Reconciling repertoire shift with affinity maturation: the role of deleterious mutations.
(33/8706)
The shift in Ab repertoire, from Abs dominating certain primary B cell responses to genetically unrelated Abs dominating subsequent "memory" responses, challenges the accepted paradigm of affinity maturation. We used mathematical modeling and computer simulations of the dynamics of B cell responses, hypermutation, selection, and memory cell formation to test hypotheses attempting to explain repertoire shift. We show that repertoire shift can be explained within the framework of the affinity maturation paradigm, only when we recognize the destructive nature of hypermutation: B cells with a high initial affinity for the Ag are less likely to improve through random mutations. (+info)
Differential expression and activation of p38 mitogen-activated protein kinase alpha, beta, gamma, and delta in inflammatory cell lineages.
(34/8706)
Four p38 mitogen-activated protein kinases (p38alpha, beta, gamma, delta) have been described. To understand the role of p38 family members in inflammation, we determined their relative expression in cells that participate in the inflammatory process. Expression was measured at the level of mRNA by reverse-transcriptase PCR and protein by Western blot analysis. p38alpha was the dominant form of p38 in monocytes; expression of p38delta was low and p38beta was undetected. In macrophages, p38alpha and p38delta were abundant, but p38beta was undetected. p38alpha and p38delta were also expressed by neutrophils, CD4+ T cells, and endothelial cells. Again, p38beta was not detected in neutrophils, although low amounts were present in CD4+ T cells. In contrast, p38beta was abundant in endothelial cells. p38gamma protein was not detected in any cell type, although p38gamma mRNA was present in endothelial cells. Immunokinase assays showed a strong activation of p38alpha and a lesser activation of p38delta in LPS-stimulated macrophages. Abs specific for mono- and dual-phophorylated forms of p38 suggested that LPS induces dual phosphorylation of p38alpha, but primarily mono-phosphorylation of p38delta. IL-1beta activated p38alpha and p38beta in endothelial cells. However, p38alpha was the more activated form based on kinase assays and phosphorylation analysis. Expression and activation patterns of p38alpha in macrophages and endothelial cells suggest that p38alpha plays a major role in the inflammatory response. Additional studies will be needed to define the contribution of p38delta to macrophage, neutrophil, and T cell functions, and of p38beta to signaling in endothelial cells and T cells. (+info)
Positive selection as a developmental progression initiated by alpha beta TCR signals that fix TCR specificity prior to lineage commitment.
(35/8706)
During positive selection, immature thymocytes commit to either the CD4+ or CD8+ T cell lineage ("commitment") and convert from short-lived thymocytes into long-lived T cells ("rescue"). By formal precursor-progeny analysis, we now identify what is likely to be the initial positive selection step signaled by alpha beta TCR, which we have termed "induction". During induction, RAG mRNA expression is downregulated, but lineage commitment does not occur. Rather, lineage commitment (which depends upon the MHC class specificity of the alpha beta TCR) only occurs after downregulation of RAG expression and the consequent fixation of alpha beta TCR specificity. We propose that positive selection can be viewed as a sequence of increasingly selective developmental steps (induction-->commitment-->rescue) that are signaled by alpha beta TCR engagements of intrathymic ligands. (+info)
Prospero distinguishes sibling cell fate without asymmetric localization in the Drosophila adult external sense organ lineage.
(36/8706)
The adult external sense organ precursor (SOP) lineage is a model system for studying asymmetric cell division. Adult SOPs divide asymmetrically to produce IIa and IIb daughter cells; IIa generates the external socket (tormogen) and hair (trichogen) cells, while IIb generates the internal neuron and sheath (thecogen) cells. Here we investigate the expression and function of prospero in the adult SOP lineage. Although Prospero is asymmetrically localized in embryonic SOP lineage, this is not observed in the adult SOP lineage: Prospero is first detected in the IIb nucleus and, during IIb division, it is cytoplasmic and inherited by both neuron and sheath cells. Subsequently, Prospero is downregulated in the neuron but maintained in the sheath cell. Loss of prospero function leads to 'double bristle' sense organs (reflecting a IIb-to-IIa transformation) or 'single bristle' sense organs with abnormal neuronal differentiation (reflecting defective IIb development). Conversely, ectopic prospero expression results in duplicate neurons and sheath cells and a complete absence of hair/socket cells (reflecting a IIa-to-IIb transformation). We conclude that (1) despite the absence of asymmetric protein localization, prospero expression is restricted to the IIb cell but not its IIa sibling, (2) prospero promotes IIb cell fate and inhibits IIa cell fate, and (3) prospero is required for proper axon and dendrite morphology of the neuron derived from the IIb cell. Thus, prospero plays a fundamental role in establishing binary IIa/IIb sibling cell fates without being asymmetrically localized during SOP division. Finally, in contrast to previous studies, we find that the IIb cell divides prior to the IIa cell in the SOP lineage. (+info)
Sibling cell fate in the Drosophila adult external sense organ lineage is specified by prospero function, which is regulated by Numb and Notch.
(37/8706)
Specification of cell fate in the adult sensory organs is known to be dependent on intrinsic and extrinsic signals. We show that the homeodomain transcription factor Prospero (Pros) acts as an intrinsic signal for the specification of cell fates within the mechanosensory lineage. The sensory organ precursors divide to give rise to two secondary progenitors - PIIa and PIIb. Pros is expressed in PIIb, which gives rise to the neuron and thecogen cells. Loss of Pros function affects the identity of PIIb and neurons fail to differentiate. Pros misexpression is sufficient for the transformation of PIIa to PIIb fate. The expression of Pros in the normal PIIb cell appears to be regulated by Notch signaling. (+info)
Apoptosis of erythroid precursors under stimulation with thrombopoietin: contribution to megakaryocytic lineage choice.
(38/8706)
Although the effect of thrombopoietin (TPO) on megakaryocyte production is well established, its role in the commitment of multipotential hematopoietic progenitors to the megakaryocytic lineage remains to be determined. In the present study, we attempted to clarify the determination process of megakaryocytic lineage as a terminal differentiation pathway under stimulation with TPO. Day 7 cultured cells grown by TPO derived from cord blood CD34+ cells were divided into four subpopulations on the basis of CD34 and CD41 expression. The CD34-/CD41- cells showed the labeling pattern of anti-CD42b and anti-CD9 antibodies closer to that of the CD34+/CD41- cells than the CD34+/CD41+ cells. Replating experiments revealed that approximately 40% of the CD34-/CD41- cells proliferated in response to a combination of growth factors, and more than 80% of them were pure erythroid precursors. However, this subpopulation failed to grow/survive and fell into apoptosis in the presence of TPO alone. In contrast, the CD34+/CD41+ cells, which predominantly contained megakaryocytic precursors, exerted a low but significant proliferative potential in the presence of TPO. The insufficient response to TPO of the CD34-/CD41- cells may result from the apparently low expression of c-MpI, as determined by flow cytometric analysis and reverse transcription-polymerase chain reaction analysis. Therefore, these results suggest that the apoptosis of hematopoietic precursors other than megakaryocytic precursors is related to the determination of the terminal differentiation under the influence of TPO. (+info)
Preliminary analysis of azoxymethane-induced colon tumorigenesis in mouse aggregation chimeras.
(39/8706)
Inbred mice exhibit differential susceptibility to colon carcinogens. The following study addresses the possibility that differences are intrinsic to colonic mucosa (cell autonomous) or are mediated by extracolonic systemic factors (e.g. liver activation of carcinogens). Our approach was to construct mouse aggregation chimeras, mice whose tissues are a mosaic of cells derived from two parental genotypes, from a susceptible (SWR) and a resistant (DBA/2) strain. Forty-five embryo aggregations yielded 11 viable pups, four of which were chimeric by coat color. Six-week-old SWR<-->BA/2 chimeras were injected i.p. with azoxymethane (AOM) once a week for 8 weeks (5 and 7.5 mg/kg body wt for 2 weeks followed by 10 mg/kg for 6 weeks) and tumor incidence in distal colon was evaluated 15 weeks after the last injection. Additional groups of parental mice received the same treatment. In the parental SWR treatment group, 1.7 +/- 0.82 tumors/colon were found. No tumors were observed in AOM-treated DBA/2 mice. In SWR<-->DBA/2 chimeras exposed to AOM, 2.8 +/- 2.1 tumors/colon were found. Tumor lineage was examined in paraffin sections stained with Dolichos biflorus agglutinin-peroxidase, a cell surface specific marker that stains intestinal endothelial cells of SWR and epithelial cells of DBA/2. Cellular lineage of tumors was further evaluated by microsatellite analysis of DNA isolated by microdissection. There was no significant difference in tumor incidence between SWR parental and chimera treatment groups. Histochemical analysis of tumor tissue in chimeras suggested that most tumors were derived from SWR. However, subsequent genetic analysis of tumors indicated mixed parental composition. These preliminary studies suggest that DBA/2 resistance mechanisms are not sufficient to protect adjacent SWR-derived epithelium from the tumorigenic effects of AOM. (+info)
The spatial relationship between stem cells and their progeny in the basal layer of human epidermis: a new view based on whole-mount labelling and lineage analysis.
(40/8706)
In order to examine the spatial organisation of stem cells and their progeny in human epidermis, we developed a method for whole-mount epidermal immunofluorescence labelling using high surface beta1 integrin expression as a stem cell marker. We confirmed that there are clusters of high beta1 integrin-expressing cells at the tips of the dermal papillae in epidermis from several body sites, whereas alpha6 integrin expression is more uniform. The majority of actively cycling cells detected by Ki67 or bromodeoxyuridine labelling were found in the beta1 integrin-dull, transit amplifying population and integrin-negative, keratin 10-positive cells left the basal layer exclusively from this compartment. When we examined p53-positive clones in sun-exposed epidermis, we found two types of clone that differed in size and position in a way that was consistent with the founder cell being a stem or transit amplifying cell. The patterning of the basal layer implies that transit amplifying cells migrate over the basement membrane away from the stem cell clusters. In support of this, isolated beta1 integrin-dull keratinocytes were more motile on type IV collagen than beta1 integrin-bright keratinocytes and EGFP-labelled stem cell clones in confluent cultured sheets were compact, whereas transit amplifying clones were dispersed. The combination of whole-mount labelling and lineage marking thus reveals features of epidermal organisation that were previously unrecognised. (+info)