Differential pharmacologic properties of the two C75 enantiomers: (+)-C75 is a strong anorectic drug. (-)-C75 has antitumour activity

C75 is a synthetic compound described as having antitumoral properties. It produces hypophagia and weight loss in rodents, limiting its use in cancer therapy but identify- ing it as a potential anti-obesity drug. C75 is a fatty acid synthase (FAS) inhibitor and, through its coenzyme A (CoA) derivative, it acts as a carnitine palmitoyltransferase (CPT) 1 inhibitor. Racemic mixtures of C75 have been used in all the previous studies; however, the potential dif- ferent biological activities of C75 enantiomers have not been examined yet. To address this question we synthesized the two C75 enantiomers separately. Our results showed that ( )- C75 inhibits FAS activity in vitro and has a cytotoxic effect on tumor cell lines, without affecting food consumption. (+)-C75 inhibits CPT1 and its administration produces anorexia, suggesting that central inhibition of CPT1 is essential for the anorectic effect of C75. The differential activity of C75 enantiomers may lead to the development of potential new specific drugs for cancer and obesity.

Differential pharmacologic properties of the two C75 enantiomers: (+)-C75 is a strong anorectic drug. (-)-C75 has antitumour activity

C75 is a synthetic compound described as having antitumoral properties. It produces hypophagia and weight loss in rodents, limiting its use in cancer therapy but identify- ing it as a potential anti-obesity drug. C75 is a fatty acid synthase (FAS) inhibitor and, through its coenzyme A (CoA) derivative, it acts as a carnitine palmitoyltransferase (CPT) 1 inhibitor. Racemic mixtures of C75 have been used in all the previous studies; however, the potential dif- ferent biological activities of C75 enantiomers have not been examined yet. To address this question we synthesized the two C75 enantiomers separately. Our results showed that ( )- C75 inhibits FAS activity in vitro and has a cytotoxic effect on tumor cell lines, without affecting food consumption. (+)-C75 inhibits CPT1 and its administration produces anorexia, suggesting that central inhibition of CPT1 is essential for the anorectic effect of C75. The differential activity of C75 enantiomers may lead to the development of potential new specific drugs for cancer and obesity.

Doxorubicin induces drug efflux pumps in Candida albicans.

Candida albicans is one of the most important opportunistic fungal pathogens. It can cause serious fungal diseases in immunocompromised patients, including those with cancer. Treatment failures due to the emergence of drug-resistant C. albicans strains have become a serious clinical problem. Resistance incidents were often mediated by fungal efflux pumps which are closely related to the human ABC transporter P-glycoprotein (P-gp). P-gp is often overexpressed in cancer cells and confers resistance to many cytotoxic drugs. We examined whether cytotoxic drugs commonly used for cancer treatment (doxorubicin and cyclophosphamide) could alter the expression of genes responsible for the development of fluconazole resistance in Candida cells in the way they can influence homologous genes in cancer cell lines. ABC transporters (CDR1 and CDR2) and other resistance genes (MDR1 and ERG11) were tested by real-time PCR for their expression in C. albicans cells at the mRNA level after induction by antineoplastic drugs. The results were confirmed by a lacZ gene reporter system and verified at the protein level using GFP and immunoblotting. We showed that doxorubicin is a potent inducer of CDR1/CDR2 expression in C. albicans at both the mRNA and protein level and thus causes an increase in fluconazole MIC values. However, cyclophosphamide, which is not a substrate of human P-gp, did not induce ABC transporter expression in C. albicans. Neither doxorubicin nor cyclophosphamide could influence the expression of the other resistance genes (MDR1 and ERG11). The induction of CDR1/CDR2 by doxorubicin in C. albicans and the resulting alteration of antifungal susceptibility might be of clinical relevance for the antifungal treatment of Candida infections occurring after anticancer chemotherapy with doxorubicin.

Des antisérums dirigés contre l'antigène carcino-embryonnaire (CEA) peuvent induire une lyse spécifique des cellules de carcinome du côlon par des lymphocytes normaux

Antibody-dependent cell-mediated cytotoxicity (ADCC) against human colon carcinoma cells grown in vitro was demonstrated with two specific rabbit anti-carcinoembryonic antigen (cea) antisera. The same antisera did not lyse the colon carcinoma cells in the presence of complement but without lymphocytes. The normal human lymphocytes in the absence of anti-CEA antiserum had a very low cytotoxic activity during the three hours 51Cr release assay used in this study. Two colon carcinoma cell lines, HT-29 and Co-115, expressing CEA on their surface as demonstrated by immunofluorescence, were significantly lysed in the ADCC test, whereas control tumor cell lines, not expressing CEA, were not affected by the anti-CEA sera and the lymphocytes. The specificity of the reaction was further demonstrated by the inhibition of antibody-dependent cell-mediated cytotoxicity after the addition of increasing amounts of purified CEA to the antiserum. The absorption of the anti-CEA antisera was controlled by radioimmunoassay. Absorption of the antisera by normal lung extracts and red cells of different blood groups did not decrease the cytotoxicity.

CD1d-antibody fusion proteins target iNKT cells to the tumor and trigger long-term therapeutic responses.

Despite the well-established antitumor activity of CD1d-restricted invariant natural killer T lymphocytes (iNKT), their use for cancer therapy has remained challenging. This appears to be due to their strong but short-lived activation followed by long-term anergy after a single administration of the CD1d agonist ligand alpha-galactosylceramide (αGC). As a promising alternative, we obtained sustained mouse iNKT cell responses associated with prolonged antitumor effects through repeated administrations of tumor-targeted recombinant sCD1d-antitumor scFv fusion proteins loaded with αGC. Here, we demonstrate that CD1d fusion proteins bound to tumor cells via the antibody fragment specific for a tumor-associated antigen, efficiently activate human iNKT cell lines leading to potent tumor cell lysis. The importance of CD1d tumor targeting was confirmed in tumor-bearing mice in which only the specific tumor-targeted CD1d fusion protein resulted in tumor inhibition of well-established aggressive tumor grafts. The therapeutic efficacy correlated with the repeated activation of iNKT and natural killer cells marked by their release of TH1 cytokines, despite the up-regulation of the co-inhibitory receptor PD-1. Our results demonstrate the superiority of providing the superagonist αGC loaded on recombinant CD1d proteins and support the use of αGC/sCD1d-antitumor fusion proteins to secure a sustained human and mouse iNKT cell activation, while targeting their cytotoxic activity and cytokine release to the tumor site.

Shikonin-loaded antibody-armed nanoparticles for targeted therapy of ovarian cancer.

Conventional chemotherapy of ovarian cancer often fails because of initiation of drug resistance and/or side effects and trace of untouched remaining cancerous cells. This highlights an urgent need for advanced targeted therapies for effective remediation of the disease using a cytotoxic agent with immunomodulatory effects, such as shikonin (SHK). Based on preliminary experiments, we found SHK to be profoundly toxic in ovarian epithelial cancer cells (OVCAR-5 and ID8 cells) as well as in normal ovarian IOSE-398 cells, endothelial MS1 cells, and lymphocytes. To limit its cytotoxic impact solely to tumor cells within the tumor microenvironment (TME), we aimed to engineer SHK as polymeric nanoparticles (NPs) with targeting moiety toward tumor microvasculature. To this end, using single/double emulsion solvent evaporation/diffusion technique with sonication, we formulated biodegradable NPs of poly(lactic-co-glycolic acid) (PLGA) loaded with SHK. The surface of NPs was further decorated with solubilizing agent polyethylene glycol (PEG) and tumor endothelial marker 1 (TEM1)/endosialin-targeting antibody (Ab) through carbodiimide/N-hydroxysuccinimide chemistry. Having characterized the physicochemical and morphological properties of NPs, we studied their drug-release profiles using various kinetic models. The biological impact of NPs was also evaluated in tumor-associated endothelial MS1 cells, primary lymphocytes, and epithelial ovarian cancer OVCAR-5 cells. Based on particle size analysis and electron microscopy, the engineered NPs showed a smooth spherical shape with size range of 120 to 250 nm and zeta potential value of -30 to -40 mV. Drug entrapment efficiency was ~80%-90%, which was reduced to ~50%-60% upon surface decoration with PEG and Ab. The liberation of SHK from NPs showed a sustained-release profile that was best fitted with Wagner log-probability model. Fluorescence microscopy and flow cytometry analysis showed active interaction of Ab-armed NPs with TEM1-positive MS1 cells, but not with TEM1-negative MS1 cells. While exposure of the PEGylated NPs for 2 hours was not toxic to lymphocytes, long-term exposure of the Ab-armed and PEGylated NPs was significantly toxic to TEM1-positive MS1 cells and OVCAR-5 cells. Based on these findings, we propose SHK-loaded Ab-armed PEGylated PLGA NPs as a novel nanomedicine for targeted therapy of solid tumors.

Proteasome-assisted identification of a SSX-2-derived epitope recognized by tumor-reactive CTL infiltrating metastatic melanoma.

The tumor Ag SSX-2 (HOM-MEL-40) was found by serological identification of Ags by recombinant expression cloning and was shown to be a cancer/testis Ag expressed in a wide variety of tumors. It may therefore represent a source of CD8(+) T cell epitopes useful for specific immunotherapy of cancer. To identify potential SSX-2-derived epitopes that can be recognized by CD8(+) T cells, we used an approach that combined: 1) the in vitro proteasomal digestion of precursor peptides overlapping the complete SSX-2 sequence; 2) the prediction of SSX-2-derived peptides with an appropriate HLA-A2 binding score; and 3) the analysis of a tumor-infiltrated lymph node cell population from an HLA-A2(+) melanoma patient with detectable anti-SSX-2 serum Abs. This strategy allowed us to identify peptide SSX-2(41-49) as an HLA-A2-restricted epitope. SSX2(41-49)-specific CD8(+) T cells were readily detectable in the tumor-infiltrated lymph node population by multimer staining, and CTL clones isolated by multimer-guided cell sorting were able to lyse HLA-A2(+) tumor cells expressing SSX-2.

Mycobacterium tuberculosis subverts innate immunity to evade specific effectors.

The macrophage is the niche of the intracellular pathogen Mycobacterium tuberculosis. Induction of macrophage apoptosis by CD4(+) or CD8(+) T cells is accompanied by reduced bacterial counts, potentially defining a host defense mechanism. We have already established that M. tuberculosis-infected primary human macrophages have a reduced susceptibility to Fas ligand (FasL)-induced apoptosis. To study the mechanisms by which M. tuberculosis prevents apoptotic signaling, we have generated a cell culture system based on PMA- and IFN-gamma-differentiated THP-1 cells recapitulating the properties of primary macrophages. In these cells, nucleotide-binding oligomerization domain 2 or TLR2 agonists and mycobacterial infection protected macrophages from apoptosis and resulted in NF-kappaB nuclear translocation associated with up-regulation of the antiapoptotic cellular FLIP. Transduction of a receptor-interacting protein-2 dominant-negative construct showed that nucleotide-binding oligomerization domain 2 is not involved in protection in the mycobacterial infection system. In contrast, both a dominant-negative construct of the MyD88 adaptor and an NF-kappaB inhibitor abrogated the protection against FasL-mediated apoptosis, showing the implication of TLR2-mediated activation of NF-kappaB in apoptosis protection in infected macrophages. The apoptosis resistance of infected macrophages might be considered as an immune escape mechanism, whereby M. tuberculosis subverts innate immunity signaling to protect its host cell against FasL(+)-specific cytotoxic lymphocytes.

Bevacizumab for recurrent ependymoma.

BACKGROUND: Ependymoma is a rare type of glioma, representing 5% of all CNS malignancies. Radiotherapy (RT) is commonly administered, but there is no standard chemotherapy. At recurrence, ependymoma is notoriously refractory to therapy and the prognosis is poor. In recurrent glioblastoma, encouraging responses with bevacizumab have been observed. METHODS: In this Institutional Review Board-approved study, we retrospectively analyzed the records of 8 adult patients treated for recurrent ependymoma and anaplastic ependymoma with bevacizumab containing chemotherapy regimens. We determined radiographic response (Macdonald criteria), median time to progression (TTP), and median overall survival (OS; Kaplan-Meier method). RESULTS: There were 4 men and 4 women with a median age of 40 years (range, 20-65). Prior treatment included surgery (n = 8), RT (8), temozolomide (5), and carboplatin (4). Bevacizumab (5-15 mg/kg every 2-3 weeks) was administered alone (2) or concurrently with cytotoxic chemotherapy including irinotecan (3), carboplatin (2), or temozolomide (1). Six patients achieved a partial response (75%) and 1 remained stable for over 8 months. Median TTP was 6.4 months (95% confidence interval 1.4-7.4) and median OS was 9.4 months (95% confidence interval 7.0-not reached), with a median follow-up of 5.2 months among 5 surviving patients (63%). CONCLUSIONS: The radiographic response rate to bevacizumab-containing regimens is high. A prospective study is warranted.

T cell recognition of hapten. Anatomy of T cell receptor binding of a H-2kd-associated photoreactive peptide derivative.

To elucidate the structural basis of T cell recognition of hapten-modified antigenic peptides, we studied the interaction of the T1 T cell antigen receptor (TCR) with its ligand, the H-2Kd-bound Plasmodium berghei circumsporozoite peptide 252-260 (SYIPSAEKI) containing photoreactive 4-azidobenzoic acid (ABA) on P. berghei circumsporozoite Lys259. The photoaffinity-labeled TCR residue(s) were mapped as Tyr48 and/or Tyr50 of complementary determining region 2beta (CDR2beta). Other TCR-ligand contacts were identified by mutational analysis. Molecular modeling, based on crystallographic coordinates of closely related TCR and major histocompatibility complex I molecules, indicated that ABA binds strongly and specifically in a cavity between CDR3alpha and CDR2beta. We conclude that TCR expressing selective Vbeta and CDR3alpha sequences form a binding domain between CDR3alpha and CDR2beta that can accommodate nonpeptidic moieties conjugated at the C-terminal portion of peptides binding to major histocompatibility complex (MHC) encoded proteins.

Allorestricted T lymphocytes with a high avidity T-cell receptor towards NY-ESO-1 have potent anti-tumor activity.

The cancer-testis antigen NY-ESO-1 has been targeted as a tumor-associated antigen by immunotherapeutical strategies, such as cancer vaccines. The prerequisite for a T-cell-based therapy is the induction of T cells capable of recognizing the NY-ESO-1-expressing tumor cells. In this study, we generated human T lymphocytes directed against the immunodominant NY-ESO-1(157-165) epitope known to be naturally presented with HLA-A*0201. We succeeded to isolate autorestricted and allorestricted T lymphocytes with low, intermediate or high avidity TCRs against the NY-ESO-1 peptide. The avidity of the established CTL populations correlated with their capacity of lysing HLA-A2-positive, NY-ESO-1-expressing tumor cell lines derived from different origins, e.g. melanoma and myeloma. The allorestricted NY-ESO-1-specific T lymphocytes displayed TCRs with the highest avidity and best anti-tumor recognition activity. TCRs derived from allorestricted, NY-ESO-1-specific T cells may be useful reagents for redirecting primary T cells by TCR gene transfer and, therefore, may facilitate the development of adoptive transfer regimens based on TCR-transduced T cells for the treatment of NY-ESO-1-expressing hematological malignancies and solid tumors.

New drugs and combinations for malignant glioma.

A new chemotherapy agent and a method for local delivery of carmustine have recently been approved for the treatment of malignant glioma. However, the increase in survival remains modest at best with only a very select patients currently benefiting truly of these treatments. Combination regimen of different alkylating agents or prior O6-alkyltransferase depletion by O6-benzylguanine or continuous temozolomide administration schedules have shown some indication for increased activity. There is preclinical rational for combining temozolomide with radiotherapy and the initial results of a phase II clinical trial were promising. Several new cytotoxic agents are currently in clinical trials in patients with recurrent glioma. More importantly, targeted therapy and antiangiogenic agents have entered the clinical development phase also for patients with glioblastoma and anaplastic astrocytoma. The optimal timing of administration of non-cytotoxic substances and their integration into the currently available treatments remains a challenge. Novel study designs and identification of surrogate markers are necessary in order to make rapid and clinically meaningful progress. This review summarises the currently available evidence of activity of the recently approved drugs against malignant glioma and mentions also agents which have failed to demonstrate a significant antitumour activity. Study endpoints are critically discussed. Combination regimens with other agents and radiation therapy are reviewed. The rational for using antiangiogenic drugs in selected ongoing trials is discussed.

The oxidative potential of differently charged silver and gold nanoparticles on three human lung epithelial cell types

Background: Nanoparticle (NPs) functionalization has been shown to affect their cellular toxicity. To study this, differently functionalized silver (Ag) and gold (Au) NPs were synthesised, characterised and tested using lung epithelial cell systems. Mehtods: Monodispersed Ag and Au NPs with a size range of 7 to 10 nm were coated with either sodium citrate or chitosan resulting in surface charges from ¿50 mV to +70 mV. NP-induced cytotoxicity and oxidative stress were determined using A549 cells, BEAS-2B cells and primary lung epithelial cells (NHBE cells). TEER measurements and immunofluorescence staining of tight junctions were performed to test the growth characteristics of the cells. Cytotoxicity was measured by means of the CellTiter-Blue ® and the lactate dehydrogenase assay and cellular and cell-free reactive oxygen species (ROS) production was measured using the DCFH-DA assay. Results: Different growth characteristics were shown in the three cell types used. A549 cells grew into a confluent mono-layer, BEAS-2B cells grew into a multilayer and NHBE cells did not form a confluent layer. A549 cells were least susceptible towards NPs, irrespective of the NP functionalization. Cytotoxicity in BEAS-2B cells increased when exposed to high positive charged (+65-75 mV) Au NPs. The greatest cytotoxicity was observed in NHBE cells, where both Ag and Au NPs with a charge above +40 mV induced cytotoxicity. ROS production was most prominent in A549 cells where Au NPs (+65-75 mV) induced the highest amount of ROS. In addition, cell-free ROS measurements showed a significant increase in ROS production with an increase in chitosan coating. Conclusions: Chitosan functionalization of NPs, with resultant high surface charges plays an important role in NP-toxicity. Au NPs, which have been shown to be inert and often non-cytotoxic, can become toxic upon coating with certain charged molecules. Notably, these effects are dependent on the core material of the particle, the cell type used for testing and the growth characteristics of these cell culture model systems.

Antigenicity and immunogenicity of Melan-A/MART-1 derived peptides as targets for tumor reactive CTL in human melanoma.

Some cancer patients mount spontaneous T- and B-cell responses against their tumor cells. Autologous tumor reactive CD8 cytolytic T lymphocyte (CTL) and CD4 T-cell clones as well as antibodies from these patients have been used for the identification of genes encoding the target antigens. This knowledge opened the way for new approaches to the immunotherapy of cancer. In this review, we describe the characterization of the structure-function properties of the melanocyte/melanoma tumor antigen Melan-A/MART-1, the assessment of the T-cell repertoire available against this antigen in healthy individuals, and the analysis of naturally acquired and/or vaccine-induced CTL responses to this antigen in patients with metastatic melanoma.

Primary γδ T cell lymphoma of the lung: report of a case with features suggesting derivation from intraepithelial γδ T lymphocytes [Primary gammadelta T cell lymphoma of the lung: report of a case with features suggesting derivation from intraepithelial gammadelta T lymphocytes].

T cell lymphoma of γδ T cell origin is a rare disease that mainly involves extranodal sites and shows aggressive clinical behavior. Here, we report a case of primary γδ T cell lymphoma of the lungs with epitheliotropism in the respiratory epithelium, a feature somewhat reminiscent of what is observed in enteropathy-associated T cell lymphoma. A 63-year-old man presented with chest pain and dyspnea on exertion, weight loss, and general weakness. On a positron emission tomography (PET) scan, multiple hypermetabolic lesions were found in both lungs. Microscopic examination of the wedge lung biopsy revealed nodular infiltration of monomorphic, medium- to large-sized atypical lymphocytes with round nuclei, coarse chromatin, and a variable amount of clear to eosinophilic cytoplasm. Of note, intraepithelial lymphocytosis by atypical lymphoid cells was observed in the respiratory epithelium within and around the nodule. Immunohistochemically, the tumor cells were CD3+, TCRβF1-, TCRγ+, CD5-, CD7+, CD20-, CD79a-, CD30-, CD4-, CD8-, CD10-, BCL6-, CD21-, CD56+, CD57-, and CD138-, and expressed cytotoxic molecules. Epstein-Barr virus (EBV) was not detected by an in situ hybridization assay for EBV-encoded RNA. Interestingly, CD103 was expressed by a subset of tumor cells, especially those infiltrating the epithelium. T cell clonality was detected by multiplex PCR analysis of TRG and TRD gene rearrangements. After 2 months of systemic chemotherapy, PET scan showed regression of the size and metabolic activity of the lesions. This case represents a unique γδ T cell lymphoma of the lungs showing epitheliotropism by CD103+ γδ T cells that is suggestive of tissue-resident γδ T cells as the cell of origin.