Search

The significant economical consequences of infections with Ascaris suum in pigs are already well documented. However, due to the subclinical nature of the disease and the lack of practical diagnostic means, ascariasis often remains undiagnosed. Here we describe the development and evaluation of a novel indirect ELISA using the purified A. suum haemoglobin (AsHb) molecule as an antigen. Initial validation using sera from 190 pigs experimentally infected twice a week with A. suum and Trichuris suis (25 and 5 eggs kg(-1)day(-1) respectively) demonstrated that the AsHb ELISA is able to detect long-term exposure to A. suum with a high sensitivity and specificity (99.5% and 100.0% respectively). Furthermore, this serological technique proved to be more sensitive than faecal examination on week 7 and 14 of the experiment (99.5% and 100% compared to 59.5% and 68.4% respectively). Cross-reactivity caused by T. suis infection was shown to be limited after analysing sera from pigs with an experimental T. suis mono-infection. Seroconversion was shown to occur from week 6 onwards in pigs receiving 100 A. suum eggs 5 times a week. Preliminary testing of the ELISA on six randomly selected farms confirmed the results obtained in the artificial infection trials, showing a higher sensitivity of the serologic method compared to faecal examination. Finally, the ELISA was used to investigate Ascaris infection rates on 101 conventional Flemish pig farms. The results showed that on 38.6% of the farms less than 20% of the tested samples were seropositive, while in 19.8% of the farms 80-100% of all pigs were seropositive. The results of this study suggest that the AsHb ELISA could provide pig farmers and veterinarians with an easier and more sensitive way to estimate the overall prevalence of A. suum on their farm., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The α-hemoglobin-derived dodecapeptide RVD-hemopressin (RVDPVNFKLLSH) has been proposed to be an endogenous agonist for the cannabinoid receptor type 1 (CB(1)). To study this peptide, we have raised mAbs against its C-terminal part. Using an immunoaffinity mass spectrometry approach, a whole family of N-terminally extended peptides in addition to RVD-Hpα were identified in rodent brain extracts and human and mouse plasma. We designated these peptides Pepcan-12 (RVDPVNFKLLSH) to Pepcan-23 (SALSDLHAHKLRVDPVNFKLLSH), referring to peptide length. The most abundant Pepcans found in the brain were tested for CB(1) receptor binding. In the classical radioligand displacement assay, Pepcan-12 was the most efficacious ligand but only partially displaced both [(3)H]CP55,940 and [(3)H]WIN55,212-2. The data were fitted with the allosteric ternary complex model, revealing a cooperativity factor value α < 1, thus indicating a negative allosteric modulation. Dissociation kinetic studies of [(3)H]CP55,940 in the absence and presence of Pepcan-12 confirmed these results by showing increased dissociation rate constants induced by Pepcan-12. A fluorescently labeled Pepcan-12 analog was synthesized to investigate the binding to CB(1) receptors. Competition binding studies revealed K(i) values of several Pepcans in the nanomolar range. Accordingly, using competitive ELISA, we found low nanomolar concentrations of Pepcans in human plasma and ∼100 pmol/g in mouse brain. Surprisingly, Pepcan-12 exhibited potent negative allosteric modulation of the orthosteric agonist-induced cAMP accumulation, [(35)S]GTPγS binding, and CB(1) receptor internalization. Pepcans are the first endogenous allosteric modulators identified for CB(1) receptors. Given their abundance in the brain, Pepcans could play an important physiological role in modulating endocannabinoid signaling.

Hemoglobin is released from lysed RBCs in numerous clinical settings. High mobility group box 1 (HMGB1) is a nuclear and cytosolic DNA-binding protein released from injured cells that has been shown to play an important role in inducing inflammation. Because both of these endogenous molecules are frequently present in sites of necrosis and inflammation, we studied their interaction on the activation of macrophages. We report in this article that hemoglobin and HMGB1 synergize to activate mouse macrophages to release significantly increased proinflammatory cytokines. Addition of microbial ligands that activate through TLR2 or TLR4 resulted in further significant increases, in a "three-way" synergy between endogenous and microbial ligands. The synergy was strongly suppressed by hemopexin (Hx), an endogenous heme-binding plasma protein. The findings suggest that hemoglobin may play an important role in sterile and infectious inflammation, and that endogenous Hx can modulate this response. Administration of Hx may be beneficial in clinical settings characterized by elevated extracellular hemoglobin and HMGB1.

Atherosclerosis initiation and progression is controlled by inflammatory molecular and cellular mediators. Cells of innate immunity, stimulated by various endogenous molecules that have undergone a transformation following an oxidative stress or nonenzymatic glycation processes, activate cells of the adaptive immunity, found at the borders of atheromas. In this way, an immune response against endogenous modified antigens takes place and gives rise to chronic low-level inflammation leading to the slow development of complex atherosclerotic plaques. These lesions will occasionally ulcerate, thus ending with fatal clinical events. Plaque macrophages represent the majority of leukocytes in the atherosclerotic lesions, and their secretory activity, including proinflammatory cytokines and matrix-degrading proteases, may be related to the fragilization of the fibrous cap and then to the rupture of the plaque. A considerable amount of work is currently focused on the identification of locally released proinflammatory factors that influence the evolution of the plaque to an unstable phenotype. A better understanding of these molecular processes may contribute to new treatment strategies. Mediators released by the immune system and associated with the development of carotid atherosclerosis are discussed., (© 2012 New York Academy of Sciences.)

Essential thrombocythemia (ET), polycythemia vera (PV) and myelofibrosis share the same acquired genetic lesion, JAK2V617F. It is believed that cytokines participate in the activation of JAK2V617F. In this study, we analyzed the plasma levels of interleukin (IL)-23, IL-10 and IL-22 in patients with PV and ET. In the same subjects we also performed analysis of the JAK2(V617F) mutation, and evaluated a possible relationship between interleukin levels and thrombotic complications or with the symptom pruritus. Plasma levels of IL-23 were significantly increased in all patients with MPN in comparison to controls. Moreover, there was a significant difference between the levels of IL-23 in patients affected by PV and those measured in controls (8.57±3.69pg/mL vs. 6.55±4.125pg/mL; p<0.03). No difference was found between IL-23 levels in ET patients and controls. No statistically significant differences were found between the levels of IL-23, Il-22 or IL-10 in PV or ET subjects with or without thrombosis, in patients with or without pruritus, or according the JAK2V617F burden. In PV patients the JAK2 burden and Hb levels correlated with occurrence of pruritus. Our study seems to point out a possible involvement of IL-23 in the pathogenesis of PV., (Copyright © 2012. Published by Elsevier Inc.)

In this Article, we present a new strategy for preparing an antihemoglobin biointerface on cellulose. The preparation method is based on functionalization of the cellulose surface by the irreversible adsorption of CMC, followed by covalent linking of antibodies to CMC. This would provide the means for affordable and stable cellulose-based biointerfaces for immunoassays. The preparation and characterization of the biointerface were studied on Langmuir-Schaefer cellulose model surfaces in real time using the quartz crystal microbalance with dissipation and surface plasmon resonance techniques. The stable attachment of antihemoglobin to adsorbed CMC was achieved, and a linear calibration of hemoglobin was obtained. CMC modification was also observed to prevent nonspecific protein adsorption. The antihemoglobin-CMC surface regenerated well, enabling repeated immunodetection cycles of hemoglobin on the same surface.

Germline-encoded naturally occurring autoantibodies (NAbs) developed about 400 to 450 million years ago to provide specificity for clearance ofbody waste in animals with 3 germ layers. Such NAbs became a necessity to selectively clear aged red blood cells (RBC) surviving 60 to 120 d in higher vertebrates. IgG NAbs to senescent RBC are directed to the most abundant integral membrane protein, the anion-transport protein or band 3 protein, but only bind firmly upon its oligomerization, which facilitates bivalent binding. The main constituent of RBC, the oxygen-carrying hemoglobin, is susceptible to oxidative damage. Oxidized hemoglobin forms hemichromes (a form of aggregates) that bind to the cytoplasmic portion of band 3 protein, induces their clustering on the cytoplasmic, as well as the exoplasmic side and thereby provides the prerequisites for the low affinity IgG anti-band 3 NAbs to bind bivalently. Bound anti-band 3 NAbs overcome their low numbers per RBC by stimulating complement amplification. An affinity for C3 outside the antigen binding region is responsible for a preferential formation of C3b(2)-IgG complexes from anti-band 3 NAbs. These complexes first bind oligomeric properdin, which enhances their affinity for factor B in assembling an alternative C3 convertase.

Polymerized Porcine Hemoglobin (pPolyHb), a hemoglobin-based oxygen carrier (HBOC), was developed as a potential red blood substitute for clinical applications. Assessment of its effects on the immune system is an important component of the overall safety evaluation of HBOC. For this purpose, we assessed three inflammation indicators, including complement C3a, IL-6, and TNF-? in cultured cells and in a rat model when pPolyHb was incubated or administrated with the cells/animals. Our results suggested that the levels of these three indicators were not statistically changed upon pPolyHb stimulation, indicating that pPolyHb is not immunotoxic to cells and animals in this aspect.

Hemoglobin (Hb) functions as a frontline defense molecule during infection by hemolytic microbes. Binding to LPS induces structural changes in cell-free Hb, which activates the redox activity of the protein for the generation of microbicidal free radicals. Although the interaction between Hb and LPS has implications for innate immune defense, the precise LPS-interaction sites on Hb remain unknown. Using surface plasmon resonance, we found that both the Hb α and β subunits possess high affinity LPS-binding sites, with K(D) in the nanomolar range. In silico analysis of Hb including phospho-group binding site prediction, structure-based sequence comparison, and docking to model the protein-ligand interactions showed that Hb possesses evolutionarily conserved surface cationic patches that could function as potential LPS-binding sites. Synthetic Hb peptides harboring predicted LPS-binding sites served to validate the computational predictions. Surface plasmon resonance analysis differentiated LPS-binding peptides from non-binders. Binding of the peptides to lipid A was further substantiated by a fluorescent probe displacement assay. The LPS-binding peptides effectively neutralized the endotoxicity of LPS in vitro. Additionally, peptide B59 spanning residues 59-95 of Hbβ attached to the surface of Gram-negative bacteria as shown by flow cytometry and visualized by immunogold-labeled scanning electron microscopy. Site-directed mutagenesis of the Hb subunits further confirmed the function of the predicted residues in binding to LPS. In summary, the integration of computational predictions and biophysical characterization has enabled delineation of multiple LPS-binding hot spots on the Hb molecule.

Hemoglobins (Hb) are the major protein components of erythrocytes circulating in the red blood, but can serve additional functions besides the transport of oxygen. Here, the cDNA of the bloody clam (Tegillarca granosa) Hb dimer (designated Tg-HbI) was cloned and was found to be 748 bp in length, consisting of an open reading frame of 441 bp encoding a polypeptide of 147 amino acids. The deduced amino acid sequence of Tg-HbI shared 81.6% similarity with HbI from two species of the genus Scapharca and 46-51% similarity with the Hb proteins from other mollusks. The 3D structure of bloody clam Tg-HbI was predicted by the SWISS-MODEL Protein Modelling Server and compared with that of Scapharca kagoshimensis. The mRNA transcript of Tg-HbI was detected in all of the clam cells/tissues examined, including haemocytes, the adductor muscle, foot, hepatopancreas, gill and mantle. The mRNA expression of Tg-HbI was significantly up-regulated after Vibrio parahaemolyticus, lipopolysaccharide and peptidoglycan challenge, indicating that Tg-HbI was involved in the immune defence responses against bacterial infection and exposure to bacterial pathogenic factors. As the first functional research on the Hb protein in bloody clam, our findings provide new insight into the innate immune defence mechanisms of T. granosa and other mollusks., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

To determine whether purified Ascaris suum haemoglobin (AsHb) is a suitable vaccine candidate for the control of Ascaris infections, pigs were vaccinated with AsHb in combination with QuilA adjuvant and challenged with A. suum eggs. The number of liver lesions and worms in the intestine was assessed on day 14, 28 and 56 post-infection (p.i.). No significant differences were found in the number of worms recovered between vaccinated and control pigs on any of these days. However, significantly more white spots were counted on the livers of vaccinated pigs on day 14 (+86%) and day 28 (+118%) p.i. compared with nonvaccinated controls. To investigate whether the increased immunoreactivity against the liver stage L3s in vaccinated pigs was triggered by and directed against AsHb, the transcription and expression of AsHb in this larval life stage was analysed by RT-PCR and immunoblotting. The results showed that neither the AsHb transcript nor protein was detectable in freshly hatched L3. However, the immunoblot analysis showed that vaccination with AsHb resulted in the production of antibodies binding to several other antigens of the L3, suggesting that these might be involved in the increased white spot development., (© 2011 Blackwell Publishing Ltd.)

The hemoglobin vesicle (HbV) is an artificial oxygen carrier in which a concentrated hemoglobin solution is encapsulated in a liposome. To apply liposome preparations in clinics, it is important to consider the accelerated blood clearance phenomenon (ABC phenomenon), which involves a loss in the long-circulation half-life after being administered repeatedly to the same animals. The objective of this study was to determine whether the ABC phenomenon is induced by repeated injection of HbV under conditions of hemorrhagic shock. We created a rat model of hemorrhagic shock and performed a pharmacokinetic study using (125)I-HbV, in which the Hb inside of HbV was labeled with (125)I. At 4 and 7 days after resuscitation from hemorrhagic shock by nonlabeled HbV (1400 mg Hb/kg), the second dose of (125)I-HbV (1400 mg Hb/kg) was rapidly cleared from the circulation compared with normal rats. Of interest, IgM against HbV was produced at 4 days after the first injection of HbV, but decreased at 7 days. In addition, phagocyte activity was increased at both 4 and 7 days after the first injection of HbV. These results suggest that repeated injections of HbV at a dose of 1400 mg Hb/kg induce the ABC phenomenon under conditions of hemorrhagic shock, which is strongly related to both the production of anti-HbV IgM and enhanced phagocyte activity. We thus conclude that it might be necessary to consider the ABC phenomenon in the dose regimen of HbV treatment in clinical settings.

The release of hemoglobin (Hb) occurs in some infectious and autoimmune diseases characterized by inflammation. As levels of haptoglobin (Hp) fall, free Hb can cause pathology. Humoral autoreactivity to human Hb was demonstrated in the sera of systemic lupus erythematosus (SLE), leishmania and malaria patients. Serum anti-murine Hb antibody levels in lupus-prone mice also exhibited an age-dependent increase, with progressive organ sequestration; significant isotypic correlation was observed with anti-dsDNA antibodies. A suggestive link between anti-Hb and anti-Sm responses was observed: Human lupus sera expressing anti-Sm antibody reactivity preferentially contained heightened levels of anti-Hb autoantibodies, and immunization of lupus-prone mice with Sm led to enhanced anti-murine Hb reactivity. Human and murine anti-Hb monoclonal antibodies were generated, some of which were preferentially reactive toward disease-associated methemoglobin. Epitope-mapping studies revealed evidence of intra-molecular cross-reactivity. One such autoantibody synergized with Hb to enhance the secretion of pro-inflammatory cytokines while eliciting the increased production of monocyte migratory signals from endothelial cells. Preferential usage of specific variable region gene segments was not observed, although somatic mutations were documented. These studies reveal that, while the etiology, specificity and sequences of anti-Hb autoreactive antibodies can vary, they occur quite frequently and can have inflammatory consequences., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Nationwide external quality assessment (EQA) of the fecal occult blood test (FOBT) in Korea was first introduced in 2007-2009. The EQA results were analyzed to assess the current status of FOBT and to plan the continuation of the EQA program., Methods: The surveys included 40 hospitals in the preliminary survey conducted in 2007, 249 general hospitals in 2008, and 389 hospitals in 2009. In the surveys, the participating hospitals provided the results of the distributed materials and replies to the questionnaire on the FOBT test procedures and quality controls., Results: In the surveys conducted between 2007 and 2009, a total of 650 institutes submitted 653 test system results; 3 institutes used 2 kinds of methods. All of the institutes used immunologic methods; 107 institutes (16.5%) used quantitative equipments and 546 institutes (84.0%) used qualitative kits. Most quantitative tests yielded consistent positive or negative results; however, their cut-off and measured values differed according to the equipments used. A low-level material tested in 2007 was negative in the quantitative methods but positive in some qualitative methods because of lower detection limits. The discordance rates among quantitative tests were 3.2% in 2007, 4.4% in 2008, and 0% in 2009 and the rates among qualitative tests were 13.8% in 2008 and 2.6% in 2009. Semi-solid EQA materials showed the ability to evaluate the overall test procedures with acceptable stability., Conclusions: In the first Korean FOBT EQA, commercially available EQA materials were proven to be stable. Continuation of the EQA program and further education of laboratory personnel are needed to reduce inconsistency in results. Further, the test kit, procedures, and result reports must be standardized.

Detection of microbial components by immune cells via Toll-like receptors (TLRs) with subsequent induction of inflammation is essential for host defense. However, an overactive immune response can cause tissue damage and sepsis. The endogenous molecule hemoglobin and its derivative heme are often released into tissue compartments where there is infection in the presence of degrading blood. We found that hemoglobin synergizes with multiple TLR agonists to induce high levels of tumor necrosis factor and interleukin-6 from macrophages and that this synergy is independent of TLR4 and MyD88. In contrast, heme synergized with some but not all TLR agonists studied. Furthermore, the synergy of both hemoglobin and heme with lipopolysaccharide was suppressed by hemopexin, a plasma heme-binding protein. These studies suggest that hemoglobin and heme may substantially contribute to microbe-induced inflammation when bacterial or viral infection coexists with blood degradation and that hemopexin may play a role in controlling inflammation in such settings.

Aim: To prepare monclonal antibody (mAb) against both hemoglobin A2 (HbA2) and hemglobin A (HbA), this antiboy can bind to delta globin chain and beta globin chain, but not to gamma globin chain., Methods: Mice was immunized with recombinant Hb delta fusion protein, and hybridoma cells were generated by cell fusion techniques followed by screening with natural HbA2 and HbA separated by ion exchange chromatography. The purified monclonal antibody was identified by indirect ELISA, Western blot based on native PAGE and SDS-PAGE, surface plasmon resonance (SPR), flow cytometry and immunohistochemistry., Results: The monoclonal antibody against both HbA2 and HbA was obtained and designated as 2C9 that shows no binding to fetal hemoglobin (HbF), alpha globin chain and recombinant zetaglobin chain., Conclusion: The mAb 2C9 was defined as specificity to hemoglobin delta globin chain and beta globin chain, which suggests that mAb 2C9 recognizes a common epitope on bothdeltaglobin chain and beta globin chain. This antibody would be expected to be an effective tool in research and clinical practice in hemoglobinopathies.

Introduction: The factors that determine the level of antibodies against N-homocysteinylated (N-Hcy) proteins have not been established so far. The clinical significance of these antibodies and their effect on cardiovascular (CV) risk in patients with end-stage renal disease (ESRD) are still unknown., Objectives: The aim of this study was to identify the factors that determine the level of antibodies against N-Hcyalbumin and N-Hcy hemoglobin in patients on long-term hemodialysis (HD)., Patients and Methods: The study involved 247 subjects on long-term HD (110 women, 137 men; age range, 23-89 years) and 60 controls matched for age, sex, and CV risk factors (serum creatinine level <140 micromol/l). Serum antibodies against N-Hcyalbumin and N-Hcyhemoglobin were determined using an in-house enzyme-linked immunosorbent assay. Total homocysteine (tHcy), folate, and 8-isoprostaglandin F2alpha (8-iso-PGF(2alpha)) were also measured., Results: Patients on HD had higher serum levels of anti-N-Hcy-albumin (absorbancy at 490 nm: 0.56 [0.49-0.623] vs. 0.259 [0.198-0.338], P <0.0001) and anti-N-Hcy-hemoglobin antibodies (0.659 [0.597-0.723] vs. 0.379 [0.289-0.442], P <0.0001) as compared with controls. The level of both antibodies correlated with tHcy (r = 0.56, P <0.0001 and r = 0.67, P <0.0001, respectively), 8-iso-PGF(2alpha) (r = 0.48, P <0.0001 and r = 0.63, P <0.0001, respectively), and folate (r = -0.18, P = 0.0054 and r = -0.38, P <0.0001, respectively), but not with HD duration, the initial cause of ESRD, and CV comorbidity., Conclusions: In HD patients, tHcy is an independent predictor of antibodies against N-Hcy proteins. Folate and 8-iso-PGF(2alpha) concentrations were not independently associated with the levels of both antibodies.

Vi capsular polysaccharide is a major virulence determinant of the human typhoid- causing pathogen Salmonella typhi; it is absent in nontyphoidal Salmonella serovars. We show in this study that through its specific interaction with the membrane recognition complex containing the prohibitin family of molecules, Vi can inhibit the production of inflammatory cytokines from mononuclear phagocytes stimulated with Salmonella flagellin. Remarkably, Vi lost this anti-inflammatory capability and switched to a proinflammatory state when cell stimulations were performed in the presence of serum. The serum-transformed proinflammatory form of Vi induced secretion of cytokines from monocytes by specifically engaging TLR-2/TLR-1. The molecule responsible for bringing about this conversion of Vi from an anti-inflammatory to a proinflammatory form was serum-derived hemoglobin. Derivatives of Vi incapable of interacting with hemoglobin did not switch to a proinflammatory state in vitro or in vivo. These findings provide compelling evidence for a role of hemoglobin in transforming the anti-inflammatory S. typhi virulence polysaccharide into an immune activator.

Crosslinking of ultrapure hemoglobin, crystalline catalase, and superoxide dismutase resulted in a soluble nanodimensional complex of polyhemoglobin-catalase-superoxide dismutase. A less expensive and more convenient way is to crosslink bovine stroma-free hemolysate (stroma-free hemolysate) that already contains hemoglobin, catalase, and superoxide dismutase into polyhemoglobin with catalase and superoxide dismutase activities (stroma-free polyhemolysate) [21]. The objective of the present study is to evaluate the immunological properties of this stroma-free polyhemolysate. Each of three groups of rats received weekly subcutaneous injections of one of the stroma-free polyhemolysate, stroma-free hemolysate, and saline for four weeks. One week after the four cycles of weekly immunization, serum and plasma were collected for C3a complement activation tests and Ouchterlony antibody-antigen precipitation tests, respectively. Results show that stroma-free polyhemolysate retained significant antioxidant enzyme activity. The C3a complement activation test and Ouchterlony test show that four weekly subcutaneous injections of bovine stroma-free polyhemolysate did not result in any immunological reaction in rats when tested this way.

Antibody phage technology has greatly facilitated the isolation of good-quality monoclonal antibodies to virtually any target antigen. Large combinatorial phage display libraries of human antibodies are routinely being used for the identification of antibody candidates for clinical applications. However, preclinical studies in rodents would benefit from the availability of good-quality single-pot mouse antibody libraries, which at present are not available. In this article, we report on the construction of three mouse antibody phage display libraries, all containing over 1 billion antibody clones and all based on a similar library design, which featured the combinatorial mutagenesis of residues in the CDR3 loops of a given antibody scaffold. While all three libraries were found to express antibodies in bacterial supernatants, only one of them (termed "PHILOtop") was shown to reliably yield good-quality antibodies towards all protein antigens used so far in selection experiments, including three tumor-associated antigens. The modular structure of the PHILOtop library facilitates a simple affinity-maturation procedure based on the combinatorial mutagenesis of CDR1 and CDR2 loops of the VH domain, which has led to the isolation of a high-affinity antibody ("H7"; Kd=6 nM) specific to the EDB domain of fibronectin, a marker of angiogenesis. The single-pot antibody library PHILOtop may thus represent a useful source of binding specificities, facilitating preclinical studies in immunocompetent syngeneic mouse models of pathology., (2010 Elsevier B.V. All rights reserved.)

Carbon nanotubes (CNTs) have a great potential for applications in medicine. However, their biocompatibility and toxicity cause a great concern. Due to the large surface area of CNTs, chemical modification can dramatically alter their physiochemical properties and hence biological activity. Using a combinatorial chemistry approach, we report the synthesis of an 80-member surface-modified nanotube library. Based upon screening of this library with respect to protein-binding capacity, cytotoxicity, and immune response, we were able to select highly biocompatible nanotubes with reduced protein-binding cytotoxicity and immune response.

Background: Animal-derived proteins are implicated in primary food allergies, but also in inhalant allergies with secondary food allergy symptoms. The objective of this study was to define the allergen(s) implicated in a case of food allergy to chicken meat, which developed in a person previously sensitized to pork after occupational exposure., Methods: A 42-year-old female with a history of occupational inhalant allergy to pork reported rhinitis, asthma, dysphonia and conjunctivitis 30 min after ingestion of chicken. Skin tests were positive to chicken meat. Protein extracts were prepared from chicken meat. Allergens were characterized by IgE immunoblotting, N-terminal sequencing and ELISA., Results: The patient showed specific IgE binding to chicken meat proteins at 12, 14, 26, 55 and 65 kDa. N-terminal amino acid sequencing identified the 12- and 14-kDa proteins as the alpha- and beta-chain of hemoglobin. ELISA and immunoblot showed specific IgE binding to hemoglobin purified from chicken blood. IgE antibodies to chicken serum albumin were detected by ELISA. Inhibition studies with chicken and porcine hemoglobin as well as with serum albumins demonstrated cross-reactive IgE antibodies., Conclusions: We report a case of confirmed occupational inhalant allergy due to pork followed by food allergy to pork and 3 years later by food allergy to chicken. Porcine and chicken hemoglobin were found to be cross-reactive allergens. Cross-reactivity between porcine and chicken serum albumin was possibly linked to a prior sensitization to cat serum albumin., (2009 S. Karger AG, Basel.)

Previously, most models that sought to explain the misregulation of immune cell function assumed molecular similarities between the disease-causing pathogens and the host's proteins. In recent time several different models have been proposed and in this study, these concepts are compared to a new hypothesis proposing another explanation for this immune dysregulation: the possibility that the mislocalization of proteins may be responsible for autoimmune activity. Based on this hypothesis, proteins are recognized as self or non-self depending on where they appear in sufficiently high concentrations. To examine this new idea, the intracellular human proteins beta-actin, GAPDH, and hemoglobin as well as the extracellular human proteins insulin and albumin, were added to human whole blood samples. After an incubation period, the activation of whole-blood T lymphocytes in the samples was measured. The observed activation pattern of the T lymphocytes fit well with the proposed hypothesis. Therefore, these data suggest that protein mislocalization and/or errors within protein trafficking might be important in the development of autoimmune diseases.

We report a novel micro-potentiometric hemoglobin (Hb) immunosensor based on electrochemically synthesized polypyrrole (PPy)-gold nanoparticles (AuNPs) composite. PPy-AuNPs film with AuNPs uniformly distributed in it was deposited on gold electrode surface by a simple and direct procedure, without the addition of any nanoparticles or reducing agent. And this generic method makes it possible to deposite different polymers on miniaturized electrodes. With the existence of AuNPs, the antibody immobilization onto the electrode surface was facilitated. Morphology study by field emission scanning electron microscope (FE-SEM) confirms the presence of AuNPs in PPy. Based on an ion-sensitive field-effect transistors (ISFETs) integrated chip, a micro-potentiometric immunosensor for Hb and hemoglobin-A1c (HbA1c) has been constructed. The sensor response was linear over the concentration range 60-180 microg/ml Hb and 4-18 microg/ml HbA1c. The Hb concentration in whole blood samples has also been analysed, with a linear dose-response behavior between 125 and 197 microg/ml and a sensitivity of 0.20 mV microg(-1)ml. The measuring ranges of the developed Hb and HbA1c immunosensors meet the clinical demand for measuring the HbA1c/Hb ratio of 5-20%. This sensor results in simple and rapid differential measurement of Hb and HbA1c, and has great potential to become an inexpensive and portable device for monitoring of diabetes.

Hemoglobin (Hb) uniquely associates with proinflammatory HDL in atherogenic mice and coronary heart disease (CHD) patients. In this paper, we report that Hb and its scavenger proteins, haptoglobin (Hp) and hemopexin (Hx) are significantly increased in apoA-1-containing particles of HDL both in mouse models of hyperlipidemia and in CHD patients, when compared with wild type mice and healthy donors, respectively. We further demonstrate that the association of Hb, Hp, and Hx proteins with HDL positively correlates with inflammatory properties of HDL and systemic inflammation in CHD patients. Interestingly, HDL from Hp(-/-) mice under atherogenic conditions does not accumulate Hb and is anti-inflammatory, suggesting that (i) Hp is required for the association of Hb with HDL and (ii) Hb x Hp complexes regulate the inflammatory properties of HDL. Moreover, treatment of apoE(-/-) mice with an apoA-1 mimetic peptide resulted in significant dissociation of Hb x Hp complexes from HDL and improvement of HDL inflammatory properties. Our data strongly suggest that HDL can become proinflammatory via the Hb x Hp pathway in mice and humans, and dissociation of Hb x Hp x Hx complexes from apoA-1-containing particles of HDL may be a novel target for the treatment of CHD.

Matrinxã (Brycon amazonicus) is a freshwater neotropical fish species with a social interaction and aggressive behavior, especially in crowded environments. This species' social structure is established by agonistic interactions, which increase significantly at the fifth hour, when compared to the first and second hours of confinement, when each dominant fish competes with other individual for a conditioned territorialism. This social relationship also induces a complex physiological response in the organism, which generates an acute stimulation from the stressor agent. A stress situation modulates the physiology of the subordinate fish, which undergo significant increases in cortisol, glucose, hematocrit, and hemoglobin, when compared to the control fish. The immune system also indicates a modulation caused by cortisol, which results in an increase in neutrophils and a significant decrease in thrombocytes in subordinate fish, in comparison with control fish; however, the dominant fish show a significant increase in monocytes and a decrease in lymphocyte levels, when compared control fish. The agonistic interactions in B. amazonicus during crowding are not favorable to the physiology and immune system of the fish, mainly subordinate fish, and should be avoided in rearing systems.

The spontaneous formation of the neurotoxic carcinogen acrylamide in a wide range of cooked foods has recently been discovered, leading to dietary exposure estimates of 30.8 microg of acrylamide day(-1) for an average 77 kg human male. This is considerably higher than the European legal limit of acrylamide in drinking water, which is approximately 0.2 microg of acrylamide person(-1) day(-1). A recent study of 62,573 women over 11.3 years has observed an increased risk of postmenopausal endometrial and ovarian cancer (but not breast cancer) with increasing dietary acrylamide intake, demonstrating significant risk to human health. As individual acrylamide exposure is affected by dietary habits, cooking methods, and cigarette consumption; accurate extrapolation from estimated dietary exposure is extremely difficult. Quantifying biomarkers of acrylamide exposure therefore remains the most effective means of rapidly determining individual exposure to acrylamide, and correlating exposure with lifestyle choices. Current methodologies for the analysis of blood biomarkers of acrylamide are focused on expensive, slower chromatographic techniques such as GC and LC coupled to mass spectrometry. This paper describes the first successful development of two monoclonal antibodies specific to acrylamide-adducted haemoglobin (IC(50) of 94 ng ml(-1) and 198 ng ml(-1)), that are suitable for use in a high-throughput biomarker immunoassay to determine individual acrylamide exposure. Further development of acrylamide-haemoglobin standards with defined levels of acrylamide adduction will enable a fully quantitative assay, and allow sensitivity comparisons with alternative chromatographic methods of analysis.

Natural regulatory T (nT(reg)) cells recognize self-peptides with high affinity, yet the understanding of how affinity influences their selection in the thymus is incomplete. We use altered peptide ligands in transgenic mice and in organ culture to create thymic environments spanning a broad range of ligand affinity. We demonstrate that the nT(reg) TCR repertoire is shaped by affinity-based selection, similar to conventional T cells. The effect of each ligand on the two populations is distinct, consistent with early nT(reg) cell lineage specification. Foxp3 expression is an independent process that does not rely on "high affinity" binding per se, but requires a high-potency agonistic interaction for its induction. The timing of ligand exposure, TGFbeta signaling, and the organization of the thymic architecture are also important. The development of nT(reg) cells is therefore a multistep process in which ligand affinity, potency, and timing of presentation all play a role in determining cell fate.

Low circulating VVH7-like immunoreactivity (VVH7 i.r) level was amazingly observed in human diabetic sera. Here, we examined the impact of diabetes type, clinico-biological features and metabolic control on circulating VVH7 i.r level in this disease. ELISA test was used to measure VVH7 i.r in sera of 120 diabetic patients (type 1 diabetes in 64, type 2 diabetes in 56). Three enzymatic tests were also applied to determine serum cathepsin D (CD), dipeptidyl peptidase IV (DPP-IV) and angiotensin-converting enzyme (ACE) activities. A subgroup of 24 type 1 diabetic patients negative for microalbuminuria and hypertension were submitted to an ambulatory blood pressure monitoring to evaluate the relationship between VVH7 i.r level and blood pressure parameters. The mean serum concentration of VVH7 i.r was drastically reduced in diabetic patients (0.91+/-0.93 micromol/l versus 5.63+/-1.11 micromol/l in controls) (p<0.001). A negative correlation between VVH7 i.r level and daytime diastolic blood pressure existed in type 1 diabetic patients. There was no association of low VVH7 i.r with either type of diabetes or HbA1c level. An increase of cathepsin D activity was found in serum of diabetic patients compared to controls (0.47 U/ml versus 0.15 U/ml, respectively) whereas DPPIV activity was significantly decreased in diabetic sera (50.81 U/ml versus 282.10 U/l respectively). Diminution of VVH7 i.r in sera of diabetic patients was confirmed but still remained unexplained. Relationships between higher systolic blood pressure and decrease of VVH7 i.r reinforce the need to investigate this pathway in this disease to elucidate its role in macro- and micro-angiopathy.

The substitution of one amino acid in the hemoglobin molecule results in sickle hemoglobin. As a result, RBCs sickle in low oxygen states causing occlusion of blood vessels, increased viscosity, and inflammation. These RBCs are prematurely removed from the circulation, resulting in a chronic hemolytic anemia. With newborn screening and early treatment, the death rate among children with SCD has declined. In addition, a variety of treatments are being introduced to help manage the various manifestations of disease. Transfusion, simple or exchange, is a mainstay of therapy, since it reduces the amount of Hgb S in circulation and suppresses erythropoiesis. Transfusion is indicated for symptomatic anemia and specifically to prevent stroke (first or recurrent), during acute stroke, and for acute chest syndrome. Unfortunately, transfusion carries risks for infectious disease transmission, as well as immunologic and inflammatory sequelae. For patients with SCD who may be chronically transfused, iron overload occurs frequently. In addition, due to differences in RBC antigens between donors and recipients, these patients are at increased risk for development of RBC alloantibodies, which can complicate further transfusion. It is, therefore, important to prevent alloimmunization by transfusing leukoreduced RBCs that match the patient for the C, E, and K1 antigens. Human progenitor cell (from bone marrow, peripheral blood stem cells, or umbilical blood) transplant can cure the disease, and is used for patients with severe disease for whom conventional therapy may not be effective.

Streptococcus suis, a major swine pathogen world-wide, can trigger macrophages to secrete large amounts of proinflammatory cytokines, which increase the permeability of the blood-brain barrier. In this study, we hypothesized that hemoglobin may potentiate the inflammatory response of human macrophages stimulated with a S. suis cell-wall preparation. Monocyte-derived macrophages were stimulated with the S. suis cell-wall preparation in the presence or absence of human hemoglobin, and the secretion of interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), IL-6, and IL-8 was analyzed by enzyme-linked immunosorbent assays. The cell-wall preparation induced dose-dependent IL-1beta, TNF-alpha, IL-6, and IL-8 responses in macrophages. Hemoglobin potentiated the cell-wall induced inflammatory response, resulting in a significantly higher secretion of all the cytokines. The S. suis cell-wall preparation in combination with hemoglobin activated macrophage intracellular kinases involved in inflammatory signaling pathways. In conclusion, hemoglobin, which may be released in vivo by the action of S. suis suilysin on red blood cells, contributes to raising the levels of pro-inflammatory mediators by acting in synergy with S. suis cell-wall components. This phenomenon may contribute to the development and the severity of meningitis.

S100A8 and S100A9, highly expressed by neutrophils, activated macrophages, and microvascular endothelial cells, are secreted during inflammatory processes. Our earlier studies showed S100A8 to be an avid scavenger of oxidants, and, together with its dependence on IL-10 for expression in macrophages, we postulated that this protein has a protective role. S-nitrosylation is an important posttranslational modification that regulates NO transport, cell signaling, and homeostasis. Relatively few proteins are targets of S-nitrosylation. To date, no inflammation-associated proteins with NO-shuttling capacity have been identified. We used HPLC and mass spectrometry to show that S100A8 and S100A9 were readily S-nitrosylated by NO donors. S-nitrosylated S100A8 (S100A8-SNO) was the preferred nitrosylated product. No S-nitrosylation occurred when the single Cys residue in S100A8 was mutated to Ala. S100A8-SNO in human neutrophils treated with NO donors was confirmed by the biotin switch assay. The stable adduct transnitrosylated hemoglobin, indicating a role in NO transport. S100A8-SNO suppressed mast cell activation by compound 48/80; intravital microscopy was used to demonstrate suppression of leukocyte adhesion and extravasation triggered by compound 48/80 in the rat mesenteric microcirculation. Although S100A8 is induced in macrophages by LPS or IFN-gamma, the combination, which activates inducible NO synthase, did not induce S100A8. Thus, the antimicrobial functions of NO generated under these circumstances would not be compromised by S100A8. Our results suggest that S100A8-SNO may regulate leukocyte-endothelial cell interactions in the microcirculation, and suppression of mast cell-mediated inflammation represents an additional anti-inflammatory property for S100A8.

Bone marrow-derived dendritic cells engineered using recombinant adenovirus to secrete high levels of IL-12p70 dramatically inhibited the growth of established CMS4 sarcomas in BALB/c mice after intratumoral administration. An analysis of splenic CD8(+) T cells in regressor mice revealed a strong, complex reactivity pattern against high-performance liquid chromatography (HPLC)-resolved peptides isolated by acid elution from single-cell suspensions of surgically resected CMS4 lesions. Mass spectrometry analyses defined two major overlapping peptide species that derive from the murine hemoglobin-beta (HBB) protein within the most stimulatory HPLC fractions. Although cultured CMS4 tumor cells failed to express HBB mRNA based on reverse transcription-PCR analyses, prophylactic vaccination of BALB/c mice with vaccines containing HBB peptides promoted specific CD8(+) T-cell responses that protected mice against a subsequent challenge with CMS4 or unrelated syngeneic (HBB(neg)) tumors of divergent histology (sarcoma, carcinomas of the breast or colon). In situ imaging suggested that vaccines limit or destabilize tumor-associated vascular structures, potentially by promoting immunity against HBB+ vascular pericytes. Importantly, there were no untoward effects of vaccination with the HBB peptide on peripheral RBC numbers, RBC hemoglobin content, or vascular structures in the brain or eye.

The trematode Fasciola hepatica (Fa.he.) is a common parasite of human and livestock. The hemoglobin (Hb) of Fa.he., a potential immunogen, was chosen for characterization in the search for an effective vaccine. Characterization of trematode Hbs show that they are intracellular single-domain globins with the following remarkable features: (1) Fa.he. expresses two Hb isoforms that differ at two amino acid sites (F1: 119Y/123Q; F2: 119F/123L). Both isoforms are monoacetylated at their N-termini; (2) the genes coding for Fa.he. and Paramphistomum epiclitum (Pa.ep.) Hbs are interrupted by two introns at the conserved positions B12.2 and G7.0.; (3) UV/VIS and resonance Raman spectroscopy identify the recombinant Fa.he. HbF2 as a pentacoordinated high-spin ferrous Hb; (4) electron paramagnetic resonance spectroscopy of cyano-met Fa.he. HbF2 proves that the endogenously bound imidazole has no imidazolate character; (5) the major structural determinants of the globin fold are present, they contain a TyrB10/TyrE7 residue pair on the distal side. Although such distal-site pair is a signature for high oxygen affinity, as shown for Pa.ep. Hb, the oxygen-binding rate parameters for Fa.he. Hb are intermediate between those of myoglobin and those of other trematode Hbs; (6) the three-dimensional structure of recombinant Fa.he. HbF2 from this study closely resembles the three-dimensional structure of Pa.ep. determined earlier. The set of distal-site polar interactions observed in Pa.ep. Hb is matched with small but significant structural adjustments; (7) despite the potential immunogenic character of the fluke Hb, vaccination of calves with recombinant Fa.he. HbF2 failed to promote protection against parasitic infection.

Background: The fetomaternal bleed assay by flow cytometry is a semiquantitative test used to determine the volume of fetomaternal hemorrhage (FMH). We have enhanced this method by combining the fetomaternal bleed assay with a D antibody in the same tube to determine the maternal D as well as the need for Rh immune globulin (RhIG) administration., Study Design and Methods: The performance of the FMB/D assay was compared against the Kleihauer-Betke (KB) acid elution method for the quantitation of fetal bleed and tube method for the maternal D determination. Peripheral blood cells from the pregnant women were fixed and permeabilized, incubated with monoclonal antibodies directed against fetal hemoglobin (Hb F) and D, and analyzed by flow cytometry. The relative concentration of Hb F-containing cells to adult cells was used to determine the volume of the FMH. In addition, the cells were identified as D+ or D-., Results: D typing of nonpregnant adult donors matched in all cases with the tube method with a mean value of 0.01% Hb F-positive cells (n = 25). D typing of pregnant or postpartum samples matched in all cases (n = 100), and the RhIG dose calculated by the percentage of Hb F for each method matched 97 percent (n = 36; p = 0.324). The one discrepant sample was consistent with the acknowledged overestimation when using the KB method., Conclusion: The highly precise anti-Hb F flow cytometric method was combined with maternal D determination by anti-D. This method correlates well with standard assays eliminating the need for additional testing without sacrificing clinical information.

To identify immunoglobulin (Ig)-producing cells with immunohistochemistry, conventional methods of preparation using chemical fixatives have problems such as the artificial diffusion of components and antigen masking. The "diffusion artifact" is caused by the translocation of soluble proteins like Ig from the serum to cytoplasm or vice versa. We have examined the immunolocalization of serum proteins, such as Ig kappa light chain (Igkappa), IgG1 heavy chain (IgG1), and albumin, in immunized mouse spleens after a peritoneal injection of human hemoglobin. Better preservation of morphology and immunoreactivity was obtained with the "in vivo cryotechnique" (IVCT) followed by freeze-substitution, than with conventional preparative methods. Although Ig-producing cells were not clearly detected in red pulp of 2-day-immunized spleens with the conventional methods, Igkappa-immunopositive cells with rich cytoplasm were detected in the red pulp with IVCT, especially in the subcapsular and peritrabecular areas, where IgG1-immunopositive cells were rarely observed. In 7-day-immunized spleens prepared with IVCT, Igkappa- or IgG1-immunopositive cells were mostly located in peritrabeculae. The development of Ig-producing cells was clarified in the specimens prepared with IVCT, which proved to be useful for analyzing the native morphology and distribution of Ig-producing cells.

The effect of low-dose methotrexate (MTX) treatment on the CD26 density on circulating monocytes and CD4(+) T lymphocytes or levels of soluble CD26 (sCD26) has not yet been described in rheumatoid arthritis (RA). While CD26 in T lymphocytes is involved in the activation and proliferation of T lymphocytes, little is known of the role of CD26 in monocytes as it has only recently been localized to monocytes. We analysed the CD26 density by flow cytometry and levels of sCD26 in plasma before initiation of MTX treatment and 12 weeks later. This was done on 34 RA patients fulfilling the 1987 American College of Rheumatology (ACR) criteria followed for 16 weeks after starting MTX treatment. CD26 density on monocytes was increased in RA patients compared with healthy controls before MTX treatment (P < 0.01). After 12 weeks of MTX treatment, the CD26 density on monocytes decreased significantly in the ACR-50% group (P = 0.03), but not in the ACR-20% and the non-responder group (P = 0.15 and 0.87). The increased CD26 density on CD4(+) T lymphocytes (P < 0.01) was unaffected by the reduction in disease activity in relation to MTX treatment. The percentage of monocytes and CD4(+) T lymphocytes among peripheral blood circulating mononuclear cells did not change during MTX treatment. No effect of MTX treatment was observed on the plasma levels of sCD26. Active chronic RA is characterized by enhanced CD26 density on circulating monocytes and CD4(+) T lymphocytes. MTX treatment decreased CD26 density on monocytes in the ACR-50% responder group and was associated with decreased disease activity. The enhanced CD26 density on CD4(+) T lymphocytes was uninfluenced by MTX treatment.

Background: Recurrence of hepatitis B virus (HBV) after orthotopic liver transplantation is very common in the absence of adequate prophylaxis and is often associated with poor prognosis because of the development of cirrhosis, fibrosing cholestatic hepatitis, or fulminant hepatitis. Therefore it is important to study the prevention of HBV reinfection after liver transplantation., Methods: Recombinant Fab (rFab) was constructed to evaluate gene therapy for post-transplantation HBV reinfection. Hepatocytes were divided into three groups: HBV-infection, rFab-blocked HBV-infection, and control. The inhibition of HBsAg adsorption test, the micro-cytotoxicity assay, and the blockade test of HBV infection were carried out. The HBsAg adsorption rate, the hepatocyte death rate and the HBV infection rate were statistically analyzed., Results: The HBsAg adsorption rate blocked by rFab in the HBsAg adsorption test was 0.3%. The hepatocyte death rate was 98.8% induced by rFab in the micro-cytotoxicity assay, 1.3% in the rFab-blocked HBV-infected group and 77% in the HBV-infected group in the blockade test of HBV., Conclusions: We found that rFab effectively blocked HBV infection in human hepatocytes. This provides an attractive alternative for hepatitis B prophylaxis.

Using the molecular imprinting approach, we have shown that polyacrylamide-based artificial antibodies against human and bovine hemoglobin have a very high selectivity, as revealed by the free-zone electrophoresis in a revolving capillary. By the same technique we have previously synthesized gel antibodies not only against proteins but also against viruses and bacteria. The synthesis is thus universal, i.e., it has the great advantage of not requiring a modification - or only a slight one - for each particular antigen. The combination synthesis of artificial gel antibodies and electrophoretic analysis reveals small discrepancies in shape and chemical composition not only of proteins, as shown here and in paper Ia, but also of viruses and bacteria, to be illustrated in papers II and III in this series. Upon rehydration, the freeze-dried gel antibodies, selective for human hemoglobin, regain their selectivity. The gel antibodies can repeatedly be used following the removal of the antigen (protein in this study) from the complex gel antibody/antigen by an SDS washing or an enzymatic degradation.

Background: Autoantibodies against N(epsilon)-homocysteinylated (N(epsilon)-Hcy) proteins at high titres have been demonstrated in patients with premature coronary artery disease (CAD) and stroke. Since recently, elevated N(epsilon)-Hcy-proteins levels have been reported in haemodialysis patients, we sought to investigate whether anti-N(epsilon)-Hcy-protein antibodies occur in such subjects and if they are associated with cardiovascular risk., Methods: We studied 43 patients, aged 27-89 (mean 58.8) years, dialysed for, on average, 50 months and 31 age- and sex-matched healthy controls. IgG antibodies against N(epsilon)-Hcy-albumin and -haemoglobin were determined using an in-house enzyme-linked immunosorbent assay., Results: Haemodialysis patients had higher plasma tHcy (23.18 +/- 1.37 vs 13.51 +/- 0.64; P < 0.0001), serum folate (29.7 +/- 6.9 vs 9.9 +/- 3.8 nmol/l; P < 0.0001) and anti-N(epsilon)-Hcy-albumin and -haemoglobin antibodies (absorbancy at 490 nm: 0.39 +/- 0.22 vs 0.34 +/- 0.12; P = 0.03 and 0.60 +/- 0.31 vs 0.42 +/- 0.09; P < 0.0001, respectively) than controls. Levels of anti-N(epsilon)-Hcy-albumin antibodies, but not those against haemoglobin, correlated negatively with the duration of haemodialysis (r = -0.39; P = 0.01). This correlation disappeared after a 6-month follow-up. Haemodialysis patients treated with folic acid on a long-term basis had similar levels of anti-N(epsilon)-Hcy-albumin and -haemoglobin antibodies compared with the minority which denied taking this vitamin (absorbancy A490: 0.35 +/- 0.22 vs 0.38 +/- 0.05; P = 0.5 and 0.63 +/- 0.30 vs 0.51 +/- 0.11; P = 0.4, respectively)., Conclusions: Our study shows that an autoimmune response to anti-N(epsilon)-Hcy-proteins occurs in patients on maintenance haemodialysis and is more pronounced than in healthy subjects.

Background: Bovine hemoglobin (Hb)-based oxygen carrier (HbOC-201; Hb glutamer-250, Hemopure, Biopure Corp.) is a blood replacement and augmentation drug that increases oxygen-carrying capacity of circulating blood in patients with anemia and acute blood loss. The objective of this study was to assess the biologic significance (cross-reactivity, hemolysis) of humoral immune responses in humans receiving repetitive HbOC-201 administrations., Study Design and Methods: Serum samples containing immunoglobulin G (IgG) anti-HbOC-201 (n = 146) or no antibody (n = 16) were collected from subjects receiving HbOC-201 in clinical studies. IgG anti-HbOC-201 levels were quantified and the extent of cross-reactivity to human hemoglobin (HuHb) was assessed in direct-binding and competitive-inhibition immunoassays. Serum samples containing the highest levels of IgG anti-HbOC-201 were studied in a complement-mediated hemolysis assay for their ability to lyse human red cells (RBCs)., Results: The IgG anti-HbOC-201 levels in the antibody-positive serum samples ranged from 0.7 to 86.8 mug per mL. Of the 146 IgG anti-HbOC-201-positive serum samples, 88.4 percent contained IgG antibodies whose binding to solid-phase HbOC-201 was competitively inhibited by incubation with soluble HuHb (11.6% [<20% inhibition]; 63% [20%-80% inhibition]; and 25.4% [>81% inhibition]). Direct-binding analysis to solid-phase HuHb confirmed that 74 percent contained IgG antibodies reactive with HuHb. Dichotomous competitive inhibition and direct-binding IgG anti-HuHb data correlated significantly (r(2) = 0.77, p < 0.001). Serum samples with the highest levels of IgG anti-HuHb, as identified from clinical studies, did not lyse human RBCs in the presence of exogenous complement or induce the direct sensitization of RBCs with human IgG or complement., Conclusion: These analyses indicate that HbOC-201 administration elicits IgG antibodies in humans that react with bovine and HuHb, but do not cause hemolysis in vitro.

Hemoglobin vesicle (HbV), a liposomal oxygen carrier containing human hemoglobin, was intravenously infused into rats. After the infusion of saline, the HbV or empty vesicle (EV), numbers of red cells, leukocytes and platelets in peripheral blood were unchanged during the observation period of one week in addition to each time point among three groups. However, the lymphocyte ratio transiently decreased and the granulocyte ratio increased in the HbV and EV groups at 6 h after the infusion. Those changes returned to the initial value one day after the infusion and those were maintained for the subsequent observation period. No dramatic change was seen in the ratio of CD4(+)/CD8(+) T cells. A transient decrease of the complement titer was observed three days after the infusion of HbV and EV, although the consumption of complement titer was not detected in rat serum by mixing HbV or EV in vitro, indicating that the transient decrease of complement titer in vivo was not due to the consumption of complement due to the interaction with HbV or EV. Multiple infusions of HbV caused the decrease of complement titer only after the first infusion and no allergic reaction was observed. No anaphylactic shock was observed in rats administered with EV several times, while ovalbumin (OVA) sensitized rats died with symptoms of respiratory distress after the second OVA administration. These results indicate that HbV could be administered without serious clinical symptoms or adverse reactions.

In this study, immunogenicity of human hemoglobin (hHb), bovine hemoglobin (bHb), porcine hemoglobin (pHb) and their glutaradehyde polymerized derivatives (hPolyHb, bPolyHb and bPolyHb, respectively) were compared. The nature of the dominant antigen determinants of the chemically polymerized proteins was studied. Glutaraldehyde chemical reaction enhanced the immunogenicity of the hemoglobin derivatives. In mice, the extent of the enhancement was largely comparable among hPolyHb, bPolyHb and pPolyHb. Using the methods of semi-quantitative western blotting and quantitative protein array, it was found that most of the polycloncal antibodies raised in rodents against glutaraldehyde polymerized hemoglobin derivatives of human, bovine or porcine species only weakly or did not cross-react with the hemoglobin derivatives of the other two species, indicating that hPolyHb, bPolyHb and bPolyHb vary significantly in their dominant antigen determinants, despite very high degree of identity in their primary amino acid sequences and high similarity in their three dimensional structures.

Hemoglobin-based oxygen carriers (HBOCs) show potential as safe, efficacious, pre-hospital resuscitation fluids. The major criticism of HBOC-201 is its vasoactive property, attributed partially to low-molecular weight (low-MW) tetrameric/dimeric (TD) hemoglobin (Hb) in HBOC solution. Here we sought to determine whether resuscitation with decreasing concentrations of low-MW Hb component of HBOC affects immune responses in hemorrhagic swine. 28 anesthetized swine underwent a soft muscle crush and controlled hemorrhage of 55% blood volume, followed by resuscitation with HBOC containing 31%, 2%, or 0.4% low-MW Hb in four 10 ml/kg infusions at 20, 30, 45 and 60 minutes before hospital arrival at 75 minutes. IL-10, cell activation and adhesion markers and CD4:CD8 ratio remained unchanged in all 3 groups compared to baseline. Leukocyte apoptosis was equally elevated across all groups. Purification from 31% to 0.4% low-MW Hb in HBOC solution did not alter immune effects in a swine model of severe controlled hemorrhagic shock.

Wischnewsky spots in the gastric mucosa are considered an important finding for the diagnosis of hypothermia-related deaths. In the present prospective histological and immunohistochemical investigation, 14 cases of fatal hypothermia presenting Wischnewsky spots at autopsy were studied. Macromorphologically, the lesions, varying in diameter from 0.1 to 0.4 cm, had a blackish-brownish color and appeared partly lofty, especially on the apex of gastric folds. Histologically, no erosions or ulcers were observed in the gastric mucosa. In some cases, hemorrhages in conjunction with infarctions of the mucosa were observed in the mucosal glands. Those regions, however, did not represent the lesions visible as Wischnewsky spots at the macroscopical level. Immunohistochemical stains were done with a specific antibody against hemoglobin (Dako, Glostrup, Denmark). Wischnewsky spots expressed immunopositivity with antihemoglobin. Concerning the pathogenesis and underlying pathophysiologic mechanisms contributing to the development of Wischnewsky spots, we hypothesize that cooling of the body in the setting of cold ambient temperatures primarily leads to circumscribed hemorrhages of the gastric glands in vivo or in the agonal period, respectively. Subsequently, due to autolysis, erythrocytes are destroyed and hemoglobin is released. Following exposure to gastric acid, hemoglobin is hematinized, leading to the typical blackish-brownish appearance of Wischnewsky spots seen at gross examination. Wischnewsky spots are not equivalent to erosions in terms of histopathological diagnosis but rather represent epiphenomena generated in vivo or in the agonal period of fatal hypothermia.

This paper discusses a membrane chromatographic immunoassay method for rapid detection and quantitative analysis of specific serum antibodies. A type of polyvinylidine fluoride (PVDF) microfiltration membrane was used in the method for its ability to reversibly and specifically bind IgG antibodies from antiserum samples by hydrophobic interaction. Using this form of selective antibody binding and enrichment an affinity membrane with antigen binding ability was obtained in-situ. This was done by passing a pulse of diluted antiserum sample through a stack of microporous PVDF membranes. The affinity membrane thus formed was challenged with a pulse of antigen solution and the amount of antigen bound was accurately determined using chromatographic methods. The antigen binding correlated well with the antibody loading on the membrane. This method is direct, rapid and accurate, does not involve any chemical reaction, and uses very few reagents. Moreover, the same membrane could be repeatedly used for sequential immunoassays on account of the reversible nature of the antibody binding. Proof of concept of this method is provided using human hemoglobin as model antigen and rabbit antiserum against human hemoglobin as the antibody source., ((c) 2005 Wiley Periodicals, Inc.)

The hemoglobin-haptoglobin (Hb-Hp) scavenger receptor CD163 is a monocyte/macrophage-restricted surface antigen, whose expression is strongly up-regulated by glucocorticoids. We have previously shown that CD163 is expressed by acute myeloid leukemia (AML) cells of monocytic lineage. Herein, we expand this finding by demonstrating constitutive and glucocorticoid-enhanced CD163 expression on French-American-British M4/M5 AML cells, and leukemic blasts of other AML subtypes and normal hematopoietic progenitor cells do not express CD163. We provide evidence that the functional characteristics of CD163 are preserved on malignant cells by showing the capability of types M4/M5 blast cells to internalize Hb-Hp by a CD163-mediated mechanism. Together, our results identify CD163 as a potential target for therapeutic intervention. It is important that CD163 does not appear to be released from leukemic blasts under noninflammatory conditions, thus reducing the probability of off-target side-effects as a result of competitive binding of potential therapeutic ligands to nonmembrane-bound CD163.

We compared plastic (polycarbonate) and high-quality glass support materials for gold-coated slides, when performing a model immunoassay against rabbit IgG using fluorescently labeled (AlexaFluor-647) anti-rabbit IgG, and detecting surface plasmon-coupled emission (SPCE) signals. Both, glass and plastic slides were simultaneously coated with a 48-nm layer of gold and protected with a 10-nm layer of silica. The maximum SPCE signal of AlexaFluor-647 was only two- to three-fold smaller on plastic slides than on glass slides. A small difference in the SPCE angles on glass (theta (F) = 55 degrees ) and plastic (theta (F) = 52.5 degrees ) slides was observed and can be explained with a slightly smaller refractive index of the plastic. We have not found any difference in the angle distribution (sharpness of the fluorescence signal at optimal SPCE angle) for the plastic slide compared to the glass slide. The kinetics of binding was monitored on the plastic slide as well as on the glass slide. Optically dense samples, a 4% red blood cell suspension and a 15% hemoglobin solution, are causing a reduction in the immunoassay SPCE signal by approximately 15% and three times, respectively, and the percentage of the reduction is the same for plastic and for glass slides. We believe that plastic substrates can be readily used in any SPCE assay, with only marginally lower total signal compared to high-quality glass slides.