Incubation Of Serum Research Articles

Recombinant MS087-based indirect ELISA for the diagnosis of Mycoplasma synoviae.

Accurate detection is a prerequisite for effective prevention and control of Mycoplasma synoviae infection. ELISA is the most popular method for the clinical detection of M. synoviae because of its convenience, low cost, and high detection rate. However, the cross-reactivity of commercially available ELISA kits with other avian pathogen-positive sera needs to be addressed. The aim of this study was to establish an ELISA method with high specificity for the detection of anti-M. synoviae antibodies in chicken serum to evaluate the M. synoviae infection status on poultry farms. The recombinant MS087 (rMS087) protein was expressed in Escherichia coli BL21 (DE3) and purified by Ni2+ affinity chromatography. An antibody against rMS087 was generated by immunizing BALB/c mice. Bioinformatic analysis revealed that MS087 was conserved among M. synoviae strains. Western blotting and indirect immunofluorescence results indicated that MS087 was not only localized in the cytoplasm and on the membrane but also secreted by the organism. For the established ELISA method based on rMS087, the optimal antigen concentration, blocking buffer, blocking duration, serum dilution, serum incubation duration, secondary antibody dilution, secondary antibody incubation duration and colorimetric reaction duration were 2 μg/mL, 1% BSA, 3 h, 1:500, 1.5 h, 1:20,000, 2 h and 5 min, respectively. Validation of the rMS087-based ELISA revealed a cut-off value of 0.5. The coefficients of variation of both the intra-batch and inter-batch methods were less than 9%. The assay was able to differentiate positive serum against M. synoviae from antisera against nine other avian pathogens and was able to recognize M. synoviae-positive sera at a dilution of 1:1,000. Compared with the commercial ELISA method, the rMS087-based ELISA has the potential to recognize more positive sera against M. synoviae. Collectively, the rMS087-based ELISA is a reproducible, specific, and sensitive serological method for detecting antibodies against M. synoviae in chicken serum and has robust potential for large-scale serological epidemiology of M. synoviae infection on poultry farms.

Technetium-99m radiolabeling of graphene quantum dots (GQDs) as a new probe for glioblastoma tumor imaging

Purpose Cancer diagnosis involves a multi-step process. Accurate identification of the tumor, staging and development of cancer cells is crucial for selecting optimal treatments to minimize disease recurrence. Quantum dots (QDs) represent an exciting class of fluorescent nanoprobes in molecular detection and targeted tumor imaging. Materials and methods In this study, graphene quantum dots (GQDs) were synthesized by pyrolysis of citric acid (CA) as a carbon precursor under high temperatures. The morphology of the obtained GQDs was first characterized using physical (TEM and DLS) and spectroscopic (fluorescence, FTIR and UV–Vis) methods. In the following,99mTc-labeled GQDs were prepared in the presence of SnCl2.2H2O as a reducing agent between 95 and 100 °C. The biodistribution and tumor targeting efficiency of radiolabeled GQDs as a novel agent for C6 glioma tumor scintigraphy in an animal model were evaluated. Furthermore, organ uptake, human serum albumin binding and tumor accumulation were measured. Results The TEM image of the prepared GQDs showed a relatively uniform size distribution in the range of diameter 6-9 nm and spherical shape. Radiolabeled GQDs showed a radiochemical yield of >97% (n = 3). Through incubation in human serum, almost 15% of 99mTc-labeled GQDs degraded after 6 h. The amount of uptake in xenograft models of glioma C6 rats was 1.10 ± 0.36% of injection dose per gram after 1 h. The kidneys, intestinal and glioma tumor sites were observed via scintigraphy imaging. Conclusion Our data suggest that 99mTc-labeled GQDs, as a new radiotracer, efficiently accumulate in the tumor site and could be included as a radiotracer for detecting glioma tumors.

H3trica: Versatile Macrocyclic Chelator for [225Ac]Ac3+ and [155/161Tb]Tb3+ Theranostics.

H3trica is a nonadentate chelating ligand intended for coordinating large radiometal ions, such as those used in nuclear medicine. This chelator, featuring a triaza-18-crown-6 macrocycle with three pendant carboxylic acid functional groups, was synthesized and characterized. Complementary nuclear magnetic resonance (NMR) spectroscopy and high-resolution electrospray-ionization mass spectroscopy (HR-ESI-MS) studies were used to explore the coordination of H3trica with metal ions such as La3+, Y3+ (as a model for Tb3+), and Lu3+ at the bulk scale. Thermodynamic solution studies provided valuable insights, highlighting robust metal complexation of H3trica with La3+, Tb3+, and Lu3+, with the most noteworthy log KML value observed for Tb3+ (log KTbL = 17.08), followed by La3+ (log KLaL = 16.64) and Lu3+ (log KLuL = 16.25). Concentration-dependent radiolabeling studies with [225Ac]Ac3+, [155Tb]Tb3+, and [161Tb]Tb3+ demonstrated rapid complexation (5-30 min) under mild conditions (pH 6-7, 25 °C). Importantly, the radiolabeled complexes exhibited stability during incubation in human serum for one-half-life of the corresponding radiometal. Thus, H3trica emerges as a valuable chelator, demonstrating its potential to coordinate the theranostic couple [225Ac]Ac3+/[155Tb]Tb3+ as well as the powerful terbium quartet ([149/152/155/161Tb]Tb3+) with efficiency and stability.

Polyvinyl alcohol-silk fibroin composite stents: A comprehensive investigation into biocompatibility and mechanical performance

Bioresorbable stents (BRS) are manufactured using biodegradable materials. As an alternative to those commonly used in commercial stents, this study explored the development of BRS using polyvinyl alcohol (PVA) and silk fibroin (SF). PVA is a promising material for the fabrication of BRS due to its biocompatibility and mechanical attributes, closely resembling those of aortic vessels. However, its application presents challenges in terms of cell adhesion and proliferation. SF has been extensively studied for its potential to enhance cell adhesion and proliferation, making it a promising biomaterial in the field of medical devices. SF was introduced by dissolving it in a PVA solution or by coating the hydrogel surface with a layer of SF. Initial tests revealed that overnight incubation of fetal bovine serum significantly increased cell viability in hydrogels. Viability assays confirmed that SF substantially improved cell viability compared to PVA alone. The method was extended to fabricate SF-coated stents, which demonstrated robust cell proliferation and improved performance compared to electrospun polycaprolactone scaffolds. In addition, the SF-coated stents displayed an increase in compressive strength, demonstrating improved biocompatibility and mechanical performance. Dynamic mechanical analysis evaluated the positive impact of SF on stent properties at physiological temperatures. The study revealed that PVA-SF stents offer a compromise between biocompatibility, mechanical strength, and elastic recovery, positioning them as a valuable alternative for cardiovascular stent applications. The dual benefits of enhanced biocompatibility and improved mechanical performance make SF-coated stents promising candidates for bioresorbable stent design.  

A Physiochemical, In Vitro, and In Vivo Comparative Analysis of Verteporfin-Lipid Conjugate Formulations: Solid Lipid Nanoparticles and Liposomes.

VisudyneⓇ, a liposomal formulation of verteporfin (benzoporphyrin derivative; BPD), is the only nanomedicine approved to date for photodynamic therapy (PDT). We have previously demonstrated that BPD conjugated to the lysophospholipid 1-arachidoyl-2-hydroxy-sn-glycero-3-phosphocholine (BPD-PC) exhibits the greatest physical stability in liposomes, while maintaining cancer cell phototoxicity, from a panel of BPD lipid conjugates evaluated. In this study, we prepared 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)-based solid lipid nanoparticles (LNPs) that stably entrap BPD-PC, which resemble the composition of the SpikevaxⓇ Moderna COVID-19 vaccine, and compared them to a DPPC based liposomal formulation (Lipo BPD-PC). We evaluated the photochemical, optical, and phototherapeutic properties of both formulations. We also investigated the in vivo distribution and tumor microdistribution of both formulations. Our results demonstrated that Lipo BPD-PC is able to generate 17% more singlet oxygen than LNP BPD-PC, while interestingly, LNP BPD-PC is able to produce 76% more hydroxyl radicals and/or peroxynitrite anion. Importantly, only 28% of BPD-PC leaches out of the LNP BPD-PC formulation during 7 days of incubation in serum at 37 °C, while 100% of BPD-PC leaches out of the Lipo BPD-PC formulation under the same conditions. Despite these differences, there was no significant difference in cellular uptake of BPD-PC or phototoxicity in CT1BA5 murine pancreatic cancer cells (derived from a genetically engineered mouse model). Interestingly, PDT using LNP BPD-PC was more efficient at inducing immunogenic cell death (calreticulin membrane translocation) than Lipo BPD-PC when using IC25 and IC50 PDT doses. In vivo studies revealed that CT1BA5 tumor fluorescence signals from BPD-PC were 2.41-fold higher with Lipo BPD-PC than with LNP BPD-PC; however, no significant difference was observed in tumor tissue selectivity or tumor penetration. As such, we present LNP BPD-PC as a unique and more stable nanoplatform to carry BPD lipid conjugates, such as BPD-PC, with a potential for future photodynamic immune priming studies and multiagent drug delivery.

Respiratory and Metabolic Responses of CD4 + T Cells to Acute Exercise and Their Association with Cardiorespiratory Fitness.

The study aimed to investigate to what extent acute endurance exercise, especially eccentric exercise and cardiorespiratory fitness, affects the metabolic profile of CD4 + cells. Fifteen male, healthy adults aged between 20 and 33 yr with a maximal oxygen uptake (V̇O 2max ) between 44 and 63 mL·kg -1 ·min -1 performed a downhill run (DR) and a level run (LR) for 45 min at 70% of their V̇O 2max on a treadmill in a crossover design. Blood samples were taken before (T0), directly after (T1), 3 h after (T3), and 24 h (T24) after each exercise for analyzing leukocyte numbers and cytokine levels. Isolated CD4 + cells were incubated for 4 h in autologous resting versus 3 h after exercise serum (T3 DR and T3 LR), and subsequently, cellular respiration, transcriptomic, and metabolomics profiles were measured. The systemic immune inflammation index increased significantly after DR and LR at T1 and T3 ( P < 0.001). In contrast, the transcriptomic and metabolic profile of CD4 + cells showed no significant alterations after incubation in T3 exercise serum. However, cardiorespiratory fitness positively correlated with the maximal mitochondrial respiration in CD4 + cells after incubation with T3 LR serum ( r = 0.617, P = 0.033) and with gene expression of oxidative phosphorylation and levels of different metabolites. Similarly, V̇O 2max was associated with an anti-inflammatory profile on RNA level. Lower lactate, methylmalonic acid, and d -gluconic acid levels were found in CD4 + cells of participants with a high V̇O 2max ( P < 0.001). Acute exercise leads to a mild proinflammatory milieu with only small changes in the metabolic homeostasis of CD4 + cells. High cardiorespiratory fitness is associated with a metabolic shift to oxidative phosphorylation in CD4 + cells. Functional relevance of this metabolic shift needs to be further investigated.

Liquid Chromatography ICP-MS to Assess the Stability of 175Lu- and natGa-Based Tumor-Targeting Agents towards the Development of 177Lu- and 68Ga-Labeled Radiopharmaceuticals.

In recent years, nuclear medicine has gained great interest, partly due to the success story of [177Lu]Lu-PSMA-617 (PluvictoTM). Still, in-depth preclinical characterization of radiopharmaceuticals mainly happens at centers that allow working with radioactive material. To support the development of novel radiopharmaceuticals, alternative non-radioactive characterization assays are highly desirable. The aim of this study was to demonstrate that inductively coupled plasma mass spectrometry (ICP-MS) associated with a chromatographic system can serve as a surrogate for the classical high-performance liquid chromatography (HPLC)-radiodetector combination for preclinical in vitro characterization of non-radioactive metal-labeled analogs of radiopharmaceuticals. In this proof-of-concept study, we demonstrate the applicability of HPLC-ICP-MS by assessing the stability of 175Lu- and natGa-labeled prostate-specific membrane antigen (PSMA)-targeting peptidomimetics, single domain antibody (sdAb) conjugates, and monoclonal antibody (mAb) conjugates. 175Lu-labeled DOTAGA-conjugated and natGa-labeled NODAGA-conjugated sdAbs and mAbs showed the highest stability with >90% still intact after 24 h. The peptidomime-tics [175Lu]Lu-PSMA-617 and [natGa]Ga-PSMA-11 showed identical in vitro serum stability as it was reported for their corresponding radioligands with >99% intact species after 24 h incubation in mouse serum, demonstrating the reliability of the method. Hence, the established HPLC-ICP-MS methods can support the development of novel radiopharmaceuticals in a classical pharmaceutical setting.

Immunoproteomics reveal increased serum IgG3/5 binding to Dermatophagoides and yeast protein antigens in severe equine asthma in a preliminary study.

Severe equine asthma (SEA) is a common, chronic respiratory disease of horses characterized by hyperreactivity to hay dust which has many similarities to severe neutrophilic asthma in humans. SEA-provoking antigens have not been comprehensively characterized, but molds and mites have been suggested as relevant sources. Here, we identified relevant antigen candidates using immunoproteomics with IgG isotype-binding analyses. Proteins from Dermatophagoides pteronyssinus (Der p) were separated by two-dimensional gel electrophoresis followed by immunoblotting (2D immunoblots) resulting in a characteristic pattern of 440 spots. After serum incubation, antibody (Ig)-binding of all Ig (Pan-Ig) and IgG isotypes (type-2-associated IgG3/5, type-1-associated IgG4/7) was quantified per each spot and compared between asthmatic and healthy horses' sera (n=5 per group). Ig binding differences were detected in 30 spots. Pan-Ig binding was higher with asthmatics compared to healthy horses' sera on four spots, and IgG3/5 binding was higher on 18 spots. Small IgG4/7 binding differences were detected on 10 spots with higher binding with asthmatics' sera on four but higher binding with healthy horses' sera on six spots. Proteins from the spots with group differences including mite and yeast proteins were identified by liquid chromatography mass spectrometry. The latter likely originated from the feeding substrate of the Der p culture. Prioritized antigen candidates amongst the proteins identified were Der p 1, Der p 11, group 15 allergens, myosin heavy chain, and uncharacterized Der p proteins. Additionally, yeast enolases, alcohol dehydrogenase (ADH), phosphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase, and heat shock proteins were prioritized. Eleven antigen candidates were tested for confirmation by ELISAs using the respective proteins separately. Differences in asthmatics vs. healthy horses' serum Ig binding to Der p 1, Der p 18, and three yeast enzymes (enolase, ADH, and PGK) confirmed these as promising antigens of immune responses in SEA. Antigens with relevance in SEA were newly identified by immunoproteomics, and yeast antigens were considered for SEA for the first time. Serum IgG3/5 binding to relevant antigens was increased in SEA and is a novel feature that points to increased type-2 responses in SEA but requires confirmation of the corresponding cellular responses.

Regulating the structural polymorphism and protein corona composition of phytantriol-based lipid nanoparticles using choline ionic liquids

Lipid-based lyotropic liquid crystalline nanoparticles (LCNPs) face stability challenges in biological fluids during clinical translation. Ionic Liquids (ILs) have emerged as effective solvent additives for tuning the structure of LCNP’s and enhancing their stability. We investigated the effect of a library of 21 choline-based biocompatible ILs with 9 amino acid anions as well as 10 other organic/inorganic anions during the preparation of phytantriol (PHY)-based LCNPs, followed by incubation in human serum and serum proteins. Small angle X-ray scattering (SAXS) results show that the phase behaviour of the LCNPs depends on the IL concentration and anion structure. Incubation with human serum led to a phase transition from the inverse bicontinuous cubic (Q2) to the inverse hexagonal (H2) mesophase, influenced by the specific IL present. Liquid chromatography-mass spectrometry (LC-MS) and proteomics analysis of selected samples, including PHY control and those with choline glutamate, choline hexanoate, and choline geranate, identified abundant proteins in the protein corona, including albumin, apolipoproteins, and serotransferrin. The composition of the protein corona varied among samples, shedding light on the intricate interplay between ILs, internal structure and surface chemistry of LCNPs, and biological fluids.

Serum Stabilities and Antiviral Activities of Chemically Modified Peptides Against Dengue Serotypes 1–4

Dengue presents a major public health concern in over 100 countries due to the absence of an effective vaccine and antiviral therapy against all four dengue virus (DENV) serotypes. Several antiviral peptides were previously reported to inhibit at least three or all four DENV serotypes. Chemical modifications such as d-amino acid substitutions, polyethylene glycol (PEG)ylation, and cyclization could be applied to peptides to improve their biological activities and stability in serum. The PEGylated peptide 3 (PEG-P3) was identified to be the most promising antiviral candidate as it demonstrated good inhibitory effects against all four DENV serotypes during the pre- and post-infection stages, Based on the RP-HPLC and LC/MS analysis, peptide 4 was identified to be more stable in human serum than peptide 3, with 78.9 % and 41.6 % of the peptides remaining after 72 h of incubation in human serum, respectively. Both peptides were also able to retain their antiviral activities against specific DENV serotypes after 72 h incubation in human serum. PEG-P3 was found to be more stable than the unmodified peptide 3 with 89.4 % of PEG-P3 remaining in the human serum after 72 h of incubation. PEG-P3 was able to retain its inhibitory effects against DENV-1 to 4 after 72 h of incubation in human serum. This study provided insights into the antiviral activities and stabilities of the unmodified and chemically modified peptides in human serum.

Initial investigation on the feasibility of porcine red blood cells from genetically modified pigs as an alternative to human red blood cells for transfusion.

The decline in blood donation rates and the ongoing shortage of blood products pose significant challenges to medical societies. One potential solution is to use porcine red blood cells (pRBCs) from genetically modified pigs as an alternative to human red blood cells (hRBCs). However, adverse immunological reactions remain a significant obstacle to their use. This study aimed to evaluate the compatibility of diverse genetically modified pRBCs with human serum. We acquired human complement-competent serum, complement 7 (C7)-deficient serum, and hRBCs from all ABO blood types. Additionally, we used leftover clinical samples from health checkups for further evaluation. pRBCs were collected from wild-type (WT) and genetically modified pigs: triple knockout (TKO), quadruple KO (QKO), and TKO/hCD55.hCD39 knockin (hCD55.hCD39KI). The extent of C3 deposition on RBCs was measured using flow cytometry after incubation in C7-deficient serum diluted in Ca++-enriched or Ca++-depleted and Mg++-enriched buffers. The binding of immunoglobulin (Ig) M/IgG antibody to RBCs after incubation in ABO-type human serum was evaluated using flow cytometry. Naïve human serum- or sensitized monkey serum-mediated hemolysis was also evaluated. Phagocytosis was assessed by incubating labeled RBCs with the human monocytic cell line THP-1 and measurement by flow cytometry. All three genetic modifications significantly improved the compatibility of pRBCs with human serum relative to that of WT pRBCs. The extent of IgM/IgG binding to genetically modified pRBCs was lower than that of WT pRBCs and similar to that of O-type hRBCs. Total and alternative pathway complement activation in all three genetically modified pRBCs was significantly weaker than that in WT pRBCs and did not differ from that in O-type hRBCs. The extent of serum-mediated hemolysis and phagocytosis of these genetically modified pRBCs was low and similar to that of O-type hRBCs. Sensitized monkey serum-mediated hemolysis in QKO and TKO/hCD55.hCD39KI pRBCs was higher than in O-type hRBCs but lower than in TKO pRBCs. The elimination of porcine carbohydrate antigens in genetically modified pigs significantly enhanced pRBC compatibility with naïve human sera, which was comparable to that of O-type hRBCs. These findings provide valuable insights into the development of pRBCs as potential alternatives to hRBCs.

Malaria sporozoites evade host complement attack.

Complement is the first line of the host innate immune response against bacterial and viral infections; however, its role in the development of the malaria liver stage remains undefined. We found that sporozoite infection by either a mosquito bite or intravenous injection activated systemic complement, but neither depletion of C3 nor knockout of C3 had a significant effect on malaria liver stage development. Incubation of mouse serum with trypsin-treated sporozoites, but not naive sporozoites, led to the deposition of a membrane attack complex (MAC) on the surface of sporozoites and greatly reduced the number of exo-erythrocytic forms (EEF). Further studies have shown that the recruitment of complement H factor (CFH) may be associated with the prevention of MAC deposition on the surface of naïve sporozoites. Our data strongly suggest that sporozoites can escape complement attacks and provide us with a novel strategy to prevent malaria infection.

The neuro-exocrine secretion: A new type of gland in tapeworms?

The phenomenon of exocrine secretion via nervous cells into the host tissue has been discovered in cestodes. In five cestode species of different orders specialized “cup-shaped” free nerve endings located in the tegument have been found. Their ultrastructure is characterized by the presence of a septate junction, a thin support ring and neurosecretory vesicles 90–110 nm in diameter, which are secreted onto the surface of the tegument through a thin pore. The phenomenon is referred to in this article as the neuro-exocrine secretion. We observed a direct relationship between neurosecretory processes in the deep subtegument and free endings in a series of ultrathin sections in two species. The peripheral neurosecretory neurons of species studied are characterized by similar ultrastructural features: size and location; diameter of neurosecretory granules; absence of microtubules and mitochondria in the neurites. The size of neurosecretory granules has been found to decrease from perikaryon towards neurosecretory terminals that lead to the tegument. In two species, we examined the neurosecretion during incubation in the host's blood serum. Depending on the time of incubation we have shown the changes a) in the diameter of the cup-shaped endings, b) in the number of secretory vesicles in the endings; c) changes in number and diameter of neurosecretory vesicles in the processes of neurosecretory neurons in the subtegument. The detected changes differ in D.dendriticus and L.interrupta and, taken together, indirectly confirm the secretory specialization of the cup-shaped endings. Supposed targets for the neurosecretory neurons in the studied cestodes are the following: (a) eccrine frontal gland ducts, especially their terminal regions involved in the release of secretory products; (b) longitudinal and circular muscles in the subtegument region; (c) the basal membrane of the tegument. Besides the discovered secretion vesicles through the cup-shaped terminals, we observed vacuoles derived from the basal membrane of the tegument containing extracellular substances released into the host tissue. Their possible role in the release of neurosecretory substances is discussed. Considering the data acquired via immunocytochemical methods, an assumption about involvement of FMRFamide-like related peptides (FaRPs) in the neuro-exocrine secretion is proposed. Possible functions of the neuro-exocrine secretion are discussed in the context of host-parasite interactions.

Hemp seed: An allergen source with potential cross-reactivity to hazelnut

The increasing exposure of the population to Cannabis sativa has revealed allergies to different parts of the plant, among which hemp seed. Nonetheless, the major hemp seed allergens remain to be identified. Several known families of allergens are present in hemp seed, including notably seed storage proteins. We therefore aimed to investigate the potential allergenicity of the hemp seed storage proteins and their potential cross-reactivity to different seeds and nuts. For this, we extracted hemp seed proteins sequentially using buffers with increasing levels of salinity (H2O, T2 and T3) to yield extracts differentially enriched in storage proteins. We used these extracts to perform immunoblots and ELISAs using sera of patients either sensitized to hemp seeds or sensitized/allergic to other seeds and nuts. Immunoblots and proteomics analyses identified vicilins and edestins as potential hemp seed allergens. Moreover, ELISA analyses revealed a correlation between sensitization to hazelnut and the hemp seed T3 extract (enriched in storage proteins). The possible cross-reactivity between hazelnut and hemp seed proteins was further strengthened by the results from inhibition ELISAs: the incubation of sera from hazelnut-sensitized individuals with increasing concentrations of the T3 extract inhibited serum IgE binding to the hazelnut extract by about 25–30%. Our study thus identifies vicilins and edestins as potential hemp seed allergens and highlights a possible cross-reactivity with hazelnut. The clinical relevance of this cross-reactivity between hemp seed and hazelnut needs to be further investigated in hazelnut-allergic individuals.

Automated radiosynthesis of [89Zr]Zr-DFOSq-Durvalumab for imaging of PD-L1 expressing tumours in vivo

Objectives89Zr-labelled proteins are gaining importance in clinical research in a variety of diseases. To date, no clinical study has been reported that utilizes an automated approach for radiosynthesis of 89Zr-labelled radiopharmaceuticals. We aim to develop an automated method for the clinical production of 89Zr-labelled proteins and apply this method to Durvalumab, a monoclonal antibody targeting PD-L1 immune-checkpoint protein. PD-L1 expression is poorly understood and can be up-regulated over the course of chemo- and radiotherapy treatment. The ImmunoPET multicentre study aims to examine the dynamics of PD-L1 expression via 89Zr-Durvalumab PET imaging before, during, and after chemoradiotherapy. The developed automated technique will enable reproducible clinical production of [89Zr]Zr-DFOSq-Durvalumab for this study at three different sites. MethodsConjugation of Durvalumab to H3DFOSqOEt was optimized for optimal chelator-to-antibody ratio. Automated radiolabelling of H3DFOSq-Durvalumab with zirconium-89 was optimized on the disposable cassette based iPHASE technologies MultiSyn radiosynthesizer using a modified cassette. Activity losses were tracked using a dose calibrator and minimized by optimizing fluid transfers, reaction buffer, antibody formulation additives and pH. The biological profile of the radiolabelled antibody was confirmed in vivo in PD-L1+ (HCC827) and PD-L1- (A549) murine xenografts. Clinical process validation and quality control were performed at three separate study sites to satisfy clinical release criteria. ResultsH3DFOSq-Durvalumab with an average CAR of 3.02 was obtained. Radiolabelling kinetics in succinate (20 mM, pH 6) were significantly faster when compared to HEPES (0.5 M, pH 7.2) with >90 % conversion observed after 15 min. Residual radioactivity in the 89Zr isotope vial was reduced from 24 % to 0.44 % ± 0.18 % (n = 7) and losses in the reactor vial were reduced from 36 % ± 6 % (n = 4) to 0.82 % ± 0.75 % (n = 4) by including a surfactant in the reaction and formulation buffers. Overall process yield was 75 % ± 6 % (n = 5) and process time was 40 min. Typically, 165 MBq of [89Zr]Zr-DFOSq-Durvalumab with an apparent specific activity of 315 MBq/mg ± 34 MBq/mg (EOS) was obtained in a volume of 3.0 mL. At end-of-synthesis (EOS), radiochemical purity and protein integrity were always >99 % and >96 %, respectively, and dropped to 98 % and 65 % after incubation in human serum for 7 days at 37 °C. Immunoreactive fraction in HEK293/PD-L1 cells was 83.3 ± 9.0 (EOS). Preclinical in vivo data at 144 h p.i. showed excellent SUVmax in PD-L1+ tumour (8.32 ± 0.59) with a tumour-background ratio of 17.17 ± 3.96. [89Zr]Zr-DFOSq-Durvalumab passed all clinical release criteria at each study site and was deemed suitable for administration in a multicentre imaging trial. ConclusionFully automated production of [89Zr]Zr-DFOSq-Durvalumab for clinical use was achieved with minimal exposure to the operator. The cassette-based approach allows for consecutive productions on the same day and offers an alternative to currently used manual protocols. The method should be broadly applicable to other proteins and has the potential for clinical impact considering the growing number of clinical trials investigating 89Zr-labelled antibodies.

Abstract 5049: Pb-203 image guided Pb-212 receptor targeted alpha particle therapy for cancer - a powerful emerging paradigm

Abstract Objective: Pb-203 and Pb-212 have emerged as a promising elementally-matched theranostic pair for imaging-guided alpha-particle radiotherapy for cancer. A somatostatin receptor 2 (SSTR2)-targeted peptide coupled with novel Pb specific chelator (PSC) has been developed (PSC-PEG2-TOC). Here, preclinical in vitro and in vivo evaluation of 203Pb/212Pb-labeled PSC-PEG2-TOC was conducted including head to head therapy of SSTR2+ tumors in mice vs standard of care beta particle therapy and first in humans clinical imaging of SSTR2+ tumors. Method: [203Pb]PSC-PEG2-TOC and [212Pb]PSC-PEG2-TOC was prepared by published methods. Radiochemical stability of [203Pb]PSC-PEG2-TOC, [212Pb]PSC-PEG2-TOC and [212Bi]PSC-PEG2-TOC in human serum was determined. Binding affinity of [203Pb]PSC-PEG2-TOC was determined by binding assays in AR42J cells. In vivo SSTR2-mediated tumor targeting of [203Pb]PSC-PEG2-TOC was determined by SPECT imaging in athymic nude mice bearing AR42J xenografts. In vivo biodistribution of [212Pb]PSC-PEG2-TOC in normal organs and potential redistribution of 212Bi daughter were determined in CD-1 Elite mice. Clinical imaging was conducted under appropriate regulations to determine the biodistribution of the agent in patients bearing SSTR2+ tumors as determined by gold standard 68Ga-DOTATOC PET imaging. Results: Rapid incorporation of 203Pb and 212Pb in PSC-PEG2-TOC was observed after reactions at temperatures as low as 4°C within 15 min. Greater than 95% radiochemical stability was observed for both [203Pb]PSC-PEG2-TOC and [212Pb]PSC-PEG2-TOC after incubation in human serum for up to 55 hours. Stable decay product [212Bi]PSC-PEG2-TOC with minimal free 212Bi was observed. Agents demonstrated superior binding affinity to SSTR2 with Kd=0.59 nM. In in vivo studies, rapid tumor uptake and renal clearance of [203Pb]PSC-PEG2-TOC were observed in athymic nude mice bearing AR42J xenografts. In the biodistribution study, nearly identical biodistribution profiles of [212Pb]PSC-PEG2-TOC and progeny [212Bi]PSC-PEG2-TOC were found. No redistribution of 212Bi activity was identified, indicating that 212Bi remained co-localized with parent [212Pb]PSC-PEG2-TOC in vivo. Therapeutic studies in this same mouse model resulted in 100% complete responses for both a single dose 4.4 MBq administration of [212Pb]PSC-PEG2-TOC or a fractionated regimen of 4 × 1.1 MBq. First in humans clinical imaging showed PK properties that included rapid tumor accumulation and retention at over 20 h post administration, coupled with fast renal clearance of residual agent. Conclusion: [203Pb]PSC-PEG2-TOC and [212Pb]PSC-PEG2-TOC have promising potential for image-guide alpha-particle therapy for SSTR2 positive tumors. Agents have received a Fastrack designation by the US FDA and a safe to proceed for a Phase 1 trial. Citation Format: Michael King Schultz, Yusuf Menda, Dijie Liu, Mengshi Li, Frances Johnson, Brianna Cagle, Nicholas Baumhover, Ivy Vance. Pb-203 image guided Pb-212 receptor targeted alpha particle therapy for cancer - a powerful emerging paradigm. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5049.

Septin-3 autoimmunity in patients with paraneoplastic cerebellar ataxia

BackgroundSeptins are cytoskeletal proteins with filament forming capabilities, which have multiple roles during cell division, cellular polarization, morphogenesis, and membrane trafficking. Autoantibodies against septin-5 are associated with non-paraneoplastic cerebellar ataxia, and autoantibodies against septin-7 with encephalopathy with prominent neuropsychiatric features. Here, we report on newly identified autoantibodies against septin-3 in patients with paraneoplastic cerebellar ataxia. We also propose a strategy for anti-septin autoantibody determination.MethodsSera from three patients producing similar immunofluorescence staining patterns on cerebellar and hippocampal sections were subjected to immunoprecipitation followed by mass spectrometry. The identified candidate antigens, all of which were septins, were expressed recombinantly in HEK293 cells either individually, as complexes, or combinations missing individual septins, for use in recombinant cell-based indirect immunofluorescence assays (RC-IIFA). Specificity for septin-3 was further confirmed by tissue IIFA neutralization experiments. Finally, tumor tissue sections were analyzed immunohistochemically for septin-3 expression.ResultsImmunoprecipitation with rat cerebellum lysate revealed septin-3, -5, -6, -7, and -11 as candidate target antigens. Sera of all three patients reacted with recombinant cells co-expressing septin-3/5/6/7/11, while none of 149 healthy control sera was similarly reactive. In RC-IIFAs the patient sera recognized only cells expressing septin-3, individually and in complexes. Incubation of patient sera with five different septin combinations, each missing one of the five septins, confirmed the autoantibodies’ specificity for septin-3. The tissue IIFA reactivity of patient serum was abolished by pre-incubation with HEK293 cell lysates overexpressing the septin-3/5/6/7/11 complex or septin-3 alone, but not with HEK293 cell lysates overexpressing septin-5 as control. All three patients had cancers (2 × melanoma, 1 × small cell lung cancer), presented with progressive cerebellar syndromes, and responded poorly to immunotherapy. Expression of septin-3 was demonstrated in resected tumor tissue available from one patient.ConclusionsSeptin-3 is a novel autoantibody target in patients with paraneoplastic cerebellar syndromes. Based on our findings, RC-IIFA with HEK293 cells expressing the septin-3/5/6/7/11 complex may serve as a screening tool to investigate anti-septin autoantibodies in serological samples with a characteristic staining pattern on neuronal tissue sections. Autoantibodies against individual septins can then be confirmed by RC-IIFA expressing single septins.

Chronic kidney disease promotes cerebral microhemorrhage formation

BackgroundChronic kidney disease (CKD) is increasingly recognized as a stroke risk factor, but its exact relationship with cerebrovascular disease is not well-understood. We investigated the development of cerebral small vessel disease using in vivo and in vitro models of CKD.MethodsCKD was produced in aged C57BL/6J mice using an adenine-induced tubulointerstitial nephritis model. We analyzed brain histology using Prussian blue staining to examine formation of cerebral microhemorrhage (CMH), the hemorrhagic component of small vessel disease and the neuropathological substrate of MRI-demonstrable cerebral microbleeds. In cell culture studies, we examined effects of serum from healthy or CKD patients and gut-derived uremic toxins on brain microvascular endothelial barrier.ResultsCKD was induced in aged C57BL/6J mice with significant increases in both serum creatinine and cystatin C levels (p < 0.0001) without elevation of systolic or diastolic blood pressure. CMH was significantly increased and positively correlated with serum creatinine level (Spearman r = 0.37, p < 0.01). Moreover, CKD significantly increased Iba-1-positive immunoreactivity by 51% (p < 0.001), induced a phenotypic switch from resting to activated microglia, and enhanced fibrinogen extravasation across the blood–brain barrier (BBB) by 34% (p < 0.05). On analysis stratified by sex, the increase in CMH number was more pronounced in male mice and this correlated with greater creatinine elevation in male compared with female mice. Microglial depletion with PLX3397 diet significantly decreased CMH formation in CKD mice without affecting serum creatinine levels. Incubation of CKD serum significantly reduced transendothelial electrical resistance (TEER) (p < 0.01) and increased sodium fluorescein permeability (p < 0.05) across the endothelial monolayer. Uremic toxins (i.e., indoxyl sulfate, p-cresyl sulfate, and trimethylamine-N-oxide) in combination with urea and lipopolysaccharide induced a marked drop in TEER compared with the control group (p < 0.0001).ConclusionsCKD promotes the development of CMH in aged mice independent of blood pressure but directly proportional to the degree of renal impairment. These effects of CKD are likely mediated in part by microglia and are associated with BBB impairment. The latter is likely related to gut-derived bacteria-dependent toxins classically associated with CKD. Overall, these findings demonstrate an important role of CKD in the development of cerebral small vessel disease.

Single-Particle Functionality Imaging of Antibody-Conjugated Nanoparticles in Complex Media.

The properties of nanoparticles (NPs) can change upon contact with serum components, occluding the NP surface by forming a biomolecular corona. It is believed that targeted NPs can lose their functionality due to this biological coating, thus losing specificity and selectivity toward target cells and leading to poor therapeutic efficiency. A better understanding of how the biomolecular corona affects NP ligand functionality is needed to maintain NP targeting capabilities. However, techniques that can quantify the functionality of NPs at a single-particle level in a complex medium are limited and often laborious in sample preparation, measurement, and analysis. In this work, the influence of serum exposure on the functionality of antibody-functionalized NPs was quantified using a straightforward total internal reflection fluorescence (TIRF) microscopy method and evaluated in cell uptake studies. The single-particle resolution of TIRF reveals the interparticle functionality heterogeneity and the substantial differences between NPs conjugated with covalent and noncovalent methods. Notably, only NPs covalently conjugated with a relatively high amount of antibodies maintain their functionality to a certain extent and still showed cell specificity and selectivity toward high receptor density cells after incubation in full serum. The presented study emphasizes the importance of single-particle functional characterization of NPs in complex media, contributing to the understanding and design of targeted NPs that retain their cell specificity and selectivity in biologically relevant conditions.

High-yield cyclotron production of 203Pb using a sealed 205Tl solid target

Introduction203Pb (t1/2 = 51.9 h, 279 keV (81 %)) is a diagnostic SPECT imaging radionuclide ideally suited for theranostic applications in combination with 212Pb for targeted alpha particle therapy. Our objectives were to develop a high-yield solid target 203Pb cyclotron production route using isotopically enriched 205Tl target material and the 205Tl(p,3n)203Pb reaction as an alternative to lower energy production via the 203Tl(p,n)203Pb reaction. Methods250 mg 205Tl metal (99.9 % isotopic enrichment) was pressed using a hardened stainless steel die. Aluminum target discs were machined with a central depression and annulus groove. The flattened 205Tl pellet was placed into the central depression of the Al disc and a circle of indium wire was laid in the machined annulus surrounding the pellet. An aluminum foil cover was then pressed onto the target disc to create an airtight bond. Targets were irradiated at 23.3 MeV for up to 516 min on a TR-24 cyclotron at currents up to 60 μA to produce 203Pb via the 205Tl(p,3n)203Pb nuclear reaction. Following a cool-down period of >12 h, the target was removed and 205Tl dissolved in 4 M HNO3. A NEPTIS Mosaic-LC synthesis unit performed automated separation using Eichrom Pb resin, and 203Pb was eluted using 8 M HCl or 1 M NH4OAc. 205Tl was diverted to a vial for recovery in an electrolytic cell. 203Pb product radionuclidic purity was assessed by HPGe gamma spectroscopy, while elemental purity was assessed by ICP-OES. Radiolabeling and stability studies were performed with PSC, TCMC, and DOTA chelators, and 203Pb incorporation was verified by radio-TLC analysis. ResultsCyclotron irradiations performed at 60 μA proton beam current and 23.3 MeV (205Tl incident energy) had a 203Pb saturated yield of 4658 ± 62 MBq/μA (n = 3). Automated NEPTIS separation took <4 h from the start of target dissolution to product elution, yielding >85 % decay-corrected [203Pb]PbCl2 with a radionuclidic purity of >99.9 %. Purified [203Pb]PbCl2 yields of up to 12 GBq 203Pb were attained (15.8 GBq at EOB). The [203Pb]PbCl2 and [203Pb]Pb(OAc)2 products contained no detectable radionuclidic impurities besides 201Pb (<0.1 %), and <0.4 ppm stable Pb. 205Tl metal was recovered with a 92 % batch yield. Aliquots of 100 μL [203Pb]Pb(OAc)2 were used for radiolabeling PSC-Bn-NCS, TCMC-NCS, and DOTA-NCS chelators at pH 4.5 and 22 °C for 30 min, with maximum respective molar activities of 461 ± 30 GBq/μmol, 195 ± 37 GBq/μmol, and 83 ± 12 GBq/μmol. PSC, TCMC, and DOTA chelators exhibited >99.9 % incorporation after a 120-hour incubation in human serum at 37 °C. ConclusionsNuclear medicine centers with access to higher energy cyclotrons can produce large 203Pb activities sufficient for clinical applications, with a convenient separation technique producing highly pure [203Pb]PbCl2 or [203Pb]Pb(OAc)2 for direct radiolabeling. This represents an attractive route to produce 203Pb for diagnostic SPECT imaging alongside 212Pb targeted alpha particle therapy. Advances in knowledge and implications for patient careOur high-yield 203Pb production technique significantly enhances 203Pb production capabilities to meet the growing preclinical and clinical demand for 203Pb radiopharmaceuticals alongside 212Pb target alpha particle therapy.