The plant diterpene epoxysiderol targets Hsp70 in cancer cells, affecting its ATPase activity and reducing its translocation to plasma membrane.

The ATP-dependent molecular chaperone Hsp70 is over-expressed in cancer cells where it plays pivotal roles in stabilization of onco-proteins, promoting cell proliferation and protecting cells from apoptosis and necrosis. Moreover, a relationship between the ability of cancer cells to migrate and the abundance of membrane-associated Hsp70 was shown. However, although Hsp70 is a promising target for cancer therapy, there is a still unsatisfied requirement of inhibitors possibly blocking its cancer-associated activities. Moving from the evidence that the plant diterpene oridonin efficiently targets Hsp70 1A in cancer cells, we set up a small kaurane diterpenoids collection and subjected it to a Surface Plasmon Resonance-screening, to identify new putative inhibitors of this chaperone. The results obtained suggested epoxysiderol as an effective Hsp70 1A interactor; therefore, using a combination of bioanalytical, biochemical and bioinformatics approaches, this compound was shown to bind the nucleotide-binding-domain of the chaperone, thus affecting its ATPase activity. The interaction between epoxysiderol and Hsp70 1A was also demonstrated to actually occur inside cancer cells, significantly reduced the translocation of the chaperone to the cell membrane, thus suggesting a possible role of epoxysiderol as an anti-metastasis agent.

Innovative Therapy, Monoclonal Antibodies and Beyond.

The seventh Edition of "Innovative Therapy, Monoclonal Antibodies and Beyond" Meeting took place in Milan, Italy, on January 27, 2017. The two sessions of the meeting were focused on: 1) Preclinical assays and novel biotargets; and 2) monoclonal antibodies, cell therapies and targeted molecules. Between these two sessions, a lecture entitled "HLA-antigens modulation and response to immune checkpoint inhibitor immunotherapy" was also presented. Despite the impressive successes in cancer immunotherapy in recent years, the response to immune based interventions occurs only in a minority of patients (∼20%). Several basic and translational mechanisms of resistance to immune checkpoint blockers (ICBs) were discussed during the meeting: 1. the impact of tumor microenvironment on the activity of immune system; 2. strategies to inhibit the cross-talk between extracellular matrix and myeloid-derived suppressor cells (MDSC) in the preclinical setting; 3. microRNA expression as a biomarker and as a target of therapy in non-small cell lung cancer (NSCLC); 4. the significance of complement activation pathways in response to immune checkpoint inhibitors; 5. the immunosuppressive activity of the microbiota by inducing IL-17 producing cells; and 6. modulation of HLA antigens as possible markers of response to ICB therapy. In order to overcome the deficiency in active anti-tumor T cells, several clinically applicable combination strategies were also discussed: 1. strategies to enhance the anticancer effects of immunogenic cell death inducing-chemotherapy; 2. the use of CAR T-cells in solid tumors; 3. the use of combination strategies involving oncolytic viruses and ICBs; 4. combinations of new ICBs with anti-PD-1/CTLA-4 therapy; and 4. combinations of targeted therapies and ICBs in melanoma. Overall, this conference emphasized the many novel strategies that are being investigated to improve the overall patient response to cancer immunotherapy. Optimization of biomarkers to accurately select patients who will respond to immunotherapy, coupled with combination strategies to improve long term patient survival remain critical challenges in the immuno-oncology field.

Targeting vasculogenesis to prevent progression in multiple myeloma.

The role of endothelial progenitor cell (EPC)-mediated vasculogenesis in hematological malignancies is not well explored. Here, we showed that EPCs are mobilized from the bone marrow (BM) to the peripheral blood at early stages of multiple myeloma (MM); and recruited to MM cell-colonized BM niches. Using EPC-defective ID1+/- ID3-/- mice, we found that MM tumor progression is dependent on EPC trafficking. By performing RNA-sequencing studies, we confirmed that endothelial cells can enhance proliferation and favor cell-cycle progression only in MM clones that are smoldering-like and have dependency on endothelial cells for tumor growth. We further confirmed that angiogenic dependency occurs early and not late during tumor progression in MM. By using a VEGFR2 antibody with anti-vasculogenic activity, we demonstrated that early targeting of EPCs delays tumor progression, while using the same agent at late stages of tumor progression is ineffective. Thus, although there is significant angiogenesis in myeloma, the dependency of the tumor cells on EPCs and vasculogenesis may actually precede this step. Manipulating vasculogenesis at an early stage of disease may be examined in clinical trials in patients with smoldering MM, and other hematological malignancies with precursor conditions.

Antisense transcription at the TRPM2 locus as a novel prognostic marker and therapeutic target in prostate cancer.

Overwhelming evidence indicates that cancer is a genetic disease caused by the accumulation of mutations in oncogenes and tumor suppressor genes. It is also increasingly apparent, however, that cancer depends not only on mutations in these coding genes but also on alterations in the large class of non-coding RNAs. Here, we report that one such long non-coding RNA, TRPM2-AS, an antisense transcript of TRPM2, which encodes an oxidative stress-activated ion channel, is overexpressed in prostate cancer (PCa). The high expression of TRPM2-AS and its related gene signature were found to be linked to poor clinical outcome, with the related gene signature working also independently of the patient's Gleason score. Mechanistically, TRPM2-AS knockdown led to PCa cell apoptosis, with a transcriptional profile that indicated an unbearable increase in cellular stress in the dying cells, which was coupled to cell cycle arrest, an increase in intracellular hydrogen peroxide and activation of the sense TRPM2 gene. Moreover, targets of existing drugs and treatments were found to be consistently associated with high TRPM2-AS levels in both targeted cells and patients, ultimately suggesting that the measurement of the expression levels of TRPM2-AS allows not only for the early identification of aggressive PCa tumors, but also identifies a subset of at-risk patients who would benefit from currently available, but mostly differently purposed, therapeutic agents.

hI-con1, a factor VII-IgGFc chimeric protein targeting tissue factor for immunotherapy of uterine serous papillary carcinoma.

BACKGROUND: Uterine serous papillary adenocarcinoma (USPC) is a highly aggressive variant of endometrial cancer. Human immuno-conjugate molecule (hI-con1) is an antibody-like molecule targeted against tissue factor (TF), composed of two human Factor VII (fVII) as the targeting domain, fused to human immunoglobulin (Ig) G1 Fc as an effector domain. We evaluated hI-con1 potential activity against primary chemotherapy-resistant USPC cell lines expressing different levels of TF. METHODS: A total of 16 formalin-fixed, paraffin-embedded USPC samples were evaluated by immunohistochemistry (IHC) for TF expression. Six primary USPC cell lines, half of which overexpress the epidermal growth factor type II (HER2/neu) receptor at 3+ levels, were assessed by flow cytometry and real-time PCR for TF expression. Sensitivity to hI-con1-dependent cell-mediated cytotoxicity (IDCC) was evaluated in 5-hour-chromium release assays. Finally, to investigate the effect of interleukin-2 (IL-2) on IDCC, 5-h (51)Cr assays were also conducted in the presence of low doses of IL-2 (i.e., 50-100 IU ml(-1)). RESULTS: Cytoplasmic and/or membrane TF expression was observed in all 16 (100%) USPC samples tested by IHC, but not in normal endometrium. High expression of TF was found in 50% (three out of six) of the USPC cell lines tested by real-time PCR and flow cytometry when compared with normal endometrial cells (NECs; P<0.001). Uterine serous papillary adenocarcinoma cell lines overexpressing TF, regardless of their high or low HER2/neu expression, were highly sensitive to IDCC (mean killing+/-s.d., 65.6+/-3.7%, range 57.5-77.0%, P<0.001), although negligible cytotoxicity against USPC was seen in the absence of hI-con1 or in the presence of Rituximab control antibody. The addition of low doses of IL-2 further increased the cytotoxic effect induced by hI-con1 against chemotherapy-resistant USPC. CONCLUSION: hI-con1 induces strong cytotoxicity against primary chemotherapy-resistant USPC cell lines overexpressing TF. The hI-con1 may represent a novel therapeutic agent for the treatment of patients harbouring advanced, recurrent and/or metastatic USPC refractory to standard treatment modalities.

In vitro activity of pertuzumab in combination with trastuzumab in uterine serous papillary adenocarcinoma.

BACKGROUND: Uterine serous papillary adenocarcinoma (USPC) is a rare but highly aggressive variant of endometrial cancer. Pertuzumab is a new humanised monoclonal antibody (mAb) targeting the epidermal growth factor type II receptor (HER2/neu). We evaluated pertuzumab activity separately or in combination with trastuzumab against primary USPC cell lines expressing different levels of HER2/neu. METHODS: Six USPC cell lines were assessed by immunohistochemistry (IHC), flow cytometry, and real-time PCR for HER2/neu expression. c-erbB2 gene amplification was evaluated using fluorescent in situ hybridisation (FISH). Sensitivity to pertuzumab and trastuzumab-induced antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) was evaluated in 5 h chromium release assays. Pertuzumab cytostatic activity was evaluated using proliferation-based assays. RESULTS: Three USPC cell lines stained heavily for HER2/neu by IHC and showed amplification of the c-erbB2 gene by FISH. The remaining FISH-negative USPCs expressed HER2/neu at 0/1+ levels. In cytotoxicity experiments against USPC with a high HER2/neu expression, pertuzumab and trastuzumab were similarly effective in inducing strong ADCC. The addition of complement-containing plasma and interleukin-2 increased the cytotoxic effect induced by both mAbs. In low HER2/neu USPC expressors, trastuzumab was more potent than pertuzumab in inducing ADCC. Importantly, in this setting, the combination of pertuzumab with trastuzumab significantly increased the ADCC effect induced by trastuzumab alone (P=0.02). Finally, pertuzumab induced a significant inhibition in the proliferation of all USPC cell lines tested, regardless of their HER-2/neu expression. CONCLUSION: Pertuzumab and trastuzumab induce equally strong ADCC and CDC in FISH-positive USPC cell lines. Pertuzumab significantly increases tratuzumab-induced ADCC against USPC with a low HER2/neu expression and may represent a new therapeutic agent in patients harbouring advanced/recurrent and/or refractory USPC.

[Study on psychiatric disorders and defensive process assessed by the "defense style questionnaire" in sterile males SAMPLE consulting in andrology]. / Etude des troubles psychiatriques et des modalités défensives évaluées par le "Defense Style Questionnaire" (DSQ) dans un echantillon d'hommes stériles consultant en andrologie.

BACKGROUND: The literature about artificial insemination and the associated psychological, psychiatric and sexual disorders is relatively rich. But the majority of these studies is made in gynaecology, with a feminine approach of the disorder. There are very few works led in andrology. This justified the investigation of new trails in order to understand better the clinical context of the sterile man. We undertake a study about the psychiatric disorders among sterile men and about the defense styles. These are a clinical entity recently introduced in the quantitative psychopathology research. The defense style questionnaire (DSQ) is a psychometric scale used in common practice in order to measure the defense styles. OBJECTIVES: We made this study in order to examine the psychiatric state of a sterile males sample consulting in andrology; to assess the defense style by means of the Bond and al DSQ-88 ; to look into a difference between the defensive process according to their clinical situation of azoospermic males or as the oligoazoospermic males and finally, to reveal a correlation between the psychiatric disorders developed in this sample of sterile males and the defensive process they used. METHOD: There were 42 people (22 azoospermic males and 20 oligoazoospermic males) aged between 23 and 49 years old in the analysed sample. These have been selected at the surgery of andrology at the RUHC of Lille, depending on their arrival order for 6 months. There was no significant difference between the two groups as far as the age and the education standard are concerned. The selection criteria were medical and somatic. Our sample population were divided into two groups: azoospermia (no spermatozoon found in the semen analysis) and oligoasthenospermia (decrease of the number and the mobility of the spermatozoa and an increase of the percentage of atypical forms). The method first consisted in the DSQ, followed by the analysis of the psychiatric state according to the DSM IV, a hetero questionnaire to collect some general information about infertility and a self questionnaire about the sexual, conjugal and social effects of infertility. The DSQ and the interviews took place in the andrology department with the same investigator trained for this job. RESULTS: We found in our sample 26.2% of psychiatric disorders according to the DSM IV with a significant over-representation of generalized anxious disorder and somatization disorder. The comparison between azoospermic males and oligoazoospermic males patients showed the absence of significative difference as far as psychiatric morbidity rate and the use of defense styles are concerned. DISCUSSION: Our sample defended himself in accordance with modalities similar to the general population and used defense mechanisms preferentially belonging to the mature defense style, such as humor, repression and anticipation. The psychiatric pathology was significantly correlated to the preferential use of withdrawal, consumption, reaction formation and lack of humor use. We also confirm in our study the fact that the subjects using especially neurotic defense styles are more likely to develop a psychiatric disorder than the others. Our male sample is a waiting population and threatened by failure. The situation of wait creates anxiety. We also know that infertility is one of the most stressful situations a couple might face. However, our study did not enable us to know the precise relations between generalized anxious disorder and infertility, especially whether the generalized anxious disorder preceded this pathology or not. The over-representation of a somatization disorder only allows us to acknowledge its existence. We can also deduce from that a possible link between infertility and psychic disorder, even if no research permitted to affirm to date the existence of interrelations linking infertility and psychic life. On the whole, this population was suffering despite 73.8% of the patients had no confirmed psychiatric disorder. It is the reason why a liaison psychiatry more inserted into highly specialized teams is interesting, especially because it includes a medical and psychological approach of such disorders. The defense mechanisms preferentially used by this population were humor, repression and anticipation. Humor can only be considered as a defense mechanism when it is applied to oneself. The population who has no psychiatric disorder more uses humor. Does humor protect against the development of a psychiatric pathology, as certain authors proved it ? On the other hand, is repression really protective? It didn't interfere in our study about the development of a psychiatric pathology. So we can suppose that repression was protective for our whole sample, but we can not prove it. However, we wonder if this mechanism works after the failure of an artificial insemination is announced. In which measure such a stress can be repressed out of the conscience field? As for anticipation, it is used by our population who is for the most part in good health. But the question is to know if our sample really envisaged all the different possible solutions or only the success of artificial insemination. As some other works, we confirm that the, psychiatric, people significantly use the neurotic style. Our psychiatric patients used less humor and more consumption, withdrawal and reaction formation than the sane people. Consumption is rarely considered as a defense mechanism by some other authors. And yet, consumption and the existence of psychiatric disorders were very closely linked. This association is found again with anxiety in other studies. The correlation between psychiatric disorder and withdrawal was veryimportant too. The DSM lV defines withdrawal as an apathetic withdrawal. It is not an apathetic withdrawal in our population because the average scores for the ,, activity >, defense mechanism remained high. In our sample, the use of this defense mechanism would encourage the expression of psychiatric troubles. The reaction formation quoted by Freud and Bergeret are both valorised in our society. What kinds of reaction formations use these men ? Are they pathological ? Our study can not answer to these questions. However, the DSQ items examining the reaction formation present its "socially promoted" aspect and forget the pathological one. It has been showed that the evaluation of the defense modalities in a certain type of population can allow the emergence of specific defense mechanisms. This can be considered as predictive factors of development of a mental pathology. The evaluation of specific mental defenses could permit to define vulnerability and affinity for given affections instead of simple personality traits or profiles. Most part of the works shows results in favour of the capacity of DSQ to assess the different defense mechanisms according to the diagnosis groups. But the insufficient numbers of studies moderate on the whole the hypothesis of the existence of specific defense mechanisms--protective factors and factors of vulnerability--linked to a given psychiatric disorder. CONCLUSION: There is not a difference of psychological effect in terms of degree of sterility. On the other hand, the existence of over-represented psychiatric disorders with sterile males compared with a control group force Consultation-Liaison psychiatrists and andrologists would be able to understand the pain beyond the need of acting by the artificial insemination. In our opinion, this justifies the fact that the patients should have the opportunity of expressing, in the department where they are treated, all the feelings inherent to their personal and conjugal drama as part of a specialized treatment. Our study confirms the difficulty to know whether some defense mechanisms are vulnerability factors for a certain psychiatric disorder or whether the defense mechanisms are an epiphenomenon of a particular psychiatric disorder. This is the reason why a lot of authors having worked with DSQ agree to conclude that additional prospective studies, which would permit to make a link between the defense mechanisms anda certain psychiatric pathology, are necessary. In the case we study, it is important to explore the defensive modalities before the infertility diagnosis and after the birth of a child, with a more important sample population. A better knowledge of the defensive modalities of such a population, used in a psychotherapeutic context could help to prevent the appearance of psychiatric disorders or, if not, to anticipate them.

Autoantibodies against a 72-kDa ductal cell membrane glycoprotein in a patient affected by Sjögren's syndrome and gastric MALT lymphoma.

Sjögren's syndrome is a chronic autoimmune disease affecting exocrine glands, resulting in xerostomia and xerophthalmia. Lymphocytic infiltration and fibrosis of exocrine glands as well as the presence of autoantibodies against organ-specific and non-organ-specific antigens are the hallmarks of the disease. We investigated whether some patients affected by Sjögren's syndrome might have autoantibodies directed against epithelial duct cell membrane proteins. We screened sera from patients affected by Sjögren's syndrome by indirect immunofluorescence on monkey salivary gland sections and FG-Met-2 cells (a pancreatic carcinoma cell line with ductal features) for the presence of antisalivary duct antibodies. Positive sera were employed in immunoprecipitation experiments on (35)S-methionine in vivo labeled and surface-biotinylated FG-Met-2 cells. The serum of a patient affected by Sjögren's syndrome and gastric mucosa-associated lymphoid tissue (MALT) lymphoma gave positive and distinct membrane immunostaining on FG-Met-2 cells. Immunoprecipitation with the patient's serum from (35)S-methionine-labeled cell extracts followed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and autoradiography showed the presence of autoantibodies against a 72-kDa protein. After biotin-surface labeling of FG-Met-2 cells, a band with identical electrophoretic mobility was immunoprecipitated by the serum, demonstrating that the 72-kDa band is a membrane glycoprotein. We demonstrated by three complementary approaches, i.e., immunocytochemistry, (35)S-methionine in vivo labeling, and cell surface biotinylation, the presence of autoantibodies directed against a duct cell membrane protein of 72-kDa in a patient affected by Sjögren's syndrome and gastric MALT lymphoma. Autoantibodies directed against this novel membrane autoantigen may be an additional serological marker in some cases of Sjögren's syndrome.

Takayasu's arteritis: therapeutic strategies.

Takayasu's arteritis (TA) is a chronic inflammatory disease of unknown origin, primarily affecting the walls of large vessels. TA can be a severe and life-threatening disease, and has relevant consequences for the patient's life-style. Mortality and morbidity depend on both the direct effect of the vascular lesions and the following complications. The mainstay of TA therapy is based on the use of glucocorticoids alone or in association with cytotoxic drugs. Unfortunately, in the majority of cases, only a partial control of the disease is obtained. The therapeutic strategy may vary in different countries, and in Japan, where the disease was first described, high dose glucocorticoids are preferred to glucocorticoids in association with cytotoxic agents. We present here a review of the pharmacologic strategies most commonly adopted for the treatment of TA in America, Italy and Japan, together with our experience on 31 TA patients, who have been followed in the last two decades. The discussion is also open on which criteria are more accurate in measuring disease activity.

Relevance of the tumor antigen in the validation of three vaccination strategies for melanoma.

Many preclinical studies of cancer immunotherapy are based on the testing of a single vaccination strategy in several tumor models. Moreover, most of those studies used xenogeneic Ags, which, owing to their high immunogenicity, may not represent realistic models for the validation of cancer immunotherapies. To address these issues, we compared the vaccination efficacy of three well established strategies (i.e., naked DNA; peptide-pulsed dendritic cells (DC), or a mixture of peptide and the Escherichia coli toxin LTR72) using the xenogeneic OVA or the naturally expressed tyrosinase-related protein 2 (TRP-2) tumor Ag in the B16 melanoma model. C57BL/6 mice received one to three s.c. injections of peptide-pulsed DC or DNA, or one to four mucosal administrations of peptide-toxin mixture. One to 2 wk later, the animals were challenged s.c. with B16 or B16 cells expressing OVA (B16-OVA). Vaccination of mice with OVA induced in all cases melanoma-specific CTL and protection against B16-OVA. When TRP-2 was used, all three vaccines elicited B16-specific CTL, but only DC pulsed with the immunodominant T cell epitope TRP-2181-188 allowed protection against B16. Even more importantly, a vaccination regimen with TRP-2-pulsed DC, started 24 h after the injection of a lethal number of B16 cells, caused a therapeutic effect in 60% of the challenged animals. Our results strongly emphasize the relevance of the tumor Ag in the definition of immunotherapeutic strategies for cancer, and support the use of peptide-pulsed DC as cancer vaccine in humans.

Thymoma associated with systemic lupus erythematosus and immunologic abnormalities.

The association between Systemic Lupus Erythematosus (SLE) and thymoma occurs with a greater frequency than dictated by coincidence alone. The immunologic effects of thymectomy on the appearance and/or the course of SLE are still to be elucidated. We report one case of SLE diagnosed at the same time as thymoma, and two cases of thymoma associated with immunologic disorders in the absence of clinical signs and symptoms diagnostic of SLE.

Apoptosis, cross-presentation, and the fate of the antigen specific immune response.

Induction of cell death by apoptosis, also called programmed cell death, and clearance of apoptotic bodies by scavenger cells has long thought to be an efficient means to dispose of unwanted cells without causing inflammatory responses able to mediate specific reactions. However, a number of evidences have been accumulated suggesting that apoptotic cell death is implicated in the pathogenesis of systemic and organ specific autoimmune diseases. In addition, recognition and engulfement of apoptotic cells by professional antigen presenting cells, such as dendritic cells, and their interaction with effector immune cells have been recently described to result in apoptotic cell-derived antigen specific tolerance. This review will summarise the most recent findings on the immunogenic potential of cells undergoing programmed death.