Using X-rays in photodynamic therapy: an overview.

Photodynamic therapy is a therapeutic option to treat cancer and other diseases. PDT is used every day in dermatology, and recent developments in the treatment of glioblastoma, mesothelioma or prostate have demonstrated the efficacy of this modality. In order to improve the efficacy of PDT, different strategies are under development, such as the use of targeted PS or nanoparticles to improve selectivity and the design of light devices to better monitor the light dose. Due to the low penetration of light into tissue, another way to improve the efficacy of PDT to treat deep tumors is the use of upconversion NPs or bi-photon absorption compounds. These compounds can be excited in the red part of the spectrum. A relatively new approach, which we will call PDTX, is the use of X-rays instead of UV-visible light for deeper penetration into tissue. The principle of this technique will be described, and the state-of-art literature concerning this modality will be discussed. First, we will focus on various photosensitizers that have been used in combination with X-ray irradiation. To improve the efficacy of this modality, nanoparticles have been designed that allow the conversion of high-energy ionizing radiation into UV-visible light; these are potential candidates for the PDTX approach. They will be discussed at the end of this review.

Classical cadherins control survival through the gp130/Stat3 axis.

Stat3 (Signal Transducer and Activator of Transcription-3) is activated by a number of receptor and nonreceptor tyrosine kinases. We recently demonstrated that engagement of E-cadherin, a calcium-dependent, cell to cell adhesion molecule which is often required for cells to remain tightly associated within the epithelium, also activates Stat3. We now examined the effect of two other classical cadherins, cadherin-11 and N-cadherin, whose expression often correlates with the epithelial to mesenchymal transition occurring in metastasis of carcinoma cells, upon Stat3 phosphorylation and activity. Our results indicate that engagement of these two cadherins also, can trigger a dramatic surge in Stat3 activity. This activation occurs through upregulation of members of the IL6 family of cytokines, and it is necessary for cell survival, proliferation and migration. Interestingly, our results also demonstrate for the first time that, in sharp contrast to Stat3, the activity of Erk (Extracellular Signal Regulated kinase) was unaffected by cadherin-11 engagement. Further examination indicated that, although IL6 was able to activate Erk in sparsely growing cells, IL6 could not induce an increase in Erk activity levels in densely growing cultures. Most importantly, cadherin-11 knock-down did allow Erk activation by IL6 at high densities, indicating that it is indeed cadherin engagement that prevents Erk activation by IL6. The fact that the three classical cadherins tested so far, E-cadherin, N-cadherin and cadherin11, which are present in essentially all tissues, actually activate Stat3 regardless of their role in metastasis, argues for Stat3 as a central survival, rather than invasion factor.

Bypassing melanocyte senescence by beta-catenin: a novel way to promote melanoma.

The Wnt/beta-catenin signaling pathway plays a key role in several cellular functions during embryonic development and adult homeostasis. The deregulation of this pathway may lead to the development of cancer, including melanoma. Deregulation of the Wnt/beta-catenin pathway occurs through either the induction/repression of, or specific mutations in, various members of this signaling pathway; this results in the stabilization of beta-catenin and its translocation from the cytoplasm to the nucleus, where it regulates transcription. Although nuclear beta-catenin is clearly involved in malignant transformation, the mechanism by which it exerts its effects remains elusive. This review focuses on the molecular and cellular mechanisms that are driven by beta-catenin and lead to melanocyte transformation. In particular, we describe how beta-catenin induces melanocyte immortalization, a novel activity of this multifunction protein. Finally, we discuss how beta-catenin-induced immortalization can cooperate with MAPKinase pathways to produce melanoma.

A new LH receptor splice mutation responsible for male hypogonadism with subnormal sperm production in the propositus, and infertility with regular cycles in an affected sister.

BACKGROUND: Inactivating LH receptor (LHR) mutations have been described so far in men as well as in women. Phenotypes in men have been variable with in nearly all cases impairment of sex differentiation or azoospermia. We report a milder reproductive phenotype both in a male patient and his sister. METHODS AND RESULTS: We describe a family that carries a homozygous mutation G-->A at position -1 at the intron 10-exon 11 boundary of the LHR gene. The male patient presented with delayed puberty, micropenis and oligospermia. Two of his sisters were homozygous for the same mutation and were infertile. Surprisingly, one of them was found to have had regular ovarian cycles for years and showed normal LH values (6.5 and 10.6 mIU/ml for LH and FSH, respectively). In vitro analysis showed that this altered splicing resulted in an LHR from which eight amino acids are deleted from the extracellular domain (Delta Tyr(317)-Ser(324)). In vitro expression has shown that the receptor was expressed and capable of LH-induced signaling, albeit with reduced potency (P < 0.001). CONCLUSIONS: LHR mutations may represent an underestimated cause of infertility in women, in addition to being responsible for male hypogonadism with reduced spermatogenesis.

Imatinib enhances human melanoma cell susceptibility to TRAIL-induced cell death: Relationship to Bcl-2 family and caspase activation.

In order to define genetic determinants of primary and metastatic melanoma cell susceptibility to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), we have applied oligonucleotide microarrays to TRAIL-sensitive primary T1 cells and TRAIL-resistant metastatic G1 cells treated or not with TRAIL. T1 and G1 cells are isogenic melanoma cell subclones. We examined 22 000 spots, 4.2% of which displayed differential expression in G1 and T1 cells. Cell susceptibility to TRAIL-mediated apoptosis was found to be correlated with gene expression signatures in this model. Some of the differentially expressed genes were identified as involved in ATP-binding and signaling pathways, based on previously published data. Further analysis provided evidences that c-kit was overexpressed in G1 cells while it was absent in T1 cells. The c-kit inhibitor, imatinib, did not restore TRAIL sensitivity, excluding a role for c-kit in TRAIL resistance in G1 cells. Surprisingly, imatinib inhibited cell proliferation and TRAIL-mediated apoptosis in melanoma cells. We investigated the possible involvement of several molecules, including c-ABL, platelet-derived growth factor receptor (PDGFR), cellular FADD-like interleukin-1 alpha-converting enzyme-like inhibitory protein (c-FLIP)(L/S), Fas-associated DD kinase, p53, p21(WAF1), proteins of B-cell leukemia/lymphoma 2 (Bcl-2) family and cytochrome c. Imatinib did not modulate the expression or activation of its own targets, such as c-ABL, PDGFRalpha and PDGFRbeta, but it did affect the expression of c-FLIP(L), BCL2-associated X protein (Bax) and Bcl-2. Moreover, c-FLIP(L) knockdown sensitized T1 cells to TRAIL-mediated apoptosis, with a sensitivity similar to that of cells previously treated with imatinib. More notably, we found that the resistance to TRAIL in G1 cells was correlated with constitutive c-FLIP(L) recruitment to the DISC and the inhibition of caspase 8, 3 and 9 processing. Moreover, c-FLIP(L) knockdown partly restored TRAIL sensitivity in G1 cells, indicating that the expression level of c-FLIP(L) and its interaction with TRAIL receptor2 play a crucial role in determining TRAIL resistance in metastatic melanoma cells. Our results also show that imatinib enhances TRAIL-induced cell death independently of BH3-interacting domain death agonist translocation, in a process involving the Bax:Bcl-X(L) ratio, Bax:Bcl-X(L)/Bcl-2 translocation, cytochrome c release and caspase activation. Our data indicate that imatinib sensitizes T1 cells by directly downregulating c-FLIP(L), with the use of an alternative pathway for antitumor activity, because PDGFRalpha is not activated in T1 cells and these cells do not express c-kit, c-ABL or PDGFRbeta. Caspase cascade activation and mitochondria also play a key role in the imatinib-mediated sensitization of melanoma cells to the proapoptotic action of TRAIL.

[Are French results in assisted reproductive techniques so bad?]. / Résultats de l'Assistance médicale à la procréation en France: sommes-nous si mauvais?

OBJECTIVE: Comparative analysis of French results in Assisted Reproductive Techniques (ART) versus those from other European countries and the United States. POPULATION AND METHODS: The study was achieved by using the officially available data. The analysis was faced with a lot of difficulties in relation with the various methods of collecting data in the different countries. RESULTS: Nevertheless, it appears clearly that French results are among the lowest in Europe with a 22% rate for pregnancy per ovum pick-up with IVF and 23.4% with ICSI, when most of the other countries report rates that are close to or over 30%. Neither the patients' pick-up recruitment nor specific practices of ART can explain this difference that certainly comes from a deficient quality of the French IVF centres. DISCUSSION AND CONCLUSION: We think that four main end-points can explain this situation: the lack of financial support, the lack of human force, the lack of transparency and finally the opposition between clinician and biologist that has blocked the set-up of integrated ART centres, and probably a poor quality culture.

Anatomical causes of difficult embryo transfer during in vitro fertilization.

OBJECTIVES: Identify, define and validate through statistical analysis the anatomical causes of difficult embryo transfers (ET). MATERIALS AND METHODS: This observational study, carried out in 306 IVF candidates, compared the frequency of anatomical anomalies of the uterus and cervix in women who underwent an easy ET with that in women who underwent a difficult ET. Anatomical anomalies were detected during an assessment of the cervix and uterus including transvaginal ultrasound, hysteroscopy and a mock transfer. Ease of ET was determined during the actual transfer procedure. RESULTS: An easy ET was achieved in 151 patients, whereas difficulties occurred in 155 patients, among whom 55 patients underwent a "very difficult" ET. The most common anatomical characteristics associated with difficult ET were abnormal crypts in the cervical canal (86%) and tortuosity of the cervical canal (68%). Less frequent causes included: internal os contractions (28%) and pronounced anteversion of the uterus (26%). Very difficult ETs were associated with the presence of several causes. CONCLUSIONS: ET is the clinical step that has the most effect on IVF outcome. Difficult transfers are associated with a fall in pregnancy rates. The anatomical causes of difficult transfer identified in this study led to major changes in transfer procedure in our department and to the development of more adapted catheters.

Transvaginal ultrasound-guided embryo transfer in IVF.

OBJECTIVES: To determine whether transvaginal ultrasound-guided embryo transfer is a technique that can be used routinely, whether it improves IVF outcomes and whether it makes difficult transfers easier and more successful. MATERIAL AND METHOD: Non-randomized retrospective study conducted between 2012 and 2016 in the fertility center of the Diaconesses-Croix St-Simon hospital group. The outcomes of 3910 transfers, performed by 5 senior operators, under transabdominal ultrasound guidance are compared with those of 800 transfers, performed by 1 senior operator under transvaginal ultrasound guidance. The criteria studied are the feasibility of the technique and the percentage of pregnancies per transfer in the two populations described, as well as in the difficult and very difficult transfer populations. RESULTS: All the transfers were feasible under transvaginal ultrasound guidance without the use of forceps or additional instruments. The percentage of pregnancies per transfer is significantly increased, when the transfer is performed under transvaginal ultrasound guidance compared with that performed under transabdominal ultrasound guidance, in the general population (38%, n=800 vs 30%, n=3910; P 0.0004) and in the reference population characterized by age <38 years and >6 oocytes collected per puncture (45%, n=490 vs 36%, n=1968; P 0.002). The percentage of pregnancies per transfer (P/T) is not significantly different in the populations of easy transfers (n 695, 38% P/T), difficult transfers (n 58, 46% P/T; P=ns) and very difficult transfers (n 47, 34% P/T; P=ns). CONCLUSIONS: Embryo transfer is a key stage in IVF, in which the quality of performance determines the outcome. In this study, transvaginal ultrasound guidance of the transfer, which is the reference procedure in gynaecological imaging, significantly increases the percentage of pregnancies per transfer, both in the general population and in the reference population, compared with transfers performed under transabdominal ultrasound guidance. Transvaginal ultrasound facilitates the performance of difficult transfers and in particular achieves outcomes in these situations that are not significantly different from those of easy transfers. Visual monitoring of transcervical passage, which is rendered more precise and less traumatic and precision of embryo deposition are the factors that probably account for the improvement in outcomes.

[Transparency of results in assisted reproductive technology]. / Transparence des résultats en Assistance médicale à la procréation.

Clear results in ART need to take into account the technical performances of the centre but also patients selection, cycles cancelling and embryo transfer policies. The risk of a partial transparency is an inappropriate selection of patients. From classical criterias like pregnancies per oocytes retrievals or embryo transfers we are heading towards more global indicators like live births of singletons by initiated cycles rate.

Melanocyte culture lines from Tyr-SV40E transgenic mice: models for the molecular genetic evolution of malignant melanoma.

Transgenic Tyr-SV40E mice previously produced on the C57BL/6 inbred-strain background, with SV40 oncogenic sequences specifically expressed in pigment cells, are predisposed to melanoma [Bradl, M., Klein-Szanto, A., Porter, S. & Mintz, B. (1991). Proc. Natl. Acad. Sci. USA, 88, 164-168]. Separate lines of these animals differ genetically only in the number of copies and chromosomal site of integration of the transgene. Skin melanocytes from young mice with no apparent skin lesions were established in continuous culture from hemizygous donors with low, medium and high numbers of transgene copies, and from a homozygous offspring of the low-copy mouse line. The standard culture conditions enable C57BL/6 wild-type melanocytes to become stably immortalized without transformation. The transgenic cell lines all changed over time in an orderly progression. However, with greater numbers of transgene copies, the cells more rapidly displayed shorter doubling times, increased anchorage independence, reduced serum and growth factor requirements, decreased tyrosinase expression and melanin content, increased oncogene expression, and capacity to form malignant melanomas when tested by grafting. Melanocytes with the lowest number of transgene copies were of special interest. They grew more rapidly than the wild-type cells from the outset, but did not become tumorigenic until an apparently small number of still-unknown genetic changes had spontaneously occurred, or until the number of transgene copies was increased slightly by homozygosity. In contrast to the hemizygous low-copy cells, the homozygous counterparts underwent striking and rapid transformational changes and early conversion to malignancy. Thus such low-copy transgenic melanocyte lines afford an exceptional opportunity for molecular analysis of somatic genetic evolution toward malignant melanoma.

IGF-II induces rapid beta-catenin relocation to the nucleus during epithelium to mesenchyme transition.

The epithelium to mesenchyme transition is thought to play a fundamental role during embryonic development and tumor progression. Loss of cell-cell adhesion and modification of both cell morphology and gene expression are the main events associated with this transition. There is a large amount of evidence suggesting that growth factors can initiate these events. Yet, the connection from growth factor induction to changes in cell adhesion and morphology is largely unknown. To elucidate this connection, we have investigated the action of IGF-II on E-cadherin/beta-catenin complex-mediated cell-cell adhesion and on beta-catenin/TCF-3 mediated gene expression. We can show that (1) IGF-II induces a rapid epithelium to mesenchymal transition; (2) IGF1R, the receptor for IGF-II, belongs to the same membrane complex as E-cadherin and beta-catenin; (3) IGF-II induces a redistribution of beta-catenin from the plasma membrane to the nucleus and an intracellular sequestration and degradation of E-cadherin; (4) IGF-II induces the transcription of beta-catenin/TCF-3 target genes. Based on the given case of IGF-II and E-cadherin/beta-catenin complex, this study reveals the backbone of a cascade connecting growth factor signaling with cell-cell adhesion during EMT.

A novel model to study the dorsolateral migration of melanoblasts.

Melanocytes derived from pluripotent neural crest cells migrate initially in the dorsolateral pathway between the ectoderm and dermomyotome. To understand the role of specific proteins involved in this cell migration, we looked for a cellular model that mimics the in vivo behavior of melanoblasts, and that allows functional studies of their migration. We report here that wild-type embryonic stem (ES) cells are able to follow the ventral and dorsolateral neural crest pathways after being grafted into chicken embryos. By contrast, a mutant ES cell line deficient for beta1 integrin subunits, proteins involved in cell-extracellular interactions, had a severely impaired migratory behavior. Interestingly, ES cells deficient for Kit, the tyrosine kinase receptor for the stem cell factor (SCF), behaved similarly to wild-type ES cells. Thus, grafting mouse ES cells into chicken embryos provides a new cellular system that allows both in vitro and in vivo studies of the molecular mechanisms controlling dorsolateral migration.

Stroke in the Lehigh Valley: racial/ethnic differences.

We investigated black/white differences in stroke rate (standardized morbidity), severity, and subtype, and the relative frequencies of 5 primary risk factors (hypertension, diabetes, myocardial infarction, other heart diseases, and transient ischemic attack [TIA]) using the Lehigh Valley Stroke Register. Blacks had a statistically significant higher, age-adjusted rate of stroke than whites. We found no differences in stroke severity using our measures but blacks had a statistically higher proportion of lacunar stroke, while whites had a higher proportion of embolic stroke. There were no differences in proportions of thrombotic stroke or intracerebral hemorrhage. The relative frequencies of hypertension, myocardial infarction, other heart diseases, and diabetes were higher for blacks, while the relative frequency of TIA was higher for whites. These observations are consistent with other reports that blacks have a higher frequency of stroke and tend to have more small-vessel cerebrovascular pathology than whites.

A new series of ellipticine derivatives (1-(alkylamino)-9-methoxyellipticine). Synthesis, DNA binding, and biological properties.

A new series of ellipticine derivatives, 1-(alkylamino)-5,11-dimethyl-9-methoxy-6H-pyrido[4,3-b]carbazoles, were synthesized as potential DNA intercalating antitumor drugs. The structure of these compounds were confirmed by 1H NMR spectroscopy and mass spectrometry. These compounds are able to bind to DNA with an affinity of about 10(6) M-1, and their intercalating characteristics (lengthening and unwinding of DNA) depend upon the length of the chain in position 1. The cytotoxicities of these compounds on L1210 and NIH-3T3 cells are quite similar, and fluorescence techniques showed that the compounds are localized mainly in the cytoplasmic granules of the cells. One of these compounds appears to show a very high antitumor activity (equivalent to the more active known ellipticine analogues: 10-[[gamma-(diethylamino)propyl]amino]-6-methyl-5H- pyrido[3',4':4,5]pyrollo[2,3-g]isoquinoleine (BD40), 1-[[gamma-(diethylamino)propyl]amino]-9-methoxyellipticine (BD84) and 2-[beta-(diethylamino)ethyl]-9-hydroxyellipticinium chloride (DEAE).

Interaction of an intercalating antitumoral agent: 9-hydroxy-2-methyl ellipticinium (NMHE) with chromatin.

In this work we study the effects of an intercalating antitumoral agent: 9-hydroxy-2-methyl ellipticinium (NMHE) on the structure of chromatin, using micrococcal nuclease and DNase 1 as structural probes. The binding of the drug to chromatin, either in vitro or in the nuclei, induces two structural changes of chromatin: (a) an unfolding of the overall structure which results in an activation of the rate of degradation of chromatin by micrococcal nuclease and (b) a disorganisation of the core particle structure leading to the unwrapping of the DNA from the histone core. Moreover, by studying the interaction of MMHE with nuclei labeled in the active regions of the genome through a nick-translation reaction, it appears that the drug is overconcentrated in these regions and does not induce any new structural changes. The interaction of NMHE with DNase 1-sensitive regions of chromatin indicates that these regions are already "open" or relaxed and represent a preferential target for the drug.

Effects of the 6-alkyl substitution of ellipticine on the uptake by NIH-3T3 cells and on the cytotoxicity.

New ellipticine derivatives of the 2-methyl ellipticinium (NME) series, i.e. 2,6-dimethylellipticinium (6-Me-NME) and 2-methyl-6-n-propylellipticinium (6-Pr-NME), have been studied as cytotoxic compounds. Their uptake by NIH-3T3 cells and efflux have been measured by a sensitive and specific high performance liquid chromatographic assay. These compounds are equitoxic if we compare their cytotoxicity by two methods: growth inhibition and cloning efficiency. However, they accumulate in NIH-3T3 cells at different steady state levels and the efflux rates are not similar. This raises the question of the mode of action of these drugs.

Hysteroscopic diagnosis of ectopic pregnancy.

Although vaginal ultrasonography combined with plasma beta-hCG determination can provide a reliable diagnosis and location of ectopic pregnancy, the results can be difficult to interpret in the early stages when hCG levels are low. Hysteroscopy can be used in such cases to differentiate between ectopic pregnancy and non-viable uterine pregnancy when viable uterine pregnancy has been ruled out. General anaesthesia and laparoscopy are avoided. We performed 60 hysteroscopic procedures between January 1989 and December 1990 in patients with suspected ectopic pregnancies. The pregnancy had been located by means of vaginal ultrasonography in every case in which the hCG was above 1500 IU/ml and in 36% of cases in which the beta-hCG was below this level. Hysteroscopy was hindered by metrorrhagia in three cases and was inconclusive in one, necessitating laparoscopy. Diagnosis was possible in all the remaining cases, as follows: ectopic pregnancy in 41 cases, with an empty uterus and occasional bleeding from an ostium; non-viable uterine pregnancy in 18 cases, with the presence of material within the cavity. Hysteroscopy therefore confirmed the diagnosis in 55% of the cases and was itself diagnostic in a further 43% of cases. Its sensitivity for the diagnosis of ectopic pregnancy was 100% and its specificity 95%. We propose a diagnostic decision tree.

Intravenous sulprostone and uterine scarring based upon 22 cases of therapeutic abortion during 2nd and 3rd trimesters of pregnancy.

A preliminary study in 22 patients with uterine scarring was undertaken using sulprostone by intravenous infusion when therapeutic abortion was deemed necessary during the 2nd and 3rd trimesters of pregnancy. The dosage used was 500 micrograms by slow infusion lasting 10 h. There were no cases of ruptured uterus. Adverse reactions were absent. Results were satisfactory. Mean induction-expulsion duration: 11 h. Expulsion rate in 24 h: 63%. With strict monitoring and in a specialized center, this technique may be suggested when a late therapeutic abortion with a scarred uterus is indicated.