Total HLA Class I Antigen Loss with the Downregulation of Antigen-Processing Machinery Components in Two Newly Established Sarcomatoid Hepatocellular Carcinoma Cell Lines.

Limited information is currently available concerning HLA class I antigen abnormalities in sarcomatoid hepatocellular carcinoma (sHCC). Here, we have analyzed the growth characteristics and HLA class I antigen status of four sHCC cell lines (sHCC29, sHCC63, sHCC74, and SAR-HCV); the first three were newly established in this study. Among the four, sHCC29 showed the highest growth rate in vitro and tumorigenicity in NOD-SCID mice. Unlike sHCC74 and SAR-HCV, both sHCC29 and sHCC63 had no detectable surface HLA class I antigen expression, alongside undetected intracellular ß 2-microglobulin (ß 2m) and marked HLA class I heavy chain and selective antigen-processing machinery (APM) component downregulation. The loss of ß 2m in sHCC29 and sHCC63 was caused by a >49 kb deletion across the B2M locus, while their downregulation of APM components was transcriptional, reversible by IFN-γ only in several components. ß 2m was also undetected in the primary HCC lesions of the patients involved, indicating its in vivo relevance. We report for the first time HLA class I antigen loss with underlying B2M gene deficiency and APM defects in 50% (2 of 4) of the sHCC cell lines tested. These findings may have implications for a proper design of T cell immunotherapy for the treatment of sHCC patients.

Molecular Characteristics of the Endometrium in Uterine Adenomyosis and Its Biochemical Microenvironment.

Adenomyosis, which manifests with focally or diffusely scattered endometrial tissue within the uterine myometrium, is an endometriosis-like disease with controversial pathogenesis and compromised reproductive outcomes. This review, including the in vitro and in vivo studies performed on human or mouse models, is aimed to summarize the specific molecular characteristics of endometrium in the biochemical microenvironments of uterine adenomyosis. Many studies attributed the endometrium as the main cause of pathogenesis, with evidence of differential genetic expression and/or epigenetic modulation as well as estrogen-induced epithelial-mesenchymal transition. However, some studies indicated that the myometrium could play a role in the development of disease, based on findings of smooth muscle metaplasia and/or fibroblast-to-myofibroblast transdifferentiation by the influence of local biochemical factors. To date, it remains unclear whether adenomyosis is a genetically determined or a microenvironmentally induced disorder or whether the dysregulation of local factors may elicit the alteration of genetic expression in the endometrium. Similarly, it is uncertain whether the endometrial characteristics would remain consistent or could change along with a woman's reproductive life. Further longitudinal studies of the epigenetic controls or system biology are needed to elucidate the pathogenesis. Discovery of effective conservative treatments to improve the reproductive outcomes of patients with adenomyosis is still warranted.

Differential expression of CD44 and CD24 markers discriminates the epitheliod from the fibroblastoid subset in a sarcomatoid renal carcinoma cell line: evidence suggesting the existence of cancer stem cells in both subsets as studied with sorted cells.

Epithelioid and fibroblastoid subsets coexist in the human sarcomatoid renal cell carcinoma (sRCC) cell line, RCC52, according to previous clonal studies. Herein, using monoclonal antibodies to CD44 and CD24 markers, we identified and isolated these two populations, and showed that CD44bright/CD24dim and CD44bright/CD24bright phenotypes correspond to epithelioid and fibroblastoid subsets, respectively. Both sorted subsets displayed different levels of tumorigenicity in xenotransplantation, indicating that each harbored its own cancer stem cells (CSCs). The CD44bright/CD24bright subset, associated with higher expression of MMP-7, -8 and TIMP-1 transcripts, showed greater migratory/invasive potential than the CD44bright/CD24dim subset, which was associated with higher expression of MMP-2, -9 and TIMP-2 transcripts. Both subsets differentially expressed stemness gene products c-Myc, Oct4A, Notch1, Notch2 and Notch3, and the RCC stem cell marker, CD105 in 4-5% of RCC52 cells. These results suggest the presence of CSCs in both sRCC subsets for the first time and should therefore be considered potential therapeutic targets for this aggressive malignancy.

Increased expression of integrin-linked kinase during decidualization regulates the morphological transformation of endometrial stromal cells.

OBJECTIVE: To study the impact of integrin-linked kinase (ILK) in endometrial stromal cells (ESCs) during decidualization. DESIGN: Laboratory study with the use of human endometrium. SETTING: University hospital. PATIENT(S): Fertile reproductive-age women who had not received hormonal treatment for 3 months before tissue collection. INTERVENTION(S): Endometrium tissue collection, in vitro decidualization of isolated ESCs, and small interfering (si) RNA transfection. MAIN OUTCOME MEASURE(S): Immunohistochemistry, ELISA, Western blot analysis, methylthiazolyl tetrazolium assay, and immunofluorescence staining. RESULT(S): In vivo expression of ILK is significantly increased in distended-fusiform stromal cells of late secretory endometrium and in cobblestone-shaped decidual cells of early pregnancy. During in vitro decidualization for up to 8 days, confluent cultures of isolated ESCs consistently displayed increased ILK expression and morphologic transformation from fibroblast-like to polygonal cells. Subsequent ILK knockdown by siRNA transfection reversed this transformation, accompanied by decreased phosphorylation of glycogen synthase kinase (GSK) 3ß and decreased viable cell numbers. Immunofluorescence staining of the decidualized ESCs demonstrated linkage of increased levels of ILK at the tips of the fan-shaped organization of actin stress fibers located in the submembranous area, which expanded the decidual cells into a typical polygonal appearance. Knock-down of ILK abrogated the polymerization and organization of actin fibers, which reverted the cells to their undecidualized morphology. CONCLUSION(S): During human endometrial decidualization, ILK is essential for morphologic transformation of ESCs through organization of the actin cytoskeleton; it may also function through subsequent GSK3ß signaling, which requires further studies.

Decreased Endometrial Expression of Leukemia Inhibitory Factor Receptor Disrupts the STAT3 Signaling in Adenomyosis During the Implantation Window.

BACKGROUND: Adenomyosis was found to have negative impacts on embryo implantation. Leukemia inhibitory factor (LIF), proposed to be a molecular marker for endometrial receptivity, works through the LIF receptor (LIFR) on both the embryo and the endometrium. We aimed to evaluate the endometrial expression of LIF and LIFR and its subsequent signaling in patients with adenomyosis during the window of implantation (WOI). METHODS: Endometrium was obtained during the WOI from patients with adenomyosis (age <45 years) who underwent hysterectomy and from age-matched controls who had no endometriosis or adenomyosis. The LIF and LIFR expressions were measured by polymerase chain reaction for messenger RNA expression, immunohistochemistry for protein intensity and localization, and immunofluorescent staining for colocalization. The ratio of signal transducer and activator of transcription 3 (STAT3) to extracellular signal-regulated kinase (ERK) phosphorylation was measured by Western blot of both the endometrium and the isolated human endometrial stromal cells (ESCs). RESULTS: Patients with adenomyosis showed significantly and parallelly reduced LIF and LIFR expressions in the eutopic endometrium during WOI as compared with the control women and subsequently with remarkably reduced activation of STAT3 and ERK signaling. The significantly increased STAT3 and ERK phosphorylation induced by the LIF treatment in the cultured ESCs supported the linkage between the LIF-LIFR reaction and the signaling cascade. CONCLUSION: Significant reduction in LIFR expression and the reduced activation of subsequent signaling strongly suggest a working model of how the implantation markers, LIF, may affect the endometrium of patients with adenomyosis. These molecular changes supported the declined implantation rates reported in patients with adenomyosis.

EBV oncogene N-LMP1 induces CD4 T cell-mediated angiogenic blockade in the murine tumor model.

Antivascular immunity may provide long-term protection by preventing neovascularization that precedes tumor progression. Although the tumorigenesis promoted by EBV-encoded oncogene latent membrane protein 1 derived from Taiwanese nasopharyngeal carcinoma (N-LMP1) has been demonstrated, the potential of N-LMP1 for inducing immune surveillance remains elusive. In this article, we describe the immunogenicity of N-LMP1 (1510) and its induction of antivascular immunity in a transplantable tumor model in immunocompetent BALB/c mice. The immunogenicity of N-LMP1 was evaluated on the basis of tumor rejection following immunization. The impact of the immunization on the dynamics of tumor angiogenesis was assessed by temporal noninvasive dynamic contrast-enhanced magnetic resonance imaging and was further confirmed by histologic study and vascular count. Through the experiments of in vivo depletion and adoptive transfer, CD4 T cells were identified as effectors that depend on IFN-γ for tumor prevention. The response was further verified by the identification of an MHC H-2 I-E(d)-restricted peptide derived from N-LMP1 and by the immunization of mice with N-LMP1 peptide-loaded dendritic cells. These studies provide insight into N-LMP1-specific immunity in vivo, which suggests that CD4 T cells may play an important role in angiogenic surveillance against LMP1-associated cancer via tumor stroma targeting.

Enhanced cytotoxicity of natural killer cells following the acquisition of chimeric antigen receptors through trogocytosis.

Natural killer (NK) cells have the capacity to target tumors and are ideal candidates for immunotherapy. Viral vectors have been used to genetically modify in vitro expanded NK cells to express chimeric antigen receptors (CARs), which confer cytotoxicity against tumors. However, use of viral transduction methods raises the safety concern of viral integration into the NK cell genome. In this study, we used trogocytosis as a non-viral method to modify NK cells for immunotherapy. A K562 cell line expressing high levels of anti-CD19 CARs was generated as a donor cell to transfer the anti-CD19 CARs onto NK cells via trogocytosis. Anti-CD19 CAR expression was observed in expanded NK cells after these cells were co-cultured for one hour with freeze/thaw-treated donor cells expressing anti-CD19 CARs. Immunofluorescence analysis confirmed the localization of the anti-CD19 CARs on the NK cell surface. Acquisition of anti-CD19 CARs via trogocytosis enhanced NK cell-mediated cytotoxicity against the B-cell acute lymphoblastic leukemia (B-ALL) cell lines and primary B-ALL cells derived from patients. To our knowledge, this is the first report that describes the increased cytotoxicity of NK cells following the acquisition of CARs via trogocytosis. This novel strategy could be a potential valuable therapeutic approach for the treatment of B-cell tumors.

Astragalus membranaceus modulates Th1/2 immune balance and activates PPARγ in a murine asthma model.

Astragalus membranaceus, a traditional Chinese herb, has been used to improve airway inflammation and asthma. The present study investigated whether A. membranaceus has immunotherapeutic effects on asthma, a chronic inflammatory mucosal disease that is associated with excess production of IgE, eosinophilia, T helper 2 (Th2) cytokines, and bronchial hyperresponsiveness. An ovalbumin (OVA)-induced, chronic inflammatory airway murine asthma model was used to examine the status of pulmonary inflammation after the administration of A. membranaceus. The IgE levels in serum and bronchoalveolar lavage fluid showed a tendency to decrease after the administration of A. membranaceus. The number of eosinophils decreased and infiltration of inflammatory cells and collagen deposition declined in lung sections after A. membranaceus administration. The RNA and protein levels of Th2 cytokines and the ratio of the GATA3/T-bet mRNA levels decreased after A. membranaceus treatment. Furthermore, the mRNA level of peroxisome proliferator-activated receptor γ (PPARγ), a nuclear hormone receptor, increased in the lung tissues of A. membranaceus-treated mice. Finally, an A. membranaceus water extract activated PPARγ activity in either human embryonic kidney 293 (HEK293) or A549 cells in a PPARγ-responsive element-containing luciferase reporter assay. These results indicate that A. membranaceus has an inhibitory effect on airway inflammation in a murine model of asthma through modulating the imbalanced relationship between Th1 and Th2 cytokines.

Syngeneic adipose-derived stem cells with short-term immunosuppression induce vascularized composite allotransplantation tolerance in rats.

BACKGROUND AIMS: A clinically applicable tolerance induction regimen that removes the requirement for lifelong immunosuppression would benefit recipients of vascularized composite allotransplantation (VCA). We characterized the immunomodulatory properties of syngeneic (derived from the recipient strain) adipocyte-derived stem cells (ADSCs) and investigated their potential to induce VCA tolerance in rats. METHODS: ADSCs were isolated from Lewis (LEW, RT1A(l)) rats; their immunomodulatory properties were evaluated by means of mixed lymphocyte reactions in vitro and VCAs in vivo across a full major histocompatibility complex mismatch with the use of Brown-Norway (BN, RT1A(n)) donor rats. Two control and four experimental groups were designed to evaluate treatment effects of ADSCs and transient immunosuppressants (anti-lymphocyte globulin, cyclosporine) with or without low-dose (200 cGy) total body irradiation. Flow cytometry was performed to quantify levels of circulating CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs). RESULTS: Cultured syngeneic ADSCs exhibited CD90.1(+)CD29(+)CD73(+)CD45(-)CD79a(-)CD11b/c(-) phenotype and the plasticity to differentiate to adipocytes and osteocytes. ADSCs dramatically suppressed proliferation of LEW splenocytes against BN antigen and mitogen, respectively, in a dose-dependent fashion, culminating in abrogation of allo- and mitogen-stimulated proliferation at the highest concentration tested. Accordingly, one infusion of syngeneic ADSCs markedly prolonged VCA survival in LEW recipients treated with transient immunosuppression; of these, 66% developed tolerance. Total body irradiation provided no additional VCA survival benefit. An important role for Tregs in tolerance induction/maintenance was suggested in vivo and in vitro. CONCLUSIONS: Treatment comprising syngeneic ADSCs and transient immunosuppression (i) increased levels of circulating Tregs and (ii) induced tolerance in 66% of recipients of major histocompatibility complex-mismatched VCAs.

Co-existence of epithelioid and fibroblastoid subsets in a sarcomatoid renal carcinoma cell line revealed by clonal studies.

BACKGROUND: The biology of sarcomatoid renal cell carcinoma (RCC) and its conversion from and to the clear cell RCC are not fully-understood. We aimed to analyze the sarcomatoid RCC cell line, RCC52, derived from a lymph node metastatic lesion consisting mostly of sarcomatoid RCC cells with occasional clear cell areas. MATERIALS AND METHODS: Representative clonal epithelioid and fibroblastoid sublines isolated from the RCC52 cell line were analyzed alongside the parental line. Cytofluorometric and western blot analyses were used for phenotypic study. Xenotransplantation and in vitro invasive assays were used to determine tumorigenicity and invasiveness. Immunohistology in conjunction with antibodies to paired box gene-2 (PAX2) were used to determine if xenografts or tumor biopsies had the clear cell component. RESULTS: RCC52 cells grown as monolayers in vitro were all PAX2-negative, and consisted mostly of epithelioid cells and partly of fibroblastoid cells as noted in a previous study, confirming the co-existence of these two cell types in the in vitro growth of exclusive sarcomatoid RCC cells. Immunohistology revealed that the parental line and all epithelioid sublines tested were able to develop into solid tumors consisting mostly of sarcomatoid cells with PAX2-positive clear cells in some areas. The RCC stem cell marker CD105 was selectively expressed by a small proportion of the epithelioid, but not fibroblastoid, sublines, which was in line with the tumorigenic property of the epithelioid sublines containing cancer stem cells (CSCs). In contrast, only fibroblastoid sublines exhibited migratory/invasive properties, as determined by in vitro assays. CONCLUSION: Our findings confirm the presence of two distinct subsets in the RCC52 line, and suggest the epithelioid subset being able to de-differentiate to clear cells, albeit partially, and harboring CSCs as an emerging therapeutic target in order to achieve effective treatment of this malignancy.

Combined treatment with regulatory T cells and vascularized bone marrow transplantation creates mixed chimerism and induces donor-specific tolerance to vascularized composite allografts without cytoreductive conditioning.

We demonstrate herein that combination treatment with regulatory T cells (Tregs) and vascularized bone marrow transplantation (VBMT) can achieve stable mixed chimerism and long-term transplantation tolerance to vascularized composite allografts (VCA) without requiring cytoreductive recipient conditioning in rats. An appreciable number of Tregs of recipient origin was shown at the interface between recipient and transplanted VCA tissues, implicating a significant role for Tregs in protecting VCA from rejection. This cytoreduction-free protocol using co-treatment with Tregs and VBMT warrants further investigation toward potential clinical application for VCA transplantation.

Improving the safety of tolerance induction: chimerism and cellular co-treatment strategies applied to vascularized composite allografts.

Although vascularized composite allografts (VCAs) have been performed clinically for a variety of indications, potential complications from long-term immunosuppression and graft-versus-host disease remain important barriers to widespread applications. Recently it has been demonstrated that VCAs incorporating a vascularized long bone in a rat model provide concurrent vascularized bone marrow transplantation that, itself, functions to establish hematopoietic chimerism and donor-specific tolerance following non-myeloablative conditioning of recipients. Advances such as this, which aim to improve the safety profile of tolerance induction, will help usher in an era of wider clinical VCA application for nonlife-saving reconstructions.

Combined treatment with regulatory T cells and vascularized bone marrow transplantation creates mixed chimerism and induces donor-specific tolerance to vascularized composite allografts without cytoreductive conditioning.

BACKGROUND: Cotreatment with regulatory T cells (T(reg)) and conventional allogeneic bone marrow transplantation (BMT) successfully induced durable chimerism and tolerance to nonvascularized skin allografts without cytoreductive conditioning in mice. We sought to determine whether T(reg) treatment combined with vascularized BMT (VBMT) could create mixed chimerism and induce tolerance to vascularized composite allografts (VCAs) without cytoreductive conditioning in rats. METHODS: Recipient Lewis rats treated (day 0) with or without naturally sorted T(reg) (3 × 10(6)) from Lewis rat spleen and lymph nodes received costimulation blockade (anti-CD154 monoclonal antibody, days 0 and 1 and CTLA-4 immunoglobin, days 2, 4, and 6), rapamycin (days -1, 0, and 2), and concurrent transplantation of fully mismatched allogeneic donor VCAs (day 0) from the Brown Norway rat hindlimb containing VBMT. The mixed chimerism level was assessed monthly using flow cytometry. Survival of VCAs and occurrence of graft-versus-host disease were assessed clinically and histologically. RESULTS: The combination of T(reg) and VBMT treatment led to long-term multilineage hematopoietic mixed chimerism (12-18%) and long-term donor-specific tolerance to VCAs (89% acceptance rate). Neither stable mixed chimerism nor VCA acceptance was observed in recipients without T(reg) treatment. Graft-versus-host disease did not occur in the VBMT recipients. CONCLUSIONS: Cotreatment with T(reg) and VBMT created stable mixed chimerism and induced long-term donor-specific tolerance to VCAs without requiring cytoreductive conditioning. This noncytoreductive T(reg)-VBMT protocol has potential for clinical application in VCAs.

The comparison of interleukin 6-associated immunosuppressive effects of human ESCs, fetal-type MSCs, and adult-type MSCs.

BACKGROUND: Although human embryonic stem cells (ESCs) and mesenchymal stem cells (MSCs) from various sources display immunomodulatory effects, direct comparisons among these classes of stem cells have not been reported. METHODS: Peripheral blood mononuclear cell suppression assays and carboxyfluorescein diacetate succinimidyl ester assays were used to assess the immunosuppressive effects of stem cells. Gene expression was measured using DNA microarrays. Enzyme-linked immunosorbent assays were used to determine interleukin (IL)-6 levels. RESULTS: We found that fetal-type MSCs proliferated significantly faster than adult-type MSCs. Compared with ESCs grown on feeder cells, ESCs grown in feeder cell-free conditions exhibited decreased immunosuppressive effects. The suppressive effects of ESCs were significantly stronger than those of MSCs, and the suppressive effects of fetal-type MSCs were significantly stronger than those of adult-type MSCs at each tested dose level. Analysis of gene expression by microarray and MetaCore pathway mapping revealed changes in eight different immune response pathways; we observed that IL-6 gene expression was highly significantly involved in all eight pathways. Significantly higher IL-6 elevation ratios (IL-6after:IL-6before) were found in ESCs compared with fetal-type MSCs, and these were also found in fetal-type MSCs compared with adult-type MSCs. Furthermore, IL-6 levels were found to correlate with cell dosages of MSCs and the suppressive effects. CONCLUSIONS: The ease of obtaining fetal-type MSCs and their rapid proliferation make these cells ideal candidates for cell-based therapies, especially for diseases associated with immune responses, given the immunosuppressive effects of these cells. IL-6 might play an important role in the immunosuppressive effects of various stem cells.

Optimizing chimerism level through bone marrow transplantation and irradiation to induce long-term tolerance to composite tissue allotransplantation.

BACKGROUND: Mixed chimerism with long-term composite tissue allotransplant (CTA) acceptance can be achieved through allogeneic bone marrow transplantation (BMT). The present study investigated the optimal chimerism level by giving different irradiation dosages to recipients to induce tolerance to CTA. METHODS: Chimera were prepared using Brown-Norway and Lewis rats with strong major histocompatibility complex incompatibility. The Lewis rats received 5 mg antilymphocyte globulin (day -1 and 10) and 16 mg/kg cyclosporine (day 0-10) and were separated into groups 1, 2, 3, 4, and 5 according to the day -1 irradiation dosage: 0, 200, 400, 600, and 950 cGy, respectively. The Lewis rats were then reconstituted with 100 × 10(6) T-cell-depleted Brown-Norway bone marrow cells (day 0) and received vascularized Brown-Norway-CTA on day 28. Chimerism was assessed monthly by flow cytometry starting on day 28 after BMT. Graft-versus-host disease (GVHD) was assessed clinically and histologically. RESULTS: Chimerism, 4 weeks after BMT, averaged 0.2%, 9.2%, 30.7%, 58%, and 99.3% in groups 1 to 5, respectively. GVHD occurred as follows: groups 1 and 2, none; group 3, 1 case of GVHD; group 4, 7 cases of GVHD (of which 3 died); and group 5, 10 cases of GVHD (of which 6 died). The percentage of long-term CTA acceptance was 0%, 0%, 90%, 70%, and 40% in groups 1 to 5, respectively. The percentage of regulatory T cells was significantly lower in high-chimerism (≥ 20%, n = 15) than in low-chimerism (<20%, n = 5) rats that accepted CTA long-term . CONCLUSIONS: The chimerism level correlated positively with GVHD occurrence and long-term CTA acceptance but correlated negatively with regulatory T-cell levels. Optimal chimerism for CTA acceptance through pre-CTA BMT and irradiation occurs at 20-50% at day 28 after BMT in the rat model.

Enhanced IL-10 production by CD4+ T cells primed in IL-15Rα-deficient mice.

In this study, we investigated the functional outcomes of CD4(+) T cells primed in the absence of IL-15 transpresentation. Compared with their WT counterparts primed in WT mice, IL-15Rα KO CD4(+) T cells primed in KO mice were found to exclusively overproduce IL-10 upon in vitro restimulation(.) The comparable expression of IL-4 and Foxp3 in CD4(+) T cells primed in the WT and IL-15Rα KO mice indicated that this was neither due to T(H) 2- nor Treg cell-differentiation. IL-10 overproduction was also observed when OVA-specific TCR transgenic CD4(+) T (OT-II) cells were primed in KO mice, excluding an intrinsic deficiency of KO CD4(+) T cells. To investigate the WT and KO microenvironment, DCs from both WT and IL-15Rα KO mice were compared. DCs from both backgrounds were indistinguishable in their steady-state survival and in their expression of MHC class II and costimulatory molecules CD80, CD86, and CD40. However, IL-15Rα KO DCs primed OT-II cells in vitro to produce higher levels of IL-10 upon their restimulation. Additionally, IL-15Rα KO DCs produced significantly more IL-10 upon activation, and IL-10 neutralization during DC-mediated in vitro priming abolished IL-10 overproduction by CD4(+) T cells. Thus, IL-15Rα KO DCs provide an IL-10-enriched environment that preferentially primes CD4(+) T cells for more IL-10 production, highlighting a regulatory role for IL-15 transpresentation in CD4(+) T-cell priming.

Tumor dormancy resulting from subcutaneous injection to SCID mice with cultured nasopharyngeal carcinoma cells is mediated via IFN-γ induction of a highly differentiated phenotype.

The mechanisms underlying tumor dormancy in human primary lesions and bone marrow metastases of nasopharyngeal carcinoma (NPC) are still not completely understood. The aim of this study was to determine differences in the fates of cultured primary NPC (P-NPC) cells, interferon-γ-transduced primary NPC (IFN-γ-P-NPC) cells, bone marrow metastatic NPC (BM-NPC), and IFN-γ-transduced BM-NPC (IFN-γ-BM-NPC) cells following xenotransplantation into these four groups of SCID mice through subcutaneous injection of 5×10(6) cells/site/animal (4 animals/group). The injected mice were monitored for tumor development at the sites of injection. In only the group injected with IFN-γ-P-NPC cells, the resulting nodules remained small throughout the 60-day observation period after injection, but gradually became palpably prickly. Histopathological examination revealed that these lesions invariably consisted of mostly structures of horny pearls and keratin bridges with occasional apoptotic and degenerative cells. In contrast, animals injected with nontransduced-P-NPC cells developed tumors progressively with occasional central necroses. In the two groups injected with IFN-γ-NPC-BM and NPC-BM cells, progressive growths of tumors were noted, with the latter being at slightly faster rates, whereas the xenografts of both groups showed a poorly differentiated phenotype with abundant vascularity. The study results highlight the high susceptibility of P-NPC but not BM-NPC following IFN-γ gene transfer to the induction of tumor dormancy, which is mediated via induced cell differentiation. Thus, induced cell differentiation could provide a new mechanism by which tumor dormancy is induced.

Induction of metastatic cancer stem cells from the NK/LAK-resistant floating, but not adherent, subset of the UP-LN1 carcinoma cell line by IFN-γ.

As an advanced status of cancer stem cells (CSCs), metastatic CSCs (mCSCs) have been proposed to be the essential seeds that initiate tumor metastasis. However, the biology of mCSCs is poorly understood. In this study, we used a lymph node (LN) metastatic CEA-producing carcinoma cell line, UP-LN1, characterized by the persistent appearance of adherent (A) and floating (F) cells in culture, to determine the distribution of CSCs and mechanisms for the induction of mCSCs. F and A cells displayed distinct phenotypes, CD44(high)/CD24(low) and CD44(low)/CD24(high), respectively. The CSC-rich nature of F cells was typified by stronger expression of multiple drug resistance genes and a 7.8-fold higher frequency of tumor-initiating cells in NOD/SCID mice when compared with A cells. F cells showed a greater depression in HLA class I expression and an extreme resistance to NK/LAK-mediated cytolysis. Moreover, the NK/LAK-resistant F cells were highly susceptible to IFN-γ-mediated induction of surface CXCR4, with concomitant downregulation of cytoplasmic CXCL12 expression, whereas these two parameters remained essentially unchanged in NK/LAK-sensitive A cells. Following the induction of surface CXCR4, enhanced migratory/invasive potential of F cells was demonstrated by in vitro assays. Confocal immunofluorescence microscopy showed the two distinct phenotypes of F and A cells could be correspondingly identified in monodispersed and compact tumor cell areas within the patient's LN tumor lesion. In response to IFN-γ or activated NK/LAK cells, the CXCR4(+) mCSCs could be only induced from the CSCs, which were harbored in the highly tumorigenic CD44(high)/CD24(low) F subset. Our results revealed the complexity and heterogeneity of the CSC of this cell line/tumor and the differential immunomodulatory roles of F and A cells. A better understanding of the interactions among different classes of CSCs and their niches may assist us in eradicating the CSCs/mCSCs through targeted immunotherapy, chemotherapy, or both.

Hydroxyethyl starch reduces high stretch ventilation-augmented lung injury via vascular endothelial growth factor.

Disruption of epithelial and endothelial barriers found in patients with acute lung injury often results in the need for the support of mechanical ventilation. High tidal volume (V(T)) mechanical ventilation can increase lung damage through lung inflammation, but the mechanisms are unclear. We hypothesized that a colloid supply with hydroxyethyl starch would decrease neutrophil infiltration, lung edema, and vascular endothelial growth factor (VEGF) production in mice exposed to high V(T) mechanical ventilation. Male C57BL/6 mice, weighing 20 g to 25 g, were exposed to high V(T) (30 mL/kg) mechanical ventilation with room air for 1 h to 5 h and infused with 15 mL/kg/h normal saline or hydroxyethyl starch intravenously at the beginning and every 30 min during ventilation. Evans blue dye, lung wet-to-dry weight ratio, histopathologic grading of lung tissue, myeloperoxidase, and inflammatory cytokine were measured to establish the extent of lung injury. Knockdown of VEGF by short interfering RNA (siRNA) was used to explore the role of VEGF. High V(T) ventilation induced the increases of microvascular permeability, neutrophil influx, expressions of VEGF mRNA and VEGF, production of VEGF protein, positive staining of VEGF in epithelium, and apoptotic epithelial cell death. Lung injury induced by high V(T) ventilation was attenuated with the supply of hydroxyethyl starch and pharmacologic inhibition of VEGF expression by siRNA. We conclude that hydroxyethyl starch reduces high V(T) mechanical ventilation-induced lung injury and neutrophil infiltration through an inhibition of VEGF expression.

Prognostic value of TNM stage and tumor necrosis for renal cell carcinoma.

Our objective was to assess the value of tumor necrosis and other factors for predicting the outcome of renal cell carcinoma (RCC). Our study comprised 328 RCC patients who were surgically treated at this hospital between 2001 and 2006. The five-year survival data was analyzed using a Kaplan-Meier statistical analysis. The prognostic factors were evaluated with a univariate analysis using a log-rank test and multivariate analysis using the Cox proportional hazards regression method. The mean follow-up period for these patients was 46.5 months (median 45.2 months). The univariate analysis revealed that age, tumor stage, TNM stage, grade, tumor necrosis, and histological type were statistically significant prognostic factors. The multivariate analysis showed that the TNM stage and tumor necrosis were the most important predictive factors in the patients' overall survival. In the TNM stage with and without tumor necrosis, the five-year overall survival rates in stages I+II were 80.5% and 89.2%, respectively (p=0.115), where as the five-year survival rates in stages III+IV were 32.7% and 84.0%, respectively (p<0.001). Collectively, our present data revealed that tumor necrosis was an important predictive factor for survival in advanced stage RCC. In conclusion, both the TNM stage and tumor necrosis provided the most important prognostic factors of survival in RCC. Tumor necrosis proved to be a poor prognostic factor in advanced RCCs.