Biosynthesis of novel desferrioxamine derivatives requires unprecedented crosstalk between separate NRPS-independent siderophore pathways.

Iron is essential to many biological processes but its poor solubility in aerobic environments restricts its bioavailability. To overcome this limitation, bacteria have evolved a variety of strategies, including the production and secretion of iron-chelating siderophores. Here, we describe the discovery of four series of siderophores from Streptomyces ambofaciens ATCC23877, three of which are unprecedented. MS/MS-based molecular networking revealed that one of these series corresponds to acylated desferrioxamines (acyl-DFOs) recently identified from S. coelicolor. The remaining sets include tetra- and penta-hydroxamate acyl-DFO derivatives, all of which incorporate a previously undescribed building block. Stable isotope labeling and gene deletion experiments provide evidence that biosynthesis of the acyl-DFO congeners requires unprecedented crosstalk between two separate non-ribosomal peptide synthetase (NRPS)-independent siderophore (NIS) pathways in the producing organism. Although the biological role(s) of these new derivatives remain to be elucidated, they may confer advantages in terms of metal chelation in the competitive soil environment due to the additional bidentate hydroxamic functional groups. The metabolites may also find application in various fields including biotechnology, bioremediation, and immuno-PET imaging.IMPORTANCEIron-chelating siderophores play important roles for their bacterial producers in the environment, but they have also found application in human medicine both in iron chelation therapy to prevent iron overload and in diagnostic imaging, as well as in biotechnology, including as agents for biocontrol of pathogens and bioremediation. In this study, we report the discovery of three novel series of related siderophores, whose biosynthesis depends on the interplay between two NRPS-independent (NIS) pathways in the producing organism S. ambofaciens-the first example to our knowledge of such functional cross-talk. We further reveal that two of these series correspond to acyl-desferrioxamines which incorporate four or five hydroxamate units. Although the biological importance of these novel derivatives is unknown, the increased chelating capacity of these metabolites may find utility in diagnostic imaging (for instance, 89Zr-based immuno-PET imaging) and other applications of metal chelators.

Chelating Effect of Siderophore Desferrioxamine-B on Uranyl Biomineralization Mediated by Shewanella putrefaciens.

In contaminated water and soil, little is known about the role and mechanism of the biometabolic molecule siderophore desferrioxamine-B (DFO) in the biogeochemical cycle of uranium due to complicated coordination and reaction networks. Here, a joint experimental and quantum chemical investigation is carried out to probe the biomineralization of uranyl (UO22+, referred to as U(VI) hereafter) induced by Shewanella putrefaciens (abbreviated as S. putrefaciens) in the presence of DFO and Fe3+ ion. The results show that the production of mineralized solids {hydrogen-uranium mica [H2(UO2)2(PO4)2·8H2O]} via S. putrefaciens binding with UO22+ is inhibited by DFO, which can both chelate preferentially UO22+ to form a U(VI)-DFO complex in solution and seize it from U(VI)-biominerals upon solvation. However, with Fe3+ ion introduced, the strong specificity of DFO binding with Fe3+ causes re-emergence of biomineralization of UO22+ {bassetite [Fe(UO2)2(PO4)2·8(H2O)]} by S. putrefaciens, owing to competitive complexation between Fe3+ and UO22+ for DFO. As DFO possesses three hydroxamic functional groups, it forms hexadentate coordination with Fe3+ and UO22+ ions via these functional groups. The stability of the Fe3+-DFO complex is much higher than that of U(VI)-DFO, resulting in some DFO-released UO22+ to be remobilized by S. putrefaciens. Our finding not only adds to the understanding of the fate of toxic U(VI)-containing substances in the environment and biogeochemical cycles in the future but also suggests the promising potential of utilizing functionalized DFO ligands for uranium processing.

MALDI-mass spectrometry imaging as a new technique for detecting non-heme iron in peripheral tissues via caudal vein injection of deferoxamine.

As one of the most common iron-chelating agents, deferoxamine (DFO) rapidly chelates iron in the body. Moreover, it does not compete for the iron characteristic of hemoglobin in the blood cells, which is common in the clinical treatment of iron poisoning. Iron is a trace element necessary to maintain organism normal life activities. Iron deficiency can lead to anemia, whereas iron overload can cause elevated levels of cellular oxidative stress and cell damage. As a consequence, detection of the iron content in tissues and blood is of great significance. The traditional techniques for detecting the iron content include inductively coupled plasma-mass spectrometry and atomic absorption spectrometry, which cannot be used for imaging purposes. Laser ablation-ICP-MS and synchrotron radiation micro-X-ray fluorescence can map the concentration and distribution of iron in tissues. However, these methods can only be used to measure the total iron levels in blood or tissues. In recent years, due to the deepening understanding of iron metabolism, diseases related to iron overload have attracted increasing attention. Therefore, we took advantage of the properties of DFO in terms of chelating iron and investigated different sampling times following DFO injection in the tail vein of mice. We used mass spectrometry imaging (MSI) technology to detect the DFO and ferrioxamine content in the blood and different tissues to indirectly characterize the non-heme iron content.

Evaluation of PET/CT imaging with [89Zr]Zr-DFO-girentuximab: a phase 1 clinical study in Japanese patients with renal cell carcinoma (Zirdac-JP).

BACKGROUND: PET/CT imaging with Zirconium-89 labeled [89Zr]Zr-DFO-girentuximab, which targets tumor antigen CAIX, may aid in the differentiation and characterization of clear cell renal cell carcinomas (RCC) and other renal and extrarenal lesions, and has been studied in European and American cohorts. We report results from a phase I study that evaluated the safety profile, biodistribution, and dosimetry of [89Zr]Zr-DFO-girentuximab in Japanese patients with suspected RCC. METHODS: Eligible adult patients received 37 MBq (± 10%; 10 mg mass dose) of intravenous [89Zr]Zr-DFO-girentuximab. Safety and tolerability profile was assessed based on adverse events, concomitant medications, physical examination, vital signs, hematology, serum chemistry, urinalysis, human anti-chimeric antibody measurement, and 12-lead electrocardiograms at predefined intervals. Biodistribution and normal organ and tumor dosimetry were evaluated with PET/CT images acquired at 0.5, 4, 24, 72 h and Day 5 ± 2 d after administration. RESULTS: [89Zr]Zr-DFO-girentuximab was administered in six patients as per protocol. No treatment-emergent adverse events were reported. Dosimetry analysis showed that radioactivity was widely distributed in the body, and that the absorbed dose in healthy organs was highest in the liver (mean ± standard deviation) (1.365 ± 0.245 mGy/MBq), kidney (1.126 ± 0.190 mGy/MBq), heart wall (1.096 ± 0.232 mGy/MBq), and spleen (1.072 ± 0.466 mGy/MBq). The mean effective dose, adjusted by the radioactive dose administered, was 0.470 mSv/MBq. The radiation dose was highly accumulated in the targeted tumor, while any abnormal accumulation in other organs was not reported. CONCLUSIONS: This study demonstrates that [89Zr]Zr-DFO-girentuximab administered to Japanese patients with suspected RCC has a favorable safety profile and is well tolerated and has a similar dosimetry profile to previously studied populations.

Improving Routine 89Zr-Immuno-PET Applications: Mild Iron Removal Can Favor the Use of Fe-DFO-N-suc-TFP Ester Over p-NCS-Bz-DFO.

A key aspect for the applicability of 89Zr-radioimmunoconjugates is inert modification and radiolabeling. The two commercially available bifunctional variants of the siderophore desferrioxamine (DFO), Fe-DFO-N-suc-TFP-ester and p-NCS-Bz-DFO, are most often used for clinical 89Zr-immuno-PET. The use of Fe-DFO-N-suc-TFP-ester is advantageous with regard to higher radiolysis stability and more facile assessment of radiochemical purity as well as chelator-to-mAb ratio. However, not all mAbs withstand the Fe-removal step at relatively low pH (4-4.5) using EDTA, which is needed after conjugation to allow 89Zr labeling. In this study, it was investigated whether hydroxybenzyl ethylenediamine (HBED) or the clinically approved deferiprone (DFP) can serve as an alternative for EDTA to establish a pH-independent mild method for Fe-removal and thereby broaden the applicability of Fe-DFO-N-suc-TFP-ester. Carrier-added [59Fe]Fe-DFO-N-suc-TFP-ester was used for mAb modification to enable direct tracking of the Fe-removal efficiency under various conditions. Whereas incomplete Fe-removal with HBED was observed at pH 5 or higher, Fe-removal with DFP was possible at a broad pH range (4-9). This provides a mild, pH-independent method for Fe-removal, improving the applicability and attractiveness of Fe-DFO-N-suc-TFP-ester for 89Zr-mAb preparation.

Developing Iron Nanochelating Agents: Preliminary Investigation of Effectiveness and Safety for Central Nervous System Applications.

Excessive iron levels are believed to contribute to the development of neurodegenerative disorders by promoting oxidative stress and harmful protein clustering. Novel chelation treatments that can effectively remove excess iron while minimizing negative effects on the nervous system are being explored. This study focuses on the creation and evaluation of innovative nanobubble (NB) formulations, shelled with various polymers such as glycol-chitosan (GC) and glycol-chitosan conjugated with deferoxamine (DFO), to enhance their ability to bind iron. Various methods were used to evaluate their physical and chemical properties, chelation capacity in diverse iron solutions and impact on reactive oxygen species (ROS). Notably, the GC-DFO NBs demonstrated the ability to decrease amyloid-ß protein misfolding caused by iron. To assess potential toxicity, in vitro cytotoxicity testing was conducted using organotypic brain cultures from the substantia nigra, revealing no adverse effects at appropriate concentrations. Additionally, the impact of NBs on spontaneous electrical signaling in hippocampal neurons was examined. Our findings suggest a novel nanochelation approach utilizing DFO-conjugated NBs for the removal of excess iron in cerebral regions, potentially preventing neurotoxic effects.

Work intensity and quality of life can be restored following double-level osteotomy in varus knee osteoarthritis.

PURPOSE: The purpose of this study was to assess changes in health-related quality of life (HRQL) and work intensity following double-level knee osteotomy (DLO). It was hypothesized that postoperative HRQL would be comparable to that of the general population and that work intensity can be restored in the short term. METHODS: Twenty-four patients (28 varus knees; mechanical tibiofemoral angle: -11.0 ± 3.0° (-6.0 to -17.0), age: 49.1 ± 9.5 (31-65) years) who underwent DLO were included. The duration the patients were unable to work was evaluated. HRQL was measured with the SF-36 questionnaire, which consists of a physical (PCS) and mental component summary score (MCS). The pre- to postoperative changes in the PCS and MCS were analysed. The PCS and MCS were also compared to those of the general population, who has a reference score value of 50 points. The work intensity measured with the REFA classification and the Tegner activity scale were assessed preoperatively and at the final postoperative follow-up examination (18.0 ± 10.0 (5-43) months). RESULTS: The duration that the patients were unable to work was 12.2 ± 4.4 (6-20) weeks. The PCS improved from 32.1 ± 11.3 (14.5-53.3) preoperatively to 54.6 ± 8.5 (25.2-63.7) (p < 0.001) at the final follow-up, and the MCS improved from 53.9 ± 11.1 (17.1-67.7) to 57.2 ± 3.1 (47.3-61.7) (n.s). The preoperative PCS was significantly lower than the reference score of the general population (p < 0.001), whereas the preoperative MCS was similar between the two groups (n.s.). At follow-up, no significant differences were observed between the PCS and the MCS of the patient group and those of the general population. Five patients who were unable to work prior to surgery due to knee symptoms returned to work with moderate (four patients) or even very heavy (one patient) workloads. The Tegner activity scale increased significantly from a median of 2.0 (0.0-5.0) to 4.0 (2.0-7.0) (p < 0.001). CONCLUSION: Our results demonstrate an improvement in quality of life and return to working activity following DLO in the short term. The HRQL can be improved by DLO in patients with varus knee osteoarthritis to the level of the general population. These results can assist surgeons in discussing realistic expectations when considering patients for DLO. LEVEL OF EVIDENCE: Study type: therapeutic, IV.

Evaluation of Chelator-to-Antibody Ratio on Development of 89Zr-iPET Tracer for Imaging of PD-L1 Expression on Tumor.

89Zr-iPET has been widely used for preclinical and clinical immunotherapy studies to predict patient stratification or evaluate therapeutic efficacy. In this study, we prepared and evaluated 89Zr-DFO-anti-PD-L1-mAb tracers with varying chelator-to-antibody ratios (CARs), including 89Zr-DFO-anti-PD-L1-mAb_3X (tracer_3X), 89Zr-DFO-anti-PD-L1-mAb_10X (tracer_10X), and 89Zr-DFO-anti-PD-L1-mAb_20X (tracer_20X). The DFO-anti-PD-L1-mAb conjugates with varying CARs were prepared using a random conjugation method and then subjected to quality control. The conjugates were radiolabeled with 89Zr and evaluated in a PD-L1-expressing CT26 tumor-bearing mouse model. Next, iPET imaging, biodistribution, pharmacokinetics, and ex vivo pathological and immunohistochemical examinations were conducted. LC-MS analysis revealed that DFO-anti-PD-L1-mAb conjugates were prepared with CARs ranging from 0.4 to 2.0. Radiochemical purity for all tracer groups was >99% after purification. The specific activity levels of tracer_3X, tracer_10X, and tracer_20X were 2.2 ± 0.6, 8.2 ± 0.6, and 10.5 ± 1.6 µCi/µg, respectively. 89Zr-iPET imaging showed evident tumor uptake in all tracer groups and reached the maximum uptake value at 24 h postinjection (p.i.). Biodistribution data at 168 h p.i. revealed that the tumor-to-liver, tumor-to-muscle, and tumor-to-blood uptake ratios for tracer_3X, tracer_10X, and tracer_20X were 0.46 ± 0.14, 0.58 ± 0.33, and 1.54 ± 0.51; 4.7 ± 1.3, 7.1 ± 3.9, and 14.7 ± 1.1; and 13.1 ± 5.8, 19.4 ± 13.8, and 41.3 ± 10.6, respectively. Significant differences were observed between tracer_3X and tracer_20X in the aforementioned uptake ratios at 168 h p.i. The mean residence time and elimination half-life for tracer_3X, tracer_10X, and tracer_20X were 25.4 ± 4.9, 24.2 ± 6.1, and 25.8 ± 3.3 h and 11.8 ± 0.5, 11.1 ± 0.7, and 11.7 ± 0.6 h, respectively. No statistical differences were found between-tracer in the aforementioned pharmacokinetic parameters. In conclusion, 89Zr-DFO-anti-PD-L1-mAb tracers with a CAR of 1.4-2.0 may be better at imaging PD-L1 expression in tumors than are traditional low-CAR 89Zr-iPET tracers.

Distal femoral osteotomy for the valgus knee: indications, complications, clinical and radiological outcome.

INTRODUCTION: The aim of this study was to describe the indications and technical aspects of medial closing and lateral opening distal femoral osteotomy (MCDFO and LODFO) for patients with a valgus knee and to report clinical and radiological outcomes and complications. METHODS: Over 6 years, 28 DFOs (22 MCDFO, 6 LODFO) were performed in 22 Patients. In this cohort study, we retrospectively analyzed clinical and radiological outcome measures as well as complications. RESULTS: The median (range) age was 47 (17-63) years, height 1.68 (1.56-1.98) m, body mass 80 (49-105) kg, and body mass index (BMI) 27.4 (18.6-37.0) kg/m2. The clinical follow-up was 21 (7-81) months, the need for total or unicompartmental knee arthroplasty (TKA/UKA) and hardware removal was followed up for 59 (7-108) months postoperatively. Preoperatively, hip-knee-ankle angle (HKA, negative values denote varus) was 7.0 (2.0-13.0)°, mechanical lateral distal femoral angle (mLDFA) was 83.7 (79.9-88.2)°, and mechanical proximal tibial angle (MPTA) was 89.0 (86.6-94.5)°. Postoperatively, HKA was -1.3 (-9.0-1.2)° and mLDFA was 90.8 (87.3-97.3)°. The incidence of minor and major complications was 25% and 14%, the incidence of delayed and nonunion was 18% and 4%, respectively. At the last follow-up, 18% of the patients had pain at rest, 25% during activities of daily living, and 39% during physical activity, and 71% were satisfied with the outcome. 7% of the cases received a TKA/UKA, 71% received a hardware removal. CONCLUSION: DFO is a reasonable treatment for lateral osteoarthritis in younger patients to avoid disease progression and the need for an UKA/TKA. However, there is a long rehabilitation time, a considerable risk for complications, and a high need for hardware removal. While many patients experienced symptoms at the long-term follow-up, most were satisfied with the outcome. Appropriate patient information is essential. Level of evidence Level IV, Case Series. Trial registration number NCT04382118, clinicaltrials.gov, May 11, 2020.

Practical syntheses of DOTAGA-DBCO and DFO-DBCO, strained alkyne pre-cursors to radioimmunoconjugates for targeted alpha therapy.

We describe practical methods to prepare DOTAGA-DBCO and DFO-DBCO from commercially available starting materials. DOTAGA-DBCO is available in five steps from cyclen with a 33 % overall yield at gram scale. Our synthesis of DFO-DBCO also proceeds in five steps from commercially available starting materials. These bifunctional molecules possess chelating functionality for the binding of medically important radiometals and a strained alkyne suitable for Huisgen cyclization with an azide. These syntheses represent an important step toward improved radioimmunoconjugates for imaging and therapeutic applications.

The use of tranexamic acid reduces blood loss in osteotomy at knee level: a systematic review.

PURPOSE: Aim of this systematic review was to evaluate the literature regarding the effect of tranexamic acid (TXA) on the outcome after knee osteotomy. METHODS: A systematic literature search was carried out in various databases on studies on the use of tranexamic acid in osteotomies around the knee. Primary outcome criterion was the hemoglobin (drop). Secondary outcome criteria were total blood loss, drainage volume, adverse effects such as thromboembolic events, blood transfusions, wound complications and clinical scores. A meta-analysis was performed for quantitative measures. The present study was registered prospectively ( www.crd.york.ac.uk/PROSPERO ; no.: CRD42021229624). RESULTS: Seven studies with 584 patients (TXA group: 282 patients, non TXA group: 302 patients) Hemoglobin decrease (1.54 g/dl vs. 2.28 g/dl), blood loss (394.49 ml vs. 595.54 ml) and drainage volume (266.5 ml vs. 359.05 ml) were significantly less in the TXA group compared to the non TXA group. No thromboembolic event was noted in any study. In the non TXA group four blood transfusions were given. Eleven wound complications occurred in the non TXA group in comparison to two wound complications in the TXA group. CONCLUSIONS: The results of the present study show that the application of TXA reduces hemoglobin drop, blood loss and drainage volume. These effects could be responsible for the lesser rate of side effects after administration of TXA during knee osteotomy.

Consistent indications, targets and techniques for double-level osteotomy of the knee: a systematic review.

PURPOSE: To systematically review and critically appraise the literature on double-level osteotomy (DLO) of the knee, and determine the indications, contraindications, targets and outcomes. MATERIALS AND METHODS: A systematic literature search was performed on PubMed, Embase®, and Cochrane for studies that reported on DLO by any technique or approach, including indications, contraindications, and targets for DLO, as well as patient-reported outcome measures (pROMS) and radiographic angles. RESULTS: Twelve eligible studies were found: 9 case series and 3 studies that compared DLO to high-tibial osteotomy (HTO). In all studies, DLO was performed by medial opening-wedge tibial osteotomy and lateral closing-wedge femoral osteotomy. Seven specified that DLO was performed if simple HTO would exceed thresholds of postoperative medial proximal tibial angle (MPTA), lateral distal femoral angle (LDFA), and/or predicted wedge size. The targets were 88°-95° for MPTA, 84°-89° for LDFA, and 0°-4° for hip-knee-ankle (HKA) angle. The 3 comparative studies reported lower MPTA after DLO (89.6°-92.5°) than after HTO (91.5°-98.3°). All 3 reported similar postoperative HKA after DLO (0.2°-4.4°) as HTO (0.4°-4.8°); only 2 compared postoperative LDFA, which was lower after DLO (85.4° and 84.9°) than HTO (88.7° and 88.8°). Two comparative studies reported postoperative overall KOOS which was slightly lower after DLO (351-403) than HTO (368-410); only 1 study reported separate items of the KOOS. CONCLUSION: There was relative consistency between studies on the indications, targets and techniques for DLO. Furthermore, while the comparative studies reported similar preoperative MPTA, LDFA and HKA, the postoperative MPTA and LDFA were lower after DLO than after HTO, though both treatments achieved equivalent postoperative HKA. LEVEL OF EVIDENCE: IV, systematic review.

Endolysosome iron restricts Tat-mediated HIV-1 LTR transactivation by increasing HIV-1 Tat oligomerization and ß-catenin expression.

HIV-1 transactivator of transcription (Tat) protein is required for HIV-1 replication, and it has been implicated in the pathogenesis of HIV-1-associated neurocognitive disorder (HAND). HIV-1 Tat can enter cells via receptor-mediated endocytosis where it can reside in endolysosomes; upon its escape from these acidic organelles, HIV-1 Tat can enter the cytosol and nucleus where it activates the HIV-1 LTR promoter. Although it is known that HIV-1 replication is affected by the iron status of people living with HIV-1 (PLWH), very little is known about how iron affects HIV-1 Tat activation of the HIV-1 LTR promoter. Because HIV-1 proteins de-acidify endolysosomes and endolysosome de-acidification affects subcellular levels and actions of iron, we tested the hypothesis that the endolysosome pool of iron is sufficient to affect Tat-induced HIV-1 LTR transactivation. Ferric (Fe3+) and ferrous (Fe2+) iron both restricted Tat-mediated HIV-1 LTR transactivation. Chelation of endolysosome iron with deferoxamine (DFO) and 2-2 bipyridyl, but not chelation of cytosolic iron with deferiprone and deferasirox, significantly enhanced Tat-mediated HIV-1 LTR transactivation. In the presence of iron, HIV-1 Tat increasingly oligomerized and DFO prevented the oligomerization. DFO also reduced protein expression levels of the HIV-1 restriction agent beta-catenin in the cytosol and nucleus. These findings suggest that DFO increases HIV-1 LTR transactivation by increasing levels of the more active dimeric form of Tat relative to the less active oligomerized form of Tat, increasing the escape of dimeric Tat from endolysosomes, and/or reducing beta-catenin protein expression levels. Thus, intracellular iron might play a significant role in regulating HIV-1 replication, and these findings raise cautionary notes for chelation therapies in PLWH.

Head-to-head comparison of DFO* and DFO chelators: selection of the best candidate for clinical 89Zr-immuno-PET.

PURPOSE: Almost all radiolabellings of antibodies with 89Zr currently employ the hexadentate chelator desferrioxamine (DFO). However, DFO can lead to unwanted uptake of 89Zr in bones due to instability of the resulting metal complex. DFO*-NCS and the squaramide ester of DFO, DFOSq, are novel analogues that gave more stable 89Zr complexes than DFO in pilot experiments. Here, we directly compare these linker-chelator systems to identify optimal immuno-PET reagents. METHODS: Cetuximab, trastuzumab and B12 (non-binding control antibody) were labelled with 89Zr via DFO*-NCS, DFOSq, DFO-NCS or DFO*Sq. Stability in vitro was compared at 37 °C in serum (7 days), in formulation solution (24 h ± chelator challenges) and in vivo with N87 and A431 tumour-bearing mice. Finally, to demonstrate the practical benefit of more stable complexation for the accurate detection of bone metastases, [89Zr]Zr-DFO*-NCS and [89Zr]Zr-DFO-NCS-labelled trastuzumab and B12 were evaluated in a bone metastasis mouse model where BT-474 breast cancer cells were injected intratibially. RESULTS: [89Zr]Zr-DFO*-NCS-trastuzumab and [89Zr]Zr-DFO*Sq-trastuzumab showed excellent stability in vitro, superior to their [89Zr]Zr-DFO counterparts under all conditions. While tumour uptake was similar for all conjugates, bone uptake was lower for DFO* conjugates. Lower bone uptake for DFO* conjugates was confirmed using a second xenograft model: A431 combined with cetuximab. Finally, in the intratibial BT-474 bone metastasis model, the DFO* conjugates provided superior detection of tumour-specific signal over the DFO conjugates. CONCLUSION: DFO*-mAb conjugates provide lower bone uptake than their DFO analogues; thus, DFO* is a superior candidate for preclinical and clinical 89Zr-immuno-PET.

Development of [89Zr]DFO-elotuzumab for immunoPET imaging of CS1 in multiple myeloma.

PURPOSE: Multiple myeloma (MM) is a bone marrow malignancy that remains mostly incurable. Elotuzumab is an FDA-approved therapeutic monoclonal antibody targeted to the cell surface glycoprotein CS1, which is overexpressed in MM cells. Identifying patients who will respond to CS1-targeted treatments such as elotuzumab requires the development of a companion diagnostic to assess the presence of CS1. Here, we evaluated [89Zr]DFO-elotuzumab as a novel PET tracer for imaging CS1 expression in preclinical MM models. METHODS: Conjugation of desferrioxamine-p-benzyl-isothiocyanate (DFO-Bz-NCS) to elotuzumab enabled zirconium-89 radiolabeling. MM.1S-CG cells were intravenously injected in NOD SCID gamma (NSG) mice. Small animal PET imaging with [89Zr]DFO-elotuzumab (1.11 MBq/mouse, 7 days post-injection), [89Zr]DFO-IgG (1.11 MBq/mouse, 7 days post-injection), and [18F]FDG (7-8 MBq, 1 h post-injection) was performed. Additionally, biodistribution of [89Zr]DFO-elotuzumab post-imaging at 7 days was also done. In vivo specificity of [89Zr]DFO-elotuzumab was further evaluated with a blocking study and ex vivo autoradiography. RESULTS: [89Zr]DFO-elotuzumab was produced with high specific activity (56 ± 0.75 MBq/nmol), radiochemical purity (99% ± 0.5), and yield (93.3% ± 1.5). Dissociation constant of 40.4 nM and receptor density of 126 fmol/mg was determined in MM.1S-CG cells. Compared to [89Zr]DFO-IgG, [89Zr]DFO-elotuzumab localized with a significantly higher standard uptake value in tumor-bearing bone tissue (8.59 versus 4.77). Blocking with unlabeled elotuzumab significantly reduced (P < 0.05) uptake of [89Zr]DFO-elotuzumab in the bones. Importantly, while [18F]FDG demonstrated similar uptake in the bone and muscle, [89Zr]DFO-elotuzumab showed > 3-fold enhanced uptake in bones. CONCLUSION: These data demonstrate the feasibility of [89Zr]DFO-elotuzumab as a companion diagnostic for CS1-targeted therapies.

Role of hepcidin in oxidative stress and cell death of cultured mouse renal collecting duct cells: protection against iron and sensitization to cadmium.

The liver hormone hepcidin regulates systemic iron homeostasis. Hepcidin is also expressed by the kidney, but exclusively in distal nephron segments. Several studies suggest hepcidin protects against kidney damage involving Fe2+ overload. The nephrotoxic non-essential metal ion Cd2+ can displace Fe2+ from cellular biomolecules, causing oxidative stress and cell death. The role of hepcidin in Fe2+ and Cd2+ toxicity was assessed in mouse renal cortical [mCCD(cl.1)] and inner medullary [mIMCD3] collecting duct cell lines. Cells were exposed to equipotent Cd2+ (0.5-5 µmol/l) and/or Fe2+ (50-100 µmol/l) for 4-24 h. Hepcidin (Hamp1) was transiently silenced by RNAi or overexpressed by plasmid transfection. Hepcidin or catalase expression were evaluated by RT-PCR, qPCR, immunoblotting or immunofluorescence microscopy, and cell fate by MTT, apoptosis and necrosis assays. Reactive oxygen species (ROS) were detected using CellROX™ Green and catalase activity by fluorometry. Hepcidin upregulation protected against Fe2+-induced mIMCD3 cell death by increasing catalase activity and reducing ROS, but exacerbated Cd2+-induced catalase dysfunction, increasing ROS and cell death. Opposite effects were observed with Hamp1 siRNA. Similar to Hamp1 silencing, increased intracellular Fe2+ prevented Cd2+ damage, ROS formation and catalase disruption whereas chelation of intracellular Fe2+ with desferrioxamine augmented Cd2+ damage, corresponding to hepcidin upregulation. Comparable effects were observed in mCCD(cl.1) cells, indicating equivalent functions of renal hepcidin in different collecting duct segments. In conclusion, hepcidin likely binds Fe2+, but not Cd2+. Because Fe2+ and Cd2+ compete for functional binding sites in proteins, hepcidin affects their free metal ion pools and differentially impacts downstream processes and cell fate.

Re-Evaluations of Zr-DFO Complex Coordination Chemistry for the Estimation of Radiochemical Yields and Chelator-to-Antibody Ratios of 89Zr Immune-PET Tracers.

(1) Background: Deferoxamine B (DFO) is the most widely used chelator for labeling of zirconium-89 (89Zr) to monoclonal antibody (mAb). Despite the remarkable developments of the clinical 89Zr-immuno-PET, chemical species and stability constants of the Zr-DFO complexes remain controversial. The aim of this study was to re-evaluate their stability constants by identifying species of Zr-DFO complexes and demonstrate that the stability constants can estimate radiochemical yield (RCY) and chelator-to-antibody ratio (CAR). (2) Methods: Zr-DFO species were determined by UV and ESI-MS spectroscopy. Stability constants and speciation of the Zr-DFO complex were redetermined by potentiometric titration. Complexation inhibition of Zr-DFO by residual impurities was investigated by competition titration. (3) Results: Unknown species, ZrHqDFO2, were successfully detected by nano-ESI-Q-MS analysis. We revealed that a dominant specie under radiolabeling condition (pH 7) was ZrHDFO, and its stability constant (logß111) was 49.1 ± 0.3. Competition titration revealed that residual oxalate inhibits Zr-DFO complex formation. RCYs in different oxalate concentration (0.1 and 0.04 mol/L) were estimated to be 86% and >99%, which was in good agreement with reported results (87%, 97%). (4) Conclusion: This study succeeded in obtaining accurate stability constants of Zr-DFO complexes and estimating RCY and CAR from accurate stability constants established in this study.

Exploration and Exploitation Zones in a Minimalist Swarm Optimiser.

The trade off between exploration and exploitation is one of the key challenges in evolutionary and swarm optimisers which are led by guided and stochastic search. This work investigates the exploration and exploitation balance in a minimalist swarm optimiser in order to offer insights into the population's behaviour. The minimalist and vector-stripped nature of the algorithm-dispersive flies optimisation or DFO-reduces the challenges of understanding particles' oscillation around constantly changing centres, their influence on one another, and their trajectory. The aim is to examine the population's dimensional behaviour in each iteration and each defined exploration-exploitation zone, and to subsequently offer improvements to the working of the optimiser. The derived variants, titled unified DFO or uDFO, are successfully applied to an extensive set of test functions, as well as high-dimensional tomographic reconstruction, which is an important inverse problem in medical and industrial imaging.

Radiolabelling of the octadentate chelators DFO* and oxoDFO* with zirconium-89 and gallium-68.

In recent years, clinical imaging with zirconium-89 (89Zr)-labelled monoclonal antibodies (Ab) by positron emission tomography (immunoPET) has been gaining significant importance in nuclear medicine for the diagnosis of different types of cancer. For complexation of the radiometal 89Zr and its attachment to the Ab, chelating agents are required. To date, only the hexadentate chelator desferrioxamine (DFO) is applied in the clinic for this purpose. However, there is increasing preclinical evidence that the [89Zr]Zr-DFO complex is not sufficiently stable and partly releases the radiometal in vivo due to the incomplete coordination sphere of the metal. This leads to unfavourable unspecific uptake of the osteophilic radiometal in bones, hence decreasing the signal-to-noise-ratio and leading to an increased dose to the patient. In the past, several new chelators with denticities > 6 have been published, notably the octadentate DFO derivative DFO*. DFO*, however, shows limited water solubility, wherefore an oxygen containing analogue, termed oxoDFO*, was developed in 2017. However, no data on the suitability of oxoDFO* for radiolabelling with 89Zr has yet been reported. In this proof-of-concept study, we present the first radiolabelling results of the octadentate, water-soluble chelator oxoDFO*, as well as the in vitro stability of the resulting complex [89Zr]Zr-oxoDFO* in comparison to the analogous octadentate, but less water-soluble derivative DFO* and the current "standard" chelator DFO. In addition, the suitability of DFO* and oxoDFO* for radiolabeling with the short-lived PET metal gallium-68 is discussed. The water-soluble, octadentate chelator oxoDFO* provides stable complexes with the positron emitter Zirconium-89. The radiolabelling can be performed at room temperature and neutral pH and thus, oxoDFO* represents a promising chelator for applications in immunoPET.

Tibial and femoral osteotomies in varus deformities - radiological and clinical outcome.

BACKGROUND: Varus deformities of the knee are frequently corrected by osteotomies, which should be performed at the level of origin. But in contrast to high tibial osteotomies (HTO), little data exists for distal femoral osteotomies (DFO). This study evaluates radiological and clinical outcomes after valgisation osteotomies in the proximal tibia and distal femur. METHODS: We used an observational cohort study design and prospectively performed preoperative long standing radiographs (LSR), lateral x-rays and clinical questionnaires (SF-36, Lysholm score, VAS). Postoperative LSR and lateral x-rays were obtained on average 18 months postoperative and postoperative clinical questionnaires at final visit (mean follow up 46 months). A subgroup analysis of the different surgical techniques (oHTO vs. cDFO) was performed, with regards to radiological and clinical outcomes. RESULTS: Finally 28 osteotomies with medial tibial opening (oHTO) or lateral femoral closing (cDFO) wedge osteotomies in 25 consecutive patients (mean age 40 years) were identified. There were 17 tibal and 11 femoral procedures. All osteotomies were performed at the origin of deformity, which was of different etiology. The average deviation of the final HKA compared to the preoperative planning was 2.4° ± 0.4°. Overall, there was a significant improvement in all clinical scores (SF-36: 61.8 to 79.4, p < 0.001; Lysholm-score: 72.7 to 90.4, p < 0.001; VAS: 3 to 1, p < 0.001). There was no significant correlation between surgical accuracy and outcome scores. CONCLUSION: Valgisation osteotomies lead to a significant improvement in all clinical scores with the demonstrated treatment protocol. An appreciable proportion of varus deformities are of femoral origin. Since cDFO provides comparable radiological and clinical results as oHTO, this is an important treatment option for varus deformities of femoral origin.