Chromosome healing in mouse embryonic stem cells.

The addition of new telomeres to the ends of broken chromosomes, termed chromosome healing, has been extensively studied in unicellular organisms; however, its role in the mammalian cell response to double-strand breaks is unknown. A system for analysis of chromosome healing, which involves the integration of plasmid sequences immediately adjacent to a telomere, has been established in mouse embryonic stem cells. This "marked" telomere contains a neo gene for positive selection in G418, an I-SceI endonuclease recognition sequence for introducing double-strand breaks, and a herpes simplex virus thymidine kinase gene for negative selection with ganciclovir for cells that have lost the telomere. Transient expression of the I-SceI endonuclease results in terminal deletions involving telomeric repeat sequences added directly onto the end of the broken chromosome. The sites of addition of the new telomeres contain short regions of complementarity to telomeric repeat sequences. The most common site of addition is the last A of the ATAA 3' overhang generated by the I-SceI endonuclease, without the loss of a single nucleotide from the end of the chromosome. The next most frequent site involved 5 bp of complementarity, which occurred after the loss of four nucleotides from the end of the chromosome. The new telomeres are generally much shorter than in the parental cell line, and most increase in size with time in culture. These results demonstrate that chromosome healing is a mechanism for repair of chromosome breaks in mammalian cells.

The monoclonal antibody TAL16.1 recognizes the aspartic acid residue at position 70 in DRB gene products.

A polymorphic monoclonal antibody (TAL16.1), raised against a mouse L-cell transfectant expressing the human DRB5*0101 gene from the HLA-DR15(2) Dw2 DR51 haplotype was shown to have a complex pattern of reactivity to DRB gene products. The antibody bound to a transfectant expressing the DRB5*0101 allele against which it was produced but not to a transfectant expressing the DRB1*1501 allele. These alleles of the DRB1 and DRB5 genes are usually coexpressed on DR15(2) Dw2 DR51 cells. A comparison of the HLA-DRB amino acid sequences of reactive and non-reactive cells identified an aspartic acid residue at position 70, conserved in all antibody-positive cells and absent in antibody-negative cells, which was postulated as being responsible for conferring the specificity of the antibody. The aspartic acid residue at position 70 is present in DRB5*0101 and DRB5*0102 alleles but absent in DRB5*0201 and DRB5*0202 alleles, allowing the antibody to distinguish between these splits of the DR51 serological specificity. TAL16.1 also binds to the product of the DRB1*0103 allele and discriminates between cells with a DR103 specificity and the other DR1 subtypes, DRB1*0101 and DRB1*0102. In this report the value of transfectants as immunogens for use in the production of monoclonal antibodies of predetermined specificity and as tools for the fine mapping of antibody specificity is discussed.

Evaluation of relationship between plasma fibrinogen concentration and tuberculin testing in red deer.

After intradermal injection of bovine purified derivative (PPD), increases in plasma fibrinogen concentration and plasma viscosity developed in red deer (Cervus elaphus) with a history of tuberculosis caused by Mycobacterium bovis. Serum haptoglobin concentrations were also found to increase under similar circumstances. The increases were reproducible and did not appear to be related to mustering, stress, or the handling associated with injection of PPD. A significant (P less than 0.05) direct relationship was found between the increase in plasma fibrinogen concentration and various markers of bovine tuberculosis infection, such as stimulation of lymphocyte transformation in response to bovine PPD and the diameter of intradermal tuberculin skin test reactions. A stronger correlation (P less than 0.01) was found with the volume of intradermal tuberculin skin test reactivity, and the strongest correlation (P less than 0.001) was with the presence of circulating antibovine PPD antibody.

Humoral immunity in the ewe. 2. The effect of pregnancy on the primary and secondary antibody response to protein antigen.

This study examines the effect of pregnancy on the quantity and isotype of antibody in ewes immunised with a novel primary antigen. The primary and secondary antibody levels to bovine serum albumin (BSA) in alum adjuvant, were compared between non-pregnant ewes and ewes immunised at different stages of pregnancy. Anti-BSA isotype specific responses were measured using an indirect ELISA. Results show the levels of immunoglobulin M (IgM) increased and persisted in response to BSA during pregnancy (P less than 0.05). Secondary immunoglobulin G1 (IgG1) titres were significantly impaired in late pregnancy and during lactation (P less than 0.05). The lower levels of immunoglobulin G2 (IgG2) were unaffected by pregnancy under these experimental conditions. Ewes were also immunised with BSA in alum adjuvant for their primary inoculum and BSA in different adjuvants for the secondary inoculation. Primary IgM persisted at higher levels during pregnancy compared with the response in non-pregnant ewes (P less than 0.05). Following the secondary injection, lower levels of anti-BSA specific IgM, IgG1 and IgG2 antibodies were produced in late pregnancy and during lactation compared with the levels in non-pregnant control ewes (P less than 0.05). Alterations in regulatory T-cell function or effector B-cell activity would most readily explain the qualitative changes in antibody titre observed following primary injection during pregnancy. The results also suggest an associated impairment of immunological memory following primary immunisation during pregnancy.

Humoral immunity in the ewe. 3. The influence of adjuvants and immunisation regimes on immune reactivity in the breeding ewe and her progeny.

Breeding ewes were immunised with clostridial vaccine using different inoculation schedules. Results, showing differences in the class of antibody produced, were heavily dependent on the vaccination regime used. Immunoglobulin M (IgM) levels were significantly lower in ewes given double doses of vaccine compared to ewes given a single inoculation or no treatment at all (P less than 0.01). Neonatal lambs showed significant de novo IgM production with interference in this antibody production in the lambs of ewes vaccinated with the clostridial vaccine (P less than 0.05). Immunoglobulin G1 (IgG1) levels were significantly increased in all lambs which had mothers vaccinated with the clostridial vaccine prior to or during pregnancy (P less than 0.025). The greatest quantity of IgG1 was transferred to lambs when their mothers were given a double injection with primary inoculation prior to conception and booster prepartum (P less than 0.025). No antigen specific immunoglobulin G2 (IgG2) was detected in the lambs. Ewes were also immunised with BSA and their isotype specific serum antibody response was compared with their respective lambs. There was no detectable anti-BSA IgM in the lambs of all groups of ewes though specific IgG1 antibodies could be readily detected in the lambs of hyperimmunised ewes. The efficiency of transfer was related directly to the ability of the adjuvant to maximise IgG1 production in the ewe. Although immunised ewes produced high levels of IgG2, this was not transferred passively to the lamb.

Humoral immunity in the ewe. 1. The influence of adjuvancy and immunogenic substitution on immune reactivity following immunisation with protein antigens.

The nature of antigen and presence of adjuvant are major factors which influence the level of immune reactivity following immunisation. This study examines the quantity and isotype of antibody produced in ewes immunised with different proteins in combination with different adjuvants. Results using indirect ELISA assays show that animals immunised with keyhole limpet haemocyanin (KLH) in adjuvant produced lower levels (P less than 0.05) of immunoglobulin M (IgM) and had increased levels and persistence of immunoglobulin G1 (IgG1) compared with ewes immunised with antigen in saline (P less than 0.005). The anti-KLH immunoglobulin G2 (IgG2) titre was significantly higher in animals given oil-emulsion adjuvant than all other groups (P less than 0.005). Ewes were also immunised with bovine serum albumin (BSA) or BSA haptenated with trinitrophenyl (TNP) for the primary injection and carrier BSA alone for the secondary inoculation. Chemical haptenation of BSA antigen reduced primary IgM (P less than 0.005), IgG1 (P less than 0.005) and IgG2 (P less than 0.005) levels compared with animals immunised with pure BSA. There was an increased secondary anti-BSA IgM response in all animals first immunised with TNP-BSA (P less than 0.05). The class of anti-hapten antibody produced to TNP determinants was influenced by the degree of TNP haptenation of the carrier BSA. Mid-range molar ratios of TNP produced the strongest IgM, IgG1 and IgG2 anti-TNP responses compared with all other groups (P less than 0.01).

Antibody responses to bacterial antigens in the pregnant ewe.

The humoral immune response to the novel antigen Brucella abortus (S19) was examined in nonpregnant ewes and in ewes at different stages of pregnancy. Both the primary and secondary responses were monitored, by measuring the levels of total antibody, Immunoglobulin G (IgG), and complement fixing antibody. Results showed that total antibody production was significantly (p<0.01) impaired in late pregnancy and that IgG titres persisted at significantly (p<0.05) higher levels in non-pregnant animals late in the primary response and following the booster inoculation. Complement fixing antibody levels were significantly higher in non-pregnant ewes and animals inoculated post-partum. These results suggest that active immunity can be altered during pregnancy, with qualitative changes in the class and biological activity of antibody.

Suspected infectious necrotic hepatitis (black disease) in Oregon cattle.

Suspected infectious necrotic hepatitis (black disease) in a herd of 436 cattle in Douglas County, Oregon, resulted in 79 deaths during a 2-week period. Although Clostridium novyi could not be isolated from hepatic lesions, the clinical course of the disease, gross and histopathologic findings, and fluorescent antibody identification of C novyi in various tissues were suggestive of the disease. The epizootic was preceded by a long drought, during which grazing conditions were sparse. A few days before the 1st dead animal was found, the drought was relieved by about 10 cm (4 in) of rainfall, resulting in the growth of young succulent grass. The cattle, attempting to eat this new grass lying close to the ground, consumed large quantities of soil. It was speculated that the soil contained C novyi and that the proliferation of these ingested organisms in necrotic tissue cuased by Fasciola hepatica resulted in fatal toxemia.