation with no chaperone activity. Live cell imaging and flow cytometry studies show that the species of β2m formed in the presence of haptoglobin are not cytotoxic and readily undergo lysosomal degradation. Analytical ultracentrifugation and dot blot studies reveal that haptoglobin interacts with prefibrillar oligomeric species formed during β2m fibril formation. Thus, haptoglobin is an extracellular chaperone with promising therapeutic implications in extracellular amyloid diseases.

The Extracellular Chaperone Haptoglobin Prevents Serum Fatty Acid-promoted Amyloid Fibril Formation of β2-Microglobulin, Resistance to Lysosomal Degradation, and Cytotoxicity

Fibril formation of β2-microglobulin and associated inflammation occur in patients on long term dialysis. We show that the plasma protein haptoglobin prevents the fatty acid-promoted de novo fibril formation of β2-microglobulin even at substoichiometric concentration. The fibrils are cytotoxic, and haptoglobin abolishes the cytotoxicity by preventing fibril formation. Haptoglobin does not alleviate the cytotoxicity of preformed fibrils. Fibrillar β2-microglobulin is resistant to lysosomal degradation. However, the species of β2-microglobulin populated in the presence of haptoglobin is susceptible to degradation. We observed that haptoglobin interacts with oligomeric prefibrillar species of β2-microglobulin but not with monomeric or fibrillar β2-microglobulin that may underlie the molecular mechanism. 1,1'-Bis(4-anilino)naphthalene-5,5'-disulfonic acid cross-linking to haptoglobin significantly compromises its chaperone activity, suggesting the involvement of hydrophobic surfaces. Haptoglobin is an acute phase protein whose level increases severalfold during inflammation, where local acidosis can occur. Our data show that haptoglobin prevents fibril formation of β2-microglobulin under conditions of physiological acidosis (between pH 5.5 and 6.5) but with relatively decreased efficiency. However, compromise in its chaperone activity under these conditions is more than compensated by its increased level of expression under inflammation. Erythrolysis is known to release hemoglobin into the plasma. Haptoglobin forms a 1:1 (mol/mol) complex with hemoglobin. This complex, like haptoglobin, interacts with the prefibrillar species of β2-microglobulin, preventing its fibril formation and the associated cytotoxicity and resistance to intracellular degradation. Thus, our study demonstrates that haptoglobin is a potential extracellular chaperone for β2-microglobulin even in moderately acidic conditions relevant during inflammation, with promising therapeutic implications in β2-microglobulin amyloid-related diseases.

Proteomic analysis of differentially expressed proteins in caprine milk during experimentally induced endotoxin mastitis

The goal of the current study was to identify proteins in goat milk before and at 18h following intramammary challenge with lipopolysaccharide (LPS). Initial evaluation of protein profiles generated using 2-dimensional gel electrophoresis on skim milk samples from a group of 6 goats collected before challenge and at 18, 24, and 48h after LPS challenge revealed little change in the abundance of casein proteins, and minimal changes in the presence or abundance of the plasma protein serum albumin, which is known to leak into milk during coliform mastitis in dairy cattle. Proteins in baseline milk samples and in milk from the same goats 18h post-LPS challenge were excised from the gels, and peptides were sequenced using nano-flow liquid chromatography coupled with tandem mass spectrometry. Despite the overwhelming presence of casein proteins and β-lactoglobulin, the lower abundance proteins β-2-microglobulin, fatty acid-binding protein, serum albumin, and retinol-binding protein were detected in skim milk samples from healthy goats. Skim milk samples 18h postchallenge were characterized by the sustained presence and abundance of the casein proteins, and by the presence of haptoglobin, serum amyloid A, lactoferrin, cathelicidin-1, and cathelicidin-3. No marked differences in the intensity of the spot corresponding to serum albumin were observed in gels of skim milk samples 18h postchallenge, which could indicate that the breakdown of the blood–milk barrier during endotoxin mastitis may not be as profound in goats as has been observed in dairy cattle. Nonetheless, the occurrence of an inflammatory response was supported by elevated somatic cell counts in the goat milk following inoculation with endotoxin, as well as by the presence of both antimicrobial and acute phase proteins. The results provide information about the composition of proteins in goat milk as well as added knowledge of the host response during endotoxin mastitis in goats.

Relevance of the amino acid conversions L144R (Zaragoza) and L159P (Zavalla) in the apolipoprotein A-I binding site for haptoglobin

The high-density lipoprotein apolipoprotein A-I (ApoA-I) stimulates the enzyme lecithin-cholesterol acyltransferase (LCAT) in the reverse cholesterol transport pathway. Two ApoA-I variants, Zaragoza (L144R) and Zavalla (L159P), are associated with low levels of HDL-cholesterol but normal LCAT activity. Haptoglobin interacts with ApoA-I, impairing LCAT stimulation. Synthetic peptides matching the haptoglobin-binding site of native or variant ApoA-I (native, P2a; variants, Zav-pep and Zar-pep) bound haptoglobin with different activity: Zar-pep>P2a>Zav-pep. They also differently rescued LCAT in vitro activity in the presence of haptoglobin (P2a=Zar-pep>Zav-pep). Therefore, both amino acid conversions affect haptoglobin binding and LCAT regulation. We highlight the role of haptoglobin in LCAT regulation in subjects with ApoA-I variants.

Haptoglobin Binding to Apolipoprotein A-I Prevents Damage from Hydroxyl Radicals on Its Stimulatory Activity of the Enzyme Lecithin-Cholesterol Acyl-Transferase

Apolipoprotein A-I (ApoA-I), a major component of HDL, binds haptoglobin, a plasma protein transporting to liver or macrophages free Hb for preventing hydroxyl radical production. This work aimed to assess whether haptoglobin protects ApoA-I against this radical. Human ApoA-I structure, as analyzed by electrophoresis and MS, was found severely altered by hydroxyl radicals in vitro. Lower alteration of ApoA-I was found when HDL was oxidized in the presence of haptoglobin. ApoA-I oxidation was limited also when the complex of haptoglobin with both high-density lipoprotein and Hb, immobilized on resin beads, was exposed to hydroxyl radicals. ApoA-I function to stimulate cholesterol esterification was assayed in vitro by using ApoA-I-containing liposomes. Decreased stimulation was observed when liposomes oxidized without haptoglobin were used. Conversely, after oxidative stress in the presence of haptoglobin (0.5 microM monomer), the liposome activity did not change. Plasma of carrageenan-treated mice was analyzed by ELISA for the levels of haptoglobin and ApoA-I, and used to isolate HDL for MS analysis. Hydroxyproline-containing fragments of ApoA-I were found associated with low levels of haptoglobin (18 microM monomer), whereas they were not detected when the haptoglobin level increased (34-70 microM monomer). Therefore haptoglobin, when circulating at enhanced levels with free Hb during the acute phase of inflammation, might protect ApoA-I structure and function against hydroxyl radicals.

Differences between normal and autoimmune T cell responses to autologous erythrocytes and haemoglobin: impairment of haptoglobin-mediated inhibition in NZB spleen cells.

Helper T cells are required for development of the autoantibody responses to native mouse erythrocytes (MRBC) that spontaneously develop in NZB mice. However, the stimulus for these Th is not known. Therefore, we compared the abilities of splenic T cells from actively autoimmune old NZB mice and preautoimmune, young NZB mice with those of T cells from nonautoimmune strains of mice to respond to autologous erythrocytes. We found that autologous RBC ghosts, washed free of haemoglobin, induced low, but statistically significant, proliferative responses in T cells from old NZB mice but not in T cells from young NZB or from normal young and old BALB/c mice. In addition, autologous RBC lysates induced proliferative responses detectable by 3H-thymidine uptake in T cells from nonautoimmune as well as autoimmune mice. CD4+ T cells accounted for most of the observed RBC lysate-induced proliferation, with virtually no response made by CD8+ T cells or B cells. T cells from actively autoimmune NZB mice were not more active in their responses to RBC lysates than T cells from normal strains of mice in terms of their level of proliferation, kinetics, or dose response. Haemoglobin was the major stimulus in the autologous RBC lysates and a similar stimulation was seen with lysates and haemoglobins from horse, human, and mouse sources. Haptoglobin, a haemoglobin-binding serum protein, inhibited T cell responses to haemoglobin and haemoglobin-containing RBC lysates but did not have the same effect on these responses in T cells from either young or old NZB mice. Therefore, either or both of the RBC stimuli from autologous RBC might account for the helper T cell activity in autoimmune NZB mice. T cells in normal mice do not respond either to RBC lysates in the presence of haptoglobin or to RBC ghosts.

A study of the mechanisms of proteinuria in nephritis and chronic renal failure.

This study was carried out on 50 patients suffering from renal disorders; 30 nephritis patients and 20 chronic renal failure patients. Twenty-four healthy persons were used as a control group. In order to cast some light on the degree of the impaired glomerular permeability with respect to the blood proteins, selectivity of proteinuria was assessed by means of the clearance of albumin, ceruloplasmin, transferrin, and haptoglobin. Disturbances in the metabolism of these proteins were observed and discussed in light of the proteinuria selectivity index. The demonstration of the selective proteinuria in the presence of haptoglobin was concluded to be indicative of the degree of impaired glomerular permeability in nephritis and chronic renal failure.

Impairment of blood-cerebrospinal fluid barrier in multiple sclerosis.

The blood-CSF barrier (BCB) function in active multiple sclerosis (MS) was studied by means of CSF proteins analysis using disc electrophoresis and immunofixation. Forty-five CSF samples were obtained by repeat lumbar punctures at various intervals, from four autopsy-proven cases and three male and nine female patients with clinically definite MS. When total protein content was increased, the percentages of prealbumin and tau fraction were decreased significantly in association with the presence of haptoglobin (Hp) polymers in nearly all the samples, as a result of increased permeability of the BCB. Even when the total protein content was normal, Hp polymers were detected in 56% of the samples, and the tau fraction tended to be decreased. Monoclonal immunoglobulin and Hp polymers were both recognized in some cases. The results suggested a more frequent occurrence of BCB impairment in MS than had formerly been revealed by CSF albumin analysis, and accorded with the recent reports of contrast-enhancing lesions of MS brain in computerized tomography.

Identification of haptoglobin in chicken serum and specificity of the chicken haptoglobin-hemoglobin complex formation

1. 1. The presence of haptoglobin in chicken serum has been demonstrated by three different techniques: gel filtration, cellulose acetate electrophoresis and fluorescence quenching. 2. 2. Chicken haptoglobin shows a narrow species specificity; it binds only avian and reptilian but not mammalian hemoglobins. 3. 3. Haptoglobin seems to have been subjected to profound changes during the course of evolution.

Serum proteins in cervical mucus

The occurrence of serum proteins in human cervical mucus was investigated in 41 cases using immunoelectrophoresis. Albumin, transferrin, and gamma globulin were always found in specimens from normal women, and in more than half of the cases alpha1 antitrypsin and prealbumin were present.There were no significant differences between specimens from the eighth to the thirteenth day, from the fifteenth to the eighteenth day, or from the twenty-first to the twenty-eighth day of the menstrual cycle. The pattern of the cervical mucus from women taking Enovid as an oral contraceptive showed only slight differences: One alpha2 globulin line was observed addition-ally in a number of cases. There was no indication of a change in the immunoelectrophoretic pattern of the cervical mucus from a surgical castrate under hormonal treatment, whereas the cytologic patterns showed typical changes.Unlike the group of normal women and those under Enovid treatment, the benzidine reaction, indicating the presence of haptoglobin, was positive in most of the cases with local pathologic changes. In cases of cervical erosion, cervicitis, or pelvic inflammatory diseases a higher number of precipitin lines was disclosed in conjunction with a remarkable increase in leukocytes.The limits of the methods are thoroughly discussed together with the significance of the findings.

Excretion of haptoglobin in normal urine.

THE presence of haptoglobin in normal urine was first suggested by Patte et al.1 and was later established by direct immuno-precipitation2. The haptoglobin found in urine gave a reaction of immunological identity with purified plasma haptoglobin. However, it could not be detected in 4 of 12 individuals whose urine was examined.

Presence of haptoglobin in synovial fluid.

ELECTROPHORETICALLY classified α- and β-glycoproteins have been shown to be normal constituents of synovial fluid1. Evidence based on immunoelectrophoresis and Cohn fractionation of human traumatic effusions also suggests the presence of most of the serum proteins in synovial fluid2,3. Individual biologically active serum proteins have also been found, namely, some enzymes4, siderophilin3, caerulo-plasmin3, and the thyroxine-binding protein5. The accepted blood-coagulating factors, however, were found to be absent from normal bovine fluid6.