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.

Melanoma cells present a MAGE-3 epitope to CD4(+) cytotoxic T cells in association with histocompatibility leukocyte antigen DR11.

In this study we used TEPITOPE, a new epitope prediction software, to identify sequence segments on the MAGE-3 protein with promiscuous binding to histocompatibility leukocyte antigen (HLA)-DR molecules. Synthetic peptides corresponding to the identified sequences were synthesized and used to propagate CD4(+) T cells from the blood of a healthy donor. CD4(+) T cells strongly recognized MAGE-3281-295 and, to a lesser extent, MAGE-3141-155 and MAGE-3146-160. Moreover, CD4(+) T cells proliferated in the presence of recombinant MAGE-3 after processing and presentation by autologous antigen presenting cells, demonstrating that the MAGE-3 epitopes recognized are naturally processed. CD4(+) T cells, mostly of the T helper 1 type, showed specific lytic activity against HLA-DR11/MAGE-3-positive melanoma cells. Cold target inhibition experiments demonstrated indeed that the CD4(+) T cells recognized MAGE-3281-295 in association with HLA-DR11 on melanoma cells. This is the first evidence that a tumor-specific shared antigen forms CD4(+) T cell epitopes. Furthermore, we validated the use of algorithms for the prediction of promiscuous CD4(+) T cell epitopes, thus opening the possibility of wide application to other tumor-associated antigens. These results have direct implications for cancer immunotherapy in the design of peptide-based vaccines with tumor-specific CD4(+) T cell epitopes.

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.

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.

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.

B7.1 expression on tumor cells circumvents the need of professional antigen presentation for in vitro propagation of cytotoxic T cell lines.

In vitro propagation of tumor-specific CTLs, to be used for identification of tumor antigens (Ag) and/or adoptive immunotherapy, is hampered by the need of large amounts of professional antigen-presenting cells (APC) used for periodical cycles of restimulation. We evaluated whether RMA T lymphoma cells, stably transfected with the cDNA encoding for the B7.1 costimulatory molecule, provided the activation signals to CD8+ T lymphocytes in the absence of professional APC and CD4+ helper cells. We demonstrate here that long-term CD8+ cell lines can be efficiently propagated in vitro by repeated cycles of stimulation with tumor cells stably expressing B7.1. Professional APC and CD4+ helper cells are not required as far as interleukin 2 is exogenously provided. Furthermore, CD8+ blasts needed both signal 1 (Ag in the contest of the MHC molecule) and signal 2 (interaction of costimulatory molecules) for restimulation. T cell blasts in the presence of signal 1 or 2 only still retained their effector potential but did not undergo clonal expansion. These results are very promising for further applications of specific immunotherapies in humans.

The immunogenicity of experimental tumors is strongly biased by the expression of dominant viral cytotoxic T-lymphocyte epitopes.

The immunogenic Friend-Moloney-Rauscher (FMR) virus-induced tumors have been used extensively to clarify the cellular and molecular mechanisms responsible for tumor rejection and to develop immunotherapeutic strategies. We characterize here the trimolecular complex MHC class I-antigenic determinant-T cell receptor involved in the induction of a protective CTL response against the RMA thymoma. This complex is mainly composed by the D(b) molecule interacting with a Rauscher virus antigen (Ag) determinant and the Vbeta5+ T cell receptor. We also show that the chemically induced EL-4 thymoma acquires the susceptibility to recognition by anti-RMA CTLs and the ability to elicit a protective anti-RMA CTL response only upon infection by a virus of the FMR family and that RMA and FMR virus infected EL-4 cells share tumor-associated Ag. The data strongly support the hypothesis that the high immunogenicity of virus-induced or infected tumors is determined by the expression of immunodominant virus-encoded Ag. The demonstration of a different outcome in the immune responses elicited in the presence or in the absence of viral Ag further open the contention of the molecular requirements for immunogenicity and should stimulate a more careful revision of unexpected cross-reactivity among tumors.

Major histocompatibility complex class I restricted cytotoxic T cells specific for natural melanoma peptides recognize unidentified shared melanoma antigen(s).

CTLs were generated in vitro from two healthy donors and one melanoma patient by stimulation of CD8+ T cells with autologous dendritic cells pulsed with natural melanoma peptides (NMPs), obtained by acid treatment of HLA-matched melanoma cells. CTLs showed MHC class I-restricted melanoma-specific cytolytic activity. Importantly, CTLs from the patient, induced with NMPs obtained from an allogeneic HLA-A-matched melanoma, killed the autologous tumor. COS-7 cells cotransfected with the cDNA of 13 melanoma antigens and the HLA-A1-restricting allele did not induce cytokines release from NMP-specific CTLs, suggesting that they recognize unidentified shared melanoma antigens and that they may be valuable for identification of new tumor antigens. These results strongly support the use of autologous and/or allogeneic NMP-pulsed dendritic cells as cancer vaccines in patients whose neoplasms do not express or have lost expression of known tumor antigens.

Induction of therapeutic T-cell immunity by tumor targeting with soluble recombinant B7-immunoglobulin costimulatory molecules.

Tumor targeting with immunomodulatory molecules is an attractive strategy to enhance the host's antitumor response. Expression of CD80 (B7-1) and CD86 (B7-2) costimulatory molecules in tumor cells has proven to be an efficient way to enhance their immunogenicity. Here, we studied the effects of tumor targeting with biotinylated recombinant soluble B7-1- and B7-2 immunoglobulin G molecules (bio-B7-IgG) using a pretargeting approach based on the sequential use of a biotinylated antitumor monoclonal antibody and avidin. Mouse RMA T-lymphoma cells bearing either bio-B7-1-IgG or bio-B7-2-IgG on their surface prime in vitro naive CD8+ CTLs, which are highly effective in adoptive immunotherapy, and induce therapeutic immunity when injected in tumor-bearing animals. In vivo targeting of established RMA tumors with bio-B7-IgG either cures tumor-bearing mice or significantly prolongs their survival. The antitumor response induced by targeted bio-B7-IgG depends on both CD4+ and CD8+ T cells. Moreover, tumor targeting with bio-B7-IgG in vivo is critical for both expansion in lymphoid organs and mobilization into the tumor of tumor-specific CD8+ CTLs. When targeting is performed on poorly immunogenic TS/A mammary adenocarcinoma, only bio-B7-1-IgG primes naive CTLs in vitro and cures or significantly prolongs the survival of tumor-bearing mice in vivo, confirming that the two costimulatory molecules are not redundant with this tumor. Altogether, these data suggest that tumor avidination and targeting with soluble bio-B7-IgG may represent a promising strategy to enhance the antitumor response in the host.

Particulate naturally processed peptides prime a cytotoxic response against human melanoma in vitro.

For an efficient antitumor cytotoxic response, tumor antigenic peptides need to be presented by professional antigen-presenting cells in association with MHC class I molecules. We established in vitro short-term human CTL lines from healthy and melanoma-bearing subjects, using as antigen-presenting cells autologous adherent cells after phagocytosis of latex beads coated with melanoma peptides. Melanoma peptides were obtained by acid extraction of melanoma cells that matched with donor peripheral blood mononuclear cells, at least for one HLA-A allele. The cytotoxic activity of the lines was specific for the melanoma from which peptides were obtained and for melanoma sharing HLA alleles. These results demonstrate that a complex mixture of naturally processed melanoma peptides conjugated to a phagocytic substrate that targets them into the MHC class I pathway of adherent cells can prime a CTL response in healthy subjects in vitro, and that peptides from allogeneic tumors may be used to propagate CTL in melanoma patients. Our data support the feasibility of active and passive vaccination procedures with nonliving vaccines in cancer patients.

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.

Role of antigen-presenting cells in cross-priming of cytotoxic T lymphocytes by apoptotic cells.

Although the mechanisms regulating recognition and phagocytosis of apoptotic cells by scavenger cells are the subject of intense investigation, little is known about the fate of the antigens contained in apoptotic cells and the constraints defining their immunogenicity. We developed a model in C57BL/6 mice to evaluate whether phagocytosis of apoptotic tumor cells yielded antigens able to get access to the MHC class I pathway and activate a specific cytotoxic T lymphocyte response. Our results demonstrate that apoptotic tumor cells are antigenic in vitro and can be immunogenic in vivo. Their immunogenicity depends on the number of cells used for immunization and the antigen-presenting cells involved in processing and presentation of antigens contained in the dying cells. The demonstration of the immunogenicity of apoptotic cells may have direct implications both in autoimmunity and cancer.

[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.

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.

Human melanoma cells transfected with the B7-2 co-stimulatory molecule induce tumor-specific CD8+ cytotoxic T lymphocytes in vitro.

Neoplastic cells express tumor-associated antigens, but tumor rejection seldom occurs in vivo. The absence of an effective immune response may be explained by the inability of tumor cells to deliver co-stimulatory signals. Indeed, transfection of either B7-1 or B7-2 co-stimulatory molecules into mouse tumor cells enhances antitumor immune responses. In this study, we stably transfected human melanoma cells with the cDNA encoding the B7-2 molecule to evaluate in vitro: (i) the induction of anti-melanoma cytotoxic T lymphocytes (CTL) by stimulation of CD8+ T cells, purified from healthy donors and a melanoma patient, with B7-2 transfected allogeneic HLA-matched melanoma cells; (ii) the tumor specificity and the HLA restriction of the induced CTL; and (iii) the feasibility to propagate long-term antimelanoma CTL lines. We found that B7-2 transfected, but not untransfected or mock-transfected, melanoma cells activated MHC-class I-restricted, melanoma-specific CD8+ CTL from healthy donors. More importantly, CD8+ tumor-associated lymphocytes, purified from a tumor-invaded lymph node of a melanoma patient and stimulated with B7-2-transfected melanoma cells, acquired a strong reactivity toward the autologous tumor. CTL lines with specific cytolytic activity could be propagated in long-term culture. These results indicate that: (i) the expression of the B7-2 molecule into human melanoma cells makes them immunogenic and able to act as antigen-presenting cells and (ii) purified CD8+ cells, stimulated with B7-2+ allogeneic HLA-matched melanoma cells, preferentially recognize melanoma-specific rather than allogeneic antigens. This study may have clinical implications for passive and/or active immunotherapy in melanoma patients.

Constitutive expression of the heat shock protein 72 kDa in human melanoma cells.

Heat shock proteins (hsp) are a family of proteins characteristically produced under stress conditions in normal cells. Overexpression of hsp 70 kDa protects tumoral cells from tumor necrosis factor cytotoxicity and is related to drug resistance. In this study we investigated whether hsp are abnormally expressed in melanoma cells in vivo. Antibodies directed against hsp 72 and hsp 90 were used to identify hsp on non-stressed neoplastic cells derived from surgical specimens of two primary and four metastatic melanomas. Hsp 72 and hsp 90 were always present at high level in the cytoplasm of melanoma cells. It is possible that the activation of hsp genes during neoplastic transformation confers a selective advantage to tumoral cells in vivo, allowing them to escape the immunological surveillance.

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.