Synthesis and preclinical evaluation of novel 18F-vancomycin-based tracers for the detection of bacterial infections using positron emission tomography.

INTRODUCTION: Bacterial infections are a major problem in medicine, and the rapid and accurate detection of such infections is essential for optimal patient outcome. Bacterial infections can be diagnosed by nuclear imaging, but most currently available modalities are unable to discriminate infection from sterile inflammation. Bacteria-targeted positron emission tomography (PET) tracers have the potential to overcome this hurdle. In the present study, we compared three 18F-labelled PET tracers based on the clinically applied antibiotic vancomycin for targeted imaging of Gram-positive bacteria. METHODS: [18F]FB-NHS and [18F]BODIPY-FL-NHS were conjugated to vancomycin. The resulting conjugates, together with our previously developed [18F]PQ-VE1-vancomycin, were tested for stability, lipophilicity, selective binding to Gram-positive bacteria, antimicrobial activity and biodistribution. For the first time, the pharmacokinetic properties of all three tracers were compared in healthy animals to identify potential binding sites. RESULTS: [18F]FB-vancomycin, [18F]BODIPY-FL-vancomycin, and [18F]PQ-VE1-vancomycin were successfully synthesized with radiochemical yields of 11.7%, 2.6%, and 0.8%, respectively. [18F]FB-vancomycin exhibited poor in vitro and in vivo stability and, accordingly, no bacterial binding. In contrast, [18F]BODIPY-FL-vancomycin and [18F]PQ-VE1-vancomycin showed strong and specific binding to Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA), which was outcompeted by unlabeled vancomycin only at concentrations exceeding clinically relevant vancomycin blood levels. Biodistribution showed renal clearance of [18F]PQ-VE1-vancomycin and [18F]BODIPY-FL-vancomycin with low non-specific accumulation in muscles, fat and bones. CONCLUSION: Here we present the synthesis and first evaluation of the vancomycin-based PET tracers [18F]BODIPY-FL-vancomycin and [18F]PQ-VE1-vancomycin for image-guided detection of Gram-positive bacteria. Our study paves the way towards real-time bacteria-targeted diagnosis of soft tissue and implant-associated infections that are oftentimes caused by Gram-positive bacteria, even after prophylactic treatment with vancomycin.

Scavenger receptor-like receptors for the binding of lipopolysaccharide and lipoteichoic acid to liver endothelial and Kupffer cells.

This study was undertaken to identify the role of scavenger receptors in the catabolism of lipopolysaccharide (LPS) and lipoteichoic acid (LTA). LPS is mainly cleared from the blood by the liver. The Kupffer cells are primarily responsible for this clearance. Although several binding sites have been described for LPS and LTA, only CD14 is involved in LPS signalling. Scavenger receptor type A (SR-A) is expressed in the liver on endothelial cells and Kupffer cells, and macrosialin (class D scavenger receptor) is expressed on Kupffer cells. Fucoidin and poly-I are both good inhibitors of scavenger receptors. Fucoidin significantly reduced the serum clearance of [125I]-LPS and decreased liver uptake of [125I]-LPS by approximately 40%. Poly-I inhibited the binding of [125I]-LPS to isolated Kupffer and endothelial cells by 75%, while poly-A, a polyanionic substrate that does not block scavenger receptors, had no effect. LPS significantly inhibited the binding of acetylated LDL and oxidized LDL (two well-described scavenger receptor ligands) to isolated Kupffer and liver endothelial cells. OxLDL and acLDL did not affect the binding of LPS to these cells. We conclude that on both endothelial cells and Kupffer cells, LPS mainly binds to scavenger receptors, but SR-A and macrosialin contribute to a limited extent to the binding of LPS. Injection of LTA into C57Bl6 mice resulted in a maximal liver uptake of 20% of the injected dose. In the liver, 50% was bound by the Kupffer cells, 20% by parenchymal cells and 30% by liver endothelial cells. The contribution of SR-A to the plasma clearance of LTA was limited. A main component in the catabolism of LTA is the interaction of LTA with plasma lipoproteins, which limit the uptake of LTA by tissues and extend the plasma half-life of LTA.

An efficient procedure for the generation of human monoclonal antibodies based on activation of human B lymphocytes by a murine thymoma cell line.

A new, efficient procedure for the generation of human monoclonal antibodies has been developed. The procedure is based on the activation of human B cells in microwells by murine thymoma EL4B5 cells. This mode of B cell stimulation leads to proliferation of at least one per eight of human B cells and to a high rate of antibody production. Subsequently, supernatants of the microwells are screened by ELISA for the presence of antibody of the desired specificity and B cells from selected wells are hybridized by electroporation. To optimize the procedure, the kinetics of the B cell expansion induced by EL4B5 cells were analysed. Counting and phenotyping of cultured cells at different time points indicated that the peak of B cell expansion occurred at day 5 for tonsil B cells (16-fold increase) and at day 7 for peripheral blood B cells (20-fold increase). The B cells did not merely proliferate but also differentiated, as indicated by loss of CD20 expression and increase of CD38 expression. At the peak of B cell expansion, B cells could be hybridized efficiently with myeloma cells. The majority of the resultant hybridomas secreted human immunoglobulin. The efficiency of the procedure is exemplified by the generation of hybridomas secreting human IgG against Haemophilus influenzae from limited numbers of either human tonsil B lymphocytes or peripheral blood B lymphocytes.

The influence of cytokines on the integrity of the blood-brain barrier in vitro.

The effects of the cytokines tumour necrosis factor-alpha (TNF-alpha), interleukin (IL)-1 beta and IL-6 on the permeability of monolayers of rat cerebral endothelial cells (RCEC) were investigated to assess potential changes in the integrity of the blood-brain barrier (BBB). RCEC were cultured to tight monolayers with a trans endothelial electrical resistance (TEER) of 100-150 ohm . cm2 on polycarbonate filters. Exposure of the RCEC to TNF-alpha, IL-1 beta and IL-6 induced a decline in the TEER, which could be completely abolished by 1 muM of indomethacin, a cyclooxygenase inhibitor. In addition, the effect of IL-1 beta on TEER across monolayers of RCEC could be completely inhibited by IL-1 receptor antagonist. In conclusion, cytokines induce a disruption of the BBB in vitro. In this process, cyclooxygenase activation within the endothelial cells seems to play a key role.

Metronidazole concentrations in human plasma, saliva, and gingival crevice fluid after a single dose.

Metronidazole concentrations were estimated in four human volunteers after a single dose of 750 mg taken orally. Samples of blood, saliva, and gingival crevice fluid were collected before intake and during the following 24 hours. The concentrations of metronidazole in plasma and saliva were measured by high-performance liquid chromatography (HPLC). The concentrations in gingival fluid were estimated by a capillary agar-diffusion assay. The results of the metronidazole measurements as obtained by both methods were significantly correlated. The peak concentrations of metronidazole in plasma and saliva were in the same range, 8.7-13.8 micrograms/mL, and similar concentrations were found in the gingival fluid samples. It is concluded that metronidazole taken orally has similar pharmacokinetics in both saliva and plasma, and that a single oral dose of 750 mg metronidazole leads to a concentration of the drug in the gingival crevice fluid that exceeds the minimal inhibitory concentration for most anaerobic oral micro-organisms.

The effect of amoxicillin on destructive periodontitis. A case report.

In a 22-year-old female patient, advanced localized periodontal destruction was observed. The planned treatment consisted of oral hygiene instructions, professional plaque control, deep scaling and root planning and finally modified Widman flap surgery. One molar had to be extracted but was left untreated initially as a control. During the treatment period of 9 months and during 1 year thereafter, samples were taken of the subgingival plaque for dark-field microscopy. The unplanned use of amoxicillin by the patient for a middle ear infection resulted in a suppression below detection level of spirochetes at the investigated sites. At the nontreated control site, the absence of spirochetes was accompanied by a 3-mm reduction of pocket depth and a 2-mm gain in clinical probing attachment, while some formation of new alveolar bone was observed. At the treated sites, clinical improvement was observed. However, a distinction between the effect of the periodontal therapy and the nonscheduled use of amoxicillin could not be made at the treated sites. It is concluded that a single course of systematically administered amoxicillin changed the composition of the subgingival microflora over a long period of time (17 months) and had a beneficial effect upon the status of the periodontium.

[Comparison of subgingival plaque buccally and interdentally localized during 3-week experimental gingivitis in humans]. / Vergleich subgingivaler Plaque an bukkalen und interdentalen Lokalisationen während einer dreiwöchigen experimentellen Gingivitis beim Menschen.

During 21 days of abolished oral hygiene and developing gingivitis the microbial changes were studied in buccal and interdental sites. The subgingival plaque of 6 dental students was analyzed by darkfield microscopy. The microbiota found in interdental sulci showed a higher complexity than that of buccal sites. Buccally and interdentally there was a significant increase in the total number of microorganisms between days 0 and 21. Yet, the development of gingivitis was slower in the buccal than in the interproximal sites. For all bacteria, but specially for more disease-associated morphotypes the accumulation rate was higher interdentally than buccally.

[Microbiological findings of subgingival plaque during long-term experimental gingivitis in humans]. / Mikrobiologische Befunde in subgingivaler Plaque während einer experimentellen Langzeitgingivitis beim Menschen.

During six month of abolished oral hygiene and concomitant development of gingivitis, the buccal subgingival microbiota was studied by darkfield microscopy as well as by cultural methods. Five dental students gave written informed consent and participated in this trial. In darkfield microscopy there was a slow reduction in the proportion of coccoid forms concomitant with an increase in the proportion of rods, while spirochetes were rarely detected during the entire experimental period. However, the cultural data revealed a decrease of the Gram-positive facultative and an increase of the Gram-negative anaerobic microorganisms after 6 months of abolished oral hygiene. The bacteriological data show, that in buccal sites--even after 6 months of abolished oral hygiene--the subgingival microflora reflects a population typical only for an initial lesion.

New scavenger receptor-like receptors for the binding of lipopolysaccharide to liver endothelial and Kupffer cells.

Lipopolysaccharide (LPS) is cleared from the blood mainly by the liver. The Kupffer cells are primarily responsible for this clearance; liver endothelial and parenchymal cells contribute to a lesser extent. Although several binding sites have been described, only CD14 is known to be involved in LPS signalling. Among the other LPS binding sites that have been identified are scavenger receptors. Scavenger receptor class A (SR-A) types I and II are expressed in the liver on endothelial cells and Kupffer cells, and a 95-kDa receptor, identified as macrosialin, is expressed on Kupffer cells. In this study, we examined the role of scavenger receptors in the binding of LPS by the liver in vivo and in vitro. Fucoidin, a scavenger receptor ligand, significantly reduced the clearance of 125I-LPS from the serum and decreased the liver uptake of 125I-LPS about 40%. Within the liver, the in vivo binding of 125I-LPS to Kupffer and liver endothelial cells was decreased 72 and 71%, respectively, while the binding of 125I-LPS to liver parenchymal cells increased 34% upon fucoidin preinjection. Poly(I) inhibited the binding of 125I-LPS to Kupffer and endothelial cells in vitro 73 and 78%, respectively, while poly(A) had no effect. LPS inhibited the binding of acetylated low-density lipoprotein (acLDL) to Kupffer and liver endothelial cells 40 and 55%, respectively, and the binding of oxidized LDL (oxLDL) to Kupffer and liver endothelial cells 65 and 61%, respectively. oxLDL and acLDL did not significantly inhibit the binding of LPS to these cells. We conclude that on both endothelial cells and Kupffer cells, LPS binds mainly to scavenger receptors, but SR-A and macrosialin contribute to a limited extent to the binding of LPS.

Gingival health and gingivitis development during puberty. A 4-year longitudinal study.

The purpose of this investigation was to follow the development of the gingival conditions during puberty and to correlate oral clinical parameters with chronological age as well as with parameters used for the determination of the pubertal development. In 22 boys and 20 girls pubertal and skeletal development, as well as plaque index (PlI) and gingival index (GI) were monitored at 1-year intervals between the ages of 11 and 15 years. During this time, the papillary bleeding index (PBI) was assessed 10 times in all interdental spaces of the dentition. The bleeding tendency, represented by whole mouth mean PBI values, as well as the % of bleeding interdental sites, was found to increase significantly with the start of the pubertal phase. It reached a peak value after 1-5 years in 35% of the children. A significant trend of decrease was noted after the age of 14 years in boys and girls. In boys, mean PBI and the % of interdental sites with bleeding were correlated with testes growth, in girls with the Tanner index for secondary sex characteristics (breast development). PlI and GI, which were only recorded annually, did not show a significant trend of increase or decrease.

Microbial and clinical measurements of periodontal pockets during sequential periods of non-treatment, mechanical debridement and metronidazole therapy.

Microbial and clinical parameters were studied in 11 subjects with chronic inflammatory periodontitis. 2 periodontal pockets per subject were studied longitudinally. The microbial parameters included counts of different subgingival micro-organisms by dark field microscopy, counts of the total colony forming units (c.f.u.) on anaerobic blood agar, the facultative anaerobic counts and counts of black-pigmented Bacteroides, Fusobacterium and E. corrodens. The clinical parameters were probing pocket depth, bleeding after probing and crevicular fluid production. Clinical and microbial observations were compared during 3 consecutive periods of non-treatment, debridement and metronidazole therapy. The experimental sites were debrided by deep scaling while no debridement was carried out at the control sites. The effect of this treatment was studied over a period of 3 months. Then, at the experimental sites, a 2nd session of debridement was followed by administration of metronidazole. The effect of metronidazole alone and combined with mechanical debridement was studied during a subsequent 3-month period. The disease activity did not correlate with the microbial parameters and was evident in the presence as well as in the absence of black-pigmented Bacteroides. A single session of subgingival debridement resulted in significant reductions in probing depth, spirochetes, motile organisms, black-pigmented Bacteroides and E. corrodens. Repopulation of the subgingival sites was observed. However, the composition of the subgingival microbiota remained significantly changed during the 3 months after debridement. The re-isolation of the same Bacteroides-species and the same B. gingivalis type after treatment indicated an outgrowth of micro-organisms remaining at these sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Free radical scavenger mechanisms in experimentally induced ischemia in the rabbit heart and protective effect of verapamil.

The protective effect of verapamil on the free radical generation in the ischemic myocardium of rabbit has been studied. A significant decrease of the lactate dehydrogenase activity in the ischemic zone was observed compared to the nonischemic control myocardial tissue. The level of malondialdehyde was found to be elevated in the ischemic zone and in other parts of the myocardium. However, there was no alteration in glutathione content in both zones. In addition, an increase in the activity of myeloperoxidase was observed in the ischemic part of the myocardium. At lower doses (30 micrograms/kg), verapamil protected the animals from ischemic changes but did not do so at higher doses (100 micrograms/kg). These results suggest that, in the rabbit, the free radical scavenging mechanism of the heart is not adversely affected during ischemia.

Vascular adhesion molecule-1 and intercellular adhesion molecule-1 expression on rat liver cells after lipopolysaccharide administration in vivo.

During sepsis the infiltration of leukocytes plays a pivotal role in tissue damage. Induction of septic shock results in an early accumulation of polymorphonuclear leukocytes in the liver (after 3 hours), which is followed by an infiltration of mononuclear phagocytes (after 30 hours). Expression of adhesion molecules may contribute to the migration of leukocytes to the site of inflammation. Therefore, in the present study we determined the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1) on hepatocytes, liver endothelial cells, and Kupffer cells after lipopolysaccharide (LPS) treatment of rats in vivo. Parenchymal cells showed no constitutive expression of VCAM-1 and the expression could not be upregulated by LPS treatment in vivo, whereas Kupffer and endothelial cells had a low basal expression of VCAM-1 and this expression was increased 40-fold by LPS treatment in vivo. All three cell types showed a basal expression of ICAM-1 and the expression on endothelial liver cells of untreated rats was two times higher than the expression on parenchymal and Kupffer cells. Stimulation with LPS increased the expression of ICAM-1 2.5 times per parenchymal cells and approximately 4 times for endothelial and Kupffer cells. It is concluded that the expression of adhesion molecules may contribute to the influx of leukocytes during septic shock and, therefore, play a role in tissue damage during septic shock.

Respiratory syncytial virus, pneumonia virus of mice, and influenza A virus differently affect respiratory allergy in mice.

BACKGROUND: Respiratory viral infections in early childhood may interact with the immune system and modify allergen sensitization and/or allergic manifestations. In mice, respiratory syncytial virus (RSV) infection during allergic provocation aggravates the allergic T helper (Th) 2 immune response, characterized by the production of IL-4, IL-5, and IL-13, and inflammatory infiltrates. However, it is unclear whether the RSV-enhanced respiratory allergic response is a result of non-specific virus-induced damage of the lung, or virus-specific immune responses. OBJECTIVE: In the present study we investigated whether RSV, pneumonia virus of mice (PVM) and influenza A virus similarly affect the allergic response. METHODS: BALB/c mice were sensitized and challenged with ovalbumin (OVA), and inoculated with virus during the challenge period. Pulmonary inflammation, lung cytokine mRNA responses, and IgE production in serum were assessed after the last OVA-challenge. RESULTS: Like RSV, PVM enhanced the OVA-induced pulmonary IL-4, IL-5, and IL-13 mRNA expression, which was associated with enhanced perivascular inflammation. In addition, PVM increased the influx of eosinophils in lung tissue. In contrast, influenza virus decreased the Th2 cytokine mRNA expression in the lungs. However, like PVM, influenza virus enhanced the pulmonary eosinophilic infiltration in OVA-allergic mice. CONCLUSION: The Paramyxoviruses RSV and PVM both are able to enhance the allergic Th2 cytokine response and perivascular inflammation in BALB/c mice, while the Orthomyxovirus influenza A is not.

Timing of infection and prior immunization with respiratory syncytial virus (RSV) in RSV-enhanced allergic inflammation.

Respiratory syncytial virus (RSV) infection has been shown to be a risk factor for the development of allergy in humans and mice. The allergy-enhancing properties of RSV may be dependent on atopic background and an individual's history of RSV infection. We examined the influence of the timing of infection and prior inoculation with RSV in a mouse model of allergic asthma. Mice were sensitized to and challenged with ovalbumin (OVA) and were inoculated with RSV either before or during the sensitization or challenge period. One group of mice was inoculated with RSV both before sensitization to OVA and during challenge with OVA. Increased pulmonary expression of interleukin (IL)-4, IL-5, and IL-13 mRNA and aggravated alveolitis and hypertrophy of mucus-producing cells were observed only when OVA-sensitized mice were inoculated with RSV shortly before or during challenge with OVA. Despite protection against viral replication, prior inoculation with RSV did not abrogate RSV-enhanced, OVA-induced expression of T helper 2 (Th2) cytokines in the lung. In conclusion, inoculation with RSV enhances allergic disease only when the immune system has already been Th2-primed by the allergen (i.e., OVA). This RSV-enhanced allergy is not completely abrogated by prior inoculation with RSV.

Differential role of transcription-coupled repair in UVB-induced G2 arrest and apoptosis in mouse epidermis.

Nucleotide excision repair (NER), apoptosis, and cell-cycle regulation are major defense mechanisms against the carcinogenic effects of UVB light. NER eliminates UVB-induced DNA photolesions via two subpathways: global genome repair (GGR) and transcription-coupled repair (TCR). Defects in NER result in the human disorders xeroderma pigmentosum (XP) and Cockayne syndrome (CS), displaying severe UV sensitivity and in the case of XP, cancer proneness. We investigated the impact of deficiencies in NER subpathways on apoptosis, hyperplasia, and cell cycle progression in the epidermis of UVB-exposed CS group B (Csb(-/-)) mice (no TCR), XP group C (Xpc(-/-)) mice (no GGR), and XP group A (Xpa(-/-)) mice (no TCR and no GGR). On UVB treatment (250 J/m(2)), Xpa(-/-) and Csb(-/-) mice revealed an extensive apoptotic response in the skin, a blockage of cell cycle progression of epidermal cells, and strong hyperplasia. Interestingly, the absence of this apoptotic response in the skin of wild-type and Xpc(-/-) mice coincided with the ability of epidermal cells to enter the S phase. However, only epidermal cells of Xpc(-/-) mice subsequently became arrested in the G(2) phase. Our data demonstrate that TCR (and/or restoration of UVB-inhibited transcription) enables damaged cells to progress through S phase and prevents the induction of apoptosis and hyperplasia. G(2) arrest is manifest only under conditions of proficient TCR in combination with deficient GGR, indicating that epidermal cells become arrested in the G(2) phase as a result of persisting damage in their genome.

Apolipoprotein E protects against bacterial lipopolysaccharide-induced lethality. A new therapeutic approach to treat gram-negative sepsis.

Septic shock is the most common cause of death in intensive care units and no effective treatment is available at present. Lipopolysaccharide (LPS) is the primary mediator of Gram-negative sepsis by inducing the production of macrophage-derived cytokines. Previously, we showed that apolipoprotein E (apoE), an established modulator of lipid metabolism, can bind LPS, thereby redirecting LPS from macrophages to hepatocytes in vivo. We now report that intravenously administered LPS strongly increases the serum levels of apoE. In addition, apoE can prevent the LPS-induced production of cytokines and subsequent death in rodents. Finally, apoE-deficient mice show a significantly higher sensitivity toward LPS than control wild-type mice. These findings indicate that apoE may have a physiological role in the protection against sepsis, and recombinant apoE may be used therapeutically to protect against LPS-induced endotoxemia.