[Pharmacotherapy for obesity]. / Farmacotherapie voor obesitas.

Obesity is a complex endocrine disease, mainly caused by environmental, behavioral and biological factors. Maintaining weight loss is extremely difficult due to the neuro-endocrine dysregulations that stimulate the body to return to the previous, increased, weight. Identifying underlying weight-gaining factors is needed, including medication-related, psychological and endocrine factors, as well as monogenic obesity. The cornerstone of treatment is optimization of lifestyle and all other contributing factors. Achieving at least 5% weight loss already has important health benefits. If combined lifestyle intervention (CLI) alone is not successful, pharmacotherapy or bariatric surgery can be added for patients with increased weight-related health risks. Recently, novel pharmacotherapy became available, among which, liraglutide 3 mg and the combination therapy naltrexone/bupropion, which leads to an additional 5-6% mean weight loss compared to CLI alone. For rare forms of obesity there are specific drugs that target defects in the regulation of hunger and satiety. Promising new pharmacotherapy for obesity is under development.

Interventional nuclear medicine: "click" chemistry as an in vivo targeting strategy for imaging microspheres and bacteria.

AIM: Pre-targeting is a proven strategy for in vivo delivery 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 vivo potential 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 vivo efficacy of strain-promoted azide-alkyne cycloaddition (SPAAC) reactions to achieve imaging and targeting of azide-functionalized macro-aggregated albumin (MAA) microspheres and Staphylococcus aureus bacteria. 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 situ and in vitro click conjugation on the -N3 moieties was studied for 20 h using a radioactivity-based assay and fluorescence microscopy. For in vivo validation, both primary entities, radiolabeled with 99mTc, were deposited into the microvasculature of the liver via intrasplenic 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. aureus served as controls. Ex vivo confocal fluorescence imaging was carried out in excised tissues to confirm the presence of functionalized MAA and bacteria. RESULTS: In vitro data 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-1 in control mice) and S. aureus-99mTc-UBI29-41-Cy5-N3 (9.3 ± 1.3%ID g-1vs. 6.0 ± 0.5%ID g-1 in control mice) resided. Ex vivo fluorescence imaging confirmed the presence of either functionalized MAA or S. aureus in 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 vivo imaging and targeting.

Interpretation of microbiota-based diagnostics by explaining individual classifier decisions.

BACKGROUND: The human microbiota is associated with various disease states and holds a great promise for non-invasive diagnostics. However, microbiota data is challenging for traditional diagnostic approaches: It is high-dimensional, sparse and comprises of high inter-personal variation. State of the art machine learning tools are therefore needed to achieve this goal. While these tools have the ability to learn from complex data and interpret patterns therein that cannot be identified by humans, they often operate as black boxes, offering no insight into their decision-making process. In most cases, it is difficult to represent the learning of a classifier in a comprehensible way, which makes them prone to be mistrusted, or even misused, in a clinical environment. In this study, we aim to elucidate microbiota-based classifier decisions in a biologically meaningful context to allow their interpretation. RESULTS: We applied a method for explanation of classifier decisions on two microbiota datasets of increasing complexity: gut versus skin microbiota samples, and inflammatory bowel disease versus healthy gut microbiota samples. The algorithm simulates bacterial species as being unknown to a pre-trained classifier, and measures its effect on the outcome. Consequently, each patient is assigned a unique quantitative estimation of which species in their microbiota defined the classification of their sample. The algorithm was able to explain the classifier decisions well, demonstrated by our validation method, and the explanations were biologically consistent with recent microbiota findings. CONCLUSIONS: Application of a method for explaining individual classifier decisions for complex microbiota analysis proved feasible and opens perspectives on personalized therapy. Providing an explanation to support a microbiota-based diagnosis could guide decisions of clinical microbiologists, and has the potential to increase their confidence in the outcome of such decision support systems. This may facilitate the development of new diagnostic applications.

Robust Microbiota-Based Diagnostics for Inflammatory Bowel Disease.

Strong 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.

In vitro and in vivo antioxidant, cytotoxic, and anti-chronic inflammatory arthritic effect of selenium nanoparticles.

The toxicity of selenium (Se) as an antioxidant supplement in the treatment of arthritis is debatable. In this study, Dextrin stabilized Se nanoparticles (SeNP) of size 64 nm ± 0.158 were used to explore its effects as a potent antioxidant with reduced toxicity in both in vitro and in vivo. In vitro toxicity of SeNP was determined using cytotoxicity assay. In vitro interactions of SeNP with DNA and protein was established. Subacute toxicity of SeNP was studied. Wistar rats with complete freunds adjuvant induced arthritis were used. Various concentrations of SeNP per kg body weight were fed orally daily upto to 21 days. Arthritic profile based on paw swelling, histopathological changes in joints, blood indices, and antioxidant enzymes level in organs such as liver, kidney, and spleen were investigated. Dextrin-SeNP when interacted with NIH-3T3 cells showed 15% cytotoxicity at 100 µg/mL whereas, bulk Se showed 95% at the same concentration. SeNP at 250 µg/mL showed protective effect on DNA. Interaction of SeNP with BSA showed increase in quenching of BSA fluorescence. SeNP did not show any subacute toxicity at concentration as high as 5 mg/kg b.w. in Wistar rats. SeNP at a concentration of 250 µg/kg b.w. acted as potent anti-inflammatory agent and significantly reduced (p < 0.05) arthritis induced parameters. The enzymatic antioxidant levels in liver, kidney, and spleen were restored significantly (p < 0.05) at 500 µg/kg b.w. while CRP was regained to normal at concentration of 100 µg/kg b.w. concluding SeNP at 500 µg/kg b.w. can be a potential antiarthritic drug supplement. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 993-1003, 2016.

Arbuscular mycorrhizal fungi: effects on plant terpenoid accumulation.

Arbuscular mycorrhizal fungi (AMF) are a diverse group of soil-dwelling fungi that form symbiotic associations with land plants. AMF-plant associations promote the accumulation of plant terpenoids beneficial to human health, although how AMF mediate terpenoid accumulation is not fully understood. A critical assessment and discussion of the literature relating to mechanisms by which AMF influence plant terpenoid accumulation, and whether this symbiosis can be harnessed in horticultural ecosystems was performed. Modification of plant morphology, phosphorus availability and gene transcription involved with terpenoid biosynthetic pathways were identified as key mechanisms associated with terpenoid accumulation in AMF-colonised plants. In order to exploit AMF-plant symbioses in horticultural ecosystems it is important to consider the specificity of the AMF-plant association, the predominant factor affecting terpenoid accumulation, as well as the end use application of the harvested plant material. Future research should focus on resolving the relationship between ecologically matched AMF genotypes and terpenoid accumulation in plants to establish if these associations are effective in promoting mechanisms favourable for plant terpenoid accumulation.

Development and prospects of dedicated tracers for the molecular imaging of bacterial infections.

Bacterial infections have always been, and still are, a major global healthcare problem. For accurate treatment it is of upmost importance that the location(s), severity, type of bacteria, and therapeutic response can be accurately staged. Similar to the recent successes in oncology, tracers specific for molecular imaging of the disease may help advance patient management. Chemical design and bacterial targeting mechanisms are the basis for the specificity of such tracers. The aim of this review is to provide a comprehensive overview of the molecular imaging tracers developed for optical and nuclear identification of bacteria and bacterial infections. Hereby we envision that such tracers can be used to diagnose infections and aid their clinical management. From these compounds we have set out to identify promising targeting mechanisms and select the most promising candidates for further development.

Prosthetic joint infections: radionuclide state-of-the-art imaging.

Prosthetic joint replacement surgery is performed with increasing frequency. Overall the incidence of prosthetic joint infection (PJI) and subsequently prosthesis revision failure is estimated to be between 1 and 3%. Differentiating infection from aseptic mechanical loosening, which is the most common cause of prosthetic failure, is especially important because of different types of therapeutic management. Despite a thorough patient history, physical examination, multiple diagnostic tests and complex algorithms, differentiating PJI from aseptic loosening remains challenging. Among imaging modalities, radiographs are neither sensitive nor specific and cross-sectional imaging techniques, such as computed tomography and magnetic resonance imaging, are limited by hardware-induced artefacts. Radionuclide imaging reflects functional rather than anatomical changes and is not hampered by the presence of a metallic joint prosthesis. As a result scintigraphy is currently the modality of choice in the investigation of suspected PJI. Unfortunately, there is no true consensus about the gold standard technique since there are several drawbacks and limitations inherent to each modality. Bone scintigraphy (BS) is sensitive for identifying the failed joint replacement, but cannot differentiate between infection and aseptic loosening. Combined bone/gallium scintigraphy (BS/GS) offers modest improvement over BS alone for diagnosing PJI. However, due to a number of drawbacks, BS/GS has generally been superseded by other techniques but it still may have a role in neutropenic patients. Radiolabelled leucocyte scintigraphy remains the gold standard technique for diagnosing neutrophil-mediated processes. It seems to be that combined in vitro labelled leucocyte/bone marrow scintigraphy (LS/BMS), with an accuracy of about 90%, is currently the imaging modality of choice for diagnosing PJI. There are, however, significant limitations using in vitro labelled leucocytes and considerable effort has been devoted to developing alternative radiotracers, such as radiolabelled HIGs, liposomes, antigranulocyte antibodies and fragments, as well as more investigational tracers such as radiolabelled antibiotics, antimicrobial peptides, bacteriophages and thymidine kinase. On the other hand, positron emission tomography (PET) is still growing in the field of PJI imaging with radiotracers such as (18)F-fluorodeoxyglucose (FDG), (18)F-FDG white blood cells and (18)F-fluoride. But unfortunately this superb tomographic technique will only receive full acceptance when specific PET uptake patterns can be successfully developed. The emergence of hybrid modality imaging using integrated single photon emission computed tomography (SPECT) and PET with computed tomography (SPECT/CT and PET/CT) may also have a contributing role for more accurate assessment of joint replacement complications, especially combined with new radiotracers such as (68)Ga and (64)Cu. Finally, in searching for infection-specific tracers, currently there is no such diagnostic agent available.

Evaluation of 99mTc-UBI 29-41 scintigraphy for specific detection of experimental multidrug-resistant Staphylococcus aureus bacterial endocarditis.

AIM: The aim of this paper was to test the ability of technetium-99m labelled synthetic peptide UBI 29-41 scintigraphy (99mTc-UBI 29-41), composed of the antimicrobial peptide ubiquicidin, specifically targets microorganisms in to discriminate between infected and uninfected endocarditis using a rat model previously validated. METHODS: 99mTc-UBI 29-41 scintigraphy was evaluated for its accumulation in infective endocarditis (IE) with multidrug resistant Staphylococcus aureus (MRSA) performed in an experimental rat model, resembling early endocarditis in humans. Serial planar scintigraphic and biodistribution analysis of infected vegetations are compared to rats with sterile vegetations. Heart-to-lung uptake ratios (T/NT ratios) were calculated in both with in-vivo scintigraphy and in ex vivo tissue samples. RESULTS: Bacterially infected vegetations were already observed at 15 min after injection of 99mTc-UBI 29-41 while no significant uptake was observed in sterile vegetations. Moreover, a good correlation (R2=0.819) was calculated between T/NT ratios of 99mTc-UBI 29-41 and the number of viable MRSA present in the infected vegetation. There was no correlation between the 99mTc-UBI 29-41 uptake and the weight of the vegetations in either case. CONCLUSION: In this experimental study in rats, planar 99mTc-UBI 29-41 scintigraphy permitted early and specific detection of MRSA induced endocarditis. Furthermore, accumulation of the tracer depends on the number of viable MRSA and not on the weight of the vegetation. This proof of principle offers much promise that 99mTc-UBI 29-41 scintigraphy can be a dedicated non-invasive imaging tool for the early detection of infective endocarditis. Finally, this model has to be further evaluated with state-of-the art small imaging modalities.

Genetic diversity of methicillin-resistant Staphylococcus aureus in a tertiary hospital in The Netherlands between 2002 and 2006.

The aim of this study was to investigate the methicillin-resistant Staphylococcus aureus (MRSA) clones isolated in a Dutch university hospital, situated near the borders of Belgium and Germany, between 2002 and 2006. MRSA strains (n = 175) were characterized using spa and SCCmec typing. The presence of Panton Valentine leukocidin (PVL) was determined. Between 2002 and 2005, ST5-MRSA-IV was predominant, and the spa type of ST5-MRSA-IV changed from t002 to t447. ST5-MRSA-I, ST5-MRSA-II, ST228-MRSA-I, and ST247-MRSA-I were also observed in this period. From 2004, the MRSA genetic background became more diverse, and in 2006, ST5-MRSA-IV was only sporadically observed. From 2005, ST5-MRSA-II, ST8-MRSA-IV, ST22-MRSA-IV, and ST45-MRSA-IV were increasingly observed. Several other MRSA clones, such as ST239-MRSA-III, were found sporadically. Four PVL-positive MRSA isolates were observed, associated with ST80-MRSA-IV and ST8-MRSA-IV. ST5-MRSA-I, ST5-MRSA-II, ST5-MRSA-IV, and ST228-MRSA-I have not been described previously in The Netherlands.

The use of technetium-99m radiolabeled human antimicrobial peptides for infection specific imaging.

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.

Can FDG-PET be used to predict growth of stage I lung cancer?

AIM: To determine the relationship between the metabolic activity measured by 2-[(18)F]-fluoro-2-deoxy-d-glucose (FDG) positron emission tomography (PET) and computed tomography (CT)-derived tumour growth rates for stage 1 lung cancer. METHODS: Stage I lung cancer patients at our institution who underwent FDG PET, and who had at least two pre-treatment chest CT examinations (n=51), were retrospectively identified. Metabolic activity was defined by maximum lesion standardized uptake value (SUV) and maximum lesion-to-mean background activity (LBR). Growth rates were determined from serial CT volume measurements and the doubling time (DT) was calculated. Tumour growth rates were divided into rapid (DT<180 days), intermediate (DT=180-270 days), and slow (DT>270 days) groups. RESULTS: Rapid, moderate, and slow DT were seen in 22, 19, and 10 patients, respectively. Means (standard deviations) of SUV in the three groups (from rapid to slow growth rate) were 8.2 (4.8), 5.5 (4.5), and 2.2 (1.1), respectively and of LBR were 22.7 (10.1), 15.1 (12.6), and 6 (2.6), respectively. There was a significant relationship between SUV and DT (p<0.05), as well as between LBR and DT (p<0.05). CONCLUSIONS: For stage I lung tumours, there is a significant relationship between growth rates, as measured by serial CT examinations, and the initial pre-treatment metabolic activities, as measured by FDG uptake. This suggests that in patients in whom it is difficult to decide on the aggressiveness on treatment, FDG-PET may be used as additional prognostic tool for determining management.

Detection of fungal infections using radiolabeled antifungal agents.

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 ((99m)Tc) and their in vivo distribution is monitored in infected mice. It has been demonstrated that (99m)Tc-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. (99m)Tc-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. albicans-infected mice could be successfully monitored using (99m)Tc-ubiquicidin. In conclusion, neither (99m)Tc-fluconazole nor the (99m)Tc-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 (99m)Tc-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.

Outcome of intensive immunosuppression and autologous stem cell transplantation in patients with severe rheumatoid arthritis is associated with the composition of synovial T cell infiltration.

OBJECTIVE: To determine clinical and immunological correlates of high dose chemotherapy (HDC) + autologous stem cell transplantation (ASCT) in patients with severe rheumatoid arthritis (RA), refractory to conventional treatment. METHODS: Serial samples of peripheral blood and synovial tissue were obtained from seven patients with RA treated with HDC and autologous peripheral blood grafts enriched for CD34+ cells. Disease activity was assessed with the Disease Activity Score (DAS), serum concentrations of C reactive protein (CRP), and human immunoglobulin (HIg) scans, and the extent of immunoablation was determined by immunophenotyping of peripheral blood mononuclear cells, and immunohistochemistry and double immunofluorescence of synovium. RESULTS: Clinical responders (n = 5) had a larger number of cells at baseline expressing CD3, CD4, CD27, CD45RA, CD45RB, and CD45RO in synovium (p < 0.05), higher activity on HIg scans (p = 0.08), and a trend towards higher concentrations of CRP in serum than non-responders (n = 2). Subsequent remissions and relapses in responders paralleled reduction and re-expression, respectively, of T cell markers. A relatively increased expression of CD45RB and CD45RO on synovial CD3+ T cells was seen after HDC + ASCT. No correlations were found between DAS and changes in B cells or macrophage infiltration or synoviocytes. CONCLUSIONS: HDC + ASCT results in profound but incomplete immunoablation of both the memory and naïve T cell compartment, which is associated with longlasting clinical responses in most patients. The findings provide strong circumstantial evidence for a role of T cells in established RA, and demonstrate a role for the synovium in post-transplantation T cell reconstitution.

Extremal dynamics and the approach to the critical state: experiments on a three dimensional pile of rice.

The evolution of the growth of a ricepile is studied in three dimensions. With time, the pile approaches a critical state with a certain slope. Assuming extremal dynamics in the evolution of the pile, the way the critical state is approached is dictated by the scaling properties of the critical state itself. Experimentally, we determine the envelope of the maximal slope, which is a measure for the distance from the critical state, as well as the growth of the average avalanche size with time. These quantities obey power-law scaling, where the experimental exponents are in good agreement with those obtained from an earlier determination of the critical state properties and extremal dynamics. Furthermore, we discuss the influence of the transient state on the avalanche size distribution, which may have applications in the prevention of large avalanches in natural systems.

99mTc-antimicrobial peptides: promising candidates for infection imaging.

This review presents the state of the art of imaging of bacterial and fungal infections in laboratory animals using antimicrobial peptides labelled with technetium-99m ((99m)Tc). The mechanistic basis of this approach is that these peptides accumulate at sites of infection, but not in sterile inflammatory lesions, because of their preferential binding to bacteria and fungi over mammalian cells. For practical reasons, such as production of large amounts of peptides under good laboratory practice conditions and favourable pharmacokinetics, synthetic peptides representing such binding domains of natural antimicrobial peptides are preferred. On the basis of their preferential in vitro and in vivo binding to microorganisms over human cells, fast and easy penetration into the target area, and rapid clearance from the circulation via the urinary tract, various (99m)Tc-antimicrobial peptides were identified. Next, it was determined whether these radiopharmaceuticals distinguish infectious foci from sites of sterile inflammation. Further experiments with (99m)Tc-ubiquicidin-derived peptides in infected laboratory animals have revealed that the radioactivity at the infectious site correlated well with the number of viable bacteria present, indicating that these (99m)Tc-labelled peptides may enable the monitoring of the efficacy of antimicrobial therapy. Together, (99m)Tc-labelled synthetic peptides derived from human ubiquicidin are promising candidates for imaging of bacterial and fungal infections in nuclear medicine.

Improved radioiodination of biomolecules using exhaustive Chloramine-T oxidation.

To improve standardization in analytical reagents we investigated Chloramine-T radioiodination (125I) of several biomolecules based on the use of a single amount of the oxidizing agent Chloramine-T as the limiting reagent being exhausted during the course of the reaction. Whenever the labeling yield resulted in less than one atom 125I/molecule, a second amount of the oxidizing agent was added. Thereafter, the integrity of the various biomolecules was assessed using radioimmunoassays, radioreceptor binding assays, or radioimmunometric assays. Purification yields were done by gel permeation (56% +/- 19%, n=230) or by precipitation with trichloroacetic acid (59% +/- 19%, n=230). Specific activity (117 +/- 61 MBq/nmol) and the degree of iodine incorporation (1.4 +/- 0.8 atoms of 125I/molecule) were achieved after 300 sec of incubation. A second addition of Chloramine-T resulted in an increased labeling yield of all biomolecules tested by a mean factor of 1.8 +/- 0.9. After the second addition of Chloramine-T, we observed for some biomolecules a significant (p<0.001) decreased effect in biological performance. In conclusion, the use of Chloramine-T as a limiting reagent resulted in molecules with appropriate immunological and biological performance. In general, tracers were minimally damaged and assessment of the shelf life as well as storing conditions showed the usefulness of the standardization of biomolecule labeling.

99mTc-labeled antimicrobial peptides for detection of bacterial and Candida albicans infections.

UNLABELLED: This study compared the possibilities and limitations of 99mTc-labeled synthetic peptides derived from two human antimicrobial peptides, namely, ubiquicidin (UBI) and lactoferrin (hLF), for the scintigraphic detection of bacterial and fungal infections in mice and rabbits. The rationale of our approach was that selected peptides accumulate in infected areas but not in sterile inflammatory lesions, because they bind preferentially to microorganisms. 99mTc-labeled human neutrophil peptides (defensins), ciprofloxacin, and human polyclonal IgG were included as control agents. METHODS: 99mTc-labeled peptides and control agents were injected intravenously into animals that had been injected intramuscularly 18 h earlier with multidrug-resistant Staphylococcus aureus, Klebsiella pneumoniae, or fluconazole-resistant Candida albicans. Sterile inflammatory sites were induced by the injection of heat-killed microorganisms or lipopolysaccharide (LPS) into the thigh muscle. Up to 4 h after injection, the accumulation of 99mTc-labeled compounds in the infected/inflamed thigh muscles was determined using scintigraphic techniques and radioactivity counts in dissected tissues. RESULTS: Scintigraphy revealed that 99mTc-labeled peptides UBI 29-41, UBI 18-35, UBI 31-38, hLF 1-11, and defensins, which showed preferential in vitro binding to microorganisms in a former study, accumulated at a significantly higher rate (P < 0.01) in bacterial and C. albicans infections in mice and rabbits than in inflamed tissues induced by heat-killed microorganisms or by LPS. No significant difference in the accumulation of 99mTc-labeled ciprofloxacin was observed between infected and sterile inflamed thigh muscles in mice. CONCLUSION: 99mTc-labeled antimicrobial peptides UBI 29-41, UBI 18-35, UBI 31-38, hLF 1-11, and defensins accumulate significantly in tissues infected with gram-positive and gram-negative bacteria and C. albicans. Significantly lower (P < 0.01) accumulation of these peptides occurs in sterile inflamed tissues. These data indicate that the peptides preferentially tag microorganisms at the site of infection, which is in agreement with their preferential binding to the microorganisms in vitro and in vivo. 99mTc-labeled ciprofloxacin does not distinguish between infections and sterile inflammatory lesions, which implies that its specificity for the detection of bacterial infections is not warranted.

A constrained EM algorithm for independent component analysis.

We introduce a novel way of performing independent component analysis using a constrained version of the expectation-maximization (EM) algorithm. The source distributions are modeled as D one-dimensional mixtures of gaussians. The observed data are modeled as linear mixtures of the sources with additive, isotropic noise. This generative model is fit to the data using constrained EM. The simpler "soft-switching" approach is introduced, which uses only one parameter to decide on the sub- or supergaussian nature of the sources. We explain how our approach relates to independent factor analysis.