Cellular basis for the age-associated increase in autoimmune reactions

The mechanisms that lead to the increased expression of autoantibodies with age are poorly understood. We have studied the number, size, and density of spleen and peritoneal cells from young and old BALB/c and C57BL/6 mice as well as the frequency of clonal precursors for antibodies to mouse erythrocytes, thyroglobulin, and IgG in these lymphoid preparations. Old mice have a 6-fold increase in the number of resident peritoneal cells and a 2-fold increase in the absolute number of Ly1-bearing B cells in this population. Furthermore, old mice have twice as many large, low density splenic B cells as young mice. The frequencies of B cell clonal precursors for anti-BrMRBC and anti-thyroglobulin antibody-forming cells in old mice were 3-10 times greater than in young mice. In the same cultures, however, no increase in the frequencies of B cell clonal precursors for anti-IgG or anti-DNA antibody forming cells was detected in old compared to young mice. These findings and other data suggest that there are at least two families of B cell autoantibody precursors, one including anti-BrMRBC and anti-thyroglobulin autoantibodies, the other including anti-IgG and anti-DNA antibodies. Studies of the differential regulation of these two families of autoantibody precursors might contribute to a greater understanding of autoimmune phenomena in age and disease.     

Stimulation of murine B lymphocytes to IgG synthesis and secretion by the mitogens lipopolysaccharide and lipoprotein and its inhibition by anti-immunoglobulin antibodies.

The two mitogens, lipopolysaccharide (LPS) and lipoprotein (LP), both stimulate to an equal extent murine B lymphocytes to develop IgG-synthesizing and IgG-secreting cells. IgG synthesis and secretion was detected either by biosynthetic labeling of stimulated B cells, followed by immunoprecipitation and polyacrylamide gel electrophoresis of labeled Ig, or by the protein A plaque assay detecting all IgG-secreting cells with IgG-specific developing antisera. B cells from spleen, lymph node, thoracic duct and fetal liver of normal and T cell-deficient nu/nu mice are all stimulated by the two mitogens to develop IgG-secreting cells. Mitogen-induced IgG secretion, therefore, can occur independently of T cells and of external antigen. Anti-Ig antibodies inhibit the mitogen-induced development of Ig-secreting cells. Antibodies with specificities for μ chains when added before or soon after mitogenic stimulation of small, resting B cells, will inhibit these cells from developing into IgG-secreting cells. Later in culture, antibodies with specificities for γ chains also become inhibitory for the mitogen-induced development of IgG-secreting cells. These results indicate that the precursors of the IgG-secreting cells carry IgM in the outer surface membrane. They also suggest that during mitogen-stimulated growth of B cell clones, IgG becomes expressed in the surface membrane in a functional way which allows the inhibition of mitogen-induced IgG secretion by anti-γ antibodies.     

B lymphocyte activation upon exclusive recognition of major histocompatibility antigens by T helper cells

This study has investigated whether exclusive recognition of I-A or I-E molecules on the B cell surface by T helper cells is sufficient to activate resting B cells. Lines and clones of long-term-cultured T helper cells with specificity for I-A or I-E antigens have been derived from mixed lymphocyte cultures between spleen cells from major histocompatibility complex (MHC)-congenic mouse strains. These cells were tested for helper activity and proved competent to induce resting B lymphocytes expressing the specific MHC antigens to polyclonal expansion and maturation to Ig secretion. B cell activation was shown to require direct recognition of I-A/E antigens by the helper cells on the responding B lymphocyte surface and it could not be achieved by soluble factors released by "third-party" helper cell activity ongoing in the same cultures. Since B lymphocyte activation occurs in the absence of antigen recognition by the responding B cells, these observations suggest that I-A and I-E molecules expressed on the B cell surface participate in the functional reception of T helper cell-derived induction signals.     

Preliminary observations on polar body extrusion and pronuclear formation in human oocytes using time-lapse video cinematography

In this study, we have used time-lapse video cinematography to study fertilization in 50 human oocytes that had undergone intracytoplasmic sperm injection (ICSI). Time-lapse recording commenced shortly after ICSI and proceeded for 17-20 h. Oocytes were cultured in an environmental chamber which was maintained under standard culture conditions. Overall, 38 oocytes (76%) were fertilized normally, and the fertilization rate and embryo quality were not significantly different from 487 sibling oocytes cultured in a conventional incubator. Normal fertilization followed a defined course of events, although the timing of these events varied markedly between oocytes. In 35 of the 38 fertilized oocytes (92%), there were circular waves of granulation within the ooplasm which had a periodicity of 20-53 min. The sperm head decondensed during this granulation phase. The second polar body was then extruded, and this was followed by the central formation of the male pronucleus. The female pronucleus formed in the cytoplasm adjacent to the second polar body at the same time as, or slightly after, the male pronucleus, and was subsequently drawn towards the male pronucleus until the two abutted. Both pronuclei then increased in size, the nucleoli moved around within the pronuclei and some nucleoli coalesced. During pronuclear growth, the organelles contracted from the cortex towards the centre of the oocyte, leaving a clear cortical zone. The oocyte decreased in diameter from 112 to 106 microm (P < 0.0001) during the course of the observation period. The female pronucleus was significantly smaller in diameter than the male pronucleus (24.1 and 22.4 microm respectively, P = 0.008) and contained fewer nucleoli (4.2 and 7.0 respectively, P < 0.0001). After time-lapse recording, oocytes were cultured for 48 h prior to embryo transfer or cryopreservation. Embryo quality was related to fertilization events and periodicity of the cytoplasmic wave, and it was found that good quality embryos arose from oocytes that had more uniform timing from injection to pronuclear abuttal and tended to have a longer cytoplasmic wave. In conclusion, we have shown that time-lapse video cinematography is an excellent tool for studying fertilization and early embryo development, and have demonstrated that human fertilization comprises numerous complex dynamic events.      

Five haplotypes account for fifty-five percent of ATM mutations in Brazilian patients with ataxia telangiectasia: seven new mutations

We have studied the molecular genetics of 27 Brazilian families with ataxia telangiectasia (AT). Five founder effect haplotypes accounted for 55.5% of the families. AT is an autosomal recessive disorder of childhood onset characterized by progressive cerebellar ataxia, ocular apraxia, telangiectasia, immunodeficiency, radiation sensitivity, chromosomal instability, and predisposition to cancer. The ATM gene spans more than 150 kb on chromosome region 11q23.1 and encodes a product of 3056 amino acids. The ATM protein is a member of the phosphatidylinositol 3-kinase (PI-3K) family of proteins and is involved in cell cycle control and DNA repair pathways. DNA was isolated from lymphoblastoid cell lines and haplotyped using four STR markers (D11S1818, NS22, D11S2179, D11S1819) within and flanking the ATM gene; all allele sizes were standardized in advance. In addition to the STR haplotypes, SNP haplotypes were determined using 10 critical polymorphisms. The entire gene was screened sequentially by protein truncation testing (PTT), single strand conformation polymorphism (SSCP), and then denaturing high performance liquid chromatography (dHPLC) to identify the disease-causing mutations. Of the expected 54 mutations, 50 were identified. All mutations but one, led to a truncated or null form of the ATM protein (nonsense, splice site, or frameshift). Five families (18.5%) carried a deletion of 3450nt (from IVS28 to Ex31), making this one of the two most common Brazilian mutations. Mutations were located throughout the entire gene, with no clustering or hotspots. Standardized STR haplotype analysis greatly enhanced the efficiency of mutation screening.