Precocious expression of T cell functional response genes in vivo in primitive thymocytes before T lineage commitment

The genes encoding effector molecules of mature T cells, IL-2, perforin and IL-4, were found to be expressed in vivo in the most primitive subsets of thymocytes of adult mice. These subsets have previously been identified by their cell surface markers and by their expression of other T lineage-associated genes. While IL-2, perforin and IL-4 are expressed in distinct patterns, all three are expressed before the induction of RAG-1 and pre-TCR alpha mRNA expression, and are confined to subsets of cells that apparently have not yet undergone commitment to the T lineage. Thus, expression of T cell response genes appears to be one of the earliest markers of lymphocyte differentiation. Activation events marked by CD69 induction occur in these early cell types, but the response gene expression by these cells is separable from CD69 expression. IL-2 and perforin are induced again much later in thymocyte development, during TCR-dependent repertoire selection. At those stages, IL-2 protein and RNA levels per cell are higher, but the fraction of cells expressing IL-2 appears to be much lower than in the most immature stages. In addition, a striking feature of the immature populations is the robust IL-2 expression by presumptive immature NK cells. These findings are discussed in terms of the developmental origins of lineage specificity in T cell response gene regulation.      

Three contiguous lipoprotein genes in Pasteurella haemolytica A1 which are homologous to a lipoprotein gene in Haemophilus influenzae type b.

An Escherichia coli clone carrying the recombinant plasmid pPH24 has been found to express highly immunoreactive antigens of Pasteurella haemolytica A1. Two or three antigens of approximately 30 kDa were located to both the inner and outer membranes of the E. coli clone and in P. haemolytica A1. From the insert DNA of 8.2 kbp on pPH24, a fragment of 4.6 kbp was found to code for these antigens. Nucleotide sequence analysis of the 4.6-kbp DNA identified three genes (designated plpA, -B, and -C) arranged in tandem which code for three proteins, Plp1, -2, and -3, with predicted molecular masses of 30.1, 30.3, and 29.0 kDa, respectively. Comparison of the nucleic acid sequence of plpA, -B, and -C with GenBank sequences showed extensive homology with a Haemophilus influenzae 28-kDa lipoprotein gene. [14C]palmitate labelling coupled with glybomycin inhibition experiments showed that Plp1, -2, and -3 are also lipoproteins. In addition, plpA, -B, and -C were found to be present only in A biotypes of P. haemolytica by Southern blot analysis. Since Plp1, -2, and -3 were found to be antigenic components in a culture supernatant vaccine, they could be candidates for further investigation as vaccine components.     

A role for G-CSF receptor signaling in the regulation of hematopoietic cell function but not lineage commitment or differentiation.

To investigate the specificity of cytokine signals in hematopoietic differentiation, we generated mice with a targeted mutation of their G-CSF receptor (G-CSFR) such that the cytoplasmic (signaling) domain of the G-CSFR is replaced with the cytoplasmic domain of the erythropoietin receptor. In homozygous mutant mice, expression of this chimeric receptor had no apparent affect on lineage commitment and was able to support the production of morphologically mature neutrophils. However, mutant neutrophils displayed reduced chemotaxis, and G-CSF-stimulated mobilization of neutrophils and hematopoietic progenitors from the bone marrow to blood was markedly impaired. Thus, the G-CSFR is generating unique signals that are required for certain specialized hematopoietic cell functions but are not required for granulocytic differentiation or lineage commitment.     

Hyperplastic polyps: a cell lineage which both synthesizes and secretes trefoil-peptides and has phenotypic similarity with the ulcer-associated cell lineage.

Hyperplastic polyps are common benign lesions of uncertain histogenesis, which occur in the colon in populations at risk for colorectal carcinoma. They contain neutral/MUC1 gene-related mucin which in turn is closely associated with the trefoil-peptide pS2, a major component of the ulcer-associated cell lineage, previously termed pseudopyloric metaplasia. We have examined 17 hyperplastic polyps for expression of the trefoil-peptides pS2 and human spasmolytic polypeptide by in situ hybridization and immunohistochemistry, as well as by using antisera to epidermal growth factor/urogastrone and its receptor and to epitopes of the product of the MUC1 gene to characterize any further similarity between these lesions and the ulcer-associated cell lineage and thus help elucidate the nature of the lesions. Our investigations show both human spasmolytic polypeptide and pS2 messenger RNA within the polyps, whereas only pS2 peptide could be demonstrated immunohistochemically. Epidermal growth factor/urogastrone, its receptor, and antisera to the MUC1 gene also showed widespread staining of these polyps. We suggest that hyperplastic polyps are formed of a lineage that both synthesizes and secretes trefoil-peptides and the MUC1 mucin and that hyperplastic polyps may be related to the phenotypically similar ulceration-associated cell lineage.     

Signals involved in CD4/CD8 lineage commitment: current concepts and potential mechanisms.

A competent immune system requires that mature T cells express TCR and CD4/CD8 coreceptors with matching MHC specificities. Such matching of TCR and coreceptor specificity is induced in the thymus at the CD4(+)8(+)stage of development and is referred to as lineage commitment. The process by which immature CD4(+)8(+)thymocytes are signaled to undergo lineage commitment continues to be the subject of intense investigation and discussion. Here, we review the major models by which lineage commitment is thought to occur and discuss the experimental results on which they were based.     

15q11-13 GABAA receptor genes are normally biallelically expressed in brain yet are subject to epigenetic dysregulation in autism-spectrum disorders

Human chromosome 15q11-13 is a complex locus containing imprinted genes as well as a cluster of three GABA(A) receptor subunit (GABR) genes-GABRB3, GABRA5 and GABRG3. Deletion or duplication of 15q11-13 GABR genes occurs in multiple human neurodevelopmental disorders including Prader-Willi syndrome (PWS), Angelman syndrome (AS) and autism. GABRB3 protein expression is also reduced in Rett syndrome (RTT), caused by mutations in MECP2 on Xq28. Although Gabrb3 is biallelically expressed in mouse brain, conflicting data exist regarding the imprinting status of the 15q11-13 GABR genes in humans. Using coding single nucleotide polymorphisms we show that all three GABR genes are biallelically expressed in 21 control brain samples, demonstrating that these genes are not imprinted in normal human cortex. Interestingly, four of eight autism and one of five RTT brain samples showed monoallelic or highly skewed allelic expression of one or more GABR gene, suggesting that epigenetic dysregulation of these genes is common to both disorders. Quantitative real-time RT-PCR analysis of PWS and AS samples with paternal and maternal 15q11-13 deletions revealed a paternal expression bias of GABRB3, while RTT brain samples showed a significant reduction in GABRB3 and UBE3A. Chromatin immunoprecipitation and bisulfite sequencing in SH-SY5Y neuroblastoma cells demonstrated that MeCP2 binds to methylated CpG sites within GABRB3. Our previous studies demonstrated that homologous 15q11-13 pairing in neurons was dependent on MeCP2 and was disrupted in RTT and autism cortex. Combined, these results suggest that MeCP2 acts as a chromatin organizer for optimal expression of both alleles of GABRB3 in neurons.     

An allele-specific,stochastic gene expression process controls the expression of multiple Ly49family genes and generates a diverse,MHC-specific NK cell receptor repertoire

Mouse NK cells express MHC class I-specific inhibitory Ly49 receptors. Since these receptors display distinct ligand specificities and are clonally distributed, their expression generates a diverse NK cell receptor repertoire specific for MHC class I molecules. We have previously found that the D ^d (or D^k )-specific Ly49A receptor is usually expressed from a single allele. However, a small fraction of short-term NK cell clones expressed both Ly49A alleles, suggesting that the two Ly49A alleles are independently and randomly expressed. Here we show that the genes for two additional Ly49 receptors (Ly49C and Ly49G2) are also expressed in a (predominantly) mono-allelic fashion. Since single NK cells can co-express multiple Ly49 receptors, we also investigated whether mono-allelic expression from within the tightly linked Ly49 gene cluster is coordinate or independent. Our clonal analysis suggests that the expression of alleles of distinct Ly49 genes is not coordinate. Thus Ly49 alleles are apparently independently and randomly chosen for stable expression, a process that directly restricts the number of Ly49 receptors expressed per single NK cell. We propose that the Ly49 receptor repertoire specific for MHC class I is generated by an allele-specific, stochastic gene expression process that acts on the entire Ly49 gene cluster.     

Changes in gene expression in the lungs of Mg-deficient mice are related to an inflammatory process.

It has been well documented that experimental hypomagnesemia in rodents evokes, as an early consequence, an inflammatory response. This also leads to the activation of cells producing reactive species of oxygen and, as a result, to the oxidative damage of tissues. Several studies have shown that lungs might be a specific target of Mg deficiency. Here, we report that 3 weeks of Mg deficiency in mice resulted in inflammatory processes in the lungs, including interstitial and perivascular pneumonia, manifested by the infiltration of leukocytes, plasmocytes and histiocytes, as well as the phenomenon of disseminated intravascular coagulation (DIC). These phenomena were accompanied by changes in gene expression assessed by cDNA array. In this study we identified 26 genes significantly changed by Mg deficiency, mostly involved in the anti-oxidative response, regulation of cell cycle and growth, apoptosis as well as cell-cell and cell-matrix interactions. We conclude that these changes are related to the phenomena of inflammatory and oxidative processes and consecutive remodeling occurring in the tissues as a result of Mg deficiency. This may have implications for at least several lung pathologies, including allergies, asthma, SIDS (Sudden Infant Death Syndrome) or facilitate formation of lung metastases.     

IL-17RA-signaling in Lgr5+ intestinal stem cells induces expression of transcription factor ATOH1 to promote secretory cell lineage commitment

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Objectivity in objective structured clinical examinations: checklists are no substitute for examiner commitment.

PURPOSE: This study explored factors that contribute to objectivity in objective structured clinical examinations (OSCEs). The authors quantified the effect of examiners on interrater reliability and separated this effect from that of station construction, determined the effect of objectification on station reliability and validity, and explored examiner factors that may contribute to interrater reliability. METHOD: Data came from examiners' mark sheets from four annual OSCEs (1997-2000). The OSCEs were conducted identically and simultaneously at three sites, within the University of Otago medical school in New Zealand, with two examiners at each station. The contribution to interrater correlations of station construction and mark sheet compared with examiners' contribution was partitioned out using a random-effects analysis of variance. For one OSCE, a multiple linear regression was used to determine the independent contributions to interrater reliability of the number of checklist items per mark sheet, examiner experience, and examiner involvement in station construction. RESULTS: Station construction and mark sheets contributed 10.1% and examiners contributed 89.9% to the variation in interrater reliability. Following multivariate analysis, the number of items per mark sheet was negatively associated, and examiner involvement in station construction was positively associated, with interrater reliability. Examiner experience in examining or in clinical medicine was not associated with interrater reliability. There was a negative, but nonsignificant, correlation between number of items per mark sheet and that station's correlation with the aggregate OSCE mark. CONCLUSIONS: The contribution of objective mark sheets to objectivity is relatively minor compared with examiners' contribution. Increasing the number of checklist items per mark sheet decreased both reliability and validity. Achieving objectivity requires diligent examiners who are involved in the whole assessment.     

Decidual macrophages are the population of decidual adherent cells which regulates perforin expression in cytolytic cells.

PROBLEM: We have shown that addition of decidual adherent cells (DAC) to the culture of decidual lymphocytes (DL) prevents the downregulation of perforin expression in these cells. Because DAC are a mixture of various cell populations, the aim is to analyze immunophenotypic characteristics of DAC and to determine which cell population is involved in the regulation of perforin expression. METHOD OF STUDY: First trimester pregnancy decidual cells were obtained by enzymatic tissue digestion. Decidual cells and peripheral blood lymphocytes (PBL) were centrifuged on Ficoll-Hypaque density gradient and cultured overnight to obtain adherent cells, which were analyzed by flow cytometry and immunocytochemically. RESULTS: Almost all peripheral blood adherent cells (PBAC) (ca 90%) expressed monocyte/macrophage markers but only 10-20% of DAC. The rest of DAC expressed markers of stromal cells. HLA-DR depleted population of DAC (stromal cells only) could not prevent downregulation of perforin expression in cultured DL and PBL. CONCLUSION: Decidual macrophages are involved in the regulation of perforin expression in DL.     

Expression of the endocannabinoid system in the bi-potential HEL cell line: commitment to the megakaryoblastic lineage by 2-arachidonoylglycerol

The role of the endocannabinoid system in haematopoietic cells is not completely understood. We investigated whether human erythroleukemia (HEL) cells were able to bind, metabolise and transport the main endocannabinoids, anandamide (AEA) and 2-arachidonoylglycerol (2-AG). We also investigated whether AEA or 2-AG could modulate HEL differentiation. Although able to internalise both endocannabinoids, HEL cells had the machinery to metabolise 2-AG only, since they were devoid of the enzymes needed to synthesise and degrade AEA. Nonetheless, the intracellular transport of exogenous AEA might be required to activate the vanilloid receptors, with yet unknown implications for vascular biology. On the contrary, 2-AG appeared to play a role in lineage determination. Indeed, 2-AG itself drove HEL cells towards megakaryocytic differentiation, as it enhanced expression of beta3 integrin subunit, a megakaryocyte/platelet surface antigen, and glycoprotein VI, a late marker of megakaryocytes; in parallel, it reduced the amount of messenger RNA encoding for glycophorin A, a marker of erythroid phenotype. All these effects were mediated by activation of CB(2) cannabinoid receptors that triggered an extracellular signal-regulated kinase-dependent signalling cascade. In addition, classical inducers of megakaryocyte differentiation reduced 2-AG synthesis (although they did not affect the binding efficiency of CB(2) receptors), suggesting that levels of this endocannabinoid may be critical for committing HEL cells towards the megakaryocytic lineage.     

Tenascin expression patterns and cells of monocyte lineage: relationship in human gliomas.

Stromal extracellular matrix (ECM) components are thought to play an important role in regulating invasion of human gliomas. Macrophages and microglial cells may heavily influence the integrity of the extracellular compartment of gliomas, and the affected ECM may play a key role in regulating migratory activity of both tumor cells and macrophages/microglia. The aim of this investigation was to study immunohistochemically the expression patterns of four ECM components: fibronectin, laminin, collagen IV, and tenascin (TN) in human gliomas, with special attention to TN. Our main goal was to study the possible correlation between TN expression and macrophagic/microglial infiltration in gliomas. Altogether, 90 gliomas were studied. Tumors included 46 glioblastomas, 19 anaplastic gliomas, 22 low grade gliomas, and 3 pilocytic astrocytomas. Vascular TN prevailed in perinecrotic areas of glioblastomas, whereas interstitial TN was more often expressed distant from necrosis and in the ECM of anaplastic and low grade gliomas. Double staining with CD68 and anti-TN antibodies showed that macrophagic/microglial density was significantly higher in TN-positive areas of most of the glioblastomas and anaplastic gliomas, whereas microglial percentage from total number of CD68-positive cells was in most of the cases significantly higher in TN-negative areas. In addition, we saw a morphologically spatial correlation between higher densities of macrophagic/microglial infiltration and TN expression in perinecrotic areas in glioblastomas. Attachment of macrophages to TN-positive basement membrane zones of newly formed stromal blood vessels was evident. On the basis of our results, we conclude that TN may play a crucial role in regulating trafficking of cells of monocyte lineage in human gliomas.     

The gamma-hemolysin locus of Staphylococcus aureus comprises three linked genes, two of which are identical to the genes for the F and S components of leukocidin.

The Staphylococcus aureus gamma-hemolysin comprises two polypeptides, whereas the gamma-hemolysin locus (hlg) contains three open reading frames. The hlgA and hlgB genes encode the gamma 1 and gamma 2 components, respectively. The HlgB protein (gamma 2) has 27% residue identity with S. aureus alpha-toxin. Surprisingly, hlgB and hlgC are 98.5 and 99.1% identical to the lukF and lukS genes, respectively, encoding the F and S components of the Panton-Valentine leukocidin.     

Cardiac neural crest in zebrafish embryos contributes to myocardial cell lineage and early heart function.

Myocardial dysfunction is evident within hours after ablation of the cardiac neural crest in chick embryos, suggesting a role for neural crest in myocardial maturation that is separate from its role in outflow septation. This role could be conserved in an animal that does not have a divided systemic and pulmonary circulation, such as zebrafish. To test this hypothesis, we used cell marking to identify the axial level of neural crest that migrates to the heart in zebrafish embryos. Unlike the chick and mouse, the zebrafish cardiac neural crest does not originate from the axial level of the somites. The region of neural crest cranial to somite 1 was found to contribute cells to the heart. Cells from the cardiac neural crest migrated to the myocardial wall of the heart tube, where some of them expressed a myocardial phenotype. Laser ablation of the cardiac premigratory neural crest at the three- to four-somite stage resulted in loss of the neural crest cells migrating to the heart as shown by the absence of AP2- and HNK1-expressing cells and failure of the heart tube to undergo looping. Myocardial function was assessed 24 hr after the cardiac neural crest ablation in a subpopulation of embryos with normal heart rate. Decreased stroke volume, ejection fraction, and cardiac output were observed, indicating a more severe functional deficit in cardiac neural crest-ablated zebrafish embryos compared with neural crest-ablated chick embryos. These results suggest a new role for cardiac neural crest cells in vertebrate cardiac development and are the first report of a myocardial cell lineage for neural crest derivatives.     

Trypanosoma cruzi carrying a monoallelic deletion of the calreticulin (TcCRT) gene are susceptible to complement mediated killing and defective in their metacyclogenesis

Trypanosoma cruzi calreticulin (TcCRT) can hijack complement C1, mannan-binding lectin and ficolins from serum thus inhibiting the classical and lectin complement pathway activation respectively. To understand the in vivo biological functions of TcCRT in T. cruzi we generated a clonal cell line lacking one TcCRT allele (TcCRT+/-) and another clone overexpressing it (TcCRT+). Both clones were derived from the TCC T. cruzi strain. As expected, TcCRT+/- epimastigotes showed impairment on TcCRT synthesis, whereas TcCRT+ ones showed increased protein levels. In correlation to this, monoallelic mutant parasites were significantly susceptible to killing by the complement machinery. On the contrary, TcCRT+ parasites showed higher levels of resistance to killing mediate by the classical and lectin but not the alternative pathway. The involvement of surface TcCRT in depleting C1 was demonstrated through restoration of serum killing activity by addition of exogenous C1. In axenic cultures, a reduced propagation rate of TcCRT+/- parasites was observed. Moreover, TcCRT+/- parasites presented a reduced rate of differentiation in in vitro assays. As shown by down- or upregulation of TcCRT expression this gene seems to play a major role in providing T. cruzi with the ability to resist complement system.     

Trypanosoma cruzi carrying a monoallelic deletion of the calreticulin (TcCRT) gene are susceptible to complement mediated killing and defective in their metacyclogenesis

Trypanosoma cruzi calreticulin (TcCRT) can hijack complement C1, mannan-binding lectin and ficolins from serum thus inhibiting the classical and lectin complement pathway activation respectively. To understand the in vivo biological functions of TcCRT in T. cruzi we generated a clonal cell line lacking one TcCRT allele (TcCRT+/?) and another clone overexpressing it (TcCRT+). Both clones were derived from the TCC T. cruzi strain. As expected, TcCRT+/? epimastigotes showed impairment on TcCRT synthesis, whereas TcCRT+ ones showed increased protein levels. In correlation to this, monoallelic mutant parasites were significantly susceptible to killing by the complement machinery. On the contrary, TcCRT+ parasites showed higher levels of resistance to killing mediate by the classical and lectin but not the alternative pathway. The involvement of surface TcCRT in depleting C1 was demonstrated through restoration of serum killing activity by addition of exogenous C1. In axenic cultures, a reduced propagation rate of TcCRT+/? parasites was observed. Moreover, TcCRT+/? parasites presented a reduced rate of differentiation in in vitro assays. As shown by down- or upregulation of TcCRT expression this gene seems to play a major role in providing T. cruzi with the ability to resist complement system.