Administration methods and dosage of poly(lactic acid)-glycol intervention to myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalitis mice.

BACKGROUND: Myelin oligodendrocyte glycoprotein-associated disorders (MOGAD) is an autoimmune central nervous system disease. Antigen-specific immune tolerance using nanoparticles such as Polylactic-co-glycolic acid (PLGA) have recently been used as a new therapeutic tolerization approach for CNS autoimmune diseases. We examined whether MOG1-125 conjugated with PLGA could induce MOG-specific immune tolerance in an experimental autoimmune encephalitis (EAE) mouse model. EAE was induced in sixty C57BL/6 J wild-type mice using MOG1-125 peptide with complete Freund's Adjuvant. The mice were divided into 12 groups (n = 5 each) to test the ability of MOG1-125 conjugated PLGA intervention to mitigate the severity or improve the outcomes from EAE with and without rapamycin compared to antigen alone or PLGA alone. EAE score and serum MOG-IgG titers were compared among the interventions.Kindly check and confirm the processed Affiliation “4” is appropriate.I confirmed the Aff 4.Affiliation: Corresponding author information have been changed to present affiliation. Kindly check and confirm.I checked and confirmed the Corresponding author's information. RESULTS: Mice with EAE that were injected intraperitoneally with MOG1-125 conjugated PLGA + rapamycin complex showed dose-dependent mitigation of EAE score. Intraperitoneal and intravenous administration resulted in similar clinical outcomes, whereas 80% of mice treated with subcutaneous injection had a recurrence of clinical score worsening after approximately 1 week. Although there was no significant difference in EAE scores between unconjugated-PLGA and MOG-conjugated PLGA, serum MOG-IgG tended to decrease in the MOG-conjugated PLGA group compared to controls. CONCLUSION: Intraperitoneal administration of PLGA resulted in dose-dependent and longer-lasting immune tolerance than subcutaneous administration. The induction of immune tolerance using PLGA may represent a future therapeutic option for patients with MOGAD.

Cutting Edge: Phagosome-associated Autophagosomes Containing Antigens and Proteasomes Drive TAP-Independent Cross-Presentation.

Activation of naive CD8-positive T lymphocytes is mediated by dendritic cells that cross-present MHC class I (MHC-I)-associated peptides derived from exogenous Ags. The most accepted mechanism involves the translocation of Ags from phagosomes or endolysosomes into the cytosol, where antigenic peptides generated by cytosolic proteasomes are delivered by the transporter associated with Ag processing (TAP) to the endoplasmic reticulum, or an endocytic Ag-loading compartment, where binding to MHC-I occurs. We have described an alternative pathway where cross-presentation is independent of TAP but remains dependent on proteasomes. We provided evidence that active proteasomes found within the lumen of phagosomes and endolysosomal vesicles locally generate antigenic peptides that can be directly loaded onto trafficking MHC-I molecules. However, the mechanism of active proteasome delivery to the endocytic compartments remained unknown. In this study, we demonstrate that phagosome-associated LC3A/B structures deliver proteasomes into subcellular compartments containing exogenous Ags and that autophagy drives TAP-independent, proteasome-dependent cross-presentation.

Proliferation history and transcription factor levels drive direct conversion.

The sparse and stochastic nature of reprogramming has obscured our understanding of how transcription factors drive cells to new identities. To overcome this limit, we developed a compact, portable reprogramming system that increases direct conversion of fibroblasts to motor neurons by two orders of magnitude. We show that subpopulations with different reprogramming potentials are distinguishable by proliferation history. By controlling for proliferation history and titrating each transcription factor, we find that conversion correlates with levels of the pioneer transcription factor Ngn2, whereas conversion shows a biphasic response to Lhx3. Increasing the proliferation rate of adult human fibroblasts generates morphologically mature, induced motor neurons at high rates. Using compact, optimized, polycistronic cassettes, we generate motor neurons that graft with the murine central nervous system, demonstrating the potential for in vivo therapies.

Lessons for the Next Generation of Scientists from the Second Annual Arthur and Sandra Irving Cancer Immunology Symposium.

The Arthur and Sandra Irving Cancer Immunology Symposium has been created as a platform for established cancer immunologists to mentor trainees and young investigators as they launch their research career in the field. By sharing their different paths to success, the senior faculty mentors provide an invaluable resource to support the development of the next generation of leaders in the cancer immunology community. This Commentary describes some of the key topics that were discussed during the 2022 symposium: scientific and career trajectory, leadership, mentoring, collaborations, and publishing. For each of these topics, established investigators discussed the elements that facilitate success in these areas as well as mistakes that can hinder progress. Herein, we outline the critical points raised in these discussions for establishing a successful independent research career. These points are highly relevant for the broader scientific community.

CMPK2 restricts Zika virus replication by inhibiting viral translation.

Flaviviruses continue to emerge as global health threats. There are currently no Food and Drug Administration (FDA) approved antiviral treatments for flaviviral infections. Therefore, there is a pressing need to identify host and viral factors that can be targeted for effective therapeutic intervention. Type I interferon (IFN-I) production in response to microbial products is one of the host's first line of defense against invading pathogens. Cytidine/uridine monophosphate kinase 2 (CMPK2) is a type I interferon-stimulated gene (ISG) that exerts antiviral effects. However, the molecular mechanism by which CMPK2 inhibits viral replication is unclear. Here, we report that CMPK2 expression restricts Zika virus (ZIKV) replication by specifically inhibiting viral translation and that IFN-I- induced CMPK2 contributes significantly to the overall antiviral response against ZIKV. We demonstrate that expression of CMPK2 results in a significant decrease in the replication of other pathogenic flaviviruses including dengue virus (DENV-2), Kunjin virus (KUNV) and yellow fever virus (YFV). Importantly, we determine that the N-terminal domain (NTD) of CMPK2, which lacks kinase activity, is sufficient to restrict viral translation. Thus, its kinase function is not required for CMPK2's antiviral activity. Furthermore, we identify seven conserved cysteine residues within the NTD as critical for CMPK2 antiviral activity. Thus, these residues may form an unknown functional site in the NTD of CMPK2 contributing to its antiviral function. Finally, we show that mitochondrial localization of CMPK2 is required for its antiviral effects. Given its broad antiviral activity against flaviviruses, CMPK2 is a promising potential pan-flavivirus inhibitor.

Dynamic Craniotomy With NuCrani Reversibly Expandable Cranial Bone Flap Fixation Plates: A Technical Report.

BACKGROUND: Dynamic craniotomy provides cranial decompression without bone flap removal along with avoidance of cranioplasty and reduced risks for complications. OBJECTIVE: To report the first clinical cases using a novel dynamic craniotomy bone flap fixation system. The NeuroVention NuCrani reversibly expandable cranial bone flap fixation plates provide dynamic bone flap movement to accommodate changes in intracranial pressure (ICP) after a craniotomy. METHODS: The reversibly expandable cranial bone flap fixation plates were used for management of cerebral swelling in a patient with a subdural hemorrhage after severe traumatic brain injury and another patient with a hemorrhagic stroke. RESULTS: Both cases had high ICP's which normalized immediately after the dynamic craniotomy. Progressive postoperative cerebral swelling was noted which was compensated by progressive outward bone flap migration thereby maintaining a normal ICP, and with resolution of the cerebral swelling, the plates retracted the bone flaps to an anatomic flush position. CONCLUSION: The reversibly expandable plates provide an unhinged cranial bone flap outward migration with an increase in ICP and retract the bone flap after resolution of brain swelling while also preventing the bone flap from sinking inside the skull.

Rapid Screen for Antiviral T-Cell Immunity with Nanowire Electrochemical Biosensors.

The ability to rapidly assess and monitor patient immune responses is critical for clinical diagnostics, vaccine design, and fundamental investigations into the presence or generation of protective immunity against infectious diseases. Recently, findings on the limits of antibody-based protection provided by B-cells have highlighted the importance of engaging pathogen-specific T-cells for long-lasting and broad protection against viruses and their emergent variants such as in SARS-CoV-2. However, low-cost and point-of-care tools for detecting engagement of T-cell immunity in patients are conspicuously lacking in ongoing efforts to assess and control population-wide disease risk. Currently available tools for human T-cell analysis are time and resource-intensive. Using multichannel silicon-nanowire field-effect transistors compatible with complementary metal-oxide-semiconductor, a device designed for rapid and label-free detection of human T-cell immune responses is developed. The generalizability of this approach is demonstrated by measuring T-cell responses against melanoma antigen MART1, common and seasonal viruses CMV, EBV, flu, as well as emergent pandemic coronavirus, SARS-CoV-2. Further, this device provides a modular and translational platform for optimizing vaccine formulations and combinations, offering quick and quantitative readouts for acquisition and persistence of T-cell immunity against variant-driven pathogens such as flu and pandemic SARS-CoV-2.

Metabolic and immunomodulatory control of type 1 diabetes via orally delivered bile-acid-polymer nanocarriers of insulin or rapamycin.

Oral formulations of insulin are typically designed to improve its intestinal absorption and increase its blood bioavailability. Here we show that polymerized ursodeoxycholic acid, selected from a panel of bile-acid polymers and formulated into nanoparticles for the oral delivery of insulin, restored blood-glucose levels in mice and pigs with established type 1 diabetes. The nanoparticles functioned as a protective insulin carrier and as a high-avidity bile-acid-receptor agonist, increased the intestinal absorption of insulin, polarized intestinal macrophages towards the M2 phenotype, and preferentially accumulated in the pancreas of the mice, binding to the islet-cell bile-acid membrane receptor TGR5 with high avidity and activating the secretion of glucagon-like peptide and of endogenous insulin. In the mice, the nanoparticles also reversed inflammation, restored metabolic functions and extended animal survival. When encapsulating rapamycin, they delayed the onset of diabetes in mice with chemically induced pancreatic inflammation. The metabolic and immunomodulatory functions of ingestible bile-acid-polymer nanocarriers may offer translational opportunities for the prevention and treatment of type 1 diabetes.

Detection of Immunogenic Cell Death in Tumor Vaccination Mouse Model.

Immunogenic cell death (ICD) is a form of regulated cell death that is capable of eliciting an immune response. In cancer, tumor cells undergoing ICD are known to emit damage associated molecular patterns (DAMPs) that are capable of recruiting and activating antigen presenting cells (APCs), which ultimately lead to the activation of an antitumor immune response. Surface translocation of intracellular chaperones such as calreticulin, release of TLR agonists such as high mobility box 1, and the secretion of type I IFN are some of the hallmark features seen in tumors succumbing to ICD. While detection of these molecules is suggestive of ICD induction, which alone does not certify that the treatment is an ICD inducer, an in vivo vaccination assay using injured tumor cells remains to be the gold standard method to functionally verify ICD. This chapter will discuss the necessary steps required to conduct an in vivo vaccination assay, focusing on the preparation of vaccine using treated tumor cells, and how these cells are then utilized in the animal model.

Aging Process of Lateral Facial Fat Compartments: A Retrospective Study.

BACKGROUND: Since 2007, when the anatomy of facial fat compartment was described, an increasing number of studies on the aging process of the compartment of cadavers has emerged. OBJECTIVES: The authors evaluated the aging changes of lateral facial fat compartments on the same person. METHODS: Sixty-three patients were included in this retrospective study. All patients had magnetic resonance imaging scans with at least 4 years apart. The authors targeted the fat compartments of the superficial temporal, subcutaneous temporal, and buccal fat pad, comparing the data on different time points. RESULTS: The thickness of the subcutaneous temporal fat did not change significantly. The 3 diameters of the superficial temporal fat compartment all became thinner on the axial view (P < 0.05). On the sagittal view, the superficial temporal fat elongated from 38.89 mm to 43.74 mm (P < 0.05). The buccal fat compartment also lengthened from 68.73 mm to 74.39 mm (P < 0.05) and had a positive correlation with follow-up duration only. CONCLUSIONS: The study revealed the fat compartment change on the same person with time. The temporal hollow mainly originates from the thinner part of the superficial temporal fat. The descending of the buccal fat pad aggravates the labiomandibular fold. By understanding the aging process more fully, we can rejuvenate our patients more naturally.

Direct Comparison of B Cell Surface Receptors as Therapeutic Targets for Nanoparticle Delivery of BTK Inhibitors.

Targeting different cell surface receptors with nanoparticle (NP)-based platforms can result in differential particle binding properties that may impact their localization, bioavailability, and, ultimately, the therapeutic efficacy of an encapsulated payload. Conventional in vitro assays comparing the efficacy of targeted NPs often do not adequately control for these differences in particle-receptor binding, potentially confounding their therapeutic readouts and possibly even limiting their experimental value. In this work, we characterize the conditions under which NPs loaded with Bruton's Tyrosine Kinase (BTK) inhibitor differentially suppress primary B cell activation when targeting either CD19 (internalizing) or B220 (noninternalizing) surface receptors. Surface binding of fluorescently labeled CD19- and B220-targeted NPs was analyzed and quantitatively correlated with the number of bound particles at given treatment concentrations. Using this binding data, suppression of B cell activation was directly compared for differentially targeted (CD19 vs B220) NPs loaded with a BTK inhibitor at a range of particle drug loading concentrations. When NPs were loaded with lower amounts of drug, CD19-mediated internalization demonstrated increased inhibition of B cell proliferation compared with B220 NPs. However, these differences were mitigated when particles were loaded with higher concentrations of BTK inhibitor and B220-mediated "paracrine-like" delivery demonstrated superior suppression of cellular activation when cells were bound to lower overall numbers of NPs. Taken together, these results demonstrate that inhibition of B cell activation can be optimized for NPs targeting either internalizing or noninternalizing surface receptors and that particle internalization is likely not a requisite endpoint when designing particles for delivery of BTK inhibitor to B cells.

Anchor, Spacer, and Ligand-Modified Engineered Exosomes for Trackable Targeted Therapy.

Exosomes have been widely demonstrated as an effective anticancer therapeutic moiety. However, their clinical translation has been limited by the requirement of prohibitively high therapeutic doses due to their lack of specificity in delivery and, consequently, short systemic half-life. To overcome these challenges, we engineered a platform for modifying exosomes with an active targeting modality composed of membrane Anchor (BODIPY)-Spacer (PEG)-targeting Ligands (cyclic RGD peptide) (ASL). Herein, we show that the intramembrane incorporation of a trackable, targeting system renders ASL exosomes (AExs) a modular platform. AExs significantly overcome challenges associated with exosome modification, including potential damage for functionalization, or destabilizing interactions between dyes and drugs. ASL-modification not only enhanced stability in imparting active targeting but also introduced a built-in bioimaging modality. Our studies show that AExs target B16F10 melanoma tumor sites by the specific interaction of cyclic RGD and integrin. Doxorubicin encapsulated AExs (dAExs) significantly inhibited the growth of melanoma in vitro and in vivo. Thus, we conclude that ASL-modification allows exosomes to be transformed into a novel therapeutic vehicle uniquely integrating in vivo tracking and robust targeting with drug delivery. We anticipate that the therapeutic, targeting, and diagnostic modularity provided by ASL will potentiate translational applications of exosome-based vehicles beyond anticancer therapy.

Continuous Label-Free Electronic Discrimination of T Cells by Activation State.

The sensitivity and speed with which the immune system reacts to host disruption is unrivaled by any detection method for pathogenic biomarkers or infectious signatures. Engagement of cellular immunity in response to infections or cancer is contingent upon activation and subsequent cytotoxic activity by T cells. Thus, monitoring T cell activation can reliably serve as a metric for disease diagnosis as well as therapeutic prognosis. Rapid and direct quantification of T cell activation states, however, has been hindered by challenges associated with antigen target identification, labeling requirements, and assay duration. Here we present an electronic, label-free method for simultaneous separation and evaluation of T cell activation states. Our device utilizes a microfluidic design integrated with nanolayered electrode structures for dielectrophoresis (DEP)-driven discrimination of activated vs naïve T cells at single-cell resolution and demonstrates rapid (<2 min) separation of T cells at high single-pass efficiency as quantified by an on-chip Coulter counter module. Our device represents a microfluidic tool for electronic assessment of immune activation states and, hence, a portable diagnostic for quantitative evaluation of immunity and disease state. Further, its ability to achieve label-free enrichment of activated immune cells promises clinical utility in cell-based immunotherapies.

Transimmunization restores immune surveillance and prevents recurrence in a syngeneic mouse model of ovarian cancer.

Ovarian cancer accounts for most deaths from gynecologic malignancies. Although more than 80% of patients respond to first-line standard of care, most of these responders present with recurrence and eventually succumb to carcinomatosis and chemotherapy-resistant disease. To improve patient survival, new modalities must, therefore, target or prevent recurrent disease. Here we describe for the first time a novel syngeneic mouse model of recurrent high-grade serous ovarian cancer (HGSOC), which allows immunotherapeutic interventions in a time course relevant to human carcinomatosis and disease course. Using this model, we demonstrate the efficacy of Transimmunization (TI), a dendritic cell (DC) vaccination strategy that uses autologous and physiologically derived DC loaded with autologous whole tumor antigens. TI has been proven successful in the treatment of human cutaneous T cell lymphoma and we report for the first time its in vivo efficacy against an intra-peritoneal solid tumor. Given as a single therapy, TI is able to elicit an effective anti-tumor immune response and inhibit immune-suppressive crosstalks with sufficient power to curtail tumor progression and establishment of carcinomatosis and recurrent disease. Specifically, TI is able to inhibit the expansion of tumor-associated macrophages as well as myeloid-derived suppressive cells consequently restoring T cell immune-surveillance. These results demonstrate the possible value of TI in the management of ovarian cancer and other intra-peritoneal tumors.

Extracorporeal Photochemotherapy: Mechanistic Insights Driving Recent Advances and Future Directions.

Dendritic cells (DCs) are professional antigen-presenting cells, necessary for the initiation and maintenance of antigen-specific immunity and tolerance. Decades of research have been driven by hopes to harness the immunological capabilities of DCs and achieve physiological partnership with the immune system for therapeutic ends. Potential applications for DC-based immunotherapy include treatments for cancer, autoimmune disorders, and infectious diseases. However, DCs have poor availability in peripheral and lymphoid tissues and have poor survivability in culture, leading to the development of multiple strategies to generate and manipulate large numbers of DCs ex vivo. Among these is Extracorporeal Photopheresis (ECP), a widely used cancer immunotherapy. Recent advancements have uncovered that stimulation of monocyte-to-DC maturation via physiologic inflammatory signaling lies at the mechanistic core of ECP. Here, we describe the landscape of DC-based immunotherapy, the historical context of ECP, the current mechanistic understanding of ex vivo monocyte-to-DC maturation in ECP, and the implications of this understanding on making scientifically driven improvements to modern ECP protocols and devices.

Platelet P-selectin initiates cross-presentation and dendritic cell differentiation in blood monocytes.

Dendritic cells (DCs) are adept at cross-presentation and initiation of antigen-specific immunity. Clinically, however, DCs produced by in vitro differentiation of monocytes in the presence of exogenous cytokines have been met with limited success. We hypothesized that DCs produced in a physiological manner may be more effective and found that platelets activate a cross-presentation program in peripheral blood monocytes with rapid (18 hours) maturation into physiological DCs (phDCs). Differentiation of monocytes into phDCs was concomitant with the formation of an "adhesion synapse," a biophysical junction enriched with platelet P-selectin and monocyte P-selectin glycoprotein ligand 1, followed by intracellular calcium fluxing and nuclear localization of nuclear factor κB. phDCs were more efficient than cytokine-derived DCs in generating tumor-specific T cell immunity. Our findings demonstrate that platelets mediate a cytokine-independent, physiologic maturation of DC and suggest a novel strategy for DC-based immunotherapies.

Rapid Production of Physiologic Dendritic Cells (phDC) for Immunotherapy.

Generation of large numbers of dendritic cells (DC) for research or immunotherapeutic purposes typically involves in vitro conversion of murine bone marrow precursors or human blood monocytes to DC via cultivation with supraphysiologic concentrations of cytokines such as GM-CSF and IL-4 for up to 7 days. Alternatively, our group has recently established a new approach, based on the underlying mechanism of action of a widely used cancer immunotherapy termed Extracorporeal Photochemotherapy (ECP). Our method of rapid and cytokine-free production of therapeutically relevant DC populations, leveraging the innate physiologic programs likely responsible for DC differentiation from blood monocytes in vivo, potentially offers a novel, inexpensive, and easily accessible source of DC for clinical and research uses. This approach involves ex vivo physiologic reprogramming of blood monocytes to immunologically tunable dendritic antigen-presenting cells, which we term "phDC," for physiological DC. To facilitate access and utilization of these new DC populations by the research community, in this chapter, we describe the use of a scaled-down version of the clinical ECP leukocyte-treatment device termed the Transimmunization (TI) chamber or plate, suitable for processing both mouse and human samples. We highlight the methodological sequences necessary to isolate mouse or human peripheral blood mononuclear cell (PBMC) from whole blood, and to expose those PBMC to the TI chamber for facilitating monocyte activation and conversion to physiological DC (phDC) through interaction with blood proteins and activated platelets under controlled flow conditions. We then provide sample protocols for potential applications of the generated DC, including their use as vaccinating antigen-presenting cells (APC) in murine in vivo antitumor models, and in human ex vivo T-cell stimulation and antigen cross-presentation assays which mimic clinical vaccination. We additionally highlight the technical aspects of loading mouse or human phDC with tumor-associated antigens (TAA) in the form of peptides or apoptotic tumor cells. We provide a simple and clinically relevant means to reprogram blood monocytes into functional APC, potentially replacing the comparatively expensive and clinically disappointing cytokine-derived DC which have previously dominated the dendritic cell landscape.

Ex vivo dendritic cell generation-A critical comparison of current approaches.

Dendritic cells (DCs) are professional antigen-presenting cells, required for the initiation of naïve and memory T cell responses and regulation of adaptive immunity. The discovery of DCs in 1973, which culminated in the Nobel Prize in Physiology or Medicine in 2011 for Ralph Steinman and colleagues, initially focused on the identification of adherent mononuclear cell fractions with uniquely stellate dendritic morphology, followed by key discoveries of their critical immunologic role in initiating and maintaining antigen-specific immunity and tolerance. The medical promise of marshaling these key capabilities of DCs for therapeutic modulation of antigen-specific immune responses has guided decades of research in hopes to achieve genuine physiologic partnership with the immune system. The potential uses of DCs in immunotherapeutic applications include cancer, infectious diseases, and autoimmune disorders; thus, methods for rapid and reliable large-scale production of DCs have been of great academic and clinical interest. However, difficulties in obtaining DCs from lymphoid and peripheral tissues, low numbers and poor survival in culture, have led to advancements in ex vivo production of DCs, both for probing molecular details of DC function as well as for experimenting with their clinical utility. Here, we review the development of a diverse array of DC production methodologies, ranging from cytokine-based strategies to genetic engineering tools devised for enhancing DC-specific immunologic functions. Further, we explore the current state of DC therapies in clinic, as well as emerging insights into physiologic production of DCs inspired by existing therapies.

Extracorporeal photochemotherapy induces bona fide immunogenic cell death.

Extracorporeal photochemotherapy (ECP) is employed for the management of cutaneous T cell lymphoma (CTCL). ECP involves the extracorporeal exposure of white blood cells (WBCs) to a photosensitizer, 8-methoxypsoralen (8-MOP), in the context of ultraviolet A (UVA) radiation, followed by WBC reinfusion. Historically, the therapeutic activity of ECP has been attributed to selective cytotoxicity on circulating CTCL cells. However, only a fraction of WBCs is exposed to ECP, and 8-MOP is inactive in the absence of UVA light, implying that other mechanisms underlie the anticancer effects of ECP. Recently, ECP has been shown to enable the physiological differentiation of monocytes into dendritic cells (DCs) that efficiently cross-present tumor-associated antigens (TAAs) to CD8+ T lymphocytes to initiate cognate immunity. However, the source of TAAs and immunostimulatory signals for such DCs remains to be elucidated. Here, we demonstrate that 8-MOP plus UVA light reduces melanoma cell viability along with the emission of ICD-associated danger signals including calreticulin (CALR) exposure on the cell surface and secretion of ATP, high mobility group box 1 (HMGB1) and type I interferon (IFN). Consistently, melanoma cells succumbing to 8-MOP plus UVA irradiation are efficiently engulfed by monocytes, ultimately leading to cross-priming of CD8+ T cells against cancer. Moreover, malignant cells killed by 8-MOP plus UVA irradiation in vitro vaccinate syngeneic immunocompetent mice against living cancer cells of the same type, and such a protection is lost when cancer cells are depleted of calreticulin or HMGB1, as well as in the presence of an ATP-degrading enzyme or antibodies blocking type I IFN receptors. ECP induces bona fide ICD, hence simultaneously providing monocytes with abundant amounts of TAAs and immunostimulatory signals that are sufficient to initiate cognate anticancer immunity.