Glucocorticoid-induced tumor necrosis factor receptor expression in patients with cervical human papillomavirus infection

Introduction The progression of human papillomavirus (HPV) infection in the anogenital tract has been associated with the involvement of cells with regulatory properties. Evidence has shown that glucocorticoid-induced tumor necrosis factor receptor (GITR) is an important surface molecule for the characterization of these cells and proposes that GITR ligand may constitute a rational treatment for many cancer types. We aimed to detect the presence of GITR and CD25 in cervical stroma cells with and without pathological changes or HPV infection to better understand the immune response in the infected tissue microenvironment. Methods We subjected 49 paraffin-embedded cervical tissue samples to HPV DNA detection and histopathological analysis, and subsequently immunohistochemistry to detect GITR and CD25 in lymphocytes. Results We observed that 76.9% of all samples with high GITR expression were HPV-positive regardless of histopathological findings. High GITR expression (77.8%) was predominant in samples with ≥1,000 RLU/PCB. Of the HPV-positive samples negative for intraepithelial lesion and malignancy, 62.5% had high GITR expression. High GITR expression was observed in both carcinoma and high-grade squamous intraepithelial lesion (HSIL) samples (p = 0.16). CD25 was present in great quantities in all samples. Conclusions The predominance of high GITR expression in samples with high viral load that were classified as HSIL and carcinoma suggests that GITR+ cells can exhibit regulatory properties and may contribute to the progression of HPV-induced cervical neoplasia, emphasizing the importance of GITR as a potential target for immune therapy of cervical cancer and as a disease evolution biomarker.

Blocking tumor exosome release using nanovectorization systems

Exosomes are small membrane vesicles secreted by most cell types, either normal or malignant and are found in most body fluids such as saliva, plasma and breast milk. In the past decade, the interest in these vesicles has been growing more and more since it was found that besides their beneficial functions such as the removal of cellular debris and unnecessary proteins during cell maturation process, they can also interact with other cells and transfer information between them, thus helping diseases like cancer to progress. The present work intended to use gold nanoparticles as vehicles for gene silencing in an attempt to reduce the tumor-derived exosome secretion, regulated by Rab27a protein, and also aimed to compare the exosome secretion between two breast cell lines, MCF7 and MDA. Changes in RAB27A gene expression were measured by Real-time Quantitative PCR and it was revealed a decreased in RAB27A gene expression, as expected. Exosomes were isolated and purified by two different methods, ultracentrifugation and the commercial kit ExoQuick™ Solution, and further characterized using Western Blot analysis. ExoQuick™ Solution was proven to be the most efficient method for exosome isolation and it was revealed that MDA cells secrete more exosomes. Furthermore, the isolated MCF7-derived exosomes were placed together with a normal bronchial/tracheal epithelial cell line (BTEC) for an additional assay, which aimed to observe the uptake of exosomes by other cells and the exosomes’ capability of promoting cell-cell communication. This observation was made based on alterations in the expression levels of c-Myc and miR-21 genes and the fact that they both have an increased expression in BTEC cells incubated with tumor-derived exosomes when compared to control cells (without incubation with the exosomes) lead us to the conclusion that the exosome uptake and exchange of information between the exosomes and the normal cells did occurred.

Retrospective evaluation of bone pain palliation after samarium-153-EDTMP therapy

PURPOSE: The aim of this study was to evaluate the degree of metastatic bone pain palliation and medullar toxicity associated with samarium-153-EDTMP treatment. METHODS: Seventy-three patients with metastatic bone pain having previously undergone therapy with samarium-153-EDTMP (1 mCi/kg) were retrospectively evaluated. Routine follow-up included pain evaluation and blood counts for 2 months after treatment. Pain was evaluated using a subjective scale (from 0 to 10) before and for 8 weeks after the treatment. Blood counts were obtained before treatment and once a week for 2 months during follow-up. Dosimetry, based upon the urinary excretion of the isotope, was estimated in 41 individuals, and the resulting radiation absorbed doses were correlated with hematological data. RESULTS: Reduction in pain scores of 75% to 100% was obtained in 36 patients (49%), with a decrease of 50% to 75%, 25% to 50%, and 0% to 25% in, respectively, 20 (27%), 10 (14%), and 7 (10%) patients. There was no significant relationship between the pain response and location of the primary tumor (breast or prostate cancer). Mild to moderate myelosuppression was noted in 75.3% of patients, usually with hematological recovery at 8 weeks. The mean bone marrow dose was 347 ± 65 cGy, and only a weak correlation was found between absorbed dose and myelosuppression (Pearson coefficient = .4). CONCLUSIONS: Samarium-153-EDTMP is a valuable method for metastatic bone pain palliation. A mild to moderate and transitory myelosuppression is the main toxicity observed after samarium therapy, showing a weak correlation with dosimetric measures.

Expression of histone methyltransferases as novel biomarkers for renal cell tumor diagnosis and prognostication

Renal cell tumors (RCTs) are the most lethal of the common urological cancers. The widespread use of imaging entailed an increased detection of small renal masses, emphasizing the need for accurate distinction between benign and malignant RCTs, which is critical for adequate therapeutic management. Histone methylation has been implicated in renal tumorigenesis, but its potential clinical value as RCT biomarker remains mostly unexplored. Hence, the main goal of this study was to identify differentially expressed histone methyltransferases (HMTs) and histone demethylases (HDMs) that might prove useful for RCT diagnosis and prognostication, emphasizing the discrimination between oncocytoma (a benign tumor) and renal cell carcinoma (RCC), especially the chromophobe subtype (chRCC). We found that the expression levels of three genes-SMYD2, SETD3, and NO66-was significantly altered in a set of RCTs, which was further validated in a large independent cohort. Higher expression levels were found in RCTs compared to normal renal tissues (RNTs) and in chRCCs comparatively to oncocytomas. SMYD2 and SETD3 mRNA levels correlated with protein expression assessed by immunohistochemistry. SMYD2 transcript levels discriminated RCTs from RNT, with 82.1% sensitivity and 100% specificity (AUC=0.959), and distinguished chRCCs from oncocytomas, with 71.0% sensitivity and 73.3% specificity (AUC: 0.784). Low expression levels of SMYD2, SETD3, and NO66 were significantly associated with shorter disease-specific and disease-free survival, especially in patients with non-organ confined tumors. We conclude that expression of selected HMTs and HDMs might constitute novel biomarkers to assist in RCT diagnosis and assessment of tumor aggressiveness.

Redirection of T cells by delivering a transgenic mouse-derived MDM2 tumor antigen-specific TCR and its humanized derivative is governed by the CD8 coreceptor and affects natural human TCR expression.

Retroviral transfer of T cell antigen receptor (TCR) genes selected by circumventing tolerance to broad tumor- and leukemia-associated antigens in human leukocyte antigen (HLA)-A*0201 (A2.1) transgenic (Tg) mice allows the therapeutic reprogramming of human T lymphocytes. Using a human CD8 x A2.1/Kb mouse derived TCR specific for natural peptide-A2.1 (pA2.1) complexes comprising residues 81-88 of the human homolog of the murine double-minute 2 oncoprotein, MDM2(81-88), we found that the heterodimeric CD8 alpha beta coreceptor, but not normally expressed homodimeric CD8 alpha alpha, is required for tetramer binding and functional redirection of TCR- transduced human T cells. CD8+T cells that received a humanized derivative of the MDM2 TCR bound pA2.1 tetramers only in the presence of an anti-human-CD8 anti-body and required more peptide than wild-type (WT) MDM2 TCR+T cells to mount equivalent cytotoxicity. They were, however, sufficiently effective in recognizing malignant targets including fresh leukemia cells. Most efficient expression of transduced TCR in human T lymphocytes was governed by mouse as compared to human constant (C) alphabeta domains, as demonstrated with partially humanized and murinized TCR of primary mouse and human origin, respectively. We further observed a reciprocal relationship between the level of Tg WT mouse relative to natural human TCR expression, resulting in T cells with decreased normal human cell surface TCR. In contrast, natural human TCR display remained unaffected after delivery of the humanized MDM2 TCR. These results provide important insights into the molecular basis of TCR gene therapy of malignant disease.

Brain-derived neurotrophic factor enhances the hippocampal expression of key postsynaptic proteins in vivo including the monocarboxylate transporter MCT2.

Brain-derived neurotrophic factor (BDNF) promotes synaptic plasticity via an enhancement in expression of specific synaptic proteins. Recent results suggest that the neuronal monocarboxylate transporter MCT2 is a postsynaptic protein critically involved in synaptic plasticity and long-term memory. To investigate in vivo whether BDNF can modulate the expression of MCT2 as well as other proteins involved in synaptic plasticity, acute injection of BDNF was performed in mouse dorsal hippocampal CA1 area. Using immunohistochemistry, it was found that MCT2 expression was enhanced in part of the CA1 area and in the dentate gyrus 6 h after a single intrahippocampal injection of BDNF. Similarly, expression of the immediate early genes Arc and Zif268 was enhanced in the same hippocampal areas, in accordance with their role in synaptic plasticity. Immunoblot analysis confirmed the significant enhancement in MCT2 protein expression. In contrast, no changes were observed for the glial monocarboxylate transporters MCT1 and MCT4. When other synaptic proteins were investigated, it was found that postsynaptic density 95 (PSD95) and glutamate receptor 2 (GluR2) protein levels were significantly enhanced while no effect could be detected for synaptophysin, synaptosomal-associated protein 25 (SNAP25), αCaMKII and GluR1. These results demonstrate that MCT2 expression can be upregulated together with other key postsynaptic proteins in vivo under conditions related to synaptic plasticity, further suggesting the importance of energetics for memory formation.

Tumor necrosis factor (TNF) signaling, but not TWEAK (TNF-like weak inducer of apoptosis)-triggered cIAP1 (cellular inhibitor of apoptosis protein 1) degradation, requires cIAP1 RING dimerization and E2 binding.

Cellular inhibitor of apoptosis (cIAP) proteins, cIAP1 and cIAP2, are important regulators of tumor necrosis factor (TNF) superfamily (SF) signaling and are amplified in a number of tumor types. They are targeted by IAP antagonist compounds that are undergoing clinical trials. IAP antagonist compounds trigger cIAP autoubiquitylation and degradation. The TNFSF member TWEAK induces lysosomal degradation of TRAF2 and cIAPs, leading to elevated NIK levels and activation of non-canonical NF-kappaB. To investigate the role of the ubiquitin ligase RING domain of cIAP1 in these pathways, we used cIAP-deleted cells reconstituted with cIAP1 point mutants designed to interfere with the ability of the RING to dimerize or to interact with E2 enzymes. We show that RING dimerization and E2 binding are required for IAP antagonists to induce cIAP1 degradation and protect cells from TNF-induced cell death. The RING functions of cIAP1 are required for full TNF-induced activation of NF-kappaB, however, delayed activation of NF-kappaB still occurs in cIAP1 and -2 double knock-out cells. The RING functions of cIAP1 are also required to prevent constitutive activation of non-canonical NF-kappaB by targeting NIK for proteasomal degradation. However, in cIAP double knock-out cells TWEAK was still able to increase NIK levels demonstrating that NIK can be regulated by cIAP-independent pathways. Finally we show that, unlike IAP antagonists, TWEAK was able to induce degradation of cIAP1 RING mutants. These results emphasize the critical importance of the RING of cIAP1 in many signaling scenarios, but also demonstrate that in some pathways RING functions are not required.

Simultaneous CD8+ T cell responses to multiple tumor antigen epitopes in a multipeptide melanoma vaccine.

The recent identification and molecular characterization of tumor-associated antigens recognized by tumor-reactive CD8+ T lymphocytes has led to the development of antigen-specific immunotherapy of cancer. Among other approaches, clinical studies have been initiated to assess the in vivo immunogenicity of tumor antigen-derived peptides in cancer patients. In this study, we have analyzed the CD8+ T cell response of an ocular melanoma patient to a vaccine composed of four different tumor antigen-derived peptides administered simultaneously in incomplete Freund's adjuvant (IFA). Peptide NY-ESO-1(157-165) was remarkably immunogenic and induced a CD8+ T cell response detectable ex vivo at an early time point of the vaccination protocol. A CD8+ T cell response to the peptide analog Melan-A(26-35 A27L) was also detectable ex vivo at a later time point, whereas CD8+ T cells specific for peptide tyrosinase(368-376) were detected only after in vitro peptide stimulation. No detectable CD8+ T cell response to peptide gp100(457-466) was observed. Vaccine-induced CD8+ T cell responses declined rapidly after the initial response but increased again after further peptide injections. In addition, tumor antigen-specific CD8+ T cells were isolated from a vaccine injection site biopsy sample. Importantly, vaccine-induced CD8+ T cells specifically lysed tumor cells expressing the corresponding antigen. Together, these data demonstrate that simultaneous immunization with multiple tumor antigen-derived peptides can result in the elicitation of multiepitope-directed CD8+ T cell responses that are reactive against antigen-expressing tumors and able to infiltrate antigen-containing peripheral sites.

Viral FLICE-inhibitory proteins (FLIPs) prevent apoptosis induced by death receptors.

Viruses have evolved many distinct strategies to avoid the host's apoptotic response. Here we describe a new family of viral inhibitors (v-FLIPs) which interfere with apoptosis signalled through death receptors and which are present in several gamma-herpesviruses (including Kaposi's-sarcoma-associated human herpesvirus-8), as well as in the tumorigenic human molluscipoxvirus. v-FLIPs contain two death-effector domains which interact with the adaptor protein FADD, and this inhibits the recruitment and activation of the protease FLICE by the CD95 death receptor. Cells expressing v-FLIPs are protected against apoptosis induced by CD95 or by the related death receptors TRAMP and TRAIL-R. The herpesvirus saimiri FLIP is detected late during the lytic viral replication cycle, at a time when host cells are partially protected from CD95-ligand-mediated apoptosis. Protection of virus-infected cells against death-receptor-induced apoptosis may lead to higher virus production and contribute to the persistence and oncogenicity of several FLIP-encoding viruses.

Anti-tumor necrosis factor drug survival in axial spondyloarthritis is independent of the classification criteria.

To compare the impact of meeting specific classification criteria [modified New York (mNY), European Spondyloarthropathy Study Group (ESSG), and Assessment of SpondyloArthritis international Society (ASAS) criteria] on anti-tumor necrosis factor (anti-TNF) drug retention, and to determine predictive factors of better drug survival. All patients fulfilling the ESSG criteria for axial spondyloarthritis (SpA) with available data on the axial ASAS and mNY criteria, and who had received at least one anti-TNF treatment were retrospectively retrieved in a single academic institution in Switzerland. Drug retention was computed using survival analysis (Kaplan-Meier), adjusted for potential confounders. Of the 137 patients classified as having axial SpA using the ESSG criteria, 112 also met the ASAS axial SpA criteria, and 77 fulfilled the mNY criteria. Drug retention rates at 12 and 24 months for the first biologic therapy were not significantly different between the diagnostic groups. Only the small ASAS non-classified axial SpA group (25 patients) showed a nonsignificant trend toward shorter drug survival. Elevated CRP level, but not the presence of bone marrow edema on magnetic resonance imaging (MRI) scans, was associated with significantly better drug retention (OR 7.9, ICR 4-14). In this cohort, anti-TNF drug survival was independent of the classification criteria. Elevated CRP level, but not positive MRI, was associated with better drug retention.

Targeting mTORC2 inhibits colon cancer cell proliferation in vitro and tumor formation in vivo.

The mammalian target of rapamycin (mTOR), which exists in two functionally distinct complexes, mTORC1 and mTORC2 plays an important role in tumor growth. Whereas the role of mTORC1 has been well characterized in this process, little is known about the functions of mTORC2 in cancer progression. In this study, we explored the specific role of mTORC2 in colon cancer using a short hairpin RNA expression system to silence the mTORC2-associated protein rictor. We found that downregulation of rictor in HT29 and LS174T colon cancer cells significantly reduced cell proliferation. Knockdown of rictor also resulted in a G1 arrest as observed by cell cycle analysis. We further observed that LS174T cells deficient for rictor failed to form tumors in a nude mice xenograft model. Taken together, these results show that the inhibition of mTORC2 reduces colon cancer cell proliferation in vitro and tumor xenograft formation in vivo. They also suggest that specifically targeting mTORC2 may provide a novel treatment strategy for colorectal cancer.

Selective abrogation of major histocompatibility complex class II expression on extrahematopoietic cells in mice lacking promoter IV of the class II transactivator gene.

MHC class II (MHCII) molecules play a pivotal role in the induction and regulation of immune responses. The transcriptional coactivator class II transactivator (CIITA) controls MHCII expression. The CIITA gene is regulated by three independent promoters (pI, pIII, pIV). We have generated pIV knockout mice. These mice exhibit selective abrogation of interferon (IFN)-gamma-induced MHCII expression on a wide variety of non-bone marrow-derived cells, including endothelia, epithelia, astrocytes, and fibroblasts. Constitutive MHCII expression on cortical thymic epithelial cells, and thus positive selection of CD4(+) T cells, is also abolished. In contrast, constitutive and inducible MHCII expression is unaffected on professional antigen-presenting cells, including B cells, dendritic cells, and IFN-gamma-activated cells of the macrophage lineage. pIV(-/-) mice have thus allowed precise definition of CIITA pIV usage in vivo. Moreover, they represent a unique animal model for studying the significance and contribution of MHCII-mediated antigen presentation by nonprofessional antigen-presenting cells in health and disease.

Tissue-specific segregation of CD1d-dependent and CD1d-independent NK T cells.

NKT cells, defined as T cells expressing the NK cell marker NK1.1, are involved in tumor rejection and regulation of autoimmunity via the production of cytokines. We show in this study that two types of NKT cells can be defined on the basis of their reactivity to the monomorphic MHC class I-like molecule CD1d. One type of NKT cell is positively selected by CD1d and expresses a biased TCR repertoire together with a phenotype found on activated T cells. A second type of NKT cell, in contrast, develops in the absence of CD1d, and expresses a diverse TCR repertoire and a phenotype found on naive T cells and NK cells. Importantly, the two types of NKT cells segregate in distinct tissues. Whereas thymus and liver contain primarily CD1d-dependent NKT cells, spleen and bone marrow are enriched in CD1d-independent NKT cells. Collectively, our data suggest that recognition of tissue-specific ligands by the TCR controls localization and activation of NKT cells.

Differential distribution of tau proteins in developing cat cerebellum.

The differential distribution and phosphorylation of tau proteins in cat cerebellum was studied with two well characterized antibodies, TAU-1 and TAU-2. TAU-1 detects tau proteins in axons, and the epitope in perikarya and dendrites is masked by phosphorylation. TAU-2 detects a phosphorylation-independent epitope on tau proteins. The molecular composition of tau proteins in the range of 45 kD to 64 kD at birth changed after the first postnatal month to a set of several adult variants of higher molecular weights in the range of 59 kD to 95 kD. The appearance of tau proteins in subsets of axons corresponds to the axonal maturation of cerebellar local-circuit neurons in granular and molecular layers and confirms previous studies. Tau proteins were also identified in synapses by immunofluorescent double-staining with synapsin I, located in the pinceau around the Purkinje cells, and in glomeruli. Dephosphorylation of juvenile cerebellar tissue by alkaline phosphatase indicated indirectly the presence of differentially phosphorylated tau forms mainly in juvenile ages. Additional TAU-1 immunoreactivity was unmasked in numerous perikarya and dendrites of stellate cells, and in cell bodies of granule cells. Purkinje cell bodies were stained transiently at juvenile ages. During postnatal development, the intensity of the phosphate-dependent staining decreased, suggesting that phosphorylation of tau proteins in perikarya and dendrites may be essential for early steps in neuronal morphogenesis during cat cerebellum development.

Giant extraskeletal myxoid chondrosarcoma of the thigh treated by wide extraarticular resection and reconstruction with a tumor prosthesis

Introduction: Extraskeletal myxoid chondrosarcoma (EMC) is a rare soft tissue tumour with a high risk for local recurrence and metastases. While this entity is resistant to radio- or chemo-therapy, wide resection remains the treatment of choice. Case report: A 60 year old man presented to our service with a large mass in his right thigh, slowly evolving over the past 7 years. His main complaint was the volume of his thigh. Imaging showed a 23x13x14 cm tumour in the quadriceps, eroding the cortical bone and with potential contamination of the knee joint. The risk of a pathological fracture was estimated considerable. A CT-guided core-needle biopsy revealed a FNCLCC grade 2 EMC. A thoraco-abdominal CT scan showed multiple pulmonary metastases. Due to the palliative situation with a very slow disease progression, a wide extraarticular resection of the distal femur and reconstruction with a megaprosthesis were performed. Extensive skin necrosis necessitated three revision procedures for débridement and confection of a pediculated lateral gastrocnemius muscle flap. No complementary treatment was possible for the pulmonary metastases. At 18 months follow-up, he walked without crutches, was able to do his activities of daily living. He was painfree and highly satisfied with the result. During the follow-up, slow progression of the pulmonary metastases was noted, which remained asymptomatic. Conclusion: Extraskeletal myxoid chondrosarcoma is a rare soft tissue tumour, and wide excision remains the treatment of choice. Whenever possible, limb salvage should be proposed to preserve function and quality of life.