Application of the Life Cycle Management of Analytical methods concept to a HPTLC-DPPH assay method for acteoside content in industrial extracts of Plantago lanceolata L.

Analytical methods used for quality control of plants and plant extracts are based on the identification and quantification of chemical markers to manage batch reproducibility and efficacy. The aim of this work was to assess the performance of a High Performance Thin Layer Chromatography (HPTLC) method developed for quality control of industrial dry extracts of ribwort plantain (P. lanceolata L.), using 2,2-diphenyl 1-picrylhydrazyle (DPPH) effect directed chemical reaction for antioxidant activity of acteoside, a phenylethanoid glycoside commonly used as a marker for P. lanceolata L., and to demonstrate the applicability of the Life Cycle Management of Analytical Methods concept to quantitative HPTLC-DPPH methods. The first step was the determination of the Analytical Target Profile (ATP) and Target Measurement Uncertainty (TMU), taking into account the quality control requirements for such extracts and the detection method applicable range. Once the desired range was established, an evaluation of the calibration function was conducted using several calibration models. Due to the lack of reference samples, spiked samples were used to evaluate the accuracy of the method by means of Total Analytical Error (TAE) determination, using prediction intervals calculation for the selected calibration functions. Measurement Uncertainty (MU) was also estimated, allowing the final choice of the calibration function to be used for quality control, giving the most fit for purpose performance level in accordance with the product specifications. As Life Cycle Management of the method also includes its routine use, the Measurement Uncertainty was checked on spiked and unspiked extract samples with different dilution levels, in order to verify the accordance of results between spiked and unspiked samples and to prepare a replication strategy to be applied during the routine use of the method.

[Adoptive immunotherapy: evaluation and perspectives in the treatment of certain cancers]. / Immunothérapie adoptive: bilan et perspectives dans le traitement de certains cancers.

INTRODUCTION: Adoptive immunotherapy was first introduced in the 1980s. This new anticancer therapeutic approach has already demonstrated promising results in both animal models and humans affected by various tumors. CURRENT KNOWLEDGE AND KEY POINTS: This review summarizes the requirements of such therapies involving either activated lymphocytes, tumor-infiltrating lymphocytes or activated macrophages. It focuses more particularly on the promising approaches that represent antigen presenting cells such as macrophages and antigen-loaded dendritic cells in the development of safe and effective cancer vaccines. FUTURE PROSPECTS AND PROJECTS: Standardized procedures for macrophages and dendritic cell generation, as well as preliminary results of clinical applications in patients with either prostate cancer or melanoma, are also discussed.

Nucleotide-metabolism and chromosome alterations in human-malignant melanoma xenografts.

The karyotypes of human melanomas exhibit multiple chromosome alterations. Recurrent deletions of 9p, 10q and 14q arms, which carry genes encoding for enzymes of purine metabolism, were also found in human gliomas, another neuroectodermal tumor previously studied for both cytogenetics and nucleotides metabolism. Postulating that this metabolism might also be modified in melanomas, the activities of eleven enzymes involved in catabolic and synthetic pathways of purine metabolism were measured, in addition to two enzymes of the pyrimidine synthesis. Assays were performed on six melanoma mestastases, five nodal and one cutaneous, after transplantation into nude mice. The purine metabolism was characterized by a more active catabolic than synthetic pathway, a possible imbalance between de novo and salvage pathways for adenylates synthesis, rather in favor of the de novo pathway, and a more active adenylate than guanylate synthesis. The skin metastasis exhibited quite different cytogenetic and metabolic patterns, when compared to the nodal metastases. Considering the relationships between cytogenetic and metabolic data, low activities of methylthioadenosine phosphorylase, adenosine kinase, adenosine monophosphate deaminase, nucleoside phosphorylase and 5'-nucleotidase were observed in melanomas, as well as frequent losses of 9p, 10q, Ip, 14q and 6q arms respectively carrying genes encoding for these enzymes, most of these rearrangements were confirmed by chromosome painting. The two enzymes exhibiting the highest activities were adenosine deaminase and adenylosuccinate lyase, encoded by genes mapped on chromosomes 20 and 22 respectively, frequently in excess in melanomas. Thus, for these tumors, the metabolic pattern roughly parallels the cytogenetic profile, even if the absence of case to case correlation suggests that gene dosage effect, if it occurs, is not the only parameter involved. The main enzymatic and cytogenetic difference between melanomas and gliomas, concerns both adenylosuccinate lyase activity and the balance of chromosome 22, high in melanomas and low in gliomas.

Pyrimidine nucleotide metabolism in human colon carcinomas: comparison of normal tissues, primary tumors and xenografts.

The activities of 5 enzymes involved in the pyrimidine metabolism were measured in xenografts of 8 human colon adenocarcinomas and in the corresponding primary tumors and normal tissues. The enzymes studied were thymidine kinase, thymidine phosphorylase, uridine kinase, uridine phosphorylase and thymidylate synthase. With the exception of the phosphorylases in one tumor, all enzyme activities were higher in primary tumors than in the corresponding normal tissues. The average activities of thymidine kinase and thymidylate synthase were of the same order of magnitude in xenografts and in primary tumors. The uridine metabolizing enzymes tend to have a higher activity in xenografts than in primary tumors. The most consistent and significant change was a sharp decrease in thymidine phosphorylase activity in xenografts as compared to primary tumors. Whether or not the difference in thymidine phosphorylase activity between xenografts and primary tumors is related to the contribution of non-cancerous cells in primary tumors remains to be determined. However, these results raise questions concerning the representativeness of xenografts with reference to primary tumors and suggest that care should be taken in the application of this model.

Purine and pyrimidine metabolism in human gliomas: relation to chromosomal aberrations.

Chromosomal aberrations in human gliomas are principally numerical. In tumours of low malignancy, karyotypes are frequently normal, but occasionally an excess of chromosome 7 and a loss of sex chromosome are observed. In highly malignant tumours, the most frequent aberrations are gain of chromosome 7, loss of chromosome 10 and less frequently losses or deletions of chromosomes 9, 22, 6, 13 and 14 or gains of chromosomes 19 and 20. To understand the meaning of these chromosome imbalances, the relationships between chromosome abnormalities and metabolic disturbances were studied. The losses or deletions observed affected principally chromosomes carrying genes encoding enzymes involved in purine metabolism. The activities of ten enzymes were measured: adenosine kinase, adenine phosphoribosyltransferase, adenylate kinase, methylthioadenosine phosphorylase, hypoxanthine phosphoribosyltransferase, adenylosuccinate lyase, inosine monophosphate dehydrogenase, adenosine deaminase, nucleoside phosphorylase and adenosine monophosphate deaminase. In parallel, two enzymes involved in pyrimidine metabolism, thymidine kinase and thymidylate synthase (TS), were studied. The activities of all these enzymes were measured on samples from 30 human primary glial tumours with low or high malignancy, six xenografted tumours at different passages, four portions of normal brain tissue and four non-glial brain neoplasms. As suggested by cytogenetic data, the enzymatic results showed a relatively low activity of purine metabolism in glial tumours when compared with normal brain and non-glial brain neoplasms. Considering the two enzymes involved in pyrimidine metabolism, only TS had higher activity in glial tumours of high malignancy than in normal brain. In comparison with normal brain, the balance between salvage and de novo pathways changes in gliomas, and even more in grafted tumours, in favour of de novo synthesis. The relation between chromosomes and metabolic imbalances does not correspond to a simple gene dosage effect in these tumours. These data suggest that the decrease of adenosine metabolism occurs before chromosomal aberrations appear, since it is observed in tumours of low malignancy when most karyotypes are still normal, and that the de novo pathway increases with tumour progression.

[Nucleotide metabolism in human glioma: comparative study of primary tumors, grafted tumors on nude mice and cell cultures]. / Métabolisme des nucléotides dans les gliomes humains: comparaison de tumeurs primaires, de tumeurs implantées sur souris nude et de lignées.

A study developed to test the hypothesis of a possible relationship between metabolic modifications and chromosomal imbalances in solid tumors leads us to investigate the metabolism of purine nucleotides in human gliomas. In order to assess the representativeness of experimental models frequently used, the activities of nine enzymes involved in the synthesis and in the catabolism of purine nucleotides were measured on samples of normal brain, primary and xenografted glial tumors and cell cultures established from human gliomas. In parallel, two enzymes involved in pyrimidine metabolism were also studied on the same samples. The results highlight the low activity of the purine metabolism in human gliomas when compared to normal brain, tissue with low proliferative activity. On the contrary, the pyrimidine metabolism in human gliomas is increased by comparison to normal brain. For the purine metabolism, few differences are observed between enzyme activities calculated in primary glial tumors, xenografts and cells in culture. In grafted tumors and cell cultures, the activity of this metabolism is similar or lower than in normal brain, except for inosine monophosphate dehydrogenase. However, for the pyrimidine metabolism, significantly differences are observed between primary glial tumors, grafted glial tumors and cell cultures. The thymidine kinase/thymidylate synthetase ratio depends on the model studied. These results point out the problem of the representativeness of these models, especially when used for experimental therapeutic studies. This metabolic study also underlines that all results should be interpreted carefully and that the limits for the use of these two experimental models should always be clearly exposed.

[Chromosome abnormalities and adenine metabolism in human glial tumors]. / Anomalies chromosomiques et métabolisme de l'adénine dans les tumeurs gliales humaines.

Most chromosome aberrations in gliomas are numerical, resulting in either gains or deficiencies of whole chromosomes. In tumors of low malignancy, the karyotype is frequently normal or exhibits a loss of sex chromosome and a gain of chromosome 7. These two anomalies may not be directly related to malignancy. In the highly malignant cases, the two most frequent aberrations are the gain of chromosome 7 and the loss of chromosome 10, other anomalies such as losses or deletions of chromosomes, 9, 22, 6, 13 and 14 being detected at various frequencies. Several of these chromosomes carry important genes of adenine metabolism: AK1 and AK3 (adenylate kinase) and MTAP (methylthioadenosine phosphorylase) for chromosome 9; ADK (adenosine kinase) and mitochondrial ATPase for chromosome 10; ADSL (adenylosuccinate lyase) for chromosome 22, NP (nucleoside phosphorylase) for chromosome 14. We performed the corresponding assays of enzyme activity on both fresh tumors and tumors grafted on nude mice, which showed that these enzymes had a relatively low activity although the tumors were proliferating. However, chromosome losses do not seem to directly cause the metabolic alterations by gene dosage effect. Interestingly, chromosome 10, frequently deficient, also carries genes of importance for glycolysis (hexokinase) and glutamate metabolism (glutamate dehydrogenase and glutamate oxaloacetate transaminase). The deficiency for these genes could be taken into account for a better type of chemotherapy by antimetabolics.

Activity of thymidylate synthetase, thymidine kinase and galactokinase in primary and xenografted human colorectal cancers in relation to their chromosomal patterns.

The relationship between chromosome anomalies and metabolic modifications in human colorectal cancers grafted into nude mice was studied. Two distinct chromosomal patterns have been demonstrated i.e., monosomic type (MT) characterized by multiple chromosome losses or deletions always involving chromosome 18, and trisomic type (TT) characterized by progressive gains of chromosomes. Grafted tumors conserve original karyotypes observed on corresponding primary tumors. Most changes involve the loss of chromosomes carrying genes encoding for enzymes of the de novo pathways and the gain of chromosomes carrying genes encoding for enzymes of the salvage pathways of nucleotide synthesis. In MT tumors the long arm (q) of chromosome 17 is frequently duplicated in association with a deletion of the short arm, forming an isochromosome 17q. The activities of 3 enzymes, thymidylate synthetase (TS) mapped on chromosome 18, thymidine kinase (TK) and galactokinase (GalK), both mapped on chromosome 17q, were studied. TS is a de novo enzyme and TK and GalK are salvage enzymes. A clear correlation could be demonstrated between tumor types and enzyme activities: MT tumors had lower TS and higher TK and GalK activities than TT tumors. These differences were too large to result from a gene dosage effect only. These data suggest that serial studies on grafted colorectal cancers give a better representation of metabolic disturbances than studies on fresh tumor samples, usually contaminated by non-cancerous cells.