Bioorthogonal Radiolabeling of Azide-Modified Bacteria Using [18F]FB-sulfo-DBCO.

Purpose: This study was motivated by the need for better positron emission tomography (PET)-compatible tools to image bacterial infection. Our previous efforts have targeted bacteria-specific metabolism via assimilation of carbon-11 labeled d-amino acids into the bacterial cell wall. Since the chemical determinants of this incorporation are not fully understood, we sought a high-throughput method to label d-amino acid derived structures with fluorine-18. Our strategy employed a chemical biology approach, whereby an azide (-N3) bearing d-amino acid is incorporated into peptidoglycan muropeptides, with subsequent "click" cycloaddition with an 18F-labeled strained cyclooctyne partner. Procedures: A water-soluble, 18F-labeled and dibenzocyclooctyne (DBCO)-derived radiotracer ([18F]FB-sulfo-DBCO) was synthesized. This tracer was incubated with pathogenic bacteria treated with azide-bearing d-amino acids, and incorporated 18F was determined via gamma counting. In vitro uptake in bacteria previously treated with azide-modified d-amino acids was compared to that in cultures treated with amino acid controls. The biodistribution of [18F]FB-sulfo-DBCO was studied in a cohort of healthy mice with implications for future in vivo imaging. Results: The new strain-promoted azide-alkyne cycloaddition (SPAAC) radiotracer [18F]FB-sulfo-DBCO was synthesized with high radiochemical yield and purity via N-succinimidyl 4-[18F]fluorobenzoate ([18F]SFB). Accumulation of [18F]FB-sulfo-DBCO was significantly higher in several bacteria treated with azide-modified d-amino acids than in controls; for example, we observed 7 times greater [18F]FB-sulfo-DBCO ligation in Staphylococcus aureus cultures incubated with 3-azido-d-alanine versus those incubated with d-alanine. Conclusions: The SPAAC radiotracer [18F]FB-sulfo-DBCO was validated in vitro via metabolic labeling of azide-bearing peptidoglycan muropeptides. d-Amino acid-derived PET radiotracers may be more efficiently screened via [18F]FB-sulfo-DBCO modification.

Determining Factors in the Success of Direct Pulp Capping: A Systematic Review.

AIM: To elucidate the factors that determine the success of direct pulp capping (DPC) in permanent teeth with pulp exposure due to dental caries. MATERIALS AND METHODS: A comprehensive electronic search from 1980 to 2023 across PubMed, Scopus, and ISI Web databases was conducted using specific keywords and MeSH terms in Q1 or Q2 journals. Only prospective/retrospective clinical studies in English on 15 or more human permanent teeth with carious pulpal exposure treated with DPC agents-mineral trioxide aggregate (MTA), Biodentine, or calcium hydroxide with a rubber dam and minimum 1-year follow-up, were considered. The factors retrieved and analyzed were based on study design, patient age, sample size, type of cavity, exposure size and location, pulp diagnosis, solutions to achieve hemostasis, hemostasis time, capping material, restoration type, follow-up period, methods of evaluation, and overall success. REVIEW RESULTS: Out of 680 articles, only 16 articles were selected for the present systematic review on application of the selection criteria. A wide age range of patients from 6 to 88 years were considered among these studies with sample sizes ranging from 15 to 245 teeth with reversible pulpitis being the predominant diagnosis of the cases. Mineral trioxide aggregate as a capping material was evaluated in 4 studies as a lone agent, while compared with other capping agents such as biodentine or calcium hydroxide in 7 studies. The follow-up period ranged from 9 days to nearly 80 months. While both clinical and radiographic evaluation was carried out in all studies, cold testing dominated the clinical tests while IOPR was the common radiograph considered. Mineral trioxide aggregate success rate was higher and similar to biodentine than calcium hydroxide. CONCLUSION: Direct pulp capping has a high and predictable success rate in permanent teeth with carious exposure to reversible and irreversible pulpitis. Currently, mineral trioxide aggregate and biodentine have better long-term results in DPC than calcium hydroxide, hence, they should be used as an alternative to calcium hydroxide. Definitive restoration within a short period improves long-term prognosis. CLINICAL SIGNIFICANCE: The significance of this review lies in its provision of evidence-based information on the effectiveness of DPC and the factors that influence its success. By considering these factors, clinicians can optimize treatment outcomes and improve the long-term prognosis of the treated teeth. This systematic review serves as a valuable resource for clinicians and researchers in the field of endodontics. How to cite this article: Gomez-Sosa JF, Granone-Ricella M, Rosciano-Alvarez M, et al. Determining Factors in the Success of Direct Pulp Capping: A Systematic Review. J Contemp Dent Pract 2024;25(4):392-401.

Evaluating the Performance of Pathogen-Targeted Positron Emission Tomography Radiotracers in a Rat Model of Vertebral Discitis-Osteomyelitis.

BACKGROUND: Vertebral discitis-osteomyelitis (VDO) is a devastating infection of the spine that is challenging to distinguish from noninfectious mimics using computed tomography and magnetic resonance imaging. We and others have developed novel metabolism-targeted positron emission tomography (PET) radiotracers for detecting living Staphylococcus aureus and other bacteria in vivo, but their head-to-head performance in a well-validated VDO animal model has not been reported. METHODS: We compared the performance of several PET radiotracers in a rat model of VDO. [11C]PABA and [18F]FDS were assessed for their ability to distinguish S aureus, the most common non-tuberculous pathogen VDO, from Escherichia coli. RESULTS: In the rat S aureus VDO model, [11C]PABA could detect as few as 103 bacteria and exhibited the highest signal-to-background ratio, with a 20-fold increased signal in VDO compared to uninfected tissues. In a proof-of-concept experiment, detection of bacterial infection and discrimination between S aureus and E coli was possible using a combination of [11C]PABA and [18F]FDS. CONCLUSIONS: Our work reveals that several bacteria-targeted PET radiotracers had sufficient signal to background in a rat model of S aureus VDO to be potentially clinically useful. [11C]PABA was the most promising tracer investigated and warrants further investigation in human VDO.

Chemoenzymatic Syntheses of Fluorine-18-Labeled Disaccharides from [18F] FDG Yield Potent Sensors of Living Bacteria In Vivo.

Chemoenzymatic techniques have been applied extensively to pharmaceutical development, most effectively when routine synthetic methods fail. The regioselective and stereoselective construction of structurally complex glycans is an elegant application of this approach that is seldom applied to positron emission tomography (PET) tracers. We sought a method to dimerize 2-deoxy-[18F]-fluoro-d-glucose ([18F]FDG), the most common tracer used in clinical imaging, to form [18F]-labeled disaccharides for detecting microorganisms in vivo based on their bacteria-specific glycan incorporation. When [18F]FDG was reacted with ß-d-glucose-1-phosphate in the presence of maltose phosphorylase, the α-1,4- and α-1,3-linked products 2-deoxy-[18F]-fluoro-maltose ([18F]FDM) and 2-deoxy-2-[18F]-fluoro-sakebiose ([18F]FSK) were obtained. This method was further extended with the use of trehalose (α,α-1,1), laminaribiose (ß-1,3), and cellobiose (ß-1,4) phosphorylases to synthesize 2-deoxy-2-[18F]fluoro-trehalose ([18F]FDT), 2-deoxy-2-[18F]fluoro-laminaribiose ([18F]FDL), and 2-deoxy-2-[18F]fluoro-cellobiose ([18F]FDC). We subsequently tested [18F]FDM and [18F]FSK in vitro, showing accumulation by several clinically relevant pathogens including Staphylococcus aureus and Acinetobacter baumannii, and demonstrated their specific uptake in vivo. Both [18F]FDM and [18F]FSK were stable in human serum with high accumulation in preclinical infection models. The synthetic ease and high sensitivity of [18F]FDM and [18F]FSK to S. aureus including methicillin-resistant (MRSA) strains strongly justify clinical translation of these tracers to infected patients. Furthermore, this work suggests that chemoenzymatic radiosyntheses of complex [18F]FDG-derived oligomers will afford a wide array of PET radiotracers for infectious and oncologic applications.

A fluorogenic micrococcal nuclease-based probe for fast detection and optical imaging of Staphylococcus aureus in prosthetic joint and fracture-related infections.

PURPOSE: Staphylococcus aureus is the most common and impactful multi-drug resistant pathogen implicated in (periprosthetic) joint infections (PJI) and fracture-related infections (FRI). Therefore, the present proof-of-principle study was aimed at the rapid detection of S. aureus in synovial fluids and biofilms on extracted osteosynthesis materials through bacteria-targeted fluorescence imaging with the 'smart-activatable' DNA-based AttoPolyT probe. This fluorogenic oligonucleotide probe yields large fluorescence increases upon cleavage by micrococcal nuclease, an enzyme secreted by S. aureus. METHODS: Synovial fluids from patients with suspected PJI and extracted osteosynthesis materials from trauma patients with suspected FRI were inspected for S. aureus nuclease activity with the AttoPolyT probe. Biofilms on osteosynthesis materials were imaged with the AttoPolyT probe and a vancomycin-IRDye800CW conjugate (vanco-800CW) specific for Gram-positive bacteria. RESULTS: 38 synovial fluid samples were collected and analyzed. Significantly higher fluorescence levels were measured for S. aureus-positive samples compared to, respectively, other Gram-positive bacterial pathogens (p < 0.0001), Gram-negative bacterial pathogens (p = 0.0038) and non-infected samples (p = 0.0030), allowing a diagnosis of S. aureus-associated PJI within 2 h. Importantly, S. aureus-associated biofilms on extracted osteosynthesis materials from patients with FRI were accurately imaged with the AttoPolyT probe, allowing their correct distinction from biofilms formed by other Gram-positive bacteria detected with vanco-800CW within 15 min. CONCLUSION: The present study highlights the potential clinical value of the AttoPolyT probe for fast and accurate detection of S. aureus infection in synovial fluids and biofilms on extracted osteosynthesis materials.

Bacteria-targeted fluorescence imaging of extracted osteosynthesis devices for rapid visualization of fracture-related infections.

PURPOSE: Fracture-related infection (FRI) is a serious complication in orthopedic trauma surgery worldwide. Especially, the distinction of infection from sterile inflammation and the detection of low-grade infection are highly challenging. The objective of the present study was to obtain proof-of-principle for the use of bacteria-targeted fluorescence imaging to detect FRI on extracted osteosynthesis devices as a step-up towards real-time image-guided trauma surgery. METHODS: Extracted osteosynthesis devices from 13 patients, who needed revision surgery after fracture treatment, were incubated with a near-infrared fluorescent tracer composed of the antibiotic vancomycin and the fluorophore IRDye800CW (i.e., vanco-800CW). Subsequently, the devices were imaged, and vanco-800CW fluorescence signals were correlated to the results of microbiological culturing and to bacterial growth upon replica plating of the imaged devices on blood agar. RESULTS: Importantly, compared to culturing, the bacteria-targeted fluorescence imaging of extracted osteosynthesis devices with vanco-800CW allows for a prompt diagnosis of FRI, reducing the time-to-result from days to less than 30 min. Moreover, bacteria-targeted imaging can provide surgeons with real-time visual information on the presence and extent of infection. CONCLUSION: Here, we present the first clinical application of fluorescence imaging for the detection of FRI. We conclude that imaging with vanco-800CW can provide early, accurate, and real-time visual diagnostic information on FRI in the clinical setting, even in the case of low-grade infections.

Variants in autoinflammatory diseases-related genes in a family with cold-induced autoinflammatory syndrome.

OBJECTIVES: To characterise what immunogenetic alterations are present in a Spanish family having several members with a familial cold-induced autoinflammatory syndrome (FCAS), a kind of autoinflammatory disease (AID). METHODS: We present the case of two sisters (cases 1 and 2) with a similar clinical picture since their childhood. The symptoms start after exposure to cold and consist of recurrent fever, papules or urticaria, and oedema in hands and fingers. The mother had similar symptomatology as her daughters, which remitted after her first pregnancy, whereas the father is healthy. Patients and their parents were genotyped in a panel of 14 candidate genes using Next-Generation Sequencing (NGS). Real-time PCR was used to quantify IL1ß mRNA levels from LPS-stimulated monocytes. ELISA was used to measure the IL1ß and IL18 concentrations in supernatants and sCD25 levels in sera. IL1ß, IL4, IL6, IL8, IL10, IL17A, IL18 and TNF-α serum levels were assessed using xMAP® Technology. RESULTS: All the genetic variants found in this family are benign with two exceptions: NLRC4 p.Leu339Pro (present in both cases and their mother) and PSTPIP1 p.Gln219His (present in Case 1 and her father). The monocytes stimulated of the individuals with the NLRC4 variant produce higher levels of IL1ß (protein and mRNA). Levels of TNF-α, IL4, and IL6 were higher in Case 1 than in the age-matched controls. CONCLUSIONS: The familial segregation and the clinical picture compatible with FCAS suggest that NLRC4 p.Leu339Pro causes the AIDs syndrome diagnosed in several family members.

An Analysis by the European Committee on Organ Transplantation of the Council of Europe Outlining the International Landscape of Donors and Recipients Sex in Solid Organ Transplantation.

Discrepancies in donation and transplantation by sex and gender have previously been reported. However, whether such differences are invariably the inevitable, unintended outcome of a legitimate process has yet to be determined. The European Committee on Organ Transplantation of the Council of Europe (CD-P-TO) is the committee that actively promotes the development of ethical, quality and safety standards in the field of transplantation in Europe. Whilst the ultimate objective is to shed light on the processes underlying potential gender inequities in transplantation, our initial goal was to represent the distribution by sex among organ donors and recipients in the CD-P-TO Member States and observer countries. Our survey confirms previous evidence that, in most countries, men represent the prevalent source of deceased donors (63.3% in 64 countries: 60.7% and 71.9% for donation after brain and circulatory death, respectively). In contrast, women represent the leading source of organs recovered from living kidney and liver donors (61.1% and 51.2% in 55 and 32 countries, respectively). Across countries, most recovered organs are transplanted into men (65% in 57 countries). These observations may be explained, at least in part, by the higher burden of certain diseases in men, childbearing related immune sensitization in women, and donor-recipient size mismatch. Future research should establish whether gender-related socially-constructed roles and socioeconomic status may play a detrimental role reducing the access of women to transplantation.

Image-guided in situ detection of bacterial biofilms in a human prosthetic knee infection model: a feasibility study for clinical diagnosis of prosthetic joint infections.

PURPOSE: Due to an increased human life expectancy, the need to replace arthritic or dysfunctional joints by prosthetics is higher than ever before. Prosthetic joints are unfortunately inherently susceptible to bacterial infection accompanied by biofilm formation. Accurate and rapid diagnosis is vital to increase therapeutic success. Yet, established diagnostic modalities cannot directly detect bacterial biofilms on prostheses. Therefore, the present study was aimed at investigating whether arthroscopic optical imaging can accurately detect bacterial biofilms on prosthetic joints. METHODS: Here, we applied a conjugate of the antibiotic vancomycin and the near-infrared fluorophore IRDye800CW, in short vanco-800CW, in combination with arthroscopic optical imaging to target and visualize biofilms on infected prostheses. RESULTS: We show in a human post-mortem prosthetic knee infection model that a staphylococcal biofilm is accurately detected in real time and distinguished from sterile sections in high resolution. In addition, we demonstrate that biofilms associated with the clinically most relevant bacterial species can be detected using vanco-800CW. CONCLUSION: The presented image-guided arthroscopic approach provides direct visual diagnostic information and facilitates immediate appropriate treatment selection.

Bacterial FtsZ protein forms phase-separated condensates with its nucleoid-associated inhibitor SlmA.

Macromolecular condensation resulting from biologically regulated liquid-liquid phase separation is emerging as a mechanism to organize intracellular space in eukaryotes, with broad implications for cell physiology and pathology. Despite their small size, bacterial cells are also organized by proteins such as FtsZ, a tubulin homolog that assembles into a ring structure precisely at the cell midpoint and is required for cytokinesis. Here, we demonstrate that FtsZ can form crowding-induced condensates, reminiscent of those observed for eukaryotic proteins. Formation of these FtsZ-rich droplets occurs when FtsZ is bound to SlmA, a spatial regulator of FtsZ that antagonizes polymerization, while also binding to specific sites on chromosomal DNA. The resulting condensates are dynamic, allowing FtsZ to undergo GTP-driven assembly to form protein fibers. They are sensitive to compartmentalization and to the presence of a membrane boundary in cell mimetic systems. This is a novel example of a bacterial nucleoprotein complex exhibiting condensation into liquid droplets, suggesting that phase separation may also play a functional role in the spatiotemporal organization of essential bacterial processes.

Access of non-residents to transplantation of deceased donor organs: practices and strategies in the European setting.

The access of non-resident patients to the deceased donor waiting list (DDWL) poses different challenges. The European Committee on Organ Transplantation of the Council of Europe (CD-P-TO) has studied this phenomenon in the European setting. A questionnaire was circulated among the Council of Europe member states to inquire about the criteria applied for non-residents to access their DDWL. Information was compiled from 28 countries. Less than 1% of recipients of deceased donor organs were non-residents. Two countries never allow non-residents to access the DDWL, four allow access without restrictions and 22 only under specific conditions. Of those, most give access to non-resident patients already in their jurisdictions who are in a situation of vulnerability (urgent life-threatening conditions). In addition, patients may be given access: (i) after assessment by a specific committee (four countries); (ii) within the framework of official cooperation agreements (15 countries); and (iii) after patients have officially lived in the country for a minimum length of time (eight countries). The ethical and legal implications of these policies are discussed. Countries should collect accurate information about residency status of waitlisted patients. Transparent criteria for the access of non-residents to DDWL should be clearly defined at national level.

Position paper on screening for breast cancer by the European Society of Breast Imaging (EUSOBI) and 30 national breast radiology bodies from Austria, Belgium, Bosnia and Herzegovina, Bulgaria, Croatia, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Israel, Lithuania, Moldova, The Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Spain, Sweden, Switzerland and Turkey.

EUSOBI and 30 national breast radiology bodies support mammography for population-based screening, demonstrated to reduce breast cancer (BC) mortality and treatment impact. According to the International Agency for Research on Cancer, the reduction in mortality is 40 % for women aged 50-69 years taking up the invitation while the probability of false-positive needle biopsy is <1 % per round and overdiagnosis is only 1-10 % for a 20-year screening. Mortality reduction was also observed for the age groups 40-49 years and 70-74 years, although with "limited evidence". Thus, we firstly recommend biennial screening mammography for average-risk women aged 50-69 years; extension up to 73 or 75 years, biennially, is a second priority, from 40-45 to 49 years, annually, a third priority. Screening with thermography or other optical tools as alternatives to mammography is discouraged. Preference should be given to population screening programmes on a territorial basis, with double reading. Adoption of digital mammography (not film-screen or phosphor-plate computer radiography) is a priority, which also improves sensitivity in dense breasts. Radiologists qualified as screening readers should be involved in programmes. Digital breast tomosynthesis is also set to become "routine mammography" in the screening setting in the next future. Dedicated pathways for high-risk women offering breast MRI according to national or international guidelines and recommendations are encouraged. KEY POINTS: • EUSOBI and 30 national breast radiology bodies support screening mammography. • A first priority is double-reading biennial mammography for women aged 50-69 years. • Extension to 73-75 and from 40-45 to 49 years is also encouraged. • Digital mammography (not film-screen or computer radiography) should be used. • DBT is set to become "routine mammography" in the screening setting in the next future.

The global diffusion of organ transplantation: trends, drivers and policy implications.

Rising incomes, the spread of personal insurance, lifestyle factors adding to the burden of illness, ageing populations, globalization and skills transfer within the medical community have increased worldwide demand for organ transplantation. The Global Observatory on Donation and Transplantation, which was built in response to World Health Assembly resolution WHA57.18, has conducted ongoing documentation of global transplantation activities since 2007. In this paper, we use the Global Observatory's data to describe the current distribution of - and trends in - transplantation activities and to evaluate the role of health systems factors and macroeconomics in the diffusion of transplantation technology. We then consider the implications of our results for health policies relating to organ donation and transplantation. Of the World Health Organization's Member States, most now engage in organ transplantation and more than a third performed deceased donor transplantation in 2011. In general, the Member States that engage in organ transplantation have greater access to physician services and greater total health spending per capita than the Member States where organ transplantation is not performed. The provision of deceased donor transplantation was closely associated with high levels of gross national income per capita. There are several ways in which governments can support the ethical development of organ donation and transplantation programmes. Specifically, they can ensure that appropriate legislation, regulation and oversight are in place, and monitor donation and transplantation activities, practices and outcomes. Moreover, they can allocate resources towards the training of specialist physicians, surgeons and transplant coordinators, and implement a professional donor-procurement network.

La hausse des revenus, le développement des assurances personnelles, les facteurs de mode de vie ajoutant à la charge de morbidité des maladies, le vieillissement des populations, la mondialisation et le transfert des compétences au sein de la communauté médicale ont augmenté la demande mondiale de transplantation d'organe. L'Observatoire Mondial du Don et de la Transplantation, qui a été fondé en réponse à la résolution WHA57.18 de l'Organisation mondiale de la Santé, a rassemblé une documentation sur les activités de transplantation dans le monde de façon continue depuis 2007. Dans cet article, nous utilisons les données de l'Observatoire Mondial pour décrire la distribution actuelle (et les tendances) des activités de transplantation et pour évaluer le rôle des facteurs de systèmes de santé et de la macroéconomie dans la diffusion des technologies de transplantation. Nous considérons ensuite les implications de nos résultats sur les politiques de santé relatives au don et à la transplantation d'organe. La majorité des États Membres de l'Organisation mondiale de la Santé s'engagent maintenant dans la transplantation d'organe et plus d'un tiers d'entre eux ont réalisé des transplantations avec des organes provenant de donneurs décédés en 2011. En général, les États Membres qui se sont engagés dans la transplantation d'organe, ont un meilleur accès aux services médicaux et des dépenses totales de santé plus élevées par habitant que les États Membres où la transplantation d'organe n'est pas réalisée. La disponibilité de la transplantation avec des organes provenant de donneurs décédés était étroitement associée avec des niveaux élevés de revenu national brut par habitant. Il existe plusieurs manières possibles pour les gouvernements de soutenir le développement éthique des programmes de don et de transplantation d'organe. En particulier, ils peuvent s'assurer que la législation, la réglementation et la surveillance sont en place, et contrôler les activités, les pratiques et les résultats des dons et des transplantations. En outre, ils peuvent affecter des ressources pour la formation des médecins spécialistes, des chirurgiens et des coordinateurs de transplantation, et mettre en œuvre un réseau professionnel de recrutement des donneurs.

El aumento de la renta, la proliferación de los seguros personales y los factores del estilo de vida, sumados a la carga de enfermedades, el envejecimiento de la población, la globalización y la transferencia de conocimientos en la comunidad médica, han aumentado la demanda mundial de trasplantes de órganos. El Observatorio Mundial de Donación y Trasplante, creado en respuesta a la resolución WHA57.18 de la Asamblea Mundial de la Salud, ha llevado a cabo una documentación continua de las actividades mundiales de trasplantes desde 2007. En este informe, se emplean los datos del Observatorio Global para describir la distribución actual (y las tendencias) de las actividades de trasplante y para evaluar el papel de los factores de los sistemas sanitarios y de la macroeconomía en la difusión de la tecnología de trasplante. A continuación, se consideraron las repercusiones de los resultados en las políticas de salud relacionadas con la donación y el trasplante de órganos. En la actualidad, la mayoría de los Estados miembros de la Organización Mundial de la Salud participa en el trasplante de órganos y más de un tercio realizó trasplantes de donantes fallecidos en 2011. En general, los Estados miembros que participan en el trasplante de órganos cuentan con mayor acceso a los servicios médicos y tienen un mayor gasto total en salud per cápita que los Estados miembros donde no se realizan el trasplantes de órganos. La prestación de los trasplantes de donantes fallecidos se asoció estrechamente con altos niveles de renta nacional bruta per cápita. Existen varias formas en que los gobiernos pueden fomentar el desarrollo ético de los programas de donación y trasplante de órganos. En concreto, pueden garantizar que se adopte una legislación, regulación y supervisión adecuadas, así como realizar un seguimiento de las actividades, las prácticas y los resultados de la donación y el trasplante. Además, pueden destinar recursos a la formación de médicos especialistas, cirujanos y coordinadores de trasplantes, así como poner en marcha una red profesional de adquisición de donantes.

Imaging the Granzyme Mediated Host Immune Response to Viral and Bacterial Pathogens In Vivo Using Positron Emission Tomography.

Understanding how the host immune system engages complex pathogens is essential to developing therapeutic strategies to overcome their virulence. While granzymes are well understood to trigger apoptosis in infected host cells or bacteria, less is known about how the immune system mobilizes individual granzyme species in vivo to combat diverse pathogens. Toward the goal of studying individual granzyme function directly in vivo, we previously developed a new class of radiopharmaceuticals termed "restricted interaction peptides (RIPs)" that detect biochemically active endoproteases using positron emission tomography (PET). In this study, we showed that secreted granzyme B proteolysis in response to diverse viral and bacterial pathogens could be imaged with [64Cu]Cu-GRIP B, a RIP that specifically targets granzyme B. Wild-type or germline granzyme B knockout mice were instilled intranasally with the A/PR/8/34 H1N1 influenza A strain to generate pneumonia, and granzyme B production within the lungs was measured using [64Cu]Cu-GRIP B PET/CT. Murine myositis models of acute bacterial (E. coli, P. aeruginosa, K. pneumoniae, and L. monocytogenes) infection were also developed and imaged using [64Cu]Cu-GRIP B. In all cases, the mice were studied in vivo using mPET/CT and ex vivo via tissue-harvesting, gamma counting, and immunohistochemistry. [64Cu]Cu-GRIP B uptake was significantly higher in the lungs of wild-type mice that received A/PR/8/34 H1N1 influenza A strain compared to mice that received sham or granzyme B knockout mice that received either treatment. In wild-type mice, [64Cu]Cu-GRIP B uptake was significantly higher in the infected triceps muscle versus normal muscle and the contralateral triceps inoculated with heat killed bacteria. In granzyme B knockout mice, [64Cu]Cu-GRIP B uptake above the background was not observed in the infected triceps muscle. Interestingly, live L. monocytogenes did not induce detectable granzyme B on PET, despite prior in vitro data, suggesting a role for granzyme B in suppressing their pathogenicity. In summary, these data show that the granzyme response elicited by diverse human pathogens can be imaged using PET. These results and data generated via additional RIPs specific for other granzyme proteases will allow for a deeper mechanistic study analysis of their complex in vivo biology.

Peptidoglycan-Targeted [18F]3,3,3-Trifluoro-d-alanine Tracer for Imaging Bacterial Infection.

Imaging is increasingly used to detect and monitor bacterial infection. Both anatomic (X-rays, computed tomography, ultrasound, and MRI) and nuclear medicine ([111In]-WBC SPECT, [18F]FDG PET) techniques are used in clinical practice but lack specificity for the causative microorganisms themselves. To meet this challenge, many groups have developed imaging methods that target pathogen-specific metabolism, including PET tracers integrated into the bacterial cell wall. We have previously reported the d-amino acid derived PET radiotracers d-methyl-[11C]-methionine, d-[3-11C]-alanine, and d-[3-11C]-alanine-d-alanine, which showed robust bacterial accumulation in vitro and in vivo. Given the clinical importance of radionuclide half-life, in the current study, we developed [18F]3,3,3-trifluoro-d-alanine (d-[18F]-CF3-ala), a fluorine-18 labeled tracer. We tested the hypothesis that d-[18F]-CF3-ala would be incorporated into bacterial peptidoglycan given its structural similarity to d-alanine itself. NMR analysis showed that the fluorine-19 parent amino acid d-[19F]-CF3-ala was stable in human and mouse serum. d-[19F]-CF3-ala was also a poor substrate for d-amino acid oxidase, the enzyme largely responsible for mammalian d-amino acid metabolism and a likely contributor to background signals using d-amino acid derived PET tracers. In addition, d-[19F]-CF3-ala showed robust incorporation into Escherichia coli peptidoglycan, as detected by HPLC/mass spectrometry. Based on these promising results, we developed a radiosynthesis of d-[18F]-CF3-ala via displacement of a bromo-precursor with [18F]fluoride followed by chiral stationary phase HPLC. Unexpectedly, the accumulation of d-[18F]-CF3-ala by bacteria in vitro was highest for Gram-negative pathogens in particular E. coli. In a murine model of acute bacterial infection, d-[18F]-CF3-ala could distinguish live from heat-killed E. coli, with low background signals. These results indicate the viability of [18F]-modified d-amino acids for infection imaging and indicate that improved specificity for bacterial metabolism can improve tracer performance.

Cortical amyloid-beta burden is associated with changes in intracortical myelin in cognitively normal older adults.

Amyloid-beta (Aß) aggregates and myelin breakdown are among the earliest detrimental effects of Alzheimer's disease (AD), likely inducing abnormal patterns of neuronal communication within cortical networks. However, human in vivo evidence linking Aß burden, intracortical myelin, and cortical synchronization is lacking in cognitively normal older individuals. Here, we addressed this question combining 18F-Florbetaben-PET imaging, cortical T1-weigthed/T2-weighted (T1w/T2w) ratio maps, and resting-state functional connectivity (rs-FC) in cognitively unimpaired older adults. Results showed that global Aß burden was both positively and negatively associated with the T1w/T2w ratio in different cortical territories. Affected cortical regions were further associated with abnormal patterns of rs-FC and with subclinical cognitive deficits. Finally, causal mediation analysis revealed that the negative impact of T1w/T2w ratio in left posterior cingulate cortex on processing speed was driven by Aß burden. Collectively, these findings provide novel insights into the relationship between initial Aß plaques and intracortical myelin before the onset of cognitive decline, which may contribute to monitor the efficacy of novel disease-modifying strategies in normal elderly individuals at risk for cognitive impairment.

Imaging the Bacterial Cell Wall Using N-Acetyl Muramic Acid-Derived Positron Emission Tomography Radiotracers.

Imaging infections in patients is challenging using conventional methods, motivating the development of positron emission tomography (PET) radiotracers targeting bacteria-specific metabolic pathways. Numerous techniques have focused on the bacterial cell wall, although peptidoglycan-targeted PET tracers have been generally limited to the short-lived carbon-11 radioisotope (t1/2 = 20.4 min). In this article, we developed and tested new tools for infection imaging using an amino sugar component of peptidoglycan, namely, derivatives of N-acetyl muramic acid (NAM) labeled with the longer-lived fluorine-18 (t1/2 = 109.6 min) radioisotope. Muramic acid was reacted directly with 4-nitrophenyl 2-[18F]fluoropropionate ([18F]NFP) to afford the enantiomeric NAM derivatives (S)-[18F]FMA and (R)-[18F]FMA. Both diastereomers were easily isolated and showed robust accumulation by human pathogens in vitro and in vivo, including Staphylococcus aureus. These results form the basis for future clinical studies using fluorine-18-labeled NAM-derived PET radiotracers.