Rabbit Red Blood Cells Research Articles

Glossina Proteolytic Lectin Does Not Require a Carbohydrate Moiety for Enzymatic or Trypanosome-Transforming Activities

Abstract The developmental cycle of the cyclically transmitted African trypanosome involves an obligatory passage through the tsetse fly, Glossina spp. This intricate relationship requires the presence of molecules within the insect vector, including a midgut lectin, that interact with the trypanosome. Recently, a gene encoding for a proteolytic lectin, with trypanosome-transforming activity, was isolated from a midgut cDNA library of Glossina fuscipes fuscipes Austen in our laboratory. Using the same approach, we have identified a similar gene from a midgut cDNA library of Glossina austeni (Newstead). The protein encoded by this gene was expressed in bacteria and a baculovirus-based expression system. The baculovirus-expressed lectin was found in the medium of baculovirus-infected Sf-21 cell cultures, indicating that the tsetse fly-derived signal peptide was recognized and cleaved by the Sf-21 cells. The baculovirus-expressed protein also was glycosylated despite the absence of classical O-linked and N-linked sugar attachment motifs. Both the baculovirus- and bacterium-expressed lectin proteins were shown to agglutinate trypanosomes and rabbit red blood cells in vitro. This agglutination was strongly inhibited by d-glucosamine. d-Glucosamine also inhibited the action of the authentic and recombinant lectins upon the chromogenic substrate Chromozym TRY. Interestingly, both baculovirus- and bacterium-expressed lectins showed no significant differences in terms of these activities, indicating that a sugar moiety is not essential for biological activity. Our results provide an important molecular tool for further characterization of Glossina proteolytic lectin.

Adjuvant effect of Achyranthes bidentata saponins on specific antibody and cellular response to ovalbumin in mice

In this study, the haemolytic activities of Achyranthes bidentata saponins (ABS) and its adjuvant potentials on the cellular and humoral immune responses of ICR mice against ovalbumin (OVA) were evaluated. We determined the haemolytic activity of ABS using 0.5% rabbit red blood cell. The concentration inducing 50% of the maximum haemolysis (HD 50) for ABS was 164.59 ± 13.41 μg/ml. ICR mice were immunized subcutaneously with OVA 100 μg alone or with OVA 100 μg dissolved in saline containing Alum (200 μg), QuilA (10 and 20 μg) or ABS (50, 100 or 200 μ g) on Days 1 and 15. Two weeks later (Day 28), concanavalin A (Con A)-, lipopolysaccharide (LPS)- and OVA-stimulated splenocyte proliferation and OVA-specific antibodies in serum were measured. ABS significantly enhanced the Con A-, LPS-, and OVA-induced splenocyte proliferation in the OVA-immunized mice especially at a dose of 100 μg ( P < 0.05 or P < 0.025). OVA-specific serum IgG, IgG1 and IgG2b antibody titers were also significantly enhanced by ABS compared with OVA control group ( P < 0.05 or P < 0.025). The results suggest that ABS showed a slight haemolytic effect and enhanced significantly a specific antibody and cellular response against OVA in mice.

Molecular characterization of a tsetse fly midgut proteolytic lectin that mediates differentiation of African trypanosomes

Differentiation of bloodstream-form trypanosomes into procyclic (midgut) forms is an important first step in the establishment of an infection within the tsetse fly. This complex process is mediated by a wide variety of factors, including those associated with the vector itself, the trypanosomes and the bloodmeal. As part of an on-going project in our laboratory, we recently isolated and characterized a bloodmeal-induced molecule with both lectin and trypsin activities from midguts of the tsetse fly, Glossina longipennis [Osir, E.O., Abubakar, L., Imbuga, M.O., 1995. Purification and characterization of a midgut lectin–trypsin complex from the tsetse fly, Glossina longipennis. Parasitol. Res. 81, 276–281]. The protein (lectin–trypsin complex) was found to be capable of stimulating differentiation of bloodstream trypanosomes in vitro. Using polyclonal antibodies to the complex, we screened a G. fuscipes fuscipes cDNA midgut expression library and identified a putative proteolytic lectin gene. The cDNA encodes a putative mature polypeptide with 274 amino acids (designated Glossina proteolytic lectin, Gpl). The deduced amino acid sequence includes a hydrophobic signal peptide and a highly conserved N-terminal sequence motif. The typical features of serine protease trypsin family of proteins found in the sequence include the His/Asp/Ser active site triad with the conserved residues surrounding it, three pairs of cysteine residues for disulfide bridges and an aspartate residue at the specificity pocket. Expression of the gene in a bacterial expression system yielded a protein ( M r ∼32,500). The recombinant protein ( Gpl) bound d(+) glucosamine and agglutinated bloodstream-form trypanosomes and rabbit red blood cells. In addition, the protein was found to be capable of inducing transformation of bloodstream-form trypanosomes into procyclic forms in vitro. Antibodies raised against the recombinant protein showed cross-reactivity with the α subunit of the lectin–trypsin complex. These results support our earlier hypothesis that this molecule is involved in the establishment of trypanosome infections in tsetse flies.

Dietary administration of bovine lactoferrin influences the immune ability of the giant freshwater prawn Macrobrachium rosenbergii (de Man) and its resistance against Aeromonas hydrophila infection and nitrite stress

Haemocyte count, phenoloxidase activity, agglutinin levels, total protein content, bacterial clearance efficiency, resistance to the pathogen Aeromonas hydrophila and nitrite stress were measured in the giant freshwater intermoult subadult prawn Macrobrachium rosenbergii (15–20 g) which had been fed diets containing bovine lactoferrin (Lf) at 50, 100, 200 mg kg −1 feed for 7 or 14 days. M. rosenbergii fed a diet containing 100 mg Lf kg −1 diet for 7 days showed significant ( P < 0.05) increase in total protein levels, agglutination titres against bacteria A. hydrophila and rabbit RBC, phenoloxidase activity, bacterial clearance (as observed through reduced number of circulating bacteria) as well as survival against A. hydrophila challenge. Increased bacterial clearance was also noticed in prawns fed Lf at 50 or 200 mg kg −1 for 14 days compared to control. Feeding of Lf at 50 mg kg −1 diet for 7 or 14 days was able to enhance only PO activity and reduce percent mortality against A. hydrophila challenge compared to its control. Total haemocyte count was higher in the lowest dose of Lf feeding, i.e. 50 mg kg −1 for 7 days. However, there was no significant alteration in the differential haemocyte population with respect to graded levels of Lf feeding for 7 or 14 days. A notable reduction in mortality percent after 120 h of nitrite stress was observed in prawn fed Lf at 100 mg kg −1 diet for 14 days. On the contrary, feeding of the highest dose of Lf, i.e. 200 mg kg −1 diet for 14 days failed to stimulate most of the innate immune parameters or reduce the percent mortality against A. hydrophila challenge or nitrite stress. It is therefore concluded that administration of Lf in the diet at 100 mg kg −1 for 7 days could enhance the immune ability of M. rosenbergii and increase its resistance to A. hydrophila infection or nitrite stress.

Cloning and expression of a novel cDNA encoding a mannose-specific jacalin-related lectin from Oryza sativa

Lectin plays an important role in defense signaling in plants. A few genes of this family have been cloned. Here we report on a mannose-specific jacalin-related lectin in rice. Using sequence information of wheat gene VER2, which we had previously cloned, we were able to amplify a cDNA of OsJAC1 from Oryza sativa by RT-PCR. The cDNA of OsJAC1 was 1172 bp and contained a 921-bp open reading frame (ORF) encoding dirigent (amino acids 26–139) and jacalin (amino acids 175–305) domains of 306 amino acids. Comparison of the OsJAC1 sequence with those of other lectins (jacalin) from rice, wheat and other species revealed that OsJAC1 had the 12 amino acid positions conserved in all mannose-binding lectins. Semi-quantitative RT-PCR revealed that OsJAC1 expression was present in stems, leaves and young spikes but not young roots; the expression was high in leaves and low in stems and spikes. And methyl jasmonate could induce the expression of OsJAC1. To test the activity of OsJAC1, the jacalin domain at the C-terminal was expressed in E. coli. BL21 (DE3). The purified recombinant protein could agglutinate red blood cells of rabbit, and the agglutination activity was strongly inhibited by mannose compared with other carbohydrates. These results indicate that lectin with dirigent and jacalin domains exists in rice as well as wheat. This is the first report of cDNA cloning of mannose-binding jacalin-related lectin with a dirigent domain in N-terminal region from O. sativa.

Immunological adjuvant effect of Glycyrrhiza uralensis saponins on the immune responses to ovalbumin in mice

In this study, the haemolytic activities of Glycyrrhiza uralensis saponins (GLS) and its adjuvant potentials on the cellular and humoral immune responses of ICR mice against ovalbumin (OVA) were evaluated. We determined the haemolytic activity of GLS using 0.5% rabbit red blood cell. Haemolytic percents of GLS-treated red blood cell were 11.20 and 5.54% at the concentrations of 500 and 250 μg/ml, respectively. ICR mice were immunized subcutaneously with OVA 100 μg alone or with OVA 100 μg dissolved in saline containing Alum (200 μg), QuilA (10 and 20 μg), or GLS (50, 100, or 200 μg) on Days 1 and 15. Two weeks later (Day 28), concanavalin A (Con A)-, lipopolysaccharide (LPS)-, and OVA-stimulated splenocyte proliferation and OVA-specific serum antibodies were measured. GLS significantly enhanced the Con A-, LPS-, and OVA-induced splenocyte proliferation in the OVA-immunized mice at a dose of 100 μg ( P < 0.025). OVA-specific IgG, IgG1, and IgG2b antibody titers in serum were also significantly enhanced by GLS compared with OVA control group ( P < 0.025). Moreover, no significant differences ( P > 0.05) were observed between enhancing effect of GLS and QuilA on the OVA-specific IgG, IgG1, and IgG2b antibody responses to OVA in mice. The results suggest that GLS showed a slight haemolytic effect and enhanced significantly a specific antibody and cellular response against OVA in mice, and deserved further researches to be developed as immunological adjuvant.

Monitoring erythrocytes in a microchip channel that narrows uniformly: Towards an improved microfluidic-based mimic of the microcirculation

The release of adenosine triphosphate (ATP) from red blood cells (RBCs) flowing through PDMS microchannels has been determined as a function of channel cross-sectional area using a design containing a channel that narrows uniformly. ATP, released from the RBCs in response to the mechanical deformation of their cell membranes, increased as the channel cross-section decreased. One sample of rabbit RBCs released 1.16 ± 0.11, 1.92 ± 0.14 and 2.09 ± 0.10 μM ATP as the median cross-sectional area decreased from 4314 to 3192 to 2052 μm 2, respectively. Numerous samples ( n = 6) displayed the same trend. Incubating a sample of RBCs with diamide, a substance known to stiffen cell membranes without harming the cell cytosol, provided evidence that no cell lysis occurred in the microchip device. This novel use of lab-on-a-chip technology allows for channel designs that enable an in vitro study of physiological events that occur in the microcirculation.

Isolation and characterization of novel lectins from Canavalia ensiformis DC and Dioclea grandiflora Mart. ex Benth. seeds

Two lectins were isolated from Canavalia ensiformis and Dioclea grandiflora seeds. Gel filtration produced a fraction corresponding to Con A or D. grandiflora lectin while erythroagglutination assays revealed a distinct fraction presenting a lectin that agglutinates human red blood cells (RBCs) but not rabbit RBCs. Hydrophobic interaction chromatography showed that the latter fraction yielded a protein that readily agglutinates human erythrocytes; the lectin was also purified by affinity chromatography on Lac-Sepharose showing similar properties to that of the Phenyl-Sepharose-purified lectin. Despite minor differences (carbohydrate content or A1%1cm), the two lectins showed similar molecular properties in that they consisted of two non-covalently linked monomers having a Mr of 29-30 kDa and their pI values indicated that both lectins were slightly acidic proteins. The C. ensiformis lectin (CEL-II) and D. grandiflora lectin (DGL-II) specifically recognised the H-type 2 blood group (alpha-L-Fuc (1-2)-beta-D-Gal (1-4)-beta-D-GlcNAc-O-R), while binding to H-type 1, H-type 3, H-type 4, Leª or Le y was weaker. Carbohydrate inhibition of erythroagglutination showed that simple sugars were weakly recognised by the lectins, if at all. The N-terminal region presented a unique sequence hitherto found only in some Diocleinae lectins (designated type II). The overall results confirmed the existence of a second distinct lectin type, phylogenetically close to Diocleinae species. The data indicate a functional similarity among lectins of this type which possesses distinctive characteristics differentiating them from "classical" Man/Glc lectins.

Haemolytic activities and adjuvant effect of Astragalus membranaceus saponins (AMS) on the immune responses to ovalbumin in mice

In this study, the haemolytic activities of Astragalus membranaceus saponins (AMS) and its adjuvant potentials on the cellular and humoral immune responses of ICR mice against OVA were evaluated. We determined the haemolytic activity of AMS using 0.5% rabbit red blood cell. AMS showed a slight haemolytic effect, with its haemolytic percents being 0.66% at the concentration of 500 μg/ml. Furthermore, the adjuvant potentials of AMS at three dose levels on the cellular and humoral immune responses of ICR mice against ovalbumin (OVA) were investigated. ICR mice were immunized subcutaneously with OVA 100 μg alone or with OVA 100 μg dissolved in saline containing Alum (200 μg), QuilA (10 and 20 μg) or AMS (50, 100 or 200 μg) on Day 1 and 15. Two weeks later (Day 28), concanavalin A (Con A)-, lipopolysaccharide (LPS)- and OVA-stimulated splenocyte proliferation and OVA-specific antibodies in serum were measured. AMS significantly enhanced the Con A-, LPS-, and OVA-induced splenocyte proliferation in the OVA-immunized mice especially at a dose of 100 μg ( P < 0.05 or P < 0.001). OVA-specific IgG, IgG1 and IgG2b antibody titers in serum were also significantly enhanced by AMS compared with OVA control group ( P < 0.01 or P < 0.001). Moreover, no significant differences ( P > 0.05) were observed between enhancing effect of AMS and QuilA on the OVA-specific IgG, IgG1 and IgG2b antibody responses to OVA in mice. In conclusion, the results suggest that AMS could be safely used as adjuvant with low or non-haemolytic effect.

A comparison of bile composition from heart-beating and non–heart-beating rabbit organ donors during normothermic extracorporeal liver perfusion: Experimental evaluation using proton magentic resonance spectroscopy

Background Organs retrieved from marginal and non–heart-beating donors (NHBDs) have sustained variable degrees of preretrieval damage that result in an increased incidence of complications. Normothermic extracorporeal liver perfusion (NELP) provides an opportunity to evaluate and resuscitate such organs. The aim of this study was to identify markers of ischemic injury in bile during perfusion of livers from heart-beating donors (HBDs) and NHBDs. Methods Livers were retrieved from New Zealand white rabbits. The HBD group ( n = 4) had no in situ warm ischemia before retrieval and the NHBD group ( n = 4), 45 minutes of in situ warm ischemia before liver retrieval. After 40 minutes of postretrieval cold ischemia, all livers were dual vessel reperfused, normothermically with oxygenated buffer solution supplemented with rabbit red blood cells, for 6 hours. Bile was collected and examined with 1HMRS. Results Perfusion bile from HBD group showed an increased concentration of bile acids, lactate, glucose, and phosphatidylcholine, but a decreased concentration of acetate as compared to retrieval bile. This trend was further enhanced in NHBD group. The mean ± SD (in μmol/L) were bile acids (10.48 ± 2.8 vs 26.05 ± 12.1 vs 44.5 ± 44.5), lactate (10.66 ± 4.5 vs 14.66 ± 5.2 vs 13.22 ± 1.8), glucose (5.37 ± 2 vs 21.2 ± 5.0 vs 29.09 ± 15.3), phosphatidylcholine (0.21 ± 0.02 vs 5.57 ± 1.7 vs 6.42 ± 0.3), and acetate (1.8 ± 0.5 vs 0.39 ± 0.1 vs 0.38 ± 0.09) for retrieval bile, HBD perfusion bile, and NHBD perfusion bile, respectively. One animal from each group did not produce any bile during perfusion. Conclusions 1HMRS of biliary constituents revealed differences with the type of ischemia. These indices may be potential markers of the extent of warm ischemic injury and the functional activity of an extracorporeally perfused liver.

Bioeffects of Low-Frequency Ultrasonic Gene Delivery and Safety on Cell Membrane Permeability Control

To develop a novel method of ultrasonic naked gene delivery (UNGD); to examine the relationship between optimal parameters of ultrasound exposure and cell membrane permeability, enzymes, and free radicals; and to find optimal control parameters that were realizable, reliable, and noncytotoxic for use in gene therapy. Suspensions of chicken, rabbit, and rat red blood cells and S180 cells were exposed to a calibrated ultrasonic field with different parameters in both the still and flowing states to obtain optimal parameters for UNGD. The optimal parameters then were used to implement UNGD. We examined morphologic characteristics, membrane permeability, enzymes, free radicals, naked gene expression efficiency, cell damage threshold, and cell viability by laser scanning confocal microscopy, fluorescent microscopy, flow cytometry, and spectrophotometry. Green fluorescent protein (GFP) as a reporter was delivered into S180 cells under the optimal parameters without cell damage or cytotoxicity. The transfection rate (mean +/- SD) was approximately 35.83% +/- 2.53% (n = 6) in viable cells, and cell viability was 90.17% +/- 1.47% (n = 6). The intensity of GFP expression with UNGD showed a higher fluorescent peak over both an adeno-associated virus vector-GFP group and a control group (P < .001). Additionally, malondialdehyde, hydroxyl free radicals, alkaline phosphatase, and acid phosphatase displayed an S-shaped growth model (r = 0.98 +/- 0.01) in response to permeability and morphologic alteration. Under optimal conditions, low-frequency ultrasound can safely deliver naked genes into cells without causing cell damage. The analytical results indicate that, except for subcavitation, free radical products are responsible for bioeffects in gene delivery. The constant E of energy deposition at 90% cell viability is the optimal control factor, and 80% viability represents the damage threshold. Optimal gene uptake by cells and safety depend on E. Constant E can be applied to control the gene delivery effect in combination with other parameters.

Vybrant™ DiO, DiI and DiD Dyes for Multiple Labeling of Red Blood Cell Populations for In Vivo Survival Studies.

Background: Studies of the circulatory time of red blood cells (RBCs) or drug delivery vehicles such as liposomes require a radiolabel (e.g., 51Cr), a fluorescent probe or an affinity tag (e.g., biotin). These techniques may be complicated by progressive loss of the probe from the labeled cells, transfer to other cells, and sometimes by the development of an immune response to the label. Long chain dialkylcarbocyanines (Vybrant DiO, DiI and DiD) are bright, stable lipophilic membrane fluorescent labels that can exhibit fluorescence resonance energy transfer (FRET) and are easily observed by flow cytometry using standard filters (FL1, FL2 and FL4 detection channels respectively). The staining is very stable in vivo and is dose-dependent, thus populations of labeled cells may have discrete fluorescence intensities. We recognized that use of these dyes in combination could permit the simultaneous observation of several different populations of cells within the same subject, together with one or more internal controls. To evaluate this technique, we developed procedures suitable for the fluorescent labeling up to 14 discrete populations of human or rabbit RBCs. The properties of the rabbit RBCs were then evaluated in vivo.Methods: Washed human RBCs were suspended at a 10% hematocrit in 20mM HEPES buffer, and added to an equal volume of freshly prepared 1, 4 or 8 μM DiO, DiI, DiD, or mixtures of two labels; 36 different combinations in total. The cells were incubated with the dye for 60 minutes at 37°C, washed twice in HEPES with 0.1% albumin and examined by flow cytometry. Aliquots from 6 to 15 differently-labeled samples were then combined, and the mixtures re-examined. For in vivo studies, the labeling procedure was repeated with autologous rabbit RBCs at dye concentrations of 1.5, 6, and 12 μM. The labeled rabbit RBCs were washed, suspended in saline and reinfused via the ear vein. Small blood samples were drawn daily for 4 days and weekly for 5 weeks, and the number of fluorescently labeled cells of each color remaining was determined by flow cytometry.Results: Of the 36 possible combinations, 14 discrete populations could be clearly distinguished from “dot-plots” of FL1 vs. FL2, FL1 vs. FL4 and FL2 vs. FL4. FRET was observed for all cells stained with two labels. In vivo evaluation of 8 different labeled rabbit RBC populations showed normal survival. A 10–20% reduction in fluorescence intensity was observed over the study period; this reduction did not compromise the clear identification of each discrete population. Rabbits that were repeatedly exposed to Vybrant-labeled autologous RBCs (up to 15 exposures over 3 years) showed no evidence of accelerated clearance, suggesting that these fluorescent labels do not exhibit significant immunogenicity.Conclusion: The use of two long-chain dialkylcarbocyanines in combination is ideal for studies which require multiple cell populations to be followed within the same experiment, and additionally allows for one or more internal controls to correct for any variability of circulation times. The technique is presently being used for studies of RBC senescence in which multiple discrete density populations of RBCs are individually labeled. If these dyes can be shown to have an acceptably low toxicity, the ability to track multiple populations could also be useful for some clinical applications, e.g., to simultaneously evaluate the survival of RBCs from many different donors in patients with multiple alloantibodies, to identify compatible units for transfusion.

Caveolin-1 binding motif of α-hemolysin: its role in stability and pore formation

We have identified a nine amino sequence in α-hemolysin (α-HL) of Staphylococcus aureus, which binds Caveolin-1. Surface plasmon resonance studies clearly show a concentration dependent interaction of α-HL with the scaffolding domain of Caveolin-1. Mutants of α-HL, devoid of Caveolin-1 recognition motif, exhibit an α-HL like proteinase K digestion profile but the resultant ‘half-like’ domains are highly susceptible to further proteolysis. They also had the same intrinsic fluorescence emission maxima as the native α-HL indicating normal folding. However, these mutants bind 1-anilino-8-naphthalene sulfonic acid probably due to exposure of their hydrophobic core. Moreover, these mutants are non-lytic and do not undergo conformational changes on rabbit RBC membrane surface. Purified Caveolin-1 blocks the hemolysis of RBCs by α-HL. Our studies indicate that the Caveolin-1 binding motif of α-HL provides stability and shields the hydrophobic core of α-HL. The motif also acts as trigger point for initiation of conformational changes.

Fate of alphaGal +/+ pancreatic islet grafts after transplantation into alphaGal knockout mice.

Important phylogenetic differences between pig and human tissues prevent xenotransplantation from becoming a clinically feasible option. Humans lack the galactose-alpha1,3-galactose (alphaGal) epitope on endothelial cell surfaces and therefore have preformed anti-alphaGal antibodies. The role of these antibodies in rejection of non-vascular xenografts remains controversial. This study investigated the role of anti-alphaGal antibodies in rejection of non-vascularized alphaGal+/+ grafts in alphaGal -/- mice. alphaGal +/+ and alphaGal -/- pancreatic islets were transplanted under the renal capsule of streptozotocin-induced diabetic (1) alphaGal -/- mice and (2) alphaGal +/+ mice. alphaGal -/- recepients were immunized with rabbit red blood cell membranes (RRBCs) to produce elevated anti-alphaGal antibody levels. Six of the 18 alphaGal -/- mice rejected the alphaGal +/+ grafts within 68 days whereas indefinite graft survival was achieved in the control groups. Animals with surviving islet grafts were challenged with alphaGal +/+ skin grafts. Although all alphaGal +/+ skin grafts were rejected within 58 days, the islet grafts remained intact. This observation correlated with the level of alphaGal expression (which was very low on islets compared to skin) rather than the actual titre of anti-alphaGal antibody. The results suggest that the level of alphaGal expression plays an important role in graft survival. Therefore, its removal is important in the development of a pig islet donor for future clinical therapy.

Adjuvant effect of Panax notoginseng saponins on the immune responses to ovalbumin in mice

In this study, the haemolytic activities of Panax notoginseng saponins (PNS) and its adjuvant potentials on the cellular and humoral immune responses of ICR mice against ovalbumin (OVA) were evaluated. We determined the haemolytic activity of PNS using 0.5% rabbit red blood cell. PNS showed a slight haemolytic effect, with its haemolytic percents being 11.59 and 3.60% at the concentrations of 500 and 250 μg/ml, respectively. Furthermore, the adjuvant potential of PNS at three dose levels on the cellular and humoral immune responses of ICR mice against ovalbumin were investigated. ICR mice were immunized subcutaneously with OVA 100 μg alone or with OVA 100 μg dissolved in saline containing aluminum hydroxide gel (Alum) (200 μg), Quil A (10 and 50 μg) or PNS (50, 100 or 200 μg) on days 1 and 15. Two weeks later (day 28), concanavalin A (Con A)-, pokeweed (PWM)- and OVA-stimulated splenocyte proliferation and OVA-specific antibodies in serum were measured. PNS significantly enhanced the Con A-, PWM-, and OVA-induced splenocyte proliferation in the OVA-immunized mice at a dose of 100 μg ( P < 0.05 or P < 0.025). OVA-specific IgG, IgG1 and IgG2b antibody levels in serum were significantly enhanced by PNS compared with OVA control group ( P < 0.025). Moreover, enhancing effect of PNS on the OVA-specific IgG2b antibody responses to OVA in mice were more significant than that of Quil A ( P < 0.025). In conclusion, the results suggest that PNS could be safely used as adjuvant with low or non-haemolytic effect.

Solid phase measurements of antibody and lectin binding to xenogenic carbohydrate antigens

Objectives: In future pig-to-man xenotransplantation it is important to master tools that identify potentially xenogenic αgalactose (Galα) antigens in the doner tissue. Design and methods: We have measured the binding potentials of Galα detecting lectins and antibodies, including a naturally occurring subfraction from human serum, to Galα containing neoglycoproteins and mouse laminin that were immobilized on microtiter plates. Results: Galα reactive antibodies with similar monosaccharide specificity have distinct structural preference for sugar ligands. Laminin and neoglycoproteins were treated with α-galactosidase and subsequently incubated with antibodies and lectins. The enzyme treatment was more deleterious on antibody binding than on lectin binding. Conclusion: Antibodies and lectins may bind to different galactose determinants on the glycoproteins. Two anti-Galα1 antibodies that both have been raised against glycans on rabbit red blood cells may recognize Galα-antigens with varying specificities. Binding results obtained after digestion with α-galactosidase indicate that some xenoreactive Galα groups are not directly accessible for removal by the enzyme.

Peritoneal cavity B cells are precursors of splenic IgM natural antibody-producing cells.

Peritoneal cavity B-1 cells are believed to produce IgM natural Abs. We have used alpha1,3-galactosyltransferase-deficient (GalT(-/-)) mice, which, like humans, produce IgM natural Abs against the carbohydrate epitope Galalpha1,3Gal (Gal), to demonstrate that peritoneal cavity B-1b cells with anti-Gal receptors produce anti-Gal IgM Abs only after LPS stimulation. Likewise, peritoneal cavity cells of GalT(-/-) and wild-type mice do not produce IgM Abs of other specificities without LPS stimulation. Development of Ab-secreting capacity is associated with loss of CD11b/CD18 (Mac-1) expression. In contrast, there are large numbers of cells producing anti-Gal and other IgM Abs in fresh splenocyte preparations from GalT(-/-) and (for non-Gal specificities) wild-type mice. These cells are Mac-1(-) but otherwise B-1b-like in their phenotype. We therefore hypothesized a pathway wherein peritoneal cavity B cells migrate into the spleen after activation in vivo and lose Mac-1 expression to become IgM Ab-producing cells. Consistent with this possibility, splenectomy reduced anti-Gal Ab production after immunization of GalT(-/-) mice with Gal-positive rabbit RBC. Furthermore, splenectomized B6 GalT(-/-), Ig micro -chain mutant ( micro (-/-)) (both Gal- and B cell-deficient) mice produced less anti-Gal IgM than nonsplenectomized controls after adoptive transfer of peritoneal cavity cells from B6 GalT(-/-) mice. When sorted GalT(-/-) Mac-1(+) peritoneal cavity B cells were adoptively transferred to B6 GalT(-/-), micro (-/-) mice, IgM Abs including anti-Gal appeared, and IgM-producing and Mac1(-) B cells were present in the spleen 5 wk after transfer. These findings demonstrate that peritoneal cavity Mac-1(+) B-1 cells are precursors of Mac-1(-) splenic IgM Ab-secreting cells.

A New Fungal Lectin Recognizing α(1–6)-linked Fucose in the N-Glycan

In this report, we describe a new lectin from the fungus Rhizopus stolonifer that agglutinates rabbit red blood cells. Agglutinating activity was detected in the extract of mycelium-forming spores cultured on agar plates but not in the mycelium-forming no spores from liquid medium. This lectin, which we designated R. stolonifer lectin (RSL), was isolated by affinity chromatography with porcine stomach mucin-Sepharose. SDS-polyacrylamide gel electrophoresis and mass spectral analysis showed that RSL is approximately 4.5 kDa, whereas gel filtration indicated a mass of 28 kDa. This indicates that the lectin is a hexamer of noncovalently associated RSL monomers. RSL activity was very stable, since it was insensitive to heat treatment at 70 degrees C for 10 min. Analysis of RSL binding specificity by both microtiter plate and precipitation assays showed that N-glycans with l-fucose linked to the reducing terminal GlcNAc residues are the most potent inhibitors of RSL binding, whereas N-glycans without alpha(1-6)-linked fucose residues are approximately 100-fold weaker inhibitors of binding. Oligosaccharides with alpha(1-2, -3, and -4) linkages showed no inhibition of binding in these assays. In a mirror resonance biosensor assay, high affinity binding was observed between RSL and the glycopeptide of bovine gamma-globulin, which has N-glycans with alpha(1-6)-linked fucose residues. However, RSL showed only a weak interaction with the glycopeptide of quail ovomucoid, which lacks fucose residues. Finally, capillary affinity electrophoresis studies indicated that RSL binds strongly to N-glycans with alpha(1-6)-linked fucose residues. Together, these results show that RSL recognizes the core structure of N-glycans with alpha(1-6)-linked l-fucose residues. This specificity could make RSL a valuable tool for glycobiological studies.

Spontaneous haemolytic activity of Atlantic halibut ( Hippoglossus hippoglossus L.) and sea bass ( Dicentrarchus labrax) serum

The spontaneous haemolytic (SH) activity of sera was compared in groups of cultured halibut and sea bass. The optimum assay temperature was determined for each species and different red blood cell donors were tested. The effects of heat inactivation, storage temperature and of different agents like EDTA, EGTA, yeast cell components and bacterial LPS were compared. Halibut sera gave optimum lysis with sheep red blood cells (RBC) at 16 °C whereas sea bass sera showed optimum lysis with rabbit RBC at 37 °C. The haemolytic activity of halibut sera was inactivated at 45 °C while sea bass sera were inactivated at 56 °C. The haemolytic activity of halibut sera was significantly reduced during short-term storage at −80 °C, whereas the sea bass sera maintained fairly good activity after 1-year storage at −80 °C. EGTA and EDTA inhibited the spontaneous haemolytic activity of sera from both the species. Zymosan and MacroGard from yeast cells also inhibited the haemolytic activity of the sera of both species, whereas LPS had a very slight effect. Considerable variation in haemolytic activity was observed within both the halibut and sea bass groups studied.

Fetal porcine thymus engraftment, survival and CD4 reconstitution in αGal‐KO mice is impaired in the presence of high levels of antibodies against αGal

Xenospecific T-cell tolerance can be induced among murine and human T-cells by porcine thymic grafting. However, anti-alpha 1,3-galactosyltranserase (alphaGal) (Galalpha1-3Galbeta1-4GlcNAc-R) natural antibodies (NAbs) pose a major barrier to porcine xenografts in humans. We used alphaGal knockout (KO) and muchain KO mice to explore the effect of natural anti-alphaGal and other xenoantibodies on porcine thymic engraftment and to examine the potential of thymic tissue to tolerize anti-alphaGal antibody-producing cells. Thymectomized [adult thymectomy (ATX)] non-immunized and rabbit red blood cell (RRBC) pre-transplant immunized alphaGal-KO (knockout), wild-type (WT) and mu chain KO B6 mice were treated with 3Gy total body irradiation (TBI), and T and natural killer (NK) cell depleting monoclonal antibodies (mAbs). These conditioned mice were grafted with fetal porcine thymus and liver (FP THY/LIV) tissue under the kidney capsule. Flow cytometric analysis was performed to follow CD4 reconstitution as a measure of FP THY engraftment and function. Only mice with >10% CD4+ peripheral blood lymphocytes (PBL) were considered successfully engrafted. Enzyme-linked immunosorbent assay (ELISA) was used to assess the kinetics of immunoglobulin M (IgM) and IgG anti-alphaGal antibodies. Anti-pig antibodies were monitored by flow cytometry (FCM). FP THY engrafted successfully in most of the immunoglobulin deficient mice (11 out of 12, 92%) and the outcome was similar in WT B6 controls (8 out of 12, 67%). Non-immunized alphaGal-KO mice grafted with FP THY had a similar success rate (7 out of 11) to that observed in non-immunized alphaGal-WT controls (2 out of 4). In contrast, alphaGal-KO mice immunized pre-transplant with RRBC, then grafted with FP THY/LIV, showed a significant reduction in the success of thymic grafting (2 out of 9, 22%) compared with pre-transplant immunized WT controls (4 out of 7; 57%) and non-immunized alphaGal-KO mice (7 out of 11, 64%). Anti-Gal and anti-pig antibody levels were not markedly augmented by porcine thymus grafts in mice with successful thymus grafts. FP THY engraftment is impaired in the presence of high levels of anti-alphaGal xenoantibodies. However, low levels of anti-alphaGal antibodies and other mouse anti-pig NAbs appear not to play a major role in the rejection of FP THY. Although grafting FP THY expressing the alphaGal epitope did not tolerize B cells producing anti-alphaGal antibodies in a T-cell independent manner, it prevented T-cell dependent sensitization by inducing T-cell tolerance to porcine antigens.