Lode Goethals

Preclinical Evaluation of TriMix and Antigen mRNA-Based Antitumor Therapy

The use of tumor-associated antigen (TAA) mRNA for therapeutic purposes is under active investigation. To be effective, mRNA vaccines need to deliver activation stimuli in addition to TAAs to dendritic cells (DC). In this study, we evaluated whether intranodal delivery of TAA mRNA together with TriMix, a mix of mRNA encoding CD40 ligand, constitutive active Toll-like receptor 4 and CD70, results in the in situ modification and maturation of DCs, hence, priming of TAA-specific T cells. We showed selective uptake and translation of mRNA in vivo by lymph node resident CD11c(+) cells. This process was hampered by codelivery of classical maturation stimuli but not by TriMix mRNA. Importantly, TriMix mRNA induced a T-cell-attracting and stimulatory environment, including recruitment of antigen-specific CD4(+) and CD8(+) T cells and CTLs against various TAAs. In several mouse tumor models, mRNA vaccination was as efficient in CTL induction and therapy response as vaccination with mRNA-electroporated DCs. Together, our findings suggest that intranodal administration of TAA mRNA together with mRNA encoding immunomodulating molecules is a promising vaccination strategy.

High rate biological treatment of sulfate-rich wastewater in an acetate-fed EGSB reactor.

An expanded granular sludge bed reactor, inoculated with acclimated sulfidogenic granular sludge, was operated at 33 °C and fed with acetic acid as COD source and sulfate as electron acceptor. The bioreactor had a sulfate conversion efficiency of 80–90% at a high sulfate loading rate of 10.4 g SO42--S/l.d after only 60 days of start-up. This was achieved by implementing a dual operational strategy. Firstly acetic acid was dosed near stoichiometry (COD over sulfur ratio = 2.0 to 2.2) which allowed almost complete sulfate removal. Secondly the pH in the bioreactor was kept slightly alkaline (7.9 ± 0.1) which limited the concentration of the inhibitory undissociated hydrogen sulfide H2S (pKa = 7). This allowed the acetotrophic sulfate reducing bacteria to predominate throughout the long term experiment. The limitations of the EGSB technology with respect to the sulfate conversion rate appeared to be related to the biomass wash-out and granule deterioration occurring at superficial upflow velocities above 10 m/h. Increasing the recirculation flow caused a drop in the sulfate reduction rate and efficiency, an increase of the suspended sludge fraction and a considerable loss of biomass into the effluent, yielding bare mainly inorganic granules. Elemental analysis revealed that a considerable amount of the granular sludge dry matter at the end of the experiment, at an upflow velocity of 20 m/h, consisted of calcium (32%), mainly in the form of carbonate deposits, while organic matter only represented 7%.

Effect of COD to sulphate ratio and temperature in expanded-granular-sludge-blanket reactors for sulphate reduction

In an ethanol-fed expanded-granular-sludge-blanket (EGSB) reactor at 33°C, 80–90% of the sulphate load was removed at a rate of 4 g S/l d, provided that at least 6 g chemical oxygen demand (COD) per g sulphate-sulphur was supplied. The reactor started up in a matter of days. Gradually decreasing the ethanol to sulphate ratio (R) to about stoichiometry, resulted in 60–70% sulphate removal at rates of 7 g S/l d. Similar tendencies were observed with ethylene glycol as sole carbon and energy source. Total COD removal never reached more than 70–75%. This was related to a rather high biomass washout. The sulphate removal efficiency decrease when R was set at levels below 6, apparently because sulphate reducing bacteria (SRB) could not compete with methane producing bacteria (MPB) for acetate produced from the substrate dosed. Thermophilic operation at 55°C, after a stepwise increase in the reactor temperature over a period of 23 days, did not favour acetotrophic sulphate reduction. Yet, operation at 48°C and subsequently returning the temperature to 33°C clearly enhanced acetate conversion by SRB. In the case of an electron donor price of 0.035–0.075 USD/kg COD, the cost for operation at R=6 was found to be competitive to that at stoichiometry, i.e. R=2, provided the biogas produced was effectively used.

High rates of microbial sulphate reduction in a mesophilic ethanol-fed expanded-granular-sludge-blanket reactor

Abstract In a mesophilic (30–35 °C), sulphidogenic, ethanol-fed expanded-granular-sludge-blanket reactor, sulphate, at loading rates of up to 10.0–12.0 g Sl−1␣day−1, was removed with an average efficiency of more than 80%. The pH was between 7.7 and 8.3 and the maximal total dissolved sulphide concentration was up to 20 mM S (650 mg S/l). The alkaline pH was maintained by either a pH-control unit with sodium hydroxide or by stripping part of the sulphide and CO2 from the recycle with nitrogen gas. The superficial upstream liquid velocity (vup) was 3.0–4.5 m/h. The ratio of ethanol to sulphur was near stoichiometry. At alkaline pH, the activity of the acetotrophic sulphate-reducing bacteria, growing on acetate, was strongly enhanced, whereas at pH below 7.7 the acetotrophic sulphate-reducing bacteria were inhibited by aqueous H2S. With regard to the removal efficiency and operational stability, external stripping with N2 and pH control were equally successful.

Healing Sacral Fracture Masquerading as Metastatic Bone Disease on a 68Ga-PSMA PET/CT.

Prostate-specific membrane antigen (PSMA) is a cell surface glycoprotein, which is frequently overexpressed on prostate cancer cells. A Ga-PSMA PET/CT can be used for early detection of lymph node or bone metastases after radical prostatectomy when there is biochemical recurrence. This report describes PSMA uptake in a healing fracture masquerading as metastatic bone disease in a patient with a history of prostate adenocarcinoma. Clinicians reporting Ga-PSMA PET/CT should be aware of this potential important pitfall.

Testicular cell transplantation into the human testes

OBJECTIVE To translate spermatogonial stem cell (SSC) transplantation towards a clinical application. DESIGN Mouse green fluorescent protein (GFP)-positive testicular cells were labeled with (99m)technetium and microbubbles. These labeled cells were injected into the rete testis of isolated human testes under ultrasound guidance. Three different conditions were tested: 1) 800 μL of a 20 million cells/mL suspension; 2) 800 μL of a 10 million cells/mL suspension; and 3) 1,400 μL of a 10 million cells/mL suspension. After injection, the human cadaver testes were analyzed with the use of single-photon-emission computerized tomography (SPECT) imaging and histology. SETTING Laboratory research environment. PATIENT(S) Cadaver testes, obtained from autopsies at the pathology department. INTERVENTION(S) Ultrasound-guided injection of mouse GFP-positive testicular cells. MAIN OUTCOME MEASURE(S) Presence of radioactive-labeled cells in the human cadaver testes and GFP-positive cells in the seminiferous tubules. RESULT(S) In all of the experimental groups, GFP-positive cells were observed in the seminiferous tubules, near and far from the rete testis, but also in the interstitium. On SPECT, significant difference was seen between the group injected with 800 μL of a 20 million cells/mL suspension (1,654.6 ± 907.6 mm³) and the group injected with 1,400 μL of a 10 million cells/mL suspension (3,614.9 ± 723.1 mm³). No significant difference was reached in the group injected with 800 μL of a 10 million cells/mL suspension. CONCLUSION(S) Injecting cells in the human cadaver testis is feasible, but further optimization is required.

Rapid hepatic clearance of 99mTc-TMEOP: a new candidate for myocardial perfusion imaging

BACKGROUND (99m)Tc labeled radiotracers used in clinical practice lack the perfect characteristics for myocardial perfusion imaging. In particular, the high liver uptake can interfere in the interpretation of the inferior myocardial wall. Within the tricarbonyl approach, we used tris(pyrazolyl)methane (99m)Tc organometallic complexes as a lead structure. Herein we present the production, in vivo and in vitro metabolic studies in rats and the first in vivo biodistribution in rats for tri-methoxy-tris-pyrazolyl-(99m)Tc-(CO)(3) ((99m)Tc-TMEOP), compared with (99m)Tc-sestamibi and (99m)Tc-tetrofosmin. METHODS The chemical identity of (99m)Tc-TMEOP was characterized by RP-HPLC. The octanol-water partition coefficient was determined under physiological conditions. In vitro stability and protein binding were determined using RP-HPLC. In vivo stability was determined in blood, heart, liver and kidney homogenates, intestine and urine using RP-HPLC. In vivo biodistribution was determined using dynamic planar acquisitions. Pinhole gated SPECT images were performed in other animals. Cardiac, liver and lung uptake were expressed as differential uptake ratios by drawing regions of interest in the organs of interest and around the total body. Heart-liver and heart-lung ratios were derived. Cardiac uptake was also expressed as percentage of injected activity. SPECT images were processed to determine the heart-liver ratio on SPECT images, to compare functional parameters between different tracers and to visualize homogeneous intracardiac tracer distribution. RESULTS (99m)Tc-TMEOP is a moderately lipophilic cation, is stable and does not undergo any transformation in vitro. (99m)Tc-TMEOP also shows a high in vivo stability. In vivo imaging shows liver kinetics faster than those of (99m)Tc-sestamibi and (99m)Tc-tetrofosmin. Cardiac uptake and functional analysis of pinhole gated SPECT data are comparable to those of (99m)Tc-sestamibi and (99m)Tc-tetrofosmin. CONCLUSION Although (99m)Tc-TMEOP shows a cardiac uptake between those of (99m)Tc-sestamibi and (99m)Tc-tetrofosmin, a better heart-liver ratio is obtained due to the faster liver washout. These results suggest possible faster cardiac perfusion imaging using (99m)Tc-TMEOP without liver activity interference.

Intratumoral delivery of TriMix mRNA results in T-cell activation by cross-presenting dendritic cells

Intratumoral injection of CTL-stimulatory agents could provide another avenue for immunotherapy. TriMix mRNA, comprising three DC-oriented stimulatory mRNAs, was examined in mouse models and provides a rationale for clinical testing in solid and accessible tumors. Modulating the activity of tumor-infiltrating dendritic cells (TiDC) provides opportunities for novel cancer interventions. In this article, we report on our study of the uptake of mRNA by CD8α+ cross-presenting TiDCs upon its intratumoral (i.t.) delivery. We exploited this property to deliver mRNA encoding the costimulatory molecule CD70, the activation stimuli CD40 ligand, and constitutively active Toll-like receptor 4, referred to as TriMix mRNA. We show that TiDCs are reprogrammed to mature antigen-presenting cells that migrate to tumor-draining lymph nodes (TDLN). TriMix stimulated antitumor T-cell responses to spontaneously engulfed cancer antigens, including a neoepitope. We show in various mouse cancer models that i.t. delivery of TriMix mRNA results in systemic therapeutic antitumor immunity. Finally, we show that the induction of antitumor responses critically depends on TiDCs, whereas it only partially depends on TDLNs. As such, we provide a platform and a mechanistic rationale for the clinical testing of i.t. administration of TriMix mRNA. Cancer Immunol Res; 4(2); 146–56. ©2015 AACR.

Aspecific Uptake of 68GA-PSMA in Paget Disease of the Bone.

Ga-PSMA plays an increasing role in prostate cancer management, but several instances of false positivity have now been recognized. We present a patient with metastatic prostatic carcinoma who also showed overexpression of PSMA in Paget disease of the humerus on Ga-PSMA PET. This probably relates to bone remodeling and increased vascularity. It is important to be aware of this aspecific uptake because its recognition may avoid overstaging and may alter the therapeutic choice.

18F-FDG PET/CT imaging of an anti-CTLA-4 antibody-associated autoimmune pancolitis.

Ipilimumab (also known as MDX-010; Bristol-Myers Squibb, New York) is a fully human monoclonal antibody that binds to and blocks CTLA-4 (cytotoxic T lymphocyteassociated antigen 4), a receptor molecule on T cells that plays a critical role in regulating natural T-cell immune responses [1]. Ipilimumab improves overall survival in pretreated patients with metastatic melanoma [2]. During ipilimumab treatment, distinct immune-related adverse events may occur, one of the most frequently observed of which is colitis [4], and others include dermatitis, hepatitis, hypophysitis, and hypothyroidism [2, 3]. Endoscopy and histopathological examination of ipilimumab-associated immune-related colitis is characterized by a diffuse active inflammation [4]. We report on the F-FDG PET/CT imaging findings of autoimmune pancolitis in a patient with stage IV-M1c melanoma. Following disease progression despite conventional dacarbazine-based chemotherapy, ipilimumab was initiated at a dose of 3 mg per kilogram of body weight. Two weeks after the fourth ipilimumab administration, the patient developed diarrhoea and abdominal pain. She had no prior history of inflammatory bowel disease. F-FDG PET/CT demonstrated a diffusely increased tracer uptake in the colon (a, c, white arrows), suggesting active pancolitis [5]. Focal tracer uptake in lung and axillary lymph nodes was indicative of metastatic disease (a, arrowheads). Concomitant CT findings of the colon demonstrated a markedly thickened colonic wall and L. Goethals Department of Radiology, Universitair Ziekenhuis Brussel, Brussels, Belgium