The superoxide scavenger TEMPOL induces urokinase receptor (uPAR) expression in human prostate cancer cells

There is little understanding of the effect that reactive oxygen metabolites have on cellular behavior during the processes of invasion and metastasis. These oxygen metabolites could interact with a number of targets modulating their function such as enzymes involved in basement membrane dissolution, adhesion molecules involved in motility or receptors involved in proliferation. We investigated the effect of increased scavenging of superoxide anions on the expression of the urokinase receptor (uPAR) in PC-3M human prostate cancer cells. Urokinase receptor is a GPI-linked cell surface molecule which mediates multiple functions including adhesion, proliferation and pericellular proteolysis. Addition of the superoxide scavenger 4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy (TEMPOL) to PC-3M cultures stimulated expression of uPAR protein peaking between 48 and 72 hours. Cell surface expression of the uPAR was also increased. Surprisingly, uPAR transcript levels increased only slightly and this mild increase did not coincide with the striking degree of protein increase. This disparity indicates that the TEMPOL effect on uPAR occurs through a post-transcriptional mechanism. TEMPOL presence in PC-3M cultures reduced intracellular superoxide-type species by 75% as assayed by NBT dye conversion; however this reduction significantly diminished within hours following TEMPOL removal. The time gap between TEMPOL treatment and peak uPAR protein expression suggests that reduction of reactive oxygen metabolites in prostate cancer cells initiates a multistep pathway which requires several hours to culminate in uPAR induction. These findings reveal a novel pathway for uPAR regulation involving reactive oxygens such as superoxide anion

A phase I pharmacokinetic study of hypoxic abdominal stop-flow perfusion with gemcitabine in patients with advanced pancreatic cancer and refractory malignant ascites

Purpose: As no curative treatment for advanced pancreatic and biliary cancer with malignant ascites exists, new modalities possibly improving the response to available chemotherapies must be explored. This phase I study assesses the feasibility, tolerability and pharmacokinetics of a regional treatment of gemcitabine administered in escalating doses by the stop-flow approach to patients with advanced abdominal malignancies (adenocarcinoma of the pancreas, n=8, and cholangiocarcinoma of the liver, n=1). Experimental design: Gemcitabine at 500, 750 and 1,125mg/m2 was administered to three patients at each dose level by loco-regional chemotherapy, using hypoxic abdominal stop-flow perfusion. This was achieved by an aorto-caval occlusion by balloon catheters connected to an extracorporeal circuit. Gemcitabine and its main metabolite 2′,2′-difluorodeoxyuridine (dFdU) concentrations were measured by high performance liquid chromatography with UV detection in the extracorporeal circuit during the 20min of stop-flow perfusion, and in peripheral plasma for 420min. Blood gases were monitored during the stop-flow perfusion and hypoxia was considered stringent if two of the following endpoints were met: pH≤7.2, pO2 nadir ratio ≤0.70 or pCO2 peak ratio ≥1.35. The tolerability of this procedure was also assessed. Results: Stringent hypoxia was achieved in four patients. Very high levels of gemcitabine were rapidly reached in the extracorporeal circuit during the 20min of stop-flow perfusion, with C max levels in the abdominal circuit of 246 (±37%), 2,039 (±77%) and 4,780 (±7.3%)μg/ml for the three dose levels 500, 750 and 1,125mg/m2, respectively. These C max were between 13 (±51%) and 290 (±12%) times higher than those measured in the peripheral plasma. Similarly, the abdominal exposure to gemcitabine, calculated as AUCt0-20, was between 5.5 (±43%) and 200 (±66%)-fold higher than the systemic exposure. Loco-regional exposure to gemcitabine was statistically higher in presence of stringent hypoxia (P<0.01 for C max and AUCt0-20, both normalised to the gemcitabine dose). Toxicities were acceptable considering the complexity of the procedure and were mostly hepatic; it was not possible to differentiate the respective contributions of systemic and regional exposures. A significant correlation (P<0.05) was found between systemic C max of gemcitabine and the nadir of both leucocytes and neutrophils. Conclusions: Regional exposure to gemcitabine—the current standard drug for advanced adenocarcinoma of the pancreas—can be markedly enhanced using an optimised hypoxic stop-flow perfusion technique, with acceptable toxicities up to a dose of 1,125mg/m2. However, the activity of gemcitabine under hypoxic conditions is not as firmly established as that of other drugs such as mitomycin C, melphalan or tirapazamine. Further studies of this investigational modality, but with bioreductive drugs, are therefore warranted first to evaluate the tolerance in a phase I study and later on to assess whether it does improve the response to chemotherap

High Frequencies of Naive Melan-a/Mart-1–Specific Cd8+ T Cells in a Large Proportion of Human Histocompatibility Leukocyte Antigen (Hla)-A2 Individuals

Using fluorescent HLA-A*0201 tetramers containing the immunodominant Melan-A/MART-1 (Melan-A) tumor-associated antigen (Ag), we previously observed that metastatic lymph nodes of melanoma patients contain high numbers of Ag-experienced Melan-A–specific cytolytic T lymphocytes (CTLs). In this paper, we enumerated and characterized ex vivo Melan-A–specific cells in peripheral blood samples from both melanoma patients and healthy individuals. High frequencies (≥1 in 2,500 CD8+ T cells) of Melan-A–specific cells were found in 10 out of 13 patients, and, surprisingly, in 6 out of 10 healthy individuals. Virtually all Melan-A–specific cells from 6 out of 6 healthy individuals and from 7 out of 10 patients displayed a naive CD45RAhi/RO− phenotype, whereas variable proportions of Ag-experienced CD45RAlo/RO+ Melan-A–specific cells were observed in the remaining 3 patients. In contrast, ex vivo influenza matrix–specific CTLs from all individuals exhibited a CD45RAlo/RO+ memory phenotype as expected. Ag specificity of tetramer-sorted A2/Melan-A+ cells from healthy individuals was confirmed after mitogen-driven expansion. Likewise, functional limiting dilution analysis and interferon γ ELISPOT assays independently confirmed that most of the Melan-A–specific cells were not Ag experienced. Thus, it appears that high frequencies of naive Melan-A–specific CD8+ T cells can be found in a large proportion of HLA-A*0201+ individuals. Furthermore, as demonstrated for one patient followed over time, dramatic phenotype changes of circulating Melan-A–specific cells can occur in vivo

Analysis of adequacy levels for human resources improvement within primary health care framework in Africa

Human resources in health care system in sub-Saharan Africa are generally picturing a lack of adequacy between expected skills from the professionals and health care needs expressed by the populations. It is, however, possible to analyse these various lacks of adequacy related to human resource management and their determinants to enhance the effectiveness of the health care system. From two projects focused on nurse professionals within the health care system in Central Africa, we present an analytic grid for adequacy levels looking into the following aspects: - adequacy between skills-based profiles for health system professionals, quality of care and service delivery (health care system /medical standards), needs and expectations from the populations, - adequacy between allocation of health system professionals, quality of care and services delivered (health care system /medical standards), needs and expectations from the populations, - adequacy between human resource management within health care system and medical standards, - adequacy between human resource management within education/teaching/training and needs from health care system and education sectors, - adequacy between basic and on-going education and realities of tasks expected and implemented by different categories of professionals within the health care system body, - adequacy between intentions for initial and on-going trainings and teaching programs in health sciences for trainers (teachers/supervisors/health care system professionals/ directors (teaching managers) of schools...). This tool is necessary for decision-makers as well as for health care system professionals who share common objectives for changes at each level of intervention within the health system. Setting this adequacy implies interdisciplinary and participative approaches for concerned actors in order to provide an overall vision of a more broaden system than health district, small island with self-rationality, and in which they operate

Large-scale identification and characterization of alternative splicing variants of human gene transcripts using 56 419 completely sequenced and manually annotated full-length cDNAs

We report the first genome-wide identification and characterization of alternative splicing in human gene transcripts based on analysis of the full-length cDNAs. Applying both manual and computational analyses for 56 419 completely sequenced and precisely annotated full-length cDNAs selected for the H-Invitational human transcriptome annotation meetings, we identified 6877 alternative splicing genes with 18 297 different alternative splicing variants. A total of 37 670 exons were involved in these alternative splicing events. The encoded protein sequences were affected in 6005 of the 6877 genes. Notably, alternative splicing affected protein motifs in 3015 genes, subcellular localizations in 2982 genes and transmembrane domains in 1348 genes. We also identified interesting patterns of alternative splicing, in which two distinct genes seemed to be bridged, nested or having overlapping protein coding sequences (CDSs) of different reading frames (multiple CDS). In these cases, completely unrelated proteins are encoded by a single locus. Genome-wide annotations of alternative splicing, relying on full-length cDNAs, should lay firm groundwork for exploring in detail the diversification of protein function, which is mediated by the fast expanding universe of alternative splicing variants

Rpb1 Sumoylation in Response to UV Radiation or Transcriptional Impairment in Yeast

Covalent modifications of proteins by ubiquitin and the Small Ubiquitin-like MOdifier (SUMO) have been revealed to be involved in a plethora of cellular processes, including transcription, DNA repair and DNA damage responses. It has been well known that in response to DNA damage that blocks transcription elongation, Rpb1, the largest subunit of RNA polymerase II (Pol II), is ubiquitylated and subsequently degraded in mammalian and yeast cells. However, it is still an enigma regarding how Pol II responds to damaged DNA and conveys signal(s) for DNA damage-related cellular processes. We found that Rpb1 is also sumoylated in yeast cells upon UV radiation or impairment of transcription elongation, and this modification is independent of DNA damage checkpoint activation. Ubc9, an E2 SUMO conjugase, and Siz1, an E3 SUMO ligase, play important roles in Rpb1 sumoylation. K1487, which is located in the acidic linker region between the C-terminal domain and the globular domain of Rpb1, is the major sumoylation site. Rpb1 sumoylation is not affected by its ubiquitylation, and vice versa, indicating that the two processes do not crosstalk. Abolishment of Rpb1 sumoylation at K1487 does not affect transcription elongation or transcription coupled repair (TCR) of UV-induced DNA damage. However, deficiency in TCR enhances UV-induced Rpb1 sumoylation, presumably due to the persistence of transcription-blocking DNA lesions in the transcribed strand of a gene. Remarkably, abolishment of Rpb1 sumoylation at K1487 causes enhanced and prolonged UV-induced phosphorylation of Rad53, especially in TCR-deficient cells, suggesting that the sumoylation plays a role in restraining the DNA damage checkpoint response caused by transcription-blocking lesions. Our results demonstrate a novel covalent modification of Rpb1 in response to UV induced DNA damage or transcriptional impairment, and unravel an important link between the modification and the DNA damage checkpoint response

Therapeutic value of scapular and pelvic girdle disarticulations in sarcoma

The authors have reviewed 22 cases of proximal disarticulations with the aim of assessing the therapeutic value, taking into account previous radio- and chemotherapy. The following criteria were especially examined: recurrences, survival, quality of life. There were 13/22 soft tissue sarcomas, 9/22 bone sarcomas. In 10 instances, the tumour was primary and treated for the first time whilst, in 12 cases, it was recurrence. Eighteen patients had been previously treated by non radical surgery, 11 by radiotherapy and 10 by chemotherapy. For upper limb tumours, six patients underwent an inter-scapulo-thoracic disarticulation and three an inter-scapulo-thoracic resection according to Tykhor-Lindberg. For lower limb tumours, seven patients were submitted to inter-ilio-abdominal disarticulation, three to coxo-femoral disarticulation and one to internal hemipelvectomy according to Eilber. Mean disease free interval has been 34.5 months and mean survival 38.5 months. Three out of 20 evaluable patients (15%) recurred locally although most of them benefited from second surgery. Quality of life has been excellent in general despite the fact that only seven patients accepted wearing a prosthesis. Karnofsky index ranged between 60 and 100%. No significant difference was seen, whether or not previous radiotherapy and/or chemotherapy had been administered.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Upper and lower limb disarticulation in soft tissue and bone sarcomas

The authors have reviewed 23 patients with proximal disarticulations with the aim of assessing the therapeutic value in sarcoma, taking into account previous radio- and chemotheray. The following criteria were especially examined: recurrences, survival, and quality of life. There were 14 soft tissue sarcomas, and 9 bone sarcomas. In 11 cases, the tumor was primary and treated for the first time while, in 12 cases, it was recurrent. Eighteen patients had been previously treated by nonradical surgery, 11 by radiotherapy, and 10 by chemotherapy. For upper limb tumors, 6 patients underwent an interscapulothoracic disarticulation, and 3 an interscapulothoracic resection according to Tikhoff-Linberg. For lower limb tumors, 8 patients were submitted to interilioabdominal disarticulation, 3 to coxofemoral disarticulation, and 1 to internal hemipelvectomy according to Eilber. The mean disease-free interval has been 34 months, and the mean survival, 38.5 months. Three (15%) of 20 evaluable patients recurred locally, although most of them benefited from second surgery. Quality of life has been excellent, in general, despite the fact that only 8 patients accepted to wear a prosthesis. The Karnofsky index ranged from 60 to 100%. No significant difference was seen, whether or not previous radio- and/or chemotherapy had been administered.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Hyperthermic isolation perfusion of the limbs with cytostatics after surgical excision of sarcomas

In order to prevent recurrence, isolation perfusion was applied in 15 patients after resection of sarcomas of the limbs. There were 6 liposarcomas, 4 synoviosarcomas, 2 malignant fibrohistiocytomas, 1 malignant mesenchynoma, 1 Kaposi, and 1 osteosarcoma (the only bone sarcoma). Eight sarcomas were recurrent after narrow surgery and/or radiotherapy. The treatment schedule was conducted in 2 steps: (a) removal of the tumor with safety margins at least outside the tumor capsule, followed 3-8 weeks later by (b) isolation perfusion with melphalan with or without actinomycin D under moderate hyperthermia at 39-41°C for 1 hour. Median follow-up time has been 30 months. Four of the 8 recurrent sarcomas recurred again and developed distal metastases. In contrast, none of the previously untreated patients recurred and 1 disseminated in the form of multicentric liposarcoma. Eleven are still alive with survival ranging from 8 to 103 months. It is concluded that isolation perfusion with melphalan with or without actinomycin D may be considered as an adjunct to surgery.SCOPUS: ar.jinfo:eu-repo/semantics/publishe