The ontogeny of diversification at the immunoglobulin heavy chain locus in Xenopus.

Since the larval and adult antibody responses are distinct and restricted in the clawed toad Xenopus, it offers a near ideal model for studying the ontogeny of antibody repertoires and the mechanisms involved. Immunoglobulin heavy chain (IgH) cDNA clones and B cell IgH DNA clones from various larval and adult libraries have been analysed in isogenic Xenopus. Some features are similar in adults and tadpoles, while others differ and explain the particularities observed previously at the protein level. Among the similarities we found are: (i) the mode of rearrangements (there are approximately 50% abortive events in B cells from both stages), (ii) VH family usage (10 of 11 known VH families are expressed proportionally to the number of VH elements per family), and (iii) JH usage (of the eight to nine Xenopus JH elements, two are used in approximately 70% of the VH regions in both stages of development). We found that there is relatively higher membrane exon expression in tadpoles compared with adults; and that most of the differences come from the diversification of CDR3 through DH usage and N diversification. Unlike in mammals, Xenopus DH elements are used with a remarkable flexibility with inversion, fusions and usage in different reading frames, but tadpoles show a strong bias for the usage of only a few DH elements and of a preferred reading frame. There is N diversification, which further increases CDR3 heterogeneity, in adult Xenopus but virtually none in tadpoles. These observations can account for the fact that larval antibody responses are less heterogeneous than those of adults.     

Cytoplasmic pH Regulation in Acer pseudoplatanus Cells: II. Possible Mechanisms Involved in pH Regulation during Acid-Load

Qualitative and quantitative aspects of the mechanisms involved in the regulation of cytoplasmic pH during an acid-load have been studied in Acer pseudoplatanus cells. Two main processes, with about the same relative importance, account for the removal of H⁺ from the cytoplasm, namely a `metabolic consumption' of protons and the excretion of protons or proton-equivalents out of the cells. The metabolic component corresponds to a change in the equilibrium between malate synthesis and degradation leading to a 30% decrease of the malate content of the cells during the period of cytoplasmic pH regulation. Various conditions which severely inhibit the activity of the plasmalemma proton pump ATPase reduce, at most by 50%, the excretion of H⁺. This suggests that, besides the plasmalemma proton-pump, other systems are involved in the excretion of proton-equivalents. Indirect information on qualitative and quantitative features of these systems is described, which suggests the involvement of Na⁺ and HCO₃⁻ exchanges in the regulation of cytoplasmic pH of acid-loaded cells.     

Autoreactive T cells in mercury-induced autoimmunity. Ability to induce the autoimmune disease

It has been previously shown that autoreactive T cells appear during mercury-induced autoimmunity in Brown-Norway (BN) rats. In the present work, it is shown that: 1) T cells and T helper cells from HgCl2-injected BN rats are able to actively transfer autoimmunity in normal BN rats; the disease transferred is exacerbated when recipients are treated with the antisuppressor/cytotoxic T cell monoclonal antibody (OX8); 2) normal T cells preincubated with HgCl2 are also able to transfer the disease in OX8-treated but not in T cell-depleted rats; and 3) T cells from HgCl2-injected BN rats also transferred the disease in both normal and T cell depleted rats. It is concluded that: 1) autoreactive T cells, and presumably anti-Ia T cells are involved in the pathogenesis of mercury-induced autoimmunity; 2) these autoreactive T cells induce suppressor/cytotoxic T cells to proliferate in normal syngeneic recipients; the fact that this T cell subset did not proliferate in HgCl2-injected BN rats suggests that HgCl2 also affects T suppressor cells; and 3) mercury-induced autoimmunity could result from the additive effect of the emergence of autoreactive T cells and of a defect at the T suppressor level.     

Low frequency of cytotoxic T lymphocytes against the novel HLA-A*0201-restricted JC virus epitope VP1(p36) in patients with proven or possible progressive multifocal leukoencephalopathy

JC virus (JCV)-specific cytotoxic T lymphocytes (CTL) in peripheral blood are associated with a favorable outcome in patients with progressive multifocal leukoencephalopathy (PML). However, the frequency of these cells in the peripheral blood mononuclear cells (PBMC) of PML patients is unknown. To develop a highly sensitive assay for detecting the cellular immune response against this virus, we performed a CTL epitope mapping study of JCV VP1 major capsid protein by using overlapping peptides. A novel HLA-A*0201-restricted epitope, the VP1(p36) peptide SITEVECFL, was characterized. The cellular immune response against JCV was assessed in 32 study subjects. By combining the results of the (51)Cr release assay on pooled peptides and staining with the HLA-A*0201/JCV VP1(p36) tetramer, VP1-specific CTL were detected in 10 of 11 PML survivors (91%) versus only 1 of 11 PML progressors (9%, P = 0.0003). VP1-specific CTL were also detected in two of two patients recently diagnosed with PML and in four of four human immunodeficiency virus-positive patients with possible PML. The frequency of CTL specific for the novel VP1(p36) and the previously described VP1(p100) epitopes was determined. In two patients, the frequency of CTL specific for the VP1(p36) or VP1(p100) epitopes, as determined by fresh blood tetramer staining (FBTS), ranged from 1/6,000 to 1/24,000 PBMC. A CTL sorting technique combining tetramer staining and selection with immunomagnetic beads allowed the detection of epitope-specific CTL in two cases that were determined to be negative by FBTS. The phenotype of these CTL in vivo was consistent with activated memory cells. These data suggest that, although present in low numbers, JCV-specific CTL may be of central importance in the containment of JCV spread in immunosuppressed individuals.     

Blockade of vascular endothelial growth factor receptor I (VEGF-RI), but not VEGF-RII, suppresses joint destruction in the K/BxN model of rheumatoid arthritis

It was recently shown that vascular endothelial growth factor (VEGF), a growth factor for endothelial cells, plays a pivotal role in rheumatoid arthritis. VEGF binds to specific receptors, known as VEGF-RI and VEGF-RII. We assessed the physical and histological effects of selective blockade of VEGF and its receptors in transgenic K/BxN mice, a model of rheumatoid arthritis very close to the human disease. Mice were treated with anti-mouse VEGF Ab, anti-mouse VEGF-RI and -RII Abs, and an inhibitor of VEGF-RI tyrosine kinase. Disease activity was monitored using clinical indexes and by histological examination. We found that synovial cells from arthritic joints express VEGF, VEGF-RI, and VEGF-RII. Treatment with anti-VEGF-RI strongly attenuated the disease throughout the study period, while anti-VEGF only transiently delayed disease onset. Treatment with anti-VEGF-RII had no effect. Anti-VEGF-RI reduced the intensity of clinical manifestations and, based on qualitative and semiquantitative histological analyses, prevented joint damage. Treatment with a VEGF-RI tyrosine kinase inhibitor almost abolished the disease. These results show that VEGF is a key factor in pannus development, acting through the VEGF-RI pathway. The observation that in vivo administration of specific inhibitors targeting the VEGF-RI pathway suppressed arthritis and prevented bone destruction opens up new possibilities for the treatment of rheumatoid arthritis.     

CoPreTHi: a Web tool which combines transmembrane protein segment prediction methods

CoPreTHi is a Java based web application, which combines the results of methods that predict the location of transmembrane segments in protein sequences into a joint prediction histogram. Clearly, the joint prediction algorithm, produces superior quality results than individual prediction schemes. The program is available at http://o2.db.uoa.gr/CoPreTHi.     

Morphological study of five bacteriophages of yellow-pigmented enterobacteria

Two bacteriophages isolated onEnterobacter cloacae (C2, C2F) and three isolated onErwinia herbicola (E3, E16P, E16B) were purified by D₂O gradient centrifugation. Phage-containing fractions were negatively stained and examined by electron microscopy. Phages C2, C2F, E3, and E16P showed an elongated head 153×51 nm and a short noncontractile tail 12 nm long terminated by at least two short fibers. These phages correspond to the rate taxonomic group C3. Big capsomeres composing the phage head were evidenced when phage suspensions in D₂O were stained. Phage E16B showed an elongated head 97×40.5 nm, and a contractile tail 89 nm long. This phage corresponds to the extremely rate group A3.     

The antibody response during amphibian ontogeny

The maturation of the anti DNP-response in adult and larval amphibians has been compared in two species of anurans,Rana catesbeiana andXenopus laevis. IgM responses were very similar in larvae and adults of both species. Specific IgG antibody synthesis, studied inRana catesbeiana only, was not obvious in larval primary response but was easily detectable in adults. Although metamorphosis results in profound changes in the lymphoid system associated with the apparent acquisition of self tolerance to new antigens, immunological memory of antigens injected during larval life ofRana andXenopus persists after the metamorphosis period.     

Implication of neutral polysaccharides associated to alginate in inhibition of murine macrophage response to Pseudomonas aeruginosa

There is evidence that exopolysaccharides (EPS) contribute to the persistence of Pseudomonas aeruginosa in cystic fibrosis lung. However, the relationship between the chemical composition of EPS and the modulation of phagocytic cells is poorly understood. In order to evaluate the role of the chemical composition of EPS in macrophage behavior changes, we pretreated macrophages with characterized EPS and assessed P. aeruginosa phagocytosis and reactive oxygen intermediate (ROI) production. The results showed that alginate and neutral polysaccharides are involved in phagocytic impairment of P. aeruginosa. Moreover, alginates were able to prime macrophages for increased P. aeruginosa-induced macrophage oxidative burst as determined by chemiluminescence. In contrast, neutral polysaccharides are responsible for the decrease of ROI by a scavenging effect evaluated by the xanthine–xanthine oxidase system. This study showed that the content of P. aeruginosa EPS in alginate, but also in neutral polysaccharides, influences the behavior of strains towards phagocytosis and macrophage oxidative burst.     

Influence of insulin treatment and feed restriction on follicular development in cyclic gilts

Crossbred gilts were used to investigate whether exogenous insulin can restore normal follicular growth in feed-restricted gilts. After an 18-day altrenogest treatment, the first day of oestrous behaviour was designed as day 0. From day 0 to 13, all gilts received the same amount of feed, calculated to meet 200% of the energy requirements for maintenance. On day 14, luteolysis was induced by injection of an analogue of prostaglandin F2alpha. All gilts were slaughtered on day 19 and their ovaries removed. In Experiment 1, gilts received a high (240% of maintenance) or low (80%) level of feeding (n=10/group) from day 14 to 18. The number of large follicles (> or = 5 mm) on day 19 was reduced in feed-restricted gilts (16.9 versus 20.6, P<0.05). The same protocol of feed restriction was used in Experiment 2 (240% versus 80% of maintenance from day 14 to 18), and some gilts received daily injections of insulin (0.6 IU live weight kg(-1)). The three experimental groups were H: 240% and no insulin (n=8); H-I: 240%+insulin (n=8) and L-I: 80%+insulin (n=7). On day 18, 4 h after insulin injection, plasma insulin was higher in insulin-treated than in untreated gilts and glucose concentrations were reduced more dramatically in L-I than in H-I gilts (P<0.05). Concentrations of IGF-I were lower in L-I than in other gilts (P<0.05) and plasma IGFBPs were not significantly affected by treatments. On day 19, the number of large follicles (> or = 5 mm) was not significantly influenced by treatments (19.4, 17.6 and 15.3 for H, H-I and L-I gilts, respectively). Insulin, IGF-I and IGFBP-2 levels in follicular fluids from large follicles did not differ between females whereas IGFBP-3 levels were lower in L-I than in H gilts (P<0.05) and intermediate in H-I gilts. Intrafollicular levels of glucose were higher in feed-restricted than in well-fed gilts (P<0.05). These results suggest that exogenous insulin does not restore final follicular growth impaired by acute undernutrition.