No abstract

No abstract

BACKGROUND: The phosphatidylinositol 3 0 -kinase (PI3K) -AKT pathway is activated in many human cancers and plays a key role in cell proliferation and survival. A mutation (E17K) in the pleckstrin homology domain of the AKT1 results in constitutive AKT1 activation by means of localisation to the plasma membrane. The AKT1 (E17K) mutation has been reported in some tumour types (breast, colorectal, ovarian and lung cancers), and it is of interest which tumour types other than those possess the E17K mutation. METHODS: We analysed the presence of the AKT1 (E17K) mutation in 89 endometrial cancer tissue specimens and in 12 endometrial cancer cell lines by PCR and direct sequencing. RESULTS: We detected two AKT1 (E17K) mutations in the tissue samples (2 out of 89) and no mutations in the cell lines. These two AKT1 mutant tumours do not possess any mutations in PIK3CA, PTEN and K-Ras. INTERPRETATION: Our results and earlier reports suggest that AKT1 mutations might be mutually exclusive with other PI3K -AKTactivating alterations, although PIK3CA mutations frequently coexist with other alterations (such as HER2, K-Ras and PTEN) in several types of tumours.

Limited T cell availability and proliferative exhaustion present major barriers to successful T cell-based immunotherapies and may potentially be overcome through the use of ''rejuvenated'' induced pluripotent stem cells derived from antigen-specific T cells (T-iPSCs). However, strict antigen specificity is essential for safe and efficient T cell immunotherapy. Here, we report that CD8ab T cells from human T-iPSCs lose their antigen specificity through additional rearrangement of the T cell receptor (TCR) a chain gene during the CD4/CD8 double positive stage of in vitro differentiation. CRISPR knockout of a recombinase gene in the T-iPSCs prevented this additional TCR rearrangement. Moreover, when CD8ab T cells were differentiated from monocyte-derived iPSCs that were transduced with an antigen-specific TCR, they showed monoclonal expression of the transduced TCR. TCR-stabilized, regenerated CD8ab T cells effectively inhibit tumor growth in xenograft cancer models. These approaches could contribute to safe and effective regenerative T cell immunotherapies.

The first step of papillomavirus infection is believed to be binding of major capsid protein L1 to the cell surface without involvement of minor capsid protein L2, but the viral infectivity can be neutralized either by anti-L1 or anti-L2 antibody. To understand the role of L2 in human papillomavirus (HPV) infection, we examined a segment of HPV type 16 (HPV16) L2, which contains a neutralization epitope common to HPV6, for its involvement in adsorption and penetration of the capsids. Preincubation of monkey COS-1 cells with a synthetic peptide having amino acids (aa) 108 to 120 of HPV16 L2 reduced the susceptibility of COS-1 cells to infection with HPV16 pseudovirions. Confocal microscopy showed that the green fluorescence protein (GFP) fused with the L2 peptide was found to bind to the surface of a HeLa cell, an HPV18-positive human cancer cell line, at 4°C and to enter the cytoplasm after subsequent incubation at 37°C. Papillomaviruses, whose nonenveloped, icosahedral, 55-nmdiameter virions are made of 72 pentameric capsomeres composed of the structural proteins L1 and L2 at an estimated molar ratio of 30 to 1, have been found in various animal species, including humans (26). Human papillomaviruses (HPVs) have to date been classified into more than 80 genotypes, which constitute two groups: cutaneous and mucosal HPVs (26). HPVs infecting the cutaneous epithelium such as types 1, 2, 4, and 8 mainly cause skin warts (26). HPVs infecting the mucosal epithelium such as types 6 and 11 cause benign condyloma, but types 16, 18, and 33 cause cervical cancer (14, 26). Among the nine major types of HPV associated with cervial cancer, HPV16 is the most prevalent type, constituting approximately 50% of cases (14, 27).Because it is almost impossible to efficiently grow HPVs in cell cultures, except HPV18 in a raft culture (16), surrogate systems have been developed for production of capsids (particles without viral DNA) and for assay of viral infectivity. The capsids produced in surrogate systems resemble morphologically and immunlogically the natural virions (7,11,12,22). When L1 alone is expressed in eukaryotic cells by recombinant baculovirus or vaccinia virus (7,11,12,22), L1 can self-assemble to form icosahedral particles (L1 capsids). When L2, which is not required for assembly, is coexpressed with L1, both L1 and L2 are incorporated into the particles (L1-L2 capsids) (7,12). Furthermore, infectious HPV pseudovirions are produced in cultured cells (19,24). Also, pseudovirions are constructed in vitro from disassembled capsids and a plasmid capable of expressing a reporter gene, and their infectivity can be assayed in COS-1 cells (9). The capsids and pseudovirions and natural bovine papillomavirus type 1 (BPV1) virions, isolated from cutaneous legions, have been used for studies of adsorption of papillomaviruses and of neutralization of their infectivity.Viral infection in vitro is supposed to start from attachment of virions to the cell surface (20,25). The adsorption appears to occur from binding of L1 to cell recep...

CD1d and CD1d-restricted natural killer T (NKT) cells serve as a natural bridge between innate and adaptive immune responses to microbes. CD1d downregulation is utilized by a variety of microbes to evade immune detection. We demonstrate here that CD1d is downregulated in human papillomavirus (HPV)-positive cells in vivo and in vitro.CD1d immunoreactivity was strong in HPV-negative normal cervical epithelium but absent in HPV16-positive CIN1 and HPV6-positive condyloma lesions. We used two cell lines for in vitro assay; one was stably CD1d-transfected cells established from an HPV-negative cervical cancer cell line, C33A (C33A/CD1d), and the other was normal human vaginal keratinocyte bearing endogenous CD1d (Vag). Flow cytometry revealed that cell surface CD1d was downregulated in both C33A/CD1d and Vag cells stably transfected with HPV6 E5 and HPV16 E5. Although the steady-state levels of CD1d protein decreased in both E5-expressing cell lines compared to empty retrovirus-infected cells, CD1d mRNA levels were not affected. Confocal microscopy demonstrated that residual CD1d was not trafficked to the E5-expressing cell surface but colocalized with E5 near the endoplasmic reticulum (ER). In the ER, E5 interacted with calnexin, an ER chaperone known to mediate folding of CD1d. CD1d protein levels were rescued by the proteasome inhibitor, MG132, indicating a role for proteasome-mediated degradation in HPV-associated CD1d downregulation. Taken together, our data suggest that E5 targets CD1d to the cytosolic proteolytic pathway by inhibiting calnexin-related CD1d trafficking. Finally, CD1d-mediated production of interleukin-12 from the C33A/CD1d cells was abrogated in both E5-expressing cell lines. Decreased CD1d expression in the presence of HPV E5 may help HPV-infected cells evade protective immunological surveillance.There are approximately 100 identified genotypes (types) of human papillomavirus (HPV). Over 40 of these are classified as genital HPV subtypes that invade the reproductive organs, including the uterine cervix, vaginal wall, vulva, and penis. Genital HPV types are further subclassified into high-risk types that are commonly associated with cervical cancer and low-risk types that cause noninvasive condyloma acuminata. Although exact classification varies among researchers, subtypes 16,18,31,33,35,39, 45, 51, 52, 56, 58, 66, and 68 are typically classified as high risk and subtypes 6,11,40,42,43,44, 54, 61, and 72 as low risk (44). Genital HPV infection involves short-term virus proliferation, followed by the long-term latent presence of a small number of copies of the viral genome within the basal cells of the genital epithelium (44). Infections with high-risk HPV subtypes result in progression to genital tract cancers (most commonly cervical) in only a small percentage of infected women and typically after a long latency period. A high percentage of high-risk HPV DNA-positive infected women resolve their infections during the proliferative phase and thereby clear the virus or progress to latency with...

Chlamydia trachomatis is an obligate intracellular pathogen that can persist in the urogenital tract. Mechanisms by which C. trachomatis evades clearance by host innate immune responses are poorly described. CD1d is MHC-like, is expressed by epithelial cells, and can signal innate immune responses by NK and NKT cells. Here we demonstrate that C. trachomatis infection down-regulates surface-expressed CD1d in human penile urethral epithelial cells through proteasomal degradation. A chlamydial proteasome-like activity factor (CPAF) interacts with the CD1d heavy chain, and CPAF-associated CD1d heavy chain is then ubiquitinated and directed along two distinct proteolytic pathways. The degradation of immature glycosylated CD1d was blocked by the proteasome inhibitor lactacystin but not by MG132, indicating that degradation was not via the conventional proteasome. In contrast, the degradation of non-glycosylated CD1d was blocked by lactacystin and MG132, consistent with conventional cellular cytosolic degradation of N-linked glycoproteins. Immunofluorescent microscopy confirmed the interruption of CD1d trafficking to the cell surface, and the dislocation of CD1d heavy chains into both the cellular cytosol and the chlamydial inclusion along with cytosolic CPAF. C. trachomatis targeted CD1d toward two distinct proteolytic pathways. Decreased CD1d surface expression may help C. trachomatis evade detection by innate immune cells and may promote C. trachomatis persistence.

Characterization of histologic pattern provides valuable information in the management of uterine carcinosarcoma.