The homodimer interfaces of costimulatory receptors B7 and CD28 control their engagement and pro-inflammatory signaling.

BACKGROUND: The inflammatory response is indispensable for protective immunity, yet microbial pathogens often trigger an excessive response, 'cytokine storm', harmful to the host. Full T-cell activation requires interaction of costimulatory receptors B7-1(CD80) and B7-2(CD86) expressed on antigen-presenting cells with CD28 expressed on the T cells. We created short peptide mimetics of the homodimer interfaces of the B7 and CD28 receptors and examined their ability to attenuate B7/CD28 coligand engagement and signaling through CD28 for inflammatory cytokine induction in human immune cells, and to protect from lethal toxic shock in vivo. METHODS: Short B7 and CD28 receptor dimer interface mimetic peptides were synthesized and tested for their ability to attenuate the inflammatory cytokine response of human peripheral blood mononuclear cells, as well as for their ability to attenuate B7/CD28 intercellular receptor engagement. Mice were used to test the ability of such peptides to protect from lethal superantigen toxin challenge when administered in molar doses far below the toxin dose. RESULTS: B7 and CD28 homodimer interfaces are remote from the coligand binding sites, yet our finding is that by binding back into the receptor dimer interfaces, short dimer interface mimetic peptides inhibit intercellular B7-2/CD28 as well as the tighter B7-1/CD28 engagement, attenuating thereby pro-inflammatory signaling. B7 mimetic peptides exhibit tight selectivity for the cognate receptor in inhibiting intercellular receptor engagement with CD28, yet each diminishes signaling through CD28. In a prominent example of inflammatory cytokine storm, by attenuating formation of the B7/CD28 costimulatory axis, B7-1 and CD28 dimer interface mimetic peptides protect mice from lethal toxic shock induced by a bacterial superantigen even when administered in doses far submolar to the superantigen. CONCLUSIONS: Our results reveal that the B7 and CD28 homodimer interfaces each control B7/CD28 costimulatory receptor engagement and highlight the protective potential against cytokine storm of attenuating, yet not ablating, pro-inflammatory signaling via these receptor domains.

In-vivo imaging for assessing tumor growth in mouse models of ocular melanoma.

Uveal melanoma (UM) and conjunctival melanoma (CM) are ocular malignancies that give rise to life-threatening metastases. Although local disease can often be treated successfully, it is often associated with significant vision impairment and treatments are often not effective against metastatic disease. Novel treatment modalities that preserve vision may enable elimination of small tumors and may prevent subsequent metastatic spread. Very few mouse models of metastatic CM and UM are available for research and for development of novel therapies. One of the challenges is to follow tumor growth in-vivo and to determine the right size for treatment, mainly of the posterior, choroidal melanoma. Hence, the purpose of this study was to establish a simple, noninvasive imaging tool that will simplify visualization and tumor follow-up in mouse models of CM and UM. Tumors were induced by inoculation of murine B16LS9 cells into the sub-conjunctival or the choroidal space of a C57BL/6 mouse eye under a surgical microscope. Five to ten days following injection, tumor size was assessed by Phoenix MicronIV™ image-guided Optical Coherence Tomography (OCT) imaging, which included a real-time camera view and OCT scan of the conjunctiva and the retina. In addition, tumor size was evaluated by ultrasound and histopathological examination of eye sections. Tumor growth was observed 5-9 days following sub-conjunctival or sub-retinal injection of seven-thousand or seventy-thousand cells, respectively. A clear tumor mass was detected at these regions using the MicronIV™ imaging system camera and OCT scans. Histology of eye sections confirmed the presence of tumor tissue. OCT allowed an accurate measurement of tumor size in the UM model and a qualitative assessment of tumor size in the CM model. Moreover, OCT enabled assessing the success rate of the choroidal tumor induction and importantly, predicted final tumor size already on the day of cell inoculation. In conclusion, by using a simple, non-invasive imaging tool, we were able to follow intraocular tumor growth of both CM and UM, and to define, already at the time of cell inoculation, a grading scale to evaluate tumor size. This tool may be utilized for evaluation of new mouse models for CM and UM, as well as for testing new therapies for these diseases.

Superantigens hyperinduce inflammatory cytokines by enhancing the B7-2/CD28 costimulatory receptor interaction.

Full T-cell activation requires interaction between the costimulatory receptors B7-2 and CD28. By binding CD28, bacterial superantigens elicit harmful inflammatory cytokine overexpression through an unknown mechanism. We show that, by engaging not only CD28 but also its coligand B7-2 directly, superantigens potently enhance the avidity between B7-2 and CD28, inducing thereby T-cell hyperactivation. Using the same 12-aa ß-strand-hinge-α-helix domain, superantigens engage both B7-2 and CD28 at their homodimer interfaces, areas remote from where these coreceptors interact, implying that inflammatory signaling can be controlled through the receptor homodimer interfaces. Short B7-2 dimer interface mimetic peptides bind diverse superantigens, prevent superantigen binding to cell-surface B7-2 or CD28, attenuate inflammatory cytokine overexpression, and protect mice from lethal superantigen challenge. Thus, superantigens induce a cytokine storm not only by mediating the interaction between MHC-II molecule and T-cell receptor but also, critically, by promoting B7-2/CD28 coreceptor engagement, forcing the principal costimulatory axis to signal excessively. Our results reveal a role for B7-2 as obligatory receptor for superantigens. B7-2 homodimer interface mimotopes prevent superantigen lethality by blocking the superantigen-host costimulatory receptor interaction.

Binding of superantigen toxins into the CD28 homodimer interface is essential for induction of cytokine genes that mediate lethal shock.

Bacterial superantigens, a diverse family of toxins, induce an inflammatory cytokine storm that can lead to lethal shock. CD28 is a homodimer expressed on T cells that functions as the principal costimulatory ligand in the immune response through an interaction with its B7 coligands, yet we show here that to elicit inflammatory cytokine gene expression and toxicity, superantigens must bind directly into the dimer interface of CD28. Preventing access of the superantigen to CD28 suffices to block its lethality. Mice were protected from lethal superantigen challenge by short peptide mimetics of the CD28 dimer interface and by peptides selected to compete with the superantigen for its binding site in CD28. Superantigens use a conserved ß-strand/hinge/α-helix domain of hitherto unknown function to engage CD28. Mutation of this superantigen domain abolished inflammatory cytokine gene induction and lethality. Structural analysis showed that when a superantigen binds to the T cell receptor on the T cell and major histocompatibility class II molecule on the antigen-presenting cell, CD28 can be accommodated readily as third superantigen receptor in the quaternary complex, with the CD28 dimer interface oriented towards the ß-strand/hinge/α-helix domain in the superantigen. Our findings identify the CD28 homodimer interface as a critical receptor target for superantigens. The novel role of CD28 as receptor for a class of microbial pathogens, the superantigen toxins, broadens the scope of pathogen recognition mechanisms.

Intracameral Injection in Rats with Low Risk of Adverse Effects.

Intracameral injection is a standard administration routine in ophthalmology. The application of intracameral injection in rodents for research is challenging due to the limiting dimensions and anatomy of the eye, including the small aqueous humor volume, the lens curvature, and lens thickness. Potential damage during intracameral injections introduces adverse effects and experimental variability. This protocol describes a procedure for intracameral injection in rats, allowing precision and reproducibility. Sprague-Dawley rats were used as experimental models. Since the lens position in rats protrudes into the anterior chamber, injecting from the periphery, as done in humans, is unfavorable. Therefore, an incision is created in the central corneal region using a 31 gauge 0.8 mm stiletto blade to form a self-sealing tunnel into the anterior chamber. An incision at an angle close to the flat allows to create a long tunnel, which minimizes the loss of aqueous humor and shallowing of the anterior chamber. A 34 gauge nanoneedle is inserted into the tunnel for injection. This enables penetration with minimal friction resistance and avoids touching the lens. Injection of trypan-blue allows visualization by slit microscopy the presence of the dye in the anterior chamber and exclude leakage. Bioavailability to the corneal endothelial layer is demonstrated by injection of Hoechst dye, which stained the nuclei of corneal endothelial cells after injection. In conclusion, this protocol implements a procedure for accurate intracameral injection in rats. This procedure may be used for intracameral delivery of various drugs and compounds in experimental rat models, increasing the efficiency and reproducibility of ophthalmic research.

Mitochondria Transplantation Promotes Corneal Epithelial Wound Healing.

Purpose: The integrity of the corneal epithelium is essential in maintaining normal corneal function. Conditions disrupting the corneal epithelial layer range from chemical burns to dry eye disease and may result in impairment of both corneal transparency and sensation. Identifying factors that regulate corneal wound healing is key for the development of new treatment strategies. Here, we investigated a direct role of mitochondria in corneal wound healing via mitochondria transplantation. Methods: Human corneal epithelial cells (hCECs) were isolated from human corneas and incubated with mitochondria which were isolated from human ARPE-19 cells. We determined the effect of mitochondria transplantation on wound healing and proliferation of hCECs. In vivo, we used a mouse model of corneal chemical injury. Mitochondria were isolated from mouse livers and topically applied to the ocular surface following injury. We evaluated the time of wound repair, corneal re-epithelization, and stromal abnormalities. Results: Mitochondria transplantation induced the proliferation and wound healing of primary hCECs. Further, mitochondria transplantation promoted wound healing in vivo. Specifically, mice receiving mitochondria recovered twice as fast as control mice following corneal injury, presenting both enhanced and improved repair. Corneas treated with mitochondria demonstrated the re-epithelization of the wound area to a multi-layer appearance, compared to thinning and complete loss of the epithelium in control mice. Mitochondria transplantation also prevented the thickening and disorganization of the corneal stromal lamella, restoring normal corneal dehydration. Conclusions: Mitochondria promote corneal re-epithelization and wound healing. Augmentation of mitochondria levels via mitochondria transplantation may serve as an effective treatment for inducing the rapid repair of corneal epithelial defects.

Addressing the challenge of solution gating in biosensors based on field-effect transistors.

Transistor-based biosensing (BioFET) is a long-enduring vision for next generation medical diagnostics. The study addresses a challenge associated with the BioFET solution gating. The standard BioFET sensing measurement involves sweeping of the solution gate (Vsol) with a concurrent measurement of the source-drain current (IDS). This IDS-Vsol sweep poses a great challenge, as Vsol does not only determine IDS, but also determines the pH levels, ion concentrations, and electric fields at the sensing area double layer accommodating the biomolecules. Therefore, inevitably, an IDS-Vsol sweep implies that the sensing area double layer is not in an electrochemical equilibrium, but rather in a continuous transient state as electrochemical potential gradients induce transient ion currents continuously affecting double layer hosting the biomolecules and the biological interactions. This challenge calls for a BioFET design which permits IDS sweeping from an off-state to an on-state while keeping Vsol constant and the double layer sensing area in electrochemical equilibrium. The study explores a BioFET design addressing this challenge by decoupling the solution potential from IDS gating. Specific and label-free sensing of ferritin in 0.5 µL drops of 1:100 diluted plasma is pursued. We show an excellent sensing performance once the solution potential and IDS gating are decoupled, with a limit-of-detection of 10 fg/ml, a dynamic range of 10 orders of magnitude in ferritin concentration and excellent linearity and sensitivity. In contrast, a poor sensing performance is recorded for the conventional Vsol sweep performed in parallel to the above. Extensive control measurements quantifying the non-specific signals are reported.

From sensing interactions to controlling the interactions: a novel approach to obtain biological transistors for specific and label-free immunosensing.

Antibody-antigen interactions are shaped by the solution pH level, ionic strength, and electric fields, if present. In biological field-effect transistors (BioFETs), the interactions take place at the sensing area in which the pH level, ionic strength and electric fields are determined by the Poisson-Boltzmann equation and the boundary conditions at the solid-solution interface and the potential applied at the solution electrode. The present study demonstrates how a BioFET solution electrode potential affects the sensing area double layer pH level, ionic strength, and electric fields and in this way shapes the biological interactions at the sensing area. We refer to this as 'active sensing'. To this end, we employed the meta-nano-channel (MNC) BioFET and demonstrate how the solution electrode can determine the antibody-antigen equilibrium constant and allows the control and tuning of the sensing performance in terms of the dynamic range and limit-of-detection. In the current work, we employed this method to demonstrate the specific and label-free sensing of Alpha-Fetoprotein (AFP) molecules from 0.5 µL drops of 1 : 100 diluted serum. AFP was measured during pregnancy as part of the prenatal screening program for fetal anomalies, chromosomal abnormalities, and abnormal placentation. We demonstrate AFP sensing with a limit-of-detection of 10.5 aM and a dynamic range of 6 orders of magnitude in concentration. Extensive control measurements are reported.

Impact of topographic hot spots on the refractive outcomes of combined DMEK and cataract surgery.

PURPOSE: To compare the refractive results of combined Descemet membrane endothelial keratoplasty (DMEK) and cataract surgery in eyes with Fuchs endothelial corneal dystrophy (FECD) with and without topographic hot spots. SETTING: "Villa Igea" Hospital, Forlì, Italy. DESIGN: Interventional case series. METHODS: 57 eyes of 52 patients with FECD undergoing combined DMEK, cataract surgery, and monofocal intraocular lens (IOL) implantation were included in this single-center study. Patients were classified based on the presence or absence of topographic hot spots on the preoperative axial power map. Prediction error (PE) was calculated as the postoperative manifest spherical equivalent (SE) refraction minus the SE predicted refraction. RESULTS: 6 months postoperatively, mean PE was +0.79 ± 1.12 diopters (D). In eyes with hot spots, mean keratometry (K), K flat, and K steep significantly decreased after surgery (all P < .05), whereas no significant changes were observed in eyes without hot spots (all P > .05). Eyes with hot spots showed a significantly more hyperopic PE than eyes without hot spots (+1.13 ± 1.23 vs +0.40 ± 0.86 D; P = .013). CONCLUSIONS: Combined DMEK and cataract surgery can result in a hyperopic refractive surprise. The presence of topographic hot spots before surgery is associated with a higher hyperopic shift.

Implantation and Evaluation of Melanoma in the Murine Choroid via Optical Coherence Tomography.

Establishing experimental choroidal melanoma models is challenging in terms of the ability to induce tumors at the correct localization. In addition, difficulties in observing posterior choroidal melanoma in vivo limit tumor location and growth evaluation in real-time. The approach described here optimizes techniques for establishing choroidal melanoma in mice via a multi-step sub-choroidal B16LS9 cell injection procedure. To enable precision in injecting into the small dimensions of the mouse uvea, the complete procedure is performed under a microscope. First, a conjunctival peritomy is formed in the dorsal-temporal area of the eye. Then, a tract into the sub-choroidal space is created by inserting a needle through the exposed sclera. This is followed by the insertion of a blunt needle into the tract and the injection of melanoma cells into the choroid. Immediately after injection, noninvasive optical coherence tomography (OCT) imaging is utilized to determine tumor location and progress. Retinal detachment is evaluated as a predictor of tumor site and size. The presented method enables the reproducible induction of choroid-localized melanoma in mice and the live imaging of tumor growth evaluation. As such, it provides a valuable tool for studying intraocular tumors.

Oxidative stress facilitates exogenous mitochondria internalization and survival in retinal ganglion precursor-like cells.

Ocular cells are highly dependent on mitochondrial function due to their high demand of energy supply and their constant exposure to oxidative stress. Indeed, mitochondrial dysfunction is highly implicated in various acute, chronic, and genetic disorders of the visual system. It has recently been shown that mitochondrial transplantation (MitoPlant) temporarily protects retinal ganglion cells (RGCs) from cell death during ocular ischemia. Here, we characterized MitoPlant dynamics in retinal ganglion precursor-like cells, in steady state and under oxidative stress. We developed a new method for detection of transplanted mitochondria using qPCR, based on a difference in the mtDNA sequence of C57BL/6 and BALB/c mouse strains. Using this approach, we show internalization of exogenous mitochondria already three hours after transplantation, and a decline in mitochondrial content after twenty four hours. Interestingly, exposure of target cells to moderate oxidative stress prior to MitoPlant dramatically enhanced mitochondrial uptake and extended the survival of mitochondria in recipient cells by more than three fold. Understanding the factors that regulate the exogenous mitochondrial uptake and their survival may promote the application of MitoPlant for treatment of chronic and genetic mitochondrial diseases.

Staphylococcal and Streptococcal Superantigens Trigger B7/CD28 Costimulatory Receptor Engagement to Hyperinduce Inflammatory Cytokines.

Staphylococcal and streptococcal superantigens are virulence factors that cause toxic shock by hyperinducing inflammatory cytokines. Effective T-cell activation requires interaction between the principal costimulatory receptor CD28 and its two coligands, B7-1 (CD80) and B7-2 (CD86). To elicit an inflammatory cytokine storm, bacterial superantigens must bind directly into the homodimer interfaces of CD28 and B7-2. Recent evidence revealed that by engaging CD28 and B7-2 directly at their dimer interface, staphylococcal enterotoxin B (SEB) potently enhances intercellular synapse formation mediated by B7-2 and CD28, resulting in T-cell hyperactivation. Here, we addressed the question, whether diverse bacterial superantigens share the property of triggering B7-2/CD28 receptor engagement and if so, whether they are capable of enhancing also the interaction between B7-1 and CD28, which occurs with an order-of-magnitude higher affinity. To this end, we compared the ability of distinct staphylococcal and streptococcal superantigens to enhance intercellular B7-2/CD28 engagement. Each of these diverse superantigens promoted B7-2/CD28 engagement to a comparable extent. Moreover, they were capable of triggering the intercellular B7-1/CD28 interaction, analyzed by flow cytometry of co-cultured cell populations transfected separately to express human CD28 or B7-1. Streptococcal mitogenic exotoxin Z (SMEZ), the most potent superantigen known, was as sensitive as SEB, SEA and toxic shock syndrome toxin-1 (TSST-1) to inhibition of inflammatory cytokine induction by CD28 and B7-2 dimer interface mimetic peptides. Thus, superantigens act not only by mediating unconventional interaction between MHC-II molecule and T-cell receptor but especially, by strongly promoting engagement of CD28 by its B7-2 and B7-1 coligands, a critical immune checkpoint, forcing the principal costimulatory axis to signal excessively. Our results show that the diverse superantigens use a common mechanism to subvert the inflammatory response, strongly enhancing B7-1/CD28 and B7-2/CD28 costimulatory receptor engagement.

Bacterial superantigen toxins induce a lethal cytokine storm by enhancing B7-2/CD28 costimulatory receptor engagement, a critical immune checkpoint.

Formation of the costimulatory axis between the B7-2 and CD28 coreceptors is critical for T-cell activation. Superantigens, Gram-positive bacterial virulence factors, cause toxic shock and sepsis by hyperinducing inflammatory cytokines. We report a novel role for costimulatory receptors CD28 and B7-2 as obligatory receptors for superantigens, rendering them therapeutic targets. We show that by engaging not only CD28 but also its coligand B7-2 directly, superantigens potently enhance the interaction between B7-2 and CD28, inducing thereby T-cell hyperactivation. Using a conserved twelve amino-acid domain, superantigens engage both B7-2 and CD28 at their homodimer interfaces, sites far removed from where these receptors interact, implying that inflammatory signaling can be controlled through the receptor homodimer interfaces. Short B7-2 and CD28 dimer interface mimetic peptides bind diverse superantigens, prevent superantigen binding to cell-surface B7-2 or CD28, attenuate inflammatory cytokine overexpression, and protect mice from lethal superantigen challenge. Thus, superantigens induce a cytokine storm by mediating not only the interaction between MHC-II molecule and T-cell receptor but critically, by promoting B7-2/CD28 coreceptor engagement, forcing the principal costimulatory axis to signal excessively. Our findings highlight the B7/CD28 interaction as a bottleneck in signaling for expression of inflammatory cytokines. B7-2 and CD28 homodimer interface mimetic peptides prevent superantigen lethality by blocking the superantigen-host costimulatory receptor interaction.

CD28: direct and critical receptor for superantigen toxins.

Every adaptive immune response requires costimulation through the B7/CD28 axis, with CD28 on T-cells functioning as principal costimulatory receptor. Staphylococcal and streptococcal superantigen toxins hyperstimulate the T-cell-mediated immune response by orders of magnitude, inducing a lethal cytokine storm. We show that to elicit an inflammatory cytokine storm and lethality, superantigens must bind directly to CD28. Blocking access of the superantigen to its CD28 receptor with peptides mimicking the contact domains in either toxin or CD28 suffices to protect mice effectively from lethal shock. Our finding that CD28 is a direct receptor of superantigen toxins broadens the scope of microbial pathogen recognition mechanisms.