Attempts to Target Staphylococcus aureus Induced Osteomyelitis Bone Lesions in a Juvenile Pig Model by Using Radiotracers.

Background [18F]FDG Positron Emission Tomography cannot differentiate between sterile inflammation and infection. Therefore, we, aimed to develop more specific radiotracers fitted for differentiation between sterile and septic infection to improve the diagnostic accuracy. Consequently, the clinicians can refine the treatment of, for example, prosthesis-related infection. METHODS: We examined different target points; Staphylococcus aureus biofilm (68Ga-labeled DOTA-K-A9 and DOTA-GSGK-A11), bone remodeling ([18F]NaF), bacterial cell membranes ([68Ga]Ga-Ubiquicidin), and leukocyte trafficking ([68Ga]Ga-DOTA-Siglec-9). We compared them to the well-known glucose metabolism marker [18F]FDG, in a well-established juvenile S. aureus induced osteomyelitis (OM) pig model. RESULTS: [18F]FDG accumulated in the OM lesions seven days after bacterial inoculation, but disappointingly we were not able to identify any tracer accumulation in OM with any of the supposedly more specific tracers. CONCLUSION: These negative results are, however, relevant to report as they may save other research groups from conducting the same animal experiments and provide a platform for developing and evaluating other new potential tracers or protocol instead.

[99mTc]-labelled interleukin-8 as a diagnostic tool compared to [18F]FDG and CT in an experimental porcine osteomyelitis model.

Osteomyelitis (OM) is an important cause of morbidity and sometimes mortality in children and adults. Long-term complications can be reduced when treatment is initiated in an early phase. The diagnostic gold standard is microbial examination of a biopsy and current non-invasive imaging methods are not always optimal. [111In]-leukocyte scintigraphy is recommended for peripheral OM, but is time-consuming and not recommended in children. [18F]FDG PET/CT is recommended for vertebral OM in adults, but has the disadvantage of false positive findings and a relatively high radiation exposure; the latter is a problem in children. [99mTc]-based tracers are consequently preferred in children. We, therefore, aimed to find a [99mTc]-marked tracer with high specificity and sensitivity for early detection of OM. Suppurating inflammatory lesions like OM caused by Staphylococcus aureus (S. aureus) will attract large numbers of neutrophils and macrophages. A preliminary study has shown that [99m Tc]-labelled IL8 may be a possible candidate for imaging of peripheral OM. We investigated [99mTc]IL8 scintigraphy in a juvenile pig model of peripheral OM and compared it with [18F]FDG PET/CT. The pigs were experimentally inoculated with S. aureus to induce OM and scanned one week later. We also examined leukocyte count, serum CRP and IL8, as well as performed histopathological and microbiological investigations. [ 99m Tc]IL8 was easily and relatively quickly prepared and was shown to be suitable for visualization of OM lesions in peripheral bones detecting 70% compared to a 100% sensitivity of [18F]FDG PET/CT. [ 99m Tc]IL8 is a promising candidate for detection of OM in peripheral bones in children.

Staphylococcus aureus infected embolic stroke upregulates Orm1 and Cxcl2 in a rat model of septic stroke pathology.

OBJECTIVE: Ischaemic brain lesions and brain abscesses are frequent in both human and animal cases of septic embolic stroke. However, existing models of brain infection do not reflect central aspects of septic embolic stroke. Our aim was to compare septic and non-septic embolic stroke in order to identify gene expressions, inflammatory mediators and brain damage in a rat model. METHODS: We created precisely located focal brain infarcts in a rat model of Staphylococcus aureus infected embolic stroke. To cause septic embolic stroke we used a fibrin-rich embolus with bacteria, while every rat in the control group received a non-infected embolus. 64 rats were randomized to receive sham-surgery, sterile embolic stroke or septic embolic stroke. All groups were compared for brain pathology, mortality, gene expressions and inflammatory mediators using histology and reverse transcription quantitative real-time PCR. RESULTS: Although infarct volumes did not differ, septic embolic stroke caused higher mortality than sterile embolic stroke (p=  0.002). Brain abscesses were observed only in the septic group. Approximately 400-500 fold increases were observed for Orm1 and Cxcl2 respectively (1.00E-08 < p < 1.92E-07) in the septic group compared to the sterile group, and these were the most dramatically regulated genes in septic embolic stroke compared to sterile embolic stroke. CONCLUSIONS: Septic embolic stroke caused brain abscesses, increased mortality and upregulated Orm1 and Cxcl2 gene expressions compared to non-infected embolic stroke. The dramatic Orm1 increase observed in the septic group is unprecedented and suggests a significant biological role of Orm1 during septic neuroinflammation.

Secretory phospholipase A2 responsive liposomes exhibit a potent anti-neoplastic effect in vitro, but induce unforeseen severe toxicity in vivo.

The clinical use of liposomal drug delivery vehicles is often hindered by insufficient drug release. Here we present the rational design of liposomes optimized for secretory phospholipase A2 (sPLA2) triggered drug release, and test their utility in vitro and in vivo. We hypothesized that by adjusting the level of cholesterol in anionic, unsaturated liposomes we could tune the enzyme specificity based on membrane fluidity, thus obtaining liposomes with an improved therapeutic outcome and reduced side effects. Cholesterol is generally important as a component in the membranes of liposome drug delivery systems due to its stabilizing effects in vivo. The incorporation of cholesterol in sPLA2 sensitive liposomes has not previously been possible due to reduced sPLA2 activity. However, in the present work we solved this challenge by optimizing membrane fluidity. In vitro release studies revealed enzyme specific drug release. Treatment of two different cancer cell lines with liposomal oxaliplatin revealed efficient growth inhibition compared to that of clinically used stealth liposomes. The in vivo therapeutic effect was evaluated in nude NMRI mice using the sPLA2 secreting mammary carcinoma cell line MT-3. Three days after first treatment all mice having received the novel sPLA2 sensitive liposome formulation were euthanized due to severe systemic toxicity. Thus the present study demonstrates that great caution should be implemented when utilizing sPLA2 sensitive liposomes and that the real utility can only be disclosed in vivo. The present studies have clinical implications, as sPLA2 sensitive formulations are currently undergoing clinical trials (LiPlaCis®).

Gastrotomy Healing After Transgastric Peritoneoscopy: A Randomized Study in a Pig Model.

INTRODUCTION: Reliable closure and infection prevention are the main barriers for implementation of pure transgastric peritoneoscopy. The primary aim of this study was to assess healing of over the scope clip (OTSC) closed gastrotomies. MATERIALS AND METHODS: Pure transgastric peritoneoscopy was performed in 7 pigs. The pigs were randomized to 14 or 28 postoperative days (POD) of follow-up. Decontamination of the access route was performed before instrumentation. A full necropsy was performed. Closure was evaluated with histopathological examination of excised gastrorrhaphies. RESULTS: Three pigs were allowed 14 POD of follow-up, and 4 pigs were allowed 28 POD of follow-up. Survival was achieved in 6 of the 7 animals; 1 pig was euthanized due to diffuse peritonitis. Based on our definition, full-thickness healing had only been achieved in a single pig allowed 28 POD. With respect to clinical relevancy, full-thickness healing was deemed achieved in 4 of 6 pigs completing follow-up and in all pigs allowed and surviving 28 POD. Access required repeated punctures and the use of several endoscopic instruments. CONCLUSIONS: Full-thickness healing of the gastrotomy was only found in a single case when adhering to the per protocol definition. Endoscopic ultrasonography-guided access was difficult. It lacks reproducibility and needs refinement. Despite a combined decontamination regimen, infectious complications still occurred.

Pure natural orifice transluminal endoscopic surgery (NOTES) with ultrasonography-guided transgastric access and over-the-scope-clip closure: a porcine feasibility and survival study.

BACKGROUND: Most natural orifice transluminal endoscopic surgery (NOTES) procedures to date rely on the hybrid technique with simultaneous laparoscopic access to protect against access-related complications and to achieve adequate triangulation for dissection. This is done at the cost of the potential benefits of this new minimally invasive technique. This study aimed to evaluate the feasibility and safety of a transgastric (TG) pure-NOTES procedure in a diagnostic setting. METHODS: A TG pure-NOTES procedure with endoscopic ultrasonograpy (EUS)-guided access and over-the-scope-clip (OTSC) closure was performed for 10 pigs in a survival and feasibility study. A full macroscopic necropsy with subsequent histologic evaluation was performed on postoperative day (POD) 14. The outcome parameters were uncomplicated follow-up assessment, survival, intraoperative complications, intraabdominal pathology, macroscopic full-thickness closure, and histology-proven full-thickness healing of the gastrotomy. RESULTS: An uncomplicated postoperative course was reported for 9 of the 10 pigs, and survival was reported for all 10 pigs. For all the pigs, EUS-guided access was performed successfully with a median duration of 25 min (range, 12-62 min) and without intraoperative complications or access-related lesions at necropsy. An OTSC closure was achieved with a median duration of 11 min (range, 3-28 min). The OTSC provided immediate closure, but according to the authors' definition of a full-thickness healing evaluated by histologic examination, this was not achieved in any of the cases. Although all the animals survived until POD 14, intraabdominal chronic abscesses were present in 3 of the 10 pigs at necropsy. CONCLUSIONS: The EUS-guided TG access proved to be feasible without access-related complications. Although OTSC provided an immediate closure, the histopathology raised concerns regarding the risk of perforation. Together with the high risk of intraabdominal infection, TG pure-NOTES is not yet ready for routine clinical practice.