Chicken Mhc alloantiserum cross-reactivity analysis by hemagglutination and flow cytometry

 The major histocompatibility complex (Mhc) haplotype in the chicken is generally determined by the use of alloantisera in a hemagglutination assay. This method restricts haplotype determination to antigens expressed on the surface of erythrocytes which includes class I (B – F) and class IV (B – G) antigens as well as any other polymorphic molecules on these cells. Alloantisera can result in complex cross-reactivity patterns. We describe here the analysis of 53 alloantisera made within Mhc-congenic lines. Each antiserum was tested by hemagglutination with erythrocytes and by flow cytometry with erythrocytes and peripheral white blood cells of seven Mhc haplotypes; B ² , B ⁵ , B ¹² , B ¹³ , B ¹⁵ , B ¹⁹ , and B ²¹ . Five types of antiserum were identified based on their reactivity to different cell subpopulations of the peripheral blood of the donor haplotype as well as in cross-reactivity for different haplotypes. RBC specific cross-reactive antigens attributed to B – G molecules were demonstrated for the B5 : B19, B12 : B19, and B19 : B21 cross-reactions. Cross-reactive antigens detected on RBC and thrombocytes attributable to B – G molecules on both types of cells were demonstrated for the B2 : B12, B2 : B15, B2 : B19, and B2 : B21 cross-reactions. In addition, cross-reactive antigens occurring on RBC and WBC were attributed to B – F (or RBC and lymphocyte-expressed B – G loci) and included the B12 : B13, B13 : B19, and B15 : B19 cross-reactions. Several antisera with specificity for B cells purportedly identifying B – L epitopes were found but their numbers were limited and cross-reactivities were not defined. The identities described here may be useful in understanding B haplotype similarities and differences in disease resistance and immune response. Received: 18 September 1995 / 15 November 1995     

Evidence for two populations of B-L (Ia-Like) molecules encoded by the chicken MHC

Two specific alloantisera detecting B-L (Ia-like) antigens on chicken lymphocytes of the B ⁶ and B ¹⁵ haplotypes were found to cross-react strongly. Anti-B-L6 and anti-B-L15 alloantisera both reacted with B-L molecules on B6 and B15 lymphocytes as demonstrated by immunofluorescence and SDS-PAGE analysis of ¹²⁵I-labeled B-L antigens isolated by incubation with anti-B-L alloantisera. Absorption studies showed that the anti-B-L alloantisera reacted with at least two kinds of antigenic determinant, one set shared by B-L6 and B-L15 molecules and another set specific for each haplotype. In spite of the absence of genetic evidence for more than one B-L locus in the chicken B complex, it was shown by sequential antibody incubations that these two different B-L antigenic determinants are associated with at least two separate species of B-L molecules, indicating the presence of at least two B-L loci within the MHC of the chicken.     

Mouse transcobalamin II: Biosynthesis and uptake by L-929 cells

Mouse fibroblast (L-929) cells, in culture, synthesized and secreted into the growth medium a vitamin B₁₂-binding substance which was identical to mouse transcobalamin II (TC II) as judged by the following criteria: (i) gel filtration on Sephadex G-200, (ii) ion-exchange chromatography on DEAE-cellulose and CM-cellulose, and (iii) the ability to facilitate cellular B₁₂ uptake by L-929 cells. The secretion of mouse fibroblast binder was blocked by cycloheximide and puromycin; and in both cases the cells' ability to secrete this binder was partially restored when the inhibitor was removed. Within 30 h after the cells were exposed to [⁵⁷Co]B₁₂ bound to mouse serum TC II (M_r ~ 38,000) the [⁵⁷Co]B₁₂ was bound to a large molecular weight intracellular binder (M_r ~ 120,000) which was not released into the culture medium. During this same incubation period, the cells released free [⁵⁷Co]B₁₂ and [⁵⁷Co]B₁₂ bound to a protein which had the same elution volume as mouse serum TC II on Sephadex G-200.     

Gastrin/cholecystokinin-like immunoreactivity in human blood cells

In the gastrin and/or cholecystokinin-like immunoreactivity (G/CCK-LI) elution patterns of blood cells in human adults, erythrocyte (RBC) elution pattern has three peaks which are coeluted with gastrin-34 (G34), gastrin-17 (G17) and V_t, and polymorphonuclear leukocyte (PMN) and mononuclear cell (MNC) elution patterns have four peaks which are coeluted with V_o, G34, G17 and V_t. The content of G/CCK-LI in RBC is 1.20±0.54 fmole/10⁸ cells (means±SD). Than in PMN and MNC is 1.44±0.67 p mole/10⁸ cells and 1.67±0.76 p mole/10⁸ cells, respectively.     

Attempt to inhibit cytotoxic alloantibody with specific anti-receptor site antiserum

Utilizing a ⁵¹Cr release cytotoxicity inhibition assay, putative anti-receptor site antisera (anti-A RS_B) were assayed for their capacity to inhibit specific alloantibody, a result predicted by the recent work of Ramseier and Lindenman. Utilizing various isogenic strains of rats, anti-RS antisera prepared by injecting appropriate F₁ hybrid animals (A × B) with either lymph node cells (A) or specific alloantiserum (A anti-B) were found to be inactive in this assay. It appears that the Ramseier-Lindenman phenomenon cannot be corroborated using a ⁵¹Cr release inhibition assay.     

Electrochemical analysis of blood cell/substrate interactions under flow conditions

Interactions of blood cells (RBCs) with a microelectrode of 50 (im diameter have been examined under flow conditions using impedance measurements at high frequencies. At such frequencies, the electrolyte resistance (R_e,) is assimilated to the real part of impedance, and interactions are associated with transient fluctuations of R_e. Sedimentation experiments suggest that one erythrocyte contributes to a 1.1% R_e, increase. Effects of wall shear rate (from 25 to 140 s¹) and RBC concentration (from 8.4 × 10⁵ to 2.7 × 10⁶ cells/ml) have been investigated; the number of interactions rapidly decreases with wall shear rate. Event frequency is proportional to RBC concentration ranging from 3.1 × 10⁶ cells/ml to 1.3 × 10⁷ cells/ml. At high concentrations of RBCs, some transient events overlap. Videotaped images help to determine how many RBCs interact with the microelectrode at the same time on separate surface areas. Under flow conditions, the contribution of one RBC on the R_e increase is similar to the mathematical value obtained by sedimentation and decreases slightly with wall shear rate.     

A strong,preferential response of mice to polymorphic antigenic determinants of the chicken MHC,analyzed with mouse hybridoma (monoclonal) antibodies

CBA/J mice were immunized with B²/B² chicken RBC's (theB locus is the major histocompatibility complex (MHC) of the chicken). Spleen cells from these mice gave a much higher plaque-forming cell response when tested with RBC's bearing B² alloantigens, than with RBC's bearing any other MHC alloantigens. Similarly, immunization with chicken RBC's of other genotypes also produced responses that were highest when tested on the genotype used for immunization. Spleen cells from mice immunized 3 days previously with B²/B² RBC's were fused with mouse myeloma cells and hybrid clones which secreted anti-B² antibodies were selected. These monoclonal antibodies could be divided into three groups: (1) those which react with all genotypes of RBC's (panreactive); (2) those which react with only certain genotypes of RBC's and detect “public” MHC antigens; and (3) those which react with only B²/B² RBC's and detect “private” MHC antigens. Monoclonal antibodies which detect a private B² alloantigen were shown to be excellent typing reagents, as all birds of an outbred population which possessed the B² allele were easily detected using a simple one-step direct agglutination assay. No “false positives” were seen. The high and preferential response of the mouse to chicken MHC alloantigens suggested that mice might possess preexisting immunity to these antigens. In agreement with this hypothesis, normal mouse serum was found to have high titres of “natural” antibody against chicken MHC antigens.     

15N NMR studies of the binding of 15N-labeled cyanide to various hemoglobins in intact erythrocyte.

¹⁵N-labeled cyanide binding to methemoglobins in intact erythrocytes has been studied by ¹⁵N NMR. The addition of C¹⁵N^? to human and dog hemoglobins in erythrocyte afforded hyperfine-shifted two ¹⁵N signals due to the C¹⁵N bound to ferric iron of the different heme-units. Single and three distinct signals were observed for rat and rabbit hemoglobins in erythrocyte. These C¹⁵N resonance positions are sensitive both to the structural difference in the hemoglobin subunits and to the variety of the animal sources. The C¹⁵N spectral difference between solution and intact hemoglobin cyanide is also discussed in relation to a possible change in the intra- and extracellular pH values.     

Hematological responses to thermal acclimation in a cold water squaliform (Heterodontus francisci girard)

Summary The hematological modifications occurring as a result of acclimation to increased temperature in the cold water horn shark,Heterodontus francisci, were evaluated. Sharks were maintained under constant conditions except for temperature (15°C and 25°C) in a closed marine system. The total red blood cell (RBC) number decreased in the 25°C sharks. In contrast, hemoglobin concentration, hematocrit, mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) significantly increased at 25°C compared to the control animals. RBC size was increased at 25°C, but the surface area/mm³ whole blood was reduced. Folic acid levels were not different between the groups. Vitamin B₁₂ levels decreased and testosterone increased at 25°C. Blood pH, number of erythroblasts, number of white blood cells (WBC) and WBC differential analyses were essentially unchanged at the two temperatures, except that the relative neutrophil number was increased. The major hematological changes occur in the erythrocytes and appear to be sequential in nature with an initial loss of RBC followed by increased hemoglobin synthesis and increased RBC size, but lack of recovery of RBC numbers.Abbreviations Hb hemoglobin - Hct hematocrit - MCH(C) mean corpuscular hemoglobin (concentration) - MCV mean corpuscular volume - RBC red blood cells - WBC white blood cells Contribution Number 359, Department of Biology     

Self-diffusion of water-soluble fullerene derivatives in mouse erythrocytes

Self-diffusion of water-soluble fullerene derivative (WSFD) C₆₀[S(CH₂)₃SO₃Na]₅H in mouse red blood cells (RBC) was characterized by ¹H pulsed field gradient NMR technique. It was found that a fraction of fullerene molecules (~13% of the fullerene derivative added in aqueous RBC suspension) shows a self-diffusion coefficient of (5.5 ± 0.8)·10⁻¹² m²/s, which is matching the coefficient of the lateral diffusion of lipids in the erythrocyte membrane (D_L = (5.4 ± 0.8)·10⁻¹² m²/s). This experimental finding evidences the absorption of the fullerene derivative by RBC. Fullerene derivative molecules are also absorbed by RBC ghosts and phosphatidylcholine liposomes as manifested in self-diffusion coefficients of (7.9 ± 1.2)·10⁻¹² m²/s and (7.7 ± 1.2)·10⁻¹² m²/s, which are also close to the lateral diffusion coefficients of (6.5 ± 1.0)·10⁻¹² m²/s and (8.5 ± 1.3)·10⁻¹² m²/s, respectively. The obtained results suggest that fullerene derivative molecules are, probably, fixed on the RBC surface. The average residence time of the fullerene derivative molecule on RBC was estimated as 440 ± 70 ms. Thus, the pulsed field gradient NMR was shown to be a versatile technique for investigation of the interactions of the fullerene derivatives with blood cells providing essential information, which can be projected on their behavior in-vivo after intravenous administration while screening as potential drug candidates.     

Studies on blood groups in the japanese quail: The common antigens possessed by red blood cells and leukocytes,and their inheritance

Two alloantigens, Ly₁ and Ly₂, were detected with alloantisera made by immunization with leukocytes. These antigens were present on red blood cells, peripheral leukocytes and spleen cells and found to be controlled by the autosomal codominant alleles. The phenotypic frequencies of Ly₁ antigen in the three quail stocks, 1, 2, and 3, were 6.7, 0, and 100 percent, respectively, and those of Ly₂ antigen were 0 percent in stocks 1 and 2, and 7.1 percent in stock 3. It was suggested that Ly₁ and Ly₂ antigens might be associated with the system controlling A(QN₁) antigen which was originally detected by the natural alloantibody. However, it remains to be investigated whether these antigens are associated with the major histocompatibility complex (MHC) of the Japanese quail.     

Water channels in membranes

A systematic programme of comparative nuclear magnetic resonance measurements of the membrane permeability for water (Pd) and of activation energy (E_a,d) of this process in red blood cells of various wild, laboratory and domestic animals was carried out here. The RBC from humans, cow, sheep and kangaroos had P_d values around 5·10^?3 cm/s at 25 °C, 7 · 10^?3 cm/s at 37 °C with E_a,d values around 25 kJ/mol. For RBC from other ten marsupial species and from mouse, rat and rabbit, the P_d values were more than twice as high as for human RBC. For mosr RBC a high value of Pd was associated with a low value of E_a,d (range from 15 to 21 kJ/mol), pointing to specialized channels for water diffusion incorporated in membrane proteins. Recently a channel-forming integral protein of 28 kDa (CHIP 28) was identified as a major water channel protein in the RBC membrane. A procedure for quantitating the purified CHIP 28 by densitometry of silver-stained polyacrylmide gel electrophoreograms was developed. The analysis of a purified fraction of CHIP 28 showed that the 28 kDa component represents approximately two-thirds of the sample with the remainder comprising the glycosylated high-molecular-weight component. A correlation between the content in CHIP 28 and the relative water permeability among RBC from different vertebrate species was attempted.     

Identification of differences between the surface proteins and glycoproteins of normal mouse (Balb/c) and human erythrocytes

Summary The topography of the external surface of the Balb/c mouse erythrocyte has been investigated and compared to the human erythrocyte by using a series of protein radiolabeling probes. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the pattern of Coomassie Blue stained proteins was very similar for mouse and human erythrocyte ghosts, as was the distribution of radioactivity in protein bands after lactoperoxidase catalyzed radioiodination. The mouse erythrocyte glycoproteins identified by periodic-acid-Schiff and Stains-All reagents, sialic acid analysis of gel slices, binding of¹²⁵I-wheat germ agglutinin and¹²⁵I-concanavalin A to the gels, and glycoprotein radiolabeling techniques, differed markedly from the sets of proteins labeled by radioiodination, and also differed from the human erythrocyte glycoproteins. Instead of the PAS I to PAS IV series of sialoglycoproteins characteristic of human erythrocytes, the mouse erythrocyte possesses a broad band of sialoglycoproteins with several peaks ranging in mol wt from 65,000 to 32,000. The same group of sialoglycoproteins were labeled by the periodate/B³H₄ ^– technique specific for terminal sialic acid, and the galactose oxidase/B³H₄ ^– method (plus neuraminidase) specific for galactosyl/N-acetylgalactosaminyl residues penultimate to sialic acid. These results emphasize the necessity to employ a variety of protein radiolabeling probes based on different labeling specificities, to study the membrane topography of cells which are poorly understood compared to the human erythrocyte membrane.     

Combinatorial Cellulose-Bound Peptide Libraries: Screening Tools for the Identification of Peptides That Bind Ligands with Predefined Specificity

We describe the synthesis of structurally different types of combinatorial peptide libraries on continuous cellulose membrane supports. These libraries consisting of tens of millions of different peptides were screened for their ability to bind given ligands such as the monoclonal antibody Tab2, transforming growth factor-β (TGFβ), nickel(II) (Ni²⁺), and technetium-99m (^99mTc). We were thus able to detect the linear transforming growth factor-α (TFGα) epitope SHFND recognized by Tab2. Other peptides that also bound Tab2 were identified within the same experiment. A first screening step for identification of peptide mixtures that bind to other ligands of biological interest is shown for peptide mixtures XB₁XB₂XX (B = defined amino acid, X = randomized position) that bind to Ni²⁺ and ^99mTc. A combinatorial linear all L- or all D-library XXB₁B₂XX and two libraries conformationally restrained either via a disulfide bridge between a C-and an N-terminal cysteine [cyclo(C¹-C⁸)-C¹XXB₁B₂XXC⁸] or an amide bond between the alpha amino group of the N-terminus and the gamma carboxyl group of a C-terminal glutamic acid [cyclo(X¹-E⁷)-X¹XB₁B₂XXE⁷] were screened with transforming growth factor-β, resulting in structurally different peptides mixtures that bound to this ligand. The results obtained indicate that chemically different types of cellulose-bound combinatorial libraries can be prepared easily, allowing the rapid and inexpensive screening of millions of peptides for selection of single molecules with predefined specificity that bind to given ligands such as proteins, metals, nucleic acids, and other molecules of biological interest.     

Increased growth of RSV-induced tumors in chickens partially tolerant to MHC alloantigens

Chickens withB ² B ² MHC genotypes were made partially tolerant to B₅ MHC cell-surface antigens and the fate of their Rous-sarcoma-virus (RSV)-induced tumors was determined.B ² B ² chickens partially tolerant to viable or lysed white blood cells (WBC) or viable red blood cells (RBC) fromB ⁵ B ⁵ chickens had a significantly higher incidence of tumor progression than untreated, PBS-treated, orB ² B ² chickens inoculated with WBC from otherB ² B ² chickens. The criteria for tolerance were absence of antibody titer to the cell type inoculated and acceptance of allografts fromB ⁵ B ⁵ donors byB ² B ² chickens. Graft-vs-host reactions occurred only inB ² B ² chickens inoculated with viable WBC fromB ⁵ B ⁵ chickens. It appears thatB ² B ² chickens partially tolerant to B₅ antigens failed to mount a successful immune response to RSV-induced tumors partly because a B₅ MHC antigen(s) cross-reacted with a tumor associated antigen(s) thereby severely limitingB ² B ² host recognition of the tumor as foreign. Since WBC and RBC cell-surface antigens appear to contribute similarly to the effect, theB-F- region of the MHC may be involved.     

Restricted expression of an MHC alloantigen in cells of the erythroid series: A specific marker for erythroid differentiation

The spectrum of reactivity with various types of cells of a monoclonal antibody (CH-4) which detects a private MHC antigen of chickens was analysed. CH-4 agglutinates only RBCs that possess the B² (MHC) haplotype. A new rosetteforming cell (RFC) assay was devised to detect individual cells (excluding RBCs) that possess the CH-4 specificity on their cell surfaces. RBCs that have CH-4 chemically coupled to their surfaces attach to, and form rosettes with, B² antigen-bearing cells. Most non-RBC RFC were detected in active erythropoietic organs (adult bone marrow and embryonic spleen), and none were found in organs where erythropoiesis does not occur: adult thymus and bursa. Preincubation of bone marrow cells with CH-4 plus complement almost completely inhibits their capacity to form CFU-E without affecting their ability to form GM-CFU. In addition, CH-4 plus complement does not inhibit the capacity of B²/B² lymphocytes to induce a graft-versus-host reaction under conditions where anti-B² lymphocyte alloantisera are completely inhibitory. Our results strongly suggest that CH-4 monoclonal antibodies detect a private specificity on a gene product of the B-G locus whose expression is restricted to erythroid stem cells and erythrocytes.     

Photoaffinity labelling of the nucleoside transporter of cultured mouse lymphoma cells

Nitrobenzylthioniosine (NBMPR), a potent and specific inhibitor of nucleoside transport, is bound reversibly by high affinity sites on nucleoside transporter proteins of erythrocyte membranes and, upon photoactivation, NBMPR molecules become covalently bonded to the sites. This study showed that [³H]NBMPR molecules reversibly bound to intact S49 and L5178Y mouse lymphoma cells became covalently bound upon exposure to UV light. Electrophoretic analysis of plasma membrane fractions from the labelled cells showed that ³H was present in polypeptides which migrated as a major band with an apparent M_r of 45000–65000.     

Uptake and removal of cortisol by canine erythrocytes in various electrolyte solutions

The uptake of free cortisol by canine RBC was studied by incubating the cells in various Ringer-Locke solutions, saline, plasma, and plasma to which EDTA was added. The uptake of cortisol by RBC was similar in all electrolyte solutions; however, uptake was significantly less when RBC were incubated in plasma. The removal of both exogenous and endogenous cortisol from RBC was studied by washing the cells in various electrolyte solutions. Although the percentages of steroid removed per wash were not significantly different when cells were washed with Ringer-Locke solutions, saline washed RBC to which no cortisol was previously added gave significantly less steroid per wash. These data indicate that Na⁺, K⁺ and Ca⁺⁺ do not affect the permeability of these cells to cortisol and that the cortisol associated with canine RBC is loosely bound.     

A Missing PD-L1/PD-1 Coinhibition Regulates Diabetes Induction by Preproinsulin-Specific CD8 T-Cells in an Epitope-Specific Manner

Coinhibitory PD-1/PD-L1 (B7-H1) interactions provide critical signals for the regulation of autoreactive T-cell responses. We established mouse models, expressing the costimulator molecule B7.1 (CD80) on pancreatic beta cells (RIP-B7.1 tg mice) or are deficient in coinhibitory PD-L1 or PD-1 molecules (PD-L1^−/− and PD-1^−/− mice), to study induction of preproinsulin (ppins)-specific CD8 T-cell responses and experimental autoimmune diabetes (EAD) by DNA-based immunization. RIP-B7.1 tg mice allowed us to identify two CD8 T-cell specificities: pCI/ppins DNA exclusively induced K^b/A_12–21-specific CD8 T-cells and EAD, whereas pCI/ppinsΔA_12–21 DNA (encoding ppins without the COOH-terminal A_12–21 epitope) elicited K^b/B_22–29-specific CD8 T-cells and EAD. Specific expression/processing of mutant ppinsΔA_12–21 (but not ppins) in non-beta cells, targeted by intramuscular DNA-injection, thus facilitated induction of K^b/B_22–29-specific CD8 T-cells. The A_12–21 epitope binds K^b molecules with a very low avidity as compared with B_22–29. Interestingly, immunization of coinhibition-deficient PD-L1^−/− or PD-1^−/− mice with pCI/ppins induced K^b/A_12–21-monospecific CD8 T-cells and EAD but injections with pCI/ppinsΔA_12–21 did neither recruit K^b/B_22–29-specific CD8 T-cells into the pancreatic target tissue nor induce EAD. PpinsΔA_12–21/(K^b/B_22–29)-mediated EAD was efficiently restored in RIP-B7.1⁺/PD-L1^−/− mice, differing from PD-L1^−/− mice only in the tg B7.1 expression in beta cells. Alternatively, an ongoing beta cell destruction and tissue inflammation, initiated by ppins/(K^b/A_12–21)-specific CD8 T-cells in pCI/ppins+pCI/ppinsΔA_12–21 co-immunized PD-L1^−/− mice, facilitated the expansion of ppinsΔA_12–21/(K^b/B_22–29)-specific CD8 T-cells. CD8 T-cells specific for the high-affinity K^b/B_22–29- (but not the low-affinity K^b/A_12–21)-epitope thus require stimulatory ´help from beta cells or inflamed islets to expand in PD-L1-deficient mice. The new PD-1/PD-L1 diabetes models may be valuable tools to study under well controlled experimental conditions distinct hierarchies of autoreactive CD8 T-cell responses, which trigger the initial steps of beta cell destruction or emerge during the pathogenic progression of EAD.     

DNA strand breakage induced by CuII and NiII, in the presence of peptide models of histone H2B

In the present study we used the plasmid relaxation assay, a very sensitive method for detection of DNA strand breaks in vitro, in order to evaluate the role of peptide fragments of histone H2B in DNA strand breakage induced by copper and nickel. We have found that in the presence of peptides modeling the histone fold domain (H2B_32-62 and H2B_63-93) as well as the N-terminal tail (H2B_1-31) of histone H2B there is an increased DNA damage by Cu²⁺/H₂O₂ and Ni²⁺/H₂O₂ reaction mixtures. On the contrary, the C-terminal tail (H2B_94-125) seems to have a protective role on the attack of ROS species to DNA. We have rendered our findings to the interactions of the peptides with DNA, as well as with the metal.