Biopsy diagnosis of intraductal carcinoma is prognostic in intermediate and high risk prostate cancer patients treated by radiotherapy.

AIM: We investigated the prognostic significance of intraductal carcinoma of the prostate (IDC-P) in biopsies and transurethral resections prior to external beam radiotherapy with or without androgen deprivation. METHODS: Cohort 1 consisted of 118 intermediate risk prostate cancer patients treated by radiotherapy, with biochemical relapse as primary end-point (median follow-up 6.5 years). Cohort 2 consisted of 132 high risk patients, enrolled in a phase III randomised trial (EORTC 22863) comparing radiotherapy alone to radiotherapy with long-term androgen deprivation (LTAD) with clinical progression free survival as primary end-point (median follow-up 9.1 years). Presence of IDC-P was identified after central review. Multivariable regression modelling and Kaplan-Meier analysis were performed with IDC-P as dichotomous variable. RESULTS: IDC-P was a strong prognosticator for early (<36 months) biochemical relapse (HR 7.3; p = 0.007) in cohort 1 and for clinical disease-free survival in both arms of cohort 2 (radiotherapy arm: HR 3.5; p < 0.0001; radiotherapy plus LTAD arm: HR 2.8, p = 0.018). IDC-P retained significance after stratification for reviewed Gleason score in the radiotherapy arm (HR 2.3; p = 0.03). IDC-P was a strong prognosticator for metastatic failure rate (radiotherapy arm: HR 5.3; p < 0.0001; radiotherapy plus LTAD arm: HR 3.6; p = 0.05). CONCLUSIONS: IDC-P in diagnostic samples of patients with intermediate or high risk prostate cancer is an independent prognosticator of early biochemical relapse and metastatic failure rate after radiotherapy. We suggest that the presence of IDC-P in prostate biopsies should routinely be reported.

Oxidative stress response to menadione and cumene hydroperoxide in the opportunistic fungal pathogen Candida glabrata

Candida glabrata is an opportunistic fungal pathogen that can cause severe invasive infections and can evade phagocytic cell clearance. We are interested in understanding the virulence of this fungal pathogen, in particular its oxidative stress response. Here we investigated C. glabrata, Saccharomyces cerevisiae and Candida albicans responses to two different oxidants: menadione and cumene hydroperoxide (CHP). In log-phase, in the presence of menadione, C. glabrata requires Cta1p (catalase), while in a stationary phase (SP), Cta1p is dispensable. In addition, C. glabrata is less resistant to menadione than C. albicans in SP. The S. cerevisiae laboratory reference strain is less resistant to menadione than C. glabrata and C. albicans; however S. cerevisiaeclinical isolates (CIs) are more resistant than the lab reference strain. Furthermore, S. cerevisiae CIs showed an increased catalase activity. Interestingly, in SP C. glabrata and S. cerevisiae are more resistant to CHP than C. albicans and Cta1p plays no apparent role in detoxifying this oxidant.

Linezolid alone or combined with rifampin against methicillin-resistant Staphylococcus aureus in experimental foreign-body infection.

We investigated the activity of linezolid, alone and in combination with rifampin (rifampicin), against a methicillin-resistant Staphylococcus aureus (MRSA) strain in vitro and in a guinea pig model of foreign-body infection. The MIC, minimal bactericidal concentration (MBC) in logarithmic phase, and MBC in stationary growth phase were 2.5, >20, and >20 microg/ml, respectively, for linezolid; 0.01, 0.08, and 2.5 microg/ml, respectively, for rifampin; and 0.16, 0.63, >20 microg/ml, respectively, for levofloxacin. In time-kill studies, bacterial regrowth and the development of rifampin resistance were observed after 24 h with rifampin alone at 1x or 4x the MIC and were prevented by the addition of linezolid. After the administration of single intraperitoneal doses of 25, 50, and 75 mg/kg of body weight, linezolid peak concentrations of 6.8, 12.7, and 18.1 microg/ml, respectively, were achieved in sterile cage fluid at approximately 3 h. The linezolid concentration remained above the MIC of the test organism for 12 h with all doses. Antimicrobial treatments of animals with cage implant infections were given twice daily for 4 days. Linezolid alone at 25, 50, and 75 mg/kg reduced the planktonic bacteria in cage fluid during treatment by 1.2 to 1.7 log(10) CFU/ml; only linezolid at 75 mg/kg prevented bacterial regrowth 5 days after the end of treatment. Linezolid used in combination with rifampin (12.5 mg/kg) was more effective than linezolid used as monotherapy, reducing the planktonic bacteria by >or=3 log(10) CFU (P < 0.05). Efficacy in the eradication of cage-associated infection was achieved only when linezolid was combined with rifampin, with cure rates being between 50% and 60%, whereas the levofloxacin-rifampin combination demonstrated the highest cure rate (91%) against the strain tested. The linezolid-rifampin combination is a treatment option for implant-associated infections caused by quinolone-resistant MRSA.

Epigenetic regulation of human cancer/testis antigen gene, HAGE, in chronic myeloid leukemia.

BACKGROUND AND OBJECTIVES Cancer testis antigens (CTA) provide attractive targets for cancer-specific immunotherapy. Although CTA genes are expressed in some normal tissues, such as the testis, this immunologically protected site lacks MHC I expression and as such, does not present self antigens to T cells. To date, CTA genes have been shown to be expressed in a range of solid tumors via demethylation of their promoter CpG islands, but rarely in chronic myeloid leukemia (CML) or other hematologic malignancies. DESIGN AND METHODS In this study, the methylation status of the HAGE CTA gene promoter was analyzed by quantitative methylation-specific polymerase chain reaction (MSP) and sequencing in four Philadelphia-positive cell lines (TCC-S, K562, KU812 and KYO-1) and in CML samples taken from patients in chronic phase (CP n=215) or blast crisis (BC n=47). HAGE expression was assessed by quantitative reverse transcriptase-polymerase chain reaction. RESULTS The TCC-S cell line showed demethylation of HAGE that was associated with over-expression of this gene. HAGE hypomethylation was significantly more frequent in BC (46%) than in CP (22%) (p=0.01) and was correlated with high expression levels of HAGE transcripts (p<0.0001). Of note, in CP-CML, extensive HAGE hypomethylation was associated with poorer prognosis in terms of cytogenetic response to interferon (p=0.01) or imatinib (p=0.01), molecular response to imatinib (p=0.003) and progression-free survival (p=0.05). INTERPRETATIONS AND CONCLUSION: The methylation status of the HAGE promoter directly correlates with its expression in both CML cell lines and patients and is associated with advanced disease and poor outcome.

Allogeneic stem cell transplantation after reduced intensity conditioning in patients with relapsed or refractory Hodgkin's lymphoma. Results of the HDR-ALLO study - a prospective clinical trial by the Grupo Español de Linfomas/Trasplante de Médula Osea (GEL/TAMO) and the Lymphoma Working Party of the European Group for Blood and Marrow Transplantation.

BACKGROUND Although Hodgkin's lymphoma is a highly curable disease with modern chemotherapy protocols, some patients are primary refractory or relapse after first-line chemotherapy or even after high-dose therapy and autologous stem cell transplantation. We investigated the potential role of allogeneic stem cell transplantation in this setting. DESIGN AND METHODS In this phase II study 92 patients with relapsed Hodgkin's lymphoma and an HLA-identical sibling, a matched unrelated donor or a one antigen mismatched, unrelated donor were treated with salvage chemotherapy followed by reduced intensity allogeneic transplantation. Fourteen patients showed refractory disease and died from progressive lymphoma with a median overall survival after trial entry of 10 months (range, 6-17). Seventy-eight patients proceeded to allograft (unrelated donors, n=23). Fifty were allografted in complete or partial remission and 28 in stable disease. Fludarabine (150 mg/m(2) iv) and melphalan (140 mg/m(2) iv) were used as the conditioning regimen. Anti-thymocyte globulin was additionally used as graft-versus-host-disease prophylaxis for recipients of grafts from unrelated donors. RESULTS The non-relapse mortality rate was 8% at 100 days and 15% at 1 year. Relapse was the major cause of failure. The progression-free survival rate was 47% at 1 year and 18% at 4 years from trial entry. For the allografted population, the progression-free survival rate was 48% at 1 year and 24% at 4 years. Chronic graft-versus-host disease was associated with a lower incidence of relapse. Patients allografted in complete remission had a significantly better outcome. The overall survival rate was 71% at 1 year and 43% at 4 years. CONCLUSIONS Allogeneic stem cell transplantation can result in long-term progression-free survival in heavily pre-treated patients with Hodgkin's lymphoma. The reduced intensity conditioning approach significantly reduced non-relapse mortality; the high relapse rate represents the major remaining challenge in this setting. The HDR-Allo trial was registered in the European Clinical Trials Database (EUDRACT, https://eudract.ema.europa.eu/) with number 02-0036.

Modeling of hybrid systems by piecewise decomposition

Piecewise linear models systems arise as mathematical models of systems in many practical applications, often from linearization for nonlinear systems. There are two main approaches of dealing with these systems according to their continuous or discrete-time aspects. We propose an approach which is based on the state transformation, more particularly the partition of the phase portrait in different regions where each subregion is modeled as a two-dimensional linear time invariant system. Then the Takagi-Sugeno model, which is a combination of local model is calculated. The simulation results show that the Alpha partition is well-suited for dealing with such a system

Identification of Leishmania major cysteine proteinases as targets of the immune response in humans.

In this study, we report the identification of two parasite polypeptides recognized by human sera of patients infected with Leishmania major. Isolation and sequencing of the two genes encoding these polypeptides revealed that one of the genes is similar to the L. major cathepsin L-like gene family CPB, whereas the other gene codes for the L. major homologue of the cysteine proteinase a (CPA) of L. mexicana. By restriction enzyme digestion of genomic DNA, we show that the CPB gene is present in multiple copies in contrast to the cysteine proteinase CPA gene which could be unique. Specific antibodies directed against the mature regions of both types expressed in Escherichia coli were used to analyze the expression of these polypeptides in different stages of the parasite's life cycle. Polypeptides of 27 and 40 kDa in size, corresponding to CPA and CPB respectively, were detected at higher level in amastigotes than in stationary phase promastigotes. Purified recombinant CPs were also used to examine the presence of specific antibodies in sera from either recovered or active cases of cutaneous leishmaniasis patients. Unlike sera from healthy uninfected controls, all the sera reacted with recombinant CPA and CPB. This finding indicates that individuals having recovered from cutaneous leishmaniasis or with clinically apparent disease have humoral responses to cysteine proteinases demonstrating the importance of these proteinases as targets of the immune response and also their potential use for serodiagnosis.

Identifying and Implementing Effective Training for Hispanic Craft Workers, American Supervisors, and DOT Inspectors: Phase III of the Hispanic Workforce Research Project, May 2006

The number of Hispanic workers in the U.S. construction industry has been steadily increasing, and language and cultural barriers have sometimes arisen on the jobsite. Due in part to these barriers, the number of fatalities among Hispanics at construction sites in 2001 jumped 24%, while construction fatalities overall dropped 3%. This study, which constitutes Phase III of the Hispanic Workforce Research Project, addresses these language and cultural barriers by investigating the most effective way to deliver training material developed in Phases I and II to Hispanic workers, American supervisors, and department of transportation (DOT) inspectors. The research methodology consisted of assessing the needs and interests of potential and current course participants in terms of exploring innovative ways to deliver the training. The training courses were then adapted and delivered to fit the specific needs of each audience. During Phase III of this project, the research team delivered the courses described in the Phase I and II reports to eight highway construction companies and two DOT groups. The courses developed in Phases I and II consist of four construction-focused language training courses that can be part of an effective training program to facilitate integration among U.S. and Hispanic workers, increase productivity and motivation at the jobsite, and decrease the existing high mortality rate for Hispanic workers. Moreover, the research team developed a course for the construction season called Toolbox Integration Course for Hispanic workers and American supervisors (TICHA), which consists of nine 45-minute modules delivered to one construction company over 11 weeks in the summer of 2005.

Emotional processing in a ten-session general psychiatric treatment for borderline personality disorder: a case study.

This study examines the effects of a borderline-specific treatment, called general psychiatric management, on emotional change, outcome and therapeutic alliance of an outpatient presenting with borderline personality disorder. Based on the sequential model of emotional processing, emotional states were assessed in a 10-session setting. The case showed an increase in expressions of distress and no change in therapeutic alliance and tended towards general deterioration. Results suggest emotional processing may play a lesser role in general psychiatric management in early phase treatment than previously hypothezised. Copyright © 2015 John Wiley & Sons, Ltd.

Frequent clonal loss of heterozygosity but scarcity of microsatellite instability at chromosomal breakpoint cluster regions in adult leukemias.

Microsatellites are important highly polymorphic genetic markers dispersed in the human genome. Using a panel of 22 (CA)n repeat microsatellite markers mapped to recurrent breakpoint cluster regions specifically involved in leukemia, we investigated 114 adult leukemias (25 acute lymphocytic leukemia [ALL], 32 acute myeloid leukemia [AML], 36 chronic lymphocytic leukemia [CLL], and 21 chronic myeloid leukemia [CML] in chronic phase) for somatic mutations at these loci. In each patient, DNA from fresh leukemia samples was analyzed alongside normal constitutive DNA from buccal epithelium. We detected loss of heterozygosity (LOH) in 81 of 114 patients (ALL 16/25, AML 25/32, CLL 30/36, CML 10/21). Deletions were most often seen in ALL at 11q23 and 19p13; in AML at 8q22 and 11q23; in CLL at 13q14.3, 11q13, and 11q23; and in CML at 3q26. Only six deletions were reported in 74 karyotypes analyzed, whereas in these same cases, 91 LOH events were detected by microsatellites. Of 26 leukemias with a normal karyotype, 16 nevertheless showed at least one LOH by microsatellite analysis. Replication errors were found in 10 of 114 patients (8.8%). Thus, microsatellite instability is rare in leukemia in contrast to many solid tumors. Our findings suggest that in adult leukemia, LOH may be an important genetic event in addition to typical chromosomal translocations. LOH may point to the existence of tumor suppressor genes involved in leukemogenesis to a degree that has hitherto been underestimated.

RsmY, a small regulatory RNA, is required in concert with RsmZ for GacA-dependent expression of biocontrol traits in Pseudomonas fluorescens CHA0.

In the plant-beneficial soil bacterium and biocontrol model organism Pseudomonas fluorescens CHA0, the GacS/GacA two-component system upregulates the production of biocontrol factors, i.e. antifungal secondary metabolites and extracellular enzymes, under conditions of slow, non-exponential growth. When activated, the GacS/GacA system promotes the transcription of a small regulatory RNA (RsmZ), which sequesters the small RNA-binding protein RsmA, a translational regulator of genes involved in biocontrol. The gene for a second GacA-regulated small RNA (RsmY) was detected in silico in various pseudomonads, and was cloned from strain CHA0. RsmY, like RsmZ, contains several characteristic GGA motifs. The rsmY gene was expressed in strain CHA0 as a 118 nt transcript which was most abundant in stationary phase, as revealed by Northern blot and transcriptional fusion analysis. Transcription of rsmY was enhanced by the addition of the strain's own supernatant extract containing a quorum-sensing signal and was abolished in gacS or gacA mutants. An rsmA mutation led to reduced rsmY expression, via a gacA-independent mechanism. Overexpression of rsmY restored the expression of target genes (hcnA, aprA) to gacS or gacA mutants. Whereas mutants deleted for either the rsmY or the rsmZ structural gene were not significantly altered in the synthesis of extracellular products (hydrogen cyanide, 2,4-diacetylphloroglucinol, exoprotease), an rsmY rsmZ double mutant was strongly impaired in this production and in its biocontrol properties in a cucumber-Pythium ultimum microcosm. Mobility shift assays demonstrated that multiple molecules of RsmA bound specifically to RsmY and RsmZ RNAs. In conclusion, two small, untranslated RNAs, RsmY and RsmZ, are key factors that relieve RsmA-mediated regulation of secondary metabolism and biocontrol traits in the GacS/GacA cascade of strain CHA0.

Gene expression profiling of human glioblastomas for the identification of predictive factors and characterisation of molecular mechanism implicated in response to therapy and outcome

ABSTRACT Poor outcome for glioblastoma patients is largely due to resistance to chemoradiation therapy. While epigenetic inactivation of MGMT mediated DNA repair is highly predictive for benefit from the alkylating agent therapy Temozolomide, additional mechanisms for resistance associated with molecular alterations exist. Furthermore, new concepts in cancer suggest that resistance to treatment may be linked to cancer stem cells that escape therapy and act as source for tumour recurrence. We determined gene expression signatures associated with outcome in glioblastoma patients enrolled in a phase II and phase III clinical trial establishing the new combination therapy of radiation plus concomitant and adjuvant Temozolomide. Correlating stable gene clusters emerging from unsupervised analysis with survival of 42 treated patients identified a number of biological processes associated with outcome. Most prominent, a gene cluster dominated by HOX genes and comprising PROM1, was associated with resistance. PROM1 encodes CD133, a marker for a subpopulation of tumour cells enriched for glioblastoma stem- like cells. The core of this correlated HOX cluster was comprised in the top genes of a "self-renewal signature" defined in a mouse model for MLL-AF9 initiated leukaemia. The association of the HOX gene cluster with tumour resistance was confirmed in two external data sets of 146 malignant glioma As additional resistance factors we identified over-expression of the epidermal growth factor receptor gene, EGFR, while increased gene expression related to biological features of tumour host interaction, including markers for tumour vascular and cell adhesion, and innate immune response, were associated with better outcome. The "self-renewal" signature associated with resistance to the new combination chemoradiation therapy provides first clinical evidence that glioma stem like cells may implicated in resistance in a uniformly treated cohort of glioblastoma patients. This study underlines the need to target the tumour stem cell compartment, and provides some testable hypothesis for biological mechanisms relevant for malignant behaviour of glioblastoma that may be targeted in new treatment approaches. Résumé Le glioblastome, tumeur cérébrale primaire maligne la plus fréquente, est connue pour son mauvais pronostique. Des avancées chimiothérapeutiques récentes avec des agents alkylants comme le témozolomide (TMZ), ont permis une amélioration notable dans la survie de certains patients. Les bénéficiaires ont la caractéristique commune de présenter une particularité génétique, la methylation du MGMT (methylguanine methyltransferase). Néanmoins, d'autres mécanismes de résistance en fonction des aberrations moléculaires existent. Nous avons établi les profils d'expressions génétiques des patients traités par irradiation et TMZ dans des études cliniques de phase II et III. En combinant des méthodes non-supervisées et supervisées, de l'étude de la cohorte des patients traités nous avons découvert des groupes de gènes associés à la survie. Un ensemble de gènes contenant les gènes Hox semble lié au mécanisme de résistance au traitement. Récemment, les gènes Hox ont été décrits comme faisant partie d"une signature d'autorenouvellement (self-renewal) des cellules souches cancéreuses de la leucémie. L'autorenouvellement est un processus grâce auquel les cellules souches se maintiennent tout au long de la vie. Cette association à la résistance est confirmée dans deux autres études indépendantes. Un autre facteur de résistance au traitement est la surexpression du gène EGFR. D'autre part, deux groupes de gènes associés à la relation entre hôte-tumeur tels que les marqueurs des vaisseaux tumoraux et de la réponse immunitaire innée s'avèrent avoir un effet positif sur la survie des patients traités. La découverte de la signature d'autorenouvellement comme facteur de résistance à la nouvelle chimio-radiothérapie offre une preuve clinique que les cellules souches cancéreuses sont impliquées dans la résistance au traitement. If est donc logique de penser que le traitement ciblé contre des cellules souches cancéreuses va dans l'avenir permettre des thérapies anticancéreuses plus performantes.

Abundant Occurrence of Basal Radial Glia in the Subventricular Zone of Embryonic Neocortex of a Lissencephalic Primate, the Common Marmoset Callithrix jacchus.

Subventricular zone (SVZ) progenitors are a hallmark of the developing neocortex. Recent studies described a novel type of SVZ progenitor that retains a basal process at mitosis, sustains expression of radial glial markers, and is capable of self-renewal. These progenitors, referred to here as basal radial glia (bRG), occur at high relative abundance in the SVZ of gyrencephalic primates (human) and nonprimates (ferret) but not lissencephalic rodents (mouse). Here, we analyzed the occurrence of bRG cells in the embryonic neocortex of the common marmoset Callithrix jacchus, a near-lissencephalic primate. bRG cells, expressing Pax6, Sox2 (but not Tbr2), glutamate aspartate transporter, and glial fibrillary acidic protein and retaining a basal process at mitosis, occur at similar relative abundance in the marmoset SVZ as in human and ferret. The proportion of progenitors in M-phase was lower in embryonic marmoset than developing ferret neocortex, raising the possibility of a longer cell cycle. Fitting the gyrification indices of 26 anthropoid species to an evolutionary model suggested that the marmoset evolved from a gyrencephalic ancestor. Our results suggest that a high relative abundance of bRG cells may be necessary, but is not sufficient, for gyrencephaly and that the marmoset's lissencephaly evolved secondarily by changing progenitor parameters other than progenitor type.

Chronic wound healing by fetal cell therapy may be explained by differential gene profiling observed in fetal versus old skin cells.

Engineering of fetal tissue has a high potential for the treatment of acute and chronic wounds of the skin in humans as these cells have high expansion capacity under simple culture conditions and one organ donation can produce Master Cell Banks which can fabricate over 900 million biological bandages (9 x 12cm). In a Phase 1 clinical safety study, cases are presented for the treatment of therapy resistant leg ulcers. All eight patients, representing 13 ulcers, tolerated multiple treatments with fetal biological bandages showing no negative secondary effects and repair processes similar to that seen in 3rd degree burns. Differential gene profiling using Affymetrix gene chips (analyzing 12,500 genes) were accomplished on these banked fetal dermal skin cells compared to banked dermal skin cells of an aged donor in order to point to potential indicators of wound healing. Families of genes involved in cell adhesion and extracellular matrix, cell cycle, cellular signaling, development and immune response show significant differences in regulation between banked fetal and those from banked old skin cells: with approximately 47.0% of genes over-expressed in fetal fibroblasts. It is perhaps these differences which contribute to efficient tissue repair seen in the clinic with fetal cell therapy.

Efficacy of daptomycin in implant-associated infection due to methicillin-resistant Staphylococcus aureus: importance of combination with rifampin.

Limited treatment options are available for implant-associated infections caused by methicillin (meticillin)-resistant Staphylococcus aureus (MRSA). We compared the activity of daptomycin (alone and with rifampin [rifampicin]) with the activities of other antimicrobial regimens against MRSA ATCC 43300 in the guinea pig foreign-body infection model. The daptomycin MIC and the minimum bactericidal concentration in logarithmic phase and stationary growth phase of MRSA were 0.625, 0.625, and 20 microg/ml, respectively. In time-kill studies, daptomycin showed rapid and concentration-dependent killing of MRSA in stationary growth phase. At concentrations above 20 microg/ml, daptomycin reduced the counts by >3 log(10) CFU/ml in 2 to 4 h. In sterile cage fluid, daptomycin peak concentrations of 23.1, 46.3, and 53.7 microg/ml were reached 4 to 6 h after the administration of single intraperitoneal doses of 20, 30, and 40 mg/kg of body weight, respectively. In treatment studies, daptomycin alone reduced the planktonic MRSA counts by 0.3 log(10) CFU/ml, whereas in combination with rifampin, a reduction in the counts of >6 log(10) CFU/ml was observed. Vancomycin and daptomycin (at both doses) were unable to cure any cage-associated infection when they were given as monotherapy, whereas rifampin alone cured the infections in 33% of the cages. In combination with rifampin, daptomycin showed cure rates of 25% (at 20 mg/kg) and 67% (at 30 mg/kg), vancomycin showed a cure rate of 8%, linezolid showed a cure rate of 0%, and levofloxacin showed a cure rate of 58%. In addition, daptomycin at a high dose (30 mg/kg) completely prevented the emergence of rifampin resistance in planktonic and adherent MRSA cells. Daptomycin at a high dose, corresponding to 6 mg/kg in humans, in combination with rifampin showed the highest activity against planktonic and adherent MRSA. Daptomycin plus rifampin is a promising treatment option for implant-associated MRSA infections.