Injection Of Antibody Research Articles

Local glucocorticoid synthesis regulates house dust mite-induced airway hypersensitivity in mice.

Extra-adrenal glucocorticoid (GC) synthesis at epithelial barriers, such as skin and intestine, has been shown to be important in the local regulation of inflammation. However, the role of local GC synthesis in the lung is less well studied. Based on previous studies and the uncontentious efficacy of corticosteroid therapy in asthma patients, we here investigated the role of 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1/Hsd11b1)-dependent local GC reactivation in the regulation of allergic airway inflammation. Airway inflammation in Hsd11b1-deficient and C57BL/6 wild type mice was analyzed after injection of lipopolysaccharide (LPS) and anti-CD3 antibody, and in acute and chronic models of airway hypersensitivity induced by house dust mite (HDM) extract. The role of 11β-HSD1 in normal and inflammatory conditions was assessed by high dimensional flow cytometry, histological staining, RT-qPCR analysis, ex vivo tissue cultures, GC-bioassays and protein detection by ELISA and immunoblotting. Here we show that lung tissue from Hsd11b1-deficient mice synthesized significantly less GC ex vivo compared with wild type animals in response to immune cell stimulation. We further observed a drastically aggravated phenotype in Hsd11b1-deficient mice treated with HDM extract compared to wild type animals. Besides eosinophilic infiltration, Hsd11b1-deficient mice exhibited aggravated neutrophilic infiltration caused by a strong Th17-type immune response. We propose an important role of 11β-HSD1 and local GC in regulating Th17-type rather than Th2-type immune responses in HDM-induced airway hypersensitivity in mice by potentially controlling Toll-like receptor 4 (TLR4) signaling and cytokine/chemokine secretion by airway epithelial cells.

Thrombospondin-1 proteomimetic polymers exhibit anti-angiogenic activity in a neovascular age-related macular degeneration mouse model.

Neovascular age-related macular degeneration (nAMD) is the leading cause of blindness in the developed world. Current therapy includes monthly intraocular injections of anti-VEGF antibodies, which are ineffective in up to one third of patients. Thrombospondin-1 (TSP1) inhibits angiogenesis via CD36 binding, and its down-regulated expression is negatively associated with the onset of nAMD. Here, we describe TSP1 mimetic protein-like polymers (TSP1 PLPs). TSP1 PLPs bind CD36 with high affinity, resist degradation, show prolonged intraocular half-lives (13.1 hours), have no toxicity at relevant concentrations in vivo (40 μM), and are more efficacious in ex vivo choroidal sprouting assays compared to the peptide sequence and Eylea (aflibercept), the current standard of care anti-VEGF treatment. Furthermore, PLPs exhibit superior in vivo efficacy in a mouse model for nAMD compared to control PLPs consisting of scrambled peptide sequences, using fluorescein angiography and immunofluorescence. Since TSP-1 inhibits angiogenesis by VEGF-dependent and independent mechanisms, TSP1 PLPs are a potential therapeutic for patients with anti-VEGF treatment-resistant nAMD.

Association between Anti-Sperm Antibody and Intra-cytoplasmic Sperm Injection Outcomes Among Male Infertility Patients

Background: Infertility, a clinical and public concern, affects both social life and the health system. Sperm abnormalities which include (Oligozoospermia), (teratozoospermia) or abnormalities related to sperm motility are essential factors in male infertility. Anti-sperm antibodies (ASA) are the leading cause of immune infertility in men (autoimmune disease). Intra-cytoplasmic Sperm Injection (ICSI) is specially designed to manage severe cases of male-factor infertility. this study aimed to determine the effect of ASA on ICSI outcomes among Normospermia and (Oligozoospermia, Asthenozoospermia and Teratozoospermia). Material and methods: This is a cross-sectional study performed at Fertility Center, Najaf-Iraq between Jan to June 2023, it included 50 couples who suffered from a minimum of 12 months of primary fertility who had attended the Fertility Center. They were divided into four groups (Normospermia, Oligospermia, Teratozoospermia, and Asthenospermia) according to their seminal fluid analysis (SFA). Sperms were collected by ICSA, immobilized, and processed. The oocytes were collected, injected, and prepared for fertilization , after that fertilization took place. Finally, pregnancy was examined in each female partner. Results: there was no significant difference in the age of the four groups, there was no relationship between ASA and Sperm concentration, and there was no relationship between ASA and Normal Sperm Morphology, fertilization , & pregnancy rates, in addition to other ICSI outcomes, were similar among the four groups.in conclusion, pregnancy rates were higher in the normospermia group than the other three groups and ICSI overcomed the presence of ASA in the semen plasma.

The D2D3 form of uPAR acts as an immunotoxin and may cause diabetes and kidney disease.

Soluble urokinase plasminogen activator receptor (suPAR) is a risk factor for kidney diseases. In addition to suPAR, proteolysis of membrane-bound uPAR results in circulating D1 and D2D3 proteins. We showed that when exposed to a high-fat diet, transgenic mice expressing D2D3 protein developed progressive kidney disease marked by microalbuminuria, elevated serum creatinine, and glomerular hypertrophy. D2D3 transgenic mice also exhibited insulin-dependent diabetes mellitus evidenced by decreased levels of insulin and C-peptide, impaired glucose-stimulated insulin secretion, decreased pancreatic β cell mass, and high fasting blood glucose. Injection of anti-uPAR antibody restored β cell mass and function in D2D3 transgenic mice. At the cellular level, the D2D3 protein impaired β cell proliferation and inhibited the bioenergetics of β cells, leading to dysregulated cytoskeletal dynamics and subsequent impairment in the maturation and trafficking of insulin granules. D2D3 protein was predominantly detected in the sera of patients with nephropathy and insulin-dependent diabetes mellitus. These sera inhibited glucose-stimulated insulin release from human islets in a D2D3-dependent manner. Our study showed that D2D3 injures the kidney and pancreas and suggests that targeting this protein could provide a therapy for kidney diseases and insulin-dependent diabetes mellitus.

The regulatory effect of NF-κB signaling pathway on biomineralization and shell regeneration in pearl oyster, Pinctada fucata

Pinctada fucata is an important pearl production shellfish in aquaculture. The formation of shells and pearls is a hot research topic in biomineralization, and matrix proteins secreted by the mantle tissues play the key role in this process. However, upstream regulatory mechanisms of transcription factors on the matrix protein genes remain unclear. Previous studies have shown that NF-κB signaling pathway regulated biomineralization process through expression regulation of specific matrix proteins, including Nacrein, Prismalin-14 and MSI60. In this study, we systematically investigated the regulatory effect of the NF-κB signaling pathway key factor Pf-Rel and inhibitory protein poI-κB on the biomineralization and shell regeneration process. We applied RNA interference and antibody injection assays to study in vivo function of transcription factor Pf-Rel and characterized shell morphology changes using scanning electron microscopy and Raman spectroscopy. We found that transcription factor Pf-Rel plays a positive regulatory role in the growth regulation of the prismatic and nacreous layers, while the function of inhibitory protein poI-κB is to prevent excessive growth and accumulation of both layers. RNA-seq was conducted based on RNA interference animal model to identify potential regulatory genes by transcription factor Pf-Rel. Shell damage repair experiments were performed to simulate shell regeneration process, and observations of newly formed shells revealed that NF-κB signaling pathway had different functions at different times. This study provides us with a more macroscopic perspective based on transcription factors to investigate biomineralization and shell regeneration.

Blocking Hemopexin With Specific Antibodies: A New Strategy for Treating Diabetic Retinopathy.

Hemopexin (HPX) is the best-characterized permeability factor in steroid-sensitive nephrotic syndrome. We have previously reported that HPX is overexpressed in the retina of patients with diabetes and induces the breakdown of the blood-retinal barrier invitro. Here, we report that intravitreal injection of anti-HPX antibodies significantly reduces vascular leakage, retinal neurodegeneration, and inflammation in diabetic murine models and that the immunoneutralization of HPX exerts a significant antiangiogenic effect invitro and in retinal explants. The blockade of HPX can be considered as a new therapy for advanced stages of diabetic retinopathy.

Fc-engineered monoclonal antibodies to reduce off-target liver uptake

BackgroundRadiolabeled-antibodies usually display non-specific liver accumulation that may impair image analysis and antibody biodistribution. Here, we investigated whether Fc silencing influenced antibody biodistribution. We compared recombinant 89Zr-labeled antibodies (human IgG1 against different targets) with wild-type Fc and with mutated Fc (LALAPG triple mutation to prevent binding to Fc gamma receptors; FcγR). After antibody injection in mice harboring xenografts of different tumor cell lines or of immortalized human myoblasts, we analyzed antibody biodistribution by PET-CT and conventional biodistribution analysis.ResultsAccumulation in liver was strongly reduced and tumor-specific targeting was increased for the antibodies with mutated Fc compared with wild-type Fc.ConclusionAntibodies with reduced binding to FcγR display lower liver accumulation and better tumor-to-liver ratios. These findings need to be taken into account to improve antibody-based theragnostic approaches.

Purified Serum IgG from a Patient with Anti-IgLON5 Antibody Cause Long-Term Movement Disorders with Impaired Dopaminergic Pathways in Mice.

Background: Anti-IgLON5 disease is a rare autoimmune disease of the central nervous system. It typically manifests as a chronic condition, characterized by cognitive impairments, movement disorders, and sleep disorders. The mechanisms underlying movement disorders in this disease remain poorly understood due to a lack of research. Furthermore, this disease exhibits both neuroimmune and neurodegenerative characteristics. The objective of this study is to explore the underlying mechanisms of movement disorders caused by anti-IgLON5 antibodies for the first time. Methods: Antibodies were purified from the serum of a confirmed patient of anti-IgLON5 disease. The passive transfer animal models were employed, where antibodies were continuously injected into the substantia nigra pars compacta (SNc) of the mouse midbrain using stereotactic injection to explore the mechanism of movement disorder. The effects of anti-IgLON5 antibodies on dopaminergic neurons in the SNc and neurodegeneration were examined through immunohistochemistry. Changes in neurotransmitter levels in the basal ganglia were assessed using high-performance liquid chromatography. Additionally, RNA-seq was employed to identify the differentially expressed genes associated with the short-term and long-term effects of anti-IgLON5 antibody on the SNc. Results: Mice injected with anti-IgLON5 antibodies in the SNc exhibited persistent movement impairments for up to 3 months. One week after antibody injection, the number of TH neurons significantly decreased compared to the control group, accompanied by reduced projection fibers in the basal ganglia and decreased dopamine levels. After 3 months of antibody injection, an increase in phosphorylated Tau was observed in the SNc of the midbrain. Additionally, long-term sustained activation of microglia was detected in the SNc. The differentially expressed genes of long-term effects of IgLON5 antibodies were different from their short-term effects on the SNc. Conclusion: Purified serum IgG from a patient with anti-IgLON5 antibodies can cause long-term movement disorder in mice. The movement disorders appear to be linked to the impaired dopaminergic pathway, and the increased p-Tau showed neurodegenerative changes induced by the anti-IgLON5 antibody.

CD147 monoclonal antibody attenuates abdominal aortic aneurysm formation in angiotensin II-Infused apoE-/- mice.

Abdominal aortic aneurysm (AAA) is a life threatening vascular disease. Our previous study reported the upregulation of CD147 expression in human aortic aneurysms. In this study, we injected apoE-/- mice intraperitoneally with CD147 monoclonal antibody or IgG control antibody to observe its effect on Angiotensin II (AngII) induced AAA formation. ApoE-/- mice were randomly divided into an AngⅡ+CD147 antibody group (n=20) and an AngⅡ+IgG antibody group (n=20). The Alzet osmotic minipump was implanted subcutaneously into the backs of mice to infuse AngII (1000ng/kg/min) for 28days and subsequently treated with CD147 monoclonal antibody or control IgG mAb (10μg/mouse/day) beginning one day after surgery. Body weight, food intake, drinking volume and blood pressure were measured weekly throughout the study. After 4weeks of injection, routine bloodwork measuring liver function, kidney function and lipid levels were recorded. Hematoxylin and eosin (H&E), Masson's trichrome, and Elastic van Gieson (EVG) staining were used to evaluate the pathological changes in blood vessels. In addition, Immunohistochemical assay was used to detect infiltration of inflammatory cells. Tandem mass tag (TMT)-based proteomic analysis was used to define differentially expressed proteins (DEPs) using a p-value<0.05 and fold change>1.2 or<0.83 as the threshold. Subsequently, we conducted protein-protein interaction (PPI) network and GO enrichment analysis to determine the core biological function altered after CD147 antibody injection. The CD147 monoclonal antibody suppresses Ang II-induced AAA formation in apoE-/- mice and reduced aortic expansion, elastic lamina degradation, and inflammatory cells accumulation. Bioinformatics analysis showed that Ptk6, Itch, Casp3, and Oas1a were the hub DEPs. These DEPs in the two group were mainly involved in collagen fibril organization, extracellular matrix organization, and muscle contraction. These data robustly demonstrated that CD147 monoclonal antibody suppresses Ang II-induced AAA formation through reduction of inflammatory response and regulation of the above defined hub proteins and biological processes. Thus, the CD147 monoclonal antibody might be a promising target in the treatment of abdominal aortic aneurysm.

Shikonin prevents mice from heat stroke-induced death via suppressing a trigger IL-17A on the inflammatory and oxidative pathways

Heat stroke (HS) is the deadliest disease. Due to the complex pathogenesis of HS, lack of effective therapeutic drugs for clinical treatment. Shikonin (SK) is the main active compound of Radix Arnebiae, which was evaluated on the HS model (temperature: (41 ± 0.5) ℃, relative humidity: (60 ± 5) %) via pathological and biochemical approaches in vivo and in vitro. Upon the dose of 10 mg.kg−1, SK delays the rising rate of core temperature, prolongs the survival time of mice, and improves organ injury and coagulation function markedly. Serum HS biomarkers interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) were decreased significantly by SK, which contribute to liver and lung protection in the models. Three pathways' responses to heat-stress were found to have a close connection with the IL-17 pathway via RNA sequencing and network analysis. WB and IHC results showed that the nuclear factor-κB (NF-κB) p65 in the SK group was down-regulated (P < 0.05). The expressions of nuclear factor erythroid 2 like 2 (NFE2L2/Nrf2) and heat shock protein 70 (HSP70) were up-regulated (P < 0.05). Additional administration of recombinant IL-17A protein on the HS model up-regulated the expression level of NF- κB p65 in the liver and lung tissue, additional intraperitoneal injection of IL-17A antibody in mice has a synergistic effect with SK in inhibiting tissue inflammatory response and protecting HS. In summary, SK was proved an effective compound for fulfilling the anti-inflammatory and antioxidative capacity of the HS model by reducing the production and inhibiting the expression of IL-17A.

Stabilized isogeometric formulation of the multi-network poroelasticity and transport model (MPET[formula omitted]) for subcutaneous injection of monoclonal antibodies

The MPET2 model couples the multi-network poroelastic theory (MPET) with solute transport equations and provides predictions of the material deformation, fluid dynamics, and solute transport in different compartments of a deformable multiple-porosity medium. MPET2 offers a comprehensive framework for understanding complex porous media across multiple disciplines. Examples of its applications include studying rock formations, soil mechanics and subsurface reservoirs, investigating biological tissues, modeling groundwater flow and contaminant transport, and optimizing the design of porous materials. Despite the wide range of applications of the model, its numerical discretization has received little attention. Here we propose a stabilized formulation of the MPET2 model. To address the unique challenges posed by the discretization of the MPET2 model, we use multiple techniques including the Fluid Pressure Laplacian stabilization, Streamline Upwind Petrov–Galerkin stabilization, and discontinuity capturing. Our spatial discretization is based on Isogeometric Analysis with higher-order continuity basis functions. The fully discretized governing equations are solved simultaneously with a monolithic algorithm. We perform a convergence study of the proposed formulation. Then, we conduct a series of simulations of subcutaneous injection of monoclonal antibodies under different injection conditions. Our simulations show that the stabilized MPET2 formulation can provide oscillation-free solutions for tissue deformation, fluid flow in the interstitial tissue, blood vessels, and lymphatic vessels, drug absorption in blood vessels and lymphatic vessels, as well as drug transport in each compartment. We also study the effects of different injection conditions on drug absorption, showing the potential of the proposed model and algorithm in the future optimization of injection strategy.

Clinical implications and mechanism of complement C1q in polymyositis.

Polymyositis (PM) is the most common autoimmune disease in neurology and among muscle disorders; it is of great significance to thoroughly understand the mechanism of PM to find new diagnosis and treatment methods. This research intends to elucidate the clinical implications and mechanisms of complement C1q in polymyositis (PM). One hundred fifteen PM patients (research group, RG) and 120 healthy subjects (control group, CG) who visited our hospital between March 2017 and March 2020 were selected. Peripheral blood C1q and creatine kinase (CK) levels of both groups were measured, and their correlations with clinical symptoms and prognostic recurrence of PM. Additionally, to further understand the mechanism of action of C1q in PM, we purchased BALB/c mice and grouped them as follows: control group with normal feeding, PM group with PM modeling, intervention group with PM modeling, and intraperitoneal injection of gC1qR monoclonal antibody 60.11, a C1q protein receptor. Inflammatory factors and muscle histopathology were detected in all groups of mice. Finally, rat macrophages (mø) were isolated, and changes in the biological behavior of mø were observed after silencing the expression of gC1qR. Serum C1q and CK were both higher in RG than in CG, with favorable diagnostic effects on PM (P < 0.05). C1q and CK increased in symptomatic anti-ribonuclear protein antibody (RNP)-positive patients but decreased in anti Jo-1 antibody (Jo-1)- and anti-neutrophil cytoplasmic antibody (ANCA)-positive patients (P < 0.05). PM mice were observed with elevated gC1qR, while model mice exhibited severe infiltration of inflammatory cells in muscle tissue, increased pro-IFs, and reduced anti-IFs, and the animals in the intervention group showed improved conditions (P < 0.05). Finally, it was found that CD68, CD86 protein, and invasion capacity of gC1qR-sh-transfected cells decreased, while CD206 and CD163 increased (P < 0.05). C1q is elevated in PM and is strongly linked to the pathological process of PM. Inhibition of gC1qR expression reduced inflammatory infiltration in PM mice.

Anti-Glycolipid Antibody Examination in Five EAE Models and Theiler's Virus Model of Multiple Sclerosis: Detection of Anti-GM1, GM3, GM4, and Sulfatide Antibodies in Relapsing-Remitting EAE.

Anti-glycolipid antibodies have been reported to play pathogenic roles in peripheral inflammatory neuropathies, such as Guillain-Barré syndrome. On the other hand, the role in multiple sclerosis (MS), inflammatory demyelinating disease in the central nervous system (CNS), is largely unknown, although the presence of anti-glycolipid antibodies was reported to differ among MS patients with relapsing-remitting (RR), primary progressive (PP), and secondary progressive (SP) disease courses. We investigated whether the induction of anti-glycolipid antibodies could differ among experimental MS models with distinct clinical courses, depending on induction methods. Using three mouse strains, SJL/J, C57BL/6, and A.SW mice, we induced five distinct experimental autoimmune encephalomyelitis (EAE) models with myelin oligodendrocyte glycoprotein (MOG)35-55, MOG92-106, or myelin proteolipid protein (PLP)139-151, with or without an additional adjuvant curdlan injection. We also induced a viral model of MS, using Theiler's murine encephalomyelitis virus (TMEV). Each MS model had an RR, SP, PP, hyperacute, or chronic clinical course. Using the sera from the MS models, we quantified antibodies against 11 glycolipids: GM1, GM2, GM3, GM4, GD3, galactocerebroside, GD1a, GD1b, GT1b, GQ1b, and sulfatide. Among the MS models, we detected significant increases in four anti-glycolipid antibodies, GM1, GM3, GM4, and sulfatide, in PLP139-151-induced EAE with an RR disease course. We also tested cellular immune responses to the glycolipids and found CD1d-independent lymphoproliferative responses only to sulfatide with decreased interleukin (IL)-10 production. Although these results implied that anti-glycolipid antibodies might play a role in remissions or relapses in RR-EAE, their functional roles need to be determined by mechanistic experiments, such as injections of monoclonal anti-glycolipid antibodies.

Computational modeling of the effect of skin pinch and stretch on subcutaneous injection of monoclonal antibodies using autoinjector devices.

Subcutaneous injection of monoclonal antibodies (mAbs) has experienced unprecedented growth in the pharmaceutical industry due to its benefits in patient compliance and cost-effectiveness. However, the impact of different injection techniques and autoinjector devices on the drug's transport and uptake is poorly understood. Here, we develop a biphasic large-deformation chemomechanical model that accounts for the components of the extracellular matrix that govern solid deformation and fluid flow within the subcutaneous tissue: interstitial fluid, collagen fibers and negatively charged proteoglycan aggregates. We use this model to build a high-fidelity representation of a virtual patient performing a subcutaneous injection of mAbs. We analyze the impact of the pinch and stretch methods on the injection dynamics and the use of different handheld autoinjector devices. The results suggest that autoinjector base plates with a larger device-skin contact area cause significantly lower tissue mechanical stress, fluid pressure and fluid velocity during the injection process. Our simulations indicate that the stretch technique presents a higher risk of intramuscular injection for autoinjectors with a relatively long needle insertion depth.

β1 integrin is essential for blood–brain barrier integrity under stable and vascular remodelling conditions; effects differ with age

BackgroundMaintaining a tight blood–brain barrier (BBB) is an important prerequisite for the preservation of neurological health, though current evidence suggests it declines with age. While extracellular matrix-integrin interactions play critical roles in regulating the balance between vascular stability and remodeling, it remains to be established whether manipulation of integrin function weakens or strengthens vascular integrity. Indeed, recent reports have generated conflicting outcomes in this regard.MethodsHere, in young (8–10 weeks) and aged (20 months) mice, we examined the impact of intraperitoneal injection of a function-blocking β1 integrin antibody, both under normoxic conditions, when the BBB is stable, and during chronic mild hypoxic (CMH; 8% O2) conditions, when a vigorous vascular remodeling response is ongoing. Brain tissue was examined by immunofluorescence (IF) for markers of vascular remodeling and BBB disruption, and microglial activation and proliferation. Data were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison post-hoc test.ResultsIn both young and aged mice, β1 integrin block greatly amplified hypoxia-induced vascular disruption, though it was much less under normoxic conditions. Interestingly, under both normoxic and hypoxic conditions, β1 integrin antibody-induced BBB disruption was greater in young mice. Enhanced BBB breakdown was associated with increased levels of the leaky BBB marker MECA-32 and with greater loss of endothelial tight junction proteins and the adherens protein VE-cadherin. Surprisingly, β1 integrin blockade did not reduce hypoxia-induced endothelial proliferation, nor did it prevent the hypoxia-associated increase in vascularity. Commensurate with the increased vascular disruption, β1 integrin blockade enhanced microglial activation both in young and aged brain, though the impact was much greater in young brain. In vitro studies revealed that β1 integrin blockade also reduced the integrity of a brain endothelial monolayer and triggered disruptions in tight junction proteins.ConclusionsThese data demonstrate that β1 integrin plays an essential role in maintaining BBB integrity, both under stable normoxic conditions and during hypoxia-induced vascular remodeling. As β1 integrin blockade had a greater disruptive effect in young brain, effectively shifting the BBB phenotype of young brain towards that of the aged, we speculate that enhancing β1 integrin function at the aged BBB may hold therapeutic potential by reverting the deteriorating BBB phenotype back towards that of the young.

Caspase-9 inhibition confers stronger neuronal and vascular protection compared to VEGF neutralization in a mouse model of retinal vein occlusion.

Retinal vein occlusion (RVO) is a sight-threatening condition typically treated with intravitreal injection of vascular endothelial growth factor (VEGF) antagonists. Treatment response to anti-VEGF therapies is highly variable, with poor visual outcomes and treatment response in patients with significant retinal nonperfusion following RVO. Recently, caspase-9 has been identified as a potent regulator of edema, gliosis, and neuronal dysfunction during acute retinal hypoxia. The purpose of this study was to compare the therapeutic effect of caspase-9 inhibition against VEGF-neutralization in an established mouse model of RVO. Adult male C57Bl/6 J mice were randomized to induction of RVO and treatment with either vehicle, intravitreal injection of anti-VEGF antibody, topical administration of a selective caspase-9 inhibitor (Pen1-XBir3), or a combination therapy. Animals were followed on days 1, 2, and 8 after RVO with fundus retinal imaging, and with optical coherence tomography (OCT) to capture retinal swelling, capillary nonperfusion (measured by disorganization of retinal inner layers, DRIL), hyperreflective foci (HRF), and retinal atrophy. Focal electroretinography (ERG) measurements were performed on day 7. Histology was performed on retinal sections from day 8. Both VEGF neutralization and caspase-9 inhibition showed significant retinal protection from RVO compared to vehicle treatment arm. Retinal reperfusion of occluded veins was accelerated in eyes receiving caspase-9 inhibitor, but not significantly different from vehicle in the anti-VEGF group. Retinal edema was suppressed in all treatment groups, with approximately 2-fold greater edema reduction with caspase-9 inhibition compared to VEGF neutralization. HRF were reduced similarly across all treatment groups compared to vehicle. Retinal detachment was reduced only in eyes treated with caspase-9 inhibitor monotherapy. Caspase-9 inhibition reduced retinal atrophy and preserved ERG response; VEGF neutralization did not prevent neurodegeneration following RVO. Caspase-9 inhibition confers stronger neuronal and vascular protection compared to VEGF neutralization in the mouse laser-induced model of RVO.

299-OR: A Combinatorial Approach with Monoclonal Antibodies to SerpinB13 and CD3 Is Therapeutically Superior to Single Antibody Use in Autoimmune Diabetes

A successful approach to autoimmune diabetes therapy may require simultaneous insulin-producing cell regeneration and T-cell mediated autoimmunity resolution in pancreatic islets. While CD3 antibody has been proved to reduce islet inflammation, the monoclonal antibody (mAb) to serpinB13 protease inhibitor, developed in our laboratory, was shown to cause regenerative changes in beta cells. Whether a combination of both antibodies is superior to single antibody use in the treatment of autoimmune diabetes is unclear. To address this non-obese diabetic (NOD) female mice at late pre-diabetic stage were injected with serpinB13 mAb (clone B29, 10, 40 or 200 micrograms/animal) and CD3 mAb (clone 145-2C11; at a suboptimal dose of 10 micrograms). All experimental injections were controlled with nonspecific hamster IgG and mouse IgG (clone HB-158). After the last antibody injection, random blood glucose levels were assessed for 20 weeks, and mice were declared diabetic after two measurements of glucose exceeding 300 mg/dL. We found that the combination of 40 micrograms of serpinB13 mAb with CD3 mAb offered a significant level of protection from diabetes (p=0.0027, Kaplan-Meyer with log rank test), while the combination with 10 micrograms of serpinB13 mAb had no effect. The higher dose of 200 micrograms of serpinB13 mAb showed a trend in favor of diabetes inhibition. Interestingly, at 6 to 7 weeks after completing the treatment with 40 micrograms of serpinB13 mAb alone, NOD mice showed a strong tendency to remain healthy, and separated from the double-negative control, which continued to progress to diabetes. Our results indicate that a combinatorial approach with mAbs to serpinB13 and CD3 is beneficial in autoimmune diabetes. The observations also suggest that an intermediate dose of serpinB13 mAb, and a sufficient time following administration of this mAb, is critical for its positive impact on diabetes prevention. Disclosure B.Szepietowska: None. S.V.Avdulov: None. Y.Kryvalap: None. J.Czyzyk: None. Funding JDRF (2-SRA-2022-1215-S-B)

PD-L1 ImmunoPET on the basis of Avidin/Biotin pre-targeted cancer imaging

This study aimed to establish the radio-immune imaging protocol on the basis of Avidin/Biotin system. The programmed death-ligand 1 (PD-L1) antibody (Atezolizumab) was employed as the primary molecule in targeting PD-L1, and the two-step strategy, consisting of the first injection of Avidin-conjugated PD-L1 monoclonal antibody (Atezolizumab) and the second injection of 7.4 MBq 68Ga-Biotin with a 60 h interval, was then verified on the colon cancer-bearing mice. PET imaging was performed at 30, 90, 180 min to measure the standard uptake value and tumor to liver ratios. Cellular binding experiments and in vivo distribution showed that the conjugation of Avidin did not affect the affinity of Atezolizumab to PD-L1 antigen. Biotin was radio-labeled with 68Ga with radiolabeling efficiency of 70.5 ± 3.5% and purification was needed to increase the radiochemical purity. For PD-L1-positive tumors, SUVmax was 0.38 ± 0.06 in the Avidin-Atezolizumab pre-treated mice at 90 min; the tumor/liver ratios of pre-targeting group were 1.06 ± 0.19 and 0.97 ± 0.16 at 30 and 90 min, while the absence of pre-treatment of Avidin was of the lower ratios as 0.88 ± 0.01 and 0.54 ± 0.11 when 68Ga-Biotin served as the radiopharmaceutical as well. In conclusion, pre-targeting immunoPET strategy can elevate the target-to-nontarget ratio, decrease the blood background and shorten the interval between injection of radiopharmaceuticals and PET scan, providing a highly PD-L1-specific and sensitive imaging method for the detection of tumorous immune micro-environment.

Faricimab for Diabetic Macular Edema in Patients Refractory to Ranibizumab or Aflibercept.

Background and Objectives: Faricimab is the first intravitreal injection of vascular endothelial growth factor-A and angiopoietin-2 bispecific monoclonal antibody. Here, we evaluate the functional and anatomical outcomes of faricimab treatment in patients with diabetic macular edema (DME) that was refractory to ranibizumab or aflibercept. Materials and Methods: We performed a retrospective, observational, consecutive-case study of patients who had DME that was refractory to treatment with ranibizumab or aflibercept and were treated with faricimab between July 2022 and January 2023 under a pro re nata regimen. All the participants were followed for ≥4 months after the initiation of faricimab. The primary outcome was a recurrence interval of ≥12 weeks, and the secondary outcomes were the changes in best-corrected visual acuity (BCVA) and central macular thickness (CMT). Results: We analyzed 18 eyes of 18 patients. The mean recurrence interval of previous anti-VEGF injection was 5.8 ± 2.5 weeks, which was significantly extended to 10.8 ± 4.9 weeks (p = 0.0005) by the switch to faricimab. Eight patients (44.4%) achieved a recurrence interval of ≥12 weeks. A history of subtenon injection of triamcinolone acetonide (p = 0.0034) and the presence of disorganization of the retinal inner layers (p = 0.0326) were found to be significantly associated with a recurrence interval of <12 weeks. The mean BCVAs were 0.23 ± 0.28 logMAR and 0.19 ± 0.23 logMAR, and the mean CMTs were 473.8 ± 222.0 µm and 381.3 ± 219.4 µm at baseline and 4 months, respectively, but these changes were not statistically significant. None of the patients experienced serious adverse events. Conclusions: Faricimab may extend the treatment interval for patients with DME that is refractory to ranibizumab or aflibercept. DME previously treated with the subtenon injection of triamcinolone acetonide or associated with disorganization of the retinal inner layers may be less likely to be associated with a longer recurrence interval after switching to faricimab.

Peripheral CCL2-CCR2 signalling contributes to chronic headache-related sensitization.

Migraine, especially chronic migraine, is highly debilitating and still lacks effective treatment. The persistent headache arises from activation and sensitization of primary afferent neurons in the trigeminovascular pathway, but the underlying mechanisms remain incompletely understood. Animal studies indicate that signalling through chemokine C-C motif ligand 2 (CCL2) and C-C motif chemokine receptor 2 (CCR2) mediates the development of chronic pain after tissue or nerve injury. Some migraine patients had elevated CCL2 levels in CSF or cranial periosteum. However, whether the CCL2-CCR2 signalling pathway contributes to chronic migraine is not clear. Here, we modelled chronic headache with repeated administration of nitroglycerin (NTG, a reliable migraine trigger in migraineurs) and found that both Ccl2 and Ccr2 mRNA were upregulated in dura and trigeminal ganglion (TG) tissues that are implicated in migraine pathophysiology. In Ccl2 and Ccr2 global knockout mice, repeated NTG administration did not evoke acute or persistent facial skin hypersensitivity as in wild-type mice. Intraperitoneal injection of CCL2 neutralizing antibodies inhibited chronic headache-related behaviours induced by repeated NTG administration and repetitive restraint stress, suggesting that the peripheral CCL2-CCR2 signalling mediates headache chronification. We found that CCL2 was mainly expressed in TG neurons and cells associated with dura blood vessels, whereas CCR2 was expressed in subsets of macrophages and T cells in TG and dura but not in TG neurons under both control and disease states. Deletion of Ccr2 gene in primary afferent neurons did not alter NTG-induced sensitization, but eliminating CCR2 expression in either T cells or myeloid cells abolished NTG-induced behaviours, indicating that both CCL2-CCR2 signalling in T cells and macrophages are required to establish chronic headache-related sensitization. At cellular level, repeated NTG administration increased the number of TG neurons that responded to calcitonin-gene-related peptide (CGRP) and pituitary adenylate cyclase activating polypeptide (PACAP) as well as the production of CGRP in wild-type but not Ccr2 global knockout mice. Lastly, co-administration of CCL2 and CGRP neutralizing antibodies was more effective in reversing NTG-induced behaviours than individual antibodies. Taken together, these results suggest that migraine triggers activate CCL2-CCR2 signalling in macrophages and T cells. This consequently enhances both CGRP and PACAP signalling in TG neurons, ultimately leading to persistent neuronal sensitization underlying chronic headache. Our work not only identifies the peripheral CCL2 and CCR2 as potential targets for chronic migraine therapy, but also provides proof-of-concept that inhibition of both peripheral CGRP and CCL2-CCR2 signalling is more effective than targeting either pathway alone.