Evolution of Antp-class genes and differential expression of Hydra Hox/paraHox genes in anterior patterning.

The conservation of developmental functions exerted by Antp-class homeoproteins in protostomes and deuterostomes suggested that homologs with related functions are present in diploblastic animals. Our phylogenetic analyses showed that Antp-class homeodomains belong either to non-Hox or to Hox/paraHox families. Among the 13 non-Hox families, 9 have diploblastic homologs, Msx, Emx, Barx, Evx, Tlx, NK-2, and Prh/Hex, Not, and Dlx, reported here. Among the Hox/paraHox, poriferan sequences were not found, and the cnidarian sequences formed at least five distinct cnox families. Two are significantly related to the paraHox Gsx (cnox-2) and the mox (cnox-5) sequences, whereas three display some relatedness to the Hox paralog groups 1 (cnox-1), 9/10 (cnox-3) and the paraHox cdx (cnox-4). Intermediate Hox/paraHox genes (PG 3 to 8 and lox) did not have clear cnidarian counterparts. In Hydra, cnox-1, cnox-2, and cnox-3 were not found chromosomally linked within a 150-kb range and displayed specific expression patterns in the adult head. During regeneration, cnox-1 was expressed as an early gene whatever the polarity, whereas cnox-2 was up-regulated later during head but not foot regeneration. Finally, cnox-3 expression was reestablished in the adult head once it was fully formed. These results suggest that the Hydra genes related to anterior Hox/paraHox genes are involved at different stages of apical differentiation. However, the positional information defining the oral/aboral axis in Hydra cannot be correlated strictly to that characterizing the anterior-posterior axis in vertebrates or arthropods.

prdl-a, a gene marker for hydra apical differentiation related to triploblastic paired-like head-specific genes.

Two homeobox genes, prdl-a and prdl-b, which were isolated from a Hydra vulgaris cDNA library, encode paired-like class homeodomains highly related to that of the aristaless-related genes. In adult polyps, prdl-b is a marker for synchronously dividing nematoblasts while prdl-a displays an expression restricted to the the nerve cell lineage of the head region. During budding and apical regeneration, an early and transient prdl-a expression was observed in endodermal cells of the stump at a time when the head organizer is established. When apical regeneration was delayed upon concomittant budding, prdl-a expression was found to be altered in the stump. Furthermore, a specific anti-prdl-a protein immunoserum revealed that prdl-a was overexpressed in adult polyps of the Chlorohydra viridissima multiheaded mutant, with an expression domain extending below the tentacle ring towards the body column. Accordingly, prdl-a DNA-binding activity was enhanced in nuclear extracts from this mutant. These results suggest that prdl-a responds to apical forming signals and might thus be involved in apical specification. When a marine hydrozoan (Podocorynae carnea) was used, the anti-prdl-a antibody showed cross-reactivity with cells located around the oral region, indicating that prdl-a function is shared by other cnidaria. The ancestral role for prdl-a-related genes in the molecular definition of the head (or oral-surrounding region) is discussed.

Cytokine gene expression in atopics: effect of IL-4 on IL-1 beta and IL-6 mRNA levels.

Mononuclear cells from atopic blood donors showed increased IL-3 steady state mRNA levels. This finding complemented our earlier observations that cells from atopics also showed increased IL-4 but decreased IFN-gamma, IL-1 beta and IL-6 mRNA levels. Therefore, we investigated the effect of human recombinant IL-4 on cytokines mRNA levels in mononuclear cells from normals and atopics. In the presence of IL-4 steady state levels of IL-1 beta and IL-6 mRNA were decreased even if cells were co-stimulated with polyclonal activators such as PMA, PWM or PHA. No influence of IL-4 on granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-3 or IFN-gamma mRNA levels was observed with the exception of a decreased IFN-gamma mRNA level in PWM stimulated cells.

Lymphokine gene expression related to CD4 T cell subset (CD45R/CDw29) phenotype conversion.

In this study, we investigated whether the phenotype-related differentiation of human 2H4+ (CD45R) naive cells to 2H4- (CDw29) memory CD4 cells corresponded to modulation of interleukin (IL) 2, IL 4, interferon (IFN)-gamma and granulocyte-monocyte colony-stimulating factor (GM-CSF) gene expression, a phenomenon which might correlate to the distinct functional activities of naive cells or memory cells. To mimic in vitro CD4 T cell subset differentiation, freshly isolated 2H4+ and 2H4- CD4 cells were stimulated with the anti-CD3 antibody (Leu-4), expanded in IL 2-containing medium and restimulated with Leu-4 after 7 and 13 days. Absence of monocyte-T cell interaction was compensated by adding monocyte supernatant to the culture medium and by cross-linking the anti-CD3 antibodies with goat anti-mouse antibody coated on culture dishes. It has been previously shown that in vitro stimulated 2H4+ cells acquire CDw29 surface antigens. Measurement of lymphokine gene expression by dot-blot hybridization revealed that although stimulated 2H4+ cells proliferated less than stimulated 2H4- cells, and expressed less actin mRNA, they expressed more IL 2 but less IL 4 and GM-CSF than 2H4- cells. No significant difference was observed between the two subsets for the expression of IFN-gamma. If subsets were restimulated with Leu-4 antibodies, expression of IL 2 was decreased and expression of IL 4 was increased in both subsets; however, the differences among the subsets persisted. They were even more enhanced for IL 2 but less pronounced for GM-CSF. Thus, in spite of phenotype conversion, CD4 T cell subsets maintained a distinct capacity to express IL 2 and IL 4 genes.

Cytokine mRNA levels in antigen-stimulated peripheral blood mononuclear cells.

Levels of cytokine mRNA coding for granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-gamma (IFN-gamma), interleukin (IL) 2, IL 1 beta, IL 4 or IL 6 have been measured by Northern blot analysis after antigen stimulation. As source for RNA we used peripheral blood mononuclear cells (PBMC) from donors which showed a proliferative response after tetanus toxoid or Candida albicans stimulation. For comparison PBMC were also stimulated with lectins and anti-CD3 antibody. With some variations among donors, antigens clearly induced measurable levels of IFN-gamma, GM-CSF and IL 2 mRNA. Increased levels for IL 6 were also detected after antigen stimulation. In contrast to polyclonal T cell stimuli, antigens showed delayed kinetics of mRNA steady-state levels and resembled in this respect more closely the stimulation with pokeweed mitogen. Thus, cytokine mRNA levels may be assessed in unfractionated PBMC after antigen stimulation. The two tested antigens also clearly show a cytokine pattern distinct from that induced in polyclonal stimulations such as anti-CD3.

Human T hybridoma-derived immunoglobulin-binding factors.

A human T hybridoma was generated lacking demonstrable low-affinity IgE surface receptors. This cell line produces spontaneously immunoglobulin-binding factors. The factors have an affinity for human IgE and IgG as well as for IgG from other species. The binding factors were purified by concanavalin A affinity chromatography, an IgE immunoaffinity column and SDS-PAGE. This purification procedure resulted in five major proteins bands at 13, 15, 30, 60 and less than 150 kilodaltons. As judged by Western blot analysis, all bands were variably glycosylated and were capable of binding IgE. Biological activity resided even within the small molecular weight (13 kilodaltons) peptide in terms of inhibiting IgE synthesis of the U266 B cell line and net IgE synthesis of B cells from atopic blood donors. Additionally, semipurified binding factor preparations also inhibited the Pokeweed mitogen-induced IgG synthesis while stimulating IgE synthesis at the same time. At present it is not clear whether the different molecular entities are dimers or polymers of the same protein, or are all derived from one larger protein (proteolytically cleaved), or whether they are completely different peptides mediating similar immunoglobulin-binding capacities as well as similar biological activities. Glycosylation of binding factors is not responsible for binding to immunoglobulins, but might be important for its biological activity.