Diversification of rabbit VH genes by gene-conversion-like and hypermutation mechanisms

Summary: Where, when and how does Vu diversification occur in the rabbit? Early diversification by gene-conversion and somatic hypermutation in rabbit appendix and chicken bursa of Fabricius are similar processes; the chicken bursa and the rabbit appendix have homologous functions. However, diversification in bursa starts during embryonic development whereas it starts in rabbit appendix about 2 weeks after birth in the presence of antigens and superantigens that ma) contribute to positive and negative selection, affect B-cell expansion and mold the repertoire. The biochemical steps leading to diversification by gene conversion are unknown. However elevated levels of RAD51 mRNA in both chicken bursa and young rabbit appendix suggest that repair of double stand breaks may be involved. The base changes found in expressed rabbit V_H sequences derived from rearrangement of known germline V_H genes followed by one or more gene conversions occur with frequencies similar to those found in analyses of somatic hypermutation. The Ser codons in CDR1 and CDR2 of rabbit V_Hl I genes are all AGY rather than TCN, suggesting that they may represent intrinsic hotspots for hypermutation comparable to those described in human and mouse V_Hr. Somatic hypermutation may further refine antibody affinities in rabbit germinal centers.     

Consumption of solutions containing sodium chloride is enhanced in female oxytocin-deficient mice

Intact and ovariectomized oxytocin (OT)-deficient (OT-/-) and wild-type (OT+/+) mice were tested for consumption of 0.5 M NaCl solution or tap water in a 2-bottle choice test. During 3 days of acclimation, voluntary ingestion of NaCl was equal between genotypes. After overnight fluid deprivation, intact OT-/- mice ingested 2 times more NaCl solution than OT+/+ mice in the 6th hr, but not the 1st hr, after reintroduction of fluid. Ovariectomized mice consumed less than intact mice after overnight fluid deprivation. When a 0.2 M NaCl solution was administered for 6 days in ovariectomized mice, OT-/- mice voluntarily consumed greater amounts than OT+/+ mice. After overnight fluid deprivation, consumption by OT-/- mice was 3 times that of OT+/+ mice at 1 hr and 2-fold greater after 6 hr. Enhanced intake of NaCl-containing solutions in female OT-/- mice suggests that central OT may be an important inhibitor of sodium consumption.     

Autoantibody synthesis in salivary glands of Sj?gren's syndrome patients

We investigated the possibility that autoantibodies are locally synthesized and secreted within salivary glands of patients with Sj?gren's syndrome by measuring specific autoantibody as a proportion of the total immunoglobulin present both in serum and saliva. Of 25 patients studied, 21 showed salivary enrichment of IgA anti-La, in three cases IgA anti-La being detected in saliva when IgG anti-La was negative (by ELISA) in serum. Twenty-four showed enrichment of salivary rheumatoid factors and IgA and/or IgM carrying the 17-109 idiotype, a marker of kappa IIIb light chains. These data suggest that autoantibodies, especially of the IgA class, are synthesized primarily in salivary gland and that they can be detected in saliva before they become apparent in the peripheral circulation. The subsequent deposition of these antibodies within salivary glands may be a contributory factor to the pathogenesis of Sj?gren's syndrome.     

Responses of single units in cat visual cortex to moving bars of light as a function of bar length

1. The median eye of the giant barnacle, B. nubilus, comprises four large photoreceptor neurones which are visible under the dissecting microscope for almost their entire length. We have studied the structure of, and the responses to light recorded in, the somata, axons, and terminal regions of these neurones.2. The photoreceptor somata, each 40-70 mum in diameter, extend numerous light-sensitive dendritic processes whose membranes form rhabdomeric microvilli. Recordings from the soma show that dim light evokes a steady, noisy depolarization; brighter light elicits a transient depolarization which decays to a maintained plateau, followed by a hyperpolarization when the light is turned off.3. Light-induced voltage changes spread decrementally along the photoreceptor axons, which average 10 mm in length and 25 mum in diameter. In distal parts of the axon, near the presynaptic terminals, depolarizations and hyperpolarizations can be as large as 50% or more of their values in the soma.4. There is no demonstrable electrical coupling between photoreceptor neurones as shown by simultaneous recordings from two receptor somata or axons.5. Each photoreceptor axon enters the mid line commissure of the supraoesophageal ganglion, bifurcates, and arborizes in a restricted zone of neuropil in each hemiganglion. The large size of the terminal processes of these neurones and their characteristic cytoplasmic inclusions enable one to trace them with the electron microscope as they branch in the neuropil.6. The terminal processes subdivide and end in 1-3 mum diameter branches which are the sites of apparently chemical synapses. Vesicle-containing, presynaptic loci on these processes of the receptor cell are invariably apposed to two post-synaptic processes from cells as yet unidentified.     

Economical, simple method for production of the gaseous environment required for cultivation of Campylobacter jejuni.

Campylobacter jejuni is an enteric pathogen recognized worldwide as a cause of diarrhea. Its isolation from stool samples requires a microaerophilic environment that heretofore has been expensive and cumbersome to create. An economical, portable, and simple method is described which involves the production of appropriate concentrations of oxygen and carbon dioxide. Inside a plastic bag are placed two cups, one containing fine steel wool (grade 0) previously soaked in a 2.5% aqueous solution of copper sulfate and the other containing an Alka-Seltzer tablet in tap water. As suggested by Jurgensen et al. (Rev. Bras. Pat. Clin. 18:58-63, 1982), we used the effervescent antacid to generate CO2. By plate counts, we found this method to be as reliable in the cultivation of 20 isolates of C. jejuni in pure and mixed fecal culture as the reference gas method (85% N2, 10% CO2, and 5% O2). Analyses of the gas mixture inside the bag after up to 24 h of incubation confirmed the creation of an atmosphere of reduced O2 and increased CO2 concentrations. This method is eminently suitable for field situations in which more costly supplies are not available.     

A subpopulation of small pre-B cells in rabbit bone marrow expresses x light chains and exhibits allelic exclusion of b locus allotypes

The expression of immunoglobulin heavy and light chains by pre-B cells and B lymphocytes was examined by two-color immunofluorescence in heterozygous b⁵b⁹ rabbits. Allelic exclusion of b5 and b9 x light chain allotypes was observed for both surface immunoglobulin-negative pre-B cells and surface immunoglobulin-positive B lymphocytes. In newborn bone marrow, pre-B cells and immature B lymphocytes expressing b9 were as numerous as those expressing b5. In contrast, circulating B cells and bone marrow plasma cells expressing the b5 marker outnumber b9⁺ cells by 2 to 1 in adult b⁵b⁹ animals. Whereas most B lymphocytes expressed x light chain b allotypes, approximately 80% of the μ heavy chain-positive pre-B cells did not. The pre-B cells that expressed detectable light chains were relatively small lymphocytes. A model is presented which includes a “transitional” pre-B cell that expresses both p chains and x chains.     

In vivo administration of anti-CD4 monoclonal antibody prolongs survival in longtailed macaques with experimental allergic encephalomyelitis

The in vivo administration of monoclonal antibody (mAb) to the CD4 antigen associated with helper T cells has been successful in prolonging the survival of nonhuman primates with experimental allergic encephalomyelitis (EAE). EAE was induced in 17 outbred longtailed macaques (Macaca fascicularis) by inoculation of homologous myelin basic protein (BP) in complete Freund's adjuvant (CFA). Treatment was begun at the onset of clinical signs. Eleven animals were treated with anti-CD4 mAb Leu3a (eight) or OKT4a (three). Of the six control animals, two received anti-CD8 mAb (Leu2a), and four were treated with saline. Specific T- and B-cell subsets which have been implicated in the development of EAE were monitored throughout the course of the disease by one- and two-color immunofluorescence (IF). The monkey anti-BP antibody and anti-mouse immunoglobulin (IgG) responses were measured by enzyme-linked immunoassay (ELISA) techniques, as were the levels of free-circulating murine IgG. The nature of the infiltrating lymphocytes in the brain was evaluated histologically post mortem. Our results indicate that anti-CD4 mAb can prolong survival and in some cases completely reverse the clinical appearance of the disease; however, relapses did occur. Treatments with Leu3a or OKT4a anti-CD4 mAbs reversed the ongoing depletion of CD4+ and CD8+ cells caused by the development of EAE and appeared to reduce the size and degree of inflammation in brain lesions. These treatments did not induce immunologic tolerance to mouse IgG since all of the anti-CD4-treated animals produced high titers of anti-mouse IgG antibodies. Treatment with Leu2a (anti-CD8) had no effect on the development of EAE. These results suggest that CD4+ cells are important to the pathogenesis of EAE in macaques and that manipulation of this subset with monoclonal antibodies may provide effective treatment of human demyelinating disease.     

Fos-like immunoreactivity in brain regions of domestic rams following exposure to rams or ewes

Limbic and basal forebrain-hypothalamic regions from male sheep differing in sexual performance were quantified for fos-like immunoreactivity. Rams classified as high-sexually performing (HP), low-sexually performing (LP), and male-oriented (MO) received noncontact sensory stimulation from either ewes in estrus (HP, n=5; LP, n=4; MO, n=4) or other males (HP, n=5; LP, n=4; MO, n=5) for a 4-h period on each of 3 consecutive days. Following exposure to stimulus animals on the third day, rams were euthanized and their brains were perfused with a 1% paraformaldehyde/1.5% glutaraldehyde solution and sections were analyzed for fos-like immunoreactivity. Brain regions analyzed were the medial amygdala (meAMY), medial preoptic area (mPOA), bed nucleus of the stria terminalis (BNST), and ventromedial hypothalamic nucleus (VMH). Fos-like immunoreactivity differed between groups in the mPOA and BNST but not in the meAMY or VMH. LP rams exposed to estrous ewes had more (P<.05) neurons staining positive for fos and fos-related antigens (FRA) in the mPOA and BNST than LP rams exposed to other rams or MO rams exposed to either estrous ewes or other rams. Numbers of neurons staining positive for FRA in the mPOA and BNST of LP rams exposed to estrous ewes, however, were not different (P>.05) from HP rams exposed to either estrous ewes or other rams. The similar fos-like immunoreactivity in areas important for the display of sexual behavior in HP and LP rams may reflect similar sensory input in these two groups of rams; however, LP rams, in contrast to HP rams, do not appear to respond similarly to the same sensory stimulus.     

Autoimmune murine thyroiditis. V. Genetic influence on the disease in BSVS and BRVR mice

Two inbred strains of mice, BSVS and BRVR, are similar in their antibody response to thyroglobulin but differ significantly with respect to thyroid gland damage. F₁ hybrids, like BSVS mice, respond well to thyroglobulin, exhibiting extensive thyroid lesions, whereas BRVR mice show minimal thyroid damage. H-2 typing of the two strains showed that BSVS mice have a type similar to H-2^th, with the H-2D-end of H-2^d and the H-2K-end of H-2^s, whereas BRVR mice are H-2^k. The cellular response to thyroglobulin appears to be influenced by a specific immune response gene located near the K-end of the H-2 complex.