Shiro Kozuma

Altered Expression of Human Leukocyte Antigen G (HLA‐G) on Extravillous Trophoblasts in Preeclampsia: Immunohistological Demonstration With Anti‐HLA‐G Specific Antibody “87G” and Anti‐cytokeratin Antibody “CAM5.2”

PROBLEM: Human leukocyte antigen‐G (HLA‐G) is suggested to be at play in the materno‐fetal immune relationship during pregnancy. In the light of current concept that disruption of the materno‐fetal immune relationship could account for several complications of pregnancy, including preeclampsia, we asked whether the expression of HLA‐G protein on the trophoblasts is altered in preeclampsia.

Evidence for an Elevation in Serum Interleukin-2 and Tumor Necrosis Factor-α Levels Before the Clinical Manifestations of Preeclampsia

PROBLEM: The purpose of this study is to clarify whether the disruption of immune regulation occurs in early pregnancy before the clinical manifestations of preeclampsia.

Human leukocyte antigen-G-expressing cells differently modulate the release of cytokines from mononuclear cells present in the decidua versus peripheral blood

PROBLEM: To better understand the role of human leukocyte antigen (HLA)‐G in regulating the T helper (Th)1/Th2 cytokine balance, one of key conditions in determining the fate of pregnancy, we asked whether the presence of HLA‐G protein altered the release of cytokines from both decidual mononuclear cells and peripheral blood mononuclear cells (PBMCs).
 METHOD OF STUDY: The amounts of cytokines released from decidual mononuclear cells and PBMCs were compared in the presence or absence of HLA‐G‐expressing cells.
 RESULTS: When cocultured with HLA‐G‐expressing cells, the amounts of tumor necrosis factor‐α and interferon‐γ released from decidual mononuclear cells and PBMCs were decreased, while the amounts of interleukin (IL)‐4 from PBMCs was increased, with IL‐4 release from decidual mononuclear cells being unchanged.
 CONCLUSIONS: Upon contact with HLA‐G, decidual mononuclear cells, and PBMCs as well, modulate their ability to release cytokines in a way that may shift the Th1/Th2 balance towards relative Th2 dominance, suggesting a role for HLA‐G in maintaining pregnancy.

Presence of HLA‐G‐Expressing Cells Modulates the Ability of Peripheral Blood Mononuclear Cells to Release Cytokines

PROBLEM: Human leukocyte antigen‐G (HLA‐G) is thought to be at play in maternal‐fetal immune interplay during pregnancy. Whether the expression of HLA‐G protein on the target cells altered the release of cytokines from effector mononuclear cells was questioned. METHOD OF STUDY: The amounts of cytokines released from peripheral blood mononuclear cells (PBMC) cocultured with or without HLA‐G‐expressing target cells were compared. RESULTS: When cocultured with HLA‐G‐expressing target cell lines, the amounts of interleukin‐3 (IL‐3) and interleukin‐lβ (IL‐1β) released from PBMC were increased, whereas the amounts of tumor necrosis factor‐α (TNF‐α) were decreased.

Highly Efficient Ex Vivo Expansion of Human Hematopoietic Stem Cells Using Delta1‐Fc Chimeric Protein

Ex vivo expansion of hematopoietic stem cells (HSCs) has been explored in the fields of stem cell biology, gene therapy, and clinical transplantation. Here, we demonstrate efficient ex vivo expansion of HSCs measured by long‐term severe combined immunodeficient (SCID) repopulating cells (SRCs) from human cord blood CD133‐sorted cells using a soluble form of Delta1. After a 3‐week culture on immobilized Delta1 supplemented with stem cell factor, thrombopoietin, Flt‐3 ligand, interleukin (IL)‐3, and IL‐6/soluble IL‐6 receptor chimeric protein (FP6) in a serum‐ and stromal cell‐free condition, we achieved approximately sixfold expansion of SRCs when evaluated by limiting dilution/transplantation assays. The maintenance of full multipotency and self‐renewal capacity during culture was confirmed by transplantation to nonobese diabetic/SCID/γcnull mice, which showed myeloid, B, T, and natural killer cells as well as CD133+CD34+ cells, and hematopoietic reconstitution in the secondary recipients. Interestingly, the CD133‐sorted cells contained approximately 4.5 times more SRCs than the CD34‐sorted cells. The present study provides a promising method to expand HSCs and encourages future trials on clinical transplantation.

ASK1 and ASK2 differentially regulate the counteracting roles of apoptosis and inflammation in tumorigenesis

Apoptosis and inflammation generally exert opposite effects on tumorigenesis: apoptosis serves as a barrier to tumour initiation, whereas inflammation promotes tumorigenesis. Although both events are induced by various common stressors, relatively little is known about the stress‐induced signalling pathways regulating these events in tumorigenesis. Here, we show that stress‐activated MAP3Ks, ASK1 and ASK2, which are involved in cellular responses to various stressors such as reactive oxygen species, differentially regulate the initiation and promotion of tumorigenesis. ASK2 in cooperation with ASK1 functioned as a tumour suppressor by exerting proapoptotic activity in epithelial cells, which was consistent with the reduction in ASK2 expression in human cancer cells and tissues. In contrast, ASK1‐dependent cytokine production in inflammatory cells promoted tumorigenesis. Our findings suggest that ASK1 and ASK2 are critically involved in tumorigenesis by differentially regulating apoptosis and inflammation.

Genotype-dependent efficacy of a dual PI3K/mTOR inhibitor, NVP-BEZ235, and an mTOR inhibitor, RAD001, in endometrial carcinomas.

The PI3K (phosphatidylinositol-3-kinase)/mTOR (mammalian target of rapamycin) pathway is frequently activated in endometrial cancer through various PI3K/AKT-activating genetic alterations. We examined the antitumor effect of NVP-BEZ235—a dual PI3K/mTOR inhibitor—and RAD001—an mTOR inhibitor—in 13 endometrial cancer cell lines, all of which possess one or more alterations in PTEN, PIK3CA, and K-Ras. We also combined these compounds with a MAPK pathway inhibitor (PD98059 or UO126) in cell lines with K-Ras alterations (mutations or amplification). PTEN mutant cell lines without K-Ras alterations (n = 9) were more sensitive to both RAD001 and NVP-BEZ235 than were cell lines with K-Ras alterations (n = 4). Dose-dependent growth suppression was more drastically induced by NVP-BEZ235 than by RAD001 in the sensitive cell lines. G1 arrest was induced by NVP-BEZ235 in a dose-dependent manner. We observed in vivo antitumor activity of both RAD001 and NVP-BEZ235 in nude mice. The presence of a MEK inhibitor, PD98059 or UO126, sensitized the K-Ras mutant cells to NVP-BEZ235. Robust growth suppression by NVP-BEZ235 suggests that a dual PI3K/mTOR inhibitor is a promising therapeutic for endometrial carcinomas. Our data suggest that mutational statuses of PTEN and K-Ras might be useful predictors of sensitivity to NVP-BEZ235 in certain endometrial carcinomas.

CD1d, a Sentinel Molecule Bridging Innate and Adaptive Immunity, Is Downregulated by the Human Papillomavirus (HPV) E5 Protein: a Possible Mechanism for Immune Evasion by HPV

ABSTRACT 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.

Transvaginal sonographic appearance of hemorrhagic functional ovarian cysts and their spontaneous regression

OBJECTIVES: To obtain information on the characteristic feature of transvaginal sonograms of hemorrhagic functional ovarian cysts (HFOCs) and their natural course. METHODS: Thirty‐four cases of suspected HFOC based on our tentative criteria were ultrasonically followed‐up for 3 months. After the study period, the sonographic findings and their changes with time were analyzed. RESULTS: Twenty‐four cases were clinically diagnosed as HFOCs, as the masses disappeared naturally. At the first examination, 5 cases (20.8%) showed a diffuse echogenic pattern which seems to consist of a blood clot, 9 (37.5%) were mixed pattern with a clearly demarcated solid part, 3 (12.5%) were hyperechoic sponge‐like reticular pattern and 7 (29.2%) were cysts including vague echo inside which looked cotton‐like. Seven masses (29.2%) disappeared within 2 weeks and the remaining had a different sonographic appearance with time until they disappeared finally within 8 weeks. CONCLUSIONS: HFOCs show the characteristic changes in transvaginal sonographic appearance and can be diagnosed by short‐term follow‐up.

Oral immunization with a Lactobacillus casei vaccine expressing human papillomavirus (HPV) type 16 E7 is an effective strategy to induce mucosal cytotoxic lymphocytes against HPV16 E7.

Although many clinical trials on human papillomavirus (HPV) therapeutic vaccines have been performed, clinical responses have not been consistent. We have addressed mucosal cytotoxic cellular immune responses to HPV16 E7 after oral immunization of mice with recombinant Lactobacillus casei expressing HPV16 E7 (LacE7). C57BL/6 mice were orally exposed to 0.1-100mg/head of attenuated LacE7 or vehicle (Lac) vaccines at weeks 1, 2, 4, and 8. Responses to subcutaneous or intramuscular injection of an HPV16 E7 fusion protein using the same timing protocol were used for comparison. Oral immunization with LacE7 elicited E7-specific IFN gamma-producing cells (T cells with E7-type 1 immune responses) among integrin alpha 4 beta 7(+) mucosal lymphocytes collected from gut mucosa. An induction of E7-specific granzyme B-producing cells (E7-CTL) exhibiting killer responses toward HPV16 E7-positive cells was also observed. The induction of T cells with specific mucosal E7-type 1 immune responses was greater after oral immunization with LacE7 when compared to subcutaneous or intramuscular antigen delivery. Oral immunization with Lactobacillus-based vaccines was also able to induce mucosal cytotoxic cellular immune responses. This novel approach at a therapeutic HPV vaccine may achieve more effective clinical responses through its induction of mucosal E7-specific CTL.