Identification of CD64 as a marker for the destructive potential of synovitis in osteoarthritis.

OBJECTIVES: OA is characterized by cartilage degeneration and persistent pain. The majority of OA patients present with synovitis, which is associated with increased cartilage damage. Activated synovial macrophages are key contributors to joint destruction. Therefore, a marker that reflects the activation of these cells could be a valuable tool to characterize the destructive potential of synovitis and benefit monitoring of OA. Here, we aimed to investigate the use of CD64 (FcγRI) as a marker to characterize the damaging potential of synovitis in OA. METHODS: Synovial biopsies were obtained from end-stage OA patients that underwent joint replacement surgery. CD64 protein expression and localization was evaluated using immunohistochemistry and immunofluorescence and quantified using flow cytometry. qPCR was performed to measure the expression of FCGR1 and OA-related genes in synovial biopsies, and in primary chondrocytes and primary fibroblasts stimulated with OA conditioned medium (OAS-CM). RESULTS: Our data exposed a wide range of CD64 expression in OA synovium and showed positive correlations between FCGR1 and S100A8, S100A9, IL1B, IL6 and MMP1/2/3/9/13 expression. CD64 protein correlated with MMP1, MMP3, MMP9, MMP13 and S100A9. Furthermore, we observed that synovial CD64 protein levels in source tissue for OAS-CM significantly associated with the OAS-CM-induced expression of MMP1, MMP3 and especially ADAMTS4 in cultured fibroblasts, but not chondrocytes. CONCLUSION: Together, these results indicate that synovial CD64 expression is associated with the expression of proteolytic enzymes and inflammatory markers related to structural damage in OA. CD64 therefore holds promise as marker to characterize the damaging potential of synovitis.

CD64 as novel molecular imaging marker for the characterization of synovitis in rheumatoid arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is one of the most prevalent and debilitating joint diseases worldwide. RA is characterized by synovial inflammation (synovitis), which is linked to the development of joint destruction. Magnetic resonance imaging and ultrasonography are widely being used to detect the presence and extent of synovitis. However, these techniques do not reveal the activation status of inflammatory cells such as macrophages that play a crucial role in synovitis and express CD64 (Fc gamma receptor (FcγR)I) which is considered as macrophage activation marker. OBJECTIVES: We aimed to investigate CD64 expression and its correlation with pro-inflammatory cytokines and pro-damaging factors in human-derived RA synovium. Furthermore, we aimed to set up a molecular imaging modality using a radiolabeled CD64-specific antibody as a novel imaging tracer that could be used to determine the extent and phenotype of synovitis using optical and nuclear imaging. METHODS: First, we investigated CD64 expression in synovium of early- and late-stage RA patients and studied its correlation with the expression of pro-inflammatory and tissue-damaging factors. Next, we conjugated an anti-CD64 antibody with IRDye 800CW and diethylenetriamine penta-acetic acid (DTPA; used for 111In labeling) and tested its binding on cultured THP1 cells, ex vivo RA synovium explants and its imaging potential in SCID mice implanted with human RA synovium explants obtained from RA patients who underwent total joint replacement. RESULTS: We showed that CD64 is expressed in synovium of early and late-stage RA patients and that FCGR1A/CD64 expression is strongly correlated with factors known to be involved in RA progression. Combined, this makes CD64 a useful marker for imaging the extent and phenotype of synovitis. We reported higher binding of the [111In]In-DTPA-IRDye 800CW anti-CD64 antibody to in vitro cultured THP1 monocytes and ex vivo RA synovium compared to isotype control. In human RA synovial explants implanted in SCID mice, the ratio of uptake of the antibody in synovium over blood was significantly higher when injected with anti-CD64 compared to isotype and injecting an excess of unlabeled antibody significantly reduced the antibody-binding associated signal, both indicating specific receptor binding. CONCLUSION: Taken together, we successfully developed an optical and nuclear imaging modality to detect CD64 in human RA synovium in vivo.

Preclinical evaluation of [11C]GW457427 as a tracer for neutrophil elastase.

INTRODUCTION: Neutrophils are part of the innate immune system and function as a first line of defense against invading microorganisms. Overactivity of the immune system may result in a devastating immuno-inflammation with extensive damage to tissue leading to organ damage and/or failure. The literature suggests several human diseases in which neutrophil elastase (NE) is postulated to be important in the pathophysiology including inflammatory bowel disease (IBD), chronic obstructive pulmonary disorder (COPD), abdominal aortic aneurysms (AAA), breast and lung cancer, and recently also in Sars-cov-2 virus infection (Covid-19). In particular, the lungs are affected by the destructive power of the protease neutrophil elastase (NE). In this paper, we report the pre-clinical development of a selective and specific positron emission tomography (PET) tracer, [11C]GW457427, as an in vivo biomarker for the study of NE, now available for human studies. METHODS: [11C]GW457427 was produced by methylation of GW447631 using [11C]methyl triflate and GMP validated production and quality control methods were developed. Chemical purity was high with no traces of the precursor GW611437 or other uv-absorbing compounds. A method for the determination of intact [11C]GW457427 in plasma was developed and the binding characteristics were evaluated in vitro and in vivo. An animal model for lung inflammation was used to investigate the specificity and sensitivity of the [11C]GW457427 tracer for neutrophil elastase (NE) in pulmonary inflammation, verified by blockade using two structurally different elastase inhibitors. RESULTS: [11C]GW457427 was obtained in approximately 45% radiochemical yield and with a radiochemical purity higher than 98%. Molar activity was in the range 130-360 GBq/µmol. Binding to NE was shown to be highly specific both in vitro and in vivo and a significantly higher uptake of tracer was found in a lipopolysaccharide mouse model of pulmonary inflammation compared with control animals. The uptake in lung tissue measured as standardized uptake value (SUV) strongly correlated with tissue NE content as measured by ELISA. In vitro studies also showed specific tracer binding in aortic tissue of patients with abdominal aorta aneurysm (AAA). The rate of metabolism in rats was appropriate considering the critical balance between available tracer for binding and requirement for blood clearance with about 40% and 20% intact [11C]GW457427 in plasma at 5 and 40 min, respectively. Radioactivity was cleared from blood and organs in control animals with mainly hepatobiliary excretion with distribution in the intestines and the urinary bladder; but without retention of the tracer in healthy organs of interests such as the lung, liver, kidneys or in the cardiovascular system. A dosimetry study in rat indicated that the whole-body effective dose was 2.2 µSv/MBq with bone marrow as the limiting organ. It is estimated that up to five PET-CT investigations could be performed in humans without exceeding a total dose of 10 mSv. CONCLUSION: [11C]GW457427 is a promising in vivo PET-biomarker for NE with high specific binding demonstrated both in vitro and in vivo. A GMP validated production method including quality control has been developed and a microdosing toxicity study performed with no adverse signs. [11C]GW457427 is currently being evaluated in a First-In-Man PET study.

Advanced imaging for quantification of abnormalities in the salivary glands of patients with primary Sjögren's syndrome.

OBJECTIVES: To assess non-invasive imaging for detection and quantification of gland structure, inflammation and function in patients with primary Sjogren's syndrome (pSS) using PET-CT with 11C-Methionine (11C-MET; radiolabelled amino acid), and 18F-fluorodeoxyglucose (18F-FDG; glucose uptake marker), to assess protein synthesis and inflammation, respectively; multiparametric MRI evaluated salivary gland structural and physiological changes. METHODS: In this imaging/clinical/histology comparative study (GSK study 203818; NCT02899377) patients with pSS and age- and sex-matched healthy volunteers underwent MRI of the salivary glands and 11C-MET PET-CT. Patients also underwent 18F-FDG PET-CT and labial salivary gland biopsies. Clinical and biomarker assessments were performed. Primary endpoints were semi-quantitative parameters of 11C-MET and 18F-FDG uptake in submandibular and parotid salivary glands and quantitative MRI measures of structure and inflammation. Clinical and minor salivary gland histological parameter correlations were explored. RESULTS: Twelve patients with pSS and 13 healthy volunteers were included. Lower 11C-MET uptake in parotid, submandibular and lacrimal glands, lower submandibular gland volume, higher MRI fat fraction, and lower pure diffusion in parotid and submandibular glands were observed in patients vs healthy volunteer, consistent with reduced synthetic function. Disease duration correlated positively with fat fraction and negatively with 11C-MET and 18F-FDG uptake, consistent with impaired function, inflammation and fatty replacement over time. Lacrimal gland 11C-MET uptake positively correlated with tear flow in patients, and parotid gland 18F-FDG uptake positively correlated with salivary gland CD20+ B-cell infiltration. CONCLUSION: Molecular imaging and MRI may be useful tools to non-invasively assess loss of glandular function, increased glandular inflammation and fat accumulation in pSS.

Quantifying disease activity in rheumatoid arthritis with the TSPO PET ligand 18F-GE-180 and comparison with 18F-FDG and DCE-MRI.

PURPOSE: While the aetiology of rheumatoid arthritis (RA) remains unclear, many of the inflammatory components are well characterised. For diagnosis and therapy evaluation, in vivo insight into these processes would be valuable. Various imaging probes have shown value including dynamic contrast-enhanced (DCE) MRI and PET/CT using 18F-fluorodeoxyglucose (18F-FDG) or tracers targeting the translocator protein (TSPO). To evaluate 18F-GE-180, a novel TSPO PET tracer, for detecting and quantifying disease activity in RA, we compared 18F-GE-180 uptake with that of 18F-FDG and DCE-MRI measures of inflammation. METHODS: Eight RA patients with moderate-to-high, stable disease activity and active disease in at least one wrist were included in this study (NCT02350426). Participants underwent PET/CT examinations with 18F-GE-180 and 18F-FDG on separate visits, covering the shoulders and from the pelvis to the feet, including hands and wrists. DCE-MRI was performed on one affected hand. Uptake was compared visually between tracers as judged by an experienced radiologist and quantitatively using the maximum standardised uptake value (SUVmax). Uptake for both tracers was correlated with DCE-MRI parameters of inflammation, including the volume transfer coefficient Ktrans using Pearson correlation (r). RESULTS: PET/CT imaging with 18F-GE-180 in RA patients showed marked extra-synovial uptake around the affected joints. Overall sensitivity for detecting clinically affected joints was low (14%). 18F-GE-180 uptake did not or only weakly correlate with DCE-MRI parameters in the wrist (r = 0.09-0.31). 18F-FDG showed higher sensitivity for detecting symptomatic joints (34%), as well as strong positive correlation with DCE-MRI parameters (SUVmax vs. Ktrans: r = 0.92 for wrist; r = 0.68 for metacarpophalangeal joints). CONCLUSIONS: The correlations between DCE-MRI parameters and 18F-FDG uptake support use of this PET tracer for quantification of inflammatory burden in RA. The TSPO tracer 18F-GE-180, however, has shown limited use for the investigation of RA due to its poor sensitivity and ability to quantify disease activity in RA.

Manganese-52: applications in cell radiolabelling and liposomal nanomedicine PET imaging using oxine (8-hydroxyquinoline) as an ionophore.

The ionophore 8-hydroxyquinoline (oxine) has been used to radiolabel cells and liposomal medicines with 111In and, more recently, 89Zr, for medical nuclear imaging applications. Oxine has also shown promising ionophore activity for the positron-emitting radionuclide 52Mn that should allow imaging of labelled cells and nanomedicines for long periods of time (>14 days). However, to date, the radiometal complex formed and its full labelling capabilities have not been fully characterised. Here, we provide supporting evidence of the formation of [52Mn]Mn(oxinate)2 as the metastable complex responsible for its ionophore activity. The cell labelling properties of [52Mn]Mn(oxinate)2 were investigated with various cell lines. The liposomal nanomedicine, DOXIL® (Caelyx) was also labelled with [52Mn]Mn(oxinate)2 and imaged in vivo using PET imaging. [52Mn]Mn(oxinate)2 was able to label various cell lines with moderate efficiency (15-53%), however low cellular retention of 52Mn (21-25% after 24 h) was observed which was shown not to be due to cell death. PET imaging of [52Mn]Mn-DOXIL at 1 h and 24 h post-injection showed the expected pharmacokinetics and biodistribution of this stealth liposome, but at 72 h post-injection showed a profile matching that of free 52Mn, consistent with drug release. We conclude that oxine is an effective ionophore for 52Mn, but high cellular efflux of the isotope limits its use for prolonged cell tracking. [52Mn]Mn(oxinate)2 is effective for labelling and tracking DOXIL in vivo. The release of free radionuclide after liposome extravasation could provide a non-invasive method to monitor drug release in vivo.

Bioenergetics and intermuscular fat in chronic obstructive pulmonary disease-associated quadriceps weakness.

INTRODUCTION: Chronic obstructive pulmonary disease (COPD) is associated with metabolic abnormalities in muscles of the lower limbs, but it is not known whether these abnormalities are generalized or limited to specific muscle groups, nor is there an easy way of predicting their presence. METHODS: Metabolism in the quadriceps and biceps of 14 COPD patients and controls was assessed during sustained contraction using 31-phosphorus magnetic resonance spectroscopy ((31) P MRS). T1 MRI was used to measure quadriceps intermuscular adipose tissue (IMAT). RESULTS: COPD patients had prolonged quadriceps phosphocreatine time (patients: 38.8 ± 12.7 s; controls: 25.2 ± 10.6 s; P = 0.006) and a lower pH (patents: 6.88 ± 0.1; controls: 6.99 ± 0.06; P = 0.002). Biceps measures were not significantly different. IMAT was associated with a nadir pH <7.0 (area under the curve = 0.84). CONCLUSIONS: Anaerobic metabolism during contraction was characteristic of quadriceps, but not biceps, muscles of patients with COPD and was associated with increased IMAT. Because IMAT can be assessed quickly by conventional MRI, it may be a useful approach for identifying patients with abnormal muscle bioenergetics.

Two complementary rat NK cell subsets, Ly49s3+ and NKR-P1B+, differ in phenotypic characteristics and responsiveness to cytokines.

Two major subsets of rat NK cells can be distinguished based on their expression of the Ly49s3 or the NKR-P1B lectin-like receptor. Ly49s3(+) NK cells, but not NKR-P1B(+) NK cells, express a wide range of Ly49 receptors. Here, we have examined differences between these two subsets in their expression of certain NK cell-associated molecules as well as their responses to cytokines. A microarray analysis suggested several differentially expressed genes, including preferential expression of NKG2A/C receptors by NKR-P1B(+) NK cells. This was confirmed by staining with tetramers of RT.BM1, the putative ligand of CD94/NKG2, indicating that Ly49 and CD94/NKG2 receptors separate into distinct NK cell compartments. Further, expression of CD25 by Ly49s3(+) NK cells was associated with more rapid proliferation in response to IL-2 as compared with NKR-P1B(+) NK cells. Thus, certain inflammatory situations may preferentially expand the Ly49s3(+) NK cells. Moreover, freshly isolated Ly49s3(+) and NKR-P1B(+) NK cells produce similar amounts of cytokines, and a minor Ly49s3(-)NKR-P1B(-) double-negative NK subset appears to be hyporesponsive based on its significantly lower IFN-gamma production. Collectively, our data demonstrate divergent profiles of NKR-P1B(+) and Ly49s3(+) NK cells, indicating distinct tasks in vivo.