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B1-anti-B2 and B2-anti-B1 allo-antisera were raised by i.v. immunization of homozygous B1/1 and B2/2 chickens, respectively, with B1/2 cells of the following sources: peripheral blood leucocytes (PBL), erythrocytes, thymus cells and bursa cells. Purified suspensions of these all produced anti-B allo-antibodies which could both haemagglutinate red blood cells and strongly inhibit the GVH reactivity of blood lymphocytes of the original donor type. Absorption studies of the sera using cells of the same four sources showed that they could all completely remove the GVH inhibitory antibodies. A fact of particular importance is that B-haplotype determined antigens which are common to erythrocytes and lymphocytes seem to include the gene products of the allo-aggression locus, as earlier defined (Simonsen 1975).

In an attempt to further map the chicken MHC (the B complex), a systematic search for genetic recombinants within the B complex was performed by serotyping the progeny from F2 crosses of chickens by means of specific anti-class I, anti-class II, and anti-class IV alloantisera. Two recombinant B-haplotypes (B21r and B15r) were found by analysing 2,656 F2 chickens representing 5,312 informative typings. In either case, the B-G (class IV) allele was recombined with both the B-F and B-L alleles of the opposite haplotype. MLC typings, tests for direct compatibility by GVH reactions, and absorption analyses confirmed the original serological typing of the two recombinant B haplotypes. No recombination between B-F (class I) and B-L (class II) loci was found. This very low frequency of recombination within the B complex as compared with recombination frequencies found in mammalian MHC's is discussed.

Autoradiographic studies have shown that radioiodinated TIGAL binds in vitro to a small but varying fraction of lymphoid cells from bursectomized, agammaglobulinemic chickens, whereas no binding of radioiodinated TGAL or a variety of other radioiodinated antigens can be observed. The binding of [125I]TIGAL is inhibited by antigen-antibody complexes, Radioiodinated antigen-antibody complexes are bound to a similar proportion of the lymphoid cells from bursectomized chickens, and this binding is inhibited by preincubation of the cells with un labeled TIGAL but not with TGAL. These results indicate a cross-reaction at the level of Fc receptors between determinants on TIGAL and on IgG.

Chicken and turkey beta 2-m were isolated from citrated plasma in sequential use of three chromatographic steps: affinity chromatography, gel filtration chromatography and anion-exchange chromatography. The purified protein was identified as beta 2-m by reaction with a beta 2-m specific monoclonal antibody and by the ability to recombine with the chicken MHC class I heavy chain. The purity was estimated by SDS-PAGE and IEF. The pI was between 5.1 and 5.3 for chicken beta 2-m and 4.7 and 4.8 for turkey beta 2-m, which fact is reflected in their different electrophoretic mobilities in agarose gel (turkey migrates in the alpha and chicken migrates in the beta region). The mol. wt of both chicken and turkey beta 2-m was 14,500 estimated by SDS-PAGE whereas calculations based on the amino acid compositions gave mol. wts of 11,000. EM280 was 15.9 for chicken beta 2-m and 16.4 for turkey beta 2-m. The amino acid compositions and sequences of the two avian beta 2-m molecules have been compared with earlier data from the literature. The sequence of the 23 N-terminal amino acids was found to be identical in our preparations from both chicken and turkey, namely DLTPKVQVYSRFPASAGTKNVLN, and is incompatible with a previously published sequence also thought to be from turkey beta 2-m. Reasons for our opinion that the molecules isolated and sequenced in this paper are the correct ones are given. Udgivelsesdato: 1986-Dec

Eggs from immunized chickens may provide a convenient and inexpensive source of antibodies. We describe two simple and efficient methods for purification of IgG from yolk. The antibody is rendered useful for most currently employed immunological techniques. Amounts of antibody corresponding to almost half a litre of antiserum may be recovered from a chicken in one month.

A simple and reliable polyethylene glycol (PEG) precipitation radioimmunoassay is described and used for examining the expression of V H allotype determinants (al) on various immunoglobulins and fragments. The alloantisera were obtained by immunization with free or antigen-complexed IgG. The results demonstrate that most of the antibody reacts with determinants which are only fully expressed in the presence of the first constant domain of the H-chain (C H l γ ), or the J γ -segment, as well as the L-chain. Only a minor part of the antibody reacts with free V H or IgA. These results may have important implications for the use of alloantibody in studies of the V H -domain.

Experiments were carried out to give information on the relative functioning, in regenerating rat liver and in spleen, of the two possible pathways for the biosynthesis of thymidine nucleotides, namely: Pathway I: uridine monophosphate → deoxyuridine monophosphate → thymidine monophosphate, and Pathway II: uridine monophosphate → cytidine monophosphate → deoxycytidine monophosphate → deoxyuridine monophosphate → thymidine monophosphate. The pyrimidines of ribonucleic acid and deoxyribonucleic acid were labelled by a single injection of [6- 14 C]orotic acid in the control rats. In the experimental animals, in addition to the labelled orotic acid, unlabelled cytidine was injected in divided doses before and after the orotic acid injection. It was noted that in the latter series the unlabelled cytidine caused a great decrease in the specific activities of cytidylic acid isolated from ribonucleic acid and of deoxycytidine from deoxyribonucleic acid, of regenerating liver and spleen, reflecting dilutions of the metabolic pools of cytidine nucleotides. In contrast, there was a comparatively small drop in the specific activity of deoxyribonucleic acid-thymidine. It is concluded that in both tissues, thymidine monophosphate is formed from uridine monophosphate to a greater extent through Pathway I without the intermediate formation of cytosine nucleotides stipulated by Pathway II.

The duration of the premeiotic DNA synthesis in oocytes of mouse embryos has been determined using a specific DNA precursor (tritiated thymidine) and autoradiography. The duration of the premeiotic S period in mouse oocytes is considerably longer than the S period in mammalian somatic cells, but in good agreement with the duration of the premeiotic S period in primary spermatocytes of the mouse [5]. Three different methods were used. The mean value of the duration of the premeiotic S period was determined by two of the methods to be 10.5 hr and 12 hr respectively. The maximum duration of premeiotic S + G2 was determined by the last method and was found to be 14 hr. G2 seems to be very short, its minimum value being less than half an hour. Some crude estimations of the duration of the early stages of the first meiotic prophase were made. Leptotene lasts from 3 to 6 hr and zygotene from 12 to 40 hr. The minimum duration of pachytene is more than 60 hr.

Crone M. and G. Waago. Transfer of radioactive iodide between mother and foetus in the rabbit. Radioiodide injected subcutaneously into rabbits disappeared more rapidly from the serum of pregnant than non-pregnant animals. To clarify the cause of this difference the 131I uptake in the embryos was measured and higher values were found than corresponded to simple diffusion between mother and foetus. The radioactivity of the foetal gastric juice increased with time. Its 131I concentration was 13 to 109 times higher than in the foetal serum. The 131I concentration in the foetal gastric juice remained high at a time when the 131I concentration had decreased to a low level in the foetal serum. This was considered to indicate that iodide is actively transported through the foetal gastric mucosa and is accumulated in the foetal gastric juice. Radioiodide was found in a higher concentration in the foetal serum and in an even higher concentration in the foetal part of the placenta than in the maternal serum. A steady state of 131 across the placenta was not obtained as the radioactivity of the maternal serum decreased rapidly. A diffusion delay in the foetus was not sufficient to explain the results, which therefore indicated that iodide is actively transported from the mother to the foetus and is concentrated in the foetal part of the placenta. These conclusions were supported by experiments where 131I was administered in such a way that the radioactivity of the maternal serum increased or was constant. Perchlorate was shown to inhibit the 131I uptake in the foetus by inhibiting the active transport across the placenta and through the foetal gastric mucosa. The iodide concentration mechanism of the foetal part of the placenta was also inhibited.

Udgivelsesdato: 1985-null

Antibodies against rabbit V H -determinants were produced by immunizing mammals and birds with rabbit IgG, IgA or V H . Most of the anti-V H produced in mammals was specific for allotype V H -determinants. but small amounts of antibody against non-allotype V H -determinants were also produced. By contrast the immunization of chickens led to the production of predominantly non-allotype V H antibody. Further analysis of the antibody specificity revealed the production of antibody against V H -determinants available in the intact immunoglobulin (H- + L-chain) as well as antibody reacting with determinants only accessible in the absence of L-chain. Antibody of the latter specificity prevailed after immunization with V H . The purified V H was not deficient in antigen determinants when compared with V H in the intact immunoglobulin (IgA). Unexpectedly, none of our chicken anti-rabbit V H antisera showed crossreactivity with V H from other mammalian species.

The F antigens, which are serologically defined Class I gene products of the major histocompatibility complex in chickens (the B complex), were analysed in outbred birds. Private specificities of the F13 antigen from the inbred CC strain were detected in 20 outbred chickens by a haemagglutination technique. In the GVH-inhibition-release test F13 antigens from outbred and inbred chickens were identical. The L antigens, which are the Class II antigens of the B complex, were detected with specific anti L13 alloantisera by indirect immunofluorescence. Antisera defining the L13 antigen(s) of the inbred CC line reacted with all F13 positive outbreds. As a test of one-way direct compatibility of the inbred and outbred animals typing F13, graft versus host reactions were performed, injecting blood of F13 positive outbreds into inbred B13/13 eggs. No GVH stimulation attributable to MHC determinants was found. In MLR, responder cells from outbred MHC heterozygous chickens, which typed F13/x, were stimulated by inbred F13/13 homozygous typing cells, and weak, but significant, reactions were found. Further analysis in the accompanying paper, however, revealed no difference in the lymphocyte activating determinants (Lads) of inbred and outbred birds typing F13 and L13. No individual has yet been found which carries one of these antigens in the absence of the other.

This paper reviews the present knowledge of B-L antigens encoded by the chicken B complex as regards to the following aspects: (1) identification and cellular expression, (2) structural studies, (3) evidence for two distinct populations of B-L antigens, (4) mapping of B-L loci of the B complex, (5) B-L and immune response, and (6) the role of the B-L antigens for the control of mixed lymphocyte reactions (MLR) and graft-versus-host (GVH) reactions. It is concluded that B-L antigens of the chicken exhibit extensive homology with mammalian Ia antigens. A genetic map of the B complex is presented.

Two specific alloantisera detecting B-L (Ia-like) antigens on chicken lymphocytes of the B6 and B15 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.

IN order to investigate the synthesis of DNA in mammalian oocytes an attempt was made to label oocytes of mice with a radioactive DNA precursor. If DNA is synthesized during the last premeiotic interphase, as it has been reported for the mouse spermatocyte1, it should be possible to label oocytes by injecting a radioactive precursor during the last third of pregnancy. Meiotic prophase in the mouse oocyte begins about the 13th day of embryonic life2–4. Tritium-labelled thymidine was therefore injected into mouse embryos on different days during the latter part of embryonic life to see: (1) whether oocytes can be labelled in this way; (2) what percentage of the oocyte population is labelled after injection on different days of embryonic life; (3) whether labelled oocytes in progressive stages of meiosis can be obtained if autoradiographs are prepared at different intervals after injection of the DNA label.

After a single injection of 3HTdR into mice shortly after birth a few labelled oocytes were observed. These were all in early diplotene, a stage of meiotic prophase in which no normal chromosome replication takes place. Several possibilities to explain this phenomenon are discussed including abnormal DNA synthesis during degeneration and DNA repair after some kind of damage or as a step in the process of genetic recombination. The first possibility is considered to be the most likely. The mechanism underlying this DNA synthesis is not known.

UNSCHEDULED synthesis of DNA has been observed in the nuclei of HeLa cells at some stage of the cell cycle other than the S-phase when cells are exposed in vitro to ultraviolet light or X-ray irradiation1. It has since been confirmed for several other cell types2–5. Part of the synthesis defined in terms of the uptake of 3H-thymidine—which was detected in HeLa cells as the result of large doses of ultraviolet irradiation—was non-semiconservative (repair replication), corresponding to the insertion of DNA precursors into extant parental DNA strands. Here I present evidence that X-irradiation of a 1 day old female guinea-pig with 5,000 rad is followed by a small but auto-radiographically detectable incorporation of 3H-thymidine into the DNA of diplotene oocytes. In the guinea-pig, the mouse and most other mammals the premeiotic DNA synthesis occurs during embryonic life6. After birth the oocytes have all entered prophase of meiosis during which no DNA synthesis is normally observed.

The binding of tritiated corticosterone in lymphocytes from thymus, bursa of Fabricius and peripheral blood from chickens were studied in vitro . A high-affinity binding of corticosterone was found in all populations of lymphocytes that were investigated. The affinity of the binding was nearly identical to that previously found in rat thymocytes. No high-affinity binding of corticosterone was found in chicken thrombocytes, in chicken erythrocytes or in sheep erythrocytes.