Immunoreactive Assay Research Articles

MIB Guides: Measuring the Immunoreactivity of Radioimmunoconjugates

Immunoglobulins, both full-length antibodies and smaller antibody fragments, have long been regarded as effective platforms for diagnostic and therapeutic radiopharmaceuticals. The construction of radiolabeled immunoglobulins (i.e., radioimmunoconjugates) requires the manipulation of the biomolecule through the attachment of a radiohalogen or the bioconjugation of a chelator that is subsequently used to coordinate a radiometal. Both synthetic approaches have historically relied upon the stochastic modification of amino acids within the immunoglobulin, a process which poses a risk to the structural and functional integrity of the biomolecule itself. Not surprisingly, radioimmunoconjugates with impaired antigen binding capacity will inevitably exhibit suboptimal in vivo performance. As a result, the biological characterization of any newly synthesized radioimmunoconjugate must include an assessment of whether it has retained its ability to bind its antigen. Herein, we provide straightforward and concise protocols for three assays that can be used to determine the immunoreactivity of a radioimmunoconjugate: (1) a cell-based linear extrapolation assay; (2) a cell-based antigen saturation assay; and (3) a resin- or bead-based assay. In addition, we will provide a critical analysis of the relative merits of each assay, an examination of the inherent limitations of immunoreactivity assays in general, and a discussion of other approaches that may be used to interrogate the biological behavior of radioimmunoconjugates.

Effectiveness of [67Cu]Cu-trastuzumab as a theranostic against HER2-positive breast cancer.

To evaluate the imaging and therapeutic properties (theranostic) of 67Cu-labeled anti-human epidermal growth factor receptor II (HER2) monoclonal antibody trastuzumab against HER2-positive breast cancer (BC). We conjugated trastuzumab with p-SCN-Bn-NOTA, 3p-C-NETA-NCS, or p-SCN-Bn-DOTA, and radiolabeled with [67Cu]CuCl2. Immunoconjugate internalization was evaluated in BT-474, JIMT-1 and MCF-7 BC cells. In vitro stability was studied in human serum (HS) and Phosphate Buffered Saline (PBS). Flow cytometry, radioligand binding and immunoreactive fraction assays were carried out. ImmunoSPECT imaging of [67Cu]Cu-NOTA-trastuzumab was done in mice bearing BT-474, JIMT-1 and MCF-7 xenografts. Pharmacokinetic wasstudiedin healthy Balb/c mice while dosimetry was done in both healthy Balb/cand inathymic nudemice bearingJIMT-1 xenograft. The therapeutic effectiveness of [67Cu]Cu-NOTA-trastuzumab was evaluated in mice bearing BT-474 and JIMT-1 xenografts after a single intravenous (i.v.) injection of ~ 16.8MBq. Pure immunoconjugates and radioimmunoconjugates (> 95%) were obtained. Internalization was HER2 density-dependent with highest internalization observed with NOTA-trastuzumab. After 5days, in vitro stabilities were 97 ± 1.7%, 31 ± 6.2%, and 28 ± 4% in HS, and 79 ± 3.5%, 94 ± 1.2%, and 86 ± 2.3% in PBS for [67Cu]Cu-NOTA-trastuzumab, [67Cu]Cu-3p-C-NETA-trastuzumab and [67Cu]Cu-DOTA-trastuzumab, respectively. [67Cu]Cu-NOTA-trastuzumab was chosen for further evaluation. BT-474 flow cytometry showed low KD, 8.2 ± 0.2nM for trastuzumab vs 26.5 ± 1.6nM for NOTA-trastuzumab. There were 2.9 NOTA molecules per trastuzumab molecule. Radioligand binding assay showed a low KD of 2.1 ± 0.4nM and immunoreactive fraction of 69.3 ± 0.9. Highest uptake of [67Cu]Cu-NOTA-trastuzumab was observed in JIMT-1 (33.9 ± 5.5% IA/g) and BT-474 (33.1 ± 10.6% IA/g) xenograft at 120h post injection(p.i.). Effectiveness of theradioimmunoconjugate was also expressed as percent tumor growth inhibition (%TGI). [67Cu]Cu-NOTA-trastuzumab was more effective than trastuzumab against BT-474 xenografts (78% vs 54% TGI after 28days), and JIMT-1 xenografts (90% vs 23% TGI after 19days). Mean survival of [67Cu]Cu-NOTA-trastuzumab, trastuzumab and saline treatedgroups were > 90, 77 and 72days for BT-474 xenografts, while that of JIMT-1 were 78, 24, and 20days, respectively. [67Cu]Cu-NOTA-trastuzumab is a promising theranostic agent against HER2-positive BC.

NEIM-01 IMMUNOSUPPRESSION IN THE GLIOMA MICROENVIRONMENT: USING PET IMAGING TO HIGHLIGHT THE PRESENCE OF TIGIT

Abstract Malignant gliomas are one of the drivers of brain tumor patient mortality as these tumors respond poorly to conventional therapies and are known to grow aggressively. Many clinical trials have evaluated the efficacy of novel immuno-therapeutics targeted to gliomas; however, these trials have largely been unsuccessful. It is believed that one of the primary reasons immunotherapies have been unsuccessful in gliomas is due to the highly immunosuppressive nature of the tumor microenvironment. T cell immunoreceptor with Ig and ITIM domain (TIGIT) is an IC receptor that is expressed on activated T cells, Natural Killer (NK) cells, and Regulatory T cells (Tregs). It has been well established that TIGIT has a primary role in down-regulating T cell and NK cell function and can serve as an inhibitor of anti-tumor immune responses. Our laboratory and others have shown that anti-TIGIT therapy may be promising for the treatment of adult gliomas via glioma patient transcriptomic data and preclinical models. In this study, our team created a radiolabeled tracer (referred to as 89Zr-DFO-TIGIT) that bound TIGIT antigen with moderate immunoreactivity (57.7% ± 3.5%). Binding affinity was determined by performing bead-based immunoreactivity assays. To create 89Zr-DFO-TIGIT, a TIGIT monoclonal antibody was conjugated to the metal chelator DFO and radiolabeled with 89Zr. Following tracer preparation, glioma-bearing mice (GL261 cell line) were injected with 89Zr-DFO-TIGIT allowing for the visualization of TIGIT in the tumor microenvironment. PET imaging analyses revealed tracer-to-tumor uptake that increased from day one to day eight while biodistribution studies confirmed that there were significantly higher amounts of 89Zr-DFO-TIGIT detected in the glioma-bearing mouse brain hemisphere compared to the non-tumor bearing hemisphere. We now intend to evaluate 89Zr-DFO-TIGIT in a glioma-bearing large animal model to determine if this method demonstrates similar efficacy in visualizing immunosuppression in the tumor microenvironment before proceeding to clinical studies.

IMG-02. VISUALIZATION OF TIGIT IN THE GLIOMA MICROENVIRONMENT: THE CREATION OF A PET IMAGING TRACER

Abstract Pediatric high-grade gliomas (HGGs) are aggressive tumors and are one of the drivers of pediatric brain tumor mortality. Few effective treatment options exist for pediatric HGGs beyond surgery and chemoradiation. Additionally, some HGGs, such as diffuse midline glioma, are often surgically inaccessible and respond poorly to chemotherapy leaving radiotherapy as the only treatment for this disease. While immune checkpoint inhibitors (ICIs) have seen success in treating cancers outside the CNS, ICIs for gliomas have shown limited efficacy. TIGIT is an IC receptor that is expressed on activated T-cells and has a critical role in suppressing T-cell and NK cell function. Prior work by our group has shown TIGIT to be a promising target for adult gliomas. These studies included analysis of transcriptomic data, and preclinical studies in glioma-bearing mice. We therefore assessed pediatric glioma transcriptomic datasets and identified via optimal cutoff analysis that at a specific mRNA threshold, TIGIT expression is associated with worsened survival. As TIGIT expression may be both prognostic and a biomarker for anti-TIGIT ICI therapy, we created a radiolabeled immunoPET tracer capable of binding TIGIT antigen that could be visualized via PET imaging. Referred to as “89Zr-DFO-TIGIT”, we found this tracer bound TIGIT with moderate immunoreactivity (57.7% ± 3.5%) as determined by bead-based immunoreactivity assays. As proof of feasibility, we injected glioma-bearing mice (GL261 cell line) with 89Zr-DFO-TIGIT which enabled the visualization of TIGIT in the tumor microenvironment via PET imaging. Thereafter, we performed biodistribution studies which found there to be significantly increased 89Zr-DFO-TIGIT in mouse tumor-bearing hemispheres as compared to non-tumor-bearing hemispheres. PET imaging also showed tracer-to-tumor uptake increased from day one to day eight. Our findings demonstrate the capability of 89Zr-DFO-TIGIT to visualize and quantify TIGIT in the glioma microenvironment, highlighting how this tracer may be of value in pediatric brain tumor immunotherapy studies.

Use of hormones in assessing reproductive physiology of humpback whales (Megaptera novaeangliae) from Juneau, Alaska

Humpback whales (Megaptera novaeangliae) in Southeast Alaska have been studied for over 50 years, and are largely considered a recovery success since the cessation of commercial whaling. Reproductive physiology is an important factor to consider in studying population health and can provide important insights into the drivers contributing to population abundance fluctuations. Validated assays for progesterone and testosterone were used on blubber biopsies from humpback whales (N = 33 whales, 71 samples) near Juneau, Alaska, in 2020 and 2021. Long-term sighting histories were used to confirm detected pregnancies with calf sightings the following year. Blubber samples were divided into two seasonal bins (early and late summer). Pregnant females sampled in both early and late summer of both 2020 and 2021 showed elevated progesterone concentrations compared to other reproductive states (p < 0.05). Progesterone concentrations in adult male whales (0.3 ± 0.2 ng/g) were not significantly different from lactating or resting female whales. Blubber testosterone concentrations in adult male humpback whales ranged from 0.05 to 1.1 ng/g, and mean concentrations were approximately double those of female whales in any reproductive state. Pregnancy was detected in 5 of 11 and 4 of 9 adult females in 2020 and 2021 respectively, yielding summer season pregnancy rates for sexually mature females at 0.45, and 0.44, respectively. Calving rates were 0.36 and 0.22 in 2020 and 2021, respectively, and the annual growth rate for this subpopulation was calculated at 2.6 % per annum. One female had successful pregnancies for four consecutive years. These results demonstrate the synergistic value of combining immunoreactive assays and long-term sighting histories to further knowledge of reproductive physiology in individual humpback whales, which can be expanded to assessing the health of a population or ecosystem.

Structural, antioxidant, and immunomodulatory activities of an acidic exopolysaccharide from Lactiplantibacillus plantarum DMDL 9010.

This study investigated the structural, antioxidant, and immunomodulatory activities of acidic exopolysaccharide (EPS-LP2) isolated from Lactiplantibacillus plantarum DMDL 9010. EPS-LP2 is composed of fucose (Fuc), arabinose (Ara), galactose (Gal), glucose (Glc), mannose (Man), and D-fructose (Fru) with a molar ratio of 0.13: 0.69: 8.32: 27.57: 62.07: 0.58: 0.46, respectively. Structural analysis of EPS-LP2 exhibited a smooth irregular lamellar surface, rod-like structure with swollen ends and slippery surfaces, and good thermal stability. Based on the methylation and NMR analysis, sugar residues including t-Manp, t-Glcp, 2-Manp, 6-Galp, 6-Glcp, and 4-Glcp were found to exist in EPS-LP2. In the 50∼400 μg/ml range, EPS-LP2 showed negligible neurotoxicity to RAW264.7 cells. Moreover, EPS-LP2 could protect RAW264.7 cells from oxidative injury by lowering the generation of reactive oxygen species (ROS), malondialdehyde (MDA), and the secretion of lactate dehydrogenase (LDH). In contrast, an increase in superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and the concentrations of glutathione (GSH) were observed. Immunoreactivity assays showed that EPS-LP2 could suppress the expression of NO, tumor necrosis factor-α (TNF-α), and interleukin 6 (IL-6) and inhibit the activation of the mitogen-activated protein kinase (MAPK)/nuclear factor-κB-gene binding (NF-κB) cell pathway. Conclusively, EPS-LP2 could be a potential natural antioxidant and immunomodulatory agent in functional foods and medicines.

Pregnancy rate and reproductive hormones in humpback whale blubber: Dominant form of progesterone differs during pregnancy

To better understand reproductive physiology of humpback whales Megaptera novaeangliae that reside in Hawai’i and Alaska, enzyme immunoassays were validated for both progesterone and testosterone in free-ranging and stranded animals (n = 185 biopsies). Concentrations were analyzed between different depths of large segments of blubber taken from skin to muscle layers of stranded female (n = 2, 1 pregnant, 1 non-pregnant) and male (n = 1) whales. Additionally, progesterone metabolites were identified between pregnant (n = 1) and non-pregnant (n = 3) females using high pressure liquid chromatography (HPLC). Progesterone concentrations were compared between juvenile (i.e., sexually immature), lactating, and pregnant females, and male whales, and pregnancy rates of sexually mature females were calculated. Based on replicate samples from ship struck animals collected at 7 depth locations, blubber containing the highest concentration of progesterone was located 1 cm below the skin for females, and the highest concentration of testosterone was in the skin layer of one male whale. HPLC of blubber samples of pregnant and non-pregnant females contain different immunoreactive progesterone metabolites, with the non-pregnant female eluate comprised of a more polar, and possibly conjugated, form of progesterone than the pregnant female. In females, concentrations of progesterone were highest in the blubber of pregnant (n = 28, 28.6 ± 6.9 ng/g), followed by lactating (n = 16, 0.9 ± 0.1 ng/g), and female juvenile (n = 5, 1.0 ± 0.2 ng/g) whales. Progesterone concentrations in male (n = 24, 0.6 ng/g ± 0.1 ng/g) tissues were the lowest all groups, and not different from lactating or juvenile females. Estimated summer season pregnancy rate among sexually mature females from the Hawai’i stock of humpback whales was 0.562 (95 % confidence interval 0.528–0.605). For lactating females, the year-round pregnancy rate was 0.243 (0.09–0.59), and varies depending on the threshold of progesterone assumed for pregnancy in the range between 3.1 and 28.5 ng/g. Our results demonstrate the synergistic value added when combining immunoreactive assays, HPLC, and long-term sighting histories to further knowledge of humpback whale reproductive physiology.

Synthesis and In Vivo Evaluation of a Site-specifically Labeled Radioimmunoconjugate for Dual-Modal (PET/NIRF) Imaging of MT1-MMP in Sarcomas.

Bone sarcomas are devastating primary bone cancers that mostly affect children, young adults, and the elderly. These aggressive tumors are associated with poor survival, and surgery remains the mainstay of treatment. Surgical planning is increasingly informed by positron emission tomography (PET), and tumor margin identification during surgery is aided by near-infrared fluorescence (NIRF) imaging, yet these investigations are confounded by probes that lack specificity for sarcoma biomarkers. We report the development of a dual-modal (PET/NIRF) immunoconjugate ([89Zr]Zr-DFO-anti-MT1-MMP-IRDye800CW) that targets MT1-MMP, a matrix metalloproteinase overexpressed in high-grade sarcomas. [89Zr]Zr-DFO-anti-MT1-MMP-IRDye800CW was synthesized via site-specific chemoenzymatic glycan modification, characterized, and isolated in high specific activity and radiochemical purity. Saturation binding and immunoreactivity assays indicated only minor perturbation of binding properties. A novel mouse model of dedifferentiated chondrosarcoma based on intrafemoral inoculation of HT1080 WT or KO cells (high and low MT1-MMP expression, respectively) was used to evaluate target binding and biodistribution. Fluorescence and Cerenkov luminescence images of [89Zr]Zr-DFO-anti-MT1-MMP-IRDye800CW showed preferential uptake in HT1080 WT tumors. Ex vivo gamma counting revealed that uptake in MT1-MMP-positive tumors was significantly higher than that in control groups. Taken together, [89Zr]Zr-DFO-anti-MT1-MMP-IRDye800CW is a promising dual-modal sarcoma imaging agent for pre-operative surgical planning and intraoperative surgical guidance.

Bothrops leucurus snake venom protein profile, isolation and biological characterization of its major toxin PLA2s-like

Bothrops leucurus is considered as a snake of medical interest in the State of Bahia, Brazil. However, so far, there are no studies that provide a refined mapping of the composition of this venom. The aim of this work was to better understand the protein composition of B. leucurus snake venom and to isolate and biologically characterize the most abundant toxin, a basic PLA2-like. Shotgun proteomics approach identified 137 protein hits in B. leucurus venom subdivided into 19 protein families. The new basic PLA2-like toxin identified was denominated Bleu-PLA2-like, it and other proteoforms represents about 25% of the total proteins in the venom of B. leucurus and induces myotoxicity, inflammation and muscle damage. Immunoreactivity assays demonstrated that B. leucurus venom is moderately recognized by bothropic and crotalic antivenoms, and on the other hand, Bleu-PLA2-like and its proteoforms are poorly recognized. Our findings open doors for future studies in order to assess the systemic effects caused by this snake venom in order to better understand the toxinological implications of this envenomation and, consequently, to assist in the clinical treatment of victims.

Immune regulation by fungal strain diversity in inflammatory bowel disease.

The fungal microbiota (mycobiota) is an integral part of the complex multikingdom microbial community colonizing the mammalian gastrointestinal tract and has an important role in immune regulation1-6. Although aberrant changes in the mycobiota have been linked to several diseases, including inflammatory bowel disease3-9, it is currently unknown whether fungal species captured by deep sequencing represent living organisms and whether specific fungi have functional consequences for disease development in affected individuals. Here we developed a translational platform for the functional analysis of the mycobiome at the fungal-strain- and patient-specific level. Combining high-resolution mycobiota sequencing, fungal culturomics and genomics, a CRISPR-Cas9-based fungal strain editing system, in vitro functional immunoreactivity assays and in vivo models, this platform enables the examination of host-fungal crosstalk in the human gut. We discovered a rich genetic diversity of opportunistic Candida albicans strains that dominate the colonic mucosa of patients with inflammatory bowel disease. Among these human-gut-derived isolates, strains with high immune-cell-damaging capacity (HD strains) reflect the disease features of individual patients with ulcerative colitis and aggravated intestinal inflammation in vivo through IL-1β-dependent mechanisms. Niche-specific inflammatory immunity andinterleukin-17A-producing T helper cell (TH17 cell) antifungal responses by HD strains in the gut were dependent on the C. albicans-secreted peptide toxin candidalysin during the transition from a benign commensal to a pathobiont state. These findings reveal the strain-specific nature of host-fungal interactions in the human gut and highlight new diagnostic and therapeutic targets for diseases of inflammatory origin.

Preparation and preclinical characterization of 111In-DTPA-Anti-MUC1 as a radioimmunoconjugate for diagnosis of breast cancer by single-photon emission computed tomography.

Targeting MUC1 antigens which are overexpressed in 80% of breast cancers can be widely used in the field of radioimmunoscintigraphy (RIS) of breast cancer. The aim of this study was to develop a new diagnostic labeled compound for breast cancer RIS. In this study, an efficient indirect labeling method of PR81 with Indium-111 was developed and preliminary preclinical qualifications were reported. 111In-DTPA-PR81 was prepared and its radiochemical purity and stabilities in human serum and in phosphate-buffered saline (PBS) buffer were surveyed. Furthermore, cellular studies including complex reactivity, binding specificity, cell toxicity, etc., were examined. Finally, biodistribution and scintigraphy of the complex were studied in normal and tumoral animals. Statistical analyses were performed using SPSS 10.0. 111In-DTPA-PR81 was prepared with a radiochemical purity of >99% at optimized conditions. Stability studies showed the radiochemical purity of >90% in PBS buffer after 96 h, while the stability in human serum showed decrement to 81% after 96 h. Reactivity of the complex with MUC1 was significantly (P < 0.005) higher than bovine serum albumin (BSA) (about 7-8 times), even though BSA concentration was about twice the MUC1. The binding specificity of the complex to the MUC1 antigen was confirmed by means of immunoreactivity assay. Cell toxicity examination showed no significant lethal effect of the radiolabeled compound on the cells. Biodistribution studies of the complex in normal rats were consistent with the biodistribution of antibodies and high accumulation was observed in the tissues expressing MUC1 antigen. The results of 111In-DTPA-PR81 scintigraphy in tumoral female BALB/c mice at 24 and 48 h after injection showed an increasement of the accumulation in the tumor site. 111In-DTPA-PR81 can be considered as a potential agent for imaging of the MUC1 +breast tumors.

The Agreement between Feline Pancreatic Lipase Immunoreactivity and DGGR-Lipase Assay in Cats-Preliminary Results.

Simple SummaryFeline pancreatitis is a common disease with diagnostics requiring a combination of clinical, laboratory and imaging methods. Measuring of pancreatic lipase concentration is the laboratory mainstay of feline pancreatitis diagnostics. It can be done using immunoenzymatic assay (feline pancreatic immunoreactivity, fPLI) or colorimetric assay based on hydrolyzing a special substrate (DGGR) to a color product. A DGGR-lipase assay is manufactured by various companies. Studies carried out in cats on the DGGR-lipase assay of a single manufacturer have shown a high agreement with fPLI, however studies in dogs indicate that the performance of assays of different manufacturers may vary considerably. One of the largest Polish veterinary laboratories working 24/7 in the largest Polish cities offers the DGGR-lipase assay of a large Polish laboratory solutions provider. The values obtained in this assay are higher than those yielded by the traditional DGGR-lipase assay validated in studies carried out so far. Therefore, we decided to evaluate the agreement between the DGGR-lipase assay of the Polish manufacturer and fPLI. The agreement proved to be as high as for the traditional assay.The colorimetric catalytic assay based on the use of 1,2-o-dilauryl-rac-glycero-3-glutaric acid-(6′-methylresorufin) (DGGR) ester as a substrate for pancreatic lipase activity is commonly used for the diagnosis of pancreatitis in dogs and cats. Even though the assay has generally been shown to yield consistent results with feline pancreatic lipase immunoreactivity (fPLI) assay, the agreement may vary between assays of different manufacturers. In this study, the chance-corrected agreement between a DGGR-lipase assay offered by one of the biggest providers of diagnostic solutions in Poland and fPLI assay was investigated. The study was carried out on 50 cats in which DGGR-lipase activity and fPLI were tested in the same blood sample. The chance-corrected agreement was determined using Gwet’s AC1 coefficient separately for the fPLI assay’s cut-off values of >3.5 μg/L and >5.3 μg/L. The DGGR-lipase activity significantly positively correlated with fPLI (Rs = 0.665; CI 95%: 0.451, 0.807, p < 0.001). The chance-corrected agreement between the fPLI assay and DGGR-lipase assay differed considerably depending on the cut-off values of the DGGR-lipase assay. When the cut-off value reported in the literature (>26 U/L) was used, it was poor to fair. It was moderate at the cut-off value recommended by the laboratory (>45 U/L), and good at the cut-off value recommended by the assay’s manufacturer (>60 U/L). The highest agreement was obtained between the fPLI assay at the cut-off value of 3.5 μg/L and the DGGR-lipase assay at the cut-off value of 55 U/L (AC1 = 0.725; CI 95%: 0.537, 0.914) and between the fPLI assay at the cut-off value of 5.3 μg/L and the DGGR-lipase assay at the cut-off value of 70 U/L (AC1 = 0.749; CI 95%: 0.577, 0.921). The study confirms that the chance-corrected agreement between the two assays is good. Prospective studies comparing both assays to a diagnostic gold standard are needed to determine which of them is more accurate.

Comparative evaluation of amino acid composition and protein profile of gliadin from different extraction protocols

AbstractSignificance and backgroundGliadin is known to trigger the celiac disease or gluten sensitivity affecting around 70 million people globally. Its detection in early stages has become a controversial issue due to lack of efficient gliadin extraction procedures. The efficiency of gliadin extraction procedure is therefore an important critical step to the results of detection of gluten‐derived products from either rapid immunoreactivity assay kit or analytical mass analysis. Hence, four gliadin extraction procedures were investigated using six wheat cultivars and analyzed for their physiochemical analysis.FindingsThe relative proportion of ω‐gliadin, α/β‐, γ‐gliadin, HMW‐NG, and amino acid concentration of gliadin varied with the extraction protocol. The higher proportion of α/β‐, γ‐gliadin was found in Weiss extraction protocol (79.74%–88.62%) and lowest in Wallace (50.70%–58.91%). The ω‐gliadin was found to be significantly (p ≤ .05) highest in DuPont (9.78%–22.54%) and lowest in Weiss protocol (0.46%–1.04%). The region between 36 and 44 kDa showed well‐resolved bands in DuPont, while a dense mass in other extraction protocols. Amino acid score was found to be significantly (p ≤ .05) higher in DuPont (79.53–91.75 g/100 g), followed by Osborne (67.17–81.16 g/100 g), Wallace (68.54–77.58 g/100 g), and Weiss (60.97–79.16 g/100 g) extracted gliadin.ConclusionThe extraction protocol was found to influence the proportion of different protein fractions as well as amino acid concentration.Significance and noveltyThe insight obtained gives a better understanding of how the same protein behaves when extracted with different extraction protocols.

Abdominal ultrasound and clinicopathologic findings in 22 cats with exocrine pancreatic insufficiency.

BackgroundAwareness of exocrine pancreatic insufficiency (EPI) in cats has increased since the development of an assay for feline trypsin‐like immunoreactivity (fTLI). Ultrasound findings in cats with EPI have only been reported rarely and described as nonspecific.Hypothesis/ObjectivesTo describe the ultrasonographic findings, clinical signs, and concurrent diseases in cats with EPI.AnimalsTwenty‐two client‐owned cats with EPI.MethodsMulticenter retrospective descriptive study including cats with serum fTLI concentration ≤8 μg/L and an abdominal ultrasound examination performed within 6 weeks of fTLI measurement. Sonographic measurements of maximal pancreatic thickness and maximal pancreatic duct diameter as well as ratios of pancreatic duct diameter to pancreatic thickness were obtained. Additional sonographic findings, concurrent conditions, and clinical signs were recorded.ResultsThe most common clinical sign was weight loss (15/22 cats). Chronic enteropathy was the most common concurrent disease (13/22 cats). In 39% of cats, the pancreas had minimal or no sonographic alterations. Pancreatic duct dilatation (>2.5 mm), pancreatic duct tortuosity with variable diameter, or both were seen in 6/13 cats. The pancreatic parenchyma was subjectively thin in 6 cats. A significant relationship was found between subjectively thin pancreatic parenchyma and increased pancreatic duct size : pancreatic thickness ratio (P = .004). Diffuse gastrointestinal dilatation with echogenic content was observed in 8/22 cats.ConclusionExocrine pancreatic insufficiency often causes minimal to no sonographic pancreatic changes. Nonetheless, the findings of thin pancreatic parenchyma, pancreatic duct dilatation, or diffuse small intestinal dilatation with echogenic contents in cats with unexplained weight loss or unformed feces should raise clinical suspicion for EPI.

Molecular and Immunological Identification of Low Allergenic Fruits among Old and New Apple Varieties.

About 50–70% of patients allergic to birch pollen suffer from sensitization after apple ingestion. Apple allergenicity was established in only few varieties. Studies were performed on apple fruits of 21 traditional and nine modern varieties organically, intensively, or integratively produced. The aim of the study was to assess whether the factors like cultivation method, maturity stage, genotype, or type of tissue place an impact on the allergenic potential of apples. To answer these questions, we used semiquantitative real-time PCR, ELISA, and immunoblotting. Apple allergen genes present divergent expression across apple cultivars. Expression of the Mal d 1.06A correlates with the Mal d 1 level and is affected by the cultivation method and maturity of the fruit. The content of the main allergen Mal d 1 varied widely across cultivars. Interestingly, in our study, the Gala variety presented a low Mal d 1 concentration regardless of the cultivation method. Based on the Mal d 1.06A expression, the Mal d 1 protein content, and the immunoreactivity assay, the Kandil Sinap, Kosztela, Rumianka from Alma-Ata, Kantówka Gdańska, Reinette Coulon, and Gala cultivars emerged as potentially hypoallergenic apple cultivars. Our study allowed distinguishing between potentially low, medium, and highly allergenic varieties.

Protective effects of nobiletin on cisplatin induced neurotoxicity in rats

Objectives This study was designed to investigate the possible antioxidant, antiapoptotic and neuroprotective effects of nobiletin on cisplatin-induced neurotoxicity rat model by evaluating neurotrophins, antioxidants and histopathology. Methods Forty male Wistar Albino rats were divided into four groups: control, cisplatin (CIS), cisplatin + nobiletin (CIS + NOB) and nobiletin + cisplatin (NOB + CIS). CIS + NOB was applied nobiletin (10 mg/kg, i.p.) during the last four days whereas NOB + CIS was applied nobiletin during the first four days of the study. Cisplatin (4 mg/kg, i.p. twice a day) was administered to the experimental groups on the 5th day of the study. All rats were sacrificed on the 10th day of the study. BDNF, NGF, G6PD, GPx, tGSH and MDA levels were determined in brain. In addition, routin histolopathological analysis and caspase-3 immunoreactivity assay were conducted. Results BDNF concentrations increased in nobiletin-administered groups, compared to Control and CIS and that the increase was statistically significant in NOB + CIS (p < 0.05). It was also found that G6PD activity increased (p < 0.05) in the nobiletin-administered groups, compared to control and CIS. Histopathologically, neuronal degeneration, oedema and gliosis increased in CIS compared to Control, and nobiletin administration decreased neuronal degeneration and oedema compared to CIS (p < 0.05). Cisplatin increased (p < 0.05) caspase-3 immunoreactivity in cerebrovascular endothelium and neurons compared to Control, while nobiletin administration decreased caspase-3 immunoreactivity in cerebrovascular endothelium. Caspase-3 immunoreactivity in neurons decreased only in NOB + CIS (p < 0.05). Conclusion Nobiletin increased BDNF concentration and G6PD activity in brain and when evaluated together with histopathological and immunohistochemical findings, it may have antioxidant, antiapoptotic and neuroprotective effects against cisplatin.

The 1,2-o-dilauryl-rac-glycero-3-glutaric acid-(6'-methylresorufin) ester (DGGR) lipase assay in cats and dogs is not specific for pancreatic lipase.

The measurement of pancreatic lipase is important for the diagnosis of feline and canine pancreatitis. Recent studies have claimed that lipase assays using the 1,2-o-dilauryl-rac-glycero-3-glutaric acid-(6'-methylresorufin) ester (DGGR) as a substrate are more specific for measuring pancreatic lipase than traditional lipase assays. However, the analytical specificity of this assay for pancreatic lipase has not been demonstrated. We aimed to determine whether hepatic and/or lipoprotein lipases can interfere with the DGGR-based assay results in cats and dogs. We, therefore, compared plasma lipase activities measured using DGGR-based and pancreatic lipase immunoreactivity (PLI) assays before and after administering heparin, known to cause the release of hepatic and lipoprotein lipases, in cats and dogs. Heparin was administered in six cats and six dogs. Blood was collected at baseline and 10, 20, 30, 60, and 120minutes after heparin administration. Lipase activity was measured using a DGGR-based assay, and PLI concentrations were measured using the Spec fPL and cPL assays for cats and dogs, respectively. Plasma lipase activities, as measured using the DGGR-based assay, increased significantly 10minutes after heparin administration in both cats (P=.003) and dogs (P=.006) and returned to baseline by 120minutes. In contrast, PLI concentrations showed no significant changes after heparin administration. DGGR is not only hydrolyzed by pancreatic lipase but also by hepatic lipase, lipoprotein lipase, or both, in cats and dogs. Since these extrapancreatic lipases are also naturally present in cats and dogs, they could contribute to the lack of analytical specificity for the DGGR-based assays.

Two common polymorphic variants of OATP4A1 as potential risk factors for colorectal cancer.

Genetic variations in the organic-anion-transporting polypeptide (OATP)-encoding solute carrier of organic anions (SLCO) genes can promote cancer development and progression. The overexpression of solute carrier organic anion transporter family member 4A1 (OATP4A1), a transporter for steroid hormones, prostaglandins, and bile acids, has been previously associated with tumor recurrence and progression in colorectal cancer (CRC). Therefore, the present study aimed to investigate the association between 2 frequent single nucleotide polymorphisms (SNPs) in SLCO4A1 (rs34419428, R70Q; rs1047099G, V78I) and CRC predisposition. Following restriction fragment length polymorphism-PCR analysis in 178 patients with CRC [Union for International Cancer Control (UICC) stage I/II] and 65 healthy controls, no significant difference was observed in allele frequency and the number of heterozygous/homozygous individuals between the groups. Notably, the R70Q minor allele was identified to be associated with the V78I minor allele in the genome. Comparing of the individual genotypes of CRC patients to clinical data, including sex, UICC-stage and relapse revealed no increased risk for CRC. In addition, the OATP4A1 immunoreactivity assay in paraffin-embedded CRC and adjacent non-tumorous mucosa sections, examined using quantitative microscopy image analysis, did not reveal any association with these polymorphisms. No significant differences were observed in the expression levels, localization, and sodium fluorescein transport capacity among the OATP4A1 variants, which was studied using functional assays in Sf9-insect and A431 tumor cells overexpressing the 2 single and a double mutant OATP4A1 SNP variants. These results suggested that the 2 most frequent polymorphisms located in the first intracellular loop of OATP4A1 do not associate with CRC predisposition and tumor recurrence. They are unlikely to affect the outcome of CRC in patients.

Corydaline and l-tetrahydropalmatine attenuate morphine-induced conditioned place preference and the changes in dopamine D2 and GluA1 AMPA receptor expression in rats

Corydalis is a Chinese herb that has been used in China for hundreds of years for analgesic and other purposes. Corydaline and l-tetrahydropalmatine (l-THP) are the main active ingredients of Corydalis. This study was aimed to study the potential utility of corydaline and l-THP in the treatment of opioid abuse and addiction and explore the possible mechanisms underlying their pharmacological actions. Conditioned place preference (CPP) was used to evaluate the rewarding effects of morphine and Western-blot immunoreactive assays were used to evaluate morphine-induced changes in dopamine D2 receptor and GluA1 AMPA receptor and GluA2 AMPA receptor expression in the brain of rats. Systemic administration of corydaline (5 mg/kg, i.p.) or l-THP (1.25, 2.5,5 mg/kg) significantly inhibited the acquisition and expression of morphine-induced CPP in a dose-dependent manner. Corydaline or l-THP alone, at the same doses, failed to produce CPP or conditioned place aversion, and didn't affect locomotor activity. We then examined the expression of dopamine D2 receptor and GluA1 AMPA receptor and GluA2 AMPA receptor subunit expression in rats after acquisition of morphine-induced CPP. We found that repeated administration of morphine produced a significant reduction in dopamine D2 receptor expression in the prefrontal cortex, hippocamps, and striatum, while an increase in the striatal GluA1 AMPA receptor expression. Pretreatment with corydaline or l-THP blocked morphine-induced dopamine D2 receptor down-regulation and GluA1 AMPA receptor up-regulation in these brain regions. Corydaline and l-THP may have therapeutic potential in prevention and treatment of opioid abuse and addiction. The underlying mechanisms may be related to their antagonism on morphine-induced changes in dopamine and glutamate transmission in the brain.

A Systematic Evaluation of Antibody Modification and 89Zr-Radiolabeling for Optimized Immuno-PET.

Immuno-PET using desferrioxamine (DFO)-conjugated zirconium-89 ([89Zr]Zr4+)-labeled antibodies is a powerful tool used for preclinical and clinical molecular imaging. However, a comprehensive study evaluating the variables involved in DFO-conjugation and 89Zr-radiolabeling of antibodies and their impact on the in vitro and in vivo behavior of the resulting radioimmunoconjugates has not been adequately performed. Here, we synthesized different DFO-conjugates of the HER2-targeting antibody (Ab)-trastuzumab, dubbed T5, T10, T20, T60, and T200-to indicate the molar equivalents of DFO used for bioconjugation. Next we radiolabeled the immunoconjugates with ([89Zr]Zr4+) under a comprehensive set of reaction conditions including different buffers (PBS, chelexed-PBS, TRIS/HCl, HEPES; ± radioprotectants), different reaction volumes (0.1-1 mL), variable amounts of DFO-conjugated Ab (5, 25, 50 μg), and radioactivity (0.2-1.0 mCi; 7.4-37 MBq). We evaluated the effects of these variables on radiochemical yield (RCY), molar activity (Am)/specific activity (As), immunoreactive fraction, and ultimately the in vivo biodistribution profile and tumor targeting ability of the trastuzumab radioimmunoconjugates. We show that increasing the degree of DFO conjugation to trastuzumab increased the RCY (∼90%) and Am/As (∼194 MBq/nmol; 35 mCi/mg) but decreased the HER2-binding affinity (3.5×-4.6×) and the immunoreactive fraction of trastuzumab down to 50-64%, which translated to dramatically inferior in vivo performance of the radioimmunoconjugate. Cell-based immunoreactivity assays and standard binding affinity analyses using surface plasmon resonance (SPR) did not predict the poor in vivo performance of the most extreme T200 conjugate. However, SPR-based concentration free calibration analysis yielded active antibody concentration and was predictive of the in vivo trends. Positron emission tomography (PET) imaging and biodistribution studies in a HER2-positive xenograft model revealed activity concentrations of 38.7 ± 3.8 %ID/g in the tumor and 6.3 ± 4.1 %ID/g in the liver for ([89Zr]Zr4+)-T5 (∼1.4 ± 0.5 DFOs/Ab) at 120 h after injection of the radioimmunoconjugates. On the other hand, ([89Zr]Zr4+)-T200 (10.9 ± 0.7 DFOs/Ab) yielded 16.2 ± 3.2 %ID/g in the tumor versus 27.5 ± 4.1 %ID/g in the liver. Collectively, our findings suggest that synthesizing trastuzumab immunoconjugates bearing 1-3 DFOs per Ab (T5 and T10) combined with radiolabeling performed in low reaction volumes using Chelex treated PBS or HEPEs without a radioprotectant provided radioimmunoconjugates having high Am/As (97 MBq/nmol; 17.5 ± 2.2 mCi/mg), highly preserved immunoreactive fractions (86-93%), and favorable in vivo biodistribution profile with excellent tumor uptake.