Conventional amphotericin B elicits markers of immunogenic cell death on leukemic blasts, mediates immunostimulatory effects on phagocytic cells, and synergizes with PD-L1 blockade.

Immunostimulatory regimens are a game changer in the fight against cancer, but still only a minority of patients achieve clinical benefit. Combination with immunomodulatory drugs and agents converting otherwise non-immunogenic forms of cell death into bona fide "immunogenic cell death" (ICD) could improve the efficacy of these novel therapies. The aim of our study was to investigate conventional Amphotericin B (AmB) as an enhancer of antitumor immune responses. In tumor cell line models, AmB induced ICD with its typical hallmarks of calreticulin (CALR) expression and release of high mobility group box 1 (HMGB1) as well as Adenosine 5'-triphosphate (ATP). Interestingly, in contrast to non-ICD inducing treatments, ICD induction led to up-regulation of PD-L1-expression by ICD experiencing cells, resulting in decreased maturation of dendritic cells (DCs). Blocking this PD-L1 expression on tumor cells could unleash full ICD effects on antigen presenting cells. Even at sub-toxic concentrations, AmB was able to enhance CALR on leukemic blasts, particularly on phagocytic monoblastic THP-1 cells, which also showed features of "M1-like" differentiation after AmB exposure. The ability of AmB to increase the immunogenicity of tumor cells was confirmed in vivo in a mouse vaccination experiment. In conclusion, we demonstrate that AmB can promote antitumor immune responses in a dose-dependent manner by ICD induction, surface translocation of CALR on leukemic blasts even at sub-toxic concentrations, and "M1-like" polarization of phagocytic cells, making it noteworthy as potential booster for cancer immunotherapy. We additionally report for the first time that PD-L1 expression may be a feature of ICD, possibly as a negative feedback mechanism regulating the maturation status of DCs and thus indirectly affecting T-cell priming.

[Professionalization of Legal Dental Experts in Germany: Results of Studies on Structured Focus Groups]. / Ergebnisse strukturierter Fokusgruppenarbeit zur Professionalisierung des zahnärztlichen Gutachterwesens in Deutschland.

BACKGROUND: Legal expert opinions are a crucial instrument of professional self-control in medicine. To give impulses for further development, focus groups were initiated to reflect upon the perspective of legal dental experts. METHODS: 5 focus group discussions on the topic "Professionalization of legal dental experts" were conducted. A total of 32 experienced legal dental experts participated in the discussions. The results were evaluated by qualitative content analysis. RESULTS: A catalogue of 68 ideas was generated for improvement and divided into 15 categories. Among these were periodic quality circles, interprofessional exchange, supervision of novices and periodic feedback for legal dental experts and dentists. CONCLUSION: Self-reflection can be included as an instrument for quality improvement of legal dental expert opinions.

SPON2, a newly identified target gene of MACC1, drives colorectal cancer metastasis in mice and is prognostic for colorectal cancer patient survival.

MACC1 (metastasis associated in colon cancer 1) is a prognostic biomarker for tumor progression, metastasis and survival of a variety of solid cancers including colorectal cancer (CRC). Here we aimed to identify the MACC1-induced transcriptome and key players mediating the MACC1-induced effects in CRC. We performed microarray analyses using CRC cells ectopically overexpressing MACC1. We identified more than 1300 genes at least twofold differentially expressed, including the gene SPON2 (Spondin 2) as 90-fold upregulated transcriptional target of MACC1. MACC1-dependent SPON2 expression regulation was validated on mRNA and protein levels in MACC1 high (endogenously or ectopically) and low (endogenously or by knockdown) expressing cells. Chromatin immunoprecipitation analysis demonstrated the binding of MACC1 to the gene promoter of SPON2. In cell culture, ectopic SPON2 overexpression induced cell viability, migration, invasion and colony formation in endogenously MACC1 and SPON2 low expressing cells, whereas SPON2 knockdown reduced proliferative, migratory and invasive abilities in CRC cells with high endogenous MACC1 and SPON2 expression. In intrasplenically transplanted NOD/SCID mice, metastasis induction was analyzed with control or SPON2-overexpressing CRC cells. Tumors with SPON2 overexpression induced liver metastasis (vs control animals without any metastases, P=0.0036). In CRC patients, SPON2 expression was determined in primary tumors (stages I-III), and survival time was analyzed by Kaplan-Meier method. CRC patients with high SPON2 expressing primary tumors demonstrated 8 months shorter metastasis-free survival (MFS) compared with patients with low SPON2 levels (P=0.053). Combining high levels of SPON2 and MACC1 improved the identification of high-risk patients with a 20-month shorter MFS vs patients with low biomarker expression. In summary, SPON2 is a transcriptional target of the metastasis gene MACC1. SPON2 induces cell motility in vitro and CRC metastasis in mice. In patients, SPON2 serves as prognostic indicator for CRC metastasis and survival, and might represent a promising target for therapeutic approaches.

[Prevention and Information for Patients Undergoing Periodontal Treatment: Potentials for Improvement from the Patients' Perspective]. / Prävention und Aufklärung im Rahmen der Parodontitisbehandlung: Verbesserungspotenziale aus Patientensicht.

2 334 patients from 29 dental practices took part in a written survey on their experiences with dental treatment in general as well as treatment of periodontal disease (response rate 80.8%). 72.6% of all participating patients fully agreed that they could recommend their dentist to their friends. 63.6% of patients undergoing treatment of periodontitis (N=328) rated this treatment as "excellent". However, for important aspects (prevention, patient information, treatment) potentials for improvement became obvious. 43.7% of patients treated for periodontitis were not completely satisfied with information on how this disease develops; 40.7% saw potentials for better information on preventive care (dental-hygiene, nutrition). An even higher percentage of patients actually not treated for periodontitis was interested in more information on prevention (51.4%). The results of the survey show that dentists should offer information and exercise on how to prevent periodontal desease more actively. There is a lack of research on the present state of affairs and potentials for improvement concerning treatment and prevention of periodontitis including the patients' perspective.

[How dentists experience legal disputes with their patients - a qualitative approach]. / Wie erleben Zahnärzte eine gerichtliche Auseinandersetzung mit Patienten? Eine qualitative Untersuchung.

BACKGROUND: A well-established doctor-patient relationship is essential for dental care. Once ­there is a legal dispute between the dentist and the patient this doctor-patient relationship has failed. Here we show for the first time how dentists experience these lawsuits. We characterise the dentist's perspectives and investigate their influence on the doctor-patient relationship. METHODS: The experience of dentists who were involved in a legal dispute due to a failed dental treatment is pictured in a pilot study on the basis of narrative and problem-oriented interviews. The narrative part of the interview was analysed with the technique of narration analysis. The problem-oriented part of the interview was evaluated content-analytically. From these data a model describing the failure of a doctor-patient relationship was developed. RESULTS: Communicated burdens and stress were: expenditure of time, duration of the proceedings, financial burdens, limited support and the feeling of being treated unfairly. Consequences taken from the legal dispute were a more detailed documentation and an improvement in patient information. The conflict in the doctor-patient relationship is transformed to a different platform when the legal profession is involved. This contradictory constellation leads to a break in the doctor-patient relationship. Advice for other affected colleagues is: remain calm, aim at an out of court settlement, seek advice from other colleagues and request legal advice, be proactive, avoid court litigation and in the end draw conclusions from the dispute. There is a need for a higher quality of legal dental experts. A contact platform provided by the dental profession is required for interactive collegial communication. CONCLUSION: Lawsuits in cases of a conflict between the doctor and the patient are very stressful and need an active coping management but also are a chance for the development of the individual dentist and the profession.

Impact of mutant ß-catenin on ABCB1 expression and therapy response in colon cancer cells.

BACKGROUND: Colorectal cancers are often chemoresistant toward antitumour drugs that are substrates for ABCB1-mediated multidrug resistance (MDR). Activation of the Wnt/ß-catenin pathway is frequently observed in colorectal cancers. This study investigates the impact of activated, gain-of-function ß-catenin on the chemoresistant phenotype. METHODS: The effect of mutant (mut) ß-catenin on ABCB1 expression and promoter activity was examined using HCT116 human colon cancer cells and isogenic sublines harbouring gain-of-function or wild-type ß-catenin, and patients' tumours. Chemosensitivity towards 24 anticancer drugs was determined by high throughput screening. RESULTS: Cell lines with mut ß-catenin showed high ABCB1 promoter activity and expression. Transfection and siRNA studies demonstrated a dominant role for the mutant allele in activating ABCB1 expression. Patients' primary colon cancer tumours shown to express the same mut ß-catenin allele also expressed high ABCB1 levels. However, cell line chemosensitivities towards 24 MDR-related and non-related antitumour drugs did not differ despite different ß-catenin genotypes. CONCLUSION: Although ABCB1 is dominantly regulated by mut ß-catenin, this did not lead to drug resistance in the isogenic cell line model studied. In patient samples, the same ß-catenin mutation was detected. The functional significance of the mutation for predicting patients' therapy response or for individualisation of chemotherapy regimens remains to be established.

Novel Clostridium perfringens enterotoxin suicide gene therapy for selective treatment of claudin-3- and -4-overexpressing tumors.

Bacterial toxins are known to be effective for cancer therapy. Clostridium perfringens enterotoxin (CPE) is produced by the bacterial Clostridium type A strain. The transmembrane proteins claudin-3 and -4, often overexpressed in numerous human epithelial tumors (for example, colon, breast, pancreas, prostate and ovarian), are the targeted receptors for CPE. CPE binding to them triggers formation of membrane pore complexes leading to rapid cell death. In this study, we aimed at selective tumor cell killing by CPE gene transfer. We generated expression vectors bearing the bacterial wild-type CPE cDNA (wtCPE) or translation-optimized CPE (optCPE) cDNA for in vitro and in vivo gene therapy of claudin-3- and -4-overexpressing tumors. The CPE expression analysis at messenger RNA and protein level revealed more efficient expression of optCPE compared with wtCPE. Expression of optCPE showed rapid cytotoxic activity, hightened by CPE release as bystander effect. Cytotoxicity of up to 100% was observed 72 h after gene transfer and is restricted to claudin-3-and -4-expressing tumor lines. MCF-7 and HCT116 cells with high claudin-4 expression showed dramatic sensitivity toward CPE toxicity. The claudin-negative melanoma line SKMel-5, however, was insensitive toward CPE gene transfer. The non-viral intratumoral in vivo gene transfer of optCPE led to reduced tumor growth in MCF-7 and HCT116 tumor-bearing mice compared with the vector-transfected control groups. This novel approach demonstrates that CPE gene transfer can be employed for a targeted suicide gene therapy of claudin-3- and -4-overexpressing tumors, leading to the rapid and efficient tumor cell killing in vitro and in vivo.

[Attitudes of dental legal expert witnesses on their present status in Germany and approaches to the development of the profession]. / Ist-Zustand und Ansätze zur Professionalisierung des zahnärztlichen Gutachterwesens in Deutschland aus der Selbstsicht der Gutachter.

BACKGROUND: Dental litigation has a key role for the autonomy of the dental profession. We conducted a study among legal dental expert witnesses in order to reflect the present situation and reveal the potential for professional development. METHODS: A questionnaire was distributed among 161 participants of the Karlsruhe training for legal dental experts between 2004 and 2009. They were asked to describe and to reflect on the present situation of dental litigation in Germany. RESULTS: 83 questionnaires were returned (51.6%). The main reason to become a legal dental expert was to "support the profession". 68 participants (85.0%) think that civil action resulting from dental treatment will become more frequent. The quality of dental expert opinions is considered to be in need of improvement. Strategies to optimise dental expert opinions and to deal with the potential growing number of claims are developed. CONCLUSION: Basic and advanced training for dental expert witnesses assures the quality of dental expert opinions and also provides a chance for the further development of the dental profession.

[Patient evaluation of dental care. Results of a written patient survey in dental practices]. / Wie bewerten Patienten ihre zahnärztliche Versorgung? Ergebnisse einer schriftlichen Befragung von Patienten bei niedergelassenen Zahnärzten.

1,317 patients from 18 dentists took part in a written survey on patient evaluation of dental care. General satisfaction was high, but patients were critical concerning some aspects of dental care. On average, the aspects rated most often with "excellent" were hygiene in the practice, the possibility to get through to the practice on the telephone and quick service in case of urgent health problems. Most critical evaluations (on average) were given for waiting times, costs of the dental treatment for the patient and the range of magazines and written information in the waiting room. The highest statistical correlation to overall satisfaction (willingness to recommend this doctor to friends) showed the patients' assessments concerning the questions whether the doctor was listening to them and took enough time, as well as the result of the dental treatment from the patients' point of view. Differences of the survey results between practices were high. On average, patients of female dentists were more satisfied than patients of their male colleagues. Patients younger than 50 years and male patients were less satisfied than older patients and female patients. The patient surveys give important clues for quality management in the participating dental practices. The results of a patient survey should be evaluated against the background of the individual situation of the practice.

Uptake, biodistribution, and time course of naked plasmid DNA trafficking after intratumoral in vivo jet injection.

Nonviral jet injection is an applicable technology for in vivo gene transfer of naked DNA. However, little is known about the biodistribution and clearance of jet-injected DNA, or about its localization within tissue and cells. Therefore, in this study we analyzed the intratumoral and systemic biodistribution of jet-injected naked DNA in human colon carcinoma-bearing NCr-nu/nu mice, which were jet-injected with the pCMVbeta plasmid DNA. Intratumoral and systemic plasmid DNA biodistribution was analyzed 5, 10, 20, and 40 min and 3, 6, 24, 48, and 72 hr after jet injection, using quantitative real-time polymerase chain reaction. In the tumors, a rapid drop in naked DNA load within 24 hr of jet injection was shown. Detailed analysis of intratumoral distribution of rhodamine-labeled DNA revealed the presence of plasmid DNA within tumor cells 5 min after jet injection and further accumulation of significant DNA amounts in the cell nuclei 30 to 60 min after jet injection. In the blood, DNA amounts rapidly dropped within 10 to 40 min of jet injection to less than 0.001 pg of plasmid per 250 ng of tissue DNA and only minimal plasmid DNA dissemination was detected in liver, lung, spleen, kidney, and ovaries, which was cleared 3 to 6 hr after jet injection. By contrast, in heart, bone marrow, and brain almost no plasmid DNA was detectable.

Pentoxifylline influences drug transport activity of P-glycoprotein and decreases mdrl gene expression in multidrug resistant mouse leukemic L1210/VCR cells.

The effects of pentoxifylline (PTX) on intracellular accumulation of doxorobicin (DOX), DOX cytotoxicity and expression of Pgp in multidrug resistant L1210/VCR cell line were investigated. PTX (100 mg/l) was able to enhance the DOX accumulation in resistant cells. The maximum intracellular levels of DOX were reached after treatment with PTX for 24 hours (total duration of PTX-treatment was 72 hours). The levels of mdrl mRNA (measured by RT-PCR method) were decreased 2-fold in the presence of 100 mg/l PTX (minimum reached within 48 hours) in comparison to control cells.

Hyperthermia-induced nuclear translocation of transcription factor YB-1 leads to enhanced expression of multidrug resistance-related ABC transporters.

Genotoxic stress leads to nuclear translocation of the Y-box transcription factor YB-1 and enhanced expression of the multidrug resistance gene MDR1. Because hyperthermia is used for the treatment of colon cancer in combination with chemoradiotherapy, we investigated the influence of hyperthermia on YB-1 activity and the expression of multidrug resistance-related genes. Here we report that hyperthermia causes YB-1 translocation from the cytoplasm into the nucleus of human colon carcinoma cells HCT15 and HCT116. Nuclear translocation of YB-1 was associated with increased MDR1 and MRP1 gene activity, which is reflected in strong efflux pump activity. However, a combination of hyperthermia and drug treatment effectively reduced cell survival of the HCT15 and HCT116 cells. These results demonstrate that activation of MDR1 and MRP1 gene expression and increased efflux pump activity after hyperthermia were insufficient to cause an increase in drug resistance in colon cancer cell lines. The ability of hyperthermia to abrogate drug resistance in the presence of an increase in functional MDR proteins may provide an explanation for the efficacious results seen in the clinic in colon cancer patients treated with a combination of hyperthermia and chemotherapy.

Nonviral in vivo gene delivery into tumors using a novel low volume jet-injection technology.

The jet-injection technology has developed as an applicable alternative to viral or liposomal gene delivery systems. In this study a novel, low-volume, 'high-speed jet injector' hand-held system was used for the direct gene transfer of naked DNA into tumors. Lewis-lung carcinoma bearing mice were jet-injected with the beta-galactosidase (LacZ), the green fluorescence (GFP) or the human tumor necrosis factor alpha (TNF-alpha) gene carrying vector plasmids. The animals received five jet injections into the tumor at a pressure of 3.0 bar, delivering 3--5 microl plasmid DNA (1 microg DNA/microl in water) per single jet injection. The jet injection of DNA leads to a widespread expression pattern within tumor tissues with penetration depths of 5--10 mm. Analysis of tumor cryosections revealed moderate LacZ or GFP expression at 48 h and strong reporter gene expression 72 h and 96 h after jet injection. The simultaneous jet injection of the TNF-alpha and LacZ carrying vectors demonstrated efficient expression and secretion of both the cytokine, as well as LacZ expression within the tumor 24 h, 48 h, 72 h, 96 h and 120 h after jet injection. These studies demonstrate the applicability of jet injection for the efficient in vivo gene transfer into tumors for nonviral gene therapy of cancer using minimal amounts of naked DNA.

Cu(II)- and Hg(II)-induced modulation of the fluorescence behavior of a redox-active sensor molecule.

Here, we report on a fluorescent 1,2,4-thiadiazole derivative (oxidized form) and its reduced form, the corresponding iminoyl thiourea. The thiadiazole displays a strong modulation of its fluorescence behavior, selectively upon addition of Cu(II), while the iminoyl thiourea functions as a chemodosimeter for Hg(II). Additionally, the Cu(II)-thiadiazole complex is characterized by HRMS, and the Hg(II)-induced desulfurization of the iminoyl thiourea is monitored by mass spectrometry.

Cloning and initial analysis of the human multidrug resistance-related MVP/LRP gene promoter.

The lung resistance-related protein (LRP) was identified as the human major vault protein (MVP), and is overexpressed in various multidrug-resistant cancer cell lines and clinical samples. We characterized DNA sequences upstream to the transcription initiation site of the MVP gene in the human non-small cell lung cancer cell line SW-1573. A 1.9-kb and a shortened 0.7-kb fragment of the 5'-upstream genomic region show strong promoter activity in chloramphenicol acetyltransferase (CAT) reporter assays. The promoter is TATA-less and contains an inverted CCAAT-box and a Sp1 site located near to a p53 binding motif. An alternative 3'-splice site of intron 1 results in a splicing variant within the 5'-untranslated region of MVP mRNA.

Viral vectors for gene transfer: a review of their use in the treatment of human diseases.

The efficient delivery of therapeutic genes and appropriate gene expression are the crucial issues for clinically relevant gene therapy. Viruses are naturally evolved vehicles which efficiently transfer their genes into host cells. This ability made them desirable for engineering virus vector systems for the delivery of therapeutic genes. The viral vectors recently in laboratory and clinical use are based on RNA and DNA viruses processing very different genomic structures and host ranges. Particular viruses have been selected as gene delivery vehicles because of their capacities to carry foreign genes and their ability to efficiently deliver these genes associated with efficient gene expression. These are the major reasons why viral vectors derived from retroviruses, adenovirus, adeno-associated virus, herpesvirus and poxvirus are employed in more than 70% of clinical gene therapy trials worldwide. Among these vector systems, retrovirus vectors represent the most prominent delivery system, since these vectors have high gene transfer efficiency and mediate high expression of therapeutic genes. Members of the DNA virus family such as adenovirus-, adeno-associated virus or herpesvirus have also become attractive for efficient gene delivery as reflected by the fast growing number of clinical trials using these vectors. The first clinical trials were designed to test the feasibility and safety of viral vectors. Numerous viral vector systems have been developed for ex vivo and in vivo applications. More recently, increasing efforts have been made to improve infectivity, viral targeting, cell type specific expression and the duration of expression. These features are essential for higher efficacy and safety of RNA- and DNA-virus vectors. From the beginning of development and utilisation of viral vectors it was apparent that they harbour risks such as toxicities, immunoresponses towards viral antigens or potential viral recombination, which limit their clinical use. However, many achievements have been made in vector safety, the retargeting of virus vectors and improving the expression properties by refining vector design and virus production. This review addresses important issues of the current status of viral vector design and discusses their key features as delivery systems in gene therapy of human inherited and acquired diseases at the level of laboratory developments and of clinical applications.

Mdr1 promoter-driven tumor necrosis factor-alpha expression for a chemotherapy-controllable combined in vivo gene therapy and chemotherapy of tumors.

Cancer gene therapy approaches are often designed as single-agent treatments; however, greater therapeutic effect might be obtained if combined with an established conventional treatment regimen such as chemotherapy. In this context, conditional promoters are useful tools, because they may be induced by therapeutic modalities. The human multidrug resistance gene (mdr1) promoter is inducible by cytostatic drugs and can be employed for the chemotherapy-regulated expression of therapeutic genes. In this in vivo study, the human mdr1 promoter fragment (-207 to +158) was used for drug-inducible expression of human tumor necrosis factor-alpha (TNF-alpha) in the vector construct pM3mdr-p-hTNF. The single doxorubicin and vincristine treatment of nude mice xenografted with pM3mdr-p-hTNF-transduced MCF-7 mammary tumors resulted in drug-induced and time-dependent elevation of intratumoral TNF-alpha expression at the mRNA and protein level. The highest drug induction was achieved at 2 days after drug application, as reflected by a maximum 25-fold increase in TNF-alpha secretion in the tumor. This drug-induced TNF-alpha expression is more effective in inhibiting tumor growth compared with the growth of tumors transduced with constitutively TNF-alpha-expressing vectors in combination with chemotherapy.

High-copy cDNA amplification of minimal total RNA quantities for gene expression analyses.

This protocol describes a PCR-based cDNA amplification technique of small total RNA quantities, optimized for determination and verification of gene expression variations in cells or tissue specimen. A proportional amplification of rare and abundant transcripts is thereby achieved by initial random hexamer-primed reverse transcription of total RNA. Compared to established oligo(dT)-primed techniques, this approach generates shorter than full length copies of long RNAs which leads to a normalized cDNA pool for a more adequate PCR-amplification. Subsequent double oligo(dA) tailing of the synthesized total cDNA strands and the utilization of heteropolymeric primers allow a highly specific, up to 500-fold PCR-amplification of the total cellular RNA amount. Thus, obstacles in availability of RNA from limited sources, such as human biopsies or microdissected histological sections, can be overcome. The amplified total cDNA (atcDNA) is shown to be applicable for confirmation of differential gene expression, as demonstrated in this protocol by expression analysis of the multidrug resistance-associated genes mdr1, mrp1 and lrp, using human cell lines as well as microdissected human tissue sections.