MART-1 TCR gene-modified peripheral blood T cells for the treatment of metastatic melanoma: a phase I/IIa clinical trial.

Background: Adoptive cell therapy with peripheral blood T cells expressing transgenic T-cell receptors (TCRs) is an innovative therapeutic approach for solid malignancies. We investigated the safety and feasibility of adoptive transfer of autologous T cells expressing melanoma antigen recognized by T cells 1 (MART-1)-specific TCR, cultured to have less differentiated phenotypes, in patients with metastatic melanoma. Materials and methods: In this phase I/IIa trial, peripheral blood T cells from HLA-A2∗02:01-positive patients with unresectable stage IIIC/IV melanoma expressing MART-1 were selected and stimulated with anti-CD3/CD28 beads, transduced with a modified MART-1(26-35)-specific 1D3 TCR (1D3HMCys) and expanded in interleukin (IL)-7 and IL-15. Patients received a single infusion of transgenic T cells in a dose-escalating manner. Feasibility, safety and objective response rate were assessed. Results: Twelve pretreated metastatic cutaneous (n = 7) and uveal (n = 5) melanoma patients were included. Patient 1 received 4.6 × 109 1D3HMCys T cells and experienced grade 5 toxicity after 9 days. Subsequent patients received 5.0 × 107 [n = 3; cohort (c) 2], 2.5 × 108 (n = 2; c3) and 1.0 × 108 (n = 6; c4) 1D3HMCys T cells. The study was prematurely terminated because of dose-dependent toxicity, concerning skin (10/12), eyes (3/12), ears (4/12) and cytokine release syndrome (5/12), with 7 patients experiencing grade 3-5 toxicity. Partial responses were seen in 2/11 (18%) assessable patients and persistence of 1D3HMCys T cells corresponded to infused cell dose. Conclusions: Production of TCR-modified cells as described leads to highly potent T cells. Partial responses were seen in 18% of patients with dose-dependent 'on-target, off-tumor' toxicity and a maximum tolerated dose of 1.0 × 108 cells.

Dataset of liver proteins changed in eu- and hypothyroid female rats upon in vivo exposure to hexabromocyclododecane (HBCD).

Female Wistar rats with different thyroid status (eu-, hypothyroid) were exposed to 0, 3 or 30 mg/kg body weight of the flame retardant HBCD for 7 days. Changes in protein patterns obtained by 2D-DIGE were evaluated, and different animal groups compared taking into account their exposure and thyroid status. Proteins significantly altered in abundance in any of these comparisons were identified by mass spectrometry. These data, together with hormone data of the animals, are discussed in "Hexa-bromocyclododecane (HBCD) induced changes in the liver proteome of eu- and hypothyroid female rats" (Miller et al., 2016) [1].

Hexabromocyclododecane (HBCD) induced changes in the liver proteome of eu- and hypothyroid female rats.

Hexabromocyclododecane (HBCD) is a brominated flame retardant known for its low acute toxicity as observed in animal experiments. However, HBCD exposure can affect liver functioning and thyroid hormone (TH) status. As exact mechanisms are unknown and only limited toxicological data exists, a gel-based proteomic approach was undertaken. In a eu- and hypothyroid female rat model, rats were exposed to 3 and 30 mg/kg bw/day HBCD for 7 days via their diet, and exposure was related to a range of canonical endpoints (hormone status, body weight) available for these animals. Alterations in the liver proteome under HBCD exposure were determined in comparison with patterns of control animals, for both thyroid states. This revealed significantly changed abundance of proteins involved in metabolic processes (gluconeogenesis/glycolysis, amino acid metabolism, lipid metabolism), but also in oxidative stress responses, in both euthyroid and hypothyroid rats. The results provide a more detailed picture on the mechanisms involved in these alterations, e.g. at the protein level changes of the proposed influence of HBCD on the lipid metabolism. Present results show that proteomic approaches can provide further mechanistic insights in toxicological studies.

Deconjugation of soy isoflavone glucuronides needed for estrogenic activity.

Soy isoflavones (SIF) are present in the systemic circulation as conjugated forms of which the estrogenic potency is not yet clear. The present study provides evidence that the major SIF glucuronide metabolites in blood, genistein-7-O-glucuronide (GG) and daidzein-7-O-glucuronide (DG), only become estrogenic after deconjugation. The estrogenic potencies of genistein (Ge), daidzein (Da), GG and DG were determined using stably transfected U2OS-ERα, U2OS-ERß reporter gene cells and proliferation was tested in T47D-ERß cells mimicking the ERα/ERß ratio of healthy breast cells and inT47D breast cancer cells. In all assays applied, the estrogenic potency of the aglycones was significantly higher than that of their corresponding glucuronides. UPLC analysis revealed that in U2OS and T47D cells, 0.2-1.6% of the glucuronides were deconjugated to their corresponding aglycones. The resulting aglycone concentrations can account for the estrogenicity observed upon glucuronide exposure. Interestingly, under similar experimental conditions, rat breast tissue S9 fraction was about 30 times more potent in deconjugating these glucuronides than human breast tissue S9 fraction. Our study confirms that SIF glucuronides are not estrogenic as such, and that the small % of deconjugation in the cell is enough to explain the slight bioactivity observed for the SIF-glucuronides. Species differences in deconjugation capacity should be taken into account when basing risk-benefit assessment of these SIF for the human population on animal data.

Transposon leads to contamination of clinical pDNA vaccine.

We report an unexpected contamination during clinical manufacture of a Human Papilomavirus (HPV) 16 E6 encoding plasmid DNA (pDNA) vaccine, with a transposon originating from the Escherichia coli DH5 host cell genome. During processing, presence of this transposable element, insertion sequence 2 (IS2) in the plasmid vector was not noticed until quality control of the bulk pDNA vaccine when results of restriction digestion, sequencing, and CGE analysis were clearly indicative for the presence of a contaminant. Due to the very low level of contamination, only an insert-specific PCR method was capable of tracing back the presence of the transposon in the source pDNA and master cell bank (MCB). Based on the presence of an uncontrolled contamination with unknown clinical relevance, the product was rejected for clinical use. In order to prevent costly rejection of clinical material, both in-process controls and quality control methods must be sensitive enough to detect such a contamination as early as possible, i.e. preferably during plasmid DNA source generation, MCB production and ultimately during upstream processing. However, as we have shown that contamination early in the process development pipeline (source pDNA, MCB) can be present below limits of detection of generally applied analytical methods, the introduction of "engineered" or transposon-free host cells seems the only 100% effective solution to avoid contamination with movable elements and should be considered when searching for a suitable host cell-vector combination.

Recent advances towards the clinical application of DNA vaccines.

DNA vaccination is an attractive method for therapeutic vaccination against intracellular pathogens and cancer. This review provides an introduction into the DNA vaccination field and discusses the pre-clinical successes and most interesting clinical achievements thus far. Furthermore, general attributes, mechanism of action and safety of DNA vaccination will be discussed. Since clinical results with DNA vaccination so far show room for improvement, possibilities to improve the delivery and immunogenicity of DNA vaccines are reviewed. In the coming years, these new developments should show whether DNA vaccination is able to induce clinically relevant responses in patients.

DNA vaccines and intradermal vaccination by DNA tattooing.

Over the past two decades, DNA vaccination has been developed as a method for the induction of immune responses. However, in spite of high expectations based on their efficacy in preclinical models, immunogenicity of first generation DNA vaccines in clinical trials was shown to be poor, and no DNA vaccines have yet been licensed for human use. In recent years significant progress has been made in the development of second generation DNA vaccines and DNA vaccine delivery methods. Here we review the key characteristics of DNA vaccines as compared to other vaccine platforms, and recent insights into the prerequisites for induction of immune responses by DNA vaccines will be discussed. We illustrate the development of second generation DNA vaccines with the description of DNA tattooing as a novel DNA delivery method. This technique has shown great promise both in a small animal model and in non-human primates and is currently under clinical evaluation.

DNA tattoo vaccination: effect on plasmid purity and transfection efficiency of different topoisoforms.

Recently, DNA tattooing was introduced as novel intradermal administration technique for plasmid DNA (pDNA) vaccines. The aim of this study was to determine if tattooing affects the integrity of pDNA (reduction in supercoiled (SC) content) and whether a change in pDNA topology would affect antigen expression and immune response. We show that 1.) in vitro tattooing of pDNA solutions results in minor damage to pDNA (or=80% SC).

GMP production of pDERMATT for vaccination against melanoma in a phase I clinical trial.

For the treatment of melanoma DNA vaccines are a promising therapeutic approach. In our institute a plasmid encoding a melanoma-associated epitope (MART-1) and an immunostimulatory sequence (tetanus toxin fragment-c) termed pDERMATT was developed. In a phase I study the plasmid will be administered intradermally using a newly developed tattoo strategy to assess the toxicity and efficacy of inducing tumor-specific T-cell immunity. To facilitate this study a Good Manufacturing Practice (GMP)-compliant plasmid manufacturing process was set up and a pharmaceutical dosage form was developed. Each batch resulted in approximately 200mg plasmid DNA of a high purity >90% supercoiled DNA, an A260/280 ratio 1.80-1.95, undetectable or extremely low residual endotoxins, Escherichia coli host cell protein, RNA, and DNA. In the manufacturing process no animal derived enzymes like RNase or potentially harmful organic solvents are used. After sterile filtration the concentration of the plasmid solution is approximately 1.1mg/mL. For the scheduled phase I study a concentration of 5mg/mL is desired, and further concentration of the solution is achieved by lyophilisation. The formulation solution is composed of 1mg/mL pDERMATT and 20mg/mL sucrose in Water for Injections. Upon reconstitution with a five times smaller volume an isotonic sucrose solution containing 5mg/mL pDERMATT is obtained. Lyophilised pDERMATT is sterile with >90% supercoiled DNA, an A260-280 ratio 1.80-1.95, content 90-110% of labeled, and residual water content <2% (w/w). The product yields the predicted profile upon restriction-enzyme digestion, is highly immunogenic as confirmed in an in vivo mouse model, and stable for at least six months at 5 degrees C. We have not only developed a reproducible process to manufacture pharmaceutical grade plasmid DNA but also a stable dosage form for the use in clinical trials.

Food-associated estrogenic compounds induce estrogen receptor-mediated luciferase gene expression in transgenic male mice.

The present paper aims at clarifying to what extent seven food-associated compounds, shown before to be estrogenic in vitro, can induce estrogenic effects in male mice with an estrogen receptor (ER)-mediated luciferase (luc) reporter gene system. The luc induction was determined in different tissues 8h after dosing the ER-luc male mice intraperitoneally (IP) or 14h after oral dosing. Estradiol-propionate (EP) was used as a positive control at 0.3 and 1mg/kg bodyweight (bw), DMSO as solvent control. The food-associated estrogenic compounds tested at non-toxic doses were bisphenol A (BPA) and nonylphenol (NP) (both at 10 and 50mg/kgbw), dichlorodiphenyldichloroethylene (p,p'-DDE; at 5 and 25mg/kgbw), quercetin (at 1.66 and 16.6mg/kgbw), di-isoheptyl phthalate (DIHP), di-(2-ethylhexyl) phthalate (DEHP) and di-(2-ethylhexyl) adipate (DEHA) all at 30 and 100mg/kgbw. In general IP dosing resulted in higher luc inductions than oral dosing. EP induced luc activity in the liver in a statistically significant dose-related way with the highest induction of all compounds tested which was 20,000 times higher than the induction by the DMSO-control. NP, DDE, DEHA and DIHP did not induce luc activity in any of the tissues tested. BPA induced luc in the liver up to 420 times via both exposure routes. BPA, DEHP and quercetin induced luc activity in the liver after oral exposure. BPA (50mg/kgbw IP) also induced luc activity in the testis, kidneys and tibia. The current study reveals that biomarker-responses in ER-luc male mice occur after a single oral exposure to food-associated estrogenic model compounds at exposure levels 10 to 10(4) times higher than the established TDI's for some of these compounds. Given the facts that (i) the present study did not include chronic exposure and that (ii) simultaneous exposure to multiple estrogenic compounds may be a realistic exposure scenario, it remains to be seen whether this margin is sufficiently high.

A synchronized amphibian metamorphosis assay as an improved tool to detect thyroid hormone disturbance by endocrine disruptors and apolar sediment extracts.

Amphibian metamorphosis assays are used to evaluate potential effects of endocrine disrupting compounds on the thyroid hormone axis. In this study, Xenopus laevis tadpoles are kept in a solution of 0.2% thiourea (TU) to arrest and synchronise them in their development. The advantage of this synchronized amphibian metamorphosis assays is that synchronised tadpoles are available at any time to start metamorphosis experiments, and experimental groups are much more homogenous at the start of experimental exposure compared with groups selected from an untreated pool of animals. The water volume per animal was kept constant throughout the experimental period to overcome the influence of declining numbers of animals per aquarium due to metamorphosis and mortality on the density dependent development of the remaining tadpoles. Clophen A50 (a technical PCB mixture), the single congener 3,3',4,4'-tetrachlorobiphenyl (PCB 77) and apolar sediment extracts that were previously tested positive in the T-Screen, an in vitro proliferation assay for thyroid hormone disruption, were tested in the Synchronized Amphibian Metamorphosis Assay. Endpoints studied were mortality, malformations, body weight, and percentage of metamorphosed froglets at the end of the 60-day experimental period, percentage of tadpoles in different developmental stages, and developmental stage-dependent awarded penalty points. Dietary exposure to Clophen A50 (0.2-50mg/kg food) resulted in a significant increased percentage of tadpoles that did not pass metamorphosis at concentrations higher than 2mg/kg food. Time until metamorphosis in those animals that were able to metamorphose after the 60-days experimental period was significantly decreased. Dietary exposure to PCB 77, a congener that can be readily metabolised, did not result in significant effects in any exposure group (2-500 microg/kg food). Apolar sediment extracts from two of the three sites that are contaminated with a wide variety of chemicals significantly decreased the percentage of metamorphosed animals and significantly increased the number of tadpoles that remained in early and late metamorphic stages. These effects already occurred when the extracts where diluted more than 1000 times (on an organic carbon base) compared to environmental concentrations. The rank of potency was comparable to results obtained with the T-screen. This suggests the presence of thyroid hormone disrupting compounds in the aquatic environment and possible effects of such compounds on animal development in the wild.

Future opportunities in preventing cisplatin induced ototoxicity.

Cisplatin is one of the most commonly used cytotoxic agents. Ototoxicity is an important and dose-limiting side-effect of cisplatin therapy. It is believed that cisplatin suppresses the formation of endogenous anti-oxidants that normally prevent the inner ear against reactive oxygen species (ROS). These ROS affect the outer hair cells (OHCs) in the organ of Corti. Results from clinical trials with amifostine, an anti-oxidant with possible otoprotective action during cisplatin therapy, were disappointing. A variety of agents with chemoprotective action against cisplatin-induced ototoxicity were successfully tested in animal models. It is important to translate these promising results from animal models into clinical practice. The possible routes of administration are systemic and transtympanic. An important condition when using such an agent systemically is that the compound may not affect the anti-tumor effect of cisplatin. The critical step at transtympanic administration is the diffusion of the compound through the round window membrane (RWM). This diffusion depends on the characteristics of the medication as on the properties of the RWM. Positive results of an otoprotector in clinical practice may increase the effectiveness of cisplatin therapy and can improve the quality of life for a large group of patients.

Lack of a distinct gradient in biomarker responses in small mammals collected at different distances from a highway.

This study describes biomarker effects in small mammals exposed to traffic emissions. Animals were collected at 10-50 m (site 1), 150-200 m (site 2), and 5 km (site 3) from a very busy highway (A2). To distinguish between routes of exposure, strictly carnivorous common shrews ( Sorex araneus) and predominantly herbivorous bank voles ( Clethrionomys glareolus) were collected. As a measure of exposure to polycyclic aromatic hydrocarbons (PAHs), aromatic DNA adduct levels were determined by (32)P-postlabeling techniques in tissue from heart, lung, and liver. Lead (Pb), cadmium (Cd), and copper (Cu) levels were analyzed in kidney as a measure of exposure to heavy metals. EROD and PROD activity and retinoid levels were determined in liver as effect biomarkers for exposure to PAHs and polyhalogenated aromatic hydrocarbons (PHAHs). Relatively high Cd levels in S. araneus and in particular elevated DNA adduct levels in C. glareolus indicated that small mammals at site 3 were exposed to more compounds than at sites 1 and 2 (3 > or = 1 > 2). The latter effect is probably due to an incidental and actual input of airborne pollutants that is deposited on plant surfaces. By consumption of above-ground vegetation, voles are chronically exposed to this pollution. Relatively high background input of PAHs probably hinders that the traffic-related gradient of airborne PAH concentrations found in an earlier study is reflected in DNA adduct levels in small mammals in the present study. Moreover, historical biomarkers for exposure to traffic emissions, such as increased kidney Pb levels, increased hepatic EROD activity, and disturbed hepatic vitamin A homeostasis are no longer applicable to indicate differences in exposure. This is a result of the ban on addition of Pb and chlorinated scavengers to gasoline and of cleaner combustion techniques, which were enforced by law over the past decade. Finally, it is advisable to use only juvenile small mammals for in situ monitoring of diffuse pollution because DNA adduct levels increased with age.

Latex laboratory-gloves: an unexpected pitfall in amphibian toxicity assays with tadpoles.

This study examined the unexpected toxic effects of protective latex laboratory gloves on developing amphibians. Mortality after exposure to rinsing water from the outside of the gloves was observed in Xenopus laevis and Rana temporaria, with R. temporaria being more sensitive. This phenomenon was further confirmed using the microtiter-version of the Microtox-Assay, an in vitro assay for general toxicity. Latex gloves from the specific brand used in the experiment, in which the toxicity to tadpoles was observed for the first time, showed the highest toxicity of all materials and brands tested. Due to the high responsiveness of amphibian tadpoles to latex-glove contaminated rinsing water, special care is necessary when cleaning aquaria during toxicological experiments with amphibians as otherwise results may be biased.

Effects of oral exposure to polychlorinated biphenyls (PCBs) on the development and metamorphosis of two amphibian species (Xenopus laevis and Rana temporaria).

This study examined the effects of polychlorinated biphenyls (PCBs) on development of families of amphibians using the African clawed frog (Xenopus laevis) and the European common frog (Rana temporaria). Amphibians were orally exposed to the technical PCB-mixture Clophen A50 or to the non-ortho-3,3',4,4',5-CB congener (PCB 126) either for a 10-day period or until metamorphosis. Occurrence and rate of malformations, mortality, period until metamorphosis and thyroid hormone levels were measured. Mortality increased in a dose-dependent manner, as did the rates of malformation. Time until metamorphic transformation was prolonged and the weight of froglets was increased. Although not statistically significant, thyroid hormone levels were also lowered. PHAHs such as PCBs may affect important aspects of amphibian fitness and may influence amphibian reproductive success.

Delayed effects of pre- and early-life time exposure to polychlorinated biphenyls on tadpoles of two amphibian species (Xenopus laevis and Rana temporaria).

This study examined the effects of polychlorinated biphenyls (PCBs) on the development of amphibians using Xenopus laevis and Rana temporaria as experimental animals. Amphibians were exposed at different life stages and via different routes to the technical mixtures Clophen A50 and Aroclor 1254 or to a non-ortho PCB congener (PCB 126). The effects of PCB exposure in amphibians, such as mortality, number and pattern of malformations, or body weight at the end of successful metamorphosis of tadpoles, depends on the route, the point of time of exposure during the complex life cycle of amphibians, and the length of the observation period. Retinoid concentrations were significantly altered in PCB dosed embryos. Presently used early-life time test systems such as the FETAX assay may underestimate toxic effects of compounds with long term response such as PCBs on amphibians.

Variant of the Lisfranc fracture-dislocation: a case report and review of the literature.

Lisfranc fracture-dislocations are uncommon injuries with several variations. We present one such variation and include a pertinent review of the literature. This case is unusual in that there was lateral tarsometatarsal disruption with neither diastasis between the first and second metatarsals nor injury to either the first or second tarsometatarsal joints. Destabilization of the lateral Lisfranc joints was secondary to fractures through the second and third metatarsal shafts. Anatomic reduction and stabilization of the lateral Lisfranc joints resulted only after open anatomic reduction and internal fixation of the metatarsal fractures. Two-year follow-up confirmed an excellent clinical and radiographic result.

The continuum of restorative materials in pediatric dentistry--a review for the clinician.

Many choices are available to the practitioner of restorative dentistry for children. With the introduction of several new classes of restorative materials in recent years, some confusion has been created about what these materials are, making it difficult to identify their appropriate clinical use. This paper reviews glass-ionomer materials, resin-modified (reinforced) glass ionomers, compomers, and composite resins for the practitioner. Definitions of these materials, a general description of their contents, and usage-selection criteria are provided. Although more choices for tooth restoration can make the selection of the right material more difficult, a better understanding of the components and the strengths and weaknesses of each category of materials offers the opportunity to select the right material for the right situation.

On-site screening for syphilis at an antenatal clinic.

OBJECTIVE: To determine the validity, predictive value and accuracy of the rapid plasma reagin card test performed on site to diagnose active syphilis in pregnant women so that immediate treatment can be offered to prevent congenital syphilis. DESIGN: Open, descriptive study. SETTING: Antenatal clinic, Mamelodi Hospital, Pretoria. PATIENTS: Four hundred and seventy-four pregnant women attending the antenatal clinic for the first time were entered into the study. METHODS: A rapid plasma reagin test was performed on site with no specialised equipment and the results were compared with those of the reference laboratory. RESULTS: In the event of rapid plasma reagin titres of 1:8 and higher, indicative of active syphilis, the on-site rapid plasma reagin test had a sensitivity of 90.5%. The test had a sensitivity of 100% if the rapid plasma reagin titres were 1:16 and higher. CONCLUSION: The on-site rapid plasma reagin test identified all women with rapid plasma reagin titres higher than 1:8. This implies that all women whose fetuses were in danger of acquiring congenital syphilis were identified at the clinic and could be treated immediately.

Carcinogenicity study of outdoor airborne particulate matter in newborn male NMRI mice.

An organic extract of airborne particulate matter (APM) was tested for carcinogenicity at two dose levels in the newborn mouse bioassay. The samples used were taken under specific polluted conditions. The doses tested corresponded with 0.75 and 1.5 times the amount of air man inhales during lifetime. Benzo(a)pyrene, which was used as a positive control, significantly increased the lung tumor incidence. No evidence was found for a carcinogenic activity of the organic extract of APM. Considering the high dose of APM applied in this animal model and the much lower actual cumulative dose to which man is exposed to in many areas, the conclusion can be drawn that exposure to APM alone probably does not represent an important cancer risk for man.