No abstract

Definitive risk factors for the development of adult T-cell leukemia (ATL) among asymptomatic human T-cell leukemia vi-

Human T cell leukemia͞lymphotropic virus type I (HTLV-I) induces adult T cell leukemia͞lymphoma (ATLL).In a small percentage of infected individuals, human T cell leukemia͞lymphotropic virus type-I (HTLV-I) causes adult T cell leukemia͞lymphoma (ATLL), an aggressive and often fatal disease (1-3). The epidemiology of ATLL suggests that cumulative genetic defects may be responsible for the acquisition of the neoplastic phenotype in a given T cell clone (4). T cell proliferation and selection following HTLV-I infection are dynamic processes that can be followed in vivo (5, 6) and in vitro (7) and generally result in the generation of clonal populations of mature CD4 ϩ , CD8 Ϫ , and CD25 ϩ ͞CD7 Ϫ T cells (8-10). In HTLV-I infection the time-dependent emergence of infected T cell clones is well documented, and ATLL results from the uncontrolled growth of a single clone.In vitro, T cell immortalization (ligand-dependent) by HTLV-I occurs within a few months of culture, whereas T cell transformation (ligand-independent) requires more time and typically results in T cell lines that display constitutive activation of the JAK͞STAT signaling pathway (11,12). In physiological conditions, activation of the JAK͞STAT pathway is triggered by cytokines through cell surface receptors (13). In the case of interferon and type I cytokines, the JAK family tyrosine kinases transduce the signal by phosphorylating the STAT proteins, which in turn dimerize and translocate to the nucleus to activate the expression of genes necessary for cell proliferation or differentiation (14). To ascertain whether the in vitro model of HTLV-I transformation has any bearing to the in vivo leukemogenesis, we investigated the JAK͞STAT activation status in uncultured ex vivo leukemic cells from 12 HTLV-I seropositive patients with ATLL.

Pseudomonas aeruginosa is an important opportunistic human pathogen. Certain strains can transmigrate across epithelial cells, and their invasive phenotype is correlated with capacity to cause invasive human disease and fatal septicemia in mice. Four multidrug efflux systems have been described in P. aeruginosa, however, their contribution to virulence is unclear. To clarify the role of efflux systems in invasiveness, P. aeruginosa PAO1 wild-type (WT) and its efflux mutants were evaluated in a Madin-Darby canine kidney (MDCK) epithelial cell monolayer system and in a murine model of endogenous septicemia. All efflux mutants except a ΔmexCD-oprJ deletion demonstrated significantly reduced invasiveness compared with WT. In particular, a ΔmexAB-oprM deletion strain was compromised in its capacity to invade or transmigrate across MDCK cells, and could not kill mice, in contrast to WT which was highly invasive (P < 0.0006) and caused fatal infection (P < 0.0001). The other mutants, including ΔmexB and ΔmexXY mutants, were intermediate between WT and the ΔmexAB-oprM mutant in invasiveness and murine virulence. Invasiveness was restored to the ΔmexAB-oprM mutant by complementation with mexAB-oprM or by addition of culture supernatant from MDCK cells infected with WT. We conclude that the P. aeruginosa MexAB-OprM efflux system exports virulence determinants that contribute to bacterial virulence.

No abstract

CpG methylation of the human T-cell leukemia virus type 1 (HTLV-1) long terminal repeat (LTR) has been implicated in proviral latency, but there is presently little information available regarding the pattern of LTR methylation and its effect on viral gene expression. To gain insight into the mechanisms of HTLV-1 latency, we have studied methylation of individual CpG sites in the U3-R region of the integrated proviral LTR by using bisulfite genomic sequencing methods. Surprisingly, our results reveal selective hypermethylation of the 5 LTR and accompanying hypomethylation of the 3 LTR in both latently infected cell lines and adult T-cell leukemia (ATL) cells having a complete provirus. Moreover, we observed a lack of CpG methylation in the LTRs of 5-defective proviruses recovered from ATL samples, which is consistent with the selective hypomethylation of the 3 LTR. Thus, the integrated HTLV-1 provirus in these carriers appears to be hypermethylated in the 5 LTR and hypomethylated in the 3 LTR. These results, together with the observation that proviral gene expression is reactivated by 5-azacytidine in latently infected cell lines, indicate that selective hypermethylation of the HTLV-1 5 LTR is common both in vivo and in vitro. Thus, hypermethylation of the 5 LTR appears to be an important mechanism by which HTLV-1 gene expression is repressed during viral latency.Human T-cell leukemia virus type 1 (HTLV-1), the first human pathogenic retrovirus isolated, is the etiologic agent of adult T-cell leukemia (ATL), tropical spastic paraparesis/ HTLV-1-associated myelopathy, and HTLV-1 uveitis. HTLV-1 is mainly transmitted through breastfeeding, and a long latency period precedes development of these diseases, which occur in carriers during middle or older age (12,18,28,29,33,34,42,44).HTLV-1-infected cells in the peripheral blood rarely express viral genes. Detection of HTLV-1 transcripts in peripheral blood mononuclear cells (PBMC) by reverse transcriptase PCR (RT-PCR) showed low levels of virus expression that are independent of the number of circulating HTLV-1-infected cells of asymptomatic carriers and tropical spastic paraparesis/ HTLV-1-associated myelopathy patients (11, 13). Furthermore, fresh ATL cells do not express viral antigens until they are cultured in the presence of fetal calf serum, when in some cases ATL cells become positive for viral antigens (9, 17, 43) or viral transcripts (11,22). However, the possibility that viral transcripts are derived from contaminating untransformed HTLV-1-infected cells was not ruled out. Thus, it appears that most HTLV-1-infected cells in vivo harbor a provirus that is transcriptionally silent. A recent study of an infection model using squirrel monkeys (Saimiri sciureus) revealed transient expression of tax/rex mRNA and early induction of latency, suggesting that primary HTLV-1 infection consists of a first transient step of reverse transcription and viral expression, followed by a latency of HTLV-1-bearing T cells (21). These observations collectively suggest that late...

No abstract

Adult T-cell leukemia (ATL) is associated with prior infection with human T-cell leukemia virus type 1 (HTLV-1); however, the mechanism by which HTLV-1 causes adult T-cell leukemia has not been fully elucidated. Recently, a functional basic leucine zipper (bZIP) protein coded in the minus strand of HTLV-1 genome (HBZ) was identified. We report here a novel isoform of the HTLV-1 bZIP factor (HBZ), HBZ-SI, identified by means of reverse transcription-PCR (RT-PCR) in conjunction with 5 and 3 rapid amplification of cDNA ends (RACE). HBZ-SI is a 206-amino-acid-long protein and is generated by alternative splicing between part of the HBZ gene and a novel exon located in the 3 long terminal repeat of the HTLV-1 genome. Consequently, these isoforms share >95% amino acid sequence identity, and differ only at their N termini, indicating that HBZ-SI is also a functional protein. Duplex RT-PCR and real-time quantitative RT-PCR analyses showed that the mRNAs of these isoforms were expressed at equivalent levels in all ATL cell samples examined. Nonetheless, we found by Western blotting that the HBZ-SI protein was preferentially expressed in some ATL cell lines examined. A key finding was obtained from the subcellular localization analyses of these isoforms. Despite their high sequence similarity, each isoform was targeted to distinguishable subnuclear structures. These data show the presence of a novel isoform of HBZ in ATL cells, and in addition, shed new light on the possibility that each isoform may play a unique role in distinct regions in the cell nucleus. Adult T-cell leukemia (ATL) is an aggressive and lethal CD4ϩ T-cell malignancy with characteristic nuclear irregularity. Human T-cell leukemia virus type 1 (HTLV-1) is a singlestranded RNA virus belonging to the subfamily Deltaretrovinae and containing reverse transcriptase. The RNA of the retrovirus is transcribed into DNA by reverse transcriptase, and is then inserted into the host genome by an integrase, forming the provirus. Since ATL is associated with prior infection with HTLV-1 (11, 27), although the mechanisms by which tumorigenesis occurs are not fully defined, the viral proteins from HTLV-1 genome have been thought to be essential for the process of leukemogenesis in ATL.The HTLV-1 genome encodes common structural and enzymatic proteins (Gag, Pol and Env) and regulatory and accessory proteins (Tax, Rex, p12 I , p13 II and p30 II ) (13). Among these HTLV-1 viral proteins, Tax protein is considered to play a central role in the early stage of leukemogenesis (8,14,21,24,25,29). However, leukemic cells frequently lack the expression of Tax due to genetic and epigenetic changes of the HTLV-1 provirus (5,23,26), suggesting that while Tax may be a necessary prerequisite for the malignant transformation of infected cells, it is not essential for the maintenance of ATL cells in vivo. In the final stage of leukemogenesis, other continuously expressed viral proteins from the HTLV-1 genome are likely to be involved in the maintenance of ATL cells, because ATL is a uniqu...