HIV infection of thymocytes inhibits IL-7 activity without altering CD127 expression

Background

Thymic function is altered in HIV infection and characterized by dysregulation of the thymic epithelial network, reduced thymic output and ultimately an impaired naïve T-cell pool. The IL-7/IL-7 receptor (IL-7R) signalling pathway is critical for the maturation and differentiation of thymocytes. HIV infection is associated with a decrease in IL-7Rα (CD127) expression and impaired CD127 signalling in circulating CD8⁺ T-cells; however, little is known about the effect of HIV on CD127 expression and IL-7 activity in the thymus. Therefore, the effect of in vitro HIV infection on CD127 expression and IL-7-mediated function in thymocytes was investigated.

Findings

In vitro HIV infection of thymocytes did not affect CD127 expression on either total thymocytes or on single positive CD4 or single positive CD8 subsets. However, HIV infection resulted in a decrease in the level of IL-7-induced STAT-5 phosphorylation and Bcl-2 expression in unfractionated thymocytes.

Conclusion

These findings indicate that HIV infection alters IL-7 responsiveness of thymocytes by a mechanism other than CD127 downregulation and potentially explain the disruption in thymopoiesis observed in HIV infection.     

The expression of the Ig H chain repertoire in developing bone marrow B lineage cells

Analyses of rearranged Ig H chain V region genes of bone marrow pre-B cells demonstrate extensive sequence diversity, particularly within the third hypervariable region (HCDR3). This diversity is constrained, however, through preferential utilization of certain D gene segments and possibly VH gene segments and a preponderance of productive rearrangements, primarily those expressing D gene segments in a preferred reading frame. The predominance of productively rearranged V genes with D regions translated in a preferred frame, is, at least in part, the consequence of selective clonal expansion encompassing at least five to six divisions subsequent to VH-D-JH rearrangement. Selection for clonal expansion appears to be dependent on recognition of the nascent H chain product of certain productively rearranged genes.     

Immature B cells can pass through a VHDJH/germ line state in the Ig H chain gene rearrangements

SPL2-1-2, an Ig- murine immature B cell line, was established by the infection of immature B cells with tsOS-59, a temperature-sensitive mutant of Abelson murine leukemia virus. Southern blot and DNA cloning and sequencing analysis showed that SPL2-1-2 had a nonproductive VHQ52.DSP2.JH3 and a germ line (G) allele (VHDJH-/G), and that D to JH joinings followed by VH to DJH joinings progressed on the G allele during culture. These results indicated the existence of novel pathway of IgH gene rearrangements: G/G----DJH/G----VHDJH/G----VHDJH/DJH----VHDJH /VHDJH. This also implied that whether DJH/G state progressed to DJH/DJH or VHDJH/G state was stochastically determined, but not strictly controlled in an ordered fashion, although DJH/G state progressed more preferentially to DJH/DJH state than to VHDJH/G state.     

Cyclosporin A inhibits creatine uptake by altering surface expression of the creatine transporter

The immunosuppressive drug cyclosporin A (CsA) inhibited the hCRT-1 cDNA-induced creatine uptake in Xenopus oocytes and the endogenous creatine uptake in cultured C(2)C(12) muscle cells in a dose- and time-dependent manner. FK506, another potent immunosuppressant, was unable to mimic the effect of CsA suggesting that the inhibitory effect of CsA was specific. To delineate the mechanism underlying, we investigated the effect of CsA on the K(m) and V(max) of creatine transport and also on the cell surface distribution of the creatine transporter. Although CsA treatment did not affect the K(m) (20-24 microm) for creatine, it significantly decreased the V(max) of creatine uptake in both oocytes and muscle cells. CsA treatment reduced the cell surface expression level of the creatine transporter in the muscle cells by approximately 60% without significantly altering its total expression level, and the reduction in the cell surface expression paralleled the decrease in creatine uptake. Taken together, our results suggest that CsA inhibited creatine uptake by altering the surface abundance of the creatine transporter. We propose that CsA impairs the targeting of the creatine transporter by inhibiting the function of an associated cyclophilin, resulting in an apparent loss in surface expression of the creatine transporter. Our results also suggest that prolonged exposure to CsA may result in chronically creatine-depleted muscle, which may be a cause for the development of CsA-associated clinical myopathies in organ transplant patients.     

STGC3 inhibits xenograft tumor growth of nasopharyngeal carcinoma cells by altering the expression of proteins associated with apoptosis

STGC3 is a potential tumor suppressor that inhibits the growth of the nasopharyngeal carcinoma cell line CNE2; the expression of this protein is reduced in nasopharyngeal carcinoma compared with normal nasopharyngeal tissue. In this study, we investigated the tumor-suppressing activity of STGC3 in nude mice injected subcutaneously with Tet/pTRE-STGC3/CNE2 cells. STGC3 expression was induced by the intraperitoneal injection of doxycycline (Dox). The volume mean of Tet/pTRE-STGC3/CNE2+Dox xenografts was smaller than that of Tet/pTRE/CNE2+Dox xenografts. In addition, Tet/pTRE-STGC3/CNE2+Dox xenografts showed an increase in the percentage of apoptotic cells, a decrease in Bcl-2 protein expression and an increase in Bax protein expression. A proteomic approach was used to assess the protein expression profile associated with STGC3-mediated apoptosis. Western blotting confirmed the differential up-regulation of prohibitin seen in proteomic analysis. These results indicate that overexpression of STGC3 inhibits xenograft growth in nude mice by enhancing apoptotic cell death through altered expression of apoptosis-related proteins such as Bcl-2, Bax and prohibitin. These data contribute to our understanding of the function of STGC3 in human nasopharyngeal carcinoma and provide new clues for investigating other STGC3-associated tumors.     

Cell lineage and gene expression in the development of polychaetes

The developmental strategies of embryos within the various taxa of polychaetes are designed to set up the fate of the cell lines. Some of these traits of pattern formation are considered to be ancestral, but we also find a number of derived developmental characteristics, some of which might be useful indicators for phylogenetic relationships. A combination of ooplasmic segregation and anisotropic cleavage rapidly establishes the fate of several larval cell lines in the polychaete embryo. The setting-up of the primary trochoblasts basically concerns the same cell line (la²–ld²) in polychaetes and even in molluscs. Such mechanisms may thus be regarded as ancestral. The determination of the mesoderm precursor occurs very late in both equally cleaving annelids and mollusks, indicating that an equal cleavage pattern may be regarded as an ancestral trait. Since both disproportionate cytoplasmic distribution (either by spindle shift or polar lobe formation) and cell cycle asynchronies appear to speed up the development of the mesoderm-forming cell line, these strategies represent derived traits. An analysis of these derived traits of early development is given and is discussed in the light of the phylogenetic relationships among the polychaetes. These data are extended by an analysis of some of the postlarval structures in polychaetes and the molecular developmenta1 circuitry involved.     

Characterization of the human c-fms gene product and its expression in cells of the monocyte-macrophage lineage.

The McDonough strain of feline sarcoma virus contains an oncogene called v-fms whose ultimate protein product (gp140v-fms) resembles a cell surface growth factor receptor. To identify and characterize the protein product of the proto-oncogene c-fms, antisera were prepared to the viral fms sequences and used to detect specific cross-reacting sequences in human choriocarcinoma cells (BeWo) known to express c-fms mRNA. Both tumor-bearing rat sera and a rabbit antiserum prepared to a segment of v-fms expressed in Escherichia coli detected a 140-kilodalton (kDa) glycoprotein in the BeWo cells. Tryptic fingerprint analysis of [35S]methionine-labeled proteins indicated that the viral fms proteins and the 140-kDa BeWo cell protein were highly related. This 140-kDa glycoprotein contained an associated tyrosine kinase activity in vitro and was labeled principally on serine after 32Pi metabolic labeling. These results suggest that the 140-kDa protein in BeWo cells is the protein product of the human c-fms proto-oncogene. This conclusion is supported by the finding that a similar protein is detectable only in other human cells that express c-fms mRNA. These other human cells include adherent monocytes and the cell line ML-1, which can be induced to differentiate along the monocyte-macrophage pathway. This is in agreement with current thought that the c-fms proto-oncogene product functions as the CSF-1 receptor specific to this pathway.     

Comparison of the effectiveness of S phase inhibitors in altering gene expression

Evidence suggests that an unidentified mechanism associated with the S phase inhibition properties of 5-azacytidine may be just as or more important than its hypomethylating properties in eliciting gene expression. To determine how important the S phase properties of this agent are to the alteration of gene expression, I compared it with other S phase inhibitors which do not affect DNA methylation for their ability to derepress the hypoxanthine phosphoribosyl transferase gene on the inactive X chromosome. Of these agents, only 5-azacytidine was able to derepress this gene in CAK cells. It appears that 5-azacytidine does alter gene expression via DNA hypomethylation. The ability of other S phase inhibitors to increase fetal globin expression may be peculiar to this genetic system.     

Muscle lineage cytoplasm can change the developmental expression in epidermal lineage cells of ascidian embryos

Cytoplasm from muscle lineage blastomeres of an ascidian embryo can cause cells of a nonmuscle lineage to produce larval tail muscle acetylcholinesterase. Muscle cytoplasm was partitioned microsurgically into epidermal lineage blastomeres at the eight-cell stage. Posterior half-embryos (the two B3 cells) of Ascidia nigra were obtained first by separating the anterior and posterior blastomere pairs at the four-cell stage. At third cleavage, the two B3 cells divide into an ectodermal cell pair that gives rise solely to epidermal tissues, and a mesodermal-endodermal blastomere pair from which the tail muscle cells are derived. When the ectodermal and mesendodermal blastomere pairs were isolated from one another by microsurgery and reared as partial embryos, only cells originating from the mesendodermal blastomeres produced a histochemical acetylcholinesterase reaction. Immediately after cleavage of the isolated B3 cells into ectodermal and mesendodermal cell pairs, the cleavage furrows could be made to disappear by pressing firmly on the mesendodermal cells with a microneedle. Repeated up and down pressure with the microneedle at a new position across the mesendodermal cells caused furrows to reestablish in the new position, thereby incorporating mesodermal cytoplasm and increasing the size of the ectodermal cells. The cytoplasmically altered ectodermal blastomere pairs, which became detached from the mesendodermal cells by this microsurgical procedure, continued to divide and were reared to “larval” stages. One-third of these epidermal partial larvae produced patches of cells containing acetylcholinesterase. These results lend further support to the theory that choice of particular differentiation pathways (embryonic determination) in ascidian embryos is mediated by segregation of specific egg cytoplasmic determinants.