Equine Influenza Infection

Grabriele A. Landolt , ... D. Paul Lunn , in Equine Infectious Diseases (Second Edition), 2014

Single Radial Hemolysis

For SRH tests, sheep erythrocytes, which have previously been incubated with influenza virus, are mixed with guinea pig complement and incorporated in agarose gels ( Fig. 13-6). Heat-inactivated serum samples are then added to wells cut into the gel, and the antibody titer is determined based on the zone of hemolysis induced by diffusion of the antibody-positive sample from the well. 138,204,205 An increase of 50% or 25 mm2 is considered evidence of recent infection. 202 Although more labor intensive than HI assays, SRH tests have been shown to be more reproducible than HI tests. 202 Since it was found that the level of antibody measured by SRH after vaccination correlates well with the level of protection, SRH may also be used to predict the level of antibody-mediated immunity and determine the need for revaccination. 72,73,165

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INDICATIONS OF ACTIVE SUPPRESSION IN MOUSE CARRIERS OF LYMPHOCYTIC CHORIOMENINGITIS VIRUS

(Communicated by G.L. Ada)Rolf Zinkernagel Peter Doherty , in Immunological Tolerance, 1974

ALLOGENEIC ABROGATION OF T CELL TOLERANCE

Tolerance of rats to sheep erythrocytes can be transiently broken by inoculation with allogeneic normal lymphocytes ( 14, 15). We have used this phenomenon to investigate the T cell status of mouse carriers of viscerotropic (WE3) LCM virus. Mice from a CBA/H (H-2k) carrier colony were injected intravenously (i.v.) at various intervals with 5.0 × 107 spleen cells from syngeneic or allogeneic normal mice. These carrier recipients were all killed on the same day, and spleen preparations (16) were assayed together for cytotoxic T cell activity. Two separate experiments are shown in Fig. 1. In both cases injection of allogeneic spleen cells resulted in generation of high levels of cell-mediated lysis. Maximal release of 51Cr was observed at 11 days after inoculation, but later intervals have not yet been examined.

Fig. 1. Carrier CBA/H mice (6–10 weeks) were injected i.v. with 5.0 × 107 CBA/H or BALB/c × C57BL/F1 spleen cells and were all killed and assayed on the same day. Carrier spleen preparations were overlaid (30:1) op 51Cr-labelled LCM-infected (o) or normal (0) L cells. %51Cr release was measured 16 hours later and expressed as mean ± SEM.

The sensitized T cells may be considered to be of carrier, not donor, origin for two reasons. Firstly, lysis of normal targets, which presumably reflects activity of transferred T cells specific for host alloantigens, had returned to background levels by day 11 (Expt. 2, Fig. 1). Apparently, these donor lymphocytes had been eliminated before maximal cytotoxicity for LCM-infected monolayers. Secondly, lysis of LCM-infected L cells occurs only when targets and T cells share at least one set of H-2 antigenic specificities (8). Lymphocytes from LCM immune BALB/c × C57BL/F1 (H-2b/d) mice do not induce specific release of 51Cr from LCM-infected L cells (H-2k). Furthermore, when spleen preparations from syngeneic or allogeneic LCM-immune mice were inoculated into CBA/H carriers, the syngeneic T cells went on to generate high levels of cytotoxic activity by day 9 (Table II). However, in carriers injected with an allogeneic immune population, specific effector activity was observed only on day 6, presumably due to the allogeneic abrogation effect.

TABLE II. CYTOTOXIC ACTIVITY OF CBA/H CARRIER SPLEEN CELLS FOLLOWING ADOPTIVE IMMUNIZATION WITH 5.0 × 107 SYNGENEIC OR ALLOGENEIC IMMUNE SPLEEN CELLS a

Cells Transferred Days after transfer %51Cr release from L cells
Infected Normal
CBA/H 3 33.5 ± 1.8 21.1 ± 1.0
6 22.7 ± 0.9 18.5 ± 1.6
9 67.1 ± 2.8 23.7 ± 1.5
12 40.5 ± 1.7 20.8 ± 2.2
BALB/c × C57BL/F1 3 27.1 ± 1.4 20.9 ± 1.1
6 41.2 ± 1.1 22.1 ± 0.8
9 24.7 ± 1.1 19.8 ± 0.8
a
This experiment was done at the same time as Expt. 1 in Fig. 1. Donors were dosed 7 days previously with 300 LD50 of WE3 LCM virus.

At present, our understanding of the conditions governing allogeneic abrogation of T cell tolerance in LCM is very incomplete. Two further attempts to induce cytotoxic activity in carrier mice at 9 and 11 days after transfer of allogeneic normal cells failed completely. Also, we have no explanation as to why, in the first experiment (Fig. 1, Table II), allogeneic normal spleen cells were much more effective than an allogeneic immune population. The phenomenon is, however, almost certainly valid. Generation of cytotoxic T cells is associated with considerable reduction of virus titres in the spleens of the carrier recipients (Table III). Elimination of LCM virus from tissues of acutely-infected mice has been shown, in cell transfer experiments, to be largely a function of sensitized thymus-derived lymphocytes (17).

TABLE III. VIRUS TITRES IN SPLEENS OF CBA/H CARRIER MICE INJECTED WITH ALLOGENEIC NORMAL SPLEEN CELLS AT VARIOUS INTERVALS

Days after inoculation Virus Titre a
5 3.8
7 4.0
9 1.6
11 0.4
No cells 4.2

This is Expt. 2 from Fig. 1.

a
log10 Ic LD50 per 106 nucleated spleen cells, assayed in WEHI mice which are uniformly susceptible to LCM.

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Introduction and History of Complement Research

GORDON D. ROSS , in Immunobiology of the Complement System, 1986

IID1 The Hemolytic Assay of Complement

A very important early development was the use of the sheep erythrocyte (E) hemolytic essay. Lysis of sheep E requires anti-sheep E antibody, all components of the classical pathway, and the terminal complement components. Hemolysis of sheep E resulted in a change in turbidity that could be easily visualized and a release of hemoglobin that could be precisely quantitated spectrophotometrically. Furthermore, stable intermediate complexes could be generated by sequential addition of the individual components, allowing the order of the reaction of the components and the mechanism of their activity to be defined. Any of the individual components could be measured specifically by use of a pool of components that was deficient in the component of interest. For example, C2 could be specifically assayed with an intermediate complex consisting of antibody-sensitized sheep E (abbreviated EA, for erythrocyte–antibody complex) coated with purified C1 and C4 (abbreviated EAC14), and a pool of the components containing C3–C9. In the presence of C2, the intermediate complex EAC142 was generated, so that the subsequent addition of C3 to C9 resulted in hemolysis of the sheep E.

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Antigen Delivery Systems I: Nonliving Microparticles, Liposomes, and Immune Stimulating Complexes (ISCOMs)

Suzanne M. Michalek , ... Guus F. Rimmelzwaan , in Mucosal Immunology (Third Edition), 2005

Parenteral immunization

It has been shown that parenteral immunization with ISCOMs containing sheep erythrocyte antigen results in an immune response in the female reproductive tract of mice. Immunization in the pelvic presacral space (which is an intramuscular site) stimulated significant anti-erythrocyte IgA titers in vaginal fluid that were higher than after intraperitoneal, subcutaneous, intravaginal, or intraperitoneal-intravaginal immunizations with the same vaccine. Specific IgG titers in vaginal washings were found to be less dependent on the route of immunization ( Thapar et al., 1991), although the intravaginal route of administration resulted in lower IgG titers.

In a more recent study, it was shown that a genital mucosal Th1 response was induced against Chlamydia trachomatis upon intramuscular immunization with ISCOMs containing the major outer membrane protein of this microorganism. Upon transfer of T cells obtained from ISCOM-immunized mice, recipients were protected against an intravaginal challenge (Igietseme and Murdin, 2000). In addition, intradermally administered ISCOMs were efficacious in eliciting both systemic and mucosal IgG and IgA antibodies in sheep, pigs, and mice (Chin et al., 1996). Also, intramuscular immunization of mice with an influenza–ISCOM preparation resulted in the development of IgA and IgG antibodies in nasal washes, which conferred partial protection against a homologous challenge infection (Ben Ahmeida et al., 1993). Thus, parenteral immunization with an ISCOM-based vaccine can induce mucosal IgA and IgG antibodies.

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ANTIGEN-SPECIFIC SUPPRESSOR FACTOR IN HUMAN LEUKOCYTE DIALYSATES: A PRODUCT OF TS CELLS WHICH BINDS TO ANTI-V REGION AND ANTI-Ia REGION ANTIBODIES

W. Borkowsky , ... H.S. Lawrence , in Immunobiology of Transfer Factor, 1983

Isolation of Cells to Prepare DLE

The nonadherent mononuclear cells were allowed to rosette with washed sheep erythrocytes for 1 hour at 4°C after a 200 g centrifugation. The pellet was gently resuspended and layered over ficoll-hypaque. The cells were spun at 400 g at 4°C for 30 minutes. The cells at the bottom of the gradient were collected. Red blood cells were subjected to hypotonic lysis with water for 20 seconds. The residual mononuclear cells were washed and collected for further assay. These "T" cells were found to be Leu 1 antigen (Becton Dickenson) positive for 96% of the cells examined after treatment with the monoclonal anti PAN T cell antibody and a rhodamine tagged goat anti-murine immunoglobulin antibody.

T cells were separated into helper and suppressor populations utilizing panning techniques and monoclonal anti-T helper cell antibodies(Leu3a & Leu2a, BectonDickenson). Briefly, bacteriologic grade Petri dishes were coated with an affinity purified goat anti-mouse IgG (ZyMed) by adding 50 μg of antibody in 2ml of H2O and coating the dish at room temperature for 2 hours. The dishes were washed with PBS and heat inactivated fetal calf serum to saturate residual protein binding sites on the plastic. Fifty million T cells were then incubated with either 1 ml of a 1/10 dilution of anti Leu2a or 1 ml of a 1/10 dilution of anti Leu3a at 4°C for 30 minutes. The cells were washed with PBS and added to the anti-mouse IgG coated Petri dishes in a volume of PBS calculated to just coat the dish. After a 45 minute incubation at 20°C, the nonadherent population was added to a second Petri dish coated with the affinity purified anti-murine IgG. The nonadherent cells were collected after another 45 minute incubation, washed and harvested. The yield of cells was approximately 10–25% of the original number of cells. The phenotype of the nonadherent T cells coated with anti-Leu2a antibody prior to panning was less than 3% Leu2a positive and greater than 85% Leu3a positive. The phenotype of the nonadherent T cells coated with anti-Leu3a antibody prior to panning was less than 5% Leu3a positive and greater than 90% Leu2a positive.

The enriched T-helper and T-suppressor cells were used to produce DLE following the method of Lawrence modified by negative pressure dialysis. DLE was prepared at a cell concentration of 5×107 lymphocyte equivalents per ml.

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Tumor Immunology and Immunotherapy – Cellular Methods Part B

Annika M. Bruger , ... Pierre van der Bruggen , in Methods in Enzymology, 2020

2.2.1 Preparation of sheep erythrocytes

a.

Use reagents equilibrated to room temperature. We use sheep erythrocytes in 50% Alsever's suspension provided by Labor Dr. Merck (E-400). 2-aminoethylisothiouronium bromide (AET) is provided by Sigma (A54601).

b.

Check the hematocrit (% erythrocytes) of the sheep erythrocytes first, using the provider's website: http://www.labormerk.com/manufacturing/blood_products/

c.

Prepare the AET solution by dissolving X mg of AET in Y mL of milli-Q water (see Table 1). Add 1   M NaOH to bring the solution to pH 9. Note that the correct pH of the solution is of particular importance.

Table 1. Reference table for the resuspension of sheep erythrocytes in AET and RPMI 1640 according to the hematocrit content.

% Hematocrit X: A.E.T. (mg) Y: H20 volume for AET (mL) Z: RPMI 1640 (mL)
10 625 13.5 15.6
11 688 14.7 17.2
12 750 16.2 18.7
13 813 17.3 20.3
14 875 18.9 21.9
15 938 20.0 23.4
16 1000 21.6 25.0
17 1063 22.7 26.6
18 1125 24.3 28.0
19 1188 25.7 29.7
20 1250 27.0 31.2
d.

Filter the AET suspension with a 0.22   μm Steriflip (Millipore, catalog ♯ SCGP00525) into a 50   mL filter Falcon tube.

e.

Resuspend sheep erythrocytes by shaking and/or pipetting, and collect 12.5   mL of the erythrocyte suspension. Wash the suspension by adding 37.5   mL of PBS and centrifuge at 910g for 8   min.

f.

Aspirate supernatant. Leave some PBS to avoid aspirating any erythrocytes. Add the AET solution and resuspend the erythrocytes. Incubate for 15   min at 37   °C.

g.

Pellet the erythrocyte suspension by centrifugation at 910g for 8   min. Remove AET supernatant. Resuspend in 50   mL PBS. The erythrocyte pellet should be of dense consistency and require repeated gentle agitation for complete resuspension.

h.

Wash with 50   mL PBS by centrifugation at 910g for 8   min three times. The pellet's consistency should become less dense with each washing step. Perform an additional washing step if the supernatant is quite red which indicates erythrocyte lysis.

i.

Resuspend the cells in Z mL of RPMI 1640 (Table 1). Store the cells at 4   °C for up to 15 days. Test the sheep erythrocytes on donor blood before use, and control the efficiency of the rosetting by flow cytometry as described in Section 2.5.

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Epstein-Barr Virus (Infectious Mononucleosis, Epstein-Barr Virus–Associated Malignant Diseases, and Other Diseases)

Eric C. Johannsen , Kenneth M. Kaye , in Mandell, Douglas, and Bennett's Principles and Practice of Infectious Diseases (Eighth Edition), 2015

Heterophile Antibodies

Heterophile antibodies, originally described by Paul and Bunnell 7 as sheep erythrocyte agglutinins, are present in about 90% of cases at some point during the illness. Beef erythrocyte hemolysins and agglutinating antibodies to horse, goat, and camel erythrocytes are also demonstrable in infectious mononucleosis. The classic heterophile antibody titer is reported as the highest serum dilution at which sheep erythrocytes are agglutinated after absorption of the test serum by guinea pig kidney ( Table 141-8). The differential absorption permits a distinction between naturally occurring Forssman antibodies, the antibodies of serum sickness, and heterophile antibodies of infectious mononucleosis. Beef red cell hemolysins do not need differential absorption for interpretation. Although titers may vary depending on laboratory techniques, a titer of 40 or greater after guinea pig absorption along with a compatible clinical presentation is strong evidence for infectious mononucleosis.

Heterophile antibodies may be seen at the onset of illness or may appear later in the course of the illness. A delayed appearance of heterophile antibodies may be associated with a more prolonged convalescence. 334 Horse red cell agglutination is more sensitive than tests for sheep red cell agglutination or beef red cell hemolysis. Horse red cell agglutinins persist for a year after diagnosis in 75% of the cases, 335 whereas sheep cell agglutinins fall to titers of less than 40 by a year in 70% of cases. False-positive titers greater than 40 of sheep and horse erythrocyte agglutinins have been found in 12% and 6.7% of sera, respectively. 336 Commercial spot kits such as the Monospot test are available and are generally specific and sensitive for heterophile antibodies. The correlation between the results obtained with the use of these kits and results of the classic tube heterophile method is quite good, although the sensitivity of the spot and slide tests is slightly greater than that of the classic tube heterophile test. Occasional false-positive Monospot test responses have been reported in patients with lymphoma or hepatitis, but the rarity of this event makes confirmation of a positive Monospot test result with EBV-specific serology unnecessary. 337-339 Three cases of false-positive Monospot tests in the setting of primary HIV infection have been reported. 340 One study of 132 patients with positive Monospot test results found no instances of primary HIV infection. 341 However, the exact rate of false-positive Monospot test results among patients with primary HIV infection is not known.

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Pulsatility in Neuroendocrine Systems

Amy Tse , Bertil Hille , in Methods in Neurosciences, 1994

Identification of Gonadotropes

Individual gonadotropes are identified by a reverse hemolytic plaque assay. Sheep erythrocytes (Colorado Serum Co; used within 21 days of collection) are first conjugated with Staphylococcus aureus-derived protein A (Sigma) using chromium chloride (0.2 mg/ml; Sigma) as the catalytic agent as described by Smith et al. (4). The pituitary cell suspension is mixed with an equal volume (200 µl) of 18% (v/v) erythrocytes in solution B at room temperature and infused via capillary action into a modified Cunningham chamber (7).

In our experiments, the bottom half of the Cunningham chamber is also used as a recording chamber. For recording current, membrane potential, or capacitance, any 35-mm plastic culture dish is suitable. However, for experiments in which an inverted microscope and fluorometry are employed to monitor intracellular calcium concentration ([Ca2+]i), it is essential that the bottom of the recording chamber be composed of thin glass instead of plastic because (i) the UV light used for excitation of fluorescent dye is largely absorbed by plastic; (ii) the large-numerical aperture oil objective typically employed for calcium measurement has a short working distance; and (iii) plastic can be dissolved by immersion oil. A thin glass-bottomed Cunningham chamber is constructed as follows. A circular hole of approximately 20 mm diameter is drilled in the bottom of a 35-mm plastic culture dish (Falcon). A 25-mm circular glass coverslip (VWR: No. 2) is cemented onto the bottom of the culture dish with Sylgard 184 elastomer (Dow Corning). The chamber is rinsed with 100% methanol and then several times with distilled water to remove any grease. To facilitate cell attachment, the bottom of the chamber is coated by adding 1 ml of 1% gelatin solution (Sigma). After 5 min, the solution is removed and the chamber is allowed to dry under UV light. The coating procedure is repeated with a 0.8 mg/ml concanavalin A solution (Sigma). We have less success with conventional polylysine coating. The roof of the Cunningham chamber is constructed by attaching a 12-mm circular coverslip onto two pieces of 3M double-sided Scotch tape (Cat. No. 136) placed approximately 10 mm apart on the bottom of the chamber. The volume of such a Cunningham chamber is approximately 8 µl.

The cell mixture (10–15 µl) is infused from one side of the chamber and slowly pulled across the chamber via capillary action with piece of Kimwipe. The chamber is incubated at 37°C for 1 hr in a 5% CO2 incubator. Solution B (20 µl) is then infused into the chamber to wash away any unattached cells, leaving a monolayer of cells stuck to the bottom of the chamber. Each pituitary cell should be surrounded by erythrocytes. Plaque formation is then initiated by infusing the chamber with 20 µl of solution B supplemented with 50 nM GnRH (Peninsula), rabbit polyclonal antibodies to rat LH (American Biochem), or bovine LH (courtesy of Dr. D. Leong, University of Virginia) at 1: 50 dilution. After a 2-hr incubation at 37°C, the chamber is infused with 20 µl of solution B, followed by 20 µl guinea-pig complement at 1:50 dilution (Gibco) in solution B. Plaques can be observed after 30 min of incubation at 37°C. The complement reaction is terminated by infusing the chamber twice with 20 µl of solution B. To facilitate the removal of the double-sided Scotch tape, about 1 ml of solution C which contains 10% horse serum (Gibco), 50 units/ml of penicillin G, and 50 µg/ml of streptomycin in DMEM is added to the chamber, and the chamber is incubated for another 15 min at 37°C. Then the Scotch tape and the coverslip that forms the roof of the chamber are removed with a pair of fine-tipped forceps. Finally another 2 ml of solution C is added to the recording chamber. Plaques remain identifiable for up to 5 days under standard culture conditions.

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MIXED HAEMADSORPTION AND IMMUNOFLUORESCENCE STUDIES ON IMMUNOCOMPETITION AT THE MEMBRANE

Astrid Fagraeus , in Membranes and Viruses in Immunopathology, 1972

Mixed Haemadsorption Test (MHT)

Indicator cells were prepared by coating sheep red cells with anti-sheep erythrocyte serum (amboceptor) of human, rabbit or mouse origin and in a second step, with sheep antiserum to the relevant species. Details about the preparation of indicator cells for human and rabbit antibody have been given earlier (4). Mouse immunoglobulin indicator cells were prepared in the same way. Two units of mouse amboceptor and 1000 units of sheep anti-mouse immunoglobulin serum were used.

Only a brief summary of the MHT will be given here as the method has been extensively described earlier (4). For the radial diffusion test confluent cultures were covered by a 4 mm thick layer of 0.75% agar and filter papers discs, 5 mm in diameter soaked with sera were placed on the agar, in some instances at a distance of 4-5 mm from each other. Serum was allowed to diffuse for 48 hours. Agar and discs were then removed and the indicator cells added. After one hour the bottle was tilted and round haemadsorption zones could be seen where indicator cells had reacted with antibodies fixed to the cultured cells (Fig. 1). The diameter of the zone was measured and knowing the dilution of the serum added to the disc the end titer of the serum could be determined.

Fig. 1. Diagram showing the principle of the radial diffusion modification of the MHT. In our tests the diffusion time is usually 48 hours. R.T. stands for room temperature.

 The drawing is made by A. Espmark.

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Rosetting Techniques

Malcolm R. MacKenzie , in Encyclopedia of Immunology (Second Edition), 1998

Erythrocyte (E) rosette

The most widely used rosetting technique in human immunology is the interaction between the sheep erythrocyte and human lymphocytes of the thymic or T cell lineage. This interaction, first appreciated by the laboratories of Brain, Coombs and Wybran, was a major step in the capacity to identify, separate and characterize the T and B cell lineages of lymphocytes in normal physiology and disease. The assay is performed by the incubation of a population of mononuclear cells obtained by a preliminary processing of blood, usually by differential density centrifugation of peripheral blood or of organ suspensions, with sufficient sheep erythrocytes to have a ratio of 50:1 or 100:1 erythrocytes to mononuclear cells. The suspension is incubated briefly at 37°C then centrifuged at 200g and a further incubation for 1–2 hours or overnight at 4°C. The cells are gently suspended and counted in a hemocytometer.

The rosettes formed are more stable in a high protein solution such as 10% human AB serum or 25% bovine serum albumin. The prior treatment of the sheep erythrocytes with neuraminidase also increases the stability. Kaplan found that prior treatment of the sheep erythrocytes with neuraminidase also increases the stability. Kaplan found that prior treatment of the sheep erythrocytes with a sulfhydryl reagent such as 2-amino-ethyisothiouronium (AET) obviates the differences between batches of sheep erythrocytes and enhances the stability of the rosette. Recently Ocklind suggested the use of 1% w/v of polyethylene glycol 10000 molecular weight in the medium allows for the detection of 97% of T cells. These modifications have proved very useful, particularly when the method is used to isolate T cells in preparative techniques.

The molecule on the surface of T cells responsible for this interaction is the 50   kDa glycoprotein CD2, present on more than 95% of thymocytes and all peripheral T cells. It binds to a protein present on sheep erythrocytes designated T11TS, which is the homolog of the human protein, lymphocyte function-associated antigen 3 (LFA-3). This latter protein is present on human erythrocytes and accounts for the observation that human erythrocytes can form rosettes with human thymocytes. The utility of the sheep erythrocyte over the human in this assay and its reaction with peripheral T cells is due to a three- to five-fold higher ligand density on its surface. The CD2 molecule is a T cell activation molecule involved in one alternate pathway. Thus T cells isolated by this technique may under certain circumstances be activated by the procedure. This phenomenon must be kept in mind when designing experiments or making conclusions about the in vivo state of such populations.

In other species E rosette formation is less successful. Limited use of the technique occurs with sheep E and primate T cells. The cat T cell forms rosettes with guinea pig and gerbil erythrocytes.

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