Targeting folate receptor beta on monocytes/macrophages renders rapid inflammation resolution independent of root causes.

Provoked by sterile/nonsterile insults, prolonged monocyte mobilization and uncontrolled monocyte/macrophage activation can pose imminent or impending harm to the affected organs. Curiously, folate receptor beta (FRß), with subnanomolar affinity for the vitamin folic acid (FA), is upregulated during immune activation in hematopoietic cells of the myeloid lineage. This phenomenon has inspired a strong interest in exploring FRß-directed diagnostics/therapeutics. Previously, we have reported that FA-targeted aminopterin (AMT) therapy can modulate macrophage function and effectively treat animal models of inflammation. Our current investigation of a lead compound (EC2319) leads to discovery of a highly FR-specific mechanism of action independent of the root causes against inflammatory monocytes. We further show that EC2319 suppresses interleukin-6/interleukin-1ß release by FRß+ monocytes in a triple co-culture leukemic model of cytokine release syndrome with anti-CD19 chimeric antigen receptor T cells. Because of its chemical stability and metabolically activated linker, EC2319 demonstrates favorable pharmacokinetic characteristics and cross-species translatability to support future pre-clinical and clinical development.

Mechanism of Nucleic Acid Sensing in Retinal Pigment Epithelium (RPE): RIG-I Mediates Type I Interferon Response in Human RPE.

Age-related macular degeneration (AMD), a degenerative disease of the outer retina, is the leading cause of blindness among the elderly. A hallmark of geographic atrophy (GA), an advanced type of nonneovascular AMD (dry AMD), is photoreceptor and retinal pigment epithelium (RPE) cell death. Currently, there are no FDA-approved therapies for GA due to a lack of understanding of the disease-causing mechanisms. Increasing evidence suggests that chronic inflammation plays a predominant role in the pathogenesis of dry AMD. Dead or stressed cells release danger signals and inflammatory factors, which causes further damage to neighboring cells. It has been reported that type I interferon (IFN) response is activated in RPE cells in patients with AMD. However, how RPE cells sense stress to initiate IFN response and cause further damage to the retina are still unknown. Although it has been reported that RPE can respond to extracellularly added dsRNA, it is unknown whether and how RPE detects and senses internally generated or internalized nucleic acids. Here, we elucidated the molecular mechanism by which RPE cells sense intracellular nucleic acids. Our data demonstrate that RPE cells can respond to intracellular RNA and induce type I IFN responses via the RIG-I (DExD/H-box helicase 58, DDX58) RNA helicase. In contrast, we showed that RPE cells were unable to directly sense and respond to DNA through the cGAS-STING pathway. We demonstrated that this was due to the absence of the cyclic GMP-AMP synthase (cGAS) DNA sensor in these cells. The activation of IFN response via RIG-I induced expression of cell death effectors and caused barrier function loss in RPE cells. These data suggested that RPE-intrinsic pathways of nucleic acid sensing are biased toward RNA sensing.

Differential Diagnosis of Sjögren Versus Non-Sjögren Dry Eye Through Tear Film Biomarkers.

PURPOSE: Systemic implications necessitate the identification of dry eye patients with Sjögren syndrome (SS). This study aims to explore the utility of tear MUC5AC and inflammatory cytokine levels in the differential diagnosis of SS-related dry eye. METHODS: A prospective, observational, case-control study was conducted on 62 patients (those with a definitive diagnosis of SS dry eye, non-SS dry eye, and age-matched healthy controls with no dry eye). Clinical evaluations included the following tests in the order listed here: noninvasive tear break-up time, osmolarity, tear sampling, Schirmer test without anesthesia, and ocular surface staining (lissamine green for conjunctiva and fluorescein for cornea). Tear MUC5AC levels were assessed with enzyme-linked immunosorbent assay, and cytokines [interferon-gamma, tumor necrosis factor alpha, interleukin (IL)-6, IL-17a, IL-1ß, IL-8, IL-10, and IL-12p70] were measured using a Luminex assay in a masked fashion. RESULTS: The Bulbar conjunctival lissamine green staining score was significantly greater in patients or controls with SS versus non-SS dry eye. This greater conjunctival staining was associated with a reduction in tear MUC5AC (B = -17.8 ng/mL, 95% confidence interval = -31.8 to -3.9, P = 0.01). Among the tear cytokines, a significant association was found between IL-8 levels (hazard ratio [HR] = 1.002, 95% confidence interval = 1.000-1.003, P = 0.03) and SS diagnosis. When patients were stratified based on tear MUC5AC levels, significantly increased tear IL-8 levels were detected in patients with SS dry eye but not with non-SS dry eye, in comparison with healthy controls. CONCLUSIONS: Tear levels of goblet cell-specific MUC5AC combined with IL-8 can potentially serve as a useful biomarker for differential diagnosis of SS dry eye from non-SS dry eye.

In vivo anti-LAP mAb enhances IL-17/IFN-γ responses and abrogates anti-CD3-induced oral tolerance.

Regulatory T cells (Tregs) play a critical role in the maintenance of immunological tolerance. The best-characterized Tregs are those expressing the transcription factor Foxp3 and in vivo modulation of Foxp3 Tregs has been employed to study their role in immune homeostasis. Latency-associated peptide (LAP) is a membrane-bound TGF-ß complex that has also been shown to play a role in Treg function and oral tolerance. We developed a novel anti-mouse LAP mAb that allowed us to investigate the effect of targeting LAP in vivo on immune function and on anti-CD3-induced oral tolerance. We found that in vivo anti-LAP mAb administration led to a decrease in the number of CD4+LAP+ Tregs in spleen and lymph nodes without affecting CD4+Foxp3+ Tregs. Spleen cells from anti-LAP-injected mice proliferated more in vitro and produced increased amounts of IL-2, IL-17 and IFN-γ. Moreover, injection of anti-LAP antibody abrogated the protective effect of oral anti-CD3 on experimental autoimmune encephalomyelitis (EAE). Finally, in vivo anti-LAP administration prior to myelin oligodendrocyte glycoprotein immunization resulted in severe EAE in the absence of pertussis toxin, which is used for EAE induction. Our findings demonstrate the importance of CD4+LAP+ T cells in the control of immune homeostasis and autoimmunity and provides a new tool for the in vivo investigation of murine LAP+ Tregs on immune function.

T and B cell hyperactivity and autoimmunity associated with niche-specific defects in apoptotic body clearance in TIM-4-deficient mice.

TIM-4, a member of the TIM family expressed on antigen-presenting cells, binds to phosphatidylserine exposed on the surface of apoptotic bodies. However, the significance of this interaction in vivo remains unknown because other receptors have been implicated in the clearance of apoptotic bodies and could compensate for the TIM-4 deficiency in vivo. In this study, we describe the generation of TIM-4-deficient mice and address whether TIM-4 serves a unique function in vivo. We show that TIM-4(-/-) peritoneal macrophages and B-1 cells do not efficiently engulf apoptotic bodies in vitro, or clear apoptotic bodies in vivo. TIM-4-deficient mice have hyperactive T and B cells, elevated levels of serum Ig, and develop antibodies to double-stranded DNA. Taken together, we show that TIM-4 is critical for the clearance of apoptotic bodies in vivo, and that lack of TIM-4 results in aberrant persistence of apoptotic bodies leading to dysregulated lymphocyte activation and signs of systemic autoimmunity.

Automated calibration of TECAN genesis liquid handling workstation utilizing an online balance and density meter.

With robotics widely used in bioanalytical assays, accurate system performance is essential to ensure the quality and productivity of the robotics. In our lab, an automated calibration procedure has been developed to evaluate the precision and accuracy of the TECAN (Research Triangle Park, NC, U.S.A.) Genesis liquid handling system in a bioanalytical laboratory setting. The calibrations were performed by transferring and weighing the solvents automatically on a microbalance controlled by a Gemini program. From the data acquired, calibration reports were generated using a template. The novel aspect of this approach is the use of an on-line balance and a density meter, both of which combine to make the calibration process simple, efficient, and precise. For quantitative bioanalysis, a variety of solvents, including methanol, water, mixed solvents, and plasma, are typically used to prepare standards and unknown samples. Density information is usually unknown for the mixed solvents, and the density of plasma can vary from species to species. However, with the use of a universal density meter, the density could be obtained in seconds. The issue of solvent evaporation during the calibration process was also addressed. Calibration curves were set up for various liquid classes. Pipetting volumes ranged from 10 microL to 900 microL. Precision and accuracy results obtained from the semiannual performance evaluations showed this procedure to be reliable and user-friendly. Using the automated calibration procedure, the calibration and performance evaluation of the robotic system is considerably more efficient, and the incidence of unacceptable precision and accuracy is greatly reduced.