Individual astrocyte morphology in the collagenous lamina cribrosa revealed by multicolor DiOlistic labeling.

Astrocytes in the lamina region of the optic nerve head play vital roles in supporting retinal ganglion cell axon health. In glaucoma, these astrocytes are implicated as early responders to stressors, undergoing characteristic changes in cell function as well as cell morphology. Much of what is currently known about individual lamina astrocyte morphology has been learned from rodent models which lack a defining feature of the human optic nerve head, the collagenous lamina cribrosa (LC). Current methods available for evaluation of collagenous LC astrocyte morphology have significant shortcomings. We aimed to evaluate Multicolor DiOlistic labeling (MuDi) as an approach to reveal individual astrocyte morphologies across the collagenous LC. Gold microcarriers were coated with all combinations of three fluorescent cell membrane dyes, DiI, DiD, and DiO, for a total of seven dye combinations. Microcarriers were delivered to 150 µm-thick coronal vibratome slices through the LC of pig, sheep, goat, and monkey eyes via MuDi. Labeled tissues were imaged with confocal and second harmonic generation microscopy to visualize dyed cells and LC collagenous beams, respectively. GFAP labeling of DiOlistically-labeled cells with astrocyte morphologies was used to investigate cell identity. 3D models of astrocytes were created from confocal image stacks for quantification of morphological features. DiOlistic labeling revealed fine details of LC astrocyte morphologies including somas, primary branches, higher-order branches, and end-feet. Labeled cells with astrocyte morphologies were GFAP+. Astrocytes were visible across seven distinct color channels, allowing high labeling density while still distinguishing individual cells from their neighbors. MuDi was capable of revealing tens to hundreds of collagenous LC astrocytes, in situ, with a single application. 3D astrocyte models allowed automated quantification of morphological features including branch number, length, thickness, hierarchy, and straightness as well as Sholl analysis. MuDi labeling provides an opportunity to investigate morphologies of collagenous LC astrocytes, providing both qualitative and quantitative detail, in healthy tissues. This approach may open doors for research of glaucoma, where astrocyte morphological alterations are thought to coincide with key functional changes related to disease progression.

"Toll"-erance in the skin.

Interactions between potentially pathogenic commensal bacteria and cutaneous immunity are poorly understood. In this issue of Immunity, Skabytska et al. (2014) show that S. aureus-derived TLR2/6 heterodimer ligands can recruit myeloid-derived suppressor cells into the skin, countering rather than promoting inflammation.

Skin codelivery of contact sensitizers and neurokinin-1 receptor antagonists integrated in microneedle arrays suppresses allergic contact dermatitis.

BACKGROUND: Allergic contact dermatitis (CD) is a chronic inflammatory skin disease caused by type 1 biased adaptive immunity for which there is an unmet need for antigen (Ag)-specific immunotherapies. Exposure to skin sensitizers stimulates secretion of the proinflammatory neuropeptides substance P and hemokinin 1, which signal via the neurokinin-1 receptor (NK1R) to promote the innate and adaptive immune responses of CD. Accordingly, mice lacking the NK1R develop impaired CD. Nonetheless, the role and therapeutic opportunities of targeting the NK1R in CD remain to be elucidated. OBJECTIVE: We sought to develop an Ag-specific immunosuppressive approach to treat CD by skin codelivery of hapten and NK1R antagonists integrated in dissolvable microneedle arrays (MNA). METHODS: In vivo mouse models of contact hypersensitivity and ex vivo models of human skin were used to delineate the effects and mechanisms of NK1R signaling and the immunosuppressive effects of the contact sensitizer NK1R antagonist MNA in CD. RESULTS: We demonstrated in mice that CD requires NK1R signaling by substance P and hemokinin 1. Specific deletion of the NK1R in keratinocytes and dendritic cells, but not in mast cells, prevented CD. Skin codelivery of hapten or Ag MNA inhibited neuropeptide-mediated skin inflammation in mouse and human skin, promoted deletion of Ag-specific effector T cells, and increased regulatory T cells, which prevented CD onset and relapses locally and systemically in an Ag-specific manner. CONCLUSIONS: Immunoregulation by engineering localized skin neuroimmune networks can be used to treat cutaneous diseases that like CD are caused by type 1 immunity.

A single subcutaneous or intranasal immunization with adenovirus-based SARS-CoV-2 vaccine induces robust humoral and cellular immune responses in mice.

Optimal vaccines are needed for sustained suppression of SARS-CoV-2 and other novel coronaviruses. Here, we developed a recombinant type 5 adenovirus vector encoding the gene for the SARS-CoV-2 S1 subunit antigen (Ad5.SARS-CoV-2-S1) for COVID-19 immunization and evaluated its immunogenicity in mice. A single immunization with Ad5.SARS-CoV-2-S1 via S.C. injection or I.N delivery induced robust antibody and cellular immune responses. Vaccination elicited significant S1-specific IgG, IgG1, and IgG2a endpoint titers as early as 2 weeks, and the induced antibodies were long lasting. I.N. and S.C. administration of Ad5.SARS-CoV-2-S1 produced S1-specific GC B cells in cervical and axillary LNs, respectively. Moreover, I.N. and S.C. immunization evoked significantly greater antigen-specific T-cell responses compared to unimmunized control groups with indications that S.C. injection was more effective than I.N. delivery in eliciting cellular immune responses. Mice vaccinated by either route demonstrated significantly increased virus-specific neutralization antibodies on weeks 8 and 12 compared to control groups, as well as BM antibody forming cells (AFC), indicative of long-term immunity. Thus, this Ad5-vectored SARS-CoV-2 vaccine candidate showed promising immunogenicity following delivery to mice by S.C. and I.N. routes of administration, supporting the further development of Ad-based vaccines against COVID-19 and other infectious diseases for sustainable global immunization programs.

Utilization of Asynchronous and Synchronous Teledermatology in a Large Health Care System During the COVID-19 Pandemic.

Background: Teledermatology offers an opportunity to continually deliver care during the coronavirus disease 2019 pandemic. Objective: To provide quantitative data about the use of teledermatology. Methods: Retrospective analysis of teledermatology consultations was performed from March 16 to May 1, 2020. The number/type of encounters, differences in diagnoses, and prescriptions between asynchronous and synchronous teledermatology visits were analyzed. Results: A total of 951 visits (36.2%) were asynchronous whereas 1,672 visits (63.8%) were synchronous. Only 131 (<5%) visits required an acute in-person follow-up. The diagnosis of acne was more frequent with asynchronous visits (p < 0.002, Bonferroni corrected). Antibiotics and nonretinoid acne medications were prescribed more with asynchronous visits, whereas immunomodulators and biologics were more commonly prescribed with synchronous visits (p < 0.02, Bonferroni corrected). Providers at our institution were split on preferred mode (54.2% synchronous, 45.8% asynchronous); however, synchronous visits were preferred for complex medical dermatology patients and return patients (p < 0.05). Limitations: This study is limited by being a single-center study. Conclusions: Asynchronous teledermatology was used more for acne management, whereas synchronous teledermatology was preferable to providers for complex medical dermatology. Postanalysis of the data collected led us to institute a hybridization of our asynchronous and synchronous teledermatology.

Inpatient eDermatology (Teledermatology) Can Help Meet the Demand for Inpatient Skin Disease.

Background: Currently, the number of inpatient dermatology providers cannot meet the overall burden of inpatient skin disease in the United States. Introduction: We seek to determine whether inpatient eDermatology can meet the need for inpatient skin disease in hospitals without access to a dermatology hospitalist. Methods: This retrospective cohort study reviewed inpatient eDermatology consults at the University of Pittsburgh eDermatology Consult Service between July 1, 2014 and June 30, 2018. This included a diverse group of 1,320 patients admitted to 10 different community hospitals. Study data were reviewed for demographics, diagnostic impressions, time to discharge, and diagnostic discordance between referring and consultant physicians. Results: Forty percent of inpatient eDermatology consults were admitted with a primary dermatologic diagnosis. Referring diagnosis most commonly was rash not otherwise specified. eDermatology consulting impressions, conversely, were specific and varied. Ninety-one percent of patients received a consultant impression by the end of day, or within 8 hours. Overall, 89.3% of patients with a referring diagnosis of "cellulitis" were given a different diagnosis by the consultant. Discussion: Although this study lacked concordance data to compare the Inpatient eDermatologist with a live Inpatient Dermatologist, overall, eDermatology consultants were able to provide rapid consult recommendations that aided patient management. Conclusions: Inpatient eDermatology appears to be an effective medium to provide dermatologic care to patients at hospitals without a dermatology presence. This delivery of health care can help prevent misdiagnosis, unnecessary costs, and inappropriate systemic therapies.

Tumor-derived high-mobility group box 1 and thymic stromal lymphopoietin are involved in modulating dendritic cells to activate T regulatory cells in a mouse model.

High-mobility group box 1 (HMGB1) is involved in the tumor-associated activation of regulatory T cells (Treg), but the mechanisms remain unknown. In a mouse tumor model, silencing HMGB1 in tumor cells or inhibiting tumor-derived HMGB1 not only dampened the capacity of tumor cells to produce thymic stromal lymphopoietin (TSLP), but also aborted the tumor-associated modulation of Treg-activating DC. Tumor-derived HMGB1 triggered the production of TSLP by tumor cells. Importantly, both tumor-derived HMGB1 and TSLP were necessary for modulating DC to activate Treg in a TSLP receptor (TSLPR)-dependent manner. In the therapeutic model, intratumorally inhibiting tumor-derived HMGB1 (causing downstream loss of TSLP production) attenuated Treg activation, unleashed tumor-specific CD8 T cell responses, and elicited CD8α+/CD103+DC- and T cell-dependent antitumor activity. These results suggest a new pathway for the activation of Treg involving in tumor-derived HMGB1 and TSLP, and have important implications for incorporating HMGB1 inhibitors into cancer immunotherapy.

Therapeutic reduction of cell-mediated immunosuppression in mycosis fungoides and Sézary syndrome.

Tumor progression is associated with progressive immunosuppression mediated in part by T regulatory cell(s) (Treg) and/or myeloid-derived suppressor cell(s) (MDSC). Development of strategies to reduce populations of immune cells with suppressive function in cancer patients may enable the induction or recovery of immunity against tumor cells, which may limit or reverse disease progression. With a goal of developing Treg and MDSC neutralizing strategies to treat mycosis fungoides (MF) and Sézary syndrome (SzS), we determined the association between disease stage and suppressor cell populations in patients with MF/SzS, including those responding to therapy. We found elevations in Treg populations, across Treg subtypes, in patients with SzS, and these Treg markedly suppressed proliferation of autologous CD4+CD25- responder T cells. Interestingly, while MDSC numbers were not increased in MF/SzS patients, MDSC from patients with stage IB and above produced significantly more reactive oxygen species than those from stage IA MF patients and control cohorts. Therapy with the CD25-targeting agent denileukin diftitox or IFN-α2b was associated with a reduction in Treg numbers or MDSC function, respectively. These studies identify potential mechanisms of action for these therapies and support the development of coordinated strategies targeting both Treg and MDSC activities in patients with MF/SzS.

Genetic vaccines to potentiate the effective CD103+ dendritic cell-mediated cross-priming of antitumor immunity.

The development of effective cancer vaccines remains an urgent, but as yet unmet, clinical need. This deficiency is in part due to an incomplete understanding of how to best invoke dendritic cells (DC) that are crucial for the induction of tumor-specific CD8(+) T cells capable of mediating durable protective immunity. In this regard, elevated expression of the transcription factor X box-binding protein 1 (XBP1) in DC appears to play a decisive role in promoting the ability of DC to cross-present Ags to CD8(+) T cells in the therapeutic setting. Delivery of DNA vaccines encoding XBP1 and tumor Ag to skin DC resulted in increased IFN-α production by plasmacytoid DC (pDC) from skin/tumor draining lymph nodes and the cross-priming of Ag-specific CD8(+) T cell responses associated with therapeutic benefit. Antitumor protection was dependent on cross-presenting Batf3(+) DC, pDC, and CD8(+) T cells. CD103(+) DC from the skin/tumor draining lymph nodes of the immunized mice appeared responsible for activation of Ag-specific naive CD8(+) T cells, but were dependent on pDC for optimal effectiveness. Similarly, human XBP1 improved the capacity of human blood- and skin-derived DC to activate human T cells. These data support an important intrinsic role for XBP1 in DC for effective cross-priming and orchestration of Batf3(+) DC-pDC interactions, thereby enabling effective vaccine induction of protective antitumor immunity.

Neurokinin-1 receptor agonists bias therapeutic dendritic cells to induce type 1 immunity by licensing host dendritic cells to produce IL-12.

Substance-P and hemokinin-1 are proinflammatory neuropeptides with potential to promote type 1 immunity through agonistic binding to neurokinin-1 receptor (NK1R). Dendritic cells (DCs) are professional antigen-presenting cells that initiate and regulate the outcome of innate and adaptive immune responses. Immunostimulatory DCs are highly desired for the development of positive immunization techniques. DCs express functional NK1R; however, regardless of their potential DC-stimulatory function, the ability of NK1R agonists to promote immunostimulatory DCs remains unexplored. Here, we demonstrate that NK1R signaling activates therapeutic DCs capable of biasing type 1 immunity by inhibition of interleukin-10 (IL-10) synthesis and secretion, without affecting their low levels of IL-12 production. The potent type 1 effector immune response observed following cutaneous administration of NK1R-signaled DCs required their homing in skin-draining lymph nodes (sDLNs) where they induced inflammation and licensed endogenous-conventional sDLN-resident and -recruited inflammatory DCs to secrete IL-12. Our data demonstrate that NK1R signaling promotes immunostimulatory DCs, and provide relevant insight into the mechanisms used by neuromediators to regulate innate and adaptive immune responses.

Dissolvable microneedle arrays for intradermal delivery of biologics: fabrication and application.

PURPOSE: Design and evaluate a new micro-machining based approach for fabricating dissolvable microneedle arrays (MNAs) with diverse geometries and from different materials for dry delivery to skin microenvironments. The aims are to describe the new fabrication method, to evaluate geometric and material capability as well as reproducibility of the method, and to demonstrate the effectiveness of fabricated MNAs in delivering bioactive molecules. METHODS: Precise master molds were created using micromilling. Micromolding was used to create elastomer production molds from master molds. The dissolvable MNAs were then fabricated using the spin-casting method. Fabricated MNAs with different geometries were evaluated for reproducibility. MNAs from different materials were fabricated to show material capability. MNAs with embedded bioactive components were tested for functionality on human and mice skin. RESULTS: MNAs with different geometries and from carboxymethyl cellulose, polyvinyl pyrrolidone and maltodextrin were created reproducibly using our method. MNAs successfully pierce the skin, precisely deliver their bioactive cargo to skin and induce specific immunity in mice. CONCLUSIONS: We demonstrated that the new fabrication approach enables creating dissolvable MNAs with diverse geometries and from different materials reproducibly. We also demonstrated the application of MNAs for precise and specific delivery of biomolecules to skin microenvironments in vitro and in vivo.

Knockdown of HMGB1 in tumor cells attenuates their ability to induce regulatory T cells and uncovers naturally acquired CD8 T cell-dependent antitumor immunity.

Although high mobility group box 1 (HMGB1) in tumor cells is involved in many aspects of tumor progression, its role in tumor immune suppression remains elusive. Host cell-derived IL-10 suppressed a naturally acquired CD8 T cell-dependent antitumor response. The suppressive activity of tumor-associated Foxp3(+)CD4(+)CD25(+) regulatory T cells (Treg) was IL-10 dependent. Neutralizing HMGB1 impaired tumor cell-promoted IL-10 production by Treg. Short hairpin RNA-mediated knockdown of HMGB1 (HMGB1 KD) in tumor cells did not affect tumor cell growth but uncovered naturally acquired long-lasting tumor-specific IFN-γ- or TNF-α-producing CD8 T cell responses and attenuated their ability to induce Treg, leading to naturally acquired CD8 T cell- or IFN-γ-dependent tumor rejection. The data suggest that tumor cell-derived HMGB1 may suppress naturally acquired CD8 T cell-dependent antitumor immunity via enhancing Treg to produce IL-10, which is necessary for Treg-mediated immune suppression.

Genetic targeting of the active transcription factor XBP1s to dendritic cells potentiates vaccine-induced prophylactic and therapeutic antitumor immunity.

In vivo dendritic cells (DC) targeting is an attractive approach with potential advantages in vaccine efficacy, cost, and availability. Identification of molecular adjuvants to in vivo "modulate " DC to coordinately render improved Th1 and CD8 T cell immunity, and attenuated deleterious Treg effects, is a critical challenge. Here, we report that in vivo genetic targeting of the active transcription factor XBP1s to DC (XBP1s/DC) potentiated vaccine-induced prophylactic and therapeutic antitumor immunity in multiple tumor models. This immunization strategy is based on a genetic vaccine encoding both cytomegalovirus (CMV)-driven vaccine Aghsp70 and DC-specific CD11c-driven XBP1s. The novel targeted vaccine induced durable Th1 and CD8 T cell responses to poorly immunogenic self/tumor antigen (Ag) and attenuated tumor-associated Treg suppressive function. Bone marrow (BM)-derived DC genetically modified to simultaneously overexpress XBP1s and express Aghsp70 upregulated CD40, CD70, CD86, interleukin (IL)-15, IL-15Rα, and CCR7 expression, and increased IL-6, IL-12, and tumor necrosis factor (TNF)-α production in vitro. XBP1s/DC elevated functional DEC205(+)CD8α(+)DC in the draining lymph nodes (DLN). The data suggest a novel role for XBP1s in modulating DC to potentiate tumor vaccine efficacy via overcoming two major obstacles to tumor vaccines (i.e., T cell hyporesponsiveness against poorly immunologic self/tumor Ag and tumor-associated Treg-mediated suppression) and improving DEC205(+)CD8α(+)DC.

Deciphering Tumor Cell Evolution in Cutaneous T-Cell Lymphomas: Distinct Differentiation Trajectories in Mycosis Fungoides and Sézary Syndrome.

Cutaneous T-cell lymphomas are a heterogeneous group of neoplasms originating in the skin, with mycosis fungoides (MF) and Sézary syndrome (SS) representing the most common variants. The cellular origin of cutaneous lymphomas has remained controversial owing to their immense phenotypic heterogeneity that obfuscates lineage reconstruction on the basis of classical surface biomarkers. To overcome this heterogeneity and reconstruct the differentiation trajectory of malignant cells in MF and SS, TCR sequencing was performed in parallel with targeted transcriptomics at the single-cell resolution among cutaneous samples in MF and SS. Unsupervised lineage reconstruction showed that Sézary cells exist as a population of CD4+ T cells distinct from those in patch, plaque, and tumor MF. Further investigation of malignant cell heterogeneity in SS showed that Sézary cells phenotypically comprised at least 3 subsets on the basis of differential proliferation potentials and expression of exhaustion markers. A T helper 1-polarized cell type, intermediate cell type, and exhausted T helper 2-polarized cell type were identified, with T helper 1- and T helper 2-polarized cells displaying divergent proliferation potentials. Collectively, these findings provide evidence to clarify the relationship between MF and SS and reveal cell subsets in SS that suggest a possible mechanism for therapeutic resistance.

3D reconstruction of skin and spatial mapping of immune cell density, vascular distance and effects of sun exposure and aging.

Mapping the human body at single cell resolution in three dimensions (3D) is important for understanding cellular interactions in context of tissue and organ organization. 2D spatial cell analysis in a single tissue section may be limited by cell numbers and histology. Here we show a workflow for 3D reconstruction of multiplexed sequential tissue sections: MATRICS-A (Multiplexed Image Three-D Reconstruction and Integrated Cell Spatial - Analysis). We demonstrate MATRICS-A in 26 serial sections of fixed skin (stained with 18 biomarkers) from 12 donors aged between 32-72 years. Comparing the 3D reconstructed cellular data with the 2D data, we show significantly shorter distances between immune cells and vascular endothelial cells (56 µm in 3D vs 108 µm in 2D). We also show 10-70% more T cells (total) within 30 µm of a neighboring T helper cell in 3D vs 2D. Distances of p53, DDB2 and Ki67 positive cells to the skin surface were consistent across all ages/sun exposure and largely localized to the lower stratum basale layer of the epidermis. MATRICS-A provides a framework for analysis of 3D spatial cell relationships in healthy and aging organs and could be further extended to diseased organs.

Resistance of Sézary cells to TNF-α-induced apoptosis is mediated in part by a loss of TNFR1 and a high level of the IER3 expression.

Failure to execute an apoptotic programme is one of the critical steps and a common mechanism promoting tumorogenesis. Immediate early responsive gene 3 (IER3) has been shown to be upregulated in several cancers. IER3 is a stress-induced gene, which upregulation leads to reduction in production of reactive oxygen species (ROS) protecting malignant cells from apoptosis. We observed that malignant lymphocytes from patients with Sézary syndrome (SzS) were resistant to pro-apoptotic dose of tumour necrosis factor-α (TNF-α). The aim of this study was to investigate the role of IER3 in the mechanism of such resistance. CD4+ CD26- lymphocytes from the peripheral blood of patients with SzS and healthy controls were negatively selected using CD4 and CD26 magnetic beads and analysed for expression of TNFR1, TNFR2, IER3 expression, and ROS production in response to TNF-α at an apoptotic dose. Sézary cells with a higher level of IER3 expression retained their viability to TNF-α. IER3 upregulation correlated with a decrease level of intracellular ROS and low TNFR1 expression on malignant cells. Targeting IER3 could be of interest for the development of future therapeutic strategies for patients with SzS.

Microfluidic Systems For Manufacturing of Microparticle-Based Drug-Delivery Systems: Design, Construction, and Operation.

Particles synthesized from biodegradable polymers hold great potential as controlled drug delivery systems. Continuous flow platforms based on microfluidics offer attractive advantages over conventional batch-emulsification techniques for the scalable fabrication of drug-loaded particles with controlled physicochemical properties. However, widespread utilization of microfluidic technologies for the manufacturing of drug-loaded particles has been hindered largely by the lack of practical guidelines toward cost-effective development and reliable operation of microfluidic systems. Here, we present a framework for rational design and construction of microfluidic systems using commercially available components for high-throughput production of uniform biodegradable particles encapsulating drugs. We also demonstrate successful implementation of this framework to devise a robust microfluidic system that is capable of producing drug-carrying particles with desired characteristics. The guidelines provided in this study will likely help broaden the applicability of microfluidic technologies for the synthesis of high-quality, drug-loaded biodegradable particles.