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An indium-111-labelled peptide binds to myeloma cells, enabling SPECT imaging of myeloma cell accumulation in lungs after intravenous administration. This is the first report of a radiolabelled cell-membrane binding peptide for cell tracking.

Hexahistidine tags (His-tags), incorporated into recombinant proteins to facilitate purification using metal-affinity chromatography, are useful binding sites for radiolabeling with [99mTc(CO)3]+ and [188Re(CO)3]+ for molecular imaging and radionuclide therapy. Labeling efficiencies vary unpredictably, and the method is therefore not universally useful. To overcome this, we have made quantitative comparisons of radiolabeling of a bespoke Celluspots array library of 382 His-tag-containing peptide sequences with [99mTc(CO)3]+ and [188Re(CO)3]+ to identify key features that enhance labeling. A selected sequence with 10-fold enhanced labeling efficiency compared to the most effective literature-reported sequences was incorporated into an exemplar protein and compared biologically with non-optimized analogues, in vitro and in vivo. Optimal labeling with either [99mTc(CO)3]+ or [188Re(CO)3]+ required six consecutive His residues in the protein sequence, surrounded by several positively charged residues (Arg or Lys), and the presence of phosphate in the buffer. Cys or Met residues in the sequence were beneficial, to a lesser extent. Negatively charged residues were deleterious to labeling. His-tags with adjacent positively charged residues could be labeled as much as 40 times more efficiently than those with adjacent negatively charged residues. 31P NMR of [Re(CO)3(H2O)3]+ and electrophoresis of solutions of [99mTc(CO)3(H2O)3]+ suggest that phosphate bridges form between cationic residues and the cationic metal synthon during labeling. The trial optimized protein, a scFv targeted to the PSMA antigen expressed in prostate cancer, was readily labeled in >95% radiochemical yield, without the need for subsequent purification. Labeling occurred more quickly and to higher specific activity than comparable non-optimized proteins, while retaining specific binding to PSMA and prostate cancer in vivo. Thus, optimized His-tags greatly simplify radiolabeling of recombinant proteins making them potentially more widely and economically available for imaging and treating patients.

Hexahistidine tags (His-tags), incorporated into recombinant proteins to facilitate purification using metal-affinity chromatography, are useful binding sites for radiolabeling with [(99m)Tc(CO)(3)](+) and [(188)Re(CO)(3)](+) for molecular imaging and radionuclide therapy. Labeling efficiencies vary unpredictably, and the method is therefore not universally useful. To overcome this, we have made quantitative comparisons of radiolabeling of a bespoke Celluspots array library of 382 His-tag-containing peptide sequences with [(99m)Tc(CO)(3)](+) and [(188)Re(CO)(3)](+) to identify key features that enhance labeling. A selected sequence with 10-fold enhanced labeling efficiency compared to the most effective literature-reported sequences was incorporated into an exemplar protein and compared biologically with non-optimized analogues, in vitro and in vivo. Optimal labeling with either [(99m)Tc(CO)(3)](+) or [(188)Re(CO)(3)](+) required six consecutive His residues in the protein sequence, surrounded by several positively charged residues (Arg or Lys), and the presence of phosphate in the buffer. Cys or Met residues in the sequence were beneficial, to a lesser extent. Negatively charged residues were deleterious to labeling. His-tags with adjacent positively charged residues could be labeled as much as 40 times more efficiently than those with adjacent negatively charged residues. (31)P NMR of [Re(CO)(3)(H(2)O)(3)](+) and electrophoresis of solutions of [(99m)Tc(CO)(3)(H(2)O)(3)](+) suggest that phosphate bridges form between cationic residues and the cationic metal synthon during labeling. The trial optimized protein, a scFv targeted to the PSMA antigen expressed in prostate cancer, was readily labeled in >95% radiochemical yield, without the need for subsequent purification. Labeling occurred more quickly and to higher specific activity than comparable non-optimized proteins, while retaining specific binding to PSMA and prostate cancer in vivo. Thus, optimized His-tags greatly simplify radiolabeling of recombinant proteins making them potentially more widely and economically available for imaging and treating patients.

INTRODUCTION AND OBJECTIVE:Medical malpractice (MP) is an important issue in our country. There has been controversy surrounding PSA testing and prostate cancer screening, specifically the United S...

68Ga-labeled urea-based inhibitors of the prostate-specific membrane antigen (PSMA), such as 68Ga-labeled N,N'-bis(2-hydroxybenzyl)ethylenediamine-N,N'-diacetic acid (HBED)-PSMA-11, are promising small molecules for targeting prostate cancer. A new radiopharmaceutical, 68Ga-labeled tris(hydroxypyridinone) (THP)-PSMA, has a simplified design for single-step kit-based radiolabeling. It features the THP ligand, which forms complexes with 68Ga3+ rapidly at a low concentration, at room temperature, and over a wide pH range, enabling direct elution from a 68Ge/68Ga generator into a lyophilized radiopharmaceutical kit in 1 step without manipulation. The aim of this phase 1 study was to assess the safety and biodistribution of 68Ga-THP-PSMA. Methods: Cohort A comprised 8 patients who had proven prostate cancer and were scheduled to undergo prostatectomy; they had Gleason scores of 7-10 and a mean prostate-specific antigen level of 7.8 μg/L (range, 5.4-10.6 μg/L). They underwent PET/CT after the administration of 68Ga-THP-PSMA. All patients proceeded to prostatectomy (7 with pelvic nodal dissection). Dosimetry from multi-time-point PET imaging was performed with OLINDA/EXM. Cohort B comprised 6 patients who had positive 68Ga-HBED-PSMA-11 PET/CT scanning results and underwent comparative 68Ga-THP-PSMA scanning. All patients were monitored for adverse events. Results: No adverse events occurred. In cohort A, 6 of 8 patients had focal uptake in the prostate (at 2 h: average SUVmax, 5.1; range, 2.4-9.2) and correlative 3+ staining of prostatectomy specimens on PSMA immunohistochemistry. The 2 68Ga-THP-PSMA scans with negative results had only 1+/2+ staining. The mean effective dose was 2.07E-02 mSv/MBq. In cohort B, 68Ga-THP-PSMA had lower physiologic background uptake than 68Ga-HBED-PSMA-11 (in the parotid glands, the mean SUVmax for 68Ga-THP-PSMA was 3.6 [compared with 19.2 for 68Ga-HBED-PSMA-11]; the respective corresponding values in the liver were 2.7 and 6.3, and those in the spleen were 2.7 and 10.5; P < 0.001 for all). In 5 of 6 patients, there was concordance in the number of metastases identified with 68Ga-HBED-PSMA-11 and 68Ga-THP-PSMA. Thirteen of 15 nodal abnormalities were subcentimeter. In 22 malignant lesions, the tumor-to-liver contrast with 68Ga-THP-PSMA was similar to that with 68Ga-HBED-PSMA (4.7 and 5.4, respectively; P = 0.15), despite a higher SUVmax for 68Ga-HBED-PSMA than for 68Ga-THP-PSMA (30.3 and 10.7, respectively; P < 0.01). Conclusion:68Ga-THP-PSMA is safe and has a favorable biodistribution for clinical imaging. Observed focal uptake in the prostate was localized to PSMA-expressing malignant tissue on histopathology. Metastatic PSMA-avid foci were also visualized with 68Ga-THP-PSMA PET. Single-step production from a Good Manufacturing Practice cold kit may enable rapid adoption.

Multimodal nanoparticulate materials are described, offering magnetic, radionuclide, and fluorescent imaging capabilities to exploit the complementary advantages of magnetic resonance imaging (MRI), positron emission tomography/single-photon emission commuted tomography (PET/SPECT), and optical imaging. They comprise Fe3O4@NaYF4 core/shell nanoparticles (NPs) with different cation dopants in the shell or core, including Co0.16Fe2.84O4@NaYF4(Yb, Er) and Fe3O4@NaYF4(Yb, Tm). These NPs are stabilized by bisphosphonate polyethylene glycol conjugates (BP-PEG), and then show a high transverse relaxivity (r2) up to 326 mM(-1) s(-1) at 3T, a high affinity to [(18)F]-fluoride or radiometal-bisphosphonate conjugates (e.g., (64)Cu and (99m)Tc), and fluorescent emissions from 500 to 800 nm under excitation at 980 nm. The biodistribution of intravenously administered particles determined by PET/MR imaging suggests that negatively charged Co0.16Fe2.84O4@NaYF4(Yb, Er)-BP-PEG (10K) NPs cleared from the blood pool more slowly than positively charged NPs Fe3O4@NaYF4(Yb, Tm)-BP-PEG (2K). Preliminary results in sentinel lymph node imaging in mice indicate the advantages of multimodal imaging.

Aim To determine whether heterogeneity of cardiac scar, as assessed by cardiovascular magnetic resonance (CMR) texture analysis, may provide insight into better risk stratification for patients with previous myocardial infarction (MI). Materials and methods Patients with previous MI (n=76) were followed for a median of 371.5 days after late gadolinium enhancement (LGE) CMR. The primary endpoint was a composite of ventricular tachycardia, ventricular fibrillation, or unexplained syncope. Areas of LGE were identified and manually segmented on a short-axis projection. The characteristics of the scar heterogeneity were evaluated via CMR texture analysis. This is a filtration-histogram technique, where images are filtered using the Laplacian of a Gaussian filter to extract features different sizes (2–6 mm in radius) corresponding to fine, medium, and coarse texture scales followed by a quantification step using histogram analysis (skewness and kurtosis). Results Patients suffering arrhythmic events during the follow-up period demonstrated significantly higher kurtosis (coarse-scale, p=0.005) and lower skewness (fine-scale, p=0.046) compared to those suffering no arrhythmic events. Furthermore, Kaplan–Meier analysis showed significantly higher coarse kurtosis (p=0.004), and lower fine skewness (p=0.035) were able to predict increased incidence of ventricular arrhythmic events. Conclusions In this pilot study, indices of texture analysis reflecting textural heterogeneity were significantly associated with a greater incidence of arrhythmic events. Further work is required to delineate the role of texture analysis techniques in risk stratification post-MI.

Cancer cell metastasis is responsible for most cancer deaths. Non-invasive in vivo cancer cell tracking in spontaneously metastasizing tumor models still poses a challenge requiring highest sensitivity and excellent contrast. The goal of this study was to evaluate if the recently introduced PET radiotracer [18F]tetrafluoroborate ([18F]BF4−) is useful for sensitive and specific metastasis detection in an orthotopic xenograft breast cancer model expressing the human sodium iodide symporter (NIS) as a reporter. In vivo imaging was complemented by ex vivo fluorescence microscopy and γ-counting of harvested tissues. Radionuclide imaging with [18F]BF4− (PET/CT) was compared to the conventional tracer [123I]iodide (sequential SPECT/CT). We found that [18F]BF4− was superior due to better pharmacokinetics, i.e. faster tumor uptake and faster and more complete clearance from circulation. [18F]BF4−-PET was also highly specific as in all detected tissues cancer cell presence was confirmed microscopically. Undetected comparable tissues were similarly found to be free of metastasis. Metastasis detection by routine metabolic imaging with [18F]FDG-PET failed due to low standard uptake values and low contrast caused by adjacent metabolically active organs in this model. [18F]BF4−-PET combined with NIS expressing disease models is particularly useful whenever preclinical in vivo cell tracking is of interest.

Background It is important to study differential inflammatory cellular migration, particularly of eosinophils and neutrophils, in asthma and how this is influenced by environmental stimuli such as allergen exposure and the effects of anti asthma therapy. Methods We isolated blood neutrophils and eosinophils from 12 atopic asthmatic human volunteers (Group 1 — four Early Allergic Responders unchallenged (EAR); Group 2 — four Early and Late Allergic Responders (LAR) challenged; Group 3 — four EAR and LAR challenged and treated with systemic corticosteroids) using cGMP CD16 CliniMACS. Cells were isolated prior to allergen challenge where applicable, labelled with 99mTc-HMPAO and then re-infused intravenously. The kinetics of cellular influx/efflux into the lungs and other organs were imaged via scintigraphy over 4 h, starting at 5 to 6 h following allergen challenge where applicable. Results Neutrophils and eosinophils were isolated to a mean (SD) purity of 98.36% (1.09) and 96.31% (3.0), respectively. Asthmatic neutrophils were activated at baseline, mean (SD) CD11bHigh cells 46 (10.50) %. Isolation and radiolabelling significantly increased their activation to > 98%. Eosinophils were not activated at baseline, CD69+ cells 1.9 (0.6) %, increasing to 38 (3.46) % following isolation and labelling. Analysis of the kinetics of net eosinophil and neutrophil lung influx/efflux conformed to a net exponential clearance with respective mean half times of clearance 6.98 (2.18) and 14.01 (2.63) minutes for Group 1, 6.03 (0.72) and 16.04 (2.0) minutes for Group 2 and 5.63 (1.20) and 14.56 (3.36) minutes for Group 3. These did not significantly differ between the three asthma groups (p > 0.05). Conclusions Isolation and radiolabelling significantly increased activation of eosinophils (CD69) and completely activated neutrophils (CD11bHigh) in all asthma groups. Net lung neutrophil efflux was significantly slower than that of eosinophils in all asthma study groups. There was a trend for pre-treatment with systemic corticosteroids to reduce lung retention of eosinophils following allergen challenge., Highlights • Isolation and radiolabelling of eosinophils or neutrophils from asthmatics significantly activates them. • Corticosteroids alter eosinophil, but not neutrophil 99mTc-HMPAO radiolabelling efficiency. • Leukocyte activation as a result of manipulation ex vivo does not measurably alter lung sequestration. • Eosinophil migration through the lungs of asthmatic patients is slower than in healthy volunteers.

DC vaccines have been used to induce tumour-specific cytotoxic T cells . However, this approach to cancer immunotherapy has had limited success. To be successful, injected DCs need to migrate to the LNs where they can stimulate effector T cells . We and others have previously demonstrated by MRI that tumour antigen-pulsed-DCs labelled ex vivo with superparamagnetic iron oxide nanoparticles migrated to the draining LNs and are capable of activating antigen-specific T cells . The results from our study demonstrated that ex vivo superparamagnetic iron oxide nanoparticles-labelled and OVA-pulsed DCs prime cytotoxic CD8(+) T-cell responses to protect against a B16-OVA tumour challenge. In the clinic, a possible noninvasive surrogate marker for efficacy of DC vaccination is to image the specific migration and accumulation of T cells following DC vaccination.

The increasing availability of the long half-life positron emitter Zr-89 (half life 78.4 h) suggests that it is a strong candidate for cell labelling and hence cell tracking using positron emission tomography. The aim was to produce a range of neutral ZrL4 lipophilic complexes for cell labelling which could be prepared under radiopharmaceutical conditions. This was achieved when the ligand was oxine, tropolone or ethyl maltol. The complexes can be prepared in high yield from zirconium(iv) precursors in hydrochloric or oxalic acid solution. The oxinate and tropolonate complexes were the most amenable to chromatographic characterisation, and HPLC and ITLC protocols have been established to monitor their radiochemical purity. The radiochemical synthesis and quality control of (89)Zr(oxinate)4 is reported as well as preliminary cell labelling data for the oxinate, tropolonate and ethyl maltolate complexes which indicates that (89)Zr(oxinate)4 is the most promising candidate for further evaluation.

Complement activation is a recognised mediator of myocardial ischaemia-reperfusion-injury (IRI) and cardiomyocytes are a known source of complement proteins including the central component C3, whose activation products can mediate tissue inflammation, cell death and profibrotic signalling. We investigated the potential to detect and quantify the stable covalently bound product C3d by external body imaging, as a marker of complement activation in heart muscle in a murine model of myocardial IRI. We used single-photon-emission-computed-tomography (SPECT) in conjunction with

Dysregulation of apoptosis and necrosis is central to many diseases and non-invasive imaging of cell death is an important clinical objective to stage disease or to monitor treatment progress. The C2A domain of rat synaptotagmin I binds to phosphatidylserine (PS) exposed during cell death and modification to its lysine residues has been shown to disrupt PS binding. Site-specifically labelled (99m)Tc(CO)3-C2AcH and (68)Ga-C2Ac have previously been investigated for single photon emission computed tomography (SPECT) and positron emission tomography (PET) imaging, respectively. We wished to design a (64)Cu-labelled counterpart due to the longer half-life of (64)Cu. Since the calcium binding sites in C2A may interfere with copper binding we sought a high affinity, fast labelling chelator. We synthesised a maleimide functionalised bis(thiosemicarbazone), H2ATSE/AMal, for the site-specific copper-64 radiolabelling of thiol-functionalised C2Ac. When radiolabelling was performed by incubation of the ligand-protein conjugate (post-labelling approach), analysis of the resultant (64)CuATSE/AMal-C2Ac revealed that the C2Ac was able to compete for radiocopper with the chelator. In contrast, the pre-labelled (64)CuATSE/AMal-C2Ac conjugate revealed good stability in serum and maintained target affinity in a red blood cell binding assay. The results suggest that due to the intrinsic copper binding properties of the protein, a pre-labelling approach is preferred for the C2Ac domain of synaptotagmin I when copper is the desired radioisotope.

Summary Cell-based therapies using natural or genetically modified regulatory T cells (Tregs) have shown significant promise as immune-based therapies. One of the main difficulties facing the further advancement of these therapies is that the fate and localization of adoptively transferred Tregs is largely unknown. The ability to dissect the migratory pathway of these cells in a non-invasive manner is of vital importance for the further development of in-vivo cell-based immunotherapies, as this technology allows the fate of the therapeutically administered cell to be imaged in real time. In this review we will provide an overview of the current clinical imaging techniques used to track T cells and Tregs in vivo, including magnetic resonance imaging (MRI) and positron emission tomography (PET)/single photon emission computed tomography (SPECT). In addition, we will discuss how the finding of these studies can be used, in the context of transplantation, to define the most appropriate Treg subset required for cellular therapy.

The development of medical imaging is a highly multidisciplinary endeavor requiring the close cooperation of clinicians, physicists, engineers, biologists, and chemists to identify capabilities, conceive challenges and solutions and apply them in the clinic. The chemistry described in this chapter illustrates how synergistic advances in these areas drive the technology and its applications forward, with each discipline producing innovations that in turn drive innovations in the others. The main thread running through the chapter is the shift from single photon radionuclide imaging toward PET, and in turn the emerging shift from PET/CT toward PET/MRI and further, combination of these with optical imaging. Chemistry to support these transitions is exemplified by building on a summary of the status quo , and recent developments, in technetium-99m chemistry for SPECT imaging, followed by a report of recent developments to support clinical application of short-lived (Ga-68) and long-lived (Zr-89) positron-emitting isotopes, copper isotopes for PET imaging, and combined modality imaging agents based on radiolabeled iron oxide-based nanoparticles.

We recently described a novel amino acid sequence, KCKLAAALEHHHHHH, for site-specific radiolabelling of proteins with [ 99m Tc(CO) 3 (OH 2 ) 3 ] + or [Re(CO) 3 (OH 2 ) 3 ] + with improved efficiency compared to conventional hexahistidine tags (His-tag). C2AH, a modification of the protein C2A (the phosphatidylserine (PS)-binding domain of rat synaptotagmin I) engineered to contain this novel C-terminal tag, was produced. Rhenium tricarbonyl conjugates of C2AH were analysed post tryptic digest by liquid chromatography-electrospray mass spectrometry (LC-MS), giving rise to a peak with the molecular weight corresponding to M + = [Re(CO) 3 + CK + LAAALEHHHHHH] + . This species arises as a result of trypsin cleavage on the C-terminus of both the lysine (Lys) residues on either side of the Cys while both fragments still remain bound to the rhenium. This confirmed that cysteine (Cys) was directly involved in the coordination of the rhenium tricarbonyl. To demonstrate the superiority of the cysteine containing His-tag sequences for binding [Re(CO) 3 ] + , two peptides CKLAAALEHHHHHH and LAAALEHHHHHH were synthesised. In a competition experiment the mixed peptides were incubated with one molar equivalent of [Re(CO) 3 (H 2 O) 3 ] + , and LC-ESMS demonstrated that 92% and 9% of CKLAAALEHHHHHH and LAAALEHHHHHH respectively were co-ordinated by one [Re(CO) 3 ] + .

We describe the design and synthesis of a new Tc-99m labeled bioconjugate for cell-death imaging, based on C2A, the phosphatidylserine (PS)-binding domain of rat synaptotagmin I. Since several lysine residues in this protein are critical for PS binding, we engineered a new protein, C2AcH, to include the C-terminal sequence CKLAAALEHHHHHH, incorporating a free cysteine (for site-specific covalent modification) and a hexahistidine tag (for site-specific radiolabeling with [99mTc(CO)3(OH2)3]+). We also engineered a second derivative, C2Ac, in which the C-terminal sequence included only the C-terminal cysteine. These proteins were characterized by electrospray mass spectrometry, SDS/PAGE, and size exclusion chromatography and radiolabeled with [99mTc(CO)3(OH2)3]+. Conjugates of the proteins with the rhenium analogue [Re(CO)3(OH2)3]+ were also synthesized. Site-specific labeling was confirmed by performing a tryptic digest of rhenium tricarbonyl-labeled C2AcH, and only peptides containing the His-tag contained the [Re(CO)3]+. The labeled proteins were tested for binding to red blood cells (RBC) with exposed PS in a calcium dependent manner. Labeling 100 microg of C2AcH with [99mTc(CO)3(OH2)3]+ at 37 degrees C for 30 min gave a radiochemical yield of96%. However, C2AcH that had first been conjugated with fluorescein maleimide or iodoacetamide via the Cys residue gave only 50% and 83% radiochemical yield, respectively, after incubation for 30 min at 37 degrees C. Serum stability results indicated that95% of radiolabeled C2AcH remained stable for at least 18 h at 37 degrees C. Site-specifically labeled C2AcH exhibited calcium-dependent binding to the PS on the RBC, whereas a nonspecifically modified derivative, C2AcH-B, in which lysines had been modified with benzyloxycarbonyloxy, did not. We conclude that (i) the combination of Cys and a His-tag greatly enhances the rate and efficiency of labeling with [99mTc(CO)3(OH2)3]+ compared to either the His-tag or the Cys alone, and this sequence deserves further evaluation as a radiolabeling tag; (ii) non-site-specific modification of C2A via lysine residues impairs target binding affinity; (iii) 99mTc-C2AcH has excellent radiolabeling, stability and PS binding characteristics and warrants in vivo evaluation as a cell-death imaging agent.

The Plasmodium falciparum apical membrane antigen 1 (AMA1) is a leading vaccine candidate and was tested for safety and immunogenicity in human Phase I Clinical Trials. PBMC from vaccine recipients were analyzed by flow cytometric methods to determine the nature of T-cell responses and AMA1-reactive memory T cells. Both CD4 and CD8 T cells produced a number of cytokines following AMA1 re-stimulation, with IL-5-producing cells at the highest frequency, consistent with a Th2 bias. The relative frequency of multifunctional cells synthesizing Th1 cytokines IFN-γ, IL-2 and TNF-α changed after each vaccination. Interestingly, median fluorescence intensity measurements revealed that cells producing more than one cytokine contributed greater quantities of each cytokine than cell populations that produced each of the cytokines alone. AMA1 vaccination also elicited the development of memory cell populations, and both central and effector memory T cells were identified concurrently after the AMA1 vaccination. The detailed profile of multifunctional T-cell responses to AMA1 presented here will advance our ability to assess the immunogenicity of human malarial vaccines.

Despite the central role of memory B cells (MBC) in protective immune responses, little is understood about how they are acquired in naive individuals in response to Ag exposure, and how this process is influenced by concurrent activation of the innate immune system’s TLR. In this longitudinal study of malaria-naive individuals, we examined the MBC response to two candidate malaria vaccines administered with or without CpG, a TLR9 ligand. We show that the acquisition of MBC is a dynamic process in which the vaccine-specific MBC pool rapidly expands and then contracts, and that CpG enhances the kinetics, magnitude, and longevity of this response. We observed that the percentage of vaccine-specific MBC present at the time of reimmunization predicts vaccine-specific Ab levels 14 days later; and that at steady-state, there is a positive correlation between vaccine-specific MBC and Ab levels. An examination of the total circulating MBC and plasma cell pools also suggests that MBC differentiate into plasma cells through polyclonal activation, independent of Ag specificity. These results provide important insights into the human MBC response, which can inform the development of vaccines against malaria and other pathogens that disrupt immunological memory.

Vaccines represent a significant potential means of decreasing global morbidity and mortality due to malaria. Clinical trials in the United States with Plasmodium falciparum Apical Membrane Antigen 1 (AMA1) showed that the vaccine induced biologically active Abs judged by an in vitro parasite growth inhibition assay (GIA). However, the same vaccine in Malian adults did not increase biological activity, although it elevated ELISA titers. Because GIA has been used to evaluate the biological activity of Abs induced by blood stage malarial vaccine candidates, we explored this discrepancy in this study. We affinity purified AMA1-specific Abs from both U.S. vaccinees and nonvaccinated individuals living in a malaria-endemic area of Mali and performed ELISA and GIA. Both AMA1-specifc Abs induced by vaccination (U.S.) and by natural infection (Mali) have comparable biological activity in GIA when the ELISA titer is normalized. However, a fraction of Malians’ IgG that did not bind to AMA1 protein (Mali-non-AMA1 IgG) reduced the biological activity of the AMA1 Abs from U.S. vaccinees; in contrast, U.S.-non-AMA1 IgGs did not show a reduction of the biological activity. Further investigation revealed that the reduction was due to malaria-specific IgGs in the Mali-non-AMA1 IgGs. The fact that both U.S.- and Mali-AMA1-specific Abs showed comparable biological activity supports further development of AMA1-based vaccines. However, the reduction of biological activity of AMA1-specific Ab by other malaria-specific IgGs likely explains the limited effect on growth-inhibitory activity of Abs induced by AMA1 vaccination in Malian adults and may complicate efforts to develop a blood stage malaria vaccine.

CpG oligodeoxynucleotides are potent immunostimulants. In this study, CPG 7909 was formulated with the recombinant Plasmodium falciparum protein AMA1-C1 adsorbed to Alhydrogel (aluminum hydroxide) and used to immunize mice. Mice receiving free CPG 7909 in a separate same site injection to the AMA1-C1/Alhydrogel had the same antibody responses as mice receiving AMA1-C1/Alhydrogel alone. For mice immunized with CPG 7909 bound to the AMA1-C1/Alhydrogel formulation, there was a bell shaped CPG 7909 dose response curve with the highest antibody response co-incident with the concentration of CPG 7909 that saturated binding to the Alhydrogel. At a higher CPG 7909 dose where 74% was unbound, there was no enhancement of response over AMA1-C1/Alhydrogel alone. Our results suggest that the adjuvant effects of CpGs are optimal when adsorbed to Alhydrogel and highlight the need for careful characterization of the vaccine formulation.

Conjugation of polysaccharides to carrier proteins has been a successful approach for producing safe and effective vaccines. In an attempt to increase the immunogenicity of two malarial vaccine candidate proteins of Plasmodium falciparum, apical membrane antigen 1 (AMA1) to a blood stage vaccine candidate and surface protein 25 (Pfs25) a mosquito stage vaccine candidate, were each independently chemically conjugated to the mutant, nontoxic Pseudomonas aeruginosa ExoProtein A (rEPA). AMA1 is a large (66kD) relatively good immunogen in mice; Pfs25 is a poorly immunogenic protein when presented on alum to mice. Mice were immunized on days 0 and 28 with AMA1- or Pfs25-rEPA conjugates or unconjugated AMA1 or Pfs25, all formulated on Alhydrogel. Remarkably, sera from mice 14 days after the second immunization with Pfs25-rEPA conjugates displayed over a 1000-fold higher antibody titers as compared to unconjugated Pfs25. In contrast, AMA1 conjugated under the same conditions induced only a three-fold increase in antibody titers. When tested for functional activity, antibodies elicited by the AMA1-rEPA inhibited invasion of erythrocytes by blood-stage parasites and antibodies elicited by the Pfs25-rEPA conjugates blocked the development of the sexual stage parasites in the mosquito midgut. These results demonstrate that conjugation to rEPA induces a marked improvement in the antibody titer in mice for the poor immunogen (Pfs25) and for the larger protein (AMA1). These conjugates now need to be tested in humans to determine if mice are predictive of the response in humans.

Background Macrophages represent a critical cell type in host defense, development and homeostasis. The ability to image non-invasively pro-inflammatory macrophage infiltrate into a transplanted organ may provide an additional tool for the monitoring of the immune response of the recipient against the donor graft. We therefore decided to image in vivo sialoadhesin (Sn, Siglec 1 or CD169) using anti-Sn mAb (SER-4) directly radiolabelled with 99mTc pertechnetate. Methods We used a heterotopic heart transplantation model where allogeneic or syngeneic heart grafts were transplanted into the abdomen of recipients. In vivo nanosingle-photon emission computed tomography (SPECT/CT) imaging was performed 7 days post transplantation followed by biodistribution and histology. Results In wild-type mice, the majority of 99mTc-SER-4 monoclonal antibody cleared from the blood with a half-life of 167 min and was located predominantly on Sn+ tissues in the spleen, liver and bone marrow. The biodistribution in the transplantation experiments confirmed data derived from the non-invasive SPECT/CT images, with significantly higher levels of 99mTc-SER-4 observed in allogeneic grafts (9.4 (±2.7) %ID/g) compared to syngeneic grafts (4.3 (±10.3) %ID/g) (p = 0.0022) or in mice which received allogeneic grafts injected with 99mTc-IgG isotype control (5.9 (±0.6) %ID/g) (p = 0.0185). The transplanted heart to blood ratio was also significantly higher in recipients with allogeneic grafts receiving 99mTc-SER-4 as compared to recipients with syngeneic grafts (p = 0.000004) or recipients with allogeneic grafts receiving 99mTc-IgG isotype (p = 0.000002). Conclusions Here, we demonstrate that imaging of Sn+ macrophages in inflammation may provide an important additional and non-invasive tool for the monitoring of the pathophysiology of cellular immunity in a transplant model. Electronic supplementary material The online version of this article (doi:10.1186/s13550-015-0146-7) contains supplementary material, which is available to authorized users.

The development of medical imaging is a highly multidisciplinary endeavor requiring the close cooperation of clinicians, physicists, engineers, biologists and chemists to identify capabilities, conceive challenges and solutions and apply them in the clinic. The chemistry described in this article illustrates how synergistic advances in these areas drive the technology and its applications forward, with each discipline producing innovations that in turn drive innovations in the others. The main thread running through the article is the shift from single photon radionuclide imaging towards PET, and in turn the emerging shift from PET/CT towards PET/MRI and further, combination of these with optical imaging. Chemistry to support these transitions is exemplified by building on a summary of the status quo, and recent developments, in technetium-99m chemistry for SPECT imaging, followed by a report of recent developments to support clinical application of short lived (Ga-68) and long-lived (Zr-89) positron emitting isotopes, copper isotopes for PET imaging, and combined modality imaging agents based on radiolabelled iron oxide based nanoparticles.

Background Following myocardial infarction (MI), patients are at risk of sudden cardiac death (SCD) due to ventricular arrhythmia, which can be prevented with an implantable cardioverter-defibrillator (ICD). Recommendations for ICD implantation is currently based on left ventricular ejection fraction (LVEF), however less than a quarter of patients who receive ICDs based on LVEF have appropriate therapy. Heterogeneity of scar has been implicated in the development of re-entrant arrhythmias and SCD. Texture analysis (TA) is a method of quantifying heterogeneity of tissues in imaging, usually using statistical-based methods to evaluate distribution of grey-level pixels. This study aimed to determine whether TA could be used to quantify heterogeneity of scar as a method of accurately risk stratifying patients. Methods This was a retrospective blinded analysis of late-gadolinium enhanced cardiac magnetic resonance (LGE-CMR) images. Twenty post-MI patients who received ICDs were followed up for up to 686 days after implantation. Ten of these patients went on to have events and were categorised as high risk, while the remaining ten had no events on follow up and were categorised as low risk. TA was performed on regions of interest delineating

Antibodies are thought to be the primary immune effectors in the defense against erythrocytic stage Plasmodium falciparum. Thus, malaria vaccines directed to blood stages of infection are evaluated based on their ability to induce antibodies with anti-parasite activity. Such antibodies may have different effector functions (e.g., inhibition of invasion or inhibition of parasite growth/development) depending on the target antigen. We evaluated four methods with regards to their ability to differentiate between invasion and/or growth inhibitory activities of antibodies specific for two distinct blood stage antigens: AMA1 and MSP142. We conclude that antibodies induced by these vaccine candidates have different modes of action that vary not only by the antigen, but also by the strain of parasite being tested. Analysis based on parasitemia and viability was essential for defining the full range of anti-parasite activities in immune sera.

Apical membrane antigen 1 (AMA1) has been shown to be a promising malaria vaccine candidate. The multiallelic AMA1-C1 vaccine currently in Phase 1 trials in the US and Mali contains an equal mixture of the ectodomain portion of recombinant AMA1 from the FVO and 3D7 clones of Plasmodium falciparum, formulated on Alhydrogel. It is hoped that inclusion of a human-optimized CpG oligodeoxynucleotide (ODN) (CPG 7909) with our existing AMA1-C1/Alhydrogel vaccine will lead to a higher concentration of functional AMA1-C1 antibodies. Preclinical studies were performed in mice, rats and guinea pigs to assess the safety, immunogenicity and functionality of the immune response to AMA1-C1 with Alhydrogel + CPG 7909 compared to antigen with Alhydrogel alone. Day 42 mean anti-AMA1 ELISA titer values derived from individual animals were compared between Alhydrogel and Alhydrogel + CPG 7909 groups at each antigen dose for each species. Sera from Alhydrogel + CPG 7909 groups displayed significantly higher antibody titers (P < 0.025) than their comparable Alhydrogel alone group. Mouse IgG isotype analysis showed that AMA1-C1/Alhydrogel induced a predominately Th2 type response while AMA1-C1/Alhydrogel + CPG 7909 gave a mixed Th1/Th2 type response. When tested for functional activity by in vitro inhibition of parasite invasion, IgG isolated from serum pools of AMA1-C1/Alhydrogel + CPG 7909 animals was more effective against both FVO and 3D7 parasites than an equal concentration of IgG from animals receiving vaccines adjuvanted with Alhydrogel alone. These promising preclinical results have recently led to the start of a Phase 1 trial in the US.

Apical membrane antigen 1 (AMA1), a polymorphic merozoite surface protein, is a leading blood-stage malaria vaccine candidate. A phase 1 trial was conducted with 30 malaria-naïve volunteers to assess the safety and immunogenicity of the AMA1-C1 malaria vaccine. AMA1-C1 contains an equal mixture of recombinant proteins based on sequences from the FVO and 3D7 clones of Plasmodium falciparum . The proteins were expressed in Pichia pastoris and adsorbed on Alhydrogel. Ten volunteers in each of three dose groups (5 μg, 20 μg, and 80 μg) were vaccinated in an open-label study at 0, 28, and 180 days. The vaccine was well tolerated, with pain at the injection site being the most commonly observed reaction. Anti-AMA1 immunoglobulin G (IgG) was detected by enzyme-linked immunosorbent assay (ELISA) in 15/28 (54%) volunteers after the second immunization and in 23/25 (92%) after the third immunization, with equal reactivity to both AMA1-FVO and AMA1-3D7 vaccine components. A significant dose-response relationship between antigen dose and antibody response by ELISA was observed, and the antibodies were predominantly of the IgG1 isotype. Confocal microscopic evaluation of sera from vaccinated volunteers demonstrated reactivity with P. falciparum schizonts in a pattern similar to native parasite AMA1. Antigen-specific in vitro inhibition of both FVO and 3D7 parasites was achieved with IgG purified from sera of vaccinees, demonstrating biological activity of the antibodies. To our knowledge, this is the first AMA1 vaccine candidate to elicit functional immune responses in malaria-naïve humans, and our results support the further development of this vaccine.

MD-2 is a secreted glycoprotein that binds to the extracellular domain of Toll-like receptor 4 (TLR4) and is required for the activation of TLR4 by lipopolysaccharide (LPS). The protein contains seven Cys residues and consists of a heterogeneous collection of disulfide-linked oligomers. To investigate the role of sulfhydryls in MD-2 structure and function, we created 17 single and multiple Cys substitution mutants. All of the MD-2 mutant proteins, including one totally lacking Cys residues, were secreted and stable. SDS/PAGE analyses indicated that most Cys residues could participate in oligomer formation and that no single Cys residue was required for oligomerization. Of the single Cys substitutions, only C95S and C105S failed to confer LPS responsiveness on TLR4 when mutant and TLR4 were cotransfected into cells expressing an NF-κB reporter plasmid. Surprisingly, substitution of both C95 and C105 partially restored activity. Structural analyses revealed that C95 and C105 formed an intrachain disulfide bond, whereas C95 by itself produced an inactive dimer. In contrast to the cotransfection experiments, only WT MD-2 conferred responsiveness to LPS when secreted proteins were added directly to TLR4 reporter cells. Our data are consistent with a model in which most, possibly all sulfhydryls lie on the surface of a stable MD-2 core structure where they form both intra- and interchain disulfide bridges. These disulfide bonds produce a heterogeneous array of oligomers, including some species that can form an active complex with TLR4.

Copper diacetyl-bis(N4-methylthiosemicarbazone), Cu(II)ATSM, is a promising agent for imaging hypoxic tissue. Here we present results that provide insight into the chemical and electronic properties underlying previously observed structure-activity relationships. Density functional theory (DFT) calculations on the electronic structures and molecular orbitals of a series of 13 Cu(II)bis(thiosemicarbazone) analogues with different alkylation patterns and with fixed geometries based on the known structure of Cu(II)PTSM showed that the LUMO and the next lowest orbital were very close in energy, and their energy order was strikingly dependent on the ligand alkylation pattern in a way that correlated with hypoxia-selectivity and redox potentials. The LUMOs of Cu(II)ATSM and other hypoxia-selective analogues were predominantly metal-based (leading to a singlet reduced species) while the LUMOs of Cu(II)PTSM and other nonselective analogues were predominantly ligand-based (leading to a triplet reduced species). Upon relaxation of the geometric constraint and full optimization in both Cu(II)ATSM and Cu(II)GTS, the metal-based orbital became the LUMO, and the singlet was the thermodynamically preferred form of the reduced species. Chemical and electrochemical investigation showed that all Cu(II) complexes were reducible, but Cu(I)PTSM and other nonselective analogues dissociated immediately upon reduction with release of ligand (detected by UV-vis) while Cu(I)ATSM and other hypoxia-selective analogues did not. Instead they were rapidly re-oxidized to the Cu(II) complex by molecular oxygen. The reversible electrochemical reduction of nonselective complexes Cu(II)PTSM and Cu(II)GTS became irreversible in the presence of weak acid, whereas that of Cu(II)ATSM was unaffected. In light of these results we present a model to explain the structure-activity relationships on the basis of electronic structure and molecular vibrations.

Copper(II) bis(thiosemicarbazone) complexes labelled with Cu-60/62/64 are useful radiopharmaceuticals for imaging blood flow and hypoxic tissues in vivo. The aim of this study was to identify structure-activity relationships within a series of analogues with different alkyl substitution patterns in the ligand, in order to design improved hypoxia imaging agents and elucidate hypoxia selectivity mechanisms. Thirteen such complexes were synthesised and characterised spectroscopically and electrochemically. The uptake of each (labelled with Cu-64) in EMT6 tumour cells in vitro under normoxic and hypoxic conditions was studied. All complexes were taken up efficiently into cells, and some showed strong hypoxia selectivity, which was highly correlated with the Cu(II/I) redox potential. Redox potentials at the low end of the range were found to be essential for hypoxia selectivity. In turn, the redox potential was strongly correlated with alkyl substitution pattern, and the most important determinant of the redox potential was the number of alkyl groups on the diimine backbone of the ligand. Several complexes in the series warrant further evaluation as hypoxia imaging agents. The radioactivity uptake/release behaviour in the cells provides insight into possible mechanisms, and a model for hypoxia-selective intracellular trapping is discussed.

Purpose 111In (typically as [111In]oxinate3) is a gold standard radiolabel for cell tracking in humans by scintigraphy. A long half-life positron-emitting radiolabel to serve the same purpose using positron emission tomography (PET) has long been sought. We aimed to develop an 89Zr PET tracer for cell labelling and compare it with [111In]oxinate3 single photon emission computed tomography (SPECT). Methods [89Zr]Oxinate4 was synthesised and its uptake and efflux were measured in vitro in three cell lines and in human leukocytes. The in vivo biodistribution of eGFP-5T33 murine myeloma cells labelled using [89Zr]oxinate4 or [111In]oxinate3 was monitored for up to 14 days. 89Zr retention by living radiolabelled eGFP-positive cells in vivo was monitored by FACS sorting of liver, spleen and bone marrow cells followed by gamma counting. Results Zr labelling was effective in all cell types with yields comparable with 111In labelling. Retention of 89Zr in cells in vitro after 24 h was significantly better (range 71 to >90 %) than 111In (43–52 %). eGFP-5T33 cells in vivo showed the same early biodistribution whether labelled with 111In or 89Zr (initial pulmonary accumulation followed by migration to liver, spleen and bone marrow), but later translocation of radioactivity to kidneys was much greater for 111In. In liver, spleen and bone marrow at least 92 % of 89Zr remained associated with eGFP-positive cells after 7 days in vivo. Conclusion [89Zr]Oxinate4 offers a potential solution to the emerging need for a long half-life PET tracer for cell tracking in vivo and deserves further evaluation of its effects on survival and behaviour of different cell types. Electronic supplementary material The online version of this article (doi:10.1007/s00259-014-2945-x) contains supplementary material, which is available to authorized users.

Reaction of PhP(CH2CH2SH)(2) with 1,2-dichloroethane and Cs2CO3 under high dilution conditions affords 1-phenyl-1-phospha-4,7-dithiacyclononane (L) as an oil in 52% yield. The geometry of L has been probed by H-1 NMR spectroscopy, which indicates an equilibrium mixture containing gauche S-C-C-S and S-C-C-P segments, and molecular mechanics calculations which find a lowest energy endodentate conformation minimum and also energetically accessible exodentate minima. Endo- and exodentate conformations are observed in the crystal structure of [Cu(eta(1)-L)(eta(3)-L)][PF6] (1) formed by the reaction of L with [Cu(NCMe)(4)[PF6]. Reaction of Hg(ClO4)(2) with L affords [HgL2][ClO4](2) (2) whose structure has also been established by X-ray diffraction. The mercury ion is in a very distorted octahedral environment with two short Hg-P bonds (average 2.404 Angstrom) and four long Hg-S bonds (average 3.092 Angstrom). Reaction of L with AgBF4 or Ni(BF4)(2) or CoBr2 or Fe(BF4)(2) affords [AgL2][BF4] (3), [NiL2][BF4](2), (4), [CoL2]BL2 (5) and FeL2][BF4](2) (6), respectively, which were characterised by FAB mass spectroscopy and elemental analysis. Cyclic voltammetry studies were used to compare the redox properties of 4, 5 and 6 with their trithiacyclononane (9S3) analogues and in compound 4 the ligand L was found to stabilise Ni in the oxidation states 0, I, Il and III. UV-Vis spectroscopic measurements show that L is a stronger field ligand than 9S3. EXAFS studies were also performed on 4, 5 and 6, which established that all three complexes are six-coordinate. (C) 1999 Elsevier Science S.A. All rights reserved.

Complexes of Re and Tc with 1,4,7-trithiacyclononane (9S3) differ from their later transition metal analogues in that their d6 form ([M(9S3)2]+) undergoes instantaneous C–S bond cleavage yielding ethene and [M(9S3)L]+ (L = SCH2CH2SCH2CH2S), a stable metal(III) thiolate complex, cleanly in aqueous solution. This contrast is interpreted as signifying increased π-back donation by Re and Tc, compared to later metals, into ligand C–S σ* orbitals. In order to validate this hypothesis within an established theoretical framework, and to compare the predicted relative C–S bond lability with relative experimental lability in a series of d6 analogues, extended Huckel theory (EHT) was used to investigate the bonding (M = Mo, Tc, Ru, Rh or Pd) while electrospray mass spectrometry (ES-MS) was used to compare ethene loss, in a series of analogous complexes (M = Tc, Re, Ru or Os). The C–S overlap populations were smaller for M = TcII and TcI than for later metal(II) analogues, and were smaller for TcI than for TcII. Fragment molecular orbitals corresponding to C–S σ* were more highly populated for M = TcII and TcI than for later analogues, and also more highly populated for TcI than for TcII. ES-MS showed that ethene loss from Tc/ReI and Tc/ReII complexes occurred at much lower energies than from the Ru/OsII analogues. EHT supports the hypothesis that C–S activation is caused by π-back donation into C–S σ* orbitals, and correctly predicts that ethene loss occurs more readily from rhenium and technetium d5 and especially d6 complexes than from later transition metal analogues.

Sialoadhesin (Sn, also known as Siglec-1 and CD169) is a macrophage-restricted cell surface receptor that is conserved across mammals. Sn is a member of the sialic acid-binding IgG-like lectin (Siglec) family of proteins characterized by affinity to specifically sialylated ligands, and under normal conditions is expressed on subsets of macrophages in secondary lymphoid tissues, such as lymph node and spleen. However, Sn-positive macrophages can also be found in a variety of pathological conditions, including (autoimmune) inflammatory infiltrates and tumours. Sn has been shown to contribute to sialylated pathogen uptake, antigen presentation and lymphocyte proliferation, and to influence both immunity and tolerance. This review presents Sn as a macrophage-specific marker of inflammation and immunoregulation with the potential to becoming an important biomarker for immunologically active macrophages and a target for therapy.

Self-associated protein aggregates or cross-linked protein conjugates are, in general, more immunogenic than oligomeric or monomeric forms. In particular, the immunogenicity in mice of a recombinant malaria transmission blocking vaccine candidate, the ookinete specific Plasmodium falciparum 25 kDa protein (Pfs25), was increased more than 1000-fold when evaluated as a chemical cross-linked protein-protein conjugate as compared to a formulated monomer. Whether alternative approaches using protein complexes improve the immunogenicity of other recombinant malaria vaccine candidates is worth assessing. In this work, the immunogenicity of the recombinant 42 kDa processed form of the P. falciparum merozoite surface protein 1 (MSP1(42)) was evaluated as a self-associated, non-covalent aggregate and as a chemical cross-linked protein-protein conjugate to ExoProtein A, which is a recombinant detoxified form of Pseudomonas aeruginosa exotoxin A. MSP1(42) conjugates were prepared and characterized biochemically and biophysically to determine their molar mass in solution and stoichiometry, when relevant. The immunogenicity of the MSP1(42) self-associated aggregates, cross-linked chemical conjugates and monomers were compared in BALB/c mice after adsorption to aluminum hydroxide adjuvant, and in one instance in association with the TLR9 agonist CPG7909 with an aluminum hydroxide formulation. Antibody titers were assessed by ELISA. Unlike observations made for Pfs25, no significant enhancement in MSP1(42) specific antibody titers was observed for any conjugate as compared to the formulated monomer or dimer, except for the addition of the TLR9 agonist CPG7909. Clearly, enhancing the immunogenicity of a recombinant protein vaccine candidate by the formation of protein complexes must be established on an empirical basis.

The NanoPET/CT represents the latest generation of commercial preclinical PET/CT systems. This article presents a performance evaluation of the PET component of the system according to the National Electrical Manufacturers Association (NEMA) NU-4 2008 standard. Methods: The NanoPET/CT consists of 12 lutetium yttrium orthosilicate:cerium modular detectors forming 1 ring, with 9.5-cm axial coverage and a 16-cm animal port. Each detector crystal is 1.12 × 1.12 × 13 mm, and 1 module contains 81 × 39 of these crystals. An optical light guide transmits the scintillation light to the flat-panel multianode position-sensitive photomultiplier tubes. Analog-to-digital converter cards and a field-programmable gate array–based data-collecting card provide the readout. Spatial resolution, sensitivity, counting rate capabilities, and image quality were evaluated in accordance with the NEMA NU-4 standard. Energy and temporal resolution measurements and a mouse imaging study were performed in addition to the standard. Results: Energy resolution was 19% at 511 keV. The spatial resolution, measured as full width at half maximum on single-slice rebinning/filtered backprojection–reconstructed images, approached 1 mm on the axis and remained below 2.5 mm in the central 5-cm transaxial region both in the axial center and at one-quarter field of view. The maximum absolute sensitivity for a point source at the center of the field of view was 7.7%. The maximum noise equivalent counting rates were 430 kcps at 36 MBq and 130 kcps at 27 MBq for the mouse- and rat-sized phantoms, respectively. The uniformity and recovery coefficients were measured with the image-quality phantom, giving good-quality images. In a mouse study with an 18F-labeled thyroid-specific tracer, the 2 lobes of the thyroid were clearly distinguishable, despite the small size of this organ. The flexible readout system allowed experiments to be performed in an efficient manner, and the system remained stable throughout. Conclusion: The large number of detector crystals, arranged with a fine pitch, results in excellent spatial resolution, which is the best reported for currently available commercial systems. The absolute sensitivity is high over the field of view. Combined with the excellent image quality, these features make the NanoPET/CT a powerful tool for preclinical research.

The partial-volume effect (PVE) can compromise the quantitative accuracy of preclinical positron emission tomogprahy (PET) images. We investigated this effect by simulating a NEMA NU4 mouse Image Quality Phantom (including calculation of a spatially variant position-dependent point spread function (PSF) ) as well as measuring it. Three different types of deconvolution-based partial- volume corrections were applied: the Lucy-Richardson (LR), Van Cittert (VC) and Wiener (WNR) algorithms. For simulation and phantom measurement, partial-volume correction was applied. Based on optimal parameters the same methods were used to correct data of a mouse study acquired with the NanoPET/CT™ preclinical scanner (Mediso Ltd. Budapest, Hungary and and Bioscan Inc., Washington DC, USA). The algorithms used successfully improve the values of recovery coefficients to varying extents both in simulation and measurement. The preliminary mouse study shows improvement in quantification. Full validation is subject to further investigation.

Efficient antigen extraction from vaccines formulated on aluminum hydroxide gels is a critical step for the evaluation of the quality of vaccines following formulation. It has been shown in our laboratory that the efficiency of antigen extraction from vaccines formulated on Alhydrogel decreased significantly with increased storage time. To increase antigen extraction efficiency, the present study determined the effect of surfactants on antigen recovery from vaccine formulations. The Plasmodium falciparum apical membrane antigen 1 (AMA1) formulated on Alhydrogel and stored at 2–8 °C for 3 years was used as a model in this study. The AMA1 on Alhydrogel was extracted in the presence or absence of 30 mM sodium dodecyl sulfate (SDS) or 20 mM cetylpyridinium chloride in the extraction buffer (0.60 M citrate, 0.55 M phosphate, pH 8.5) using our standard antigen extraction protocols. Extracted AMA1 antigen was analyzed by 4–20% Tris–glycine SDS-PAGE followed by silver staining or western blotting. The results showed that inclusion of SDS or cetylpyridinium chloride in extraction buffer increased the antigen recovery dramatically and can be used for efficient characterization of Alhydrogel vaccines.

A new tripodal tris(hydroxypyridinone) bifunctional chelator for gallium allows easy production of (68)Ga-labelled proteins rapidly under mild conditions in high yields at exceptionally high specific activity and low concentration.

We describe the design and synthesis of a new Tc-99m labeled bioconjugate for imaging activated complement, based on Short Consensus Repeats 1 and 2 of Complement Receptor 2 (CR2), the binding domain for C3d. To avoid non specific modification of CR2 and the potential for modifying lysine residues critical to the CR2/C3d contact surface, we engineered a new protein, recombinant CR2 (rCR2), to include the C-terminal sequence VFPLECHHHHHH, a hexahistidine tag (for site-specific radiolabeling with [(99m)Tc(CO)(3)(OH(2))(3)](+)). The protein was characterized by N-terminal sequencing, SDS-PAGE and size exclusion chromatography. To test the function of the recombinant CR2, binding to C3d was confirmed by enzyme-linked immunosorbent assay (ELISA). The function was further confirmed by binding of rCR2 to C3d(+) red blood cells (RBC) which were generated by deposition of human or rat C3d and analyzed by fluorescence microscopy and flow cytometry. The affinity of rCR2 for C3d(+), in presence of 150 mM NaCl, was measured using surface plasma resonance giving rise to a K(D)≈500 nM. Radiolabeling of rCR2 or an inactive mutant of rCR2 (K41E CR2) or an unrelated protein of a similar size (C2A) with [(99m)Tc(CO)(3)(OH(2))(3)](+) at gave radiochemical yields >95%. Site-specifically radiolabeled rCR2 bound to C3d to C3d(+) RBC. Binding of radiolabeled rCR2 to C3d was inhibited by anti-C3d and the radiolabeled inactive mutant K41E CR2 and C2A did not bind to C3d(+) RBCs. We conclude that rCR2-Tc(99m) has excellent radiolabeling, stability and C3d binding characteristics and warrants in vivo evaluation as an activated complement imaging agent.

The normal function of lymphatic vessels is to facilitate the trafficking of antigen presenting cells to draining lymph nodes where they evoke an immune response. Donor lymphatic vessels are not connected to that of recipients' during organ transplantation. The pathophysiology of this disruption has received little attention. Murine heterotopic cardiac transplantation has been used extensively in transplantation research. Following vascularized organ transplantation, the main site of allosensitization is thought to be in the spleen of the recipient as a result of migration of donor passenger leukocytes via blood. Here, using Single Photon Emission Computed Tomography/Computerized Tomography (SPECT/CT) lymphoscintigraphy, we studied the pattern of lymphatic flow from mouse heterotopic abdominal cardiac grafts and identified mediastinal lymph nodes as the draining nodes for the donor graft. Staining with HY tetramer after transplantation of HY mismatched heart grafts and ELISPOT following allogeneic grafts to detect donor specific T cells revealed them as important sites for allosensitization. Our data indicates that mediastinal lymph nodes play a crucial role in the alloimmune response in this model, and should be used for ex vivo and adoptive transfer studies after transplantation in addition to the spleen.

Regulatory T cells (Tregs) were identified several years ago and are key in controlling autoimmune diseases and limiting immune responses to foreign antigens, including alloantigens. In vivo imaging techniques including intravital microscopy as well as whole body imaging using bioluminescence probes have contributed to the understanding of in vivo Treg function, their mechanisms of action and target cells. Imaging of the human sodium/iodide symporter via Single Photon Emission Computed Tomography (SPECT) has been used to image various cell types in vivo. It has several advantages over the aforementioned imaging techniques including high sensitivity, it allows non-invasive whole body studies of viable cell migration and localisation of cells over time and lastly it may offer the possibility to be translated to the clinic. This study addresses whether SPECT/CT imaging can be used to visualise the migratory pattern of Tregs in vivo. Treg lines derived from CD4(+)CD25(+)FoxP3(+) cells were retrovirally transduced with a construct encoding for the human Sodium Iodide Symporter (NIS) and the fluorescent protein mCherry and stimulated with autologous DCs. NIS expressing self-specific Tregs were specifically radiolabelled in vitro with Technetium-99m pertechnetate ((99m)TcO(4)(-)) and exposure of these cells to radioactivity did not affect cell viability, phenotype or function. In addition adoptively transferred Treg-NIS cells were imaged in vivo in C57BL/6 (BL/6) mice by SPECT/CT using (99m)TcO(4)(-). After 24 hours NIS expressing Tregs were observed in the spleen and their localisation was further confirmed by organ biodistribution studies and flow cytometry analysis. The data presented here suggests that SPECT/CT imaging can be utilised in preclinical imaging studies of adoptively transferred Tregs without affecting Treg function and viability thereby allowing longitudinal studies within disease models.

A Phase 1 trial was conducted in malaria-naïve adults to evaluate the recombinant protein vaccine apical membrane antigen 1-Combination 1 (AMA1-C1) formulated in Montanide ISA 720 (SEPPIC, France), a water-in-oil adjuvant. Vaccinations were halted early due to a formulation issue unrelated to stability or potency. Twenty-four subjects (12 in each group) were enrolled and received 5 or 20 microg protein at 0 and 3 months and four subjects were enrolled and received one vaccination of 80 microg protein. After first vaccination, nearly all subjects experienced mild to moderate local reactions and six experienced delayed local reactions occurring at Day 9 or later. After the second vaccination, three subjects experienced transient grade 3 (severe) local reactions; the remainder experienced grade 1 or 2 local reactions. All related systemic reactogenicity was grade 1 or 2, except one instance of grade 3 malaise. Anti-AMA1-C1 antibody responses were dose dependent and seen following each vaccination, with mean antibody levels 2-3 fold higher in the 20 microg group compared to the 5 microg group at most time points. In vitro growth-inhibitory activity was a function of the anti-AMA1 antibody titer. AMA1-C1 formulated in ISA 720 is immunogenic in malaria-naïve Australian adults. It is reasonably tolerated, though some transient, severe, and late local reactions are seen.

A double blind, randomized and controlled Phase 1 clinical trial was conducted to assess the safety and immunogenicity in malaria-exposed adults of the Plasmodium falciparum blood stage vaccine candidate Apical Membrane Antigen 1-Combination 1 (AMA1-C1)/Alhydrogel with and without the novel adjuvant CPG 7909. Participants were healthy adults 18-45 years old living in the village of Donéguébougou, Mali. A total of 24 participants received 2 doses one month apart of either 80 microg AMA1-C1/Alhydrogel or 80 microg AMA1-C1/Alhydrogel + 564 microg CPG 7909. The study started in October 2007 and completed follow up in May 2008. Both vaccines were well tolerated, with only mild local adverse events and no systemic adverse events judged related to vaccination. The difference in antibody responses were over 2-fold higher in the group receiving CPG 7909 for all time points after second vaccination and the differences are statistically significant (all p0.05). This is the first use of the novel adjuvant CPG 7909 in a malaria-exposed population.

A Phase 1 study was conducted in 24 malaria naive adults to assess the safety and immunogenicity of the recombinant protein vaccine apical membrane antigen 1-Combination 1 (AMA1-C1)/Alhydrogel with CPG 7909 in two different formulations (phosphate buffer and saline), and given at two different dosing schedules, 0 and 1 month or 0 and 2 months. Both formulations were well tolerated and frequency of local reactions and solicited adverse events was similar among the groups. Peak antibody levels in the groups receiving CPG 7909 in saline were not significantly different than those receiving CPG 7909 in phosphate. Peak antibody levels in the groups vaccinated at a 0,2 month interval were 2.52-fold higher than those vaccinated at a 0,1 month interval (p=0.037, 95% CI 1.03, 4.28). In vitro growth inhibition followed the antibody level: median inhibition was 51% (0,1 month interval) versus 85% (0,2 month interval) in antibody from samples taken 2 weeks post-second vaccination (p=0.056).