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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.

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

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 ] + .

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.

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.

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.

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 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.

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 double blind, randomized, controlled Phase 2 clinical trial was conducted to assess the safety, immunogenicity, and biologic impact of the vaccine candidate Apical Membrane Antigen 1- Combination 1 (AMA1-C1), adjuvanted with Alhydrogel®. Participants were healthy children 2-3 years old living in or near the village of Bancoumana, Mali. A total of 300 children received either the study vaccine or the comparator. No impact of vaccination was seen on the primary endpoint, the frequency of parasitemia measured as episodes >3000 per μL per day at risk. There was a negative impact of vaccination on the hemoglobin level during clinical malaria, and mean incidence of hemoglobin

In this paper we report our efforts to enhance the immunogenicity of Pfs28, a transmission blocking vaccine candidate of Plasmodium falciparum, using a strategy of chemical conjugation. With an improved procedure, Pfs28 was covalently coupled to the mutant and non-toxic ExoProtein A of Pseudomonas aeruginosa by the reaction between thiolated antigen and maleimide modified carrier protein. The optimized process resulted in a higher antigen-carrier conjugation ratio, and the conjugation product could be purified using single-step size-exclusion chromatography. A significant increase in immunogenicity measured by ELISA was observed in mice immunized with conjugated Pfs28 as compared to unconjugated Pfs28.

Background Apical Membrane Antigen 1 (AMA1) of Plasmodium falciparum merozoites is a leading blood-stage malaria vaccine candidate. Protection of Aotus monkeys after vaccination with AMA1 correlates with antibody responses. Study Design/Results A randomized, controlled, double-blind phase 1 clinical trial was conducted in 54 healthy Malian adults living in an area of intense seasonal malaria transmission to assess the safety and immunogenicity of the AMA1-C1 malaria vaccine. AMA1-C1 contains an equal mixture of yeast-expressed recombinant proteins based on sequences from the FVO and 3D7 clones of P. falciparum, adsorbed on Alhydrogel. The control vaccine was the hepatitis B vaccine (Recombivax). Participants were enrolled into 1 of 3 dose cohorts (n = 18 per cohort) and randomized 2:1 to receive either AMA1-C1 or Recombivax. Participants in the first, second, and third cohorts randomized to receive AMA1-C1 were vaccinated with 5, 20 and 80 µg of AMA1-C1, respectively. Vaccinations were administered on days 0, 28, and 360, and participants were followed until 6 months after the final vaccination. AMA1-C1 was well tolerated; no vaccine-related severe or serious adverse events were observed. AMA1 antibody responses to the 80 µg dose increased rapidly from baseline levels by days 14 and 28 after the first vaccination and continued to increase after the second vaccination. After a peak 14 days following the second vaccination, antibody levels decreased to baseline levels one year later at the time of the third vaccination that induced little or no increase in antibody levels. Conclusions Although the AMA1-C1 vaccine candidate was well-tolerated and induced antibody responses to both vaccine and non-vaccine alleles, the antibody response after a third dose given at one year was lower than the response to the initial vaccinations. Additionally, post-vaccination increases in anti-AMA1 antibody levels were not associated with significant changes in in vitro growth inhibition of P. falciparum. Trial Registration ClinicalTrials.gov NCT00343005

Plasmacytoid dendritic cells (DC) are professional APC and a major source of type I IFN following viral infection. We previously showed that histamine alters the cytokine profiles of maturing monocyte-derived DC resulting in a change from Th1 to Th2 in their T cell polarizing function. In this study, we show that human plasmacytoid DC, activated by either CpG oligodeoxynucleotides or viral infection, also respond to histamine through H2 receptors, leading to a marked down-regulation of IFN-α and TNF-α and a moderate switch in their capacity to polarize naive T cells. Our findings provide an explanation for low levels of type I IFN frequently observed in atopic individuals.

Background Chimeric antigen receptors (CARs) are fusion molecules that re-direct T-cell specificity against a selected cell surface target. Recent successes in the treatment of haematological malignancies using CD19-specific CAR T cells has moved this approach into the clinical mainstream. On target, off tumour toxicity, inadequate homing, and limited survival of CAR T cells are important challenges to the adaptation of this promising technology to treat solid tumours. We aimed to address these limitations while developing a clinically applicable T-cell immunotherapy for the commonest male malignancy, prostate cancer. Targeting has been directed against prostate-specific membrane antigen (PSMA), a cell-surface glycoprotein that is overexpressed by more than 80% of cases, with highest levels in castration-resistant disease Methods A tripartite cassette of genes named PIN4 was developed to re-direct human T cells against prostate cancer. PIN4 mediates the stoichiometric co-expression of: P28ζ, a CAR specific for PSMA; the human sodium iodide symporter (hNIS), which allows real-time PET/SPECT imaging of T cells; and 4αβ, a chimeric cytokine receptor that permits ex-vivo expansion and enrichment of PIN4 -positive T cells using interleukin (IL) 4. To promote the survival and trafficking of PIN4 T cells to the tumour, methods for targeting IL4 to tumour-associated stroma are being developed. To test these immunotherapeutic strategies in vivo, we developed a metastatic model of prostate cancer using PSMA–firefly luciferase-engineered PC3-LN3 (PLP) cells, inoculated subcutaneously in immune compromised SCID/beige mice. Tumour metastases in this model were imaged with bioluminescence imaging. SPECT was used for serial imaging of T cells, after administration of technetium-99m pertechnetate ( 99m TcO4). Findings Ex-vivo expansion accompanied by selective enrichment of PIN4 + T cells occurred consistently in response to IL4, whereas comparable enrichment was not seen in response to the non-selective signal provided by IL2. IL4-expanded PIN4 + T cells retained type 1 polarity and showed potent P28ζ-dependent anti-tumour activity against a panel of PSMA-positive prostate cancer cell lines. Function of hNIS in PIN4 + T cells was first confirmed in vitro by 99m TcO4 uptake studies. Then, we infused PIN4 + or control T cells in mice with metastatic PSMA+ PLP prostate cancer. We showed that PIN4 + T cells could be repeatedly imaged by SPECT, after the administration of 99m TcO4. Evidence of proliferation of PIN4 + T cells at sites of metastatic PLP deposits was observed. Interpretation We have a highly effective strategy for prostate cancer therapy with PIN4 , which overcomes the challenges of ex-vivo expansion and T-cell survival. The goal of this work is to further improve the therapeutic window for PIN4 therapy, with a clear view to clinical translation. Funding The Prostate Cancer Charity, Academy of Medical Sciences, King's Health Partners Biomedical Research Centre.

Background Hitherto, in vivo studies of human granulocyte migration have been based on indiscriminate labeling of total granulocyte populations. We hypothesized that the kinetics of isolated human neutrophil and eosinophil migration through major organs in vivo are fundamentally different, with the corollary that studying unseparated populations distorts measurement of both. Methods Blood neutrophils and eosinophils were isolated on 2 separate occasions from human volunteers by using Current Good Manufacturing Practice CD16 CliniMACS isolation, labeled with technetium 99m–hexamethylpropyleneamine oxime, and then reinfused intravenously. The kinetics of cellular efflux were imaged over 4 hours. Results Neutrophils and eosinophils were isolated to a mean purity of greater than 97% and greater than 95%, respectively. Activation of neutrophils measured as an increase in their CD11b mean fluorescence intensity in whole blood and after isolation and radiolabeling was 25.98 ± 7.59 and 51.82 ± 17.44, respectively, and was not significant ( P = .052), but the mean fluorescence intensity of CD69 increased significantly on eosinophils. Analysis of the scintigraphic profile of lung efflux revealed exponential clearance of eosinophils, with a mean half-life of 4.16 ± 0.11 minutes. Neutrophil efflux was at a significantly slower half-life of 13.72 ± 4.14 minutes ( P = .009). The migration of neutrophils and eosinophils was significantly different in the spleen at all time points ( P = .014), in the liver at 15 minutes ( P = .001), and in the bone marrow at 4 hours ( P = .003). Conclusions The kinetics of migration of neutrophils and eosinophils through the lung, spleen, and bone marrow of human volunteers are significantly different. Study of mixed populations might be misleading.

Introduction To date, our knowledge of in vivo migration of neutrophils and eosinophils in homeostasis and disease states is based on granulocytes. Here we present a pilot study using purified human eosinophils or neutrophils and demonstrate their differential in vivo kinetics in asthmatic and healthy volunteers. Methods: On two separate occasions 100 ml of blood was obtained from eight human volunteers (4 mild stable asthmatics and 4 non asthmatic, healthy volunteers) Granulocytes were separated using gradient Ficoll-Paque PLUS 1.084 centrifugation. Superparamagnetic particles coupled to a monoclonal antibody against CD16, a surface marker present in neutrophils, were incubated with the granulocytes (containing eosinophils and neutrophils). CliniMACS system (Miltenyi biotec, Bergisch-Gladbach, Germany; and Becton-Dickinson, Oxford, UK) was used to obtain highly purified (>93% pure) human blood eosinophils or neutrophils (>; 97%). Purified cells were labelled with Tc-99m HMPAO (Ceretec, GE Healthcare) under aseptic cGMP conditions and 75–100 MBq of labelled cells were administered intravenously. Dynamic lung images were acquired for the first 30 minutes. Further static scans of 5 minutes each were acquired at 1; 2 and 4 hours. Results: We were able to obtain highly purified neutrophils (positive selection) or eosinophils (negative selection). Kinetics of eosinophils in lung, liver and spleen differed significantly from kinetics of neutrophils. Initial dynamic lung images revealed a significant difference in the time activity curves for eosinophils and neutrophils. Migration of eosinophils from the lungs followed a monexponential clearance (t1/2) of 4.16 min. While neutrophil had significantly different clearance half-lives of 13.72 min (p=0.0019). There were significant differences in eosinophil and neutrophil migration and distribution in the liver and spleen (p Conclusions For the first time it has been possible to identify distinct patterns of neutrophil and eosinophil migration through lung, liver and spleen in both healthy volunteers and stable asthmatics. This technique provides the opportunity for rapid throughput screening of novel therapeutic agents designed to alter leukocyte migration in disease conditions, or to further phenotype disease such as asthma.

Dendritic cells (DCs) generated in vitro to present tumour antigens have been injected in cancer patients to boost in vivo anti-tumour immune responses. This approach to cancer immunotherapy has had limited success. For anti-tumour therapy, delivery and subsequent migration of DCs to lymph nodes leading to effective stimulation of effector T cells is thought to be essential. The ability to non-invasively monitor the fate of adoptively transferred DCs in vivo using magnetic resonance imaging (MRI) is an important clinical tool to correlate their in vivo behavior with response to treatment. Previous reports of superparamagnetic iron oxides (SPIOs) labelling of different cell types, including DCs, have indicated varying detrimental effects on cell viability, migration, differentiation and immune function. Here we describe an optimised labelling procedure using a short incubation time and low concentration of clinically used SPIO Endorem to successfully track murine DC migration in vivo using MRI in a mouse tumour model. First, intracellular labelling of bone marrow derived DCs was monitored in vitro using electron microscopy and MRI relaxometry. Second, the in vitro characterisation of SPIO labelled DCs demonstrated that viability, phenotype and functions were comparable to unlabelled DCs. Third, ex vivo SPIO labelled DCs, when injected subcutaneously, allowed for the longitudinal monitoring by MR imaging of their migration in vivo. Fourth, the SPIO DCs induced the proliferation of adoptively transferred CD4(+) T cells but, most importantly, they primed cytotoxic CD8(+) T cell responses to protect against a B16-Ova tumour challenge. Finally, using anatomical information from the MR images, the immigration of DCs was confirmed by the increase in lymph node size post-DC injection. These results demonstrate that the SPIO labelling protocol developed in this study is not detrimental for DC function in vitro and in vivo has potential clinical application in monitoring therapeutic DCs in patients with cancer.

Background Merozoite surface protein 142 (MSP142) is a leading blood stage malaria vaccine candidate. In order to induce immune responses that cover the major antigenic polymorphisms, FVO and 3D7 recombinant proteins of MSP142 were mixed (MSP142-C1). To improve the level of antibody response, MSP142-C1 was formulated with Alhydrogel plus the novel adjuvant CPG 7909. Methods A Phase 1 clinical trial was conducted in healthy malaria-naïve adults at the Center for Immunization Research in Washington, D.C., to evaluate the safety and immunogenicity of MSP142-C1/Alhydrogel +/− CPG 7909. Sixty volunteers were enrolled in dose escalating cohorts and randomized to receive three vaccinations of either 40 or 160 µg protein adsorbed to Alhydrogel +/− 560 µg CPG 7909 at 0, 1 and 2 months. Results Vaccinations were well tolerated, with only one related adverse event graded as severe (Grade 3 injection site erythema) and all other vaccine related adverse events graded as either mild or moderate. Local adverse events were more frequent and severe in the groups receiving CPG. The addition of CPG enhanced anti-MSP142 antibody responses following vaccination by up to 49-fold two weeks after second immunization and 8-fold two weeks after the third immunization when compared to MSP142-C1/Alhydrogel alone (p

Background Apical Membrane Antigen 1 (AMA1), a polymorphic merozoite surface protein, is a leading blood-stage malaria vaccine candidate. This is the first reported use in humans of an investigational vaccine, AMA1-C1/Alhydrogel, with the novel adjuvant CPG 7909. Methods A phase 1 trial was conducted at the University of Rochester with 75 malaria-naive volunteers to assess the safety and immunogenicity of the AMA1-C1/Alhydrogel+CPG 7909 malaria vaccine. Participants were sequentially enrolled and randomized within dose escalating cohorts to receive three vaccinations on days 0, 28 and 56 of either 20 microg of AMA1-C1/Alhydrogel+564 microg CPG 7909 (n = 15), 80 microg of AMA1-C1/Alhydrogel (n = 30), or 80 microg of AMA1-C1/Alhydrogel+564 microg CPG 7909 (n = 30). Results Local and systemic adverse events were significantly more likely to be of higher severity with the addition of CPG 7909. Anti-AMA1 immunoglobulin G (IgG) were detected by enzyme-linked immunosorbent assay (ELISA), and the immune sera of volunteers that received 20 microg or 80 microg of AMA1-C1/Alhydrogel+CPG 7909 had up to 14 fold significant increases in anti-AMA1 antibody concentration compared to 80 microg of AMA1-C1/Alhydrogel alone. The addition of CPG 7909 to the AMA1-C1/Alhydrogel vaccine in humans also elicited AMA1 specific immune IgG that significantly and dramatically increased the in vitro growth inhibition of homologous parasites to levels as high as 96% inhibition. Conclusion/significance The safety profile of the AMA1-C1/Alhydrogel+CPG 7909 malaria vaccine is acceptable, given the significant increase in immunogenicity observed. Further clinical development is ongoing. Trial registration ClinicalTrials.gov NCT00344539.

Background Apical Membrane Antigen-1 (AMA1) is one of the leading blood stage malaria vaccine candidates. AMA1-C1/Alhydrogel® consists of an equal mixture of recombinant AMA1 from FVO and 3D7 clones of P. falciparum, adsorbed onto Alhydrogel®. A Phase 1 study in semi-immune adults in Mali showed that the vaccine was safe and immunogenic, with higher antibody responses in those who received the 80 µg dose. The aim of this study was to assess the safety and immunogenicity of this vaccine in young children in a malaria endemic area. Design This was a Phase 1 dose escalating study in 36 healthy children aged 2–3 years started in March 2006 in Donéguébougou, Mali. Eighteen children in the first cohort were randomized 2∶1 to receive either 20 µg AMA1-C1/Alhydrogel® or Haemophilus influenzae type b Hiberix® vaccine. Two weeks later 18 children in the second cohort were randomized 2∶1 to receive either 80 µg AMA1-C1/Alhydrogel® or Haemophilus influenzae type b Hiberix® vaccine. Vaccinations were administered on Days 0 and 28 and participants were examined on Days 1, 2, 3, 7, and 14 after vaccination and then about every two months. Results to Day 154 are reported in this manuscript. Results Of 36 volunteers enrolled, 33 received both vaccinations. There were 9 adverse events related to the vaccination in subjects who received AMA1-C1 vaccine and 7 in those who received Hiberix®. All were mild to moderate. No vaccine-related serious or grade 3 adverse events were observed. There was no increase in adverse events with increasing dose of vaccine or number of immunizations. In subjects who received the test vaccine, antibodies to AMA1 increased on Day 14 and peaked at Day 42, with changes from baseline significantly different from subjects who received control vaccine. Conclusion AMA-C1 vaccine is well tolerated and immunogenic in children in this endemic area although the antibody response was short lived. Trial Registration Clinicaltrials.gov NCT00341250