Rabbit Red Blood Cells Research Articles

Hemolytic activity of alternative complement pathway as an indicator of innate immunity in pacu (Piaractus mesopotamicus)

The objective of this study was to evaluate the methodology to establish the hemolytic activity of alternative complement pathway as an indicator of the innate immunity in Brazilian fish pacu (Piaractus mesopotamicus), in addition to verifying the influence of β-glucan as an immunostimulant. Fish were fed with diets containing 0, 0.1 and 1% β-glucan, during seven days, and then inoculated with Aeromonas hydrophila. Seven days after the challenge, they were bled for serum extraction. The methodology consisted of a kinetic assay that allows calculating the required time for serum proteins of the complement to promote 50% lysis of a rabbit red blood cell suspension. The method developed in mammals was successfully applied for pacu and determined that the hemolytic activity of the proteins of the complement system (alternative pathway) increased after the pathogen challenge, but was not influenced by the β-glucan treatment.

Analysis of species-dependent hydrolysis and protein binding of esmolol enantiomers

The stereoselective hydrolysis of esmolol in whole blood and in its separated components from rat, rabbit and human was investigated. Blood esterase activities were variable in different species in the order of rat>rabbit>human. Rat plasma showed the high esterase activity and had no stereoselectivity to enantiomers. Rabbit red blood cell (RBC) membrane, RBC cytosol and plasma all hydrolyzed esmolol but with different esterase activity, whereas the hydrolysis in RBC membrane and cytosol showed significant stereoselectivity towards R-(+)-esmolol. Esterase in RBC cytosol from human blood mainly contributed to the esmolol hydrolysis, which was demonstrated with no stereoselctivity. Esterase in human plasma showed a low activity, but a remarkable stereoselectivity with R-(+)-esmolol. In addition, the protein concentration affected the hydrolysis behavior of esmolol in RBC suspension. Protein binding of esmolol enantiomers in human plasma, human serum albumin (HSA) and α1-acid glycoprotein (AGP) revealed that there was a significant difference in bound fractions between two enantiomers, especially for AGP. Our results indicated that the stereoselective protein binding might play a role in the different hydrolysis rates of esmolol enantiomers in human plasma.

Identification of Anti-Alpha Toxin Monoclonal Antibodies That Reduce the Severity of Staphylococcus aureus Dermonecrosis and Exhibit a Correlation between Affinity and Potency

Staphylococcus aureus alpha toxin (AT) is an important virulence determinant and may be a valid target for immunoprophylaxis against staphylococcal disease. Here we report the identification of potent inhibitory anti-AT monoclonal antibodies (MAbs) derived using B-cell hybridoma technology from VelocImmune mice engineered to produce IgG with a human variable domain. A small panel of inhibitory MAbs blocked AT-mediated lysis of rabbit red blood cells, A549 human lung epithelial cells, and THP-1 human monocytic cells, in a dose-dependent manner. Binding studies indicated that these MAbs recognize a similar epitope on AT and exhibit dissociation constants (K(D)) ranging from 0.50 to 15 nM. In an S. aureus dermonecrosis model, mice passively immunized with anti-AT inhibitory MAbs exhibited significant reductions of lesion size relative to mice treated with an irrelevant IgG control. Interestingly, there was a correlation between MAb affinity for a single epitope, the 50% inhibitory concentration (IC(50)) in the AT hemolytic assay, and lesion size reduction in the dermonecrosis model. A representative high-affinity MAb, 2A3.1, was demonstrated to significantly reduce lesion size following infection with three different clinical isolates (USA300, CC30, and CC5). Taken together, these results indicate that in vitro potency of anti-AT MAbs predicts in vivo potency in this model, supporting their continued preclinical evaluation as molecules for immunoprophylaxis against staphylococcal skin and soft tissue infections caused by diverse clinical isolates.

Detection and characterization of natural and inducible lectins in human serum

This study was performed to detect and characterise the possible occurrence of natural and inducible lectins in human serum by hemagglutination method, wherein, the serum was treated using exogenous elicitors, namely, proteases and detergents.Natural and inducible lectins were detected and characterised in human serum. Untreated serum agglutinated buffalo and rabbit RBC, while serum treated with pronase, trypsin, α-chymotrypsin or SDS for the very first time, agglutinated hen/hen and sheep RBC within 15min in a dosimetric manner. Cross adsorption test revealed that both trypsin and α-chymotrypsin-treated serum showed similar RBC adsorption pattern. The lectin activity in untreated, pronase-treated serum was cation independent and moderately sensitive/insensitive to calcium chelator EDTA, whereas, trypsin-treated serum was cation dependent as well as EDTA sensitive (sheep RBC), cation independent and EDTA insensitive (hen RBC). Hemagglutination of untreated serum was inhibited by certain glycosides and di-, oligo-saccharides, whereas, activity in pronase-treated serum was inhibited by hexosamines. By contrast, hemagglutination of trypsin-treated serum showed specificity for acetylated mannosamine as well as sialic acid for sheep RBC and certain glycoproteins for hen RBC.Thus, we have detected inducible lectins with distinct ligand binding specificity, upon treatment of human serum with proteases, namely, pronase and trypsin. Nevertheless, lectin activity was found in untreated human serum too with different ligand specificity.

Reply to Mangold and Ankersmit

We appreciate the comments [1] of Drs Mangold and Ankersmit on our article [2]. Our group proved that genipin fixation is a novel alternative to conventional glutaraldehyde fixation, and the addition of decellularization, organic solvent treatment and detoxification prevent calcification of glutaraldehyde/genipinfixed bovine pericardium. We also found that the titer of anti-α-Gal(Galα1-3Galβ1-4GlcNAc-R) antibodies (immunoglobulin G) increases after the implantation of bovine pericardium to rabbit, and in vivo calcification results are consistent with the increase in anti-α-Gal antibodies titer [2]. Although the cause and significance of the xenoreactive antibody response between the different species in α-Gal concordant animal model have not been exactly known, some possible suggestions may exist: (i) by a species-specific expression of these conjugated compounds including α-Gal epitopes; (ii) a tissue-specific expression of these conjugated compounds including α-Gal epitopes and (iii) the differences in the fine specificity of natural anti-Gal antibodies in various species which recognize various ‘facets’ of the α-Gal epitope in its three-dimensional form. A species-specific expression of these conjugated compounds including α-Gal epitopes has been well demonstrated for so long. Thyroglobulin, fibrinogen and immunoglobulins from various species express varying amounts of Galα1-3Galβ14GlcNAc residues between 0.01 and 11 residues per molecule [3]. Rabbit red blood cells contain a range of glycolipids of varying lengths that terminate with the Galα1-3Galβ1-4GlcNAc-R structure. These glycolipids vary in size by increments of five monosaccharides, and each increment forms an additional branch. Although rat did not contain Galα1-3Galβ1-4GlcNAc neutral glycosphingolipids in the kidney, pig and sheep contained sialic acid-containing glycolipids (gangliosides) with the Galα1-3Galβ1-4GlcNAc-R structure, most likely the same branched, ganglioside identified in bovine red blood cells [4]. Thymus tissue from sheep, pig and rabbit also contained a range (compounds with 5–11 sugars) of neutral glycolipids terminating with the Galα1-3Galβ1-4GlcNAc-R structure demonstrating a species-specific expression of these compounds. Although the kidney and thymus from sheep, pig and rabbit express Galα1-3Galβ1-4GlcNAc-R glycolipids, these glycolipids were not detected in brain tissue demonstrating a tissue-specific expression of these compounds [5]. The detailed analysis of N-linked glycans from porcine kidney demonstrated that Galα1-3Galβ14GlcNAc-R termini were found on a variety of complex bi-, triand tetra-antennary N-glycans [6]. As we evaluated recently the immune response in the implantation of bovine pericardium to mice in which the α1,3-galactosyltransferase gene was knocked out (Gal/ mice), the titer of anti-α-Gal antibodies much increased. But interestingly, we also observed the increase in anti-α-Gal antibodies in wild-type mouse and rat as well as rabbit, although much less than Gal/ mice. We think that further transplantation or implantation experiments are needed to validate more exact xenoreactive immunologic responses between the different species.

Isolation of Galectin-1 from Human Platelets: Its Interaction with Actin

Galectins are a family of animal lectins defined by their β-galactoside-binding specificity and a consensus sequence in their carbohydrate-recognition domain. Galectin-1 (Gal-1) is expressed as a non-covalently linked homodimer present in a variety of tissues. Here we describe its isolation from human platelets by a procedure involving ionic exchange chromatography and affinity chromatography on lactose-agarose. Platelet Gal-1 co-purifies with actin, forming an actin-Gal-1 complex which does no dissociate even after treatment with sodium dodecyl sulfate. The presence of both proteins was confirmed by Western blot and by trypsin digestion followed by mass spectrometry identification. By hemagglutination assays we studied the response of recombinant Gal-1/actin, mixed and pre-incubated in different proportions, and then tested against neuraminidase treated rabbit red blood cells. The complex formation was confirmed by confocal microscopy, showing that both proteins co-localised in resting platelets as well as in thrombin-activated ones. These results suggest that endogenous Gal-1 forms an intracellular complex with monomeric actin and that, after platelet activation, Gal-1 could play a role in the polymerization-depolymerization process of actin, which concludes in platelet aggregation.

Screening and appraisal for immunological adjuvant-active fractions from Platycodon grandiflorum total saponins

In this study, the total saponins from the root of Platycodon grandiflorum (PGSt) was subjected to D101 macroreticular resin column chromatography to afford four fractions (PGS30, PGS50, PGS75 and PGS95). PGSt and its four fractions were evaluated and compared for the haemolytic activities and adjuvant potentials on the specific cellular and humoral immune responses of ICR mice against recombinant hepatitis B surface antigen (HBsAg). PGSt, PGS30, PGS50, PGS75, and PGS75 showed a slight haemolytic effect, with their concentration inducing 50% of the maximum haemolysis (HD50) being 16.13 ± 0.81, >200, 17.53 ± 0.24, 20.16 ± 0.76, 76.31 ± 2.20 μg/mL against 0.5% rabbit red blood cell, respectively. PGSt, PGS50, and PGS75 significantly not only enhanced the Con A-, lipopolysaccharide-, and HBsAg-induced splenocyte proliferation, but promoted the killing activities of natural killer (NK) cells from splenocytes in HBsAg-immunized mice (P < 0.01 or P < 0.001). HBsAg-specific IgG, IgG1, IgG2a, and IgG2b antibody levels in serum were also significantly enhanced by PGSt, PGS50, and PGS75 compared with HBsAg control group (P < 0.05, P < 0.01, or P < 0.001). Moreover, the adjuvant effects of PGS50 and PGS75 on the cellular immune responses and HBsAg-specific IgG2a and IgG2b antibody responses were more significant than those of Alum, PGS30, and PGS95. The results indicated that PGS50 and PGS75 could improve both cellular and humoral immune responses, and elicit a balanced Th1/Th2 response to HBsAg in mice, and that PGS75 may be developed as an ideal candidate adjuvant for hepatitis B vaccine.

The effects of propolis on antibody production by laying hens

Propolis is a honeybee product showing several biological properties that enhance the immune response, depending on the concentration and intake period. Because propolis possesses an immunomodulatory action on mammals, the objective of our study was to investigate the effects of propolis on the humoral immune response of laying hens by evaluating antibody production. Laying hens (ISA Brown) were divided into 5 groups with 7 birds each. Group 1 was a nonimmunized control, whereas birds in group 2 were immunized intravenously with SRBC, and those in groups 3, 4, and 5 were treated intraperitoneally with propolis (2, 10, and 50 mg/kg, respectively) on 3 consecutive days and then inoculated intravenously with SRBC. Hematological and serological analyses were carried out on d 0, 3, and 38. Natural and specific antibody levels were determined by hemagglutination with rabbit red blood cells and SRBC, respectively. Propolis-treated birds (50 mg/kg) showed a significant decline in heterophils and in the heterophil:lymphocyte ratio. After SRBC immunization, significant increases in levels of IgG were observed in groups 4 and 5. Furthermore, higher levels of natural antibodies were observed in propolis-treated laying hens. The administration of propolis to laying hens increased the production of IgG specific to SRBC and natural antibodies, and could be used to increase antigen-specific antibody responses to vaccines.

Characterization of a natural hemolysin in the serum of a hermit crab Clibanarius longitarsus (Crustacea: Decapoda)

A naturally occurring hemolysin (HL) was detected in the serum of the hermit crab Clibanarius longitarsus using mammalian RBC as indicator cells. The serum gave the highest HL titer with rabbit RBC. Cross adsorption of C. longitarsus serum with fixed rabbit RBC resulted in the complete removal of the lytic activity from the serum. The hemolytic activity in the serum of C. longitarsus was independent of divalent cations, reversibly sensitive to EDTA, thermolabile, stable between the pH 6-8. Further studies demonstrated that the HL activity is proteinaceous as it was precipitable by conventional deproteinizing agents and susceptible to the action of proteases and 2-mercapthoethanol. The HL-inhibition assay performed with several carbohydrates (mono, di, oligo, polysaccharides and Lipo polysaccharides) revealed that the serum HL was capable of specifically recognizing laminarin (a homopolymer of β 1, 3-glucan). Thus this hemolysin appears to be unique among all the known crustacean hemolysins.

Characterization of a natural hemolysin in the serum of a hermit crab Clibanarius longitarsus (Crustacea: Decapoda)

A naturally occurring hemolysin (HL) was detected in the serum of the hermit crab Clibanarius longitarsus using mammalian RBC as indicator cells. The serum gave the highest HL titer with rabbit RBC. Cross adsorption of C. longitarsus serum with fixed rabbit RBC resulted in the complete removal of the lytic activity from the serum. The hemolytic activity in the serum of C. longitarsus was independent of divalent cations, reversibly sensitive to EDTA, thermolabile, stable between the pH 6-8. Further studies demonstrated that the HL activity is proteinaceous as it was precipitable by conventional deproteinizing agents and susceptible to the action of proteases and 2-mercapthoethanol. The HL-inhibition assay performed with several carbohydrates (mono, di, oligo, polysaccharides and Lipo polysaccharides) revealed that the serum HL was capable of specifically recognizing laminarin (a homopolymer of β 1, 3–glucan). Thus this hemolysin appears to be unique among all the known crustacean hemolysins. Keywords: Hemolysin, serum, Clibanarius longitarsus, laminarin.

Haemolytic action of N-alkylpolymethylenediamines.

The haemolytic action of various N-alkyl derivatives (lauryl; C12H25-, myristyl; C14H29-, palmityl; C16H33-) of 1,3-diaminopropane, 1,4-diaminobutane, 1,5-diaminopentate, 1,6-diaminohexane, 1,7-diaminoheptane, 1,8-diaminooctane was examined using rabbit red blood cells. The activities of the various derivatives were compared with those of antiplaque agents commonly used as mouthwashes; cetylpyridinium chloride (CP) and chlorhexidine acetate (CH). The haemolytic activities of these agents were dependent on the length of the N-alkyl chain, whereas the number of methylene groups between the nitrogen atoms had little effect. The order of potency was CP, N-palmityl derivatives, N-myristyl derivatives greater than N-lauryl derivatives greater than CH which was similar to the order of the antiplaque effect evaluated in-vitro.

The development, characterization, and demonstration of a novel strategy for purification of recombinant proteins expressed in plants

Plants have attracted increasing attention as an expression platform for the production of pharmaceutical proteins due to its unlimited scalability and low cost potential. However, compared to other expression systems, plants accumulate relatively low levels of foreign proteins, thus necessitating the development of efficient systems for purification of foreign proteins from plant tissues. We have developed a novel strategy for purification of recombinant proteins expressed in plants, based on genetic fusion to soybean agglutinin (SBA), a homotetrameric lectin that binds to N-acetyl-D-galactosamine. Previously it was shown that high purity SBA could be recovered from soybean with an efficiency of greater than 90% following one-step purification using N-acetyl-D-galactosamine-agar columns. We constructed an SBA fusion protein containing the reporter green fluorescent protein (GFP) and transiently expressed it in N. benthamiana plants. We achieved over 2.5% of TSP accumulation in leaves of N. benthamiana. Confocal microscopic analysis demonstrated in vivo activity of the fused GFP partner. Importantly, high purity rSBA-GFP was recovered from crude leaf extract with ~90% yield via one-step purification on N-acetyl-D-galactosamine-agar columns, and the purified fusion protein was able to induce the agglutination of rabbit red blood cells. Combined with this, tetrameric assembly of the fusion protein was demonstrated via western blotting. In addition, rSBA-GFP retained its GFP signal on agglutinated red blood cells, demonstrating the feasibility of using rSBA-GFP for discrimination of cells that bear the ligand glycan on their surface. This work validates SBA as an effective affinity tag for simple and rapid purification of genetically fused proteins.

Adjuvant effects of Astragalus saponins Macrophyllosaponin B and Astragaloside VII

The present study was undertaken to evaluate the hemolytic activities of two immunomodulator Astragalus saponins [Macrophyllosaponin B (MacB) from Astragalus oleifolius DC. and Astragaloside VII (Ast VII) from Astragalus trojanus Stev.], and their adjuvant potentials on the cellular and humoral immune responses of Swiss albino mice against BSA. The hemolytic activity of Mac B and Ast VII was determined using 0.5% rabbit red blood cell. For adjuvant activity, Swiss albino mice were immunized subcutaneously with BSA 100 μg alone or with BSA 100 μg dissolved in saline containing Ast VII (30, 60, 120 and 240 μg), Mac B (30, 60, 90 and 120 μg) or Freund's adjuvant on Days 1 and 15. Sera and splenocytes were collected 2 weeks after the last immunization for concanavalin A (Con A)-, lipopolysaccharide (LPS)- and BSA-stimulated splenocyte proliferation assay and measurement of BSA-specific antibodies in serum. Mac B and Ast VII showed a slight hemolytic effect, with 0.42% and 0.54% values, respectively, at the highest concentration of 500 μg/ml. Mac B and Ast VII significantly enhanced the Con A-, LPS-, and BSA-induced splenocyte proliferation in the BSA-immunized mice especially at 120 and 240 μg (P<0.001), and 60, 90 and 120 μg (P<0.05, P<0.01 or P<0.001) doses, respectively. BSA-specific IgG, IgG1 and IgG2b antibody titers in serum were also significantly enhanced by Ast VII (120 μg), Mac B (90 μg) and Freund's as compared to the control group (P<0.01 or P<0.001). Moreover, the IFN-γ and IL-4 levels in the sera were detected using ELISA two weeks after the last immunization. Ast VII and Mac B were also found to stimulate IFN-γ production such as Freund's, two weeks after the last immunization at doses of 120 μg and 90 μg, respectively, as compared to the control. Results show that Ast VII and Mac B generate important specific antibody and cellular response against BSA in mice, proving their potentials as a new class saponin adjuvant.

High Molecular Weight Lectin Isolated from the Mucus of the Giant African SnailAchatina fulica

To understand better the host defense mechanisms of mollusks against pathogens, we examined the anti-microbial activity of mucus from the giant African snail Achatina fulica. Hemagglutination activity of the mucus secreted by the integument of snails inoculated with Escherichia coli was observed to increase and to cause hemagglutination of rabbit red blood cells. Purification of the snail mucus lectin by sequential column chromatography revealed that the relative molecular mass of the lectin was 350 kDa. The hemagglutination activity of the lectin was Ca(2+)-dependent and was inhibited by galactose. Growth arrest tests showed that the lectin did not inhibit bacterial growth, but did induce agglutination of gram-positive and gram-negative bacteria. Tissue distribution analyses using a polyclonal antibody revealed that the lectin was expressed in the tissues of the mantle collar. The lectin isolated from the mucus of the snail appeared to contribute to its innate immunity.

TT30, a Novel Human Complement Inhibitor in Development for Paroxysmal Nocturnal Hemoglobinuria and Other Hemolytic Disorders, Demonstrates Red Blood Cell Surface Targeting and Retention In a Model of Complement Alternative Pathway-Mediated Hemolysis

AbstractAbstract 638Polymorphisms and mutations that promote Complement Alternative Pathway (CAP) activity are associated with human diseases, especially genetically linked hemolytic disorders such as paroxysmal nocturnal hemoglobinuria (PNH) and thrombotic microangiopathy (TMA) disorders such as atypical hemolytic uremic syndrome (aHUS) and thrombotic thrombocytopenic purpura. The complement system can be activated through three unique pathways (classical, lectin/mannose and alternative). In PNH, the lack of CD55 on RBC allows CAP-initiated complement C3 activation by C3 convertases, while the lack of CD59 allows C5 activation by C5 convertase to proceed to formation of the membrane attack complex (MAC; C5b-9), resulting in intravascular hemolysis (IVH). Treatment of patients with the anti-C5 monoclonal antibody (mAb) eculizumab abrogates IVH; however, because eculizumab does not inhibit CAP activity prior to C5, covalently bound C3 fragments (C3frag) and both C3 and C5 convertases continue to accumulate on PNH red blood cells (RBCs). Clearance of PNH RBCs that are C3frag-coated by complement receptors within the reticuloendothelial system (RES) is the putative cause of continued extravascular hemolysis (EVH) in patients who receive eculizumab. Continued anemia and transfusion requirements are found in a substantial proportion of eculizumab-treated patients, and correlate with PNH RBC-bound C3frag. High levels of C5 convertases on the same cells may also contribute to intermittent escape from eculizumab control of IVH due to pharmacodynamic breakthrough. To selectively modulate CAP activity on PNH RBC and replace the CD55-mediated control of CAP activation, we developed TT30, a novel therapeutic fusion protein linking the C3frag-binding domain of human complement receptor type 2 (CR2/CD21) with the CAP inhibitory domain of human factor H (fH). TT30 delivers cell surface-targeted (via CR2) inhibition of CAP activity (via fH) and blocks the ex vivo hemolysis of PNH RBCs, while at the same time retaining the normal ability of the complement system to efficiently activate C3 through the classical and lectin pathways. We studied the mechanism of TT30 prevention of hemolysis by control of CAP activity in human serum using: 1) an in vitro model of CAP-mediated hemolysis in which rabbit RBCs are exposed to normal human serum under conditions promoting CAP activation (Mg++/EGTA) and the extent of hemolysis is quantified by measuring hemoglobin release; 2) flow cytometric phenotyping of C3frag accumulation on rabbit RBCs exposed to normal or C5-deficient human serum using mAbs specific for human iC3b (A710, Quidel) or C3d (A702, Quidel); 3) flow cytometric demonstration of TT30 binding to C3frag+ rabbit RBCs with a noncompeting mAbs against CR2 (HB5, Taligen) or fH (A255, Quidel); and 4) an in vitro model of CAP-mediated MAC formation in which human serum is exposed to an LPS-coated surface in the presence of Mg++/EGTA and CAP activation through to the MAC is quantified by detection of a neoantigen in poly-C9 by ELISA. The results demonstrate that TT30 efficiently inhibits CAP-mediated MAC formation (IC50 of 3.2 ug/ml) and hemolysis (IC50 of 50.1 ug/ml) and that both of these activities are dependent upon targeting to C3frag+ surfaces by CR2, as evidenced by complete reversal of TT30 inhibitory activity in the presence of a 2-fold molar excess of a competing anti-CR2 mAb (1048, Taligen). Rabbit RBCs were shown to become coated with C3frag in the presence of normal and C5-deficient serum and to undergo lysis with normal serum. TT30 was readily demonstrated to be bound to C3frag+ RBCs during prevention of hemolysis and to remain detectable on RBCs for at least 24 hours. The amount of bound TT30 was proportional to the accumulation of C3frags. Collectively, these results demonstrate that TT30 displays targeted control of cell surface CAP activation, with both effective and prolonged blockade of MAC formation, and dose-dependent inhibition of hemolysis. Therefore, the CAP-specific novel therapeutic TT30 has potential utility for the treatment of human complement-mediated diseases, such as PNH and aHUS, in which modulation of CAP activation is predicted to be clinically beneficial. Disclosures:Fridkis-Hareli:Taligen Therapeutics: Employment. Storek:Taligen Therapeutics: Employment. Risitano:Taligen Therapeutics: Consultancy, Research Funding. Lundberg:Taligen Therapeutics: Employment, Membership on an entity's Board of Directors or advisory committees. Horvath:Taligen Therapeutics: Employment. Holers:Taligen Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Isolation and identification of pelteobagrin, a novel antimicrobial peptide from the skin mucus of yellow catfish (Pelteobagrus fulvidraco)

Fish skin mucus has recently been recognized to be a potential source of antimicrobial peptides, which provides the first line of defense against invading pathogens. This study reports the purification and characterization of a novel linear antimicrobial peptide, pelteobagrin, from the skin mucus of yellow catfish (Pelteobagrus fulvidraco, Richardson). Pelteobagrin is 20 amino acids in length (GKLNLFLSRLEILKLFVGAL) and shows no clear homology with any known bioactive peptides. MALDI-TOF MS indicated the molecular mass of the purified peptide was 2244.4 Da, which is in good agreement with pelteobagrin's predicted molecular weight of 2244.8 Da. Pelteobagrin exhibited antibacterial activity against Gram-positive and Gram-negative bacteria as well as fungi, and the activity was relatively salt-insensitive as it was not affected by NaCl concentrations of up to 137 mM. Moreover, pelteobagrin displayed no hemolytic activity to rabbit red blood cells. Transmission electron microscopy suggested that pelteobagrin might kill bacteria via acting on both the cell wall and the cytoplasmic membrane of bacteria. These results suggest that pelteobagrin might be involved in the innate defense system in yellow catfish.

Old blood, new blood or better stored blood?

Blood transfusion has controversial origins shrouded in legend. In ancient times blood was considered “vital lymph” - “sanguis vita vitae” - and was probably used as a potion or a tonic, and already the possibility of transferring it from one body to another for therapeutic or rejuvenating purposes had been taken into consideration1. However, it was only in 1628, the year in which the circulation of blood was scientifically first demonstrated, that transfusing blood became a concrete possibility. The subsequent “experimental” period, a mixture of successes and failures, extended from 1628 to the end of the XVII century and was followed by the so-called “therapeutic” period from the mid 1800s to the present1. After the anatomic demonstration of blood circulation, nearly 300 years passed before Karl Landsteiner discovered blood groups, the pharmacologist Luigi Sabbatani discovered the anticoagulant properties of sodium citrate1, and, in 1915, the first red blood cell (RBC) storage solution (a mixture of citrate and glucose) was developed for storing rabbit RBC for use in a heterophile agglutination test for syphilis2.

Hydroxyurea stimulates the release of ATP from rabbit erythrocytes through an increase in calcium and nitric oxide production

Hydroxyurea, a proven therapy for sickle cell disease, is known to improve blood flow and reduce vaso-occlusive crises, although its exact mechanism of action is not clear. The objective of this study was to determine if hydroxyurea results in an increase of ATP release from the red blood cell (RBC) via the drug's ability to stimulate nitric oxide (NO) production in these cells. A system enabling the flow of RBCs through microbore tubing was used to investigate ATP release from the RBC. Incubation of rabbit RBCs (7% hct) with 50 μM hydroxyurea resulted in a significant increase in the release of ATP from these cells. This level of ATP release was not detected in the absence of flow. Studies also showed that increments in hydroxyurea and NO (from spermine NONOate) resulted in an initial increase in ATP release, followed by a decrease in this release at higher concentrations of hydroxyurea and the NO donor. Incubation with L-NAME abolished the effect of the hydroxyurea, suggesting that NO production by the RBC was involved. Indeed, in the presence of 50 μM hydroxyurea, the amount of total Ca 2+ measured (by atomic absorption spectroscopy) in a 7% solution of RBCs increased from 363 ± 47 ng/ml and 530 ± 52 ng/ml. Finally, EPR studies suggest that an increase in nitrosylated Hb in the RBC is only measured for those studies involving hydroxyurea and a Ca 2+-containing buffer.

SpirulinaImproves Antioxidant Status by Reducing Oxidative Stress in Rabbits Fed a High-Cholesterol Diet

The beneficial effect of Spirulina (Spirulina platensis) on tissue lipid peroxidation and oxidative DNA damage was tested in the hypercholesterolemic New Zealand White rabbit model. After hypercholesterolemia was induced by feeding a high cholesterol (0.5%) diet (HCD) for 4 weeks, then HCD supplemented with 1% or 5% Spirulina (SP1 or SP5, respectively) was provided for an additional 8 weeks. Spirulina supplementation significantly reduced the increased lipid peroxidation level in HCD-fed rabbits, and levels recovered to control values. Oxidative stress biomarkers such as glutathione, glutathione peroxidase, glutathione reductase, and glutathione S-transferase were significantly improved in the liver and red blood cells of rabbits fed SP1. Furthermore, SP5 induced antioxidant enzyme activity by 3.1-fold for glutathione, 2.5-fold for glutathione peroxidase, 2.7-fold for glutathione reductase, and 2.3-fold for glutathione S-transferase in liver, compared to the HCD group. DNA damage in lymphocytes was significantly reduced in both the SP1 and SP5 groups, based on the comet assay. Findings from the present study suggest that dietary supplementation with Spirulina may be useful to protect the cells from lipid peroxidation and oxidative DNA damage.

Detection of Insect Immunity Substances (Lectins) in the Midgut Extracts from Larvae and Adult Red Palm Weevil Rhynchophorus ferrugineus (Olivier) in Al-Ahsa, Saudi Arabia

The hemagglutination activities of midgut (MG) fractions of red palm weevil, Rhynchophorus ferrugineus against mammalian erythrocytes (RBC) from man (ABO), rabbit, horse and sheep were studied. The highest titers were seen with rabbit RBC (p < 0.05) followed by human group B, human group O, horse, human group A, human group AB and sheep, respectively. Insect age and nutritional status significantly influenced the agglutinating activities. Some of the unfed insects were having low activities (p > 0.01) than the well fed insects (p < 0.05). The agglutination activities were linearly correlated with the insect age. The agglutination activities were higher in old insects than the young insects (p < 0.05). Further studies are needed to investigate the lectins in the hemolymph of the red palm weevil, Rhynchophorus ferrugineus beside demonstrating their agglutination activities against the natural entomopathogenes organisms of this economically important insect for use in its biological control.