[99mTc]Tc-PentixaTec: development, extensive pre-clinical evaluation, and first human experience.

PURPOSE: The clinical success non-invasive imaging of CXCR4 expression using [68 Ga]Ga-PentixaFor-PET warrants an expansion of the targeting concept towards conventional scintigraphy/SPECT with their lower cost and general availability. To this aim, we developed and comparatively evaluated a series of 99mTc-labeled cyclic pentapeptides based on the PentixaFor scaffold. METHODS: Six mas3-conjugated CPCR4 analogs with different 4-aminobenzoic acid (Abz)-D-Ala-D-Arg-aa3 linkers (L1-L6) as well as the corresponding HYNIC- and N4-analogs of L6-CPCR4 were synthesized via standard SPPS. Competitive binding studies (IC50 and IC50inv) were carried out using Jurkat T cell lymphoma cells and [125I]FC-131 as radioligand. Internalization kinetics were investigated using hCXCR4-overexpressing Chem-1 cells. Biodistribution studies and small animal SPECT/CT imaging (1 h p.i.) were carried out using Jurkat xenograft bearing CB17/SCID mice. Based on the preclinical results, [99mTc]Tc-N4-L6-CPCR4 ([99mTc]Tc-PentixaTec) was selected for an early translation to the human setting. Five patients with hematologic malignancies underwent [99mTc]Tc-N4-L6-CPCR4 SPECT/planar imaging with individual dosimetry. RESULTS: Of the six mas3-conjugated peptides, mas3-L6-CPCR4 (mas3-dap-r-a-Abz-CPCR4) showed the highest CXCR4 affinity (IC50 = 5.0 ± 1.3 nM). Conjugation with N4 (N4-L6-CPCR4) further improved hCXCR4 affinity to 0.6 ± 0.1 nM. [99mTc]Tc-N4-L6-CPCR4 also showed the most efficient internalization (97% of total cellular activity at 2 h) and the highest tumor accumulation (8.6 ± 1.3% iD/g, 1 h p.i.) of the compounds investigated. Therefore, [99mTc]Tc-N4-L6-CPCR4 (termed [99mTc]Tc-PentixaTec) was selected for first-in-human application. [99mTc]Tc-PentixaTec was well tolerated, exhibits a favorable biodistribution and dosimetry profile (2.1-3.4 mSv per 500 MBq) and excellent tumor/background ratios in SPECT and planar imaging. CONCLUSION: The successive optimization of the amino acid composition of the linker structure and the N-terminal 99mTc-labeling strategies (mas3 vs HYNIC vs N4) has provided [99mTc]Tc-PentixaTec as a novel, highly promising CXCR4-targeted SPECT agent for clinical application. With its excellent CXCR4 affinity, efficient internalization, high uptake in CXCR4-expressing tissues, suitable clearance/biodistribution characteristics, and favorable human dosimetry, it holds great potential for further clinical use.

Ants avoid superinfections by performing risk-adjusted sanitary care.

Being cared for when sick is a benefit of sociality that can reduce disease and improve survival of group members. However, individuals providing care risk contracting infectious diseases themselves. If they contract a low pathogen dose, they may develop low-level infections that do not cause disease but still affect host immunity by either decreasing or increasing the host's vulnerability to subsequent infections. Caring for contagious individuals can thus significantly alter the future disease susceptibility of caregivers. Using ants and their fungal pathogens as a model system, we tested if the altered disease susceptibility of experienced caregivers, in turn, affects their expression of sanitary care behavior. We found that low-level infections contracted during sanitary care had protective or neutral effects on secondary exposure to the same (homologous) pathogen but consistently caused high mortality on superinfection with a different (heterologous) pathogen. In response to this risk, the ants selectively adjusted the expression of their sanitary care. Specifically, the ants performed less grooming and more antimicrobial disinfection when caring for nestmates contaminated with heterologous pathogens compared with homologous ones. By modulating the components of sanitary care in this way the ants acquired less infectious particles of the heterologous pathogens, resulting in reduced superinfection. The performance of risk-adjusted sanitary care reveals the remarkable capacity of ants to react to changes in their disease susceptibility, according to their own infection history and to flexibly adjust collective care to individual risk.

Anti-pathogen protection versus survival costs mediated by an ectosymbiont in an ant host.

The fitness effects of symbionts on their hosts can be context-dependent, with usually benign symbionts causing detrimental effects when their hosts are stressed, or typically parasitic symbionts providing protection towards their hosts (e.g. against pathogen infection). Here, we studied the novel association between the invasive garden ant Lasius neglectus and its fungal ectosymbiont Laboulbenia formicarum for potential costs and benefits. We tested ants with different Laboulbenia levels for their survival and immunity under resource limitation and exposure to the obligate killing entomopathogen Metarhizium brunneum. While survival of L. neglectus workers under starvation was significantly decreased with increasing Laboulbenia levels, host survival under Metarhizium exposure increased with higher levels of the ectosymbiont, suggesting a symbiont-mediated anti-pathogen protection, which seems to be driven mechanistically by both improved sanitary behaviours and an upregulated immune system. Ants with high Laboulbenia levels showed significantly longer self-grooming and elevated expression of immune genes relevant for wound repair and antifungal responses (ß-1,3-glucan binding protein, Prophenoloxidase), compared with ants carrying low Laboulbenia levels. This suggests that the ectosymbiont Laboulbenia formicarum weakens its ant host by either direct resource exploitation or the costs of an upregulated behavioural and immunological response, which, however, provides a prophylactic protection upon later exposure to pathogens.

Social transfer of pathogenic fungus promotes active immunisation in ant colonies.

Due to the omnipresent risk of epidemics, insect societies have evolved sophisticated disease defences at the individual and colony level. An intriguing yet little understood phenomenon is that social contact to pathogen-exposed individuals reduces susceptibility of previously naive nestmates to this pathogen. We tested whether such social immunisation in Lasius ants against the entomopathogenic fungus Metarhizium anisopliae is based on active upregulation of the immune system of nestmates following contact to an infectious individual or passive protection via transfer of immune effectors among group members--that is, active versus passive immunisation. We found no evidence for involvement of passive immunisation via transfer of antimicrobials among colony members. Instead, intensive allogrooming behaviour between naive and pathogen-exposed ants before fungal conidia firmly attached to their cuticle suggested passage of the pathogen from the exposed individuals to their nestmates. By tracing fluorescence-labelled conidia we indeed detected frequent pathogen transfer to the nestmates, where they caused low-level infections as revealed by growth of small numbers of fungal colony forming units from their dissected body content. These infections rarely led to death, but instead promoted an enhanced ability to inhibit fungal growth and an active upregulation of immune genes involved in antifungal defences (defensin and prophenoloxidase, PPO). Contrarily, there was no upregulation of the gene cathepsin L, which is associated with antibacterial and antiviral defences, and we found no increased antibacterial activity of nestmates of fungus-exposed ants. This indicates that social immunisation after fungal exposure is specific, similar to recent findings for individual-level immune priming in invertebrates. Epidemiological modeling further suggests that active social immunisation is adaptive, as it leads to faster elimination of the disease and lower death rates than passive immunisation. Interestingly, humans have also utilised the protective effect of low-level infections to fight smallpox by intentional transfer of low pathogen doses ("variolation" or "inoculation").

Two pathways ensuring social harmony.

Reproductive division of labour is a characteristic trait of social insects. The dominant reproductive individual, often the queen, uses chemical communication and/or behaviour to maintain her social status. Queens of many social insects communicate their fertility status via cuticle-bound substances. As these substances usually possess a low volatility, their range in queen-worker communication is potentially limited. Here, we investigate the range and impact of behavioural and chemical queen signals on workers of the ant Temnothorax longispinosus. We compared the behaviour and ovary development of workers subjected to three different treatments: workers with direct chemical and physical contact to the queen, those solely under the influence of volatile queen substances and those entirely separated from the queen. In addition to short-ranged queen signals preventing ovary development in workers, we discovered a novel secondary pathway influencing worker behaviour. Workers with no physical contact to the queen, but exposed to volatile substances, started to develop their ovaries, but did not change their behaviour compared to workers in direct contact to the queen. In contrast, workers in queen-separated groups showed both increased ovary development and aggressive dominance interactions. We conclude that T. longispinosus queens influence worker ovary development and behaviour via two independent signals, both ensuring social harmony within the colony.

Optimization of the Pharmacokinetic Profile of [99mTc]Tc-N4-Bombesin Derivatives by Modification of the Pharmacophoric Gln-Trp Sequence.

Current radiolabeled gastrin-releasing peptide receptor (GRPR) ligands usually suffer from high accumulation in GRPR-positive organs (pancreas, stomach), limiting tumor-to-background contrast in the abdomen. In novel N4-bombesin derivatives this was addressed by substitutions at the Gln7-Trp8 site within the MJ9 peptide (H-Pip5-phe6-Gln7-Trp8-Ala9-Val10-Gly11-His12-Sta13-Leu14-NH2) either by homoserine (Hse7), ß-(3-benzothienyl) alanine (Bta8) or α-methyl tryptophan (α-Me-Trp8), with the aim of optimizing pharmacokinetics. We prepared and characterized the peptide conjugates 6-carboxy-1,4,8,11-tetraazaundecane (N4)-asp-MJ9, N4-asp-[Bta8]MJ9, N4-[Hse7]MJ9 and N4-[α-Me-Trp8]MJ9, and evaluated these compounds in vitro (GRPR affinity via IC50,inverse; internalization; lipophilicity via logD7.4) and in vivo (biodistribution and µSPECT/CT studies at 1 h post injection (p.i.) in PC-3 tumor-bearing CB17-SCID mice). 99mTc-labeling resulted in radiochemical yields (RCYs) > 95%. All 99mTc-labeled MJ9 analogues showed comparable or higher GRPR affinity than the external reference [99mTc]Tc-Demobesin 4. Receptor-bound fractions were noticeably higher than that of the reference. Despite a slightly enhanced lipophilicity, all novel MJ9 derivatives revealed improved in vivo pharmacokinetics compared to the reference. The Bta8-modified ligand revealed the most favorable tumor-to-abdomen contrast at 1 h p.i. Substitutions at the Gln7-Trp8 site within GRPR ligands hold great potential to modify pharmacokinetics for improved imaging.

CXCR4 peptide-based fluorescence endoscopy in a mouse model of Barrett's esophagus.

BACKGROUND: Near-infrared (NIR) fluorescence imaging has been emerging as a promising strategy to overcome the high number of early esophageal adenocarcinomas missed by white light endoscopy and random biopsy collection. We performed a preclinical assessment of fluorescence imaging and endoscopy using a novel CXCR4-targeted fluorescent peptide ligand in the L2-IL1B mouse model of Barrett's esophagus. METHODS: Six L2-IL1B mice with advanced stage of disease (12-16 months old) were injected with the CXCR4-targeted, Sulfo-Cy5-labeled peptide (MK007), and ex vivo wide-field imaging of the whole stomach was performed 4 h after injection. Before ex vivo imaging, fluorescence endoscopy was performed in three L2-IL1B mice (12-14 months old)  by a novel imaging system with two L2-IL1B mice used as negative controls. RESULTS: Ex vivo imaging and endoscopy in L2-IL1B mice showed that the CXCR4-targeted MK007 accumulated mostly in the dysplastic lesions with a mean target-to-background ratio > 2. The detection of the Sulfo-Cy5 signal in dysplastic lesions and its co-localization with CXCR4 stained cells  by confocal microscopy further confirmed the imaging results. CONCLUSIONS: This preliminary preclinical study shows that CXCR4-targeted fluorescence endoscopy using MK007 can detect dysplastic lesions in a mouse model of Barrett's esophagus. Further investigations are needed to assess its use in the clinical setting.

Preclinical comparison of four [18F, natGa]rhPSMA-7 isomers: influence of the stereoconfiguration on pharmacokinetics.

INTRODUCTION: Radiohybrid (rh) ligands, a novel class of prostate-specific membrane antigen (PSMA)-targeted radiopharmaceuticals, can be labeled either with [18F]fluorine via isotopic exchange or with radiometals (such as [68Ga]Gallium, [177Lu]Lutetium, [225Ac]Actinium). Among these, [18F, natGa]rhPSMA-7 has recently entered clinical assessment. AIM: Since [18F, natGa]rhPSMA-7 is composed of four stereoisomers ([18F, natGa]rhPSMA-7.1, -7.2, -7.3 and -7.4), we initiated a preclinical selection process to identify the isomer with the most favorable pharmacokinetics for further clinical investigation. METHODS: A synthetic protocol for enantiopure [19F, natGa]rhPSMA-7 isomers has been developed. The comparative evaluation of the four isomers comprised human serum albumin binding, lipophilicity, IC50, internalization and classical biodistribution studies and competition experiments in LNCaP tumor-bearing CB-17 SCID mice. In addition, a radio high-performance liquid chromatography-based method was developed allowing quantitative, intraindividual comparison of [18F, natGa]rhPSMA-7.1 to -7.4 in LNCaP tumor-bearing mice. RESULTS: Cell studies revealed high PSMA affinity and internalization for [18/19F, natGa]rhPSMA-7.2, -7.3 and -7.4, whereas [18/19F, natGa]rhPSMA-7.1 showed approximately twofold lower values. Although the biodistribution profile obtained was typical of PSMA inhibitors, it did not allow for selection of a lead candidate for clinical studies. Thus, an intraindividual comparison of all four isomers in LNCaP tumor-bearing mice was carried out by injection of a diastereomeric mixture, followed by analysis of the differential uptake and excretion pattern of each isomer. Based on its high tumor accumulation and low uptake in blood, liver and kidneys, [18F, natGa]rhPSMA-7.3 was identified as the preferred isomer and transferred into clinical studies. CONCLUSION: [18F, natGa]rhPSMA-7.3 has been selected as a lead compound for clinical development of a [18F]rhPSMA-based candidate. The intraindividual differential uptake and excretion analysis in vivo allowed for an accurate comparison and assessment of radiopharmaceuticals.

Coordination and Conversion of Urea at Dinuclear &mgr;-Acetato Nickel(II) Complexes with Symmetric and Asymmetric Cores.

A series of symmetric and asymmetric pyrazolate-based dinuclear Ni(II) complexes relevant to the active site of urease is reported, which have acetate ions as secondary bridges and which feature variations in the type (N or S) and number of donor sites provided within the individual coordination compartments of the primary pyrazolate ligand matrixes. X-ray crystallographic structures of the acetone adduct [L(1)Ni(2)(&mgr;-OAc)(acetone)(2)](ClO(4))(2) (1) as well as of the urea adducts [L(1)Ni(2)(&mgr;-OAc)(benzylurea)(2)](ClO(4))(2) (2c), [L(2)Ni(2)(&mgr;-OAc)(urea)](ClO(4))(2) (3a), and [L(3)Ni(2)(&mgr;-OAc)(N,N'-dimethylurea)(2)(MeOH)(2)](ClO(4))(2) (4) have been determined. They reveal that the urea substrates are tied up with the bimetallic cores by both O-coordination to the metal centers and hydrogen bonding between the urea NH and the O atoms of the bridging acetate. In a related complex [L(3)Ni(2)(&mgr;-OAc)(OAc)(2)Na]BPh(4) (5) a sodium ion is associated with the dinickel framework via binding to one O atom of each of the three acetates. The nickel(II) ions in 1 and 2a are weakly antiferromagnetically coupled (J = -2.6 and -1.9 cm(-)(1)), where the magnitude of the coupling appears to correlate with the tilting of the acetate moiety with respect to the plane of the pyrazolate. The superexchange in 3a and 4 is even weaker. The ability of the new complexes to mediate the ethanolysis of urea is examined and is found to be dependent on the number and stereochemical arrangement of the accessible coordination sites at the dinuclear core: the asymmetric species 3a is not capable of inducing any solvolysis of the substrate, and the activity of the symmetric systems 1 and 2b is less than stoichiometric, whereas 4 displays higher activity, albeit this is still very low and possibly proceeds via a one metal ion mechanism.

Ants disinfect fungus-exposed brood by oral uptake and spread of their poison.

To fight infectious diseases, host immune defenses are employed at multiple levels. Sanitary behavior, such as pathogen avoidance and removal, acts as a first line of defense to prevent infection before activation of the physiological immune system. Insect societies have evolved a wide range of collective hygiene measures and intensive health care toward pathogen-exposed group members. One of the most common behaviors is allogrooming, in which nestmates remove infectious particles from the body surfaces of exposed individuals. Here we show that, in invasive garden ants, grooming of fungus-exposed brood is effective beyond the sheer mechanical removal of fungal conidiospores; it also includes chemical disinfection through the application of poison produced by the ants themselves. Formic acid is the main active component of the poison. It inhibits fungal growth of conidiospores remaining on the brood surface after grooming and also those collected in the mouth of the grooming ant. This dual function is achieved by uptake of the poison droplet into the mouth through acidopore self-grooming and subsequent application onto the infectious brood via brood grooming. This extraordinary behavior extends the current understanding of grooming and the establishment of social immunity in insect societies.

In vitro comparison of tibial plateau leveling osteotomy with and without use of a tibial plateau leveling jig.

OBJECTIVE: To evaluate the influence of a tibial plateau leveling jig on osteotomy orientation, fragment reduction, and postoperative tibial plateau angle (TPA) during tibial plateau leveling osteotomy (TPLO). STUDY DESIGN: In vitro experimental study. ANIMALS: Large-breed canine cadavers (n=20). METHODS: TPLO was performed on 40 hindlimbs using 4 methods. Group 1: Jig; dogs in dorsal recumbency with the osteotomy parallel to the distal jig pin. Groups 2-4: No jig; dogs in lateral recumbency with the osteotomy in a vertical orientation (group 2: tibia parallel to the table top; group 3: controlled superimposition of the femoral condyles; group 4: internal rotation of the tibia). Postoperative TPA, fragment reduction, and osteotomy orientation relative to the tibial plateau were compared. Positive or negative values denoted deviation from parallel relative to the tibial plateau. RESULTS: Postoperative TPA, fragment reduction, and proximodistal osteotomy orientation were not significantly different between groups. Craniocaudal osteotomy orientation was significantly different (P<.005) from the tibial plateau. Median deviations were -4.0 degrees (group 1), 11.8 degrees (group 2), 11.2 degrees (group 3), and 0.2 degrees (group 4). Group 1 was not significantly different from group 4. CONCLUSIONS: A jig is not essential for osteotomy orientation, tibial plateau rotation, or fragment reduction. Comparable results were achieved performing a vertical osteotomy with the tibia slightly internally rotated (10 degrees -15 degrees) and parallel to the table surface. CLINICAL RELEVANCE: TPLO without use of a jig reduces surgical trauma, is less time consuming, and reduces cost.

Robot-assisted arm trainer for the passive and active practice of bilateral forearm and wrist movements in hemiparetic subjects.

OBJECTIVE: To determine whether use of a robotic arm trainer for bilateral exercise in daily repetitive training for a 3-week period reduced spasticity and improved motor control in the arm of severely affected, chronic hemiparetic subjects. DESIGN: Before-after trial. SETTING: Community rehabilitation center in Germany. PARTICIPANTS: Consecutive sample of 12 chronic hemiparetic patients; minimum stroke interval 6 months; patients could maximally protract the affected shoulder, hold the extended arm, or slightly flex and extend the elbow. INTERVENTIONS: Additional daily therapy of 15 minutes with the arm trainer for 3 weeks; the 1 degree of freedom trainer enabled the bilateral passive and active practice of a forearm pronation and supination and wrist dorsiflexion and volarflexion; impedance control guaranteed a smooth movement. MAIN OUTCOME MEASURES: Patients' impressions, the Modified Ashworth Scale (MAS) score (range, 0-5) to assess spasticity, and the arm section of the Rivermead Motor Assessment (RMA) score (range, 0-15) to assess motor control were rated before therapy, after each 3-week interval, and at follow-up 3 months later. RESULTS: All patients had favorable impressions: the extremity felt more vivid, and 8 subjects noticed a reduction in spasticity, an ease of hand hygiene, and pain relief. The MAS score of the wrist and fingers joints decreased significantly (P<.0125) from a median of 3 (2-3) and 3 (3-4) to 2 (1-2) and 2.5 (2-3). The RMA score minimally increased in 5 cases without improvement in functional tasks. The median RMA score before therapy was 2.0 (1-2) and 2.0 (1-3.75) after therapy. There were no side effects. At follow-up, the effects had waned. CONCLUSIONS: The arm trainer made possible intensive bilateral elbow and wrist training of severely affected stroke patients. Future studies should address the treatment effect in subacute stroke patients and determine the optimum treatment intensity.