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OBJECTIVES/GOALS: Donor hearts are transported in cold storage (CS) and undergo ischemia-reperfusion injury (IRI) when transplanted. IRI injures microvascular endothelial cells (EC), heightens the immune response, and has been associated with increased autophagy. We aim to understand the changes in autophagy during CS and IRI and its impact on EC immunogenicity. METHODS/STUDY POPULATION: To study autophagy changes during IRI, immunoblotting for autophagy markers was performed in mouse cardiac ECs (MCECs) lysates. MCECs were in a cold preservation solution in a hypoxic chamber for 6 hours(h) and warm conditions with culture medium for 24 h. MCECs, under standard conditions, served as controls. Secreted interferon-gamma (IFN-γ) levels were quantified via ELISA to study autophagy and EC immunogenicity. MCEC-sensitized CD8+ T-cells were isolated from C57BL/6 spleens and co-cultured with MCECs pre-treated for 16 h with rapamycin or starvation, autophagy inducers, or chloroquine, an autophagy inhibitor under normal or IRI conditions. MCECs without any treatment served as controls. RESULTS/ANTICIPATED RESULTS: To determine autophagy levels in IRI, immunoblotting of MCEC lysates revealed a significant increase (P

The structural diversity of metazoic heparan sulfate (HS) composed of unique sulfated domains is remarkably preserved among various vertebrates and invertebrate species. Interestingly the sulfated moieties of HS have been known as the key determinants generating extraordinary ligand binding sites in the HS chain to regulate multiple biological functions and homeostasis. One such ligand for 3-O sulfation in the HS chain is a glycoprotein D (gD) from an ancient herpesvirus, herpes simplex virus (HSV). This interaction between gD and 3-O sulfated HS leads to virus-cell fusion to promote HSV entry. It is quite astonishing that HSV-1, which infects two-thirds of the world population, is also capable of causing severe diseases in primates and non-primates including primitive zebrafish. Supporting evidence that HSV may cross the species barrier comes from the fact that an enzymatic modification in HS encoded by 3-O sulfotransferase-3 (3-OST-3) from a vertebrate zoonotic species enhances HSV-1 infectivity. The latter phenomenon suggests the possible role of sulfated-HS as an entry receptor during reverse zoonosis, especially during an event when humans encounter domesticated animals in proximity. In this mini-review, we explore the possibility that structural diversity in HS may have played a substantial role in species-specific adaptability for herpesviruses in general including their potential role in promoting cross-species transmission.

Checkpoint inhibitors treat a variety of tumor types with significant benefits. Unfortunately, these therapies come with diverse adverse events. Skin rash is observed early into treatment and might serve as an indicator of downstream responses to therapy. We studied the cellular composition of cutaneous eruptions and whether their contribution varies with the treatment applied. Skin samples from 18 patients with cancer and 11 controls were evaluated by mono- and multiplex imaging, quantification, and statistical analysis. T cells were the prime contributors to skin rash, with T cells and macrophages interacting and proliferating on site. Among T cell subsets examined, type 1 and 17 T cells were relatively increased among inflammatory skin infiltrates. A combination of increased cytotoxic T cell content and decreased macrophage abundance was associated with dual checkpoint inhibition over PD1 inhibition alone. Importantly, responders significantly separated from nonresponders by greater CD68+ macrophage and either CD11c+ antigen-presenting cell or CD4+ T cell abundance in skin rash. The microenvironment promoted epidermal proliferation and thickening as well. The combination of checkpoint inhibitors used affects the development and composition of skin infiltrates, whereas the combined abundance of two cell types in cutaneous eruptions aligns with responses to checkpoint inhibitor therapy.

Mutations underlying disease in tuberous sclerosis complex (TSC) give rise to tumors with biallelic mutations in TSC1 or TSC2 and hyperactive mammalian target of rapamycin complex 1 (mTORC1). Benign tumors might exhibit de novo expression of immunogens, targetable by immunotherapy. As tumors may rely on ganglioside D3 (GD3) expression for mTORC1 activation and growth, we compared GD3 expression in tissues from patients with TSC and controls. GD3 was overexpressed in affected tissues from patients with TSC and also in aging Tsc2+/– mice. As GD3 overexpression was not accompanied by marked natural immune responses to the target molecule, we performed preclinical studies with GD3 chimeric antigen receptor (CAR) T cells. Polyfunctional CAR T cells were cytotoxic toward GD3-overexpressing targets. In mice challenged with Tsc2–/– tumor cells, CAR T cells substantially and durably reduced the tumor burden, correlating with increased T cell infiltration. We also treated aged Tsc2+/– heterozygous (>60 weeks) mice that carry spontaneous Tsc2–/– tumors with GD3 CAR or untransduced T cells and evaluated them at endpoint. Following CAR T cell treatment, the majority of mice were tumor free while all control animals carried tumors. The outcomes demonstrate a strong treatment effect and suggest that targeting GD3 can be successful in TSC.

The drive to withstand environmental stresses and defend against invasion is a universal trait extant in all forms of life. While numerous canonical signaling cascades have been characterized in detail, it remains unclear how these pathways interface to generate coordinated responses to diverse stimuli. To dissect these connections, we followed heparanase (HPSE), a protein best known for its endoglycosidic activity at the extracellular matrix but recently recognized to drive various forms of late-stage disease through unknown mechanisms. Using herpes simplex virus-1 (HSV-1) infection as a model cellular perturbation, we demonstrate that HPSE acts beyond its established enzymatic role to restrict multiple forms of cell-intrinsic defense and facilitate host cell reprogramming by the invading pathogen. We reveal that cells devoid of HPSE are innately resistant to infection and counteract viral takeover through multiple amplified defense mechanisms. With a unique grasp of the fundamental processes of transcriptional regulation and cell death, HPSE represents a potent cellular intersection with broad therapeutic potential.

Vitiligo is an autoimmune skin disease characterized by melanocyte destruction. Regulatory T cells (Tregs) are greatly reduced in vitiligo skin, and replenishing peripheral skin Tregs can provide protection against depigmentation. Ganglioside D3 (GD3) is overexpressed by perilesional epidermal cells, including melanocytes, which prompted us to generate GD3-reactive chimeric antigen receptor (CAR) Tregs to treat vitiligo. Mice received either untransduced Tregs or GD3-specific Tregs to test the hypothesis that antigen specificity contributes to reduced autoimmune reactivity in vitro and in vivo. CAR Tregs displayed increased IL-10 secretion in response to antigen, provided superior control of cytotoxicity towards melanocytes, and supported a significant delay in depigmentation compared to untransduced Tregs and vehicle control recipients in a TCR transgenic mouse model of spontaneous vitiligo. The latter findings were associated with a greater abundance of Tregs and melanocytes in treated mice versus both control groups. Our data support the concept that antigen-specific Tregs can be prepared, used, and stored for long-term control of progressive depigmentation.

Vitiligo is a T-cell mediated skin disorder characterized by progressive loss of skin color. In individuals genetically predisposed to the disease, various triggers contribute to the initiation of vitiligo. Precipitating factors can stress the skin, leading to T-cell activation and recruitment. Though hereditary factors are implicated in the pathogenesis of vitiligo, it is unknown whether precipitating, stressful events play a role in vitiligo. To understand this, we utilized a validated perceived stress scale (PSS) to measure this parameter in vitiligo patients compared to persons without vitiligo. Additionally, we probed a clinical database, using a knowledge linking software called ROCKET, to gauge stress-related conditions in the vitiligo patient population. From a pool of patients in an existing database, a hundred individuals with vitiligo and twenty-five age- and sex-matched comparison group of individuals without vitiligo completed an online survey to quantify their levels of perceived stress. In parallel, patients described specifics of their disease condition, including the affected body sites, the extent, duration and activity of their vitiligo. Perceived stress was significantly higher among vitiligo individuals compared to those without vitiligo. ROCKET analyses suggested signs of metabolic-related disease (i.e., 'stress') preceding vitiligo development. No correlation was found between perceived stress and the stage or the extent of disease, suggesting that elevated stress may not be a consequence of pigment loss alone. The data provide further support for stress as a precipitating factor in vitiligo development.

Herpes simplex virus type 1 (HSV-1) is an important factor for vision loss in developed countries. A challenging aspect of the ocular infection by HSV-1 is that common treatments, such as acyclovir, fail to provide effective topical remedies. Furthermore, it is not very clear whether the viral glycoproteins, required for HSV-1 entry into the host, can be targeted for an effective therapy against ocular herpes in vivo. Here, we demonstrate that HSV-1 envelope glycoprotein gD, which is essential for viral entry and spread, can be specifically targeted by topical applications of a small DNA aptamer to effectively control ocular infection by the virus. Our 45-nt-long DNA aptamer showed high affinity for HSV-1 gD (binding affinity constant [Kd] = 50 nM), which is strong enough to disrupt the binding of gD to its cognate host receptors. Our studies showed significant restriction of viral entry and replication in both in vitro and ex vivo studies. In vivo experiments in mice also resulted in loss of ocular infection under prophylactic treatment and statistically significant lower infection under therapeutic modality compared to random DNA controls. Thus, our studies validate the possibility that targeting HSV-1 entry glycoproteins, such as gD, can locally reduce the spread of infection and define a novel DNA aptamer-based approach to control HSV-1 infection of the eye.

Herpes simplex virus-1 (HSV-1) causes lifelong recurrent pathologies without a cure. How infection by HSV-1 triggers disease processes, especially in the immune-privileged avascular human cornea, remains a major unresolved puzzle. It has been speculated that a cornea-resident molecule must tip the balance in favor of pro-inflammatory and pro-angiogenic conditions observed with herpetic, as well as non-herpetic, ailments of the cornea. Here, we demonstrate that heparanase (HPSE), a host enzyme, is the molecular trigger for multiple pathologies associated with HSV-1 infection. In human corneal epithelial cells, HSV-1 infection upregulates HPSE in a manner dependent on HSV-1 infected cell protein 34.5. HPSE then relocates to the nucleus to regulate cytokine production, inhibits wound closure, enhances viral spread, and thus generates a toxic local environment. Overall, our findings implicate activated HPSE as a driver of viral pathogenesis and call for further attention to this host protein in infection and other inflammatory disorders.

Etiology, transmission and protection: Herpes simplex virus-2 (HSV-2) is a leading cause of sexually transmitted infections with recurring manifestations throughout the lifetime of infected hosts. Currently no effective vaccines or prophylactics exist that provide complete protection or immunity from the virus, which is endemic throughout the world. Pathology/Symptomatology: Primary and recurrent infections result in lesions and inflammation around the genital area and the latter accounts for majority of genital herpes instances. Immunocompromised patients including neonates are susceptible to additional systemic infections including debilitating consequences of nervous system inflammation. Epidemiology, incidence and prevalence: More than 500 million people are infected worldwide and most reported cases involve the age groups between 16-40 years, which coincides with an increase in sexual activity among this age group. While these numbers are an estimate, the actual numbers may be underestimated as many people are asymptomatic or do not report the symptoms. Treatment and curability: Currently prescribed medications, mostly nucleoside analogs, only reduce the symptoms caused by an active infection, but do not eliminate the virus or reduce latency. Therefore, no cure exists against genital herpes and infected patients suffer from periodic recurrences of disease symptoms for their entire lives. Molecular mechanisms of infection: The last few decades have generated many new advances in our understanding of the mechanisms that drive HSV infection. The viral entry receptors such as nectin-1 and HVEM have been identified, cytoskeletal signaling and membrane structures such as filopodia have been directly implicated in viral entry, host motor proteins and their viral ligands have been shown to facilitate capsid transport and many host and HSV proteins have been identified that help with viral replication and pathogenesis. New understanding has emerged on the role of autophagy and other innate immune mechanisms that are subverted to enhance HSV pathogenesis. This review summarizes our current understanding of HSV-2 and associated diseases and available or upcoming new treatments.

An extraordinary binding site generated in heparan sulfate (HS) structures, during its biosynthesis, provides a unique opportunity to interact with multiple protein ligands including viral proteins, and therefore adds tremendous value to this master molecule. An example of such a moiety is the sulfation at the C3 position of glucosamine residues in HS chain via 3-O sulfotransferase (3-OST) enzymes, which generates a unique virus-cell fusion receptor during herpes simplex virus (HSV) entry and spread. Emerging evidence now suggests that the unique patterns in HS sulfation assist multiple viruses in invading host cells at various steps of their life cycles. In addition, sulfated-HS structures are known to assist in invading host defense mechanisms and initiating multiple inflammatory processes; a critical event in the disease development. All these processes are detrimental for the host and therefore raise the question of how HS-sulfation is regulated. Epigenetic modulations have been shown to be implicated in these reactions during HSV infection as well as in HS modifying enzyme sulfotransferases, and therefore pose a critical component in answering it. Interestingly, heparanase (HPSE) activity is shown to be upregulated during virus infection and multiple other diseases assisting in virus replication to promote cell and tissue damage. These phenomena suggest that sulfotransferases and HPSE serve as key players in extracellular matrix remodeling and possibly generating unique signatures in a given disease. Therefore, identifying the epigenetic regulation of OST genes, and HPSE resulting in altered yet specific sulfation patterns in HS chain during virus infection, will be a significant a step toward developing potential diagnostic markers and designing novel therapies.

Heparan sulfate (HS) is ubiquitously expressed on mammalian cells. It is a polysaccharide that binds growth factors, cytokines, and chemokines, and thereby controls several important physiological functions. Ironically, many human pathogens including viruses interact with it for adherence to host cells. HS functions can be regulated by selective modifications and/or selective cleavage of the sugar chains from the cell surface. In mammals, heparanase (HPSE) is the only known enzyme capable of regulating HS functions via a selective endoglycosidase activity that cleaves polymeric HS chains at internal sites. During homeostasis, HPSE expression and its endoglycosidase activity are tightly regulated; however, under stress conditions, including infection, its expression may be upregulated, which could contribute directly to the onset of several disease pathologies. Here we focus on viral infections exemplified by herpes simplex virus, dengue virus, human papillomavirus, respiratory syncytial virus, adenovirus, hepatitis C virus, and porcine respiratory and reproductive syncytial virus to summarize recent advances in understanding the highly significant, but emerging roles, of the enzyme HPSE in viral infection, spread and pathogenesis.