Hemolysis Of Sheep Red Blood Cells Research Articles

INNATE IMMUNE FUNCTION IN LAKE ERIE WATERSNAKES (NERODIA SIPEDON INSULARUM) WITH OPHIDIOMYCOSIS.

Ophidiomycosis, caused by the fungus Ophidiomyces ophidiicola, poses a threat to the health of wild and managed snakes worldwide. Variation in snake innate immunity, the primary defense against infection in reptiles, may explain the observed variation in ophidiomycosis clinical disease severity among snakes. In this study, two components of the innate immune response were examined in snake plasma. We investigated whether complement activity, as measured by sheep red blood cell hemolysis, and chitotriosidase activity were associated with ophidiomycosis disease severity and time in captivity in Lake Erie watersnakes (Nerodia sipedon insularum). There was no difference in complement-mediated hemolysis or chitotriosidase activities between snakes with varying levels of ophidiomycosis clinical severity sampled in the field. However, among snakes with skin lesions kept in captivity, chitotriosidase activity was significantly higher in snakes with mild disease, compared with snakes with severe disease, and hemolysis activity increased with time in captivity. Overall, Lake Erie watersnakes had higher complement activity, but lower chitotriosidase activity, compared with other reptile species. To our knowledge, this study is the first to describe chitotriosidase activity in a snake species. These results provide mixed evidence of associations between innate immune function and ophidiomycosis severity, and more work is needed to investigate differences among snake species.

Comparison of complement system activity amongst wild and domestic animals

Multiple mechanisms have evolved for the defensive recognition of foreign components, such as microorganisms. The majority of immunological studies with vertebrates have been focused on endothermic species, and relatively little attention has been directed toward ectothermic vertebrates. We employed a colorimetric assay designed to compare plasma hemolytic activities based on the serum complement system (CS) activities amongst some representative reptiles, wild and domestic birds, and mammals. Results obtained from the hemolytic assays conducted with plasma derived from all of the animal species used showed that broad-snouted caiman had the highest activity, and no differences were observed in the hemolytic activities of plasma from birds or the other reptile species. In contrast, the CS activity obtained with mammalian plasma was markedly lower than that from the other taxa. This assay has many advantages, such as the requirement of small sample volume, reproducible results, and low cost. In addition, unsensitized sheep red blood cell hemolysis can be successfully used for the evaluation of innate immune system activities in non-mammalian species; however, for mammals, it should be combined with other immunological determinates to evaluate integral innate immunocompetence.

Characterization of serum complement immune activity in the prairie rattlesnake (Crotalus viridis)

BackgroundReptile populations face a growing number of threats including global climate change and emerging pathogens. Unfortunately, research investigating the reptile immune system lags behind other taxa groups, hindering our ability to predict or mitigate species’ response to threats. Baseline studies are critical for our understanding of reptile immune response and overall health of wild populations.ResultsTreatment of unsensitized sheep red blood cells (SRBCs) with plasma collected from captive prairie rattlesnakes (Crotalus viridis) resulted in volume-dependent hemolysis, with a CH50 of 0.23 ± 0.01 mL. Kinetic analyses at different temperatures revealed that the hemolysis was relatively rapid, with 50% of hemolytic activity occurring within 15 min (25 °C), 18 min (35 °C), or 23 min (15 °C), and near maximum hemolysis within 60–90 min at all three temperatures. A comparative thermal profile revealed that complement activity was low at 5 °C, but increased sharply at 10 and 15 °C, and was maximal at 20–30 °C. A steep decline in activity was noted at temperatures > 30 °C. Mild heat treatment of the snake plasma (56 °C, 30 min) or treatment with proteases completely abolished the hemolytic activity. In addition, inclusion of 5 mM EDTA inhibited 90% of the hemolysis, but the activity could be reconstituted with the addition of 15 mM Ca2+ or Mg2+, but not Ba2+, Cu2+, or Fe2+. Furthermore, the hemolysis was unaffected in the presence of 20 mM methylamine, indicating that the alternative mechanism of complement activation is responsible for the observed activities.ConclusionsRattlesnakes show a relatively robust innate immune response as measured by hemolysis of SRBCs. However, hemolytic activity is reduced at high temperatures, indicating that rising global temperatures may have immune consequences for snake species, making them more vulnerable to known and emerging pathogens.

In vivo and in vitro immunomodulatory potential of swertiamarin isolated from Enicostema axillare (Lam.) A. Raynal that acts as an anti-inflammatory agent.

Swertiamarin is a secoiridoid glycoside found in Enicostema axillare (Lam) A. Raynal, a medicinal plant used as a depurative in the Indian system of traditional medicine. The present study evaluated the immunomodulatory activity of isolated swertiamarin. In vivo immunomodulatory activity of swertiamarin (2, 5, and 10 mg/kg b.w.) was evaluated in a model of sheep red blood cells (SRBC) by assessing its effect on organ weight, hemagglutinating antibody titer (HA), plaque-forming cells (PFC), quantitative hemolysis of SRBC, and delayed type hypersensitivity (DTH). In vitro immunomodulatory potential was studied on isolated splenocytes, neutrophils, and peritoneal macrophages. In silico immunomodulatory effects were evaluated by docking of swertiamarin on proinflammatory cytokines to confirm its potential. In in vivo studies, the animals treated with swertiamarin showed a significant (P ≤ 0.05) increase in antibody titer, plaque-forming cells, and also in weight of the thymus and spleen. A decreased response to DTH reaction was recorded with the treatment of swertiamarin. In in vitro studies, treatment with swertiamarin modulated the messenger RNA (mRNA) and protein expression of IFN-γ, IL-10, and IL-4 significantly (P ≤ 0.05) and also favored Th2-mediated response on concanavalin A (Con A)-induced splenocytes. The compound inhibited the release of free radicals significantly (P ≤ 0.05) in phytohemagglutinin (PHA)-induced neutrophils and also ameliorated the mRNA and protein expression of proinflammatory cytokines (TNF-α, IL-1β, and IL-6) in lipopolysaccharide (LPS)-induced macrophages. In in silico, the best docked pose of swertiamarin with the target proteins (TNF-α, IL-1β, and IL-6) was confirmed that swertiamarin acted as an anti-inflammatory mediator.

Characterization of Serum Complement Activity in Three Species of Crocodilians from Southeast Mexico

Aims: To characterize the serum complement innate immune system in three species of crocodilians native to southeast Mexico. Methodology: Plasma collected from three wild crocodilian species native to southeast Mexico were exposed to sheep red blood cells (SRBCs) to measure hemolysis, which is used as an indication of serum complement immune activity. Results: Incubation of different volumes of plasma from Crocodylus acutus, Crocodylus moreletii, and Caiman crocodilus resulted in a volume-dependent increase in SRBC hemolysis. However, while maximum hemolysis for C. acutus and C. moreletii were both approximately five-fold higher than that of Ca. crocodilus. A kinetic study revealed that the hemolysis was rapid, with near-maximum activity recorded at 30 min for C. acutus and C. moreletii. However, Ca. crocodilus activity exhibited a significant increase (P<.5) only between one and two hours. A thermal analysis showed that the SRBC hemolysis was maximal at temperatures to which these species thermoregulate. The thermal profiles were similar for all three species, although the activity was lower for Ca. crocodilus Original Research Article International Journal of Biochemistry Research & Review, 4(4): 295-305, 2014 296 (P<.01). The SRBC hemolysis was strongly inhibited by mild heat treatment (56°C,30 min) and also by EDTA, indicating that the hemolytic activity was probably due to the presence of crocodilian serum complement activity. The EDTA-inhibited activity was restored by the addition, of a 20mM excess of Caor Mg, but not FeorCu, thus exhibiting the specific need for CaorMg. Conclusions: The serum complement activities of C. acutus and C. moreletii are much higher (P<.01) than Ca. crocodilus, which may be related to the maximum sizes and increased aggressive natures of C. acutus and C. moreletii.

Serum Complement Activity in Two Species of Divergent Central African Crocodiles

Serum complement in the serum of two divergent Central African crocodiles, the African dwarf crocodile (Osteolaemus tetraspis) and the slender-snouted crocodile (Mecistops cataphractus) was assessed using a sheep red blood cell (SRBC) hemolytic assay. The hemolysis for both crocodilian species was serum volume-, time-, and temperature-dependent. Although the serum volume-dependent activities were similar for both species (CH50 = 81 μL for Osteolaemus tetraspis and 96 μL for Mecistops cataphractus), the kinetic curves show a greater amount cooperativity, and thus more rapid SRBC lysis, for Osteolaemus tetraspis. In addition, the hemolytic activities were very similar at 10 – 35°C, but the serum from Osteolaemus tetraspis was more active than that of Mecistops cataphractus at the temperature extremes tested (5°C and 40°C). The activities for both species were almost completely inhibited by 1 mM EDTA, indicating the dependency on divalent metal ions. However, the EDTA-inhibited hemolysis could be restored by the addition of excess Mg2+ and Ca2+, but not Ba2+, Cu2+, or Fe2+, which exhibited the specificity for Mg2+or Ca2+. These data indicate that these sympatric, but evolutionarily and ecologically divergent crocodile species have similar SRBC hemolysis activities with similar mechanisms, thus reinforcing the idea that serum complement is an ancient, innate immunity host defense system.

Changes in the Gut Microbiome of the Sea Lamprey during Metamorphosis

Vertebrate metamorphosis is often marked by dramatic morphological and physiological changes of the alimentary tract, along with major shifts in diet following development from larva to adult. Little is known about how these developmental changes impact the gut microbiome of the host organism. The metamorphosis of the sea lamprey (Petromyzon marinus) from a sedentary filter-feeding larva to a free-swimming sanguivorous parasite is characterized by major physiological and morphological changes to all organ systems. The transformation of the alimentary canal includes closure of the larval esophagus and the physical isolation of the pharynx from the remainder of the gut, which results in a nonfeeding period that can last up to 8 months. To determine how the gut microbiome is affected by metamorphosis, the microbial communities of feeding and nonfeeding larval and parasitic sea lamprey were surveyed using both culture-dependent and -independent methods. Our results show that the gut of the filter-feeding larva contains a greater diversity of bacteria than that of the blood-feeding parasite, with the parasite gut being dominated by Aeromonas and, to a lesser extent, Citrobacter and Shewanella. Phylogenetic analysis of the culturable Aeromonas from both the larval and parasitic gut revealed that at least five distinct species were represented. Phenotypic characterization of these isolates revealed that over half were capable of sheep red blood cell hemolysis, but all were capable of trout red blood cell hemolysis. This suggests that the enrichment of Aeromonas that accompanies metamorphosis is likely related to the sanguivorous lifestyle of the parasitic sea lamprey.

Serum complement activity in the three-toed amphiuma (Amphiuma tridactylum)

Some animals routinely endure serious injuries from predators or during intraspecific territorial conflicts. Such is the case for Amphiuma tridactylum, an aquatic salamander that lives in an environment rich in potentially infectious microbes, apparently with rare or no pathogenic infection. Some vertebrates possess innate immune mechanisms, but whether this is the case for Amphiuma is unknown. To assess this potential, plasma from 19 A. tridactylum was pooled and used for characterisation of serum complement activity. The ability of A. tridactylum plasma to hemolyse unsensitised sheep red blood cells (SRBCs) was titer-dependent, with low activity observed even at high plasma titers. The kinetic characterisation of SRBC hemolysis revealed that significant activity could be measured within 10min of incubation, and maximal activity occurred within 60min. The SRBC hemolysis by A. tridactylum plasma was also temperature-dependent, with maximal activity at 30°C. In addition, this activity was sensitive to mild heat treatment, with 96% of activity inhibited by incubation at 56°C for 30min. The SRBC hemolysis could also be inactivated by pretreatment of the plasma with proteases, indicating that this activity was protein dependent. The activity required divalent metals ions, with activity inhibited by EDTA, citrate, or phosphate. However, the chelator-inhibited activity could be restored by the addition of excess Ca(2+) or Mg(2+), but not Cu(2+) or Ba(2+), indicating specificity of the divalent metal ion requirement. The sensitivity to heat, proteases, and divalent metal ion chelators strongly suggests that A. tridactylum plasma-mediated hemolysis of SRBCs is mediated by the serum complement system of proteins.

Characterization of serum complement activity of saltwater ( Crocodylus porosus) and freshwater ( Crocodylus johnstoni) crocodiles

We employed a spectroscopic assay, based on the hemolysis of sheep red blood cells (SRBCs), to assess the innate immune function of saltwater and freshwater crocodiles in vitro. Incubation of serum from freshwater and saltwater crocodiles with SRBCs resulted in concentration-dependent increases in SRBC hemolysis. The hemolytic activity occurred rapidly, with detectable activity within 2 min and maximum activity at 20 min. These activities, in both crocodilian species, were heat sensitive, unaffected by 20 mM methylamine, and completely inhibited by low concentrations of EDTA, suggesting that the alternative serum complement cascade is responsible for the observed effects. The hemolytic activities of the sera were inhibited by other chelators of divalent metal ions, such as phosphate and citrate. The inhibition of SRBC hemolysis by EDTA could be completely restored by the addition of 10 mM Ca 2+ or Mg 2+, but not Ba 2+, Cu 2+ or Fe 2+, indicating specificity for these metal ions. The serum complement activities of both crocodilians were temperature-dependent, with peak activities occurring at 25–30 °C and reduced activities below 25 °C and above 35 °C.

A novel artemisinin derivative, 3-(12-beta-artemisininoxy) phenoxyl succinic acid (SM735), mediates immunosuppressive effects in vitro and in vivo1

To study the immunosuppressive activity of SM735 {[3-(12-beta-artemisininoxy)] phenoxyl succinic acid}, a synthetic artemisinin derivative with nonsteroidal anti-inflammatory drug structure, with the aim of finding potential immunosuppressive agents. Concanavalin A (ConA), lipopolysaccharide (LPS), and mixed lymphocyte reaction (MLR), were used to induce the proliferation of splenocytes, and [3H]-thymidine incorporation was used to evaluate the proliferation of splenocytes. Cytokine production was promoted with ConA, LPS, or PMA plus ionomycin, and was detected with the enzyme-linked immunosorbent assay. Dinitrofluorobenzene (DNFB) and sheep red blood cells (SRBC) were used to induce delayed-type hypersensitivity and quantitative hemolysis of SRBC (QHS) mouse models, as criteria for the evaluation of in vivo immune activity. SM735 strongly inhibited the proliferation of splenocytes induced by ConA, LPS, or MLR, with IC(50) values of 0.33 micromol/L, 0.27 micromol/L, and 0.51 micromol/L, respectively. When compared with a CC(50) value of 53.1 micromol/L, SM735 had a favorable safety range. SM735 dose-dependently inhibited proinflammatory cytokine production [including interleukins (IL)-12, interferon (IFN)-gamma and IL-6] induced by LPS or PMA plus ionomycin. Upon ConA stimulation, SM735 suppressed IFN-gamma in a dose-dependent manner, but did not affect IL-2 secretion. SM735 also strongly suppressed both T-cell-mediated delayed-type hypersensitivity (DTH) and B-cell-mediated QHS reactions. SM735 had strong immunosuppressive activity in vitro and in vivo, suggesting a potential role for SM735 as an immunosuppressive agent, and established the groundwork for further research on SM735.

Identification of alternative pathway serum complement activity in the blood of the American alligator ( Alligator mississippiensis)

Incubation of different dilutions of alligator serum with sheep red blood cells (SRBCs) that had not been sensitized with antibodies resulted in concentration-dependent hemolytic activity. This hemolytic activity was not affected by the presence of ammonium hydroxide and methylamine, known inactivators of the classical complement cascade. However, the hemolytic activities were inhibited by EDTA and salicylaldoxime, indicating that the alternate pathway is primarily responsible for these activities. Immunofixation of electrophoretically-resolved alligator serum proteins with antihuman C3 polyclonal antibodies resulted in detection of a protein antigenically similar to human C3 in alligator serum. SDS-PAGE, followed by Western blot analysis, revealed the presence of two alligator serum proteins with nearly identical molecular weights as human C3α and C3β. SRBC hemolysis and antibacterial activity by alligator serum was significantly reduced in the presence of antihuman C3 antibodies. The hemolytic effect of alligator serum was shown to occur rapidly, with significant activity within 5 min and maximal activity occurring at 15 min. SRBC hemolysis was also temperature-dependent, with reduced activity below 15 °C and above 30 °C. These data suggest that the antibiotic properties of alligator serum are partially due to the presence of a complement-facilitated humoral immune response analogous to that described in mammalian systems.

Functional human monoclonal antibodies of all isotypes constructed from phage display library-derived single-chain Fv antibody fragments

We have constructed a series of eukaryotic expression vectors that permit the rapid conversion of single chain (sc) Fv antibody fragments, derived from semi-synthetic phage display libraries, into intact fully human monoclonal antibodies (mAb) of each isotype. As a model, a scFv fragment specific for sheep red blood cells (SRBC) was isolated from a semi-synthetic phage antibody (Ab) display library, and used to produce human mAbs of IgM, IgG1–IgG4, IgA1, IgA2m(1) and IgE isotype in vitro in stably transfected cells. N-terminal protein sequence analysis of purified immunoglobulin heavy (H) and light (L) chains revealed precise proteolytic removal of the leader peptide. Biochemical analysis of purified recombinant human mAbs demonstrated that properly glycosylated molecules of the correct molecular size were produced. The IgG and IgA mAbs retained SRBC-binding activity, interacted with different Fc receptor-transfectants, and induced complement-mediated hemolysis and Ab-dependent phagocytosis of SRBC by neutrophils in a pattern consistent with the immunoglobulin (Ig) H chain isotype. We conclude that in vitro produced recombinant human mAbs constructed from phage display library-derived scFv fragments mirror their natural counterparts and may represent a source of mAbs for use in human therapy.

Effect of synthetic thymic hormones on the cocaine-induced inhibition of the primary immune response in mice

The effects of thymosyn α1 (Tα1) and thymopentin (TP5) on the cocaine-induced impairment of the primary antibody response to sheep red blood cells (SRBC) was studied. The administration of cocaine from day −4 to the day of immunization (day 0) induced a significant impairment of the response to the T-dependent antigen SRBC, as evaluated on day 5 post-immunization by the Splenocyte-Induced SRBC Hemolysis (SIH) assay. The analysis of the responses to immunogen elicited from each single mouse indicated that, under the experimental conditions used, cocaine acted by exerting more an “all or nothing” effect rather than by modulating the strength of the immune response. Both Tα1 and TP5, injected into mice during cocaine administration and for 4 days after, induced a significant recovery of the response to SRBC. Our experiments did not show any great differences in the overall efficacy of the two drugs, although they showed quite a different dose-response effect. The results of the present investigation demonstrated the capability of TP5 and Tα1 to reverse the cocaine-induced impairment of the response to SRBC and suggested that the effect of the two peptides may be related to their immunomodulating activities on T-cell functions.

Equine alternative pathway activation by unsensitized rabbit red blood cells

The equine alternative complement pathway has been partially characterized and compared to the equine classical activation pathway. A dose-dependent lysis of RbRBC was observed with peak lytic values noted within 10 minutes at 37°C when rabbit red blood cells (RbRBC) were used as an alternative pathway activator. Sheep red blood cells (SRBC) sensitized with rabbit hemolysin or partially purified equine IgM antibodies were equally sensitive to lysis. Dilution of the commercial hemolysin by 1 5 reduced lysis from 90% to 38% in the presence of constant cell numbers. Hemolysis of SRBC peaked at 10 minutes and the majority of lysis occurred within 10 minutes. Dilution of equine sera by as little as 1 5 decreased hemolytic activity for SRBC to 21.5% from greater than 90% with undiluted sera. The alternative pathway protein, equine factor B, was tested using RbRBC and monitored by its differential susceptibility to heat treatment at 50°C. This treatment led to almost complete inactivation after a 15-minute incubation. An apparent heat-dependent decay of certain classical pathway components was also observed after 50°C treatment. This sensitivity was indicated by a reduction in the lytic activity for sensitized SRBC. Treatment for 15 minutes at 56°C with either RbRBC or SRBC was sufficient to abolish hemolytic activity in all equine sera tested. Chelation of cations with 0.04 M EDTA blocked expression of alternative and classical pathway activation; however, chelation of Ca ++ ions with 10 mM EGTA containing 1 mM Mg ++ ions permitted lysis of the RbRBC but not the SRBC. A dose-related Mg ++-ion dependence for RbRBC hemolytic activity was observed as the concentration of Mg ++ was increased to 1.0 mM. In addition, our results obtained with pre-colostral foal serum strongly suggest that natural antibody to RbRBC was of little importance in the lysis observed with these cells. These results also show that the equine alternative pathway activation may require Ca ++ ions. If Ca ++ ions are required, the equine alternative pathway is quite different from any other mammalian complement system so far described. Our results suggest that the alternative pathway of activation is of major importance in the equine complement system. Confirmation of this hypothesis requires both purification of the components involved as well as further characterization.

Spectrophotometric determination of lymphocyte mediated sheep red blood cell hemolysis in vitro

An assay has been developed in which the degree of in vitro spleen cell-mediated sheep red blood cell (SRBC) hemolysis is quantitated spectrophotometrically (QHS). Immune mouse spleen cells, SRBC and guinea pig complement were incubated at 37°C for 1 h in buffer, centrifuged, and the supernatant solutions read at 413 nm. The greatest degree of cell-mediated SRBC hemolysis was observed in day +5 harvested spleens following a day 0, 5% SRBC i.p. injection. In the assay system, it was found that a stimulator SRBC dose of 0.2% resulted in maximal stimulation with little non-specific hemolysis. This assay is presented as an alternative to the well established plaque forming and rosette assays.