Aim. Implant infections caused by Staphylococcus aureus are difficult to treat due to biofilm formation, which complicates surgical and antibiotic treatment. Herewith we introduce an alternative approach using monoclonal antibodies (mAbs) targeting S. aureus and provide the biodistribution and specificity in a mouse implant infection model. Methods. 4497-IgG1targeting S. aureus Wall Teichoic Acid was labeled to Indium-111 using “CHXA” as a chelator. SPECT-CT scans were performed at 24, 72 and 120 hours after administration in Balb/cAnNCrl mice with a subcutaneous implant pre-colonized with biofilm of S. aureus. Biodistribution over the various organs of this labelled antibody was visualized and quantified using SPECT-CT imaging and compared to uptake at the target tissue with implant infection. Results. Uptake of the . 111. In-4497 mAbs (half-life 59 hours) at the infected implant gradually increased from 8.34%ID/g at 24 hours to 9.22%ID/g at 120 hours. Uptake at the heart/blood pool decreased over time from 11.60 to 7.58%ID/g whereas the uptake in other organs decreased from 7.26 to less than 4.66%ID/g at 120 hours. Conclusion. 111. In-4497 mAbs was found to specifically detect S. aureus and its biofilm with excellent and prolonged accumulation at the colonized implant site. Therefore, it holds great promise as a drug delivery system for diagnostic and bactericidal treatment of biofilm. However, high activity in the blood pool must be considered as it could pose a risk to healthy tissue

Aim. Prosthetic joint infections (PJI) remain a great challenge in orthopedic surgery with a high mortality rate. It is particularly complicated by biofilms and infections caused by Methicillin-resistant Staphylococcus aureus (MRSA). It concurrently shields bacteria from host immune responses and confers resistance to antibiotics. This study aims to investigate the efficacy of radioimmunotherapy as an innovative therapeutic modality to address the challenges posed by MRSA and its biofilm. Method. We induced specific monoclonal antibodies 4497-IgG1 as carriers, which target wall teichoic acids (WTA) existing on MRSA and its biofilm. Radionuclides actiniumr-225 (. 225. Ac, α-emitter) and lutetium-177 (. 177. Lu, β-emitter) were conjugated with mAbs using DOTA as chelator. Quality control was assessed using thin layer chromatography and immunoreactivity assays. . 225. Ac- and . 177. Lu-labelled 4497-IgG1 were employed to evaluate the susceptibility of MRSA and its biofilm to the radioimmunotherapy in vitro. Planktonic MRSA and biofilms, at concentrations of 10. 8. and 10. 7. CFU/mL, were incubated at 37°C for 60 minutes in PBS containing either . 225. Ac-mAb (0 - 14.8 kBq) or . 177. Lu-mAb (0 - 14.8 MBq). Radiolabelled dunituximab and free radionuclides serve as isotype-matched negative control. The bacterial viability and metabolic activity were subsequently quantified using CFU and XTT assays. Results. The radiochemical purity of the . 225. Ac-mAbs and . 177. Lu-mAbs complex were determined to be 95.4% and 96.16%. Immunoreactivity fractions of them were measured at 81.8% and 80.8%. . 225. Ac-mAbs and . 177. Lu-mAbs exhibited significant and dose-dependent antimicrobial effects on both planktonic MRSA and biofilm. . 225. Ac- and . 177. Lu-4497IgG1 at doses of 7.4 kBq and 7.4 MBq resulted in more than 4-log reduction in bacterial counts. In biofilms, 2-log reduction at the highest . 225. Ac radioactivity of 14,8kBq. The . 177. Lu complex showed a strong dose-dependent effect, with a reduction of up to 4-log. The XTT assay confirmed these findings, showing a decrease in metabolic activity corresponding to a decrease in bacterial counts, and a slight increase in metabolic activity at the lower dose. Conclusions. Our study demonstrates the efficacy of . 225. Ac and . 177. Lu-labelled 4497-IgG1 antibodies in mediating dose-dependent bactericidal effects against planktonic MRSA and biofilms in vitro. This indicates that radioimmunotherapy could be a potential targeted therapeutic strategy against MRSA and its biofilm. Further research in preclinical and clinical settings is warranted to validate and refine these findings on biofilm-associated implant infections

Aim. “Implant associated Staphylococcus aureus or S. epidermidis infections are often difficult to treat due to the formation of biofilms on prosthetic material. Biofilms are bacterial communities adhered to a surface with a self-made extracellular polymeric substance that surrounds resident bacteria. In contrast to planktonic bacteria, bacteria in a biofilm are in an adherent, dormant state and are insensitive to most antibiotics. In addition, bacteria in a biofilm are protected from phagocytic cells of the immune system. Therefore, complete surgical removal and replacement of the prosthetic implant is often necessary to treat this type of infections. Neutrophils play a crucial role in clearing bacterial pathogens. They recognize planktonic bacteria via immunoglobulin (Ig) and complement opsonisation. In this project, we aim to evaluate the role of IgG and complement in the recognition and clearance of staphylococcal biofilms by human neutrophils. Furthermore, we evaluate if monoclonal antibodies (mAbs) targeting biofilm structures can enhance recognition and clearance of staphylococcal biofilms by the human immune system.”. Method. “We produced a set of 20 recombinant mAbs specific for staphylococcal antigens. Using flow cytometry and ELISA-based methods we determined the binding of these mAbs to planktonic staphylococci and in vitro staphylococcal biofilms. Following incubation with IgG/IgM depleted human serum we determined whether mAbs can react with the human complement system after binding to biofilm. Confocal microscopy was used to visualize the location of antibody binding in the biofilm 3D structure.”. Results. “We show that mAbs directed against several staphylococcal surface targets such as wall teichoic acid (a glycopolymer on the S. aureus/S. epidermidis cell wall) and polymeric-N-acetyl-glucosamine (major constituent of the S. epidermidis biofilm extracellular matrix) bind biofilms in a dose-dependent manner. This interaction was specific since no binding was observed for control antibodies (recognizing the hapten DNP). Furthermore we show that these antibodies can penetrate the complete 3D structure of an in vitro biofilm. Products of complement activation via the classical pathway were detected upon incubation with human serum and the biofilm binding mAbs.”. Conclusions. “Having established that our mAbs can bind biofilms and induce complement opsonisation via C3b deposition, we will now study if we can engineer these antibodies to enhance complement deposition. A combination of enhanced complement and antibody opsonisation may improve recognition and clearance of biofilms by phagocytic immune cells. These mAbs could be used to boost the immune system to clear implant associated infections, without the need to replace the implant via invasive surgical procedures.”

Over 80% of patients with advanced breast cancer will develop bone metastases for which there is no cure. Although thought to involve a complex cascade of cell-cell interactions, the factors controlling the development of bone metastases are still poorly understood. Osteoblasts may have an important role in mediating homing and proliferation of breast cancer cells to the bony environment. This study aimed to examine the potential role osteoblasts have in the migration of circulating tumour cells to bone and the factors involved in this attraction. Culture of osteoblasts and MDA-MB-231 breast cancer cells was performed. Breast cancer cell migration in response to osteoblasts was measured using Transwell Migration Inserts. Potential mediators of cell migration were detected using ChemiArray & ELISA assays. A luminometer based Vialight assay was used to measure breast cancer cell proliferation in response to factors secreted by osteoblasts. There was a 3-4 fold increase of MDA-MB-231 migration in response to osteoblasts. ChemiArray analysis of osteoblast-conditioned medium revealed a range of secreted chemokines including IL-6 & 8, TIMP 1 & 2 and MCP-1. Initially, MCP-1 was quantified at 282 pg/ml, but rose to over 9000 pg/ml when osteoprogenitor cells were differentiated into mature osteoblasts. Inclusion of a monoclonal antibody to MCP-1 in osteoblast-conditioned medium resulted in a significant decrease in breast cancer cell migration to osteoblasts. There was no significant change in proliferation of MDA-MB 231 cells when exposed to osteoblast-conditioned medium. Osteoblasts are capable of inducing breast cancer cell migration mediated at least in part by chemokine secretion. MCP-1 produced by the osteoblasts was shown to play a central role in mediating homing of the breast cancer cells. Increased understanding of the pathways involved in the development of bone metastases may provide new targets for therapeutic intervention

Aim. To assess the role of Tc-99m labelled anti granulocyte monoclonal antibody Fab' fragment (Sulesomab) in the diagnosis of bone and joint infections. Methods. We analysed the results of 95 patients referred with a clinical suspicion of bone and joint infections. There were 47 male and 48 female patients with a mean age of 60 years (range=16 to 89). Referrals were made for suspected infection of prosthetic total joint replacements (38), long bones (32), primary joints (12) and feet (13). Sulesomab imaging was done with 650 MBq of 99mTcSulesomab. The final diagnosis was determined by conclusive microbiology, culture and/or histology, intra-operative findings, aspiration, complementary investigations like CT/MRI and long term clinical follow-up. The findings of 99mTcSulesomab images were compared with the clinical outcome to arrive at the decision of True Positive/ False positive/ True negative/ False negative results. Using the above definitions sensitivity, specificity and diagnostic accuracy of 99mTcSulesomab for suspected bone and joint infection were calculated. Results. 58/95 patients had normal or equivocal blood results. Plain radiographs revealed no abnormality or were inconclusive of infection in more than half of the patients. Outcome classification revealed 29 true positives, 56 true negatives, 9 false positives and one false negative. The overall sensitivity was 96.66% and specificity 86.15% with a negative predictive value of 98.24%. The individual sensitivity and specificity of each category was compared. Diagnostic accuracy for long bone infections (96.87%) was the highest than any other group. Conclusion. 99mTc Sulesomab imaging has a high sensitivity and specificity in a heterogeneous group of orthopaedic bone and joint infections. With a high negative predictive value, this test seems to be useful in excluding orthopaedic infection rather than confirming it

Aims

Bacterial infection activates neutrophils to release neutrophil extracellular traps (NETs) in bacterial biofilms of periprosthetic joint infections (PJIs). The aim of this study was to evaluate the increase in NET activation and release (NETosis) and haemostasis markers in the plasma of patients with PJI, to evaluate whether such plasma induces the activation of neutrophils, to ascertain whether increased NETosis is also mediated by reduced DNaseI activity, to explore novel therapeutic interventions for NETosis in PJI in vitro, and to evaluate the potential diagnostic use of these markers.