Treatment with low doses of polyclonal immunoglobulin improves B cell function during immune reconstitution in a murine model.

PURPOSE: [corrected] After autologous stem cell transplantation (ASCT) the immunological B cell compartment recovers slowly. Delays on the recovery of B cell function after autologous stem cell transplantation are due to the low lymphocytes count and to their intrinsic dysfunction. METHODS: We studied the in vivo B cell reconstitution after ASCT examining the independent effect of polyclonal IgG (PolyIg), Fab or Fc fragments infusions in a murine animal model during a period of 12 weeks. These molecules were used in low doses, mimicking the recommended use of IVIg in the case of hypogammaglobulinemia in humans. Flow cytometry analysis and ELISA tests were conducted to monitor the reconstitution of B cells and serum immunoglobulin production. Panama blot and PCA factor 1 analysis were used to study the kinetics of immunoglobulin repertoires reconstitution. Mechanistic studies were also performed using in vitro cell culture. RESULTS: During follow-up after ASCT, peripheral B cells expand independently of treatment, correcting the immediate increase in sBAFF (soluble B cell activating factor) induced by previous intense myeloablation. Treatments with Fab and Fc fragments infusions promote significant IgM and IgG production comparing to control. Although the complete recovery of antibody repertoire is only achieved at the end of follow-up after ASCT, there is an earlier and significantly stronger recovery in the treated mice, which is evident at 9 weeks after ASCT. At 30 weeks after ASCT, normal values of antibody repertoire were detected in all individuals. Mechanistic studies show that Fab and Fc fragments promote IgG1 production by indirect pathways. CONCLUSIONS: The results presented here demonstrate that polyclonal immunoglobulin indirectly improves the function of the reconstituted B cells and their IgG production by means of Fc-mediated effects on bystander cells. These results further stimulate the discussion about the advantages of IVIg therapy during immune reconstitution after human ASCT.

Differential expression and co-regulation of carrot AOX genes (Daucus carota).

Alternative oxidase (AOX) is a mitochondrial protein encoded by the nuclear genome. In higher plants AOX genes form a small multigene family mostly consisting of the two subfamilies AOX1 and AOX2. Daucus carota L. is characterized by a unique extension pattern of AOX genes. Different from other plant species studied so far it contains two genes in both subfamilies. Therefore, carrot was recently highlighted as an important model in AOX stress research to understand the evolutionary importance of both AOX subfamilies. Here we report on the expression patterns of DcAOX1a, DcAOX1b and DcAOX2a and DcAOX2b. Our results demonstrate that all of the four carrot AOX genes are expressed. Differential expression was observed in organs, tissues and during de novo induction of secondary root phloem explants to growth and development. DcAOX1a and DcAOX2a indicated a differential transcript accumulation but a similar co-expression pattern. The genes of each carrot AOX sub-family revealed a differential regulation and responsiveness. DcAOX2a indicated high inducibility in contrast to DcAOX2b, which generally revealed low transcript abundance and rather weak responses. In search for within-gene sequence differences between both genes as a potential reason for the differential expression patterns, the structural organization of the two genes was compared. DcAOX2a and DcAOX2b showed high sequence similarity in their open reading frames (ORFs). However, length variability was observed in the N-terminal exon1 region. The predicted cleavage site of the mitochondrial targeting sequence in this locus is untypical small for both genes and consists of 35 amino acids for DcAOX2a and of 21 amino acids for DcAOX2b. The importance of structural gene organization and the relevancy of within-gene sequence variations are discussed. Our results strengthen the value of carrot as a model plant for future studies on the importance of AOX sub family evolution.