Comparison of the Phytochemical Variation of Non-Volatile Metabolites within Mother Tinctures of Arnica montana Prepared from Fresh and Dried Whole Plant Using UHPLC-HRMS Fingerprinting and Chemometric Analysis.

Arnica montana L. has been recognized for centuries as an herbal remedy to treat wounds and promote healing. It also has a long tradition of use in homeopathy. Depending on its medicinal utilization, standardization regulations allow different manufacturing processes, implying different raw materials, such as the whole arnica plant in its fresh or dried state. In this study, an untargeted metabolomics approach with UHPLC-HRMS/MS was used to cross-compare the phytochemical composition of mother tinctures of A. montana that were prepared from either fresh whole plant (fMT) matter or from oven-dried whole plant (dMT) matter. The multivariate data analysis showed significant differences between fMT and dMT. The dereplication of the HRMS and MS/MS spectra of the more discriminant compounds led to annotated quinic acid, dicaffeoyl quinic acids, ethyl caffeate, thymol derivatives and dehydrophytosphingosine, which were increased in fMT, while Amadori rearrangement products (ARP) and methoxyoxaloyl-dicaffeoyl quinic acid esters were enhanced in dMT. Neither sesquiterpene lactones nor flavonoids were affected by the drying process. This is the first time that a sphingosine, ethyl caffeate and ARP are described in A. montana. Moreover, putative new natural products were detected as 10-hydroxy-8,9-epoxy-thymolisobutyrate and an oxidized proline fructose conjugate, for which isolation and full structure elucidation will be necessary to verify this finding.

Benefits of Homeopathic Complementary Treatment in Patients With Breast Cancer: A Retrospective Cohort Study Based on the French Nationwide Healthcare Database.

BACKGROUND: Complementary therapy in oncology aims to help patients better cope with the illness and side effects (SEs) of cancer treatments that affect their quality of life (QOL). This study aimed to assess the benefits of homeopathic treatment on the health-related QOL (HRQOL) of patients with non-metastatic breast cancer (BC) prescribed in postsurgical complementary therapy. PATIENTS AND METHODS: An extraction from the French nationwide healthcare database targeted all patients who underwent mastectomy for newly diagnosed BC between 2012 and 2013. HRQOL was proxied by the quantity of medication used to palliate the SEs of cancer treatments. RESULTS: A total of 98,009 patients were included (mean age: 61 ± 13 years). Homeopathy was used in 11%, 26%, and 22% of patients respectively during the 7 to 12 months before surgery, the 6 months before, and 6 months after. Thereafter, the use remained stable at 15% for 4 years. Six months after surgery, there was a significant overall decrease (RR = 0.88, confidence interval (CI)95 = 0.87-0.89) in the dispensing of medication associated with SEs in patients treated with ≥ 3 dispensing of homeopathy compared to none. The decrease appeared to be greater for immunostimulants (RR = 0.79, (CI)95 = 0.74-0.84), corticosteroids (RR = 0.82, (CI)95 = 0.79-0.85), and antidiarrheals (RR = 0.83, (CI)95 = 0.77-0.88). CONCLUSION: The study showed an increasing use of homeopathy in patients with BC following diagnosis. This use was maintained after surgery and seemed to play a role in helping patients to better tolerate the SEs of cancer treatments.

Improvement of HSV-1 based amplicon vectors for a safe and long-lasting gene therapy in non-replicating cells.

A key factor for developing gene therapy strategies for neurological disorders is the availability of suitable vectors. Currently, the most advanced are adeno-associated vectors that, while being safe and ensuring long-lasting transgene expression, have a very limited cargo capacity. In contrast, herpes simplex virus-based amplicon vectors can host huge amounts of foreign DNA, but concerns exist about their safety and ability to express transgenes long-term. We aimed at modulating and prolonging amplicon-induced transgene expression kinetics in vivo using different promoters and preventing transgene silencing. To pursue the latter, we deleted bacterial DNA sequences derived from vector construction and shielded the transgene cassette using AT-rich and insulator-like sequences (SAm technology). We employed luciferase and GFP as reporter genes. To determine transgene expression kinetics, we injected vectors in the hippocampus of mice that were longitudinally scanned for bioluminescence for 6 months. To evaluate safety, we analyzed multiple markers of damage and performed patch clamp electrophysiology experiments. All vectors proved safe, and we managed to modulate the duration of transgene expression, up to obtaining a stable, long-lasting expression using the SAm technology. Therefore, these amplicon vectors represent a flexible, efficient, and safe tool for gene delivery in the brain.

Silencing Status Epilepticus-Induced BDNF Expression with Herpes Simplex Virus Type-1 Based Amplicon Vectors.

Brain-derived neurotrophic factor (BDNF) has been found to produce pro- but also anti-epileptic effects. Thus, its validity as a therapeutic target must be verified using advanced tools designed to block or to enhance its signal. The aim of this study was to develop tools to silence the BDNF signal. We generated Herpes simplex virus type 1 (HSV-1) derived amplicon vectors, i.e. viral particles containing a genome of 152 kb constituted of concatameric repetitions of an expression cassette, enabling the expression of the gene of interest in multiple copies. HSV-1 based amplicon vectors are non-pathogenic and have been successfully employed in the past for gene delivery into the brain of living animals. Therefore, amplicon vectors should represent a logical choice for expressing a silencing cassette, which, in multiple copies, is expected to lead to an efficient knock-down of the target gene expression. Here, we employed two amplicon-based BDNF silencing strategies. The first, antisense, has been chosen to target and degrade the cytoplasmic mRNA pool of BDNF, whereas the second, based on the convergent transcription technology, has been chosen to repress transcription at the BDNF gene. Both these amplicon vectors proved to be effective in down-regulating BDNF expression in vitro, in BDNF-expressing mesoangioblast cells. However, only the antisense strategy was effective in vivo, after inoculation in the hippocampus in a model of status epilepticus in which BDNF mRNA levels are strongly increased. Interestingly, the knocking down of BDNF levels induced with BDNF-antisense was sufficient to produce significant behavioral effects, in spite of the fact that it was produced only in a part of a single hippocampus. In conclusion, this study demonstrates a reliable effect of amplicon vectors in knocking down gene expression in vitro and in vivo. Therefore, this approach may find broad applications in neurobiological studies.

Chronic myeloproliferative disorders with rearrangement of the platelet-derived growth factor alpha receptor: a new clinical target for STI571/Glivec.

Two cases of atypical chronic myeloid leukaemia (CML) carrying the t(4;22)(q12;q11) translocation involving the breakpoint cluster region (BCR) and platelet-derived growth factor alpha receptor (PDGFRA) genes have been recently characterized. We report a third case of atypical CML with the same translocation but with a distinct breakpoint fusing BCR exon 1 with PDGFRA exon 13. The patient had a clinical presentation of CML with progressive transformation in B-cell acute lymphoblastic leukaemia. The involvement of PDGFRA led us to treat the patient with the small organic compound imatinib mesylate/STI571 (Glivec) that blocks the ATP binding site of tyrosine kinases such as Abelson, KIT and platelet-derived growth factor receptors. The patient subsequently achieved a rapid clinical and molecular response clearly demonstrating, for the first time, that Glivec is active against PDGFRA in vivo. Therefore, our study expands the list of Glivec targets and has direct biological and also clinical implications.

Differential effects of X-ALK fusion proteins on proliferation, transformation, and invasion properties of NIH3T3 cells.

Majority of anaplastic large-cell lymphomas (ALCLs) are associated with the t(2;5)(p23;q35) translocation, fusing the NPM (nucleophosmin) and ALK (anaplastic lymphoma kinase) genes (NPM-ALK). Recent studies demonstrated that ALK may also be involved in variant translocations, namely, t(1;2)(q25;p23), t(2;3)(p23;q21), t(2;17)(p23;q23) and inv(2)(p23q35), which create the TPM3-ALK, TFG-ALK5, CLTC-ALK, and ATIC-ALK fusion genes, respectively. Although overexpression of NPM-ALK has previously been shown to transform fibroblasts, the transforming potential of variant X-ALK proteins has not been precisely investigated. We stably transfected the cDNAs coding for NPM-ALK, TPM3-ALK, TFG-ALK, CLTC-ALK or ATIC-ALK into nonmalignant NIH3T3 cells. All X-ALK variants are tyrosine phosphorylated and their subcellular distribution was in agreement with that observed in tumors. Moreover, our results show that the in vitro transforming capacity of NIH3T3-transfected cells are in relation to the level of X-ALK fusion proteins excepted for TPM3-ALK for which there is an inverse correlation. The differences between the five X-ALK variants with regard to proliferation rate, colony formation in soft agar, invasion, migration through the endothelial barrier and tumorigenicity seem to be due to differential activation of various signaling pathways such as PI3-kinase/AKT. These findings may have clinical implications in the pathogenesis and prognosis of ALK-positive ALCLs.

Gene expression profiling defines molecular subtypes of classical Hodgkin's disease.

Although the prognosis of Hodgkin's disease is relatively good, around 20% of patients do not benefit from current therapies and succumb to their disease. A large-scale molecular characterization of disease might help improve HD management. Using cDNA arrays, we studied the mRNA expression levels of approximately 1000 selected genes in 34 benign and malignant lymphoid samples including 21 classical Hodgkin's disease (HD) tissue samples. Hierarchical clustering identified three main molecular groups of HD tumours relevant with respect to histology and clinical outcome (response to therapy and survival). Samples from all bad outcome HD (BOHD) patients clustered in one group whereas the two other groups contained most good outcome HD (GOHD) cases. The nodular sclerosis GOHD samples overexpressed genes involved in apoptotic induction and cell signalling, including cytokines, while the BOHD samples were characterized by the upregulation of genes involved in fibroblast activation, angiogenesis, extracellular matrix remodelling, cell proliferation, and the downregulation of tumour suppressor genes. Our results establish a molecular taxonomy of HD correlating with response to therapy and clinical outcome, thereby suggesting the possibility of improving the current prognostic classification.

Targeted oncolytic herpes simplex virus type 1 eradicates experimental pancreatic tumors.

As many other cancers, pancreatic ductal adenocarcinoma (PDAC) progression is associated with a series of hallmark changes for cancer cells to secure their own growth success. Yet, these very changes render cancer cells highly sensitive to viral infection. A promising strategy may rely on and exploit viral replication for tumor destruction, whereby infection of tumor cells by a replication-conditional virus may lead to cell destruction and simultaneous release of progeny particles that can spread and infect adjacent tumor cells, while sparing healthy tissues. In the present study, we used Myb34.5, a second-generation replication-conditional herpes simplex virus type 1 (HSV-1) mutant in which ICP6 gene expression is defective and expression of the HSV-1 γ134.5 gene is regulated by the cellular B-myb promoter. We found that B-myb is present in experimental PDAC and tumors, and is overexpressed in patients' tumors, as compared with normal adjacent pancreas. Myb34.5 replicates to high level in human PDAC cell lines and is associated with cell death by apoptosis. In experimental models of PDAC, mice receiving intratumoral Myb34.5 injections appeared healthy and tumor progression was inhibited, with evidence of tumor necrosis, hemorrhage, viral replication, and cancer cell death by apoptosis. Combining standard-of-care chemotherapy with Myb34.5 successfully led to a very impressive antitumoral effect that is rarely achieved in this experimental model, and resulted in a greater reduction in tumor growth than chemotherapy alone. These promising results warrant further evaluation in early phase clinical trial for patients diagnosed with PDAC for whom no effective treatment is available.

In Hodgkin's disease Reed-Sternberg cells and normal B-lymphocytes are infected by related Epstein-Barr virus strains.

In Hodgkin's disease (HD), both neoplastic Reed-Sternberg (RS) cells and bystander B-lymphocytes may be infected by Epstein-Barr virus (EBV). We postulated that if tumorigenic EBV strains did exist, they would be preferentially found in consistently EBV-associated tumors, such as RS cells, and differ significantly from the strains present in other, non-pathological sites of the same patients. In the present study we have compared LMP1-BNLF1 polymorphism of EBV strains infecting RS cells and B-lymphocytes in lymph nodes effected by HD on the one hand, and bystander B-lymphocytes in reactive lymph nodes on the other. It appeared that viral strains detected in HD tissues including RS cells and bystander B-lymphocytes were infected by different, but related EBV strains and were four times more polymorphic than EBV strains infecting bystander B-lymphocytes of reactive lymph nodes. The question arises as to the biological significance of these observations and the origin and chronology of multiple infections in the same patient. Since RS cells are derived from B-lymphocytes it is conceivable that the latter events could have occurred during the proliferation of bystander B-lymphocytes and their EBV episome following an antigenic stimulation.

Gene expression profiling in anaplastic large cell lymphoma and Hodgkin's disease.

Recent efforts have been made to isolate molecular targets that could explain different outcome between histological subtypes of lymphomas and to understand the molecular mechanisms underlying oncogenic events. Using the SSH technique, we compared the transcriptome of 2 cases of ALK+ and ALK- anaplastic large cell lymphoma (ALCL) and of 2 cases of classical Hodgkin's lymphoma (cHL) with opposite behavior. Regarding ALCL, we showed that ALK-positive tumors overexpressed genes involved in different signaling pathways such as activation or signaling of T-cells, regulation of apoptosis, phospholipase Cgamma and phosphatidyl inositol-3 Kinase. In addition, the characterization of a specific molecular signature may be of clinical relevance since ALK+ tumors generally have a better prognosis than ALK- ones. Similar problems of differential prognosis is observed in cases of cHL, which in addition, may be morphologically and immunologically indistinguishable. Therefore, we applied the same SSH technique to 2 cHL samples from patients with favorable and poor outcome, respectively. Forty-four cDNAs were significantly overexpressed in the poor outcome case. In addition to the defender against death cell 1 (DAD1) gene, overexpressed clones corresponded mostly to expressed sequence tags (ESTs). Interestingly, the present study identifies new genes which may be involved in the pathogenesis and/or clinical outcome of cHL and deserve further investigations.

Gelsemium low doses protect against serum deprivation-induced stress on mitochondria in neuronal cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Gelsemium dynamized dilutions (GDD) are known as a remedy for a wide range of behavioral and psychological symptoms of depression and anxiety at ultra-low doses, yet the underlying mechanisms of the mode of action of G. sempervirens itself are not well understood. AIM OF THE STUDY: The present study was designed to examine the neuroprotective effects of Gelsemium preparations in counteracting stress-related mitochondrial dysfunctions in neuronal cells. MATERIALS AND METHODS: We started by studying how serum deprivation affects the mitochondrial functions of human neuroblastoma (SH-SY5Y) cells. Next, we looked into the potential of various Gelsemium dilutions to improve cell survival and ATP levels. After identifying the most effective dilutions, 3C and 5C, we tested their ability to protect SH-SY5Y cells from stress-induced mitochondrial deficits. We measured total and mitochondrial superoxide anion radicals using fluorescent dyes dihydroethidium (DHE) and the red mitochondrial superoxide indicator (MitoSOX). Additionally, we assessed total nitric oxide levels with 4,5-diaminofluorescein diacetate (DAF-2DA), examined the redox state using pRA305 cells stably transfected with a plasmid encoding a redox-sensitive green fluorescent protein, and analyzed mitochondrial network morphology using an automated high-content analysis device, Cytation3. Furthermore, we investigated bioenergetics by measuring ATP production with a bioluminescence assay (ViaLighTM HT) and evaluated mitochondrial respiration (OCR) and glycolysis (ECAR) using the Seahorse Bioscience XF24 Analyzer. Finally, we determined cell survival using an MTT reduction assay. RESULTS: Our research indicates that Gelsemium dilutions (3C and 5C) exhibited neuroprotective effects by: - Normalizing total and mitochondrial superoxide anion radicals and total nitric oxide levels. - Regulating the mitochondrial redox environment and mitochondrial networks morphology. - Increasing ATP generation as well as OCR and ECAR levels, thereby reducing the viability loss induced by serum withdrawal stress. CONCLUSIONS: These findings highlight that dynamized Gelsemium preparations may have neuroprotective effects against stress-induced cellular changes in the brain by regulating mitochondrial functions, essential for the survival, plasticity, and function of neurons in depression.

Isolation of differentially expressed genes in NPM-ALK-positive anaplastic large cell lymphoma.

In this study, we used subtractive suppression hybridization to compare gene expression between an ALK-positive anaplastic large cell lymphoma (ALCL)-derived cell line and a clinical case of ALK-negative ALCL. Construction and screening of a subtracted library resulted in the cloning of 29 cDNAs which were differentially expressed. Most of these clones corresponded to novel genes with unknown function (EST) or to genes implicated in the differentiation, activation or signalling of T cells such as Ran/TC4, interleukin 1-receptor, thymosin beta4, thymosin beta10, moesin and cytohesin-1. Other genes involved in the regulation of apoptosis, such as human inhibitor of apoptosis-1 (HIAP-1), Bax inhibitor-1 and MCL-1, or DNA repair, such as poly (ADP-ribose) polymerase 1 (PARP-1), X-associated protein-1 (XAP-1), SUMO-1 (sentrin-1) and RanGTPase-activating protein 1 (RanGAP-1), were isolated. Interestingly, we found that both RNA and protein levels of human sterol isomerase (hSI), also referred to as emopamil binding protein (EBP), were overexpressed in ALK+ tumours. This protein is involved in the biosynthesis of cholesterol and may be activated by NPM-ALK. Overall, our results suggest that all the genes described above are upregulated in the NPM-ALK-driven transformation process, and that moesin and cytohesin-1 may be more specifically implicated in a signalling pathway involving PLCgamma and PI3K.

Evidence for early infection of nonneoplastic natural killer cells by Epstein-Barr virus.

We examined lymph nodes and tonsils from patients with infectious mononucleosis by combined detection of EBV-encoded RNA and a specific marker of natural killer (NK) cells, PEN5. A small number of Epstein-Barr virus (EBV) latently infected nonneoplastic NK cells were detected. Our data demonstrate that NK cells are natural targets of EBV and that infection of these cells is an early event observed during primary EBV infection.