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Aim: Pre-targeting is a proven strategy for in vivodelivery of a diagnostic or therapeutic payload. The pre-targeting concept can be realized through various conjugation strategies, one of which is based on copper-free “click” chemistry. Copper-free click reactions have shown in vivopotential for imaging and radionuclide therapy, but this conjugation strategy has not yet been explored in combination with microspheres or unicellular organisms. This study aims to evaluate the in vivoefficacy of strain-promoted azide–alkyne cycloaddition (SPAAC) reactions to achieve imaging and targeting of azide-functionalized macro-aggregated albumin (MAA) microspheres and Staphylococcus aureusbacteria. Methods: MAA microspheres (diameter 10–90 μm) were functionalized with a biorthogonal Cy5 fluorophore, bearing an azide functionality (N3), to generate MAA-Cy5-N3. S. aureus(diameter ∼1 μm) were functionalized with 99mTc-UBI29–41-Cy5-N3, generating S. aureus-99mTc-UBI29–41-Cy5-N3. In situand in vitroclick conjugation on the –N3moieties was studied for 20 h using a radioactivity-based assay and fluorescence microscopy. For in vivovalidation, both primary entities, radiolabeled with 99mTc, were deposited into the microvasculature of the liver viaintrasplenic injections. Secondary targeting was realized following the intravenous administration of indium-111-radiolabeled diethylenetriaminepentaacetic acid-dibenzocyclooctyne (111In-DTPA-DBCO). To assess click reaction efficiency in vivo, 99mTc and 111In-biodistributions were measured (SPECT and %ID g−1). Use of 111In-DTPA-DBCO in mice without MAA deposits or mice infected with non-functionalized S. aureusserved as controls. Ex vivoconfocal fluorescence imaging was carried out in excised tissues to confirm the presence of functionalized MAA and bacteria. Results: In vitrodata confirmed effective click reactions on both the MAA particles and the bacterial membrane. SPECT imaging and biodistribution studies revealed significantly (p< 0.05) increased accumulation of 111In-DTPA-DBCO at the sites where MAA-Cy5-N3(7.5 ± 1.5%ID g−1vs. 3.5 ± 0.5%ID g−1in control mice) and S. aureus-99mTc-UBI29–41-Cy5-N3(9.3 ± 1.3%ID g−1vs.6.0 ± 0.5%ID g−1in control mice) resided. Ex vivofluorescence imaging confirmed the presence of either functionalized MAA or S. aureusin excised spleens and livers of mice. Conclusion: Copper-free click chemistry between a DBCO moiety and Cy5-N3-functionalized microspheres or bacterial entities in the liver can be used to realize in vivoimaging and targeting.

ABSTRACTStrong evidence suggests that the gut microbiota is altered in inflammatory bowel disease (IBD), indicating its potential role in noninvasive diagnostics. However, no clinical applications are currently used for routine patient care. The main obstacle to implementing a gut microbiota test for IBD is the lack of standardization, which leads to high interlaboratory variation. We studied the between-hospital and between-platform batch effects and their effects on predictive accuracy for IBD. Fecal samples from 91 pediatric IBD patients and 58 healthy children were collected. IS-pro, a standardized technique designed for routine microbiota profiling in clinical settings, was used for microbiota composition characterization. Additionally, a large synthetic data set was used to simulate various perturbations and study their effects on the accuracy of different classifiers. Perturbations were validated in two replicate data sets, one processed in another laboratory and the other with a different analysis platform. The type of perturbation determined its effect on predictive accuracy. Real-life perturbations induced by between-platform variation were significantly greater than those caused by between-laboratory variation. Random forest was found to be robust to both simulated and observed perturbations, even when these perturbations had a dramatic effect on other classifiers. It achieved high accuracy both when cross-validated within the same data set and when using data sets analyzed in different laboratories. Robust clinical predictions based on the gut microbiota can be performed even when samples are processed in different hospitals. This study contributes to the effort to develop a universal IBD test that would enable simple diagnostics and disease activity monitoring.

Bacterial resistance to conventional antibiotics poses a challenge medicine to search for alternatives. Cationic antimicrobial peptides (AMPs) are promising for the development of a new class of antibiotics. This review focuses on the use of technetium-99m labeled synthetic AMPs, derived from human natural cationic AMPs, for target-delivery to and in vivo detection of infection sites caused by (drug-resistant) micro-organisms. The scintigraphic approach has proven to be a reliable method for evaluating AMPs in pharmacological studies and for optimizing target-delivery of radiolabeled AMPs to pathological sites in animals and humans. In addition, the effect of alterations in amphipathicity, amino acid substitution, and dimerization on the biological performance of AMPs is reported. Radiolabeled AMPs offer good perspectives for diagnosis of infections, for monitoring therapy, and, most importantly, for the ability to discriminate between infections and sterile inflammatory processes.

The outcome of antifungal therapy depends on the progression of the infection at the start of therapy. Unfortunately, most patients are diagnosed once the fungal infection has progressed considerably as a result of the non-specific clinical signs of fungal infections in immunocompromised patients and the poor sensitivity of current mycological diagnostic tests. This review will highlight current fungal diagnostic techniques and will focus on scintigraphic methods for the specific detection of fungal infections in mice. For this purpose, antifungal components (e.g. fluconazole and antifungal peptides) are radiolabeled e.g. with technetium-99m (99mTc) and their in vivo distribution is monitored in infected mice. It has been demonstrated that 99mTc-fluconazole is an excellent tracer to detect Candida albicans infections in mice as it distinguishes these infections from bacterial infections and sterile inflammations. However, this radiopharmaceutical only poorly detects infections with Aspergillus fumigatus in mice. 99mTc-peptides derived from antifungal peptides/ proteins, such as human ubiquicidin and lactoferrin, can distinguish C. albicans and A. fumigatus infections from sterile inflammations, but not from bacterial infections, in mice. Furthermore, the efficacy of fluconazole in C. albicansinfected mice could be successfully monitored using 99mTc-ubiquicidin. In conclusion, neither 99mTc-fluconazole nor the 99mTc-peptides tested are optimal tracers for fungal infections. Nonetheless, since early initiation of antifungal therapy for candidemia reduces its high mortality rate, a positive result with 99mTc-fluconazole scintigraphy is of clinical relevance. Finally, the possibility that other (radiolabeled) antifungal agents, e.g. voriconazole, caspofungin, antifungal plant or insect defensins, can be useful for detection of fungal infections should be considered.

ABSTRACTSince human lactoferrin (hLF) binds to bacterial products through its highly positively charged N terminus, we investigated which of the two cationic domains is involved in its bactericidal activity. The results revealed that hLF lacking the first three residues (hLF−3N) was less efficient than hLF in killing of antibiotic-resistant Staphylococcus aureus, Listeria monocytogenes, and Klebsiella pneumoniae. Both hLF preparations failed to kill Escherichia coliO54. In addition, hLF−3Nwas less effective than hLF in reducing the number of viable bacteria in mice infected with antibiotic-resistant S. aureusand K. pneumoniae. Studies with synthetic peptides corresponding to the first 11 N-terminal amino acids, designated hLF(1–11), and fragments thereof demonstrated that peptides lacking the first three N-terminal residues are less effective than hLF(1–11) in killing of bacteria. Furthermore, a peptide corresponding to residues 21 to 31, which comprises the second cationic domain, was less effective than hLF(1–11) in killing of bacteria in vitro and in mice having an infection with antibiotic-resistant S. aureusor K. pneumoniae. Using fluorescent probes, we found that bactericidal hLF peptides, but not nonbactericidal peptides, caused an increase of the membrane permeability. In addition, hLF killed the various bacteria, most probably by inducing intracellular changes in these bacteria without affecting the membrane permeability. Together, hLF and peptides derived from its N terminus are highly effective against infections with antibiotic-resistant S. aureusand K. pneumoniae, and the first two arginines play an essential role in this activity.

At present, it is difficult to distinguish between bacterial infections and sterile inflammatory processes using radiopharmaceuticals. This is so for a variety of reasons, including binding to bacteria with low affinity (e.g. infection) and binding to a specific micro-organism (e.g. radiolabelled monoclonal antibodies or F(ab)2fragments thereof against micro-organisms). In this review, we propose that radiolabelled antimicrobial peptides should be the first choice in the development of new radiopharmaceuticals for imaging of bacterial infections. Antimicrobial peptides are a recently discovered component of the innate defence system of plants, animals and humans. These peptides, which now number more than 100, with proven microbicidal activity against a variety of micro-organisms, share certain properties, such as their small size and cationic charge. The latter allows them to bind preferentially to a broad spectrum of microorganisms. We have recently demonstrated that radiolabelled human defensins allow the rapid visualization of bacterial infections in mice. Furthermore, binding of this antimicrobial peptide to bacteria is the major factor contributing to the accumulation of this tracer in bacterial infections. Based on these considerations, we believe that radiolabelled antimicrobial peptides will be an important asset in the imaging of infections in patients.

Our objective was to investigate the mechanism of accumulation of 99Tcm-labelled non-specific polyclonal human immunoglobulin (99Tcm-HIG) in inflamed synovial tissue (ST) in an experimental animal model of arthritis. Following 99Tcm-HIG scintigraphy, the in vivolocalization of 99Tcm-HIG in the ST of knee joints of rats with adjuvant arthritis was studied using immunohistochemical techniques. In addition, the in vitro binding of 99Tcm-HIG to extracellular matrix proteins was analysed by means of immunohistochemistry and enzyme-linked immunosorbent assay (ELISA). After 99Tcm-HIG scintigraphy, 99Tcm-HIG was detected in the ST of rats with adjuvant arthritis. 99Tcm-HIG was diffusely distributed and not bound to cells. In vitroincubation of 99Tcm-HIG on the ST of rats with adjuvant arthritis revealed binding of 99Tcm-HIG to inflamed, but not to non-inflamed, ST. In addition, specific binding of 99Tcm-HIG to fibronectin, fibrin, collagen type I and III was demonstrated by ELISA. We conclude that the accumulation of 99Tcm-HIG in inflamed ST can be explained by the binding of 99Tcm-HIG to extracellular matrix proteins.

The aim of this study was to determine the contribution of various IgG subclasses to the scintigraphic detection of a staphylococcal infection. An experimental thigh infection in mice was used to determine the accumulation of the various 99Tcm-labelled IgG preparations with enriched IgG1, IgG2 or IgG4 subclass. Multiple-regression analysis was used to investigate a relationship between the IgG subclasses and the time-dependent accumulation in infected sites.

In this paper we consider space-times containing matter expanding or contracting according to a time-dependent scale factor. Cosmologies with vanishing, positive or negative cosmological constant are considered. In the case of vanishing or negative cosmological constant open and closed spatial surfaces are solutions while in the case of positive cosmological constant only closed surfaces exist. The gravitational field is solved explicitly in the case of 1 or 2 particles, 1 black hole, and 1 black hole vacuum state.

In Madagascar, a fungal disease was observed in the migratory locust, Locusta migratoria capito, which was caused by the deuteromycete Sorosporella sp. Multiplication of the fungus in locusts results in the formation of brick-red resting spores filling the body of the insect, and in a pale and fragile cuticle which breaks easily, releasing these spores. The conidia of the fungus (only observed in artificial cultures) are cylindrical in shape, measuring 11.0 2.8 mu m. The growth of the fungus was compared on several solid and liquid media, but was invariably slow. The best growth was obtained on a medium containing 3% ground rice, 3% malt extract, 0.3% peptone and 1.5% agar. Attempts were made to initiate artificial infections in L. migratoria migratorioides and Schistocerca gregaria from a laboratory stock. This was carried out by feeding resting spores or through contact with Sorosporella material from agar cultures. However, it rarely resulted in mortalities with typical symptoms. Inoculations by injection were more successful, and resulted in the formation of resting spores in the cadavers 12 to 21 days after inoculation. In Madagascar, Sorosporella sp. seems to be an important, and frequently occurring, locust pathogen. However, the natural mode of infection and the ecology of the fungus are still unclear. Une mycose causee par le champignon deuteromycete Sorosporella sp. a ete observee a Madagascar chez le criquet migrateur Locusta migratoria capito. La multiplication du champignon dans le corps des criquets entraine la formation de spores rouge-briques, qui emplissent celui-ci. Les individus infestes developpent en outre une cuticule pale et fragile, qui se brise facilement, laissant ainsi les spores se disseminer. Les conidies (observees uniquement en cultures artificielles) mesurent 11.0 2.8 mum. La croissance du champignon reste lente sur tous les milieux solides et liquides compares. La meilleure croissance a ete obtenue sur un milieu contenant 3% de riz, 3% d'extrait de malt, 0.3% de peptone et 1.5% d'agar. Des essais d'infection par ingestion de spores d'une part, par contact avec du materiel de culture sur agar d'autre part, ont etes effectues avec L. migratoria migratorioides et Schistocerca gregaria (mate riel d'elevage). Ces essais n'ont abouti que rarement a une mortalite avec des symptomes typiques. Des resultats plus positifs ont ete obtenus au moyen d'inoculations par injection. Ils se forment dans ce cas des spores dans les cadavres des criquets 12 a 21 jours apres l'inoculation. Sorosporella sp. semble etre un agent pathogene des criquets important et frequent a Madagascar. Le mode d'infection naturel, ainsi que l'ecologie de ce champignon restent cependant peu connus.

Abstract: Five strains ofMetarhizium anisopliae (Metsch.) Sorokin and one strain ofMetarhizium flavoviride Gams & Rozsypal originally isolated in Madagascar were studied. Measurements of conidia and, for the first time, also of blastospores produced in a liquid medium were used for species and variety determination. Blastospores ofM. flavoviride were more homogenous in their size than those ofM. anisopliae. Growth at high temperatures between 25° and 40°C showed that 4 isolates ofM. anisopliae grew at 36°C andM. flavoviride grew at 38°C. Using alternating day/night temperatures (8/16 h) the three strains tested could also tolerate 40°/25°C. In bioassays, fiveMetarhizium spp. isolates were tested against third and fourth instar larvae ofLocusta migratoria (L.) at two alternating day/night temperatures of 30°/25°C and 36°/25°C. In the cooler regime, all strains caused a mortality of 50% within 5.9 to 8.5 days (median lethal time), while in the 36°/25°C treatment only the thermophilicM. flavoviride and oneM. anisopliae strain isolated from a soil sample gave comparable results with median lethal times of 6.8 and 7.3 days, respectively.

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